【作者】 李鹏云；

【导师】 曾晓荣；

【作者基本信息】 泸州医学院 ， 生理学， 2006， 硕士

【摘要】 目的:大电导钙激活钾通道(BKCa通道)是血管平滑肌舒缩调控机制中的重要靶点。平滑肌细胞肌浆网上三磷酸肌醇受体(inositol1,4,5-trisphosphate receptors,IP3Rs)和兰诺定受体(ryanodine receptors,RyRs)介导的Ca2+释放可引起细胞内局部[Ca2+]显著增加,激活邻近胞膜上BKCa通道的开放产生自发性瞬时外向电流(spontaneous transient outwardcurrents,STOCs),进而引起细胞膜超极化,电压依赖性Ca2+内流减少,负反馈调节平滑肌的收缩。目前多数研究已经证实在血管平滑肌,RyRs介导的Ca2+释放与BKCa通道之间的耦联对于血管舒缩活性的调控具有重要意义,而三磷酸肌醇(inositol 1,4,5-trisphosphate,IP3)作为一种重要的胞内第二信使,其调控血管平滑肌BKCa通道的机制还存在较多争论。前期研究已证实了IP3对猪冠状动脉平滑肌细胞BKCa的单通道活性具有激活作用,但其对于全细胞构型时BKCa通道的调控机制目前尚不清楚。由于IP3极易降解不稳定,且目前国内外缺乏可透膜的IP3衍生物,本实验拟采用IP3的前体物质尿嘧啶三磷酸(uridine 5’-triphosphate,UTP)作为IP3替代物,研究其对于急性酶分离的猪冠状动脉平滑肌细胞STOCs的作用,探讨IP3对全细胞构型时BKCa通道的作用机制,为下一步结合激光扫描共聚焦显微成像技术研究细胞内Ca2+信号的动力学与STOCs之间的相关性奠定基础。方法:更多还原

【Abstract】 Objective: Large-conductance Ca2+-activated potassiumchannels (BKCa channels) play a pivotal role in the regulation of vasomotion.Inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs)endowed with sarcoplasmic reticulum (SR) are involved in the regulation ofSTOCs. It has been well established that Ca2+ release mediated by RyRsprovides a negative feedback mechanism for contraction by hyperpolarizing thecell membrane and thereby reducing activated Ca2+ entry throughvoltage-dependent L-type Ca2+ channels. As a crucial intracellular secondmessenger, inositol 1,4,5-trisphosphate (IP3) interacts with IP3 receptors (IP3Rs),which then leads to local intracellular Ca2+ transients and thereby to be involvedpotentially in the regulation of BKCa channels. However, the underlyingmechanisms seem controversial. Our previous study has demonstrated thatBKCa channels in excised patches were significantly activated by IP3 in freshlyisolated porcine coronary artery smooth muscle cells. However, little is knownabout its effect on BKCa channels in whole-cell patches. In present study,considering no membrane-penetrable IP3 is available as well as its unstablefeature, we applied IP3-generating agonist-uridine 5’-triphosphate(UTP) insteadof IP3 to study indirectly the regulation of IP3 on STOCs (i.e. BKCa channels) inthe same preparation. Methods: The coronary artery was excised from the freshporcine heart and cut into small segments (2mm×5mm) and then transferred toenzymatic dissociation solution for incubation. Single smooth muscle cellswere obtained by two-step enzyme digestion at 37οС. STOCs were recorded bya whole-cell, amphotericin-perforated configuration of patch-clamp techniques.The currents were amplified and filtered by patch clamp amplifier (Axopatch200B), and then the digitized data were recorded by a Lenovo-compatiblepersonal computer running pClamp9.0 software and further analyzed byMiniAnalysis6.0 program. The followings were studied: ①The characteristicsof STOCs in porcine coronary artery smooth muscle cells were observed.②Recorded and analyzed the effect of UTP on STOCs. ③Studied the effect ofU73122(PLC blocker)on STOCs regulated by UTP. ④ Ca2+ entry throughvoltage-dependent L-type Ca2+ channels as well as intracellular Ca2+ releasewere studied to explore their possible effect on the underlying mechanisms ofSTOCs regulated by UTP. Results: 1.The characteristics of STOCs in freshlyisolated porcine coronary artery smooth muscle cells were as follows: ①Thecurrents were voltage-dependent and extracellular calcium-dependent and hadhighly variable amplitude and frequency;STOCs superimposed stochasticallyonto whole-cell BKCa currents.② STOCs activity was completely abolished byChTX(200nM)(n=5);or by removal of extracellular Ca2+(n=5).WhileCdCl2(200μM) and Verapamil(20μM), specific inhibitors of voltage-dependentL-type Ca2+ channels, had little effect on STOCs(P>0.05;n=5). 2. IntracellularCa2+ release was involved in the regulation of STOCs by UTP.① STOCsactivity was completely blocked by ryanodine(50μM)(n=6);2APB(40μM),amembrane-penetrable IP3Rs antagonist, also greatly suppressed the activity ofSTOCs(P<0.05;n=14), STOCs amplitude and frequency decreased by 25.88±9.60% and 29.22±12.75% respectively.② UTP (40μM), an IP3-generatingagonist, led to conspicuous increases in STOCs amplitude by 57.54±5.34%and frequency by 77.46±8.42% (P<0.01;n=38).③ In the presence of UTP,2APB(40μM) suppressed STOCs amplitude by 24.08±3.97%(P<0.05;n=8),whereas it had little effect on STOCs frequency(P>0.05;n=8).Furtherapplication of 2APB(80μM) caused a significantly decrease in STOCsamplitude and frequency by 31.43 ± 6.34% and 40.59 ± 19.01%respectively(P<0.05;n=6). In addition, UTP (40μM) failed to activate STOCs inthe presence of 2APB(40μM) or ryanodine (50μM).④ U73122 itself caused asignificantly decrease in STOCs amplitude and frequency by 31.04±7.46%and 41.65±16.59% respectively(P<0.05;n=10), and subsequent exposing cellsto UTP (40μM) failed to reactivate STOCs. U73122(5μM) also suppressedSTOCs activity initiated by UTP by 52.92±4.72%(P<0.05;n=9) and 66.53±9.89% respectively(P<0.01;n=9).⑤ CdCl2(200μM) and Verapamil(20μM)had little effect on UTP-induced STOCs activation(P>0.05;n=8).Conclusions:1. STOCs in freshly isolated porcine coronary artery smooth muscle cells arisedfrom BKCa channels, which were voltage-dependent and extracellularcalcium-dependent. However, voltage-dependent L-type Ca2+ channels playedlittle role in the regulation of STOCs activity. 2. IP3-generating agonist-UTPstimulated PLC-IP3 signal transduction pathways. Complex Ca2+-mobilizingpathways, including internal Ca2+ release through IP3Rs and RyRs, wereinvolved in STOCs regulation as well as UTP mediated STOCs enhancement.更多还原