Cardiovascular Research Advance Access originally published online on January 28, 2009
Cardiovascular Research 2009 82(1):133-142; doi:10.1093/cvr/cvp034
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Effect of uncoupling endothelial nitric oxide synthase on calcium homeostasis in aged porcine endothelial cells
1 Institut de Recherches SERVIER, 11 rue des Moulineaux, 92150 Suresnes, France.
2 UMR CNRS 8601 Université Paris Descartes, Paris, France
* Corresponding author. 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 requirement of endothelial NO synthase (NOS3) calcium to produce NO is well described, although the effect of NO on intracellular calcium levels [Ca2+]i is still confusing. Therefore, NO and [Ca2+]i cross-talk were studied in parallel in endothelial cells possessing a functional or a dysfunctional NO pathway.
Methods and 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, BH4 intracellular availability, cGMP, and superoxide anion production. Imaging of the Ca2+ indicator FURA 2-AM was recorded and sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump activity was analysed by 45Ca2+ uptake into cells. 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 and the decrease in NO bioavailability were 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.
Conclusion: 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 the calcium-dependent functions.
KEYWORDS Endothelial nitric oxide synthase; Nitric oxide; Tetrahydrobiopterin; [Ca2+] signalling; Ageing
Time for primary review: 42 days