The formation of plasma membrane blebs in hepatocytes exposed to agents that increase cytosolic Ca2+ is mediated by the activation of a non-lysosomal proteolytic system

Exposure of isolated hepatocytes to extracellular ATP, cystamine or ionophore A23187 was associated with an increase in cytosolic Ca2+ concentration, a stimulation of intracellular proteolysis, and the appearance of plasma membrane blebs which preceded the loss of cell viability. Both bleb formation and cell killing were prevented when inhibitors of Ca2+-activated neutral proteases, such as antipain or leupeptin, were included in the incubation medium, whereas inhibitors of lysosomal proteases had no effect. Thus, the activation of a Ca2+-dependent, non-lysosomal proteolytic system appears to be responsible for the plasma membrane blebbing and, ultimately, the cytotoxicity associated with treatment of hepatocytes with agents that disrupt intracellular Ca2+ homeostasis.     

Activation of the plasma membrane Ca2+ pump during agonist stimulation of pancreatic acini.

The role of internal stores and plasma membrane Ca2+ pumps in controlling [Ca2+]i during agonist stimulation and their regulation by agonists are not well understood. We report here measurements of intracellular ([Ca2+]i) and extracellular ([Ca2+]o) Ca2+ concentrations in agonist-stimulated pancreatic acini in an effort to directly address these questions. Stimulation of acini suspended in Ca(2+)-free or Ca(2+)-containing medium with Ca2+ mobilizing agonists resulted in a typical transient increase in [Ca2+]i. Thapsigargin, a specific inhibitor of internal Ca2+ pumps, inhibited the rate of [Ca2+]i reduction after agonist stimulation by approximately 40%. Under the same conditions, thapsigargin had no effect on the rate of the unidirectional Ca2+ efflux across the plasma membrane as revealed by measurements of [Ca2+]o. These findings suggest that internal Ca2+ pumps actively remove Ca2+ from the cytosol during continued agonist stimulation. The correlation between the reduction in [Ca2+]i and the increase in [Ca2+]o showed that Ca2+ efflux from cells stimulated with agonist and thapsigargin represent Ca2+ efflux across the plasma membrane. Inhibition of cells exposed to agonist and thapsigargin with a specific antagonist sharply reduced the rates of the [Ca2+]i decrease and the accompanied [Ca2+]o increase. Hence, at comparable [Ca2+]i, Ca2+ efflux from stimulated cells was about 3-fold faster than that from resting cells, indicating that agonists directly activate the plasma membrane Ca2+ pump. To study the role of [Ca2+]i increase in plasma membrane Ca2+ pump activation the acini were loaded with 1,2-bis-(2-aminophenoxyethane-N,N,N',N')-tetraacetic acid (BAPTA), and [Ca2+]o was measured during agonist stimulation. Surprisingly, although BAPTA completely prevented the increase in [Ca2+]i, Ca2+ efflux rate was reduced by only 34%. These findings provide the first evidence for Ca(2+)-independent activation of the plasma membrane Ca2+ pump by Ca2+ mobilizing agonists.     

The sinusoidal domain of the plasma membrane of rat hepatocytes contains an amiloride-sensitive Na+/H+ antiport

ATP-dependent trapping of [14C]methylamine was demonstrated in vesicles selectively derived from the sinusoidal plasma membrane of rat hepatocytes; activity was lacking in vesicles prepared from the canalicular domain of the plasma membrane of rat hepatocytes. The proton movement was inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, strophanthidin, vanadate, amiloride, and absence of sodium. 22Na efflux from sinusoidal membrane vesicles increased inversely to extravesicular pH. The results indicate that the sinusoidal plasma membrane of rat hepatocytes contains a Na+/H+ antiport.     

Studies of the interaction between glucagon and alpha-adrenergic agonists in the control of hepatic glucose output

alpha-Adrenergic stimulation of hepatocytes prevented, in a dose-dependent manner, the stimulation of [U-14C]lactate conversion to [14C]glucose by glucagon and exogenously added cAMP and Bt2cAMP. The inhibition was referable to an interaction with adrenergic receptors which resulted in a small decrease in hepatic cAMP levels. Low concentrations of epinephrine (10 nM) were able to inhibit phosphorylase activation and glucose output elicited by low doses of glucagon (5 X 10(-11) M to 2 X 10(-10) M). The ability of epinephrine (acting via alpha 1-adrenergic receptors), vasopressin, and angiotensin II to elicit calcium efflux was inhibited by glucagon, suggesting that intracellular redistributions of Ca2+ are importantly involved in the gluconeogenic process. It is proposed that vasopressin, angiotensin II, and catecholamines, acting primarily via alpha 1-adrenergic receptors, are responsible for inhibition of glucagon mediated stimulation of gluconeogenesis by altering subcellular calcium redistribution and decreasing cAMP levels.     

The mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity: role of intracellular calcium

The mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity to isolated hepatocytes was studied. MPTP was more toxic to hepatocytes than its major metabolite, 1-methyl-4-phenylpyridine (MPP+); this may, in part, be explained by the lesser permeability of the hepatocyte plasma membrane to the cation compared to its parent compound, MPTP. Loss of cell viability was preceded by plasma membrane bleb formation and disturbance of intracellular Ca2+ homeostasis. MPTP caused a rapid depletion of the mitochondrial Ca2+ pool which was followed by a marked and sustained elevation of cytosolic free Ca2+ concentration. This increase of cytosolic Ca2+ level appeared to be associated with the impairment of the cell's Ca2+ extrusion system since the plasma membrane Ca2+-ATPase was markedly inhibited in MPTP-treated hepatocytes. Preincubation of hepatocytes with inhibitors of monoamine oxidase type B, but not A, protected the cells from MPTP-induced cytotoxicity. Moreover, the monoamine oxidase B inhibitor, pargyline, prevented the rise in cytosolic free Ca2+ concentration and partially protected the plasma membrane Ca2+-ATPase from inhibition by MPTP. As observed with MPTP, MPP+ caused an extensive loss of mitochondrial Ca2+ and significantly decreased the rate of Ca2+ efflux from hepatocytes. However, MPP+ was without effect on the plasma membrane Ca2+-ATPase. In conclusion, our studies demonstrate that MPTP caused a substantial elevation of cytosolic Ca2+ which preceded loss of cell viability and we propose that calcium ions are of major importance in the mechanism of MPTP- and MPP+-induced toxicity in hepatocytes.     

Effect of bile acids on calcium efflux from isolated rat hepatocytes and perfused rat livers

The changes in intracellular Ca2+ concentration [( Ca2+]i) of hepatocytes induced by certain bile acids are biphasic: an initial increase is followed by a more gradual decrease. This latter decline in [Ca2+]i may be due to an efflux of Ca2+ across the plasma membrane. This hypothesis was tested by studying the effect of different bile acids on the efflux of 45Ca from preloaded rat hepatocytes and isolated perfused rat livers. The following bile acids were studied: cholic (C), ursodeoxycholic (UDC), chenodeoxycholic (CDC), and deoxycholic (DC) acids; their taurine (T) conjugates (TC, TUDC, TCDC, and TDC); and the taurine, sulfate (S), and glucuronide (Glu) derivatives of lithocholic acid (TLC, LS, TLS, and LGlu, respectively). At 0.3 mM, all bile acids except C, TC, TCDC, UDC, and TUDC significantly increased 45Ca efflux from preloaded hepatocytes without affecting cell viability. Dose-response studies revealed that the minimum effective concentration needed to induce 45Ca efflux was 0.06 mM for LS, 0.8 mM for TCDC, and 10 mM for TC. Efflux of 86Rb from preloaded hepatocytes was not significantly altered by 0.1 mM LS, indicating relative specificity for calcium. TDC and DC, but not TC, increased 45Ca efflux from preloaded perfused rat livers. These results showed that bile acids known to increase [Ca2+]i (CDC, DC, TDC, and TLC) also increased 45Ca efflux from hepatocytes and perfused livers and that efflux was also stimulated by LS, TLS, and LGlu. The extent of this efflux was related to the hydrophobicity of the steroid nucleus of the bile acid. It is speculated that bile acid-induced increases in [Ca2+]i activate the plasma membrane Ca2+ pump resulting in increased Ca2+ efflux.     

