Emptying Of Intracellular Ca2 Research Articles

Involvement of STIM1 and Orai1 in EGF-mediated cell growth in retinal pigment epithelial cells

BackgroundIn non-excitable cells, one major route for calcium entry is through store-operated calcium (SOC) channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ store. STIM1 and Orai1 are major regulators of SOC channels. In this study, we explored the functions of STIM1 and Orai1 in epidermal growth factor (EGF)-induced cell proliferation and migration in retinal pigment epithelial cells (ARPE-19 cell line).ResultsEGF triggers cell proliferation and migration in ARPE-19 cells. Cell proliferation and migration involve STIM1 and Orai1, as well as phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2, and Akt. Pharmacological inhibitors of SOC channels and siRNA of Orai1 and STIM1 suppress cell proliferation and migration. Pre-treatment of mitogen-activated protein kinase kinase (MEK) inhibitors and a phosphatidylinositol 3 kinases (PI3K) inhibitor attenuated cell proliferation and migration. However, inhibition of the SOC channels failed to prevent EGF-mediated ERK 1/2 and Akt phosphorylation.ConclusionsOur results showed that STIM1, Orai1, ERK 1/2, and Akt are key determinants of EGF-mediated cell growth in ARPE-19 cells. EGF is a potent growth molecule that has been linked to the development of PVR, and therefore, STIM1, Orai1, as well as the MEK/ERK 1/2 and PI3K/Akt pathways, might be potential therapeutic targets for drugs aimed at treating such disorders.

Sites of action of hydrogen peroxide on ion transport across rat distal colon

The aim of this study was the identification of the mechanism of oxidant-induced intestinal secretion. The action of H2O2 on ion transport across rat distal colon was evaluated in Ussing chambers. Changes in cytosolic Ca2+ concentration were measured using fura-2. H2O2 concentration-dependently induced an increase in short-circuit current (Isc), which was due to a stimulation of Cl(-) secretion. The effect of H2O2 was dependent on the presence of serosal Ca2+. It was inhibited after emptying of intracellular Ca2+ stores by cyclopiazonic acid or blockade of ryanodine receptors by ruthenium red, whereas a blocker of inositol 1,4,5-trisphosphate receptors was less effective. Fura 2-experiments confirmed an increase in the cytosolic Ca2+ concentration in the presence of H2O2. Measurements of Cl- currents across the apical membrane at basolaterally depolarized epithelia revealed the activation of a glibenclamide-sensitive, SITS-resistant Cl- conductance by the oxidant. The activation of this conductance was inhibited after blockade of protein kinases with staurosporine. When the apical membrane was permeabilized with nystatin, two sites of action of H2O2 were identified at the basolateral membrane. The oxidant stimulated a basolateral tetrapentylammonium-sensitive K+ conductance and increased the current generated by the Na+-K+ pump. Pretreatment of the tissues with H2O2 reduced the action of subsequently administered Ca2+-, cAMP- and cGMP-dependent secretagogues demonstrating a long-term downregulation after the initial secretory response evoked by the oxidant. H2O2 affects colonic anion secretion by action sites at both the apical, as well as the basolateral membrane.

Store-operated calcium channels and pro-inflammatory signals

In non-excitable cells such as T lymphocytes, hepatocytes, mast cells, endothelia and epithelia, the major pathway for calcium [Ca2+] entry is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, however, neither the gating mechanism nor the downstream targets of these channels has been clear established. Here, I review some of the proposed gating mechanisms of store-operated Ca2+ channels and the functional implications in regulating pro-inflammatory signals.

Store-Operated Calcium Channels

In electrically nonexcitable cells, Ca(2+) influx is essential for regulating a host of kinetically distinct processes involving exocytosis, enzyme control, gene regulation, cell growth and proliferation, and apoptosis. The major Ca(2+) entry pathway in these cells is the store-operated one, in which the emptying of intracellular Ca(2+) stores activates Ca(2+) influx (store-operated Ca(2+) entry, or capacitative Ca(2+) entry). Several biophysically distinct store-operated currents have been reported, but the best characterized is the Ca(2+) release-activated Ca(2+) current, I(CRAC). Although it was initially considered to function only in nonexcitable cells, growing evidence now points towards a central role for I(CRAC)-like currents in excitable cells too. In spite of intense research, the signal that relays the store Ca(2+) content to CRAC channels in the plasma membrane, as well as the molecular identity of the Ca(2+) sensor within the stores, remains elusive. Resolution of these issues would be greatly helped by the identification of the CRAC channel gene. In some systems, evidence suggests that store-operated channels might be related to TRP homologs, although no consensus has yet been reached. Better understood are mechanisms that inactivate store-operated entry and hence control the overall duration of Ca(2+) entry. Recent work has revealed a central role for mitochondria in the regulation of I(CRAC), and this is particularly prominent under physiological conditions. I(CRAC) therefore represents a dynamic interplay between endoplasmic reticulum, mitochondria, and plasma membrane. In this review, we describe the key electrophysiological features of I(CRAC) and other store-operated Ca(2+) currents and how they are regulated, and we consider recent advances that have shed insight into the molecular mechanisms involved in this ubiquitous and vital Ca(2+) entry pathway.

The Role of Mitochondria for Ca2+ Refilling of the Endoplasmic Reticulum

Endoplasmic reticulum (ER) Ca2+ refilling is an active process to ensure an appropriate ER Ca2+ content under basal conditions and to maintain or restore ER Ca2+ concentration during/after cell stimulation. The mechanisms to achieve successful ER Ca2+ refilling are multiple and built on a concerted action of processes that provide a suitable reservoir for Ca2+ sequestration into the ER. Despite mitochondria having been found to play an essential role in the maintenance of capacitative Ca2+ entry by buffering subplasmalemmal Ca2+, their contribution to ER Ca2+ refilling was not subjected to detailed analysis so far. Thus, this study was designed to elucidate the involvement of mitochondria in Ca2+ store refilling during and after cell stimulation. ER Ca2+ refilling was found to be accomplished even during continuous inositol 1,4,5-trisphosphate (IP3)-triggered ER Ca2+ release by an agonist. Basically, ER Ca2+ refilling depended on the presence of extracellular Ca2+ as the source and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) activity. Interestingly, in the presence of an IP3-generating agonist, ER Ca2+ refilling was prevented by the inhibition of trans-mitochondrial Ca2+ flux by CGP 37157 (7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one) that precludes the mitochondrial Na+/Ca2+ exchanger as well as by mitochondrial depolarization using a mixture of oligomycin and antimycin A. In contrast, after the removal of the agonist, ER refilling was found to be largely independent of trans-mitochondrial Ca2+ flux. Under these conditions, ER Ca2+ refilling took place even without an associated Ca2+ elevation in the deeper cytosol, thus, indicating that superficial ER domains mimic mitochondrial Ca2+ buffering and efficiently sequester subplasmalemmal Ca2+ and consequently facilitate capacitative Ca2+ entry. Hence, these data point to different contribution of mitochondria in the process of ER Ca2+ refilling based on the presence or absence of IP3, which represents the turning point for the dependence or autonomy of ER Ca2+ refilling from trans-mitochondrial Ca2+ flux.

Ca2+-Calmodulin-dependent Facilitation and Ca2+ Inactivation of Ca2+ Release-activated Ca2+ Channels

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.

Close Functional Coupling between Ca2+ Release-activated Ca2+ Channels, Arachidonic Acid Release, and Leukotriene C4 Secretion

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types, particularly of hemopoietic origin, store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. However, little is known about the downstream consequences of CRAC channel activation. Here, we report that Ca2+ entry through CRAC channels stimulates arachidonic acid production, whereas Ca2+ release from the stores is ineffective even though the latter evokes a robust intracellular Ca2+ signal. We find that arachidonic acid released by Ca2+ entering through CRAC channels is used to synthesize the potent paracrine proinflammatory signal leukotriene C4 (LTC4). Both pharmacological inhibitors of CRAC channels and mitochondrial depolarization, which impairs CRAC channel activity, suppress arachidonic acid release and LTC4 secretion. Thus, arachidonic acid release is preferentially stimulated by elevated subplasmalemmal Ca2+ levels due to open CRAC channels, suggesting that the enzyme is located close to the CRAC channels. Our results also identify a novel role for CRAC channels, namely the activation of a downstream signal transduction pathway resulting in the secretion of LTC4. Finally, mitochondria are key determinants of the generation of both intracellular (arachidonic acid) and paracrine (LTC4) signals through their effects on CRAC channel activity.

Activation of the store-operated calcium current ICRAC can be dissociated from regulated exocytosis in rat basophilic leukaemia (RBL-1) cells.

In many cell types, the emptying of intracellular Ca2+ stores results in the opening of store-operated Ca2+ channels in the plasma membrane. However, the nature of the signal that couples store content to the opening of these Ca2+ channels is unclear. One model proposes that the Ca2+ channels are initially stored in cytoplasmic vesicles but inserted into the plasma membrane upon store depletion via a regulated exocytoytic mechanism (vesicular fusion model). Using the whole-cell patch-clamp technique to measure the store-operated Ca2+ current ICRAC and the capacitance method to monitor vesicular fusion, an indicator of exocytosis, we have investigated the effects of interfering with regulated exocytosis on the ability of ICRAC to activate. We find that the recombinant protein alpha-SNAP1-285, an inhibitor of exocytosis in many systems, suppresses such fusion but has no impact on the activation of ICRAC. A variety of other manoeuvres that interfere with vesicle trafficking and exocytosis were also without effect on ICRAC. Impairing constitutive exocytosis with brefeldin A reduced the extent of ICRAC, but this effect was less pronounced when current density was considered instead. Activation of ICRAC can therefore be clearly dissociated from an exocytotic mechanism, a finding that is not easily reconcilable with the vesicular fusion model.

Effects of inhibitors of the lipo-oxygenase family of enzymes on the store-operated calcium current I(CRAC) in rat basophilic leukaemia cells.

In non-excitable cells, the major Ca2+ entry pathway is the store-operated pathway in which emptying of intracellular Ca2+ stores activates Ca2+ channels in the plasma membrane. In many cell types, store-operated influx gives rise to a Ca2+-selective current called I(CRAC) (Ca2+ release-activated Ca2+ current). Using both the whole-cell patch clamp technique to measure I(CRAC) directly and fluorescent Ca2+ imaging, we have examined the role of the lipo-oxygenase pathway in the activation of store-operated Ca2+ entry in the RBL-1 rat basophilic leukaemia cell-line. Pretreatment with a variety of structurally distinct lipo-oxygenase inhibitors all reduced the extent of I(CRAC), whereas inhibition of the cyclo-oxygenase enzymes was without effect. The inhibition was still seen in the presence of the broad protein kinase blocker staurosporine, or when Na+ was used as the charge carrier through CRAC channels. The lipo-oxygenase blockers released Ca2+ from intracellular stores but this was not associated with subsequent Ca2+ entry. Lipo-oxygenase blockers also reduced both the amount of Ca2+ that could subsequently be released by the combination of thapsigargin and ionomycin in Ca2+-free solution and the Ca2+ influx component that occurred when external Ca2+ was re-admitted. The inhibitors were much less effective if applied after I(CRAC) had been activated. This inhibition of I(CRAC) could not be rescued by dialysis with 5(S)-hydroxyperoxyeicosa-6E,8Z,11Z,14Z,tetraenoic acid (5-HPETE), the first product of the 5-lipo-oxygenase pathway. Our findings indicate that exposure to pharmacological tools that inhibit the lipo-oxygenase enzymes all decrease the extent of activation of the current. Our results raise the possibility that a lipo-oxygenase might be involved in the activation of I(CRAC). Alternative explanations are also discussed.

Phospholipase A2 Is Involved in Thapsigargin-Induced Sodium Influx in Human Lymphocytes

Previously, we reported that emptying of intracellular Ca2+ pools with endoplasmatic Ca2+-ATP-ase inhibitor thapsigargin leads to the Na+ influx in human lymphocytes (M. Tepel et al., 1994, J. Biol. Chem. 269, 26239–26242). In the present study we examined the mechanism underlying the thapsigargin-induced Na+ entry. We found that the thapsigargin-induced increase in Na+ concentration was effectively inhibited by three structurally unrelated phospholipase A2 (PLA2) inhibitors, p-bromophenacyl bromide, 3-(4-octadecyl)-benzoylacrylic acid (OBAA), and bromoenol lactone (BEL). The thapsigargin-induced Na+ influx could be mimicked by PLA2 exogenously added to the lymphocyte suspension. In addition, thapsigargin stimulated formation of arachidonic acid (AA), the physiological PLA2 product. AA induced Na+ entry in a time- and concentration-dependent fashion. Both, thapsigargin-induced Na+ influx and AA liberation were completely inhibited in the presence of tyrosine kinase inhibitor genistein but not in the absence of extracellular Ca2+. Collectively, these data show that thapsigargin-induced Na+ entry is associated with tyrosine kinase-dependent stimulation of PLA2.

Characteristics of Ca2+ Oscillations in Ileal Longitudinal Muscle Cells of Guinea Pig

We studied the mechanisms and characteristics of the spontaneously evoked intracellular Ca2+ changes (Ca2+ oscillations) in ileal longitudinal smooth muscle from guinea pig. Two-dimensional images of Ca2+ oscillations were obtained at 33-ms intervals with a Ca2+-sensitive fluorescence probe, fluo-3 using the intensified CCD camera. Nicardipine (10 −7 M) significantly decreased the maximum level of fluorescence intensity of the Ca2+ oscillations, inhibited the frequency of the oscillations and tended to decrease the basal level of fluorescence intensity. However, tetrodotoxin (3 × 10−7 M) did not affect these oscillations. Phorbol 12,13-dibutyrate (10 −7 M) significantly increased the maximum level of fluorescence intensity and the frequency of Ca2+ oscillations, and it changed them to steady and chronometric Ca2+ oscillations. Cyclopiazonic acid (3 × 10−5 M) also significantly increased the frequency of Ca2+ oscillations. Acetylcholine (10−8 M) increased the basal and maximum level of fluorescence intensity and the frequency of Ca2+ oscillations, and accelerated their onset. The increase of basal level of fluorescence intensity was then decreased by cyclopiazonic acid treatment. These results suggest that the augmentation of Ca2+ oscillations is mainly due to the activation of L-type Ca2+ channels, which is modulated by protein kinase C, and that the emptying of intracellular Ca2+ stores may activate the Ca2+ oscillations mediated through the increase of Ca2+ influx in ileal smooth muscle of guinea pig.

ATP-triggered calcium signals in human neutrophils.

There is evidence that ATP is an important extracellular factor regulating neutrophil functions. Indeed, extracellular ATP has been shown to stimulate neutrophil migration into sites of inflammation as well as adhesion of circulating cells to the endothelium1. This nucleotide also acts as a secretagogue for enhanced exocytosis of primary granules from neutrophils. Finally, it does improve the release of superoxide induced by fMLP, thereby acting as a priming agent for this response2. Extracellular ATP causes a transient increase in cytosolic level of Ca2+, which is known to be used as a second messenger to control many cellular processes including secretion, enzyme control, gene regulation, and apoptosis3. The Ca 2+ signal is a complex event that involves both intracellular Ca2+ pool release and extracellular Ca entry. Emptying of intracellular Ca2+ pools is the major trigger for activation of Ca entry during the generation of receptor-mediated Ca2+ signal. This capacitative mechanism, that has been termed store-operated Ca2+ entry, may be the basis by which the cells maintain elevated cytosolic free calcium concentration and replenish their intracellular Ca2+ stores in response to agonist stimulation. The importance of this signalling pathway has been recognised in numerous investigations and has received a great deal of attention.

Urocortin hyperpolarizes stomach smooth muscle via activation of Ca2+-sensitive K+ currents.

The effect of urocortin (Uro), a recently discovered neuropeptide with selectivity towards corticotropin-releasing hormone type 2 receptor, was tested on whole cell currents expressed by guinea-pig gastric antrum smooth muscle cells. Uro (1 pmol/l-1 nmol/l) caused a concentration-dependent increase of Ca2+-sensitive K currents (I(K)) up to 500% as compared to control currents and did not affect the kinetics and voltage-dependence of inward Ca2+ currents. The I(K)-increasing effect of Uro was fully antagonized by preliminary emptying of intracellular Ca2+ stores with ryanodine and cyclopiazonic acid, as well as by bath application of selective blockers of adenylyl cyclase and cAMP-dependent protein kinase (PKA), but not by inhibitors of guanylyl cyclase, cGMP-dependent protein kinase, and protein kinase C. Comparable I(K) increase was obtained by forskolin (activator of adenylyl cyclase), Sp-cAMPS (activator of PKA), or by intracellular application of the catalytic subunit of PKA. It was concluded that Uro binds to a selective receptor in antral smooth muscle cells where it stimulates I(K) via PKA-dependent increase of Ca2+ concentration near the plasma membrane due to enhanced release from intracellular calcium stores.

Relationship between NaF- and thapsigargin-induced endothelium-dependent hyperpolarization in rat mesenteric artery.

1. In isolated rat mesenteric artery with endothelium, NaF caused slowly developing hyperpolarization. The hyperpolarizing effect was unchanged in the presence of N(G)-nitro-L-arginine (L-NOARG) and indomethacin, but was markedly reduced by high K+. In Ca2+ -free medium or in the presence of Ni2+, NaF failed to produce hyperpolarization. 2. NaF-induced hyperpolarization was substantially unaffected by deferoxamine, an Al3+ chelator, okadaic acid and calyculin A, phosphatase inhibitors, and preincubation with pertussis toxin, suggesting that neither the action of fluoroaluminates as a G protein activator nor inhibition of phosphatase activity contributes to the hyperpolarizing effect. 3. The selective inhibitors of the Ca2+ -pump ATPase of endoplasmic reticulum, thapsigargin and cyclopiazonic acid, elicited hyperpolarization, whose properties were very similar to those of NaF. When intracellular Ca2+ stores had been depleted with these inhibitors, NaF no longer generated hyperpolarization. 4. In Ca2+ -free medium, NaF (or thapsigargin) caused a transient increase in the cytosolic Ca2+ concentration ([Ca2+]i) in cultured porcine aortic endothelial cells, and subsequent application of thapsigargin (or NaF) failed to increase [Ca2+]i. 5. In arterial rings precontracted with phenylephrine, NaF produced endothelium-dependent relaxation followed by sustained contraction even in the presence of L-NOARG and indomethacin. The relaxant response was abolished by high K+ or cyclopiazonic acid. 6. These results indicate that NaF causes endothelium-dependent hyperpolarization, thereby leading to smooth muscle relaxation of rat mesenteric artery. This action appears to be mediated by the promotion of Ca2+ influx into endothelial cells that can be triggered by the emptying of intracellular Ca2+ stores, as proposed for those of thapsigargin and cyclopiazonic acid.

Emptying of intracellular Ca2+ stores stimulates Ca2+ entry in mouse pancreatic beta-cells by both direct and indirect mechanisms.

1. In non-excitable cells, the depletion of intracellular Ca2+ stores triggers Ca2+ influx by a process called capacitative Ca2+ entry. In the present study, we have investigated how the emptying of these stores by thapsigargin (1 microM) influences Ca2+ influx in electrically excitable pancreatic beta-cells. The cytoplasmic Ca2+ concentration ([Ca2+]i) was monitored in clusters of mouse beta-cells or in whole islets loaded with fura-2. 2. The membrane was first held hyperpolarized by diazoxide, an opener of ATP-sensitive K+ (KATP) channels, in the presence of 4.8 mM K+. Alternating between Ca(2+)-free medium and medium containing 2.5 mM Ca2+ caused a minor rise in [Ca2+]i (approximately 14 nM) in clusters of beta-cells. A larger rise (approximately 65 nM), resistant to the blockade of voltage-dependent Ca2+ channels by D600, occurred when extracellular Ca2+ was readmitted after emptying intracellular Ca2+ stores with thapsigargin or acetylcholine. Thus there exists a small capacitative Ca2+ entry in beta-cells. 3. When the membrane potential was clamped at depolarized levels with 10, 20 or 45 mM K+ in the presence of diazoxide, [Ca2+]i increased to different plateau levels ranging between 100 and 900 nM. Thapsigargin consistently caused a further transient rise in [Ca2+]i, but had little (at 10 mM K+) or no effect on the plateau level. This confirms that the capacitative Ca2+ entry is small. 4. In clusters of cells whose membrane potential was not clamped with diazoxide, 15 mM glucose (in 4.8 mM K+) induced [Ca2+]i oscillations by promoting Ca2+ influx through voltage-dependent Ca2+ channels. The application of thapsigargin accelerated these oscillations and increased their amplitude, sometimes causing a sustained elevation of [Ca2+]i. Similar results were obtained from whole islets perifused with a medium containing > or = 6 mM glucose. The effect of thapsigargin was always much larger than expected from the capacitative Ca2+ entry, probably because of a potentiation of Ca2+ influx through voltage-dependent Ca2+ channels. 5. This potentiating effect of thapsigargin did not result from an acceleration of cell metabolism since the drug did not affect glucose-induced changes in NAD(P)H fluorescence. It is also unlikely to involve the inhibition of KATP channels because thapsigargin steadily elevated [Ca2+]i in cells in which [Ca2+]i oscillations persisted in the presence of a maximally effective concentration of tolbutamide. 6. In conclusion, the emptying of intracellular Ca2+ stores in beta-cells induces a small capacitative Ca2+ entry and activates a depolarizing current which potentiates glucose-induced Ca2+ influx through voltage-dependent Ca2+ channels.

