Steroid sulfation occurs in nervous tissue and endogenous sulfated steroids can act as positive or negative modulators of N-methyl-D-aspartate (NMDA) receptor function. In the current study, structure-activity relationships for sulfated steroids were examined in voltage-clamped chick spinal cord and rat hippocampal neurons in culture and in Xenopus laevis oocytes expressing NR1(100) and NR2A subunits. The ability of pregnenolone sulfate (a positive modulator) and epipregnanolone sulfate (a negative modulator) to compete with each another, as well as with other known classes of NMDA receptor modulators, was examined. The results show that steroid positive and negative modulators act at specific, extracellularly directed sites that are distinct from one another and from the spermine, redox, glycine, Mg2+, MK-801, and arachidonic acid sites. Sulfated steroids are effective as modulators of ongoing glutamate-mediated synaptic transmission, which is consistent with their possible role as endogenous neuromodulators in the CNS.
Release of the excitatory neurotransmitter glutamate and the excessive stimulation of N-methyl-Daspartate (NMDA)-type glutamate receptors is thought to be responsible for much of the neuronal death that occurs following focal hypoxia-ischemia in the central nervous system. Our laboratory has identified endogenous sulfated steroids that potentiate or inhibit NMDA-induced currents. Here we report that 3␣-ol-5-pregnan-20-one hemisuccinate (3␣5HS), a synthetic homologue of naturally occurring pregnanolone sulfate, inhibits NMDA-induced currents and cell death in primary cultures of rat hippocampal neurons. 3␣5HS exhibits sedative, anticonvulsant, and analgesic properties consistent with an action at NMDA-type glutamate receptors. Intravenous administration of 3␣5HS to rats (at a nonsedating dose) following focal cerebral ischemia induced by middle cerebral artery occlusion significantly reduces cortical and subcortical infarct size. The in vitro and in vivo neuroprotective effects of 3␣5HS demonstrate that this steroid represents a new class of potentially useful therapeutic agents for the treatment of stroke and certain neurodegenerative diseases that involve over activation of NMDA receptors.Exposure of neurons to the excitatory neurotransmitter glutamate causes an increase in the concentration of intracellular free Ca 2ϩ (1) and initiates the process of excitotoxic cell death (2). There are at least three pharmacologically distinct ionotropic glutamate receptors that differ in their sensitivity to the selective agonists N-methyl-D-aspartate (NMDA), ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate. Whereas excessive activation of NMDA, AMPA, or kainate receptors results in neurotoxicity, specific inhibition of NMDA receptors is sufficient to attenuate most of the neuronal death that develops after in vitro hypoxia, exposure to glutamate, in vivo ischemia, or hypoglycemia (3). As Ca 2ϩ passes through the NMDA receptor operated ion channel, it is believed that this receptor subserves a crucial role in mediating excitotoxicity. Therefore, it has been proposed that over activation of NMDA receptors may be an obligatory phase preceding neuronal death that occurs following focal cerebral ischemia induced stroke in humans.Neuroactive steroids have been shown to directly modulate excitatory and inhibitory amino acid receptor function (4-7). Therefore the possibility is raised that certain steroids might be useful neuroprotective agents. The endogenous neurosteroid pregnenolone sulfate probably acts as a positive allosteric modulator of the NMDA receptor by enhancing NMDAinduced inward currents and the subsequent rise in cytoplasmic free calcium (8, 9), whereas 3␣-ol-5-pregnan-20-one sulfate (3␣5S) is a negative modulator of NMDA-induced currents (6) and inhibits NMDA-stimulated increases in intracellular calcium (10). The present study examines the in vitro and in vivo neuroprotective activity of 3␣-ol-5-pregnan-20-one hemisuccinate (3␣5HS) ( Fig. 1; a synthetic analog of the endogenou...
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