BAY 41-2272, a potent activator of soluble guanylyl cyclase, stimulates calcium elevation and calcium-activated potassium current in pituitary GH cells

The effects of BAY 41-2272, a nitric oxide-independent activator of soluble guanylyl cyclase, on Ca2+ signalling and ion currents were investigated in pituitary GH3 cells. Intracellular Ca2+ concentrations ([Ca2+]i) in these cells were increased by BAY 41-2272. Removing extracellular Ca2+ abolished the BAY 41-2272-induced increase in [Ca2+]i. After [Ca2+]i was elevated by BAY 41-2272 (300 nmol/L), subsequent application of 1-benzyl-3-(5'-hydroxymethyl-2'-furyl) indazole (YC-1; 1 micromol/L) did not increase [Ca2+]i further. In whole-cell recordings, BAY 41-2272 reversibly stimulated Ca2+-activated K+ current (I(K(Ca))) with an EC50 of 225 +/- 8 nmol/L. At 3 micromol/L, BAY 41-2272 slightly and significantly decreased L-type Ca2+ current.In the cell-attached configuration, BAY 41-2272 (300 nmol/L) enhanced the activity of large-conductance Ca2+-activated K+ (BK(Ca)) channels. After BK(Ca) channel activity was stimulated by spermine NONOate (30 micromol/L) or YC-1 (10 micromol/L) in cell-attached patches, subsequent application of BAY 41-2272 (300 nmol/L) further increased the channel open probability. In the inside-out configuration, BAY 41-2272 applied to the intracellular surface of excised patches enhanced BK(Ca) channel activity. Unlike 1 micromol/L paxilline, 1H-[1,2,4]oxadiazolol-[4,3a] quinoxalin-1-one (ODQ; 10 micromol/L) or heme (10 micromol/L) had no effect on BAY 41-2272-stimulated channel activity. BAY 41-2272 caused no shift in the activation curve of BK(Ca) channels; however, it did increase the Ca2+ sensitivity of these channels. At 300 nmol/L, BAY 41-2272 reduced the firing rate of spontaneous action potentials stimulated by thyrotropin-releasing hormone (10 micromol/L). The BK(Ca) channel activity was also enhanced by 300 nmol/L BAY 41-2272 in neuroblastoma IMR-32 cells. Therefore, the BAY 41-2272-induced increase in [Ca2+]i is primarily explained by an increase in Ca2+ influx. The BAY 41-2272-mediated simulation of IK(Ca) may result from direct activation of BKCa channels and indirectly as a result of elevated [Ca2+]i.     

Fendiline mobilizes intracellular Ca2+ in Chang liver cells

1. The effects of the antianginal drug fendiline (N-[3,3-diphenylpropyl]-alpha-methyl-benzylamine) on intracellular free Ca2+ levels ([Ca2+](i)) in Chang liver cells were evaluated using fura-2 as a fluorescent Ca2+ indicator. 2. Fendiline (1-100 micromol/L) increased [Ca2+](i) in a concentration-dependent manner, with an EC50 of 25 micromol/L. 3. The [Ca2+](i) response was composed of an initial rise and a slow decay to a sustained phase. Removal of extracellular Ca2+ partly reduced the [Ca2+](i) signals. 4. Fendiline (10 micromol/L)-induced release of intracellular Ca2+ was reduced by 65% following pretreatment with 1 micromol/L thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete Ca2+ stored in the endoplasmic reticulum. 5. After pretreatment with 10 micromol/L fendiline in Ca2+-free medium for several minutes, addition of 3 mmol/L Ca2+ induced an increase in [Ca2+](i) of a magnitude four-fold greater than control. This increase in [Ca2+](i) was not reduced by 10 micromol/L SKF96365, econazole, nifedipine or verapamil. 6. Fendiline (10 micromol/L)-induced release of intracellular Ca2+ was not altered by inhibition of phospholipase C with 2 micromol/L 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino) hexyl)-1H-pyrrole-2,5-dione (U73122). 7. The results of the present study show that fendiline induces an increase in [Ca2+](i) in Chang liver cells by releasing stored Ca2+ in an inositol 1,4,5-trisphosphate-independent manner and by causing extracellular Ca2+ influx.     

