RhoA/ROCK信号通路参与2型糖尿病小鼠主动脉收缩高反应的机制北大核心CSCD

目的探讨RhoA/ROCK信号通路在2型糖尿病(type 2 diabetes,T2DM)小鼠主动脉收缩功能异常中的作用及机制。方法实验分成对照组(db/m小鼠)和T2DM组(db/db小鼠)。采用血管张力测定技术,在不同条件下测定两组小鼠主动脉环收缩反应。用Western blot方法测定小鼠主动脉平滑肌相关蛋白表达变化。结果T2DM组小鼠血糖和体质量水平明显升高,心功能异常(P<0.01);苯肾上腺素(Phe)等收缩剂诱导T2DM组主动脉收缩反应明显增加(P<0.05);L-型钙通道、钙释放和钙库操纵性钙通道(store-operated Ca^(2+)channel,SOCC)介导T2DM组收缩明显增加(P<0.05);ROCK抑制剂Y27632对T2DM组Phe作用的抑制率更明显(P<0.05),PKC抑制剂G 6983对两组作用无差异(P>0.05);Y27632明显抑制SOCC介导的主动脉收缩(P<0.05);T2DM组主动脉组织中的RhoA、ROCK1、Cav1.2和Orai1蛋白表达增加(P<0.05),α1-AR表达无差异(P>0.05);孵育10μmol·L^(-1) Phe的主动脉组织中Cav1.2和Orai1蛋白表达无差异(P>0.05);孵育1μmol·L^(-1)硝苯地平的主动脉组织中RhoA、ROCK1蛋白表达无差异(P>0.05)。结论2型糖尿病小鼠主动脉平滑肌对收缩剂的高反应性,与RhoA/ROCK通路介导钙信号增强密切相关。     

L-型钙通道参与大鼠动脉收缩反应的异质性

目的研究不同动脉对同一血管收缩剂的反应性是否存在差异,及其与L-型钙通道的关系。方法采用离体血管张力测定实验方法,测定在累积浓度血管收缩剂处理下的大鼠胸主动脉、肾内动脉或冠状动脉的张力变化以及L-型钙通道阻断剂硝苯地平(nifedipine)的影响。结果苯肾上腺素(phenylephrine,Phe)对冠状动脉无明显影响,但可诱导胸主动脉和肾内动脉产生明显的浓度依赖性收缩,胸主动脉的pEC_(50)明显大于肾内动脉(P<0.05);给予硝苯地平孵育后,再给予累积浓度的Phe,胸主动脉收缩的下降幅度大于肾内动脉(P<0.05)。5-羟色胺(5-hydroxy tryptamine,5-HT)对胸主动脉的收缩作用不明显,但可浓度依赖性诱导肾内动脉和冠状动脉收缩;给予硝苯地平孵育后,再给予累积浓度的5-HT,冠状动脉收缩的下降幅度明显大于肾内动脉(P<0.05)。胸主动脉、肾内动脉和冠状动脉均对血栓素A2类似物(9,11-dideoxy-11α,9α-epoxymethanoprostaglandin,U46619)产生浓度依赖性收缩,肾内动脉收缩量效曲线的E_(max)最小(P<0.05),胸主动脉收缩量效曲线pEC_(50)最大(P<0.05)。给予硝苯地平孵育后,再给予累积浓度的U46619,冠状动脉收缩的下降幅度最大,胸主动脉收缩的下降幅度最小。结论大鼠胸主动脉、肾内动脉及冠状动脉对同一血管收缩剂的反应存在异质性,其中L-型钙通道介导的收缩作用也不同。     

硝苯地平和环匹阿尼酸对易卒中型肾性高血压大鼠血管平滑肌收缩功能的影响

观察了易卒中型肾性高血压大鼠(SPRHR)主动脉环血管平滑肌在术?后血压升高不同阶段收缩反应性. 与假手术组相比，随着术后血压的升高，低浓度KCl 10～20 μmol·L^-1使SPRHR主动脉环的收缩反应明显增强；苯肾上腺素（Phe）在无钙Krebs液中所致的收缩反应强度降低，而复钙后的收缩张力明显增强；SPRHR主动脉环在无钙Krebs液中温育1 h后，重新复钙后可致收缩反应且为硝苯地平（Nif）所抑制；肌浆网Ca²⁺泵抑制剂环匹阿尼酸（CPA）在SPRHR主动脉环上所致的持续收缩反应也明显强于对照并为Nif所阻断；连续给予Nif，卡托普利180±11 mg·kg^-1·d^-1 8 wk后，SPRHR的血压均明显下降. 在术后1, 4 wk, Nif对部分SPRHR主动脉环在无钙液中复钙所致的收缩反应并无抑制作用且可进一步引起收缩反应. 结果表明，肾性高血压大鼠血管平滑肌收缩功能的高反应性与高血压状态下Ca²⁺转运的失调有关，不同高血压时期的血管平滑肌细胞膜上的Ca²⁺通道特性可能存在差异性.     

布比卡因增强α_1受体介导大鼠胸主动脉收缩反应中血管内皮的作用

目的分析血管内皮在布比卡因(bupivacaine,BUP)增强苯肾上腺素(phenylephrine,Phe)诱发血管收缩反应中的作用。方法制备大鼠离体胸主动脉血管环,采用机械损伤或工具药干扰血管内皮的舒张功能。记录Phe作为α1受体激动剂诱发的动脉收缩反应。结果 BUP(30μmol·L~(-1))与内皮完整血管标本孵育20 min后,Phe诱发的血管收缩Emax值为(2.50±0.05)g,明显高于对照组标本的Emax值[(2.22±0.07)g,P<0.01]。孵育时间缩短至5、10或15min时,BUP无此增强效应。在内皮损伤动脉标本,同浓度BUP孵育20 min时,轻度但明显抑制低浓度Phe诱发的血管收缩反应(P<0.05)。在吲哚美辛、Ch TX、apamin和LNAME预处理的内皮完整血管标本上,ACh诱发的血管舒张反应消失;此时BUP(30μmol·L~(-1)孵育20 min)对Phe诱发的血管收缩反应无明显影响(P>0.05)。结论在大鼠离体胸主动脉,BUP对α1受体介导收缩的增强作用与其抑制血管内皮的舒张功能密切相关,这种抑制作用间接导致Phe诱发的血管收缩反应明显增强。     

哇巴因对豚鼠主动脉环平滑肌作用及与Ca2+、去甲肾上腺素的相互关系

目的观察哇巴因(ouabain,Oua)对豚鼠血管平滑肌的作用,及其与Ca2+和去甲肾上腺素(NE)的相互作用关系.方法利用豚鼠离体胸主动脉环,观察药物对其张力的影响.结果无论有无内皮存在,Oua均能剂量依赖性地直接收缩血管平滑肌.在无钙溶液中,Oua不能引起血管收缩.Oua可使NE的量效曲线平行左移,Emax不变;Oua可使CaCl2的量效曲线左移上移,Emax增大.硝苯地平和维拉帕米可使Oua所致的血管收缩曲线下降.结论Oua所致的血管收缩作用不依赖于血管内皮的存在,但依赖于细胞外钙,并且能被钙拮抗剂所拮抗;NE与Oua,Ca2+对血管平滑肌的收缩呈协同作用.     

心肌细胞钙通道的结构和功能

钙通道既存在于心肌的肌膜上 ,又存在于肌质网膜上。本文讨论的是钙通道的结构 ,相关蛋白质和功能的关系。肌膜的钙通道对于基本的细胞电生理特性和心肌收缩性能的调控 (通过兴奋收缩耦联 )都是非常关键的。1　简介心肌细胞膜上的钙通道 (Ｌ -型和Ｔ -型 )主要是引起钙离子的内流。钙离子的内流可以引起一个使膜电位更正的内向电流 ,同时这种钙离子的内流也是兴奋收缩耦联过程的重要第二信使 ,从而导致细胞的收缩。存在于肌浆网和内质网上的钙释放通道则引起胞内钙的释放 ,从肌浆网中释放的钙可以极大地增强在兴奋收缩过程中从肌膜进入细胞…     

