CHARACTERIZATION OF NA+ H+ EXCHANGE ACTIVITY IN CULTURED RAT HIPPOCAMPAL ASTROCYTES/

Astrocytes actively maintain their intracellular pH (pH(i)) more alkal ine than expected by passive distribution of H+, Acid extruding transp orters such as the amiloride-sensitive Na+/H+ exchanger (NHE) are nece ssary for pH regulation, Currently, four mammalian NHEs (NHE1-NHE4) ha ve 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 criter ia, In the absence of HCO3-, amiloride blocked pH(i) recovery after an acid load with an IC50 of similar to 3.18 mu M, similar to values rep orted for the amiloride-sensitive isoforms NHE1 and NHE2, Immunoblotti ng with a highly specific antibody for NHE1 identified a 100 kDa prote in, indicating the presence of NHE1 in whole brain, hippocampus, and c ultured hippocampal astrocytes, Further probing for an additional amil oride-sensitive NHE failed to detect evidence of the presence of NHE4. Surprisingly, application of the potent analog of amiloride, ethyliso propylamiloride (EIPA), caused a reversible alkalinization of pH(i), s uggesting the presence of an additional acid/base transport mechanism that is EIPA-sensitive. (C) 1996 Wiley-Liss, Inc.