Inhibition of aftercontractions and phasic calcium release by yohimbine in ferret papillary muscle

Phasic release of calcium from the sarcoplasmic reticulum occurs in all mammalian cardiac preparations when the intracellular calcium concentration is sufficiently high. The phasic calcium release is often sufficient to trigger electrophysiological responses and aftercontractions. These can be detrimental to normal cardiac function. We induced phasic calcium release in ferret papillary muscles loaded with the calcium indicator aequorin. Development of phasic calcium release was associated with an increase in resting and peak [Ca²⁺]_i. Inhibiting sodium channels with yohimbine reduced resting [Ca²⁺]_i and prevented phasic calcium release. We propose a mechasism where by reduced [Na⁺]_i, and the subsequent increased efflux of calcium via sodium/calcium exchange reduced [Ca²⁺]_i.     

细胞钙转运系统研究进展*

钙离子是重要的细胞信号分子。胞内和胞外、胞浆和细胞器之间的Ca2+转运都依赖于钙转运系统的精确调控。钙转运系统包括钙泵、钙通道、Na+/Ca2+交换体及膜结构域,如质膜的PMCA、NCX、VGCC、LGCC和TRP通道;内质网膜的SERCA、IP3Rs和RyRs等;线粒体的MCU和NCLX;内溶酶体的TPCs;联结质膜和内质网膜的CRAC通路;联结内质网膜和线粒体膜的MAM结构域等。本文对定位于质膜、内质网和线粒体的钙转运系统的种类、功能、组织分布、相关疾病以及抑制剂作综述。     

川芎嗪对心肌细胞核钙转运功能异常的保护

目的 :观察异丙肾上腺素 (ISO)致大鼠心肌缺血损伤时心肌细胞核钙转运功能的变化及川芎嗪对其影响。方法 :Wistar大鼠皮下注射 5mg·kg- 1ISO液 ,造成心肌缺血损伤模型。超速离心分离纯化心肌细胞核。酶学方法鉴定核纯度和测定核膜Ca2 + ATP酶活性 ,同位素法观测核钙的摄取。结果 :与正常对照组比较 ,心肌缺血组 (实验组 )心肌细胞核膜Ca2 + 依赖性ATP酶活性降低 18.1%(P <0 .0 5 ) ,4 5Ca2 + 摄取效率也显著降低 ,其最大反应速度(Vmax)降低 5 4 .6 %,细胞核Ca2 + 依赖性ATP酶的最大反应速度 (Vmax)降低 33.0 %(P <0 .0 5 ) ,平衡常数 (Km)降低 4 2 .8%(P <0 .0 1) ;川芎嗪保护组心肌细胞核Ca2 + ATP酶活性及核4 5Ca2 + 摄取与正常对照组比较均未见显著性改变。结论 :川芎嗪对ISO致大鼠心肌缺血损伤时心肌细胞核钙转运功能降低有保护作用。     

Endothelium-independent vasorelaxation by ticlopidine and clopidogrel in rat caudal artery

Objectives Thienopyridines are prodrugs currently used as anti‐aggregating agents. The aim of this study was to determine if these compounds might have vascular activity independent of hepatic bioactivation. Methods The direct activity of thienopyridines was studied in rat caudal arterial rings and aortic smooth muscle cells in culture. Key findings Both compounds (0.01 µm –100 µm ) showed a concentration‐dependent vasorelaxation in arterial tissues precontracted with phenylephrine, 5‐hydroxytryptamine and KCl. The relaxation induced by 100 µm ticlopidine and clopidogrel was greater than 80%. The relaxation by ticlopidine was compared with the activity of acetylcholine. These two agents showed similar potency, although ticlopidine was slightly more active. Pretreatment with the nitric oxide synthase inhibitor L‐NAME inhibited the relaxation by acetylcholine but not that by ticlopidine. To further study vasorelaxation by ticlopidine, other pharmacological inhibitors including propranolol, nifedipine and suramin were used. These compounds lacked inhibitory effects on the vasorelaxation by ticlopidine. In vascular smooth muscle cells, 1 µm ticlopidine induced a decrease in cell proliferation, while incubation with both ticlopidine and ADP or 2‐methioADP led to an additive effect. Conclusions The data suggest that ticlopidine and clopidogrel cause relaxation of arterial tissues and influence vascular smooth muscle cell proliferation directly without hepatic biotransformation. Furthermore, the arterial relaxation induced in vitro by thienopyridines is endothelium independent, and β‐adrenergic and P2 receptors are not involved.     

Store-operated calcium channels and pro-inflammatory signals

In non-excitable cells such as T lymphocytes, hepatocytes, mast cells, endothelia and epithelia, the major pathway for calcium [Ca2+] entry is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, however, neither the gating mechanism nor the downstream targets of these channels has been clear established. Here, I review some of the proposed gating mechanisms of store-operated Ca2+ channels and the functional implications in regulating pro-inflammatory signals.     

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.     

钙感受器STIM1在小鼠主动脉平滑肌收缩反应中的作用研究

目的探讨钙感受器STIM1在小鼠主动脉平滑肌收缩反应中的作用。方法采用Cre-lox重组技术制备平滑肌特异性STIM1敲除小鼠(sm-STIM1-KO);采用离体血管张力测定方法,测定sm-STIM1-KO小鼠主动脉对不同血管收缩剂反应,并给予不同的钙通道阻断剂,观察血管收缩变化。结果sm-STIM1-KO小鼠制备成功。sm-STIM1-KO小鼠钙库操纵的钙通道(SOCC)介导的血管收缩消失;与野生型相比,sm-STIM1-KO小鼠对Phe、5-HT和U46619总的收缩反应无明显变化,但在有钙和无钙的K-H溶液中,经硝苯地平孵育后,两组血管收缩均被抑制,且sm-STIM1-KO小鼠收缩明显低于野生型小鼠(P<0.01);在含硝苯地平的高钾溶液中,Phe引起的快相收缩没有变化,慢相收缩下降(P<0.01);sm-STIM1-KO小鼠肌浆网钙释放介导的血管收缩达峰速度和下降速度明显加快(P<0.05)。结论STIM1是SOCC介导的血管收缩的必须组成成分,且参与肌浆网钙释放介导的血管收缩反应。     

川芎嗪对大鼠缺血心肌细胞核钙转运异常的影响

目的 :观察大鼠缺血心肌细胞核钙转运功能的变化及川芎嗪的作用。方法 :将Wistar大鼠随机分成对照组、缺血组和川芎嗪保护组。采用皮下注射异丙肾上腺素 (ISP,5mg·kg- 1 ) ,2 4h后再注射相同剂量一次 ,造成心肌缺血模型。超速离心纯化细胞核 ,酶学法鉴定核纯度和测定核膜Ca2 + ATPase活性 ,同位素法观测核钙的摄取。结果 :缺血组心肌细胞核膜Ca2 + ATPase活性较对照组下降 2 1 .4% (P<0 .0 1 ) ,4 5Ca2 + 摄取率也呈显著性降低 ,其最大反应速度 (Vmax)降低 48.5 % ,细胞核膜Ca2 + 依赖性ATP酶的Vmax 降低 3 4.1 % ,Km 值降低 42 .2 % (P <0 .0 1 ) ,川芎嗪保护组心肌细胞核Ca2 + ATP酶活性及核4 5Ca2 + 摄取率较缺血组均有显著性升高 ,且有剂量依赖关系。结论 :川芎嗪对ISP所致的缺血心肌细胞核钙转运功能下降有拮抗作用     

VASORELAXATION OF RAT THORACIC AORTA CAUSED BY 14-DEOXYANDROGRAPHOLIDE

1. The pharmacological effects of 14-deoxyandrographolide on rat isolated thoracic aorta were examined. 2. 14-Deoxyandrographolide (2.5-120 μmiol/L) inhibited contractions induced by phenylephrine (PE; 0.1 μmol/L) and high K⁺ (80mmol/L) in a concentration-dependent manner in endothelium-intact aorta. The effect was attenuated in endo-thelium-denuded aorta without modifying the maximal response. Like verapamil, 14-deoxyandrographolide produced a much greater vasorelaxant effect in aorta precontracted by KCI than by PE. 14-Deoxyandrographolide (20-60 μmol/L) also inhibited responses of the rat aorta to PE. 3. In Ca²⁺-free medium (KCI 55mmol/L), 14-deoxyandrographolide (20-80 μmol/L) antagonized Ca²⁺-induced vasocon-traction in a concentration-dependent manner and transient contractions induced by both caffeine (10mmol/L) and noradrenaline (1μmol/L) were suppressed or almost abolished by 14-deoxyandrographolide. 4. The vasorelaxant effect of 14-deoxyandrographolide was partially antagonized by N^G-nitro-L -arginine methyl ester (25 μmol/L), a specific and competitive nitric oxide synthase (NOS) inhibitor, and methylene blue (10 μmol/L), a soluble guanylate cyclase inhibitor, but was not affected by indomethacin (20 μmol/L), a cyclo-oxygenase inhibitor, or glibenclamide (10 μmol/L), an ATP-sensitive K⁺-channel blocker. 5. These results suggest that the vasorelaxant activity of 14-deoxyandrographolide may be mediated via the activation of NOS and guanylate cyclase, as well as the blockade of Ca²⁺ influx through both voltage- and receptor-operated Ca²⁺ channels.     

