Rho Kinase Inhibitors Are Involved in L‐Type Voltage‐dependent Calcium Channel Pathway in Vascular Smooth Muscle Cells

Abstract

Voltage‐dependent L‐type calcium channels (L‐VDCC) and the rho/rho kinase pathway are two predominate intracellular pathways that regulate renal microvascular tone and reactivity. Traditionally, these two pathways have been thought to act independently; however, recent evidence suggests these pathways are convergent. We hypothesized that the rho kinase inhibitors influenced L‐VDCC signaling. The effects of rho kinase inhibitors Y‐27632 or RKI‐1447 on KCl‐depolarization were assessed in afferent arterioles using the in vitro blood‐perfused rat juxtamedullary nephron preparation. Superfusion of KCl (30, 60, and 90mM) led to concentration‐dependent vasoconstriction of afferent arterioles reducing the diameter to 95±6, 56±4 and 42±3% of the baseline, respectively (p<0.05). Addition of Y‐27632 (1, 5 and 10 μM) or RKI‐1447 (0.1, 1 and 10 μM) significantly increased starting diameter by 16–65% (n=6/each, p<0.05). The KCl‐induced vasoconstriction with 1 μM Y‐27632 or 0.1 and 1 μM RKI‐1447 was similar to control, but markedly attenuated with 5 and 10 μM Y‐27632, and 10 μM RKI‐1447 (p<0.05 vs KCl alone). The afferent arteriole diameter averaged 99±2, 83±2, 71±4%, and 98±0.8, 89±5, 81±6% of the baseline for 5 and 10 μM Y‐27632, and 97±2, 89±3 and 88±4% of the baseline for 10 μM RKI‐1447, respectively (n=6/each, p<0.05 vs KCl alone). Changes in intracellular calcium concentration [Ca2+]i were estimated by fura‐2‐AM fluorescence (ratio F340/380) during KCl‐depolarization in cultured A7r5 cells (n=46–118/each group) and in freshly isolated renal microvascular smooth muscle cells (RMVSMCs, n=32–118/each group). Administration of 90 mM KCl increased fura‐2 fluorescence markedly in both cell types. The KCl‐induced increase of the F340/F380 ratio was unaltered by 0.1 or 0.5 μM Y‐27632 but was significantly attenuated by Y‐27632 between 1–10 μM by 50–56% (p<0.05 vs KCl alone) in A7r5 cells but only at 10 μM in RMVSMCs by 42% (p<0.05 vs KCl alone). RKI‐1447 however, significantly attenuated KCl‐mediated elevation of [Ca2+]i in both cell types regardless of the concentrations applied (0.1–10 μM, p<0.05), indicating that Y‐27632 and RKI‐1447 decrease L‐VDCC function. These studies indicate that the rho kinase inhibitors, Y‐27632 and RKI‐1447 partially inhibit L‐VDCC function and participate in L‐VDCC signaling.Support or Funding InformationThis study was supported by a Grant‐in‐Aid (15GRNT25240015) from the American Heart Association Great Southeast Affiliate and a grant from NIH (DK106500) to ZG, and by grants from NIH (DK044628, HL074167, HL098135 and HL095499) to EWI.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.