Jan-Robert Schwark

S3226, a novel inhibitor of Na+/H+ exchanger subtype 3 in various cell types

Abstract Inhibition of Na+/H+ exchange (NHE) subtypes has been investigated in a study of the mouse fibroblast L cell line (LAP1) transfected with human (h) NHE1, rabbit (rb) NHE2, rat (rt) or human (h) NHE3 as well as an opossum kidney cell line (OK) and porcine renal brush-border membrane vesicles (BBMV). S3226 {3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydro-chloride} was the most potent and specific NHE3 inhibitor with an IC50 value of 0.02 µmol/l for the human isoform, whereas its IC50 value for hNHE1 and rbNHE2 was 3.6 and @80 µmol/l, respectively. In contrast, amiloride is a weak NHE3 inhibitor (IC50>100 µmol/l) with a higher affinity to hNHE1 and rbNHE2. Cariporide (4-isopropyl-3-methylsulphonyl-benzoyl-guanidine methane-sulphonate), which has an IC50 for NHE3 of approximately 1 mmol/l, is a highly selective NHE1 inhibitor (0.08 µmol/l). Therefore, S3226 is a novel tool with which to investigate the physiological and pathophysiological roles of NHE3 in animal models.

Selective inhibition of the Na+/H+ exchanger type 3 activates CO2/H+-sensitive medullary neurones.

Abstract Hypercapnia as well as lowered intracellular pH (pHi) increase the bioelectric activity of CO2/H+-sensitive neurones (VLNcs) of the ventrolateral medulla oblongata. Here we describe that immunoreactive Na+/H+ exchanger (NHE3) is present in ventrolateral neurones from medullary organotypic cultures (obex level). To test whether VLNcs can be acidified and thereby activated by inhibition of NHE3, we used the novel high-affinity NHE3-inhibitors S1611 and S3226. Both drugs raised the firing rates of VLNcs to at least 150% of the control values, and depolarized membrane potential by up to 15 mV at concentrations (0.5–1 µmol/l) suitable for selective inhibition of NHE3. The changes in bioelectric activity strongly resembled the responses to hypercapnia (PCO2: 60–100 mmHg). In BCECF-AM-loaded cultures a subfraction of ventrolateral VLNcs was found to be intracellularly acidified by 0.05–0.1 pH units following treatment with S1611; the time course of this acidification was similar to that evoked by hypercapnia. All drug effects were sustained and readily reversible upon washing. Non-CO2/H+-responsive medullary neurones as well as hippocampal CA3 neurones were unaffected by up to 20 µmol/l S1611. It is concluded that the selective inhibition of NHE3 acidifies and activates CO2/H+-sensitive neurones within the ventrolateral medulla oblongata.