Mediation of adenylyl cyclase sensitization by PTX-insensitive GαoA, Gαi1, Gαi2 or Gαi3

Chronic activation of mu-opioid receptors, which couple to pertussis toxin-sensitive Gai/o proteins to inhibit adenylyl cyclase (AC), leads to a compensatory sensitization of AC. Pertussis toxin-insensitive mutations of Gai/o subtypes, in which the pertussis toxin-sensitive cysteine is mutated to isoleucine (Ga i=o), were used to determine whether each of the Gai/o subtypes is able to mediate sensitization of AC. Ga oA, Ga CI i1 , Ga CI i2 or Ga CI i3 were individually transiently transfected into C6 glioma cells stably expressing the muopioid receptor, or transiently co-expressed with the muopioid receptor into human embryonic kidney (HEK)293T cells. Cells were treated with pertussis toxin to uncouple endogenous Gai/o proteins, followed by acute or chronic treatment with the mu-opioid agonist, [D-Ala,N-Me-Phe, Gly-ol]enkephalin (DAMGO). Each Gai/o subtype mediated acute DAMGO inhibition of AC and DAMGO-induced sensitization of AC. The potency for DAMGO to stimulate sensitization was independent of the Gai/o subtype, but the level of sensitization was increased in clones expressing higher levels of Gai/o subunits. Sensitization of AC mediated by a component of fetal bovine serum, which was also dependent on the level of functional Gai/o subunits in the cell, was observed. This serum-mediated sensitization partially masked mu-opioid-mediated sensitization when expressed as percentage overshoot due to an apparent increase in AC