Intracellular pH regulation in IEC-6 cells, a cryptlike intestinal cell line.
Regulation of intracellular pH, pHi, was studied using microspectrofluorimetry of the pH-sensitive, fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in the rat intestinal crypt cell line, IEC-6. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solutions with pHi 7.25, treatment with a pulse of NH4Cl caused cells to acidify and then recover to control level. Because recovery was Na dependent, blocked by 1 mM amiloride, and unaffected by the presence and absence of Cl, it was likely because of a Na+-H+ exchanger. Cells were also acid loaded by changing from HEPES to HCO3-CO2-buffered solutions. pHi again recovered, but 1 mM amiloride reduced the rate of H+ efflux by only 47%. This HCO3-dependent, amiloride-insensitive H efflux required Na+ but not Cl- and was completely blocked by 200 microM [H2] 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). We conclude that a Na+-HCO3- cotransporter was operative. Cl-free solutions caused pHi to increase from 7.19 to 7.41; this effect required the presence of exogenous HCO3-CO2 but not Na and was blocked by 200 microM [H2]DIDS. A Cl- -HCO3- exchanger is the most likely explanation for these data. All the pHi regulatory mechanisms are operative in NaCl-HCO3-CO2-buffered solutions. The Na+-H+ and Na+-HCO3- mechanisms are acid extruders, whereas the Cl- -HCO3- exchanger is an acid loader. These transporters may be important for generating HCO3 secretion by intestinal crypt cells.
Wenzl
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Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Sjaastad
M D
MD
Weintraub
W H
WH
Machen
T E
TE
D000143:Acids / Q000494:pharmacology; D000818:Animals; D001639:Bicarbonates / Q000494:pharmacology; D002021:Buffers; D002245:Carbon Dioxide / Q000494:pharmacology; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D006531:HEPES / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D011522:Protons; D012964:Sodium / Q000378:metabolism; D012996:Solutions
D000143:Acids; D001639:Bicarbonates; D002021:Buffers; D002712:Chlorides; D011522:Protons; D012996:Solutions; D002245:Carbon Dioxide; D012964:Sodium; D006531:HEPES