Activation of p42/p44 mitogen-activated protein kinase and contraction by prostaglandin F-2a, ionomycin, and thapsigargin in cat iris sphincter smooth muscle: Inhibition by PD98059, KN-93, and isoproterenol

In the present study we investigated the cross talk between the Ca2+ mobili zation pathway and the mitogen-activated protein (MAP) kinase pathway and c ontraction in the cat iris sphincter smooth muscle. Three Ca2+-mobilizing a gonists, namely, prostaglandin F-2 alpha (PGF(2 alpha)), ionomycin, and tha psigargin, and three specific inhibitors, PD98059, a p42/p44 MAP kinase inh ibitor; KN-93, a Ca2+-calmodulin-dependent protein kinase II (CaMKII) block er; and isoproterenol, a cAMP-elevating agent, were used. Changes in tensio n in response to the agonists were recorded isometrically and MAP kinase ph osphorylation and activation were monitored by Western blotting and by in s itu myelin basic protein phosphorylation, respectively. We found that 1) st imulation of the sphincter muscle with PGF(2 alpha), ionomycin, or thapsiga rgin resulted in rapid phosphorylation and activation of p42/p44 MAP kinase and contraction; and 2) treatment of the muscles with PD98059, KN-93, or i soproterenol resulted in inhibition of the Ca2+-mobilizing agonist-induced responses. The contractile responses induced by PGF(2 alpha), ionomycin, an d thapsigargin were (mg of tension/mg of wet weight tissue) 15.2, 15.4, and 16.2, respectively; the increases in MAP kinase phosphorylation by these a gonists were 228, 203, and 190%, respectively; and the increases in MAP kin ase activation by the agonists were 212, 191, and 162%, respectively. The s timulatory effects of the agonists on contraction and on MAP kinase phospho rylation and activation were blocked by preincubation of the muscle with PD 98059, KN-93, or isoproterenol. These data demonstrate that in the iris sph incter phosphorylation and activation of p42/p44 MAP kinases by PGF(2 alpha ), ionomycin, or thapsigargin require intracellular Ca2+ either from extrac ellular sources or from internal stores, that CaMKII plays an important rol e in the regulation of contraction, that CaMKII acts upstream of MAP kinase to control its activation, and that the MAP kinase signaling pathway can p lay a significant role in mediating the cellular effects of these Ca2+-mobi lizing agonists.