ORIGINAL CONTRIBUTION Basic Res Cardiol 93: 250 – 256 (1998) © Steinkopff Verlag 1998 T. Bachetti L. Comini L. Agnoletti P. Pedersini G. Gaia A. Cargnoni M. Bellet S. Curello R. Ferrari Effects of chronic noradrenaline on the nitric oxide pathway in human endothelial cells Abstract Altered endothelium-depen- dent vasodilation has been observed in congestive heart failure (CHF), a disease characterized by a sustained adrenergic activation. The purpose of our study was to test the hypothesis that chronically elevated catechola- mines influence the nitric oxide (NO) pathway in the human endothelium. Human umbilical vein endothelial cells (HUVEC) were exposed for 7 days to a concentration of noradrenaline (NA,1 ng/mL) similar to that found in the blood of patients with CHF. Kinetics of endothelial constitutive NO synthase (ecNOS) and inducible NO synthase (iNOS) activity, measured by [ 3 H]L- arginine to [ 3 H]L-citrulline conversion, and protein expression of ecNOS and iNOS, assessed by Western blot analy- sis, were unaffected by chronic NA treatment. Furthermore, no changes in subcellular fraction-associated ecNOS were found; this indirectly shows that chronic NA did not cause phosphoryla- tion of the enzyme. Moreover, [ 3 H]L- arginine transport through the plasma membrane was conserved in chroni- cally NA-treated cells. The data demonstrate that prolonged in vitro exposure to pathologic CHF-like NA does not affect the L-arginine: NO path- way in human endothelial cells. Key words Noradrenaline – nitric oxide – nitric oxide synthase – endothelial cells Dr. T. Bachetti () · L. Comini · L. Agnoletti P. Pedersini · G. Gaia · A. Cargnoni Foundation Salvatore Maugeri Cardiovascular Pathophysiology Research Centre Via Pinidolo 23 25064 Gussago, Brescia, Italy E-mail: curello@master.cci.unibs.it M. Bellet · S. Curello · R. Ferrari Chair of Cardiology Spedali Civili University of Brescia Brescia, Italy Received: 11 July 1997 Returned for 1. revision: 13 August 1997 1. Revision received: 6 October 1997 Returned for 2. revision: 17 November 1997 2. Revision received: 5 January 1998 Accepted: 26 January 1998 BRC 087 Introduction Chronic congestive heart failure (CHF) is a complex clinical syndrome which includes activation of the adrenergic system and endothelial dysfunction. As endothelial cells express both  and  receptors (12, 14, 20, 21, 27, 29), it has been suggested that adrenergic agents might modulate the L-arginine:NO path- way, and a link between these events has been assumed. It is well-known that acute in vitro exposure of the endothelium to sympathomimetic agonists enhances the basal release of nitric oxide (NO) (20, 21). In experimental and human CHF, how- ever, the opposite is true: the chronic increase of the adrener- gic tone is associated with a reduction of endothelium-derived NO (1, 9, 7, 16, 18, 30, 32), suggesting that catecholamines elicit different effects on the endothelium according to the duration of exposure. In the literature, however, there are no data on endothelial function after prolonged in vitro stimula- tion with catecholamines. To overcome this gap, we incubated human endothelial cells with noradrenaline (NA) for 7 days at a concentration reflecting that circulating in the blood of patients with CHF. The effects of NA on the L-arginine:NO pathway were evaluated in terms of: 1) kinetics of endothelial constitutive NO synthase (ecNOS) and inducible NO synthase (iNOS) activities; 2) expression of ecNOS and iNOS proteins; 3) transmembrane transport of L-arginine.