Decreased level of calpain inhibitor activity in red blood cells from Milan hypertensive rats

In mature red cells of rats from Milan Normal (MNS) and Hypertensive Strains (MHS), the soluble Ca2+-dependent neutral proteinase (calpain) is present in similar amounts as the form requiring 0.1-0.2 mM Ca2+ for maximum catalytic activity. The amount of the endogenous calpain inhibitor, however, differs greatly in the red cells of the two strains. In red cells from hypertensive rats the activity of the inhibitor is 10 times less with a ratio of inhibitor to calpain activity (unit/unit) of 0.2; compared to red cells from normal rats, in which this ratio is approximately 2. This is the first demonstration of the existence, in a mammalian cell, of such a low ratio of calpain to inhibitor and implies the occurrence of a potentially "unregulated" intracellular soluble proteinase. This abnormal condition may be responsible for some of the structural and metabolic changes reported in rats of the genetically determined MHS strain.     

Activation of the Ca2+-ATPase of human erythrocyte membrane by an endogenous Ca2+-dependent neutral protease

Limited proteolysis of the plasma membrane calcium transport ATPase (Ca2+-ATPase) from human erythrocytes by trypsin produces a calmodulin-like activation of its ATP hydrolytic activity and abolishes its calmodulin sensitivity. We now demonstrate a similar kind of activation of the human erythrocyte membrane Ca2+-ATPase by calpain (calcium-dependent neutral protease) isolated from the human red cell cytosol. Upon incubation of red blood cell membranes with purified calpain in the presence of Ca2+ the membrane-bound Ca2+-ATPase activity was increased and its sensitivity to calmodulin was lost. In contrast to the action of other proteases tested, proteolysis by calpain favors activation over inactivation of the Ca2+-ATPase activity, except at calpain concentrations more than 2 orders of magnitude higher. Exogenous calmodulin protects the Ca2+-ATPase against calpain-mediated activation at concentrations which also activate the Ca2+-ATPase activity. Calcium-dependent proteolytic modification of the Ca2+-ATPase could provide a mechanism for the irreversible activation of the membrane-bound enzyme.     

Activation of intracellular calcium-activated neutral proteinase in erythrocytes and its inhibition by exogenously added inhibitors

Intracellular calcium-activated neutral proteinase (CANP) in rabbit erythrocytes was activated by an influx of Ca2+ into the cells. The catalytic large subunit changed from the original 79 kDa from to the 77 kDa and 76 kDa forms on activation just in the same manner as occurs in the autolytic activation of purified CANP in vitro. The activation required both extracellular Ca2+ and A23187, and was accompanied by the degradation of some membrane proteins and morphological changes in erythrocyte shape from discocytes to echinodisks, echinocytes, and spherocytes. Exogenously added Cbz-Leu-Leu-Leu-aldehyde inhibited the activation of intracellular CANP as well as the degradation of membrane proteins and the morphological changes indicating that the latter two processes are due to the action of CANP. Leupeptin and E64d were without effect on intracellular CANP.     

Inactivation of c-Yes tyrosine kinase by elevation of intracellular calcium levels.

We have previously shown that the c-Src tyrosine kinase is activated four- to fivefold when cultured keratinocytes differentiate following the elevation of intracellular calcium levels. In contrast to c-Src, another Src family tyrosine kinase, c-Yes, was rapidly inactivated in these same cells, despite its marked similarity in structure and enzymatic activity to c-Src. The inactivation of c-Yes was independent of the protein kinase C pathway, which is usually activated by elevation of intracellular calcium levels. The protein levels of c-Src and c-Yes were not altered, but the phosphotyrosine content of both proteins was greatly reduced. As has been demonstrated for c-Src, in vitro dephosphorylation of c-Yes by incubation with protein tyrosine phosphatases also resulted in its activation, not inactivation. In vitro reconstitution experiments showed that c-Yes can be inactivated by preincubation with a Ca(2+)-supplemented cell extract and that this inhibition was reversed by the addition of EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. Gradient sedimentation of cell lysates showed that in cells treated with calcium and ionophore, c-Yes formed complexes with two distinct cellular proteins, whereas similar complexes were not seen in c-Src immunoprecipitates. One of these two proteins has the ability to inhibit c-Yes kinase activity in vitro. Finally, the Ca(2+)-dependent inactivation of c-Yes was observed in kidney tubular cells and fibroblasts, suggesting that the Ca(2+)-dependent regulation of c-Yes tyrosine kinase is not unique to keratinocytes. We postulate that c-Yes is inactivated through a Ca2+ -dependent association with cellular proteins, which seems to override its activation resulting from tyrosine dephosphorylation.     

The role of Ca2+-dependent biochemical changes in the ageing process in normal red cells and in the development of irreversibly sickled cells

During the ageing process of normal red cells and in the formation of irreversibly sickled cells (ISCs) there is a progressive increase in the intracellular concentration of Ca2+. This is parallelled by the development of a variety of morphological and biochemical changes in older fractions of normal cells and in ISCs which are similar to those seen in normal cells exposed to Ca2+ ionophore. These changes include cell shrinkage, loss of membrane lipid and degradation of cytoskeletal proteins and polyphosphoinositides. In this paper we consider the ways in which the Ca2+-dependent biochemical changes may be related to the morphological alterations which are characteristic of ageing and irreversible sickling.     

Different susceptibility of red cell membrane proteins to calpain degradation.

The presence of low levels of calpastatin activity in erythrocytes of hypertensive rats affects regulation of calpain activity so it is highly susceptible to activation within physiological fluctuations in [Ca2+]. Under identical conditions, in red cells of normotensive rats, calpain activation is efficiently controlled by the high levels of calpastatin activity, and a progressive increase in proteinase activity can only be observed in parallel with a decrease in the level of calpastatin. In intact erythrocytes from hypertensive rats exposed to small variations in [Ca2+], degradation of anion transport protein (band 3) and Ca(2+)-ATPase appears as a primary event indicating that these two transmembrane proteins are probably early recognized as targets of intracellular calpain activity. Furthermore, band 3 protein seems to be structurally modified in erythrocytes from hypertensive rats, as indicated by its increased susceptibility to degradation in the presence of 10-50 microM Ca2+. In addition, when exposed to progressive and limited increases in [Ca2+], erythrocytes from hypertensive rats, but not those from normotensive rats, show a high degree of fragility that can be restored to normal values by inhibition of calpain. These results indicate that, within fluctuations in [Ca2+] close to physiological values, regulation of calpain activity is efficiently accomplished in normal erythrocytes but is completely lost in cells from hypertensive animals. Regulation is of critical importance in maintaining normal structural and functional properties of selective red cell membrane and cytoskeletal proteins, among which band 3 and Ca(2+)-ATPase appear to be the substrates with highest susceptibility to digestion by calpain.     

Oxidative inactivation of the calcium-stimulated neutral proteinase from human red blood cells by divicine and intracellular protection by reduced glutathione

Calpain, the micromolar Ca2+-requiring form of Ca2+-stimulated neutral proteinase purified from human red cells, is remarkably inactivated during autoxidation of divicine (2,6-diamino-4,5-dihydroxypyrimidine), an aglycone implicated in the pathogenesis of favism. Inactivation of purified calpain is produced, in decreasing order of efficiency, by transient, probably semiquinonic species arising from autoxidation of divicine, by the H2O2 that is formed upon autoxidation itself, and by quinonic divicine, respectively. Purified procalpain, the millimolar Ca2+-requiring form that can be converted to the fully active calpain form by a variety of mechanisms, is less susceptible than calpain itself to inactivation by the same by-products of divicine autoxidation. When intact red cells are exposed to autoxidizing divicine, procalpain undergoes a significant loss of activity. At 1 mM divicine, intracellular inactivation is observed with procalpain only, while the activity of a number of red cell enzymes is unaffected. Inactivation of procalpain is consistently greater in red cells from glucose-6-phosphate dehydrogenase-deficient subjects than in normal cells. Restoration of normal levels of glucose-6-phosphate dehydrogenase activity by means of entrapment of homogeneous human glucose-6-phosphate dehydrogenase in the deficient red cells results in normal stability of intracellular reduced glutathione; decreased susceptibility of procalpain to inactivation by autoxidizing divicine. These findings suggest that in the glucose-6-phosphate dehydrogenase-deficient red cells the procalpain-calpain system is a major target of divicine cytotoxicity.     

