PHARMACOLOGICAL CHARACTERIZATION OF STABLY TRANSFECTED NA+ H+ ANTIPORTER ISOFORMS USING AMILORIDE ANALOGS AND A NEW INHIBITOR EXHIBITING ANTIISCHEMIC PROPERTIES/

A fibroblast mutant cell line devoid of Na+/H+ exchange was used to st ably express cDNAs encoding the NHE1, NHE2, and NHE3 Na+/H+ antiporter s. Pharmacological studies using amiloride and two of its 5-N-substitu ted derivatives, 5-N-dimethyl amiloride and 5-N-(methyl-propyl)amilori de (MPA), demonstrate that the NHE1 isoform is the ubiquitously expres sed amiloride-sensitive Na+/H+ antiporter (K(i) of 0.08 muM for MPA), whereas the NHE2 and NHE3 isoforms exhibit a lower affinity for these inhibitors (K(i) of 0.5 muM and 10 muM, respectively, for MPA) and are therefore likely to be members of the epithelial Na+/H+ exchanger's f amily. In addition, we have used this system to test a new Na+/H+ exch anger inhibitor possessing anti-ischemic properties on myocardial cell s [(3-methylsulphonyl-4-piperidinobenzoyl) guanidine methanesulphonate ]. This compound inhibits competitively NHE1 (K(i) of 0.1 6 muM) with a much greater affinity than NHE2 and NHE3 (K(i) of 5 muM and 650 muM, respectively) and therefore appears to be much more discriminative be tween these two classes of antiporter isoforms than the amiloride-rela ted molecules. These results suggest an explanation for the observed d ifference of physiological effects between amiloride and HOE694, and i dentify this new inhibitor as a useful tool for studies of Na+/H+ exch ange.