Effect of caffeine on response of rabbit isolated corpus cavernosum to high K+ solution, noradrenaline and transmural electrical stimulation

1. Caffeine has wide-ranging activities on smooth muscles, including contractile and relaxant effects. The aim of the present study was to examine the activity of caffeine on rabbit corpus cavernosum (RCC). 2. The effects of caffeine (0.5-4.0 mmol/L) on the response of RCC to high K+ solution, noradrenaline (NA) and transmural electrical stimulation (EFS) were studied in a tissue bath system. 3. Caffeine did not contract the RCC. However, 0.5-4.0 mmol/L caffeine caused concentration-dependent relaxation of tension development in high-K+ (120 mmol/L) solution in contrast with the solvent control. At 4.0 mmol/L caffeine, high-K+ solution-induced tone of the RCC was reduced by 73.4 +/- 7.3%. Caffeine (0.5-4.0 mmol/L) also concentration-dependently relaxed NA (12.5 micro mol/L)-induced tonic contraction of the RCC. At 4.0 mmol/L caffeine, NA-induced tone of the RCC was reduced by 41.1 +/- 7.0%. Incubation of RCC in 2.0 mmol/L caffeine for 30 min prior to EFS (1-40 Hz) caused a marked rightward shift in the frequency-response curve. 4. The results of the present study suggest that caffeine exhibits relaxant activity on rabbit cavernosal smooth muscle and the mechanism of this activity possibly involves inhibition of Ca2+ signalling.     

Effects of econazole on Ca2+ levels in and the growth of human prostate cancer PC3 cells

1. Econazole is used clinically as an antifungal drug with many different in vitro effects. However, the effects of econazole on prostate cancer cells are unknown. The effects of econazole on intracellular Ca2+ concentrations ([Ca2+]i) in and the proliferation of human PC3 prostate cancer cells was explored in the present study using fura-2 and tetrazolium as fluorescent dyes. 2. At a concentration of 0.1 micromol/L, econazole started to increase [Ca2+]i in a concentration-dependent manner. The econazole-induced increase in [Ca2+]i was reduced by 48% by removal of extracellular Ca2+, suggesting that the econazole-induced increase in [Ca2+]i was composed of extracellular Ca2+ influx and intracellular Ca2+. 3. This econazole-induced Ca2+ influx was via an L-type Ca2+ channel-like pathway. In Ca2+-free medium, 1 micromol/L thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic increase in [Ca2+]i, after which the effect of econazole to increase [Ca2+]i was substantially inhibited. Conversely, pretreatment with 5 micromol/L econazole to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+. 4. The phospholipase C (PLC) inhibitor U73122 (2 micromol/L) abolished the increase in [Ca2+]i induced by 10 micromol/L ATP (a Ca2+ mobilizer that needs inositol 1,4,5-trisphosphate). 5. Overnight incubation with 1-30 micromol/L econazole inhibited proliferation of PC3 cells in a concentration-dependent manner. 6. These findings suggest that, in PC3 cells, econazole increases [Ca2+]i by stimulating Ca2+ influx into cells and Ca2+ release from the endoplasmic reticulum via a PLC-independent mechanism. Econazole is cytotoxic at submicromolar concentrations.     

Spontaneous oscillatory contractions in aortas of rats with arterial pressure lability caused by sinoaortic denervation

1. The spontaneous variation of blood pressure is defined as arterial pressure lability. Sinoaortic denervation (SAD) is characterized by arterial pressure lability without sustained hypertension. 2. The phenomenon of spontaneous oscillatory contractions (SOCs) occurs more frequently in the vascular beds of hypertensive animals. In large arteries, such as the aorta, SOCs occur only occasionally or they can be initiated by application of chemical stimuli. 3. In the present study, we investigated whether the arterial pressure lability evoked by SAD could be related to the emergence of SOCs in the aorta of rats submitted to SAD compared with sham-operated rats (SO). Three days after surgery (SAD or SO), aortic rings were placed in an organ chamber and the incidence (percentage of rats presenting SOCs), frequency (number of SOCs in 10 min) and amplitude (mN) of SOCs were measured. The participation of external Ca(2+) and K(+) channels in the maintenance of SOCs was also verified. 4. The incidence and frequency of SOCs were higher in endothelium-denuded aortas from SAD rats (82% and 38 +/- 4 SOCs/10 min, respectively) than in aortas from SO rats (40% and 14 +/- 2 SOCs/10 min, respectively). In aortas from SAD rats, verapamil (0.2 micromol/L), pinacidil (0.3 micromol/L) and tetraethylammonium (TEA; 5 mmol/L) totally inhibited SOCs, whereas increasing the CaCl(2) concentration to 2.0 and 2.5 mmol/L increased the frequency of SOCs. Interestingly, increasing the concentration of CaCl(2) to 3.5 mmol/L inhibited these contractions in aortas from SAD rats. 5. These results show that although SAD rats did not become hypertensive, their aortas were capable of initiating SOCs without the application of any chemical stimuli. The SOCs seem to be dependent on Ca(2+) influx sensitive to verapamil and also involve K(+) channels sensitive to pinacidil and TEA.     

POTENTIATION BY ALDOSTERONE OF VASOPRESSIN-INDUCED CALCIUM KINETICS IN VASCULAR SMOOTH MUSCLE CELLS

1. The present study was undertaken to examine the effect of aldosterone on arginine vasopressin (AVP)-induced Ca2+ kinetics in cultured rat vascular smooth muscle cells (VSMC). The pre-incubation of cells with 1 x 10(-6) mol/L aldosterone for 24 h did not affect the basal cytosolic free Ca2+ [( Ca2+]i) but enhanced the AVP-induced mobilization of [Ca2+]i (1 x 10(-8) mol/L AVP; 287 vs 401, 1 x 10(-6) mol/L AVP; 430 vs 714 nmol/L). 2. The pre-incubation of cells with 1 x 10(-7) mol/L aldosterone for 24 h did not show this enhancing effect on the AVP-induced mobilization of [Ca2+]i. Without the preincubation, aldosterone did not change the basal [Ca2+]i or the AVP-induced mobilization of [Ca2+]i. This enhancement was still observed in the Ca2+-free solution containing 0.1 mmol/L EGTA (1 x 10(-8) mol/L AVP; 169 vs 341 nmol/L). 3. The enhancement by aldosterone of the AVP-mobilized [Ca2+]i was completely blocked by the simultaneous administration of 1 x 10(-4) mol/L spironolactone (1 x 10(-8) mol/L AVP; 258 vs 265 nmol/L). The treatment with aldosterone also stimulated the AVP-produced [45Ca2+] efflux during a 3 min period (1 x 10(-8) mol/L AVP; 32 vs 49, 1 x 10(-6) mol/L AVP; 50 vs 58% released from the resting intracellular [45Ca2+]-contents). 4. The present results indicate that aldosterone enhances the vascular action of AVP mediated through the stimulation of Ca2+ kinetics which may be dependent on the changes in the cellular signal transduction systems.     

Effect of the antidepressant maprotiline on Ca2+ movement and proliferation in human prostate cancer cells

1. The effect of maprotiline, an antidepressant, on human prostate cells is unclear. In the present study, the effect of maprotiline on [Ca2+]i and growth in PC3 human prostate cancer cells was measured using the fluorescent dyes fura-2 and tetrazolium, respectively. 2. Maprotiline caused a rapid, concentration-dependent increase in [Ca2+]i (EC50 = 200 micromol/L). The maprotiline-induced [Ca2+]i increase was reduced by removal of extracellular Ca2+ or pretreatment with nicardipine. 3. The maprotiline-induced Mn2+ influx-associated fura-2 fluorescence quench directly suggests that maprotiline caused Ca2+ influx. 4. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca2+]i increase, after which the effects of maprotiline of increasing [Ca2+]i were abolished. In addition, pretreatment with maprotiline reduced a major portion of the thapsigargin-induced increase in [Ca2+]i. 5. U73122, an inhibitor of phospholipase C, abolished the ATP (but not maprotiline)-induced increase in [Ca2+]i. 6. Overnight incubation with 1-10 micromol/L maprotiline did not alter cell proliferation, although incubation with 30-50 micromol/L maprotiline decreased cell proliferation. 7, These findings suggest that maprotiline rapidly increases [Ca2+]i in human prostate cancer cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release and that it may modulate cell proliferation in a concentration-dependent manner.     

