Morphine receptors as regulators of adenylate cyclase activity.

Abstract

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.

THE BINDING OF [3H]‐PROSTACYCLIN TO MEMBRANES OF A NEURONAL SOMATIC HYBRID

“…Enzyme activity was determined by a modification (Sharma, Nirenberg & Klee, 1975) of method C of Salomon, Londos & Rodbell (1974 (Nicolaou, Bamette, Gasic, Magolda & Sipio, 1977) to give [1 1,3-3H]-PGI2 as its sodium salt. The chemical identity of the stable hydrolysis product (6-oxo-PGF1G,) was determined by gas chromatography-mass spectrometry, and the biological activity compared with that of authentic PGI2 by adenylate cyclase activation in homogenates of the NCB-20 cell line (Blair et al, 1980b (Pert & Snyder, 1973;MacDermot & Nirenberg, 1978 …”

Receptor-mediated shifts in cGMP and cAMP levels in neuroblastoma cells.

“…7E and F (18), activates adenylate cyclase activity in homogenates (19), including those prepared from neuroblastoma X glioma hybrid cells (20), and inhibits the vasopressin-dependent activation of adenylate cyclase (21).…”

“…Each filter was placed in a scintillation vial with 1 ml of 2-methoxyethanol; after filters dissolved (0.5-1 hr) 15 ml of Aquasol (New England Nuclear) were added. Radioactivity was determined [12][13][14][15][16][17][18][19][20][21][22][23][24] hr later in a Beckman liquid scintillation counter (3H plus 14C isoset).…”

Prostacyclin‐dependent Activation of Adenylate Cyclase in a Neuronal Somatic Cell Hybrid: Prostanoid Structure‐activity Relationships

“…Prostaglandin El (PGEI) increases adenylate cyclase (ATP: pyrophosphate-lyase (cyclizing); EC 4.6.1.1) activity in rat brain homogenates (Abdulla & McFarlane, 1972;Collier & Roy, 1974;Duffy & Powell, 1975), many neuroblastoma (Brunton, Wiklund, Van Arsdale & Gilman, 1976) and two neuronal hybrid cell lines (Sharma, Nirenberg & Klee, 1975;MacDermot, Higashida, Wilson, Matsuzawa, Minna & Nirenberg, 1979). A regulatory function of E-prostaglandins at nerve terminals has been proposed (Hedqvist, 1977), with inhibition of noradrenaline release from peripheral autonomic nerves.…”