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pHi in piglet cerebral microvascular endothelial cells: recovery from an acid load
P Hsu1, J Haffner, M L Albuquerque
1Laboratory for Research in Neonatal Physiology, Department of Physiology, University of Tennessee, Memphis 38163, USA.
Insights
Cerebral endothelial cells use Na+-H+ exchange and Na+-dependent HCO3-/Cl- exchange to recover from acidosis. These mechanisms are crucial for maintaining brain function, especially in newborns facing metabolic or respiratory challenges.
Area of Science:
- Physiology
- Cell Biology
- Biochemistry
Background:
- Cerebral microvascular endothelial cells form a critical blood-brain barrier.
- Acidosis, particularly in newborns, can challenge endothelial cell function.
- Understanding pH recovery mechanisms is vital for neurological health.
Purpose of the Study:
- Investigate the mechanisms of intracellular pH (pHi) recovery in cerebral endothelial cells following acid load.
- Identify the specific ion transport systems involved in pHi regulation.
Main Methods:
- Primary piglet cerebral microvascular endothelial cells were cultured.
- Intracellular pH was monitored using the fluorescent dye BCECF and dual-wavelength spectroscopy.
- Cells were exposed to acid loads (propionate, elevated PCO2) and inhibitors (amiloride, H2DIDS) in various ionic conditions (Na+-free, Cl--free).
Main Results:
- Basal pHi was 7.18 +/- 0.02.
- Amiloride and H2DIDS inhibited basal pHi, suggesting Na+-H+ and HCO3-/Cl- exchangers.
- Removal of Na+ or Cl- significantly decreased pHi.
- Recovery from propionate-induced acidosis was inhibited by amiloride and H2DIDS.
- Recovery from elevated PCO2-induced acidosis was blocked by amiloride, H2DIDS, and Na+-free conditions.
Conclusions:
- Cerebral endothelial cells utilize both amiloride-sensitive Na+-H+ exchange and Na+-dependent HCO3-/Cl- anion exchange for pHi recovery.
- The relative contribution of these mechanisms depends on the type of acid load.
- These findings are crucial for understanding brain protection during acidosis.
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.