Predominant involvement of mu-rather than delta- or kappa-opiate receptors in LH secretion

Opiate alkaloids and opioid peptides have been shown to suppress plasma LH and FSH levels via a naloxone sensitive mechanism in several species including man. Three subtypes of opiate receptors have been characterized: mu, delta and kappa. The present study was designed to investigate their role in gonadotropin release. Three highly selective opioid ligands, DAGO, MRZ and DTE12 (a dimeric tetrapeptide enkephalin), were injected intraventricularly into chronically ovariectomized rats. Injection of the mu-agonist at doses of 1 and 10 nmol produced a significant suppression of LH secretion, while the delta- and kappa-agonists had no significant effect. Thus, the mu-receptor seems to be the primary opiate receptor involved in the regulation of LH secretion. None of the opiate agonists employed had an effect on FSH secretion.     

Functional dissociation of mu opioid receptor signaling and endocytosis: implications for the biology of opiate tolerance and addiction.

Opiate analgesia, tolerance, and addiction are mediated by drug-induced activation of the mu opioid receptor. A fundamental question in addiction biology is why exogenous opiate drugs have a high liability for inducing tolerance and addiction while native ligands do not. Studies indicate that highly addictive opiate drugs such as morphine are deficient in their ability to induce the desensitization and endocytosis of receptors. Here, we demonstrate that this regulatory mechanism reveals an independent functional property of opiate drugs that can be distinguished from previously established agonist properties. Moreover, this property correlates with agonist propensity to promote physiological tolerance, suggesting a fundamental revision of our understanding of the role of receptor endocytosis in the biology of opiate drug action and addiction.     

Specific protection of the binding sites of D-Ala2-D-Leu5-enkephalin (delta-receptors) and dihydromorphine (mu-receptors).

Phenoxybenzamine causes a long-lasting inactivation of the opiate receptors of the mu- and delta-type in homogenates of guinea-pig brain. The effect is selectively prevented when, before exposure to phenoxybenzamine, the homogenate is pre-incubated with ligands of high affinity for either of the two binding sites, i.e. dihydromorphine for the mu-receptor and Tyr-D-Ala-Gly-Phe-D-Leu for the delta-receptor. In contrast, Tyr-D-Ala-Gly-Phe-L-Leu amide, which has high affinities for both binding sites, protects both receptor sites.     

Body temperature effects of opioids administered into the periaqueductal grey area of rat brain

The ability of opioids to influence rectal temperature after injection into the periaqueductal grey region (PAG) of rat brain was investigated. Both morphine and beta-endorphin caused a dose-dependent increase in rectal temperature of up to 2 degrees C. By using selective ligands of the subclasses of opiate receptor such as [D-Ala2,D-Leu5]enkephalin for delta-receptors and ethylketocyclazocine, dynorphin(1-17) and dynorphin(1-8) for kappa-receptors, it was possible to show that neither the delta- nor the kappa-opiate receptor was involved in the hyperthermic response. However, [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO), a mu-receptor ligand, did produce a dose-dependent hyperthermia. The ability of naltrexone, an opiate receptor antagonist, to reverse the hyperthermia induced by beta-endorphin and DAGO suggests that the opioid-stimulated increase in body temperature via the PAG is mediated through the mu-opiate receptor. Since the application of opioids to the PAG produces a hyperthermic response, it is possible that this brain site may have a role in the peptidergic control of body temperature.     

The development of opiate tolerance may dissociate from dependence

Experiments on opiate sensitive peripheral tissue preparations such as the mouse vas deferens and the guinea-pig ileum have demonstrated the ability to induce very high degrees of selective tolerance towards particular opiate agonists. Interestingly, the highly tolerant mouse vas deferens failed to display any sign of dependence as judged by the inability of naloxone to precipitate a withdrawal sign. In analogy, the present studies on the guinea-pig ileum revealed a striking dissociation in the degree of tolerance and dependence developed. Opiate receptor binding studies on both tissues point to distinct differences in the opiate-induced effector mechanisms. It is concluded that adaptational changes upon chronic opiate receptor activation may occur at multiple sites within the effector system of the opiate receptor.     

