Sonic hedgehog is associated with H+-K+-ATPase-containing membranes in gastric parietal cells and secreted with histamine stimulation.
Sonic hedgehog (Shh) is found within gastric parietal cells and processed from a 45-kDa to a 19-kDa bioactive protein by an acid- and protease-dependent mechanism. To investigate whether Shh is associated with the parietal cell membrane compartment that becomes exposed to both acid and proteolytic enzymes during acid secretion, the cellular location of Shh within resting and stimulated gastric parietal cells was examined. Immunofluorescence microscopy of rabbit stomach sections showed that Shh colocalized predominantly with parietal and pit, not chief/zymogen or neck, cell markers. In resting and histamine-stimulated rabbit gastric glands Shh was expressed only in parietal cells close to H+-K+-ATPase-containing tubulovesicular and secretory membranes with some colocalizing with gamma-actin at the basolateral membrane. Gastric gland microsomal membranes were prepared by differential and sucrose gradient centrifugation and immunoisolation with an anti-H+-K+-ATPase-alpha subunit antibody. The 45- and 19-kDa Shh proteins were detected by immunoblot in immunopurified H+-K+-ATPase-containing membranes from resting and stimulated gastric glands, respectively. Incubating glands with a high KCl concentration removed Shh from the membranes. Histamine stimulated 19-kDa Shh secretion from gastric glands into the medium. In human gastric cancer 23132/87 cells cultured on permeable membranes, histamine increased 19-kDa Shh secretion into both apical and basolateral media. These findings show that Shh is a peripheral protein associated with resting and stimulated H+-K+-ATPase-expressing membranes. In addition, Shh appears to be expressed at or close to the basolateral membrane of parietal cells.
Zavros
Yana
Y
Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, PO Box 670576, Cincinnati, OH 45267-0576, USA. yana.zavros@uc.edu
Orr
Melissa A
MA
Xiao
Chang
C
Malinowska
Danuta H
DH
D000818:Animals; D015415:Biomarkers; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D005753:Gastric Mucosa / Q000166:cytology / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism*; D053823:Hedgehog Proteins / Q000378:metabolism*; D006632:Histamine / Q000494:pharmacology*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010295:Parietal Cells, Gastric / Q000378:metabolism*; D021381:Protein Transport; D011817:Rabbits
D015415:Biomarkers; D053823:Hedgehog Proteins; D006632:Histamine; D017506:H(+)-K(+)-Exchanging ATPase