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Effect of external salinity change on the adenylate energy charge in the brackish bivalve Corbicula japonica
Camp. Biochem. Physiol. Vol. 77A, No. Printed in Great Bntain
EFFECT ADENYLATE
0300-9629!84 S3.00 + 0.00 ,S 1984 Pergamon Press Ltd
I, pp. 57-61, 1984
OF EXTERNAL SALINITY CHANGE ON THE ENERGY CHARGE IN THE BRACKISH BIVALVE CORBICULA
JAPONICA
0. MATSUSHIMA, H. KATAYAMA, K. YAMADA and Y. KADO Zoological
Institute,
Faculty of Science, Hiroshima University, Hiroshima 730 (Tel.: 0822 41-1221) Suzugamine Women’s College, Hiroshima 733, Japan
and
(Received 29 March 1983) Abstract1. The energy charge in the brackish clam Corbicula japonica changed within the range of 0.6-0.8 during exposure to osmotic or anoxic stress. 2. The energy charge elevated transiently at the initial day of hypoosmotic stress and at the third day of hyperosmotic stress. Exposure to anoxia resulted in a small but significant decline in energy charge. The transient elevation during osmotic stress may be a reflection of aerobic metabolism. 3. During exposure to these stresses, activities of key enzymes involved in amino acid metabolism may be regulated by the energy charge.
change in FAA pool size. In addition, change in water content of tissue, which is considered to be an index of cell volume regulation, was examined to compute the net quantity of adenine nucleotides excluding the effect of tissue hydration or dehydration.
INTRODUCTION known that in many aquatic molluscs the concentration of intracellular free amino acids (FAA) changes in response to salinity change in external medium (Virkar and Webb, 1970; Pierce, 1971; Gainey, 1978). The change in FAA concentration is considered to be important in cell volume regulation or isosmotic intracellular regulation during acclimation to salinity change. Although the biochemical mechanism underlying the FAA fluctuation is obscure, glutamate dehydrogenase (GDH) is assumed to be one of the important enzymes involved in FAA accumulation during acclimation to salinity increase (Gainey, 1978; Baginski and Pierce, 1978). We reported previously that GDH activities of the bivalves Corbicula juponica and C. leana were markedly affected by the adenylate energy charge in reaction mixture (Matsushima and Kado, 1983). Atkinson (1968, 1977) has proposed that the energy level in the adenylate system can be quantitatively represented by the energy charge (ATP+ l/2 ADP)/(ATP+ADP+AMP) and that activity of the key enzymes involved in energy metabolism and biosynthesis is under control of this parameter. Some enzymes are reported to respond to the energy charge (Atkinson and Walton, 1967; Thompson and Atkinson, 1971), though the response pattern is dependent upon some other factors (Purich and Fromm, 1972, 1973). As a condition influencing the energy charge in intact animals, anoxic stress has been known to reduce its value markedly in some animals (Ridge, 1972; Wijsman, 1976; Ellington, 1981). However, little is known about the effect of osmotic stress on the energy charge. The present study was primarily designed to examine whether the energy charge in uiuo could account for the physiological significance of the in oitro energy charge response of GDH. In this study, the effect of both hypo- and hyperosmotic stresses on the energy charge of the brackish bivalve, Corbicula japonica, was investigated in the context of intracellular osmoregulation mediated by It is well
MATERIALS AND METHODS Acclimation
of animal
Specimens of Corbicula japonica (shell length of 2-3 cm) were collected from a brackish river in Hiroshima City, where salinity of water was remarkably changed by tidal cycle. At low tide, osmotic concentrations of water and filtrate of the bottom mud in the habitat were about 100 mOsm and 300400mOsm, respectively. The animals were acclimated to 35% sea water (340 mOsm) or to fresh water (3-7 mOsm), which was recirculated and aerated, for more than two weeks at water temperature of 2C-26°C. In order to examine the effect of osmotic stress, the animals acclimated to 340 mOsm were transferred to fresh water and the fresh water acclimated animals to 340 mOsm. At specified intervals, concentrations of adenine nucleotides in total tissue (whole animal except for valves and adductors) were determined as described below. This species tolerated both osmotic stresses and survived in the respective media for several months. For the experiments on anoxic stress, the animals were treated in two ways; the 340 mOsm acclimated animals were transferred to a vessel containing the same medium (6 individuals/l) constantly bubbled with N, gas or to a closed vessel containing N, gas only without water. Two days after the transfer to the anoxic conditions, concentrations of the adenine nucleotides in total tissue were determined. In all experiments conducted here, valves were not wedged open. Controls were simultaneously worked up using the animals without stress. Medium for acclimation was prepared bydilutingfiltered natural sea water with tap water and osmotic concentration of the medium was determined with an osmometer (Advanced, DII). Analytical reayents Enzyme products of phosphoglycerate kinase (PGK)glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mixture, lactate dehydrogenase (LDH), pyruvate kinase (PK) and myokinase (MK), nucleotides of ATP, ADP and AMP for 57
0.
58
MATSUSH~MAet al
standards, substrates of glycerate-3-phosphate and phosphoenolpyruvate, and triethanolamine hydrochloride were obtained from Boehringer. Cofactor NADH was purchased from Sigma. All other chemicals used were of reagent grade. Determination
of adenine nucleotides
Tissue extract was prepared by a method similar to that of Wijsman (1976). In brief, excised total tissue was promptly blotted with filter paper and dropped in liquid nitrogen. It took about 1 min from excision to freezing. The frozen tissue (1-2 g) was removed from liquid nitrogen, rapidly weighed to the nearest 0.01 g and homogenized in 5 vol of ice-cold 7% trichloroacetic acid (TCA) using an Ultra-Turrax homogenizer before the frozen tissue melted. The homogenate was centrifuged at 25,000 9 for 60 min. The supernatant was neutralized with 5 M K,CO, and analysed for ATP, ADP and AMP. Factor of dilution effect of K,CO, was taken into account (1.04). In order to examine the recovery of the three nucleotides in this method, frozen tissue was homogenized together with a standard sample of known concentration of nucleotide mixture. For each nucleotide, a high recovery percentage of almost 100% was obtained. Concentrations of ATP, ADP and AMP were determined enzymatically by the method ofJaworek et (II. (1974a,b). Final volume of reaction mixture was 3.0 ml for ATP assay and 2.8 ml for ADP and AMP assay, in which 0.2 ml sample and 20 pl of respective enzyme products were included. Activities of enzymes added to the assay mixture were 8 U of both PGK and GAPDH for ATP determination and 50 U of LDH, 40 U of PK and 35 U of MK for ADP and AMP determination. Decrease in absorbance (AE,,o = 6.3 x 103) by oxidation of NADH was measured using 1 cm quartz cuvettes and a spectrophotometer (Hitachi 100-20) equipped with a thermostatted chamber (30°C). A series of known concentration of nucleotides were assayed in comparison between the presence and absence of TCA. In the presence of TCA, ATP was underestimated as reported by Wijsman (1976). Therefore, inhibition factor was included in the calculation of concentration (1.08). Further, TCA delayed completion of MK reaction for AMP assay, but it did not affect the endpoint. For the experiments on osmotic stress, nucleotide levels were expressed as pmol/g dry wt by including the water content of tissue in the calculation. Water content At specified times after the osmotic shock, the animals were removed, and acclimating water on the shell surface was wiped away with tissue paper. Then, valves were pried open by cutting the adductors and the total tissue was excised. The tissue was blotted with filter paper, weighed and dried overnight in a 95°C drying oven. Water content was calculated from the difference between the wet and dry wt and expressed as o/0of wet wt. Statistics Student’s t-test was used to compare experimental groups with controls and P < 0.05 was used as the level of significance. RESULTS Volume
regulation
Time course of volume means of the water content
Days Fig. 1. Changes in water content of total tissue of Corbicula japonica during acclimation to hypo-(a) and hyperosmotic(b) stress. (a) Clams acclimated to 340 mOsm and transferred to 340 mOsm (0) or to fresh water (a). (b) Clams acclimated to fresh water and transferred to fresh water (0) or to 340 mOsm (0). Points t_ vertical lines are the means +SE (n = 68).
recovered to 83.2% and after three days to 80.6%. This value of 80.6% was not different statistically from that of animals acclimated to fresh water for two weeks (P > 0.5), but the water content never returned to the original level of control animals kept in 340 mOsm ; it was significantly higher than controls at the 5th day (0.01 < P < 0.02) and at the 7th day (P < 0.01). When the animals acclimated to fresh water were transferred to 340 mOsm, they did not show an evident process of volume regulation (Fig. lb). Water content of the control animals kept in fresh water was maintained within the range of 80.1-81.8x during the experimental period. Upon transfer to 340 mOsm the animal tissue lost water rapidly; after 5 hr the water content decreased significantly to a level lower than that of controls (P < 0.001) and reached the lowest level of 74.8% after one day. The water content of the experimental animals was maintained significantly lower than that of controls throughout the experimental period (P < 0.001 at each time). Effect
regulation
was examined
by
in total tissue of the animals exposed to hypo- (Fig. la) and hyperosmotic stress (Fig. lb). As shown in Fig. l(a), water content of the control animals acclimated to 340 mOsm was 77-78x of wet tissue wt. When the animals were transferred to fresh water, the water content increased remarkably within 5 hr and reached the maximum of 86.4% in 10 hr. Thereafter, the content began to decrease towards the control level; after one day the water content
of osmotic
stress
on the energy
charge
In the animals acclimated to 340 mOsm, the energy charge of the total tissue was around 0.6 at each specified time of sampling (Fig. 2). When the animals were transferred to fresh water, the energy charge increased significantly to 0.75 after one day (P < 0.001). However, (P > 0.5),
the
increase
was
transient;
at
the
3rd
5th (P > 0.5) and 7th day (P > 0.05) difference in the energy charge between the experimental animals and controls was not significant. This change in energy charge reflected the change in each
Osmotic/anoxic
59
stress affects Bivalve energy charge 1.0
1
t Energy
01, ADP
charge
’
’
’
?
u
m
i
5-
Total
pool
z E X
ATP
OL
01234567
‘0
1
2
3
4
5
6
7
Days Fig. 2. Changes of adenine nucleotide levels in total tissue of Corhiculajaponica and the calculated parameter of the adenylate pool during acclimation to hypoosmotic stress. Clams acclimated to 340 mOsm and transferred to 340 mOsm (0) or to fresh water (0). Points + vertical lines are the means + SE (n = 5-8). For those points without vertical lines, standard errors are less than the size of the points.
the experimental animals could not be distinguished statistically from that of the controls. Changes in quantity of ATP were completely reverse to those of ADP and AMP in both hypo- and hyperosmotic acclimation. The values of the energy charge were considerably different from one individual to another, though scarcely below 0.6. At specific times (the initial day of hyperosmotic stress and the 3rd day of hyperosmotic stress), however, most individuals showed high values of the energy charge.
nucleotide; a marked increase (l&fold) in ATP accompanied by a decrease in ADP and AMP was observed after one day, but thereafter (3,5 and 7 days) the level of each nucleotide was nearly the same in the control and experimental animals. In the reverse osmotic stress, the energy charge of the control groups kept in fresh water was O.&O.7 throughout the experimental period (Fig. 3). At the 3rd day after transfer to 340 mOsm, the energy charge of the experimental animals was significantly higher than that of controls (P < O.OOl), reflecting an increase in ATP and decreases in ADP and AMP. However, this increase was transient; at the 1st (P > 0.1) 5th (P > 0.5) and 7th day (P > 0.2) the energy charge of
E$ect of anoxic stress on the energy charge The energy charge of the animals which were kept in 340 mOsm sea water gassed with N, (P < 0.02) or in l.Or
Energy
charge
u ol
5;
i
ATP
Total
pool
iz
% Oo
1
2
3
4
5
6
7
Days Fig. 3. Changes of adenine nucleotide levels in total tissue of Corhiculajaponicaand the calculated parameter of the adenylate pool during acclimation to hyperosmotic stress. Clams acclimated to fresh water and transferred to fresh water (0) or to 340 mOsm (0). Points k vertical lines are the means f SE (n = 5-8). For those points without vertical lines, standard errors are less than the size of the points.
