Volume 126, Issue 1, Pages 182-195 (January 2004) Divalent cations regulate acidity within the lumen and tubulovesicle compartment of gastric parietal cells  Andrea Gerbino, Aldebaran M. Hofer, Breda McKay, Bonnie W. Lau, David I. Soybel  Gastroenterology  Volume 126, Issue 1, Pages 182-195 (January 2004) DOI: 10.1053/j.gastro.2003.10.068

Figure 1 Digital images in gray scale of an isolated rabbit gastric gland, loaded with LysoSensor Blue-Yellow DND-160 (4 μmol/L for 25 minutes) and now perfused with standard Ringer’s solution. (A) Image of the gland during fluorescence excitation at 340 nm. (B) Image of the gland during fluorescence excitation at 340 nm with low-visible light background to show cells and gland structure. Note the more intense staining of gland lumen and apical regions of cells in the plane of focus. (Original magnification 30×.) Gastroenterology 2004 126, 182-195DOI: (10.1053/j.gastro.2003.10.068)

Figure 2 (A) Calibration of LysoSensor Blue-Yellow DND-160 in situ using nigericin (7 μmol/L) in Ringer’s solution containing high K+ concentration. Record begins with glands perfused by standard Ringer’s solution (R). Regions of interest (ROI) shown in dark lines are lumen areas (n = 3); regions shown in light lines are cells (n = 6). (B) Calibrations similar to that performed in A in 6 different glands on 6 different days. Each graph represents the mean emission intensity of cell and lumen ROIs. Note that the baseline acidity of the TV/L compartment is between pH 3 and pH 4. Gastroenterology 2004 126, 182-195DOI: (10.1053/j.gastro.2003.10.068)

Figure 3 Superimposed recordings of acidity within the TV/L compartment during exposure of gastric glands to omeprazole (100 μmol/L) or SCH28080 (10 μmol/L). Recording begins with a 3-minute baseline period of perfusion with Ringer’s solution, exposure for 6 minutes to inhibitor plus vehicle or vehicle alone, and then exposure to Ringer’s solution for 6 minutes, followed by perfusion with nigericin (7 μmol/L) in high-K+ Ringer’s solution. Each record represents the mean response of a single gland (lumen and cell ROIs). At the end of each experiment, acidity in the TV/L compartment can be clamped at pH 2.0, demonstrating that alkalization is not due to dye loss or unresponsiveness. Gastroenterology 2004 126, 182-195DOI: (10.1053/j.gastro.2003.10.068)

Figure 4 Measurements of acidity in the TV/L compartment during exposure to different concentrations of TPEN [tetrakis-(2-pyridylmethyl) ethylenediamine] in Ringer’s solutions nominally free of Ca2+. Recordings begin with tissues perfused with standard Ringer’s solution, followed by Ringer’s solution nominally free of Ca2+, then Ca2+-free Ringer’s solution containing TPEN. Measurements are recorded in individual cells (light lines) or segments of lumen (dark lines) within the gland. Gastroenterology 2004 126, 182-195DOI: (10.1053/j.gastro.2003.10.068)

Figure 5 Measurements of acidity in the TV/L compartment during exposure to different concentrations of TPEN. Studies were performed as in Figure 4. Each bar represents the mean of responses observed after 20 minutes of exposure to TPEN in individual glands (n = 4 in each group, 6–10 ROIs per gland). Cell and lumen ROIs were averaged to provide an integrated measurement. Results expressed as mean ± SD, ∗P < 0.05 compared with control. Gastroenterology 2004 126, 182-195DOI: (10.1053/j.gastro.2003.10.068)

Figure 6 Measurements of acidity in the TV/L compartment during exposure to TPEN [tetrakis-(2-pyridylmethyl) ethylenediamine] in a Ca2+-containing Ringer’s solution. Recordings begin with gland perfused with standard Ringer’s solution. The gland is then exposed to Ringer’s containing 1.5 mmol/L Ca2+ and 0.5 mmol/L TPEN. Assuming a Kd of TPEN for Ca2+ of between 50 μmol/L and 150 μmol/L in Ringer’s solution at pH 7.4 (see text), the free concentration of Ca2+ would be ∼1.0 mmol/L and that of TPEN would be between 20 and 50 μmol/L, comparable to concentrations of TPEN used in nominally Ca2+-free Ringer’s solution in Figure 4. Measurements are recorded in individual cells (light lines, n = 9) or segments of lumen (dark lines, n = 2) within the gland. Gastroenterology 2004 126, 182-195DOI: (10.1053/j.gastro.2003.10.068)

Figure 7 Gland permeabilized with α-toxin and perfused with intracellular buffer (ICB), showing compartmentalization of Lyso-Sensor in lumen and tubulovesicle compartment (A) and responses to nigericin (7 μmol/L)-mediated calibration (B). Reversible susceptibility to ATP depletion (500 to 0 μmol/L; C) indicates successful permeabilization. In B and C, dark lines indicate lumen ROIs; light lines indicate cell ROIs. Gastroenterology 2004 126, 182-195DOI: (10.1053/j.gastro.2003.10.068)

Figure 8 Acidity of TV/L compartment during depletion of divalent cations in cytoplasmic compartment of permeabilized glands. Recording begins with glands briefly exposed to low ATP (50 μmol/L) to demonstrate success of permeabilization. The gland is perfused with intracellular buffer containing EGTA then BAPTA and then TPEN in the presence of BAPTA. Alkalization is observed only in the presence of TPEN, indicating that it is due to depletion of divalent cations in the TV/L compartment. Dark lines indicate lumen ROIs, light lines cell ROIs. Gastroenterology 2004 126, 182-195DOI: (10.1053/j.gastro.2003.10.068)

Figure 9 Freshly isolated parietal cell loaded with LysoSensor DND-160 and perfused on a coverslip with standard Ringer’s solution. (A) Under light microscopy. (B) Fluorescence during excitation at 340 nm. Gastroenterology 2004 126, 182-195DOI: (10.1053/j.gastro.2003.10.068)

Figure 10 Measurements of acidity in the TV/L compartment of 3 freshly isolated parietal cells during exposure to TPEN. Recordings begin with tissues perfused with standard Ringer’s solution, followed by Ringer’s nominally free of Ca2+ then Ca2+-free Ringer’s solution containing TPEN. Substantial recovery of compartment acidity is observed after removal of TPEN. Gastroenterology 2004 126, 182-195DOI: (10.1053/j.gastro.2003.10.068)

Figure 11 Measurements of acidity in the TV/L compartment during exposure to BAPTA-AM. Recordings begin with gland perfused with standard Ringer’s solution. The gland is then exposed to Ringer’s solution containing 1 mmol/L Ca2+ and 100 μmol/L BAPTA-AM. At 24 minutes, the gland is exposed to high-K+-Ringer’s solution containing nigericin (pH 2). Measurements are recorded in individual cells (light lines, n = 6) or segments of lumen (dark lines, n = 2) within the gland. Gastroenterology 2004 126, 182-195DOI: (10.1053/j.gastro.2003.10.068)

Figure 12 Acidity of TV/L compartment during simultaneous depletion of divalent cations in the TV/L compartment and inhibition of H+/K+ ATPase. Recordings begin with tissues perfused with standard Ringer’s solution (R), which is then replaced with Ringer’s solution containing no added Ca2+, followed by Ringer’s solution containing SCH28080 (10 μmol/L) and then TPEN (50 μmol/L) (A) and then in the opposite order (B). Note in the lower panel that, after TPEN is removed, the effect is still not fully reversible until after SCH28080 is removed from the perfusate. Individual recordings from cells (light lines) and luminal segments (dark lines) indicate similar effects in both regions. Gastroenterology 2004 126, 182-195DOI: (10.1053/j.gastro.2003.10.068)