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Uptake and accumulation of amino acids in the brackish-water bivalve Corbicula japonica Prime during high salinity acclimation
J. Exp. Mar. Biol. Ecol., 1988, Vol. 123, pp. 201-210 Elsevier
201
JEM 01164
Uptake and accumulation of amino acids in the brackish-water bivalve Corbicula japonica Prime during high salinity acclimation 0. Matsushima
and Y. S. Hayashi
Zoological Institute, Faculty of Science, Hiroshima University, Hiroshima. Japan (Received
18 April 1988; revision
received
11 August
1988; accepted
22 August
1988)
r_-alanine in the external medium was taken up more rapidly by intact animals of the brackishwater bivalve Corbicula japonica Prime transferred from freshwater to 250 mOsm than to 2 mOsm. The animals transferred to 250 mOsm took up L-alanine at a rate comparable to that of elevation of the concentration of ninhydrin positive substances (NPS) in tissues. However, the tissue NPS accumulation in animals exposed to the increased salinity did not differ between the presence and absence of external L-alanine. When isolated gill and foot tissues were incubated under a hyperosmotic condition, accumulation of NPS was augmented by external L-alanine at concentrations as low as 0.5 mM. Such a remarkable augmentation by exogenous L-alanine was not found in the tissues incubated under the isosmotic condition. Uptake of amino acids appears to contribute, at least partly, to intracellular osmoregulation during high salinity acclimation. Abstract:
Key words: Amino
acid uptake;
Bivalve;
Free amino acid; Salinity
acclimation;
Volume regulation
INTRODUCTION
It is well known that marine and brackish-water molluscs utilize free amino acids as the chief solute for intracellular osmoregulation (Pierce & Greenberg, 1972, 1973), but the source of amino acids for enlargement of the intracellular pool during high salinity acclimation is not known. Bishop (1976) has reviewed three possible mechanisms for amino acid accumulation, i.e., ammonia fixation, amino acid uptake and protein degradation. Crowe (1981) has suggested that uptake of amino acids both from the environment and from the hemolymph should not be precluded as a candidate for the source of amino acid accumulation during high salinity acclimation. Pequignat (1973) investigated autoradiographically amino acid uptake in Mytilus edulis and suggested that mantle-gill area was the main site for direct absorption of dissolved organic solutes. Jorgensen (1983) also showed that externally labelled taurine was accumulated mainly in gills of M. edulis. Wright (198 5) reported that there appeared to be at least four pathways for epidermal amino acid transport, all Na-dependent, in gills of Mytilus cal$mzianus and suggested that the transport process plays a role in the reaccumulation of amino acids lost from the surface cells through passive diffusion. Correspondence address: Hiroshima 730, Japan. 0022-0981/88/$03.50
0
0. Matsushima,
1988 Elsevier
Zoological
Science
Institute,
Publishers
Faculty of Science, Hiroshima
B.V. (Biomedical
Division)
University,
202
O.MATSUSHIMAANDY.S.HAYASHI
In the present study, we examined the effects of external amino acid (r_-alanine) on accumulation of intracellular free amino acids in intact animals and isolated tissues of the brackish-water bivalve Corbiculu juponica Prime. Further, uptake of L-alanine at the concentration, detected in sediment interstitial water of the habitat of this species, was studied using intact animals exposed to artificial waters of increased or unchanged salinity. The possible involvement of amino acid uptake from the environment and from the hemolymph in intracellular osmoregulation during high salinity acclimation is discussed.
MATERIALS COLLECTION
AND
ANDMETHODS
ACCLIMATION
Specimens of brackish-water bivalve Corbicula japonica were collected in Hiroshima City and used for experiments after acclimation to freshwater (5-10 mOsm) for lo-30 days at 17-20 “C. This period was sufficient for complete acclimation because the animals opened their shell valves and extruded foot and siphons just a few hours after the transfer to freshwater. TISSUE
NPS CONCENTRATION
Intact clams, acclimated to freshwater, were transferred to either artificial pond water (APW, 2 mOsm) or diluted artificial seawater (DASW, 250 mOsm). Changes in concentrations of ninhydrin-positive substances (NPS) in tissues were examined with the respective media in the presence and absence of 0.1 mM L-alanine. Following transfer to the experimental media, animals were sampled over 7 days at Days 1, 3, and 7 for APW-incubated animals and at hours 5 and 10 and Days 1,2, 3, 5, and 7 for DASWincubated animals. The foot muscle and gill were dissected out from each animal, blotted lightly with filter paper and weighed to the nearest 0.1 mg. The tissues were heated at 90 ‘C for 15 min with 80% ethanol and homogenized with a glass-glass homogenizer. The homogenate was centrifuged at 20000 x g for 15 min and the supernatant was collected and evaporated at 40 “C under reduced pressure. The residue was dissolved in deionized water and assayed for NPS by the method of Yemm & Cocking (1955) with L-alanine as a standard. A standard curve was prepared for each assay. Amino acids in the tissue extracts were analysed by two-dimensional thin-layer chromatography (TLC), using microcrystalline cellulose plates (20 x 20 cm) and the following solvents: n-butanol : acetone : diethylamine : water (10 : 10 : 2 : 5) for the first dimensional development (3 h) and isopropanol : formic acid : water (20 : 1: 5) for the second dimensional development (6 h). After development the plates were immersed in ninhydrin solution, dried and allowed to stand overnight in darkness. Ninhydrin-positive spots were identified by comparing the developing position with that of standard amino acids or by developing a mixture of the sample and the predicted amino acids.
203
UPTAKE OF AMINO ACIDS IN CLAM
The details of this method The compositions
have been described
of APW
(Hanson
& Dietz,
previously
(Matsushima
1976) and DASW
et al., 1984).
