Naltriben Research Articles

Involvement of TRPM7 Channel on the Induction of Diabetic Neuropathic Pain in Mice: Protective Role of Selenium and Curcumin.

Excessive levels of the mitochondrial reactive oxygen radical (mitSOX) and Ca2+ influx were found to cause neuropathic pain in patients with diabetes mellitus (DM). Naltriben (NLT) and mitSOX activate the transient receptor (TRP) melastatin 7 (TRPM7) channel, but antioxidants and carvacrol inhibit it. Selenium (Se) and curcumin (CRC) have been thoroughly studied for their modulator effects on streptozotocin (STZ)-induced neuropathic pain, apoptosis, and oxidative stress through the blockage of TRP channels in dorsal root ganglion (DRG) neurons. It has not yet been fully understood how Se and CRC protect against STZ-induced neuropathic pain by modulating TRPM7. Here, we assessed how Se and CRC affected the Ca2+ influx, mitSOX-mediated oxidative damage, and apoptosis in the DRGs of mice through modifying TRPM7 activity. Seven groups (control, Se, CRC, STZ, STZ + Se, STZ + CRC, and STZ + Se + CRC) were induced from the 56 male mice. We observed that the STZ-induced stimulation of TRPM7 increased mechanical neuropathic pain (von Frey), thermal neuropathic pain (hot plate), cytosolic Ca2+, TRPM7 current density, TRPM7 expression, lipid peroxidation, mitSOX, cytosolic ROS, apoptosis, caspase-3, caspase-8, and caspase-9 concentrations, whereas Se and CRC therapies diminished the alterations. The STZ-mediated decreases of DRG viability, brain glutathione, glutathione peroxidase, vitamin A, and vitamin E concentrations were also upregulated in the treatment groups by the therapies. These findings collectively imply that an imbalance of neuropathic pain, oxidative neurotoxicity, and apoptosis in the mice is caused by the STZ-mediated activation of TRPM7. However, the downregulation of TRPM7 activity caused by the injections of Se and CRC reduced the neurotoxicity and apoptosis.

Development of Novel δ Opioid Receptor Inverse Agonists without a Basic Nitrogen Atom and Their Antitussive Effects in Mice.

Our previous results showed that naltrindole (NTI) derivatives with certain types of electron-withdrawing groups as an N-substituent showed δ opioid receptor (DOR) inverse agonistic activities. We therefore synthesized N-acylated NTI derivatives 3a-e and observed that N-benzoyl and N-cyclopropanecarbonyl derivatives SYK-736 (3b) and SYK-623 (3c) were DOR full inverse agonists and the N-acryloyl derivative 3d was a DOR partial inverse agonist. SKY-623 was over 110-fold more potent than the reference compound ICI-174,864. Both naltriben (NTB) and 7-benzylidenenaltrexone (BNTX) derivatives with N-benzoyl and N-cyclopropanecarbonyl groups were also DOR full inverse agonists. These N-acylated inverse agonists are interesting compounds because they have no basic nitrogen atom, which has been demonstrated to be an important pharmacophore. NTI and BNTX-type DOR inverse agonists SYK-623 and SYK-723 (12c) showed dose-dependent antitussive effects in a mouse cough model induced by citric acid exposure. The antitussive effects by SYK-623 and SYK-723 were significantly attenuated by pretreatment with DOR agonist SNC80.

Activation of transient receptor potential melastatin 7 (TRPM7) channel increases basal autophagy and reduces amyloid β-peptide

Cerebral accumulation of amyloid β-peptide (Aβ), which is produced from amyloid precursor protein (APP), is the primary cause of Alzheimer's disease (AD). Autophagy recycles cellular components and digests intracellular components including Aβ. The Ca2+- and Mg2+-permeable transient receptor potential melastatin 7 (TRPM7) channel underlies the constitutive Ca2+ influx in some cells. Since we already reported that TRPM7 channel-mediated Ca2+ influx regulates basal autophagy, we hypothesize that the activation of TRPM7 channel could increase basal autophagy and consequently decrease Aβ. In this study, we showed that naltriben (NTB), a specific TRPM7 channel activator, induced Ca2+ influx and activated autophagic signaling in neuroblastoma SH-SY5Y cells. NTB also promoted co-localization of LC3 and APP, and reduced Aβ. Furthermore, we found that an early-onset familial AD-associated presenilin1 ΔE9 (PS1 ΔE9) mutant cells had attenuated basal autophagy. NTB was able to recover autophagy and decrease Aβ in PS1 ΔE9 cells. Our results show that the activating TRPM7 channel may prevent AD-related Aβ neuropathology via modulating Ca2+-regulated basal autophagy.