超氧阴离子对牛主动脉平滑肌细胞收缩性的影响

目的观察超氧阴离子对培养的牛动脉平滑肌细胞内钙及收缩性的影响。方法原代培养牛动脉平滑肌,使用数字荧光显微镜技术测定细胞内钙,采用胶原凝胶收缩方法分析收缩性。结果超氧阴离子对高钾溶液及A-23187诱导的钙内流无明显影响(P>0.05),但可使凝胶收缩面积减小。超氧阴离子可使高钾诱导的胶原凝胶收缩面积从18.32%±2.11%减小至5.13%±0.45%(P<0.01),使A-23187诱导的胶原凝胶收缩面积从17.16%±1.10%减小至4.31%±0.04%(P<0.01)。结论超氧阴离子可明显抑制牛主动脉平滑肌细胞收缩性,但对电压依赖性钙通道引起的钙内流及A-23187诱导的细胞内钙变化无影响。     

丹酚酸B镁抑制ATP和KCl诱导大鼠主动脉平滑肌细胞内钙的升高

目的研究丹酚酸B镁(M agnesium lithosperm ate B,MLB)对去内皮离体血管舒缩反应以及对血管平滑肌细胞内游离钙浓度[Ca2+]i的影响。方法去内皮大鼠胸主动脉血管环等张收缩实验和采用钙离子荧光指示剂F luo-3,运用F-4500阳离子测定系统动态检测胸主动脉平滑肌细胞[Ca2+]i。结果血管舒缩实验显示,无钙或常钙条件下MLB对血管基础张力均无作用。MLB 50~200μmol.L-1预给药组抑制无钙条件下苯肾上腺素(PE)1μmol.L-1诱导的血管收缩以及常钙条件下KC l 60 mmol.L-1诱导的血管收缩,并呈浓度相关性。而钙离子通道阻滞剂维拉帕米(Ver)10μmol.L-1则完全阻断KC l诱导的血管收缩。在复钙实验中观察到,MLB 50~200μmol.L-1不仅抑制PE 1μmol.L-1诱导的内钙依赖性血管收缩,而且对复钙后外钙依赖性的血管收缩也有抑制作用。细胞内钙测定实验表明,MLB预孵育的AVSMCs静息态[Ca2+]i没有变化。无钙条件下,MLB 50、100和200μmol.L-1抑制ATP20μmol.L-1诱导内钙释放引起的[Ca2+]i升高,抑制率分别为17.4%、32.4%和61.1%,显示较好的浓度相关性。AVSMCs于常钙条件下用Thapsigargin耗竭钙库后,KCl 60 mmol.L-1诱发外钙内流,引起[Ca2+]i升高,10μmol.L-1的Ver则能完全阻断这种外钙内流。在MLB预给药组,KCl诱导的[Ca2+]i升高降低,抑制率分别为20.0%、32.8%和52.6%。结论MLB能够抑制PE、高K+和复Ca2+诱导的血管收缩,并能抑制ATP和KCl诱导的血管平滑肌细胞内钙的升高,提示MLB对血管平滑肌细胞内钙的影响可能与抑制细胞内钙释放和电压依赖性钙通道有关。     

