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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.

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.

According to current concepts, agonists can effect the down-regulation of cell surface receptors, whereas antagonists can cause their up-regulation. We have discovered that the opioid antagonists naltrexone, naloxone, and ICI174864 induce a transient down-regulation of delta-opioid receptors before up-regulation, in NG108-15 cells. The possibility of an apparent loss of sites due to blockade by residual antagonist was ruled out by several lines of evidence. The reduction in delta receptors was time, temperature, and antagonist concentration dependent. This down-regulation could not be induced by either the highly mu-selective opioid antagonist cyclic D-Phe-Cys-Try-D-Trp-Arg-Thr-Pen-Thr-amide or the muscarinic antagonist atropine. In the same neurohybrid cells, the opioid agonist [D-Ala2,D-Leu5]enkephalin (0.1 microM, 60 min) effected a greater down-regulation of delta-opioid receptors. Similar qualitative changes in opioid binding of subcellular fractions were elicited with [D-Ala2,D-Leu5]enkephalin and naltrexone. However, the agonist was 2-fold more effective in reducing the heavy membrane population of receptors and 4-fold more potent in increasing the light membrane sites. Because heavy membranes are enriched in plasma membrane, whereas light membranes contain intracellular sites, these findings indicate that internalization occurs in both instances. Naltrexone and the delta-specific antagonists ICI174864 and naltrindole also diminished specific activities of two lysosomal enzymes, whereas opioid agonist-induced down-regulation was accompanied by an increase in their specific activities. Pretreatment of cell cultures with concanavalin A blocked both down-regulation and alterations in the lysosomal enzyme activities elicited by agonists and antagonists, suggesting that the latter is an opioid receptor-mediated process. The up-regulation of delta-opioid receptors by antagonists appears, then, to entail down-regulation that differs from that of agonists.

To examine the kinetics of opioid receptor binding, the agonists [D-Ala2-D-Leu5]enkephalin (DADL) and [D-Ala2-MePhe4-Gly-ol5]enkephalin (DAGO) and the antagonists diprenorphine and naltrexone were used with bovine hippocampal synaptic plasma membranes. By computer modeling of equilibrium binding displacement curves utilizing the LIGAND program, we found opioid peptides bind with high affinity to single populations of synaptic plasma membranes receptors, whereas opiate alkaloids bind to multiple sites. Initial kinetic experiments revealed that agonist rates of association were radioligand concentration-independent. Pseudo first-order rate constants for DADL, DAGO, diprenorphine, and naltrexone association were estimated to be 5.63 X 10(5), 5.08 X 10(5), 4.60 X 10(6), and 2.3 X 10(6) mol-1 X s-1, respectively. After preincubation of 0.2-1 nM radioligand for variable time intervals, dissociation was initiated by addition of 1 microM unlabeled ligand. If saturation binding was achieved before dissociation was initiated, then nearly monophasic dissociation of DADL, DAGO, and diprenorphine and a biphasic off-rate for naltrexone were observed. When association times were reduced to pre-equilibrium intervals, the kinetics of dissociation of agonists became biphasic and association time-dependent, but that for antagonists did not change significantly. Comparisons by both graphical methods and computerized nonlinear regression analyses of rate constants revealed that the fraction of the rapid component of agonist dissociation decreases and that of the slow component is elevated with increasing receptor occupancy. In the presence of 100 mM NaCl, DADL dissociation became association time-independent. These data are consistent with the idea that the Na+ effect is brought about by a change of receptor to an antagonist-like conformation. On the basis of both association and dissociation kinetic data, opioid agonists appear to interact in a multistep process in which a rapid, reversible association is followed by the formation of a more tightly bound complex.

The rat hippocampus contains the major types of opioid receptors, delta, mu, and kappa, as determined by autoradiographic and membrane binding analyses. Chronic exposure to excessive amounts of opioid antagonists results in a doubling of the number of binding sites. However, the direct electrophysiological significance of this increased number of opioid receptors in the central nervous system remains uncharacterized. We examined the effects of an opioid peptide with high affinity and high specificity for mu receptors, DAMGO (D-ala2-mePhe4-gly-ol5 enkephalin), under normal conditions and after 1 or 2 weeks of continuous infusion of the opiate antagonist naltrexone. Chronic infusion of naltrexone administered to the whole animal resulted in significant up-regulation (71%) of mu opioid receptors in the rat hippocampus. Slices of the hippocampus were perfused with artificial cerebrospinal fluid while recording population spikes in stratum pyramidale, excitatory postsynaptic potentials in stratum radiatum and while stimulating afferents in the Schaffer collaterals. Superfusion of slices with DAMGO produced a concentration-dependent increase in the amplitude of population spikes. No significant change was observed in the simultaneously recorded excitatory postsynaptic potential slope. This selective increase in population spike amplitude led to a leftward shift (19%) in the derived input-output curve. In addition, DAMGO superfusion produced extra spiking at higher stimulus intensities. Naltrexone reversed the DAMGO-induced increase in excitability, as well as prevented additional spikes. DAMGO superfusion of slices taken from chronically treated rats produced a much greater shift (42%) in the input-output curve than it did in untreated controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Earlier, we demonstrated that agonist binding to synaptic plasma membranes involves a multi-step association process. In this study, high affinity binding kinetics of an agonist, [3H]D-Ala2-D-Leu5-enkephalin (DADLE), to delta sites on bovine hippocampal microsomal and synaptic plasma membranes (SPM) were compared. delta site selectivity of DADLE was ensured by suppressing undesirable mu site binding with 20 nM unlabeled D-Ala2-MePhe4-Glyol5-enkephalin. The kinetics of receptor binding to microsomal delta sites are generally more rapid than those of SPMs. Furthermore, the association time-dependent rate of dissociation, which is readily observed with SPMs, was not detected for microsomal binding sites. Although the apparent KD of DADLE did not differ significantly from that in SPMs, kinetic analysis indicated that little or no formation of the high affinity, slowly dissociating, complex occurred with microsomes. The absence of this complex, shown previously in SPMs to be most sensitive to guanine nucleotides, appeared to account for the attenuated effect of guanyl 5'-yl-imidodiphosphate [Gpp(NH)p] on dissociation from microsomes. Nevertheless, the presence in microsomes of inhibitory guanine nucleotide binding proteins was demonstrated by specific 32P-labeling by pertussis toxin of bands at 39 and 41 kDa, attributable to the alpha subunit of Go and Gi, respectively. The action of 100 mM Na+ to increase the off-rate is similar for both preparations. In contrast, addition of Mn2+ reduced the rates of association and dissociation for both subcellular fractions. The off-rate in the presence of Mn2+ is similar for SPMs and microsomes, displaying association time-dependent rates of dissociation for both. To determine whether Mn2+ promotes coupling in microsomes, the effect of Gpp(NH)p was examined. After a 60-min association, Gpp(NH)p did not affect microsomal kinetics but increased the off-rate from SPMs. The actions of both Na+ and Mn2+ appear to be mediated at early steps in the association process.

