Calcium-dependent and calcium-independent mechanisms of irreversible cell injury in cultured hepatocytes

It has been proposed that alterations in intracellular calcium homeostasis mediate the genesis of lethal cell injury with an acute oxidative stress. It is shown here, however, that such changes can be dissociated by two different means from the cell death occurring with the exposure of cultured hepatocytes to hydrogen peroxide generated either in the medium by glucose oxidase or intracellularly by the mechanism of menadione. The chelation of intracellular ferric iron with deferoxamine inhibits the formation of hydroxyl radicals from hydrogen peroxide and prevents cell killing. Deferoxamine did not prevent, however, an elevation of the cytosolic Ca2+ ion concentration detected as an activation of phosphorylase alpha. Sulfhydryl reagents inhibited the rise in phosphorylase alpha activity in deferoxamine-pretreated hepatocytes. Conversely, cultured hepatocytes were depleted of Ca2+ ions by treatment with EGTA in a calcium-free medium. Calcium-depleted cells were not resistant to the toxicity of hydrogen peroxide despite the virtual elimination of the activation of phosphorylase alpha. In contrast, it was possible to kill cultured hepatocytes by a mechanism dependent upon a disordered intracellular calcium homeostasis using hepatocytes pretreated in calcium-free medium with the ionophore A23187. These cells were killed in a dose-dependent manner by the addition of calcium ions to the culture medium in concentrations ranging from 0.1 to 2.0 mM. There was a similar dose-dependent activation of phosphorylase alpha, but phosphorylase alpha activities were higher than with H2O2 at comparable cell killing. Deferoxamine pretreatment and sulfhydryl reagents had no effect on the loss of viability with this calcium-dependent cell killing.     

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.     

Increases in cytosolic calcium ion concentration can be dissociated from the killing of cultured hepatocytes by tert-butyl hydroperoxide

Digital imaging fluorescence microscopy was used to study the effect of tert-butyl hydroperoxide (TBHP) on the cytosolic free calcium concentration ([Ca2+]i) of single rat hepatocytes in primary culture. Within minutes of the addition of TBHP, individual hepatocytes displayed one or more peaks of increased [Ca2+]i that promptly returned to the prestimulation level. This was followed by a slower increase of [Ca2+]i that reached a plateau of 696 +/- 260 nM (basal 194 +/- nM) after 20 min. Another rise in [Ca2+]i, abrupt and much larger, preceded the death of the cells after about 45 min. Pretreatment of the hepatocytes with deferoxamine, a ferric iron chelator, or the addition of the antioxidants N,N'-diphenyl-p-phenylenediamine or catechol prevented the loss of viability. Neither the number of hepatocytes displaying the initial [Ca2+]i transients nor the magnitude of these oscillations was affected by deferoxamine, N,N'-diphenyl-p-phenyl-enediamine, or catechol. However, both the plateau phase and the abrupt rise in [Ca2+]i were prevented. Treatment of the hepatocytes with TBHP in a low calcium buffer (less than 2 microM Ca2+) reduced or abolished the initial [Ca2+]i transients and eliminated both the plateau phase and abrupt rise in [Ca2+]i. The onset of cell death was delayed by 10 min in the low calcium medium. Addition of 3.5 mM EGTA to the cultures lowered the basal calcium concentration, prevented both the initial [Ca2+]i spikes and the delayed changes, and further prolonged the onset of cell death. These data indicate that the killing of the cultured hepatocytes by TBHP can be dissociated from changes in intracellular calcium homeostasis. An influx of extracellular Ca2+ ions may aggravate somewhat the mechanisms of cell injury by an oxidative stress and accelerate the time of onset of cell death.     

Effect of dimethyl sulfoxide on cytosolic ionized calcium concentration and cytoskeletal organization of hepatocytes in a primary culture

The addition of dimethyl sulfoxide (DMSO) to a chemically defined, serum free medium prolonged hepatocytes survival in primary culture. DMSO exposure had a remarkable effect on morphological change and F-actin filaments distribution of hepatocytes. When hepatocytes were cultured in a medium containing 2% DMSO, the cells showed a compact and cubical shape and intracellular F-actin filaments were mainly observed in a ring-like fashion around the intercellular space. After exposure to DMSO, fibronectin fibers in the interspace between cell and substratum were not apparent. Exposing the hepatocytes to DMSO also caused a sharp increase in cytosolic free ionized calcium ([Ca2+]). The initial increase in [Ca2+]i following the addition of DMSO was not attenuated by the chelation of extracellular Ca2+ with EGTA. The Ca2+ signal in the absence of extracellular Ca2+ was transient and returned to the basal levels within 1-2 min, while it was maintained at a high steady state in the presence of extracellular Ca2+. These results suggest that DMSO may be able to increase [Ca2+]i by two mechanisms, by the release of the ion from intracellular pools and, by the stimulation of influx across the plasma membrane. The increase in [Ca2+]i induced by DMSO treatment may play a role in prolonging hepatocyte survival in culture, since [Ca2+]i is one of the most important dynamic second messengers in various cellular metabolic processes.     

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.     

Cyclosporine augments receptor-mediated cellular Ca2+ fluxes in isolated hepatocytes

The immunosuppressive agent, cyclosporine, has been found to augment receptor-stimulated calcium fluxes in isolated hepatocytes. After treatment of Quin 2-loaded hepatocytes with cyclosporine, both the amplitude and duration of the vasopressin-induced rise in the cytosolic free Ca2+ are increased. These effects are dependent upon the concentration and time of exposure of the cells to cyclosporine. Cyclosporine increases both 45Ca2+ influx across the plasma membrane and the cellular calcium content. The total cellular magnesium, sodium, and potassium contents are not affected by cyclosporine. However, cyclosporine treatment, per se, has no apparent effect on the cytosolic free Ca2+ concentration as assayed by Quin 2 fluorescence. The increase in total cell calcium is associated with progressive increases in the calcium content of the endoplasmic reticular and mitochondrial calcium pools. The vasopressin-induced net efflux of Ca2+ from hepatocytes was 2-fold greater after treatment with 10 micrograms/ml cyclosporine for 10 min, but the lag time prior to the onset of Ca2+ efflux was not affected. These results are interpreted on the basis of cyclosporine having a primary effect on increasing the permeability of the plasma membrane to Ca2+, thereby leading to an increase of the calcium content of the hormone-sensitive intracellular calcium pool.     

Toxic injury from mercuric chloride in rat hepatocytes

The relationship between cytosolic free Ca2+, mitochondrial membrane potential, ATP depletion, pyridine nucleotide fluorescence, cell surface blebbing, and cell death was evaluated in rat hepatocytes exposed to HgCl2. In cell suspensions, 50 microM HgCl2 oxidized pyridine nucleotides between 1/2 and 2 min, caused ATP depletion between 2 and 5 min, and produced an 89% loss of cell viability after 20 min. Rates of cell killing were identical in high (1.2 mM) and low (2.6 microM) Ca2+ buffers. Cytosolic free Ca2+ was determined in 1-day cultured hepatocytes by ratio imaging of Fura-2 employing multiparameter digitized video microscopy. In high Ca2+ medium, HgCl2 caused a 3-4-fold increase of free Ca2+ beginning after 6-7 min, but free Ca2+ did not change in low Ca2+ medium. Bleb formation occurred after about 4-5 min in both buffers prior to any increase of free Ca2+. Subsequently, in high Ca2+ medium, blebs became hot spots of free Ca2+ (greater than 600 nM). After about 2 min of exposure to HgCl2, rhodamine 123 fluorescence redistributed from mitochondrial to cytosolic compartments signifying collapse of the mitochondrial membrane potential. The results taken together demonstrate that bleb formation, ATP depletion, and the onset of cell death are not dependent on an increase of cytosolic free Ca2+. HgCl2 toxicity appears to be a consequence of inhibition of oxidative phosphorylation leading to ATP depletion and cell death.     

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.     

