Signaling between intracellular Ca2+ stores and depletion-activated Ca2+ channels generates [Ca2+]i oscillations in T lymphocytes

Stimulation through the antigen receptor (TCR) of T lymphocytes triggers cytosolic calcium ([Ca2+]i) oscillations that are critically dependent on Ca2+ entry across the plasma membrane. We have investigated the roles of Ca2+ influx and depletion of intracellular Ca2+ stores in the oscillation mechanism, using single-cell Ca2+ imaging techniques and agents that deplete the stores. Thapsigargin (TG; 5-25 nM), cyclopiazonic acid (CPA; 5-20 microM), and tert- butylhydroquinone (tBHQ; 80-200 microM), inhibitors of endoplasmic reticulum Ca(2+)-ATPases, as well as the Ca2+ ionophore ionomycin (5-40 nM), elicit [Ca2+]i oscillations in human T cells. The oscillation frequency is approximately 5 mHz (for ATPase inhibitors) to approximately 10 mHz (for ionomycin) at 22-24 degrees C. The [Ca2+]i oscillations resemble those evoked by TCR ligation in terms of their shape, amplitude, and an absolute dependence on Ca2+ influx. Ca(2+)- ATPase inhibitors and ionomycin induce oscillations only within a narrow range of drug concentrations that are expected to cause partial depletion of intracellular stores. Ca(2+)-induced Ca2+ release does not appear to be significantly involved, as rapid removal of extracellular Ca2+ elicits the same rate of [Ca2+]i decline during the rising and falling phases of the oscillation cycle. Both transmembrane Ca2+ influx and the content of ionomycin-releasable Ca2+ pools fluctuate in oscillating cells. From these data, we propose a model in which [Ca2+]i oscillations in T cells result from the interaction between intracellular Ca2+ stores and depletion-activated Ca2+ channels in the plasma membrane.     

Muscarinic acetylcholine receptors stimulate Ca2+ influx in PC12D cells predominantly via activation of Ca2+ store-operated channels

Activation of muscarinic acetylcholine receptors (mAChRs) causes the rapid release of Ca2+ from intracellular stores and a sustained influx of external Ca2+ in PC12D cells, a subline of the widely studied cell line PC12. Release of Ca2+ from intracellular stores and a sustained influx of Ca2+ are also observed following exposure to thapsigargin, a sesquiterpene lactone that depletes intracellular Ca2+ pools by irreversibly inhibiting the Ca2+ pump of the endoplasmic reticulum. In this study, we show that carbachol and thapsigargin empty the same intracellular Ca2+ stores, and that these stores are a subset of intracellular stores depleted by the Ca2+ ionophore ionomycin. Intracellular Ca2+ stores remain depleted during continuous stimulation of mAChR with carbachol in medium containing 2 mM extracellular Ca2+, but rapidly refill following inhibition of mAChRs with atropine. Addition of atropine to carbachol-stimulated cells causes intracellular Ca2+ levels to return to baseline levels in two steps: a rapid decrease that correlates with the reuptake of Ca2+ into internal stores and a delayed decrease that correlates with the inhibition of a Mn2+-permeable Ca2+ channel. Several lines of evidence suggest that carbachol and thapsigargin stimulate Ca2+ influx by a common mechanism: (i) pretreatment with thapsigargin occludes atropine-mediated inhibition of Ca2+ influx, (ii) carbachol and thapsigargin applied individually or together are equally efficient at stimulating the influx of Mn2+, and (iii) identical rates of Ca2+ influx are observed when Ca2+ is added to cells pretreated with carbachol, thapsigargin, or both agents in the absence of extracellular Ca2+. Taken together, these data suggest that the sustained influx of extracellular Ca2+ observed following activation of mAChRs in PC12D cells is mediated primarily by activation of a Mn2+-permeable, Ca2+ store-operated Ca2+ channel.     

Mechanisms for activation and subsequent removal of cytosolic Ca2+ in bradykinin-stimulated neuronal and glial cell lines

Mechanisms for activation and for removal of cytosolic Ca2+ after stimulation with bradykinin were investigated in two neural cell lines by measuring cytosolic Ca2+ activity and 45Ca2+ fluxes. In the neuronal (neuroblastoma x glioma hybrid) and in the glial (rat glioma) cell lines, the transient, bradykinin-induced rise in cytosolic Ca2+ activity (determined by fura-2 or indo-1 fluorescence) was blocked by a bradykinin B2 receptor antagonist. Ca2+ ionophores (ionomycin and 4-Br-A23187) caused a comparable transient rise in cytosolic Ca2+ activity. After addition of ionophores, the Ca2+ response to bradykinin was reduced or completely blocked in both cell lines. At the concentrations used, the ionophores primarily depleted intracellular Ca2+ stores and prevented refilling of the stores. Thus, the bradykinin-induced rise of cytosolic Ca2+ activity seems to be mostly due to Ca2+ release from internal stores. In the neuronal but not in the glial cell line, a brief stimulation by bradykinin of 45Ca2+ uptake was followed by a long-lasting inhibition below control values. Thus, in the neuronal cells bradykinin presumably blocks Ca2+ channels by a readily reversible, pertussis toxin-insensitive mechanism. Excess cytosolic Ca2+ of the bradykinin-stimulated cells is mostly not resequestered into the internal Ca2+ pool accessible to bradykinin, but is mainly extruded through the plasma membrane, as indicated by (i) stimulation of 45Ca2+ release by bradykinin, (ii) quick reduction by bradykinin of cellular 45Ca2+ content of cells preequilibrated with 45Ca2+, and (iii) diminution of the ionophore-inducible Ca2+ response after the addition of bradykinin.     

Na+-Ca2+ exchange and Ca2+ efflux in transfected Chinese hamster ovary cells

The objective of this study was to assess the contribution of Na+-Ca2+ exchange activity to Ca2+ efflux at various cytosolic Ca2+ concentrations ([Ca2+]i) in transfected Chinese hamster cells expressing the bovine cardiac Na+-Ca2+ exchanger. Ionomycin was added to fura-2 loaded cells and the resulting [Ca2+]i transient was monitored in Ca2+-free media with or without extracellular Na+. The presence of Na+ reduced both the amplitude and duration of the [Ca2+]i transient. Na+ had similar effects when the peak of the [Ca2+]i transient was buffered to 100 nM by cytosolic EGTA, or when Ca2+ was slowly released from internal stores with thapsigargin. Ca2+ efflux following ionomycin addition was directly measured with extracellular fura-2 and followed a biphasic time course (t(1/2) approximately = 10 s and 90s). The proportion of total efflux owing to the rapid phase was increased by Na+ and reduced by EGTA-loading. Na+ accelerated the initial rate of Ca2+ efflux by 65% in unloaded cells but only by 16% in EGTA-loaded cells. In both cases, the stimulation by Na+ was less than expected, given the pronounced effects of Na+ on the [Ca2+]i transient. We conclude that the exchanger contributes importantly to Ca2+ efflux activity at all [Ca2+]i values above 40 nM. We also suggest that Ca2+ efflux pathways may involve non-cytosolic or local routes of Ca2+ traffic.     

Interaction between mitogens upon intracellular Ca2+ pools in murine fibroblasts.

A comparison of the effect of platelet-derived growth factor (PDGF) and bombesin on intracellular Ca2+ stores was carried out in Swiss 3T3 cells loaded with Fura-2. It was found that the tumor promoter thapsigargin (Tg) almost completely inhibited both the PDGF- and the bombesin-induced intracellular Ca2+ concentration ([Ca2+]i) rise, indicating that the two mitogens mobilize Ca2+ from intracellular pool(s) sensitive to the tumor promoter. It was also found that pre-treatment with PDGF almost totally and persistently (up to at least 30 min) inhibited the bombesin-, Tg- and ionomycin-induced rise in [Ca2+]i, whereas pre-treatment with bombesin had only a partial inhibitory effect on the PDGF, Tg and ionomycin [Ca2+]i response, both in the absence and in the presence of external Ca2+. On the other hand, vasopressin and bradykinin, which also stimulate hydrolysis of phosphoinositides in these cells, did not affect the [Ca2+]i response induced by the same agents. These results indicate that, despite the poor production of inositol 1,4,5-trisphosphate (InsP3), PDGF was capable of totally discharging and maintaining discharged the InsP3-sensitive stores of intracellular Ca2+, regardless of whether extracellular Ca2+ was present in the medium. Bombesin only partially caused this effect. On the contrary, bradykinin and vasopressin, after releasing intracellular Ca2+ allowed an almost total refilling of the pools. It is interesting to note that, at variance with PDGF and bombesin, neither bradykinin nor vasopressin are able to induce a mitogenic response in Swiss 3T3 cells.     

Regulation of Ca2+ influx during mitosis: Ca2+ influx and depletion of intracellular Ca2+ stores are coupled in interphase but not mitosis.

Activation of a wide variety of membrane receptors leads to a sustained elevation of intracellular Ca2+ ([Ca2+]i) that is pivotal to subsequent cell responses. In general, in nonexcitable cells this elevation of [Ca2+]i results from two sources: an initial release of Ca2+ from intracellular stores followed by an influx of extracellular Ca2+. These two phases, release from intracellular stores and Ca2+ influx, are generally coupled: stimulation of influx is coordinated with depletion of Ca2+ from stores, although the mechanism of coupling is unclear. We have previously shown that histamine effects a typical [Ca2+]i response in interphase HeLa cells: a rapid rise in [Ca2+]i followed by a sustained elevation, the latter dependent entirely on extracellular Ca2+. In mitotic cells only the initial elevation, derived by Ca2+ release from intracellular stores, occurs. Thus, in mitotic cells the coupling of stores to influx may be specifically broken. In this report we first provide additional evidence that histamine-stimulated Ca2+ influx is strongly inhibited in mitotic cells. We show that efflux is also strongly stimulated by histamine in interphase cells but not in mitotics. It is possible, thus, that in mitotics intracellular stores are only very briefly depleted of Ca2+, being replenished by reuptake of Ca2+ that is retained within the cell. To ensure the depletion of Ca2+ stores in mitotic cells, we employed the sesquiterpenelactone, thapsigargin, that is known to affect the selective release of Ca2+ from intracellular stores by inhibition of a specific Ca(2+)-ATPase; reuptake is inhibited. In most cells, and in accord with Putney's capacitative model (1990), thapsigargin, presumably by depleting intracellular Ca2+ stores, stimulates Ca2+ influx. This is the case for interphase HeLa cells. Thapsigargin induces an increase in [Ca2+]i that is dependent on extracellular Ca2+ and is associated with a strong stimulation of 45Ca2+ influx. In mitotic cells thapsigargin also induces a [Ca2+]i elevation that is initially comparable in magnitude and largely independent of extracellular Ca2+. However, unlike interphase cells, in mitotic cells the elevation of [Ca2+]i is not sustained and 45Ca2+ influx is not stimulated by thapsigargin. Thus, the coupling between depletion of intracellular stores and Ca2+ influx is specifically broken in mitotic cells. Uncoupling could account for the failure of histamine to stimulate Ca2+ influx during mitosis and would effectively block all stimuli whose effects are mediated by Ca2+ influx and sustained elevations of [Ca2+]i.     

Effect of diethylstilbestrol on Ca2+ handling and cell viability in human breast cancer cells

In human breast cancer cells, the effect of the widely prescribed estrogen diethylstilbestrol (DES) on intracellular Ca2+ concentrations ([Ca2+]i) and cell viability was explored by using fura-2 and trypan blue exclusion, respectively. DES caused a rise in [Ca2+]i in a concentration-dependent manner (EC50 = 15 microM). DES-induced [Ca2+]i rise was reduced by 80 % by removal of extracellular Ca2+. DES-induced Mn(2+)-associated quench of intracellular fura-2 fluorescence also suggests that DES induced extracellular Ca2+ influx. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of DES on [Ca2+]i was greatly inhibited. Conversely, pretreatment with DES to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+, whereas ionomycin added afterward still released some Ca2+. These findings suggest that in human breast cancer cells, DES increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum. Acute trypan blue exclusion studies suggest that 10-20 NM DES killed cells in a time-dependent manner.     

Intracellular Ca2+ stores regulate muscarinic receptor stimulation of phospholipase C in cerebellar granule cells

Muscarinic receptor activation of phosphoinositide phospholipase C (PLC) has been examined in rat cerebellar granule cells under conditions that modify intracellular Ca2+ stores. Exposure of cells to medium devoid of Ca2+ for various times reduced carbachol stimulation of PLC with a substantial loss (88%) seen at 30 min. A progressive recovery of responses was observed following the reexposure of cells to Ca2+-containing medium (1.3 mM). However, these changes did not appear to result exclusively from changes in the cytosolic Ca2+ concentration ([Ca2+]i), which decreased to a lower steady level (approximately 25 nM decrease in 1-3 min after extracellular omission) and rapidly returned (within 1 min) to control values when extracellular Ca2+ was restored. Only after loading of the intracellular Ca2+ stores through a transient 1-min depolarization of cerebellar granule cells with 40 mM KCl, followed by washing in nondepolarizing buffer, was carbachol able to mobilize intracellular Ca2+. However, the same treatment resulted in an 80% enhancement of carbachol activation of PLC. In other experiments, partial depletion of the Ca2+ stores by pretreatment of cells with thapsigargin and caffeine resulted in an inhibition (18 and 52%, respectively) of the PLC response. Furthermore, chelation of cytosolic Ca2+ with BAPTA/AM did not influence muscarinic activation of PLC in either the control or predepolarized cells. These conditions, however, inhibited both the increase in [Ca2+]i and the PLC activation elicited by 40 mM KCl and abolished carbachol-induced intracellular Ca2+ release in predepolarized cells. Overall, these results suggest that muscarinic receptor activation of PLC in cerebellar granule cells can be modulated by changes in the loading state of the Ca2+ stores.     

Functional homogeneity of the non-mitochondrial Ca2+ pool in intact mouse lacrimal acinar cells.

In the absence of extracellular Ca2+, treatment of mouse lacrimal acinar cells with maximal concentrations of methacholine released Ca2+ from intracellular stores. No additional Ca2+ was mobilized by subsequent application of the intracellular Ca(2+)-ATPase inhibitor, thapsigargin, the stable inositol 1,4,5-trisphosphate ((1,4,5)IP3) analog, inositol 2,4,5-trisphosphate ((2,4,5)IP3) (by microinjection), or the Ca2+ ionophore, ionomycin. However, following prolonged activation of cells by methacholine in the presence of extracellular Ca2+, Ca2+ accumulated into a pool which was released by ionomycin but not by thapsigargin. This latter accumulation was blocked by prior microinjection of ruthenium red, indicating that it represents mitochondrial uptake. In saponin-permeabilized lacrimal cells, two Ca(2+)-sequestering pools were detected: (i) a ruthenium red-sensitive, thapsigargin-insensitive pool, presumed to be the mitochondria; and (ii) a ruthenium red-insensitive, thapsigargin-sensitive pool. Only the thapsigargin-sensitive pool accumulated Ca2+ at concentrations similar to those in unstimulated cells. The thapsigargin-sensitive Ca2+ pool was sensitive to (1,4,5)IP3; however, in contrast to findings in intact cells, only 44% of this pool was releasable by (1,4,5)IP3 or (2,4,5)IP3. These data indicate that, in intact lacrimal acinar cells, all exchangeable (ionomycin-sensitive) Ca2+ residues in a pool which responds homogeneously to agonists, (1,4,5)IP3, and thapsigargin. Prolonged elevation of [Ca2+]i results in Ca2+ accumulation into a second, ruthenium red-sensitive pool, presumably mitochondria. Finally, permeabilization of the cells fragments the non-mitochondrial pool, resulting in two pools, one sensitive and one insensitive to (1,4,5)IP3.     

Dissociation of Ca2+ entry and Ca2+ mobilization responses to angiotensin II in bovine adrenal chromaffin cells

In fura-2-loaded bovine adrenal chromaffin cells, 0.5 microM angiotensin II (AII) stimulated a 185 +/- 19 nM increase of intracellular-free calcium [( Ca2+]i) approximately 3 s after addition. The time from the onset of the response until achieving 50% recovery (t 1/2) was 67 +/- 10 s. Concomitantly, AII stimulated both the release of 45Ca2+ from prelabeled cells, and a 4-5-fold increase of [3H]inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) levels. In the presence of 50 microM LaCl3, or when extracellular-free Ca2+ [( Ca2+]o) was less than 100 nM, AII still rapidly increased [Ca2+]i by 95-135 nM, but the t 1/2 for recovery was then only 23-27 s. In medium with 1 mM MnCl2 present, AII also stimulated a small amount of Mn2+ influx, as judged by quenching of the fura-2 signal. When [Ca2+]o was normal (1.1 mM) or low (less than 60 nM), 1-2 microM ionomycin caused [Ca2+]i to increase 204 +/- 26 nM, while also releasing 45-55% of bound 45Ca2+. With low [Ca2+]o, ionomycin pretreatment abolished both the [Ca2+]i increase and 45Ca2+ release stimulated by AII. However, after ionomycin pretreatment in normal medium, AII produced a La3+-inhibitable increase of [Ca2+]i (103 +/- 13 nM) with a t 1/2 of 89 +/- 8 s, but no 45Ca2+ release. No pretreatment condition altered AII-induced formation of [3H]Ins(1,4,5)P3. We conclude that AII increased [Ca2+]i via rapid and transient Ca2+ mobilization from Ins(1,4,5)P3- and ionomycin-sensitive stores, accompanied (and/or followed) by Ca2+ entry through a La3+-inhibitable divalent cation pathway. Furthermore, the ability of AII to activate Ca2+ entry in the absence of Ca2+ mobilization (i.e. after ionomycin pretreatment) suggests a receptor-linked stimulus other than Ca2+ mobilization initiates Ca2+ entry.     

The thapsigargin-sensitive intracellular Ca2+ pool is more important in plasma membrane Ca2+ entry than the IP3-sensitive intracellular Ca2+ pool in neuronal cell lines

In NG108-15 cells, bradykinin (BK) and thapsigargin (TG) caused transient increases in a cytosolic free Ca2+ concentration ([Ca2+]i), after which [Ca2+]i elevated by TG only declined to a higher, sustained level than an unstimulated level. In PC12 cells, carbachol (CCh) evoked a transient increase in [Ca2+]i followed by a sustained rise of [Ca2+]i, whereas [Ca2+]i elevated by TG almost maintained its higher level. In the absence of extracellular Ca2+, the sustained elevation of [Ca2+]i induced by each drug we used was abolished. In addition, the rise in [Ca2+]i stimulated by TG was less affected after CCh or BK, whereas CCh or BK caused no increase in [Ca2+]i after TG. TG neither increased cellular inositol phosphates nor modified the inositol phosphates format on stimulated by CCh or BK. We conclude that TG may release Ca2+ from both IP3-sensitive and -insensitive intracellular pools and that some kinds of signalling to link the intracellular Ca2+ pools and Ca2+ entry seem to exist in neuronal cells.     

Thapsigargin increases cytoplasmic free Ca2+ without influencing steroidogenesis in chicken granulosa cells.

The effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) and progesterone production were determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye Fura-2. Thapsigargin stimulated a 4.6 +/- 0.2-fold increase in [Ca2+]i from a resting level of 55 +/- 6 nM up to 233 +/- 23 nM (n = 8) in 100% of the cells tested (n = 86). However, two different response patterns were observed. Dependent on the cell populations, a maximally effective concentration of thapsigargin (100 nM) stimulated either a rapid (within 16 +/- 2 s) transient increase in [Ca2+]i or a slowly (99 +/- 20 s) developing and sustained increase in [Ca2+]i. Both [Ca2+]i responses were concentration (0.001-1 microM)-dependent with an EC50 around 40 nM. The transient [Ca2+]i response occurred in the absence of extracellular Ca2+ and was unaffected by pretreating the cells with the Ca2+ channel blockers methoxyverapamil (50 microM) or lanthanum (1 mM). The plateau phase of the sustained [Ca2+]i response returned to resting level in the absence of extracellular Ca2+, but remained elevated in the presence of methoxyverapamil (50 microM) or lanthanum (1 mM). Despite its ability to cause transient or prolonged increases in [Ca2+]i, thapsigargin (0.001-1 microM) did not affect basal or luteinizing hormone-stimulated progesterone production by chicken granulosa cells.     

The antidepressant mirtazapine-induced cytosolic Ca2+ elevation and cytotoxicity in human osteosarcoma cells

The effect of the antidepressant mirtazapine on cytosolic free Ca2+ concentration ([Ca2+]i) and viability has not been explored in any cell type. This study examined whether mirtazapine alters Ca2+ levels and causes cell death in osteoblast-like cells using MG63 human osteosarcoma cells as a model. [Ca2+]i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Mirtazapine at concentrations above 250 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 60% by removing extracellular Ca2+. The mirtazapine-induced Ca2+ influx was sensitive to blockade of nifedipine and verapamil. In Ca(2+)-free medium, after pretreatment with 1.5 mM mirtazapine, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 2 microM CCCP (a mitochondrial uncoupler), and 1 microM ionomycin failed to release more stored Ca2+; conversely, pretreatment with thapsigargin, CCCP and ionomycin abolished mirtazapine-induced Ca2+ release. Inhibition of phospholipase C with 2 microM U73122 did not change mirtazapine-induced [Ca2+]i, increase. Seal of Ca2+ movement across the plasma membrane with 50 microM extracellular La3+ enhanced 1 microM thapsigargin-induced [Ca2+]i increase, suggesting that Ca2+ efflux played a role in lowering thapsigargin-induced [Ca2+]i increase; however, the same La3+ treatment did not alter mirtazapine-induced [Ca2+]i increase. At concentrations of 500 microM and 1000 microM, mirtazapine killed 30% and 60% cells, respectively. The cytotoxicity was not reversed by chelating cytosolic Ca2+ with BAPTA. Collectively, in MG63 cells, mirtazapine induced a [Ca2+]i increase by causing Ca2+ release from stores and Ca2+ influx from extracellular space. Furthermore, mirtazapine caused cytotoxicity at higher concentrations in a Ca(2+)-dissociated manner.     

Swelling-induced Ca2+ release from intracellular calcium stores in rat submandibular gland acinar cells

The effects of osmotically-induced cell swelling on cytoplasmic free Ca2+ concentration ([Ca2+]i) were studied in acinar cells from rat submandibular gland using microspectrofluorimetry. Video-imaging techniques were also used to measure cell volume. Hypotonic stress (78% control tonicity) caused rapid cell swelling reaching a maximum relative volume of 1.78 +/- 0.05 (n = 5) compared to control. This swelling was followed by regulatory volume decrease, since relative cell volume decreased significantly to 1.61 +/- 0.08 (n = 5) after 10 min exposure to hypotonic medium. Osmotically induced cell swelling evoked by medium of either 78% or 66% tonicity caused a biphasic increase of [Ca2+]i. The rapid phase of this increase in [Ca2+]i was due to release of Ca2 + from intracellular stores, since it was also observed in cells bathed in Ca2+-free solution. The peak increase of [Ca2+]i induced by cell swelling was 3.40 +/- 0.49 (Fura-2 F340/F380 fluorescence ratio, n = 11) and 3.17 +/- 0.43 (n = 17) in the presence and the absence of extracellular Ca2+, respectively, corresponding to an absolute [Ca2+]i of around 1 microm. We found that around two-thirds of cells tested still showed some swelling-induced Ca2+ release (SICR) even after maximal concentrations (10(-5) M - 10(-4) M) of carbachol had been applied to empty agonist-sensitive intracellular Ca2+ stores. This result was confirmed and extended using thapsigargin to deplete intracellular Ca2+ pools. Hypotonic shock still raised [Ca2+]i in cells pretreated with thapsigargin, confirming that at least some SICR occurred from agonist-insensitive stores. Furthermore, SICR was largely inhibited by pretreatment of cells with carbonyl cyanide m-cholorophenyl hydrazone (CCCP) or ruthenium red, inhibitors of mitochondrial Ca2+ uptake. Our results suggest that the increase in [Ca2+]i, which underlies regulatory volume decrease in submandibular acinar cells, results from release of Ca2+ from both agonist-sensitive and mitochondrial Ca2+ stores.     

A role for Ca2+ in mediating hormone-induced biphasic pepsinogen secretion from the chief cell determined by luminescent and fluorescent probes and X-ray microprobe

In isolated chief cells from the guinea pig, cholecystokinin (10 nM) and a high concentration of ionomycin each caused a biphasic pattern of pepsinogen secretion. The initial fast response to cholecystokinin was not dependent on medium Ca2+ ans was mimicked by low concentration of ionomycin (100 nM). Inositol 1,4,5-trisphosphate caused a similar fast release from permeabilized cells. The slow component of release was dependent on medium Ca2+, however, and was mimicked by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) (100 nM) or the diacylglycerol analogue 1-oleoyl-2-acetylglycerol (OAG) (100 microM). Ionomycin (100 nM) and TPA (and/or OAG), when applied together, reproduced the biphasic pattern of pepsinogen secretion, suggesting that the signalling pathways utilized by both types of agonist contribute to the response evoked by cholecystokinin-hormone stimulation. Both fura-2 and aequorin were used to monitor changes of intracellular Ca2+. Three pathways were found to contribute to the Ca2+ transient. A rapid release of Ca2+ from intracellular store(s), a rapid Ca2+ entry from the extracellular space, and a more sustained Ca2+ entry from the extracellular space. Cholecystokinin induced a rapid increase in cytoplasmic Ca2+ ([Ca2+]i) as estimated with fura-2 and aequorin. This rise was reduced but not abolished upon removal of extracellular Ca2+, suggesting that both Ca2+ entry from the extracellular space and Ca2+ mobilization from the intracellular store(s) contribute to the initial, fast component of the Ca2+ transient. A second, more sustained component of the Ca2+ transient induced by cholecystokinin was abolished by lanthanum. TPA and OAG induced a biphasic Ca2+ transient that could be detected only with aequorin. The late, sustained component of this response was again abolished by lanthanum as well as by removal of extracellular Ca2+. It appears that the late component of the Ca2+ transient is dependent on Ca2+ influx from the extracellular space and is too localized to be detected by fura-2. Prestimulation of cells with TPA or OAG prevented the aequorin transient caused by cholecystokinin and vice versa, suggesting that TPA, OAG and cholecystokinin activate the same pathways of Ca2+ entry into the cytosol from the intracellular store(s) or the extracellular space. The stimulation-sensitive Ca2+ pool was examined with electron probe X-ray microanalysis. It appears to be restricted to an area enriched in secretory granules or peripheral endoplasmic reticulum just beneath the apical plasma membrane and in close association with the microtubular-microfilamentous system.(ABSTRACT TRUNCATED AT 400 WORDS)     

2,5-Di-(tert-butyl)-1,4-benzohydroquinone mobilizes inositol 1,4,5-trisphosphate-sensitive and -insensitive Ca2+ stores

In permeabilized rat hepatocytes a maximal concentration (25 microM) of 2,5-di-(tert-butyl)-1,4-benzohydroquineone (tBuBHQ) mobilized 70% of sequestere Ca2+ and a half-maximal effect was produced by 1.7 microM tBuBHQ. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) stimulated release of about 40% of the intracellular Ca2+ stores. Combined applications of a range of tBuBHQ concentrations with a maximal concentration of Ins(1,4,5)P3 demonstrated that tBuBHQ has slight selectivity for the Ca2+ transport process of the Ins(1,4,5)P3-sensitive stores. We conclude that the Ins(1,4,5)P3-sensitive stores are a subset of those sensitive to tBuBHQ and that the latter is therefore unlikely to prove useful as a tool to discriminate Ins(1,4,5)P3-sensitive and -insensitive Ca2+ stores though it may provide opportunities to design more selective agents.     

