SIGNALING BY VASOPRESSIN--ARTERIAL SMOOTH MUSCLE CELLS

[精氨酸8]-加压素（AVP）是一种肽类激素，在血压降低或血浆渗透压升高时从垂体后叶释放。AVP既是一种强有力的血管收缩剂，又是血管平滑肌的有丝分裂原。 AVP的这些作用不仅对于血流和压力的生理调节是重要的，而且还可能在疾病中起作用，例如高血压和动脉粥样硬化，其分别涉及血管平滑肌收缩性和增殖的改变。 本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胞浆Ca ~（2+）浓度（[Ca ~（2+）]i）的增加。 在A7 r5大鼠主动脉平滑肌细胞中，AVP与V1 a亚型的单一类别的加压素受体结合。然而，AVP对A7 r5细胞中的[Ca 2 +]i具有两种不同的作用：低浓度的AVP（10-500 pM）刺激[Ca 2 +]i的振荡（Ca 2+尖峰）;较高浓度（0.5-100 nM）抑制Ca 2+尖峰，但引起[Ca 2 +]i的双相升高，这是由于细胞内Ca 2+储存的释放和跨质膜的Ca 2+内流增加。 这两种效应涉及不同的机制。 AVP刺激的Ca 2+尖峰来自依赖于通过L型电压敏感性Ca 2+通道的Ca 2+进入的动作电位。 高浓度AVP引起的细胞内Ca ~（2+）的释放是由于磷脂酶C激活产生的三磷酸肌醇所致。拟议的研究项目将研究AVP刺激的Ca 2+尖峰信号转导机制。初步研究表明，激活磷脂酶D（PLD）是必需的刺激钙尖峰AVP。[Ca2用fura-2或indo-1测定细胞膜电位和钙离子浓度，并采用电生理学方法研究AVP对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而增加细胞膜Ca ~（2+）峰电位，从而导致细胞膜去极化和电压敏感性Ca ~（2+）通道激活的假说。PLD和非受体酪氨酸激酶PYK 2参与K+通道抑制也将使用免疫沉淀技术进行研究。 AVP刺激的PYK 2磷酸化和Ca 2+尖峰被蛋白激酶C（PKC）抑制剂阻止。 另外的实验将研究特定的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[Ca 2 +]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.