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Vol. 293, Issue 3, 747-754, June 2000
Laboratory of Molecular Neurobiology, Department of Pharmacology, Boston University School of Medicine, Boston, Massachusetts
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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Modulation of N-methyl-D-aspartate (NMDA)
receptor function by a series of sulfated steroids and dicarboxylic
acid ester analogs of pregnenolone sulfate and pregnanolone sulfate was
investigated in cultured hippocampal neurons. The "bent" steroid
ring structure associated with 5
-stereochemistry favors receptor
inhibition, whereas the more planar ring structure of the pregn-5-enes
and 5
-pregnanes favors potentiation of NMDA-induced
[Ca2+] increases and neuronal cell death. The nature of
the negatively charged group attached to the steroid C3 position is
important for both the neuroprotection afforded by pregnane steroids
and the exacerbation of NMDA-induced neuronal death by pregn-5-enes. Dicarboxylic acid hemiesters of various lengths can substitute for the
sulfate group of the positive modulator pregnenolone sulfate and the
negative modulator pregnanolone sulfate. This result suggests that
precise coordination with the oxygen atoms of the sulfate group is not
critical for modulation and that the steroid recognition sites can
accommodate bulky substituents at C3. The capacity of charged steroids
to enhance or protect against NMDA-induced death of hippocampal neurons
is strongly correlated with modulation of NMDA-induced Ca2+
accumulation, indicating that direct enhancement or inhibition of NMDA
receptor function is responsible for the proexcitotoxic or
neuroprotective effects of these steroids.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Glutamate
is the major excitatory neurotransmitter in the mammalian central
nervous system (CNS). However, excessive exposure to glutamate and its
analogs can result in neuronal death through a process termed
excitotoxicity (Olney, 1986
). Although the
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and
kainate-type ionotropic glutamate receptors mediate excitotoxicity
under certain conditions (Koh et al., 1990), the N-methyl-D-aspartate (NMDA) receptor
has been the major focus of attention.
Under normal physiological conditions, the NMDA receptor plays a vital
role in the synaptic plasticity thought to underlie learning and
memory (Collingridge and Bliss, 1987), most likely as a result of its
ability to transport Ca2+ (Mac Dermott et al.,
1986), which acts as an intracellular second messenger. However,
overstimulation of NMDA receptors can disrupt Ca2+ homeostasis, resulting in an elevated
[Ca2+]i, which can
trigger a number of deleterious Ca2+-dependent
enzymatic cascades, including activation of proteases (Siman and
Noszek, 1988; Rami et al., 1997) and endonucleases (Joseph et al.,
1993) and generation of oxygen free radicals (Beckman et al., 1990;
Dawson et al., 1991; Heinzel et al., 1992), ultimately resulting in
cell death.
NMDA receptor-mediated neuronal death has been linked to certain
neurodegenerative diseases (Choi, 1988; Young et al., 1988; Greenamyre
and Young, 1989; Greenamyre and O'Brien, 1991) as well as to the
neurodegeneration associated with hypoxic-ischemic events (Rothman and
Olney, 1986) and trauma (Gómez-Pinilla et al., 1989). Selective
NMDA receptor antagonists are able to inhibit the
Ca2+-dependent neuronal death caused by
hypoxia-ischemia and glutamate exposure (Choi et al., 1988).
Various steroids, including 17-estradiol (Behl et al., 1995; Miura
et al., 1996) and certain synthetic 21-aminosteroids (Monyer et al.,
1990), have been shown to have neuroprotective properties, and are
thought to antagonize excitotoxicity by scavenging free radicals.
However, steroids also may modulate neurotransmitter receptors
directly. Pregnenolone sulfate (PS), an abundant neurosteroid (Corpéchot et al., 1983), potentiates NMDA-induced currents (Wu et al., 1991; Bowlby, 1993), whereas pregnanolone sulfate
(3-hydroxy-5-pregnan-20-one sulfate; 35S) and
3-hydroxy-5-pregnan-20-one sulfate (35S) inhibit
NMDA-induced currents (Park-Chung et al., 1994; Yaghoubi et al., 1998).
