Role of reactive oxygen species (ROS) in angiotensin II-induced stimulation of the cardiac Na+/HCO3− cotransport

The sarcolemmal Na+/HCO3− cotransporter (NBC) plays an important role in intracellular pH (pHi) regulation in the heart. In the present work we studied, in isolated cat ventricular myocytes, the role of Angiotensin II (Ang II) and reactive oxygen species (ROS) production as potential activators of the NBC. pHi was measured in single cells in a medium with HCO3− using the fluorescent pH indicator BCECF. The NH4+ pulse method was used to induce an intracellular acid load and the acid efflux (JH) in the presence of the Na+/H+ exchanger blocker HOE642 (10 μM) was calculated as indicator of NBC activity. The following JH data are presented at pHi of 6.8 (⁎ and # indicate p < 0.05 after ANOVA vs. control and Ang II, respectively). The basal JH (1.03 ± 0.12 mM/min, n = 11) was significantly increased in the presence of 100 nM Ang II (1.70 ± 0.15 mM/min, n = 8⁎). This effect of Ang II was abolished when we added to the extracellular solution 2 mM MPG (ROS scavenger; 0.80 ± 0.08 mM/min, n = 11#), 300 μM apocynin (NADPH oxidase blocker; 0.80 ± 0.13 mM/min, n = 6#), 500 μM 5-hydroxidecanoate (mitochondrial ATP dependent K+ channel, mKATP, blocker; 0.97 ± 0.21 mM/min, n = 9#), or the inhibitor of the MAP kinase ERK pathway U0126 (10 μM; 0.56 ± 0.18 mM/min, n = 6#). We also determined the phosphorylation of ERK during the first min of acidosis and we detected that Ang II significantly enhanced the ERK phosphorylation levels, an effect that was cancelled by scavenging ROS with MPG. In conclusion, we propose that Ang II enhances the production of ROS through the activation of the NADPH oxidase, which in turn triggers mKATP opening and mitochondrial ROS production (“ROS-induced ROS-release mechanism”). Finally, these mitochondrial ROS stimulate the ERK pathway, leading to the activation of the NBC.