Functional aspects of opioid and alpha2-adrenergic receptor activation: Involvement of specific G proteins

Spinal coadministration of opioid and alpha2-adrenergic agonists results in analgesia which is greater than the sum of the analgesic effects of each of the drugs when administered separately. The mechanisms mediating the interactions which produce the greater-than-additive (synergistic) analgesia are not well understood, but may indicate important therapeutic targets for pain management. This project investigated the role of heterotrimeric G proteins on the functional interactions produced when opioid- and alpha 2-receptor agonists are used together.;The first part of this project identified Gi1alpha, G i2alpha, Gi3alpha, Goalpha (two forms), Gsalpha, Gzalpha, Gq/11alpha, G 12alpha, and Gbeta in mouse spinal neuronal membranes. The second part examined the association of the Galpha subunits with receptors in vitro, by measuring agonist-enhanced incorporation of [alpha- 32P]GTP-azidoanilide ([alpha-32P]GTP-AA) into Galpha. Selective mu-opioid and alpha2-agonists enhanced incorporation of [alpha-32P]GTP-AA into Go1alpha . When membranes were incubated with [alpha-32P]GTP-AA in the presence of both mu-opioid and alpha2-agonists, the amount of [alpha-32P]GTP-AA incorporated into Go1alpha was additive.;The third part investigated the role of Gzalpha or G oalpha in antinociceptive synergism. Spinally injected antisense oligodeoxynucleotides (ODN) directed against Gzalpha attenuated alpha2- as well as delta-opioid-agonist-induced antinociception, but did not affect antinociception induced by morphine, or the selective mu- or kappa-opioid agonists. In contrast, pretreatment with antisense ODN to Goalpha attenuated both morphine- and clonidine-induced antinociception. Pretreatment with antisense ODN to Gzalpha or Goalpha did not affect antinociceptive synergism between the two agonists.;The fourth part of the project investigated the role of G proteins in morphine and clonidine tolerance. Tolerance to these drugs did not affect the expression of Galpha or Gbeta subunits. However, tolerance to morphine increased the activity of Go1alpha as indicated by increased incorporation of [alpha-32P]GTP-AA into G o1alpha.;These findings suggest that spinal opioid and alpha2-adrenergic agonists associate with multiple G proteins to produce antinociception. Functional interactions between opioid and alpha2-agonists are additive for the GTP binding to Go1alpha. Increased activity of G o1alpha may also be part of intracellular adaptive mechanisms that occur during the development of morphine tolerance.