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     

Ca2+ stores regulate ryanodine receptor Ca2+ release channels via luminal and cytosolic Ca2+ sites

1. In muscle, intracellular calcium concentration, hence skeletal muscle force and cardiac output, is regulated by uptake and release of calcium from the sarcoplasmic reticulum. The ryanodine receptor (RyR) forms the calcium release channel in the sarcoplasmic reticulum. 2. The free [Ca2+] in the sarcoplasmic reticulum regulates the excitability of this store by stimulating the Ca2+ release channels in its membrane. This process involves Ca2+-sensing mechanisms on both the luminal and cytoplasmic sides of the RyR. In the cardiac RyR, these have been shown to be a luminal Ca2+ activation site (L-site; 60 micromol/L affinity), a cytoplasmic activation site (A-site; 0.9 micromol/L affinity) and a cytoplasmic Ca2+ inactivation site (I2-site; 1.2 micromol/L affinity). 3. Cardiac RyR activation by luminal Ca2+ occurs by a multistep process dubbed 'luminal-triggered Ca2+ feed-through'. Binding of Ca2+ to the L-site initiates brief (1 msec) openings at a rate of up to 10/s. Once the pore is open, luminal Ca2+ has access to the A-site (producing up to 30-fold prolongation of openings) and to the I2-site (causing inactivation at high levels of Ca2+ feed-through). 4. The present paper reviews the evidence for the principal aspects of the 'luminal-triggered Ca2+ feed-through' model, the properties of the various Ca2+-dependent gating mechanisms and their likely role in controlling sarcoplasmic reticulum (SR) Ca2+ release in cardiac muscle. 5. The model makes the following important predictions: (i) there will be a close link between luminal and cytoplasmic regulation of RyRs and any cofactor that prolongs channel openings triggered by cytoplasmic Ca2+ will also promote RyR activation by luminal Ca2+; (ii) luminal Mg2+ (1 mmol/L) is essential for the control of SR excitability in cardiac muscle by luminal Ca2+; and (iii) the different RyR isoforms in skeletal and cardiac muscle will be controlled quite differently by the luminal milieu. For example, Mg2+ in the SR lumen (approximately 1 mmol/L) can strongly inhibit RyR2 by competing with Ca2+ for the L-site, whereas RyR1 is not affected by luminal Mg2+.     

Agonist-dependent difference in the mechanisms involved in Ca2+ sensitization of smooth muscle of porcine coronary artery

This study was undertaken to explore possible signal-transduction mechanisms involved in the Ca2+-sensitizing effects of carbachol and endothelin-1 (ET-1) by using beta-escin-skinned smooth muscle of porcine coronary artery. Pretreatment with C3 exoenzyme of Clostridium botulinum, which selectively inactivates rho p21 by adenosine diphosphate (ADP) ribosylation, resulted in a significant inhibition of ET-1-induced Ca2+ sensitization, but had no effect on carbachol-induced Ca2+ sensitization. Whereas the protein kinase C (PKC) inhibitors calphostin C and staurosporine did not affect the Ca2+-sensitizing effect of carbachol, the tyrosine kinase inhibitors genistein and tyrphostin 25 greatly but incompletely suppressed it. In contrast, the Ca2+-sensitizing effect of ET-1 was significantly inhibited by either calphostin C or genistein. Although the inhibitory effect of calphostin C on ET-1-induced Ca2+ sensitization was less than that of genistein, the effects of calphostin C and genistein were additive. The genistein-sensitive component of ET-1-induced Ca2+ sensitization appeared to include the C3-sensitive one. However, a substantial enhancement by ET-1 of the Ca2+-induced contraction was observed even in the presence of the two inhibitors. In beta-escin-skinned smooth muscle of rabbit mesenteric artery, ET-1-induced Ca2+ sensitization was marginally affected by C3 pretreatment, calphostin C, and genistein. We conclude that, although PKC activation and rho p21 protein-dependent and -independent tyrosine phosphorylation each plays an important role in an increase in myofilament Ca2+ sensitivity, the contributions of these signaling pathways to Ca2+ sensitization are different depending on receptor agonists and tissues used. Furthermore, these data suggest the existence of an as yet undefined signal-transduction mechanism involved in Ca2+ sensitization caused by receptor agonists.     

Ca2+ mobilization in the aortic endothelium in streptozotocin-induced diabetic and cholesterol-fed mice

1. Experiments were performed to compare Ca²⁺ mobilization in the aortic endothelium in streptozotocin (STZ)-induced diabetic and cholesterol-fed mice with that in age-matched controls.
2. The intracellular free Ca²⁺ ([Ca²⁺]_i) in the fura PE-3 loaded endothelium of aortic rings was dose-dependently increased by cumulative administration of acetylcholine (ACh). ACh caused a transient rise in [Ca²⁺]_i in Ca²⁺-free medium. The ACh-induced increase in [Ca²⁺]_i in normal or Ca²⁺-free medium was significantly weaker in both STZ-induced diabetic and cholesterol-fed mice.
3. The weaker [Ca²⁺]_i response in Ca²⁺-containing medium in STZ-induced diabetic and cholesterol-fed mice was normalized by chronic administration of cholestyramine.
4. The increased low density lipoprotein (LDL) levels seen in both STZ-induced diabetic and cholesterol-fed mice were normalized by the same chronic administration of cholestyramine (300 mg kg⁻¹, p.o. daily for 10 weeks). Chronic administration of cholestyramine had no effect on the plasma glucose level.
5. Lysophosphatidylcholine (LPC) decreased the [Ca²⁺]_i responses to ACh in the aortic endothelium from normal mice.
6. These results suggest that ACh increases both Ca²⁺ influx and Ca²⁺ release from storage in the aortic endothelium. The weaker [Ca²⁺]_i influx seen in the endothelium of aortae from both STZ-induced diabetic and cholesterol-fed mice was improved by the chronic administration of cholestyramine, and we suggest that this improvement is due, at least in part, to a lowering of the plasma LDL level. It is further suggested that LPC may have an important influence over Ca²⁺ mobilization in the endothelium.

     

异紫堇啡碱对血管平滑肌钙内流和钙释放的影响

目的研究异紫堇啡碱（ＩＳＯＣ）对血管平滑肌钙内流和钙释放的影响，以初步阐明其作用方式。方法利用兔胸主动脉螺旋条标本观察ＩＳＯＣ对去甲肾上腺素（ＮＡ）及ＫＣｌ量效曲线的影响和对无钙液中ＮＡ、ＣａＣｌ２复钙、咖啡因缩血管效应的影响。结果ＩＳＯＣ１０μｍｏｌ·Ｌ－１对ＮＡ的量效曲线呈非竞争性拮抗作用，而对高钾的作用则不明显。在无钙液中，ＩＳＯＣ１００μｍｏｌ·Ｌ－１能明显抑制ＮＡ所致的收缩及复钙后外钙内流诱发的收缩，ＩＳＯＣ１０及３０μｍｏｌ·Ｌ－１则只作用于前者；各实验浓度的ＩＳＯＣ对咖啡因在无钙液中的缩血管作用均无影响。结论ＩＳＯＣ抑制受体中介的钙释放和钙内流，但不是典型的钙拮抗剂。     

普鲁托品对兔血小板内钙的影响

AIM: To study the influence of protopine (Pro) on the cytoplasmic free Ca2+ concentration ([Ca2+]i) in rabbit platelets. METHODS: Measurement of [Ca2+]i of platelets in vitro by Fura 2-AM fluorescence technique. RESULTS: In the presence of CaCl2 1 mmol.L-1, Pro 10, 20, and 40 mumol.L-1 attenuated the rise in [Ca2+]i evoked by ADP from (420 +/- 57) to (320 +/- 26), (264 +/- 21), and (180 +/- 14) nmol.L-1, respectively, by arachidonic acid (AA) from (280 +/- 36) to (210 +/- 17), (184 +/- 21), and (143 +/- 16) nmol.L-1, respectively, and by platelet-activating factor (PAF) from (350 +/- 42) to (282 +/- 31), (223 +/- 30), and (165 +/- 15) nmol.L-1, respectively. In the presence of egtazic acid 1 mmol.L-1, Pro 10, 20, and 40 mumol.L-1 reduced the Ca2+ release induced by ADP, AA, and PAF, respectively. Pro 10, 20, and 40 mumol.L-1 also decreased ADP-, AA-, and PAF-induced Ca2+ influx. CONCLUSION: Pro inhibited not only Ca2+ release but also the influx of Ca2+.     