Rapidly exchanging Ca2+ stores of non-muscle cells

The rapid and transient redistribution of calcium from intracellular stores is a key event of cell activation. The nature and molecular composition of intracellular Ca2+ stores of non-muscle cells are the object of intense investigation. In this paper, we review: (a) the experimental evidence in favor of the existence of intracellular, membrane-bound compartments specialized for uptake, storage and release of calcium, (b) the main protein components of rapidly exchanging Ca2+ stores, i.e. Ca2+ pump, intralumenal Ca2+ binding proteins (calsequestrin, calreticulin, etc.) and Ca2+ channels sensitive to either inositol 1,4,5-trisphosphate or Ca2+, caffeine and ryanodine, and (c) the relationship between Ca2+ stores and the endoplasmic reticulum.     

Reduction of the Ca2+ transport systems in mouse young brown fat cells

Principal differences in the kinetics and amplitude of Ca2+ response to norepinephrine were found between freshly isolated young differentiated brown fat cells. An increase in the Ca2+ concentration in the cytoplasm ([Ca2+]i) in the young cells was unusually slow (A[Ca2+]i = 0.03 nM/s) in comparison with that in the differentiated cells, and the Ca2+ influx from the outside was not induced by Ca2+ mobilization agents, such as thapsigargin and ionomycin. Ionomycin increased [Ca2+]i up to 150 nM in a Ca2+-free medium and up to 270 nM in the normal medium. This results in that the intracellular Ca2+ stores in freshly isolated young cells are rather poor, and the mechanism of capacitive calcium entry does not virtually function. The data on chemical modification of Ca2+ channels in the plasma membrane by thimerosal suggest that the conductance of these channels is low and/or their number in young brown fat cells is insignificant.     

NAADP induces pH changes in the lumen of acidic Ca2+ stores

NAADP (nicotinic acid-adenine dinucleotide phosphate)-induced Ca2+ release has been proposed to occur selectively from acidic stores in several cell types, including sea urchin eggs. Using fluorescence measurements, we have investigated whether NAADP-induced Ca2+ release alters the pH(L) (luminal pH) within these acidic stores in egg homogenates and observed their prompt, concentration-dependent alkalinization by NAADP (but not beta-NAD+ or NADP). Like Ca2+ release, the pH(L) change was desensitized by low concentrations of NAADP suggesting it was secondary to NAADP receptor activation. Moreover, this was a direct effect of NAADP upon the acidic stores and not secondary to increases in cytosolic Ca2+ as it was not mimicked by IP3 (inositol 1,4,5-trisphosphate), cADPR (cyclic adenine diphosphoribose), ionomycin, thapsigargin or by direct addition of Ca2+, and was not blocked by EGTA. The results of the present study further support acidic stores as targets for NAADP and for the first time reveal an adjunct role for NAADP in regulating the pH(L) of intracellular organelles.     

Role of Ca(2+)-ATPase in spontaneous oscillations of cytosolic free Ca2+ in GH3 rat pituitary cells.

The relative contribution of voltage-sensitive Ca2+ channels, Ca(2+)-ATPases, and Ca2+ release from intracellular stores to spontaneous oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) observed in secretory cells is not well characterized owing to a lack of specific inhibitors for a novel thapsigargin (Tg)-insensitive Ca(2+)-ATPase expressed in these cells. We show that spontaneous [Ca2+]i oscillations in GH3 cells were unaffected by Ca2+ depletion in inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores by the treatment of Tg, but could be initiated by application of caffeine. Moreover, we demonstrate for the first time that these spontaneous [Ca2+]i oscillations were highly temperature dependent. Decreasing the temperature from 22 to 17 degrees C resulted in an increase in the frequency, a reduction in the amplitude, and large inhibition of [Ca2+]i oscillations. Furthermore, the rate of ATP-dependent 45Ca2+ uptake into GH3-derived microsomes was greatly reduced at 17 degrees C. The effect of decreased temperatures on extracellular Ca2+ influx was minor because the frequency and amplitude of spontaneous action potentials, which activate L-type Ca2+ channels, was relatively unchanged at 17 degrees C. These results suggest that in GH3 secretory cells, Ca2+ influx via L-type Ca2+ channels initiates spontaneous [Ca2+]i oscillations, which are then maintained by the combined activity of Ca(2+)-ATPase and Ca(2+)-induced Ca2+ release from Tg/IP3-insensitive intracellular stores.