Cellular regulation of cochlear blood flow

描述（由申请人提供）：耳蜗的主要血液供应来自基底动脉，经小脑前下动脉和螺旋模侧动脉。了解耳蜗血流的调节机制具有重要的临床意义，因为许多听力障碍，如感音神经性听力损失、急性声损伤、噪声性听力损失、耳鸣、梅尼埃氏病和耳聋老年性等，都可能由局部血管痉挛引起或加重，从而在病理上限制耳蜗血流并引起短暂性缺血事件。钙是这个建议的主要焦点，因为它对血管收缩是必需的。血管平滑肌细胞内的细胞质钙浓度受到严格控制，并被有效地划分为介导血管收缩的钙池和介导血管舒张的钙池。介导血管收缩的钙通常以钙波的形式释放，介导血管舒张的钙通常以钙火花的形式释放。一般来说，胞质钙浓度是通过胞质膜的进出口和胞质钙储存库的进出来控制的。细胞外钙浓度可能不仅是钙的简单储存库，而且是一个重要的旁分泌信使，迄今为止很少受到关注。对于这一建议，我们针对两个主要领域集中在钙，因为它涉及到耳蜗血流的细胞调节。大多数实验将使用离体的螺旋型臼齿动脉的超级制备进行。血管直径将通过视频显微镜和共聚焦显微荧光法测定细胞质钙。受体和离子通道的存在将通过使用功能测定的药理学方法、分子生物学方法和蛋白质生化方法的组合来确定。在Specific Aim 1中，我们将确定不同的钙从细胞质储存中释放是否分别引起血管舒张和收缩。在具体目标2下，我们将确定平滑肌细胞如何感知细胞外钙浓度。这些特异性目标的完成将极大地增强我们对耳蜗血流调节的细胞机制的理解，这是合理设计对抗多种听力障碍所需的药物干预的必要前提。

DESCRIPTION (provided by applicant): The cochlea receives its main blood supply from the basilar artery, via the anterior inferior cerebellar artery and the spiral modiolar artery. It is of clinical importance to understand the mechanisms that regulate cochlear blood flow, since many hearing disorders, such as sensorineural hearing loss, acute acoustic trauma, noise-induced hearing loss, tinnitus, Meniere's disease and presbyacusis may be caused or aggravated by localized vasospasms that pathologically limit cochlear blood flow and cause transient ischemic events. Calcium is the major focus of this proposal since it is obligatory for vasoconstrictions. The cytosolic Calcium concentration in vascular smooth muscle cells is tightly controlled and effectively compartmentalized into pools that mediate vasoconstriction and pools that mediate vasodilation. Calcium that mediates vasoconstriction is commonly released as Calcium waves and calcium that mediates vasodilations is commonly released as Calcium sparks. In general, the cytosolic Calcium concentration is controlled via export/import across the plasma membrane and in and out of cytosolic Calcium stores. The extracellular Calcium concentration may not only serve as a simple reservoir for Calcium but constitute an important paracrine messenger that has so far received little attention. For this proposal we have targeted two major areas focused on Calcium as it relates to the cellular regulation of cochlear blood flow. Most experiments will be performed using an isolated in vitro superfused preparation of the spiral modiolar artery. Vascular diameter will be measured by videomicroscopy and cytosolic Calcium by confocal microfluorometry. The presence of receptors and ion channels will be determined by a combination of a pharmacological approach using functional assays, a molecular biological approach and a protein biochemical approach. Under Specific Aim 1, we will determine whether different modes of Calcium release from cytosolic stores cause vasodilations and constrictions, respectively. Under Specific Aim 2 we will determine how smooth muscle cells sense the extracellular Calcium concentration. Completion of these Specific Aims will greatly enhance our understanding of the cellular mechanisms of cochlear blood flow regulation, which is a necessary prerequisite for the rational design of pharmacological interventions needed to combat a multitude of hearing disorders.