Nitric Oxide-Induced Decrease in Calcium Sensitivity of Resistance Arteries Is Attributable to Activation of the Myosin Light Chain Phosphatase and Antagonized by the RhoA/Rho Kinase Pathway

Abstract

Background— NO-induced dilations in resistance arteries (RAs) are not associated with decreases in vascular smooth muscle cell Ca 2+ . We tested whether a cGMP-dependent activation of the smooth muscle myosin light chain phosphatase (MLCP) resulting in a Ca 2+ desensitization of the contractile apparatus was the underlying mechanism and whether it could be antagonized by the RhoA pathway. Methods and Results— The Ca 2+ sensitivity of RA was assessed as the relation between changes in diameter and [Ca 2+ ] i in depolarized RA (120 mol/L K + ) exposed to stepwise increases in Ca 2+ ex (0 to 3 mmol/L). Effects of 10 μmol/L sodium nitroprusside (SNP) on Ca 2+ sensitivity were determined before and after application of the soluble guanylate cyclase inhibitor ODQ (1 μmol/L) and the MLCP inhibitor calyculin A (120 nmol/L) and in presence of the RhoA-activating phospholipid sphingosine-1-phosphate (S1P, 12 nmol/L). SNP-induced dilations were also studied in controls and in RAs pretreated with the Rho kinase inhibitor Y27632 or transfected with a dominant-negative RhoA mutant (N19RhoA). Constrictions elicited by increasing Ca 2+ ex were significantly attenuated by SNP, which, however, left associated increases in [Ca 2+ ] i unaffected. This NO-induced attenuation was blocked by ODQ, calyculin A, and S1P. The S1P-induced translocation of RhoA indicating activation of the GTPase was not reversed by SNP. Inhibition of RhoA/Rho kinase by N19RhoA or Y27632 significantly augmented SNP-induced dilations. Conclusions— NO dilates RA by activating the MLCP in a cGMP-dependent manner, thereby reducing the apparent Ca 2+ sensitivity of the contractile apparatus. MLCP inactivation via the RhoA/Rho kinase pathway antagonizes this Ca 2+ -desensitizing effect that, in turn, can be restored using RhoA/Rho kinase inhibitors.