Membrane transport in helminth parasites: A review

EXPERIMENTAL

PARASITOLOGY

37, 469530

(1975)

PARASITOLOGICAL Membrane

Transport

REVIEW

in Helminth

Parasites:

A Review

PETER W. PAPPAS Department of Zoology, College of Biological Sciences, The Ohio State University, Columbus, Ohio 43210

AND CLARK P. READ 1 Department

of Biology,

Rice University,

Houston,

PAPPAS, PETER W., AND READ, CLARK P. 1975. Membrane parasites: A review. Experimental Pamsitology 37, 469-530. I. Introduction II. Amino Acid Absorption: A. The Cestoda: 1. Hymenokpis diminuta and H. citelli 2. Calliobothrium verticillatum 3. Taeniu crassiceps larvae 4. General Considerations B. The Acanthocephala C. The Trematoda: 1. Fasciolu hepatica and Fascidoides magna 2. Schistosoma mansoni D. The Nematoda 111. Carbohydrate Absorption: A. The Cestoda: 1. Hymenolepis diminuta and H. microstoma 2. Callioboth7ium vertidlutum 3. Taeniu crassiceps larvae 4. Taenia (= Hydatigera) taeniaeformis 5. General Considerations B. The Trematoda: 1. Fasciola hepatica 2. Schistosoma mansonf C. The Nematoda IV. Purine, Pyrimidine, and Nucleoside Absorption: A. The Cestoda B. The Trematoda V. Fatty Acid Absorption: A. The Cestoda B. The Trematoda C. The Nematoda VI. Vitamin Absorption: A. The Cestoda B. The Nematoda VII. Relation of Surface Enzymes to Transport

1 Deceased,

December

24, 1973. 469

Copyright All rights

1975 by Academic Press, o3 reproduction in any form

Inc. reserved.

Texas i’i’OO1 transport

in helminth 470 473 473 473 484 486 488 489 491 491

491 494 495 495 495 498 499 499 500 504 504 505 506 507 507 512 514 514 516 516 517 517 518 518

470

PAPPAS AND READ VIII. IX.

Significance of Mediated Transport 521 Summary 523 Acknowledgments 525 References 525 IKDEX DESCRIPTORS: Membrane transport; Active transport; Facilitated diffusion; Diffusion; Ion-coupled transport; Trematoda; Cestoda; Acanthocephala; Nematoda; Hymenolepis diminuta; Hymenolepis cite& Hymenolepis microstoma; Taenia erasverticillatum; siceps larvae; Taenia (= Hydatigem) taeniaeformis; Calliobothrium Schistosoma mansoni; Fasciola hepatica; Fascioloides magna; Moniliformis dubius; Macracanthorhynchus hirudinaceus; Ascaris; Amino acids; Carbohydrates; Monosaccharides; Fatty acids; Purines; Pyrimidines; Nncleosides; Vitamins, water-soluble; Enzymes, tegumentary.

I. INTRODUCTION

During the past 15 yr it has become increasingly apparent that parasitic helminths ( i.e., the Trematoda, Cestoda, Acanthocephala, and Nematoda) possess specific systems for the mediated uptake of organic solutes from the ambient medium. Although several books and review articles dealing solely, or in part, with nutritional mechanisms of parasitic helminths have been published (Read and Simmons 1963; Read 1966, 1968, 1970; Smyth 1966, 1969; von Brand 1952, 1973; Lee 1965; and others), these contributions have included quite limited considerations of transport phenomena in parasitic helminths. In addition, the many published reviews dealing with transport phenomena in mammalian and bacterial systems (see Oxender 1972, for a partial list of references) have ignored the literature dealing with transport in parasitic helminths, though, in many instances, the processes and mechanisms involved are strikingly similar. The purpose of the present review is not solely the presentation of a summary of transport phenomena in parasitic helminths. A second major purpose is a presentation of materials supporting the hypothesis that in the evolution of parasitic helminths there has been a diversification, rather than a simplification, of those systems involved in transmembrane movement of low molecular weight organic compounds. Of the four major parasitic groups mentioned above, the Cestoda have been stud-

ied most extensively. Since cestodes (and acanthocephalans, also) lack a digestive tract all absorbed materials must enter these worms through the external body the external covering. Not surprisingly, body covering, or tegument, of cestodes is cellular in nature and modified morphologically to resemble the mammalian intestinal brush border. Although the acanthocephalan tegument differs morphologically from that of cestodes, it also is obviously modified as an absorptive surface. Trematodes possess two potential absorptive surfaces-the gut epithelium and external tegument. As in cestodes and acanthocephalans, the trematode tegument is cellular and apparently modified for absorptive purposes; published studies dealing with absorptive mechanisms in trematodes have dealt solely with transport across the external tegument. That the teguments of cestodes, acanthocephalans, and trematodes are readily permeable to numerous low molecular weight compounds has been demonstrated by numerous investigators, and these studies are discussed in the following sections. Nematodes typically possess a complete digestive tract and, therefore, two potential absorptive surfaces. However, the outer layers of the nematode body covering (generally referred to as a “cuticle,” but, in fact, composed of modified and keratinized cells (Lumsden 1975) ) are usually not differentiated as an absorptive surface. In addition, there are few experimental data available which indicate clearly that the

MEMBRANE

TRANSPORT

cuticle of most nematodes is readily permeable to metabolically important compounds (some exceptions to this are found among the entomophilic nematodes, and are discuss.ed later in this review). On the contrary, many studies have shown that the typical nematode cuticle is only shghtly permeable to water-soluble organic compounds. For example, the cuticle of Ascuris suum 2 is essentially impermeable to glucose (Mueller 1929; Cavier and Save1 1952; Castro and Fairbairn 1969), vitamin Blz (Zam et al. 1963) and amino acids (Read 1966) ; transcuticular glucose uptake accounts for less than 2% of the total glucose absorbed by adult A. suum (Castro and Fairbairn 1969). According to Weatherly et al. ( 1963), the cuticle of Ascaridiu galli is permeable to glucose and alanine. However, the physiological significance of the small quantities ‘of glucose and alanine which moved across the cuticle of A. galli has been questioned (Read 1966). (Readers unfamiliar with the ultrastructural details of trematode, cestode, and acanthocephalan teguments, and the nematode cuticle, should consult the excellent review by Lumsden (1975) for further details.) Provisionally, the cuticle of most nematodes (entomophilic nematodes excepted) should be considered impermeable to physiologically significant amounts of amino acids and monosaccharides. Unlike the nematode cuticle, the intestine of nematodes is lined with a single Iayer of columnar cells bearing microvilli at the luminal surface. Underlying these cells is a basal lamina. Since nematodes lack a gut musculature the pseudocoelomic fluid is in direct contact with the basal Iamina (see Bird 1971, for a review of the structure of the nematode gut). Therefore, the nematode intestine represents an ideal mono2 Some publications dealing with transport in Ascaris have referred to the species A. lumbricoides. However, since A. lumbricoides and A. suum appear to be distinct species (Ansel and Thibaut 1973), and since all these studies have used material of porcine origin, the species is referred to as A. suum throughout this review.

IN

HELMINTH

PARASITES

471

layer of cells across which transport can be measured. In addition to their lack of a digestive tract, cestodes represent excellent material for studies of transport because many species can be reared in the laboratory under controlled conditions. That is, these systems are amenable to standardization. In 1963, Read and his co-workers conducted numerous experiments attempting to standardize experimental procedures to measure transport phenomena in Hymenolepk diminuta. The results of these investigators’ efforts demonstrated that controlling such factors as number of parasites per host, parasite age, host sex, and age yielded parasites which gave reproducibIe transport data. It has been the present authors’ experience that data obtained from such standardized H. diminuta infections are comparable, regardless of where or when the experiments were conducted. A detailed description of the experiments conducted by Read et al. (1963) is beyond the scope of this review, and interested readers are urged to consult the original publication. techniques More recently, standardized similar to those described by Read et al. (1963) have been applied to studies dealing with transport in other cestode species microstoma and Taenia ( HymenoZepis crassiceps larvae), acanthocephalans (Moniliformis dubius) and trematodes ( Schistosoma marhsoni) . The importance of standardized techniques in the study of transport phenomena can not be overemphasized. However, not all parasites can be raised in the laboratory. For example, the numerous studies on CaL liobothrium verticillatum have used material from naturally infected hosts (Mustelus canis), since the lifecycle of this tetraphyllidean cestode is unknown. However, C. verticillatum is in a sense, “selfstandardizing.” The adult worms are hyperapolytic, and the strobila contains no &avid -proglottids which may confound biochemical and kinetic data. Unlike C. verticillatum, Fasciola hepatica can be

472

PAPPAS

maintained (with some difficulty) in the laboratory. However, the studies dealing with transport in F, hepatica have used infected hosts organisms from naturally only. Therefore, although kinetic data obtained from one experiment may be useful, comparisons of different experiments, or data obtained from different laboratories, may pose a problem due to the variable nature of the hosts and parasites. Typically, transport data can be analyzed using the classical Michaelis-Menten relationship derived for enzymatic reactions. That fact has led investigators to postulate the presence of distinct “carriers” within cell membranes, these carriers being analogous to the active site of an enzyme. Inherent in this carrier hypothesis is the assumption that the discrete carriers are motile and can, therefore, shuttle solute (substrate) molecules across the cell membrane in either direction (see Stein 1967, for a general discussion of the carrier hypothesis). However, the movement of solutes into a cell following MichaelisMenten (saturation) kinetics can also be described by a second hypothesis, the “association-induction hypothesis.” This latter hypothesis states that “the entry of solutes into living cells following Michaelis-Menten kinetics merely indicates the presence of specific adsorption sites for the entrant solute near the cell surface” (Ling et al. 1973). Since both theories require the binding of a solute to some membrane component (either a carrier or “adsorption site”), the primary factor separating these two theories is the question of the presence of discrete carriers. The data supporting either hypothesis of membrane transport is not overwhelming, and both theories allow for transport phenomena to be discussed in terms of Michaelis-Menten kinetics. Therefore, the data presented in the present review have been interpreted in terms of the carrier hypothesis, since this hypothesis is probabIy more familiar to the readers. Some specific types of transport phenomena should be defined before pursuing

AND

READ

the specific transport studies dealing with helminth parasites. Di@sion is the movement of a solute due only to the kinetic energy of the solute molecules; net movement of solute by diffusion occurs only from a region of higher solute concentration to a region of lower solute concentration. The diffusion rate of a given solute through a membrane will depend on certain physical and chemical properties of both the solute and membrane, thus anomalies may result when diffusion through a membrane is compared with diffusion in free solution. However, with most diffusion systems there is a broad solute concentration range in which the rate of absorption and solute concentration will be a linear function (Fig. 1). Unlike active transport (se.e below), diffusion requires the expenditure of no energy on behalf of the organism (cell) absorbing the solute, and stereospecificity is lacking. Examples of helminth parasites absorbing specific compounds by diffusion are discussed in the following sections. Facilitated difusion is similar to diffusion in that solute is not accumulated against a concentration difference. That is, solute moves in relation to the prevailing concentration difference, and no energy is required. However, rather than being a linear function of solute concentration, the rate of absorption due to facilitated diffusion characteristically follows saturation kinetics (Fig. 1). In addition, facilitated diffusion is inhibited by chemicals of identical or similar structures (i.e., the process is stereospecific), and by some metabolic poisons. The absorption of compounds by some parasitic helminths has been attributed to facilitated diffusion, but in almost all instances apparently involving this mediated process the solute being absorbed is metabolized at an extremely rapid rate and not accumulated against a concentration difference. Therefore, before it can be stated absolutely that the absorptive process involved in these latter cases is indeed facilitated diffusion, it is necessary to

MEMBRANE

TRANSPORT

demonstrate that the solute is not accumulated when metabolism of the solute is inhibited. Active transport is identical to facilitated diffusion, with one important addition. A solute that is absorbed by active transport moves against a concentration difference and is, therefore, accumulated. For example, if an organism is placed in a solution containing a solute at 1 mM, the organism may absorb enough solute to raise the internal concentration of solute to greater than 1 mM (I pmole/mI tissue water). Such “uphill” movement of solute requires energy to be expended. The reader should bear in mind that a basic assumption must be made when speaking of solute accumulation, that being that the solute is in free solution in the tissue water. Intracellular binding of solute is a possibility, but no studies are available which indicate that intracellular binding of solute does, or does not Occur in helminths: Some parasitic helminths may absorb a compound by a combination of mediated uptake (facilitated diffusion or active transport) and diffusion. In such cases, the rate of absorption is not linear with respect to substrate concentration at low substrate concentrations, but becomes linear at higher substrate concentrations. Subtracting the apparent diffusion component in such cases results typically in saturation kinetics (Fig. 1). The fact that mediated uptake can be inhibited by structurally similar compounds, while diffusion can not, has been used extensively in discerning the presence of two distinct transport mechanisms for the absorption of a single solute. II.

AMINO

ACID

ABSORPTION

A. The Cestoda 1. Hymenolepis diminuta and H. cite& It is appropriate to begin with a discussion of amino acid transport in Hymenolepis diminuta, since the first experiments exploring the possibility of mediated trans-

IN

HELMINTH

473

PARASITES

/A

I

1’

I

I

/

/

,’

/’

.’

2 s

B C D

/’ /’ /’ 1’ /’ r /’ SUBSTRATE

CONCENTRATION

FIG. 1. A summary of graphic representation of absorption mechanisms. A = diffusion, where the rate of absorption is a linear function of the substrate (solute) concentration. B = saturation kinetics, characteristic of facilitated diflusion and active transport. C = a combination of mediated uptake (i.e., facilitated diffusion or active transport) and diffusion, with the rate of absorption being nonlinear at low substrate concentrations, and linear at high substrate concentrations. D = line C following correction for the apparent diffusion component ( shaded area).

port mechanisms in helminths were conducted with this organism. Daugherty (1957a, b) presented evidence that amino acid uptake by H. diminuta may involve som.e mechanism other than simple diffusion. The absorption of methionine 8 and cysteine by H. diminuta was found to be temperature dependent and stereospecific; methionine uptake was inhibited by alanine, glycine, valine, serine, and lysine (later experiments have not confirmed the inhibition of methionine uptake by lysine), and cysteine uptake was inhibited by glytine, methionine, alanine, and p-alanine. Glutamic, aspartic, and a-ketoglutaric acids inhibited the uptake of neither methionine nor cysteine. These data led Daugherty (1957b) to suggest the presence of at least two systems for amino acid uptake in H. diminuta, one for acidic amino acids, and another for neutral amino acids. In addition, methionine and cysteine uptakes by Raillientina cesticillus were inhibited by neutral amino acids, and the inhibitory actions of amino acids on methionine uptake by H. diminuta and R. cesticillus were de3 Unless noted otherwise, all amino the naturally occurring L-isomers.

acids

are

474

PAPPAS AND READ TABLE A Summary

I

of Kinetic Parameters Describing Mediated Amino Acid Uptake in Hymenolepis. All Amino Acids are L-Isomers Unless Noted Otherwise Reference

Solute (substrate) Hymenolepis

diminuta

Methionine n-Methionine Aspartie acid Glutamic acid Arginine Lysine Alanine

0.368 0.308 1.6 0.88 0.44 0.11 0.42

1110 183a -

@-Alanine Glycine Histidine Isoleucine Leucine Proline

0.30 0.56 0.18 0.35 0.21 0.44

-

Proline Phenylalanine Serine Threonine

0.26gb 0.14 0.94 0.57

10gc -

Valine Cycloleucine cu-Aminoisobutyric

0.50 0.25 0.55

17oc -

0.27d 2.43

1.7gd 1ooc

Read et al. 1963 Read et al. 1963 Read et aE. 1963 Read et al. 1963 Read et al. 1963 Read et al. 1963 Read et al. 1963 Read et al. 1963 Read et al. 1963 Read et al. 1963 Read et al. 1963 Read et al. 1963 Read et al. 1963 Kilejian 196Ga Read et al. 1963 Read et al. 1963 Read et al. 1963 Read et al. 1963 Harris and Read 1968 Harris and Read 1968 Arme and Coates 1973 Laws and Read 1969

437JJ 436a

Senturia Senturia

acid

cu-Aminoisobutyric acid N-methyl-a-aminoisobutyric

acid

Hymenolepis Met,hionine D-Methionine

0.61 0.64

citelli 1964 1964

a gmoles absorbed/g dry wt/hr. b Kilejian (1966a) attributed the difference in her Kc and that of Read et al. (1963) to the fact that the worms she used were slightly larger. c pmoles absorbed/g ethanol extracted dry wt/hr. d Data for cysticercoid larvae. V,,, units = nmoles absorbed/100 larvae/2 min.

creased at low temperatures (Daugherty and Foster 1958). Subsequent investigations have shown that all amino acids absorbed by H. diminuta are absorbed, in part, by mediated processes. That is, uptake is saturable and stereospecific (Tables I and II). Some additional facts demonstrate that methionine uptake by H. diminuta is, in fact, mediated. Methionine uptake was pH sensitive (pH optimum = 8.4) and inhibited by iodoacetate, iodoacetamide, azide, and arsenite, but not by cholate, eserine, phlorizin, pchloromercuribenzoate, ouabin ( Strophan-

thin G), or 2,4-dinitrophenol (Read et al. 1963). The mechanism(s) of the actions of these various inhibitors remains uncertain, but the fact that methionine uptake was inhibited by some metabolic poisons suggests a mediated transport system. The role of diffusion in amino acid uptake by H. diminuta is at present unclear. Diffusion apparently is involved, in part, in proline (Kilejian 1966a) and histidine (Woodward and Read 1969) uptake, while lysine is absorbed entirely through a mediated system (Uglem unpublished). It is

MEMBRANE

TRANSPORT

IN

HELMINTH

TABLE A Summary of Inhibitor Constants Acid Uptake in Hymenolepis. Solute (substrate) Methionine Methionine Methionine Methionine Methionine Methionine Methionine Methionine Methionine Methionine Methionine Met.hionine Methionine Methionine Methionine Methionine Methionine Methionine Methionine Methionine Methionine Methionine Methionine Methionine Methionine Methionine Tyrosine Tyrosine Phenylalanine Phenylalanine Phenylalanine Phenylslanine Cycloleucine Cycloleucine a-Aminoisobutyric cY-Aminoisobutyric

II

(Ki) for Various Amino Acids Actang as Inhibitors of Amino All Amino Acids are LIsomers Unless Noted Otherwise

Ki (d)

Inhibitor

(H. diminuta)

acid acid

475

PARASITES

Methionine n-Methionine Threonine Asparagine Leucine Aspartic acid Glutamic acid Arginine Lysine Alanine @-Alanine Glycine Histidine Isoleucine Proline Phenylalanine Serine Tryptophan Tyrosine Valine Hydroxyproline Cysteine Glutamine Norvaline Norleucine Ethionine Methionine Phenylalanine Methionine Tyrosine Cycloleucine wAminoisobutyric Cycloleucine a-Aminoisobutyric Cycloleucine ol-Aminoisobutyric

0.55s 0.77” 0.85a 2.7” 19.0” coa W” 1.3a 16.0” 33.00 2.5” 1.3” 3.0” 15.0” 1.oa 3.18 3.6” 1.9” 5.2a co” -

acid acid acid

0.297” 0.258” 0.292” 0.250” 0.75c 0.850 0.w 0.2oc 0.60c 0.6jc

IG Wf) (H. cite&) O.SlOb 0.776b 1.176 1.49b 1.55b 6.14b 4.34b 4.90b 2.66” 6.16b 5.90b 2.93b 2.93b 3.096 1.706 0.74b 2.14b 1.15b -

LLData from Read et al. (1963). b Data from Senturia (1964). c Data from Harris and Read (1968).

not known whether H. diminuta absorbs other amino acids, in part, by diffusion. Only a few amino acids are known to be absorbed by active transport by H. diminuta. H. diminuta accumulated methionine (Read et al. 1963; Pappas et al. 1974), proline (Kilejian 1966b), histidine (Woodward and Read 1969), cycloleucine and CY-

aminoisobutyric acid (Harris and Read 1968 ) against a concentration difference (Fig. 2); in the strict sense, these four amino acids are absorbed by active transport. Attempts to demonstrate accumulation of other amino acids have, apparently, not been undertaken, so it may be that other amino acids ar.e accumulated as well.

476

PAPPAS AND READ

20

40

60

TIME

FIG. 2. The internal concentration of amino acid ( [AMINO ACID], pmoles/mI worm water) following incubation of Hymenolepis diminuta for increasing time periods (mm). Line A = incubation of worms in 2mM methionine (redrawn from Pappas et al. 1974); Line B = incubation in 2 mA4 proline (redrawn from Kilejian 1966b); Line C = incubation of worms in 2 mM methionine (redrawn from Read et al. 1963); Line D = incubation of worms in 0.1 mM histidine (in this instance the internal amino acid concentration is reported as pmoles/g ethanol extracted dry wt; thus, the histidine concentration at 60 min is approx 1.1 mM) (redrawn from Woodward and Read 1969); Line E = incubation of worms in 0.1 mM cycloleucine (redrawn from Harris and Read 1968). The single point represents the internal concentration of cr-aminoisobutyric acid following incubation of worms in 0.1 mM n-aminoisobutyric acid for 60 min (data from Harris and Read 1968). Note th’at in all cases, the concentration of amino acid inside H. diminut~ exceeds that of the incubation media.

It is apparent that H. diminuta is limited with regard to the amount of a specific amino acid accumulated. When incubated in 2 mM methionine for 40 min or longer, the internal methionine concentration of worms reached a steady state (efflux = influx) ‘of about 7.5 mM (Read et ~2. 1963) or 10.5 mM (Pappas et al. 1974); the latter value agrees well with the value of 10.3 mM derived by Read et al. (1963) for the steady state distribution ratio of methionine based on short-term (2 min) incubations of H. diminuta in radioactive methionine. When incubated in 2 mM proline for 45 min or longer, the internal proline concen-

tration of H. diminuta reached a steady state of about 11 mM (Kilejian 196613). That a steady state was, in fact, reached after a 60 min incubation in 0.5 mM proline was shown by the observation that experimentally derived efflux and influx rates were equal (Kilejian 1966a). Although it is clear that H. diminuta absorbs all amino acids tested, in part, by mediated processes, and that some amino acids are absorbed by active transport, the number of distinct amino acid transport systems remains somewhat unclear. Probably the most clearly definable amino acid system of H. diminutu is that for basic amino acids. The presence of this system was first suggested by the data of Read et al. ( 1963), which showed that arginine and lysine uptake rates were nonlinear with respect to concentration ( saturable), and that these basic amino acids were mutual competitive inhibitors of each other. Also, of the numerous neutral, basic, and aromatic amino acids, and amino acid derivatives and dipeptides tested as inhibitors of lysine uptake by H. diminuta, ‘only arginine, lysine, ornithine, canavanine, and histidine inhibited uptake. Clearly, the basic amino acids do not interact with neutral, acidic or aromatic amino acids during absorption. Hymenolepis diminutn also has a distinct transport system with a high affinity for dicarboxylic amino acids, and a low affinity for some neutral amino acids as well. The data supporting the existence of this system are as follows. Glutamic acid uptake was inhibi’ted by methionine, leutine, and proline, while methionine uptake was inhibited by glutamic acid; glutamic acid uptake was not inhibited by histidine, but histidine inhibited the uptake of several neutral amino acids, the latter of which inhibited ghrtamic acid uptake (hence, the dicarboxylic amino acid system has an affinity for neutral amino acids, but not histidine, and histidine inhibits neutral amino acid uptake through a system other

MEMBRANE

TRANSPORT

than the dicarboxylic amino acid system) ; aspartic acid was a better inhibitor of glutamic acid uptake than of methionine or histidine uptake (Read et al. 1963). Data available currently indicate the presence of at least tu;o distinct systems for neutral amino acid transport by H. diwere minuta. Tyrosine and methionine strong inhibitors of phenylalanine uptake by H. diminuta, and phenylalanine and methionine were strong inhibitors of tyrosine uptake. But phenylalanine and tyrosine were only weak inhibitors of methionine uptake by H. diminuta (Read et al. 1963). Also, the Ki (inhibitor constant, the reciprocal of which is a measure of the affinity of an inhibitor for a transport system) of methionine acting as an inhibitor of phenylalanine or tyrosine uptake approximated the Ki of methionine acting as an inhibitor of methionine uptake (Table II), and of neutral amino acids acting as inhibitors of methionine uptake (Read et al. 1963). Read et al. (1963) also tes’ted 13 phenylalanine and tyrosine derivatives as inhibitors of phenylalanine, tyrosine, and methionine uptake. All inhibitors were found to inhibit tyrosine and phenylalanine uptake, while only four of these derivatives inhibited methionine uptake. Therefore, as postulated by Read et al. (1963), there appears to be at least two neutral amino acid transport systems, one with a high affinity for aromatic amino acids and a low afhnity for neutral amino acids, and another with a high affinity fmor neutral amino acids. The presence of two neutral amino acid systems in H. diminuta was indicated further by the data of Harris and Read ( 1968). These authors found that the uptake of labeled cycloleucine (which was absorbed by active transport and not metabolized) was inhibited competitively by a-aminoisobutyric acid, alanine, proline, valine, leucine, glutamic acid, phenylalanine, and histidine, but not by lysine or arginine. Cycloleucine inhibited competi-

IN

HJZLMINTH

PARASITES

477

tively the uptakes of leucine, valine, (Yaminoisobutyric acid, phenylalanine, alanine, histidine, and glutamic acid, but had no effect on lysine uptake. These data demonstrated clearly that cycloleucine interacted quite strongly with most neutral amino acids, but ‘only weakly with glutamic acid. A calculation of respective inhibitor constants (K,) demonstrated that at least two transport systems were involved in cycloleucine absorption. The Kt for cycloleucine uptake approximated the Ki of cycloleucine acting as an inhibitor of cycloleucine or a-aminoisobutyric acid uptake, while the Kt of a-aminoisobutyric acid uptake approximated the K6 of this amino acid acting as an inhibitor for aaminoisobutyric acid or cycloleucine uptake. However, the Kt for phenylalanine uptake was only about 20% of the Ki of phenylalanine acting as an inhibitor of cycloleucine or a-aminoisobutyric acid uptake (i.e., cycloleucine and a-aminoisobutyric acid were better inhibitors of the uptake of each other than was phenylalanine) (Tables I and II). In addition, if proline or phenylalanine was added to a system in which cyclosleucine or a-aminoisobutyric acid uptake was already maximally inhibited by phenylalanine or proline, respectively, the inhibition of amino acid uptake was increased. Thus, cycloleucine and aaminoisobutyric acid entered H. diminuta through two distinct systems, one inhibited by phenylalanine, and the other by proline (Harris and Read 1968). More recent studies by MacInnis, Graff, Kilejian, and Read (unpublished), in which the reciprocal interactions omfall possible pairs of 14 amino acids were studied, have suggested the presence of at least two additional neutral amino acid transport systems in H. diminuta (Table III). The four postulated systems possess a rather broad specificity for neutral amino acids. In the preceding discussion several references have been made to the fact that the basic amino acid transport system of H.

478

PAPPAS

AND

TABLE The Am&o

READ

III

dcid Transport Systems Identified (from Madnnis, Grafl, Kilejian

in the Cestode, Hgmenolepis

dimiwuta

and Read, Unpublished)

~______ System Dicarboxylic

Glycine

Major amino acids interacting

Aspartic Glutamic Methionine

Glycine Methionine

Other amino acids

Serine Alanine Glycine

Serine Threonine Alanine

diminuta displays a high degree of specificity, since neutral and dicarboxylic amino acids do not interact with the basic amino acids, and vice versa. The one exception to this is the amino acid histidine which inhibited the uptake of both neutral and basic amino acids (Read et aZ. 1963). This

@HIDING

FIG. 3. The velocity (pmoles absorbed/g ethanol extracted dry wt/Z min) of histidine uptake by Hymenolepis diminuta as a function of histidine concentration (mM). Line A = histidine uptake in the absence of inhibitor. Line B = histidine uptake in the presence of a constant 4 mM phenylalanine. Line C = histidine uptake in the presence of a constant 4 mM arginine. Line D = histidine uptake in the presence of 4 mM phenylalanine + 4 mM arginine. The shaded area represents the apparent diffusion component for histidine uptake by H. diminuta. Redrawn from Woodward and Read ( 1969).

Serine

Leucine

Phenylalanine

Dibasic

Serine Alanine Threonine Methionine Valine I’roline

Leucine Isoleucine Methionine

Phenylalanine Tryosine Histidine Methionine

Arginine Lysine Histidine

Glycine

Glycine Serine Threonine Alanine Valine

Leucine Isoleucine

overlapping affinity of histidine for two distinct systems was studied by Woodward and Read (1969). These authors demonstrated that, in additi’on to a small diffusion component, histidine entered H. diminuta through the basic amino acid system (inhibited by arginine), and one of the neutral amino acid systems (inhibited by phenylalanine). Since phenylalanine and arginine did not interact, labeled histidine uptake through the neutral or basic amino acid system could be studied in the presence of high concentrations of arginine or phenylalanine, respectively; the contributions of these two amino acid transport systems during histidine uptake were additive in that their sum equaled histidine uptake in the absence of inhibitor (Fig. 3). Apparently, only one of the neutral amino acid systems was involved in histidine uptake, since in the presence of saturating eoncentrations of phenylalanine, or phenylalanine plus arginine, leucine did not inhibit histidine uptake (Woodward and Read 1969). That two transport systems were involved in histidine absorption by H. diminuta was supported further by efllux experiments conducted by Woodward and Read ( 1969)) and discussed later in this review. The studies of MacInnis et al, (unpublished) have recently substantiated the

MEMBRANE

TRANSPORT

presence of the amino acid transport systems discussed in the above paragraphs. Thus, there appears to be (1) a basic amino acid transport system which also transports histidine, (2) a dicarboxylic amino acid system with a low affinity for some neutral amino acids, and (3) at least four (not two) neutral amino acid transport systems, one of which also transports aromatic amino acids and histidine. The interactions of amino acids with these various transport systems is summarized in Table III.

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PARASITES

479

Although experiments utilizing single inhibitors and/or substrates in transport studi.es are helpful in determining the specificity of transport systems, these studies provide no information concerning how these systems may function in more complex environments, such as in the presence of several potential inhibitors. Read et al. (1963) derived an equation from classical Michaelis-Menten enzyme theory describing the effects of a complex amino acid mixture on the uptake of a single amino acid :

V max

VI = &+1+

(Kt) KW) + UL) KfLl) +. . .0-G)KfLl) ’ (Kd (cm) Wtd (cm) uw (Cfck)

where: Vr = velocity of the inhibited uptake; V,,, = maximum velocity of the uninhibited uptake; Kt = transport constant of the amino acid of which the uptake is being measured; IL, Ktz, etc. = transport constants of the inhibitors; [S] = concentration of amino acid of which the uptake is being measured; [S,], [S,], etc. =concentration of inhibitors. To determine whether this derived equation described accurately the inhibitory effects of an amino acid mixture on the uptake of a single labeled amino acid, Read et al. (1963) tested a number of amino acid mixtures on the uptake of radioactive methionine by H. diminuta. These authors found that if Ki values (inhibitor constants) for the inhibitors were used, rather than Kt values (transport constants), this equation described accurately the effects of specified amino acid mixtures on methionine uptake. When Kt values for all competing amino acids were substituted into the above equation, an accurate prediction of the effect was not possible. The data presented in Tables I-III for amino acid transport in H. diminuta have been derived from studies of inhibitory interactions of various amino acids, and

it can not be concluded that all amino acids interacting with a particular system are also translocated by that system. It is possible that an amino acid may bind reversibly with a specific transport system without subsequent translocation across the tegument. Such a phenomenon has been termed nonproductive (abortive) binding, and although no specific instances of this have been demonstrated in amino acid transport in H. diminuta (probably because it has not been investigated), there are examples of nonproductive binding of purine and pyrimidine bases in H. diminuta (Pappas et al. 1973a). The differences in Kt and Ki values for various amino acids reported by Read et al. (1963) may be attributed to nonproductive binding; the value of K, is directly related to solute translocation, while the value of Ki is influenced by both translocation and nonproductive binding. Physiologically, however, nonproductive binding would be important in determining the influx rates of some individual components in an amino acid mixture. Therefore, the nonproductive binding phenomenon would also explain the observation of Read et al. (1963) that a reliable result was obtained by the use of

480

PAPPAS

Ki, rather than Kt, values in their equation predicting the uptake of a single amino acid from a complex mixture. The phenomenon of nonproductive binding in amino acid transport in H. diminuta warrants further investigation. Although each of the postulated six amino acid transport systems of H. diminuta apparently bind several amino acids, the systems, taken together, are quite specific for a-amino acids. The uptake of radioactive methionine by H. diminuta was not inhibited by di- or tripeptides (Read et al. 1963), and proline uptake was not inhibited by glucose (Kilejian 1966a ). Blocking the a-amino group of a potential inhibitor destroyed its inhibitory action; while both valine and methionine inhibited radioactive methionine uptake by H. diminuta, N-acetyl-m-valine and N-acetyl-mmethionine did not affect methionine uptake. In addition, moving the o-amino group of a potential inhibitor to a different carbon atom decreased dramatically the affinity of the inhibitor for the amino acid transport systems; o(- and p-aminoisobutyric and y-aminobutyric acids inhibited methionine uptake by H. diminuta 89, 33, and aspartic O%h, respectively, and inhibited acid uptake 51, 33, and OS, respectively (Read et aZ. 1963). The data presented by Laws and Read (1969) confirmed the importance of an unsubstituted a-amino group for maximum interaction of amino acids with the amino acid transport systems. These authors demonstrated that N-methyla-aminoisobutyric and a-aminoisobutyric acids were absorbed by H. diminuta through the same transport system(s), but that the affinity of this (these) system(s) for N-methyl-a aminoisobutyric acid was approximately five times less than for aaminoisobutyric acid (Table I). In addition, the charge, or lack thereof, on the a-amino group was demonstrated to be important, since oxamic acid and acetamidoxine had no inhibitory effect on neutral amino acid uptake by H. diminuta (the

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a-amino groups of oxamic acid and acetamidoxine are uncharged at pH 7.4, unlike the a-amino groups of naturally occurring amino acids). Technical difficulties in synthesizing additional derivatives have prevented further study along this line. The data discussed in the above paragraphs were obtained using the standardized techniques of Read et al. ( 1963), techniques which are still used widely today. Basically, these techniques involve lo-day-old (11-day-old, in some cases) H. diminuta reared in 60-100 g male rats. The importance of using standardized techniques was shown by Read et al. (1963) when they demonstrated that the kinetic parameters of methionine transport in H. diminuta varied with worm age. Kilejian (1966a) demonstrated a similar effect of worm size on the K, for proline uptake by this cestode (Table I). The effects of some inhibitors were also age dependent; p-alanine inhibited methionine uptake in 9- and lo-day-old worms, but had little effect on methionine uptake by ll- and 12-day-old worms (Read et al. 1963). The host species in which H. diminuta was reared affected significantly the amino acid transport systems of worms. When H. diminuta was reared in rats or golden hamsters (each with 30 worm infections), and the worms harvested 10 days postexposure, not only was the apparent affinity for methionine different in the two groups of worms, but marked decreases in the inhibition of methionine uptake by hydroxyproline, glycine, histidinc, phenylalanine, and aspartic acid were noted in worms reared in hamsters (Read et al. 1963). The basis of these alterations in amino acid transport systems during worm maturation, or worm growth in different hosts, is unknown, but these results demonstrated the necessity of standardized techniques. The literature dealing with amino acid influx (uptake) is quite s’traightforward, since it is very simple to control the chemical composition of the incubation medium;

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thus, experiments designed to demonstrate the presence or absence of a specific phenomenon are easily controlled. Defining the conditions of efflux experiments, on the other hand, is more difficult, and these experiments usually contain some inherent problems. Efflux experiments are generally conducted by first incubating worms in a specific radioactive substrate for a given time period, and then transferring these preloaded worms to various media of defined composition for a predetermined time period. The effects of various media on amino acid efflux are then determined by measuring the amount of radioactive amino acid remaining in the worms following efflux, and comparing this value with that from worms which were preloaded and placed in saline for the efflux period. The mos’t difficult problem involved in efflux experiments is defining the physicochemical state of the solute within the worm’s tissues; is the solute in free solution, or is it bound (adsorbed) to some cellular component? One must also consider this same question in regard to the cellular water; is the intracellular water available for solute dissolution, or is some of the water ordered (due to interaction with hydrophilic moieties) and not available for solute dissolution? Although attempts have been made to answer these questions in other systems (see Hazelwood 1973, and references therein), no data are available concerning the physicochemical state of water and solutes in parasitic helminths. Last, one must consider the localization of solute within the worm’s tissues after preloading in radioactive solute. Once the solute is absorbed, is it readily available for efflux, or is the solute compartmentalized? With regard to this last problem, both H. diminuta and Taenia crassiceps larvae apparently compartmentalize proline. Kilejian (1966b) demonstrated that of the 24 pmoles/g dry wt of proline absorbed by H. diminuta in 60 min, only 15 pmoles/g dry wt were readily exchangeable with proline

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in the ambient medium. Pappas and Read (1973) found that T. crassiceps larvae absorbed proline against an apparent concentration difference by a nonmediated system (diffusion). In both cases the authors reasoned that proline was compartmentalized and, therefore, not contributing to the free proline pool of worms. There is ample evidence that amino acid efflux from H. diminuta is mediated (this, of course, must be the case if the carrier hypothesis is accepted as valid). Read et al. (1963) first demonstrated mediated amino acid efflux by preincubating worms in radioactive methionine for 2 min, and then placing these worms in either saline (controls), or saline containing a single amino acid, and measuring methionine efflux. These authors found good agreement of the calculated l/KS values for each amino acid acting as an inhibitor of methionine uptake (i.e., the effectiveness of the amino acids as inhibitors) and the relative activity of each amino acid in exchanging with absorbed methionine. The data ‘of Read et aZ. (1963) represent an excellent example of exchange diffusion (mediated efflux). These authors also attempted to demonstrate a second form of mediated e&x, namely counterflow (countertransport). After a 1-min incubation in 0.1 mM labeled methionine, worms were transferred to a solution containing 0.1 mM radioactive methionine and a single unlabeled amino acid. In the presence of 1 mM serine, phenylalanine or isoleucine in the efflux medium, radioactive methionine effluxed from the worms. However, whether this methionine .efflux was against a methionine concentration difference, a necessary prerequisite for counter-flow, is uncertain. This is discussed further below. When H. diminuta was preloaded in 0.1 mM radioactive histidine and subsequently transferred to media containing unlabeled arginine, phenylalanine, or histidine, efllux of labeled histidine from worms was stimulated over that of controls which were pre-

482

PAPPAS AND READ

I .I

I .3

I .5 AMINO

I .l

I 1.0

ACID

FIG. 4. The effect of external amino acid (abscissa, mM) on the efflux of absorbed histidine from Hymenolepis diminuta. Sample were preincubated in 0.1 m&4 [“Clhistidine for 15 min, and then placed in the efflux medium for 15 min; values of the amount of radioactive histidine remaining after the 15 min e&x period are reported as pmoles/g ethanol extracted dry wt. Line A = arginine or phenylalanine in the efflux medium. Line B = arginine plus phenylalanine (each at the indicated concentration) in the efflux medium. Line C = histidine in the efflux medium. Redrawn from Woodward and Read ( 1969).

incubated and placed in saline. As was the case with absorption data, the effects of arginine and phenylalanine on labeled histidine efflux were additive, in that the sum of the effects of saturating concentrations of arginine and phenylalanine on histidine efflux were identical to the effect of unlabeled histidine alone (Woodward and Read 1969) (Fig. 4). These data demonstrated histidine efflux through two kinetically distinct systems. Kilejian (1966a) found that proline efflux from H. diminutu was stimulated in the presence of amino acids in the external medium, and that intracellular proline and methionine competed during efflux. Both Woodward and Read (1969) and Kilejian (1966a) suggested that mediated amino acid efflux from H. diminutu occurred through the process of counterflow, since previously absorbed amino acids had no effect on subsequent amino acid uptake. Although the data of these authors demonstrated mediated amino acid efllux, their interpretation

of the efflux mechanism may require reevaluation, as discussed below. Increased amino acid efflux in the presence of aminmo acids in ambient medium was also demonstrated by Hopkins and Callow ( 1965) and Arme and Read ( 1969). In both studies it was found that amino acid efflux from H. diminuta was stimulated when preloaded worms were placed in a medium having an amino acid composition similar to that found in the rat intestine. In addition, Arme and Read (1969) demonstrated stimulated cycloleutine efllux from H. diminuta in the presence of single amino acids, but not monosaccharides, in the ambient medium. Apparently, amino acid efflux is stimulated only by amino acids. It is of interest to ask whether results obtained with H. diminuta in vitro reflect the true characteristics of amino acid fluxes of H. diminuta in uiuo. The available literature indicates that, in fact, such fluxes in vivo and in vitro are similar when the simplified chemical nature of the in vitro medium is taken into account. Hopkins and Callow (1965) demonstrated that when H. diminuta was preloaded for 10 min in 2 mM radioactive methionine and placed in a solution with an amino acid composition similar to that found in the rat intestine, the efllux of methionine from worms was similar to that of worms preloaded and implanted surgically into the rat intestine. Arme and Read (1969) obtained results similar to those of Hopkins and Callow ( 1965) when studying radioactive cycloleucine efflux from H. diminuta, and both studies demonstrated that amino acid efflux in vivo was an exponential function of time. Arme and Read (1969) demonstrated further that the rate of cycloleucine efflux from preloaded worms implanted surgically into the rat intestine was intermediate between the efflux rates of preloaded worms placed in amino acid mixtures with final amino acid concentrations of 4.06 and 8.12 mM; the experimentally determined total amino

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acid concentration in the rat intestine varied from 6 to 21 mM. In this latter instance, efflux rates in viva and in vitro were comparable. Kilejian (196613) and Arme and Read (1969) also studied amino acid efflux in vivo by feeding infected rats large doses of single amino acids, amino acid mixtures, or carbohydrates. Kilejian (196613) fed rats infected with H. diminuta ( 1) 100 mg proline, (2) 400 mg proline, or (3) 100 mg proline plus 100 mg of an amino acid mixture (containing hydroxyproline, alanine, a-aminoisobutyric acid, threonine, leucine, methionine, glycine, and serine) three times at hourly intervals. Hymenolepis diminuta removed from the proline-fed rats had an increased proline concentration proportional to the amount of proline fed the rats. Worms from rats fed proline plus the amino acid mixture had a greatly reduced internal proline concentration, presumably resuhing from increased proline efflux and inhibited proline uptake by worms. When infected rats, previously injected with labeled cycloleucine (this amino acid was metabolized by neither rats nor worms), were fed 250 mg lysine, proline, fructose, glucose, or acetate, only proline and lysine produced demonstrable effects on the quantity of cycloleucine in H. diminuta recovered from these rats; the amount of radioactive cycloleucine increased in worms from rats fed lysine, and decreased in worms from rats fed proline (Arme and Read 1969). (The total concentration of amino acids in the rat intestine also increased significantly after administration of proline or lysine. Although the absolute concentrations of amino acids in the rat intestine changed, their relative concentrations remained fairly constant [Anne and Read 1969; see also Read 1970; Mettrick 19721. ) The effects of proline and lysine on cycloleucine fluxes in H. diminuta in vivo were explained as follows. Since proline inhibited cycloleucine influx and stimulated cycloleucine efflux in vitro, the

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cycloleucine level in H. diminutu from rats fed proline was lowered. In dysine-fed rats the amount of labeled cycloleucine in worms increased since lysine stimulated cycloleucine efflux from rat intestine in vitro, but did not affect cycloleucine fluxes in H. diminuta in vitro. Therefore, cycloleucine effluxed from the rat intestine and was absorbed by the worms in the lysinefed rats ( Arme and Read 1969). In the above discussion several references have been made to the phenomenon of counterflow ( countertransport). Although several investigators showed mediated amino acid efflux from H. diminutu in the presence of amino acids in the external medium (Read et al. 1963; Hopkins and Callow 1965; Kilejian 1966a; Woodward and Read 1969; Arme and Read 1969), the experiments of these authors have not always differentiated clearly the phenomena of counterflow and exchange diffusion. Read et al. (1963) presented data which were interpreted as indicating counterflow of methionine in H. diminutu. Following a 1-min incubation in 0.1 mM radioactive methionine, worms were transferred to media containing the same concentration of radioactive methionine plus 1 mM serine, phenylalanine, or isoleucine. In these experiments labeled methionine effluxed from worms. Although Read et al. (1963) suggested that this efflux was against a methionine concentration difference (a necessary prerequisite for counterflow ) , this appears not to have been the case. According to Read et al. (1963, see their Table 8) uptake of 0.1 mM methionine (as determined from 2-min incubations) was 50 pmoles/g dry wt/hr, or approximately 12.5 pmoles/g worm water/hr. Following a 1-min incubation the internal methionine concentration would, therefor.e, be expected to be at least 0.21 mM if methionine was distributed homogeneously within the worm water. Since homogeneous distribution after a lmin incubation is unlikely, the actual methionine concentration was probably some-

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what higher. Thus, efflux of methionine as described by Read et ~2. ( 1963), although stimulated in the presence of external amino acids, was not against a methionine concentration difference. Woodward and Read (1969) presented data demonstrating both counterflow and exchange diffusion of amino acids in N. diminuta. Worms were preloaded in 0.1 mM radioactive histidine for 5 min resulting in an internal concentration of approximately 0.5 mM (2 ymoles/g dry wt ). When thes.e worms were placed in solutions containing increasing concentrations of histidine, or histidine plus phenylalanine and/ or arginine, efflux of histidine occurred against an apparent histidine concentration difference. Efflux of histidine was also stimulated in the presence of arginine and/ or phenylalanine when histidine was not present in the external medium. Thus, these experiments demonstrated both counterflow and axchange diffusion. Woodward and Read (1969) presented additional evidence af functional exchange diffusion in that the effects of external histidine, phenylalanine, and arginine on histidine efflux from H. diminuta r.eached a maximum with increasing concentration, demonstrating saturation of the efflux system. Hopkins and Callow (1965) and Kilejian (1966a) demonstrated mediated amino acid efllux in H. diminuta, and Kilejian (196617) suggested that counterflow, rather than exchange diffusion, was responsible for proline efflux. As pointed out by Woodward and Read ( 1969), the phenomena of counterflow and exchange diffusion can be accounted for by identical mechanisms, and may indeed be a single phenomenon. By simply altering the experimental conditions each phenomenon can be demonstrated independently of the other, Therefore, one should not conclude that amino acid efllux from H. diminuta occurs by exchange diffusion or counterflow, but that either mechanism of mediated amino acid efflux day b.e functional depending on the experimental design.

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Like H. diminuta adults, H. diminuta larvae (cysticercoids) absorb a-aminoisobutyric acid by active transport. Uptake of acid was saturable a-aminoisobutyric (Table I) and was inhibited competitively by alanine and aspartic acid (Arme and Coates 1971, 1973). Uptake of or-aminoisobutyric acid was also inhibited by cyanide, iodoacetate, and neutral, aromatic, and dicarboxylic amino acids. Uptake was not inhibited by lysine, arginine, monosaccharides, purines, pyrimidines, or fatty acids. Hymenolepis diminuta cysticercoids accumulated a-aminoisobutyric acid against a concentration difference; following a 30min incubation in 0.4, 0.8, or 2 mM radioactive a-aminoisobutyric acid, larvae accumulated a-aminoisobutyric acid 16.5-, ll-, or &S-fold, respectively; the amino acid was not metabolized (Arme and Coates 1973). These data suggest a similarity between the amino acid transport system(s) of H. diminuta adults and cysticercoids, but additional studies are needed to elucidate further the amino acid transport system(s) of larvae. Hymenolepis cite& reared in golden hamsters, actively transports methionine. Methionine uptake was saturable (Table I), and inhibited competitively by neutral and aromatic amino acids (Table II). Dicarboxylic and basic amino acids, and di- and tripeptides were without effect on methionine uptake, Hymenolepis citelli accumulated methionine against a concentration difference; following a 60-min incubation in 1 mM methionine, the internal methionine concentration of worms reached a steady state of approximately 18 mM ( Senturia 1964). Using the equation derived by Read et al. (1963), Senturia (1964) showed that the effects of amino acid mixtures on the uptake of methionine by H. cite& could be predicted accurately, thus demonstrating the applicability of this equation to other cestodes. 2. Calliobotkrium verticillatum. Valine and leucine uptake by Calliobothrium ver-

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IN

tidatum is mediated. In addition to temperature dependence (Qlo > 3, 5-20 C), and pH sensitivity (optimum = pH 7.4), valine and leucine uptakes followed saturation kinetics (Table IV). Leucine and valine were reciprocal competitive inhibitors, with Ki values similar to their respective Kt values (Table IV and V ) , suggesting a common transport system for these two amino acids. Uptake of both amino acids was inhibited by 2,4-dinitrophenol and iodoacetate, but not by glucose in the ambient medium (Read et al. 1960b). Valine and leucine were accumulated against a concentration difference, thus both amino acids were absorbed by active transport. Experiments testing the effects of various inhibitors on the uptake of radioactive amino acids were conducted by Read et al. (196Oa, b). Valine and serine were reciprocal competitive inhibitors, and threonine and alanine were competitive inhibitors of valine uptake by C. verticillatum. Basic amino acids had no effect on neutral amino acid uptake. Efflux of labeled valine from preloaded C. verticiElatum was enhanced in the presence of valine or alaTABLE

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TABLE

IV

A Summaq of Kinetic Parameters Describing Amino Acid Uptake in Calliobothrium verticillatum and Taenia crassiceps Larvae. All Amino Acids are tilsomers Unless Noted Otherwise Solute (substrate)

v

Reference

(f&

max

Calliobothrzum 1 2

Leucine Valine

verticillatum -

Read et al. 1960a Read et al. 1960a

Taenia crassiceps larvaea Phenylalanine Arginine Lysine

0.43 0.38 0.8

27 27 20

Methionine

0.4

57

Pappas et cd. 1973b Pa.ppas et al. 197313 Unpublished (data from Pappas and Read 1973) Unpublished (data from Pappas and Read 1973)

a Data from Haynes and Taylor (1968) and Haynes (1970) have not been included in this table, since they were not corrected for diffusion. V,,, units = rmoles absorbed/g ethanol extracted dry &/hr.

nine in the ambient medium, demonstrating mediated amino acid e&x (Read et al. 1960a). These data suggested the presence V

A Summary of Inhibitor Constants (Ki) for Various Amtino Acids Acting as Inhibitors Amino Acid Uptake in Calliobothrium verticillatum and Taenia crassiceps Larvae. All Amino Acids are Msomers Unless Noted Otherwise Solute (substrate)

Inhibitor

Calliobothrium Leucine Valine

Valine Leucine

Ki (mM)

of

Reference

verticillatum l-2 2.5

Read et al. 1960a Read et al. 1960a

Taenia crassiceps larvaea Phenylalanine Phenylalanine Phenylalanine Phenylalanine Phenylalanine Phenylalanine Arginine Arginine

Tyrosine Histidine Alanine Methionine Leucine Serine Lysine Ornithine

0.76 0.86 4.17 4.76 5.26 7.14 1.49 3.22

Pappas Pappas Pappas Pappas Pappas Pappas Pappas Pappas

et al. et al. et al. et al. et al. et al. et al. et al.

1973b 1973b 197313 1973b 1973b 1973b 1973b 1973b

5 The data of Hanes (1970) have not been included since they were derived from uptake data not corrected for diffusion.

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of at least two transport systems in C. oerticillatum, one for neutral amino acids, and another for basic amino acids. Conclusions regarding the existence of additional amino acid transport systems in this cestode are unwarranted due to the lack of additional data. Read and Simmons (1962) demonstrated that the uptake of valine, serine, or threonine was inhibited competitively by a mixture of 15 amino acids. However, addition of methionine, previously shown to be an inhibitor of vahne uptake (Read et al. 196Ob), to this amino acid mixture did not alter the effects of the mixture on valine uptake by C. verticillatum. As pointed out by Read and Simmons (1962), these observations demonstrate that data derived from simple systems, such as in. vitro incubations with a single amino acid, may not be directly applicable to more complex systems. However, it would be of interest to reexamine this lack of increased inhibition in the presence of methionine plus an amino acid mixture using the equation of Read et al. ( 1963), and determine whether methionine would be expected to make a significant additional contribution to the inhibition of valine uptake. The gut of Mustelus canis (the host of C. vetiicillatum) contains approximately 300 mM urea, and approximately 470 of the dry weight of C. verticillatum is urea (Read et al. 1959). Since urea was shown to be an important constituent in maintaining the osmotic balance in C. verticillatum by Read et al. ( 1959), the kinetics of urea permeation in C. verticillatum were studied. Ur.ea permeation into C. verticillatum was independent of pH and Qlo values (O-21 C) were less than 1.5. In addition, the rate of urea permeation was linear with respect to urea concentration, and accumulation of urea was not demonstrable (Simmons et al. 1960). Therefore, urea influx into C. verticillatum is an excellent example of absorption by diffusion, 3. Taenia crassiceps larvae. Haynes and TayIor (1968) reported mediated amino

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acid transport in Taeniu crassiceps larvae (cysticerci). However, the data of these authors must be interpreted cautiously for two reasons: ( 1) The incubation periods used by Haynes and Taylor (1968) were relatively long (up to 32 min and longer) in some instances, and it is possible that the uptake rates over these long incubation periods were not an accurate measure of the initial uptake velocity; (2) Haynes and Taylor (1968) did not recognize the contribution of diffusion in amino acid uptake by T. crassiceps larvae, and the validity of graphical analyses without correcting for diffusion must be questioned. Haynes (1970) reported subsequently the active transport of several amino acids by T. crassiceps larvae. Haynes’ (1970) conclusions were based on the observations that amino acid uptake by larvae displayed saturation kinetics, and that amino acids were apparently accumulated against a concentration difference. However, these experiments involved quite limited substrate concentration ranges (0.1-0.5 mM or l-5 mM), and no correction appears to have been made for diffusion. Further, the conclusion of Haynes (1970) that amino acids were accumulated by larvae was based solely on the distribution ratios of radioactivity ( [radioactivity/ml worm waincubation ter]/[radioactivity/ml medium] ), without consideration of possible amino acid metabolism. Hence, the data of Haynes and Taylor (1968) and Haynes (1970) must be accepted with some reservations. The uptake of methionine, phenylalanine, arginine, and lysine by T. crassiceps larvae is mediated; influx of these amino acids was not linear with respect to concentration at low substrate concentraitons (Table IV). At higher substrate concentrations uptake of these four amino acids occurred predominately by diffusion (i.e., uptake became a linear function of substrate concentration) (Haynes and Taylor 1968; Haynes 1970; Pappas and Read 1973; Pappas et al. 1973b). Methionine and phenyl-

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alanine were accumulated against an apparent concentration difference, but accucould not be mulation of arginine demonstrated since it was rapidly metabolized to ornithine, proline, and an unidentified compound; following a 90-min incubation in 0.1 mM radioactive arginine, only 7.5% of the ethanol-extractable radioactivity of larvae was present as free arginine (Pappas et al. 1973b). Apparently, methionine and phenylalanine were absorbed by active transport, while the process r.esponsible for arginine absorption must be tentatively identified as facilitated diffusion. On the basis of distribution ratios of radioactivity, accumulation of lysine, tyrosine (Haynes 1970), valine, serine, glytine, and leucine (Haynes and Taylor 1968) by T. crassiceps larvae has been reported. However, the extent of metabolism in these experiments was uncertain. Amino acid uptake by larvae was pH dependent (optimum about pH 7), and temperature dependent ( QIo = 2.6, ll-43C) (Haynes and Taylor 1968). Lysine uptake by T. crassiceps larvae was inhibited by lysine, ornithine, arginine and, to a slight extent, histidine (Haynes 1970; Pappas and Read 1973) ; omithine and lysine were completely competitive inhibitors of arginine uptake (Table V) (Pappas et al. 1973b). Neutral, dicarboxylic and aromatic acids did not inhibit basic amino acid uptake suggesting the presence of a transport system in T. crassiceps larvae specific for basic amino acids (Haynes 1970; Pappas and Read 1973). The inhibition of phenylalanine uptake by serine, leucine, tryptophan, phenylalanine, methionine, histidine, tyrosine, and alanine was completely competitive (Table V), but significant inhibition of methionine uptake by phenylalanine or tryptophan was not demonstrable (Pappas and Read 1973; Pappas et al, 1973b). Taenia crassiceps larvae possibly possess at least two neutral amino acid transport systems, one with a high affinity for aliphatic amino acids and a low affinity for aromatic amino acids, and

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another showing an inverse relationship. Since phenylalanine uptake was inhibited significantly by methionine and tryptophan, but methionine uptake was not inhibited significantly by phenylalanine or tryptophan, it was suggested (Pappas et al. 1973b) that the binding of methionine to the phenylalanine-preferring system of T. crassiceps larvae was nonproductive. Additional studies are needed to clarify this latter point. Proline and glutamic acid apparently enter T. crassiceps larvae by diffusion only. Uptake of both amino acids was a linear function of substrate concentration over a 20-fold substrate concentration range, and the uptake of neither proline nor glutamic acid was inhibited by their own respective molecular species (Pappas and Read 1973). Further, the observation that dicarboxylic organic acids (e.g., malic and fumaric acids) inhibited glutamic acid uptake (Haynes and Taylor 1968) could not be confirmed (Pappas and Read 1973). The effectiveness of an inhibitor of neutral amino acid uptake by T. crassiceps larvae was reduced significantly by acetylation of the a-amino group. For exam.pIe, lysine uptake by larvae was inhibited significantly by lysine and arginine, but not by N-acetyl-m-arginine; methionine uptake was inhibited significantly by D- and Lvaline and D- and L-methionine, bu,t not by either N-acetyl-nL-methionine or N-acetylm-valine. As in H. diminuta, moving the amino group from the a-carbon decreased the inhibitory powers of a potential inhibitor. Methionine uptake by T. crassiceps larvae was inhibited 78, 58, and 0% by alanine, p-alanine, and propionic acid, respectively (Pappas et al. 1973b). Clearly, the presence of an unsubstituted a-amino group is essential for an inhibitor to interact with the neutral amino acid transport system(s) of T. cmwiceps larvae. Using kinetic parameters derived from their experiments, Haynes and Taylor (1968) attempted to demonstrate the applicability of the relationship derived by

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Read et al. (1963) for determining the effect of an amino acid mixture on the uptake of a single amino acid by T. crassiceps larvae. However, of the three experiments reported by Haynes and Taylor (196S), only one yielded an exp.erimental value similar to the calculated expected value. These discrepancies probably arose from the inaccurate transport constants (K,) derived by Haynes and Taylor (1968), and the use of Kt values instead of Ki values in the equation. As stressed by Read et al. ( 1963), Kc must be used in this equation, since it approximates more closely a measure of affinity of an inhibitor and a transport system. 4. General considerations. From the preceding discussion it is apparent that the amino acid transport systems of the four cestode species discussed above are similar in some respects. The available data indicate that all species possess more than one transport system, with as many as six distinct system being identified in H. diminuta, and three systems in T. crassiceps larvae. The amino acid transport systems of cestodes appear to be unique, when compared with generalized mammalian systems, in two respects. These are (1) the stereospecificity of cestode transport systems for Dand L-amino acids, and (2) the relationship of solute transport and transmembrane movement of ions. The amino acid transport systems of most mammals characteristically display a high affinity for L-amino acids, as opposed to n-amino acids. Some amino acid transport systems of cestodes, on the other hand, have an equal affinity for D- and L-amino acids, or in some instances, a higher affinity for n-amino acids. The Kt values for D- and L-methionine uptake by H. diminuta and H. citelli were similar indicating an equal affinity of the transport systems for both isomers (Table I). Arme and Coates (1973) found that n-amino acids were equal in th.eir ability to inhibit a-aminoisobutyric acid uptake by H. diminuta cysticercoids when compared

AND

READ

with their respective L-isomers (Table VI). In T. crawiceps larvae, n-isomers of neutral amino acids were better inhibitors of L-methionine uptake than were their respective L-isomers (Table VI). Clearly, the neutral amino acid trans’port system(s) of H. diminutn adults and cysticercoids, H. citelli adults and T. crassiceps larvae display a high affinity for n-amino acids. However, this apparent lack of specificity may not be a characteristic of all cestode transport systems, for L-amino acids were better inhibitors of L-lysine uptake by T. crassiceps larvae than were their nisomers (Table VI). Additional studies are needed before any generalized conclusions regarding the stereospecificity of cestode transport systems can be drawn. The second unique aspect of cestode amino acid transport systems is the apparent lack of ion-coupled active transport. In many mammalian systems, amino acid (and monosaccharide) transport is coupled to the movements of Nat and/or an anion (such as Cl-) (see Pappas et al. 1974, and references therein). According to the “ion-gradient hypothesis” an organic solute is accumulated intracellularly because the cell can maintain a low concentration of co-transported ion. Thus, the organic solute and ion move in relation to the prevailing concentration difference in the co-transported ion; as long as the concentration difference in the co-transported ion is maintained (through the active extrusion of the ion from the cell), transport will continue and solute (but not ion) will be accumulated. The limited data available indicate that neither cations nor anions are required for amino acid influx and accumulation in cestodes. Replacing the Na+ in the ambient medium with either choline or K+, or replacing the Cl- with CH,,COOor NOS-, had no effect on the initial uptake rate of labeled methionine by H. diminuta (Read et al. 1963; Pappas et al. 1974); similarly replacement of Na+ with tris (hydroxymethyl)aminomethane had no effect on the initial uptake rate of methionine,

MEMBRANE

TRANSPORT

IN

HELMINTH

TABLE A Summary

mM wAminoisobutyric or-Aminoisohutyric ol-Aminoisobutyric mM ru-Aminoisobutyric mM or-Aminoisobutyric mM a-Aminoisobutyric mM mM

VI

of the Data Demonstrating the Afinity of Cestode Amino Acid Transport Systems for D- and x.-Amino Acids

Solute (substrate)

0.4 0.4 0.4 0.4 0.4 0.4

Inhibitor

acid acid acid acid acid acid

Percentage inhibition

Hymenolepis

diminuta

4 4 4 4

n-Methionine bMet.hionine n-Alanine LAlanine

mM mM m&f mM 4 mM 4 mM

Reference

cysticercoids

D-Vahe

LValine

Hymenolepis 0.1 mM L-Methionine 0.1 mM L-Methionine

489

PARASITES

91.1 85.9 92.5 61.0 48.5

Arme Arme Arme Arme Arme Arme

52 54

Senturia Senturia

58 47 32 100 97 100 72 99 78 0 80 45 92

Haynes Haynes Haynes Pappas Pappas Pappas Pappas Pappas Pappas Pappas Pappas Pappas Pappas

80.8

and and and and and and

Coates Coates Coates Coates Coates Coates

1973 1973 1973 1973 1973 1973

cite&

1 mM n-Methionine 1 mM L-Methionine

1964 1964

Taenia crassiceps larvae 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

mM mM mM mM mM mM mM mM mM mM mM mM mM

LMethionine L-Methionine LMethionine LMethionine LMethionine L-Methionine LMethionine LMethionine GMethionine L-Lysine L-Lysine LLysine L-Lysine

1 mM 1 mM 1 mM 10 mM 10 mM 10 mM 10 mM 10 mM 10 mM 10 mM 10 mM 10 mM 10 mM

n-Alanine DtAlanine LAlanine n-Methionine tMethionine n-Valine L-Valine n-Alanine tAlanine n-Lysine L-Lysine n-Arginine LArginine

phenylalanine, or arginine by T. crassiceps larvae (Pappas et al. 1973b). In Nat-free media T. crassiceps larvae accumulated phenylalanine and methionine, and H. diminuta accumulated methionine (Pappas et al. 1973b, 1974). Hymenolepis diminuta accumulated methionine in Cl--free media also (Pappas et al. 1974). Although accumulative systems which are not ion-dependent are though,t to be the exception (see Schultz and Curran 1970, for a review), the available data indicate that ion-insensitive amino acid transport and accumulation may be the rule, rather than the exception, in cestodes. In this regard, a question of fundamental importance which remains to be answered is the origin of the “driving force” for accumulation in the absence of ion-dependency.

and Taylor 1968 and Taylor 1968 and Taylor 1968 et al. 1973h et al. 1973b et al. 1973b et al. 1973b et al. 1973b et al. 1973b et al. 1973b et al. 1973b et al. 197313 et al. 1973b

B. The Acanthocephala The Acanthocephala, unlike the Cestoda, are dioecious and pseudoco.elomate. These two facts make acanthocephalans somewhat more difficult material than cestodes for transport studies. Care must be taken to distinguish the sexes, and the presence of a pseudocoelom may complicate kinetic data due to compartmentalization of absorbed solutes. Like the cestodes, acanthocephalans ‘absorb amino acids ,by mediated systems. The uptake of methionine, leucine, isoleucine, serine, and alanine by Moniliformis dubius males and females, and Macrucanthorhynthus hirudinaceus males and females was saturable (Table VII) (Rothman and Fisher 1964) ; at high concentrations, ala-

490

PAPPAS AND READ TABLE

VII

A Summary of the Kinetic Parameters Describing Amino Acid Transport in the Acanthocephala. Ail Amino Acids are &Isomers. Data Taken from Rothman and Fisher (1964), with the one Exception Noted in the Table Solute (substrate) Moniliformis Females Alanine Alanine Isoleucine Leucine Methionine Serine

Males

Females III ales

1.80

1.30

348

0.30 0.27 0.69 0.93

0.18 0.45 0.81 1.30

30 52 70 156

Macracanthorhynchus

Alanine Isoleucine Leucine Methionine Serine

dubius

342 200* 172 114 86 272

hirudinaceus

Females

iWales

0.4 0.73 0.9 1.0 -

0.28 0.9 10.0

Females Alales 14 18 40 30 -

28 42 706

B pmoles absorbed/g ethanol extracted dry wt/hr. 6 This value was approximated from the data of Uglem and Read (1973). The obvious discrepancy between the two values is probably due to the fact that the data of Rothman and Fisher (1964) were not corrected for diffusion.

on the basis of distribution ratios of radioactivity, but Uglem and Read (1973) found that M. dubius males rapidly metabolized alanine, with only 38% of the ethanolextractable radioactivity being present as alanine following a 60-min incubation in radioactive alanine. Since Branch’s (1970) data did not take into account the possibility of metabolism, they must be interpreted with caution. It appears that methionine absorption by M. dubius and &I. hirudinaceus, and leucine absorption Iby M. dubius, occurs by active transport, while alanine absorption by M. dubius must be tentatively classified as facilitated diffusion. Deletion of Na+ from the external medium had no effect on lysine, glutamic acid, methionine, or leucine absorption by M. dubius males, and 22Na+ influx in males TABLE

A Summary qf Inhibitor Constants (K;) for Various Amino Acids Acting as Inhibitors of Amino Acid Uptake in the Acanthocephala. All Amino Acids are L-Isomers. Data Taken jrom Rothwlan and Fisher (1064) Solute (substrate)

Inhibitor

MoniliJormis

nine and leucine entered M. dubius males predominately by diffusion (Uglem and Read 1973). Methionine uptake by both sexes of both species was inhibited comp.etitively by leucine, isoleucine, serine, and alanine; the uptake of leucine, serine, and alanine by M. dub&s males and females and M. hirudinaceus females was inhibited by methionine (Table VIII) ( Rothman and Fisher 1964; Edmonds 1965; Uglem and Read 1973). Moniliformis dubius and M. hirudinuceus males and females accumulated methionine (Rothman and Fisher 1964), and M. dubius males accumulated leucine against an apparent concentration difference (Uglem and Read 1973). Branch ( 1970) reported accumulation of alanine, serine, and leucine by M. dubiw females

VIII

Alanine Isoleucine Leucine Methionine Methionine Methionine Methionine Serine

Met,hionine Methionine Methionine Alanine Isoleucine Leucine Serine Methionine

Macracanthorhynchus

Ki C&O

dub&s Females

Males

5.20 0.46 0.75 0.45 0.65 0.55 0.64 5.30

Il.6 1.3 0.98 0.45 0.86 0.36 0.32 2.4

hirudinaceus Females

Alanine Isoleucine Leucine Methionine Methionine Methionine Met,hionine Serine

Methionine Methionine Methionine Alanine Isoleucine Leucine Serine Met,hionine

0.55 19.0 30.0 0. -5 1.0 9.0 -

Males 1.3 1.9 1.0 0.5 0.95 3.5

MEMBRANE

TRANSPORT

was not increased in the presence of leucine in the ambient medium. These data suggest that amino acid uptake in M. dubius is not ion-sensitiv.e, however, it is not known whether leucine accumulation is ion-sensitive as well. If accumulation is ion-insensitive, one is again faced with the question of ,the “driving force” for solute accumulation in the absence of ion-dependency (Uglem and Read 1973). Using the equation of Read et al. ( 1963), Rothman and Fisher ( 1964) demonstrated that the effect of an amino acid mixture on methionine uptake by both male and female M. dub&s could be predicted accurately, provided certain kinetic parameters for amino acid permation were known. Thus, the applicability of this equation to organisms other than cestodes was demonstrated. Only a single attempt has been made to demonstrate mediated amino acid efflux in acanthocephalans. Uglem and Read (1973) were unable to demonstrate increased leutine efflux from M. dubius males in the presence of leucine in the ambient medium. Amino acid efflux in acanthocephalans requires further study. C. The Trematoda 1. Fasciola hepatica and Fascioloides magna. Quite limited data are available regarding amino acid absorption by trematodes. Amino acid absorption by Fasciola hepatica and Fascioloides magna, two trematodes which are large and easy to handle, was studied by Isseroff and Read ( 1969). These authors demonstrated that the uptake of radioactive cycloleucine was the same with ligated and nonligated F. hepatica and F. magna during a 2-min incubation, and concluded that a 2-min incubation measured absorption across the tegument. The uptake of cycloleucine by both species was linear over a 40-fold concentration range; proline, arginine, and methionine uptake rates in F. hepatica were linear with respect to concentration also. Cycloleucine uptake by both species

IN

HELMINTH

PARASITES

491

was not inhibited by glycine, valine, methionine, a-aminoisobutyric acid, glutamic acid, or arginine, and no inhibition was detected when cycloleucine, proline, methionine and arginine were tested as mutual inhibitors of one another in F. heputica. Cycloleucine was not accumulated by F. hepatica in vivo, and the Qlo for cycloleucine uptake by both species was less than 2 (Isseroff and Read 1969). Although the above data indicated strongly the lack of mediated amino acid uptake systems in the teguments of F. hepatica and F. magna, Isseroff and Read (1969) presented data which suggested that diffusion may account for only the initial tegumentary absorption of amino acids. When F. hepatica was preloaded in labeled cycloleucine for 30 min and subsequent efAux of this amino acid in saline measured, a distinct biphasic curve was obtained; 50% of the absorbed amino acid efflux in 2 min, after which a sharp decline in the efflux rate occurred. Isseroff and Read (1969) suggested that some of the absorbed amino acid was compartmentalized within the worms, and that this compartmentalization may have involved some mediated process. However, since the F. hepatica used in the latter experiments were not ligated, one can not rule out the possibility that the biphasic efflux curve resulted from labeled amino acid in the intestine of these worms. 2. Schistosoma mansoni. Amino acids appear to enter Schistosoma mansoni adults, when incubated in vitro, predominately through the tegument. Several experiments in which amino acid uptake by ligated and nonligated S. mansoni was compared failed to detect a significant difference in uptake (Asch and Read 1975a; Chappell 1974). Also, treatment of S. mansoni with physostigmine (a compound which induces “vomiting” in schistosomes) following incubation in labeled amino acid demonstrated the presence of no radioactivity in the worm’s gut (Chappell 1974).

