Alterations in the binding of [3H]leucine-enkephalin to striatal membranes by endogenous factors.
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Animals↗
Biomedical subjects
R B Rothman
Publications and source records attributed to R B Rothman.
A review of the effects of dopaminergic agents on humans, animals, and drug-seeking behavior, and its implications for medication development. Focus on GBR 12909.
Medication development for cocaine abuse has focused on potential mechanisms of action related to the abuse of cocaine. The hypothesis that mesolimbic dopamine (DA) is the key neurochemical mediator of cocaine's addictive and reinforcing effects is well supported by a wide variety of data from animal studies. On the other hand, medications that increase DA or block its action in humans can produce effects that appear incompatible with this hypothesis. This article reviews these incompatibilities between animal and human data with a focus on the DAergic actions of drugs, including DA reuptake inhibitors, direct DA agonists, DA increasers, and DA antagonists. Possible reasons for these discrepancies are discussed, and the potential role of high-affinity DA uptake inhibitors, such as GBR12909, for pharmacotherapies for treating cocaine addiction in humans is likely to come from understanding its mechanisms of action, it is clear that further research on the effects of cocaine in humans and animals will be critical to the medication development effort.
Animals↗
Neurotoxicity, drugs and abuse, and the CuZn-superoxide dismutase transgenic mice.
Administration of methamphetamine (METH) to animals causes loss of DA terminals in the brain. The manner by which METH causes these changes in neurotoxicity is not known. We have tested the effects of this drug in copper/zinc (CuZn)-superoxide dismutase transgenic (SOD Tg) mice, which express the human CuZnSOD gene. In nontransgenic (non-Tg) mice, acute METH administration causes significant decreases in DA and dihydroxyphenylacetic acid (DOPAC) in the striata of non-Tg mice. In contrast, there were on significant decreases in striatal DA in the METH administration caused decreases in striatal DA and DOPAC in the non-Tg mice, but not in the SOD-Tg mice. Similar studies were carried out with 1-methyl-1,2,3,6-tetrahydropyridine (MPTP), which also causes striatal DA and DOPAC depletion. As in the case of METH, MPTP causes marked depletion of DA and DOPAC in the non-Tg mice, but not in the SOD Tg mice. These results suggest that the mechanisms of toxicity of both METH and MPTP involved superoxide radical formation.
3,4-Dihydroxyphenylacetic Acid↗
Increased cortical serotonin-2 (5-HT-2) receptors in the iminodipropionitrile (IDPN)-model of persistent dyskinesia in the rat.
Chronic treatment with IDPN causes a persistent behavioral syndrome which is reminiscent of the head shakes, backward locomotion, and hyperactivity observed after acute injection of 5-HT agonists in rodents. Evaluation of the characteristics of 5-HT receptors, using mianserin to label 5-HT-2 receptors, revealed a significant increase in the number of binding sites in the cortices of rats which demonstrated the IDPN-induced syndrome. There was an associated decrease in the affinity of the receptors. These results add more evidence to support the idea that the serotonergic system may play a significant role in the manifestation of the persistent abnormalities induced by IDPN.
Animals↗
Rat brain PCP receptors: alterations in binding parameters following chronic administration of opiate agonists and antagonists.
Phencyclidine (PCP) is a widely abused drug of the arylcyclohexylamine class which is capable of producing symptoms of acute psychosis in man. PCP interacts with a specific CNS receptor, for which a putative endogenous peptide ligand has been identified. We have investigated whether PCP receptor binding parameters are modulated by activity in central opiate pathways. We have found that chronic administration of both an opiate agonist (etonitazene) and an opiate antagonist (naloxone) are able to decrease the affinity of the PCP receptor for TCP, a thienyl derivative of PCP. Furthermore, naloxone, but not etonitazene, resulted in a significant increase in the Bmax of TCP binding to the PCP receptor. These results suggest that neural activity mediated by CNS opioids systems is capable of affecting the binding parameters of the PCP receptor.
Animals↗
Unexpectedly high levels of opioid peptides in rat brain membranes.
Several considerations led us to directly measure, using radioimmunoassay, the levels of dynorphin-A(1-8), cholecystokinin-8, and Met5-enkephalin-Arg6-Gly7-Leu8 (MERGL) in lysed-P2 membranes, membranes incubated 60 min at 25 degrees C in 50 mM TRIS-HCl at pH 7.4, and membranes preincubated 60 min at 25 degrees C in TRIS buffer containing 0.4 M NaCl. Lysed-P2 membranes contained neuropeptides at levels between 0.253 and 1.727 pmol/mg protein. Incubating membranes in buffer alone decreased membrane peptide levels by 40 to 60%. Preincubations in the presence of 0.4 M NaCl resulted in even greater reductions in the peptide content of membranes (between 58 and 90%), without changing [3H]etorphine binding. Detailed comparisons of the two binding sites labeled by [3H]naloxone revealed changes in binding parameters induced by preincubation with NaCl. Taken collectively, these studies demonstrate that lysed-P2 membranes contain sequestered peptides that are not bound to opiate receptors.
Animals↗
Autoradiographic evidence for two classes of mu opioid binding sites in rat brain using [125I]FK33824.
Previous studies demonstrated that pretreatment of brain membranes with the irreversible mu antagonist, beta-funaltrexamine (beta-FNA), partially eliminated mu binding sites [25,35], consistent with the existence of two mu binding sites distinguished by beta-FNA. This paper tests the hypothesis that the FNA-sensitive and FNA-insensitive mu binding sites have different anatomical distributions in rat brain. Prior to autoradiographic visualization of mu binding sites, [3H]oxymorphone, [3H]D-ala2-MePhe4, Gly-ol5-enkephalin (DAGO), and [125I]D-ala2-Me-Phe4-met(o)-ol]enkephalin (FK33824) were shown to selectively label mu binding sites using slide mounted sections of molded minced rat brain. As found using membranes, beta-FNA eliminated only a portion of mu binding sites. Autoradiographic visualization of mu binding sites using the mu-selective ligand [125I]FK33824 in control and FNA-treated sections of rat brain demonstrated that the proportion of mu binding sites sensitive to beta-FNA varied across regions of the brain, particularly the dorsal thalamus, ventrobasal complex and the hypothalamus, providing anatomical data supporting the existence of two classes of mu binding sites in rat brain.
Animals↗
Interaction of endogenous opioid peptides and other drugs with four kappa opioid binding sites in guinea pig brain.
Guinea pig brain membranes depleted of mu and delta receptors by pretreatment with the site-directed acylating agents, 2-(4-ethoxybenzyl)-1- diethylaminoethyl-5-isothiocyanatobenzimidazole.HCl (BIT) and N-phenyl-N-[1-(2-(4-isothiocyanato)phenethyl)-4- piperidinyl]-propanamide.HCl (FIT), were used in this study to test the hypothesis that guinea pig brain possesses subtypes of kappa receptors. Pretreatment of membranes with either (-)-(1S,2S)-U50,488 or the kappa selective acylating agent, (1S,2S)-trans-2-isothiocyanato-N-methyl-N-[2-(1- pyrrolidinyl)cyclohexyl]benzeneacetamide, caused a wash-resistant inhibition of kappa 1 binding sites labeled by [3H]U69,593 binding, but not kappa 2 binding sites labeled by [3H]bremazocine. Binding surface analysis of [3H]bremazocine binding resolved two binding sites, termed kappa 2 and kappa 2b, present at densities of 212 and 225 fmol/mg protein, which had low affinity for (-)-(1S,2S)-U50,488 and U69,593. The kappa 2b site had high affinity for beta-endorphin(1-31) (Kd = 5.5 nM) and [D-Ala2,D-Leu5]enkephalin (Kd = 14 nM), and lower affinity for [D-Ala2-MePhe4,Gly-ol5]enkephalin (Kd = 147 nM) and [Leu5]enkephalin (Kd = 46.0 nM). Binding surface analysis of [3H]U69,593 binding also resolved two binding sites, termed kappa 1a and kappa 1b, present at densities of 6.0 and 40.0 fmol/mg protein. The kappa 1a binding site was characterized by very high affinity for alpha-neoendorphin. Quantitative autoradiographic studies demonstrated that kappa 2a and kappa 2b binding sites are heterogeneously distributed in guinea pig brain, and that the anatomical distribution of kappa 1 binding sites reported in the literature is different from that observed in this study for the kappa 2 binding sites. Viewed collectively, these data provide evidence for four kappa receptor subtypes in guinea pig brain.
