G(0) MEDIATES THE COUPLING OF THE MU-OPIOID RECEPTOR TO ADENYLYL CYCLASE IN CLONED NEURAL CELLS AND BRAIN

In membranes from SH-SY5Y human neuroblastoma cells differentiated wit h retinoic acid, the mu-selective agonist Tyr-D-Ala-Gly-N-Me-Phe-Gly-o l (DAMGO) inhibited cAMP formation with an IC50 of 26 nM. Two separate antibodies raised against distinct regions of the G(oalpha) sequence attenuated the effect of DAMGO by 50-60%, whereas antibodies to G(ialp ha1,2) or G(ialpha3) reduced the mu-opioid signal insignificantly or t o a lesser extent. In contrast, inhibition of adenylyl cyclase by the delta-opioid agonist Tyr-D-Pen-Gly-Phe-D-Pen-OH (DPDPE; Pen = penicill amine) was very sensitive to the G(ialpha1,2) antibody. In membranes f rom rat brain striatum, coupling of the mu opioid receptor to adenylyl cyclase was also maximally blocked by antibodies to G(oalpha). After long-term treatment of the cells with DAMGO, the content of G(oalpha) was reduced by 26%, whereas the levels of G(ialpha1,2), G(ialpha3), an d G(salpha) were unaltered. Addition of G(o), purified from bovine bra in, to membranes from pertussis toxin-treated SH-SY5Y cells restored t he inhibition of adenylyl cyclase by DAMGO to 70% of that in toxin-unt reated cells. To comparably restore the effect of DPDPE, much higher c oncentrations of G(o) were required. By demonstrating mediation of cAM P-dependent signal transduction by G(o), these results describe (i) an additional role for this G protein present at a high concentration in brain, (ii) preferential, although not exclusive, interaction of mu a nd delta opioid receptors with different G protein subtypes in couplin g to adenylyl cyclase, and (iii) reduced levels of G(o) following chro nic opioid treatment of SH-SY5Y cells with mu opioids.