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J W Spain

Publications and source records attributed to J W Spain.

At least 19 recordsLinked to original sources
PubMed1991

Chronic opioids impair acquisition of both radial maze and Y-maze choice escape.

Chronic morphine impaired acquisition of two dissimilar behavioral tasks. In the radial maze, the performance of saline-treated and morphine-treated groups diverged with the latter failing to improve despite extensive training. In contrast, rats treated with naltrexone became skilled in the procedure 2-4 times as rapidly as saline controls. Withdrawal of treatment significantly improved performance of morphine-treated rats, with no change for rats treated with saline or naltrexone. When a second group of rats was extensively trained prior to instituting chronic morphine treatment, performance scores were not affected, suggesting that morphine does not impair spatial working memory despite subjective evidence of other gross behavioral effects, such as ataxia. In the Y-maze choice escape task, acquisition of a response strategy was significantly impaired in rats that had been previously treated with morphine for 17-21 days, despite clear indications that morphine-treated rats were sensitive to the aversive stimulus.

Animals↗
PubMed1989

Cross-linking of [125I]beta-endorphin to mu-opioid receptors during development.

Radioiodinated human beta-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 Mr 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 mu-receptor: the binding of [125I]beta-endorphin to this band was displaced by mu-selective ligands and blocked by alkylation of the receptor by mu-specific, but not delta-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 [125I]beta-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 delta-receptor isolated from NG108-15 neuroblastoma-glioma cells, competition studies suggest this protein is not the delta-receptor. The 65 kDa band, tentatively identified as the mu-receptor, was present in adults but not detected in neonates, despite competition binding data indicating the presence of mu-sites. The results suggest that the 55 kDa band found in the 1-day-old neonate may be an immature form of the mu-opioid receptor that undergoes posttranslational modification, perhaps glycosylation, during development.

Alkylation↗
PubMed1988

Binding kinetics of delta opioid receptors differ for microsomal and synaptic sites.

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.

Animals↗
PubMed1987

Multiple interconvertible affinity states for the delta opioid agonist-receptor complex.

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.

Animals↗
PubMed1987

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.

Animals↗
PubMed1987

Guanine nucleotide and cation regulation of mu, delta, and kappa opioid receptor binding: evidence for differential postnatal development in rat brain.

A study of the onset of cation and guanine nucleotide regulation of delta, mu, and kappa 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 microM guanosine-5'-(beta, gamma-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 delta sites was stimulated by Mn2+, whereas that to mu sites was not affected and kappa binding was inhibited. The postnatal development of Mn2+ regulation for the three receptor subtypes was distinctly different. The largest effects were seen on delta sites detected in the early neonatal period, Mn2+ eliciting a 68% stimulation of binding over controls at day 1. Significant inhibition of kappa site binding by Mn2+ was detected only after the third postnatal week. Mn2+ caused a significant reversal of Gpp(NH)p inhibition of delta binding in the early neonatal period, exceeding that in the absence of regulators. Inhibition of mu and delta 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

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 N18TG2 neuroblastoma cells. This investigation examines the possible role of opioid receptors in the cannabimimetic response. Opioid receptors of the delta subtype were found on N18TG2 membranes using [3H]D-Ala2-D-Leu5-enkephalin. No mu or kappa receptors were detected using selective ligands for these sites. The delta 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 delta 9-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.

Adenylyl Cyclase Inhibitors↗
PubMed1986

A comparison of microsomal and synaptic delta site binding kinetics.

High-affinity binding of the agonist, [3H]D-ala2-D-leu5-enkephalin (DADLE), to delta sites on bovine hippocampal synaptic plasma membranes (SPM) entails a multi-step association process. Microsomal binding sites, which are thought to originate from internal membranes (golgi, ser, etc.), display kinetic patterns that differ from SPMs. This is evidenced by the absence of an association time dependent rate of dissociation from microsomal binding sites. Although high affinity steady state binding of agonists to microsomes occurs, kinetic analysis indicates little or no formation of the high affinity slowly dissociating complex. This slowly dissociating complex of SPMs is most sensitive to guanine nucleotides. Consequently, the effect of Gpp(NH)p on dissociation is significantly less for microsomes.

