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Biomedical subjects

R B Rothman

Publications and source records attributed to R B Rothman.

At least 235 records · Page 13Linked to original sources
PubMed1984

Visualization of rat brain receptors for the neuropeptide, substance P.

Biochemical analysis of the binding of [125I]Bolton-Hunter coupled substance P [( 125I]BH-SP) to slide-mounted sections of rat brain demonstrated that [125I]BH-SP labels a binding site with a structure-activity profile characteristic of a substance P receptor. Under optimized preincubation and incubation conditions, the locations of substance P (SP) receptors were visualized by film and emulsion autoradiography. Receptor densities were quantified by computer-assisted densitometry. SP receptors are widely but discretely distributed throughout sensory, limbic and cortical areas of rat brain, though several motor areas also possess SP receptors. No receptors were detected in the substantia nigra and interpeduncular nucleus, which are innervated by SPergic nerves; these regions of the brain may possess a low affinity SP receptor not detectable with this assay. Findings are discussed in the framework of an overall notion of the role of neuropeptides in the biochemistry of emotion.

Animals↗
PubMed1984

Tritiated-6-beta-fluoro-6-desoxy-oxymorphone: a highly selective ligand for the opiate mu receptor whose binding is characterized by low nonspecific binding.

In this paper we examine the binding of [3H]FOXY (tritiated-6-beta-fluoro-6-desoxy-oxymorphone) to membranes of rat brain. Using the site-directed alkylating agents BIT and FIT, evidence is presented that [3H]FOXY selectively labels mu opiate binding sites in vitro. Further, BIT and FIT did not significantly affect [3H]bremazocine binding to kappa receptors. Scatchard plots of [3H]FOXY binding were somewhat curvilinear, suggesting the presence of two classes of mu binding sites. At concentrations up to 19 nM, 90 percent of the total binding was specific. The combination of high mu-selectivity and low nonspecific binding suggests the [3H]FOXY may prove to be a powerful tool for studying the opiate mu receptor.

Animals↗
PubMed1984

Autoradiographic localization of a novel peptide binding site in rat brain using the substance P analog, eledoisin.

Using slide mounted sections of rat brain sausage, we have characterized the binding of [125I]Bolton Hunter conjugated eledoisin and [125I]Bolton Hunter conjugated substance P. Structure activity studies suggest that the two radiolabeled peptides bind to different binding sites. Autoradiographic studies support this notion. Whereas [125I]BH-SP sparsely labels the interpeduncular nucleus and does not label the substantia nigra at all, [125I]BH-ED densely labels the former and sparsely labels the latter structure. Further, the cortical labeling patterns obtained with the two peptides are strikingly different. These data support the hypothesis that there exist two classes of tachykinin binding sites in rat nervous tissue.

Animals↗
PubMed1984

Preparation of rat brain membranes greatly enriched with either type-I-delta or type-II-delta opiate binding sites using site directed alkylating agents: evidence for a two-site allosteric model.

Although it is widely accepted that radiolabeled prototypic delta receptor agonists label two binding sites in vitro, the mechanism by which mu ligands inhibit peptide binding as well as the identity of the binding sites remains unsettled (Rothman and Westfall, Mol. Pharmacol. 21:538-547, 1982 ; Bowen et al., Proc. Natl. Acad. Sci. U.S.A. 78:4818-4822, 1981). Using the site directed, receptor selective alkylating agents, BIT and FIT (Rice et al., Science 220:314-316, 1983), we describe the preparation of membranes devoid of high affinity binding sites and demonstrate that the mu agonist oxymorphone noncompetitively inhibits the binding of [3H]DADL to the residual lower affinity binding sites.

Alkylating Agents↗
PubMed1984

Morphine noncompetitively inhibits [3H]leucine enkephalin binding to membranes lacking type-II delta binding sites: evidence for a two-site allosteric model.

Using the site-directed, delta-selective alkylating agent FIT (Rice et al., Science 220, 314-316, 1983), membranes devoid of detectable higher affinity delta binding sites were prepared. As compared to control membranes, the IC50 of morphine required to inhibit [3H]LE binding to FIT-treated membranes was two orders of magnitude lower. Further, morphine was a noncompetitive inhibitor of [3H]LE binding to FIT-treated membranes, supporting the notion that the lower affinity delta binding site is the delta binding site of the opiate receptor complex.

Allosteric Regulation↗
PubMed1984

Ionic conditions differentially affect 3H-DADL binding to type-I and type-II opiate delta receptors in vitro.

It is widely accepted that the prototypic delta agonist DADL (D-ala2-D-leu5-enkephalin) labels two binding sites in vitro. Using the site directed, receptor selective alkylating agents, BIT and FIT (Rice et al.. Science 220:314-316, 1983), we recently described (Rothman et al, Neuropeptides, in press) the preparation of membranes possessing only lower affinity 3H-DADL binding sites (FIT-treated membranes, type-I delta sites) as well as membranes greatly enriched with higher affinity binding sites (BIT-treated membranes, type-II delta sites). In this paper we report that ionic conditions differentially affect the binding of 3H-DADL to FIT- and BIT-treated membranes, supporting the notion that 3-H-DADL labels two distinct delta binding sites.

Animals↗
PubMed1984

Effect of beta-FNA on opiate delta receptor binding.

beta-FNA, the beta-fumaramate methyl ester of naltrexone, has been shown to antagonize irreversibly the actions of morphine on the guinea pig ileum and mouse vas deferens bioassays but does not affect the actions of delta-receptor ligands on the mouse vas deferens bioassay, suggesting that the compound does not irreversibly bind to the delta receptor. In this paper we examine the effect of beta-FNA on the binding of the prototypic delta agonists, Leu-enkephalin and D-Ala2-D-Leu5-enkephalin, its metabolically stable analogue, and show that treatment of membranes with beta-FNA does lead to alterations in the in vitro properties of delta receptors.

Animals↗
PubMed1983

Multiple opioid receptors: an examination of the dissociation of [3H]leucine enkephalin from rat brain membranes.

The experiments reported in this paper address the hypothesis that [3H]leucine enkephalin labels both mu and delta receptors. As reported by other workers, this peptide dissociates from rat brain membranes in a biphasic manner. This is consistent with a two site binding model which hypothesizes that the peptide labels both opioid mu and delta receptors from which it dissociates at different rates. To test this hypothesis, we determined the dissociation of bound ligand from rat brain membranes incubated to equilibrium with [3H]leucine enkephalin in the absence and presence of 100 nM morphine. The data were not significantly different. We conclude that the biphasic off-kinetics of [3H]leucine enkephalin is not evidence for a two-site binding model.

Animals↗
PubMed1983

Interaction of leucine enkephalin with (3H)naloxone binding in rat brain: evidence for an opioid receptor complex.

We recently presented evidence that distinct morphine and enkephalin receptors coexist in an opioid receptor complex (Mol. Pharmacol. 21:548-557, 1982). In this paper, we present data which demonstrate that in the presence of sodium leucine enkephalin noncompetitively inhibits the binding of [3H]naloxone to a crude particulate fraction of rat brain. Since the binding site labeled by [3H]naloxone in the presence of sodium may be an alternate conformation of the morphine receptor, these data provide further evidence that morphine and enkephalin receptors are allosterically coupled.

Animals↗
PubMed1983

Analysis of binding surfaces: a methodology appropriate for the investigation of complex receptor mechanisms and multiple neurotransmitter receptors.

In this study we describe a novel experimental design: analysis of three-dimensional binding surfaces. Evidence is presented that a binding surface provides greater information content than does a single binding isotherm. This approach to the design and analysis of ligand binding studies should facilitate quantitative studies of the opiate receptor.

Animals↗
PubMed1983

Multidimensional analysis of ligand binding data: application to opioid receptors.

The existence of distinct mu and delta opioid receptors is now well accepted. Most investigators favor the hypothesis that these receptors are physically distinct and that the enkephalins are only 2-10 fold selective for the delta receptor. Rothman and Westfall (Mol. Pharmacol. 21:548-557) recently challenged this hypothesis, proposing that at least some population of mu and delta receptors coexist in an opioid receptor complex and that the enkephalins are at least 100 fold selective for the delta receptor. In this paper we describe a generally applicable method we have used to design and analyze ligand binding experiments which distinguish between the two different models.

Animals↗
PubMed1983

Binding of radiolabeled opiates to slide-mounted sections of molded minced rat brain: a novel method for conducting radioreceptor assays.

To facilitate the quantitative study of the opiate receptor, we have developed a novel variation of the ligand binding technique. The binding of 3H-opiates to rat brain membranes is compared with the binding to slide-mounted sections of molded minced rat brain. The latter method is characterized by a high signal and a superior signal-to-noise ratio over a wide range of ligand concentrations. The results are discussed in reference to other ligand binding methodologies.

Animals↗
PubMed1982

Mu and delta receptors: their role in analgesia in the differential effects of opioid peptides on analgesia.

Utilizing the mouse tail-flick assay, the rank order of analgesic potency for various opioids (i.c.v.) is beta h-endorphin greater than D-Ala2-D-Leu5-enkephalin greater than morphine greater than D-Ala2-met-enkephalinamide much greater than met-enkephalin much greater than leu-enkephalin. Assuming mu receptor mediation of analgesia, there is an affinity and analgesic potency (ie: D-Ala2-Leu5-enkephalin has 1/7 the affinity of morphine for the mu receptor but is 18X more potent as an analgesic). Additionally, sub-analgesic doses of various opioid peptides have opposite effects on analgesic responses. Leu-enkephalin, D-Ala2-D-Leu5-enkephalin or beta h-endorphin potentiate morphine or D-Ala2-met-enkephalinamide analgesia whereas met-enkephalin or D-Ala2-met-enkephalinamide antagonize opioid-induced analgesia. Using the enkephalins as the prototypic delta ligands (100 fold selective) and based on their effects on analgesia, we suggest that Leu-enkephalin-like peptides interact with the delta receptor as an "agonist" to facilitate and met-enkephalin-like peptides as an "antagonist" to attenuate analgesia. Given the biochemical evidence of a coupling between mu and delta receptors, we suggest that the mechanism of facilitation or attenuation of analgesia by the enkephalins is a direct in vivo consequence of this coupling. Further, the analgesic potencies of various opioid ligands can be better correlated to the combination of their simultaneous occupancy of mu and delta receptors.

Animals↗
PubMed1982

Interaction of naloxone with the opioid receptor complex in vitro.

Previous work from this laboratory suggests that distinct morphine and enkephalin receptors coexist in an opioid receptor complex. In this paper the interaction of naloxone with the receptor complex is studied. The results further strengthens the hypothesis that leucine enkephalin binds poorly to the morphine receptor and that morphine and enkephalin receptors are allosterically coupled.

Animals↗
PubMed1982

Allosteric coupling between morphine and enkephalin receptors in vitro.

In a recent paper from our laboratory [Mol. Pharmacol 21:538-547 (1982)] evidence was presented which suggested that [3H]leucine enkephalin labels a single class of binding sites (the enkephalin receptor) and that morphine allosterically induces a masking of enkephalin receptors as a consequence of binding to a receptor (the morphine receptor) not labeled by the 3H-peptide. Evidence is presented in this paper that [3H]etorphine can be used to label selectively the morphine receptor and that the inhibitory dissociation constants (KI) of morphine, etorphine, and human beta-endorphin for the [3H]etorphine binding site closely approximate the concentration of these drugs which produce a half-maximal decrease in the number of enkephalin receptors. Furthermore, an examination of the interaction of leucine enkephalin and methionine enkephalin with the morphine receptor has demonstrated that the pentapeptides are not competitive inhibitors of [3H] etorphine binding, and that they have much lower affinities for the morphine receptor than previously thought. On the basis of these data, a working hypothesis has been formulated which postulates that distinct morphine and enkephalin receptors coexist in an opioid-receptor complex.

Allosteric Site↗
PubMed1982

Morphine allosterically modulates the binding of [3H]leucine enkephalin to a particulate fraction of rat brain.

Equilibrium binding studies have demonstrated that [3H]leucine enkephalin labels a single class of binding sites in a particulate fraction of rat brain with a dissociation constant (KD) of 3.2 +/- 0.1 nM. Methionine enkephalin was a competitive inhibitor of [3H]leucine enkephalin binding, changing the KD to 14.1 +/- 1.5 nM. In contrast, Scatchard analysis of the binding of [3H]leucine enkephalin in the absence and presence of 10, 50, and 100 nM morphine demonstrated that these concentrations of morphine decreased the number of binding sites by 23%, 32%, and 42%, respectively, with no change in the KD. In contrast, morphine at 500 nM caused a 45% decrease in the number of binding sites and an increase in the KD. On the basis of these data, the inhibitory dissociation constant (KI) of morphine was calculated to 400 +/- 17 nM. The noncompetitive inhibition by morphine of [3H]leucine enkephalin binding was shown to be rapidly reversible, ruling out pseudoirreversible binding of morphine to the enkephalin binding site as the underlying mechanism. Computer analysis of the displacement [3H]leucine enkephalin binding by various concentrations of morphine has demonstrated that an allosteric model, not a two-site model, best describes the observed data. We conclude that at low concentration morphine binds to a receptor not labeled by [3H]leucine enkephalin and by doing so allosterically induces an apparent loss of enkephalin receptors.

Allosteric Site↗
PubMed1981

Allosteric modulation by leucine-enkephalin of [3H]naloxone binding in rat brain.

The interaction of morphine and leucine-enkephalin with the binding site labeled by [3H]naloxone in the presence of sodium was compared. The effect of fixed concentrations of morphine and leucine enkephalin on the saturation binding of [3H]naloxone demonstrated that whereas morphine was a competitive inhibitor, leucine enkephalin caused a dose-dependent masking of binding sites. From these data we conclude that the enkephalin receptor is allosterically coupled to the morphine receptor.

Allosteric Site↗