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Methionine uptake by larval and adult Schistosoma mansoni
METI-IIONINE
UPTAKE BY LARVAL AND SCHISTOSOMA ML4 NSONZ L.
Molten0
Institute,
H.
ADULT
CHAPPELL*
Downing
Street,
( Received22 nugrrst
Cambridge,
England
1973)
L. H. 1974. Methionine uptake by larval and adult Sehisfosoma mansoni. Internafional Journalfor Parasitology 4: 361-369. The uptake of methyl-laC-methionine by schistosomula, 21 day-old and adult Schistosoma mansoni in vitro has been investigated. Ligation of the adult pharynx and comparison between the kinetics of uptake of schistosomula and adults suggests that methionine is absorbed, in the system described, primarily via the tegument. All stages of the parasite examined absorb methionine by, at least, two kinetically distinguishable systems, one which is saturable and a second which appears to be simple diffusion. The saturable system has relatively low specificity for amino acids but shows no affinity for other classes of compounds. The effects of pH, temperature, metabolic inhibitors, and sodium ion concentration have been examined. The results are discussed with reference to schistosome gut function and also to the differential response to chemotherapy according to age of infection. Abstract-CHAPPELL
INDEX uptake;
KEY WORDS: Schistosoma mansoni: schistosomes; transport; tegument.
INTRODUCTION MECHANISMS
* Present address: Aberdeen
Dept. of Zoology,
methionine;
amino
acids;
schistosomes to antimony chemotherapy depending upon the age of the infection: one-week-old worms and adults are more susceptible to antimony therapy than are three-week-old worms. An explanation for this might be that there is a variation in the degree of permeability of the tegument to solutes according to worm age. It is the aim of the work described below to determine whether such a permeability difference does exist using the uptake of a labelled amino acid by various developmental stages of S. n7ar7soni ir7 vitro.
by which schistosomes obtain amino acids from the definitive host are not completely understood. While the worm possesses an alimentary tract there is no evidence of complete proteolysis within the gut (Grant & Senft, 1971) and it is not known whether the absorption of peptides originating from the incomplete hydrolysis of globin is carried out by the mucosa; neither is there any evidence of pinocytotic absorption of whole globin (Dike, 1971). Support for the hypothesis that free amino acids are absorbed from the host blood via the tegument comes from the work of Senft (1968), who studied the distribution of ‘T-proline in adult S. mar7soni using autoradiography, and from the work of Clegg (1965) who observed that schistosomula of S. mansor7i in culture required amino acid supplements for optimum growth during the first five days of cultivation, during which time the gut of the schistosomulum is thought to be in closure. Additionally Clegg (1972) pointed out that the dramatic proliferation of the tegument and the resulting increase in surface area that occurs during development in the final host are consistent with a tegumental absorption function. Chappell & Coles (unpublished data) have corroborated the earlier findings of Schubert (1948), Standen (1955), Bruce at al. (1962) and Stohler & Frey (1963) that there is a differential response of THE
schistosomula;
MATERIALS I. Maintenance
AND
METHODS
of the parasite and collection qf material
The Wellcome sti-ain of S. mansoni was maintained in the laboratory in Biomphaluria glabrafa at 25’C and in Hough strain albino male mice (J. Hough, Lydgate, Lanes.). Mice weighing 15-25 g were infected by the abdominal ring technique after Nembutal anaesthesia using freshly collected cercariae. Three developmental stages of the parasite were used in this study; adult worms and three-week-old worms were obtained from infected mice by perfusion with cold 0.9 y0 NaCl containing I .5% trisodium citrate, and O-day-old schistosomula were obtained in vitro by cercarial penetration through excised mouse skin using the technique of Clegg & Smithers (1972). Adult worms were harvested at eight weeks postinfection from an exposure of 200 cercariae/mouse; this gave a mean recovery of 50 per cent. Three-week worms were harvested from mice given an infection dose of 2000 cercariae/mouse, with a yield of between I5 and 30 per cent at 21 days post-infection. Zero-day schistosomula were collected, following skin penetration, by gentle centrifugation; 200&4000 cercariae were used per
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At39 2TN. 361
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penetration tube with a penetration time of 3 h. The use of eight
penetration
tubes afforded
a recovery
of up to
20,000 schistosomula, but a mean yield of only 28 per cent was obtained. All cultures of schistosomula were examined before experimentation and those contaminated with an excess of 5 per cent cercariae were discarded.
2. Amino acid uplake
procedwes
Worms were collected as outlined above and were washed several times in 25 mM Krebs-Ringer buffered with trissmaleic acid (KRT of Read e/ al., 1963), containing I mg/ml glucose, at pH 7.4 and at room temperature. Adult schistosomes were partially separated into the sexes by shaking in KRT at 37°C for 5-10 min, final separation being obtained manually. At no time were adult worms of the particular sex required actually handled during separation as it was not possible to separate worms manually without the attendant risk of damaging the tegument. Consequently one of the sexes was always discarded after separation allowing, therefore, the use of fine forceps to aid final separation. No attempt was made to sex or separate male and female 3-week-old worms. Worms collected on any one day were grouped together and then randomly redistributed into tubes where they were maintained in a preincubation medium of KRTglucose. Preincubation in all cases was carried out at 37’C for 15 min. For studies on amino acid uptake the following sample sizes were chosen: IO-1 5 adult males (0.6-0.9 mg dry wt), 15-20 adult females (0.3-0.5 mg dry wt), 50-100 three-week worms (0.3-0.6 mg dry wt) and 2000 schistosomula (I.8 mg dry wt). Experimental incubations were carried out at 37’C unless otherwise specified with worms suspended in KRT-glucose plus methyl-‘“C-methionine and any other additions as required, to give a final volume of 2 ml for each sample. All incubations were under air unless otherwise stated. Incubations with adult or three week worms were terminated by the addition of excess, cold (4 C) KRT and by cooling the tubes in ice. Four additional washes of IO ml each wash with KRT were sufficient to remove any methionine-“C. Schistosomula were non-absorbed treated in a similar manner but were washed on an 8 Mm cellulose filter with four volumes of KRT containing 40 mM stable methionine. Filter controls were used in the latter procedure at all times to allow for correction to include any non-absorbed methionine-“C that was adsorbed to the filter. Methionine-“C absorbed by the worms was extracted with 70% ethanol for 24 h at room temperature and was quantitatively determined by liquid scintillation counting on a Packard Tri-Carb counter employing a liquid scintillator (hat contained per litre, 700 ml toluene, 300 ml Triton-X 100, 5 g PPO (2,5-diphenyloxarole) and 0.5 g POPOP (I ,4-di[2-(phenyloxazolyl)]-benzene). Counting efficiency was estimated to be 85 per cent. With adult and three-week worms the dry wt of each sample was determined by drying overnight at 95 C after ethanolic extraction, whereas. for schistosomula, the numbers in each sample were determined and their dry weights estimated by reference to a predetermined scale. This was necessitated by the filtration-washing procedure used for schistosomula; the washed larvae were counted directly on the filter paper and were not recoverable for weight determinations.
According to the supplier methyl-“C-methionine in aqueous solution decomposes at a rate of 10-15’2; pel month even at -2O’C. It was thus necessary first, to purchase stocks in small quantities at regular intervals and secondly, to determine the radiochemical purity of existing stock solutions routinely. Purity was determined either by high voltage electrophoresis (2500 V for I h with DH I.9 formic/acetic buffer) and liauid scintillation couniing or by scanning two dimensional thin layer plates [cellulose MN 300HR; first solvent, butanol/acetic acid/water (6:1 :5 v/v); second solvent, butanol/acetone/ diethylamine/water(lO:lO:7:5 v/v)] with a Geiger-Miillet tube. 4. Amino
acid determiuutiotl
The free amino acids of S. marwoni were determined by routine automatic analysis (Technicon or Locarte analysers) of ethanolic (70%) extracts of fresh tissue. Confirmation of peak identity was made by co-chromatography. 5. Chemic,al.s Methyl-“C-methionine (> 50 mCi/mmol) was obtained from the Radiochemical Centre and was made up in aqueous solution containing stable methionine carrier to IO mM (0.5 pCi/umole). All other chemicals used were
of analytical grade where commercially available.
RESULTS
I. Fwa unlitw acids of S. mansoni The free amino acids of adult and larval S. manswri are shown in Table I. The large standard error given is thought to represent genuine variation in amino acid levels rather than any technical shortcomings. The data for cercarial amino acids have been omitted as variation between replicate samples was unacceptably large despite the precaution of freezing in liquid nitrogen immediately after additiolt of ethanol and extraction in the cold to prevent autolysis. Even so the cercarial free amino acid pool was considerably higher than that of any other stage of the life cycle and there appears to be a trend for a decrease in free amino acid content with age. Cysteic acid was present in all stages in relatively large amounts but it could not be separated from an unknown contaminating peak so that no quantity is given in Table I.
