阿片κ-受体和ORL1受体二聚化的初步研究

为探讨κ-受体(κ-opioid receptor,KOR)和阿片受体样受体(opioid receptor like-1 receptor,ORL1 receptor)是否能够形成异源性受体二聚体,在原代培养的大鼠神经元细胞和用带有HA(hemagglutinin,血细胞凝集素)、Myc或Flag标签的KOR和ORL1质粒共同转染的中国仓鼠卵巢(CHO)细胞、人胚肾上皮(HEK293)细胞上,采用免疫荧光和免疫共沉淀的方法,研究KOR和ORL1之间的共定位以及是否存在相互作用。结果表明:在原代培养的海马和皮质神经元上,KOR和ORL1的免疫荧光在细胞膜上有重叠。同样,在HA-KOR和Myc-ORL1共同瞬时转染的CHO和HEK293细胞上也有类似的发现。另外,在共同表达Flag-KOR和Myc-ORL1的CHO细胞裂解液中,KOR与ORL1的受体蛋白能够被相互免疫共沉淀。这些研究结果提示,作为阿片受体不同亚型的KOR和ORL1受体之间有可能存在着异源二聚体,这也为进一步解释阿片受体结构的多样性和功能的复杂性提供了新的实验依据。     

孤啡肽及其片段的合成、痛觉调节作用和对免疫功能的影响

目的：研究孤啡肽(NC)与其4个片段(NC(1-15)NH₂, NC(1-13)NH₂, NC(1-11)NH₂, NC(1-5)NH₂) 在痛觉调节和免疫活性上的变化,探讨NC的构效关系。方法：固相多肽合成法合成NC及其片段;甩尾法测定它们对小鼠的痛敏作用和对吗啡镇痛作用的拮抗;T细胞玫瑰花结形成百分率和红细胞免疫粘附能力评测对免疫功能的影响。结果：虽然NC及其片段均有痛敏作用并可拮抗吗啡的镇痛作用,但NC(1-11)NH₂和NC(1-5)NH₂比母体活性约降低100倍,而NC(1-13)NH₂和NC(1-15)NH₂与母体有相同的活性。NC及片段(0.3～3 nmol.kg^-1)对T细胞免疫功能均有促进作用;NC(1-11)NH₂(0.3 nmol.kg^-1)对红细胞的免疫粘附能力有促进作用;NC(1-5)NH₂(0.3～30 nmol.kg^-1)不影响红细胞的免疫功能。结论：C端在NC的构效关系中有重要的作用。     

糖尿病肾病患者血清N/OFQ、IL-6的表达变化及相关性分析

目的 观察糖尿病肾病(DKD)患者血清孤啡肽(N/OFQ)、白细胞介素-6(IL-6)水平变化及其相关性。方法 选择2021年3月至2022年3月确诊的2型糖尿病(T2DM)患者180例,根据尿白蛋白/肌酐比值(ACR)分为正常白蛋白尿组(ACR<30 mg/g)、微量白蛋白尿组(30 mg/g≤ACR≤300 mg/g)和大量白蛋白尿组(ACR>300 mg/g),每组60例。记录3组的一般资料及相关并发症情况,监测空腹血糖(FBG)、糖化血红蛋白(HbA1c)、尿微量白蛋白(mALB)、尿白蛋白/肌酐比值(ACR)、估算肾小球滤过率(eGFR)、总胆固醇(CHO)、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)、中性粒细胞计数(NEUT)、中性粒细胞百分比(NEU%)。使用ELISA法测定3组血清N/OFQ、IL-6水平。分析上述指标并比较3组血清N/OFQ、IL-6水平的差异及其与DKD的相关性。采用logistic回归分析T2DM并发DKD的独立危险因素。采用受试者工作特征(ROC)曲线分析N/OFQ、IL-6单项及二者联合检测...     

内吗啡肽—1的镇痛作用

目的：研究内吗啡肽－1（EM－1）的镇痛作用。方法：采用电刺激鼠尾－嘶叫法、扭体法、佐剂性关节炎以及神经源性疼痛等多种疼痛模型,观察腹腔注射EM－1的镇痛作用,并和脊髓蛛网膜下腔注射和侧脑室注射EM－1的镇痛作用进行比较。结果：1）EM－1能剂量依赖地提高大鼠电刺激鼠尾－嘶叫法的痛阈;能抑制醋酸引起的小鼠扭体反应;在佐剂性关节炎所致的炎症性痛觉过敏及坐骨神经部分结扎所引起的神经源性痛觉过敏中,EM－1与有镇痛作用。2）中枢给EM－1的镇痛作用比外击给药出现得较快,而且较强。3）阿片受体拮抗选择性拮抗剂cyprodime也能翻转EM－1的镇痛作用;反复给予EM－1具有确切的镇冯作用,其镇痛作用由中枢μ阿片受体介导。     

孤啡肽、内吗啡肽-1和-2与无脊椎动物免疫和神经系统的μ_3阿片受体无相互作用(英文)

AIM: To determine if endomorphin-1,-2 and nociceptin (orphanin FQ) bind to the μ3 opiate receptor subtype or release nitric oxide as μ3 selective ligands do. METHODS: These opioid peptides were examined for their ability to displace [3H]dihydromorphine (DHM) binding from the invertebrate (immunocytes and pedal ganglia)μ3 opiate receptor in membrane homogenates. The ligands were also tested for their ability to release nitric oxide from the same intact tissues utilizing an amperometric probe that measures nitric oxide in real-time. RESULTS: Endomorphin-1,-2 and nociceptin do not displace [3H]DHM binding from immunocyte or pedal ganglia membrane homogenates nor do they release nitric oxide from these tissues. CONCLUSION: Since these newly discovered opioid peptides do not interact with the μ3 opiate receptor subtype, endogenous morphine' s significance is enhanced because it appears to be the only naturally occurring opiate ligand for the receptor. Furthermore, since this study involves invertebrate t     

降钙素基因相关肽在神经性痛大鼠扣带回内的痛觉调节作用及其机制

目的研究降钙素基因相关肽(CGRP)在神经性痛大鼠前扣带回(ACC)内的痛觉调节作用。方法采用热板法和Randall Selitto法分别测定大鼠对热刺激和机械刺激的缩爪反应潜伏期(HWL),评价大鼠对伤害性刺激的反应。对大鼠左侧坐骨神经进行轻度结扎,建立神经性痛模型。在正常大鼠及神经性痛大鼠扣带回内,注射CGRP以及注射CGRP后注射CGRP的拮抗剂CGRP8-37,观察对伤害性刺激的反应。采用逆转录聚合酶链反应(RT-PCR)和Western印迹检测神经性疼痛对ACC中CLR m RNA水平和CLR蛋白水平的影响。结果 CGRP在神经性痛大鼠ACC中能引起显著的镇痛作用,并呈剂量依赖的关系。在神经性痛大鼠中,CGRP诱导的镇痛作用高于正常大鼠。在神经性痛大鼠的ACC内注射CGRP受体拮抗CGRP8-37,明显抑制CGRP引起的镇痛作用,表明CGRP受体参与ACC中CGRP的镇痛作用。与正常大鼠相比,神经性痛大鼠ACC中的CLR m RNA和蛋白水平显著升高,这支持了上述结果,在神经性痛大鼠ACC中在CGRP诱导的镇痛作用发生了明显的变化。结论在神经性痛大鼠扣带回内注射降钙素基因相关肽引起显著的镇痛作用,CGRP受体参与其中。与正常大鼠相比,神经性痛大鼠ACC中CGRP诱导的镇痛作用发生了明显的变化,这与CLR的表达发生显著变化有关。     

磷脂酰乙醇胺结合蛋白对阿片受体信号转导及阿片依赖的调节作用

药物成瘾是一种慢性、反复发作性脑疾病,其机制尚未阐明。磷脂酰乙醇胺结合蛋白(PEBP)是近来发现的一种多功能蛋白,能够调节信号转导功能,也能通过水解产生的海马胆碱能神经刺激肽(HCNP)增强胆碱乙酰基转移酶(ChAT)活性而影响隔-海马胆碱能系统功能。但是,PEBP在药物成瘾中有何作用尚未见文献报道。我们采用蛋白质组学研究技术首次发现吗啡慢性处理能够上调大鼠海马内PEBP的表达,提示其与阿片成瘾可能有一定的相关性。鉴于PEBP可通过磷酸化与非磷酸化形式的转化而调节ERK和GRK的活性,我们首先研究了PEBP对μ阿片受体信号转导的影响。结果发现,μ阿片受体激活不影响PEBP磷酸化与非磷酸化之间的转化;过表达PEBP不影响μ阿片受体与激动剂处理诱导的G蛋白的偶联,也不影响μ阿片受体的磷酸化和脱敏,但是降低μ阿片受体激活后对腺苷酸环化酶活性的抑制和对ERK的激活,提示PEBP对μ阿片受体的信号转导有一定的调节作用。整体动物水平的研究发现,吗啡慢性处理导致大鼠海马内PEBP蛋白的表达水平特异性的上调,并呈现时间及剂量依赖性;而戒断3 d时海马内PEBP表达水平恢复至接近基础水平,之后其表达水平逐渐上调并维持至28 d。采用反义核酸的策略下调海马内PEBP表达,能够加重吗啡依赖的形成。进一步的调节机制研究发现,吗啡依赖及戒断引起海马内ChAT活性出现变化,其变化趋势与PEBP表达水平的变化完全一致,提示PEBP可能通过剪切成HCNP影响胆碱能系统功能而调节阿片依赖。因此,本文研究不仅发现PEBP对μ阿片受体信号转导具有一定的调节作用,而且发现PEBP参与了阿片依赖过程,机制可能与影响海马内ChAT活性、进而影响胆碱能神经系统功能有关。     

侧脑室注射内吗啡肽-1对麻醉大鼠血压的影响

目的　观察侧脑室注射内吗啡肽-1(EM-1)对麻醉大鼠血压的影响,并初步探讨其作用机理。方法　侧脑室埋植导管给药,颈动脉插管测血压。结果　icv EM-1剂量依赖、纳洛酮敏感地降低麻醉大鼠的血压。icv或iv酚妥拉明、普萘洛尔和iv L-NNA对EM-1引起的血压降低反应无影响;给予阿托品(icv 25 μg·kg^-1 ;或iv 50 μg·kg^-1)和切断双侧迷走神经减弱EM-1引起的血压降低反应。结论　icv EM-1可引起麻醉大鼠血压降低;此效应由阿片受体介导,有中枢M受体的参与,通过兴奋迷走神经所致     

吗啡对体外培养的人乳腺细胞雌激素受体mRNA表达的影响

目的：研究吗啡对雌激素诱导的体外培养人的正常乳腺细胞的雌激素受体（ER）mRNA表达的影响。方法：购买人正常乳腺纤维细胞株进行培养，给予17-β雌二醇（E2），吗啡（Mor）及纳洛酮干预。通过RT—PCR的方法半定量分析吗啡对正常乳腺细胞ER mRNA表达的影响。结果：ER与内参照B—actin分别在249 bp和270 bp处出现特征性条带。雌激素组、吗啡组与对照组比较，差异有显著性（P〈0．05），E2＋Mor组与对照组比较，差异无显著性（P〉0．05）。吗啡作用24h呈剂量依赖性抑制乳腺细胞ERmRNA的表达，3组吗啡的终浓度分别为1×10^-6mol／L、1×10^-5mol／L、1×10^-4 mol／L，差异有显著性（P〈0，05）。结论：从mRNA水平证明了外源性阿片类药物吗啡可直接影响雌激素对乳腺的促增殖作用，同时还可剂量依赖性下调雌激素受体mRNA的表达。     

内吗啡肽-1对麻醉大鼠左心室功能的影响

目的：观察内吗啡肽-1（endomorphin-1,EM-1）静脉注射和侧脑室注射对麻醉大鼠左心室功能的影响,并初步探讨其可能的作用机理。方法：Wistar大鼠麻醉后,静脉注射或侧脑室注射EM-1,经右颈总动脉左心室插管测定左心室功能,并测定血清和心肌组织NO和T-NoS含量。结果：静脉注射、侧脑室注射EM-1均剂量依赖性地降低麻醉大鼠左心室功能,预先给予阿片受体阻断剂纳洛酮或特异性肚受体阻断剂cyprodime可阻断EM-1引起的心功能降低反应;预先给予胆碱能M受体阻断剂阿托品可减弱EM-1引起的心功能降低反应。静脉注射EM-1血清中NO和T-NOS比对照组有所增加,而心肌组织的NO和T-NOS无明显变化;侧脑室注射EM-1的血清和心肌组织的NO和T-NOS均无明显变化。结论：静脉注射、侧脑室注射EM-1可引起麻醉大鼠在体左心室功能下降,此效应由阿片受体介导,可能有胆碱能M受体参加。     

The ORL-1 (NOP1) receptor ligand nociceptin/orphanin FQ (N/OFQ) inhibits neurogenic dural vasodilatation in the rat

The effects of the ORL-1 (NOP(1)) receptor ligand nociceptin (N/OFQ) and the nociceptin antagonists [Nphe(1)]N/OFQ-(1-13)-NH(2) (Nphe) and nocistatin (NST) on neurogenic dural vasodilatation (NDV) in the rat dura mater evoked by electrical stimulation of a closed cranial window were studied. The middle meningeal artery was visualised using intravital microscopy, and the vessel diameter analysed using a video dimension analyser. N/OFQ (1, 10, 100 nmol kg(-1); i.v., n=10) significantly and dose-dependently suppressed NDV maximally by 65% (P<0.01). Neither Nphe (100 nmol kg(-1); n=5) nor NST (100 nmol kg(-1); n=4) alone had an effect on NDV (P>0.05). Baseline vessel diameter was not significantly affected by application of N/OFQ, NST or Nphe. Application of the selective N/OFQ antagonist Nphe (10, 100 nmol kg(-1) i.v., n=8) dose-dependently and significantly (P<0.01) reversed the inhibition of NDV induced by application of N/OFQ (10 nmol kg(-1)). NST (10, 100 nmol kg(-1); n=7) failed to reverse the effects elicited by N/OFQ. Application of N/OFQ elicited a dose-dependent transient decrease in arterial blood pressure (P<0.01). Nphe dose-dependently reversed the cardiovascular effects induced by application of N/OFQ (10 nmol kg(-1)) (P<0.01),while NST did not alter the blood pressure reaction elicited by N/OFQ. The results show that N/OFQ inhibits NDV, an effect which is antagonised by Nphe, but not by NST. ORL-1 (NOP(1)) receptors located on trigeminal sensory fibres may be involved in the regulation of dural vessel diameter and hence may play a role in migraine pathophysiology.     

孤啡肽对大鼠顶叶皮层神经元延迟整流钾电流的抑制作用

目的研究孤啡肽(orphanin FQ/nociceptin,OFQ/N)对大鼠顶叶皮层神经元延迟整流钾电流(IK)的影响,初步探讨其干扰学习记忆过程的离子机制。方法采用全细胞膜片钳技术,观察OFQ/N对急性分离的大鼠顶叶皮层神经元IK的作用。结果①OFQ/N明显抑制IK,并呈剂量依赖性(P<0.05)。②0.1μmol.L-1OFQ/N使IK的电流-电压(I-V)曲线降低(n=8,P<0.01)。③0.1μmol.L-1OFQ/N使IK激活曲线的半数激活电压(V1/2)和斜率因子(k)分别由给药前的(-43.4±6.1)mV和(11.5±1.1)mV变为给药后的(-19.1±4.6)mV和(17.3±3.2)mV(n=8,P<0.01)。④0.1μmol.L-1OFQ/N使IK失活曲线的半数失活电压(V1/2)和斜率因子(k)分别由给药前的(-68.8±2.6)mV和(16.5±1.7)mV变为给药后的(-76.8±2.8)mV(n=5,P<0.01)和(15.7±3.1)mV(n=5,P>0.05)。结论OFQ/N可抑制大鼠顶叶皮层神经元IK,使IK激活曲线右移,失活曲线左移。     

The endogenous OFQ/N/ORL-1 receptor system regulates the rewarding effects of acute cocaine

Previous studies have shown that orphanin FQ/nociceptin (OFQ/N), the endogenous ligand of the opioid receptor-like (ORL-1) receptor, reduces the rewarding and addictive properties of cocaine and other drugs of abuse. In the present study, using the conditioned place preference (CPP) paradigm, as an animal model of drug reward, we assessed whether the rewarding action of acute cocaine would be altered in mice lacking the ORL-1 receptor or in wild type mice treated with J-113397, an ORL-1 receptor antagonist, relative to their saline-treated controls. On day 1, mice were tested for their baseline place preferences, in which each mouse was placed in the neutral chamber of a three-chambered CPP apparatus, allowed to freely explore all the chambers and the amount of time that a mouse spent in each conditioning chamber was recorded for 15 min. On days 2-3, mice received once daily alternate-day saline/cocaine (15 or 30 mg/kg) conditioning for 30 min. On day 4, mice were tested for their postconditioning preferences, as described for day 1. In a subsequent study, the effect of J-113397 (3 mg/kg) on the rewarding action of acute cocaine (15 mg/kg) was also examined in wild type mice. Our results showed that mice lacking the ORL-1 receptor expressed greater CPP than their wild type littermates. Furthermore, the rewarding action of cocaine was enhanced in the presence of J-113397 in wild type mice. Together, the present results suggest that the endogenous OFQ/N/ORL-1 receptor system is involved in the rewarding action of acute cocaine.     

Alterations in the level of OFQ/N-IR in rat brain regions by cocaine

We have previously shown that administration of orphanin FQ/nociceptin (OFQ/N), the endogenous ligand of the opioid receptor-like (ORL-1) receptor, into the lateral ventricles or VTA blocked cocaine sensitization. In the present study, we determined the effect of acute and chronic cocaine treatment on the level of endogenous OFQ/N in rat brain regions. Male Sprague Dawley rats were tested for motor activity in response to saline or cocaine (20 mg/kg) injection once daily for three consecutive days. To determine the effect of single or repeated cocaine administration on the level of OFQ/N, rats were sacrificed 1 h following saline or cocaine injection either on day 1 or 3, respectively. Additional groups of rats were treated similarly with saline or cocaine on days 1-3 and sacrificed or tested for locomotor sensitization on day 8. Consistent with previous studies, repeated cocaine administration induced locomotor sensitization to a challenge dose of cocaine (7.5 mg/kg) given on day 8. Measurements of tissue content of OFQ/N-IR using radioimmunoassay indicated that the rat hypothalamus and striatum, respectively, contained the highest and lowest levels of the peptide among the brain regions tested. Acute cocaine decreased the level of OFQ-IR in the rat midbrain and to a lesser extent in the striatum. On the other hand, the level of OFQ/N was higher in rats treated with cocaine on days 1-3 and sacrificed on day 8. These findings suggest that endogenous OFQ/N may be involved in the actions of cocaine and possibly in cocaine-induced motor stimulation and locomotor sensitization.     

中枢阿片受体介导鞘内注射吗啡对大鼠缺血后心肌的保护作用

目的探讨鞘内注射吗啡预处理对缺血/再灌注心肌的影响及中枢神经系统阿片受体在其中的作用。方法建立大鼠鞘内注射和心脏缺血/再灌注损伤的动物模型。分为对照组(NS)和鞘内注射吗啡预处理组(M),M组分4个剂量组(M1,10μg·kg-1;M2,1μg·kg-1;M3,0.1μg·kg-1;M4,0.01μg·kg-1)。鞘内注射吗啡的方法是分别在缺血/再灌注前30 min鞘内注射吗啡5 min,间隔5 min共3次。在M2组鞘内注射吗啡前鞘内注射3种选择性阿片受体拮抗剂Naltrindole(NTD,δ阿片受体阻断剂,15 nmol(nM),NTD+M2组)、nor-Binaltorphimine(nor-BNI,κ阿片受体阻断剂,15 nmol.L-1,nor-BNI+M2组)和CTOP(μ阿片受体阻断剂,15 nM,CTOP+M2组)。观察指标包括:平均动脉压(MAP)、心率(HR)、计算平均动脉压和心率乘积(RPP);缺血危险区(AAR)、梗死区(IS)的体积、心肌梗死面积以IS/AAR来表示。结果M1组、M2组和M3组的IS/AAR均低于NS组(P<0.05,P<0.01),M4组与NS组的IS/AAR相似(P>0.05);NTD+M2组、nor-BNI+M2组和CTOP+M2组与自身对照组(NS+NTD组,NS+nor-BNI组和NS+CTOP组)及NS组相比差异无显著性(P>0.05),而均高于M2组(P<0.05,P<0.01)。各组在各时点的MAP、HR和RPP差异无显著性(P>0.05)。结论鞘内注射吗啡对在体大鼠心肌缺血/再灌注损伤有保护作用,中枢神经系统的δ,κ和μ三种阿片受体都参与介导了其保护作用。     

Small-molecule agonists and antagonists of the opioid receptor-like receptor (ORL1, NOP): Ligand-based analysis of structural factors influencing intrinsic activity at NOP

The recently discovered fourth member of the opioid receptor family, the nociceptin receptor (NOP) and its endogenous ligand, the heptadecapeptide nociceptin, are involved in several central nervous system pathways, such as nociception, reward, tolerance, and feeding. The discovery of small-molecule ligands for NOP is being actively pursued for several therapeutic applications. This review presents a brief overview of the several recently reported NOP ligands, classified as NOP agonists and antagonists, with an emphasis on the analysis of the structural features that may be important for modulating the agonist/antagonist profile (intrinsic activity) of these ligands. Structure-activity relationships in our own series of dihydroindolinone-based NOP ligands and those of the various reported ligands indicate that the lipophilic substituent on the common basic nitrogen present in all NOP ligands plays a role in determining the agonist/antagonist profile of the NOP ligand. This analysis provides a basis for the rational drug design of NOP ligands of desired intrinsic activity and provides a framework for developing pharmacophore models for high affinity binding and intrinsic activity at the NOP receptor. Since NOP agonists and antagonists both have therapeutic value, rational approaches for obtaining both within a high-affinity binding class of compounds are very useful for designing potent and selective NOP ligands with the desired profile of intrinsic efficacy.     

Activities of mixed NOP and mu-opioid receptor ligands

BACKGROUND AND PURPOSE: Compounds that activate both NOP and mu-opioid receptors might be useful as analgesics and drug abuse medications. Studies were carried out to better understand the biological activity of such compounds. EXPERIMENTAL APPROACH: Binding affinities were determined on membranes from cells transfected with NOP and opioid receptors. Functional activity was determined by [(35)S]GTPgammaS binding on cell membranes and using the mouse vas deferens preparation in vitro and the tail flick antinociception assay in vivo. KEY RESULTS: Compounds ranged in affinity from SR14150, 20-fold selective for NOP receptors, to buprenorphine, 50-fold selective for mu-opioid receptors. In the [(35)S]GTPgammaS assay, SR compounds ranged from full agonist to antagonist at NOP receptors and most were partial agonists at mu-opioid receptors. Buprenorphine was a low efficacy partial agonist at mu-opioid receptors, but did not stimulate [(35)S]GTPgammaS binding through NOP. In the mouse vas deferens, each compound, except for SR16430, inhibited electrically induced contractions. In each case, except for N/OFQ itself, the inhibition was due to mu-opioid receptor activation, as determined by equivalent results in NOP receptor knockout tissues. SR14150 showed antinociceptive activity in the tail flick test, which was reversed by the opioid antagonist naloxone. CONCLUSIONS AND IMPLICATIONS: Compounds that bind to both mu-opioid and NOP receptors have antinociceptive activity but the relative contribution of each receptor is unclear. These experiments help characterize compounds that bind to both receptors, to better understand the mechanism behind their biological activities, and identify new pharmacological tools to characterize NOP and opioid receptors.     

