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.     

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.     

Suppression of Ca2+ oscillations in cultured rat hepatocytes by chemical hypoxia

The model of "chemical hypoxia" with KCN plus iodoacetic acid mimics the ATP depletion and reductive stress of hypoxia. Here, we examined the effects of chemical hypoxia on cytosolic free Na+ and Ca2+ in single cultured rat hepatocytes by multiparameter digitized video microscopy and ratio imaging of sodium-binding furan indicator (SBFI) and Fura-2. Intracellular Na+ increased from about 10 mM to more than 100 mM after 20 min of chemical hypoxia, whereas cytosolic free Ca2+ remained virtually unchanged. In normoxic hepatocytes, phenylephrine (50 microM) and Arg-vasopressin (20-40 nM) induced Ca2+ oscillations in 70 and 40% of cells, respectively. These Ca2+ oscillations were suppressed after one spike following the onset of chemical hypoxia. Phenylephrine and vasopressin also increased inositol phosphate formation by 22 and 147%, respectively. This effect was suppressed by KCN plus iodoacetate. Intracellular acidosis is characteristic of chemical hypoxia. Intracellular acidosis induced by 40 mM Na-acetate suppressed Ca2+ oscillations but did not inhibit hormone-induced inositol phosphate formation. Cytosolic alkalinization also suppressed Ca2+ oscillations. However, prevention of intracellular acidosis with monensin (10 microM) did not prevent suppression of Ca2+ oscillations during chemical hypoxia. Mitochondrial depolarization with uncoupler did not change free Ca2+ levels during chemical hypoxia, indicating that mitochondria do not regulate free Ca2+ during chemical hypoxia. From these results, we conclude: 1) chemical hypoxia does not block Na+ influx across the plasma membrane; 2) Chemical hypoxia inhibits hormone-stimulated Ca2+ flux pathways across cellular membranes by two different mechanisms: (a) by ATP depletion, which disrupts hormone-myo-inositol 1,4,5-triphosphate coupling, and (b) by intracellular acidosis, which inhibits myo-inositol 1,4,5-triphosphate-stimulated Ca2+ release from intracellular stores; 3) during ATP depletion by chemical hypoxia, mitochondria do not take up Ca2+ to maintain cytosolic free Ca2+ at low concentrations.     

Measurement of mitochondrial and non-mitochondrial Ca2+ in isolated intact hepatocytes: a critical re-evaluation of the use of mitochondrial inhibitors.

Isolated rat hepatocytes treated with mitochondrial inhibitors FCCP or antimycin A release discrete amounts of Ca2+ in a Ca(2+)-free extracellular medium as revealed by changes in the absorbance of the Ca2+ indicator arsenazo III. The process is completed in 2 min and the amount of Ca2+ released is not affected by the type of the mitochondrial poison employed. The subsequent treatment with the cation ionophore A23187 causes a further release of Ca2+ that does not appear related to the specificity of the previous treatment with FCCP or antimycin A. Both FCCP and antimycin A cause a progressive loss of cellular ATP associated with a decrease in the ATP/ADP ratio from 6 to 2-1.5. However, this decrease does not significantly prevent 45Ca2+ accumulation in isolated liver microsomes. Moreover, the decrease of the ATP/ADP ratio to 1, does not promote a significant release of 45Ca2+ from 45Ca(2+)-preloaded microsomes. Finally, experiments with Fura-2-loaded hepatocytes reveal that agents specifically releasing Ca2+ from non-mitochondrial stores (vasopressin and 2,5-di-tert-butyl-1-4-benzohydroquinone) are still able to increase the cytosolic Ca2+ concentration in FCCP-treated cells. Taken together, these findings demonstrate that, in freshly isolated hepatocytes, FCCP specifically releases Ca2+ from mitochondrial stores without significantly affecting active Ca2+ sequestration in other cellular pools. For these reasons, FCCP can be used to release and quantitate mitochondrial Ca2+ in liver cells.     