It was initially necessary to determine the relative contributions of the gut and of the tegument in the uptake of methionine under the i/l vitro conditions described. Adult male worms were selected for ligation because of their size and the ease with which a ligature could be applied. The uptake of methionine in worms with pharyngeal ligation (i.e. the region between oral and ventral suckers) was compared with that of normal worms. A number of ligatures
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Methionine
TABLE
Amino
acid
Cysteic
acid
1~+ 4.4 7.2 IO.6 22.8 16.7 4.1 33.2 0 9.8 3.2 6.2 17.8 I.2 4.4 2.2 2.1 18.9 5.9 3.7
Aspartate Threonine Serine Glutamate Proline Glycine Alanine Cystine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine Histidine Ornithine Lysine Tryptophan Arginine Total
II
pool
I-THE
Schistosomula
uptake
FREE AMINO
in schistosomes ACIDS
21 day worms
-t + $ i It i_ + :i
t
0.6 0.7 I.2 7.4 3.2 0.6 8.1
$ + + + * i i $ $ + $
1.1 0.4 0.7 4.6 0.1 0.3 0.2 0.1 2.1 0.6 0.6
177.1 5
6.5 6.4 9.8 21.0 6.2 8.2 19.5 0.2 2.5 0.7 I.3 2.8 I.0 I.0 0.8 I.8 6.0 0 I.7
OF S.
363
mansoni
Adult
males
Adult
L I.7 + 0.8 + I.8 i 5.7 + 2.4 + 2.2 + 2.5 .$ 0 i 0.1 + 0 $ 0.1 $ 0.3 f 0.1 + 0.1 + 0.2 i 0.1 1~ 0.7 + 0.7
95.6 6
2.4 2.1 3.3 8.7 2.2 3.4 9.2 I.4 I.0 0.7 0.6 I.3 0.9 0.7 0.9 0 2.0 0 0.1
f f I i + $ + + _i + i 11 i + +
0.3 0.1 0.1 0.5 0.4 0.5 I.6 0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.1
+
0.1
6.5 3.0 3.0 4.6 3.0 3.7 5.3 3.9 2.2 0.8 I.2 2.2 I.0 0.2 0.9 0 3.8 0 0
_f 0
40.8 6
3. Clmruct~ristics
of’ metl~ionine
absorption
To establish conditions for unidirectional flux of a transported moiety it is necessary to determine the duration of the initial rate of influx. This was found to extend to 5 min, shown in Fig. I and the convenient incubation time of 2 min was therefore chosen for subsequent experiments.
$ + f $ i + 1 _i 1 f _t +
0.6 0.4 0.6 0.6 0.4 0.6 0.5 0.1 0.3 0 0.3 0.4 0.1
t 0 1~ 0.2
t
0.2
45.3 6
Values are umoles/g dry wt (mean i S.E. of 5 or 6 replicate samples). ~presence of cysteic acid unresolved from an adjacent, unidentified peak.
was tested the most suitable being human hair. In incubations from 30 set to 30 min with a range of external methionine concentrations from 0.1 to 5.0 mM no difference in uptake was recorded between the two groups. Evans blue was used routinely as an indicator of damage to the tegument resulting from ligation (Halton & Arme, 1971) and, with practice and careful manipulation, no tissue damage was effected during ligation. No obvious method was available for determining the efficacy of the ligature as a barrier to solutes. However, physostigmine at IO-“M may be used to induce vomiting in schistosomes (Bueding, 1952) and this was administered post-incubation, but, again, revealed no difference in uptake between ligated and normal worms. While the above cannot be regarded as unequivocal evidence for the lack of alimentary involvement in the absorption of methionine it is considered that, in the short-term incubations employed primarily in this study, the role of the gut is minimal. Consequently, in subsequent experiments ligation of adult worms was not used and it would be clearly impractical to attempt ligation of three week worms and impossible with schistosomula.
females
i
I I
I
Refers to the
I
I
20
IO Incubation
time,
min
FIN. I.
Time curve for methionine uptake in adult male S. mansoni. Each point is the mean -~ S.E. of 8 replicate samples. Methionine concentration 7 0.5 mM.
The curves for methionine uptake in larval and adult schistosomes as a function of methionine concentration in the medium (Fig. 2) tend towards saturation and are clearly not linear over the concentration range examined. Quantitatively schistosomula absorb methionine at a rate one order of magnitude less than older worms, while female adults absorb methionine somewhat more rapidly than males. Extension of the concentration of methionine in the medium 50 mM reveals an apparent biphasic mode of uptake (Fig. 3). At low concentrations uptake occurs by a system which is apparently saturable while at higher concentrations uptake
364
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CHAPPFLI.
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a mediated transport system with specific binding affinity and transporting capability for methionine and, perhaps, related amino acids. Were this hypothesis valid it should be possible to inhibit methionine transport by the addition of generically similar molecules, in this case neutral amino acids. The effects of the addition of 25 individual amino acids on the rate of methionine transport were examined; the potentially inhibitory acids were included individually in a forty-fold excess (20 mM) of methionine (0.5 mM). The results of these inhibitions are summarised in Table 2, in which the added amino acids are arranged in ranking order of inhibitory activity for female adult schistosomes. Almost all amino acids tested possessed some inhibitory function, the least active being those with the greatest spatial separation of the a.amino group and the a-carboxyl group (i.e. p-and y-amino acids) and the molecular species that lack one of these groups (i.e. urea, the imino acids) and the acidic amino acids. The overall pattern of methionine inhibition by other amino acids in adult worms, prepatent worms and schistosomula was essentially similar as judged by reference to selected examples (Table 2). A number of the above inhibitions were examined by the method of Lineweaver & Burk (1934) to determine their precise nature. All amino acid inhibitions examined in this way (thre, leu, phe, arg, orn, his and lys) were fully competitive inhibitors of methionine uptake. The inhibitor constants (K,) for basic ammo acids were larger than those for neutral acids (e.g. Ki’“’ _ 5.4; KiPhe : 4.5; Kyrg 47,s; 36.3) suggesting much lower affinity of K(‘Y‘ basics for the methionine system than shown by neutrals. Preliminary experiments using combinations of inhibitory amino acids representing different classes suggest that methionine may enter via two distinct systems (Table 3), a neutral system and a basic system, but more detailed examination would be required to establish this. The specificity of the methionine transporting process was examined addit ionally by testing representatives of other classes of compounds as potential inhibitors of methionine uptake. These included, (I) organic acids (citrate, fumarate. lactate, oxalacetate, pyruvate and succinate); (2) carbohydrates (arabinose 2-deoxygtucose, fructose, glucose, malose, mannose, rhamnose, ribose, sorbose, sucrose, trehalose, and xylose), and (3) free fatty acids (acetate, propionate and butyrate). None of these was inhibitory. represent
Methionine
concentration,
mM
FIG. 2. Methionine uptake as a function of n~ethiollil~e concentration in the medium in larval and adult S. n~ansoni. Each point is the mean of, at least, 10 replicate samples. Incubation time 2 min.
Totol 4
uptoka/*
I
60-
I
IO
20 Methlontne
30
“0
40
concentrotfon,
rr!d
Fro
3. Extended concentration curve of methionine uptake in adult male S. ~u~?s~/?;.Each point is the mean of 8 replicate samples. incubation time 2 min.
occurs by a second, apparently non-saturable process. Assuming this second system to be Fickian diffusion it is permissible to distinguish between the two systems graphically as in Fig. 3, to show the relative contributions of each system over a range of methionine concentrations. The alternative possibility tnat the linear component of methionine uptake might represent a second saturable process experimentwhose If,,, had not been encountered ally has not been discounted and this is discussed later. 4. Inlribitiott ~~‘nx~tlric~tlitrc~ uptake Reference to the above data suggests that the saturable component of methionine uptake may
5. Metaboiic itrhibitors To evaluate the possible dependence of methionine transport on energy metabolism the effects of a variety of inhibitors of aerobic and anaerobic metabolism were investigated. None of those tested (2,4-dinitrophenol, cyanide, azide, iodoacetate, oua-
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4. 1974 TABLE
Methionine 2-PERCENTAGE
INHIBITION
Amino acid inhibitor
uptake in schistosomes
OF METHIONINE
UPTAKE
IN s.
