SIGNALING BY VASOPRESSIN--ARTERIAL SMOOTH MUSCLE CELLS

加压素发出的信号——动脉平滑肌细胞

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/6184234
• 关键词: arginine vasopressin; calcium channel; calcium flux; calcium indicator; cell proliferation; electrophysiology; enzyme activity; enzyme inhibitors; hormone receptor; hormone regulation /control mechanism; immunoprecipitation; laboratory rat; membrane potentials; phospholipase D; protein kinase C; protein tyrosine kinase; receptor binding; tissue /cell culture; vascular smooth muscle; vasomotion; voltage gated channel

[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] -Vasopressin（AVP）是一种肽激素，它因血压降低或血浆渗透压的增加而从垂体后腺释放。 AVP既是有效的血管收缩剂，又是血管平滑肌的有丝分裂原。 AVP的这些影响不仅对血流和压力的生理调节很重要，而且还可能在疾病中起作用，例如高血压和动脉粥样硬化，这些疾病分别涉及血管平滑肌收缩力和增殖的改变。 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+浓度的增加（[Ca2+] I）。 在A7R5大鼠主动脉平滑肌细胞中，AVP与V1A亚型的一类加压素受体结合。但是，AVP对A7R5细胞中的[Ca2+] I有两个不同的影响：低浓度的AVP（10-500 pm）刺激[Ca2+] I（Ca2+Spikes）中的振荡；较高的浓度（0.5-100 nm）抑制了Ca2+尖峰，但引起[Ca2+] I的双相升高是由于细胞内Ca2+储存量的释放而导致的，并且在整个质膜上的Ca2+涌入增加。 这两个效果涉及不同的机制。 AVP刺激的Ca2+尖峰是由通过L型电压敏感Ca2+通道取决于Ca2+进入的动作电位。 较高浓度的AVP释放细胞内Ca2+归因于磷脂酶C的激活产生的肌醇三磷酸。所提出的研究项目将检查参与AVP刺激的Ca2+尖峰的信号转导机制。初步研究表明，AVP刺激Ca2+尖峰需要磷脂酶D（PLD）的激活。 [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. PLD和非受体酪氨酸激酶PYK2的参与也将使用免疫沉淀技术研究。 蛋白激酶C（PKC）抑制剂可预防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，将在来自大鼠肠系膜动脉的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。