pH i in piglet cerebral microvascular endothelial cells

Recovery from an acid load (44014)

Pauline Hsu, John Haffner, Maria Luiza C. Albuquerque, Charles Leffler

Research output: Contribution to journalArticle

25 Citations (Scopus)

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 acldosls, 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-sensitlve fluorescent dye BCECF (2′,7′-bis-2-carboxyethyl-5(6)-carboxy-fluorescein acetoxymethyl ester), with dual wavelength fluorescence spectroscopy. Endothelial cells attached to coversllps and continuously superfused with HCO 3 - /CO 2 containing medium (25 mM HCO 3 - , 5% CO 2 ; pH 7.40) have a steady state of pH i of 7.18 ± 0.02. Under basal conditions, amiloride (100 μM) and H 2 DIDS (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 HCO 3 - -Cl - anion exchanger in endothelial cells. Proplonate and high PCO 2 cause rapid intracellular acidification at constant pH . The cells recover to control pH i over 10 min. Recovery from propionate was largely inhibited by amiloride, slightly inhibited by H 2 DIDS, and completely prevented by the combination. pH i recovery during elevated PCO 2 was blocked by amiloride, H 2 DIDS, or Na + -free media. These results indicate that recovery from intracellular acidosis can involve amiloride-sensitive Na + -H + exchange and a Na + -dependent HCO 3 - /Cl - anion exchange. Relative contributions of pumps and their independence appears to depend on the nature of the acid load.

Original languageEnglish (US)
Pages (from-to)256-262
Number of pages7
JournalExperimental Biology and Medicine
Volume212
Issue number3
StatePublished - Jul 1 1996

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Endothelial cells
Amiloride
Endothelial Cells
Recovery
Acids
Carbon Monoxide
Anions
Sodium-Hydrogen Antiporter
Acidification
Propionates
Fluorescence spectroscopy
Fluorescein
Fluorescent Dyes
Cell culture
Brain
Esters
Blood
Pumps
Wavelength
Chloride-Bicarbonate Antiporters

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

pH i in piglet cerebral microvascular endothelial cells : Recovery from an acid load (44014). / Hsu, Pauline; Haffner, John; Albuquerque, Maria Luiza C.; Leffler, Charles.

In: Experimental Biology and Medicine, Vol. 212, No. 3, 01.07.1996, p. 256-262.

Research output: Contribution to journalArticle

Hsu, Pauline ; Haffner, John ; Albuquerque, Maria Luiza C. ; Leffler, Charles. / pH i in piglet cerebral microvascular endothelial cells : Recovery from an acid load (44014). In: Experimental Biology and Medicine. 1996 ; Vol. 212, No. 3. pp. 256-262.
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AB - 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 acldosls, 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-sensitlve fluorescent dye BCECF (2′,7′-bis-2-carboxyethyl-5(6)-carboxy-fluorescein acetoxymethyl ester), with dual wavelength fluorescence spectroscopy. Endothelial cells attached to coversllps and continuously superfused with HCO 3 - /CO 2 containing medium (25 mM HCO 3 - , 5% CO 2 ; pH 7.40) have a steady state of pH i of 7.18 ± 0.02. Under basal conditions, amiloride (100 μM) and H 2 DIDS (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 HCO 3 - -Cl - anion exchanger in endothelial cells. Proplonate and high PCO 2 cause rapid intracellular acidification at constant pH • . The cells recover to control pH i over 10 min. Recovery from propionate was largely inhibited by amiloride, slightly inhibited by H 2 DIDS, and completely prevented by the combination. pH i recovery during elevated PCO 2 was blocked by amiloride, H 2 DIDS, or Na + -free media. These results indicate that recovery from intracellular acidosis can involve amiloride-sensitive Na + -H + exchange and a Na + -dependent HCO 3 - /Cl - anion exchange. Relative contributions of pumps and their independence appears to depend on the nature of the acid load.

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