ACTIVATION OF A CYCLIC AMP-GUANINE EXCHANGE FACTOR IN HEPATOCYTES DECREASES NITRIC OXIDE SYNTHASE EXPRESSION

Adenosine 3'5'-cyclic adenosine monophosphate (cAMP) activates intracellular signaling by regulating protein kinase A, calcium influx, and cAMP-binging guanine nucleotide exchange factors (Epac [exchange protein directly activated by cAMP] or cAMP-GEF). Cyclic adenosine monophosphate inhibits cytokine-induced expression of inducible nitric oxide synthase (iNOS) in hepatocytes by a protein kinase A independent mechanism. We hypothesized that Epac mediates this effect. A cyclic AMP analog that specifically activates Epac, 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3'5'-cyclic monophosphate (OPTmecAMP), and overexpression of liver-specific Epac2 both inhibited interteukin 1 beta/interferon gamma-induced iNOS expression and nitrite production. OPTmecAMP inactivated Raf1/MEK/ERK signaling, but ERK had no effect on iNOS expression. OPTmecAMP induced a persistent Akt phosphorylation in hepatocytes that lasted up to 8 h. Overexpression of a dominant-negative Aid blocked the inhibitory effect of OPTmecAMP on iNOS production. A specific PI3K inhibitor, LY294002, attenuated the inhibition of nitrite production and iNOS expression produced by overexpressing a liver-specific Epac2 (LEpac2). OPTmecAMP also induced c-Jun N-terminal kinase (JNK) phosphorylation in hepatocytes. Overexpression of dominant-negative JNK enhanced cytokine-induced iNOS expression and nitrite production and reversed the inhibitory effects of LEpac2 on nitrite production and iNOS expression. We conclude that Epac regulates hepatocyte iNOS expression through an Aid- and JNK-mediated signaling mechanism.