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Regulation of Kv Channels by Anorexigens

Anorexigens 对 Kv 通道的调节

基本信息

批准号：
7198058
负责人：
Bruce Robert Pitt
金额：
$ 27.1万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-04-01 至 2009-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7198058
关键词：
4-Aminopyridine 5&apos Flanking Region Acute Amino Acids Appetite Depressants Binding Biological Assay Blood Vessels Cell Line Cell membrane Cells Chinese Hamster Ovary Cell Databases Deletion Mutation Down-Regulation Elements Exhibits Exposure to Fenfluramine Fluoxetine Gene Expression Genes Genetic Transcription Goals Homeostasis Immunoblot Analysis Incidence Indium Isomerism Lung Mammalian Cell Mediating Membrane Messenger RNA Metabolic Molecular Molecular Target National Research Service Awards Patients Pharmaceutical Preparations Phentermine Physiologic pulse Prevention Process Protein Inhibition Protein Kinase Proteins Pulmonary Hypertension Pulse taking Rattus Regulation Reporter Smooth Muscle Myocytes Stimulus Structure of parenchyma of lung System Testing Voltage-Gated Potassium Channel Xenopus oocyte cell growth density kinase inhibitor mutant patch clamp polypeptide primary pulmonary hypertension promoter research study transcription factor vasoconstriction voltage

项目摘要

Voltage-gated K+ (Kv) channels in pulmonary arterial smooth muscle cells (PASMCs) have been implicated in the initiation of pulmonary hypertension: inhibition of these channels results in membrane depolarization and an increase in intracellular Ca2+ concentration, leading to vasoconstriction and cell growth / remodeling. The use of anorexic agents (phentermine, fenfluramine and their related drugs) is associated with an increased incidence of pulmonary hypertension. These drugs also decrease the activity and expression of Kv channels in PASMCs. Thus, understanding the mechanisms by which these identified stimuli produce alterations in the function and level of these channels may provide clues for prevention and treatment of primary pulmonary hypertension. The anorexic agents reduce Kv channel activity at multiple steps. They acutely inhibit 4-aminopyridine (4-AP)-sensitive Kv current in PASMCs. Furthermore, long-term treatment of PASMCs with fenfluramine leads to decreases in Kv current density and the expression of Kv1.5 mRNA. Lung tissues from patients with primary, but not secondary, pulmonary hypertension also exhibit reduced expression of Kv1.5 mRNA. These findings suggest acute and long-term exposures to these drugs influence 4-AP-sensitive Kv channels at plasma membrane and transcription of Kv channel subunit genes, respectively. Using Xenopus oocyte expression system, we found that fenfluramine and phentermine inhibit Kv1.5, Kv2.1 and Kv4.2, but not Kv3.1b, current. Using cultured rat PASMCs and heterologous expression systems, we have analyzed molecular mechanisms underlying the anorexigen-induced changes in the activity and expression of Kv channels. First, exposure to fenfluramine decreased endogenous Kv2.1 proteins in PASMCs. The drug also reduced heterologously expressed Kv2.1, but not Kv1.5 or Kv4.3, proteins in a mammalian cell line. In addition, the non-selective kinase inhibitor staurosporin mimicked and occluded the fenfluramine-induced decrease in the channel protein level in PASMCs. Second, the anorexic drugs caused significant decreases in the level of endogenous Kv1.5 mRNA and reporter gene expression driven by the Kv1.5 promoter in PASMCs. Reductions in the channel promoter activity were also seen in A7r5 smooth muscle cells, but not in CHO or HEK293 cells. Finally, fenfluramine and phentermine rapidly and reversibly inhibited Kv1.5, Kv2.1 and Kv4.2, but not Kv3.1b, currents in Xenopus oocytes. Thus, the anorexigen-induced pulmonary hypertension may be mediated by their multitude of actions to produce acute and long-term inhibition of PASMC Kv channels. Hence, this proposal is to identify molecular mechanisms for anorexigen-induced inhibition of Kv channels at the three levels: a slow decrease in Kv2.1 proteins, inhibition of Kv1.5 gene transcription and blockade of Kv currents at plasma membrane.

肺动脉平滑肌细胞（PASMCs）中的电压门控K+ (Kv)通道与肺动脉高压的开始有关：这些通道的抑制导致膜去极化和细胞内Ca2+浓度的增加，导致血管收缩和细胞生长/重塑。使用厌食剂（芬特明、芬氟拉明及其相关药物）与肺动脉高压发生率增加有关。这些药物还能降低pasmc中Kv通道的活性和表达。因此，了解这些已识别的刺激产生这些通道功能和水平改变的机制可能为预防和治疗原发性肺动脉高压提供线索。厌氧剂通过多个步骤降低Kv通道活性。它们严重抑制pasmc中4-氨基吡啶（4-AP）敏感的Kv电流。此外，长期使用芬氟拉明治疗PASMCs可导致Kv电流密度和Kv1.5 mRNA表达降低。原发性而非继发性肺动脉高压患者的肺组织也表现出Kv1.5 mRNA的表达降低。这些发现表明，急性和长期暴露于这些药物分别影响质膜上4- ap敏感的Kv通道和Kv通道亚基基因的转录。利用爪蟾卵母细胞表达系统，我们发现芬氟拉明和芬特明对Kv1.5、Kv2.1和Kv4.2均有抑制作用，但对Kv3.1b无抑制作用。利用培养的大鼠PASMCs和异源表达系统，我们分析了缺氧诱导Kv通道活性和表达变化的分子机制。首先，暴露于芬氟拉明会降低pasmc的内源性Kv2.1蛋白。该药物还降低了哺乳动物细胞系中异种表达的Kv2.1蛋白，但不降低Kv1.5或Kv4.3蛋白。此外，非选择性激酶抑制剂staurosporin模拟并阻断了芬氟拉明诱导的PASMCs通道蛋白水平的降低。其次，厌食药导致PASMCs内源性Kv1.5 mRNA水平和Kv1.5启动子驱动的报告基因表达显著降低。通道启动子活性的降低在A7r5平滑肌细胞中也可见，但在CHO或HEK293细胞中未见。最后，芬氟拉明和芬特明能快速、可逆地抑制爪蟾卵母细胞中Kv1.5、Kv2.1和Kv4.2电流，但不能抑制Kv3.1b电流。因此，厌氧性肺动脉高压可能是由它们的多种作用介导的，从而产生对pasmckv通道的急性和长期抑制。因此，本研究旨在从三个层面确定厌氧诱导的Kv通道抑制的分子机制：Kv2.1蛋白的缓慢下降、Kv1.5基因转录的抑制和质膜上Kv电流的阻断。