GEA 857 Blocks Potassium Channels in the Membrane and,Thereby, Prolongs Muscarinic Cholinergic Responses in N1E-115 Neuroblastoma Cells

Abstract: GEA 857 [2-(4-chlorophenyl)-1,1-dimethylethyl 2-amino-3-methylbutanoate], a structural analogue of the serotonin (5-HT) uptake inhibitor alaproclate but without effects on the 5-HT uptake, was shown to potentiate muscarinic cholinergic responses in N1E-115 neuroblastoma cells. In intracellular recording experiments, GEA 857 (1 μM) increased the cell input resistance and prolonged the action potential. It also prolonged the cellular response to carbachol acting on muscarinic receptors in a manner mimicked by potassium channel blockers such as 4-aminopyridine and TEA. GEA 857 did not affect the carbachol stimulated uptake of ⁴⁵Ca, but depressed the carbachol activated outflow of ⁸⁶Rb from neuroblastoma cells. The conclusion drawn from these results is that GEA 857 reduces potassium conductances in the membrane in N1E-115 neuroblastoma cells and, thereby, prolongs muscarinic agonist-induced responses.     

动脉粥样硬化兔血管平滑肌细胞大电导钙激活钾通道NO直接激活研究

目的:研究NO是否可以直接激活VSMC BKCa以及AS时血管平滑肌细胞BKCa对NO的反应性是否发生改变,为治疗AS提供新思路.方法:3月龄新西兰兔20只,雌雄各半,随机分为实验组(AS组)和对照组(正常组),每组10只.实验组兔喂高脂饲料8周以建立AS模型,对照组兔喂普通饲料8周.以ISMN为外源性NO供体,应用单通道膜片钳技术检测NO对兔VSMC BKCa的直接激活作用及两组兔对NO的反应性差别.结果:在inside-out patches(膜电位 40 mV)下,ISMN显著增加BKCa通道开放事件数,缩短Tc,增加Po,且具有浓度依赖性.10-6 mol·L-1的ISMN(膜电位 40 mV)可分别使AS组和对照组BKCa的Po增加4.917±1.475倍和9.616±3.227倍(P＜0.01).结论:NO对VSMCBKCa有直接激活作用,但在AS时VSMC BKCa对NO的激活敏感性是降低的.     

吗啡依赖与毒蕈碱型乙酰胆碱受体的适应

毒蕈碱型乙酰胆碱(muscarinic acetylcholine)能神经在阿片依赖中的作用可以追溯到30年代,当时认为吗啡可以抑制迷走神经.到60年代认为吗啡镇痛耐受与毒蕈碱乙酰胆碱能神经的慢性适应有关;70年代有作者认为吗啡戒断时毒蕈碱能神经兴奋[1].本文系统地介绍M受体介导吗啡镇痛耐受、吗啡戒断反应和吗啡精神依赖的作用机制.     

Activators of Sodium,Calcium and Potassium Channels Modulate the Cardiac Effects of Quinidine in vitro

Abstract: Quinidine (25.5 μmol/l) reduced the beating frequency of isolated right guinea-pig atria, caused a negative inotropic effect in papillary muscles and slightly raised the contractile force of left atria. The functional refractory period of both tissues was prolonged. A 20% increase of the extracellular sodium concentration did not reverse the effects of quinidine. The Na-channel activator BDF 9148 (1 μmol/l) and the Ca-channel agonist Bay-K-8644 (0.5 μmol/l) further increased the contractile force and caused an additional prolongation of the functional refractory period in quinidine-pretreated atria. Only Bay-K-8644 was able to reverse the negative inotropic effect of quinidine in papillary muscles. The influence of Bay-K-8644 on the contractile force in quinidine-pretreated muscles was not attenuated by lemacalim (3 μmol/l), an activator of ATP-dependent potassium channels, but the duration of the functional refractory period was significantly reduced. These results suggest that a combination of a calcium channel activator and a potassium channel opener might be able to improve the treatment of quinidine intoxications.     

