MYOSIN LIGHT-CHAIN KINASE PHOSPHORYLATION IN SWINE CAROTID-ARTERY CONTRACTION AND RELAXATION

We investigated the role of myosin light chain kinase (MLCK) phosphory lation in regulating the sensitivity of vascular smooth muscle myosin light chain (MLC) phosphorylation to intracellular Ca2+ concentration ([Ca2+](i)). (PO4)-P-32-loaded swine carotid arteries were stimulated with histamine or high K+, MLCK was isolated, and the relative phospho rylation of tryptic peptides was measured. In nonlabeled tissues, we m easured [Ca2+](i) with aequorin, MLCK activity ratio, MLC phosphorylat ion, and force. A comparison of MLCK phosphorylation on peptide A (mol P in site A/mol MLCK) and MLCK activity ratio showed an inverse relat ion, suggesting that MLCK site A phosphorylation can regulate the Ca2-sensitivity of MLCK. MLCK site A phosphorylation and MLCK activity ra tio depended on [Ca2+](i). Histamine stimulation yielded greater MLC p hosphorylation than high K+ stimulation over a range of [Ca2+](i); how ever, there were no apparent stimulus-dependent differences in MLCK ph osphorylation, suggesting that stimulus-dependent differences in the C a2+ sensitivity of MLC phosphorylation are not based on differences in MLCK phosphorylation. We also determined whether MLCK phosphorylation was involved in adenosine 3',5'-cyclic monophosphate-mediated relaxat ion. In histamine-contracted tissues, forskolin decreased [Ca2+](i), M LC phosphorylation, and force. MLCK phosphorylation decreased to an ex tent consistent with the decrease in [Ca2+](i). In KCl-stimulated tiss ues, forskolin did not alter [Ca2+](i) or increase MLCK phosphorylatio n but forskolin did decrease MLC phosphorylation. Thus, in swine carot id artery, MLCK phosphorylation appears to be regulated exclusively by Ca2+ and plays little role in stimulus-dependent differences in Ca2sensitivity of MLC phosphorylation or in mediating forskolin-induced r elaxation.