Extracellular Ca2+ is obligatory for ouabain-induced potentiation of cardiac basal energy expenditure

1. The method of action of cardiac glycosides is commonly explained by the 'pump-inhibition hypothesis': inhibition of the Na+/K+-ATPase allows [Na+]i to rise, eventually reversing Na+/Ca2+ exchange. The resulting influx of Ca2+o increases [Ca2+]i, thereby activating intracellular Ca2+-dependent ATPases and, hence, energy demand. This sequence has been presumed to occur during diastole as well as systole. However, it has been reported that dihydro-ouabain-induced potentiation of heat production by quiescent ventricular trabeculae persists in the absence of Ca2+o. This implies that the pump-inhibition hypothesis is inapplicable during diastole. 2. We tested this implication by: (i). measuring the rate of oxygen consumption (Vo2) of arrested guinea-pig whole-hearts; (ii). measuring[Ca2+]i in quiescent ventricular trabeculae; and (iii). mathematical modelling using software (Oxsoft Heart, Oxford Software, Oxford, UK) based on DiFrancesco-Noble formalism. 3. Upon induction of arrest, whole heart Vo2 fell to one-quarter of its 'beating' value. Subsequent perfusion with ouabain (20 micromol/L), in the presence of Ca2+o, increased Vo2 fourfold. This increase was prevented by withholding Ca2+o. Comparable results were obtained in quiescent trabeculae: ouabain increased [Ca2+]i only if Ca2+o was present. Mathematical modelling readily simulated these experimental results. 4. We conclude that influx of Ca2+o is mandatory for potentiation of cardiac basal metabolism by cardiac glycosides.     

K(Ca) channel-opening activity of Ginkgo Biloba extracts and ginsenosides in cultured endothelial cells

1. Extracts of Ginkgo biloba (EGb) and ginsenosides (GS) have been reported to induce vasorelaxation. In the present study, the role of K+ channels in the action of EGb and GS to activate nitric oxide synthase (NOS) activity was investigated in cultured endothelial cells. 2. Nitric oxide synthase activity of cultured endothelial cells detected by the reduced nicotinamide adenine dinucleotide phosphate (NADPH) histochemistry method was significantly increased after treatment with 20 microg/mL EGb or 40 microg/mL GS plus 10 mmol/L L-arginine. The effect was completely abolished by the addition of 0.5 micromol/L Nomega-nitro-L-arginine, an inhibitor of NOS, to the incubation medium and partially inhibited by 10 micromol/L tetraethylammonium (TEA), an inhibitor of Ca2+-activated K+ (KCa) channels. 3. Application of EGb to the intracellular surface of excised inside-out patches activated K+ channels in a concentration-dependent manner in the concentration range 1-100 microg/mL. Channel activity was also activated by application of GS at concentrations ranging from 1 to 300 microg/mL. The modulation of channel activity was inhibited by 0.5 mmol/L TEA but not by 0.5 mmol/L glibenclamide, an inhibitor of ATP-sensitive K+ channels. 4. Thus, in cultured endothelial cells, the increase in NOS activity induced by EGb or GS depends on the activity of KCa channels. These compounds may regulate nitric oxide release by changing the cell membrane potential in vascular endothelial cells.     

CAFFEINE-AND POTASSIUM-INDUCED CONTRACTURES OF MOUSE ISOLATED SOLEUS MUSCLE: EFFECTS OF VERAPAMIL, MANGANESE, EGTA AND CALCIUM WITHDRAWAL

