Regulation of the stress-like protein adenotin in PC 12 cells by ethanol exposure

The effects of chronic ethanol exposure on the stress-like protein adenotin were investigated using the radioligand [3H]-5'-N-ethylcarboxamidoadenosine ([3H]NECA). A 4-day exposure to 150 mM ethanol increased both the KD and the density of [3H]NECA binding sites. These changes were not due to residual ethanol as the acute addition of ethanol did not alter [3H]NECA binding. Chronic ethanol exposure of A126-1B2-1 cells, which are a mutant PC 12 cell line deficient in protein kinase A (PKA), increased the cellular density of adenotin, but did not affect the KD for the radioligand. Conversely, when PC 12 cells were exposed to 10 microM forskolin for either 2 or 4 days, the cellular density of adenotin was not altered, but the affinity of adenotin for [3H]NECA was reduced significantly. An increase in KD was not observed after a 1-hr exposure of PC 12 cells to forskolin, indicating that the reduction in affinity for the radioligand was not due simply to a PKA-mediated phosphorylation of adenotin. The present study demonstrated that chronic ethanol regulates adenotin through two different mechanisms. The ethanol-induced increase in the density of adenotin does not involve PKA, while the reduction in affinity ap pears to involve a cAMP-dependent mechanism.