Effects of Hypoxia on Energy Metabolism in Goldfish Hepatocytes

Comp. Biochem. Physiol. Vol. 117B, No. 1, pp. 151–158, 1997 Copyright  1996 Elsevier Science Inc.

ISSN 0305-0491/97/$17.00 PII S0305-0491(96)00318-5

Effects of Hypoxia on Energy Metabolism in Goldfish Hepatocytes M. Dorigatti,* G. Krumschnabel,* P. J. Schwarzbaum,† and W. Wieser* ¨ kophysiologie, Universita¨t Innsbruck, A-6020 Innsbruck, Austria, *Institut fu¨r Zoologie, Abteilung fu¨r O ´ ´ and †Instituto de Quimica y Fisicoquimica Biolo´gicas (Facultad de Farmacia y Bioqui´mica), Universidad de Buenos Aires, 1113 Buenos Aires, Argentina ABSTRACT. The present study addresses the question whether long-term acclimation to hypoxia of the whole animal is accompanied by a chronic re-organization of cellular function and metabolism. To this end, long- and short-term effects of hypoxia on energy metabolism were studied in hepatocytes isolated from goldfish acclimated to normoxia or hypoxia (10% air saturation). Aerobic (oxygen consumption) and anaerobic (lactate production under chemical anoxia) ATP turnover was not affected by acclimation to hypoxia. The initial ATP content, a crude measure of energy status, was elevated in hypoxia-acclimated cells compared with normoxic controls but returned to control levels within 3 hr of normoxic exposure. Na1 pump activity and the rate of protein synthesis were estimated from inhibitor sensitive rates of oxygen consumption. Neither of these two major ATP consumers of the cell was significantly altered by hypoxia acclimation, although Na1 pump activity showed a tendency to be elevated in the acclimated cells. During exposure to chemical anoxia, a pronounced decrease of lactate production was observed in hepatocytes that may have been related to metabolic acidosis. The decrease of anaerobic energy output was not accompanied by a decrease of cellular [ATP] and is therefore believed to reflect metabolic depression. The anaerobic energy metabolism of goldfish hepatocytes relies on ample glycogen reserves and does not involve ethanol formation as a way to minimize acidotic load. comp biochem physiol 117B;1:151–158, 1997.  1997 Elsevier Science Inc. KEY WORDS. Oxygen consumption, lactate production, ATP, chemical anoxia, metabolic depression, ethanol formation, glycogen

INTRODUCTION Among vertebrates, the goldfish (Carassius auratus L.) is one of the most tolerant species toward environmental anoxia. This has repeatedly been shown in whole animal studies [for review, see (27)] and recently also in studies on goldfish hepatocytes (10,11). Compared with a closely related species, the Crucian carp (Carassius carassius L.), however, the anoxic endurance of the goldfish is limited, allowing survival of fully anoxic conditions for a maximum of a few days at 4°C (32). Whereas the Crucian carp is more likely to encounter long-term anoxia when overwintering in icecovered lakes (16,22), the ecologically more relevant situation for the goldfish is the experience of severe hypoxia in warm eutrophic ponds. The latter situation can be tolerated for extended periods as shown in acclimation studies by van Address reprint requests to: W. Wieser, Institut fu¨r Zoologie, Abteilung fu¨ r ¨ kophysiologie, Universita¨t Innsbruck, Technikerstraβe 25, A-6020 InnsO bruck, Austria. Tel. (0)512-507-6181; Fax (0)512-507-2930; E-mail: [email protected]. Abbreviations–Vo2 tot, total oxygen consumption; osVo2 , ouabain-sensitive oxygen consumption; csVo2 , cycloheximide-sensitive oxygen consumption; NA, normoxia acclimation; HA, hypoxia acclimation. Received 4 May 1996; accepted 4 November 1996.

den Thillart et al. (24) and van den Thillart and Smit (25). On the organismic level, hypoxia tolerance may be achieved by an increase in oxygen uptake by increased ventilation rates and/or increased blood flow through the gills (18). However, these and other strategies aimed at maintaining a high oxygen supply to the tissues are limited in themselves because they pose an additional energy demand on the fish. Thus, below a critical level of environmental oxygen, the sole route to long-term survival is via a decrease of oxygen consumption without full activation of anaerobic energy turnover (8). It is this very capability of shutting down energy supply and energy demand in a concerted manner that has been found to render the goldfish anoxia tolerant. In the present study, we address the question whether tolerance toward environmental hypoxia can be traced to the cellular level, that is, does hypoxic exposure trigger cellular adaptations and/or reorganization of cellular metabolism that render the whole organism fitter for survival under these conditions. To answer this question, we compared long- and short-term effects of exposure to 10% air saturated water, which is a critical level of oxygen for goldfish (23), on total and function-related energy turnover, energetic

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state and fuel reserves of goldfish hepatocytes. Long-term effects were studied in hepatocytes isolated from goldfish acclimated to hypoxic conditions for 3–5 weeks, whereas short-term effects were examined by subjecting cells from normoxia- and hypoxia-acclimated animals to chemical anoxia. Comparative experiments were conducted with hepatocytes isolated from rainbow trout, aimed at determining the impact of incubation with 10% air saturated medium on the energetic state of anoxia-sensitive hepatocytes. One key characteristic of the anaerobic metabolism of the goldfish and two other teleost species is their capability of producing ethanol from pyruvate during hypoxia and anoxia (28). The responsible enzyme for ethanol production, alcohol dehydrogenase, has been detected in both muscle and liver tissue, but only the muscle isozyme seems to possess the kinetic characteristics necessary for quantitative ethanol production (15). Nevertheless, it has been shown that ethanol accumulates to significant amounts in the liver and in other tissues as well (21). To clarify whether ethanol can be taken up from the bloodstream or is produced in the liver, we determined ethanol in isolated liver cells maintained under anoxic conditions. MATERIALS AND METHODS Animals Goldfish, C. auratus (about 80g), and rainbow trout, Onchorynchus mykiss (about 250g), were obtained from local suppliers. Goldfish were acclimated to 15°C in large aquaria for at least 1 month before use; trout were kept in large outside tanks at water temperatures fluctuating between 10 and 15°C. Animals were fed trout pellets (EWOS Aquaculture International) ad libitum once a day. Normoxia/Hypoxia Acclimation Groups of 15–20 goldfish were transferred to 120-l aquaria in a separate room to avoid stress of the animals not related to the acclimation procedure. Animals assigned to the normoxia acclimation (NA) treatment were supplied with a continuous flow of aerated tap water. The aquaria used for hypoxia acclimation (HA) were covered with a glass screen and received fresh water according to the Po2 prevailing in the tanks. This was achieved by connecting a stirred oxygen electrode placed in the aquarium to a magnetic valve that controlled the inlet of water to the acclimation chamber. Because the rate of water exchange in these tanks was low, hypoxic water was continuously recycled over an outside filter to secure the appropriate water quality. After introduction of the fish into these chambers, the Po 2 was lowered from normoxia to 10% air saturation over a period of 10 days. When the desired level of hypoxia was reached, the animals were maintained at this condition for another 3–5 weeks before they were killed for hepatocyte isolation.

Isolation of Hepatocytes Goldfish hepatocytes were isolated according to Birnbaum et al. (2), as described by Schwarzbaum et al. (20). The isolation procedure was carried out with cold normoxic media except for the collagenase digestion step, which was done with medium warmed to room temperature. The time that elapsed between excision of the liver and incubation of the cells ranged from 45 to 60 min. The final cell pellets were suspended in media containing (for goldfish, in mM) 10 HEPES, 135 NaCl, 3.8 KCl, 1.3 CaCl2, 1.2 KH2PO4 , 1.2 MgSO4, 10 NaHCO3 , pH 7.6 at 20°C, including 2% bovine serum albumin and (for trout, in mM) 10 HEPES, 136.9 NaCl, 5.4 KCl, 1 MgSO4 , 0.33 Na2HPO4 , 0.44 KH2PO4, 5 NaHCO3 , 1.5 CaCl2, 5 glucose and 2% bovine serum albumin, pH 7.6 at 20°. Cell viability as determined by Trypan Blue exclusion averaged .95% and was maintained throughout the experiments described. After isolation, the cells were incubated in 40-ml flasks in a shaking water bath at 15°C. Incubation conditions were either normoxia, chemical anoxia or 10% air saturation. For the latter treatment, the incubation flasks were closed with a rubber cap through which two 18-gauge syringe needles were inserted. Through one needle a humidified hypoxic gas mixture (90% N2 –10% air) was perfused over the cells, whereas the other needle served as an outlet for the gas. For chemical anoxia, the cells were maintained under normoxia in the presence of 2 mM NaCN. Oxygen Consumption and Lactate Production Aerobic and anaerobic ATP generation was measured as described by Krumschnabel et al. (10). Na1pump activity and protein synthetic activity were estimated from ouabainsensitive oxygen consumption (osVo2, 1 mM ouabain final concentration) and cycloheximide-sensitive oxygen consumption (csVo2, 15 mM cycloheximide final concentration), respectively. ATP, Ethanol and Glycogen For the determination of cellular ATP contents, we followed the luciferin/luciferase method described by Brown (3). Ethanol and glycogen were measured using standard analytical methods given by Bernt and Gutmann (1) and by Keppler and Decker (9), respectively. Each metabolite was determined in triplicate samples of cells incubated in the absence or presence of 2mM NaCN or under hypoxic conditions as indicated throughout the text. Statistics All data are presented as means 6 SEM of the number of n independent preparations. Differences between treatments were evaluated with Student’s t-test with P , 0.05 considered as significant.

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TABLE 1. Aerobic and anaerobic energy turnover, glycogen and ATP contents of hepatocytes maintained under normoxic

conditions for 1 hr after isolation from goldfish acclimated to normoxia or hypoxia NA Vo 2 tot osVo 2 csVo 2 Lactate production Glycogen content ATP content

0.456 6 0.050 0.103 6 0.011 0.198 6 0.032 0.338 6 0.060 3468.7 6 457.2 3.591 6 0.213

P

HA (8) (8) (8) (5) (7) (7)

0.514 6 0.042 0.149 6 0.016 0.207 6 0.023 0.329 6 0.025 4807.9 6 459.4 6.618 6 0.953

(9) (9) (9) (8) (12) (6)

0.39 0.04 0.81 0.88 0.07 0.01

Rates are given as nmol ⋅ 106 cells21 ⋅ min21; glycogen contents are nmol glycosyl units ⋅ 106 cells21; ATP contents are nmol ⋅ 106 cells 21. Shown are the means 6 SEM of the number of experiments given in parentheses. NA, HA and P denote normoxia acclimation, hypoxia acclimation and the level of significance obtained in the Student’s t-test.

RESULTS Table 1 summarizes various parameters characterizing energy metabolism of freshly isolated hepatocytes of NA and HA goldfish. For these measurements, cells were allowed to recover from the isolation procedure for 1 hr at 15°C under normoxic conditions and were then used for the determination of Vo2 , lactate production, glycogen and ATP contents. Total Vo2 and protein synthesis (as estimated from csVo2 ), as well as lactate production were unaltered by hypoxia acclimation, whereas Na 1pump activity (estimated from osVo2 ) was significantly elevated in the HA group. Glycogen content of the hepatocytes had increased during hypoxia acclimation by 39%, but this increase was not quite significant. Cellular ATP contents were significantly higher in the HA than in the NA cells. To test whether the elevated ATP content might protect the cells under fully anoxic conditions, the ATP levels of cells from NA and HA goldfish were measured over 3 hr of normoxia and chemical anoxia. As shown in Fig. 1A, the NA cells maintained ATP levels constant for 3 hr under normoxic conditions and lost about 45% during 3 hr of chemical anoxia. In contrast, the ATP content of normoxic HA cells was initially about 50% higher than in the NA cells (Fig. 1B) but fell continuously to approach the value of NA controls at the end of the 3-hr period. Chemical anoxia appeared to have induced a slightly steeper decrease of [ATP], but this difference was not statistically significant. From these data, we hypothesized that the elevated initial ATP content of HA cells was caused by exposing the cells to normoxic conditions after isolation from the whole animal. Thus, another experiment was conducted in which cells from both NA and HA goldfish were exposed to hypoxic conditions (10% air saturation) either immediately (HA) or 1 hr after (NA) the isolation procedure (Fig. 2). Time zero in this experiment again refers to 1 hr after isolation for both groups. Under these conditions, HA cells kept [ATP] constant over the entire period, whereas NA cells showed an initial increase of [ATP] at 30 min of incubation and returned to near control values after 3 hr. For the sake of comparison and to evaluate whether the chosen level of hypoxia was adequate to affect energy metabolism in iso-

lated cells of a less hypoxia-tolerant species, a hypoxia experiment was also conducted with hepatocytes of rainbow trout. In these cells, hypoxia led to a significant reduction of [ATP] after 3 hr of exposure (Fig. 3). To gain additional insight into the dynamics of other parameters, besides [ATP], of the energetics of the hepatocytes, we followed the time course of aerobic (Vo2) and an-

FIG. 1. ATP contents of hepatocytes isolated from normoxia- (A) and hypoxia- (B) acclimated goldfish. Cells were incubated under normoxia for 1 hr after isolation before use. At time 0, 2mM NaCN was added to one aliquot of the cells. Data points represent means 6 SEM of five to seven independent preparations. *Significantly different from controls, P , 0.05.

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FIG. 2. ATP contents of hepatocytes isolated from normoxia- and hypoxia-acclimated goldfish. Hypoxic incubation (10% air saturation) commenced immediately after isolation (HA cells) and 1 hr after isolation (NA cells). Time 0 refers to 1 hr after isolation for both groups. Data points represent means 6 SEM of four to six independent preparations. *Significantly different from controls at time 0 and from respective value of hypoxia-acclimated cells, P , 0.05.

FIG. 4. Total (VO2tot, A), ouabain-sensitive (osVO2 , B), and

cycloheximide-sensitive (csVO 2, C) oxygen consumption of hepatocytes isolated from normoxia (h) and hypoxia- ( ) acclimated goldfish. Bars represent means 6 SEM of three independent preparations, except for control values at 8 hr where n 5 2. Cells were incubated under normoxia and at the times indicated an aliquot of the suspensions was taken and used for oxygen consumption measurements. Inhibitorsensitive fractions were calculated from the difference in oxygen consumption before and after the addition of the inhibitor.

FIG. 3. ATP contents of hepatocytes isolated from nor-

moxia-acclimated rainbow trout. One hour after isolation, one aliquot of each preparation was subjected to hypoxia (10% air saturation). Data points represent means 6 SEM of three to nine independent preparations. *Significantly different from controls, P , 0.05.

aerobic metabolism (lactate production) over a period of 8 hr. These data are summarized in Figs 4 and 5. For the determination of aerobic metabolism, hepatocytes were incubated under normoxic conditions and total oxygen consumption (Vo2tot), os Vo2 and cs Vo2 were measured in aliquots of the cell suspensions after 0, 2, 4 and 8 hr of incubation. Total Vo2 showed a decrease of about 30% in the control cells after 8 hr, whereas it was slightly better preserved in the HA cells (Fig. 4A). However, the difference between the two groups was not statistically significant. The same was true for Na 1pump activity (osVo2 , Fig

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FIG. 5. Lactate accumulation in suspensions of goldfish he-

patocytes. At time 0, 2mM NaCN were added to cell suspensions, and after 0, 2, 4 and 8 hr, triplicate samples were taken for lactate determinations (A). (B) One aliquot of each preparation was taken after 4 hr of incubation and the medium exchanged. Then further samples were taken after 4, 6 and 8 hr. The small shift in time of these samples is due to the time required for washing and resuspending these cells. Data points represent means 6 SEM of nine (A) and four (B) independent preparations.

4B) and rate of protein synthesis (csVo2 , Fig. 4C), although there was a general trend for osVo2 of HA cells to be elevated compared with the NA cells. Anaerobic metabolism was assessed in cells exposed to 2 mM NaCN. Although the addition of NaCN inhibited oxygen consumption by .95%, the inhibition persisting for the entire 8-hr period (data not shown), it did not at all effect cell viability as determined by Trypan Blue exclusion (Table 2). Anaerobic ATP production (lactate accumulation) under chemical an-

TABLE 2. Viability of goldfish hepatocytes incubated under

normoxia or chemical anoxia for 8 hrs Time 0 hr 4 hr 8 hr

Normoxia

Chemical anoxia

97.93 6 0.83 97.17 6 0.81 96.47 6 0.82

98.05 6 0.54 97.92 6 0.69 97.77 6 0.47

Values are means 6 SEM of six independent preparations. Viability was assessed by Trypan Blue exclusion and is given as fraction of cells excluding Trypan Blue relative to total number of cells.

FIG. 6. ATP contents of hepatocytes isolated from normoxia- (A) and hypoxia- (B) acclimated goldfish. Treatments were as in Fig. 1. Data points represent means 6 SEM of two (A) and three (B) independent preparations.

oxia was similar in both groups. Thus, Fig. 5A combines the data of NA and HA cells. It can be seen that lactate production decreased with time, following a hyperbolic function with t1/2 5 160 min. To test whether saturation kinetics was the consequence of lactate accumulation to nonphysiologically high levels, we conducted another experiment in which the incubation medium was exchanged after 4 hr in one aliquot of the cells while it was left unchanged in another aliquot. As shown in Fig. 5B, this treatment significantly increased lactate production (t1/2 changed from approximately 270 to 490 min) but failed to reestablish the rates of the first 2 hr. Because under anoxic conditions anaerobic glycolysis is the only source of metabolic energy, it was expected that the decrease of anaerobic ATP output after 4 hr would affect the ATP content of the cells. This, however, was not the case, as shown in Fig. 6. In both NA and HA cells, ATP levels stabilized at 40–60% of the normoxic controls after 3 hr of chemical anoxia and remained at this level for the rest of the incubation period. At this point, it became of interest to enquire whether a so far unconsidered source of anaerobically generated ATP, namely ethanol formation, could have contributed to the maintenance of ATP levels. Figure 7 shows an experiment in which lactate and ethanol contents of cell suspensions

156

FIG. 7. Lactate accumulation, ethanol levels and glycogen

contents in suspensions of goldfish hepatocytes. At time 0, 2mM NaCN was added to cell suspensions, and after 0, 1, 2, 3 and 4 hr, triplicate samples were taken. Data points represent means 6 SEM of three independent preparations.

were measured during 4 hr of NaCN exposure. Again, lactate production showed a decrease with time. However, ethanol was not detectable for the entire period. The same was true for 8 hr of chemical anoxia, as well as for physiological anoxia, that is, when the medium was kept at near zero oxygen (data not shown). Also included in Fig. 7 is the glycogen content of the cells that did not change significantly during chemical anoxia. DISCUSSION The effects of long-term HA in goldfish have been studied before (24,25). In these studies, hypoxia was found to affect metabolic characteristics such as adenylate content, energy charge and enzyme patterns of the liver. These and other changes were interpreted to be of adaptive value, because the resistance to anoxia seemed to be enhanced in HA fish compared with controls (23). The present study was performed to find out whether these changes, which were obtained on tissue homogenates, can be traced to adaptations on the cellular level. Moreover, because the previously observed changes of tissue enzyme patterns during HA appeared rather inconsistent and difficult to interpret, we wanted to find out whether the study of cellular energetics provided a clearer picture. A first estimate of the consequences of HA was derived from the data of freshly isolated hepatocytes, as summarized in Table 1. From these data, it appears that neither aerobic nor anaerobic ATP generation was significantly affected by the acclimation treatment. Of the two major ATP consuming functions, Na1 pumping and protein synthesis, the for-

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mer was significantly elevated in the HA cells, whereas the latter was not different between the two groups. The lack of an effect of HA on csVo2 contradicts suggestions made by van den Thillart and Smit (25). On the basis of an altered liver enzyme pattern, these authors predicted a decrease of the rate of protein synthesis in HA fish. However, neither the interpretation of enzyme patterns nor the indirect estimate of protein synthesis from csVo2 may be appropriate measures to clarify the actual situation prevailing in the living animal. In the intact liver, protein synthesis is under hormonal control, a steering mechanism lost in isolated cells that cannot be evaluated from enzyme activities measured in tissue homogenates. The most prominent effect of HA was a significant increase of [ATP] in the hepatocytes. This was also found in the study of van den Thillart et al. (24), and there only in the liver, not in red and white muscle. However, in our study, this difference disappeared after 3 hr of incubation of the cells at normoxia (Fig. 1B). We interpret this to reflect a transient imbalance of ATP generation and consumption in cells adapted to severely restricted oxygen supply during HA and suddenly exposed to a fully oxygenated medium. Thus, when HA hepatocytes were given the time to recover from the isolation procedure under hypoxia, the ATP content of the cells was both constant and no longer different from that of NA cells (Fig. 2). Moreover, when cells from NA goldfish were exposed to hypoxia, we also observed a short-lived increase of [ATP] (Fig. 2). In other words, acute normoxia in HA cells and acute hypoxia in NA cells both caused a temporary increase of [ATP]. Although the mechanisms underlying these observations remain obscure in the present study, the literature provides some indication as to how they might be explained. First, the temporary imbalance of ATP turnover in HA cells might reflect an underlying rearrangement of cellular energy metabolism, indicative of an adaptive response to long-term hypoxia. This adaptation is expected to reside at the level of mitochondrial respiration (25), which would account for the high sensitivity of ATP generation toward variations in Po2. Second, in the case of NA cells producing an ATP-overshoot during acute hypoxia, the situation is different. It is known that protein synthesis is generally oxygen-conforming in the liver, being severely depressed at oxygen levels as high as 50% air saturation (a Po2 of 80 mm Hg) in rat hepatocytes (14). This depression was found to be independent of alterations in cellular ATP content, and it may thus well be that the decrease of a major cellular ATP-consuming process at a possibly unaltered rate of ATP production resulted in the observed transient elevation of cellular [ATP]. It is worthwhile noting that recent studies by Land et al. (13) and Kwast and Hand (12) suggest that oxygen-sensing proteins are involved in the regulation of metabolic depression of anoxia-tolerant cells and organisms. Preliminary studies in our own laboratory tend to support the existence of such oxygen-sensing proteins in goldfish hepatocytes (unpub-

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TABLE 3. Generation of ATP in hepatocytes under nor-

moxia and chemical anoxia Time

Normoxia

Chemical anoxia

% Depression

0–4 hr 4–8 hr

2.97 2.22

0.75 0.18

75 92

ATP generation (in nmol ⋅ 106 cells21 ⋅ min21) was calculated by converting oxygen consumption and lactate production to ATP turnover rates using the following stoichiometries 6 ATP/O 2 , 1.5 ATP/lactate, assuming glycogen as the sole source of anaerobic energy metabolism.

lished observation). Whether these proteins are capable of affecting ATP production and consumption simultaneously is not known. However, if there is any difference in the dynamics of metabolic suppression, a downregulation of ATP consumers preceding that of ATP production appears to be more likely, which would also account for our findings reported in Fig. 2. Neither short- (Table 1) nor long-term (Fig. 5) experiments revealed any difference in anaerobic capacity between NA and HA cells. An interesting observation, however, was that lactate production of the hepatocytes, irrespective of acclimation history, showed a pronounced decrease with time between 4 and 8 hr of NaCN exposure. Because this was neither accompanied by a corresponding decrease of cellular ATP (Fig. 6) nor by a loss of cell viability (Table 2), we consider this to represent a real metabolic depression, the extent of which is shown in Table 3. The initial decrease of metabolic rate by about 75% compares well to the calorimetrically determined value of 76% found in hepatocytes of the anoxia tolerant turtle, Chrysemys bella pictii (5), but is less than the 90% metabolic depression calculated from lactate production rates of these cells under anoxia (4). No decrease of lactate production was seen in the turtle hepatocytes during 10 hr of anoxia (4). A tentative explanation for the decrease of lactate production in goldfish hepatocytes is derived from the experiment shown in Fig. 5B in which lactate accumulation rate was partially restored by exchanging the incubation medium after 4 hr of NaCN exposure. This points toward metabolic acidosis as being responsible for the depression of glycolytic flux. Such an effect is not uncommon, because metabolic acidosis has repeatedly been associated with metabolic depression and is known to prolong survival of hepatocytes under anoxia (6,19). It has also been observed in white muscle of goldfish exposed to anoxia (26,29) where metabolic depression amounted to 70% compared with the normoxic metabolic rate (30,31). It is tempting to speculate that the decrease of [ATP] in the hepatocytes seen during the first phase of anoxia also contributes to acidification of the cell, because the net effect of a drop in [ATP] implies a liberation of protons (7,17). Two other reasons that might account for the decreased rate of lactate accumulation were ruled out in additional

experiments. First, ethanol production is not involved in the anaerobic energy budget of goldfish hepatocytes, neither under chemical anoxia (Fig. 7) nor under physiological anoxia. The accumulation of ethanol observed in the liver of anoxic goldfish in previous studies must therefore have been due to ethanol uptake from the bloodstream. Second, glycogen did not become rate limiting as a substrate in the anoxic cells, because its level was only marginally affected over 4 (Fig. 7) and 8 hr (data not shown) of incubation. The content of cell glycogen in goldfish hepatocytes was extremely high (Table 1) when compared with the glycogen content of the anoxia-intolerant trout hepatocytes (9.34 6 2.39 nmol glycosyl units ⋅ 106 cells21, n 5 7) (Biasi, unpublished observation). This work was supported by the Fonds zur Fo¨rderung der wissenschaf¨ sterreich, Project no. 10113-BIO. P.J.S. was tlichen Forschung in O supported by a grant from the Universidad de Buenos Aires, CONICET and the International Foundation for Science (Sweden). P.J.S. is career investigator from CONICET. We thank Dr. S.C. Hand for helpful comments in the early phase of this study.

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