Robert J. McHale

β‐Endorphin Is a Potent Inhibitor of Thymidine Incorporation into DNA via μ and κ‐Opioid Receptors in Fetal Rat Brain Cell Aggregates in Culture

Abstract: Thymidine incorporation into DNA was inhibited dose‐dependently by β‐endorphin in rat fetal brain cell aggregate cultures. The inhibition was reversed partially by μ (cyclic D‐Phe‐Cys‐Tyr‐D‐Trp‐Orn‐Thr‐Pen‐Thr amide) or k (norbinaltorphimine) antagonists. Complete blockade of the β‐endorphin inhibitory effect was achieved only on concomitant exposure to both antagonists. Eadie–Hofstee analysis revealed that β‐endorphin inhibited thymidine incorporation noncompetitively. In the presence of protease inhibitors, β‐endorphin decreased thymidine incorporation with an IC50 of 0.7 nM. Truncated and N‐acetylated β‐endorphin derivatives, which bind with low affinity to opioid receptors, did not affect thymidine incorporation. These findings indicate that β‐endorphin at physiological concentrations can regulate thymidine incorporation in cultured brain cells.

Dynorphins Modulate DNA Synthesis in Fetal Brain Cell Aggregates

Abstract: Previously, opioid peptide analogues, β‐endorphin, and synthetic opiates were found to inhibit DNA synthesis in 7‐day fetal rat brain cell aggregates via κ‐and μ‐opioid receptors. Here dynorphins and other endogenous opioid peptides were investigated for their effect on DNA synthesis in rat and guinea pig brain cell aggregates. At 1 µM, all dynorphins tested and β‐endorphin inhibited [3H]thymidine incorporation into DNA by 20–38% in 7‐day rat brain cell aggregates. The putative ε‐antagonist β‐endorphin (1–27) did not prevent the effect of β‐endorphin, suggesting that the ε‐receptor is not involved in opioid inhibition of DNA synthesis. The κ‐selective antagonist norbinaltorphimine blocked dynorphin A or B inhibition of DNA synthesis, implicating a κ‐opioid receptor. In dose‐dependency studies, dynorphin B was three orders of magnitude more potent than dynorphin A in the attenuation of thymidine incorporation, indicative of the mediation of its action by a discrete κ‐receptor subtype. The IC50 value of 0.1 nM estimated for dynorphin B is in the physiological range for dynorphins in developing brain. In guinea pig brain cell aggregates, the κ‐receptor agonists U50488, U69593, and dynorphin B reduced thymidine incorporation by 40%. When 21‐day aggregates were treated with dynorphins, a 33–86% enhancement of thymidine incorporation was observed. Because both 7‐ and 21‐day aggregates correspond to stages in development when glial cell proliferation is prevalent and glia preferentially express κ‐receptors in rat brain, these findings support the hypothesis that dynorphins modulate glial DNA synthesis during brain ontogeny.

A monoclonal anti-idiotypic antibody to μ and δ opioid receptors

A mouse monoclonal, anti-idiotypic, anti-opioid receptor antibody (Ab2-AOR) has been generated from monoclonal anti-morphine antibodies (Ab1). Hybridoma culture supernatants were screened by a solid phase radioimmunoassay (RIA), based on their competition with radiolabelled morphine for Ab1. One of the Ab2s that gave a positive RIA also competed at rat brain opioid receptors with tritiated opioid ligands dihydromorphine (DHM), naloxone, etorphine, Tyr-D-Ala-Gly-Phe-D-Leu (DADLE), Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAMGE) and Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE). SDS-PAGE revealed Ab2-AOR to be highly purified after successive affinity and protein A-Sepharose chromatography. Ab2-AOR at concentrations of 10-100 nM competed with both mu- and delta-selective specific ligands for brain opioid receptors. Less than 13 micrograms/ml Ab2-AOR completely inhibited specific opioid radioligand binding to both soluble and membrane-bound opioid receptors. To demonstrate its anti-delta receptor activity further, a double-antibody ELISA procedure was developed that is based on the binding of Ab2-AOR to immobilized NG 108-15 cells (which contain only delta opioid receptors). Dose-dependent, opioid peptide- and opiate alkaloid-competitive binding of Ab2-AOR-containing ascites fluid to NG 108-15 cells was observed. A mu opioid agonist effect was demonstrated for Ab2-AOR, in that it decreased by 70% [3H]thymidine incorporation into DNA of fetal brain cell aggregates. This agonist-like action of Ab2-AOR was blocked by naltrexone. The antibody bound specifically to brain tissue sections and the presence of diprenorphine blocked this interaction. Hence, an Ab2 with mu and delta specificity has been characterized.