Alterations in intracellular calcium compartmentation following inhibition of calcium efflux from isolated hepatocytes

Addition of ATP to the incubation medium of freshly isolated rat hepatocytes causes a marked inhibition of the efflux of Ca2+ from the cells, and its accumulation in intracellular compartments. After an initial rise in cytosolic free Ca2+ concentration, as indicated by the activation of phosphorylase, Ca2+ is preferentially sequestered in the mitochondria, without any apparent contribution by the endoplasmic reticulum. Impairment of mitochondrial Ca2+ homeostasis by pyridine nucleotide oxidation associated with tert-butyl hydroperoxide metabolism, prevents the ATP-dependent cellular Ca2+ accumulation and causes a release of Ca2+ from the hepatocytes into the medium. Conversely, maintenance of the mitochondrial pyridine nucleotides in a more reduced state, e. g. in presence of 3-hydroxybutyrate in the medium, prevents this hydroperoxide-induced release of intracellular Ca2+. Under conditions of impaired mitochondrial Ca2+ sequestration, there appears to be a redistribution of a minor fraction of the intracellular Ca2+ from the mitochondria to the endoplasmic reticulum. Our results provide additional evidence for the critical involvement of the plasma membrane Ca2+-extruding system in the physiological regulation of the cytosolic free Ca2+ concentration in hepatocytes, and suggest that the mitochondria play a more important role than the endoplasmic reticulum in the regulation of the cytosolic free Ca2+ level when the plasma membrane Ca2+ pump is inhibited.     

Cystamine induces toxicity in hepatocytes through the elevation of cytosolic Ca2+ and the stimulation of a nonlysosomal proteolytic system

Infusion of cystamine into the isolated, perfused rat liver resulted in tissue damage preceded by the formation of cystamine-protein mixed disulfides which were mainly detected in the plasma membrane fraction. Hepatotoxicity was prevented when dithiothreitol was infused after cystamine or when the calcium antagonist, verapamil, was co-infused with the disulfide. In isolated hepatocytes, the formation of cystamine-protein mixed disulfides was associated with an inhibition of plasma membrane Ca2+-ATPase activity and a decreased rate of Ca2+ efflux from the cells. This resulted in intracellular Ca2+ accumulation which was followed by a stimulation of both phospholipid hydrolysis and proteolysis, as indicated by enhanced rates of release of radioactivity from hepatocytes prelabeled with [14C]arachidonate and [14C]valine, respectively. Preincubation of hepatocytes with the calmodulin inhibitor, calmidazolium, or with the phospholipase inhibitors, chlorpromazine and dibucaine, inhibited the stimulation of [14C]arachidonate release by cystamine. However, none of these agents prevented the onset of cystamine toxicity in hepatocytes. In contrast, pretreatment of the cells with antipain or leupeptin, two inhibitors of Ca2+-activated proteases, abolished the stimulation of proteolysis by cystamine and also protected the cells from cystamine toxicity. Our results suggest that the perturbation of intracellular Ca2+ homeostasis by cystamine is caused by the inhibition of Ca2+ efflux associated with the formation of cystamine-protein mixed disulfides in the plasma membrane and that subsequent cytotoxicity results from Ca2+-activation of a nonlysosomal proteolytic system.     

The effects of anoxia on cytosolic free calcium, calcium fluxes, and cellular ATP levels in cultured kidney cells

The effect of anoxia and substrate removal on cytosolic free calcium (Ca2+i), cell calcium, ATP content, and calcium efflux was determined in cultured monkey kidney cells (LLC-MK2) exposed to 95% N2, 5% CO2 for 60 min. In the control period, the basal Ca2+i level was 70.8 +/- 9.4 nM. During 1 h of anoxia without substrate, ATP content decreased 70%, Ca2+i and calcium efflux increased 2.5-fold, while the total cell calcium did not change. When the cells were perfused again with O2 and 5 mM glucose, the ATP concentration, Ca2+i, and calcium efflux returned to control levels within 15-20 min. In the presence of 20 mM glucose, anoxia did not produce any change in ATP, in Ca2+i or in calcium efflux. An important source of calcium contributing to the rise in Ca2+i induced by anoxia appears to be extracellular because the rate of rise in Ca2+i is proportional to the extracellular calcium concentration, and because La3+ which blocks calcium influx greatly reduces the rise in Ca2+i. Mitochondria appear to control Ca2+i as well since the early rise in Ca2+i cannot be blocked by La3+ during the initial phase of anoxia, and since the mitochondrial inhibitor carbonyl cyanide p-trifluoromethoxyphenylhydrazone increases Ca2+i further during reoxygenation and slows the return of Ca2+i to control levels.     

