Ammonia promotes endothelial cell survival via the heme oxygenase-1-mediated release of carbon monoxide

Abstract

Although endothelial cells produce substantial quantities of ammonia during cell metabolism, the physiologic role of this gas in these cells is not known. In this study, we investigated if ammonia regulates the expression of heme oxygenase-1 (HO-1), and if this enzyme influences the biological actions of ammonia on endothelial cells. Exogenously administered ammonia, given as ammonium chloride or ammonium hydroxide, or endogenously generated ammonia stimulated HO-1 protein expression in cultured human and murine endothelial cells. Dietary supplementation of ammonia also induced HO-1 protein expression in murine arteries. The increase in HO-1 protein by ammonia in endothelial cells was first detected 4h after ammonia exposure and was associated with the induction of HO-1 mRNA, enhanced production of reactive oxygen species (ROS), and increased expression and activity of NF-E2-related factor-2 (Nrf2). Ammonia also activated the HO-1 promoter and this was blocked by mutating the antioxidant responsive element or by overexpressing dominant-negative Nrf2. The induction of HO-1 expression by ammonia was dependent on ROS formation and prevented by N-acetylcysteine or rotenone. Finally, prior treatment of endothelial cells with ammonia inhibited tumor necrosis factor-α-stimulated cell death. However, silencing HO-1 expression abrogated the protective action of ammonia and this was reversed by the administration of carbon monoxide but not bilirubin or iron. In conclusion, this study demonstrates that ammonia stimulates the expression of HO-1 in endothelial cells via the ROS-Nrf2 pathway, and that the induction of HO-1 contributes to the cytoprotective action of ammonia by generating carbon monoxide. Moreover, it identifies ammonia as a potentially important signaling gas in the vasculature that promotes endothelial cell survival.

Keywords: Ammonia; Carbon monoxide; Cytoprotection; Endothelial cells; Heme oxygenase-1.

Figure 1

Ammonia increases HO-1 expression in HUVECs. (A) NH₄Cl (5.0mM) stimulates a time-dependent increase in HO-1 protein. (B) NH₄Cl exposure for 24 hours stimulates a concentration-dependent increase in HO-1 protein. (C) NH₄OH exposure for 24 hours stimulates a concentration-dependent increase in HO-1 protein. (D) NaOH exposure for 24 hours has no effect on HO-1 protein expression. (E) NH₄Cl (5.0mM) or NH₄OH (5.0mM) exposure for 8 hours stimulates HO-1 mRNA. (F) NH₄Cl (5mM) or NH₄OH (5mM) exposure for 24 hours elevates HO activity. Protein expression was quantified by scanning laser densitometry, normalized with respect to β-actin, and expressed relative to that of control, untreated cells (open bars). Results are means ± SEM (n=3–5). *Statistically significant effect of NH₄Cl or NH₄OH.

Figure 2

Endogenous ammonia (NH₃) production regulates HO-1 expression in HUVECs. (A) Glutamine (GTN;500µM) stimulates NH₃ formation in GTN-deprived endothelial cells. The NH₃ scavenger methyl pyruvate (MP;10mM) blocks basal and GTN-stimulated NH₃ synthesis. (B) GTN (500µM) stimulates HO-1 protein in an NH₃-dependent manner. MP (10mM) inhibits basal and GTN-stimulated expression of HO-1 protein. Results are means±SEM (n=3) *Statistically significant effect of GTN.

Figure 3

Ammonia stimulates HO-1 protein in MAECs, MASMCs, and murine arteries. (A) NH₄Cl exposure for 24 hours stimulates HO-1 protein expression in MAEC. (B) NH₄Cl exposure for 24 hours stimulates HO-1 protein expression in MASMCs. (C and D) Administration of NH₄Cl (0.28M) in the drinking water of mice for 7 days increases plasma ammonia concentration and arterial HO-1 protein expression. Results are means± SEM (n=3).*Statistically significant effect of NH₄Cl.

Figure 4

Ammonia stimulates HO-1 promoter activity via the Nrf2/ARE complex in HUVECs. (A) Ammonia-mediated HO-1 gene expression requires de novo RNA synthesis. Effect of actinomycin D (ActD; 0.10µg/ml) on NH₄Cl (5.0mM for 8 hour)-mediated increase in HO-1 protein. (B) Ammonia stimulates HO-1 promoter activity. Cells were transfected with a HO-1 promoter construct (E1) or a mutated HO-1 promoter construct (M739) and a Renilla luciferase construct, treated with NH₄Cl (5.0mM for 6 hours), and then analyzed for luciferase activity. In some instances, a dominant-negative Nrf2 (dnNrf2) construct was co-transfected into the cells. (C) Time-course of Nrf2 protein expression in native non-transfected cells after administration of NH₄Cl (5.0mM). (D) Time-course of Nrf2 mRNA expression native non-transfected after administration of NH₄Cl (5.0mM). (E) Ammonia stimulates Nrf2 activity. Native non-transfected cells were treated with NH₄Cl (5.0mM) for 6 hours and nuclear extracts analyzed for Nrf2 binding by ELISA. O.D., optical density. (F) Effect of ammonia on GSK3β activity. Native non-transfected cells were treated with NH₄Cl (5.0mM) for various times (0–24 hours) or with the phosphatidylinositol-3-kinase inhibitor LY2942002 (LY;10µM) for 4 hours. Protein and mRNA expression was quantified by scanning laser densitometry and normalized with respect to β-actin or GSK3β and 18 S rRNA, respectively, and expressed relative to that of control, untreated cells (open bars). Results are means ± SEM (n=3–5). *Statistically significant effect of NH₄Cl or LY.

Figure 5

Ammonia stimulates oxidative stress in HUVECs. (A) NH₄Cl exposure for 8 hours increases intracellular ROS production in a concentration-dependent manner. (B) NH₄Cl (5.0mM) exposure increases ROS production in a time-dependent fashion. (C) NH₄Cl exposure for 24 hours had no effect on endothelial cell viability. Results are means ± SEM (n=6). *Statistically significant effect of NH₄Cl.

Figure 6

Ammonia-induced HO-1 expression is dependent on oxidative stress in HUVECs. (A) Effect of N-acetyl-L-cysteine (NAC; 10mM) or rotenone (Rot;5µM) on NH₄Cl (5.0mM for 8 hours)-mediated intracellular ROS production. (B) Effect of NAC (10mM) on NH₄Cl (5.0mM for 24 hours)-mediated HO-1 protein expression. (C) Effect of NAC (10mM) on NH₄Cl (5.0mM for 6 hours)-mediated Nrf2 activation. O.D., optical density. (D) Effect of Rot (5µM) on NH₄Cl (5.0mM for 24 hours)-mediated HO-1 protein expression. (E) Effect of apocynin (Apo;300µM) on NH₄Cl (5.0mM for 24 hours)-mediated HO-1 protein expression. (F) Effect of allopurinol (Allo;100µM) on NH₄Cl (5.0mM for 24 hours)-mediated HO-1 protein expression. HO-1 protein was quantified by scanning laser densitometry and normalized with respect to β-actin, and expressed relative to that of control, untreated cells (open bars). Results are means ± SEM (n=3–6). *Statistically significant effect of NH₄Cl.

Figure 7

Ammonia inhibits inflammatory stress-induced cell death in HUVECs via the HO-1-mediated production of CO. (A). Ammonia promotes endothelial cell survival via the induction of HO-1 and release of CO. HUVEC were transfected with HO-1 siRNA (0.1 µM) or NT siRNA (0.1µM), treated with NH₄Cl (5.0mM) for 24 hours, and then exposed to TNFα (10ng/ml) and cycloheximide (CX;2µg/ml) for 8 hours in the presence or absence of CO (200ppm), bilirubin (10µM), or FeSO₄ (10µM). (B) HO-1 protein expression in cells transfected with HO-1 siRNA (0.1µM) or non-targeting (NT) siRNA (0.1µM) in the presence and absence of NH₄Cl (5.0mM) for 24 hours. (C) CO inhibits cytokine-mediated apoptosis. HUVEC were treated with TNFα (10ng/ml) and CX (2µg/ml) for 8 hours in the presence or absence of CO (200ppm), bilirubin (BR;10µM), or FeSO₄ (10µM). Results are means ± SEM (n=3–6). *Statistically significant effect of TNFα and CX. ⁺Statistically significant effect of NH₄Cl or CO.