Regulation of Kv Channels by Anorexigens

Anorexigens 对 Kv 通道的调节

• 批准号: 6867416
• 负责人: KOICHI TAKIMOTO TAKIMOTO
• 金额: $ 28.86万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6867416
• 关键词: Xenopus; anorexic agent; calcium flux; cardiovascular pharmacology; enzyme inhibitors; fenfluramine; fluoxetine; genetic transcription; laboratory rat; membrane potentials; phentermine; potassium channel; protein kinase; protein localization; protein metabolism; pulmonary hypertension; vascular smooth muscle

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 (PASMC) 中的电压门控 K+ (Kv) 通道与肺动脉高压的发生有关：抑制这些通道会导致膜去极化和细胞内 Ca2+ 浓度增加，从而导致血管收缩和细胞生长/重塑。使用抗食欲药物（芬特明、芬氟拉明及其相关药物）与肺动脉高压发病率增加有关。这些药物还会降低 PASMC 中 Kv 通道的活性和表达。因此，了解这些已识别的刺激导致这些通道的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。厌食剂可通过多个步骤降低 Kv 通道活性。它们可急性抑制 PASMC 中 4-氨基吡啶 (4-AP) 敏感的 Kv 电流。此外，长期用芬氟拉明治疗 PASMC 会导致 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 表达降低。这些发现表明，急性和长期接触这些药物分别会影响质膜上的 4-AP 敏感 Kv 通道和 Kv 通道亚基基因的转录。利用非洲爪蟾卵母细胞表达系统，我们发现芬氟拉明和芬特明抑制Kv1.5、Kv2.1和Kv4.2，但不抑制Kv3.1b电流。使用培养的大鼠 PASMC 和异源表达系统，我们分析了厌食素诱导的 Kv 通道活性和表达变化的分子机制。首先，接触芬氟拉明会减少 PASMC 中的内源性 Kv2.1 蛋白。该药物还减少哺乳动物细胞系中异源表达的 Kv2.1 蛋白，但不减少 Kv1.5 或 Kv4.3 蛋白。此外，非选择性激酶抑制剂星孢菌素模拟并阻断芬氟拉明诱导的 PASMC 通道蛋白水平下降。其次，厌食药物导致 PASMC 中由 Kv1.5 启动子驱动的内源 Kv1.5 mRNA 和报告基因表达水平显着降低。在 A7r5 平滑肌细胞中也观察到通道启动子活性的降低，但在 CHO 或 HEK293 细胞中未观察到。最后，芬氟拉明和芬特明快速且可逆地抑制爪蟾卵母细胞中的 Kv1.5、Kv2.1 和 Kv4.2，但不抑制 Kv3.1b 电流。因此，厌食剂诱导的肺动脉高压可能是通过其多种作用介导的，从而对 PASMC Kv 通道产生急性和长期的抑制。因此，本提议旨在确定厌食素诱导的 Kv 通道在三个水平上的抑制的分子机制：Kv2.1 蛋白的缓慢减少、Kv1.5 基因转录的抑制和质膜 Kv 电流的阻断。