Effectors of amino acid transport processes in animal cell membranes

03~~-9629~85 $3.00 + 0.00 t“ 1985 Per~amon Press Ltd

REVIEW

EFFECTORS OF AMINO ACID TRANSPORT PROCESSES IN ANIMAL CELL MEMBRANES JOSEPH LERNER De~drtrn~nt

of Chemistry,

Box 5065, Tennessee Technological University, Telephone: (615) 5283118

(Received

19 November

Cookeville.

TN 38505, USA.

1984)

Abstract-l. Various effecters, which act upon ion gradients, protein synthesis, membrane components or cellular functionai groups. have been employed to provide insights into the nature of amino acid-membrane transport processes in animal cells. Such effecters, for example, include ions. hormones, metabolites and various organic reagents and their judicious use has allowed the following list of conclusions. 2. Sodium ion has been found to stimulate amino acid transport in a wide variety of cell systems, although depending on the tissue and/or substrate, this ion may have no effect on such transport, or even inhibit it. 3. Amino acid transport can be stimulated in some cell systems by other ions such as K +, Li r. H _ or Cl-. Both H * and K+ have been found to be inhibitory in other systems. 4. Amino acid transport is dependent in many cell systems upon an inwardly directed Na*’ gradient and is stimulated by a membrane potential (negative cell interior). In some cell systems an inwardly directed Cl-- and H * gradient or an outwardly directed K+ gradient can energize transport. 5. Structurally dissimilar effecters such as ouabain, Clostridium enterotoxin, aspirin and amiloride inhibit amino acid transport presumably through dissipation of the Na” gradient. Inhibition by certain sugars or metabolic interm~iates of the tricarboxy~ic acid cycle may compete with the substrate for the energy of the Na j gradient or interact with the substrate at the carrier level either allosterically or at a common site. Stimulation of transport by other sugars or intermediates may result from their catabolism to furnish energy for transport. 6. Insulin and glucagon stimulate transport of amino acids in a variety of cell systems by a mechanism which involves protein synthesis. Microtubules may be involved in the regulation of transport by insulin or glucagon. Some reports also suggest that insulin has a direct effect on membranes. 7. In addition, a number of growth hormones and factors have stimulatory effects on amino acid transport which are also mediated by protein synthesis. 8. Steroid hormones have been noted to enhance or diminish transport of amino acids depending on the nature of the hormone. These agents appear to function at the level of protein synthesis. While stimulation may involve increased carrier synthesis. inhibition probably involves synthesis of a labile protein which either decreases the rate of synthesis or increases the rate of degradation of a component of the transport system. 9. Cdtecholamines directly stimulate amino acid transport through alpha-adrenergic mediation by a process which involves protein synthesis. 10. Protein hormone secretagogues such as caerulein inhibit amino acid transport by a process that is mediated by Cal+ I I. Cyclic nucleotides have been shown to have both stimulatory and inhibitory effects on amino acid transport. depending on the cell system. Some studies suggest that protein synthesis is involved in the stimulation by cyclic nucleotides, although in one cell system protein synthesis was ruled out as a cause of the stimulation. Stimulation of phosphorylation of membrane components by a cyclic nucleotide may be a mechanism to regulate amino acid transport. 12. Glycoprotein glycosylation appears to be required for amino acid transport. Recent results suggest that a glycoprotein component of system A must be continually synthesized to sustain an increase in transport activity. 13. Microfilament accumulation which occurs as a compact network beneath the cell membrane in some systems is responsible for inhibition of amino acid transport, 14. Beta-adrenergic agonism effected by isoproterenol evokes a Ca? +-dependent stimulation of amino acid transport and an associated increase in polyamine levels (e.g. putrescine). 15. Cell membrane thiol groups as well as cytosolic ones appear to be involved in amino acid transport, 16. Gamma-~lutamyl transpeptidase appears to be involved in the transport of a variety of amino acids in animal cell systems. 17. The inhibitory effects on amino acid transport of trypsin. polyanions (e.g. dextrdn sulfate and heparin), phloretin. retinol, bile salts, pyridoxal phosphate. platinum co-ordination complexes. nicotine and ethanol and the stimulatory effects of polycations (e.g. neomycin), phorbol ester, serum albumin and indomethacin are described in this report.

JOSEPH LERNEK

714 INTRODUCTION

but no effect was seen in toad cornea. Glycine transport is stimulated by Nat in Dicentrurchus The underlying mechanisms of cell-membrane translubrus intestine, rabbit kidney, mouse Ehrlich cells. port have proved not to be readily amenable to our human erythrocytes, toad embryo and rat brain but understanding principally because of the vectorial not in the squid giant axon. Alanine transport has nature of such processes. Nevertheless the use of been found to be stimulated by Na in Pseudovesicles in the last decade or so has allowed a pleuronectes americanus intestine and kidney. human substantial advance in our understanding of such intestine, human red blood cells, marine polychaete mechanisms. since results can now be analysed indeintegument and cat pancreas; no effect of this ion pendently of the influence of cytosohc contents on was observed in the transport of alanine in Testdo transport. Another highly significant development in graecu intestine or sheep red blood cells. Transport of the last decade has been the much broader use of lysine or other basic amino acids via system Ly ’ is chemicals as probes to explore transport mechanisms. enhanced by Na ’ in Macrohruchium rosenhergii midThe purpose of this report is to describe the actions gut (high K,, carrier), rat kidney, rabbit kidney and of such agents on amino acid transport in an effort mouse blastocyte but not in M. rosenhergii midgut to elucidate the underlying mechanism. Despite the (low K,,, carrier), rabbit jejunum, human erythrocytes, state-of-the-art of transport physiology. too few orsheep erythrocytes. mouse Friend cells or human skin ganisms and cell and tissue types have been explored. fibroblasts. Busse (1978) has reported that arginine Perhaps by illustration in this report of what has been transport in rabbit renal brush-border membrane achieved in studies on a small number of cell systems, vesicles is inhibited by added Na + Leucine transport comparative physiologists will be enlightened to exis stimulated by Na + in rainbow trout intestine but tend such studies to other species and cell types. not in mouse Friend cells or rat soleus muscle; it is inhibited by Na + in toad embryo. Valine transport is enhanced by Na + in rat jejunum. In addition to the A REVIEW OF THE RECENT LITERATURE findings for leucine and phenylalanine, L system Role of’ ions in energizing transport substrates have not been found to be influenced by Ion gradients play major roles in energizing the Na’ in rabbit jejunum and human red blood cells. movement of amino acids across animal cell memMethionine transport is also unaffected by Na ’ in branes The specificity of various ions for activation sea urchin sperm and tryptophan transport in human of amino acid transport is considered below. Alred blood cells. A variety of other systems and substrates are stimulated by Na _ . e.g. the imino acid though Na ’ has been studied extensively, in recent system in rabbit jejunum, beta-alanine transport in years attention has been focused on the role that several other ions such as Cl play in such transport, rabbit kidney, taurine transport in rat kidney. the The use of ionophores to study the role of ion proline transport system and the ASC system in gradients in amino acid transport is also considered human red blood cells, system N in rat hepatocytes in the sections below. Techniques which employ and y-aminobutyric acid (GABA) uptake in rat membrane vesicles have significantly improved such brain. Glutamate transport is stimulated by Na ’ in rabbit and rat kidney, rat liver. rat brain and squid studies. giant axon. Ion qffi~ct0r.s of’ umino ucid transport A small amount of data is available on the ability A great deal of effort over the years has gone into of Li + to influence amino acid transport. Transport investigating the effects of Na + on amino acid transof phenylalanine is stimulated by this ion in mouse intestine, as is that of arginine in rat kidney, acidic port and its role in the mechanism of nutrient transamino acids in rat liver and system N substrates in rat location across the plasma membrane (Lerner, 1978). Table I shows that a variety of tissues and cell types hepatocytes. Lithium ion has no effect on transport have been used to study the effects of this ion on of amino acids by systems L, Ly ’ or the imino acid transport; in addition a number of studies have system in rabbit jejunum. centered on other ions such as K ’ . Hi . Li +1 Hydrogen ions do not affect the transport of lysine by either high- or low-affinity systems in the shrimp choline + and Cl (see also section on “chloride ion midgut; nor do they influence transport of methrequirement”). Please refer to Table I for references ionine in sea urchin sperm or transport of basic corresponding to tissue and cell types given in this section. In terms of ion involvement in amino acid amino acids in human skin tibroblasts. Stimulation of glutamate transport by protons has been observed in transport, they may serve as cosubstrates and stimulate uptake, they may destabilize carriers or change rabbit kidney, although under the same conditions in this organ it inhibits arginine transport. membrane permeability of specific cosubstrate ions (Benjamin and Quastel, 1977) and hence inhibit Potassium ion has been found to stimulate transport of phenylalanine in Lepidopterun larvae midgut transport, or in some cases they may have no influence on transport. and mouse intestine, glutamate transport in rabbit Phenylalanine transport has been observed to be kidney, arginine transport in rat kidney and glutamate transport in rat liver. This ion has an inenhanced by medium Na’ in Lepidopterun larvae hibitory effect on transport of glutamate and arginine midgut and in the mouse intestine; however, this ion has no effect on phenylalanine transport in Bomh,r~.\- in rabbit kidney. and on acidic amino acid and mori larvae midgut. With reference to substrate AIB, glycine uptake in rat brain. Transport of alanine in stimulation of transport in the presence of Na + has Testudo grueca intestine is unaffected by K _. as is methionine transport in sea urchin sperm and glubeen demonstrated in Boops .dpu intestine, mouse tamine uptake in rat brain. Friend erythroleukemia cells and rat soleus muscle,

Na K

Na

Alanine

Alanine

Motrobrachium

(Marine fish) Intestine BBMV Rabbit renal BBMV Rabbit renal BB.MV

Diwnrrarchus

Ratjejunum BBMV

1ahru.r

Human intestine BBMV Rabbit jejunum BBMV

Sabno gairdnerii Rainbow trout intestine Mouse intestine BBMV

Shrimp midgut

rovenhwgii

Pseudupl~~urone~rc~s umericanus

Intestine Kidney BBMV

Na Li Na Li Na Li Na Li Na

L system

+ + + + K Na H K

Na

Glutamate

Glutamate

Nelson L’(~1. (1983)

Schneider and Sachtor (1980)

Boge ef al. (1981)

+ + Na Cl

Will and Hopfer (1979)

0

0

0

Lucke er al. (1977)

+

+

+

4.

Bertrloot et ol. (1982)

+ + +

Brick and Ahearn (1978)

Eveloff PI al. (1980)

Nassar rl al. (1980)

Eoge and Rigai (1981)

Sacchi et nf. (1981)

PI ul. (1982)

ingham and Arme (1977)

0 0 0

0

0

0

0 0

0

Giordana

Rel&tlce

+

+

+

+

-I-

+ +

Etlixts of ion*

Glycine AIB

Several neutral amino acid systems Imino acid system Valine

Ly + system

Alanine

Na K Li Na

Phenylalanine

Na

H Na H

Na

Na Cl

AIB Glycine

(Marine fish) Intestine EBMV Terruria grawn Intestine Epithelium Serosal Membrane

Lysine (High 6, carrier) Lysine (Low K,,, carrier) Leucine

Na

PhenyIa~nine

Eoops salpa

K Na

Phenylalanine

Ion

Larvee Midgut BBMVt &m/?~s ?nori Larvae Midgut

Substrate

Table I. Ion effecters of amino acid transport

Lqkfoptrwm

CdllTiSSW

+

Na C1 sarcosine and proline system

Valille

Rat hepatocytes

System AX system L System L Tryptophan Ly + system ASC system Alanine cysteme and lysine system Taurine GABA Beta-alanine

Glycine,

Cysteine

Rat liver membrane vesicles

ceils

+ + + +

Na Na

Glycine Giycine

Na Na Na Na K Li Nil

Na Na Na Na Na Na Na Na Na Na

Na Cl

Na CI

+ + f +

+ + +

+

+ i-t

Johnstone (1978)

+

Na

Glutamate

0

0 0

0

0

0

0

0

0

0

Sacktor PI al. (1981)

+ + -t + +

Na K Li Choline Na

Arginine

Cl

Chewy

+

Na

Taurine PI al.

(1983)

Seglen and Solheim

Sips PI 01. (1982)

(197X)

Gordon and Rubm (1982)

Goldstein and Boyd (1978)

Young er al. (1980) Fugelli and Rohrs (1980)

Young er ul. (1980)

Ellory er al. (1981)

Al-Saieh and Wheeler (1982) Ellory CI al. (1980)

kdn

et al. (1983)

and SdCktor (1982)

Hilden and Sacktor (1981) Fass et al. (1977) Mircheff ef al. (1982)

Hammerman

+ + + + + +

Reference

Arginine Alanine Lysine MeAIB Beta-alanine Glycine

Busse (1978)

-

_-_-

of ion* .:Effects __

Arginine

IOtl

Table I. ronfinued

H Na K Na Na Na Na Na Na

Substrate

Leucine Lysine AIB Aspartate and glutamate

Skate RBC Mouse Friend erythroleukemia

Roja erinacw

Flounder RBC

Platichrh.vs fresus

Sheep RBC

Human RBC

Human RBC

Rat rend1 Basolateraf MV Mouse Ehrlich cells Human RBCS Human RBC

Rabbit renal BBMV Rat renal BBMV Rat renal BBMV

Rabbit renal Rabbit renal Rabbit renal BBMV

~__ Rabbit renal BBMV

Cell/Tissue

vesicles

of transport;

0. no effect on transport;

_

Reference

+ , stimulation

Glutamate AIB Leucine Alanine Cationic amino acids

- , inhibition

K K K K Na Cl Na Na Na Na Na H

Glutamate Aspartate Glvcine

of transport.

+

+ + + +

Aragon

+ Na

Kanner

+

Cl

Tyrosine

Mayor

+ +

Na Cl

and Sharon (1978) and Kohn (1977)

(1978)

(1983)

(1977)

Tyrakowski er al. (1978) White er al. (1982)

Kanner Cooper

Kanner

and Quastel

et al. (1981)

and Sharon

(1980)

and Arme (1979)

ef al. (1981)

Benjamin

(1980)

(1983)

and Lea (1978)

and Vacquier

Richards

Caldwell

Glycine

+

Na Na Na

Glycine Glutamate Amino acids

+

CI ul. (1982)

Mlot er al. (1978)

Klekamp

Van Winkle (1981) McGahan (1981)

Kilberg ef al. (1980) Vadgama and Christensen

Stevens and Preston

0

Effects of ion*

Na

+

+ + + +

Gache

Rat brain synaptosomes Rat soleus muscle

*When effector is present in medium. tBrush-border membrane vesicles. $Red blood cells.

and system N

Ion %i Na Li Na Na

Na Na Na K H

GABA

Cat pancreas membrane Human skin libroblasts

histidine

1. conrinued

Leucine Isoleucine Methionine

Rat brain synaptosomes

Lvtechinus &I”.7 Sea urchin Sperm Glwra dibranchiara Marine Po/.vchaere Integument Squid giant AX”” Eisenia forrido Lumbricid Earthworm integument Rat brain membrane vesicles Rat brain membrane vesicles Rat brain membrane vesicles Rat brain cortex

asparagine,

Table

Na

Lysine AIB

Mouse blastocyte Bufo murinus Toad cornea XrnO~US laeuis Toad embryo

Substrate

Glycine

Glutamine Glutamine,

Rat hepatocytes Rat hepatocytes

Cell/Tissue

718

JOSEPHLERNER

Stimulatory effects of CI on amino acid transport are considered in detail in another section of this report. Of interest. this ion has been shown to stimulate transport of certain neutral amino acids in human red blood cells, but not that of alanine or cysteine (Table 1). Chloride ion stimulation of amino acid transport has also been demonstrated for marine fish intestine, rat intestine and rat brain. Sodium ion has been postulated to serve as a cosubstrate in the transport of amino acids across cell membranes (for a review, see Lerner, 1978). It either enhances the ability of substrate amino acid to bind to the carrier molecule or enhances the rate-limiting step of the translocation process. Such affinity and/or velocity effects may come about through conformational changes in the carrier molecule. The driving force for transport arises from the action of the Nat. K +-ATPase, which pumps three Na + molecules out of the cell for every two K ’ molecules it pumps into it; this electrogenic effect results in the generation of a net negative charge at the inner membrane face. The movement of Na+ into the cell as a cosubstrate on the amino acid carrier occurs in response to the negative membrane potential. In some systems, depending on the nature of the substrate, the sodium ion carrier binding stoichiometry is greater than one. Sodium ion-independent transport of amino acids is carrier mediated in the systems reported in Table I; such catalysed transport occurs without the energization of the Na+, K +-ATPase. Such transport presumably takes place on carriers which do not possess Na + binding sites. It is unclear why Na + has been reported to be a negative effector of transport by several groups studying basic or neutral amino acid uptake. .Eectors

qf ion

gradients

Table 2 summarizes results showing that different ion gradients can drive amino acid transport in a variety of systems, and provides information on the inhibition of such transport by ionophores which are capable of abolishing ion gradients. Please refer to Table 2 for references in this section. Of the ionophores employed, valinomycin enables K+ to pass through the membrane (Sips er al., 1982) carbonyl cyanide m-chlorophenylhydrazone and carbonyl cyanide p-trifluoromethoxyphenylhydrazone to dissipate the proton potential (Kanner and Sharon, 1978; Mohri et al., l983), nigericin and gramicidin to collapse both Na + and K + gradients (Busse, 1978; Heinz et al., 1981; Kanner, 1978) and monensin to catalyse the electroneutral exchange of external Na + for internal protons (Smith and Austic, 1980). The uptake of glycine in rat brain membrane vesicles was found to be stimulated by a membrane potential (interior negative) as demonstrated by the effects of valinomycin and carbonyl cyanide m-chlorophenylhydrazone and anions of different permeabilities (Mayor et al., 1981). The importance of the Na+ gradient was shown by the action of nigericin which completely inhibits glycine uptake. Similarly, tyrosine uptake by rat brain vesicles is dependent on an inwardly directed Na + gradient and is stimulated by a membrane potential (negative inside) (Aragon et a/.. 1981). Moreover, GABA

transport in this system can be driven either by an inwardly directed Na + gradient or by a gradient of Cl (Kanner, 1978). Transport of GABA was found to be absolutely dependent on the simultaneous presence of both types of ions in the medium. It was inhibited by a proton ionophore and by ionophores which collapse Na ’ gradients. Transport was stimulated by valinomycin when vesicles were loaded with in the K ’ or when they were exposed to SCN presence of a Na+ gradient. Kanner and Sharon (1978) have reported that glutamic acid transport into synaptosomal rat brain fractions requires the presence of external Na+ and internal K . : either a Na + gradient (out > in) or a K ’ gradient (in > out) or both can be used to energize transport. Transport and is stimulated by valinomycin or external SCN can be inhibited by the proton ionophore. carbonyl cyanide m-chlorophenylhydrazone, and nigericin. A Na ’ electrochemical gradient (out > in) was found to stimulate uptake of beta-alanine into rabbit renal brush-border membrane vesicles (Hammerman and Sacktor, 1978). This stimulation was specific TOI Na+. Gramicidin abolished the overshoot of this substrate, presumably by accelerating the electrogenie uptake of Na + into the vesicles. Hammerman and Sacktor (1982) similarly found that the uptake ol glycine in these vesicles could be enhanced by a Nn electrochemical gradient (out > in), though such stimulation could not be induced by imposition of gradients of K. , Li + or choline ’ Sodium ion stimulated glycine uptake could be diminished by dissipation of the Na+ gradient with gramicidin. In addition to these results Fass et N/. (1977) have shown that stimulation of alanine uptake in these vesicles is specific for Na + ; valinomycin enhanced the uptake ol this substrate provided an outwardly directed K ’ gradient was present. Uptake of glutamate by such vesicles is dependent upon an inwardly directed Na ’ gradient; this uptake could be increased by an outwardly directed K + gradient (Schneider and Sacktor. 1980). The increase in Na’ gradient-dependent uptake by the K + gradient was completely inhibited by nigericin which dissipated both cation gradients. The K+ gradient could provide the energy for uphill transport in the absence of a Na + gradient. provided that Na + was present. The effect of the K ’ gradient in stimulating uptake had an absolute requirement for Na +. The action of the K ’ gradient was not attributable to a change in the membrane potential and these workers have suggested that potassium ion interacts directly with the carrier. These results suggest further that the cotransport of Na + and glutamate is coupled to the transmembrane flux of K In a more recent study. Nelson et rrl. (1983) have reported that a low external pH stimulates Na ’ -dependent glutamate transport into rabbit renal brush-border membrane vesicles and a low internal pH inhibits it. In the absence of other driving forces. glutamate accumulation can be driven by a pH difference (interior alkaline). This process requires Na+ but is not due to generation of a Na ’ gradient in response to a pH differential. Internal K stimulates glutamate accumulation but is not absolutely required for transport. Internal H ’ inhibits gtustimulation. tamate uptake by decreasing the K Moreover. external K + inhibits glutamate influx and

Table 2. Effecters

(in 1 out) (in > out)t

H gradient K gradient Na gradient

Na gradient

Alanine

Arginine

Taurine

Aspartate. Glutamate

wphulus

CI ~1.. 1978)

MO”t?“Si”

Na gradient K gradient (in > out)* H gradient (m > out):

Ala”l”e

added.

Carbonyl cyanide p-trifluoromethoxyphenylhydrarone

H gradient (out > in) Na gradient not significant

Lysine

*Valinomyci” added. Wramicidin added. ZCarbonyl cyamde p-triHuoromethoxyphe”ylhydrarone

(Tyrakowski

Mullet renal BBMV (Lee and Pritchard, 1983) Cat pancreas membrane vesicles

Mugil

Carbonyl cyamde p-trifluoromethoxyphenylhydrazone

Na gradient K gradlent (out > in)

Phenylalanine

(in > out)*

K gradient

Glutamate

(Sips et a/., 1982) Mouse intestine BBMV (Berteloot (‘I u[., 1982)

Na gradient K gradient (in > out)

Glycine Beta-alanine

Rabbit renal BBMV (Hammerman and Sacktor. 1982; Hammerman and Sacktor. 1978) Rabbit renal BBMV (Schneider and Sacktor. 1980) Rabbit renal BBMV (Fass ef al.. 1977) Rabbit renal BBMV (Busse, 1978) Rat renal BBMV (Chewy er al., 1983) Rat liver membrane vesxlea Nigericin

Na gradient Na gradient

Glutamate

Carbonyl cyanide m-chlorophenylhydrazone Nigericin Gramicidin Carbonyl cyanide m-chlorophenylhydrarone Nigericin Gramicidin Gramicidin

Na gradient Cl gradient SCN graduznt K gradient (in tout)* Na gradient K gradient (I” > out)’

Inhibited

by:

m-chlorophenylhydrarone

GABA

cyanide

Carbonyl

Na gradient

Tyrosine

Transport Nigericin

by:

Na gradient

driven

of ion gradients Transport

Glycine

Substrate

Rat brain synaptosomes (Kanner and Sharon, 1978)

Rat brain membrane vesicles (Mayor et al., 1981) Rat brain membrane vesicles (Aragon e, nl., 1981) Rat brain synaptosomes (Kanner, 1978)

Cell/Tissue

720

JOSEPHLERNEK

this inhibition can be overcome by lowering the external pH. These results suggest that H + is cotransported with glutamate. In this connection leucine transport into vesicles prepared from Chang liver cells has been reported to be stimulated by an inward H + gradient and is essentialy independent of the Na+ gradient (Mohri et al., 1983). The stimulatory effect was inhibited by the presence of carbonyl The cyanide p-trifluoromethoxyphenylhydrazone. results suggest that an electrochemical gradient of protons can serve as a driving force for transport of leucine and that the movement of protons is coupled to that of leucine. The uptake of lysine was also found not to be significantly stimulated by a Na ’ gradient in brush-border membrane vesicles isolated from mullet (Mugil c+u/us). a marine teleost (Lee and Pritchard, 1983). Uptake is, however, transiently stimulated in the absence of Na ’ when a proton gradient (out > in) was imposed across the membrane. Uptake under these conditions was inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Maximal transport flux was also shown to increase with an increase in available protons. Under the same conditions no change was seen in the apparent transport affinity. When the membrane potential was increased (instde negative) with valinomycin, a similar but smaller stinluiation of lysine uptake occurred. These results suggest that the proton motive force can provide a driving force for lysine uptake in this system. Arginine uptake by isolated brush-border membrane vesicles from rabbit kidney cortex was shown to be stimulated to produce an overshoot when a membrane potential was induced by an increase in pH of the medium or by the presence of gramicidin in the medium which enhances an outward movement of K ’ (Busse. 1978). Arginine uptake was faster in the presence of an inwardly directed Na + gradient than with a K ’ gradient. Both ion gradients reduced uptake compared with uptake in a mannitol medium. Uptake was faster in a Na ’ than in a K’ medium after the membrane potential was minimized by equilibration of the vesicles in NaCl or KC1 in the presence of gramicidin. Taurine is actively transported into rat renal epithelial membrane vesicles by a Na ’ -dependent transport system; this transport is enhanced by the membrane potential under conditions of hyperpolarization in the presence of valinomycin and decreased by dissipation of the Na + gradient by gramicidin (Chesney et u/.. 1983). The uphill transport of aspartate and glutamate in membrane vesicles from rat liver is driven by a Na +-concentration gradient (out > in); this transport is relatively specific for Na+. since only Li ’ could replace it; K + is also required for optimal functioning of the system (Sips rf al., 1982). Alanine transport has a similar requirement for the Na ’ gradient: no uptake is observed in the absence of this gradient even though the membrane potential may be intact. Valinomycin substantially increases alanine transport in this tissue. Phenylalanine transport in mouse intestinal brushborder membrane vesicles exhibited an overshoot phenomenon indicative of active transport in the presence of a Na ’ or K + gradient (out > in) (Berteloot cutal., 1982). Lithium ion was also demonstrated to elicit active transport of phenylalanine.

Potassium ion-dependent amino acid transport has also been found in insect midgut (see Table I). The Na ’ - and K + -driven phenylalanine transports in the mouse were shown to be dependent on the presence of a membrane potential, since short-circuiting the membrane with carbonyl /~-trifluoromethoxyphenylhydrazone reduced the amplitude of the overshoots seen with both ions. The K ‘-induced overshoot, however, is not due to the presence of a membrane potential alone because a gradient of choline chloride failed to produce it.

The uptake of glycine and AIB by intestinal brushborder membrane vesicles of the Mediterranean fish. Diccntrarchus luhras, has been found to be dependent upon both Na ’ and Cl (Boge er (II., 1983). The chloride requirement is influenced by pH in that increasing medium pH reduces the role played by chloride. This anion causes a reduction of transport K,,, and an increase in If,,,. Intravesicular Cl also enhances transport. The effects of Cl on transport may involve the membrane potential due to the rapid diffusion of this anion and/or to a change in carrier affinity in its presence. The uptake ofA1B and glycine in intestinal brush-border membrane vesicles of the marine fish Boops sulpu is stimulated by a Na ’ gradient (out > in); however, an additional requirement for transport besides this gradient is the presence of chloride ion on the external membrane face (Boge and Rigal. 1981). In the absence of CL .3 transport is not stimulated by the Na + gradient, nor is it influenced by an electrical membrane potential generated by SCN- or by a K + diffusion potential. Chloride is also required for glycine transport in both the pigeon and human erythrocyte (Ellory (II al.. 1980). Other anions that were able to substitute for chIoride in the human red cell include, in the following order of effectiveness: bromide > nitrate > sulfate > acetate. Bumetanide, an inhibitor of the Cl -dependent Na +/K +-cotransport system. inhibited Cl -dependent glycine uptake insignificantly. The uptake of glycine in membrane vesicles from rat brain has been shown additionally to be strictly dependent on the presence of Cl in the medium and the process can be driven either by a Na’ gradient (out > in) or Cl gradient (out > in) when the other essential ion is present (Mayor et ul., 1981). The role of Cl in the process may be to create a membrane potential (interior negative) or to activate the carrier. Furthermore, GABA transport into synaptic plasma membrane vesicles isolated from rat brain depends in an absolute sense upon both Na’ and Cl (Radian and Kanner, 1983). Transport V,,,,, is increased and K,,, decreased by either Na ’ or Cl The requirement is not due to its ability to serve as a for Cl permeant species, according to these workers. because transport of GABA still requires external chloride when a K + diffusion potential (interior negative) is imposed across the membrane with valinomycin. In support of these findings the concentrative uptake of GABA into isolated superior cervical ganglia of the rat has been shown to be depressed by removal of external chloride ions (Bowery er ~1.. 1979) and the transport of proline in isolated membrane vesicles

Effecters of amino acid transport

processes in animal cell membranes

from rat brain was found to be optimal in the presence of medium Cl (Kanner and Sharon, 1980). In a number of cell systems replacement of Cl- by other ions leads to an increase or decrease in relative transport. Thus the Na + -mediated stimulation of alanine transport in plasma membrane vesicles isolated from liver parenchymal cells of the rat is abolished by replacement of chloride with sulfate anion (Quinlan et cd., 1982). The pattern of stimulation of alanine transport by anions (SCN _ > Cl > SO: ) reflects their lipophilic nature. Similarly alanine uptake by intestinal and renal brush-border membrane vesicles of the winter flounder, Pseudopleuronectes americanus, was stimulated by replacement of Cl with more permanent anions such as SCN (Eveloff et al., 1980); in the presence of a Na + gradient, SCN caused an increase in alanine transport in brush-border membrane vesicles isolated from human small intestine, whereas the nearly impermeant sulfate anion decreased uptake compared with uptake in the presence of Cl (Luke et al., 1977). Moreover. Nishino et nl. (1980) employing a vesicular system reconstituted from proteins derived from plasma membranes isolated from mouse fibroblasts and exogenous phosphohpids, showed that SCN _ could promote a greater uptake of AIB than either Cl or SO:-. In distinction to the results of these reports, Berteloot et a/. (1982) discovered that substitution of Cl _ by more lipophilic anions such as NO, and SCN produced an inhibition of phenylalanine uptake in mouse intestinal brush-border membrane vesicles. Also glycine transport in human and sheep erythrocytes has been observed to be highest in SOimedium, lower in Cl- and lowest in NO, medium (Young et ul., 1981). Glycine influxes in the three media were inversely related to the affinities of these anions for the Band 3 anion-exchange transport system which serves the exchange of chloridee bicarbonate and has affinity for organic acids such as lactate or pyruvate. The half-maximal inhibition of glycine transport by 4-acetamido-4’-isothiocyanostilbene-2.2’-disulfonate (SITS) in SOior Clmedia was comparable with the affinity of SITS for the Band 3 transport system. Also, the SITS-sensitive component of glycine uptake was greater in SOithan in Cl ~. These workers have proposed that a significant fraction of glycine uptake into red blood cells occurs via the anion-exchange system. Little information is available on the influence of FI on transport of amino acids. This anion has been shown to increase uptake of amino acids in the human erythrocyte (Al-Saleh and Wheeler, 1982). _!$ect.s of’metubolic

intermediates

Citrate, malate, succinate, fumarate, alphaketoglutarate and oxaloacetate each produce biphasic effects, stimulation and inhibition of AIB transport in isolated renal tubules of the rabbit (Kippen et al., 1980). A given intermediate at high levels presumably inhibits at the membrane level by competition for shared transport systems, or competition may occur for energy provided by Na+ or electrical gradients. Stimulation of transport by such intermediates may result from their transport into the cell and catabolism to furnish an increased level of ATP. Stimulation of the Na + , K + -ATPase

721

by ATP results in an increase in the Na+ electrochemical potential gradient leading to an increased Na+-dependent uptake. Heinz et ul. (1981) suggest that metabolic substrates such as lactate and pyruvate may provide metabolic energy for transport of AIB in Ehrlich cells apart from that provided by the Na + -coupled mechanism. Eflects qf’ dinitrophenol Dinitrophenol has generally been regarded as an inhibitor of transport of amino acids through its effects on mitochondrial ATP production and consequent reduction in Na’ , K + -ATPase activity (Lerner, 1978). However, the results of Sacchi et al. (1981), showing that phenylalanine uptake by the isolated midgut of Bombyx mori larva is a Na + -independent process which can be inhibited by dinitrophenol, perhaps suggest a direct effect of this compound on the membrane carrier.

The influx of amino acids into guinea-pig intestine is inhibited by monosaccharides (Alvarado and Robinson, 1979). Two hypotheses have been suggested to explain this inhibition: (1) there is an allosteric interaction between sugars and amino acids which bind to separate but related sites on the brush border membrane; (2) there is a partial dissipation of the sodium ion gradient due to the cotransport of each substrate with sodium ion. These workers contend that the second hypothesis can be ruled out on the basis of the following observation. Galactose is a stronger inhibitor of phenylalanine transport in this system than is beta-methylglucoside despite the fact that the latter monosaccharide carries more Na + into the cell than galactose and thereby should cause a greater membrane depolarization. In another study, isolated intestinal epithelial cells of the rat showed a higher transport of leucine, isoleucine, valine, alanine, phenylalanine, tryptophan and histidine in the presence of fructose than in its absence (Reiser and Hallfrisch, 1977). Glucose generally inhibited these amino acids and sorbose had no effect on leucine transport. The stimulation appears to be relatively specific since basic amino acid transport was not generally stimulated. Leucine transport was optimally stimulated by preloading cells with fructose. Stimulation may occur, as mentioned above, by provision of metabolic energy to the transport process. Effects of ouabain Ouabain, a cardiac glycoside which is a potent inhibitor of Na +, K +-ATPase, inhibits Na +dependent transport of amino acids by dissipating the Na+ gradient upon which coupled transport depends. This glycoside exerted an inhibitory effect on methionine, valine and glycine uptake when exposed to the parenchymal extracellular space of sliced intestinal worms (Hymenolepis diminutu) but had no effect when exposed only to brush-border (Lussier et al., 1979). These results suggest that the ouabainsensitive site of amino acid uptake is at the basal pole of the epithelial cells. Different concentrations of ouabain were required to inhibit uptake of Nat -dependent amino acid transport. For example,

727

JOSEPHLERNER

glycine required a concentration that was IOO-fold greater than that necessary to inhibit uptake of alanine and valine. Similarly the transport of taurine in dogfish kidney slices is completely inhibited by ouabain (Schrock tjr u(., 1982). An interesting consequence of the effects of ouabain in blocking amino acid transport has been reported by White and Christensen (1983). They found that cellular accumulation of Na _ produced by ouabain in cultured hepatoma cells reduced the ability of MeAIB to repress system A because its intracellular concentration is prevented from rising due to the dissipated Na + gradient. Apparently the repressive signal appears to come from the internal level of the amino acid rather than from binding to the carrier. As might be expected, Na _-independent transport of amino acids has been found to be ouabaininsensitive. As an example of such transport, Na ’ independent leucine uptake in isolated bovine retinal and brain microvessels was found to be resistant to inhibition by ouabain (Hjelle ct al.. 1978). Also transintegumentary uptake of amino acids by the lumbricid earthworm. Eiseniu f&~tidu, is not influenced by Na ’ levels and is unaffected by ouabain (Richards and Arme, 1979). As an interesting exception. high-a~nity uptake of lysine by a Na ’ -dependent process across the mucosal border of the midgut of the freshwater shrimp. Mu~rohrccc~ltiun~rosmhergii. was shown to be unresponsive to ouabain (Bricks and Ahearn. 1978). 4fi’tci.r of‘ Clostridium

enwott~.~ie

~~~)~tr~~~urnfmf i.iyps

enterotoxin (molecular weight 35,000) rapidly depresses AIB transport in cultured rat hepatocytes (Giger and Pariza. 1978). This action was noted to be reversible with the degree of recovery being inversely related to the length of exposure time. The etTect was reduced by reacting this protein with a specific antiserum or by heating it. The depression of transport has been correlated with a rapid increase in intracellular Na * . which may be due to membrane damage (Giger and Pariza, 1980). although Na +-independent amino acid transport was decreased little under these conditions.

Aspirin reduces the mucosal to serosal fiux ol alanine in rat jejunum; it also inhibits net active Na ’ flux (Cooke et al.. 1979). By its effect on Na ’ transport aspirin may be inhibiting amino acid transport through dissipation of the Na + electrochemical gradient. Q@c*tr 0f‘ ~~zj~(~ri{i~ Amiloride has been found to inhibit MeAIB transport and that of Na ’ in isolated rat hepatocytes (Fehlmann or a/., 1981). The activity of the Na+-independent L system was unaffected by this compound. Amiloride may alter Na +-dependent transport of amino acids by an effect on the transmembrane Na+ gradient which energizes uptake.

gjkts

i?#immoml upzrs

The effects of a wide variety of humoral agents on amino acid transport are reported in Table 3. The influence of insulin on amino acid transport has been

studied extensively in a number of tissue and cell types. This hormone has been found to stimulate transport of AIB in rat hepatocytes (LeCam (‘1 crl.. 1979; Prentki et uI., 198l), human tibroblasts (Martin and Pohl. 1979). mouse fibroblasts (Yuli fl (II.. 19X2). chick embryo heart cell aggregates (Santora et ul.. 1979: Wheeler rt 01.. 1978). mouse soleus muscle (Marchand-Brustel rt trl.. 19X?), kidney cortex ot lambs and piglets (Scharrer and Landes, lY78) and in rat thymocytes (Kwock, 1981). Insulin has also been shown to enhance Na -independent uptake of cystine in rat hepatocytes (Takada and Bannai. lYX4). In contrast to these findings on stimulatio?l of transport. Mann and coworkers (iY83) have reported that perfusion of the rat pancreas with insulin for 30 min leads to a decrease in the uptake of AIB, serinc and leucine. Also system N in rat hepatocytcs for glutamine. asparagine and histidine has been shown bq Vadeama and Christensen (IYX?) to be unaffected by insuiin. Little is currently known about the nature of insulin receptors which are involved in the mechanism of stimulation of amino acid transport processes. In this regard. cultured l4-day embryonic chick heart cells have been shown to possess at least two classes of receptors which bind insulin: occupancy of a lower-inanity class correlates with ini;ulin stimulation of AIB transport (Santoru t’t d.. 1979). Stimulation of AIB transport by insulin in the rat hepatocyte can be reduced by preexposing the cells to trypsin, which has no effect on basal AIB uptake (Dolais-Kitabgi CJ~ni.. 19X1). Perhaps trypsin digests the insulin receptor site on the cell membrane. A few rccrgt studies have provided inform~~tion on the nature of the insulin efrect in terms of change in transport kinetic parameters. The uptake of AIR in soleus muscle of mice was shown to be increased by insulin through an cll’ect on maximal Rux ( I ‘,,,.,,) without an alteration in k;,: of transport (MarchandBrustel c’f ui.. 1982). Sitnil~lrly insulin increases the transport V,,,;,,of RIB with no change in transport k;,, in kidney cortex of young lambs and piglets (Scharrer and Landes. 1978). On the other hand, insulin stimulates AIB uptake by decreasing K,,, without a change in C;,,E, in cultured human fibroblasts derived from skin biopsies (Martin and Pohl. 1979). In a variety of cell and tissue systems the insulin efrect is mediated. at least in part, by ;I mechanism which involves protein synthesis. since the efrect is reduced or abolished by inhibitors of prolein or RNA synthesis (Table 4). Thus the insulin response appears to involve protein synthesis for transport of cystinc in rat hepatocytes. McAIB in mouse fibrohlasts. AIR in lamb and pig kidney cortex and system A substrates in chick embryo heart cells. Insulin has been found to stimulate system A transport in chick heart cell aggregates from both 7- and l4-day embryos and to prolong the half-life of transport actlvlty in the presence of cycloheximide in the 7-da> old embryo cell aggregates (Wheeler ct d.. 1978). At 14 da>is. cycloheximide reduces the insulin response substantially. The effect of insulin appears to shift from the post-translational level at seven days to a syrthetic level by 14 days. In another stud) the effect ot Insulin on the uptake of AIR in soleus muscle of mice has been shown to occur within 30 min and is incom-

AIB

Weaniing

trout intestine

heart cells

Rabbit intestine Turtle intestine Human placenta Rat hepatocytes

Rat s&us muscle Sheep dictyate oocytes Xenopus lreris embryos

Rainbow

Chick embryo

Acetylcholine Epinephrine

Leucine

AIB AIB

Ovine placental lactogen Ovine growth hormone Ovine placental lactogen Ovine growth hormone Rat growth hormone Human placental lactogen Ovine prolactin Ovine placental lactogen Ovine proiactin human

Insulin

LelXine Cycloleucine system L System A Lysine

acids

acids

peptide

agents on transport

Prostaglandins F+lpha, D2. E,, F, alpha, Thromboxane BI Hydrocortisone Insulin Bovine growth hormone

Gastrin releasing Cholecystokimn Caerulein Gastrin Testosterone Insulin Glucagon

ElTector

Table 3. Elfects of humoral

Bovine insulin Chicken insulin Guinea-pig insulin 17-Alplramethyltestosterone 17-Betdestradiol Insuhn “Follicular factor” Triiodo-t_-thyronine Trhodo-L-thyronine Prostaglandin E,

AIB

Amino

Fetal rat diaphragm

rat diaphragm

AIB AIB AIB Amino

libroblasts

AIR Histtdine Asparagine Glutamine (System N) AIB

AIB

AIB

Human dermai fibroblasts Human skm fibroblasts Rat diaphragm Prenatal rat diaphragm Postnatal rat diaphragm

Human

Mouse pancreas Acini Rat brain Rat hepatocytes

AWli

Mouse pancreas

CehTissue

-0

Vadgdma

+

+

0

0

0 0

U/.

(1979)

er al. (1983)

el

and Handwerger

and Handwerger

er tri. (IYXZ)

(1982)

(1983)

(1983)

(1983)

Isaksson

Rowell and Rama Sastry (1978) Pdrira c’r L//. (1977)

Nassar

(1983)

(1977)

Cooper and Kohn (1977) Moor and Smith (1978) Mlot r, ctl. (1980)

Habibi

SdntOKl

Freeman

Freeman

and Handwerger

Freeman

+

0 +

Russell PI rrf. (1982) Martin and Pohl (1979) Albertsson-Wikland and Riggs e/ (I/. (1978) Freeman and Handwerger

and Kawamura

(1977) and Christensen

and Williams

(1980)

Reference rt rtl. (1983)

+ + +

Murota

Litterra

0

Iwamoto

Iwamoto

t

Response

(1978)

Cell/Tissue

Regenerating rat hver (Walker and Whitfield.

1978)

1984)

System L System A High affinity Glycine System AIB

AIB

Rat hepatoma (McDonald and Gelehrter. Rat hepatoma (Nadzlejko and Rachherg.

1977)

System A

System A

Rat hrpatoma cell lines (Kelley and Potter. 1979)

1982)

AIB System A System A

Rat hepatocyte (Fehlmann er a/, 1979) Rat hepatocyte (Handlogten and Kilberg. Fetal rat hepatocytes (Handlogten and Kilberg.

1984)

AIB

Rat hepatocyte (Kelley ef al., 1980)

AIB

AIB

(Barber CI ul.. 1983) Rat hepatocyte

(L&am and Freychet, 1978) Rat hepatocyte (Kelley and Potter, 1978)

System A

1984)

Substrate

Cystine (Na+independent

AIB

Alanine AIB Alanine

(Takada and Bannai. Rat hepatocyte

(Fehlmann et ul.. 1981) Rat hepatocyte

Rat hepatocyte (Samson and Fehlmann, 1982) Rat hepatocyte (Edmondson and Lumeng, 1980) Rat hepatocyte

system)

inactivating

Partial

hepatcctom~

Dexamethasone

Dexamethasone

Amino acid starvatmn Insulin Glucago” Dexamethasone Amlno acid starvation

“Transport

protein”

involvine

svnthesis

by Ca’ +)

orotein

(el%ct enhanced

Effector

plus glucagon

acid starvation

Glucagon Catecholamines Dexamethasone Fasted animals

Amino

Epinephrine Phenylephrine Isoproterenol

Amino aad starvation Glucagon Dexamethasone Insulin Vasopressin

Insulin Dexamethasone

In&

Glucagon

DBcAMP

Table 4. EtTectors of tra”s”ort

+

0

0

0

+

_

+

+

Response (Change in transport activity)

Actinomycin D Cycloheximidr AIB Cycloheximide Actinomyci” D Cycloheximide Actinomycin D Insulin (partialI>

Puromycin Cycloheximide Actinomycin D Cycloheximide Cycloheximide

re\rl-w

Cycloheximide, Puromycin, AIB, Actinomycin D Protein synthesis inhibitors

Cycloheximide, Actinomycin D

Tunicamycln

by:

el‘lilcl on S>wzrn A)

lnhlhitora

nullified

synthesis

Response

RNA and protein

Amiloride

Cycloheximide

Cycloheximide

Cell:Tissue

Lamb/Pig kidney cortex (Scharrer and Landes, 1978) Hamster ovary cells (Chinese) (Shotwell et al., 1981) Chick embryo heart cells (Wheeler el al., 1978)

Plant lectins (Phytohema~lutinin)

Proline, Actinomycin D Puromycin, Cycloheximide Cycloheximide

+

Protein-free amino acid-free medium Insulin “Transport

System A

AIB protein”

System A Substrates Cycloheximide

+

inactivating

Cycloheximide

+ Starvation

System A

Inhibitors of protein synthesis

Cycloheximide Actinomycin D Cordycepin Inhibitors of protein and RNA synthesis

Cycloheximide Puromycin Actinomycin D Cycloheximide

+

+

+ +

+

AIB Alanine Proline AIB

stimutating activity

Multiplication

System A

Chick embryo fibroblasts (Derr and Smith, 1980) Human blood I~phocytes (Segei and Lichtman, 1981)

Medium devoid of System A substrates System A substrated Dialysed serum (or undialysed)

Medium devoid of glucose

Proline System A Proline AI6

MeAIB

AIB

system

Cystme-glutamate

Actinomycin D Cycloheximide Cycloheximide Glucose

system

Cystine-giutalnate Cystine starvation

Response nullified by: I_ Inhibitors of protein synthesis Inhibitors of early mRNA synthesis Inhibitors of early mRNA synthesis RNA and protein synthesis inhibitors

+

Amino acid starvation System A amino acids Eieetrophilic agents (Diethyl Maleate, etc.)

_

System A

ERector Human platelet derived growth factor

Substrate

System A

Chick embryo fibroblasts (Dall’asta CI al., 1978) Chick embryo fibroblasts (Borghetti et al., 1979)

Human fibroblasts (Owen el nl., 1982) Human fibroblasts (Ciazzola et al.. 1981) Human fibroblasts (Bannai, 1984) Human fibroblasts (Bannai and Kitamura, 1982) Hamster fibtoblasts (Nishino ec 01.. 1978; Christopher er ul., 1979) Mouse fibroblasts 3T3 cells (Grunfeid and Jones, 19833

Response (Change in transport activity)

726

JOSEPH LERNER

pletely prevented by inhibitors of protein synthesis, suggesting that it is the resultant of synthesis of new carrier proteins as well as possibly of a direct effect on the carrier to increase its rate of translocation (Marchand-Brustel et a/., 1982). Several reports in the literature indicate that tissues can become refractory to the effects of insulin on amino acid transport. Chronic exposure to insulin has been found to induce resistance of AIB uptake in cultured human fibroblasts by decreasing the apparent affinity for insulin (Martin and Pohl, 1979). Moreover, insulin causes an increase in the uptake of AIB in dexamethasone-treated rat hepatoma cells with a maximal effect seen 2-4 hr after insulin addition (Heaton and Gelehrter, 1980). Subsequently there is a decrease in responsiveness to control levels by 24 hr of incubation, suggesting a loss of sensitivity to the hormone. Diaphragms from rats less than I day old do not show the increased transport rate of AIB found in older animals in response to insulin presumably because the high levels of insulin in the donor animals rendered the day-old tissue insulin resistant (Riggs et cd., 1978). Insulin has been postulated to have a direct effect on the cell membrane since it may oxidize membrane components (Kwock. 1981). For example. uptake of AIB by membrane vesicles from rat thymocytes can be activated by insulin or diamide, both of which induce disulfide formation. Uptake is also modulated by glutathione in these cells, with reduced glutathione being inhibitory and its oxidized form being stimulatory. This information suggests that AIB uptake involves a plasma membrane sulfiydryl-containing protein. Kwock (1981) has proposed that when such sulfhydryl groups are in the disulfide form, the potential energy barrier for transport is lowered. whereas. when oxidized, the barrier is increased. Another study proposes that the internalization of insulin is not required for the expression of its effects on amino acid transport. In this regard, insulin stimulation of AIB transport in the rat hepatocyte is not influenced by methylamine, which is known to inhibit clustering and internalization of the hormonereceptor complex (Le Cam ct a/.. 1979). On the other hand. Prentki et nl. (1981) have found evidence to support the idea that microtubules are involved in the regulation of AIB transport by insulin in hepatocytes. Microtubules might be part of the mechanism in the transfer of hormonal information from the plasma membrane to the site of synthesis of the carrier protein. Colchicine, a microtubule inhibitor. inhibits caused by the increase in V,,,‘,, of AIB transport insulin in these cells. Alternatively this drug, or vinblastine (another inhibitor of insulin-stimulated transport and microtubular action), may interfere with the intracellular transport of the carrier to the plasma membrane. Glucagon, in studies done with liver cells, has been shown to stimulate system A transport (Handlogten and Kilberg, 1984) as well as the uptake of leucine by a Na ’ -independent system (Mohri and Sasaki. 1982) and that of cationic amino acids (Handlogten and Kilberg, 1984). In contrast, system N in rat hepatocytes (Vadgama and Christensen, 1983) is unaffected by glucagon. Edmondson and Lumeng (1980) have shown that this hormone stimulates

uptakes of AIB and alanine in two phases in rat hepatocytes. The initial stage of stimulation can be abolished by incubation of the cells in a medium containing I2 meqjl K ’ ; the later phase. however. IS little altered by incubation of cells in this medium. Moreover. cycloheximide does not alter the first phase but abolishes the second. The biphasic stimulation by glucagon appears to depend on cell menbrane hyperpolarization in the first phase and on protein synthesis in the second. The effect of glucagon to increase AIB transport in rat hepatocytes can be inhibited by microtubule inhibitors such as colchicine and vinblastine (Prentki vt al., 1981). Colchicine inhibits the increase in the I’,,,,, of a high-affinit) component of transport caused by this hormone without affecting a low-affinity component. These results suggest that microtubules are involved in the regulation of this transport by glucagon and ma) affect transfer of hormonal information from the cell membrane to the site of carrier-protein synthesis. The stimulation of leucine transport in Chang liver cells by glucagon can be inhibited by preincubation of cells with colchicine or cytochalasin B, which arc known inhibitors of cytoskeleton formation (Mohri and Sasaki. 1982). Glucagon treatment was shown to affect v”,,,,,,of a high-affinity leucine transport system and the K,,, of a low-affinity leucine system. Kelley ct cd. (1980) have found that protein synthesis is required for the stimulation of AIB transport by glucagon in the rat hepatocyte; mobilization of cellular calcium by glucagon either directly or through cyclic AMP mediates the stimulation. A number of growth hormones and growth factor\ have a general stimulatory efrect on amino acid transport. Diaphragm muscle from IS-day old rats has a biphasic response to growth hormone (Nutting and Coates. 1977). Administration of the hormone br riw acutely enhances the uptake of AIB; houe~cr. 4 hr after administration the rate of AIB uptake i5 no longer increased and the transport system becomeh refractory to additional hormone irk vitro. Similarly. bovine growth hormone administered i/7 13iw itiduces a transient stimulation of AIB transport in diaphragms of IX-day old rats studied irl c.i//~j (Albertsson-Wikland et ul.. 1980). Three hours aftcladministration of the hormone amino acid transport returned to normal. Five to seven hours after administration transport became completely refractor> tn the hormone it7 rim. Bovine growth hormone has been found in another study to restore transport 01 AIB in rat tissues such as diaphragm. skeletal mttsclc. isolated from vitamin intestine and kidney B,-deficient animals (Heindel and Riggs. 1978): ho\\ever, the hormone had no further efrect aboce that produced by pyridoxine administration. It appears that the decreased amino acid transport found in rat tissues with a dietary deficiency of vitamin B,, ih caused by a decreased amount of growth hormone resulting from a lack of the vitamin. Ovine growth hormone and ovine placental Iactogen, polypeptide hormones with similar biological actions in tissues of postnatal animals, stimulate amino acid transport equally in diaphragms of postnatal rats (Freeman and Handwerger. 1983). Amino acid uptake was shown to be stimulated by ovine placental lactogen in diaphragms from l’ctal ratx.

Effectorb

of amino

acid transport

processes in animal cell membranes

while ovine growth hormone, human placental lactogen, ovine prolactin and rat growth hormone were without effect. Human platelet-derived growth factor, a mitogenic polypeptide carried on the alpha-granules of platelets, is released during clotting (Owen et a/., 1982). It is a cationic protein with a molecular weight of 32,000-35,000 daltons and consists of two subunits, both of which are required for biological activity. This protein induces an increase in amino acid uptake by system A in human diploid fibroblasts. Transport stimulation requires protein synthesis. Antiserum to this factor blocks factor-stimulated DNA synthesis and also blocks factor-stimulated amino acid transport. Platelet-derived growth factor and epidermal growth factor have been found to prevent the cyclic AMP-dependent stimulation of amino acid transport in rat hepatocytes by glucagon and the cyclic AMPindependent stimulation by catecholamines, but not the stimulation by insulin (Auberger et ul., 1983). Multiplication-stimulating activity (a polypeptide, insulin-like growth factor of about 10,000 daltons molecular weight which is related to the somatomedin family of growth regulatory proteins) stimulates the proliferation of quiescent cultures of chicken embryo fibroblasts, stimulates DNA synthesis and enhances AIB transport by the A system with little or no change in the activities of transport systems ASC, L or Ly + (Derr and Smith, 1980). The increased transport activity is due to a change in V,,,, alone and inhibitors of RNA and protein synthesis inhibit the response. Multiplication-stimulating activity causes an increase in amino acid transport in dexamethasone-treated rat hepatoma cells (Heaton et ccl.. 1980). Additionally AIB uptake was found to be stimulated in serum-starved L6 myoblasts after exposure to this growth factor. the effect being seen on I’,,,,, alone (Merrill et ul., 1977). Nerve growth factor, a polypeptide required for the survival and maturation of sensory neurons, has been observed to stimulate AIB transport in clonal PC12 pheochromocytoma cells in culture, the effect being an increase in V,,,, with no change in K,, (MC&ire and Greene, 1979). Steroid hormones have been found in a variety of studies to either enhance or diminish transport of amino acids, depending on the nature of the hormone. These agents most probably function at the level of protein synthesis. With reference to sex hormones, testosterone proprionate causes an increase in active transport of AIB in a wide variety of brain regions of the immature rat 5 hr after i.p. injection (Litteria, 1977). The effectiveness of the steroid was found to decrease with age. In another study addition of l7-alpha-methyltestosterone to incubation media increased the transmural transport of leucine in everted sacs of rainbow trout (Habibi ct cd., 1983). Addition of l7-beta-estradiol however, was without effect on transport of leucine. Dexamethasone. a synthetic glucocorticoid, additively enhances the uptake of cystine by a Na’-independent system in adult rat hepatocytes (Takada and Bannai. 1984). On the other hand, dexamethasone has been shown to inhibit the activity of a Na +-independent transport system for leucine, phenylalanine and 2-aminonorbornane-2-carboxylic

727

acid in rat hepatoma cells (Nadziejko and Reichberg, 1984). In rat hepatoma cells glycine uptake occurs by way of at least two systems (Reichberg and Gelehrter, 1980). One system, which appears to be analogous to the A system. is inhibited by dexamethasone in less than ten hours and has low affinity and high capacity for glycine. The other system has a low capacity for glycine and requires more than twenty hours of exposure for full inhibition by this hormone. McDonald and Gelehrter (1977) have demonstrated that dexamethasone reversibly inhibits AIB transport in rat hepatoma cells. This effect can be inhibited by cycloheximide and actinomycin D, suggesting that protein synthesis is involved in the action of the hormone. Colcemid and cytochalasin B do not inhibit transport nor interfere with the effect of dexamethasone, ruling out an involvement of microtubules and microfilaments in the hormonal effect. These workers have postulated that dexamethasone induces the synthesis of a labile protein which either decreases the rate of synthesis or increases the rate of degradation or both of a component of the transport system. In contrast to these results, proline transport in human dermal fibroblasts has been found to be increased when cells are grown in hydrocortisone (Russell et al., 1982). Fibroblasts derived from keloid tissue (a benign tumor) are stimulated to a greater degree than normal fibroblasts. Progesterone has been observed to inhibit the hydrocortisone-induced increase in transport. Pariza et ul. (1977) have demonstrated that epinephrine induces AIB transport in primary cultures of adult rat liver cells by a process which is independent of cyclic AMP. Thus, epinephrine and isoproterenol both induce large increases in cyclic AMP in this system within l-2 min, whereas epinephrine. but not isoproterenol, induces an increase in AIB transport within 224 hr after a 1 hr lag. Propranolol abolishes the increase in cyclic AMP levels caused by both compounds but does not block the induction of AIB transport by epinephrine. Dihydroergotamine and phentolamine do not affect the stimulation of cyclic AMP levels by epinephrine but they inhibit the induction of AIB transport by epinephrine. Subsequently, LeCam and Freychet (1978) reported that a 2-hr exposure to epinephrine, phenylephrine or isoproterenol increased AIB influx in suspensions of isolated rat hepatocytes. The order of potency of stimulation was epinephrine > norepinephrine > phenylephrine > isoproterenol. Epinephrine increased V,,,;,, with no change in K,,,. Cycloheximide, and to a lesser extent actinomycin D, inhibited this stimulation. Phentolamine, an alpha-antagonist, abolished the stimulation whereas propranolol. a beta-antagonist, had little effect. The effect of isoproterenol was partially inhibited by both antagonists. It is concluded that catecholamines directly stimulate the A system (no effect was seen on the L system) through alpha-adrenergic mediation which involves protein synthesis. Acetylcholine, the synaptic transmitter for the preganglionic and some postganglionic neurons of the autonomic system, has been shown to inhibit AIB transport in isolated villi from human term placenta (Rowe11 and Rama Sastry, 1978). The protein hormone secretagogues caerulein, cho-

JOSEPHLERNER

728

lecystokinin and gastrin and the cholinergic agent carbachol inhibited AIB uptake in isolated mouse pancreatic acini (Iwamoto and Williams, 1980). In the presence of Ca’ +, the calcium ionophore A23 187 mimicked the effect of caerulein on AIB uptake. In the absence of Ca’ +. AIB uptake was reduced and both caerulein and A23187 had no further effects. It would appear therefore that secretagogues known to induce enzyme release in the pancreas by an effect on cellular Ca*’ also reduce AIB uptake and that this effect on uptake occurs by mediation involving Ca” In a related study Iwamoto et a/. (1983) showed that AIB uptake can be inhibited in mouse pancreatic acini by gastrin-releasing peptide, a recently isolated gut hormone. While this hormone can mimic the biological effects of cholecystokinin (a powerful stimulant of pancreatic secretion and an inhibitor of AIB uptake in mouse acini) and acetylcholine, its effects appear to be mediated through distinct receptors from those either of cholecystokinin or cholinergic agents. Thyroxine has been found by Riggs et al. (1984) to accelerate the development of neutral, alpha-amino acid transport systems in the rat brain during the first six days after birth. In contrast triiodo-L-thyronine had very little effect on the transport of L system amino acids in embryos of the South African clawed toad, Xenopzrs Iueois, while the transport of amino acids by the Na + -dependent A system was found to be inhibited (Mlot et al., 1980). Prostaglandins having an hydroxyl functional group in an intramolecular 5-membered ring showed an inhibitory effect on AIB uptake in cultured human fibroblasts (Murota and Kawamura, 1977). The potency of such compounds decreased in the following order: prostaglandin FZ a,pha> F, a,phi> D3 > Ez > thromboxane Bz. Inhibition by prostaglandin Fz alph., was maximal after I hr of exposure and persisted at least up to 6 hr; this compound did not enter the cells even after incubation for 24 hr. The double bond located between carbon 5 and 6 in PGE, accounts for the inhibitory activity of this compound since its analog, PGE?, is inactive. The hydroxyl functional group in the five-membered ring appears to be an indispensible moiety for inhibition. In another study lysine influx in rabbit and turtle small intestines can be reduced by the presence of relative!y large pharmacological doses of prostaglandin E, in the incubation medium (Nassar et ul., 1982). Because of the requirement for a preincubation period. the inhibition is probably not caused by a direct efTect on the membrane. Cyclic

nucleotides

und trunsport

Perfusion of rat heart with dibutyryl cyclic AMP (DbcAMP) or theophylline, a phosphodiesterase inhibitor that inhibits the rate of enzymatic hydrolysis of cyclic AMP, resulted in a stimulation of taurine transport (Huxtable and Chubb, 1977). Dibutyrl cyclic AMP has also been found to directly stimulate, without preincubation, the uptake of AIB by separated rabbit kidney tubules and oxygen consumption suggesting that the transport stimulation may occur by a linkage with renal oxidative metabolism (Kippen rt a/., 1979). Moreover, the stimulation of AIB transport was not affected by cycloheximide. ruling

out protein synthesis as a cause of the stimulation. This nucleotide also increased the uptake of AIB into renal brush-border membrane vesicles; under these conditions, however. the transports of proline and leucine were inhibited. Sodium ion-independent transport of leucine in Chang liver cells is stimulated by glucagon, an activator of adenyl cyclase, or by DbcAMP (Mohri and Sasaki, 1982). Dibutyryl cyclic AMP promoted transport by affecting both V,,,,,, and K,,,. Colchicine or cytochalasin B inhibit the stimulation of transport by glucagon or DbcAMP. suggesting that the effects of these agents require functioning of the cytoskeleton. Several studies indicate that protein synthesis is involved in the stimulation of transport by cyclic nucleotides. Treatment of cultured glial cells with a mixture of DbcAMP and 8-bromocyclic GMP for I or 2 days results in an increase in the V,‘,,,,, of glutamate uptake by way of a high-affinity system in the mouse (Borg et ~1.. 1979). Similarly, 2 days of exposure was required for a maximal effect of DbcAMP in stimulating proline transport in Kirsten sarcoma virus-transformed BALBj3T3 cells (Peterkofsky and Prather, 1979). No effect of the nucleotide was seen on transport of either glycine or glutamine. The results are consistent with the notion that DbcAMP induces the synthesis of a component of the A system. Likewise. vesicles prepared from hepatocytes exposed to DbcAMP retain an increased maximal uptake of alanine (Samson and Fehlmann. 1982). This stimulation requires a 2-3 hr exposure 01 cells and is totally inhibited by cycloheximide. These results support the hypothesis that this nucleotide increases the number of carrier molecules. Nilsen-Hamilton and Hamilton (1979) have shown that incubation of membrane vesicles from mouse fibroblasts (3T3 cells) with ATP, cyclic AMP and cyclic AMP-dependent protein kinase, or ATP with the catalytic subunit of this kinase, results in inhibition of AIB transport. The cyclic AMPdependent protein kinase and its catalytic subunit phosphorylate a number of membrane proteins: these results suggest the possibility that stimulation of phosphorylation of membrane components by the cyclic nucleotide may be a mechanism by which 3T7 cells regulate amino acid transport.

Tunicamycin, a glucosamine-containing antibiotic that specificially inhibits dolichol pyrophosphatemediated glycosylation of asparaginyl residues 01 glycoproteins, has been found to inhibit transport of AIB and cycloleucine in chick embryo fibroblasts (Olden ct al., 1979). Glycoprotein glycosylation. from these results, appears to be required for membrane transport. Tunicamycin also inhibits system Amediated amino acid transport in rat hepatocytcs (Barber et al., 1983). The basal activity of system A was decreased by the antibiotic when cells were cultured for 24 hr in its presence. The glycine transport system was also sensitive but the activities of systems L,, L2 and N were relatively resistant and that of ASC was only moderately affected. This inhibitor also blocked the increase in system A caused by incubation in the absence of amino acids (adaptive control), as well as the stimulation by glucagon.

Effecters

of amino

acid transport

processes

insulin or vasopressin. Addition of tunicamycin to cells which were pretreated with glucagon and dexamethasone blocked any further increase in transport indicating that a glycoprotein component of system A must be continually synthesized to sustain the increase in activity. Preincubation of quiescent BALB/3T3 cells with uridine diphosphate N-acetylglucosamine in conditioned medium, but not with other nucleotide sugars, caused a stimulation of AIB uptake over that seen in conditioned medium alone (Natraj and Datta, 1978). Furthermore, incubation of quiescent cells with UDP-[‘H]N-acetylglucosamine caused an incorporation of labeled N-acetylglucosamine into cell surface receptors, suggesting that the cellular acceptors of quiescent cells are under-glycosylated and that stimulation of transport may be a consequence of restoration of the amino sugar residues on the oligosaccharide chains of acceptors on the cell membrane. Perhaps the acceptor molecules are glycoproteins. dexamethasone,

Eflhcts qf electrophilic

agents

The transport activity of cystine and glutamate by a common system in cultured human diploid fibroblasts is enhanced by low concentrations of various electrophilic agents such as diethyl maleate, cyclohex-2-en- l-one, ethacrynic acid, 1,2-epoxy-3(p -nitrophenyl)propane and sulfobromophthalein (Bannai, 1984). These compounds cause an increase in transport VmaXwith little change in apparent K,,. The enhancement of transport requires RNA and protein synthesis. Inhibitors

qf microtubular

and microjilament

,function

Inhibitors of microtubular function, the vinca alkaloids vincristine and vinblastine, depress uptake of AIB in Ehrlich cells with no concurrent change in the chemical gradients of Na +, K + or H + (Goldman et u/., 1977). Another inhibitor of microtubular function, colchicine, has been shown to prevent cells from initiating DNA synthesis and also abolishes the increased uptake of AIB in rat hepatocytes initiated by partial hepatectomy (Walker and Whitfield, 1978). Colchicine may also interfere with the translocation of rRNA across the nuclear membrane, since this process involves the microtubular network. or the compound may disrupt microtubule-dependent movement of receptors on the cell membrane (Walker and Whitfield. 1978). Colchicine has been shown to inhibit transport of AIB, methionine and leucine in rat hepatoma cells (Tauber and Reutter, 1980). The drug has been postulated to affect transport by an action on the movement of the carrier protein within the plasma membrane. Insulin or glucagon stimulation of AIB transport in rat hepatocytes can be inhibited by colchicine and vinblastine (Prentki c)ta/., 1981). Colchicine inhibits the increase in V,,,,, of a high-affinity component of transport caused by the hormone without affecting the uptake by a lowaffinity component. These results indicate that microtubules are involved in the regulation of AIB transport by insulin or glucagon. In another investigation, serum-stimulated AIB uptake in avian embryonic skeletal muscle was noted to be sensitive to colchicine and vincristine (Vandenburgh and Kaufman, 1982). In addition to these results, the transport of AIB

in animal

cell membranes

729

in adult rat hepatocytes in early culture (day 2) was little affected by cytochalasin D, which can induce depolymerization of microfilaments by binding to a membrane protein complex and disturbing the interaction and association between the membrane and the microfilaments (Mak and Pitot, 1981). Cells in early culture contain few microfilaments compared to late cultures of hepatocytes (day 6) which have extensive accumulation. Such microfilaments contain actin and their accumulation is due to, in part, the synthesis of this protein. These findings suggest that the microfilament accumulation which occurs as a compact network almost exclusively beneath the cell membrane is responsible for inhibition of transport and why day-6 cultures demonstrated enhanced transport when exposed to cytochalasin D. These workers have hypothesized that cytochalasin D binds to the plasma membrane, induces the dissociation and disruption of microfilaments and thereby alters the fluidity of the membrane, hence affecting transport. In another recent study, colchicine was noted to increase MeAIB transport in undifferentiated mouse fibroblasts (Grunfield and Jones, 1983). This compound showed little effect on insulin-stimulated transport in differentiated fat cells. It is of interest in this connection that tubulin and actin levels decrease dramatically during differentiation of the fibroblast. Effects ofplunt

lectins

Plant lectins stimulate the uptake of AIB, alanine and proline into human blood lymphocytes (Segel and Lichtman, 1981). De noco protein synthesis was found to be necessary for stimulation of AIB transport. Borghetti et al. (1981) have demonstrated that addition of phytohaemagglutinin-P to quiescent peripheral pig lymphocytes causes the emergence of system A and stimulation of system ASC, which results from a large change in the maximal velocity of the latter system without a significant change in apparent transport affinity. Adrenergic

betu-receptor

ugents und trunsport

Taurine transport by rat heart is immediately stimulated by addition of isoproterenol to the perfusion medium (Huxtable and Chubb, 1977). This effect is seen kinetically as an increase in both V,,,, and K,,,. The major pharmacological action of isoproterenol is beta-adrenergic agonism. Perfusion of the beta-adrenergic antagonist propranolol was without effect but did block the isoproterenol-induced stimulation of influx. The results indicate that taurine transport in the heart is regulated by beta-adrenergic activation. More recently, Koenig and coworkers ( 1983) showed that isoproterenol evokes a rapid Ca* + -dependent stimulation of amino acid transport in mouse renal cortex proximal tubule cells which is associated with increased Cal+ fluxes and a mobilization of mitochondrial calcium, suggesting that the stimulus-transport coupling is mediated by cytosolic Ca2 + Isoproterenol also rapidly increases ornithine decarboxylase activity causing a sustained increase in polyamine levels. Alpha-difluoromethyl-ornithine, an inhibitor of ornithine decarboxylase, suppressed a testosterone-induced increase in polyamine levels and amino acid transport and blocked the isoproterenol-

730

JOSEPHLEKNEK

induced increase in Ca’ +. Putrescine abolished the effects of alpha-difluoromethyl-ornithine and restored the increment in polyamines, Ca” and amino acid transport. Significantly, these results implicate polyamine synthesis in amino acid transport. Transport of AIB in mouse kidney cortex slices is rapidly stimulated by isoproterenol (Goldstone e’t al., 1983). This response is abolished in a calcium-free medium, by the Ca’ c chelator EGTA, by the Ca’ + antagonist La’ ’ and the Ca’+ transport inhibitor verapamil and by propranolol, again suggesting both calcium- and beta-adrenergic-receptor dependence. Isoproterenol rapidly stimulates both the influx and elflux of Ca’+ in cortex slices, decreases the mitochondrial content of Ca’+ and increases the cytosolic content of calcium. The calcium ionophore A23187 mimicks isoproterenol in stimulating AIB transport. These results suggest that beta-adrenergic stimulation of amino acid transport involves an increased influx of mitoof extracellular Ca’ ’ and a mobilization chondrial Ca” ; the resulting increase in cytosolic Ca’ ’ content appears to be the regulatory signal for amino acid transport. Kelley et cl/. (1980) employing rat hepatocytes in culture. have shown that mobilization of cellular calcium through cyclic AMP mediates stimulation of AIB transport. This mechanism is not likely to account for rapid changes in transport, however, since it involves protein synthesis. Isoproterenol. moreover. induces a large increase in cyclic AMP in rat liver cells but does not induce an increase in AIB transport (Pariza (‘t uI., 1977). Propranolol has been shown to induce cellular loss of K+ in Ehrlich cells and alters the membrane potential in a K +-dependent manner (Pershadsingh activates a Ca’ ’ ct al.. 1978). It presumably dependent K + channel which increases membrane hyperpermeability to K ’ . resulting in membrane polarization (Valdeolmillos et a/., 1982). In contrast to the inhibitory effects of propranolol seen on isoproterenol-stimulated transport in the Ehrlich cell, it stimulates amino acid influx at low K + (external concentration) (Pershadsingh ct ul.. 1978). Mechanistically, the ‘increase in the electrochemical gradient for Na + that results from membrane hyperpolarization stimulates amino acid uptake through the coupled Na +-dependent A system. Thus, propranolol in Na +-free medium does not affect the uptake of substrates by the Na ‘-independent L system (Valdeolmillos et N/., 1982).

N-Ethylmaleimide (NEM), p-chloromercuribenzoate (PCMB) and p-chloromercuriphenylsulfonate (PCMBS) have been found to inhibit GABA transport in rat brain synaptosomes (Troeger et d., 1984). 5,5’-Dithiobis-2-nitrobenzoate (DTNB), mercaptoproprionate and N-nitroethylenediamine were much less effective or ineffective. The ability of reagents such as PCMBS or PCMB, which are only partially membrane permeable though somewhat lipophilic, to inhibit indicates perhaps the presence of reactive sulthydryl groups on the carrier protein. Compounds with the least lipophilicity, mercaptoproprionate and were ineffective perhaps N-nitroethylenediamine,

because the reactive sulfhydryl groups are not completely exposed to the medium. The concentrative uptake of AIB in Leishmunia tropicu has been shown to be sensitive to NEM, PCMB and iodoacetate (Lepley and Mukkada, 1983). Alanine influx in sheep erythrocytes was found to be inhibited by HgC&, PCMBS, azodicarboxylic acid bisdimethylamide (diamide), NEM and r-butylhydroperoxide (Young pt al., 1980). lodoacetamide and DTNB had no effect. Three classes of cellular thiol groups appear to be essential for alanine transport. Class 1 thiols react with PCMBS and are located on the outer surface of the cell membrane in the region of the transport site. Class 2 thiols are affected by NEM and diamide but not by r-butylhydroperoxide. Class 3 thiols are oxidized by t-butylhydroperoxide and are either intracellular glutathione or reactive thiols which form mixed disulhdes with oxidized glutathione. With reference to the human erythrocyte. PCMBS. thimerosal and NEM were found to inhibit Na ’ dependent alanine transport (Ellory rt trl., 1980). Moreover, sulfhydryl groups appear to be involved at the site of action of a transport system for tryptophan in the human red blood cell, since strong inhibition of transport was found with NEM and PCMBS (Rosenberg. 1981). 5,5’Dithiobis-2-nitrobenzoate was found to be a moderate inhibitor. N-Ethylmaleimide has been reported to stimulate Na ’ -independent uptake of leucine at low concentrations in Chang liver cells (Takadera and Mohri. 1982). Pretreatment with NEM in the same fashion depressed the Na ’ -dependent uptake of glycine. Diamide has been shown to have a direct inhibitory effect on the uptake of glycine by isolated rat renal brush-border membrane vesicles (Reynolds et (I/.. 1979). Will and Hopfer (1979) have reported that the Na ’ gradient-energized overshoot of valine can be eliminated by pretreatment with PCMBS in brush-border membrane vesicles prepared from rat jejunum. On the other hand, this mercurial failed to inhibit nonenergized valine transport. indicating that it has no direct effect on the carrier. Since PCMBS is known to of biological memincrease the Na + conductance branes, in the vesicle system it could reduce or abolish the Na’ gradient and result in inhibition of solute influx. Membrane vesicles obtained from basal lateral membranes of the rat intestinal epithelium contain, in addition to several Na ’ -dependent systems for alanine transport, a Na ’ -independent system for this substrate (Mircheff clt d.. 1980). These systems are all blocked by PCMBS and significantly inhibited by dithiopyridine and NEM. Uptake of AIB by membrane vesicles from rat thymocytes can be modulated by glutathione; reduced glutathione. for example. inhibits uptake whereas oxidized glutathione increases uptake (Kwock. 1981). Insulin and diamide. both of which induce disultide formation. activate transport. These findings suggest that AIB uptake involves a putative plasma membrane sulfhydryl-containing protein. Kwock has proposed that when such membrane sulfhydryl groups are in the disulfide form. the poten-

Effecters

of amino

acid transport

processes

tial energy barrier is lowered whereas when reduced, the potential energy barrier for transport is increased. Glututhione and amino acid transport Evidence from a number of experiments suggests that glutathione and gamma-glutamyltranspeptidase are involved in the transport of a certain amino acids in animal cell systems. For example, the absorption of basic amino acids by renal proximal tubules in the rat is inhibited by methionine sulfoximine plus ATP plus MgCI, (Craan and Bergeron, 1979). Methionine sulfoximine or ATP or MgCl? alone had no effect, suggesting that the former compound was not competing directly with the basic amino acid carrier system. Since methionine sulfoximine is an effective inhibitor of gamma-glutamylcysteine synthetase in the presence of its cofactors ATP and MgCl,, it is likely that the inhibition is due to suppression of the renewal of intracellular glutathione. Moreover, treatment of human lymphoid cells with the chloroketone glutamine analog, L-2-amino-4-oxo-5-chloropentanoate, leads to a rapid and complete depletion of intracellular glutathione without affecting cell viability and produces an irreversible inhibition of glutamine transport (Novogrodsky et al., 1979). The chloroketone also may cause a decline in cellular glutathione by inhibition of gamma-glutamylcysteine synthetase. Additionally the compounds N-tosylt_-lysine chloromethyl ketone and L-l-tosylamido2-phenylethyl chloromethyl ketone, first studied as inhibitors of protein synthesis, have been demonstrated to inhibit amino acid uptake in Morris hepatomas (Lea and Koch. 1979). There is some evidence that certain chloromethyl ketones may react with gamma-glutamyltranspeptidase, a membrane-bound enzyme that catalyses the transfer of the gammaglutamyl group of glutathione and other gammaglutamyl compounds to a variety of amino acid and peptide acceptors. Thus leucine chloromethyl ketone, when preincubated with sarcoma 37 murine ascites tumor cells. was found to inhibit both the A and L transport systems (Matthews et cd.. 1980). This analog was found to be concentrated by these cells, was a competitive inhibitor of leucine transport and in the labeled form was incorporated into a plasma membrane protein fraction comigrating on a DEAE-cellulose column with gamma-glutamyltranspeptidase activity, suggesting alkylation of the membrane. Leucine was found to partially protect against preincubation inhibition caused by the analog, indicating that leucine chloromethylketone probably interacts with the systems in a manner similar to the interaction of the systems with normal substrates. There is additional evidence provided by the experiments of Kalra et u/. (1981) to suggest that the transpeptidase can mediate the transport of amino acids. They showed that the uptake of glutamic acid and alanine into human erythrocytes could be stimulated by implanting purified hog kidney cortex gamma-glutamyltranspeptidase into the membranes. Uptake of the amino acids by the implanted system was sensitive to inhibitors of transpeptidase activity, e.g. serine-borate, azaserine and antibody to gamma-glutamyltranspeptidase. Sikka and Kalra (1980) have also been successful in being able to demonstrate uptake of glutamic acid, but not of

in animal cell membranes

731

proline, in phospholipid vesicles containing gammaglutamyltranspeptidase. In this system the uptake of glutamic acid was inhibited by serine-borate, azaserine and 6-diazo-5-oxo-t-norleucine, the latter compound also being an inhibitor of the transpeptidase. Stimulation of’ transport by phenazine methosdfute! uscorbate Garcia-Sancho et ul. (1977) have shown that Ehrlich cells pretreated with dinitrophenol and iodoacetate rapidly recover transport of MeAIB on treatment with phenazine methosulfate plus ascorbate (an artificial electron donor system that acts at site II of the respiratory chain in mitochondria); during the recovery period transport is restored before the cells recover their ATP levels and re-establish their ion alkali gradients. Pyruvate, moreover, restores uptake in the absence of ion gradients, an effect which can be inhibited by rotenone. Interestingly, Na ’ independent transport is affected in the same manner. Quinacrine (atebrin), an inhibitor of plasma membrane NADH reductase, also inhibits MeAIB transport even when ATP levels are maintained by pyruvate under normal ion gradient conditions. These workers have proposed that amino acid transport can be energized by reducing equivalents (NADH) which may reach the plasma membrane from the mitochondrion when the energy source is pyruvate. Furthermore, Ohsawa et ul. (1980) found that phenazine methosulfatejascorbate enhances amino acid transport by plasma membrane vesicles of Ehrlich cells from which mitochondria are absent, suggesting more definitively that this electron donor system can serve as a donor to redox activity at the plasma membrane. The phenazine methosulfate/ascorbate system has been shown by Yamamoto and Kawasaki (1981) to stimulate AIB uptake into Ehrlich cells by an increase in maximal flux with no change in apparent K,,, under conditions in which glycolysis and mitochondrial electron transport were blocked by iodoacetate and KCN and the cellular ATP concentration was maintained below 0.1 mM. Ascorbate could be replaced by NADH but not by NADPH. Proton conductors did not affect this stimulation, but the stimulatory phenomenon required the presence of a Na gradient and was accompanied by an increase in Na i influx. Quinacrine inhibited both the enhanced AIB uptake and that of Na + influx. This effect was seen only with Na+-dependent substrates but not with leucine or threonine, in contrast to the results of Garcia-Sancho et ul. (1977). In comparison to the interpretations given above, Valdeolmillos et al. (1982) have proposed that phenazine methosulfate/ascorbate stimulates Na + dependent transport of AIB in the Ehrlich cell by activation of a Ca’+-dependent K + channel which increases membrane permeability to K ‘, resulting in membrane hyperolarization. This effect is prevented by quinine, which is a known inhibitor of the Ca’+-dependent channel in this cell. The increase in the electrochemical gradient for Na’ that results from the membrane hypolarization stimulates amino acid uptake through the coupled Na +-dependent A system.

732

JOSEPHLERNER

.Effticts of anaesthetics Anaesthetics have been found to either inhibit, stimulate or have no effect on amino acid transport. A supra-anaesthetic concentration of methohexitone increased the uptake of o-aspartate and GABA by rat thalmic slices (Kendall and Minchin, 1982). On the other hand pentobarbital, but not phenobarbital, was found to inhibit the uptake of GABA into mouse astrocytes in primary culture (Hertz and Sastry, 1978). Furthermore, the Na +-dependent, insulinsensitive uptake of AIB by rat soleus muscle has been found to be inhibitable by the local anaesthetic tetracaine (Cooper and Kohn, 1977). However, the uptake of leucine, which is Na+-independent and insulin-insensitive, was found to be unaffected by tetracaine. Moreover uptake of amino acids in rat brain slices was shown to be weakly inhibited, if at all, by the anaesthetics thiopentone, pentobarbitone, methohexitone, hydroxydione, alphaxalone/alphadolone, ketamine. alpha-chloralose and urethane (Minchin, 1981). Taurine uptake in cultured human lymphoblastoid cells is strongly inhibited in an uncompetitive fashion by the psychoactive drugs and membrane stabilizers chlorpromazine and imipramine (Tallan et al., 1983). Tetracaine and chlorpromazine have also been shown to suppress AIB uptake in rat soleus muscle (Cooper and Kohn, 1980). Membrane stabilizing drugs such as local anaesthetics and barbiturates affect Na + conductance (Kohn and Watt, 1980) and may specifically increase membrane permeability at low concentrations. At high concentrations amino acid movement across the plasma membrane is associated with a loss of cell K + and a rise in cell Na + , suggesting a possible loss of membrane integrity. According to Kohn and Watt (1980) the heterogeneous effects observed among the different classes of effecters, as well as between the different substrates. could reflect specific interaction with those regions of the membrane where specific lipids are associated with carrier proteins; presumably it is less probable that such effects could result from a general change in lipid bilayer structure.

Hsu et a/. (1982) showed that trypsin treatment of isolated rat renal brush-border membrane vesicles decreased the maximal velocity of the high-affinity transport systems of leucine and proline with little effect on the low-affinity systems of these substrates. Ultrastructural studies on the effects of trypsin indicated that surface protein was removed from the microvillar membrane, although the treatment appears to leave the vesicular membrane intact since no alkaline phosphatase activity is released. Such vesicles are osmotically normal and show a loss in leucine aminopeptidase activity which parallels the loss in uptake activity of leucine and proline. _E@cts qf‘polyanions Treatment of Ehrlich cells with calf thymus DNA inhibits glycine uptake, an effect which can be prevented by pretreating the DNA with DNase, suggesting that the inhibition is not caused by nucleotides (Johnson and Johnstone, 1981). Similar effects

have been reported for added dextran sulfate and heparin, substances with anionic characteristics. Treating trypsinized cells with DNase reverses a similar inhibition of transport caused by trypsin exposure. While trypsin has been demonstrated to lower cellular ATP levels and abolish cation gradients, vesicles prepared from trypsin-treated cells retain essentially normal uptake in the presence of ion gradients. These workers have hypothesized that such compounds may interact with membranes through electrostatic interactions with zwitterionic phospholipids and proteins; furthermore, Ca’+ may be involved in the formation of cross-links. Alternatively, polyphosphates may alter lateral mobility of membrane glycoproteins. The polyanions appear to remove membrane-bound Ca*+ which results in a loss of cell components such as K +. Addition of serum plus Ca*+ restores transport activity, suggesting that protein-bound Ca’+ may be readily accessible to the cell surface for restoration. .Fflects of pol.ycations Kessler et al. (1978) have reported that the uptake of amino acids by rat intestine from animals that had been treated for 8 days with neomycin or its Nmethyl and N-acetylated derivatives was greater than uptake in controls. The effects of neomycin were reproduced by its N-methyl derivative, which does not have antibiotic properties. Both substances have similar polycationic properties. No effect was seen with the N-acetylated derivative of neomycin which is devoid of polycationic properties. These results suggest that the effects may be related to the polycationic properties of neomycin and can be compared with the inhibitory actions of polyanions discussed elsewhere. l$ects

qf’ phloretin

and naringenin

Phloretin, the aglucon of the glycoside phlorhizin. has been found by Rosenberg (1979) to be a potent competitive inhibitor of leucine transport in the human erythrocyte with an inhibition constant similar to the dissociation constant for the high-affinity binding of phloretin to protein binding sites on the human erythrocyte membrane. These results suggest that the inhibition of leucine transport is the result of an interaction between the aglucone and membrane proteins and that these proteins play a role in the transport of leucine. Rosenberg et a/. (1980) have also reported for human red blood cells an aromatic amino acid transport system which has high selectivity for tryptophan. is Na +-independent and is inhibited by phloretin. Robinson (1979) has reported that naringenin (4,5,7-trihydroxy-flavanone) inhibits the influx of amino acids in the guinea-pig small intestine. This compound has a structure that is superficially similar to phloretin although its action on phenylalanine transport in this system is of the fully competitive type, while that of phloretin appears to be of the partially competitive type. .!Zffects o#’a phorbol ester Transport of AIB in cultured bovine lymphocytes has been demonstrated to be rapidly accelerated by the phorbol ester 12-O-tetradecanoylphorbol-

Effecters of amino acid transport processes in animal cell membranes I3-acetate, a potent tumor-promoting agent and comitogen (Kensler rt ul., 1979). The action of the ester on transport is largely insensitive to inhibition of RNA and protein synthesis by actinomycin D and cycloheximide. Retinoic acid, an antagonist of the tumor-promoting and comitogenic actions of such esters, also inhibits the stimulation of uptake by this agent. This ester, furthermore, has been shown to stimulate the capping phenomenon suggesting that it facilitates membrane movement with interchange of components. For example, it may change lipid associations around specific membrane proteins to either release or activate them. Such membrane changes may be responsible for induction of genetic or developmental changes and hence tumor promotion. lTfects

qf‘ retinal, retinul und citumin A

In relatively large pharmacological doses, retinal, retinal and vitamin A inhibit the transport of alanine in rabbit ileum through a reduction in transport affinity (Hajjar et cd., 1977). The action of vitamin A is not direct on the transport system because a lag period is observed before inhibition occurs; vitamin A in this regard has been reported to cause membrane alterations such as changes in stability and structure. _Ejfects qf bile salts Taurocholate and taurodeoxycholate depressed essential amino acid absorption by the human duodenum in perfusion experiments (Dimagno et (II., 1977). The addition of mono-olein or lecithin to a perfusion mixture containing taurocholate restored absorption to normal. Apparently when either lecithin or mono-olein is present less bile salt is available to disrupt the cell surface membrane. Similarly deoxycholate inhibits alanine transport in isolated brush-border membrane vesicles of hamster jejunum (Beesley and Faust, 1980). Deoxycholate but not cholate stimulated Na+ influx and alanine influx in the absence of a Na+ gradient (passive influx). The bile salt appeared to inhibit Na+-coupled transport by enhancing the rate of dissipation of the Na+ gradient. The order of inhibitory potency of different bile salts appears to be correlated roughly with their tendency to partition into the cell membrane. Dihydroxy bile salts were more inhibitory than trihydroxy bile salts and the unconjugated form of a given salt was more inhibitory than the glycine or taurine conjugate. Other properties of the bile salts, perhaps their effectiveness in perturbating lipid-lipid or protein-lipid interactions, may also play a part in such inhibitions. EfJects qf’pyidoxal

5’-phosphate

Tunnicliff (1980) has found that pyridoxal 5’-phosphate can inhibit the uptake of GABA by mouse brain synaptosomes in a competitive fashion. Preincubation with pyridoxal 5’-phosphate, followed by treatment with sodium borohydride, leads to an irreversible inhibition of subsequent GABA uptake. This inhibition was reduced if GABA was present during the preincubation with the inhibitor. Both types of inhibition may result from the interference of pyridoxal S-phosphate with the binding of the amino acid to its carrier. Since pyridoxal 5’-phosphate can form a Schiffs base with lysine residues of proteins,

Tunnicliff has suggested present at the GABA protein.

733

that a lysine residue may be binding site on the carrier

EfSects CI~serum albumin Serum albumin has been demonstrated to stimulate proline uptake by synaptosomes (Raghupathy et a/., 1978). N-terminal peptide fragments of serum albumin do not stimulate such uptake whereas fragments derived from the C-terminal region has stimulatory capacities similar to that of parent albumin. The site responsible for the stimulatory effect has been shown to reside in the sequence region 377-504. Effects qj’ platinum coordination

complexes

The monoaquo derivative of the antineoplastic agent cisplatin (a platinum coordination complex) binds irreversibly to murine lymphoid leukemia cells and immediately inhibits Nat-dependent uptake of AIB and methionine (Scanlon et al., 1983). Inhibition of transport by cisplatin, on the other hand, requires a preincubation time consistent with its conversion to the monoaquo derivative. Inhibition by cisplatin thus may be due to a direct effect of its derivative on the carrier and indirectly to a reduction of the Na ’ gradient, since exposure to cisplatin results in an inhibition of the Na + , K + -ATPase. Effects qf indomethacin The nonsteroidal, anti-inflammatory drug. indomethacin, was found to immediately and reversibly stimulate Na + -independent uptake of 2-aminonorbornane-2-carboxylic acid and to inhibit Na + dependent AIB transport in a delayed fashion in rat hepatoma cells in culture (Bayer et ul., 1980). These effects involve changes in transport V,,,,, alone. Effects qf‘ nicotine Transport of AIB in isolated villi from human term placenta is inhibited by nicotine (Rowe11 and Rama Sastry, 1978). Nicotine may act by causing the massive release of acetylcholine, which is a known inhibitor of transport. Similar inhibitory effects are seen when endogenously released acetylcholine is spared by inhibiting cholinesterase with phospholine. Nicotine, administered to female mice by addition to the drinking water for several weeks before breeding and throughout gestation or by injection. decreased net uptake of AIB into placenta in vitro (Rowe11 and Clark, 1982). Effects of’ ethanol Alanine influx in rabbit jejunum was found to be inhibited by 3% (v/v) ethanol at concentrations which also decreased the electrical potential difference, short-circuit current and inhibited active Na+ transport (Kuo and Shanbour, 1978). This concentration also increased the permeability of the mucosa for Cl-. Absorption of methionine by perfused intestine of the intact rat was inhibited by ethanol; however, a mixture of amino acids protected against this inhibition (Jacobs et a/., 1980). Ethanol in the perfusate was found to enhance the absorption of glutamic acid and histidine when they were administered in a mixture. Ethanol (and acetaldehyde) at a level of 2-20mM inhibit AIB transport by human placenta

734

JOSEPHLERNEK

(Fisher et ul., 1981). Placental AIB transfer was also observed to be lowered in the rat following the ingestion

of 20”,,

ethanol,

indicating

that

maternal

alcohol ingestion during pregnancy may affect fetal nutrient accumulation by a direct effect on the placenta

(Baran,

1982).

REFERENCES Albertsson-Wikland K. and Isaksson 0. (1978) Time course of the effect of growth hormone in ritr-u on amino acid and monosaccharide transport and on protein synthesis in diaphragm of young normal rats. En&crino/o~~~ 102, 1445-1452. Albertsson-Wikland K.. Eden S. and Ahren K. (1980) Analysis of refractoriness to the effects of growth hormone on amino acid transport and protein synthesis in diaphragms of young normal rats. Endoc~rim~/q~~~ 106, 289-305. Al-Saleh E. A. and Wheeler K. P. (1982) Transport of neutral amino acids by human crythrocytcs. B;o&ir~?. hiophrs. Acra 684, 157~171. Alvarado F. and Robinson J. W. L. (lY7Y) A kmetlc study of the interactions between amino acids and monosaccharides at the intestinal brush-border membrane. J. PI~~sioI.,

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295, 457-475.

Aragon M. C.. Gtmenez C.. Mayor F.. Marvizon J. G. and Valdivieso F. (1981) Tyrosine transport bv membrane vesicles isolated from rat brain. Bioc/?im. /&&~.F. Acftr 646, 465-470. Auberger P.. Samson M. and LeCam A. (1983) Effects of growth factors on hormonal stimulation of amino acid transport in primary cultures of rat hepatocytes. Bioclzem. J. 210, 361-366. Bannai S. and Kitamura E. (1982) Adaptive enhancement of cystine and glutamate uptake in human diploid fibroblasts in culture, Biocllinl. hic/p/ly.c. Acrcr 721, I-IO. Bannai S. (1984) Induction of cystine and glutamate transport activity in human fibroblasts by diethyl maleate and other electrophilic agents. J. hid. Chem. 259, 2435-2440. Baran D. T. (1982) Alcohol-induced inhibition of fetal ?5-[ZH]hydroxyvitamin D and alpha-(‘4C]aminoisobutyr*c acid accumulation in the pregnant rat. Endocrino/o?q 111, 1114-1109. Barber E. F.. Handlogten M. E. and Kilberg M. S. (1983) Induction of amino acid transport system A in rat hepatocytes is blocked by tunicamycin. J. hid Chem. 258, 11851-l 1855. Bayer B. M.. Lo T. N. and Beaven M. A. (1980) Antiinflammatory drugs alter amino acid transport in HTC cells. J. hid. Chew. 255, 8784-8790. Beesley R. C. and Faust R. G. (1980) Bile-salt inhibition of sodium ion-coupled D-glucose and L-alanine accumulation by brush border membrane vesicles from hamster jejunum. Biochrm. J. 190, 731-736. Benjamin A. M. and Quastel J. H. (1977) Elects of acetylcholine on potassium-induced changes of GABA and taurine uptakes and release in cerebral cortex slices from the rat. Cut?. J. Ph?siol. Plrarmrc. 55, 356-362. Berteloot A.. Khan A. H. and Ramaswamy K. (1982) K _ - and Na + -gradient-dependent transport of L-phenylalanine by mouse intestinal brush-border membrane vesicles. Biochim. hioph~.s. Actu 691, 321-33 I. Blum J. J. (1982) Effects of cycloheximide and actinomycin D on the amino acid transport system of Tetrah~mmcr. J. w/I. Ph.vsio/. 11 I, IO4- 1 IO. Boge G. and Rigal A. (1981) A chloride requirement for Na+-dependent amino-acid transport by brush-border membrane vesicles isolated from the intestine of a Mediterranean teleost (Boops S+(I). Biochirn. hiophys. Ac[u 649, 455-46 I. Boge G.. Rigal A. and Peres G. (1983) Analysis of two

chloride requirements for sodium-dependent amino acid and glucose transport by intestinal brush-border mcmbrane vesicles of tish. Bioclrin~. hiol~/~~x. .A(.~o 729, 209-Z I 8. Borg 1.. Balcar V. J. and Mandel P. (1979) Effect of cqcl~c nucleotides on high affinity uptake of L-glutamate and taurine in glial and neuroblastoma cells. Brtrirl Rcr. 166, Il3-120. Borghetti A. F.. Tramacere M.. Piedlmonte G. and Gtndottl G. G. (1979) Amino acid transport in chick embryo fibroblasts: Evidence for transcriptional regulation 01 transport following serum addition. J. cc//. f’/?>,.vicd. 9X, 307-314. Borghetti A. F.. Tramacerc M.. Ghiringhelli P.. Sevcrlnt A. and Kay J. E. (1981) Amino acid transport in pig lymphocytes. Enhanced activity of transport system AS? following mitogenic stimulation. Rio&i/i?. hi&l,.\. I(,/0 646, 1 I X-230. Bowery N. G.. Brown D. A.. White R. D. and Yaminl G. (1979) [‘Hjgamma-aminobutyric acid uptake Into ncuroglial cells of rat superior cervical sympathetic ganglia. j.

P/~~~.riol., Loncl. 243, 5 I-74.

