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Sayoko E. Moroi, Yibai Hao, Ari Sitaramayya; Nitric Oxide Attenuates α2-Adrenergic Receptors by ADP-ribosylation of Giα in Ciliary Epithelium. Invest. Ophthalmol. Vis. Sci. 2001;42(9):2056-2062.
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purpose. To determine the mechanism by which nitric oxide (NO) regulatesα 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
Giα protein subunit were evaluated by Western blot
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—Gi1α,
Gi2α, and Gi3α—in bovine ciliary
conclusions. NO attenuates α2-adrenergic receptor–mediated inhibition
of adenylyl cyclase in ciliary epithelium through ADP-ribosylation of
the Giα subunit. The findings demonstrate heterologous
regulation between the NO and cAMP signaling pathways in ciliary
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