492

PAPPAS AND READ TABLE

IX

A Summary of Kinetic Parameters Describing Mediated Transport of Various Solutes in Trematades. The Amino Acids and Monosaccharides Listed are L and D-Isomers, Respectively Solute (substrate)

Kt bw

V mnxe

Reference

Fasciota hepatica 3-O-methylglucose Frutose Glucose Galactose Mannose Glucosamine Ribose

2.5 2.5 0.7-1.3b 2-4b 1.4 2.0 4.5

-

Isseroff Isseroff Isseroff Isseroff Isseroff Isseroff Isseroff

and and and and and and and

Read Read Read Read Read Read Read

1974 1974 1974 1974 1974 1974 1974

Schistosoma mansonic

Glucose Galactose 2-Deoxyglucose Methionine Methionine Glycino Proline Arginine Glutamic acid Trytophan Hypoxanthine Adenosine Uridine Adenine

0.8 0.24 1.72 0.33 1.9 0.6-1.05 1.7-2.0 0.1-0.35 0.3-0.5 0.5-1.0 0.87 0.49 0.36 0.05

147.2 10.1 270.3 1X@ 192e 165-22je 75@ 45-60’ 750 go-18Oe 45.3 51.9 22.2 9.6

Uglem and Read, unpublished Uglem and Read, unpublished Uglem and Read, unpublished Chappell 1974 Asch and Read 1974b &ch and Read 197413 Asch and Read 1974b Asch and Read 1974h Asch and Read 197413 Asch and Read 1974h Levy and Read, unpublished Levy and Read, unpublished Levy and Read, unpublished Levy and Read, unpublished

5 pmoles absorbed/g Lowry protein/hr, unless noted ot,herxise. b Values reported to vary with worm size. 6 Values of Isseroff et al. (1972) not reported, since (1) the data were not corrected for diffusion, only five Kt values were reported for six monosaccharides. d pmoles absorbed/g ethanol extract.ed dry wt./hr. 0 Data originally reported in terms of uptake/2 min, but have been converted to uptake/hr.

uptake by male and female Methionine S. wzansoni occurs by a combination of mediated uptake and diffusion. Methionine uptake was nonlinear with respect to concentration (Table IX), and inhibited by numerous amino acids (Table X) (Chappell 1974; Asch and Read 197513) . Leucine, phenylalanine, arginine, and Iysine were completely competitive inhibitors of methionine uptake by S. mansoni, with Ki values of 5.4, 4.5, 47.7, and 36.3 mM, respectively. Methionine uptake was not inhibited by metabolic poisons (2,4-dinitrophenol, cyanide, azide, iodoacetate), ouabain, potassium antimony tartrate, monosaccharides, fatty or other organic

and (2)

acids. However, methionine uptake was temperature and pH sensitive, with a pH optimum of 7.4; methionine uptake as a function of temperature increased up to 45 C (Chappell 1974). Asch and Read (1975b) and Chappell (1974) suggested that S. munsoni may possess two transport systems for methionine. The former authors demonstrated that when methionine uptake was inhibited maximally with aspartate, the addition of phenylalanine resulted in increased inhibition of methionine uptake. The effects of saturating concentrations of phenylalanine plus aspartate as inhibitors were similar, suggesting ‘that phenylalanine inhibited up-

MEMBRANE

TRANSPORT

IN

HELMINTH

TABLE A Summary

of amino acid inhibitor

X

studies jor Schistosoma mansoni Adult Malesa

Inhibitor

Substrate Methionine

Alanine @Alanine Arginine Asp&ate Citrulline Cysteic acid Cystine Glutamate Gly tine Histidine Hydroxyproline Isoleucine Leucine Lysine Methionine Ornithine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

493

PARASITES

Glycine

E”, ++ + (++I ++(t-1 (+++I f(++I (+I d= +-l++ (+++I (+) ++ (+++) ++++ (+++I ++ ++++ (+++) + e-T-+, ++ z,

+++ + ++ -

0 ++ +++ +++ ++ +++ 0 +++ +++ + +++ -

+++ -

Proline

Glutamate

++ +++ ++ -

++ * + f f + + f + + ++ f +\ + f -

Arginine

1: ++ +++ ++ ++-I++ +++ ++ f ++ f -

++t -

+++

5 The relative inhibitions are reported as follows : 0 = no inhibition; f = l-10% inhibition; + = 11-25% inhibition; + + = 26-7’570 inhibition; + f + = greater than 757, inhibition. A (-) indicates that those combinations of inhibitor and substrate were not tested. b Data in parentheses from Chappell (1974), with remainder of data coming from Asch and Read (1974b). Since both Chappell (1974) and Asch and Read (1974b) conducted inhibitor experiments with methionine as the substrate, both sets of data have been incorporated into the table. In cases where only data from Asch and Read (1974b) are reported, either Chappell (1974) did not test that combination of inhibitor and substrate, or his results agreed. In cases where only the data of Chappell (1974) are reported, this combination was not tested by Arch and Read (1974b). In cases where both sets of data are presented, the data of the two studies did not agree.

take through two amino acid transport sysuptake tems, while aspartate inhibited through a single system. Chappell (1974) demonstralted that methionine uptake in the presence of saturating concentrations of threonine as an inhibitor was unaffected by addition of glutamate, but that the addition ,of histidine or arginine (in addition to the threonine) resulted in increased inhibition of methionine uptake. Glutamate, glycine, arginine, alanine, and tryptophan entered S. mansoni males by a combination of a mediated system and dif-

fusion, as indicated by the obervation that the uptake of these amino acids as a function of concentration was nonlinear at low substrate concentra,tions, but became linear at higher substrate concentrations. Proline entered worms by a mediated process only (Table IX). The uptake of each of these amino acids was stereospecific, in that uptake was inhibited #by other amino acids (Table X). Cysteine entered worms by diffusion; uptake was a linear function of concentration, and the uptake of radioac-

494

PAPPAS

tive cysteine was not inhibited by unlabeled cysteine ( Asch and Read 1975b). Chappell ( 1974) and Asch and Read (197513) conducted extensive inhibitor experiments, studies which demonstrated the complex nature of the amino acid transport systems of S. mansoni. AIthough the data, at present, are insufficient for determining the number of distinct amino acid transport systems, several conclusions are evident from the data of these two studies. As suggested by Chappell (1974) and Asch and Read ( 1975b), methionine probably enters S. mamoni through two distinct amino acid systems. These systems have a high affinity for amino acids only, since rnethionine uptake was not inhibited by other organic compounds, and, as in cestodes, moving the amino group to carbon atoms adjacent to the a-carbon decreased the affinity of the inhibitor for the transport system(s) (Chappell 1974). A third amino acid transport system was suggested by Asch and Read ( 1975b), a system with relatively high specificity, since proline uptake by S. manxoni males was inhibited only by proline, methionine, and alanine. These authors suggested further that the inhibition of proIine uptake by these amino acids (methionine and alanine) was due to nonproductive Mnding, since proline was only a very weak inhibitor of the uptake of the two amino acids (Table X). Also, the specificity of the amino acid transport system(s) of S. mansoni is quite different than in ‘cestodes. For example, arginine uptake by S. mans& was inhibited by both neutral and basic amino acids, as was methionine uptake (Table X) (Chappell 1974; Asch and Read 1975b ) . This is clearly different from cestodes, in which neutral and basic amino acids do not interact. The exact mechanism of amino acid absorption in S. mansoni is unknown, but Chappell (1974) d emonstrated that methionine uptake ,by worms was decreased in media when the Na+ was replaced with tris, choline, K+, or Li+. Although these data are suggestive of a Na+ requirement for amino

AND

READ

acid uptake by S. mans&, further studies are necessary to determine the interactions of cations and amino acids in schistosomes. D. The Nematoda Unlike the cuticle of most nematodes, the gut epithelium of nematodes is readily permeable to amino acids and monosaccharides. However, extensive kinetic data are not available. Read (1966), using “ribbon preparations,” reported that histidine, methionine, glycine, and valine uptake by Ascaris suum gut was nonlinear with respect to concentration, and that the uptake of each of these amino acids was stereospecific. Although uptake was apparently mediated, it was not determined whether any amino acids were accumulated. Further data concerning amino acid transport in A. suum gut are lacking. Harris et al. (1972) demonstrated that ovary-oviduct preparations of A. suum accumulated radioactive cycloleucine against a concentration difference, and that in 0.1 mA4 cycloleucine a steady state was reached in approximately 30 min with a final distribution ratio of radioactivity of 5: 1 (the amino acid was not metabolized); body muscle preparations of A. suum did not accumulate cycloleucine in 60 min. Radioactive cydoleucine uptake by reproductive tissues was inhibited by leucine, while cycloleucine uptake by muscle was not. Reciprocal experiments in which the uptake of radioactive leucine by both types of tissue was measured in the presence of cycloleucine as an inhibitor yielded similar results. Apparently, cycloleucine entered reproductive tissues by active transport, and entered body muscIes by diffusion. Amino acid uptake by A. Suum reproductive tissue and muscle was the same in anaerobic (95% Na-5% CO?) and aerobic CO,) environments, and (95% air-5% was unaffected by preincubation of tissues in glucose (Harris et al. 1972), Further studies of amino acid transport in A. suum, and nematodes in general, are certainly warranted.

MEMBRANE

III.

CARBOHYDRATE

TRANSPORT

IN

HELMINTH

TABLE

ABSORPTION

A. The Cestoda 1. Hyrnenolepic diminuta and H. microstoma Glucose ’ uptake by Nymenolepis diminuta is a process of active transport. Glucose uptake was temperature dependent ( Qlo > 2.4, E-40 C) (Phifer 1960a) and saturable (Table XI). Glucose uptake was stereospecific, since uptake was inhibited by structurally similar monosaccharides (Tables XII and XIII), and glucose was accumulated by worms against a concentration difference (Phifer 1960b; Pappas et al. 1974); folIowing a 60-min incubation in 5 mM glucose, the internal glucose concentration of worms was approximately 25 mM (Pappas et al. 1974). Only a negligible diffusion component for glucose uptake by H. diminuta was detected (McCracken and Lumsden 1974a ) . Glucose uptake by H. diminuta during 60-min incubations was inhibited by piodoacetate, and chloromercuribenzoate, 2,4-dinitrophenol. However, the latter two inhibitors had no effect during 1-min incubations unless worms were first preincubated in the inhibitor (Phifer 1960a, b). Although the effects of these inhibitors on glucose uptake by H. diminuta suggest an energy requirement, the mechanism of the actions remains unknown. The data of Phifer (19600~) also suggested a distinct energy requirement for glucose uptake by H. diminuta. Using worms from starved hosts, Phifer (1960~) showed that preincubation of worms in a metabolizable monosaccharide (e.g., glucose or galactose) resulted in a significant increase in radioactive glucose uptake over that of controls which wer.e not preincubated. Preincubation of worms from starved hosts in a nonmetabolizable monosaccharide (e.g., fructose) yielded results similar to control incubations. Additional evidence for mediated glucose uptake by H. diminuta, rather than 4 Unless noted otherwise, all monosaccharides are the naturally occurring D-isomers.

495

PARASITES

XI

A Summary of Kinetic Parameters Describing Carbohydrate Absorption in Cestodes. All Solutes are n-Isomers Solute (substrate)

Kt (m&f)

Reference

V,,,o

Hymenolepis Glucose Glucose Glucose

1.6 1.4 0.74

789b 400

Glucose

1

585

Glucose Galactose Glycerol Glycerol

1.54c 5.0 0.6gd 0.24e

19Bb*c 91d 84e

Hymenolepis Glucose

2

diminuta Read 1961a Read et al, 1974 McCracken and Lumsden 1974a McCracken and Lumsden 1974b Arme et al. 1973 Read 1961a Uglem et al. 1974 Uglem et al. 1974

microstoma

lOSOb

Pappas and Freeman 1974

Taenia crassiceps larvae Glucose Galactose

0.26 0.75

20 70

Calliobolhrium Glucose

0.61

610

Pappas Pappas

etal. L973c etaZ. 1973c

verticillatum Pappas and Read 1972a

a rmoles absorbed/g ethanol extracted dry wt/hr unless noted otherwise. b Uptake rates in original publication given in terms of uptake/2 min. These values have been converted to uptake/hr. c Data for cysticercoid> larvae. V,,, units = nmoles absorbed/LOO larvae/hr. d Natindependent system. 6 Natdependent system.

simple diffusion, was shown by the fact that glucose uptake per unit dry weight was significantly greater in 12-day-old worms than in 8-, 16-, 20-, or 90-day-old worms. Although these data may suggest a quantitative difference in the transport system as a funotion of age, they do not allow inferences concerning qualitative changes. Starling and Roberts (personal communication) have obtained evidence that the Kt for glucose transport changes during strobilar development of H. dimi-

496

PAPPAS AND FEAD TABLE

A Summary

XII

oj Inhibitor Constants (Ki) for Various Compounds Acting as Inhibitors of Monosaccharide Uptake in Cestodes. All Substrates are D-Isomers Unless Noted Otherwise

Sub&ate

Inhibitor

Glucose Galactose Glucosea

Phlorizin Phlorizin Galactose”

Hymenolepis

Glucose Galactose Phlorizin Phloretin

5 Data for cysticercoid

McCracken and Lumsden McCracken and Lumsden Arme et al. 1973

1974a 1974a

microstoma Pappas Pappas Pappas Pappas

1.4 5.3 16 X lo+ 0.23

and and and and

Freeman Freeman Freeman Freeman

1974 1974 1974 1974

larvae.

TABLE A Summary

diminuta

3 x 10-a 1.4 x 10-Z 1.19a Hymenolepis

Glucose Glucose Glucose Glucose

Reference

Ki (mM)

XIII

of the Actions of Various Monosaccharides and Other Compounds as Inhibitors of Glucose Uptake in Various Cestode Speciesa

Inhibitor

Hymenolepis diminuta

Hymenolepis

Taenia

microstoma

crassiceps

Calliobothrium verticillatum

larvae Glucose a-Methylglucoside 0-1Methylglucoside 1-Deoxyglucose 2-Deoxyglucose 3-O-methylglucose 6-Deoxyglucose Glucosamine N-acetylglucosamine Galactose 2-Deoxygalactose 6-Deoxygalactose Galacitol Allose Mannose 1,5-Anhydro-o-mannitol Fructose Phlorizin Phloretin Ouabain a A (+) indicates that the inhibitor inhibited Those combinations of inhibitor and substrate b The data for this table were taken from Phifer 1960a, McCracken and Lumsden 1974a, 1974; Taenia crassiceps larvae, Pappas et al.

+ + + + + + 0 + 0 0 + 0 0

+ + + 0 0 0 + 0 0 0 -

+ + +

+ + -

-

0

0 0 + + 0 -

0 -

0 0

0 + + 0

+ 0

glucose uptake, and a (0) indicates no significant inhibition. which have not been tested are denoted as (-). the following sources : Hymenolepis diminuta, Read 1961a, Dike and Read 1971b; H. microstoma, Pappas and Freeman 1973c; Calliobothrium verticillatum, Fisher and Read 1971.

MEMBRANE

TRANSPORT

nuta; this may indicate qualitative changes in the transport mechanism. Overturf (1966) attempted to determine the kinetic parameters for glucose uptake by H. diminuta in viva using intact intestinal loops of infected rats. However, in vitro and in vivo results wer.e not comparable for, as Overturf (1966) pointed out, data from in viva studies were difficult to interpret since the individual contributions of host and parasite could not be determined accurately. More recently Podesta and Mettrick (1974) indicated that glucose transport in H. diminuta in vivo may be composed of two distinct components, these being “solvent drag” and “aotive transport.” Although the data of these authors demonstrate that the individual contributions of the rat intestine and tapeworm can be separated when studying absorptive processes in the rat-H. diminuta host-parasite relationship, the data are not amenable to kinetic analysis. Hymenolepis diminuta cysticercoids also absorb glucose through a mediated system. Glucose uptake by larvae was saturable (Table XI) and inhibited competitively by galactose ( Table XII). Apparently, diffusion made an insignificant contribution to glucose uptake by larvae. Glucose uptake was inhibited by 2,4-dinitrophenol, cyanide, iodoacetate, and phlorizin. The fact that glucose uptake was inhibited only 32% in the absence of external Na+, and 34% in the presence of 0.1 mM phlorizin, suggested ‘that glucose uptake by H. diminuta larvae is only partially Na+-sensitive ( Arme et al. 1973). However, since glucose uptake was inhibited 97% by galactose, two transport systems may be functional in larvae, only one of them being Na+-sensitive. This requires further study. Galactose uptake by H. diminuta also .occurs by aotive transport, and both glucose and galactose appear to enter this cestode via a single monosaccharide transport system. This conclusion is based on the following observations: Galactose uptake by worms was saturable (Table XI); galactose

IN

HELMINTH

PARASITES

497

was accumulated by worms (Read 1967); galactose uptake was inhibited competitively by phlorizin, as was glucose uptake (Table XII) (McCracken and Lumsden 1974a; Read et aZ. 1974); glucose and galactose were mutual competitive inhibitors of each other (Read 1961a). Two distinct mechanisms are involved in glycerol uptake by H. diminuta, these being a mediated component and diffusion. Uptake of radioactive glycerol by worms was nonlinear with respect to concentration below 0.5 mM glycerol, (but at concentrations greater than 0.5 mM uptake was linear; correcting these data for the apparent diffusion component resulted in typical saturation kinetics (Pittman and Fisher 1972; Uglem et al. 1974). That glycerol uptake was mediated was also shown by the observations that (1) uptake was pH and temperature dependent (optima at pH 7-9 and 37 C), (2) uptake was affected by worm age, with uptake by 12- and 13-dayold worms being less than by 6, 9-, lo-, or 11-day-old worms, and (3)
498

PAPPAS

GLYCEROL

5. The velocity of glycerol uptake (V, pmoles absorbed/g ethanol extracted dry wt/hr ) by Hymenolepis diminuta in Krebs-Ringer saline ([Na+] = 154 mM), and Na+-free saline (lines A and B, respectively), as a function of glycerol concentration (mM). All data are corrected for diffusion. The area below Line B represents the Na+-independent glycerol uptake, while the shaded area represents the Na+-dependent glycerol uptake. Redrawn from Uglem et al. ( 1974). FIG.

as an inhibitor (93 76 inhibition), i,t appeared that 23. diminuta possessed two glycerol transport systems, one of which was Nat-sensitive and inhibited by 1,2-propandiol, and another which was not Na+sensitive and not inhibited by 1,2-propandiol. Uglem et al. (1974) demonstrated subsequently that H. diminuta has two distinct glycerol transport systems. Glycerol uptake by worms in Nat-free media (with K+, Li’, or choline as the replacement cation) was mediated, but not inhibited by I,&-propandiol; the Na+-sensitive glycerol transport system was inhibited by 1,2-propandiol. Both transport systems were inhibited competitively by glycerol and phloretin (Table XI, Fig. 5). Attempts to demonstrate coupled movements of Na+ and gIycero1 were unsuccessful (UgIem et al. 1974). Ahhough Uglem and his coworkers (1974) demonstrated clearly the presence of these two glycerol transport systems in H. diminuta, the significance of a helminth parasite having both a Na+sensitive and Na+-insensitive glycerol transport system remains unknown. Hymenolepis microstoma absorbs glucose by aotive transport. Glucose uptake

