Changes in extra- and intracellular pH in hepatocytes exposed to gabexate mesilate.

Gabexate mesilate (GM) is a synthetic inhibitor of plasmatic and pancreatic serine proteases licensed for the treatment of pancreatitis. Here we show that in suspensions of isolated hepatocytes, profound changes in extracellular, cytoplasmic, and vesicular pH occur after addition of GM. Isolated hepatocytes obtained by collagenase perfusion of rat liver were pre-incubated with 1, 2, and 4 mM GM. Extracellular pH (pH in the incubation medium) was measured by a conventional pH electrode, cytosolic and vesicular pH were measured by fluorescence changes of 2',7'-biscarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and fluorescein dextran, respectively. Incubation of hepatocytes with GM resulted in a dose-dependent decrease of extracellular pH. Cytosolic pH decreased rapidly and markedly in a dose-dependent manner during the first minutes and gradually returned towards baseline. Simultaneously, GM induced a rapid alkalinization of acidic vesicles. The presence of bis-(p-nitrophelyl) phosphate (BNPP), an esterase inhibitor, reduced the extent of extracellular acidification. Incubation of hepatocytes in the presence of dimethylamiloride, an Na+/H+ exchanger inhibitor, or in a sodium-free medium, did not modify the rate and extent of extracellular acidification. GM, a commercially available pharmacological agent, could be useful to manipulate extra- and intracellular pH.

Italian Society for Rheumatology recommendations for the management of hand osteoarthritis.

Hand osteoarthritis (OA) is a common and potentially disabling disease, with different features from hip and knee OA so that a specific therapeutic approach is required. Evidence based recommendations for the management of hand OA were developed by the European League Against Rheumatism (EULAR) in 2006. The Italian Society for Rheumatology (SIR) aimed to update, adapt to national contest and disseminate the EULAR recommendations for the management of hand OA. The multidisciplinary group of experts included specialists involved in the management of patients with hand OA. In order to maintain consistency with EULAR recommendations, a similar methodology was utilized by the Italian group. The original propositions were reformulated in terms of a search query and for every recommendation a systematic search was conducted updating EULAR recommendations' review. The propositions were translated in Italian and reformulated basing on collected evidences and expert opinion. The strength of recommendation was measured for each proposition with the EULAR ordinal and visual analogue scales. The original 11 propositions of EULAR recommendations were translated and adapted to Italian context. Further evidences were collected about non-pharmacological therapies, local treatments, intra-articular injection with SYSADOA and corticosteroids, and surgery. The SIR has developed updated recommendations for the management of hand OA adapted to the Italian healthcare system. Their implementation in clinical practice is expected to improve the management of patients with hand OA.

Subnormothermic machine perfusion protects against rat liver preservation injury: a comparative evaluation with conventional cold storage.

UNLABELLED: Hypothermic machine perfusion (MP) of the liver has been reported to improve graft function reclaiming marginal livers, such as those from non-heart-beating donors. Livers from obese donors often have fatty infiltrates and are more susceptible to hypothermic conditions. No data exist about MP at temperatures >4 degrees C. This study evaluated liver function after organ preservation by comparing MP at 20 degrees C with conventional cold storage. METHODS: For MP, rat livers were perfused for 6 hours using an oxygenated Krebs-Henseleit (KH) solution at 20 degrees C (pH 7.4). For cold storage, livers were perfused in situ and preserved with Celsior solution at 4 degrees C for 6 hours. The reperfusion period with KH (2 hours at 37 degrees C) was performed under the same conditions both among livers preserved by MP or cold storage. Hepatic enzyme release (aspartate aminotransferase [AST], alanine aminotransferase [ALT], lactate dehydrogenase [LDH], and gamma-glutamyl transferase [GGT]), bile production, and ATP levels were measured during MP and reperfusion. RESULTS: At the end of reperfusion, livers preserved by MP showed significantly decreased liver damage compared with cold storage: AST, 18 +/- 4 vs. 45 +/- 6 mU/mL (P < .01); ALT, 1.5 +/- .07 vs. 6 +/- 0.5 mU/mL (P < .01); and LDH, 82 +/- 2 vs. 135 +/- 29 mU/mL (P < .05). No difference was observed between bile production between MP and cold storage. High levels of biliary GGT and LDH were found in cold preserved livers. ATP levels were higher in livers preserved with MP compared with those preserved by cold storage. CONCLUSIONS: MP at 20 degrees C resulted in a better quality of liver preservation, improving hepatocyte survival, compared with conventional cold storage. This may provide a new method for successful utilization of marginal livers, in particular fatty livers.