were as follows,
APW: 0.5 mM NaCl, 0.4 mM CaCl,, 0.05 mM KCl, and 0.2 mM NaHCO,;
DASW:
112 mM NaCl, 2.5 mM KCl, 2.6 mM CaCl,, 12.4 mM MgCl,, and 0.2 mM NaHCO,. Osmotic concentrations of the media were determined with a freezing point depression osmometer (Advanced, DII). INCUBATION
OF ISOLATED
TISSUES
Foot and gill tissues were excised from the freshwater-acclimated animals, lightly blotted and weighed. Then the tissues were incubated in the media (4 ml medium/tissue 50-100 mg wet wt) of RFW (Ringer’s for freshwater-acclimated animals, 64 mOsm) or DAS W (250 mOsm) with different concentrations of L-alanine. During incubation the medium was constantly aerated and kept at 25 ‘C. After the 6-h incubation the tissues were subjected to measurement of NPS concentrations as described above. The composition of Ringer’s solution (RFW) for isosmotic incubation was determined according to the osmotic concentration of hemolymph from the freshwater-acclimated C. japonica (60-65 mOsm, Matsushima et al., 1986). NPS IN INTERSTITIAL
WATER
The substratum (mud) of the habitat of C. japonica was collected at low tide and filtered with filter paper. The filtrate was centrifuged at 20000 x g for 20 min, and the supernatant was subjected to ultrafiltration with Centrisart (Sartorius, SM13229E) by centrifugation at 2000 x g for 30 min (5 “C). The ultraliltrate was examined for NPS as described above and also used for TLC examination after desalting with Amberlite column. INCUBATION
FOR MEASUREMENT
OF L-ALANINE
UPTAKE
To examine the uptake of r_-alanine by clams, intact animals, previously acclimated to freshwater, were separately placed in 200 ml of APW or DASW containing 0.1 mM L-alanine and incubated at 23 “C with constant aeration. The respective incubation media were changed daily, and r_-alanine concentration was determined by the method described below. Since the t_-alanine concentration changed by evaporation during 24 h incubation, only the medium was also run for that correction. Another control was incubation of shell valves except for any soft part. Further, kanamycin (50 mg/l), an antibiotic, was added to exclude a possible bacterial effect on L-alanine depletion, and this was not harmful for the clams because they siphoned actively. After the experiments, the whole soft tissues were excised and dried overnight in a 95 “C drying oven. The water content of the tissue was determined from the wet and dry weight difference.
204 DETERMINATION
Concentrations
0. MATSUSHIMA
AND Y. S. HAYASHI
OF L-ALANINE
of L-alanine in the medium (APW or DASW),
in which intact animals
or valves were incubated for 24 h, were determined enzymatically by the method of Grass1 & Supp (1985). The final volume of assay mixture was 2.92 ml, in which 2.0 ml of sample, 55 U of lactate dehydrogenase, and 8 U of glutamate-pyruvate transaminase were included. Final concentrations of NADH and cr-ketoglutarate in the incubation mixture were 0.17 and 6.8 mM, respectively. Enzyme reaction for the assay was performed at 30 “C and completed within 60 min for APW and 90 min for DASW. Kanamycin scarcely influenced the L-alanine determination. STATISTICS
The data were analysed significance.
by Student’s
t test and P < 0.01 was used as the level of
RESULTS
In order to study the possible contribution of amino acid uptake to intracellular free amino acid accumulation, changes in tissue NPS concentrations were examined in the presence or absence of 0.1 mM L-alanine (Fig. 1). The NPS concentration in interstitial water of the sediments in the habitat was 0.062 mM & 0.010 ( k SD, n = 4), and only the alanine spot was detected on the TLC plate. When the animals acclimated to freshwater were transferred to DASW (250 mOsm), tissue NPS concentrations increased from z 10 pmol/g wet wt to z 60 pmol/g in the foot and to z 40 pmol/g in the gill. A large part of the NPS accumulation occurred within 2 days. The amount of NPS accumulated in response to this salinity increase did not differ between animals kept in the presence and absence of external L-alanine throughout the experimental period (P > 0.2 at any time). On the other hand, when the animals were transferred to APW (2 mOsm), NPS concentration did not increase in either foot or gill. Further, there was no effect of the addition of ambient L-alanine on the tissue NPS concentrations. Amino acid compositions of the NPS pool were tested in animals transferred to DASW (250 mOsm) with or without 0.1 mM r_-alanine. Two-dimensional thin-layer chromatography (TLC) revealed that there was no distinct difference in amino acid species accumulated in the tissues between the control (without alanine) and experimental (with alanine) animals. In the gill, alanine and glutamate were major, and glutamine, glycine, ornithine and aspartate were minor on the first day. However, on the 7th day of transfer, only two spots of major alanine and minor glutamate were detected. The results of TLC on foot were essentially the same as those on gill, but in foot tissue glycine and aspartate were not observed. These results suggest that although some minor amino acids accumulate during high salinity acclimation, two amino acids (alanine and glutamate), which accumulate ultimately, are selected as the intracellular osmolytes by C. japonica.
UPTAKE
OF AMINO
ACIDS
IN CLAM
205
Fig. 2 shows the effects of external L-alanine concentration in isolated tissues. The NPS concentration in foot of freshwater-acclimated animals was E 10 pmol/g wet wt. When the isolated foot from the freshwater-acclimated animals was incubated for 6 h in DASW (250 mOsm), NPS concentration increased to x 30pmol/g even in the absence of external L-alanine. When L-alanine was added to the incubation medium, NPS accumulation at all the concentrations > 0.1 mM (0.02 < P < 0.05) was significantly higher than that of the control (0 mM): P < 0.002 at 0.5 mM; P < 0.001 at 1,2, and 5 mM. Further, the increments in NPS accumulation were much greater than those expected from the concentrations of added L-ahurine. On the other hand, foot tissues incubated in RFW (64 mOsm), of which osmotic concentration was almost equivalent to that of the hemolymph of animals acclimated to freshwater, did not
I)
60-
P
5040$
30-
%
zo-
Foot
30-
I I o- ’ 01234567
I
1
I
I
Days Fig. 1. Changes in tissue NPS concentration of intact animals after transfer to APW (2 mOsm, triangle) or to DASW (250 mOsm, circle). Closed and open symbols represent the presence and absence of 0.1 mM L-alanine, respectively. The vertical bars indicate & SD (n = 3-4).
206
increase
0. MATSUSHIMA
NPS
concentration
concentration
in the absence
of external
r_-alanine.
in the presence of 2 and 5 mM L-alanine was significantly
of the control (P < O.Ol), the increments L-alanine
AND Y. S. HAYASHI
of NPS concentration
Though
NPS
higher than that
did not exceed the added
level.
6
250 mOsm
DASW
250 mOsm DASW
4
3 4
i
I +L
Foot
011 012345
I
I
0 Alanine
Gill -
I
1
2
3
4
5
( mM )
Fig. 2. NPS concentration in isolated tissues after 6 h incubation in DASW (250 mOsm, closed symbols) or RFW (64 mOsm, open symbols). Foot and gill tissues were isolated from the freshwater-acclimated animals and incubated for 6 h in media containing various concentrations of L-alanine. The vertical bars indicate + SD. The figure on each point represents the number of tissues examined.
When the gill tissues were incubated in DASW, accumulated NPS was remarkably increased by the addition of r_-alanine showing significantly higher levels than the control (P < 0.002 at 0.1 mM; P < 0.001 at 0.5, 1, 2, and 5 mM). The increments by L-alanine were greater than those in foot. The NPS concentration in gills incubated in RFW slightly increased by addition of r_-alanine; only significant at 1 mM (P< 0.005) and 5 mM (P-c0.001). Fig. 3 shows L-alanine consumption during 24-h incubation of an approximately equal-sized intact animal or shell valve in 200 ml of APW (2 mOsm) or DASW (250 mOsm) containing 0.1 mM (20 pmol) L-alanine. When shells were incubated in either APW or DASW, a great part of L-alanine was depleted 2-3 days after the onset of incubation. However, the addition of an antibiotic, kanamycin (50 mg/l), markedly reduced the depletion of L-alanine from the medium of shell incubation. Incubation of the medium without shells or intact animals did not result in any decrease in L-alanine concentration. These results evidently indicate that some bacteria attached to valves
UPTAKE
consumed
L-alanine.
depletion
of L-alanine
course coincided
OF AMINO
When intact animals increased
ACIDS
207
IN CLAM
were incubated
in APW with the antibiotic,
with the lapse of time after the transfer,
with that of the shell incubated
with kanamycin.
and the time
In APW, therefore,
0 shell without kana I shell with kana. m intact with kana.
DASW
1
ldl1 36
O-l
2-3
4-5 Days
o-1
after
2-3
4-5
transfer
Fig. 3. Changes in rates of depletion of 200 ml of 0.1 mM L-alanine by an intact animal (hatched column) or shell from one animal (open and solid columns) after the transfer to APW (left) or DASW (right). The animal and shell were incubated at 23 “C in 200 ml of the respective media containing 0.1 mM L-alanine, which were daily renewed; with (solid and hatched columns) or without (open column) kanamycin (50 mg/l). Data were obtained from 24 h incubation of O-1, 2-3, and 4-5 days after the transfer. The vertical bars indicate SD. The figure on each column of O-l day indicates the number of animals or shells examined.
a major
part of consumed L-alanine seemed to be attributed to kanamycin-resistant bacteria or other microorganisms. On the other hand, when the intact animals were transferred to DASW with kanamycin, a considerable amount of L-alanine was depleted and the time course of the depletion was independent of that of the shell incubated with the antibiotic. Even in the presence of kanamycin, some microorganisms, which could be perhaps mold, were observed to be adherent to the shell surface on and after 4-5 days of transfer in both incubations of the shell and intact animal and in both APW and DASW. Probably, this accounted for the increase in r_-alanine depletion on days 4-5 (Fig. 3, solid columns in both APW and DASW). Therefore, the comparison of the depletion rate between the intact animals transferred to APW and to DASW seemed valid only on the 1st day, because a marked increase in tissue NPS and a minimal influence by bacteria or other microorganisms were observed. The rate of depletion of
208
L-alanine
0. MATSUSHIMA
AND Y. S. HAYASHI
was 5 1.4 + 20.1 pmol/g dry wt per day for animal transferred
8.4 + 5.6 for the APW-animal (+ SD, n = 5 in each case), the difference
to DASW
and
being signiti-
cant (P < 0.005). DISCUSSION
The accumulation of NPS in isolated tissues exposed to an increased salinity (250 mOsm) was augmented by the addition of L-alanine; the augmentation far exceeded the concentration of exogenous L-alanine especially in gills. The isosmotic condition of incubation (RFW, 64 mOsm), however, did not stimulate NPS accumulation, and the addition of L-alanine caused a little increase in tissue NPS concentration. Further, under the condition of 250 mOsm, a remarkable increment in tissue NPS accumulation was induced by exogenous r_-alanine at a low concentration (< 0.5 mM). These results suggest that some active mechanism to take up L-alanine exists and is activated by the salinity increase as reported in other bivalve species (Stewart & Bamford, 1976; Wright, 1985). It has been reported that total free amino acid concentrations in blood of Modiolus demissus changed within the range of several (Strange & Crowe, 1979) or several tens of pmol/ml (Bartbarger & Pierce, 1976) during adaptation to salinity changes. Further, Gainey (1978) reported that the blood free amino acid concentrations in Polymesoda caroliniana (Corbiculidae) increased from x 0.4 to 0.8 pmol/ml in the first 10 h following transfer from IO%,, to freshwater and stabilized at 0.076 pmol/ml by 30 days, and that the principal amino acid in the blood was alanine as in the foot muscle. The brackishwater clam Corbicula japonica (Corbiculidae) in the present study also utilizes alanine as the chief intracellular osmotic effector (Matsushima et al., 1984) and, in response to osmotic changes, alters the concentration and composition of intracellular amino acid pool in a manner similar to P. caroliniana and Corbicula manilensis (Gainey, 1978). The range of added L-alanine concentration (< 0.5 mM), which greatly affected NPS accumulation
in isolated tissues exposed to hyperosmotic
salinity,
agrees with the range of
hemolymph amino acid concentration change in P. caroliniana described above. This indicates that the uptake of hemolymph amino acids into cells occurs physiologically and comprises a significant part of hyperosmotically-induced free amino acid accumulation in addition to intracellular metabolism (Bishop et al., 1981). The balance between influx and efflux of amino acids through the cell membrane may be important for conservation of amino acids within cells and could be altered by changing the concentration of Ca and Na ions (Matsushima, 1988). The experiments on intact animals revealed that r_-alanine in the external media was taken up more rapidly in 250 mOsm than in freshwater (2 mOsm). The concentration of external L-alanine in this experiment (0.1 mM) was not much different from NPS concentration in interstitial water from the sediments (0.062 mM); alanine was its main component. Stephens (1975) found the mean concentration of 0.05 mM primary amines in the interstitial water of inshore sediments in the habitat of annelids, Capitella capitata
UPTAKE OF AMINO ACIDS IN CLAM
and Nereis diversicolor. Crowe et al. (1977) reported of the sediment
interstitial
in the sediment
with a maximal
water in the habitat concentration
that primary
209
amine concentration
of Modiolus demissus varied with depth of 0.11 mM. Therefore,
the uptake
of
interstitial amino acids seems to be significant for such infaunal species as C. japonica, though the concentrations of primary amines in nearshore surface seawater (North, 1975) or of free amino acids in lake water (Jorgensen, 1987) are in two orders of magnitude smaller than those in interstitial waters. The rate of r_-alanine uptake by intact C. japonica transferred to 250 mOsm was FZ50 pmol/g dry wt per day, which was significantly higher than that of animals transferred to 2 mOsm, and it can be roughly converted to 10 pmol/g fresh wt per day. On the other hand, NPS accumulation in the intact animals transferred to 250 mOsm was 30-50 pmol/g fresh wt, indicating that the uptake from the external medium might comprise at least a part of hyperosmotically induced accumulation of intracellular amino acids. In spite of this rationale, NPS accumulation did not differ either quantitatively or qualitatively (TLC) between animals incubated with and without 0.1 mM L-alanine. These apparently contradictory results may be explained as follows. Although the animals exposed to a high salinity take up L-alanine in amounts comparable to those of intracellular accumulation, the introduced L-alanine could affect the intracellular metabolism of free amino acid production so as to maintain the proper rate of amino acid accumulation. The activation of L-alanine uptake by high salinity indicates its involvement in cell volume regulation. Stewart & Bamford (1976) have reported also that uptake of L-alanine by isolated gill ofMya arenaria is activated by increased salinity. Another explanation for these results is immediate degradation of L-alanine taken up without any contribution to volume regulation. However, this is unlikely because ammonia excretion markedly decreased in response to an increased salinity (Matsushima, 1982), and increased by a decreased salinity (Hayashi, 1987). Strange & Crowe (1979) have observed a decrease in hemolymph amino acid concentration concomitant with a rapid increase in intracellular Modiolus demissus when transferred to 100% seawater
amino acid concentration following a 6-h exposure
in to
500 mOsm and suggested that hemolymph amino acids may be utilized for cell volume regulation during high salinity acclimation. However, confirmation of this hypothesis would need additional experimental data, i.e., volume ratio of hemolymph to tissue mass in a certain species. In conclusion, it seems likely that the intracellular metabolic production of amino acids is the main source for hyperosmotic amino acid accumulation because in the absence of exogenous L-alanine amino acids were accumulated in tissues. However, uptake of amino acids at both organismal (from environment) and cellular (from hemolymph to cells) levels seems to contribute significantly to hyperosmotic volume regulation.
