Conventional PKCs regulate the temporal pattern of Ca2+ oscillations at fertilization in mouse eggs

In mammalian eggs, sperm-induced Ca2+ oscillations at fertilization are the primary trigger for egg activation and initiation of embryonic development. Identifying the downstream effectors that decode this unique Ca2+ signal is essential to understand how the transition from egg to embryo is coordinated. Here, we investigated whether conventional PKCs (cPKCs) can decode Ca2+ oscillations at fertilization. By monitoring the dynamics of GFP-labeled PKCalpha and PKCgamma in living mouse eggs, we demonstrate that cPKCs translocate to the egg membrane at fertilization following a pattern that is shaped by the amplitude, duration, and frequency of the Ca2+ transients. In addition, we show that cPKC translocation is driven by the C2 domain when Ca2+ concentration reaches 1-3 microM. Finally, we present evidence that one physiological function of activated cPKCs in fertilized eggs is to sustain long-lasting Ca2+ oscillations, presumably via the regulation of store-operated Ca2+ entry.     

Oxytocin-induced Ca2+ responses in human myometrial cells

Complex spatiotemporal changes in intracellular Ca2+ were monitored in an immortalized human myometrial cell line (PHM1-41) and first-passage human myometrial cells after oxytocin stimulation (1. 0-1000 nM). Laser cytometry revealed intracellular Ca2+ oscillations in both culture systems starting at 1.0 nM, which were followed by repetitive Ca2+ transients by 10-15 min that lasted for at least 90 min. The amplitude of the initial Ca2+ spike was dose dependent, while the frequency of Ca2+ oscillations identified by Fast Fourier Transform (FFT) tended to increase with dose. Removal of oxytocin resulted in termination of oscillations. Analysis of the sources of the Ca2+ involved in oscillations indicated that the major contribution to oscillation frequencies of 相似文献   

The cytosol of human erythrocytes contains a highly Ca2+-sensitive thiol protease (calpain I) and its specific inhibitor protein (calpastatin)

The cytosol of human erythrocytes was found to contain a Ca2+-dependent thiol protease (calpain) and its specific inhibitor (calpastatin) by DEAE-cellulose chromatography at pH 8.0, although no proteolytic activity toward casein was detected in the unfractionated hemolysate. The protease required only 40 microM Ca2+ for 50% activation, indicating that it belongs to the highly Ca2+-sensitive type of calpain, namely, calpain I. It was not inactivated by heating at 58 degrees C for 10 min at pH 7.2, the optimal pH for its action on casein. The inhibitor comprised major and minor components, calpastatin H (Mr = 280,000) and caplastatin L (Mr = 48,000). Both were heat-stable proteins which were readily inactivated by tryptic digestion. The inhibition of erythrocyte calpain by erythrocyte calpastatin H or L was not due to sequestering of Ca2+ from the reaction medium by the inhibitor protein. The calpain preparation preferentially digests bands III and IVa of human erythrocyte membrane proteins, with little or no cleavage of the bands corresponding to spectrin.     

A role for the intracellular protease calpain in the activation of store-operated calcium entry in human platelets

Here, we report a novel role for the cysteine protease calpain in store-operated calcium entry. Several structurally and mechanistically unrelated inhibitors of calpain inhibited Ca2+ entry activated in human platelets by thapsigargin-evoked Ca2+ store depletion or the physiological agonist thrombin, whereas inhibitors of other cysteine proteases were without effect. The use of the cell-permeable fluorogenic calpain substrate 7-amino-4-chloromethylcoumarin, t-BOC-l-leucyl-l-methionine amide revealed rapid activation of calpain which was closely temporally correlated with Ca2+ store depletion even in the absence of a rise in cytosolic [Ca2+]. Calpain inhibition prevented the tyrosine phosphorylation of several proteins upon Ca2+ store depletion, suggesting that calpain may lie upstream of protein tyrosine phosphorylation that is known to be required for the activation of store-operated Ca2+ entry in human platelets. Earlier studies using calpain inhibitors may need reinterpretation in the light of this finding that calpain plays a role in the activation of physiological Ca2+ entry pathways.     