Ryanodine receptors in liver

The ryanodine receptor has been mainly regarded as the Ca2+ release channel from sarcoplasmic reticulum controlling skeletal and cardiac muscle contraction. However, many studies have shown that it is widely expressed, with functions not restricted to muscular contraction. This study examined whether ryanodine receptor plays a role in calcium signaling in the liver. RT-PCR analysis of isolated hepatocytes showed expression of a truncated type 1 ryanodine receptor, but no type 2 or type 3 message was detected. We also detected binding sites for [3H]ryanodine in the microsomal cellular fraction and in permeabilized hepatocytes. This binding was displaced by caffeine and dantrolene, but not by ruthenium red, heparin or cyclic ADP-Ribose. Ryanodine, by itself, did not trigger Ca2+ oscillations in either primary cultured hepatocytes or hepatocytes within the intact perfused rat liver. In both preparations, however, ryanodine significantly increased the frequency of the cytosolic free [Ca2+] oscillations evoked by an alpha1 adrenergic receptor agonist. Experiments in permeabilized hepatocytes showed that both ryanodine and cyclic ADP-ribose evoked a slow Ca2+ leak from intracellular stores and were able to increase the Ca2+-released response to a subthreshold dose of inositol 1,4,5-trisphosphate. Our findings suggest the presence of a novel truncated form of the type 1 ryanodine receptor in rat hepatocytes. Ryanodine modulates the pattern of cytosolic free [Ca2+] oscillations by increasing oscillation frequency. We propose that the Ca2+ released from ryanodine receptors on the endoplasmic reticulum provides an increased pool of Ca2+ for positive feedback on inositol 1,4,5-trisphosphate receptors.     

Intracellular Ca2+ regulation in CD3 stimulated Jurkat T cells involves H+ fluxes.

Triggering the CD3/TCR complex of T lymphocytes induces a rapid rise in cytosolic free calcium followed by a slowly declining plateau. The level of this plateau depends on external pH, the more alkalinized media leading to higher values. Neither a pH-dependent binding of mAb, nor a perturbation of internal pH can account for this effect. In a sodium-free medium, or in the presence of dimethylamiloride Ca2+, elevation is accompanied by an acidification of the cells; both of them depend, to the same extent, on external calcium concentration. TPA inhibits CD3-, but not ionomycin-induced Ca2+ and H+ raises, indicating that it acts more probably on Ca2+ influx, rather than on its efflux. These results suggest that intracellular calcium could be regulated by a Ca2+/H+ ATPase which drives H+ in and Ca2+ out. In the presence of external Na+, H+ should return to the medium by the Na+/H+ exchanger.     

Calcium dependence of bleb formation and cell death in hepatocytes

Calcium dependence of bleb formation and cell death was evaluated in rat hepatocytes following ATP depletion by metabolic inhibition with KCN and iodoacetate ('chemical hypoxia'). Cytosolic free Ca2+ was measured in single cells by ratio imaging of Fura-2 fluorescence using multiparameter digitized video microscopy. Cells formed surface blebs within 10 to 20 minutes after chemical hypoxia and most cells lost viability within an hour. An increase of cytosolic free Ca2+ was not required for bleb formation to occur. One to a few minutes prior to the onset of cell death, free Ca2+ increased rapidly in high Ca2+ buffer (1.2 mM) but not in low Ca2+ buffer (less than 1 microM). In either buffer, the rate of cell killing was the same. As the onset of cell death was approached in both high and low Ca2+ buffers, Fura-2 began to leak from the cells at an accelerating rate indicating rapidly increasing plasma membrane permeability. In high Ca2+ buffer, cytosolic free Ca2+ increased in parallel with dye leakage. No regional changes in cytosolic free Ca2+ were observed during this metastable period of increased membrane permeability. In many experiments, actual rupture of cell surface blebs could be observed which led to micron-size discontinuities of the cell surface and cell death. We conclude that a metastable period characterized by increasing plasma membrane permeability marked the onset of cell death in cultured hepatocytes which culminated in rupture of a cell surface bleb. An increase of cytosolic free Ca2+ was not required for the metastable state to develop or cell death to occur.     