Moreover, the steroid negative modulators 17-estradiol (Weaver et
al., 1997b) and pregnanolone hemisuccinate (35HS; Weaver et al.,
1997a) are neuroprotective, whereas the steroid positive modulator PS
enhances excitotoxicity (Weaver et al., 1998).
We have shown previously that sulfated steroid positive and negative
modulators of the NMDA receptor act through distinct sites to modulate
ligand-gated ion channel activity (Park-Chung et al., 1997). In this
study, we examine how two different types of structural modifications
affect the ability of steroids to interact with these sites to modulate
NMDA-induced Ca2+ accumulation and cell death in
cultures of hippocampal neurons. Results demonstrate that there is a
strong correlation between these two measures of NMDA receptor
modulation, and that a more planar versus bent structure is an
important determinant of selectivity between the positive and negative
modulatory sites associated with the NMDA receptor. In addition,
carboxylic acid esters of various lengths in some cases can substitute
for sulfate at the C3 position. This is of use in the design of
therapeutic agents because the substitution of the hemisuccinate group
for sulfate results in a drug that can cross the blood-brain barrier
and protect against middle cerebral artery occlusion-induced
degeneration of cortical tissue (Weaver et al., 1997a).
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Materials.
Steroids were used at 100 µM, except where
otherwise stated. PS and 17-estradiol were obtained commercially
from Steraloids (Wilton, NH).
20°C and acetic anhydride (1.9 ml) added dropwise over a
30-min period with stirring. The reaction mixture was then warmed to
0°C for 30 min and then the reaction stopped by the addition of
methanol (1.0 ml). After evaporation of the solvents in vacuo, the
residue was partitioned between ethyl acetate (10 ml) and aqueous 1 N
HCl. The organic phase was washed twice with 1 N HCl (10 ml) and water
(10 ml) and evaporated to dryness. The product was crystallized twice
from a mixture of acetone and hexane. The hemioxalate esters were
prepared as described above, but with 568 mg of oxalic acid instead of
formic acid. The hemiglutarate esters were prepared as follows: to a solution of steroid (400 mg) in dry pyridine (6 ml), we added glutaric
anhydride (400 mg) and 4-pyrolidinopyridine (40 mg). The mixture was
allowed to stand at room temperature in the dark for 4 days, when
thin-layer chromatography showed complete disappearance of the steroid
starting material. The reaction mixture was then poured into ice water
(20 ml) and the product extracted with ethyl acetate (20 ml), and the
extract washed with aqueous 1 N HCl (5 ml) and water (5 ml). After
drying the ethyl acetate solution over anhydrous sodium sulfate, the
product was treated with activated charcoal (200 mg) and crystallized
from a mixture of ethyl acetate and hexane. The hemisuccinate esters
were prepared as described above, except that succinic anhydride (225 mg) was used in place of glutaric anhydride, and the reaction required
7 days for completion at room temperature.
Steroids were initially dissolved in 100% dimethyl sulfoxide (DMSO),
then diluted into assay buffer at a final DMSO concentration of 0.5%
and sonicated for 20 min. All other solutions also contained 0.5%
DMSO. Except where otherwise noted, the final steroid concentration was
100 µM. Pregnenolone and 35 were used at 20 and 50 µM,
respectively, because higher concentrations tended to precipitate in
the assay buffer.
Cell Culture.
Predominantly neuronal cultures were prepared
from hippocampal tissue of fetal Sprague-Dawley rats on day 18 of
embryonic development as described in Brewer and Cotman (1989).
Briefly, hippocampal cells were dissociated by trituration in
Ca2+/Mg2+-free Hanks'
basic salt solution (Life Technologies, Gaithersburg, MD)
supplemented with 4.2 mM bicarbonate, 1 mM sodium pyruvate, 20 mM
HEPES, and 3 mg/ml BSA. Dissociated cells were then pelleted by
centrifugation (500g; 4 min). The resulting pellet was
suspended in Dulbecco's modified Eagle's medium (DMEM; Life
Technologies) supplemented with 2.4 mg/ml BSA, 26.5 mM sodium
bicarbonate, 1 mM sodium pyruvate, 20 mM HEPES, 10% fetal bovine serum
(Life Technologies), 100 U/ml penicillin, 100 µg/ml streptomycin
(Life Technologies), and a modification of Brewer's B16 defined
components (with 250 nM vitamin B12 and without
catalase, glutathione, and superoxide dismutase; Pike et al., 1993).
Cells were then plated onto poly(L-lysine)-coated
24-well culture dishes (Nunclon, Naperville, IL) at a density of 15,000 cell/cm2 and maintained in a humidified
atmosphere containing 5% CO2, 95% air at
37°C. After 48 h, non-neuronal cell division was inhibited by a
48-h exposure to 1 µM cytosine arabinoside. Cultures were subsequently maintained in serum-free DMEM plus defined components and
were used for experiments 16 to 24 days after plating. Cultures prepared in this manner contained ~80% neurons, as indicated by staining of non-neuronal cells with antibody to glial fibrilary acidic
protein and staining of neurons with antibody to neuron-specific enolase.
Intracellular Calcium Concentration Measurements.
NMDA-induced increases in
[Ca2+]i were measured
with the Ca2+-sensitive fluorescent dye Fluo-3 AM
(Molecular Probes, Eugene, OR) and a Cytofluor 2350 (Perceptive
Biosystems, Cambridge, MA) fluorescence plate reader, with excitation
and emission filters of 485 and 530 nm, respectively. Hippocampal
neurons were loaded with dye by incubating cultures with 10 µM Fluo-3
AM and 0.05% (w/v) Pluronic F-127 (Molecular Probes), a nonionic
detergent, for 2 h at 37°C. Fluo-3 AM and Pluronic F-127 were
dissolved in DMSO (final concentration of 0.5%). Cultures were then
washed three times with assay buffer (120 mM NaCl, 5.4 mM KCl, 1.8 mM
CaCl2, 15 mM glucose, 25 mM Tris-HCl, and 0.5 µM tetrodotoxin; pH 7.4) to remove excess dye. For the purposes of
calibration, other plate wells were rinsed instead with assay buffer in
which 1.8 mM MnCl2 replaced 1.8 mM
CaCl2 (Fmin buffer).
[Ca2+]i was calculated
with the equation:
[Ca2+]i = KD[F Fmin]/[Fmax F], where
F is the fluorescence measured, Fmin is the
fluorescence in the absence of calcium (determined in
Fmin buffer after the addition of 10 µM the
Ca2+ ionophore A-23187),
Fmax is the fluorescence of the
Ca2+-saturated dye (determined in assay buffer
after the addition of 10 µM A-23187), and
KD = 320 nM (the equilibrium
dissociation constant for the binding of Ca2+ to
Fluo-3 AM). Fluorescence measurements were made before and 40 s
after the addition of NMDA. Steroid or vehicle (0.5% DMSO) was added
10 min before the addition of NMDA. NMDA was dissolved in DMEM;
steroids and A-23187 were dissolved in DMSO. DMSO also was added to
controls to maintain a constant final DMSO concentration of 0.5%. Data
are expressed as the percentage change in the NMDA-induced increase in
[Ca2+]i in the presence
of the indicated steroid. None of the steroids tested significantly
altered [Ca2+]i in the
absence of NMDA.
NMDA-Induced Cell Death. Primary cultures of rat hippocampal neurons were exposed to NMDA (dissolved in DMEM) for 15 min (acute exposure) or 16 h (chronic exposure). In acute exposure experiments, cultures were treated with steroid, MK-801 (Research Biochemicals International; dissolved in DMEM), or vehicle during and/or after NMDA exposure. Steroids were dissolved in DMSO (0.5% final concentration), and all treatment media contained 0.5% DMSO. In chronic exposure experiments, cultures were additionally treated with steroid, vehicle, SR-95531 (Research Biochemicals International; dissolved in DMEM), 6,7-dinitroquinoxaline-2,3-dione (Research Biochemicals International; dissolved in DMEM), or MK-801 (dissolved in DMEM) during NMDA exposure. After exposure, cultures were washed three times with medium from sister cultures (conditioned medium). After the final wash, steroid or vehicle was reintroduced. Drugs were added to cultures in 25 µl of conditioned medium to yield a final volume of 0.25 ml/well. Except where otherwise noted, final steroid concentration was 100 µM.
