pHi in Piglet Cerebral Microvascular Endothelial Cells: Recovery from an Acid Load

Abstract

Cerebral microvascular endothelial cells form a barrier between the blood and brain, which is critical for normal neuronal functions. These endothelial cells can be challenged by metabolic and respiratory acidosis, especially in newborn babies. We investigated mechanism(s) by which cerebral endothelial cells recover intracellular pH (pHi) when challenged with an intracellular acid load. pHi in piglet cerebral microvascular endothelial cells in primary culture was monitored using the pH-sensitive fluorescent dye BCECF (2',7'-bis-2-carboxyethyl-5(6)-carboxy-fluorescein acetoxymethyl ester), with dual wavelength fluorescence spectroscopy. Endothelial cells attached to coverslips and continuously superfused with HCO3-/CO2 containing medium (25 mM HCO3-, 5% CO2; pH 7.40) have a steady state of pHi of 7.18 +/- 0.02. Under basal conditions, amiloride (100 microMol) and H2DIDS (0.5 mM) decreased pHi 0.12 +/- 0.01 and 0.05 +/- 0.01 pH units, respectively. Removal of external Na+ lowered pHi 0.18 +/- 0.02pH units, while Cl- free medium decreased pHi 0.16 +/- 0.03pH units. These data suggest the presence of an amiloride-sensitive Na+-H+ exchanger and a Na+-dependent HCO3- -Cl- anion exchanger in endothelial cells. Propionate and high PCO2 cause rapid intracellular acidification at constant pH. The cells recover to control pHi over 10 min. Recovery from propionate was largely inhibited by amiloride, slightly inhibited by H2DIDS, and completely prevented by the combination. pHi recovery during elevated PCO2 was blocked by amiloride, H2DIDS, or Na+-free media. These results indicate that recovery from intracellular acidosis can involve amiloride-sensitive Na+-H+ exchange and a Na+-dependent HCO3-/Cl- anion exchange. Relative contributions of pumps and their independence appears to depend on the nature of the acid load.

Clonidine Transport at the Mouse Blood—Brain Barrier by a New H+ Antiporter that Interacts with Addictive Drugs

“…In vitro experiments have shown that a change in the extracellular medium can lead first to an abrupt, sustained change in pH i . Later-acting cell mechanisms then attempt to restore the basal pH i as illustrated by in vitro BBB experiments (Hsu et al, 1996;Nicola et al, 2008;Taylor et al, 2006). We therefore adapted the in situ brain perfusion procedures to perform acute pH i changes at the mouse BBB, as these alterations were more predictable, and take place on the same time scale as the brain perfusion time.…”

“…This 'carbonate/no carbonate' change was dramatically reduced when ACTZ (10 mmol/L), a permeating CA inhibitor, was added to the carbonate-free HEPES (pH e 7.40; Figure 4A) Acidification of the Brain Endothelial Cells: Decreasing the pH i alters the strength of the proton gradient. Removing Na + from the extracellular medium rapidly reduces the pH i of the BBB by increasing the acid load (Hsu et al, 1996). We acidified the BBB by removing the vascular Na + to study the effect of endothelial cell acidification on [ 3 H]clonidine transport.…”

“…The anion exchanger HCO 3 À /Cl À , also called the AE transporter, restores the pH i at the BBB (Nicola et al, 2008). However, a lack of chloride in the extracellular medium leads to the immediate and sustained acidification of the brain endothelial cells by a molecular mechanism that is presently unknown (Hsu et al, 1996;Nicola et al, 2008). When we replaced the chloride in the Krebs carbonate buffer (pH e 7.40) with gluconate and nitrate the [ 3 H]clonidine transport, K in (7.60±0.51 mL/sec per g; n = 4; P < 0.05) was significantly (1.3-fold) greater than that for control mice perfused with Krebs carbonate fluid (5.95 ± 0.52 mL/sec per g), as might be expected after a decrease in the BBB pH i .…”

Na+/H+ Exchange Inhibitor SM-20220 Improves Endothelial Dysfunction Induced by Ischemia-Reperfusion

“…Hsu et al (11) reported that the pHi in piglet cerebral microvascular endothelial cells superfused with HCO 3 -/CO2-containing medium was 7.18. The slightly lower pHi we observed in BBMCs may have been due to the different kind of superfusing medium rather than the different species; in fact, blocking the Na + -dependent HCO3 --Cl -anion exchanger via the application of inhibitor or Cl --free culture medium slightly lowers the steady-state pHi (11).…”

“…It is well known that endothelial cells play an important role in the physiologic homeostasis of the cerebral circulation (9,10). The NHE and Na + /Ca 2+ exchangers are present on cerebral vascular endothelial cells and maintain the ion homeostasis of these cells (11,12). In this study, we investigated the effect of the NHE inhibitor SM-20220 on endothelial cell dysfunction induced by the ischemic condition.…”