T型钙通道和心血管及神经系统疾病

低电压T型钙通道广泛分布在各种类型细胞中,包括心血管和神经元细胞中,与高电压钙通道不同,在接近膜静息电位的低度去极化时即能被激活,因而有利于心脏起博和神经元细胞在生理状态下接近静息时,对兴奋和电反应的调节。但对T型钙通道在疾病中的作用所知有限。近年来因为克隆出3种T型钙通道α1亚单位的基因,(Cav3.1,Cav3.2和Cav3.3),使深入研究实验动物及人体疾病时T型钙通道的性质、药理、体内分布、基因调节成为可能。并且为新药研发提供有力工具。转基因动物实验已证明T型钙通道不仅是治疗肾性高血压、心律失常也是治疗意识丧失型癫痫及神经性疼痛的重要药物靶点。此外,细胞内钙超载还与房颤、心衰、偏头痛、阿尔采末病、睡眠障碍等的发病有关,所以盼望有新的T型钙通道阻断剂出现。有报道Efonidipine能选择性地抑制低电压T型钙通道,有望成为选择性阻断剂。此外,近年来调控T型钙通道活性的分子机制的研究取得新的达展,对新药的开发有所启迪。作者建议,对治疗心血管及神经系统疾病的药物靶点T型钙通道及其阻断剂的研究给予更多的关注。     

The antidepressant-like effect induced by the sigma(1) (sigma(1)) receptor agonist igmesine involves modulation of intracellular calcium mobilization

RATIONALE: Activation of the neuronal sigma(1) (sigma(1)) receptor potentiates calcium mobilization, leading to effective modulation of postsynaptic responses to neurotransmitters. At the behavioral level, sigma(1) agonists modulate learning, response to stress and depression. In particular, the selective sigma(1) agonist igmesine reduced immobility in the forced swimming test. OBJECTIVES AND METHODS: We investigated the effect of modulators of Ca(2+) influx and mobilization, administered intracerebroventricularly at doses ineffective alone, on the igmesine effect. The tricyclic antidepressant desipramine was also studied for comparison. RESULTS: The calcium chelator EGTA blocked both igmesine and desipramine-induced decreases of immobility duration, indicating the importance of extracellular Ca(2+) influx in the initial action of each compound. Both L- and N-type voltage-dependent calcium channel (VDCC) appeared involved in the sigma(1) agonist effect. Verapamil, an L-type VDCC antagonist or omega-conotoxin GVI, a N-type VDCC antagonist, blocked whereas (-)-Bay K8644, a L-type VDCC agonist, potentiated the igmesine effect. Mobilization of intracellular Ca(2+) stores is involved selectively in the effect mediated by the sigma(1) receptor, since the membrane permeable intracellular Ca(2+) chelator EGTA/AM affected only the igmesine effect. Inositol 1,4,5-trisphosphate (InsP(3)) receptor-sensitive Ca(2+) pools appeared primarily involved, rather than Ca(2+)/caffeine-sensitive Ca(2+) pools. Indeed, the InsP(3) receptor positive modulator bradykinin potentiated, whereas the InsP(3) receptor antagonist xestospongin C blocked the igmesine effect. The ryanodine receptor agonist caffeine failed to affect the efficacy of igmesine, whereas the antagonist ryanodine reduced it. CONCLUSIONS: The sigma(1) receptor-mediated behavioral effect is dependent not only on rapid Ca(2+) influx, as observed for a classical antidepressant, but also on intracellular Ca(2+) mobilization.     

