Non-capacitative Ca2 Research Articles

Contribution of capacitative and non-capacitative Ca 2+-entry to M 3-receptor-mediated contraction of porcine coronary smooth muscle

We studied the contribution of store-operated or capacitative Ca 2+-entry (SOCE or CCE, respectively) through store-operated Ca 2+ channels (SOCCs) and the contribution of Ca 2+-entry through receptor-operated, non-selective cation channels (ROCCs or NSCCs, respectively), on the M 3-receptor-mediated (270 nM Ach) contractile response of porcine coronary smooth muscle strips by means of the respective inhibitors. In the presence of L-VOCC blockade (1 μM verapamil), LOE 908 (inhibition of NSCCs) decreased the contractile response to 75 ± 5% ( p < 0.01, n = 6), 2-APB (inhibition of SOCCs) and SK&F 96365 (inhibition of SOCCs and of NSCCs) decreased the response to 45 ± 4% ( p < 0.001, n = 10) and to 23 ± 2% ( p < 0.001, n = 5), respectively (control: Ach response in the presence of verapamil alone). In the absence of L-VOCC blockade, LOE 908 reduced the Ach-response to 49 ± 7% ( p < 0.001, n = 8) and SK&F 96365 to 3 ± 2% ( p < 0.001, n = 4) of control, whereas 2-APB transiently increased the response (peak effect: 130 ± 11%; p < 0.05, n = 8). We conclude: (1) the main source of activator Ca 2+ during the M 3-receptor-mediated contractile response is the Ca 2+ influx through L-VOCCs; (2) however, in the presence of L-VOCC blockade, the contractile response is mainly due to Ca 2+-entry through SOCCs; (3) NSCCs may be considerably involved in M 3-receptor-mediated contraction as they may serve to depolarize the membrane potential and, thus, to open L-VOCCs; (4) in primary tissue of vascular smooth muscle, both, SOCE and Ca 2+-entry through NSCCs are activated during M 3-receptor stimulation.

Different phospholipase-C-coupled receptors differentially regulate capacitative and non-capacitative Ca2+ entry in A7r5 cells

Several receptors, including those for AVP (Arg8-vasopressin) and 5-HT (5-hydroxytryptamine), share an ability to stimulate PLC (phospholipase C) and so production of IP3 (inositol 1,4,5-trisphosphate) and DAG (diacylglycerol) in A7r5 vascular smooth muscle cells. Our previous analysis of the effects of AVP on Ca2+ entry [Moneer, Dyer and Taylor (2003) Biochem. J. 370, 439-448] showed that arachidonic acid released from DAG stimulated NO synthase. NO then stimulated an NCCE (non-capacitative Ca2+ entry) pathway, and, via cGMP and protein kinase G, it inhibited CCE (capacitative Ca2+ entry). This reciprocal regulation ensured that, in the presence of AVP, all Ca2+ entry occurred via NCCE to be followed by a transient activation of CCE only when AVP was removed [Moneer and Taylor (2002) Biochem. J. 362, 13-21]. We confirm that, in the presence of AVP, all Ca2+ entry occurs via NCCE, but 5-HT, despite activating PLC and evoking release of Ca2+ from intracellular stores, stimulates Ca2+ entry only via CCE. We conclude that two PLC-coupled receptors differentially regulate CCE and NCCE. We also address evidence that, in some A7r5 cells lines, AVP fails either to stimulate NCCE or inhibit CCE [Brueggemann, Markun, Barakat, Chen and Byron (2005) Biochem. J. 388, 237-244]. Quantitative PCR analysis suggests that these cells predominantly express TRPC1 (transient receptor potential canonical 1), whereas cells in which AVP reciprocally regulates CCE and NCCE express a greater variety of TRPC subtypes (TRPC1=6>2>3).

