The effect of high calcium intake on intracellular free [Ca2+] and Na(+)-H+ exchange in DOC-NaCl-hypertensive rats.

The effects of calcium supplementation on blood pressure, intracellular free calcium concentration ([Ca2+]i) and rate of Na(+)-H+ exchange were studied in DOC-NaCl-hypertensive rats. All the animals were uninephrectomized and divided into two main groups: the first group received deoxycorticosterone (DOC) (25 mg/kg, s.c.) once a week and had 0.7% NaCl as drinking fluid while the other received equal volumes of saline and tap water to drink. The animals were further divided according to dietary calcium intake: in the Control and DOC groups the chow contained 1.1% calcium, in the Calcium and DOC+Calcium groups, 2.5%. After 6 and 8 weeks, blood pressure in the DOC group was higher than in the Control group; on the other hand, the development of hypertension was attenuated in the DOC+Calcium compared with the DOC group. The Control and Calcium groups did not differ from each other. Platelets and lymphocytes were used as experimental models to study changes in the regulation of [Ca2+]i, evaluated by fluorescent indicators indo-1 and quin-2. In lymphocytes, basal [Ca2+]i was highest in the DOC group, but similar in DOC+Calcium and Control groups. In platelets, both basal and thrombin-stimulated [Ca2+]i were higher in the DOC and DOC+Calcium groups than in the Control group. In both cell types [Ca2+]i was similar in Control and Calcium groups. In addition, platelets were used to study the ability of the cells to recover from intracellular acidification by first blocking the Na(+)-H+ exchange in a Na(+)-free medium and then restarting the exchange mechanism by increasing the extracellular Na+ concentration at constant speed.(ABSTRACT TRUNCATED AT 250 WORDS)     

积雪草总苷对大鼠系膜细胞游离钙的影响

目的:探讨积雪草提取物对大鼠肾小球系膜细胞增殖和游离钙离子[Ca2+];水平的影响.方法:将血管紧张素Ⅱ(AngⅡ)作用肾小球系膜细胞,加入不同浓度的积雪草提取物,采用MTT法和Fura-2/AM荧光指示剂负载测定系膜细胞增殖和细胞内[Ca2+]i水平的变化.结果:系膜细胞一经AngⅡ刺激后增殖显著,且[Ca2+]i水平明显升高,而积雪草提取物对AngⅡ刺激下的系膜细胞增殖和Ca2+水平有抑制和降低作用,并呈一定的剂量依赖性.结论:积雪草提取物可能通过降低系膜细胞内[Ca2+]i的水平,从而达到抑制系膜细胞增殖的目的.     

积雪草总苷对大鼠系膜细胞游离钙的影响

目的:探讨积雪草提取物对大鼠肾小球系膜细胞增殖和游离钙离子[Ca2+];水平的影响.方法:将血管紧张素Ⅱ(AngⅡ)作用肾小球系膜细胞,加入不同浓度的积雪草提取物,采用MTT法和Fura-2/AM荧光指示剂负载测定系膜细胞增殖和细胞内[Ca2+]i水平的变化.结果:系膜细胞一经AngⅡ刺激后增殖显著,且[Ca2+]i水平明显升高,而积雪草提取物对AngⅡ刺激下的系膜细胞增殖和Ca2+水平有抑制和降低作用,并呈一定的剂量依赖性.结论:积雪草提取物可能通过降低系膜细胞内[Ca2+]i的水平,从而达到抑制系膜细胞增殖的目的.     

Effects of amiloride in guinea-pig and rat left atrial contraction as affected by frequency of stimulation and [Ca2+]0-[Na+]0 ratio: role of Na+/Ca2+ exchange.

