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 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.     

Initiation of cultured rat hepatocyte proliferation does not involve Na+-dependent plasma membrane Ca2+ fluxes

Adult rat hepatocytes in primary culture were examined to determine if Na+-dependent transmembrane Ca2+ fluxes precede reinitiation of DNA synthesis. Studies with 45Ca2+ and atomic absorption measurements of 40Ca2+ showed that hepatocytes lack plasma membrane Na+-Ca2+ exchange activity. Under chemically defined conditions, combinations of mitogens - EGF, insulin, and glucagon - failed to induce transmembrane Ca2+ fluxes early in the prereplicative phase. In addition, a Ca2+ ionophore, A23187, was non-mitogenic. Thus, plasma membrane Na+-Ca2+ exchange is not a mitogenic signal for hepatocytes. Elevated intracellular Ca2+ levels are thought to mediate early prereplicative events required for animal cell proliferation. These conclusions stem partly from findings that A23187, a Ca2+ ionophore, stimulates transmembrane Ca2+ fluxes and proliferation in several cell systems (reviewed in Boynton et al., 1982). Sodium ion fluxes also are implicated as "initiating" mitogenic signals (Koch and Leffert, 1979). In particular, amiloride-sensitive Na+ influxes, stimulated by growth factors, may be necessary to initiate DNA synthesis in rat hepatocytes, mouse and human fibroblasts, rat liver derived cell lines, mouse sympathetic neurons, human lymphocytes, and monkey kidney epithelial cells (reviewed in Leffert, 1982). Several investigators, using cells from electrically excitable tissues (Schellenberg and Swanson, 1981; Eckert and Grosse, 1982), have reported that plasma membrane Na+-Ca2+ exchange carriers regulate intracellular Na+ and Ca2+ concentration. It is unclear if this exchange system exists in non-electrically excitable membranes, especially with regard to hepatocytes (Judah and Ahmed, 1964; van Rossum, 1970). We have here investigated the possible association of Na+ influxes with transmembrane Ca2+ movement following reinitiation of hepatocyte growth.     

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.     

Cytochrome P450 may regulate plasma membrane Ca2+ permeability according to the filling state of the intracellular Ca2+ stores.

The filling state of the intracellular Ca2+ stores of rat thymocytes regulates plasma membrane permeability to Mn2+, used here as a Ca2+ surrogate for plasma membrane Ca2+ channels. Emptying of the Ca2+ stores accelerated Mn2+ entry about 10-fold, and refilling with Ca2+ restored low Mn2+ permeability. The acceleration of Mn2+ entry observed in cells with empty intracellular Ca2+ stores was prevented by cytochrome P450 inhibitors. Imidazole antimycotics, especially econazole and miconazole, were the most potent inhibitors (IC50 approximately equal to 10(-6) M). The inhibitor sensitivity profile was similar to IA-type cytochrome P450. Calmodulin antagonists increased the plasma membrane permeability to Mn2+ in cells with filled Ca2+ stores, and this effect was also blocked by imidazole antimycotics. On this basis, we propose a model in which activation of a cytochrome P450, situated at the Ca2+ stores, opens a plasma membrane Ca2+ pathway. This activity would be inhibited by Ca2+ inside the stores by a calmodulin-dependent mechanism.     

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.     

Pharmacological characterization of NAADP-induced Ca2+ signals in starfish oocytes

The recently discovered second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) is central to the onset of intracellular Ca2+ signals induced by several stimuli, including fertilization. The nature of the Ca2+ pool mobilized by NAADP is still controversial. Depending on the cell type, NAADP may target either an acidic compartment with lysosomal properties or ryanodine receptors (RyRs) on endoplasmic reticulum. In addition, NAADP elicits a robust Ca2+ influx into starfish oocytes by activating a Ca2+-mediated current across the plasma membrane. In the present study, we employed the single-electrode intracellular recording technique to assess the involvement of either acidic organelles or RyRs in NAADP-elicited Ca2+ entry. We found that neither drugs which interfere with acidic compartments nor inhibitors of RyRs affected NAADP-induced depolarization. These data further support the hypothesis that a yet unidentified plasma membrane Ca2+ channel is the target of NAADP in starfish oocytes.     

Effect of extracellular Ca2+ on plasma membrane Ca2+ inflow and cytoplasmic free Ca2+ in isolated hepatocytes

An initial rapid phase and a subsequent slow phase of 45Ca2+ uptake were observed following the addition of 45Ca2+ to Ca2+-deprived hepatocytes. The magnitude of the rapid phase increased 15-fold over the range 0.1-11 mM extracellular Ca2+ (Ca2+o) and was a linear function of [Ca2+]o. The increases in the rate of 45Ca2+ uptake were accompanied by only small increases in the intracellular free Ca2+ concentration. In cells made permeable to Ca2+ by treatment with saponin, the rate of 45Ca2+ uptake (measured at free Ca2+ concentrations equal to those in the cytoplasm of intact cells) increased as the concentration of saponin increased from 1.4 to 2.5 micrograms per mg wet weight cells. Rates of 45Ca2+ uptake by cells permeabilized with an optimal concentration of saponin were comparable with those of intact cells incubated at physiological [Ca2+o], but were substantially lower than those for intact cells incubated at high [Ca2+o]. It is concluded that Ca2+ which enters the hepatocyte across the plasma membrane is rapidly removed by binding and transport to intracellular sites and by the plasma membrane (Ca2+ + Mg2+)-ATPase and the plasma membrane Ca2+ inflow transporter is not readily saturated with Ca2+o.     

Stimulation of intracellular Ca2+ oscillations by diacylglycerol in human myometrial cells

Stimulation of G-protein coupled membrane receptors linked to phospholipase C results in production of the second messengers diacylglycerol and inositol-1,4,5-trisphosphate (IP3). IP3 releases Ca2+ from the endoplasmic reticulum, which triggers increased Ca2+ influx across the plasma membrane, so-called capacitative calcium entry. DAG can also activate plasma membrane calcium-permeable channels but the mechanism is still not fully understood. In the pregnant human myometrial cell line PHM1 and in primary myometrial cells, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analogue of diacylglycerol, induced variable oscillatory patterns of intracellular free Ca2+. Similar behavior was seen with Sr2+ entry. The Ca2+ oscillations were not blocked by a broad spectrum of protein kinase C inhibitors, including chelerytrine, bisindolylmaleimide I and calphostin C, and were enhanced and prolonged by RHC-80267, an inhibitor of diacylglycerol lipase. The OAG-induced oscillatory response was not dependent on Ca2+ release from the endoplasmic reticulum but required extracellular Ca2+. Our results indicate that diacylglycerol directly activates cation channels in PHM1 and primary myometrial cells and promotes intracellular Ca2+ oscillations by actions independent of intracellular Ca2+ -ATPase activity and protein kinase C involvement.     

Cyclopiazonic acid depletes intracellular Ca2+ stores and activates an influx pathway for divalent cations in HL-60 cells.

The filling state of intracellular Ca2+ stores has been proposed to regulate Ca2+ influx across the plasma membrane in a variety of tissues. To test this hypothesis, we have used three structurally unrelated inhibitors of the Ca(2+)-ATPase of intracellular Ca2+ stores and investigated their effect on Ca2+ homeostasis in HL-60 cells. Without increasing cellular inositol (1,4,5)trisphosphate levels, all three inhibitors (cyclopiazonic acid, thapsigargin, and 2,5-Di-tert-butylhydroquinone) released Ca2+ from intracellular stores, resulting in total depletion of agonist-sensitive Ca2+ stores. The Ca2+ release was relatively slow with a lag time of 5 s and a time to peak of 60 s. After a lag time of approximately 15 s, all three Ca(2+)-ATPase inhibitors activated a pathway for divalent cation influx across the plasma membrane. At a given concentration of an inhibitor, the plasma membrane permeability for divalent cations closely correlated with the extent of depletion of Ca2+ stores. The influx pathway activated by Ca(2+)-ATPase inhibitors conducted Ca2+, Mn2+, Co2+, Zn2+, and Ba2+ and was blocked, at similar concentrations, by La3+, Ni2+, Cd2+, as well as by the imidazole derivate SK&F 96365. The divalent cation influx in response to the chemotactic peptide fMLP had the same characteristics, suggesting a common pathway for Ca2+ entry. Our results support the idea that the filling state of intracellular Ca2+ stores regulates Ca2+ influx in HL-60 cells.     

