Carbachol-induced protein phosphorylation changes in bovine tracheal smooth muscle.
The changes in protein phosphorylation associated with bovine tracheal smooth muscle contraction were studied by labeling intact muscle strips with [32P]PO4(3-) and analyzing the phosphoproteins by two-dimensional gel electrophoresis. Among 20 to 30 phosphoproteins resolvable with the two-dimensional electrophoresis system, the phosphorylation of 12 proteins was reproducibly affected by treatment with carbachol, in a time-dependent manner. Five of these proteins have been identified as 20-kDa myosin light chain, caldesmon, synemin, and two isoelectric variants of desmin. The other 7 are low molecular weight (Mr less than 40,000) cytosolic proteins. One cytosolic protein and myosin light chain are quickly but transiently phosphorylated by carbachol, the peak of myosin light chain phosphorylation being at about 1 min after agonist addition. In contrast, both variants of desmin, synemin, caldesmon, and 5 cytosolic proteins are phosphorylated at varying rates and remain phosphorylated for the duration of carbachol action. These "late" phosphorylation changes occur simultaneously with the dephosphorylation of one cytosolic protein. These carbachol-induced phosphorylation changes, like the contractile response, appear to be calcium-dependent. The addition of 12-deoxyphorbol 13-isobutyrate, a protein kinase C activator, causes a dose-dependent, sustained contraction of tracheal smooth muscle which develops more slowly than that induced by carbachol. This contractile response is associated with the same protein phosphorylation changes as those observed after prolonged carbachol treatment. In contrast, forskolin, an adenylate cyclase activator and a potent smooth muscle relaxant, induces the phosphorylation protein 3 and one variant of desmin. These observations strongly suggest that different phosphoproteins may be mediators of tension development and tension maintenance in agonist-induced contraction of tracheal smooth muscle.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology*; D002417:Cattle; D005576:Colforsin / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D003893:Desmin / Q000378:metabolism; D007381:Intermediate Filament Proteins; D007526:Isoelectric Point; D007700:Kinetics; D009119:Muscle Contraction / Q000187:drug effects*; D009124:Muscle Proteins / Q000378:metabolism; D009130:Muscle, Smooth / Q000502:physiology*; D009218:Myosins / Q000378:metabolism; D010703:Phorbol Esters / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D014132:Trachea / Q000502:physiology
D002148:Calmodulin-Binding Proteins; D003893:Desmin; D007381:Intermediate Filament Proteins; D009124:Muscle Proteins; D010703:Phorbol Esters; D010750:Phosphoproteins; C430456:desmuslin; D005576:Colforsin; C039067:12-deoxyphorbol 13-isobutyrate; D002217:Carbachol; D009218:Myosins; D002118:Calcium