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.

Dynorphins modulate DNA synthesis in fetal brain cell aggregates.

Previously, opioid peptide analogues, beta-endorphin, and synthetic opiates were found to inhibit DNA synthesis in 7-day fetal rat brain cell aggregates via kappa- and mu-opioid receptors. Here dynorphins and other endogenous opioid peptides were investigated for their effect on DNA synthesis in rat and guinea pig brain cell aggregates. At 1 microM, all dynorphins tested and beta-endorphin inhibited [3H]thymidine incorporation into DNA by 20-38% in 7-day rat brain cell aggregates. The putative epsilon-antagonist beta-endorphin (1-27) did not prevent the effect of beta-endorphin, suggesting that the epsilon-receptor is not involved in opioid inhibition of DNA synthesis. The kappa-selective antagonist norbinaltorphimine blocked dynorphin A or B inhibition of DNA synthesis, implicating a kappa-opioid receptor. In dose-dependency studies, dynorphin B was three orders of magnitude more potent than dynorphin A in the attenuation of thymidine incorporation, indicative of the mediation of its action by a discrete kappa-receptor subtype. The IC50 value of 0.1 nM estimated for dynorphin B is in the physiological range for dynorphins in developing brain. In guinea pig brain cell aggregates, the kappa-receptor agonists U50488, U69593, and dynorphin B reduced thymidine incorporation by 40%. When 21-day aggregates were treated with dynorphins, a 33-86% enhancement of thymidine incorporation was observed. Because both 7- and 21-day aggregates correspond to stages in development when glial cell proliferation is prevalent and glia preferentially express kappa-receptors in rat brain, these findings support the hypothesis that dynorphins modulate glial DNA synthesis during brain ontogeny.

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.

Opioids inhibit endothelin-mediated DNA synthesis, phosphoinositide turnover, and Ca2+ mobilization in rat C6 glioma cells.

Opioid agonists inhibit DNA synthesis in C6 rat glioma cells that express opioid receptors, induced by desipramine (DMI). This inhibition was not observed in cells that were not treated with DMI, and thus did not express opioid-binding sites. Endothelin, a known mitogen, increased thymidine incorporation dose dependently (up to 1.7-fold) in DMI-treated C6 cells. This increase was reversed by an anti-idiotypic antibody to opioid receptors, Ab2AOR, which has opioid agonist properties. The opioid antagonist naltrexone blocked the inhibition caused by Ab2AOR. Endothelin also stimulated phosphoinositide (PI) turnover and this effect was inhibited by morphine (50%) or by Ab2AOR (72%) in DMI-treated but not in DMI-untreated C6 cells. These actions of morphine and Ab2AOR were reversed by naltrexone. The inhibition of PI turnover and of thymidine incorporation by Ab2AOR or morphine was insensitive to pertussis toxin (PTX). Since PI turnover is known to induce Ca2+ mobilization, it was of interest to examine the effects of the applied opioids on intracellular Ca2+ concentrations. Endothelin increased the concentration of cytosolic free Ca2+ in the cells while Ab2AOR, morphine, and beta-endorphin reversed the endothelin-induced Ca2+ mobilization in DMI-treated but not in DMI-untreated C6 cells. The effect of these agonists was also blocked by naltrexone. The results indicate that glial cells can be a target of an opioid receptor-mediated antimitogenic action and that an abatement in PI turnover and Ca2+ mobilization may be associated with this mechanism.

In vitro and in vivo expression of opioid and sigma receptors in rat C6 glioma and mouse N18TG2 neuroblastoma cells.

Mouse N18TG2 neuroblastoma and rat C6 glioma cell lines were injected into male nude mice, and the tumors were passaged serially. At each generation, tumors were analyzed for delta opioid binding using [3H][D-Ala2,D-Leu5]enkephalin and for sigma 1 and sigma 2 binding with 1,3-[3H]di-o-tolylguanidine in the presence and absence of 1 microM pentazocine. Receptor density (Bmax) and affinity (KD) were estimated by homologous competition binding assays. Opioid and sigma Bmax values in the solid tumors were significantly lower than their original levels in vitro. KD values for opioid/sigma ligands were similar in vitro and in vivo. With successive passages in the murine host, delta opioid and sigma 1 binding of the neuroblastoma-derived solid tumors became undetectable. In contrast, sigma 2 receptor Bmax values were unchanged with successive passages of the neuroblastoma-derived tumors and doubled in the nude mouse-borne gliomas. When neuroblastoma-derived solid tumors that were devoid of delta opioid binding were returned to culture, opioid receptors appeared to be up-regulated as compared with their original in vitro levels. Serial passaging of these recultured cells in vivo again resulted in a rapid decline in opioid receptor content. The opioid data are consistent with our prior findings on opioid binding diminution in human brain tumors. The pattern of change for sigma binding was more complex, with the sigma 2 response in late passages of the glioma being reminiscent of the formerly observed increase in number of sigma sites in transformed human meninges, kidney, and colon tissue.

