Nicotine-induced adrenal beta-arrestin1 upregulation mediates tobacco-related hyperaldosteronism leading to cardiac dysfunction

Figure 3 Effect of βarrestin1 knockdown on tobacco-induced aldosterone production in adrenocortical zona glomerulosa cells in vitro. A: Immunoblotting for βarrestin1 in H295R cell extracts 48 h post-transfection with βarrestin1-sepcific (Arrb1) siRNA or scrambled (Scr) siRNA to test the efficiency of the βarrestin1 siRNA-mediated knockdown. A representative blot of three independent cell extracts run in duplicate is shown, along with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as loading control, confirming an > 80% βarrestin1 protein knockdown; B: Aldosterone secretion into the culture medium from H295R cells in response to a 6-hour-long 100 nmol/L AngII challenge, as measured 24 h post-treatment with 10 µmol/L nicotine or 10µmol/L cotinine or vehicle (DMSO) in H295R cells having Arrb1 siRNA or not (scrambled siRNA-transfected, Scr). Drug treatments were carried out 48 h after siRNA transfection (i.e., AngII was added at 72 h post-transfection). Data are expressed as % of the AngII response in DMSO-treated cells. ^aP < 0.05, vs DMSO (Arrb1 siRNA or Scr); ^bP < 0.05, vs Scr; n = 5 independent measurements per treatment performed in duplicate; C: StAR mRNA levels in these cells; D-E: Protein levels in these cells. For StAR immunoblotting, representative blots are shown in (D), along with GAPDH as loading control, and the densitometric quantitation of three independent cell extracts per treatment condition run in duplicate (and normalized to GAPDH) is shown in (E). ^aP < 0.05, vs DMSO (vehicle); ^bP < 0.05, vs Scr; n = 3 independent experiments/treatment in duplicate. DMSO: Vehicle; Scr: Scrambled; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; Arrb1: Arrestin1-sepcific.