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 G_i/o proteins. Three members of the G_i subfamily (G_i1, G_i2 and G_i3) were modified into PTX-insensitive G_i proteins (G_i1CG, G_i2z and G_i3CG). Together with the wild type PTX-insensitive G_z, G_i1CG, G_i2z or G_i3CG 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...[ Read more ]

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 G_i/o proteins. Three members of the G_i subfamily (G_i1, G_i2 and G_i3) were modified into PTX-insensitive G_i proteins (G_i1CG, G_i2z and G_i3CG). Together with the wild type PTX-insensitive G_z, G_i1CG, G_i2z or G_i3CG 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 G_i 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 G_z, or endogenous G_i/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 G_i or G_z 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.