Stimulation of ß2-adrenoceptors (ß2AR) on vascular endothelium causes vasorelaxation through endothelial cell release of nitric oxide, with no change in endothelial cell Ca2+ concentration (Ferro et al., 1999). We hypothesised that this might be explained by ß2AR-mediated serine phosphorylation of endothelial-type nitric oxide synthase (NOS3). The aim of the present work was to examine changes in serine phosphorylation of NOS3 in response to ß2AR stimulation in human umbilical vein endothelial cells (HUVEC), and whether such changes could be explained by the action of protein kinase A (PKA) and/or protein kinase B (PKB, Akt).
HUVEC were isolated
from umbilical cords derived from healthy uncomplicated pregnancies, and
cultured to confluence at passage 3, as previously described (Ferro et al., 1999). Following extensive washing, and equilibration in Krebs buffer,
HUVEC were incubated with the NOS inhibitor NG-monomethyl-L-arginine
(L-NMMA) 10-4M or corresponding vehicle.
Additionally, incubations were performed with the PKA inhibitor H-89 10-7M,
the phosphatidylinositol 3-kinase inhibitor wortmannin 500nM, Akt inhibitor
or corresponding vehicle. All incubations were for 15 min, after which
the selective ß2AR agonist albuterol
The phosphoserine/total NOS3 densitometric ratio in untreated cells was 0.41±0.07 arbitrary units, and this increased to 0.82±0.04 in response to albuterol (P<0.01). This increase was abolished when H-89 was co-incubated (0.47±0.02, P<0.05 as compared with albuterol alone), but only partially attenuated in the presence either of wortmannin or of Akt inhibitor (densitometric ratios 0.64±0.01 and 0.65±0.05 respectively; P<0.05 for each as compared with vehicle and also as compared with albuterol alone). Forskolin also increased phosphoserine/total NOS3 densitometric ratio, to 0.72±0.02 (P<0.05 as compared with vehicle). The increases to albuterol and to forskolin were not attenuated by L-NMMA.
In conclusion, ß2AR stimulation with albuterol or adenylyl cyclase activation with forskolin each cause an increase in serine phosphorylation of NOS3 in HUVEC. The increase in response to ß2AR stimulation is not affected by NOS inhibition, but is partially attenuated by inhibition of the Akt pathway and is abolished by PKA inhibition, suggesting that both PKA and Akt are important in this response.
Ferro, A. et al.
(1999) Br. J. Pharmacol. 126, 1872-1880.