Arachidonic acid mediates non‐capacitative calcium entry evoked by CB1‐cannabinoid receptor activation in DDT1 MF‐2 smooth muscle cells

Cannabinoid CB1-receptor stimulation in DDT1 MF-2 smooth muscle cells induces a rise in [Ca2+]i, which is dependent on extracellular Ca2+ and modulated by thapsigargin-sensitive stores, suggesting capacitative Ca2+ entry (CCE), and by MAP kinase. Non-capacitative Ca2+ entry (NCCE) stimulated by arachidonic acid (AA) partly mediates histamine H1-receptor-evoked increases in [Ca2+]i in DDT1 MF-2 cells. In the current study, both Ca2+ entry mechanisms and a possible link between MAP kinase activation and increasing [Ca2+]i were investigated. In the whole-cell patch clamp configuration, the CB-receptor agonist CP 55, 940 evoked a transient, Ca2+-dependent K+ current, which was not blocked by the inhibitors of CCE, 2-APB, and SKF 96365. AA, but not its metabolites, evoked a transient outward current and inhibited the response to CP 55,940 in a concentration-dependent manner. CP 55,940 induced a concentration-dependent release of AA, which was inhibited by the CB1 antagonist SR 141716. The non-selective Ca2+ channel blockers La3+ and Gd3+ inhibited the CP 55,940-induced current at concentrations that had no effect on thapsigargin-evoked CCE. La3+ also inhibited the AA-induced current. CP 55,940-induced AA release was abolished by Gd3+ and by phospholipase A2 inhibition using quinacrine; this compound also inhibited the outward current. The CP 55,940-induced AA release was strongly reduced by the MAP kinase inhibitor PD 98059. The data suggest that in DDT1 MF-2 cells, AA is an integral component of the CB1 receptor signaling pathway, upstream of NCCE and, via PLA2, downstream of MAP kinase.

Arachidonic acid inhibits capacitative Ca 2+ entry and activates non-capacitative Ca 2+ entry in cultured astrocytes

Arachidonic acid (AA) plays important physiological or pathophysiological roles. Here, we show in cultured rat astrocytes that: (i) endothelin-1 or thapsigargin (Tg) induces store-depleted activated Ca 2+ entry (CCE), which is inhibited by 2-aminoethoxydiphenyl borane (2-APB) or La 3+; (ii) AA (10 μM) and other unsaturated fatty acids (8,11,14-eicosatrienoic acid and γ-linoleic acid) have an initial inhibitory effect on the CCE, due to AA- or fatty acid-induced internal acid load; (iii) after full activation of CCE, AA induces a further Ca 2+ influx, which is not inhibited by 2-APB or La 3+, indicating that AA activates a second Ca 2+ entry pathway, which coexists with CCE; and (iv) Tg or AA activates two independent and co-existing non-selective cation channels and the Tg-induced currents are initially inhibited by addition of AA or weak acids. A possible pathophysiological effect of the AA-induced [Ca] i overload is to cause delayed cell death in astrocytes.

Obligatory Role of Src Kinase in the Signaling Mechanism for TRPC3 Cation Channels

Members of the canonical transient receptor potential (TRPC) subfamily of cation channels are candidates for capacitative and non-capacitative Ca2+ entry channels. When ectopically expressed in cell lines, TRPC3 can be activated by phospholipase C-mediated generation of diacylglycerol or by addition of synthetic diacylglycerols, independently of Ca2+ store depletion. Apart from this mode of regulation, little is known about other receptor-dependent signaling events that modulate TRPC3 activity. In the present study the role of tyrosine kinases in receptor- and diacylglycerol-dependent activation of TRPC3 was investigated. In HEK293 cells stably expressing TRPC3, pharmacological inhibition of tyrosine kinases, and specifically of Src kinases, abolished activation of TRPC3 by muscarinic receptor stimulation and by diacylglycerol. Channel regulation was lost following expression of a dominant-negative mutant of Src, or when TRPC3 was expressed in an Src-deficient cell line. In both instances, wild-type Src restored TRPC3 regulation. We conclude that Src plays an obligatory role in the mechanism for receptor and diacylglycerol activation of TRPC3.

Regulation of capacitative and non-capacitative Ca2+ entry in A7r5 vascular smooth muscle cells

A capacitative Ca2+ entry (CCE) pathway, activated by depletion of intracellular Ca2+ stores, is thought to mediate much of the Ca2+ entry evoked by receptors that stimulate phospholipase C (PLC). However, in A7r5 vascular smooth muscle cells, vasopressin, which stimulates PLC, empties intracellular Ca2+ stores but simultaneously inhibits their ability to activate CCE. The diacylglycerol produced with the IP3 that empties the stores is metabolized to arachidonic and this leads to activation of nitric oxide (NO) synthase, production of NO and cyclic GMP, and consequent activation of protein kinase G. The latter inhibits CCE. In parallel, NO directly activates a non-capacitative Ca2+ entry (NCCE) pathway, which is entirely responsible for the Ca2+ entry that occurs in the presence of vasopressin. This reciprocal regulation of two Ca2+ entry pathways ensures that there is sequential activation of first NCCE in the presence of vasopressin, and then a transient activation of CCE when vasopressin is removed. We suggest that the two routes for Ca2+ entry may selectively direct Ca2+ to processes that mediate activation and then recovery of the cell.

Calcineurin Directs the Reciprocal Regulation of Calcium Entry Pathways in Nonexcitable Cells

The reciprocal regulation of noncapacitative and capacitative (or store-operated) Ca2+ entry in nonexcitable cells (Mignen, O., Thompson, J. L., and Shuttleworth, T. J. (2001) J. Biol. Chem. 276, 35676-35683) represents a switching between two distinct Ca2+-selective channels: the noncapacitative arachidonate-regulated Ca2+ channels (ARC channels) and the store-operated Ca2+ channels (SOC channels). This switch is directly associated with the change from oscillatory to sustained Ca2+ signals as agonist concentrations increase and involves a Ca2+-dependent inhibition of the ARC channels. Here we show that this process is mediated via a calcineurin-dependent inhibition of the noncapacitative ARC channels. Pharmacological and molecular inhibition of calcineurin activity (using cyclosporin or the FK506 analogue ascomycin, and a transfected C-terminal domain of the calcineurin inhibitory protein CAIN, respectively) results in a complete reversal of the Ca2+-dependent inhibition of the ARC channels. Agonist concentrations that result in oscillatory Ca2+ signals and specifically activate Ca2+ entry through the ARC channels fail to increase calcineurin activity. However, agonist concentrations that activate the store-operated Ca2+ channels and produce prolonged increases in cytosolic Ca2+ concentrations increase calcineurin activity. Thus, calcineurin is the key mediator of the reciprocal regulation of these co-existing channels, allowing each to play a unique and non-overlapping role in Ca2+ signaling.

Calcium entry and the control of calcium oscillations.

During oscillatory Ca(2+) signals, the agonist-induced enhanced entry of extracellular Ca(2+) plays a critical role in modulating the frequency of the oscillations. Although it was originally assumed that the entry of Ca(2+) under these conditions occurred via the well-known, and apparently ubiquitous, store-operated mechanism, subsequent studies suggested that this was unlikely. It is now known that, in many cell types, a novel non-capacitative Ca(2+)-selective pathway whose activation is dependent on arachidonic acid is responsible, and the channels involved [ARC channels (arachidonate-regulated Ca(2+) channels)] have been characterized. These ARC channels co-exist with the store-operated CRAC channels (Ca(2+)-release-activated Ca(2+) channel) in cells, but each plays a unique and non-overlapping role in Ca(2+) signalling. In particular, it is the ARC channels that are specifically activated at the low agonist concentrations that give rise to oscillatory Ca(2+) signals and provide the predominant mode of Ca(2+) entry under these conditions. The indications are that Ca(2+) entry through the ARC channels increases the likelihood that low concentrations of Ins(1,4,5)P(3) will trigger repetitive Ca(2+) release. At higher agonist concentrations, store-depletion is more complete and sustained resulting in the activation of CRAC channels. At the same time the ARC channels are turned off, resulting in what we have described as a reciprocal regulation of these two distinct Ca(2+) entry pathways.

Role of sarcoplasmic reticulum Ca2+ content in Ca2+ entry of bovine airway smooth muscle cells.

