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

Figure 1. Effect of tobacco compounds on βarrestin1 levels in AZG cells. A: βarrestin1 mRNA levels in H295R cells treated for 24 h with 10 μmol/L nicotine or 10 μmol/L cotinine or vehicle. B-C: Protein levels in H295R cells treated for 24 h with 10 μmol/L nicotine or 10 μmol/L cotinine or vehicle. Representative western blots are shown in (B), along with glyceraldehyde 3-phosphate dehydrogenase as loading control, and the densitometric quantitation of three independent cell extracts per condition run in duplicate (and normalized with glyceraldehyde 3-phosphate dehydrogenase levels) is shown in (C). aP < 0.05 vs vehicle, n = 3 independent experiments/treatment in duplicate. DMSO: Vehicle; Cot: Cotinine; Nic: Nicotine; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Figure 2. Effect of tobacco compounds on AngII-dependent aldosterone synthesis and secretion in adrenocortical zona glomerulosa cells. A: Aldosterone secretion into the culture medium from H295R cells in response to a 6-hour-long 100 mmol/L AngII challenge, as measured 24 h post-treatment with 10 μmol/L nicotine or 10 μmol/L cotinine or vehicle (DMSO). Data are expressed as % of the AngII response in DMSO-treated cells. aP < 0.05, vs DMSO; n = 5 independent measurements per treatment performed in duplicate; B: StAR mRNA levels in H295R cells treated for 24 h with 10 μmol/L nicotine (Nic) or 10 μmol/L cotinine (Cot) or vehicle (DMSO). C-D: Protein levels in H295R cells treated for 24 h with 10 mol/L nicotine (Nic) or 10 μmol/L cotinine or DMSO. Representative western blots are shown in (C), along with glyceraldehyde 3-phosphate dehydrogenase as loading control, and the densitometric quantitation of three independent cell extracts per condition run in duplicate (and normalized to glyceraldehyde 3-phosphate dehydrogenase) is shown in (D). aP < 0.05, vs DMSO; n = 3 independent experiments/treatment in duplicate. DMSO: Vehicle; Cot: Cotinine; Nic: Nicotine; GAPDH: Glyceraldehyde 3-phosphate…

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:…

Figure 4. Effect of adrenal βarrestin1 knockdown on nicotine-dependent hyperaldosteronism and cardiac dysfunction in vivo. A: Circulating aldosterone levels in rats injected i.p. with 1 mg/kg per day nicotine or saline (control) for 7 consecutive d. aP < 0.05; n = 5 rats/group; B: Ejection fraction [EF (%)] of these animals at the end of the saline or nicotine treatments. aP < 0.05; n = 5 rats/group; C: Immunoblotting for βarrestin1 in adrenal protein extracts isolated from rats at 7 d post-injection with βarrestin1-specific siRNA or scrambled (Scr) siRNA directly into their adrenal glands. A representative blot of three independent rat adrenal protein extracts is shown, along with glyceraldehyde 3-phosphate dehydrogenase as loading control, confirming an > 90% βarrestin1 protein knockdown in the adrenal glands in vivo; D: Circulating aldosterone levels of rats having adrenal βarrestin1 knocked-down (rrestin1-specific siRNA) or not (scrambled siRNA-injected, Scr) and treated with 1 mg/ kg per day i.p. nicotine or saline (control) for 7 consecutive d, at the end of these treatments. E: EF (%) of rats having adrenal βarrestin1 knocked-down (rrestin1-specific siRNA) or not (scrambled…