蛋白酪氨酸激酶抑制剂对脑血管平滑肌细胞Ca~(2+)池操纵性Ca~(2+)内流的影响

目的　研究蛋白酪氨酸激酶和蛋白酪氨酸磷酸酶抑制剂对牛脑血管平滑肌细胞 (CSMC)Ca2 + 池操纵性Ca2 + 内流的影响。方法　采用培养的CSMC ,在生物荧光双波长影像分析系统用Fura 2 /Am荧光探针测定单个细胞内游离Ca2 + 浓度。结果　 (1)蛋白酪氨酸激酶抑制剂 (genistein ,2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低内皮素 1(ET 1,10 -7mol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为5 6%± 2 .9%、2 5 6%± 3 9%、48 9%± 3 7% ;蛋白酪氨酸磷酸酶抑制剂 (vanadate ,2 ,4,8μmol·L-1)能浓度依赖性升高CPA刺激引起的CSMCCa2 + 内流 ,增加比率分别为8 2 %± 3 9%、18 8%± 4 9%、46 6%± 6 9% ;(2 ) genistein(2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低ATP(10 μmol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为 6 7%±2 6%、2 4 6%± 6 5 %、5 1 3 %± 6 9% ;vanadate (2 ,4,8μmol·L-1)能浓度依赖性升高ATP刺激引起的CSMCCa2 +内流 ,增加比率分别为 4 8%± 2 0 %、2 8 5 %± 4 6%、49 6%± 3 3 % ;(3 ) genistein (2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低环匹阿尼酸 (Cyclopiazonicacid ,CPA ,10 μmol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为 6 5 %± 3 0 %、2 2 5 %± 5 2 %、     

褪黑素对谷氨酸所致大鼠神经细胞Ca~(2+)内流的影响

目的 :研究褪黑素对谷氨酸引起的神经细胞Ca2 +内流的抑制作用。方法 :用Ca2 +敏感荧光指示剂Fura -2/AM负载大鼠脑细胞 ,测定神经细胞内游离Ca2 +浓度。结果 :谷氨酸500μmol/L可促进Ca2 +内流 ,显著增加细胞内游离Ca2 +浓度 (P<0. 01) ;应用褪黑素10 -5mol/L后可显著抑制Ca2 +内流 ,降低细胞内游离Ca2 +浓度 (P<0. 01)。结论 :褪黑素可通过抑制谷氨酸引起的Ca2 +内流保护神经细胞。     

谷氨酸触发大鼠大脑皮质神经元Ca~(2+)内流与PTK的关系

目的　研究谷氨酸 (glutamate,Glu)触发大鼠大脑皮质神经元Ca2 + 内流特性 ,蛋白酪氨酸激酶 (PTK)抑制剂genistein及蛋白酪氨酸磷酸酶 (PTP)抑制剂vanadate对其影响 ,揭示PTK与Glu触发大鼠大脑皮质神经元Ca2 + 内流的内在联系。方法　采用Fura 2 /AM荧光测定胞浆Ca2 + 变化技术 ,在原代培养的大鼠大脑皮质神经元上观察药物对Glu触发Ca2 + 内流的影响。结果　Glu触发的Ca2 + 内流不受电压依赖性钙通道 (VDCC)阻断剂尼莫地平影响 ,亦不受非VDCC阻断剂SK&F96 36 5影响 ,但可被PTK抑制剂genis tein抑制 ,被PTP抑制剂vanadate增强。genistein(1～ 30μmol·L-1)呈浓度依赖性抑制Glu触发的Ca2 + 内流。vana date则浓度依赖性增强Glu触发的Ca2 + 内流。结论　对尼莫地平敏感的VDCC及对SK&F96 36 5敏感的非VDCC没有参与Glu触发的Ca2 + 内流。PTK激活参与了Glu触发的Ca2 + 内流     

