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 alpha(2)-adrenergic receptor coupling to adenylyl cyclase in bovine ciliary epithelium.

METHODS

Ciliary epithelial explants were dissected, cultured, and labeled with [(3)H]adenine. [(3)H]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 [(32)P]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 [(3)H]cAMP content was 13.4 +/- 1.3 picomoles/mg protein (SEM). Both isoproterenol and forskolin stimulated [(3)H]cAMP accumulation to 36.0 +/- 3.9 and 73.2 +/- 17.5 picomoles/mg protein, respectively. Clonidine did not affect basal [(3)H]cAMP levels, but attenuated both isoproterenol- and forskolin-mediated [(3)H]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 [(32)P]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 inhibition of adenylyl cyclase in ciliary epithelium through ADP-ribosylation of the G(i)alpha subunit. The findings demonstrate heterologous regulation between the NO and cAMP signaling pathways in ciliary epithelium.