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Fate of endogenous free amino acids in osmotic adjustment of Crassostrea virginica (Gmelin)
Camp.Biochem.Physiol.Vol. 82A, No. 3, pp. 571-576, 1985
0300-9629/X5 163.00+ 0.00
! 1985
Printed in Great Britain
Pergamon
Press Ltd
FATE OF ENDOGENOUS FREE AMINO ACIDS IN OSMOTIC ADJUSTMENT OF CRASSOSTREA VIRGINICA (GMELIN)” BARBARA WEGENER HEAVERS and C. S. HAMMEN Department
of Zoology,
University
of Rhode
Island, 792-2372
Kingston,
RI 02881,
USA.
Telephone:
(401)
(Received 21 Februurv 1985) Abstract-l. Free amino acids (FAA) in mantle tissue of whole oysters, adapted to sea-water or one-half salinity, were determined by automatic amino acid analyzer; isolated tissue was incubated with ‘%-labeled substrates, transferred to different media, and labeled metabolites were determined by liquid scintillation counter. 2. FAA accounted for l6.7-19.7% of osmotically active substances; taurine made up 57~71”:,, alanine, aspartic acid and glycine 19-32’4,. and all other amino acids together about 10%. 3. After 3 hr in reduced salinity, tissue FAA decreased 32% and ‘%02 production increased 2.7-fold; in increased salinity, FAA increased 2.8-fold and “‘CO2 production decreased by 12%. 4. Significant efflux and biosynthesis of FAA occurred within I hr of transfer. but did not account for all loss or gain.
shown FAA regularly constitute 5-20’2: of nitrogenous compounds released (Hammen, 1969). A study of isolated hearts of Modiolus demissus, exposed to a medium of lower salinity for 3 hr, showed that FAA loss was sufficient to account for all of the reduction in tissue concentration (Pierce and Greenberg, 1972). On the other hand, a marked decrease in taurine content occurred in Mya arenaria placed in water of reduced salinity, but no taurine appeared in the medium (Allen and Garrett, 1972). One goal of the research reported here was to determine whether oysters release FAA as such on exposure to lower salinity media. Pathways of amino acid degradation and biosynthesis may be activated by changes in salinity (Bishop, 1976). In particular, the glutamate dehydrogenase reaction, which is directly affected by salt concentration in crustaceans, is now known to occur in bivalves, but there is still no evidence that it is important in osmotic adjustment (Bishop et al., 1983). Increases in FAA with adjustment to higher salinity could occur by means of new biosynthesis, uptake from the environment, or depolymerization of polypeptide. There is no information on which of these processes are used by oysters. In this study, we have examined early changes in the FAA pool size in oysters subjected to short-term salinity stress. Aspects we have considered include: (1) changes in catabolic or anabolic activities reflected in O? consumption and CO, production from labelled substrate, (2) the rate of change in the tissue FAA pool during short-term exposure and adaptation, and (3) the fate of endogenous FAA during hyposmotic stress either by direct release, hemolymph shifts and/or catabolism to ammonia and CO,.
INTRODUCTION
The American oyster, Crassostrea virginica, is an euryhaline osmoconformer, thriving in estuarine situations where salinity is lower than ocean water and varies seasonally (Galtsoff, 1964). Physiological chemists in the period 1901-1905 discovered that the tissues of marine mollusks contain less chloride than present in hemolymph or sea-water, and that the deficit is made up in part by free amino acids (FAA) such as taurine and glycine (Krogh, 1939). In the Portuguese oyster, Crassostrea angulata, FAA made up an average of 30% of the total osmotically active substances (TOS) in adductor muscles of animals adapted to waters of both 1188 and 594mosM (Bricteux-Gregoire et al., 1964). In muscle of C. virginica, FAA accounted for 20.9% of the total osmotic effect in water of 785mosM, and those undergoing the greatest changes with salinity were taurine, alanine, glycine and proline (Lynch and Wood, 1966). It is well known that in mollusks, FAA make a dominant contribution to intracellular osmolarity, and are varied or adjusted more than mineral cations (Somero and Bowlus, 1983). The means for accomplishing these adjustments, however, are not well known. Processes of cellular respiration, intermediary metabolism and nitrogenous excretion are intertwined to make adjustment “a complicated process” (Bishop et al., 1983). One possible way to adjust to lower salinity is simply to release the surplus amino acids. Analysis of excretion water of C. virginica has
*This work is derived from a dissertation submitted to the University of Rhode Island by the senior author in partial fulfillment of the degree of Doctor of Philosophy. Partial support of this research was provided by a NSF Summer Traineeship from the University of Rhode Island and a grant from the Membrane Physiology Laboratory of Duke University, Durham, North Carolina.
MATERIALS
AND METHODS
Oysters used in this study were collected at Bird Shoal, Beaufort, North Carolina during two summers and one fall 571
512
BARBARA WEGENER HEAVERS and C. S. HAMMEN
season. Weights ranged from 50 to 150 g. They were maintained at Duke University Marine Laboratory in running sea-water or in aerated 50% sea-water tanks. Temperatures ranged from 19 C in summer, to 8’C in fall. Salinities were 31-340/w for sea-water (SW) and 17~19X for 50% sea-water (SW/Z). Animals were considered SW/2-adapted after 3 weeks holding in 5O”/0 sea-water. Experimental saline solutions were prepared from local sea-water. They were filtered and buffered to pH 7.5 with H,BO, (0.0088 M). SW/2 was prepared by mixing equal volumes of glass-distilled water and full strength sea-water. Room temperature was 20-22-C. Pyruvate- I-14C and alanine-U-‘4C were obtained from Sigma Chemical Company. Samples of excised mantle tissue (25-125 mg) included a portion of the lobulated border along with a small portion of the thin, adjacent, interior area. Samples of fluid were removed from the pericardial cavity by syringe. The percentage of water composition was determined by the difference in wet and dry weights of mantle tissue of SW and SW/2-adapted animals. SW-adapted animals were exposed to SW/2 for 6 and 24 hr. Excised mantle from SW animals was exposed to SW/2 for 10min.
The total osmolality of the pericardial fluid was determined on 0.5-ml samples with an Advanced Osmometer. Model 65-3 1. Ammonia
unalysis
Tissue ammonia levels and excretion rates were determined by placing paired portions of excised mantle tissue from a single oyster in separate 5-ml samples of aerated water for 4 hr. Afterward, each tissue was blotted, weighed and homogenized in 2 ml of cold 50% trichloroacetic acid in a glass grinder. The homogenate was washed into a 1Oml volumetric flask and diluted to 1 in 10 with ammonia-free water. Dilutions were centrifuged at 25,OOOg for 10 min and the supernatant was analyzed for ammonia by the microdiffusion method of Conway (1957). The incubation media were similarly analyzed. FAA unalysis Pooled samples (N = 10) of pericardial fluid were prepared for each group of animals. These were centrifuged at 25,000 g for 30 min, and the supernatant was dialyzed. The FAA fraction was evaporated to near dryness, transferred by rinsings, then completely dried. Mantle tissue samples (N = IO) were placed in boiling water for 15min. cooled, homogenized and dialyzed. The FAA fraction was evaporated to near dryness and the remainder transferred to a vial along with rinsings (4 x 2 ml). The sample was reduced to dryness and diluted again for analysis. Amino acids were analyzed on a Beckman Model 116 Amino Acid Analyzer.
