title: pHi 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 (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.
authors.affiliation: Laboratory for Research in Neonatal Physiology, Department of Physiology, University of Tennessee, Memphis 38163, USA.
authors.forename: P
authors.initials: P
authors.lastname: Hsu
authors.forename: J
authors.initials: J
authors.lastname: Haffner
authors.forename: M L
authors.initials: ML
authors.lastname: Albuquerque
authors.forename: C W
authors.initials: CW
authors.lastname: Leffler
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives; D000138:Acidosis / Q000139:chemically induced / Q000503:physiopathology*; D000584:Amiloride; D000818:Animals; D000831:Animals, Newborn; D001812:Blood-Brain Barrier / Q000502:physiology*; D001921:Brain / Q000098:blood supply*; D002245:Carbon Dioxide; D002478:Cells, Cultured; D002712:Chlorides / Q000502:physiology; D004730:Endothelium, Vascular / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006935:Hypercapnia / Q000139:chemically induced / Q000503:physiopathology; D008833:Microcirculation / Q000378:metabolism; D011422:Propionates; D012964:Sodium / Q000502:physiology; D013552:Swine
chemical_list: D002712:Chlorides; D011422:Propionates; D002245:Carbon Dioxide; C018564:dihydro-DIDS; D000584:Amiloride; D012964:Sodium; C029658:propionic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid