Mechanism of vascular relaxation by thaligrisine: functional and binding assays

In the present study we examine the mechanism by which thaligrisine, a bisbenzyltetrahydroisoquinoline alkaloid, inhibits the contractile response of vascular smooth muscle. The work includes functional studies on rat isolated aorta and tail artery precontracted with noradrenaline or KCl. In other experiments rat aorta was precontracted by caffeine in the presence or absence of extracellular Ca2+. In order to assess whether thaligrisine interacts directly with calcium channel binding sites or with alpha-adrenoceptors we examined the effect of the alkaloid on [3H]-(+)-cis diltiazem, [3H]-nitrendipine and [3H]-prazosin binding to cerebral cortical membranes. The functional studies showed that the alkaloid inhibited in a concentration-dependent manner the contractile response induced by depolarization in rat aorta (IC50 = 8.9+/-2.9 microM, n=5) and in tail artery (IC50 = 3.04+/-0.3 microM, n=6) or noradrenaline induced contraction in rat aorta (IC50 = 23.0+/-0.39 microM, n=9) and in tail artery (IC50 = 3.8+/-0.9 microM, n=7). In rat aorta, thaligrisine concentration-dependently inhibited noradrenaline-induced contraction in Ca2+-free solution (IC50 = 13.3 microM, n=18). The alkaloid also relaxed the spontaneous contractile response elicited by extracellular calcium after depletion of noradrenaline-sensitive intracellular stores (IC50 = 7.7 microM, n=4). The radioligand receptor-binding study showed that thaligrisine has higher affinity for [3H]-prazosin than for [3H]-(+)-cis-diltiazem binding sites, with Ki values of 0.048+/-0.007 microM and 1.5+/-1.1 microM respectively. [3H]-nitrendipine binding was not affected by thaligrisine. The present work provides evidence that thaligrisine shows higher affinity for [3H]-prazosin binding site than [3H]-(+)-cis-diltiazem binding sites, in contrast with tetrandrine and isotetrandrine that present similar affinity for both receptors. In functional studies thaligrisine, acted as an alpha1-adrenoceptor antagonist and as a Ca2+ channel blocker, relaxing noradrenaline or KCl-induced contractions in vascular smooth muscle. This compound specifically inhibits the refilling of internal Ca2+-stores sensitive to noradrenaline, by blocking Ca2+-entry through voltage-dependent Ca2+-channels.     

Thrombin activation of the Na+/H+ exchanger in vascular smooth muscle cells. Evidence for a kinase C-independent pathway which is Ca2+-dependent and pertussis toxin-sensitive

The mechanism by which human alpha-thrombin activates the Na+/H+ exchanger was studied in cultured neonatal rat aortic smooth muscle cells. Thrombin (0.4 unit/ml) caused a rapid cell acidification followed by a slow, amiloride-inhibitable alkalinization (0.10-0.14 delta pHi above base line). In protein kinase C down-regulated cells (exposed to phorbol 12-myristate 13-acetate for 24 or 72 h), the delta pHi induced by thrombin was only partially attenuated. This protein kinase C-independent activation of the Na+/H+ exchanger was blocked by pertussis toxin (islet activating protein (IAP)), reducing delta pHi by 50%. IAP did not directly inhibit Na+/H+ exchange activity as assessed by the response to intracellular acid loading. Thrombin also stimulated arachidonic acid release by 2.5 fold and inositol trisphosphate release by 6.2 fold. IAP inhibited both of these activities by 50-60%. Intracellular Ca2+ chelation with 120 microM quin2 prevented the thrombin-induced Ca2+ spike, inhibited thrombin-induced arachidonic acid release by 75%, and inhibited thrombin-induced activation of the Na+/H+ exchanger in protein kinase C-deficient cells by 65%. Increased intracellular [Ca2+] alone was not sufficient to activate the Na+/H+ exchanger, since ionomycin (0.3-1.5 microM) failed to elevate cell pH significantly. 10 microM indomethacin inhibited thrombin-induced delta pHi in both control and protein kinase C down-regulated cells by 30-50%. Thus, thrombin can activate the Na+/H+ exchanger in vascular smooth muscle cells by a Ca2+-dependent, pertussis toxin-sensitive pathway which does not involve protein kinase C.     

