Adenylyl cyclase (AC) 'superactivation' has long held a central position in models of opioid tolerance. The enclosed application will explore a new, complementary hypothesis that opioid tolerance also results from changes in the consequences of opioid receptor activation of Gi. Specifically, it is postulated that chronic opioid treatment induces a shift from opioid receptor- Galphai inhibition to Gbetagamma (Gi-derived) stimulation of AC activity. This would result from the induction of Gbetagamma-stimulated ACs and increases in AC isoform-specific phosphorylation after chronic morphine. In chronic morphine-treated tissue and cell lines, stimulatory responsiveness of some AC isoforms, assessed in the absence of exogenous opioid, is significantly reduced. The switch from inhibitory to stimulatory opioid receptor signaling would compensate for this attenuated activity. Consequently, despite the continued presence of inhibitory concentrations of opioid, 'normal' activity of these isoforms would be maintained, i.e., opioid tolerance would ensue. Although the myenteric plexus has been of enormous value in formulating the above hypothesis, its proof will require the use of simpler, cell culture systems such as CHO and HEK 293 cell lines, stably transfected with the mu opioid receptor. The specific aims are: (1) Determine the effect of chronic opioid treatment on levels of mRNA encoding Gbetagamma-stimulated AC isoforms and AC protein. (2) Determine the effect of chronic opioid treatment on inhibitory vs stimulatory opioid receptor-AC signaling. (3) Determine if the chronic morphine-induced shift from inhibitory to stimulatory opioid receptor signaling is mediated via augmented Gbetagamma stimulation of AC. (4) Determine the specific AC isoform(s) (I, II, IV VII) and sites therein that manifest augmented phosphorylation following chronic morphine. Altered content of G proteins and opioid receptor coupling thereto has been a predominant focus of attempts to elucidate neurochemical underpinnings of tolerance. The formulation that chronic morphine induces changes in the relative abundance and phosphorylation state of specific AC isoforms which in turn alters the consequences of opioid receptor activation of Gi is novel. It represents a new approach to probing narcotic tolerance which could result in more effective pharmacotherapies for managing pain.