Depletion of intracellular Ca(2+) stores induces the opening of an unknown Ca(2+ )entry pathway to the cell. We measured the intracellular free-Ca(2+) concentration ([Ca(2+)]i) at different sarcoplasmic reticulum (SR) Ca(2+) content in fura-2-loaded smooth muscle cells isolated from bovine tracheas. The absence of Ca(2+) in the extracellular medium generated a time-dependent decrement in [Ca(2+)]i which was proportional to the reduction in the SR-Ca(2+) content. This SR-Ca(2+) level was indirectly determined by measuring the amount of Ca(2+) released by caffeine. Ca(2+) restoration at different times after Ca(2+)-free incubation (2, 4, 6 and 10 min) induced an increment of [Ca(2+)]i. This increase in [Ca(2+)]i was considered as Ca(2+) entry to the cell. The rate of this entry was slow (~0.3 nM/s) when SR-Ca(2+) content was higher than 50% (2 and 4 min in Ca(2+)-free medium), and significantly ( p<0.01) accelerated (>1.0 nM/s) when SR-Ca(2+) content was lower than 50% (6 and 10 min in Ca(2+)-free medium). Thapsigargin significantly induced a higher rate of this Ca(2+) entry ( p<0.01). Variations in Ca(2+) influx after SR-Ca(2+) depletion were estimated more directly by a Mn(2+) quench approach. Ca(2+) restoration to the medium 4 min after Ca(2+) removal did not modify the Mn(2+) influx. However, when Ca(2+) was added after 10 min in Ca(2+)-free medium, an increment of Mn(2+) influx was observed, corroborating an increase in Ca(2+) entry. The fast Ca(2+) influx was Ni(2+) sensitive but was not affected by other known capacitative Ca(2+) entry blockers such as La(3+), Mg(2+), SKF 96365 and 2-APB. It was also not affected by the blockage of L-type Ca2(+) channels with methoxyverapamil or by the sustained K(+)-induced depolarisation. The slow Ca(2+) influx was only sensitive to SKF 96365. In conclusion, our results indicate that in bovine airway smooth muscle cells Ca(2+) influx after SR-Ca(2+) depletion has two rates: A) The slow Ca(2+) influx, which occurred in cells with more than 50% of their SR-Ca(2+) content, is sensitive to SKF 96365 and appears to be a non-capacitative Ca(2+) entry; and B) The fast Ca(2+) influx, observed in cells with less than 50% of their SR-Ca(2+) content, is probably a capacitative Ca(2+) entry and was only Ni(2+)-sensitive.

Phospholipase C and phosphatidylinositol 3-kinase signaling are involved in the exogenous arachidonic acid-stimulated respiratory burst in human neutrophils.

To define the role of phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI-3K), signaling pathways in arachidonic acid (AA)-stimulated respiratory burst in human neutrophils, the AA-stimulated respiratory burst, Ins(1,4,5)P(3) production, PI-3K activation, and cytoplasmic Ca(2+) mobilization were investigated. It was found that Ins(1,4,5)P(3) production and PI-3K activity in AA-stimulated cells were increased in a dose-dependent manner. U73122, the PLC inhibitor, effectively inhibited the AA-stimulated respiratory burst and Ca(2+) release from the intracellular calcium store but not the activity of PI-3K, indicating the independence of PI-3K signaling on PLC activation. Wortmannin, the PI-3K inhibitor, at the concentration sufficient to inhibit PI-3K activity, can only partially inhibit Ca(2+) release from the internal store, indicating a partial regulation of PLC signaling by PI-3K and the existence of two pathways initiated by different PLC subfamilies. One is regulated by PI-3K activation, and the other is independent of PI-3K signaling. It was observed that AA could still induce a noncapacitative Ca(2+) entry in the cells when Ca(2+) release from the intracellular store was blocked by a PLC inhibitor, or a capacitative Ca(2+) entry was induced by preincubation with thapsigargin. However, the AA-mediated, noncapacitative Ca(2+) entry seems to play a little, if any, role in the stimulated respiratory burst. The present study suggests that the PLC signaling pathway, which may be activated by PLC(beta) and PLC(gamma), respectively, and the PI-3K signaling pathway are involved in the AA-stimulated respiratory burst in human neutrophil.

