Jean M. Bidlack

Differential Targeting of Gßγ-Subunit Signaling with Small Molecules

G protein βγ subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gβγ subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gβγ subunit functions. Several compounds bound to Gβγ subunits with affinities from 0.1 to 60 μM and selectively modulated functional Gβγ-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor–dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein–coupled receptor signaling that may represent an important therapeutic strategy.

Evidence for opioid receptors on cells involved in host defense and the immune system

Although the role of opiates and opioids in the physiological and pathological function of the immune system is only beginning to be unraveled, converging lines of evidence indicate that the opioid receptors expressed by immune cells are often the same or similar to the neuronal subtypes, particularly delta and kappa. Recent studies also point to the existence of novel opioid receptors and/or binding sites on immune cells that are selective for morphine. Opioids and their receptors, particularly those with high affinity for delta agonists, appear to function in an autocrine/paracrine manner. Thus, opioid peptides generated from immune-derived proenkephalin A act as cytokines, capable of regulating myriad functions of both granulocytes and mononuclear cells. Further identification and characterization of receptors and signal transduction pathways that account for some of the unique properties of opiate binding and immunomodulation (e.g., dose-dependent effects of morphine that occur at exceptionally low concentrations relative to the Kds of the neuronal mu receptor or the morphine binding site reported on activated human T-cells) represents one of the major research challenges ahead. Elucidating mechanisms, such as these, may provide unique therapeutic opportunities through the application of opioid immunopharmacology to disorders involving immune responses in peripheral organs and the central nervous system.

Detection and Function of Opioid Receptors on Cells from the Immune System

Opioid alkaloids and peptides, such as morphine and the endogenous opioid peptides, including β-endorphin and the dynorphin peptides, modulate the function of lymphocytes and other cells involved in host defense and immunity. In recent years, investigations from several laboratories have indicated

Nalmefene Induced Elevation in Serum Prolactin in Normal Human Volunteers: Partial Kappa Opioid Agonist Activity?

In humans, mu- and kappa-opioid receptor agonists lower tuberoinfundibular dopamine, which tonically inhibits prolactin release. Serum prolactin is, therefore, a useful biomarker for tuberoinfundibular dopamine. The current study evaluated the unexpected finding that the relative mu- and kappa-opioid receptor selective antagonist nalmefene increases serum prolactin, indicating possible kappa-opioid receptor agonist activity. In all, 33 healthy human volunteers (14 female) with no history of psychiatric or substance use disorders received placebo, nalmefene 3 mg, and nalmefene 10 mg in a double-blind manner. Drugs were administered between 0900 and 1000 on separate days via 2-min intravenous infusion. Serial blood specimens were analyzed for serum levels of prolactin. Additional in vitro studies of nalmefene binding to cloned human kappa-opioid receptors transfected into Chinese hamster ovary cells were performed. Compared to placebo, both doses of nalmefene caused significant elevations in serum prolactin (p<0.002 for nalmefene 3 mg and p<0.0005 for nalmefene 10 mg). There was no difference in prolactin response between the 3 and 10 mg doses. Binding assays confirmed nalmefenes affinity at kappa-opioid receptors and antagonism of mu-opioid receptors. [35S]GTPγS binding studies demonstrated that nalmefene is a full antagonist at mu-opioid receptors and has partial agonist properties at kappa-opioid receptors. Elevations in serum prolactin following nalmefene are consistent with this partial agonist effect at kappa-opioid receptors. As kappa-opioid receptor activation can lower dopamine in brain regions important to the persistence of alcohol and cocaine dependence, the partial kappa agonist effect of nalmefene may enhance its therapeutic efficacy in selected addictive diseases.

Sites and mechanisms for nicotine's action in the brain

A variety of pharmacologic, behavioral, and receptor-binding studies were performed in an effort to determine the mechanism and site of action of nicotine on the rat brain. When nicotine was given into the lateral or fourth ventricles or directly into the lateral vestibular nuclei of rats, it produced a characteristic prostration often accompanied by tonic seizures and body rotation along a longitudinal axis. Of a variety of brain areas studied, the prostration response could only be elicited from the lateral and, to a lesser extent, medial vestibular nuclei. The response could not be produced by a variety of cholinergic agonists or antagonized with nicotinic cholinergic antagonists, with the possible exception of mecamylamine. A good correlation was observed between the ability of nicotine analogues to antagonize the nicotine-induced prostration and their ability to compete with 3H-nicotine binding to rat brain membranes. 3H-nicotine binding had a high affinity, was stereoselective and concentrated in nerve endings and such brain regions as the thalamus, cerebrum, and hippocampus. When nicotine was administered intraventricularly to rats, it significantly elevated the threshold to an aversive shock. It was concluded that many of the central actions of nicotine could not be explained on the basis of traditional nicotinic cholinergic mechanisms.

Stereospecific 3H-nicotine binding to intact and solubilized rat brain membranes and evidence for its noncholinergic nature

3H-nicotine binding was performed on intact and solubilized rat brain membranes as well as membranes from the electric organ of the Torpedo fish. The Kd for binding to intact and solubilized rat brain membranes was 5.6 × 10−9 M and 1.1 × 10−8M respectively, and the binding capacity 2.0 × 10−14 and 3.0 × 10−13 moles /mg protein respectively. The Kd for Torpedo membranes was 3.1 × 10−7M and the binding capacity 6.8 × 10−13 moles/mg protein. The binding was stereospecific with the affinity of the (−)-nicotine being about 8 times greater than the (+)-nicotine with all three preparations. The relative affinity for the nicotine binding site of nicotinic cholinergic drugs was considerably less in rat brain than in Torpedo membranes, where the sites are mainly cholinergic. A comparison was made of the ability of a variety of cholinergic drugs and nicotine derivatives to compete with 3H-nicotine binding and their relative pharmacologic potency to produce or inhibit a characteristic prostration syndrome caused by (−)-nicotine administered intraventricularly to rats. From such studies it was concluded that nicotine, in part, may be interacting at noncholinergic sites in rat brain.

κ-Opioid binding sites on a murine lymphoma cell line

Abstract As a first step in determining whether any subset of lymphocytes expresses opioid receptors, membranes prepared from mouse lymphoma cell lines were screened for [3H]naloxone binding sites. Membranes from the R1.1 cell line specifically bound [3H]naloxone. The Hill coefficient for [3H]naloxone binding was 0.93 ± 0.18, and nonlinear regression analysis indicated that a one-site model was the best fit of the [3H]naloxone saturation binding data. Low concentrations of κ-selective opioids, but neither μ nor δ opioids, inhibited [3H]naloxone binding. Saturation binding studies with the κ-selective compound [3H]U69,593 revealed a single binding site with a KD value of 0.204 ± 0.039 nM and a Bmax value of 31.7 ± 3.1 fmol/mg of membrane protein. The Hill coefficient for [3H]U69,593 binding was 1.03 ±0.11, indicative of a single site. Time courses for the association and dissociation of [3H]U69,593 binding at 25°C exhibited properties consistent with a single class of binding sites. Low concentrations of κ-selective opioids, including dynorphin peptides, inhibited [3H]U69,593 binding, while high concentrations of μ opioids were needed to inhibit binding, and the δ-selectivc ligands were ineffective at concentrations up to 10 μM. Stereosclectivity of the binding site was demonstrated by the finding that the Ki value for ( − )-pentazocine in inhibiting [3H]U69,593 binding was 25 times less than for the ( + )-isomer. Based on its high affinity for U69,593, α-neo-endorphin, and dynorphin B, the κ opioid binding site on R1.1 cell membranes belongs to the k1b subtype. As observed with brain κ opioid binding sites, sodium inhibited [3H]U69,593 binding to R1.1 cell membranes in a concentration-dependent manner. These data demonstrate that the murine lymphoma cell line R1.1 expresses κ opioid binding sites that are very similar to brain κ opioid binding sites.

Opioid Receptors and Signaling on Cells from the Immune System

This review discusses the criteria for determining whether a binding site or functional response is directly mediated by either the mu, delta, or kappa opioid receptors. In 1988, Sibinga and Goldstein published the first review that addressed whether cells from the immune system express opioid receptors. The criteria that they used, namely, structure–activity relationships, stereoselectivity, dose- and concentration-dependence, and saturability are still relevant criteria today for determining if an immunological response is mediated by either the mu, delta or kappa opioid receptors. Radioligand receptor binding studies and functional studies that clearly show the presence of an opioid receptor on immunocytes are presented. Selective agonists and antagonists for the mu, delta, and kappa opioid receptors are discussed, and the need for their use in experiments is emphasized. Conditions used in functional assays are very important. Receptor desensitization and downregulation occur within minutes after the application of an agonist. However, many immunological assays are applying an agonist for days before measuring an immunological effect. The results obtained may reflect changes that are results of receptor desensitization and/or downregulation instead of changes that are observed with acute activation of the receptor. The future of receptor pharmacology lies in the crosstalk and dimerization of G protein-coupled receptors. In transfected systems, opioid receptors have been shown to dimerize with chemokine and cannabinoid receptors, resulting in crosstalk between different types of receptors.

A Novel Gβγ-Subunit Inhibitor Selectively Modulates μ-Opioid-Dependent Antinociception and Attenuates Acute Morphine-Induced Antinociceptive Tolerance and Dependence

The Gβγ subunit has been implicated in many downstream signaling events associated with opioids. We previously demonstrated that a small molecule inhibitor of Gβγ-subunit-dependent phospholipase (PLC) activation potentiated morphine-induced analgesia (Bonacci et al., 2006). Here, we demonstrate that this inhibitor, M119 (cyclohexanecarboxylic acid [2-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)-(9Cl)]), is selective for μ-opioid receptor-dependent analgesia and has additional efficacy in mouse models of acute tolerance and dependence. When administered by an intracerebroventricular injection in mice, M119 caused 10-fold and sevenfold increases in the potencies of morphine and the μ-selective peptide, DAMGO, respectively. M119 had little or no effect on analgesia induced by the κ agonist U50,488 or δ agonists DPDPE or Deltorphin II. Similar results were obtained in vitro, as only activation of the μ-opioid receptor stimulated PLC activation, whereas no effect was seen with the κ- and δ-opioid receptors. M119 inhibited μ-receptor-dependent PLC activation. In studies to further explore the in vivo efficacy of M119, systemic administration M119 also resulted in a fourfold shift increase in potency of systemically administered morphine. Of particular interest, M119 was also able to attenuate acute, antinociceptive tolerance and dependence in mice treated concomitantly with both M119 and morphine. These studies suggest that small organic molecules, such as M119, that specifically regulate Gβγ subunit signaling may have important therapeutic applications in enhancing opioid analgesia, while attenuating the development of tolerance and dependence.

Opioids Activate Brain Analgesic Circuits Through Cytochrome P450/Epoxygenase Signaling

To assess the importance of brain cytochrome P450 (P450) activity in μ opioid analgesic action, we generated a mutant mouse with brain neuron–specific reductions in P450 activity; these mice showed highly attenuated morphine antinociception compared with controls. Pharmacological inhibition of brain P450 arachidonate epoxygenases also blocked morphine antinociception in mice and rats. Our findings indicate that a neuronal P450 epoxygenase mediates the pain-relieving properties of morphine.