Enhanced cyclic AMP responses in GH3 pituitary cells pretreated with muscarinic receptor agonists

The effect of prolonged activation of cholinergic receptors on the subsequent cAMP responses in GH3 pituitary cells was examined. Whereas acetylcholine pretreatment had no effect on basal cAMP levels, it transiently enhanced vasoactive intestinal peptide-stimulated cAMP accumulation in the cells and this effect was abolished by atropine. Inclusion of eserine in the incubation medium, however, sustained and reinforced the enhancing effect of acetylcholine on forskolin-stimulated cAMP synthesis over 24 h. Similar enhancing effect was observed by oxotremorine or carbachol pretreatment. Furthermore, cAMP production in response to forskolin in the absence or presence of 3-isobutyl-1-methylxanthine was similarly augmented by acetylcholine pretreatment. The enhancement of forskolin-stimulated cAMP accumulation was arrested by pertussis toxin whether it was added before or during the oxotremorine pretreatment. Although cholera toxin pretreatment increased greatly the forskolin-stimulated cAMP levels, concomitant addition of oxotremorine was able to augment the response further in GH3 cells. In addition, cholera toxin- or pertussis toxin-catalysed ADP-ribosylation of G proteins as well as immunoreactive Gs alpha or Gi alpha levels in membranes obtained from oxotremorine-pretreated cells were not different from those from non-treated cells. Thus, the present study has demonstrated that continuous muscarinic receptor activation leads to increased adenylate cyclase activity in GH3 cells. Although a functional Gi/Go seems to be required for the development of this enhanced activity, neither Gi/Go nor Gs appears to be altered.