Nitric oxide attenuates alpha(2)-adrenergic receptors by ADP-ribosylation of G(i)alpha in ciliary epithelium.
OBJECTIVE
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.
Moroi
S E
SE
Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor 48105, USA. smoroi@umich.edu
Hao
Y
Y
Sitaramayya
A
A
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D002924:Ciliary Body / Q000187:drug effects / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009924:Organ Culture Techniques; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
D000067956:Adenylyl Cyclase Inhibitors; D020030:Nitric Oxide Donors; D018341:Receptors, Adrenergic, alpha-2; D000246:Adenosine Diphosphate Ribose; D009569:Nitric Oxide; D000242:Cyclic AMP; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000262:Adenylyl Cyclases