Diverse effects of monensin on capacitative Ca2+ entry and release of stored Ca2+ in vascular smooth muscle cells

The effects of monensin, an activator of Na(+)/H(+) exchanger (NHE), on capacitative Ca(2+) entry (CCE) were investigated using A7r5 cells. Capacitative Ca(2+) entry was induced by elevation of extracellular Ca(2+) concentrations of A7r5 cells in which stored Ca(2+) had been depleted by previous administration of thapsigargin. Capacitative Ca(2+) entry was abolished by pretreatment of the cells with SKF-96365 (1-[beta-(3-[4-methoxyphenyl]propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride) but was not affected by pretreatment with verapamil. Monensin significantly increased capacitative Ca(2+) entry. On the other hand, 5-hydroxytryptamine-induced inositol monophosphate accumulation and subsequent intracellular Ca(2+) release from its stores were significantly inhibited by monensin, while thapsigargin-induced Ca(2+) release was not affected by monensin. These results suggest that monensin has diverse actions on capacitative Ca(2+) entry and agonist-induced release of stored Ca(2+) in vascular smooth muscle cells.     

Novel pyrazole compounds for pharmacological discrimination between receptor-operated and store-operated Ca2+ entry pathways

Background and purpose

Pyrazole derivatives have recently been suggested as selective blockers of transient receptor potential cation (TRPC) channels but their ability to distinguish between the TRPC and Orai pore complexes is ill-defined. This study was designed to characterize a series of pyrazole derivatives in terms of TRPC/Orai selectivity and to delineate consequences of selective suppression of these pathways for mast cell activation.

Experimental approach

Pyrazoles were generated by microwave-assisted synthesis and tested for effects on Ca²⁺ entry by Fura-2 imaging and membrane currents by patch-clamp recording. Experiments were performed in HEK293 cells overexpressing TRPC3 and in RBL-2H3 mast cells, which express classical store-operated Ca²⁺ entry mediated by Orai channels. The consequences of inhibitory effects on Ca²⁺ signalling in RBL-2H3 cells were investigated at the level of both degranulation and nuclear factor of activated T-cells activation.

Key Results

Pyr3, a previously suggested selective inhibitor of TRPC3, inhibited Orai1- and TRPC3-mediated Ca²⁺ entry and currents as well as mast cell activation with similar potency. By contrast, Pyr6 exhibited a 37-fold higher potency to inhibit Orai1-mediated Ca²⁺ entry as compared with TRPC3-mediated Ca²⁺ entry and potently suppressed mast cell activation. The novel pyrazole Pyr10 displayed substantial selectivity for TRPC3-mediated responses (18-fold) and the selective block of TRPC3 channels by Pyr10 barely affected mast cell activation.

Conclusions and Implications

The pyrazole derivatives Pyr6 and Pyr10 are able to distinguish between TRPC and Orai-mediated Ca²⁺ entry and may serve as useful tools for the analysis of cellular functions of the underlying Ca²⁺ channels.     

Stimulation of cellular free Ca2+ elevation and inhibition of store-operated Ca2+ entry by kazinol B in neutrophils

Kazinol B, a natural isoprenylated flavan, stimulated the [Ca²⁺]_i elevation in the presence or absence of Ca²⁺ in the medium. Treatment with chymotrypsin or phorbol 12-myristate 13-acetate to shedding of l-selectin had no effect on subsequent kazinol B-induced Ca²⁺ response. Upon initial cyclopiazonic acid (CPA) treatment in the absence of external Ca²⁺, the subsequent [Ca²⁺]_i rise followed by challenge with kazinol B was greatly diminished. The ryanodine receptor blockers, 8-bromo-cyclic ADP-ribose and ruthenium red did not affect kazinol B-evoked Ca²⁺ release from internal stores. However, the inhibitors of sphingosine kinase, dimethylsphingosine, but not dihydrosphingosine, inhibited kazinol B-induced Ca²⁺ release. Kazinol B-induced [Ca²⁺]_i rise was not affected by two nitric oxidase inhibitors, N-(3-aminomethyl)benzylacetamidine (1400W) and 7-nitroindazole, cytochalasin B and Na⁺-deprivation. This response was slightly attenuated by 2-aminoethyldiphenyl borate (2-APB), a d-myo-inositol 1,4,5-trisphosphate (IP₃) receptor blocker, and by genistein, a general tyrosine kinase inhibitor. However, the Ca²⁺ response was greatly diminished by two actin filament reorganizers, calyculin A and jasplakinolide, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), an inhibitor of phosphoinositide 3-kinase, N-(3-aminomethyl)benzylacetamidine (SB 203580), the p38 mitogen-activated protein kinase inhibitor, 1-[6-[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), the inhibitor of phospholipase C-coupled processes, and by 0.3 mM La³⁺ or Ni²⁺. Kazinol B did not evoke any appreciable Ba²⁺ and Sr²⁺ entry into cells. The Ca²⁺ entry blockers, 1-[-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365), but not cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12,330A), inhibited a kazinol B-induced [Ca²⁺]_i rise. Kazinol B had no effect on the pharmacologically isolated plasma membrane Ca²⁺-ATPase activity. In a Ca²⁺-free medium, kazinol B inhibited the subsequent Ca²⁺ addition, resulting in robust entry in CPA- and formyl peptide-activated cells. Kazinol B produced a concentration-dependent reduction in the mitochondrial membrane potential. These results indicate that kazinol B stimulates Ca²⁺ release from internal Ca²⁺ store, probably through the sphingosine 1-phosphate and IP₃ signaling, and activates external Ca²⁺ influx mainly through a non-store-operated Ca²⁺ entry (non-SOCE) pathway. Inhibition of SOCE by kazinol B is probably attributable to a break in the Ca²⁺ driven force of mitochondria.     

Heparin specifically inhibits the inositol 1,4,5-trisphosphate-induced Ca2+ release from skinned rat aortic smooth muscle cells in primary culture

Summary By measuring the ⁴⁵Ca²⁺ movement in saponin-skinned primary cultured rat aortic smooth muscle cells, we examined the specificity of the inhibitory effect of heparin on the IP₃-induced Ca²⁺ release. IP₃ (100 mol/l) markedly (98%) decreased the MgATP-dependent ⁴⁵Ca²⁺ content in the non-mitochondrial Ca²⁺ stores in the presence of 1 mol/l free Ca²⁺. Heparin (1–100 g/ml) dose-dependently inhibited this Ca²⁺ release by IP₃. In Ca²⁺-free solution, heparin (100 g/ml) inhibited the increases in ⁴⁵Ca²⁺ efflux rate evoked by 10 mol/l IP₃. De-N-sulfated heparin did not inhibit the IP₃-induced Ca²⁺ release. Hyaluronic acid, heparan sulfate, chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C and 2,6-disulfated d-glucosamine had no inhibitory effects on the IP₃-induced Ca²⁺ release. High concentrations (over 1 mg/ml) of heparin inhibited the ⁴⁵Ca²⁺ influx and decreased the Ca²⁺ content in skinned cells. These results suggest that heparin (1–100 g/ml) specifically inhibits the IP₃-induced increase in Ca²⁺ permeability of Ca²⁺ stores and that three sulfate groups at different locations on the molecule of heparin, two at the d-glucosamine and one at the iduronic acid, may be important for this action, in skinned vascular smooth muscle cells, in culture. Send offprint requests to H. Kanaide at the above address     

Orai1-STIM1 formed store-operated Ca2+ channels (SOCs) as the molecular components needed for Pb2+ entry in living cells

Heavy metal lead (Pb2+) is a pollutant and causes severe toxicity when present in human tissues especially the nervous system. Recent reviews have suggested that Pb2+ can target Ca2+-related proteins within neurons and that Ca2+ channels might be a candidate for Pb2+ entry. This study's main aim was to identify the functional entry pathway of Pb2+ into living cells. We firstly characterized the endogenous expression of Orai1 and STIM1 mRNA together with the level of thapsigargin (TG) stimulated capacitative Ca2+ entry in PC12 and HeLa cells; this was done by RT-PCR and time-lapse Ca2+ imaging microscopy, respectively. Our data supported Orai1 and STIM1 as contributing to store-operated Ca2+ channel (SOC) basal activity. Secondly, using the indo-1 quenching method with the SOC blocker 2-APB, we observed that Pb2+ was able to enter cells directly through unactivated SOCs without TG pretreatment. Thirdly, we further demonstrated that co-expression of Orai1 and STIM1 differentially enhanced SOC functional activity (4-fold with PC12 and 5-fold with HeLa cells) and Pb2+ entry (5- to 7-fold with PC12 and 2-fold with HeLa cells). Furthermore, after a 1 h of Pb2+ exposure, the depolarization- and histamine-induced Ca2+ responses were significantly decreased in both PC12 and HeLa cells in a dose-dependent manner. This result indicated that the decreased Ca2+ responses were, in part, due to Pb2+ entry. In summary, our results suggest that SOCs are responsible for Pb2+ permeation and that the Orai1-STIM1 protein complex formed by functional SOCs is one of the molecular components involved in Pb2+ entry.     