The calmodulin-stimulated (Ca2+ + Mg2+)-ATPase in hemoglobin S erythrocyte membranes: effects of sickling and oxidative agents

A decrease in the reactivity of erythrocyte membrane (Ca2+ + Mg2+)-ATPase to calmodulin stimulation has been observed in aging red cells and in various types of hemolytic anemias, particularly in sickle red cell membranes. Unlike the aging process, the defect in the (Ca2+ + Mg2+)-ATPase from SS red blood cells is not secondary to a decrease in calmodulin activity and is already present in the least dense SS red blood cells separated on a discontinuous density gradient. Deoxygenated AS red cells were forced to sickle by lowering the pH, raising the osmolarity of the buffer (sickling pulse). Under these conditions an inhibition of the calmodulin-stimulated enzyme was observed only if several cycles of oxygenation/deoxygenation were applied. No alteration of the enzyme could be detected after submitting AS red blood cells to other conditions or in AA red blood cells submitted to the same treatments. This suggests that oxidative processes are involved in the alterations of the (Ca2+ + Mg2+)-ATPase activity. Treatment of membranes from AA erythrocytes by thiol group reagents and malondialdehyde, a by-product of auto-oxidation of membrane unsaturated lipids and a cross-linking agent of cytoskeletal proteins, led to a partial inhibition of the calmodulin-stimulated (Ca2+ + Mg2+)-ATPase. We postulate that the hyperproduction of free radicals described in the SS red blood cells and involved in the destabilization of the membrane may be also responsible for the (Ca2+ + Mg2+)-ATPase failure.     

Ca2+-dependent neutral proteinase from human erythrocytes: activation by Ca2+ ions and substrate and regulation by the endogenous inhibitor

Ca2+-dependent neutral proteinase purifies from human erythrocytes as an inactive proenzyme, that can be converted in an active low Ca2+ requiring form either by high concentrations of Ca2+ (0.1-1 mM) in the absence of the substrate, or by low concentrations of Ca2+ (1-5 microM) in the presence of digestible substrates. Activation requires dissociation to constituent inactive proenzyme subunits which are then converted to the active proteinase species still retaining their monomeric structure. The activation process produced by high Ca2+ concentrations is controlled by the endogenous inhibitor which also dissociates into constituent subunits in order to exert its inhibitory effect. An additional regulation of the activated proteinase involves an autoproteolytic process, Ca2+ and substrate dependent, producing enzyme inactivation.     

Activation of Ca(2+)-dependent currents in cultured rat dorsal root ganglion neurones by a sperm factor and cyclic ADP-ribose.

The effects of intracellular application of two novel Ca2+ releasing agents have been studied in cultured rat dorsal root ganglion (DRG) neurones by monitoring Ca(2+)-dependent currents as a physiological index of raised free cytosolic Ca2+ ([Ca2+]i). A protein based sperm factor (SF) extracted from mammalian sperm, has been found to trigger Ca2+ oscillations and to sensitize unfertilized mammalian eggs to calcium induced calcium release (CICR). In this study intracellular application of SF activated Ca(2+)-dependent currents in approximately two-thirds of DRG neurones. The SF induced activity was abolished by heat treatment, attenuated by increasing the intracellular Ca2+ buffering capacity of the cells and persisted when extracellular Ca2+ was replaced by Ba2+. In addition, activity could be triggered or potentiated by loading the cells with Ca2+ by activating a series of voltage-gated Ca2+ currents. Ca(2+)-activated inward current activity was also generated by intracellular application of cyclic ADP-ribose (cADPR), a metabolite of NAD+, which causes Ca2+ release in sea urchin eggs. This activity could also be enhanced by loading the cells with Ca2+. The cADPR induced activity, but not the SF induced activity, was abolished by depleting the caffeine sensitive Ca2+ store. Ruthenium red markedly attenuated SF induced activity but had little action on cADPR induced activity or caffeine induced activity. Our results indicate that both SF and cADPR release intracellular Ca2+ pools in DRG neurones and that they appear to act on subtly distinct stores or distinct intracellular Ca2+ release mechanisms, possibly by modulating CICR.     

Ca2+-activated Na+ fluxes in human red cells. Amiloride sensitivity

The effect of Ca2+ on the ouabain- and bumetanide-resistant Na+ fluxes in intact red cells was studied at relatively constant internal Ca2+, membrane potential, and cell volume. The red cell calcium concentration was modified using the ionophore A23187. In fresh red cells, the Na+ influx and efflux (1.2 +/- 0.13 and 0.26 +/- 0.07 mmol/liter cells x h, respectively) were not affected by amiloride (1 mM). When external Ca2+ was raised from 0 to 150 microM, in the presence of A23187, both the Na+ influx and efflux were stimulated (about 3.5-fold). The Ca2+-activated Na+ efflux and influx had an apparent Km for activation by Ca2+o of about 25 microM. The Ca2+-dependent Na+ transport was inhibited 30-60% by amiloride (ID50 = 17.3 +/- 8 microM). Amiloride, however, had no effect on the Ca2+-dependent K+ influx. The amiloride-sensitive (AS) transport pathway was a linear function of the Na+o concentration in the range from 0 to 75 mM. The Ca2+i activation seems to depend on the metabolic integrity of red cells. 1) It does not take place in ATP-depleted red cells; 2) ATP-repletion of ATP-depleted red cells fully restored AS Na influx; and 3) ATP-enrichment (ATP-red cells) enhanced the AS Na influx by about 100%. The Ca2+-activated AS Na+ influx was not affected by either DIDS or trifluoperazine. The present results indicate that in human erythrocytes an increase in internal Ca2+ activates on otherwise silent AS Na+-transport system, which is dependent on the metabolic integrity of the red cells.     

Isozymes of protein kinase C in human neutrophils and their modification by two endogenous proteinases

Two major protein kinase C (PKC) isozymes, accounting for approximately 95% of the total activity in human neutrophils, were separated by hydroxyapatite chromatography and were identified as beta-PKC (60% of the total) and alpha-PKC (35% of the total). No gamma-PKC was detected. A minor Ca2+/phospholipid requiring kinase that eluted from hydroxyapatite after alpha-PKC did not react significantly with any of the specific antisera employed for identification. Modification of beta-PKC or the minor PKC isozyme by calpain yielded Ca2+/phospholipid-independent forms (PKM) that retained only 50% of the original activities. In contrast, PKM formed from alpha-PKC retained full catalytic activity. For each native isozyme the rate of conversion by calpain was accelerated in the presence of Ca2+ and the lipid effectors, and the PKM form generated in each case was resistant to further digestion by calpain. All three PKC isozymes were also modified by a neutral serine proteinase isolated from human neutrophils, with this proteinase the major effect being loss of kinase activity, via a transient production of a Ca2+/phospholipid-independent form. This neutral serine proteinase appears to be localized at sites of interaction of cytoskeletal proteins with the cell membrane. Following stimulation of intact neutrophils with phorbol 12-myristate 13-acetate complete loss of native cytosolic kinase activity was observed, with recovery of approximately 30% of the original activity as a cytosolic Ca+/phospholipid independent form, presumably PKM. Loss of native PKC activity was greatest for the beta-isozyme. In cells stimulated by fMet-Leu-Phe approximately 60% of the original PKC activity was recovered as native cytosolic PKC and 30% as cytosolic PKM. Inhibitors of calpain reduced the extent of down-regulation of PKC, increased the proportion of PKC that remained associated with the plasma membrane and significantly reduced the proteolytically generated fully active PKM. Taken together, the in vitro and in vivo results suggest that calpain is involved primarily in the conversion of the PKC isozymes to the irreversibly activated PKM forms, and that the neutral serine proteinase may be the enzyme responsible for down-regulation, possibly via PKM as an intermediate.     

ATPase and phosphatase activities from human red cell membranes: II. The effects of phospholipases on Ca2+-dependent enzymic activities.

Treatment of human red cell membranes with pure phospholipase A2 results in a progressive inactivation of both Ca2+-dependent and (Ca2+ + K+)-dependent ATPase and phosphatase activities. When phospholipase C replaces phospholipase A2, Ca2+-dependent ATPase activity and Ca2+-dependent phosphorylation of red cell membranes are lost, while Ca2+-dependent phosphatase activity is enhanced and its apparent affinity for Ca2+ is increased about 20-fold. Activation of Ca2+-dependent phosphatase following phospholipase C treatment was not observed in sarcoplasmic reticulum preparation. Phospholipase C increases the sensitivity of the phosphatase to N-ethylmaleimide but has little effect on the kinetic parameters relating the phosphatase activity to substrate and cofactors, suggesting that no extensive structural disarrangement of the Ca2+-ATPase system has occurred after incubation with phospholipase C.     

Purification of the Ca2+-dependent proteinase inhibitor from bovine cardiac muscle and its interaction with the millimolar Ca2+-dependent proteinase

A protein inhibitor of the Ca2+-dependent proteinase has been purified from bovine cardiac muscle by using the following steps in succession: salting out 17,600 X gmax supernatants from muscle homogenates in 50 mM Tris acetate, pH 7.5, 4 mM EDTA between 25 and 65% ammonium sulfate saturation; eluting between 25 and 120 mM KCl from a DEAE-cellulose column at pH 7.5; salting out between 30 and 60% ammonium sulfate saturation; Ultrogel-22 gel permeation chromatography at pH 7.5; heating to 80 degrees C followed by immediate cooling to 0 degree C; 6% agarose gel permeation chromatography in 4 M urea, pH 7.5; and elution from a phenyl-Sepharose hydrophobic column between 0.7 and 0.5 M ammonium sulfate. Approximately 1.16-1.69 mg of purified Ca2+-dependent proteinase inhibitor are obtained from 1 kg of bovine cardiac muscle, fresh weight. Bovine cardiac Ca2+-dependent proteinase inhibitor has an Mr of 115,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a pI of 4.85-4.95, very little alpha-helical structure, a very low specific absorbance of 1.647 (A1% 280), and very low contents of histidine, tryptophan, phenylalanine, and tyrosine. Bovine cardiac Ca2+-dependent proteinase inhibitor probably contains a single polypeptide chain in nondenaturing solvents. One 115-kDa inhibitor polypeptide inactivates 10 110-kDa millimolar Ca2+-requiring proteinase (millimolar Ca2+-dependent proteinase) molecules in assays of purified proteins. Inhibition of millimolar proteinase by the proteinase inhibitor did not change in the pH range 6.2-8.6. The inhibitor requires Ca2+ to bind to millimolar Ca2+-dependent proteinase. The Ca2+ concentration required for one-half-maximum binding of millimolar Ca2+-dependent proteinase to the inhibitor was 0.53 mM, compared with a Ca2+ concentration of 0.92 mM required for one-half maximum activity of millimolar Ca2+-dependent proteinase in the absence of the proteinase inhibitor. Unless millimolar Ca2+-dependent proteinase is located subcellularly in a different place than the proteinase inhibitor or unless the proteinase/inhibitor interaction is regulated, millimolar proteinase could never be active in situ.     

The reversible activation by Mn2+ ions of the Ca2+-requiring neutral proteinase of human erythrocytes

Mn2+ (50 microM) satisfies the requirement for activity of the purified Ca2+-dependent neutral proteinase from human erythrocytes. Unlike the activation by Ca2+ [E. Melloni et al. (1984) Biochem. Int. 8, 477-489], the effect of Mn2+ is fully reversible and does not involve autodigestion of the native 80-kDa catalytic subunit. However, the native dimeric proenzyme (procalpain), which contains both the 80-kDa subunit and a smaller 30-kDa subunit, is not activated by Mn2+ alone but also requires the presence of micromolar concentrations of Ca2+. Under these conditions, 40% of the maximum activity is expressed without dissociation of the 80- and 30-kDa subunits. Mn2+, but not micromolar Ca2+, can also partially satisfy the metal requirement of the native 80-kDa subunit isolated after dissociation of the heterodimer. This activity is further enhanced by the addition of 5 microM Ca2+, which is ineffective in the absence of Mn2+. After procalpain is converted to active calpain by incubation with Ca2+ and substrate [S. Pontremoli et al. (1984) Biochem. Biophys. Res. Commun. 123, 331-337] full activity is observed with 5 microM Mn2+, which now substitutes completely for Ca2+. Activation of procalpain by Mn2+ represents a new mechanism for modulation of the Ca2+-dependent proteinase activity.     