Liver damage during ischemia/reperfusion and glutathione: implications for potential organ donors.

Free radicals play a central role in the development of liver ischemia/reperfusion (I/R) injury. Reduced glutathione (GSH) is the main hepatic free radical scavenger. Brain-dead patients exhibit abnormalities of endocrine status. Many clinicians administer thyroid hormones to improve the transplantation outcomes. We previously reported that thyroxine (T(4)) pretreatment decreased rat liver tissue GSH, which was associated with increased liver I/R-induced damage. In this study, we investigated whether the reduction in GSH by T(4) pretreatment affected cell viability during anoxia or oxidative stress in suspensions of isolated hepatocytes. Furthermore, we evaluated the levels of GSH in isolated livers from hypothyroid rats preserved at 0-1 degrees C and reperfused. Thyroid hormone modulation was obtained by T(4) or 6-propylthiouracil (PTU) treatment. Isolated hepatocytes from T(4)-pretreated rats that underwent anoxia and oxidative stress, which was induced by tert-butylhydroperoxide, displayed progressive, time-dependent loss of cell viability, which was greater than that in hepatocytes in non-T(4)-pretreated rats. A significant decrease in GSH levels was observed in isolated hepatocytes obtained from hyperthyroid rats compared with those from euthyroid rats. In contrast, administration of the antithyroid drug PTU increased liver concentrations of GSH at the end of reperfusion thereby improving liver function after cold storage. These results may yield new protective strategies in the management of brain-dead organ donors.

Production of matrix metalloproteinases and their inhibitors in osteoarthritic patients undergoing mud bath therapy.

Several studies have demonstrated that matrix metalloproteinases (MMPs) are frequently implicated in the destruction of articular cartilage in arthritis. The control of MMP activity is dependent on the local concentration of tissue inhibitors of metalloproteinases (TIMPs), and the imbalance of the enzyme-to-inhibitor ratios plays an important role in the remodeling of articular tissues. Some cytokines such as interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha which regulate leukocyte activities, promote MMP secretion and, as a consequence, cartilage degradation. The aim of the present study was to investigate whether a natural treatment is effective in reducing cartilage inflammation and degradation by influencing MMP and TIMP serum levels. Eighty patients with osteoarthritis (OA) were enrolled in the trial and were divided into group A (30 patients who did not undergo mud bath therapy), group B (28 patients repeating mud bath therapy more than 5 times and less than 10) and group C (22 patients repeating mud bath therapy more than 10 times). Blood samples were obtained from all the patients for assay of MMP-1, -2, -3, -8 and -9 and TIMP-1 and -2. The parameters were determined by an ELISA technique. Statistical indexes were calculated for each parameter and mean values were compared. The differences between mean values of MMP-3, -8 and -9 were statistically significant between group A and the treated groups (B and C). Analysis of variance established a significant difference (p < 0.05) between groups A and C in mean serum levels of MMP-8, MMP-9 showed a statistically significant difference (p < 0.05) in mean serum concentration between groups A and B. Regression analysis showed a very high R2 between MMP-2 and TIMP-2. One of the most interesting findings in this study was that MMP-3 serum levels were significantly lower in the treated groups, since this enzyme plays an important role in cartilage degradation, suggesting that mud bath therapy contributes to matrix integrity in OA cartilage. In contrast, MMP-8 and -9 were higher in the treated subjects and no correlation with TIMPs was evident. One possible explanation is that these enzymes are required for the efficient degradation and removal of already compromised cartilage matrix and that they operate as part of a matrix turnover and repair process. In conclusion, our data suggest that mud bath therapy alone is not able to influence chondrocyte metabolic activity in the advanced phases of OA. There could be a synergic and sequential association with pharmacologic therapy and/or interventions.

Both serum receptors of tumor necrosis factor are influenced by mud pack treatment in osteoarthrotic patients.

Several authors have demonstrated the pivotal role of proinflammatory cytokines in inducing progressive cartilage degradation and secondary inflammation of the synovial membrane in osteoarthritis (OA). It has recently been established that tumor necrosis factor (TNF)-alpha plays a well-defined role in the pathophysiology of inflammatory joint diseases and that binding to circulating soluble TNF-alpha receptors can inactivate it. We investigated the influence of mud pack treatment, which is able to diminish TNF-alpha serum values, on specific TNF receptor (sTNF-R) levels. Thirty-six patients with OA were enrolled and randomized into two groups. Group A underwent mud pack treatment and group B underwent thermal bath treatment. A group of 20 healthy untreated subjects was used as a control. Blood samples were collected at baseline and after treatment, and assays of sTNF-R55 and sTNF-R75 were performed in both groups. We found small changes in sTNF-Rs serum values but these were not statistically significant. sTNF-R55 serum values decreased by 0.4% after the therapy in group A, while in group B the decrease was -17.7%. sTNF-R75 was reduced by -21.17% in group A and by -10.6% in group B. In conclusion, through its thermic and ant/inflammatory activity mud pack treatment shows complex interaction with the most common factors of inflammatory and cartilage degradation. Our results suggest that the thermic component of this natural treatment is mainly involved in modulating inflammatory reaction and cartilage damage through binding of the circulating TNF, which controls the activation of the cells responsible for the production of proinflammatory cytokines.

Cold-induced apoptosis in isolated rat hepatocytes: protective role of glutathione.

Liver conservation for transplantation is usually made at 2-4 degrees C. We studied the effect of rewarming to 37 degrees C for up to 3 h of rat hepatocytes kept at 4 degrees C for 20 h, modulating intracellular glutathione (GSH) concentration either with a GSH precursor (N-acetyl-L-cysteine, NAC), or with GSH depleting agents (diethylmaleate and buthionine sulfoximine, DEM/BSO). Untreated hepatocytes showed time-dependent production of reactive oxygen species (ROS), lipid peroxidation, chromatin condensation and membrane blebbing, decrease in GSH concentration, and protein sulfhydryl groups. Fluorochromatization with Propidium Iodide (PI) and Annexin V (AnxV) of cells rewarmed for 1 h caused an increase of AnxV-positive cells without PI staining and any observed lactate dehydrogenase leakage. TUNEL and DNA-laddering tests were negative for all times and treatments, indicating that apoptosis may occur without DNA fragmentation. Cold preservation and rewarming in the presence of NAC induced a significant improvement in the morphology, less oxidative stress and apoptosis. Conversely, DEM/BSO caused a marked deterioration of morphology, increase of oxidative stress and apoptosis. These results suggested that marked changes in GSH status might play a critical role in triggering apoptosis during cold preservation of isolated rat hepatocytes. NAC, added before rewarming, might represent a therapeutic approach for preventing the early events of apoptosis during cold storage.

Selective blockade of mGlu5 metabotropic glutamate receptors protects rat hepatocytes against hypoxic damage.

Western blot analysis of protein extracts from rat liver revealed the presence of the mGlu5 receptor, one of the G-protein-coupled receptors activated by glutamate (named "metabotropic glutamate receptors" or mGlu receptors). mGlu5 expression was particularly high in extracts from isolated hepatocytes, where levels were comparable with those seen in the rat cerebral cortex. The presence of mGlu5 receptors in hepatocytes was confirmed by reverse-transcription polymerase chain reaction (RT-PCR) analysis, immunohistochemistry in neonate or adult rat liver, as well as by immunocytochemical analysis in HepG2 hepatoma cells, where the receptor appeared to be preferentially distributed in cell membranes. Interestingly, mGlu1 receptors (which are structurally and functionally homologous to mGlu5 receptors) were never found in rat liver or hepatocytes. In hepatocytes exposed to anoxic conditions for 90 minutes, glutamate, (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid (1S,3R-ACPD) and quisqualate, which all activate mGlu5 receptors, accelerated the onset and increased the extent of cell damage, while 4-carboxy-3-hydroxyphenylglycine (4C3HPG), an agonist of mGlu2/3 receptors, was inactive. 2-methyl-6-(2-phenyl-1-ethynyl)-pyridine (MPEP), a novel, noncompetitive, highly selective mGlu5 receptor antagonist, not only abolished the toxic effect of 1S,3R-ACPD, but, unexpectedly, was protective by itself against anoxic damage. This suggests that hepatocytes express mGlu5 receptors and that activation of these receptors by endogenous glutamate facilitates the development of anoxic damage in hepatocytes.

Mud bath therapy influences nitric oxide, myeloperoxidase and glutathione peroxidase serum levels in arthritic patients.