Functional interaction between HIV-gp120 and opioid system in the preoptic anterior hypothalamus

BackgroundRecently we found that fever (part of HIV-related wasting) is induced by the action of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (gp120) in the preoptic anterior hypothalamus (POAH). As the opioid system plays a role in the pathogenesis of HIV-1, in the present study we sought to examine the capacity of the opioid system to regulate the febrile response induced by gp120. MethodsStainless steel cannulas were stereotactically into the POAH, and a biotelemetry system was used to monitor the body temperature (Tb changes). We examined the in vivo effects of naloxone as well as highly opioid-selective receptor antagonists, on gp120-induced fever. ResultsPretreatment with naloxone or the mu-opioid receptor-selective antagonist, cyclic d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP), significantly delayed the febrile response induced by gp120. In contrast, naltriben (NTB), a selective antagonist for the delta-2 opioid receptor, did not cause any effect on gp120-induced fever. ConclusionThese results (1) provide pharmacologic evidence of a functional in vivo interaction between the opioid system and this viral protein in the POAH and (2) show that mu-opioid receptors can regulate gp120-induced fever.

Sympatholytic delta-2 opioid receptors moderate ganglionic vasomotor control

This study tested the hypothesis that enkephalin increases femoral vascular conductance via the delta-2 phenotype of the opioid receptor (DOR-2) within peripheral sympathetic ganglia. Graded pulses of methionine-enkephalin (ME) were administered (0.03-10 μg/kg) into the terminal aorta of anesthetized dogs proximal to lumbar arteries that perfuse vasomotor ganglia regulating femoral blood flow. Femoral vascular conductance increased sharply (ED50 = 2.6 × 10(-9) mol/kg) accompanied by declines in arterial pressure and femoral vascular resistance. A dose-related increase in arterial pressure preceded each subsequent fall in pressure. The DOR-2 antagonist, naltriben (NTB), abrogated the hyperemic effect of ME (ID50 = 1.4 × 10(-9) mol/kg). DOR-1 blockade (BNTX) was five-fold less effective. The hyperemic effect of ME was also enhanced when sympathetic activity was reflexly increased by bilateral carotid occlusion. The DOR-2 agonist, deltorphin II, produced exaggerated increases in conductance compared with ME that were also reduced by DOR-2 blockade. DOR-1 blockade eliminated the initial pressor responses, exaggerated the subsequent depressor response, increased baseline femoral conductance 10-fold and shifted the ME-mediated hyperemic threshold one dose lower from 0.3 to 0.1 μg/kg, providing indirect support for a competing DOR-1-mediated constriction. Extended exposure to DOR-1 blockade lowered the maximal ME increase in conductance by 30%, suggesting that BNTX reduces the available pool of DOR receptors. In summary, enkephalin mediates a robust hyperemic effect through sympatholytic ganglionic DOR-2 receptors and DOR-1 antagonist studies provide indirect evidence for constituent opposition from a proposed DOR-1-mediated sympathotonic constrictor pathway.

Comparative analysis of cardioprotective and antiarrhythmic properties of opioid receptor agonists in a rat model of myocardial infarction

We examined the effect of opioid receptor (OR) agonists/antagonists and inhibitors on infarct size (IS) and arrhythmia in rats following coronary artery occlusion and reperfusion. Pretreatment with the specific δ2‐OR agonist Deltorphin II reduced the IS and was prevented by treatment with naltrexone, naloxone and the specific δ2‐OR antagonist naltriben (NTB). The infarct‐limiting effect of Deltorphin II was also abolished by inhibition of Protein Kinase C (PKC) and potassium ATP‐sensitive K (KATP) channels but not by the specific δ1‐OR antagonist, 7‐Benzylidennaltrexone (BNTX). All deltorphins tested exhibited antiarrhythmic properties during phase 1b of ischemia, but only Deltorphin II prevented ventricular arrhythmias during phase 1a and phase 1b. The antiarrhythmic effect of deltorphin II was abolished by treatment with naltrexone, naloxone methiodide and the selective δ2‐OR antagonist NTB but not by the selective δ1‐OR antagonist BNTX. PKC inhibition, but not KATP channel inhibition, eliminated the antiarrhythmic effect of Deltorphin II. Pretreatment with other opioids D‐Ala2, N‐Me‐Phe4, Gly5‐ol‐Enkephalin, Dermorphin H, D‐Pen2,5 Enkephalin, Deltorphin‐D, Deltorphin‐E, and U‐50,488, nociception did not reduce the IS or arrhythmias. We conclude that peripheral δ2‐OR activation induces infarct size reduction and prevents ischemia induced ventricular arrhythmias.

Acute and chronic heroin dependence in mice: Contribution of opioid and excitatory amino acid receptors

Opioid and excitatory amino acid receptors contribute to morphine dependence, but there are no studies of their role in heroin dependence. Thus, mice injected with acute or chronic heroin doses in the present study were pretreated with one of the following selective antagonists: 7-benzylidenenaltrexone (BNTX), naltriben (NTB), nor-binaltorphimine (nor-BNI; δ 1, δ 2, and κ opioid receptors, respectively), MK-801, or LY293558 (NMDA and AMPA excitatory amino acid receptors, respectively). Naloxone-precipitated withdrawal jumping frequency, shown here to be a reliable index of heroin dependence magnitude, was reduced by BNTX or NTB in mice injected with both acute and chronic heroin doses. In contrast, nor-BNI did not alter jumping frequencies in mice injected with an acute heroin dose but significantly increased them in mice receiving chronic heroin injections. Continuous MK-801 or LY293558 infusion, but not injection, reduced jumping frequencies during withdrawal from acute heroin treatment. Their delivery by injection was nonetheless effective against chronic heroin dependence, suggesting mechanisms not simply attributable to NMDA or AMPA blockade. These data indicate that whereas δ 1, δ 2, NMDA, and AMPA receptors enable acute and chronic heroin dependence, κ receptor activity limits the dependence liability of chronic heroin. With the exception of δ 1 receptors, the apparent role of these receptors to heroin dependence is consistent with their contribution to morphine dependence, indicating that there is substantial physiological commonality underlying dependence to both heroin and morphine. The ability of κ receptor blockade to differentially alter acute and chronic dependence supports previous assertions from studies with other opioids that acute and chronic opioid dependence are, at least in part, mechanistically distinct. Elucidating the substrates contributing to heroin dependence, and identifying their similarities and differences with those of other opioids such as morphine, may yield effective treatment strategies to the problem of heroin dependency.

Role of δ-opioid receptor subtypes in anxiety-related behaviors in the elevated plus-maze in rats

Recent studies have shown that endogenous opioid systems are associated with the regulation of emotional responses. In particular, it has been reported that delta-opioid receptors act naturally to inhibit stress and anxiety. The present study was designed to examine the possible involvement of opioid delta-receptor subtypes in the anxiety-related behavior in the elevated-plus-maze test. Six-week-old male Lewis rats were used. The total numbers of visits to the closed and open arms and the cumulative time spent and visits in the open arms were determined. Plasma corticosterone levels were measured by enzyme immunoassay. Naltrindole (NTI), a delta-opioid receptor antagonist (3 mg/kg s.c.), induced a significant decrease in the percentages of time spent and visits in the open arms. Naltriben (NTB), a delta2-opioid receptor antagonist (3 mg/kg s.c.), but not 7-benzylidenenaltrexone, a delta1-opioid receptor antagonist, produced similar anxiety-related behaviors in the elevated plus-maze. These effects of NTI and NTB were antagonized by pretreatment with (+)-4-[(aR)-a-((2S,5R)-4-allyl-2,5-dimethyl-1piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80), a delta-opioid receptor agonist. Furthermore, after exposure to the elevated plus-maze, the maximal increase in the plasma corticosterone level in NTI-treated rats was clearly higher than that in vehicle-treated rats. However, when NTI and SNC80 were coadministered, higher levels of plasma corticosterone were not seen after exposure to the elevated plus-maze. These results suggest that endogenous delta2-opioid-receptor-mediated systems are involved in the regulation of anxiety-related behaviors and might play a physiologically important role in the regulation of adrenocortical activity.

