Ethyl isopropylamiloride downregulates Na,K-ATPase gene expression which confers cytotoxicity in primary proximal tubule cell cultures

Hui Min Chiu, Hsi Hui Lin, Ming-Jer Tang

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Our original attempt was to examine whether inhibition of Na/H exchange in proximal tubule would affect the expression of basolateral membrane protein Na,K-ATPase. Three amiloride analogues were tested within the range of 10 -6 M to 10 - M in primary cultures of proximal tubule cells. Only ethyl-isopropyl amiloride (EIPA) dose-dependently downregulated Na,K-ATPase activity in cultured proximal tubule cells. The time course study revealed that EIPA (10 -4 M) significantly decreased Na,K-ATPase α- and α-mRNA abundance within 4 hr and suppressed Na,K-ATPase α- and β-mRNA levels by 76.3±4.5% and 85.5±5.8%, respectively, within 24 hr. The decrease in Na,K- ATPase mRNA was followed by a decrease in Na,K-ATPase activity by 22.5±10.8% and 48.8±5.9% within 12 and 24 hr, respectively, which could be reflected by a coordinate decrease in levels of both α- and mature β-protein. The cell viability was not affected until 20 hr of EIPA treatment, when an increase in LDH release and cell detachment was observed. Because EIPA rapidly decreased intracellular pH (pHi) to 6.7 within 2 hr and raising phi to 6.6 by metabolic acidosis could not elicit changes in Na,K-ATPase activity, EIPA-induced downregulation of Na,K-ATPase should not be mediated through H + . In view of the time course of EIPA effects on Na,K-ATPase subunit mRNA, protein, activity and cell toxicity, the cytotoxic effect is likely resulted from a decrease in Na,K-ATPase activity. Taken together, we conclude that EIPA induces downregulation of Na,K-ATPase expression via both pre- and post- translational mechanisms, which confers cytotoxic effects on proximal tubule cells.

Original languageEnglish
Pages (from-to)195-202
Number of pages8
JournalChinese Journal of Physiology
Volume41
Issue number4
Publication statusPublished - 1998 Jan 1

Fingerprint

Amiloride
Down-Regulation
Cell Culture Techniques
Gene Expression
Messenger RNA
sodium-translocating ATPase
Protein Subunits
Acidosis
Cell Survival
Membrane Proteins

All Science Journal Classification (ASJC) codes

  • Physiology
  • Physiology (medical)

Cite this

@article{84395c30cb9345808dcf3017f085a093,
title = "Ethyl isopropylamiloride downregulates Na,K-ATPase gene expression which confers cytotoxicity in primary proximal tubule cell cultures",
abstract = "Our original attempt was to examine whether inhibition of Na/H exchange in proximal tubule would affect the expression of basolateral membrane protein Na,K-ATPase. Three amiloride analogues were tested within the range of 10 -6 M to 10 - M in primary cultures of proximal tubule cells. Only ethyl-isopropyl amiloride (EIPA) dose-dependently downregulated Na,K-ATPase activity in cultured proximal tubule cells. The time course study revealed that EIPA (10 -4 M) significantly decreased Na,K-ATPase α- and α-mRNA abundance within 4 hr and suppressed Na,K-ATPase α- and β-mRNA levels by 76.3±4.5{\%} and 85.5±5.8{\%}, respectively, within 24 hr. The decrease in Na,K- ATPase mRNA was followed by a decrease in Na,K-ATPase activity by 22.5±10.8{\%} and 48.8±5.9{\%} within 12 and 24 hr, respectively, which could be reflected by a coordinate decrease in levels of both α- and mature β-protein. The cell viability was not affected until 20 hr of EIPA treatment, when an increase in LDH release and cell detachment was observed. Because EIPA rapidly decreased intracellular pH (pHi) to 6.7 within 2 hr and raising phi to 6.6 by metabolic acidosis could not elicit changes in Na,K-ATPase activity, EIPA-induced downregulation of Na,K-ATPase should not be mediated through H + . In view of the time course of EIPA effects on Na,K-ATPase subunit mRNA, protein, activity and cell toxicity, the cytotoxic effect is likely resulted from a decrease in Na,K-ATPase activity. Taken together, we conclude that EIPA induces downregulation of Na,K-ATPase expression via both pre- and post- translational mechanisms, which confers cytotoxic effects on proximal tubule cells.",
author = "Chiu, {Hui Min} and Lin, {Hsi Hui} and Ming-Jer Tang",
year = "1998",
month = "1",
day = "1",
language = "English",
volume = "41",
pages = "195--202",
journal = "Chinese Journal of Physiology",
issn = "0304-4920",
publisher = "Chinese Physiological Society",
number = "4",

}

Ethyl isopropylamiloride downregulates Na,K-ATPase gene expression which confers cytotoxicity in primary proximal tubule cell cultures. / Chiu, Hui Min; Lin, Hsi Hui; Tang, Ming-Jer.

