Search PubMed⌕ Search

Biomedical subjects

R B Rothman

Publications and source records attributed to R B Rothman.

At least 163 records · Page 9Linked to original sources
PubMed1992

Endogenous opioids accumulate in plasma in a rat model of acute cholestasis.

To obtain data on the degree to which the opioid system is changed in cholestasis, endogenous opioid activity in plasma of rats with acute cholestasis was determined 5 days after bile duct resection. Total plasma opioid activity was determined using a radioreceptor technique that measured the displacement of the opiate receptor ligand [3H]-DAMGO from lysed synaptosomal fractions of normal rat brain. Plasma total opioid activity was threefold greater in bile duct-resected rats than in sham-operated and unoperated controls (P less than or equal to 0.05). Plasma levels of the individual endogenous opioid, methionine-enkephalin, were determined using a sensitive radioimmunoassay, and the specificity of the assay was confirmed using high-performance liquid chromatography. In cholestatic rats, plasma methionine-enkephalin levels were more than six-fold greater than in sham-operated controls (P less than or equal to 0.001) and more than 17-fold greater than in unoperated controls (P less than or equal to 0.001). However, plasma methionine-enkephalin levels accounted for less than 5% of total plasma opioid activity after bile duct resection. Plasma methionine-enkephalin levels in both cholestatic plasma and plasma from sham-operated animals were stable when incubated in vitro despite the presence of undiminished activity of the major enkephalin-degrading enzymes. Thus, protection of methionine-enkephalin from degradation may be a factor contributing to the elevated plasma levels of methionine-enkephalin found in cholestasis. The magnitude of the increase in plasma endogenous opioid activity in bile duct-resected rats provides support for the hypothesis that endogenous opioids contribute to the pathophysiology of cholestasis.

Acute Disease↗
PubMed1992

Cocaine and GBR12909 produce equivalent motoric responses at different occupancy of the dopamine transporter.

The motoric-stimulating effect of dopamine (DA) reuptake blockers is thought to result from the increase in synaptic dopamine levels, which occurs as a consequence of blockade of DA reuptake. The present study tested measured occupancy of the DA transporter in vivo produced by behaviorally equivalent doses of the DA reuptake blockers GBR12909 (20 mg/kg), cocaine (20 mg/kg), WIN35-065-2 (1 mg/kg), and nomifensine (5 mg/kg). Two methods were used to measure in vivo occupancy of the DA transporter: a) an ex vivo method, in which the ability of whole brain supernatants, prepared from rats administered the test drugs, were tested for their ability to inhibit the reuptake of [3H]DA by striatal synaptosomes; and b) an in vivo binding assay using [3H]N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine ([3H]BTCP) to label the striatal DA transporter in vivo. Considerable data support the notion that this measurement is predictive of transporter occupancy in the nucleus accumbens. Similar results were obtained with both methods: The order of potency for apparent transporter occupancy was GBR12909 >> nomifensine > WIN35-065-2 = cocaine. These data indicate that it takes greater occupancy of the DA transporter by GBR12909 to produce behavioral effects equivalent to those produced by cocaine at lower transporter occupancy. The data of the present study suggest, therefore, that studies relating the effects of DA reuptake inhibitors on DA-mediated motoric behaviors to DA transporter occupancy might facilitate the identification of novel compounds potentially useful for the pharmacotherapy of cocaine abuse.

Animals↗
PubMed1992

Central mu-opioid receptors are down-regulated in a rat model of cholestasis.

Ameliorations of the pruritus of cholestasis by opioid antagonists are consistent with this form of pruritus being centrally mediated by the opioid system. To determine whether the central opioid system is altered in cholestasis, the specific binding of a selective mu-opioid receptor ligand, 3H-DAMGO, to mu-opioid receptors was studied in rats with acute cholestasis due to bile duct resection. Using whole brain membranes and subcellular mitochondrial-synaptosomal fractions the density of mu-receptor sites was 30% (p less than 0.01) and 22% (p = 0.03) less in bile-duct-resected rats than in sham-resected rats. Using membranes from individual brain regions specific binding of 3H-DAMGO was reduced by 43-53% in the cerebral cortex, hippocampus and caudate nucleus of bile-duct-resected rats. Thus mu-opioid receptors in the brain are down-regulated in a classical model of cholestasis. This alteration of the central opioid system could be a consequence of increased exposure of opioid receptors to endogenous opioids in cholestasis and may reflect an important mechanism in the pathogenesis of the pruritus of cholestasis.

Animals↗
PubMed1992

Characterization of the enantiomers of cis-N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1- pyrrolidinyl)cyclohexylamine (BD737 and BD738): novel compounds with high affinity, selectivity and biological efficacy at sigma receptors.

A novel class of compounds with very high affinity and selectivity for sigma receptors has been discovered. BD614 [(+/-)-cis-N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1- pyrrolidinyl)cyclohexylamine] and its optically pure 1S,2R-(-)-[BD737] and 1R,2S-(+)-[BD738]enantiomers bound to sigma receptors of guinea pig brain with Ki = 2.0 +/- 0.4, 1.3 +/- 0.3 and 6 +/- 3 nM, respectively. These compounds exhibited little or no affinity for dopamine-D2, kappa opiate or phencyclidine receptors and displayed high biological efficacy in assays of sigma receptor function, ability to produce alterations in motor behavior and inhibition of the muscarinic cholinergic phosphoinositide response. Microinjection of BD614 into the rat red nucleus or substantia nigra produced a dose-dependent alteration in head position and contralateral circling, respectively. BD614, BD737 and BD738 inhibited stimulation of inositol phosphate production by carbachol or oxotremorine-M in a dose-dependent manner. Thus, N-substituted cis-2-(1-pyrrolidinyl)cyclohexylamines may prove useful in studies of sigma receptor structure and function.

Analgesics↗
PubMed1991

Interaction of beta-funaltrexamine with [3H]cycloFOXY binding in rat brain: further evidence that beta-FNA alkylates the opioid receptor complex.

beta-Funaltrexamine (beta-FNA) is an alkylating derivative of naltrexone. In addition to acting as an irreversible inhibitor of mu-receptor-mediated physiological effects, intracerebroventricular (i.c.v.) administration of beta-FNA to rat attenuates the ability of selective delta receptor antagonists and naloxone to reverse delta receptor-mediated effects. Moreover, recent work demonstrated that i.c.v. administration of beta-FNA alters the conformation of the opioid receptor complex, as inferred by a decrease in the Bmax of the lower affinity [3H][D-ala2,D-leu5]enkephalin binding site. Consistent with the decreased potency of naloxone as an inhibitor of delta receptor mediated effects, beta-FNA doubled the naloxone IC50 for displacing [3H][D-ala2,D-leu5]enkephalin from its lower affinity binding site. These data collectively support the hypothesis that the opioid receptor complex postulated to mediate mu-delta interactions in vivo is identical to the opioid receptor complex as defined by vitro ligand binding studies. A direct prediction of this hypothesis is that beta-FNA should increase the Kd of antagonists for the mu binding site (mu cx) of the receptor complex. The data reported in this paper demonstrate that beta-FNA doubled the IC50 of the potent narcotic antagonist, 6-desoxy-6 beta-fluoronaltrexone (cycloFOXY) for displacing [3H][D-ala2,D-leu5]enkephalin from its lower affinity binding site, and doubled the Kd of [3H]cycloFOXY for its mu binding site, providing additional data that the mu binding site labeled by [3H]cycloFOXY is the mu binding site of the opioid receptor complex. beta-FNA also altered the kappa binding site labeled by [3H]cycloFOXY, and when administered intrathecally to mice, beta-FNA produced a longlasting antinociception in the acetic acid writhing test.

Alkylating Agents↗
PubMed1991

[3H]1-[2-(2-thienyl)cyclohexyl]piperidine labels two high-affinity binding sites in human cortex: further evidence for phencyclidine binding sites associated with the biogenic amine reuptake complex.

Previous work demonstrated two high-affinity PCP binding sites in guinea pig brain labeled by [3H]TCP (1-(1-[2-thienyl]cyclohexyl)piperidine): site 1 (N-methyl-D-aspartate [NMDA]-associated) and site 2 (dopamine-reuptake complex associated). The present study examined brain membranes prepared from various species, including human, for the presence of site 2, defined as binding in the presence of (+)-5-methyl-10,11-dihydro-5H-dibenzo [a, d]cyclohepten-5,10-imine maleate ((+)-MK801) minus binding in the presence of 10 microM TCP (nonspecific binding). Studies were conducted in absence of sodium which was found to be inhibitory to [3H]TCP binding. The results demonstrated detectable levels of site 2 in brain membranes of guinea pig, rabbit, pig, mouse, sheep, and human but not in the rat or chicken. Using human cortical membranes, site 2 was the predominant binding site. Detailed studies conducted with human cortical tissue showed that high-affinity dopamine (1-[2- [bis(4-fluorophenyl)-methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR12909)], [1,2]benzo(b)thiophenylcyclo-hexylpiperidine (BTCP), and serotonin (fluoxetine) uptake inhibitors produced a wash-resistant inhibition of [3H]TCP binding to site 2, but not site 1. Preincubation of guinea pig brain membranes with BTCP was shown to produce an increase in the dissociation rate of [3H]TCP from PCP site 2. Structure activity studies with various uptake inhibitors showed that GBR12909, benztropine, fluoxetine, and BTCP have higher affinity for site 2 than for site 1. (+)-MK801, ketamine, and tiletamine were very selective for site 1, whereas dexoxadrol and TCP were moderately selective for site 1. These results suggest that human cortex possesses high-affinity PCP binding sites associated with biogenic reuptake binding sites, and that guinea pig brain, but not rat brain, may be an appropriate animal model for studying PCP site 2 in human brain.