δ-Opioid receptor antagonists inhibit GIRK channel currents in acutely dissociated brainstem neurons of rat

In this study, we investigated the effects of δ-opioid receptor antagonists on the G protein-coupled inwardly rectifying potassium (GIRK) channel currents induced by serotonin (5-HT) and noradrenaline (NAd) in the dorsal raphe and the locus coeruleus neurons, respectively. Perforated patch and conventional whole-cell patch clamp recording techniques were used for the study. Neurons were acutely dissociated from neonatal rats. Both naltrindole (NTI) and naltriben (NTB), which are selective δ-antagonists possessing antitussive activity in in vivo animal studies, reversibly inhibited the 5-HT-induced GIRK channel currents ( I 5-HT) in dorsal raphe neurons. This inhibition was concentration-dependent and voltage-independent. The half-maximum inhibitory concentration (IC 50) on I 5-HT was 9.84×10 −5 M for NTI and 1.28×10 −5 M for NTB. The inhibition was not reversed by 10 −5 M DPDPE, a selective δ-opioid receptor agonist. NTI did not affect 50% effective concentration (EC 50) on the concentration–response relationship for 5-HT but inhibited the maximum response. In neurons internally perfused with GTPγS, both NTI and NTB also inhibited the GIRK channel currents irreversibly activated by 5-HT. Furthermore, these antagonists concentration dependently inhibited 10 −6 M NAd-induced currents ( I NAd) in locus coeruleus neurons. The IC 50 of NTI on I NAd was 8.44×10 −5 M, which was close to that on I 5-HT. The results suggest that NTI and NTB, which are δ-opioid receptor antagonists possessing antitussive activity, may inhibit GIRK channel currents through a non-opioid action, and give further support to our idea previously proposed that centrally acting non-narcotic antitussives have a common characteristic of the inhibitory action on GIRK channels.

Profound spinal tolerance after repeated exposure to a highly selective mu-opioid peptide agonist: role of delta-opioid receptors.

Recent studies suggest that delta-opioid receptors play a role in the development of opioid tolerance and led us to hypothesize that highly selective mu-opioid agonists may produce less tolerance. H-2',6'-dimethyltyrosine-D-Arg-Phe-Lys-NH(2) ([Dmt(1)]DALDA) has extraordinary selectivity for mu-receptors (K(i)(delta)/K(i)(mu) > 14,000). Daily administration of [Dmt(1)]DALDA (5 times ED(50); s.c.) for 7 days increased ED(50) 3.6-fold from 0.16 to 0.58 micromol/kg. A higher dose of [Dmt(1)]DALDA (10 times ED(50), every 12 h) for 2.5 days resulted in a 11.7 times increase in the ED(50) (1.9 micromol/kg). Complete cross-tolerance to morphine was observed, with a 3.4- and 15.1-fold shift in the morphine ED(50), respectively. We also compared the extent of spinal versus supraspinal tolerance after repeated s.c. [Dmt(1)]DALDA administration. Five doses of [Dmt(1)]DALDA (10 times ED(50), every 12 h) resulted in a 3.4 times shift in the i.c.v. ED(50) (15.4 versus 4.6 pmol/mouse) but a 44 times shift in the i.t. ED(50) (52.9 versus 1.2 pmol/mouse). Tolerance to [Dmt(1)]DALDA was associated with 30 to 35% reduction in [(3)H][Dmt(1)]DALDA binding in brain and spinal cord. Coadministration of [Dmt(1)]DALDA with delta-antagonist naltriben (NTB) reduced spinal tolerance by 50%. Even after spinal tolerance had been established, addition of a delta-antagonist (NTB or H-Tyr-TicPsi[CH(2)NH]Phe-Phe-OH) significantly enhanced the potency of i.t. [Dmt(1)]DALDA 2- to 4-fold. These results suggest that agonist activation of delta-receptors is not necessary for the development of opioid tolerance; however, delta-receptors play a modulatory role in the maintenance of the tolerant state.

