Transporters involved in regulation of intracellular pH in primary cultured rat brain endothelial cells

Abstract

Fluid secretion across the blood-brain barrier, critical for maintaining the correct fluid balance in the brain, entails net secretion of HCO 3 − , which is brought about by the combined activities of ion transporters situated in brain microvessels. These same transporters will concomitantly influence intracellular pH (pH i ). To analyse the transporters that may be involved in the maintenance of pH i and hence secretion of HCO 3 − , we have loaded primary cultured endothelial cells derived from rat brain microvessels with the pH indicator BCECF and suspended them in standard NaCl solutions buffered with Hepes or Hepes plus 5% CO 2 /HCO 3 − . pH i in the standard solutions showed a slow acidification over at least 30 min, the rate being less in the presence of HCO 3 − than in its absence. However, after accounting for the difference in buffering, the net rates of acid loading with and without HCO 3 − were similar. In the nominal absence of HCO 3 − the rate of acid loading was increased equally by removal of external Na + or by inhibition of Na + /H + exchange by ethylisopropylamiloride (EIPA). By contrast, in the presence of HCO 3 − the increase in the rate of acid loading when Na + was removed was much larger and the rate was then also significantly greater than the rate observed in the absence of both Na + and HCO 3 − . Removal of Cl − in the presence of HCO 3 − produced an alkalinization followed by a resumption of the slow acid gain. Removal of Na + following removal of Cl − increased the rate of acid gain. In the presence of HCO 3 − and initial presence of Na + and Cl − , DIDS inhibited the changes in pH i produced by removal of either Na + or Cl − . These are the expected results if these cells possess an AE-like Cl − /HCO 3 − exchanger, a 'channel-like' permeability allowing slow influx of acid (or efflux of HCO 3 − ), a NBC-like Cl − -independent Na + −HCO 3 − cotransporter, and a NHE-like Na + /H + exchanger. The in vitro rates of HCO 3 − loading via the Na + −HCO 3 − cotransporter could, if the transporter is located on the apical, blood-facing side of the cells, account for the net secretion of HCO 3 − into the brain.

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

“…Several mechanisms are responsible for keeping the pH i of the endothelial cells forming the BBB more acidic than the surrounding medium under basal condition (Taylor et al, 2006). In vitro experiments have shown that a change in the extracellular medium can lead first to an abrupt, sustained change in pH i .…”

“…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 inversion of the proton gradient is effective at the BBB because the brain endothelial cells have both cytosolic and luminal membrane-bound CA, which catalyze the rapid change in the pH i and transformation of CO 2 in the vascular bed to HCO 3 À , which diffuses poorly (Ghandour et al, 1992;Taylor et al, 2006). Our ACTZ inhibition experiment indicates that the intracellular alkalinization produced by removing carbonate requires CA, as it does in vitro (Taylor et al, 2006). Similarly, we used only a short-time exposure to NH 4 Cl to rapidly increase the pH i and consequently reduce clonidine brain transport.…”

“…The carbonate-free extracellular medium causes the intracellular CO 2 to diffuse out of endothelial cells to establish a new equilibrium between the intracellular and extracellular media. The loss of intracellular CO 2 results in HCO 3 À and H + being converted into CO 2 by carbonic anhydrase (CA), which then diffuses toward the vascular space (Ennis et al, 1996;Taylor et al, 2006). Perfusion with the bicarbonate-free buffer thus led to an abrupt, rapid increase in pH i that required CA within the cells and at the luminal membrane of the BBB (Taylor et al, 2006 Figure 4A).…”

“…The loss of intracellular CO 2 results in HCO 3 À and H + being converted into CO 2 by carbonic anhydrase (CA), which then diffuses toward the vascular space (Ennis et al, 1996;Taylor et al, 2006). Perfusion with the bicarbonate-free buffer thus led to an abrupt, rapid increase in pH i that required CA within the cells and at the luminal membrane of the BBB (Taylor et al, 2006 Figure 4A). 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.…”