Phosphorylation of type II regulatory subunit of cAMP-dependent protein kinase in intact smooth muscle.
A monoclonal antibody was used to quantitate changes in the extent of phosphorylation of the type II regulatory subunit of cAMP-dependent protein kinase in intact bovine tracheal smooth muscle. The autophosphorylated and nonphosphorylated forms of the regulatory subunit (RII) were separated in sodium dodecyl sulfate-polyacrylamide gels and identified by immunoblot analysis. Addition of cAMP to tissue extracts resulted in rapid dephosphorylation of RII (t 1/2 = 20s at 4 degrees C) while addition of MgATP caused complete conversion to the phosphorylated form. Under basal conditions, 56% of RII in intact muscle was phosphorylated when the tissue was homogenized under conditions which fully inhibit protein kinase and phosphatase activities. Incubation with isoproterenol caused a dose-dependent decrease in the phosphorylation state of RII (EC50 = 5 X 10(-8) M). Incubation with high concentrations of isoproterenol, 1-methyl-3-isobutylxanthine, or forskolin caused maximal decreases in the phosphorylated form to 12-18% of the total RII. The effect of isoproterenol was rapid (t 1/2 = 15 s at 37 degrees C), reversible, and could be blocked with the antagonist propranolol. Contraction of the smooth muscle with K+ or low (less than 1 microM) concentrations of carbachol had no effect on the phosphorylation level. A decrease in the basal phosphorylation level to 41% was observed with 10 microM carbachol which was additive with the dephosphorylation produced by isoproterenol. The time course of isoproterenol-induced dephosphorylation of RII paralleled that of muscle relaxation, consistent with a role of cAMP-dependent protein kinase activation in relaxation of smooth muscle.
Scott
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Mumby
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D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology*; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000187:drug effects / Q000201:enzymology*; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D012969:Sodium Fluoride / Q000494:pharmacology; D014132:Trachea / Q000378:metabolism
D000255:Adenosine Triphosphate; D002217:Carbachol; D012969:Sodium Fluoride; D000242:Cyclic AMP; D011494:Protein Kinases; D007545:Isoproterenol; D011188:Potassium