Cyclic AMP has previously been shown to promote cell survival in a variety of cell types, but the downstream signaling pathway(s) of this anti-apoptotic effect is unclear. Thus, the role of cAMP signaling through protein kinase A (PKA) and cAMP regulated guanine nucleotide exchange factors (cAMP-GEF's) in cAMP's anti-apoptotic action was investigated in the present study. Cyclic AMP's protective effect against bile acid, Fas ligand and tumor necrosis factor- (TNF-) induced apoptosis in rat hepatocytes was largely unaffected by the selective PKA inhibitor, Rp-8-4- chlorophenythio-cAMP (Rp-cAMP). In contrast, a novel cAMP analogue, 8-(4-chloro-phenylthio)- 2'-O-methyladenosine-3'-5'-cyclic monophosphate, (CPT-2-Me-cAMP), which activated cAMP-GEF's in hepatocytes without activating PKA, protected hepatocytes against apoptosis induced by bile acids, Fas ligand and TNF-. The role of cAMP-GEF and PKA on activation of Akt, a kinase implicated in cAMP survival signaling was investigated. Inhibition of PKA with RP-cAMP had no effect on cAMP mediated Akt phosphorylation, while CPT-2-Me-cAMP, which did not activate PKA, induced phosphoinositide-3-kinase (PI3K) dependent activation of Akt. Pretreatment of hepatocytes with the PI3K inhibitor, Ly 294002, prevented CPT-2-Me-cAMP's protective effect against bile acid and Fas ligand, but not TNF-, mediated apoptosis. Glucagon, CPT-cAMP and CPT-2-Me-cAMP all activated Rap1, a downstream effector of cAMP-GEF. Taken together, these results suggest that a PKA independent cAMP/cAMP-GEF/Rap pathway exists in hepatocytes and that activation of cAMP-GEF's promotes Akt phosphorylation and hepatocyte survival. Thus, a cAMP/cAMP-GEF/Rap/PI3K/Akt signaling pathway may confer protection against bile acid and Fas induced apoptosis in hepatocytes.