Silencing regulator of G protein signaling-2 (RGS-2) increases angiotensin II signaling: insights into hypertension from findings in Bartter's/Gitelman's syndromes.
OBJECTIVE
Regulator of G-protein signaling (RGS)-2 is a regulator of angiotensin II (Ang II) signaling. In Bartter's syndrome/Gitelman's syndrome (BS/GS), we have demonstrated increased RGS-2 levels and blunted Ang II signaling which contribute to their reduced vasomotor tone and remodeling. The present study investigates the effect of silencing RGS-2 in fibroblasts from six BS/GS patients on intracellular Ca2+ (CaI2+) mobilization and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, established Ang II-mediated responses.


METHODS
Fibroblasts were RGS-2 silenced by transfecting chemically synthesized small interfering RNA. Silencing efficiency and Ang II-induced ERK 1/2 phosphorylation were evaluated by western blot and Ang II-induced Cai2+ using Fura-2 AM.


RESULTS
RGS-2 expression in not silenced BS/GS fibroblasts from patients is increased compared with healthy controls [0.34 +/- 0.02 vs. 0.19 +/- 0.01 densitometric units (d.u.), P = 0.0005]. Silencing RGS-2 in BS/GS patients was achieved to the level of controls. Ang II-induced Cai2+ release and ERK 1/2 phosphorylation were reduced in not silenced cells from BG/GS patients compared with controls (112.16 +/- 13.2 vs. 130.33 +/- 13.64 mmol/l, P = 0.011 and 0.64 +/- 0.08 vs. 0.91 +/- 0.03 mmol/l, P < 0.006, respectively). Silencing RGS-2 in BS/GS patients increased Ang II-induced Cai2+ release and ERK 1/2 phosphorylation in silenced cells compared with not silenced cells [59.3 +/- 10.8 (peak-basal) vs. 40.5 +/- 14.1 nmol/l, P = 0.017 and 0.84 +/- 0.06 vs. 0.64 +/- 0.08 nmol/l, P < 0.03, respectively], whereas they were not different compared with controls (60.1 +/- 4.3 and 0.91 +/- 0.03 nmol/l). Integrating the Cai2+ response over time showed increased Cai2+ area under the curve (AUC) of BS/GS silenced cells compared with that of not silenced cells (P = 0.013).


CONCLUSIONS
This is the first report of silencing RGS-2 effect on Ang II signaling in a human clinical condition of altered vascular tone regulation and remodeling and establishes RGS-2 as a key regulatory element of Ang II signaling in humans.
Calò
Lorenzo A
LA
Department of Clinical and Experimental Medicine, Clinica Medica 4, University of Padova, Padova, Italy. renzcalo@unipd.it
Pagnin
Elisa
E
Ceolotto
Giulio
G
Davis
Paul A
PA
Schiavo
Silvia
S
Papparella
Italia
I
Semplicini
Andrea
A
Pessina
Achille C
AC
D000328:Adult; D000804:Angiotensin II / Q000502:physiology*; D001477:Bartter Syndrome / Q000503:physiopathology*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D015331:Cohort Studies; D005260:Female; D005347:Fibroblasts / Q000502:physiology; D020868:Gene Silencing; D053579:Gitelman Syndrome / Q000503:physiopathology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D010766:Phosphorylation; D020710:RGS Proteins / Q000378:metabolism*; D015398:Signal Transduction
D020710:RGS Proteins; C087182:RGS2 protein, human; D000804:Angiotensin II; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D002118:Calcium