SIGNALING BY VASOPRESSIN--ARTERIAL SMOOTH MUSCLE CELLS

加压素发出的信号——动脉平滑肌细胞

基本信息

批准号：
6184234
负责人：
KENNETH L BYRON
金额：
$ 21.68万
依托单位：
LOYOLA UNIVERSITY CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-04-01 至 2003-03-31
项目状态：
已结题

项目摘要

[Arginine8]-vasopressin (AVP) is a peptide hormone which is released from the posterior pituitary gland in response to a decrease in blood pressure or an increase in plasma osmolality. AVP is both a potent vasoconstrictor and a mitogen for vascular smooth muscle. These effects of AVP are important not only for physiological regulation of blood flow and pressure, but may also play a role in diseases, such as hypertension and atherosclerosis, which involve alterations in vascular smooth muscle contractility and proliferation, respectively. The long term objectives of this project are to understand the molecular mechanisms involved in these responses of arterial smooth muscle to AVP. Both contraction and proliferation of arterial smooth muscle cells involve an increase in cytosolic Ca2+ concentration ([Ca2+]i). In A7r5 rat aortic smooth muscle cells, AVP binds to a single class of vasopressin receptors of the V1a subtype. However, AVP has two distinct effects on [Ca2+]i in A7r5 cells: low concentrations of AVP (10-500 pM) stimulate oscillations in [Ca2+]i (Ca2+ spikes); higher concentrations (0.5-100 nM) inhibit Ca2+ spiking, but elicit a biphasic elevation of [Ca2+]i resulting from release of intracellular Ca2+ stores and increased Ca2+ influx across the plasma membrane. These two effects involve different mechanisms. AVP-stimulated Ca2+ spiking results from action potentials that depend on Ca2+ entry through L-type voltage-sensitive Ca2+ channels. The release of intracellular Ca2+ by higher concentrations of AVP is attributed to inositol trisphosphate produced as a result of activation of phospholipase C. The proposed research project will examine the signal transduction mechanisms involved in AVP-stimulated Ca2+ spiking. Preliminary studies suggest that activation of phospholipase D (PLD) is required for stimulation of Ca2+ spiking by AVP. [Ca2+]i measurements with fura-2 or indo-1 and electrophysiological methods will be used to examine the effects of AVP on whole cell membrane currents and membrane potential to test the hypothesis that AVP increases Ca2+ spiking activity by inhibiting delayed rectifier K+ currents, resulting in membrane depolarization and activation of voltage-sensitive Ca2+ channels. The involvement of PLD and the non-receptor tyrosine kinase, PYK2, in K+ channel inhibition will also be investigated using immunoprecipitation techniques. AVP-stimulated PYK2 phosphorylation and Ca2+ spiking are prevented by protein kinase C (PKC) inhibitors. Additional experiments will examine the role of specific PKC isoforms in the AVP signaling pathway and their relationship to PLD activation. Finally, the effects of AVP and other hormones on [Ca2+]i and membrane currents will be examined in freshly isolated smooth muscle cells from rat mesenteric arteries.

[精氨酸8]-加压素 (AVP) 是一种肽激素，在血压降低或血浆渗透压升高时从垂体后叶释放。 AVP 既是一种有效的血管收缩剂，也是血管平滑肌的有丝分裂剂。 AVP 的这些作用不仅对于血流和压力的生理调节很重要，而且还可能在高血压和动脉粥样硬化等疾病中发挥作用，这些疾病分别涉及血管平滑肌收缩性和增殖的改变。该项目的长期目标是了解动脉平滑肌对 AVP 的这些反应所涉及的分子机制。动脉平滑肌细胞的收缩和增殖都涉及胞质 Ca2+ 浓度 ([Ca2+]i) 的增加。在 A7r5 大鼠主动脉平滑肌细胞中，AVP 与 V1a 亚型的单一类别加压素受体结合。然而，AVP 对 A7r5 细胞中的 [Ca2+]i 有两种不同的影响：低浓度 AVP (10-500 pM) 刺激 [Ca2+]i 振荡（Ca2+ 尖峰）；较高浓度 (0.5-100 nM) 会抑制 Ca2+ 尖峰，但会由于细胞内 Ca2+ 储存的释放和 Ca2+ 跨质膜流入的增加而引起 [Ca2+]i 的双相升高。这两种效应涉及不同的机制。 AVP 刺激的 Ca2+ 尖峰源自动作电位，而动作电位依赖于 Ca2+ 通过 L 型电压敏感 Ca2+ 通道进入。高浓度 AVP 释放细胞内 Ca2+ 归因于磷脂酶 C 激活产生的肌醇三磷酸。拟议的研究项目将研究 AVP 刺激 Ca2+ 尖峰所涉及的信号转导机制。初步研究表明，AVP 刺激 Ca2+ 尖峰需要激活磷脂酶 D (PLD)。使用 fura-2 或 indo-1 进行的 [Ca2+]i 测量和电生理学方法将用于检查 AVP 对全细胞膜电流和膜电位的影响，以检验 AVP 通过抑制延迟整流 K+ 电流来增加 Ca2+ 尖峰活性的假设，导致膜去极化和电压敏感 Ca2+ 通道的激活。 PLD 和非受体酪氨酸激酶 PYK2 在 K+ 通道抑制中的作用也将使用免疫沉淀技术进行研究。蛋白激酶 C (PKC) 抑制剂可阻止 AVP 刺激的 PYK2 磷酸化和 Ca2+ 尖峰。其他实验将检查特定 PKC 同工型在 AVP 信号通路中的作用及其与 PLD 激活的关系。最后，将在大鼠肠系膜动脉中新鲜分离的平滑肌细胞中检查 AVP 和其他激素对 [Ca2+]i 和膜电流的影响。