Role of Na+/H+ exchanger during O2 deprivation in mouse CA1 neurons

To determine the role of membrane transporters in intracellular pH (pH(i)) regulation under conditions of low microenvironmental O-2, we monitored pHi in isolated single CAI neurons using the fluorescent indicator carboxyseminaphthorhodafluor-1 and confocal microscopy. After total O-2 deprivation or anoxia (PO2 congruent to 0 Torr), a large increase in pHi was seen in CAI neurons in HEPES buffer, but a drop in pH(i), albeit small, was observed in the presence of HCO3-. Ionic substitution and pharmacological experiments showed that the large anoxia-induced pHi increase in HEPES buffer was totally Na+ dependent and was blocked by HOE-694, strongly suggesting the activation of the Na+/H+ exchanger (NHE). Also, this pH(i) increase in HEPES buffer was significantly smaller in Na+/H+ exchanger isoform 1 (NHE1) null mutant CAI neurons than in wild-type neurons, demonstrating that NHE1 is responsible for part of the pH(i) increase following anoxia. Both chelerythrine and H-89 partly blocked, and H-7 totally eliminated, this anoxia-induced pH(i) increase in the absence of HCO3-. We conclude that 1) O-2 deprivation activates Na+/H+ exchange by enhancing protein kinase activity and 2) membrane proteins, such as NHE, actively participate in regulating pHi during low-O-2 states in neurons.