Biomedical subjects

R B Rothman

Publications and source records attributed to R B Rothman.

At least 217 records · Page 12Linked to original sources

PubMed1988

beta-FNA binds irreversibly to the opiate receptor complex: in vivo and in vitro evidence.

beta-Funaltrexamine (beta-FNA) is an alkylating derivative of naltrexone. Considerable data support its use as an irreversible mu receptor antagonist. However, pretreatment of rats with beta-FNA attenuates the ability of delta antagonists and naloxone to reverse delta receptor-mediated physiological effects, suggesting that physically adjacent mu and delta receptors interact in vivo. The purpose of this study was to determine which opiate receptor subtype is altered by i.c.v. injections of beta-FNA, as well as by in vitro incubations with beta-FNA, and then to examine the hypothesis that pretreatment of rats with beta-FNA increases the IC50 for naloxone at the altered binding site. The results demonstrate that beta-FNA alters the conformation of the opiate receptor complex, as evidenced by a decrease in the Bmax of the lower affinity [3H]D-Ala2-D-Leu5-enkephalin binding site and a doubling of the naloxone IC50 for displacing [3H]D-Ala3-D-Leu5-enkephalin from this site. [3H]D-Ala2-MePhe4,Gly-ol5-enkephalin binding sites were not detectably altered by i.c.v. injections of beta-FNA. These data collectively support the concept of coupling among opioid receptor subtypes.

Animals↗

PubMed1987

Synthesis and absolute configuration of optically pure enantiomers of a kappa-opioid receptor selective agonist.

The enantiomers of U50,488, ligands highly selective for kappa-opioid receptors, have been prepared by a refined procedure and their optical purity demonstrated. The absolute configuration of (+)-trans-2-pyrrolidinyl-N-methylcyclohexylamine, a chemically versatile intermediate for synthesis of analogs of kappa-opioid receptor ligands with defined chirality, has been determined to be 1S,2S by X-ray crystallographic analysis. This intermediate has been used to synthesize the optically pure U50,488 enantiomers with known absolute configuration.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1987

Chronic morphine upregulates a mu-opiate binding site labeled by [3H]cycloFOXY: a novel opiate antagonist suitable for positron emission tomography.

CycloFOXY (17-cyclopropylmethyl-3,14-dihydroxy-4,5-alpha-epoxy-6-beta- fluoromorphinan) is a novel opiate antagonist synthesized as a ligand suitable for in vivo visualization of opiate receptors using positron emission transaxial tomography. In this paper we report that [3H]cycloFOXY labels two distinct opiate binding sites in rat brain membranes, tentatively identified as mu and kappa. Furthermore, chronic administration of morphine results in a selective up-regulation of the mu binding site. The implications of this finding for models of the opioid receptors and the mechanism of the sodium effect are discussed.

Alkylating Agents↗

PubMed1987

Distribution of opiate receptor subtypes and enkephalin and dynorphin immunoreactivity in the hippocampus of squirrel, guinea pig, rat, and hamster.

The distribution of enkephalin and dynorphin immunoreactivity in the hippocampus of four rodent species (gray squirrel, guinea pig, rat, and hamster) is compared with the pattern of opiate receptor subtypes (mu, delta, and kappa). The distribution of opioid peptides is fairly consistent in the anterior hippocampus of these four species. Intense immunoreactivity for dynorphin and enkephalin is found in the hilus of the dentate gyrus and in the mossy fiber system. Occasional immunoreactive processes are seen in the dentate molecular layer and scattered throughout the CA1 and CA3 fields. In the rat and hamster, an additional plexus of enkephalinergic fibers straddles both sides of the hippocampal fissure. Cells immunoreactive for both opioid peptides are located in and just superficial to the dentate granule cell layer. Opiate receptors are variably distributed in these rodent species. In the squirrel, guinea pig, and hamster, mu and kappa binding is dense in the stratum lucidum of CA3 and the molecular layer of the dentate gyrus. In the rat, dense mu and kappa binding is localized within and adjacent to the pyramidal and granule cell layers. Delta receptor patterns show additional species differences. In the rat, the delta distribution is similar to the mu and kappa patterns. In the other species, the delta binding pattern is generally the inverse of the mu/kappa pattern: most areas of the hippocampus are enriched in delta sites, whereas the stratum lucidum and the pyramidal cell layer are receptor-sparse. Thus, the stratum lucidum--site of dense terminations of mossy fibers containing opioid peptides--is characterized by selectively sparse delta receptors in four species and by selectively dense kappa receptors in three species. The three receptor subtypes, taken either individually or together and compared to the peptides, are more variably and more widely distributed throughout the hippocampus and fail to show a correspondence with opioid-peptide-containing terminals. The mismatches suggest that receptor locations and densities are organized without relation to the sites of relevant transmitter release.

