title: Intracellular distribution and functional importance of vesicle-associated membrane protein 2 in gastric parietal cells.
abstract: OBJECTIVE
  Acid secretion by parietal cells involves secretagogue-dependent recycling of the H+-K+-ATPase. Proteins called soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) have been implicated as participants in membrane trafficking, docking, and fusing processes. Here we studied the intracellular distribution and functional importance of one SNARE protein, vesicle associated membrane protein-2 (VAMP-2), in gastric parietal cells.
  
  
  METHODS
  Using an adenoviral recombinant expression system encoding VAMP-2 (synaptobrevin-2) fused to the green fluorescent protein (GFP), we expressed the GFP-VAMP-2 protein in primary cultures of rabbit parietal cells, which enables us to visualize the dynamics of GFP-VAMP-2 in a variety of functional states by fluorescence microscopy. To ascertain the function of VAMP-2 in parietal cell activation, streptolysin-O permeabilized gastric glands were treated with tetanus toxin, a potent and preferential protease for VAMP-2, and acid secretion was measured.
  
  
  RESULTS
  In resting parietal cells GFP was detected throughout the cytoplasm in a pattern of distribution that was very similar to that of H+-K+-ATPase. After stimulation, we observed that the GFP-VAMP-2 translocated to the apical plasma membrane along with the H+-K+-ATPase. A relatively high degree of co-localization was detected between GFP-VAMP-2 and H+-K+-ATPase. Tetanus toxin inhibited cAMP/ATP-stimulated acid secretion by about 45% in permeabilized gastric glands with a concomitant reduction in the level of immunoreactive VAMP-2.
  
  
  CONCLUSIONS
  Adenovirus-based GFP reporter fusion proteins can be used to efficiently study the functional dynamics of SNAREs. VAMP-2 is associated with tubulovesicle membranes in the parietal cell and plays a role in stimulation-associated membrane recruitment and acid secretion.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200, USA.
authors.forename: Serhan
authors.initials: S
authors.lastname: Karvar
authors.forename: Xuebiao
authors.initials: X
authors.lastname: Yao
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Duman
authors.forename: Kevin
authors.initials: K
authors.lastname: Hybiske
authors.forename: Yuechueng
authors.initials: Y
authors.lastname: Liu
authors.forename: John G
authors.initials: JG
authors.lastname: Forte
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured; D005744:Gastric Acid / Q000378:metabolism; D049452:Green Fluorescent Proteins; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism; D006632:Histamine / Q000494:pharmacology; D007202:Indicators and Reagents; D007425:Intracellular Membranes / Q000378:metabolism*; D008164:Luminescent Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D010295:Parietal Cells, Gastric / Q000378:metabolism*; D050683:R-SNARE Proteins; D011817:Rabbits; D013744:Tetanus Toxin / Q000494:pharmacology; D014018:Tissue Distribution
chemical_list: D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008565:Membrane Proteins; D050683:R-SNARE Proteins; D013744:Tetanus Toxin; D049452:Green Fluorescent Proteins; D006632:Histamine; D017506:H(+)-K(+)-Exchanging ATPase; D015056:1-Methyl-3-isobutylxanthine