title: Myosin light chain kinase phosphorylation in swine carotid artery contraction and relaxation.
abstract: We investigated the role of myosin light chain kinase (MLCK) phosphorylation in regulating the sensitivity of vascular smooth muscle myosin light chain (MLC) phosphorylation to intracellular Ca2+ concentration ([Ca2+]i). 32PO4-loaded swine carotid arteries were stimulated with histamine or high K+, MLCK was isolated, and the relative phosphorylation of tryptic peptides was measured. In nonlabeled tissues, we measured [Ca2+]i with aequorin, MLCK activity ratio, MLC phosphorylation, and force. A comparison of MLCK phosphorylation on peptide A (mol P in site A/mol MLCK) and MLCK activity ratio showed an inverse relation, suggesting that MLCK site A phosphorylation can regulate the Ca2+ sensitivity of MLCK. MLCK site A phosphorylation and MLCK activity ratio depended on [Ca2+]i. Histamine stimulation yielded greater MLC phosphorylation than high K+ stimulation over a range of [Ca2+]i; however, there were no apparent stimulus-dependent differences in MLCK phosphorylation, suggesting that stimulus-dependent differences in the Ca2+ 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 relaxation. In histamine-contracted tissues, forskolin decreased [Ca2+]i, MLC phosphorylation, and force. MLCK phosphorylation decreased to an extent consistent with the decrease in [Ca2+]i. In KCl-stimulated tissues, forskolin did not alter [Ca2+]i or increase MLCK phosphorylation but forskolin did decrease MLC phosphorylation. Thus, in swine carotid artery, MLCK phosphorylation appears to be regulated exclusively by Ca2+ and plays little role in stimulus-dependent differences in Ca2+ sensitivity of MLC phosphorylation or in mediating forskolin-induced relaxation.
authors.affiliation: Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Van Riper
authors.forename: B A
authors.initials: BA
authors.lastname: Weaver
authors.forename: J T
authors.initials: JT
authors.lastname: Stull
authors.forename: C M
authors.initials: CM
authors.lastname: Rembold
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D017536:Carotid Artery, Common / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D005576:Colforsin / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D009119:Muscle Contraction / Q000187:drug effects; D009126:Muscle Relaxation; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D010449:Peptide Mapping; D010710:Phosphates / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D013552:Swine
chemical_list: D010710:Phosphates; D010748:Phosphopeptides; D010761:Phosphorus Radioisotopes; D005576:Colforsin; D011189:Potassium Chloride; D006632:Histamine; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; D002118:Calcium