Myosin light chain phosphorylation in 32P-labeled rabbit aorta stimulated by phorbol 12,13-dibutyrate and phenylephrine.
The mechanism(s) of force development in vascular smooth muscle following pharmacological activation of protein kinase C by phorbol esters are not known. In this study, we examined the myosin light chain phosphorylation response following stimulation by phorbol 12,13-dibutyrate (PDB) or phenylephrine in rabbit aorta which had been incubated with 32PO4 in order to label ATP pools. Through tryptic phosphopeptide mapping of myosin light chain from intact tissue and comparison to controls using purified components, we inferred that Ca2+-dependent force stimulated by PDB was associated with small increases in serine-19 phosphorylation, consistent with a contractile mechanism involving indirect activation of myosin light chain kinase. Additional residues, consistent with the in vitro substrate specificity of protein kinase C, were also observed to be phosphorylated in response to PDB and represented proportionately a larger fraction of the total phosphorylated myosin light chain in Ca2+-depleted tissues. Stimulation by an alpha 1-adrenergic agonist (phenylephrine) resulted in phosphorylation of residues which were consistent with an activation mechanism involving myosin light chain kinase only. These results indicate that in rabbit aorta the contractile effects of PDB may be partially mediated by Ca2+-dependent activation of myosin light chain kinase. However, the data do not rule out a component of the PDB-stimulated contractile response which is independent of myosin light chain phosphorylation on the serine-19 residue. In addition, activation by a more physiological stimulus, phenylephrine, does not result in protein kinase C-mediated myosin light chain phosphorylation.
Singer
H A
HA
Sigfried and Janet Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.
Oren
J W
JW
Benscoter
H A
HA
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D014644:Genetic Variation; D066298:In Vitro Techniques; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D015879:Myosin Subfragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D010449:Peptide Mapping; D010656:Phenylephrine / Q000494:pharmacology*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010748:Phosphopeptides / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011817:Rabbits; D011865:Radioisotope Dilution Technique; D014357:Trypsin
D000596:Amino Acids; D015879:Myosin Subfragments; D010748:Phosphopeptides; D010761:Phosphorus Radioisotopes; D010656:Phenylephrine; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D009219:Myosin-Light-Chain Kinase; D014357:Trypsin