Animals↗
PubMed1991

Preliminary ligand binding data for subtypes of the delta opioid receptor in rat brain membranes.

Delta opioid binding sites were assayed using [3H][D-ala2,D-leu5]enkephalin and rat brain membranes depleted of mu binding sites with the site-directed acylating agent, 2-(p-ethoxybenzyl)-1-diethylaminoethyl-5-isothiocyanatobenzimid a zole-HCI. [D-Pen2,D-Pen5]enkephalin (DPDPE), [D-Pen2,L-Pen5]enkephalin, [D-Ala2]deltorphin-I and [D-Ala2]deltorphin-II inhibition curves were characterized by slope factors (Hill coefficients) less than 1. The low slope factor of DPDPE persisted in the presence of 50 microM 5'-guanylyimidodiphosphate in the assay Quantitative analysis of [D-ala2,D-leu5]enkephalin, DPDPE and [D-Ala2]deltorphin-I binding surfaces resolved two binding sites. Whereas [D-ala2,D-leu5]enkephalin had equal affinity for both sites, DPDPE and [D-Ala2]deltorphin-I had high affinity for the high capacity binding site, and low affinity for the low capacity binding site. These data support pharmacological studies demonstrating delta receptor subtypes which mediate antinociception.

Amino Acid Sequence↗
PubMed1991

RTI-4614-4: an analog of (+)-cis-3-methylfentanyl with a 27,000-fold binding selectivity for mu versus delta opioid binding sites.

The objective of this study was to determine the binding affinities of (+/-)-cis-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]- N-phenylpropanamide-HCI (RTI-4614-4), which is an analog of (+)-cis-3-methylfentanyl for opioid receptor subtypes. The Ki values (nM) of this agent for opioid receptor subtypes were as follows: mu (0.0055), delta (148), kappa 1 (84.8), kappa 2a (2275), and kappa 2b (22.3). The selectivity of this agent for the mu binding site was 27,000 vs. the delta binding site, 15,400 vs. the kappa 1 binding site, 413,700 vs the kappa 2a and 4,054 vs the kappa 2b binding site. In contrast, two other fentanyl analogs, N-(2-(4-methylpyridinyl))-N-(1-phenethyl-4-piperidinyl) 2-furamide and N-(2-pyrazinyl)-N-(1-phenethyl-4-piperdinyl)2-furamide had considerably higher Ki values at, and were less selective for, the mu binding site. Since RTI-4614-4 is composed of a mixture of four stereoisomers, the resolution of these isomers should permit identification of an extremely potent and selective agent for the opioid mu receptor.

Animals↗
PubMed1991

(+)-cis-3-methylfentanyl and its analogs bind pseudoirreversibly to the mu opioid binding site: evidence for pseudoallosteric modulation.

Previous studies demonstrated that preincubation of membranes from the brain of the rat with 1 microM (+)-cis-3-methylfentanyl produced a wash-resistant inhibition of mu receptor binding. The present study was designed to: (1) determine the mechanism by which (+)-cis-3-methylfentanyl produced a wash-resistant inhibition of mu receptor binding, and (2) to generate a structure-activity study, using wash-resistant inhibition as the end-point. Pretreatment of membranes with 500 nM (+)-cis-3-methylfentanyl increased the Kd of binding sites for [3H]ohmefentanyl, without altering the Bmax. The increase in the Kd was only partially due to the presence of residual drug and was accompanied by an increase in the dissociation rate of the binding of [3H]ohmefentanyl. Therefore, pretreatment of membranes with (+)-cis-3-methylfentanyl resulted in a lower affinity interaction of [3H]ohmefentanyl with the mu binding site, consistent with a model postulating pseudoallosteric modulation of mu binding sites by (+)-cis-3-methylfentanyl and its analogs. The rank order of potencies for wash-resistant inhibition of the binding of [3H]6 beta-fluoro-6-desoxyoxymorphone or [3H]ohmefentanyl, was lofentanil greater than (+)-cis-3-methylfentanyl greater than ohmefentanyl greater than sufentanil. All other opioids tested (1 microM morphine, 1 microM naloxone, 1 microM fentanyl, 1 microM (+)-cyclazocine, 1 microM (-)-cis-3-methylfentanyl) did not act as wash-resistant inhibitors of mu binding sites. Although the pseudoirreversible IC50 of these agents did not correlate with their ED50 values for producing antinociception, after intravenous administration, the authors speculate that this property, termed "pseudoirreversible inhibition", might contribute to the extraordinary potency of (+)-cis-3-methylfentanyl and its analogs as antinociceptive agents.

Allosteric Regulation↗
PubMed1991

GBR12909 antagonizes the ability of cocaine to elevate extracellular levels of dopamine.

Rats were administered various IP doses of the high-affinity dopamine (DA) reuptake inhibitor 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-[3-phenylpropyl]piperazine (GBR12909). The caudate nuclei were removed 60 min after drug administration and stored at -70 degrees C. Striatal membranes were prepared later. The results demonstrated that GBR12909 produced a dose-dependent decrease in the binding of [3H]cocaine or [3H]GBR12935 to the DA transporter (ED50 about 10 mg/kg). Saturation binding studies with [3H]GBR12935 showed that this was due to both an increase in the Kd, due to residual drug, and to a decrease in the Bmax. At a dose of 25 mg/kg IP, GBR12909 produced a 50% decrease in the Bmax, and a 3.4-fold increase in the Kd. In the in vivo microdialysis studies, GBR12909 (25 mg/kg IP) produced a modest, long-lasting and stable elevation of extracellular DA. Administration of cocaine through the microdialysis probe to rats pretreated with either saline or GBR12909 (25 mg/kg IP) produced a dose-dependent increase in extracellular DA in both groups. GBR12909 inhibited cocaine-induced increases in extracellular DA by about 50% at all doses. These data collectively indicate that at a dose sufficient to decrease by 50% the Bmax of [3H]GBR12935 binding sites, GBR12909 antagonizes the ability of cocaine to elevate extracellular DA by 50%. Further studies will be needed to evaluate a possible role for GBR12909 in the medical treatment of cocaine addiction.

Animals↗
PubMed1991

Labeling by [3H]1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands.

Equilibrium binding studies with the sigma receptor ligand [3H]1,3-di(2-tolyl)guanidine ([3H]DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. [Life Sci. 45:1721-1732 (1989)]. Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and low affinity for most other sigma ligands. Kinetic experiments demonstrated that [3H]DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of [3H]DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of [3H]DTG from site 2, suggesting an association of this binding site with calcium channels.

Animals↗
PubMed1990

The potent opioid agonist, (+)-cis-3-methylfentanyl binds pseudoirreversibly to the opioid receptor complex in vitro and in vivo: evidence for a novel mechanism of action.

The present study demonstrates that pretreatment of rat brain membranes with (+)-cis-3-methylfentanyl [(+)-cis-MF], followed by extensive washing of the membranes, produces a wash-resistant decrease in the binding of [3H]-[D-ala2,D-leu5]enkephalin to the d binding site of the opioid receptor complex (delta cx binding site). Intravenous administration of (+)-cis-MF (50 micrograms/kg) to rats produced a pronounced catalepsy and also produced a wash-resistant masking of delta cx and mu binding sites in membranes prepared 120 min post-injection. Administration of 1 mg/kg i.v. of the opioid antagonist, 6-desoxy-6 beta-fluoronaltrexone (cycloFOXY), 100 min after the injection of (+)-cis-MF (20 min prior to the preparation of membranes) completely reversed the catatonia and restored masked delta cx binding sites to control levels. This was not observed with (+)-cycloFOXY. The implications of these and other findings for the mechanism of action of (+)-cis-MF and models of the opioid receptors are discussed.

Animals↗
PubMed1990

High affinity dopamine reuptake inhibitors as potential cocaine antagonists: a strategy for drug development.

The addictive and euphorogenic effects of cocaine are thought to result primarily from inhibition of dopamine reuptake. Although the potency of cocaine-like drugs as inhibitors of DA reuptake is highly correlated with their potency as reinforcers in animals, several potent DA reuptake blockers (bupropion, nomifensine, benztropine, and mazindol) have not been reported to produce addiction or euphoria in humans. Based on these observations in humans, DA reuptake inhibitors are classified into two groups; type 1 blockers, which produce addiction and euphoria, and type 2 blockers, which do not. Given that type 1 and type 2 blockers act at the same site (the DA transporter), the author suggests that type 2 agents may antagonize the effects of cocaine, and might prove useful in the treatment of cocaine addiction.

Benztropine↗
PubMed1990

Pseudoallosteric modulation by (+)-MK801 of NMDA-coupled phencyclidine binding sites.

Two high affinity phencyclidine (PCP) binding sites, labelled by [3H] 1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP), have been identified in guinea pig brain, with one site coupled to the N-methyl-D-aspartate (NMDA) receptor (site 1) and the other site associated with the dopamine reuptake carrier complex (site 2). In this study, PCP enhanced the dissociation of [3H]TCP from PCP site 1 and site 2, while (+)-MK801 only enhanced dissociation of [3H]TCP from PCP site 1. Although additional studies will be required to determine the exact mechanism(s) of these effects, these data demonstrate that the interactions of PCP with both site 1 and site 2 are more complex than previously appreciated.

Allosteric Regulation↗
PubMed1990

Wash-resistant inhibition of phencyclidine- and haloperidol-sensitive sigma receptor sites in guinea pig brain by putative affinity ligands: determination of selectivity.

Several putative affinity ligands, based on the structures of phencyclidine etoxadrol, 5-methyl-10,11-dihydro-5H-dibenzo[a,d] cycloheptene-5,10-imine (MK801) and 1,3-di-(2-methylphenyl)guanidine (DTG) were evaluated in vitro for their ability to produce a wash-resistant inhibition of phencyclidine and sigma receptor sites in homogenates of the brain of the guinea pig. All the phencyclidine-based ligands, including 1-[1-(3-isothiocyanatophenyl)cyclohexyl]piperidine (Metaphit) and (+/-)-N-(2-isothiocyanatoethyl) MK801 [(+/-)-MK801-NCS], produced a wash-resistant inhibition of binding sites for phencyclidine, labelled by [3H]-1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) and sigma binding sites, labelled by [3H]DTG. The DTG-based ligands, 1-(4-isothiocyanato-2-methylphenyl)-3-(2-methylphenyl)guanidine (DIGIT) and 1-(4-[2-(2-isothiocyanatoethoxy)ethoxy]-2-methyl-phenyl)-3-(2- methylphenyl)guanidine (DIGIE), produced a wash-resistant inhibition of sigma sites, at concentrations as small as 1 microM and also inhibited binding sites for phencyclidine at larger concentrations (100 microM). Both 1-(3-isothiocyanatophenyl)-1-ethyl-4-(2-piperidyl)-1,3-dioxolane (ETOX-NCS) and 1-[1-(3-bromoacetyloxyphenyl)cyclohexyl]-1,2,3,6-tetrahydropyri din e (Bromoacetyl-PCP) were the most potent and selective inhibitors of the binding of [3H]TCP, while DIGIT was the most selective inhibitor of the binding of [3H]DTG. Future studies will examine the selectivity of these agents in vivo after intracerebroventricular administration.

Affinity Labels↗
PubMed1990

Pretreatment of rats with the irreversible mu-receptor antagonist, beta-FNA, fails to prevent naltrexone-induced upregulation of mu-opioid receptors.

This study examined the effect of beta-funaltrexamine (beta-FNA), an irreversible mu-receptor antagonist, on naltrexone-induced upregulation of mu-(mu cx + mu nex) and delta nex-opioid receptors. [The subscripts 'cx' and 'nex' denote binding sites 'in' (cx) and 'not in' (nex) the opioid receptor complex.] Rats were treated according to the following protocol. Two naltrexone or two placebo pellets were implanted subcutaneously in a nylon mesh on day 1. and were removed intact on day 8. Rats were given either saline or 20 nmol of beta-FNA in 10 microliters of saline (i.c.v.) on days 1, 3, 5 and 6, 60 min prior to implantation of the pellet. On day 9 frozen lysed-P2 membranes were prepared for assay of mu binding sites. In other experiments, membranes were depleted of mu-receptors by pretreatment with the site-directed acylating agent 2-(4-ethoxybenzyl)-l-diethylaminoethyl-5-isothiocyanatobenzimid azole.HCl (BIT) for assay of delta nex binding sites, using [3H] [D-ala2, D-leu5]enkephalin. The results demonstrated that beta-FNA did not upregulate the mu binding sites and also did not prevent naltrexone-induced upregulation of mu binding sites. Both beta-FNA and naltrexone increased the Bmax of delta nex binding sites and their effects were additive. These data suggest that the mechanism(s) responsible for antagonist-induced upregulation of opioid receptors are more complex than previously appreciated.

Animals↗