The involvement of altered vesicle transport in redistribution of Ca2+, Mg2+-ATPase in cholestatic rat liver

Vectorial sorting of plasma membrane protein-containing vesicles is essential for the establishment and maintenance of cell polarity. In the present study, the involvement of altered vesicle transport in the redistribution of membrane-bound Ca2+, Mg2+-ATPase resulting from cholestasis was investigated in hepatocytes. Cholestasis was induced in rat liver by common bile duct ligation. Ca2+, Mg2+-ATPase activity was demonstrated histochemically at the light and electron microscopical levels. Microtubules, an important factor for transcellular transport of vesicles, were studied in situ by immunofluorescence microscopy and electron microscopy in detergent-extracted preparations. The results showed that microtubules underwent significant changes after common bile duct ligation. The most pronounced alteration was focal accumulation of -tubulin in the cytoplasm of hepato cytes after 7 days of common bile duct ligation. At the electron microscopical level, the number of microtubules was increased considerably. In control livers, the activity of Ca2+, Mg2+-ATPase was localized only at the apical plasma membrane of hepatocytes, but it was also present at the basolateral plasma membrane after common bile duct ligation. The number of intracellular vesicles containing Ca2+, Mg2+-ATPase activity was increased strikingly, and some of them were associated with lateral membrane domains in which Ca2+, Mg2+-ATPase activity was found. It is concluded that common bile duct ligation induces the rearrangement of microtubules, which may disturb vectorial transport of Ca2+, Mg2+-ATPase-containing vesicles in hepatocytes, leading to the redistribution of Ca2+, Mg2+-ATPase. © 1998 Chapman & Hall     

Phospholipase C-gamma1 is required for the activation of store-operated Ca2+ channels in liver cells

Repetitive hormone-induced changes in concentration of free cytoplasmic Ca2+ in hepatocytes require Ca2+ entry through receptor-activated Ca2+ channels and SOCs (store-operated Ca2+ channels). SOCs are activated by a decrease in Ca2+ concentration in the intracellular Ca2+ stores, but the molecular components and mechanisms are not well understood. Some studies with other cell types suggest that PLC-gamma (phospholipase C-gamma) is involved in the activation of receptor-activated Ca2+ channels and/or SOCs, independently of PLC-gamma-mediated generation of IP3 (inositol 1,4,5-trisphosphate). The nature of the Ca2+ channels regulated by PLC-gamma has not been defined clearly. The aim of the present study was to determine if PLC-gamma is required for the activation of SOCs in liver cells. Transfection of H4IIE cells derived from rat hepatocytes with siRNA (short interfering RNA) targeted to PLC-gamma1 caused a reduction (by approx. 70%) in the PLC-gamma1 protein expression, with maximal effect at 72-96 h. This was associated with a decrease (by approx. 60%) in the amplitude of the I(SOC) (store-operated Ca2+ current) developed in response to intracellular perfusion with either IP(3) or thapsigargin. Knockdown of STIM1 (stromal interaction molecule type 1) by siRNA also resulted in a significant reduction (approx. 80% at 72 h post-transfection) of the I(SOC) amplitude. Immunoprecipitation of PLC-gamma1 and STIM1, however, suggested that under the experimental conditions these proteins do not interact with each other. It is concluded that the PLC-gamma1 protein, independently of IP3 generation and STIM1, is required to couple endoplasmic reticulum Ca2+ release to the activation of SOCs in the plasma membrane of H4IIE liver cells.     