Vasorelaxant effect of the flavonoid galangin on isolated rat thoracic aorta

Here we investigated the effect of the flavonoid galangin in isolated rat thoracic aortic rings. Galangin (0.1-100 microM) induced relaxation in rings pre-contracted with phenylephrine (PE 1 microM) or with KCl (100 mM) or pre-treated with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 100 microM), the cyclooxygenase inhibitor indomethacin (10 microM) and the adenylate cyclase inhibitor, SQ 22,536 (100 microM). In another set of experiments, rat aortic rings were incubated with galangin (1-100 microM) and the contractile responses to PE (0.001-3 microM) or to KCl (60 mM) were evaluated. We also evaluated the effect of galangin (100 microM) on PE (10 microM)-induced contraction in a Ca2+-free medium. Galangin relaxed aortic rings with or without endothelium. Galangin effect was significantly inhibited by L-NAME. Galangin inhibited the contractile response to PE, either in presence or in absence of external calcium, and to KCl. In the end, we also found that galangin caused nitric oxide (NO) release from aortic rings and abolished the increase in [Ca2+]i triggered by PE or KCl in aortic smooth muscle cells, either in presence and in absence of external Ca2+. Our results suggest that galangin reduces the contractility of rat aortic rings through an endothelium-dependent mechanism, involving NO, and also through an endothelium-independent mechanism, inhibiting calcium movements through cell membranes.     

Vascular effects of caffeic acid phenethyl ester (CAPE) on isolated rat thoracic aorta

This study was aimed to investigate the vascular activity of caffeic acid phenethylester (CAPE), one of the major components of honeybee propolis. Experiments were performed on rat thoracic aortic rings, mounted in an isolated organ bath and connected to an isometric force transducer. The effect of CAPE (0.1-300 microM) was evaluated on tissue pre-contracted with phenylephrine (PE, 1 microM) or with KCl (100 mM). In another set of experiments, tissue was incubated with CAPE (1-100 microM) and responses to PE (0.01-3 microM) or KCl (60 mM) were evaluated. The effect of CAPE on cytosolic Ca(2+) concentration in aortic smooth muscle cells stimulated with PE or KCl was also evaluated. CAPE (0.1-300 microM) caused a concentration-dependent relaxation (pEC(50) 4.99 +/- 0.19; Emax 100.75 +/- 1.65%; n = 4) of tissue pre-contracted with PE that was reduced by endothelium removal or by incubation with N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM). CAPE also relaxed KCl-precontracted tissue (pEC(50) 4.40 +/- 0.08; n = 4). CAPE inhibited contractile responses to PE or to KCl, and also inhibited the contractile response to PE obtained in a Ca(2+)-free medium. In addition, CAPE inhibited the increase in cytosolic Ca(2+) concentration triggered by stimulation of aortic smooth muscle cells with PE or KCl. Our results demonstrate a vascular activity for CAPE, that is only partially dependent on nitric oxide. Indeed, at high concentrations, CAPE vasorelaxant effect occurs also in absence of endothelium and it is likely due to an inhibitory effect on calcium movements through cell membranes.     

5-Oxo-ETE regulates tone of guinea pig airway smooth muscle via activation of Ca2+ pools and Rho-kinase pathway

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a proinflammatory mediator, but its effects on airway smooth muscle (ASM) have never been assessed. Tension measurements performed on guinea pig ASM showed that 5-oxo-ETE induced sustained concentration-dependent positive inotropic responses (EC50 = 0.89 microM) of somewhat lower amplitude than those induced by carbamylcholine and the thromboxane A2 (TXA2) agonist U-46619. Transient inotropic responses to 5-oxo-ETE were recorded in Ca2+-free medium, suggesting mobilization of intracellular Ca2+. Meanwhile, the sustained contraction, which required Ca2+ entry, was partially blocked by 1 microM nifedipine (an L-type Ca2+ channel blocker) but relatively insensitive to 100 microM Gd3+. The 5-oxo-ETE responses were also inhibited by indomethacin and SC-560 [a cyclooxygenase (COX-1) inhibitor] pretreatments but not by NS-398 (a selective COX-2 inhibitor). The contractile effects of 5-oxo-ETE on ASM were inhibited by the selective TXA2 receptor (TP receptor) antagonist SQ-29548 (-75%) and by 2-(p-amylcinnamoyl) amino-4-chlorobenzoic acid pretreatment, a phospholipase A2 inhibitor (-66%), suggesting that the major part of its effect is mediated by the release of TXA2. ASM responses to 5-oxo-ETE were also blocked by the Rho-kinase inhibitor Y-27632, which also partially inhibited the response to the TP receptor agonist U-46619, suggesting that the contractile response is due in part to Ca2+ sensitization of ASM cell myofilaments.     

Genistein inhibits lysosomal enzyme release by suppressing Ca2+ influx in HL-60 granulocytes

The tyrosine kinase inhibitor genistein (5-200 microM) suppressed Ca(2+)-dependent fMLP (1 microM) and ATP (100 microM)-induced release of the lysosomal enzyme, beta-glucuronidase from neutrophil-like HL-60 granulocytes. Agonist-induced Ca2+ mobilization resulted from the release of intracellular Ca2+ stores and the influx of extracellular Ca2+. Genistein (200 microM) suppressed fMLP (1 microM) and ATP (100 microM)-induced Ca2+ mobilization, by 30-40%. Ca2+ release from intracellular stores was unaffected by genistein, however, genistein abolished agonist-induced Ca2+ (Mn2+) influx. Consistent with these findings, genistein (200 microM) or removal of extracellular Ca2+ (EGTA 1 mM), inhibited Ca(2+)-dependent agonist-induced beta-glucuronidase release by similar extents (about 50%). In the absence of extracellular Ca2+, genistein had a small additional inhibitory effect on fMLP and ATP-induced beta-glucuronidase release, suggesting an additional inhibitory site of action. Genistein also abolished store-operated (thapsigargin-induced) Ca2+ (Mn2+) influx. Neither fMLP nor ATP increased the rate of Mn2+ influx induced by thapsigargin (0.5 microM). These data indicate that agonist-induced Ca2+ influx and store-operated Ca2+ influx occur via the same genistein-sensitive pathway. Activation of this pathway supports approximately 50% of lysosomal enzyme release induced by either fMLP or ATP from HL-60 granulocytes.     

Spontaneous Ca^2＋ oscillations in subcellular compartments of vascular smooth muscle cells rely on different Ca^2＋ pools

INTRODUCTION In vascular smooth muscle, as in other types of muscle,an increase in intracellular Ca2 is the immediate triggerfor contraction, which ultimately determines vascular toneand peripheral resistance. In the past 12 years, investiga-tors have …     

Phorbol ester and 1-oleoyl-2-acetylglycerol inhibit angiotensin activation of phospholipase C in cultured vascular smooth muscle cells

