Influence of Gz and Gi2 transducer proteins in the affinity of opioid agonists to μ receptors

The affinity displayed by different opioids to mu receptors (ORs) was determined in mouse brain membranes incubated with antibodies directed to Gee subunits of the guanine nucleotide-binding proteins Gi2 and Gz. Assays were conducted with 10 pM I-125-Tyr(27)-beta-endorphin in the presence of 300 nM N,N-diallyl-Tyr-(alpha-aminoisobutyric acid)2-Phe-Leu-OH (ICI-174 864), which prevented the binding of the iodinated neuropeptide to delta-ORs. Gpp(NH)p or the preincubation of mouse brain membranes with IgGs to G(i2)alpha or G(z)alpha subunits, promoted reductions in the affinity exhibited by the labelled probe. The potencies of beta-endorphin, [D-Ala(2),N-MePhe(4),Gly-ol(5)]-enkephalin (DAMGO) and [D-Pen(2,5)]enkephalin (DPDPE) were reduced after impairing the coupling of mu-ORs to Gi2 or Gz proteins. Morphine showed a loss of affinity towards the mu-OR after preincubation of membranes with IgGs to G(z)alpha subunits. However, it retained its potency after treatment with the anti-G(i2)alpha IgGs. Conversely, [D-Ala(2), D-Leu(5)]enkephalin (DADLE) and [D-Ser(2), Leu(5)] enkephalin-Thr(6) (DSLET) showed decreased affinity to mu-ORs after treatment with anti-G(i2)alpha IgGs, with no noticeable change following the use of IgGs to G(z)alpha subunits. The affinity exhibited by the opioid antagonists naloxone, naltrexone, naloxonazine and [Cys(2),Tyr(3), Orn(5), Pen(7) amide]somatostatin analogue (CTOP) remained unchanged after either treatment. Therefore, the affinity exhibited by opioid agonists of mu-ORs, but not antagonists, depends on the nature of the G-protein coupled to these receptors.