Adenylyl cyclase supersensitivity in opioid-withdrawn NG108-15 hybrid cells requires Gs but is not mediated by the Gsα subunit

On the cellular level, opioid dependence is characterized by a significant elevation of adenylyl cyclase (AC) activity after drug withdrawal, a regulatory phenomenon termed "AC supersensitivity" or "cAMP overshoot." The present study examines the role of the stimulatory G protein (G(s)) in the expression of naloxone precipitated opioid withdrawal in chronically morphine (10 mu M; 3 days) treated neuroblastoma X glioma (NG108-15) hybrid cells. Determination of high-affinity [H-3]forskolin binding to intact cells, which provides a direct parameter for the binding of the activated cr-subunit of G(s) (G(s)alpha) to AC, revealed that the enhancement of AC activity after opioid withdrawal is not caused by an increased stimulation of effector activity by G(s)alpha. Although not a direct function of G(s), the expression of AC supersensitivity required G(s)alpha-mediated stimulation of AC, because 1) the enhancement of AC activity after opioid withdrawal was observed only in the presence of low, but not of high concentrations of forskolin, and 2) chemical inactivation of G(s)alpha by low pH pretreatment abolished the induction of AC supersensitivity. Moreover, the regulatory mechanism,underlying AC supersensitivity not only required the presence of activated G(s)alpha per se, but functional intact stimulatory signal transduction pathways. Indeed, blockade of prostaglandin E-1 receptcr/G(s) interaction in situ with a site-specific anti-G(s)alpha antibody, as well as uncoupling of prostaglandin E-1 receptor signaling by cholera toxin-catalyzed ADP-ribosylation of G(s)alpha, prevented the expression of AC supersensitivity in membranes from opioid-withdrawn cells. These results suggest that the enhancement of AC activity in opioid-dependent cells, triggered by drug withdrawal, is not a direct G(s)alpha effect, but involves a secondary regulatory event that requires costimulation of AC by acutely receptor-activated G(s)alpha.