Selective Delta-opioid Agonist Research Articles

P-209 Implication of delta opioid receptor in the in vitro maturation of oocytes and its effects on subsequent fertilization and embryo development in mice

Abstract Study question Could opiods modulate in vitro maturation of oocyte and improve fertilization rates? Summary answer Delta opioid receptor was present in mice oocytes, changing its expression pattern depending on the maturation stage and helping oocytes to reach blastocyst stage. What is known already The molecular mechanisms responsible for oocyte maturation are not completely understood and different molecules have been reported to be implicated as modulators of this process. The endogenous opioid peptides could be interesting candidates because they are involved in the regulation of reproductive physiology at multiple sites. When those receptors are activated, the adenylyl cyclase is inhibited, thus reducing cyclic AMP and blocking PKA. Moreover, opioids are also able to activate proteins involved in maturation. There is evidence suggesting a role for opioids during oocyte maturation because the signaling exerted by opioids is similar to that necessary for the meiosis resumption. Study design, size, duration Immature cumulus–oocyte complexes (COCs) from female 8 to 10 week old WT (C57BL/6xCBA) were retrieved after EGF stimulation. Mouse in vitro maturation culture was performed. In vitro grown oocytes, their corresponding cumulus (CC) and granulosa cells (GC) were classified at germinal vesicle stage (GV), at metaphase 1 (MI) and at metaphase 2 (MII). Participants/materials, setting, methods The presence of DOR was analysed in granulosa cells and oocytes at each stage of maturation by qRT-PCR and immunocytochemistry. AKT and MAPK signalling pathway (ERK 1/2) was studied by immunocytochemistry. Similarly, COCs were matured in vitro in the absence and presence of DPDPE and naltrindole at various concentrations. After IVM, in vitro fertilisation (IVF) was performed and development was observed until the blastocyst stage was reached, analysing fertilisation and embryo production percentages (p < 0.05). Main results and the role of chance Our findings showed that OPRD1 was present in mice oocytes and granulosa cells, changing its expression pattern depending on the maturation stage. The evaluation of RT-PCR examination and immunohistochemical (IHC) staining revealed the presence of the transcript for Oprd1 gene in the different stages of the mouse oocyte's maturation, as well as in the granulosa cells at the moment of the extraction from the ovary. The obtained transcript was compared with the transcript for Oprd1 of mouse cerebral cortex as a positive control. The expected 482 bp fragment was detected in the germinal vesicle stage and in granulosa cells, whereas in MI and MII stage the signal was lower. Immunofluorescence analysis revealed that OPRD1 protein was present in mouse oocytes but, its localization differed at the different stages of the maturation process. Furthermore, the selective delta opioid agonist DPDPE, modulating PI3K/Akt and MAPK pathways, helped oocytes to reach blastocyst stage. Finally, we examined that the observed effect of DPDPE during oocyte maturation in the in vitro fertilization and subsequent embryo development could be blocked by the opioid antagonist naltrindole. Limitations, reasons for caution Although we have obtained higher rates of blastocysts, the quality of these embryos still needs to be analysed and transferred to recipient females to evaluate the offspring. Wider implications of the findings Our study has clinical implications for the improvement of oocyte in vitro maturation techniques (IVM). OPRD1 could be a possible therapeutic target for in vitro maturation culture medium, as it could improve the blastocyst rates obtained in the actual reproduction assisted techniques. Trial registration number not applicable

Activation of Kappa Opioid Receptor Regulates the Hypothermic Response to Calorie Restriction and Limits Body Weight Loss.

Mammals maintain a nearly constant core body temperature (Tb) by balancing heat production and heat dissipation. This comes at a high metabolic cost that is sustainable if adequate calorie intake is maintained. When nutrients are scarce or experimentally reduced such as during calorie restriction (CR), endotherms can reduce energy expenditure by lowering Tb [1-6]. This adaptive response conserves energy, limiting the loss of body weight due to low calorie intake [7-10]. Here we show that this response is regulated by the kappa opioid receptor (KOR). CR is associated with increased hypothalamic levels of the endogenous opioid Leu-enkephalin, which is derived from the KOR agonist precursor dynorphin [11]. Pharmacological inhibition of KOR, but not of the delta or the mu opioid receptor subtypes, fully blocked CR-induced hypothermia and increased weight loss during CR independent of calorie intake. Similar results were seen with DIO mice subjected to CR. In contrast, inhibiting KOR did not change Tb in animals fed ad libitum (AL). Chemogenetic inhibition of KOR neurons in the hypothalamic preoptic area reduced the CR-induced hypothermia, whereas chemogenetic activation of prodynorphin-expressing neurons in the arcuate or the parabrachial nucleus lowered Tb. These data indicate that KOR signaling is a pivotal regulator of energy homeostasis and can affect body weight during dieting by modulating Tb and energy expenditure.

Novel diazabicycloalkane delta opioid agonists

Here we report the investigation of diazabicycloalkane cores as potential new scaffolds for the development of novel analogues of the previously reported diazatricyclodecane selective delta (δ) opioid agonists, as conformationally constrained homologues of the reference δ agonist (+)-4-[(αR)-α((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80).In particular, we have simplified the diazatricyclodecane motif of δ opioid agonist prototype 1a with bridged bicyclic cores. 3,6-diazabicyclo[3.1.1]heptane, 3,8-diazabicyclo[3.2.1]octane, 3,9-diazabicyclo[3.3.1]nonane, 3,9-diazabicyclo[4.2.1]nonane, and 3,10-diazabicyclo[4.3.1]decane were adopted as core motifs of the novel derivatives. The compounds were synthesized and biologically assayed as racemic (3–5) or diastereoisomeric (6,7) mixtures.All the novel compounds 3–7 showed δ agonism behaviour and remarkable affinity to δ receptors. Amongst the novel derivatives, 3,8-diazabicyclo[3.2.1]octane based compound 4 evidenced improved δ affinity and selectivity relative to SNC80.

Opiate-agonist induced taste aversion learning in the Fischer 344 and Lewis inbred rat strains: Evidence for differential mu opioid receptor activation

The Fischer 344 (F344) and Lewis (LEW) inbred rat strains react differently to morphine in a number of behavioral and physiological preparations, including the acquisition of aversions induced by this compound. The present experiment tested the ability of various compounds with relative selectivity at kappa, delta and mu receptor subtypes to assess the relative roles of these subtypes in mediating the differential aversive effects of morphine in the two strains. In the assessment of the role of the kappa receptor in morphine-induced aversions, animals in both strains were given access to saccharin followed by varying doses of the kappa agonist (−)-U50,488H (0.0, 0.28, 0.90 and 1.60 mg/kg). Although (−)-U50,488H induced aversions in both strains, no strain differences emerged. A separate subset of subjects was trained with the selective delta opioid agonist, SNC80 (0.0, 5.6, 10.0 and 18.0 mg/kg), and again although SNC80 induced aversions, there were no strain differences. Finally, a third subset of subjects was trained with heroin (0.0, 3.2, 5.6 and 10.0 mg/kg), a compound with activity at all three opiate receptor subtypes. Although heroin induced aversions in both strains, the aversions were significantly greater in the F344 strain, suggesting that differential activation of the mu opioid receptor likely mediates the reported strain differences in morphine-induced aversion learning. These data were discussed in terms of strain differences in opioid system functioning and the implications of such differences for other morphine-induced behavioral effects reported in F344 and LEW rats.

