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Transintegumentary absorption of acidic amino acids in the oligochaete annelid Enchytraeus albidus
Comp. Biochem. Physiol.. I.'ol. 61A. pp. 55 to 60 © Per!lamtm Pres~ Lid 1978. Printed in Great Britain
0300-9629/78/0F,01-0055502.00/0
TRANSINTEGUMENTARY ABSORPTION OF ACIDIC AMINO ACIDS IN THE OLIGOCHAETE ANNELID E N C H Y T R A E U S ALBIDUS DIETRICH SIEBERS and UTA EHLERS Biologische Anstalt Helgoland (Zentrale), D-2000 Hamburg 50, Palmaille 9, West Germany
(Received 27 October 1977) Abstract--l. Transintegumentary absorption of acidic amino acids in Enchytraeus albidus amounts
to only a few per cent of neutral amino acid uptake. 2. Influx of acidic amino acids is composed of a mediated route with high affinity and an apparent diffusional entry. 3. Glutamic acid influx is susceptible to metabolic inhibition, reduced salinity and Na+-levels and the presence of additional acidic amino acids. 4. Inhibition experiments suggest the presence of a distinct integumentary uptake system for acidic amino acids.
INTRODUCTION
transported across the body surface (Siebers & Bulnheim, 1977), glutamic acid and aspartic acid are absorbed at comparably low rates (present paper), evoking the authors' interest in further characterization of the uptake system.
Absorption of small organic molecules from very low ambient concentrations across the body surface is a widespread phenomenon in soft-bodied marine and brackish water invertebrates (Stephens, 1972, Jorgensen, 1976; Sepers, 1977). Such absorptive capacities seem to be absent in arthropods. Among annelids, transintegumentary solute uptake is comparatively well investigated in polychaetes (Stephens, 1963a, b, 1964; Chapman & Taylor, 1968; Ernst & Goerke, 1969; Reish & Stephens, 1969; Taylor, 1969; Ahearn & Gomme, 1972, 1975; G o m m e & Ahearn, 1972; Testerman, 1972). Enchytraeus albidus is the first oligochaete, for which absorption of dissolved organic molecules across the body surface has been demonstrated (Siebers & Bulnheim, 1976, 1977; Siebers, 1976). This fact is obviously due to difficulties in experimental handling of oligochaetes because of the smallness and sensitivity of most species. Absorption of acidic amino acids has been shown to occur by means of a saturable process across a variety of animal membranes, including preparations of subcellular organelles, single cells, organs, and the body surface of intact parasitic and free-living invertebrates (cf. Lerner & Steinke, 1977). It was demonstrated in a few cases that absorption of amino acids depends to a certain extent on the charge of the molecule. In contrast to neutral amino acids, intestinal absorption of acidic amino acids does not take place against a concentration gradient in mammalian everted gut sacs (Wiseman, 1953; Lin et al., 1962). Intestinal absorption of glutamic acid in the chicken is strongly reduced in comparison to leucine and lysine influx (Lerner & Steinke, 1977), and transintegumentary uptake of dissolved glutamic acid and aspartic acid amounts to only a few per cent of neutral amino acid uptake in the polychaete Nereis diversicolor at equal external concentrations (Stephens, 1975). In the oligochaete annelid Enchytraeus albidus the same phenomenon is obvious: unlike neutral exogenous amino acids, which are rapidly
MATERIALS AND METHODS
Enchytraeus albidus Henle was cultivated in moistened earth and fed with oat flakes. Before experimental use the oligochaetes were directly transferred to the bottom filter of aquaria (15°C, 20%0 salinity) and fed with flaky artificial fish food (Tetra Min). Under these conditions the worms can be maintained for over a year. After at least 2 weeks of acclimatization to submersal life the oligochaetes were investigated for uptake of acidic amino acids. The test assays contained about 50 mg fresh weight (about 15 individuals), L-[14C]amino acid (Amersham) and unlabeled amino acid(s) in a final water volume of l0 ml (15°C, 20%0 salinity). Incubations lasted 0.5 hr and were terminated by removal of water and rinsing the oligochaetes twice with seawater. The worms were then transferred to counting vials and stored at -25°C. After dissolution of animal material for 1.5 hr at 60°C in 0.5 ml of soluene-350 (Packard) and addition of l0 ml of counting solution, the samples were assayed for absorbed [~4C]label by liquid scintillation counting with quench corrections by internal standard. Details on preparation of experimental seawater are described by Siebers & Rosenthal (1977). Special care was devoted to the problem of unknown amino acid traces in seawater. They were controlled by direct fluorimetric determination with fluorescamine (North, 1975; Stephens, 1975) and in no case exceeded 1% of the experimentally applied amounts. To test the integrity of glutamic acid during the process of absorption from l0/aM solution for 0.5 hr, five 50 mg samples were pooled and homogenized with 80% methanol. Total low molecular weight organic material was isolated by procedures described by Siebers (1972) for crustacean hemolymph. Extracts were assayed for free amino acids by means of automated amino acid analysis (Beckman). The effluent was fractionated and counted for absorbed [14C]label. 55
56
DIETRICH SIEBERSand UTA EHLERS
Experiments concerning amino-acid uptake by mucuscovering bacteria or drinking were not undertaken. Numerous investigations in this respect have been performed by other authors, employing antibiotics or ligatures. All experiments showed a negligible participation of bacteria or drinking in transintegumentary uptake of amino acids by soft-bodied invertebrates. Uptake rates (nmoles/g fresh weight per hr) were calculated from the amounts of absorbed [*4C]radioactivity. Mean values between varying experimental groups (4-21 replicates) were compared by means of the t-test.
800
700
6OO o
o-
500
--~
400
RESULTS
Influx of labeled acidic L-amino acids Influx of [14C]L_aspartic acid from 10/~M solution is linear with time up to at least 45 min, covering the general incubation period of 0.5 hr, and deviating slightly from linearity thereafter (Fig. 1). The gradient of L-glutamic acid between 10/~M ambient concentration and intracellular levels amounted to about 1 : 1.3 x 103. Calculation of this gradient based on an L-glutamic acid concentration of 2.9 mmoles per g fresh weight at a salinity of 20°~ as derived from automated amino acid analysis of alcoholic extracts. We furthermore assumed that all free amino acids in worms were dissolved in intracellular water, since in marine invertebrates free amino acid concentrations of body fluids are generally very low in comparison to intracellular levels (Florkin, 1969). A reliable value for intracellular water was calculated from data published by Freeman & Shuttleworth (1977) for the polychaete annelid Arenicola marina, amounting to 21.9% of wet weight. Uptake of L-aspartic acid and L-glutamic acid amounts to only about 5~o of glycine and L-valine influx at comparable ambient concentrations (Fig. 2). L-aspartic acid uptake as a function of concentration is presented in Fig. 3 with an enlarged scale. It differs only inconsiderably from the respective L-glutamic acid curve. Figure 3 reveals the biphasic nature of L-aspartic acid uptake, being curvilinear up to about 50/tM and linear above this ~mbient concentration. This indicates the presence of at least two separate uptake systems. The first operates at lower concentrations and reveals saturation kinetics. The second 20
3OO 4-o 20O
o_ I O0
L I 0 I0 25
I 75
I 100
Concenfro'f'ion
L
150
250
,
Fig. 2. Rate of absorption of L-glutamic acid (0) and L-aspartic acid (O) in comparison to uptake of L-valine (A) and glycine (&) as a function of exogenous concentration. Values represent means + S.D. (vertical bars) from a sample size of four (neutral amino acids) and eight determinations (acidic amino acids). For standard deviations see Fig. 3. Curves for neutral amino acids are modified from Siebers & Bulnheim (1977). one, which operates at higher concentrations, is linear in relation to ambient concentration. The rates of these non-saturable, apparently diffusional entries were calculated from the slope of the linear portion of the uptake curve, amounting to 0.08 (L-aspartic acid) and 0.1 (L-glutamic acid) nmoles/g.hr per /~M exogenous acidic amino acid. The saturable components of the uptake curves, obtained by subtraction of apparent diffusional influx, had maximum uptake rates (Vm.Oof 16.0 (L-aspartic acid) and 18.4 nmoles/g per hr (L-glutamic acid) and transport constants o~ as
c
I 50
40
o. ~n
30
E
20
~o /
• ~
5
o J 1,5
J 30
l 45 rncubotion
i 120
60 time
,
,o 2~
5o
A
,oo
Concent"rofion,
Jso
z~o ~M
min
Fig. 1. Time course of [z4C]-aspartic acid influx from 10/~M solution. Results are expressed as means _+ S.D.
