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

Abstract

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 ‫ס‬ 30 ± 4 mM for Na + . Addition of either one of the four inhibitors decreased the acidification rate. The IC 50 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 IC 50 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.

pH Regulation in the Intracellular Malaria Parasite, Plasmodium falciparum

“…All inhibitors were purchased from Sigma with the exception of EMD 96785 (17,18), which was a generous gift from Dr. Norbert Beier, Merck KGaA. Stock solutions of amiloride, EIPA, EMD 96785, and bafilomycin A 1 were prepared in dimethyl sulfoxide (Me 2 SO); DCCD was dissolved in ethanol; N-ethylmaleimide (NEM) and vanadate were dissolved in water.…”

“…3, B and C, respectively). However, EMD 96785, a more potent and more selective inhibitor of at least some of the mammalian NHE isoforms (17,18), caused only a slight (although significant) decrease in pH i when added at a concentration (500 M) more than 3 orders of magnitude higher than its IC 50 values for the inhibition of mammalian NHE1 and NHE2 (Refs. 17 and 18; Fig.…”

Characteristics of acid extrusion from Chinese hamster ovary cells expressing different prostaglandin EP receptors

“…The microphysiometer was developed as a pH sensor to detect the changes of cell activity in response to a variety of stimuli (McConnell et al, 1992;Owicki et al, 1990;Fischer et al, 1999). In the original manual of this apparatus, pH changes in the extracellular medium were measured as total acidification during ''flow off'' of a relatively long period (60 -180 s) (McConnell et al, 1992).…”

P2Y₁ Receptor Activation Enhances the Rate of Rat Pinealocyte-Induced Extracellular Acidification via a Calcium-Dependent Mechanism

“…Its molecular function is the electroneutral, reversible exchange of Na + against H + . The activation of Na + /H + antiporter results in intracellular alkalization due to the transport of H + out of the cell [16]. Therefore, it is possible that following the pattern described for noradrenaline [15], the increase in [Ca 2+ ] i activates Na + /H + antiporter.…”