Narcotics affect adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.8.1.1J in two opposing ways, both mediated by the opiate receptor. The first process is the readily reversible inhibition of the enzyme by narcotics; the second is a compensatory increase in enzyme activity which is delayed in onset and relatively stable. Late positive regulation of the enzyme counteracts the inhibitory influence of morphine and is responsible for narcotic dependence and tolerance. The coupled inhibitory and positive regulatory mechanisms for adenylate cyclase provide a means of activating and deactivating neural circuits hours after the initial event and thus may play a role in a memory process. Recent observations with neuroblastoma X glioma hybrid cells indicate that the binding of morphine and other opiates to narcotic receptors results in an inhibition of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity (1-3) and a decrease in cAMP levels in intact cells (1, 2, 4, 5). Similar observations have been made with brain (6, 9) but the heterogeneity of cell types present and apparent lability of the brain enzyme may be responsible for conflicting observations (8)(9)(10). Both dependence upon opiates and tolerance to these compounds were hypothesized to result from either an increase in the number of molecules of adenylate cyclase or a long-lived factor which affects the rates of adenylate cyclase activity or turnover (1). In this report, we describe the results of experiments which were designed to test the hypothesis illustrated diagramatically in Fig. 1. The addition of morphine results in the rapid inhibition of adenylate cyclase activity and a resultant decrease in intracellular cAMP levels. Further incubation reveals a second regulatory process which involves a compensatory increase in adenylate cyclase activity termed late positive regulation. The increase in adenylate cyclase activity counteracts the inhibition of enzyme activity by morphine and cAMP levels are restored to the normal value. Cells now are tolerant to morphine and are also dependent upon the narcotic, since withdrawal of the drug or the addition of a specific narcotic antagonist will raise cAMP levels to abnormally high values and secondarily produce a gradual return to the normal level of adenylate cyclase activity.In this communication we report data which demonstrate a rapid inhibition and a late positive regulation of adenylate cyclase which are dependent upon narcotics and account for the phenomena of narcotic dependence and tolerance. METHODS AND MATERIALSThe source of each chemical and the medium and growth conditions for neuroblastoma X glioma hybrid NG108-15 were described previously (1).Assay of cAMP in Intact Cells and Medium. Growth medium was changed 12 hr before assay. Narcotic was added to the growth medium [Dulbecco's modification of Eagle's medium (DMEM), hypoxanthine-aminopterinthymidine (HAT), and 10% fetal bovine serum] and plates were incubated in a humidified atmosphere of 90% air-10% C02. A...

Morphine inhibits adenylate cyclase (EC 4.6.1.1) activity of neuroblastoma X glioma hybrid cells. The inhibition is stereospecific and is reversed by the antagonist, naloxone. The relative affinities of narcotics for the opiate receptor agree well with their effectiveness as inhibitors of adenylate cyclase. Morphine-sensitive and -insensitive cell lines were found, and the degree of sensitivity was shown to be dependent upon the abundance of narcotic receptors. Thus, morphine receptors are functionally coupled to adenylate cyclase. A molecular mechanism for narcotic addiction and tolerance is proposed.A neuroblastoma X glioma hybrid cell line with morphine receptors has been described (1). The receptors are stereospecific with respect to narcotic binding, and receptor affinities for narcotics closely resemble those of rat brain (2-4) and correlate well with their pharmacologic potencies.Collier and Roy have reported that prostaglandin E dependent cAMP synthesis is inhibited by narcotics in rat brain homogenates (5,6 Adenylate Cyclase Assay. Cells that had been washed three times were homogenized in 0.32 M sucrose, 10 mM Tris * HCl, pH 7.4 (15 mg of protein per ml) with 10-15 strokes, by hand, of a ground-glass homogenizer. Enzyme activity was determined by a modification of method C of Salomon et al.(11). Each tube contained: 45 mM Tris-HCl, pH 7.4; 5 mM MgCl2; 160 mM sucrose; 20 mMl creatine phosphate; 10 U of creatine kinase; 1 mM cAMP; 0.5 mM Ro20-1724 (0.5% ethanol, final concentration); 1 mM [a-32P]ATP (3 to 5 X 106 cpm); and 100-300 jg of homogenate protein in a final volume of 100 u.d The reaction was terminated by addition of 50 ,ul of 15% trichloroacetic acid. For product characterization, the 32p product purified through the alumina column step (11) was subjected to Dowex-1 formate column chromatography. The 2 M formic acid eluate was lyophilized, and the radioactivity was characterized by paper or thin-layer chromatography in: (A) isopropanol-NH40H-0.1 M boric acid (7:1:2); (B) 1 M ammonium acetate-95% ethanol (3:7); (C) isobutyric acid-2 M NH40H (2:1); and (D) H20-washed polyethyleneimine-cellulose thin-layer plates developed sequentially with H20 and 0.25 M LiCl. Greater than 90% of the radioactivity recovered from the alumina columns was cAMP.Opiate Binding.

