Blockade of maitotoxin-induced endothelial cell lysis by glycine and L-alanine.
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 Ca(2+) concentration ([Ca(2+)](i)), which is followed by the biphasic uptake of vital dyes. The initial phase of dye entry reflects activation of large pores and correlates with surface membrane bleb formation; 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 [Ca(2+)](i) or on the first phase of vital dye uptake but produced a concentration-dependent (EC(50) approximately 1 mM) inhibition of the second phase of dye uptake. No cytoprotective effect was observed with l-valine, l-proline, or d-alanine, whereas l-alanine was equieffective to glycine. Furthermore, glycine had no effect on MTX-induced bleb formation. To test the hypothesis that glycine specifically blocks formation of a lytic "pore," the loss of fluorescence from BAECs transiently expressing GFP and concatemers of GFP ranging in size from 27 to 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. The data suggest that the second "lytic" phase of MTX-induced endothelial cell death reflects 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.
Estacion
Mark
M
Rammelkamp Center for Education and Research, MetroHealth Medical Center, Cleveland, Ohio 44109-1998, USA.
Weinberg
Justin S
JS
Sinkins
William G
WG
Schilling
William P
WP
D000409:Alanine / Q000494:pharmacology*; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000502:physiology; D002417:Cattle; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D019610:Cytoprotection; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D005998:Glycine / Q000494:pharmacology*; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000493:pharmacokinetics; D008164:Luminescent Proteins / Q000493:pharmacokinetics; D008387:Marine Toxins / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D035964:Oxocins / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D010539:Permeability / Q000187:drug effects; D013997:Time Factors; D000095702:Polyether Toxins
D000409:Alanine; D005998:Glycine; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008387:Marine Toxins; D035964:Oxocins; C020394:maitotoxin; D000095702:Polyether Toxins