蛋白激酶C调节的1,4,5-三磷酸肌醇受体磷酸化在胆囊收缩素介导的胃窦平滑肌细胞钙动员中的作用

The role of protein kinase C-mediated phosphorylation of type HI inositol 1,4,5-triphosphate receptor in cholecystokinin octapeptide induced calcium mobilization in gastric antral smooth muscle cells

目的研究八肽胆囊收缩素(CCK-8)对大鼠胃窦平滑肌细胞(SMC)胞内钙释放和胞外钙内流的作用及对其具体相关机制的探讨。方法 (1)多导生理记录仪记录大鼠离体胃窦肌条在不同条件下的收缩活动;(2)免疫印迹法和免疫沉淀法检测胃窦 SMC 的三型1,4,5-三磷酸肌醇受体(Ins P_3R3)及其磷酸化水平;(3)Fura-2/AM 标记胃窦 SMC,观察 CCK-8S 对胞内钙离子浓度([Ca^(2+)]i)的影响;(4) 全细胞膜片钳检测胃窦 SMC 的 L-型电压门控钙通道电流(I_(Ca-L))的变化情况。结果(1)CCK-8S作用下胃窦肌条收缩幅值和频率改变明显[增长率分别为(62±13)%和(58±17)%,均P<0.01],可被 CCK-A 受体拮抗剂和钙泵抑制剂所阻断;(2)蛋白激酶 C(PKC)上调 InsP_3 R3磷酸化水平,抑制 CCK-8S 介导的内钙释放;(3)CCK-8S 引起的[Ca^(2+)]i 显著升高[从(69±7)mol/L 升至(472±36)nmol/L,P<0.01]分别可被 CCK-A 受体或胞内钙泵抑制剂和 PKC 激动剂所阻断;去除外钙或给予 L-型钙通道阻滞剂时 CCK-8S 仍可引起[Ca^(2+)]i 升高;(4)CCK-8S 显著增强胃窦 SMC 的I_(Ca-L)[从(-56±7)pA 升至(-89±6)pA,P<0.01],可分别被 I_(Ca-L)阻滞剂、胞内钙泵抑制剂和钙依赖性氯通道阻滞剂所阻断。结论 CCK-8S 引起的大鼠胃窦 SMC 的[Ca^(2+)]i 升高依赖于 PKC 介导的InsP_3R3磷酸化作用调节下的细胞内钙离子释放。胞内钙释放可激活 I_(Cl-Ca),引起细胞膜去极化而活化 I_(Ca-L)引起胞外钙内流,最终引起 SMC 收缩效应。

Objective To study the effects of sulfated cholecystokinin octapeptide （sCCK-SS） on intracellular calcium release and extracellular calcium influx in gastric antral smooth muscle cells （ SMC ） and the mechanism thereof. Methods （ 1 ） Longitudinal muscle （LM） and circular muscle （CM） strips of gastric antrum and pylorus were isolated from SD rats and suspended in a tissue chamber to record the contractile responses by polyphysiogTaphy. （2） Immunoprecipitation, electrophoresis, and immunoblotting were used to detect the phosphorylation of type Ⅲ inositol 1,4, 5- triphosphate receptor （ InsP3 R3 ） in the SMCs. （3） The responsiveness of gastric SMC to CCK-SS was examined by using fura-2-1oaded microfluorimetric measurement of intracellular calcium concentration （ [ Ca^2＋ ] i）. （4） The current of L-type calcium channels （ICaL） was recorded by using patch-clamp techniques. Results （ 1 ） Significant changes to CCK-SS were found in the mean contractile amplitude of the CIM and frequency of LM of gastric antrum and could be suppressed by CCK-A receptor （CCK-AR） antagonist and ATPase inhibitors. （2） CCK-8S stimulation of SMC resulted in PKC-depondent phosphorylation of the InsP3R3. （3） CCK-SS-evoked significant increase in [ Ca^2 ＋ ] i [ from （69 ± 7 ） mol/L to （472 ± 36 ） nmol/L, P 〈 0. 01 ] could be suppressed by CCK-AR antagonist, ATPase inhibitors and protein kinase C （PKC） activator; whereas on condition that extracellular calcium was removed or L-type calcium inhibitor nifidipine was added a small but significant increase of [Ca^2＋ ] i could be still elicited by CCK-8S. （4） CCK-SS-intensified calcium current [ from（ -56 ±7）pA to（ -89 ± 6）pA,P 〈0.01 ] could be apparently inhibited by respective administration of nifidipine, ATPase inhibitors, and calcium dependent chloride channel （ICl-Ca） blocker （ all P 〈 0.01 ）. Conclusion CCK-SS-evoked [ Ca^2＋] i increase in gastric antral SMCs depends on the release of intracellular calcium stores, which is regulated by PKC mediated phosphorylation of InsP3 R3. The released intracellular calcium in turn activates the L-type voltage-dependent calcium channels （VDCC） through the activation of calcium dependent chloride channels, and ultimately results in the occurrence of contraction response of smooth muscles.