60
0. MATSUSHIMA et al. a) 340 m&m aerated b) 340 mOsm gassed with N2 c) Nz gas
0.8 3 i 0.6 2
I1 0.4
b
1
:
0.2
a bc
: AD
C
.~
AMP
TcItal po
0
Energy charge
Fig. 4. Effect of anoxic stress (48 hr) on concentration of adenine nucleotides in total tissue of Corbicula japonica and the calculated parameter of the adenylate pool. Clams acclimated to 340 mOsm and transferred to (a) 340 mOsm aerated ; (b) 340 mOsm gassed with N, ; and (c) N, gas only without water. Columns + vertical lines are the means + SE (n = 5-6).
N2 gas without water (P < 0.01) for two days was slightly but significantly lower than that of the control animals acclimated to well aerated 340 mOsm sea water (Fig. 4). The two kinds of anoxic stresses caused to the same extent a decrease in concentration of ATP and increase in ADP and AMP. The decrease in ATP was completely accounted for by the sum of increase in ADP and AMP, and this resulted in unchanged pool size of total adenylates (P > 0.5). Although some of the animals placed in 340 mOsm gassed with N, were observed to open their valves remarkably during the course of experiment, they responded well to mechanical stimulation. DISCUSSION
Concentration of total adenylate pool in total tissues of Corbicula japonica was quite similar to that in total tissue, hepatopancreas and mantle of Myths edulis (Wijsman, 1976), but lower than the concentration in adductors of M. edulis (Wijsman, 1976), Chlamys opercularis (Grieshaber, 1978) and Lima hiuns (Gade, 1981). Beis and Newsholme (1975) investigated the contents of adenine nucleotides in muscle tissues from many animal species of vertebrates and invertebrates and found that the lowest concentrations of ATP and total adenylates were determined in the molluscan muscles except for Pecten maximus. The high concentration of ATP and total adenylates in adductor muscles of Chlamys, Lima and Pecten were related to swimming behavior of these species. The energy charge values of C. juponica ranged from 0.6 to 0.8 during acclimation to salinity change. This range of energy charge was similar to that determined for M. edulis (Wijsman, 1976) and the sea anemone Bunodosomn cauernata (Ellington, 1981), which were exposed to anoxic stress. However, the control group of C. japonicu without stress exhibited lower energy charge (O&-0.7) than that of the above species. Many species of intertidal bivalves are known to depend conspicuously upon anaerobic metabolism which produces alanine as one of the major end-products
(Hochachka and Mustafa, 1972; de Zwaan and Wijsman, 1976; de Zwaan, 1977). Mangum and Burnett (1975) observed that Rungia cuneutu and Myu urenuriu extracted only a small part of oxygen from water which was ventilated by the animals under normoxic condition. The normoxic anaerobiosis may account for the low level of the energy charge observed in control animals of C. japonicu. In fact, the energy charge of the animals exposed to anoxic stress fell by only 0.1 and this decrement by anoxic stress was much smaller than that determined for the foregoing species of the sea mussel and sea anemone. Corbiculu juponicu inhabits the bottom mud of river near the estuary, whose environment is labile in salinity, temperature and oxygen concentration and even ebbs away at low tide. As pointed out by Mangum and Burnett (1975), adaptation to anaerobiosis may be the result that intracellular osmoregulation mediated by free amino acids would have been more serious problem to these animals than efficiency of energy production. The energy charge of C. juponicu elevated transiently at the initial day of hypoosmotic stress but at the third day of hyperosmotic stress. Such a different time course between hypo- and hyperosmotic stress was observed in other responses of this species (Matsushima, 1982). That is, during acclimation to salinity decrease the osmotic pressure of the mantle fluid and concentration of ninhydrin positive substances (NPS) in tissue were decreased within a day to the level of fresh water acclimated animals. During acclimation to salinity increase, on the other hand, it took 3-5 days for those values to increase to almost the level of the animals acclimated to high salinity. Thus, the change in energy charge presented here is closely related to both organismal and cellular responses to osmotic stress. Since the change in osmotic concentration of the mantle fluid reflects the ventilatory activity of the clam, the time required for osmotic equilibrium between the mantle fluid and external medium would be related to the resumption of oxygen uptake after osmotic shock. In fact, Rungiu cuneuta resumed oxygen uptake more rapidly after hypo-
Osmoticianoxic
stress affec:ts Bivalve energy charge
osmotic transfer than after hyperosmotic transfer (Henry and Mangum, 1980; Henry et al., 1980). From this point of view, the transient conversion from anaerobic metabolism to aerobic one appears to account for the elevation in energy charge during osmotic stress. According to the theory of Atkinson (1968), the activity of key enzymes related to energy metabolism and biosynthesis is affected by energy charge within the range of change in C. japonica after osmotic shock. Mustafa and Hochachka (1971) have reported that pyruvate kinase of Crassostrea gigas, the enzyme that participates in alanine production by catalyzing conversion from phosphoenolpyruvate to pyruvate, is inhibited by ATP. Further, we have found that glutamate dehydrogenase (GDH) of C. japonica responds typically to the energy charge; reduction in the energy charge in reaction mixture including 3 mM total adenylate pool resulted in activation of the GDH (Matsushima and Kado, 1983). It can be considered, therefore, that activity of both enzymes in ~ivo would be closely related to change in the energy charge during acclimation to salinity change. Since alanine is the major component of FAA pool in C. japonica (Matsushima et al., 1982), change in the energy charge during osmotic stress may play an important role in intracellular osmoregulation by controlling these enzyme activities, REFERENCES
Atkinson D. E. (1968) The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 7,403@-4034. Atkinson D. E. (1977) Cellular Energy Metabolism and its Regulation, pp. 85-107. Academic Press, New York. Atkinson D. E. and Walton G. M. (1967) Adenosine triphosphate conversion in metabolic regulation. J. biol. Chem. 242, 3239-3241. Baginski R. M. and Pierce S. K. (1978) A comparison of amino acid accumulation during high salinity adaptation with anaerobic metabolism in the ribbed mussel, Modiolus demissus demissus. J. exp. Zool. 203,419428. Beis 1. and Newsholme E. A. (1975) The contents of adenine nucleotides, phosphagens and some glycolytic intermediates in resting muscles from vertebrates and invertebrates. Biochem. J. 152, 23-32. Ellington W. R. (1981) Effect of anoxia on the adenylates and the energy charge in the sea anemone, Bunodosoma cauernata (BOSC). Physiol. Zool. S&415422. Gade G. (1981) Energy production during swimming in the adductor muscle of the bivalve Lima mans: Comparison with the data from other bivalve mollusks. Physiol. Zool. 54,40%406. Gainey L. F. (1978) The response of the Corbiculidae (Mollusca: Bivalvia) to osmotic stress: The cellular response. Physiol. Zool. 51, 79-91. Grieshaber M. (1978) Breakdown and formation of highenergy phosphates and octopine in the adductor muscle of the scallop, Chlamys opercularis (L.), during escape swimming and recovery. J. romp. Physiol. 126, 269-276.
61
Henry R. P. and Mangum C. P. (1980) Salt and water balance in the oligohaline clam, Rangia cuneata. III. Reduction of the free amino acid pool during low salinity adaptation. J. exp. 2001. 211, 25-32. Henry R. P., Mangum C. P. and Webb K. L. (1980) Salt and water balance in the oligohaline clam, Rangia cuneata. II. Accumulation of intracellular free amino acids during high salinity adaptation. J. exp. Zoo/. 211, 1 l-24. Hochachka P. W. and Mustafa T. (1972) Invertebrate facultative anaerobiosis. Science, N.Y. 178, 10561060. Jaworek D., Gruber W. and Bergmeyer H. U. (1974a) Adenosine-S-triphosphate. Determination with 3-phosphoglycerate kinase. In Methods of Enzymatic Analysis (Edited by Bergmeyer H. U.), Vol. 4, pp, 2097-2101. Academic Press, New York. Jaworek D., Gruber W. and Bergmeyer H. U. (1974b) Adenosine-5’-diphosphate and adenosine-5’-monophosphate. In Methods of Enzymatic Analysis (Edited by Bergmeyer H. U.), Vol. 4. pp. 2 127-2 13 1.Academic Press, New York. Mangum C. P. and Burnett L. E. (1975) The extraction of oxygen by estuarine invertebrates. In Physiological Ecology olEstuarine Organisms (Edited by Vernberg F. J.), pp. 147-163. Univ. So. Carolina Press, Columbia. Matsushima 0. (1982) Comparative studies on responses to osmotic stresses in brackish and freshwater clams. J. Sci. Hiroshima Uniu. (B-l) 30, 173-192. Matsushima 0. and Kado Y. (1983) Effect of adenine nucleotides on glutamate dehydrogenase activities of the brackish and freshwater clams, Corbicula japonica and C. leana. Annot. 2001. Japon. 56, 3-9. Matsushima O., Sakka F. and Kado Y. (1982) Free amino acid involved in intracellular osmoregulation in the clam, Corbicula. J. Sci. Hiroshima Uniu. (B-l), 30, 213-219. Mustafa T. and Hochachka P. W. (1971) Catalytic and regulatory properties of pyruvate kinases in tissue of marine bivalve. J. biol. Chem. 246, 31963203. Pierce S. K. (1971) A source of solute for volume regulation in marine mussels. Camp. Biochem. Physiol. 38A, 619-635. Purich D. L. and Fromm H. J. (1972) Studies on factors influencing enzyme responses to adenylate energy charge. J. biol. Chem. 247, 249-255. Purich D. L. and Fromm H. J. (1973) Additional factors influencing enzyme responses to the adenylate energy charge. J. biol. Chem. 248,461466. Ridge J. W. (1972) Hypoxia and the energy charge of the cerebral adenylate pool. Biochem. J. 127,351-355. Thompson F. M. and Atkinson D. E. (1971) Response of nucleoside diphosphate kinase to the adenylate energy charge. Biochem. biophys. Res. Commun. 45, 1581-1585. Virkar R. A. and Webb K. L. (1970) Free amino acid composition of the soft-shell clam Mya arenaria in relation to salinity of the medium. Camp. Biochem. Physiol. 32,775783. Wijsman T. C. M. (1976) Adenosine phosphates and energy charge in different tissues of Mytilus edulis L. under aerobic and anaerobic conditions. J. camp. Physiol. 107, 129-140. Zwaan A. de (1977) Anaerobic energy metabolism in bivalve molluscs. Oceanogr. mar. biol. Ann. Reo. 15, 103-187. Zwaan A. de and Wijsman T. C. M. (1976) Anaerobic metabolism in bivalvia (Mollusca). Characteristics of anaerobic metabolism. Camp. Biochem. Physiol. 54B, 313324.
EFFECT ADENYLATE
0300-9629!84 S3.00 + 0.00 ,S 1984 Pergamon Press Ltd
I, pp. 57-61, 1984
OF EXTERNAL SALINITY CHANGE ON THE ENERGY CHARGE IN THE BRACKISH BIVALVE CORBICULA
JAPONICA
0. MATSUSHIMA, H. KATAYAMA, K. YAMADA and Y. KADO Zoological
Institute,
Faculty of Science, Hiroshima University, Hiroshima 730 (Tel.: 0822 41-1221) Suzugamine Women’s College, Hiroshima 733, Japan
and
(Received 29 March 1983) Abstract1. The energy charge in the brackish clam Corbicula japonica changed within the range of 0.6-0.8 during exposure to osmotic or anoxic stress. 2. The energy charge elevated transiently at the initial day of hypoosmotic stress and at the third day of hyperosmotic stress. Exposure to anoxia resulted in a small but significant decline in energy charge. The transient elevation during osmotic stress may be a reflection of aerobic metabolism. 3. During exposure to these stresses, activities of key enzymes involved in amino acid metabolism may be regulated by the energy charge.
change in FAA pool size. In addition, change in water content of tissue, which is considered to be an index of cell volume regulation, was examined to compute the net quantity of adenine nucleotides excluding the effect of tissue hydration or dehydration.
INTRODUCTION known that in many aquatic molluscs the concentration of intracellular free amino acids (FAA) changes in response to salinity change in external medium (Virkar and Webb, 1970; Pierce, 1971; Gainey, 1978). The change in FAA concentration is considered to be important in cell volume regulation or isosmotic intracellular regulation during acclimation to salinity change. Although the biochemical mechanism underlying the FAA fluctuation is obscure, glutamate dehydrogenase (GDH) is assumed to be one of the important enzymes involved in FAA accumulation during acclimation to salinity increase (Gainey, 1978; Baginski and Pierce, 1978). We reported previously that GDH activities of the bivalves Corbicula juponica and C. leana were markedly affected by the adenylate energy charge in reaction mixture (Matsushima and Kado, 1983). Atkinson (1968, 1977) has proposed that the energy level in the adenylate system can be quantitatively represented by the energy charge (ATP+ l/2 ADP)/(ATP+ADP+AMP) and that activity of the key enzymes involved in energy metabolism and biosynthesis is under control of this parameter. Some enzymes are reported to respond to the energy charge (Atkinson and Walton, 1967; Thompson and Atkinson, 1971), though the response pattern is dependent upon some other factors (Purich and Fromm, 1972, 1973). As a condition influencing the energy charge in intact animals, anoxic stress has been known to reduce its value markedly in some animals (Ridge, 1972; Wijsman, 1976; Ellington, 1981). However, little is known about the effect of osmotic stress on the energy charge. The present study was primarily designed to examine whether the energy charge in uiuo could account for the physiological significance of the in oitro energy charge response of GDH. In this study, the effect of both hypo- and hyperosmotic stresses on the energy charge of the brackish bivalve, Corbicula japonica, was investigated in the context of intracellular osmoregulation mediated by It is well
MATERIALS AND METHODS Acclimation
of animal
Specimens of Corbicula japonica (shell length of 2-3 cm) were collected from a brackish river in Hiroshima City, where salinity of water was remarkably changed by tidal cycle. At low tide, osmotic concentrations of water and filtrate of the bottom mud in the habitat were about 100 mOsm and 300400mOsm, respectively. The animals were acclimated to 35% sea water (340 mOsm) or to fresh water (3-7 mOsm), which was recirculated and aerated, for more than two weeks at water temperature of 2C-26°C. In order to examine the effect of osmotic stress, the animals acclimated to 340 mOsm were transferred to fresh water and the fresh water acclimated animals to 340 mOsm. At specified intervals, concentrations of adenine nucleotides in total tissue (whole animal except for valves and adductors) were determined as described below. This species tolerated both osmotic stresses and survived in the respective media for several months. For the experiments on anoxic stress, the animals were treated in two ways; the 340 mOsm acclimated animals were transferred to a vessel containing the same medium (6 individuals/l) constantly bubbled with N, gas or to a closed vessel containing N, gas only without water. Two days after the transfer to the anoxic conditions, concentrations of the adenine nucleotides in total tissue were determined. In all experiments conducted here, valves were not wedged open. Controls were simultaneously worked up using the animals without stress. Medium for acclimation was prepared bydilutingfiltered natural sea water with tap water and osmotic concentration of the medium was determined with an osmometer (Advanced, DII). Analytical reayents Enzyme products of phosphoglycerate kinase (PGK)glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mixture, lactate dehydrogenase (LDH), pyruvate kinase (PK) and myokinase (MK), nucleotides of ATP, ADP and AMP for 57
0.
58
MATSUSH~MAet al
standards, substrates of glycerate-3-phosphate and phosphoenolpyruvate, and triethanolamine hydrochloride were obtained from Boehringer. Cofactor NADH was purchased from Sigma. All other chemicals used were of reagent grade. Determination
of adenine nucleotides
Tissue extract was prepared by a method similar to that of Wijsman (1976). In brief, excised total tissue was promptly blotted with filter paper and dropped in liquid nitrogen. It took about 1 min from excision to freezing. The frozen tissue (1-2 g) was removed from liquid nitrogen, rapidly weighed to the nearest 0.01 g and homogenized in 5 vol of ice-cold 7% trichloroacetic acid (TCA) using an Ultra-Turrax homogenizer before the frozen tissue melted. The homogenate was centrifuged at 25,000 9 for 60 min. The supernatant was neutralized with 5 M K,CO, and analysed for ATP, ADP and AMP. Factor of dilution effect of K,CO, was taken into account (1.04). In order to examine the recovery of the three nucleotides in this method, frozen tissue was homogenized together with a standard sample of known concentration of nucleotide mixture. For each nucleotide, a high recovery percentage of almost 100% was obtained. Concentrations of ATP, ADP and AMP were determined enzymatically by the method ofJaworek et (II. (1974a,b). Final volume of reaction mixture was 3.0 ml for ATP assay and 2.8 ml for ADP and AMP assay, in which 0.2 ml sample and 20 pl of respective enzyme products were included. Activities of enzymes added to the assay mixture were 8 U of both PGK and GAPDH for ATP determination and 50 U of LDH, 40 U of PK and 35 U of MK for ADP and AMP determination. Decrease in absorbance (AE,,o = 6.3 x 103) by oxidation of NADH was measured using 1 cm quartz cuvettes and a spectrophotometer (Hitachi 100-20) equipped with a thermostatted chamber (30°C). A series of known concentration of nucleotides were assayed in comparison between the presence and absence of TCA. In the presence of TCA, ATP was underestimated as reported by Wijsman (1976). Therefore, inhibition factor was included in the calculation of concentration (1.08). Further, TCA delayed completion of MK reaction for AMP assay, but it did not affect the endpoint. For the experiments on osmotic stress, nucleotide levels were expressed as pmol/g dry wt by including the water content of tissue in the calculation. Water content At specified times after the osmotic shock, the animals were removed, and acclimating water on the shell surface was wiped away with tissue paper. Then, valves were pried open by cutting the adductors and the total tissue was excised. The tissue was blotted with filter paper, weighed and dried overnight in a 95°C drying oven. Water content was calculated from the difference between the wet and dry wt and expressed as o/0of wet wt. Statistics Student’s t-test was used to compare experimental groups with controls and P < 0.05 was used as the level of significance. RESULTS Volume
regulation
Time course of volume means of the water content
Days Fig. 1. Changes in water content of total tissue of Corbicula japonica during acclimation to hypo-(a) and hyperosmotic(b) stress. (a) Clams acclimated to 340 mOsm and transferred to 340 mOsm (0) or to fresh water (a). (b) Clams acclimated to fresh water and transferred to fresh water (0) or to 340 mOsm (0). Points t_ vertical lines are the means +SE (n = 68).
recovered to 83.2% and after three days to 80.6%. This value of 80.6% was not different statistically from that of animals acclimated to fresh water for two weeks (P > 0.5), but the water content never returned to the original level of control animals kept in 340 mOsm ; it was significantly higher than controls at the 5th day (0.01 < P < 0.02) and at the 7th day (P < 0.01). When the animals acclimated to fresh water were transferred to 340 mOsm, they did not show an evident process of volume regulation (Fig. lb). Water content of the control animals kept in fresh water was maintained within the range of 80.1-81.8x during the experimental period. Upon transfer to 340 mOsm the animal tissue lost water rapidly; after 5 hr the water content decreased significantly to a level lower than that of controls (P < 0.001) and reached the lowest level of 74.8% after one day. The water content of the experimental animals was maintained significantly lower than that of controls throughout the experimental period (P < 0.001 at each time). Effect
regulation
was examined
by
in total tissue of the animals exposed to hypo- (Fig. la) and hyperosmotic stress (Fig. lb). As shown in Fig. l(a), water content of the control animals acclimated to 340 mOsm was 77-78x of wet tissue wt. When the animals were transferred to fresh water, the water content increased remarkably within 5 hr and reached the maximum of 86.4% in 10 hr. Thereafter, the content began to decrease towards the control level; after one day the water content
of osmotic
stress
on the energy
charge
In the animals acclimated to 340 mOsm, the energy charge of the total tissue was around 0.6 at each specified time of sampling (Fig. 2). When the animals were transferred to fresh water, the energy charge increased significantly to 0.75 after one day (P < 0.001). However, (P > 0.5),
the
increase
was
transient;
at
the
3rd
5th (P > 0.5) and 7th day (P > 0.05) difference in the energy charge between the experimental animals and controls was not significant. This change in energy charge reflected the change in each
Osmotic/anoxic
59
stress affects Bivalve energy charge 1.0
1
t Energy
01, ADP
charge
’
’
’
?
u
m
i
5-
Total
pool
z E X
ATP
OL
01234567
‘0
1
2
3
4
5
6
7
Days Fig. 2. Changes of adenine nucleotide levels in total tissue of Corhiculajaponica and the calculated parameter of the adenylate pool during acclimation to hypoosmotic stress. Clams acclimated to 340 mOsm and transferred to 340 mOsm (0) or to fresh water (0). Points + vertical lines are the means + SE (n = 5-8). For those points without vertical lines, standard errors are less than the size of the points.
the experimental animals could not be distinguished statistically from that of the controls. Changes in quantity of ATP were completely reverse to those of ADP and AMP in both hypo- and hyperosmotic acclimation. The values of the energy charge were considerably different from one individual to another, though scarcely below 0.6. At specific times (the initial day of hyperosmotic stress and the 3rd day of hyperosmotic stress), however, most individuals showed high values of the energy charge.
nucleotide; a marked increase (l&fold) in ATP accompanied by a decrease in ADP and AMP was observed after one day, but thereafter (3,5 and 7 days) the level of each nucleotide was nearly the same in the control and experimental animals. In the reverse osmotic stress, the energy charge of the control groups kept in fresh water was O.&O.7 throughout the experimental period (Fig. 3). At the 3rd day after transfer to 340 mOsm, the energy charge of the experimental animals was significantly higher than that of controls (P < O.OOl), reflecting an increase in ATP and decreases in ADP and AMP. However, this increase was transient; at the 1st (P > 0.1) 5th (P > 0.5) and 7th day (P > 0.2) the energy charge of
E$ect of anoxic stress on the energy charge The energy charge of the animals which were kept in 340 mOsm sea water gassed with N, (P < 0.02) or in l.Or
Energy
charge
u ol
5;
i
ATP
Total
pool
iz
% Oo
1
2
3
4
5
6
7
Days Fig. 3. Changes of adenine nucleotide levels in total tissue of Corhiculajaponicaand the calculated parameter of the adenylate pool during acclimation to hyperosmotic stress. Clams acclimated to fresh water and transferred to fresh water (0) or to 340 mOsm (0). Points k vertical lines are the means f SE (n = 5-8). For those points without vertical lines, standard errors are less than the size of the points.
60
0. MATSUSHIMA et al. a) 340 m&m aerated b) 340 mOsm gassed with N2 c) Nz gas
0.8 3 i 0.6 2
I1 0.4
b
1
:
0.2
a bc
: AD
C
.~
AMP
TcItal po
0
Energy charge
Fig. 4. Effect of anoxic stress (48 hr) on concentration of adenine nucleotides in total tissue of Corbicula japonica and the calculated parameter of the adenylate pool. Clams acclimated to 340 mOsm and transferred to (a) 340 mOsm aerated ; (b) 340 mOsm gassed with N, ; and (c) N, gas only without water. Columns + vertical lines are the means + SE (n = 5-6).
N2 gas without water (P < 0.01) for two days was slightly but significantly lower than that of the control animals acclimated to well aerated 340 mOsm sea water (Fig. 4). The two kinds of anoxic stresses caused to the same extent a decrease in concentration of ATP and increase in ADP and AMP. The decrease in ATP was completely accounted for by the sum of increase in ADP and AMP, and this resulted in unchanged pool size of total adenylates (P > 0.5). Although some of the animals placed in 340 mOsm gassed with N, were observed to open their valves remarkably during the course of experiment, they responded well to mechanical stimulation. DISCUSSION
Concentration of total adenylate pool in total tissues of Corbicula japonica was quite similar to that in total tissue, hepatopancreas and mantle of Myths edulis (Wijsman, 1976), but lower than the concentration in adductors of M. edulis (Wijsman, 1976), Chlamys opercularis (Grieshaber, 1978) and Lima hiuns (Gade, 1981). Beis and Newsholme (1975) investigated the contents of adenine nucleotides in muscle tissues from many animal species of vertebrates and invertebrates and found that the lowest concentrations of ATP and total adenylates were determined in the molluscan muscles except for Pecten maximus. The high concentration of ATP and total adenylates in adductor muscles of Chlamys, Lima and Pecten were related to swimming behavior of these species. The energy charge values of C. juponica ranged from 0.6 to 0.8 during acclimation to salinity change. This range of energy charge was similar to that determined for M. edulis (Wijsman, 1976) and the sea anemone Bunodosomn cauernata (Ellington, 1981), which were exposed to anoxic stress. However, the control group of C. japonicu without stress exhibited lower energy charge (O&-0.7) than that of the above species. Many species of intertidal bivalves are known to depend conspicuously upon anaerobic metabolism which produces alanine as one of the major end-products
(Hochachka and Mustafa, 1972; de Zwaan and Wijsman, 1976; de Zwaan, 1977). Mangum and Burnett (1975) observed that Rungia cuneutu and Myu urenuriu extracted only a small part of oxygen from water which was ventilated by the animals under normoxic condition. The normoxic anaerobiosis may account for the low level of the energy charge observed in control animals of C. japonicu. In fact, the energy charge of the animals exposed to anoxic stress fell by only 0.1 and this decrement by anoxic stress was much smaller than that determined for the foregoing species of the sea mussel and sea anemone. Corbiculu juponicu inhabits the bottom mud of river near the estuary, whose environment is labile in salinity, temperature and oxygen concentration and even ebbs away at low tide. As pointed out by Mangum and Burnett (1975), adaptation to anaerobiosis may be the result that intracellular osmoregulation mediated by free amino acids would have been more serious problem to these animals than efficiency of energy production. The energy charge of C. juponicu elevated transiently at the initial day of hypoosmotic stress but at the third day of hyperosmotic stress. Such a different time course between hypo- and hyperosmotic stress was observed in other responses of this species (Matsushima, 1982). That is, during acclimation to salinity decrease the osmotic pressure of the mantle fluid and concentration of ninhydrin positive substances (NPS) in tissue were decreased within a day to the level of fresh water acclimated animals. During acclimation to salinity increase, on the other hand, it took 3-5 days for those values to increase to almost the level of the animals acclimated to high salinity. Thus, the change in energy charge presented here is closely related to both organismal and cellular responses to osmotic stress. Since the change in osmotic concentration of the mantle fluid reflects the ventilatory activity of the clam, the time required for osmotic equilibrium between the mantle fluid and external medium would be related to the resumption of oxygen uptake after osmotic shock. In fact, Rungiu cuneuta resumed oxygen uptake more rapidly after hypo-
Osmoticianoxic
stress affec:ts Bivalve energy charge
osmotic transfer than after hyperosmotic transfer (Henry and Mangum, 1980; Henry et al., 1980). From this point of view, the transient conversion from anaerobic metabolism to aerobic one appears to account for the elevation in energy charge during osmotic stress. According to the theory of Atkinson (1968), the activity of key enzymes related to energy metabolism and biosynthesis is affected by energy charge within the range of change in C. japonica after osmotic shock. Mustafa and Hochachka (1971) have reported that pyruvate kinase of Crassostrea gigas, the enzyme that participates in alanine production by catalyzing conversion from phosphoenolpyruvate to pyruvate, is inhibited by ATP. Further, we have found that glutamate dehydrogenase (GDH) of C. japonica responds typically to the energy charge; reduction in the energy charge in reaction mixture including 3 mM total adenylate pool resulted in activation of the GDH (Matsushima and Kado, 1983). It can be considered, therefore, that activity of both enzymes in ~ivo would be closely related to change in the energy charge during acclimation to salinity change. Since alanine is the major component of FAA pool in C. japonica (Matsushima et al., 1982), change in the energy charge during osmotic stress may play an important role in intracellular osmoregulation by controlling these enzyme activities, REFERENCES
Atkinson D. E. (1968) The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 7,403@-4034. Atkinson D. E. (1977) Cellular Energy Metabolism and its Regulation, pp. 85-107. Academic Press, New York. Atkinson D. E. and Walton G. M. (1967) Adenosine triphosphate conversion in metabolic regulation. J. biol. Chem. 242, 3239-3241. Baginski R. M. and Pierce S. K. (1978) A comparison of amino acid accumulation during high salinity adaptation with anaerobic metabolism in the ribbed mussel, Modiolus demissus demissus. J. exp. Zool. 203,419428. Beis 1. and Newsholme E. A. (1975) The contents of adenine nucleotides, phosphagens and some glycolytic intermediates in resting muscles from vertebrates and invertebrates. Biochem. J. 152, 23-32. Ellington W. R. (1981) Effect of anoxia on the adenylates and the energy charge in the sea anemone, Bunodosoma cauernata (BOSC). Physiol. Zool. S&415422. Gade G. (1981) Energy production during swimming in the adductor muscle of the bivalve Lima mans: Comparison with the data from other bivalve mollusks. Physiol. Zool. 54,40%406. Gainey L. F. (1978) The response of the Corbiculidae (Mollusca: Bivalvia) to osmotic stress: The cellular response. Physiol. Zool. 51, 79-91. Grieshaber M. (1978) Breakdown and formation of highenergy phosphates and octopine in the adductor muscle of the scallop, Chlamys opercularis (L.), during escape swimming and recovery. J. romp. Physiol. 126, 269-276.
61
Henry R. P. and Mangum C. P. (1980) Salt and water balance in the oligohaline clam, Rangia cuneata. III. Reduction of the free amino acid pool during low salinity adaptation. J. exp. 2001. 211, 25-32. Henry R. P., Mangum C. P. and Webb K. L. (1980) Salt and water balance in the oligohaline clam, Rangia cuneata. II. Accumulation of intracellular free amino acids during high salinity adaptation. J. exp. Zoo/. 211, 1 l-24. Hochachka P. W. and Mustafa T. (1972) Invertebrate facultative anaerobiosis. Science, N.Y. 178, 10561060. Jaworek D., Gruber W. and Bergmeyer H. U. (1974a) Adenosine-S-triphosphate. Determination with 3-phosphoglycerate kinase. In Methods of Enzymatic Analysis (Edited by Bergmeyer H. U.), Vol. 4, pp, 2097-2101. Academic Press, New York. Jaworek D., Gruber W. and Bergmeyer H. U. (1974b) Adenosine-5’-diphosphate and adenosine-5’-monophosphate. In Methods of Enzymatic Analysis (Edited by Bergmeyer H. U.), Vol. 4. pp. 2 127-2 13 1.Academic Press, New York. Mangum C. P. and Burnett L. E. (1975) The extraction of oxygen by estuarine invertebrates. In Physiological Ecology olEstuarine Organisms (Edited by Vernberg F. J.), pp. 147-163. Univ. So. Carolina Press, Columbia. Matsushima 0. (1982) Comparative studies on responses to osmotic stresses in brackish and freshwater clams. J. Sci. Hiroshima Uniu. (B-l) 30, 173-192. Matsushima 0. and Kado Y. (1983) Effect of adenine nucleotides on glutamate dehydrogenase activities of the brackish and freshwater clams, Corbicula japonica and C. leana. Annot. 2001. Japon. 56, 3-9. Matsushima O., Sakka F. and Kado Y. (1982) Free amino acid involved in intracellular osmoregulation in the clam, Corbicula. J. Sci. Hiroshima Uniu. (B-l), 30, 213-219. Mustafa T. and Hochachka P. W. (1971) Catalytic and regulatory properties of pyruvate kinases in tissue of marine bivalve. J. biol. Chem. 246, 31963203. Pierce S. K. (1971) A source of solute for volume regulation in marine mussels. Camp. Biochem. Physiol. 38A, 619-635. Purich D. L. and Fromm H. J. (1972) Studies on factors influencing enzyme responses to adenylate energy charge. J. biol. Chem. 247, 249-255. Purich D. L. and Fromm H. J. (1973) Additional factors influencing enzyme responses to the adenylate energy charge. J. biol. Chem. 248,461466. Ridge J. W. (1972) Hypoxia and the energy charge of the cerebral adenylate pool. Biochem. J. 127,351-355. Thompson F. M. and Atkinson D. E. (1971) Response of nucleoside diphosphate kinase to the adenylate energy charge. Biochem. biophys. Res. Commun. 45, 1581-1585. Virkar R. A. and Webb K. L. (1970) Free amino acid composition of the soft-shell clam Mya arenaria in relation to salinity of the medium. Camp. Biochem. Physiol. 32,775783. Wijsman T. C. M. (1976) Adenosine phosphates and energy charge in different tissues of Mytilus edulis L. under aerobic and anaerobic conditions. J. camp. Physiol. 107, 129-140. Zwaan A. de (1977) Anaerobic energy metabolism in bivalve molluscs. Oceanogr. mar. biol. Ann. Reo. 15, 103-187. Zwaan A. de and Wijsman T. C. M. (1976) Anaerobic metabolism in bivalvia (Mollusca). Characteristics of anaerobic metabolism. Camp. Biochem. Physiol. 54B, 313324.