ACTIVATION OF MAP KINASES IN AIRWAY SMOOTH-MUSCLE

To test the hypothesis that mitogen-activated protein (MAP) kinases ar e activated by contractile agonists in intact nonproliferating airway smooth muscle, kinase activities were compared in resting and stimulat ed canine tracheal smooth muscle. Kinase activities in sodium dodecyl sulfate extracts were assayed by a gel renaturation method. Myelin bas ic protein kinase activities corresponding to ERK1 and ERK2 immunoreac tive proteins were activated twofold above the basal level within 5 mi n by 1 mu M carbachol. MAP kinase activity assayed in crude homogenate s using a synthetic peptide substrate (APRTPGGRR) also increased twofo ld above basal in muscles stimulated with 1 l-IM carbachol. Two protei n kinases separated by Mono-Q chromatography were identified on Wester n 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+-ind uced contraction. The results show that ERK MAP kinases are activated after stimulation of muscarinic receptors in airway smooth muscle, whi ch is consistent with coupling of MAP kinases to phosphorylation of ca ldesmon in vivo.