Differential effects of opioid agonists on G protein expression in CHO cells expressing cloned human opioid receptors

Recent evidence indicates that agonist ligands of G protein coupled receptors (GPCR) can activate different signaling systems. Such “agonist-directed” signaling also occurs with opioid receptors. Previous work from our laboratory showed that chronic morphine, but not DAMGO, up-regulates the expression of Gα12 and that both morphine and DAMGO decreased Gαi3 expression in CHO cells expressing the cloned human mu opioid receptor. In this study, we tested the hypothesis that chronic opioid regulation of G protein expression is agonist-directed. Following a 20 h treatment of CHO cells expressing the cloned human mu (hMOR-CHO), delta (hDOR-CHO) or kappa (hKOR-CHO) opioid receptors with various opioid agonists, we determined the expression level of Gα12 and Gαi3 by Western blots. Among five mu agonists (morphine, etorphine, DADLE, DAMGO, herkinorin) tested with hMOR-CHO cells, only chronic morphine and etorphine up-regulated Gα12 expression. All five mu agonists decreased Gαi3 expression. Among six delta agonists (SNC80, DPDPE, deltorphin-1, morphine, DADLE, etorphine) tested with hDOR-CHO cells, all six agonists down-regulated Gαi3 expression or moderately up-regulated Gα12 expression. Among five kappa agonists, ((−)-ethylketocyclazocine, salvinorin A, U69,593, etorphine, (−)-U50,488) tested with hKOR-CHO cells, only chronic (−)-U50,488 and (−)-EKC up-regulated Gα12 expression. All kappa agonists decreased Gαi3 expression. These data demonstrate that chronic opioid agonist regulation of G protein expression depends not only on the agonist tested, but also on the type of opioid receptor expressed in a common cellular host, providing additional evidence for agonist-directed signaling.     

Modulation of humoral immune response by central administration of leucine-enkephalin: Effects of μ, δ and κ opioid receptor antagonists

The effect of leucine-enkephalin (Leu-Enk) on primary humoral immune response was investigated following intracerebroventricular (i.c.v.) administration of the peptide in the rat. Leu-Enk stimulated plaque-forming cell (PFC) response in rats i.c.v. injected with 0.1 and 1 μg/kg, whereas doses of 20 and 50 μg/kg exerted immunosuppressive effects. I.c.v. treatment of rats with δ opioid receptor antagonist ICI 174864 and κ opioid receptor antagonist nor-binaltorphimine (nor-BNI) blocked stimulation and suppression of PFC response induced by Leu-Enk, respectively. The μ opioid receptor antagonist β-funaltrexamine (β-FNA) reversed both immunomodulatory effects produced by Leu-Enk. Since β-FNA alone had no effect on PFC response (unlike ICI 174 864 and nor-BNI), these data showed that central effects of Leu-Enk on PFC response were mediated by brain μ opioid receptors, and suggested a possible involvement of δ and κ opioid receptors.     

Opioid research in amphibians: an alternative pain model yielding insights on the evolution of opioid receptors

This review summarizes the work from our laboratory investigating mechanisms of opioid analgesia using the Northern grass frog, Rana pipiens. Over the last dozen years, we have accumulated data on the characterization of behavioral effects after opioid administration on radioligand binding by using opioid agonist and antagonist ligands in amphibian brain and spinal cord homogenates, and by cloning and sequencing opioid-like receptor cDNA from amphibian central nervous system (CNS) tissues. The relative analgesic potency of mu, delta, and kappa opioids is highly correlated between frogs and other mammals, including humans. Radioligand binding studies using selective opioid agonists show a similar selectivity profile in amphibians and mammals. In contrast, opioid antagonists that are highly selective for mammalian mu, delta, and kappa opioid receptors were not selective in behavioral and binding studies in amphibians. Three opioid-like receptor cDNAs were cloned and sequenced from amphibian brain tissues and are orthologs to mammalian mu, delta, and kappa opioid receptors. Bioinformatics analysis of the three types of opioid receptor cDNAs from all vertebrate species with full datasets gave a pattern of the molecular evolution of opioid receptors marked by the divergence of mu, delta, and kappa opioid receptor sequences during vertebrate evolution. This divergence in receptor amino acid sequence in later-evolved vertebrates underlies the hypothesis that opioid receptors are more type-selective in mammals than in nonmammalian vertebrates. The apparent order of receptor type evolution is kappa, then delta, and, most recently, the mu opioid receptor. Finally, novel bioinformatics analyses suggest that conserved extracellular receptor domains determine the type selectivity of vertebrate opioid receptors.     