硝苯地平和环匹阿尼酸对易卒中型肾性高血压大鼠血管平滑肌收缩功能的影响

观察了易卒中型肾性高血压大鼠（ＳＰＲＨＲ）主动脉环血管平滑肌在术后血压升高不同阶段收缩反应性．与假手术组相比，随着术后血压的升高，低浓度ＫＣｌ１０～２０μｍｏｌ·Ｌ－１使ＳＰＲＨＲ主动脉环的收缩反应明显增强；苯肾上腺素（Ｐｈｅ）在无钙Ｋｒｅｂｓ液中所致的收缩反应强度降低，而复钙后的收缩张力明显增强；ＳＰＲＨＲ主动脉环在无钙Ｋｒｅｂｓ液中温育１ｈ后，重新复钙后可致收缩反应且为硝苯地平（Ｎｉｆ）所抑制；肌浆网Ｃａ２＋泵抑制剂环匹阿尼酸（ＣＰＡ）在ＳＰＲＨＲ主动脉环上所致的持续收缩反应也明显强于对照并为Ｎｉｆ所阻断；连续给予Ｎｉｆ，卡托普利１８０±１１ｍｇ·ｋｇ－１·ｄ－１８ｗｋ后，ＳＰＲＨＲ的血压均明显下降．在术后１，４ｗｋ，Ｎｉｆ对部分ＳＰＲＨＲ主动脉环在无钙液中复钙所致的收缩反应并无抑制作用且可进一步引起收缩反应．结果表明，肾性高血压大鼠血管平滑肌收缩功能的高反应性与高血压状态下Ｃａ２＋转运的失调有关，不同高血压时期的血管平滑肌细胞膜上的Ｃａ２＋通道特性可能存在差异性．     

硝苯地平对小白鼠离体小肠收缩活动的影响

目的探讨硝苯地平对小白鼠消化道平滑肌收缩的影响。方法以1%丙酮溶液0.2ml作对照组,采用离体器官灌流的实验方法,分别滴加0.02%硝苯地平0.05ml、0.10ml、0.15ml、0.20ml,观察小白鼠离体小肠收缩曲线下面积和自发收缩活动频率的变化。结果滴加0.02%硝苯地平0.05ml后对离体小肠收缩活动的影响不显著。0.02%硝苯地平在0.10～0.20ml后均能引起离体小肠收缩曲线下面积下降(P<0.05,P<0.01),自发性收缩频率下降(P<0.01),与0.02%硝苯地平0.05ml、1%丙酮溶液0.2ml比较,差异均有统计学意义(P<0.05,P<0.01)。但0.02%硝苯地平在0.10～0.20ml范围内引起离体小肠收缩活动变化差异无统计学意义。结论硝苯地平用量达一定程度对小白鼠离体小肠平滑肌收缩具有抑制作用。     

钙通道在心房颤动时表达及功能变化的研究进展

细胞内钙超载在心房颤动(AF)的发生和维持中的作用被广泛关注。AF时快速心房率引起细胞内钙超载,细胞内钙通道的表达和功能改变,使AF维持。T型钙通道参与基础钙的调节,而心肌收缩时,L型钙通道在钙释放过程中起到触发作用。收缩期胞浆内90%的Ca2+由SR上的RyR2释放;舒张期胞浆内70%～75%的Ca2+由SR上的Ca2+-ATP酶摄取贮存。本文对AF时L型、T型钙通道以及SR上RyR2和Ca2+-ATP酶的表达和功能变化进行概述,为AF的防治提供更多的理论依据。     

Ca~(2+)运动对α_1-肾上腺素受体引起内源性ClC-3氯通道表达的作用

目的　研究Ca2 +运动对α1 肾上腺素受体引起ClC 3氯通道表达的作用。方法　在A10细胞上 ,用RT PCR和Westernblot检测ClC 3mRNA和蛋白质表达情况。结果　苯肾上腺素 ( 10 μmol·L-1)和thapsigargin( 0 1μmol·L-1)可促进ClC 3mRNA和蛋白质的表达 ;SK&F963 65 ( 10 μmol·L-1)和genistein( 10 μmol·L-1)可抑制苯肾上腺素引起的ClC 3的表达 ,nifedipine( 10 μmol·L-1)无此作用。结论　在A10细胞上 ,通过钙池操纵性Ca2 +通道 (SOCC)的Ca2 +内流参与了α1 肾上腺素受体引起的内源性ClC 3氯通道的表达 ,而通过电压依赖性Ca2 +通道 (VDCC)的Ca2 +内流可能与此无关。蛋白酪氨酸激酶参与了ClC 3氯通道的表达     

