Do Neurosteroids Act As Endogenous Modulators Of Glutamate Receptor Function

Two important unresolved questions are whether neurosteroids play a modulatory role in the normal functioning of the nervous system, and whether neurosteroids contribute to CNS pathology. Neuromodulatory effects on GABAa or NMDA receptors have been reported for many neurosteroids. On the other hand, most of the reported modulatory effects of neurosteroids occur in the micromolar concentration range (except for poten-tiation of the GABAA receptor by allopregnanolone, which can be observed at submicromolar concentrations). Because bulk concentrations of steroids in the CNS are typically in the nanomolar range (2), some mechanism of concentration or focal release would seem to be required for steroids to play a role in the normal functioning of the nervous system. Primary cultures of glial cells containing astrocytes and oligodendro-cytes synthesize cholesterol and PREG from mevalonalactone (90), and mitochondria derived from oligodendrocytes convert cholesterol to PREG (3). This suggests that PREGS may be synthesized and stored in oligodendrocytes. PREGS carries a full negative charge and should not readily diffuse across a lipid bilayer, raising the possibility that it may be concentrated and sequestered in glial cells. Focal release of PREGS from glia could then give rise to local concentrations sufficient to modulate ligand gated ion channels. Thus, PREGS and other sulfated steroids may represent a means of communication between glia and neurons.

Alternatively, it is possible that high CNS steroid levels might occur in particular circumstances. The concentrations of PREGS and other neurosteroids in brain tissue have been shown to increase in response to changes in physiological state. Neurosteroid levels increase in male rats following surgical stress (91,92), electroshock (93), and sexual encounters with female rats (94). In female rats, brain neurosteroid levels fluctuate during the estrous cycle and pregnancy (95). Pregnanolone sulfate, a negative modulator of the NMDA receptor, is the sulfated form of the neurosteroid pregnanolone, which is a potent positive modulator of the GABAA receptor. Pregnanolone sulfate and its stere-oisomers allopregnanolone sulfate and epiallopregnanolone sulfate are major metabolites of PROG (96-98), whereas epipregnanolone sulfate appears to be a minor metabolite (99). The plasma concentrations of these sulfated steroids in women closely parallel the levels of PROG, peaking around the time of parturition. Concentrations of pregnanolone sulfate and allopregnanolone sulfate reach 1-2 ^M in the peripheral circulation of woman during the late stages of pregnancy (96-98). Because they exist as negatively charged species, sulfated steroids would not be expected to easily enter the brain from the periphery in the absence of an active transport system. However the enzymes required for the conversion of PROG to allopregnanolone and the addition of sulfate to neurosteroids are present in the brain (100,101), and it is possible that sulfated steroids are produced either de novo or from circulating PROG. Systemic administration ofPREGS to rats causes an increase in PREGS content within the brain. It has been proposed that PREGS in the circulation may have sulfate removed by the action of peripheral steroid sulfatases, cross the blood brain barrier as PREG, and then be resulfated by steroid neuronal sulfotransferases (102). A similar mechanism may enable sulfated PROG metabolites to enter and accumulate in the CNS.

Sulfated steroids, including PREGS, have been measured in brain (1,91,94,101), but the concentrations of sulfated pregnane steroids have not been determined. If present in the brain at sufficient concentration, PREGS may constitute an additional mechanism for modulation of neuronal activity. Elevation of PREGS levels during CNS damage could constitute an innate neuroprotective mechanism. PREG and pregnanolone do not modulate NMDA-induced currents (19,56), and we have shown that neither of these steroids modulates NMDA-induced neuronal death (C. E. Weaver, Jr., unpublished data). Sulfation therefore converts an inactive neurosteroid to an active form, suggesting that steroid sulfotransferases and sulfatases may play an important role in the modulation of NMDA receptor function in the CNS. It is interesting to note that the apparent KMs for PREG sulfatase measured in adult hippocampus and hippocampal cultures are in the low micromolar range (10 and 14 ^M respectively; C. E. Weaver, Jr., unpublished data). Under physiological conditions the substrate/Km ratio for enzymes is typically between 0.01 and 1 (103). This suggests that the physiological concentration of PREGS is between 0.1 and 10 ^M. This is a concentration range at which PREGS would be expected to have modu-latory effects on NMDA-induced currents and NMDA-induced cell death.

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