Abstract 674: Coupling Factor 6 Activates c-Src By Intracellular Acidosis And Enhances Calcium Signaling In Resistance Arteriole Smooth Muscle Cells
In vascular endothelial cells, coupling factor 6 (CF6) binds to the β subunit of plasma membrane ATP synthase and attenuates prostacyclin generation by inducing intracellular acidosis. However, intracellular signaling in resistance arterioles that is directly related to vascular tonus and hypertension has not been determined yet. We investigated the direct effect of CF6 on Ca2+ signaling, involvement of c-Src in it, and the role of CF6 in the pathogenesis of hypertension. CF6 at 10−7M induced a monophasic increase in intracellular free Ca2+ concentration ([Ca2+ ]i) in A7r5 cells (rat vascular smooth muscle cells (VSMC) expressing voltage-gated Ca2+ channel). When CF6 at 10−7M was added after pretreatment with nifedipine at 10−5M, the sustained increase in [Ca2+ ]i was abolished. In cultured resistance arteriole VSMC obtained from the mesenteric artery network of spontaneously hypertensive rats (SHR, n = 8) and Wistar Kyoto rats (WKY, n = 8), angiotensin II (Ang II) at 10−7M caused a spike increase in [Ca2+]i followed by a sustained increase. When Ang II at 10−7M was added after pre-administration of CF6 at 10−7M, the spike phase of [Ca2+]i was enhanced and it was greater in SHR than in WKY (412 ± 26 vs 162 ± 14 nM, p < 0.05). Pretreatment with PP1 at 50 μM, a c-Src inhibitor, blocked not only the CF6-induced Ca2+ influx but the CF6-induced increase in the AngII-mediated spike phase of [Ca2+]i. CF6 receptor of VSMC was the β-subunit of ATP synthase, and the affinity was higher in SHR than in WKY (Kd: 3.6 ± 0.3 vs 9.6 ± 0.3 nM, p < 0.05). The increase in ATPase activity by CF6 was greater in SHR than in WKY (p < 0.05), and the decrease in intracellular pH was greater in SHR than in WKY (0.26 ± 0.03 vs 0.13 ± 0.02, p < 0.05). CF6 activated c-Src at 15 minutes by 2 folds greater in SHR than in WKY, and it was blocked by efrapeptine at 10−5M, an ATPase inhibitor. In mesenteric arterioles obtained from CF6-transgenic mice (2 fold over-expressed), AngII caused greater vasoconstriction by 2 folds than in those from wild mice (p < 0.05), but it was abolished by pretreatment with PP1. These suggest that CF6 activates c-Src by inducing intracellular acidosis and enhances Ca2+ signaling in resistance arteriole VSMC. Since the c-Src activation was enhanced in SHR, CF6 might be involved in the genesis of hypertension.