Divalent cations regulate acidity within the lumen and tubulovesicle compartment of gastric parietal cells.
OBJECTIVE
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.
Gerbino
Andrea
A
Department of Surgery, Brigham and Women's Hospital, VA Boston Health Care System, Boston, MA 02115, USA.
Hofer
Aldebaran M
AM
McKay
Breda
B
Lau
Bonnie W
BW
Soybel
David I
DI
D000818:Animals; D002413:Cations, Divalent / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005029:Ethylenediamines / Q000494:pharmacology; D005744:Gastric Acid / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D066298:In Vitro Techniques; D009853:Omeprazole / Q000494:pharmacology; D010295:Parietal Cells, Gastric / Q000378:metabolism*; D054328:Proton Pump Inhibitors; D011817:Rabbits; D014018:Tissue Distribution
D002413:Cations, Divalent; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D005029:Ethylenediamines; D007093:Imidazoles; D054328:Proton Pump Inhibitors; C035235:Sch 28080; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D009853:Omeprazole; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine