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Articles in PresS, published online ahead of print December 11, 2002
Am J Physiol Cell Physiol, 10.1152/ajpcell.00258.2002
Submitted on May 31, 2002
Accepted on November 27, 2002
1 Rammelkamp Center for Education and Research, MetroHealth Medical Center, Cleveland, OH, USA
2 Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, USA
3 Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, USA; Rammelkamp Center for Education and Research, MetroHealth Medical Center, Cleveland, OH, USA
* To whom correspondence should be addressed. E-mail: wschilling{at}metrohealth.org.
The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial cells (BAECs) is a model for oncotic/necrotic cell death. The cascade is initiated by an increase in cytosolic free Ca2+ concentration ([Ca2+]i) which is followed by the biphasic uptake of vital dyes. The initial phase of dye entry reflects MTX-induced activation of large pores and correlates with surface membrane bleb formation. The second phase, which reflects cell lysis, correlates with lactate dehydrogenase (LDH) release and dramatic bleb dilation (Estacion and Schilling, BMC Physiology 1: 2, 2001). 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 first phase of vital dye uptake, but produced a concentration-dependent (EC50~1 mM) inhibition of the second phase of dye uptake and greatly attenuated LDH release. No cytoprotective effect was observed with L-valine, L-proline, or D-alanine, whereas L-alanine was equi-effective to glycine. Furthermore, glycine had no effect on MTX-induced bleb formation or dilation. Thus, the cytoprotective effect was selective and stereospecific. To test the hypothesis that glycine specifically blocks formation of a lytic "pore" and to determine if pore dimensions change with time, the loss of fluorescence from BAECs transiently expressing GFP and concatemers of GFP ranging in molecular weight from 27-162 kDa, was examined using time-lapse videomicroscopy. MTX-induced loss of GFP was rapid, correlated with the second phase of dye uptake, and was relatively independent of molecular size. The MTX-induced loss of GFP from BAECs was completely blocked by glycine and the cytoprotection extended to at least 48 hrs after MTX treatment. The data suggest that the second "lytic" phase of MTX-induced endothelial cell death represents the formation of a novel permeability pathway that allows macromolecules such as GFP or LDH to escape, yet can be prevented by the cytoprotective agents, glycine and L-alanine.
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