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Equilibrium and kinetic measurements of muscarinic receptor antagonism on living cells using bead injection spectroscopy

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ANALYTICAL CHEMISTRY 2000年第14期72卷 3109-3115页

作者： Hodder, PS Beeson, C Ruzicka, J Univ Washington Dept Chem Seattle WA 98195 USA

Bead injection spectroscopy (BIS) techniques are introduced for automated measurement of pharmacological antagonism by functional assay. Chinese hamster ovary cells that express the rat type 1 muscarinic receptor are cultured on microbeads and used as a renewable biological target for muscarinic receptor antagonist ligands. A now injection instrument is used to reproducibly sample and capture the cells in a jet ring chamber. The effect of the antagonist pirenzepine on the carbachol-induced intracellular calcium response of the cells is measured with a fluorescence microscope photometry system. The BTS functional assay is used to quantify both equilibrium and kinetic pharmacological values for pirenzepine. In addition, two muscarinic receptor antagonists (pirenzepine and atropine) are assayed to compare their relative efficacy at diminishing the calcium response. Due to the precision of the automated fluid/bead handling protocols, and reproducibility of the measured calcium response, the quantification of useful pharmacological information from living cells by BIS techniques is demonstrated.

关键词：结合竞争性/药物作用 CHO细胞仓鼠亚科指示剂和试剂动力学毒蕈碱拮抗剂/药理学受体毒蕈碱/化学分光光度法动物大鼠

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Lipid-dependent activation of protein kinase C-α by normal alcohols

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第48期274卷 34036-34044页

作者： Shen, YMA Chertihin, OI Biltonen, RL Sando, JJ Univ Virginia Hlth Sci Ctr Dept Pharmacol Charlottesville VA 22903 USA Univ Virginia Hlth Sci Ctr Biophys Program Charlottesville VA 22903 USA Univ Virginia Hlth Sci Ctr Ctr Canc Charlottesville VA 22903 USA

Significant stimulation of protein kinase C-alpha (PKC alpha) by n-alcohols was observed in characterized lipid systems composed of phosphatidylcholine/phosphatidylserine/dioleoylglycerol (PC/PS/DO), The logarithm of the alcohol concentrations to achieve half-maximal PKC stimulation (ED50) and of the maximal PKC stimulation by alcohols were both linear functions of alcohol chain length, consistent with the Meyer-Overton effect. Binding of phorbol esters to PKC was not significantly affected by octanol, Octanol increased, up to 4-fold, the affinity of PKC binding to the lipid bilayers in both the absence and presence of DO. However, octanol increased PKC activity much more significantly than it enhanced binding of the enzyme to the lipid bilayers, suggesting that the stimulation of PKC is not merely a reflection of the increase in PKC bilayer binding affinity. P-31 NMR experiments did not reveal formation of non-lamellar phases with octanol, Differential scanning calorimetry suggested that alcohols, like diacylglycerol, induce formation of compositionally distinct domains and the maximal enzyme activity with alcohol resided roughly in the putative domain-coexistence region. These results suggest that alcohols are mimicking diacylglycerol in activating PKC, not by binding to the high affinity phorbol ester binding site, but by altering lipid structure and by enhancing PCK-bilayer binding.

关键词：醇类/化学醇类/药理学结合竞争性/药物作用甘油二酯类/化学二肉豆蔻磷脂酰胆碱/化学剂量效应关系药物酶激活/药物作用同工酶类/化学同工酶类/代谢脂双层/化学脂双层/代谢脂类/化学膜流动性辛醇类/药理学戊醇类/药理学佛波醇酯类/代谢磷脂酰丝氨酸类/化学蛋白质结合/药物作用蛋白激酶C/化学蛋白激酶C/代谢蛋白激酶Cα 温度

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A subtype of the γ-aminobutyric acid_B receptor regulates cholinergic twitch response in the guinea pig ileum

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JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 2000年第1期293卷 42-47页

作者： Marcoli, M Scarrone, S Maura, G Bonanno, G Raiteri, M Dipartimento Med Sperimentale Sez Farmacol & Tossicol I-16148 Genoa Italy

The pharmacological profile of the gamma-aminobutyric acid (GABA), receptor regulating cholinergic twitch contraction in the guinea pig ileum myenteric plexus-longitudinal muscle preparation was investigated. GABA and (-)-baclofen inhibited the contraction, exhibiting quite close potencies (pD(2) for GABA = 5.70;pD(2) for (-)-baclofen = 5.33). The compound CGP 47656 also reduced the cholinergic twitch concentration (pD(2) = 5.42), but its efficacy was significantly lower than that of (-)-baclofen or GABA. Added at varying concentrations, CGP 47656 modified the concentration-response curve of (-)-baclofen as expected for a partial agonist. Phaclofen, CGP 36742, CGP 35348, and CGP 52432 behaved as competitive antagonists of (-)-baclofen, exhibiting the following pA(2) values: 3.90, 4.88, 5.02, and 7.82, respectively. The compound CGP 56999 behaved as a potent noncompetitive GABA, receptor antagonist. In comparing the pharmacological profile of the ileal receptor with those of the previously characterized pharmacological subtypes of the GABA, receptor present in the central nervous system, it can be seen that the GABA, receptor inhibiting cholinergic twitch contraction in guinea pig ileum myenteric plexus-longitudinal muscle mostly resembles the receptor located on somatostatin human neocortex nerve terminals.

关键词：巴氯芬/类似物和衍生物巴氯芬/拮抗剂和抑制剂巴氯芬/药理学结合竞争性/药物作用电刺激 GABA激动剂/药理学 GABA拮抗剂/药理学回肠/药物作用回肠/生理学配体肌收缩/药物作用肌平滑/药物作用肌平滑/生理学肠肌丛/药物作用副交感神经系统/药物作用副交感神经系统/生理学受体 GABA-B/药物作用受体 GABA-B/代谢脊髓/药物作用脊髓/代谢动物豚鼠男(雄)性

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Novel biotinylated phenylarsonous acids as bifunctional reagents for spatially close thiols:: Studies on reduced antibodies and the agonist binding site of reduced Torpedo nicotinic receptors

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BIOCONJUGATE CHEMISTRY 1999年第4期10卷 629-637页

作者： Moaddel, R Sharma, A Huseni, T Jones, GS Hanson, RN Loring, RH Northeastern Univ Dept Pharmaceut Sci Boston MA 02115 USA

We synthesized three novel organoarsenicals as prototype bifunctional reagents for spatially close thiols, N-(4-arsenosopheny) hexahydro-2-oxo-(3aS,4S,6aR)-1H-thieno[3,4-d]imidazole-4-pentamide (1), 2-[4-[(4-arsenosophenyl)amino]-1,4-dioxobutyl] hydrazide, (3aS,4S,GaR)-hexahydro-2-oxo- 1H-thieno[3,4-d] imidazole-4-pentanoic acid (2), and [4-[[12-[[5-[(3aS,4S,GaR)-hexahydro-2-ox d]imidazol-4-yl]-1-oxopentyl]amino]-1-oxododecyl]amino]phenyl]-arsonous acid (3) containing both biotin and arsenic with intervening varying length spacers extending from 2 to 15 Angstrom beyond biotin bound to streptavidin. Conceptually, the arsenical group can form a stable, covalent ring structure with appropriately spaced thiols and thereby anchor the reagent to a macromolecule, while biotin allows for the detection of the reagent-macromolecule complex via avidin binding. Because the alpha-subunits of all characterized nicotinic receptors contain an easily reducible disulfide bond between adjacent cysteine residues, the reduced a-subunit is an attractive site for labeling. Compounds 1-3 all simultaneously bound streptavidin and dithiols, and all three decreased the number of [I-125]alpha- bungarotoxin-binding sites in reduced Torpedo nicotinic receptors (IC50s 10-300 nM). Moreover, arsenylation of the receptors prevented their reoxidation with dithio-bis(nitrobenzoic acid), was reversible with 2,3-dimercaptopropanesulfonic acid, and protected the receptor from irreversible alkylation by bromoacetylcholine. However, in no case did 1-3 allow simultaneous binding to reduced nicotinic receptors and to [I-125]streptavidin, although 3 alone allowed simultaneous labeling of a spatially close dithiol located in reduced antibodies.