The ability of neurons to exclude trypan blue was used to quantitate cell viability (Dawson et al., 1991). Twenty-four hours after acute and
16 h after chronic exposure to NMDA, culture medium was replaced
by 0.4% trypan blue in 0.1 M PBS (pH 7.4) and placed in a humidified
incubator for 10 min. Cultures were then fixed with 4%
paraformaldehyde in PBS for 30 min, at which time the fixative was
replaced with PBS. The number of stained and unstained neurons were
counted in four high-power fields per culture well with an inverted
phase contrast microscope under both bright field and phase contrast
settings. At this stage in vitro, neurons were easily distinguishable
from non-neuronal cells in vitro by the presence of oval, phase-bright
somata and by the morphology of their processes, as confirmed by
experiments in which neurons and non-neuronal cells were stained with
antibodies as described above. Experiments were performed in
triplicate, and all assessments were made with the experimenter blind
to the treatment of each culture well. Percentage of cell death is
expressed as follows: (number of trypan blue stained neurons)/(total
number of neurons) × 100%. The basal level of neuronal death
(termed background), measured in controls lacking NMDA and PS, was 0 to
10% and was subtracted from the raw data in each experiment.
Statistical Analysis.
The degree of modulation of
NMDA-induced Ca2+ influx and cell death is
expressed as the percentage of change, defined as (I'/I 1) × 100%, where I and I' are the NMDA-induced responses in the absence
and presence of modulator, respectively. All data are expressed as
mean ± S.E. Statistical significance was evaluated with 95% CL
unless otherwise noted.
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Effect of Geometry on Modulation of NMDA Neurotoxicity by Sulfated
Steroids.
We have identified several reduced metabolites of
progesterone that modulate currents evoked by NMDA (Farb and Gibbs,
1996). 35S was the first steroid found to inhibit NMDA-induced
currents in cultured neurons (Park-Chung et al., 1994). Consistent with this negative modulation of the NMDA response, 35S (100 µM) reduces the 5 µM NMDA-evoked Ca2+ influx by
32 ± 5% (n = 8) (Fig.
1A). Furthermore, 35S protects neurons from the cell death produced by acute (15 min) exposure to
NMDA, raising the EC50 for NMDA-induced neuronal
death from 28 to 71 µM and lowering the maximal NMDA-induced
excitotoxicity from 80 to 63% cell death (Fig. 1B). This effect is
dose dependent, with an EC50 of 45 µM and a
97% maximal inhibition of the cell death induced by 30 µM NMDA (Fig.
1C). The neuronal death caused by chronic (16 h) NMDA treatment also is
attenuated by 35S, which, under these conditions, reduces the
NMDA Emax from 86 to 70% cell death
without affecting the NMDA EC50 (Fig. 1D).
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5S
inhibits currents elicited by AMPA and kainate (Park-Chung et
al., 1994), raising the possibility that the effect of the steroid on
NMDA-induced neuronal death might not be specific to the NMDA receptor.
10 µM 6,7-dinitroquinoxaline-2,3-dione [DNQX, a selective non-NMDA
glutamate receptor antagonist (Honore et al., 1988)] and 100 µM
SR-95531 (a selective 
-aminobutyric acidA
receptor antagonist; Farrant and Webster, 1989) have no effect on
NMDA-induced neuronal death (data not shown), arguing that the
-aminobutyric acidA, AMPA, and kainate
receptor types do not play a significant role in this process.
Although 35S nearly eliminates the toxic effects of an acute
exposure to 30 µM NMDA, its stereoisomer
3-hydroxy-5-pregnan-20-one sulfate (35S) is only half as
effective, producing a 47 ± 12% (n = 4)
inhibition of neuronal death (Fig. 2).