20-HETE inotropic effects involve the activation of a nonselective cationic current in airway smooth muscle.

20-Hydroxyeicosatetraenoic acid (20-HETE) controls several mechanisms such as vasoactivity, mitogenicity, and ion transport in various tissues. Our goal was to quantify the effects of 20-HETE on the electrophysiological properties of airway smooth muscle (ASM). Isometric tension measurements, performed on guinea pig ASM, showed that 20-HETE induced a dose-dependent inotropic effect with an EC50 value of 1.5 microM. This inotropic response was insensitive to GF-109203X, a PKC inhibitor. The sustained contraction, requiring Ca2+ entry, was partially blocked by either 100 microM Gd3+ or 1 microM nifedipine, revealing the involvement of noncapacitative Ca2+ entry and L-type Ca2+ channels, respectively. Microelectrode measurements showed that 3 microM 20-HETE depolarized the membrane potential in guinea pig ASM by 13 +/- 2mV(n = 7), as did 30 microM 1-oleoyl-2-acetyl-sn-glycerol. Depolarizing effects were also observed in the absence of epithelium. Patch-clamp recordings demonstrated that 1 microM 20-HETE activated a nonselective cationic inward current that may be supported by the activation of transient receptor potential channels. The presence of canonical transient receptor potential mRNA was confirmed by RT-PCR in guinea pig ASM cells.

Nitric oxide co-ordinates the activities of the capacitative and non-capacitative Ca2+-entry pathways regulated by vasopressin.

In A7r5 vascular smooth muscle cells vasopressin, via arachidonic acid, regulates two Ca(2+)-entry pathways. Capacitative Ca(2+) entry (CCE), activated by empty Ca(2+) stores, is inhibited by arachidonic acid, and non-capacitative Ca(2+) entry (NCCE) is stimulated by it. This reciprocal regulation ensures that all Ca(2+) entry is via NCCE in the presence of vasopressin, while CCE mediates a transient Ca(2+) entry only after removal of vasopressin. We demonstrate that type III NO synthase (NOS III) is expressed in A7r5 cells and that NO inhibits CCE. Inhibition of CCE by vasopressin requires NOS III and the requirement lies downstream of arachidonic acid. Activation of soluble guanylate cyclase by NO and subsequent activation of protein kinase G are required for inhibition of CCE. Stimulation of NCCE by vasopressin also requires NOS III, but the stimulation is neither mimicked by cGMP nor blocked by inhibitors of soluble guanylate cyclase or protein kinase G. We conclude that arachidonic acid formed in response to vasopressin stimulates NOS III. NO then directly stimulates Ca(2+) entry through NCCE and, via protein kinase G, it inhibits CCE. The additional amplification provided by the involvement of guanylate cyclase and protein kinase G ensures that CCE will always be inhibited when vasopressin activates NCCE.

Mg(2+)-sensitive non-capacitative basolateral Ca(2+) entry secondary to cell swelling in the polarized renal A6 epithelium.

