title: Mannitol at clinical concentrations activates multiple signaling pathways and induces apoptosis in endothelial cells.
abstract: OBJECTIVE
  Hyperosmotic mannitol therapy is widely used in the clinical setting for acute and subacute reduction in brain edema, to decrease muscle damage in compartment syndrome, and to improve renal perfusion. Though beneficial rheological effects commonly are attributed to mannitol, its direct effects on endothelial cells are poorly understood.
  
  
  METHODS
  We studied the effect of hypertonic and hypotonic stress on bovine aortic endothelial (BAE) cells, using mannitol, urea, and sodium chloride and medium dilution in vitro.
  
  
  RESULTS
  Exposure to incremental osmolar concentrations of 300 mOsm of each osmotic agent increased apoptosis in BAE cells (mannitol congruent withNaCl>urea). Induced programmed cell death was detected by DAPI staining of intact cell nuclei, and by TUNEL and DNA fragmentation ladder assays. Mannitol-induced apoptosis exhibited dose dependence (42% of cells at 300 mOsm [P<0.0001] compared with 1.2% of control cells) and was also observed in bovine smooth muscle cells. Mannitol-induced apoptosis was attenuated approximately 50% in the presence of cycloheximide or actinomycin D. Hypertonic mannitol and NaCl, but not urea, increased tyrosine phosphorylation of the focal adhesion contact-associated proteins paxillin and FAK. Hypotonic medium, which did not lead to apoptosis, increased protein tyrosine phosphorylation of FAK but not of paxillin. Addition of mannitol or NaCl also produced sustained increases in c-Jun NH2-terminal kinase (JNK) activity. In addition, hypertonic mannitol increased intracellular free [Ca2+] in a dose-dependent manner. Chelation of intracellular Ca2+ with quin2-AM (10 micromol/L) inhibited mannitol-induced apoptosis approximately 50%, as to a lesser extent did inhibition of tyrosine kinase activity with herbimycin (1 micromol/L).
  
  
  CONCLUSIONS
  We have shown that hypertonic mannitol exposure induces endothelial cell apoptosis, accompanied by activation of tyrosine and stress kinases, phosphorylation of FAK and paxillin, and elevation of intracellular free [Ca2+]. The apoptosis is attenuated by inhibition of transcription or translation, by inhibition of tyrosine kinases, or by intracellular Ca2+ buffering. These data suggest that clinical use of the osmotic diuretic mannitol may exert direct deleterious effects on vascular endothelium.
authors.affiliation: Department of Neurosurgery, Brigham & Women's Hospital, Children's Hospital, and Harvard Medical School, Boston, Mass., USA. ammalek@bics.bwh.harvard.edu
authors.forename: A M
authors.initials: AM
authors.lastname: Malek
authors.forename: G G
authors.initials: GG
authors.lastname: Goss
authors.forename: L
authors.initials: L
authors.lastname: Jiang
authors.forename: S
authors.initials: S
authors.lastname: Izumo
authors.forename: S L
authors.initials: SL
authors.lastname: Alper
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D004234:Diuretics, Osmotic / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D006982:Hypertonic Solutions / Q000494:pharmacology; D007038:Hypotonic Solutions / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases; D008353:Mannitol / Q000494:pharmacology*; D020928:Mitogen-Activated Protein Kinases; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects; D009994:Osmolar Concentration; D010750:Phosphoproteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D012965:Sodium Chloride / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism; D014508:Urea / Q000494:pharmacology
chemical_list: D004234:Diuretics, Osmotic; D006982:Hypertonic Solutions; D007038:Hypotonic Solutions; D010750:Phosphoproteins; D008353:Mannitol; D014443:Tyrosine; D012965:Sodium Chloride; D014508:Urea; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium