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(Circulation Research. 2007;100:121.)
© 2007 American Heart Association, Inc.

Cellular Biology

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

George J. Dimopoulos^*, Shingo Semba^*, Kazuyo Kitazawa, Masumi Eto, Toshio Kitazawa

From the Boston Biomedical Research Institute (G.J.D., S.S., K.K., T.K.), Watertown, Mass; and Department of Physiology (M.E.), Jefferson Medical College, Philadelphia, Pa.

Correspondence to Toshio Kitazawa, Boston Biomedical Research Institute, 64 Grove St, Watertown, MA 02472. E-mail Kitazawa{at}bbri.org

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 0.38±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.

Key Words: CPI-17 • MYPT1 • PKC • Rho-kinase

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