Stimulation by ATP of Inositol Trisphosphate Accumulation and Calcium Mobilization in Cultured Adrenal Chromaffin Cells

Effects of ATP on accumulation of inositol phosphates and Ca2+ mobilization were investigated in cultured bovine adrenal chromaffin cells. When the cells were stimulated with 30 microM ATP, a rapid and transient rise in intracellular Ca2+ concentration was observed. At the same time, ATP rapidly increased accumulation of inositol phosphates. The concentration-response curve for the ATP-induced Ca2+ mobilization was similar to that for inositol trisphosphate (IP3) accumulation. ATP exerted its maximal effects at 30 microM for either IP3 accumulation or Ca2+ mobilization. The order of the efficacy of the agonists for IP3 accumulation and Ca2+ mobilization at 100 microM was ATP greater than ADP greater than AMP approximately adenosine, AMP (100 microM) and adenosine (300 microM) failed to induce IP3 accumulation and Ca2+ mobilization. Although 100 microM GTP and 100 microM UTP also induced IP3 accumulation and Ca2+ mobilization, their efficacy was less than that of ATP. CTP (100 microM) induced a slight IP3 accumulation, but it did not induce Ca2+ mobilization. Nifedipine (10 microM), a Ca2+ channel antagonist, and theophylline (100 microM), a P1-purinergic receptor antagonist, failed to inhibit the ATP-induced IP3 accumulation and Ca2+ mobilization. The above two cellular responses induced by ATP were also observed in the Ca2+-depleted medium. ATP induced a rapid and transient accumulation of 1,4,5-IP3 (5s), followed by a slower accumulation of 1,3,4-IP3. These results suggest that ATP induces the formation of 1,4,5-IP3 through the P2-purinergic receptor and consequently promotes Ca2+ mobilization from intracellular storage sites in cultured adrenal chromaffin cells.     

Calcium stimulates ATP-Mg/Pi carrier activity in rat liver mitochondria

Adenine nucleotide transport over the carboxyatractyloside-insensitive ATP-Mg/Pi carrier was assayed in isolated rat liver mitochondria with the aim of investigating a possible regulatory role for Ca2+ on carrier activity. Net changes in the matrix adenine nucleotide content (ATP + ADP + AMP) occur when ATP-Mg exchanges for Pi over this carrier. The rates of net accumulation and net loss of adenine nucleotides were inhibited when free Ca2+ was chelated with EGTA and stimulated when buffered [Ca2+]free was increased from 1.0 to 4.0 microM. The unidirectional components of net change were similarly dependent on Ca2+; ATP influx and efflux were inhibited by EGTA in a concentration-dependent manner and stimulated by buffered free Ca2+ in the range 0.6-2.0 microM. For ATP influx, increasing the medium [Ca2+]free from 1.0 to 2.0 microM lowered the apparent Km for ATP from 4.44 to 2.44 mM with no effect on the apparent Vmax (3.55 and 3.76 nmol/min/mg with 1.0 and 2.0 microM [Ca2+]free, respectively). Stimulation of influx and efflux by [Ca2+]free was unaffected by either ruthenium red or the Ca2+ ionophore A23187. Calmodulin antagonists inhibited transport activity. In isolated hepatocytes, glucagon or vasopressin promoted an increased mitochondrial adenine nucleotide content. The effect of both hormones was blocked by EGTA, and for vasopressin, the effect was blocked also by neomycin. The results suggest that the increase in mitochondrial adenine nucleotide content that follows hormonal stimulation of hepatocytes is mediated by an increase in cytosolic [Ca2+]free that activates the ATP-Mg/Pi carrier.     

Role of a nonmitochondrial Ca2+ pool in the synergistic stimulation by cyclic AMP and vasopressin of Ca2+ uptake in isolated rat hepatocytes.

