High glucose and arteriolar Kv channels

描述（由申请人提供）：高血糖和糖尿病与冠状动脉血管内皮依赖性扩张受损有关，部分原因是血管扩张剂一氧化氮因产生过量的超氧化物而猝灭。相比之下，关于氧化应激对超极化介导的血管舒张的影响知之甚少。这一点很重要，因为冠状动脉扩张，尤其是人类，较少依赖于一氧化氮，更多依赖于通过打开K +通道的超极化。电压依赖性（Kv） K +通道是冠状血管平滑肌（VSM）中高表达的几种K +通道之一。它们在调节静息张力和参与生理性扩张中起重要作用。许多与糖尿病和高血糖相关的血管功能障碍是由于活性氧（ROS）的过量产生。然而，通过K +通道和超极化介导的血管舒缩反应在多大程度上受到葡萄糖升高的影响，以及这种影响是否涉及ROS的产生尚不清楚。本应用程序的总体目的是确定高葡萄糖（无论是在分离血管的组织培养基中还是在糖尿病大鼠的血清中）对高极化介导的冠状动脉扩张和冠状动脉VSM Kv通道活性的影响，并确定超氧化物在通道活性改变和高极化诱导的冠状动脉扩张中的机制作用。视频显微镜、膜电位、贴片夹紧和Western blot分析分离的冠状动脉将在大鼠和狗模型中使用，以测试三个特定目的。在目的1中，我们将检验高葡萄糖降低大鼠冠状动脉中Kv通道活性和相关的异丙肾上腺素和福斯克林扩张的假设。在目标2中，我们将确定暴露于高葡萄糖后，超氧化物对Kv通道活性降低的贡献。在目标3中，我们将确定暴露于高葡萄糖或高血糖后血管超氧化物生成的来源。在正常或高葡萄糖环境中培养的大鼠冠状动脉，或从糖尿病大鼠和对照大鼠或狗身上分离出来的大鼠冠状动脉，要么安装在微移液管上进行直径测量，要么分离成单个VSM细胞进行K +电流测量。同样制备的血管也保存用于Western blot分析。这种血管和细胞功能的药理学、电生理和分子评估的结合为研究超极化依赖的血管调节过程提供了强有力的机制。这些研究将对糖尿病的冠状动脉血管舒缩调节机制具有重要意义，并提供冠状动脉血管调节改变的新机制。该结果可能为糖尿病患者微血管功能障碍的治疗提供新的治疗途径。

DESCRIPTION (provided by applicant): Hyperglycemia and diabetes are associated with impaired endothelium-dependent dilation of coronary conduit vessels, due in part to quenching of the vasodilator nitric oxide by production excess superoxide. In contrast, little is known regarding the effect of oxidative stress on hyperpolarization-mediated vasodilation. This is important since coronary arteriolar dilation, especially in humans, depends less upon nitric oxide and more upon hyperpolarization via opening of K + channels. Voltage-dependent (Kv) K + channels are one of several K + channels highly expressed in coronary vascular smooth muscle (VSM). They play an important role in regulating resting tone and participate in physiological dilation. Much of the vascular dysfunction associated with diabetes and hyperglycemia is due to excess production of reactive oxygen species (ROS). However the extent to which vasomotor responses mediated through K + channels and hyperpolarization are affected by elevations in glucose, and whether this influence involves the production of ROS is not known. The overall aim of this application is to determine the effect of high glucose (either in the tissue medium bathing isolated vessels or in the serum of diabetic rats) on hyperpolarization-mediated coronary dilation and coronary VSM Kv channel activity, and to establish a mechanistic role for superoxide in both the altered channel activity and hyperpolarization-induced dilation of coronary arterioles. Videomicroscopy, membrane potential, patch clamping, and Western blot analysis of isolated coronary arterioles will be used in both rat and dog models to test three specific aims. In aim 1 we shall examine the hypothesis that high glucose reduces both Kv channel activity and the associated dilation to isoproterenol and forskolin in rat coronary arterioles. In aim 2 we shall determine the contribution of superoxide to the reduced Kv channel activity following exposure to high glucose. In aim 3 we shall identify the source of vascular superoxide generation following exposure to high glucose or hyperglycemia. Rat coronary arterioles incubated in normal or high glucose or isolated from diabetic and control rats or dogs are either mounted onto micropipettes for diameter measurements or dissociated into single VSM cells for K + current measurements. Vessels from the same preparations are also saved for Western blot analysis. This combination of pharmacological, electrophysiological and molecular assessment of vascular and cellular function provides a powerful mechanism for studying hyperpolarization-dependent vascular regulatory process. These studies will have important implications with regard to mechanisms of coronary vasomotor regulation in diabetes and provide novel mechanisms of altered coronary vasoregulation. The results may suggest new therapeutic approaches for the treatment of microvascular dysfunction in patients with diabetes.