Geraniol Blocks Calcium and Potassium Channels in the Mammalian Myocardium: Useful Effects to Treat Arrhythmias

Geraniol is a monoterpene present in several essential oils, and it is known to have a plethora of pharmacological activities. In this study, we explored the contractile and electrophysiological properties of geraniol and its antiarrhythmic effects in the heart. The geraniol effects on atrial contractility, L‐type Ca²⁺ current, K⁺ currents, action potential (AP) parameters, ECG profile and on the arrhythmia induced by ouabain were evaluated. In the atrium, geraniol reduced the contractile force (~98%, EC = 1,510 ± 160 μM) and diminished the positive inotropism of CaCl₂ and BAY K8644. In cardiomyocytes, the I_Ca,L was reduced by 50.7% (n = 5) after perfusion with 300 μM geraniol. Moreover, geraniol prolonged the AP duration (APD) measured at 50% (n = 5) after repolarization, without changing the resting potential. The increased APD could be attributed to the blockade of the transient outward K⁺ current (I_to) (59.7%, n = 4), the non‐inactivation K⁺ current (I_ss) (39.2%, n = 4) and the inward rectifier K⁺ current (I_K1) (33.7%, n = 4). In isolated hearts, geraniol increased PRi and QTi without affecting the QRS complex (n = 6), and it reduced both the left ventricular pressure (83%) and heart rate (16.5%). Geraniol delayed the time to onset of ouabain‐induced arrhythmias by 128%, preventing 30% of the increase in resting tension (n = 6). Geraniol exerts its negative inotropic and chronotropic responses in the heart by decreasing both L‐type Ca²⁺ and voltage‐gated K⁺ currents, ultimately acting against ouabain‐induced arrhythmias.     

Association of solubilized angiotensin II receptors with phospholipase C-alpha in murine neuroblastoma NIE-115 cells.

The peptide angiotensin II (AngII) has been reported to stimulate phosphoinositide-specific phospholipase C (PLC) activity in the murine neuroblastoma cell line N1E-115. In the present study, polyclonal antibodies raised against a PLC isoenzyme, PLC-alpha, reacted with a 60-kDa protein present in both membrane and cytosolic fractions of differentiated N1E-115 cells. In order to examine the possible association of PLC-alpha with cell surface AngII receptors (AngII-Rs), membranes from differentiated N1E-115 cells were solubilized, using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). CHAPS (1%) solubilized AngII-Rs, from N1E-115 cells, that maintained their high affinity for agonists. Gel filtration analysis of the solubilized membranes revealed that the majority of the specific binding of 125I-AngII eluted as a large protein complex with a molecular mass of 380 kDa and that agonist binding was partially reduced by guanosine-5'-O-(3-thio)triphosphate (GTP gamma S), within this complex. CHAPS also effectively solubilized immunoreactive PLC-alpha, from N1E-115 cell membranes, that was similarly present within the 380-kDa AngII-binding complex. Anti-PLC-alpha antisera immunoprecipitated approximately 16% of the total phosphatidylinositol-4,5-bisphosphate-specific PLC activity in the 1% CHAPS extract and 40% of cytosolic PLC activity. Moreover, a 60-kDa 35S-Trans S-labeled protein, comigrating with immunoreactive PLC-alpha, was immunoprecipitated from the 1% CHAPS extract by the antisera. In addition, anti-PLC-alpha antisera immunoprecipitated approximately 20% of solubilized AngII-Rs prebound with 125I-AngII but failed to precipitate receptors prebound with the antagonist 125I-Sarc1,Ile8-AngII. The anti-PLC-alpha antisera also immunoprecipitated AngII-Rs when intact membranes were labeled with 125I-AngII before solubilization in 1% CHAPS, suggesting that the AngII-R interaction with PLC-alpha was not the result of detergent-promoted protein-protein interaction. On the other hand, monoclonal antibodies against another PLC isozyme, PLC-gamma, did not precipitate AngII-Rs in solubilized N1E-115 membranes. Finally, the formation of the immunoprecipitated AngII-R-PLC-alpha complex was disrupted by the nonhydrolyzable guanine nucleotide analog GTP gamma S, suggesting that the interaction between AngII-Rs and PLC-alpha is likely to involve a heterotrimeric guanine nucleotide-binding protein in neuron-like cells.     

