Inositol phosphate formation in fMet-Leu-Phe-stimulated human neutrophils does not require an increase in the cytosolic free Ca2+ concentration.

The accumulation of inositol phosphates in myo-[3H]inositol-labelled human neutrophils stimulated with the chemotactic peptide fMet-Leu-Phe was measured. The challenge with the chemotactic peptide caused the generation of inositol monophosphate (InsP), inositol bisphosphate (InsP2) and inositol trisphosphate (InsP3). The formation of the three inositol phosphates followed a differential time course: InsP3 accumulated very rapidly and transiently, whereas InsP increased steadily for more than 2 min. Inositol phosphate formation was only partially decreased by procedures which prevented the fMet-Leu-Phe-dependent increase of cytosolic free Ca2+ concentration.     

Activation of protein kinase C in human neutrophils attenuates agonist-stimulated rises in cytosolic free Ca2+ concentration by inhibiting bivalent-cation influx and intracellular Ca2+ release in addition to stimulating Ca2+ efflux.

Stimulation of fura-2-loaded human neutrophils with formylmethionyl-leucyl-phenylalanine (FMLP) or ionomycin elevated the cytosolic free Ca2+ concentration, [Ca2+], to a maintained elevated level. Activation of protein kinase C (C-kinase) with phorbol 12-myristate 13-acetate, 4 beta-phorbol 12,13-didecanoate or dioctanoylglycerol caused decreases in [Ca2+]i from this level. 4 alpha-Phorbol didecanoate, which does not activate C-kinase, had no effect. These results confirm previous reports that C-kinase activation decreases neutrophil [Ca2+]i by stimulating removal of Ca2+ from the cytosol. Further experiments showed that activation of C-kinase attenuated the component of the FMLP-stimulated [Ca2+]i rise that was dependent on external Ca2+. C-kinase activation also inhibited FMLP-stimulated entry of the quenching cation, Mn2+, used as an indicator of bivalent-cation entry. In contrast, C-kinase activation caused only a partial inhibition of FMLP-stimulated release of Ca2+ from intracellular stores. 4 alpha-Phorbol didecanoate was ineffective in inhibiting Ca2+ entry, Mn2+ entry and intracellular Ca2+ release. Addition of FMLP also stimulated a decrease in the ionomycin-elevated [Ca2+]i, and this effect was blocked by staurosporine, a protein kinase inhibitor. These results show that, in addition to stimulating Ca2+ efflux, C-kinase activation in neutrophils inhibits FMLP-stimulated entry of bivalent cations, and partially inhibits intracellular release of Ca2+. Further, FMLP itself can modulate [Ca2+]i by activation of C-kinase.     

Rapid kinetics of agonist-evoked changes in cytosolic free Ca2+ concentration in fura-2-loaded human neutrophils.

The initial kinetics of agonist-evoked rises in the cytosolic Ca2+ concentration [Ca2+]i were investigated in fura-2-loaded human neutrophils by stopped-flow fluorimetry. The rises in [Ca2+]i evoked by chemotactic peptide (fMet-Leu-Phe), platelet-activating factor and ADP all lagged behind agonist addition by 1-1.3 s. Lag times were not significantly different in the presence and in the absence of external Ca2+. Stimulation of the cells in the presence of extracellular Mn2+ resulted in a quench of fluorescence with a similar lag time to [Ca2+]i rise. The delay in onset of the rise in [Ca2+]i evoked by fMet-Leu-Phe was dependent on concentration, becoming longer at lower concentrations of agonist. These results indicate that both the agonist-evoked discharge of the intracellular Ca2+ stores and the generation of bivalent-cation influx lag behind agonist-receptor binding in neutrophils. Both pathways thus appear to be mediated by indirect mechanisms, rather than by a directly coupled process such as a receptor-operated channel. The temporal coincidence of the onset of store discharge with the commencement of bivalent-cation influx suggests that the two events may be causally linked.     