μ-Opioid receptor activation enhances DNA synthesis in immature oligodendrocytes

Opioids disrupt nervous system development by inhibiting the proliferation of neuronal and glial progenitors. These studies explored the hypothesis that μ opioid receptors are expressed by immature oligodendrocytes (OLs) and are functionally related to growth. Antibodies identifying the cloned μ opioid receptor demonstrated that cultured OLs expressed μ opioid receptor immunoreactivity very early during development. Cultures were treated with the selective μ opioid receptor agonist H-Tyr-Pro-Phe (N-Me)- -Pro-NH₂ (PL017; 1 μM), or PL017 (1 μM) plus the antagonist naloxone (3 μM). Opioid-dependent changes in DNA synthesis were assessed by determining the proportion of bromodeoxyuridine (BrdU)-labeled O4-immunoreactive OLs. Treatment with PL017 caused a 311% increase in the proportion of O4-immunoreactive OLs incorporating BrdU compared to untreated controls, and these effects were prevented by co-administering naloxone. These preliminary results indicate that (i) immature OLs express μ opioid receptors and that (ii) the activation of this receptor type is functionally coupled to DNA synthesis and the cell division cycle. The expression of opioid receptors by OLs suggests that the endogenous opioid system is widely distributed among glial types.     

Quantitative autoradiographic mapping of the ORL1, μ-, δ- and κ-receptors in the brains of knockout mice lacking the ORL1 receptor gene

Until recently the opioid receptor family was thought to consist of only the μ-, δ- and κ-receptors. The cloning of opioid receptor like receptor (ORL1) and its endogenous ligand nociceptin/orphanin FQ, which displayed anti-opioid properties, has raised the issue of functional co-operativity of this system with the classical opioid system. ORL1 receptor knockout mice have been successfully developed by homologous recombination to allow the issue of potential heterogeneity of this receptor and also of compensatory changes in μ-, δ- or κ-receptors in the absence of ORL1 to be addressed. We have carried out quantitative autoradiographic mapping of these receptors in the brains of mice that are wild-type, heterozygous and homozygous for the deletion of the ORL1 receptor. ORL1, μ-, δ- and κ-receptors were labelled with [³H] leucyl-nociceptin (0.4 nM), [³H] DAMGO (4 nM), [³H] deltorphin-I (7 nM), and [³H] CI-977 (2.5 nM) respectively. An approximately 50% decrease in [³H] leucyl-nociceptin binding was seen in heterozygous ORL1 mutant mice and there was a complete absence of binding in homozygous brains indicating the single gene encodes for the ORL1 receptor and any putative subtypes. No significant gross changes in the binding to other opioid receptors were seen across genotypes in the ORL1 mutant mice demonstrating a lack of major compensation of classical opioid receptors in the absence of ORL1. There were a small number of region specific changes in the expression of classical opioid receptors that may relate to interdependent function with ORL1.     

Antisense oligodeoxynucleotides to the κ-1 receptor block the antinociceptive effects of Δ-THC in the spinal cord

Intrathecal pretreatment of mice with an antisence oligodeoxynucleotide directed against the κ-1 receptor significantly reduced the antinociceptive effects of the kappa receptor agonist U50,488 as well as Δ⁹-THC, the major psychoactive ingredient found in cannabis. A mismatched oligodeoxynucleotide which contained four switched bases did not block the antinociception produced by U50,488 orΔ⁹-THC. Furthermore, κ-1 antisense did not alter the antinociceptive effects of either the mu receptor-selective opioid DAMGO, or the delta receptor-selective opioid DPDPE. By using κ-1 antisense, we were able to demonstrate that an interaction occurs between the cannabinoids and opioids in the spinal cord.     