关键词：砷剂/化学结合部位结合竞争性/药物作用生物素/化学环蛇毒素类/代谢半乳糖苷酶类/化学免疫印迹法指示剂和试剂模型分子烟碱激动剂/化学烟碱激动剂/药理学氧化还原受体烟碱/药物作用抗生蛋白链菌素/化学抗生蛋白链菌素/药理学巯基化合物/化学电鳐/代谢动物

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Natural electrophoresis of norepinephrine and ascorbic acid

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BIOPHYSICAL JOURNAL 2000年第1期79卷 370-376页

作者： Dillon, PF Root-Bernstein, RS Sears, PR Olson, LK Michigan State Univ Dept Physiol E Lansing MI 48823 USA

The electric field produced by cell membranes, extending only a few nanometers, is 1000 times stronger than the electric fields required to produce dissociation of molecular complexes. Using the complex formed by norepinephrine (NE) and ascorbic acid (AA), we have demonstrated the quantitative binding of AA to NE, the use of capillary electrophoresis to measure quantitative binding of nonelectrolyte complexes, the determination of a dissociation constant (Kd) from electric field-dissociation constants (Ke), and a model for natural dissociation of the NE-AA complex due to the electric field generated by a cell membrane. NE-AA dissociation constants show little effect of NE concentration or pH changes. NE-related compounds also bind AA: epinephrine > norepinephrine > tyrosine > histamine > phenylalanine. Serotonin does not bind AA. Phosphorylated AA and glucose also bind NE at 0.05 and 0.08 of the AA binding, respectively. Natural electrophoresis of molecular complexes allows compounds to travel through the body in a protected state and still be available for physiological activity upon reaching a membrane.

关键词：抗坏血酸/化学结合竞争性/药物作用细胞膜/生理学剂量效应关系药物电泳毛细管静电学肾上腺素/化学葡萄糖/化学组胺/化学氢离子浓度模型生物学去甲肾上腺素/化学去甲肾上腺素/药理学苯丙氨酸/化学磷酰化血清素/化学酪氨酸/化学

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Identification and characterization of the high-affinity choline transporter

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NATURE NEUROSCIENCE 2000年第2期3卷 120-125页

作者： Okuda, T Haga, T Kanai, Y Endou, H Ishihara, T Katsura, I Univ Tokyo Fac Med Dept Neurochem Bunkyo Ku Tokyo 1130033 Japan Japan Sci & Technol Corp CREST Bunkyo Ku Tokyo 1130033 Japan Kyorin Univ Sch Med Dept Pharmacol & Toxicol Tokyo 1818611 Japan Japan Sci & Technol Corp PRESTO Tokyo 1818611 Japan Grad Univ Adv Studies Sch Life Sci Dept Genet Mishima Shizuoka 4118540 Japan Grad Univ Adv Studies Natl Inst Genet Struct Biol Ctr Mishima Shizuoka 4118540 Japan

In cholinergic neurons, high-affinity choline uptake in presynaptic terminals is the rate-limiting step in acetylcholine synthesis. Using information provided by the Caenorhabditis elegans Genome Project, we cloned a cDNA encoding the high-affinity choline transporter from C. elegans (cho-1). We subsequently used this clone to isolate the corresponding cDNA from rat (CHT1). CHT1 is not homologous to neurotransmitter transporters, but is homologous to members of the Na+-dependent glucose transporter family. Expression of CHT1 mRNA is restricted to cholinergic neurons. The characteristics of CHT1-mediated choline uptake essentially match those of high-affinity choline uptake in rat brain synaptosomes.

关键词：氨基酸序列结合竞争性/药物作用 CHO细胞新小杆线虫漂亮载体蛋白质类/化学载体蛋白质类/遗传学载体蛋白质类/代谢细胞膜/代谢胆碱/代谢胆碱/药代动力学克隆分子仓鼠亚科 DNA 互补/遗传学半胆碱3/药理学膜转运蛋白质类分子序列数据单糖转运蛋白质类/遗传学神经元/代谢神经递质摄取抑制药/药理学卵母细胞/细胞学卵母细胞/代谢器官特异性种系发生 RNA 信使/生物合成序列同源性氨基酸钠/代谢动物大鼠

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The design, synthesis and activity of non-ATP competitive inhibitors of pp60^c-src tyrosine kinase.: Part 2:: Hydroxyindole derivatives

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BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 2000年第5期10卷 483-486页

作者： Milkiewicz, KL Marsilje, TH Woodworth, RP Bifulco, N Hangauer, MJ Hangauer, DG SUNY Buffalo Sch Pharm Dept Med Chem Buffalo NY 14260 USA

As part of a continuing effort to identify novel scaffolds that inhibit the pp60(c-src) protein tyrosine kinase, a series of hydroxyindole amides was rationally designed and synthesized. The most potent derivative was... 详细信息