Incubation with alaninr-U-‘4C Two pieces of tissue, one from each side of the mantle of an oyster, were placed in aerated SW containing alanine-U14C, ZpCi/ml and 20mM L-alanine for 90min. Each was then moved through a separate series of rinsing vials containing 2.5 ml at 5-min intervals for 180 min. One piece was then exposed for 60 min to 5 ml of SW, and the other to 5 ml of SW,‘2. They were blotted, weighed and prepared for FAA analysis. The sample was diluted again for amino acid analysis and counting. The media for the 60 min incubation were reduced to dryness and diluted for analysis of FAA and radioactivity measurement. In a second set of experiments, excised tissue was moved at 5 min intervals through rinsing vials contiaining 2.5 ml SW for 180 min then into vials containing 2.5 ml SW12 at 5 min intervals for an additional 120min. These saline rinsings were prepared for counting by adding 10 ml of a scintillation mixture of 2 parts O.Oll~; POPOP and 0.4”; PPO in toluene and 1 part Triton X-100. Samples were counted for 2 min (0.2”,, error). RESULTS
Mantle tissue taken from SW-adapted animals transferred to SW/2 had water content equal to 76.18’:z; of total weight initially, 80.39% after 6 hr, and 80.75’:,, after 24 hr. Samples of isolated mantle tissue showed an increase in water content from 76.19”,; to 80.52;,, in 10 min. These differences in water content were reproducible, highly significant by t-test and were used to correct calculated concentrations of FAA. Chmges in FAA concentrutions in isolated muntle tissue ,fiorn S W-adupted und S W/2-adupted animals The total FAA content of mantle tissue of oysters adapted to SW was 145.9pmol/g, and the FAA content of SW/2 mantle was 91.4pmol/g (Table 1). If I g of SW tissue contains 0.76 ml water, the concentration becomes 192 mosM or 16.70,; of the total osmotic substances (TOS) in SW mantle tissue. If I g of SW/2 tissue contains 0.805 ml water, concentration is 113.5 mosM or 19.7”,; of TOS. In another group of oysters, mantle tissue incubated with alanine-U-“C showed somewhat higher FAA concentrations after 1 hr with the substrate. The FAA content of SW tissues was 167.7 pmol/g and the SW/2 tissue contained 112.7 pmol/g or 19.2’:, and 24.3’:; TOS, respectively (Table 5).
Incubation with pyruvate-l-‘4C Oxygen consumption and 14C0, production were measured on a Gilson respirometer by placing pairs of excised mantle tissue from SW-adapted and SW/Z-adapted animals in solutions containing labeled pyruvate for 3 hr at 25°C. The SW contained 0.4 mM pyruvate-l-V, 2 pCi/ml with a specific activity of 2.359 x 105dpm/~mol, and the SW/2 had specific activity 2.626 x 10Sdpm/~mol and pyruvate 9.6mM. “‘CO2 was collected in 0.5ml of hydroxide of hyamine in the center well of the Warburg flask. Afterwards, the tissues were prepared for FAA analysis, as described above. Hyamine samples were transferred to scintillation vials along with rinsings with methanol. A scintillation mixture of 2 ml methanol, 4ml toluene and 5 ml PPO-POPOP from Packard Instruments was added to the vials. These were counted on a Beckman Model LS-100 Liquid Scintillation Counter.
Table I. Free amino acids in mantle tmur in SW and SW,‘2 adapted oysters after 2 weeks SW-adapted (1150mosM)
Free amlno acid Taunne
Asp&c acid Serine Glutamic acid Proline Glycine Alanme Others Total
mN:g) 94.80 I6 54 I.60 6 90 4.45 1.44 I2 70 I 93 145.X6
Values are based upon analysts of pooled wnples each condition.
SW,‘Z-adapted (575 mosM) l/llIlOl’g) 66.24 13.04 I .4s 3.X0 + I .x0 2.14 4.32 93.39 of IO oysters for
Free
AA
in osmotic
adjustment
Exposure to SW/2 (hr) 0 3 6 12 24 72 4 weeks
1000+ 701.87 846.12 628.70 709.90 769 02 574.90
Tissue Free amino
SW
acid
Total *Indicates
SW
SW/2
4.017 I .oo 0.130 0.052 mj I.207
10.869 0.460 0.07 I 0.036 0.0143 0.5813
Radioactive metaholites
SW:2
~sw
‘JCO, Alanine Aspartic acid Glutamic acid Taarine Sum of amino acids
5.686 0.152 0.0047 0.002 + 0. I587
‘4CO: Alanine Asparuc acid Glutamic acid TaLlr1ne Sum of amino acids
~- 4.974 0.390 0.1 IO 0.049 0.0939 0.6329
The total osmotic concentration of pericardial fluid of these animals decreased markedly within 3 hr, reached a minimum of 629 mosM in 12 hr, remained nearly the same for 3 days, then decreased further to 575 mosM after 4 weeks (Table 2). The total FAA of the fluid was about 1.O mosM at the start, O.I’;/, of total osmotic substances. An increase to 2.2 mosM in 24 hr was followed by a decline to 1.25mosM after 3 days, and to 1.O mosM after 4 weeks. Taurine made up about 60% of FAA, and taurine, glycine and alanine about 75%.
of pyruvate- 1-‘4C
Mantle tissue from SW and SW/2-adapted utilized the substrate pyruvate-1-Y mainly Table
65.05 9.76’ 1.896 6.825 18.684’ 6.311* 4.176
167.676 labelled compounds
SW
112.702
oysters in pro-
Mantle tissue adapted to SW lost one third of its FAA following 1 hr exposure to SW/2 (Table 5). The total FAA tissue complement was reduced from 167.7 to 112.7p moI/g tissue. The pool size and compostion after 1 hr was similar to that observed in mantle tissue following three weeks’ adaptation to SW/2 (Table 1). Of the 55pmol lost, about 29% (16.1 pmol) were found in the medium, mostly (89%) taurine. Radioactivity in the tissues was located in alanine, aspartic acid and glutamic acid in both SW and SW/2 conditions. Incorporation of the label in alanine and aspartic acid in SW was double that in SW/2. A rapid, spontaneous release of radioactivity into the saline occurred when SW-adapted tissue was placed in SW/2 (Fig. 1). Alanine was the only labeled FAA found, and it accounted for about 30% of the radioactivity and excretion.
SW-adapted
Taurine Aspartic Glutamic Alanine Glycine Serinr Others
acid acid
SW
SW/Z-adapted SW/2
S
52.2 114.24 8.2 3976.2 7.58 1601.85 20.0 I 1748.7 18.2 _ 1.76 I.0 2.32 -
16.098
Distribution of alanine-U-V in mantle tissue jbllowing exposure to SW and SW/2
Free
C
0.165
14.27 0.142 0.106 0 190 I .39* _
(flmmol/g).
