Delta-opioid Receptor Antagonist Research Articles

Nucleus accumbens facilitates nociception

We have previously demonstrated an opioid link in nucleus accumbens (NAc) that mediates antinociception produced by a novel ascending pain modulation pathway. For example, noxious stimulation induces heterosegmental antinociception that is mediated by both mu- and delta-opioid receptors in NAc. However, spinal intrathecal administration of the mu-receptor agonist [ d-Ala 2, N-Me-Phe 4,Gly 5-ol]-enkephalin (DAMGO) also induces heterosegmental antinociception. The aim of the present study in the rat was to identify the intra-NAc opioid receptors that mediate the antinociceptive effects of spinally administered DAMGO and also to determine the effect of NAc efferent activity on nociception. Intra-NAc administration of either the mu-opioid receptor antagonist Cys 2,Tyr 3, Orn 5,Pen 7amide (CTOP) or the delta-opioid receptor antagonist naltrindole blocked the antinociceptive effect of spinally administered DAMGO on the jaw-opening reflex (JOR). Injection of quaternary lidocaine (QX-314) attenuated the JOR, suggesting that the output of NAc is pronociceptive. In support of this, intra-NAc injection of the excitatory amino acid agonist kainate enhanced the JOR. Thus, it is possible to modulate activity in NAc to bidirectionally attenuate or enhance nociception, suggesting a potential role for NAc in setting nociceptive sensitivity.

Delta opioid peptide [ d-Ala2, d-Leu5] enkephalin (DADLE) triggers postconditioning against transient forebrain ischemia

Preconditioning with selective delta opioid peptide [ d-Ala2, d-Leu5] enkephalin (DADLE) provides ischemic tolerance following transient forebrain ischemia in rats. However, whether DADLE postconditioning retains its neuroprotective efficacy and the underlying molecular mechanism in ischemic brain is largely unknown. We investigated DADLE postconditioning protection of hippocampal CA1 neurons against transient forebrain ischemia. 6 days after being implanted with cannula at the right lateral ventricle, rats underwent 10 min of forebrain ischemia by four vessel occlusion. Hippocampal CA1 neuronal survival and degeneration were measured in the hippocampi of rats at 3 days after ischemia. The behavioral and cognitive improvements of DADLE treatment in rats were also evaluated on days 5–9 using open-field and Morris water maze tests. The results showed that DADLE at doses of 0.25 and 2.5 nmol, but not 25 nmol, could significantly protect CA1 neurons against ischemia/reperfusion injury. Co-administration with the delta-opioid receptor antagonist naltrindole or pretreatment with the Akt antagonist LY294002 completely abolished the DADLE postconditioning effect. Furthermore, DADLE postconditioning exhibited cognitive benefits in rats with transient forebrain ischemia. The study thus suggested a therapeutic opportunity of postconditioning neuroprotection by DADLE and also provided important information in understanding the mechanism of DADLE action in the ischemic brain.

The delta opioid receptor antagonist, SoRI-9409, decreases yohimbine stress-induced reinstatement of ethanol-seeking

A major problem in treating alcohol use disorders (AUDs) is the high rate of relapse due to stress and re-exposure to cues or an environment previously associated with alcohol use. Stressors can induce relapse to alcohol-seeking in humans or reinstatement in rodents. Delta opioid peptide receptors (DOP-Rs) play a role in cue-induced reinstatement of ethanol-seeking; however, their role in stress-induced reinstatement of ethanol-seeking is not known. The objective of this study was to determine the role of DOP-Rs in yohimbine-stress-induced reinstatement of ethanol-seeking. Male, Long-Evans rats were trained to self-administer 10% ethanol in daily 30-minute operant self-administration sessions using a FR3 schedule of reinforcement, followed by extinction training. Once extinction criteria were met, we examined the effects of the DOP-R antagonist, SoRI-9409 (0-5 mg/kg, i.p.) on yohimbine (2 mg/kg, i.p.) stress-induced reinstatement. Additionally, DOP-R-stimulated [(35) S]GTPγS binding was measured in brain membranes and plasma levels of corticosterone (CORT) were determined. Pre-treatment with SoRI-9409 decreased yohimbine stress-induced reinstatement of ethanol-seeking but did not affect yohimbine-induced increases in plasma CORT levels. Additionally, yohimbine increased DOP-R-stimulated (35) [S]GTPγS binding in brain membranes of ethanol-trained rats, an effect that was inhibited by SoRI-9409. This suggests that the DOP-R plays an important role in yohimbine-stress-induced reinstatement of ethanol-seeking behavior, and DOP-R antagonists may be promising candidates for further development as a treatment for AUDs.

Discovery and pharmacological characterization of a selective delta opiate receptor antagonist (CP-646,777)

CP-646,777 (compound 2a) is identified as a potent and selective delta opioid receptor antagonist. The synthesis, pharmacological evaluation, disposition characteristics and pharmacokinetic properties of this compound are reported herein. An approach for reducing clearance as measured by human liver microsomes is demonstrated. The significance of p-glycoprotein efflux on CNS disposition and on the pharmacological action of CP-646,777 is also discussed.

Effects of delta opioid receptors activation on a response inhibition task in rats

Response inhibition, a primary symptom of many psychiatric disorders, is mediated through a complex neuropharmacological network that involves dopamine, serotonin, glutamate, noradrenaline, and cannabinoid mechanisms. Recently, we identified an opioidergic contribution to response inhibition by showing that deletion of mu or delta opioid receptors in mice alters motor impulsivity. We investigated this phenomenon further by testing whether pharmacological activation of opioid receptors disrupts the ability to inhibit a motor response. Long-Evans rats were trained to withhold a lever-pressing response for sucrose until a discriminative stimulus (lever light) was presented. The delay to the discriminative stimulus (1 to 60s) was varied, so animals could not predict, on any given trial, the length of the pre-response phase. Motor impulsivity was assessed as the inability to inhibit lever pressing prior to the discriminative stimulus. Rats were tested following an injection of the mu opioid receptor agonist morphine (0, 0.5, 1, 2, 4, 6, 8, or 10mg/kg) or the delta receptor agonist SNC80 (0, 2.5, 5, or 10mg/kg). SNC80 (10mg/kg) increased premature responses and locomotor activity, but had no effect on the speed of responding or non-reinforced presses. The SNC80-induced decrease in accuracy was blocked by the delta opioid receptor antagonist naltrindole. Morphine had no effect on accuracy but increased locomotor activity (2mg/kg). These findings point to a role for delta, but not mu, opioid receptors in disinhibition as measured in the response inhibition task. The results appear to contradict those of previous opioid receptor deletion studies; possible sources of these discrepant results are discussed.

Opioid receptors and the discriminative stimulus effects of ethanol in squirrel monkeys: Mu and delta opioid receptor mechanisms

Mu and delta opioid receptors modulate the reinforcing effects of ethanol, however, their role in the subjective effects of ethanol is not well understood. This study evaluated the contribution of mu and delta opioid receptors to the subjective effects of ethanol using drug discrimination procedures. Monkeys were trained to discriminate ethanol from saline under a schedule of food delivery. In tests, ethanol engendered increases in drug-lever responding, reaching a maximum of > 80%. The mu opioid receptor agonists fentanyl and buprenorphine and the delta opioid receptor agonists SNC 80 and SNC 162 did not substitute for the discriminative stimulus effects of ethanol. As pretreatments, the full agonists fentanyl and SNC 80 enhanced the effects of low doses of ethanol and fentanyl attenuated the effects of the ethanol training dose. Although the possibility of pharmacological antagonism of the effects of ethanol cannot be ruled out, a more likely alternative is that the diminished effects of ethanol were due to perceptual masking of the ethanol stimulus. In contrast, the partial agonists buprenorphine and SNC 162 did not alter ethanol's effects. Finally, the discriminative stimulus effects of ethanol were attenuated following administration of presumably mu-selective doses of the antagonist naltrexone, but not after administration of the delta opioid receptor antagonist naltrindole. The ability of naltrexone to block the discriminative stimulus effects of ethanol likely reflects its capacity to attenuate ethanol-induced increases in endogenous opioids, in particular beta-endorphin, because attenuation of the ethanol stimulus was not accompanied by significant suppression of response rate.

Remote pharmacological post‐conditioning by intrathecal morphine: cardiac protection from spinal opioid receptor activation

Intrathecal morphine pre-conditioning attenuates cardiac ischemia-reperfusion injury via activation of central opioid receptors. We hypothesized that intrathecal morphine also post-conditions the myocardium in the rat. Intrathecal morphine at 0.3 μg/kg (LMPC), 3 μg/kg (MMPC) or 30 μg/kg (HMPC) was administered for 5 min before 120-min reperfusion following 30-min ischemia. Infarct size as a percentage of area at risk (IS/AAR) was determined using triphenyltetrazolium staining. MMPC was repeated following the intrathecal administration of nor BNI, NTD, CTOP, or naloxone methiodide (NM), kappa, delta, mu and non-specific opioid receptor antagonists, respectively. The role of peripheral opioid, adenosine and calcitonin gene-related peptide (CGRP) receptors was examined by the intravenous administration of NM, 8-ρ-sulfophenyl theophylline (8-SPT) and human CGRP fragment (CGRP(8-37)), respectively. Morphine post-conditioning at all three doses was cardioprotective (IS/AAR of LMPC=37 ± 4%, MMPC=35 ± 5%, HMPC=32 ± 4%, control=50 ± 5%, P<0.01). The prior administration of opioid receptor antagonists intrathecally, as well as intravenous 8-SPT and CGRP(8-37) receptor antagonists, abolished this effect (nor BNI+MMPC=47 ± 7%, NTD+MMPC=49 ± 7%, CTOP+MMPC=45 ± 9%, NM+MMPC=47 ± 6% 8-SPT+MPC=46 ± 5% & CGRP(8-37)+MPC=53 ± 6%, P=0.63). However, the intravenous administration of NM did not prevent the protective effect (34 ± 4%, P<0.01). Intrathecal morphine administration can induce pharmacological cardiac post-conditioning as it involves opioid receptor centrally but non-opioid receptors peripherally.

Peripheral antinociceptive effects of the cyclic endomorphin-1 analog c[YpwFG] in a mouse visceral pain model

We previously described a novel cyclic endomorphin-1 analog c[Tyr- D-Pro- D-Trp-Phe-Gly] (c[YpwFG]), acting as a mu-opioid receptor (MOR) agonist. This study reports that c[YpwFG] is more lipophilic and resistant to enzymatic hydrolysis than endomorphin-1 and produces preemptive antinociception in a mouse visceral pain model when injected intraperitoneally (i.p.) or subcutaneously (s.c.) before 0.6% acetic acid, employed to evoke abdominal writhing (i.p. ED 50 = 1.24 mg/kg; s.c. ED 50 = 2.13 mg/kg). This effect is reversed by the selective MOR antagonist β-funaltrexamine and by a high dose of the mu 1-opioid receptor-selective antagonist naloxonazine. Conversely, the kappa-opioid receptor antagonist nor-binaltorphimine and the delta-opioid receptor antagonist naltrindole are ineffective. c[YpwFG] produces antinociception when injected i.p. after acetic acid (ED 50 = 4.80 mg/kg), and only at a dose of 20 mg/kg did it elicit a moderate antinociceptive response in the mouse, evaluated by the tail flick assay. Administration of a lower dose of c[YpwFG] (10 mg/kg i.p.) apparently produces a considerable part of antinociception on acetic acid-induced writhes through peripheral opioid receptors as this action is fully prevented by i.p. naloxone methiodide, which does not readily cross the blood–brain barrier; whereas this opioid antagonist injected intracerebroventricularly (i.c.v.) is not effective. Antinociception produced by a higher dose of c[YpwFG] (20 mg/kg i.p.) is partially reversed by naloxone methiodide i.c.v. administered. Thus, only at the dose of 20 mg/kg c[YpwFG] can produce antinociception through both peripheral and central opioid receptors. In conclusion, c[YpwFG] displays sufficient metabolic stability to be effective after peripheral administration and demonstrates the therapeutic potential of endomorphin derivatives as novel analgesic agents to control visceral pain.

Disruption of morphine‐conditioned place preference by a δ2‐opioid receptor antagonist: study of μ‐opioid and δ‐opioid receptor expression at the synapse