Reversible activation of human neutrophil calpain promoted by interaction with plasma membranes

Human neutrophil calpain is a monomer of 85 kDa molecular weight. The proteinase shows an absolute requirement for Ca2+ with maximal catalytic activity at 0.1-0.2 mM Ca2+ and negligible activity at 1-5 microM Ca2+. At this concentration of Ca2+ neutrophil calpain becomes active and reaches 65% of its maximal catalytic activity following interaction with plasma membranes. The activation is fully reversible since the enzyme returns to its native, high Ca2+ requiring form following removal of the membranes. Membrane phospholipids appear to be the physiological compounds responsible for the promotion of such reversible activation. Unlike other Ca2+ dependent proteinases, neutrophil calpain does not undergo conversion to a low Ca2+ requiring form by limited autoproteolysis.     

Effects of Na+/Ca2+ exchange induced by SR Ca2+ release on action potentials and afterdepolarizations in guinea pig ventricular myocytes

In cardiac cells, evoked Ca2+ releases or spontaneous Ca2+ waves activate the inward Na+/Ca2+ exchange current (INaCa), which may modulate membrane excitability and arrhythmogenesis. In this study, we examined changes in membrane potential due to INaCa elicited by sarcoplasmic reticulum (SR) Ca2+ release in guinea pig ventricular myocytes using whole cell current clamp, fluorescence, and confocal microscopy. Inhibition of INaCa by Na+-free, Li+-containing Tyrode solution reversibly abbreviated the action potential duration at 90% repolarization (APD90) by 50% and caused SR Ca2+ overload. APD90 was similarly abbreviated in myocytes exposed to the Na+/Ca2+ exchange inhibitor KB-R7943 (5 microM) or after inhibition of SR Ca2+ release with ryanodine (20 microM). In the absence of extracellular Na+, spontaneous SR Ca2+ releases caused minimal changes in resting membrane potential. After the myocytes were returned to Na+-containing solution, the potentiated intracellular Ca2+ concentration ([Ca2+]i) transients dramatically prolonged APD90 and [Ca2+]i oscillations caused delayed and early afterdepolarizations (DADs and EADs). Laser-flash photolysis of caged Ca2+ mimicked the effects of spontaneous [Ca2+]i oscillations, confirming that APD prolongation, DADs, and EADs could be ascribed to intracellular Ca2+ release. These results suggest that Na+/Ca2+ exchange is a major physiological determinant of APD and that INaCa activation by spontaneous SR Ca2+ release/oscillations, depending on the timing, can account for both DADs and EADs during SR Ca2+ overload.     

Role of voltage-dependent Na+ channels for rhythmic Ca2+ signalling in glucose-stimulated mouse pancreatic beta-cells.

The putative role of voltage-dependent Na+ channels for glucose induction of rhythmic Ca2+ signalling was studied in mouse pancreatic beta-cells with the use of the Ca2+ indicator fura-2. A rise in glucose from 3 to 11 mM resulted in slow oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i). These oscillations, as well as superimposed transients seen during forskolin-induced elevation of cAMP, remained unaffected in the presence of the Na+ channel blocker tetrodotoxin. During exposure to 1-10 microM veratridine, which facilitates the opening of voltage-dependent Na+ channels, the slow oscillations were replaced by repetitive and pronounced [Ca2+]i transients arising from the basal level. The effects of veratridine were reversed by tetrodotoxin. The veratridine-induced [Ca2+]i transients were critically dependent on the influx of Ca2+ and persisted after thapsigargin inhibition of the endoplasmic reticulum Ca2+-ATPase. Both tolbutamide and ketoisocaproate mimicked the action of glucose in promoting [Ca2+]i transients in the presence of veratridine. It is suggested that activation of voltage-dependent Na+ channels is a useful approach for amplifying Ca2+ signals for insulin release.     

Ca2+ transients of cardiomyocytes from senescent mice peak late and decay slowly

Ventricular myocytes were isolated from either young (2 months, "young myocytes") or senescent (20-26 months, "senescent myocytes") mice. Ca2+ transients were evoked by 40ms voltage-clamp pulses depolarising at 0.4, 1, 2, 4 or 8Hz. At 8Hz, Ca2+ transients from senescent cells peaked later (39ms versus 23ms) to smaller systolic [Ca2+](c) (667nM versus 1110nM) and decayed at slower rate (16s(-1) versus 33s(-1)) to higher end-diastolic [Ca2+](c) (411nM versus 220nM) than those from young myocytes. These differences were less pronounced at lower frequencies of pulsing and could not be explained by differences of the time integral of Ca2+ inward current. Since concentrations of SERCA2a and SERCA2b proteins were similar in young and senescent cells, slow rate of Ca2+ decay and high diastolic [Ca2+]c are explained on the assumption that the usual Ca2+ stimulation of SERCA2 activity is attenuated in senescent cells. The prolonged time-to-peak [Ca2+]c is discussed to result from insufficient SR Ca2+ filling by SERCA2 and, in context with confocal images, from a shift of the SERCA2b distribution to the subsarcolemmal space. The age-related changes of the Ca2+ transients are discussed to cause systolic and diastolic failure if senescent mouse hearts beat at high frequencies.     