关键词：腺苷三磷酸/代谢结合竞争性/药物作用药物设计酶抑制剂/化学合成酶抑制剂/药理学吲哚类/化学合成吲哚类/药理学模型分子 src族激酶类/拮抗剂和抑制剂人类

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Overexpression of angiotensin II type I receptor in cardiomyocytes induces cardiac hypertrophy and remodeling

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2000年第2期97卷 931-936页

作者： Paradis, P Dali-Youcef, N Paradis, FW Thibault, G Nemer, M Clin Res Inst Montreal Lab Dev & Differenciat Cardiaques Montreal PQ H2W 1R7 Canada Univ Montreal Dept Pharmacol Montreal PQ H3C 3J7 Canada Clin Res Inst Montreal MRC Multidisciplinary Res Grp Hypertens Montreal PQ H2W 1R7 Canada

Angiotensin II (AII) is a major determinant of arterial pressure and volume homeostasis, mainly because of its vascular action via the All type 1 receptor (AT1R), All has also been implicated in the development of cardiac hypertrophy because angiotensin I-converting enzyme inhibitors and AT1R antagonists prevent or regress ventricular hypertrophy in animal models and in human. However, because these treatments impede the action of All at cardiac as well as vascular levels, and reduce blood pressure, it has been difficult to determine whether AII action on the heart is direct or a consequence of pressure-overload. To determine whether AII can induce cardiac hypertrophy directly via myocardial AT1R in the absence of vascular changes, transgenic mice overexpressing the human AT1R under the control of the mouse alpha-myosin heavy chain promoter were generated. Cardiomyocyte-specific overexpression of AT1R induced, in basal conditions, morphologic changes of myocytes and nonmyocytes that mimic those observed during the development of cardiac hypertrophy in human and in other mammals. These mice displayed significant cardiac hypertrophy and remodeling with increased expression of ventricular atrial natriuretic factor and interstitial collagen deposition and died prematurely of heart failure. Neither the systolic blood pressure nor the heart rate were changed. The data demonstrate a direct myocardial role for AII in the development of cardiac hypertrophy and failure and provide a useful model to elucidate the mechanisms of action of AII in the pathogenesis of cardiac diseases.

关键词： 1-肌氨酸-8-异亮氨酸血管紧张素Ⅱ/代谢血管紧张素受体拮抗剂心钠素/遗传学心钠素/代谢结合竞争性/药物作用印迹法 RNA 心脏扩大/遗传学心脏扩大/病理学基因表达调控心房/化学心房/代谢心房/病理学心室/化学心室/代谢心室/病理学咪唑类/药理学免疫组织化学洛沙坦/药理学小鼠转基因心肌/细胞学心肌/代谢心肌/病理学表型吡啶类/药理学 RNA 信使/遗传学 RNA 信使/代谢放射配体测定受体血管紧张素 1型受体血管紧张素 2型受体血管紧张素/遗传学受体血管紧张素/代谢组织分布转基因/遗传学心室重构/遗传学动物人类小鼠

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A novel angiotensin analog with subnanomolar affinity for angiotensin-converting enzyme

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JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 2000年第1期293卷 260-267页

作者： Krebs, LT Hanesworth, JM Sardinia, MF Speth, RC Wright, JW Harding, JW Washington State Univ Dept VCAPP Pullman WA 99164 USA

This study demonstrates that a novel angiotensin I analog, angiotensinogen 3-11(Lys(11)), possesses a high affinity for angiotensin-converting enzyme (ACE), which is substantially greater than the endogenous substrates. This assessment is based on data derived from a variety of techniques. First, the binding characteristics of I-125-angiotensinogen 3-11(Lys(11)) were examined. Equilibrium saturation isotherms utilizing guinea pig lung membranes revealed that I-125-angiotensinogen 3-11(Lys(11)) bound a single high-affinity site in the presence of EDTA exhibiting a K sigma of 0.15 +/- 0.02 nM with a B-max = 4295 +/- 535 fmol/mg of protein. Competition studies revealed the following rank order of binding affinity: I-125-angiotensinogen 3-11(Lys(11))>> bradykinin >> angiotensin I. Next, SDS-polyacrylamide gel electrophoresis analysis revealed that chemically cross-linked I-125-angiotensinogen 3-11(Lys(11)) specifically bound a protein of M-r 173,000 that had the same molecular weight as ACE. Utilizing in vitro autoradiography, the binding distributions of I-125-angiolensinogen 3-11(Lys(11)) and the ACE inhibitor, I-125-351A, were also compared. These experiments demonstrated that the binding distributions of I-125-angiotensinogen 3-11(Lys(11)) and I-125-351A are identical in the guinea pig lung and testes. Finally, the purification of ACE from guinea pig serum was monitored with I-125-angiotensinogen 3-11(Lys(11)) and I-125-351A binding. These results demonstrated that the binding site for I-125-angiotensinogen 3-11(Lys(11)) and I-125-351A copurified. These experiments indicate that the novel angiotensin I analog, I-125-angiotensinogen 3-11(Lys(11)) binds to ACE and suggest that there are critical binding sites outside the catalytic domains of ACE that determine binding specificity and affinity.