Fig. 3. Concentration-dependent influx of [14C]-L-aspartic acid (O). Results are expressed as means _+ S.D. from a sample size of eight determinations. The lower curve (O)
(vertical lines), obtained from a sample size of eight determinations.
represents the saturable component of uptake after correction for diffusion.
Uptake of acidic amino acids in I O0
@
80
60
\
4o • 2c
I
,O I 0
I I00
I I000
i 500 Concent"rGtion
,
¢xM
Fig. 4. Effects of increasing concentrations of L-aspartic acid on lO,uM glutamic acid uptake (©) and of L-glutamic acid on 10~M L-aspartic acid uptake (I). Results are means from a sample size of five determinations.
Enchytraeus
57.
( K , ) - - o b t a i n e d from Lineweaver-Burk p l o t s - - o f 5 10 (L-aspartic acid) and 3-6 (L-glutamic acid) #M. It is not obvious from Fig. 3, whether or not the nonsaturable c o m p o n e n t represents simple diffusion or belongs to a second facilitated system, which is nonsaturable within the applied concentration range. Further evidence supporting the existence of an apparent diffusional c o m p o n e n t is obtained from the fact that increasing concentrations of L-aspartic acid do not reduce 10/aM L-glutamic acid uptake (and vice versa) to zero levels (Fig. 4). Extracts of low molecular weight substances were prepared from worms after 0.5hr of 10#M ['4C]Lglutamic acid uptake and subjected to automated amino acid analysis. Roughly 70% of absorbed radioactivity c o - c h r o m a t o g r a p h with L-glutamic acid, while about 30% elute from the column, before the first amino acids appear, presumably in the form of organic acids. This result suggests that the majority of t-glutamic acid molecules must have been translocated unchanged in chemical nature.
Table 1. Effects of various concentrations of amino acids, carbohydrates, and organic acids on uptake of L-aspartic acid from 10/~M solution
Added substances
50/~M Uptake of L-aspartic acid (nmoles/g per hr) n = 4
Concentration of added substance 250/~M 500 #M Uptake of Uptake of L-aspartic acid L-aspartic acid Inhibition (nmoles/g per hr) Inhibition (nmoles/g per hr) (~o) n = 3 (%) n= 3
Inhibition
(%)
Acidic amino acids L-Cysteic acid o-Phospho-L-serine L-Glutamic acid Taurine
12.7 12.1 14.3 14.7
+ + + +
1.2" 1.0" 0.6* 1.4"
33 36 25 23
6.6 + 0.4* 10.5 + 1.0" 12.1 ___ 1.1" 13.8 + 1.3"
65 45 36 27
6.7 + 7.2 + 10.5 + 12.0 +
0.2* 0.4* 0.6* 0.9*
65 62 45 37
17.1 + 1.6 20.6 + 1.1
10 8
15.6 + 1.3" 18.2 -t- 2.0
18 4
14.7 + 1.6" 17.1 + 1.7
23 10
17.9 + 1.1 19.7 + 1.3 18.4 + 1.1
6 +4 3
18.0 + 1.0 18.5 _ 0.7 18.3 + 0.6
5 3 4
17.6 + 0.2 17.7 + 1.5 18.2 + 1.0
7 7 4
2.5 1.3 0.8 0.8 0.3
7 2 6 2 4
18.0 + 1.1 17.9 + 2.6 19.0 _+ 1.6 18.5 + 2.8 18.1 + 2.2
5 6 0 3 5
17.8 + 0.4 18.0 + 0.9 18.1 + 2.0 18.2 ___2.2 19.0 + 0.1
6 5 5 4 0
19.9 + 0.7 19.1 + 1.0 19.6 + 1.6
+5 +1 +3
20.4 + 1.4 18.5 + 0.9 18.7 + 2.4
+8 3 2
18.0 + 1.2 18.4 ___0.9 18.4 + 0.9
5 3 3
19.4 + 0.7 18.2 + 2.2 19.0 _ 2.0
+2 4 0
19.3 + 1.8 18.5 ___2.4 18.9 _ 0.8
+2 3 1
18.4 + 0.5 18.8 + 1.1 20.1 + 1.2
3
Amino acid amides L-Glutamine L-Asparagine Basic amino acids L-Arginine L-Lysine L-Ornithine Neutral amino acids L-Valine L-Alanine Glycine L-Serine L-Histidine
17.6 + 18.7 + 17.8 + 18.6 + 18.2 +
Carbohydrates Do(+ )-Glucose D-(-)-Ribose D-( -- )-Fructose Organic acids L-Lactic acid L-Malic acid x-Keto glutaric acid
Absorption in absence of a second substrate (controls, n = 4) amounted to 19.0 + 0.5 nmoles/g per hr. Values represent means + S.D. The level of significance in comparison to controls is P < 0.01 (*). All other differences are insignificant.
1
+6
DIETRICH S1EBERS a n d UTA EHLERS
58
Specificity
az
Susceptibility of L-aspartic acid influx to substances analogous in chemical structure provides further evidence for the presence of a saturable uptake component (Table l). Of all substances tested only additional acidic amino acids in the incubation water significantly inhibited L-aspartic acid uptake, while neutral and basic amino acids, carbohydrates, and organic acids showed no significant effects, if any. An indication for the assumption that the charge of the additional amino acid is responsible for the inhibition of e-aspartic acid uptake, is obtained from missing inhibitory effects of L-serine. In contrast, o-phosphoL-serine with a negative charge from an additional phosphate group inhibits L-aspartic acid uptake between 36 and 62'~. Special attention was devoted to the detection of possible interactions between acidic amino acids and 7-aminobutyric acid (GABA), since GABA and L-glutamic acid are known to function as neurotransmitters in invertebrates. As shown in Fig. 5 increasing concentrations (up to 100-fold) of GABA had no significant effect on 10/~M L-glutamic acid influx. In general: as described for neutral 0c-amino acids we also found no interactions between non-~-amino acids (fl-alanine, GABA) on uptake of L-glutamic acid or L-aspartic acid. Inhibition of L-glutamic acid influx from increasing concentrations (5~40 ~M) by a fixed concentration of L-aspartic acid (10/~M) is shown in Fig. 6. By plotting the reciprocal uptake (corrected for diffusion) against the reciprocal concentration two straight lines were obtained. The common intercept of the inhibited and the non-inhibited plot shows competitive inhibition. The same result was found for inhibition of L-aspartic acid by L-glutamic acid. The inhibitor constant (K~) obtained from uptake of L-glutamic acid from two concentrations (5 and 15 ~M) in the presence of increasing concentrations (5-20,uM) of L-aspartic acid amounted to 2.5/~M (Fig. 7), which is not far from the Krvalue of 3~/~M. I0 - r- tO0 o
a~ ~
0
0.2
~
~
-'
. ~ , ~" 0
,
005
I
0 I
02 I
Reciprocal conc.,
/~moles
Fig. 6. Lineweaver Burk plots for L-glutamic acid influx in absence (O) and in the presence (O) of 10/tM L-aspartic acid. E a c h value represents the mean of four determinations.
