Background and purpose: Amiloride derivatives are blockers of the Na +/H+ exchanger (NHE) and at micromolar concentrations have protective effects on cardiac and brain ischaemia/reperfusion injury but at higher concentrations also induce apoptosis. Here, we aimed to elucidate the mechanism related to this cytotoxic action. Experimental approach: We quantified the expression of genes associated with endoplasmic reticulum (ER) stress and measured changes in luminal ER Ca2+ concentration ([Ca 2+]ER) with a 'cameleon' indicator, D1ER. Key results: Amiloride derivatives induced apoptosis in vascular endothelial cells, an effect that increased at alkaline extracellular pH. The potency order for cytotoxicity was 5-(N,N-hexamethylene)-amiloride (HMA) > 5-(N-methyl-N-isobutyl) amiloride > 5-(N-ethyl-N-isopropyl) amiloride (EIPA) >> amiloride. HMA dose-dependently increased the transcription of the ER stress genes GADD153 and GADD34 and rapidly depleted [Ca2+]ER, mimicking the effects of the sarco/endoplasmic reticulum ATPase (SERCA) inhibitor thapsigargin. The NHE1-specific inhibitor HOE 694 inhibited NHE activity by 87% but did not alter [Ca2+]ER. The decrease in [Ca 2+]ER evoked by amiloride derivatives was also observed in HeLa cells and was mirrored by an increase in cyto/ic Ca2+ concentration. Conclusions and implications: Amiloride derivatives disrupt ER and cyto/ic Ca2+ homeostasis by a mechanism unrelated to NHE inhibition, most likely by interfering with the activity of SERCA. We propose that ER Ca2+ depletion and subsequent ER stress provide a rationale framework for the apoptotic effects of amiloride derivatives.
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