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

Ca ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca 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 Ca transient, we compared Ca 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 0.38 0.04 mol of Pi/mol within 7 seconds following stimulation, similar to the rapid time course of Ca rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca -dependent protein kinase C. This was followed by a slow Ca -independent and Rho-kinase/protein kinase C–dependent phosphorylation of CPI-17. In contrast, MYPT1 phosphorylation had only a slow component that increased from 0.29 0.09 at rest to the peak of 0.68 0.14 mol of Pi/mol at 1 minute, similar to the time course of contraction. Thus, there are 2 components of the Ca sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca rise induces a rapid inhibition of MLC phosphatase coincident with the Ca -induced MLC kinase activation to synergistically initiate a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content. (Circ Res. 2007;100:121-129.)