1. The effect of amiloride (0.5 mM) on guinea-pig and rat left atria driven at various rates of stimulation and different [Ca2+]0-[Na+]0 ratios has been studied. 2. Amiloride elicited a positive inotropic response in guinea-pig left atria driven at 0.1 Hz, 0.5 Hz and 1 Hz when [Ca2+]0 was 3.6 mM, 1.8 mM and 0.9 mM respectively but not when [Ca2+]0 was 2.7 mM at 0.1 Hz, 0.9 mM at 0.5 Hz and 0.45 mM at 1 Hz. 3. A positive inotropic response was obtained in guinea-pig left atria driven at 0.1 Hz and 1 Hz when [Ca2+]0-[Na+]0(2) was increased respectively from 8 x 10(-5) to 16 x 10(-5) and from 2 x 10(-5) to 8 x 10(-5). The positive inotropic effect was evident only when the ratio was increased by increasing [Ca2+]0 and not by decreasing [Na+]0. 4. In the presence of amiloride, the force of contraction of guinea-pig left atria decreased instead of increasing, when the rate of stimulation was lowered from 1 Hz to 0.01 Hz. Amiloride inhibited the post-rest potentiation. 5. In rat left atria amiloride was devoid of any effect in all the above-mentioned experimental conditions. 6. It is suggested that the pattern of cardiac actions of amiloride can be explained by the inhibition of the Na+/Ca2+ exchange system.     

Effect of Ca2+ antagonists on high-K+ evoked increase in [Ca2+]i in rat cerebral synaptosomes and hippocampal neurons.

By fura-2 fluorometry, we investigated the direct effects of Ca2+ antagonists including a new benzothiazepine, clentiazem, on the high-K(+)-evoked increase in the concentration of cytosolic free Ca2+ ([Ca2+]i) in rat cerebral synaptosomes and cultured hippocampal neurons. In both preparations, metal ions inhibited the high-K(+)-induced increase in [Ca2+]i, in the following order: La3+ greater than Cd2+ much greater than Ni2+. Although flunarizine and nicardipine inhibited the K(+)-induced increase in [Ca2+]i in synaptosomes, other Ca2+ antagonists, including clentiazem and nitrendipine, had little effect at 10 microM. In hippocampal neurons, clentiazem inhibited the K(+)-induced increase in [Ca2+]i at 10 microM, as did flunarizine and nicardipine. However, nifedipine and nitrendipine had little effect in either cultured neurons or in synaptosomes.     

Endothelin-1 decreases [Ca2+]i via Na+/Ca2+ exchanger in CHO cells stably expressing endothelin ETA receptor

Endothelin ET(A) receptor couples to Gq/11 protein that transduces a variety of receptor signals to modulate diverse cellular responses including Ca2+ mobilization. Stimulation of endothelin ETA receptor with endothelin-1 is generally believed to induce an increase in intracellular Ca2+ concentration ([Ca2+]i) via Gq/11 protein. Here we provide the first convincing evidence that endothelin-1 elicited Gq/11 protein-dependent and -independent 'decrease' in [Ca2+]i via Na+/Ca2+ exchanger (NCX) in Chinese hamster ovary (CHO) cells stably expressing human endothelin ETA receptor. In the cells treated with 1 microM thapsigargin, an inhibitor of endoplasmic Ca2+ pump, that induces an increase in [Ca2+]i via capacitative Ca2+ entry, endothelin-1 induced a decrease in [Ca2+]i which was partially inhibited by YM-254890, a specific inhibitor of Gq/11, indicating that Gq/11-dependent and independent pathways are involved in the decrease. The endothelin-1-induced decrease in [Ca2+]i was markedly suppressed by 3',4'-dichlorobenzamil hydrochloride, a potent NCX inhibitor, and also by a replacement of extracellular Na+ with Li+, which was not transported by NCX, indicating a major role of NCX operating in the forward mode in the endothelin-1-induced decrease in [Ca2+]i. Molecular approach with RT-PCR demonstrated the expression of mRNA for NCX1, NCX2 and NCX3. These results suggest that stimulation of endothelin ETA receptor with endothelin-1 activates the forward mode NCX through Gq/11-dependent and -independent mechanisms: the NCX exports Ca2+ out of the cell depending on Na+ gradient across the cell membrane, resulting in the decrease in [Ca2+]i.     