The addictive properties of morphine limit its clinical use. Learned associations that develop between the abused opiate and the environment in which it is consumed are engendered through Pavlovian conditioning processes. Disruption of the learned associations between the opiate and environmental cues may be a therapeutic approach to prevent morphine dependence. Although a role for the delta-opioid receptor in the regulation of the rewarding properties of morphine has already been shown, in this study we further characterized the role of the delta-opioid receptor in morphine-induced conditioned responses by examining the effect of a selective delta2-opioid receptor antagonist (naltriben), using a conditioned place preference paradigm in rats. Additionally, we used a subcellular fractionation technique to analyze the synaptic localization of mu-opioid and delta-opioid receptors in the hippocampus, in order to examine the molecular mechanisms that may underlie this morphine-induced conditioned behavior. Our data show that the administration of 1 mg/kg naltriben (but not 0.1 mg/kg) prior to morphine was able to block morphine-induced conditioned place preference. Interestingly, this naltriben-induced disruption of morphine conditioned place preference was associated with a significant increase in the expression of the delta-opioid receptor dimer at the postsynaptic density. In addition, we also observed that morphine conditioned place preference was associated with an increase in the expression of the mu-opoid receptor in the total homogenate. Overall, these results suggest that modulation of the delta-opioid receptor expression and its synaptic localization may constitute a viable therapeutic approach to disrupt morphine-induced conditioned responses.

Mu-opioid receptors transiently activate the Akt-nNOS pathway to produce sustained potentiation of PKC-mediated NMDAR-CaMKII signaling.

BackgroundIn periaqueductal grey (PAG) matter, cross-talk between the Mu-opioid receptor (MOR) and the glutamate N-methyl-D-Aspartate receptor (NMDAR)-CaMKII pathway supports the development of analgesic tolerance to morphine. In neurons, histidine triad nucleotide binding protein 1 (HINT1) connects the regulators of G protein signaling RGSZ1 and RGSZ2 to the C terminus of the MOR. In response to morphine, this HINT1-RGSZ complex binds PKCγ, and afterwards, the interplay between PKCγ, Src and Gz/Gi proteins leads to sustained potentiation of NMDAR-mediated glutamate responses.Methodology/Principal FindingsFollowing an intracerebroventricular (icv) injection of 10 nmol morphine, Akt was recruited to the synaptosomal membrane and activated by Thr308 and Ser473 phosphorylation. The Akt activation was immediately transferred to neural Nitric Oxide Synthase (nNOS) Ser1417. Afterwards, nitric oxide (NO)-released zinc ions recruited PKCγ to the MOR to promote the Src-mediated phosphorylation of the Tyr1325 NMDAR2A subunit. This action increased NMDAR calcium flux and CaMKII was activated in a calcium-calmodulin dependent manner. CaMKII then acted on nNOS Ser847 to produce a sustained reduction in NO levels. The activation of the Akt-nNOS pathway was also reduced by the binding of these proteins to the MOR-HINT1 complex where they remained inactive. Tolerance to acute morphine developed as a result of phosphorylation of MOR cytosolic residues, uncoupling from the regulated G proteins which are transferred to RGSZ2 proteins. The diminished effect of morphine was prevented by LNNA, an inhibitor of nNOS function, and naltrindole, a delta-opioid receptor antagonist that also inhibits Akt.Conclusions/SignificanceAnalysis of the regulatory phosphorylation of the proteins included in the study indicated that morphine produces a transient activation of the Akt/PKB-nNOS pathway. This activation occurs upstream of PKCγ and Src mediated potentiation of NMDAR activity, ultimately leading to morphine tolerance. In summary, the Akt-nNOS pathway acts as a primer for morphine-triggered events which leads to the sustained potentiation of the NMDAR-CaMKII pathway and MOR inhibition.

Adenosine A1 receptor agonist N6-cyclohexyl-adenosine induced phosphorylation of delta opioid receptor and desensitization of its signaling

To define the effect of adenosine A(1) receptor (A(1)R) on delta opioid receptor (DOR)-mediated signal transduction. CHO cells stably expressing HA-tagged A(1)R and DOR-CFP fusion protein were used. The localization of receptors was observed using confocal microscope. DOR-mediated inhibition of adenylyl cyclase was measured using cyclic AMP assay. Western blots were employed to detect the phosphorylation of Akt and the DOR. The effect of A(1)R agonist N(6)-cyclohexyladenosine (CHA) on DOR down-regulation was assessed using radioligand binding assay. CHA 1 micromol/L time-dependently attenuated DOR agonist [D-Pen(2,5)]enkephalin (DPDPE)-induced inhibition of intracellular cAMP accumulation with a t(1/2)=2.56 (2.09-3.31) h. Pretreatment with 1 micromol/L CHA for 24 h caused a right shift of the dose-response curve of DPDPE-mediated inhibition of cAMP accumulation, with a significant increase in EC(50) but no change in E(max). Pretreatment with 1 micromol/L CHA for 1 h also induced a significant attenuation of DPDPE-stimulated phosphorylation of Akt. Moreover, CHA time-dependently phosphorylated DOR (Ser363), and this effect was inhibited by A(1)R antagonist 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) but not by DOR antagonist naloxone. However, CHA failed to produce the down-regulation of DOR, as neither receptor affinity (K(d)) nor receptor density (B(max)) of DOR showed significant change after chronic CHA exposure. Activation of A(1)R by its agonist caused heterologous desensitization of DOR-mediated inhibition of intracellular cAMP accumulation and phosphorylation of Akt. Activation of A(1)R by its agonist also induced heterologous phosphorylation but not down-regulation of DOR.