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READ

was saturable (Table XI) and inhibited by monosaccharides, phlorizin, and phloretin (Tables XII and XIII). In addition, ~glucose was accumulated against a concentration difference ,by H. microstoma; following a 60-min incubation in 1 mM glucose, the internal glucose concentration of worms reached 18 mM. Diffusion of glucose into H. microstoma was negligible (Pappas and Freeman 1975). As noted in Table XIII, phloretin (the aglycone of phlorizin) inhibited glucose uptake in H. microstoma, but had no effect on glucose uptake in other cestode species. In addition, the inhibition produced by phloretin was characterized as mixed in nature, suggesting that phloretin may interact with the glucose transport system of H. microstoma, in addition to a separate site which also affects glucose uptake (Pappas and Freeman 1975). Phlorizin inhibited glucose uptake in H. diminuta (McCracken and Lumsden 1974a ) and H. microstoma (Pappas and Freeman 1974). However, in H. diminuta the inhibition of glucose uptake by phlorizin was characterized as completely competitive (Read et al. 1974; McCracken and Lumsden 1974a), while the inhibition in H. microstoma was characterized as partially competitive. Although the action of phlorizin as an inhibitor ‘of glucose uptake in H. diminuta appeared to involve a direct interaction with the glucose transport system (McCracken and Lumsden 1974a), the mechanisms by which phlorizin and phloretin inhibit glucose uptake in H. microstoma remains unknown. 2. CaZliobothrium verticilkztum. Like H. diminuta and H. microstoma, Calliobothrium verticiltatum absorbs glucose through the process of active transport. Glucose uptake by C. verticillatum was saturable (Table XI) (Fisher and Read 1971; Pappas and Read 1972a), and inhibited competitively by several monosaccharides, phlorizin, and ouabain (Table XIII); salitin, arbutin, iodoacetate, and 2-4-dinitro-

MEMBRANE

TRANSPORT

phenol also inhibited glucose uptake, but the nature of the inhibition produced by these latter four compounds was not determined (Fisher and Read 1971). Uptake of radioactive glucose by C. verticillatum was not inhibited by phloretin, rhamnose (the glycone of ouabain), lactose, sorbose, sucrose, some monosaccharides (Table XIII), or numerous amino acids (Fisher and Read 1971) . (Fisher and Read [ 19711 reported that the disaccharides cellobiose and maltose inhibited glucose uptake by C. verticillatum. However, Read and Pappas [unpublished] were unable to confirm these results, nor couId they detect hydrolysis of either disaccharide in the presence of C. verticillatum.) Calliobothrium verticillatum accumulated glucose to a significant extent; following a 30-min incubation in 0.5 mM glucose, the internal glucose concentration increased from approximately 5 mM to lo-12 mM (Fisher and Read 1971; Pappas and Read 1972a). Although the above data demonstrate clearly tha’t active transport of glucose occurs in C. verticillatum, there are additional data supporting the mediated nature of glucose uptake in this cestode. Glucose uptake was temperature and pH dependent (optima at 20 C and pH 9.2), and efflux of was stimulated in the glucose from worms presence of those compounds which inhibited glucose uptake; hence, e&x of glucose appeared mediated as well (Fisher and Read 1971). Fisher and Read ( 1971) also indicated that gaIactose was accumuIated by C. verticillatum, and that glucose and galactose entered worms through a single transport system by identical mechanisms, i.e., active transport. However, 3-0-methylglucose absorption by C. verticillatum was a linear function of sugar concentration, and this monosaccharide was not accumulated. Therefore, the mechanism for S-O-methylglucose uptake appeared nonmediated (diffusion). 3. Taenia crassiceps larvae. The uptake of glucose and galactose by Taenia crassi-

IN

HELMINTH

PARASITES

499

ceps larvae is mediated. Absorption of both hexoses was nonlinear with respect to concentration below 2.5 mM; correcting the data for the apparent diffusion component resulted in typical saturation kinetics (Table XI) (M urrell 1968; Pappas et al. 1973c). Even at low substrate concentrations diffusion was responsible for a relatively large proportion of glucose and galactose uptake; diffusion accounted for 15 and 19% of the uptake of 0.1 mM glucose and galactose, respectively (Pappas et al. 1973c). Glucose and galactose were reciprocal competitive inhibitors, and the uptake of both hexoses was inhibited by a- and pmethylgluooside, fucose (6-deoxygalactose), and phlorizm. Uptake of neither hexose was inhibited by amino acids, mannose, glycerol galacitol, sugar alcohols, disaccharides, or ouabain (Table XIII). The fact that the relative effects of inhibitors on glucose and galactose uptake by T. crassiceps larvae were similar, and the fact that glucose and galactose were mutual competitive inhibitors, suggested that these monosaccharides enter.ed larvae through a common transport system which was separate from the amino acid system(s) (Pappas and Read 1973; Pappas et al. 197313,c). Uptake of glucose by T. crassiceps larvae occurs by aotive transport. Following a 90min incubation in 5mM glucose, the internal glucose concentration of larvae increased from 7.4 to 10.4 mM. Galactose accumulation by larvae has not been demonstrated (Pappas et aE. 1973c). Fructose apparently enters larvae by diffusion. The uptake of radioactive fructose was linear with respect to concentration over the concentration range of 0.05-20 mM. In addition, the uptake of 0.05 mM radioactive fructose was not inhibited by 5 mLM unlabaled fructose, tagatose, or Lsorbose (Pappas et al. 1973c). 4. Taenia ( = Hydatigera ) taeniaeformis. Although considerable data on glucose uptake by Taenia ( = Hydatigera) taeniaeformis larvae (strobilocerci) and adults

500

PAPPAS

have been accumulated by von Brand and his co-workers, many of the data cannot be evaluated in kinetic terms because of the extended incubation periods (generally 1 hr or longer). However, there is evidence for a Nat-dep.endent glucose transport system in T. taeniaeformis larvae and adults. Adult T. taeniaeformis accumulated glucose against an apparent concentration difference (whether larvae accumulated glucose is unknown), and glucose uptake and accumulation by adults was inhibited almost completely in the presence of phlorizin (von Brand et al. 1964). In addition, no glucose was absorbed by either larvae or adults of T. taeniaeformis in Nab-free media, and glucose uptake was independent of Ca2+, Mg*+, and K+ concentrations in media with a normal Na+ concentration (von Brand and Gibbs 1966). Additional evidence for a mediated system was provided by the observations of von Brand et al. (1964) showing that glucose leakage (efflux) from larvae and adults was stimulated in the presence of glucose or galactose in the external medium. These data are suggestive of exchange diffusion. That glucose uptake by larvae and adults of T. taeniacformis was Na+-sensitive was indicated further by the data of von Brand et al. (1964) and von Brand and Gibbs (1966) which showed that glucose efflux from larvae and adults was stimulated in the absence of external Na+. However, it was not determined whether glucose effluxed against a concentration difference in Na’-free media. Von Brand et al. (1964) reported that the rate of gIucose uptake by larvae and adults was independent of the glucose concentration, a result which, as Read (1967) pointed out, could have resulted from extended incubation periods. Von Brand et al. (1964) also reported that galactose uptake was proportional to the external galactose concentration. The fact that glucose uptake was Nat-sensitive and inhibited by phlorizin, and the fact that glucose was accumulated by adults, allows 11s to pos-

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HEAD

tulate that glucose uptake by larvae and adults occurs by active transport, possibly through a Na+-coupled system. Further characterization of the glucose transport system, and the quantitative interactions of Na+ and glucose, are needed in order to determine the mechanism of glucose uptake in T. taenineformis. Unlike glucose absorption, glycerol absorption by T. taeniaeformis larvae and adults was only partially Na+-sensitive, and uptake was phlorizin-insensitive. The uptake of glycerol was dependent on the external glycerol concentration and temperature, and glucose in the external medium had no effect on glycerol uptake. Glucose e&x from adults and larvae was not stimulated in the presence of glycerol (von Brand et nl. 1966). These observations led von Brand and his co-workers (1966) to postulate that glycerol uptake by T. taeniaeformis larvae and adults occurs through a glycerol specific mediated system, plus diffusion. Since accumulation of glycerol was not demonstrable, probably because of its rapid rate of metabolism, von Brand et nl. (1966) suggested a mechansm of facilitated diffusion, rather than active transport. Further studies of the kinetics of gIycero1 uptake are necessary before more definite conclusions regarding the mechanism of glycerol uptake can be drawn. 5. General considerations. Studies dealing with carbohydrate transport in cestodes have dealt mainly with the transport of glucose. This is not surprising since glucose appears to be the main monosaccharide from which many cestodes derive energy, and the only glycogenic monosaccharide in other cestode species (Read and Simmons 1963; Read 1967; van Brand 1973; Smyth 1969). A comparison of the data discussed in the preceding sections demonstrates that all cestodes studied to date utilize the same process for glucose transport, namely, active transport. No published data are available which demonstrate that any cestode possesses multiple transport systems for glucose.

MEMBRANE

TRANSPORT

IN

Therefore, if it is assumed that only one glucose transport system is present in these cestode species, it is apparent that the glucose transport systems of various cestode species do not differ markedly (Table XIII}. Although some differences in the activities of various compounds as inbibitors of glucose uptake are apparent, the data summarized in Table XIII are not sufficient for drawing generalized conclusions regarding the specificities of glucose transport systems in cestodes. Unlike amino acid transport and accumulation in c&odes which is apparentIy Na+insensitive (see Amino Acid Absorption, The Cestoda, General Considerations), glncosc transport in all cestode species studied to date is dependent on Nat in the ambient medium. In addition, coupling of Na- and glucose fluxes has been demonstrated in two cestode species (see below). The first report of Na+-sensi’tive glucose uptake in cestodes was presented by von Brand and Gibbs ( 1966). These authors demonstrated that Tagnia ( = Hydutigern f taentieformis larvae and adults failed to absorb glucose in Nat-free media; phlorizin, a potent inhibitor of many Na+-dependent glucose transport systems (Schultz and Curran 1970), also inhibited glucose uptake and accumulation by larvae and adults (von Brand et al. 1964). Pappas et al. (1973c) obtained similar results with Taenin crassiceps larvae. In media in which the lNa+ was replaced with K’, choline, or tris( hydroxymethyl)aminomethane, glucose uptake by larvae corresponded to the diffusion component for glucose uptake, and choline, tris, or K+ did not activate glucose uptake by T. crass&pa larvae in the absence of Na+ in the externa1 medium. At low concentrations, K’ was a competitiv*e inhibitor of Na+ activation of glucose transport, suggesting that K’ may bind in a manner similar to that of Na+, but not activate the transport of glucose. As in T. taeniaeformis, phlorizin inhibited completely the uptake of glucose by T. c?Y&ceps larvae. Attempts to demonstrate

HE1 MIKTH

PARASI‘IZS

501

coupled fluxes of Nat and glucose in T. tnen.iaeforn~is or T. crassiceps do not appear in the literature. Glucose uptake in Calliobothrium verticillatum, Hymenolepis dim&&a, and Hymendepis microstomu has also been demonstrated to be Na+-dependent. In these species, replacement of external Na+ with K+, choline or tris resulted in almost cornplete inhibition of mediated glucose uptake. Replacement of Na+ with Li+ also inhibited glucose uptake by these species; however, contrary to other cations, high concentrations of Lit appeared to replace partially Na+ in that glucose uptake was not inhibited maximally. (Table XlV). In H. diminuta and H. micros&ma, the effects of Na+ deletion were demonstrated to be reversible, since replacement of the Na+ in the external medium resulted in normal glucose uptake (Read et nE. 1974; Pappas and Freeman 1975). The reversibility of Na’ deletion in other cestode species has not been tested. H. diminnutu and C. -tiertic&turn transported small amounts of glucose in the absence of external Na+ (Pappas and Read 1972a; Read et al. 1974). A thorough experimental examination of this latter finding is lacking, but Read et al. (1974) suggested that H. dim& nutw may possess a Na+-insensitive glucose transport system. This system, if functional, would account for only a small percentage of the glucose absorbed by worms, Glucose uptake by C. ti~icillc~tum and H. diminu&z was a hyperbolic function of both glucose and Na+ concentrations of the ambient medium. In both species the major effect of lowered glucose or Na+ concentrations on glucose uptake was a decrease in the maximal velocity (V,,,) of glucose uptake. Decreasing ‘the Na+ or glucose concentration of the ambient medium decreased significantly the V,,, for glucose uptake by both speces, but had only a small effect on the transport constant (K, ) for glucose uptake (Pappas and Read 1972a; Read et al. 1974).

502

PAPPAS

TABLE

XIV

The Effects oJ NatDeletion on the Bate of Glucose Influx &moles/g Ethanol IS&acted dry wtihr) in Hymenolepis diminuta, Hymenolepis microstoma and Calliobothri~~m verticillatum. In All Experiments, Other l’han Controls, the Na+ in the Incubation Medium. was lieplaced Comyletel!/ with the Indicated Cation _--~~ ~ (ilrlcoae

Replacement cation It. diminuta” None (control) Kf Choline Tris Li+

15S.60 2.,58 2.28

0.93

__

16.80

- -

infltlx

H. micro- c. vertistoma” cillatzimc 54.3 0.36 0.34 0.4.i

647 32 73 7s

183 1.t;!) ~-~~~-~- ~-.

a Glucose concentration = 0.5 mJl. I)ata from Read et al. (1974), and originally reported as llptake/2 min. * Glucose concentration = 0.1 mU. !Jata from Pappas and Freeman (1974), and originally reported as uptake/2 min. c Glucose concentration = .5 m:M. Data from Pappas and Read (1972n).

The movements of Na+ and glucose across the tegument of H. diminuta and C. verticillatum are coupled. Incubation of C. verticilkztum in 0.2 mM glucose in saline ([Na’] = 250 mM) for 30 min resulted in an increase in the internal Nat concentration from 172 to 234 mM, and a decrease in the internal K+ concentration from 87 to 70 mM; incubation of worms in ouabain had similar effects on the intracellular Na’ and K+ concentrations (Fisher and Read 1971). The influx of Na+ in H. diminutu and C. verticillatum (as determined by short-term incubations) was stimulated significantly in the presence of glucose in the ambient medium (Read et al. 1974; Pappas and Read 1972a, respectively), and Na+ e%lux from C. verticillatum was inhibited by ouabain (Fisher and Read 1971). In addition to the dependency of glucose influx on Na+ in the ambient medium, and coupling of Na+ and g1ucos.e movement in some cestodes, glucose accumulation is also

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inhibited completely in Na+-free media. Na+-dependent glucose accumulation has been demonstrated in H. diminuta by Pappas et al. (1974) and H. microstoma by Pappas and Freeman (1975). These observations are consistent with Crane’s (1965) hypothesis for Na+-coupled glucose movements in mammalian tissues, a hypothesis which states that Na’coupled glucose movements results from the prevailing Na+ concentration difference, rather than a glucose concentration difference. If this is the case, then a reversal of the Na+ concentration difference (such as when worms are placed in Na+-free media) should allow worms to efflux glucose against a concentration difference. This phenomenon of “transconcentration” was demonstrated by Pappas and Read (1972a) when they prcincubated C. verticillutum in glucose in normal saline, and then transferred these preloaded worms to media of modified cation composition, all of which contained high concentrations of glucose. In normal saline and low-Na+ media, preloaded worms continued to accumulate glucose. In Na+free media there was an apparent net efflux of glucose from worms against a glucose demonstrating concentration difference, transconcentration effects of cation concentrations. Although the basic mechanism of glucose uptake in C. verticillatum and H. diminuta appears similar, some important differences exist. When simultaneous influxes of glucose and Na+ in C. verticilZatum were measured, it was found that 2 moles of Na+ were transported for every mole of glucose. This coupling coefficient ( [Na+ influx]/[ glucose influx] ) was independent of the .external Na+ concentrations (Table XV) (Pappas and Read 1972a). However, in H. diminuta, the coupling coefficient was found to be an inverse function of the Na+ concentration, such that as the Na+ concentration decreased the coupling coefficient increased (Table XV) (Read et al. 1974). Separate models were developed in

MEMBRANE

TRANSPORT

an attempt to explain the interaotions of Na+ and glucose in C. verticillatum and H. diminuta. The model postulated for C. verticillatum suggested that each “carrier” could bind a total of three atoms of Na+ and one molecule of glucose, but that only one Na+ was necessary for translocation of the Nat-glucose-“carrier” (ternary) complex across the tegument. Also, it was postulated that the binding of one Na+ to the “carrier” was independent of any other Na+ binding to the “carrier,” so that the “carrier” was just as likely to be translocated with one, two of three bound Na+ atoms. Thus, the translocation of these different “carrier”-complexes would result in an average coupling coefficient of two (Pappas and Read 1972a). The model postulated for H. diminuta also included a “carrier” with three Na+ binding sites. However, in order to account for the observation that the coupling coefficient increased as the Na+ concentration decreased it was necessary to postulate that one of the Na+ binding sites had a very high affinity for Na’, and was therefore saturated at very low Na* concentrations, but that binding of the Na+ to this high-affinity site did not result in translocation of the “carrier.” Thus, at low Na+ concentrations, the high-affinity Na+ binding site on the “carrier” would be saturated, but the binding of Na+ to the two sites responsible for translocation of glucose would be far Erom saturated, and glucose translocation would be below maximum. In such a system, increasing the Na+ concen8tration would result in increased binding of Na+ with the sites which are responsible for activating glucose translocation. Since the “nonactivating” site would be saturated over a broad Na’ concentration range, while the “activating” sites would not, increasing Na+ concentrations would yield a decreasing coupling coefficient approaching unity (Read et al. 1974 ) . Although the models postulated for Na+coupled glucose transport in C. verticil-

IN

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503

PARASITES

TABLE

XV

Summary of the Cou,pling Coeficients for Na+Coupled Glucose Transport in Hymenolepis di-

minuta and Calliobothrium verticillatum as a Function of the Naf Concentration c$ the h’xternal Medium (Data,from Read et al. [197.&I and Pappas and Read [1,97%‘a], Respectively). Note that in H diminuta the Coupling Coeficient is an Inverse Function of the Na+ Concentration, While in C. verticillatum it is Independent of the Na+ ConcentratiorL

-

Na+ concentrationa

Coupling coefficientb

Hymenolepis

diminuta

0.87 1.60 1.90

50

25 3 CaUiobothrium

verticillatum

50 33 20 10 a Reported as mequivlliter

1.78 1.89 1.96 1.76 for H. diminuta and

mM for C. verticillatum.

b The coupling coefficient equals : (Glucose influx)/ ([Na+ influx in the presence of glucose]-[Na+ influx in the absence of glucose]).

latum and H. diminuta do explain the observed data, neither model has been tested experimentally. The possibility of Na+coupled glucose transport has not even been examined in other cestode species. There is limited evidence that glucose transport in H. diminuta may be anionsensitive, also. Replacement of Cl- in the ambient medium with NOj- or CH,COOresulted in a decreased glucose influx rate; accumulation of glucose in Cl--free media was demons,trated, although the steady state internal glucose concentration was lower than that maintained by H. diminuta incubated in normal saline ( [Cl-] = 130 mM) (Pappas et al. 1974). The possibility of Cl--sensitive glucose transport in other cestode species has not been investigated. Clearly, the interactions of cations, anions, and glucose in absorptive processes

504

I'API'AS

AND HEAD

TABLE

XVI

SllbStl%le

Inhibit,or

_~.:i-0-nlethyiglrl~ose

~-

:3-O-methylglllcose ~~~ -. .~~~.-.~~

C;hl(!(JW

81

(+lwose ::7 .(+alact~ose 0 32 Alannose 90 Glllcosamine 7:: Frlu%ose 0 0 Sorboae 0 0 Ribose 0 2-Deoxyribose Arabinose Xylose 0 ___~ ~ .-~ ~~ .~ ~- ~~~ (LValLles are listed as percentage inhibition of tIptake; ( -) s&strate not tested. I)ata from Issetoff and Read (1974).

in cestodes is an area which requires additional investigative work before the mechanisms are elucidated. B. The Tremutoda 1. Fasciolu hepaticn. Isseroff and Read (1974) showed that the uptake of 3-O methylglucose, glucose, fructose, galactose, mannose, glucosamine, and ribose across the tegument of Fasciola hepatica was nonlinear with respect to concentration (Table IX) and stereospecific (Table XVI). However, F. hepatica did not accumulate 3-0methlyglucose; following a 90-min incubation in radi0activ.e 3-0-methylglucose a steady state was attained at an internal 30-methylglucose concentration below that of the incubation medium (this monosaccharide was not metabolized). Radioactive glucose, on the other hand, appeared to be accumulated by F. hepatica during 2-min incubations, based on the distribution ratios of radioactivity, but the rapid rate of glucose metabolism (70-80s metabolized in 2 min) precluded accumulation during these short-term incubations. Glucose uptake was Na+- and phlorizin insensitive, as well. Since it is unknown whether F. he-

( ::tlac~tose

Frrlrtose

Ribose

(Xi

TB

-

64

65 32 55 6

fiti 2s

:%.I 42 0

18 0

0 0 0

0

indicates

those cwmhinations

Its 1:: of inhibi tar arid

pat& will accumulate glucose during longer incubation periods, the mechanism of 3-0-methylglucose and glucose absorption by F. hepatica must be characterized provisionally as facilitated diffusion. Based on reciprocal inhibitor studies, Isseroff and Read (1974) postulated the presence of at least two distinct monosaccharide transport systems in F. hepatica. One system, referred to as the “glucose site,” appeared responsible for glucose, galactos.e, 3-0-methylglucose, glucosamine, and mannose transport. A second system, referred to as the “fruotose site” was postulated since fructose uptake was inhibited by aldohexoses, but fructose did not inhibit the uptake of thes.e same aldohexoses. Therefore, the data suggested that glucose, 3-0-methylglucose and galactose were bound nonproductively at the fructose site, and inhibited fructose uptake. Since the binding of the aldohexoses was postulated to be nonproductive, fructose would not be expected to inhibit the uptake of these aldohexoses. Mannose and glucosamine may also bind nonproductively at the fructose site, but the data are insufficient for a definite conclusion. The inhibition of

MEMBRANE

TRANSPORT

ribose uptake by fructose, glucose, and galactose suggested that ribose may also en’ter through the fructose system. However, further studies are needed to verify this point. A summary of the inhibitor data presented by Isseroff and Read (1974) is presented in Table XVI. Xylose appeared to enter F. hepatica by diffusion; uptake of this aldopentose was linear with respect to concentration, and was not inhibited by any monosaccharide which inhibited ribose uptake (Isseroff and Read 1974). Further studies are required to determine exactly the interactions of the various monosaccharides with the two postulated monosaccharide transport systems of F. hepatica. 2. Schistosomu munsoni. Bueding ( 1962) concluded that glucose uptake by the dioecious Schistosoma mansoni was mediated, since benzylic diamines inhibited glucose utilization by worms without affecting the enzymes involved in glucose metabolism. Subsequent studies by Isseroff et al. (1972) and Uglem and Read (unpublished) have substantiated Bueding’s original hypothesis, and provided additional data regardng monosaccharide transport in pairs. However, the reS. mansoni worm ports of Isseroff et al. (1972) and Uglem and Read ( unpublished) differ significantly in several aspects, as discussed below. Isseroff et al. (1972) reported that S. mansoni worm pairs absorbed fructose, galactose, glucose, glucosamine, ribose, and 3-0-methylglucose by “facilitated diffusion.” In support of this statement these authors presented Lineweaver-Burk plots (l/V vs l/[S] ) of absorption data, plots which indicate that at high substrate concentrations diffusion accounted for a considerable portion of monosaccharide absorption. None of the data of Isseroff et al. ( 1972) were corrected for this diffusion component. Isseroff et al. (1972) also stated that p-methylglucoside entered worms by diffusion, yet no data were reported to substantiate this claim.