The subcellular distribution of 45Ca2+ accumulated by isolated rat hepatocytes exposed to dibutyryl cyclic AMP (dbcAMP) followed by vasopressin (Vp) was studied by means of a nondisruptive technique. When treated with dbcAMP followed by vasopressin, hepatocytes obtained from fed rats accumulated an amount of Ca2+ approximately fivefold higher than that attained under control conditions. Ca2+ released from the mitochondrial compartment by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) accounted for only a minor portion of the accumulated Ca2+. The largest portion was released by the Ca2+ ionophore A23187 and was attributable to a nonmitochondrial compartment. DbcAMP + Vp-treatment also caused a maximal stimulation of glucose production and a twofold increase in cellular glucose 6-phosphate levels. In hepatocytes obtained from fasted rats, dbcAMP + Vp-stimulated Ca2+ accumulation was lower, although with the same subcellular distribution, and was associated with a minimal glucose production. In the presence of gluconeogenetic substrates (lactate plus pyruvate) hepatocytes from fasted rats were comparable to cells isolated from fed animals. However, Ca2+ accumulation and glucose 6-phosphate production could be dissociated in the absence of dbcAMP, in the presence of lactate/pyruvate alone. Under this condition in fact Vp induced only a minimal accumulation of Ca2+ in hepatocytes isolated from fasted rats, although glucose production was markedly increased. Moreover, treatment of fed rat hepatocytes with 1 mM ATP caused a maximal activation of glycogenolysis, but only a moderate stimulation of cellular Ca2+ accumulation. In this case, sequestration of Ca2+ occurred mainly in the mitochondrial compartment. By contrast, the addition of ATP to dbcAMP-pretreated hepatocytes induced a large accumulation of Ca2+ in a nonmitochondrial pool. Additional experiments using the fluorescent Ca2+ indicator Fura-2 showed that dbcAMP pretreatment can enlarge and prolong the elevation of cytosolic free Ca2+ caused by Vp. A nonmitochondrial Ca2+ pool thus appears mainly responsible for the Ca2+ accumulation stimulated by dbcAMP and Vp in isolated hepatocytes, and cyclic AMP seems able to activate Ca2+ uptake in such a nonmitochondrial pool.     

Stimulation of inositol trisphosphate formation in hepatocytes by vasopressin, adrenaline and angiotensin II and its relationship to changes in cytosolic free Ca2+.

At maximally effective concentrations, vasopressin (10(-7) M) increased myo-inositol trisphosphate (IP3) in isolated rat hepatocytes by 100% at 3 s and 150% at 6 s, while adrenaline (epinephrine) (10(-5) M) produced a 17% increase at 3 s and a 30% increase at 6 s. These increases were maintained for at least 10 min. Both agents increased cytosolic free Ca2+ [( Ca2+]i) maximally by 5 s. Increases in IP3 were also observed with angiotensin II and ATP, but not with glucagon or platelet-activating factor. The dose-responses of vasopressin and adrenaline on phosphorylase and [Ca2+]i showed a close correspondence, whereas IP3 accumulation was 20-30-fold less sensitive. However, significant (20%) increases in IP3 could be observed with 10(-9) M-vasopressin and 10(-7) M-adrenaline, which induce near-maximal phosphorylase activation. Vasopressin-induced accumulation of IP3 was potentiated by 10mM-Li+, after a lag of approx. 1 min. However the rise in [Ca2+]i and phosphorylase activation were not potentiated at any time examined. Similar data were obtained with adrenaline as agonist. Lowering the extracellular Ca2+ to 30 microM or 250 microM did not affect the initial rise in [Ca2+]i with vasopressin but resulted in a rapid decline in [Ca2+]i. Brief chelation of extracellular Ca2+ for times up to 4 min also did not impair the rate or magnitude of the increase in [Ca2+]i or phosphorylase a induced by vasopressin. The following conclusions are drawn from these studies. IP3 is increased in rat hepatocytes by vasopressin, adrenaline, angiotensin II and ATP. The temporal relationships of its accumulation to the increases in [Ca2+]i and phosphorylase a are consistent with it playing a second message role. Influx of extracellular Ca2+ is not required for the initial rise in [Ca2+]i induced by these agonists, but is required for the maintenance of the elevated [Ca2+]i.     

