Hypoxic sensitivity of the sodium-hydrogen exchanger in isolated mammalian ventricular myocytes. The role of mitochondria

Hilmi Burak Kandilci1,2, Chae Hun Leem3, Pawel Swietach1, Richard D. Vaughan-Jones1. 1Department of Physiology, Anatomy and Genetics, Oxford University, Oxford, United Kingdom. 2Department of Biophysics, Faculty of Medicine, Ankara University, Ankara, Turkey, 3College of Medicine, University of Ulsan, Seoul, Korea, Republic of.

Intracellular pH (pHi) is a major regulator of cell physiology. Sodium/hydrogen exchange (NHE) is one of the principal acid-extruders, implicated in pHi regulation. NHE activity was measured in isolated rat ventricular myocytes, loaded with the pH-reporter dye, BCECF. To stimulate NHE, cells were acid-loaded by means of an ammonium prepulse (20mM NH4Cl, 4-5 min) under nominally CO2/HCO-3-free conditions. Acute anoxia (5-9 min, 1mM Na-dithionite, 100% N2) induced background acid loading (0.5 mM/min, n = 3-6) and slowed the pHi recovery rate by up to 75% (n = 7). Reoxygenation immediately (∼15 sec, n = 5) reversed anoxic inhibition. Anoxia did not alter intrinsic buffering capacity (n = 6) or Cl--dependent intracellular acid-loading (n = 6). Real hypoxia (%100 N2, ∼1 mmHg PO2) also reduced pHi recovery rate by 53% (n = 4). Anoxia-induced slowing of NHE was mimicked by oxidative inhibitors (myxothiazol, rotenone (1 M); 53% and 50% inhibition respectively, n = 3-7) and also by the glycolytic and oxidative phosphorylation inhibitor iodoacetate by 75% (n = 4). Anoxia slowly depolarized mitochondrial membrane potential, relative to mitochondrial uncouplers (FCCP, 5 M) (∼15 fold difference, n = 3-4). In conclusion, both dithionite-induced anoxia and real hypoxia attenuated NHE activity rapidly and reversibly. This observation is important while understanding the molecular mechanisms that take place during myocardial ischemia. Moreover, there seems to be a correlation between mitochondrial activity and hypoxia/anoxia-response of NHE.