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）：我们实验室的先前数据表明，急性暴露于乔兰衍生物（胆汁酸和合成类似物）会增加大型电导率，CA2+敏感的K+（BK）通道在动脉平滑肌中。平滑肌BK通道的激活被认为是由胆汁酸衍生物，而且是其他与生理相关的类固醇（例如雌二醇，孕酮和雄激素）引起的主要机制。相比之下，其他人，我们发现胆固醇水平升高会导致平滑肌BK通道抑制。该建议的长期目标是确定类固醇分子中的结构决定因素和导致BK通道类固醇激活的通道蛋白区域。从电生理学（斑块钳），分子生物学和药理学中结合方法，我们将专门解决：1）cholane衍生物分子中哪种化学决定因素对于激活平滑肌BK通道至关重要； 2）哪个通道亚基（Alpha vs. Beta）对于乔兰衍生物作用是必需的/足够的； 3）河道亚基中哪些蛋白质区域负责赋予离子通道复合物的乔兰敏感性； 4）乔兰衍生物对BK通道活性的调节是否是由这些类固醇引起的平滑肌松弛的基础。我们将回答这些问题，从孤立的动脉研究，以评估组织生理学的药物作用到使用在Xenopus卵母细胞中表达的工程离子通道亚基来解决应用程序更机械方面的研究。我们最初将使用大鼠脑动脉来新鲜分离出电阻动脉段，以测量由类固醇引起的张力变化，并解离肌细胞以探测天然BK通道的类固醇调节。将来可以将相同的整体策略应用于其他相关船只。我们选择大鼠脑动脉/肌细胞模型是因为：a）明确证明了BK通道活性作为动脉张力的调节剂的关键作用； b）我们有研究调节剂对孤立大鼠脑动脉的主动/被动音调的影响的经验； c）我们已经在爪蟾卵母细胞中分离，克隆并成功表达了大鼠脑动脉BK通道的孔子亚基； d）对平滑肌本身作用的新脑血管扩张剂的治疗需求，即在没有完整的内皮功能的情况下可以有效。精确指出类固醇 - 脑血管BK通道相互作用所涉及的分子机制将为潜在治疗使用的类固醇血管扩张剂的基本原理设计带来基本见解，而没有激素副作用。