Increases in cytosolic free Ca2+ induced by ATP, complement and beta-lipoprotein in mouse L fibroblasts

By means of Ca2+- and K+-selective microelectrodes, the changes in intracellular free Ca2+ and K+ were measured during the hyperpolarizing responses induced by ATP, complement and beta-lipoprotein in mouse fibroblastic L cells. The cytoplasmic Ca2+ concentration [( Ca]i) was about 0.4 microM in the resting state. The hyperpolarizing responses always coincided with a phasic increase in [Ca]i. ATP or beta-lipoprotein induced about a 2-fold rise in [Ca]i, and complement did up to 3-fold. Both the hyperpolarizing responses and [Ca]i increases were prevented by removal of external Ca2+ or by application of a Ca-channel blocker, nifedipine. Quinine, a Ca-activated K-channel inhibitor, suppressed the hyperpolarizing responses but not the [Ca]i increases. During the hyperpolarizing response, the intracellular free K+ concentration gradually decreased from about 120 to 110 mM. Thus, it is concluded that ATP, complement and beta-lipoprotein caused a transient elevation of cytoplasmic free Ca2+ due to Ca2+ influxes, thereby inducing electrical membrane responses through activation of Ca-dependent K-channels in the fibroblasts.     

Mechanisms of regulation of intracellular distribution of Ca2+ in adipose tissue

The kinetics of influx and efflux of 45Ca and its accumulation by the subcellular membranes of adipose tissue have been studied. The initial rate of Ca2+ efflux does not depend on the intracellular concentration of Na+ and K+. The rate of exchange between intracellular 45Ca and 40Ca of the incubation medium is independent on concentration of Na+ and K+ in the incubation mixture. This suggests the absence of Na,Ca-transmembrane exchange in the adipocytes. The changes in the ratio of intracellular concentration of Na+ and K+ by the factors inhibiting the activity ofNa,K-ATPase cause redistribution of Ca in the intracellular pools of the adipocytes. The lypolytic agents (adrenalin, adrenocorticotropic hormone, caffeine) but not dibutytyl-3' : 5'-AMP, accelerate Ca2+ efflux from the adipocytes. At physiological concentrations of ATP, succinate and Pi the highest Ca-accumulating activity is observed in adipose tissue mitochondria. The highest initial rate of Ca uptake, as in the case of contractile tissues, is detected in the endoplasmic reticulum membranes. In contrast to the plasma membranes and reticulum, in which the Ca-accumulating capacity is independent of ATP concentration up to 0.5 mM, the Ca-accumulating capacity of mitochondria decreases 8--9-fold with a reduction in ATP concentration from 4 down to 1 mM. The physiological significance of this phenomenon in the action mechanism of lipolytic agents, which reduce the ATP content in the adipocytes, is discussed.     

Silver ion triggers Ca2+ release from intracellular store sites in saponin-treated HL-60 cells

We examined the effect of silver ion on Ca2+ mobilization from intracellular stores in permeabilized HL-60 cells using a filtration method and 45Ca2+. In HL-60 cells preloaded with Ca2+ in the presence of ATP, micromolar concentrations of AgNO3 elicited marked Ca2+ release within 1 min. The AgNO3-induced Ca2+ release was not affected by the free Ca2+ concentration in the medium. Equivalent concentrations of AgNO3 inhibited energy-dependent Ca2+ uptake as well as oxalate-supported Ca2+ uptake. In passive Ca2+ release experiments when ATP was completely depleted in the solution, AgNO3 also triggered Ca2+ release. Sulfhydryl protecting agents such as 2-mercaptoethanol, dithiothreitol, and glutathione (reduced form) blocked the AgNO3-induced Ca2+ release. From these results, we conclude that the apparent Ca2+ release induced by AgNO3 is mainly due to inhibition of the Ca2+ pump with increased permeability for Ca2+ and partly due to a direct effect on the Ca2+ release channel, probably by modification of sulfhydryl groups on these proteins.     