Strikingly, whereas 35S reduces NMDA-induced currents and
neuronal death (86 ± 3% inhibition; n = 6), its
5-isomer 3-hydroxy-5-pregnan-20-one sulfate (35S), both
potentiates NMDA-induced currents (Park-Chung, 1997) and exacerbates
neuronal death by 40 ± 7% (n = 17). This shows
that, as with their effects on NMDA-evoked currents, the neuroprotective effects of these sulfated steroids are contingent on
the stereochemistry of the A-B ring junction, whereas stereochemistry at C3 appears to be important only for 5-isomers.
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Effect of Charge and Chain Length on Modulatory Actions of
35S Analogs.
To elucidate further the structure-activity
relationships for modulation of the NMDA receptor by steroids, we
synthesized a series of carboxylic acid derivatives of 35 (Fig.
3A). The three negatively charged
derivatives, 35HS, pregnanolone hemioxylate (35HO), and
pregnanolone hemiglutarate (35HG) are about equally effective,
inhibiting the NMDA-induced rise in
[Ca2+]i by ~40% in
primary hippocampal cultures, whereas the uncharged 35 and
pregnanolone formate (35F) have no significant effect on
NMDA-induced Ca2+ accumulation (Fig. 3B).
Similarly, 35HO, 35HS, and 35HG are neuroprotective,
reducing neuronal death caused by acute exposure to 30 µM NMDA by
35 ± 6 (n = 10), 54 ± 3 (n = 24), and 38 ± 6% (n = 9), respectively (Fig.
3C), whereas 35 and 35F do not exhibit significant
neuroprotective activity.
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Effect of Charge and Chain Length on Modulatory Effects of PS
Analogs.
PS is a potent positive modulator of NMDA receptor
function (Wu et al., 1991; Bowlby, 1993). To evaluate the role of the
sulfate group at C3, we examined the effects of pregnenolone,
pregnenolone formate (PF), pregnenolone hemioxylate (PHO), pregnenolone
hemisuccinate (PHS), and pregnenolone hemiglutarate (PHG; Fig.
4A).
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Modulation of Ca2+ Accumulation Correlates with
Excitotoxicity.
The excitotoxicity produced by excessive NMDA
receptor stimulation has been implicated in the neurodegeneration
associated with a number of CNS diseases and insults (Rothman and
Olney, 1986; Gómez-Pinilla et al., 1989; Greenamyre, 1991;
Greenamyre and O'Brien, 1991; Weaver et al., 1997a). Evidence
indicates that neuronal death results from NMDA receptor-mediated
activation of a Ca2+-dependent enzymatic cascade
involving lipid peroxidation and protein and DNA degradation (Choi,
1992; Chan, 1996). Our present results, demonstrating that modulation
by steroids of NMDA-induced Ca2+ uptake is
highly correlated with modulation of NMDA-induced neuronal death
(Fig. 5), support this view, and
indicate that this rapid functional assay can be usefully used to
identify steroids with neuroprotective activity.
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5S also markedly inhibits
NMDA-induced changes in
[Ca2+]i and neuronal
death under both acute and chronic exposure conditions, consistent with
our previous finding that 35S inhibits NMDA-induced currents in
neurons maintained in primary culture (Park-Chung et al., 1994). It is
interesting that, in acute treatments, 35S reduces both the NMDA
EC50 and Emax
for causing cell death, while only reducing the
Emax in chronic treatments. The reason
for this difference is unclear, but it may indicate metabolic
conversion of 35S, such as through the action of a steroid
sulfatase, during the course of the chronic treatment, or an adaptive
change at the NMDA receptor itself.
Stereochemistry of NMDA Receptor Modulation.
To investigate
the structural requirements for steroid inhibition of NMDA-induced
neuronal death, 35 and stereoisomers of 35S were assayed
for activity, as were several related synthetic pregnane steroids.
35S is as effective as 35S at protecting against the
neuronal death produced by acute exposure to NMDA. This suggests that
the stereochemistry at C3 is not critical for inhibition of
NMDA-induced neuronal death by the C5-pregnane isomers. Notably, the
isomer with 5-stereochemistry, 35S, exhibits reduced
neuroprotective activity compared with the 5-isomer. 35S is
about half as effective as 35S and 35S in protecting against NMDA-induced cell death, whereas 35S actually exacerbates the toxicity of NMDA and potentiates the NMDA-induced elevation of
[Ca2+]i. These results
are in agreement with previous electrophysiological studies of
voltage-clamped chick spinal cord neurons in primary culture (Fig. 2),
in which 35S and 35S are strong inhibitors of the
NMDA-induced current, 35S is a weaker inhibitor, and 35S
weakly potentiates the NMDA response (Park-Chung et al., 1997).