Effects of low extracellular sodium on cytosolic ionized calcium. Na+-Ca2+ exchange as a major calcium influx pathway in kidney cells

The effects of extracellular Na+ (Na+o) on cytosolic ionized calcium (Ca2+i) and on calcium and sodium fluxes were measured in monkey kidney cells (LLC-MK2). Ca2+i was measured with aequorin and the ion fluxes with 45Ca and 22Na. Na+-free media rapidly increased Ca2+i from 60 to a maximum of about 700 nM in 2-3 min. After the peak, Ca2+i declined and reached a plateau of about twice the resting Ca2+i. The peak Ca2+i was inversely proportional to Na+o and directly proportional to the extracellular calcium concentration (Ca2+o). On the other hand, a pH of 6.8 reduced and Ca2+o substitution with Sr2+ completely blocked the Ca2+i response to low Na+o. A Na+-free medium stimulated calcium efflux from the cells 4-5-fold, a response which was abolished in the absence of extracellular Ca2+. Na+-free media also stimulated calcium influx and sodium efflux. The cell calcium content, however, was not increased. These results indicate that removal of extracellular Na+ increases Ca2+i by stimulating calcium influx and not by inhibiting calcium efflux; the increased calcium influx takes place on the Na+-Ca2+ antiporter operating in the reverse mode in exchange for sodium efflux. The increased calcium efflux occurs as a consequence of the rise in Ca2+i and presumably takes place on the (Ca2+-Mg2+) ATPase-dependent calcium pump.     

Planarian regeneration: in vivo and in vitro effects of calcium and calmodulin on DNA synthesis

Regeneration does not occur when planarians are grown in Ca2+-free medium. The possible effect of calcium upon DNA synthesis was therefore studied using cultured planarian cells and regenerating planarian fragments. In the cultures, DNA synthesis was Ca2+-dependent and required a minimum of 10(-6) M Ca2+ in the medium. It was gradually decreased in cells grown in Ca2+-free medium. Addition of Ca2+ to these cultures raised DNA synthesis. The time lag between addition of Ca2+ and stimulation of DNA synthesis varied with culture age. The triggering effect of Ca2+ was amplified by ionophore A 23187. A calcium binding protein, ram testis calmodulin, intensified the stimulatory effect of calcium, but EGTA blocked this effect. In the presence of trifluoperazine (TFP), DNA synthesis was not stimulated by Ca2+. This inhibition by TFP was overcome by adding calmodulin to the medium. Ca2+ therefore triggered DNA synthesis in vitro, and this role might have been potentiated by calmodulin. In vivo, DNA synthesis was shown to be dependent on the Ca2+ concentration in the medium in which intact or regenerating planarians were grown. In 12-h regenerates, the Ca2+ concentration in the medium was no longer critical. Total calcium content decreased just after sectioning until completion of healing (at 6 h) and then rose significantly to a peak at 12 h which coincided with the first peak of DNA synthesis. The calmodulin content gradually diminished during the first 6 h after sectioning. After a transient rise at 12 h, calmodulin content further decreased until 48 h. The results demonstrate the crucial role of Ca2+ in triggering DNA synthesis in planarian cells in vitro and in regenerating fragments. Calmodulin, whose concentration is very low in planarians compared to vertebrates, might help to induce the first peak of DNA synthesis at 12 h after sectioning, but is probably not the main Ca2+-binding protein involved in the regeneration process.     

Oxidative stress studied in intact mammalian cells

Exposure of isolated rat hepatocytes to toxic doses of menadione (2-methyl-1,4-naphthoquinone) results in enhanced formation of active oxygen species, depletion of cellular glutathione and protein thiols, and perturbation of intracellular calcium ion homeostasis. An increase in cytosolic Ca2+ concentration, resulting from inhibition of the plasma membrane Ca2+ translocase by menadione metabolism, appears to be critically involved in the development of cytotoxicity.     

Calcium metabolism in rat hepatocytes.

