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 (pHi) of the endothelium. Cerebral microvascular endothelial cells form the blood–brain barrier (BBB) and pHi 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 (H2O2) on the pH regulation in primary rat brain capillary endothelial (RBCE) cells. The RBCE monolayers were loaded with the fluorescent pH indicator BCECF and pHi was monitored by detecting the fluorescent changes. 2. The steady-state pHi 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 NH4Cl to induce intracellular acidification and then the recovery to resting pH was studied. Half-recovery time after NH4Cl 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, HCO3 −/Cl− exchange mechanism is also involved in the restoration of pHi 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 pHi 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 pHi were 6.37 ± 0.02 and 6.60 ± 0.02, respectively. 4. No detectable change was observed in the steady-state pHi in the presence of 100 μM H2O2; however, recovery from NH4Cl prepulse-induced intracellular acid load was inhibited when H2O2 was present in 50 or 100 μM concentration in the HEPES-buffered medium during NH4Cl washout. These data suggest that H2O2 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.