365
mUiWOtli
BY OTHER
AMINO ACIDS
Adult females
Adult males
21 day worms
Schistosomula
100 16.7 17.0 17.5 18.8 20.2 24.5 26.5 26.6 27.0 27.5 28.0 29.2 32.1 32.6 39.3 43.2 51.0 64.3 66.8 80.6 83.2 65.0 86.3 93.0 93.6
100 23.2 18.0 17.3 31.0 49.5 16.0 24.2 30.3 26.6 29.0 53.6 36.0 20.8 27.7 24, I 86.6 35.5 44. I 78.0 54.0 79.6 82.0 52.2 87. I 85.9
100 15.9 9.0 16.0
loo 20.9 25.3 31.5
36.5 22.7
19.5 20.1
None Phenylalanine Leucine Threonine Tryptophan Arginine Histidine lsoleucine Lysine Serine Alanine Ornithine Valine a-amino butyrate Glycine Citrulline OH-proline Aspartate Glutamate Proline p-amino-isobutyrate Cysteic acid Urea p-alanine y-amino butyratc Cystine
-
-
62.3
64.3 60.9 54.3
40.0 69.5 58.7 61.1 -
86.6 86.2 _ Values are the mean percentage uptake of 6 replicate samples. Dashed line indicates no inhibition examined. MethionineJIC concentration 7 0.5 mM, inhibitor concentration 20 mM. TABLE
90.6 91.4
~-EFFECT OF COMBINATIONS OF INHIBITORY AMINO ACIDS ON METHIONINE UPTAKE BY ADULT S. mansoni (MALE WORMS)
inhibitor
None Threonine Histidine Threonine Threonine Threonine Threonine
(20 mM)
Methionine
L Arginine t Glutamate I Glutamate
Methionine-14C concentration -7 0.5 mM, inhibitor concentration mM. Values are the mean i S.E. of 6 replicate samples. bain
and
potassium
antimony
dependence
of’ntetltiortine
tartrate)
inhibited
uptake. 6. Sodium
uptake
Despite lack of sensitivity to ouabain methionine uptake in all stages of S.
the rate of was dependent upon the concentration of sodium ions in the medium. When sodium was replaced with isosmolar tris, potassium, lithium or choline methionine uptake was reduced as shown in Fig. 4a while an increase in sodium ion concentration in the medium resulted in an exponential increase in uptake (Fig. 4b). Because of experimental protocol it was not possible to delete completely sodium in incubations with either of the larval groups and a ntattsotti
--
5.4 f 0.7 I.5 i 0.2 I.0 i 0.3 I.4 i o-3 0.9 i 0.1 0.8 i 0.1 I.4 i 0.3 0.8 _t 0.1 0.8 1 0.1
(20 mM) (40 rnM) (60 mM)
~a Histidine I- Arginine
uptake (wmoles/g/2 min)
20
maximum depletion of 50 per cent was used, producing the expected decrease in methionine uptake (Table 4) though quantitatively not to the same degree as in adults. 7.
Temperature
attd
pH
depettdettce
of’ tnetltiottinr
uptake
Methionine uptake in larval and adult S. ntattsotti was temperature sensitive (Fig. 5); in adults the response was approximately linear over the entire temperature range whereas in both larval groups uptake was greatly enhanced at higher temperatures with a non-linear response. The rate of methionine uptake in adult worms that had been killed by heat treatment (60 C for 20 min) was considerably
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366
depressed compared with normal worms (Fig. 6). Over a pH range from 3 to 1 I.5 a small but significant variation in methionine uptake rate was recorded in all worm groups, with an optimum pH
0
Adult
VOL.
4. 1974
moles
LI .1
at 7.4.
Sodturn
free
O-Cloy
schlstosomulo
0 b
FIG. 5. Effect of temperature on methionine uptake in larval and adult 5’. mansoni. Each point is the mean of 8 2 min. Methionine replicate samples. Incubation time I.0 mM; (2) threeconcentration: (I) adult worms 5 nlM. week-old worms 5 mM; (3) schistosomula
I
20
I
I
60
40
Percentage
of
I
I
80
sodturn
I”
I00 medaum
(b)
5
4. Effect of sodium on methionine uptake in adult male S. munsoni. (a) Effect of complete removal of sodium; (b) Eff~t of increasing sodium ion concentration. Each point is the mean ~. SE. of 8 replicate samples. Incubation time 2 min.
Methlonlne
concentrotlon,
rn~
FIG.
TA8Lt
4---REDUCTION
S. mamoni
IN
MtTHIONINt
wsuLT1~0
FROM
FIG. 6. Methionine uptake by heat-killed adult male S. ma,~soni. Each point represents the mean 1 S.E. of 8 replicate samples. Worms were killed by exposure to 2 min. 60 C for 20 min. Incubation time UPTAKE
Percentage
-
IN
INCUBATION
Adult males Three week worms Schistosomula Values are the mean t S.L. of 8 replicate 2 min. I mM, incubation time
LARVAL IN
507;
reduction 61.4 39.3 41.5
samples.
AND
ADULl
sootuM
in methionine
uptake
.
2.4 + I.8 j 2.1 Methionine
concentration
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Radiochemical
Methionine purity
ad
,fbte of’ absorbed
uptake in schistosomes
ttwthio-
ttinr-‘K
Thin layer chromatography of stock methionine never revealed more than a single ninhydrin-positive, radioactive spot, indicating that, under the conditions of storage and usage, little or no detectable decomposition of methionine occurred.Furthermore, similar analysis of ethanolic extracts of larval and adult worms incubated in methionine-‘$C (plus carrier) for up to 30 min revealed only a single radioactive spot that cochromatographed with methionine, suggesting lack of methionine metabolism to any significant extent. DISCUSSION Surprisingly, the nutrition of schistosomes is poorly understood, particularly with regard to the route(s) of entry of nutritional requirements. Characteristically all adult worms removed from the host show the alimentary tract to be replete with black pigmented material usually accepted as representing the breakdown products of host red cell haemoglobin. There is a paucity of information on the fate of any products of globin hydrolysis released into the schistosome gut. Robinson (1961) considered that haemoglobin represented one source of amino acids for S. mattsoni in culture but also suggested that the requirement for amino acids was probably supplemented by absorption of serum amino acids. Unfortunately no evidence was provided to support this. Cheever & Weller (1958), growing schistosomula of S. mattsoni in vitro, noted that the stimulation of growth brought about by the addition of whole red cells also occurred with isolated red cell membranes and with the red cell soluble fraction. This, they conjectured, was indicative that the growth stimulating effect resided in the non-haemoglobin fraction of the red cell. The addition of purified globin to their cultures extended worm viability reflecting, perhaps, the hydrolysis of globin and absorption of the products by the schistosomula. Recent reports describe the occurrence of a single proteolytic enzyme isolated from the gut of S. mattsoni (Grant & Senft, 1971; Sauer & Senft, 1972). Studies on partially purified preparations of this enzyme provide no evidence for the complete hydrolysis of globin to yield free amino acids. Accordingly Grant & Senft ( 1971) have raised the question of whether schistosomes are capable of absorbing peptides through the mucosa of the gut. Earlier studies by Senft (1968) showed that IrCproline appeared to enter adult male S. ntansorri via both the gut and the tegument as judged by autoradiographic evidence, while the female showed reduced level of uptake at both sites. The interpretation of autoradiographs is unfortunately limited and cannot provide unequivocal evidence as to the primary route of entry of a solute. Dike (1971) examined the morphology of the oesophagus of S. ntattsotti and reported that the extracellular
367
interaction between material in the lumen and the plasma membrane of the mucosa was consistent with a digestive-absorptive function. No evidence for pinocytosis of globin was forthcoming from this study. There is clearly, then, some doubt as to the precise function of the schistosome gut in digestion and absorption and the role of the tegument must be considered in nutrient absorption. The morphology of the adult schistosome tegument is not dissimilar from that of cestodes and there can be no doubt of the absorptive role of the latter since cestodes lack a gut. Additionally it must be recalled that the mammalian phase of the schistosome is provided with a rich supply of available nutrient molecules of host origin, continuously bathing the external surface of the worm. Attention has been drawn to the marked and progressive increase in the surface area of schistosomes during development in the final host (Clegg, l972), an observation consistent with the view that the tegument may be absorptive. There are obvious technical difficulties in demonstrating an absorptive function of the tegu,nent. The careful use of ligatures and treatment with emetic post-incubation described above, do provide data that add support to the proposition that methionine uptake it? vitro occurs primarily via the tegument. One further line of evidence that may be introduced relates to the observations of Clegg (1965) who recorded significant stimulation of growth of S. tnattsotti in culture following the addition of lactalbumin hydrolysate; in the same study Clegg showed that schistosomula in culture do not ingest red cells until the fifth day, suggesting that the gut of the young worm may initially be in closure. Yet at this time free amino acids are required for growth and would presumably enter via the tegument. In the present study schistosomula absorbed methionine, yet at a rate ten times slower than that of older worms. While this is recognised as ambiguous in terms of the current debate, it does show that the tegument is apparently perm:able to methionine at this stage whereas preliminary observations suggested that cercariae absorb considerably less methionine than schistosomula (Chappell. unputlished data). Subsequent increasing permeability to methioninz could, therefore, be related to increased surface area on the one hand, development of gut function on the other, or indeed to both. However, sine: I have been unable to detect any basic difference in the kinetic; of methionine uptake in relation to worm age it is reasonable to postulate that, under the conditions described, the tegument provides the major route of methionine entry. Precisely how this might relate to the situatioll itt viva known. Preliminary experiments is not (Chappell, unpublished) show that methionine-“C injected intraperitoneally into mice is absorbed by adult schistosomes within IO min. but it would neither be feasible to investigate the kinetics of uptake itr viva nor would it be possible to determir.e
368
L. H.
CHAPPELL
the route of entry of solutes in this way. There are similarities between the kinetic characteristics of methionine absorption in S. mansoni and those of the cestode Hymenolepis diminuta (Read et al., 1963), particularly with regard to the relatively low degree of specificity encountered. There is also quantitative agreement in the kinetic constants of the saturable systems (K, for H. diminuttr -~- 0.31 mM, K, for S. munsoni z 0.33 mM: V,,, for H. diminrttn y 6.1 umoles/g/2 min (original rate given per h) and V,,,, for S. mansoni 4.0 umoles/g/ 2 min). Transport of neutral amino acids in many microbial and mammalian systems is unaffected or only slightly inhibited by other classes of amino acids (Eavenson & Christensen, 1967; Gazzola et al., 1972) while other systems show varying degrees of overlap between the different classes of amino acids (Hampton, 1970; Yamaguchi et al., I971 ; Eccleston & Kelly, 1972). Read er al. (1963) demonstrated inhibition ofmethionine uptake in H. dimimrta by histidine, aspartate, proline and hydroxyproline in addition to inhibitions by neutral amino acids; the least effective inhibitors were g-alanine, glutamate, lysine, cysteine and arginine. Methionine, on the other hand, inhibited glutamate uptake in H. diminuta. The methionine transport system of S. munsoni is not peculiar, then, in its low degree of specificity. Reiser & Christiansen (1969) attributed the inhibition of lysine uptake in rat everted intestinal sacs by leucine and methionine to allosteric alteration of the basic amino acid system by the binding of neutral amino acids to a discrete but specific region on a single or polyfunctional carrier (sic). There is no evidence to support this contention for the cross inhibitions observed in S. munsoni, though the fact that basic acids have a lower affinity for the methionine system than do neutral acids may indicate that allosteric effects are not important. The essential absence of qualitative differences between the uptake of methionine by adult and by juvenile worms is of interest; this, in conjunction with the quantitative difference between schistcsomula and older worms, renders the hypothesis of differential permeability to solutes as unlikely to account for the varying response of worms of different age to antimony or other drugs. The observation that three-week-old worms are less susceptible to antimony therapy than older or younger worms remains unexplained, although it may not be permissible to extrapolate from amino acid uptake to the absorption of potassium antimony tartrate. The precise location of worms of different ages in the host may account for this differential response to antimony; detoxication of antimony compounds occurs in the liver and both migrating schistosomula and adult worms in the mesenteric veins lie some distance from that organ. The present study has led to the view that permeability increases during the early stages of tegumental proliferation but not during the later stages of development and it is
I.J.P. VOL. 4. 1974
not clear at present whether there is any direct relationship between these two phenomena. Hockley & McLaren (1973) provide evidence that the adult schistosome tegument may be continually renewed and suggest that such complexity of structure may be related rather to the binding of host antigens than to any digestive or absorptive function. Further study is required to elucidate the function(s) of the schistosome surface. The minor differences in methionine uptake between adult males and females would seem to contrast with the suggestion of Senft (1968) that the male may furnish the female with amino acids in viva. However, it could be worthwhile to study in detail the permeability constants for a large number of amino acids in schistosomes of both sexes since these values may well be different. Additionally comparison of amino acid uptake by paired and single schistosomes should provide evidence on the question of nutritional synergism between adult paired worms. The biphasic mode of methionine entry described for S. mnnsoni, comprising saturable and apparently non-saturable components, is indicative that at low, probably physiological concentrations the majority of methionine absorbed enters via the saturable system. No evidence was obtained for active transport and the internal methionine concentration in adult males removed from mice was very low (0.14 mM). In contrast to this lsseroff & Levy (1972) reported the uptake of a number of amino acids, including methionine, by adult S. munsoni to occur by active processes. No explanation for this discrepancy is available at present. Furthermore, I have observed a marked dependence of methionine uptake on the concentration of sodium ions but insensitivity to ouabain, whereas lsseroff & Levy recorded sensitivity both to sodium ion concentration and to the presence of ouabain. It was thought possible that the saturable component of methionine uptake described above might represent adsorption to the tegument rather than absorption while true uptake would be represented by the non-saturable, diffusion component. Removal of the tegument by the freeze-thaw technique (Kusel, 1972), however, revealed that over 95 per cent of labelled methionine was located in subtegumental tissues. The occurrence of a large diffusion component is well documented in transport studies (Eavenson & Christensen, 1967; Woodward & Read, 1969; Hampton, 1970). It has been argued by Christensen & Liang (1966) that such non-saturable systems represent, in reality, mediated systems which were not measurably saturated under the experimental conditions adopted. This has not been substantiated and it is reasonable to question the possible selective advantage to any organism or cellular system in the possession of a transport process saturating at solute concentrations far in excess of those normally encountered.
I.J.P. VOL.4. 1974
Methionine
uptake in schistosomes
Meth~onine uptake rate in both larval and adult schistosomes was markedly affected by temperature. The differences in response by juveniles and by adults might be best explained in terms of physical properties of the tegument. Leakiness of larval membranes at higher temperatures is an unlikely explanation since heat killed adults and juveniles all absorbed less methionine than normal worms. There is no satisfactory explanation for the wide variability seen in the free amino acid pools of all stages of schistosomes. There is clearly a trend for a decrease in the size of the free pool during development from cercariae to adults, and this aspect of schistosome bicchemistry deserves further attention. Ackno+&&emenrsThe technical assistance of Mrs. B. Smvth. Miss S. Gardiner and Mr. S. Pocock is aratefullv acknowledged. Amino acids were analysed thr&gh the co-operation of Drs. D. W. T. Crompton, G. A. M. Cross and C. Milstein. Dr. G. C. Coles kindly read and commented on the manuscript. This study was supported by a grant from the Overseas Development Administration and thanks are extended to Dr. B. A. Newton for the provision of laboratory facilities.
BUEDI~QC~ E.
REFERENCES 1952. Acetylcholinesterase
of
The prophylactic and curative activity of sodium antimony dimercapto succinate (TWSb) in experimental infections with Schistosoma mansoni. American Jownal of Tropical Medicine and Hygiene 11: 25-30. CHEEVER A, W. Sr WELLERT. H. 1958. Observations on the growth and nutritional requirements of Schistosoma munsoni in vitro. American Jomxal of’ Hygietje 68: 322-339. CHRISTENSEN H. N. & LI~NC; M. 1966. On the nature of
the “non-saturable” migration of amino acids into Ehrlich cells and into rat jejLinum. ~~#r~~~~zj~~&clpl?ysica Arta 112: 524-531. CLEGG J. A. 1965. In vitro cultivation of Schisto.soma Experimental
Parasitology
16: 133-147.
CLFGGJ. A. 1972. The schistosome surface in relation to parasitism. Symposia j&r the British Society for Parusitology 10 : Fti~tcti[)ffaI 23-40. CLEGG J. A. & SMITIIERS S.
Aspects
of Parasite
Surjizt-rs
R. 1972. The effects of immune Rhesus monkey serum on schistosomula of Schistu.wma mansoni during cultivation in vitro. International
Jorrrr:al /iw Parasitolqqy
2: 79-98.
DIKE S. C. 1971. Ultrastructure of the esophagoel region in Scbisto.s(~ma manso,ti. The Amzricarr J~~~tr~za/ of Tropical
Mediei,le
and Hygiene
20: 552.--568.
EAVENS~N E. & CHRISTENSENH. N. 1967. Transport systems for neutral amino acids in the pigeon erythrocyte. Journal of Biological Chemistry 242: 53865396.
EC~LEST~NM. & KELLY D. P. 1972. Competition among amino acids for incorporation into ~ethyIoro~~f~~
capsulattts. Journal of General Microbiology 13: 303314. GRANT C. T. & SFNFT A. W. 1971. Schistosome proteo-
C(?mparative
Bioeher;ti~try
and Physiology
38: 663-678.
GAZZOLAG. C., FRANCHIR., SAIBENEV., RONCHI P. & GUIDO~TI G. G. 1972. Regulation of amino acid transport in chick embryo heart cells-l. Adaptive system of mediation for neutral amino acids. Biochimiea Bi~~ph.vsica Acta 266:
407-421.
HALTOND. W. & ARME C. 1971. In vitro technique for detecting tegument damage in Diclidophora merlangi: possible screening method for selection of undamaged tissue of organisms prior to physiological investigation. Experimental
Purasito/ogy
30: 54-57.
HAMPTONJ. R. 1970. Lysine transport in the culture form of ~r~vpa~u.~oma cruzi: kinetics and inhibition of uptake by structural analogues. Interna/ional Journal of Biochemistry
1: 706-7
14.
HOCKLEYD. J. & MCLAREN D. J. 1973. Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm. lttternutio??u/ JottrnaI f&r Parasitology 3: 13-25. ISSEROFFH. LPLLEVY M. 1972. Amino acid transport in Schistosoma
mansoni. American
Zoologbt
12: 681.
KUSEL J. R. 1972. Protein composition and protein synthesis in the surface membranes of Schktosoma t~a/z~o~i. ~ara.~ifoIo~_v 65: 55-69.
LINEWEAVERH. & BURK D. 1934. The determination of enzyme dissociation constants. Journal qfthe Americwt Ch&icaI
activity
Schisiosoma munsoni. British Joutwal of Pharmacolo~,v and Chentotherapy 7: 563-566. BRUC-E J. I., SADUN E. H. & SCHOENBECHL.ER M. J. 1967.
mansoni.
lytic enzyme.
369
Society 56: 658-666.
READ C. P.. ROTHMANA. H. & SIMMONSJ. E. 1963. Studies on membrane transport, with special reference to parasite-host integration. Ama/.sof’ the New York Academv of Sciences 113: 154-205. REISERS. *&&+RISTIANSEN P. A. 1969. A cross-inhibition of basic amino acid transport by neutral acids. Biochimica Biophysiea Acta 183: 61 I -624. ROBINSONII. L. H. 1961. Amino acids of Schisfosoma mattsoni. Anrrals of Tropical 55: 403-6.
Medicine
and Para.sifoIo~<~y
SAUER M. C. V. & SENFT A. W. 1972. Properties proteolytic enzyme from Schistosoma mnnsoni. parative
Biochemistry
and Physiology
of a Cotn-
42: 205-220.
S~I~UBERT M. 1948. Effects of drugs during earliest stagesofexperimental Schistosomiasis mansoni in mice. American
J~~t~rttal of Tropical
~edie~ne
28: 157- 162.
SENFT A, W. 1968. Studies in proline metabolism by Schistosoma mansoniI. Radioautography following in vitro exposure to radioproline-“C. Comparative Biochemisiry and Physiology 27: 25 l-261.
STANDEN0. D. 1955. The treatment of experimental schistosomiasis in mice: sexual maturity and drug resoonse. An:ta/.s of’ TropiraI Medicim~ and Parasitolo~_v 491 183-192. ~ STOHLERH. R. & FREY J. R. 1963. Chemotherapy of exp-rimental schistosomiasis mansoni: prophylactic and protective activity of clinically active compounds in mice and hamsters. Annals of Tropical l~e~/ieine and Parasitok~gy
57: 466-42’0.
WOODWARC) C. K. & READC. P. 1969. Studies on membrane transport VII. Transport of histidine through two distinct systems in the tapeworm Hymenolepis dikninuta. 30: 1161-l
Comparative 177.
Biochemistry
and
Physioiogy
YAMAC~UCHI T., YAMAGU~HI M. & LAJT~~AA. 1972. S:lective inhibition by heat of amino acid transport in slices of mouse brain. Biochimica Biophysicu Acta 266: 422 -43.5.
UPTAKE BY LARVAL AND SCHISTOSOMA ML4 NSONZ L.
Molten0
Institute,
H.
ADULT
CHAPPELL*
Downing
Street,
( Received22 nugrrst
Cambridge,
England
1973)
L. H. 1974. Methionine uptake by larval and adult Sehisfosoma mansoni. Internafional Journalfor Parasitology 4: 361-369. The uptake of methyl-laC-methionine by schistosomula, 21 day-old and adult Schistosoma mansoni in vitro has been investigated. Ligation of the adult pharynx and comparison between the kinetics of uptake of schistosomula and adults suggests that methionine is absorbed, in the system described, primarily via the tegument. All stages of the parasite examined absorb methionine by, at least, two kinetically distinguishable systems, one which is saturable and a second which appears to be simple diffusion. The saturable system has relatively low specificity for amino acids but shows no affinity for other classes of compounds. The effects of pH, temperature, metabolic inhibitors, and sodium ion concentration have been examined. The results are discussed with reference to schistosome gut function and also to the differential response to chemotherapy according to age of infection. Abstract-CHAPPELL
INDEX uptake;
KEY WORDS: Schistosoma mansoni: schistosomes; transport; tegument.
INTRODUCTION MECHANISMS
* Present address: Aberdeen
Dept. of Zoology,
methionine;
amino
acids;
schistosomes to antimony chemotherapy depending upon the age of the infection: one-week-old worms and adults are more susceptible to antimony therapy than are three-week-old worms. An explanation for this might be that there is a variation in the degree of permeability of the tegument to solutes according to worm age. It is the aim of the work described below to determine whether such a permeability difference does exist using the uptake of a labelled amino acid by various developmental stages of S. n7ar7soni ir7 vitro.
by which schistosomes obtain amino acids from the definitive host are not completely understood. While the worm possesses an alimentary tract there is no evidence of complete proteolysis within the gut (Grant & Senft, 1971) and it is not known whether the absorption of peptides originating from the incomplete hydrolysis of globin is carried out by the mucosa; neither is there any evidence of pinocytotic absorption of whole globin (Dike, 1971). Support for the hypothesis that free amino acids are absorbed from the host blood via the tegument comes from the work of Senft (1968), who studied the distribution of ‘T-proline in adult S. mar7soni using autoradiography, and from the work of Clegg (1965) who observed that schistosomula of S. mansor7i in culture required amino acid supplements for optimum growth during the first five days of cultivation, during which time the gut of the schistosomulum is thought to be in closure. Additionally Clegg (1972) pointed out that the dramatic proliferation of the tegument and the resulting increase in surface area that occurs during development in the final host are consistent with a tegumental absorption function. Chappell & Coles (unpublished data) have corroborated the earlier findings of Schubert (1948), Standen (1955), Bruce at al. (1962) and Stohler & Frey (1963) that there is a differential response of THE
schistosomula;
MATERIALS I. Maintenance
AND
METHODS
of the parasite and collection qf material
The Wellcome sti-ain of S. mansoni was maintained in the laboratory in Biomphaluria glabrafa at 25’C and in Hough strain albino male mice (J. Hough, Lydgate, Lanes.). Mice weighing 15-25 g were infected by the abdominal ring technique after Nembutal anaesthesia using freshly collected cercariae. Three developmental stages of the parasite were used in this study; adult worms and three-week-old worms were obtained from infected mice by perfusion with cold 0.9 y0 NaCl containing I .5% trisodium citrate, and O-day-old schistosomula were obtained in vitro by cercarial penetration through excised mouse skin using the technique of Clegg & Smithers (1972). Adult worms were harvested at eight weeks postinfection from an exposure of 200 cercariae/mouse; this gave a mean recovery of 50 per cent. Three-week worms were harvested from mice given an infection dose of 2000 cercariae/mouse, with a yield of between I5 and 30 per cent at 21 days post-infection. Zero-day schistosomula were collected, following skin penetration, by gentle centrifugation; 200&4000 cercariae were used per
The University,
At39 2TN. 361
367
L.
H. ~‘HAPPtl.l.
I.J.1’.
VOL..
4. 1974
penetration tube with a penetration time of 3 h. The use of eight
penetration
tubes afforded
a recovery
of up to
20,000 schistosomula, but a mean yield of only 28 per cent was obtained. All cultures of schistosomula were examined before experimentation and those contaminated with an excess of 5 per cent cercariae were discarded.
2. Amino acid uplake
procedwes
Worms were collected as outlined above and were washed several times in 25 mM Krebs-Ringer buffered with trissmaleic acid (KRT of Read e/ al., 1963), containing I mg/ml glucose, at pH 7.4 and at room temperature. Adult schistosomes were partially separated into the sexes by shaking in KRT at 37°C for 5-10 min, final separation being obtained manually. At no time were adult worms of the particular sex required actually handled during separation as it was not possible to separate worms manually without the attendant risk of damaging the tegument. Consequently one of the sexes was always discarded after separation allowing, therefore, the use of fine forceps to aid final separation. No attempt was made to sex or separate male and female 3-week-old worms. Worms collected on any one day were grouped together and then randomly redistributed into tubes where they were maintained in a preincubation medium of KRTglucose. Preincubation in all cases was carried out at 37’C for 15 min. For studies on amino acid uptake the following sample sizes were chosen: IO-1 5 adult males (0.6-0.9 mg dry wt), 15-20 adult females (0.3-0.5 mg dry wt), 50-100 three-week worms (0.3-0.6 mg dry wt) and 2000 schistosomula (I.8 mg dry wt). Experimental incubations were carried out at 37’C unless otherwise specified with worms suspended in KRT-glucose plus methyl-‘“C-methionine and any other additions as required, to give a final volume of 2 ml for each sample. All incubations were under air unless otherwise stated. Incubations with adult or three week worms were terminated by the addition of excess, cold (4 C) KRT and by cooling the tubes in ice. Four additional washes of IO ml each wash with KRT were sufficient to remove any methionine-“C. Schistosomula were non-absorbed treated in a similar manner but were washed on an 8 Mm cellulose filter with four volumes of KRT containing 40 mM stable methionine. Filter controls were used in the latter procedure at all times to allow for correction to include any non-absorbed methionine-“C that was adsorbed to the filter. Methionine-“C absorbed by the worms was extracted with 70% ethanol for 24 h at room temperature and was quantitatively determined by liquid scintillation counting on a Packard Tri-Carb counter employing a liquid scintillator (hat contained per litre, 700 ml toluene, 300 ml Triton-X 100, 5 g PPO (2,5-diphenyloxarole) and 0.5 g POPOP (I ,4-di[2-(phenyloxazolyl)]-benzene). Counting efficiency was estimated to be 85 per cent. With adult and three-week worms the dry wt of each sample was determined by drying overnight at 95 C after ethanolic extraction, whereas. for schistosomula, the numbers in each sample were determined and their dry weights estimated by reference to a predetermined scale. This was necessitated by the filtration-washing procedure used for schistosomula; the washed larvae were counted directly on the filter paper and were not recoverable for weight determinations.