慢性染铅对大鼠海马区神经细胞Ca2+浓度及Ca2+-ATP酶活性的影响

目的：观察慢性染铅对大鼠海马区神经细胞Ｃａ２＋浓度及Ｃａ２＋－ＡＴＰ酶活性的影响。方法：用０．１５％醋酸铅饲养大鼠建立慢性染铅动物模型,参照Ｄｉｌｄｙ法和徐友涵法测定海马神经细胞Ｃａ２＋浓度及Ｃａ２＋－ＡＴＰ酶活性。结果发现：细胞内Ｃａ２＋浓度,染铅组（２０３．８３±３０．５０）ｎｍｏｌ／Ｌ,对照组（９７．６２±１９．８３）ｎｍｏｌ／Ｌ,ｔ＝８．３１Ｐ＜０．００５;Ｃａ２＋－ＡＴＰ酶活性,染铅组（３２６．４２±４０．０６）ｎｍｏｌ（Ｐｉ）·ｍｇ－１·ｍｉｎ－１,对照组（２５３．０７±２５．４０）ｎｍｏｌ（Ｐｉ）·ｍｇ－１·ｍｉｎ－１,ｔ＝３．５４,Ｐ＜０．０１。结论：慢性染铅可使大鼠海马区神经细胞内Ｃａ２＋浓度升高,Ｃａ２＋－ＡＴＰ酶活性增强     

Intracellular smooth muscle [Ca2+] in acetylcholine and nitric oxide-mediated relaxation of human small arteries

In human resistance arteries the role of intracellular calcium during receptor agonist and nitric oxide (NO)-mediated vasorelaxation is almost unknown. We examined changes in smooth muscle calcium concentration ([Ca2+]i) caused by acetylcholine and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) in isolated human subcutaneous small arteries. In arteries constricted with 50 mM KCl, acetylcholine and SNAP induced relaxation without any change in [Ca2+]i, whereas in noradrenaline constricted vessels, both acetylcholine and to a lesser degree also SNAP-mediated relaxation were associated with a decrease in [Ca2+]i. Furthermore incubation with SNAP (1 microM) induced a rightward shift in the [Ca2+]i-force relationship. These results suggest that relaxation mediated by endothelium derived hyperpolarizing factors (EDHF) is associated with reduction in [Ca2+]i, whereas NO-mediated relaxation can take place without changes in [Ca2+]i. This finding seems to be, at least partly, due to NO-mediated desensitization of the contractile apparatus to calcium.     

Na+/H+ exchange inhibition with cardioplegia reduces cytosolic [Ca2+] and myocardial damage after cold ischemia

Cold cardioplegia protects against reperfusion damage. Blocking Na+/H+ exchange may be as protective as cardioplegia by improving the left ventricular pressure (LVP)-[Ca2+] relationship after cold ischemia. In guinea pig isolated hearts subjected to cold ischemia (4 h, 17 degrees C) and reperfusion, the cardioprotective effects of a Krebs-Ringer (KR) solution, a cardioplegia solution, a KR solution containing the Na+/H+ exchange inhibitor eniporide (1 microM), and a cardioplegia solution containing eniporide were compared. Treatments were given before and initially after cold ischemia. Systolic and diastolic [Ca2+] were calculated from indo-1 fluorescence transients recorded at the LV free wall. During ischemia, diastolic [Ca2+] increased in each group but more so in the KR group. Peak systolic and diastolic [Ca2+] on initial reperfusion were highest after KR and smallest after cardioplegia + eniporide. After reperfusion, systolic-diastolic LVP (% of baseline) and infarct size (%), respectively, were KR, 47 +/- 3%, 37 +/- 4%; cardioplegia, 71 +/- 5%*, 20 +/- 2.2%*; KR + eniporide, 73 +/- 5%*, 11 +/- 3%* dagger; and cardioplegia + eniporide 77 +/- 3%*, 10 +/- 1.4%* dagger (*P 相似文献   

Effect of capsazepine on [Ca2+]i in MDCK renal tubular cells

The current study explored whether capsazepine changed basal cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) levels in suspended Madin Darby canine kidney (MDCK) cells cells by using fura‐2 as a Ca²⁺‐selective fluorescent dye. At concentrations of 10–200 µM, capsazepine increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was partially reduced by 40% by removing extracellular Ca²⁺. Capsazepine induced Mn²⁺ quench of fura‐2 fluorescence, indirectly implicating Ca²⁺ entry. Capsazepine‐induced Ca²⁺ influx was unchanged by L‐type Ca²⁺ entry inhibitors and protein kinase C modulators [phorbol 12‐myristate 13‐acetate (PMA) and GF109203X]. In Ca²⁺‐free medium, 100 µM capsazepine‐induced Ca²⁺ release was substantially suppressed by pretreatment with thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor). Pretreatment with capsazepine nearly abolished thapsigargin‐induced Ca²⁺ release. Inhibition of phospholipase C with U73122 did not change capsazepine‐induced [Ca²⁺]_i rises. Collectively, in MDCK cells, capsazepine induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via non‐L‐type Ca²⁺ channels. Drug Dev Res 72: 323–329, 2011. © 2010 Wiley‐Liss, Inc.     