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We immunized rabbits with purified guanine nucleotide-binding proteins (G proteins) from bovine brain and obtained an antiserum, RV3, that reacts specifically with the a subunit (39 kDa) of a G protein of unknown function, termed Go, as well as with the P subunit (35 kDa) common to all G proteins. RV3 showed no crossreactivity with the a subunits of the stimulatory (Gj) or inhibitory (Gi) G proteins associated with adenylate cyclase, nor with that of the rod outer segment G protein, transducin. Immunoblots with crude and affinitypurified antiserum showed that RV3 specifically recognizes the

Reactions mediated by the opiate receptors that inhibit adenylate cyclase (EC 4.6 (1-3). However, exposure of cells to an opiate for 12 or more hours results in a compensatory increase in adenylate cyclase activity that is long-lived and not readily reversed by withdrawal of the opiate (4-6). Similar dual regulations of adenylate cyclase activity are mediated by a-adrenergic receptors (7) and excitatory muscarinic acetylcholine receptors (S. K. Sharma and M. Nirenberg, unpublished data; ref. 8). Thus, dual regulation may be a general regulatory mechanism which alters the responsiveness of adenylate cyclase to activations mediated by other species of receptors. Because the mechanism of coupling inhibition of adenylate cyclase with a subsequent increase in enzyme activity is unknown, we examined the properties of adenylate cyclase from control cells and cells treated with opiates. MATERIALS AND METHODSThe source of each chemical, growth conditions for culturing neuroblastoma X glioma NG108-15 hybrid cells, and preparation of homogenates have been described (2). Adenylate cyclase activity was measured by the procedure of Salomon et al. RESULTSEffect of Activators of Adenylate Cyclase. If the increase in adenylate cyclase activity of opiate-treated NG108-15 cells were due primarily to an increase in the number of molecules of adenylate cyclase, the relatively high enzyme activity of opiate-treated cells should be maintained under other assay conditions. For this reason, we studied the effects of activators and inhibitors on adenylate cyclase of cells growmn in the presence or absence of morphine or etorphine for S35 days. Sodium fluoride is known to activate basal adenylate cyclase and inhibit activation of the enzyme by hormones, presumably by uncoupling the enzyme from the hormone-receptor complex (10).The effects of NaF upon basal and prostaglandin El (PGE1)-stimulated adenylate cyclase activity from control or etorphine-treated NG108-15 cells as a function of time are shown in Fig. 1. In the presence of 8 mM NaF, the basal activities of adenylate cyclase from control and opiate-treated cells were increased 4.3-and 2.6-fold, respectively, and the specific activities of the two enzyme preparations then were similar.Conversely, the activities of PGEI-stimulated adenylate cyclase from control and opiate-treated cells were inhibited 44% and 60%, respectively, by NaF and the final specific activities were similar. These results show that adenylate cyclase from control cells is more responsive to activation by NaF than adenylate cyclase of opiate-treated cells and raise the possibility that the relatively high activity of adenylate cyclase of opiate-treated cells may be due to enzyme activation rather than to an increase in the number of enzyme molecules.