3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
Probing the opioid receptor complex with (+)-trans-superfit. I. Evidence that [D-Pen2,D-Pen5]enkephalin interacts with high affinity at the delta cx binding site.
A variety of data support the existence of an opioid receptor complex composed of distinct but interacting mu cx and delta cx binding sites, where "cx" indicates "in the complex." The ability of subantinociceptive doses of [Leu5]enkephalin and [Met5]enkephalin to potentiate and attenuate morphine-induced antinociception, respectively, is thought to be mediated via their binding to the delta cx binding site. [D-Pen2,D-Pen5]Enkephalin also modulates morphine-induced antinociception, but has very low affinity for the delta cx binding site in vitro. In the present study, membranes were depleted of their delta ncx binding sites by pretreatment with the site-directed acylating agent, (3S,4S)-(+)-trans-N-[1-[2-(4-isothiocyanato)phenyl)-ethyl]-3-methy l-4- piperidyl]-N-phenylpropaneamide hydrochloride, which permits selective labeling of the delta cx binding site with [3H][D-Ala2,D-Leu5]enkephalin. The major findings of this study are that with this preparation of rat brain membranes: a) there are striking differences between the delta cx and mu binding sites; and b) both [D-Pen2,D-Pen5]enkephalin and [D-Pen2,L-Pen5]enkephalin exhibit high affinity for the delta cx binding site.
Animals↗
Upregulation of the opioid receptor complex by the chronic administration of morphine: a biochemical marker related to the development of tolerance and dependence.
Studies conducted after the development of the rapid filtration assay for opiate receptors, and before the recognition of multiple opioid receptors, failed to detect changes in opioid receptors induced by chronic morphine. Recent experiments conducted in our laboratories were designed to examine the hypothesis that only one of several opioid receptor types might be altered by chronic morphine. Using binding surface analysis and irreversible ligands to increase the "resolving power" of the ligand binding assay, the results indicated that chronic morphine increased both the Bmax and Kd of the opioid receptor complex, labeled with either [3H][D-Ala2,D-Leu5]enkephalin, [3H][D-Ala2-MePhe4,Gly-ol5]enkephalin or [3H]6-desoxy-6 beta-fluoronaltreone. In the present study rats were pretreated with drugs known to attenuate the development of tolerance and dependence [the irreversible mu-receptor antagonist, beta-funaltrexamine (beta-FNA), and the inhibitor of tryptophan hydroxylase, para-chlorophenylalanine], prior to subcutaneous implantation of morphine pellets. The results demonstrated that 1) unlike chronic naltrexone, beta-FNA failed to upregulate opioid receptors and 2) both beta-funaltrexamine and PCPA pretreatment attenuated the chronic morphine-induced increase in the Bmax, but not the Kd, of the opioid receptor complex. These results provide evidence that naltrex-one-induced upregulation of the opioid receptor complex might occur indirectly as a consequence of interactions at beta-funaltrexamine-insensitive opioid receptors and that morphine-induced upregulation (increased Bmax) of the opioid receptor complex is a relevant in vitro marker related to the development of tolerance and dependence. These data collectively support the hypothesis that endogenous antiopiate peptides play an important role in the development of tolerance and dependence to morphine.
Amino Acid Sequence↗
Probing the opioid receptor complex with (+)-trans-SUPERFIT. II. Evidence that mu ligands are noncompetitive inhibitors of the delta cx opioid peptide binding site.
Previous studies delineated two classes of delta binding sites; a delta binding site not associated with the opioid receptor complex, termed the delta ncx site, and a delta site associated with the opioid receptor complex, termed the delta cx site. The delta ncx site has high affinity for [D-Pen2,D-Pen5]enkephalin, and is synonymous with what is now identified as the delta 1 binding site. Pretreatment of membranes with the delta-selective acylating agents FIT, or (+)-trans-SUPERFIT, deplete membranes of the delta ncx binding site, which permits the selective labeling of the delta cx binding site with [3H][D-Ala2,Leu5]enkephalin. The present study compared the properties of the delta cx binding site present in brain membranes pretreated with (+)-trans-SUPERFIT with the properties of the delta cx site present in untreated membranes. The major findings are: 1) pretreatment of membranes with (+)-trans-SUPERFIT decreased the IC50 values of delta-preferring drugs, and increased the IC50 values of mu-preferring drugs, for the delta cx binding site; 2) the degree of delta selectivity was highly correlated with the magnitude of the (+)-trans-SUPERFIT-induced shift in the IC50 values; 3) the ligand-selectivity patterns of the mu and delta cx sites present in (+)-trans-SUPERFIT-pretreated membranes were poorly correlated; 4) whereas mu-preferring drugs were noncompetitive inhibitors of [3H][D-Ala2,Leu5]enkephalin binding to the delta cx site, delta-preferring drugs were competitive inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Interaction of opioid peptides and other drugs with multiple delta ncx binding sites in rat brain: further evidence for heterogeneity.
Recent pharmacological data strongly support the hypothesis of delta receptor subtypes as mediators of both supraspinal and spinal antinociception (delta 1 and delta 2 receptors). In vitro ligand binding data, which are fully supportive of the in vivo data, are still lacking. A previous study indicated that [3H][D-Ala2,D-Leu5]enkephalin labels two binding sites in membranes depleted of mu binding sites by pretreatment with the site-directed acylating agent, 2-(p-ethoxybenzyl)-1-diethylaminoethyl-5-isothiocyanatobenzimid azole-HCI (BIT). The main goal of the present study was to develop a ligand-selectivity profile of the two delta ncx binding sites. The data indicated that naltrindole and oxymorphindole were relatively selective for site 1 (20-fold). [D-Ser2,Thr6]Enkephalin and deltorphin-II were only 2.7-fold and 2.2-fold selective for site 1. [D-Pen2,D-Pen5]Enkephalin and deltorphin-I were 80-fold and 38-fold selective for site 2. 3-Iodo-Tyr-D-Ala-Gly-Phe-D-Leu was 52-fold selective for site 1. Morphine had moderate affinity for site 1 (Ki = 16 nM), and was about 11-fold selective for site 1. Thus, of the 10 drugs studied, only DPDPE and DELT-I were selective for site 2. Viewed collectively with other data, it is likely that the delta 1 receptor and the delta ncx binding site are synonymous.
Acylation↗
Interaction of opioid peptides and other drugs with multiple kappa receptors in rat and human brain. Evidence for species differences.
Previous experiments resolved four kappa binding sites in guinea pig brain termed kappa 1a, kappa 1b, and kappa 2b. The present study was undertaken to examine the occurrence of kappa receptor subtypes in rat and human brain. [3H]U69,593 and [3H]bremazocine were used to label kappa 1 and kappa 2 binding sites, respectively, present in brain membranes depleted of mu and delta binding sites by pretreatment with the irreversible ligands, BIT and FIT. Low levels of [3H]U69,593 binding precluded a detailed quantitative study of kappa 1 binding sites in these species. Quantitative examination of [3H]bremazocine binding resolved two kappa 2 binding sites in both rat and human brain whose ligand selectivity patterns differed from that of the guinea pig. These observations suggest that there may be considerable variation in the ligand recognition site of kappa receptor subtypes among mammalian species.
3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
A study of the effect of the irreversible delta receptor antagonist [D-Ala2,Leu5,Cys6]-enkephalin on delta cx and delta ncx opioid binding sites in vitro and in vivo.
Several lines of data support the existence of two classes of delta receptors: the delta cx binding site, which is the delta binding site of the mu-delta opioid receptor complex, and the delta ncx, which is the noncomplexed delta receptor. [D-Ala2,Leu5,Cys6]Enkephalin (DALCE) is an extended analog of [Leu5]enkephalin, which has been shown to bind irreversibly to delta receptors via the terminal cysteine by formation of a disulfide bond with the receptor. In vivo studies have shown that DALCE produces short-lived antinociceptive actions, followed by long-term antagonism of delta receptor-mediated antinociception. The major goal of the present study was to examine the effect of DALCE on the delta cx and delta ncx binding sites in vitro and in vivo. Intracerebroventricular administration of 40 micrograms DALCE failed to decrease [3H][D-Ala2,D-Leu5]enkephalin binding to the delta cx and delta ncx binding sites. Pretreatment of membranes with DALCE in vitro greatly reduced the Bmax of the delta ncx binding site, without significantly altering the Bmax of the delta cx binding site. These findings suggest that when administered in vivo, DALCE fails to distribute uniformly throughout the brain, and that it therefore binds covalently to opioid receptors mostly in the periventricular regions. Viewed collectively, these data support the hypothesis that DALCE acts as a selective delta ncx antagonist, and that the delta ncx binding site, which is sensitive to DALCE, is most likely synonymous with the recently described delta 1 receptor.
Animals↗
Phenylpiperidine opioid antagonists that promote weight loss in rats have high affinity for the kappa 2B (enkephalin-sensitive) binding site.
Certain opioid antagonists of the phenylpiperidine series (PPAs), such as LY255582, seem uniquely efficacious at producing weight loss in lean and meal-fed obese Zucker rats. Comparison of the pharmacological and receptor binding profile of PPAs that promote marked weight loss with those that do not has failed to find any obvious differences between these two groups of narcotic antagonists, which might explain the differences in their biological activities. The potent stimulatory effect of dynorphin, and other kappa agonists, on feeding behavior suggests that the antagonists that promote weight loss might have high affinity for kappa receptors. The recent demonstration by several laboratories of kappa receptor heterogeneity prompted us to test the hypothesis that the antagonists that promote weight loss might have high affinity for a subtype of kappa binding sites. In the present study, therefore, we determined the Ki values of five PPAs, naloxone, and naltrexone at mu, delta, kappa 1, kappa 2a, and kappa 2b binding sites. The data indicate that antagonists have subnanomolar Ki values and high selectivity for the kappa 2b binding site (relative to the kappa 2a binding site) are efficacious at promoting weight loss.
Animals↗
Differential binding of opioid peptides and other drugs to two subtypes of opioid delta ncx binding sites in mouse brain: further evidence for delta receptor heterogeneity.