Regulatory volume decrease by SV40-transformed rabbit corneal epithelial cells requires ryanodine-sensitive Ca2+-induced Ca2+ release.

The relationship between relative cell volume and time-dependent changes in intracellular Ca2+ concentration ([Ca2+]i) during exposure to hypotonicity was characterized in SV-40 transformed rabbit corneal epithelial cells (tRCE) (i). Light scattering measurements revealed rapid initial swelling with subsequent 97% recovery of relative cell volume (characteristic time (tauvr) was 5.9 min); (ii). Fura2-fluorescence single-cell imaging showed that [Ca2+]i initially rose by 216% in 30 sec with subsequent return to near baseline level after another 100 sec. Both relative cell volume recovery and [Ca2+]i transients were inhibited by either: (a) Ca2+-free medium; (b) 5 mM Ni2+ (inhibitor of plasmalemma Ca2+ influx); (c) 10 microM cyclopiazonic acid, CPA (which causes depletion of intracellular Ca2+ content); or (d) 100 microM ryanodine (inhibitor of Ca2+ release from intracellular stores). To determine the temporal relationship between an increased plasmalemma Ca2+ influx and the emptying of intracellular Ca2+ stores during the [Ca2+]i transients, Mn2+ quenching of fura2-fluorescence was quantified. In the presence of CPA, hypotonic challenge increased plasmalemma Mn2+ permeability 6-fold. However, Mn2+ permeability remained unchanged during exposure to either: 1.100 microM ryanodine; 2.10 microM CPA and 100 microM ryanodine. This report for the first time documents the time dependence of the components of the [Ca2+]i transient required for a regulatory volume decrease (RVD). The results show that ryanodine sensitive Ca2+ release from an intracellular store leads to a subsequent increase in plasmalemma Ca2+ influx, and that both are required for cells to undergo RVD.

Muscarinic Stimulation Increases Na+Entry in Pancreatic B-Cells by a Mechanism Other than the Emptying of Intracellular Ca2+Pools

Stimulation of muscarinic (M3) receptors depolarizes pancreatic B-cells by increasing Na+influx. Here, we measured [Na+]iand [Ca2+]iin B-cell clusters to investigate whether depletion of intracellular Ca2+pools triggers this unusual transduction pathway for muscarinic receptors. Acetylcholine emptied Ca2+pools less completely than did the SERCA pump inhibitors, thapsigargin, and cyclopiazonic acid. However, the rise in [Na+]iproduced by acetylcholine was not mimicked by thapsigargin or cyclopiazonic acid and was not prevented by previous depletion of Ca2+pools. Depolarization of B-cells by acetylcholine stimulates Ca2+influx and steadily increases [Ca2+]i. In the presence of glucose and extracellular Ca2+, B-cells treated with thapsigargin or cyclopiazonic acid displayed large [Ca2+]ioscillations. Subsequent application of acetylcholine was followed by a sustained rise in [Ca2+]ias in untreated cells. In conclusion, intracellular Ca2+pool depletion does not mediate acetylcholine stimulation of Na+entry and of subsequent events. We propose that the muscarinic receptors are coupled to Na+channels in B-cells.

4-Hydroxynonenal Triggers Ca2+Influx in Isolated Rat Hepatocytes

Addition of micromolar concentrations of 4-hydroxynonenal (4-HNE), a reactive end-product of lipid peroxidation, to isolated rat hepatocytes was found to cause an early and transient increase in cytosolic Ca2+concentration followed by a more pronounced and progressive elevation. Such a late effect of 4-HNE was prevented by chelation of extracellular Ca2+with EGTA or by the addition of GdCl3, which is known to block the activity of store operated Ca2+channels in the hepatocyte plasma membrane. Moreover, the preincubation of isolated hepatocytes with the phospholipase C inhibitor U73122 resulted in a complete inhibition of both the early increase of cytosolic Ca2+and the subsequent Ca2+inflow. When 4-HNE was added to the hepatocytes 5 min after the empting of intracellular Ca2+pools by thapsigargin, the aldehyde caused a further increase in the accumulation of Ca2+which was prevented in the presence of GdCl3. Taken together these results indicate that in hepatocytes 4-HNE causes Ca2+inflow across GdCl3-sensitive Ca2+channels. The mechanism responsible for such an effect is triggered by the emptying of intracellular Ca2+pools likely resulting from 4-HNE mediated stimulation of phospholypase C, but 4-HNE also appears to interfere with the channel protein(s) or with the mechanism(s) regulating capacitative Ca2+inflow.

T cell receptor‐mediated Ca2+ signaling: Release and influx are independent events linked to different Ca2+ entry pathways in the plasma membrane

In this study, we showed that cross-linking CD3 molecules on the T cell surface resulted in Ca2+ release from the intracellular stores followed by a sustained Ca2+ influx. Inhibition of release with TMB-8 did not block the influx. However, inhibition of phospholipase C activity suppressed both Ca2+ release and influx. Once activated, the influx pathway remained open in the absence of further hydrolysis of PIP2. Thapsigargin, a microsomal Ca(2+)-ATPase inhibitor, stimulated Ca2+ entry into the cells by a mechanism other than emptying Ca2+ stores. In addition, Ca2+ entry into the Ca(2+)-depleted cells was stimulated by low basal level of cytosolic Ca2+, not by the emptying of intracellular Ca2+ stores. Both the Ca2+ release and influx were dependent on high and low concentrations of extracellular Ca2+. At low concentrations, Mn2+ entered the cell through the Ca2+ influx pathway and quenched the sustained phase of fluorescence; whereas, at higher Mn2+ concentration both the transient and the sustained phases of fluorescence were quenched. Moreover, Ca2+ release was inhibited by low concentrations of Ni2+, La3+, and EGTA, while Ca2+ influx was inhibited by high concentrations. Thus, in T cells Ca2+ influx occurs independently of IP3-dependent Ca2+ release. However, some other PIP2 hydrolysis-dependent event was involved in prolonged activation of Ca2+ influx. Extracellular Ca2+ influenced Ca2+ release and influx through the action of two plasma membrane Ca2+ entry pathways with different pharmacological and biochemical properties.

Ca2+ entry activated by emptying of intracellular Ca2+ stores in ileal smooth muscle of the rat.

1. The effects of depletion of intracellular Ca2+ stores on muscle tension and the intracellular Ca2+ concentration ([Ca2+])i were studied in fura-2 loaded longitudinal smooth muscle cells of the rat ileum. 2. After exposure to a Ca(2+)-free solution, application of Ca2+ caused a small contraction and a rise in [Ca2+]i, both of which were potentiated when the muscle was challenged with carbachol or caffeine before the addition of Ca2+. 3. Cyclopiazonic acid (CPA), a specific inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase, dose-dependently decreased tension development and the rises in [Ca2+]i induced by carbachol and caffeine in the Ca(2+)-free solution, but conversely increased the Ca(2+)-induced responses even in the presence of the voltage-dependent Ca2+ channel blockers, methoxyverapamil and nifedipine. 4. The contraction and rise in [Ca2+]i evoked by Ca2+ gradually declined with time after removal of CPA, while the reverse was the case for the responses to carbachol and caffeine. 5. The Ca(2+)-induced contraction and rise in [Ca2+]i in the presence of CPA were inhibited by the replacement of Na+ with K+ or Cs+, and by the addition of Cd2+, Ba2+, Ni2+ or La3+. 6. The influx of Mn2+ was much greater in extent in the presence of CPA than in its absence. 7. These results suggest that the emptying of intracellular Ca2+ stores may activate Ca2+ influx not associated with voltage-dependent Ca2+ channels in the rat ileal smooth muscle.