Korean J Physiol Pharmacol.  2021 Mar;25(2):159-166. 10.4196/kjpp.2021.25.2.159.

NOX4/Src regulates ANP secretion through activating ERK1/2 and Akt/GATA4 signaling in beating rat hypoxic atria

• Wu Cz
• Li X
• Hong L
• Han Zn
• Liu Y
• Wei Cx
• Cui X

• KMID: 2512959
• DOI: http://doi.org/10.4196/kjpp.2021.25.2.159

Abstract

Nicotinamide adenine dinucleotide phosphate oxidases (NOXs) are the major enzymatic source of reactive oxygen species (ROS). NOX2 and NOX4 are expressed in the heart but its role in hypoxia-induced atrial natriuretic peptide (ANP) secretion is unclear. This study investigated the effect of NOX on ANP secretion induced by hypoxia in isolated beating rat atria. The results showed that hypoxia significantly upregulated NOX4 but not NOX2 expression, which was completely abolished by endothelin-1 (ET-1) type A and B receptor antagonists BQ123 (0.3 µM) and BQ788 (0.3 µM). ET-1-upregulated NOX4 expression was also blocked by antagonists of secreted phospholipase A2 (sPLA2; varespladib, 5.0 µM) and cytosolic PLA2 (cPLA2; CAY10650, 120.0 nM), and ET-1-induced cPLA2 expression was inhibited by varespladib under normoxia. Moreover, hypoxia-increased ANP secretion was evidently attenuated by the NOX4 antagonist GLX351322 (35.0 µM) and inhibitor of ROS N-Acetyl-D-cysteine (NAC, 15.0 mM), and hypoxia-increased production of ROS was blocked by GLX351322. In addition, hypoxia markedly upregulated Src expression, which was blocked by ET receptors, NOX4, and ROS antagonists. ET-1-increased Src expression was also inhibited by NAC under normoxia. Furthermore, hypoxiaactivated extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (Akt) were completely abolished by Src inhibitor 1 (1.0 µM), and hypoxia-increased GATA4 was inhibited by the ERK1/2 and Akt antagonists PD98059 (10.0 µM) and LY294002 (10.0 µM), respectively. However, hypoxia-induced ANP secretion was substantially inhibited by Src inhibitor. These results indicate that NOX4/Src modulated by ET-1 regulates ANP secretion by activating ERK1/2 and Akt/GATA4 signaling in isolated beating rat hypoxic atria.

Keyword

Atrial natriuretic peptide; Endothelin-1; Hypoxia; NADPH oxidase; Src tyrosine kinase

Figure

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  Fig. 1 Effect of hypoxia on ANP secretion (A) and dynamics (B) in isolated beating rat atria. Data were expressed as mean ± standard error of the mean (n = 6). Cont, control; Hy, hypoxia. *p < 0.05 vs. control.

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  Fig. 2 Effect of hypoxia on NOX2 and NOX4 expression and its role in the regulation of ANP secretion in isolated beating rat atria. Data were expressed as the mean ± standard error of the mean (A and B, n = 5; C and D, n = 6). Cont, control; Hy, hypoxia; 123, BQ123, an antagonist of ETRA; 788, BQ788, an antagonist of ETRB; GLX, 351322, an antagonist of NOX4. *p < 0.05 vs. control; #p < 0.05 vs. hypoxia.

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  Fig. 3 Effects of ETR and PLA2 antagonists on NOX4 (A, B & D) and pcPLA2 (C) expression in beating rat atria under normoxic conditions. Data were expressed as the mean ± standard error of the mean (n = 5). Cont, control; ET, ET-1; Var, varespladib, an antagonist of sPLA2; CAY, CAY10650, an antagonist of cPLA2. *p < 0.05 vs. control; &p < 0.05 vs. ET-1.

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  Fig. 4 Effect of hypoxia on H2O2 production and its role in ANP secretion in beating rat atria. Data were expressed as the mean ± standard error of the mean (A, n = 5; B, n = 6). Cont, control; Hy, hypoxia; GLX, GLX351322, an antagonist of NOX4; NAC, N-Acetyl-D-cysteine, an antagonist of ROS. *p < 0.05 vs. control; #p < 0.05 vs. hypoxia.

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  Fig. 5 Effects of ET-1, NOX4 and ROS on Src expression in beating rat atria under hypoxic (A–C) and normoxic (D) conditions. Data were expressed as the mean ± standard error of the mean (n = 5). Cont, control; Hy, hypoxia; GLX, GLX351322; NAC, N-Acetyl-D-cysteine; ET, ET-1. *p < 0.05 vs. control; #p < 0.05 vs. hypoxia; &p < 0.05 vs. ET-1.

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  Fig. 6 Effect of Src on ERK1/2 and Akt expression (A, B) and its role in the regulation of GATA4 (C) and ANP secretion (D) in hypoxic beating rat atria. Data were expressed as the mean ± standard error of the mean (A–C, n = 5; D, n = 6). Cont, control; Hy, hypoxia; SrcI, Src inhibitor 1; PD, PD98059, an antagonist of ERK1/2; LY, LY294002, an antagonist of Akt. *p < 0.05 vs. control; #p < 0.05 vs. hypoxia.

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