ChemInform Abstract: Novel Dmt-Tic Dipeptide Analogues as Selective Delta-Opioid Receptor Antagonists.

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Alteration of intravenous nicotine self-administration by opioid receptor agonist and antagonists in rats

The role played by endogenous opioids in mediating the reinforcing properties of nicotine is unclear. As with preclinical studies, clinical trials with naloxone, a prototypic opioid receptor antagonist have yielded equivocal findings with regard to its efficacy in reducing cigarette smoking. The aim of the present study was to examine the effects of three opioids that exhibit relative selectivity at mu-, kappa- and delta-opioid receptors on nicotine self-administration in male hooded Lister rats. Graded doses (0.3, 1.0, and 3.0 mg/kg IP) of each opioid agonist or antagonist were tested in different groups of rats repeatedly over three consecutive nicotine intravenous nicotine-self administration (0.03 mg/kg/infusion) sessions. The same treatments were tested in parallel groups of rats trained to respond for food reinforcement. Naloxone was very effective in attenuating the levels of nicotine self-administered across all doses tested. The selective kappa-opioid receptor agonist U50,488, reduced nicotine self-administration in doses of 1 and 3 mg/kg, while the 0.3 mg/kg dose produced a small increase in nicotine intake. Finally, the specific delta-opioid receptor antagonist, naltrindole did not significantly modify nicotine self-administration behaviour. In contrast, all three opioids failed to modify behaviour maintained by food reinforcement. These findings suggest endogenous opioids are crucial in mediating the reinforcing effects of nicotine and that the mu-opioid receptor subtype may represent a potential target for selectively reducing nicotine-taking behaviour as part of a pharmacological approach to develop smoking cessation aids.

The central antidepressant‐like and antinociceptive effects of a delta opioid receptor agonist SNC80 in rats

Animal studies have indicated that systemic administration of delta opioid receptor (DOR) agonists or tricyclic antidepressants (TCA) produced both antidepressant‐like and antinociceptive effects. The aim of the study was to investigate the role of DORs in the supraspinal regions by comparing behavioral effects of DOR agonist SNC80 and TCA amitriptyline in rats. The forced swim test and formalin‐induced flinches were used to quantify the antidepressant‐like and antinociceptive effects of test compounds, respectively. Following intracerebroventricular (ICV) administration, both SNC80 and amitriptyline dose‐dependently decreased immobility in the rat forced swim test, indicating of antidepressant‐like effects. ED50 values of ICV SNC80 and amitriptyline for decreasing immobility were 5.1 and 38.1 μg, respectively. In addition, ICV SNC80 dose‐dependently produced antinociception, but ICV amitriptyline tested up to 100 μg failed to attenuate formalin‐induced nociceptive responses. More importantly, pretreatment with a DOR antagonist naltrindole (1 mg/kg, SC) only blocked antidepressant‐like effects produced by ICV SNC80, but not those by ICV amitriptyline. These results suggest that central DOR is involved in the regulation of both mood and pain, and that DOR agonists may have the therapeutic potential as antidepressants or/and analgesics (Supported by NSC grant NSC‐97‐2628‐H‐004‐089‐MY2).

Remifentanil post‐conditioning attenuates cardiac ischemia–reperfusion injury via κ or δ opioid receptor activation

Ischemic pre- or post-conditioning of the heart has been shown to involve opioid receptors. Remifentanil, an ultra-short-acting selective mu opioid receptor agonist in clinical use, pre-conditions the rat heart against ischemia-reperfusion injury. This study investigates whether remifentanil post-conditioning is also cardioprotective. Remifentanil post-conditioning (5-min infusion at 1, 5, 10 or 20 microg/kg/min) or ischemic post-conditioning (three cycles of a 10 s reperfusion interspersed with a 10 s ischemia) was induced in an open-chest rat heart model of ischemia and reperfusion injury, in the presence or absence of nor-binaltorphimine, naltrindole or CTOP, specific kappa, delta and mu opioid receptor antagonists, respectively. The same sequence of experiments was repeated in the isolated heart model using the maximal protective dose of remifentanil from the dose-response studies. Both ischemic and remifentanil post-conditioning reduced the myocardial infarct size relative to the control group in both models. This cardioprotective effect for both post-conditioning regimes was prevented by the prior administration of nor-binaltorphimine and naltrindole but not CTOP. The sole administration of the antagonists had no effect on the size of myocardial infarction. These results indicate that remifentanil post-conditioning protects the heart from ischemia-reperfusion injury to a similar extent as of ischemic post-conditioning. This protection involves kappa and delta but not mu opioid receptor activation. This drug has great potential as a clinical post-conditioning modality as it can be given in large doses without prolonged opioid-related side effects.

Involvement of the κ-opioid receptor in nitrous oxide-induced analgesia in mice

Nitrous oxide (N(2)O)-induced analgesia is thought to be mediated by endogenous opioids. We previously showed that the mu-opioid receptor is not required for the analgesic action of N(2)O in mice using a gene knockout approach. In this study, we examined the effect of kappa- (KOP)- or delta-opioid receptor (DOP)-selective antagonists on N(2)O-induced analgesia. The analgesic effect of N(2)O was evaluated using a writhing test. Male C57BL/6 mice aged 7-8 weeks were assigned to control, N(2)O, KOP agonist, and DOP agonist groups. According to the group assignment, mice were pretreated with a KOP antagonist, nor-binaltorphimine (nor-BNI), a DOP antagonist, naltrindole hydrochloride (NTI), a KOP agonist U50488, and a DOP agonist SNC80. Mice in the control, KOP agonist, and DOP agonist groups were exposed to 25% oxygen/75% nitrogen for 30 min, and mice in the N(2)O group were exposed to 25% oxygen/75% N(2)O for 30 min. Nor-BNI [10 mg kg(-1), subcutaneously (s.c.)] significantly suppressed the analgesic effect of N(2)O and U50488. In contrast, NTI (10 mg kg(-1) s.c.) did not significantly affect the analgesic action of N(2)O, but almost completely inhibited the analgesic effect of SNC80. These results suggest that KOP plays an important role in the analgesic effect of N(2)O in mice.

Antinociceptive effects of galanin in the central nucleus of amygdala of rats, an involvement of opioid receptors

The central nucleus of amygdala (CeA) is a very important brain structure involved in multiple physiological functions, especially in pain modulation. There are high densities of galanin and galanin receptors found in the CeA. The present study was performed to explore the antinociceptive effects of galanin in the CeA of rats, and possible involvements of opioid receptors in the galanin-induced antinociception. Intra-CeA injection of galanin induced dose-dependent increases in hindpaw withdrawal latencies (HWLs) to noxious thermal and mechanical stimulations in rats. Interestingly, the amtinociceptive effect induced by intra-CeA injection of galanin was blocked by intra-CeA injection of naloxone, a common opioid receptor antagonist, indicating an involvement of opioid receptors in the galanin-induced antinociception in the CeA of rats. Moreover, intra-CeA injection of either selective mu-opioid receptor antagonist β-funaltrexamine (β-FNA) or delta-opioid receptor antagonist naltrindole, but not kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI), significantly attenuated the galanin-induced increases in HWLs in the CeA of rats. Taken together, the results demonstrate that galanin induces antinociceptive effects in the CeA of rats, and both mu- and delta-opioid receptors are involved in the galanin-induced antinociception.

Delta opioid receptor on equine sperm cells: subcellular localization and involvement in sperm motility analyzed by computer assisted sperm analyzer (CASA).

