title: Mechanisms of hypotonic inhibition of the sodium, proton exchanger type 1 (NHE1) in a biliary epithelial cell line (Mz-Cha-1).
abstract: OBJECTIVE
  To elucidate the cellular events that results in inhibition of Na(+), H(+) exchanger type 1 (NHE1) by hypotonicity.
  
  
  METHODS
  Intracellular pH (pH(i)) was measured in biliary epithelial cells, with the pH-sensitive fluorochrome 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) using a spectrophotometer. Regulatory volume decrease (RVD) was analysed from confocal images. Changes in NHE1 membrane content were visualized by confocal laser scanning microscopy after transfection of Mz-Cha-1 cells with a NHE1-cMyc fusion protein.
  
  
  RESULTS
  In Mz-Cha-1 cells hypotonicity (-80 mmol L(-1) NaCl) inhibited endogenous Na(+), H(+) exchange. Tyrosine and serine kinase inhibitors were incapable to prevent inhibition. As several signalling pathways influence Na(+), H(+) exchange, we tested the effect of the Ca(++), Calmodulin, protein kinase C or the cAMP, protein kinase A system on inhibition of Na(+), H(+) exchange by hypotonic challenge, but neither system was involved. In contrast, cytoskeleton did influence the effect of hypotonicity. Inhibition of microtubule polymerization by colchicine prevented inhibition of NHE1, and also restored Na(+), H(+) exchange kinetics. Specific inhibition of Src kinases with PP2, attenuated pH(i) recovery rate from 1.93 +/- 0.16 pH units min(-1) (normotonic environment) to 1.02 +/- 0.50 pH units min(-1) (hypotonic environment). Membrane staining of NHE1-cMyc fusion protein was maintained after hypotonic exposure in colchicine pre-treated cells as was RVD. Microfilament inhibition by cytochalasin preserved NHE1 activity. Inhibition of phosphatidylinositol-3'-kinase was unable to restore Na(+), H(+) exchange activity.
  
  
  CONCLUSIONS
  We conclude that regulation of Na(+), H(+) exchange during RVD is mediated by cytoskeletal elements. This receptor independent pathway is regulated by Src.
authors.affiliation: Department of Gastroenterology, St Elisabeth Hospital, Dorsten, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Elsing
authors.forename: I
authors.initials: I
authors.lastname: Gosch
authors.forename: J C
authors.initials: JC
authors.lastname: Hennings
authors.forename: C A
authors.initials: CA
authors.lastname: Hübner
authors.forename: T
authors.initials: T
authors.lastname: Herrmann
mesh_terms: D001659:Biliary Tract / Q000166:cytology; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D027682:Cation Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D003599:Cytoskeleton; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D009994:Osmolar Concentration; D010766:Phosphorylation; D011493:Protein Kinase C; D015398:Signal Transduction; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D027682:Cation Transport Proteins; D008565:Membrane Proteins; C490997:Slc9a1 protein, mouse; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D011493:Protein Kinase C; D002118:Calcium