Luminal perfusion of isolated gastric glands

Abstract

We have extended to rabbit gastric glands the technique for perfusing single isolated renal tubules. We isolated glands by hand dissection and used concentric glass pipettes to hold them and perfuse their lumina. Parietal cells (PCs), which tended to be located toward the gland opening, were identified by their pyramidal shape, large size, and autofluorescence. Chief cells (CCs) were identified by their round shape and smaller size. In some experiments, we perfused the lumen with hydroxypyrenetrisulfonate, a pH-sensitive fluorophore, at pH 7.4 and used digital image processing to monitor luminal pH (pH1). Solutions were buffered with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid to pH 7.4 at 37 degrees C. With fast perfusion, we found no evidence of decreased pH1, even with stimulation by 10 microM carbachol. With slow perfusion, pH1 often fell below the dye's sensitive range (pH < 5), especially at low perfusate buffering power. In other experiments, we loaded cells with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and monitored intracellular pH (pHi) in multiple individual PCs and CCs in a single gland. Mean pHi was 7.21 +/- 0.02 (n = 136 cells) for PCs and 7.27 +/- 0.03 (n = 103) for CCs. To examine the response to decreased pH1 and basolateral pH (pHb), we lowered pHb to 6.4 or lowered pH1 to 3.4 or 1.4. Lowering pHb to 6.4 for approximately 1 min caused pHi to fall reversibly by 0.39 +/- 0.05 (n = 53) in PCs and 0.58 +/- 0.03 (n = 50) in CCs. Lowering pH1 to 3.4 or 1.4 caused no significant pHi changes in PCs (n = 38 and 82) or in CCs (n = 44 and 77). Carbachol did not affect the response to changes in pH1 or pHb. We conclude that the apical surfaces of PCs and CCs are unusually resistant to extreme pH gradients.

Acid-Base Transport by the Renal Proximal Tubule

“…These were the first membranes shown to have negligible gas permeability. We also found that lowering luminal pH to 1 failed to alter pH i (22). We similarly observed null pH i shifts, rather than the expected pH i increase, when perfusing lumens of gastric gland (24) or colonic crypts (25) In fact, it would be interesting to see whether the apical membranes of the medullary thick ascending limb, like those of the gastric gland, indeed are impermeable to CO 2 and NH 3 .…”

The Stomach Divalent Ion-sensing Receptor SCAR Is a Modulator of Gastric Acid Secretion

“…The tissues were then transferred to the stage of a dissecting microscope. Individual glands were isolated using a hand dissection technique as described previously (11). After isolation, the glands were allowed to adhere to coverslips precoated with Cell-Tak (Collaborative Research, Bedford, MA) and transferred to a thermostatically controlled chamber maintained at 37°C on either a laser confocal microscope or on a video imaging system for the duration of the experiment.…”

ΔF508 Mutation Results in Impaired Gastric Acid Secretion

“…Following laparotomy a total gastrectomy was performed, and the stomach was immediately rinsed with ice-cold HEPES-buffered Ringer solution (125 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1.2 mM MgCl 2 , 32.2 mM HEPES, and 5 mM glucose, pH 7.4) ( Table 1) to eliminate any residual food matter, and kept in HEPES solution on ice until use. 0.5-cm square sections were cut from the corpus and transferred to the stage of a dissecting microscope where individual gastric glands were hand-dissected using a previously described technique (66). After isolation, the glands were transferred to coverslips precoated with adhesive Cell-Tak (BD Cell-Tak Cell and Tissue Adhesion, BD Biosciences) and mounted in a thermostatically controlled chamber maintained at 37°C on an inverted microscope (Olympus IX50) attached to a digital imaging system (Universal Imaging, Downingtown, PA) for the duration of the experiment.…”