Effects of salinity, sodium, and metabolic inhibition As shown in Table 2 L-aspartic acid absorption across the body surface of E. albidus from l0/~M solution was positively correlated to ambient salinity levels. Experiments were performed after acclimatization of the oligochaetes to the respective salinities (0-40%o) for at least 2 weeks. Uptake rates were also susceptible to Na-depletion (Table 3). Investigations were performed after 4hr preincubations in appropriate media. When 20.°oo seawater was replaced by 348 mM NaC1 (equal in salinity to 200oo seawater), uptake rates were reduced by 60%. If, however, 75 mole-~o of Na + in the pure NaCl-solution were replaced by K + or Li+, further reductions in L-aspartic acid influx were observed. The results shown in Tables 2 and 3 provide evidence that transintegumentary L-aspartic acid influx considerably depends on one or several still unknown seawater components and additionally on the external Na+-concentration. The metabolic inhibitors NaCN (50-250 uM) and ouabain (100-1000/tM), applied during a 4hr preincubation and subsequent experimental period of 0.5hr, strongly reduce 10/IM L-aspartic acid influx (Table 4), implying the expenditure of metabolic energy and the involvement of the monovalent cation .c ~
t~
04
5--50
0.8
0.6
c
~-
0.4
o .~-
02
o
/
O
0--0
J I
tO0
I
I
500
Concentration,
~000
uM
Fig. 5. Effects of increasing concentrations of y-aminobutyric acid on influx of 10 ~M L-glutamic acid. Results are expressed as means + S.D. from a sample size of five determinations.
--K~
o
5
Inhibitor
I0 conc,,
15
20
/~M
Fig. 7. Uptake of L-glutamic acid from 5 (O) and 15 (O) #M solution in the presence of increasing concentrations of L-aspartic acid (inhibitor). Each value represents the mean of four determinations.
Uptake of acidic amino acids in Enchytraeus Table 2. Effect of salinity on 10/~M L-aspartic acid uptake Salinity ('.%o)
Uptake of L-aspartic acid (nmoles/g per hr)
0 10 20 30
0.7 _+ 0.2 (8) 5.3 _+ 0.7 (21) 10.2 _+ 1.6 (20) 12.4 _+ 1.0 (13)
Worms had been maintained in the bottom-filter of aquaria containing water of respective salinities for at least 2 weeks. Data are means + S.D.; the number of replicates is given in parentheses. Table 3. Effects of Na-depletion on 10/~M L-aspartic acid uptake after preincubation in appropriate media for 4hr Composition of incubation media
Uptake of L-aspartic acid (nmoles/g per hr)
%
10.6 + 0.1
100
348 mM NaCI
4.3 + 0.3
40
87 mM NaCI +261 mM KCI
3.1 _+ 0.3
31
87 mM NaCI +261 mM LiC1
2.1 + 0.2
20
20/°~° seawater
Values are means + S.D. from a sample size of seven determinations.
pump in the absorption process. These reductions are regarded to concern the saturable uptake component, since identical concentrations of metabolic inhibitors decrease e-aspartic acid uptake considerably less at higher exogenous concentrations (250 ~M), where diffusion as compared to saturable entry is much higher than at lower (10/~M) concentrations (Fig. 3). DISCUSSION Influx of a [14C]amino acid does not represent net flux, which is composed of influx and efflux. The fluorescamine reagent (North, 1975; Stephens, 1975), which forms stable fluorescent derivates with low
59
amino-acid concentrations in aqueous solution, is suitable for measuring net fluxes of rapidly accumulated amino acids, but fails to demonstrate low uptake rates. At an ambient concentration of 50 ~M, L-aspartic acid influx amounted to 19.0nmoles/g per hr (Fig. 3). If this represented net flux, 5 0 ~ M of L-aspartic acid would be reduced by 100 mg of worms in a volume of 10ml seawater during an incubation period of 1 hr by 0.4~o. This small difference is scarcely detectable. When we measured net flux of L-aspartic acid, the concentration (50/~M) was insignificantly reduced by about 1~o (1 hr, 10 ml incubation volume, 100 mg wet weight), suggesting that no efflux of any amino acids had occurred at rates exceeding influx. Whether or not the data presented for transintegumentary influx of acidic [14C]amino acids in E. albidus represent net fluxes, is uncertain. Only with respect to the findings that influx of [~*C]glycine is nearly identical to glycine net flux as measured with the fluorescamine reagent (Siebers & Bulnheim, 1977), we feel justified in using influx data also for the description of acidic amino acid transport in terms of Michaelis-Menten kinetics. In Enchytraeus albidus uptake systems for amino acids are charge-dependent. Absorption of neutral amino acids is strongly inhibited by additional neutral amino acids and to minor degrees by basic amino acids. Additional acidic amino acids have no effects (Siebers & Bulnheim, 1977). Basic amino acid uptake strongest interacts with additional basic amino acids and less with neutral amino acids, while acidic amino acids also exert no effects (Siebers, 1976). As demonstrated in the present paper, acidic amino acids only interact with additional acidic amino acids, suggesting the existence of an uptake system, which is clearly distinct from neutral and basic amino acid transport. Inhibition of L-glutamic acid influx by L-aspartic acid (and vice versa) is of competitive nature, suggesting the presence of an uptake system common to acidic amino acids. Charge-dependent uptake systems of amino acid transporting membranes are known for at least 3 decades (cf. Christensen, 1975). Only in recent years have these specificities been stated also for the solute transporting integuments of marine invertebrates (Jorgensen, 1976). The transport constant of L-glutamic acid influx (3-6/~M) in Enchytraeus albidus belongs to the lowest
Table 4. Effects of metabolic inhibitors (NaCN, ouabain) on uptake rates of L-aspartic acid from 10 and 250,uM solution after preincubation in appropriate inhibitor concentrations for 4 hr
Inhibitor concentration Controls 50#M NaCN 100/~M NaCN 250/~M NaCN 100/~M Ouabain 500/~M Ouabain 1000#M Ouabain
10/~M L-aspartic acid Uptake rate Inhibition (nmoles/g per hr) (~o)
250/~M L-aspartic acid Uptake rate Inhibition (nmoles/g per hr) (0/,,,)
10.6 + 2.0 7.2 _ 1.0 4.3 _+ 0.8 3.0 + 0.9
32 60 72
35.8 28.3 25.2 21.3
+ 3.3 _+ 1.7 + 1.7 + 1.5
21 30 40
6.8 + 0.6 5.1 + 0.9 4.5 _ 0.7
36 52 58
34.3 + 1.6n.s. 24.6 _+ 1.1 27,3 + 5.7
4 31 24
Values are means + S.D. obtained from a sample size of five determinations. As compared to controls all differences are significant (P < 0.01) except one, which was indicated as n.s. (not significant, P > 0.05).
