INTRACELLULAR PH REGULATION OF HUMAN COLONIC CRYPT CELLS

In order to investigate the regulation of intracellular pH (pH(i)) 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 (KBS) a baseline pH(i) 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,-buffered solution (BBS) led to a transient intracellular acidification (7.29 +/- 0.09), followed by a recovery to a final resting pH(i) 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 (H,DIDS)-sensitive HCO3--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. With beta(i) = 80 mmol.l(-1) pH unit(-1) for the pH interval ranging from 6.9 to 7.1 (n = 8) the colonocytes exhibited a relatively high intrinsic buffering in comparison with other cell types. In conclusion, the freshly isolated human colonocytes express a Na+/ H+ exchanger, a Na+/HCO3- cotransporter and a Na+-dependent CI-M[CO; exchanger, all of them likely to be involved in the regulation of pH homeostasis in vivo in the presence of widely varying extracellular conditions. Maintenance of a stable pH, of human colonocytes seems to be facilitated by a comparatively high Pi at physiological pH values.