Binding properties of C-truncated delta opioid receptors

目的：研究δ阿片受体Ｃ末端在受体结合配体的亲和力及选择性中的作用．方法：在中国苍鼠卵巢细胞（ＣＨＯ细胞）中分别稳定表达Ｃ末端截短３１个氨基酸残基及野生型δ阿片受体，用受体结合分析法研究表达产物与配体的结合特征．结果：表达得到典型突变受体克隆ＣＨＯＴ及野生型受体克隆ＣＨＯＷ．ＣＨＯＴ结合［３Ｈ］ｄｉｐｒｅｎｏｒｐｈｉｎｅ（Ｄｉｐ）及［３Ｈ］［ＤＡｌａ２，ＤＬｅｕ５］ｅｎｋｅｐｈａｌｉｎ（ＤＡＤＬＥ）的Ｋｄ值与ＣＨＯＷ一致，δ选择性激动剂对二者与［３Ｈ］Ｄｉｐ的结合均有很强的抑制作用，且Ｋｉ相似；而μ及κ选择性激动剂则对二者均无抑制作用．结论：δ阿片受体的Ｃ末端与受体结合配体的亲和力及选择性无关．     

Stoichiometry of δ subunit containing GABAA receptors

Background and Purpose

Although the stoichiometry of the major synaptic αβγ subunit-containing GABA_A receptors has consensus support for 2α:2β:1γ, a clear view of the stoichiometry of extrasynaptic receptors containing δ subunits has remained elusive. Here we examine the subunit stoichiometry of recombinant α4β3δ receptors using a reporter mutation and a functional electrophysiological approach.

Experimental Approach

Using site-directed mutagenesis, we inserted a highly characterized 9′ serine to leucine mutation into the second transmembrane (M2) region of α4, β3 and δ subunits that increases receptor sensitivity to GABA. Whole-cell, GABA-activated currents were recorded from HEK-293 cells co-expressing different combinations of wild-type (WT) and/or mutant α4(L297S), β3(L284S) and δ(L288S) subunits.

Key Results

Recombinant receptors containing one or more mutant subunits showed increased GABA sensitivity relative to WT receptors by approximately fourfold, independent of the subunit class (α, β or δ) carrying the mutation. GABA dose–response curves of cells co-expressing WT subunits with their respective L9′S mutants exhibited multiple components, with the number of discernible components enabling a subunit stoichiometry of 2α, 2β and 1δ to be deduced for α4β3δ receptors. Varying the cDNA transfection ratio by 10-fold had no significant effect on the number of incorporated δ subunits.

Conclusions and Implications

Subunit stoichiometry is an important determinant of GABA_A receptor function and pharmacology, and δ subunit-containing receptors are important mediators of tonic inhibition in several brain regions. Here we demonstrate a preferred subunit stoichiometry for α4β3δ receptors of 2α, 2β and 1δ.     

Central cholinergic regulation of respiration： nicotinic receptors

Nicotinic acetylcholine receptors （nAChRs） are expressed in brainstem and spinal cord regions involved in the control of breathing. These receptors mediate central cholinergic regulation of respiration and effects of the exogenous ligand nicotine on respiratory pattern. Activation of α4＊ nAChRs in the preBotzinger Complex （preBotC）, an essential site for normal respiratory rhythm generation in mammals, modulates excitatory glutamatergic neurotransmission and depolarizes preBotC inspiratory neurons, leading to increases in respiratory frequency, nAChRs are also present in motor nuclei innervating respiratory muscles. Activation of post- and/or extra-synaptic α4＊ nAChRs on hypoglossal （XII） motoneurons depolarizes these neurons, potentiating tonic and respiratory-related rhythmic activity. As perinatal nicotine exposure may contribute to the pathogenesis of sudden infant death syndrome （SIDS）, we discuss the effects of perinatal nicotine exposure on development of the cholinergic and other neurotransmitter systems involved in control of breathing. Advances in understanding of the mechanisms underlying central cholinergic/nicotinic modulation of respiration provide a pharmacological basis for exploiting nAChRs as therapeutic targets for neurological disorders related to neural control of breathing such as sleep apnea and SIDS.     