Inhibition of store-operated Ca(2+) channels prevent ethanol-induced intracellular Ca(2+) increase and cell injury in a human hepatoma cell line

Elevated intracellular Ca²⁺ content is implicated in ethanol-induced hepatocyte apoptosis and necrosis. Extracellular Ca²⁺ influx has been suggested to play a role in this process. However, the exact Ca²⁺-permeable channel involved in the plasma membrane is still unclear. This study investigated the role of store-operated calcium entry (SOCE) in ethanol-induced cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) increase and hepatotoxicity. Ethanol (25-800 mM) dose-dependently increased [Ca²⁺]_i content and hepatocyte damage in HepG2 cells. 2-aminoethoxydiphenyl borate (2-APB), the proved efficient antagonist of SOCs, dose-dependently suppressed the ethanol (200 nM)-increased [Ca²⁺]_i content and protected against ethanol-induced viability loss and transaminase leakage. Exposure to 200 mM ethanol for 24 h significantly upregulated the mRNA and protein expression of calcium release-activated calcium channel protein 1 (CRACM1, Orai1) and stromal interaction molecule 1 (STIM1), the two main molecular constituents of SOCs, which was sustained for at least 72 h. In addition, small interfering RNA knockdown of STIM1 attenuated the ethanol-increased [Ca²⁺]_i content and hepatotoxicity. Taken together, these data indicate that the Ca²⁺ channel of SOCE may be involved in the pathogenesis of ethanol-induced intracellular Ca²⁺ elevation and consequent hepatocyte damage.     

Role of sarcoplasmic reticulum Ca<Superscript>2+</Superscript> content in Ca<Superscript>2+</Superscript> entry of bovine airway smooth muscle cells

Depletion of intracellular Ca²⁺ stores induces the opening of an unknown Ca²⁺ entry pathway to the cell. We measured the intracellular free-Ca²⁺ concentration ([Ca²⁺]i) at different sarcoplasmic reticulum (SR) Ca²⁺ content in fura-2-loaded smooth muscle cells isolated from bovine tracheas. The absence of Ca²⁺ in the extracellular medium generated a time-dependent decrement in [Ca²⁺]i which was proportional to the reduction in the SR-Ca²⁺ content. This SR-Ca²⁺ level was indirectly determined by measuring the amount of Ca²⁺ released by caffeine. Ca²⁺ restoration at different times after Ca²⁺-free incubation (2, 4, 6 and 10 min) induced an increment of [Ca²⁺]i. This increase in [Ca²⁺]i was considered as Ca²⁺ entry to the cell. The rate of this entry was slow (~0.3 nM/s) when SR-Ca²⁺ content was higher than 50% (2 and 4 min in Ca²⁺-free medium), and significantly (p<0.01) accelerated (>1.0 nM/s) when SR-Ca²⁺ content was lower than 50% (6 and 10 min in Ca²⁺-free medium). Thapsigargin significantly induced a higher rate of this Ca²⁺ entry (p<0.01). Variations in Ca²⁺ influx after SR-Ca²⁺ depletion were estimated more directly by a Mn²⁺ quench approach. Ca²⁺ restoration to the medium 4 min after Ca²⁺ removal did not modify the Mn²⁺ influx. However, when Ca²⁺ was added after 10 min in Ca²⁺-free medium, an increment of Mn²⁺ influx was observed, corroborating an increase in Ca²⁺ entry. The fast Ca²⁺ influx was Ni²⁺ sensitive but was not affected by other known capacitative Ca²⁺ entry blockers such as La³⁺, Mg²⁺, 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 Ca²⁺ influx was only sensitive to SKF 96365. In conclusion, our results indicate that in bovine airway smooth muscle cells Ca²⁺ influx after SR-Ca²⁺ depletion has two rates: A) The slow Ca²⁺ influx, which occurred in cells with more than 50% of their SR-Ca²⁺ content, is sensitive to SKF 96365 and appears to be a non-capacitative Ca²⁺ entry; and B) The fast Ca²⁺ influx, observed in cells with less than 50% of their SR-Ca²⁺ content, is probably a capacitative Ca²⁺ entry and was only Ni²⁺-sensitive.     