Nitric oxide (NO) has recently been proposed as an important mediator in inflammatory phases and in loss of cartilage. In inflammatory arthritis NO levels are correlated with disease activity and articular cartilage is able to produce large amounts of NO with the appropriate inducing factors such as cytokines and/or endotoxin. Neutrophils also play an important role in inflammatory reactions and the level of myeloperoxidase, a constituent of neutrophil granules, is related to the intensity of the inflammation. Because there is evidence that suggests that mud packs influence the main cytokines involved in cartilage damage, we tried to determine whether NO and myeloperoxidase are involved in the mechanisms of action of mud bath treatment. We enrolled 37 subjects and randomly assigned them to two groups: 19 patients underwent mud bath treatment (group A) while 18 patients underwent bath treatment alone. Blood samples were obtained before and after the treatment cycles to assay serum levels of NO, myeloperoxidase (MPO) and glutathione (GSH)-peroxidase. The results showed a statistically significant decrease in NO and myeloperoxidase serum values in groups A and B, while GSH-peroxidase was not significantly increase in either of the groups; no correlation was found between NO, myeloperoxidase and GSH-peroxidase serum values. Mud bath treatment can exert beneficial effects on cartilage homeostasis and inflammatory reactions, influencing NO and decreasing myeloperoxidase serum values. The increase in GSH-peroxidase was not correlated with the reduction of other biochemical markers, suggesting that mud bath treatment has different mechanisms of action.

Haloperidol-induced changes in glutathione and energy metabolism: effect of nicergoline.

The aim of this study was to evaluate the possible effects of nicergoline, a semisynthetic ergot derivative, on the biochemical changes observed during chronic treatment with haloperidol in male Sprague-Dawley rats. Chronic treatment with haloperidol induced a significant decrease in the cellular glutathione (GSH) content in selected areas of the brain (cerebellum, striatum and cortex) and in the liver. Prolonged nicergoline administration was able to antagonize the haloperidol-induced GSH decrease, maintaining the GSH concentration at levels comparable to those observed in the control group. Analysis of the energy charge revealed changes similar to those observed for GSH: haloperidol induced a significant decrease in ATP and energy charge that was completely reversed by repeated nicergoline administration. In conclusion, chronic treatment with the classical antipsychotic haloperidol induces profound biochemical changes in the brain and in the liver. Nicergoline treatment is able to counteract the haloperidol-induced decrease in GSH levels and energy charge, suggesting a potential role of the drug in the treatment of neuroleptic-induced side effects.

The effects of thyroid hormone modulation on rat liver injury associated with ischemia-reperfusion and cold storage.

UNLABELLED: We investigated the effects of thyroid hormone modulation on liver injury associated with ischemia-reperfusion (I-R) and cold storage in rats. First, euthyroid and thyroxine (T4)-pretreated rats were exposed in vivo to 20-min global liver ischemia, then 30-min reperfusion. Liver injury was assessed by measuring serum alanine aminotransferase (ALT) levels. Liver concentrations of adenine nucleotides, reduced glutathione (GSH), and oxidized glutathione were evaluated. Second, rats were given the antithyroid drug propylthiouracil (PTU). Livers stored at 0-1 degrees C in Euro-Collins' solution for 20 h were reperfused at 37 degrees C for 15 min. Lactate dehydrogenase (LDH) in the effluent perfusate and bile flow were evaluated during reperfusion. Serum ALT levels increased after ischemia and I-R. ALT increased significantly more in T4-pretreated than in euthyroid rats after ischemia and I-R. Preischemic levels of adenosine triphosphate (ATP) were significantly lower in livers from T4-pretreated than in euthyroid rats (6.22 +/- 0.7 and 11 +/- 0.9 nmol/mg protein, respectively; P < 0.05). After ischemia, liver ATP was similarly reduced in T4-pretreated and euthyroid rats. After reperfusion, ATP partially recovered in euthyroid rats but remained low in T4-pretreated rats (6.7 +/- 1.0 and 1.91 +/- 0.7 nmol/mg protein, respectively; P < 0.05). Preischemic levels of liver GSH decreased to 44% in T4-pretreated rats. After ischemia, GSH decreased similarly in euthyroid and T4-pretreated rats. GSH recovered promptly after reperfusion in euthyroid rats but remained low in T4-pretreated rats (13.9 +/- 3.3 and 3.9 +/- 0.9 nmol/mg protein, respectively; P < 0.02). During reperfusion after cold storage, LDH in effluent perfusate was significantly lower and bile flow higher in livers from PTU-pretreated rats than from euthyroid rats. The histopathological changes observed after I-R and cold storage confirmed the biochemical findings. Our results suggest that T4 administration exacerbates pretransplant liver damage by increasing liver susceptibility to I-R, whereas PTU administration reduces the liver injury associated with cold storage. IMPLICATIONS: We studied the effects of thyroid hormone modulation on liver injury associated with ischemia-reperfusion and cold storage in rats. Thyroxine administration increased susceptibility to ischemia-reperfusion injury, whereas the antithyroid agent propylthiouracil reduced the deleterious effects associated with cold storage.