关键词：血管紧张素Ⅰ/类似物和衍生物血管紧张素Ⅰ/药理学血管紧张素转换酶抑制药/药理学血管紧张素类/药理学放射自显影术结合竞争性/药物作用缓激肽/药理学化学物理交联试剂电泳聚丙烯酰氨凝胶指示剂和试剂碘放射性同位素/诊断应用肺/药物作用肺/代谢肽基二肽酶A/血液肽基二肽酶A/代谢蛋白质结合动物豚鼠男(雄)性

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Pancreatic β-cell K_ATP channel activity and membrane-binding studies with nateglinide:: A comparison with sulfonylureas and repaglinide

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JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 2000年第2期293卷 444-452页

作者： Hu, SL Wang, SY Fanelli, B Bell, PA Dunning, BE Geisse, S Schmitz, R Boettcher, BR Novartis Inst Biomed Res Metab & Cardiovasc Dis Dept Summit NJ 07901 USA Novartis Pharma Res Core Technol Area Basel Switzerland

Nateglinide (A-4166) is an amino acid derivative with insulinotrophic action in clinical development for treatment of type 2 diabetes. The aim of this study was to determine whether nateglinide's interaction at the K-ATP channel/sulfonylurea receptor underlies its more rapid onset and shorter duration of action in animal models. Binding studies were carried out with membranes prepared from RIN-m5F cells and HEK-293 cells expressing recombinant human sulfonylurea receptor 1 (SUR1). The relative order for displacement of [H-3]glibenclamide in competitive binding experiments with RIN-m5F cell membranes was glibenclamide > glimepiride > repaglinide > glipizide > nateglinide > L-nateglinide > tolbutamide. The results with HEK-293/recombinant human SUR1 cells were similar with the exception that glipizide was more potent than repaglinide. Neither nateglinide nor repaglinide had any effect on the dissociation kinetics for [H-3]glibenclamide, consistent with both compounds competitively binding to the glibenclamide-binding site on SUR1. Finally, the inability to measure [H-3]nateglinide binding suggests that nateglinide dissociates rapidly from SUR1. Direct interaction of nateglinide with K-ATP channels in rat pancreatic beta-cells was investigated with the patch-clamp method. The relative potency for inhibition of the K-ATP channel was repaglinide. glibenclamide. nateglinide. Kinetics of the inhibitory effect on K-ATP current showed that the onset of inhibition by nateglinide was comparable to glibenclamide but more rapid than that of repaglinide. The time for reversal of channel inhibition by nateglinide was also faster than with glibenclamide and repaglinide. These results suggest that the unique characteristics of nateglinide are largely the result of its interaction at the K-ATP channel.

关键词： ATP结合匣式转运子结合竞争性/药物作用氨甲酸酯类/药代动力学氨甲酸酯类/药理学细胞膜/药物作用细胞膜/代谢细胞分离细胞培养的环己烷类/药理学葡萄糖/代谢格列本脲/药理学糖基转移酶类低血糖症/血液低血糖症/化学诱导降血糖药/药理学胰岛素/代谢胰岛/代谢 KATP通道动力学膜蛋白质类膜片钳术苯丙氨酸/类似物和衍生物苯丙氨酸/药理学哌啶类/药代动力学哌啶类/药理学钾通道阻滞剂钾通道/药物作用钾通道内向整流大鼠 Sprague-Dawley 阻遏蛋白质类/生物合成阻遏蛋白质类/遗传学酿酒酵母蛋白质类磺酰脲化合物/药代动力学磺酰脲化合物/药理学动物人类男(雄)性大鼠

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