Effects of calcitonin gene-related peptide (CGRP) on Ca2+-channel current of isolated smooth muscle cells from rat vas deferens

Summary Effects of calcitonin gene-related peptide (CGRP), a putative non-adrenergic non-cholinergic neutrotransmitter on the electrical properties of the cell membrane, were investigated in enzymically dispersed smooth muscle cells from rat vas deferens. Under current clamp conditions, CGRP (up to 10^–7 M) did not induce significant changes in membrane potentials or input resistance in the resting state. The configurations of action potentials elicited by depolarizing current pulses were also unaffected, except that a prolongation of the duration of the action potentials by a high dose (10^–7 M) of CGRP was observed in some of the cells. Under whole cell voltage clamp conditions, the transient and sustained K⁺ currents, activated by depolarizing voltage-steps, were apparently decreased in the presence of 10^–9 to 10^–7 M CGRP. The peptide increased the voltage-gated Ca²⁺ current in cells loaded with 145 mM Cs⁺ solution in order to block the K+ currents. The voltage-dependency of the peak Ca²⁺ current was not changed by CGRP. Ba²⁺ (10.8 mM) was used as a charge carrier for the Ca²⁺-channel current to clarify further the effects of CGRP on the properties of the current. CGRP (10^–8 M) delayed the inactivation time course of the Ca²⁺-channel current and slowed the recovery from inactivation. The peptide did not affect the steady-state inactivation measured by changing the holding potential. The Ca²⁺-channel current in the presence of CGRP was suppressed by nicardipine (10^–6 M) to the same extent as the current under control conditions. The results suggest that CGRP modifies the L-type Ca²⁺ channel in smooth muscle cells. Correspondence to N. Matsuki at the above address     

Ca2+ signalling by P2Y receptors in cultured rat aortic smooth muscle cells

Background and purpose:

P2Y receptors evoke Ca²⁺ signals in vascular smooth muscle cells and regulate contraction and proliferation, but the roles of the different P2Y receptor subtypes are incompletely resolved.

Experimental approach:

Quantitative PCR was used to define expression of mRNA encoding P2Y receptor subtypes in freshly isolated and cultured rat aortic smooth muscle cells (ASMC). Fluorescent indicators in combination with selective ligands were used to measure the changes in cytosolic free [Ca²⁺] in cultured ASMC evoked by each P2Y receptor subtype.

Key results:

The mRNA for all rat P2Y receptor subtypes are expressed at various levels in cultured ASMC. Four P2Y receptor subtypes (P2Y1, P2Y2, P2Y4 and P2Y6) evoke Ca²⁺ signals that require activation of phospholipase C and comprise both release of Ca²⁺ from stores and Ca²⁺ entry across the plasma membrane.

Conclusions and implications:

Combining analysis of P2Y receptor expression with functional analyses using selective agonists and antagonists, we isolated the Ca²⁺ signals evoked in ASMC by activation of P2Y1, P2Y2, P2Y4 and P2Y6 receptors.     

Ceramide 1-(2-cyanoethyl) phosphate enhances store-operated Ca2+ entry in thyroid FRTL-5 cells

Sphingolipid derivatives cause diverse effects towards the regulation of intracellular free Ca(2+) concentrations ([Ca(2+)](i)) in a multitude of nonexcitable cells. In the present investigation, the effect of C-8 ceramide-1-(2-cyanoethyl) phosphate (C1CP) on store-operated Ca(2+) (SOC) entry was investigated. C1CP evoked a modest increase in [Ca(2+)](i). The increase was inhibited by the SOC channel antagonist 1-(beta-[3-(4methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole (SKF96365) but not by overnight pretreatment of the cells with pertussis toxin. C1CP did not invoke the production of inositol phosphates. When cells were stimulated with both C1CP and thapsigargin, the thapsigargin-invoked increase in [Ca(2+)](i) was enhanced in comparison to control cells. When Ca(2+) was added to cells treated with both C1CP and thapsigargin in a Ca(2+)-free buffer, the increase in [Ca(2+)](i) was enhanced in comparison to control cells. In patch-clamp experiments, C1CP hyperpolarized the membrane potential (E(m)) of the cells and attenuated the thapsigargin-invoked depolarization of the E(m). The effects of C1CP came, in part, as a result of a decreased conductance of the cell membrane towards Cl(-) ions, as C1CP in a Cl(-)-free solution also enhanced Ca(2+) entry. Barium 2-cyanoethylphosphate (Ba2Cy), which also contains the 2-cyanoethyl group, did not modulate thapsigargin-invoked changes in [Ca(2+)](i) nor did it modulate the E(m). In conclusion, C1CP enhances SOC entry, in part, via hyperpolarization of the E(m) and attenuation of the thapsigargin-invoked membrane depolarization, thus increasing the electrochemical gradient for Ca(2+) ions. Hence, C1CP may be a useful reagent for investigating the cellular effects of ceramide derivatives.     