60
DIETRICH SIEBERSand UTA EHLERS
values reported for animal tissues. Lerner & Steinke (1977) found a K, of 4-8/~M for L-glutamic acid transport across the chicken intestine. Krvalues for L-glutamic acid uptake by marine bacterial populations vary between 0.021 and 0.156#M (Sepers, 1977); in the integument of the parasitic trematode Schistosoma mansoni K t amounts to 140/~M (Isseroff et al., 1976), suggesting an adaptation of affinities to ambient concentrations in bacteria and trematode parasites. For interstitial waters of detritus-rich marine sediments Stephens (1975)reported concentrations of dissolved amino acids of about 50/~M. Krvalues of a few # M - - a s reported for amino acid uptake in several polycbaetes--seemingly reflect an adaptation of affinities to ambient concentrations in infaunal species, as represented by numerous polychaete and oligochaete annelids. Whether or not marine invertebrates actually obtain substantial nutritional benefits from transintegumentary solute uptake, is still a matter for discussion. While net uptake from 50/~M L-aspartic acid and L-glutamic acid did not differ in magnitude from uptake of glycine and L-serine in the polychaete Capitella capitata, acidic amino acid transport in the polychaete Nereis diversicolor amounted to only about 5~o of the respective data for neutral amino acids (Stephens, 1975). We reported here the same findings for the oligochaete E. albidus. In this worm transport of rapidly accumulated neutral a m i n o acids is susceptible to reduced salinity and Na+-levels and to the presence of metabolic inhibitors (Siebers & Bulnheim, 1977). Since influx of acidic amino acids underlies the same susceptibilities (present paper), these phenomena alone cannot explain the mentioned differences in charge-dependent uptake rates of amino acids. REFERENCES
AHEARN G. A. & GOMMEJ. (1972) Integumentary uptake, internal distribution, and metabolic degradation of exogenous glucose by Nereis diversicolor (Annelida, Polychaeta). Am. Zool. 12, 208 (Abstract). AHEARN G. A. & GOMMEJ. 0975) Transport of exogenous D-glucose by the integument of a polychaete worm (Nereis diversicolor Miiller). J. exp. Biol. 62, 243-264. CHAPMAN G. & TAYLOR A. G. 0968) Uptake of organic solutes by Nereis virens. Nature 217, 763-764. CHRISTENSENH. N. (1975) Biological Transport. Benjamin, Reading, MA. ERNST W. & GOERKE H. (1969) Aufnahme und Umwandlung gel6ster Glucose-[~4C] durch Lanice conchileya (Polychaeta, Terebellidae). Verfff. Inst. Meeresforsch. Bremerh. ll, 313-326. FLORKIN M. (1969) Nitrogen metabolism. In Chemical Zoology (Edited by FLORKIN M. & SCHEER B. T.), Vol. 4, pp. 147-162. Academic Press, New York. FREEMANR. F. H. & SHUTTLEWORTHT. J. (1977) Distribution of water in Arenicola marina (L.) equilibrated to diluted sea water. J. mar. biol. Ass. U.K. 57, 501-519. GOMMEJ. & AHEARNG. A. (1972) Transport of exogenous glucose across epidermal cell membranes of a polychaete worm. Am. Zool. 12, 207 (Abstract).
ISSEROFFH. I., ERTEL J. C. • LEVY M. G. (1976) Absorption of amino acids by Schistosoma mansoni. Comp. Biochem. Physiol. 54B, 125-133. JORGENSENC. B. (1976) August PiJtter, August Krogh, and modern ideas on the use of dissolved organic matter in aquatic environments. Biol. Rev. 51, 291 328. LERNER J. & STEINKE O. L. (1977) Intestinal absorption of glutamic acid in the chicken. Comp. Biochem. Physiol. 57A, l l 16. LIN E. C. C., HAGIHIRAH. & WILSON T. n. (1962) Specificity of the transport system for neutral amino acids in the hamster intestine. Am. d. Physiol. 202, 919-925. NORTH B. B. (1975) Primary amines in California coastal waters: utilization by phytoplankton. Limnol. Oceanogr. 20, 20-27. REISH D. J. & STEPHENSG. C. (1969) Uptake of Organic material by aquatic invertebrates. V. The influence of age on the uptake of glycine-C ~4 by the polychaete Neanthes arenaceodemata. Mar. Biol. 3, 352 355. SEVERS A. I. J. (1977) The utilization of dissolved organic compounds in aquatic environments. Hydrobiolooia 52, 39-54. SIEBERSD. 0972) Mechanismen der intrazellul/iren isosmotischen Regulation der Aminos/iurekonzentration bei dem Flul3krebs Orconectes limosus. Z. verol. Physiol. 76, 97-114. SIEBERS D. (1976) Absorption of neutral and basic amino acids across the body surface of two annelid species. Helgolihlder wiss. Meeresunters. 78, 456-466. S~EBERSD. & BULNHEIMH.-P. (1976) Salzgehaltsabh~ingigkeit der Aufnahme gelBster Aminos~iuren bei dem Oligochaeten Enchytraeus albidus. Verh. dt. Tool. Ges. 69, 212. SIEBERS D. & BULNHE1MH.-P. (1977) Salinity dependence. uptake kinetics, and specificity of amino-acid absorption across the body surface of the oligochaete annelid Enchytraeus albidus. Helgolih~der wiss. Meeresunters. 29, 473~,92. SIEBERSD. & ROSENI"HALH. (1977) Amino acid absorption by developing herring eggs. Helgolhnder wiss. Meeresunters. 29, 464~,72. S~PHENS G. C. (1963a) The influence of salinity on the uptake of glycine by euryhaline annelids, lnt. Congr. Zool. 16(2), 41. STEVHEYS G. C. (1963b) Uptake of organic material by aquatic invertebrates. II. Accumulation of amino acids by the bamboo worm, Clymenella torquata. Comp. Biochem. Physiol. |0, 191-202. STEPHENSG. C. (1964) Uptake of organic material by aquatic invertebrates. III. Uptake of glycine by brackishwater annelids. Biol. Bull. 126, 150--162. S'rEPHENS G. C. (1972) Amino acid accumulation and assimilation in marine organisms. In Nitrogen Metabolism and the Enviromnent (Edited by CAMPBELLJ. W. t~ GOLDSTEIN L.), pp. 318. Academic Press, New York. STEPHENS G. C. (1975) Uptake of naturally occurring primary amines by marine annelids. Biol. Bull. 149, 397~107. TAYLOR A. G. (1969) The direct uptake of amino acids and other small molecules from sea water by Nereis virens Sars. Comp. Biochem. Physiol. 29, 243-250. TES'rERMAN J. K. (1972) Accumulation of free fatty acids from sea water by marine invertebrates. Biol. Bull. 142, 160-177. WlSEMAN G. (1953) Absorption of amino acids using an in vitro technique. J. Physiol., Lond. 120, 63-72.
