Horace H. Loh

Pertussis toxin treatment modifies opiate action in the rat brain striatum

In this report we present evidence that a guanine nucleotide regulatory protein, Gi, mediates opiate action in the rat brain striatum. Opiates inhibit basal adenylate cyclase activity in rat brain striatum. This effect on adenylate cyclase is dose-dependently attenuated by pretreatment of membranes with pertussis toxin, which ADP-ribosylates a protein with a molecular mass of 41,000 daltons. This protein co-migrates with the GTP-binding subunit of Gi, which mediates inhibition of adenylate cyclase. Several brain regions were compared for the extent of radiolabeling and effects on adenylate cyclase activity. Although Gi was found in each region examined, opiate inhibition of adenylate cyclase is clearly seen only in the striatum.

Decrease in μ-opioid receptor binding capacity in rat brain after chronic PL017 treatment

In previous studies, we have demonstrated that chronic treatment of rats with either etorphine or D-Ala2, D-Leu5-enkephalin (DADLE) resulted in the reduction of opioid receptor binding activities during the course of tolerance development. In both cases, mu-opioid receptor binding capacity was attenuated together with the delta-opioid receptor binding capacity. Because both etorphine and DADLE are relatively non-specific opioid ligands, interacting with both mu and delta receptors, these studies could not determine whether down-regulation of a specific receptor type is possible. Therefore, in the current studies, animals were rendered tolerant to the mu-opioid receptor-selective ligand PL017 and the receptor binding capacity was measured afterwards. Treating Sprague-Dawley rats with increasing doses of PL017 (2.5-20 micrograms/kg) i.c.v. for 5 days resulted in a 30- to 40-fold increase in the AD50 of the peptide to elicit the antinociceptive response and about 14-fold increase in the ED50 of the peptide to elicit the catatonic effect. When mu- and delta-binding was determined using [3H]diprenorphine in the presence of morphiceptin or DPDPE respectively, a significant decrease (20-30%) in the mu-opioid receptor binding but not in delta-opioid receptor binding was observed in all the brain areas tested after 5 days of PL017 treatment. Scatchard analysis of the [3H]DAMGO saturation binding data revealed a decrease in Bmax values and no change in the Kd values. Hence, mu-opioid receptors can be specifically regulated by ligand in the brain as delta-receptors are in neuroblastoma x glioma NG 108-15 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid requirement for μ opioid receptor binding

Abstract: We have previously shown that a partially purified μ opioid receptor from bovine brain requires lipids to exhibit full binding activity. In the present report, we have determined the specificity of this lipid requirement. Lipids active in this regard were found always to contain an acidic head group and a fatty acid with two or more double bonds. Free, polyunsaturated fatty acids were also able to confer high binding activity on the partially purified opioid receptor. The possible roles lipids play in opioid binding are discussed in light of these data.

Involvement of both inhibitory and stimulatory guanine nucleotide binding proteins in the expression of chronic opiate regulation of adenylate cyclase activity in NG108-15 cells

Abstract: Chronic etorphine treatment of neuroblastoma × glioma NG108‐15 cells results in both an increase in adenylate cyclase activity (upon addition of the opiate antagonist naloxone) as well as an homologous desensitization of the opiate receptor. The continued ability of opiate agonists to regulate adenylate cyclase activity following opiate receptor desensitization can be understood by proposing that the catalytic subunit of adenylate cyclase in NG108‐15 cells is under tonic regulation by both guanine nucleotide regulatory (Ni) and stimulatory (Ns) components. Inactivation of Ni by pertussis toxin (PT) treatment resulted in elevated adenylate cyclase activities comparable to those observed in control cells following chronic opiate treatment. This increased enzymatic activity could not be further induced by PT treatment of cells exposed to opiate previously. In addition, procedures that prevented receptor‐mediated activation of Ns, i.e., treatment with NaF or desensitization of the stimulatory receptors (prostaglandin E1, adenosine) eliminated the increase in adenylate cyclase activity induced by naloxone following chronic opiate exposure. Hence, the increase in enzymatic activity observed following chronic opiate treatment may be due to a loss in tonic inhibitory regulation of adenylate cyclase mediated through Ni resulting in the unimpeded expression of Ns activity. This tonic inhibition of adenylate cyclase activity is one of the multiple mechanisms by which Ni regulates adenylate cyclase in this cell line.

Effect of dynorphin-(1–13) and related peptides on respiratory rate and morphine-induced respiratory rate depression

Previous studies from our laboratory have shown that the opioid peptide dynorphin-(1-13), although not analgesic when given by itself, can inhibit morphine-induced analgesia in naive mice and potentiate it in morphine tolerant mice. In the present study, we examined the effect of dynorphin-(1-13) with two other dynorphin-like peptides, alpha-neoendorphin and dynorphin-(1-10) amide, on respiration. Our results show that none of the peptides studied had any significant activity on the respiratory rate in mice when given alone. However, in the presence of morphine, dynorphin-(1-13) antagonized the morphine-induced respiratory rate depression in morphine-tolerant animals; alpha-neoendorphin enhanced the morphine-induced respiratory rate depression in naive but had no effect in morphine-tolerant animals and dynorphin-(1-10) amide had no modulatory effect on the morphine-induced respiratory rate depression in either group of animals.

Specific opioid-amphetamine interactions in the caudate putamen

Bilateral microinjection of morphine (0.003–3 μg/side) into the caudate putamen enhances the behavior induced by the IP injection of 1 mg/kg d-amphetamine phosphate in a dose-related manner. The duration of activity was prolonged and ambulation was changed to d-amphetamine stereotypy, a behavior normally associated with higher doses of d-amphetamine. The opioid activity was stereospecific in that levorphanol was active, whereas dextrorphan was not. The enhancement of d-amphetamine-induced behavior by the opioids was blocked by naloxone. d-ala2-met-Enkephalin also enhanced the amphetamine-induced behavior. This enhancement appears to be specific to the caudate putamen because the oral stereotypy observed appears to be a unique action of amphetamine in this region of the brain.

A monoclonal antibody that inhibits opioid binding to rat brain membranes

To understand the structure-function relationship and to probe the molecular characteristics of the purified opioid receptor, monoclonal antibodies (mab) were raised against a purified opioid receptor protein. After intensive screening of almost 1500 hybridoma cell lines, only 7 clones were shown to have very high immunoreactivity against the purified receptor. Moreover, out of these 7 clones, only 2, 3B4F11 and 3A27G, were found to inhibit the ligand binding property of the mu-opioid receptor. The mab 3B4F11 was found to inhibit 3H-diprenorphine binding to the purified receptor in a dose dependent manner with a maximal inhibition of 100% achieved with 20 micrograms of the antibody. Likewise, Fab fragments prepared from the mabs 3B4F11 inhibited 3H-diprenorphine binding to P2 membranes in a dose-dependent manner. In addition, it was found that the binding of 3H-DAGO, 3H-DPDPE and 3H-EKC was inhibited with approximately equal potency, suggesting that the Fabs prepared from the mab 3B4F11 interact with all 3 receptor types.

Different molecular weight forms of opioid receptors revealed by polyclonal antibodies

Polyclonal antibodies were raised against a purified opioid receptor from bovine brain (Cho, et. al., 1986), and shown to inhibit 3H-diprenorphine binding to this receptor in a dose-dependent fashion. These antibodies were then used to characterize opioid-binding material present in rat brain and in NG108-15 neuroblastoma-glioma hybrid cells. Western blot analysis revealed that the antibodies reacted with a single species of 58,000 molecular weight in rat brain membranes; this closely corresponds in size to the bovine opioid receptor used to raise the antibodies. In contrast, the polyclonal antibodies reacted with a 45,000 molecular weight species in NG108-15 neuroblastoma-glioma hybrid cells; moreover, this band was specifically reduced in NG108-15 cells in which opioid receptors had been down-regulated by incubation with D-ala2-D-leu5-enkephalin for 24 hours. Thus at least two distinct opioid receptor molecules have been identified, which have antigenic similarities.

Modification of opioid agonist binding by pertussis toxin

Membrane fractions prepared from rat striate, cortex and midbrain were treated with pertussis toxin, which has been shown to adenosine diphosphate (ADP)-ribosylate the GTP-binding protein Gi, reducing its coupling with receptors. In striatal membranes, treatment with 40 micrograms toxin per mg membrane protein labeled 60% of the Gi present and 70% of another G protein, Go; this treatments reduced binding of the opioid agonist [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE) 20-50%, with the decrease largely reflecting a decreased affinity. In cortex, toxin treatment reduced [3H]DADLE binding by 35-70%, corresponding to ADP-ribosylation of 50% of Gi and 40% of Go. In midbrain, [3H]DADLE binding was unaffected by toxin treatment that ADP-ribosylated 86% of the Gi and 72% of the Go. These results provide further evidence that opioid receptors are associated with GTP-binding proteins in striatum and cortex, where they have also been shown to inhibit adenylate cyclase. Despite the presence of Gi and Go in midbrain, however, there appears to be no coupling between them and opioid receptors.

Cell-Free Desensitization of Opioid Inhibition of Adenylate Cyclase in Neuroblastoma × Glioma NG108–15 Hybrid Cell Membranes

Abstract: When membranes from neuroblastoma |MX glioma NG108–15 hybrid cells were incubated in a cell‐free system with opioid agonists, a time‐, temperature‐, and dose‐dependent desensitization to opioid inhibition of adenylate cyclase activity was observed. The composition of the system during the incubation was manipulated to elucidate the biochemical mechanisms of desensitization. Receptor coupling appeared to be a prerequisite for desensitization, because both magnesium and sodium, which are necessary for coupling, were required for desensitization. Removal of ATP and addition of cyclic AMP or cyclic GMP had no effect on desensitization.