OPIOID INHIBITION OF ADENYLYL-CYCLASE IN MEMBRANES FROM PERTUSSIS TOXIN-TREATED NG108-15 CELLS

G(i)/G(0) proteins are uncoupled from receptors by ADP-ribosylation wi th pertussis toxin (PTX). However, PTX treatment of delta opioid recep tor-containing NG108-15 cells reduces, but does not eliminate, opioid inhibition of adenylyl cyclase. The present study explored potential m echanisms of this residual inhibition. Overnight treatment of NG108-15 cells with 100 ng/ml PTX eliminated both PTX-catalyzed [adenylyl-P-32 ]NAD(+)-labeling of G proteins and agonist stimulation of low K-m GTPa se in membranes. Although PTX-treatment decreased the maximal opioid i nhibition of adenylyl cyclase by 50-65%, the inhibition that remained was concentration-dependent and antagonist-reversible. This inhibition persisted in the absence of GTP (even though opioid inhibition of ade nylyl cyclase in untreated membranes was GTP-dependent), but was elimi nated by hydrolysis-resistant guanine nucleotide analogs, indicating t hat G-proteins were still involved in the coupling mechanism. However, assays of agonist-stimulated [S-35]GTP gamma S binding in the presenc e of excess GDP indicated that PTX pretreatment eliminated stimulation of guanine nucleotide exchange by opioid agonists. These results sugg est that in membranes from PTX-treated NG108-15 cells, a subpopulation of G proteins may transduce an inhibitory signal from agonist-bound o pioid receptors without involvement of guanine nucleotide exchange.