AT2 receptor activation regulates myocardial eNOS expression via the calcineurin-NF-AT pathway.
The role of AT2-receptors has recently been subject of considerable debate. We investigated the influence of AT2-stimulation/inhibition on myocardial endothelial NO-synthase (eNOS, NOS-III) promoter activity and eNOS protein expression. Stimulation of rat cardiomyocytes with angiotensin II (AngII) increased eNOS protein expression 3.3-fold. This was blocked by Cyclosporin A (CsA). Inhibition of the AT1-receptor did not reduce AngII-mediated eNOS protein expression, whereas AT2 stimulation increased it 2.4-fold and AT2 inhibition suppressed it. The modulatory effects of the AT2-receptor on eNOS expression was confirmed in mice with a genetic deletion of the AT2-receptor (AT2-KO). In gel shift assays two putative NF-AT sites in a 1.6 kb eNOS promoter fragment showed NF-AT binding and a supershift by NF-AT2(-c1)-specific antibodies. Stimulation of transfected cells with AngII or specific AT2-receptor agonists resulted in a significant increase in eNOS promoter activity, which was blocked by CsA, MCIP1, and mutation of an upstream NF-AT site.


CONCLUSIONS
1) AngII-stimulation of the myocardium, both in vivo and in vitro, is accompanied by increased expression of eNOS. 2) This effect is mediated by the calcineurin pathway and is induced by the AT2-receptor. 3) These results define a calcineurin/NF-AT/eNOS pathway as downstream effector of AT2-receptor activation in the myocardium.
Ritter
Oliver
O
Department of Medicine, University of Wuerzburg, Germany.
Schuh
Kai
K
Brede
Marc
M
Röthlein
Nicola
N
Burkard
Natalie
N
Hein
Lutz
L
Neyses
Ludwig
L
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019703:Calcineurin / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D050778:NFATC Transcription Factors; D019001:Nitric Oxide Synthase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D051381:Rats; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D000804:Angiotensin II; D008156:Luciferases; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; C496316:Nos3 protein, rat; D019703:Calcineurin