Modulation by external Ca2+ and nicardipine of Ca2+ influx and cytosolic concentration in human erythrocytes

Variations of Ca2+ influx (evaluated by the initial rate of 45Ca2+ uptake) and cytosolic free Ca2+ concentration ([Ca2+]i, measured with fura-2) were investigated in human erythrocytes. When external Ca2+ concentration ([Ca2+]o) rose from 1 to 2 mM, the initial rate of Ca2+ influx nearly doubled whereas [Ca2+]i increased only by 15%. Nicardipine dose-dependently decreased both initial rate of Ca2+ influx and [Ca2+]i (up to 53 and 18%. respectively at 10(-6) M). The less marked changes in [Ca2+]i than in Ca2+ influx indicate a partial adjustment of the Ca2+ extruding-pump activity to of Ca2+ influx. In vivo administration of nicardipine reduced [Ca2+]i only when its initial value exceeded 80 nM and prevented the rise in [Ca2+]i induced by the increase in [Ca2+]o. Our results indicate that nicardipine may reduce Ca2+ influx in human erythrocytes and participate in the control of [Ca2+]i when elevated.     

Biphasic regulation of mitochondrial Ca2+ uptake by cytosolic Ca2+ concentration

A rise in cytosolic Ca(2+) concentration is used as a key activation signal in virtually all animal cells, where it triggers a range of responses including neurotransmitter release, muscle contraction, and cell growth and proliferation [1]. During intracellular Ca(2+) signaling, mitochondria rapidly take up significant amounts of Ca(2+) from the cytosol, and this stimulates energy production, alters the spatial and temporal profile of the intracellular Ca(2+) signal, and triggers cell death [2-10]. Mitochondrial Ca(2+) uptake occurs via a ruthenium-red-sensitive uniporter channel found in the inner membrane [11]. In spite of its critical importance, little is known about how the uniporter is regulated. Here, we report that the mitochondrial Ca(2+) uniporter is gated by cytosolic Ca(2+). Ca(2+) uptake into mitochondria is a Ca(2+)-activated process with a requirement for functional calmodulin. However, cytosolic Ca(2+) subsequently inactivates the uniporter, preventing further Ca(2+) uptake. The uptake pathway and the inactivation process have relatively low Ca(2+) affinities of approximately 10-20 microM. However, numerous mitochondria are within 20-100 nm of the endoplasmic reticulum, thereby enabling rapid and efficient transmission of Ca(2+) release into adjacent mitochondria by InsP(3) receptors on the endoplasmic reticulum. Hence, biphasic control of mitochondrial Ca(2+) uptake by Ca(2+) provides a novel basis for complex physiological patterns of intracellular Ca(2+) signaling.     

Ca2+ homeostasis in permeabilized human neutrophils. Characterization of Ca2+-sequestering pools and the action of inositol 1,4,5-triphosphate

The regulation of Ca2+ transport by intracellular compartments was studied in digitonin-permeabilized human neutrophils, using a Ca2+-selective electrode. When incubated in a medium containing ATP and respiratory substrates, the cells lowered within 6 min the ambient [Ca2+] to a steady state of around 0.2 microM. A vesicular ATP-dependent and vanadate-sensitive non-mitochondrial pool maintained this low [Ca2+] level. In the absence of ATP, a higher Ca2+ steady state of 0.6 microM was seen, exhibiting the characteristics of a mitochondrial Ca2+ "set point." Both pools were shown to act in concert to restore the previous ambient [Ca2+] following its elevation. Thus, the mitochondria participate with the other pool(s) in decreasing [Ca2+] to the submicromolar range whereas only the nonmitochondrial pool(s) lowers [Ca2+] to the basal level. The action of inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a few cell types was studied. IP3 released (detectable within 2 s) Ca2+ accumulated in the ATP-dependent pool(s) but had no effect on the mitochondria. The response was transient and resulted in desensitization toward subsequent IP3 additions. Under experimental conditions in which the ATP-dependent Ca2+ influx was blocked, the addition of IP3 resulted in a very large Ca2+ release from nonmitochondrial pool. The results strongly suggest that IP3 is a second messenger mediating intracellular Ca2+ mobilization in human neutrophils. Furthermore, the nonmitochondrial pool appears to have independent influx and efflux pathways for Ca2+ transport, a Ca2+ ATPase (the influx component) and an IP3-sensitive efflux component activated during Ca2+ mobilization.     

Inhibition by nicardipine of endothelin-mediated inositol phosphate formation and Ca2+ mobilization in smooth muscle cell

We have investigated the effects of endothelin on phosphoinositide metabolism and Ca2+ mobilization in cultured A10 cells. Endothelin stimulated a significant increase in inositol phosphate formation in a time- and dose-dependent manner. IP3 was significantly elevated by 30 sec and reached a 2.0-fold above control at 1 min. The EC50 for endothelin was 0.5 nM. The initiation of inositol phosphate formation was independent of extracellular Ca2+, and the Ca2+ ionophore, A23187, did not stimulate IP3 formation. However, the sustained elevation of inositol phosphates was partially inhibited by incubating cells in buffer lacking Ca2+ or in buffer containing nicardipine. Endothelin mobilized both intracellular and extracellular Ca2+ reaching a peak intracellular concentration of 350 +/- 11 nM by 1 min when cells were bathed with Ca2+-complete buffer. Intracellular Ca2+ remained 2-fold above baseline for at least 15 min. In contrast, when cells were exposed to endothelin in Ca2+-free buffer, the peak value of [Ca2+]i was 195 +/- 20 nM and returned to baseline by 2 min. Nicardipine completely blocked the influx of extracellular Ca2+ but did not interfere with the mobilization of intracellular stores. We conclude that endothelin produces a rapid and sustained elevation in inositol phosphate formation. The rapid production of IP3 is consistent with the time course for mobilization of intracellular Ca2+. Elevated cytosolic Ca2+ levels are maintained by the influx of extracellular Ca2+ through a nicardipine-sensitive Ca2+ channel and are involved in the sustained formation of inositol phosphates. These data provide an explanation for the sustained, nicardipine-inhibitable contraction of coronary artery strips induced by endothelin.     

Raising the cytosolic Ca2+ concentration increases the membrane capacitance of maize coleoptile protoplasts: Evidence for Ca2+-stimulated exocytosis

Enhanced elongation of coleoptile cells has been proposed to be related to a rise in secretory activity. Therefore, to obtain a direct measurement of exocytotic events in maize (Zea mays L.) coleoptile protoplasts we used the patch-clamp method to record changes in membrane capacitance (Cm) as a parameter proportional to fluctuations of the membrane surface area. The secretory activity of protoplasts was correlated with the cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt): dialyzing protoplasts with 1 M [Ca²⁺]_cyt caused a steady rise in Cm of 3.3 ± pF·s^–1. In contrast, dialysis with a solution containing <20 nM Ca²⁺ produced a small and persistent decrease in Cm. This demonstrates that secretory activity in coleoptile cells can be controlled by factors which modulate [Ca²⁺]_cyt.Abbreviation Cm membrane capacitance This work was made possible by a visiting grant from the Research Council of Slovenia and financial support of the Deutsche Forschungsgemeinschaft to G.T. We are grateful to Dr. W. Diekmann (University of Göttingen) for teaching us the preparation of coleoptile protoplasts.     

Synergistic responses in human platelets. Comparison between aggregation, secretion and cytosolic Ca2+ concentration

Synergistic interaction between ADP, adrenaline, 5-hydroxytryptamine (5HT) and [8-arginine]vasopressin is not observed for the aggregatory response of aspirin-treated human platelets when this response is estimated directly from the decrease in the number of single platelets in the suspension. This finding is in marked contrast with prior reports of synergistic interaction between these agonists when the rate and extent of the aggregometer response is estimated from the increase in the light transmittance of the suspension, using a platelet aggregometer. We propose that the apparent synergistic response detected using the aggregometer results from the inability of this instrument to respond during the initial phase of aggregation. Significant synergistic interaction is observed for the increase in cytosolic [Ca2+] induced by addition of the ADP/5HT and, to a lesser extent, of the ADP/vasopressin agonist pairs as compared with that caused by addition of the individual agonists. This effect is not, however, typical of the system since increases in cytosolic [Ca2+] induced by addition of the ADP/thrombin or 5HT/vasopressin agonist pairs are no greater than the sum of the responses to these agonists added separately. Addition of collagen prior to ADP or 11,9-epoxymethanoprostaglandin H2 (U46619) fails to enhance the increase in cytosolic [Ca2+] induced by these latter agonists. Adrenaline, when added prior to non-saturating concentrations of U46619, thrombin, vasopressin or ADP, significantly enhances the increase in cytosolic [Ca2+] induced by these agonists in platelets suspended in media containing less than 0.1 microM or 1 mM Ca2+. However, adrenaline fails to enhance the increase in cytosolic [Ca2+] induced by the divalent cation ionophore, ionomycin. Enhancement by adrenaline of Ca2+ influx induced by U46619, thrombin and ADP has been shown by using Mn2+ as probe. Adrenaline also enhances the extent of [3H]5HT secretion induced by U46619, thrombin and vasopressin but fails to increase that induced by ADP in this aspirin-treated preparation. These results are in part consistent with the postulate that adrenaline, acting via an alpha 2-adrenoceptor, modulates receptor--phospholipase-C coupling. However, such modulation does not appear to involve inhibition of adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)     

2,5-Di-(tert-butyl)-1,4-benzohydroquinone rapidly elevates cytosolic Ca2+ concentration by mobilizing the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool

2,5-Di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), a potent inhibitor of liver microsomal ATP-dependent Ca2+ sequestration (Moore, G. A., McConkey, D. J., Kass, G. E. N., O'Brien, P. J., and Orrenius, S. (1987) FEBS Lett. 224, 331-336), produced a concentration-dependent, rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) in isolated rat hepatocytes (EC50 = 1-2 microM). The amplitude of the [Ca2+]i increase was essentially identical with that produced by vasopressin, but the tBuBHQ-stimulated [Ca2+]i increase remained sustained for 15-20 min. Vasopressin added 2-3 min after tBuBHQ caused [Ca2+]i to rapidly return to basal levels; however, tBuBHQ added after vasopressin resulted in a Ca2+ transient rather than a sustained [Ca2+]i elevation. Ca2+ influx was not stimulated in tBuBHQ-treated hepatocytes, but was markedly enhanced upon addition of vasopressin. Depletion of the endoplasmic reticular Ca2+ pool by the addition of vasopressin to hepatocytes incubated in low Ca2+ medium virtually abolished the tBuBHQ-mediated [Ca2+]i rise and vice versa. In saponin-permeabilized hepatocytes, tBuBHQ released Ca2+ from the same nonmitochondrial, ATP-dependent Ca2+ pool which was released by inositol 1,4,5-trisphosphate. Furthermore, tBuBHQ-induced Ca2+ release in saponin-permeabilized cells was not inhibited by neomycin, and tBuBHQ did not produce any apparent accumulation of inositol phosphates in intact hepatocytes. The rate of passive efflux of Ca2+ from Ca2+-loaded hepatic microsomes was unaltered by tBuBHQ. Thus, tBuBHQ inhibits ATP-dependent Ca2+ sequestration via a direct effect on the endoplasmic reticulum Ca2+ pump, resulting in net Ca2+ release and elevation of [Ca2+]i. Taken together, our results show that in the absence of hormonal stimuli, excess Ca2+ is only slowly cleared from the hepatocyte cytosol, indicating that the basal rate of Ca2+ removal by the plasma membrane Ca2+ pump and mitochondria is slow. Furthermore, Ca2+-mobilizing hormones appear to stimulate an active process of Ca2+ removal from hepatocyte cytosol which does not depend on re-uptake into the endoplasmic reticulum.     

Characterisation of the serotonin efflux induced by cytosolic Ca2+ and Na+ concentration increase in human platelets.

BACKGROUND/AIM: The present study aimed at elucidating the mechanism(s) of serotonin (5-HT) efflux induced by thapsigargin from human platelets in the absence of extra-cellular Ca2+. METHODS: Efflux of pre-loaded radiolabeled serotonin was generally determined by filtration techniques. Cytosolic concentrations of Ca2+, Na+ and H+ were measured with appropriate fluorescent probes. RESULTS: 5-HT efflux from control or reserpine-treated platelets--where reserpine prevents 5-HT transport into the dense granules--was proportional to thapsigargin evoked cytosolic [Ca2+]c increase. Accordingly factors as prostacyclin, aspirin and calyculin which reduced [Ca2+]c-increase also inhibited the 5-HT efflux. Thapsigargin, which also caused a remarkable increase in cytosolic [Na+]c, promoted less 5-HT release, in parallel to lower [Na+]c and [Ca2+]c increase, when added to platelet suspensions containing low [Na+]. The Na+/H+ exchanger monensin increased the [Na+]c and induced 5-HT efflux without affecting the Ca2+ level. The 5-HT efflux induced by both [Ca2+] or [Na+]c increase did not depend on pH or membrane potential changes, whereas it decreased in the absence of extra-cellular K+, and increased in the absence of Cl- or Na+. CONCLUSION: Increases in [Ca2+]c and [Na+]c independently induce serotonin efflux through the outward directed plasma membrane serotonin transporter SERT. This event might be physiologically important at the level of capillaries or narrowed arteries where platelets are subjected to high shear stress which causes [Ca2+]c increase followed by 5-HT release which might exert vasodilatation.     

