Effects of the Na⁺/H⁺-exchange inhibitor Hoe 642 on intracellular pH, calcium and sodium in isolated rat ventricular myocytes

Abstract

 The inhibitors of the Na⁺/H⁺-exchange (NHE1) system Hoe 694 and Hoe 642 possess cardioprotective effects in ischaemia/reperfusion. It is assumed that these effects are due to the prevention of intracellular sodium (Na_i) and calcium (Ca_i) overload. The purpose of the present study was to investigate the effects of Hoe 642 on intracellular pH, Na⁺ and Ca²⁺ (pH_i, Na_i and Ca_i) in isolated rat ventricular myocytes under anoxic conditions or in cells in which oxidative phosphorylation had been inhibited by 1.5 mmol/l cyanide. In cells which were dually loaded with the fluorescent dyes 2,7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and Fura-2, anoxia caused acidification of the cells (from pH_i 7.2 to pH_i 6.8) and an increase in Ca_i from about 50 nmol/l to about 1 μmol/l. The decrease in pH_i began before the cells underwent hypoxic (rigor) contracture, whereas Ca_i only began to rise after rigor shortening had taken place. After reoxygenation, pH_i returned to its control value and Ca_i oscillated and then declined to resting levels. It was during this phase that the cells rounded up (hypercontracture). When 10 μmol/l Hoe 642 was present from the beginning of the experiment, pH_i and Ca_i were not significantly different from control experiments. At reoxygenation, pH_i did not recover, but Ca_i oscillated and returned to its resting level. To monitor Na_i, the cells were loaded with the dye SBFI. After adding 1.5 mmol/l cyanide or 100 μmol/l ouabain, Na_i increased from the initial 8 mmol/l to approximately 16 mmol/l. Hoe 642 or Hoe 694 (10 μmol/l) did not prevent the increase in Na_i. In contrast, the blocker of the persistent Na⁺ current R56865 (10 μmol/l) attenuated the CN^–-induced rise in Na_i. The substance ethylisopropylamiloride was not used because it augmented considerably the intensity of the 380 nm wavelength of the cell’s autofluorescence. In conclusion, the specific NHE1 inhibitor Hoe 642 did not attenuate anoxia-induced Ca_i overload, nor CN^–-induced Na_i and Ca_i overload. Hoe 642 prevented the recovery of pH_i from anoxic acidification. This low pH_i maintained after reoxygenation may be cardioprotective. Other possible mechanisms of NHE1 inhibitors, such as prevention of Ca²⁺ overload in mitochondria, cannot be ruled out. The increase in Na_i during anoxia is possibly due to an influx of Na⁺ via persistent Na⁺ channels.