PH(I) IN PIGLET CEREBRAL MICROVASCULAR ENDOTHELIAL-CELLS - RECOVERY FROM AN ACID LOAD

Cerebral microvascular endothelial cells form a barrier between the bl ood and brain, which is critical for normal neuronal functions. These endothelial cells can be challenged by metabolic and respiratory acido sis, especially in newborn babies. We investigated mechanism(s) by whi ch cerebral endothelial cells recover intracellular pH (pH(i)) when ch allenged with an intracellular acid load, pH(i), in piglet cerebral mi crovascular endothelial cells in primary culture was monitored using t he pH-sensitive fluorescent dye BCECF (2',7'-bis-2-carboxyethyl-5(6)-c arboxy-fluorescein acetoxymethyl ester), with dual wavelength fluoresc ence spectroscopy, Endothelial cells attached to coverslips and contin uously superfused with HCO3-/CO2 containing medium (25 mM HCO3-, 5% CO 2; pH 7.40) have a steady state of pH(i) of 7.18 +/- 0.02. Under basal conditions, amiloride (100 mu M) and H2DIDS (0.5 mM) decreased pH(i) 0.12 +/-0.01 and 0.05 +/- 0.01 pH units, respectively. Removal of exte rnal Na+ lowered pH(i) 0.18 +/- 0.02 pH units, while Cl--free medium d ecreased 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-- Cl- anion exchanger in endothelial cells. Propionate and high PCO2 cau se rapid intracellular acidification at constant pH(o). The cells reco ver 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 indicat e that recovery from Intracellular acidosis can involve amiloride-sens itive Na+- H+ exchange and a Na+-dependent HCO3-/Cl- anion exchange. R elative contributions of pumps and their independence appears to depen d on the nature of the acid load.