Platelet-activating factor increases inositol phosphate production and cytosolic free Ca2+ concentrations in cultured rat Kupffer cells

Platelet-activating factor (PAF) stimulates glycogenolysis in perfused livers but not in isolated hepatocytes [(1984) J. Biol. Chem. 259, 8685-8688]. PAF-induced glycogenolysis in liver is associated closely with a pronounced constriction of the hepatic vasculature [(1986) J. Biol. Chem. 261, 644-649]. These and other observations suggest that PAF stimulates glycogenolysis in liver indirectly by interactions with cells other than hepatocytes. We have evaluated effects of PAF on hepatic Kupffer cells, which regulate flow through the hepatic sinusoids. Application of PAF to [3H]inositol-labeled Kupffer cells produced dose-dependent increases in [3H]inositol phosphates with an EC50 value of 4 x 10(-10) M. Increases in inositol phosphate production in response to PAF were inhibited by a specific PAF receptor antagonist, SRI 63-675 (2 x 10(-7) M), and stimulus of protein kinase C, phorbol 12-myristate 13-acetate (1 x 10(-7) M). Measurements of cytosolic free Ca2+ concentrations ([Ca2+]i) in single Kupffer cells loaded with Fura-2 demonstrated that application of PAF (2 x 10(-9) M) resulted in significant increases in [Ca2+]i. These observations lead us to propose that interactions of PAF with Kupffer cells may result in the hemodynamic and metabolic responses to PAF in liver.     

Activation of cytosolic phospholipase A2 in permeabilized human neutrophils

Neutrophils (PMN) contain two types of phospholipase A₂ (PLA₂), a 14 kDa ‘secretory’ Type II PLA₂ (sPLA₂) and an 85 kDa ‘cytosolic’ PLA₂ (cPLA₂), that differ in a number of key characteristics: (1) cPLA₂ prefers arachidonate (AA) as a substrate but hydrolyzes all phospholipids; sPLA₂ is not AA specific but prefers ethanolamine containing phosphoacylglycerols. (2) cPLA₂ is active at nM calcium (Ca²⁺) concentrations; sPLA₂ requires μM Ca²⁺ levels. (3) cPLA₂ activity is regulated by phosphorylation; sPLA₂ lacks phosphorylation sites. (4) cPLA₂ is insensitive to reduction; sPLA₂ is inactivated by agents that reduce disulfide bonds. We utilized PMN permeabilized with Staphylococcus aureus α-toxin to determine whether one or both forms of PLA₂ were activated in porated cells under conditions designed to differentiate between the two enzymes. PMN were labeled with [³H]AA to measure release from phosphatidylcholine and phosphatidylinositol; gas chromatography-mass spectrometry was utilized to determine total AA release (mainly from phosphatidylethanolamine) and to asses oleate and linoleate mass. A combination of 500 nM Ca²⁺, a guanine nucleotide, and stimulation with n-formyl-met-leu-phe (FMLP) were necessary to induce maximal AA release in permeabilized PMN measured by either method; AA was preferentially released. [³H]AA and AA mass release occurred in parallel over time. A hydrolyzable form of ATP was necessary for maximum AA release and staurosporin inhibited PLA₂ activation. Dithiothreitol treatment had little affect on [³H]AA release and metabolism but inhibited AA mass release. Assay of cell supernatants after cofactor addition did not detect sPLA₂ activity and the cytosolic buffer utilized did not support activity of recombinant sPLA₂. These results strongly suggested that cPLA₂ was the enzyme activated in the permeabilized cell model and this is the first report which unambiguously demonstrates AA release in response to activation of a specific type of PLA₂ in PMN.     