4. Concentrations (c) and specific activities (s) of FAA in pairs of mantle tissue after incubation with pyruvate-l-‘4C. Specific activity is expressed as counts/min/~mol of FAA
amino acid
Medium SW/2
0. I55 0.01 ~~ ~ ~ ~~
with
duction of CO, (Table 3). Conversion of pyruvate into amino acids and CO, amounted to 1.74-1.94 pmol/hr/g tissue in three out of the four categories of experiment. The conversion rate was 3.62pmol/hr/g in SW tissue placed in SW/2, representing a 50% decrease in FAA labeling and a 2.7-fold increase in CO, production. In SW/2, synthesis of FAA from pyruvate was only 137: of the rate in SW. On placing SW/2-adapted tissue in SW, the rate increased four-fold, but still accounted for < 1% of the net increase in FAA content (Tables 3 and 4). Changes in specific activity were consistent with those of pyruvate utilization (Table 4). Alanine showed the greatest specific activity, followed by aspartic acid, glutamic acid, and the least labeling in taurine. There was no significant difference in oxygen consumption in any direction. In sea-water the rate was 11.296pmol/hr/g tissue, and in SW/2 it was 12.202.
Changes in composition of pericardial fluid of SWudapted oysters abruptly trunsjkrred to SW/2
Eff;ts of sulinity on the utilixtion by isoluted mantle tissue
SW/?
95.50 14.57* 2.595 9.177 30.827* 8.360’ 6.647
Taarine Aspartic acid Swine Glycine Alanine Glutamic acid Nine others
Table 3. Effect of salinity on utilization of pyruvate1% by mantle tissue. Values represent ~mmoles of pyruvate incorporated into compounds by I g of tissue after 3 hr incubation in saline with labelled substrate. First column 1s medium of adaptation and second column is medium of testing salinity response Radioactive metabolites
573
oirginica
Table 5. Concentrations of FAA in mantle tissue first treated ‘%Y-alanine then immersed in SW or SW/2 for I hr
Table 2. Effect of salinitv srress on osmolalitv Osmolality of pericardial fluld (mosM)
of Crassostrea
C
SW
SW/2 S
37.8 99.33 5.3 3518.9 5.7 1661.2 10.7 11392.5 6.1 1.50 1.70
C
S
26.5 I.2 0.2 3.3 0.896 0.41 0.21
tr 1023.2 2098.4 11964.0 -
C
S
71.6 276.34 6.6 3919.4 4.2 2762.7 6.0 15236.7 I.9 I.4 6.34 -
3 hr
BARBARA WEGENER HEAVERS and C. S. HAMMEN
574
Fig. I. Pattern of release of “C-labelled compounds by isolated mantle tissue transferred from sea water to SW/2. The rate is expressed in counts released/5 min/mg tissue. The ordinate scale is logarithmic.
Ammonia
production
and excretion
concentrations increased Tissue ammonia markedly when SW-adapted mantle was transferred to SW/2, but the rate of ammonia release was not significantly changed (Table 6). Changes in ammonia concentration and release accounted for only 2% of the FAA loss from mantle tissue during SW to SW/2 transfers. In summary, the routes studied during hypotonic adjustment account for FAA reduction as follows: (1) Oxidation to CO? equals 0.761 pmol/hr/g, (2) ammonia production equals 7.93 pmol/hr/g, (3) ammonia excretion equals 0.40pmol/hr/g, (4) FAA release to pericardial fluid equals 1.2 pmol/hr/ml, and (5) FAA release to the medium equals 16.098 pmol/hr/g. The total of 26.39 pmol/hr/g represents 47.80/:, of the decrease in tissue free amino acids in the first hour. DISCUSSION
When an oyster is subjected to osmotic stress its tissues actively adjust the intracellular osmotic constituents to the new osmotic concentration of the body fluids and the diluted sea-water, a process called “isosmotic intracellular regulation” by Jeuniaux et al. (1961). A substantial part of this adjustment consists of release of salts, which was not studied in this work.
Our attention was directed at the part played by FAA in this regulation. We confirmed that uptake of water was small. The largest change observed was 37g;, reduction in the free amino acid (FAA) pool of mantle tissue in SW/Z-adapted animals, as compared with SW-adapted individuals. Excised mantle tissue showed a similar reduction in the FAA pool of 33% after 1 hr in hypotonic saline. These reductions are equivalent to 52.5 and .55.0pmol/g tissue, respectively. The small change in water content (4.50;) reflects the effectiveness of the reduction in osmotic components in oyster mantle during both short-term and long-term adjustments. The FAA adjustments associated with isosmotic intracellular regulation observed in the oyster resemble responses seen in some other conforming mollusks. For example, the blue mussel, Myths edulis, adapted to 30%0 sea-water, showed a 367/, reduction in FAA after 21 days’ exposure to 15% sea-water (Livingstone et al., 1979). The reduction in the FAA pool size differed with respect to the specific free amino acids undergoing adjustment. In the oyster, the FAA which decreased most on adaptation to reduced salinity were alanine, glycine, glutamic acid, taurine and aspartic acid, in that order. In Mytilus edulis, taurine declined and alanine and glutamic acid increased (Livingstone et al.. 1979). In the present study, the rapid efflux of taurine into the saline, which accounts for 26% of the pool size reduction in mantle, is similar to the loss of taurine by adductor muscle of Mytilus edulis in SOD,:,seawater (Gilles. 1972). Lange (1963) suggested that taurine plays a sparing role in isosmotic adjustment in bivalves, reducing the loss of essential FAA. In the oyster, the reduced rate of taurine synthesis in hypotonic conditions and the reverse shown by pyruvate-l-‘4C incorporation in hyperosmotic conditions, suggest that not only is taurine an important osmotic effector in the short term, but it can be synthesized for accumulation as an osmotic effector. In another euryhaline bivalve, A4ya arenaria, which normally loses taurine during adaptation to low salinity, taurine is not restored when a starved animal is moved from dilute to normal sea-water, suggesting that taurine or metabolic precursors come from the diet (Allen and Garrett, 1972). In the long-term adjustment to hypotonic saline, oyster mantle shows a 45-757; decline in alanine, glutamic acid and glycine, as well as a l&30% decline in aspartic acid, serine and taurine. The salinityinduced variation in the incorporation of pyruvate-l‘“C into the FAA pool by isolated mantle tissue can be linked to the metabolic adjustments of alanine, glutamic acid and aspartic acid, in particular. Their decline, coupled with increased “‘CO2 production in hypotonic saline suggests probable rapid conversion of pyruvate to acetyl CoA and a small reduction in
Table 6. ElTect of salimty on tissue ammonia concentrations release bv mantle tissue
and rates of ammon~
Medium
Tissue NH, (,umol/g)
SW SW/‘2
I I .82 f 2.28 I9 75 * 1.75
2.03 f 0.73 2.43 + I .22
SW;2 SW
13.39 + 2.17 10.88 + 1.26
3.38 * I .34 I .70 + 0.60
Released NH, (pmol/hr/g)