Cl~-通道阻断剂对5-HT和CPA引起的兔脑椎基底动脉平滑肌细胞Ca~(2+)内流的影响

目的　在培养的兔脑椎基底动脉平滑肌细胞上观察5 HT和CPA诱导的Ca2 + 内流的特性 ,电压依赖性Ca2 + 通道 (VDC)抑制药尼莫地平 ,非电压依赖性Ca2 + 通道抑制药SK&F963 65及Cl-通道阻断剂DIDS、NPPB对两种激动剂引起 [Ca2 + ]i 反应的影响 ,以探讨脑血管平滑肌细胞中 5 HT引起Ca2 + 内流的特性、Cl-通道与Ca2 + 内流的关系。方法　采用生物荧光双波长影像分析系统瞬即测定单细胞胞质[Ca2 + ]i 技术。结果　① 5 HT和CPA均能诱导平滑肌细胞[Ca2 + ]i 呈双相升高 ,并且 5 HT诱导的Ca2 + 释放是环匹阿尼酸 (CPA)敏感Ca2 + 池的一部分 ;②尼莫地平对 5 HT和CPA触发的Ca2 + 内流无明显影响 ,而SK&F963 65可阻止二者触发的Ca2 + 内流 ;③Cl-通道阻断剂DIDS、NPPB呈浓度依赖性抑制Ca2 + 内流 ,在SK&F963 65最大限度抑制Ca2 + 内流后 ,DIDS、NPPB可进一步抑制Ca2 + 内流 ;而Ca2 +内流被DIDS、NPPB分别最大抑制后 ,SK&F963 65也可进一步抑制Ca2 + 内流。结论　 5 HT引起的Ca2 + 内流是经SK&F963 65敏感的非VDC ,其中包含Ca2 + 释放引起的Ca2 + 内流 (CRAC)成分与非CRAC成分 ,并且这两部分Ca2 +内流均与DIDS、NPPB敏感的Cl-通道开放有关     

牛磺酸对H-2O_2引起的大鼠血管平滑肌细胞核[Ca~(2+)]及胞浆[Ca~(2+)]变化的影响

目的　毒性剂量H2 O2 可引起细胞坏死或凋亡 ,此过程是否与H2 O2 引起的核 [Ca2 +]([Ca2 +]n)变化有关未见报导。本文研究牛磺酸对H2 O2 引起的血管平滑肌细胞(VSMC) [Ca2 +]n 与胞浆 [Ca2 +]([Ca2 +]c)的变化影响。方法　应用激光共聚焦显微镜对负载Fluo 3的培养大鼠VSMC的 [Ca2 +]n 及 [Ca2 +]c 变化进行研究。结果　在0 5 %H2 O2 作用下 ,[Ca2 +]n 与 [Ca2 +]c 持续升高 ,用抗氧化细胞保护剂 牛磺酸 2 0mmol·L-1预处理细胞 ,可降低H2 O2 引起的 [Ca2 +]n升幅 (P <0 0 5 ) ,但不影响 [Ca2 +]c 升幅 (P >0 0 5 ) ,表明 [Ca2 +]n 变化与 [Ca2 +]c 变化对牛磺酸反应性存在差异。结论　VSMC的核Ca2 +调控与胞浆Ca2 +调控各自具有一定的独立性。牛磺酸对H2 O2 引起的大鼠VSMC核Ca2 +的变化具有保护作用     

牛磺酸对脑挫伤大鼠氧应激与Ca~(2+)超载损伤的影响

目的 :探讨牛磺酸对大鼠脑挫伤导致的氧应激和细胞Ca2 + 超载损伤的影响。方法 :将SD大鼠随机分为正常对照组、脑挫伤模型组、牛磺酸 (高、中、低剂量 )治疗组与尼莫地平组。在给药7d后进行脑挫伤造模 ,造模后2h切取活体脑片以荧光标记法测定胞内Ca2 +水平 ;造模后24h分离大鼠患侧大脑皮层 ,以比色法测定脑匀浆中超氧化物歧化酶 (SOD )活性与丙二醛 (MDA )含量。结果 :与模型组比较 ,牛磺酸各剂量组皮层胞内Ca2 + 、MDA水平显著降低 ,高剂量牛磺酸组皮层SOD水平显著升高。结论 :牛磺酸对大鼠脑挫伤导致的氧应激和细胞Ca2 +超载损伤有较好的保护作用。     

酪氨酸激酶抑制剂对hTrp3蛋白介导的α_(1B)-AR引起的Ca~(2+)内流的影响

目的　探讨Trp3(transientreceptorpotential 3)蛋白是否参与α1B AR引起的Ca2 + 内流以及酪氨酸激酶对其调控作用。方法　采用脂质体转染 ,将hTrp3cDNA分别转染到HEK2 93细胞和已有α1B受体稳定表达的HEK2 93细胞 ;Westernblot方法检测Trp3蛋白表达情况 ;Fura 2 /AM荧光分光光度法 ,测定胞浆游离Ca2 + 浓度。结果　HEK2 93细胞上可检测到hTrp3的内源性表达 ,转染后其表达增加。α1B HEK2 93细胞转染hTrp3cDNA后 ,α1B AR引起的Ca2 +内流显著增加 (P <0 0 1) ;转染hTrp3cDNA对thapsigargin诱导的Ca2 + 内流无作用。 5～ 30 μmol·L-1genistein对转染细胞α1B AR诱发的Ca2 + 内流有抑制作用 ,最大抑制率达(75 2± 12 6 ) %。结论　Trp3cDNA转染可能主要通过非CRAC(calciumreleaseactivatedcalcium )途径增加α1B AR引起的Ca2 + 内流 ,这一过程很大程度上依赖酪氨酸激酶的调控     

人参皂苷Rg_2对培养心肌细胞内游离Ca~(2+)含量的影响

众所周知 ,Ca2 + 在心肌的舒缩过程中具有调控作用 ,文献[1] 曾研究了人参皂苷Rg2 对休克动物心功能的改善作用 ,该作用是否与Ca2 + 有关 ,故观察了Rg2 对体外培养心肌细胞内游离Ca2 + 含量的影响。1　材料与方法　　药物 :人参皂苷Rg2 (Rg2 )由吉林省中医中药研究院制备 ,纯度为 98 5 % ,Fura 2 /AM和DME/F12培养基购自Sigma公司。动物 :新生 4d的Wistar大鼠乳鼠 ,由中山医科大学实验动物中心提供。心肌细胞培养参照文献[2 ] 进行 ,取乳鼠心肌 ,用胰蛋白酶反复消化 ,制成单细胞悬液。用DME/F12培养液调细胞浓度 ,接种培养皿 ,形成…     

大明胶囊对糖尿病大鼠心肌L型Ca~(2+)通道蛋白mRNA表达的影响

目的探讨大明胶囊对2型糖尿病大鼠心肌L型Ca2+通道蛋白mRNA表达的影响。方法建立2型糖尿病大鼠模型,筛选空腹血糖值大于16.7mmol·L-1的大鼠随机分组:大明胶囊大(200mg·kg-1·d-1)、中(100mg·kg-1·d-1)、小(50mg·kg-1·d-1)剂量组、糖尿病模型组、苯乙双胍(75mg·kg-1·d-1)组,连续用药14d,用快速血糖仪测空腹血糖值。提取心肌总RNA,采用逆转录聚合酶链式反应(RTPCR)的方法,观察大明胶囊治疗后2型糖尿病大鼠心肌L型Ca2+通道蛋白mRNA水平的变化。结果2型糖尿病组大鼠心肌L型Ca2+通道蛋白mRNA表达高于正常组大鼠(P<0.01),经大明胶囊治疗后的心肌L型Ca2+通道蛋白mRNA表达降低(P<0.05),空腹血糖也明显下降。结论大明胶囊对2型糖尿病大鼠有明显的降糖作用,并且可以降低2型糖尿病大鼠心肌L型Ca2+通道蛋白mRNA的表达。     

Cl~-通道在内皮素-1引起的血管平滑肌细胞增殖中的作用

目的　研究Cl-通道在内皮素 1(endothelin 1,ET 1)引起的血管平滑肌细胞增殖中的作用 ,并探讨其可能的作用机制。方法　通过细胞计数和3H TdR参入实验 ,并结合fura 2 /AM荧光测定胞浆游离Ca2 + 浓度 ([Ca2 + ]i)等技术 ,观察了Cl-通道阻断剂对ET 1引起的 [Ca2 + ]i 变化及血管平滑肌细胞增殖的影响。结果　Cl-通道阻断剂DIDS可呈浓度依赖性地抑制 10nmol·L-1ET 1引起的血管平滑肌细胞增殖 ,其它Cl-通道阻断剂如IAA 94、NPPB、SITS、DPC和速尿均无此作用 ,DIDS也能抑制 10nmol·L-1ET 1引起的内流相 [Ca2 + ]i 升高 ,而对ET 1引起的Ca2 + 释放无影响 ;预先将细胞与 1μmol·L-1nifedipine作用后 ,3μmol·L-1DIDS对 10nmol·L-1ET 1引起的内流相 [Ca2 + ]i 升高及血管平滑肌细胞增殖不再有效 ,将细胞与 10 μmol·L-1SK&F96 36 5预孵后 ,DIDS却能进一步抑制ET 1的上述作用 ;3μmol·L-1DIDS对 30mmol·L-1KCl引起的胞浆[Ca2 + ]i升高无影响。结论　DIDS可通过阻断Cl-通道来抑制ET 1因促发Cl-通道开放经电压依赖性钙通道的Ca2 +内流及细胞增殖 ,DIDS敏感的Cl-通道可能在ET 1促发的Ca2 + 内流及血管平滑肌细胞增殖的调控上都起着重要的作用     

Store-operated calcium entry in vascular smooth muscle

In non-excitable cells, activation of G-protein-coupled phospholipase C (PLC)-linked receptors causes the release of Ca(2+) from intracellular stores, which is followed by transmembrane Ca(2+) entry. This Ca(2+) entry underlies a small and sustained phase of the cellular [Ca(2+)](i) increases and is important for several cellular functions including gene expression, secretion and cell proliferation. This form of transmembrane Ca(2+) entry is supported by agonist-activated Ca(2+)-permeable ion channels that are activated by store depletion and is referred to as store-operated Ca(2+) entry (SOCE) and represents a major pathway for agonist-induced Ca(2+) entry. In excitable cells such as smooth muscle cells, Ca(2+) entry mechanisms responsible for sustained cellular activation are normally considered to be mediated via either voltage-operated or receptor-operated Ca(2+) channels. Although SOCE occurs following agonist activation of smooth muscle, this was thought to be more important in replenishing Ca(2+) stores rather than acting as a source of activator Ca(2+) for the contractile process. This review summarizes our current knowledge of SOCE as a regulator of vascular smooth muscle tone and discusses its possible role in the cardiovascular function and disease. We propose a possible hypothesis for its activation and suggest that SOCE may represent a novel target for pharmacological therapeutic intervention.     

哮喘豚鼠气道平滑肌细胞内钙释放通道的变化

目的检测哮喘豚鼠气道平滑肌细胞(ASMCs)内钙释放通道功能的改变,探讨与支气管哮喘的关系,同时,寻找传代培养ASMCs的方法。方法以Flou-3/AM为细胞内钙离子示踪剂,观察ASMCs在工具药作用下细胞内钙离子浓度([Ca2+]i)的改变。结果①在ASMCs外无钙情况下,不同浓度ryanodine(5×10-5,10-4,2×10-4mol·L-1)作用于原代培养正常与哮喘ASMCs,[Ca2+]i迅速升高,哮喘组明显高于正常组(P<0·01)。10-4mol·L-1的组织胺(hista-mine)作用于原代培养的正常组与哮喘组ASMCs,[Ca2+]i升高无差异(P>0·05)。②传代培养哮喘ASMCs在10-4、2×10-4mol·L-1ryanodine作用下,[Ca2+]i迅速升高,与原代细胞比较无差异(P>0·05)。在浓度为5×10-5mol·L-1时,原代明显高于传代(P<0.01)。哮喘组传代ASMCs对10-4mol·L-1的histamine反应不明显。结论哮喘豚鼠ASMCs内钙释放通道(RyRs)功能升高,特定条件下,哮喘传代细胞仍然保持原代细胞内钙释放通道的特性。     

糖尿病发病过程中大鼠主动脉血管平滑肌细胞钙泵活性以及血流动力学变化的研究

目的探讨糖尿病发病过程中大鼠主动脉平滑肌细胞钙泵活性变化与血流动力学变化之间的联系以及对糖尿病大血管病变产生和发展的影响。方法用链脲佐菌素(STZ)诱导糖尿病模型,第4、8、12、16周用彩色多普勒对各组主动脉进行血流参数测量。然后处死各组动物取其主动脉并分离得到肌浆网蛋白和胞膜蛋白,分别测定SERCA和PMCA的活性。结果4wk开始随着病程延长糖尿病组大鼠腹主动脉峰值血流速度、平均血流速度较对照组明显降低(P<0.05),而搏动指数和阻力指数则明显增高(P<0.05);12wk开始血管内径逐渐减小(P<0.05),16wk时内膜中膜厚度明显增加(P<0.05);PMCA的活性在早期(4wk)轻度增高,从12wk开始明显降低(P<0.05);SERCA活性在8wk起较对照组明显降低(P<0.05)。结论随着糖尿病病程逐渐延长,糖尿病大鼠主动脉血管结构发生改变,功能损坏;PMCA和SERCA活性改变导致的胞内钙离子调控紊乱参与了糖尿病大血管病变的发生和发展。     

酪氨酸激酶抑制剂对cyclopiazoni cacid引起的大鼠主动脉血管环收缩效应的影响

目的　探讨酪氨酸激酶在Ca2 +池操纵性Ca2 +内流中的作用。方法　记录大鼠胸主动脉环收缩反应。结果　①不同剂量酪氨酸激酶抑制剂 genistein (1～ 10 0 μmol·L-1)和tyrphostin 2 5 (Tyr 2 5 ,1～ 30 μmol·L-1)均以浓度依赖性抑制cyclopiazonicacid (CPA)引起的平滑肌收缩平台期。Tyr 2 5的最大作用浓度为 10 μmol·L-1,抑制率为 42 %±11%。② 10 μmol·L-1Tyr 2 5和 1μmol·L-1nifedipine(Nif)对CPA引起电压依赖性Ca2 +通道 (VDCC)开放过程的抑制作用存在部分交叉。③ 1μmol·L-1Nif预处理阻断VDCC作用后 ,10 μmol·L-1Tyr 2 5只能部分阻断CPA引起的Ca2 +池操纵性Ca2 +通道 (SOCC)开放过程 ,再加入 6 0 μmol·L-1SK&F96 36 5可完全阻断SOCC的开放过程。结论　CPA引起平滑肌的收缩过程中 ,蛋白质酪氨酸激酶参与了开启VDCC和SOCC的信号转导过程     

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.     

