Nitric oxide attenuates alpha(2)-adrenergic receptors by ADP-ribosylation of G(i)alpha in ciliary epithelium

PURPOSE. To determine the mechanism by which nitric oxide (NO) regulates al pha (2)-adrenergic receptor coupling to adenylyl cyclase in bovine ciliary epithelium. METHODS. Ciliary epithelial explants were dissected, cultured, and labeled with [H-3]adenine. [H-3]Adenosine 3 ' ,5 ' -cyclic monophosphate (CAMP) was measured under basal conditions and after exposure to forskolin, isoproter enol, clonidine, yohimbine, pertussis toxin, and the NO donor spermine-NO. Endogenous and NO-stimulatcd ADP-ribosylation of ciliary epithelial membran e proteins was determined by [P-32]nicotinamide adenosine diphosphate (NAD) labeling and autoradiography. The three isoforms of the G(i)alpha protein subunit were evaluated by Western blot analysis. RESULTS. Basal [H-3]cAMP content was 13.4 +/- 1.3 pico-moles/mg protein (SE M). Both isoproterenol and forskolin stimulated [H-3] CAMP accumulation to 36.0 +/- 3.9 and 73.2 +/- 17.5 picomoles/mg protein, respectively. Clonidin e did not affect basal [H-3]cAMP levels, but attenuated both isoproterenol- and forskolin-mediated [H-3]cAMP accumulation to 23.2 +/- 4.4 and 31.6 +/- 4.6 picomoles/mg; protein, respectively. Yohimbine antagonized the clonidi ne-mediated adenylyl cyclase inhibition. Pertussis toxin blocked the effect of clonidine. In the presence of the NO donor spermine-NO, the clonidine-m ediated inhibition of forskolin- and isoproterenol-stimulated CAMP accumula tion was attenuated completely. NO significantly stimulated endogenous [P-3 2]ADP-ribosylation of a 40-kDa membrane protein. Western blot analysis with specific antibodies revealed expression of all three G(i) subtypes - G(i1) alpha, G(i2)alpha, and G(i3)alpha - in bovine ciliary epithelium. CONCLUSIONS. NO attenuates alpha (2)-adrenergic receptor-mediated inhibitio n of adenylyl cyclase in ciliary epithelium through ADP-ribosylation of the G(i)alpha subunit. The findings demonstrate heterologous regulation betwee n the NO and CAMP signaling pathways in ciliary epithelium.