0300-9629/78/0F,01-0055502.00/0
TRANSINTEGUMENTARY ABSORPTION OF ACIDIC AMINO ACIDS IN THE OLIGOCHAETE ANNELID E N C H Y T R A E U S ALBIDUS DIETRICH SIEBERS and UTA EHLERS Biologische Anstalt Helgoland (Zentrale), D-2000 Hamburg 50, Palmaille 9, West Germany
(Received 27 October 1977) Abstract--l. Transintegumentary absorption of acidic amino acids in Enchytraeus albidus amounts
to only a few per cent of neutral amino acid uptake. 2. Influx of acidic amino acids is composed of a mediated route with high affinity and an apparent diffusional entry. 3. Glutamic acid influx is susceptible to metabolic inhibition, reduced salinity and Na+-levels and the presence of additional acidic amino acids. 4. Inhibition experiments suggest the presence of a distinct integumentary uptake system for acidic amino acids.
INTRODUCTION
transported across the body surface (Siebers & Bulnheim, 1977), glutamic acid and aspartic acid are absorbed at comparably low rates (present paper), evoking the authors' interest in further characterization of the uptake system.
Absorption of small organic molecules from very low ambient concentrations across the body surface is a widespread phenomenon in soft-bodied marine and brackish water invertebrates (Stephens, 1972, Jorgensen, 1976; Sepers, 1977). Such absorptive capacities seem to be absent in arthropods. Among annelids, transintegumentary solute uptake is comparatively well investigated in polychaetes (Stephens, 1963a, b, 1964; Chapman & Taylor, 1968; Ernst & Goerke, 1969; Reish & Stephens, 1969; Taylor, 1969; Ahearn & Gomme, 1972, 1975; G o m m e & Ahearn, 1972; Testerman, 1972). Enchytraeus albidus is the first oligochaete, for which absorption of dissolved organic molecules across the body surface has been demonstrated (Siebers & Bulnheim, 1976, 1977; Siebers, 1976). This fact is obviously due to difficulties in experimental handling of oligochaetes because of the smallness and sensitivity of most species. Absorption of acidic amino acids has been shown to occur by means of a saturable process across a variety of animal membranes, including preparations of subcellular organelles, single cells, organs, and the body surface of intact parasitic and free-living invertebrates (cf. Lerner & Steinke, 1977). It was demonstrated in a few cases that absorption of amino acids depends to a certain extent on the charge of the molecule. In contrast to neutral amino acids, intestinal absorption of acidic amino acids does not take place against a concentration gradient in mammalian everted gut sacs (Wiseman, 1953; Lin et al., 1962). Intestinal absorption of glutamic acid in the chicken is strongly reduced in comparison to leucine and lysine influx (Lerner & Steinke, 1977), and transintegumentary uptake of dissolved glutamic acid and aspartic acid amounts to only a few per cent of neutral amino acid uptake in the polychaete Nereis diversicolor at equal external concentrations (Stephens, 1975). In the oligochaete annelid Enchytraeus albidus the same phenomenon is obvious: unlike neutral exogenous amino acids, which are rapidly
MATERIALS AND METHODS
Enchytraeus albidus Henle was cultivated in moistened earth and fed with oat flakes. Before experimental use the oligochaetes were directly transferred to the bottom filter of aquaria (15°C, 20%0 salinity) and fed with flaky artificial fish food (Tetra Min). Under these conditions the worms can be maintained for over a year. After at least 2 weeks of acclimatization to submersal life the oligochaetes were investigated for uptake of acidic amino acids. The test assays contained about 50 mg fresh weight (about 15 individuals), L-[14C]amino acid (Amersham) and unlabeled amino acid(s) in a final water volume of l0 ml (15°C, 20%0 salinity). Incubations lasted 0.5 hr and were terminated by removal of water and rinsing the oligochaetes twice with seawater. The worms were then transferred to counting vials and stored at -25°C. After dissolution of animal material for 1.5 hr at 60°C in 0.5 ml of soluene-350 (Packard) and addition of l0 ml of counting solution, the samples were assayed for absorbed [~4C]label by liquid scintillation counting with quench corrections by internal standard. Details on preparation of experimental seawater are described by Siebers & Rosenthal (1977). Special care was devoted to the problem of unknown amino acid traces in seawater. They were controlled by direct fluorimetric determination with fluorescamine (North, 1975; Stephens, 1975) and in no case exceeded 1% of the experimentally applied amounts. To test the integrity of glutamic acid during the process of absorption from l0/aM solution for 0.5 hr, five 50 mg samples were pooled and homogenized with 80% methanol. Total low molecular weight organic material was isolated by procedures described by Siebers (1972) for crustacean hemolymph. Extracts were assayed for free amino acids by means of automated amino acid analysis (Beckman). The effluent was fractionated and counted for absorbed [14C]label. 55
56
DIETRICH SIEBERSand UTA EHLERS
Experiments concerning amino-acid uptake by mucuscovering bacteria or drinking were not undertaken. Numerous investigations in this respect have been performed by other authors, employing antibiotics or ligatures. All experiments showed a negligible participation of bacteria or drinking in transintegumentary uptake of amino acids by soft-bodied invertebrates. Uptake rates (nmoles/g fresh weight per hr) were calculated from the amounts of absorbed [*4C]radioactivity. Mean values between varying experimental groups (4-21 replicates) were compared by means of the t-test.
800
700
6OO o
o-
500
--~
400
RESULTS
Influx of labeled acidic L-amino acids Influx of [14C]L_aspartic acid from 10/~M solution is linear with time up to at least 45 min, covering the general incubation period of 0.5 hr, and deviating slightly from linearity thereafter (Fig. 1). The gradient of L-glutamic acid between 10/~M ambient concentration and intracellular levels amounted to about 1 : 1.3 x 103. Calculation of this gradient based on an L-glutamic acid concentration of 2.9 mmoles per g fresh weight at a salinity of 20°~ as derived from automated amino acid analysis of alcoholic extracts. We furthermore assumed that all free amino acids in worms were dissolved in intracellular water, since in marine invertebrates free amino acid concentrations of body fluids are generally very low in comparison to intracellular levels (Florkin, 1969). A reliable value for intracellular water was calculated from data published by Freeman & Shuttleworth (1977) for the polychaete annelid Arenicola marina, amounting to 21.9% of wet weight. Uptake of L-aspartic acid and L-glutamic acid amounts to only about 5~o of glycine and L-valine influx at comparable ambient concentrations (Fig. 2). L-aspartic acid uptake as a function of concentration is presented in Fig. 3 with an enlarged scale. It differs only inconsiderably from the respective L-glutamic acid curve. Figure 3 reveals the biphasic nature of L-aspartic acid uptake, being curvilinear up to about 50/tM and linear above this ~mbient concentration. This indicates the presence of at least two separate uptake systems. The first operates at lower concentrations and reveals saturation kinetics. The second 20
3OO 4-o 20O
o_ I O0
L I 0 I0 25
I 75
I 100
Concenfro'f'ion
L
150
250
,
Fig. 2. Rate of absorption of L-glutamic acid (0) and L-aspartic acid (O) in comparison to uptake of L-valine (A) and glycine (&) as a function of exogenous concentration. Values represent means + S.D. (vertical bars) from a sample size of four (neutral amino acids) and eight determinations (acidic amino acids). For standard deviations see Fig. 3. Curves for neutral amino acids are modified from Siebers & Bulnheim (1977). one, which operates at higher concentrations, is linear in relation to ambient concentration. The rates of these non-saturable, apparently diffusional entries were calculated from the slope of the linear portion of the uptake curve, amounting to 0.08 (L-aspartic acid) and 0.1 (L-glutamic acid) nmoles/g.hr per /~M exogenous acidic amino acid. The saturable components of the uptake curves, obtained by subtraction of apparent diffusional influx, had maximum uptake rates (Vm.Oof 16.0 (L-aspartic acid) and 18.4 nmoles/g per hr (L-glutamic acid) and transport constants o~ as
c
I 50
40
o. ~n
30
E
20
~o /
• ~
5
o J 1,5
J 30
l 45 rncubotion
i 120
60 time
,
,o 2~
5o
A
,oo
Concent"rofion,
Jso
z~o ~M
min
Fig. 1. Time course of [z4C]-aspartic acid influx from 10/~M solution. Results are expressed as means _+ S.D.
