The pH of spontaneously beating cultured rat heart cells is regulated by an ATP-calmodulin-dependent Na+/H+ antiport

Abstract

We investigated the mechanisms by which spontaneously beating cultured rat ventricular cells regulate intracellular pH (pH(i)). Specifically, the relative contributions of the Na+/H+ antiport, Cl-/HCO3- exchange, ATP, and calmodulin-dependent processes in regulating the pH(i) of cells loaded with the intracellular fluorescent pH indicator BCECF were investigated. The pH(i) of ventricular cells bathed in HEPES-buffered medium averaged 7.30 ± 0.02. Subsequent exposure of the cells to CO2-HCO3--buffered medium resulted in intracellular acidification followed by recovery to pH(i) levels approximately 0.1 pH units lower than in controls. Recovery was inhibited by the Na+/H+ antiport inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA). The recovery from intracellular acidification, induced by a 15-mM ammonium chloride prepulse, was also dependent solely upon activation of the Na+/H+ antiport. Recovery was dependent upon extracellular sodium, was completely inhibited by EIPA, and could be modulated by changes in extracellular pH (pH(o)). Al low pH(o) values (6.3) the recovery of pH(i) was greatly attenuated, while at high pH(o) (8.0) the recovery process was accelerated. The final pH(i) to which the cells recovered was also dependent upon pH(o). Preincubation of the cells with 2-deoxy-D-glucose to deplete cellular ATP levels reduced pH(i) by approximately 0.2 pH units and greatly impaired the cells' ability to recover from 15-mM ammonium chloride-induced acid load. Similarly, preincubation of cells with the calmodulin inhibitors W-7 and trifluoperazine also impaired their ability to recover from the acid load. The Cl--HCO3- exchange played no role in the cells' ability to recover from intracellular acidosis. However, the presence of HCO3- significantly increased the resistance of myocardial cells to changes in pH(i) by approximately doubling their buffer capacity. These results demonstrated that a Na+/H+ antiport is the major pH(i)-regulating system in spontaneously beating rat ventricular cells. The ability of the Na+/H+ antiport to regulate myocardial pH(i) is dependent upon the cells' ability to maintain adequate levels of ATP. The antiport's dependency on ATP, in conjunction with its dependency on calmodulin, suggests that activation of the antiport in ventricular cells involves phosphorylation processes.