Background & aims: Until recently, it has not been possible to evaluate factors that regulate the acidity of the microenvironment within the tubulovesicles and luminal (TV/L) spaces of the gastric gland. The goal of this study was to develop a method for monitoring the mechanisms that regulate acidity in the TV/L compartment.
Methods: Isolated rabbit gastric glands (intact or permeabilized with S. aureus alpha-toxin) were loaded with a recently characterized fluorescent dye, LysoSensor Yellow-Blue DND 160 (Molecular Probes, Eugene, OR), which localizes to highly acidic compartments and can be used to monitor acidity ratiometrically.
Results: In resting glands, the pH of the TV/L compartment was approximately 3.4. Moderate alkalizations ( approximately 0.5 to 1.0 pH unit alkalization) were observed during exposure to inhibitors of the apical H(+)/K(+) ATPase (omeprazole and SCH28080), thereby unmasking a stable, low-level leak of H(+) ions from the TV/L compartment. Similar changes were observed in alpha-toxin permeabilized glands following depletion of ATP in the cytoplasm. In intact and permeabilized glands, we used the cell-permeant, divalent cation chelator, tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) to probe the effects of lowering divalent cation content of the TV/L compartment. Exposure to relatively low concentrations (20 micromol/L, 50 micromol/L) of TPEN reversibly promoted H(+) leakage. At these concentrations, simultaneous inhibition using SCH28080 led to marked enhancement of the rate of alkalization.
Conclusions: The effects of low-dose TPEN suggests that acidity within the TV/L compartment of the gastric gland may be regulated, at least in part, by its content of divalent cations such as Zn(2+), for which TPEN has high affinity.