Regulation of intracellular pH in crypt cells from rabbit distal colon

Abstract

H+ secretory mechanisms and intrinsic intracellular buffering capacity were studied in crypt cells from rabbit distal colon. To this end crypts of Lieberkühn were isolated by microdissection, and intracellular pH (pHi) was measured using digital imaging fluorescence microscopy and the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)- 5(6)-carboxyfluorescein. In the absence of HCO(3-)-CO2 and presence of Na+, resting pHi was 7.51 +/- 0.04 (n = 237/23, cells/crypts). However, 6 min after superfusion with a solution containing zero Na+, 1 x 10(5) M Sch-28080 and 5 x 10(-8) M bafilomycin A1, pHi in cells at the bottom of the crypts was significantly reduced, whereas pHi in cells at the top of the crypts remained unchanged. The intrinsic buffering capacity of cells from the middle to the top portion of crypts was significantly higher in the pHi range 7.2-7.6 than of cells at the bottom of the crypt. H+ secretion after an NH(4+)-NH3 pulse amounted to 245 +/- 53 microM/s (n = 73/7) at pHi 7.1 and was largely Na+ dependent and ethylisopropylamiloride sensitive. The Na(+)-independent recovery of pHi after an acid load was insensitive to Sch-28080 and bafilomycin A1. In conclusion, pHi in colonic crypt cells is regulated through Na+/H+ exchange activity in the absence of HCO3-. In addition, intracellular buffering capacity varied with the position along the crypt axis, whereas Na+/H+ exchange activity and pHi did not.

Maintenance and regulation of the pH microclimate at the luminal surface of the distal colon of guinea‐pig

“…Even after an experimental decrease of intracellular pH the pHs was not, or at least very little, affected, independently of whether or not K¤-H¤-ATPase was inhibited (authors' unpublished observations). Although Abrahamse et al (1992Abrahamse et al ( , 1994 have postulated a contribution of the K¤-H¤-ATPase to intracellular pH regulation, we conclude that the K¤-H¤-ATPase in the distal colon of guinea-pig may not be sufficiently active under physiological conditions to cause acidification at the luminal surface. In this respect it is important to consider that mechanisms involved in intracellular pH regulation of enterocytes in the distal colon of guinea-pig as well as in the polarized layer of colonic tumour cells (Busche et al 1993(Busche et al , 1997 are located primarily in the basolateral membrane, not in the apical membrane.…”

cAMP-mediated regulation of murine intestinal/pancreatic Na+/HCO 3 − cotransporter subtype pNBC1

“…Fluorescence microscopy for determination of pHi was performed as described (5,30), similar to previously applied techniques (1,20). After crypts were loaded for 30 min at room temperature with 5 M BCECF in buffer A, crypts were fixed between a glass coverslip and a 0.3-m polycarbonate membrane (Osmonics, Minnetonka, MN) on the stage of an inverted microscope (Nikon Diaphot TMD, Nikon, Dü sseldorf, Germany) and superfused follow-ing the appropriate protocol with buffer A, buffer B (40 mM NaCl replaced by NH 4Cl), and buffer C [containing 120 mM tetramethyl ammonium chloride (TMA) instead of NaCl].…”

Regulation of cytosolic pH and lactic acid release in mesangial cells overexpressing GLUT1

“…4E). As illustrated in Figure 4 C to E, the Na ϩdependent pH regulation was fully inhibited by treatment with 0.1 or 2 mol/L EIPA, an inhibitor of the Na ϩ /H ϩ exchanger [20]. Following a 3-hour treatment with EIPA, pH i was no more significantly different between GLUT1 cells (pH i ϭ 6.78 Ϯ 0.10, N ϭ 6) and LacZ cells (pH i ϭ 6.77 Ϯ 0.08, N ϭ 5), indicating that the difference depended on functional Na ϩ /H ϩ exchanger.…”