Angiotensin II acts on cultured rat aortic vascular smooth muscle cells (VSMC) to induce the rapid, phospholipase C-mediated generation of inositol trisphosphate from phosphatidylinositol 4,5-bisphosphate and mobilization of intracellular Ca2+. sn-1,2-Diacylglycerol, the other major product of inositol phospholipid breakdown, is known to activate protein kinase C, but its role in angiotensin II action on VSMC has not been defined. We report herein that, in cultured VSMC prelabeled with [3H]myoinositol, brief incubations (2-5 min) with 4 beta-phorbol 12-myristate 13-acetate (PMA) (1-100 nM) or 1-oleoyl-2-acetylglycerol (10-100 microM), two potent activators of protein kinase C, inhibit subsequent angiotensin II (100 nM)-induced increases in phosphatidylinositol 4,5-bisphosphate breakdown and inositol trisphosphate formation. In addition, pretreatment of VSMC with either PMA (IC50 approximately 1 nM) or 1-oleoyl-2-acetylglycerol (IC50 approximately 7.5 microM) also markedly inhibits angiotensin II (1 nM)-stimulated increases in cytosolic free Ca2+, as measured with the calcium-sensitive fluorescent indicator quin 2, or 45Ca2+ efflux. Neither PMA nor 1-oleoyl-2-acetylglycerol initiated phosphatidylinositol 4,5-bisphosphate breakdown or Ca2+ flux by itself. PMA treatment (10 nM, 5 min) did not influence the number or affinity of 125I-angiotensin II-binding sites in intact cells. These data suggest that one function of angiotensin II-generated sn-1,2-diacylglycerol in vascular smooth muscle may be to modulate, by protein kinase C-mediated mechanisms, angiotensin II receptor coupling to phospholipase C.     

Calcium mobilization as the endothelium-independent mechanism of action involved in the vasorelaxant response induced by the aqueous fraction of the ethanol extract of Albizia inopinata G. P. Lewis (AFL) in the rat aorta.

In a previous work, we demonstrated that, in normotensive rats, AFL induced a marked hypotension due to a decrease in total peripheral resistances (TPR), partially secondary to the release of NO by the endothelium. NO did not, however, account for the total vasodilation produced by AFL in these rats. The aim of this study was to determine the involvement of the intracellular calcium mobilization in the vasorelaxant action induced by AFL in the rat aorta. In aorta of normotensive rats AFL (10, 20, 40 and 80 microg/ml) inhibited the sustained contractions induced by KCl (80 and 30 mM) and phenylephrine (Phe, 1 microM) with similar IC50 values (54 +/- 6, 52 +/- 4 and 65 +/- 4 microg/ml, respectively). The relaxing response induced by AFL against Phe-induced contractions was modified significantly by the endothelium removal (IC50 = 132 +/- 23 and 65 +/- 4 microg/ml, endothelium removed and intact endothelium aortic rings, respectively). Nevertheless, removal of the endothelium did not significantly change IC50 values when KCl (30 and 80 mM) was used as the contractile agent. The inhibitory effect induced by AFL on high (64.5 mM) K+-induced contraction was potentiated slightly (p < 0.05) by the decrease (from 2.5 to 0.3 mM, Ca2+) and attenuated by the increase (from 2.5 to 7.5 mM Ca2+) in the external [Ca2+]. In addition, in aortas from normotensive rats, AFL antagonized transient contractions induced in Ca2+-free media induced by 1 microM noradrenaline in a concentration-dependent manner, but not those induced by 20 mM caffeine. It is suggested that the remaining vasodilator effect of AFL in normotensive rats is probably due to an inhibition of Ca2+ influx and/or inhibition of intracellular Ca2+ mobilization from the noradrenaline-sensitive stores.     

Effect of t-butyl hydroperoxide on Ca2+ movement in PC12 pheochromocytoma cells

The effect of the oxidant t-butyl hydroperoxide on intracellular free levels of Ca2+ ([Ca2+]i) in PC12 pheochromocytoma cells was examined by using fura-2 as a fluorescent dye. t-Butyl hydroperoxide induced an increase in [Ca2+]i in a concentration-dependent fashion between 50-250 microM with an EC50 of 100 microM. The [Ca2+]i signal consisted of a slow rise and a sustained phase. The response was decreased by 65% by removal of extracellular Ca2+. In Ca(2+)-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) abolished 150 microM t-butyl hydroperoxide-induced [Ca2+]i increase, and conversely, pretreatment with t-butyl hydroperoxide abrogated thapsigargin-induced [Ca2+]i increase. The 150 microM t-butyl hydroperoxide-induced [Ca2+]i increase in Ca2+ medium was reduced by 42 +/- 5% by pretreatment with 0.1 microM nicardipine but not by 10 microM verapamil, nifedipine, nimodipine or diltiazem, or by 50 microM La3+ or Ni2+. Pretreatment with 10 microM t-butyl hydroperoxide for 40 min did not affect 10 microM ATP-induced [Ca2+]i increase. Together, the results show that t-butyl hydroperoxide induced significant [Ca2+]i increase in PC12 cells by causing store Ca2+ release from the thapsigargin-sensitive endoplasmic reticulum pool in an inositol 1,4,5-trisphosphate-independent manner and by inducing Ca2+ influx via a nicardipine-sensitive pathway.     