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.     

The Effect of Inhibitors of Na/H Exchange and Na/Ca Exchange on Airway Smooth Muscle Contractility

Summary: We have previously shown that amiloride, an inhibitor of several cell membrane sodium exchangers and channels including Na/H exchange and Na/Ca exchange, inhibits receptor-operated contraction of bovine airway smooth muscle. However, the precise mechanism of action of amiloride is unknown. To evaluate the mechanism whereby amiloride reduces airway smooth muscle contractility, we compared the effects of amiloride with 5-N-methyl isobutyl amiloride and 5-N,N-hexamethylene amiloride, selective inhibitors of Na/H exchange, and 5-N(N-4-chlorobenzyl)-2,4-dimethyl benzamil, a selective inhibitor of Na/Ca exchange on histamine-induced contraction of bovine trachea. Unlike amiloride, none of the amiloride analogues, 5-N-methyl isobutyl amiloride (10 μmol/l), 5-N,N-hexamethylene amiloride (10 μmol/l) nor 5-N(N-4-chlorobenzyl)-2,4-dimethyl benzamil (20 μmol/l), inhibited histamine-induced contraction. Our results do not support the hypothesis that Na/H exchange or Na/Ca exchange are involved in histamine-induced contraction of airway smooth muscle.     

Effect of APGW-amide on [Ca2+]i in rat pheochromocytoma PC12 cells

In order to determine whether Ala-Pro-Gly-Try-NH2 (APGW-amide) could affect mammalian excitable cells, we investigated the effect of APGW-amide in PC12 cells. APGW-amide caused a rapid [Ca2+]i elevation, which was completely prevented by elimination of extracellular Ca2+ with EGTA and inhibited by two L-type Ca2+ channel blockers. [Ca2+]i elevation was also blocked by a specific PKC inhibitor and prolonged pretreatment of cells with PMA. These results indicate that APGW-amide elevates [Ca2+]i in PC12 cells, possibly by Ca2+ influx via L-type Ca2+ channel activated by PKC.     

Effect of dronedarone on Na+, Ca2+ and HCN channels

Previous studies showed that amiodarone causes state-dependent inhibition of Na(+) channels thereby mediating an atrial-selective drug effect. The aim of the present study was to investigate the impact of the new antiarrhythmic compound dronedarone on Na(+), Ca(2+) and hyperpolarization-activated cyclic nucleotide-gated ion channels. Monophasic action potentials (MAP) and effective refractory period (ERP) were studied in arterially perfused left atria and ventricular wedge preparations of the pig. Fast Na(+) and Ca(2+) currents in isolated guinea pig ventricular myocytes as well as human HCN4 channels expressed in Chinese hamster ovary (CHO) cells were investigated with the patch-clamp technique. In left atrial epicardial tissue, dronedarone (3?μM) had no effect on the MAP duration, but the drug caused a significant prolongation of the ERP from 145?±?9 to 184?±?17?ms (n?=?6; p?相似文献   

The Effect of Ageing and in vitro Exposure to Xylene and KC1 on [Ca2+]i in Synaptosomes from Rats Exposed Prenatally to Xylene

Abstract: Female rats (Mol: WIST) were exposed prenatally to 500 p.p.m. of technical xylene on days 7-20. At the age of fourteen months the rats were sacrified and the synaptosomal fraction prepared for in vitro studies. The cytosolic calcium concentration was measured using the FURA-2 technique. The cytosolic calcium was increased in synaptosomes from old rats compared to those from rats at the age of three months, but no effect of prenatal exposure was seen. When synaptosomes were incubated with xylene, potassium or both, the cytosolic calcium concentration was changed identically in all groups of rats. When synaptosomes were incubated simultaneously to xylene and potassium a dramatical leakage of FURA-2 was observed. The mechanisms behind the membrane leakage are discussed.     

慢性铝染毒对大鼠海马细胞内[Ca2+]i和CaMKⅡ蛋白表达的影响

目的通过进一步研究慢性铝暴露引起大鼠海马细胞内[Ca2 ]i和钙调蛋白激酶激酶Ⅱ(CaMⅡ)蛋白表达的变化,来探讨铝损害学习记忆的作用机制。方法选择断乳后Wistar大鼠,以含有不同浓度AlCl3的水进行饲养。3个月后,取海马测定细胞内[Ca2 ]i,用Western blotting方法检测CaMKⅡ的蛋白表达。结果(1)各染铝组的[Ca2 ]i与对照组比较明显降低,差异有统计学意义(P<0.01),但各染铝组间差异无统计学意义;(2)各染铝组CaMKⅡ蛋白表达与对照组比较也显著降低,并成剂量关系,差异有统计学意义(P<0.01)。结论铝能够降低大鼠海马细胞内的[Ca2 ]i浓度并造成CaMKⅡ的蛋白表达降低,从而损伤学习记忆功能。     

Effect of protriptyline on [Ca2+]i and viability in MG63 human osteosarcoma cells

Tricyclic antidepressants (TCA) have been clinically prescribed in the auxiliary treatment of cancer patients. Although protriptyline, a type of TCA, was used primarily in the clinical treatment of mood disorders in cancer patients, the effect of protriptyline on physiology in human osteosarcoma is unknown. This study examined the effect of protriptyline on cytosolic free Ca^2+?concentrations ([Ca²⁺]_i) and viability in MG63 human osteosarcoma cells. Protriptyline between 50 and 250?μM evoked [Ca²⁺]_i rises concentration-dependently. Protriptyline induced influx of Mn²⁺, indirectly implicating Ca^2+?influx. Protriptyline-evoked Ca^2+?entry was inhibited by nifedipine by 20% but was not altered by econazole, SKF96365, GF109203X, and phorbol-12-myristate-13-acetate (PMA). In Ca²⁺-free medium, treatment with protriptyline inhibited the endoplasmic reticulum Ca^2+?pump inhibitor thapsigargin-evoked [Ca²⁺]_i rises. Conversely, treatment with thapsigargin inhibited 45% of protriptyline-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 failed to alter protriptyline-evoked [Ca²⁺]_i rises. Protriptyline at 50–250?μM decreased cell viability, which was not reversed by pretreatment with the Ca^2+?chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Collectively, our data suggest that in MG63 cells, protriptyline induced [Ca²⁺]_i rises by evoking Ca^2+?release from the endoplasmic reticulum and other stores in a PLC-independent manner, and Ca^2+?entry via a nifedipine-sensitive Ca^2+?pathway. Protriptyline also caused Ca²⁺-independent cell death.     

Maturation attenuates the effects of cGMP on contraction, [Ca2+]i and Ca2+ sensitivity in ovine basilar arteries.

The present study explores the hypothesis that age-related variations in cerebrovascular responses to vasodilators reflect corresponding age-dependent differences in the mechanisms coupling changes in cytosolic cGMP to vasorelaxation. The experiments focused on cGMP's ability to decrease either [Ca2+]i or myofilament Ca2+ sensitivity, because both effects can contribute to cGMP-induced vasodilation. Use of the cGMP analog 8-pCPT-cGMP minimized problems associated with limited cell permeation or cGMP hydrolysis. In fetal basilars contracted with 10 microM serotonin, the EC30 for 8-pCPT-cGMP-induced relaxation was 6 microM. In fura-2 loaded fetal basilars, pretreatment with 6 microM 8-pCPT-cGMP significantly depressed the sensitivity of [Ca2+]i to 5HT, and also myofilament sensitivity to calcium, but only in fetal arteries. In fetal basilar arteries contracted with 120 mM potassium, the EC30 for 8-pCPT-cGMP-induced relaxation was 25 microM. In fura-2 loaded ovine arteries, pretreatment with 25 microM 8-pCPT-cGMP had no effect on the ability of graded concentrations of potassium to elevate [Ca2+]i but reduced potassium's ability to induce contraction and attenuated myofilament calcium sensitivity; these latter effects were significant only in fetal arteries. In alpha-toxin permeabilized preparations, 25 microM 8-pCPT-cGMP significantly depressed both basal- and agonist-stimulated myofilament calcium sensitivity, only in fetal but not in adult basilars. Together, these results demonstrate that: (1) sensitivity to cGMP is greater in fetal than adult sheep arteries independent of method of contraction; (2) cGMP can reduce [Ca2+]i but only in agonist-contracted and not in potassium-contracted arteries; (3) and cGMP attenuates myofilament calcium sensitivity regardless of method of contraction. Overall, the data demonstrate that variations in the ability of cGMP to produce vasodilatation reflect age-, artery-, and agonist-dependent differences in the combination of mechanisms mediating responses to cGMP.     

