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. 2021 Jan 27;7:617038. doi: 10.3389/fcvm.2020.617038

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Copyright © 2021 Kandilci, Richards, Fournier, Şimşek, Chung, Lakhal-Littleton and Swietach.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Cardiac NHE1 activity is rapidly inhibited by acute anoxia. (A) Measurement of oxygen tension in the superfusion chamber, showing anoxic conditions are attainable by solutions bubbled with N₂ and supplemented with the oxygen-scavenger dithionite (1 mM). (B) Exemplar trace of paired ammonium prepulses, first performed in normoxia and then in anoxia, in a rat ventricular myocyte loaded with cSNARF1. Recovery of pH_i from the acid load is greatly attenuated under anoxic conditions, but becomes re-activated upon re-oxygenation. (C) Exemplar time course showing lack of background acid-loading at low pH_i under anoxic conditions, revealed in the presence of cariporide (30 μM) to block NHE1. (D) NHE1-generated H⁺-flux measured by cSNARF1 or (E) BCECF in normoxia (cSNARF1: n = 22 cells from 5 rats; BCECF: n = 9 cells from 4 rats) or 9 min anoxia (cSNARF: n = 9 cells from 2 rats; BCECF: n = 7 cells from 2 rats). In the case of BCECF experiments, the effect of anoxia was also tested after 2 min exposure (n = 5 cells from 3 rats), 5 min exposure to anoxia (n = 5 cells from 3 rats). Two-way ANOVA: effect of 9 min anoxia vs. control was significant (P < 0.0001 for cSNARF1 and P < 0.01 for BCECF datasets); effect of 5 min anoxia vs. control was significant (P < 0.0001); effect of 2 min anoxia was not significant (P = 0.93). Interaction between pH and treatment was not significant in any of the cases; the effect of pH_i was significant (P < 0.0001 for all datasets). Insets plot flux in anoxia (9 min) vs. normoxia for matching pH_i; the parallel shift is indicative of a change in affinity. (F) Effect of 15 min 3 Torr hypoxia (~1% O₂) attained by including 0.1 mM dithionite in N₂-bubbled solutions. Data recorded with cSNARF1. NHE1 activity was inhibited by hypoxia (n = 26 cells, 3 rats) relative to controls (n = 55, 3 rats). Two-way ANOVA: significant effect of hypoxia (P < 0.001); of pH_i (P < 0.0001); and interaction (P < 0.0001). Inset plots flux in anoxia vs. normoxia for matching pHi. The observed parallel shift is indicative of a change in affinity.