Reconstitution of acid secretion in digitonin-permeabilized rabbit gastric glands. Identification of cytosolic regulatory factors.
When isolated rabbit gastric glands were permeabilized with digitonin, they lost their ability to secrete acid, as monitored by [14C]aminopyrine accumulation, and they never recovered by supplement with cytosol prepared from gastric mucosa. However, the permeabilized glands elicited acid secretion when brain cytosol was supplemented. Fractionation of gastric cytosol by gel filtration revealed that the fraction at 30 kDa stimulated permeabilized glands by itself, whereas the 200-kDa fraction potently inhibited brain cytosol-stimulated acid secretion. Brain cytosol contained only the former stimulatory factor. With further gel filtration, the 30-kDa activator was separated into two components, 20 kDa (peak 1) and 1.8 kDa (peak 2), both of which are necessary for full activity. We purified peak 1 from bovine brain, and phosphatidylinositol transfer protein (PITP) was identified as the main component of the activity. The stimulating activity in brain and gastric mucosa correlated with the contents of PITP, and recombinant PITP mimicked the effect of peak 1, suggesting that PITP is one of the essential components in gastric acid secretion. When gastric glands were stimulated, the inhibitory activity, but not stimulatory activity, in the cytosol was increased. This suggests a regulatory mechanism such as stimulation translocates the inhibitory component from the secretory site on the membrane to cytosol. These results demonstrate a high degree of usefulness for our present model, the reconstituted digitonin-permeabilized gastric glands.
Akagi
K
K
Laboratory of Pharmacology & Toxicology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.
Nagao
T
T
Urushidani
T
T
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002417:Cattle; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D003600:Cytosol / Q000378:metabolism*; D004072:Digitonin / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005744:Gastric Acid / Q000378:metabolism*; D005753:Gastric Mucosa / Q000378:metabolism*; D008074:Lipoproteins / Q000302:isolation & purification / Q000378:metabolism; D008565:Membrane Proteins; D010716:Phosphatidylinositols / Q000378:metabolism; D049294:Phospholipid Transfer Proteins; D010770:Phosphotransferases; D020033:Protein Isoforms; D011817:Rabbits; D051381:Rats; D013270:Stomach / Q000187:drug effects / Q000502:physiology
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; C492235:Cdk5r1 protein, rat; D008074:Lipoproteins; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D049294:Phospholipid Transfer Proteins; D020033:Protein Isoforms; D010770:Phosphotransferases; D004072:Digitonin