Mechanisms of miconazole-induced rise in cytoplasmic calcium concentrations in Madin Darby canine kidney (MDCK) cells

The effect of miconazole on intracellular calcium levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was studied using fura-2 as the Ca2+ indicator. Miconazole increased [Ca2+]i dose-dependently at concentrations of 5-100 microM. The [Ca2+]i transient consisted of an initial rise, a gradual decay and an elevated plateau (220 s after addition of the drug). Removal of extracellular Ca2+ partly reduced the miconazole response. Mn2+ quench of fura-2 fluorescence confirmed that miconazole induced Ca2+ influx. The miconazole-sensitive intracellular Ca2+ store overlapped with that sensitive to thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump, because 20 microM miconazole depleted the thapsigargin (1 microM)-sensitive store, and conversely, thapsigargin abolished miconazole-induced internal Ca2+ release. Miconazole (20-50 microM) partly inhibited the capacitative Ca2+ entry induced by 1 microM thapsigargin, measured by depleting intracellular Ca2+ store in Ca(2+)-free medium followed by addition of 10 mM CaCl2. Miconazole induced capacitative Ca2+ entry on its own. Pretreatment with 0.1 mM La3+ partly inhibited 20 microM miconazole-induced Mn2+ quench of fura-2 fluorescence and [Ca2+]i rise, suggesting that miconazole induced Ca2+ influx via two pathways separable by 0.1 mM La3+. Miconazole-induced internal Ca2+ release was not altered when the cytosolic level of inositol 1,4,5-trisphosphate (IP3) was substantially inhibited by the phospholipase C inhibitor U73122.     

Capsazepine, a vanilloid antagonist, abolishes tonic responses induced by 20-HETE on guinea pig airway smooth muscle

The aim of this study was to delineate the mode of action of 20-hydroxy-eicosatetraenoic acid (20-HETE) in airway smooth muscle (ASM) cells. ASM metabolizes arachidonic acid by various enzymatic pathways, including the cytochrome P-450 (CYP-450) omega-hydroxylase, which leads to the production of 20-HETE, a bronchoconstrictive eicosanoid. The present study demonstrated that 20-HETE induced concentration-dependent tonic responses in ASM, whereas transient responses were recorded in Ca2+-free solution, suggesting an intracellular Ca2+ release process. 20-HETE inotropic responses were abolished by 36 microM 2-aminoethoxydiphenyl borate or 1 microM thapsigargin but were insensitive to 10 microM ryanodine, indicating that inositol triphosphate receptors likely control the release of intracellular Ca2+. Sustained tension, which required Ca2+ entry, was partially blocked by 1 microM nifedipine (an L-type) and 100 microM Gd3+ (a nonselective cationic channel blocker). Moreover, in the absence of selective 20-HETE receptor antagonists, 20-HETE tonic responses were inhibited in a concentration-dependent manner (0.1-10 microM) by capsazepine, a well-characterized vanilloid receptor antagonist. Capsazepine was also observed to reverse cumulative responses to 20-HETE and capsaicin, a TRPV1 agonist. In addition, capsazepine pretreatment largely modified the sustained inotropic responses to 20-HETE, suggesting that 20-HETE cross-reacted with TRPV1 receptors with a low affinity (microM) or that its specific receptor was inhibited by the vanilloid antagonist. Data obtained using RHC-80267, ONO-RS-082, and eicosatetraynoic acid, respective inhibitors of diacylglycerol-lipase, phospholipase A2, and CYP-450 omega-hydroxylase, reveal that intracellular arachidonic acid production and its 20-HETE metabolite may be responsible for the activation of nonselective cationic channels and tonic responses.     

