Blockade of maitotoxin-induced endothelial cell lysis by glycine and L-alanine

The maitotoxin (MTX)-induced cell deathcascade in bovine aortic endothelial cells (BAECs) is a model foroncotic/necrotic cell death. The cascade is initiated by an increase incytosolic free Ca²⁺ concentration([Ca²⁺]_i), which is followed by the biphasicuptake of vital dyes. The initial phase of dye entry reflectsactivation of large pores and correlates with surface membrane blebformation; the second phase reflects cell lysis. In the present study,the effect of the cytoprotective amino acid glycine was examined.Glycine had no effect on MTX-induced change in[Ca²⁺]_i or on the first phase of vital dyeuptake but produced a concentration-dependent (EC₅₀ ~1mM) inhibition of the second phase of dye uptake. No cytoprotectiveeffect was observed with L-valine, L-proline,or D-alanine, whereas L-alanine wasequieffective to glycine. Furthermore, glycine had no effect onMTX-induced bleb formation. To test the hypothesis that glycinespecifically blocks formation of a lytic "pore," the loss offluorescence from BAECs transiently expressing GFP and concatemers ofGFP ranging in size from 27 to 162 kDa was examined using time-lapsevideomicroscopy. MTX-induced loss of GFP was rapid, correlated with thesecond phase of dye uptake, and was relatively independent of molecularsize. The MTX-induced loss of GFP from BAECs was completely blocked byglycine. The data suggest that the second "lytic" phase ofMTX-induced endothelial cell death reflects formation of a novelpermeability pathway that allows macromolecules such as GFP or LDH toescape, yet can be prevented by the cytoprotective agents glycine andL-alanine.