Spermine. A regulator of mitochondrial calcium cycling

Steady-state free Ca2+ concentrations have been measured with a Ca2+ electrode using suspensions of isolated rat liver mitochondria or saponin-treated hepatocytes. Mitochondria, when incubated in the presence of Mg2+ and MgATP2-, maintain a steady-state pCa2+ (-log [Ca2+]) of approximately 6.1 (0.8 microM). Addition of spermine lowered this value to a pCa2+ of 6.6 (0.25 microM). Spermine was the most effective polyamine, giving half-maximal effects at 170 microM and maximal effects at 400 microM. With saponin-permeabilized hepatocytes, spermine addition similarly showed that the mitochondria buffered the steady-state medium-free Ca2+ at a level approximating the cytosolic free Ca2+ concentration of intact hepatocytes. The initial rate of Ca2+ uptake by the mitochondrial Ca2+ uniporter was investigated using Ca2+-depleted mitochondria incubated in the presence of succinate and 0.3 mM free Mg2+. Under control conditions, Ca2+ uptake was not observed at free Ca2+ concentrations below 0.5 microM. Spermine (350 microM) increased the rate of Ca2+ uptake at all Ca2+ concentrations below 4.5 microM, but at higher Ca2+ concentrations, it was inhibitory. Spermine also affected mitochondrial Ca2+ efflux by decreasing the apparent Km from 16 to 3.8 nmol of Ca2+/mg of mitochondrial protein with no change of Vmax. Experiments with 45Ca2+ confirmed that spermine increased mitochondrial Ca2+ cycling at 0.2 microM free Ca2+. Hepatic spermine contents are reported to be about 1 mumol/g, wet weight, suggesting that this polyamine may have an important physiological role in intracellular calcium homeostasis.     

Alterations in inositol phosphate production during oxidative stress in isolated hepatocytes

The level of inositol phosphates was measured in rat hepatocytes treated with 2-methyl-1,4-naphthoquinone (menadione) or tert-butyl hydroperoxide, which cause Ca2+ mobilization from intracellular stores and an increase in cytosolic free Ca2+ concentration. Although neither agent produced any apparent changes in the resting level of inositol phosphates, pretreatment of hepatocytes with either menadione or tert-butyl hydroperoxide, as well as with several sulfhydryl reagents, markedly inhibited the increase in inositol phosphates induced by both hormonal and nonhormonal stimuli. Addition of dithiothreitol to menadione- or tert-butyl hydroperoxide-treated hepatocytes reversed this inhibition and reestablished responsiveness to extracellular stimuli. Our findings suggest that the inhibition of the inositol phosphate response by menadione and tert-butyl hydroperoxide occurs through the modification of critical sulfhydryl group(s) and that the alterations in intracellular Ca2+ homeostasis occurring during the metabolism of menadione and tert-butyl hydroperoxide in hepatocytes are not mediated by inositol phosphates.     

Differential functional properties of Ca2+ stores in pulmonary arterial conduit and resistance myocytes

In smooth muscle cells, oscillations of intracellular Ca2+ concentration ([Ca2+]i) are controlled by inositol 1,4,5-trisphosphate (InsP3) and ryanodine (Ry) receptors on the sarcoplasmic reticulum (SR). Here we show that these Ca2+ oscillations are regulated differentially by InsP3 and Ry receptors in cells dispersed from the main trunk of the pulmonary artery (conduit myocytes) or from tertiary and quaternary arterial branches (resistance myocytes). Ry receptor antagonists inhibit either spontaneous or ATP-induced Ca2+ oscillations in resistance myocytes but they do not affect the oscillations in most conduit myocytes. In contrast, agents that inhibit InsP3 production or activation of InsP3 receptors do not alter the oscillations is resistance myocytes but block them in conduit myocytes. We have also examined the degree of overlap of Ry- and InsP3-sensitive stores in myocytes along the pulmonary arterial tree. In conduit myocytes, depletion of Ry-sensitive stores with repeated application of caffeine in the presence of Ry or in Ca2+ free solutions did not prevent the ATP-induced Ca2+ release from InsP3-dependent stores. However, responsiveness to ATP was completely abolished in resistance myocytes subjected to the same experimental protocol. Thus, InsP3- and Ry-dependent stores appear to be separated in conduit myocytes but joined in resistance myocytes. These data demonstrate for the first time differential properties of intracellular Ca2+ stores and receptors in myocytes distributed along the pulmonary arterial tree and help to explain the distinct functional responses of large and small pulmonary vessels to vasoactive agents.     

ATP-Evoked Ca2+ Mobilisation and Prostanoid Release from Astrocytes: P2-Purinergic Receptors Linked to Phosphoinositide Hydrolysis

Astrocyte cultures prelabelled with either [3H]inositol or 45Ca2+ were exposed to ATP and its hydrolysis products. ATP and ADP, but not AMP and adenosine, produced increases in the accumulation of intracellular 3H-labelled inositol phosphates (IP), efflux of 45Ca2+, and release of thromboxane A2 (TXA2). Whereas ATP-stimulated 3H-IP accumulation was unaffected, its ability to promote TXA2 release was markedly reduced by mepacrine, an inhibitor of phospholipase A2 (PLA2). ATP-evoked 3H-IP production was also spared following treatment with the cyclooxygenase inhibitor, indomethacin. We conclude that ATP-induced phosphoinositide (PPI) breakdown and 45 Ca2+ mobilisation occurred in parallel with, if not preceded, the release of TXA2. Following depletion of intracellular Ca2+ with a brief preexposure to ATP in the absence of extracellular Ca2+, the release of TXA2 in response to a subsequent ATP challenge was greatly reduced when compared with control. These results suggest that mobilisation of cytosolic Ca2+ may be the stimulus for PLA2 activation and, thus, TXA2 release. Stimulation of alpha 1-adrenoceptors also caused PPI breakdown and 45 Ca2+ efflux but not TXA2 release. The effects of ATP and noradrenaline (NA) on 3H-IP accumulation were additive, but their combined ability to increase 45Ca2+ efflux was not. Interestingly, in the presence of NA, ATP-stimulated TXA2 release was reduced. Our data provide evidence that functional P2-purinergic receptors are present on astrocytes and that ATP is the first physiologically relevant stimulus found to initiate prostanoid release from these cells.     

Release of calcium from the endoplasmic reticulum by bile acids in rat liver cells

The effects of four bile acids on cell Ca2+ were examined in suspensions of isolated rat hepatocytes. Taurolithocholate and lithocholate which inhibit bile secretion increased the cytosolic Ca2+ concentration (ED50, 25 microM), as measured by the fluorescent indicator quin2, and promoted a net loss of Ca2+ from the cells. This effect resulted from rapid mobilization of Ca2+ from an intracellular Ca2+ store. This store corresponds to the one that is permeabilized by the inositol (1,4,5)trisphosphate-dependent hormone vasopressin. However, taurolithocholate and lithocholate, unlike the hormone, did not induce a significant accumulation of inositol trisphosphate fraction in isolated hepatocytes. In addition, these agents did not alter the cell and the mitochondria membrane permeability to ions. When applied to saponin-permeabilized cells, taurolithocholate and lithocholate released Ca2+ (ED50, 20 microM) from an ATP-dependent, nonmitochondrial pool which is sensitive to inositol (1,4,5)trisphosphate. In contrast, the bile acids taurocholate and cholate, which increase bile secretion, had no effect on cell Ca2+ in intact hepatocytes or in saponin-permeabilized hepatocytes. It is suggested that taurolithocholate and lithocholate permeabilize the endoplasmic reticulum to Ca2+ and that the resulting permeabilization of this compartment may be involved in the inhibition of bile secretion in mammalian liver.     

Role of calcium homeostasis in gastric mucosal injury and protection.

Using a human gastric mucosal cell line, known as AGS cells, we determined the role that perturbations in intracellular Ca2+ concentration [Ca2+]i might play in cellular injury induced by various damaging agents. For deoxycholate (CD) and ethanol (EtOH) induced damage, a concentration related increase in [Ca2+]i was noted that preceded and closely paralleled the magnitude of injury. Thus, the higher the concentration of DC or EtOH, the more profound were the changes in [Ca2+]i and the resultant degree of cellular injury. Pretreatment with a low concentration of DC (50 microM; called a mild irritant) that was not damaging by itself attenuated injury induced by a damaging concentration (i.e. 250 microM) of DC, and appeared to elicit this protective action through mechanisms that resisted intracellular Ca2+ accumulation. Additional studies indicated that the mechanism of aspirin damage may be similar and that other protective agents such as prostaglandins and growth factors appear to mediate their protective properties through prevention of intracellular Ca2+ alterations. We propose that agents that prevent mucosal injury mediate this activity through a cellular response (involving active Ca2+ efflux) that subsequently provides a protective action by limiting the magnitude of intracellular Ca2+ accumulation.     

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.     

Modulation of Neuronal Signal Transduction Systems by Extracellular ATP

The secretion of ATP by stimulated nerves is well documented. Following repetitive stimulation, extracellular ATP at the synapse can accumulate to levels estimated to be well over 100 microM. The present study examined the effects of extracellular ATP in the concentration range of 0.1-1.0 mM on second-messenger-generating systems in cultured neural cells of the clones NG108-15 and N1E-115. Cells in a medium mimicking the physiological extracellular environment were used to measure 45Ca2+ uptake, changes in free intracellular Ca2+ levels by the probes aequorin and Quin-2, de novo generation of cyclic GMP and cyclic AMP from intracellular GTP and ATP pools prelabeled with [3H]guanosine and [3H]adenine, respectively, and phosphoinositide metabolism in cells preloaded with [3H]inositol and assayed in the presence of LiCl. Extracellular ATP induced a concentration-dependent increase of 45Ca2+ uptake by intact cells, which was additive with the uptake induced by K+ depolarization. The increased uptake involved elevation of intracellular free Ca2+ ions, evidenced by measuring aequorin and Quin-2 signals. At the same concentration range (0.1-1.0 mM), extracellular ATP induced an increase in [3H]cyclic GMP formation, and a decrease in prostaglandin E1-stimulated [3H]cyclic AMP generation. In addition, extracellular ATP (1 mM) caused a large (15-fold) increase in [3H]inositol phosphates accumulation, and this effect was blocked by including La3+ ions in the assay medium. In parallel experiments, we found in NG108-15 cells surface protein phosphorylation activity that had an apparent Km for extracellular ATP at the same concentration required to produce half-maximal effects on Ca2+ uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP stimulates inositol phosphates accumulation and calcium mobilization in a primary culture of rat aortic myocytes

The effects of extracellular ATP on phosphoinositide metabolism and intracellular Ca2+ concentration were studied in a primary culture of rat aortic myocytes. ATP increases the level of inositol phosphates, the putative second messenger for Ca2+ mobilization. No saturation of inositol phosphates accumulation is obtained (up to 10(-2) M ATP). Under the same conditions, ATP rapidly mobilizes intracellular Ca2+ in fura-2 loaded myocytes. The mobilization of intracellular Ca2+ is dose-dependent (maximal at 10(-4) M ATP), and is not affected by addition of EGTA. It is concluded that the receptors mediating the cytosolic increase of Ca2+ are of the P2-purinoceptor subtype. The physiological functions of these receptors are not presently known.