According to the supplier methyl-“C-methionine in aqueous solution decomposes at a rate of 10-15’2; pel month even at -2O’C. It was thus necessary first, to purchase stocks in small quantities at regular intervals and secondly, to determine the radiochemical purity of existing stock solutions routinely. Purity was determined either by high voltage electrophoresis (2500 V for I h with DH I.9 formic/acetic buffer) and liauid scintillation couniing or by scanning two dimensional thin layer plates [cellulose MN 300HR; first solvent, butanol/acetic acid/water (6:1 :5 v/v); second solvent, butanol/acetone/ diethylamine/water(lO:lO:7:5 v/v)] with a Geiger-Miillet tube. 4. Amino
acid determiuutiotl
The free amino acids of S. marwoni were determined by routine automatic analysis (Technicon or Locarte analysers) of ethanolic (70%) extracts of fresh tissue. Confirmation of peak identity was made by co-chromatography. 5. Chemic,al.s Methyl-“C-methionine (> 50 mCi/mmol) was obtained from the Radiochemical Centre and was made up in aqueous solution containing stable methionine carrier to IO mM (0.5 pCi/umole). All other chemicals used were
of analytical grade where commercially available.
RESULTS
I. Fwa unlitw acids of S. mansoni The free amino acids of adult and larval S. manswri are shown in Table I. The large standard error given is thought to represent genuine variation in amino acid levels rather than any technical shortcomings. The data for cercarial amino acids have been omitted as variation between replicate samples was unacceptably large despite the precaution of freezing in liquid nitrogen immediately after additiolt of ethanol and extraction in the cold to prevent autolysis. Even so the cercarial free amino acid pool was considerably higher than that of any other stage of the life cycle and there appears to be a trend for a decrease in free amino acid content with age. Cysteic acid was present in all stages in relatively large amounts but it could not be separated from an unknown contaminating peak so that no quantity is given in Table I.
It was initially necessary to determine the relative contributions of the gut and of the tegument in the uptake of methionine under the i/l vitro conditions described. Adult male worms were selected for ligation because of their size and the ease with which a ligature could be applied. The uptake of methionine in worms with pharyngeal ligation (i.e. the region between oral and ventral suckers) was compared with that of normal worms. A number of ligatures
I.J.P.
VOL.
4. 1974
Methionine
TABLE
Amino
acid
Cysteic
acid
1~+ 4.4 7.2 IO.6 22.8 16.7 4.1 33.2 0 9.8 3.2 6.2 17.8 I.2 4.4 2.2 2.1 18.9 5.9 3.7
Aspartate Threonine Serine Glutamate Proline Glycine Alanine Cystine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine Histidine Ornithine Lysine Tryptophan Arginine Total
II
pool
I-THE
Schistosomula
uptake
FREE AMINO
in schistosomes ACIDS
21 day worms
-t + $ i It i_ + :i
t
0.6 0.7 I.2 7.4 3.2 0.6 8.1
$ + + + * i i $ $ + $
1.1 0.4 0.7 4.6 0.1 0.3 0.2 0.1 2.1 0.6 0.6
177.1 5
6.5 6.4 9.8 21.0 6.2 8.2 19.5 0.2 2.5 0.7 I.3 2.8 I.0 I.0 0.8 I.8 6.0 0 I.7
OF S.
363
mansoni
Adult
males
Adult
L I.7 + 0.8 + I.8 i 5.7 + 2.4 + 2.2 + 2.5 .$ 0 i 0.1 + 0 $ 0.1 $ 0.3 f 0.1 + 0.1 + 0.2 i 0.1 1~ 0.7 + 0.7
95.6 6
2.4 2.1 3.3 8.7 2.2 3.4 9.2 I.4 I.0 0.7 0.6 I.3 0.9 0.7 0.9 0 2.0 0 0.1
f f I i + $ + + _i + i 11 i + +
0.3 0.1 0.1 0.5 0.4 0.5 I.6 0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.1
+
0.1
6.5 3.0 3.0 4.6 3.0 3.7 5.3 3.9 2.2 0.8 I.2 2.2 I.0 0.2 0.9 0 3.8 0 0
_f 0
40.8 6
3. Clmruct~ristics
of’ metl~ionine
absorption
To establish conditions for unidirectional flux of a transported moiety it is necessary to determine the duration of the initial rate of influx. This was found to extend to 5 min, shown in Fig. I and the convenient incubation time of 2 min was therefore chosen for subsequent experiments.
$ + f $ i + 1 _i 1 f _t +
0.6 0.4 0.6 0.6 0.4 0.6 0.5 0.1 0.3 0 0.3 0.4 0.1
t 0 1~ 0.2
t
0.2
45.3 6
Values are umoles/g dry wt (mean i S.E. of 5 or 6 replicate samples). ~presence of cysteic acid unresolved from an adjacent, unidentified peak.
was tested the most suitable being human hair. In incubations from 30 set to 30 min with a range of external methionine concentrations from 0.1 to 5.0 mM no difference in uptake was recorded between the two groups. Evans blue was used routinely as an indicator of damage to the tegument resulting from ligation (Halton & Arme, 1971) and, with practice and careful manipulation, no tissue damage was effected during ligation. No obvious method was available for determining the efficacy of the ligature as a barrier to solutes. However, physostigmine at IO-“M may be used to induce vomiting in schistosomes (Bueding, 1952) and this was administered post-incubation, but, again, revealed no difference in uptake between ligated and normal worms. While the above cannot be regarded as unequivocal evidence for the lack of alimentary involvement in the absorption of methionine it is considered that, in the short-term incubations employed primarily in this study, the role of the gut is minimal. Consequently, in subsequent experiments ligation of adult worms was not used and it would be clearly impractical to attempt ligation of three week worms and impossible with schistosomula.
females
i
I I
I
Refers to the
I
I
20
IO Incubation
time,
min
FIN. I.
Time curve for methionine uptake in adult male S. mansoni. Each point is the mean -~ S.E. of 8 replicate samples. Methionine concentration 7 0.5 mM.
The curves for methionine uptake in larval and adult schistosomes as a function of methionine concentration in the medium (Fig. 2) tend towards saturation and are clearly not linear over the concentration range examined. Quantitatively schistosomula absorb methionine at a rate one order of magnitude less than older worms, while female adults absorb methionine somewhat more rapidly than males. Extension of the concentration of methionine in the medium 50 mM reveals an apparent biphasic mode of uptake (Fig. 3). At low concentrations uptake occurs by a system which is apparently saturable while at higher concentrations uptake
364
L.
H.
CHAPPFLI.
I.J.P.
VOL.
4. 1974
a mediated transport system with specific binding affinity and transporting capability for methionine and, perhaps, related amino acids. Were this hypothesis valid it should be possible to inhibit methionine transport by the addition of generically similar molecules, in this case neutral amino acids. The effects of the addition of 25 individual amino acids on the rate of methionine transport were examined; the potentially inhibitory acids were included individually in a forty-fold excess (20 mM) of methionine (0.5 mM). The results of these inhibitions are summarised in Table 2, in which the added amino acids are arranged in ranking order of inhibitory activity for female adult schistosomes. Almost all amino acids tested possessed some inhibitory function, the least active being those with the greatest spatial separation of the a.amino group and the a-carboxyl group (i.e. p-and y-amino acids) and the molecular species that lack one of these groups (i.e. urea, the imino acids) and the acidic amino acids. The overall pattern of methionine inhibition by other amino acids in adult worms, prepatent worms and schistosomula was essentially similar as judged by reference to selected examples (Table 2). A number of the above inhibitions were examined by the method of Lineweaver & Burk (1934) to determine their precise nature. All amino acid inhibitions examined in this way (thre, leu, phe, arg, orn, his and lys) were fully competitive inhibitors of methionine uptake. The inhibitor constants (K,) for basic ammo acids were larger than those for neutral acids (e.g. Ki’“’ _ 5.4; KiPhe : 4.5; Kyrg 47,s; 36.3) suggesting much lower affinity of K(‘Y‘ basics for the methionine system than shown by neutrals. Preliminary experiments using combinations of inhibitory amino acids representing different classes suggest that methionine may enter via two distinct systems (Table 3), a neutral system and a basic system, but more detailed examination would be required to establish this. The specificity of the methionine transporting process was examined addit ionally by testing representatives of other classes of compounds as potential inhibitors of methionine uptake. These included, (I) organic acids (citrate, fumarate. lactate, oxalacetate, pyruvate and succinate); (2) carbohydrates (arabinose 2-deoxygtucose, fructose, glucose, malose, mannose, rhamnose, ribose, sorbose, sucrose, trehalose, and xylose), and (3) free fatty acids (acetate, propionate and butyrate). None of these was inhibitory. represent
Methionine
concentration,
mM
FIG. 2. Methionine uptake as a function of n~ethiollil~e concentration in the medium in larval and adult S. n~ansoni. Each point is the mean of, at least, 10 replicate samples. Incubation time 2 min.
