孤啡肽和阿片类配体与阿片孤儿受体相互作用的机制研究(英文)

目的:研究孤啡肽和阿片类配体与阿片孤儿受体相 互作用的分子机制。方法:用分子动力学方法计算孤啡肽的最低能构象;通过分子对接程序将孤啡肽、阿片类配体对接到阿片孤儿受体的结合口袋中;通过结合能的计算研究配体对受体的亲和力与它们的结合能之间的关系。结果:孤啡肽(1-4)残基位于结合口袋的底部,孤啡肽(5-7)残基位于结合口袋的顶部,孤啡肽(8-17)残基与孤儿受体的第二膜外环区结合;阿片类配体和孤儿受体的结合方式与孤啡肽的情况类似,区别在于孤儿受体参与配体结合的残基种类和数量不同,因而亲和力不同;配体-受体的结合能与配体的亲和力之间有很好的相关性;预测了洛芬太尼四个异构体与阿片孤儿受体的亲和力。结论:该研究能够解释许多实验事实,有助于进一步理解阿片受体与配体相互作用的分子机制并设计新的分子生物学实验。     

二氢埃托啡对大鼠脑阿片受体的结合特性

本文在大鼠脑匀浆P_2膜上,观察了二氢埃托啡(DHE)对[~3H]纳洛酮,[~3H]DPDPE和[~3H]埃托啡(预先用30nmol/L吗啡和100nmol/L DADLE阻断μ和δ受体)与阿片受体结合的抑制强度。结果表明:DHE对[~3H]纳洛酮与阿片受体结合的抑制强度远远大于对[~3H]DPDPE和[~3H]埃托啡(预先阻断μ和δ受体后)。DHE对μ,δ和κ受体的相对亲和力之比为1951:2:1,提示DHE为μ受体相对选择性配体。     

冠脉壁上阿片及苯环利定受体的自显影观察

为了了解冠状动脉上苯环利定（Ｐｈｅｎｃｙ－ｃｌｉｄｉｎｅ，Ｐｈｅ）受体及阿片受体的分布，利用猪冠状动脉的大、小支作了离体血管放射自显影研究，显微镜下观察到，猪冠脉壁上有与［３Ｈ］Ｐｈｅ和［３Ｈ］Ｅｔｏｒ－ｐｈｉｎｅ特异结合位点，它们均主要分布于冠脉的外膜上。通过［３Ｈ］Ｏｈｍｅｆｅｎｔａｎｙｌ自显影观察，冠脉上未见特异结合颗粒，提示冠脉上的阿片受体不是μ型。     

家兔尾核神经元对微电泳羟戊甲吗啡、纳洛酮及电针的反应

本实验用多管微电极离子微电泳的方法,记录家兔尾核神经元的自发活动并测试其对微电泳吗啡类物质羟戊甲吗啡及其拮抗剂纳洛酮的反应,从161个单位的测试中,观察到有61个被羟戊甲吗啡所抑制,这种抑制可被纳洛酮所阻断(59/61)。对羟戊甲吗啡敏感的单位大多数分布在尾核头部背侧和边缘部分,与感觉调制的部位一致。纳洛酮可使部分处于安静状态的单位活动(7/34)。在46个单位中,观察到有24个对电针和羟戊甲吗啡的反应一致,其中16个均表现为自发活动的抑制。电针的抑制作用也可被纳洛酮所阻断(4/8)。支持电针作用部分与尾核内阿片肽系统有关的看法。     

Effect of α2 adrenergic agents upon central etorphine antinociception in the cat

Systemic (s.c.) administration of α₂ agonists clonidine (25–100 μg/kg) or guanfacine (50–400 μg/kg) elicited antinociception as assessed by the cat tail-flick model and potentiated in a dose-dependent manner the antinociceptive effect of etorphine (2.5 μg) administered directly into the periaqueductal gray. Conversely, systemic yohimbine (1 mg/kg) attenuated the effects of central etorphine, and diminished potentiation of etorphine by theα₂ agonists. Prior microinjection of clonidine (5μg) or guanfacine (5 μg) into the locus coeruleus (LC) reduced the intensity of central etorphine antinociception whereas central yohimbine (20 μg) pretreatment increased peak antinociceptive activity and prolonged the duration of etorphine. Thus, systemicα₂ agonists are inherently antinociceptive and potentiate central narcotic antinociception; however, the site of interaction betweenα₂ agonists and opiates does not appear to be the LC inasmuch asα₂ agonists attenuate the antinociceptive effect of etorphine when administered directly into the LC. A spinal site of action is suggested based upon known LC-spinal projections and our experimental observations.     

Opiate receptor binding-effect relationship: Sufentanil and etorphine produce analgesia at the μ-site with low fractional receptor occupancy

The analgesic activity of the opiate agonists etrophine and sufentanil and the antagonistic effects of diprenorphine and naloxone have been related to the occupancy of 3 classes of opiate binding sites previously defined in vivo²⁹in order to establish their pharmacological significance. Sufentanil binds specifically in vivo to the first type of site (site 1), exhibiting 1100-fold selectivity over site 2, whereas etorphine displays 20-fold selectivity for site 1 over site 2. Neither agonist has measurable affinity to the third type of binding site. The binding data suggest that site 1 is analogous to the μ site previously identified in vitro²⁹. Both agonists produce analgesia in the rat tail flick test at the same low fractional occupancy of site 1 ( 2% at the ED₅₀) while they display much lower and quite different occupancies at site 2. Both of the opiate antagonists naloxone and diprenorphine reduce the potency of sufentanil and etorphine by a factor of 2 at 50% occupancy of site 1 alone. These results provide strong evidence that these 4 drugs exert their effects by interaction with site 1 (μ sites) which therefore may be regarded as the receptor responsible for analgesic action in this test. The assumption of a direct relationship between antagonistic effect and fractional occupancy appears to be valid for naloxone and diprenorphine at site 1, while the agonists exert their action at a very low fractional occupancy implying a non-linear binding-effect process.     