Fig. 3. Concentration-dependent influx of [14C]-L-aspartic acid (O). Results are expressed as means _+ S.D. from a sample size of eight determinations. The lower curve (O)
(vertical lines), obtained from a sample size of eight determinations.
represents the saturable component of uptake after correction for diffusion.
Uptake of acidic amino acids in I O0
@
80
60
\
4o • 2c
I
,O I 0
I I00
I I000
i 500 Concent"rGtion
,
¢xM
Fig. 4. Effects of increasing concentrations of L-aspartic acid on lO,uM glutamic acid uptake (©) and of L-glutamic acid on 10~M L-aspartic acid uptake (I). Results are means from a sample size of five determinations.
Enchytraeus
57.
( K , ) - - o b t a i n e d from Lineweaver-Burk p l o t s - - o f 5 10 (L-aspartic acid) and 3-6 (L-glutamic acid) #M. It is not obvious from Fig. 3, whether or not the nonsaturable c o m p o n e n t represents simple diffusion or belongs to a second facilitated system, which is nonsaturable within the applied concentration range. Further evidence supporting the existence of an apparent diffusional c o m p o n e n t is obtained from the fact that increasing concentrations of L-aspartic acid do not reduce 10/aM L-glutamic acid uptake (and vice versa) to zero levels (Fig. 4). Extracts of low molecular weight substances were prepared from worms after 0.5hr of 10#M ['4C]Lglutamic acid uptake and subjected to automated amino acid analysis. Roughly 70% of absorbed radioactivity c o - c h r o m a t o g r a p h with L-glutamic acid, while about 30% elute from the column, before the first amino acids appear, presumably in the form of organic acids. This result suggests that the majority of t-glutamic acid molecules must have been translocated unchanged in chemical nature.
Table 1. Effects of various concentrations of amino acids, carbohydrates, and organic acids on uptake of L-aspartic acid from 10/~M solution
Added substances
50/~M Uptake of L-aspartic acid (nmoles/g per hr) n = 4
Concentration of added substance 250/~M 500 #M Uptake of Uptake of L-aspartic acid L-aspartic acid Inhibition (nmoles/g per hr) Inhibition (nmoles/g per hr) (~o) n = 3 (%) n= 3
Inhibition
(%)
Acidic amino acids L-Cysteic acid o-Phospho-L-serine L-Glutamic acid Taurine
12.7 12.1 14.3 14.7
+ + + +
1.2" 1.0" 0.6* 1.4"
33 36 25 23
6.6 + 0.4* 10.5 + 1.0" 12.1 ___ 1.1" 13.8 + 1.3"
65 45 36 27
6.7 + 7.2 + 10.5 + 12.0 +
0.2* 0.4* 0.6* 0.9*
65 62 45 37
17.1 + 1.6 20.6 + 1.1
10 8
15.6 + 1.3" 18.2 -t- 2.0
18 4
14.7 + 1.6" 17.1 + 1.7
23 10
17.9 + 1.1 19.7 + 1.3 18.4 + 1.1
6 +4 3
18.0 + 1.0 18.5 _ 0.7 18.3 + 0.6
5 3 4
17.6 + 0.2 17.7 + 1.5 18.2 + 1.0
7 7 4
2.5 1.3 0.8 0.8 0.3
7 2 6 2 4
18.0 + 1.1 17.9 + 2.6 19.0 _+ 1.6 18.5 + 2.8 18.1 + 2.2
5 6 0 3 5
17.8 + 0.4 18.0 + 0.9 18.1 + 2.0 18.2 ___2.2 19.0 + 0.1
6 5 5 4 0
19.9 + 0.7 19.1 + 1.0 19.6 + 1.6
+5 +1 +3
20.4 + 1.4 18.5 + 0.9 18.7 + 2.4
+8 3 2
18.0 + 1.2 18.4 ___0.9 18.4 + 0.9
5 3 3
19.4 + 0.7 18.2 + 2.2 19.0 _ 2.0
+2 4 0
19.3 + 1.8 18.5 ___2.4 18.9 _ 0.8
+2 3 1
18.4 + 0.5 18.8 + 1.1 20.1 + 1.2
3
Amino acid amides L-Glutamine L-Asparagine Basic amino acids L-Arginine L-Lysine L-Ornithine Neutral amino acids L-Valine L-Alanine Glycine L-Serine L-Histidine
17.6 + 18.7 + 17.8 + 18.6 + 18.2 +
Carbohydrates Do(+ )-Glucose D-(-)-Ribose D-( -- )-Fructose Organic acids L-Lactic acid L-Malic acid x-Keto glutaric acid
Absorption in absence of a second substrate (controls, n = 4) amounted to 19.0 + 0.5 nmoles/g per hr. Values represent means + S.D. The level of significance in comparison to controls is P < 0.01 (*). All other differences are insignificant.
1
+6
DIETRICH S1EBERS a n d UTA EHLERS
58
Specificity
az
Susceptibility of L-aspartic acid influx to substances analogous in chemical structure provides further evidence for the presence of a saturable uptake component (Table l). Of all substances tested only additional acidic amino acids in the incubation water significantly inhibited L-aspartic acid uptake, while neutral and basic amino acids, carbohydrates, and organic acids showed no significant effects, if any. An indication for the assumption that the charge of the additional amino acid is responsible for the inhibition of e-aspartic acid uptake, is obtained from missing inhibitory effects of L-serine. In contrast, o-phosphoL-serine with a negative charge from an additional phosphate group inhibits L-aspartic acid uptake between 36 and 62'~. Special attention was devoted to the detection of possible interactions between acidic amino acids and 7-aminobutyric acid (GABA), since GABA and L-glutamic acid are known to function as neurotransmitters in invertebrates. As shown in Fig. 5 increasing concentrations (up to 100-fold) of GABA had no significant effect on 10/~M L-glutamic acid influx. In general: as described for neutral 0c-amino acids we also found no interactions between non-~-amino acids (fl-alanine, GABA) on uptake of L-glutamic acid or L-aspartic acid. Inhibition of L-glutamic acid influx from increasing concentrations (5~40 ~M) by a fixed concentration of L-aspartic acid (10/~M) is shown in Fig. 6. By plotting the reciprocal uptake (corrected for diffusion) against the reciprocal concentration two straight lines were obtained. The common intercept of the inhibited and the non-inhibited plot shows competitive inhibition. The same result was found for inhibition of L-aspartic acid by L-glutamic acid. The inhibitor constant (K~) obtained from uptake of L-glutamic acid from two concentrations (5 and 15 ~M) in the presence of increasing concentrations (5-20,uM) of L-aspartic acid amounted to 2.5/~M (Fig. 7), which is not far from the Krvalue of 3~/~M. I0 - r- tO0 o
a~ ~
0
0.2
~
~
-'
. ~ , ~" 0
,
005
I
0 I
02 I
Reciprocal conc.,
/~moles
Fig. 6. Lineweaver Burk plots for L-glutamic acid influx in absence (O) and in the presence (O) of 10/tM L-aspartic acid. E a c h value represents the mean of four determinations.