Anxiolytic effects of 5-HT₁A receptors and anxiogenic effects of 5-HT₂C receptors in the amygdala of mice

The aim of the present study is to test a hypothesis that 5-HT(1A) and 5-HT(2C) receptors in the amygdala play an important role in the regulation of anxiety behaviors. We examined alterations in anxiety-like behaviors after manipulation of the expression of 5-HT(1A) and 5-HT(2C) receptors in the amygdala using recombinant adenovirus approaches. Recombinant adenoviruses containing a 5-HT(1A) promoter-controlled 5-HT(1A) antisense sequence or a 5-HT(2C) promoter-controlled 5-HT(2C) sense sequence were injected into the amygdala. Elevated plus-maze (EPM) and open field tests were conducted to determine anxiety-like behavior and locomotor activity. Reductions in the expression of 5-HT(1A) receptors in the amygdala significantly attenuated the time spent in the open arms of EPM and time spent in the center of an open field. Reduction in the percent of time spent in the open arms of EPM is negatively correlated with the density of 5-HT(1A) receptors in the central amygdala. On the other hand, increased expression of 5-HT(2C) receptors reduced the time spent in the open arms of EPM and time spent in the center of an open field. The reductions in the time spent and distance traveled in the open arms of EPM were correlated to the density of 5-HT(2C) receptors in the basolateral nucleus of amygdala. These data suggest that amygdaloid 5-HT(1A) receptors produce anxiolytic and 5-HT(2C) receptors produce anxiogenic effects. Together, the present results demonstrate the important role of the amygdaloid 5-HT(1A) and 5-HT(2C) receptors in the regulation of anxiety-like behaviors. This article is part of a Special Issue entitled 'Anxiety and Depression'.     

Modulation of phosphatidylinositol turnover on central nicotinic receptors

目的：研究磷脂酰肌醇代谢对中枢烟碱受体功能的调节作用，以分析脑烟碱受体与磷脂酰肌醇代谢之间的关系．方法：在小鼠上观察肌醇磷酸酶抑制剂氯化锂对烟碱诱发惊厥作用的影响．结果：氯化锂２５－１０ｍｍｏｌ·ｋｇ－１预处理后，烟碱诱发小鼠惊厥的量效关系发生变化，在高于半数效量的剂量下，烟碱诱发惊厥的作用显著增强．但氧颤莫林００５－０２０ｍｇ·ｋｇ－１预处理后，烟碱诱发小鼠惊厥的量效关系无显著变化．在小鼠上每日注射一次氯化锂５０ｍｍｏｌ·ｋｇ－１７ｄ后，烟碱诱发惊厥的作用显著减弱，半数效量由０５８增至０９７ｍｇ·ｋｇ－１．结论：磷脂酰肌醇代谢可调节中枢烟碱受体的功能．     

