大鼠胃底平滑肌细胞5-HT受体的信号转导通路的研究

目的　以 5 HT引起胞内Ca2 + 变化为指标 ,研究大鼠胃底平滑肌细胞 5 HT受体的信号转导机制。方法　采用Ca2 + 指示剂Fluo - 3/AM负载培养的胃底平滑肌细胞 (SF SMC) ,共聚焦显微术检测细胞内钙荧光强度的变化。结果　基础状态下SFSMCCa2 + ]i 荧光强度 (fluorescenceintensi ty ,FI)为 2 6 4± 1 5 ,5 HT 10 μmol·L-1使荧光强度缓慢升高 ;这一作用与细胞外钙内流和内钙释放有关 ;10 μmol·L-1米安舍林 (mianserin)可部分拮抗 5 HT升高 Ca2 + ]i 的作用 ,钙拮抗剂拉西地平 (lacidipine)、G蛋白拮抗剂NEM均能完全抑制 5 HT升高 Ca2 + ]i 的作用 ;5 HT引起 Ca2 + ]i的升高被PLC抑制剂部分地取消 ;PKC激动剂拮抗了 5 HT引起的钙动员 ,而PKC特异性抑制剂D 鞘氨醇取消了这一抑制作用。结论　 5 HT部分通过激动了胃底平滑肌细胞的G 蛋白偶联的 5 HT2B受体引起细胞去极化 ,从而激发L型钙通道使外Ca2 + 内流 ,并促进SR上的ryanodine受体的开放 ,引起 Ca2 + ]i 升高。而这一升高 Ca2 + ]i 的作用又受到PLC和PKC的调控

Mechanism of 5-hydroxytryptamine induced calcium signaling in cultured rat stomach fundus smooth muscle cells

AIM To get an insight into intracellular signaling steps, a very early step in the signaling cascade, the biphasic Ca 2+ elicited by 5 HT in rat stomach fundus smooth muscle cells was investigated. METHODS Cells were cultured and loaded with Fluo 3 AM. Ca 2+ ] i was measured by fluorescent intensity (FI) in each cell with confocal microscopy. RESULTS The resting FI level of SFSMC was 264±15. Stimulation of SFSMCs by 5 HT produced an elevation of Ca 2+ ] i; Depletion of external Ca 2+ by addition of EGTA led to a significant attenuation of Ca 2+ ] i change induced by 5 HT; Pre treatment of SFSMCs with ryanodine (10 μmol·L -1 , 5 min) in D Hanks, the effect of 5 HT was completely inhibited; The stimulation of SFSMCs by 5 HT was partly attenuated by miaserin(10 μmol·L -1 ), however, L type Ca 2+ channel antagonist lacidipine and G protein inhibitor NEM completely abolished the increase of Ca 2+ ] i mediated by 5 HT; 5 HT mediated Ca 2+ release was reduced by phospholipase C specific inhibitor compound 48/80(1 2 μg·ml -1 ); When protein kinase C was activated by phorbol 12 myristate 13 acetate (PMA 0 1 μmol·L -1 , 5 min) the effect of 5 HT was inhibited, and the inhibitory effect of PMA was reversed by D sphingosine, a PKC inhibitor. CONCLUSION Our data suggest that G protein coupled 5 HT 2B receptor in the rat stomach fundus modulates 5 HT stimulated Ca 2+ increase, and it is coupled to calcium influx through L type calcium channels, and also intracellular calcium release by the opening of ryanodine receptor. The 5 HT 2B receptor mediated signal of 5 HT is transduced by PLC and PKC.