Polarized renal A6 epithelia respond to hyposmotic shock with an increase in transepithelial capacitance (C(T)) that is inhibited by extracellular Mg(2+). Elevation of free cytosolic [Ca(2+)] ([Ca(2+)](i)) is known to increase C(T). Therefore, we examined [Ca(2+)](i) dynamics and their sensitivity to extracellular Mg(2+) during hyposmotic conditions. Fura-2-loaded A6 monolayers, cultured on permeable supports were subjected to a sudden reduction in osmolality at both the basolateral and apical membranes from 260 to 140 mosmol (kg H(2)O)(-1). Reduction of apical osmolality alone did not affect [Ca(2+)](i). In the absence of extracellular Mg(2+), the hyposmotic shock induced a biphasic rise in [Ca(2+)](i). The first phase peaked within 40 s and [Ca(2+)](i) increased from 245 +/- 12 to 606 +/- 24 nM. This phase was unaffected by removal of extracellular Ca(2+), but was abolished by activating P2Y receptors with basolateral ATP or by exposing the cells to the phospholipase C (PLC) inhibitor U73122 prior to the osmotic shock. Suramin also severely attenuated this first phase, suggesting that the first phase of the [Ca(2+)](i) rise followed swelling-induced ATP release. The PLC inhibitor, the ATP treatment or suramin did not affect a second rise of [Ca(2+)](i) to a maximum of 628 +/- 31 nM. The second phase depended on Ca(2+) in the basolateral perfusate and was largely suppressed by 2 mM basolateral Mg(2+). Acute exposure of the basolateral membrane to Mg(2+) during the upstroke of the second phase caused a rapid decline in [Ca(2+)](i). Basolateral Mg(2+) inhibited Ca(2+) entry in a dose-dependent manner with an inhibition constant (K(i)) of 0.60 mM. These results show that polarized A6 epithelia respond to hyposmotic shock by Ca(2+) release from inositol trisphosphate-sensitive stores, followed by basolateral Ca(2+) influx through a Mg(2+)-sensitive pathway. The second phase of the [Ca(2+)](i) response is independent of the initial intracellular Ca(2+) release and therefore constitutes non-capacitative Ca(2+) entry.

Regulation of capacitative and noncapacitative receptor-operated Ca2+ entry by rho-kinase in tracheal smooth muscle.

To determine the mechanisms of Ca2+ mobilization induced by receptor agonists, we examined the role of Rho-kinase on the sarcoplasmic reticulum (SR) Ca2+ stores-dependent and -independent Ca2+ influx in guinea pig tracheal smooth muscle (TSM). Isometric tension and intracellular Ca2+ concentration ([Ca2+]i) were simultaneously measured using fura-2-loaded tissues. Depletion of the SR Ca2+ stores by thapsigargin caused an increase in [Ca2+]i and contraction, demonstrating capacitative Ca2+ entry (CCE). Because CCE was not inhibited by nifedipine, voltage-operated Ca2+ channels are not involved in CCE. Under the condition that CCE is fully activated, methacholine (MCh) and histamine caused further increases in [Ca2+]i and tension, demonstrating noncapacitative receptor-operated Ca2+ entry (non-CCE). The Ca2+ influx and contraction via non-CCE was inhibited by Y-27632, a Rho-kinase inhibitor, in a concentration-dependent fashion. In contrast, Y-27632 did not affect thapsigargin-induced CCE. Cytochalasin D, which disrupts actin cytoskeleton, inhibited contraction induced by CCE or MCh with no change in [Ca2+]i. Our results indicate that not only CCE but also non-CCE exist in TSM and that the latter is regulated by Rho-kinase, independent of actin cytoskeleton. In conclusion, Ca2+ influx regulated by the RhoA/Rho-kinase pathway may play a functional role in contraction by agonists.

Reciprocal regulation of capacitative and non-capacitative Ca2+ entry in A7r5 vascular smooth muscle cells: only the latter operates during receptor activation

In A7r5 vascular smooth muscle cells, Arg8-vasopressin (AVP) stimulates phospholipase C leading to activation of two distinct Ca2+ entry pathways. The capacitative Ca2+ entry (CCE) pathway is activated by depletion of Ca2+ stores, is permeable to Mn2+, Ba2+ and Ca2+, and is selectively blocked by Gd3+ (1μM). A7r5 cells also express a non-capacitative Ca2+ entry (NCCE) pathway, which is activated by arachidonic acid that is released by the sequential activities of phospholipase C and diacylglycerol lipase. This pathway is permeable to Sr2+, Ba2+ and Ca2+ and selectively blocked by (R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate ('LOE-908'). We use these selective tools to show that AVP, via the same signalling pathway that leads to activation of NCCE, also inhibits CCE and that the inhibition is not due to depolarization of the plasma membrane. Using the selective inhibitors to resolve the contributions of each Ca2+ entry pathway during stimulation with AVP, we establish that reciprocal regulation of CCE and NCCE by arachidonic acid ensures that only NCCE is active in the presence of AVP, whereas CCE is active only after its removal. NCCE and CCE are therefore activated in a strict temporal sequence: NCCE first and then CCE. Because Ca2+ passing through different Ca2+ entry pathways can selectively regulate different responses, reciprocal regulation of CCE and NCCE may allow a stimulus to first evoke a response and then recruit actively a different response when the stimulus is removed.

Reciprocal regulation of capacitative and non-capacitative Ca2+ entry in A7r5 vascular smooth muscle cells: only the latter operates during receptor activation.

In A7r5 vascular smooth muscle cells, Arg(8)-vasopressin (AVP) stimulates phospholipase C leading to activation of two distinct Ca(2+) entry pathways. The capacitative Ca(2+) entry (CCE) pathway is activated by depletion of Ca(2+) stores, is permeable to Mn(2+), Ba(2+) and Ca(2+), and is selectively blocked by Gd(3+)(1 microM). A7r5 cells also express a non-capacitative Ca(2+) entry (NCCE) pathway, which is activated by arachidonic acid that is released by the sequential activities of phospholipase C and diacylglycerol lipase. This pathway is permeable to Sr(2+), Ba(2+) and Ca(2+) and selectively blocked by (R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate ("LOE-908"). We use these selective tools to show that AVP, via the same signalling pathway that leads to activation of NCCE, also inhibits CCE and that the inhibition is not due to depolarization of the plasma membrane. Using the selective inhibitors to resolve the contributions of each Ca(2+) entry pathway during stimulation with AVP, we establish that reciprocal regulation of CCE and NCCE by arachidonic acid ensures that only NCCE is active in the presence of AVP, whereas CCE is active only after its removal. NCCE and CCE are therefore activated in a strict temporal sequence: NCCE first and then CCE. Because Ca(2+) passing through different Ca(2+) entry pathways can selectively regulate different responses, reciprocal regulation of CCE and NCCE may allow a stimulus to first evoke a response and then recruit actively a different response when the stimulus is removed.

Two Modes of Secretion in Pancreatic Acinar Cells: Involvement of Phosphatidylinositol 3-Kinase and Regulation by Capacitative Ca2+ Entry

In pancreatic acinar cells, muscarinic agonists stimulate both the release of Ca2+ from intracellular stores and the influx of extracellular Ca2+. The part played by Ca2+ released from intracellular stores in the regulation of secretion is well established; however, the role of Ca2+ influx in exocytosis is unclear. Recently, we observed that supramaximal concentrations of acetylcholine (≥10 μM) elicited an additional component of exocytosis despite reducing Ca2+ influx. In the present study, we found that supramaximal exocytosis was substantially inhibited (∼70%) by wortmannin (100 nM), an inhibitor of phosphatidylinositol 3-kinase. In contrast, exocytosis evoked by a lower concentration of acetylcholine (1 μM) was potentiated (∼45%) by wortmannin. Exocytosis stimulated by 1 μM acetylcholine in the absence of extracellular Ca2+ was, like supramaximal exocytosis, inhibited by wortmannin. The switch to a wortmannin-inhibitable form of exocytosis depended upon a reduction in Ca2+ entry through store-operated Ca2+ channels, as the switch in exocytotic mode could also be brought about by the selective blockade of these channels by Gd3+ (2 μM), but not by inhibition of noncapacitative Ca2+ entry by SB203580 (10 μM). We conclude that supramaximal doses of acetylcholine lead to a switch in the mode of zymogen granule exocytosis by inhibiting store-dependent Ca2+ influx.

Reciprocal Regulation of Capacitative and Arachidonate-regulated Noncapacitative Ca2+ Entry Pathways

