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首页> 外文期刊>Neurochemistry International: The International Journal for the Rapid Publication of Critical Reviews, Preliminary and Original Research Communications in Neurochemistry >Comparative pharmacology of adrenergic alpha(2C) receptors coupled to Ca(2+) signaling through different Galpha proteins.
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Comparative pharmacology of adrenergic alpha(2C) receptors coupled to Ca(2+) signaling through different Galpha proteins.

机译：肾上腺素α（2C）受体通过不同的Galpha蛋白偶联到Ca（2+）信号传导的比较药理学。

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摘要

Adrenergic alpha(1), alpha(2) and beta receptors are members of the G-protein-coupled receptor families (GPCRs) mediating physiological responses to adrenaline (epinephrine) and noradrenaline (norepinephrine). Since GPCRs are major targets for potential therapeutic agents, development of robust, reliable and cost effective functional screening methods for these receptors is in the focus of pharmacological research. For this reason, the aim of the present study was to develop an intracellular calcium assay for investigating the pharmacology of the alpha(2C) type of adrenergic receptors (alpha(2C)-AR). Although activation of alpha(2C)-AR is not linked to calcium mobilization, co-expression of these receptors with the chimeric Galpha(qi5) protein, containing the five carboxyl-terminal amino acids from G(i), or promiscuosus Galpha(16) protein can divert receptor signaling to the G(q) pathway generating Ca(2+) release from intracellular stores. In order to assess the functional potency of alpha(2)-AR agonists and antagonists, we established a fluorometric Ca(2+) assay using cell lines stably and constitutively co-expressing alpha(2C)-AR and Galpha(qi5) or Galpha(16) proteins (Galpha(qi5)/alpha(2C) and Galpha(16)/alpha(2C)). As part of the pharmacological characterization, we measured the changes in cytoplasmic Ca(2+) levels due to activation of the chimeric Galpha(qi5) or Galpha(16) coupled recombinant alpha(2C) receptors as a function of increasing concentration of several agonists (noradrenaline, brimonidine, oxymetazoline, clonidine, moxonidine) and antagonists (MK912, yohimbine). The binding affinities of alpha(2)-AR agonist and antagonists and the inhibition of the forskolin-stimulated cAMP accumulation in alpha(2C)-AR expressing cells were also measured. These results confirmed that the Galpha(qi5)/alpha(2C) and Galpha(16)/alpha(2C) recombinant systems can be useful for modelling the native G(i)-coupled system. Our results indicate that a plate-reader based fluorometric Ca(2+) assay may be suitable in high-throughput screening for alpha(2C)-AR ligands as well.
机译：肾上腺素α（1），α（2）和β受体是G蛋白偶联受体家族（GPCR）的成员，介导对肾上腺素（肾上腺素）和去甲肾上腺素（去甲肾上腺素）的生理反应。由于GPCR是潜在治疗剂的主要靶标，因此针对这些受体的鲁棒，可靠且经济高效的功能筛选方法的开发一直是药理学研究的重点。出于这个原因，本研究的目的是开发一种细胞内钙测定法，用于研究α（2C）型肾上腺素能受体（alpha（2C）-AR）的药理作用。尽管alpha（2C）-AR的激活与钙动员没有联系，但这些受体与嵌合Galpha（qi5）蛋白的共表达，其中包含来自G（i）或Promiscuosus Galpha（16）的五个羧基末端氨基酸）蛋白可以将受体信号转移到G（q）途径，从而从细胞内存储中释放Ca（2+）。为了评估α（2）-AR激动剂和拮抗剂的功能效价，我们建立了使用细胞系稳定且组成型共表达α（2C）-AR和Galpha（qi5）或Galpha的荧光Ca（2+）测定法（16）蛋白（Galpha（qi5）/ alpha（2C）和Galpha（16）/ alpha（2C））。作为药理学表征的一部分，我们测量了由于嵌合Galpha（qi5）或Galpha（16）耦合的重组alpha（2C）受体的激活所致的细胞质Ca（2+）水平的变化，这是几种激动剂浓度增加的函数（去甲肾上腺素，溴莫尼定，羟甲唑啉，可乐定，莫索尼定）和拮抗剂（MK912，育亨宾）。还测量了α（2）-AR激动剂和拮抗剂的结合亲和力，以及在表达α（2C）-AR的细胞中受福司可林刺激的cAMP积累的抑制作用。这些结果证实，Galpha（qi5）/ alpha（2C）和Galpha（16）/ alpha（2C）重组系统可用于建模本地G（i）耦合系统。我们的结果表明，基于板读数器的荧光Ca（2+）分析可能也适用于alpha（2C）-AR配体的高通量筛选。

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