Δ-opioid Antagonist Naltrindole Research Articles

Endogenous Opioid Receptor Activation in the Caudal Medullary Raphe Depresses Respiratory Rate in Decerebrate Rabbits

Background The caudal medullary raphe (CMR) is an essential structure in controlling pain, cardiovascular, and respiratory function (1). Partial reversal of systemic opioid-induced respiratory depression was achieved with naloxone injection into the CMR (2). This study investigated the presence of endogenous opioid activity in the CMR. Methods The study was approved by the local Animal Care Committee and conformed to NIH standards. Adult New Zealand White rabbits (3-4kg) were anesthetized, tracheotomized, ventilated (FiO2 0.6, PCO2 45-55 mmHg), decerebrated and vagotomized. Phrenic nerve activity was recorded from the c5 rootlet, time averaged and used to calculate inspiratory (TI) and expiratory (TE) duration, breaths per minute (BPM), and peak phrenic activity (PPA). Using a multibarrel glass micropipette, we performed the following injection protocols into the CMR in the midline between 2 mm caudal and 1.5 mm rostral to the functionally identified preBötzinger Complex (preBC, (3)) and into the middle medullary raphe (middle MR (2), 2.5-3.5mm rostral to the preBC) in separate sets of animals: i) α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 50 μM, 70 nl), stepsize 0.5mm rostro-caudal, 0.5mm dorso-ventral. ii) The non-selective opioid antagonist naloxone (1-5 mM, 700 nl), stepsize 0.5mm rostro-caudal, 1st injection at the ventral edge of neuronal activity, 2nd injection 1.5-2mm dorsal; and iii) the µ-opioid antagonist CTAP and δ-opioid antagonist naltrindole (each 1 mM, 350-700 nl), stepsize same as ii). Statistical comparison with Wilcoxon signed rank test. Median (25-75% range). Results i) In 6 animals, AMPA injection into the CMR caudal to and at the level of the preBC increased respiratory rate by up to 59% and PPA by up to 38% at multiple dorso-ventral levels. This was due to a decrease in TI and TE. In the middle MR, AMPA injection depressed respiratory rate, at times to apnea, through an increase in TI and TE, and also depressed PPA. ii) Naloxone injection into the CMR increased respiratory rate from 30 (23-34) to 37 (30-6) BPM (p=0.031, n=6) through a decrease in TI from 0.9 (0.8-1.0) to 0.7 (0.6-0.8) sec (p=0.031) and TE from 1.2 (1.0-1.6) to 0.9 (0.7-1.2) sec (p=0.031). We observed effects with injections caudal to, at the level of and rostral to the preBC. iii) CTAP injection increased respiratory rate from 26 (22-27) to 34 (29-34) BPM, and subsequent naltrindole injection increased respiratory rate to 43 (37-44) BPM (n=3). In the middle MR, CTAP + naltrindole increased respiratory rate between 2-5 BPM. Conclusion Excitation of the CMR increased respiratory rate over an area of several millimeters. During quiet breathing under moderate hyperoxic hypercapnia in our decerebrate rabbit preparation, respiratory raphe neurons were depressed by endogenous opioid activity. It will be important to further investigate the role of the CMR in opioid-induced respiratory depression. (1) Holtman et al., J Neurosci 1986, 6: 1185-93 (2) Zhang et al., Anesthesiology 2007, 107: 288-97 (3) Cook-Snyder et al., Resp Physiol Neurobiol 2019, 260: 37-52

Delta- and mu-opioid pathways are involved in the analgesic effect of Ocimum basilicum L in mice.

Ocimum basilicum L. is a perennial herb that has been used in traditional Asian Indian medicine for thousands of years as a natural anti-inflammatory, antibiotic, diuretic, and analgesic. The present study was conducted to investigate the analgesic effects of basil essential oil (BEO) in inflammatory pain models and identify underlying mechanisms. We further investigated whether BEO affects physiological pain and motor coordination. The analgesic effects of BEO were assessed in various mouse experimental pain models using formalin, acetic acid, heat, and carrageenan as stimuli. BEO was administered by intraperitoneal injection or inhalation. The involvement of various pathways in the analgesic effect of BEO was assessed by pretreating mice with selective pharmacological inhibitors, administered intraperitoneally. Opioid pathways were tested using the κ-opioid antagonist 5'-guanidinonaltrindole (GNTI; 0.3mg/kg), δ-opioid antagonist naltrindole (NTD; 5mg/kg) and μ-opioid antagonist naloxone (NAL; 8mg/kg); nitric oxide (NO) pathways were tested using the NO synthase inhibitor N-nitro l-arginine methyl ester (L-NAME; 37.5mg/kg) and NO precursor L-arginine (L-Arg; 600mg/kg); and KATP channel pathways were tested using the ATP-sensitive K+ channel blocker, glibenclamide-hippuric acid (GHA, 2mg/kg). Potential effects of BEO on motor coordination were assessed using a rotarod test. BEO exerted analgesic effects in all pain models. Notably, pretreatment with naltrindole, naloxone, or L-arginine significantly reduced the analgesic effects of BEO in the formalin test. BEO increased mean withdrawal latencies in a thermal plantar test at a high dose, but not at lower doses. BEO had no effect on motor coordination. Our findings indicate that the analgesic effects of BEO are primarily mediated by delta- and mu-opioid pathways and further suggest that BEO has potential for development as an analgesic agent for the relief of inflammatory pain.

