Effects of siRNA knock-down of TRPC6 and InsP(3)R1 in vasopressin-induced Ca(2+) oscillations of A7r5 vascular smooth muscle cells.
Effects of siRNA knock-down of TRPC6 and InsP(3)R1 in vasopressin-induced Ca(2+) oscillations of A7r5 vascular smooth muscle cells.
复制标题
DOI：
10.1016/j.phrs.2008.09.004
复制
发表时间：
2008-11
影响因子：
9.3
通讯作者：
Manxiang Li;J. Zacharia;Xiu-zhen Sun;W. Wier
中科院分区：
文献类型：
--
作者：
Manxiang Li;J. Zacharia;Xiu-zhen Sun;W. Wier
We used post-transcriptional gene silencing (with small interfering RNA) to examine specifically the roles of Type 1 inositol tris-phosphate receptors (InsP3R1) and transient receptor potential channel 6 (TRPC6) in Ca2+oscillations induced by arginine vasopressin (AVP), a typical G-protein coupled receptor agonist. Ca2+oscillations were observed in individual A7r5 cells with confocal imaging of fluo-4 fluorescence, and SR-releasable Ca2+was assessed by exposure to cyclopiazonic acid (CPA). In control cells, both AVP (100nM) and a direct activator of TRPC6 (OAG, l-oleoyl-2-acetyl-glycerol, 100μM) caused Ca2+oscillations in the majority of cells (e.g. AVP: 85%, 0.97±0.05/min; OAG: 83%, 1.00±0.07/min). Partial knock-down of TRPC6 (to <27% protein expression) was more effective than partial knock-down of InsP3R1 (to <30% protein expression) in reducing the fraction of cells that produced Ca2+oscillations in response to AVP or OAG (22% and 83% of cells showing oscillations, respectively, in response to AVP; 31% and 72% of cells showing oscillation, respectively, in response to OAG). CPA-induced SR Ca2+release was unaffected by siRNA transfection. Inhibition of InsP3R with Xestospongin C abolished both AVP and OAG-induced Ca2+oscillations. Nifedipine (10μM) had no effect. The key results, including the effects of partial (as opposed to complete) knock-down of InsP3R1 and TRPC6, and the (unexpected) finding of OAG-induced Ca2+oscillations, are predicted by a canonical mathematical model of Ca2+oscillations in which InsP3R1 functions as the SR Ca2+release channel and TRPC6 as the receptor-operated Ca2+influx channel. These results indicated that TRPC6 functioning as a major type of receptor-operated Ca2+channel played a critical role in Ca2+oscillations of A7r5 cells’ response to AVP or OAG, and partial knock-down of TRPC6 was more effective than partial knock-down of InsP3R1 in reducing Ca2+oscillations.
