7-benzylidenenaltrexone and norbinaltorphimine

We studied the role of opioid receptor subtypes in improvement of the functional state of the heart during reperfusion after adaptation to continuous normobaric hypoxia. To this end, male Wistar rats were subjected to continuous normobaric hypoxia (12% O

Topics: Adaptation, Physiological; Animals; Benzylidene Compounds; Creatine Kinase; Hypoxia; Male; Myocardial Reperfusion Injury; Myocardium; Naloxone; Naltrexone; Narcotic Antagonists; Oligopeptides; Organ Culture Techniques; Peptides; Rats; Rats, Wistar; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Tetrahydroisoquinolines

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.

Topics: Animals; Benzylidene Compounds; Carcinogens; Dose-Response Relationship, Drug; Enkephalin, Methionine; Hydrogen Peroxide; Macrophages, Peritoneal; Male; Naltrexone; Narcotic Antagonists; Rats; Rats, Wistar; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Tetradecanoylphorbol Acetate

Opioid receptors display basal signaling (constitutive, agonist-independent activity), which seems to be regulated by agonist exposure. Whereas agonist pretreatment desensitizes receptors to subsequent agonist stimulation, basal signaling of mu-opioid receptor (MOR) was shown to increase. Moreover, agonist pretreatment converts the neutral antagonists naloxone and naltrexone into inverse agonists, suppressing basal signaling, whereas analogs with reduced C6-position, e.g., 6beta-naltrexol, remain neutral antagonists at MOR under any condition. This study compares the regulation of basal signaling of MOR, delta-(DOR), and kappa-(KOR) opioid receptors after pretreatment with morphine or receptor-selective agonists, in transfected human embryonic kidney 293 cell membranes. Moreover, naloxone, naltrexone, and related antagonists were compared for binding potency and effect on basal and agonist-stimulated receptor signaling, measuring guanosine 5'-O-(3-[35S]thio)triphosphate binding. The results demonstrate basal activity for each opioid receptor, which is modulated by pretreatment with agonists. Even closely related opioid antagonists display distinct patterns of neutral and inverse effects before and after agonist pretreatment, including distinct efficacies between naloxone and naltrexone at agonist-pretreated DOR and KOR. Pretreatment with different agonists has varying effects on inverse and neutral activities of some analogs tested. These results demonstrate that antagonist efficacy is context-dependent, possibly accounting for paradoxical pharmacological effects. Activity profiles at the three opioid receptors under different conditions could lead to antagonists with optimal clinical properties in treatment of addiction and adverse opioid effects.

Topics: Benzylidene Compounds; Cells, Cultured; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Morphine; Naltrexone; Narcotic Antagonists; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Signal Transduction

The existence of heterodimeric opioid receptors has introduced greater complexity to the in vivo characterization of pharmacological selectivity of agonists by antagonists. Because of the possibility of cooperativity between receptors organized as heterodimers, it is conceivable that selective antagonists may antagonize an agonist bound to a neighboring, allosterically coupled receptor. As a consequence, the in vivo selectivity of an opioid antagonist may depend on the organizational state of receptors that mediate analgesia. In this regard, phenotypic delta- and kappa-opioid receptors have been proposed to arise from different organizational states that include oligomeric delta-kappa heterodimers and homomeric delta and kappa receptors. In view of the evidence for analgesia mediated by delta-kappa heterodimers in the spinal cord, but not the brain, we have investigated the selectivity of pharmacologically selective delta and kappa antagonists in mice by both i.t. and i.c.v. routes of administration to evaluate changes in selectivity. Using pharmacologically selective delta (benzylidenenaltrexone, naltrindole, and naltriben) and kappa (norbinaltorphimine) antagonists versus delta ([D-Pen(2),D-Pen(5)]-enkephalin and deltorphin II) and kappa [3,4-dichloro-N-methyl-N-[(1R,2R)-2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide (U50488) and bremazocine] agonists, the delta-1/delta-2 selectivity ratios were found to be dependent on the route of administration (i.t. versus i.c.v.). The data from different routes of administration suggest that differences in molecular recognition between spinal delta-kappa heterodimers and supraspinal homomeric delta and kappa receptors may contribute to the divergent selectivity ratios of selective antagonists. In view of the observed tissue-dependent selectivity, we suggest that multiple opioid antagonists be employed routinely in establishing agonist selectivity in vivo.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Benzylidene Compounds; Enkephalin, D-Penicillamine (2,5)-; Injections, Intraventricular; Injections, Spinal; Ligands; Male; Mice; Mice, Inbred ICR; Naltrexone; Narcotic Antagonists; Receptors, Opioid, delta; Receptors, Opioid, kappa

