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PURPOSE: To determine the mechanism by which nitric oxide (NO) regulates α2-adrenergic receptor coupling to adenylyl cyclase in bovine ciliary epithelium. METHODS: Ciliary epithelial explants were dissected, cultured, and labelled with [3H]adenine. [3H]Adenosine 3',5'-cyclic monophosphate (cAMP) was measured under basal conditions and after exposure to forskolin, isoproterenol, clonidine, yohimbine, pertussis toxin, and the NO donor spermine-NO. Endogenous and NO-stimulated ADP-ribosylation of ciliary epithelial membrane proteins was determined by [32P]nicotinamide adenosine diphosphate (NAD) labelling and autoradiography. The three isoforms of the G1α protein subunit were evaluated by western blot analysis. RESULTS: Basal [3H]cAMP content was 13.4 ± 1.3 picomoles/mg protein (SEM). Both isoproterenol and forskolin stimulated [3H]cAMP accumulation to 36.0 ± 3.9 and 73.2 ± 17.5 picomoles/mg protein, respectively. Clonidine did not affect basal [3H]cAMP levels, but attenuated both isoproterenol- and forskolin-mediated [3H]cAMP accumulation to 23.2 ± 4.4 and 31.6 ± 4.6 picomoles/mg protein, respectively. Yohimbine antagonized the clonidine-mediated adenylyl cyclase inhibition. Pertussis toxin blocked the effect of clonidine. In the presence of the NO donor spermine-NO, the clonidine-mediated inhibition of forskolin- and isoproterenol-stimulated cAMP accumulation was attenuated complete NO significantly stimulated endogenous [32P]ADP-ribosylation of a 40-kDa membrane protein. Western blot analysis with specific antibodies revealed expression of all three Gi subtypes - Gi1α, Gi2α, and Gi3α - in bovine ciliary epithelium. CONCLUSIONS: NO attenuates α2-adrenergic receptor-mediated inhibition of adenylyl cyclase in ciliary epithelium through ADP-ribosylation of the Giα subunit. These findings demonstrate heterologous regulation between the NO and cAMP signalling pathways in ciliary epithelium.
Dr S.E. Moroi, Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, MI, USA
2.9 Ciliary body (Part of: 2 Anatomical structures in glaucoma)
11.14 Investigational drugs; pharmacological experiments (Part of: 11 Medical treatment)