Binding, pharmacological and immunological profiles of the δ-selective opioid receptor antagonist HS 378

HS 378 is a recently developed indolomorphinan with high selectivity and antagonist potency at the δ-opioid receptor. The present study was performed to characterize the opioid binding properties and pharmacological and immunological activity of HS 378 and to compare them with those of two well-known δ-opioid receptor antagonists, naltrindole (NTI) and naltriben (NTB). In vitro opioid receptor binding profiles were determined in rat brain homogenates. HS 378 showed 4.7- and 2.4- fold higher μ/δ selectivity compared to NTI and NTB, respectively. In the [ 35S]GTPγS functional assay carried out in cell lines expressing cloned human opioid receptors, HS 378 was found to be a pure δ-opioid receptor antagonist. In vitro, exposure of HS 378 resulted in an apparent dose-related suppression of concanavalin A induced rat T-lymphocyte proliferation with an IC 50 value of 0.54 μM. NTI showed also immunosuppression with an IC 50 value of 6.93 μM, whereas NTB had no effect. The IC 50 of HS 378 was 13 times lower than that of NTI and 8 times higher than that of cyclosporin A. Taken together, our findings indicate that the small molecule HS 378 has properties that may be of therapeutic value in the setting of human inflammatory diseases.

Contribution of Endogenous Enkephalins to the Enhanced Analgesic Effects of Supraspinal μ Opioid Receptor Agonists after Inflammatory Injury

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.

Pharmacological effects of naltriben as a ligand for opioid μ and κ receptors in rat cerebral cortex

Naltriben (NTB) has been used to differentiate the subtypes of δ opioid receptors, δ 1 and δ 2. 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 μ and κ 2 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 δ but also as a noncompetitive antagonist for μ receptors, and as an agonist for κ 2 receptors in rat cerebral cortex.

Ligand binding profiles of δ-opioid receptor in human cerebral cortex membranes: Evidence of δ-opioid receptor heterogeneity

In this study, receptor binding profiles of opioid ligands for subtypes of opioid δ-receptors were examined employing [ 3H]D-Pen 2,D-Pen 5-enkephalin ([ 3H]DPDPE) and [ 3H]Ile 5,6-deltorphin II ([ 3H]Ile-Delt II) in human cerebral cortex membranes. [ 3H]DPDPE, a representative ligand for δ 1 sites, labeled a single population of binding sites with apparent affinity constant (Kd) of 2.72 ± 0.21 nM and maximal binding capacity (Bmax) value of 20.78 ± 3.13 fmol/mg protein. Homologous competition curve of [ 3H]Ile-Delt II, a representative ligand for δ 2 sites, was best fit by the one-site model (Kd = 0.82 ± 0.07 nM). Bmax value (43.65 ± 2.41 fmol/mg) for [ 3H]Ile-Delt II was significantly greater than that for [ 3H]DPDPE. DPDPE, [D-Ala 2,D-Leu 5]enkephalin (DADLE) and 7-benzylidenaltrexone (BNTX) were more potent in competing for the binding sites of [ 3H]DPDPE than for those of [ 3H]Ile-Delt II. On the other hand, deltorphin II (Delt II), [D-Ser 2,Leu 5,Thr 6]enkephalin (DSLET), naltriben (NTB) and naltrindole (NTI) were found to be equipotent in competing for [ 3H]DPDPE and [ 3H]Ile-Delt II binding sites. These results indicate that both subtypes of opioid δ-receptors, δ 1 and δ 2, exist in human cerebral cortex with different ligand binding profiles.

Enhanced Contractile Responsiveness to Cytosolic Ca2+by Delta-2 Opioid Agonist Deltorphin in Intact Guinea Pig Hearts

S. Fujita, S. C. Smart and D. F. Stowe. Enhanced Contractile Responsiveness to Cytosolic Ca2+by Delta-2 Opioid Agonist Deltorphin in Intact Guinea Pig Hearts. Journal of Molecular and Cellular Cardiology (2000) 32, 1647–1659. Opioid receptor subtypes, δ and κ, are found in cardiac tissue and may play a role in cardiac function. We explored if the synthetic opioidδ2 [D-Ala2]-deltorphin (DTP) and μ peptide agonist [D-Ala2]-enkephalin (DAMGO) alter the left ventricular pressure (LVP) [Ca2+]irelationship in isolated guinea pig hearts. LV phasic [Ca2+]iwas measured from dual fluorescence signals using indo 1. Ca2+transients were corrected and calibrated to n m[Ca2+]i. Diastolic (d), systolic (s) [Ca2+]i, and s–d[Ca2+]iwere plotted v LVP at 0.3 to 6.8 m m[CaCl2]eto assess the association of contractility to Ca2+. Also given were naltriben (NTB) and CTOP, δ2and μ antagonists, and nifedipine (NIF) and thapsigargin (THAP). From a control of 880±95 n m (SEM), DTP decreased s–d[Ca2+]maxto 525±82 n m after DTP and to 405±84 n m after NIF, whereas THAP increased s–d[Ca2+]maxto 1605±275 n m. NTB, 795±33 n m, NTB+DTP, 820±98 n m, DAMGO, 970±82 n m, and DAMGO+CTOP, 830±93 n m, gave values similar to controls. From a control value of 61±4 mmHg, LVPmaxwas increased by DTP to 73±3 mmHg and by THAP to 77±2 mmHg, was unchanged by DAMGO at 48±6 mmHg, and was decreased by NIF to 24±2 mmHg. Compared to the control value of 594±18 n m, less s–d[Ca2+]iwas required to attain 50% s–dLVPmax(curve left shift) with increasing [CaCl2]efor DTP, 407±17 n m, and more was required for THAP, 737±35 n m. DTP raised the slope max of s–dLVPmax(100%) v s–d[Ca2+]iby 2.7-fold. This indicates DTP enhances cardiac performance by enhancing responsiveness to cytosolic Ca2+rather than by raising diastolic Ca2+and subsequently released Ca2+, as does THAP.

Highly delta selective antagonists in the RVM attenuate the antinociceptive effect of PAG DAMGO.

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.

Constitutive activity of the delta-opioid receptor expressed in C6 glioma cells: identification of non-peptide delta-inverse agonists.

1. G-protein coupled receptors can exhibit constitutive activity resulting in the formation of active ternary complexes in the absence of an agonist. In this study we have investigated constitutive activity in C6 glioma cells expressing either the cloned delta-(OP1) receptor (C6delta), or the cloned mu-(OP3) opioid receptor (C6mu). 2. Constitutive activity was measured in the absence of Na+ ions to provide an increased signal. The degree of constitutive activity was defined as the level of [35S]-GTPgammaS binding that could be inhibited by pre-treatment with pertussis toxin (PTX). In C6delta cells the level of basal [35S]-GTPgammaS binding was reduced by 51.9+/-6.1 fmols mg-1 protein, whereas in C6mu; and C6 wild-type cells treatment with PTX reduced basal [35S]-GTPgammaS binding by only 10.0+/-3.5 and 8.6+/-3.1 fmols mg-1 protein respectively. 3. The delta-antagonists N, N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864), 7-benzylidenenaltrexone (BNTX) and naltriben (NTB), in addition to clocinnamox (C-CAM), acted as delta-opioid receptor inverse agonists. Naloxone, buprenorphine, and naltrindole were neutral antagonists. Furthermore, naltrindole blocked the reduction in [35S]-GTPgammaS binding caused by the inverse agonists. The inverse agonists did not inhibit basal [35S]-GTPgammaS binding in C6mu; or C6 wild-type cell membranes. 4. Competition binding assays in C6delta cell membranes revealed a leftward shift in the displacement curve of [3H]-naltrindole by ICI 174,864 and C-CAM in the presence of NaCl and the GTP analogue, GppNHp. There was no change in the displacement curve for BNTX or NTB under these conditions. 5. These data confirm the presence of constitutive activity associated with the delta-opioid receptor and identify three novel, non-peptide, delta-opioid inverse agonists.

Selective in vivo binding of [formula omitted]naltriben to δ-opioid receptors in mouse brain

Naltriben (NTB) is a selective antagonist for the putative δ 2-opioid receptor. We have determined the regional kinetics and pharmacological profile of [ 3 H] naltriben in vivo in mouse brain. After i.v. administration to CD1 mice, [ 3 H] naltriben uptake and retention were high in striatum, cortical regions and olfactory tubercles, and low in superior colliculi and cerebellum. Robust rank order correlation was found between [ 3 H] naltriben uptake in discrete brain regions and prior δ-opioid receptor binding determinations in vitro and in vivo. [ 3 H] Naltriben binding in vivo was saturable, and was blocked by the δ-opioid receptor antagonist naltrindole, but not by the μ-opioid receptor antagonist cyprodime or the κ-opioid receptor agonist ( trans)-(±)-3,4-dichloro- N-methyl- N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide mesylate (U50,488H). ( E)-7-Benzylidenenaltrexone (BNTX), a selective antagonist for the putative δ 1-opioid receptor, was 9.6- to 12.9-fold less potent than naltriben as an inhibitor of [ 3 H] naltriben binding. Thus, the sites labeled by [ 3 H] naltriben in vivo may correspond to the δ 2-opioid receptor subtype. Such assignment is not definitive, particularly considering the 4-fold higher brain uptake of naltriben as compared to ( E)-7-benzylidenenaltrexone. Moreover, the regional distribution of [ 3 H] naltriben in brains from CXB-7/BY (CXBK) mice, a strain that shows supraspinal δ 1- but not δ 2-opioid receptor agonist effects, was quite similar to that found for CD1 mice.

Role of spinal δ1 and δ2 opioid receptors in the antinociception produced by microinjection of l-glutamate in the ventromedial medulla of the rat

This study examined the contribution of spinal δ 1 and δ 2 opioid receptors to the antinociception produced by microinjection of l-glutamate in either the nucleus raphe magnus (NRM) or the nucleus reticularis gigantocellularis pars α (NGCp α) of the rat. Intrathecal (i.t.) pretreatment with 1 μg of 7-benzylidinenaltrexone (BNTX), a δ 1 opioid receptor antagonist, did not antagonize the increase in tail flick latency (TFL) produced by microinjection of l-glutamate in either the NRM or the NGCp α. In contrast, i.t. pretreatment with 3 μg of naltriben (NTB), a δ 2 opioid receptor antagonist, completely antagonized the increase in TFL evoked by microinjection of l-glutamate in the NRM, but did not antagonize the increase in TFL evoked from the NGCp α. These results suggest that the antinociception produced by activation of these bulbospinal pathways is predominantly mediated by spinal δ 2 opioid receptors and that there is little, if any, contribution by spinal δ 1 opioid receptors.

Involvement of δ 1 and δ 2 Opioid Receptor Subtypes in the Development of Physical Dependence on Morphine in Mice

The effects of the highly selective δ opioid receptor antagonists naltrindole (NTI) for δ 1 and δ 2 naltriben (NTB) and naltrindole 5′-isothiocyanate (5′-NTII) for δ 2 and 7-benzylidenenaltrexone (BNTX) for δ 1 on the development of physical dependence on morphine were investigated in mice. Neither NTI (3 mg/kg, sc), NTB (0.5 mg/kg, sc), 5′-NTII (0.5 mg/kg, sc) nor BNTX (0.5 mg/kg, sc) suppressed the antinociception induced by morphine (10 mg/kg, sc). Pretreatment with NTI (3 mg/kg, sc), NTB (0.5, 1.0 mg/kg, sc) or 5′-NTII (0.5, 1.0 mg/kg, sc) during chronic treatment with morphine for 5 days significantly suppressed naloxone-induced body-weight loss in morphine-dependent mice. The incidence of jumping and body shakes in morphine-dependent mice that were pretreated with NTI, NTB or 5′-NTII were significantly lower than with morphine alone. Pretreatment with BNTX (0.5, 1.0 mg/kg, sc) during chronic treatment with morphine also significantly suppressed naloxone-induced body-weight loss in morphine-dependent mice, but this suppression was weaker than that by the antagonists. In contrast to mice that had been pretreated with NTI, NTB or 5′-NTII, the incidence of several withdrawal signs, such as jumping and body shakes, was not significantly affected in morphine-dependent mice that were pretreated with BNTX. These findings suggest that both δ 2 and δ 1 opioid receptors may play important roles in modulating the development of physical dependence on morphine.