Characterization of Ca2+ channels involved in endothelin-1-induced contraction of rabbit basilar artery

This study attempted to characterize Ca2+ channels involved in endothelin-1-induced contraction of rabbit basilar artery using whole-cell patch-clamp and measurement of intracellular free Ca2+ concentration. Endothelin-1 activates two types of Ca2+-permeable nonselective cation channels (NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC) in addition to the voltage-operated Ca2+ channel (VOCC). These channels can be discriminated using Ca2+ channel blockers, SK&F 96365 and LOE 908. Tension study was conducted to clarify the Ca2+ channels involved in endothelin-1-induced contraction of basilar artery. Endothelin-1-induced basilar artery contraction is fully dependent on extracellular Ca2+ influx. Based on sensitivity to nifedipine, an L-type VOCC blocker, VOCCs have a minor role in endothelin-1-induced contraction. Both LOE 908 and SK&F 96365 inhibit endothelin-1-induced contraction in a concentration-dependent manner, and their combination abolished it. The median inhibitory concentrations of these blockers for endothelin-1-induced contraction correlated well with those of the endothelin-1-induced [Ca2+]i responses. Thus, the inhibitory action of these blockers on endothelin-1-induced contraction may be mediated by blockade of NSCC-1, NSCC-2, and the SOCC. Extracellular Ca2+ influx through NSCC-1, NSCC-2, and SOCC may be essential for endothelin-1-induced basilar artery contraction.     

Tonic potentiation and attenuation produced by membrane depolarization in guinea-pig trachealis

1. We studied how membrane depolarization directly affected intracellular Ca2+ signalling when voltage-operated Ca2+ channels (VOCC) were not available in guinea-pig tracheal smooth muscle. To block VOCC, we used 3 micromol/L verapamil, which completely abolished high K+ (20-60 mmol/L)-induced contraction, and elevation of fura-2 signal. 2. Muscle tone was generated by adding Ca2+ to the extracellular Ca2+-free solution containing prostaglandin (PG)E2 (100 nmol/L) after abolishing basal tone with indomethacin (1 micromol/L). 3. In the absence of verapamil, high K+ (20-60 mmol/L) solution potentiated 2.4 mmol/l Ca2+-induced sustained contractions. Even in the presence of 3 micromol/L verapamil, replacement with 20 and 40 mmol/L K+ solution induced tonic potentiation, which was changed to attenuation with a higher K+ solution (60 mmol/L), lower extracellular Ca2+ concentration ([Ca2+]o) and pretreatment with cyclopiazonic acid (10 micromol/L), a Ca2+ sequestration inhibitor. 4. These results indicate that the balance between depolarization-dependent Ca2+ release and receptor-operated cation channel inhibition may determine whether tonic potentiation or attenuation is manifested, depending on the availability of VOCC, the magnitude of the depolarization, [Ca2+]o and Ca2+ content in the sarcoplasmic reticulum.     

Effects of phosphoinositide 3-kinase on endothelin-1-induced activation of voltage-independent Ca2+ channels and vasoconstriction

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and a store-operated Ca(2+) channel (SOCC) in rabbit basilar artery (BA) vascular smooth muscle cells (VSMCs). In this study, we investigated the effects of phosphoinositide 3-kinase (PI3K) on ET-1-induced activation of these channels and BA contraction by using PI3K inhibitors, wortmannin and LY 249002. To determine which Ca(2+) channels are activated via PI3K, monitoring of intracellular Ca(2+) concentration was performed. Role of PI3K in ET-1-induced vasoconstriction was examined by tension study using rabbit BA rings. Only NSCC-1 was activated by ET-1 in wortmannin- or LY 294002-pretreated VSMCs. In contrast, addition of these drugs after ET-1 stimulation did not suppress Ca(2+) influx. Wortmannin inhibited the ET-1-induced contraction of rabbit BA rings that depends on the Ca(2+) influx through NSCC-2 and SOCC. The IC(50) values of wortmannin for the ET-1-induced Ca(2+) influx and vasoconstriction were similar to those for the ET-1-induced PI3K activation. These results indicate that (1) NSCC-2 and SOCC are stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated via PI3K-independent cascade; (2) PI3K is required for the activation of the Ca(2+) entry, but not for its maintenance; and (3) PI3K is involved in the ET-1-induced contraction of rabbit BA rings that depends on the extracellular Ca(2+) influx through SOCC and NSCC-2.     

Tetrandrine is not a selective calcium channel blocker in vascular smooth muscle.

The effects of tetrandrine (Tet) on the contractile properties of rat aortic ring preparations were studied to test the hypothesis that Tet is a Ca2+ antagonist acting on voltage-operated Ca2+ channels (VOC). The tests were performed on contractions induced by depolarizing concentrations of KCl and by alpha 1-adrenoceptor agonist, phenylephrine (Phe). These vascular effects of Tet were compared to those of nifedipine (Nif). We found that Tet behaved qualitatively similar to, but less potent than, Nif in that it inhibited KCl-induced contraction in a concentration-dependent fashion and its inhibitory effect was long-lasting. However, the effects on Phe-induced contraction of Tet was different from those of Nif in that the extracellular Ca(2+)-dependent contraction was inhibited by Tet, but not by Nif. Tet (60 mumol.L-1) completely inhibited the 45Ca2+ uptake induced by KCl and Phe in rat aortic muscle strips. When the aortic muscle contractile response was induced by addition of Ca2+ following depletion of intracellular stores by Phe in the presence of sarcoplasmic reticulum Ca(2+)-pump inhibitor, cyclopiazonic acid, Tet (60 mumol.L-1) was more effective than Nif 1 mumol.L-1 in inhibiting such a response to extracellularly added Ca2+. Furthermore, Tet, but not Nif, also significantly inhibited the contraction to Phe in Ca(2+)-free medium. Collectively, these results led us to conclude that Tet does not behave as a selective VOC blocker like Nif.     

ROLE OF EXTRACELLULAR Na+, Ca2+-ACTIVATED Cl- CHANNELS AND BK CHANNELS IN THE CONTRACTION OF Ca2+ STORE-DEPLETED TRACHEAL SMOOTH MUSCLE

• 1 In the present study, we investigated the series of events involved in the contraction of tracheal smooth muscle induced by the re‐addition of Ca²⁺ in an in vitro experimental model in which Ca²⁺ stores had been depleted and their refilling had been blocked by thapsigargin.
• 2 Mean (±SEM) contraction was diminished by: (i) inhibitors of store‐operated calcium channels (SOCC), namely 100  µ mol/L SKF‐96365 and 100  µ mol/L 1‐(2‐trifluoromethylphenyl) imidazole (to 66.3 ± 4.4 and 41.3 ± 5.2% of control, respectively); (ii) inhibitors of voltage‐gated Ca²⁺ channels Ca_V1.2 channels, namely 1  µ mol/L nifedipine and 10  µ mol/L verapamil (to 86.2 ± 3.4 and 76.9 ± 5.9% of control, respectively); and (iii) 20  µ mol/L niflumic acid, a non‐selective inhibitor of Ca²⁺‐dependent Cl^? channels (to 41.1 ± 9.8% of control). In contrast, contraction was increased 2.3‐fold by 100 nmol/L iberiotoxin, a blocker of the large‐conductance Ca²⁺‐activated K⁺ (BK) channels.
• 3 Furthermore, contraction was significantly inhibited when Na⁺ in the bathing solution was replaced by N‐methyl–d ‐glucamine (NMDG⁺) to 39.9 ± 7.2% of control, but not when it was replaced by Li⁺ (114.5 ± 24.4% of control). In addition, when Na⁺ had been replaced by NMDG⁺, contractions were further inhibited by both nifedipine and niflumic acid (to 3.0 ± 1.8 and 24.4 ± 8.1% of control, respectively). Nifedipine also reduced contractions when Na⁺ had been replaced by Li⁺ (to 10.7 ± 3.4% to control), the niflumic acid had no effect (116.0 ± 4.5% of control).
• 4 In conclusion, the data of the present study demonstrate the roles of SOCC, BK channels and Ca_V1.2 channels in the contractions induced by the re‐addition of Ca²⁺ to the solution bathing guinea‐pig tracheal rings under conditions of Ca²⁺‐depleted sacroplasmic reticulum and inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase. The contractions were highly dependent on extracellular Na⁺, suggesting a role for SOCC in mediating the Na⁺ influx.

