Palytoxin-induced increase in endothelial Ca2+ concentration in the rabbit aortic valve

Palytoxin (PTX) is one of the most potent toxins isolated from marine coelenterates of the genus Palythoa. It induces depolarization in various types of cells by increasing the permeability for monovalent cations. It has been reported that PTX induces endothelium-dependent relaxation of vascular smooth muscle. In this study, we examined the effect of PTX on the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in the endothelium of rabbit aortic valves loaded with fluorescent Ca²⁺ indicators, fura-PE3 or fluo-3. PTX (10pM-300nM) irreversibly increased endothelial [Ca²⁺]_i in a concentration-dependent manner. ATP and thapsigargin also increased [Ca²⁺]_i. Imaging of [Ca²⁺]_i with a confocal microscope revealed that PTX increased [Ca²⁺]_i in all endothelial cells studied (n=13). An inorganic Ca²⁺ entry blocker, La³⁺ (30μM), had no effect on the increase in [Ca²⁺]_i induced by PTX whereas it inhibited the sustained phase of the increase in [Ca²⁺]_i induced by ATP or thapsigargin. The PTX-induced increase in [Ca²⁺]_i was partially inhibited by ouabain and was abolished by removal of external Ca²⁺ although decrease of Na⁺ concentration in the incubation medium was ineffective. Activation of protein kinase C by 1μM 12-deoxyphorbol 13-isobutyrate or inhibition of phosphatase by 10nM calyculin-A had no effect on the increase in [Ca²⁺]_i induced by PTX, whereas both agents inhibited the sustained phase of the increase in [Ca²⁺]_i induced by ATP or thapsigargin. Mn²⁺ influx, measured by the quenching of fura-PE3 fluorescence, was accelerated by ATP or thapsigargin, but not by PTX. These results suggest that PTX increases [Ca²⁺]_i in the endothelium of the rabbit aortic valve by increasing Ca²⁺ influx through a pathway which is different from that activated by ATP or thapsigargin. Received: 28 February 1997     

Involvement of different calcium channels in K+- and veratridine-induced increases of cytosolic calcium concentration in rat cerebral cortical synaptosomes

Intracellular calcium ion concentrations ([Ca²⁺]_i) in rat cerebral cortical synaptosomes were measured, using the calcium chelating fluorescence dye fura-2. The synaptosomes were depolarized by elevation of the extracellular K⁺ concentration or by addition of veratridine, which opens voltage-dependent Na⁺-channels and prevents their inactivation. Both enhancement of the concentration of extracellular K⁺ (up to 60 mM) and veratridine (1–100 μM) increased the [Ca²⁺]_i in a concentration-dependent manner. In the absence of extracellular Ca²⁺, the K⁺- and veratridine-induced increases in [Ca²⁺]_i were abolished, indicating that the increase in [Ca²⁺]_i was due to an influx of extracellular Ca²⁺. Tetrodotoxin (TTX), a blocker of the voltage-dependent Na⁺ channel, inhibited the veratridine-induced (10 μM) Ca²⁺ influx by more than 80%, while the K⁺-evoked (30 mM) increase of [Ca²⁺]_i was TTX-resistant. Both the K⁺- and the veratridine-induced Ca²⁺ influx were not reduced by nifedipine (1 μM), a blocker of L-type Ca²⁺ channels. Blockade of the voltage dependent N-type Ca²⁺ channels with ω-conotoxin GVIA (ω-CTx GVIA; 0.1 μM) and of the voltage-dependent P/Q-type channels with ω-agatoxin IVA (ω-AgaTx IVA; 0.2 μM) inhibited the K⁺-induced increase in [Ca²⁺]_i by about 30 and 55%, respectively; these effects were additive. ω-Conotoxin MVIIC (ω-CTx MVIIC) at a concentration of 0.2 μM, which may be assumed to block predominantly the Q-type Ca²⁺ channel, inhibited the K⁺-induced increase in [Ca²⁺]_i by 50%. The veratridine-induced increase in [Ca²⁺]_i was reduced by about 25% by ω-CTx GVIA (0.1 μM), but was resistant to ω-AgaTx IVA (0.2 μM) and ω-CTx MVIIC (0.2 μM). Mibefradil (former designation Ro 40-5967), a Ca²⁺ antagonist which blocks all types of voltage-dependent Ca²⁺ channels including the T and R channels, led to a concentration-dependent inhibition of the K⁺- and veratridine-induced increase in [Ca²⁺]_i (abolition at 10 μM mibefradil). Ifenprodil, another non-specific blocker of voltage-dependent Ca²⁺ channels, also inhibited the K⁺- and veratridine-induced increase in [Ca²⁺]_i in concentration-dependent manner and abolished it at 320 μM ifenprodil. In contrast, KB-R 7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate; 1 and 3 μM), a highly potent and selective inhibitor of the Na⁺/Ca²⁺ exchanger (NCX1), failed to inhibit the K⁺- and veratridine-induced increase in [Ca²⁺]_i. It is concluded that the K⁺-induced increase in free cytosolic Ca²⁺ results from Ca²⁺ influx through voltage-dependent N- and, above all, Q-type Ca²⁺ channels. N-type Ca²⁺ channels also play a minor role in the veratridine-induced increase in [Ca²⁺]_i, but P/Q-type channels do not appear to be involved at all. The inhibition of the veratridine-induced, ω-CTx GVIA- and ω-AgaTx IVA-resistant increase in [Ca²⁺]_i by mibefradil and the failure of KB-R 7943 to inhibit this response are compatible with the suggestion that in rat cerebral cortical synaptosomes, Ca²⁺ influx via the R-type Ca²⁺ channel and/or another so far uncharacterized Ca²⁺ channel may substantially contribute to the veratridine-induced increase in [Ca²⁺]_i. Received: 7 March 1997 / Accepted: 9 September 1997     

In U-937 promonocytes, misteltoe lectin I increases basal [Ca2+]i, enhances histamine H1- and complement C5a-receptor-mediated rises in [Ca2+]i, and induces cell death

Mistletoe lectin I (ML I) from Viscum album inhibits cell growth and induces apoptosis (programmed cell death) in several cell types. Because increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) constitute a signal for the induction of apoptosis, we studied the effects of ML I on basal [Ca²⁺]_i, receptor-mediated rises in [Ca²⁺]_i and cell viability, using human U-937 promonocytes as model system. Treatment of U-937 cells with ML I (30–100 ng/ml) significantly increased basal [Ca²⁺]_i. ML I (10–30 ng/ml) enhanced histamine-induced rises in [Ca²⁺]_i up to five-fold. The effect of histamine was inhibited by clemastine but not by famotidine, indicative for its mediation via H₁-receptors. ML I additionally enhanced the stimulatory effect of complement C5a on [Ca²⁺]_i, whereas the effect of ATP was unaffected. ML I did not induce responsiveness of U-937 cells towards a bacteria-derived chemotactic peptide. ML I up to 10 ng/ml did not affect cell viability and growth of U-937 cells. ML I at 30 ng/ml moderately inhibited cell growth and reduced cell viability. At 100 ng/ml, ML I was strongly cytotoxic. Our data support the view that Ca²⁺ plays a role as intracellular signal molecule in the induction of apoptosis and point to an accelerating role of H₁- and C5a-receptors in the regulation of this process. Received: 1 July 1996 / Accepted: 11 October 1996     

Stimulation of Erythrocyte Cell Membrane Scrambling by Nystatin

The antifungal ionophore nystatin dissipates the Na⁺ and K⁺ gradients across the cell membrane, leading to cellular gain of Na⁺ and cellular loss of K⁺. The increase of cellular Na⁺ concentration may result in Ca²⁺ accumulation in exchange for Na⁺. Increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i) and loss of cellular K⁺ foster apoptosis‐like suicidal erythrocyte death or eryptosis, which is characterised by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the erythrocyte surface. The present study explored whether nystatin stimulates eryptosis. Cell volume was estimated from forward scatter (FSC), phosphatidylserine exposure from annexin V binding and [Ca²⁺]_i from Fluo3‐fluorescence in flow cytometry. A 48‐hr exposure to nystatin (15 μg/ml) was followed by a significant increase of [Ca²⁺]_i, a significant increase of annexin V binding and a significant decrease of FSC. The annexin V binding after nystatin treatment was significantly blunted in the nominal absence of extracellular Ca²⁺. Partial replacement of extracellular Na⁺ with extracellular K⁺ blunted the nystatin‐induced erythrocyte shrinkage but increased [Ca²⁺]_i and annexin V binding. Nystatin triggers cell membrane scrambling, an effect at least partially due to entry of extracellular Ca²⁺.     

Neuropeptide Y as a stimulator of Na+-dependent Ca2+ efflux from freshly isolated adult rat cardiomyocytes

Several physiological stimuli cause a rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiomyocytes. This increased [Ca²⁺]_i must be restored to physiological resting level to ensure response to further stimuli. In the present study, we examined the effect of neuropeptide Y (NPY), which is secreted from certain adrenergic or non-adrenergic neurons, on Ca²⁺ efflux from freshly isolated, quiescent adult rat cardiomyocytes. The isolated cardiomyocytes were preloaded with ⁴⁵CaCl₂ for 1 h. Then, the fractional release of ⁴⁵Ca²⁺ from the cells was measured. NPY stimulated the efflux of ⁴⁵Ca²⁺ from isolated adult rat cardiomyocytes in a concentration-dependent manner (10^–8 M to 10^–6 M). NPY (10^–6 M)-induced Ca²⁺ efflux was 2.0 ± 0.16% of the total cellular content. The ⁴⁵Ca²⁺ efflux from the cells was also stimulated by Y₁ receptor agonist, [Leu³¹, Pro³⁴]NPY, but not by Y₂ receptor agonist, NPY_13–36. The effect of NPY was inhibited by a peptide NPY inhibitor, NPY_18–36 and a non-peptide NPY inhibitor, benextramine to a similar extent. From these results, it is conceivable that the effect of NPY on Ca²⁺ efflux from cardiomyocytes is mediated through Y₁ receptors. It was also observed that NPY caused a rise in [Ca²⁺]_i to almost 150 nM. NPY-stimulated ⁴⁵Ca²⁺ efflux was not affected by removal of extracellular Ca²⁺, but was dependent on the presence of extracellular Na⁺. Moreover, NPY caused a ²²Na⁺ influx into the cells of about 1.6-fold over the basal value which was inhibited by amiloride and 5-(N,N-dimethyl)-amiloride, known Na⁺/Ca²⁺ exchange inhibitors. In addition, isoproterenol also caused ⁴⁵Ca²⁺ efflux from the cells and which was enhanced by the addition of NPY. These results suggest that NPY stimulates extracellular Na⁺-dependent ⁴⁵Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on plasma membrane Y₁ receptors with which NPY may couple during Na⁺/Ca²⁺ exchange. Received: 21 May 1997 / Accepted: 26 August 1997     

The role of Na+, K+-ATPase in the hypoxic vasoconstriction in isolated rat basilar artery

Hypoxia-induced cerebrovascular dysfunction is a key factor in the occurrence and the development of cerebral ischemia. Na⁺, K⁺-ATPase affects the regulation of intracellular Ca^2 + concentration and plays an important role in vascular smooth muscle function. However, the potential role of Na⁺, K⁺-ATPase in hypoxia-induced cerebrovascular dysfunction is unknown. In this study, we found that the KCl-induced contraction under hypoxia in rat endothelium-intact basilar arteries is similar to that of denuded arteries, suggesting that hypoxia may cause smooth muscle cell (SMC)-dependent vasoconstriction in the basilar artery. The Na⁺, K⁺–ATPase activity of the isolated basilar artery with or without endothelium significantly reduced with prolonged hypoxia. Blocking the Na⁺–Ca^2 + exchanger with Ni^2 + (10^− 3 M) or the L-type Ca^2 + channel with nimodipine (10^− 8 M) dramatically attenuated KCl-induced contraction under hypoxia. Furthermore, prolonged hypoxia significantly reduced Na⁺, K⁺-ATPase activity and increased [Ca^2 +]_i in cultured rat basilar artery SMCs. Hypoxia reduced the protein and mRNA expression of the α₂ isoform of Na⁺, K⁺-ATPase in SMCs in vitro. We used a low concentration of the Na⁺, K⁺-ATPase inhibitor ouabain, which possesses a high affinity for the α₂ isoform. The contractile response in the rat basilar artery under hypoxia was partly inhibited by ouabain pretreatment. The decreased Na⁺, K⁺-ATPase activity in isolated basilar artery and the increased [Ca^2 +]_i in SMCs induced by hypoxia were partly inhibited by pretreatment with a low concentration of ouabain. These results suggest that hypoxia may educe Na⁺, K⁺-ATPase activity in SMCs through the α₂ isoform contributing to vasoconstriction in the rat basilar artery.     

Correlation between catecholamine release and sodium pump inhibition in the perfused adrenal gland of the cat

1 Ca²⁺ reintroduction to retrogradely perfused and ouabain (10^-4 M)-treated cat adrenal glands caused a catecholamine secretory response which was greater the longer the time of exposure to the cardiac glycoside. Such a response was proportional to the external Na⁺ concentration [Na⁺]_o.

2 A qualitatively similar, yet smaller response was observed when glands were perfused with Krebs solution lacking K⁺ ions; thus, K⁺ deprivation mimicked the secretory effects of ouabain. Catecholamine secretion evoked by Ca²⁺ reintroduction in K⁺-free solution (0-K⁺) was also proportional to [Na⁺]_o and greater the longer the time of exposure of the gland to 0-K⁺ solution.

3 The ionophore X537A also mimicked the ouabain effects, since Ca²⁺ reintroduction to glands treated with this agent (25 μM) caused a sharp secretory response. When added together with X537A, ouabain (10^-4 M) did not modify the response to the ionophore.

4 N-ethylmaleimide (NEM), another Na⁺, K⁺-ATPase inhibitor, did not evoke the release of catecholamines; on the contrary, NEM (10^-4 M) inhibited the catecholamine secretory response to high [K⁺]_o, acetylcholine, Ca²⁺ reintroduction and ouabain.

5 Ouabain (10^-4 M) inhibited the uptake of ⁸⁶Rb into adreno-medullary tissue by 60%. Maximal inhibition had already occurred 2 min after adding the drug, indicating a lack of temporal correlation between ATPase inhibition and the ouabain secretory response, which took longer (about 30-40 min) to reach its peak. NEM (10^-4 M) blocked ⁸⁶Rb uptake in a similar manner.

6 The results are further evidence in favour of the presence of a Na⁺-Ca²⁺ exchange system in the chromaffin cell membrane, probably involved in the control of [Ca²⁺]_i and in the modulation of catecholamine secretion. This system is activated by increasing [Na⁺]_i, either directly (ionophore X537A, increased [Na⁺]_o) or indirectly (Na⁺ pump inhibition). However, the simple inhibition of Na⁺ pumping does not always lead to a catecholamine secretory response; such is the case for NEM.

     

Mechanisms of carvedilol-induced [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> rises and death in human hepatoma cells

The effect of the cardiovascular drug carvedilol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability has not been explored in human hepatoma cells. This study examined whether carvedilol altered [Ca²⁺]_i and caused cell death in HA59T cells. [Ca²⁺]_i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Carvedilol at concentrations ≥1 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 20 μM. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Carvedilol induced Mn²⁺ quench of fura-2 fluorescence, implicating Ca²⁺ influx. The Ca²⁺ influx was sensitive to La³⁺, econazole, nifedipine, and SKF96365. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), carvedilol-induced [Ca²⁺]_i rises were abolished; and conversely, carvedilol pretreatment inhibited a major part of thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change carvedilol-induced [Ca²⁺]_i rises. At concentrations between 1 and 50 μM, carvedilol killed cells in a concentration-dependent manner. The cytotoxic effect of 1 μM (but not 30 μM) carvedilol was fully reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Apoptosis was induced by 30 (but not 1) μM carvedilol. Collectively, in HA59T hepatoma cells, carvedilol induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase-C-independent manner and Ca²⁺ influx via store-operated Ca²⁺ channels. Carvedilol-caused cytotoxicity was mediated by Ca²⁺ and apoptosis in a concentration-dependent manner.     

Effects of ouabain on human bronchial muscle in vitro

The effects of ouabain, an inhibitor of the plasmalemmal Na⁺/K⁺-ATPase activity, were examined in human isolated bronchus. Ouabain produced concentration-dependent contraction with –logEC₅₀=7.16±0.11 and maximal effect of 67±4% of the response to acetylcholine (1 mM). Ouabain (10 M)-induced contraction was epithelium-independent and was not depressed by inhibitors of cyclooxygenase and lipoxygenase, antagonists of muscarinic, histamine H₁-receptors and -adrenoceptors, or neuronal Na⁺ channel blockade. The inhibition of ouabain contraction in tissues bathed in K⁺-free medium, and the inhibition by ouabain of the K⁺-induced relaxation confirm that the contractile action of ouabain is mediated by inhibition of Na⁺/K⁺-ATPase. Furthermore, depolarization (16.4±0.9 mV) was observed in human isolated bronchus by intracellular microelectrode recording. Ouabain (10 M)-induced contractions were abolished by a Ca²⁺-free solution but not by blockers of L-type Ca²⁺ channels. In human cultured bronchial smooth muscle cells, ouabain (10 M) produced a sustained increase in [Ca²⁺]_i (116±26 nM) abolished in Ca²⁺-free medium. Incubation with a Na⁺-free medium or amiloride (0.1 mM) markedly inhibited the spasmogenic effect of ouabain thus suggesting the role of Na⁺/Ca²⁺ exchange in ouabain contraction while selective inhibitors of Na⁺/H⁺-antiport, Na⁺/K⁺/Cl^–-antiport, or protein kinase C had no effect. Ouabain (10 M) failed to increase inositol phosphate accumulation in human bronchus. Ouabain (10 M) did not alter bronchial responsiveness to acetylcholine or histamine but inhibited the relaxant effects of isoprenaline, forskolin, levcromakalim, or sodium nitroprusside. These results indicate that ouabain acts directly to produce contraction of human airway smooth muscle that depends on extracellular Ca²⁺ entry unrelated to L-type channels and involving the Na⁺/Ca²⁺-antiporter.     

Action of ouabain on the contraction of ileal longitudinal muscle by manganese ions in a Ca-free, high-K, Na-sufficient or Na-deficient solution

The action of ouabain, an external Na⁺-dependent inhibitor of active glucose transport, on the contraction by Mn²⁺in a Ca²⁺-free, Na⁺-sufficient or Na⁺-deficient medium was examined in ileal muscle. Ouabain ( 9 × 10⁻⁵m) inhibited the contraction by 5 mm Mn²⁺in a Ca²⁺-free, 60 mm K⁺, Na⁺-sufficient medium in ileal muscle. The addition of pyruvate or lactate, utilized independently in the external Na⁺in the pretreatment with ouabain, induced the dose-dependent contraction and manganese uptake by Mn²⁺in the Ca²⁺-free, 60 mm K⁺, Na⁺-sufficient medium. These results suggest that Mn²⁺-induced ileal contraction in a high-K⁺, Na⁺-sufficient medium is maintained by an active glucose transport, dependent on external Na⁺. While, in contrast, Mn²⁺-induced contraction in a high-K⁺, Na⁺-deficient medium is maintained by an external Na⁺-independent glucose transport, and is insensitive to ouabain.     

Gossypol stimulates opening of a Ca2+‐ and Na+‐permeable but Ni2+‐ and Co2+‐impermeable pore in bEND.3 endothelial cells

Gossypol, a polyphenolic dialdehyde toxin isolated from cotton seed, has anti‐cancer properties and has recently shown some success in the treatment of glioma. Its effects on brain neurons and blood vessels are poorly understood. In this work we examined the effects of gossypol on cytosolic Ca²⁺ concentration ([Ca²⁺]_i) of mouse brain bEND.3 endothelial cells. Cell viability tests revealed that after 3 hour and 18 hour exposures, 10 µmol/L gossypol caused 23% and 65% cell death, respectively; 3 µmol/L gossypol caused no and 21% cell death, respectively. [Ca²⁺]_i was raised concentration‐dependently by 1‐10 µmol/L gossypol. We then explored the Ca²⁺ signalling triggered by 3 µmol/L gossypol, which inflicted minimal toxicity: the Ca²⁺ signal was composed largely of Ca²⁺ influx and to a small extent, intracellular Ca²⁺ release. Such Ca²⁺ influx was much larger than store‐operated Ca²⁺ influx triggered by maximal Ca²⁺ pool depletion. The Ca²⁺ influx triggered by 3 and 10 µmol/L gossypol caused NO release and cell death, respectively. Gossypol also triggered influx of Mn²⁺ and Na⁺, but not Ni²⁺ and Co²⁺. Gossypol‐triggered Ca²⁺ signal was inhibited only by 14% and 37% by 100 µmol/L La³⁺ and 10 µmol/L nimodipine, respectively; and not suppressed at all by 5 mmol/L Ni²⁺. Gossypol‐triggered Ca²⁺ signal was suppressed by 78% by 30 µmol/L ruthenium red, suggesting gossypol may act on TRPV channels. Our results suggest gossypol triggered opening of a non‐selective cation pore, possibly a member of the TRPV family.     

Functional expression of voltage-gated Na+ and Ca2+ channels during neuronal differentiation of PC12 cells with nerve growth factor or forskolin

Voltage-gated ion channels and morphological differentiation were studied in rat PC12 pheochromocytoma cells after treatment with nerve growth factor (NGF) or forskolin. Ca²⁺ and Na⁺ channels were analyzed by electrophysiological techniques (using Ba²⁺ as charge carrier through Ca²⁺ channels) and by binding studies with specific ligands. With NGF, Na⁺ current (I _Na) density increased in parallel with neurite extension. Ba²⁺ current (I _Ba) density and Ca²⁺ channel numbers were both enhanced after a 2-day latency period. The tyrosine kinase inhibitor genistein blocked NGF-induced neurite extension but not the increase in I _Na density. With forskolin, neurite outgrowth was linked to an apparent increase in I _Ba density similar to the one induced by NGF, while no change in I _Na was observed. Dihydropyridine-sensitive (L-type) as well as ω-conotoxin-sensitive (N-type) currents contributed to this effect. In spite of its stimulating effect on I _Ba, binding studies with radiolabeled ligands in forskolin-treated cells showed no change in N-type and an apparent loss of high affinity L-type Ca²⁺ channel binding. Our results suggest that induction of individual voltage-dependent channel types as well as morphological differentiation each require the activation of different signaling pathways. NGF and forskolin both enhanced current flow through voltage-dependent Ca²⁺ channels. However, only NGF increased channel expression while forskolin appeared to modulate channel kinetics. Received: 15 December 1998 / Accepted: 15 February 1999     

Effect of clomiphene on [Ca2+]i rises and cell viability in rabbit corneal epithelial cells

The effect of clomiphene a first‐line therapy for WHO group II (eu‐estrogenic) infertility on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability has not been explored in rabbit corneal epithelial cells (SIRC). This study examined whether clomiphene altered [Ca²⁺]_i levels and caused cell death in SIRC cells. [Ca²⁺]_i and cell viability were measured using the fluorescent dyes fura‐2 and WST‐1, respectively. Clomiphene at concentrations ≥5 µM increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The clomiphene‐induced Ca²⁺ influx was insensitive to blockade of L‐type Ca²⁺ channel blockers. In Ca²⁺‐free medium, after pretreatment with 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), clomiphene failed to increase [Ca²⁺]_i. Inhibition of phospholipase C with 2 µM U73122 did not change clomiphene‐induced [Ca²⁺]_i rises. At concentrations of 0.5–20 µM, clomiphene killed cells in a concentration‐dependent manner. The cytotoxic effect of 15 µM clomiphene was not reversed by prechelating cytosolic Ca²⁺ with BAPTA/AM. Collectively, in SIRC cells, clomiphene‐induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx from non‐L‐type Ca²⁺ channels. Clomiphene‐caused cytotoxicity was not mediated by a preceding [Ca²⁺]_i rise. Drug Dev Res 69:272–278, 2008 ©2008 Wiley‐Liss, Inc.     

Bitter tastants induce relaxation of rat thoracic aorta precontracted with high K+

相似文献   

Mechanisms of actions of hydrogen sulphide on rat distal colonic epithelium

BACKGROUND AND PURPOSE

The aim of this study was to clarify the mechanisms by which hydrogen sulphide (H₂S) affects ion secretion across rat distal colonic epithelium.

EXPERIMENTAL APPROACH

Changes in short-circuit current induced by the H₂S-donor, sodium hydrosulphide (NaHS; 10 mmol·L⁻¹), were measured in Ussing chambers after permeabilization of the apical membrane with nystatin. Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and Ca²⁺ in intracellular stores were measured with fluorescent dyes. Changes in mitochondrial membrane potential were estimated with rhodamine 123.

KEY RESULTS

NaHS had a biphasic effect on overall currents across the basolateral membrane: an initial inhibition followed by a secondary stimulation. Both a scilliroside-sensitive action on the Na⁺-K⁺-ATPase and modulation of glibenclamide-sensitive and tetrapentylammonium-sensitive (i.e. ATP-sensitive and Ca²⁺-dependent) basolateral K⁺ channels were involved in this action. Experiments with rhodamine 123 revealed that NaHS induced a hyperpolarization of the mitochondrial membrane. NaHS evoked a biphasic change in [Ca²⁺]_i, an initial decrease followed by a secondary increase, known to be mediated by the release of stored Ca²⁺. Initial falls in [Ca²⁺]_i were not mediated by a sequestration of Ca²⁺ in intracellular Ca²⁺ storing organelles, as the Mag-Fura-2 signal was unaffected by NaHS. Falls in [Ca²⁺]_i were inhibited by 2′,4′-dichlorobenzamil, an inhibitor of the Na⁺-Ca²⁺-exchanger, and attenuated in Na⁺-free buffer, suggesting a transient stimulation of Ca²⁺ outflow by this transporter, directly demonstrated by Mn²⁺ quenching experiments.

CONCLUSIONS AND IMPLICATIONS

ATP-sensitive and Ca²⁺-dependent basolateral K⁺ conductances, the basolateral Na⁺-K⁺-pump as well as Ca²⁺ transporters were involved in the action of H₂S in regulating colonic ion secretion.     

Effects of paraquat,dinoseb and 2,4-D on intracellular calcium and on vasopressin-induced calcium mobilization in isolated hepatocytes

 The effects of the herbicides paraquat, dinoseb and 2,4-D on intracellular Ca²⁺ levels and on vasopressin-induced Ca²⁺ mobilization were investigated in intact isolated hepatocytes. Incubation of rat hepatocytes with paraquat (5 mM for 60 min) and dinoseb (10 μM) resulted in a time-dependent loss of viability by approximately 25%. Viability of cells treated with 2,4-D decreased significantly, dropping to about 20% at 10 mM and 60 min incubation. Exposure of hepatocytes to paraquat (1–10 mM) for 60 min had no effect on the basal level of [Ca²⁺] _i . Additionally, exposure to paraquat had no effect on the magnitude and on the duration of the [Ca²⁺] _i response to vasopressin. In the presence of 2,4-D (1–10 mM), basal [Ca²⁺] _i increases as a function of herbicide concentration. The magnitude of the Δ[Ca²⁺] _i response decreases from 256±8 nM in control to 220±5 nM, at 10 mM 2,4-D. Exposure of hepatocytes to dinoseb (1–10 μM) had no effect on the basal level of [Ca²⁺] _i . However, a strong concentration-dependent decrease in the magnitude of Δ[Ca²⁺] _i in response to vasopressin was noticed at 60 min incubation. Dinoseb markedly inhibited the stimulation of the production of inositol phosphates by vasopressin stimulus. The present study demonstrates that paraquat, 2,4-D and dinoseb cause cell death in hepatocytes by mechanisms not related to an early increase in [Ca²⁺] _i . Additionally, it has been shown for the first time that dinoseb disturbs the transduction mechanism promoted by vasopressin by inhibiting the formation of IP₃. Received: 11 October 1994/Accepted: 5 December 1994     

Effect of capsaicin on Ca2+ fluxes in Madin‐Darby canine renal tubular cells

The effect of capsaicin, a transient receptor potential vanniloid‐1 (TRPV1) receptor agonist, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether capsaicin changed basal [Ca²⁺]_i levels in suspended MDCK cells by using fura‐2 as a Ca²⁺‐sensitive fluorescent dye. Capsaicin at concentrations between 10–100 µM increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced by 80% by removing extracellular Ca²⁺. Capsacin induced Mn²⁺ influx, leading to quench of fura‐2 fluorescence suggesting Ca²⁺ influx. This Ca²⁺ influx was inhibited by phospholipase A2 inhibitor aristolochic acid and the non‐selective Ca²⁺ entry blocker La³⁺, but not by store‐operated Ca²⁺ channel blockers nifedipine, econazole, and SK&F96365, and protein kinase C/A modulators. In Ca²⁺‐free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin abolished capsaicin‐induced Ca²⁺ release. Conversely, pretreatment with capsaicin partly reduced thapsigargin‐induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not alter capsaicin‐induced [Ca²⁺]_i rise. The TRPV1 receptor antagonist capsazepine also induced significant Ca²⁺ entry and Ca²⁺ release. Collectively, in MDCK cells, capsaicin induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via phospholipase A2‐regulated, La³⁺‐sensitive Ca²⁺ channels in a manner dissociated from stimulation of TRPV1 receptors. Drug Dev Res, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of thimerosal on Ca2+ movement and apoptosis in PC3 prostate cancer cells

The present study evaluated the effects of thimerosal, a vaccine preservative, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in human prostate cancer cells (PC3). Thimerosal (10–200 µM) increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Thimerosal‐induced Ca²⁺ influx was inhibited by econazole, SKF963656, the phospholipase A₂ inhibitor aristolochic acid, and protein kinase C modulators [phorbol 12‐myristate 13‐acetate (PMA) and GF109203X]. In Ca²⁺‐free medium, a 200‐µM thimerosal‐induced [Ca²⁺]_i rise was partly inhibited after pretreatment with 2,5‐di‐tert‐butylhydroquinone (BHQ) (an endoplasmic reticulum Ca²⁺ pump inhibitor). Thimerosal at 1–7 µM induced cell death in a concentration‐dependent manner that was not reversed when cytosolic Ca²⁺ was chelated with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA). Propidium iodide staining suggests that apoptosis played a role in the death. Collectively, in PC3 cells, thimerosal induced [Ca²⁺]_i rise by causing Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via store‐operated Ca²⁺ channels in a manner regulated by protein kinase C and phospholipase A2. Thimerosal also induced cell death in a Ca²⁺‐independent apoptotic manner. Drug Dev Res 72: 330–336, 2011. © 2011 Wiley‐Liss, Inc.     

Triggering of Suicidal Erythrocyte Death by the Antibiotic Ionophore Nigericin

The K⁺,H⁺ ionophore and antibiotic nigericin has been shown to trigger apoptosis and is thus considered for the treatment of malignancy. Cellular mechanisms involved include induction of oxidative stress, which is known to activate erythrocytic Ca²⁺‐permeable unselective cation channels leading to Ca²⁺ entry, increase in cytosolic Ca²⁺ activity ([Ca²⁺]_i) and subsequent stimulation of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. This study explored whether and how nigericin induces eryptosis. Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3 fluorescence, pH_i from BCECF fluorescence, ceramide abundance utilizing antibodies and reactive oxygen species (ROS) formation from DCFDA‐dependent fluorescence. A 48‐hr exposure of human erythrocytes to nigericin significantly increased the percentage of annexin‐V‐binding cells (0.1–10 nM), significantly decreased forward scatter (0.1–1 nM), significantly decreased cytosolic pH (0.1–1 nM) and significantly increased Fluo3 fluorescence (0.1–10 nM). Nigericin (1 nM) slightly, but significantly, increased ROS, but did not significantly modify ceramide abundance. The effect of nigericin on annexin V binding was significantly blunted, but not abolished by removal of extracellular Ca²⁺. The nigericin‐induced increase in [Ca²⁺]_i and annexin V binding was again significantly blunted but not abolished by the Na⁺/H⁺ exchanger inhibitor cariporide (10 μM). Nigericin triggers eryptosis, an effect paralleled by ROS formation, in part dependent on stimulation of Ca²⁺ entry, and involving the cariporide‐sensitive Na⁺/H⁺ exchanger.     

Effect of calmidazolium on [Ca2+]i and viability in human hepatoma cells

The effect of calmidazolium on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability has not been explored in human hepatoma cells. This study examined whether calmidazolium altered [Ca²⁺]_i and caused cell death in HA59T cells. [Ca²⁺]_i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Calmidazolium at concentrations ≥1 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 1.5 μM. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Calmidazolium induced Mn²⁺ quench of fura-2 fluorescence implicating Ca²⁺ influx. The Ca²⁺ influx was insensitive to L-type Ca²⁺ entry blockers, but was inhibited partly by enhancing or inhibiting protein kinase C activity. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), calmidazolium-induced [Ca²⁺]_i rises were largely inhibited; and conversely, calmidazolium pretreatment totally suppressed thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change calmidazolium-induced [Ca²⁺]_i rises. At concentrations between 1 and 15 μM, calmidazolium induced apoptosis-mediated cell death. Collectively, in HA59T hepatoma cells, calmidazolium induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca²⁺ influx via protein kinase C-regulated Ca²⁺ entry pathway. Calmidazolium caused cytotoxicity via apoptosis.