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-isopro pylidene-2-methyl-acrylamide dihydro-chloride¿ was the most potent and specific NHE3 inhibitor with an IC50 value of 0.02 micromol/l for the human isoform, whereas its IC50 value for hNHE1 and rbNHE2 was 3.6 and approximately = 80 micromol/l, respectively. In contrast, amiloride is a weak NHE3 inhibitor (IC50>100 micromol/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 micromol/l). Therefore, S3226 is a novel tool with which to investigate the physiological and pathophysiological roles of NHE3 in animal models.

Ezrin regulates NHE3 translocation and activation after Na + -glucose cotransport

“…Consistent with the reported IC 50 for NHE3 of 0.02 M, 0.1 M S3226, a dose that inhibits NHE3 partially but does not inhibit NHE1 or NHE2 (20), inhibited cytoplasmic alkalinization after initiation of Na ϩ -glucose cotransport by 41 Ϯ 8% in cells not transfected with exogenous ezrins (1). Increasing S3226 to 1 M, a dose that inhibits NHE3 more completely but may partially inhibit NHE1 (20), increased this inhibition to 66 Ϯ 1% (1), but further increases in S3226 dose did not inhibit alkalinization further.…”

“…Increasing S3226 to 1 M, a dose that inhibits NHE3 more completely but may partially inhibit NHE1 (20), increased this inhibition to 66 Ϯ 1% (1), but further increases in S3226 dose did not inhibit alkalinization further. The residual S3226-resistant alkalinization response was not due to other Na ϩ -H ϩ exchangers, because 1 M S3226 and 50 M HOE694 (to inhibit NHE1 and NHE2) in combination inhibited alkalinization by 77 Ϯ 6%, only 11% more than 1 M S3226 without HOE694 (Fig.…”

Splicing variation of Long-IRBIT determines the target selectivity of IRBIT family proteins

“…LongV2 and LongV4 slightly inhibited NHE3 activity, but no significant difference was observed. To eliminate the effect of endogenous NHE1 activity, we performed the same experiment in the presence of 30 μM amiloride (AML), which strongly inhibits NHE1 and partially inhibits NHE3 (21). In the presence of 30 μM AML, we observed gradual acidification in IRBIT KO MEF cells expressing mRFP alone (Fig.…”

An animal homolog of plant Mep/Amt transporters promotes ammonia excretion by the anal papillae of the disease vector mosquito,Aedes aegypti

“…A leucine amino acid residue substitution in the NHE3 of A. aegypti renders this exchanger amiloride insensitive (Pullikuth et al, 2006). Application of S3226, a specific inhibitor of NHE3 in mammals (Schwark et al, 1998), to larval mosquitoes resulted in decreased NH 4 + efflux at the anal papillae (Fig. 6A), suggesting that an NHE, likely NHE3, plays a role in ammonia excretion.…”