Ca2+ dependent rapid Ca2+ sensitization of contraction in arterial smooth muscle

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 alpha(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 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 0.29 +/- 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 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 a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content.