Effects of Prostaglandin F2αand Carbachol on MAP Kinases, Cytosolic Phospholipase A2and Arachidonic Acid Release in Cat Iris Sphincter Smooth Muscle Cells

Abstract

The signal transduction pathways initiated by Ca2+-mobilizing agonists, such as prostaglandin F2α(PGF2α) and carbachol (CCh), leading to activation of cytosolic phospholipase A2(cPLA2) and arachidonic acid (AA) release in a wide variety of tissues remain obscure. To further define the role of protein kinases in receptor mediated stimulation of cPLA2and consequently AA release we have investigated the role of mitogen-activated protein (MAP) kinases and protein kinase C (PKC) in PGF2α- and CCh-induced cPLA2phosphorylation and AA release in cat iris sphincter smooth muscle (CISM) cells. The cells were prelabeled with [3H]AA for 24hr and incubated in the absence or presence of the agonist for 5–10min as indicated. MAP kinases activities and cPLA2phosphorylation were determined in immunoprecipitates obtained by using anti-p38 MAP kinase and anti-cPLA2antibodies. We found that: (a) PGF2αand CCh increased p38 MAP kinase activity by 197 and 215%, respectively, and increased p42/p44 MAP kinase activity by 200 and 125%, respectively. (b) SB202190, a p38 MAP kinase specific inhibitor, inhibited PGF2α- and CCh-induced cPLA2phosphorylation by 92 and 85%, respectively, and AA release by 62 and 78%, respectively. (c) PD98059, a p42/p44 MAP kinase inhibitor, inhibited CCh-induced cPLA2phosphorylation by 70% and AA release by 71%, but had no effect on that of PGF2α. (d) Inhibition of PKC activity by RO 31-8220 inhibited both PGF2α- and CCh-stimulation of p38 MAP kinase, p42/p44 MAP kinases and cPLA2phosphorylation. We conclude from these results that in CISM cells PGF2α-induced cPLA2phosphorylation and AA release is mediated through p38 MAP kinase, but not through p42/p44 MAP kinases, whereas that of CCh is mediated through both p38 MAP kinase and p42/p44 MAP kinases. These effects of PGF2αand CCh are regulated by the MAP kinases in a PKC-dependent manner. Studies aimed at elucidating the role of protein kinases in the coupling mechanism between the activation of PGF2αand muscarinic receptors, and the stimulation of cPLA2and AA release in the smooth muscles of the iris-ciliary body will provide important information about the role of protein kinases signaling pathways in smooth muscle function, as well as about the mechanism of the intraocular pressure-lowering effects of PGF2αand its analog, latanoprost, in glaucoma therapy.