1. The total calcium concentration in rat hepatocytes was 7.9 microgram-atoms/g dry wt.; 77% of this was mitochondrial. Approx. 20% of cell calcium exchanged with 45Ca within 2 min. Thereafter incorporation proceeded at a low rate to reach 28% of total calcium after 60 min. Incorporation into mitochondria showed a similar time course and accounted for 20% of mitochondrial total calcium after 60 min. 2. The alpha-adrenergic agonists phenylephrine and adrenaline + propranolol stimulated incorporation of 45Ca into hepatocytes. Phenylephrine was shown to increase total calcium in hepatocytes. Phenylephrine inhibited efflux fo 45Ca from hepatocytes perifused with calcium-free medium. 3. Glucagon, dibutryl cyclic AMP and beta-adrenergic agonists adrenaline and 3-isobutyl-1-methyl-xanthine stimulated calcium efflux from hepatocytes perifused with calcium-free medium. The effect of glucagon was blocked by insulin. Insulin itself had no effect on calcium efflux and it did not affect the response to dibutyryl cyclic AMP. 4. Incorporation of 45Ca into mitochondria in hepatocytes was stimulated by phenylephrine and inhibited by glucagon and by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The effect of glucagon was blocked by insulin. 5. Ionophore A23187 stimulated hepatocyte uptake of 45Ca, uptake of 45Ca into mitochondria in hepatocytes and efflux of 45Ca into a calcium-free medium.     

Cyclosporin A modifies cytoplasmic calcium levels in isolated hepatocytes exposed to oxidative stress due to tert-butyl hydroperoxide

Within the framework of our studies on hypertension in various rat strains, we have examined the effect of cyclosporin A (CsA) on intracellular calcium signaling under conditions of oxidative stress. For these preliminary experiments, we have chosen isolated hepatocytes of normotensive rats as a model system for the study of the role of intracellular calcium. We used tert-butyl hydroperoxide (t-BHP, 1 mmol x l(-1)) as an prooxidant agent. When compared to the controls, we found increased levels of cytosolic free calcium concentration (Ca2+i) during 120 min incubation. The preincubation of hepatocytes with CsA in the concentration of 0.5 micromol x l(-1)] did not change the physiological level of cytosolic calcium. However, a dual action of CsA on elevated Ca2+i was observed during oxidative injury of hepatocytes: while in the first period of incubation CsA increased Ca2+i, CsA reduced the effect of t-BHP on Ca2+i during the next period of incubation. This indicates the ability of CsA to modify oxidative stress, but further studies are necessary to explain these findings.     

Development of sarcoplasmic reticulum in cultured chicken muscle.

The development of sarcoplasmic reticulum membranes was studied in vivo and in tissue culture in chicken pectoralis muscle cells. The concentration of the calcium- and magnesium-activated ATPase measured by selective labeling of the enzyme with [32P]ATP in whole muscle homogenates was found to increase in developing chicken pectoralis muscle in vivo from 0.01 nmol/mg of protein in 12-day embryos to 0.3 to 0.4 nmol/mg of protein in 1-month-old chicks, where it constitutes about 3% of the total protein content of muscle. In cultured muscle cells the concentration of calcium-sensitive phosphoprotein increased from 0.015 nmol/mg of protein at 2 days to 0.04 to 0.05 nmol/mg of protein after 5 days of culture. This amount represents about 0.5% of the protein content of the muscle cells. The accumulation of Ca2+ transport ATPase began during fusion and continued with a linear rate during 8 days of culture. The density of 75 A intramembranous particles seen by freeze-etch electron microscopy on fracture faces of sarcoplasmic reticulum membranes is about 4,000/mum2 in adult chick pectoralis muscle but only 400/mum2 in cultured muscle cells in rough proportion to the concentration of Ca2+-sensitive phosphoprotein. The Ca2+, Na+, and K+ concentration of the medium and addition of ouabain, caffeine, or the calcium ionophores A23187 and X537A sharply influence the concentration of calcium transport ATPase in cultured muscle cells, parallel with their effect upon cell fusion and growth. These observations are consistent with the proposition that the gene expression leading to the accumulation of Ca2+ transport ATPase during development in culture may be regulated by intracellular ion concentrations.