Subcellular mechanism of desensitization of the ATP-induced Ca2+ mobilization in human umbilical vein endothelial cells: role of intracellular Ca2+ stores

Confluent monolayers of human umbilical vein endothelial cells subcultured on glass coverslips were loaded with the fluorescent Ca2+ indicator, fura-2. Changes in fura-2 fluorescence were detected by means of a fluorescence spectrophotometer. Both ATP and ADP (0.3-100 microM) caused a concentration-dependent transient peak response of the intracellular free calcium concentration ([Ca2+]i), followed by a lower sustained response. AMP and adenosine did not induce detectable changes in [Ca2+]i. The sustained response to ATP was abolished by superfusion with the Ca2(+)-free solution (with 1 mM EGTA), while the transient peak response was uninfluenced. The transient peak response to ATP (30 microM) was inhibited by pre-exposure to ATP in a graded manner depending on the concentration of ATP. The response to ATP recovered after washout for 20 min with the solution containing Ca2+, but not with the Ca2(+)-free solution. The transient peak response to ATP was markedly reduced by preceding exposure to histamine, while the response to histamine was not influenced by pre-exposure to ATP. These findings indicate that depletion and refilling of the ATP-sensitive intracellular Ca2+ store may be responsible for the desensitization and recovery of the ATP-induced [Ca2+]i response. The pharmacological characteristics of the ATP-sensitive intracellular Ca2+ store seem different from those of the histamine-sensitive store.     

Differential regulation of Ca2+ mobilization in human thymocytes by coaggregation of surface molecules.

Variations in intracellular Ca2+ levels in developing thymocytes are likely to play a major role in both the activation-associated differentiation of thymocytes and in the selection or clonal deletion of cells. Here we examine the role of CD4, CD8, CD2, and CD45 in the regulation of intracellular Ca2+ levels in mature and immature thymocytes. Mature and immature thymocytes, distinguished on the basis of their CD5 expression, were analyzed simultaneously for their ability to mobilize Ca2+ after coaggregation of their CD3/TCR with other thymic surface Ag. Flow cytometric analysis by using Indo-1 showed that coaggregation of CD4, CD8, and CD2 with CD3/TCR clearly enhances a minimal signal delivered via CD3/TCR on immature thymocytes. Coaggregation with class I MHC had no discernible effect. The responsiveness of immature thymocytes correlated strictly with CD3 surface expression, such that loss of responsiveness occurred with reduced CD3 cell-surface density. However, even thymocytes with very low CD3 expression were able to respond to triggering via CD3 under optimal conditions, indicating that the CD3 signal-transducing mechanism is functional on early thymic cells. Intracellular increases in Ca2+ concentrations induced via CD3, could effectively be inhibited by cross-linking of CD45 and CD3 on immature thymocytes. Although triggering via CD2 alone induced a strong Ca2+ flux, prolonged incubation with activating anti-CD2 antibodies made thymocytes refractory to subsequent triggering. Refractoriness was associated with partial loss of surface CD3 and CD3 zeta. Our results indicate that thymic surface Ag are differentially involved in the regulation of intracellular Ca2+ levels in immature as well as mature thymocytes.     

Umami changes intracellular Ca2+ levels using intracellular and extracellular sources in mouse taste receptor cells

Recently, candidates for umami receptors have been identified in taste cells, but the precise transduction mechanisms of the downstream receptor remain unknown. To investigate how intracellular Ca(2+) increases in the umami transduction pathway, we measured changes in intracellular Ca(2+) levels in response to umami stimuli monosodium glutamate (MSG), IMP, and MSG + IMP in mouse taste receptor cells (TRCs) by Ca(2+) imaging. Even when extracellular Ca(2+) was absent, 1/3 of umami-responsive TRCs exhibited increased intracellular Ca(2+) levels. When intracellular Ca(2+) was depleted, half of the TRCs retained their response to umami. These results suggest that umami-responsive TRCs increase their intracellular Ca(2+) levels through two pathways: by releasing Ca(2+) from intracellular stores and by an influx of Ca(2+) from extracellular sources. We conclude that the Ca(2+) influx from extracellular source might play an important role in the synergistic effect between MSG and IMP.     

The T-cell antigen receptor regulates sustained increases in cytoplasmic free Ca2+ through extracellular Ca2+ influx and ongoing intracellular Ca2+ mobilization.

Signal transduction by the T-cell antigen receptor involves the turnover of polyphosphoinositides and an increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is due initially to the release of Ca2+ from intracellular stores, but is sustained by the influx of extracellular Ca2+. To examine the regulation of sustained antigen-receptor-mediated increases in [Ca2+]i, we studied the relationships between extracellular Ca2+ influx, the mobilization of Ca2+ from intracellular stores, and the contents of inositol polyphosphates after stimulation of the antigen receptor on a human T-cell line, Jurkat. We demonstrate that sustained antigen-receptor-mediated increases in [Ca2+]i are associated with ongoing depletion of intracellular Ca2+ stores. When antigen-receptor-ligand interactions are disrupted, [Ca2+]i and inositol 1,4,5-trisphosphate return to basal values over 3 min. Under these conditions, intracellular Ca2+ stores are repleted if extracellular Ca2+ is present. There is a tight temporal relationship between the fall in [Ca2+]i, the return of inositol 1,4,5-trisphosphate to basal values, and the repletion of intracellular Ca2+ stores. Reversal of the increase in [Ca2+]i preceeds any fall in inositol tetrakisphosphate by 2 min. These studies suggest that sustained antigen-receptor-induced increases in [Ca2+]i, although dependent on extracellular Ca2+ influx, are also regulated by ongoing inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ mobilization. In addition, an elevated concentration of inositol tetrakisphosphate in itself is insufficient to sustain an increase in [Ca2+]i within Jurkat cells.     

A quantitative kinetic model for ATP-induced intracellular Ca2+ oscillations

A quantitative kinetic model is proposed to simulate the ATP-induced intracellular Ca(2+) oscillations. The quantitative effect of ATP concentration upon the oscillations was successfully simulated. Our simulation results support previous experimental explanations that the Ca(2+) oscillations are mainly due to interaction of Ca(2+) release from the endoplasmic reticulum (ER) and the ATP-dependent Ca(2+) pump back into the ER, and the oscillations are prolonged by extracellular Ca(2+) entry that maintains the constant Ca(2+) supplies to its intracellular stores. The model is also able to simulate the sudden disappearance phenomenon of the Ca(2+) oscillations observed in some cell types by taking into account of the biphasic characteristic of the Ca(2+) release from the endoplasmic reticulum (ER). Moreover, the model simulation results for the Ca(2+) oscillations characteristics such as duration, peak [Ca(2+)](cyt), and average interval, etc., lead to prediction of some possible factors responsible for the variations of Ca(2+) oscillations in different types of cells.     

Differential Ca2+ and Sr2+ regulation of intracellular divalent cations release in ventricular myocytes

The regulation of the Ca2+ -induced Ca2+ release (CICR) from intracellular stores is a critical step in the cardiac cycle. The inherent positive feedback of CICR should make it a self-regenerating process. It is accepted that CICR must be governed by some negative control, but its nature is still debated. We explore here the importance of the Ca2+ released from sarcoplasmic reticulum (SR) on the mechanisms that may control CICR. Specifically, we compared the effect of replacing Ca2+ with Sr2+ on intracellular Ca2+ signaling in intact cardiac myocytes as well as on the function of single ryanodine receptor (RyR) Ca2+ release channels in panar bilayers. In cells, both CICR and Sr2+ -induced Sr2+ release (SISR) were observed. Action potential induced Ca2+ -transients and spontaneous Ca2+ waves were considerably faster than their Sr2+ -mediated counterparts. However, the kinetics of Ca2+ and Sr2+ sparks was similar. At the single RyR channel level, the affinities of Ca2+ and Sr2+ activation were different but the affinities of Ca2+ and Sr2+ inactivation were similar. Fast Ca2+ and Sr2+ stimuli activated RyR channels equally fast but adaptation (a spontaneous slow transition back to steady-state activity levels) was not observed in the Sr2+ case. Together, these results suggest that regulation of the RyR channel by cytosolic Ca2+ is not involved in turning off the Ca2+ spark. In contrast, cytosolic Ca2+ is important in the propagation global Ca2+ release events and in this regard single RyR channel sensitivity to cytosolic Ca2+ activation, not low-affinity cytosolic Ca2+ inactivation, is a key factor. This suggests that the kinetics of local and global RyR-mediated Ca2+ release signals are affected in a distinct way by different divalent cations in cardiac muscle cells.     

Endoplasmic reticulum Ca2+ store: regulation of Ca2+ release and reuptake by intracellular and extracellular Ca2+ in pancreatic acinar cells

We investigated the effect of cytosolic and extracellular Ca2+ on Ca2+ signals in pancreatic acinar cells by measuring Ca2+ concentration in the cytosol([Ca2+]c) and in the lumen of the ER([Ca2+]Lu). To control buffers and dye in the cytosol, a patch-clamp microelectrode was employed. Acetylcholine released Ca2+ mainly from the basolateral ER-rich part of the cell. The rate of Ca2+ release from the ER was highly sensitive to the buffering of [Ca2+]c whereas ER Ca2+ refilling was enhanced by supplying free Ca2+ to the cytosol with [Ca2+]c clamped at resting levels with a patch pipette containing 10 mM BAPTA and 2 mM Ca2+. Elevation of extracellular Ca2+ to 10 mM from 1 mM raised resting [Ca2+]c slightly and often generated [Ca2+]c oscillations in single or clustered cells. Although pancreatic acinar cells are reported to have extracellular Ca2+-sensing receptors linked to phospholipase C that mobilize Ca2+ from the ER, exposure of cells to 10 mM Ca2+ did not decrease [Ca2+]Lu but rather raised it. From these findings we conclude that 1) ER Ca2+ release is strictly regulated by feedback inhibition of [Ca2+]c, 2) ER Ca2+ refilling is determined by the rate of Ca2+ influx and occurs mainly in the tiny subplasmalemmal spaces, 3) extracellular Ca2+-induced [Ca2+]c oscillations appear to be triggered not by activation of extracellular Ca2+-sensing receptors but by the ER sensitised by elevated [Ca2+]c and [Ca2+]Lu.     

Effect of gossypol on intracellular Ca2+ regulation in human hepatoma cells

Gossypol is a natural toxicant present in cottonseeds, and is hepatotoxic to animals and human. The effect of gossypol on cytosolic free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatocytes was explored using fura-2 as a fluorescent Ca2+ indicator. Gossypol increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 2 microM. The Ca2+ signal was reduced by removing extracellular Ca2+ or by 10 microM La3+, but was not affected by nifedipine, verapamil or diltiazem. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ partly reduced 10 microM gossypol-induced Ca2+ release; and conversely pretreatment with gossypol abolished thapsigargin-induced Ca2+ release. The Ca2+ release induced by 10 microM gossypol was not changed by inhibiting phospholipase C with 2 microM U73122 or by depleting ryanodine-sensitive Ca2+ stores with 50 microM ryanodine. Together, the results suggest that in human hepatocytes, gossypol induced a [Ca2+]i increase by causing store Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and by inducing Ca2+ influx.     

Suppression of EGF-induced cell proliferation by the blockade of Ca2+ mobilization and capacitative Ca2+ entry in mouse mammary epithelial cells

The role of intracellular Ca2+ stores and capacitative Ca2+ entry on EGF-induced cell proliferation was investigated in mouse mammary epithelial cells. We have previously demonstrated that EGF enhances Ca2+ mobilization (release of Ca2+ from intracellular Ca2+ stores) and capacitative Ca2+ entry correlated with cell proliferation in mouse mammary epithelial cells. To confirm their role on EGF-induced cell cycle progression, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), a reversible inhibitor of the Ca2+ pump of intracellular Ca2+ stores, and SK&F 96365, a blocker of capacitative Ca2+ entry, on mitotic activity induced by EGF. Mitotic activity was examined using an antibody to PCNA for immunocytochemistry. SK&F 96365 inhibited capacitative Ca2+ entry in a dose-dependent manner (I50: 1-5 microM). SK&F 96365 also inhibited EGF-induced cell proliferation in the same range of concentration (I50: 1-5 microM). DBHQ suppressed [Ca2+]i response to UTP and thus depleted completely Ca2+ stores at 5 microM. DBHQ also inhibited EGF-induced cell proliferation at an I50 value of approximately 10 microM. The removal of these inhibitors from the culture medium increased the reduced mitotic activity reversibly. Using a fluorescent assay of DNA binding of ethidium bromide, no dead cells were detected in any of the cultures. These results indicate that the inhibitory effects of SK&F 96365 and DBHQ on cell proliferation were due to the inhibition of capacitative Ca2+ entry and Ca2+ mobilization suggesting the importance of capacitative Ca2+ entry and Ca2+ mobilization in the control of EGF-induced cell cycle progression in mouse mammary epithelial cells.     

Effect of intracellular Na+ on platelet aggregation and Ca2+ mobilization

The effects of ionophores, which can carry alkali metal cations, on platelet aggregation were examined. At an alkaline extracellular pH, alkali metal cation/H+ exchanger nigericin accelerated aggregation in K+-enriched medium, whereas it rather inhibited aggregation in Na+-enriched medium, even though the intracellular pH was only slightly alkaline. The inhibitory effect of Na+ on platelet aggregation was more clearly shown with the alkali metal cation exchanger gramicidin D. The ionophore had no effect or a slightly accelerative effect on aggregation in K+-enriched medium, whereas it significantly inhibited aggregation induced by thrombin, ADP and platelet activating factor in Na+-enriched medium. Fluorescence studies on fura-2-labeled platelets revealed that in Na+-enriched medium gramicidin D inhibited agonist-induced Ca2+ mobilization both in the presence and absence of extracellular Ca2+. These results suggest that the intracellular Na+ inhibits platelet aggregation by inhibiting Ca2+ mobilization.     

Na+/Ca2+ exchange in catfish retina horizontal cells: regulation of intracellular Ca2+ store function

The role of theNa⁺/Ca²⁺exchanger in intracellular Ca²⁺regulation was investigated in freshly dissociated catfish retinalhorizontal cells (HC).Ca²⁺-permeable glutamate receptorsand L-type Ca²⁺ channels as wellas inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitiveintracellular Ca²⁺ stores regulateintracellular Ca²⁺ in these cells.We used the Ca²⁺-sensitive dyefluo 3 to measure changes in intracellularCa²⁺ concentration([Ca²⁺]_i)under conditions in whichNa⁺/Ca²⁺exchange was altered. In addition, the role of theNa⁺/Ca²⁺exchanger in the refilling of the caffeine-sensitiveCa²⁺ store followingcaffeine-stimulated Ca²⁺ releasewas assessed. Brief applications of caffeine (1-10 s) producedrapid and transient changes in[Ca²⁺]_i.Repeated applications of caffeine produced smallerCa²⁺ transients until no furtherCa²⁺ was released. Store refillingoccurred within 1-2 min and required extracellularCa²⁺. Ouabain-induced increases inintracellular Na⁺ concentration([Na⁺]_i)increased both basal free[Ca²⁺]_iand caffeine-stimulated Ca²⁺release. Reduction of external Na⁺concentration([Na⁺]_o)further and reversibly increased[Ca²⁺]_iin ouabain-treated HC. This effect was not abolished by the Ca²⁺ channel blocker nifedipine,suggesting that increases in[Na⁺]_ipromote net extracellular Ca²⁺influx through aNa⁺/Ca²⁺exchanger. Moreover, when[Na⁺]_owas replaced by Li⁺, caffeine didnot stimulate release of Ca²⁺ fromthe caffeine-sensitive store afterCa²⁺ depletion. TheNa⁺/Ca²⁺exchanger inhibitor 2',4'-dimethylbenzamil significantlyreduced the caffeine-evoked Ca²⁺response 1 and 2 min after store depletion.

     

Differentiation between Ca2+ transport and ATP-induced Ca2+ binding by sarcoplasmic reticulum

The Ca²⁺ actively accumulated by sarcoplasmic reticulum isolated from skeletal muscle is composed of two fractions; one represented by intravesicular free Ca²⁺ and another represented by Ca²⁺ selectively bound to the membranes. Both of these Ca²⁺ fractions depend on ATP, although it is not clear whether ATP hydrolysis is essential for accumulation of the second Ca²⁺ fraction. The existence of the membrane-bound Ca²⁺ induced by ATP is clearly shown in experiments in which the Ca²⁺ retention by sarcoplasmic reticulum is measured in the presence and in the absence of X-537A, a Ca²⁺ ionophore, which makes the membrane permeable to Ca²⁺. Thus, in the presence of X-537A all Ca²⁺ accumulated due to ATP is bound to the membranes. This membrane-bound Ca²⁺ represents about 30 nmol/mg protein in the range of external $\text{p}\text{Ca}$ values of 7 to 3.5. The magnitude of this Ca²⁺ fraction is slightly higher whether or not the experiments are performed in the presence of oxalate, which greatly increased the intravesicular Ca²⁺ accumulation. Furthermore, taking advantage of the impermeability of sarcoplasmic reticulum to EGTA, it is possible to show the existence of the membrane-bound Ca²⁺ as a distinct fraction from that which exists intravesicularly.     

Alpha 2-adrenergic receptor stimulation mobilizes intracellular Ca2+ in human erythroleukemia cells

Human erythroleukemia cells are a model system for studies of alpha 2-adrenergic receptors and their coupling to inhibition of adenylate cyclase (McKernan, R. M., Howard, M. J., Motulsky, H. J., and Insel, P. A. (1987) Mol. Pharmacol. 32, 258-265). Using Fura-2, we show that alpha 2-adrenergic receptor stimulation also increases intracellular Ca2+ in these cells by 80-250 nM. Although epinephrine only inhibited forskolin-stimulated cAMP generation when beta-adrenergic receptors were blocked, the Ca2+ increase was not affected by beta-adrenergic receptor blockade. The Ca2+ increase was not affected by forskolin or 8-bromo-cAMP. Thus, alpha 2-adrenergic receptors independently couple to elevation of intracellular Ca2+ and adenylate cyclase inhibition. Chelating all extracellular Ca2+ did not reduce the response, demonstrating mobilization of intracellular, rather than influx of extracellular Ca2+. The epinephrine-stimulated Ca2+ mobilization occurred prior to any detectable increase in inositol-(1,4,5)-trisphosphate. It was abolished by pretreatment with pertussis toxin (which blocks some G protein-mediated processes), but not by aspirin and indomethacin (which inhibit cyclooxygenase), nordihydroguaiaretic acid (which inhibits lipoxygenase), or Na+-free buffer (to block any Na+H+ exchange). We conclude, therefore, that alpha 2-adrenergic receptors on human erythroleukemia cells couple to mobilization of intracellular Ca2+ via a (pertussis toxin-sensitive) G protein-mediated mechanism that is independent of inhibition of adenylate cyclase.     

Caffeine does not inhibit substance P-evoked intracellular Ca2+ mobilization in rat salivary acinar cells

We used theCa²⁺-sensitive fluorescent dyefura 2, together with measurements of intracellularD-myo-inositol1,4,5-trisphosphate [Ins(1,4,5)P₃],to assess the inhibitory effects of caffeine on signal transduction viaG protein-coupled receptor pathways in isolated rat mandibular salivaryacinar cells. ACh, norepinephrine (NE), and substance P (SP) all evokedsubstantial increases in the intracellular freeCa²⁺ concentration([Ca²⁺]_i).Responses to ACh and NE were markedly inhibited by prior application of20 mM caffeine. The inhibitory effect of caffeine was not reproduced byphosphodiesterase inhibition with IBMX or addition of cell-permeantdibutyryl cAMP. In contrast to the ACh and NE responses, the[Ca²⁺]_iresponse to SP was unaffected by caffeine. Despite this, SP and AChappeared to mobilize Ca²⁺ from acommon intracellular pool. Measurements of agonist-induced changes inIns(1,4,5)P₃levels confirmed that caffeine inhibited the stimulus-response couplingpathway at a point beforeIns(1,4,5)P₃ generation. Caffeine did not, however, inhibit[Ca²⁺]_iresponses evoked by direct activation of G proteins with 40 mMF. These data show thatcaffeine inhibits G protein-coupled signal transduction in these cellsat some element that is common to the muscarinic and -adrenergicsignaling pathways but is not shared by the SP signaling pathway. Wesuggest that this element might be a specific structural motif on the Gprotein-coupled muscarinic and -adrenergic receptors.     

Depolarization of the membrane potential decreases the ATP-induced influx of extracellular Ca2+ and the refilling of intracellular Ca2+ stores in rat thyroid FRTL-5 cells.

The aim of the present study was to investigate the effect of membrane depolarization on ATP-induced changes in intracellular Ca2+ ([Ca2+]i) and the refilling of intracellular Ca2+ stores in thyroid follicular FRTL-5 cells. Depolarizing the cells with 50 mM K+, an amount sufficient to almost totally depolarize the cells as determined by bisoxonal, significantly reduced the ATP-induced uptake of 45Ca2+. This effect was not dependent on an enhanced efflux of Ca2+, as no difference in the ATP-induced efflux of 45Ca2+ was obtained between control cells and depolarized cells. The ATP-induced transient increase in [Ca2+]i in Fura-2 loaded cells was not altered by depolarization, whereas the ATP-induced plateau in [Ca2+]i was decreased compared with control cells. Furthermore, in cells stimulated with ATP in a Ca(2+)-free buffer, readdition of Ca2+ after the termination of the ATP response induced a decreased response in [Ca2+]i in depolarized cells. Refilling of intracellular Ca2+ stores was investigated by first stimulating the cells with noradrenaline (NA). The effect of NA was then terminated with prazosin, and the cells restimulated with ATP. In cells depolarized with high K+, the response to ATP was decreased compared with that seen in control cells. The results thus suggest that both the ATP-induced influx of extracellular Ca2+ and the refilling of intracellular Ca2+ stores is decreased in depolarized FRTL-5 cells.     

Translocation of alpha subunits of stimulatory guanine nucleotide-binding proteins through stimulation of the prostacyclin receptor in mouse mastocytoma cells.

The translocation of the alpha subunits of Gs from the membrane to the cytosol by iloprost, a stable prostacyclin analogue, was studied in mouse mastocytoma P-815 cells. In the presence of guanosine 5'-O-(thiotriphosphate) (GTP gamma S), iloprost stimulated the adenylate cyclase activity, caused the release of both 42- and 45-kDa proteins reactive with the anti Gs alpha carboxyl-terminal antibody, RM/1, from the membrane and attenuated cholera toxin-catalyzed ADP-ribosylation of the 42- and 45-kDa proteins in the membrane. The iloprost-stimulated adenylate cyclase activity and release of Gs alpha from the membrane were markedly suppressed by RM/1. Cholera toxin treatment also stimulated the adenylate cyclase activity and release of Gs alpha from the membrane, and iloprost synergistically potentiated these actions of cholera toxin. In mastocytoma cells, iloprost induced the translocation of both 42- and 45-kDa Gs alpha from the membrane to the cytosol, 45-kDa Gs alpha remaining in the cytosol for a longer time than 42- kDa Gs alpha. Whereas 42-kDa Gs alpha in the cytosol was eluted at the position of Mr = approximately 40,000 45-kDa Gs alpha was eluted at the position of Mr = approximately 120,000 from a Superose 12 gel filtration column. In contrast, both 42- and 45-kDa Gs alpha released in vitro from the membrane by iloprost plus GTP gamma S were eluted at the position of Mr = approximately 40,000, but only 45-kDa Gs alpha was eluted at the position of Mr = approximately 120,000 when it was incubated with cytosol. These results taken together demonstrate that iloprost induces the translocation of both 42- and 45-kDa Gs alpha from the membrane to the cytosol and that only the 45-kDa Gs alpha released exists in the cytosol as a soluble complex with unidentified component(s) in mastocytoma cells.     

Modulation of CCK-8-evoked intracellular Ca2+ waves by hydrogen peroxide in mouse pancreatic acinar cells.

In the present study we have employed single cell imaging analysis to monitor the propagation of cholecystokinin-evoked Ca(2+) waves in mouse pancreatic acinar cells. Stimulation of cells with 1 nM CCK-8 led to an initial Ca(2+) release at the luminal cell pole and subsequent spreading of the Ca(2+) signal towards the basolateral membrane in the form of a Ca(2+) wave. Inhibition of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) activity by 1 microM thapsigargin, preincubation in the presence of 100 microM H(2)O(2) or inhibition of PKC with either 5 microM Ro31-8220 or 3 microM GF-109203-X all led to a faster propagation of CCK-8-induced Ca(2+) signals. The propagation of CCK-8-evoked Ca(2+) signals was slowed down by activation of PKC with 1 microM PMA, and preincubation of cells in the presence of H(2)O(2) counteracted the effect of PKC inhibition. The protonophore FCCP (100 nM) and the inhibitor of the mitochondrial Ca(2+)-uniporter Ru360 (10 microM) led to an increase in the propagation rate of CCK-8-evoked Ca(2+) waves. Finally, depolymerisation of actin cytoskeleton with cytochalasin D (10 microM) led to a faster propagation of CCK-8-evoked Ca(2+) signals. Stabilization of actin cytoskeleton with jasplakinolide (10 microM) did not induce significant changes on CCK-8-evoked Ca(2+) waves. Preincubation of cells in the presence of H(2)O(2) counteracted the effect of cytochalasin D on CCK-8-evoked Ca(2+) wave propagation. Our results suggest that spreading of cytosolic Ca(2+) waves evoked by CCK-8 can be modulated by low levels of oxidants acting on multiple Ca(2+)-handling mechanisms.     

Inositol 1,4,5-trisphosphate-induced Ca2+ release from permeabilized mastocytoma cells

The effect of inositol 1,4,5-trisphosphate (IP₃) on Ca²⁺ release in the transformed murine mast cells, mastocytoma P-815 cells permeabilized with digitonin was studied. Ca²⁺ was sequestered by intracellular organelles in the presence of ATP until the medium free Ca²⁺ concentration was lowered to a new steady-state level. The subsequent addition of IP₃ caused a rapid Ca²⁺ release, which was followed by a slow re-uptake of Ca²⁺. Fifty percent of the sequestered Ca²⁺ was released by 10 μM IP₃. Maximal Ca²⁺ release occurred at 10 μM and half maximal activity was at 1.3 μM. These results indicate that IP₃ may function as a messenger of intracellular Ca²⁺ mobilization in mastocytoma cells.     

Endothelin-induced intracellular Ca2+ mobilization through its specific receptors in murine peritoneal macrophages.

We studied the presence of specific binding sites for endothelin (ET) and the effect of ET on cytosolic free Ca2+ concentration ([Ca2+]i) in murine thioglycolate-activated peritoneal macrophages. Scatchard analysis for binding experiments using [125I]ET-1 or [125I]ET-3 revealed the existence of a single class of binding sites. The binding parameters (Kd and Bmax) for [125I]ET-1 were almost identical to those for [125I]ET-3. In addition, unlabeled 3 ET isopeptides (ET-1, ET-2 and ET-3) inhibited the specific binding of both ET-1 and ET-3 with similar inhibitory potencies. All 3 ET isopeptides caused an increase in [Ca2+]i in the same dose-dependent manner (0.01-100 nM). These results demonstrate the existence of an ET receptor with the same affinity for all isoforms that mediates the ET-induced intracellular Ca2+ mobilization in murine peritoneal macrophages.