Free AA in osmotic adjustment of Crassosfrea airginica FAA synthesis. In reporting the rise in alanine and glutamic acid in Myths edulis on abrupt transfer to dilute salinity. Livingstone et al. (1979) suggested a rise in tissue free ammonia from the deamination of other amino acids promoted reductive amination via glutamic dehydrogenase and alanine dehydrogenase. In the oyster, although tissue ammonia levels were comparable to those reported for Mytilus edulis, no increase in reductive amination of either pyruvate to alanine, or oxaloacetate to glutamic acid, occurred. The presence of labeling in alanine, glutamic acid and aspartic acid with both pyruvate-1-“C and alanine-U-“‘C in hypertonic and hypotonic conmditions supports the evidence for alanine and aspartate aminotransferases (AT) in the oyster (Hammen, 1969). Several metabolic routes of FAA synthesis are implicated by the labeling pattern in the pyruvate experiment. These include transamination, carboxylation of pyruvate to malate and Krebs’ cycle interconversions leading to glutamic acid production. Carboxylation of pyruvate has already been described as a mode of CO, fixation in the oyster (Hammen, 1969). Thus, pyruvate utilization ultimately results in the labelling of aspartate via oxaloacetate, since conversion of malate to oxaloacetate is extremely rapid. Labeled oxaloacetate can also provide a source of labeled glutamate via ketoglutarate. The redistribution of the label during incubation of mantle tissue with alanine-U-‘4C suggests deamination of alanine by means of alanineAT to pyruvate, which is then utilized to produce both aspartic acid and glutamic acid. The utilization of pyruvate by mantle tissue does not appear to be oxygen dependent. The relationship between the free amino acids and anaerobic glucose metabolism has been explored by several authors. Baginski and Pierce (1978) reported that glycogen is the major source of carbon for alanine during hyperosmotic adjustment in Modiohs demissus, and the process is not influenced by anaerobiosis. In this study. a large amount of radioactivity released by the excised mantle tissue incubated in hypotonic saline remained unidentified. The unknown substance may be alanopine, known to be formed from alanine and pyruvate by tissues of C. gigas and possibly useful in osmotic adjustment (Fields, 1983). Ammonia is the normal primary end product of nitrogen metabolism in the oyster (Hammen, 1969). Small short-term changes in the rate of both ammonia excretion and tissue levels of ammonia were detected in response to salinity. Catabolism of FAA is a possible source of excreted ammonia during FAA pool reduction. However, the net change in ammonia production accounts for only 2:/o of the total net loss of free amino acids. Changes in the FAA concentration of the pericardial fluid during osmotic stress reflect some movement of FAA across the cell membrane. The rapid decrease in the osmolality of the pericardial fluid during hypotonic conditions is accompanied by a movement of FAA into the pericardial fluid at a rate of about 1.2 ~mol/ml/hr. The routes examined by this study account for 47.8”” of the decrease in tissue free amino acids in the first hour of exposure of mantle tissue to hypotonic saline. Processes that may account for the remaining
575
52.2% include polymerization and production of unidentified amino acid derivatives. The first process is suggested by the observation that the clam Rangia cuneata accumulates “C-glycine and converts it to osmotically inactive compounds within 1 hr (Anderson, 1975). The second process is suggested by the presence of alanopine dehydrogenase, mentioned earlier. This study has shown that the metabolism and permeability changes in isolated tissue can account for a large part of the FAA changes observed in tissues of whole oysters when subjected to variations in salinity.
REFERENCES
Allen J. A. and Garrett M. R. (1972) Studies on taurine in the euryhaline bivalve h4ya arenaria. Camp. Biochem. PIrysiol. 41A, 307-3 17. Anderson J. W. (1975) The uptake and incorporation of glycine by the gill of Rangia cuneafa (Mollusca: Bivalvia) in response to variations in salinity and sodium. In Physiological Ecology of E.siurtuarineOrganisms (Edited by Vernberg F. J.). University South Carolina Press, Columbia. Baginski R. M. and Pierce S. K. Jr. (1978) A comparison of amino acid accumulation during high salinity adaptation with anaerobic metabolism in the ribbed mussel, Modiobs demissus demissus. J. esp. Zool. 203, 419428. Bishop S. H. (1976) Nitrogen metabolism and excretion: Regulation of intracellular amino acid concentrations. In Estuarine Processes (Edited by Wiley M.), Vol. I, pp. 414-431. Academic Press. New York. Bishop S. H., Ellis L. L. and Burcham J. M. (1983) Amino acid metabolism in molluscs. In The Molluscu (Edited by Hochachka P. W.). Vol. 1, pp. 2433327. Academic Press, New York. Bricteux-Gregoire S., Duchateau-Bosson Gh., Jeuniaux C. H. and Florkin M. (1964) Constituants osmotiquement actifs des muscles adducteurs de Gryphaecr angulara adaptee a l’eau de mer ou a I’eau saumatre. Arc/r. hrr. Ph_vsiol. Biochim. 72, 835-842. Conway E. J. (1957) Microd@sion Anal.v~Gs and Volumetric Error, 4th edn. Lockwood. London. Fields J. H. A. (1983) Alternatives to lactic acid: possible advantages. J. exp. Zool. 228, 4455457. Galtsoff P. S. (1964) The American oyster, Crassostrea cirpinicrr. Gmelin. Fish BUN 64. US Deut of the Interior. Washington DC. Gilles R. (1972) Osmoregulation in three molluscs: .4canthochitonu discrepans (Brown), Glycymeris gl_vcymeris (L.) and Mytilus rdulis (L.) Biol. Bull. 142, 25-35. Hammen C. S. (1969) Metabolism of the oyster, Crussosrrea virginica. Am. Zool. 9. 309-318. Jeun&x Ch., Bricteux-Gregoire S. and Florkin M. (1961) Contribution des acides amines libres a la regulation osmotique intracellulaire chez deux crustaces euryhaline, Leander serratus F., et Leander syuilla L. Cahiers Biol. Mur. 2, 373-380. Krogh A. (1939) Osmotic Regulation in Aquatic Animals. Cambridge University Press, Cambridge. Lange R. (I 963) The osmotic function of amino acids and taurine in the mussel, Myrilus edulis. Camp. Biochem. Physiol. 10, 173%179. Livingstone D. R., Widdows J. and Fieth P. (1979) Aspects of nitrogen metabolism of the common mussel Myrilus edulis: adaptation to abrupt and fluctuating changes in salinity. Mar. Biol. 53, 41-55. Lynch M. P. and Wood L. (1966) Effects of environmental salinity on free amino acids of Crassostrea airginica, Gmelin. Comp. Biochem. Physiol. 19, 783-790.
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BARBARA WEGENER HEAVEKS and C. S. HAMMEN
Pierce S. K. and Greenberg M. J. (1972) The nature of cellular volume regulation in marine bivalves. J. exp. Biol. 51, 681-692. Somero G. N. and Bowlus R. D. (1983) Osmolytes and
metabolic end products of molluscs: the design of compatible solute systems. In The Molluscu (Edited by Hochachka P. W.), Vol. 2, pp. 77- 100 Academic Press, New York.
0300-9629/X5 163.00+ 0.00
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FATE OF ENDOGENOUS FREE AMINO ACIDS IN OSMOTIC ADJUSTMENT OF CRASSOSTREA VIRGINICA (GMELIN)” BARBARA WEGENER HEAVERS and C. S. HAMMEN Department
of Zoology,
University
of Rhode
Island, 792-2372
Kingston,
RI 02881,
USA.
Telephone:
(401)
(Received 21 Februurv 1985) Abstract-l. Free amino acids (FAA) in mantle tissue of whole oysters, adapted to sea-water or one-half salinity, were determined by automatic amino acid analyzer; isolated tissue was incubated with ‘%-labeled substrates, transferred to different media, and labeled metabolites were determined by liquid scintillation counter. 2. FAA accounted for l6.7-19.7% of osmotically active substances; taurine made up 57~71”:,, alanine, aspartic acid and glycine 19-32’4,. and all other amino acids together about 10%. 3. After 3 hr in reduced salinity, tissue FAA decreased 32% and ‘%02 production increased 2.7-fold; in increased salinity, FAA increased 2.8-fold and “‘CO2 production decreased by 12%. 4. Significant efflux and biosynthesis of FAA occurred within I hr of transfer. but did not account for all loss or gain.
shown FAA regularly constitute 5-20’2: of nitrogenous compounds released (Hammen, 1969). A study of isolated hearts of Modiolus demissus, exposed to a medium of lower salinity for 3 hr, showed that FAA loss was sufficient to account for all of the reduction in tissue concentration (Pierce and Greenberg, 1972). On the other hand, a marked decrease in taurine content occurred in Mya arenaria placed in water of reduced salinity, but no taurine appeared in the medium (Allen and Garrett, 1972). One goal of the research reported here was to determine whether oysters release FAA as such on exposure to lower salinity media. Pathways of amino acid degradation and biosynthesis may be activated by changes in salinity (Bishop, 1976). In particular, the glutamate dehydrogenase reaction, which is directly affected by salt concentration in crustaceans, is now known to occur in bivalves, but there is still no evidence that it is important in osmotic adjustment (Bishop et al., 1983). Increases in FAA with adjustment to higher salinity could occur by means of new biosynthesis, uptake from the environment, or depolymerization of polypeptide. There is no information on which of these processes are used by oysters. In this study, we have examined early changes in the FAA pool size in oysters subjected to short-term salinity stress. Aspects we have considered include: (1) changes in catabolic or anabolic activities reflected in O? consumption and CO, production from labelled substrate, (2) the rate of change in the tissue FAA pool during short-term exposure and adaptation, and (3) the fate of endogenous FAA during hyposmotic stress either by direct release, hemolymph shifts and/or catabolism to ammonia and CO,.
INTRODUCTION
The American oyster, Crassostrea virginica, is an euryhaline osmoconformer, thriving in estuarine situations where salinity is lower than ocean water and varies seasonally (Galtsoff, 1964). Physiological chemists in the period 1901-1905 discovered that the tissues of marine mollusks contain less chloride than present in hemolymph or sea-water, and that the deficit is made up in part by free amino acids (FAA) such as taurine and glycine (Krogh, 1939). In the Portuguese oyster, Crassostrea angulata, FAA made up an average of 30% of the total osmotically active substances (TOS) in adductor muscles of animals adapted to waters of both 1188 and 594mosM (Bricteux-Gregoire et al., 1964). In muscle of C. virginica, FAA accounted for 20.9% of the total osmotic effect in water of 785mosM, and those undergoing the greatest changes with salinity were taurine, alanine, glycine and proline (Lynch and Wood, 1966). It is well known that in mollusks, FAA make a dominant contribution to intracellular osmolarity, and are varied or adjusted more than mineral cations (Somero and Bowlus, 1983). The means for accomplishing these adjustments, however, are not well known. Processes of cellular respiration, intermediary metabolism and nitrogenous excretion are intertwined to make adjustment “a complicated process” (Bishop et al., 1983). One possible way to adjust to lower salinity is simply to release the surplus amino acids. Analysis of excretion water of C. virginica has
*This work is derived from a dissertation submitted to the University of Rhode Island by the senior author in partial fulfillment of the degree of Doctor of Philosophy. Partial support of this research was provided by a NSF Summer Traineeship from the University of Rhode Island and a grant from the Membrane Physiology Laboratory of Duke University, Durham, North Carolina.
MATERIALS
AND METHODS
Oysters used in this study were collected at Bird Shoal, Beaufort, North Carolina during two summers and one fall 571
512
BARBARA WEGENER HEAVERS and C. S. HAMMEN
season. Weights ranged from 50 to 150 g. They were maintained at Duke University Marine Laboratory in running sea-water or in aerated 50% sea-water tanks. Temperatures ranged from 19 C in summer, to 8’C in fall. Salinities were 31-340/w for sea-water (SW) and 17~19X for 50% sea-water (SW/Z). Animals were considered SW/2-adapted after 3 weeks holding in 5O”/0 sea-water. Experimental saline solutions were prepared from local sea-water. They were filtered and buffered to pH 7.5 with H,BO, (0.0088 M). SW/2 was prepared by mixing equal volumes of glass-distilled water and full strength sea-water. Room temperature was 20-22-C. Pyruvate- I-14C and alanine-U-‘4C were obtained from Sigma Chemical Company. Samples of excised mantle tissue (25-125 mg) included a portion of the lobulated border along with a small portion of the thin, adjacent, interior area. Samples of fluid were removed from the pericardial cavity by syringe. The percentage of water composition was determined by the difference in wet and dry weights of mantle tissue of SW and SW/2-adapted animals. SW-adapted animals were exposed to SW/2 for 6 and 24 hr. Excised mantle from SW animals was exposed to SW/2 for 10min.
The total osmolality of the pericardial fluid was determined on 0.5-ml samples with an Advanced Osmometer. Model 65-3 1. Ammonia
unalysis
Tissue ammonia levels and excretion rates were determined by placing paired portions of excised mantle tissue from a single oyster in separate 5-ml samples of aerated water for 4 hr. Afterward, each tissue was blotted, weighed and homogenized in 2 ml of cold 50% trichloroacetic acid in a glass grinder. The homogenate was washed into a 1Oml volumetric flask and diluted to 1 in 10 with ammonia-free water. Dilutions were centrifuged at 25,OOOg for 10 min and the supernatant was analyzed for ammonia by the microdiffusion method of Conway (1957). The incubation media were similarly analyzed. FAA unalysis Pooled samples (N = 10) of pericardial fluid were prepared for each group of animals. These were centrifuged at 25,000 g for 30 min, and the supernatant was dialyzed. The FAA fraction was evaporated to near dryness, transferred by rinsings, then completely dried. Mantle tissue samples (N = IO) were placed in boiling water for 15min. cooled, homogenized and dialyzed. The FAA fraction was evaporated to near dryness and the remainder transferred to a vial along with rinsings (4 x 2 ml). The sample was reduced to dryness and diluted again for analysis. Amino acids were analyzed on a Beckman Model 116 Amino Acid Analyzer.