Ca2+ channel blocking effect of iso-S-petasin in rat aortic smooth muscle cells

The purpose of the present study was to examine the mechanisms underlying the putative hypotensive actions of iso-S-petasin, a sesquiterpene extract of Petasites formosanus through both in vivo and in vitro experiments. Intravenous administration of iso-S-petasin elicited dose-dependent (0.1-1.5 mg/kg) hypotensive and bradycardiac responses in anesthetized rats. Isometric tension recording in isolated thoracic aorta revealed that iso-S-petasin (0.01-100 microM) inhibited KCl- or Bay K 8644 (1,4-dihydro-2,6-dimethyl-5-nitro-4-[2'-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid methyl ester)-induced vasoconstriction independent of endothelium. Iso-S-Petasin also attenuated Ca(2+)-induced vasoconstriction in a concentration-dependent manner in Ca(2+)-depleted/high K(+)-depolarized ring segments, indicating that iso-S-petasin inhibited Ca(2+) influx into vascular smooth muscle cells. This was confirmed by whole-cell patch-clamp recording in cultured vascular smooth muscle cells where iso-S-petasin (10-100 microM) appeared to directly inhibit the L-type voltage-dependent Ca(2+) channel (VDCC) activity. Intracellular Ca(2+) concentration ([Ca(2+)](i)) measurements using the fluorescent probe fura-2/AM (1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-amino-5'-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid pentaacetoxymethyl ester) showed suppression of the KCl-stimulated increase in [Ca(2+)](i) by iso-S-petasin (10, 100 microM). In conclusion, these results suggest that Ca(2+) antagonism of the L-type VDCC in vascular smooth muscle cells might largely account for the hypotensive action of iso-S-petasin.     

Blockade of calcium entry in smooth muscle cells by the antidepressant imipramine

The present study was designed to evaluate the effects of antidepressants on smooth muscle contractile activity. In rat aortic rings, the antidepressants imipramine, mianserin and sertraline provoked concentration-dependent inhibitions of the mechanical responses evoked by K+ (30 mM) depolarization. These myorelaxant effects were not modified by the presence of glibenclamide or 80 mM K+ in the bathing medium. Moreover, the vasodilator properties of imipramine were not affected by atropine, phentolamine and pyrilamine. Radioisotopic experiments indicated that imipramine failed to enhance 86Rb outflow from prelabelled and perifused aortic rings whilst counteracting the increase in 45Ca outflow provoked by a rise in the extracellular K+ concentration. Simultaneous measurements of contractile activity and fura-2 fluorescence revealed that, in aortic rings, imipramine reduced the mechanical and fluorimetric response to K+ challenge. In A7r5 smooth muscle cells, whole cell recordings further demonstrated that imipramine inhibited the inward Ca2+ current. Under different experimental conditions, the ionic and relaxation responses to the antidepressants were reminiscent of those mediated by the Ca2+ entry blocker verapamil. Lastly, it should be pointed out that imipramine exhibited a myorelaxant effect of similar amplitude on rat aorta and on rat distal colon. All together, these findings suggest that the myorelaxant properties of imipramine, and probably also setraline and mianserin, could result from their capacity to inhibit the voltage-sensitive Ca2+ channels.     

Relaxation induced by calcium ionophore is impaired in carotid arteries from 2K-1C rats due to failed effect of nitric oxide on the smooth muscle cells

Vascular endothelium generates nitric oxide (NO) in large vessels and induces relaxation of vascular smooth muscle cells (VSMC). The aim of this study was to evaluate the contribution of NO produced in the endothelial cells (EC) to the relaxation induced by the Ca²⁺ ionophore A23187 and whether this relaxation is impaired in renal hypertensive (2K-1C) rat arteries. Concentration-effect curves for A23187 were constructed in intact endothelium isolated carotid rings from 2K-1C and normotensive (2K) in the absence or in the presence of the extracellular NO scavenger haemoglobin or inhibitors of NO-synthase (NOS, L-NOARG), guanylyl-cyclase (GC, ODQ). In carotid rings loaded with Fluo-3AM, both EC and VSMC were simultaneously imaged by a confocal microscope and [Ca²⁺]_c was derived from fluorescence intensities (IF). The maximal relaxation (ME) induced by A23187 was lower in 2K-1C than in 2K arteries. A23187-induced relaxation was abolished by haemoglobin and L-NOARG in both groups. ODQ reduced the ME to A23187 in 2K and abolished its relaxation in 2K-1C. A23187 increased [Ca²⁺]_c in a similar way in 2K and 2K-1C EC, and decreased [Ca²⁺]_c in VSMC, which effect was higher in 2K than in 2K-1C arteries. L-NOARG inhibited the effect of A23187 in VSMC from 2K and abolished it in 2K-1C rats. On the other hand, L-NOARG did not modify the effect of A23187 in EC from 2K and 2K-1C rats.The basal content of cGMP was higher in 2K than in 2K-1C arterial rings that was similarly increased by A23187. In conclusion, the Ca²⁺ ionophore A23187 increases Ca²⁺, activates NOS and NO production in the EC activating GC in VSMC and [Ca²⁺]_c decrease. All these effects are higher in 2K, which contribute to the impaired relaxation to A23187 in 2K-1C rat arteries.     

2-Benzyloxybenzaldehyde inhibits formyl peptide-stimulated increase in intracellular Ca2+ in neutrophils mainly by blocking Ca2+ entry

2-Benzyloxybenzaldehyde (CCY1a) inhibited the formyl-Met-Leu-Phe (fMLP)-induced elevation of cytosolic [Ca²⁺] ([Ca²⁺]_i) in rat neutrophils. The late plateau phase, but not the initial Ca²⁺ spike, of the fMLP-induced [Ca²⁺]_i change was inhibited by CCY1a. In the absence of external Ca²⁺, CCY1a had no appreciable effect on either the fMLP- or cyclopiazonic acid (CPA)-induced [Ca²⁺]_i elevation. CCY1a failed to inhibit [Ca²⁺]_i changes induced by N-ethylmaleimide, GEA3162, ionomycin or sphingosine, but slightly inhibited the Ca²⁺ signals elicited by ATP or interleukin-8 (IL-8). In a classical Ca²⁺ readdition protocol, addition of CCY1a after cell activation strongly inhibited the [Ca²⁺]_i response to fMLP, whilst that to CPA was only slightly reduced. CCY1a nearly abrogated the fMLP-stimulated Mn²⁺ influx but was less effective on the CPA-induced response. CCY1a attenuated the levels of tyrosine-phosphorylated bands in the 70–85 kDa molecular mass range. CCY1a had no effect on the basal [Ca²⁺]_i level, the pharmacologically isolated plasma membrane Ca²⁺-ATPase activity or on the mitochondrial membrane potential. Thus, CCY1a blocks fMLP-induced Ca²⁺ entry into neutrophils probably by blocking the relevant Ca²⁺ channel directly or, alternatively, indirectly through the attenuation of tyrosine phosphorylation of some cellular proteins.     

Fluoxetine inhibits ATP-induced [Ca(2+)](i) increase in PC12 cells by inhibiting both extracellular Ca(2+) influx and Ca(2+) release from intracellular stores

Fluoxetine, a widely used antidepressant, has additional effects, including the blocking of voltage-gated ion channels. We examined whether fluoxetine affects ATP-induced calcium signaling in PC12 cells using fura-2-based digital calcium imaging, an assay for [3H]-inositol phosphates (IPs) and whole-cell patch clamping. Treatment with ATP (100 microM) for 2 min induced increases in intracellular free Ca(2+) concentrations ([Ca(2+)](i)). Treatment with fluoxetine (100 nM to 30 microM) for 5 min inhibited the ATP-induced [Ca(2+)](i) increases in a concentration-dependent manner (IC(50) = 1.85 microM). Treatment with fluoxetine (1.85 microM) for 5 min significantly inhibited the ATP-induced responses following the removal of extracellular Ca(2+) or depletion of intracellular Ca(2+) stores. Whereas treatment for 10 min with nimodipine (1 microM) significantly inhibited the ATP-induced [Ca(2+)](i) increase, treatment with fluoxetine further inhibited the ATP-induced response. Treatment with fluoxetine significantly inhibited [Ca(2+)](i) increases induced by 50 mM K(+). In addition, treatment with fluoxetine markedly inhibited ATP-induced inward currents in a concentration-dependent manner. However, treatment with fluoxetine did not inhibit ATP-induced [3H]-IPs formation. Therefore, we conclude that fluoxetine inhibits ATP-induced [Ca(2+)](i) increases in PC12 cells by inhibiting both the influx of extracellular Ca(2+) and the release of Ca(2+) from intracellular stores without affecting IPs formation.     

