Alterations in Arachidonic Acid (AA) Release, Inostil Phosphates Formation and Phospholipase C-beta1 (PLC-beta1) Expression in Glaucomatous Human Ciliary Muscle (HCM) Cells

Abstract

Abstract: : Purpose:Prostaglandin F_2alpha (PGF_2alpha ) as well as other Ca₂₊-mobilizing agonists, such as carbachol (CCh) and endothelin-1 (ET-1), induce an increase in uveoscleral outflow in part through receptor -mediated mechanisms in the ciliary muscle. Since changes in uveoscleral outflow across the ciliary muscle could cause elevation of intraocular pressure (IOP) in glaucoma patients, the purpose of the present study was to investigate the possibility that agonist-induced second messenger generation may be altered in glaucomatous HCM (g-HCM) cells as compared to normal HCM (n-HCM) cells. Methods:[₃H] AA release was measured by scintillation counting, PGE₂ release was measured by Radioimmunoassay, [₃H]myo-inositol phosphates production was measured by ion-exchange chromatography, and PLC-beta₁ expression was monitored by Western blot using antibodies specific for PLC-beta₁. Results:Both n-HCM and g-HCM cells exhibited similar morphological characteristics and immunoreactivities. The effects of the agonists on AA release in both n-HCM and g-HCM cells were in the following order: PGF_2alpha ≷ ET-1 ≷ CCh, their effects on PGE₂ release were in the following order: PGF_2alpha ≷ CCh ≷ ET-1, and their effects on inositol phosphates production were in the following order CCh ≷ ET-1 ≷ PGF_2alpha . The data obtained can be summarized as follows: (a) Both the basal- and stimulated release of AA is significantly higher in the g-HCM cells than in the n-HCM cells, (b) the basal release of PGE₂ in g-HCM cells is 2-5 fold higher than that of n-HCM cells, (c) agonist-induced inositol phosphates production in g-HCM cells is considerably lower than that of n-HCM cells, and (d) the amount of phospholipase C-beta₁ (PLC-beta₁) expressed in g-HCM cells, as compared to n-HCM cells, is markedly reduced. Conclusion: These data are the first to show that basal and agonists-induced AA release and inositol phosphates production as well as expression of PLC-beta₁ are altered in g-HCM cells as compared to n-HCM cells. Comparative studies on the signaling pathways in g-HCM and n-HCM cells may provide important information about the regulation of uveoscleral outflow and glaucoma pathology.

Keywords: 577 second messengers: pharmacology/physiology • 392 eicosanoids • 348 ciliary body