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 (pHi). Specifically, the relative contributions of the Na+/H+ antiport, Cl-/HCO3- exchange, ATP, and calmodulin-dependent processes in regulating the pHi of cells loaded with the intracellular fluorescent pH indicator BCECF were investigated. The pHi 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 pHi 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 (pHo). At low pHo values (6.3) the recovery of pHi was greatly attenuated, while at high pHo (8.0) the recovery process was accelerated. The final pHi to which the cells recovered was also dependent upon pHo. Preincubation of the cells with 2-deoxy-D-glucose to deplete cellular ATP levels reduced pHi 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 pHi by approximately doubling their buffer capacity. These results demonstrated that a Na+/H+ antiport is the major pHi-regulating system in spontaneously beating rat ventricular cells. The ability of the Na+/H+ antiport to regulate myocardial pHi 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.

The effect of a calmodulin inhibitor on intracellular [Ca2+] and contraction in isolated rat ventricular myocytes.

“…The possibility that \V7 wvas decreasing myofilament Ca2+ sensitivity by producing an intracellular acidosis (e.g. see Orchard & Kentish, 1990) by inhibiting the Na+-H+ exchange mechanismn (Weissberg et al 1989) seems unlikely because our results with BC(ECF-loaded myocytes suggest that neither the magnitude nor time course of the change of p14j in W7 were appropriate to explain the observed changes in the size of the twitch.…”

Hydrogen Peroxide Decreases pH i in Human Aortic Endothelial Cells by Inhibiting Na + /H + Exchange

“…27 Indeed, ATP depletion results in decreased activity of Na ϩ /H ϩ exchange by all 3 exchanger isoforms. 25,28 Although it has been suggested that ATP depletion affects basal Na ϩ /H ϩ exchanger activity by decreasing phosphorylation of the antiporter, 22,45,46 this concept has been challenged, since ATP depletion rapidly inhibits the Na ϩ /H ϩ exchanger in NHE-1-transfected fibroblasts without affecting NHE-1 phosphorylation. 27 The mechanism of ATP depletion may be important in determining the effect on exchanger activity.…”

Changes in intracellular pH associated with glutamate excitotoxicity

“…Operation of the Na+-H+ antiporter depends upon the Na gradient but metabolic energy may contribute to its pumping ability and modulation by [H+], (Cassel et al, 1986;Grinstein and Rothstein, 1986;Weissberg et al, 1989;Wakabayashi et al, 1992). With reduction of cellular ATP, the exchanger decreases its affinity for hydrogen at the internal regulatory site (Cassel et al, 1986;Wakabayashi et al, 1992).…”

“…Indirect evidence suggests that protein kinase C may stimulate the antiporter via a phosphorylationdependent regulatory site in neuroblastoma cells and lymphocytes (Moolenaar et al, 1984;Grinstein et al, 1985a). Regulatory phosphorylation sites have also been implicated by antiporter sensitivity to calmodulin antagonists in cardiac ventriculocytes (Weissberg et al, 1989). With the loss of highenergy phosphate sources that accompanies excitotoxic damage (Kass and Lipton, 1982;Rothman et al, 1987), this regulatory site may become dephosphorylated, resulting in a slower rate of hydrogen extrusion.…”