1. The effects of verapamil, manganese, EGTA and Ca2+-withdrawal on caffeine and potassium-induced contractures of the mouse isolated soleus muscle have been studied. All four treatments profoundly inhibited, in concentration-dependent manner, both K+- and caffeine-induced contractures, and recovery from these treatments was slight. 2. Caffeine (5.14-51.4 mmol/l), elicited biphasic contractures, characterized by an initial phasic, and subsequent tonic contractures. K+-induced contractures were monophasic except at the higher concentrations (53.4-214 mmol/l), when biphasic responses were also recorded. The biphasic K+ contracture had a time course similar to the caffeine-induced response. 3. Both phases of the caffeine and K+ responses were abolished by the introduction of CA2+-free Krebs'-Henseleit solution (KHS). Increasing [Ca2+]0 from 1 to 5 mmol/l, markedly inhibited the amplitude of caffeine and K+ contractures. 4. In high [K+]0 KHS, peak contractures to caffeine were reduced to about 50% of those in normal KHS. In the presence of 15.4 mmol/l caffeine, the responses to lower concentrations of K+ (13.4-26.8 mmol/l) increased, while responses evoked by the higher K+ concentrations were depressed compared with controls in normal KHS. 5. Pancuronium or D-tubocurarine severely inhibited caffeine-induced contractures, but only slightly inhibited K+-induced responses. 6. It is suggested that both caffeine- and K+-induced contractures of the mouse soleus are dependent upon the presence of [Ca2+]0, and that these contractures possibly occurred as a result of extracellular Ca2+-influx.     

小檗碱对培养大鼠神经细胞内游离Ca^2＋的影响

以Ｆｕｒａ２／ＡＭ为细胞内钙离子的荧光指示剂，用ＡＲＣＭＭＩＣ阳离子测定系统，直接测定了体外培养的新生大鼠神经细胞内游离钙（［Ｃａ２＋］ｉ）值，并观察了小檗碱（Ｂｅｒ）的影响。结果表明，Ｂｅｒ对神经细胞静息［Ｃａ２＋］ｉ无明显影响，Ｂｅｒ１～１００μｍｏｌ·Ｌ－１能剂量依赖地抑制去甲肾上腺素和Ｈ２Ｏ２引起的［Ｃａ２＋］ｉ升高，其ＩＣ５０分别为３９．９和１７．９μｍｏｌ·Ｌ－１。高剂量Ｂｅｒ（１０～１００μｍｏｌ·Ｌ－１）能抑制高Ｋ＋引起的［Ｃａ２＋］ｉ升高。姐果提示，Ｂｅｒ对去甲肾上腺素，高Ｋ＋及Ｈ２Ｏ２引起的［Ｃａ２＋］ｉ升高的抑制作用可能是其抗脑缺血作用机制之一。     

Role of CA(2+)-activated K+ channels in the regulation of basilar arterial tone in spontaneously hypertensive rats

1. Ionic channels appear to play an important role in contractile responses of the cerebral arteries and, thereby, contribute to the regulation of cerebral circulation. In the present study, we investigated the role of large-conductance Ca(2+)-activated K+ (BK(Ca)) channels in the regulation of cerebral arterial tone during chronic hypertension. 2. Ring segments of the basilar artery from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were placed in bath chambers and the isometric tension of each ring was measured. 3. Application of inhibitors of BK(Ca) channels, namely tetraethylammonium (TEA; > or = 0.1 mmol/L) and charybdotoxin (CTX; > or = 0.1 nmol/L), produced spontaneous contraction with rhythmic oscillation in the basilar artery from SHR. 4. The oscillatory contraction was not induced by 5-hydroxytryptamine (0.01-10 micromol/L) or depolarization by external high K+ (20-60 mmol/L). 5. The rhythmic contraction was completely abolished by either the removal of external Ca(2+) or the application of nicardipine (10 nmol/L). 6. The oscillation was not affected by the substitution of external Cl(-) by various equimolar anions (i.e. acetate, benezenesulphonate, bromide and isethianate). 7. The amplitude of the oscillation was dose-dependently increased by the vasodilators forskolin and sodium nitroprusside, as well as by stimulation of the endothelium with histamine and acetylcholine, whereas the frequency was decreased. 8. In contrast, the oscillation was eliminated by depletion of Ca(2+) stores by caffeine. Neither TEA (10 mmol/L) nor CTX (10 nmol/L) produced any significant contraction of the basilar artery in WKY rats. 9. These results suggest that BK(Ca) channels may play an important role in regulating the resting tone of the cerebral artery in SHR.     