High Throughput Assays of Cloned Adrenergic,Muscarinic, Neurokinin,and Neurotrophin Receptors in Living Mammalian Cells

Abstract: Many receptors stimulate proliferation of NIH 3T3 cells in a ligand dependent fashion. Based on this observation, we developed a high throughput assay of cloned receptor pharmacology. In this assay, receptors are transiently co-expressed with the marker enzyme β-galactosidase. Receptors that induce cellular proliferation select and amplify the cells that also express the marker, thus the ability of ligands to alter receptor activity are reported as changes in enzyme activity. In the present study, we used this assay to evaluate the ability of agonist ligands to stimulate four cloned receptors. The agonists phenylephrine, carbachol, substance P and nerve growth factor selectively stimulated cells transfected with the α-1b adrenergic, m4 muscarinic, NK1 neurokinin and trkA neurotrophin receptors, respectively. These data demonstrate that a high throughput colorimetric assay performed in 96 well plates can be used to evaluate the pharmacology of ligands for cloned receptors belonging to a wide range of functional and pharmacological classes.     

Muscarinic receptors activate calcium channels and calcium dependent potassium channels in NlE-115 neuroblastoma cells

Responses of NlE-115 neuroblastoma cells to application of carbachol were studied using intracellular recording techniques. Activation of muscarinic cholinergic receptors by carbachol resulted in a depolarization of the cells. The response was blocked by pirenzepine (1 microM) and by CoCl2 (5 mM), verapamil (10 microM) and gallopamil (10 microM), and prolonged by quinine (5 mM). It is suggested that muscarinic receptors increase the membrane calcium permeability, and that the influx of calcium activates calcium dependent potassium channels.     

The Domain and Conformational Organization in Potassium Voltage-Gated Ion Channels

Potassium ion channels play critical roles in cell function, providing the maintenance of the membrane, repolarization of action potentials, and the regulation of firing frequency. Mutations in genes that interfere with K_v ion channel function cause severe inherited diseases, such as episodic ataxia type 1, deafness, epilepsy, or cardiac arrhythmia. Because of their critical role in the central nervous system, all ion channels are targets for multiple pharmacologically active compounds. Better understanding of the structure and function of K_v channels may eventually contribute to a more effective design of drugs. In this review, we show the recent data about domain organization of eukaryotic potassium voltage-gated ion channels. We are giving special attention to the interaction between the domains and the corresponding conformational changes upon activation of the channel. Meeting presentation: Russian-American Nanomedicine Workshop, Moscow, 10–12 December, 2007.     

豚鼠气道平滑肌的急性分离及K_(ATP)通道单通道电流的记录

目的:建立急性分离豚鼠气道平滑肌的方法,并初步分析ATP敏感钾通道(KATP通道)单通道电流的性质。方法:急性分离出豚鼠3~4级支气管,并用链霉蛋白酶E分散气道平滑肌细胞,应用膜片钳技术的内面向外式记录方法,研究气道平滑肌KATP通道单通道电学性质。结果:成功记录到电导为112.4±5.14 pS的、可被优降糖所阻断的KATP通道单通道电流。钳制电压在0~-60 mV之间,通道电流无整流现象,且未见时间依赖性失活。结论:建立了急性分离豚鼠气道平滑肌的方法,并成功记录KATP通道单通道电流,为进一步研究气道平滑肌KATP通道在呼吸道疾病中的作用提供了基础。     

Potassium Channels: Structures,Diseases, and Modulators

Potassium channels participate in many critical biological functions and play important roles in a variety of diseases. In recent years, many significant discoveries have been made which motivate us to review these achievements. The focus of our review is mainly on three aspects. Firstly, we try to summarize the latest developments in structure determinants and regulation mechanism of all types of potassium channels. Secondly, we review some diseases induced by or related to these channels. Thirdly, both qualitative and quantitative approaches are utilized to analyze structural features of modulators of potassium channels. Our analyses further prove that modulators possess some certain natural‐product scaffolds. And pharmacokinetic parameters are important properties for organic molecules. Besides, with in silico methods, some features that can be used to differentiate modulators are derived. There is no doubt that all these studies on potassium channels as possible pharmaceutical targets will facilitate future translational research. All the strategies developed in this review could be extended to studies on other ion channels and proteins as well.     

Interaction between Opioid and Muscarinic Receptors in the Guinea-Pig Ileum Preparation: A Mathematical Model

Abstract: Fentanyl and pethidine are opioid agonists and muscarinic antagonists in the guinea-pig ileum preparation. In this preparation an opioid agonist reduces the release of acetylcholine. Therefore an opiate may influence the potency of an anticholinergic drug. A mathematical model was developed to characterize this putative interaction between opioid and muscarinic receptors. The model is based on the assumption that the drugs interact with the receptors in a competitive manner according to the law-of-mass action. Concentration-response experiments were performed in the guinea-pig ileum preparation to test the model. The mathematical model describes the concentration-response curves very well and estimates the IC₅₀ values for the two components with good precision. The study shows that an opioid agonist can potentiate the effect of an anticholinergic drug substantially. This is interesting with regard to the central anticholinergic syndrome. The conclusion is that the model describes the interaction adequately.     