Long-term exposure to opioid antagonists up-regulates prodynorphin gene expression in rat brain

We investigated the effect of long-term administration of opioid antagonists on the regulation of prodynorphin gene expression in rat brain. Intracerebroventricular (i.c.v.) injections for seven days of nor-binaltorphimine (nor-BNI), the highly selective κ opioid antagonist, naloxone and its longer acting analog naltrexone, both relatively selective antagonists for the μ opioid receptor, markedly raised prodynorphin mRNA levels in rat hypothalamus, hippocampus and striatum. Peptides, namely immunoreactive-dynorphin A (ir-dyn A), were unaffected after chronic treatment with all antagonists, in the same tissues. These results, taken together with our previous observations, suggest that chronic opioid antagonists, acting on κ and μ opioid receptors, clearly up-regulate prodynorphin gene expression in discrete rat brain regions, activating its biosynthesis. Moreover, our data support the hypothesis that the endogenous opioid system plays a role in the mechanisms underlying the development of opiate tolerance.     

Expression of the conditioned NK cell activity is β-endorphin dependent

We are interested in identifying the pathways which are responsible for triggering the conditioned enhancement of natural killer (NK) cell activity. Earlier studies have suggested that central opioid(s) are involved in eliciting the expression of the conditioned NK cell activity. The purpose of this study was to identify the central opioid peptides that allow the central nervous system (CNS) to communicate with the immune system. Mediators that activate the efferent pathway of communication between the CNS and immune system was examined by injection of the mediator via the cisterna magna (CM). Conditioning was used as a tool to show that the bi-directional communication between the CNS and the immune system does take place. We found that β-endorphin but not dynorphin could stimulate NK cell activity, when β-endorphin or dynorphin was injected into the CM. In addition, when anti-β-endorphin or anti-dynorphin antibody was injected into the conditioned animals via CM the conditioned response was blocked by anti-β-endorphin but not by anti-dynorphin antibody. These observations suggest that β-endorphin appears to be one of the signals that is induced in the brain at the CS recall step of the conditioned response to trigger the elevation of NK cell activity.     

Analysis of central opioid receptor subtype antagonism of hypotonic and hypertonic saline intake in water-deprived rats

Intake of either hypotonic or hypertonic saline solutions is modulated in part by the endogenous opioid system. Morphine and selective mu and delta opioid agonists increase saline intake, while general opioid antagonists reduce saline intake in rats. The present study evaluated whether intracerebroventricular administration of general (naltrexone) and selective mu (beta-funaltrexamins, 5–20 μg), mu₁ (naloxonazine, 50 μg), kappa (nor-binaltorphamine, 5–20 μg), delta (naltrindole, 20 μg), or delta, (DALCE, 40 μg) opioid receptor subtype antagonists altered water intake and either hypotonic (0.6%) or hypertonic (1.7%) saline intake in water-deprived (24 h) rats over a 3-h time course in a two-bottle choice test. Whereas peripheral naltrexone (0.5–2.5 mg/kg) significantly reduced water intake and hypertonic saline intake, central naltrexone (1–50 μg) significantly reduced water intake and hypotonic saline intake. Water intake was significantly reduced following mu and kappa receptor antagonism, but not following mu₁, delta, or delta₁ receptor antagonism. In contrast, neither hypotonic nor hypertonic saline intake was significantly altered by any selective antagonist. These data are discussed in terms of opioid receptor subtype control over saline intake relative to the animal's hydrational state and the roles of palatability and/or salt appetite.     

Opioid receptor-mediated suppression of humoral immune response in vivo and in vitro: involvement of κ opioid receptors

The selective κ opioid receptor agonist MR 2034 exerted pronounced suppression of plaque-forming cell (PFC) response following intraperitoneal (i.p.) administration in the rat. Pretreatment with preferential κ and μ opioid receptor antagonists MR 2266 and naloxone, respectively, revealed that this effect was mediated mainly by κ, and to a low extent by μ opioid receptors. Intracerebroventricular (i.c.v.) administration of quaternary naltrexone (QNtx) moderately attenuated, whereas i.p. given QNtx completely prevented the suppressive effect of MR 2034, suggesting a peripheral mechanism of action, and only minor involvement of brain opioid receptors. MR 2034 markedly decreased the PFC response of spleen cells obtained from in vivo immunized rats, treated in vitro with the opiate. The immunosuppressive action of MR 2034 in vitro was completely and partially blocked by equimolar concentrations of MR 2266 and naloxone, respectively. Antagonists alone produced stimulation of PFC following i.p. administration in the rat, but did not affect PFC response upon in vitro treatment. These results suggest that peripheral k opioid receptors down-regulate primary humoral immune response in the rat, and that this effect may be produced by direct interference with plasma cell activity.     

Infusion of β-FNA into the thalamus attenuates morphine-induced c-Fos induction in the rat caudate putamen

The medial thalamus contains μ opioid receptors and sends a glutamatergic projection to the caudate putamen (CPu) in rat. Morphine-induced c-Fos expression in the CPu has been shown to be blocked by pretreatment with antagonists to N-methyl- -aspartate receptors, indicating the involvement of glutamate in this morphine-induced response. The importance of the glutamatergic projections from the thalamus was assessed by infusing the μ opioid receptor antagonist, β-funaltrexamine (β-FNA), prior to systemic morphine injection. Infusion of β-FNA near specific medial thalamic nuclei attenuated morphine-induced c-Fos expression in the CPu.     

Opioid receptors in midbrain dopaminergic regions of the rat II. Kappa and delta receptor autoradiography

Summary Opiates and opioid peptides are known to influence the dopaminergic (DA) neurons in the midbrain. The purpose of this study was to map and quantify the density of kappa and delta opioid receptor subtypes in the retrorubral field, substantia nigra, and ventral tegmental area and related nuclei, which contain DA nuclei A8, A9, and A10, respectively. Sections through the rostral-caudal extent of the rat midbrain were stained with an antibody against tyrosine hydroxylase, as a DA cell marker, and comparable sections were processed for in vitro receptor autoradiography using the kappa-selective ligand, U-69593, and the delta-selective ligand, D-Pen², D-Pen⁵-enkephalin. In general, both kappa and delta ligands exhibited low levels of specific binding in regions occupied by the midbrain DA neurons.Kappa binding (4–8 fmol/mg tissue) was high throughout the rostral-caudal extent of the substantia nigra, in rostral portions of the ventral tegmental area, and in the nucleus paranigralis; low binding occurred in the retrorubral field and central linear nucleus raphe.Delta binding (6–18 fmol/mg tissue) was high in the caudal portion of the substantia nigra pars reticulata, and in the medial terminal nucleus of the accessory optic system (a region previously shown to contain DA dendrites). The kappa and delta receptor binding is heterogeneously distributed in regions occupied by midbrain dopaminergic neurons, and several fold lower than the binding of mu opioid receptors in the same brain regions.     