Effects of salinity, sodium, and metabolic inhibition As shown in Table 2 L-aspartic acid absorption across the body surface of E. albidus from l0/~M solution was positively correlated to ambient salinity levels. Experiments were performed after acclimatization of the oligochaetes to the respective salinities (0-40%o) for at least 2 weeks. Uptake rates were also susceptible to Na-depletion (Table 3). Investigations were performed after 4hr preincubations in appropriate media. When 20.°oo seawater was replaced by 348 mM NaC1 (equal in salinity to 200oo seawater), uptake rates were reduced by 60%. If, however, 75 mole-~o of Na + in the pure NaCl-solution were replaced by K + or Li+, further reductions in L-aspartic acid influx were observed. The results shown in Tables 2 and 3 provide evidence that transintegumentary L-aspartic acid influx considerably depends on one or several still unknown seawater components and additionally on the external Na+-concentration. The metabolic inhibitors NaCN (50-250 uM) and ouabain (100-1000/tM), applied during a 4hr preincubation and subsequent experimental period of 0.5hr, strongly reduce 10/IM L-aspartic acid influx (Table 4), implying the expenditure of metabolic energy and the involvement of the monovalent cation .c ~
t~
04
5--50
0.8
0.6
c
~-
0.4
o .~-
02
o
/
O
0--0
J I
tO0
I
I
500
Concentration,
~000
uM
Fig. 5. Effects of increasing concentrations of y-aminobutyric acid on influx of 10 ~M L-glutamic acid. Results are expressed as means + S.D. from a sample size of five determinations.
--K~
o
5
Inhibitor
I0 conc,,
15
20
/~M
Fig. 7. Uptake of L-glutamic acid from 5 (O) and 15 (O) #M solution in the presence of increasing concentrations of L-aspartic acid (inhibitor). Each value represents the mean of four determinations.
Uptake of acidic amino acids in Enchytraeus Table 2. Effect of salinity on 10/~M L-aspartic acid uptake Salinity ('.%o)
Uptake of L-aspartic acid (nmoles/g per hr)
0 10 20 30
0.7 _+ 0.2 (8) 5.3 _+ 0.7 (21) 10.2 _+ 1.6 (20) 12.4 _+ 1.0 (13)
Worms had been maintained in the bottom-filter of aquaria containing water of respective salinities for at least 2 weeks. Data are means + S.D.; the number of replicates is given in parentheses. Table 3. Effects of Na-depletion on 10/~M L-aspartic acid uptake after preincubation in appropriate media for 4hr Composition of incubation media
Uptake of L-aspartic acid (nmoles/g per hr)
%
10.6 + 0.1
100
348 mM NaCI
4.3 + 0.3
40
87 mM NaCI +261 mM KCI
3.1 _+ 0.3
31
87 mM NaCI +261 mM LiC1
2.1 + 0.2
20
20/°~° seawater
Values are means + S.D. from a sample size of seven determinations.
pump in the absorption process. These reductions are regarded to concern the saturable uptake component, since identical concentrations of metabolic inhibitors decrease e-aspartic acid uptake considerably less at higher exogenous concentrations (250 ~M), where diffusion as compared to saturable entry is much higher than at lower (10/~M) concentrations (Fig. 3). DISCUSSION Influx of a [14C]amino acid does not represent net flux, which is composed of influx and efflux. The fluorescamine reagent (North, 1975; Stephens, 1975), which forms stable fluorescent derivates with low
59
amino-acid concentrations in aqueous solution, is suitable for measuring net fluxes of rapidly accumulated amino acids, but fails to demonstrate low uptake rates. At an ambient concentration of 50 ~M, L-aspartic acid influx amounted to 19.0nmoles/g per hr (Fig. 3). If this represented net flux, 5 0 ~ M of L-aspartic acid would be reduced by 100 mg of worms in a volume of 10ml seawater during an incubation period of 1 hr by 0.4~o. This small difference is scarcely detectable. When we measured net flux of L-aspartic acid, the concentration (50/~M) was insignificantly reduced by about 1~o (1 hr, 10 ml incubation volume, 100 mg wet weight), suggesting that no efflux of any amino acids had occurred at rates exceeding influx. Whether or not the data presented for transintegumentary influx of acidic [14C]amino acids in E. albidus represent net fluxes, is uncertain. Only with respect to the findings that influx of [~*C]glycine is nearly identical to glycine net flux as measured with the fluorescamine reagent (Siebers & Bulnheim, 1977), we feel justified in using influx data also for the description of acidic amino acid transport in terms of Michaelis-Menten kinetics. In Enchytraeus albidus uptake systems for amino acids are charge-dependent. Absorption of neutral amino acids is strongly inhibited by additional neutral amino acids and to minor degrees by basic amino acids. Additional acidic amino acids have no effects (Siebers & Bulnheim, 1977). Basic amino acid uptake strongest interacts with additional basic amino acids and less with neutral amino acids, while acidic amino acids also exert no effects (Siebers, 1976). As demonstrated in the present paper, acidic amino acids only interact with additional acidic amino acids, suggesting the existence of an uptake system, which is clearly distinct from neutral and basic amino acid transport. Inhibition of L-glutamic acid influx by L-aspartic acid (and vice versa) is of competitive nature, suggesting the presence of an uptake system common to acidic amino acids. Charge-dependent uptake systems of amino acid transporting membranes are known for at least 3 decades (cf. Christensen, 1975). Only in recent years have these specificities been stated also for the solute transporting integuments of marine invertebrates (Jorgensen, 1976). The transport constant of L-glutamic acid influx (3-6/~M) in Enchytraeus albidus belongs to the lowest
Table 4. Effects of metabolic inhibitors (NaCN, ouabain) on uptake rates of L-aspartic acid from 10 and 250,uM solution after preincubation in appropriate inhibitor concentrations for 4 hr
Inhibitor concentration Controls 50#M NaCN 100/~M NaCN 250/~M NaCN 100/~M Ouabain 500/~M Ouabain 1000#M Ouabain
10/~M L-aspartic acid Uptake rate Inhibition (nmoles/g per hr) (~o)
250/~M L-aspartic acid Uptake rate Inhibition (nmoles/g per hr) (0/,,,)
10.6 + 2.0 7.2 _ 1.0 4.3 _+ 0.8 3.0 + 0.9
32 60 72
35.8 28.3 25.2 21.3
+ 3.3 _+ 1.7 + 1.7 + 1.5
21 30 40
6.8 + 0.6 5.1 + 0.9 4.5 _ 0.7
36 52 58
34.3 + 1.6n.s. 24.6 _+ 1.1 27,3 + 5.7
4 31 24
Values are means + S.D. obtained from a sample size of five determinations. As compared to controls all differences are significant (P < 0.01) except one, which was indicated as n.s. (not significant, P > 0.05).