Atrial natriuretic peptide attenuates elevations in Ca2+ and protects hepatocytes by stimulating net plasma membrane Ca2+ efflux

Elevations in intracellular Ca(2+) concentration and calpain activity are common early events in cellular injury, including that of hepatocytes. Atrial natriuretic peptide is a circulating hormone that has been shown to be hepatoprotective. The aim of this study was to examine the effects of atrial natriuretic peptide on potentially harmful elevations in cytosolic free Ca(2+) and calpain activity induced by extracellular ATP in rat hepatocytes. We show that atrial natriuretic peptide, through protein kinase G, attenuated both the amplitude and duration of ATP-induced cytosolic Ca(2+) rises in single hepatocytes. Atrial natriuretic peptide also prevented stimulation of calpain activity by ATP, taurolithocholate, or Ca(2+) mobilization by thapsigargin and ionomycin. We therefore investigated the cellular Ca(2+) handling mechanisms through which ANP attenuates this sustained elevation in cytosolic Ca(2+). We show that atrial natriuretic peptide does not modulate the release from or re-uptake of Ca(2+) into intracellular stores but, through protein kinase G, both stimulates plasma membrane Ca(2+) efflux from and inhibits ATP-stimulated Ca(2+) influx into hepatocytes. These findings suggest that stimulation of net plasma membrane Ca(2+) efflux (to which both Ca(2+) efflux stimulation and Ca(2+) influx inhibition contribute) is the key process through which atrial natriuretic peptide attenuates elevations in cytosolic Ca(2+) and calpain activity. Moreover we propose that plasma membrane Ca(2+) efflux is a valuable, previously undiscovered, mechanism through which atrial natriuretic peptide protects rat hepatocytes, and perhaps other cell types, against Ca(2+)-dependent injury.     

Controlled Proteolysis Activates the Plasma Membrane Ca2+ Pump of Higher Plants (A Comparison with the Effect of Calmodulin in Plasma Membrane from Radish Seedlings)

The effects of calmodulin and of controlled trypsin treatments on the activity of the Ca2+ pump were investigated in plasma membrane purified from radish (Raphanus sativus L.) seedlings. Treatment of the plasma membrane with ethylenediaminetetra-acetate (EDTA), which removed about two-thirds of the plasma membrane-associated calmodulin, markedly increased the stimulation of the Ca2+ pump by calmodulin. In EDTA-treated plasma membrane, stimulation by calmodulin of the Ca2+ pump activity was maximal at low free Ca2+ (2-5 [mu]M) and decreased with the increase of free Ca2+ concentration. The Ca2+ pump activity was stimulated also by a controlled treatment of the plasma membrane with trypsin: the effect of trypsin treatment depended on the concentration of both trypsin and plasma membrane proteins and on the duration of incubation. Stimulation of the Ca2+ pump activity by trypsin treatment of the plasma membrane was similar to that induced by calmodulin both in extent and in dependence on the free Ca2+ concentration in the assay medium. Moreover, the Ca2+ pump of trypsin-treated plasma membrane was insensitive to further stimulation by calmodulin, suggesting that limited proteolysis preferentially cleaves a regulatory domain of the enzyme that is involved in its activation by calmodulin.     

