Calcitonin receptor-mediated CFTR activation in human intestinal epithelial cells

J Cell Mol Med. 2011 Dec;15(12):2697-705. doi: 10.1111/j.1582-4934.2011.01264.x.

Abstract

High levels of calcitonin (CT) observed in medullary thyroid carcinoma and other CT-secreting tumours cause severe diarrhoea. Previous studies have suggested that CT induces active chloride secretion. However, the involvement of CT receptor (CTR) and the molecular mechanisms underlying the modulation of intestinal electrolyte secreting intestinal epithelial cells have not been investigated. Therefore, current studies were undertaken to investigate the direct effects of CT on ion transport in intestinal epithelial cells. Real time quantitative RT-PCR and Western blot analysis demonstrated the expression of CTR in intestinal epithelial T84 cells. Exposure of T84 cells to CT from the basolateral but not from apical side significantly increased short circuit current (I(SC) ) in a dose-dependent manner that was blocked by 1 μM of CTR antagonist, CT8-32. CT-induced I(SC) was blocked by replacing chloride in the bath solutions with equimolar gluconate and was significantly inhibited by the specific cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor, CFTR(127inh). Further, biotinylation studies showed that CT increased CFTR levels on the apical membrane. The presence of either the Ca(2+) chelator, bis(2-aminophenoxy)ethane tetraacetic acid-acetoxymethyl (BAPTA-AM) ester or the protein kinase A (PKA) inhibitor, H89, significantly inhibited I(SC) induced by CT (∼32-58% reduction). Response to CT was retained after permeabilization of the basolateral or the apical membranes of T84 cells with nystatin. In conclusion, the activation of CTR by CT induced chloride secretion across T84 monolayers via CFTR channel and the involvement of PKA- and Ca(2+) -dependent signalling pathways. These data elucidate the molecular mechanisms underlying CT-induced diarrhoea.

Publication types

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

MeSH terms

  • Biotinylation
  • Blotting, Western
  • Calcitonin / metabolism*
  • Cell Membrane / metabolism
  • Cell Proliferation
  • Cells, Cultured
  • Chelating Agents / pharmacology
  • Cyclic AMP / pharmacology
  • Cyclic AMP-Dependent Protein Kinases / antagonists & inhibitors
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Cystic Fibrosis Transmembrane Conductance Regulator / genetics
  • Cystic Fibrosis Transmembrane Conductance Regulator / metabolism*
  • Egtazic Acid / analogs & derivatives
  • Egtazic Acid / pharmacology
  • Epithelial Cells / drug effects
  • Epithelial Cells / metabolism*
  • Humans
  • Intestinal Mucosa / metabolism*
  • Intestines / cytology
  • Intestines / drug effects
  • Ion Transport
  • RNA, Messenger / genetics
  • Real-Time Polymerase Chain Reaction
  • Receptors, Calcitonin / genetics
  • Receptors, Calcitonin / metabolism*
  • Signal Transduction

Substances

  • CFTR protein, human
  • Chelating Agents
  • RNA, Messenger
  • Receptors, Calcitonin
  • Cystic Fibrosis Transmembrane Conductance Regulator
  • 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester
  • Egtazic Acid
  • Calcitonin
  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases