Effects of siRNA knock-down of TRPC6 and InsP3R1 in vasopressin-induced Ca2+ oscillations of A7r5 vascular smooth muscle cells

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 (100 nM) and a direct activator of TRPC6 (OAG, l-oleoyl-2-acetyl-glycerol, 100 μM) 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 (10 μM) 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.