pH i 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 (pH_i) when challenged with an intracellular acid load. pH_i 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 pH_i of 7.18 ± 0.02. Under basal conditions, amiloride (100 μ M) and H₂DIDS (0.5 mM) decreased pH_i 0.12 ± 0.01 and 0.05 ± 0.01 pH units, respectively. Removal of external Na⁺ lowered pH_i 0.18 ± 0.02 pH units, while Cl^--free medium decreased pH_i 0.16 ± 0.03 pH units. These data suggest the presence of an amiloride-sensitive Na⁺ -H⁺ exchanger and a Na⁺-dependent HCO₃ -CI anion exchanger in endothelial cells. Propionate and high PC0₂ cause rapid intracellular acidification at constant pH_e. The cells recover to control pH_i over 10 min. Recovery from propionate was largely inhibited by amiloride, slightly inhibited by H₂DIDS, and completely prevented by the combination. pH_I 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.