A fibroblast mutant cell line devoid of Na/H exchange was used to stably express cDNAs encoding the NHE1, NHE2, and NHE3 Na/H antiporters. Pharmacological studies using amilor-ide and two of its 5-N-substituted derivatives, 5-N-dimethyl ami-bride and 5-N-(methyl-propyl) amiloride(MPA), demonstrate that the NHE1 isoform is the ubiquitously expressed amiloride-sen-sitive Na/H antiporter(K, of 0.08 zM for MPA), whereas the NHE2 and NHE3 isoforms exhibit a lower affinity for these inhibitors (K of 0.5 tM and 1 0 M, respectively, for MPA) and are therefore likely to be members of the epithelial Na/W exchanger’s family.

In addition, we have used this system to test a new Na/W exchanger inhibitor possessing anti-ischemic properties on myocardial cells [(3-methylsulphonyl-4-piperidinobenzoyl) guanidine methanesulphonate]. This compound inhibits competitively NHE1 (K, of 0.1 6 zM) with a much greater affinity than NHE2 and NHE3 (K, of 5 pM and 650 pM, respectively) and therefore appears to be much more discriminative between these two classes of antiporter isoforms than the amilonde-related molecules. These results suggest an explanation for the observed difference of physiological effects between amiloride and H0E694, and dentify this new inhibitor as a useful tool for studies of Na/W exchange.