Kovac Jason R, Preiksaitis Harold G, Sims Stephen M
Dept. of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada.
Am J Physiol Gastrointest Liver Physiol. 2005 Dec;289(6):G998-1006. doi: 10.1152/ajpgi.00529.2004. Epub 2005 Jul 14.
Excitation of human esophageal smooth muscle involves the release of Ca(2+) from intracellular stores and influx. The lower esophageal sphincter (LES) shows the distinctive property of tonic contraction; however, the mechanisms by which this is maintained are incompletely understood. We examined Ca(2+) channels in human esophageal muscle and investigated their contribution to LES tone. Functional effects were examined with tension recordings, currents were recorded with patch-clamp electrophysiology, channel expression was explored by RT-PCR, and intracellular Ca(2+) concentration was monitored by fura-2 fluorescence. LES muscle strips developed tone that was abolished by the removal of extracellular Ca(2+) and reduced by the application of the L-type Ca(2+) channel blocker nifedipine (to 13 +/- 6% of control) but was unaffected by the inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). Carbachol increased tension above basal tone, and this effect was attenuated by treatment with CPA and nifedipine. Voltage-dependent inward currents were studied using patch-clamp techniques and dissociated cells. Similar inward currents were observed in esophageal body (EB) and LES smooth muscle cells. The inward currents in both tissues were blocked by nifedipine, enhanced by Bay K8644, and transiently suppressed by acetylcholine. The molecular form of the Ca(2+) channel was explored using RT-PCR, and similar splice variant combinations of the pore-forming alpha(1C)-subunit were identified in EB and LES. This is the first characterization of Ca(2+) channels in human esophageal smooth muscle, and we establish that L-type Ca(2+) channels play a critical role in maintaining LES tone.
人类食管平滑肌的兴奋涉及细胞内钙库释放Ca(2+)以及Ca(2+)内流。食管下括约肌（LES）具有强直性收缩的独特特性；然而，维持这种特性的机制尚未完全明确。我们研究了人类食管肌肉中的Ca(2+)通道，并探讨了它们对LES张力的作用。通过张力记录检测功能效应，采用膜片钳电生理学记录电流，利用逆转录聚合酶链反应（RT-PCR）探索通道表达，并通过fura-2荧光监测细胞内Ca(2+)浓度。LES肌条产生的张力在去除细胞外Ca(2+)后消失，应用L型Ca(2+)通道阻滞剂硝苯地平后降低（降至对照的13±6%），但不受环匹阿尼酸（CPA）抑制肌浆网Ca(2+)-ATP酶的影响。卡巴胆碱使张力高于基础张力，CPA和硝苯地平处理可减弱这种效应。使用膜片钳技术和分离细胞研究电压依赖性内向电流。在食管体（EB）和LES平滑肌细胞中观察到类似的内向电流。两种组织中的内向电流均被硝苯地平阻断，但被Bay K8644增强，并被乙酰胆碱短暂抑制。利用RT-PCR探索Ca(2+)通道的分子形式，在EB和LES中鉴定出相似的孔形成α(1C)-亚基剪接变体组合。这是首次对人类食管平滑肌中的Ca(2+)通道进行表征，我们证实L型Ca(2+)通道在维持LES张力中起关键作用。
