||Chronic morphine consumption induces cellular adaptations leading to addict ion. Adenylyl cyclase (AC) supersensitivity is the enhancement of the enzyme's responsiveness to forskolin stimulation observed upon chronic opioid treatment, and it is regulated by opioid receptors (δ-, κ- and μ-) and pertussis toxin (PTX)-sensitive Gi/o proteins. Three members of the Gi subfamily (Gi1, Gi2 and Gi3) were modified into PTX-insensitive Gi proteins (Gi1CG, Gi2z and Gi3CG). Together with the wild type PTX-insensitive Gz, Gi1CG, Gi2z or Gi3CG were stably co-transfected with opioid receptors (δ-, κ- or μ-) into 293 cells. Subsequently, each stable cell line was challenged with acute or chronic opioid stimulation with or without PTX. Results revealed that acute activation of all these PTX-insensitive Gi proteins by opioids inhibited forskolin-stimulated cAMP levels in a PTX-insensitive manner. However, their chronic activation did not lead to AC supersensitivity, despite their ability to functionally interact with all three types of opioid receptors to suppress AC activity. Blockade of [beta gamma]-complex-regulated mitogen-activated protein kinase (MAPK) pathway by the MEK specific inhibitor (PD98059) in 293 cells expressing δ-, κ- or μ-opioid receptors mimicked chronic opioid treatment and induced AC supersensitivity, indicating that MAPK cascade can regulate AC activity. Acute stimulation of δ-, κ- or μ-opioid receptors induced phosphorylation of ERK1/2, a downstream effector of MAPK, through Gz, or endogenous Gi/o proteins, and it was completely blocked by chronic opioid treatment. Acute or chronic opioid treatment had no effect on CREB phosphorylation. Collectively, the present results indicated that (1) chronic activation of individual Gi or Gz proteins is insufficient to trigger AC supersensitivity; (2) MAPK cascade can regulate AC activity and it may be involved in the regulation of AC supersensitivity; and (3) AC supersensitivity has no additive or synergistic effect on ERKl/2 and CREB phosphorylation states.