Pregnenolone sulfate potentiation of NMDA-mediated increases in intracellular calcium in cultured chick cortical neurons

Abstract

Pregnenolone sulfate (PS) has been reported to selectively augment glutamate-induced depolarizations mediated by the NMDA subtype of the glutamate receptor. The present study examines the ability of this neuroactive steroid to potentiate NMDA-mediated increases in intracellular calcium in cultured chick cortical neurons using the fluorescent dye Fura2. PS, in the absence of NMDA and glycine, significantly elevated intracellular calcium at 250 and 500 μM. This increase in free calcium was significantly attenuated at 250 μM PS by the prior addition of 50 μM CNQX, 10 μM dizocilpine or 1 μM nimodipine. NMDA and glycine, when added to the cells in saturating concentrations of 500 and 50 μM, respectively, consistently increased intracellular free calcium over baseline levels. In the presence of NMDA and glycine, both 50 and 100 μM PS produced a further significant rise in intracellular free calcium. The prior addition of CNQX, dizocilpine or both compounds together significantly inhibited this elevation in free calcium. The application of the endogenous polyamine spermine (250 μM) significantly potentiated the response of chick cortical neuronal cells to NMDA and glycine. PS, in the presence of NMDA, glycine and spermine, produced a further increase in intracellular free calcium at concentrations of 50 and 100 μM. The prior application of CNQX, dizocilpine or both compounds together significantly attenuated this rise in free calcium. These data confirm that PS is a positive allosteric modulator of the NMDA receptor and provide evidence that this neurosteroid does not interact with the polyamine modulatory site.