Direct block of Ca2+ channels by calmidazolium in cultured vascular smooth muscle cells.
Direct block of Ca2+ channels by calmidazolium in cultured vascular smooth muscle cells.
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卡米达唑直接阻断培养的血管平滑肌细胞中的 Ca2+ 通道。
DOI：
10.1097/00005344-199910000-00003
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发表时间：
1999
影响因子：
3
通讯作者：
Sperelakis,N
中科院分区：
文献类型：
--
作者：
Sunagawa,M;Yokoshiki,H;Seki,T;Nakamura,M;Laber,P;Sperelakis,N
采用全细胞电压钳技术，研究钙调素（CaM）抑制剂钙咪唑（calmidazolium，CMZ）对培养的血管平滑肌（vascularsmoothmuscle，VSM）细胞（A7 r5细胞株）L型钙电流（ICa（L））的作用。所有实验均在室温（24-25 ℃）下进行。通过从− 60 mV的保持电位到+ 10 mV的测试电位，每15 s诱发一次峰值I Ba（5 mM Ba 2+作为电荷载体的Ca 2+通道电流）。为了将细胞内游离Ca 2+浓度（[Ca] i）提高至pCa 6.5，移液管溶液含有Ca 2+-EGTA缓冲液（pCa 6.5）以允许与细胞平衡。1 μM CMZ使I Ba峰振幅降低36.7±4.9%（n= 8），最大作用出现在7-8 min内。即使在10分钟的洗脱后也未观察到I Ba的恢复。即使在移液器溶液中存在CaM依赖性蛋白激酶-II的肽抑制剂（5.2 μM），CMZ也可抑制I Ba至27.8±5.3%（n= 7）。为了排除其他Ca 2+/CaM依赖性激酶和磷酸酶可能调节Ca 2+通道活性的可能性，我们用Ca 2+/EGTA缓冲液检测了CMZ对[Ca] i降低时I Ba的影响。在pCa ~（10）时，CMZ对IBa的抑制率为33.4±5.9%（n= 14），半数抑制浓度（IC 50）为0.29 μM。激活曲线（pCa ≥ 10）正向移动6.3 mV;灭活曲线负向移动5.0 mV。CMZ在重复阶跃去极化过程中逐渐降低I Ba。CMZ不会减慢失活的恢复速率。CMZ对Ca 2+通道电流的抑制作用具有使用依赖性。这种抑制作用不依赖于CaMK-Ⅱ和其它Ca 2+/CaM依赖性通路。这可能与CMZ直接阻断Ca 2+通道有关. CMZ可降低细胞外表面电荷，阻断Ca 2+通道的开放状态.
We investigated the action of calmidazolium (CMZ), an inhibitor of calmodulin (CaM), on the L-type Ca 2+ currents (I Ca (L)) of cultured vascular smooth muscle (VSM) cells (A7r5 cell line), by using the whole-cell voltage-clamp method. All experiments were conducted at room temperature (24-25 C). The peak I Ba (Ca 2+ channel current with 5 mM Ba 2+ as charge carrier) was evoked every 15 s by a test potential to+ 10 mV from a holding potential of− 60 mV. To elevate intracellular free Ca 2+ concentration ([Ca] i) to pCa 6.5, the pipette solution contained a Ca 2+-EGTA buffer (pCa 6.5) to allow equilibration with the cells. Bath application of 1 μM CMZ reduced the peak amplitude of I Ba to 36.7±4.9%(n= 8); maximal effect occurred within 7-8 min. Peak I Ba continued to decrease even after washing out the CMZ. Recovery of I Ba was not observed even after 10 min of washout. Even in presence of an peptide inhibitor of CaM-dependent protein kinase-II (5.2 μM) in the pipette solution, CMZ inhibited I Ba to 27.8±5.3%(n= 7). To exclude the possibility that other Ca 2+/CaM-dependent kinases and phosphatases may regulate Ca 2+ channel activity, we examined the effect of CMZ on I Ba when [Ca] i was reduced by use of Ca 2+/EGTA-buffered pipette solutions. At pCa≈ 10 (10 mM EGTA and only contaminant Ca 2+), CMZ inhibited I Ba to 33.4±5.9%(n= 14) with a median inhibitory concentration (IC 50) value of 0.29 μM. The activation curve (pCa≈ 10) was shifted in the positive direction by 6.3 mV; the inactivation curve was shifted in the negative direction by 5.0 mV. CMZ decreased I Ba progressively during repetitive step depolarizations. CMZ did not slow the rate of recovery from inactivation. In conclusion, CMZ inhibits Ca 2+ channel current in a use-dependent manner. This inhibition is independent of CaMK-II and other Ca 2+/CaM-dependent pathways. Therefore it is likely due to direct blockade of Ca 2+ channels by CMZ. CMZ may reduce the outer surface charge and block the open state of the Ca 2+ channels.