60
DIETRICH SIEBERSand UTA EHLERS
values reported for animal tissues. Lerner & Steinke (1977) found a K, of 4-8/~M for L-glutamic acid transport across the chicken intestine. Krvalues for L-glutamic acid uptake by marine bacterial populations vary between 0.021 and 0.156#M (Sepers, 1977); in the integument of the parasitic trematode Schistosoma mansoni K t amounts to 140/~M (Isseroff et al., 1976), suggesting an adaptation of affinities to ambient concentrations in bacteria and trematode parasites. For interstitial waters of detritus-rich marine sediments Stephens (1975)reported concentrations of dissolved amino acids of about 50/~M. Krvalues of a few # M - - a s reported for amino acid uptake in several polycbaetes--seemingly reflect an adaptation of affinities to ambient concentrations in infaunal species, as represented by numerous polychaete and oligochaete annelids. Whether or not marine invertebrates actually obtain substantial nutritional benefits from transintegumentary solute uptake, is still a matter for discussion. While net uptake from 50/~M L-aspartic acid and L-glutamic acid did not differ in magnitude from uptake of glycine and L-serine in the polychaete Capitella capitata, acidic amino acid transport in the polychaete Nereis diversicolor amounted to only about 5~o of the respective data for neutral amino acids (Stephens, 1975). We reported here the same findings for the oligochaete E. albidus. In this worm transport of rapidly accumulated neutral a m i n o acids is susceptible to reduced salinity and Na+-levels and to the presence of metabolic inhibitors (Siebers & Bulnheim, 1977). Since influx of acidic amino acids underlies the same susceptibilities (present paper), these phenomena alone cannot explain the mentioned differences in charge-dependent uptake rates of amino acids. REFERENCES
AHEARN G. A. & GOMMEJ. (1972) Integumentary uptake, internal distribution, and metabolic degradation of exogenous glucose by Nereis diversicolor (Annelida, Polychaeta). Am. Zool. 12, 208 (Abstract). AHEARN G. A. & GOMMEJ. 0975) Transport of exogenous D-glucose by the integument of a polychaete worm (Nereis diversicolor Miiller). J. exp. Biol. 62, 243-264. CHAPMAN G. & TAYLOR A. G. 0968) Uptake of organic solutes by Nereis virens. Nature 217, 763-764. CHRISTENSENH. N. (1975) Biological Transport. Benjamin, Reading, MA. ERNST W. & GOERKE H. (1969) Aufnahme und Umwandlung gel6ster Glucose-[~4C] durch Lanice conchileya (Polychaeta, Terebellidae). Verfff. Inst. Meeresforsch. Bremerh. ll, 313-326. FLORKIN M. (1969) Nitrogen metabolism. In Chemical Zoology (Edited by FLORKIN M. & SCHEER B. T.), Vol. 4, pp. 147-162. Academic Press, New York. FREEMANR. F. H. & SHUTTLEWORTHT. J. (1977) Distribution of water in Arenicola marina (L.) equilibrated to diluted sea water. J. mar. biol. Ass. U.K. 57, 501-519. GOMMEJ. & AHEARNG. A. (1972) Transport of exogenous glucose across epidermal cell membranes of a polychaete worm. Am. Zool. 12, 207 (Abstract).
ISSEROFFH. I., ERTEL J. C. • LEVY M. G. (1976) Absorption of amino acids by Schistosoma mansoni. Comp. Biochem. Physiol. 54B, 125-133. JORGENSENC. B. (1976) August PiJtter, August Krogh, and modern ideas on the use of dissolved organic matter in aquatic environments. Biol. Rev. 51, 291 328. LERNER J. & STEINKE O. L. (1977) Intestinal absorption of glutamic acid in the chicken. Comp. Biochem. Physiol. 57A, l l 16. LIN E. C. C., HAGIHIRAH. & WILSON T. n. (1962) Specificity of the transport system for neutral amino acids in the hamster intestine. Am. d. Physiol. 202, 919-925. NORTH B. B. (1975) Primary amines in California coastal waters: utilization by phytoplankton. Limnol. Oceanogr. 20, 20-27. REISH D. J. & STEPHENSG. C. (1969) Uptake of Organic material by aquatic invertebrates. V. The influence of age on the uptake of glycine-C ~4 by the polychaete Neanthes arenaceodemata. Mar. Biol. 3, 352 355. SEVERS A. I. J. (1977) The utilization of dissolved organic compounds in aquatic environments. Hydrobiolooia 52, 39-54. SIEBERSD. 0972) Mechanismen der intrazellul/iren isosmotischen Regulation der Aminos/iurekonzentration bei dem Flul3krebs Orconectes limosus. Z. verol. Physiol. 76, 97-114. SIEBERS D. (1976) Absorption of neutral and basic amino acids across the body surface of two annelid species. Helgolihlder wiss. Meeresunters. 78, 456-466. S~EBERSD. & BULNHEIMH.-P. (1976) Salzgehaltsabh~ingigkeit der Aufnahme gelBster Aminos~iuren bei dem Oligochaeten Enchytraeus albidus. Verh. dt. Tool. Ges. 69, 212. SIEBERS D. & BULNHE1MH.-P. (1977) Salinity dependence. uptake kinetics, and specificity of amino-acid absorption across the body surface of the oligochaete annelid Enchytraeus albidus. Helgolih~der wiss. Meeresunters. 29, 473~,92. SIEBERSD. & ROSENI"HALH. (1977) Amino acid absorption by developing herring eggs. Helgolhnder wiss. Meeresunters. 29, 464~,72. S~PHENS G. C. (1963a) The influence of salinity on the uptake of glycine by euryhaline annelids, lnt. Congr. Zool. 16(2), 41. STEVHEYS G. C. (1963b) Uptake of organic material by aquatic invertebrates. II. Accumulation of amino acids by the bamboo worm, Clymenella torquata. Comp. Biochem. Physiol. |0, 191-202. STEPHENSG. C. (1964) Uptake of organic material by aquatic invertebrates. III. Uptake of glycine by brackishwater annelids. Biol. Bull. 126, 150--162. S'rEPHENS G. C. (1972) Amino acid accumulation and assimilation in marine organisms. In Nitrogen Metabolism and the Enviromnent (Edited by CAMPBELLJ. W. t~ GOLDSTEIN L.), pp. 318. Academic Press, New York. STEPHENS G. C. (1975) Uptake of naturally occurring primary amines by marine annelids. Biol. Bull. 149, 397~107. TAYLOR A. G. (1969) The direct uptake of amino acids and other small molecules from sea water by Nereis virens Sars. Comp. Biochem. Physiol. 29, 243-250. TES'rERMAN J. K. (1972) Accumulation of free fatty acids from sea water by marine invertebrates. Biol. Bull. 142, 160-177. WlSEMAN G. (1953) Absorption of amino acids using an in vitro technique. J. Physiol., Lond. 120, 63-72.