Progesterone induction of phospholipid methylation and arachidonic acid turnover during the first meiotic division in amphibian oocytes

Progesterone is the physiological stimulus that acts at the amphibian oocyte plasma membrane to induce the meiotic divisions. Rana oocytes were preincubated with [3H]-arachidonic acid, [3H]-methionine and/or [14C]choline. Total and plasma membrane phospholipids were monitored during the first 2 h after induction with progesterone. A transient increase in methylation of phosphatidylethanolamine during the first 10 minutes coincided with an increased Ca2+ efflux and was followed by increased arachidonic acid incorporation into phosphatidylcholine during a period of increasing membrane conductance. The labeled phospholipids disappeared sequentially 5-90 min after the hormone stimulus, suggesting that activation of phospholipases A2 and/or C occur as part of a cascade of membrane events.     

Uptake of lactate by the liver: effect of red blood cell carriage

Multiple-indicator dilution experiments with labeled lactate were performed in the livers of anesthetized dogs. A mixture of (51)Cr-labeled erythrocytes, [(3)H]sucrose, and L-[1-(14)C]lactate or a mixture of (51)Cr-labeled erythrocytes, [(14)C]sucrose, and L-[2-(3)H]lactate was injected into the portal vein, and samples were obtained from the hepatic vein. Data were evaluated using a model comprising flow along sinusoids, exchange of lactate between plasma and erythrocytes and between plasma and hepatocytes, and, in the case of L-[1-(14)C]lactate, metabolism to H[(14)C]O(-)(3) within hepatocytes. The coefficient for lactate efflux from erythrocytes was 0.62 +/- 0.24 s(-1), and those for influx into and efflux from hepatocytes were 0.44 +/- 0.13 and 0.14 +/- 0.07 s(-1), respectively. The influx permeability-surface area product of the hepatocyte membrane for lactate (P(in)S, in ml x s(-1) x g(-1)) varied with total flow rate (F, in ml s(-1) x g(-1)) according to P(in)S = (3.1 +/- 0.5)F + (0.021 +/- 0.014). Lactate in plasma, erythrocytes, and hepatocytes was close to equilibrium, whereas lactate metabolism was rate limiting.     

Protease inhibitors do not prevent the killing of cultured hepatocytes by cystamine

A study was made of the conditions of the killing of cultured hepatocytes by the reactive disulfide cystamine. Six to 12 mM cystamine killed up to 60% of the hepatocytes within 3 hours. The cytosolic calcium ion concentration rose prior to the loss of viability. Treatment with EGTA in a Ca2+-free medium lowered the initial Ca2+ concentration and prevented the rise in response to cystamine. However, there was no change in the number of dead cells. Furthermore, the sensitivity of cultured hepatocytes to cystamine was unaffected by the concentration of calcium in the culture medium. Addition to the culture medium of 3 protease inhibitors, leupeptin, antipain, or chymostatin, did not reduce the extent of cell killing by cystamine despite an inhibition of protein degradation. These data do not support the hypothesis that the toxicity of cystamine is necessarily mediated by proteases activated by a rise in the cytosolic calcium ion concentration.     

Changes in 45Ca and 109Cd uptake, membrane potential and cell pH in Saccharomyces cerevisiae provoked by Cd2+

The effect of Cd2+ poisoning of Saccharomyces cerevisiae on 45Ca, 109Cd and [14C]tetraphenylphosphonium (TPP) uptake and cell pH was examined. At Cd2+ concentrations that produced substantial K+ efflux the rates of uptake of 45Ca, 109Cd and [14C]TPP increased progressively during incubation of the cells with Cd2+, and the cell pH was lowered concomitantly. The initial rates of uptake of the divalent cations and of TPP were increased in cells pre-loaded with Cd2+, which shows that stimulation of the ion fluxes was exerted by the Cd2+ that accumulated in the cells. The distribution ratio of TPP between cells and medium, however, was decreased by Cd2+. Although hyperpolarization of the cell membrane by Cd2+ cannot be excluded, it is argued that Cd2+ primarily stimulated divalent cation uptake by increasing the cation permeability of the cell membrane allowing the cations to enter the cells more easily.     

Sodium-dependent calcium efflux from adrenal chromaffin cells following exocytosis. Possible role of secretory vesicle membranes.