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505

During short-term incubations (2 min), S. mansoni worms pairs accumulated both glucose and glucosamine, based on the distribution ratios of radioactivity. However, glucose was rapidly metabolized by worm pairs, thus precluding accumulation during these short-term incubations (Isseroff et al. 1972). Since Isseroff et al. ( 1972) did not determine the extent of glucosamine metabolism, it is possible that glucosamine was accumulated against a concentration difference, although these authors stated that this monosaccharide was absorbed by “facilitated diffusion.” No attempt was made to demonstrate accumulation of ribose, galactose, and fructose, so active transport of these monosaccharides is a possibility. Unpublished studies by Uglem and Read and Levy and Read confirmed the mediated nature of glucose, galactose, and ribose absorption by S. mansoni worm pairs (Table IX), and demons’trated that there was a significant diffusion component for the absorption of these monosaccharides at higher substrate concentrations. Uglem and Read (unpublished) also showed, as did Isseroff et al. ( 1972), that glucose was not accumulated due to its rapid rate of metabolism. However, Uglem and Read indicated that the absorption of 3-0methylglucose and fructose by S. mansoni worm pairs occurred by diffusion, rather than a mediated process, since uptake omf both of these monosaccharides was linear with respect to concentration, and not inhibited by their own moIecular species. Glucose and 2-deoxyglucose were mutual competitive inhibitors, and the uptake of glucose and 2-deoxyglucose by S. mansoni worm pairs was inhibited by the same monosaccharides. Since glucose and 2deoxygIucose apparentIy entered worms through the same transport system, and since 2-deoxyglucose was metabolized relatively slowly ( 10% in 15 min), as compared to glucose (70-8Oyi’ in 2 min), Uglem and Read (unpublished) partially characterized the glucose transport system

506

PAPPAS

by studying 2-deoxyglucose uptake by S. mansoni worm pairs. These authors found that 2-deoxyglucose was accumulated by worm pairs, indicative of active transport, and concluded that glucose may be accumulated if not metabolized so rapidly. Uptake of 2-deoxyglucose was inhibited in Na+-free media, and by ouabain, phlorizin, and phloretin (Isseroff et al. [ 19721 had reported glucose uptake to be insensitive to both ouabain and phlorizin), but neither Na+-deletion nor ouabain inhibited completely the accumulation of 2-deoxyglucose. The movements of 2-deoxyglucose and Na+ were shown to be coupled, with an experimemally determined coupling coefficient of 0.73 (Uglem and Read unpublished). These data suggest that glucose uptake by S. mnnsoni occurs, in part, through a Na+-coupled transport system, possibly similar to tha’t occurring in cestodes (see Carbohydrate Absorption, The Cestoda, General Considerations). A complete characterization of the monosaccharide transport system of S. mansoni will require much additional experimentation. C. The Nematoda Unlike amino acid transport by body tissues of Ascaris suum (Harris et al. 1972), transport of monosaccharides across “sac preparations” of A. suum gut (i.e., movement of solutes from the luminal solution to the external incubation medium) was significantly lower when a metabolizable monosaccharide was not present in either the luminal or incubation medium. That is, there appear,ed to be a distinct energy requirement for monosaccharide transport, derivable from either glucose or fructose in the luminal or incubation fluids (Senhueza et nl. 1968; Beames 1971). Also, monosaccharide transport was CO, dependent, and inhibited significantly in the presence of 0,. Therefore, a precise definition of the experimental conditions involved in the study of A. suum transport is essential.

AND

READ

The first indication that glucose uptake by nematodes involved a mediated process was provided by Bueding et al. (1961). These authors demonstrated that dithiazanine inhibited glucose uptake by Trichuris culpis, and also caused decreased levels of adenosine triphosphate, free glucose, and storage carbohydrates. Subsequent studies showed that glucose uptake by A. suum gut was inhibited by dithiazanine (Fisher. unpublished, cited by Read 1966) and mebendazole (Van der Bossche 1972; Van den Bossche and De Nollin 1973). The inhibitory action of mebendazolc was reported to be reversible, but detailed studies of the mechanism of inhibition do not appear in the literature. In A. suum intestinal preparations, glucose was transported from the luminal to pseudocoelomic (serosal) fluid (Fisher, unpublished, cited by Read 1966). Glucose transport by A. suum gut apparently occurred by a mediated process, since glueosc riptakc appeared sattnable \vith a \‘ll,;,h and Kt of 9.2 nmoles/mg wet wt/min and 9.1 mM, respectively (Senhueza et d. 1968). Intestinal cells of “ribbon preparation” accumulated glucose against a concentration difference (Castro and Fairbairn 1969) however, attempts to demonstrate glucose accumulation by sac preparations of A. suum gut have been unsuccessful (Senhueze et al. 1968). Glucose uptake by sac preparations of A. suum gut was Nat-sensitive and inhibited by phlorizin, suggesting the presence of a Nat-coupled transport system (Senhueza et al. 1968). Like glucose, fructose was readily absorbed and metabolized by A. suum gut preparations, while these same preparations appeared essentially impermeable to galactose ( Scnhueza et al. 1968; Beames 1971). Unlike glucose and fructose, 3-O-methylglucose was not metabolized by A. swum gut (Beames 1971). Thus, the low rates of 3-0-methylglucose absorption reported for ribbon and sac preparations of A. .suum gut by Castro and Fairbairn (1969) and Sen-

MEMBRANE

TRANSPORT

hueza et al. ( 1968 ), respectively, must be viewed with some caution, since Beames (1971) showed that 3-0-methylglucose absorption by sac preparations was increased significantly in the presence of glucose in the external incubation medium. Beames (1971) demonstrated further that 3-0methylglucose was transported from the luminal solution of sac preparations into the external medium against a concentration difference, as long as glucose was present in the external solution; if glucose was removed from the external solution, or replaced with 3-0-methylglucose, 3-O-methylglucose did not accumulate in the external medium. These facts, and the observation of Schanbacher and Beames (1973) that the rate of 3-0-methylglucose was ‘different in different sections of A. suum gut, are active suggestive of 3-0-methylglucose transport. The movement of 3-0-methylglucose out of A. suum gut sac preparations appeared to be unidirectonal in that 3-O-methylglucos,e in the external medium was not transported into the luminal solution even in the presence of glucose in the luminal solution (Beames 1971). Senhueza et al. (1968) showed that large amounts of glucose, fructose, and xylose were absorbed from the external solution by A. suum gut preparations, but they apparently did not analyze the luminal solution for the presence of these monosaccharides. Thus, it is not clear whether glucose, fructose, and xylose fluxes are unidirectional in A. suum gut. The brush border of A. suum gut contains intrinsic disaccharidases which act only on those disaccharides composed of glucose and fructose (maltose, palatinose, trehalose, and sucrose) ; disaccharides composed of glucose and galactose (lactose) are not hydrolyzed (Gentner et al. 1972; Van den Bossche and Borgers 1973). These findings correlate well with the ability of A. suum gut to absorb glucose and fructose, but not galactose. In addition to studies on the kinetics of monosaccharide and amino acid

IN

HELMINTH

507

PARASITES

transport by A. suum gut, experiments should be conducted to determine whether these intrinsic disaccharidases confer a “kinetic advantage” for monosaccharide transport, as is the case with the surface phosphohydrolases of H. diminuta and glucose transport by this cestode (see Relation of Surface Enzymes to Transport ) . Some nematodes have developed unique morphological characters which appear to function in absorption. For example, the cuticle of Bnzdynema sp. is composed of distinct microvillar projections (Riding 1970) reminiscent of the cestode tegument. Whether the cuticle of Bradynema sp. does, in fact, perform an absorptive function remains to be shown experimentally. In Sphaerulariu bombi the uterus is everted through the vulva, and a recent electronmicroscopical study (Poinar and Hess 1972) has suggested that the everted uterus performs an absorptive function. Again, direct evidence is lacking. In Mermis nigrescens the cuticle appears to be absorptive in nature. Rutherford and Webster (1974) demonstrated that the cuticle of larval M. nigrescens was readily permeable to radioaotive glucose, and that glucose uptake by larvae was nonlinear with respect to concentration. In addition, glucose uptake was inhibited by phloretin and 2,Cdinitrophenol. The data of Rutherford and Webster (1974) are suggestive of a mediated glucose transport system in larval M. nigrescens, but further studies are ne.eded if the mechanism of glucose uptake is to be elucidated. IV.

PURINE, NUCLEOSIDE

PYRIMJDINE,

AND

ABSORPTION

A. The Cestoda Of the transport systems known currently in Hymenolepis diminuta, the purine-pyrimidine transport system is the most complex by virtue of the fact that at least three distinct loci (“carriers”) are apparently involved, two of which contain multiple substrate binding sites.

508

PAPPAS

AND

TABLE A Summary

of the Interactions

of Purines

READ

XVII and Pyrimidines

Thymine

Thymine Uracil 5-Bromouracil Hypoxanthine Adenine Guanine

Uracil

5-Bromouracil

5-Aminouracil

f f 0 -

It

+ + +

f f + 0 0 0

a A (+) indicates that the “effect,or” uptake of substrate was inhibited. ,4 “0” substrate. A (+) indicates the “effector” the “effector”:substjrate ratio. Data from

+’ -

I 2.5

I

0 0 0

&Methyluracil -

Hypexanthine

Adenine

Purine

-

-

-

* -

0 0

stimulated substrate absorption, while a (-) indicates that the indicates that the “effector” had no effect on the upt,ake of the either stimulated or inhibited sltbstrate absorption, depending on Pappas et al. 1973a.

MacInnis et al. (1965) presented the first data suggesting the extremely complex nature of this transport system. In addition to demonstrating a mediated system (plus diffusion) for the uptake of uracil, adenine, and hypoxanthine, these authors showed that both purines and pyrimidines inhibited the uptake of uracil by H. diminuta, while only purines inhibited hypoxanthine uptake. In addition, these authors noted that uracil stimulated or inhibited uptake of labeled uracil by H. diminuta, depending

I I

diminutaa

“Effector”

Substrate

I

in Hymenolepis

I

I

1.5

10

r:l FIG. 6. The velocity of 0.1 mM radioactive uracil uptake (V, &moles absorbed/g ethanol extracted dry wet/hr) by Hymenolepis diminuta in the presence of increasing concentrations of “effector” ([El, m&f). ( l )-Thymine as the effector; (0)-uracil as the effector; ( W )-hypoxanthine as the effector. Redrawn from Pappas et al. ( 1973a).

on the inhibitor: substrate (I : S ) ratio, and also showed that thymine was a “competitive stimulator” (as opposed to a competitive inhibitor) of uracil uptake. Additional studies on the apparent stimulation of uracil uptake by uracil and thymine were conducted by MacInnis and Ridley ( 1969). These studies demonstrated conclusively that uracil uptake by H. diminuta was stimulated by some pyrimidines, and that thymine uptake as a function of thymine concentration displayed sigmoid kinetics. Analyses of the data by Hill plots indicated the presence of at least two substrate binding sites for uracil and thymine, leading MacInnis and Ridley (1969) to suggest that an allosteric mechanism might be involved. These authors also showed that the molecular configuration of a potential “effector” was important in determining its effects on uracil uptake; additions or substitutions of various chemical groups at the no. 6 carbon of uracil altered significantly the effects of these uracil derivatives on uracil uptake by H. diminuta. The studies by MacInnis et al. (1965) and MacInnis and Ridley (1969) showed the complex nature of the purine-pyrimidine transport system, and a recent report (Pappas et al. 1973a) has presented a model derived from the measurements of

MEMBRANE

TRANSPORT

the effects of numerous purines and pyrimidines on the uptake of radioactive uracil, thymine, 5-bromouracil, hypoxanthine, adenine, and guanine by H. diminuta. In the presence of increasing ooncentrations of either uracil or thymine, labeled uracil uptake by H. diminuta was stimulated; at low I: S ratios, uptake of radioactive uracil was inhibited by uracil, thymine, and 5-bromouracil, followed by &imulation of uptake at higher 1:s ratios (Fig. 6; Table XVII). Similar results were obtained when thymine, uracil, and S-bromouracil were tested as effecters of radioactive thymine uptake. The uptake of labeled thymine and uracil by H. diminuta was stimulated by 5-aminouracil, Ei-bromouracil, and thymine, and inhibited by 6methyluracil, hypoxanthine, adenine, and purine (MacInnis and Ridley 1969; Pappas et al. 1973a) (Table XVII). The lack of an inhibitory effect of low I: S ratios of 5-bromouracil and thymine on 5-bromouracil uptake, and the lack of any effect of uracil as an effector, suggested that the interaction of 5-bromouracil with the thymine-uracil transport locus was different from either the interaction of thymine or uracil with this locus (Pappas et al. 1973a). Although the data of MacInnis and Ridley (1969) did not ,show inhibition of labeled uracil or thymine uptake lby H. diminuta, since only higher I : S ratios were used, Maclnnis et al. (1965) noted that uptake of 0.05 mM radioactive uracil was inhibited by 0.5 mM uracil. Additional inhibistor studies (Pappas et al. 1973a) showed that uptake of radioactive thymine or uracil was unaffected by pyrimidine, cytosine, 5-methylcytosine, alloxan, 5-carboxyuracil (iso-erotic acid), thymidine, and uridine; 5-bromouracil uptake was unaffected by pyrimidine, cytosine, and 5-carboxyuracil. As was suggested by MacInnis and Ridley ( 1969), the stimulation of uracil uptake by relaited pyrimidines indicated at least two binding sites for uracil, these binding sites being denoted as an “activator” and

IN HELMINTH

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509

a “transport” site. The above data indicated that at low concentrations of effector uptake of radioactive substrate through the transport site was inhibited. As the concentration of effector increased the activator site (with a relatively low affinity for pyrimidines) was bound and uptake of substrate thereby increased due to either (1) an increased binding affinity of the transport site for the substrate, or (2) an increased translocation rate of the substrate (Pappas et al. 1973a). The data indicated further that thymine had a greater affinity for this transport locus than uracil, and that purines and 6-methyluracil interacted only with the transport site since they did not stimulate pyrimidine uptake (MacInnis and Ridley 1969; Pappas et al. 1973a). Hypoxanthine uptake by H. diminuta was inhibited by purines (adenine, hypoxanthine, and purine ) , 6-methyluracil, 5-bromouracil, and uracil, but not by thymine, 5-aminouracil, cytosine, thymidine, uridine, or pyrimidine (MacInnis et al. 1965; Pappas et al. 1973a) (Table XVII). When the uptake of radioactive hypoxanthine as a function of increasing concentrations of hypoxanthine or uracil was compared, it was apparent that hypoxanthine was a more effective inhibitor of labeled hypoxanthine uptake than was uracil. When the uptake of hypoxanthine by H. diminuta in the presence of constant 10 mM uracil was measured as a function of increasing concentrations of hypoxanthine, the data indicated the presence of two transport systems (loci), only one of which was inhibited by uracil (Fig. 7). The effects of uracil and adenine on the uptake of radioactive hypoxanthine in the presence of 10 mM uracil were additive, in that adenine inhibited hypoxanthine uptake in the presence of excess uracil. Apparently, the hypoxanthine locus which interacted with hypoxanthine, bat not uracil, interacted with adenine also ( Fig. 8 ) . AIthough the above data may be interpreted as indicating that hypoxanthine, uracil, and thymine all entered H. diminuta

510

PAPPAS AND READ THYMINEURACIL LOCUS

I/ I 1

I

I

2.5

I

I

7.5

10

T-T u-u SBU-3BU 5ilu A II (1’) (6MLJ)

HYPOXANTHINE LOCUS no. 1

HYPOXANTHINE LOCUS no. 2 I

II H A (P)

i;

FIG. 7. The velocity of 0.1 mM radioactive hypoxanthine uptake (V, pmoles absorbed/g ethanol extracted dry wt/hr) by Hymenolepis diminuta in the presence of various effecters ([El, mhl). ( l )-uracil as the effector; (0)-adenine as the effector; (A )-hypoxanthine as the effector; ( n )-constant 10 rnnl uracil plus increasing concentrations of adenine as the effecters; ( q )constant 10 mM uracil plus increasing concentrations of hypoxanthine as the effecters. Redrawn from Pappas et (11. ( 1973a).

through a common locus, since hypoxanthine, uracil, and thymine uptake was inhibited by adenine, purine, hypoxanthine, and 6-methyluracil, and since hypoxanthine uptake was not inhibited by uracil or 5bromouracil (Table XVII), this apparently was not the case. As discussed below, hypoxanthine appeared to enter worms through two distinct loci, both of which were distinct from the thymine-uracil locus. One locus bound hypoxanthine, uracil, and 5-bromouracil (but not 5-aminouracil), and the other locus bound hypoxanthine, adenine, and purine (Fig. 8). With .the exception of purine, those inhibitors which inhibited hypoxanthine uptake by H. diminuta also inhibited guanine uptake (Table XVII). Therefore, guanine appeared to share a and hypoxanthine common locus, this being one of the hypoxanthine l’oci. The fact that purine failed to inhibit guanine uptake supported the hypothesis of two hypoxanthine loci, only one of which bound guanine (Pappas et al. 1973a). Labeled adenine uptake by H. diminuta was sigmoid with respect to adenine con-

FIG. 8. A summary of the interactions of purine and pyrimidine bases with the transport loci of Hymenokpis diminuta. T = thymine; U = uracil; 5BU = 5-bromouracil; 5AU = 5-aminouracil; 6’MU = 6-methyluracil; H = hypoxanthine; A = adcnine; G = guanine; P = purine. Compounds noted in italics are nonproductively bound to the indicated locus. The nature of the binding of those compounds in parentheses is uncertain. The remaining purines and pyrimidines are productively bound to the indicated locus. Compounds noted as dimers (e.g., T-T and U-U) appear to bind at two sites on the indicated locus since they display allosteric properties. This model makes no inference as to the total number of binding sites at each locus. Redrawn from Pappas et al. ( 1973a).

centration, sugges,ting an allosteric mechanism. In the presence of increasing concentrations of unlabeled adenine, radioactive adenine uptake was stimulated at low I: S ratios, followed by inhibition of uptake

FIG. 9. The velocity of 0.1 mM radioactive adenine uptake (V, pmoles absorbed/g ethanol extracted dry wt/hr) by Hymenolepis diminuta in the presence of various effecters ([El, mM). (0)-Adenine as the effector; ( l )-hypoxanthine as the effector; ([7)-constant 0.25 mM adenine plus increasing concentrations of hypoxanthine. Redrawn from Pappas et al. ( 1973a).

MEMBRANE

TRANSPORT

at higher I: S ratios (Fig. 9). Adenine uptake was also inhibited by hypoxanthine, 6-me~thyluracil, uracil, Sbromouracil, but not by thymine, 5-aminouracil, or purine ( Table XVII). The effects of unlabeled adenine on the uptake of labeled adenine suggested that at low concentrations of effector adenine uptake was stimulated, possibly by binding of the effector at an activator site. As the concentration of effector increased, the activator site was apparently saturated, and, at high effector concentrations, the “stimulated” adenine uptake was inhibited by binding of the effector at the transport site. This hypothesis was supported by the fact that hypoxanthine inhibited adenine uptake more effectively than adenine. In addition, when the uptake of 0.1 mM radioactive adenine was measured in the presence of a constant 0.25 mM adenine (that concentration of unlabeled adenine which resulted in maximal stimulation of uptake) and increasing concentrations of hypoxanthine, the effects were similar to when hypoxanthine alone was tested as an effector (Fig. 9). Therefore, hypoxanthine appeared to bind at both the activator and transport sites, thereby inhibiting the stimulation of adenine uptake by adenine (Pappas et al. 1973a). The effects of all effecters tested against hypoxanthine, adenine, and guanine uptake, except purine, were essentially identical (Table XVII) suggesting that these in part, purines entered H. diminuta, through a common locus. The fact that purine failed to inhibit labeled adenine or hypoxanguanine uptake, but inhibited thine uptake, supported the hypothesis of two hypoxanthine loci, only one of which bound purine (in addition to hypoxanthine), and a second locus which bound hypoxanthine, adenine and guanine, but not purine. From these observations a model for purine-pyrimidine transport in H. diminuta was constructed (Pappas et al. 1973a), a model which took into account the fol-

IN HELMINTH

PARASITES

511

lowing general considerations. It was apparent that thymine and uracil were transported through a single locus in H. diminuta. The uptake of each of these pyrimidines was affected by each other, and thymine uptake was sigmoid with respect to concentration ( MacInnis and Ridley 1969; Pappas et al. 1973a). These data strengthened the original hypothesis of MacInnis and Ridley ( 1969) that “the carrier transporting uracil into Hymenolepb diminuta contains a minimum of two binding sites.” As indicated by the effects ti various pyrimidines and purines on the uptake of uracil and thymine, these two classes of compounds displayed varying affinities for the uracil-thymine locus. Pyrimidines appeared to bind to both the transport and activator sites of the locus, while purines were bound only at the transport site. An exception to this was the pyrimidine 6-methyluracil. The nature of the chemical group occupying the no. 5 and no. 6 carbons of the uracil ring was shown to be important in determining the effects of uracil derivatives on uracil uptake by H. diminuta. Uracil, uracil derivatives, and thymine (5-methyluracil ) all stimulated uracil transport, while 6-methyluracil inhibited transport (MacInnis and Ridley 1969). These data demonstrated conclusively that the effects of 6-methyluracil on uracil and thymine uptake by H. diminuta paralleled those of purines rather than pyrimidines. Apparently, 5-bromouracil entered H. diminuta, in part, through the thymine-uracil locus. The effects of purines on the uptake of S-bromouracil, thymine and uracil were similar, and Sbromouracil, uracil, and thymine were mutual effecters of each other (Table XVII). H owever, 5-bromouracil, like uracil, also entered worms through one of the hypoxanthine loci (Fig. 8). A determination of the exact nature of the interaction of 5-bromouracil with the thymineuracil locus requires further investigation. The additive inhibitory effects of uracil and adenine on hypoxanthine uptake indi-