Totol 4
uptoka/*
I
60-
I
IO
20 Methlontne
30
“0
40
concentrotfon,
rr!d
Fro
3. Extended concentration curve of methionine uptake in adult male S. ~u~?s~/?;.Each point is the mean of 8 replicate samples. incubation time 2 min.
occurs by a second, apparently non-saturable process. Assuming this second system to be Fickian diffusion it is permissible to distinguish between the two systems graphically as in Fig. 3, to show the relative contributions of each system over a range of methionine concentrations. The alternative possibility tnat the linear component of methionine uptake might represent a second saturable process experimentwhose If,,, had not been encountered ally has not been discounted and this is discussed later. 4. Inlribitiott ~~‘nx~tlric~tlitrc~ uptake Reference to the above data suggests that the saturable component of methionine uptake may
5. Metaboiic itrhibitors To evaluate the possible dependence of methionine transport on energy metabolism the effects of a variety of inhibitors of aerobic and anaerobic metabolism were investigated. None of those tested (2,4-dinitrophenol, cyanide, azide, iodoacetate, oua-
I.J.P.
VOL.
-_
4. 1974 TABLE
Methionine 2-PERCENTAGE
INHIBITION
Amino acid inhibitor
uptake in schistosomes
OF METHIONINE
UPTAKE
IN s.
365
mUiWOtli
BY OTHER
AMINO ACIDS
Adult females
Adult males
21 day worms
Schistosomula
100 16.7 17.0 17.5 18.8 20.2 24.5 26.5 26.6 27.0 27.5 28.0 29.2 32.1 32.6 39.3 43.2 51.0 64.3 66.8 80.6 83.2 65.0 86.3 93.0 93.6
100 23.2 18.0 17.3 31.0 49.5 16.0 24.2 30.3 26.6 29.0 53.6 36.0 20.8 27.7 24, I 86.6 35.5 44. I 78.0 54.0 79.6 82.0 52.2 87. I 85.9
100 15.9 9.0 16.0
loo 20.9 25.3 31.5
36.5 22.7
19.5 20.1
None Phenylalanine Leucine Threonine Tryptophan Arginine Histidine lsoleucine Lysine Serine Alanine Ornithine Valine a-amino butyrate Glycine Citrulline OH-proline Aspartate Glutamate Proline p-amino-isobutyrate Cysteic acid Urea p-alanine y-amino butyratc Cystine
-
-
62.3
64.3 60.9 54.3
40.0 69.5 58.7 61.1 -
86.6 86.2 _ Values are the mean percentage uptake of 6 replicate samples. Dashed line indicates no inhibition examined. MethionineJIC concentration 7 0.5 mM, inhibitor concentration 20 mM. TABLE
90.6 91.4
~-EFFECT OF COMBINATIONS OF INHIBITORY AMINO ACIDS ON METHIONINE UPTAKE BY ADULT S. mansoni (MALE WORMS)
inhibitor
None Threonine Histidine Threonine Threonine Threonine Threonine
(20 mM)
Methionine
L Arginine t Glutamate I Glutamate
Methionine-14C concentration -7 0.5 mM, inhibitor concentration mM. Values are the mean i S.E. of 6 replicate samples. bain
and
potassium
antimony
dependence
of’ntetltiortine
tartrate)
inhibited
uptake. 6. Sodium
uptake
Despite lack of sensitivity to ouabain methionine uptake in all stages of S.
the rate of was dependent upon the concentration of sodium ions in the medium. When sodium was replaced with isosmolar tris, potassium, lithium or choline methionine uptake was reduced as shown in Fig. 4a while an increase in sodium ion concentration in the medium resulted in an exponential increase in uptake (Fig. 4b). Because of experimental protocol it was not possible to delete completely sodium in incubations with either of the larval groups and a ntattsotti
--
5.4 f 0.7 I.5 i 0.2 I.0 i 0.3 I.4 i o-3 0.9 i 0.1 0.8 i 0.1 I.4 i 0.3 0.8 _t 0.1 0.8 1 0.1
(20 mM) (40 rnM) (60 mM)
~a Histidine I- Arginine
uptake (wmoles/g/2 min)
20
maximum depletion of 50 per cent was used, producing the expected decrease in methionine uptake (Table 4) though quantitatively not to the same degree as in adults. 7.
Temperature
attd
pH
depettdettce
of’ tnetltiottinr
uptake
Methionine uptake in larval and adult S. ntattsotti was temperature sensitive (Fig. 5); in adults the response was approximately linear over the entire temperature range whereas in both larval groups uptake was greatly enhanced at higher temperatures with a non-linear response. The rate of methionine uptake in adult worms that had been killed by heat treatment (60 C for 20 min) was considerably
I.J.P.
L. ft. C’IiAPP~LL
366
depressed compared with normal worms (Fig. 6). Over a pH range from 3 to 1 I.5 a small but significant variation in methionine uptake rate was recorded in all worm groups, with an optimum pH
0
Adult
VOL.
4. 1974
moles
LI .1
at 7.4.
Sodturn
free
O-Cloy
schlstosomulo
0 b
FIG. 5. Effect of temperature on methionine uptake in larval and adult 5’. mansoni. Each point is the mean of 8 2 min. Methionine replicate samples. Incubation time I.0 mM; (2) threeconcentration: (I) adult worms 5 nlM. week-old worms 5 mM; (3) schistosomula
I
20
I
I
60
40
Percentage
of
I
I
80
sodturn
I”
I00 medaum
(b)
5
4. Effect of sodium on methionine uptake in adult male S. munsoni. (a) Effect of complete removal of sodium; (b) Eff~t of increasing sodium ion concentration. Each point is the mean ~. SE. of 8 replicate samples. Incubation time 2 min.
Methlonlne
concentrotlon,
rn~
FIG.
TA8Lt
4---REDUCTION
S. mamoni
IN
MtTHIONINt
wsuLT1~0
FROM
FIG. 6. Methionine uptake by heat-killed adult male S. ma,~soni. Each point represents the mean 1 S.E. of 8 replicate samples. Worms were killed by exposure to 2 min. 60 C for 20 min. Incubation time UPTAKE
Percentage
-
IN
INCUBATION
Adult males Three week worms Schistosomula Values are the mean t S.L. of 8 replicate 2 min. I mM, incubation time
LARVAL IN
507;
reduction 61.4 39.3 41.5
samples.
AND
ADULl
sootuM
in methionine
uptake
.
2.4 + I.8 j 2.1 Methionine
concentration
I.J.P. VOL.
8.
4. 1974
Radiochemical
Methionine purity
ad
,fbte of’ absorbed
uptake in schistosomes
ttwthio-
ttinr-‘K
Thin layer chromatography of stock methionine never revealed more than a single ninhydrin-positive, radioactive spot, indicating that, under the conditions of storage and usage, little or no detectable decomposition of methionine occurred.Furthermore, similar analysis of ethanolic extracts of larval and adult worms incubated in methionine-‘$C (plus carrier) for up to 30 min revealed only a single radioactive spot that cochromatographed with methionine, suggesting lack of methionine metabolism to any significant extent. DISCUSSION Surprisingly, the nutrition of schistosomes is poorly understood, particularly with regard to the route(s) of entry of nutritional requirements. Characteristically all adult worms removed from the host show the alimentary tract to be replete with black pigmented material usually accepted as representing the breakdown products of host red cell haemoglobin. There is a paucity of information on the fate of any products of globin hydrolysis released into the schistosome gut. Robinson (1961) considered that haemoglobin represented one source of amino acids for S. mattsoni in culture but also suggested that the requirement for amino acids was probably supplemented by absorption of serum amino acids. Unfortunately no evidence was provided to support this. Cheever & Weller (1958), growing schistosomula of S. mattsoni in vitro, noted that the stimulation of growth brought about by the addition of whole red cells also occurred with isolated red cell membranes and with the red cell soluble fraction. This, they conjectured, was indicative that the growth stimulating effect resided in the non-haemoglobin fraction of the red cell. The addition of purified globin to their cultures extended worm viability reflecting, perhaps, the hydrolysis of globin and absorption of the products by the schistosomula. Recent reports describe the occurrence of a single proteolytic enzyme isolated from the gut of S. mattsoni (Grant & Senft, 1971; Sauer & Senft, 1972). Studies on partially purified preparations of this enzyme provide no evidence for the complete hydrolysis of globin to yield free amino acids. Accordingly Grant & Senft ( 1971) have raised the question of whether schistosomes are capable of absorbing peptides through the mucosa of the gut. Earlier studies by Senft (1968) showed that IrCproline appeared to enter adult male S. ntansorri via both the gut and the tegument as judged by autoradiographic evidence, while the female showed reduced level of uptake at both sites. The interpretation of autoradiographs is unfortunately limited and cannot provide unequivocal evidence as to the primary route of entry of a solute. Dike (1971) examined the morphology of the oesophagus of S. ntattsotti and reported that the extracellular
367
interaction between material in the lumen and the plasma membrane of the mucosa was consistent with a digestive-absorptive function. No evidence for pinocytosis of globin was forthcoming from this study. There is clearly, then, some doubt as to the precise function of the schistosome gut in digestion and absorption and the role of the tegument must be considered in nutrient absorption. The morphology of the adult schistosome tegument is not dissimilar from that of cestodes and there can be no doubt of the absorptive role of the latter since cestodes lack a gut. Additionally it must be recalled that the mammalian phase of the schistosome is provided with a rich supply of available nutrient molecules of host origin, continuously bathing the external surface of the worm. Attention has been drawn to the marked and progressive increase in the surface area of schistosomes during development in the final host (Clegg, l972), an observation consistent with the view that the tegument may be absorptive. There are obvious technical difficulties in demonstrating an absorptive function of the tegu,nent. The careful use of ligatures and treatment with emetic post-incubation described above, do provide data that add support to the proposition that methionine uptake it? vitro occurs primarily via the tegument. One further line of evidence that may be introduced relates to the observations of Clegg (1965) who recorded significant stimulation of growth of S. tnattsotti in culture following the addition of lactalbumin hydrolysate; in the same study Clegg showed that schistosomula in culture do not ingest red cells until the fifth day, suggesting that the gut of the young worm may initially be in closure. Yet at this time free amino acids are required for growth and would presumably enter via the tegument. In the present study schistosomula absorbed methionine, yet at a rate ten times slower than that of older worms. While this is recognised as ambiguous in terms of the current debate, it does show that the tegument is apparently perm:able to methionine at this stage whereas preliminary observations suggested that cercariae absorb considerably less methionine than schistosomula (Chappell. unputlished data). Subsequent increasing permeability to methioninz could, therefore, be related to increased surface area on the one hand, development of gut function on the other, or indeed to both. However, sine: I have been unable to detect any basic difference in the kinetic; of methionine uptake in relation to worm age it is reasonable to postulate that, under the conditions described, the tegument provides the major route of methionine entry. Precisely how this might relate to the situatioll itt viva known. Preliminary experiments is not (Chappell, unpublished) show that methionine-“C injected intraperitoneally into mice is absorbed by adult schistosomes within IO min. but it would neither be feasible to investigate the kinetics of uptake itr viva nor would it be possible to determir.e
368
L. H.
CHAPPELL
the route of entry of solutes in this way. There are similarities between the kinetic characteristics of methionine absorption in S. mansoni and those of the cestode Hymenolepis diminuta (Read et al., 1963), particularly with regard to the relatively low degree of specificity encountered. There is also quantitative agreement in the kinetic constants of the saturable systems (K, for H. diminuttr -~- 0.31 mM, K, for S. munsoni z 0.33 mM: V,,, for H. diminrttn y 6.1 umoles/g/2 min (original rate given per h) and V,,,, for S. mansoni 4.0 umoles/g/ 2 min). Transport of neutral amino acids in many microbial and mammalian systems is unaffected or only slightly inhibited by other classes of amino acids (Eavenson & Christensen, 1967; Gazzola et al., 1972) while other systems show varying degrees of overlap between the different classes of amino acids (Hampton, 1970; Yamaguchi et al., I971 ; Eccleston & Kelly, 1972). Read er al. (1963) demonstrated inhibition ofmethionine uptake in H. dimimrta by histidine, aspartate, proline and hydroxyproline in addition to inhibitions by neutral amino acids; the least effective inhibitors were g-alanine, glutamate, lysine, cysteine and arginine. Methionine, on the other hand, inhibited glutamate uptake in H. diminuta. The methionine transport system of S. munsoni is not peculiar, then, in its low degree of specificity. Reiser & Christiansen (1969) attributed the inhibition of lysine uptake in rat everted intestinal sacs by leucine and methionine to allosteric alteration of the basic amino acid system by the binding of neutral amino acids to a discrete but specific region on a single or polyfunctional carrier (sic). There is no evidence to support this contention for the cross inhibitions observed in S. munsoni, though the fact that basic acids have a lower affinity for the methionine system than do neutral acids may indicate that allosteric effects are not important. The essential absence of qualitative differences between the uptake of methionine by adult and by juvenile worms is of interest; this, in conjunction with the quantitative difference between schistcsomula and older worms, renders the hypothesis of differential permeability to solutes as unlikely to account for the varying response of worms of different age to antimony or other drugs. The observation that three-week-old worms are less susceptible to antimony therapy than older or younger worms remains unexplained, although it may not be permissible to extrapolate from amino acid uptake to the absorption of potassium antimony tartrate. The precise location of worms of different ages in the host may account for this differential response to antimony; detoxication of antimony compounds occurs in the liver and both migrating schistosomula and adult worms in the mesenteric veins lie some distance from that organ. The present study has led to the view that permeability increases during the early stages of tegumental proliferation but not during the later stages of development and it is
I.J.P. VOL. 4. 1974
not clear at present whether there is any direct relationship between these two phenomena. Hockley & McLaren (1973) provide evidence that the adult schistosome tegument may be continually renewed and suggest that such complexity of structure may be related rather to the binding of host antigens than to any digestive or absorptive function. Further study is required to elucidate the function(s) of the schistosome surface. The minor differences in methionine uptake between adult males and females would seem to contrast with the suggestion of Senft (1968) that the male may furnish the female with amino acids in viva. However, it could be worthwhile to study in detail the permeability constants for a large number of amino acids in schistosomes of both sexes since these values may well be different. Additionally comparison of amino acid uptake by paired and single schistosomes should provide evidence on the question of nutritional synergism between adult paired worms. The biphasic mode of methionine entry described for S. mnnsoni, comprising saturable and apparently non-saturable components, is indicative that at low, probably physiological concentrations the majority of methionine absorbed enters via the saturable system. No evidence was obtained for active transport and the internal methionine concentration in adult males removed from mice was very low (0.14 mM). In contrast to this lsseroff & Levy (1972) reported the uptake of a number of amino acids, including methionine, by adult S. munsoni to occur by active processes. No explanation for this discrepancy is available at present. Furthermore, I have observed a marked dependence of methionine uptake on the concentration of sodium ions but insensitivity to ouabain, whereas lsseroff & Levy recorded sensitivity both to sodium ion concentration and to the presence of ouabain. It was thought possible that the saturable component of methionine uptake described above might represent adsorption to the tegument rather than absorption while true uptake would be represented by the non-saturable, diffusion component. Removal of the tegument by the freeze-thaw technique (Kusel, 1972), however, revealed that over 95 per cent of labelled methionine was located in subtegumental tissues. The occurrence of a large diffusion component is well documented in transport studies (Eavenson & Christensen, 1967; Woodward & Read, 1969; Hampton, 1970). It has been argued by Christensen & Liang (1966) that such non-saturable systems represent, in reality, mediated systems which were not measurably saturated under the experimental conditions adopted. This has not been substantiated and it is reasonable to question the possible selective advantage to any organism or cellular system in the possession of a transport process saturating at solute concentrations far in excess of those normally encountered.
I.J.P. VOL.4. 1974
Methionine
uptake in schistosomes
Meth~onine uptake rate in both larval and adult schistosomes was markedly affected by temperature. The differences in response by juveniles and by adults might be best explained in terms of physical properties of the tegument. Leakiness of larval membranes at higher temperatures is an unlikely explanation since heat killed adults and juveniles all absorbed less methionine than normal worms. There is no satisfactory explanation for the wide variability seen in the free amino acid pools of all stages of schistosomes. There is clearly a trend for a decrease in the size of the free pool during development from cercariae to adults, and this aspect of schistosome bicchemistry deserves further attention. Ackno+&&emenrsThe technical assistance of Mrs. B. Smvth. Miss S. Gardiner and Mr. S. Pocock is aratefullv acknowledged. Amino acids were analysed thr&gh the co-operation of Drs. D. W. T. Crompton, G. A. M. Cross and C. Milstein. Dr. G. C. Coles kindly read and commented on the manuscript. This study was supported by a grant from the Overseas Development Administration and thanks are extended to Dr. B. A. Newton for the provision of laboratory facilities.
BUEDI~QC~ E.
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