Although cytosolic Ca2+ transients are known to influence the magnitude and duration of hormone and neurotransmitter release, the processes regulating the decay of such transients after cell stimulation are not well understood. Na(+)-dependent Ca2+ efflux across the secretory vesicle membrane, following its incorporation into the plasma membrane, may play a significant role in Ca2+ efflux after stimulation of secretion. We have measured an enhanced 45Ca2+ efflux from cultured bovine adrenal chromaffin cells following cell stimulation with depolarizing medium (75 mM K+) or nicotine (10 microM). Such stimulation also causes Ca2+ uptake via voltage-gated Ca2+ channels and secretion of catecholamines. Na+ replacement with any of several substitutes (N-methyl-glucamine, Li+, choline, or sucrose) during cell stimulation inhibited the enhanced 45Ca2+ efflux, indicating and Na(+)-dependent Ca2+ efflux process. Na+ deprivation did not inhibit 45Ca2+ uptake or catecholamine secretion evoked by elevated K+. Suppression of exocytotic incorporation of secretory vesicle membranes into the plasma membrane with hypertonic medium (620 mOsm) or by lowering temperature to 12 degrees C inhibited K(+)-stimulated 45Ca2+ efflux in Na(+)-containing medium but did not inhibit the stimulated 45Ca2+ uptake. Enhancement of exocytotic secretion with pertussis toxin resulted in an enhanced 45Ca2+ efflux without affecting calcium uptake. The combined results suggest that Na(+)-dependent Ca2+ efflux across secretory vesicle membranes, following their incorporation into the plasma membrane during exocytosis, plays a significant role in regulating calcium efflux and the decay of cytosolic Ca2+ in adrenal chromaffin cells and possibly in related secretory cells.     

Ca2+o-Independent Veratridine-Evoked Acetylcholine Release from Striatal Slices Is Not Inhibited by Vesamicol (AH5183): Mobilization of Distinct Transmitter Pools

The effect of 2-(4-phenylpiperidino)cyclohexanol (AH5183 or vesamicol), a compound known to block the uptake of acetylcholine (ACh) into cholinergic synaptic vesicles, on the release of endogenous and [14C]ACh from slices of rat striatum was investigated. ACh release was evoked either by electrical stimulation or by veratridine. The effect of electrical stimulation was entirely dependent on external Ca2+. By contrast, veratridine (40 microM) also enhanced ACh release in the absence of Ca2+. Indeed, with veratridine two components were clearly distinguished: one dependent on external Ca2+ and the other not. Vesamicol inhibited [14C]ACh release evoked by both veratridine and electrical stimulation in the presence of external Ca2+, provided it was added to the tissue prior to loading with [14C]choline. With the same treatment vesamicol only slightly affected the release of endogenous ACh. Under the same conditions the Ca2(+)-independent [14C]ACh release evoked by veratridine was not prevented by vesamicol. The differential responsiveness to vesamicol suggests that ACh pools involved in Ca2+o-dependent ACh release are different from those mobilized during Ca2+o-independent ACh release.     

Liver glucose-6-phosphatase activity is modulated by physiological intracellular Ca2+ concentrations

The effect of varying concentrations of free Ca2+ on the formation of Pi from mannose-6-P or of Pi and [U-14C]glucose from [U-14C]glucose-6-P was investigated in isolated fasted rat hepatocytes made permeable by freezing and in liver microsomes. Free Ca2+ concentration was adjusted by the use of Ca-EGTA buffers. In permeabilized cells, glucose-6-phosphatase (EC 3.1.3.9) activity was inhibited up to 50% and in intact microsomes up to 70% by increasing free Ca2+ concentrations from 0.01 to 10 microM. The inhibition was reversible and competitive with respect to glucose-6-P. Treatment of microsomes with 0.4% deoxycholate exposed 90% of latent mannose-6-phosphatase activity which was insensitive to Ca2+. The results indicate that Ca2+ affects the glucose-6-P translocase rather than the phosphohydrolase component. It is concluded that the glucose-6-phosphatase system is modulated by changes in Ca2+ concentrations in the range of those occurring in the liver cell upon hormonal stimulation.     

