Mannitol at Clinical Concentrations Activates Multiple Signaling Pathways and Induces Apoptosis in Endothelial Cells

Abstract

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 (mannitolХNaClϾurea). 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 [PϽ0.0001] compared with 1.2% of control cells) and was also observed in bovine smooth muscle cells. Mannitol-induced apoptosis was attenuated Ϸ50% 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 [Ca 2ϩ ] in a dose-dependent manner. Chelation of intracellular Ca 2ϩ with quin2-AM (10 mol/L) inhibited mannitol-induced apoptosis Ϸ50%, 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 free [Ca 2ϩ ]. The apoptosis is attenuated by inhibition of transcription or translation, by inhibition of tyrosine kinases, or by intracellular Ca 2ϩ buffering. These data suggest that clinical use of the osmotic diuretic mannitol may exert direct deleterious effects on vascular endothelium. (Stroke. 1998;29:2631-2640.)

Cell shrinkage and monovalent cation fluxes: Role in apoptosis

“…Mannitol-induced apoptosis in bovine aortic endothelial cells was accompanied by activation of tyrosine and stress kinases, phosphorylation of focal adhesion contact-associated proteins paxillin and FAK, and elevation of intracellular free calcium [13], suggesting the activation of multiple signaling pathways under hyperosmotic conditions. Hyperosmolality was shown to activate the Akt pathway that prevented mild hyperosmolalityinduced apoptosis in MDCK cells [14].…”

Paxillin: Adapting to Change

“…In each of these scenarios, profound alterations in the cytoskeleton are effected that are essential to activate and regulate host defenses. Finally, many different modes of cellular stress such as membrane depolarization (123,260), tissue injury (89), hypertonicity (168), stretch (253,317), shear stress (83), increased cell density (13), ␤-amyloid exposures (17), and treatment with the phorbol ester phorbol 12-myristate 13-acetate (PMA) (140) have also been reported to induce paxillin tyrosine phosphorylation.…”

Stress-induced corneal epithelial apoptosis mediated by K+ channel activation

“…Hyperosmotic stress induces cell apoptosis by activating JNK and p38 signaling pathways in various cell types (Qin et al, 1997;Bilney and Murray, 1998;Malek et al, 1998;Hoover et al, 2000;Lu et al, 2000;Morrison et al, 2003;Reinehr et al, 2003). In corneal epithelial cells, the effect of hyperosmotic stress on apoptosis is characterized by measuring cell viability and activations of JNK and p38 signaling pathways.…”