Pregnenolone sulfate, a naturally occurring excitotoxin involved in delayed retinal cell death

The present study was designed to investigate the neurosteroid pregnenolone sulfate (PS), known for its ability to modulate NMDA receptors and interfe re with acute excitotoxicity, in delayed retinal cell death. Three hours af ter exposure of the isolated and intact retina to a 30-min PS pulse, DNA fr agmentation as assessed by genomic DNA gel electrophoresis and a modified i n situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end- labeling (TUNEL) method appeared concurrently with an increase in superoxid e dismutase (SOD) activity and thiobarbituric acid-reactive substances (TBA RS) levels. At 7 h, the increased amount of DNA laddering was accompanied b y a higher number of TUNEL-positive cells in the inner nuclear and ganglion cell layers. Necrotic signs were characterized by DNA smear migration, lac tate dehydrogenase (LDH) release, and damage mainly in the inner nuclear la yer. PS-induced delayed cell death was markedly reduced by the NMDA recepto r antagonists 4-(3-phosphonopropyl)-2-piperazinecarboxylic acid and 3 alpha -hydroxy-5 beta-pregnan-20-one sulfate but completely blocked after concomi tant addition of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxalin e-2,3-dione. Steroids with antioxidant properties (progesterone, dehydroepi androsterone and its sulfate ester, and 17 beta-estradiol) differently prev ented PS-induced delayed cell death. Cycloheximide treatment protected agai nst DNA fragmentation and LDH release but failed to prevent the rise in SOD activity and TBARS level. We conclude that a brief PS pulse causes delayed cell death in a slowly evolving apoptotic fashion characterized by a cyclo heximide-sensitive death program downstream of reactive oxygen species gene ration and lipid peroxidation, turning into secondary necrosis in a retinal cell subset.