In the human colon the removal of HCO₃^– from the bathing medium reduces the cAMP-dependent secretory response by 60 % (Taylor et al. 2001). As this HCO₃^–-dependent secretion is independent of Cl^– and inhibited by serosal 4,4′-diisothiocyano-stilbene-2,2′-disulfonate (DIDS) it is likely that it is mediated via a basolateral NaHCO₃ cotransporter. Therefore, we have used measurements of intracellular pH (pH_i) to determine if a NaHCO₃ cotransporter is present in the secretory cells of the human colon.

All measurements used crypts isolated from colonoscopy biopsies collected from the descending and sigmoid colon. Isolated crypts were loaded with the pH-sensitive fluorescent dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluoroscein (BCECF) and pH_i was measured in the basal third of the crypts at 37 °C. To characterize the pH_i regulatory mechanisms, cells were acidified with an NH₄Cl pre-pulse and the activity of pH_i regulatory mechanism quantified by the initial rate of recovery of pH_i, expressed as ΔpH units min^-1. All data are given as means ± S.E.M. and n = number of crypts. The experiments were approved by the Otago Ethics Committee, Dunedin, New Zealand.

In HCO₃^–-free Ringer solution, pH_i recovery was dependent upon the presence of Na⁺ (ΔpH units min^-1 in presence = 0.19 ± 0.02, n = 12 and absence of Na⁺ = -0.022 ± 0.007, n = 15; P < 0.05, Student’s unpaired t test). It was also inhibited in a dose-dependent manner by amiloride (K_i = 8 µM) with complete inhibition occurring at 0.5 mM (ΔpH units min^-1 = -0.004 ± 0.002, n = 4). In the presence of HCO₃^–/CO₂, recovery of pH_i was also dependent upon Na⁺ (ΔpH units min^-1 in presence = 0.100 ± 0.013, n = 6 and absence of Na⁺ = -0.023 ± 0.006, n = 11; P < 0.01, ANOVA, Dunnett’s post test). However, although the recovery was inhibited by 0.5 mM amiloride (ΔpH units min^-1 0.0215 ± 0.005, n = 6, P < 0.01, ANOVA, Dunnett’s post test), there was evidence of amiloride-insensitive pH_i recovery. This amiloride-insensitive pH_i recovery was inhibited by DIDS. Following exposure to NH₄Cl and Na⁺-free Ringer solution the rate of recovery of pH_i in the presence of 0.5 mM amiloride was 0.0789 ± 0.009 pH units min^-1 (n = 18), whereas in the presence of amiloride plus 250 µM DIDS this was reduced to 0.0176 ± 0.005 pH units min^-1 (n = 9, P < 0.01, unpaired Student’s t test).

These data demonstrate that the dominant mechanism for regulation of pH_i in human colonic crypts under basal conditions is a Na⁺/H⁺ exchanger. There is also a small contribution from a HCO₃^–-dependent transporter, most likely a NaHCO₃ cotransporter. It remains to be established whether this is stimulated by secretagogues and so contributes to the secretory response of the intact colon.

This work was funded by the University of Otago.