Molecular dissection of HCl secretion in gastric parietal cells using streptolysin O permeabilization

Methods Mol Biol. 2008:440:217-26. doi: 10.1007/978-1-59745-178-9_16.

Abstract

Histamine-stimulated gastric acid secretion involves a transient elevation of intracellular Ca(2+) and the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) cascade through phosphorylation, the actions of which ultimately result in the fusion of vesicles containing H,K-ATPase (adenosine triphosphatase) to the apical plasma membrane of parietal cells. To dissect the signaling events underlying gastric acid secretion, we have developed a permeabilized gastric gland model using streptolysin O (SLO). The advantage of this model is its ability to retain cytosolic components that are required for the secretory machinery while granting accessibility for the introduction of macromolecules into the cytoplasm. Our studies showed that acid secretion in SLO-permeabilized glands is a cAMP-dependent process and involves the recruitment of H,K-ATPase-rich tubulovesicles into the apical plasma membrane as judged by biochemical assays. These studies established a functional permeabilized gland model in which the resting-to-secreting transition can be triggered by second messengers, while the manipulation of the cytoplasmic environment can be achieved with ease.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Aminopyrine / metabolism
  • Animals
  • Bacterial Proteins / pharmacology
  • Biological Assay / methods*
  • Carbon Radioisotopes
  • Cell Fractionation
  • Cell Membrane Permeability / drug effects*
  • Cyclic AMP / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Gastric Acid / metabolism*
  • H(+)-K(+)-Exchanging ATPase / metabolism*
  • Membrane Fusion
  • Parietal Cells, Gastric / drug effects
  • Parietal Cells, Gastric / enzymology
  • Parietal Cells, Gastric / metabolism*
  • Potassium Chloride / metabolism
  • Rabbits
  • Signal Transduction / drug effects*
  • Streptolysins / pharmacology*
  • Subcellular Fractions / drug effects
  • Subcellular Fractions / metabolism
  • Transport Vesicles / drug effects
  • Transport Vesicles / enzymology
  • Transport Vesicles / metabolism*

Substances

  • Bacterial Proteins
  • Carbon Radioisotopes
  • Streptolysins
  • streptolysin O
  • Aminopyrine
  • Potassium Chloride
  • Adenosine Triphosphate
  • Cyclic AMP
  • H(+)-K(+)-Exchanging ATPase