Brian R. DeCosta

Evidence for a δ-class opioid receptor on cells of the immune system

Abstract Opioid receptors have been reported to be present on cells of the immune system. Both opioid receptor-ligand interactions which are reversible by naloxone (classical) and those interactions which are irreversible by naloxone (nonclassical) appear to alter various immunologic parameters through the ligand-receptor interactions. In an effort to characterize opioid receptors (which operate with classical interactions) on cells of the immune system, we employed the δ-class opioid receptor-specific ligand, cis -(+)-3-methylfentanylisothiocyanate (SUPERFIT). We herein report the specific labeling of a protein with a molecular weight of 70,000 Da under reducing conditions (58,000 Da under nonreducing conditions) from both B and T cell-enriched murine splenocytes and peripheral blood lymphocytes. Similar results are demonstrated by human peripheral blood lymphocytes and plastic-adherent (monocyte-enriched) leukocytes.

Corticotropin-releasing hormone augments natural killer cell activity through a naloxone-sensitive pathway

Overnight treatment of murine leukocytes with corticotropin-releasing hormone (CRH) and arginine vasopressin enhances natural killer cell activity. Moreover, the opioid receptor antagonist, naloxone, as well as the delta-class opioid receptor antagonist, naltrindole, can block this effect. The responsivity of murine leukocytes to CRH is both dose- and time-dependent. The effector cells are both MAC-1 and Thy-1.2 antigen-positive. Whereas beta-endorphin is also shown to enhance natural killer cell activity in a naloxone-reversible manner, adrenocorticotropic hormone (ACTH) has a negligible effect. Macrophage depletion prior to incubation with CRH blocks the CRH-induced natural killer cell augmentation. These results suggest hypothalamic-releasing hormones such as CRH may have a biologically relevant role in the modulation of immune cells either directly or indirectly through the induction of neuropeptide hormones known to have immunomodulatory capabilities.

Autoradiographic distribution of [3H](+)-pentazocine and [3H]1,3-di-o-tolylguanidine (DTG) binding sites in guinea pig brain: a comparative study

Binding studies suggested the selectivity of (+)-pentazocine for sigma receptors, and subsequent synthesis and testing of [3H](+)-pentazocine confirmed its high potency and selectivity for sigma sites. Newer data have demonstrated the selectivity of (+)-pentazocine for a subtype of the sigma receptor called sigma-1. Based on these findings, the distribution of [3H](+)-pentazocine binding sites in the guinea pig brain was examined using in vitro autoradiography. [3H](+)-Pentazocine binding was high in the cingulate cortex, dorsal diagonal band, periaqueductal gray, cerebellum and cranial nerve nuclei. It was relatively low in the nucleus accumbens, neocortical areas and caudate nucleus. A significant correlation was found between the binding of [3H](+)-pentazocine and [3H]1,3-di-o-tolylguanidine, a selective sigma ligand across brain regions. However, certain nuclei exhibited markedly different ratios of binding of the two ligands. Since DTG is not selective for the sigma subtypes, while (+)-pentazocine is selective for the sigma-1 type, the data are suggestive of relative differences in the distributions of sigma-1 and sigma-2 sites.

Differential effect of opioids on immunoglobulin production by lymphocytes isolated from Peyer's patches and Spleen

The mucosal immune system plays an important role in blocking the penetration of invasive organisms into various mucosal surfaces. Evidence now suggests neuroendocrine peptide hormones have immunomodulatory properties, including the ability to alter mucosal immunity. The potential for opioid compounds and corticotropic hormone (ACTH) to modulate mucosal immune function was investigated. We have found beta-endorphin, ACTH, and naltrindole (delta-class opioid receptor antagonist) to significantly suppress concanavalin A-stimulated Peyers patch lymphocyte immunoglobulin production of IgA, IgG, and IgM isotypes. Oxymorphindole, a delta class opioid receptor agonist, significantly decreased IgM but not IgA or IgG production by the mitogen-stimulated Peyers patch lymphocytes. Both oxymorphindole and naltrindole modestly reduced interleukin-2 receptor expression of concanavalin A- (Con A)-stimulated splenic and Peyers patch lymphocytes. Neither compound appreciably affected immunoglobulin production by lipopolysaccharide-stimulated Peyers patch lymphocytes. Collectively, these results indicate stress-related peptides such as ACTH and opioids may be involved in the regulation of immunoglobulin synthesis by Peyers patch lymphocytes.

Enantioselective kappa opioid binding sites on the macrophage cell line, P388d1

A kappa (kappa) opioid binding site has been characterized on the macrophage cell line, P388d1, using the kappa selective affinity ligand, [3H] (1S,2S)-(-)-trans-2-isothiocyanato-N-methyl-N-[2-(1- pyrrolidinyl) cyclohexyl] benzeneacetamide (-)BD166). The kappa site has a relative molecular mass (Mr) of 38,000 under nonreducing conditions and 42,000 under reducing conditions. Moreover, it exhibits enantioselectivity in that 1S,2S-(-)-trans-3,4-dichloro-N-methyl-N-[2-1-pyrrolidinyl)cyclohexyl] benzeneacetamide ((-)-U-50,488) blocks [3H](5 alpha, 7 alpha, 8 beta)-(-)-N-methyl-N-[7-(1- pyrrolidinyl)-1-oxaspiro-(4,5)-dec-8-yl]benzeneacetamide (U-69,593) binding to P388d1 cells with an IC50 = 7.0 nM whereas 1R,2R-(+)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide ((+)U-50,488) blocks [3H]U-69,593 binding to P388d1 cells with an IC50 = 7000 nM.

The in vitro pharmacological characterization of naloxone benzoylhydrazone

On the basis of its in vivo activity and binding affinity, naloxone benzoylhydrazone has been characterized as a kappa 3-opioid receptor agonist and a mu-opioid receptor antagonist. This paper continues its pharmacological characterization with the help of isolated tissue preparations. Naloxone benzoylhydrazone was found to have partial agonist activity in the guinea pig ileum longitudinal muscle/myenteric plexus preparation. As an antagonist, naloxone benzoylhydrazone is similar to naloxone, with pA2 values of 8.8, 7.8, and 7.8 for mu-, delta-, and kappa 1-opioid receptors, respectively. Its agonist activity in the guinea pig ileum preparation was not influenced by beta-funaltrexamine treatment but was reversed by the selective kappa-opioid receptor antagonist nor-binaltorphimine and by the irreversible kappa 1-opioid receptor blocker UPHIT (1S,2S)-trans-2-isothiocyanato-4,5-dichloro-N-methyl-N-[2-(1- pyrrolidinyl)-cyclohexyl] benzeneacetamide. The presence of kappa 3-opioid receptors could not be demonstrated by [3H]naloxone benzoylhydrazone binding in the guinea pig ileum longitudinal muscle/myenteric plexus preparation. From these studies it is concluded that the partial agonist activity of naloxone benzoylhydrazone in this bioassay is probably due to the activation of the kappa 1-opioid receptors.

Immunoaffinity-purified opiate receptor specifically binds the δ-class opiate receptor ligand, cis-(+)-3-methylfentanylisothiocyanate, SUPERFIT

Using an antibody generated against the opiate receptor on NG108‐15 cells, we recently purified the putative receptor from this hybrid cell line. We herein report that the purified receptor complex specifically binds tritiated cis‐(+)‐3‐methylfentanylisothiocyanate (SUPERFIT), with the predominant binding associated with a 58 kDa polypeptide chain. Consistent with these findings is the in situ labeling of a 58 kDa protein with [3H]SUPERFIT on NG108‐15 cells.

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

The enantiomers of U50,488 (1, in fig. 1), ligands highly selective for ϰ‐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 ϰ‐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.

Anti-Opioid Receptor Antibody Recognition of a Binding Site on Brain and Leukocyte Opioid Receptors

Opioid receptors reportedly exist on neuronal tissue of central and peripheral origin as well as on cells of the immune system. Previously, an opioid receptor has been purified from the neuroblastoma x glioma hybrid cell line, NG108-15 cells. In an effort to compare these results with opioid receptors isolated from primary neuronal tissue, we employed a methodology based on the molecular recognition theory to develop a monoclonal antibody which was used to isolate and biochemically characterize murine brain opioid receptors. We herein report the purification of an opioid receptor from mouse brain with a molecular weight of 65,000 daltons (range was 62-70 kD under reducing conditions) using a monoclonal antibody to an (the) opioid receptor. In situ labeling experiments with the delta-class selective opioid receptor affinity ligand, cis-(+)-3-methylfentanylisothiocyanate (SUPERFIT) of brain membrane confirmed these observations. Moreover, SUPERFIT, when coupled to the binding site, could block the recognition of the receptor by the monoclonal antibody. However, the selective, mu-class opioid receptor affinity reagent, 2-(p-ethoxybenzyl)-1-N,N-diethylaminoethyl-5-isothiocyanatobenz imidazole was ineffective at masking the binding site from recognition by the monoclonal antibody. Likewise, opioid-like receptors were purified from murine leukocytes which migrated at a molecular weight of 58,000 daltons under nonreducing conditions and 70,000 daltons under reducing conditions. In addition, immunoaffinity-purified receptor is shown to specifically bind the delta-class-selective opioid ligand, cis-(+)-3-methylfentanylisothiocyanate as well as the endogenous opioid peptides, beta-endorphin and [Met]-enkephalin.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple [3H]DTG binding sites in guinea pig cerebellum: evidence for the presence of non-specific binding

The characteristics of the low affinity component of 1,3-di(2-[5-3H]tolyl)guanidine binding to the guinea pig cerebellum were investigated. Saturation binding assays where sigma 1 receptors were masked with dextrallorphan indicated that 1,3-di(2-[5-3H]tolyl)guanidine bound to cerebellar membranes in a fashion best described by a 1 site+non-specific binding model with a low density of specific binding sites (Bmax approximately 200 fmol/mg protein). Boiling the cerebellar membranes before addition to the saturation assay had no effect on the density of 1,3-di(2-[5-3H]tolyl)guanidine binding. In contrast, both the Kd and Bmax for 1,3-di(2-[5-3H]tolyl)guanidine binding to liver membranes was significantly reduced by boiling, as was the density of [3H](+)-pentazocine binding to cerebellum and liver. Thus, a substantial component of 1,3-di(2-[5-3H]tolyl)guanidine binding in the guinea pig cerebellum is to non-specific, proteinaceous binding sites with some of the pharmacological characteristics of the sigma 2 binding site.