Intracellular pH regulation of human colonic crypt cells.
In order to investigate the regulation of intracellular pH (pHi) in freshly isolated human colonocytes, we have used a newly developed technique for the rapid isolation and covalent attachment of these cells to glass surfaces and microspectrofluorimetric measurement of the pH-sensitive fluorescence of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded specimens in a perfusion chamber (37 degrees C). In N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic-acid-(HEPES)-buffered Ringer solution (HBS) a baseline pHi of 7.35 +/- 0.03 (mean +/- SD; n = 42) was found for human colonocytes and in HBS, NH4Cl-prepulse-induced intracellular acidification in colonocytes is reversed rapidly by the ubiquitous amiloride-sensitive (1 mmol/l) Na+/H+ exchanger. Switching from HBS to HCO(3-)-buffered solution (BBS) led to a transient intracellular acification (7.29 +/- 0.09), followed by a recovery to a final resting pHi of 7.43 +/- 0.03. One-third of the acid extrusion in BBS is amiloridesensitive; the remaining two-thirds are caused by the dihydroderivative of 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (H2DIDS)-sensitive HCO(3-)-dependent mechanisms. The functional activity of an acid-extruding Na+/HCO3- cotransporter in human colonocytes was observed in response to the reintroduction of Na+ into amiloride-containing Na+/Cl(-)-free BBS. In addition, the mechanism leading to alkalinization (7.56 +/- 0.05) in Cl(-)-free BBS was identified as Na(+)-dependent Cl-/HCO3- exchange, by its H2DIDS sensitivity and the specific requirement for Cl- and Na+. The intrinsic buffering capacity (beta i) of the human colonocytes was calculated from pH changes induced by sequential NH4Cl-loading steps during blockage of acid/base transporters.(ABSTRACT TRUNCATED AT 250 WORDS)
Teleky
B
B
Department of Surgery I, University of Vienna, Medical School, Austria.
Hamilton
G
G
Cosentini
E
E
Bischof
G
G
Riegler
M
M
Koperna
T
T
Feil
W
W
Schiessel
R
R
Wenzl
E
E
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000136:Acid-Base Equilibrium / Q000502:physiology; D001639:Bicarbonates / Q000378:metabolism; D002021:Buffers; D002478:Cells, Cultured; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007474:Ion Exchange; D012964:Sodium / Q000378:metabolism
D001639:Bicarbonates; D002021:Buffers; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006859:Hydrogen; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid