Permeation of NH3/NH4+ and cell pH in colonic crypts of the rat.
Colon cells are subjected to high concentrations of NH3 and NH4+, and a sizeable portion of this buffer is absorbed. The flux of these components into cells causes opposite effects on their pH; this effect is largely used to induce an acid load and to observe the mechanism of acid extrusion from cells. We studied cells of microdissected colon crypts loaded with BCECF and superfused with NH4Cl-containing Krebs-Ringer solution. We found a marked transient reduction in pH measured by ratiometric fluorescence microscopy, from a control value of 7.51 +/- 0.041 to 7.15 +/- 0.041 (n = 21), instead of the initial alkalinization found in most cells. This pH was reached at a rate of 0.95 +/- 0.07 pH units/min. Addition of 1 mmol/l furosemide, a blocker of Na+,K+,2Cl- cotransport, to the ammonium solution inverted this acidification toward alkalinization (pH 7.89 +/- 0.041, n = 5), and superfusion with furosemide plus 0.1 mmol/l hexamethylene amiloride, a specific blocker of Na+/H+ exchange, increased this initial alkalinization further to 8.10 +/- 0.117 (n = 7). When Krebs-Ringer with 0 Cl- containing (NH4)2SO4 instead of NH4Cl was superfused, the acid transient was also reverted to alkalinization; however, a higher degree of alkalinization was observed either when 1 mmol/l furosemide was added to the superfusing sulfate solution (when a pH of 7.78 +/- 0.010 was reached), or when ammonium gluconate was used instead of ammonium sulfate. The addition of Ba2+ to the superfusion solution did not alter the initial acidification. These data indicate that in colon crypt cells, basolateral membrane transporters, in particular the Na+,K+,2Cl- cotransporter and the Na+/H+ exchanger (but not Ba(2+)-sensitive K+ channels), mediate the predominant influx of NH4+ ions leading to the initial transient acidification.
Ramirez
M
M
Depto. de Fisiologia e Biofísica, Univ. São Paulo, Brasil.
Fernandez
R
R
Malnic
G
G
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000641:Ammonia / Q000378:metabolism*; D000818:Animals; D001464:Barium / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D004338:Drug Combinations; D005665:Furosemide / Q000494:pharmacology; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008297:Male; D010539:Permeability / Q000187:drug effects; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D002712:Chlorides; D004338:Drug Combinations; D000644:Quaternary Ammonium Compounds; C057360:5-(N,N-hexamethylene)amiloride; D001464:Barium; D000641:Ammonia; D000584:Amiloride; D005665:Furosemide; D006859:Hydrogen