Ca2-Dependent Rapid Ca2 Sensitization of Contraction in Arterial Smooth Muscle

Abstract—Ca2 ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca2 with calmodulin activates myosin light chain (MLC) kinase to initiate a rapid MLC phosphorylation and contraction. To test the hypothesis that regulation of MLC phosphatase is involved in the rapid development of MLC phosphorylation and contraction during Ca2 transient, we compared Ca2 signal, MLC phosphorylation, and 2 modes of inhibition of MLC phosphatase, phosphorylation of CPI-17 Thr38 and MYPT1 Thr853, during 1 agonist–induced contraction with/without various inhibitors in intact rabbit femoral artery. Phenylephrine rapidly induced CPI-17 phosphorylation from a negligible amount to a peak value of 0.380.04 mol of Pi/mol within 7 seconds following stimulation, similar to the rapid time course of Ca2 rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca2 release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca2-dependent protein kinase C. This was followed by a slow Ca2-independent and Rho-kinase/protein kinase C–dependent phosphorylation of CPI-17. In contrast, MYPT1 phosphorylation had only a slow component that increased from 0.290.09 at rest to the peak of 0.680.14 mol of Pi/mol at 1 minute, similar to the time course of contraction. Thus, there are 2 components of the Ca2 sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca2 rise induces a rapid inhibition of MLC phosphatase coincident with the Ca2-induced MLC kinase activation to synergistically initiate