Dopamine partial receptor agonists reduce ethanol intake in the rat.

Dopamine neurotransmission is an important neuropharmacological component of ethanol reinforcement in rodents. A recently characterized class of compounds, dopamine partial receptor agonists, appears to possess a unique pharmacological profile on dopamine neurotransmission. The aim of the present study was to test the effects of systemic administration of terguride and SDZ 208-911 (N-[(8 alpha)-2,6-dimethylergoline-8-yl]-2,2-diethylpropanamide), two prototype dopamine partial receptor agonists, in free-feeding, non-deprived rats trained to drink ethanol (10% w/v) and water in 'free-choice' limited access conditions. Both acute and chronic administration of terguride and SDZ 208-911 significantly reduced ethanol intake while water intake was not significantly affected, thus ruling out possible non-specific effects of these drugs on fluid intake. These results suggest that dopamine partial receptor agonists reduce the reinforcing properties of ethanol in the rat, an effect similar to that previously observed with cocaine. Therefore, the pharmacological profile of dopamine partial receptor agonists and their effects in animal models of dependence provide preclinical support to the hypothesis that these compounds may represent a novel pharmacological strategy for intervention in various forms of drug addiction.

Thyroxine pretreatment and halothane administration alter Ca2+ transport and transmembrane potential in rat liver mitochondria. An additional mechanism for halothane-induced liver damage in the hyperthyroid rat model.

Male rats pretreated with thyroid hormones and exposed to halothane in non-hypoxic conditions develop acute liver damage. In order to investigate the mechanisms leading to liver damage in this animal model, the effects of thyroxine (T4) pretreatment and halothane administration on Ca2+ transport and transmembrane potential were studied in isolated rat liver mitochondria. Five-day T4-pretreatment reduced the mitochondrial Ca2+ loading capacity and increased the rate of Ca2+ cycling across the mitochondrial membrane. Halothane administration further increased Ca2+ cycling and produced a time- and dose-dependent loss of transmembrane potential which was more pronounced in mitochondria from T4-pretreated rats than in euthyroid animals. When mitochondria from T4-pretreated rats were incubated in the presence of the Ca2+ chelator EGTA, membrane potential was well preserved. In contrast, when Ca2+ concentration in the extramitochondrial medium was increased, mitochondria deenergization occurred earlier. These findings confirm that alterations in Ca2+ transport and mitochondrial function can be interrelated events and suggest that a Ca(2+)-dependent, halothane-induced loss of transmembrane potential could participate in generating acute liver damage in hyperthyroid rats exposed to halothane in non-hypoxic conditions.

Demonstration of nuclear compartmentalization of glutathione in hepatocytes.

The intracellular distribution of glutathione (GSH) in cultured hepatocytes has been investigated by using the compound monochlorobimane (BmCl), which interacts specifically with GSH to form a highly fluorescent adduct. Image analysis of BmCl-labeled hepatocytes predominantly localized the fluorescence in the nucleus; the nuclear/cytoplasmic concentration gradient was approximately three. This concentration gradient was collapsed by treatment of the cells with ATP-depleting agents. The uneven distribution of BmCl fluorescence was not attributable to (i) nonspecific interaction of BmCl with protein sulfhydryl groups, (ii) any selective nuclear localization of the GSH transferase(s) catalyzing formation of the GSH-BmCl conjugate, or (iii) any apparent alterations in cell morphology from culture conditions, suggesting that this distribution did, indeed, reflect a nuclear compartmentalization of GSH. That the nuclear pool of GSH was found more resistant to depletion by several agents than the cytoplasmic pool supports the assumption that GSH is essential in protecting DNA and other nuclear structures from chemical injury.

