Loss of K+Homeostasis in Trout Hepatocytes during Chemical Anoxia: A Screening Study for Potential Causes and Mechanisms

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 353, No. 2, May 15, pp. 199 –206, 1998 Article No. BB980646

Loss of K1 Homeostasis in Trout Hepatocytes during Chemical Anoxia: A Screening Study for Potential Causes and Mechanisms1 Gerhard Krumschnabel, Michael E. Frischmann, Pablo J. Schwarzbaum,* and Wolfgang Wieser2 ¨ kophysiologie, Universita¨t Innsbruck, Technikerstrasse 25, A-6020 Innsbruck, Austria; Institut fu¨r Zoologie, Abteilung fu¨r O and *Instituto de Quı´mica y Fisicoquı´mica Biolo´gicas, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Junin 956, 1113 Buenos Aires, Argentina

Received September 25, 1997, and in revised form February 12, 1998

In isolated trout hepatocytes intoxication with CN2 (chemical anoxia) leads to a rapid breakdown of K1 homeostasis. In the present study an attempt has been made to identify the causes and mechanisms underlying this phenomenon. Our results indicate that neither Ca21 elevation nor cell swelling, both of which occurred during chemical anoxia and could be prevented by exposure to Ca21 chelating agents or to hyperosmotic conditions, respectively, is solely responsible for the breakdown of K1 homeostasis. From a number of inhibitors of dissipative K1 fluxes tested, only BaCl2, an inhibitor of voltage-gated K1 channels, proved to be effective in significantly reducing K1 efflux during chemical anoxia. The KCl cotransporter known to be involved in regulatory volume decrease after hypoosmotic shock does not seem to be activated during CN2-induced cell swelling. © 1998 Academic Press Key Words: Oncorhynchus mykiss; calcium; cell swelling; conductive K1 movement; glycolysis.

Among the numerous detrimental effects of anoxic exposure on cellular metabolism the breakdown of ion homeostasis is one of the most prominent (1). The adverse consequences of, e.g., the loss of transmembrane K1 gradients, such as a decrease of membrane potential and perturbation of intracellular pH buffering capacity, as well as secondary effects of these al1 Supported by the Fonds zur Fo¨rderung der wissenschaftliche ¨ sterreich, Project 11975-BIO. P.J.S. is career invesForschung in O tigator from CONICET and was supported by UBA and CONICET of Argentina and IFS (Sweden). 2 To whom correspondence should be addressed. Fax: (0)512 507 2930.

0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

terations of the internal cell milieu, have been extensively studied (1). However, causes and mechanisms underlying the breakdown of K1 homeostasis in the first place have not been characterized as well. Recently we have shown that in isolated trout hepatocytes, as in other anoxia-sensitive cells, intoxication with CN2 (chemical anoxia) leads to a rapid breakdown of K1 homeostasis (2, 3). Considering that, on the one hand, a functioning Na,K-ATPase is of critical importance in maintaining a steady-state K1 gradient across the cell membrane and that, on the other hand, inhibition of oxidative phosphorylation induces a drastic depletion of ATP contents in these cells (2, 3), the latter observation might be easily identified as the primary cause for the decoupling of K1 uptake (95% of which is directly driven by the Na,K-ATPase in these cells) and passive K1 efflux and, as a consequence, for the loss of K1 homeostasis. This situation could be further aggravated if trout hepatocytes were to possess ATP-sensitive K1 channels. KATP channels have been identified in various tissues and have been found to open when intracellular ATP levels fall (4, 5). Under chemical anoxia cooccurring with the breakdown of K1 homeostasis, the concentration of cytosolic free Ca21 (Ca21 i ) was found to increase. Interestingly, we have also observed that under fully aerobic condiwith the use of the Ca21 ionotions elevation of Ca21 i phore A23187 exerted a decoupling effect on unidirectional K1 fluxes similar to that of chemical anoxia. are In a number of cell types, the levels of Ca21 i inversely correlated with the activity of the sodium pump (6). At the same time it is known that Ca21 is a potent modulator of K1 efflux in cells possessing Gardos channels, i.e., Ca21-activated K1 channels (7). Thus, taking into account that an increase of Ca21 199