Noradrenaline induces peripheral antinociception by endogenous opioid release.

The aim of this study was to investigate this involvement in not inflammatory model of pain and which opioid receptor subtype mediates noradrenaline-induced peripheral antinociception. Noradrenaline is involved in the intrinsic control of pain-inducing pro-nociceptive effects in the primary afferent nociceptors. However, inflammation can induce various plastic changes in the central and peripheral noradrenergic system that, upon interaction with the immune system, may contribute, in part, to peripheral antinociception. Hyperalgesia was induced by intraplantar injection of prostaglandin E2 (PGE2, 2μg) into the plantar surface of the right hind paw and the paw pressure test to evaluated the hyperalgesia was used. Noradrenaline (NA) was administered locally into right hind paw of Wistar rat (160-200g) alone and after either agents, α2-adrenoceptor antagonist yohimbine, α1-adrenoceptor antagonist prazosin, β-adrenoceptor antagonist propranolol, μ-opioid antagonist clocinnamox, δ-opioid antagonist naltrindole and κ-opioid antagonist nor-binaltorfimina. In addition, the enkephalinase inhibitor bestatin was administered prior to NA low dose. Intraplantar injection of NA induced peripheral antinociception against hyperalgesia induced by PGE2. This effect was reversed, in dose dependent manner, by intraplantar injection of yohimbine, prazosin, propranolol, clocinnamox and naltrindole. However, injection of nor-binaltorfimina did not alter antinociception of NA after PGE2 hyperalgesia. Bestatin intensified the antinociceptive effects of low-dose of NA. Besides the α2-adrenoceptor, the present data provide evidence that, in absence of inflammation, NA activating α1 and β-adrenoceptor induce endogenous opioid release to produce peripheral antinociceptive effect by μ and δ opioid receptors.

Opioid receptors in the prelimbic cortex modulate restraint stress-induced cardiovascular responses in the rat

The prelimbic cortex (PL) is involved in the control of behavioral and autonomic responses to stress. The present study aimed to investigate whether opioid neurotransmission in the PL modulates autonomic responses evoked by restraint stress (RS). Bilateral microinjection of 0.03, 0.3 and 3 nmol/100 nL of the nonselective opioid antagonist naloxone into the PL reduced pressure and tachycardiac responses evoked by RS. However, no effects were observed after its injection at doses of 0.003 and 30 nmol/100 nL, thus resulting in an inverted U-shaped dose–inhibition curve. Similar to naloxone, the selective μ-opioid antagonist CTAP, and the selective κ-opioid antagonist nor-BNI, also reduced MAP and HR increases induced by RS when injected into the PL, whereas treatment with the selective δ-opioid antagonist naltrindole did not affect the pressor and tachycardiac response caused by RS. Blockade of opioid neurotransmission in the PL did not affect the fall in tail temperature and increase in body temperature induced by RS. The present results confirm the involvement of PL opioid neurotransmission in the modulation of cardiovascular responses evoked during the exposure to an aversive situation, and suggest that responses observed after the blockade of local opioid receptors is due to alterations in PL neuronal activity. Furthermore, these results suggest that a distinct circuitry is involved in modulation of the sympathetic output to different vascular territories.

Role of the endogenous opioid system in modulation of urinary bladder activity by spinal nerve stimulation

The role of the endogenous opioid system in modulation of urinary bladder activity by spinal nerve (SN) stimulation was studied in anesthetized female rats, using the rat model of isovolumetric bladder contraction. SN stimulation at a fixed frequency of 10 Hz attenuated bladder contraction frequency; the magnitude of the inhibition was directly proportional to the current intensity. Neither the κ-opioid antagonist nor-binaltorphimine (2 mg/kg iv) nor the δ-opioid antagonist naltrindole (5 mg/kg iv) attenuated the bladder inhibitory response to SN stimulation. In contrast, the μ-opioid receptor antagonist naloxone (NLX; 0.03 mg/kg iv) blocked the inhibitory responses evoked by SN stimulation at therapeutic current intensities at ≤1 × motor threshold current (Tmot). An action at spinal and supraspinal centers was further confirmed by the ability of intrathecal or intracerebroventricular administration of NLX methiodide to attenuate the bladder inhibitory effects of 1 × Tmot SN stimulation. The magnitude of SN-mediated neuromodulation using therapeutically relevant stimulation intensity (Tmot) is equivalent to 0.16 mg/kg of systemically administered morphine, which produces 50% inhibition of bladder contraction frequency. These results suggest that the inhibitory effects of lower intensity SN stimulation may be mediated through the release of endogenous μ-opioid peptides. Additionally, these data suggest that neuromodulation may offer a mode of treating the symptoms of overactive bladder with efficacy equal to the opioid drugs but without their liability for abuse and dependence.

Opioid receptor characterisation of neuronally stimulated isolated human vas deferens

Rodent vas deferens is routinely used as a native tissue preparation to assess opioid pharmacology of new compounds. The aim of this study was to investigate the effects of a selected number of opioid compounds in the human vas deferens. Stable contractions to electrical field stimulation (EFS) were inhibited by guanethidine (1μM) and tetrodotoxin (TTX, 1μΜ), confirming neuronally induced contractions. Contractile responses to EFS were inhibited by the selective δ-opioid agonists (DPDPE ([D-Pen2,5]enkephalin), PF-391459 (3-{4-[(R)-[(2S,5R)-4-benzyl-2,5-dimethylpiperazin-1-yl](3-hydroxyphenyl)methyl]phenyl}propanoic acid) and SNC-80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide)) (tested from 1ρM to 10μM or maximum), and the μ-opioid agonists DAMGO ([D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin), loperamide and SC-50484 (N-{2-[(N-acetyl-L-phenylalanyl)amino]-1,1-dimethylethyl}-L-tyrosinamide). There was no effect using the selective κ agonist U50488 (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide) (tested from 1ρM to 10μM). The selective δ-opioid antagonist naltrindole (10nM) surmountably antagonised the responses to DPDPE, but not to PF-3911459. Responses to DAMGO were completely abolished in the presence of the μ-opioid antagonist CTAP (3μM), which only weakly antagonised responses to SC-50484. We conclude that under these conditions, δ and μ-opioid receptors, but not κ-opioid receptors, are functional in the neuronally stimulated longitudinal human vas deferens. Additionally, the human vas deferens preparation can be used as part of a drug discovery screening project to assess opioid potency, efficacy and selectivity at native human tissues, thus providing more confidence in translation.

Agonist activity of N-desmethylclozapine at δ-opioid receptors of human frontal cortex

The clozapine metabolite N-desmethylclozapine (NDMC) has been recently shown to act at different neurotransmitter receptors and to display both antagonist and agonist activities. We have previously reported that in cells over-expressing the recombinant δ-opioid receptor NDMC behaved as partial agonist with high intrinsic activity, but its action at the receptors naturally expressed in human brain remained to be investigated. In the present study, we examined whether NDMC was able to bind to and activate δ-opioid receptors in membranes of post-mortem human frontal cortex. In radioligand binding assays, NDMC competition curves displayed high- ( K i = 26 nM) and low-affinity ( K i = 3 µM) components, whose proportion was regulated by guanine nucleotides in an agonist-like fashion. In functional assays, NDMC stimulated [ 35S]GTPγS binding (EC 50 = 905 nM) and inhibited cyclic AMP formation (EC 50 = 590 nM) as effectively as δ-opioid agonists, whereas clozapine was much less potent and efficacious and clozapine N-oxide was completely inactive. The NDMC agonist activity was potently antagonized by the δ-opioid antagonist naltrindole, but not by the µ-opioid receptor antagonist CTAP (D-phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH 2) or the κ-opioid antagonist nor-binaltorphimine. Moreover, blockade of either acetylcholine muscarinic, dopamine D 2 or serotonin 5HT 1A receptors failed to affect NDMC agonist activity. These data demonstrate that at clinically relevant concentrations NDMC behaves as an efficacious agonist at δ-opioid receptors of human frontal cortex.

Mechanistic Investigation of N,N-Diethyl-4-(phenyl-piperidin-4-ylidenemethyl)-benzamide–Induced Insulin Depletion in the Rat and RINm5F Cells

These studies describe the effect of N,N-diethyl-4-(phenyl-piperidin-4-ylidenemethyl)-benzamide (AR-M100390), a delta-opioid agonist, on the pancreas and its mechanisms for pancreatic toxicity. Rats were treated with 5, 100, and 600 micromol/kg of AR-M100390 for 3 and/or 7 days; another group of rats treated with 600 micromol/kg of compound were allowed to recover for 14 days. AR-M100390 (600 micromol/kg) caused vacuolation in the beta-cell of the rat pancreas that was associated with depletion of insulin and hyperglycemia after 7 days of dosing. The loss of insulin by AR-M100390 was due to specific inhibition of rat insulin2 mRNA transcription in vivo. Insulin depletion and hyperglycemia were reversible. The effects of AR-M100390 in rats were reproduced in the rat pancreatic beta-cell line RINm5F, where it inhibited intracellular insulin content and secretion without affecting cell survival. Loss of insulin in vitro was also a result of specific inhibition of insulin2 mRNA transcription and was reversible. Pretreatment of cells with the delta-opioid antagonist naltrindole or pertussis toxin did not reverse loss of insulin in AR-M100390-treated cells suggesting that the effects were not mediated by the delta-opioid receptor. AR-M100390 inhibited KCl-mediated calcium mobilization in RINm5F cells, suggesting that L-type calcium channels found in these cells and in pancreatic beta-cells may partially play a role in the inhibition of insulin secretion by this compound. In summary, the in vitro and in vivo studies suggest that inhibition of insulin by AR-M100390 is due to a combination of inhibition of insulin synthesis and/or release.

Involvement of opioid receptors in the oxytocin-induced antinociception in the central nervous system of rats

Recent studies showed that oxytocin and opioid peptides play important roles in pain modulation at different levels in the central nervous system. The present study was performed to explore whether opioid system is involved in the oxytocin-induced antinociception in the brain of rats. The results showed that: (1) intracerebroventricular injection of oxytocin induced dose-dependent increases in hindpaw withdrawal latencies (HWL) to noxious thermal and mechanical stimulation in rats. (2) The antinociceptive effect of oxytocin was attenuated dose-dependently by intracerebroventricular injection of naloxone, indicating an involvement of opioid system in the oxytocin-induced antinociception. (3) It is interesting that the antinociceptive effect of oxytocin was attenuated by subsequent intracerebroventricular injection of the μ-opioid antagonist β-funaltrexamine (β-FNA) and the κ-opioid antagonist nor-binaltorphimine (nor-BNI), but not the δ-opioid antagonist naltrindole. The results indicate that oxytocin plays an antinociceptive role in the brain of rats; μ- and κ-opioid receptors, not δ-receptors, are involved in the oxytocin-induced antinociception in the central nervous system of rats.

Antinociceptive role of oxytocin in the nucleus raphe magnus of rats, an involvement of μ-opioid receptor

Recent studies showed that oxytocin plays an important role in nociceptive modulation in the central nervous system. The present study was undertaken to investigate the role of oxytocin in antinociception in the nucleus raphe magnus (NRM) of rats and the possible interaction between oxytocin and the opioid systems. Intra-NRM injection of oxytocin induced dose-dependent increases in hindpaw withdrawal latencies (HWLs) to noxious thermal and mechanical stimulation in rats. The antinociceptive effect of oxytocin was significantly attenuated by subsequent intra-NRM injection of the oxytocin antagonist 1-deamino-2- d-Tyr-(Oet)-4-Thr-8-Orn-oxytocin. Intra-NRM injection of naloxone dose-dependently antagonized the increased HWLs induced by preceding intra-NRM injection of oxytocin, indicating an involvement of opioid receptors in oxytocin-induced antinociception in the NRM of rats. Furthermore, the antinociceptive effect of oxytocin was dose-dependently attenuated by subsequent intra-NRM injection of the μ-opioid antagonist β-funaltrexamine (β-FNA), but not by the κ-opioid antagonist nor-binaltorphimine (nor-BNI) or the δ-opioid antagonist naltrindole. The results demonstrated that oxytocin plays an antinociceptive role in the NRM of rats through activating the oxytocin receptor. Moreover, μ-opioid receptors, not κ and δ receptors, are involved in the oxytocin-induced antinociception in the NRM of rats.

Deltorphin transport across the blood-brain barrier.

