SIGNALING BY VASOPRESSIN--ARTERIAL SMOOTH MUSCLE CELLS

[精氨酸8]-加压素(AVP)是一种在血压降低或血浆渗透压升高时由脑下垂体释放的多肽激素。AVP既是一种有效的血管收缩药，又是一种血管平滑肌有丝分裂原。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，这些疾病分别涉及血管平滑肌收缩和增殖的变化。本项目的长期目标是了解动脉平滑肌对AVP的这些反应的分子机制。动脉平滑肌细胞的收缩和增殖都与细胞内钙离子浓度([Ca~(2+)]i)升高有关。在A7r5大鼠主动脉平滑肌细胞中，AVP与V1a亚型的单一类型的加压素受体结合。然而，AVP对A7r5细胞[Ca~(2+)]_i有两种不同的作用：低浓度(10~500 pm)AVP刺激[Ca~(2+)]i振荡(Ca~(2+)峰)；高浓度(0.5~100 nM)抑制Ca~(2+)峰，但通过释放细胞内的Ca~(2+)和增加跨质膜的Ca~(2+)内流，引起细胞内[Ca~(2+)]i的双相升高。这两种效应涉及不同的机制。AVP刺激的钙峰电流是由动作电位引起的，这种动作电位依赖于钙离子通过L型电压敏感钙通道的内流。较高浓度的AVP引起的细胞内钙的释放归因于磷脂酶C激活产生的三磷酸肌醇。该研究将探讨AVP刺激的细胞内钙信号转导机制。初步研究表明，激活磷脂酶D(PLD)是AVP刺激Ca~(2+)峰所必需的。[CA2]用Fura-2或Indo-1和电生理方法检测AVP对全细胞膜电流和膜电位的影响，以验证AVP通过抑制延迟整流钾电流，从而增加膜去极化和激活电压敏感的钙通道，从而增加钙激活活性的假说。PLD和非受体酪氨酸激酶PYK2在钾通道抑制中的作用也将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磷酸化和钙尖峰。其他实验将研究特定的PKC亚型在AVP信号通路中的作用及其与PLD激活的关系。最后，AVP和其他激素对新鲜分离的大鼠肠系膜动脉平滑肌细胞的[钙]i和膜电流的影响将被检测。

[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.