Opioid‐induced preconditioning is dependent on caveolin‐3 expression

Caveolae are flask‐like invaginations of the sarcolemmal membrane. Recently, we have shown that caveolar structural proteins, caveolins, are essential in cardiac protection and that cardiac specific overexpression of caveolin‐3 (Cav‐3 OE) results in innate cardiac protection. Additionally, we identified that opioid‐induced preconditioning (OPC) is absent when caveolae are disrupted in vitro. However, the impact of caveolin‐3 and caveolae on OPC in vivo has not been investigated. 8‐12 week old Cav‐3 OE, caveolin‐3 knockout mice (Cav‐3 KO), and control mice were exposed to 30 min ischemia/2 hr reperfusion (I/R). Cav‐3 OE and control mice were treated with naloxone, a non‐selective opioid antagonist, 10 min prior to I/R to determine whether basal protection was abolished. Cav‐3 KO and control mice were treated with SNC, a delta‐selective opioid agonist 15 min prior to I/R to induce OPC. Cav‐3 OE mice displayed endogenous cardiac protection from I/R when compared to control mice (20±2% vs. 41±2%, n=8, respectively). This endogenous protection in Cav‐3 OE mice was abolished with naloxone pretreatment (41±2%). Conversely, control mice were protected in the presence of SNC (28±1%), although no protection was observed in Cav‐3 KO mice (38±5%). These results suggest that OPC is dependent on caveolin‐3 expression and endogenous protection induced in Cav‐3 OE mice may be opioid dependent.

Enhancement of Spinal N -Methyl-d-aspartate Receptor Function by Remifentanil Action at δ-Opioid Receptors as a Mechanism for Acute Opioid-induced Hyperalgesia or Tolerance

Intraoperative remifentanil infusions have been associated with postoperative opioid-induced hyperalgesia and tolerance. Using a previously identified subpopulation of spinal neurons that displays an augmentation in N-methyl-D-aspartate (NMDA) receptor current after chronic morphine, investigations were undertaken to determine whether remifentanil induces acute increases in NMDA responses that are concentration dependent and receptor subtype dependent. Electrophysiologic recordings of NMDA current were made from cultured rat dorsal horn neurons treated with remifentanil at various concentrations for 60 min. Selective mu- or delta-opioid receptor inhibitors and agonists were used to determine the site of action of remifentanil. Remifentanil at 4, 6, and 8 nM, but not higher or lower concentrations, caused significant mean increases in NMDA peak current amplitude of 37.30% (P < 0.001), 30.19% (P < 0.001), and 23.52% (P = 0.025), respectively, over control conditions. This occurred by 36 min of remifentanil perfusion and persisted throughout its washout. Inhibition by 100 nM naloxone or 1 nM naltrindole attenuated the remifentanil-induced NMDA response increase. Selective delta-opioid agonists [D-Pen(2), D-Pen(5)]enkephalin and deltorphin II displayed a similar bell-shaped concentration-response relation for the enhancement of NMDA responses, and 10 nM deltorphin II occluded the effects of 4 nM remifentanil on NMDA responses. Clinically relevant concentrations of remifentanil induce rapid, persistent increases in NMDA responses that mirror the development of remifentanil-induced hyperalgesia and tolerance. NMDA enhancement by remifentanil is dependent on the activation of both mu- and delta-opioid receptors and is inducible solely by delta-opioid receptor activation. Therefore, selective delta-opioid inhibition may attenuate acute paradoxical increases in pain and tolerance to opioids.

The selective delta opioid agonist SNC80 enhances amphetamine-mediated efflux of dopamine from rat striatum

The highly selective delta opioid agonist, SNC80, elicits dopamine-related behaviors including locomotor stimulation and conditioned place-preference. In contrast, it has been reported that SNC80 fails to promote dopamine efflux from the striatum of freely moving rats. However, SNC80 does enhance behavioral responses to the stimulants, amphetamine and cocaine, suggesting an interaction between delta opioids and psychostimulants. Since the increase in locomotor activity elicited by amphetamine and related stimulants acting at the dopamine transporter is associated with increases in extracellular concentrations of dopamine within the striatum, we hypothesized that SNC80 enhances this activity by potentiating the overflow of dopamine through the transporter. To test this hypothesis, striatal preparations from Sprague Dawley rats were assayed for dopamine efflux in response to amphetamine challenge. SNC80 was given either in vivo or in vitro directly to rat striatal tissue, prior to in vitro amphetamine challenge. Both in vivo and in vitro administration of SNC80 enhanced amphetamine-mediated dopamine efflux in a concentration- and time-dependent manner. However, SNC80 in either treatment paradigm produced no stimulation of dopamine efflux in the absence of amphetamine. The effect of SNC80 on amphetamine-mediated dopamine overflow, but not the effect of amphetamine alone, was blocked by the delta selective antagonist, naltrindole and was also observed with other delta agonists. The results of this study demonstrate that even though SNC80 does not stimulate dopamine efflux alone, it is able to augment amphetamine-mediated dopamine efflux through a delta opioid receptor mediated action locally in the striatum.

