dihydromorphine and Glioma

In synaptosomal membranes from rat brain cortex, the mu selective agonist [3H]dihydromorphine in the absence of sodium, and the nonselective antagonist [3H]naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with Kd values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 microM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP[gamma-35S] binding sites by 90% and low Km, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP[gamma S] was diminished. High-affinity (Kd, 0.72 nM), guanine nucleotide-sensitive agonist binding was reconstituted by polyethylene glycol-induced fusion of the alkali-treated membranes with (opioid receptor devoid) C6 glioma cell membranes. Also restored was opioid agonist-stimulated, naltrexone-inhibited GTPase activity. In contrast, antagonist binding in the fused membranes was unaltered. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low Km GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor-guanine nucleotide binding protein complex.

Topics: Animals; Binding, Competitive; Cell Line; Cell Membrane; Cells, Cultured; Cerebral Cortex; Dihydromorphine; Glioma; GTP Phosphohydrolases; Guanosine 5'-O-(3-Thiotriphosphate); Kinetics; Male; Membrane Fusion; Naloxone; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, mu; Synaptosomes

In the course of our studies on lipidoses induced by amphiphilic drugs, we have investigated the ef- of desipramine, a tricyclic antidepressant, on glial cells in culture. We noted that the addition of desipramine to the culture medium of C6 glioma cells resulted in the modification of the lipid profile of the cell membranes. Of particular interest was the presence, in the desipramine-treated cells, of an additional lipid comigrating on thin layer chromatography with sulfogalactosylceramide (S-GalCer). Addition of radiolabelled sulfuric acid in the culture medium of the desipramine-treated cells resulted in the incorporation of [35S]sulfate in the newly synthesized lipid. Furthermore, this lipid was localized selectively by indirect immunofluorescence using a specific rabbit anti-S-GalCer antibody on the cell surface of desipramine-treated, but not control, C6 cells. Desipramine also increased the activity of 3'-phosphoadenosine-5'-phosphosulfate sulfotransferase (the enzyme responsible for the synthesis of S-GalCer). Since it has been suggested that S-GalCer may be involved in opiate receptors, we looked for opiate binding sites on C6 glioma cells after exposure to desipramine. We found that dihydromorphine was able to bind to the desipramine-treated C6 cell membrane. The binding of [3H]dihydromorphine (180 fmol/mg protein) was stereospecific and had a KD of 30-60 nM. Furthermore, morphine reduced both the basal and isoproterenol-stimulated cyclic AMP levels of the desipramine-treated C6 cells. This effect was blocked by naloxone. In these respects, the opiate binding sites induced after treatment of C6 glioma cells with desipramine fulfill the requirements of a true opiate receptor.

Topics: Animals; Cerebrosides; Desipramine; Dihydromorphine; Fluorescent Antibody Technique; Galactosylceramides; Glioma; Kinetics; Receptors, Opioid; Time Factors

Incubation for 48 hours of C6 glioma cell cultures with 10(-4)M tricyclic antidepressant desipramine gave rise to a quantitative increase of total lipids and to qualitative modifications of glycosphinegolipids involving detection by thin-layer chromatography of spots migrating according to cerebroside and sulfatide and presence of an abnormal ganglioside pattern. These lipid modifications were associated with the appearance of stereospecific binding of opiates (dihydromorphine) with a dissociation constant of 30-60 nM. These results favor an important role of lipids in opioid receptor function.

Topics: Animals; Cell Transformation, Neoplastic; Desipramine; Dihydromorphine; Glioma; Lipid Metabolism; Neuroblastoma; Rats; Receptors, Opioid; Stereoisomerism