Nongenomic bile acid on smooth muscle BK channels

平滑肌 BK 通道上的非基因组胆汁酸

基本信息

批准号：
6891407
负责人：
ALEX M. DOPICO
金额：
$ 18.25万
依托单位：
UNIVERSITY OF TENNESSEE HEALTH SCI CTR
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：
2004-05-01 至 2008-04-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Previous data from our laboratory demonstrated that acute exposure to cholane derivatives (bile acids and synthetic analogs) increases the activity of large conductance, Ca2+-sensitive K+ (BK) channels in arterial smooth muscle. Activation of smooth muscle BK channels is thought to be a major mechanism underlying vasodilation caused by not only bile acid derivatives, but also other physiologically relevant steroids, such as estradiol, progesterone, and androgens. In contrast, others and we found that increased cholesterol levels lead to smooth muscle BK channel inhibition. The long-term goal of this proposal is to identify both the structural determinants in the steroid molecule and the channel protein regions that are responsible for steroid activation of BK channels. Combining methodologies from Electrophysiology (patch-clamp), Molecular Biology, and Pharmacology, we will specifically address: 1) which chemical determinants in the cholane derivative molecule are critical for activating smooth muscle BK channels; 2) which channel subunit (alpha vs. beta) is necessary/sufficient for cholane derivative action; 3) which protein regions in the channel subunits are responsible for conferring cholane sensitivity to the ion channel complex; 4) whether modulation of BK channel activity by cholane derivatives underlies smooth muscle relaxation caused by these steroids. We will answer these questions going from studies in isolated arteries to evaluate drug action on tissue physiology to studies that used engineered ion channel subunits expressed in Xenopus oocytes to address the more mechanistic aspects of the application. We will initially use the rat cerebral artery to freshly isolate both a resistive artery segment to measure changes in tone caused by steroids, and dissociated myocytes to probe steroid modulation of native BK channels. The same overall strategy can be applied to other relevant vessels in the future. We chose the rat cerebral artery/myocyte model because: a) the key role of BK channel activity as regulator of arterial tone was clearly demonstrated; b) we have experience studying the effect of modulators on the active/passive tone of isolated rat cerebral arteries; c) we have isolated, cloned and successfully expressed in Xenopus oocytes the pore forming subunit of the rat cerebral artery BK channel; d) the therapeutic need for new cerebral vasodilators that act on the smooth muscle itself, i.e., that can be effective in the absence of intact endothelial function. Pinpointing the molecular mechanisms involved in the steroid-cerebrovascular BK channel interaction will bring fundamental insight for the rationale design of steroidal vasodilators of potential therapeutic use, devoid of hormonal side effects.

描述（由申请人提供）：我们实验室先前的数据表明，急性接触胆烷衍生物（胆汁酸和合成类似物）会增加动脉平滑肌中大电导、Ca2+ 敏感的 K+ (BK) 通道的活性。平滑肌 BK 通道的激活被认为是血管舒张的主要机制，不仅由胆汁酸衍生物引起，还由其他生理相关类固醇（如雌二醇、孕酮和雄激素）引起。相反，其他人和我们发现胆固醇水平升高会导致平滑肌 BK 通道抑制。该提案的长期目标是确定类固醇分子中的结构决定因素以及负责类固醇激活 BK 通道的通道蛋白区域。结合电生理学（膜片钳）、分子生物学和药理学的方法，我们将具体解决：1）胆烷衍生物分子中的哪些化学决定簇对于激活平滑肌 BK 通道至关重要； 2) 哪个通道亚基（α 与 β）对于胆烷衍生物作用是必要/充分的； 3) 通道亚基中的哪些蛋白质区域负责赋予离子通道复合物胆烷敏感性； 4) 胆烷衍生物对 BK 通道活性的调节是否是这些类固醇引起平滑肌松弛的基础。我们将回答这些问题，从评估药物对组织生理学作用的离体动脉研究，到使用爪蟾卵母细胞表达的工程离子通道亚基来解决应用的更多机械方面的研究。我们首先将使用大鼠大脑动脉新鲜分离电阻性动脉段以测量类固醇引起的音调变化，并分离肌细胞以探测类固醇对天然 BK 通道的调节。未来同样的总体策略可以应用于其他相关船舶。我们选择大鼠脑动脉/肌细胞模型，因为：a）BK通道活性作为动脉张力调节器的关键作用得到了清楚的证明； b) 我们有研究调节剂对离体大鼠脑动脉主动/被动张力影响的经验； c)我们分离、克隆了大鼠大脑动脉BK通道的成孔亚基，并在爪蟾卵母细胞中成功表达； d) 对作用于平滑肌本身的新型脑血管扩张剂的治疗需求，即在缺乏完整内皮功能的情况下可以有效。查明类固醇-脑血管 BK 通道相互作用的分子机制将为具有潜在治疗用途且无激素副作用的类固醇血管扩张剂的基本原理设计提供基础见解。