Brick R. W. and Ahearn C;. A. (1978) Lvsinc transm~rt across the mucosal border of the perfused midgut in the freshwater shrimp, Ma~rohr~lc~hilm~ ro.tmhr,;y~/. J. ccarp. Ph>.sio/. 124, 169. 179. Busse D. (1978) Transport of L-arginine In brush border vesicles derived from rabbit kidney cortex. Adz\ Hiothen/. Biq?l~~~.\. 191, 55 I 560. Caldwell P. C. and Lea T. J. (1978) Glycine tluxcs in \quld giant axons. J. P/~wio/.. Land. 278, I 25. Chesney R. W.. Gusowski N. and Friedman A. L. (IYXZ) Renal adaptation to altered dietary sulfur ammo acid intake occurs at luminal brushborder membrane. kitbrczl. Inr. 24, 588-594. Christopher C. W.. Nishino H.. Schiller R. M.. Isselbachcr K. J. and Kalckar H. M. (1979) Catabolic control of the enhanced alanine-preferring system for amino acid transport in glucose-starved hamster cells requires protein synthesis. Prw. ncrtn. .AM~. SC,/. L:.S.A. 76, 187X IXXI. Cooke H. J.. Arvanitakis C.. Folscroft J. and Bornstcin J. (I 979) Effect of aspirin on sugar, amino acid and sodium transport in rat jejunum. Am. J. Phy.Ml. 236, E495-~E499. Cooper G. J. and Kohn P. G. (1977) The mechanism of 2-amino-isobutyric acid efflux from rat soleus muscle and its modification by the membrane stabilizer. tctracaInc. J. Phy.riu/.. Land. 271, 2Opmm2 I p. Cooper G. J. and Kohn P. G. ( 1980) Alpha-aminoisobutyrlc acid transport in rat soleus muscle and its modification hq membrane stabilizers and insulin. ./. P/~~~.~/,,/..Lontl. 302, X9-105. Craan A. G. and Bergeron M. (lY79) Nonpartlclpatlon 01 extracellular glutathione in renal transport of diba\ic amino acids. Gun. J. Physiol. Phtrrmw. 57, 116X I 17 I Dali’ asta V., Gazsola G. C. and Guidotti Ci. G. (1978) Adaptive regulation of amino acid transport in cultured avian fibroblasts. Influence of the amino acid composition of the culture media. Bioch//~. hiop/?~.s. .4(,(n 507, 165 174. Derr J. T. and Smith G. L. ( 1980) Regulation of amino acid transport in chicken embryo fihroblasts hq purified multiplication-stimulating activity (MSA). J. w/l. Ph).vird 102, 55-62.

Dimagno E. P., Malagelada J. R. and Go V. L. W. (1077) Effects of bile acids. lecithm, and mono-olein on amino acid absorption from the human duodenum. Proc So. ‘_,-/I. Bid.

Md.

1545, 325 -330.

Dolais-Kitabgi J.. Rcy J. F.. Fehlmann M.. Morm 0. and Freychct P. (1981) Effect of insulin and glucagon on amino acid transport in isolated hepatocytes after partial hepatectomy in the rat. Endo~i&~~~.r 109, 86% X75. Edmondson J. W. and Lumeng L. (1980) Biphasic ctlmulation of amino acid uptake by glucagon in hcpatocyte\ B~oc~/wr~~.hir@~c\. RI,\. Crmrmun. 96. 6 I 6X.

Effecters

of amino

acid transport

Ellory J. C., Jones S. E. M., Rink T. J., Wolowyk M. W. and Young J. D. (1980) The role of sodium in amino acid transport by human erythrocytes. J. Physiol., Lond. 308, 52P-53P. Ellory J. C., Jones S. E. M. and Young J. D. (1981) Cloride-activated sodium-dependent glycine transport in human erythrocytes. J. Physiol., Lond. 310, 22~. Eveloff J.. Field M.. Kinne R. and Murer H. (1980) Sodium-cotransport systems in intestine and kidney of the winter flounder. J. camp. Physiol. 135, 1755182. Fass S. J., Hammerman M. R. and Sacktor B. (1977) Transport of amino acids in renal brush-border membrane-vesicles. J. hiol. Chem. 252, 5833590. Fehlmann M.. LeCam A., Kitabgi P., Rey J. F. and Freychet P. (I 979) Regulation of amino acid transport in the-liver. J. hiol. C/W~I. 254, 401-407. Fehlmann M.. Samson M.. Koch K. S.. Leffert H. L. and Freychet P. (I 981) Amiloride inhibits protein synthesis in isolated rat hepatocytes. LiJe Sci. 28, 12951302. Fisher S. E., Atkinson M., Van Thiel D. H., Rosenblum E., David R. and Holzman 1. (1981) Selective fetal malnutrition: the effect of ethanol and acetaldehyde upon in c’irro uptake of alpha amino isobutyric acid by human placenta. Life Sci. 29, 1283-1288. Freeman M. ‘and Handwerger S. (1982) Ovine placental lactogen stimulates amino acid transport in rat diaphragm. Emlocrbwlo~~~ 110. 2201-2203. Freeman M. and Handwerger S. (1983) Ovine placental lactogen. but not growth hormone, stimulates amino acid transport in fetal rat diaphragm. Endocrinok~gj, 112, 402-404.

Fugelli K. and Rohrs H. (1980) The effect of Na+ and osmolality on the influx and steady state distribution of taurine gamma-aminobutyric acid in flounder (P/litichrh~~sflesus) erythrocytes. c’omp. Biochem. Physiol. 67A, 545-55 I. Cache C. and Vacquier V. D. (1983) Transport of methionine in sea urchin sperm by a neutral amino-acid carrier. Eur. J. Biochem. 133, 341-347. Garcia-Sancho J., Sanchez A., Handlogten M. E. and Christensen A. N. (1977) Unexpected additional mode of energization of amino-acid transport into Ehrlich cells. Proc. nufn. Acad. Sci. U.S.A. 74. 1488-1491. Gazzola G. C.. Dali’ Asta V. and Guidotti G. G. (1981) Adaptive regulation of amino acid transport in cultured human Iibroblasts. J. hiol. Chrm. 256. 3191-3198. Giger 0. and Pariza M. W. (1978) Depression of amino acid transport in cultured rat hepatocytes by purified enterotoxin from Clastridium prrfringens. Biochem. hiophrs. Rrs. Commun. 82, 378383. Giger 0. and Pariza M. W. (1980) Mechanism of action of CIostridium pe~fringms enterotoxin. Effects on membrane permeability and amino acid transport in primary cultures of adult rat hepatocytes. B&him. hiophys. Actu. 595, 264-276.

Giordana B.. Sacchi F. V. and Intestinal amino acid absorption Biochim.

hiophys.

Acta

Hanozet G. M. (1982) in lepidopteran larvae,

692, 81-88.

Goldman I. D., Fyfe M. J.. Bowen D., Loftfield S. and Schafer J. A. (1977) The effect of microtubular inhibitors on transport of alpha-aminoisobutyric acid. Inhibition of uphill transport without changes in transmembrane gradients of Na *. K + , or H . Biochim. hionhvs. Acta 467. ‘ 1x5-191. Goldstein L. and Boyd T. A. (1978) Regulation of betaalanine transport in skate (Ruju erinocar) erythrocytes. Camp. Biochem. Physiol. 6OA, 319-325. Goldstone A. D., Koenig H.. Lu C. Y. and Trout J. J. (1983) Beta-adrenergic stimulation evokes a rapid, Ca’--dependent stimulation of endocytosis. hexose and amino acid transport associated with increased Ca’+ fluxes in mouse kidney cortex. Biochem. hiophys. Res. Commun.

114, 9 13-92 1.

processes

in animal

cell membranes

135

Gordon P. B. and Rubin M. S. (1982) Membrane transport during erythroid differentiation. J. Mrmh. Biol. 64, I l-21. Grunfeld C. and Jones D. S. (1983) Insulin-stimulated methylaminoisobutyric acid uptake in 3T3-LI fibroblasts and fat cells. Endocrinology 113, 1763-l 770. Habibi H., Ince B. W. and Matty A. J. (1983) Effects of 17-alpha-methyltestosterone and l7-beta-oestradiol on intestinal transport and absorption of L-(C’4)-leucine in l,itro in rainbow trout (S&IO guirdrwrii). J. c’omp. PhJsiol. 151, 247-252. Hajjar J. J., Nassar K. and Bikhazi A. (1977) Effect of vitamin A on in vitro alanine transport in isolated rabbit ileum. Cotp. Biochem. Ph~~.~io/. %A, 28 l-284. Hammerman M. R. and Sacktor B. (1978) Transport of beta-alanine in renal brush-border membrane vesicles. Biochim.

hiophys.

Actor 509, 338-347.

Hammerman M. R. and Sacktor B. (I 982) Na + -dependent transport of glycine in renal brush border membrane vesicles. Evidence for a single specific transport system. Biochim. hiophys. Acts 686, 1899196. Handlogten M. E. and Kilberg M. S. (1982) Transport system A is not responsive to hormonal stimulation in primary cultures of fetal rat hepatocytes. Biochrm. hiophw Res. Commun. 108, 1113~1119. Hanhlogten M. E. and Kilberg M. S. (1984) Induction and decay of amino acid transport in the liver. Turnover of transport activity in isolated hepatocytes after stimulation by diabetes or glucagon. J. hiol. Chem. 259, 3519-3535. Heaton J. H. and Gelehrter T. D. (1980) Regulation of insulin responsiveness in rat hepatoma cells. B~odxw~. hioplq~. Rex. Common. 92, 7955802. Heaton J. H.. Schilling E. E.. Gelehrter T. D., Rechlcr M. M., Spencer C. J. and Nissley S. P. (1980) Induction of tyrosine aminotransferase and amino acid transport in rat hepatoma cells by insulin and the insulin-like growth factor, multiplication-stimulating activity. Bioc~hrrn. hioph.w Actu 632, 192-203. Heindel J. J. and Riggs T. R. (1978) Amino acid transport in vitamin B,,-deficient rats: dependence on growth hormone supply. Am. J. Ph>,.sio/. 235, E316-E323. Heinz A., Jackson J. W.. Richey B. E.. Sachs G. and Schafer J. A. (1981) Amino acid active transport and stimulation by substrates in the absence of a Na ’ clcctrochemical potential gradient. J. Mrmh. Biol. 62, 1499160. Hertz L. and Sastry B. R. (1978) Inhibition of gammaaminobutyric acid uptake into astrocytes bv pcntobarbital. Con. J. Phys’iol. Phcrrmcrc. 56, 1083-l I%?. Hilden S. A. and Sacktor B. (1981) L-Argininc uptake into renal brush-border membrane vesicles. Arc/n Biochem. Biophys.

210, 289-297.

Hjelle J. T.. Baird-Lambert J.. Cardinale G.. Spector S. and Udenfriend S. (1978) Isolated microvessels: The blood-brain barrier in t,itro. Proc. notn. Aud. Sri. L’.S..‘l. 75, 45444548.

Hsu B. Y. L.. Corcoran S. M., Marshall C. M. and Segal S. (1982) The effect on amino acid transport of trypsin treatment of rat renal brush-border membranes. Biochim. hiophys. Actrr 689, l81ll93. Huxtable R. and Chubb J. (1977) Adrenergic stimulation of taurine transport by the heart. Science N. Y. 198,409941 I Ingham L. and Arme C. (1977) Intestinal absorption of amino acids by rainbow trout. Sulmo zoirdnerii (Richardson). J. co&p. Phrsiol. 117, 3233334. ” Iwamoto Y. and Williams J: A. (1980) Inhibition of nancreatic alpha-aminoisobutyric acid uptake by cholecystokinin and other secretagogues. Am. J. Plr~siol. 238, G440-G444.

Iwamoto Y., Nakamura R. and Akanuma Y. (1983) Elfects of porcine gastrin-releasing peptide on amylasc release. 2-deoxyglucose uptake. and alpha-aminoisobutyric acid uptake in mouse pancreatic acini. Endowirtolog~~ 113, 21062112. Jacobs F. A., Crandall J. C. and Fabel C. B. (I 980) An c&t

736

JOSEPH

of ethanol on the bidirectional intestinal llux of amino acids. Nuw. Rep. Int. 21, 3977403. Jean T., Ripoche P. and Poujeol P. (1983) A sodiumindependent mechanism for L-arginine uptake by rat renal brush-border membrane vesicles. Mrmh. Riochem. 5, lll8. Johnson P. A. and Johnstone R. M. (1981) Alterattons in membrane permeability with trypsin treatment. Cwt. J. Biochem. 59, 6688675. Johnstone R. M. (1978) The basic asymmetry of Na +-dependent glycine transport in Ehrlich cells. Bioc/rim. hiophjx. Acts 512, 199-213. Kalra V. K.. Sikka S. C. and Sethi G. S. (1981) Transport of amino acids in gamma-glutamyl transpeptidaseimplanted human erythrocytes. J. hid. Chem. 256, 556775571. Kanner B. I. (197X) Active transport of gammaaminobutyric acid by membrane vesicles isolated from rat brain. Biochemkrry 17, 1207~121 1. Kanner B. I. and Sharon I. (1978) Active transport of t-glutamate by membrane vesicles isolated from rat brain. Biochemis/r~~

17, 3949-3953.

Kanner B. I. and Sharon I. (19X0) Active transport of t-proline by membrane vesicles isolated from rat brain. Biochim. hiophrs. Actrr 600, I85- 194. Kelley D. S. and Potter V. R. (lY78) Regulation of amino acid transport systems by amino acid depletion and supplementation in monolayer cultures of rat hepatocytes. J. hid. Chem. 253, 9009-9017. Kelley D. S. and Potter V. R. (1979) Repression. derepression, transinhibition. and trans-stimulation of amino acid transport in rat hepatocytes and four rat hepatoma cell lines in culture. J. hid. Chem. 254, 669 l-6697. Kelley D. S., Evanson T. and Potter V. R. (1980) Calciumdependent hormonal regulation of amino acid transport and cyclic AMP accumulation in rat hepatocytc monolayer cultures. Proc. nrrrn. A~ci. Sci. Y.S.A. 77, 5953-5957. Kendall T. J. G. and Minchin M. C. W. (19X2)The effects of anaesthetics on the uptake and release of amino acid neurotransmitters in thalamic slices. Br. J. Phrwmrrc~. 75, 219-227. Kensler T. W.. Wertz P. W. and Mueller G. C. (1979) Inhibition of phorbol ester-accelerated amino acid transport in bovine lymphocytes. Biochim. hiophjx. ilcrcc 585, 43- 52.

Kessler J. I.. Sehgdl A. K. and Turcotte R. (1978) Effect of neomycin on amino acid uptake and on synthesis and release of lipoproteins by rat intestine. Cwt. J. Ph~siol. Phurmuc.

56, 420-427.

Kilberg M. S., Handlogten M. E. and Christensen H. N. (1980) Characteristics of an amino acid transport system in rat liver for glutamine. asparagine. histidine, and closely related analogs. J. hid. C/tern. 255, 401 I-4019. Kippen I., Hirayama B., Klingenberg J. R. and Wright E. M. (1979) Effects of dibutyryl cyclic AMP on the transport of alpha-methyl-o-glucoside and alphaaminoisobutyric acid in separated tubules and brushborder membranes from rabbit kidney. Biochim. hiophys. Acra 558,

126-135.

Kippen I., Klinenberg J. R. and Wright E. M. (1980) Effects of metabolic intermediates on sugar and amino acid uptake in rabbit renal tubules and brush border membranes. J. Physiol.. Land 304, 373-387. Klekamp M.. Prahlad K. V. and Hampel A. (1982) Inhibition by sodium of sodium-independent amino acid transport. Camp. Biochem. Phy.d. 72A, 143-147. Koenig H., Goldstone A. D. and Lu C. Y. (1983) Betaadrenergic stimulation of Ca’ * fluxes, endocytosis. hexose transport, and amino acid transport in mouse kidney cortex is mediated by polyaminc synthesis. Proc. rwtn. Acud. .%i. 80, 72 1O-72 14.

L.ERNER Kohn P. G. and Watt D. E. (1980) Membrane stabilizers and cycloleucine efflux in rat soleus muscle. J. Ph,,sio/., Lend. 306, 44P. Kuo Y. J. and Shanbour L. L. ( 1978) Effects of ethanol on sodium, 3-O-methyl glucose. and t.-alanine transport in the jejunum. Dig. Dis. 23, 51-55. Kwock L. (1981) Sulthydryl group involvement in the modulation of neutral amino acid transport in thymocyte membrane vesicles. J. c,e//. Phy.yio/. 106, 279-282. Lea M. A. and Koch M. R. (1979) Selective effects of two chloromethyl ketones on amino acid and phosphate uptake in rat liver and tumors. .J. nuts. Ccmcw In.sr. 62, lXlll85. LeCam A. and Frechet P. (lY78) Etfcct of catecholamtnes on amino acid transport in isolated rat hepatocytcs. Edocrinolo~y 102, 379. 385. LeCam A., A., Maxlield F.. Willingham M. and Pastan I. (1979) Insulin stimulation of amino acid transport in isolated rat hepatocytes is independent of hormone internalization. Biochem. hiop/z~,.s. Res. Commun. 88, 87388 I. Lee S. H. and Pritchard J. B. (1983) Proton-coupled t.-lysine uptake by renal brush-border membrane vesicles from mullet (Muxil cephu/u.c ). J. Memh. Bid. 75, I7lll 78. Lepley P. R. and Mukkada A. J. (1983) Characteristics of an uptake system for alpha-aminoisobutyric acid in Leishmrrnirr fropic,rr promastigotes. _/. Plo/o:o0/. 30, 41-46. Lerner J. ( 1978) A Rev/w of Amirro Ac,id Trcrn.vpor/ Prw wsse’s in Animd Cells cmd Tissues. University of Maine at Ororio Press, Orono. Litteria M. (1977) The effects of neonatal androgenization on the in riro transport of alpha-aminoisobutyric acid into specific regions of the rat brain. Brtrin Rev. 132, 2877299. Lucke H.. Haase W. and Murer H. (1977) Amino acid transport in brush-border membrane vesicles isolated from human small intestine. Biochem. J. 168, 529 532. Lussier P. E.. Podesta R. B. and Mettrick D. F. ( 1979) H~menolepis diminutcr : Na +-independent components of neutral amino acid transport. J. P~ru.si/o/. 65, X42~X48. Mak W. W. N. and Pitot H. C. (1981) Microfilament accumulation and the transport of amino acids and glucose in adult rat hepatocytes cultured on collagen gel/nylon mesh. Biodzcm. hiopl~~s. Rex Commwz. 98, 203-2 IO. Mann G. E.. Wilson S. M. and Yudilevich D. L. (1983) Characteristics of a high-alhnity t.-lysine transport system in the basolaterdl membrane of cat salivary epithelium. J. Ph~xiol..

Land.

340, 32P.

Marchand-Brustel Y. L.. Moutard N. and Freychcr I’. (I 982) Aminoisobutyric acid transport in soleus muscles of lean and gold thioglucose-obese mice. A/n. J. Phrcid. 243, E74-E7Y. Martin M. S. and Pohl S. L. (1979) Insulin-induced insuhn resistance of alpha-aminoisobutyric acid transport in cultured human skin tibroblasts. ./. h/o/. C’hrw. 254, 99769978. Matthews R. H.. Lewis N. J., Hes J.. Im J. H. and Milo G. E. (1980) Inhibition of S37 ascites cell amino acid transport systems by alpha-chloromethylketone analogs. Biochim.

hioph~x.

Acto

601, 640~-653.

Mayor F.. Marvizon J. G.. Ardgon M. C.. Gimcncr C‘. and Valdivieso F. (1981) Glycinc transport into plasmamembrane vesicles derived from rat brain synaptosome\. Biochem.

J. 198, 535-541.

McDonald R. A. and Gelehrtcr T. D. (1977) Glucocorticoid inhibition of amino acid transport in rat hepatoma cells. Biochem. hic>p/ly.v. Res. Commun. 78, 1304 I3 IO. McGahan M. C. (1981) Alpha-aminoisobutyric acid etllux from the cornea of the toad. BU/O murin~.t. .I P/z~~\/td. Lord.

315, 253.-266.

McGuire J. C. and Greene L. A. (1979) Rapid stimulatton by nerve growth factor of amino acid uptake by clonal

Effecters

of amino

acid transport

processes

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Effecters

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Young J. D.. Jones S. E. M. and Ellory J. C. (1981) Amino acid transport via the red cell anion transport system. Biochim. hioplrys. Acta 645, 157-160. Yuli I., Incerpi S., Luly P. and Shinitzky M. (1982) Insulin

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of glucose and amino acid transport in mouse with elevated membrane microviscosity. E.uperientiu 38, I1 14-l 115.