In: Chinese Journal of Physiology, Vol. 41, No. 4, 01.01.1998, p. 195-202.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Ethyl isopropylamiloride downregulates Na,K-ATPase gene expression which confers cytotoxicity in primary proximal tubule cell cultures

AU - Chiu, Hui Min

AU - Lin, Hsi Hui

AU - Tang, Ming-Jer

PY - 1998/1/1

Y1 - 1998/1/1

N2 - Our original attempt was to examine whether inhibition of Na/H exchange in proximal tubule would affect the expression of basolateral membrane protein Na,K-ATPase. Three amiloride analogues were tested within the range of 10 -6 M to 10 - M in primary cultures of proximal tubule cells. Only ethyl-isopropyl amiloride (EIPA) dose-dependently downregulated Na,K-ATPase activity in cultured proximal tubule cells. The time course study revealed that EIPA (10 -4 M) significantly decreased Na,K-ATPase α- and α-mRNA abundance within 4 hr and suppressed Na,K-ATPase α- and β-mRNA levels by 76.3±4.5% and 85.5±5.8%, respectively, within 24 hr. The decrease in Na,K- ATPase mRNA was followed by a decrease in Na,K-ATPase activity by 22.5±10.8% and 48.8±5.9% within 12 and 24 hr, respectively, which could be reflected by a coordinate decrease in levels of both α- and mature β-protein. The cell viability was not affected until 20 hr of EIPA treatment, when an increase in LDH release and cell detachment was observed. Because EIPA rapidly decreased intracellular pH (pHi) to 6.7 within 2 hr and raising phi to 6.6 by metabolic acidosis could not elicit changes in Na,K-ATPase activity, EIPA-induced downregulation of Na,K-ATPase should not be mediated through H + . In view of the time course of EIPA effects on Na,K-ATPase subunit mRNA, protein, activity and cell toxicity, the cytotoxic effect is likely resulted from a decrease in Na,K-ATPase activity. Taken together, we conclude that EIPA induces downregulation of Na,K-ATPase expression via both pre- and post- translational mechanisms, which confers cytotoxic effects on proximal tubule cells.

AB - Our original attempt was to examine whether inhibition of Na/H exchange in proximal tubule would affect the expression of basolateral membrane protein Na,K-ATPase. Three amiloride analogues were tested within the range of 10 -6 M to 10 - M in primary cultures of proximal tubule cells. Only ethyl-isopropyl amiloride (EIPA) dose-dependently downregulated Na,K-ATPase activity in cultured proximal tubule cells. The time course study revealed that EIPA (10 -4 M) significantly decreased Na,K-ATPase α- and α-mRNA abundance within 4 hr and suppressed Na,K-ATPase α- and β-mRNA levels by 76.3±4.5% and 85.5±5.8%, respectively, within 24 hr. The decrease in Na,K- ATPase mRNA was followed by a decrease in Na,K-ATPase activity by 22.5±10.8% and 48.8±5.9% within 12 and 24 hr, respectively, which could be reflected by a coordinate decrease in levels of both α- and mature β-protein. The cell viability was not affected until 20 hr of EIPA treatment, when an increase in LDH release and cell detachment was observed. Because EIPA rapidly decreased intracellular pH (pHi) to 6.7 within 2 hr and raising phi to 6.6 by metabolic acidosis could not elicit changes in Na,K-ATPase activity, EIPA-induced downregulation of Na,K-ATPase should not be mediated through H + . In view of the time course of EIPA effects on Na,K-ATPase subunit mRNA, protein, activity and cell toxicity, the cytotoxic effect is likely resulted from a decrease in Na,K-ATPase activity. Taken together, we conclude that EIPA induces downregulation of Na,K-ATPase expression via both pre- and post- translational mechanisms, which confers cytotoxic effects on proximal tubule cells.

UR - http://www.scopus.com/inward/record.url?scp=0032418309&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032418309&partnerID=8YFLogxK

M3 - Article

VL - 41

SP - 195

EP - 202

JO - Chinese Journal of Physiology

JF - Chinese Journal of Physiology

SN - 0304-4920

IS - 4

ER -