Regulation of Kv Channels by Anorexigens

肺动脉平滑肌细胞(PASMCs)上的电压门控性K+(Kv)通道参与了肺动脉高压的发生：抑制这些通道导致膜去极化和细胞内钙离子浓度升高，导致血管收缩和细胞生长/重构。使用厌食药(芬太尼、芬氟拉明及其相关药物)会增加肺动脉高压的发生率。这些药物还可降低PASMCs的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变化的机制可能为预防和治疗原发性肺动脉高压提供线索。厌食剂在多个步骤降低Kv通道的活动。它们能显著抑制PASMCs对4-氨基吡啶(4-AP)敏感的Kv电流。长期应用芬氟拉明可使PASMCs的Kv电流密度降低，Kv1.5基因表达降低。原发性而非继发性肺动脉高压患者的肺组织中Kv1.5基因的表达也降低。这些发现表明，急性和长期接触这些药物分别影响了质膜上的4-AP敏感的Kv通道和Kv通道亚单位基因的转录。利用非洲爪哇卵母细胞表达系统，我们发现芬氟拉明和芬太尼抑制Kv1.5、Kv2.1和Kv4.2的电流，但不抑制Kv3.1b的电流。利用培养的大鼠PASMC和异源表达系统，我们分析了厌氧诱导Kv通道活性和表达变化的分子机制。首先，芬氟拉明降低了PASMCs内源性Kv2.1蛋白。该药物还减少了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形孢菌素模拟并阻断了芬氟拉明引起的PASMCs通道蛋白水平的下降。其次，厌食药使PASMCs中Kv1.5启动子驱动的内源性Kv1.5 mRNA和报告基因表达水平显著降低。通道启动子活性在A7r5平滑肌细胞中也有降低，但在CHO或HEK293细胞中未见。芬氟拉明和芬太尼可快速、可逆地抑制非洲爪哇卵母细胞的Kv1.5、Kv2.1和Kv4.2电流，但不抑制Kv3.1b电流。因此，厌食素诱导的肺动脉高压可能是通过它们产生急性和长期抑制PASMC Kv通道的多种作用而介导的。因此，本研究拟从三个水平探讨厌食素抑制Kv通道的分子机制：Kv2.1蛋白的缓慢减少、Kv1.5基因转录的抑制和质膜上Kv电流的阻断。

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.