ANTIHYPERTENSIVE EFFECT OF H,K ATPASE INHIBITION

申请人的实验室在……领域处于领先地位

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.