In Vitro and In Vivo Expression of Opioid and σ Receptors in Rat C6 Glioma and Mouse N18TG2 Neuroblastoma Cells

Abstract: Mouse N18TG2 neuroblastoma and rat C6 glioma cell lines were injected into male nude mice, and the tumors were passaged serially. At each generation, tumors were analyzed for δ opioid binding using [3H][d‐Ala2,d‐Leu5]enkephalin and for σ1 and σ2 binding with 1,3‐[3H]di‐o‐tolylguanidine in the presence and absence of 1 µM pentazocine. Receptor density (Bmax) and affinity (KD) were estimated by homologous competition binding assays. Opioid and σBmax values in the solid tumors were significantly lower than their original levels in vitro. KD values for opioid/σ ligands were similar in vitro and in vivo. With successive passages in the murine host, δ opioid and σ1 binding of the neuroblastoma‐derived solid tumors became undetectable. In contrast, σ2 receptor Bmax values were unchanged with successive passages of the neuroblastoma‐derived tumors and doubled in the nude mouse‐borne gliomas. When neuroblastoma‐derived solid tumors that were devoid of δ opioid binding were returned to culture, opioid receptors appeared to be up‐regulated as compared with their original in vitro levels. Serial passaging of these recultured cells in vivo again resulted in a rapid decline in opioid receptor content. The opioid data are consistent with our prior findings on opioid binding diminution in human brain tumors. The pattern of change for σ binding was more complex, with the σ2 response in late passages of the glioma being reminiscent of the formerly observed increase in number of σ sites in transformed human meninges, kidney, and colon tissue.

Naltrexone induces down- and upregulation of δ opioid receptors in rat brain regions

Opioid antagonists such as naltrexone, naloxone, and ICI174864 induce a transient downregulation of delta opioid receptors prior to upregulation in NG108-15 cells. Here we show that naltrexone can also elicit a transient downregulation of delta 2 opioid receptors preceding upregulation in brain. A 1 h treatment of rats with naltrexone (IP, 10 mg/kg) resulted in lowered 3H-[D-Ser2,L-Leu5]enkephalyl-Thr Bmax values in hindbrain, but not in striatum, hippocampus, or cortex. The decrease in hindbrain delta 2 receptor density was not accompanied by changes in Kd values, indicating that downregulation rather than receptor blockade occurred. Longer naltrexone treatment (48 h), caused twofold upregulation of delta opioid binding in all four regions. These data suggest that the process of upregulation of delta opioid receptors by antagonists in vivo can entail an initial, transient downregulation.

Dynorphin B inhibits thymidine incorporation into DNA in fetal rat and guinea pig brain cell aggregates

Abstract Numerous studies suggest that opioids have a modulatory role in the regulation of neural cell proliferation (1). We have found that μ opioid receptor agonists inhibit thymidine incorporation into DNA of neural cells in rat fetal brain cell aggregates (2). This inhibition is culture age-dependent. Moreover, morphine inhibited glial cell proliferation in a concentration dependent manner (3). In contrast, U50488, a κ opioid receptor agonist, exerted either an inhibitory or stimulatory action on thymidine incorporation depending on the culture duration (4). Here we addressed the question of the effect of the endogenous κ-selective opioid peptide, Dynorphin B, on thymidine incorporation in rat or guinea pig aggregates expressing more than 50% of μ (rat) or κ (guinea pig) receptors. In both cultures Dynorphin B inhibited DNA synthesis by 30–40%.

Nuclear matrix associated opioid binding sites are increased upon D-Ala2-D-Leu5-enkephalin-induced down-regulation in NG108-15 cells

Abstract Nuclear matrix preparations obtained from purified nuclei of NG108-15 cells, display high affinity opioid antagonist binding (1). Under conditions of opioid agonist-induced desensitization of cell surface receptors, nuclear matrix associated opioid binding was abolished, but expectedly binding densities in P 20 (membranes sedimenting at 20,000 g) were unaffected. Here, binding changes were monitored in P 20 and nuclear matrix fractions during agonist-induced down-regulation. D-Ala 2 -D-Leu 5 -enkephalin (DADLE, 0.1 μM, 1 h) treatment of NG108-15 cells caused an increase in nuclear matrix-associated binding, while diminishing that in P 20 . Taken together with other findings, these results suggest that a subpopulation of opioid binding sites, originally in the plasma membrane, are internalized and migrate to the nuclear matrix.