High concentration of neutral endopeptidase (enkephalinase E.C. 3.4.24.11) in a malignant tumor: rat hepatoma 3924A

The activity of the membrane-bound neutral endopeptidase 24.11 was low in the normal liver (21 +/- 3 pmol/h/mg protein, mean +/- SE) but it increased 56-fold in rapidly-growing rat hepatoma 3924A. The identity of the enzyme in the tumor was established by immunoprecipitation and by using a specific inhibitor of neutral endopeptidase. The endopeptidase concentration in the differentiating and regenerating liver was lower than in normal tissue, 39 and 8% of the corresponding control. The activity of a plasma membrane marker enzyme carboxypeptidase M in the normal liver was 1.0 +/- 0.2 nmol/h/mg protein, it increased about 2-fold in the rapidly-growing hepatoma and in the differentiating liver, but was unchanged in regenerating liver. The function of the strikingly increased neutral endopeptidase activity in the rapidly growing hepatoma may relate to activation of autocrine or exocellular growth factors or to inactivation of cell proliferation-inhibitory factors. Such a biochemical change should confer selective advantages to the cancer cells.     

Endogenous and exogenous Ca2+ buffers differentially modulate Ca2+-dependent inactivation of Ca(v)2.1 Ca2+ channels

Voltage-gated Ca2+ channels undergo a negative feedback regulation by Ca2+ ions, Ca2+-dependent inactivation, which is important for restricting Ca2+ signals in nerve and muscle. Although the molecular details underlying Ca2+-dependent inactivation have been characterized, little is known about how this process might be modulated in excitable cells. Based on previous findings that Ca2+-dependent inactivation of Ca(v)2.1 (P/Q-type) Ca2+ channels is suppressed by strong cytoplasmic Ca2+ buffering, we investigated how factors that regulate cellular Ca2+ levels affect inactivation of Ca(v)2.1 Ca2+ currents in transfected 293T cells. We found that inactivation of Ca(v)2.1 Ca2+ currents increased exponentially with current amplitude with low intracellular concentrations of the slow buffer EGTA (0.5 mm), but not with high concentrations of the fast Ca2+ buffer BAPTA (10 mm). However, when the concentration of BAPTA was reduced to 0.5 mm, inactivation of Ca2+ currents was significantly greater than with an equivalent concentration of EGTA, indicating the importance of buffer kinetics in modulating Ca2+-dependent inactivation of Ca(v)2.1. Cotransfection of Ca(v)2.1 with the EF-hand Ca2+-binding proteins, parvalbumin and calbindin, significantly altered the relationship between Ca2+ current amplitude and inactivation in ways that were unexpected from behavior as passive Ca2+ buffers. We conclude that Ca2+-dependent inactivation of Ca(v)2.1 depends on a subplasmalemmal Ca2+ microdomain that is affected by the amplitude of the Ca2+ current and differentially modulated by distinct Ca2+ buffers.     

Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels

An analysis of the relationship between electrical membrane activity and Ca2+ influx in differentiated GnRH-secreting (GT1) neurons revealed that most cells exhibited spontaneous, extracellular Ca(2+)-dependent action potentials (APs). Spiking was initiated by a slow pacemaker depolarization from a baseline potential between -75 and -50 mV, and AP frequency increased with membrane depolarization. More hyperpolarized cells fired sharp APs with limited capacity to promote Ca2+ influx, whereas more depolarized cells fired broad APs with enhanced capacity for Ca2+ influx. Characterization of the inward currents in GT1 cells revealed the presence of tetrodotoxin-sensitive Na+, Ni(2+)-sensitive T-type Ca2+, and dihydropyridine-sensitive L-type Ca2+ components. The availability of Na+ and T-type Ca2+ channels was dependent on the baseline potential, which determined the activation/inactivation status of these channels. Whereas all three channels were involved in the generation of sharp APs, L-type channels were solely responsible for the spike depolarization in cells exhibiting broad APs. Activation of GnRH receptors led to biphasic changes in cytosolic Ca2+ concentration ([Ca2+]i), with an early, extracellular Ca(2+)-independent peak and a sustained, extracellular Ca(2+)-dependent phase. During the peak [Ca2+]i response, electrical activity was abolished due to transient hyperpolarization. This was followed by sustained depolarization of cells and resumption of firing of increased frequency with a shift from sharp to broad APs. The GnRH-induced change in firing pattern accounted for about 50% of the elevated Ca2+ influx, the remainder being independent of spiking. Basal [Ca2+]i was also dependent on Ca2+ influx through AP-driven and voltage-insensitive pathways. Thus, in both resting and agonist-stimulated GT1 cells, membrane depolarization limits the participation of Na+ and T-type channels in firing, but facilitates AP-driven Ca2+ influx.     

Phosphatidylinositol-specific phospholipase C activity of chromaffin granule-binding proteins

Using [U-14C]phosphatidylinositol as substrate, Ca2+-dependent phospholipase C activity was detected in a group of bovine adrenal medullary proteins that bind to chromaffin granule membranes in the presence of Ca2+ ("chromobindins," Creutz, C. E., Dowling, L. G., Sando, J. J., Villar-Palasi, C., Whipple, J. H., and Zaks, W. J. (1983) J. Biol. Chem. 258, 14664-14674). The activity was maximal at neutral pH and represented an 80- to 240-fold enrichment of adrenal medullary cytosol phospholipase C activity measured at pH 7.3. The stimulation of activity by Ca2+ was complex; no activity was present in the absence of Ca2+, 25% activation occurred at 1 microM Ca2+, and full activation at 5 mM Ca2+. The enzyme bound to chromaffin granule membranes in the presence of 2 mM Ca2+ but was released at 40 microM Ca2+, suggesting that intrinsic enzyme activity may be regulated by [Ca2+] at 1 microM, but additional activation at higher concentrations of Ca2+ is seen in vitro as a result of Ca2+-dependent binding of the active enzyme to substrate-containing membranes. This enzyme may generate diacylglycerol and phosphorylated inositol to act as intracellular messengers in the vicinity of the chromaffin granule membrane during the process of exocytosis.     

Role of calcium and calcium-activated proteases in CYP2E1-dependent toxicity in HEPG2 cells.

The objective of this work was to investigate whether CYP2E1- and oxidative stress-dependent toxicity in HepG2 cells is mediated by an increase of cytosolic Ca2+ and activation of Ca2+-modulated processes. HepG2 cells expressing CYP2E1 (E47 cells) or control cells not expressing CYP2E1 (C34 cells) were preloaded with arachidonic acid (AA, up to 10 microm) and, after washing, incubated with iron-nitrilotriacetic acid (up to 100 microm) for variable periods (up to 12 h). Toxicity was greater in E47 cells than in C34 cells at all times and combinations of iron/AA tested. Cytosolic calcium increased with incubation time in both cell lines, but the increase was higher in E47 cells than in C34 cells. The rise in calcium was an early event and preceded the developing toxicity. Toxicity in E47 cells and the increase in Ca2+ were inhibited by omission of Ca2+ from the extracellular medium, and toxicity was restored by reincorporation of Ca2+. An inhibitor of Ca2+ release from intracellular stores did not prevent the toxicity or the increase in Ca2+, reflecting a role for the influx of extracellular Ca2+ in the toxicity. Reactive oxygen production was similar in media with or without calcium, indicating that calcium was not modulating CYP2E1-dependent oxidative stress. Toxicity, lipid peroxidation, and the increase of Ca2+ in E47 cells exposed to iron-AA were inhibited by alpha-tocopherol. E47 cells (but not C34 cells) exposed to iron-AA showed increased calpain activity in situ (40-fold). The toxicity in E47 cells mirrored calpain activation and was inhibited by calpeptin, suggesting that calpain activation plays a causal role in toxicity. These results suggest that CYP2E1-dependent toxicity in this model depends on the activation of lipid peroxidation, followed by an increased influx of extracellular Ca2+ and activation of Ca2+-dependent proteases.