Capacitative Ca2+ entry associated with α1-adrenoceptors in rat aorta

In rat aorta, depletion of internal Ca²⁺ stores by addition of noradrenaline (1μM) induces a biphasic response (an initial phasic response and a tonic one) mediated by two different intracellular Ca²⁺ pools. This response cannot be repeated, suggesting a depletion of internal Ca²⁺ stores sensitive to noradrenaline. In absence of the agonist, this depletion is the signal for the entry of extracellular Ca²⁺, not only to refill the stores but also, under our experimental conditions, to activate the contractile proteins thus inducing an increase in the resting tone (IRT) that constitutes functional evidence of this Ca²⁺ entry. The ionic channels involved in the mechanism of the IRT have been studied in the present work. The fact that the addition of nimodipine (10^–15– 10^–11M) selectively inhibits the IRT suggests that this mechanical response is mediated by Ca²⁺ influx through dihydropyridine-sensitive Ca²⁺ channels. Moreover, the inhibitory action of nimodipine is attenuated by glibenclamide (10μM). Cromakalim (10^–10–10^–6M) also inhibits the IRT concentration dependently, and this inhibition is antagonized by glibenclamide (10μM). These results relate the ATP-dependent K⁺ channels to the mechanism of the IRT. The refilling of the two internal Ca²⁺ compartments sensitive to noradrenaline was, like the IRT, altered in presence of the compounds tested, since the subsequent contractile response to noradrenaline was decreased. The present results suggest that nimodipine treatment inhibits the refilling of the Ca²⁺ compartment responsible for the tonic contraction induced by noradrenaline in Ca²⁺-free medium, whereas the refilling of the Ca²⁺ pool responsible for the phasic response to noradrenaline remained unaltered. Both the phasic and tonic responses to noradrenaline in Ca²⁺-free medium decreased after treatment with cromakalim. We can therefore assume that the refilling of both Ca²⁺ compartments sensitive to noradrenaline was inhibited. In conclusion, these results are consistent with the contraction of the rat aorta in response to noradrenaline in Ca²⁺-free medium consisting of an initial phasic response and a tonic one. The former is due to the release of internal Ca²⁺ from a compartment refilled through a special channel that is cromakalim but not dihydropyridine sensitive. The tonic response is due to Ca²⁺ release from another compartment refilled through a cromakalim- and dihydropyridine-sensitive Ca²⁺ channel. The Ca²⁺ entry through this latter channel intervenes in the IRT observed during the refilling of these stores previously depleted by noradrenaline, and the opening state of this channel is also modulated by ATP-dependent K²⁺ channels. Received: 7 May 1996 / Accepted: 30 January 1997     

Multiple effects of caffeine on contraction and cytosolic free Ca2+ levels in vascular smooth muscle of rat aorta

Summary 1. Effects of caffeine on cytosolic Ca²⁺ level ([Ca²⁺]_cyt), measured simultaneously with muscle tension using fura-2-Ca²⁺ fluorescence, were examined in isolated smooth muscle of rat aorta. 2. Caffeine (20 mmol/l) induced a large transient increase in [Ca²⁺]_cyt followed by a plateau which was higher than resting level. However, muscle tension showed a transient increase followed by a decrease to or below the resting level. In Ca²⁺-free solution, caffeine induced only a transient increase in both [Ca²⁺]_cyt, and muscle tension. 3. At low temperature (22°C), high K⁺ (72.7 mmol/l) induced sustained increase in both [Ca²⁺]_cyt and muscle tension which were smaller than those observed at 37°C. At 22°C, however, caffeine-induced transient changes were greater than those observed at 37°C. 4. Ryanodine (10 mol/l) inhibited the transient changes due to caffeine but showed little effects on the sustained changes due to high K⁺. 5. During the sustained increase in [Ca²⁺]_cyt induced by noradrenaline (10 gmmol/l) or high K⁺ (140 mmol/l), addition of caffeine transiently increased [Ca²⁺]_cyt followed by a decrease to a level slightly lower than that before the addition of caffeine. In contrast to this, muscle tension transiently increased and then decreased to or below the resting level. 6. These results suggest that caffeine-induced contraction is due to the release of Ca²⁺ from cellular store. Caffeine also has an inhibitory effect which is partly attributable to decrease in [Ca²⁺ _cyt, and partly to the decrease in the sensitivity to Ca²⁺ of the contractile elements.Send offprint requests to H. Ozaki at the above address     

