Characterization of Na+/H+ exchange activity in cultured rat hippocampal astrocytes.
Astrocytes actively maintain their intracellular pH (pHi) 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 pHi recovery after an acid load with an IC50 of approximately 3.18 microM, 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 pHi, suggesting the presence of an additional acid/base transport mechanism that is EIPA-sensitive.
Pizzonia
J H
JH
Department of Surgery (Neurosurgery), Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Ransom
B R
BR
Pappas
C A
CA
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001639:Bicarbonates / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D015870:Gene Expression; D006624:Hippocampus / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D051381:Rats; D017207:Rats, Sprague-Dawley; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*
D001639:Bicarbonates; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1; D000584:Amiloride; C039614:ethylisopropylamiloride