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

J Physiol. 2006 Nov 1;576(Pt 3):769-85. doi: 10.1113/jphysiol.2006.117374. Epub 2006 Aug 17.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / pharmacology
  • Amiloride / analogs & derivatives
  • Amiloride / pharmacology
  • Ammonium Chloride / pharmacology
  • Animals
  • Blood-Brain Barrier / physiology
  • Cells, Cultured
  • Chloride-Bicarbonate Antiporters / physiology*
  • Endothelium, Vascular / cytology*
  • Endothelium, Vascular / drug effects
  • Endothelium, Vascular / physiology*
  • Hydrogen-Ion Concentration
  • Mathematics
  • Neuroprotective Agents / pharmacology
  • Rats
  • Rats, Wistar
  • Sodium Chloride Symporters / physiology*
  • Sodium-Bicarbonate Symporters / physiology*
  • Sodium-Hydrogen Exchangers / physiology*
  • Sodium-Potassium-Exchanging ATPase / physiology


  • Chloride-Bicarbonate Antiporters
  • Neuroprotective Agents
  • Sodium Chloride Symporters
  • Sodium-Bicarbonate Symporters
  • Sodium-Hydrogen Exchangers
  • Ammonium Chloride
  • Amiloride
  • Sodium-Potassium-Exchanging ATPase
  • 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid
  • ethylisopropylamiloride