Myosin light chain kinase phosphorylation in swine carotid artery contraction and relaxation.
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.
Van Riper
D A
DA
Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Weaver
B A
BA
Stull
J T
JT
Rembold
C M
CM
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
D010710:Phosphates; D010748:Phosphopeptides; D010761:Phosphorus Radioisotopes; D005576:Colforsin; D011189:Potassium Chloride; D006632:Histamine; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; D002118:Calcium