Failure of Bay K 8644 to induce RhoA kinase-dependent calcium sensitization in rabbit blood vessels

Background and purpose: RhoA kinase (ROCK) participates in K+ depolarization (KCl)-induced Ca2+ sensitization of contraction. Whether constitutive, depolarization- or Ca2+-activated ROCK plays the major role in this signalling system remains to be determined. Here, we determined whether Bay K 8644, a dihydropyridine that promotes Ca2+ channel clusters to operate in a persistent Ca2+ influx mode, could cause ROCK-dependent Ca2+ sensitization. Experimental approach: Renal and femoral artery rings from New Zealand white rabbits were contracted with Bay K 8644. Tissues were frozen and processed to measure active RhoA and ROCK substrate (myosin phosphatase targeting subunit, MYPT1) and myosin light chain (MLC) phosphorylation, or loaded with fura-2 to measure intracellular free Ca2+ ([Ca2+](i)). Effects of selective inhibitors of contraction were assessed in resting (basal) tissues and those contracted with Bay K 8644. Key results: Bay K 8644 produced strong increases in [Ca2+](i), MLC phosphorylation and tension, but not in MYPT1 phosphorylation. ROCK inhibition by H-1152 abolished basal MYPT1-pT853, diminished basal MLC phosphorylation and inhibited Bay K 8644-induced increases in MLC phosphorylation and tension. MLC kinase inhibition by wortmannin abolished Bay K 8644-induced contraction and increase in MLC phosphorylation but did not inhibit basal MYPT1-pT853. H-1152 and wortmannin had no effect on MYPT1-pT696, but 1 mu M staurosporine inhibited basal MYPT1-pT853, MYPT1-pT696 and MLC phosphorylation. Conclusions and implications: These data suggest that the constitutive activities of ROCK and a staurosporine-sensitive kinase regulate basal phosphorylation of MYPT1, which participates along with activation of MLC kinase in determining the strength of contraction induced by the Ca2+ agonist, Bay K 8644.