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Keywords:

  • cytochrome P450scc;
  • GABAA receptors;
  • neurotoxicity;
  • NMDA receptors;
  • pregnenolone and pregnenolone sulphate synthesis;
  • R (+)-p-aminoglutethimide

Abstract

Here we investigated the possible regulation of neurosteroidogenesis by N-methyl-d-aspartic acid (NMDA) receptor activation and addressed the hypothesis that neurosteroid synthesis may be involved in acute excitotoxicity. In the isolated retina, exposure to NMDA modified pregnenolone and pregnenolone sulphate formation. This effect was dose and time dependent, the synthesis being increased by relatively moderate NMDA doses (1–100 μm) within 30 min exposure and reduced to its control value by 60 min or by raising drug concentrations. NMDA-stimulated neurosteroid synthesis was blocked by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine hydrogen maleate (MK-801) and 3(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (CPP), depended on extracellular calcium and reproduced by glutamate. Lactate dehydrogenase (LDH) release and morphological analysis revealed that retinal cell viability was not significantly affected after 30 min exposure to 50 μm NMDA, but severe cell damage occurred by 60 min. When the GABAA (γ-aminobutyric acid) receptor agonist muscimol (1–1000 μm), known to activate retinal neurosteroidogenesis, was added together with NMDA, no additional increase in neurosteroid synthesis was observed, and NMDA-induced LDH release remained unchanged. However, exposure to a high concentration of muscimol alone (500 μm) provoked a similar degree of toxicity to NMDA. By contrast, bicuculline abolished the increase in neurosteroidogenesis and LDH release. Similarly, pretreatment with R (+)-p-aminoglutethimide (AMG), an inhibitor of cholesterol side-chain cleavage cytochrome P450, attenuated acute retinal cell damage. The inhibitory nature of AMG on NMDA-stimulated neurosteroidogenesis was confirmed in the observation that drug treatment reduced pregnenolone content and did not affect the bindings of [3H] MK-801 and [3H] muscimol. The results demonstrate that NMDA receptors regulate neurosteroidogenesis through a transneuronal mechanism, which implies GABAA receptor activation. The early NMDA-mediated stimulation of neurosteroid synthesis seems to play a critical role in acute excitotoxicity; consequently, its inhibition is likely to delay neuronal cell death.