Mediation of adenylyl cyclase sensitization by PTX-insensitive Gα oA , Gα i1 , Gα i2 or Gα i3

Chronic activation of mu-opioid receptors, which couple to pertussis toxin-sensitive Gα i/o proteins to inhibit adenylyl cyclase (AC), leads to a compensatory sensitization of AC. Pertussis toxin-insensitive mutations of Gα i/o subtypes, in which the pertussis toxin-sensitive cysteine is mutated to isoleucine (G ), were used to determine whether each of the Gα i/o subtypes is able to mediate sensitization of AC. G , G , G or G were individually transiently transfected into C6 glioma cells stably expressing the mu-opioid receptor, or transiently co-expressed with the mu-opioid receptor into human embryonic kidney (HEK)293T cells. Cells were treated with pertussis toxin to uncouple endogenous Gα i/o proteins, followed by acute or chronic treatment with the mu-opioid agonist, [D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]enkephalin (DAMGO). Each Gα i/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 Gα i/o subtype, but the level of sensitization was increased in clones expressing higher levels of Gα i/o subunits. Sensitization of AC mediated by a component of fetal bovine serum, which was also dependent on the level of functional Gα i/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 activity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65707/1/j.1471-4159.2006.04176.x.pd