Ethanol impairs hormone-stimulated cAMP production in a number of cell types, yet the effects of ethanol on downstream responses mediated by cAMP-dependent protein kinase (PKA) are not understood. Here we have investigated the effects of ethanol feeding on cAMP-mediated inhibition of tumor necrosis factor- (TNF-) synthesis in rat Kupffer cells. Male Wistar rats were fed liquid diets containing 36% of calories as ethanol for 4 wk or were pair fed a control diet. Stimulation of cAMP production by the adenosine A₂ receptor agonist 5'-(N-ethylcarboxamido)-adenosine (NECA), prostaglandin E₂, or forskolin was decreased to 25% of control values in Kupffer cells isolated from ethanol-fed rats. This decrease was associated with a reduction in the quantity of immunoreactive G_s protein in ethanol-fed rats, with no changes observed in G_i or G. TNF- production was higher in ethanol-fed rats in response to stimulation with lipopolysaccharide or latex beads. Despite the profound reduction in the ability of hormone to increase cAMP production, NECA and prostaglandin E₂ inhibited TNF- production to an equivalent degree in Kupffer cells from ethanol- and pair-fed rats. Total activity and immuoreactive protein quantity of PKA did not differ between groups. Activation of PKA in response to a 15-min treatment with 1 µM NECA was reduced by 50% in ethanol-fed rats compared with control. Despite this reduction in activation, translocation of the catalytic subunit of PKA to the nucleus and phosphorylation of cAMP response element binding protein in response to activation were observed in Kupffer cells from both ethanol- and pair-fed rats. These data demonstrate that there is a dissociation between ethanol-induced desensitization of hormone-stimulated cAMP production in rat Kupffer cells and the downstream inhibition of TNF- production mediated by cAMP.

G protein; tumor necrosis factor- ; desensitization; lipopolysaccharide; macrophage; adenosine 3',5'-cyclic monophosphate

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