James W. Spain

An Assessment of the Role of Opioid Receptors in the Response to Cannabimimetic Drugs

Cannabimimetic drugs have been shown to inhibit adenylate cyclase activity in NI8TG2 neuroblastoma cells. This investigation examines the possible role of opioid receptors in the cannabimimetic response. Opioid receptors of the δ subtype were found on N18TG2 membranes using [3H]D‐Ala2‐D‐Leu5‐enkephalin. No δ or K receptors were detected using selective ligands for these sites. The δ binding affinity and capacity were unaltered by cannabimimetic drugs. To test if cannabimimetic drugs may modulate opioid effector mechanisms, cyclic AMP metabolism was determined in intact cells and in membranes. N18TG2 adenylate cyclase was inhibited by the cannabimimetic drugs Δ9‐tetrahydrocannabinol and desacetyllevonantradol, and by the opioid agents morphine, etorphine, and D‐Ala2‐Met5‐enkephalinamide. The opioid inhibition was reversed by naloxone and naltrexone; however, the cannabimimetic response was unaffected. Both cannabimimetic and opioid drugs decreased cyclic AMP accumulation in intact cells, but opioid antagonists blocked the response only to the latter. Thus, cannabimimetic effects are observed even though opioid receptors are blocked by antagonist drugs. The interaction between desacetyllevonantradol and etorphine was neither synergistic nor additive at maximal concentrations, suggesting that these two drugs operate via the same effector mechanism. Other neuronal cell lines having an opioid response were also examined. The cannabimimetic inhibition of cyclic AMP accumulation in NG108‐15 neuroblastoma x glioma cells was not as great as the response in N18TG2. N4TG1 neuroblastoma cells did not respond to cannabimimetic drugs under any conditions tested. Thus, the cannabimimetic inhibition of adenylate cyclase is not universally observed, and the efficacy of the cannabimimetic response does not correlate with the efficacy of the opioid response.

Guanine Nucleotide and Cation Regulation of μ, δ, and k Opioid Receptor Binding: Evidence for Differential Postnatal Development in Rat Brain

Abstract: A study of the onset of cation and guanine nucleotide regulation of δ, μ, and k rat brain opioid receptors during postnatal development was undertaken. Site‐specific binding assays were utilized for each receptor type and the effects of 0.5 mM MnCl2, 100 mM NaCl, and/or 50 μM guanosine‐5′‐(β,γ‐imido) triphosphate [GPP(NH)P] were assessed. The most pronounced changes of opioid binding were seen in the presence of Mn2+. In adults, agonist binding to δ sites was stimulated by Mn2+, whereas that to μ. sites was not affected and k binding was inhibited. The postnatal development of Mn2+ regulation for the three receptor subtypes was distinctly different. The largest effects were seen on δ sites detected in the early neonatal period, Mn2+ eliciting a 68% stimulation of binding over controls at day 1. Significant inhibition of k site binding by Mn2+ was detected only after the third postnatal week. Mn2+ caused a significant reversal of Gpp(NH)p inhibition of δ binding in the early neonatal period, exceeding that in the absence of regulators. Inhibition of μ and δ receptor binding by Na+ was greater, and the Mn2+ reversal of this effect was smaller, in the first 2 postnatal weeks than in adults. Gpp(NH)p + Na+ regulation did not change appreciably during the postnatal period. However, Mn2+ reversal of the considerable inhibition elicited by the combination of Na+ and Gpp(HN)p was developmental time‐dependent. The data are discussed in terms of multiple sites of interaction for guanine nucleotides and cations. Our results demonstrate that the characteristics and postnatal development of guanine nucleotide and cat‐ionic regulation of μ, δ, and k binding are distinctly different. Furthermore, neonates may serve as a model for the examination of individual regulatory effects on opioid receptors.

Differential up-regulation of microsomal and synaptic membrane μ opioid receptors

Naltrexone was administered to rats for 7 days by osmotic minipump (5 mg/kg/day) and thereupon, forebrain mu opioid receptor levels in subcellular fractions were monitored by homologous displacement of [3H]D-ala2-mePhe4-gly-ol5 enkephalin binding. Microsomes displayed increases in mu receptor concentrations that were twofold greater than those associated with synaptic plasma membrane fractions (92 vs. 51%). Levels in crude membranes rose 77%. Binding affinities were unchanged.