The effect of 12-O-tetradecanoyl-phorbol 13-acetate on the ribonuclease activity of circulating human lymphocytes.

12-O-Tetradecanoyl-phorbol 13-acetate is a very effective tumor promotor and inflammatory agent and can act as a mitogen for a subset of T lymphocytes. We report here that even short exposure of lymphocytes to 12-O-tetradecanoyl-phorbol 13-acetate changes the balance between the levels of neutral ribonuclease and ribonuclease inhibitor. The most dramatic change occurs in a B-lymphocyte-enriched population. We find that most, if not all, of the neutral ribonuclease activity in circulating lymphocytes is associated with this population and that this activity is lost with exposure to 12-O-tetradecanoyl-phorbol 13-acetate. Both 12-O-tetradecanoyl-phorbol 13-acetate and phytohaemagglutinin increase the level of ribonuclease inhibitor in T cells. However, phytohaemagglutinin has no effect on the ribonuclease or inhibitor level of the B-cell-enriched population.     

Opiate receptor: irreversible inactivation by an alkylating local anesthetic.

The mouse brain opiate receptor is irreversibly inactivated by incubation with an alkylating local anesthetic, a chloroethyl analog of lidocaine. The inactivation reaction is strongly temperature dependent and is undetectable at 0°C. The fraction of opiate receptors which is not inactivated in a given period of time retains its original affinity for naltrexone. Opiate agonists and antagonists are equally effective in protecting the receptor from inactivation by this compound.     

The sensitivity of opiate receptors and ligands to short wavelength ultraviolet light

The sensitivity of the opiate receptor, opiates and opioid ligands to ultraviolet light has been examined with the view of using this property to aid in characterizing the receptor. It is demonstrated that the opiate receptor is very sensitive to short wavelength ultraviolet light which causes rapid destruction of opiate binding activity. It is also shown that tritiated and cold opiate ligands are sensitive to light of this wavelength and when the receptor and opiate ligands are irradiated together, irreversible binding occurs. Along with the sensitivity of the receptor system to proteases and phospholipase the photosensitivity will aid in receptor isolation as well as in development of specific photoaffinity labels.     

Opiate receptor phenomenon: proconvulsant action of morphine in the mouse

Opiate narcotics lowered the chemoconvulsive threshold in the mouse in a manner that is characteristic of an opiate receptor mediated process. The proconvulsant action was dose-dependent, stereoselective for narcotics possessing the absolute configuration of D(-)-morphine, and selectively antagonized by naloxone. Chronic morphine treatment produced tolerance and cross-tolerance to the convulsant actions of narcotics. The chemconvulsant threshold provides a convenient and reproducible measure of opiate receptor-drug interaction and the development of narcotic tolerance.     

罂粟中阿片依赖机制及药物治疗进展

阿片为罂粟中的主要成分 ,阿片依赖性机制可能涉及脑内奖赏中心、多巴胺 (DA)通路、阿片受体及内源性阿片肽及多种神经递质系统。阿片依赖性治疗主要包括脱毒治疗及防复吸治疗 ,防复吸是目前该领域的研究重点 ,探讨阿片类依赖性机制以开发新药是今后的研究方向。     

Molecular size of opiate (enkephalin) receptors in neuroblastoma-glioma hybrid cells as determined by radiation inactivation analysis

Opiate receptor binding decayed exponentially in mouse neuroblastoma-rat glioma (NG108-15) hybrid cell preparations following exposure to increasing doses of ionizing radiation (0.2 to 7.0 Mrads; 2.0 Mrads/min). Target size analysis revealed that [3H][D-Ala2, D-Leu5]enkephalin (agonist) and [3H]naloxone (antagonist) bound specifically to a component with an apparent molecular size of 200,000 +/- 20,000. Lyophilization of cells for the irradiation procedure did not significantly alter receptor affinity or binding capacity for these ligands. Furthermore, the loss of opiate receptor binding in irradiated cell samples could not be attributed to reduced receptor affinity since increasing concentrations of radiolabeled ligand failed to reverse the inhibition; nonspecific binding decreased only slightly under identical experimental conditions. The value of determining molecular size by radiation inactivation analysis was confirmed by showing that apparent target sizes for two representative lysosomal enzymes (beta-galactosidase and alpha-mannosidase) were consistent with results obtained previously using conventional methods. Thus, the data suggest that the ligand binding component of delta-opiate (enkephalin) receptors in NG108-15 cells has a minimum functional size of approximately 200,000.     

Receptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor

Opiate tolerance and dependence are major clinical and social problems. The anti-opiate neuropeptides FF and AF (NPFF and NPAF) have been implicated in pain modulation as well as in opioid tolerance and may play a critical role in this process, although their mechanism of action has remained unknown. Here we describe a cDNA encoding a novel neuropeptide Y-like human orphan G protein-coupled receptor (GPCR), referred to as HLWAR77 for which NPAF and NPFF have high affinity. Cells transiently or stably expressing HLWAR77 bind and respond in a concentration-dependent manner to NPAF and NPFF and are also weakly activated by FMRF-amide (Phe-Met-Arg-Phe-amide) and a variety of related peptides. The high affinity and potency of human NPFF and human NPAF for HLWAR77 strongly suggest that these are the cognate ligands for this receptor. Expression of HLWAR77 was demonstrated in brain regions associated with opiate activity, consistent with the pain-modulating activity of these peptides, whereas the expression in adipose tissue suggests other physiological and pathophysiological activities for FMRF-amide neuropeptides. The discovery that the anti-opiate neuropeptides are the endogenous ligands for HLWAR77 will aid in defining the physiological role(s) of these ligands and facilitate the identification of receptor agonists and antagonists.     

Regulated endocytosis of opioid receptors: cellular mechanisms and proposed roles in physiological adaptation to opiate drugs

Opiate drugs such as morphine and heroin are among the most effective analgesics known. Prolonged or repeated administration of opiates produces adaptive changes in the nervous system that lead to reduced drug potency or efficacy (tolerance), as well as physiological withdrawal symptoms and behavioral manifestations such as craving when drug use is terminated (dependence). These adaptations limit the therapeutic utility of opiate drugs, particularly in the treatment of chronically painful conditions, and are thought to contribute to the highly addictive nature of opiates. For many years it has been proposed that physiological tolerance to opiate drugs is associated with a modification of the number or functional activity of opioid receptors in specific neurons. We now understand certain mechanisms of opioid receptor desensitization and endocytosis in considerable detail. However, the functional roles that these mechanisms play in the complex physiological adaptation of the intact nervous system to opiates are only beginning to be explored.     

Actions of opiate agonists, naloxone, and paraben preservatives in the rat lung circulation

Previous studies have documented direct vascular effects of opiate substances in the systemic circulation. Because opiate receptors have been identified in the lung, we wondered whether opiate substances might affect vasoreactivity in the lung circulation. We studied the pulmonary vascular effects of three opiate agonists: morphine, leucine-enkephalin, and dynorphin, as well as the opiate receptor antagonist naloxone, in isolated rat lungs perfused with a cell- and plasma-free salt solution. Because of previous reports of the smooth muscle effects of the methyl- and propylparaben preservatives in the naloxone preparation, we also studied the pulmonary vascular effects of these preservatives in the rat lung circulation. We found that morphine, a mu-receptor agonist, leucine-enkephalin, a delta-receptor agonist, and dynorphin, a kappa-receptor agonist, caused no immediate vascular effect when injected into the pulmonary artery. In addition, morphine did not affect the pulmonary vasoconstrictions induced by hypoxia, angiotensin II, or potassium chloride. The commercial preparation of naloxone, Narcan, caused a marked vasodilation during hypoxic pulmonary vasoconstriction. However, this effect was entirely attributable to the preservatives methyl- and propylparaben, as pure naloxone had no effect on either the baseline pulmonary vascular tone or the vasoconstrictive response to hypoxia. We conclude that opiate receptor agonists and antagonists do not affect vasoreactivity in the rat lung circulation and that the methyl- and propylparaben preservatives in Narcan are pulmonary vasodilators.     

Opiate receptor blockade in man reduces 2-deoxy-d-glucose-induced food intake but not hunger,thirst, and hypothermia

Opioid peptides may act as neuromodulators in the central nervous system to conserve energy stores and water in mammals. To examine this hypothesis in man, the effect of opiate receptor blockade with naloxone on the hunger, thirst, and hypothermic response to 2-deoxy-D-glucose-induced glucoprivic stress was assessed. Opiate receptor blockade decreased stress-induced food intake but did not reduce marked increases in hunger produced by glucoprivation. Naloxone infusions did not change the hypercortisolemic, polydipsic, hypothermic, and thermogenic response to 2-deoxy-D-glucose. While these results do not suggest a major role for a β-endorphin modulation of stress-induced hunger, hypothermia and water conservation, the reduction of food intake could be due to augmented satiety, perhaps associated with retardation of gastric emptying during opiate receptor blockade.     

Opioids, feeding, and anorexias

This review summarizes recent work that focuses on the role of endogenous opioids (EOs) and opiate receptors in the control of food intake. Although the anorexic effect of opiate antagonists are now well accepted, the exact EO, site(s), and mechanism(s) of action remain to be established. However, accumulating evidence suggests that dynorphin, an endogenous ligand for kappa-type opiate receptors, is an important regulator (stimulant) of appetite. The roles of other EOs, such as beta-endorphin, are less clear. EOs appear to be involved in maintaining normal feeding behavior and are likely responsible for the overconsumption of fat in genetically obese and stressed subjects. Opiate antagonists block overconsumption of palatable foods, thus offering a promising approach to weight reduction for some overweight individuals. Anorexias may follow from a deficiency of kappa-type opioid activity, and surprisingly, can also result from excess opioid activity. Indeed, opiate antagonists of the mu type (naloxone) can enhance eating and weight gain in certain anorexic conditions. Therefore, it appears that excess opioid agonist activity may result in hyperphagia or anorexia (depending on the opiate receptor type). Finally, opiate antagonists may help normalize both types of pathological feeding states.     

Upregulation of the opioid receptor complex by the chronic administration of morphine: a biochemical marker related to the development of tolerance and dependence.

Studies conducted after the development of the rapid filtration assay for opiate receptors, and before the recognition of multiple opioid receptors, failed to detect changes in opioid receptors induced by chronic morphine. Recent experiments conducted in our laboratories were designed to examine the hypothesis that only one of several opioid receptor types might be altered by chronic morphine. Using binding surface analysis and irreversible ligands to increase the "resolving power" of the ligand binding assay, the results indicated that chronic morphine increased both the Bmax and Kd of the opioid receptor complex, labeled with either [3H][D-Ala2,D-Leu5]enkephalin, [3H][D-Ala2-MePhe4,Gly-ol5]enkephalin or [3H]6-desoxy-6 beta-fluoronaltreone. In the present study rats were pretreated with drugs known to attenuate the development of tolerance and dependence [the irreversible mu-receptor antagonist, beta-funaltrexamine (beta-FNA), and the inhibitor of tryptophan hydroxylase, para-chlorophenylalanine], prior to subcutaneous implantation of morphine pellets. The results demonstrated that 1) unlike chronic naltrexone, beta-FNA failed to upregulate opioid receptors and 2) both beta-funaltrexamine and PCPA pretreatment attenuated the chronic morphine-induced increase in the Bmax, but not the Kd, of the opioid receptor complex. These results provide evidence that naltrex-one-induced upregulation of the opioid receptor complex might occur indirectly as a consequence of interactions at beta-funaltrexamine-insensitive opioid receptors and that morphine-induced upregulation (increased Bmax) of the opioid receptor complex is a relevant in vitro marker related to the development of tolerance and dependence. These data collectively support the hypothesis that endogenous antiopiate peptides play an important role in the development of tolerance and dependence to morphine.     

Localization of human placental opiate binding sites on the syncitial brush border membrane

Opiate binding sites were measured in different placental membrane fractions which were characterized by marker enzyme analysis and electron microscopic examination. The distribution pattern of opiate binding sites in the different fractions closely parallels that of placental alkaline phosphatase. These results clearly show thatopiate binding sites are mainly located on the syncitial brush border membrane. The opiate binding sites found on microvillus membrane fraction have the same pharmacological characteristics as the Kappa opiate binding site previously characterized on placental crude membrane fraction.     

Opioid-mediated suppression of cultured peripheral blood mononuclear cell respiratory burst activity

Opiate addiction and stress have been associated with altered immune responses. In this study, we evaluated the influence of morphine and the stress responsive opioid peptide beta-endorphin (beta-END) on O-2 and H2O2 production by cultured human peripheral blood mononuclear cells. Exposure of these cells during 48 hr of culture to morphine and beta-END at pharmacologically (10(-8) M) and physiologically (10(-12) M) relevant concentrations, respectively, markedly suppressed peripheral blood mononuclear cell O-2 and H2O2 release in response to the respiratory burst stimuli opsonized zymosan and phorbol myristate acetate. Both opioids also induced a minimal, but statistically significant, increase in resting O-2 and H2O2 generation. The modulatory effects of morphine and beta-END on peripheral blood mononuclear cell oxygen metabolism appeared to involve a classical opioid receptor, because opioid activity was blocked by naloxone and was not observed with N-acetylated-beta-END. Using purified lymphocyte and monocyte preparations, we determined that although opioids directly increase monocyte-resting oxygen metabolism, lymphocytes are the primary target cell in opioid-mediated suppression of monocyte respiratory burst activity. The release of a suppressive product from opioid-triggered lymphocytes was inhibited by cyclosporine. Based on the results of this study, we propose that opioid-mediated suppression of mononuclear phagocyte respiratory burst activity is another factor to be considered in the immunodeficiency of opiate addiction and stress.     

Assessment of in situ host immunity to syngeneic tumors utilizing the multicellular spheroid model

By the 1g sedimentation method using discontinuous gradients of Ficoll solution (concentrations of 6 to 14%), keyhole limpet hemocyanin (KLH)-primed spleen cells of C3H/He or DBA/2 mice were fractionated into 4 to 10 populations after IgG antibody-coated erythrocytes (EA gamma) rosetting and then treatment with anti-Thy-1.2 + complement (C). No significant difference was observed in the distribution of isotype specificities of surface immunoglobulins on B cells in each population thus fractionated, when determined by indirect immunofluorescence staining. The mixture of the 12 and 14% Ficoll fractions contained 95% of B cells bearing Fc receptor for IgG (FcR+ gamma) and 3.58% of antigen-binding cells (ABC) for KLH, while the 8% Ficoll fraction included 15% of FcR+ gamma B cells and 1.53% of ABC. Nevertheless, the FcR- gamma B-cell-enriched populations caused intensive plaque-forming cell (PFC) responses to dinitrophenol (DNP), whereas FcR+ gamma B-cell-enriched populations generated weak responses. Noteworthy is that 4 days preculture of a population containing 95% FcR+ gamma B cells resulted in the appearance of precursor activity which was ascertained by a further 4 days culture of these cells with antigen, DNP-dextran. These findings suggest that FcR gamma bearing B cells intrinsically possess precursor activity for IgM/IgG antibody-forming cells, but lose it transiently by binding immune complexes (IC). Moreover, the titer of a factor suppressing anti-DNP PFC responses (suppressive B-cell factor, SBF) was higher in the 24-hr culture supernatants of the FcR+ gamma B-cell-enriched fraction than of the FcR- gamma B-cell-enriched fraction, suggesting that SBF is produced by FcR+ gamma B cells themselves. Thus, IC seems to play an important role for the negative feedback regulation of antibody production by stimulating FcR gamma bearing B cells.