The mechanism of honokiol-induced and magnolol-induced inhibition on muscle contraction and Ca2+ mobilization in rat uterus

The effects of honokiol and magnolol extracted from the Magnolia officinalis on muscular contractile responses and intracellular Ca²⁺ mobilization were investigated in the non-pregnant rat uterus. Honokiol and magnolol (1–100 mol/l) were observed to inhibit spontaneous and uterotonic agonists (carbachol, PGF₂, and oxytocin)-, high K⁺-, and Ca²⁺ channel activator (Bay K 8644)-induced uterine contractions in a concentration-dependent manner. The inhibition rate of honokiol on spontaneous contractions appeared to be slower than that of magnolol-induced response. The time periods that were required for honokiol and magnolol, at 100 mol/l, to abolish 50% spontaneous contractions were approximately 6 min. Furthermore, honokiol and magnolol at 10 mol/l also blocked the Ca²⁺-dependent oscillatory contractions. Consistently, the increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) induced by PGF₂ and high K⁺ were suppressed by both honokiol and magnolol at 10 mol/l. After washout of these treatments, the rise in [Ca²⁺]_i induced by PGF₂ and high K⁺ was still partially abolished. In conclusion, the inhibitory effects of honokiol and magnolol on uterine contraction may be mediated by blockade of external Ca²⁺ influx, leading to a decrease in [Ca²⁺]_i. Honokiol and magnolol may be considered as putative Ca²⁺ channel blockers and be of potential value in the treatment of gynecological dysfunctions associated with uterine muscular spasm and dysmenorrhea.     

Post-transcriptional silencing of TRPC1 ion channel gene by RNA interference upregulates TRPC6 expression and store-operated Ca entry in A7r5 vascular smooth muscle cells

This study investigates functional consequences of TRPC1 ion channel downregulation observed in aging rat aorta by employing RNA interference in cultured vascular smooth muscle cells. For this purpose, A7r5 aortic smooth muscle cells were used in quantitative gene and protein expression as well as in functional analyses. According to quantitative RT-PCR results, TRPC3, TRPC4 and TRPC5 mRNAs were not at detectable levels. In siTRPC1-transfected cells, TRPC1 mRNA and protein levels were decreased by 40% and 64%; however, those of TRPC6 were drastically increased by 100% and 200%, respectively. In fura-2-loaded TRPC1 knockdown cells, despite the decreased TRPC1 levels, cyclopiazonic acid-induced Ca²⁺ entry and store-operated Ca²⁺ entry following Ca²⁺ addition were elevated by 77% and 135%, respectively. Results suggest that decrease in TRPC1 may be compensated by upregulated TRPC6 that possibly takes part in store-operated Ca²⁺ entry in vascular smooth muscle cells.     

The interactions of 1,4-dihydropyridines bearing a 2-(2-aminoethylthio)methyl substituent at voltage-dependent Ca+ channels of smooth muscle,cardiac muscle and neuronal tissues

Summary The Ca²⁺ channel antagonistic potencies of tiamdipine [2-(2-aminoethylthio)methyl-3-carboethoxy-5-carbomethoxy-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine] and nifedipine [2,6-dimethyl-3,5-dicarbomethoxy-4-(2nitrophenyl)-1,4-dihydropyridine] analogs bearing phenyl ring substituents were studied using pharmacologic and radioligand binding techniques. Additionally, analogs of tiamdipine possessing (2-aminoethylthio)methyl-, (2-acetamidoethylthio)methyl-and (2-pyrrolidinylmethylthio)methyl-groups at the C₂ position of the 1,4-dihydropyridine ring have been studied.Tiamdipine and nifedipine analogs inhibited K⁺-induced contractile responses in rat tail artery. IC₅₀ values of 4-phenyl ring substituted 2-(2-aminoethylthio)methyl tiamdipine analogs ranged from 10^–7 mol/l to 10^–8 mol/l. However, the corresponding 4-phenyl ring substituted nifedipine analogs covered a wider range of potency from 10^–6 mol/l to 10^–9 mol/l. K, values of the corresponding tiamdipine analogs for the inhibition of specific [³H]PN 200-110 [( I- ) [³H]isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-5-methoxycarbonyl-2,6-dimethyl-3-pyridinecarboxylate] binding-ranged from 10^–7 mol/l to 10^–9 mol/l in guinea pig ileal and rat heart membranes and rat brain synaptosomes.The two stereoisomers of tiamdipine and its analog 2-(2acetamidoethylthio)methyl-3-carboethoxy-5-carbomethoxy-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine, and the four stereoisomers of 2-(2-pyrrolidinylmethylthio)methyl-3carboethoxy-5-carbomethoxy-6-methyl-4-(3-nitrophenyl)1,4-dihydropyridine showed high stereoselectivity ratios of approximately (–)/(+) = 100 and 1000 in pharmacologic and binding experiments, respectively.The inhibitory actions of 2-(2-aminoethylthio)methyltiamdipine analogs against K⁺-induced contractile responses in rat tail artery developed very slowly requiring at least 2 h for maximum effect. The recoveries of response to K⁺ depolarization were also correspondingly slow. However, recovery was greatly accelerated by the presence of the 1,4-dihydropyridine activator Bay K 8644 [2,6-dimethyl-3carbomethoxy-5-nitro-4-(2-trifluoromethyl)-1,4-dihydropyridine, 5 × 10^–6 mol/l] immediately prior to the K⁺ challenge. The 2-(2-acetamidoethylthio)methyl tiamdipine derivative and nifedipine produced maximum inhibitory effects within 10 min, and responses recovered rapidly upon washing.The slow kinetics of onset and offset of action of the tiamdipine analogs and the reduced effects of 4-phenyl substitution relative to agents of the nifedipine series suggest that these two series of 1,4-dihydropyridines exhibit different modes of interaction with the Ca²⁺ channel. At least part of this difference is to be attributed to the presence of a charged group in the basic tiamdipine series. Trapping of these agents within the membrane phase likely contributes to their observed slow kinetics of action.     