蚓激酶的心肌保护作用及机制

目的研究蚓激酶对心肌缺血的保护作用，并进一步探讨其可能机制。方法采用结扎大鼠左冠状动脉前降支制备急性心肌缺血模型，观察蚓激酶对心肌缺血的保护作用；应用全细胞膜片钳和激光扫描共聚焦技术，研究蚓激酶对L-型钙电流(I_Ca-L)和细胞内游离钙离子浓度的影响。结果蚓激酶80，40和20 mg·kg^-1剂量组均可缩小心肌梗死面积。膜片钳研究结果表明，当刺激电压为+10 mV时，10和50 μmol·L^-1蚓激酶使I_Ca-L降低共聚焦结果显示，在静息状态下，10 μmol·L^-1蚓激酶对［Ca²⁺］_i无明显影响；但10 μmol·L^-1蚓激酶对60 mmol·L^-1 KCl诱导的［Ca²⁺］_i升高却有明显抑制作用，并且在整个实验过程中(240 s)并未出现明显的峰值。结论蚓激酶对大鼠心肌缺血具有保护作用，其机制可能与抑制I_Ca-L及下调［Ca²⁺］_i有关。     

粉防己碱对培养乳牛基底动脉平滑肌细胞内游离钙浓度的影响

目的：研究粉防己碱对培养乳牛基底动脉平滑肌细胞游离钙浓度（[Ca^2 ]i)的影响。方法：利用AR－CM－MIC阳离子测定系统,采用Fura 2-AM为指示剂,测量单个细胞内[Ca^2 ]i。结果：粉防己碱10－100μmol/L对培养乳牛基底动脉平滑肌细胞静息[Ca^2 ]i无明显影响。在细胞外钙为1．3mmol/L,粉防己碱可浓度依赖性地抑制KC1引起[Ca^2 ]i的升高。咖啡因10mmol/L可诱导一次[Ca^2 ]i瞬间快速升高,随后自发回复到静息水平,粉防己碱10和30μmol/L对咖啡因诱导的[Ca^2 ]i瞬间升高没有作用,但高浓度（100μmol/L）粉防己碱抑制了[Ca^2 ]i瞬间升高。在细胞外钙为1.3mmol/L,苯肾上腺素10μmol/L可引起双相[Ca^2 ]i变化,包括快速升高相和持续升高相。在细胞外钙为零,苯肾上腺素仅引起[Ca^2 ]i的快速升高相。粉防己碱可浓度依赖性地抑制苯肾上腺素引起[Ca^2 ]i快速升高相。结论：在培养乳牛基底动脉平滑肌细胞,粉防己碱可能通过影响电压依赖性和苯肾上腺素受体介导的钙通道而抑制钙内流。高浓度粉防己碱也可能影响肌浆网钙释放或钙摄取。     

EFFECTS OF BKCa CHANNELS ON THE REDUCTION OF CYTOSOLIC Ca2+ IN cGMP-INDUCED RELAXATION OF GUINEAPIG TRACHEA

1. In order to examine the mechanisms of cGMP-induced relaxation in airway smooth muscle, the effects of atrial natriuretic peptide (ANP) and 8-brom cGMP on muscle tone were studied by measuring isometric tension, while the effects on cytosolic Ca²⁺ concentrations were studied by measuring the spectra of fura-2 loaded in guinea-pig tracheal strips. 2. Atrial natriuretic peptide and 8-brom cGMP caused a concentration-dependent inhibition of spontaneous tone in the guinea-pig trachea. The relaxant effects of these agents on spontaneous tone were markedly suppressed in the presence of iberiotoxin (IbTX), a selective inhibitor of large-conductance Ca²⁺-activated K⁺ (BKca) channels. Iberiotoxin (30 nmol/L) markedly affected the maximal effect induced by ANP and 8-brom cGMP and augmented EC₇₀ values for ANP and EC₅₀values for 8-brom cGMP approximately 27- and 17-fold, respectively. The inhibitory effects of IbTX on relaxation induced by these agents were diminished in the presence of 1 μmol/L nifedipine, an antagonist of voltage-operated Ca²⁺channels (VOCC). 3. The inhibitory action of ANP and 8-brom cGMP on spontaneous tone was not affected by the presence of 10 μmol/L glibenclamide, an inhibitor of ATP-sensitive K⁺ channels, and 100 nmol/L apamin, an inhibitor of small-conductance Ca²+-activated K⁺ channels. When these agents were applied to tissues precontracted by high (40mmol/L) K⁺, the relaxant effects of these agents markedly diminished. 4. The extracellular Ca²⁺-dependent contraction was inhibited in the presence of 0.3 μmoI/L ANP or 0.1 mmol/L 8-brom cGMP. Concentration—response curves to extracellular Ca²⁺ (0.03—2.4 mmol/L) were markedly diminished by exposure to these agents. The maximal effect induced by extracellular Ca²⁺ was affected by these agents. 5. Atrial natriuretic peptide caused an inhibition of spontaneous tone accompanied by a reduction in the intracellular Ca²⁺ concentration. In the presence of IbTX, the elimination of both muscle tone and cytosolic Ca²⁺ by ANP was suppressed. 6. We conclude that ANP and 8-brom cGMP activate BKca channels and that the inhibition of Ca²⁺ influx through VOCC, mediated by BKca channel activation, may be involved in cGMP-dependent bronchodilation.     

Regulation of K+ channels underlying the slow afterhyperpolarization in enteric afterhyperpolarization-generating myenteric neurons: role of calcium and phosphorylation

1. Myenteric afterhyperpolarization-generating myenteric (AH) neurons serve as intrinsic primary afferent neurons of the enteric nervous system and generate prolonged or slow afterhyperpolarizing potentials (slow AHP). The slow AHP is generated by an increase in a Ca2+-activated K+ conductance (gK-Ca) and is inhibited by enteric neurotransmitters leading to increased excitability. 2. Using cell-attached patch-clamp recordings from AH neurons, we have shown that K+ channels with an intermediate unitary conductance (IK channels) open following action potential firing. 3. In excised patches from AH neurons, we have identified an IK-like channel that can be activated by submicromolar levels of cytoplasmic Ca2+ and is not voltage dependent. 4. Application of the catalytic subunit of cAMP-dependent protein kinase to the cytoplasmic surface of inside-out patches inhibits the opening of IK-like channels previously activated by Ca2+. 5. The IK-like channels are resistant to external tetraethylammonium (5 mmol/L) and apamin (0.3-1 micro mol/L), but are inhibited by clotrimazole (10 micro mol/L). 6. Our present data support the idea that an increase in the open probability of IK-like channels in AH neurons following an increase in cytoplasmic [Ca2+] is responsible for the slow AHP and their opening is modulated by kinases.     

Endothelin-induced myoplasmic Ca2+ responses and tyrosine phosphorylation in coronary smooth muscle

This study investigated the role of tyrosine phosphorylation and source of Ca2+ in prolonged endothelin-1 (ET-1)-induced potentiation of myoplasmic free Ca2+ ([Ca2+]m) responses to depolarization in coronary smooth muscle cells. Fura-2 microfluorometry showed typical increases in [Ca2+]m in response to 80 mM K+ (80K) and 0.01 microM endothelin. After washout of ET-1 80K-induced [Ca2+]m increases were augmented (potentiated) 31%. Time to peak [Ca2+]m response to 80K was less after ET-1 exposure than before. ET-1 potentiation of 80K-induced [Ca2+]m responses by decreased sarcoplasmic reticulum (SR) buffering of [Ca2+]m or Ca2+-induced Ca2+ release was ruled out by lack of potentiation by 5 mM caffeine and 1 microM thapsigargin. Diltiazem abolished potentiation, providing evidence for Ca2+ influx through voltage-gated Ca2+ channels (VGCC). Genistein (30 microM) and methyl 2,5-dihydroxycinnamate (1 microM, MDHC) abolished potentiation of Ca2+ influx. Single cell phosphotyrosine measured directly by immunofluorescence was increased 95% in cells treated with ET-1 compared to control, genistein, and MDHC treated cells. ET-1 increased tyrosine phosphorylation of an 80-85 kDa protein, but not the 240 kDa alpha1C subunit of the VGCC. Tyrosine phosphorylation of proteins other than VGCC is necessary for prolonged potentiation by ET-1 of depolarization-induced Ca2+ influx.     

小檗胺对高钾,去甲肾上腺素及咖啡因引起大鼠心肌细胞内钙动拮？…

AIM: To study the effects of berbamine (Ber) on intracellular calcium concentration ([Ca2+]i) mobilized by KCl depolarization, norepinephrine (NE), and caffeine. METHODS: [Ca2+]i was measured with fluorescent intensity (FI) by confocal microscope in single cultured cardiomyocytes of newborn rats loaded with Fluo 3-AM 2 mumol.L-1. RESULTS: FI value of [Ca2+]i in control level was 248 +/- 70 in the presence of extracellular calcium 1.5 mmol.L-1 and was not changed by Ber 3-30 mumol.L-1. KCl (60 mmol.L-1)- and NE (30 mumol.L-1)-induced [Ca2+]i mobilizations were inhibited (P < 0.01) by Ber 30 mumol.L-1, similar to that of verapamil (Ver). The inhibitory effect of Ber on [Ca2+]i induced by KCl was further increased (P < 0.05) in the presence of egtazic acid 3 mmol.L-1, but that on [Ca2+]i induced by NE was not changed. The [Ca2+]i mobilized by caffeine 80 and 160 mumol.L-1 in D-Hanks' solution was not affected (P > 0.05) by Ber and Ver. CONCLUSION: Ber possessed the antagonistic effects on [Ca2+]i increases via voltage-dependent Ca2+ channel and receptor-operated Ca2+ channel in newborn rat cardiomyocytes, but without effect on intracellular Ca2+ release.     

(-)-Epigallocatechin gallate attenuates glutamate-induced cytotoxicity via intracellular Ca modulation in PC12 cells

1. The effects of (-)-epigallocatechin gallate (EGCG), a green tea polyphenol, on glutamate-induced increases in intracellular Ca2+ concentrations ([Ca2+]i) and cytotoxicity in PC12 cells were investigated. 2. Changes in [Ca2+]i were measured using Fura-2/AM calcium indicator dye and cellular viabilities were determined by a viable cell count and a 3-(4,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. 3. Glutamate increased [Ca2+]i in PC12 cells in a dose-dependent manner. (-)-Epigallocatechin gallate attenuated this glutamate (30 mmol/L)-induced [Ca2+]i increase and EGCG (50 micromol/L) increased the viability of PC12 cells against glutamate-induced cytotoxicity. The EGCG effect was also found to be independent of its general anti-oxidant mechanism. In contrast, EGCG directly suppressed both N-methyl-D-aspartate (50 mmol/L)- and kainate (20 mmol/L)-mediated Ca2+ influx, but not metabotropic receptor-mediated Ca2+ release. 4. These results suggest that EGCG reduces the glutamate-induced [Ca2+]i increase by attenuating ionotropic Ca2+ influx and that this promotes the viability of PC12 cells.     

小檗胺对培养的HeLa细胞内游离钙浓度的作用

AIM: To study the involvement of Ca2+ signaling and the effects of berbamine (Ber) on intracellular calcium concentration ([Ca2+]i) elevated in cultured HeLa cells. METHODS: [Ca2+]i was measured by confocal microscopy in single HeLa cell loaded with Fluo 3-AM. The change of [Ca2+]i was represented by fluorescent intensity (FI). RESULTS: (1) In the presence of extracellular Ca2+ 1.3 mmol.L-1, the resting level of FI was 186 +/- 44, n = 49 cells from all control experiments, and KCl, NE, caffeine, and calcimycin (Cal) all induced [Ca2+]i elevations in cultured HeLa cells. (2) The resting level of FI was not affected by pretreatment with Ber. The FI increased by KCl 60 mmol.L-1, NE 100 micromol.L-1, and Cal 30 micromol.L-1 were attenuated (P < 0.05 or P < 0.01), the slope and the time to peak of FI increase were decreased and prolonged. (3) In the absence of extracellular Ca2+, caffeine 80 mmol.L-1-induced [Ca2+]i mobilization was not inhibited by Ber 100 micromol.L-1 pretreatment. (4) These effects of Ber were similar to those of verapamil (Ver) 10 mumol.L-1. CONCLUSION: Although it was derived from cervical cancer, the HeLa cells which were belong to the nonexcitable cell possessed the similar biological properties with excitable cells, and Ca2+ also played a crucial role in signal transduction processes.