【摘要】 应用生物换能技术和Ca2+通道特异性阻断剂观察并记录大鼠离体远端结肠平滑肌收缩张力的变化,分析库容性 Ca2+内流(capacitative Ca2+ entry,CCE)是否与ACh诱导的离体远端结肠平滑肌收缩反应有关。结果表明,以无钙的Krebs 液灌流或应用EGTA螯合细胞外Ca2+后,高K+及ACh引起的远端结肠平滑肌收缩几乎完全消失。电压操纵性Ca2+通道阻断剂veiapamil也能减弱高K+及ACh引起的远端结肠平滑肌收缩,其减弱的程度分别为74％和41％。在无钙的Krebs液中, 5 μmol／L ACh可引起离体肠管瞬时性收缩,这是由肌质网(sarcoplasmic reticulum,SR)释放钙所致;然后加入10 μmol／L阿托品(atropine),并在此基础上恢复细胞外Ca2+(2．5 mmol／L),结肠平滑肌则出现持续性收缩,待收缩反应达峰值时,加入5μmol／L verapamil,收缩无明显变化,且该收缩反应对钙库操纵性通道(store-operated Ca2+ channel,SOCC)阻断剂La3+ 敏感,20,50和100 μmol／L的La3+使上述收缩张力分别降低15％,23％和36％,且呈浓度依赖性,但对Cd2+不敏感。研究结果提示,细胞外Ca2+内流对高K+及ACh介导的离体远端结肠平滑肌持续性收缩是必需的,由ACh诱导的远端结肠平滑肌收缩至少包括SR释放钙引起的短暂性收缩及受体操纵性Ca2+通道(receptor-operated Ca2+ channel,ROCC)、电压操纵性Ca2+通道(voltage-operated Ca2+ channel,VOCC)和CCE介导的胞外Ca2+内流等途径。这将从通道水平进一步分析消化管平滑肌收缩的机制和特征,亦将为预防和控制因胃肠动力紊乱所致的消化管疾病寻求有针对性的药物干预和治疗提供理论依据。更多还原

【Abstract】 Contraction of smooth muscle cells is triggered by an increase in cytosolic Ca2+ upon agonist stimulation. Ca2+ influx across the plasma membrane constitutes a major component of the agonist-induced response in smooth muscle cells. Traditionally, voltage-operated Ca2+ channel (VOCC) is considered as the channel mediating the Ca2+ entry. However, this view has been challenged by recent discoveries, which demonstrated that other types of ion channels, such as store-operated and/or receptor-operated Ca2+ channels (SOCC and/or ROCC), also participate in Ca2+ response induced by agonists in smooth muscle cells. SOCC is defined as the channel activated in response to the depletion of the internal Ca2+ stores, an event secondary to G protein coupled receptor or receptor tyrosine kinase stimulation. The Ca2+ flow mediated by SOCC is termed as capacitative Ca2+ entry (CCE). Previous study from other group has demonstrated that VOCC played a predominant role in ACh-induced contraction of distal colon smooth muscle in guinea pig. However, whether SOCC participates in the agonist-induced contractile response in this particular tissue is unknown. The present study was performed to investigate the role of CCE in ACh-induced mechanical activity of distal colon smooth muscle in rats. The contractile function of the smooth muscle was assessed by measuring isometric force of isolated rat distal colon rings. We showed that both high extracellular K+ (40 mmol/L) and ACh (5 μmmol/L) evoked striking contraction of the smooth muscle. The contractile responses were almost abolished by removal of extracellular Ca2+ with ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’ tetraacetic acid (EGTA), suggesting a critical contribution of extracellular source of Ca2+ to the contraction. Verapamil (5 μmol/L), an L-type VOCC blocker, significantly attenuated, but didn’t completely eliminate the high K+- and ACh-induced contraction (74% and 41% for high K+ and ACh, respectively), indicating that additional channels might be involved in the contractile mechanism. Furthermore, ACh only induced transient contractions in the absence of extracellular Ca2+. Readmission of Ca2+ into the extracellular compartment resulted in a significant and sustained increase in the tension of the smooth muscle. This response was not affected by verapamil (5 μmol/L) and Cd2+ (5 μmol/ L), both of which efficiently block VOCC at the doses. However, La3+, a known inhibitor of SOCC, significantly suppressed the Ca2+ readdition-induced contraction in a dose-dependent manner. On the basis of these results, we conclude that contraction of smooth muscle in the distal colon is regulated by multiple Ca2+ channels. In addition to VOCC-mediated Ca2+ influx, SOCC-mediated CCE participates in agonist-induced contractile response of distal colon smooth muscle in rats.更多还原