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.     

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.     

Isolation and characterization of a plasma membrane calcium pump from Dictyostelium discoideum.

During the aggregation and differentiation of amoebae of Dictyostelium discoideum, changes in free cytosolic Ca2+ appear to regulate a number of physiological processes. To understand the mechanisms regulating free intracellular Ca2+ in this organism, we have isolated and characterized an ATP/Mg2+-dependent, high-affinity Ca2+ pump. When homogenates of 2 h starved cells were fractionated on Percoll/KCl gradients, one peak of high-affinity Ca2+-pumping activity was detected. This activity was resolved from enzyme markers of the mitochondrion and the rough endoplasmic reticulum but it cosedimented with the plasma membrane marker, alkaline phosphatase. Further studies suggested that the pump was associated with 'inside-out' plasma membrane vesicles. Like plasma membrane Ca2+-transport ATPases from other systems, this isolated Ca2+ pump: (1) was Mg2+-dependent, (2) displayed a high specificity for ATP as an energy source, (3) exhibited a high affinity for free Ca2+ with a Km of 0.3 microM, and (4) was very sensitive to inhibition by vanadate (IC50 2 microM) but was unaffected by mitochondrial inhibitors, ouabain and Ca2+-channel blockers. Unlike plasma membrane Ca2+ pumps from most other systems, this enzyme appeared not to be regulated by calmodulin. During development, non-mitochondrial, vanadate-sensitive, high-affinity Ca2+-pumping activity in crude lysates remained relatively constant for at least 15 h. These observations suggest that this plasma membrane Ca2+ pump probably functions in Dictyostelium to maintain Ca2+ homeostasis by extruding free cytosolic Ca2+ from the cells.     

Susceptibility of lysosomes to rupture is a determinant for plasma membrane disruption in tumor necrosis factor alpha-induced cell death

Since a release of intracellular contents can induce local inflammatory responses, mechanisms that lead to loss of plasma membrane integrity in cell death are important to know. We showed previously that deficiency of the plasma membrane Ca2+ ATPase 4 (PMCA4) in L929 cells impaired tumor necrosis factor alpha (TNF-alpha)-induced enlargement of lysosomes and reduced cell death. The lysosomal changes can be determined by measuring the total volume of intracellular acidic compartments per cell (VAC), and we show here that inhibition of the increase in VAC due to PMCA4 deficiency not only reduced cell death but also converted TNF-alpha-induced cell death from a process involving disruption of the plasma membrane to a cell demise with a nearly intact plasma membrane. The importance of the size of lysosomes in determining plasma membrane integrity during cell death was supported by the observations that chemical inhibitors that reduce VAC also reduced the plasma membrane disruption induced by TNF-alpha in wild-type L929 cells, while increases in VAC due to genetic mutation, senescence, cell culture conditions, and chemical inhibitors all changed the morphology of cell death from one with an originally nearly intact plasma membrane to one with membrane disruption in a number of different cells. Moreover, the ATP depletion-mediated change from apoptosis to necrosis is also associated with the increases of VAC. The increase in lysosomal size may due to intracellular self-digestion of dying cells. Big lysosomes are easy to rupture, and the release of hydrolytic enzymes from ruptured lysosomes can cause plasma membrane disruption.     

Cytosolic free Ca2+ level in isolated hepatocytes: the effect of insulin

Cytosolic free Ca2+ level was estimated in rat hepatocytes using the method described by Murphy et al. (1980). For control hepatocytes, a value of 0.20 +/- 0.06 mumol/l was obtained. Insulin increased cytosolic free Ca2+ level to 0.63 +2- 0.24 mumol/l. No net fluxes of Ca2+ across the plasma membrane were observed during incubation of hepatocytes with insulin. Mitochondria were shown to be the main Ca2+ buffering system. FCCP released 77-88% of releasable calcium from the cell. Dibucaine increased cytosolic free Ca2+ level to 1.16 mumol/l.     

