enkephalin--ala(2)-mephe(4)-gly(5)- and methylnaltrexone

This study characterized the pharmacology of the peripherally restricted opioid receptor antagonists, alvimopan, its metabolite, ADL 08-0011, and methylnaltrexone. The activities of the compounds were investigated with respect to human or guinea pig opioid receptor binding and function in recombinant cell lines and mechanical responsiveness of the guinea pig ileum. Alvimopan and ADL 08-0011 had higher binding affinity than methylnaltrexone at human mu opioid receptors (pK (i) values of 9.6, 9.6, and 8.0, respectively). The compounds had different selectivities for the mu receptor over human delta and guinea pig kappa opioid receptors. ADL 08-0011 had the highest mu receptor selectivity. With respect to their mu opioid receptor functional activity ([(35)S]GTPgammaS incorporation), methylnaltrexone had a positive intrinsic activity, consistent with partial agonism, unlike alvimopan and ADL 08-0011, which had negative intrinsic activities. Alvimopan, ADL 08-0011, and methylnaltrexone antagonized inhibitory responses mediated by the mu opioid agonist, endomorphin-1 (pA (2) values of 9.6, 9.4, and 7.6, respectively) and by U69593, a kappa opioid agonist (pA (2) values of 8.4, 7.2, and 6.7, respectively). In morphine-naive guinea pig ileum, methylnaltrexone reduced, while alvimopan and ADL 08-0011 increased, the amplitude of electrically evoked contractions and spontaneous mechanical activity. In tissue from morphine-dependent animals, alvimopan and ADL 08-0011 increased spontaneous activity to a greater degree than methylnaltrexone. The data suggested that alvimopan-induced contractions resulted predominantly from an interaction with kappa opioid receptors. It is concluded that alvimopan, ADL 08-0011, and methylnaltrexone differ in their in vitro pharmacological properties, particularly with respect to opioid receptor subtype selectivity and intrinsic activity. The clinical significance of the data from this study remains to be determined.

Topics: Analgesics, Opioid; Animals; Benzeneacetamides; CHO Cells; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guanosine 5'-O-(3-Thiotriphosphate); Guinea Pigs; Humans; Ileum; In Vitro Techniques; Male; Morphine; Muscle Contraction; Naltrexone; Narcotic Antagonists; Oligopeptides; Piperidines; Pyrrolidines; Quaternary Ammonium Compounds; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Recombinant Proteins; Transfection

Endothelial cell (EC) barrier dysfunction (i.e., increased vascular permeability) is observed in inflammatory states, tumor angiogenesis, atherosclerosis, and both sepsis and acute lung injury. Therefore, agents that preserve vascular integrity have important clinical therapeutic implications. We examined the effects of methylnaltrexone (MNTX), a mu opioid receptor (mOP-R) antagonist, on human pulmonary EC barrier disruption produced by edemagenic agents including morphine, the endogenous mOP-R agonist DAMGO, thrombin, and LPS. Pretreatment of EC with MNTX (0.1 muM, 1 h) or the uncharged mOP-R antagonist naloxone attenuated morphine- and DAMGO-induced barrier disruption in vitro. However, MNTX, but not naloxone, pretreatment of EC inhibited thrombin- and LPS-induced barrier disruption, indicating potential mOP-R-independent effects of MNTX. In addition, intravenously delivered MNTX attenuated LPS-induced vascular hyperpermeability in the murine lung. We next examined the mechanistic basis for this MNTX barrier protection and observed that silencing of mOP-R attenuated the morphine- and DAMGO-induced EC barrier disruption, but not the permeability response to either thrombin or LPS. Because activation of the sphingosine 1-phosphate receptor, S1P(3), is key to a number of barrier-disruptive responses, we examined the role of this receptor in the permeability response to mOP-R ligation. Morphine, DAMGO, thrombin, and LPS induced RhoA/ROCK-mediated threonine phosphorylation of S1P(3), which was blocked by MNTX, suggesting S1P(3) transactivation. In addition, silencing of S1P(3) receptor expression (siRNA) abolished the permeability response to each edemagenic agonist. These results indicate that MNTX provides barrier protection against edemagenic agonists via inhibition of S1P(3) receptor activation and represents a potentially useful therapeutic agent for syndromes of increased vascular permeability.

Topics: Analgesics, Opioid; Animals; Capillary Permeability; Cells, Cultured; Electrophysiology; Endothelial Cells; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Morphine; Naloxone; Naltrexone; Narcotic Antagonists; Protein Serine-Threonine Kinases; Quaternary Ammonium Compounds; Receptors, Lysosphingolipid; Receptors, Opioid, mu; rho-Associated Kinases; rhoA GTP-Binding Protein; RNA, Small Interfering; Thrombin; Transcriptional Activation

3-O-Methylnaltrexone (3-MNTX), a putative antagonist of morphine-6-beta-d-glucuronide (M6G) receptors, has been reported to block the behavioral effects of heroin at doses that do not block those of morphine, suggesting that M6G receptors may play a unique role in the addictive properties of heroin. This study investigated the effects of 3-MNTX in monkeys trained to discriminate i.v. heroin from vehicle or to self-administer i.v. heroin under a progressive-ratio schedule. Additional in vitro studies determined the effects of 3-MNTX and reference drugs on adenylyl cyclase activity in caudate-putamen membranes of monkeys and rats. In drug discrimination experiments, heroin, morphine, and M6G substituted for heroin in all subjects, whereas 3-MNTX substituted for heroin in one-half the monkeys tested. In these latter monkeys, the effects of 3-MNTX were antagonized by naltrexone, and pretreatment with 3-MNTX enhanced the effects of heroin, M6G, and morphine, indicative of micro-agonist activity. In monkeys showing no substitution of 3-MNTX for heroin, 3-MNTX antagonized the effects of heroin, M6G, and morphine. In self-administration experiments, heroin and 3-MNTX maintained injections per session significantly above those maintained by vehicle when the initial response requirement (IRR) was low; only heroin maintained significant self-administration when the IRR was high. In vitro, 3-MNTX inhibited adenylyl cyclase activity in both monkey and rat brain membranes. The degree of inhibition produced by 3-MNTX was less than that produced by the full agonist [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO). The results suggest that 3-MNTX functions primarily as a partial agonist at micro-receptors in monkeys and do not support a singular role for M6G receptors in the abuse-related effects of heroin.

Topics: Adenylyl Cyclases; Animals; Cocaine; Discrimination Learning; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Heroin; Heroin Dependence; Macaca mulatta; Male; Morphine; Morphine Derivatives; Naltrexone; Narcotics; Quaternary Ammonium Compounds; Receptors, Opioid, mu; Self Administration

Studies completed in both humans and animals have shown that opioids have significant effects on the immune system via pharmacological interactions with the opioid receptor. However, the type of opioid receptor at which morphine binding produces changes in immune status has not been well characterized. To determine the type of opioid receptor involved in opioid-induced immune alterations, the present study assessed the effects of agonists selective for the mu-, delta-, and kappa-opioid receptors. The site of action (i.e., peripheral vs central) at which opioids produce immune changes was investigated by injecting the agonists directly into the left lateral ventricle of the brain. Specifically, Lewis rats received an intracerebroventricular administration of [d-Ala(2),N-Me-Phe(4), Gly-ol(5)]enkephalin (DAMGO), a mu-receptor selective agonist, [D-Pen(2,5)]enkephalin (DPDPE), a delta-opioid receptor agonist, or U69,593, a kappa-receptor agonist. Immune assessments completed 1 h following drug administration showed that the mu-receptor selective agonist DAMGO produced a dose-dependent decrease in natural killer cell activity and T-lymphocyte proliferation to the mitogen concanavalin A (Con A); no immunological changes were found following DPDPE or U69,593 treatment. Calculation of the number of white blood cells per sample showed no differences between rats treated with saline and rats treated with any of the selective agonists. Administration of the opioid antagonist N-methylnaltrexone prior to DAMGO treatment attenuated the DAMGO-induced changes in immune status. Results from the present study indicate that the immunomodulatory effects of opioids can be attributed to interactions with the mu-opioid receptor.