The cytoskeleton as a target in quinone toxicity.

The exposure of mammalian cells to toxic concentrations of redox cycling and alkylating quinones causes marked changes in cell surface structure known as plasma membrane blebbing. These alterations are associated with the redistribution of plasma membrane proteins and the disruption of the normal organization of the cytoskeletal microfilaments which appears to be due mainly to actin cross-linking and dissociation of alpha-actinin from the actin network. The major biochemical mechanisms responsible for these effects seem to involve the depletion of cytoskeletal protein sulfhydryl groups and the increase in cytosolic Ca2+ concentration following the alkylation/oxidation of free sulfhydryl groups in several Ca2+ transport systems. Depletion of intracellular ATP is also associated with quinone-induced plasma membrane blebbing. However, ATP depletion occurs well after the onset of the morphological changes, and thus it does not seem to be causatively related to their appearance. Thiol reductants, such as dithiothreitol, efficiently prevent the oxidation of cytoskeletal protein thiols, the increase in cytosolic free Ca2+ concentration and cell blebbing induced by redox cycling, but not alkylating, quinones. These results demonstrate that alkylating and redox cycling quinones cause similar structural and biochemical modifications of the cytoskeleton by means of different mechanisms, namely alkylation and oxidation of critical sulfhydryl groups.

1,2-Dichloropropane (DCP) toxicity is correlated with DCP-induced glutathione (GSH) depletion and is modulated by factors affecting intracellular GSH.

Acute 1,2-dichloropropane (DCP) poisoning in humans is relatively frequent in Italy, where DCP is widely diffused as a constituent of commercial solvents and dry cleaners. In this study we have investigated the effects of DCP on intracellular glutathione (GSH) content in main target tissues of male Wistar rats, i.e. liver, kidney and blood, in order to establish if a correlation between DCP-induced GSH depletion and tissue damage exists. Administration of DCP (2 ml/kg body weight orally) caused a dramatic loss of tissue GSH occurring 24 h after DCP intoxication, followed by a slow restoration approaching physiological levels after 96 h. GSH depletion was associated with a marked increase in serum GOT, GPT, 5'-nucleotidase, gamma-glutamyl transpeptidase, alkaline phosphatase, urea and creatinine, and a significant degree of hemolysis. When animals were pretreated with a GSH depleting agent, buthionine-sulfoximine (BSO) (0.5 g/kg body weight) i.p. 4 h before DCP intoxication, an increase of overall mortality was found, significantly different from the group of animals treated with DCP alone. On the contrary, the administration of a GSH precursor, N-acetylcysteine (NAC) i.p. (250 mg/kg body weight) 2 and 16 h after DCCP intoxication prevented the dramatic loss of cellular GSH and reduced the extent of injury in target tissues, as demonstrated by laboratory indices. Furthermore, statistical analysis of the data revealed a correlation between: (1) depletion of liver GSH and increase in serum GOT, GPT, 5'-nucleotidase, (2) depletion of kidney GSH and increase in serum urea and creatinine and (3) depletion of blood GSH and the occurrence of hemolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

On the role of mitochondria in cell injury caused by vanadate-induced Ca2+ overload.

Incubation of isolated rat hepatocytes with vanadate (0.25, 0.5 and 1 mM) resulted in progressive accumulation of Ca2+ in the intracellular compartments. Vanadate- induced Ca2+ accumulation was related to inhibition of the plasma membrane Ca2+-extruding system, but did not involve either enhanced plasma membrane permeability to Ca2+ or the enhanced operation of a putative Na+/Ca2+ exchanger. After an initial rise in the cytosolic free Ca2+ concentration, as revealed by phosphorylase activation, Ca2+ was sequestered predominantly by the mitochondria with little contribution from the endoplasmic reticulum. As the amount of Ca2+ in the mitochondria increased, a progressive decrease in mitochondrial membrane potential occurred, together with an impairment of the ability of these organelles to further sequester Ca2+. Associated with this, there was a decrease in intracellular ATP level, formation of surface blebs and cytotoxicity. Addition of an uncoupler to vanadate-treated hepatocytes dramatically accelerated the appearance of plasma membrane blebs and toxicity. Our results demonstrate that under conditions in which the plasma membrane Ca2+ pump is inhibited, mitochondria play an important role in protecting hepatocytes against damage induced by Ca2+ overload.