GSK3β inhibition and KATP channel opening mediate acute opioid-induced cardioprotection at reperfusion

Both glycogen synthase kinase 3beta (GSK3beta) and the ATP-dependant potassium channel (K(ATP)) mediate opioid-induced cardioprotection (OIC). However, whether direct K(ATP) channel openers induce cardioprotection prior to reperfusion and their signaling cascade position with respect to GSK3beta inhibition is unknown. Therefore, we investigated the role of K(ATP) channel opening at reperfusion in OIC, and the interaction between the GSK signaling axis and K(ATP) channels in cardioprotection.Male Sprague-Dawley rats underwent 30 minutes ischemia with 2 hours of reperfusion and infarct size was determined. Rats given the nonselective opioid agonist, morphine (0.3 mg/kg), or the selective delta opioid agonist, BW373U86 (1.0 mg/kg), 5 minutes prior to reperfusion reduced infarct size (40.3+/-1.6*, 39.7+/-1.9* versus 60.0+/-1.1%, respectively, * P<0.001%). This protection was abrogated with prior administration of the putative sarcolemmal K(ATP) antagonist, HMR-1098 (6 mg/kg), or the putative mitochondrial K(ATP) antagonist, 5-HD (10 mg/kg). The putative sK(ATP) channel opener, P-1075 (1microg/kg) or the putative mK(ATP) channel opener, BMS-191095 (1 mg/kg) given 5 minutes prior to reperfusion also reduced infarct size (41.8+/-2.4*, 43.4+/-1.4*) and protection was abrogated by prior administration of the PI3k inhibitor wortmannin (60.0+/-1.7, 64.0+/-2.6%, respectively, * P<0.001). Cardioprotection afforded by the GSK inhibitor SB216763 (0.6 mg/kg) given 5 minutes prior to reperfusion was also partially blocked by either HMR or 5-HD and completely blocked when HMR and 5-HD were given in combination (40.8+/-1.6*, 50.4+/-1.6;; 49.4+/-1.7;, 61.6+/-1.6%, respectively, * or ; P<0.001). These data indicate that both the sK(ATP) and mK(ATP) channel are involved in acute OIC and the GSK signaling axis regulates cardioprotection via K(ATP) channel opening.

Effects of the selective delta opioid agonist SNC80 on cocaine- and food-maintained responding in rhesus monkeys

Delta agonists such as SNC80 ((+)-4-[(aR)-a-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]- N, N-diethylbenzamide) produce some cocaine-like behavioral effects and warrant evaluation as candidate “agonist” medications for cocaine abuse. The present study examined acute and chronic effects of the systemically active delta agonist SNC80 on cocaine- and food-maintained responding in rhesus monkeys. Acute SNC80 (0.32–3.2 mg/kg, i.m.) pretreatment dose-dependently decreased cocaine self-administration (0.0032 mg/kg/injection), but doses of SNC80 that decreased cocaine self-administration also decreased food-maintained responding. In chronic studies, SNC80 (0.32–3.2 mg/kg/h, i.v.) was delivered for 7 days, and food or cocaine (0.01 mg/kg/injection) was available during 4 daily components of food availability and 4 daily components of drug availability. Chronic SNC80 (1.8 mg/kg/h) tended to decrease cocaine self-administration but produced greater reductions in food-maintained responding. A higher dose of 3.2 mg/kg/h SNC80 eliminated both cocaine- and food-maintained responding and produced profound sedation in one monkey and was not tested in other monkeys. These findings indicate that SNC80 produced dose-dependent and non-selective reductions in cocaine self-administration. These results suggest that SNC80 is unlikely to be useful as a treatment for cocaine dependence.