Modulation of carbachol-induced [Ca2+]i oscillations by Ca2+ influx in single intestinal smooth muscle cells.

1. Oscillations of cytosolic Ca2+ concentration ([Ca2+]i) evoked by carbachol (CCh; 2 microM), a muscarinic agonist, were detected as oscillatory changes of muscarinic receptor-coupled cationic current (Icat) in guinea-pig ileal smooth muscle cells by the whole cell patch-clamp technique. 2. Reduction of extracellular Ca2+ from 2 mM to 0.2 or 0.05 mM, during CCh-induced Icat oscillations, caused them to disappear or to decrease markedly in frequency. A return to 2 mM Ca2+ concentration restored the initial Icat oscillations. 3. Application of nifedipine (1-3 microM) or D600 (2-5 microM) to block the voltage-gated Ca2+ channel (VGCC) decreased the frequency of the ongoing Icat oscillations in the cells held at -20 mV, but it was without effect in cells held at -60 mV. 4. Displacement of the holding potential of -20 mV to -60 mV to deactivate VGCC produced a decrease, an increase or no noticeable change in the frequency of the Icat oscillations in different cells. Displacement to 20 mV to inactivate VGCC invariably produced a decrease in the frequency. In nifedipine-treated cells, the Icat oscillations varied in frequency voltage-dependently in a reverse and linear way within the range -80 to 40 mV. 5. Application of thapsigargin (1 or 2 microM), an inhibitor of Ca(2+)-ATPase in the membrane of internal Ca2+ stores, caused CCh-induced Icat oscillations to disappear with a progressing phase during which their amplitude, but not frequency, declined. 6. The results suggest that membrane Ca2+ entry has a crucial role to play in regulation of the frequency of CCh-induced [Ca2+]i oscillations in addition to persistence of their generation, and that the effect is brought about by a potential mechanism independent of Ca2+ store replenishment. They also provide evidence that two types of Ca2+ permeant channels, VGCC and an as yet unidentified channel, are involved in the Ca2+ entry responsible for modulation of [Ca2+]i oscillations.     

Influence of authentic nitric oxide on basal cytosolic [Ca2+] and Ca2+ release from internal stores in human platelets.

1. Nitric oxide (NO) donors inhibit platelet function and Ca2+ mobilization evoked by different agonists. This led us to investigate the direct effects of authentic NO on basal cytosolic Ca2+ concentration ([Ca2+]i) and on Ca2+ mobilization induced by thrombin or by two inhibitors of intracellular Ca(2+)-ATPases, thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone (t-BuBHQ). 2. Cytosolic Ca2+ concentration was evaluated with Fura-2, in the absence of Ca2+ influx. Addition of 5 microM NO increased by 48% the basal cytosolic [Ca2+] of resting human platelets whereas a lower concentration (0.1 microM) did not induce significant modifications. This NO-induced Ca2+ increase was inversely correlated with the basal level of cytosolic [Ca2+]. 3. NO pretreatment for 30 to 120 s decreased by 42 to 57% the transient [Ca2+]i peak evoked by 0.10 u ml-1 thrombin and strongly attenuated the initial rate of [Ca2+]i rise induced by 1 microM thapsigargin or by 20 microM t-BuBHQ. The two components of the thapsigargin response, the Ca2+ release due to inhibition of Ca2+ pumps and the thromboxane A2-dependent self-amplification mechanism, were inhibited by NO. The observation that a subsequent stimulation was still capable of eliciting Ca2+ release suggests the presence of NO-insensitive Ca2+ stores. 4. These findings indicate that nitric oxide can modulate basal cytosolic [Ca2+] in unstimulated human platelets and decrease the Ca2+ mobilization from NO-sensitive internal stores evoked by stimulation of receptors or by Ca(2+)-ATPase inhibitors. This underlines the important role of nitric oxide in the modulation of platelet Ca2+ handling.