Effects of prostaglandin F-2 alpha and carbachol on MAP kinases, cytosolicphospholipase A(2) and arachidonic acid release in cat iris sphincter smooth muscle cells

The signal transduction pathways initiated by Ca2+-mobilizing agonists, suc h as prostaglandin F-2 alpha (PGF(2 alpha)) and carbachol (CCh), leading to activation of cytosolic phospholipase A(2) (cPLA(2)) 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 cPLA(2) an d consequently AA release we have investigated the role of mitogen-activate d protein (MAP) kinases and protein kinase C (PKC) in PGF(2 alpha)- and CCh -induced cPLA(2) phosphorylation and AA release in cat iris sphincter smoot h muscle (CISM) cells. The cells were prelabeled with [H-3]AA for 24 hr and incubated in the absence or presence of the agonist for 5-10 min as indica ted. MAP kinases activities and cPLA(2) phosphorylation were determined in immunoprecipitates obtained by using anti-p38 MAP kinase and anti-cPLA(2) a ntibodies. We found that: (a) PGF(2 alpha) and 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 spe cific inhibitor, inhibited PGF(2 alpha)- and CCh-induced. cPLA(2) phosphory lation by 92 and 85 %, respectively, and AA release by 62 and 78 %, respect ively. (c) PD98059, a p42/p44 MAP kinase inhibitor, inhibited CCh-induced c PLA(2) phosphorylation by 70 % and AA release by 71 %, but had no effect on that of PGF(2 alpha). (d) Inhibition of PKC activity by RO 31-8220 inhibit ed both PGF(2 alpha)- and CCh-stimulation of p38 MAP kinase, p42/p44 MAP ki nases and cPLA(2) phosphorylation. We conclude from these results that in C ISM cells PGF(2 alpha)-induced cPLA(2) phosphorylation and AA release is me diated 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 kinase s. These effects of PGF(2 alpha) and CCh are regulated by the MAP kinases i n a PKC-dependent manner. Studies aimed at elucidating the role of protein kinases in the coupling mechanism between the activation of PGF(2 alpha) an d muscarinic receptors, and the stimulation of cPLA(2) and AA release in th e smooth muscles of the iris-ciliary body will provide important informatio n about the role of protein kinases signaling pathways in smooth muscle Fun ction, as well as about the mechanism of the intraocular pressure-lowering effects of PGF(2 alpha) and its analog, latanoprost, in glaucoma therapy. ( C) 2001 Academic Press.