(Stroke. 1998;29:2631-2640.)
© 1998 American Heart Association, Inc.
Original Contributions |
Mannitol at Clinical Concentrations Activates Multiple Signaling Pathways and Induces Apoptosis in Endothelial Cells
From the Department of Neurosurgery, Brigham & Women's Hospital, Children's Hospital, and Harvard Medical School (A.M.M.), and the Molecular Medicine and Renal Units (G.G.G., L.J., S.L.A.) and Division of Cardiology (S.I.), Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Mass.
Correspondence to Adel M. Malek, MD, PhD, UCSF Medical Center, Rm L352, Box 0628, 505 Parnassus Avenue, San Francisco, CA 94143. E-mail ammalek{at}bics.bwh.harvard.edu
Background and Purpose—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
NaCl>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
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 µmol/L) inhibited mannitol-induced apoptosis
50%, as to a lesser extent did inhibition of tyrosine kinase
activity with herbimycin (1 µmol/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.
Editorial Comment
Department of Neurology, Washington University School of Medicine, St Louis, Missouri
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