Different effects of norepinephrine and KCl on the cytosolic Ca2+-tension relationship in vascular smooth muscle of rat aorta

Norepinephrine and KCl induced a concentration-dependent increase in the cytosolic Ca2+ level [( Ca2+]cyt) measured either by intracellular fura 2-Ca2+ fluorescence or by 45Ca2+ uptake. Muscle contraction in isolated rat aortic strips was also increased although a greater contraction was induced by norepinephrine than by KCl at the same [Ca2+]cyt. This result suggests that the contraction of vascular smooth muscle is regulated by [Ca2+]cyt and also by other factor(s).     

Gender difference in the role of endothelium-derived relaxing factors modulating renal vascular reactivity

This study analyzed the role of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in gender differences in the renal vascular reactivity of rats. Renal responses to vasoconstrictors and vasodilators were studied in isolated kidneys from male and female rats under basal conditions, after NO and EDHF blockade or after endothelium removal. Female rat kidneys had reduced responsiveness to vasoconstrictors. The blockade of NO or of EDHF did not completely abolish the differences, but the simultaneous blockade of both factors or endothelium removal abolished gender differences. Male and female kidneys showed a similar responsiveness to endothelium-dependent and -independent vasodilators under basal conditions and after NO or EDHF blockade. In conclusion: (a) the attenuated response to vasoconstrictors in female kidneys is related to an increased production of NO and EDHF; and (b), the contributions of NO and EDHF to endothelium-dependent vasodilation are similar in the male and female renal vasculature.     

Pharmacologic differentiation between inositol-1,4,5-trisphosphate-induced Ca2+ release and Ca2+- or caffeine-induced Ca2+ release from intracellular membrane systems

Various known Ca2+ channel blockers and intracellular Ca2+ antagonists have been tested for effects of inositol-1,4,5-trisphosphate (IP3)-induced Ca2+ release from isolated canine brain microsomes. In agreement with previous reports, heparin, p-chloromercuribenzoic acid, W-7, cinnarizine, flunarizine, certain local anesthetics, La3+, and Ca2+ inhibit the release of Ca2+ induced by addition of IP3. In addition, we report here pronounced inhibition of IP3-induced Ca2+ release by low levels of Cd2+, by relatively high concentrations of TMB-8, and by phytic acid. In contrast, a number of blockers of other Ca2+ channels (nifedipine, verapamil, dantrolene, dithiothreitol, and ruthenium red) have relatively little or no effect on IP3-induced Ca2+ release from brain microsomes. The relative ineffectiveness of substances that inhibit Ca2+- or caffeine-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum suggests that release of Ca2+ from caffeine- and IP3-sensitive neuronal Ca2+ stores is likely to be mediated by different channels. Further evidence that different channels are involved is presented by way of demonstration of the lack of Ca2+-induced Ca2+ release from these brain microsomes and the lack of effect on sarcoplasmic reticulum caffeine-induced Ca2+ release of certain inhibitors of IP3-induced Ca2+ release used here. Among IP3-induced Ca2+ release blockers, La3+ appeared to be exceptional in its ability to stimulate microsomal Ca2+ uptake sufficiently to attenuate release of Ca2+ induced by IP3. Most blockers of IP3-induced Ca2+ release appear not to function by way of inhibiting K+ counter-ion movements (valinomycin does not reverse the inhibition) but rather by way of direct interaction with the IP3 receptor or the Ca2+ channel that mediates the IP3-induced Ca2+ release. Inhibition of [3H]IP3 binding to the microsomes by phytic acid, heparin, pyrophosphate, p-chloromercuribenzoic acid, and Ca2+ could be demonstrated but not by the other substances tested.     

Mg2+ and caffeine-induced intracellular Ca2+ release in human vascular endothelial cells.

Interaction of ionized magnesium ([Mg2+]o) and caffeine in regulation of intracellular free calcium concentration ([Ca2+]i) in human aortic endothelial cells was studied using fura-2 and digital imaging microscopy. In 1.2 mM [Mg2+]o, basal [Ca2+]i was 73.7 +/- 22.4 nM, with a heterogeneous distribution within the cells. No significant changes of basal [Ca2+]i were found either when cells were treated with 10 mM caffeine or when [Mg2+]o was lowered from 1.2 mM to 0.3 mM. However, a combined superfusion of the cells with 0.3 mM [Mg2+]o and 10 mM caffeine resulted in a significant elevation of [Ca2+]i to 382.8 +/- 57.1 nM, probably by release of Ca2+ from internal stores, which was attenuated by NiCl2 (1 mM). These results suggest that a Ca(2+)-induced Ca2+ release mechanism is involved in regulation of [Ca2+]i in endothelial cells, which may be either regulated or modulated by Mg2+.     

Different effects of verapamil on cytosolic Ca2+ and contraction in norepinephrine-stimulated vascular smooth muscle

Effects of verapamil on cytosolic Ca2+ levels ([Ca2+]cyt) and contraction in fura-2-loaded rat aorta were examined. Norepinephrine (NE) induced a greater contraction than KCl for a given increase in [Ca2+]cyt. Cumulative addition of verapamil decreased the NE-stimulated [Ca2+]cyt more strongly than contraction whereas verapamil decreased high K(+)-stimulated [Ca2+]cyt and contraction in parallel. In the presence of verapamil at a concentration needed to completely inhibit the high K(+)-induced increments, NE induced a transient increase, followed by a small sustained increase in [Ca2+]cyt which averaged 25% of that in the absence of verapamil. These changes were followed by a sustained contraction which averaged 60% of that in the absence of verapamil. In Ca2(+)-free solution, NE induced only a transient increase in [Ca2+]cyt whereas it induced a transient contraction, followed by a small sustained contraction. The second application of NE induced a small sustained contraction (10% of that in the presence of Ca2+) without increasing [Ca2+]cyt. These changes were not affected by verapamil. These results suggest that verapamil inhibits NE-induced increase in [Ca2+]cyt, but not the Ca2(+)-sensitization or Ca2(+)-independent contraction, and this may be the reason why the NE-induced contraction is less sensitive to verapamil than that induced by high K+.     

Inhibitory effects of cadralazine and its metabolite, ISF-2405, on contractions and the level of cytosolic Ca2+ in vascular smooth muscle

The inhibitory effects of a hypotensive agent, cadralazine and its metabolite, ISF-2405, on the level of cytosolic Ca2+ ([Ca2+]cyt) and on contractions were examined in isolated vascular smooth muscle. Cadralazine slightly inhibited the transient norepinephrine-induced contraction in rabbit aorta and canine femoral, renal and mesenteric arteries and saphenous vein, and prostaglandin F2 alpha-induced contractions in canine basilar and coronary arteries. In contrast, ISF-2405 inhibited the contractions induced by prostaglandin F2 alpha in canine basilar and coronary arteries and those induced by norepinephrine in canine renal and femoral arteries and rabbit aorta. In aorta, ISF-2405 inhibited the increase in [Ca2+]cyt and muscle tension caused by norepinephrine. A Ca2+ channel blocker, verapamil, inhibited the norepinephrine-stimulated increase in [Ca2+]cyt more potently than it inhibited the increase in muscle tension, and ISF-2405 inhibited the verapamil-resistant part of the contraction. In Ca2(+)-free solution, norepinephrine induced transient increases in [Ca2+]cyt and muscle tension. ISF-2405 inhibited these changes. However, ISF-2405 did not inhibit the transient contraction induced by caffeine in the aorta. These results suggest that cadralazine is metabolized to ISF-2405 and inhibits vascular smooth muscle contraction by inhibiting receptor-mediated Ca2+ influx, Ca2+ release and Ca2+ sensitization of contractile elements.     

Protamine induces elevation of cytosolic free Ca2+ in cultured porcine aortic endothelial cells.

To test the hypothesis that protamine influences calcium movement in endothelial cells, we measured the concentration of intracellular free calcium ([Ca2+]i) in cultured porcine aortic endothelial (PAE) cells in Krebs solution (2.5mM Ca2+, pH 7.4) at 37 degrees C, by fura-2 fluorimetry. The basal [Ca2+]i of PAE cells was 113+/-18 nM (n=6). Protamine increased [Ca2+]i in a concentration-dependent manner (EC50, the concentration having 50% of the maximum effect, 1.4+/-0.3 microg mL(-1), n=6). The response of PAE cells to 100 microg mL(-1) protamine (330+/-80 nM, n=6) was blocked by a Ca2+ chelator, 5 mM glycoletherdiaminetetraacetic acid (EGTA; 131+/-16 nM, n=6), and by a non-selective Ca2+ channel blocker, 3 mM Co2+ (134+/-14 nM, n=6). These results suggest that Ca2+ influx through cell-membrane Ca2+ channels is mainly responsible for the protamine-induced Ca2+ elevation.     