Receptor-activated Ca(2+) entry is usually thought to occur via capacitative or store-operated Ca(2+) channels. However, at physiological levels of stimulation, where Ca(2+) store depletion is only transient and/or partial, evidence has suggested that an arachidonic acid-dependent noncapacitative Ca(2+) entry is responsible. Recently, we have described a novel arachidonate-regulated Ca(2+)-selective (ARC) conductance that is entirely distinct from store-operated conductances in the same cell. We now show that these ARC channels are indeed specifically activated by low agonist concentrations and provide the predominant route of Ca(2+) entry under these conditions. We further demonstrate that sustained elevations in cytosolic Ca(2+), such as those resulting from activation of store-operated Ca(2+) entry by high agonist concentrations, inhibit the ARC channels. This explains earlier failures to detect the presence of this noncapacitative pathway in experiments where store-operated entry had already been fully activated. The result is that the respective activities of ARC and store-operated Ca(2+) channels display a unique reciprocal regulation that is related to the specific nature of the [Ca(2+)](i) signals generated at different agonist concentrations. Importantly, these data show that at physiologically relevant levels of stimulation, it is the noncapacitative ARC channels that provide the predominant route for the agonist-activated entry of Ca(2+).

Permeation of Monovalent Cations through the Non-capacitative Arachidonate-regulated Ca2+ Channels in HEK293 Cells: COMPARISON WITH ENDOGENOUS STORE-OPERATED CHANNELS

In a manner similar to voltage-gated Ca(2+) channels and Ca(2+) release-activated Ca(2+) (CRAC) channels, the recently identified arachidonate-regulated Ca(2+) (ARC) channels display a large monovalent conductance upon removal of external divalent cations. Using whole-cell patch-clamp recording, we have characterized the properties of these monovalent currents in HEK293 cells stably transfected with the m3 muscarinic receptor and compared them with the corresponding currents through the endogenous store-operated Ca(2+) (SOC) channels in the same cells. Although the monovalent currents seen through these two channels displayed certain similarities, several marked differences were also apparent, including the magnitude of the monovalent current/Ca(2+) current ratio, the rate and nature of the spontaneous decline in the currents, and the effects of external monovalent cation substitutions and removal of internal Mg(2+). Moreover, monovalent ARC currents could be activated after the complete spontaneous inactivation of the corresponding SOC current in the same cell. We conclude that the non-capacitative ARC channels share, with voltage-gated Ca(2+) channels and store-operated Ca(2+) channels (e.g. SOC and CRAC the general property of monovalent ion permeation in the nominal absence of extracellular divalent ions. However, the clear differences between the properties of these currents through ARC and SOC channels in the same cell confirm that these represent distinct conductances.

Protein kinase C activates non-capacitative calcium entry in human platelets.

1. In many non-excitable cells Ca2+ influx is mainly controlled by the filling state of the intracellular Ca2+ stores. It has been suggested that this store-mediated or capacitative Ca2+ entry is brought about by a physical and reversible coupling of the endoplasmic reticulum with the plasma membrane. Here we provide evidence for an additional, non-capacitative Ca2+ entry mechanism in human platelets. 2. Changes in cytosolic Ca2+ and Sr2+ were measured in human platelets loaded with the fluorescent indicator fura-2. 3. Depletion of the internal Ca2+ stores with thapsigargin plus a low concentration of ionomycin stimulated store-mediated cation entry, as demonstrated upon Ca2+ or Sr2+ addition. Subsequent treatment with thrombin stimulated further divalent cation entry in a concentration-dependent manner. 4. Direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol also stimulated divalent cation entry, without evoking the release of Ca2+ from intracellular stores. Cation entry evoked by thrombin or activators of PKC was abolished by the PKC inhibitor Ro-31-8220. 5. Unlike store-mediated Ca2+ entry, jasplakinolide, which reorganises actin filaments into a tight cortical layer adjacent to the plasma membrane, did not inhibit divalent cation influx evoked by thrombin when applied after Ca2+ store depletion, or by activators of PKC. Thrombin also activated Ca2+ entry in platelets in which the release from intracellular stores and store-mediated Ca2+ entry were blocked by xestospongin C. 6. These results indicate that the non-capacitative divalent cation entry pathway is regulated independently of store-mediated entry and does not require coupling of the endoplasmic reticulum and the plasma membrane. These results support the existence of a mechanism for receptor-evoked Ca2+ entry in human platelets that is independent of Ca2+ store depletion. This Ca2+ entry mechanism may be activated by occupation of G-protein-coupled receptors, which activate PKC, or by direct activation of PKC, thus generating non-capacitative Ca2+ entry alongside that evoked following the release of Ca2+ from the intracellular stores.