Topics: Animals; Brain; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Immune System; Male; Naltrexone; Narcotic Antagonists; Quaternary Ammonium Compounds; Rats; Rats, Inbred Lew; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu

In this study, we evaluated the gastric effects of methylnaltrexone, an opioid receptor antagonist that does not cross the blood-brain barrier in vivo, on mu, kappa and delta opioid agonists induced brainstem unitary responses in an in vitro neonatal rat brainstem-gastric preparation. Single units in the medial subnucleus of the nucleus tractus solitarius (NTS), responding to electrical stimulation of subdiaphragmatic vagal fibers, were recorded. Selective opioid receptor agonists and antagonists were applied only to the gastric compartment of the bath chamber and thus, the brainstem functions of the preparation were not affected by the drugs. The peripheral gastric effects of a mu opioid receptor agonist, DAMGO, and a kappa opioid receptor agonist, U-50,488H, were evaluated on 58 tonic units that received the subdiaphragmatic vagal inputs. For approximately 78% of the units observed, DAMGO (1.0 microM) and U-50,488H (1.0 microM) induced a concentration-dependent inhibition of 62.1+/-9.3% (mean +/- SE) and 49.2+/-6.5% of the control level of the NTS neuronal activity, respectively. Methylnaltrexone competitively antagonized the DAMGO-induced brainstem neuronal effects. Methylnaltrexone at an 18.8-fold higher concentration also reversed U-50,488H-induced NTS neuronal responses. Naloxone, a non-selective opioid receptor antagonist, reversed the inhibitory effects of DAMGO and U-50,488H at much lower concentrations (3.8% and 0.5%, respectively) compared to methylnaltrexone. Only 18% of the NTS neurons evaluated showed inhibitory responses to a delta receptor agonist, DPDPE, (19.7+/-5.0% at 10 microM), and this inhibition could not be reversed by methylnaltrexone in the concentration range we tested. In addition, when methylnaltrexone (1.0 microM) alone was applied to the gastric compartment, there was an activation (8.5+/-2.1%) of the NTS neurons receiving subdiaphragmatic vagal inputs, suggesting an endogenous gastric opioid action in the modulation of brainstem neuronal activities.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Animals, Newborn; Brain Stem; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Morphine; Naltrexone; Narcotic Antagonists; Neurons; Quaternary Ammonium Compounds; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Solitary Nucleus; Stomach; Vagus Nerve

Previous studies have shown that administration of morphine results in alterations of splenic macrophage nitric oxide production. The present studies were conducted to determine the subtype of opioid receptor involved in the modulation of macrophage nitric oxide production. Moreover, the present work was directed at determining whether nitric oxide production is regulated through opioid receptors in the central nervous system (CNS) or via opioid receptors found directly on splenocytes. The study shows that intracerebroventricular (i.c.v.) administration of the mu-selective opioid agonist, DAMGO, to rats dose-dependently increases the production of nitric oxide by splenocytes stimulated with toxic shock syndrome toxin (TSST-1). The effect of DAMGO is blocked by prior i.c.v. administration of N-methylnaltrexone. In contrast, i.c.v. administration of the kappa-selective agonist, U69,593, and the delta-selective agonist, DPDPE, have no significant effect on the production of nitric oxide. Furthermore, the in vitro administration of DAMGO, DPDPE, or U69,593 to splenocytes cultures does not significantly alter the production of nitric oxide by splenocytes. In addition, the present work shows that elevation of nitric oxide production by i.c.v. administration of DAMGO produces functional changes in splenic lymphocytes. Collectively, these results indicate that mu-opioid receptors within the CNS are involved in the regulation of splenic nitric oxide production.

Topics: Analgesics; Analgesics, Opioid; Animals; Bacterial Toxins; Benzeneacetamides; Cell Division; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Enterotoxins; Enzyme Inhibitors; Injections, Intraventricular; Lymphocytes; Male; Naltrexone; Narcotic Antagonists; Neuroimmunomodulation; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Pyrrolidines; Quaternary Ammonium Compounds; Rats; Rats, Inbred Lew; Receptors, Opioid, mu; Spleen; Superantigens

The opioid receptors involved in the mediation of gastric acid secretory effects were studied in the pylorus-ligated rat. The effects of i.c.v. and i.v. administration of morphine and mu ([D-Ala2, NMePhe4, Gly5-ol]enkephalin and Tyr-Pro-NMePheD-Pro-NH2)-, delta ([D-Pen2,D-Pen5]enkephalin)- and kappa-selective [trans-3,4-dichloro-N-methyl-N-[2-91-pyrrolidinyl)-cyclohexyl]- benzeneacetamide methanesulfonate (U-50,488H), dynorphin-(1-9), dynorphin-(1-17), nalorphine, alpha-neoendorphin and ethyl-ketocyclazocine) opioid receptor agonists on gastric volume and acid output were examined. Morphine, [D-Ala2, NMePhe4, Gly5-ol]enkephalin and Tyr-Pro-NMePhe-D-Pro-NH2 decreased gastric acid secretion more potently after i.c.v. than after i.v. administration. The inhibitory effect of i.v. administered morphine on gastric acid secretion was not blocked by the quaternary opioid antagonist naltrexone methylbromide when given s.c. However, when naltrexone methylbromide was administered i.c.v., it blocked completely the effects of i.c.v. morphine and partially antagonized the effects of i.v. morphine, indicating a central site of action for morphine. The delta-selective agonist [D-Pen2,D-Pen5]enkephalin did not alter gastric acid secretion after i.c.v. or i.v. administration. The kappa-selective opioid agonist U-50,488H produced a dose-dependent increase in gastric acid secretion after i.v. but not i.c.v. administration. The other kappa-selective agonists tested did not produce a significant increase in gastric acid secretion after i.c.v. or i.v. administration. The increase in gastric acid secretion produced by U-40,488H was blocked by pretreatment with the opioid receptor antagonist naloxone, the nonselective muscarinic receptor antagonist atropine and the M1 selective muscarinic receptor antagonist pirenzepine.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Brain; Cyclazocine; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Ethylketocyclazocine; Gastric Acid; Male; Morphine; Naltrexone; Pyrrolidines; Quaternary Ammonium Compounds; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu

Enkephalin-containing neuronal fibers and perikarya, and opioid receptors are present in the A10 dopamine (DA) region, and many studies have implicated enkephalin as a neuromodulator of A10 DA neurons projecting to the prefrontal cortex and certain limbic nuclei. Footshock stress is known to activate the A10 DA neurons projecting to the prefrontal cortex and nucleus accumbens, and the present study was designed to evaluate the possibility that footshock-induced release of enkephalin into the A10 DA region may play a role in activating the DA neurons. Microinjection of the quaternary opioid antagonist, naltrexone methobromide (NMB), into the ventral tegmental area (VTA; subnucleus of the A10 DA region) significantly attenuated the increase in DA metabolism produced by exposure to footshock (0.2 mA; 200 ms on; 800 ms off for 20 min) in the prefrontal cortex and nucleus accumbens. Rats were exposed to footshock for 5, 10 or 20 min and a time-dependent decrease in the level of immunoreactive Met-enkephalin was measured in the midline A10 region, but not in the lateral A10 region. It has been shown that daily exposure to footshock enhances the motor stimulant effect of intra-VTA injection of the enkephalin analogue, [D-Ala2,Met]-enkephalinamide (DALA). Rats were pretreated with an intra-VTA injection of NMB prior to daily exposure to footshock, and it was found that NMB abolished the potentiating effect of daily footshock on subsequent intra-VTA injection of DALA. Taken together, these data indicate that footshock stress enhances the release of enkephalin into the A10 region, and that this enkephalin activates A10 DA neurons projecting to the prefrontal cortex and nucleus accumbens.

Topics: Animals; Dopamine; Electroshock; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Methionine; Enkephalins; Frontal Lobe; Male; Naltrexone; Narcotic Antagonists; Nucleus Accumbens; Quaternary Ammonium Compounds; Rats; Rats, Inbred Strains; Septal Nuclei; Stress, Physiological; Tegmentum Mesencephali