Abstract
We examined neuroprotective effects of β-estradiol, dehydroepiandrosterone (DHEA), and dehydroepiandrosterone sulfate (DHEA-S) against N-methyl-d-aspartate (NMDA)-induced neurotoxicity in primary cultured rat hippocampal neurons. All three steroids demonstrated neuroprotective effects. Time-course studies revealed that steroid cotreatment for only 15 min at the same time as exposure to NMDA, but neither pretreatment nor addition of steroids for 24 h after NMDA-mediated neuroprotective effects. This indicates that short-term actions of these steroids are critical for this process. Acute treatment with β-estradiol dose dependently inhibited NMDA-induced intracellular Ca2+ increases, which strongly correlated with its neuroprotective effect via L-type voltage-gated calcium channels. Acute treatment with DHEA, but not with DHEA-S, significantly inhibited nitric oxide (NO) production and Ca2+-sensitive NO synthase (NOS) activity caused by NMDA stimulation. An NOS inhibitor, NG-monomethyl-l-arginine acetate was also protective against NMDA-induced neurotoxicity. These data indicate that β-estradiol may exert neuroprotective effects mainly by reducing Ca2+ increases but that DHEA may act by inhibiting NOS activity. Treatment with the σ-1 receptor (Sig-1R) antagonists rimcazole or BD1063 (1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine dihydrochloride) partially, but significantly, reversed the neuroprotective effect of DHEA-S against NMDA-induced neurotoxicity, whereas muscimol, a GABA-A-receptor agonist, did not. This suggests that the neuroprotective effect of DHEA-S may be mediated via Sig-1R, at least in part. Together, our data suggest that the neurosteroid family members β-estradiol, DHEA, and DHEA-S exert neuroprotective effects through different nongenomic mechanisms.
Footnotes
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This work was supported by a grant-in-aid for Nervous and Mental Disorders from the Ministry of Health and Welfare of Japan and Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation.
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doi:10.1124/jpet.104.067629.
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ABBREVIATIONS: NMDA-R, N-methyl-d-aspartate receptor; NOS, nitric-oxide synthase; NO, nitric oxide; NMDA, N-methyl-d-aspartate; DHEA, dehydroepiandrosterone; DHEA-S, dehydroepiandrosterone sulfate; CNS, central nervous system; GABA-A-R, γ-aminobutyric acid type A receptor; Sig-1R, σ-1 receptor; Sig-2R, σ-2 receptor; l-NMMA, NG-monomethyl-l-arginine acetate; BD1063, 1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine dihydrochloride; MK-801, (–)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; DMSO, dimethyl sulfoxide; SNP, sodium nitroprusside; BSS, balanced salt solution; WST-8, tetrazolium salt; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; ANOVA, analysis of variance; VGCC, voltage-gated calcium channel; nNOS, neuronal nitric-oxide synthase; eNOS, endothelial nitric-oxide synthase; PKC, protein kinase C.
- Received February 25, 2004.
- Accepted May 28, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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