Abstract 16460: Enhanced Trpc Channel-Mediated Ca2+ Influx in the Cells With Phospholipase C-δ1 Overexpression

Abstract

Background: We recently showed that ergometrine induces coronary spasm in the mice with enhanced phospholipase C (PLC)-δ1 activity, which was detected in patients with coronary spasm (Circulation 2012), and PLC-mediated Ca2+ entry is involved in the genesis of coronary spams. We examined the role of enhanced PLC-δ1 in extracellular Ca2+ entry and its underlying mechanisms in human embryonic kidney (HEK)-293 cells and human coronary arteries smooth muscle cells (CASMC). Methods and Results: The cells were stimulated with acetylcholine (ACh) in a extracellular Ca2+-free condition, and the increase in intracellular free Ca2+ concentration ([Ca2+]i) after addition of extracellular Ca2+ was defined as Ca2+ influx. [Ca2+]i was measured by fura-2. In HEK-293 cells, ACh-induced Ca2+ influx (nM ) was 21±2 in the control and 52±6 in the cells with PLC-δ1 overexpression (p<0.05). ACh-induced Ca2+ influx in the cells with PLC-δ1 overexpression was suppressed by nifedipine in a dose-dependent manner but was partially by 36±13% at 10-5M (p<0.05), thereby even after treatment with nifedipine at 10-5M, ACh-induced Ca2+ influx was increased by 2.9±0.1 times by enhanced PLC-δ1 compared with the control (p<0.05). To clarify the role of diacylglycerol (DAG) in Ca2+ influx, the effect of blockers for DAG-activated transient receptor potential (TRPC) channel was examined. ACh-induced Ca2+ influx in the cells with PLC-δ1 overexpression was suppressed by 2-APB, an inhibitor of non-selective cation channel TRPC, in a dose-dependent manner, and was almost completely blocked by 89±12% at 10-4M (p<0.05). While TRPC3 siRNA did not affect ACh-induced Ca2+ influx (59±27 vs 75±23 nM, p=ns), TRPC6 siRNA suppressed Ca2+ influx to 37±28 nM (p<0.05). In human CASMC, ACh-induced Ca2+ influx was 41±11 in the control and 64±15 in the cells with PLC-δ1 overexpression (p<0.05). Like HEK-293 cells, pretreatment with nifedipine partially suppressed Ca2+ influx, whereas either 2-APB or TRPC6 siRNA almost completely blocked it. Conclusion: ACh-induced Ca2+ influx, mediated by both voltage-gated Ca2+ channels and non-selective cation channels TRPC6, is enhanced by PLC-δ1 overexpression. Inhibition of TRPC may be effective in enhanced PLC-δ1-mediated coronary spasm.