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

Detection of kappa opioid receptors on mouse thymocyte phenotypic subpopulations as assessed by flow cytometry

ABSTRACT Recent studies have shown kappa opioid receptor labeling on the R1EGO thymoma cell line by indirect immunofluorescence and flow cytometric analysis. The present study used a fluorescein-labeled arylacetamide (FITC-AA), a kappa opioid ligand, in conjunction with biotin-conjugated anti-fluorescein IgG and extravidin-R-phycoerythrin (PE), along with doublelabeling with antibodies against specific immune cell surface markers to determine which subpopulation(s) of thymocytes express the kappa opioid receptor. Thymocytes, isolated from 6-to 8-week-old C57BL/6ByJ mice, incubated with FITC-AA followed by the PE amplification procedure, demonstrated labeling of the kappa opioid receptor. This labeling was inhibited 55 Ϯ 4% above background by excess nor-binaltorphimine (nor-BNI), a kappa selective antagonist. This kappa opioid receptor positive population consisted of 58 Ϯ 2% of all gated thymocytes. Phenotypic characterization determined that not only were 64 Ϯ 3% of the gated thymocytes CD4 ϩ / kappa opioid receptor positive, but 60 Ϯ 1% of all thymocytes were CD8 ϩ / kappa opioid receptor positive. Two subpopulations of CD3 ϩ thymocytes, consisting of both mature and immature cells, also displayed labeling for the kappa opioid receptor. Double-labeling of thymocytes with anti-CD4 and anti-CD8 antibodies demonstrated 82 Ϯ 0.5% of these cells were of the double-positive phenotype. Therefore, these findings demonstrate that the thymocytes, which express the kappa opioid receptor, are predominantly of the immature CD4 ϩ /CD8 ϩ phenotype. Collectively, these findings not only establish the presence of the kappa opioid receptor on immune cells involved in opioid responsiveness, but further indicate that this technique allows for the identification of distinct lymphocyte subpopulations which express the receptor

Stage-specific inhibition of IL-7R expression by the kappa opioid receptor in double negative thymocytes

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Pharmacology and Physiology, 2009.Opioids are known to affect both immune cell functions and development. Administration of morphine as well as knockout of the kappa-, but not mu-opioid receptor has been shown to decrease total thymic numbers and alter the distribution of thymocyte subpopulations. The mechanisms underlying opioid receptor-mediated changes during thymopoiesis remain unknown. A previous study demonstrated that the direct activation of kappa opioid receptor (KOR) on mixed thymocytes decreases the level of expression of IL-7 receptor (IL-7R), the receptor that plays a major role in early T-cell development by promoting thymocyte survival and proliferation. The IL-7R level is precisely controlled during T-cell development and we assumed that KOR-mediated regulation of IL-7R level might be important for thymocyte maturation. To further elucidate the influence of the KOR in IL-7R expression, we investigated the effect of κ-selective opioid agonist on the transcription of IL-7Rα-chain at different stages of thymocyte development and in related cell lines. The κ-selective opioid agonist, U50,488, decreased the number of cells expressing the IL-7R (up to 50%) and the density of the receptor expression in double negative (DN) 1 and 2 thymocyte subpopulations, while U50,488 did not alter IL-7R expression in more mature thymocyte subsets and in T cells from peripheral lymph nodes. U50,488 also decreased the density of IL-7R expression in DN1-related EL4 cell line. The observed U50,488-dependent inhibition of IL-7R expression was completely blocked by the k-selective antagonist nor-binaltorphimine, suggesting the involvement of a classical KOR. In vivo, U50,488 administration to mice decreased the number of cells expressing IL-7R during double DN1 and DN2 stages by 40%. Further analysis demonstrated that promoter region containing AP-1 response element was shown to be responsible for U50,488-induced inhibition of IL-7Rα expression. In addition, U50,488 decreased c-Fos mRNA level. However, point mutation analysis did not reveal the involvement of AP-1 transcription factor in KOR-mediated decrease in IL-7Rα expression. These findings provide evidence that the inhibitory effect of a kappa-opioid agonist on IL-7R expression is stage-specific and occurred only at DN1 and DN2 stages. Taking into account that it is during early DN1 and DN2 stages that IL-7R signaling is critically important for thymocyte survival and that KOR is preferably expressed on immature thymocytes, our data suggest that KOR signaling plays a role in early thymocyte development through transcriptional regulation of IL-7R expression on DN1 and DN2 thymocytes.

Cytokine-mediated regulation of kappa opioid receptor expression and a role for kappa agonists in inflammation

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Pharmacology and Physiology, 2008.In addition to numerous neurological effects, opioids also elicit responses from the immune system. Effects of opioids have been seen on a variety of immune cell types; however, studies have been unable to fully analyze the expression of opioid receptors on human immune cells. Individual genetic variability, lack of specific antibodies for opioid receptors, low density of receptors on immune cells, and expression of receptors only on small subsets of immune cells all hinder the detection of opioid receptors in the immune system. In addition, some studies have pointed to detectable levels of opioid receptors being present in immune cells only after induction by cytokine/chemokine stimulation. Preliminary data strongly suggested the presence of the kappa opioid receptor on a human monocyte cell line, U-937. This study aimed to verify the presence of this receptor and to further characterize the regulation of its expression after exposure to inflammatory stimuli. RT-PCR analysis showed that exposure to a bacterial endotoxin, lipopolysaccharide, induced a 4-fold increase in KOR mRNA in the U-937 cell line. Further investigation revealed that this induction was mediated by autocrine/paracrine effects due to the release of the cytokine, interferon-γ. Based on past studies in murine mononuclear leukocytes, this study also hypothesized that KOR activation in the U-937 cell line would lead to inhibition of pro-inflammatory cytokine production. It was observed that κ-selective agonists inhibited production of TNF-α by impeding NFκB activation. These effects were blocked by the κ-selective antagonist, nor-binaltorphimine. This study presents the first direct evidence for regulation of KOR transcripts by cytokines/chemokines and the first direct functional support for its presence on human monocytes. The characterization of the KOR in the U-937 cell line provides a useful model for studies on the effects of opioids in peripheral inflammation and gives insight into mechanisms behind neuroinflammatory diseases. The results of this study also aid in understanding the multifaceted circumstances that determine opioid receptor expression on cells of the immune system.

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 δ and κ. 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 δ 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 K d's of the neuronal μ 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