ALTERATIONS IN THE EXPRESSION OF G-PROTEINS AND REGULATION OF ADENYLATE-CYCLASE IN HUMAN NEUROBLASTOMA SH-SY5Y CELLS CHRONICALLY EXPOSED TO LOW-EFFICACY MU-OPIOIDS

Western-blot analysis of human neuroblastoma SH-SY5Y cells (mu- and delta-receptors) revealed the presence of the following G-protein subunits: G(i)alpha1, G(i)alpha2, G(s)alpha, G(o)alpha, G(z)alpha, and Gbeta, a pattern resembling that observed in central nervous tissue. Chronic treatment of differentiated [all-trans-retinoic acid (10 muM; 6 days)] SH-SY5Y cells With D(-)-morphine (10 muM; 3 days) significantly increased the abundance of all G-protein subunits identified. Co-incubation of morphine-exposed cells together with naloxone (10 muM; 3 days) or the mu-selective opioid antagonist CTOP (10 muM; 3 days), but not with the delta-selective antagonist ICI-174,864 (10 muM; 3 days), completely abolished this effect, suggesting that the increase in G-protein abundance is specifically mediated by mu-receptors. Moreover, the biologically inactive enantiomer L(+)-morphine (10 muM; 3 days) failed to produce a similar effect. G-protein up-regulation developed in a time- and dose-dependent manner and is most likely due to enhanced protein synthesis de novo, since concomitant treatment of the cells with cycloheximide (100 mug/ml; 3 days) prevented this effect. Chronic treatment with the low-efficacy mu-selective opioid peptide morphiceptin (10 muM: 3 days), but not with the highly potent mu-agonist DAGO (0.1 muM, 3 days) produced a comparable increase in G-protein abundance. Coincident with quantitative effects on G-protein levels in morphine-tolerant/dependent SH-SY5Y cells, we found elevated levels of basal, forskolin (1 muM)- and prostaglandin-E1 (1 muM)-stimulated adenylate cyclase activities. Reconstitution experiments using S49 cyc- lymphoma-cell membranes suggest that this increase is most likely due to elevated levels of functionally intact G(s). Chronic treatment with both morphine and DAGO induces high degrees of tolerance in this cell line. However, the intrinsic activity of G(i) was unchanged, as assessed in functional studies with low-nanomolar concentrations of guanosine 5'-[betagamma-imido]triphosphate. Our data demonstrate that chronic treatment of SH-SY5Y cells with low-efficacy mu-opioids increases G-protein abundance, a phenomenon which might contribute to the biochemical mechanisms underlying opioid tolerance/dependence.