Involvement of µ-opioid receptors in the antitussive effects of pentazocine

Summary The effect of pentazocine on the capsaicininduced cough reflex in rats was investigated. Intraperitoneal injection of pentazocine, in doses from 1 to 10 mg/kg, significantly decreased the number of coughs in a dose-dependent manner. The antitussive effect of pentazocine (10 mg/kg, i.p.) was significantly reduced by prior injection of naloxone (0.3 mg/kg, i.p.), but it was unaffected by Mr-2266 BS (5 mg/kg, i.p.), an antagonist of -opioid receptors. The antinociceptive potency of pentazocine (30 mg/kg, i.p.), as determined by the formalin test, was significantly reduced by pretreatment with Mr-2266 BS (5 mg/kg, i.p.), whereas naloxane (0.3 mg/ kg, i.p.) had no significant effect on the antinociceptive effect of pentazocine. The antitussive effects of pentazocine (3 mg/kg) and morphine (0.1 mg/kg) were significantly enhanced in rats treated chronically with naloxone (5 mg/kg/day, 5 days), whereas the antitussive effect of U-50,488 H (1 mg/kg, i.p.), a selective -opioid agonist, was not enhanced in these rats. By contrast, the antinociceptive effect of morphine (0.01 mg/kg, i.p.) was significantly enhanced in rats that had been pretreated chronically with naloxone. However, the antinociceptive effects induced by pentazocine (3 mg/kg, i.p.) and U-50,488 H (1 mg/kg, i.p.) were unchanged. These results suggest that pentazocine-induced antitussive effects in rats are mediated via stimulation of µ-opioid receptors. Send offprint requests to J. Kamei at the above address     

Lack of involvement ofδ-opioid receptors in mediating the rewarding effects of cocaine

The non-selective opioid antagonist naltrexone and the partial agonist buprenorphine have been reported to reduce cocaine self-administration (SA) and relapse in both humans and rhesus monkeys. Data suggesting an involvement of-opioid receptors in modulating the conditioned rewarding effects of cocaine were also recently presented. In view of such findings, the present SA and place conditioning studies were conducted to examine the influence of the selective-opioid receptor antagonist naltrindole upon the rewarding effects of cocaine. Sprague-Dawley rats were trained to self-administer cocaine (1.0 mg/kg per infusion) on an FR2 schedule of reinforcement. Dose-response and antagonist testing commenced once stable rates of cocaine SA were achieved. For antagonist testing, rats received naltrindole (0.03–10.0 mg/kg, IP) 30 min prior to the start of 2-h SA sessions. SA behavior in response to cocaine delivery (0.25 and 1.0 mg/kg per infusion) was then determined. Naltrindole in doses of 0.03–3.0 mg/kg did not alter the number of cocaine infusions taken by the rats. A higher dose of naltrindole (10.0 mg/kg), which markedly depressed locomotor activity, resulted in a 16% reduction of cocaine (0.25 mg/kg per infusion) SA behavior. When SA sessions were terminated and naltrindole (1.0 mg/kg) was administered repeatedly for 3 days, no alterations in the re-acquisition of cocaine SA were seen. Place conditioning studies also failed to find an effect of naltrindole (0.1–3.0 mg/kg) on cocaine (10 mg/kg) — induced conditioned place preferences. Naltrindole, by itself, did not induce significant place conditioning. These data fail to indicate a role of-opioid receptors in modulating either the positive reinforcing or conditioned rewarding effects of cocaine. Furthermore, they suggest that the therapeutic actions of naloxone, naltrexone and buprenorphine on cocaine SA behavior may not result from the specific blockade of-opioid receptors.     

Irreversible blockade of β-adrenergic receptors with a bromoacetyl derivative of pindolol

Summary A potent irreversible -adrenergic derivative of pindolol possessing a chemically reactive group (Br-AAM-pindolol) was synthesized. This compound devoid of agonist properties, competed for all (³H)-dihydroalprenolol (³H-DHA) binding sites in C₆ glioma cell and rat cerebellum membranes. Pretreatment of C₆ glioma cell membranes with Br-AAM-pindolol and subsequent washing resulted in a time- and dose-dependent blockade of -adrenergic receptors. A 50% blockade was achieved in the presence of 1.6 nM Br-AAM-pindolol.This blockade occurs specifically at the -adrenergic receptor level, as: 1) it induced a decrease of maximal isoproterenol stimulated adenylate cyclase activity with no modification of basal and sodium fluoride stimulated activity and 2) decreases of (³H)-DHA binding and stimulation of adenylate cyclase activity by the agonist were suppressed in the presence of isoproterenol, a -adrenergic agonist. Furthermore, Br-AAM-pindolol treatment did not affect (³H)-diazepam binding in C₆ glioma cell membranes.Pretreatment of C₆ glioma cells with Br-AAM-pindolol also reduced the response of adenylate cyclase to isoproterenol and the number of -adrenergic receptors. The blockade of -adrenergic receptors of C₆ glioma cells by Br-AAM-pindolol was non-competitive, whereas the blockade obtained with AM-pindolol, a derivative of pindolol devoid of alkylating properties, was competitive.The irreversible blockade of -adrenergic receptors by Br-AAM-pindolol in rat erythrocyte membranes was substantiated by the demonstration that no recovery of -adrenergic receptors occurred during long term incubation of the membranes (48 h) following Br-AAM-pindolol treatment and subsequent washing.Double reciprocal plotting of equiactive isoproterenol concentrations in dose-response curves of adenylate cyclase from membranes of control and Br-AAM-pindolol treated C₆ glioma cells permitted calculation of the dissociation constant for isoproterenol from its binding sites (1.5±0.2×10^–7 M, n=11). This is very close to the dissociation constant of the agonist derived from binding experiments (1.7±0.5×10^–7 M, n=13).These results suggest that Br-AAM-pindolol is a potent irreversible -adrenergic antagonist and may be useful for pharmacological and physiological studies of -adrenergic receptors.     

Effects of the α subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors

1. Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (α4β2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co-expression of a Drosophila melanogaster neuronal α subunit (SAD) with the chicken β2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)-epibatidine, (−)-nicotine and acetylcholine) were compared on both recombinant nicotinic AChRs by use of two-electrode, voltage-clamp electrophysiology.
2. Imidacloprid alone of the 4 agonists behaved as a partial agonist on the α4β2 receptor; (+)-epibatidine, (−)-nicotine and acetylcholine were all full, or near full, agonists. Imidacloprid was also a partial agonist of the hybrid Drosophila SAD chicken β2 receptor, as was (−)-nicotine, whereas (+)-epibatidine and acetylcholine were full agonists.
3. The EC₅₀ of imidacloprid was decreased by replacing the chicken α4 subunit with the Drosophila SAD α subunit. This α subunit substitution also resulted in an increase in the EC₅₀ for (+)-epibatidine, (−)-nicotine and acetylcholine. Thus, the Drosophila (SAD) α subunit contributes to the greater apparent affinity of imidacloprid for recombinant insect/vertebrate nicotinic AChRs.
4. Imidacloprid acted as a weak antagonist of ACh-mediated responses mediated by SADβ2 hybrid receptors and as a weak potentiator of ACh responses mediated by α4β2 receptors. This suggests that imidacloprid has complex effects upon these recombinant receptors, determined at least in part by the α subunit.

     

Ligands of the melanocortin receptors, 2002 – 2003 update

α-Melanocyte-stimulating hormone (α-MSH), an endogenous ligand for the melanocortin receptors, has been long recognised as a mediator of numerous physiological processes including, among others, energy homeostasis, immunity, inflammation, sexual function, pigmentation and neurite outgrowth. Compounds mimicking or suppressing actions of α-MSH could therefore be useful in the treatment of many clinically important conditions. Since the cloning of the five melanocortin receptors, medicinal chemistry efforts have centred on the development of melanocortin-4 receptor-specific agonists for the treatment of obesity and erectile dysfunction. Yet, the growing research interest in the other α-MSH functions, reflected in part by the constantly increasing repertoire of ligands specific for the other melanocortin receptors, suggests that some medicinal chemistry efforts might soon be directed towards identification of compounds suitable for treating other diseases. In previous reviews in this journal, the pharmacology of the melanocortin receptors and melanocortin receptor ligands has been discussed in detail. This article will only report the newest ligands, those disclosed in patents published at the end of 2002 and in 2003.     

