AJP - Cell Physiology, Vol 259, Issue 4 C631-C639, Copyright © 1990 by American Physiological Society
Protein kinase C activation and myosin light chain phosphorylation in 32P-labeled arterial smooth muscle
H. A. Singer
Sigfried and Janet Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.
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.