Ontogeny of benzomorphan-selective (κ) sites: A computerized analysis

Abstract In an investigation of the postnatal development of κ opiate receptors, the affinity and capacity of 0.5 nM [3H]-ethylketocyclazocine (EKC) binding in crude rat brain homogenates was measured by displacement with unlabeled EKC, morphine, or D-ala2-D-leu5-enkephalin (DADL). Displacement curves were analyzed using a weighted, non-linear regression, curve fitting computer program. At all stages of development, [3H]-EKC binding fit a two site model significantly better than a one site model. Affinities of EKC, morphine, or DADL for the high affinity [3H]-EKC binding site did not change during the postnatal period. The density of the high affinity [3H]-EKC binding site increased linearly with age, whereas the levels of the low affinity site rose more rapidly during the second week.

Exogenous cholecystokinin (CCK) reduces neonatal rat brain opioid receptor density and CCK levels

Newborn rats were given saline or cholecystokinin8 (CCK8) (5 micrograms/kg, twice daily) i.p. for 3 weeks. On day 21, effects on brain development were assessed. CCK-like immunoreactivity was measured in 7 brain regions; a small (12-18%) but significant decrease in endogenous levels of this peptide was detected in cerebral cortex, medulla and pons of the CCK-treated rats. Morphometric measurements revealed a slight reduction in thickness of most cerebral cortical sections within the CCK-treated group. The area of a midsagittal section of the cerebellum was unchanged except for the Purkinje/granule cell layer, which was smaller in CCK-treated animals. Levels of mu-, delta- and kappa-opioid receptors were estimated by homologous displacement binding assays using selective radioligands. The CCK treatment resulted in a significant decrease in levels of mu- (11%) and delta- (13%)-sites in the cerebral cortex. Neither binding affinities nor kappa-receptor densities were altered. Other animals received the same treatment regimens for 21 days and were maintained for an additional 29 days without treatment; these rats had reductions only in cortical mu-sites (15%). Chronic intraventricular administration of CCK (0.1 microgram/h) to adult rats did not elicit a similar down-regulation of cortical mu or delta receptors, suggesting that the effects observed in neonates reflected developmental processes.

Cross-linking of [125I]β-endorphin to μ-opioid receptors during development

Abstract Radioiodinated human β-endorphin was cross-linked to opioid receptors from rat brain membranes using the bifunctional reagents bis-[2-(succinimidooxycarbonyloxy)ethyl] sulfone (BSCOES) and disuccinimidyl suberate (DSS). Major radiolabeled bands migrated with M r values of 65,000, 55,000 and 33,000, however the presence of the 55 kDa band was variable. The 65 kDa band was characterized as the μ-receptor: the binding of [ 125 I]β-endorphin to this band was displaced by μ-selective ligands and blocked by alkylation of the receptor by μ-specific, but not δ-specific alkylating agents. The cross-linked receptor underwent alterations in mol. wt. during development. Early in development, embryonic day 18 and postnatal day 1, the [ 125 I]β-endorphin-labeled material migrated as a single band of mol. wt. 55 kDa. By day 21 postnatally the higher mol. wt. band of 65 kDa was present, as was material of 53, 47 and 43 kDa. Although the protein labeled early in development migrated with a mol. wt. of 55 kDa similar to the δ-receptor isolated from NG108-15 neuroblastoma-glioma cells, competition studies suggest this protein is not the δ-receptor. The 65 kDa band, tentatively identified as the μ-receptor, was present in adults but not detected in neonates, despite competition binding data indicating the presence of μ-sites. The results suggest that the 55 kDa band found in the 1-day-old neonate may be an immature form of the μ-opioid receptor that undergoes posttranslational modification, perhaps glycosylation, during development.

Gpp(NH)p promotes the formation of a low affinity state for the δ opioid agonist-receptor complex

Abstract We previously demonstrated an association time-dependent dissociation of the δ opioid agonist, DADL, from its receptor that suggested multi-step formation of a high affinity complex. We have now examined the effect of the GTP analog, Gpp(NH)p, on the dissociation of [ 3 H]DADL from these same sites on bovine hippocampal synaptic membranes. Gpp(NH)p accelerates dissociation of this complex in a dose-dependent manner resulting in the transformation of a high affinity form to a lower affinity state. The Gpp(NH)p effect was more pronounced when it was added at the time of initiation of dissociation. Under these conditions Gpp(NH)p engendered biphasic off-rates that were no longer association time-dependent. The addition of Gpp(NH)p at the onset of association did not prevent (but retarded) the formation of the high affinity state. Steady state homologous displacement binding curves demonstrated single site [ 3 H]DADL binding in the absence of Gpp(NH)p but in its presence an improved fit for a two site model was obtained. In summary, on the basis of both kinetic and equilibrium binding data Gpp(NH)p appears to transform the δ opioid agonist-receptor complex into a low affinity state.