Effects of phenothiazine-class antipsychotics on the function of α7-nicotinic acetylcholine receptors

The effects of phenothiazine-class antipsychotics (chlorpromazine, fluphenazine, phenothiazine, promazine, thioridazine, and triflupromazine) upon the function of the cloned α? subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes were tested using the two-electrode voltage-clamp technique. Fluphenazine, thioridazine, triflupromazine, chlorpromazine, and promazine reversibly inhibited acetylcholine (100 μM)-induced currents with IC?? values of 3.8; 5.8; 6.1; 10.6 and 18.3 μM, respectively. Unsubstituted phenothiazine did not have a significant effect up to a concentration of 30 μM. Inhibition was further characterized using fluphenazine, the strongest inhibitor. The effect of fluphenazine was not dependent on the membrane potential. Fluphenazine (10 μM) did not affect the activity of endogenous Ca2?-dependent Cl? channels, since the extent of inhibition by fluphenazine was unaltered by intracellular injection of the Ca2? chelator BAPTA and perfusion with Ca2?-free bathing solution containing 2 mM Ba2?. Inhibition by fluphenazine, but not by chlorpromazine was reversed by increasing acetylcholine concentrations. Furthermore, specific binding of [12?I] α-bungarotoxin, a radioligand selective for α?-nicotinic acetylcholine receptor, was inhibited by fluphenazine (10 μM), but not by chlorpromazine in oocyte membranes. In hippocampal slices, epibatidine-evoked [3H] norepinephrine release was also inhibited by fluphenazine (10 μM) and chlorpromazine (10 μM). Our results indicate that phenothiazine-class typical antipsychotics inhibit, with varying potencies, the function of α?-nicotinic acetylcholine receptor.     

Involvement of 5-HT₇ receptors in the pathogenesis of temporal lobe epilepsy

The 5-hydroxytryptamine 7 (5-HT(7)) receptor is the most recently classified member of the serotonin receptor family. The localization of 5-HT(7) receptors and the biological activity of its ligands have suggested that 5-HT(7) receptors might be involved in the pathogenesis of epilepsy. In the present study, we investigated the correlation between temporal lobe epilepsy and 5-HT(7) receptors using pilocarpine-induced rat models of temporal lobe epilepsy and surgical samples of temporal neocortex from intractable epilepsy patients. An analysis of electroencephalogram (EEG) and behavioral changes before and after the treatment of SB269970 hydrochloride (a selective 5-HT(7) receptor antagonist, 10 mg/kg, i.p.) and AS19 (a selective 5-HT(7) receptor agonist, 10 mg/kg, s.c.) demonstrated that in epileptic rats the activation of 5-HT(7) receptors could increase the number of seizures, which could be reduced by a 5-HT(7) receptor antagonist. Moreover, the expression of 5-HT(7) receptors was higher in the epilepsy group compared with the nonepileptic group in both rat and human brain tissues. The present results suggested that 5-HT(7) receptors participate in the pathogenesis of temporal lobe epilepsy, and a 5-HT(7) receptor antagonist may be used as a therapeutic alternative for temporal lobe epilepsy.     

Interaction models of 3-methylfentanyl derivatives with μ opioid receptors

研究３-甲基芬太尼衍生物与μ阿片受体的作用模型．方法：经过系统构象搜寻，用比较分子力场分析法（ＣｏＭＦＡ）研究三维定量构效关系．结果：①６种ＣｏＭＦＡ模型具有良好的预测活性，且每种模型均对应于１３个被研究化合物的低能构象；②μ药效基团的几何参数ｄ１（），ｄ２（），ｄ３（），ｄ４（），ｄ５（）和ｄ６（）分别为模型Ａ：５２，５４，４９，１０．６，１０２和５８；模型Ｂ：５２，６５，３６，１０６，１１６和５８；模型Ｃ：５２，４６，４９，１１６，９２和６５；模型Ｄ：５２，５４，４９，１０５，１０３和５８；模型Ｅ：３６，５４，４９，５７，７５和５７；模型Ｆ：５２，４７，４９，１１２，９５和６４．结论：可能存在几种活性构象与μ受体相互作用，并且不一定是最低能量构象．     

Effects of tetrahydroprotoberberines on dopamine D_2 receptors in ventral tegmental area of rat

阐明四氢原小檗碱同类物（ＴＨＰＢ）对大鼠中脑腹侧被盖区（ＶＴＡ）多巴胺（ＤＡ）受体的作用特性，并比较它们的作用强度．方法：采用大鼠在体胞外单位放电记录．结果：观察了１１个ＴＨＰＢ均可完全地翻转ＤＡ受体激动剂阿扑吗啡（２０μｇ·ｋｇ－１）所产生的放电抑制作用，为Ｄ２受体拮抗剂的作用特性．ＴＨＰＢ对Ｄ２受体的作用与Ｃ２位上的ＯＨ基团有密切的关系．它们的作用强度（ＥＤ５０，μｇ·ｋｇ－１）：ＴＨＰＢ１４３（５６）＞ＳＰＤ（８５）＞Ｉｓｏ（１７０）＞ＴＨＰ（３３）＞ＴＨＢ（４８）＞ＴＨＰＢ１８（６６）＞ＴＨＰＢ１（１７９）＞ＴＨＰＢ１９（４０８）＞ＴＨＰＢ１２６（５１０）＞ＴＨＰＢ１０４（１０１９）＞ＴＨＰＢ１０（４８１５）．结论：１１个ＴＨＰＢ均为ＶＴＡＤ２受体拮抗剂，以Ｃ２位上有ＯＨ基团的ＴＨＰＢ１４３作用最强．     

Allosteric modulators of the α4β2 subtype of neuronal nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors are ligand-gated ion conducting transmembrane channels from the Cys-loop receptor super-family. The α4β2 subtype is the predominant heteromeric subtype of nicotinic receptors found in the brain. Allosteric modulators for α4β2 receptors interact at a site other than the orthosteric site where acetylcholine binds. Many compounds which act as allosteric modulators of the α4β2 receptors have been identified, with both positive and negative effects. Such allosteric modulators either increase or decrease the response induced by agonist on the α4β2 receptors. Here we discuss the concept of allosterism as it pertains to the α4β2 receptors and summarize the important features of allosteric modulators for this nicotinic receptor subtype.     

Involvement of μ-opioid receptors and α-adrenoceptors in the immunomodulatory effects of dihydroetorphine

The present study investigated the effects of acutely administered dihydroetorphine on mitogen-stimulated lymphocytes proliferation and lyrnphokine production in mice.These immune functions were significantly suppressed by dihydroetorphine at 24μg·kg~(-1) and 128μ·g-kg~(-1) in a dose-dependent fashion.This study further examined the involvement of μ-opioid receptors and     

Stable overexpression of human β2-adrenergic receptors in mammalian cells

Summary Human ₂-adrenergic receptors were overexpressed in chinese hamster ovary (CHO) and human epitheloid carcinoma (HeLa) cells. Stable expression in these cells was achieved by transfection of a vector containing the cDNAs for the human ₂-adrenergic receptor as well as for dihydrofolate reductase. By stepwise increases of the concentration of methotrexate — an inhibitor of dihydrofolate reductase — the expression in CHO cells could be increased to levels of almost 200 pmol/mg membrane protein, which is more than 1% of the total membrane protein. In contrast, overexpression of the receptors in HeLa cells by the same technique led to cell death.The receptors produced in overexpressing CHO cells were correctly processed and were fully functional with respect to their ligand binding and signalling properties. The adenylyl cyclase activity of membranes from these cells responded with extremely high sensitivity to the -adrenergic receptor agonist isoproterenol. The receptors could be purified from these membranes to apparent homogeneity by solubilization and chromatography on a single affinity column. Thus, the expression system described here allows the preparation of human ₂-adrenergic receptors in quantities sufficient for pharmacological and biochemical investigations.