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.     

The long-term inhibitory effect of a Ca2+ channel blocker, nisoldipine, on cytosolic Ca2+ and contraction in vascular smooth muscle.

The mechanism of the long-term inhibitory effect of a dihydropyridine Ca2+ channel blocker, nisoldipine, on contraction and cytosolic Ca2+ level ([Ca2+]i) was examined in isolated rat aorta. Nisoldipine inhibited the [Ca2+]i and muscle tension induced by high K+. The inhibitory effects were antagonized by a Ca2+ channel activator, 100 nM Bay k8644, and by a high concentration of Ca2+ (6.5 mM). Ultraviolet light, which has been shown to decompose dihydropyridines, attenuated the effects of nisoldipine. After nisoldipine had been removed from muscle bath, the inhibitory effect faded away slowly. The residual inhibitory effects on [Ca2+]i and muscle tension were antagonized by Bay k8644, high Ca2+ and ultraviolet light. These results suggest that the inhibitory effect of nisoldipine is caused by a decrease in [Ca2+]i as a result of inhibition of L-type Ca2+ channels, and that the residual inhibitory effects are caused by the same mechanism as the inhibitory effects of nisoldipine, namely the tight binding of nisoldipine to Ca2+ channels even after washout.     

Changes in the cytosolic Ca2+ concentration and Ca(2+)-sensitivity of the contractile apparatus during angiotensin II-induced desensitization in the rabbit femoral artery

1. To investigate the underlying mechanism for the angiotensin II-induced desensitization of the contractile response during the prolonged stimulation of the vascular smooth muscle, we determined the effects of angiotensin-II on (1) cytosolic Ca2+ concentration ([Ca2+]i) and tension using fura-2-loaded medial strips of the rabbit femoral artery, (2) 45Ca2+ influx in ring preparations, and (3) Ca(2+)-sensitivity of the contractile apparatus in alpha-toxin permeabilized preparations. 2. In the presence of extracellular Ca2+, high concentrations of angiotensin-II elicited biphasic increases in [Ca2+]i and tension, which consisted of initial transient and subsequent lower and sustained phases. 3. The 45Ca2+ influx initially increased after the application of 10(-6) M angiotensin-II, and thereafter gradually decreased. At 20 min after the application, there was a discrepancy between the level of [Ca2+]i and the extent of 45Ca2+ influx. 4. The relationships between [Ca2+]i and tension suggested that the angiotensin-II-induced increase in the Ca(2+)-sensitivity of the contractile apparatus was maintained during the desensitization of smooth muscle contraction. 5. When 10(-6) M angiotensin-II was applied during the sustained phase of contraction induced by 118 mm K(+)-depolarization, at 10 min after the application, the [Ca2+]i levels were significantly lower and the tension levels were significantly higher than those prior to the application of angiotensin-II. 6. In conclusion, the decrease in [Ca2+]i, which is partially due to the inhibition of the Ca2+ influx, is mainly responsible for the desensitization evoked by high concentrations of angiotensin-II, and angiotensin-II seems to activate additional mechanisms which inhibit Ca2+ signaling during prolonged stimulation.     

Correlation between cytosolic Ca2+ concentration and cytotoxicity in hepatocytes exposed to oxidative stress

To investigate the relationship between alterations of cytosolic Ca2+ concentration and development of cytotoxicity, isolated rat hepatocytes were loaded with the fluorescent indicator Quin-2 AM and then incubated with non-toxic or toxic levels of menadione (2-methyl-1,4-naphthoquinone) or tert-butyl hydroperoxide (t-BH). The resulting changes in cytosolic Ca2+ concentration were compared to those seen upon exposure of the hepatocytes to an alpha 1-adrenergic agonist, phenylephrine, as well as to those induced by menadione and t-BH in hepatocytes pretreated with agents that modify their toxicity. Exposure of hepatocytes to phenylephrine or non-toxic levels of menadione caused a moderate and transient increase in cytosolic Ca2+ (less than or equal to 0.7 microM), whereas a toxic concentration of menadione produced a marked, sustained increase in Ca2+ which fully saturated the binding capacity of Quin-2 (greater than 1.5 microM). Treatment of the hepatocytes with the protective agent, dithiothreitol, prevented both the increase in cytosolic Ca2+ and the cytotoxicity induced by menadione. On the other hand, pretreatment of cells with diethylmaleate to deplete intracellular glutathione made otherwise non-toxic concentrations of menadione cause both a sustained increase in cytosolic Ca2+ and cytotoxicity. Similarly, toxic concentrations of t-BH also caused a sustained increase in cytosolic Ca2+. The iron chelator, desferrioxamine, and dithiothreitol (DTT), which protected the cells from t-BH toxicity, also prevented the sustained elevation of cytosolic Ca2+. Our findings provide further support for the hypothesis that a perturbation of intracellular Ca2+ homeostasis is an early and critical event in the development of toxicity in hepatocytes exposed to oxidative stress.     