M5受体参与中脑奖赏的研究进展

在五种毒蕈碱受体亚型中,M5毒蕈碱受体是最后被发现的,M5受体在大脑和内脏组织中含量最少[1]。至今未找到M5受体的高亲和性的选择性配体和毒素,也没有发现哪个大脑区域强烈表达M5受体mRNA[2]。因此,尽管对M1～M4受体的药理学作用有详尽的了解,但对M5受体的药理学特性却知之甚少。全脑免疫组化研究可以粗略地了解大脑M5受体的含量,但不能反映其在解剖学上的详尽分布[1]。另外,有研究者用树眼睛蛇毒素和AQ-RA741封闭M1～M4受体,了解各个脑区和一些内脏组织的剩余M5受体的分布状况[2]。即便是这样,残存的约15%的M3受体仍可能干扰对M5受…     

A Stable and Highly Potent Hexamethonium Derivative Which Modulates Muscarinic Receptors Allosterically in Guinea-pig Hearts

Abstract— W84 (N, N,N′,N′-tetramethyl-N,N′-bis-(3-phthalimidopropyl)-N,N′-hexane-1,6-diyl-bis-ammomum dibromide) is a potent stabilizer of antagonist binding to muscarinic receptors; however, W84 hydrolyses in aqueous buffered medium. The synthesis of the stable derivative CHIN3/6 is presented containing a 2-phenyl-quinazolinone instead of the labile phthalimide substituent. This compound retarded [³H]N-methylscopolamine-dissociation in guinea-pig cardiac membranes with slightly higher potency than W84, the EC50 values amounting to 7·5 × 10^?7and 13× 10^?7 m , respectively. Molecular modelling revealed differences in the electron density of the substituents and in their molecular shape. It is suggested that the derivatives use partially different sites of attachment when occupying the allosteric binding site of the receptor protein.     

Guide to Receptors and Channels (GRAC), 5th edition

The Fifth Edition of the 'Guide to Receptors and Channels' is a compilation of the major pharmacological targets divided into seven sections: G protein-coupled receptors, ligand-gated ion channels, ion channels, catalytic receptors, nuclear receptors, transporters and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside suggestions for further reading. Available alongside this publication is a portal at http://www.GuideToPharmacology.org which is produced in close association with NC-IUPHAR and allows free online access to the information presented in the Fifth Edition.     

Muscarinic Receptors Modulate the Intrinsic Excitability of Infralimbic Neurons and Consolidation of Fear Extinction

There is considerable interest in identifying pharmacological compounds that could be used to facilitate fear extinction. Recently, we showed that the modulation of M-type K⁺ channels regulates the intrinsic excitability of infralimbic (IL) neurons and fear expression. As muscarinic acetylcholine receptors inhibit M-type K⁺ channels, cholinergic inputs to IL may have an important role in controlling IL excitability and, thereby, fear expression and extinction. To test this model, we combined whole-cell patch-clamp electrophysiology and auditory fear conditioning. In prefrontal brain slices, muscarine enhanced the intrinsic excitability of IL neurons by reducing the M-current and the slow afterhyperpolarization, resulting in an increased number of spikes with shorter inter-spike intervals. Next, we examined the role of endogenous activation of muscarinic receptors in fear extinction. Systemic injected scopolamine (Scop) (muscarinic receptor antagonist) before or immediately after extinction training impaired recall of extinction 24-h later, suggesting that muscarinic receptors are critically involved in consolidation of extinction memory. Similarly, infusion of Scop into IL before extinction training also impaired recall of extinction 24-h later. Finally, we demonstrated that systemic injections of the muscarinic agonist, cevimeline (Cev), given before or immediately after extinction training facilitated recall of extinction the following day. Taken together, these findings suggest that cholinergic inputs to IL have a critical role in modulating consolidation of fear extinction and that muscarinic agonists such as Cev might be useful for facilitating extinction memory in patients suffering from anxiety disorders.