Rho kinase inhibitors reduce voltage-dependent Ca2+ channel signaling in aortic and renal microvascular smooth muscle cells

Abstract

Voltage-dependent L-type Ca²⁺ channels (L-VDCCs) and the RhoA/Rho kinase pathway are two predominant intracellular signaling pathways that regulate renal microvascular reactivity. Traditionally, these two pathways have been thought to act independently; however, recent evidence suggests that these pathways could be convergent. We hypothesized that Rho kinase inhibitors can influence L-VDCC signaling. The effects of Rho kinase inhibitors Y-27632 or RKI-1447 on KCl-induced depolarization or the L-VDCC agonist Bay K8644 were assessed in afferent arterioles using an in vitro blood-perfused rat juxtamedullary nephron preparation. Superfusion of KCl (30-90 mM) led to concentration-dependent vasoconstriction of afferent arterioles. Administration of Y-27632 (1, 5, and 10 µM) or RKI-1447 (0.1, 1, and 10 µM) significantly increased the starting diameter by 16-65%. KCl-induced vasoconstriction was markedly attenuated with 5 and 10 µM Y-27632 and with 10 µM RKI-1447 (P < 0.05 vs. KCl alone). Y-27632 (5 µM) also significantly attenuated Bay K8644-induced vasoconstriction (P < 0.05). Changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) were estimated by fura-2 fluorescence during KCl-induced depolarization in cultured A7r5 cells and in freshly isolated preglomerular microvascular smooth muscle cells. Administration of 90 mM KCl significantly increased fura-2 fluorescence in both cell types. KCl-mediated elevation of [Ca²⁺]_i in A7r5 cells was suppressed by 1-10 µM Y-27632 (P < 0.05), but 10 µM Y-27632 was required to suppress Ca²⁺ responses in preglomerular microvascular smooth muscle cells. RKI-1447, however, significantly attenuated KCl-mediated elevation of [Ca²⁺]_i. Y-27632 markedly inhibited Bay K8644-induced elevation of [Ca²⁺]_i in both cell types. The results of the present study indicate that the Rho kinase inhibitors Y-27632 and RKI-1447 can partially inhibit L-VDCC function and participate in L-VDCC signaling.

Keywords: Bay K8644; RKI-1447; Y-27632; afferent arteriole; calcium signaling; voltage-dependent L-type Ca2+ channels.