Characterization of a polyclonal antibody to the μ opioid receptor

Active opioid receptors have been solubilized from bovine striatal synaptosomal membranes and purified approximately 4000-fold using a combination of affinity and hydroxyapatite chromatography. The affinity column was constructed by attaching hybromet, a newly synthesized opioid ligand with high affinity for the μ receptor, to a solid support matrix. A polyclonal antibody was generated to opioid receptors by injection of the purified receptor preparation into female New Zealand rabbits. The specificity of the antiserum was demonstrated by receptor competition and immunoprecipitation studies. Immunological titration of opioid binding activity from rat brain showed that the antibody was able to displace specific binding of [³H]etorphine (universal opioid) and [³H]dihydromorphine (μ opioid) from rat membranes, but was ineffective against the binding of [³H]ethylketocyclazocine (κ opioid), [³H]d-Ala²,d-Leu⁵-enkephalin (δ opioid) or [³H]phencyclidine (phencyclidine/σ receptor ligand). The antibody was able to precipitate the M_r 94 000 component of the ¹²⁵I-labeled affinity-purified receptor, a finding which suggests that this subunit may be an opioid recognition component. By indirect immunofluorescence, the antibody was shown to bind specifically to the plasma membranes of the neurotumor cell line NCB-20 (neuroblastoma × Chinese hamster brain hybrid cells), which has high affinity opioid receptors. The observed fluorescence in the neuroblastoma cells was prevented by pre-adsorption of the antibody with purified receptor from rat brain. These results indicate that the antibody is specific for opioid receptors and may prove useful in the precise localization of opioid receptors in the central and peripheral nervous systems by immunohistochemical procedures.     

Methionine-enkephalin modulation of hydrogen peroxide (H2O2) release by rat peritoneal macrophages involves different types of opioid receptors

We investigated the involvement of specific types of opioid receptors in methionine-enkephalin (MET)-induced modulation of hydrogen peroxide (H2O2) release by rat macrophages primed with sub-optimal concentrations of phorbol myristate acetate (PMA). Peritoneal macrophages in vitro treated with different concentrations of MET were tested for H2O2 release in phenol red assay. In the antagonistic study macrophages were treated with MET and one opioid receptor antagonist, or combination of MET and two or three opioid receptor antagonists. MET decreased H2O2 release in eight individual macrophage samples, and increased it in 10 samples. The increase of H2O2 release induced by MET in macrophages was blocked with combination of opioid receptor antagonists specific delta1,2 and mu receptors, as well as with combination of antagonists specific for delta1,2 and kappa opioid receptors. MET-induced decrease of the H2O2 release in macrophages was prevented by opioid receptor antagonists specific for delta1,2 or mu receptors, and also with combination of two or three opioid receptor antagonists. MET-induced enhancement of H2O2 release was mediated via delta1 or delta2 opioid receptor subtypes, or by mu-kappa opioid receptor functional interactions, while MET-induced suppression involved functional interactions between delta1 and mu, delta2 and mu, or delta1 and kappa opioid receptors. It is possible that individual differences in basal or induced macrophage capacity to produce H2O2 might shape the repertoire of opioid receptors expression and in that way pre-determine the direction of MET-induced changes after the in vitro treatment.     

Autoradiographic Localization of β-Endorphin Binding in the Pancreas

Autoradiographic localization of ¹²⁵I-labeled β-endorphin binding in the rabbit pancreas demonstrated specific binding in the pancreatic islet cells. Binding was inhibited by (1) nonradioactive β-endorphin, (2) the opioid antagonist naloxone, (3) the μ receptor agonists morphine and [ -Ala², (Me)Phe⁴, Gly(ol)⁵]enkephalin, (4) the δ receptor agonist [ -penicillamine², -penicillamine⁵]-enkephalin, (5) the μ and δ agonist met-enkephalin and (6) the δ and κ agonist dynorphin. Specific binding was not clearly demonstrable in the acinar portion of the rabbit pancreas. The binding characteristics of ¹²⁵ I-β-endorphin in the pancreatic islets were comparable with those of μ and δ opioid receptors in the rabbit brain. In the pancreas, β-endorphin binding appeared to be concentrated in discrete areas in the islets. Combined immunohistochemistry and autoradiography demonstrated that β-endorphin binding was primarily concentrated in the glucagon-containing alpha and somatostatin-containing delta cells, but was also found in the insulin-containing beta cells to a lesser extent, Given the intraislet location of the opioid binding sites, and our previous finding of immunoreactive β-endorphin in the pancreatic beta cells and the inhibitory effect of β-endorphin on insulin secretion, it appears that β-endorphin may serve a paracrine or autocrine function in the regulation of pancreatic hormone secretion.