Calcium homeostasis in trigeminal ganglion cell bodies

In primary sensory afferent neurons, Ca2+ plays a vital role in the regulation of cellular processes including receptor and synaptic plasticity, neurotransmitter and trophic factor release and gene regulation. Current understanding of the mechanisms underlying Ca2+ homeostasis of primary sensory afferent neurons is mostly derived from studies on dorsal root ganglia and nodose ganglia neuron cell bodies. Little is known about Ca2+ homeostasis in trigeminal ganglion neurons (TGNs). To determine what cellular processes contribute to electrically-evoked Ca2+ transients in TGNs, we probed Ca2+ regulatory mechanisms in TGN cell bodies from the ophthalmic division with a panel of pharmacological reagents. Ca2+ transients were evoked in fura-2 loaded TGNs by depolarizing the plasma membrane with brief (500 ms) puffs of 50 mM KCl. Cyclopiazonic acid (CPA; 5 microM), an inhibitor of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), significantly decreased the peak amplitude, and slowed the decay, of the KCl-evoked Ca2+ transients in TGNs. The mitochondrial protonophore, carbonyl cyanide 3-chloro-phenylhydrazone (CCCP; 5 microM) significantly increased the peak amplitude of KCl-evoked Ca2+ transients. These data demonstrate that Ca2+ stores do play a major role in Ca2+ homeostasis in TGN cell bodies. To determine the role of the sodium-calcium exchanger (NCX) in KCl-evoked Ca2+ transients in TGNs, we inhibited the exchanger with KB-R7943 (10 microM), or by replacing Na+ with Li+. NCX inhibition did not affect either the peak amplitude or the decay kinetics of the KCl-evoked Ca2+ transients. Therefore, the NCX does not play a significant role in removing cytosolic Ca2+ from TGNs. To test whether the plasma membrane calcium-ATPase (PMCA) contributes to Ca2+ extrusion, we inhibited its activity by a shift to alkaline pH (9.0). At pH 9.0, both the peak amplitude and decay time of the KCl-evoked Ca2+ transient were increased significantly. These data suggest that, in TGNs, the PMCA is the major mechanism for removing cytosolic Ca2+ following electrical activity.     

Differential effects of phospholamban and Ca2+/calmodulin-dependent kinase II on [Ca2+]i transients in cardiac myocytes at physiological stimulation frequencies

In cardiac myocytes, the activity of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is hypothesized to regulate Ca(2+) release from and Ca(2+) uptake into the sarcoplasmic reticulum via the phosphorylation of the ryanodine receptor 2 and phospholamban (PLN), respectively. We tested the role of CaMKII and PLN on the frequency adaptation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients in nearly 500 isolated cardiac myocytes from transgenic mice chronically expressing a specific CaMKII inhibitor, interbred into wild-type or PLN null backgrounds under physiologically relevant pacing conditions (frequencies from 0.2 to 10 Hz and at 37 degrees C). When compared with that of mice lacking PLN only, the combined chronic CaMKII inhibition and PLN ablation decreased the maximum Ca(2+) release rate by more than 50% at 10 Hz. Although PLN ablation increased the rate of Ca(2+) uptake at all frequencies, its combination with CaMKII inhibition did not prevent a frequency-dependent reduction of the amplitude and the duration of the [Ca(2+)](i) transient. High stimulation frequencies in the physiological range diminished the effects of PLN ablation on the decay time constant and on the maximum decay rate of the [Ca(2+)](i) transient, indicating that the PLN-mediated feedback on [Ca(2+)](i) removal is limited by high stimulation frequencies. Taken together, our results suggest that in isolated mouse ventricular cardiac myocytes, the combined chronic CaMKII inhibition and PLN ablation slowed Ca(2+) release at physiological frequencies: the frequency-dependent decay of the amplitude and shortening of the [Ca(2+)](i) transient occurs independent of chronic CaMKII inhibition and PLN ablation, and the PLN-mediated regulation of Ca(2+) uptake is diminished at higher stimulation frequencies within the physiological range.     

Kinetics of calcium binding to fluo-3 determined by stopped-flow fluorescence

The kinetics of Ca2+ dissociation from fluo-3 was measured using stopped flow fluorimetry. Analysis of dissociation revealed, in contrast to other commonly used fluorescent Ca2+ indicators, a biexponential behaviour with two distinct dissociation rates of 550 s-1 and 200 s-1 at physiological pH and room temperature. The dissociation rate constant of the fast phase increases to 700 s-1 at physiological temperature, whereas that of the slow phase does not change markedly. While the rate constants do not depend on pH between 6.6 and 7.8, the dissociation turns out to be monoexponential at pH 5.86. The association rate of Ca2+ to fluo-3 could not be measured within the mixing dead time and is estimated to be above 10(9) M-1 s-1. Since the rate constants of fluo-3 are larger than those of other fluorescent Ca2+ indicators, fluo-3 is well suited for investigations of Ca2+ oscillations in biological systems.     

The calpain-calpastatin system in mammalian cells: properties and possible functions.

All mammalian cells contain a calcium-dependent proteolytic system, composed by a proteinase, calpain, and an inhibitor, calpastatin. In some cell types an activator protein has also been identified. Moreover, two calpain isoforms, distinguishable on the basis of a different calcium requirement, can be present in a single cell. Both calpain forms are heterodimers composed of a heavy subunit (80 kDa) that contains the catalytic site and a smaller (regulatory?) subunit (30 kDa). Calpain I expresses full activity at 10-50 microM Ca2+, whereas calpain II requires calcium concentrations in the millimolar range. The removal by autoproteolysis of a fragment from the N-terminus of both calpain subunits generates a proteinase form that can express catalytic activity at concentrations of Ca2+ close to the physiological range. This process is significantly accelerated in the presence of cell membranes or phospholipid vesicles. Calpastatin, the specific inhibitor of calpain, prevents activation and the expression of catalytic activity of calpain. It is in itself a substrate of the proteinase and undergoes a degradation process which correlates with the general mechanism of regulation of the intracellular proteolytic system. The natural calpain activator specifically acts on calpain II isoform, by reducing the Ca2+ required for the autoproteolytic activation process. Based on the general properties of the calpain-calpastatin system and on the substrate specificity, its role in the expression of specific cell functions can be postulated.     

Hystereses in the force-length relation and regulation of cross-bridge recruitment in tetanized rat trabeculae

Various mechanisms have been suggested to explain cardiac force-length Ca2+ relations. The existence of a cooperativity mechanism, whereby cross-bridge (XB) recruitment is affected by the number of active XBs, suggests that the force response to length oscillations should lag length oscillations. Consequently, the oscillatory force response should be larger during shortening than during lengthening. To test this prediction, force responses to large-sarcomere length (SL) oscillations (36.7 +/- 16.0 nm) at different SLs (n = 6) and frequencies (n = 7) were studied in intact tetanized trabeculae dissected from rat right ventricle (n = 13). Stable tetani were obtained by utilizing 30 microM cyclopiazonic acid in Krebs-Henseleit solution containing 6 mM extracellular Ca(2+) at 25 degrees C. SL was measured by laser diffraction techniques (Dalsa). Force was measured by silicone strain gauge. Instantaneous dynamic stiffness during large oscillations was measured by superimposing additional fast (50 or 200 Hz) and small-amplitude (2.25 +/- 0.25 nm) oscillations. The force responses lagged the SL oscillations at slow frequencies (112 +/- 41 ms at 1 Hz), and counterclockwise hystereses were obtained in the force-length plane: the force was higher during shortening than during lengthening. The delay in the force response decreased as the frequency of the SL oscillation was increased. Clockwise hysteresis, where the force preceded the SL, was obtained at frequencies >4 Hz. Similar hysteresis characteristics were obtained in the force-SL and stiffness-SL planes. Maximal lag was observed at the shortest SL, and the delay decreased with sarcomere elongation: 131.1 +/- 31.7 ms at 1.78 +/- 0.03 microm vs. 14.7 +/- 18.5 ms at 1.99 +/- 0.015 microm. The results establish the ability of cardiac fiber to adapt XB recruitment to changes in prevailing loading conditions. This study supports the stipulated existence of a cooperativity mechanism that regulates XB recruitment and highlights an additional method to characterize regulation of the force-length relation.     

Dynamic and static calcium gradients inside large snail (Helix aspersa) neurones detected with calcium-sensitive microelectrodes

We have used quartz Ca2+-sensitive microelectrodes (CASMs) in large voltage-clamped snail neurones to investigate the inward spread of Ca2+ after a brief depolarisation. Both steady state and [Ca2+]i transients changed with depth of penetration. When the CASM tip was within 20 microm of the far side of the cell the [Ca2+]i transient time to peak was 4.4+/-0.5s, rising to 14.7+/-0.7s at a distance of 80 microm. We estimate that the Ca2+ transients travelled centripetally at an average speed of 6 microm2 s(-1) and decreased in size by half over a distance of about 45 microm. Cyclopiazonic acid had little effect on the size and time to peak of Ca2+ transients but slowed their recovery significantly. This suggests that the endoplasmic reticulum curtails rather than reinforces the transients. Injecting the calcium buffer BAPTA made the Ca2+ transients more uniform in size and increased their times to peak and rates of recovery near the membrane. We have developed a computational model for the transients, which includes diffusion, uptake and Ca2+ extrusion. Good fits were obtained with a rather large apparent diffusion coefficient of about 90+/-20 microm2 s(-1).This may assist fast recovery by extrusion.     

Role of mitochondria in kainate-induced fast Ca2+ transients in cultured spinal motor neurons

Motor neuron death in amyotrophic lateral sclerosis (ALS) has been linked to selective vulnerability towards AMPA receptor-mediated excitotoxicity. We investigated intracellular mechanisms leading to impairment of motor neuron Ca2+ homeostasis with near physiological AMPA receptor activation. Using fast solution exchange on patch-clamped cultured neurons, kainate (KA) was applied for 2s. This induced a transient increase in the cytosolic Ca2+ concentration ([Ca2+]c) for seconds. Inhibition of the mitochondrial uniporter by RU-360 abolished the decay of the Ca2+ transient and caused immediate [Ca2+]c overload. Repetitive short KA stimulation caused a slowing of the decay of the Ca2+ transient and a gradual increase in peak and baseline [Ca2+]c in motor neurons, but not in other neurons, indicating saturation of the mitochondrial buffer. Furthermore, mitochondrial density was lower in motor neurons and, in a network of neurons with physiological synaptic AMPA receptor input, RU-360 acutely induced an increase in Ca2+ transients. We conclude that motor neurons have an insufficient mitochondrial capacity to buffer large Ca2+ elevations which is partly due to a reduced mitochondrial density per volume compared to non-motor neurons. This may exert deleterious effects in motor neuron disease where mitochondrial function is thought to be compromised.     

Comparison of calpain I and calpain II from carp muscle

1. The content of calpain II is 3.4 times more than that of calpain I when estimated by the elution profiles from a column of DEAE-cellulose. 2. Calpain I required 1 mM Ca2+ and calpain II required 5 mM Ca2+ to show the full activities. These data demonstrated that Ca2+-sensitivities of both calpains were lower than those of mammalian calpains, respectively. 3. The optimum caseinolytic activity was pH 7.2 for calpain I and pH 7.5 for calpain II. 4. The molecular weight of calpain I was estimated to be 110 k and that of calpain II to be 120 k by gel filtration. 5. Calpain I was much more heat-stable than calpain II around 50-60 degrees C. 6. Both calpains were sensitive to calpastatin, an endogenous inhibitor for calpain.     

Rapid inhibition of Ca2+ influx by neurosteroids in murine embryonic sensory neurones

The non-genomic role of neuroactive steroids on [Ca2+]i transients induced by GABA receptor activation was investigated in cultured dorsal root ganglia (DRG) neurones at embryonic stage E13. [Ca2+]i measurements were performed with Fura-2 fast fluorescence microfluorimetry. Application of the GABAA receptor agonist muscimol (Musci) evoked an increase in [Ca2+]i, confirming the excitatory effect of GABA at this embryonic stage. The muscimol-induced [Ca2+]i response was inhibited by progesterone (Proges) and its primary metabolite allopregnanolone (Allo) in a rapid, reversible and dose-dependent manner. These calcium transients were suppressed in the absence of external Ca2+ or in the presence of Ni2+ + Cd2+ suggesting an involvement of voltage-activated Ca2+ channels. In contrast, none of these steroids affected the resting [Ca2+]i nor exhibited any inhibitory effect on 50 mM KCl-induced [Ca2+]i increases. In view of the well-established potentiation of GABAA receptor by direct binding of neurosteroids, the inhibitory effects described in this study seem to involve distinct mechanisms. This new inhibitory effect of progesterone is observed at low and physiological concentrations, is rapid and independent of RU38486, an antagonist of the classic progesterone receptor, probably involving a membrane receptor. Using RT-PCR, we demonstrated the expression of progesterone receptor membrane component 1 (Pgrmc1), encoding 25-Dx, a membrane-associated progesterone binding protein in DRG neurones at different stages of development. In conclusion, we describe for the first time a rapid effect of progestins on embryonic DRG neurones involving an antagonistic effect of progesterone and allopregnanolone on GABAA receptors.     

Purification and characterization of a calpain activator from human platelets.

A calpain (Ca(2+)-activated neutral protease) activator was purified from human platelets by ammonium sulfate fractionation, gel-filtration, ion-exchange chromatography, followed by heat-treatment. The purified calpain activator with a Mr of 47.5 kDa was a heat-stable protein as demonstrated in other cells. The calpain activator did not change the Ca2+ sensitivity of calpain but activated calpain activity about 2-fold. This calpain activator may play an important role in the activation of the protease system leading to the Ca(2+)-mediated physiological process of platelets.     

Calcium-induced weakening of skeletal muscle Z-disks

Structural changes in the Z disk were sensitively detected by measuring fragmentation indexes of myofibrils. The Ca2+-induced weakening of Z disks and the Z-disk removal by muscle calpain could be clearly distinguished by using muscle calpastatin, an endogenous inhibitor of muscle calpain. The Ca2+-induced weakening of Z disks occurred without concomitant release of alpha-actinin and had maxima at 10(-4) M Ca2+ and 45 degrees C and a minimum at pH 6.5, while the Z-disk removal by calpain had similar optima to the caseinolytic activity of calpain, at 10(-3) M Ca2+, 20 degrees C and pH 7.0. The Ca2+-induced weakening of Z disks is therefore not due to the proteolytic action of calpain. In postmortem muscle, moreover, the Ca2+-induced weakening of Z disks was inferred to be predominate over calpain proteolysis, and therefore to be the major factor in the characteristic weakening of Z disks.     

Modulation of histamine-induced Ca2+ release by protein kinase C. Effects on cytosolic and mitochondrial [Ca2+] peaks

In HeLa cells, histamine induces production of inositol 1,4,5-trisphosphate (InsP3) and release of Ca2+ from the endoplasmic reticulum (ER). Ca2+ release is typically biphasic, with a fast and brief initial phase, followed by a much slower and prolonged one. In the presence of inhibitors of protein kinase C (PKC), including staurosporine and the specific inhibitors GF109203X and Ro-31-8220, the fast phase continued until the ER became fully empty. On the contrary, treatment with phorbol 12,13-dibutyrate inhibited Ca2+ release. Staurosporine had no effect on InsP3-induced Ca2+ release in permeabilized cells and did not modify either histamine-induced InsP3 production. These data suggest that histamine induces Ca2+ release and with a short lag activates PKC to down-regulate it. Consistently, Ca2+ oscillations induced by histamine were increased in amplitude and decreased in frequency in the presence of PKC inhibitors. We show also that mitochondrial [Ca2+] was much more sensitive to changes in ER-Ca2+ release induced by PKC modulation than cytosolic [Ca2+]. PKC inhibitors increased the histamine-induced mitochondrial [Ca2+] peak by 4-fold but increased the cytosolic [Ca2+] peak only by 20%. On the contrary, PKC activation inhibited the mitochondrial [Ca2+] peak by 90% and the cytosolic one by only 50%. Similarly, the combination of PKC inhibitors with the mitochondrial Ca2+ uniporter activator SB202190 led to dramatic increases in mitochondrial [Ca2+] peaks, with little effect on cytosolic ones. This suggests that activation of ER-Ca2+ release by PKC inhibitors could be involved in apoptosis induced by staurosporine. In addition, these mechanisms allow flexible and independent regulation of cytosolic and mitochondrial [Ca2+] during cell stimulation.