MANNITOL AT CLINICAL CONCENTRATIONS ACTIVATES MULTIPLE SIGNALING PATHWAYS AND INDUCES APOPTOSIS IN ENDOTHELIAL-CELLS

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 ren al perfusion. Though beneficial rheological effects commonly are attri buted 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 to NaCl>urea). In duced programmed cell death was detected by DAPI staining of intact ce ll 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 attenuat ed approximate to 50% in the presence of cycloheximide or actinomycin D. Hypertonic mannitol and NaCl, but not urea, increased tyrosine phos phorylation 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-te rminal kinase (JNK) activity. In addition, hypertonic mannitol increas ed intracellular free [Ca2+] in a dose-dependent manner. Chelation of intracellular Ca2+ with quin2-AM (10 mu mol/L) inhibited mannitol-indu ced apoptosis approximate to 50%, as to a lesser extent did inhibition of tyrosine kinase activity with herbimycin (1 mol/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 fr ee [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 d iuretic mannitol may exert direct deleterious effects on vascular endo thelium.