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during chemical anoxia might inhibit K1 uptake by the Na1 pump and concurrently stimulate K1 efflux via Ca21-activated K1 channels, the CN2-induced breakdown of K1 homeostasis may well be due to effects of Ca21 in addition to the lack of metabolic energy driving the exchange of cations. A number of other mechanisms, some not even related directly to the energetic state of the cell, might also contribute to the loss of K1 homeostasis under anoxic conditions. For example, Bianchini et al. (8) have shown that cell swelling induced by a hyposmotic shock in trout hepatocytes leads to a drastic rise in K1 membrane permeability, with efflux exceeding uptake by as much as sixfold. If, as seen in rat hepatocytes exposed to chemical anoxia (9), CN2 causes cell swelling in trout hepatocytes as well, the decoupling of K1 fluxes might reflect a compensatory phenomenon involved in the process of regulatory volume decrease (RVD).3 RVD has been reported to occur in liver cells of all species tested so far (8, 10, 11). Furthermore, most cell types from anoxia-sensitive organisms such as rainbow trout are generally dependent on mitochondrial ATP synthesis. Thus, when cut off from this ATP source during anoxia, they may be barely capable of adequately redistributing and utilizing glycolytic ATP. In the present study, which attempted to identify the causes and mechanisms underlying the breakdown of K1 homeostasis in anoxic trout hepatocytes, all of these possibilities were addressed. MATERIALS AND METHODS Experimental animals and hepatocyte preparation. Rainbow trout, Oncorhynchus mykiss (200 – 400 g), were obtained from local suppliers. Fish were acclimated to 15°C in the lab and were fed trout pellets (EWOS Aquaculture) ad libitum once a day. Hepatocytes were prepared as previously described (2) and were incubated in a shaking waterbath at 15°C for 60 min before further use. Incubation medium contained (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 1% bovine serum albumin, pH 7.6, at 20°C. Cell viability as determined by trypan blue exclusion averaged .95%. Determination of cell weight. Cell weight of trout hepatocytes was determined in triplicate samples withdrawn from cell suspensions incubated in isoosmotic or hyperosmotic media under normoxia or under chemical anoxia. Hyperosmotic conditions were established by transferring cells to saline containing 300 mM sucrose at the time chemical anoxia was initiated. Samples were pipetted into preweighed microfuge tubes and centrifuged for 1 min at 6000 g. Supernatants were carefully aspired and the tubes containing cell pellets were weighed again. Other methods. Methods applied to the determination of cellular 1 uptake, and K1 efflux have been ATP content, lactate, Ca21 i , Rb described previously (2, 3).

3 Abbreviations used: BAPTA-AM, bis(o-aminophenoxy)ethaneN, N, N9, N9-tetraacetic acid/tetra(acetoxymethyl) ester; dGlc, 2-deoxyglucose; DMSO, dimethyl sulfoxide; IAA, iodoacetic acid; RVD, regulatory volume decrease.

Incubation protocols. Chemical anoxia was induced by addition of 2 mM NaCN from a neutralized stock solution to cells maintained in aerobic incubation medium and lasted for 30 min for all treatments applied, except where noted otherwise. BAPTA-AM loading [50 mM dissolved in dimethyl sulfoxide (DMSO)] of hepatocytes was started 30 min after initiation of Fura-2 AM loading (5 mM in DMSO). The latter commenced immediately after isolation and lasted for 60 min at 15°C. After this period cells or unidirectional were washed and used for determinations of Ca21 i K1 fluxes. For some experiments cells were suspended in a medium in which glucose had been replaced by 15 mM fructose during both loading and exposure to CN2. Aliquots of hepatocyte suspensions assigned to Ca21-free treatment were washed in nominally Ca21-free medium containing 0.5 mM EGTA, were resuspended in the same saline, and were then loaded with Fura-2 AM and BAPTA-AM as described above. Thapsigargin was used at a concentration of 100 nM (dissolved in ethanol). Amiloride (1 mM in DMSO) and apamin (50 nM in ethanol), when applied, were added to cell suspensions 5 and 10 min prior to initiation of chemical anoxia, respectively. Glibenclamide (100 mM in DMSO) and BaCl2 (5 mM in saline) were given 30 min before CN2 addition. Cells in which glycolysis was inhibited with 20 mM 2-deoxyglucose (dGLc) were incubated with this agent in glucose-free saline for 60 min before further experimentation. For measurements of K1 fluxes in Cl2-free medium, cells were 2 washed in saline containing NO2 3 salts instead of Cl salts and were incubated in the same saline for 1 h before further use. Chemicals. Fura 2-AM, BAPTA-AM, and inhibitors of metabolism and ion transport were purchased from Sigma. Other chemicals were obtained from Merck.

RESULTS

Effect of chemical anoxia on cell energetics and Ca21 i . In agreement with previous observations, addition of CN2 to trout hepatocytes induced a rapid decrease in cellular ATP contents, an increase in lactate production, and a substantial rise in Ca21 (Fig. 1). Ca21 began i i 2 to rise within 1 min after CN application, peaked around 15 min later, and slowly returned toward control levels within the next 90 min (Fig. 1C). Effects of Ca21 chelation on anaerobic cell energetics. A comparison of the effects of chemical anoxia on cellular ATP contents and on lactate production was made for hepatocytes incubated either in standard saline or in Ca21-free medium including BAPTA-AM. When after 60 min of incubation in the respective saline CN2 was added to the cell suspensions, no difference in the decrease of ATP contents could be observed (Fig. 2A). Lactate production decreased with time, following a hyperbolic function with t1/2 being 50 and 13 min in the absence and the presence of Ca21, respectively (Fig. 2B). These values were not significantly different (P 5 0.12). Interaction of Ca21 with K1 homeostasis during i chemical anoxia. In control cells incubated in Ca21-containing medium CN2 induced a rise in Ca21 to about twice i the resting level, with the absolute increase in Ca21 i amounting to 98 6 21 nM (Table I). Nearly identical results were obtained with cells preloaded with the membrane-

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permeant Ca21 chelator BAPTA-AM. In contrast, although incubation in Ca21-free medium did not affect resting levels 2 21 of Ca21 i , it significantly dampened the CN -evoked Ca increase to 25% of the value observed in the presence of extracellular Ca21. The simultaneous application of both intracellular and extracellular Ca21 chelating agents significantly lowered both the resting level of Ca21 and the increase during chemical anoxia (Table I). A similar result

FIG. 2. Cellular ATP content (A) and lactate production (B) in trout hepatocytes under chemical anoxia. Cells were incubated for 60 min in standard saline or in Ca21-free medium including 50 mM BAPTA-AM before addition of 2 mM NaCN. Duplicate samples were withdrawn at times indicated and analyzed for ATP content and lactate as in Fig. 1. Values represent means 6 SE of three independent preparations.

FIG. 1. Cellular ATP content, lactate production, and Ca21 in trout i hepatocytes under chemical anoxia or inhibition of glycolysis. ATP content and lactate production were determined in duplicate samples as described by Brown (47) and Gutmann and Wahlefeld (48), respectively. Chemical anoxia was initiated by addition of 2 mM NaCN; inhibition of glycolysis was achieved with 20 mM dGlc administered to cells maintained in glucose-free saline. (C) A represenduring chemical anoxia and inhibition of tative tracing of Ca21 i glycolysis as determined in Fura-2-loaded cells is shown. Values of (A) and (B) represent means 6 SE of three to seven independent preparations.

was obtained when thapsigargin, an inhibitor of the endoplasmic Ca21-ATPase, was added to either control cells or cells incubated in Ca21-free saline with BAPTA-AM. In this case the resting levels of Ca21 were 91 6 18 and 28 6 7 nM i (n 5 4; P , 0.05) in controls and in hepatocytes treated with Ca21 chelators, respectively. Absolute increases in Ca21 i upon addition of thapsigargin amounted to 115 6 26 (controls) and 23 6 3 nM (EGTA1BAPTA-AM-treated cells; P , 0.05). Next we tried to evaluate the impact of these treatments on unidirectional K1 fluxes during chemical anoxia. Since inclusion of fructose in the incubation medium has previously been found to significantly reduce the Ca21 increase in CN2-intoxicated trout hepatoi cytes (3), the effect of this treatment was also tested. Furthermore, hepatocytes were incubated with apamin, a selective inhibitor of Ca21-activated K1 channels prior to chemical anoxia. Figure 3 summarizes these experiments. In control cells, exposed to standard conditions (i.e., Ca21-containing saline with no Ca21 chelators present) and run in parallel to each individual treatment, uni-

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Response of Ca to Chemical Anoxia in Trout Hepatocytes Incubated in the Presence or the Absence of Ca21-Chelating Agents Addition

Controls

1CN2

Increase

No addition BAPTA-AM EGTA BAPTA-AM1EGTA

100.0 6 16.2 65.5 6 10.6 86.9 6 29.2 47.8 6 11.3†

197.9 6 28.7* 143.1 6 13.5* 105.9 6 28.6* 58.9 6 9.8*,†

97.7 6 21.2 77.7 6 15.0 19.0 6 5.9† 11.3 6 3.7†

Note. Cells were exposed to 2 mM CN2 in the absence or the presence of Ca21 chelators. Incubation conditions were NaCN in standard glucose saline (no addition), NaCN in standard glucose saline after preloading with 50 mM BAPTA-AM (BAPTA-AM), NaCN after 60 min of incubation in nominally Ca21-free medium containing 0.5 mM EGTA (EGTA), and NaCN in nominally Ca21-free medium containing 0.5 mM EGTA after preloading with 50 mM BAPTA-AM in the same saline (BAPTA-AM1EGTA). Values are given in nM and are means 6 SE of four to nine independent values before and peak preparations. Controls and CN2 denote Ca21 i levels observed after CN2 addition. Ca21 i * P , 0.05 versus control value. † P , 0.05 versus values with no addition.

directional K1 fluxes were perfectly balanced, with Rb1 uptake rates equaling those of K1 efflux. Exposure of hepatocytes to 30 min of chemical anoxia induced a pronounced decrease of Rb1 uptake without significantly altering K1 efflux, thus leading to a net loss of K1 of the cells. This decoupling of opposing K1 fluxes was also seen in cells preloaded with BAPTA-AM, in either the presence or the absence of extracellular Ca21, and in cells incubated with 15 mM fructose. Similarly, application of apamin was without effect on Rb1 uptake or on K1 efflux during anoxia. Cell weight during chemical anoxia under isoosmotic or hyperosmotic conditions. Under control conditions hepatocytes maintained constant cell weight for at least 2 h (Fig. 4). In contrast, when cells were incubated with CN2 in isoosmotic saline an increase in cell weight to nearly 30% above the control value was observed. Cell weight peaked at around 30 min after the onset of chemical anoxia and showed a tendency to recover within the next 90 min. In the presence of 300 mM sucrose added at the same time as CN2 the increase in cell weight was significantly delayed, reaching less than 5% during 30 min of chemical anoxia. Within the following hour, however, cell weight increased to about the same value as seen under isoosmotic anoxic conditions. Effect of inhibition of glycolysis on cell energetics. Inhibition of glycolysis by exposure of trout hepatocytes to 20 mM dGlc in glucose-free saline did not affect cellular ATP levels (Fig. 1A) and left lactate contents unaltered (Fig. 1B) over a period of 2 h. In addition, Ca21 was maintained at control levels under these i

conditions for at least 1 h (Fig. 1C), confirming previous results with the glycolytic inhibitor iodoacetic acid (IAA) (3). Effects of dGlc, hyperosmotic exposure, and inhibitors of specific K1 channels on unidirectional K1 fluxes. As was to be expected from the lack of effect of dGlc on cell energetics, inhibition of glycolysis also failed to affect unidirectional K1 fluxes, which were as equally well balanced as in the controls (Fig. 5). Hyperosmotic exposure, while significantly delaying CN2-induced cell swelling, did not protect against the CN2-induced decoupling of Rb1 uptake and K1 efflux, the latter exceeding the former 3.8-fold after 30 min of chemical anoxia.

FIG. 3. Rb1 uptake (A) and K1 efflux (B) in trout hepatocytes after exposure to 30 min of normoxia or chemical anoxia. Unidirectional K1 fluxes were determined as previously described (2). Open bars represent control cells in standard saline with no addition; hatched bars are values from cells subjected to the treatment indicated. Incubation conditions were NaCN in standard glucose saline (Glc), NaCN in medium containing 15 mM fructose (Fru), NaCN after incubation for 60 min in nominally Ca21-free medium containing 0.5 mM EGTA (EGTA), NaCN in Ca21-free medium after loading with 50 mM BAPTA-AM in the absence of extracellular Ca21 (BAPTA1EGTA), and NaCN in standard saline after preincubation with 50 nM apamin for 10 min (Apa). Values are means 6 SE of three to five independent preparations. *P , 0.05 compared to controls; §P , 0.05 compared to respective Rb1 uptake rate.

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chemical anoxia Rb1 uptake was 0.125 6 0.055 nmol z 106 cells21 z min21, significantly different from the controls, whereas K1 efflux was unaltered, amounting to 0.424 6 0.112 nmol z 106 cells21 z min21. DISCUSSION

FIG. 4. Relative cell weight of trout hepatocytes incubated under normoxia or under chemical anoxia in isoosmotic or hyperosmotic conditions. NaCN and 300 mM sucrose, when present, were given at time 0. Cell weight was determined in triplicate as described under Materials and Methods. Cell weight was expressed relative to the initial weight (1.681 6 0.288 mg fresh wt 3106 cells21, n 5 13) determined at time 0. Values are means 6 SE of three (controls), six (CN2), and five (CN2 1 300 mM sucrose) independent preparations.

From the inhibitors of specific K1 channels tested, only BaCl2, an inhibitor of voltage-gated K1 channels, had a significant effect on K1 efflux, lowering it by 55% compared to that of untreated controls. We also tested the effect of ouabain on K1 fluxes. While this specific inhibitor of the Na1 pump should not interfere with ATP-providing pathways, it should nonetheless reduce membrane potential and therefore stimulate voltage-gated K1 channels. In fact, although immediately after addition of ouabain, K1 efflux was reduced by 21 6 4% (n 5 6, P , 0.01), after 30 min of incubation with ouabain, K1 efflux was as great as in untreated controls (Fig. 5). Ouabain inhibited Rb1 uptake by over 95%, similar to previously published observations (2, 8). These data indicated to us that the imbalance of K1 fluxes created by anoxia could be related to a decrease in membrane potential caused by both loss of K1 and uncoupled uptake of Na1. The latter could result not only from inhibition of the Na1 pump but also from activation of Na1/H1 exchange at low pH (12). A drop in intracellular pH is a phenomenon that coincides with anoxia in many cell types (13–16). Thus we conducted a series of experiments in which this exchanger was inhibited by use of the drug amiloride. However, as shown in Fig. 5, amiloride did not alter the effect of CN2 on Rb1 uptake or on K1 efflux of trout hepatocytes when added prior to chemical anoxia. Finally, since in trout hepatocytes KCl cotransport plays a prominent role in K1 movements across the cell membrane during osmotic shock (8), Rb1 uptake/K1 efflux experiments were conducted in saline in which Cl2 had been replaced by NO2 3 . Under these conditions Rb1 uptake and K1 efflux in control cells were 0.337 6 0.051 and 0.344 6 0.086 nmol z 106 cells21 z min21 (n 5 3), respectively. After 30 min of

Effect of Ca21 chelation on Ca21 during chemical i anoxia. For a number of cell types beneficial effects of suppressing Ca21 elevation evoked by anoxia or exposure to toxicants have been described. These positive effects include inhibition of the mitochondrial permeability transition (17), better preservation of cellular glutathione status (18), prevention of DNA fragmenta-

FIG. 5. Rb1 uptake (A) and K1 efflux (B) in trout hepatocytes after exposure to various agents. Unidirectional K1 fluxes were determined as previously described (2). Open bars represent control cells in standard saline with no addition; hatched bars are values from cells subjected to the treatment indicated. Incubation conditions were 60 min with 20 mM dGlc in glucose-free saline (dGlc), 30 min with NaCN in hyperosmotic medium containing 300 mM sucrose (Suc1CN2), 30 min with NaCN after preincubation with 100 mM glibenclamide for 30 min (Glib1CN2), 30 min with NaCN after preincubation with 5 mM BaCl2 for 30 min (BaCl21CN2), 30 min with 1 mM ouabain (OB), and 30 min with NaCN after preincubation with 1 mM amiloride for 5 min (Am1CN2). Values are means 6 SE of three to five independent preparations. *P , 0.05 compared to controls; §P , 0.05 compared to respective Rb1 uptake rate.

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tion (19), and prolonged reactivity toward neural stimulation (20), in each case ultimately leading to improved preservation of cell viability during the toxic insult. Following our previous observation that in trout coincided with a loss of hepatocytes an increase of Ca21 i K1 homeostasis (3), the present study was originally designed to investigate whether the balance of unidirectional K1 fluxes could be similarly ameliorated by a suppression of the Ca21 increase induced by chemical anoxia in these cells. To this end a number of intervention strategies have been applied with the aim of suppressing the CN2-induced Ca21 elevation, in this way separating the effects of Ca21 elevation on K1 fluxes from those of energetic limitations due to inhibition of mitochondrial ATP generation. As documented in Table I, incubation of cells in the presence of EGTA alone or EGTA plus BAPTA-AM significantly suppressed the anoxia-induced elevation of Ca21 i . Since we have previously shown that anoxic elevation is due to a release from intracellular Ca21 i stores (3), it appears that the incubation of cells with EGTA for 1 h before initiation of chemical anoxia was sufficient to partially deplete intracellular Ca21 stores, thus leaving less Ca21 to be released upon CN2 addition. In principle, the same holds for the experiments with both EGTA and BAPTA-AM, except for the fact that in this case both the control level and the increase of Ca21 upon addition of CN2 were significantly lowered. In line with this interpretation is the fact that thapsigargin released significantly more Ca21 from intracellular stores in controls compared to cells treated with Ca21-chelating agents. In our hands incubation of cells with a 50 mM concentration of the membrane-permeant Ca21-chelator BAPTA-AM was not effective in diminishing the increase of Ca21 i , a fact that contradicts the findings of Reader et al. (21), who found that the same concentration of BAPTA-AM effectively suppressed the Ca21 increase evoked by the toxicant tri-n-butylin chloride in trout hepatocytes. Apart from the problems known to be associated with the use of this Ca21 chelator (leakage out of the cells), part of this difference may be explained by the fact that in the study of Reader et al. was only half of that the relative increase of Ca21 i observed in our anoxic trout cells. with K1 homeostasis during Interaction of Ca21 i chemical anoxia. An assessment of the impact of suppression of the Ca21 elevation evoked by CN2 is summarized in Fig. 3. In brief, none of the treatments applied had an appreciable effect on K1 homeostasis in CN2-intoxicated trout hepatocytes. This holds both for treatments using Ca21 chelators and for the application of fructose, which, by some not yet identified mechanism, significantly reduced Ca21 elevation in anoxic hepatocytes of both rat (22) and trout (3).

In a separate experiment apamin, a known inhibitor of Ca21-sensitive K1 channels, did not prevent the CN2-induced decoupling of K1 fluxes. In summary these data tend to exclude a significant role for Ca21 in the control of K1 homeostasis during anoxia. Even an indirect impact of Ca21 elevation on K1 homeostasis via, e.g., an inhibitory effect of high on anaerobic glycolysis, appears to be rather Ca21 i unlikely. If anything, a stimulatory effect of Ca21 on glycolytic ATP production was observed (Fig. 2B). Loss of ATP, on the other hand, was identical in hepatocytes irrespective of whether Ca21 was chelated prior to anoxic exposure or not (Fig. 2A). Similarly, an involvement of KATP channels in the anoxic decoupling of K1 fluxes in trout cells could not be shown, which is in agreement with findings by Johansson and Nilsson (23) on trout brain tissue. In cultured rat hepatocytes Sakaida et al. (24) have shown that exposure to CN2 resulted in an accelerated rate of phospholipid degradation which was not mediated by a rise of Ca21. Hence, it might be argued that after 30 min of chemical anoxia membrane damage could have resulted in a general, nonspecific increase in membrane permeability leading to the decoupling of K1 fluxes observed here. However, in this case we would expect (i) K1 efflux to rise dramatically instead to show a continuous of remaining unchanged, (ii) Ca21 i increase in cells not treated with Ca21 chelators instead of returning toward the control level (Fig. 1A), and (iii) a significant increase in trypan blue staining of the hepatocytes, all of which was not found in the present study (viability data not shown). In line with this argument is the fact that the internal K1 concentration diminished to only 86% of the control value after 30 min of chemical anoxia (unpublished observation, P. J. Schwarzbaum, M. E. Frischmann, and G. Krumschnabel). Cell swelling and K1 homeostasis during chemical anoxia. Having demonstrated that the anoxia-inincrease is not the primary cause for the duced Ca21 i decoupling of K1 fluxes across the plasma membrane of trout hepatocytes, we next investigated if the anoxic loss of K1 was in some way linked to volume regulation. This idea was based on the fact that in many cell types inhibition of the Na1 pump, as observed in the present study, results in cell swelling (25–27). Moreover, cell swelling has been demonstrated to occur in rat hepatocytes subjected to chemical anoxia (by inhibition of glycolysis and oxidative phosphorylation) (9). Results in Fig. 4 show that trout hepatocytes indeed swelled when exposed to CN2, followed by a decrease of volume toward initial levels. Thus, the question arises whether the net K1 efflux under anoxia would be a mechanism to retard or compensate swelling, as has been reported for rat hepatocytes (see Ref. 28). How-

K1 HOMEOSTASIS IN ANOXIC TROUT HEPATOCYTES

ever, this idea seems difficult to reconcile with results shown in Figs. 4 and 5, where it can be seen that exposure of cells to a hypertonic medium (by addition of sucrose) indeed abolishes the initial phase of swelling but proves ineffective in preventing or diminishing the decoupling of K1 fluxes. A qualitatively similar counteraction of hypertonic medium on cell swelling was seen in rat hepatocytes by Gores et al. (9). Thus, swelling induced by metabolic inhibition does not seem to be the primary stimulus for K1 loss in anoxic trout hepatocytes. This conclusion is further supported by the results obtained in Cl2-free media. In trout hepatocytes K1 efflux triggered by hypoosmotic shock appears to occur via a KCl cotransporter and could therefore be blocked by replacement of Cl2 with NO2 3 (8). In the present study such a replacement of Cl2 did not affect K1 efflux during chemical anoxia, suggesting that a different mechanism for K1 efflux is activated under these conditions. Interestingly, however, exposure to Cl2-free conditions diminished unidirectional K1 fluxes by approximately 50% in normoxic controls compared to K1 exchange rates in standard saline, indicative of an important role of Cl2 for K1 movements across the cell membrane under steadystate conditions. Similar observations have been reported for rat hepatocytes (29). K1 efflux and membrane depolarization. Metabolic inhibition induced by anoxia is supposed to cause depolarization of the cell membrane (30), the reason being that while the Na1 pump is inhibited an unopposed Na1 influx/K1 efflux proceeds, which tends to relax transmembrane cation gradients. Therefore, at least as a short-term strategy, an ongoing decoupled K1 efflux could be seen as a mechanism to export positive charge and to repolarize the membrane. To test whether such a mechanism is involved in the response of trout hepatocytes to energy limitation, we incubated anoxic cells with BaCl2, a known inhibitor of conductive K1 movements. It can be seen that, under chemical anoxia, incubation with BaCl2 partially reduced the K1 efflux (Fig. 5), so that the K1 efflux/Rb1 influx ratio was changed from approximately 5.5 in the absence of BaCl2 to about 2.7 in its presence. In line with this evidence is the fact that, during anoxia, exposure of cells to a medium containing 300 mM sucrose enhanced the net efflux of K1 to twice the value found under isoosmotic anoxia (Fig. 5). Since hyperosmotic conditions established by the addition of sucrose have been reported to depolarize the plasma membrane of mouse hepatocytes (31), the idea that the degree of K1 decoupling depends on the magnitude of membrane depolarization gains support. A similar conclusion is derived from the results obtained when ouabain was present in the incubation medium. In the short term, i.e., within a few minutes after addition of ouabain, K1 efflux responded to inhibition of the Na1 pump with a 20%

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reduction, pointing to a certain degree of coupling between uptake and efflux, as reported for various transporting epithelia (32–34). However, after 30 min of pump inhibition, which, as known from other cell preparations (35, 36), causes depolarization of the cell membrane, K1 efflux had again increased to its initial level (Fig. 5). In conclusion, while the existence of voltage-operated K1 channels has, to our knowledge, not yet been documented for trout hepatocytes, these data led us to postulate the presence and functionality of this mechanism in these cells. As a different approach to this problem we tested whether the postulated compensatory K1 efflux could be prevented by inhibiting Na1 influx promoted by the Na1/H1 exchanger present in the trout hepatocyte plasma membrane (37). This exchange mechanism has been found to be activated by intracellular acidification and in rat hepatocytes addition of amiloride to anoxic cells resulted in prolongation of cell survival (13). However, a study directed more directly toward the function of Na1/H1 exchange in anoxic rat hepatocytes revealed that this mechanism is not operative during anoxia (14). The same appears to be true for trout hepatocytes, since Rb1/K1 fluxes were totally unaffected by amiloride, both during normoxia (data not shown) and during chemical anoxia (Fig. 5). Effects of inhibition of glycolysis. Inhibition of glycolysis has been repeatedly shown to have profound effects on cell energetics (38, 39). Notably, in some cell types glycolytic ATP production was preferentially coupled to the activity of ion-transporting ATPases, as, e.g., to the Na1 pump (40 – 42) or to the endoplasmic reticulum Ca21-ATPase (43). In another teleost hepatocyte preparation the decrease in both cellular ATP and Na1 pump activity in the presence of the glycolytic inhibitor IAA was as great as in the presence of CN2 (44). In trout hepatocytes glycolytic ATP production appears to be of minor importance under aerobic conditions so that during glycolytic inhibition cellular ATP (Fig. 1A), Rb1 uptake (Fig. 5), and Ca21 (Fig. 1C) could i all be maintained at control levels. Since no extracellular substrate was supplied under these conditions, these cells were obviously capable of mobilizing sufficient endogenous substrate for nonglycolytic ATP production. This conclusion is supported by the virtual absence of IAA-inhibitable oxygen consumption in trout hepatocytes (unpublished observation, C. Biasi and G. Krumschnabel). Conversely, however, this apparent lack of coupling between ion transport and glycolysis seems to persist under anoxia, in this situation favoring the breakdown of ion gradients due to the inability of glycolysis to support the Na1 pump. In summary, taking into account the results discussed herein, the following sequence of events is sug-

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gested to take place upon initiation of chemical anoxia in trout hepatocytes: (i) Inhibition of mitochondrial ATP production causes rapid depletion of cellular ATP contents. (ii) Due to this decrease Na1 pump activity cannot be maintained at control levels, causing a continuous net gain of positive charges in the form of Na1 influx. (iii) The increase in intracellular Na1 concentration leads to a decrease in membrane potential, causing the opening of a conductive K1 efflux mechanism which serves to reestablish membrane potential. The overall effect of this process might be to maintain a high driving force for Na1 entry, which is a prerequisite for the functioning of a number of Na1-coupled transport mechanisms. Among these is the Na1/Ca21 exchanger operating in the reversed mode (i.e., Ca21 extrusion) (45, 46), a mechanism serving to prolong anoxic survival of the cells. ACKNOWLEDGMENTS The authors gratefully acknowledge the expert technical assistance of Susanne Steu and Christina Biasi.

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