[PubMed] [Google Scholar]Suto T, Losonczy G, Qiu C, et al. inhibitors. These findings claim that low endothelial NOS activity might donate to hypertension in end stage renal disease sufferers. 1995). Hypertension takes place in mice with knockout from the endothelial nitric oxide synthase (eNOS) gene (Huang 1995) and in man with certain eNOS gene polymorphisms (Soma 1999). There is evidence that regional vascular endothelial NO production is defective in some patients with primary and secondary hypertension (Baylis & Vallance 1996). Therefore, insufficient NO production from eNOS may play a role in some forms of hypertension in man. Hypertension is a major complication of end stage renal disease (ESRD) (Rostand 1991) and although in part, caused by volume overload, may also involve NO deficiency. Indeed, we have reported reductions in total NO synthesis (from 24 h NO2 + NO3 = NOproduction) in both peritoneal dialysis (PD) and haemodialysis (HD) patients (Schmidt Neferine 1999a, b). Patients with ESRD accumulate endogenous circulating compounds which may competitively inhibit the l-arginine : NO pathway (Vallance 1992). The purpose of this study was to assess the effects of uraemic plasma on NOS activity in cultured vascular endothelial cells. The majority of studies were on human dermal microvascular endothelium although Neferine some experiments were done on human glomerular endothelial cells and bovine thoracic aortic endothelium. METHODS Human dermal microvascular endothelial cells (HDMEC) and endothelium growth medium (EGM-MV) were obtained from Clonetics Corporation (San Diego, CA). Human glomerular endothelial Neferine cells (HGEC) and CS-C growth medium were from Mouse monoclonal to Alkaline Phosphatase Cell System Corporation (Kirkland, WA). The bovine thoracic aortic endothelial cells (BAEC) were established by us in primary culture. Human plasma was from PD patients, pre- and immediately posthaemodialysis (pre-HD and post-HD) and normal controls. These studies were performed with the consent of each subject and permission of the West Virginia University Institutional Review Board. Clinical Neferine characteristics of the study populations are shown in Table 1. Each type of plasma was pooled from two to three patients, stored frozen at C80 C, and thawed immediately prior to use. All HD patients were dialysed with polysulfone membranes on F-80 dialysers (Fresenius USA, Lexington, MA). Table 1 The clinical characteristics of the patients with end stage renal disease and normal control = 5)117 672 410 30.8 0.20.45 0.10NonePD (= 6)127 474 542 6*10.1 3.2*2.16 0.27*BB, CEI, DHD (= 5) Pre/post136 8*69 374 8*10.2 1.4*4.13 0.78*BB,CEI, D32 4*#3.2 0.4*#1.46 0.44*# Open in a separate window ?Antihypertensive treatment: BB, Beta-blocker; CEI, angiotensin converting enzyme inhibitor; D, diuretic drugs. *< 0.05 vs. control. #After haemodialysis and < 0.05 vs. control. Cell culture HDMEC (passage 4C7) were maintained in EGM-V media containing 10 pg mLC1 human recombinant epidermal growth factor, 1 (1993), using Dowex 50WX8-400 resin (Na+ form) to remove unconverted l-[3H]arginine. Determination of nitric oxide synthase activity in fractionated endothelial cells Confluent endothelial cells grown in T-75 flasks were disrupted by freeze-thawing and the NOS activity in the cell lysate was determined by conversion rate of 3H-l-arginine to 3H-l-citrulline (Bredt & Synder 1994). Measurement of NO production from nitrate + nitrite = NOx level To obtain a sufficient amount of NOfor analysis, cells were grown to confluence in T25 flasks, then incubated for 6 h with 20% normal.