Cyclic GMP-dependent protein kinase activation in canine tracheal smooth muscle by methacholine and sodium nitroprusside.
Methacholine (3 microM) and sodium nitroprusside (300 microM) increased cGMP-dependent protein kinase activity ratios (activity without cGMP divided by activity with 2 microM cGMP) in canine tracheal smooth muscle from a control value of 0.47 to 0.55 and 0.71, respectively. This correlates with 3-fold and 6-fold increases in cGMP concentrations in response to methacholine and sodium nitroprusside, respectively. Addition of charcoal to the homogenizing buffer prior to homogenization had no significant effect on the cGMP-dependent protein kinase response to either agent, suggesting that activation of the enzyme was not occurring as a result of cGMP release during homogenization. In order to limit cGMP dissociation from cGMP-dependent protein kinase during the assay procedure, it was necessary to perform assays at a reduced temperature (0 degree C) and with an abbreviated incubation time (2.5 min). When assayed at 30 degrees C, activated cGMP-dependent protein kinase rapidly lost activity. This inactivation occurred whether the enzyme had been activated exogenously, by exposing a supernatant fraction of canine trachealis to 0.1 microM cGMP, or endogenously, by treating intact canine trachealis with methacholine or sodium nitroprusside. By assaying instead at 0 degree C, the inactivation of cGMP-dependent protein kinase was minimized. Therefore, the activity ratio obtained by this new modified assay provided an estimate of the endogenous activation state of cGMP-dependent protein kinase. The data indicate that cGMP responses in canine trachealis to both methacholine and sodium nitroprusside are functionally linked to activation of cGMP-dependent protein kinase and are consistent with the hypothesis that cGMP, via cGMP-dependent protein kinase activation, regulates smooth muscle contractility.
D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology; D004285:Dogs; D004789:Enzyme Activation; D005292:Ferricyanides / Q000494:pharmacology*; D007700:Kinetics; D016210:Methacholine Chloride; D008688:Methacholine Compounds / Q000494:pharmacology*; D009130:Muscle, Smooth / Q000201:enzymology*; D009599:Nitroprusside / Q000494:pharmacology*; D010710:Phosphates / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D013816:Thermodynamics; D014132:Trachea / Q000201:enzymology
D005292:Ferricyanides; D008688:Methacholine Compounds; D010710:Phosphates; D016210:Methacholine Chloride; D009599:Nitroprusside; D000242:Cyclic AMP; D011494:Protein Kinases