The effect of oxidative stress on Ca2+ release and capacitative Ca2+ entry in vascular endothelial cells

Oxidative stress imposed by the accumulation of oxygen free radicals (reactive oxygen species, ROS) has profound effects on Ca2+ homeostasis in the vascular endothelium, leading to endothelial dysfunctions and the development of cardiovascular pathologies. We tested the effect of the oxidant and ROS generator tert-butyl-hydroperoxide (tBuOOH) on Ca2+ signaling in single cultured calf pulmonary artery endothelial (CPAE) cells loaded with the fluorescent Ca2+ indicator indo-1. Acute brief (5 min) exposures to tBuOOH had no effect on basal cytosolic free Ca2+ ([Ca2+](i)), agonist (ATP)-induced Ca2+ release from the endoplasmic reticulum (ER) and on Ca(2+) store depletion-dependent capacitative Ca2+ entry (CCE). Prolonged (60 min) exposure to tBuOOH did not affect intracellular Ca2+ release, but caused a profound inhibition of CCE. After 120 min of treatment with tBuOOH not only was CCE further reduced, but also ATP-induced Ca2+ release due to a slow depletion of the stores that resulted from CCE inhibition. The antioxidant Trolox (synthetic vitamin E analog) prevented the inhibition of CCE by tBuOOH and attenuated the increase of [ROS](i), indicating that inhibition of CCE was due to the oxidant effects of tBuOOH. The data suggest that in vascular endothelial cells oxidative stress primarily affects Ca2+ influx in response to Ca2+ loss from internal stores. [Ca2+](i) is an important signal for the production and release of endothelium-derived factors such as nitric oxide (NO). Since CCE is the preferential Ca2+ source for NO synthase activation, the finding that oxidative stress inhibits CCE may explain how oxidative stress contributes to endothelial dysfunction-related cardiovascular pathologies.     

Evidence on the operation of ATP-induced capacitative calcium entry in breast cancer cells and its blockade by 17beta-estradiol

Little is known about the regulation of cytosolic calcium Ca(2+) levels ([Ca(2+)](i)) in breast cancer cells. We investigated the existence of capacitative calcium entry (CCE) in the tumorigenic cell line MCF-7 and its responsiveness to ATP. MCF-7 cells express purinergic receptors as well as estrogen receptors (ER). Depletion of calcium stores with thapsigargin (TG, 500 nM) or ATP (10 microM) in the absence of extracellular Ca(2+), resulted in a rapid and transient elevation in [Ca(2+)](i). After recovery of basal levels, Ca(2+) readmission (1.5 mM) to the medium increased Ca(2+) influx (twofold over basal), reflecting pre-activation of a CCE pathway. Cells pretreated with TG were unable to respond to ATP, thus indicating that the same Ca(2+) store is involved in their response. Moreover, IP(3)-dependent ATP-induced calcium mobilization and CCE were completely blocked using compound U-73122, an inhibitor of phospholipase C. Compound 2-APB (75 microM) and Gd(3+) (10 microM), antagonists of the CCE pathway, completely prevented ATP-stimulated capacitative Ca(2+) entry. CCE in MCF-7 cells was highly permeable to Mn(2+) and to the Ca(2+) surrogate Sr(2+). Mn(2+) entry sensitivity to Gd(3+) matched that of the Ca(2+) entry pathway. 17Beta-estradiol blocked ATP-induced CCE, but was without effect on TG-induced CCE. Besides, the estrogen blockade of the ATP-induced CCE was completely abolished by preincubation of the cells with an ER monoclonal antibody. ER alpha immunoreactivity could also be detected in a purified plasma membrane fraction of MCF-7 cells. These results represent the first evidence on the operation of a ATP-responsive CCE pathway in MCF-7 cells and also indicate that 17beta-estradiol interferes with this mechanism by acting at the cell surface level.     

Mitochondria efficiently buffer subplasmalemmal Ca2+ elevation during agonist stimulation

In endothelial cells, local Ca(2+) release from superficial endoplasmic reticulum (ER) activates BK(Ca) channels. The resulting hyperpolarization promotes capacitative Ca(2+) entry (CCE), which, unlike BK(Ca) channels, is inhibited by high Ca(2+). To understand how the coordinated activation of plasma membrane ion channels with opposite Ca(2+) sensitivity is orchestrated, the individual contribution of mitochondria and ER in regulation of subplasmalemmal Ca(2+) concentration ([Ca(2+)](pm)) was investigated. For organelle visualization, cells were transfected with DsRed and yellow cameleon targeted to mitochondria and ER. The patch pipette was placed far from any organelle (L1), close to ER (L3), or mitochondria (L2) and activity of BK(Ca) channels was used to estimate local [Ca(2+)](pm). Under standard patch conditions (130 mm K(+) in the bath), histamine increased [Ca(2+)](pm) at L1 and L3 to approximately 1.6 microm, whereas close to mitochondria [Ca(2+)](pm) remained unchanged. If mitochondria moved apart from the pipette or in the presence of carbonyl cyanide-4-trifluoromethoxyphenylhyrazone, [Ca(2+)](pm) at L2 increased in response to histamine. Under standard patch conditions Ca(2+) entry was negligible due to cell depolarization. Using a physiological patch approach (5.6 mm K(+) in the bath), changes in [Ca(2+)](pm) to histamine could be monitored without cell depolarization and, thus, in conditions where Ca(2+) entry occurred. Here, histamine induced an initial transient Ca(2+) elevation to > or =3.5 microm followed by a long lasting plateau at approximately 1.2 microm in L1 and L3, whereas mitochondria kept neighboring [Ca(2+)](pm) low during stimulation. Thus, superficial mitochondria and ER generate local domains of low and high Ca(2+) allowing simultaneous activation of BK(Ca) and CCE, despite their opposite Ca(2+) sensitivity.     

Hypoxia-induced alterations in Ca(2+) mobilization in brain microvascular endothelial cells

To investigate the possible cellular mechanisms of the ischemia-induced impairments of cerebral microcirculation, we investigated the effects of hypoxia/reoxygenation on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine brain microvascular endothelial cells (BBEC). In the cells kept in normal air, ATP elicited Ca(2+) oscillations in a concentration-dependent manner. When the cells were exposed to hypoxia for 6 h and subsequent reoxygenation for 45 min, the basal level of [Ca(2+)](i) was increased from 32.4 to 63.3 nM, and ATP did not induce Ca(2+) oscillations. Hypoxia/reoxygenation also inhibited capacitative Ca(2+) entry (CCE), which was evoked by thapsigargin (Delta[Ca(2+)](i-CCE): control, 62.3 +/- 3.1 nM; hypoxia/reoxygenation, 17.0 +/- 1.8 nM). The impairments of Ca(2+) oscillations and CCE, but not basal [Ca(2+)](i), were restored by superoxide dismutase and the inhibitors of mitochondrial electron transport, rotenone and thenoyltrifluoroacetone (TTFA). By using a superoxide anion (O(2)(-))-sensitive luciferin derivative MCLA, we confirmed that the production of O(2)(-) was induced by hypoxia/reoxygenation and was prevented by rotenone and TTFA. These results indicate that hypoxia/reoxygenation generates O(2)(-) at mitochondria and impairs some Ca(2+) mobilizing properties in BBEC.     

Capacitative calcium entry induces hippocampal long term potentiation in the absence of presenilin-1

Presenilins, whose mutant forms are the most common cause of early onset familial Alzheimer's disease, are involved in two very distinct processes: (i) proteolytic activity as gamma-secretase acting on amyloid precursor protein to produce amyloid peptides and (ii) storage of Ca2+ in the endoplasmic reticulum (ER). In particular, absence of presenilin-1 (PS1) was claimed to potentiate capacitative calcium entry (CCE), i.e. the mechanism of replenishment of ER Ca2+ stores. However, until now, evidence in favor of the latter role has been obtained only in isolated or cultured cells and not on neurons in situ. Here, we studied the strength of the synapses between Schaffer's collaterals and CA1 neurons in hippocampal slices when they were submitted first to Ca(2+)-free medium containing thapsigargin and subsequently to normal artificial cerebrospinal fluid, a procedure known to trigger CCE. We demonstrate that Ca2+ influx via the CCE mechanism is sufficient to trigger robust long term potentiation of the synapses in hippocampal slices from transgenic mice with a postnatal, neuron-specific ablation of PS1, but remarkably not from wild-type mice. Our data establish for the first time in neurons confined in normal neuronal networks that PS1 acts on the refilling mechanism of ER Ca2+ stores.     

Expression of Trp3 determines sensitivity of capacitative Ca2+ entry to nitric oxide and mitochondrial Ca2+ handling: evidence for a role of Trp3 as a subunit of capacitative Ca2+ entry channels

The role of Trp3 in cellular regulation of Ca(2+) entry by NO was studied in human embryonic kidney (HEK) 293 cells. In vector-transfected HEK293 cells (controls), thapsigargin (TG)-induced (capacitative Ca(2+) entry (CCE)-mediated) intracellular Ca(2+) signals and Mn(2+) entry were markedly suppressed by the NO donor 2-(N,N-diethylamino)diazenolate-2-oxide sodium salt (3 microm) or by authentic NO (100 microm). In cells overexpressing Trp3 (T3-9), TG-induced intracellular Ca(2+) signals exhibited an amplitude similar to that of controls but lacked sensitivity to inhibition by NO. Consistently, NO inhibited TG-induced Mn(2+) entry in controls but not in T3-9 cells. Moreover, CCE-mediated Mn(2+) entry into T3-9 cells exhibited a striking sensitivity to inhibition by extracellular Ca(2+), which was not detectable in controls. Suppression of mitochondrial Ca(2+) handling with the uncouplers carbonyl cyanide m-chlorophenyl hydrazone (300 nm) or antimycin A(1) (-AA(1)) mimicked the inhibitory effect of NO on CCE in controls but barely affected CCE in T3-9 cells. T3-9 cells exhibited enhanced carbachol-stimulated Ca(2+) entry and clearly detectable cation currents through Trp3 cation channels. NO as well as carbonyl cyanide m-chlorophenyl hydrazone slightly promoted carbachol-induced Ca(2+) entry into T3-9 cells. Simultaneous measurement of cytoplasmic Ca(2+) and membrane currents revealed that Trp3 cation currents are inhibited during Ca(2+) entry-induced elevation of cytoplasmic Ca(2+), and that this negative feedback regulation is blunted by NO. Our results demonstrate that overexpression of Trp3 generates phospholipase C-regulated cation channels, which exhibit regulatory properties different from those of endogenous CCE channels. Moreover, we show for the first time that Trp3 expression determines biophysical properties as well as regulation of CCE channels by NO and mitochondrial Ca(2+) handling. Thus, we propose Trp3 as a subunit of CCE channels.     

Uncoupling protein 3 (UCP3) modulates the activity of Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) by decreasing mitochondrial ATP production

The uncoupling proteins UCP2 and UCP3 have been postulated to catalyze Ca(2+) entry across the inner membrane of mitochondria, but this proposal is disputed, and other, unrelated proteins have since been identified as the mitochondrial Ca(2+) uniporter. To clarify the role of UCPs in mitochondrial Ca(2+) handling, we down-regulated the expression of the only uncoupling protein of HeLa cells, UCP3, and measured Ca(2+) and ATP levels in the cytosol and in organelles with genetically encoded probes. UCP3 silencing did not alter mitochondrial Ca(2+) uptake in permeabilized cells. In intact cells, however, UCP3 depletion increased mitochondrial ATP production and strongly reduced the cytosolic and mitochondrial Ca(2+) elevations evoked by histamine. The reduced Ca(2+) elevations were due to inhibition of store-operated Ca(2+) entry and reduced depletion of endoplasmic reticulum (ER) Ca(2+) stores. UCP3 depletion accelerated the ER Ca(2+) refilling kinetics, indicating that the activity of sarco/endoplasmic reticulum Ca(2+) (SERCA) pumps was increased. Accordingly, SERCA inhibitors reversed the effects of UCP3 depletion on cytosolic, ER, and mitochondrial Ca(2+) responses. Our results indicate that UCP3 is not a mitochondrial Ca(2+) uniporter and that it instead negatively modulates the activity of SERCA by limiting mitochondrial ATP production. The effects of UCP3 on mitochondrial Ca(2+) thus reflect metabolic alterations that impact on cellular Ca(2+) homeostasis. The sensitivity of SERCA to mitochondrial ATP production suggests that mitochondria control the local ATP availability at ER Ca(2+) uptake and release sites.     

Highly cooperative dependence of sarco/endoplasmic reticulum calcium ATPase SERCA2a pump activity on cytosolic calcium in living cells

Sarco/endoplasmic reticulum (SR/ER) Ca(2+)-ATPase (SERCA) is an intracellular Ca(2+) pump localized on the SR/ER membrane. The role of SERCA in refilling intracellular Ca(2+) stores is pivotal for maintaining intracellular Ca(2+) homeostasis, and disturbed SERCA activity causes many disease phenotypes, including heart failure, diabetes, cancer, and Alzheimer disease. Although SERCA activity has been described using a simple enzyme activity equation, the dynamics of SERCA activity in living cells is still unknown. To monitor SERCA activity in living cells, we constructed an enhanced CFP (ECFP)- and FlAsH-tagged SERCA2a, designated F-L577, which retains the ATP-dependent Ca(2+) pump activity. The FRET efficiency between ECFP and FlAsH of F-L577 is dependent on the conformational state of the molecule. ER luminal Ca(2+) imaging confirmed that the FRET signal changes directly reflect the Ca(2+) pump activity. Dual imaging of cytosolic Ca(2+) and the FRET signals of F-L577 in intact COS7 cells revealed that SERCA2a activity is coincident with the oscillatory cytosolic Ca(2+) concentration changes evoked by ATP stimulation. The Ca(2+) pump activity of SERCA2a in intact cells can be expressed by the Hill equation with an apparent affinity for Ca(2+) of 0.41 ± 0.0095 μm and a Hill coefficient of 5.7 ± 0.73. These results indicate that in the cellular environment the Ca(2+) dependence of ATPase activation is highly cooperative and that SERCA2a acts as a rapid switch to refill Ca(2+) stores in living cells for shaping the intracellular Ca(2+) dynamics. F-L577 will be useful for future studies on Ca(2+) signaling involving SERCA2a activity.     

Phosphatidylinositol 3-kinase regulates Ca2+ signaling in pancreatic acinar cells through inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase

Calcium is a key mediator of hormone-induced enzyme secretion in pancreatic acinar cells. At the same time, abnormal Ca(2+) responses are associated with pancreatitis. We have recently shown that inhibition of phosphatidylinositol 3-kinase (PI3-kinase) by LY-294002 and wortmannin, as well as genetic deletion of PI3-kinase-gamma, regulates Ca(2+) responses and the Ca(2+)-sensitive trypsinogen activation in pancreatic acinar cells. The present study sought to determine the mechanisms of PI3-kinase involvement in Ca(2+) responses induced in these cells by CCK and carbachol. The PI3-kinase inhibitors inhibited both Ca(2+) influx and mobilization from intracellular stores induced by stimulation of acini with physiological and pathological concentrations of CCK, as well as with carbachol. PI3-kinase inhibition facilitated the decay of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) oscillations observed in individual acinar cells. The PI3-kinase inhibitors decreased neither CCK-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] production nor Ins(1,4,5)P(3)-induced Ca(2+) mobilization, suggesting that the effect of PI3-kinase inhibition is not through Ins(1,4,5)P(3) or Ins(1,4,5)P(3) receptors. PI3-kinase inhibition did not affect Ca(2+) mobilization induced by thapsigargin, a specific inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). Moreover, SERCA blockade with thapsigargin abolished the effects of pharmacological and genetic PI3-kinase inhibition on [Ca(2+)](i) signals, suggesting SERCA as a downstream target of PI3-kinase. Both pharmacological PI3-kinase inhibition and genetic deletion of PI3-kinase-gamma increased the amount of Ca(2+) in intracellular stores during CCK stimulation. Finally, addition of the PI3-kinase product phosphatidylinositol 3,4,5-trisphosphate to permeabilized acini significantly attenuated Ca(2+) reloading into the endoplasmic reticulum. The results indicate that PI3-kinase regulates Ca(2+) signaling in pancreatic acinar cells through its inhibitory effect on SERCA.     

Sensing and refilling calcium stores in an excitable cell.

Inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ mobilization leads to depletion of the endoplasmic reticulum (ER) and an increase in Ca2+ entry. We show here for the gonadotroph, an excitable endocrine cell, that sensing of ER Ca2+ content can occur without the Ca2+ release-activated Ca2+ current (Icrac), but rather through the coupling of IP3-induced Ca2+ oscillations to plasma membrane voltage spikes that gate Ca2+ entry. Thus we demonstrate that capacitative Ca2+ entry is accomplished through Ca(2+)-controlled Ca2+ entry. We develop a comprehensive model, with parameter values constrained by available experimental data, to simulate the spatiotemporal behavior of agonist-induced Ca2+ signals in both the cytosol and ER lumen of gonadotrophs. The model combines two previously developed models, one for ER-mediated Ca2+ oscillations and another for plasma membrane potential-driven Ca2+ oscillations. Simulations show agreement with existing experimental records of store content, cytosolic Ca2+ concentration ([Ca2+]i), and electrical activity, and make a variety of new, experimentally testable predictions. In particular, computations with the model suggest that [Ca2+]i in the vicinity of the plasma membrane acts as a messenger for ER content via Ca(2+)-activated K+ channels and Ca2+ pumps in the plasma membrane. We conclude that, in excitable cells that do not express Icrac, [Ca2+]i profiles provide a sensitive mechanism for regulating net calcium flux through the plasma membrane during both store depletion and refilling.     

Ca(2+) removal mechanisms in freshly isolated rabbit aortic endothelial cells

Calcium removal from the cytoplasm was investigated in freshly isolated aortic endothelial cells by monitoring changes in intracellular calcium ([Ca(2+)](i)) using ratiometric fura-2 fluorimetry. Blockade of the Na(+)/Ca(2+) exchanger (NCX) by replacement of external sodium with equi-molar N-methyl-D-glutamine (0Na PSS) decreased the removal rate by 52%. Blockade of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) by cyclopiazonic acid (CPA) decreased the removal rate by 50%. Simultaneous application of CPA and 0Na PSS did not reduce the removal rate any further (53%). The lack of additivity of these two procedures, suggests that SERCA and the NCX function in series to lower [Ca(2+)](i). In addition, in the absence of extracellular Ca(2+), removal of external Na(+) markedly reduced the rate of loss of Ca(2+) from the ER further supporting the hypothesis that NCX is functionally linked to ER calcium release channels, and thus, plays an important role in ER calcium unloading. To investigate the mechanism for the coupling of NCX and SERCA, the same protocols as described above were repeated after treating the cells with cytochalasin D, which disrupts the cytoskeleton. This treatment uncoupled the NCX from SERCA, as evidenced by the resulting additive inhibitory effects of application of CPA and removal of extracellular Na(+) on the rate of Ca(2+) removal from the cytoplasm. These data suggest that in endothelial cells NCX and SERCA function in series to remove about half of the free Ca(2+) from the cytosol, while PMCA contributes to the other half of the Ca(2+) removal process.     

A homolog of voltage-gated Ca(2+) channels stimulated by depletion of secretory Ca(2+) in yeast

In animal cells, capacitative calcium entry (CCE) mechanisms become activated specifically in response to depletion of calcium ions (Ca(2+)) from secretory organelles. CCE serves to replenish those organelles and to enhance signaling pathways that respond to elevated free Ca(2+) concentrations in the cytoplasm. The mechanism of CCE regulation is not understood because few of its essential components have been identified. We show here for the first time that the budding yeast Saccharomyces cerevisiae employs a CCE-like mechanism to refill Ca(2+) stores within the secretory pathway. Mutants lacking Pmr1p, a conserved Ca(2+) pump in the secretory pathway, exhibit higher rates of Ca(2+) influx relative to wild-type cells due to the stimulation of a high-affinity Ca(2+) uptake system. Stimulation of this Ca(2+) uptake system was blocked in pmr1 mutants by expression of mammalian SERCA pumps. The high-affinity Ca(2+) uptake system was also stimulated in wild-type cells overexpressing vacuolar Ca(2+) transporters that competed with Pmr1p for substrate. A screen for yeast mutants specifically defective in the high-affinity Ca(2+) uptake system revealed two genes, CCH1 and MID1, previously implicated in Ca(2+) influx in response to mating pheromones. Cch1p and Mid1p were localized to the plasma membrane, coimmunoprecipitated from solubilized membranes, and shown to function together within a single pathway that ensures that adequate levels of Ca(2+) are supplied to Pmr1p to sustain secretion and growth. Expression of Cch1p and Mid1p was not affected in pmr1 mutants. The evidence supports the hypothesis that yeast maintains a homeostatic mechanism related to CCE in mammalian cells. The homology between Cch1p and the catalytic subunit of voltage-gated Ca(2+) channels raises the possibility that in some circumstances CCE in animal cells may involve homologs of Cch1p and a conserved regulatory mechanism.     

A mathematical model predicts that calreticulin interacts with the endoplasmic reticulum Ca(2+)-ATPase.

A robust mathematical model developed from single cell calcium (Ca(2+)) dynamics has enabled us to predict the consequences of over-expression of endoplasmic reticulum-located chaperones. Model predictions concluded that calreticulin interacts with the lumenal domain of the sarcoplasmic and endoplasmic reticulum Ca(2+)-activated ATPase (SERCA) pump, altering pump affinity for Ca(2+) (K(1/2) switches from 247 to 431 nM) and hence generating Ca(2+) oscillations. Expression of calreticulin in the ER generated an average of six transient-decline oscillations during the Ca(2+) recovery phase, upon exposure to maximal levels of the agonist ATP. In contrast, normal cells produced a single Ca(2+) transient with few or no oscillations. By conditioning the model to experimental data, parameters for generation and decay of IP(3) and SERCA pump kinetics were determined. To elucidate the possible source of the oscillatory behavior three possible oscillators, 1) IP(3), 2) IP(3)R, and 3) SERCA pump, were investigated and parameters constrained by experimental data to produce the best candidate. Each of the three oscillators generated very good fits with experimental data. However, converting a normal exponential recovery to a transient-decline oscillator predicted that the SERCA pump is the most likely candidate for calreticulin-mediated Ca(2+) release, highlighting the role of this chaperone as a signal protein within the endoplasmic reticulum.     

Calcium entry mediated by SOCs and TRP channels: variations and enigma

Ca(2+) signals in response to receptors mediate and control countless cellular functions ranging from short-term responses such as secretion and contraction to longer-term regulation of growth, cell division and apoptosis. The spatial and temporal details of Ca(2+) signals have been resolved with great precision in many cells. Ca(2+) signals activated by phospholipase C-coupled receptors have two components: Ca(2+) release from endoplasmic reticulum (ER) stores mediated by inositol 1,4,5-trisphosphate (InsP(3)) receptors, and Ca(2+) entry from outside the cell. The latter remains largely a molecular and mechanistic mystery. The activation of "store-operated" Ca(2+) channels is believed to account for the entry of Ca(2+). However, debate now focuses on how much of a contribution emptying of stores plays to the activation of Ca(2+) entry in response to physiological activation of receptors. Here we discuss recent information and ideas on the exchange of signals between the plasma membrane (PM) and ER that results in activation of Ca(2+) entry channels following receptor stimulation and/or store emptying.     

Interactions between calcium release pathways: multiple messengers and multiple stores

The discovery of cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) as Ca(2+) releasing messengers has provided additional insight into how complex Ca(2+) signalling patterns are generated. There is mounting evidence that these molecules along with the more established messenger, myo-inositol 1,4,5-trisphosphate (IP(3)), have a widespread messenger role in shaping Ca(2+) signals in many cell types. These molecules have distinct structures and act on specific Ca(2+) release mechanisms. Emerging principles are that cADPR enhances the Ca(2+) sensitivity of ryanodine receptors (RYRs) to produce prolonged Ca(2+) signals through Ca(2+)-induced Ca(2+) release (CICR), while NAADP acts on a novel Ca(2+) release mechanism to produce a local trigger Ca(2+) signal which can be amplified by CICR by recruiting other Ca(2+) release mechanisms. Whilst IP(3) and cADPR mobilise Ca(2+) from the endoplasmic reticulum (ER), recent evidence from the sea urchin egg suggests that the major NAADP-sensitive Ca(2+) stores are reserve granules, acidic lysosomal-related organelles.In this review we summarise the role of multiple Ca(2+) mobilising messengers, Ca(2+) release channels and Ca(2+) stores, and the interplay between them, in the generation of specific Ca(2+) signals. Focusing upon cADPR and NAADP, we discuss how cellular stimuli may draw upon different combinations of these messengers to produce distinct Ca(2+) signalling signatures.     

Analysis of Mn(2+)/Ca(2+) influx and release during Ca(2+) oscillations in mouse eggs injected with sperm extract

Repetitive Ca(2+) release from the endoplasmic reticulum (ER) is necessary for activation of mammalian eggs. Influx and release of Mn(2+) and Ca(2+) during Ca(2+) oscillations induced by injection of sperm extract (SE) into mouse eggs were investigated by Mn(2+)-quenching of intracellular Fura-2 after adding Mn(2+) to external medium. Mn(2+)/Ca(2+) influx was detected at the resting state. A marked Mn(2+)/Ca(2+) influx occurred during the first Ca(2+) release upon SE injection, and persistently facilitated Mn(2+)/Ca(2+) influx was observed during steady Ca(2+) oscillations. As intracellular Mn(2+) concentration ([Mn(2+)](i)) increased progressively, periodic [Mn(2+)](i) rises appeared, corresponding to each Ca(2+)transient but taking a slower time course. A numerical simulation based on continuous Mn(2+)/Ca(2+) influx-extrusion across the plasma membrane and release-uptake across the ER membrane in a competitive manner mimicked well the Mn(2+) oscillations calculated from experimental data, strongly suggesting that repetitive Mn(2+) release develops after Mn(2+) entry and uptake into the ER. In other experiments, a marked Mn(2+) influx occurred upon Mn(2+) addition to Ca(2+)-free medium after depletion of the ER using an ER Ca(2+) pump inhibitor plus repeated injection of inositol 1,4,5-trisphosphate (InsP(3)). No significant increase in Mn(2+) influx was induced by injection of SE, InsP(3), or Ca(2+), when Ca(2+) release was prevented by pre-injection of an antibody against the InsP(3) receptor. We concluded that Ca(2+) influx is activated during the initial large Ca(2+)release possibly by a capacitative mechanism and kept facilitated during steady Ca(2+) oscillations. The finding that repetitive Mn(2+) release is caused by continuous Mn(2+) entry suggests that continuous Ca(2+) influx may play a critical role in refilling the ER and, thereby, maintaining Ca(2+)oscillations in mammalian fertilization.     

Store-operated Ca2+ entry suppresses distention-induced ATP release from the urothelium

Epithelial cells in the urinary bladder (urothelium) trigger sensory signals in micturition by releasing ATP in response to distention of the bladder wall. Our previous study revealed the distinct roles of extracellular Ca(2+) and the Ca(2+) stores in the endoplasmic reticulum (ER) in urothelial ATP release. In the present study, we investigated the regulation of urothelial ATP release by Ca(2+) influx from the extracellular space and Ca(2+) release from the ER using a distention assay of the mouse bladder wall in a small Ussing chamber. Stimulation of Ca(2+) release from the ER in the mucosal side of the bladder induced significant ATP release without distention. Blockade of the inositol 1,4,5-triphosphate receptor reduced distention-induced ATP release, suggesting that Ca(2+) release from the ER is essential for the induction of urothelial ATP release. On the other hand, blockade of store-operated Ca(2+) entry (SOCE) from the extracellular space significantly enhanced distention-induced ATP release. Thus Ca(2+) release from the ER causes urothelial ATP release and depletion of Ca(2+) stores in the ER, which in turn causes the depletion-inducing SOCE to suppress the amount of urothelial ATP released.     

Role of STIM and Orai proteins in the store-operated calcium signaling pathway

Ca(2+) signals are universal among cells in regulating a spectrum of cellular responses. Phospholipase C-coupled receptors activate two components of Ca(2+) signals--rapid Ca(2+) release from ER stores, followed by slower Ca(2+) entry from outside the cell. The coupling process between ER and PM to mediate this "store-operated" Ca(2+) entry process remained until recently a molecular mystery. The recent discovery of the necessity for STIM1 and Orai proteins in this process has provided crucial information on the coupling mechanism between stores and PM Ca(2+) entry. STIM1 is a single spanning membrane protein with an unpaired Ca(2+) binding EF-hand and appears to function as the sensor of ER luminal Ca(2+), and, through redistribution in the ER, transduces information directly to the PM. Orai1 is a tetra-spanning PM protein and functions as the highly Ca(2+)-selective channel in the PM that is gated through interactions with the store-activated ER Ca(2+) sensor. Recent evidence shows the two proteins together are necessary and sufficient for the function of store-operated Ca(2+) entry. However, many questions arise about how and where the interactions of the STIM1 and Orai1 proteins occur within cells. Here we discuss recent information and ideas about the coupling between these proteins that leads to store-operated channel activation.