210
O.MATSUSHIMAANDY.S.HAYASHI REFERENCES
BARTBERGER, C. A.& S.K.PIERCE, 1976. Relationship between ammonia excretion rates and hemolymph nitrogenous compounds of a euryhaline bivalve during low salinity acclimation. Biol. Bull. (Woods Hole, Muss.), Vol. 150, pp. l-14. BISHOP, S. H., 1976. Nitrogen metabolism and excretion: regulation of intracellular amino acid concentration In, Estuarine processes, Vol. I, edited by M. Wiley, Academic Press, New York, pp. 414-431. BISHOP, S. H., D.E. GREENWALT & J.M. BURCHAM, 1981. Amino acid cycling in ribbed mussel tissues subjected to hyperosmotic shock. J. Exp. Zool., Vol. 215, pp. 277-287. CROWE, J. H., 198 1. Transport ofexogenous substrate and cell volume regulation in bivalve molluscs. J. Exp. Zool., Vol. 215, pp. 363-370. CROWE, J. H., K.A. DICKSON, J. L. Orro, R. D. COLON & K. K. FARLEY, 1977. Uptake of amino acids by the mussel Modiolus demissus. J. Exp. Zool., Vol. 202, pp. 323-332. GAINEY, L. F., 1978. The response of the Corbiculidae (Mollusca: Bivalvia) to osmotic stress: the cellular response. Physiol. Zool., Vol. 5 1, pp. 79-91. GRASSL, M. & M. SUPP, 1985. Determination with alanine aminotransferase and lactate dehydrogenase. In, Methods of enzymatic analysis, Vol. 8, edited by H.U. Bergmeyer, Academic Press, New York, pp. 345-349. HANSON, J.A. & T. H. DIEIZ, 1976. The role offree amino acids in cellular osmoregulation in the freshwater bivalve Ligumia subrostrata (Say). Can. J. Zool, Vol. 54, pp. 1927-1931. HAYASHI, Y.S., 1987. Some properties of glutamate dehydrogenase from the brackish-water bivalve Corbicula japonica (Prime). J. Exp. Mar. Biol. Ecol., Vol. 114, pp. 11 l-121. JORGENSEN, C. B., 1983. Patterns of uptake of dissolved amino acids in mussels (Mytilus edulis). Mar. Biol., Vol. 73, pp. 177-182. JORGENSEN, N. O.G., 1987. Free amino acids in lakes: concentrations and assimilation rates in relation to phytoplankton and bacterial production. Limnol. Oceanogr., Vol. 32, pp. 97-l 11. MATSUSHIMA, O., 1982. Comparative studies on responses to osmotic stresses in brackish and fresh-water clams. J. Sci. Hiroshima Univ., Vol. 30, pp. 173-192. MATSUSHIMA, O., 1988. Accumulation and conservation of free amino acids in isolated bivalve foot muscle exposed to hyperosmotic conditions. Camp. Biochem. Physiol., Vol. 90A, pp. 349-353. MATSUSHIMA, O., H. KATAYAMA, K. YAMADA & Y. KADO, 1984. Occurrence offree L-alanine and alanine racemase activity in bivalve molluscs with special reference to intracellular osmoregulation. Mar. Biol. Left., Vol. 5, pp. 217-225. MATSUSHIMA, O., N. SHIGA, Y. OMURA, H. KATAYAMA & K. YAMADA, 1986. Effect ofmetabolic inhibitors on hypoosmotically induced free amino acid et&x from the isolated foot of the brackish water bivalve Corbicula japonica. Camp. Biochem. Physiol., Vol. 84A, pp. 169-174. NORTH, B. B., 1975. Primary amines in California coastal waters: utilization by phytoplankton. Limnol. Oceanogr., Vol. 20, pp. 20-27. PEQUIGNAT, E., 1973. A kinetic and autoradiographic study of the direct assimilation of amino acids and glucose by organs of the mussel Mytilus edulis. Mar. Biol., Vol. 19, pp. 227-244. PIERCE, S. K. & M. J. GREENBERG, 1972. The nature of cellular volume regulation in marine bivalves. J. Exp. Biol., Vol. 51, pp. 681-692. PIERCE, S. K. & M. J. GREENBERG, 1973. The initiation and control of free amino acid regulation of cell volume in salinity-stressed bivalves. J. Exp. Biol., Vol. 59, pp. 435-446. STEPHENS, G.C., 1975. Uptake of naturally occurring primary amines by marine annelids. Biol. Bull., Vol. 149, pp. 397-407. STEWART, M. G. & D. R. BAMFORD, 1976. The effect of environmental factors on the absorption of amino acids by isolated gill tissue of the bivalve, Mya arenaria (L.). J. Exp. Mar. Biol. Ecol., Vol. 24, pp. 205-212. STRANGE, K. B. & J. H. CROWE, 1979. Acclimation to successive short term salinity changes by the bivalve Modiolus demissus. II. Nitrogen metabolism. J. Exp. Zool., Vol. 210, pp. 227-236. WRIGHT, S. H., 1985. Multiple pathways for amino acid transport in Mytilus gill. J. Comp. Physiol., Vol. 156, pp. 259-267. YEMM, E. W. & E.C. COCKING, 1955. The determination of amino-acids with ninhydrin. Analyst, Vol. 80, pp. 209-2 13.