Epidermal growth factor-induced increases in inositol trisphosphates, inositol tetrakisphosphates, and cytosolic Ca2+ in a human hepatocellular carcinoma-derived cell line

A human hepatocellular carcinoma-derived cell line, PLC/PRF/5, was examined for its ability to respond to epidermal growth factor (EGF) exposure with increased phosphatidylinositol 4,5-bisphosphate hydrolysis. Upon addition of EGF (25 ng/ml), a rapid (10-15 s) but transient increase in Ins(1,4,5)P3 levels and large, prolonged (2 min) increases in Ins(1,3,4,5)P4 and Ins(1,3,4)P3 levels were detected. Increases in cytosolic Ca2+ were observed after a 10 to 20 s lag, reaching peak value at 1 min, and remaining elevated for 10 min. The initial burst of cytosolic Ca2+ occurred in the absence of extracellular Ca2+ and probably reflects mobilization of intracellular Ca2+ stores. In cells pretreated with EGTA, the sustained component of the Ca2+ response was not observed. Comparison of the inositol phosphate and Ca2+ responses of PLC/PRF/5 cells to responses reported in other cell types indicates that this cell line is a good model for EGF action in liver.     

fMet-Leu-Phe-induced activation of phospholipase D in human neutrophils. Dependence on changes in cytosolic free Ca2+ concentration and relation with respiratory burst activation.

In this study, we have investigated the Ca2+ requirements for the activation of phospholipase D by the tripeptide fMet-Leu-Phe (fMLP) in human neutrophils. EGTA inhibited the activation of phospholipase D (PLD) by 55% (n = 4). When the initial transient rise in [Ca2+]i was prevented by loading the cells with limited amounts of the Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), PLD activation was inhibited by 92% (n = 4). In the presence of both chelators, PLD activation was only 4% of control. In electropermeabilized neutrophils, too, the activation of PLD after the addition of fMLP strongly depends on the Ca2+ concentration, being almost absent with 100 nM free Ca2+ present and reaching maximum activation with a free [Ca2+] of 500 nM. We subsequently investigated the relationship between PLD activation and the activation of the respiratory burst. In neutrophils loaded with BAPTA/AM (10 microM), in which PLD activation was almost absent, a respiratory burst could be induced by fMLP, albeit with a much longer lag time. A respiratory burst could also be elicited by fMLP in electropermeabilized neutrophils incubated with 100 nM free Ca2+. This response, however, was strongly enhanced in the presence of 1 microM Ca2+. Our results indicate that changes in [Ca2+]i are essential for the activation of PLD by fMLP, but probably do not constitute the sole activation signal. In addition, our data provide evidence that PLD activation is important, but not necessary, for activation of the neutrophil respiratory burst.     

Tributyltin increases cytosolic free Ca2+ concentration in thymocytes by mobilizing intracellular Ca2+, activating a Ca2+ entry pathway, and inhibiting Ca2+ efflux.

The immunotoxic environmental pollutant tri-n-butyltin (TBT) kills thymocytes by apoptosis through a mechanism that requires an increase in intracellular Ca2+ concentration. The addition of TBT (EC50 = 2 microM) to fura-2-loaded rat thymocytes resulted in a rapid and sustained increase in the cytosolic free Ca2+ concentration ([Ca2+]i) to greater than 1 microM. In nominally Ca(2+)-free medium, TBT slightly but consistently increased thymocyte [Ca2+]i by about 0.11 microM. The subsequent restoration of CaCl2 to the medium resulted in a sustained overshoot in [Ca2+]i; similarly, the addition of MnCl2 produced a rapid decrease in the intracellular fura-2 fluorescence in thymocytes exposed to TBT. The rates of Ca2+ and Mn2+ entry stimulated by TBT were essentially identical to the rates stimulated by 2,5-di-(tert.-butyl)-1,4-benzohydroquinone (tBuBHQ), which has previously been shown to empty the agonist-sensitive endoplasmic reticular Ca2+ store and to stimulate subsequent Ca2+ influx by a capacitative mechanism. The addition of excess [ethylenebis(oxyethylenenitrilo)]tetraacetic acid to thymocytes produced a rapid return to basal [Ca2+]i after tBuBHQ treatment but a similar rapid return to basal [Ca2+]i was not observed after TBT treatment. In addition, TBT produced a marked inhibition of both Ca2+ efflux from the cells and the plasma membrane Ca(2+)-ATPase activity. Also, TBT treatment resulted in a rapid decrease in thymocyte ATP level. Taken together, our results show that TBT increases [Ca2+]i in thymocytes by the combination of intracellular Ca2+ mobilization, stimulation of Ca2+ entry, and inhibition of the Ca2+ efflux process. Furthermore, the ability of TBT to apparently mobilize the tBuBHQ-sensitive intracellular Ca2+ store followed by Ca2+ and Mn2+ entry suggests that the TBT-induced [Ca2+]i increase involves a capacitative type of Ca2+ entry.     

Luminal Ca2+ controls the activation of the inositol 1,4,5-trisphosphate receptor by cytosolic Ca2+.

Luminal Ca2+ controls the sensitivity of the intracellular Ca2+ stores to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Ins(1,4,5)P3-induced Ca2+ release is also controlled by cytosolic Ca2+; low concentrations of Ca2+ stimulate the release. The aim of this work was to investigate whether luminal Ca2+ would affect the stimulation of the Ins(1,4,5)P3 receptor by cytosolic Ca2+ in permeabilized A7r5 smooth muscle cells. We also report that the Ins(1,4,5)P3 receptor in A7r5 cells is activated by low concentrations of cytosolic Ca2+. Cytoplasmic Ca2+ increases the Ins(1,4,5)P3 sensitivity without affecting the cooperativity. The increase in Ins(1,4,5)P3 sensitivity becomes relatively more pronounced when the Ca2+ content of the stores decreases. This modulatory effect of luminal Ca2+ on the responsiveness to cytosolic Ca2+ is an intrinsic property of the Ins(1,4,5)P3 receptor.     

The size of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores depends on inositol 1,4,5-trisphosphate concentration.

An explanation of the complex effects of hormones on intracellular Ca2+ requires that the intracellular actions of Ins(1,4,5)P3 and the relationships between intracellular Ca2+ stores are fully understood. We have examined the kinetics of 45Ca2+ efflux from pre-loaded intracellular stores after stimulation with Ins(1,4,5)P3 or the stable phosphorothioate analogue, Ins(1,4,5)P3[S]3, by simultaneous addition of one of them with glucose/hexokinase to rapidly deplete the medium of ATP. Under these conditions, a maximal concentration of either Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 evoked rapid efflux of about half of the accumulated 45Ca2+, and thereafter the efflux was the same as occurred under control conditions. Submaximal concentrations of Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 caused a smaller rapid initial efflux of 45Ca2+, after which the efflux was similar whatever the concentration of Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 present. The failure of submaximal concentrations of Ins(1,4,5)P3 and Ins(1,4,5)P3[S]3 to mobilize fully the Ins(1,4,5)P3-sensitive Ca2+ stores despite prolonged incubation was not due either to inactivation of Ins(1,4,5)P3 or to desensitization of the Ins(1,4,5)P3 receptor. The results suggest that the size of the Ins(1,4,5)P3 sensitive Ca2+ stores depends upon the concentration of Ins(1,4,5)P3.     

Ca2+-induced exocytosis in individual human neutrophils: high- and low-affinity granule populations and submaximal responses.

We have investigated Ca2+-induced exocytosis from human neutrophils using the whole cell patch-clamp capacitance technique. Microperfusion of Ca2+ buffer solutions (<30 nM to 5 mM free Ca2+) through the patch-clamp pipette revealed a biphasic activation of exocytosis by Ca2+. The first phase was characterized by high affinity (1.5-5 microM) and low apparent cooperativity (<=2) for Ca2+, and the second phase by low affinity (approximately 100 microM) and high cooperativity (>6). Only the second phase was accompanied by loss of myeloperoxidase, suggesting that the low-affinity exocytosis reflected release of peroxidase-positive (primary) granules, while the high-affinity exocytosis reflected release of peroxidase-negative (secondary and tertiary) granules. At submaximal Ca2+ concentrations, only a fraction of a given granule population was released. This submaximal release cannot simply be explained by Ca2+ modulation of the rate of exocytosis, and it suggests that the secretory response of individual cells is adjusted to the strength of the stimulus. The Ca2+ dependence of the high- and low-affinity phases of neutrophil exocytosis bears a resemblance to endocrine and neuronal exocytosis, respectively. The occurrence of such high- and low-affinity exocytosis in the same cell is novel, and suggests that the Ca2+ sensitivity of secretion is granule-, rather than cell-specific.