ORL1 receptor-mediated inhibition by nociceptin of noradrenaline release from perivascular sympathetic nerve endings of the rat tail artery

In the present study the effect of the opioid heptadecapeptide nociceptin, also termed orphanin FQ, an endogenous ligand for the orphan receptor named ORL₁ (opioid receptor-like 1) receptor, was investigated on [³H]noradrenaline release induced by electrical field stimulation (24 pulses at 0.4 Hz, 200 mA, 0.3 ms duration) in the rat tail artery in the absence and presence of an α₂-adrenoceptor antagonist, rauwolscine 3 μM. Nociceptin inhibited the electrically-evoked tritiated noradrenaline release in a concentration-dependent manner from rat tail arteries. This inhibitory effect of nociceptin was enhanced in the presence of the α₂-adrenoceptor antagonist rauwolscine (maximum inhibition by 25% and 50% in the absence and presence of rauwolscine, respectively). At a supramaximal concentration (10 μM), the inhibitory action of DAGO, a selective μ-opioid receptor agonist, was less pronounced than that of nociceptin. The inhibitory effect of nociceptin was counteracted by naloxone benzoylhydrazone (3 μM) which by itself did not change the stimulation-evoked noradrenaline overflow. Naloxone (10 μM), a non-selective opioid receptor antagonist, did not affect the inhibitory effect of nociceptin whereas it abolished that of DAGO. In conclusion, these results suggest that nociceptin modulates noradrenergic neurotransmission by acting on prejunctional ORL₁ receptors located on nerve terminals innervating the rat tail artery. They also demonstrate that prejunctional ORL₁ receptors interact with prejunctional α₂-adrenoceptors. The physiological significance of this phenomenon remains to be determined. Received: 27 August 1998 / Accepted: 29 September 1998     

Nociceptin inhibits noradrenaline release in the mouse brain cortex via presynaptic ORL1 receptors

A fourth type of opioid receptor, termed ORL₁, has been cloned and nociceptin (also known as orphanin FQ) has been identified as an endogenous ligand at this receptor. We examined whether nociceptin affects the release of noradrenaline in the brain. For this purpose, cerebral cortex slices from the mouse, rat or guinea-pig were preincubated with [³H]noradrenaline and then superfused with medium containing desipramine and rauwolscine. Tritium overflow was evoked electrically (0.3 Hz) or by introduction of Ca²⁺ 1.3 mM into Ca²⁺-free K⁺-rich (15 mM) medium. Nociceptin 1 μM reduced the electrically evoked tritium overflow from mouse, rat and guinea-pig brain cortex slices by 80, 71 and 36%, respectively. Naloxone 10 μM did not change the effect of nociceptin. All subsequent experiments were performed on mouse brain cortex slices and in the presence of naloxone 10 μM. The concentration-response curve of nociceptin (maximum inhibition by 80%, pEC₅₀ 7.5) was shifted to the right by the non-selective ORL₁ receptor antagonist naloxone benzoylhydrazone and the selective ORL₁ receptor antagonist [Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ (pA₂ 6.6 and 7.2, respectively). Naloxone benzoylhydrazone did not affect the evoked overflow by itself whereas [Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ caused an inhibition by maximally 35% (pEC₅₀ 7.0; intrinsic activity α 0.45). The inhibitory effect of [Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ was counteracted by naloxone benzoylhydrazone. Nociceptin also reduced the Ca ²⁺ -evoked tritium overflow in mouse brain cortex slices superfused in the presence of tetrodotoxin. This effect was also antagonized by naloxone benzoylhydrazone, which, by itself, did not affect the evoked tritium overflow. In conclusion, nociceptin inhibits noradrenaline release more markedly in the mouse than in the rat or guinea-pig brain cortex. The effect of nociceptin in the mouse brain cortex involves ORL₁ receptors, which are located presynaptically on noradrenergic neurones. Received: 19 June 1998 / Accepted: 17 July 1998