An assay technique modified from existing procedures was developed for measuring receptor-mediated cyclic AMP formation by intact murine neuroblastoma cells (N1E-115) and was used to study some of the pharmacological characteristics of the prostaglandin E1 (PGE1) receptor and the effects that acute exposure to ethanol has on PGE1 receptor-mediated cyclic AMP formation. The technique involved radioactively labeling the intracellular stores of ATP by incubating the cells with [3H]adenine and then isolating the cyclic [3H]AMP by a two-step chromatography method using a cation exchange resin (Dowex-50+) column followed by an alumina column. The time course for PGE1-mediated cyclic [3H]AMP formation by intact cells in the presence of 100 mM ethanol showed an instantaneous increase in the rate of cyclic [3H]AMP formation. Ethanol added shortly before PGE1 caused a concentration-related potentiation of PGE1-mediated cyclic [3H]AMP formation, but not basal cyclic [3H]AMP formation over a wide range of concentrations (40 mM-1.1 M). One hundred millimolar ethanol increased receptor-mediated cyclic [3H]AMP synthesis about 50 to 70% above control levels after 10 min; 600 mM ethanol, about 400% above control levels. Methanol had similar effects but was less potent than ethanol. In the presence of 100 or 200 mM ethanol, the PGE1 dose-response curve was not shifted, but the maximal response was increased. When controls for the effect of ethanol on the osmolality of the incubation medium were made the effect of ethanol was abolished and increasing medium osmolality with nonpermeable or slowly permeable solutes such as sugars (sucrose, glucose and mannitol) or salts (NaCl and CsCl) mimicked the effect of ethanol. In addition, ethanol stimulated only slightly (about 6 to 18% above control levels) PGE1-mediated adenylate cyclase activity in a membranal preparation from these cells and sucrose inhibited this activity at all concentrations tested. These data suggest that ethanol causes its effect mainly at a site beyond the agonist-receptor complex and that the mechanism of this effect may be related to changes in cellular osmolality.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Pharmacology and Experimental Therapeutics|
|State||Published - 1982|
ASJC Scopus subject areas
- Molecular Medicine