Effects of the selective delta opioid agonist SNC80 on cocaine‐ and food‐maintained responding in rhesus monkeys

Delta agonists such as SNC80 produce some cocaine-like behavioral effects and warrant evaluation as candidate “agonist” medications for cocaine abuse. The present study examined acute and chronic effects of SNC80 on cocaine- and food-maintained responding in rhesus monkeys. In acute studies, SNC80 (0.32–3.2 mg/kg, i.m.) was administered i.m. 15 min before 2 hr, multiple-component sessions during which food or cocaine was available under a FR30 schedule. SNC80 dose-dependently decreased cocaine self-administration, but doses of SNC80 that decreased cocaine self-administration also decreased food-maintained responding. In chronic studies, SNC80 (0.32–3.2 mg/kg/hr) was delivered by continuous i.v. infusion for 7 days, and food or cocaine (0.01 mg/kg/inj) were available during four daily 1hr food components and four 1hr drug components under a FR4(VR16:S) schedule. SNC80 doses up to 1.8 mg/kg/hr had little effect on cocaine self-administration, although 1.8 mg/kg/hr SNC80 transiently decreased food-maintained responding. A higher dose of 3.2 mg/kg/hr SNC80 eliminated all responding and produced profound sedation for 2 days in the first monkey tested. Further treatment was terminated in this monkey, and other monkeys were not tested. These findings indicate that SNC80 produced a dose-dependent and non-selective reduction in cocaine self-administration. These results contrast with the selective reductions in cocaine self-administration produced by other compounds (e.g. amphetamine, low-efficacy mu agonists) and suggest that SNC80 is unlikely to be useful as a treatment for cocaine dependence. Support: RO1-DA11460, RO1-DA02519 and K05-DA000101.

The selective delta opioid agonist SNC80 increases amphetamine‐mediated release of dopamine

The delta opioid agonist SNC80 produces dopamine-related behaviors including locomotor stimulation, antidepressant and anti-Parkisonian like effects. In contrast, it has been reported that SNC80 fails to release dopamine from the striatum of freely moving rats (Longoni et al., 1998 Behav. Pharmacol., 9: 9). To address this apparent inconsistency, we first showed that SNC80 enhanced locomotor stimulation produced by the psychomotor stimulant amphetamine (AMPH). Second, since the locomotor activity elicited by AMPH is associated with an increase in extracellular concentrations of dopamine within the striatum, we hypothesized that SNC80 enhances AMPH-induced locomotor activity by potentiating AMPH-stimulated release of dopamine. To test this hypothesis, rats were given varying doses (3.2–32 mg/kg, i.p.) of SNC80 and 3 h later were sacrificed, striatal slices were prepared and assayed for the release of dopamine in response to AMPH challenge. SNC80 enhanced AMPH-mediated dopamine release in a dose-dependent manner. The effect of SNC80 on AMPH-mediated dopamine release, but not the effect of AMPH alone, was blocked by a delta selective dose of the antagonist naltrindole. Similarly, addition of SNC80 (1–100μM) to striatal preparations of naïve rats, 30 min before AMPH challenge, resulted in an enhancement of dopamine release. The results of this study demonstrate that even though SNC80 is reported not to release dopamine alone, it is able to augment AMPH-mediated dopamine release by a local action in the striatum. Supported by NIDA grants DA04087 (JRT) and 07267.

