ANTIHYPERTENSIVE EFFECT OF H,K ATPASE INHIBITION

描述：申请人的实验室是先行者 证明血管内皮细胞中存在H+，K+-ATPase 和平滑肌细胞。此前的研究表明， 胃、肠和肾小管细胞中的酶。在……里面 血管平滑肌细胞，这种酶负责泵入K+ 进入细胞和H+输出，从而维持相对较高的细胞pH值。 这种新的ATPase似乎在激动剂诱导的 血管平滑肌的收缩反应和刺激 一氧化氮的产生。这种酶似乎有助于 一些模型中的高血压，以及抑制H，K- ATPase将有效地治疗高血压病 测试过。本项目的目标是提供一个更彻底的 H，K-ATPase系统的表征及其机制作用 血管平滑肌细胞和内皮细胞。该项目将 测试一种特定的酶抑制剂SCH 28080(一种衍生物)的效果 咪唑并[1，2]吡啶)对肾脏和心血管功能的影响。两者都有 短期和长期效果将使用培养的细胞进行评估 除了对老鼠和狗的整体动物研究。具体目标 1.量化H，K-ATPase对硝酸(NO)的影响 在培养的血管内皮细胞和平滑肌细胞中形成。 新分离的细胞将与来自人类的培养细胞进行比较 脐静脉和狗冠状动脉。除了急性 抑制，H，K-ATPase处理的大鼠将获得主动脉 几个星期的缓释剂。NO将被检测为NO2和NO3 代谢物根据格雷斯反应。2.定义关系 H，K-ATPase抑制过程中细胞pH和钙对NO生成的影响。 在一些制剂中，细胞的pH值将通过使用质子- 在外加介质中加入黑色素和醋酸盐。这个 将通过测量ATP来测试对ATP和钙的可能的调节作用， NADPH和钙离子浓度，抑制糖酵解(6- 磷酸果糖激酶)。3.H，K-ATPase抑制作用分析 大鼠血管平滑肌pH与收缩(张力)的关系 大动脉。4.评价H，K-ATPase对肾脏的抑制作用 功能。清除和血流研究将在#年进行 麻醉犬评价其对肾小球滤过率的影响 肾小球滤过率(GFR)、肾血浆流量、尿液电解质和水排泄，以及 肾素释放。将分析NO可能参与的情况 观察L服药前后对SCH-28080的反应 名字。5.测定血压对H，K-ATPase的反应 高血压动物(狗和大鼠)中的抑制剂。动脉压 将在有意识的、无压力的条件下被连续记录。 SCH复合制剂将在之前和之后急性和慢性地给药 抑制NO生成期间(与L同名)。高血压模型 将包括自发性高血压大鼠(SHR)、血管紧张素II 灌注性高血压与Goldblatt肾一肾和二肾模型 大鼠动脉夹闭性高血压和犬血管紧张素II。

DESCRIPTION: The applicant's laboratory has been a forerunner in demonstrating the presence of a H+, K+-ATPase in vascular endothelial and smooth muscle cells. Previous studies had shown the existence of the enzyme in stomach and intestine and renal tubular cells. In vascular smooth muscle cells, the enzyme is responsible for pumping K+ into cells and H+ out, thereby maintaining a relatively high cell pH. This novel ATPase appears to play and important role in agonist-induced contractile response in vascular smooth muscle, and in stimulating nitric oxide production. The enzyme appears to contribute to hypertension in some models, and the hypothesis that inhibition of H,K- ATPase will be efficacious in the treatment of hypertension will be tested. The goal of the present project is to provide a more thorough characterization of the H, K-ATPase system and mechanistic actions in vascular smooth muscle cells and endothelial cells. The project will test the effects of a specific enzyme inhibitor, SCH 28080 (a derivative of imidazo [1,2] pyridine) on renal and cardiovascular function. Both short and long term effects will be evaluated using cultured cells in addition to whole animal studies of rats and dogs. The specific aims are: 1. Quantify the effect of H, K-ATPase on nitric acid (NO) formation in cultured vascular endothelial and smooth muscle cells. Freshly isolated cells will be compared with cultured cells from human umbilical vein and dog coronary artery. In addition to the acute inhibition, aorta will be obtained from rats treated with the H,K-ATPase inhibitor for several weeks. NO will be assayed as NO2 and NO3 metabolites according to the Greiss reaction. 2. Define the relation of cell pH and calcium to NO production during H,K-ATPase inhibition. In some preparations, cell pH will be held constant using the proton- ophore nigericin in combination with acetate n the external media. The possible mediation of ATP and calcium will be tested by measuring ATP, NADPH and Ca2+ concentrations, inhibiting glycolysis (6- phosphofructokinase). 3. Analyze the effect of H,K-ATPase inhibition on vascular smooth muscle pH and contractility (tension) in isolated aorta. 4. Evaluate the effects of H,K-ATPase inhibition of renal function. Clearance and blood flow studies will be performed in anesthetized dogs to assess the effects on glomerular filtration rate (GFR), renal plasma flow, urinary electrolyte and water excretion, and renin release. The possible involvement of NO will be analyzed by observing responses to SCH 28080 before and during administration of L- NAME. 5. Determine blood pressure responses to the H,K-ATPase inhibitor in hypertensive animals (dogs and rats). Arterial pressure will be recorded continuously under conscious, unstressed conditions. The SCH compound will be given acutely and chronically in the before and during inhibition of NO production (with L-NAME). Hypertensive models will include spontaneously hypertensive rat (SHR), angiotensin II infusion hypertension, one and two-kidney models of Goldblatt renal artery-clip hypertension in the rat and angiotensin II in the dog.