Identification of Na+/H+ exchange on the apical side of surface colonocytes using BCECF.
Colonocytes must regulate intracellular pH (pHi) while they transport H+ and HCO3-. To investigate the membrane transport processes involved in pHi regulation, colonocyte pHi was measured with 2,'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in intact segments of rat distal colon mounted on a holder that fits into a standard fluorometer cuvette and allows independent superfusion of mucosal and serosal surfaces. When NCECF-acetoxymethyl ester was in the mucosal solution only, BCECF loaded surface colonocytes with a high degree of selectivity. In HEPES-buffered solutions, basal pHi was 7.31 +/- 0.01 (n = 68), and pHi was dependent on extracellular Na+. Cells acidified in Na(+)-free solution, and pHi rapidly corrected when Na+ was returned. pHi recovered at 0.22 +/- 0.01 pH/min (n = 6) when Na+ was introduced into the mucosal solution and at 0.02 +/- 0.01 pH/min (n = 7) when Na+ was absent from the mucosal solution. The presence or absence of Na+ in the serosal solution did not affect pHi. This indicated that the Na(+)-dependent pHi recovery process is located in the apical cell membrane, but not in the basolateral membrane. Because amiloride (1 mM) inhibited Na(+)-dependent pHi recovery by 75%, Na+/H+ exchange appears to be present in the apical membrane. Because Na(+)-independent pHi recovery was not affected by K(+)-free media, 50 microM SCH-28080, 100 nM bafilomycin A1, or Cl(-)-free media, this transport mechanism does not involve a gastriclike H(+)-K(+)-ATPase, a vacuolar H(+)-ATPase, or a Cl-/base exchanger. In summary, pHi was selectively measured in surface colonocytes by this technique. In these cells, the Na+/H+ exchange activity involved in pHi regulation was detected in the apical membrane, but not in the basolateral membrane.
King
G G
GG
Department of Medicine, McGuire Veterans Affairs Medical Center, Richmond 23249.
Lohrmann
W E
WE
Ickes
J W
JW
Feldman
G M
GM
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014018:Tissue Distribution
D005452:Fluoresceins; D005456:Fluorescent Dyes; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium