Inhibition of the adenylate cyclase activity in homogenates of mouse neuroblastoma-glioma hybrid cells (NG108-15) by the opioid peptide [-Ala2,Metsjenkephalin amide (AMEA) requires the presence of Na+ and GTP. In this process, the selectivity for monovalent cations is Na+ > Li+ > K+ > choline+; ITP will replace GTP but ATP, UTP, or CTP will not. The apparent Km for Na+ is 20 mM and for GTP it is I .&M. Under saturating Na+ and GTP conditions, the apparent Ki for AMEA-directed inhibition is 20 nM for basal and 100 nM for prostaglandin E1-activated adenylate cyclase activity. For both cyclase activities, maximal inhibition is only partial (i.e., -55% of control in each case). In intact viable NG108-15 cells, the decrease in basal and prostaglandin El-stimulated intracellular cyclic AMP concentrations by AMEA is also dependent upon extracellular Na+. Thee ephalin-directed reductions in cyclic AMP co riss are at least 75%. The specificity of the monovalent cation requirement for enkephalin action on intact cells is the same as for enkephalin regulation of homogenate adenylate cyclase activity. Based on these data, a model is presented in which the transfer of information from opiate receptors to adenylate cyclase requires active separate membrane components, which correspond to the sites of action of Na+ and GTP in this process. Opiates decrease the intracellular cyclic AMP (cAMP) concentrations of intact mouse neuroblastoma-rat glioma hybrid cells (NG108-15) by inhibiting the activity of the adenylate cyclase of these cells (1-3). This regulation of cAMP requires occupation of highly specific receptors by opiate agonists and is blocked by opiate antagonists. Recently, these opiate receptors were found to be sensitive to guanine nucleotides (4, 5) and Na+ (4-7). The effects of Na+ and nucleotides on NG108-15 opiate receptors parallel the effects of Na+ (8-14) and nucleotides (15, 16) on brain opiate receptors. Although nucleotides and Na+ have nonspecific effects on NG108-15 and brain opiate receptors, the actions of ions and nucleotides can be used to discriminate between agonist and antagonist interactions with opiate receptors.The selective nucleotide and ion regulation of agonist interactions with opiate receptors is reminiscent of the welldocumented guanine nucleotide regulation of agonist interaction with hormonal receptors that mediate activations of adenylate cyclase (17-20). In the latter case, agonist occupation of the receptor can take place without any nucleotides present; however, in order for the agonist-receptor complex to cause an increase in adenylate cyclase activity, the presence of a guanine nucleotide is required. Biochemical (21), genetic (22, 23), cell fusion (24, 25), and reconstitution (26-37) studies have shown that the receptor and catalytic units of adenylate cyclase are separate proteins and that another membrane component(s) (here termed the "coupler") is actually required for receptordirected activation of the catalytic unit.The published observations of Na+ and nucleotide sen...

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