New Drugs for the Na⁺/H⁺ Exchanger. Influence of Na⁺ Concentration and Determination of Inhibition Constants with a Microphysiometer

Abstract.

The NHE-1 isoform of the Na⁺/H⁺ exchanger is excessively activated in cardiac cells during ischemia. Hence NHE-1 specific inhibitors are being developed since they could be of beneficial influence under conditions of cardiac ischemia and reperfusion. In this study, the Cytosensor™ microphysiometer was used to measure the potency of four new drug molecules, i.e., EMD 84021, 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 hamster 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 Michaelis-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 values 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 642 at 138 mm NaCl, in good agreement with more elaborate biological assays. The IC₅₀ values increased with the NaCl concentration indicating competitive binding of the inhibitor. The microphysiometer approach is a fast and simple method to measure the activity of the Na⁺/H⁺ antiporter and allows a quantitative kinetic analysis of the proton excretion rate.