BackgroundOpioid receptors and endogenous opioid peptides act not only in the control of nociceptive pathways, indeed several reports demonstrate the effects of opiates on sperm cell motility and morphology suggesting the importance of these receptors in the modulation of reproduction in mammals. In this study we investigated the expression of delta opioid receptors on equine spermatozoa by western blot/indirect immunofluorescence and its relationship with sperm cell physiology.MethodsWe analyzed viability, motility, capacitation, acrosome reaction and mitochondrial activity in the presence of naltrindole and DPDPE by means of a computer assisted sperm analyzer and a fluorescent confocal microscope. The evaluation of viability, capacitation and acrosome reaction was carried out by the double CTC/Hoechst staining, whereas mitochondrial activity was assessed by means of MitoTracker Orange dye.ResultsWe showed that in equine sperm cells, delta opioid receptor is expressed as a doublet of 65 and 50 kDa molecular mass and is localized in the mid piece of tail; we also demonstrated that naltrindole, a delta opioid receptor antagonist, could be utilized in modulating several physiological parameters of the equine spermatozoon in a dose-dependent way. We also found that low concentrations of the antagonist increase sperm motility whereas high concentrations show the opposite effect. Moreover low concentrations hamper capacitation, acrosome reaction and viability even if the percentage of cells with active mitochondria seems to be increased; the opposite effect is exerted at high concentrations. We have also observed that the delta opioid receptor agonist DPDPE is scarcely involved in affecting the same parameters at the employed concentrations.ConclusionsThe results described in this paper add new important details in the comprehension of the mammalian sperm physiology and suggest new insights for improving reproduction and for optimizing equine breeding.

Mu-opioid receptors are not necessary for nortriptyline treatment of neuropathic allodynia

Tricyclic antidepressants (TCAs) are among the first line treatments clinically recommended against neuropathic pain. However, the mechanism by which they alleviate pain is still unclear. Pharmacological and genetic approaches evidenced a critical role of delta-opioid receptors (DORs) in the therapeutic action of chronic TCA treatment. It is however unclear whether mu-opioid receptors (MORs) are also necessary to the pain-relieving action of TCAs. The lack of highly selective MOR antagonists makes difficult to conclude based on pharmacological studies. In the present work, we thus used a genetic approach and compared mutant mice lacking MORs and their wild-type littermates. The neuropathy was induced by unilateral sciatic nerve cuffing. The threshold for mechanical response was evaluated using von Frey filaments. MOR-deficient mice displayed the same baseline for mechanical sensitivity as their wild-type littermates. After sciatic nerve cuffing, both wild-type and MOR-deficient mice displayed an ipsilateral mechanical allodynia. After about 10 days of treatment, nortriptyline suppressed this allodynia in both wild-type and MOR-deficient mice. MORs are thus not critical for nortriptyline action against neuropathic pain. An acute injection of the DOR antagonist naltrindole induced a relapse of neuropathic allodynia in both wild-type and MOR-deficient mice, thus confirming the critical role of DORs in nortriptyline action. Moreover, morphine induced an acute analgesia in control and in neuropathic wild-type mice, but was without effect in MOR-deficient mice. While MORs are crucial for morphine action, they are not critical for nortriptyline action. Our results highlight the functional difference between DORs and MORs in mechanisms of pain relief.

Chronic treatment with agonists of β 2-adrenergic receptors in neuropathic pain

Expression of β 2-adrenoceptors (β 2-ARs) within the nociceptive system suggested their potential implication in nociception and pain. Recently, we demonstrated that these receptors are essential for neuropathic pain treatment by antidepressant drugs. The aim of the present study was to investigate whether the stimulation of β 2-ARs could in fact be adequate to alleviate neuropathic allodynia. Neuropathy was induced in mice by sciatic nerve cuffing. We demonstrate that chronic but not acute stimulation of β 2-ARs with agonists such as clenbuterol, formoterol, metaproterenol and procaterol suppressed neuropathic allodynia. By using a pharmacological approach with the β 2-AR antagonist ICI 118,551 or a transgenic approach with mice deficient for β 2-ARs, we confirmed that the antiallodynic effect of these agonists was specifically related to their action on β 2-ARs. We also showed that chronic treatment with the β 1-AR agonist xamoterol or with the β 3-AR agonist BRL 37344 had no effect on neuropathic allodynia. Chronic stimulation of β 2-ARs, but not β 1- or β 3-ARs, by specific agonists is thus able to alleviate neuropathic allodynia. This action of β 2-AR agonists might implicate the endogenous opioid system; indeed chronic clenbuterol effect can be acutely blocked by the delta-opioid receptor antagonist naltrindole. Present results show that β 2-ARs are not only essential for the antiallodynic action of antidepressant drugs on sustained neuropathic pain, but also that the stimulation of these receptors is sufficient to relieve neuropathic allodynia in a murine model. Our data suggest that β 2-AR agonists may potentially offer an alternative therapy to antidepressant drugs for the chronic treatment of neuropathic pain.