Reconstitution of acid secretion in digitonin-permeabilized rabbit gastricglands - Identification of cytosolic regulatory factors

When isolated rabbit gastric glands were permeabilized with digitonin, they lost their ability to secrete acid, as monitored by [C-14]aminopyrine accu mulation, 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 ge l filtration revealed that the fraction at 30 kDa stimulated permeabilized glands by itself, whereas the 200-kDa fraction potently inhibited brain cyt osol-stimulated acid secretion. Brain cytosol contained only the former sti mulatory factor. With further gel filtration, the 30-kDa activator was sepa rated into two components, 20 kDa (peak 1) and 1.8 kDa (peak 2), both of wh ich 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 m ucosa correlated with the contents of PITP, and recombinant PITP mimicked t he effect of peak 1, suggesting that PITP is one of the essential component s in gastric acid secretion. When gastric glands were stimulated, the inhib itory 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. Th ese results demonstrate a high degree of usefulness for our present model, the reconstituted digitonin-permeabilized gastric glands.