P5.013 The selective delta opioid agonist SNC 80 potentiates cocaine-induced locomotor activity without concomitant increase in dopamine release in the nucleus accumbens

Corticosterone inhibits the proliferation of hippocampal neural stem cells (NSCs). The removal of corticosterone-induced inhibition of NSCs proliferation has been reported to contribute to neural regeneration. Leptin has been shown to regulate brain development, improve angiogenesis, and promote neural regeneration; however, its effects on corticosterone-induced inhibition of NSCs proliferation remain unclear. Here we reported that leptin significantly promoted the proliferation of hippocampal NSCs in a concentration-dependent pattern. Also, leptin efficiently reversed the inhibition of NSCs proliferation induced by corticosterone. Interestingly, pre-treatment with non-specific NMDA antagonist MK-801, specific NR2B antagonist Ro 25-6981, or small interfering RNA (siRNA) targeting NR2B, significantly blocked the effect of leptin on corticosterone-induced inhibition of NSCs proliferation. Furthermore, corticosterone significantly reduced the protein expression of NR2B, whereas pre-treatment with leptin greatly reversed the attenuation of NR2B expression caused by corticosterone in cultured hippocampal NSCs. Our findings demonstrate that leptin reverses the corticosterone-induced inhibition of NSCs proliferation. This process is, at least partially mediated by increased expression of NR2B subunits of NMDA receptors.

SNC 80 and related delta opioid agonists.

The discovery of the selective delta (delta) opioid agonists SNC 80 and BW373U86, which possess a diarylmethylpiperazine structure unique among opioids, was a major advance in the field of delta-opioid ligands. Much research has been performed to uncover the structure-activity relationships (SAR) of this class of ligands and also to compare the diarylmethylpiperazines with the traditional morphinan-based delta opioids. This review focuses on the development of the SAR of this unique series of ligands, and discusses questions which remain unanswered.

Opioid-induced cardioprotection occurs via glycogen synthase kinase beta inhibition during reperfusion in intact rat hearts.

Glycogen synthase kinase (GSK) inhibition produced by ischemic preconditioning has been previously shown to be regulated through phosphatidylinositol-3 kinase (PI3K). Therefore, we determined whether opioid-induced cardioprotection (OIC) occurs during reperfusion by altering GSK phosphorylation through PI3K and target of rapamycin (TOR). Furthermore, we determined if selective GSK inhibitors, SB216763(SB21) or SB415286(SB41), emulate OIC. Rats were treated with the nonselective opioid agonist, morphine (MOR, 0.3 mg/kg), the delta-selective opioid agonist BW373U86 (BW, 1 mg/kg), or the GSK inhibitors, SB21 (0.6 mg/kg) or SB41(1.0 mg/kg), either 10 minutes before ischemia or 5 minutes before reperfusion. Five minutes before opioid or SB21 treatment, some rats received either the PI3K inhibitor wortmannin (15 microg/kg) or LY294002 (0.3 mg/kg) or the TOR inhibitor rapamycin (3 microg/kg). After 30 minutes of ischemia followed by 2 hours of reperfusion, infarct size was assessed. MOR, BW, SB41, and SB21 reduced infarct size compared with vehicle when administered before ischemia (42.9+/-2.6, 40.3+/-2.3, 46.6+/-1.6, 42.2+/-1.8 versus 60.0+/-1.1%, respectively; P<0.001) and showed similar protection when administered 5 minutes before reperfusion (43.6+/-2.3, 40.2+/-2.6, 44.8+/-2.8, 39.4+/-0.8%, respectively; P<0.001). Wortmannin, LY294002, and rapamycin were found to inhibit OIC; however, they did not abrogate SB21-induced infarct size reduction. At 5 minutes of reperfusion, both MOR and BW increased P-GSKbeta at Ser9 in the ischemic zone compared with vehicle (181+/-20, 178+/-15 versus 75+/-17 DU, respectively; P<0.05), and this effect was abrogated by prior administration of wortmannin or rapamycin in MOR-treated rats. Furthermore, no differences were seen in phosphorylation of GSKalpha (Ser21 or Tyr279) or phosphorylation of GSKbeta (Tyr216). These data indicate that OIC occurs via the phosphorylation of GSKbeta at Ser9 during reperfusion.

SNC-80-induced preconditioning: selective activation of the mitochondrial adenosine triphosphate-gated potassium channel.

Pharmacologic preconditioning by delta-opioid agonists occurs via activation of an adenosine triphosphate (ATP)-gated potassium channel (I(KATP)). Opening of mitochondrial I(KATP) confers pharmacologic preconditioning whereas opening the sarcolemmal I(KATP) shortens action potential duration and is proarrhythmic. This study investigated whether SNC-80, a selective delta-opioid agonist, is associated with development of ventricular arrhythmia due to activation of I(KATP). Rabbit isolated hearts were subjected to 12 min of hypoxia and 40 min of reoxygenation after pretreatment with SNC-80 (1 microM, n = 6), pinacidil (1.25 microM, n = 12), or BMS-191095 (6.0 microM, n = 4). Nine additional hearts served as controls. The cytoprotective effects of SNC-80 at a concentration of 1 microM were confirmed using 30 min of regional ischemia followed by 120 min of reperfusion. Ventricular fibrillation (VF) developed in 11 of 12 pinacidil-treated hearts whereas none of the SNC-80-treated (zero of six) hearts developed VF (P < 0.001 compared with pinacidil pretreatment) and zero of four BMS-191095-pretreated hearts developed VF. Similarly, zero of nine control hearts developed VF. SNC-80 reduced infarct size expressed as a percentage of the area at risk from 33 +/- 4% to 14 +/- 3% (P = 0.004) compared with control. SNC-80, which selectively activates the delta-opioid receptor, provided cytoprotection but did not induce VF after hypoxia reoxygenation. The results indicate that pinacidil-induced nonselective activation of I(KATP) results in proarrhythmia that is dependent on activation of the sarcolemmal I(KATP). Selectivity for the mitochondrial I(KATP) is necessary to prevent induction of a proarrhythmic state.

Kinin-induced anion-dependent secretion in porcine ileum: characterization and involvement of opioid- and cannabinoid-sensitive enteric neural circuits.

The intestinal secretory actions of the proinflammatory peptide kallidin (lysyl-bradykinin) are mediated partially by enteric neurons. We hypothesized that kallidin produces neurogenic anion secretion through opioid- and cannabinoid-sensitive enteric neural pathways. Changes in short-circuit current (I(sc)) across sheets of porcine ileal mucosa-submucosa mounted in Ussing chambers were measured in response to kallidin (1 microM) or drugs added to the contraluminal bathing medium. Kallidin transiently increased I(sc), an effect reduced after inhibition of neuronal conduction by 0.1 microM saxitoxin, cyclooxygenase inhibition by 10 microM indomethacin, or kinin B(2) receptor blockade by 1 microM d-arginyl-l-arginyl-l-prolyl-trans-4-hydroxy-l-prolylglycyl-3-(2-thienyl)-l-alanyl-l-seryl-d-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl-l-(2alpha,3beta,7alphabeta)-octahydro-1H-indole-2-carbonyl-l-arginine (HOE-140). Its action was dependent upon extracellular Cl(-) or HCO(3)(-) ions, but was resistant to 10 microM bumetanide or 0.3 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, and seemed to involve luminal alkalinization as measured by pH-stat titration. Kallidin-induced I(sc) elevations were sensitive to saxitoxin in tissues bathed in Cl(-)-, but not HCO(3)(-)-deficient media. Tissues pretreated with 0.1 microM [d-Pen(2,5)]-enkephalin, a selective delta-opioid agonist, displayed reduced I(sc) responses to kallidin; this effect was prevented by the delta-opioid antagonist naltrindole. At a contraluminal concentration of 1 microM, the cannabinoid receptor agonist (6aR)-trans-3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol (HU-210) also attenuated responses to kallidin. Proinflammatory kinins seem to stimulate neurogenic anion secretion in porcine ileum by activating enteric neural circuits expressing inhibitory opioid and possibly cannabinoid receptors.

Activity of mu- and delta-opioid agonists in vas deferens from mice deficient in MOR gene.