Gallopamil阻滞猫心室肌钙通道的状态依赖性分析

根据闸门相关受体假说及模型,应用计算机模拟分析了gallopamil(D600)阻滞猫心室肌钙通道的动力学特点及其状态依赖性。结果表明D600频率依赖性阻滞钙通道的动力学特点是起效快而静息阻滞恢复慢。其阻滞的状态依赖性为开放态阻滞,激活门关闭可将药物滞留于通道内。其阻滞恢复除受闸门影响外亦有其它影响因素。以闸门相关受体的观点对D600的状态依赖性作了新的解释,认为其阻滞钙通道的作用与失活过程无关。     

Cardiac alternans and intracellular calcium cycling

Cardiac alternans refers to a condition in which there is a periodic beat‐to‐beat oscillation in electrical activity and the strength of cardiac muscle contraction at a constant heart rate. Clinically, cardiac alternans occurs in settings that are typical for cardiac arrhythmias and has been causally linked to these conditions. At the cellular level, alternans is defined as beat‐to‐beat alternations in contraction amplitude (mechanical alternans), action potential duration (APD; electrical or APD alternans) and Ca²⁺ transient amplitude (Ca²⁺ alternans). The cause of alternans is multifactorial; however, alternans always originate from disturbances of the bidirectional coupling between membrane voltage (V_m) and intracellular calcium ([Ca²⁺]_i). Bidirectional coupling refers to the fact that, in cardiac cells, V_m depolarization and the generation of action potentials cause the elevation of [Ca²⁺]_i that is required for contraction (a process referred to as excitation–contraction coupling); conversely, changes of [Ca²⁺]_i control V_m because important membrane currents are Ca²⁺ dependent. Evidence is mounting that alternans is ultimately caused by disturbances of cellular Ca²⁺ signalling. Herein we review how two key factors of cardiac cellular Ca²⁺ cycling, namely the release of Ca²⁺ from internal stores and the capability of clearing the cytosol from Ca²⁺ after each beat, determine the conditions under which alternans occurs. The contributions from key Ca²⁺‐handling proteins (i.e. surface membrane channels, ion pumps and transporters and internal Ca²⁺ release channels) are discussed.     

Adenosine A2 receptor activation facilitates Ca uptake by rat brain synaptosomes

Adenosine has been shown to increase the release of neurotransmitters by stimulation of adenosine A₂ receptors. This effect probably depends on Ca²⁺ entry into presynaptic nerve terminals. In the present work the ability of the mixed adenosine A₁/A₂ agonist, 2-chloroadenosine, to stimulate Ca²⁺ uptake into rat brain synaptosomes was investigated. ⁴⁵Ca²⁺ uptake was induced by 20 μM veratridine. In the absence of other drugs, 2-chloroadenosine (1 μM) decreased ⁴⁵Ca²⁺ uptake into synaptosomes. Blocking the adenosine A₁ receptor with 100 nM of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), 2-chloroadenosine (1 μM) increased rather than decreased the uptake of ⁴⁵Ca²⁺ into synaptosomes. The excitatory effect of 2-chloroadenosine observed in the presence of DPCPX was reversed by 200 nM of ω-agatoxin-IVA, a specific P-type Ca²⁺ channel antagonist, but not by L-type (nifedipine, 100 nM to 1 μM; methoxyverapamil 1-10 μM) or N-type (ω-conotoxin GVIA, 500 nM) Ca²⁺ channel antagonists. The adenosine A_2A selective agonist, 2-p-(2-carboxyethyl)-phenethylamino-5′-N-ethyl-carboxamido-adenosine (CGS 21680), did not significantly modify Ca²⁺ uptake induced by veratridine. In contrast, the selective adenosine A₂ receptor agonist, N⁶-(2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl)-adenosine (DPMA), in concentrations ranging from 10 nM to 1 μM increased Ca²⁺ uptake induced by veratridine. The selective adenosine A₂ receptor antagonist 3,7-dimethyl-1-propargylxanthine (DPMX) at a concentration of 10 μM antagonized the stimulatory effect of DPMA (0.1 μM) on ⁴⁵Ca²⁺ uptake. In conclusion, activation of adenosine A₂ receptors increases Ca²⁺ uptake by synaptosomes depolarized by veratridine, which could explain the increase of neurotransmitter release observed when A₂ receptors are activated.     

Dependence of the cardiac uptake of digitalis glucosides on the extracellular calcium concentration in guinea pig isolated hearts

The purpose of the present study was to investigate, at the myocardial level, teh divergent influences of Ca²⁺ on the action of various cardiotonic steroids. Therefore, the cardiac uptake of ³H-digitoxin and ³H-digoxin was studied in experiments on guinea pig isolated hearts. The following results were obtained: with digitoxin the increase of the extracellular calcium concentration from 0.45 upto 7.2 mM resulted in a concomitant decrease of the myocardial glycoside uptake from about 1.8 nmoles/g wet weight down to about 1.2 nmoles/g wet weight. Similar results were obtained when digoxin uptake was studied under the same conditions: with 0.45 mM Ca²⁺ about 0.6 nmoles/g wet weight were bound, increasing the calcium concentration upto 7.2 mM lead to a concomitant decrease of the cardiac digoxin uptake down to about 0.45 nmoles/g wet weight. Despite the different physiochemical behaviour of these two drugs and the different amounts of drug present in the hearts the influence of Ca²⁺ was almost identical if calculated on a relative basis. So far, no experimental based explanation can be given for the above discrepancies. Other possible interpretation discussed.     

Alpha-naphthoflavone induces vasorelaxation through the induction of extracellular calcium influx and NO formation in endothelium

The effect of -naphthoflavone (-NF) on vascular function was studied in isolated ring segments of the rat thoracic aorta and in primary cultures of human umbilical vein endothelial cells (HUVECs). -NF induced concentration-dependent relaxation of the phenylephrine-precontracted aorta endothelium-dependently and -independently at lower and higher concentrations, respectively. The cGMP, but not cAMP, content was increased significantly in -NF-treated aorta. Pretreatment with N -nitro-l-arginine methyl ester (L-NAME) or methylene blue attenuated both -NF induced vasorelaxation and the increase of cGMP content significantly. The increase of cGMP content induced by -NF was also inhibited by chelating extracellular Ca²⁺ with EGTA. These results suggest that the endothelium-dependent vasorelaxation induced by -NF is mediated most probably through Ca²⁺-dependent activation of NO synthase and guanylyl cyclase. In HUVECs, -NF induced concentration-dependent formation of NO and Ca²⁺ influx. -NF-induced NO formation was abolished by removal of extracellular Ca²⁺ and by pretreatment with the Ca²⁺ channel blockers SKF 96365 and Ni²⁺, but not by the L-type Ca²⁺ channel blocker verapamil. The Ca²⁺ influx, as measured by ⁴⁵Ca²⁺ uptake, induced by -NF was also inhibited by SKF 96365 and Ni²⁺. Our data imply that -NF, at lower concentrations, induces endothelium-dependent vasorelaxation by promoting extracellular Ca²⁺ influx in endothelium and the activation of the NO-cGMP pathway.     

Ca~(2+)经钙激活非选择性阳离子通道进入ECV304内皮细胞

目的　研究钙离子进入ECV30 4内皮细胞株的途径和血管紧张素Ⅱ (AⅡ )对钙内流的影响。方法　用膜片钳的细胞贴附式和全细胞方式记录ECV30 4内皮细胞的通道活动。结果　 (1 )在记录单通道电流时电极液含 1 2 0mmol·L- 1 CaCl2 ,细胞浴液不含K+ 、Na+ 时 ,Ca2 + 经非选择性阳离子通道 (CAN)内流的电导为γ0 =(1 2 90± 2 1 1 ) pS(n =4)。1× 1 0 - 7mol·L- 1 AⅡ可显著增强通道电流幅度和延长通道开放时 ,其电导增大为γ1 =(2 2 1 8± 2 2 9)pS(n =4)。全细胞记录得到的结果与单通道的一致。 (2 )用全细胞方式记录到ECV30 4内皮细胞的电压依赖性钙通道电流 ,记录到该峰值电流为 (2 9 32± 3 56)pA(n =4) ,2 0 μmol·L- 1 nifedepine能抑制这个峰值电流 ,被抑制后的电流峰值为 (6 0 0± 3 94)pA(n =4)。 2 μmol·L- 1 BayK8644能显著激活通道活动。结论　Ca2 + 经CAN进入ECV30 4细胞 ,AⅡ可显著增强CAN的钙流     

糖尿病大鼠冠状动脉平滑肌细胞中大电导钙离子激活钾通道电流和钙离子浓度变化

目的探讨糖尿病大鼠冠状动脉平滑肌细胞中大电导钙离子激活钾通道(BK通道)电流及钙离子浓度的变化。方法 40只SD大鼠随机均分为正常对照组(A组)和糖尿病组(B组)。采用链脲霉素腹腔内注射建立糖尿病大鼠模型,酶消化法分离冠状动脉平滑肌细胞,全细胞膜片钳实验和荧光测定方法分别检测冠状动脉平滑肌细胞BK通道电流和钙离子浓度。结果与A组相比,当刺激电压大于60mV时,B组冠状动脉平滑肌细胞BK通道电流密度明显下降(P<0.05);A组冠状动脉平滑肌细胞内钙离子浓度明显低于B组[(103±23)nmol/L vs.(193±22)nmol/L](P<0.05)。结论冠状动脉平滑肌细胞中BK通道电流下降及细胞内钙离子浓度升高可能是糖尿病冠状动脉功能损伤的重要原因。