Animals↗

PubMed1987

Intracerebroventricular administration of superFIT and its enantiomer to rats: evidence for in vivo acylation of [3H]DADL binding sites.

SuperFIT is an high affinity acylating ligand derived from fentanyl. Previous studies suggested that a selective acylation of delta receptors (J. Med. Chem. 29:1087-1093, 1986) resulted from exposure of membranes to this and structurally related compounds. We report in this preliminary study that intracerebroventricular administration of either superFIT or its enantiomer 18 to 24 hours prior to sacrifice decreased the subsequent binding of [3H]DADL to both its higher and lower affinity binding sites.

Acylation↗

PubMed1987

[3H]cyclofoxy, a ligand suitable for positron emission tomography, labels mu and kappa opioid receptors.

The autoradiographic distribution of [3H]cyclofoxy (6-deoxy-6 beta-fluoronaltrexone) after in vivo administration or in vitro incubation suggests that it labels mu and kappa opioid receptors. In the rat, the pattern of [3H]cyclofoxy binding is similar to the distribution of mu receptors, however labeling is also present in the neural lobe of the pituitary, the central nucleus of the amygdala and the hypothalamus, areas where kappa receptors outnumber mu receptors. In the guinea pig, [3H]cyclofoxy binding sites are dense in the deep layers of the cortex, an area enriched in kappa receptors. These results are consistent with [3H]cyclofoxy binding to mu and kappa receptors.

Animals↗

PubMed1987

A postmortem study of the effect of chronic opiate abuse on psychotomimetic binding sites of human frontal cortex.

Membranes were prepared from area 8 of human frontal cortex harvested from five controls and five opiate addicts. Psychotomimetic binding sites were assayed using tritiated 1-(1-[2-thiethyl]cyclohexyl)piperidine ([3H]TCP) to label phencyclidine binding sites and 1,3-Di(2-[5-3H]tolyl)guanidine ([3H]DTG) to label the "haloperidol-sensitive sigma binding site." The results demonstrated no significant differences between the control and experimental groups, suggesting that in the region of the human brain sampled in this study, chronic abuse of opiates is not accompanied by alterations in psychotomimetic binding sites.

Adult↗

PubMed1986

Decreased striatal opiate delta-receptors in the rat model of persistent dyskinesia induced by iminodipropionitrile.

Chronic administration of iminodipropionitrile (IDPN) causes a persistent behavioral syndrome which consists of hyperactivity, vertical neck dyskinesias and lateral head twitches. D-Ala-D-Leu-enkephalin binding revealed a 26% decrease in the number but no change in the affinity of delta-opiate receptors in the striata of IDPN-treated rats. These findings are similar to those seen in the brains of patients with Huntington's disease. Further studies are needed to clarify the relationship of these findings to the phenomenology of the IDPN-induced dyskinetic abnormalities.

Animals↗

PubMed1986

A comparative autoradiographic study of the distributions of substance P and eledoisin binding sites in rat brain.

The relative potencies of tachykinin peptide analogs competing for binding of [125I]Bolton Hunter-conjugated substance P ([125I]BH-SP) or [125I]Bolton Hunter-conjugated eledoisin ([125I]BH-ED) in slide-mounted rat brain sections are very different, indicating the presence of two distinct tachykinin binding sites. The structure-activity profiles resemble those described in peripheral bioassay studies in which two tachykinin receptors have been postulated. Autoradiography of the two iodinated ligands bound with selective and one-site in vitro incubation conditions shows two discrete and distinctly different distribution patterns in brain. Binding sites for [125I]BH-ED are densely distributed in the accessory olfactory bulb, intermediate layers of the cerebral neocortex, portions of the hippocampal CA fields, hypothalamic supraoptic and paraventricular nuclei, central portions of the interpeduncular nucleus, sphenoid nucleus, medial subdivision of the solitary tract complex, and the substantia gelatinosa of the spinal cord. Binding sites for [125I]BH-SP are present in many of these same structures, but the densities and distribution patterns are different. In addition, [125I]BH-SP binds in numerous structures not labeled by [125I]BH-ED. Neither pattern matches the locations of terminations of endogenous tachykinin pathways marked by immunohistochemistry. The results suggest that it would be inappropriate to name brain tachykinin receptors according to the endogenous ligand which binds with highest affinity.

Animals↗

PubMed1986

Opiate receptors in rat pituitary are confined to the neural lobe and are exclusively kappa.

The distribution and density of opiate receptor subtypes in rat pituitary were examined by quantitative autoradiography of tritiated ligand-binding to slide-mounted sections under conditions optimized to label mu, delta, or kappa opiate receptors. Mu and delta receptor-binding was virtually undetectable in the pituitary. Kappa receptor-binding was confined to the neural lobe where it was densest in the external rim. Autoradiographic silver grains in emulsion-coated, Nissl-stained sections were preferentially located between cells, suggesting kappa receptor localization on nerve terminals and/or processes of pituicytes.

Animals↗

PubMed1986

Autoradiographic localization of mu- and delta-opiate receptors in the forebrain of the rat.

The autoradiographic distributions of mu opiate receptors, labeled in vitro by [125I]D-Ala2-MePhe4-Met(o)5-ol-enkephalin (FK), and delta-opiate receptors, labeled by [3H]D-Ala2-D-Leu5-enkephalin (DADLE) in the presence of oxymorphone to block high affinity binding to the mu site, were examined and compared in the forebrain of the rat. The mu- and delta-receptors were differentially distributed in most structures. mu Binding sites were found in nearly all gray matter structures and showed heterogeneous patterns of density that were correlated with cytoarchitecture and neuronal connections. Laminar density profiles were seen in laminated structures such as olfactory bulb, cerebral cortex and hippocampus. Highest mu binding densities were in striatal patches and the habenular streak. delta Sites had distinct laminar patterns in the main olfactory bulb and cortex which differed from the mu patterns. The external plexiform layer of the main olfactory bulb had the greatest density of delta binding sites; cortex and striatum were also densely labeled. The septum, globus pallidus, preoptic area and hypothalamus were lightly labeled by both ligands. The magnocellular hypothalamic nuclei had negligible mu and delta labeling. The thalamus had dense mu but sparse delta sites. mu And delta binding sites were both present in the amygdala but had different distributions. Two fiber tracts--optic tract and fasciculus retroflexus--had FK labeling. In contrast, a portion of the corpus callosum was labeled by DADLE and not by FK. The results suggest an association of mu-opiate receptors with sensory, especially olfactory, and limbic projections in the forebrain, and delta-opiate receptors with intrinsic and commissural forebrain pathways.

Amygdala↗

PubMed1986

Morphine tolerance increases mu-noncompetitive delta binding sites.

In light of more recent knowledge concerning endogenous opioid peptides and their multiple opiate receptors, we reevaluated the effects of morphine tolerance on opiate receptor binding parameters. Rats were implanted with morphine or placebo pellets, and [3H][D-Ala2,D-Leu5]enkephalin ([3H]DADL) was used to label brain membranes. Utilizing the technique of binding surface analysis, we observed a selective 47% up-regulation of lower affinity [3H]DADL binding sites (mu-noncompetitive delta binding sites) in morphine pelleted rats. To corroborate these results, we treated brain membranes with the site directed alkylating agent FIT (N-phenyl-N-[1-(2-p-isothiocyanato)phenyl-ethyl)-4-piperidinyl] propanamide), which results in membranes highly enriched with the lower affinity [3H]DADL binding site. Scatchard plots of [3H]DADL binding to FIT-treated membranes also revealed that chronic morphine treatment produced a 60-65% up-regulation of the mu-noncompetitive delta binding site. These data indicate that chronic morphine alters a selective subpopulation of opiate receptors that may play a role in the mechanisms of opiate tolerance and physical dependence.

Animals↗

PubMed1986

Chronic iminodipropionitrile (IDPN) causes no changes in the rat brain phencyclidine (PCP) receptor.

Chronic IDPN treatment leads to a persistent stereotypic and dyskinetic behavioral syndrome which is reminiscent of that caused by PCP in mammals. Since the neuropharmacological profile of the two syndromes are very similar, the status of the PCP binding site was studied in rats who were suffering from the IDPN-induced syndrome. The characteristics of the receptor were not altered in either the striata or the hippocampi of the animals. These results suggest that the development of chronic stereotypies is not intimately linked to any perturbation of the PCP binding site in rat brain.

Animals↗

PubMed1986

Upregulation of the mu-noncompetitive delta binding site by chronic morphine administration: effect of preincubating membranes in 400 nM sodium chloride.

Rats treated chronically with morphine were found to have an increased number of mu-noncompetitive delta binding sites. The increase in the Bmax was 39% and the enhancement was blocked by pre-incubation in 50 mM Tris, pH 7.4 with 400 mM NaCl for one hour at 25 degrees C. These findings are consistent with the removal of newly manifested binding sites being a specific property of sodium, by analogy with previous studies where sodium has been shown to extract extrinsic proteins from red cell ghosts.

Animals↗

PubMed1985

Leucine enkephalin noncompetitively inhibits the binding of [3H]naloxone to the opiate mu-recognition site: evidence for delta----mu binding site interactions in vitro.

Using quantitative methods, this study examined the hypothesis that delta-ligands are noncompetitive inhibitors at a population of mu-binding sites. Evidence is presented that with the defined set of in vitro assay conditions utilized, [3H]naloxone labels two binding sites: the mu binding site and a second site tentatively identified as a kappa binding site, and that leucine enkephalin is a noncompetitive inhibitor at the mu recognition site.

Animals↗

PubMed1985

Evidence that the delta-selective alkylating agent, fit, alters the mu-noncompetitive opiate delta binding site.

Considerable evidence supports the notion that the prototypic delta agonist [3H]D-ala2-D-leu5-enkephalin labels two binding sites on brain membranes in vitro. Recent studies have demonstrated that treatment of brain membranes with the delta-selective, site-directed, alkylating agent, FIT (Rice et al., Science 220, 314-316, 1983) results in a membrane preparation devoid of detectable higher affinity [3H]D-ala2-D-leu5-enkephalin binding sites, but contain residual lower affinity binding sites at which mu-ligands are apparent noncompetitive inhibitors (Rothman et al., Neuropeptides 4:210-215, 1984). In this paper we extend these data by showing that although FIT eliminates the higher affinity binding site, it also alters the properties of the residual lower affinity binding sites.

Affinity Labels↗

PubMed1985

Preparation of rat brain membranes highly enriched with opiate kappa binding sites using site-directed acylating agents: optimization of assay conditions.

The goal of this study was to determine optimal conditions with which to measure opiate kappa binding sites in rat brain. Membranes were pretreated with mu-selective (BIT) and delta-selective (FIT) site-directed acylating agents (Rice et al., Science 220, 314-316), and the binding of [3H]bremazocine to the residual binding sites was defined as the kappa binding site. The binding of [3H]bremazocine to BIT/FIT-treated membranes was greatly increased by conducting the assay at 0 degrees C in the presence of 0.4 M NaCl. Using this 0 degrees C/NaCl assay condition, the binding of [3H]bremazocine was best described by a one-site binding model with a KD of 0.45 nM and a Bmax of 378 fmol/mg protein. Autoradiographic studies demonstrated that, using this assay condition, [3H]bremazocine densely labeled the deep layers of guinea pig cortex, an area known to be enriched with kappa binding sites. These and additional data suggest that the binding of [3H]bremazocine to the kappa binding site of rat brain is optimally assayed at 0 degrees C in the presence of 0.4 M NaCl using BIT/FIT-treated membranes and that rat brain is endowed with a high level of kappa binding sites.

Alkylating Agents↗

PubMed1985

A quantitative study of [3H]D-Ala2-D-Leu5-enkephalin binding to rat brain membranes. Evidence that oxymorphone is a noncompetitive inhibitor of the lower affinity delta-binding site.

The mechanism by which mu ligands inhibit the binding of prototypic delta agonists to preparations of brain membranes is controversial. Most investigators assume competitive inhibition. In this study, we examine the interaction of the mu agonist oxymorphone and delta agonist DSTLE (D-Ser2-Thr6-Leu-enkephalin) with [3H]D-Ala2-D-Leu5-enkephalin (DADL) binding to membranes of rat brain. According to the two-site competitive model, mu ligands are competitive inhibitors at both sites. The two-site allosteric model supposes that mu ligands are competitive inhibitors at one binding site, and noncompetitive inhibitors at the other binding site. Quantitative analysis of DSTLE and oxymorphone binding demonstrated that the two-site allosteric model fit the data significantly better than did the two-site competitive model, and that oxymorphone is a noncompetitive inhibitor of the lower affinity [3H]DADL-binding site. Autoradiographic studies demonstrated that the lower affinity [3H]DADL-binding site (mu-noncompetitive binding site) had an anatomical distribution apparently indistinguishable from that obtained with [3H]oxymorphone (type I pattern), supporting the hypothesis that the lower affinity delta-binding site is the delta-binding site of an opiate receptor complex consisting of interacting mu- and delta-binding sites.

Animals↗