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(Investigative Ophthalmology and Visual Science. 2001;42:2056-2062.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

Nitric Oxide Attenuates {alpha}2-Adrenergic Receptors by ADP-ribosylation of Gi{alpha} in Ciliary Epithelium

Sayoko E. Moroi1, Yibai Hao1 and Ari Sitaramayya2

1 From the Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor; and 2 Eye Research Institute, Oakland University, Rochester, Michigan.

PURPOSE. To determine the mechanism by which nitric oxide (NO) regulates {alpha}2-adrenergic receptor coupling to adenylyl cyclase in bovine ciliary epithelium.

METHODS. Ciliary epithelial explants were dissected, cultured, and labeled 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) labeling and autoradiography. The three isoforms of the Gi{alpha} 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 completely. 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—Gi1{alpha}, Gi2{alpha}, and Gi3{alpha}—in bovine ciliary epithelium.

CONCLUSIONS. NO attenuates {alpha}2-adrenergic receptor–mediated inhibition of adenylyl cyclase in ciliary epithelium through ADP-ribosylation of the Gi{alpha} subunit. The findings demonstrate heterologous regulation between the NO and cAMP signaling pathways in ciliary epithelium.






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