Cellular regulation of cochlear blood flow

描述(申请人提供)：耳蜗的主要血液供应来自基底动脉，通过小脑前下动脉和螺旋蜗牛动脉。许多听力障碍，如感音神经性耳聋、急性听力损伤、噪声性听力损失、耳鸣、梅尼埃病和老年性耳聋，都可能是由局部血管痉挛引起或加重的，这些血管痉挛在病理性上限制了耳蜗血流量，导致一过性脑缺血事件，因此了解耳蜗血流量的调节机制具有重要的临床意义。钙是这项提案的主要焦点，因为它对血管收缩是必需的。血管平滑肌细胞内的胞浆钙浓度受到严格控制，并被有效地划分为介导血管收缩的池和介导血管扩张的池。介导血管收缩的钙通常以钙波的形式释放，而介导血管扩张的钙通常以钙火花的形式释放。一般说来，胞质钙浓度是通过跨质膜的进出口以及胞质钙库的进出来控制的。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，到目前为止还很少受到关注。对于这项建议，我们瞄准了两个主要领域，重点是钙，因为它与耳蜗血流量的细胞调节有关。大多数实验将使用体外分离的螺旋蜗牛动脉超融合制剂进行。血管直径将通过视频显微镜测量，细胞内钙离子将通过共聚焦显微荧光法测量。受体和离子通道的存在将通过结合使用功能分析的药理学方法、分子生物学方法和蛋白质生化方法来确定。在具体目标1下，我们将确定胞浆钙释放的不同模式是否分别导致血管扩张和收缩。在特定目标2下，我们将确定平滑肌细胞如何感知细胞外钙浓度。这些特定目标的完成将极大地提高我们对耳蜗血流量调节的细胞机制的理解，这是合理设计抗击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.