REGULATION OF INTRACELLULAR PH IN CRYPT CELLS FROM RABBIT DISTAL COLON

H+ secretory mechanisms and intrinsic intracellular buffering capacity were studied in crypt cells from rabbit distal colon. To this end cry pts of Lieberkuhn were isolated by microdissection, and intracellular pH (pH(i)) was measured using digital imaging fluorescence microscopy and the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6 )-c arboxyfluorescein. In the absence of HCO3--CO2 and presence of Na+, re sting pH(i) was 7.51 +/- 0.04 (n = 237/23, cells/crypts). However, 6 m in after superfusion with a solution containing zero Na+, 1 x 10(5) M Sch-28080 and 5 x 10(-8) M bafilomycin A(1), pH(i) in cells at the bot tom of the crypts was significantly reduced, whereas pHi in cells at t he top of the crypts remained unchanged. The intrinsic buffering capac ity of cells from the middle to the top portion of crypts was signific antly higher in the pH(i) range 7.2-7.6 than of cells at the bottom of the crypt. H+ secretion after an NH4+-NH3 pulse amounted to 245 +/- 5 3 mu M/s (n = 73/7) at pH(i) 7.1 and was largely Na+ dependent and eth ylisopropylamiloride sensitive. The Na+-independent recovery of pH(i) after an acid load was insensitive to Sch-28080 and bafilomycin A(1). In conclusion, pH(i) in colonic crypt cells is regulated through Na+/H + the crypt axis, whereas Na+/H+ exchange activity and pH(i) did not.