a-亚基蛋白磷酸化修饰与胆固醇代谢紊乱兔BKca通道活性及SO功能调节

Cholesterol metabolic disturbance and cholestatic cholecystitis is the base of gallstone formation. Our previous findings demonstrated that cholesterol can cause SO (sphincter of Oddi, SO)dysfunction and induce cholestatic cholecystitis in rabbits, and SO dysfunction is closely related to [Ca2+]i overload induced by down-regulation of BKca channel activity. As Ca2+/CaM dependent protein kinase, CaMK-Ⅱ is the key intracellular components to activate the BKca channel in SO cell. Our experimental results showed that the cellular expression of SO is normal with the up-regulated activity of CaMK-Ⅱ in rabbits of cholesterol metabolic disturbance under the [Ca2+]i overload, and the expression of tyrosine phosphorylation in α-subunit of BKca channel. It is suggested that the abnormal phosphorylation modification of SO cell in rabbits of cholesterol metabolic disturbance has occurred. The study reveal the changes in channel phosphorylation of α-subunit, channel K+,Ca2+ current, membrane potential and SO tension in rabbits of cholesterol metabolic disturbance using a variety of methods such as molecular biology, and explore the relationship between phosphorylation and regulatory activity of BKca channel protein in rabbits of cholesterol metabolic disturbance and the role in functional regulation of SO, to provide experimental and theoretical basis for clarifying mechanism of SO dysfunction in rabbits of cholesterol metabolic disturbance.

胆固醇代谢紊乱及胆囊淤胆是胆囊结石形成的基础。已经证实，胆固醇可致兔胆管SO功能紊乱并诱发胆囊淤胆，而SO功能紊乱与BKca通道活性下调导致的[Ca2+]i超载密切相关。CaMK-Ⅱ是Ca2+/CaM依赖性激酶，是激活SO细胞BKca通道的关键细胞内组分。我们实验结果显示，[Ca2+]i超载状态下，胆固醇代谢紊乱兔SO细胞CaM表达正常，CaMK-Ⅱ活性已经上调，但BKca通道a-亚基酪氨酸磷酸化蛋白表达量下调,提示胆固醇代谢紊乱兔SO细胞 BKca通道蛋白磷酸化修饰已发生异常。本实验采用分子生物学等多种方法，检测胆固醇代谢紊乱兔SO细胞BKca通道a-亚基磷酸化修饰、通道K+、Ca2+电流及膜电位和SO肌张力变化，探讨胆固醇代谢紊乱兔SO细胞BKca通道蛋白磷酸化修饰与通道活性调节关系及其在SO功能调节中的地位和作用，为阐明胆固醇代谢紊乱兔SO功能紊乱发生机制提供实验和理论基础。

壶腹括约肌（sphincter of Oddi, SO）在维护胆管内流体环境的稳定和调节胆汁流动中起关键作用。胆囊胆汁淤积是胆囊结石形成的动力学基础，而胆囊是否淤胆与SO的功能状态密切相关。前期研究证实，高胆固醇血症（hypercholesterol, HC）可诱发兔胆囊淤胆并形成结石，这与其导致SO胞内钙离子浓度（intracellular calcium concentration, [Ca2+]i）超载及BKCa通道蛋白磷酸化修饰异常，进而引发SO功能紊乱关系密切，但其内在分子机制尚不明确。.本课题表明，在HC条件下，兔SO BKCa通道α-和β1-亚基表达无显著变化，而α-亚基酪氨酸磷酸化明显降低。肌环张力实验结果显示，HC可引起乙酰胆碱预收缩的兔SO肌环张力升高。加入BKCa通道激活剂（NS1619）或酪氨酸磷酸酶抑制剂（Na3VO4，促进酪氨酸磷酸化）时，都会引起HC兔的SO肌环张力降低。而添加酪氨酸激酶抑制剂（Genistein，抑制酪氨酸磷酸化）会引起HC兔SO肌环张力升高。且电生理实验结果表明，HC引起[Ca2+]i 超载，但兔SO BKCa通道活性下调。当促进通道蛋白酪氨酸磷酸化时可增强HC组SO BKCa通道活性；而升高SO细胞的[Ca2+]i（加钙通道激动剂Bayk8644）可增强CON组BKCa通道活性。但抑制通道蛋白酪氨酸磷酸化或SO的[Ca2+]i（加钙通道抑制剂,nifedipine）时，两组的BKCa通道活性都会下调。此外，共聚焦实验表明，HC条件下，增加BKCa通道α-亚基酪氨酸磷酸化水平可使SO细胞的[Ca2+]i减少，而降低α-亚基酪氨酸磷酸化水平会升高SO的[Ca2+]i。由此可见，BKCa通道α-亚基酪氨酸磷酸化水平正常或上调时，升高的[Ca2+]i才可以激活SO BKCa通道，且HC兔SO的[Ca2+]i与BKCa通道α-亚基酪氨酸磷酸化修饰密切相关。.综上所述，本课题表明兔SO BKCa通道α-亚基蛋白酪氨酸磷酸化修饰不仅很可能是通道活性及SO功能调节的关键因子，更有可能是胆固醇代谢紊乱兔SO细胞[Ca2+]i超载的根本原因。因此，本课题有助于明确SO功能调节的内在机制，为SOD和胆囊胆固醇胆结石形成的分子机制研究奠定了理论基础，对此类病症的预防和治疗有重要的临床意义。