Some Pharmacological Properties of the Combination of Etorphine and Acepromazine (Immobilon®) and the Morphine antagonist,Diprenorphine, in the Dog *

Abstract: The analgesic effect of immobilon® (etorphine 125 μg/ml and acepromazine 500 μg/ml) has been compared with that of its two components and of morphine. The following values were found (ED 50 μg/kg subcutaneously; acetic acid test, mice): immobilon (etorphine 0.3 + acepromazine 1.2); etorphine 1.2; acepromazine 40 and morphine 350. The action of immobilon and of the antagonist, diprenorphine (revivon®) on systolic/diastolic blood pressure, heart rate, ECG, respiration and rectal temperature was checked during clinical neuroleptanalgesia in seven dogs. No effect on the systolic and a slight fall in the diastolic blood pressure were observed. Furthermore a significant bradycardia was noted during the neuroleptanalgesia as well as a significant tachycardia during arousal produced by diprenorphine. The former could readily be antagonized by atropine, and the latter by propranolol. It is concluded that the effect of immobilon®/revivon® on cardiovascular and respiratory parameters does not preclude its clinical use in dogs.     

3H]-etorphine and [3H]-diprenorphine receptor binding in vitro and in vivo: differential effect of Na+ and guanylyl imidodiphosphate

The in vivo and in vitro cerebral receptor binding kinetics of the opiate agonist etorphine and the antagonist diprenorphine were investigated in the rat. Although of similar receptor affinity in vitro in Tris buffer brain homogenates, etorphine exhibited considerably less affinity than diprenorphine in vivo. The hypothesis was tested whether the opiate receptor regulators, Na⁺ and GTP, are responsible for the low in vivo receptor affinity of the agonist. [³H]Etorphine and [³H]diprenorphine dissociation curves were similarly affected by Na⁺ and guanylyl imidodiphosphate (GPP(NH)P), a hydrolysis resistant GTP analog when added separately in vitro. However, the combination of Na⁺ and GPP(NH)P greatly accelerated only the [³H]etorphine off-rate over that with Na⁺ alone and reproduced the rapid dissociation half-life observed in vivo (t_1/2 < 1 min). In contrast, the receptor dissociation rate of the antagonist was not further accelerated by Na⁺ plus GPP(NH)P over that with Na⁺ alone. Moreover, Na⁺ plus GPP(NH)P decreased [³H]etorphine, but not [³H]diprenorphine, equilibrium binding in vitro. These results suggested that the lower in vivo affinity of etorphine than of diprenorphine is predominantly caused by the combined action of Na⁺ and GTP. Furthermore, the data are consistent with the hypothesis that both etorphine and diprenorphine bind to the opiate receptor in its high affinity form, but that only the agonist etorphine is capable of converting the high affinity form to one of low affinity in the presence of Na⁺ and guanine nucleotide. Assuming that production of the low affinity form reflects biological activation of the opiate receptor system, then this hypothesis is consistent with the high pharmacological potency of etorphine (agonistic ED₅₀ ～ 1μg/kg) relative to its low apparent in vivo receptor affinity, as well as with the low fractional receptor occupancy of etorphine (～ 2%) at its analgesic ED₅₀. Finally, in vivo [³H]etorphine and [³H]diprenorphine displacement curves obtained with unlabeled diprenorphine and etorphine showed that [³H]etorphine labels only a subpopulation of the total [³H]diprenorphine binding sites. It remains to be determined which subsites of the opiate receptor system mediate the agonistic actions of etorphine.     

Continuous opioid agonist treatment dose-dependently regulates mu-opioid receptors and dynamin-2 in mouse spinal cord

Continuous opioid agonist treatment produces tolerance and in some cases mu opioid receptor (muOR) down-regulation. Previous studies indicate that down-regulation of muOR is more likely with high-efficacy opioid agonists (e.g., etorphine), whereas lower efficacy agonists (e.g., morphine) do not regulate muOR density. It has been suggested that muOR down-regulation may depend upon increases in Dynamin-2 (DYN-2) proteins. Therefore, the present study examined the effect of various infusion doses of etorphine on muOR density, DYN-2 protein, and DYN-2 mRNA abundance in mouse spinal cord. Mice were implanted sc with an osmotic pump that infused etorphine (50-250 microg/kg/day). Controls were implanted with inert placebo pellets. At the end of 7 days, mice were sacrificed, spinal cord removed and processed for radioligand binding, quantitative Western blotting, or RT-PCR assay. Results indicate that etorphine induced dose-dependent regulation of muOR density, DYN-2 proteins, and mRNA abundance in mouse spinal cord. Higher infusion doses significantly down-regulated muOR density, increased DYN-2 protein abundance, and decreased DYN-2 mRNA. Analysis of these results indicated a significant correlation between muOR down-regulation and DYN-2 abundance in mouse spinal cord. Taken together, muOR regulation may depend on changes in DYN-2 abundance induced by high-efficacy opioid agonists in mouse spinal cord.     

Synthesis of deuterium‐labelled etorphine and dihydroetorphine

The synthesis of deuterium‐labelled [²H₆]‐etorphine and [²H₆]‐dihydroetorphine from codeine is described. The isotopically labelled compounds are used as internal standards in gas chromatography–mass spectrometry (GC–MS) assays. Copyright © 2007 John Wiley & Sons, Ltd.