Inhibitory effects of (1R,9S)-β-hydrastine on calcium transport in PC12 cells

(1R,9S)-beta-Hydrastine (BHS), at 100 microM, has been shown to mainly reduce the K+-induced dopamine release and Ca2+ influx by blocking the L-type Ca2+ channel and inhibit the caffeine activated store-operated Ca2+ channels, but not those activated by thapsigargin, in PC12 cells. In this study, the effects of BHS on Ca2+ transport from Ca2+ stores in the absence of external Ca2+ were investigated in PC12 cells. BHS decreased the basal intracellular Ca2+ concentration ([Ca2+]i) in the absence of external Ca2+ in PC12 cells. In the absence of external Ca2+, pretreating PC12 cells with 100 microM BHS reduced the rapid increase in the [Ca2+]i elicited by 20 mM caffeine, but not that by 1 microM thapsigargin. In addition, BHS inhibited the increase in the [Ca2+]i elicited by restoration of 2 mM CaCl2 after the Ca2+ stores had been depleted by 20 mM caffeine, but not those depleted by 1 microM thapsigargin, in the absence of external Ca2+. These results suggested that BHS mainly inhibited Ca2+ leakage from the Ca2+ stores and the caffeine-stimulated release of Ca2+ from the caffeine-sensitive Ca2+ stores in PC12 cells.     

Dual effect of hydrogen peroxide on store-mediated calcium entry in human platelets

Redox regulation is important for the modulation of cytosolic Ca(2+) concentration. Hence, we have investigated the effect of H(2)O(2) on store-mediated Ca(2+) entry (SMCE). In fura-2-loaded human platelets treatment with H(2)O(2) resulted in a concentration-dependent increase in Ca(2+) release from intracellular stores, while the effect on Ca(2+) entry was biphasic. In addition, 1mM H(2)O(2) reduced SMCE induced by agonists. The inhibitory effect of 1mM H(2)O(2) was prevented by inhibition of actin polymerization with cytochalasin D. Consistent with this, we found that 10microM H(2)O(2) and store depletion by treatment with thapsigargin plus ionomycin induced a similar temporal sequence of actin reorganization, while exposure to 1mM H(2)O(2) shifted the dynamics between polymerization and depolymerization in favor of the former. One millimolar H(2)O(2)-induced polymerization was reduced by treatment with methyl 2,5-dihydroxycinnamate and farnesylthioacetic acid, inhibitors of tyrosine kinases and Ras superfamily proteins, respectively. Finally, exposure to 1mM H(2)O(2) significantly increased store depletion-induced p60(src) activation. We conclude that H(2)O(2) exerted a biphasic effect on SMCE. The inhibitory role of high H(2)O(2) concentrations is mediated by an abnormal actin reorganization pattern involving both Ras- and tyrosine kinases-dependent pathways.     

Effect of calmodulin on ginseng saponin-induced Ca2+-Activated CI-channel activation inXenopus laevis oocytes

We previously demonstrated the ability of ginseng saponins (active ingredients of Panax ginseng) to enhance Ca2+-activated Cl- current. The mechanism for this ginseng saponin-induced enhancement was proposed to be the release of Ca2+ from IP3-sensitive intracellular stores through the activation of PTX-insensitive Galpha(q/11) proteins and PLC pathway. Recent studies have shown that calmodulin (CaM) regulates IP3 receptor-mediated Ca2+ release in both Ca2+-dependent and -independent manner. In the present study, we have investigated the effects of CaM on ginseng saponin-induced Ca2+-activated Cl- current responses in Xenopus oocytes. Intraoocyte injection of CaM inhibited ginseng saponin-induced Ca2+-activated Cl- current enhancement, whereas co-injection of calmidazolium, a CaM antagonist, with CaM blocked CaM action. The inhibitory effect of CaM on ginseng saponin-induced Ca2+-activated Cl- current enhancement was dose- and time-dependent, with an IC50 of 14.9 +/- 3.5 microM. The inhibitory effect of CaM on saponin's activity was maximal after 6 h of intraoocyte injection of CaM, and after 48 h the activity of saponin recovered to control level. The half-recovery time was calculated to be 16.7 +/- 4.3 h. Intraoocyte injection of CaM inhibited Ca2+-induced Ca2+-activated Cl- current enhancement and also attenuated IP3-induced Ca2+-activated Cl- current enhancement. Ca2+/CaM kinase II inhibitor did not inhibit CaM-caused attenuation of ginseng saponin-induced Ca2+-activated Cl- current enhancement. These results suggest that CaM regulates ginseng saponin effect on Ca2+-activated Cl current enhancement via Ca2+-independent manner.     

Inhibition of store-operated calcium entry-mediated superoxide generation by histamine trifluoromethyltoluide independent of histamine receptors

Store-operated calcium entry (SOCE) plays an important role in shaping the Ca(2+) response of various tissues and cell types. In this report, we show that thapsigargin (TG)-induced SOCE was inhibited by the histamine receptor agonist, histamine-trifluoromethyltoluide (HTMT), in U937 and HL-60 human promyelocytes. Preincubation of HTMT resulted in a significant inhibition of subsequent TG-induced Ca(2+) elevation without affecting Ca(2+) release from intracellular stores. HTMT also inhibited TG-induced Ca(2+) current and Ba(2+)/Mn(2+) influx in a concentration-dependent manner. In contrast with HTMT, other H1 histamine receptor agonists, histamine, 2-methylhistamine and 2-thiazolylethylamine, did not affect TG-induced SOCE. In addition, HTMT also attenuated TG-induced cytosolic superoxide generation. Taken together, our data clearly suggest that the anti-inflammatory effect of HTMT may occur through direct inhibition of SOCE.     

2-Aminoethoxydiphenyl borate perturbs hormone-sensitive calcium stores and blocks store-operated calcium influx pathways independent of cytoskeletal disruption in human A549 lung cancer cells

Recent studies have identified novel actions for 2-aminoethoxydiphenyl borate (2-APB) in triggering calcium release and enhancing calcium influx induced by the depletion of intracellular calcium stores. In this study, we have examined the effects of 2-APB on the human lung adenocarcinoma A549 cell line, which we have previously shown displays a unique calcium influx response, when ER calcium stores are depleted by thapsigargin (TG) treatment. Here, we show that low concentrations of 2-APB failed to induce the rapid augmentation of TG-activated calcium influx previously reported for other cell types. We observed that store-operated calcium (SOC) channels in the A549 cell line exhibited short-term sensitivity to low doses of 2-APB, perhaps reflecting a delayed augmentation of SOC channel activity or the recruitment of 2-APB-insensitive SOC channels. In both intact and permeabilized cells, 2-APB effectively discharged a subset of A549 calcium pools corresponding to the hormone-sensitive intracellular calcium stores. The 2-APB-induced calcium release produced a long-lasting perturbation of the adenosine triphosphate (ATP)-releasable calcium pools, effectively uncoupling ATP-activated calcium release even, when stores are replenished with calcium. In contrast to previous reports, we found that disruption of either the actin or microtubule-based cytoskeleton failed to block the 2-APB-induced effects on calcium signaling in A549 cells. Our study describes novel cytoskeletal-independent effects of 2-APB on Ca2+-signaling pathways, revealing differentially sensitive Ca2+-influx pathways and long-term perturbation of hormone-sensitive Ca2+ stores.     

Distinct effects of N-ethylmaleimide on formyl peptide- and cyclopiazonic acid-induced Ca2+ signals through thiol modification in neutrophils

In this study, we demonstrate that N-ethylmaleimide (NEM), a cell permeable thiol-alkylating agent, enhanced the [Ca2+]i rise caused by stimulation with cyclopiazonic acid (CPA), a sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibitor, in rat neutrophils. In addition, NEM attenuated the formyl-Met-Leu-Phe (fMLP)-induced [Ca2+]i rise whether NEM was added to cells prior to or after fMLP stimulation. Moreover, application of NEM after fMLP activation in the absence of external Ca2+ inhibited the Ca2+ signal upon addition of Ca2+ to the medium. Similar patterns were also obtained by using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), a cell impermeable dithiol-oxidizing agent, which replaced NEM in the CPA- and fMLP-induced [Ca2+]i rise experiments. Treatment with dithiothreitol (DTT), a cell permeable dithiol-reducing agent, N-acetyl-l-cysteine (NAC), a cell permeable monothiol-reducing agent, and tris-(2-carboxyethyl)phosphine (TCEP), a cell impermeable reductant without a thiol group, all rescued the fMLP-induced Ca2+ signal from NEM. Rat neutrophils express the mRNA encoding for transient receptor potential (TRP) C6, inositol trisphosphate receptor (IP3R) 2 and IP3R3. NEM had no effect on the mitochondrial membrane potential. NEM could restore the polarization and F-actin accumulation of fMLP-treated cells to those of the control. In the absence of external Ca2+, NEM rendered the CPA-induced [Ca2+]i elevation persistently but inhibited the fMLP-induced Ca2+ spike, which was reversed by tris-(2-cyanoethyl)phosphine (TCP), a cell permeable reductant without a thiol group. DTNB did not affect the Ca2+ spike caused by fMLP. These results indicate that through protein thiol oxidation, NEM affects the receptor-activated and the store depletion-derived Ca2+ signals in an opposing manner.     

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.     

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.     

Octyl Gallate Inhibits ATP-induced Intracellular Calcium Increase in PC12 Cells by Inhibiting Multiple Pathways

Phenolic compounds affect intracellular free Ca²⁺ concentration ([Ca²⁺]_i) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced Ca²⁺ signaling in PC12 cells using fura-2-based digital Ca²⁺ imaging and whole-cell patch clamping. Treatment with ATP (100 µM) for 90 s induced increases in [Ca²⁺]_i in PC12 cells. Pretreatment with octyl gallate (100 nM to 20 µM) for 10 min inhibited the ATP-induced [Ca²⁺]_i response in a concentration-dependent manner (IC₅₀=2.84 µM). Treatment with octyl gallate (3 µM) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular Ca²⁺ with nominally Ca²⁺-free HEPES HBSS or depletion of intracellular Ca²⁺ stores with thapsigargin (1 µM). Treatment for 10 min with the L-type Ca²⁺ channel antagonist nimodipine (1 µM) significantly inhibited the ATP-induced [Ca²⁺]_i increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the [Ca²⁺]_i increase induced by 50 mM KCl. Pretreatment with protein kinase C inhibitors staurosporin (100 nM) and GF109203X (300 nM), or the tyrosine kinase inhibitor genistein (50 µM) did not significantly affect the inhibitory effects of octyl gallate on the ATP-induced response. Treatment with octyl gallate markedly inhibited the ATP-induced currents. Therefore, we conclude that octyl gallate inhibits ATP-induced [Ca²⁺]_i increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of Ca²⁺ from extracellular space and P2Y receptor-induced release of Ca²⁺ from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced Ca²⁺ responses by inhibiting the secondary activation of voltage-gated Ca²⁺ channels.     

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

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

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.     

Histamine H3-receptor signaling in cardiac sympathetic nerves: Identification of a novel MAPK-PLA2-COX-PGE2-EP3R pathway

We hypothesized that the histamine H(3)-receptor (H(3)R)-mediated attenuation of norepinephrine (NE) exocytosis from cardiac sympathetic nerves results not only from a Galpha(i)-mediated inhibition of the adenylyl cyclase-cAMP-PKA pathway, but also from a Gbetagamma(i)-mediated activation of the MAPK-PLA(2) cascade, culminating in the formation of an arachidonate metabolite with anti-exocytotic characteristics (e.g., PGE(2)). We report that in Langendorff-perfused guinea-pig hearts and isolated sympathetic nerve endings (cardiac synaptosomes), H(3)R-mediated attenuation of K(+)-induced NE exocytosis was prevented by MAPK and PLA(2) inhibitors, and by cyclooxygenase and EP(3)-receptor (EP(3)R) antagonists. Moreover, H(3)R activation resulted in MAPK phosphorylation in H(3)R-transfected SH-SY5Y neuroblastoma cells, and in PLA(2) activation and PGE(2) production in cardiac synaptosomes; H(3)R-induced MAPK phosphorylation was prevented by an anti-betagamma peptide. Synergism between H(3)R and EP(3)R agonists (i.e., imetit and sulprostone, respectively) suggested that PGE(2) may be a downstream effector of the anti-exocytotic effect of H(3)R activation. Furthermore, the anti-exocytotic effect of imetit and sulprostone was potentiated by the N-type Ca(2+)-channel antagonist omega-conotoxin GVIA, and prevented by an anti-Gbetagamma peptide. Our findings imply that an EP(3)R Gbetagamma(i)-induced decrease in Ca(2+) influx through N-type Ca(2+)-channels is involved in the PGE(2)/EP(3)R-mediated attenuation of NE exocytosis elicited by H(3)R activation. Conceivably, activation of the Gbetagamma(i) subunit of H(3)R and EP(3)R may also inhibit Ca(2+) entry directly, independent of MAPK intervention. As heart failure, myocardial ischemia and arrhythmic dysfunction are associated with excessive local NE release, attenuation of NE release by H(3)R activation is cardioprotective. Accordingly, this novel H(3)R signaling pathway may ultimately bear therapeutic significance in hyper-adrenergic states.     

Stimulation of intracellular Ca2+ elevation in neutrophils by thiol-oxidizing phenylarsine oxide

Phenylarsine oxide (PAO), a trivalent arsenical compound, stimulated [Ca2+]i elevation in rat neutrophils in a Ca2+-containing medium but caused no appreciable response in a Ca2+-free medium. PAO also induced external Mn2+ entry, which was inhibited by N-acetyl-L-cysteine (NAC), but failed to elicit any appreciable Ba2+ and Sr2+ entry. Pretreatment of neutrophils with thiol-reducing agents including dithiothreitol (DTT), NAC, 2,3-dimercapto-1-propanol (DMP), 2,3-dimercaptopropane-1-sulfonic acid (DMPS) and tris-(2-carboxyethyl)phosphine (TCEP), all greatly inhibited PAO-induced [Ca2+]i elevation. Addition of Ni2+ or La3+ followed by PAO stimulation also attenuated the Ca2+ signals in a concentration-dependent manner. PAO had no significant effect on the production of reactive oxygen intermediates (ROI) and nitric oxide (NO) nor did it decrease cellular low molecular weight thiols levels. PAO-induced [Ca2+]i elevation was significantly inhibited by 1-[6-[17beta-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, genistein, a general tyrosine kinase inhibitor, phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, calyculin A, a cortical actin stabilizer, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), a phosphoinositide 3-kinase inhibitor, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365), and cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12,330A), the blockers of receptor-gated and store-operated Ca2+ channels, whereas there was no appreciable effect exerted by aristolochic acid, a phospholipase A2 inhibitor, 7-nitroindazole and N-(3-aminomethyl)benzylacetamidine (1400W), the blockers of NO synthase, and by suspension in a Na+-deprived medium. In contrast, 2-aminoethoxydiphenyl borane (2-APB), the blocker of IP3 receptor and Ca2+ influx, enhanced the PAO-induced response. PAO had no effect on the plasma membrane Ca2+-ATPase (PMCA) activity in the pharmacological isolated neutrophil preparation and the neutrophil membrane fractions. These results indicate that PAO stimulates [Ca2+]i rise in rat neutrophils mainly through the oxidation of vicinal thiol groups on the cell surface membrane to activation of a non-store operated Ca2+ entry (non-SOCE) without affecting the activity of PMCA and the plasmalemmal Na+/Ca2+ exchanger.     

Involvement of Ca(2+)-induced Ca2+ releasing system in interleukin-1beta-associated adenosine release

Interleukin-1beta (IL-1beta) plays an important role in neuroprotective and neurodegenerative events in the central nervous system. To clarify the mechanism of controversial actions of IL-1beta, we determined the effect of IL-1beta, as well as the interaction between IL-1beta and Ca(2+)-induced Ca2+ releasing system (CICR), on adenosine releases in mice hippocampus using mini-slices method. Basal and K(+)-stimulated adenosine releases were regulated by two types of CICRs, including inositol-1,4,5-trisphosphate (IP3) receptor and ryanodine receptor. Lower concentration of IL-1beta increased both adenosine releases, whereas higher concentration did not affect their releases. The stimulatory effect of IL-1beta on basal adenosine release was reduced by removal of extracellular Ca2+ and IP3 receptor inhibitor, while the stimulatory effect of IL-1beta on K(+)-stimulated adenosine release was reduced by ryanodine receptor inhibitor. These results suggest that the potent effect of IL-1beta upon adenosine release might contribute to the neuroprotective action of IL-1beta, whereas IL-1beta-induced neurodegeneration might be due to the overload response of Ca2+ mobilization and the inactivation of adenosine exocytosis.     

