Activation of MAP kinases in airway smooth muscle.
To test the hypothesis that mitogen-activated protein (MAP) kinases are activated by contractile agonists in intact nonproliferating airway smooth muscle, kinase activities were compared in resting and stimulated canine tracheal smooth muscle. Kinase activities in sodium dodecyl sulfate extracts were assayed by a gel renaturation method. Myelin basic protein kinase activities corresponding to ERK1 and ERK2 immunoreactive proteins were activated twofold above the basal level within 5 min by 1 microM carbachol. MAP kinase activity assayed in crude homogenates using a synthetic peptide substrate (APRTPGGRR) also increased twofold above basal in muscles stimulated with 1 microM carbachol. Two protein kinases separated by Mono-Q chromatography were identified on Western blots as ERK1 and ERK2 MAP kinases. Carbachol stimulation increased caldesmon phosphorylation in intact muscle, and purified caldesmon was a substrate for activated murine ERK2 MAP kinase. Activated ERK2 MAP kinase added to Triton X-100-permeabilized fibers potentiated Ca2+-induced contraction. The results show that ERK MAP kinases are activated after stimulation of muscarinic receptors in airway smooth muscle, which is consistent with coupling of MAP kinases to phosphorylation of caldesmon in vivo.
Gerthoffer
W T
WT
Department of Pharmacology, University of Nevada School of Medicine, Reno 89557-0046, USA.
Yamboliev
I A
IA
Pohl
J
J
Haynes
R
R
Dang
S
S
McHugh
J
J
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002845:Chromatography; D004285:Dogs; D004789:Enzyme Activation; D005260:Female; D006652:Histological Techniques; D066298:In Vitro Techniques; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048053:Mitogen-Activated Protein Kinase 6; D020928:Mitogen-Activated Protein Kinases; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D017830:Octoxynol / Q000494:pharmacology; D010766:Phosphorylation; D014132:Trachea / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology
D002148:Calmodulin-Binding Proteins; D017830:Octoxynol; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048053:Mitogen-Activated Protein Kinase 6; D020928:Mitogen-Activated Protein Kinases