Characterization of Na⁺/H⁺ Exchange Activity in Cultured Rat Hippocampal Astrocytes

Astrocytes actively maintain their intracellular pH (pH_i) more alkaline than expected by passive distribution of H⁺. Acid extruding transporters such as the amiloride-sensitive Na⁺/H⁺ exchanger (NHE) are necessary for pH regulation. Currently, four mammalian NHEs (NHE1-NHE4) have been cloned, with a fifth (NHE5) partially cloned. We attempted to determine which isoform(s) of NHE was present in cultured hippocampal astrocytes using amiloride sensitivity and immunospecificity as criteria. In the absence of HCO₃^-, amiloride blocked pH(i) recovery after an acid load with an IC₅₀ of ~3.18 μM, similar to values reported for the amiloride-sensitive isoforms NHE1 and NHE2. Immunoblotting with a highly specific antibody for NHE1 identified a 100 kDa protein, indicating the presence of NHE1 in whole brain, hippocampus, and cultured hippocampal astrocytes. Further probing for an additional amiloride-sensitive NHE failed to detect evidence of the presence of NHE4. Surprisingly, application of the potent analog of amiloride, ethylisopropylamiloride (EIPA), caused a reversible alkalinization of PH(i), suggesting the presence of an additional acid/base transport mechanism that is EIPA-sensitive.