1. Mice lacking the mu-opioid receptor have been recently generated. Centrally mediated responses of mu-opioid agonists are suppressed whereas some of the delta-opioid responses are preserved in these mutant mice. 2. The vas deferens bioassay has been used in this study to investigate the functional activity at a peripheral level of mu- and delta-opioid agonists in mice lacking mu-opioid receptors. 3. The different mu-opioid agonists evaluated, morphine, DAMGO, dermorphin and [Lys(7)]-dermorphin produced an inhibitory response in vas deferens from wild-type mice but had no relevant activity on vas deferens from mutant mice. 4. The selective delta-opioid agonists DPDPE, BUBU, deltorphin I, deltorphin II and [D-Met(2)]-deltorphin induced inhibitory effects in vas deferens from both wild-type and mutant mice. However, the biological activities of these ligands were slightly reduced in preparations from mutant mice. The inhibitory responses of all these delta-opioid agonists were prevented by the administration of the selective delta-opioid antagonist naltrindole. 5. These data indicate that delta-opioid agonists, but not mu-opioid agonists, are biologically active in vas deferens from mice lacking mu-opioid receptors. The decreased response of delta-agonists in mutant mice suggests that some cooperativity may exist between mu- and delta-opioid receptors in these vas deferens preparations.

Development of novel pain relief agents acting through the selective activation of the delta-opioid receptor

A continuing effort in the development of non-peptide delta-opioid agonists as possible safe and effective pain relief agents has been seen in the last years. Novel potent and selective delta-opioid agonists based on the pirrolomorphinan framework have been designed, synthesised and biologically characterised in our laboratories. In particular, compound 7 (SB 235863) proved the concept that a selective delta-opioid agonist may be a valuable pain relief agent free of the unwanted side-effects usually associated with narcotic analgesics such as morphine.

A DPDPE-induced enhancement of inward rectifier potassium current via opioid receptor in neuroblastoma×glioma NG108-15 cells

The effect of a delta-selective opioid agonist, DPDPE([D-Pen(2, 5)]-enkephalin), on the inward rectifier potassium current (I(KIR)) of NG108-15 cell was studied by whole cell voltage-clamp technique. It was found that microM DPDPE increased the amplitude and delayed the activation and inactivation of I(KIR) rapidly and reversibly. These effects could be reversed by naloxone, but were still obtained in pertussis toxin (PTX) preincubated cells or when using GDP-betas (guanosine 5'-o-[2-thio] diphoaphate) containing electrodes to block the G-protein coupled events. The above results suggest that DPDPE-induced change of I(KIR) is mediated by delta-opioid receptor but does not involve G-proteins.

Reduction of stress-induced analgesia but not of exogenous opioid effects in mice lacking CB1 receptors.

CB1 cannabinoid receptors are widely distributed in the central nervous system where they mediate most of the cannabinoid-induced responses. Here we have evaluated the interactions between the CB1 cannabinoid receptors and the endogenous opioid system by assaying a number of well-characterized opioid responses, e.g. antinociception and stress-mediated effects, on mutant mice in which the CB1 receptor gene was invalidated. The spontaneous responses to various nociceptive stimuli (thermal, mechanical and visceral pain) were not changed in mutant CB1 mice. Furthermore, the absence of the CB1 cannabinoid receptor did not modify the antinociceptive effects induced by different opioid agonists: morphine (preferential mu opioid agonist), D-Pen2-D-Pen5-enkephalin (DPDPE) and deltorphin II (selective delta opioid agonists), and U-50,488H (selective kappa opioid agonist) in the hot-plate and tail-immersion tests. In contrast, the stress-induced opioid mediated responses were modified in CB1 mutants. Indeed, these mutants did not exhibit antinociception following a forced swim in water at 34 degrees C and presented a decrease in the immobility induced by the previous exposure to electric footshock. However, the antinociception induced by a forced swim in water at 10 degrees C was preserved in CB1 mutants. These results indicate that CB1 receptors are not involved in the antinociceptive responses to exogenous opioids, but that a physiological interaction between the opioid and cannabinoid systems is necessary to allow the development of opioid-mediated responses to stress.