201
JEM 01164
Uptake and accumulation of amino acids in the brackish-water bivalve Corbicula japonica Prime during high salinity acclimation 0. Matsushima
and Y. S. Hayashi
Zoological Institute, Faculty of Science, Hiroshima University, Hiroshima. Japan (Received
18 April 1988; revision
received
11 August
1988; accepted
22 August
1988)
r_-alanine in the external medium was taken up more rapidly by intact animals of the brackishwater bivalve Corbicula japonica Prime transferred from freshwater to 250 mOsm than to 2 mOsm. The animals transferred to 250 mOsm took up L-alanine at a rate comparable to that of elevation of the concentration of ninhydrin positive substances (NPS) in tissues. However, the tissue NPS accumulation in animals exposed to the increased salinity did not differ between the presence and absence of external L-alanine. When isolated gill and foot tissues were incubated under a hyperosmotic condition, accumulation of NPS was augmented by external L-alanine at concentrations as low as 0.5 mM. Such a remarkable augmentation by exogenous L-alanine was not found in the tissues incubated under the isosmotic condition. Uptake of amino acids appears to contribute, at least partly, to intracellular osmoregulation during high salinity acclimation. Abstract:
Key words: Amino
acid uptake;
Bivalve;
Free amino acid; Salinity
acclimation;
Volume regulation
INTRODUCTION
It is well known that marine and brackish-water molluscs utilize free amino acids as the chief solute for intracellular osmoregulation (Pierce & Greenberg, 1972, 1973), but the source of amino acids for enlargement of the intracellular pool during high salinity acclimation is not known. Bishop (1976) has reviewed three possible mechanisms for amino acid accumulation, i.e., ammonia fixation, amino acid uptake and protein degradation. Crowe (1981) has suggested that uptake of amino acids both from the environment and from the hemolymph should not be precluded as a candidate for the source of amino acid accumulation during high salinity acclimation. Pequignat (1973) investigated autoradiographically amino acid uptake in Mytilus edulis and suggested that mantle-gill area was the main site for direct absorption of dissolved organic solutes. Jorgensen (1983) also showed that externally labelled taurine was accumulated mainly in gills of M. edulis. Wright (198 5) reported that there appeared to be at least four pathways for epidermal amino acid transport, all Na-dependent, in gills of Mytilus cal$mzianus and suggested that the transport process plays a role in the reaccumulation of amino acids lost from the surface cells through passive diffusion. Correspondence address: Hiroshima 730, Japan. 0022-0981/88/$03.50
0
0. Matsushima,
1988 Elsevier
Zoological
Science
Institute,
Publishers
Faculty of Science, Hiroshima
B.V. (Biomedical
Division)
University,
202
O.MATSUSHIMAANDY.S.HAYASHI
In the present study, we examined the effects of external amino acid (r_-alanine) on accumulation of intracellular free amino acids in intact animals and isolated tissues of the brackish-water bivalve Corbiculu juponica Prime. Further, uptake of L-alanine at the concentration, detected in sediment interstitial water of the habitat of this species, was studied using intact animals exposed to artificial waters of increased or unchanged salinity. The possible involvement of amino acid uptake from the environment and from the hemolymph in intracellular osmoregulation during high salinity acclimation is discussed.
MATERIALS COLLECTION
AND
ANDMETHODS
ACCLIMATION
Specimens of brackish-water bivalve Corbicula japonica were collected in Hiroshima City and used for experiments after acclimation to freshwater (5-10 mOsm) for lo-30 days at 17-20 “C. This period was sufficient for complete acclimation because the animals opened their shell valves and extruded foot and siphons just a few hours after the transfer to freshwater. TISSUE
NPS CONCENTRATION
Intact clams, acclimated to freshwater, were transferred to either artificial pond water (APW, 2 mOsm) or diluted artificial seawater (DASW, 250 mOsm). Changes in concentrations of ninhydrin-positive substances (NPS) in tissues were examined with the respective media in the presence and absence of 0.1 mM L-alanine. Following transfer to the experimental media, animals were sampled over 7 days at Days 1, 3, and 7 for APW-incubated animals and at hours 5 and 10 and Days 1,2, 3, 5, and 7 for DASWincubated animals. The foot muscle and gill were dissected out from each animal, blotted lightly with filter paper and weighed to the nearest 0.1 mg. The tissues were heated at 90 ‘C for 15 min with 80% ethanol and homogenized with a glass-glass homogenizer. The homogenate was centrifuged at 20000 x g for 15 min and the supernatant was collected and evaporated at 40 “C under reduced pressure. The residue was dissolved in deionized water and assayed for NPS by the method of Yemm & Cocking (1955) with L-alanine as a standard. A standard curve was prepared for each assay. Amino acids in the tissue extracts were analysed by two-dimensional thin-layer chromatography (TLC), using microcrystalline cellulose plates (20 x 20 cm) and the following solvents: n-butanol : acetone : diethylamine : water (10 : 10 : 2 : 5) for the first dimensional development (3 h) and isopropanol : formic acid : water (20 : 1: 5) for the second dimensional development (6 h). After development the plates were immersed in ninhydrin solution, dried and allowed to stand overnight in darkness. Ninhydrin-positive spots were identified by comparing the developing position with that of standard amino acids or by developing a mixture of the sample and the predicted amino acids.
203
UPTAKE OF AMINO ACIDS IN CLAM
The details of this method The compositions
have been described
of APW
(Hanson
& Dietz,
previously
(Matsushima
1976) and DASW
et al., 1984).