Evidence from studies employing radioactively labelled fatty acids that the stimulation of flux through the diacylglycerol pool is an early action of vasopressin on hepatocytes.

1. In isolated hepatocytes prelabelled with [14C]-arachidonic, -stearic, -linoleic, -oleic or -palmitic acids, vasopressin increased the amount of radioactivity present in diacylglycerols. The largest increase was observed in cells labelled with arachidonic or stearic acids. 2. In cells prelabelled with [14C]- or [3H]-arachidonic acid, the onset of the increase in radioactivity in diacylglycerols induced by vasopressin was slow, the increase was partly dependent on the presence of extracellular Ca2+, and was associated with an increase in radioactivity present in phosphatidic acid which was more rapid in onset. Vasopressin decreased the amount of [3H]arachidonyl-phosphatidylinositol 4,5-bisphosphate, but the magnitude of this decrease was less than 10% of the observed increase in radioactivity in [3H]arachidonyl-diacylglycerol. 3. The concentration of vasopressin which gave half-maximal increase in [14C]arachidonyl-diacylglycerol at low extracellular Ca2+ was 10-fold higher than that which gave half-maximal stimulation of 45Ca2+ efflux. Phenylephrine, but not glucagon, also increased the amount of [14C]arachidonyl-diacylglycerol. 4. It is concluded that an early action of vasopressin on the liver cell is to increase the flux of carbon from phospholipids, including the phosphoinositides, to diacylglycerols.     

Activation of insulin-secreting cells by pyruvate and halogenated derivatives.

Addition of pyruvate to rat islets perifused in the presence of 5 mM-glucose elicited an immediate pronounced biphasic stimulation of insulin secretion. At lower concentrations of glucose (2.5 mM), only the initial, transient, phase of secretion was observed. Pyruvate inhibited 45Ca2+ efflux from islets at 2.5 mM-glucose and stimulated efflux at 5 mM-glucose. Pyruvate also decreased the rate of efflux of 86Rb+ from perifused islets. A marked stimulation of insulin secretion and 45Ca2+ efflux rate was observed in response to 3-fluoropyruvate and 3-bromopyruvate, compounds which inhibited oxidative metabolism of [14C]glucose and [14C]pyruvate in islets. The stimulatory effects of 3-fluoro- and 3-bromo-pyruvate were associated with enhanced 86Rb+ efflux. Withdrawal of pyruvate or halogenated analogues from the perfusate resulted in a secondary stimulation of insulin release, 45Ca2+ efflux and, to some extent, 86Rb+ efflux rates. Pyruvate, 3-fluoropyruvate and 3-bromopyruvate were all effective in promoting intracellular acidification and a rise in cytosolic Ca2+ concentration, as judged from fluorescence measurements in HIT-T15 cells loaded with 2',7'-biscarboxyethyl-5'(6')-carboxyfluorescein and Quin 2 respectively. It is proposed that oxidative metabolism of pyruvate is not a prerequisite for its stimulatory actions on pancreatic beta-cells. An alternative mechanism of activation by pyruvate and its halogenated derivatives is proposed, based on the possible electrogenic flux of these anions across the cell membrane.     

The effects of adrenalectomy on the alpha-adrenergic regulation of cytosolic free calcium in hepatocytes

