New drugs for the Na+/H+ exchanger. Influence of Na+ concentration and determination of inhibition constants with a microphysiometer

The NHE-1 isoform of the Na+/H+ exchanger is excessively activated in cardi ac cells during ischemia. Hence NHE-1 specific inhibitors are being develop ed since they could be of beneficial influence under conditions of cardiac ischemia and reperfusion. In this study, the Cytosensor(TM) microphysiomete r was used to measure the potency of four new drug molecules, i.e., EMD 840 21, EMD 94309, EMD 96785 and HOE 642 which are inhibitors of the isoform 1 of the Na+/H+ exchanger. The experiments were performed with Chinese hamste r ovary cells (CHO K1) which are enriched in the NHE-1 isoform of the Na+/H + antiporter. The Na+/H+ exchanger was stimulated with NaCl and the rate of extracellular acidification was quantified with the Cytosensor. The proton exchange rate was measured as a function of the NaCl concentration in the range of 10-138 mM NaCl stimulation. The proton exchange rate followed Mich aelis-Menten kinetics with a K-M = 30 +/- 4 mM for Na+. Addition of either one of the four inhibitors decreased the acidification rate. The IC50 value s of the four compounds could be determined as 23 +/- 7 nM for EMD 84021, 5 +/- 1 nM for EMD 94309, 9 +/- 2 nM for EMD 96785 and 8 +/- 2 nM for HOE 64 2 at 138 mM NaCl, in good agreement with more elaborate biological assays. The IC50 values increased with the NaCl concentration indicating competitiv e binding of the inhibitor. The microphysiometer approach is a fast and sim ple method to measure the activity of the Na+/H+ antiporter and allows a qu antitative kinetic analysis of the proton excretion rate.