Animals↗
PubMed1986

Detection and characterization of beta-adrenergic receptors and adenylate cyclase in coated vesicles isolated from bovine brain.

To assess whether internalization of beta-adrenergic receptor occurs in the CNS, we have isolated clathrin-coated vesicles from bovine forebrain and examined them for the presence of beta-adrenergic receptor binding and adenylate cyclase activities. A coated vesicle enriched preparation isolated by successive D2O-Ficoll density gradient centrifugations was applied to a glass bead permeation column to achieve further purification. Two major peaks of protein were eluted from the column and monitored by electron microscopy and SDS-PAGE. Peak II contained almost exclusively coated vesicles (98%), whereas peak I, which appeared in the void volume, contained larger smooth vesicles and few coated vesicles. beta-Adrenergic receptor binding to peaks I and II was measured with 125I-cyanopindolol (CYP) as ligand in Sepharose 4B column assays. 125I-CYP was found to bind specifically and saturably to both peaks I and II with a Bmax of 28 +/- 4 and 32 +/- 3 fmol/mg protein, respectively. 3H-CGP 12177, a hydrophilic beta-adrenergic receptor ligand, did not label receptors present in peak II, but it specifically bound to synaptic plasma membranes (SPM) prepared from bovine hippocampus and, to a lesser extent, to peak I. These results suggest that receptors present in coated vesicles are cryptic in nature. In the displacement of 125I-CYP binding by (-)-isoproterenol, addition of 50 microM GppNHp caused a significant "right shift" with SPM and peak I but not the peak II preparation. Adenylate cyclase activities could also be detected in both peaks I and II (specific activities, 21 +/- 0.6 and 24 +/- 0.5 pmol cAMP/mg protein/min, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1985

Differential up-regulation of microsomal and synaptic membrane mu 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.

Animals↗
PubMed1985

[3H]Spiperone binding in the rat striatum during the development of physical dependence on phencyclidine and after withdrawal.

Binding of [3H]spiperone to dopamine D2 receptors was measured in the striatum of male rats that were infused with phencyclidine (PCP, 45 mg/kg per day) or vehicle (saline) via an intrajugular cannula for 1, 3.5, or 7 days. The 7-day PCP infusion, which was shown previously to induce physical dependence, produced a significant 30% decrease in receptor density (Bmax). Two days after termination of the 7-day PCP infusion, Bmax values were no longer significantly lower than those of saline-infused controls. The acute administration of PCP (20 mg/kg, i.p.) did not alter receptor density 45 min later.

Animals↗
PubMed1985

Stereospecific opiate-binding sites occur in coated vesicles.

We prepared clathrin-coated vesicles from bovine forebrain utilizing sucrose or deuterium oxide-Ficoll density gradient centrifugation followed by permeation chromatography. Homogeneity was monitored by electron microscopy (EM) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). EM revealed that the predominant (up to 98% of the total) organelles were coated vesicles and empty hexagonal baskets. Diameters of the coated vesicles ranged from 37 to 120 nm with a mean of 65.2 +/- 2.2. Upon SDS-PAGE of the coated vesicle fraction, the most prominent band appeared at 180,000 daltons. There were also three additional bands at 100,000, 50,000 and 35,000 daltons, giving the overall pattern characteristic of coated vesicles. Both 0.5 nM tritiated naltrexone and etorphine displayed specific binding to coated vesicles. Naltrexone binding in coated vesicles from gradient fractions was increased 2.5-fold over the original 100,000 X g pellet. An additional 4-fold enrichment in specific binding was observed after permeation chromatography which was concomitant with an increase in the volume density of coated vesicles in electron micrographs. Naltrexone binding was stereospecific and etorphine binding was inhibited by 100 mM NaCl (40%). Both naltrexone and etorphine binding were inhibited by 50 microM guanyl-5'-yl imidodiphosphate (40 to 50%). In summary, purified bovine brain-coated vesicles contained high affinity stereospecific opiate alkaloid-binding sites with characteristic opioid binding properties.

Animals↗
PubMed1985

Differential ontogeny of multiple opioid receptors (mu, delta, and kappa).

We investigated the postnatal ontogeny of opioid receptors in rat brain under assay conditions which, when combined with computerized analysis, effectively reflect the developmental profile of high affinity binding to mu, delta, and kappa subpopulations. Concentrations of mu sites were assessed with the selective ligand 3H-[D-ala2,mePhe4,gly-ol5]enkephalin (DAGO). The other two sites were analyzed in binding assays with less selective radioligands but in the presence of specific unlabeled ligands which suppress cross-reactivity. We utilized 3H-[D-ala2,D-leu5]enkephalin (DADL) in the presence of 10 nM DAGO to label delta sites and 3H-ethylketocyclazocine (EKC) in the presence of 100 nM DADL + 100 nM [D-ala2,mePhe4,Met(0)ol5]enkephalin to detect kappa receptors. After birth, the density (femtomoles per milligram of wet weight) of mu sites declined for several days and then rose sharply over the next 2 weeks, increasing 2-fold by adulthood. Delta (delta) sites appeared in the second week postnatal and increased more than 8-fold in the next 2 weeks. Levels of kappa receptors were relatively low at birth and increased slowly (2-fold, overall). Computerized analyses of binding data revealed that DAGO and DADL were binding to single populations of sites throughout the postnatal period. DAGO and EKC affinities did not fluctuate in this period, whereas DADL affinities were low for the first week and then rose to adult levels. In summary, mu, kappa, and delta receptors exhibit differential postnatal developmental profiles. The former two are present at birth, whereas the latter appears in the second week. The postnatal increase for all three sites appear to be preceded by the previously demonstrated emergence of opioid peptides.

Animals↗
PubMed1985

Continuous intravenous infusion of phencyclidine in unrestrained rats results in the rapid induction of tolerance and physical dependence.

The continuous infusion of 45 mg/kg/24 hr of phencyclidine (PCP) into the jugular vein of unrestrained rats induced tolerance to PCP-induced impairment of forced motor activity and physical dependence in 3.5 and 7 days, respectively. In drug-naive rats, an i.v. 2-mg/kg PCP test dose abolished rotarod performance for more than 20 min which returned to pretreatment values at 40 min. Eight hours after the termination of 3.5 days of infusion, rotarod performance of PCP-infused rats was significantly less impaired by the PCP test dose at 20 min than that of saline-infused controls. After infusion of PCP for 7 days, the duration of performance abolition produced by the PCP test dose (given 8 hr after the termination of infusion) was shortened further with performance significantly better than that of saline-infused controls at both 10 and 20 min. The results showed a greater than 2-fold tolerance development to this PCP effect and suggest the observed tolerance to be mainly functional in nature. Abrupt withdrawal of PCP after infusion for 7 days resulted in an abstinence syndrome with the following signs: piloerection, increased susceptibility to audiogenic seizures, transient weight loss and reductions in exploratory activity and rotarod performance. The first withdrawal signs were noted 4 hr after the termination of infusion. At 24 hr of abstinence, most of the withdrawal signs had subsided. The reduced rotarod performance, associated with withdrawal, could be reversed by a single i.v. dose of 2 mg/kg of PCP. The reversibility of this sign supports the interpretation of impaired rotarod performance after withdrawal as being an abstinence sign and adds to the experimental evidence that physical dependence on PCP is inducible within 7 days in rats.

Animals↗
PubMed1984

Kinetic evidence for differential agonist and antagonist binding to bovine hippocampal synaptic membrane opioid receptors.

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.

Animals↗