Transient down-regulation of neonatal rat brain mu-opioid receptors upon in utero exposure to buprenorphine.

Gestational actions of the mixed agonist-antagonist buprenorphine on mu- and kappa 1-opioid binding in neonatal and maternal rat brain were investigated. Upon exposure of pregnant rats to 0.5 mg/kg buprenorphine for 7 days prior to birth, postnatal day-one (P1) and P7 offspring brain mu-binding parameters (Kd and Bmax) were assessed with 3H-labeled [D-Ala2,Mephe4,Gly-ol5] enkephalin (DAMAGE). DAMAGE binding was attenuated by 64% in P1 membranes, whereas P7 preparations showed no changes. The same buprenorphine regimen resulted in diminished DAMGE Bmax values in mothers' brains, 2 but not 7 days after cessation of drug administration. Receptor density changes were not accompanied by alteration of mu-binding affinities. Although the postnatal developmental profile of kappa 1 opioid receptors in rat brain measured with [3H]U69593 revealed the presence of an ample number of sites for detection, their binding parameters in P1, P7 pups and mothers were unaffected by 0.5 mg/kg buprenorphine. In summary, buprenorphine administration to pregnant rats transiently down-regulates mu opioid receptors in neonatal and maternal brain.

A monoclonal anti-idiotypic antibody to opioid receptors labels desipramine-induced opioid binding sites on rat C6 glioma cells and attenuates thymidine incorporation into DNA.

Treatment of rat C6 glioma cells with the tricyclic antidepressant desipramine induces opioid binding. Here the distribution of these opioid-binding sites on C6 cell membranes and a functional property were investigated. Immunohistochemical examination of C6 cells was performed using a monoclonal anti-idiotypic antibody to opioid receptors (Ab2AOR). Ab2AOR uniformly labeled > 97% of the cells exposed to desipramine over their entire surface. The opioid-receptor antagonist naltrexone completely blocked Ab2AOR binding. Ab2AOR, which has opioid agonist properties, also inhibited DNA synthesis in desipramine-treated but not in naive C6 cells. Similarly, morphine blocked C6 cell proliferation only after desipramine treatment. The antineurotrophic action of Ab2AOR was reversed by naltrexone and was insensitive to pertussis toxin. These findings demonstrate that Ab2AOR suppresses the proliferation of C6 glioma cells by binding to desipramine-induced opioid receptors.

Differential enhancement of benzophenanthridine alkaloid content in cell suspension cultures of Sanguinaria canadensis under conditions of combined hormonal deprivation and fungal elicitation.

An elicitation protocol, resulting in the accumulation of sanguinarine in suspension cultures of Papaver bracteatum, was assessed for induction of the same alkaloid in Sanguinaria canadensis. Although only a trace constituent of P. bracteatum plants, sanguinarine is a major alkaloid (1-3% dry wt) of S. canadensis rhizomes. By combining hormonal deprivation for various intervals and a 3-day fungal (Verticillium dahliae) elicitation, benzophenanthridine alkaloid accumulation was induced in S. canadensis cell suspensions. Chelirubine content increased (0.1-1.3% dry wt) consistently in elicited cell cultures while chelerythrine (0.01-0.10% dry wt) and sanguinarine (0-0.02% dry wt) levels were considerably less. Alkaloid accumulation always occurred upon removal of hormone but only at certain time intervals in the log phase upon fungal elicitation. Levels of dopamine, a precursor of the alkaloids, fluctuated over the incubation period, but displayed a 2- to 6-fold increase in cell suspensions grown without hormone. In some experiments dopamine accumulated to levels > 20% dry wt, and these increases were enhanced by the addition of fungal elicitor. Although the same fungal elicitor induces benzophenanthridines in taxonomically related S. canadensis and P. bracteatum, it did not elicit the accumulation of the same alkaloid in the two different plant cultures.

Differential down- and up-regulation of rat brain opioid receptor types and subtypes by buprenorphine.

The induction of opioid receptor adaptation by mixed agonist-antagonists such as buprenorphine has not been investigated. To this end, neonatal rats were given injections of buprenorphine (0.1-2.5 mg/kg/day) and mu binding (Kd and Bmax) to brain membranes was measured with [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin. At doses of buprenorphine of > or = 0.5 mg/kg, mu sites were reduced 47-75%, without changes in affinity. Chronic administration of the structurally related partial agonist diprenorphine (2.5-75 mg/kg) failed to alter mu binding. Apparent loss of sites due to receptor blockade by residual buprenorphine was ruled out by several lines of evidence. Bmax values for delta ([3H][D-Ser2,L-Leu5]enkephalyl-Thr) and kappa ([3H]U69593) binding were elevated 1.9-4.2-fold by buprenorphine treatment. In adult rats buprenorphine (0.5-2.5 mg/kg) reduced mu-opioid binding to forebrain membranes dose dependently, by 25-77%. [3H][D-Ser2,L-Leu5] Enkephalyl-Thr-labeled delta subtype receptors and kappa sites in adult forebrain membranes were up-regulated 2-3-fold. The delta subtype receptors that bind [3H][D-Pen2,D-Pen5]enkephalin in neonatal or adult brain membranes were unaffected by 0.5-2.5 mg/kg buprenorphine treatment. Down-regulation (70-74%) of mu sites and up-regulation (1.9-6.7 fold) of delta and kappa receptors were also observed in synaptic plasma membrane-enriched and microsomal fractions from buprenorphine-treated adult rat brain. Because agonist-induced opioid receptor down-regulation is difficult to elicit in adult mammalian brain, these data indicate that buprenorphine is a useful tool to study brain opioid receptor adaptation in vivo.

Up-regulation of delta opioid receptors in neuroblastoma hybrid cells: evidence for differences in the mechanisms of action of sodium butyrate and naltrexone.

Opioid binding in subcellular fractions from neurohybrid cells was assessed using two models of up-regulation. Homologous up-regulation was achieved by treating NG108-15 cells with the opioid antagonist naltrexone. Na butyrate was added to NCB-20 cell cultures to affect heterologous up-regulation. In both paradigms light and heavy membranes were resolved by concanavalin A (con A) pretreatment of cells followed by density centrifugation. [3H][D-Ala2,D-Leu5]enkephalin (DADLE) and [3H]diprenorphine Bmax values for these fractions increased without changes in affinity. In contrast to 48 h of antagonist treatment, 5 min of exposure to naltrexone down-regulated heavy membrane delta sites. Under both conditions of up-regulation, inhibition of LM [3H]DADLE specific binding by 5'-guanylylimidodiphosphate was enhanced suggesting greater receptor coupling to guanine nucleotide binding regulatory proteins. Although attenuated by addition of cycloheximide, [3H]DADLE binding to total homogenates increased upon naltrexone treatment of NG108-15 cells. Heavy membrane Bmax values were also augmented in the presence of cycloheximide and naltrexone for 48 h. Activities of beta-glucuronidase and beta-hexoseaminidase were diminished in total homogenates and subcellular fractions from naltrexone-treated cells, suggesting an opioid-induced alteration in lysosomal enzyme trafficking. Comparable receptor down- and up-regulation and attenuation of lysosomal enzyme activity were elicited by the delta-selective opioid peptide antagonist (allyl)2 Tyr-Aib-Aib-Phe-Leu-OH. These results suggest that homologous up-regulation entails initial down-regulation and blockade of receptor degradation.

A monoclonal anti-idiotypic antibody to mu and delta opioid receptors.

A mouse monoclonal, anti-idiotypic, anti-opioid receptor antibody (Ab2-AOR) has been generated from monoclonal anti-morphine antibodies (Ab1). Hybridoma culture supernatants were screened by a solid phase radioimmunoassay (RIA), based on their competition with radiolabelled morphine for Ab1. One of the Ab2s that gave a positive RIA also competed at rat brain opioid receptors with tritiated opioid ligands dihydromorphine (DHM), naloxone, etorphine, Tyr-D-Ala-Gly-Phe-D-Leu (DADLE), Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAMGE) and Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE). SDS-PAGE revealed Ab2-AOR to be highly purified after successive affinity and protein A-Sepharose chromatography. Ab2-AOR at concentrations of 10-100 nM competed with both mu- and delta-selective specific ligands for brain opioid receptors. Less than 13 micrograms/ml Ab2-AOR completely inhibited specific opioid radioligand binding to both soluble and membrane-bound opioid receptors. To demonstrate its anti-delta receptor activity further, a double-antibody ELISA procedure was developed that is based on the binding of Ab2-AOR to immobilized NG 108-15 cells (which contain only delta opioid receptors). Dose-dependent, opioid peptide- and opiate alkaloid-competitive binding of Ab2-AOR-containing ascites fluid to NG 108-15 cells was observed. A mu opioid agonist effect was demonstrated for Ab2-AOR, in that it decreased by 70% [3H]thymidine incorporation into DNA of fetal brain cell aggregates. This agonist-like action of Ab2-AOR was blocked by naltrexone. The antibody bound specifically to brain tissue sections and the presence of diprenorphine blocked this interaction. Hence, an Ab2 with mu and delta specificity has been characterized.