Endothelial nitric oxide synthase activity is inhibited by the plasma membrane calcium ATPase in human endothelial cells.
OBJECTIVE
Nitric oxide (NO) plays a pivotal role in the regulation of cardiovascular physiology. Endothelial NO is mainly produced by the endothelial nitric oxide synthase (eNOS) enzyme. eNOS enzymatic activity is regulated at several levels, including Ca(2+)/calmodulin binding and the interaction of eNOS with associated proteins. There is emerging evidence indicating a role for the plasma membrane calcium ATPase (PMCA) as a negative regulator of Ca(2+)/calmodulin-dependent signal transduction pathways via its interaction with partner proteins. The aim of our study was to investigate the possibility that the activity of eNOS is regulated through its association with endothelial PMCA.


RESULTS
We show here a novel interaction between endogenous eNOS and PMCA in human primary endothelial cells. The interaction domains were located to the region 735-934 of eNOS and the catalytic domain of PMCA. Ectopic expression of PMCA in endothelial cells resulted in an increase in phosphorylation of the residue Thr-495 of endogenous eNOS. However, disruption of the PMCA-eNOS interaction by expression of the PMCA interaction domain significantly reversed the PMCA-mediated effect on eNOS phosphorylation. These results suggest that eNOS activity is negatively regulated via interaction with PMCA. Moreover, NO production by endothelial cells was significantly reduced by ectopic expression of PMCA.


CONCLUSIONS
Our results show strong evidence for a novel functional interaction between endogenous PMCA and eNOS in endothelial cells, suggesting a role for endothelial PMCA as a negative modulator of eNOS activity, and, therefore, NO-dependent signal transduction pathways.
Holton
MaryLouisa
M
Molecular Pharmacology Group, Department of Pharmacy, Research Institute in Healthcare Sciences, School of Applied Sciences, University of Wolverhampton, Wulfruna Street, Wolverhampton WV1 1SB, UK.
Mohamed
Tamer M A
TM
Oceandy
Delvac
D
Wang
Weiguang
W
Lamas
Santiago
S
Emerson
Michael
M
Neyses
Ludwig
L
Armesilla
Angel L
AL
D019703:Calcineurin / Q000378:metabolism; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D015536:Down-Regulation; D042783:Endothelial Cells / Q000201:enzymology*; D004789:Enzyme Activation; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D015398:Signal Transduction; D014162:Transfection
D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D019703:Calcineurin; C507517:ATP2B4 protein, human; D053499:Plasma Membrane Calcium-Transporting ATPases; C506958:ATP2B2 protein, human; D006152:Cyclic GMP