ANTIHYPERTENSIVE EFFECT OF H,K ATPASE INHIBITION

描述：申请人的实验室一直是 证明在血管内皮细胞中存在H+，K+-ATP酶， 和平滑肌细胞。 先前的研究表明， 胃、肠和肾小管细胞中的酶。 在 血管平滑肌细胞，这种酶负责泵送K+ 进入细胞和H+出，从而保持相对高的细胞pH。 这种新的ATP酶似乎在激动剂诱导的细胞凋亡中起重要作用。 血管平滑肌的收缩反应，以及刺激 一氧化氮生成 这种酶似乎有助于 高血压在一些模型中，和假设，抑制H，K- ATP酶将有效地治疗高血压， 测试. 本项目的目标是提供一个更全面的 H，K-ATP酶系统的表征和机械作用 血管平滑肌细胞和内皮细胞。 该项目将 测试特定酶抑制剂SCH 28080（衍生物）的作用 咪唑并[1，2]吡啶）对肾和心血管功能的影响。 两 短期和长期效果将使用培养的细胞进行评估， 除了大鼠和犬的整体动物研究之外。 具体目标 是：1. 定量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。 将新鲜分离的细胞与来自人的培养细胞进行比较。 脐静脉和犬冠状动脉。 除了急性 抑制，主动脉将从用H，K-ATP酶处理的大鼠获得 抑制剂数周。 NO将以NO2和NO3的形式进行分析 根据Greiss反应的代谢物。 2. 定义关系 细胞pH和Ca ~（2+）对NO生成的影响。 在某些制备中，细胞pH值将使用质子保持恒定- 在外部介质中与乙酸盐组合的菌丝体尼日利亚菌素。 的 ATP和钙的可能介导将通过测量ATP来测试， NADPH和Ca 2+浓度，抑制糖酵解（6- 磷酸果糖激酶）。 3. 分析H，K-ATP酶抑制剂的作用 对血管平滑肌pH和收缩性（张力）的影响 主动脉 4. H，K-ATP酶抑制剂对肾功能的影响 功能 清除率和血流研究将在 麻醉犬，以评估对肾小球滤过率的影响 (GFR)、肾血浆流量、尿电解质和水排泄，以及 肾素释放 NO的可能参与将被分析， 在L-给药前和给药期间观察SCH 28080的反应 名字 5. 确定血压对H，K-ATP酶的反应 高血压动物（狗和大鼠）中的抑制剂。 动脉压 会在清醒无压力的情况下持续记录 SCH化合物将在给药前和给药后进行急性和慢性给药， 在抑制NO产生期间（用L-NAME）。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，Goldblatt肾移植单肾和双肾模型 大鼠动脉夹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.