Mitogen-activated protein kinase inhibitors suppress prostaglandin F-2 alpha-induced myosin-light chain phosphorylation and contraction in iris sphincter smooth muscle

The purpose of this study was to investigate the potential role of mitogen- activated protein (MAP) kinase in contraction by monitoring MAP kinase phos phorylation (activation) and contraction during agonist stimulation of cat iris sphincter smooth muscle. Changes in tension in response to prostagland in F-2 alpha, latanoprost, a prostaglandin F-2 alpha analog used as an anti -glaucoma drug, and carbachol were recorded isometrically, and MAP kinase a ctivation was monitored by Western blot using a phosphospecific p42/p44 MAP kinase antibody. We found that treatment of the muscle with 2'-Amino-3'-me thoxyflavone (PD98059) (10 muM), a specific inhibitor of MAP kinase kinase (MEK), inhibited significantly prostaglandin F-2 alpha- and latanoprost-ind uced phosphorylation and contraction, but had little effect on those evoked by carbachol. Prostaglandin F-2 alpha increased MAP kinase phosphorylation in a concentration-dependent manner with EC50 value of 1.1 x 10(-8) M and increased contraction with EC50 of 0.92 x 10(-9) M. The MAP kinase inhibito rs PD98059, Apigenin and 1,4-Diamino-3,3-dicyano-1,4bis(2-aminophenylthio)b utadiene (UO126) inhibited prostaglandin F-2 alpha-induced contraction in a concentration-dependent manner with IC50 values of 2.4, 3.0 and 4.8 muM, r espectively. PD98059 had no effect on prostaglandin F-2 alpha- or on carbac hol-stimulated inositol-1,4,5-trisphosphate (IP3) production. In contrast, the MAP kinase inhibitor inhibited prostaglandin F-2 alpha-induced myosin-l ight chain (MLC) phosphorylation, but had no effect on that of carbachol. N -[2-(N-(4-Chloro-cinnamyl)-N-methylaminomethyl)phenyl]-N-[2-hydroxyethyl] - 4-methoxybenzenesulfonamide (KN-93) (10 muM), a Ca2+-calmodulin-dependent p rotein kinase inhibitor, and Wortmannin (10 muM), an MLC kinase inhibitor, inhibited significantly (by 80%) prostaglandin F-2 alpha- and carbachol-ind uced contraction. It can be concluded that in this smooth muscle p42/p44 MA P kinases are involved in the mechanism of prostaglandin F-2 alpha-, but no t in that of carbachol, induced contraction. In addition, these data clearl y indicate that the stimulation of the iris sphincter with prostaglandin F- 2 alpha and carbachol activate two distinct pathways, the MAP kinase pathwa y and the Ca2+ mobilization pathway. (C) 2000 Elsevier Science B.V. All rig hts reserved.