Potentiation of oxidative cell injury in hepatocytes which have accumulated Ca2+

Incubation of freshly isolated rat hepatocytes with exogenous ATP, but not with succinate, resulted in intracellular Ca2+ accumulation which was partly prevented when the inhibitor of mitochondrial Ca2+ sequestration, ruthenium red, was also present in the medium. Although the bulk of the accumulated Ca2+ was sequestered by the mitochondria, formation of surface blebs and stimulation of phosphorylase alpha activity during incubation of the hepatocytes with ATP indicate that this treatment was also associated with an increase in cytosolic free Ca2+ concentration. When hepatocytes loaded with Ca2+ by preincubation with ATP were exposed to either 2-methyl-1,4-naphthoquinone or t-butyl hydroperoxide, the cytotoxicity of both agents was markedly potentiated. Our results suggest that ATP-induced Ca2+ accumulation in hepatocytes is not due to contamination of the cell suspension with damaged cells or free intracellular organelles and that the intracellular Ca2+ concentration can affect the response to toxic agents.     

Control mechanisms governing the infectivity of Chlamydia trachomatis for HeLa cells: the role of calmodulin

Adhesion of the obligate intracellular bacterium Chlamydia trachomatis to host cells is associated with a flux of Ca2+ across the cell membrane, and infection is enhanced by treatment of host cells with Ca2+ ionophore. The possibility that Ca2+ might interact with host cell Ca2+ regulatory proteins to promote chlamydial infection was investigated. Treatment of HeLa 229 cells with the calmodulin inhibitors pimozide, trifluoperazine, chlorpromazine, promethazine or haloperidol reduced chlamydial infectivity as measured by inclusion counting or the specific incorporation of [3H]threonine. The inhibitory effect was reversible, dose-related and shown to be associated with impairment of chlamydial adhesion and uptake by the host cells. This effect was clearly distinguished from the delayed maturation of chlamydiae due to continuous exposure to calmodulin inhibitors which may result from a decrease in the availability of high energy compounds from the host cells necessary for chlamydial growth. The possible mechanisms for calmodulin-mediated chlamydial endocytosis are discussed.     

Effects of lysophospholipids on Ca2+ transport in rat liver mitochondria incubated at physiological Ca2+ concentrations in the presence of Mg2+, phosphate and ATP at 37 degrees C.

Lysophospholipids caused the release of 45Ca2+ from isolated rat liver mitochondria incubated at 37 degrees C in the presence of low concentrations of free Ca2+, ATP, Mg2+, and phosphate ions. The concentrations of lysophosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidic acid and lysophosphatidylinositol which gave half-maximal effects were 5, 26, 40 and 56 microM, respectively. The effects of lysophosphatidylethanolamine were not associated with a significant impairment of the integrity of the mitochondria as monitored by measurement of membrane potential and the rate of respiration. Lysophosphatidylethanolamine did not induce the release of Ca2+ from a microsomal fraction, or enhance Ca2+ inflow across the plasma membrane of intact cells, but did release Ca2+ from an homogenate prepared from isolated hepatocytes and incubated under the same conditions as isolated mitochondria. The proportion of mitochondrial 45Ca2+ released by lysophosphatidylethanolamine was not markedly affected by altering the total amount of Ca2+ in the mitochondria, the concentration of extramitochondrial Mg2+, by the addition of Ruthenium Red, or when oleoyl lysophosphatidylethanolamine was employed instead of the palmitoyl derivative. The effects of 5 microM-lysophosphatidylethanolamine were reversed by washing the mitochondria. The possibility that lysophosphatidylethanolamine acts to release Ca2+ from mitochondria in intact hepatocytes following the binding of Ca2+-dependent hormones to the plasma membrane is briefly discussed.