Intracellular free calcium transients induced by norepinephrine in rat aortic smooth muscle cells in primary culture

In cultured rat arterial smooth muscle cells treated with quin 2, cytosolic Ca2+ transients induced by norepinephrine were recorded microfluorometrically. In the presence or absence of extracellular Ca2+, norepinephrine induced transient and dose-dependent elevations in cytosolic Ca2+, with a similar time course, the peak levels being observed at 2 min. These transient elevations in cytosolic Ca2+ were dose-dependently inhibited by alpha-adrenergic antagonists, the order of potency being prazosin greater than phentolamine greater than yohimbine, irrespective of the presence of extracellular Ca2+. We propose that with or without extracellular Ca2+, norepinephrine activates mainly alpha-1 adrenoceptors leading to a release of Ca2+ from intracellular stores. This would explain the transient elevation in cytosolic Ca2+ in rat aortic vascular smooth muscle cells in primary culture.     

Effects of valinomycin on calcium mobilization in vascular smooth muscle cells induced by angiotensin II

The effect of the specific potassium (K+) ionophore valinomycin on increase in intracellular calcium concentration [( Ca2+]i) was studied in vascular smooth muscle cells (VSMC). Valinomycin at more than 10(-9) M dose-dependently suppressed phasic increase in [Ca2+]i in VSMC induced by angiotensin II (AII) in both control and Ca2+-free solution, indicating that it suppressed the release of Ca2+ from intracellular Ca2+ stores. Nicorandil and cromakalim, which are both K+ channel openers, also suppressed the increases in [Ca2+]i induced by AII in the Ca2+ free solution. However, valinomycin did not suppress AII-induced production of inositol 1,4,5-trisphosphate (IP3), which is known to mediate the release of Ca2+. These results indicate that decrease of intracellular K+ induced by valinomycin suppressed the release of Ca2+ from intracellular Ca2+ stores induced by IP3.     

Chronic muscarinic stimulation of SH-SY5Y neuroblastoma cells suppresses inositol 1,4,5-trisphosphate action. Parallel inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ mobilization and inositol 1,4,5-trisphosphate binding.

The possibility that chronic activation of the phosphoinositide-mediated signaling pathway modifies the Ca(2+)-mobilizing action of inositol 1,4,5-trisphosphate (InsP3) was examined. SH-SY5Y human neuroblastoma cells were exposed to carbachol, permeabilized electrically, loaded with 45Ca2+, and 45Ca2+ mobilization in response to exogenous InsP3 was assessed. In control permeabilized cells, InsP3 released 65 +/- 2% of sequestered 45Ca2+ (EC50 = 0.32 +/- 0.05 microM). Pre-treatment with carbachol reduced both maximal InsP3-induced 45Ca2+ release (to 34 +/- 3%, with half-maximal and maximal inhibition at approximately 3 and 6 h, respectively) and the potency of InsP3 (EC50 = 0.92 +/- 0.13 microM). This inhibitory effect of carbachol was half-maximal at approximately 5 microM, was mediated by muscarinic receptors, and was reversible following withdrawal of agonist. Pretreatment with phorbol 12,13-dibutyrate did not alter the maximal effect of InsP3 but doubled its EC50. Evidence suggesting that the inhibitory effects of carbachol pretreatment resulted from altered Ca2+ homeostasis was not forthcoming; both 45Ca2+ uptake and release induced by ionomycin and thapsigargin were identical in control and pretreated permeabilized cells, as were the characteristics of reuptake of released Ca2+. In contrast, carbachol pretreatment, without altering the affinity of InsP3 (Kd = 64 +/- 7 nM), reduced the density of [32P]InsP3-binding sites from 2.0 +/- 0.1 to 1.0 +/- 0.1 pmol/mg protein with a time course essentially identical to that for the reduction in responsiveness to InsP3. This effect was not mimicked by pretreatment of cells with phorbol 12,13-dibutyrate. These data indicate that chronic activation of phosphoinositide hydrolysis can reduce the abundance of InsP3 receptors and that this causes a reduction in size of the InsP3-sensitive Ca2+ store. This modification, possibly in conjunction with a protein kinase C-mediated event, appears to account for the carbachol-induced suppression of InsP3 action. As intracellular InsP3 mass remained elevated above basal for at least 24 h after addition of carbachol, suppression of the Ca(2+)-mobilizing activity of InsP3 represents an important adaptive response to cell stimulation that can limit the extent to which intracellular Ca2+ is mobilized.     

The regulation of the cytoplasmic free Ca2+ concentration in aortic smooth muscle cells (A7r5 line) after stimulation by vasopressin and bombesin

The characteristics of intracellular Ca2+ transient induced by vasopressin and bombesin in aortic smooth muscle cells were studied using flow cytometric analysis of indo-1 loaded cells. The two hormones induced a rapid and transient rise in [Ca2+]i. This Ca2+ transient was independent of the presence of extracellular Ca2+. Addition of bombesin to cells that have already been stimulated by vasopressin (or conversely the addition of vasopressin to bombesin-stimulated cells) results in a second Ca2+ transient that has a smaller amplitude. This transient is the same when the external Ca2+ concentration is lowered from 1.8 mM to 50 nM, suggesting that the agonist-sensitive pool reloaded using the Ca2+ that has been previously released into the cytoplasm. Intracellular Ca2+ pools that have been depleted by a prolonged incubation of the cells in a low Ca2+ medium can be refilled by shifting cells to a high Ca2+ medium. The reloading was analyzed in detail and found to be a slow process. It is hardly affected by Ni2+ or by (-)D888, a potent inhibitor of the voltage-dependent Ca2+ channels. It is accelerated when Ca2+ uptake by the Na+/Ca2+ exchange system is stimulated. The results suggest that Ca2+ homeostasis in aortic smooth muscle cells is achieved using mechanisms that are distinct from those operating in various acini and in striated muscles.     

Endothelin-1 increases intracellular calcium mobilization but not calcium uptake in rabbit vascular smooth muscle cells

Conflicting evidence has been reported regarding the role of endothelin-1, a potent vasconstrictor peptide, in stimulating extracellular calcium influx in rabbit vascular smooth muscle. The objective of this study was to elucidate the effects of endothelin-1 on transmembrane 45Ca2+ influx and intracellular calcium mobilization in cultured rabbit aortic smooth muscle cells. In calcium containing buffer, endothelin-1 induced a concentration-dependent 45Ca2+ efflux response over the range of 10 pM to 100 nM with an EC50 of approximately 60 pM. Maximum endothelin-stimulated 45Ca2+ efflux was not affected by the absence of extracellular calcium or the presence of 1 microM verapamil. Endothelin-1 did not induce transplasmalemmal 45Ca2+ uptake at times up to 30 min. These findings suggest that an alteration in intracellular calcium handling, rather than extracellular calcium influx, is responsible for the endothelin-stimulated increase in intracellular calcium concentration in rabbit aortic smooth muscle cells.     

The extracellular zinc-sensing receptor mediates intercellular communication by inducing ATP release

Taste and salivary secretion disorders have been linked to zinc deficiency, indeed zinc is found in secretory granules in the salivary gland. The signaling role for the zinc release in this tissue, however, is poorly understood. Here, we address the signaling pathways and physiological role of the zinc-sensing receptor, ZnR, in the ductal salivary gland cell line, HSY. Exposure of these cells to zinc triggered intracellular Ca2+ release from thapsigargin-sensitive stores. The G alpha q inhibitor, YM-254890 (1 microM), eliminated the Zn2+-dependent Ca2+ response, demonstrating that ZnR is a G alpha q-coupled receptor. Dose-response curves yielded an apparent K0.5 of 36 microM and a Hill coefficient of 7 in the absence of extracellular Ca2+, and K0.5 of 55 microM with a Hill coefficient of 3 in its presence. This indicates that although Zn2+ is essential for ZnR activation, Ca2+ may affect the receptor co-operativity. The homologous desensitization pattern of ZnR was characterized by pre-exposure of cells to Zn2+ at concentrations found to activate the receptor. Re-exposure of cells to Zn2+ elicited an attenuated Zn2+-dependent Ca2+ response for at least 3 h, indicating that the ZnR is strongly desensitized by Zn2+. Finally, we studied the paracrine affects of ZnR using a co-culture consisting of the HSY cells and vascular smooth muscle cells (VSMCs). While no Zn2+-dependent Ca2+ release was observed in VSMC alone, application of Zn2+ to the co-culture induced a Ca2+ rise in both HSY cells and VSMC. This Ca2+ rise was inhibited by the ATP scavenger, apyrase. Taken together, our results demonstrate that ZnR activity is monitored in salivary cells and is modulated by extracellular Ca2+. We further show that ZnR enhances secretion of ATP, thereby linking zinc to key signaling pathways involved in modification of salivary secretions by the ductal cells.     

The effects of EGTA on vascular smooth muscle contractility in calcium-free medium

The effect of EGTA, commonly present in Ca2+-free physiological saline solution, on the contractile responses induced by Ca2+ and phenylephrine was studied in dog mesenteric arteries and aortas of rats and rabbits. EGTA substantially enhanced the contractile responses of these vascular strips or rings to added Ca2+ after a prolonged preincubation period in the Ca2+-free medium. The maximal level of the enhanced contractile responses was independent of EGTA concentration, but the rate of the maximal responses was faster at higher EGTA concentration, presumably as a result of faster removal of intracellular Ca2+. Such a Ca2+-induced response was sensitive to the Ca2+ antagonist, nifedipine. EGTA present at low concentrations (50 and 400 microM) in Ca2+-free medium also inhibited the phenylephrine-induced contractile response more prominently for the longer preincubation periods of the vascular tissues in Ca2+-free medium. Our results suggest that EGTA, even when added at low concentrations to the vascular smooth muscle for a sufficiently long period in Ca2+-free medium, may cause destabilization of the cell membranes leading to increased permeability to subsequently added Ca2+. EGTA may also remove the superficially bound Ca2+ and subsequently reduce the intracellular Ca2+ pool via extraction of the intracellular Ca2+ at the cell membrane surfaces.     

Receptor-Coupled Phosphoinositide Hydrolysis in Human Retinal Pigment Epithelium

Carbachol and histamine stimulated phosphoinositide (PPI) hydrolysis in cultured human retinal pigment epithelium (RPE), as reflected by an accumulation of 3H-inositol phosphates in the presence of 10 mM Li+. Carbachol increased PPI hydrolysis to greater than 600% of basal with an EC50 of 60 microM; stimulation was linear up to 60 min. This activation likely occurred via the M3 muscarinic cholinergic receptor based on the IC50 values for 4-diphenylacetoxy-N-methylpiperidine methiodide (0.47 nM), pirenzepine (280 nM), and 11-[[2-[(diethylamino)methyl]-1-piperidinyl]-acetyl]-5,11- dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one (1.4 microM). Carbachol-mediated PPI hydrolysis was decreased by 80% in the absence of extracellular Ca2+. Histamine stimulated PPI turnover in a linear manner by 180% with an EC50 of 20 microM by the H1 histaminergic receptor. Serotonin, glutamate, norepinephrine, and dopamine were inactive. In human RPE, the resting cytoplasmic Ca2+ concentration, as determined by fura-2 fluorescence, was 138 +/- 24 nM. On the addition of carbachol, there was a 180% increase in peak intracellular Ca2+; addition of histamine increased intracellular Ca2+ by 187%. These results suggest receptor-mediated, inositol lipid hydrolysis is coupled to intracellular Ca2+ flux in human RPE.