enkephalin--ala(2)-mephe(4)-gly(5)- and naltrindole-benzofuran

In view of the co-localization of spinal delta- and kappa-opioid receptors, we have investigated the interaction of selective opioid receptor agonists and antagonists in the spinal cord of mice in order to determine if these receptors are organized as heteromers. The finding that norbinaltorphimine (kappa) antagonized [D-Pen(2,5)]enkephalin (delta(1)), but not deltorphin II (delta(2)), strongly suggests that the putative delta(1)-subtype is a delta-kappa heteromer. Studies with selective opioid receptor (ant)agonists support this conclusion.

Topics: Animals; Benzylidene Compounds; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Immune Sera; Mice; Naltrexone; Oligopeptides; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Somatostatin; Spinal Cord

In the intestine, opioids produce antidiarrhoeal and constipating actions that are mediated by enteric neurones. Through interactions with opioid receptors (ORs) on submucosal neurones, opioids suppress active ion transport evoked by transmural electrical stimulation (TES) in mucosa-submucosa sheets from the porcine ileum. In this study, we examined the pharmacological characteristics of the previously described OR, which is sensitive to the delta1-OR antagonist 7-benzylidenenaltrexone and modulates neurogenic transepithelial ion transport in this tissue preparation. Increases in short-circuit current (Isc, a measure of active anion transport) evoked by TES in ileal mucosa-submucosa sheets were inhibited by opioid agonists possessing high selectivity for either delta- or micro-ORs including [d-Pen2,5]enkephalin (DPDPE), [d-Ala2, Glu4]deltorphin II, and [d-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO). As determined by the Schild analysis, the actions of these agonists were competitively inhibited by 7-benzylidenenaltrexone. The nonequilibrium micro-OR antagonist beta-funaltrexamine inhibited the actions of DAMGO only at a high concentration (1 microm) but did not alter DPDPE or deltorphin II action. At concentrations up to 10 microm, the nonequilibrium delta-OR antagonist naltrindole 5'-isothiocyanate did not alter the actions of delta- or micro-OR agonists. Radioligand binding analyses of neuronal homogenates from the ileal submucosa revealed that the nonselective OR ligand [3H]diprenorphine bound to two populations of specific binding sites. One of these sites possessed binding characteristics similar to the delta-OR. In summary, neurogenic ion transport in the porcine intestine is modulated by an OR which shares pharmacological characteristics of both micro- and delta-ORs and may represent a novel receptor entity.

Topics: Amphibian Proteins; Animals; Anions; Benzamides; Benzylidene Compounds; Biological Transport, Active; Carrier Proteins; Diprenorphine; Dose-Response Relationship, Drug; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Female; Gastrointestinal Motility; Ileum; Intestinal Mucosa; Male; Naltrexone; Neurons, Afferent; Oligopeptides; Piperazines; Quinolines; Receptors, Opioid, delta; Receptors, Opioid, mu; Swine; Tritium

Naltriben (NTB) has been used to differentiate the subtypes of delta opioid receptors, delta1 and delta2. However, there is considerable evidence suggesting that NTB may act on other types of opioid receptors too. We examined the effects of NTB on the specific binding of radiolabeled ligands for opioid mu and kappa2 receptors, and the effects on the release of [3H]norepinephrine ([3H]NE) in rat cerebral cortex slices. NTB displaced the specific binding of [3H]DAMGO with Ki value of 19.79 +/- 1.12 nM in rat cortex membranes. Specific binding of [3H]diprenorphine ([3H]DIP) was inhibited by NTB with Ki value of 82.75 +/- 6.32 nM in the presence of DAMGO and DPDPE. High K+ (15 mM)-stimulated release of [3H]NE was attenuated by DAMGO in rat cerebral cortex slices. NTB (30 nM) shifted the dose-response curve of DAMGO to the right and attenuated the maximal effect. In the meantime, NTB inhibited high K+-stimulated [3H]NE release at concentrations above 100 nM. The inhibitory effect of NTB was not attenuated by CTAP (10 nM) and naloxone (3 nM) but by higher concentration of naloxone (30 nM), nor-BNI (300 nM) and bremazocine (3 nM). These results indicate that NTB, depending on the dosage, could acts not only as an antagonist at delta but also as a noncompetitive antagonist for mu receptors, and as an agonist for kappa2 receptors in rat cerebral cortex.

Topics: Animals; Cerebral Cortex; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Ligands; Male; Naloxone; Naltrexone; Narcotic Antagonists; Norepinephrine; Peptide Fragments; Peptides; Potassium; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Somatostatin; Tritium

This study examined a mechanism responsible for the enhanced antihyperalgesic and antinociceptive effects of the mu opioid receptor agonist (ORA) [D-Ala(2), NMePhe(4), Gly(5)-ol]enkephalin (DAMGO) microinjected in the rostroventromedial medulla (RVM) of rats with inflammatory injury induced by injection of complete Freund's adjuvant (CFA) in one hindpaw. In rats injected with CFA 4 hr earlier, microinjection of the mu opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP) in the RVM antagonized both the marginal enhancement of the potency of DAMGO and its antinociceptive effect. The delta opioid receptor antagonist naltriben (NTB) was without effect. In rats injected with CFA 2 weeks earlier, CTAP antagonized the effects of DAMGO to a lesser extent. However, NTB completely prevented the enhancement of the potency of DAMGO, whereas it did not antagonize DAMGO's antinociceptive effects. Microinjection of NTB alone, but not CTAP in the RVM of CFA-treated rats, enhanced the hyperalgesia present in the ipsilateral hindpaw and induced hyperalgesia in the contralateral, uninjured hindpaw. These results suggest that persistent inflammatory injury increased the release in the RVM of opioid peptides with preferential affinity for the delta opioid receptor, which can interact in a synergistic or additive manner with an exogenously administered mu opioid receptor agonist. Indeed, the levels of [Met(5)]enkephalin and [Leu(5)]enkephalin were increased in the RVM and in other brainstem nuclei in CFA-treated rats. This increase most likely presents a compensatory neuronal response of the CNS of the injured animal to mitigate the full expression of inflammatory pain and to enhance the antinociceptive and antihyperalgesic effects of exogenously administered mu opioid receptor analgesics.

Topics: Animals; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Inflammation; Male; Microinjections; Naltrexone; Pain; Peptide Fragments; Peptides; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Somatostatin

Two highly selective mu-opioid receptor agonists, endomorphin-1 and endomorphin-2, have been identified and postulated to be endogenous ligands for mu-opioid receptors. Intrathecal (i.t.) administration of endomorphin-1 and endomorphin-2 at doses from 0.039 to 5 nmol dose-dependently produced antinociception with the paw-withdrawal test. The paw-withdrawal inhibition rapidly reached its peak at 1 min, rapidly declined and returned to the pre-injection levels in 20 min. The inhibition of the paw-withdrawal responses to endomorphin-1 and endomorphin-2 at a dose of 5 nmol observed at 1 and 5 min after injection was blocked by pretreatment with a non-selective opioid receptor antagonist naloxone (1 mg/kg, s.c.). The antinociceptive effect of endomorphin-2 was more sensitive to the mu (1)-opioid receptor antagonist, naloxonazine than that of endomorphin-1. The endomorphin-2-induced paw-withdrawal inhibition at both 1 and 5 min after injection was blocked by pretreatment with kappa-opioid receptor antagonist nor-binaltorphimine (10 mg/kg, s.c.) or the delta(2)-opioid receptor antagonist naltriben (0.6 mg/kg, s.c.) but not the delta(1)-opioid receptor antagonist 7-benzylidine naltrexone (BNTX) (0.6 mg/kg s.c.). In contrast, the paw-withdrawal inhibition induced by endomorphin-1 observed at both 1 and 5 min after injection was not blocked by naloxonazine (35 mg/kg, s.c.), nor-binaltorphimine (10 mg/kg, s.c.), naltriben (0.6 mg/kg, s.c.) or BNTX (0.6 mg/kg s.c.). The endomorphin-2-induced paw-withdrawal inhibition was blocked by the pretreatment with an antiserum against dynorphin A-(1-17) or [Met(5)]enkephalin, but not by antiserum against dynorphin B-(1-13). Pretreatment with these antisera did not affect the endomorphin-1-induced paw-withdrawal inhibition. Our results indicate that endomorphin-2 given i.t. produces its antinociceptive effects via the stimulation of mu (1)-opioid receptors (naloxonazine-sensitive site) in the spinal cord. The antinociception induced by endomophin-2 contains additional components, which are mediated by the release of dynorphin A-(1-17) and [Met(5)]enkephalin which subsequently act on kappa-opioid receptors and delta(2)-opioid receptors to produce antinociception.