We have previously published that bilateral adrenalectomy in the rat reduces the Ca2+-mediated alpha-adrenergic activation of hepatic glycogenolysis, while it increases the cellular calcium content of hepatocytes. In the experiments presented here, the concentration of cytosolic free calcium (Ca2+i) at rest and in response to epinephrine was measured in aequorin-loaded hepatocytes isolated from sham and adrenalectomized male rats. We found that in adrenalectomized rats the resting Ca2+i was elevated, the rise in Ca2+i evoked by epinephrine was reduced, and the rise in 45Ca efflux that follows such stimulation was depressed. Furthermore, the slope of the relationship between Ca2+i and calcium efflux was decreased 60% in adrenalectomized. Adrenalectomy did not change Ca2+ release from intracellular calcium pools in response to IP3 in saponin-permeabilized hepatocytes. The EC50 for inositol 1,4,5-triphosphate and the maximal Ca2+ released were similar in both sham and adrenalectomized animals. Finally, the liver calmodulin content determined by radioimmunoassay was not significantly different between sham and adrenalectomized rats. These results suggest that 1) adrenalectomy reduces calcium efflux from the hepatocyte, probably by an effect on the plasma membrane (Ca2+-Mg2+)-ATPase-dependent Ca2+ pump and thus alters cellular calcium homeostasis; 2) adrenalectomy decreases the rise in Ca2+i in response to epinephrine; 3) this decreased rise in Ca2+i is not due to defects in the intracellular Ca2+ storage and mobilization processes; and 4) the effects of adrenalectomy on cellular calcium metabolism and on alpha-adrenergic activation of glycogenolysis are not caused by a reduction in soluble calmodulin.     

Effects of ATP and adenosine addition on activity of oxoglutarate dehydrogenase and the concentration of cytoplasmic free Ca2+ in rat hepatocytes

Addition of ATP (100 microM) to hepatocytes from starved rats incubated with 5 mM [1-14C]glutamine caused a stimulation of glucose formation; the magnitude of the concomitant increases in 14CO2 production and glutamine consumption indicate that flux from glutamine to glucose was increased. ATP also caused a simultaneous decrease in the cell content of oxoglutarate; together with the increased flux this is consistent with an activation of oxoglutarate dehydrogenase. In corroboration of this, a stimulation by ATP of gluconeogenesis and a decrease in oxoglutarate was also observed with 5 mM proline as substrate. ATP caused an increase in hepatocyte cytoplasmic free Ca2+ concentration, [Ca2+]c, as indicated by the increase in the fluorescence of cytoplasmically trapped quin2, from a resting value of about 0.2 microM to greater than 1 microM. The mechanism of oxoglutarate dehydrogenase activation may be via an increase in mitochondrial Ca2+ content as a consequence of the increase in [Ca2+]c. The effects of 100 microM adenosine were also investigated. An increase in flux from glutamine to glucose was observed together with a decrease in the cell oxoglutarate, thus indicating that adenosine addition to hepatocytes could also activate oxoglutarate dehydrogenase. The activation by adenosine was less than that produced by ATP. Adenosine caused a small apparent increase in [Ca2+]c to 0.3-0.4 microM; it remains to be established if this effect, which is small relative to that of ATP, is sufficient to elicit the activation of oxoglutarate dehydrogenase: alternative mechanisms may exist.     

Ca2+ homeostasis and cytotoxicity in isolated hepatocytes: studies with extracellular adenosine 5'-triphosphate

The incubation of isolated rat hepatocytes with extracellular adenosine 5'-triphosphate (ATP) resulted in an inhibition of Ca2+ efflux. The ATP-induced Ca2+ accumulation as determined by the increase in phosphorylase a activity and the Ca2+-sensitive fluorescent indicator (2-[(2-bis-[carboxymethyl]-amino-5-methylphenoxy)-methyl]-6-methoxy-8- bis-[carboxymethyl]aminoquinoline-tetrakis-[acetoxymethyl]ester) (Quin 2-AM) was associated with both the hydrolysis of ATP and the phosphorylation of a 110 kDa protein. No significant alteration in the intracellular ATP level was observed. The appearance of surface blebs and cytotoxicity followed the rise in cytosolic Ca2+, suggesting that the increased free Ca2+ may be responsible for the loss of viability. When a calmodulin inhibitor, 1-[bis(4-chlorophenyl)methyl]-3-[ 2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxy] ethyl]-1H- imidazolium chloride (calmidazolium), was included in the medium prior to ATP addition, bleb formation was reduced and the loss of viability was completely prevented, indicating that a Ca2+-calmodulin process may be involved in the initiation of cytotoxicity.