Buprenorphine differentially alters opioid receptor adaptation in rat brain regions.

Previous in vivo studies revealed that the mixed agonist-antagonist buprenorphine can down-regulate mu and up-regulate delta 2 and kappa 1 opioid receptors in rat brain. In this report brain regional differences in opioid receptor adaptation were addressed. Rats received i.p. injections with buprenorphine (0.5-2.5 mg/kg) and were killed 20 h later. Membranes from 7 brain regions were analyzed for mu (3H-[D-Ala2,N-mephe4,Gly-ol5] enkephalin), kappa 1 (3H-U-69593), delta 1 (3H-[D-Pen2, D-Pen5] enkephalin) and delta 2 (3H-deltorphin II) receptor binding parameters. Buprenorphine induced down-regulation of mu receptors in frontal cortex, occipital cortex, thalamus, hippocampus, striatum and brain stem. Kd values for 3H-[D-Ala2,N-mephe4,Gly-ol5] enkephalin were unchanged from controls. Up-regulation of kappa 1 receptors was observed in frontal, parietal, occipital cortexes and striatum. Binding to delta 2 sites was elevated in frontal and parietal cortexes. Buprenorphine did not alter delta 1 binding in any of the regions examined. Changes in opioid receptor adaptation induced by buprenorphine were further supported by data from cross-linking of 125I-beta-endorphin to cortical membrane preparations. A reduction in a 60- to 65-kDa band was detected in frontal and occipital cortices in which binding assays revealed down-regulation of mu receptors. In parietal cortex neither the 60- to 65-kDa product nor Bmax changes were observed. These results indicate that buprenorphine is a useful tool to study brain opioid receptor adaptation in vivo and the information accrued may be relevant to the mode of action of this drug in the treatment of heroin and cocaine abuse.

Modulation of opioid binding associated with nuclear matrix and nuclear membranes of NG108-15 cells.

Opioid binding sites were found in nuclear matrix preparations from NG108-15 neurohybrid cells. Binding parameters of delta-specific radioligands indicated that high-affinity binding sites discovered in purified nuclei were present in nuclear membranes and nuclear matrix fractions. Agonists bind with low affinity, if at all, to nuclear matrix preparations. Neither sensitivity of agonist binding to the GTP analog 5-guanylylimidodiphosphate nor adenylyl cyclase activity were detected in this fraction, suggesting the presence of guanine nucleotide binding regulatory protein/effector uncoupled sites. Opioid inhibition of basal and forskolin-stimulated adenylyl cyclase activity was found in nuclear membrane preparations. Cycloheximide treatment of cells inhibited opioid binding to nuclear membrane fractions to a greater extent than that associated with membranes sedimenting at 20,000 x g (P20) or nuclear matrix. Colchicine, a microtubule disrupter and inhibitor of receptor internalization, caused up-regulation of nuclear membrane and P20 opioid receptors and a loss in nuclear matrix associated sites. Taxol, a microtubule stabilizing agent, prevented the effect of colchicine. Etorphine-elicited down-regulation increased nuclear matrix associated binding while diminishing that in nuclear membranes and P20 fractions. Agonist-induced desensitization completely abolished nuclear matrix binding. In vitro preincubation of nuclear matrix preparations with protein kinase A catalytic subunit mimicked the desensitization effect. Forskolin treatment of cells potentiated nuclear matrix and P20 binding. These data suggest that nuclear membrane opioid receptors represent newly synthesized molecules en route to the cell surface, whereas nuclear matrix contains internalized delta sites.