SIGNALING BY VASOPRESSIN--ARTERIAL SMOOTH MUSCLE CELLS

[精氨酸8]-抗利尿激素（AVP）是一种肽激素，当血压下降或血浆渗透压升高时，由垂体后叶释放。AVP是一种有效的血管收缩剂和血管平滑肌的有丝分裂原。AVP的这些作用不仅对血流和血压的生理调节很重要，而且可能在高血压和动脉粥样硬化等疾病中发挥作用，这两种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反应的分子机制。动脉平滑肌细胞的收缩和增殖都涉及细胞质Ca2+浓度的增加（[Ca2+]i）。在A7r5大鼠主动脉平滑肌细胞中，AVP与一类V1a亚型的抗利尿激素受体结合。然而，AVP对A7r5细胞中的[Ca2+]i有两种不同的影响：低浓度的AVP （10-500 pM）刺激[Ca2+]i的振荡（Ca2+峰值）；较高的浓度（0.5-100 nM）抑制Ca2+峰值，但引起[Ca2+]i的双相升高，这是由于细胞内Ca2+储存的释放和Ca2+在质膜上的内流增加。这两种效应涉及不同的机制。avp刺激的Ca2+尖峰是由依赖于通过l型电压敏感Ca2+通道的Ca2+进入的动作电位引起的。较高浓度的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+尖峰的信号转导机制。初步研究表明，AVP刺激Ca2+峰值需要磷脂酶D （PLD）的激活。使用fura-2或indo-1和电生理方法测量[Ca2+]i将用于检查AVP对全细胞膜电流和膜电位的影响，以验证AVP通过抑制延迟整流K+电流增加Ca2+尖峰活性的假设，从而导致膜去极化和激活电压敏感的Ca2+通道。PLD和非受体酪氨酸激酶PYK2在K+通道抑制中的作用也将通过免疫沉淀技术进行研究。avp刺激的PYK2磷酸化和Ca2+尖峰被蛋白激酶C （PKC）抑制剂阻止。进一步的实验将研究特定PKC异构体在AVP信号通路中的作用及其与PLD激活的关系。最后，AVP和其他激素对[Ca2+]i和膜电流的影响将在新鲜分离的大鼠肠系膜动脉平滑肌细胞中进行检查。

[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.