Research into the functional role of the opioid delta receptor has intensified with the recent in vivo identification of delta receptor subtypes, termed delta 1 and delta 2, which mediate antinociception in the mouse. A variety of data also support the hypothesis of an opioid receptor complex composed of distinct, yet interacting, mu, delta, and perhaps kappa binding sites. This model postulates two classes of delta binding sites: a delta binding site not associated with the opioid receptor complex, termed the delta ncx site, and a delta site associated with the receptor complex, termed the delta cx site. A major purpose of this study was to clarify the relationship between the delta ncx binding sites and the delta 1 and delta 2 receptors. Mouse brain membranes were depleted of mu sites by pretreatment with the site-directed acylating agent, BIT, and the delta ncx binding sites were labeled with [3H][D-Ala2,D-Leu5]enkephalin. Binding surface analysis readily resolved two binding sites (delta ncx-1 and delta ncx-2) in the absence and presence of 100 mM NaCl. Control experiments with guanine nucleotides and the ligand-selectivity analysis indicated that the two sites were not two states of a single receptor. Pretreatment of membranes with DALCE, but not [Cys4]deltorphin, decreased [3H] [D-Ala2,D-Leu5]enkephalin and [3H][D-Ser2,Thr6]enkephalin binding. Ligand-selectivity analysis of the two binding sites suggested that neither delta ncx binding site had the characteristics expected of the delta 2 receptor, and that the delta ncx-1 site, but not the delta ncx-2 site, was synonymous with the delta 1 receptor. Moreover, our finding that the racemic nonpeptide delta agonist, BW373U86, had high affinity at and selectivity for the delta ncx-2 site suggests that this site may be a novel delta receptor that mediates some of the effects of BW373U86.
Alkylating Agents↗
Chronic intracerebroventricular infusion of the antiopioid peptide, Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (NPFF), downregulates mu opioid binding sites in rat brain.
Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (NPFF), an endogenous mammalian antiopioid peptide, has been shown by other laboratories to attenuate the acute antinociceptive effects of morphine, the development of morphine tolerance, and naloxone-induced withdrawal in morphine-dependent rats. The present study determined the effect of chronic NPFF on mu opioid receptors and mRNA for the endogenous opioids dynorphin and enkephalin. Rats received ICV infusions of either saline or NPFF (5 micrograms/h) for 13 days via Alzet 2002 osmotic minipumps. Homogenate binding studies, which used whole brain membranes, demonstrated that NPFF decreased the Bmax of mu binding sites (labeled by [3H][D-Ala2-MePhe4,Gly-ol5]enkephalin) from 262 +/- 12 to 192 +/- 12 fmolmg protein, and increased the Kd from 1.1 to 2.3 nM. Quantitative receptor autoradiography and in situ hybridization experiments were conducted with sections collected at the level of the striatum. The density of mu opioid binding sites labeled by [3H][D-Ala2-MePhe4,Gly-ol5]enkephalin was decreased in all brain areas measured except the corpus callosum, and there was no change in dynorphin mRNA or enkephalin mRNA in the caudate, the nucleus accumbens, or the ventral pallidum. Rats chronically administered ICV morphine sulfate (20 micrograms/h) for 14 days developed tolerance to morphine and a low degree of dependence, as measured by naloxone-precipitated withdrawal. Chronic administration of NPFF concurrently with morphine sulfate did not significantly alter naloxone-induced withdrawal signs or the development of morphine tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Selective labeling of kappa 2 opioid receptors in rat brain by [125I]IOXY: interaction of opioid peptides and other drugs with multiple kappa 2a binding sites.
Recent studies from our laboratory resolved two subtypes of the kappa 2 binding site, termed kappa 2a and kappa 2b, using guinea pig, rat, and human brain membranes depleted of mu and delta receptors by pretreatment with the site-directed acylating agents BIT (mu-selective) and FIT (delta-selective). 6 beta-Iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5 alpha-epoxymorphinan (IOXY), an opioid antagonist that has high affinity for kappa 2 sites, was radioiodinated to maximum specific activity (2200 Ci/mmol) and purified by high pressure liquid chromatography and used to characterize multiple kappa 2 binding sites. The results indicated that [125I]IOXY, like [3H]bremazocine, selectively labels kappa 2 binding sites in rat brain membranes pretreated with BIT and FIT. Using 100 nM [D-Ala2-MePhe4,Gly-ol5]enkephalin to block [125I]IOXY binding to the kappa 2b site, two subtypes of the kappa 2a binding site were resolved, both in the absence and presence of 50 microM 5'-guanylyimidodiphosphate. Viewed collectively, these results provide further evidence for heterogeneity of the kappa opioid receptor, which may provide new targets for drug design, synthesis, and therapeutics.
Animals↗