     

Ca2+ entry channels involved in contractions of rat aorta induced by endothelin-1, noradrenaline,and vasopressin

Endothelin-1 (ET-1) has been shown to activate three types of Ca2+ channel, namely two Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC), and that these channels can be discriminated by Ca2+ channel blockers such as LOE 908 (a blocker of NSCC-1 and NSCC-2) and SK&F 96365 (a blocker of NSCC-2 and SOCC). This study pharmacologically compared Ca2+ entry channels involved in contractions of rat thoracic aorta without endothelium induced by ET-1, noradrenaline (NA), or arginine-vasopressin (AVP). These agonists-induced contractions of aortic rings without endothelium and increases in the intracellular free Ca2+ concentration ([Ca2+]i) of cultured aortic smooth muscle cells were abolished by removal of extracellular Ca2+. A blocker of L-type voltage-operated Ca2+ channel (VOCC), nifedipine had no effect on the responses to ET-1, but it suppressed the responses to NA and AVP to 70% and 65% of control responses, respectively. LOE 908 partially suppressed the nifedipine-resistant responses to ET-1 and AVP, but not those to NA. SK&F 96365 also partially suppressed the nifedipine-resistant responses to ET-1 and AVP, whereas it abolished the responses to NA. LOE 908 in combination with SK&F 96365 abolished the nifedipine-resistant responses to either of the agonists. These results show that the contraction of rat aorta involves different Ca2+ entry channel depending on agonists: (a) NSCC-1, NSCC-2, and SOCC for ET-1; (b) VOCC and SOCC for NA; and (c) VOCC, NSCC-1, NSCC-2, and SOCC for AVP.     

Ca2+ buffering action of sarcoplasmic reticulum on Bay k 8644-induced Ca2+ influx in rat femoral arterial smooth muscle

We examined the Ca2+ buffering action of sarcoplasmic reticulum during the stimulation of arterial smooth muscle with Bay k 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyr idine-5-carboxylate]. The effects of Bay k 8644 on tension and cellular Ca2+ level were first determined in endothelium-denuded strips of rat femoral artery. The Ca2+ buffering action was examined by using cyclopiazonic acid and thapsigargin to inhibit Ca2+-ATPase of sarcoplasmic reticulum and ryanodine to deplete Ca2+ stored in sarcoplasmic reticulum. The addition of Bay k 8644 (0.3-300 nM) to the resting strips almost failed to cause a contraction. When the strips were preincubated with 10 microM cyclopiazonic acid, Bay k 8644 induced a concentration-dependent contraction that is antagonized by nifedipine. The maximum contraction induced by Bay k 8644 in the presence of cyclopiazonic acid was comparable to the maximum contraction induced by 65.9 mM K+-depolarization and the ED50 value for Bay k 8644 was around 5 nM. Similar results were obtained when the strips were preincubated with 30 nM thapsigargin or 10 microM ryanodine. Bay k 8644 also induced a strong contraction when the extracellular K+ concentration was elevated. During the stimulation with 100 nM Bay k 8644, the Ca2+ influx was increased. We conclude that in rat femoral arterial smooth muscle, (1) the Ca2+ influx induced by Bay k 8644 is completely buffered by Ca2+ uptake into the sarcoplasmic reticulum, and (2) this sarcoplasmic reticulum can buffer a large amount of Ca2+ that induces a maximum contraction.     

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.