Incubation with alaninr-U-‘4C Two pieces of tissue, one from each side of the mantle of an oyster, were placed in aerated SW containing alanine-U14C, ZpCi/ml and 20mM L-alanine for 90min. Each was then moved through a separate series of rinsing vials containing 2.5 ml at 5-min intervals for 180 min. One piece was then exposed for 60 min to 5 ml of SW, and the other to 5 ml of SW,‘2. They were blotted, weighed and prepared for FAA analysis. The sample was diluted again for amino acid analysis and counting. The media for the 60 min incubation were reduced to dryness and diluted for analysis of FAA and radioactivity measurement. In a second set of experiments, excised tissue was moved at 5 min intervals through rinsing vials contiaining 2.5 ml SW for 180 min then into vials containing 2.5 ml SW12 at 5 min intervals for an additional 120min. These saline rinsings were prepared for counting by adding 10 ml of a scintillation mixture of 2 parts O.Oll~; POPOP and 0.4”; PPO in toluene and 1 part Triton X-100. Samples were counted for 2 min (0.2”,, error). RESULTS
Mantle tissue taken from SW-adapted animals transferred to SW/2 had water content equal to 76.18’:z; of total weight initially, 80.39% after 6 hr, and 80.75’:,, after 24 hr. Samples of isolated mantle tissue showed an increase in water content from 76.19”,; to 80.52;,, in 10 min. These differences in water content were reproducible, highly significant by t-test and were used to correct calculated concentrations of FAA. Chmges in FAA concentrutions in isolated muntle tissue ,fiorn S W-adupted und S W/2-adupted animals The total FAA content of mantle tissue of oysters adapted to SW was 145.9pmol/g, and the FAA content of SW/2 mantle was 91.4pmol/g (Table 1). If I g of SW tissue contains 0.76 ml water, the concentration becomes 192 mosM or 16.70,; of the total osmotic substances (TOS) in SW mantle tissue. If I g of SW/2 tissue contains 0.805 ml water, concentration is 113.5 mosM or 19.7”,; of TOS. In another group of oysters, mantle tissue incubated with alanine-U-“C showed somewhat higher FAA concentrations after 1 hr with the substrate. The FAA content of SW tissues was 167.7 pmol/g and the SW/2 tissue contained 112.7 pmol/g or 19.2’:, and 24.3’:; TOS, respectively (Table 5).
Incubation with pyruvate-l-‘4C Oxygen consumption and 14C0, production were measured on a Gilson respirometer by placing pairs of excised mantle tissue from SW-adapted and SW/Z-adapted animals in solutions containing labeled pyruvate for 3 hr at 25°C. The SW contained 0.4 mM pyruvate-l-V, 2 pCi/ml with a specific activity of 2.359 x 105dpm/~mol, and the SW/2 had specific activity 2.626 x 10Sdpm/~mol and pyruvate 9.6mM. “‘CO2 was collected in 0.5ml of hydroxide of hyamine in the center well of the Warburg flask. Afterwards, the tissues were prepared for FAA analysis, as described above. Hyamine samples were transferred to scintillation vials along with rinsings with methanol. A scintillation mixture of 2 ml methanol, 4ml toluene and 5 ml PPO-POPOP from Packard Instruments was added to the vials. These were counted on a Beckman Model LS-100 Liquid Scintillation Counter.
Table I. Free amino acids in mantle tmur in SW and SW,‘2 adapted oysters after 2 weeks SW-adapted (1150mosM)
Free amlno acid Taunne
Asp&c acid Serine Glutamic acid Proline Glycine Alanme Others Total
mN:g) 94.80 I6 54 I.60 6 90 4.45 1.44 I2 70 I 93 145.X6
Values are based upon analysts of pooled wnples each condition.
SW,‘Z-adapted (575 mosM) l/llIlOl’g) 66.24 13.04 I .4s 3.X0 + I .x0 2.14 4.32 93.39 of IO oysters for
Free
AA
in osmotic
adjustment
Exposure to SW/2 (hr) 0 3 6 12 24 72 4 weeks
1000+ 701.87 846.12 628.70 709.90 769 02 574.90
Tissue Free amino
SW
acid
Total *Indicates
SW
SW/2
4.017 I .oo 0.130 0.052 mj I.207
10.869 0.460 0.07 I 0.036 0.0143 0.5813
Radioactive metaholites
SW:2
~sw
‘JCO, Alanine Aspartic acid Glutamic acid Taarine Sum of amino acids
5.686 0.152 0.0047 0.002 + 0. I587
‘4CO: Alanine Asparuc acid Glutamic acid TaLlr1ne Sum of amino acids
~- 4.974 0.390 0.1 IO 0.049 0.0939 0.6329
The total osmotic concentration of pericardial fluid of these animals decreased markedly within 3 hr, reached a minimum of 629 mosM in 12 hr, remained nearly the same for 3 days, then decreased further to 575 mosM after 4 weeks (Table 2). The total FAA of the fluid was about 1.O mosM at the start, O.I’;/, of total osmotic substances. An increase to 2.2 mosM in 24 hr was followed by a decline to 1.25mosM after 3 days, and to 1.O mosM after 4 weeks. Taurine made up about 60% of FAA, and taurine, glycine and alanine about 75%.
of pyruvate- 1-‘4C
Mantle tissue from SW and SW/2-adapted utilized the substrate pyruvate-1-Y mainly Table
65.05 9.76’ 1.896 6.825 18.684’ 6.311* 4.176
167.676 labelled compounds
SW
112.702
oysters in pro-
Mantle tissue adapted to SW lost one third of its FAA following 1 hr exposure to SW/2 (Table 5). The total FAA tissue complement was reduced from 167.7 to 112.7p moI/g tissue. The pool size and compostion after 1 hr was similar to that observed in mantle tissue following three weeks’ adaptation to SW/2 (Table 1). Of the 55pmol lost, about 29% (16.1 pmol) were found in the medium, mostly (89%) taurine. Radioactivity in the tissues was located in alanine, aspartic acid and glutamic acid in both SW and SW/2 conditions. Incorporation of the label in alanine and aspartic acid in SW was double that in SW/2. A rapid, spontaneous release of radioactivity into the saline occurred when SW-adapted tissue was placed in SW/2 (Fig. 1). Alanine was the only labeled FAA found, and it accounted for about 30% of the radioactivity and excretion.
SW-adapted
Taurine Aspartic Glutamic Alanine Glycine Serinr Others
acid acid
SW
SW/Z-adapted SW/2
S
52.2 114.24 8.2 3976.2 7.58 1601.85 20.0 I 1748.7 18.2 _ 1.76 I.0 2.32 -
16.098
Distribution of alanine-U-V in mantle tissue jbllowing exposure to SW and SW/2
Free
C
0.165
14.27 0.142 0.106 0 190 I .39* _
(flmmol/g).
