Amiloride derivatives induce apoptosis by depleting ER Ca2+ stores in vascular endothelial cells

K. S. Park, D. Poburko, C. B. Wollheim, N. Demaurex

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1296-1304
Number of pages9
JournalBritish Journal of Pharmacology
Volume156
Issue number8
DOIs
Publication statusPublished - 2009 Apr 1

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Amiloride
Endoplasmic Reticulum
Endothelial Cells
Apoptosis
Sodium-Hydrogen Antiporter
Endoplasmic Reticulum Stress
Adenosine Triphosphatases
Thapsigargin
Reperfusion Injury
Brain Ischemia
HeLa Cells
Homeostasis
Gene Expression

All Science Journal Classification (ASJC) codes

  • Pharmacology

Cite this

Park, K. S. ; Poburko, D. ; Wollheim, C. B. ; Demaurex, N. / Amiloride derivatives induce apoptosis by depleting ER Ca2+ stores in vascular endothelial cells. In: British Journal of Pharmacology. 2009 ; Vol. 156, No. 8. pp. 1296-1304.
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abstract = "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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Amiloride derivatives induce apoptosis by depleting ER Ca2+ stores in vascular endothelial cells. / Park, K. S.; Poburko, D.; Wollheim, C. B.; Demaurex, N.

In: British Journal of Pharmacology, Vol. 156, No. 8, 01.04.2009, p. 1296-1304.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Amiloride derivatives induce apoptosis by depleting ER Ca2+ stores in vascular endothelial cells

AU - Park, K. S.

AU - Poburko, D.

AU - Wollheim, C. B.

AU - Demaurex, N.

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N2 - 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.

AB - 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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