Azaspiracid-4 inhibits Ca2+ entry by stored operated channels in human T lymphocytes

Azaspiracids (AZs) are a new group of phycotoxins discovered in the Ireland coast that includes the isolated analogues: AZ-1, AZ-2, AZ-3, AZ-4 and AZ-5 and the recently described AZ-6-11. Azaspiracid toxic episodes show gastrointestinal illness, but neurotoxic symptoms are also observed in mouse bioassay. Despite their great importance in human health, so far its mechanism of action is largely unknown. In this report, we present the first data about the effect of AZ-4 on cytosolic calcium concentration [Ca2+]i in freshly human lymphocytes. Cytosolic Ca2+ variations were determined by fluorescence digital imaging microscopy using Fura2 acetoxymethyl ester (Fura2-AM). AZ-4 did not modify cytosolic Ca2+ in resting cells. However, the toxin dose-dependent inhibited the increase in cytosolic Ca2+ levels induced by thapsigargin (Tg). AZ-4 decreased Ca2+-influx induced by Tg but did not affect the Ca2+-release from internal stores induced by this drug. The effects of AZ-4 on Ca2+-influx induced by Tg were reversible and not regulated by adenosine 3',5'-cyclic monophosphate (cAMP) pathway. When AZ-4 was added before, after or together with nickel, an unspecific blocker of Ca2+ channels, the effects were indistinguishable and additive. AZ-4 also inhibited maitotoxin (MTX)-stimulated Ca2+-influx by 5-10%. Thus, AZ-4 appeared to be a novel inhibitor of plasma membrane Ca2+ channels, affecting at least to store operated channels, showing an effect clearly different from other azaspiracid analogues.     

ML-9, a myosin light chain kinase inhibitor, reduces intracellular Ca2+ concentration in guinea pig trachealis

We investigated the effects of ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine], a myosin light chain kinase (MLCK) inhibitor, on intracellular Ca2+ concentration ([Ca2+]i), contraction induced by high K+ and an agonist, and capacitative Ca2+ entry in fura-2-loaded guinea pig tracheal smooth muscle. ML-9 inhibited both the increase in [Ca2+]i and the contraction induced by 60 mM K+, 1 microM methacholine or 1 microM thapsigargin, an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase. However, another MLCK inhibitor, wortmannin (3 microM), inhibited the contraction elicited by these stimuli without affecting [Ca2+]i. Under the condition that the thapsigargin-induced contraction was fully suppressed by 3 microM wortmannin, 30 microM ML-9 caused a further decrease in [Ca2+]i. The inhibitory effects of ML-9 on [Ca2+]i and the contraction elicited by methacholine were similar to those of SKF-96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride), a Ca2+ channel blocker. These results indicate that ML-9 acts as a potent inhibitor of Ca2+-permeable channels independently of MLCK inhibition in tracheal smooth muscle.     

Direct inhibition of inositol-1,4,5-trisphosphate-induced Ca2+ release from brain microsomes by K+ channel blockers

Tetraethylammonium and 9-tetraethylammonium have previously been reported to inhibit inositol-1,4,5-trisphosphate (IP3)-induced Ca2+ release from brain microsomes, purportedly by blocking potassium channels [Biochem. J. 258:617-620 (1988)]. The effects of these and other K+ channel blockers have been studied here in greater detail using a spectrophotometric assay for Ca2+ movements into and out of canine brain microsomes. IP3-induced Ca2+ release was inhibited by substitution of K+ in the medium with nominally impermeant cations or by addition of most of the K+ channel blockers tested. Nevertheless, addition of valinomycin to the medium (to provide an alternative pathway for counter-ion K+ movements) failed to alleviate the inhibition of IP3-induced Ca2+ release caused by K+ channel blockers. To determine whether these substances act by inhibition of IP3 binding or by direct interaction with the Ca2+ channel of the internal store that promotes IP3-induced Ca2+ release, their effect on [3H]IP3 binding was investigated. None of the K+ channel blockers tested inhibited [3H]IP3 binding. Nearly all the K+ channel blockers appear to interact directly with a Ca2+ channel of the intracellular stores or perhaps interfere with its coupling to the IP3 receptor. Because of their multiplicity of actions, these substances cannot be presumed to be either selective K+ channel blockers or selective inhibitors of IP3-induced Ca2+ release from internal stores. Three of them were even found to partially inhibit valinomycin-stimulated 86Rb uptake into liposomes.