Responsiveness of beta-escin-permeabilized rabbit gastric gland model: effects of functional peptide fragments

Abstract

We established a beta-escin-permeabilized gland model with the use of rabbit isolated gastric glands. The glands retained an ability to secrete acid, monitored by [14C]aminopyrine accumulation, in response to cAMP, forskolin, and histamine. These responses were all inhibited by cAMP-dependent protein kinase inhibitory peptide. Myosin light-chain kinase inhibitory peptide also suppressed aminopyrine accumulation, whereas the inhibitory peptide of protein kinase C or that of calmodulin kinase II was without effect. Guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) abolished cAMP-stimulated acid secretion concomitantly, interfering with the redistribution of H+-K+-ATPase from tubulovesicles to the apical membrane. To identify the targets of GTPgammaS, effects of peptide fragments of certain GTP-binding proteins were examined. Although none of the peptides related to Rab proteins showed any effect, the inhibitory peptide of Arf protein inhibited cAMP-stimulated secretion. These results demonstrate that our new model, the beta-escin-permeabilized gland, allows the introduction of relatively large molecules, e.g., peptides, into the cell, and will be quite useful for analyzing signal transduction of parietal cell function.