Methyl-beta-cyclodextrin prevents Ca2+-induced Ca2+ release in smooth muscle cells of mouse urinary bladder

We examined the effects of methyl-beta-cyclodextrin (MbetaCD) on Ca(2+)-induced Ca(2+) release (CICR) in smooth muscle cells (SMCs) of mouse urinary bladder (UB). Short depolarization of UBSMCs under voltage-clamp elicited several local Ca(2+) transients (Ca(2+) hot spots) via CICR within 20 ms in discrete sub-sarcolemmal areas. Then, the Ca(2+) wave spread to whole areas. The pretreatment with 10 mM MbetaCD significantly attenuated Ca(2+) hot spots in UBSMCs and reduced contraction by single direct electrical pulse stimulation in UBSM strips. MbetaCD may prevent CICR by attenuating the coupling between voltage-dependent Ca(2+) channels and ryanodine receptors in Ca(2+) hot spot areas.     

Differential roles of ryanodine- and thapsigargin-sensitive intracellular CA2+ stores in excitation-contraction coupling in smooth muscle of guinea-pig taenia caeci

1. To explore roles of intracellular Ca(2+) stores in excitation-contraction coupling in smooth muscle, we examined the effects of ryanodine, a fixer of ryanodine receptor-Ca(2+) channels to an open state, and thapsigargin, a selective inhibitor of the Ca(2+) pump in the intracellular stores, on smooth muscle contraction in the presence and absence of extracellular Ca(2+) in guinea-pig taenia caeci. 2. In Ca(2+) -free solution, contractions induced by 0.1 mmol/L carbachol and 0.1 mmol/L histamine were reduced to approximately 65% of control by either 1 micro mol/L thapsigargin or 10 micro mol/L ryanodine. In contrast, caffeine-induced contraction was reduced to approximately 40% of control by ryanodine, but was not affected by thapsigargin. 3. In the presence of extracellular Ca(2+), thapsigargin slowly induced a large and sustained contraction. In contrast, ryanodine did not induce an apparent contraction, but increased the sensitivity of contractile responses to receptor agonists (carbachol, AHR-602 and histamine) or depolarizing high K(+) with no changes in the maximal contraction. 4. These results suggest that there are pharmacological and physiological differences between ryanodine- and thapsigargin-sensitive intracellular Ca(2+) stores in excitation-contraction coupling in smooth muscle, which may be responsible for their differential effects on the Ca(2+) -influx pathway.     

Hypertonic saline inhibits airway smooth muscle contraction by inhibiting Ca2+ sensitization

The effects of hypertonic solution on airway smooth muscle (ASM) contraction and the underlying mechanisms are largely unknown. We found that hypertonic saline (HS) inhibited acetylcholine (ACh)‐induced contraction of ASM from the mouse trachea and human bronchi. In single mouse ASM cells (ASMCs), ACh induced an increase in intracellular Ca²⁺ that was further enhanced by 5% NaCl, indicating that the HS‐induced inhibition of ASM contraction was not mediated by a decrease in cytosolic Ca²⁺. The Rho‐associated kinase (ROCK) inhibitor Y‐27632 relaxed ACh‐induced precontraction of mouse tracheal rings. However, such inhibition was not observed after the relaxation induced by 5% NaCl. Moreover, the incubation of mouse tracheal rings with 5% NaCl decreased ACh‐induced phosphorylation of myosin light chain 20 and myosin phosphatase target subunit 1. These data indicate that HS inhibits the contraction of ASM by inhibiting Ca²⁺ sensitization, not by decreasing intracellular Ca²⁺.