Topics: Analgesics; Animals; Benzylidene Compounds; Dose-Response Relationship, Drug; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Methionine; Immune Sera; Injections, Spinal; Injections, Subcutaneous; Male; Mice; Naloxone; Naltrexone; Narcotic Antagonists; Oligopeptides; Pain; Pain Measurement; Pain Threshold; Peptide Fragments; Time Factors

Exposure to stressful or fear-inducing environmental stimuli activates descending antinociceptive systems resulting in a decreased pain response to peripheral noxious stimuli. Stimulating mu opioid receptors in the basolateral nucleus of the amygdala (BLA) in anesthetized rats produces antinociception that is similar to environmentally induced antinociception in awake rats. Recent evidence suggests that both forms of antinociception are mediated via projections from the amygdala to the ventral periaqueductal gray (PAG). In the present study, we examined the types of neurochemicals released in the ventral PAG that may be important in the expression of antinociception produced by amygdala stimulation in anesthetized rats. Microinjection of a mu opioid receptor agonist into the BLA resulted in a time dependent increase in tail flick latency that was attenuated by preadministration of a mu opioid receptor or a neurotensin receptor antagonist into the ventral PAG. Microinjection of a delta(2) opioid receptor antagonist or an NMDA receptor antagonist into the ventral PAG was ineffective. These findings suggest that amygdala stimulation produces antinociception that is mediated in part by opioid and neurotensin release within the ventral PAG.

Topics: Amygdala; Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Male; Naltrexone; Narcotic Antagonists; Neural Pathways; Neurons; Neurotensin; Nociceptors; Pain; Peptides; Periaqueductal Gray; Rats; Rats, Long-Evans; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotensin; Receptors, Opioid, delta; Receptors, Opioid, mu; Time Factors; Valine

The present study tested the hypothesis that endogenous opioid peptides acting at the delta-opioid receptor (DOR) in the rostral ventromedial medulla (RVM) contribute to the antinociception elicited by the mu-opioid receptor (MOR) agonist DAMGO in the midbrain periaqueductal gray (PAG). Following microinjection of DAMGO into the PAG, either the highly selective DOR antagonist TIPP[psi] or the DOR2 antagonist naltriben (NTB) was microinjected into the RVM. Both TIPP[psi] (1.0 microg) and NTB (5.0 ng) significantly attenuated the analgesic effect of PAG DAMGO but had no effect when given before PAG saline. These results confirm and extend previous studies suggesting that PAG mu-opioids activate a descending system with a DOR mediated endogenous opioid link in the RVM.

Topics: Analgesics; Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Male; Medulla Oblongata; Naltrexone; Narcotic Antagonists; Neural Pathways; Oligopeptides; Periaqueductal Gray; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu; Tetrahydroisoquinolines

Our laboratory has previously shown that delta-opioid receptors are involved in the cardioprotective effect of ischemic preconditioning in the rat heart. However, this class of receptors consists of two subtypes, delta1, and delta2, and mu- or kappa-opioid receptors may also exist in the heart. Therefore, the purpose of the present study was to test the hypothesis that ischemic preconditioning is mediated through stimulation of one or both delta-opioid receptor subtypes.. Anesthetized, open chest, male Wistar rats were assigned to 1 of 14 groups. All animals were subjected to 30 minutes of occlusion and 2 hours of reperfusion. Ischemic preconditioning was elicited by three 5-minute occlusion periods interspersed with 5 minutes of reperfusion. Two doses of 7-benzylidenenaltrexone (BNTX; 1 and 3 mg/kg i.v.), a selective delta1-opioid receptor antagonist, or naltriben (NTB; 1 and 3 mg/kg i.v.), a selective delta2-opioid receptor antagonist, were given before ischemic preconditioning. To test for a role of mu-opioid receptors, rats were pretreated with beta-funaltrexamine (beta-FNA; 15 mg/kg s.c), an irreversible mu-opioid receptor antagonist, 24 hours before ischemic preconditioning or given the mu-opioid receptor agonist D-Ala,2N-Me-Phe,4glycerol5-enkephalin (DAMGO) as three 5-minute infusions (1, 10, and 100 microg/kg per infusion i.v., respectively) interspersed with 5-minute drug-free periods before the prolonged ischemic and reperfusion periods (lowDAMGO, medDAMGO, and hiDAMGO, respectively). The involvement of kappa-opioid receptors was tested by administering one of two doses of nor-binaltorphimine (nor-BNI; 1 and 5 mg/kg i.v.) before ischemic preconditioning. Infarct size (IS) as a percent of the area at risk (AAR) was measured by triphenyltetrazolium stain. Ischemic preconditioning markedly reduced IS/AAR (14+/-4%, P<.05) compared with control (55+/-4%). NTB, beta-FNA, and nor-BNI were unable to block the cardioprotective effect of ischemic preconditioning. In addition, DAMGO had no effect on IS/AAR. However, the high dose of BNTX (3 mg/kg i.v.) significantly attenuated the cardioprotective effect of ischemic preconditioning (39+/-5%; P<.05 versus control and ischemic preconditioning).. These results indicate that delta1-opioid receptors play an important role in the cardioprotective effect of ischemic preconditioning in the rat heart.

Topics: Animals; Benzylidene Compounds; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Hemodynamics; Ischemic Preconditioning, Myocardial; Male; Myocardial Infarction; Naltrexone; Narcotic Antagonists; Rats; Rats, Wistar; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu

As part of the continuing investigation of the role of spinal delta opioid receptors in antinociception, this study characterized the ability of 7-benzylidenenaltrexone (BNTX), a selective delta-1 opioid receptor antagonist, to antagonize the antinociception produced in the rat by intrathecal (i.t.) administration of the respective delta-1 and delta-2 opioid receptor agonists, DPDPE and [D-Ala2, Glu4]deltorphin (DELT), or the mu receptor agonist DAMGO. In the tail flick test, 10-min pretreatment with 1 microgram of BNTX, increased the ED50 value of DPDPE from 27.5 micrograms (42.6 nmol) to 114.8 micrograms (177.8 nmol), but did not increase the ED50 values of either DELT or DAMGO. Increasing the dose of BNTX to 3 micrograms did not produce a significantly greater antagonism of the antinociceptive effects of DPDPE and did not antagonize the antinociceptive effects of DAMGO. However, it did enhance the antinociceptive effects of DELT decreasing its ED50 from 5.3 to 0.18 micrograms in the tail flick test. In the hot plate test, 10 min pretreatment with 1 microgram of BNTX selectively antagonized the antinociceptive effects of DPDPE, but did not antagonize the actions of DAMGO or DELT. Increasing the dose of BNTX to 3 micrograms also did not produce a significantly greater antagonism of the antinociceptive effects of DPDPE in the hot plate test, but did antagonize both the increase in hot plate latency and the modest decrement in motor function produced by 30 micrograms i.t. of DELT. However, the antagonism of these effects of DELT occurred much later in time than BNTX's antagonism of the antinociceptive effects of DPDPE.

Topics: Analgesics; Animals; Benzylidene Compounds; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Spinal; Male; Naltrexone; Narcotic Antagonists; Oligopeptides; Pain Threshold; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Spinal Cord

The role of spinal delta opioid receptors in mediating antinociception was studied by using the carrageenan-induced model of thermal hyperalgesia. Intrathecal administration of [D-Ala2, Glu4]-deltorphin (DELT), a delta-2 receptor agonist, or DPDPE, a delta-1 receptor agonist, produced a dose-dependent increase in paw-flick latency (PFL) with an ED50 of 14.0 micrograms for DELT and 30.4 micrograms for DPDPE. DAMGO, a mu receptor agonist, also increased the PFL in a dose-dependent manner when administered intrathecally with an ED50 of 0.02 microgram. Each opioid agonist increased the PFL to values that exceeded base-line latencies determined before the injection of carrageenan. However, DELT and DPDPE increased the PFL to a greater extent than did DAMGO. Coadministration of 30 micrograms of naltrindole shifted the dose-effect line of DELT to the right by 3.5-fold and that of DPDPE to the right by 2.5-fold, consistent with its characterization as a mixed delta-1/delta-2 receptor antagonist. Coadministration of 3 micrograms of naltriben (NTB) shifted the dose-effect line of DELT to the right by 3.2-fold, whereas 10 micrograms of NTB shifted the dose-effect line of DELT at least 15-fold to the right. Neither dose of NTB antagonized the effects of DPDPE. These data are consistent with characterization of NTB as a selective delta-2 receptor antagonist. The antinociception produced by DAMGO was noncompetitively antagonized by 30 micrograms of naltrindole and it was competitively antagonized by 10 micrograms of NTB. Thus, although NTB distinguishes between delta-1 and delta-2 opioid receptors, high doses may not effectively distinguish between delta and mu receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carrageenan; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Hyperalgesia; Male; Naltrexone; Oligopeptides; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Spinal Cord

The antinociception induced by beta-endorphin given intracerebroventricularly (i.c.v.) has been previously demonstrated to be mediated by the release of Met-enkephalin and subsequent stimulation of delta receptors in the spinal cord for antinociception. The present study was designed to determine what type of opioid receptor, delta 1 or delta 2, in the spinal cord is involved in i.c.v. beta-endorphin-induced antinociception. Antinociception was assessed by the tail-flick test in male ICR mice. NTB (0.2-20 nmol) and NTI (0.22-2.2 nmol), selective delta 2 receptor antagonists, given intrathecally (i.t.) dose-dependently attenuated i.c.v. beta-endorphin-induced inhibition of the tail-flick response. On the other hand, BNTX (0.02-2.2 nmol), a selective delta 1 receptor antagonist, given i.t., did not block i.c.v. beta-endorphin-induced antinociception. The tail-flick inhibition induced by DAMGO, a mu receptor agonist, or U50,488H, a kappa receptor agonist, was not blocked by i.t. BNTX, NTB or NTI. It is concluded that delta 2 but not delta 1 receptors in the spinal cord are involved in i.c.v. beta-endorphin-induced antinociception.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Benzylidene Compounds; beta-Endorphin; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Injections, Intraventricular; Injections, Spinal; Male; Mice; Mice, Inbred ICR; Naltrexone; Narcotic Antagonists; Nociceptors; Pain Measurement; Pyrrolidines; Receptors, Opioid, delta; Spinal Cord

This study characterized the antinociception produced by intrathecal (i.t.) administration of the respective delta-2 and delta-1 receptor-selective agonists, [D-Ala2, Glu4]deltorphin (DELT) and DPDPE or the mu receptor selective agonist DAMGO in the rat. It also determined whether the antinociception produced by these opioid agonists was differentially affected by i.t. coadministration of the delta-2 receptor-selective antagonist, naltriben (NTB). In the tail-flick test, the ED50 values of DELT and DPDPE were 2.7 micrograms (3.4 nmol) and 19.0 micrograms (29.4 nmol), respectively. Coadministration of 3 micrograms (6.4 nmol) of NTB increased the ED50 of DELT at least 25-fold, but did not significantly increase the ED50 of DPDPE. These findings suggest that: 1) DELT and DPDPE act at different delta opioid receptor subtypes in the rat spinal cord; 2) 3 micrograms of NTB can distinguish these receptor subtypes and 3) activation of either delta-1 or delta-2 receptors is sufficient to produce antinociception in the tail-flick test. Although NTB did not antagonize the increase in tail-flick latency produced by 0.1 to 0.3 microgram of DAMGO, it did antagonize the increase produced by 0.03 microgram of DAMGO resulting in a steeper dose-response relationship. Unlike DPDPE or DAMGO, DELT did not increase hot-plate latency except at a dose that produced adverse motor effects. Coadministration of 3 micrograms of NTB antagonized the increase in hot-plate latency produced by DPDPE, but not DAMGO, suggesting that this delta-1 receptor-selective agonist may also have efficacy at delta-2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Spinal; Male; Naltrexone; Narcotic Antagonists; Oligopeptides; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu; Reflex; Spinal Cord