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Cardiovascular Research Advance Access [Accepted Manuscript] published online on January 28, 2009
Cardiovascular Research, doi:10.1093/cvr/cvp034
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2009. For permissions please email: journals.permissions@oxfordjournals.org.

Effect of uncoupling eNOS on calcium homeostasis in aged porcine endothelial cells

Emeline Perriera, Marie-Pierre Fournet-Bourguignona,*, Emilie Royerea, Stephanie Moleza, Helene Reurea, Ludovic Lesagea, Willy Gosgnach, Yves Frapartb, Jean-Luc Boucherb, Nicole Villeneuvea and Jean-Paul Vilainea

a Institut de Recherches SERVIER, Suresnes, France
b UMR CNRS 8601 Université Paris Descartes, Paris, France

* Corresponding author: MP Fournet-Bourguignon Institut de Recherches Servier 11 rue des Moulineaux 92150 Suresnes France. Tel: +33 1 55 72 22 15 Fax: +33 1 55 72 24 30 E-mail address: marie-pierre.bourguignon{at}fr.netgrs.com

Aims: The endothelial NO synthase (NOS3) calcium requirement to produce NO is well described while the effect of NO on intracellular calcium levels [Ca2+]i is still confusing. Therefore, NO and [Ca2+]i crosstalk were studied in parallel in endothelial cells possessing a functional or a dysfunctional NO pathway.

Methods: Dysfunctional porcine endothelial cells were obtained either in vitro by successive passages or in vivo from regenerated endothelium one month after coronary angioplasty. NOS3 activity was characterized by conversion of arginine to citrulline, BH4 intracellular availability, cGMP and superoxide anion production. Imaging of the Ca2+ indicator FURA-AM was recorded and sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump activity was analyzed by 45Ca2+ uptake into cells.

Results: In endothelial cells with a functional NO pathway, NOS3 inhibition increased [Ca2+]i, and, conversely, an NO donor decreased it. In aged cells with an uncoupled NOS3 as shown by the reduced BH4 level, the increase in superoxide anion and the lower production of cGMP, the decrease in NO bioavailability was linearly correlated with the increase in basal [Ca2+]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 Ca2+ homeostasis. In aged cells, uncoupled NOS3 produced NO that was insufficient to control the [Ca2+]i. Consequently, under resting conditions, SERCA activity decreased and [Ca2+]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 calcium-dependent functions.

KEYWORDS endothelial nitric oxide synthase; nitric oxide; tetrahydrobiopterin; [Ca2+] signalling; aging

Time for primary review: 42 Days


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