Identification of a functional Ca2+-sensing receptor in normal human gastric mucous epithelial cells

Michael Rutten, Kathy D. Bacon, Katie L. Marlink, Mark Stoney, Camie L. Meichsner, Fred P. Lee, Susan A. Hobson, Karin D. Rodland, Brett Sheppard, Donald Trunkey, Karen Deveney, Clifford Deveney

Research output: Contribution to journalArticle

62 Citations (Scopus)

Abstract

The purpose of the present study was to determine whether human gastric mucous epithelial cells express a functional Ca2+-sensing receptor (CAR). Human gastric mucous epithelial cells were isolated from surgical tissues and cultured on glass coverslips, plastic dishes, or porous membrane filters. Cell growth was assessed by the MTT assay, CaR localization was detected by immunohistochemistry and confocal microscopy, CaR protein expression was assessed by Western immunoblotting, and intracellular Ca2+ concentration ([Ca2+](i)) was determined by fura 2 spectrofluorometry. In paraffin sections of whole stomach, we found strong CaR immunohistochemical staining at the basolateral membrane, with weak CaR-staining at the apical membrane in mucous epithelial cells. Confocal microscopy of human gastric mucous epithelial cell cultures showed abundant CaR immunofluorescence at the basolateral membrane and little to no CaR immunoreactivity at the apical membrane. Western immunoblot detection of CaR protein in cell culture lysates showed two significant immunoreactive bands of 140 and 120 kDa. Addition of extracellular Ca2+ to preconfluent cultures of human gastric mucous epithelial cells produced a significant proliferative response. Changes in [Ca2+](i) were also observed in response to graded doses of extracellular Ca2+ and Gd3+. The phospholipase C inhibitor U-73122 specifically inhibited Gd3+induced changes in [Ca2+](i) in the gastric mucous epithelial cell cultures. In conclusion, we have identified the localization of a functional CaR in human gastric mucous epithelial cells.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume277
Issue number3 40-3
StatePublished - Sep 1999

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Calcium-Sensing Receptors
Stomach
Epithelial Cells
Membranes
Cell Culture Techniques
Confocal Microscopy
Western Blotting
Staining and Labeling
Fluorescence Spectrometry
Type C Phospholipases
Paraffin
Plastics
Glass
Fluorescent Antibody Technique
Mucous Membrane
Proteins
Immunohistochemistry
Growth

Keywords

  • Cell culture
  • Confocal microscopy
  • Fura 2
  • Gadolinium
  • Immunohistochemistry
  • Intracellular calcium
  • Proliferation
  • Signal transduction
  • Stomach
  • U- 73122
  • Western immunoblotting

ASJC Scopus subject areas

  • Physiology
  • Gastroenterology
  • Physiology (medical)

Cite this

Identification of a functional Ca2+-sensing receptor in normal human gastric mucous epithelial cells. / Rutten, Michael; Bacon, Kathy D.; Marlink, Katie L.; Stoney, Mark; Meichsner, Camie L.; Lee, Fred P.; Hobson, Susan A.; Rodland, Karin D.; Sheppard, Brett; Trunkey, Donald; Deveney, Karen; Deveney, Clifford.

In: American Journal of Physiology - Gastrointestinal and Liver Physiology, Vol. 277, No. 3 40-3, 09.1999.

Research output: Contribution to journalArticle

Rutten, Michael ; Bacon, Kathy D. ; Marlink, Katie L. ; Stoney, Mark ; Meichsner, Camie L. ; Lee, Fred P. ; Hobson, Susan A. ; Rodland, Karin D. ; Sheppard, Brett ; Trunkey, Donald ; Deveney, Karen ; Deveney, Clifford. / Identification of a functional Ca2+-sensing receptor in normal human gastric mucous epithelial cells. In: American Journal of Physiology - Gastrointestinal and Liver Physiology. 1999 ; Vol. 277, No. 3 40-3.
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