Proceedings of The Physiological Society

University of Manchester (2010) Proc Physiol Soc 19, C48

Oral Communications

Interaction of nitric oxide synthase type-3 with beta catenin in human umbilical vein endothelial cells

N. Chen1,2, V. Snetkov2, J. Ward2, A. Ferro1

1. Department of Clinical Pharmacology, Cardiovascular Division, King's College London, London, United Kingdom. 2. Division of Asthma, Allergy and Lung Biology, King?

Endothelium-derived nitric oxide is an important vasorelaxant and anti-atherogenic mediator, generated from L-arginine by the action of nitric oxide synthase type 3 (NOS-3). NOS-3 can undergo activation classically through the Ca2+-calmodulin pathway; and more recently Ca2+-insensitive activation has been shown to occur, especially through phosphorylation of key residues and association with a number of other proteins (including heat shock protein-90, caveolin-1 and beta-actin). We have previously shown a direct association between NOS-3 and beta-catenin in endothelial cells, which is increased in response to a number of standard NOS-3 agonists. Here we demonstrate for the first time that such agonists cause translocation of beta-catenin into the nucleus, which is sensitive to inhibition by NG-nitro-L-arginine methyl ester (L-NAME, an L-arginine analogue which inhibits NOS activity) in the case of Ca2+-insensitive but not Ca2+-sensitive agonists. Human umbilical vein endothelial cells (HUVEC), isolated from umbilical cords obtained following healthy uncomplicated pregnancies, were cultured to confluence at passage 3; at which point they were exposed to L-NAME (100μM) or corresponding vehicle for 30 min, followed by addition of histamine (100μM), thrombin (1U/ml), salbutamol (1μM), adenosine (100μM), or corresponding vehicle (Krebs), all for 2 min, at37°C. The reactions were terminated by washing with cold (4°C) PBS, and nuclear lysates were prepared. These were then run on SDS-PAGE and immunoblotted for NOS-3 and beta-catenin, as well as the nuclear housekeeping protein lamin A/C. The resulting bands were analysed by scanning densitometry, and band densities were compared using repeated measures one-way ANOVA. p<0.05 (two-tailed) was taken as indicating statistical significance. All data are presented as mean +/- SEM. Immunoblotting analysis of nuclear extracts demonstrated an increase in nuclear beta-catenin in response to significantly increased by adenosine (70±37.4%), histamine (153±105%), salbutamol (90±61%) and thrombin (166±128%) (p<0.01 for each, n=5~8). The nuclear translocation of beta-catenin induced by either adenosine or salbutamol (both Ca2+-insensitive agonists) was abolished by co-incubation with L-NAME, but that induced by histamine or thrombin (both Ca2+-sensitive agonists) was unaffected. Ionomycin treatment also induced the translocation of beta catenin. NOS-3 agonists induce translocation of beta-catenin into the nucleus. Moreover, agents which induce an increase in intracellular Ca2+ can increase nuclear translocation. The precise functional consequences of this phenomenon remain to be determined.

Where applicable, experiments conform with Society ethical requirements