We investigated whether delta- and kappa-opioid agonists alter myocardial function, intracellular Ca(2+) concentration ([Ca(2+)](i)), and myofilament Ca(2+) sensitivity in intact guinea pig beating hearts and whether these effects are mediated by an opioid receptor. Intact guinea pig hearts were perfused with modified Krebs Ringer solution containing delta- (TAN-67) and kappa- (ICI-199441) opioid agonists in the absence and presence of delta- (BNTX) and kappa- (nor-BNI) opioid antagonists, respectively, while functional variables and [Ca(2+)](i) were recorded. TAN-67 (1 microM) and ICI-199441 (1 microM) decreased heart rate (P < 0.05). TAN-67 (1 microM) and ICI-199441 (1 micro M) decreased available [Ca(2+)](i) without changing developed left ventricular pressure (LVP) (P < 0.05). TAN-67 (1 microM) and ICI-199441 (1 microM) also caused a leftward shift in the curve of developed LVP as a function of available [Ca(2+)](i) (P < 0.05). ICI-199441 (1 microM) produced a steeper slope in the relation curve compared with baseline (P < 0.05). BNTX (1 microM) and nor-BNI (1 microM) blocked the effects of TAN-67 and ICI-199441, respectively. delta- and kappa-opioid agonists enhance myofilament Ca(2+) sensitivity despite decreasing available [Ca(2+)](i) in intact isolated guinea pig hearts, and these effects are mediated by delta- and kappa-opioid receptor stimulation.. Our results indicate that delta- and kappa-opioid agonists enhance myofilament Ca(2+) sensitivity despite decreasing available intracellular Ca(2+) concentrations in intact isolated guinea pig beating hearts, and these effects are mediated by delta- and kappa-opioid receptor stimulation.

Topics: Actin Cytoskeleton; Animals; Benzylidene Compounds; Blood Pressure; Calcium; Coronary Circulation; Guinea Pigs; Heart; Heart Rate; In Vitro Techniques; Kinetics; Naltrexone; Narcotic Antagonists; Pyrrolidines; Quinolines; Receptors, Opioid, delta; Receptors, Opioid, kappa; Stimulation, Chemical; Ventricular Function, Left

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

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

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

Pain thresholds are elevated during gestation and following the simulation of pregnancy blood levels of estrogen and progesterone (hormone simulated pregnancy; HSP). The analgesia associated with both conditions is opioid-mediated and results from the activation of spinal cord kappa and delta (but not mu) opiate receptors. Blockade of spinal kappa or delta opiate receptors, individually, can abolish the antinociception associated with either gestational day 20 or day 19 of HSP. Surprisingly, during either physiological pregnancy or HSP, the magnitude of reduction in the increment in jump thresholds following the combined intrathecal application of suboptimum concentrations of kappa and delta antagonists is indistinguishable from that observed following their individual intrathecal application. These data indicate that gestational and ovarian sex steroid-induced antinociception is not simply the sum of the independent analgesic effects of spinal kappa and delta opioid systems but requires their coincident activation. It is suggested that the synergy that has been reported following the exogenous intrathecal application of kappa and delta opioids also occurs between their endogenous counterparts and underlies the intrinsic analgesia associated with each condition. Utilization of such a mechanism allows for significant physiological effects (analgesia) to be achieved with doses of relevant substrates (dynorphin and enkephalin) which alone would produce minimal receptor activation (and analgesia). This would minimize tolerance and dependence formation.

Topics: Animals; Benzylidene Compounds; Dose-Response Relationship, Drug; Estrogens; Female; Naltrexone; Narcotic Antagonists; Ovary; Pain Threshold; Pregnancy; Pregnancy, Animal; Progesterone; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, kappa; Spinal Cord

The effects of selective mu-, delta- and kappa-opioid receptor agonists and antagonists on the discriminative stimulus properties of methamphetamine were examined in rats that had been trained to discriminate between methamphetamine (0.4 mg/kg) and saline. Methamphetamine produced a dose-related increase in methamphetamine-appropriate responses in all of the rats. In generalization tests, neither morphine (a mu-opioid receptor agonist: 0.3-10 mg/kg) nor 3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexo]benzeneacetamide (U50,488H: a kappa-opioid receptor agonist: 1.0-8.0 mg/kg) generalized to the discriminative stimulus properties of methamphetamine. A newly synthesized non-peptide selective delta-opioid receptor agonist 2-methyl-4aalpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aalpha- octahydroquinolino(2,3,3,-g)isoquinoline (TAN-67: 32 mg/kg) partially generalized (70% methamphetamine-appropriate responses) to the discriminative stimulus properties of methamphetamine. In combination tests, pretreatment with the mu- and kappa-opioid receptor antagonists, beta-funaltrexamine (9.0 mg/kg) and nor-binaltorphimine (10 mg/kg), respectively, had little or no influence on the discriminative stimulus properties of methamphetamine. In contrast, pretreatment with naltrindole (a non-selective delta-opioid receptor antagonist: 3.0 mg/kg) or naltriben (a selective delta2-opioid receptor antagonist: 1.0 mg/kg), but not with 7-benzylidenenaltrexone (a selective delta1-opioid receptor antagonist: 0.5 and 1.0 mg/kg), significantly attenuated the discriminative stimulus properties of methamphetamine. However, naltrindole (3.0 mg/kg) did not significantly attenuate the discriminative stimulus properties of methamphetamine at a higher training dose (1.0 mg/kg). Our findings may have some bearing on the relative importance of the role of delta-opioid (especially delta2-opioid) receptors in the discriminative stimulus properties of a low dose of methamphetamine.

Topics: Animals; Benzylidene Compounds; Central Nervous System Stimulants; Discrimination, Psychological; Dose-Response Relationship, Drug; Male; Methamphetamine; Naltrexone; Quinolines; Rats; Rats, Inbred F344; Receptors, Opioid, delta

The antinociception induced by beta-endorphin given supraspinally has been previously demonstrated to be mediated by the release of [Met5]enkephalin acting on delta-opioid receptors in the spinal cord. The present study was designed to determine what type of opioid receptors in the spinal cord is involved in beta-endorphin-induced antinociception in the rat. Antinociception was induced by beta-endorphin (0.6 nmol) given into nucleus raphe obscurus and was assessed by the tail-flick test in pentobarbital-anesthesized rats. Naltriben (0.6-6.0 nmol), a selective delta 2-opioid receptor antagonist, given intrathecally dose-dependently attenuated beta-endorphin-induced inhibition of the tail-flick response. On the other hand, 7-benzylidene naltrexone (2.1-64.3 nmol), CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, 0.09-2.8 nmol), or nor-binaltorphimine (1.4-40.8 nmol), selective delta 1-, mu-, and kappa-opioid receptor antagonists, respectively, did not block beta-endorphin-induced antinociception. The results of present study in rats are consistent with previous experiments in mice indicating that spinal delta 2-, but not delta 1-, mu- or kappa-opioid receptors are involved in beta-endorphin-induced inhibition of the tail-flick response.

Topics: Amino Acid Sequence; Analgesia; Animals; Benzylidene Compounds; beta-Endorphin; Dose-Response Relationship, Drug; Enkephalin, Methionine; Injections, Intraventricular; Injections, Spinal; Male; Molecular Sequence Data; Naltrexone; Narcotic Antagonists; Pentobarbital; Raphe Nuclei; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Somatostatin; Spinal Cord; Tail

beta-Endorphin(1-27) (i.c.v.) has been reported to inhibit the antinociceptive activity of i.c.v. administered beta-endorphin in mice. In this study the antagonist activity of beta-endorphin(1-27) has been confirmed and the antagonism appears to be mediated at delta 1 opioid receptors. At higher doses than that used for antagonism, i.c.v. administered beta-endorphin(1-27) was a full antinociceptive agonist. The antinociceptive activity of beta-endorphin is attributed to the release of met-enkephalin in the spinal cord and is antagonized by the selective delta 2 opioid receptor antagonist, naltriben (NTB) but not by the selective delta 1 opioid receptor antagonist, 7-benzylidenenaltrexone (BNTX). In contrast, the antinociceptive activity of i.c.v. administered beta-endorphin(1-27) was not affected by either NTB or BNTX administered i.c.v. or i.t. Also, the antinociceptive activity of beta-endorphin(1-27) was unaffected by the selective mu opioid receptor antagonist, beta-funaltrexamine (beta-FNA) or the selective kappa opioid receptor antagonist, norbinaltorphimine (norBNI). Thus, beta-endorphin(1-27) appears to mediate antinociception supraspinally through the interaction of a unique receptor, i.e. a receptor that is different from mu, kappa, delta 1 or delta 2 opioid receptors. Alternatively, a non-opioid mechanism may be considered.

Topics: Animals; Benzylidene Compounds; beta-Endorphin; Male; Mice; Naltrexone; Narcotic Antagonists; Nociceptors; Peptide Fragments; Receptors, Opioid