4. Concentrations (c) and specific activities (s) of FAA in pairs of mantle tissue after incubation with pyruvate-l-‘4C. Specific activity is expressed as counts/min/~mol of FAA
amino acid
Medium SW/2
0. I55 0.01 ~~ ~ ~ ~~
with
duction of CO, (Table 3). Conversion of pyruvate into amino acids and CO, amounted to 1.74-1.94 pmol/hr/g tissue in three out of the four categories of experiment. The conversion rate was 3.62pmol/hr/g in SW tissue placed in SW/2, representing a 50% decrease in FAA labeling and a 2.7-fold increase in CO, production. In SW/2, synthesis of FAA from pyruvate was only 137: of the rate in SW. On placing SW/2-adapted tissue in SW, the rate increased four-fold, but still accounted for < 1% of the net increase in FAA content (Tables 3 and 4). Changes in specific activity were consistent with those of pyruvate utilization (Table 4). Alanine showed the greatest specific activity, followed by aspartic acid, glutamic acid, and the least labeling in taurine. There was no significant difference in oxygen consumption in any direction. In sea-water the rate was 11.296pmol/hr/g tissue, and in SW/2 it was 12.202.
Changes in composition of pericardial fluid of SWudapted oysters abruptly trunsjkrred to SW/2
Eff;ts of sulinity on the utilixtion by isoluted mantle tissue
SW/?
95.50 14.57* 2.595 9.177 30.827* 8.360’ 6.647
Taarine Aspartic acid Swine Glycine Alanine Glutamic acid Nine others
Table 3. Effect of salinity on utilization of pyruvate1% by mantle tissue. Values represent ~mmoles of pyruvate incorporated into compounds by I g of tissue after 3 hr incubation in saline with labelled substrate. First column 1s medium of adaptation and second column is medium of testing salinity response Radioactive metabolites
573
oirginica
Table 5. Concentrations of FAA in mantle tissue first treated ‘%Y-alanine then immersed in SW or SW/2 for I hr
Table 2. Effect of salinitv srress on osmolalitv Osmolality of pericardial fluld (mosM)
of Crassostrea
C
SW
SW/2 S
37.8 99.33 5.3 3518.9 5.7 1661.2 10.7 11392.5 6.1 1.50 1.70
C
S
26.5 I.2 0.2 3.3 0.896 0.41 0.21
tr 1023.2 2098.4 11964.0 -
C
S
71.6 276.34 6.6 3919.4 4.2 2762.7 6.0 15236.7 I.9 I.4 6.34 -
3 hr
BARBARA WEGENER HEAVERS and C. S. HAMMEN
574
Fig. I. Pattern of release of “C-labelled compounds by isolated mantle tissue transferred from sea water to SW/2. The rate is expressed in counts released/5 min/mg tissue. The ordinate scale is logarithmic.
Ammonia
production
and excretion
concentrations increased Tissue ammonia markedly when SW-adapted mantle was transferred to SW/2, but the rate of ammonia release was not significantly changed (Table 6). Changes in ammonia concentration and release accounted for only 2% of the FAA loss from mantle tissue during SW to SW/2 transfers. In summary, the routes studied during hypotonic adjustment account for FAA reduction as follows: (1) Oxidation to CO? equals 0.761 pmol/hr/g, (2) ammonia production equals 7.93 pmol/hr/g, (3) ammonia excretion equals 0.40pmol/hr/g, (4) FAA release to pericardial fluid equals 1.2 pmol/hr/ml, and (5) FAA release to the medium equals 16.098 pmol/hr/g. The total of 26.39 pmol/hr/g represents 47.80/:, of the decrease in tissue free amino acids in the first hour. DISCUSSION
When an oyster is subjected to osmotic stress its tissues actively adjust the intracellular osmotic constituents to the new osmotic concentration of the body fluids and the diluted sea-water, a process called “isosmotic intracellular regulation” by Jeuniaux et al. (1961). A substantial part of this adjustment consists of release of salts, which was not studied in this work.
Our attention was directed at the part played by FAA in this regulation. We confirmed that uptake of water was small. The largest change observed was 37g;, reduction in the free amino acid (FAA) pool of mantle tissue in SW/Z-adapted animals, as compared with SW-adapted individuals. Excised mantle tissue showed a similar reduction in the FAA pool of 33% after 1 hr in hypotonic saline. These reductions are equivalent to 52.5 and .55.0pmol/g tissue, respectively. The small change in water content (4.50;) reflects the effectiveness of the reduction in osmotic components in oyster mantle during both short-term and long-term adjustments. The FAA adjustments associated with isosmotic intracellular regulation observed in the oyster resemble responses seen in some other conforming mollusks. For example, the blue mussel, Myths edulis, adapted to 30%0 sea-water, showed a 367/, reduction in FAA after 21 days’ exposure to 15% sea-water (Livingstone et al., 1979). The reduction in the FAA pool size differed with respect to the specific free amino acids undergoing adjustment. In the oyster, the FAA which decreased most on adaptation to reduced salinity were alanine, glycine, glutamic acid, taurine and aspartic acid, in that order. In Mytilus edulis, taurine declined and alanine and glutamic acid increased (Livingstone et al.. 1979). In the present study, the rapid efflux of taurine into the saline, which accounts for 26% of the pool size reduction in mantle, is similar to the loss of taurine by adductor muscle of Mytilus edulis in SOD,:,seawater (Gilles. 1972). Lange (1963) suggested that taurine plays a sparing role in isosmotic adjustment in bivalves, reducing the loss of essential FAA. In the oyster, the reduced rate of taurine synthesis in hypotonic conditions and the reverse shown by pyruvate-l-‘4C incorporation in hyperosmotic conditions, suggest that not only is taurine an important osmotic effector in the short term, but it can be synthesized for accumulation as an osmotic effector. In another euryhaline bivalve, A4ya arenaria, which normally loses taurine during adaptation to low salinity, taurine is not restored when a starved animal is moved from dilute to normal sea-water, suggesting that taurine or metabolic precursors come from the diet (Allen and Garrett, 1972). In the long-term adjustment to hypotonic saline, oyster mantle shows a 45-757; decline in alanine, glutamic acid and glycine, as well as a l&30% decline in aspartic acid, serine and taurine. The salinityinduced variation in the incorporation of pyruvate-l‘“C into the FAA pool by isolated mantle tissue can be linked to the metabolic adjustments of alanine, glutamic acid and aspartic acid, in particular. Their decline, coupled with increased “‘CO2 production in hypotonic saline suggests probable rapid conversion of pyruvate to acetyl CoA and a small reduction in
Table 6. ElTect of salimty on tissue ammonia concentrations release bv mantle tissue
and rates of ammon~
Medium
Tissue NH, (,umol/g)
SW SW/‘2
I I .82 f 2.28 I9 75 * 1.75
2.03 f 0.73 2.43 + I .22
SW;2 SW
13.39 + 2.17 10.88 + 1.26
3.38 * I .34 I .70 + 0.60
Released NH, (pmol/hr/g)
Free AA in osmotic adjustment of Crassosfrea airginica FAA synthesis. In reporting the rise in alanine and glutamic acid in Myths edulis on abrupt transfer to dilute salinity. Livingstone et al. (1979) suggested a rise in tissue free ammonia from the deamination of other amino acids promoted reductive amination via glutamic dehydrogenase and alanine dehydrogenase. In the oyster, although tissue ammonia levels were comparable to those reported for Mytilus edulis, no increase in reductive amination of either pyruvate to alanine, or oxaloacetate to glutamic acid, occurred. The presence of labeling in alanine, glutamic acid and aspartic acid with both pyruvate-1-“C and alanine-U-“‘C in hypertonic and hypotonic conmditions supports the evidence for alanine and aspartate aminotransferases (AT) in the oyster (Hammen, 1969). Several metabolic routes of FAA synthesis are implicated by the labeling pattern in the pyruvate experiment. These include transamination, carboxylation of pyruvate to malate and Krebs’ cycle interconversions leading to glutamic acid production. Carboxylation of pyruvate has already been described as a mode of CO, fixation in the oyster (Hammen, 1969). Thus, pyruvate utilization ultimately results in the labelling of aspartate via oxaloacetate, since conversion of malate to oxaloacetate is extremely rapid. Labeled oxaloacetate can also provide a source of labeled glutamate via ketoglutarate. The redistribution of the label during incubation of mantle tissue with alanine-U-‘4C suggests deamination of alanine by means of alanineAT to pyruvate, which is then utilized to produce both aspartic acid and glutamic acid. The utilization of pyruvate by mantle tissue does not appear to be oxygen dependent. The relationship between the free amino acids and anaerobic glucose metabolism has been explored by several authors. Baginski and Pierce (1978) reported that glycogen is the major source of carbon for alanine during hyperosmotic adjustment in Modiohs demissus, and the process is not influenced by anaerobiosis. In this study. a large amount of radioactivity released by the excised mantle tissue incubated in hypotonic saline remained unidentified. The unknown substance may be alanopine, known to be formed from alanine and pyruvate by tissues of C. gigas and possibly useful in osmotic adjustment (Fields, 1983). Ammonia is the normal primary end product of nitrogen metabolism in the oyster (Hammen, 1969). Small short-term changes in the rate of both ammonia excretion and tissue levels of ammonia were detected in response to salinity. Catabolism of FAA is a possible source of excreted ammonia during FAA pool reduction. However, the net change in ammonia production accounts for only 2:/o of the total net loss of free amino acids. Changes in the FAA concentration of the pericardial fluid during osmotic stress reflect some movement of FAA across the cell membrane. The rapid decrease in the osmolality of the pericardial fluid during hypotonic conditions is accompanied by a movement of FAA into the pericardial fluid at a rate of about 1.2 ~mol/ml/hr. The routes examined by this study account for 47.8”” of the decrease in tissue free amino acids in the first hour of exposure of mantle tissue to hypotonic saline. Processes that may account for the remaining
575
52.2% include polymerization and production of unidentified amino acid derivatives. The first process is suggested by the observation that the clam Rangia cuneata accumulates “C-glycine and converts it to osmotically inactive compounds within 1 hr (Anderson, 1975). The second process is suggested by the presence of alanopine dehydrogenase, mentioned earlier. This study has shown that the metabolism and permeability changes in isolated tissue can account for a large part of the FAA changes observed in tissues of whole oysters when subjected to variations in salinity.
REFERENCES
Allen J. A. and Garrett M. R. (1972) Studies on taurine in the euryhaline bivalve h4ya arenaria. Camp. Biochem. PIrysiol. 41A, 307-3 17. Anderson J. W. (1975) The uptake and incorporation of glycine by the gill of Rangia cuneafa (Mollusca: Bivalvia) in response to variations in salinity and sodium. In Physiological Ecology of E.siurtuarineOrganisms (Edited by Vernberg F. J.). University South Carolina Press, Columbia. Baginski R. M. and Pierce S. K. Jr. (1978) A comparison of amino acid accumulation during high salinity adaptation with anaerobic metabolism in the ribbed mussel, Modiobs demissus demissus. J. esp. Zool. 203, 419428. Bishop S. H. (1976) Nitrogen metabolism and excretion: Regulation of intracellular amino acid concentrations. In Estuarine Processes (Edited by Wiley M.), Vol. I, pp. 414-431. Academic Press. New York. Bishop S. H., Ellis L. L. and Burcham J. M. (1983) Amino acid metabolism in molluscs. In The Molluscu (Edited by Hochachka P. W.). Vol. 1, pp. 2433327. Academic Press, New York. Bricteux-Gregoire S., Duchateau-Bosson Gh., Jeuniaux C. H. and Florkin M. (1964) Constituants osmotiquement actifs des muscles adducteurs de Gryphaecr angulara adaptee a l’eau de mer ou a I’eau saumatre. Arc/r. hrr. Ph_vsiol. Biochim. 72, 835-842. Conway E. J. (1957) Microd@sion Anal.v~Gs and Volumetric Error, 4th edn. Lockwood. London. Fields J. H. A. (1983) Alternatives to lactic acid: possible advantages. J. exp. Zool. 228, 4455457. Galtsoff P. S. (1964) The American oyster, Crassostrea cirpinicrr. Gmelin. Fish BUN 64. US Deut of the Interior. Washington DC. Gilles R. (1972) Osmoregulation in three molluscs: .4canthochitonu discrepans (Brown), Glycymeris gl_vcymeris (L.) and Mytilus rdulis (L.) Biol. Bull. 142, 25-35. Hammen C. S. (1969) Metabolism of the oyster, Crussosrrea virginica. Am. Zool. 9. 309-318. Jeun&x Ch., Bricteux-Gregoire S. and Florkin M. (1961) Contribution des acides amines libres a la regulation osmotique intracellulaire chez deux crustaces euryhaline, Leander serratus F., et Leander syuilla L. Cahiers Biol. Mur. 2, 373-380. Krogh A. (1939) Osmotic Regulation in Aquatic Animals. Cambridge University Press, Cambridge. Lange R. (I 963) The osmotic function of amino acids and taurine in the mussel, Myrilus edulis. Camp. Biochem. Physiol. 10, 173%179. Livingstone D. R., Widdows J. and Fieth P. (1979) Aspects of nitrogen metabolism of the common mussel Myrilus edulis: adaptation to abrupt and fluctuating changes in salinity. Mar. Biol. 53, 41-55. Lynch M. P. and Wood L. (1966) Effects of environmental salinity on free amino acids of Crassostrea airginica, Gmelin. Comp. Biochem. Physiol. 19, 783-790.
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BARBARA WEGENER HEAVEKS and C. S. HAMMEN
Pierce S. K. and Greenberg M. J. (1972) The nature of cellular volume regulation in marine bivalves. J. exp. Biol. 51, 681-692. Somero G. N. and Bowlus R. D. (1983) Osmolytes and
metabolic end products of molluscs: the design of compatible solute systems. In The Molluscu (Edited by Hochachka P. W.), Vol. 2, pp. 77- 100 Academic Press, New York.