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 interfere with acute excitotoxicity, in delayed retinal cell death. Three hours after exposure of the isolated and intact retina to a 30-min PS pulse, DNA fragmentation as assessed by genomic DNA gel electrophoresis and a modified in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method appeared concurrently with an increase in superoxide dismutase (SOD) activity and thiobarbituric acid-reactive substances (TBARS) levels. At 7 h, the increased amount of DNA laddering was accompanied by a higher number of TUNEL-positive cells in the inner nuclear and ganglion cell layers. Necrotic signs were characterized by DNA smear migration, lactate dehydrogenase (LDH) release, and damage mainly in the inner nuclear layer. PS-induced delayed cell death was markedly reduced by the NMDA receptor antagonists 4-(3-phosphonopropyl)-2-piperazinecarboxylic acid and 3alpha-hydroxy-5beta-pregnan-20-one sulfate but completely blocked after concomitant addition of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. Steroids with antioxidant properties (progesterone, dehydroepiandrosterone and its sulfate ester, and 17beta-estradiol) differently prevented PS-induced delayed cell death. Cycloheximide treatment protected against 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 cycloheximide-sensitive death program downstream of reactive oxygen species generation and lipid peroxidation, turning into secondary necrosis in a retinal cell subset.
Cascio
C
C
Istituto di Biologia dello Sviluppo, CNR, Università di Palermo, Palermo, Italy.
Guarneri
R
R
Russo
D
D
De Leo
G
G
Guarneri
M
M
Piccoli
F
F
Guarneri
P
P
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D003513:Cycloheximide / Q000494:pharmacology; D053938:DNA Fragmentation; D019314:Dehydroepiandrosterone Sulfate / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D015227:Lipid Peroxidation / Q000187:drug effects; D008297:Male; D009498:Neurotoxins / Q000494:pharmacology*; D011284:Pregnenolone / Q000494:pharmacology*; D011374:Progesterone / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology; D012160:Retina / Q000737:chemistry / Q000166:cytology* / Q000201:enzymology; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis / Q000378:metabolism
D000276:Adjuvants, Immunologic; D009498:Neurotoxins; D011500:Protein Synthesis Inhibitors; D017382:Reactive Oxygen Species; D016194:Receptors, N-Methyl-D-Aspartate; D017392:Thiobarbituric Acid Reactive Substances; C018370:pregnenolone sulfate; D013481:Superoxides; D011374:Progesterone; D004958:Estradiol; D019314:Dehydroepiandrosterone Sulfate; D011284:Pregnenolone; D003513:Cycloheximide; D007770:L-Lactate Dehydrogenase; D013482:Superoxide Dismutase