Janine A. Danks

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.

Morphine tolerance increases μ-noncompetative δ binding sites☆

Abstract 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 [ 3 H][D-Ala 2 ,D-Leu 5 ]enkephalin ([ 3 H]DADL) was used to label brain membranes. Utilizing the technique of binding surface analysis, we observed a selective 47% up-regulation of lower affinity [ 3 H]DADL binding sites (μ-noncompetitive δ 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 [ 3 H]DADL binding site. Scatchard plots of [ 3 H]DADL binding to FIT-treated membranes also revealed that chronic morphine treatment produced a 60–65% up-regulation of the μ-noncompetitive δ 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.

Chronic administration of morphine and naltrexone up-regulate[3H][d-ala2,d-leu5]enkephalin binding sites by different mechanisms

Previous studies have demonstrated that chronic administration of morphine up-regulated the lower affinity binding site for [3H][D-ala2,D-leu5]enkephalin, without producing a detectable alteration in the higher affinity binding site for [3H][D-ala2,D-leu5]enkephalin (Rothman et al., Eur. J. Pharmac. 124: 113-119, 1986). The experiments reported in this paper tested the hypothesis that chronic administration of morphine and naltrexone up-regulated the binding sites for [3H][D-ala2,D-leu5]enkephalin by different mechanisms. Rats were given either morphine or naltrexone chronically. Chronic administration of morphine up-regulated the lower affinity site, while chronic administration of naltrexone up-regulated both the higher and lower affinity binding sites for [3H][D-ala2,D-leu5]enkephalin. Unlike the lower affinity binding site for [3H][D-ala2,D-leu5]enkephalin present in membranes prepared from rats treated with placebo pellets, the lower affinity binding sites which were up-regulated by naltrexone and morphine were partially (naltrexone) or completely (morphine) labile to preincubation for 60 min at 25 degrees C in 50 mM Tris-HCl, pH 7.4, containing 0.4 M NaCl. These data suggest that chronic administration of morphine and naltrexone up-regulate binding sites for [3H][D-ala2,D-leu5]enkephalin through different mechanisms, and that the lower affinity binding sites for [3H][D-ala2, D-leu5]enkephalin which are up-regulated by chronic administration of morphine and naltrexone might differ biochemically from the lower affinity binding sites present in membranes treated with placebo.

S-[18F]Acetylcyclofoxy: a useful probe for the visualization of opiate receptors in living animals

Opiate receptor PET 3‐[18F]Acetylcyclofoxy Stereospecificity Naloxone

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.

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.

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.

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.

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.

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.