Flubendiamide, a novel Ca2+ channel modulator, reveals evidence for functional cooperation between Ca2+ pumps and Ca2+ release

Flubendiamide, developed by Nihon Nohyaku Co., Ltd. (Tokyo, Japan), is a novel activator of ryanodine-sensitive calcium release channels (ryanodine receptors; RyRs), and is known to stabilize insect RyRs in an open state in a species-specific manner and to desensitize the calcium dependence of channel activity. In this study, using flubendiamide as an experimental tool, we examined an impact of functional modulation of RyR on Ca2+ pump. Strikingly, flubendiamide induced a 4-fold stimulation of the Ca2+ pump activity (EC50=11 nM) of an insect that resequesters Ca2+ to intracellular stores, a greater increase than with the classical RyR modulators ryanodine and caffeine. This prominent stimulation, which implies tight functional coupling of Ca2+ release with Ca2+ pump, resulted in a marginal net increase in the extravesicular calcium concentration despite robust Ca2+ release from the intracellular stores by flubendiamide. Further analysis suggested that luminal Ca2+ is an important mediator for the functional coordination of RyRs and Ca2+ pumps. However, kinetic factors for Ca2+ pumps, including ATP and cytoplasmic Ca2+, failed to affect the Ca2+ pump stimulation by flubendiamide. We therefore conclude that the stimulation of Ca2+ pump by flubendiamide is mediated by the decrease in luminal calcium, which may induce calcium dissociation from the luminal Ca2+ binding site on the Ca2+ pump. This mechanism should play an essential role in precise control of intracellular Ca2+ homeostasis.     

Effects of semotiadil fumarate, a novel Ca2+ antagonist, on cytosolic Ca2+ level and force of contraction in porcine coronary arteries.

1. The mechanisms of action of semotiadil fumarate, a novel Ca2+ antagonist, were examined by measuring the cytosolic Ca2+ level ([Ca2+]i) and force of contraction in porcine coronary arteries, and by determining [3H]-pyrilamine binding to bovine cerebellar membranes. 2. Semotiadil or verapamil (0.1 and 1 microM) inhibited both the high KCl-induced increases in [Ca2+]i and force in a concentration-dependent manner. 3. Histamine (30 microM) produced transient increases followed by sustained increases in [Ca2+]i and force, which were inhibited by semotiadil and verapamil (1 and 10 microM). The agents were different in that semotiadil reduced the maximum [Ca2+]i and force responses to histamine, but not pD2 values, whereas verapamil did reduce the pD2 values for histamine, but not the maximum responses. 4. Verapamil (10 microM), but not semotiadil, inhibited histamine-induced increases in [Ca2+]i and force in Ca(2+)-free solution. Neither semotiadil nor verapamil affected the increases in [Ca2+]i and force induced by caffeine. Semotiadil even at the higher concentration (10 microM) did not displace specific binding of [3H]-pyrilamine to bovine cerebellar membranes. 5. These results suggest that semotiadil inhibits both KCl- and histamine-induced contractions mainly by blocking voltage-dependent L-type Ca2+ channels.     

Endothelin dissociates muscle tension from cytosolic Ca2+ in vascular smooth muscle of rat carotid artery

Endothelin induced rapid increase followed by a decrease in cytosolic Ca2+ [( Ca2+]i) and a slow increase in muscle tension in the vascular smooth muscle strip of rat carotid artery. Thus, the endothelin-induced contraction was smaller, and it became gradually greater than high K-induced contraction at a given [Ca2+]i. In Ca2+-free solution, endothelin induced a transient increase in [Ca2+]i and a sustained contraction. These results suggest that endothelin-induced contraction is due to the increase in [Ca2+]i, the time-dependent change in Ca2+-sensitivity of contractile elements, and the mechanism which is independent of the increment in [Ca2+]i.