title: Effect of uncoupling endothelial nitric oxide synthase on calcium homeostasis in aged porcine endothelial cells.
abstract: OBJECTIVE
  The requirement of endothelial NO synthase (NOS3) calcium to produce NO is well described, although the effect of NO on intracellular calcium levels [Ca(2+)](i) is still confusing. Therefore, NO and [Ca(2+)](i) cross-talk were studied in parallel in endothelial cells possessing a functional or a dysfunctional NO pathway.
  
  
  RESULTS
  Dysfunctional porcine endothelial cells were obtained either in vitro by successive passages or in vivo from regenerated endothelium 1 month after coronary angioplasty. Activity of NOS3 was characterized by conversion of arginine to citrulline, BH(4) intracellular availability, cGMP, and superoxide anion production. Imaging of the Ca(2+) indicator FURA 2-AM was recorded and sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) pump activity was analysed by (45)Ca(2+) uptake into cells. In endothelial cells with a functional NO pathway, NOS3 inhibition increased [Ca(2+)](i) and, conversely, an NO donor decreased it. In aged cells with an uncoupled NOS3 as shown by the reduced BH(4) level, the increase in superoxide anion and the lower production of cGMP and the decrease in NO bioavailability were linearly correlated with the increase in basal [Ca(2+)](i). Moreover, when stimulated by bradykinin, the calcium response was reduced while its decay was slowed down. These effects on the calcium signalling were abolished in calcium-free buffer and were similarly induced by SERCA inhibitors. In aged cells, NO improved the reduced SERCA activity and tended to normalize the agonist calcium response.
  
  
  CONCLUSIONS
  In control endothelial cells, NO exerts a negative feedback on cytosolic Ca(2+) homeostasis. In aged cells, uncoupled NOS3 produced NO that was insufficient to control the [Ca(2+)](i). Consequently, under resting conditions, SERCA activity decreased and [Ca(2+)](i) increased. These alterations were reversible as exogenous NO, in a cGMP-independent way, refilled intracellular calcium stores, reduced calcium influx, and improved the agonist-evoked calcium response. Therefore, prevention of the decrease in NO in dysfunctional endothelium would normalize the calcium-dependent functions.
authors.affiliation: Institut de Recherches SERVIER, 11 rue des Moulineaux, 92150 Suresnes, France.
authors.forename: Emeline
authors.initials: E
authors.lastname: Perrier
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Fournet-Bourguignon
authors.forename: Emilie
authors.initials: E
authors.lastname: Royere
authors.forename: Stephanie
authors.initials: S
authors.lastname: Molez
authors.forename: Helene
authors.initials: H
authors.lastname: Reure
authors.forename: Ludovic
authors.initials: L
authors.lastname: Lesage
authors.forename: Willy
authors.initials: W
authors.lastname: Gosgnach
authors.forename: Yves
authors.initials: Y
authors.lastname: Frapart
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Boucher
authors.forename: Nicole
authors.initials: N
authors.lastname: Villeneuve
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Vilaine
mesh_terms: D000818:Animals; D001120:Arginine / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D001920:Bradykinin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D016922:Cellular Senescence; D002956:Citrulline / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006706:Homeostasis; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013552:Swine; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; D013481:Superoxides; D001708:Biopterins; D002956:Citrulline; D009569:Nitric Oxide; D001120:Arginine; D052250:Nitric Oxide Synthase Type III; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C003402:sapropterin; D006152:Cyclic GMP; D001920:Bradykinin; D002118:Calcium