Abstract
Neurosteroids are endogenous steroids acting in the central nervous system. They participate in synaptic plasticity, memory and learning, Alzheimer's disease, and certain drug reward. Some mechanisms behind these effects are thought to be nongenomic, e.g., they modulate the function of the N-methyl-d-aspartate (NMDA) receptor complex. In this study, we used a Chinese hamster ovary cell line stably transfected with NMDA receptor constituents NR1/NR2B, to investigate the effects of nanomolar concentrations of the neurosteroids pregnenolone sulfate (PS) and pregnanolone sulfate (3α5βS) on binding of the radioligand [3H]ifenprodil. Neither of the steroids displaced [3H]ifenprodil, but both induced a shift in its fit from one to two binding sites. The effects of the neurosteroids were also measured as changes in intracellular calcium ([Ca2+]i) after glutamate stimulation. Although the steroids did not alter the response to glutamate, they influenced the extent of ifenprodil blockade of the receptor: PS increased and 3α5βS decreased this effect. The coincubation of several NMDA receptor ligands in the assay indicated that PS and 3α5βS act via different binding sites from those for glutamate, glycine, and dithiothreitol. Combining the two steroids revealed that they do not share a common binding site. In conclusion, these results substantiate previous evidence of the allosteric modulatory effect induced by PS and 3α5βS on NMDA receptors at nanomolar concentrations. The neurosteroid-mediated modulation of the receptor is also reflected in an altered glutamate stimulated [Ca2+]i, in response to ifenprodil.
Footnotes
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This study was funded by the Swedish Medical Research Council Grant 9459 and Gunvor och Josef Anérs stiftelse.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.130518.
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ABBREVIATIONS: NMDA, N-methyl-d-aspartate; NR, N-methyl-d-aspartate receptor; PS, pregnenolone sulfate (5-pregnen-3β-ol-20-one sulfate); 3α5βS, pregnanolone sulfate (5β-pregnan-3α-ol-20-one sulfate); CHO, Chinese hamster ovary; APV, dl-2-amino-5-phosphonopentaoic acid; BSS, balanced salt solution; DTT, dithiothreitol; Fura-2 AM, Fura-2 acetomethoxy ester; ANOVA, analysis of variance.
- Received August 28, 2007.
- Accepted November 14, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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