Impaired regulation of pH homeostasis by oxidative stress in rat brain capillary endothelial cells

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Abstract

1. Endothelial cells are permanently challenged by altering pH in the blood, and oxidative damage could also influence the intracellular pH (pH i) of the endothelium. Cerebral microvascular endothelial cells form the blood-brain barrier (BBB) and pH i regulation of brain capillary endothelial cells is important for the maintenance of BBB integrity. The aim of this study was to address the pH regulatory mechanisms and the effect of an acute exposure to hydrogen peroxide (H 2O 2) on the pH regulation in primary rat brain capillary endothelial (RBCE) cells. The RBCE monolayers were loaded with the fluorescent pH indicator BCECF and pH i was monitored by detecting the fluorescent changes. 2. The steady-state pH i of RBCE cells in HEPES-buffer (6.83 ± 0.1) did not differ significantly from that found in bicarbonate-buffered medium (6.90 ± 0.08). Cells were exposed to NH 4Cl to induce intracellular acidification and then the recovery to resting pH was studied. Half-recovery time after NH 4Cl prepulse-induced acid load was significantly less in the bicarbonate-buffered medium than in the HEPES-medium, suggesting that in addition to the Na +/H + exchanger, HCO 3 -/Cl - exchange mechanism is also involved in the restoration of pH i after an intracellular acid load in primary RBCE cells. We used RT-PCR-reactions to detect the isoforms of Na +/H + exchanger gene family (NHE). NHE-1 -2, -3 and -4 were equally present, and there was no significant difference in the relative abundance of the four transcripts in these cells. 3. No pH i recovery was detected when the washout after an intracellular acid load occurred in nominally Na +-free HEPES-buffered medium or in the presence of 10 μM 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a specific inhibitor of Na +/H + exchanger. The new steady-state pH i were 6.37 ± 0.02 and 6.60 ± 0.02, respectively. 4. No detectable change was observed in the steady-state pH i in the presence of 100 μM H 2O 2; however, recovery from NH 4Cl prepulse-induced intracellular acid load was inhibited when H 2O 2 was present in 50 or 100 μM concentration in the HEPES-buffered medium during NH 4Cl washout. These data suggest that H 2O 2 is without effect on the activity of Na +/H + exchanger at rest, but could inhibit the function of the exchanger after an intracellular acid load.

Original languageEnglish
Pages (from-to)141-151
Number of pages11
JournalCellular and Molecular Neurobiology
Volume25
Issue number1
DOIs
Publication statusPublished - Feb 2005

Fingerprint

Oxidative stress
Endothelial cells
HEPES
Rats
Brain
Sodium-Hydrogen Antiporter
Oxidative Stress
Homeostasis
Endothelial Cells
Acids
Recovery
Bicarbonates
Acidification
Hydrogen Peroxide
Restoration
Blood-Brain Barrier
Monolayers
Buffers
Protein Isoforms
Blood

Keywords

  • BCECF
  • Blood-brain barrier
  • Hydrogen peroxide
  • Intracellular pH
  • Rat brain capillary endothelial cells
  • Sodium/hydrogen exchange

ASJC Scopus subject areas

  • Neuroscience(all)
  • Clinical Biochemistry
  • Cell Biology
  • Genetics

Cite this

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title = "Impaired regulation of pH homeostasis by oxidative stress in rat brain capillary endothelial cells",
abstract = "1. Endothelial cells are permanently challenged by altering pH in the blood, and oxidative damage could also influence the intracellular pH (pH i) of the endothelium. Cerebral microvascular endothelial cells form the blood-brain barrier (BBB) and pH i regulation of brain capillary endothelial cells is important for the maintenance of BBB integrity. The aim of this study was to address the pH regulatory mechanisms and the effect of an acute exposure to hydrogen peroxide (H 2O 2) on the pH regulation in primary rat brain capillary endothelial (RBCE) cells. The RBCE monolayers were loaded with the fluorescent pH indicator BCECF and pH i was monitored by detecting the fluorescent changes. 2. The steady-state pH i of RBCE cells in HEPES-buffer (6.83 ± 0.1) did not differ significantly from that found in bicarbonate-buffered medium (6.90 ± 0.08). Cells were exposed to NH 4Cl to induce intracellular acidification and then the recovery to resting pH was studied. Half-recovery time after NH 4Cl prepulse-induced acid load was significantly less in the bicarbonate-buffered medium than in the HEPES-medium, suggesting that in addition to the Na +/H + exchanger, HCO 3 -/Cl - exchange mechanism is also involved in the restoration of pH i after an intracellular acid load in primary RBCE cells. We used RT-PCR-reactions to detect the isoforms of Na +/H + exchanger gene family (NHE). NHE-1 -2, -3 and -4 were equally present, and there was no significant difference in the relative abundance of the four transcripts in these cells. 3. No pH i recovery was detected when the washout after an intracellular acid load occurred in nominally Na +-free HEPES-buffered medium or in the presence of 10 μM 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a specific inhibitor of Na +/H + exchanger. The new steady-state pH i were 6.37 ± 0.02 and 6.60 ± 0.02, respectively. 4. No detectable change was observed in the steady-state pH i in the presence of 100 μM H 2O 2; however, recovery from NH 4Cl prepulse-induced intracellular acid load was inhibited when H 2O 2 was present in 50 or 100 μM concentration in the HEPES-buffered medium during NH 4Cl washout. These data suggest that H 2O 2 is without effect on the activity of Na +/H + exchanger at rest, but could inhibit the function of the exchanger after an intracellular acid load.",
keywords = "BCECF, Blood-brain barrier, Hydrogen peroxide, Intracellular pH, Rat brain capillary endothelial cells, Sodium/hydrogen exchange",
author = "Ildik{\'o} Sipos and B. T{\"o}rőcsik and L. Tretter and V. {\'A}d{\'a}m-Vizi",
year = "2005",
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T1 - Impaired regulation of pH homeostasis by oxidative stress in rat brain capillary endothelial cells

AU - Sipos, Ildikó

AU - Törőcsik, B.

AU - Tretter, L.

AU - Ádám-Vizi, V.

PY - 2005/2

Y1 - 2005/2

N2 - 1. Endothelial cells are permanently challenged by altering pH in the blood, and oxidative damage could also influence the intracellular pH (pH i) of the endothelium. Cerebral microvascular endothelial cells form the blood-brain barrier (BBB) and pH i regulation of brain capillary endothelial cells is important for the maintenance of BBB integrity. The aim of this study was to address the pH regulatory mechanisms and the effect of an acute exposure to hydrogen peroxide (H 2O 2) on the pH regulation in primary rat brain capillary endothelial (RBCE) cells. The RBCE monolayers were loaded with the fluorescent pH indicator BCECF and pH i was monitored by detecting the fluorescent changes. 2. The steady-state pH i of RBCE cells in HEPES-buffer (6.83 ± 0.1) did not differ significantly from that found in bicarbonate-buffered medium (6.90 ± 0.08). Cells were exposed to NH 4Cl to induce intracellular acidification and then the recovery to resting pH was studied. Half-recovery time after NH 4Cl prepulse-induced acid load was significantly less in the bicarbonate-buffered medium than in the HEPES-medium, suggesting that in addition to the Na +/H + exchanger, HCO 3 -/Cl - exchange mechanism is also involved in the restoration of pH i after an intracellular acid load in primary RBCE cells. We used RT-PCR-reactions to detect the isoforms of Na +/H + exchanger gene family (NHE). NHE-1 -2, -3 and -4 were equally present, and there was no significant difference in the relative abundance of the four transcripts in these cells. 3. No pH i recovery was detected when the washout after an intracellular acid load occurred in nominally Na +-free HEPES-buffered medium or in the presence of 10 μM 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a specific inhibitor of Na +/H + exchanger. The new steady-state pH i were 6.37 ± 0.02 and 6.60 ± 0.02, respectively. 4. No detectable change was observed in the steady-state pH i in the presence of 100 μM H 2O 2; however, recovery from NH 4Cl prepulse-induced intracellular acid load was inhibited when H 2O 2 was present in 50 or 100 μM concentration in the HEPES-buffered medium during NH 4Cl washout. These data suggest that H 2O 2 is without effect on the activity of Na +/H + exchanger at rest, but could inhibit the function of the exchanger after an intracellular acid load.

AB - 1. Endothelial cells are permanently challenged by altering pH in the blood, and oxidative damage could also influence the intracellular pH (pH i) of the endothelium. Cerebral microvascular endothelial cells form the blood-brain barrier (BBB) and pH i regulation of brain capillary endothelial cells is important for the maintenance of BBB integrity. The aim of this study was to address the pH regulatory mechanisms and the effect of an acute exposure to hydrogen peroxide (H 2O 2) on the pH regulation in primary rat brain capillary endothelial (RBCE) cells. The RBCE monolayers were loaded with the fluorescent pH indicator BCECF and pH i was monitored by detecting the fluorescent changes. 2. The steady-state pH i of RBCE cells in HEPES-buffer (6.83 ± 0.1) did not differ significantly from that found in bicarbonate-buffered medium (6.90 ± 0.08). Cells were exposed to NH 4Cl to induce intracellular acidification and then the recovery to resting pH was studied. Half-recovery time after NH 4Cl prepulse-induced acid load was significantly less in the bicarbonate-buffered medium than in the HEPES-medium, suggesting that in addition to the Na +/H + exchanger, HCO 3 -/Cl - exchange mechanism is also involved in the restoration of pH i after an intracellular acid load in primary RBCE cells. We used RT-PCR-reactions to detect the isoforms of Na +/H + exchanger gene family (NHE). NHE-1 -2, -3 and -4 were equally present, and there was no significant difference in the relative abundance of the four transcripts in these cells. 3. No pH i recovery was detected when the washout after an intracellular acid load occurred in nominally Na +-free HEPES-buffered medium or in the presence of 10 μM 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a specific inhibitor of Na +/H + exchanger. The new steady-state pH i were 6.37 ± 0.02 and 6.60 ± 0.02, respectively. 4. No detectable change was observed in the steady-state pH i in the presence of 100 μM H 2O 2; however, recovery from NH 4Cl prepulse-induced intracellular acid load was inhibited when H 2O 2 was present in 50 or 100 μM concentration in the HEPES-buffered medium during NH 4Cl washout. These data suggest that H 2O 2 is without effect on the activity of Na +/H + exchanger at rest, but could inhibit the function of the exchanger after an intracellular acid load.

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KW - Blood-brain barrier

KW - Hydrogen peroxide

KW - Intracellular pH

KW - Rat brain capillary endothelial cells

KW - Sodium/hydrogen exchange

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U2 - 10.1007/s10571-004-1379-6

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JO - Cellular and Molecular Neurobiology

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