Previously we demonstrated that rates of dissociation of [3H-D-Ala2-D-Leu5]enkephalin [( 3H]DADL) from bovine hippocampal synaptic plasma membranes (SPMs) varied depending upon association time, suggesting a multistep binding process. To characterize different kinetic intermediates, we examined the effects of guanine nucleotide on dissociation rate. Control off-rates were compared to those obtained when guanyl-5'-yl-imidodiphosphate (Gpp(NH)p) (50 microM) was added either coincident with the radioligand at association or with 1 microM unlabeled DADL which initiated dissociation. delta site selectivity of [3H]DADL was ensured by addition of 20 nM unlabeled [D-Ala2-Me-Phe4-Gly-Ol5]enkephalin which suppressed mu site cross-reactivity in this preparation. Addition of Gpp(NH)p at the onset of dissociation increased the off-rate to a much greater extent after steady state binding was reached (60 min) compared to that following an association time of only 7 or 20 min. A slowly formed high affinity state appeared to be rapidly converted to a lower affinity state under these conditions. When Gpp(NH)p was present throughout the association period, the slowly dissociating state was no longer observed. Also, the off-rate following a 7-min association is linear and much faster than control, suggesting that Gpp(NH)p may affect an initial intermediate state as well as the high affinity complex. Pretreatment of the membranes with N-ethylmaleimide (NEM) eliminated the association time-dependent dissociation rates, apparently preventing time-dependent formation of a high affinity state. This state is thought to be possibly a ligand-receptor complex interacting with a GTP binding protein. However, the rate of dissociation from NEM-treated membranes was accelerated by addition of Gpp(NH)p and the effect was not association time-dependent. NEM treatment resulted in an increased potency for Gpp(NH)p inhibition of [3H]DADL steady state binding. These results suggest the occurrence of at least three steps in the association of DADL to bovine hippocampal synaptic membranes.

Enzymes involved in catecholamine metabolism were assayed in the presence of a new class of naturally occurring tetrahydroisoquinoline alkaloids, the norlaudanosolinecarboxylic acids (NLCAs). NLCAs inhibited tyrosine hydroxylase noncompetitively with respect to its substrate, tyrosine and the cofactor, 6-methyltetrahydropterin (NLCA Kj = 4 x 10(-4) M; 3',4'-deoxynorlaudanosolinecarboxylic acid (DNLCA) Kj = 1.5 x 10(-4) M). Adrenal dopamine-beta-hydroxylase was also inhibited by NLCAs [3'O-methylnorlaudanosolinecarboxylic acid (MNLCA) Kj = 1.2 x 10(-4) M] and NLCA is a competitive inhibitor of norepinephrine methylation by hepatic catechol-O-methyltransferase (NLCA Kj = 5.6 x 10(-5) M). While a slight reduction of rat adrenal monoamine oxidase by MNLCA was also observed, NLCA did not affect the oxidation of tyrosine by D-amino acid oxidase. Kinetic patterns of tyrosine aminotransferase and aromatic amono acid decarboxylase from rat liver were not altered by addition of 1 to 10 x 10(-5) M NLCA or its 3'-O-methyl ether (MNLCA). In vivo studies of brain tyrosine metabolism in mouse neonates corroborated results on the in vitro effect of DNLCA on tyrosine hydroxylase. The potential of high-pressure liquid chromatography was demonstrated in both enzyme assays and radiometric studies of in vivo metabolism.

The intracellular location of a cytochrome P-450-dependent monoterpene hydroxylase from the higher plant, Catharanthus roseus, has been investigated. By differential and sucrose density gradient centrifugation, utilizing marker enzymes and electron microscopy, the monooxygenase was demonstrated to be associated with vesicles having a membrane thickness of 40-60 nm. The vesicles could be distinguished from endoplasmic reticulum, Golgi apparatus, mitochondria, and plasma membrane and were found in light membrane fractions containing provacuoles. Most definitive results were obtained when seedlings were ground in the presence of sand and in a medium containing sorbitol. Upon subjection of the 20,000-g pellet preparation to linear sucrose density gradient centrifugation, a threefold enrichment in hydroxylase activity was afforded in a yellow band having vesicles varying in size from 0.1 to 0.8 mum in diam and having a density of 1.09 to 1.10 g/cm3. Since the monooxygenase has been implicated in indole alkaloid biosynthesis in this plant, the data suggest the compartmentalization of at least a part of this pathway.

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.

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.