Ca2+ entry through reverse Na+/Ca2+ exchanger in NCI-H716, glucagon-like peptide-1 secreting cells

Glucagon like peptide-1 (GLP-1) released from enteroendocine L-cells in the intestine has incretin effects due to its ability to amplify glucose-dependent insulin secretion. Promotion of an endogenous release of GLP-1 is one of therapeutic targets for type 2 diabetes mellitus. Although the secretion of GLP-1 in response to nutrient or neural stimuli can be triggered by cytosolic Ca²⁺ elevation, the stimulus-secretion pathway is not completely understood yet. Therefore, the aim of this study was to investigate the role of reverse Na⁺/Ca²⁺ exchanger (rNCX) in Ca²⁺ entry induced by muscarinic stimulation in NCI-H716 cells, a human enteroendocrine GLP-1 secreting cell line. Intracellular Ca²⁺ was repetitively oscillated by the perfusion of carbamylcholine (CCh), a muscarinic agonist. The oscillation of cytosolic Ca²⁺ was ceased by substituting extracellular Na⁺ with Li⁺ or NMG⁺. KB-R7943, a specific rNCX blocker, completely diminished CCh-induced cytosolic Ca²⁺ oscillation. Type 1 Na⁺/Ca²⁺ exchanger (NCX₁) proteins were expressed in NCI-H716 cells. These results suggest that rNCX might play a crucial role in Ca²⁺ entry induced by cholinergic stimulation in NCI-H716 cells, a GLP-1 secreting cell line.     

Calcium mobilization induced by endothelins and sarafotoxin in cultured canine tracheal smooth muscle cells

Endothelins (ETs)- and sarafotoxin (S6b)-induced rises in intracellular Ca²⁺ concentration ([Ca²⁺]_i) were monitored in cultured canine tracheal smooth muscle cells by using a fluorescent Ca²⁺ indicator fura-2. ET-1, ET-2, ET-3 and S6b elicited an initial transient peak and followed by a sustained elevation of [Ca²⁺]_i, with half-maximal effect (EC₅₀) of 18, 20, 38 and 21 nM, respectively. BQ-123, an ET_A receptor antagonist, had a high affinity to block the rise in [Ca2⁺]_i response to ET-1, ET-2, and S6b, as well as a low affinity for ET-3. Removal of external Ca²⁺ by addition of EGTA during the sustained phase, caused a rapid decline in [Ca²⁺]_i to the resting level. In the absence of external Ca²⁺, only an initial transient peak of [Ca²⁺]_i was seen, the sustained elevation of [Ca²⁺]. could then be evoked by addition of 1.8 mM Ca²⁺. Ca²⁺ influx was required for the changes of [Ca²⁺]_i, since the Ca²⁺-channel blockers, diltiazem, verapamil, and Ni²⁺, decreased both the initial and sustained elevation of [Ca²⁺]_i response to these peptides. ETs exhibited homologous desensitization of the Ca²⁺ response, but partial heterologous desensitization of the Ca²⁺ response mediated by carbachol to different extents. In contrast, ETs did not desensitize the Ca²⁺ response induced by ATP or vice versa. These data demonstrate that the initial detectable increase in [Ca2⁺]_i stimulated by these peptides is due to the activation of ET_A receptors and subsequently the release of Ca²⁺ from internal stores, whereas the contribution of external Ca²⁺ follows and partially involves a diltiazem- and verapamil-sensitive process. There is a cross-regulation among ETs and other receptor-coupling signal transduction pathways through PI hydrolysis in canine tracheal smooth muscle cells. Correspondence to: C. Mao Yang at the above address