In vivo antinociception studies demonstrate that deltorphins are opioid peptides with an unusually high blood-brain barrier penetration rate. In vitro, isolated bovine brain microvessels can take up deltorphins through a saturable nonconcentrative permeation system, which is apparently distinct from previously described systems involved in the transport of neutral amino acids or of enkephalins. Removing Na+ ions from the incubation medium decreases the carrier affinity for deltorphins (-25%), but does not affect the Vmax value of the transport. The nonselective opiate antagonist naloxone inhibits deltorphin uptake by brain microvessels, but neither the selective delta-opioid antagonist naltrindole nor a number of opioid peptides with different affinities for delta- or mu-opioid receptors compete with deltorphins for the transport. Binding studies demonstrate that mu-, delta-, and kappa-opioid receptors are undetectable in the microvessel preparation. Preloading of the microvessels with L-glutamine results in a transient stimulation of deltorphin uptake. Glutamine-accelerated deltorphin uptake correlates to the rate of glutamine efflux from the microvessels and is abolished by naloxone.

Immunomodulatory responses to δ-opioid receptor ligands in human lymphocytes

In phytohemagglutinin (PHA) activated human peripheral blood mononuclear cells, [ 3 H ]thymidine uptake and interferon γ production were increased by the δ-opioid receptor agonist, deltorphin-I (10 −14–10 −10 M) and by the δ-opioid antagonist naltrindole (10 −13–10 −9 M). Combination of 10 −9 M naltrindole with deltorphin-I (10 −12–10 −8 M) significantly inhibited the proliferative response but did not affect interferon production.

CCK A, but not CCK B, agonists suppress the hyperlocomotion induced by endogenous enkephalins, protected from enzymatic degradation by systemic RB 101

Interactions between CCKergic and enkephalinergic systems were studied in mice using behavioral responses measured in Animex. The hyperlocomotion induced by 5 mg/kg of RB 101, a mixed inhibitor of enkephalin-degrading enzymes able to cross the blood-brain barrier, was previously shown to be mediated by δ-opioid receptor stimulation. The IP administration of a CCK A agonist, Boc-Tyr-Lys-(CONH- o-tolyl)-Asp-Phe- NH 2 (0.1, 1, 10 μg/ kg), suppressed the hyperlocomotion produced by IV injection of 5 mg/kg of RB 101. The effect of the CCK A agonist was suppressed by a selective CCK A antagonist, devazepide, injected IP at doses of 20 and 200 μg/kg and was potentiated by the selective δ-opioid antagonist naltrindole at the doses of 0.03 mg/kg. IP injection of the selective CCK B agonist BC 264 (0.1–1 mg/kg) did not modify the RB 101-induced hyperlocomotor effect. These results reinforce the observed physiological antagonism between the endogenous CCK and opioid systems but are at variance with the responses measured in stressful conditions. It is concluded that CCK A, but not CCK B, receptor activation counteracts the opioid-related hyperlocomotion.

The increase in morphine antinociceptive potency produced by carrageenan-induced hindpaw inflammation is blocked by naltrindole, a selective δ-opioid antagonist

Carrageenan-induced inflammation of the rat hindpaw has been used as a model for persistent pain of inflammatory origin. The induction of inflammation resulting from carrageenan injection in the rat hindpaw has been shown to elicit an increase in the antinociceptive potency of morphine, an effect postulated to be related to reduced levels of spinal cholecystokinin (CCK). Recent findings have related the anti-opioid effect of CCK to a decrease in activation of δ-opioid receptors. For this reason, we have examined the effects of the δ-opioid antagonist naltrindole (NTI) on the modulation of morphine antinociceptive potency resulting from carrageenan-induced inflammation. Rats with carrageenan-induced hindpaw inflammation received several doses of morphine in the absence or presence of NTI and were tested in the hot plate (HP) and tail flick (TF) tests. These results were compared to those of non-carrageenan injected rats. Morphine was significantly more potent in inflamed, than in control, rats in both tests. While NTI did not affect morphine antinociceptive potency in control rats in either test, this opioid δ antagonist blocked the increase in morphine potency resulting from carrageenan inflammation in nearly every case. The blockade of the enhancement of morphine potency was such that the effect of a given dose of morphine was similar in control rats and carrageenan-injected rats with NTI. We suggest that carrageenan-induced indlammation may alter endogenous enkephalin levels, perhaps by a decrease in CCK availability. The enhancement of morphine antinociceptive potency may result from the well-established synergism seen following the activation of opioid δ receptors by enkephalins.