512

PAPPAS AND READ

cated the presence of two hypoxanthine loci. Hypoxanthine, guanine, and adenine all appeared to be transported through one of these loci, since these purines were mutual effecters of each other, and since all effecters (except purine) had identical effects on the uptake of these labeled substrates (Table XVII). The fact that purine inhibited hypoxanthine uptake, but not adenine and guanine uptake, supported the hypothesis of two hypoxanthine loci. These loci w.ere characterized as follows: Hypoxanthine locus no. 1 transported hypoxanthine, guanine, and adenine (the latter by an allosteric mechanism), but did not bind locus no. 2 transpurine; hypoxanthine ported hypoxanthine and also bound purine. Uracil and Sbromouracil apparently interacted with hypoxanthine locus no. 1 by virtue of their inhibitory action on the uptake of guanine, adenine, and hypoxanthine. In the presence of saturation concentrations of uracil (10 mM) hypoxanthine uptake was inhibited by adenine, an observation which appears to contradict the above description of hypoxanthine locus no. 2. The data can be explained, however, if one considers that hypoxanthine was productively bound (i.e., bound and transported) at locus no. 2, while adenine was nonproductively bound (i.e., bound but not transported) at this same locus. This would explain the inhibition of hypoxanthine uptake by adenine in the presence of 10 mh1 uracil, and the lack of an effect of purine on adenine uptake (Table XVII; Fig. 8). Adenine and hypoxanthine apparently bound nonproductively to the thymineuracil locus of H. diminuta. Both adenine and hypoxanthine inhibited the uptake of uracil and thymine, but thymine had no effect on the uptake of either hypoxanthine or adenine. The inhilbitory effect of uracil on hypoxanthine and adenine uptake was due to nonproductive binding of uracil at one of the hypoxanthine loci. Purine and 6methyluraciI also interacted with the thy-

mine-uracil locus, but the nature of these interactions was not determined (Fig. 8). The purine-pyrimidine loci are specific for purines and pyrimidines, but the possibility exists that nucleosides may interact to a limited extent (see last paragraph of this section). Amino acids with ring structures similar to purines and pyrimidines had no effect on the uptake of either uracil or hypoxanthine by H. diminuta (Pappas et al. 1973a). Also, thymine appeared to have a stimulatory effect only on the pyrimidine locus of H. diminuta, since glucose, isoleucine, and phenylalanine were unaffected by thymine (MacInnis and Ridley 1969). The interactions of the various purines and pyrimidines with the hypothesized loci are summarized in Fig. 8. Nucleoside transport in H. diminuta is mediated, but extensive kinetic data are lacking. Radioactive uridine uptake by H. diminuta was saturable (Table XVIII) and inhibited by unlabeled uridine, adenosine, deoxyuridine, cytidine, inosine, adenosine monophosphate, and adenosine triphosphate (Table XIX) ( MacInnis and Ridley 1969; Pappas and Read 1974). The fact that labeled uridine uptake was inhibited 98+% by high concentrations of uridine or adenosine indicated that diffusion of uridine was negligible (Pappas and Read 1974). Labeled uridine uptake was stimulated by thymine, but unaffected by hypoxanthine or uracil (MacInnis and Ridley 1969). These data are not sufficient to determine whether nucleosides interact with the purine-pyrimidine loci of H. diminuta, or whether purines and pyrimidines interact with a nucleoside transport system in this cestode. B. The Trematoda The only data available concerning purine, pyrimidine, and nucleoside transport in trematodes are the unpublished data of Levy and Read dealing with transport of these compounds in Schistosoma mansoni

MJZMBFL~NE

TRANSPORT

TABLE A Summary

Substrate Uracil Hypoxanthine Acetate Acetated Butyrate Palmitate Palmitate Uridine

IN

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513

PARASITES

XVIII

of the Kinetic Parameters Describing Mediated Various Substrates by Hymenolepis diminuta

Uptake of

Kt (mM)

V maxa

Reference

0.025 0.029 0.92 1.15 1.5 0.71e 1.W 0.082

0.43 2.68 54.6c 2 x 10-4 15@ 14.40eJ 42erf 26.6

MacInnis et al. 1965b MacInnis et al. 1965b Arme and Read 1968 Arme et al. 1973 Arme and Read 1968 Chappell et aZ. 1969 Chappell et al. 1969 Pappas and Read 1974

apmoles absorbed/g ethanol extracted dry wt/hr, unless noted otherwise. b MacInnis et al. (1965) reported kinetic parameters for other purines and pyrimidines, but these are the only data that were corrected for diffusion. c Originally reported in terms of uptake/min, but converted to uptake/hr. d Da.ta for cysticercoid larvae. V mal: = pmoles/lOO cysticercoids/2 min. e The kinetic parameters describing palmitate absorption varied greatly, depending on the concentration of the “stock” palmitate solution. ’ Originally reported in terms of uptake/2 min, but converted to uptake/hr.

worm pairs. These authors found that uptake of pyrimidines (uracil, thymine, and cytosine) by S. mansoni occurred solely by diffusion. The uptake rate of each of these pyrimidines was linear with respect to concentration, and uptake was unaffected by their own molecular species when tested as inhibitors. Purines (adenine, guanine, and hypoxanthine) were absorbed by S. munsoni worm pairs by a combination of a mediated system and diffusion (Table IX). Adenine and guanine were completely competitive inhibitors of each other suggesting transport through a single system. The evidence obtained by Levy and Read (unpublished) indicated that nucleosides ( purine ribosides) also interacted with the adenineguanine transport system. The mediated uptake of labeled adenine by S. munsoni worm pairs was inhibited only partially by unlabeled adenosin.e, and in reciprocal experiments, the mediated uptake of labeled adenosine was inhibited only partially by unlabeled adenine. Thus, these investigators postulated tha’t adenine and guanine were transported, in part, through a single distinct system, while adenosine was

transported in part through a second system. Apparently, the remainder of the mediated adenine and adenosine uptake occurred through a third system which transported both adenine and adenosine. The data of Levy and Read (unpublished) demonstrated further that uridine uptake by S. munsoni was saturable (Table IX) and inhibited by adenosine. At high concentrations, uridine was absorbed, in part, through simple diffusion. Mediated uridine uptake was inhibited completely by adenosine, but uridine inhibited radioactive adenosine uptake incompletely. Since adenosine uptake by S. mansoni was not inhibited completely by uridine, uridine transport apparen,tIy occurred through only one of the adenosine transport systems, that being the system which transported adenosine only (as opposed to the system which transported both adenosine and adenine ) . Levy and Read (unpublished) also tested the possibility that nucleosides may be transported through the ribose transport system of S. mansoni. No data were obtained to substantiate this possibility,

514

PAPPAS

TABLE A Summary

of Inhibitor Co&ants Uptake of Different

Substrate Uracil Uraeil Hypoxanthine Hypoxanthine Hypoxanthine Adenine Acetate Acetate Acetate Acetate Acetate Acetate Acetate Butyrate Palmitate Palmitate Palmitate Palmitate Palmitate Palmitate Palmitate PaImitate Palmitate Palmitate Uridine Uridine Uridine

AND

READ

XIX

Describing the Effects of Various Inhibitors S,ubstrates by Hymenolepis diminutn

on the

Inhibitor

K, (mM)

Reference

Hypoxant’hine Thymine Hypoxanthine Purine Adenine Hypoxanthine Formate Propionat’e Propionate Butyrate Butyrate Valerate Octanoate Acetate Pentadecanoate Heptadecanoate Stearate Arachidate Oleate Oleate Linoleate Linoleate Linolenate Linolenate Adenosine Adenosine monophosphate Adenosine triphosphate

1.15 -4.22= 0.84 4.54 5.95 0.95 6.9 1.25 5.36 3.6 w 5.0 5.1 5.4 4.9 2.3 2.3 3.5 -4.5c 4.5c -3.8c 3.8c - 5.6c 5.6c 0.085 0.104 0.285

Madnnis et al. 1965 MacInnis et al. 1965 MacInnis et al. 1965 Maclnnis et al. 1965 Maclnnis et al. 1965 Maclnnis et al. 1965 Arme and Read 1968 arme and Read 1968 Arme et al. 1973 Arme and Read 1968 Arme et al. 1973 Arme and Read 1968 Arme and Read 1968 Arme and Read 1968 Chappell et al. 1969 Chappell et al. 1969 Chappell et al. 1969 Chappell et al. 1969 Chappdl et al. 1969 Chappell et al. 1969 Chappell et al. 1969 Chappell et al. 1969 Chappell et al. 1969 Chappell et al. 1969 Pappas and Read 1974 Pappas and Read 1974 Pappas and Read 1974

a Classified as a “competit,ive stimulator” by MacInnis et al. (1965), since it stimulated tnacil when present at high concentjratjions. * Data for cysticercoid larvae. c These long chain fatty acids either stimulated or inhibited the uptake of palmitate, depending concentration of inhibitor in the medium.

V. FATTY

ACID

ABSORPTION

A. The Cestoda Using radioactive acetate as a substrate, Arme and Read (1968) demonstrated a mediated system in Hymenolepis diminuta for the uptake of short chain fatty acids. The absorption of acetate was nonlinear with respect to concentration at low concentrations, At higher acetate concentrations, uptake occurred mainly by diffusion (i.e., uptake was linear). Typical saturation kinetics resulted following correction for the apparent diffusion component (Table XVIII). Arme and Read (1968) were un-

uptake

on the

able to demonstrate acetate accumulation by H. diminuta. Following a 40-min incubation in 0.1 mM labeled acetate, the apparent internal acetate concentration, as determined from radioactivity, was 0.175 mhl, 60% of which was acetate. These authors suggested that mediated aceta,te uptake may involve facilitated diffusion rather than active transport. The uptake of radioactive acetate was inhibited by unlabeled propionate (Table XIX). At a 500/l I: S ratio the rate of acetate uptake corresponded to the expected diffusion component, suggesting that the linear rate of acetate uptake at high con-

MEMBRANE

TRANSPORT

centrations was not a second transport system. Labeled acetate was inhibited by other short chain fatty acids (Table XIX), however, only the inhibitions produced by formate and acetate were charaoterized as fully competitive in nature (Arme and Rea’d 1968). Acetate absorption was unaffected by amino acids, monosaccharides, uracil, adenine, ouabain, phlorizin, 2,4-diand deoxycholate, showing nitrophenol, that the short chain fatty acids were absorbed through a locus which is distinct from other transport loci (Arme and Read 1968 ) . Acetate absorption by H. diminuta was pH dependent. Acetate uptake at pH 7.4 was nonlinear with respect to concentration; at pH 6.2, acetate uptake was higher than at pH 7.4, and was linear with respect to concentration, Also, the effectiveness of unlabeled propionate as an inhibitor of labeled acetate uptake increased with increasing pH, leading Arme and Read (1968) to speculate that the undissociated form of the fatty acid entered mainly by diffusion, while the dissociated form was involved in mediated transport. The uptake of labeled butyrate by H. diminuta was studied in a few experiments by Arme and Read ( 1968). Butyrate uptake was saturable and inhibited by the same short chain fatty acids which ‘inhibited acetate uptake (Tables XVIII and XIX). Two observations of Arme and Read (1968) indicated that acetate and butyra,te were absorbed through a common locus. First, acetate and butyrate were mutual competitive inhibitors, and second, the relative effects of the various short chain fatty acids on the uptake of labeled acetate and butyrate were almost identical. The nature of the interactions of other short chain fatty acids with the transport locus remains unknown. Using the equation derived by Read et al. (1963), Arme and Read ( 1968) demonstrated that the effect of a complex mixture of short chain fatty acids on the uptake of a single fatty acid could be predicted

IN

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515

with reasonable certainty, provided that certain kinetic parameters were known for these fatty acids. This demonstrated the usefulness of this equation in systems other than those involving amino acids. As in H. diminuta adults, acetate absorption by H. diminuta cysticercoids appeared to be mediated at low acetate concentrations; at high acetate concentrations uptake occurred mainly by diffusion (Table XVIII). Butyrate and propionate were competitive inhibitors of acetate uptake (Table XIX), but stereate, and other nutrients (not listed by authors ) did not inhibit acetate uptake ( Arme et ab. 1973). Therefore, H. diminuta larvae also possess a short chain fatty acid transport system. Unlike short chain fatty acids, which are readily soluble in water, long chain fatty acids (C : 12 and longer) present a difficult solubility problem. Even so, Chappell et al. (1969) accumulated significant data on long chain fatty acid transport in H. diminuta. The uptake of radioactive palmitate appeared to be saturable over the concentration range of O-4 mM (Table XVIII), and uptake was inhibited by other long chain fatty acids (Table XIX). At high I : S ratios the inhibition produced by these long chain fatty acids was not complete suggesting the presence of a significant diffusion component. Uptake of labeled palmitate was not inhibited by short chain fatty acids, nor by dicarboxylic acids, amino acids, mono-, and disaccharides, adenine, uracil, ouabain, or 2,Cdinitrophenol. Clearly, the short chain fatty acid and long chain fatty acid systems are distinct (Chappell et al. 1969). The effects of laurate on the uptake of labeled palmitate by H. diminuta were quite different from those of other fatty acids; the uptake of 0.025 mM labeled palmitate was stimulated in the presence of increasing concentrations of laurate, with a maximum stimulation of 77% at 2 mM laurate. Stimulation of labeled palmitate (0.025 mM) uptake was also noted in the

516

PAPPAS

AND

READ

presence of 0.025 mM oleate, linoleate, and linolenate, but higher concentrations of these unsaturated fatty acids inhibited palmitate uptake. Low concentrations of palmitoleate had no effect on the uptake of palmitate (Chappell et al. 1969). The stimulation of palmitate uptake by unsaturated fatty acids suggests an “allosteric” mechanism, possibly similar to that postulated for purine and pyrimidine uptake by H. diminutu (MacInnis and Ridley 1969; Pappas et al. 1973a). Extensive studies will be needed to characterize this system. The inhibition of palmitate uptake by saturated and unsaturated long chain fatty acids, and stimulation by laurate, was characterized as partially competitive in nature by Chappell et al. (1969). However, the apparent partial nature of these competitions may involve allosteric relationships as suggested above. The uptake of labeled palmitate was pH dependent, with an optimum at pH 6.8, and was also dependent on the concentration of deoxycholate (used to solubilize the fatty acids) in the medium. Palmitate uptake was affected little by deoxycholate concentrations ranging from 0 to 2 mM, while deoxycholate concentrations greater than 2 mM nearly doubled the uptake rate of palmitate by H. diminuta. A thorough discussion of this phenomenon was presented by Chappell et al. ( 1969), but these authors were unable to determine whether these results were due to a direct effect of deoxycholate on the tegumental membranes (unlikely since this bile salt had no effect on acetate absorption), or the formation of choleic acids or a micellar solution,

rocal inhibitions were tested at 100/l I: S ratios, no inhibition of any substrate was observed. Absorption of all substrates was ouabain insensitive, and succinate uptake was unaffected by pyridoxal, horse serum, or host bile in the medium. The Qlc, (2838 C) for succinate uptake was less than 2. From these data, Isseroff and Walczak (1971) suggested that these substrate entered F. hepatica by diffusion. Wright and Isseroff (1973) demonstrated that the stimulator-y effects of citrate and propionate on acetate uptake by F. hepatica, as reported previously by Isseroff and Walczak ( 1971)) were due to alterations in the pH of the incubation medium, and that these stimulatory effects could be duplicated by altering the pH of the incubation medium. Wright and Isseroff (1973) demonstrated further that when the pH of all media was carefully controlled acetate uptake by F. hepatica was nonlinear with respect to concentration, with a Kt of about 20 mM. In addition, acetate uptake was not affected by palmitate, stearate, iodoacetate, or Nat-deletion, but was inhibited by butyrate, valerate, and propionate at 100/l inhibitor: substrate ratios. Due to its rapid rate of metabolism, acetate was not accumulated by F. hepatica even after a 1-hr incubation. Clearly, the data of Wright and Isseroff (1973) suggest that acetate enters F. hepatica, in part through facilitated diffusion, and not simple diffusion as suggested by Isseroff and Walczak ( 1971). Also, since butyrate, valerate, and propionate inhibited acetate uptake, these short chain fatty acids may also enter through a mediate system.

B. The Trematodu

C. The Nematoda

Very little is known of the kinetics or the mechanisms of absorption of fatty and other organic acids by trematode parasites. Isseroff and Walczak (1971) showed that the rate of absorption of acetate, butyrate, malate, pyruvate and succinate by Fasciola hepatica was linear up to 10 mM substrate concentration, and when all possible recip-

Using everted Ascaris suum gut sac preparations, Beames and King (1972) demonstrated that the movement of albumin-complexed labeled palmitic acid into mucosal cells was not inhibited by oxygen, iodoacetate, or the absence of glucose. However, the movement of albumin-complexed labeled palmitic acid from the external me-

MEMBRANE

TRANSPORT

dium (mucosal side) to the pseudocoelomic side of sac preparations was inhibited in the absence of glucose, and in the presence of oxygen and iodoacetate; Van den Bossche and de Nollin (1973) reported that glucose enhanced the absorption of labeled palmitic acid by intact A. suum. Thus, the m’ovement of palmitic acid ilzto the mucosal cells appeared passive, while movement of the absorbed palmitic acid out of the mucosal cells into the pseudocoelomic fluid appeared to require energy (i.e., possibly mediated) which was obtained from carbohydrate catabolism. The effects of ‘bile salts on fatty acid absorption by simple sac preparations of A. suum were studied using a micellar solution of glycochenodeoxycholate and labeled palmitic, oleic or linoleic acid. Complexing these fatty acids with glycochenodeoxycholate increased their rate of absorption (i.e., movement from the luminal solution into the external medium) over that of fatty acids complexed with albumin. The absence of glucose inhibited the movement of glycochenodeoxycholate-complexed fatty acids. Although the rate of absorption of complexed fatty acids increased with increasing concentration, no evidence for saturation of a transport system was found (Beames and King 1972). Although some of the available data suggest mediated fatty acid absorption by A. suum gut, the data are insufficient for a firm conclusion. Beames et al. (1974b) reported that sac ‘preparations of A. suum gut were essentially impermeable to glycerol-tri [ 1-14C]palmitate, and that this triglyceride was not hydrolyzed by the intestine. In addition, these authors showed that radioactive monoolein was readily absorbed by A. suum gut, but that monoglycerides were probably first hydrolyzed and not absorbed intact. Although considerable amounts of radioactive monoolein were incorporated into polar lipids and triglycerides of A. suum intestinal tissue during experimental incubations, 8%96% of the radioactivity

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in the pseudocoelomic solution was present as free fatty acid. Apparently, the free fatty acid was the major transported substrate (Beames et al. 1974b). A. suum gut was also shown to be permeable to both cholesterol and P-sitosterol ( Beames et al. 1974a), however, kinetic data regarding the absorption of these sterols is apparently lacking. VI.

ABSORPTION

OF

VITAMINS

A. The Cestook One of the best examples of a helminth parasite with a special predilection for a specific nutrient is found in the case of Dibothriocephalus (= Diphyllabothrium) Eatus and vitamin Brz ( cobalamine ) , Although data on the kinetics of cobalamine absorption by D. l&us do not seem available, previous studies indicate that D. latus accumulates significant amounts of this vitamin (Brante and Emberg 1957, 1958; von BonsdorfI 1956; Nyberg 1958). It is not uncommon for specimens of D. Zatus to have a distinct pink color due to the accumulation of large quantities of cobalamine. Mueller (1968) reported that spargana ( plerocercoid larvae) of Spirometra mansonoides may occasionally be pink, and suggested that this pink color may be due to an accumulation of cobalamine. The kinetics of cobalamine absorption in these two pseudophyllidean cestodes should be studied, since previous studies indicate the presence of a system for active transport of this vitamin. The only data available on the kinetics of vitamin absorption by cestodes involve the absorption of water-soluble vitamins by Hymenolepis diminuta. The rate of labeled thiamine uptake by H. diminuta was linear with respect to concentration over the range of 0.05-100 mM suggesting the lack of a mediated system. However, the uptake of 0.1 mA4 radioactive thiamine was inhibited significantly by unlabeled thiamine indicating the presence of a mediated component which was saturated at very low

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thiamine concentrations. Evidence of a mediated system was also evident from additional inhibitor studies; labeled ,thiamine uptake was inhibited by thiamine monophosphate, oxythiamine, neopyrithiamine, and iodoacetate. Thiamine uptake was not inhibited by thiamine pyrophosphate, amino acids, monosaccharides, pyrimidines, other water-soluble vitamins (riboflavin, fohc acid, nicotinamide ), or 2,4-dinitrophenol, indicating a distinct mediated system for thiamine uptake (Pappas and Read 1972b ) . Two components (i.e., diffusion and a mediated system) have also been demonstrated for riboflavin uptake by H. diminuta. The mediated nature of riboflavin uptake was demonstrated by the facts that uptake of radioactive riboflavin was not linear with respect to concentration, and riboflavin uptake was inhibited by riboflavin, flavin mononucleotide, and %avin adenine dinucleotide. However, radioactive riboflavin uptake was not inhibited by amino acids, monosaccharides, adenosine triphosphate, nicotinamide, folic acid, pyridoxine, or thiamine (Pap,pas and Read I972c). Therefore, H. diminuta possesses at leas’t #two specific systems for vitamin absorption, one for thiamine, and another for riboflavin, In contrast to thiamine and riboflavin, pyridoxine, and nicotinamide transport systems are apparently lacking in H. diminuta. Uptake of both of these water-soluble vitamins was linear with respect to concentration over a wide concentration range (Pappas 1972; Pappas and Read 1972c). Uptake of labeled pyridoxine was not inhibited by unlabeled pyridoxine, pyridoxal, pyridoxamine, pyridoxal-S-phosphate, pyridoxamine-5’-phosphate, or deoxypyridoxine (Pappas and Read 1972c). The uptake of radioactive nicotinamide was not inhibited by unlabeled nicotinamide ( Pappas 1972). The lack of inhibition of pyridoxine and nicotinamide uptake by identical and structurally related molecular species indicates

that diffusion is the only process operating in the absorption of these vitamins. It is interesting to note that while H. diminuta apparently lacks a mediated transport system for pyridoxine absorption, this cestode has a specific pyridoxine requirement (Platzer and Roberts 1969; Roberts and Mong 1973 ) _ It would seem, therefore, that the pyridoxine requirement of H. diminuta can be met by simple diffusion of this vitamin into the worms, and that, in this instance, a mediated system is unnecessary for nutrient acquisition. B. The Nematoda Although no kinetic data are available regarding vitamin absorption by nematodes, Zam et aZ. (1963) presented data suggesting that Ascaris suum can accumulate significant concentrations of F°C,o-vitamine Bl?. When male A. suum were incubated in a solution of 2 pg G°Co-vitamin Blz/ml for 12 hr, the final concentration of vitamin B12 in worms was 10 @g/g wet wt, or about 12 pg/ml worm water. With some female A. suum the final B12.concentration rose as high as 25 pg/g wet wt. Zam et al. (1963) demonstrated that the cuticle of A. suum was impermeable to vitamin B12, thus absorption must have occurred across the intestinal brush border. Obviously, more studies are needed to evaluate the mechanisms of vitamin absorption in A. suum and other nematodes. VII.

RELATION OF SURFACEENZYMES TO TRANSPORT

As is evident from the preceding discussion, the cestode tegument as an absorptive surface has been studied in some detail. Only recently, however, has the digestive role of the cestode tegument been examined in any detail. Phifer (196Oc) demonstrated that intact Hymenolepis diminuta hydrolyzed p-nitrophenylphosphate and suggested that hydrolysis took place at the cestode surface. Using electronmicroscopy and histochemical methods, Rothman (1966), Lumsden et al. (1968), and Dike

MEMBRANE

TRANSPORT

and Read (1971a) verified Phifer’s hypothesis by localizing nucleotide, hexose phosphate and p-nitrophenylphosphate hydrolysis at the worm surface. Arme and Read (1970) presented chemical data which also supported Phifer’s hypothesis, when they showed that H. diminuta hydrolyzed fructose 1,6-diphosphate with fructose accumulating in the medium. Since H. diminuta was also shown to be virtually impermeable to fructose, Anne and Read (1970) concluded that hydrolysis occurred at the worm surface. Lumsden et al. (1968) showed that the phosphohydrolases associated with H. diminuta hydrolyed several hexose and triose phosphate esters, and the nucleotides of adenosine, guanosine, and cytidine. These authors suggested that phosphohydrola,se hydrolysis products may interact with the various transport systems of H. diminuta, and Dike and Read (1971b) conducted an experimental examination of this possibility. These latter authors showed that “apparent” glucose-6-phosphate uptake by H. diminuta was Na+-sensitive, and that when H. diminuta was incubated in the presence of glucose-6-phosphate in Nat-free media, glucose accumulated in the medium. In addition, Dike and Read (1971b) showed that those phosphohydrolase inhibitors which were not glucose derivatives (e.g., fructose phosphates, mono- and triphosphate nucleotides, and ammonium moIybdate) inhibited the “apparent” uptake of glucose-8phosphate, but were without effect on glucose uptake. Thosse co’mpounds which inhibited glucose uptake (e.g., glucose and galactose) had no effect on surface phosphohydrolase activity. Compounds which inhibited either glucose uptake or phosphohydrolase activity inhibited “apparent” glucose-6-phosphate uptake. Dike and Read (1971b) concluded that the phosphohydrolases and glucose transport system of H. diminuta were separate, and that apparent hexose phosphate absorption occurred through two distinct followed processes, i.e., dephosphorylation

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519

by absorption. Since glucose oxidase in the external medium did not inhibit “apparent” glucose-6-phosphate uptake, but did inhibit glucose uptake (the worms were subsequently shown to be impermea,ble to radioactive gluconic acid), and since the effects of glucose oxidase were readily reversible by rinsing the worms, Dike and Read (1971b) concluded that the phosphohydrolases and glucose transport system of H. diminuta must be in such close spatial proximity tha.t glucose liberated from the hydrolysis of glucose phosphates was rapidly absorbed rather than diffusing into the medium. (If the glucose liberated from glucose phosphate hydrolysis had diffused into the medium in the experiment described above, then the glucose oxidase would have oxidized the glucose to gluconic acid, a compound to which the worms were shown to be impermeable. ) Since Lumsden et al. (1968) had shown that H. diminuta tissue homogenates hydrolyzed various nucleotides, Pappas and Read ( 1974) investigated the possibility of the interaction of nucleotide hydrolysis and nucleoside transport in H. diminuta. It was shown that adenosine monophosphate and adenosine triphosphate were oompletely competitive inhibitors of radioactive uridine uptake by worms, as were unlabeled adenosine and uridine (Table XIX), and the inhibition of labeled uridine uptake by a,denosine triphosphate was relieved in the presence of phosphohydrolase inhibitors (these same inhibitors had no effect on uridine uptake). Pappas and Read (1974) concluded that the nucleoside transport system and phosphohydrolases of H. diminuta were located in such a relation that products liberated from adenosine triphosphate hydrolysis interacted with the nucleoside transport system o,f worms rather than diffusing into the ambient medium. Hymenolepis diminuta also possesses, what appears to be, an intrinsic tegumentary ribonuclease (Pappas et al. 1973d). However, the significance of this enzyme

520

PAPPAS

remains uncertain, for it does not seem to function in supplying phosphohydrolase substrates (i.e., nucleotides ) (Pappas unpublished). There is also evidence that the tegument of H. diminuta may contain a functiona monoglyceride hydroIase (Bailey and Fairbairn 1968). However, an experimental examination of this possibility is lacking. Although intact H. diminuta lacks detectable aminopeptidase or dipeptidase activity, the acanthocephalan, Moniliformis dubius, has significant aminopeptidase activity (Read et al. 1963; Uglem et al. 1973). Uglem et al. ( 1973) showed that M. dubius hydrolyzed several leucine and alanine containing peptides, and that the amino acids liberated from peptide hydrolysis were readily absorbed by the worms. In the presence of excess methionine in the ambient medium (as an inhibitor of amino amino acids liberated acid absorption), from dipeptide hydroIysis accumulated in the medium. The uptake of radioactive leucine by M. dubius was inhibited by leucylleucine and alanylalanine in the ambient medium; the inhibition of labeled leucine uptake by both dipeptides was relieved by Pb”+, a potent inhibitor of aminopeptidase activity. Apparently, the aminoand leucine-alanine peptidase activity transport system of M. dubius are separate, bu,t in close spatial proximity such that amino acids liberated from peptide hydrolysis are preferentially absorbed (Uglem et al. 1973). Uglem and his co-workers (1973) also demonstrated that activated larvae (cystacanths) of M. dubius hydrolyzed dipeptides, but that the litberated amino acids did not inhibit the mediated uptake of radioactive amino acids. These authors speculated that, since adults possess welldeveloped surface pits while larvae do not, the aminopeptidase and amino acid transport systems of adult M. dub& are localized in these surface pits, thus resulting in an “unstirred” region where hydrolysis and absorption take place. The data of Uglem

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READ

et aZ. (1973) also suggested that the aminopeptidase of M. dubius was, in fact, an intrinsic enzyme of worm origin, since cystacanths also possessed the enzyme. In larvae, however, the enzyme was apparently inactivated by a lipid-like Iayer surrounding the larvae. Aminopeptidase in larvae was demonstrable only after larvae were treated with taurocholate, Triton X100, Tween-80 or lipase (i.e., substances active on lipids). It is apparent that the surface phosphohydrolases of H. diminuta and aminopeptidase activity of M. dubius are located in close spatial proximity to certain transport systems of these parasites. Therefore, liberated hydrolysis products are rapidly absorbed rather than diffused into the surin a rounding medium, this resulting “kinetic advantage” for absorption of hydrolysis products. Since many helminths, including H. diminuta and Al. dubius, possess abbreviated metabohc pathways, or lack these pathways altogether (Read and Simmons 1963; von Brand 1973; Smyth 1969), it is not difficult to see that such a kinetic advantage might be significant in allowing a parasite to compete more effectively with the host for necessary nutrients and metabolic intermediates. In additimon to intrinsic membrane-bound enzymes associated with some intestinal helminths, recent studies have shown that some heIrninths also interact with enzymes of host origin, resulting in alteration in enzyme activity. Several species of cestodes adsorb o-amylase in vitro, and it has been shown that this adsorption results i’n an increased amyl,olytic activity (Taylor and Thomas 196S; Read 1973). In addition, hl. dubius adsorbs large amounts of rat (Yamylase in viva (Ruff et al. 1973). The significance of increased amlolytic activity in the immediate vicinity of a parasite is apparent when one considers that glucose is the main energy source for many helminths (Read and Simmons 1963; von Brand 1973; Read 1967; Smyth 1966, 1969). However, the results of Mead and Roberts (1972)

MEMBRANE TRANSPORT IN HELMINTH

suggested that increased amylolytic activity may not occur in the rat intestine parasitized with H. diminuta. This apparent discrepancy will be resolved only with further experimentation. Hymenolepis diminuta also interacts with trypsin, a- and p-chymotrypsin, and lipase in vitro, However, these enzymes are either irreversibly inactivated (trypsin and chymotrypsins) (Pappas and Read 1972d, e), or inhibited (lipase) (Ruff and Read 1973) by worms. Although protease inactivation might appear unprofitable in terms of the worm’s ability to obtain amino acids, protease inactivation may be important in protecting the worms from proteolytic activity (Pappas and Read 1972d, e). The significance of lipase inhibition remains obscure (Ruff and Read 1973). VIII.

SIGNIFICANCE OF MEDIATED TRANSPORT

Parasitism is generally defined in nutritional terms, including movement of organic solutes from the host body fluids into the parasite soma. In such a conceptual framework the structures and mechanisms involved in the absorption ‘of organic solutes by parasites must be of primary significance in the parasitic relationship. In the case of forms absorbing sign’ificant quantities of nutrients or metabolites through tegumental structures, such as cestodes, and some trematodes, acanthocephalans, nematodes, the distal tegument with accom,panying glycocalyx (“fuzzy coat”) must be regarded as what has been termed the “host-parasite interface” by Read et al. ( 1963). This must be qualified by recognition that some organic solutes may be acted upon by hydrolytic enzymes intrinsic to, or adsorbed to, the helminth tegument, while interaction of other host digestive enzymes with the parasite tegument may inhibit, or otherwise alter, their enzymatic activities. Thus, the term interface may be an unfortunate one since it implies a surface, whereas the tegumental events of digestion

PARASITES

521

and absorption probably occur in a space distal to and including the tegumental plasmalemma. The term “host-par’asite inter-facial space” would seem to be a more accurate descriptor of this region. The events of absorption occur through the interfacial space, and we can only assume that the rate-limiting step occurs at the tegumentary surface membranes. There is, however, no evidence that the latter assumption is valid. In the case of forms such as Ascaris, in which absorption does not occur through tegumentary structures, but through the gut, the interfacial space must include the lumen of the parasite’s digestive tract. Ascaris has some exocrine-secretory capacities, an intestinal flora, and a multitude of digestive enzymes, and the absorption of nutrients occurs from this luminal space. Some other parasitic nematodes, such as Ascaridia, probably resemble Ascaris in this regard. On the other hand, the trematodes (e.g., Schistosoma and Fasciola), and some parasitic nematodes (e.g., Bradynema, Sphaerulariu, and Mermis) may absorb nutrients through the cuticle (or tegument), gut lumen, and/or other modified structures. With these latter forms, definition and study of the host-parasite interfacial space is more complex than with such helminths as cestodes and acanthocephalans. The foregoing remarks are intended to lay a foundation for a meaningful exploration of the significance of med’iated transport in helminth parasites. A host-parasite interfacial space showing selectivity in allowing the entry of organic solutes would serve to exclude nonfavored molecular species. In Hymenolepis diminuta, for example, the monosaocharide fructose is virtually excluded (Arme and Read 1970) by the specificity of the hexose transport system, and this hexose is not metabolized to a significant extent by this cestode (Read and Simmons 1963). Many other molecular species are excluded by the specificity of the hexose transport system,

522

PAPPAS AND READ

and the same conclusion is justified with respect to a number of other transport systems described in the preceding pages. All mediated transport systems examined seem to enhance the rate of influx of stereospecifically favored substrates when such suibstrates are present at low concentrations. This is particularly obvious when there is a significant diffusion component at higher substrate concentrations, as shown in the generalized case in Fig. 1. This influx enhancement would occur in either facilitate’d diffusion or active transport systems. It may be hypothesized that in the host such enhancement would favor the acquisition of nutrients by the parasite. Transport systems showing any selectivity would function in regulating the composition of internal pools of free metabolites. However, in most instances, this would not be a simple and direct function in determining pool levels of a given metabolite. Systems involved in synthesis of the metabolite, or in removal by degradation or by incorporation into some other molecular species, would operate, in conjunction with influx mechanisms, in determining the pool level of that given metabolite in a vectorial mode. To this might be added internal nonspecific binding of the metabolite. To further complicate a descriptive view of pool regulation, the activity of mediated systems in exchange diffusion and/or counterflow must be taken into account. In this regard it is important to point out that, in the host, the hostparasite interfacial space is exposed to highly complex mixtures of organic solutes. Further, any or all of the systems involved in determining pool composition may be subject to modulation, depending on the physiological state of the parasite, and the host. Although modulation of catabolic, anabolic, or transport systems in helminths does not appear to have been studied in detail, epigenetic changes in time are known (Fairbairn 1970), and studies of modulation of enzyme systems of parasites are in progress in several laboratories.

Each ‘of a number of transport systems described in the preceding pages show broad affinity for a variety of analogous compounds of probable nutritional significance. For example, the amino acid transport systems show affinities for two or more amino acids known to be constituents of proteins. Two possible advantages of such broad affinities seem apparent. First, such sys’tems would operate to inhibit flooding of an internal pool by some individual compound present at high concentrations in the ambient medium. For example, if an amino acid A were present at an arbitrary concentration of 1.0, and each of ten other amdno acids was also present at a concentration of 1.0, the rate of transport of A would be sharply reduced by inhibitory interactions of the ten amino acids with the transport system(s) involved in the transport of A. Data reviewed in preceding pages indicate that some, or all, osfthe amino acids in the mixture might be expected to act as competitive inhibitors of A influx. Further, the presence of the ten amino acids would be expected to lower the steady state level of A in the parasite by enhancing efflux of A. Second, the broad affinity of a transport system for several structurally related compounds would allow for a minimizing of the number of transport systems to be coded and synthesized. The advantage of such minimizing may follow from Lwoff’s ( 1944) hypothesis that auxotrophic mutants would have a selective advantage over prototrophic parents because of the energetic economy effected by the deletion of a biosynthetic pathway. A somewhat related hypothesis concerning the selective advantage of a simplified energy metabolism in parasitic helminths was presented by Read (1961b). Zamenhof and Eichorn (1967) examined experimentally Lwolf’s (1944) hypothesis. These workers studied the growth of each of several auxotrophic mutants in competition with the protoBacillus trophic strain of a bacterium, subtilis, in media which were adequate

MEMBRANE

TRANSPORT

for the auxotrophs. The data showed that mutants requiring histidine, indole, or tryptophan had a distinct selective advantage over the parental strain when grown in media containing the nutrient required by the mutant. Further, it was shown that a mutant with a block early in the pathway for tryptophan synthesis had a selective advantage over a mutant blocked further along the pa,thway. Zamenhof and Eichorn (1967) pointed out that the cases studied by them were gene inactivations resulting from point mutations. Thus, the economy effected did not include synthesis of the structural gene and messenger RNA. They postulated that, if mutations were deletions resulting in loss of a DNA segment, the additional advantage in a suitable medium may include faster replication of the chromosome and savings on sugars, amino acids, bases, and enery required by the prototroph to synthesize DNA, messenger RNA, and protein. The same reasoning may be applied to mediated transport systems. An organism having a few transport systems for amino acids should have an energetic advantage over an organism having a special system for each of the protein amino acids, particularly if the ,protein amino acids are all regularly present in the environmental medium at reasonable concentrations. This would be especially pertinent in the case of a parasitic helminth furnished a large array of prefabricated metabolites at host-regulated concentrations. Having presented arguments for the advantages of transport systems showing some breadth of affinities, it is appropriate to ask what advantages there may be in maintaining a multiplicity of sites with each showing molecular class specificity. The advantages of class speoificity seem obvious in some cases. The maintenance of a “foodgathering” transport system for major energy sources, such as sugars, qualitatively distinct from the systems involved in the transport of amino acids or vitamins, would seem to represent an economic scission of function in the utilization of external re-

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PARASITES

sources. On the other hand, the maintenance of two separate fatty acid transport systems in H. diminuta is not obviously advantageous, although it is possibly related to the fact that this worm is almost totally dependent on the host for fatty aoids incorporated into fats (Girrger and Fatirbairn 1966; Jacobsen and Fairbaim 1967). The advantages ‘of maintaining two separate glycerol transport systems, one of which is Nat-dependent and the other of which is Nat-independent, by H. diminuta is less apparent. On the other hand, what is the significance of multiple amino acid transport systems having very broad but quantitativel y different overlapping affinities? It may be postulated that this represents a mechanism for modulating amino acid fluxes. By changing the relative numbers of such multiple systems, the parasite may respond to (1) the environments furnished by more than one host species, (2) changes in the physiology of the host associated with age or intercurrent disease, or (3) the presence of other parasites in the host. Of these three possi~bilities, there is some evidence that a parasite may show such responses in d’ifferent host species (Read et al. 1963). The remaining two possibilities have not been examined experimentally. All of the above discussion points to the concIusion that the significance of mediated transport in parasitic helminths is to be found in the biochemical fitness of parasites for the environments in which they dwell. However, it is too early to develop a “program” which will fully describe such fitness in any single parasitic species. IX.

SUMMARY

Parasitic helminths absorb numerous low molecular weight organic compounds. In the Cestoda and Acanthocephala, absorption of these compounds occurs across the external cellular tegument, since these helminths lack a digestive tract. In the Nematoda, absorption of water-soluble com-

524

PAPPAS

pounds occurs almost entirely across the microvillous brush border of the intestine, except, possibly, in those entomophilic nematodes which have specialized morphological modifications apparently related to absorption. The cuticle of nematodes appears relatively impermeable to water-soluble compounds. The Trematoda possess two absorptive surfaces, the gut epithelium and external tegument; however, only those processes involved in transtegumental movement of solutes have been studied. The absorptive mechanisms utilized by parasitic helminths include active transpor.t, facilitated diffusion, and diffusion. In some species a combination of mediated (“carrier”) uptake (i.e., facilitated diffusion or active transport) and diffusion occurs, with mediated uptake being predominant at low substrate concentrations. Examples of each of these absorptive mechanisms are the following: active transport (with negligible diffusion) appears to be the mechanism of glucose uptake in Hymenolepis diminuta and Hymenolepis microstoma; active transport, with a significant diffusion component at higher substrate concentrations, is involved in the absorption of glucose and some amino acids by Taenia crassiceps larvae and histidine absorption by H. diminuta; albsorption of acetate by H. diminuta and glucose by Fasciola hepatica and Schistosoma mansoni occurs by facilitated diffusion, in addition to a significant dliffusion component; glutamate and proline appear to enter T. cmssiceps larvae by diffusion, and this is also the case with pyridoxine and nicotinam’ide absorption by H. diminuta and amino acid absorption by F. hepatica. Parasitic helminths have maintained a “full complement” of absorptive mechanisms, and the mechanisms involved in the absorption of a specific nutrient by various helminths may be essentially identical. For example, cestodes and nematodes actively transport glucose through a Na+-dependent system, and trematodes may also utilize a Na+-dependent active transport mechanism,

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RJUD

since there is some suggestion of Na*-sensitivity of glucose uptake. However, helminths have also maintained a certain “individuality” with respect to their transport systems. For example, amino acid transport in acanthocephalans and cestodes appears to involve mediated systems, and the same appears true for the trematode S. mansoni and nematode (Ascaris suum) gut. In the trematode F. hepatica, however, amino acids enter by diffusion. More subtle differences are apparent when the kinetic parameters describing solute transport, or the activity of various inhibitors, are compared among the various groups of parasitic helminths. Transport mechanisms in some helminths are strikingly simlar to those found in vertebrate tissues. The most extensively studied instance of this is the similarity of the Na+-dependent glucose transport mechanisms in cestodes and vertebrate intestinal tissues. However, some transport systems of parasitic helminths are unique when compared with vertebrate systems, and two examples of this are the complicated purine-pyrimidine system of H. diminuta, and the two glycerol transport systems (one of which is Nat-dependent) in this same cestode. Also, the apparent lack of Nat( cation) -dependent amino acid influx and accumulation in some cestodes and acanthocephalans is quite different from many vertebrate tissues. At present our understanding of these unique systems is so limited that it is difficult to speculate as to their physiological significance. The wide variety of transport systems found in parasitic helminths is demonstrated by the numerous transport systems which have been identified in H. diminuta, the most extensively studied helminth. These include ( 1) six amino acid systems (or, if one wishes, one amino acid system with six distinct “carriers”), (2) one monosaccharide system, (3) two glycerol systems, (4) three purine-pyrimidine transport systems, (5) two fatty acid transport systems, one for short chain fatty acids, and

MEMBRANE

TRANSPORT

another for long chain fatty acids (the allosteric nature of long chain fatty acid absorption suggests additional “carriers”), and (6) two water-soluble vitamin systems; this cestode probably possesses additional tranpsort systems which have not yet been demonstrated. Only limited data are available concerning the number of transport systems in other parasitic helminths, but it is not unreasonable to speculate that these other species also possess numerous, distinct systems. Although parasitic helminths have lost some synthetic capabilities, they have maintained (or evolved) complex absorptive mechanisms which allow them to compete with the host for nutrients. These systems appear to be relatively specific in the type of molecule they transport (i.e., a system may transport amino acids, but not monosaccharides), and relatively nonspecific within that class of compounds ( i.e., an amino acid system may transport several different amino acids). Thus, the evolution of these transport systems may have involved a compromise between ( 1) the maintenance of a large number of systems which serve as both “food-gathering” systems and homeostatic mechanisms for internal pool regulation, and (2) a reduction in the number of transport systems which must be maintained (in the genetic code) and synthesized at the expense of other biochemical functions. ACKNOWLEDGMENTS Much of the work summarized in this review was supported by continuing Grants (2-TOlAI00106 and AI-01384) from The National Institutes of Health, U. S. Public Health Service, awarded to the late Dr. Clark P. Read. During the initial phases of writing this article, the senior author was supported by a N.I.H. Postdoctoral Fellowship (5-F02-AI-45108-02 ). The authors wish to thank those individuals who supplied unpublished data and/or manuscripts in press. These included: Drs. R. D. Lumsden, R. 0. McCracken, G. L. Uglem, L. Chappell, J. M. Webster, J. A. Starling, L. S. Roberts, H. L. Asch, and Messrs. T. A. Rutherford and M. G. Levy.

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Note added in proof: A recent article (McManus, D. P., and James, B. L. 1975. The absorption of sugars and organic acids by the daughter sporocysts of Microphallus similis (Jag). International Journal for Parasitology 5, 33-38) has reported active transport of monosaccharides in a larval trematode. The major findings of this study were as follows: Glucose, galactose and fructose entered daughter sporocysts of M. similis through a mediated system. Inhibitor studies suggested that all three monosaccharides entered through a single system, a system which may also interact with a-methyl-n-glucoside, fucose, mannose and 3-O-methylglucose. Since glucose was accumulated against an apparent concentration difference, the absorptive process was characterized as active transport. The kinetic parameters describing monosaccharide transport in daughter sporocysts of M. sir&s were as follows: Kt values for glucose, galactose and fructose were 1.38, 5.0 and 4.35 m&4, respectively, and V,,, values were 37.04, 76.92 and 47.50 pmoles/g dry wt/hr, respectively. Inhibitor constants (K$) for mannose, galactose, 3-O-methylglucose, fructose, a-methyl-n-glucoside and fucose acting as inhibitors of glucose uptake were 4.129, 4.361, 4.684, 6.207, 9.315 and 9.645 m&I, respectively. Pyruvate, acetate, citrate and succinate appeared to enter daughter sporocysts by the process of diffusion only. An additional article (Borgers, M., and De Nollin, S. 1975. Ultrastructural changes in Ascaris suum intestine after mebendazole treatment in vivo. Journal of Parasitology 61, 110-122) has shown that the initial effects of mebendazole on Ascaris intestinal cells is that of altering the cellular organelles involved in secretion and absorption. These morphological modifications may play some role in the action of mebendazole on glucose uptake by Ascaris intestine. The “unpublished data” of Uglem and Read pertaining to glucose absorption by Schistosoma mamoni has been submitted for publication (Uglem, G. L., and Read, C. P. Sugar transport and metabolism in Schistosoma mansoni. Journal of Parasitology). The “unpublished data” of Levy and Read pertaining to purine, pyrimidine and nucleoside absorption, and surface enzyme activity in Schistosoma mansoni has been submitted for publication in the form of two manuscripts (Levy, M. G., and Read, C. P. Purine and pyrimidine transport in Schistosoma mansoni. Journal of Parasitology, and Relation of tegumentary phosphohydrolases to purine and pyrimidine transport in Schistosoma mansoni. Journal of Parasitology). REFERENCES ANSEL, M., AND THIBAUT, M. 1973. Value of the specific distinction between Ascaris lumbri-

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