were as follows,
APW: 0.5 mM NaCl, 0.4 mM CaCl,, 0.05 mM KCl, and 0.2 mM NaHCO,;
DASW:
112 mM NaCl, 2.5 mM KCl, 2.6 mM CaCl,, 12.4 mM MgCl,, and 0.2 mM NaHCO,. Osmotic concentrations of the media were determined with a freezing point depression osmometer (Advanced, DII). INCUBATION
OF ISOLATED
TISSUES
Foot and gill tissues were excised from the freshwater-acclimated animals, lightly blotted and weighed. Then the tissues were incubated in the media (4 ml medium/tissue 50-100 mg wet wt) of RFW (Ringer’s for freshwater-acclimated animals, 64 mOsm) or DAS W (250 mOsm) with different concentrations of L-alanine. During incubation the medium was constantly aerated and kept at 25 ‘C. After the 6-h incubation the tissues were subjected to measurement of NPS concentrations as described above. The composition of Ringer’s solution (RFW) for isosmotic incubation was determined according to the osmotic concentration of hemolymph from the freshwater-acclimated C. japonica (60-65 mOsm, Matsushima et al., 1986). NPS IN INTERSTITIAL
WATER
The substratum (mud) of the habitat of C. japonica was collected at low tide and filtered with filter paper. The filtrate was centrifuged at 20000 x g for 20 min, and the supernatant was subjected to ultrafiltration with Centrisart (Sartorius, SM13229E) by centrifugation at 2000 x g for 30 min (5 “C). The ultraliltrate was examined for NPS as described above and also used for TLC examination after desalting with Amberlite column. INCUBATION
FOR MEASUREMENT
OF L-ALANINE
UPTAKE
To examine the uptake of r_-alanine by clams, intact animals, previously acclimated to freshwater, were separately placed in 200 ml of APW or DASW containing 0.1 mM L-alanine and incubated at 23 “C with constant aeration. The respective incubation media were changed daily, and r_-alanine concentration was determined by the method described below. Since the t_-alanine concentration changed by evaporation during 24 h incubation, only the medium was also run for that correction. Another control was incubation of shell valves except for any soft part. Further, kanamycin (50 mg/l), an antibiotic, was added to exclude a possible bacterial effect on L-alanine depletion, and this was not harmful for the clams because they siphoned actively. After the experiments, the whole soft tissues were excised and dried overnight in a 95 “C drying oven. The water content of the tissue was determined from the wet and dry weight difference.
204 DETERMINATION
Concentrations
0. MATSUSHIMA
AND Y. S. HAYASHI
OF L-ALANINE
of L-alanine in the medium (APW or DASW),
in which intact animals
or valves were incubated for 24 h, were determined enzymatically by the method of Grass1 & Supp (1985). The final volume of assay mixture was 2.92 ml, in which 2.0 ml of sample, 55 U of lactate dehydrogenase, and 8 U of glutamate-pyruvate transaminase were included. Final concentrations of NADH and cr-ketoglutarate in the incubation mixture were 0.17 and 6.8 mM, respectively. Enzyme reaction for the assay was performed at 30 “C and completed within 60 min for APW and 90 min for DASW. Kanamycin scarcely influenced the L-alanine determination. STATISTICS
The data were analysed significance.
by Student’s
t test and P < 0.01 was used as the level of
RESULTS
In order to study the possible contribution of amino acid uptake to intracellular free amino acid accumulation, changes in tissue NPS concentrations were examined in the presence or absence of 0.1 mM L-alanine (Fig. 1). The NPS concentration in interstitial water of the sediments in the habitat was 0.062 mM & 0.010 ( k SD, n = 4), and only the alanine spot was detected on the TLC plate. When the animals acclimated to freshwater were transferred to DASW (250 mOsm), tissue NPS concentrations increased from z 10 pmol/g wet wt to z 60 pmol/g in the foot and to z 40 pmol/g in the gill. A large part of the NPS accumulation occurred within 2 days. The amount of NPS accumulated in response to this salinity increase did not differ between animals kept in the presence and absence of external L-alanine throughout the experimental period (P > 0.2 at any time). On the other hand, when the animals were transferred to APW (2 mOsm), NPS concentration did not increase in either foot or gill. Further, there was no effect of the addition of ambient L-alanine on the tissue NPS concentrations. Amino acid compositions of the NPS pool were tested in animals transferred to DASW (250 mOsm) with or without 0.1 mM r_-alanine. Two-dimensional thin-layer chromatography (TLC) revealed that there was no distinct difference in amino acid species accumulated in the tissues between the control (without alanine) and experimental (with alanine) animals. In the gill, alanine and glutamate were major, and glutamine, glycine, ornithine and aspartate were minor on the first day. However, on the 7th day of transfer, only two spots of major alanine and minor glutamate were detected. The results of TLC on foot were essentially the same as those on gill, but in foot tissue glycine and aspartate were not observed. These results suggest that although some minor amino acids accumulate during high salinity acclimation, two amino acids (alanine and glutamate), which accumulate ultimately, are selected as the intracellular osmolytes by C. japonica.
UPTAKE
OF AMINO
ACIDS
IN CLAM
205
Fig. 2 shows the effects of external L-alanine concentration in isolated tissues. The NPS concentration in foot of freshwater-acclimated animals was E 10 pmol/g wet wt. When the isolated foot from the freshwater-acclimated animals was incubated for 6 h in DASW (250 mOsm), NPS concentration increased to x 30pmol/g even in the absence of external L-alanine. When L-alanine was added to the incubation medium, NPS accumulation at all the concentrations > 0.1 mM (0.02 < P < 0.05) was significantly higher than that of the control (0 mM): P < 0.002 at 0.5 mM; P < 0.001 at 1,2, and 5 mM. Further, the increments in NPS accumulation were much greater than those expected from the concentrations of added L-ahurine. On the other hand, foot tissues incubated in RFW (64 mOsm), of which osmotic concentration was almost equivalent to that of the hemolymph of animals acclimated to freshwater, did not
I)
60-
P
5040$
30-
%
zo-
Foot
30-
I I o- ’ 01234567
I
1
I
I
Days Fig. 1. Changes in tissue NPS concentration of intact animals after transfer to APW (2 mOsm, triangle) or to DASW (250 mOsm, circle). Closed and open symbols represent the presence and absence of 0.1 mM L-alanine, respectively. The vertical bars indicate & SD (n = 3-4).
206
increase
0. MATSUSHIMA
NPS
concentration
concentration
in the absence
of external
r_-alanine.
in the presence of 2 and 5 mM L-alanine was significantly
of the control (P < O.Ol), the increments L-alanine
AND Y. S. HAYASHI
of NPS concentration
Though
NPS
higher than that
did not exceed the added
level.
6
250 mOsm
DASW
250 mOsm DASW
4
3 4
i
I +L
Foot
011 012345
I
I
0 Alanine
Gill -
I
1
2
3
4
5
( mM )
Fig. 2. NPS concentration in isolated tissues after 6 h incubation in DASW (250 mOsm, closed symbols) or RFW (64 mOsm, open symbols). Foot and gill tissues were isolated from the freshwater-acclimated animals and incubated for 6 h in media containing various concentrations of L-alanine. The vertical bars indicate + SD. The figure on each point represents the number of tissues examined.
When the gill tissues were incubated in DASW, accumulated NPS was remarkably increased by the addition of r_-alanine showing significantly higher levels than the control (P < 0.002 at 0.1 mM; P < 0.001 at 0.5, 1, 2, and 5 mM). The increments by L-alanine were greater than those in foot. The NPS concentration in gills incubated in RFW slightly increased by addition of r_-alanine; only significant at 1 mM (P< 0.005) and 5 mM (P-c0.001). Fig. 3 shows L-alanine consumption during 24-h incubation of an approximately equal-sized intact animal or shell valve in 200 ml of APW (2 mOsm) or DASW (250 mOsm) containing 0.1 mM (20 pmol) L-alanine. When shells were incubated in either APW or DASW, a great part of L-alanine was depleted 2-3 days after the onset of incubation. However, the addition of an antibiotic, kanamycin (50 mg/l), markedly reduced the depletion of L-alanine from the medium of shell incubation. Incubation of the medium without shells or intact animals did not result in any decrease in L-alanine concentration. These results evidently indicate that some bacteria attached to valves
UPTAKE
consumed
L-alanine.
depletion
of L-alanine
course coincided
OF AMINO
When intact animals increased
ACIDS
207
IN CLAM
were incubated
in APW with the antibiotic,
with the lapse of time after the transfer,
with that of the shell incubated
with kanamycin.
and the time
In APW, therefore,
0 shell without kana I shell with kana. m intact with kana.
DASW
1
ldl1 36
O-l
2-3
4-5 Days
o-1
after
2-3
4-5
transfer
Fig. 3. Changes in rates of depletion of 200 ml of 0.1 mM L-alanine by an intact animal (hatched column) or shell from one animal (open and solid columns) after the transfer to APW (left) or DASW (right). The animal and shell were incubated at 23 “C in 200 ml of the respective media containing 0.1 mM L-alanine, which were daily renewed; with (solid and hatched columns) or without (open column) kanamycin (50 mg/l). Data were obtained from 24 h incubation of O-1, 2-3, and 4-5 days after the transfer. The vertical bars indicate SD. The figure on each column of O-l day indicates the number of animals or shells examined.
a major
part of consumed L-alanine seemed to be attributed to kanamycin-resistant bacteria or other microorganisms. On the other hand, when the intact animals were transferred to DASW with kanamycin, a considerable amount of L-alanine was depleted and the time course of the depletion was independent of that of the shell incubated with the antibiotic. Even in the presence of kanamycin, some microorganisms, which could be perhaps mold, were observed to be adherent to the shell surface on and after 4-5 days of transfer in both incubations of the shell and intact animal and in both APW and DASW. Probably, this accounted for the increase in r_-alanine depletion on days 4-5 (Fig. 3, solid columns in both APW and DASW). Therefore, the comparison of the depletion rate between the intact animals transferred to APW and to DASW seemed valid only on the 1st day, because a marked increase in tissue NPS and a minimal influence by bacteria or other microorganisms were observed. The rate of depletion of
208
L-alanine
0. MATSUSHIMA
AND Y. S. HAYASHI
was 5 1.4 + 20.1 pmol/g dry wt per day for animal transferred
8.4 + 5.6 for the APW-animal (+ SD, n = 5 in each case), the difference
to DASW
and
being signiti-
cant (P < 0.005). DISCUSSION
The accumulation of NPS in isolated tissues exposed to an increased salinity (250 mOsm) was augmented by the addition of L-alanine; the augmentation far exceeded the concentration of exogenous L-alanine especially in gills. The isosmotic condition of incubation (RFW, 64 mOsm), however, did not stimulate NPS accumulation, and the addition of L-alanine caused a little increase in tissue NPS concentration. Further, under the condition of 250 mOsm, a remarkable increment in tissue NPS accumulation was induced by exogenous r_-alanine at a low concentration (< 0.5 mM). These results suggest that some active mechanism to take up L-alanine exists and is activated by the salinity increase as reported in other bivalve species (Stewart & Bamford, 1976; Wright, 1985). It has been reported that total free amino acid concentrations in blood of Modiolus demissus changed within the range of several (Strange & Crowe, 1979) or several tens of pmol/ml (Bartbarger & Pierce, 1976) during adaptation to salinity changes. Further, Gainey (1978) reported that the blood free amino acid concentrations in Polymesoda caroliniana (Corbiculidae) increased from x 0.4 to 0.8 pmol/ml in the first 10 h following transfer from IO%,, to freshwater and stabilized at 0.076 pmol/ml by 30 days, and that the principal amino acid in the blood was alanine as in the foot muscle. The brackishwater clam Corbicula japonica (Corbiculidae) in the present study also utilizes alanine as the chief intracellular osmotic effector (Matsushima et al., 1984) and, in response to osmotic changes, alters the concentration and composition of intracellular amino acid pool in a manner similar to P. caroliniana and Corbicula manilensis (Gainey, 1978). The range of added L-alanine concentration (< 0.5 mM), which greatly affected NPS accumulation
in isolated tissues exposed to hyperosmotic
salinity,
agrees with the range of
hemolymph amino acid concentration change in P. caroliniana described above. This indicates that the uptake of hemolymph amino acids into cells occurs physiologically and comprises a significant part of hyperosmotically-induced free amino acid accumulation in addition to intracellular metabolism (Bishop et al., 1981). The balance between influx and efflux of amino acids through the cell membrane may be important for conservation of amino acids within cells and could be altered by changing the concentration of Ca and Na ions (Matsushima, 1988). The experiments on intact animals revealed that r_-alanine in the external media was taken up more rapidly in 250 mOsm than in freshwater (2 mOsm). The concentration of external L-alanine in this experiment (0.1 mM) was not much different from NPS concentration in interstitial water from the sediments (0.062 mM); alanine was its main component. Stephens (1975) found the mean concentration of 0.05 mM primary amines in the interstitial water of inshore sediments in the habitat of annelids, Capitella capitata
UPTAKE OF AMINO ACIDS IN CLAM
and Nereis diversicolor. Crowe et al. (1977) reported of the sediment
interstitial
in the sediment
with a maximal
water in the habitat concentration
that primary
209
amine concentration
of Modiolus demissus varied with depth of 0.11 mM. Therefore,
the uptake
of
interstitial amino acids seems to be significant for such infaunal species as C. japonica, though the concentrations of primary amines in nearshore surface seawater (North, 1975) or of free amino acids in lake water (Jorgensen, 1987) are in two orders of magnitude smaller than those in interstitial waters. The rate of r_-alanine uptake by intact C. japonica transferred to 250 mOsm was FZ50 pmol/g dry wt per day, which was significantly higher than that of animals transferred to 2 mOsm, and it can be roughly converted to 10 pmol/g fresh wt per day. On the other hand, NPS accumulation in the intact animals transferred to 250 mOsm was 30-50 pmol/g fresh wt, indicating that the uptake from the external medium might comprise at least a part of hyperosmotically induced accumulation of intracellular amino acids. In spite of this rationale, NPS accumulation did not differ either quantitatively or qualitatively (TLC) between animals incubated with and without 0.1 mM L-alanine. These apparently contradictory results may be explained as follows. Although the animals exposed to a high salinity take up L-alanine in amounts comparable to those of intracellular accumulation, the introduced L-alanine could affect the intracellular metabolism of free amino acid production so as to maintain the proper rate of amino acid accumulation. The activation of L-alanine uptake by high salinity indicates its involvement in cell volume regulation. Stewart & Bamford (1976) have reported also that uptake of L-alanine by isolated gill ofMya arenaria is activated by increased salinity. Another explanation for these results is immediate degradation of L-alanine taken up without any contribution to volume regulation. However, this is unlikely because ammonia excretion markedly decreased in response to an increased salinity (Matsushima, 1982), and increased by a decreased salinity (Hayashi, 1987). Strange & Crowe (1979) have observed a decrease in hemolymph amino acid concentration concomitant with a rapid increase in intracellular Modiolus demissus when transferred to 100% seawater
amino acid concentration following a 6-h exposure
in to
500 mOsm and suggested that hemolymph amino acids may be utilized for cell volume regulation during high salinity acclimation. However, confirmation of this hypothesis would need additional experimental data, i.e., volume ratio of hemolymph to tissue mass in a certain species. In conclusion, it seems likely that the intracellular metabolic production of amino acids is the main source for hyperosmotic amino acid accumulation because in the absence of exogenous L-alanine amino acids were accumulated in tissues. However, uptake of amino acids at both organismal (from environment) and cellular (from hemolymph to cells) levels seems to contribute significantly to hyperosmotic volume regulation.
210
O.MATSUSHIMAANDY.S.HAYASHI REFERENCES
BARTBERGER, C. A.& S.K.PIERCE, 1976. Relationship between ammonia excretion rates and hemolymph nitrogenous compounds of a euryhaline bivalve during low salinity acclimation. Biol. Bull. (Woods Hole, Muss.), Vol. 150, pp. l-14. BISHOP, S. H., 1976. Nitrogen metabolism and excretion: regulation of intracellular amino acid concentration In, Estuarine processes, Vol. I, edited by M. Wiley, Academic Press, New York, pp. 414-431. BISHOP, S. H., D.E. GREENWALT & J.M. BURCHAM, 1981. Amino acid cycling in ribbed mussel tissues subjected to hyperosmotic shock. J. Exp. Zool., Vol. 215, pp. 277-287. CROWE, J. H., 198 1. Transport ofexogenous substrate and cell volume regulation in bivalve molluscs. J. Exp. Zool., Vol. 215, pp. 363-370. CROWE, J. H., K.A. DICKSON, J. L. Orro, R. D. COLON & K. K. FARLEY, 1977. Uptake of amino acids by the mussel Modiolus demissus. J. Exp. Zool., Vol. 202, pp. 323-332. GAINEY, L. F., 1978. The response of the Corbiculidae (Mollusca: Bivalvia) to osmotic stress: the cellular response. Physiol. Zool., Vol. 5 1, pp. 79-91. GRASSL, M. & M. SUPP, 1985. Determination with alanine aminotransferase and lactate dehydrogenase. In, Methods of enzymatic analysis, Vol. 8, edited by H.U. Bergmeyer, Academic Press, New York, pp. 345-349. HANSON, J.A. & T. H. DIEIZ, 1976. The role offree amino acids in cellular osmoregulation in the freshwater bivalve Ligumia subrostrata (Say). Can. J. Zool, Vol. 54, pp. 1927-1931. HAYASHI, Y.S., 1987. Some properties of glutamate dehydrogenase from the brackish-water bivalve Corbicula japonica (Prime). J. Exp. Mar. Biol. Ecol., Vol. 114, pp. 11 l-121. JORGENSEN, C. B., 1983. Patterns of uptake of dissolved amino acids in mussels (Mytilus edulis). Mar. Biol., Vol. 73, pp. 177-182. JORGENSEN, N. O.G., 1987. Free amino acids in lakes: concentrations and assimilation rates in relation to phytoplankton and bacterial production. Limnol. Oceanogr., Vol. 32, pp. 97-l 11. MATSUSHIMA, O., 1982. Comparative studies on responses to osmotic stresses in brackish and fresh-water clams. J. Sci. Hiroshima Univ., Vol. 30, pp. 173-192. MATSUSHIMA, O., 1988. Accumulation and conservation of free amino acids in isolated bivalve foot muscle exposed to hyperosmotic conditions. Camp. Biochem. Physiol., Vol. 90A, pp. 349-353. MATSUSHIMA, O., H. KATAYAMA, K. YAMADA & Y. KADO, 1984. Occurrence offree L-alanine and alanine racemase activity in bivalve molluscs with special reference to intracellular osmoregulation. Mar. Biol. Left., Vol. 5, pp. 217-225. MATSUSHIMA, O., N. SHIGA, Y. OMURA, H. KATAYAMA & K. YAMADA, 1986. Effect ofmetabolic inhibitors on hypoosmotically induced free amino acid et&x from the isolated foot of the brackish water bivalve Corbicula japonica. Camp. Biochem. Physiol., Vol. 84A, pp. 169-174. NORTH, B. B., 1975. Primary amines in California coastal waters: utilization by phytoplankton. Limnol. Oceanogr., Vol. 20, pp. 20-27. PEQUIGNAT, E., 1973. A kinetic and autoradiographic study of the direct assimilation of amino acids and glucose by organs of the mussel Mytilus edulis. Mar. Biol., Vol. 19, pp. 227-244. PIERCE, S. K. & M. J. GREENBERG, 1972. The nature of cellular volume regulation in marine bivalves. J. Exp. Biol., Vol. 51, pp. 681-692. PIERCE, S. K. & M. J. GREENBERG, 1973. The initiation and control of free amino acid regulation of cell volume in salinity-stressed bivalves. J. Exp. Biol., Vol. 59, pp. 435-446. STEPHENS, G.C., 1975. Uptake of naturally occurring primary amines by marine annelids. Biol. Bull., Vol. 149, pp. 397-407. STEWART, M. G. & D. R. BAMFORD, 1976. The effect of environmental factors on the absorption of amino acids by isolated gill tissue of the bivalve, Mya arenaria (L.). J. Exp. Mar. Biol. Ecol., Vol. 24, pp. 205-212. STRANGE, K. B. & J. H. CROWE, 1979. Acclimation to successive short term salinity changes by the bivalve Modiolus demissus. II. Nitrogen metabolism. J. Exp. Zool., Vol. 210, pp. 227-236. WRIGHT, S. H., 1985. Multiple pathways for amino acid transport in Mytilus gill. J. Comp. Physiol., Vol. 156, pp. 259-267. YEMM, E. W. & E.C. COCKING, 1955. The determination of amino-acids with ninhydrin. Analyst, Vol. 80, pp. 209-2 13.