Protein kinase C activation and myosin light chain phosphorylation in 32P-labeled arterial smooth muscle.
Experiments using 32P-labeled strips of swine carotid artery medial smooth muscle were performed to define the relative contribution of myosin light chain (MLC) phosphorylation as an activation mechanism mediating contractile responses stimulated by phorbol dibutyrate (PDB). Tryptic phosphopeptide mapping of phosphorylated MLC indicated that near-maximal force responses were associated with increases in functional MLC phosphorylation of less than 10% of the total MLC content following tonic (45 min) stimulation by PDB. Significant phosphorylation of MLC residues, consistent with the specificity of protein kinase C, occurred in response to high concentrations of PDB (greater than 0.1 microM). Histamine (10 microM)-induced MLC phosphorylation after 2 min (72.5% of total MLC) or 45 min (61.7%) was restricted to serine residues on peptides thought to contain serine19. Although agonist (histamine)-induced responses were eliminated under conditions of Ca2+ depletion, near-maximal force in response to 10 microM PDB (89.4% of a standard KCl response) was associated with monophosphorylation of less than 9% of the total MLC on peptides interpreted as containing serine19. A substantial fraction of this was localized to threonine residues. The quantitative analysis of the relation between PDB-stimulated force and the residues in MLC phosphorylated supports the concept that PDB stimulation results in activation of arterial smooth muscle cross bridges by MLC-phosphorylation-independent mechanisms.
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Sigfried and Janet Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.
D000818:Animals; D001345:Autoradiography; D002118:Calcium / Q000378:metabolism; D002339:Carotid Arteries / Q000201:enzymology*; D004789:Enzyme Activation; D066298:In Vitro Techniques; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D015879:Myosin Subfragments / Q000302:isolation & purification / Q000378:metabolism*; D010449:Peptide Mapping; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D013552:Swine
D015879:Myosin Subfragments; D010761:Phosphorus Radioisotopes; D011493:Protein Kinase C; D002118:Calcium