-stereochemistry on the
structure of the steroid molecule is to flatten out the ring system
into a more planar configuration, much like the effect of the C5-C6
double bond in the pregn-5-ene series (Fig. 2). Because competition
experiments indicate that positive and negative modulation by steroids
are mediated by distinct sites (Park-Chung et al., 1997), it seems
likely that the more planar ring structure of the pregn-5-enes and
5-pregnanes improves the fit of the steroid molecule to the
potentiating site and/or impairs its fit into the inhibitory site.
Importance of a Negatively Charged Group at C3.
To investigate
the structural requirements for the C3 ester, a carboxylic acid
chain-length series was synthesized in which the sulfate group of PS or
35S was replaced by a formate, hemioxylate, hemisuccinate, or
hemiglutarate group. The parent compound, 35, is without effect
on NMDA-induced Ca2+ influx and neuronal death,
as is its uncharged formate derivative 35F. However, the three
charged derivatives, 35HO, 35HS, and 35HG, reduce
Ca2+ accumulation and cell death of hippocampal
neurons resulting from NMDA application.
5 and 35F is consistent with our
previous observation that 35HS, but not the uncharged
pregnanolone hemisuccinate methyl ester, inhibits NMDA-induced current
in chick spinal cord neurons (Park-Chung et al., 1997). Therefore,
inhibition of NMDA receptor function requires the presence of a
negative charge adjacent to the C3 position, and it is not a
nonspecific effect of the 35 steroid nucleus. A degree of
tolerance for the geometry of the charged group at C3 is suggested by
the fact that a range of lengths (from the relatively short hemioxalate group to the five-carbon hemiglutarate group) is able to confer inhibitory activity.
As with the pregnane steroids, the group esterified at the C3 hydroxyl
group plays a crucial role in determining activity of pregn-5-ene
derivatives. The uncharged parent compound pregnenolone and its
uncharged derivative PF do not potentiate NMDA-stimulated Ca2+ accumulation or exacerbate NMDA-induced
neuronal death. In contrast, the negatively charged PHO, PHS, and PHG
potentiate the action of NMDA in both assays. Interestingly, PHO,
although negatively charged, is significantly less effective than PHG
in enhancing Ca2+ accumulation by hippocampal
neurons, suggesting that precise positioning of the negative charge is
important for potentiation by pregn-5-ene derivatives. The lower
activity of PHO is a bit surprising because PHO has the shortest chain
length, such that the position of its charged carboxyl might be
expected to be most similar to that of the even smaller sulfate
group. However, molecular modeling indicates that the
hemisuccinate and hemiglutarate groups have much greater conformational
freedom than the hemioxylate group, perhaps allowing more favorable
positioning of the negatively charged carboxyl group in the binding site.
The finding that negatively charged carboxylic acid esters can
substitute for the sulfate ester at the C3 position offers prospects
for modifying the steroid nucleus to optimize pharmacological and
pharmacokinetic properties. Carboxylic acid derivatives of neuroactive
steroids may offer improved penetration into the CNS without
susceptibility to hydrolysis by sulfatases. This supposition is
supported by the observation that 35HS is effective as a hypnotic, anticonvulsant, and analgesic, and reduces the neuronal death
that results from middle cerebral artery occlusion in rats, a model of
stroke (Weaver et al., 1997a). Thus, it may be possible to rationally
design steroidal-based and nonsteroidal therapeutics for the treatment
of stroke and disorders arising from the overactivation of the NMDA
receptor. Carboxylic acid derivatives of the pregn-5-ene steroids have
not been tested in vivo, but based on the present results might be
expected to exhibit memory-enhancing effects such as have been
described for pregnenolone sulfate (Isaacson et al., 1994; Flood et
al., 1995; Vallee et al., 1997).
| |
Footnotes |
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Accepted for publication February 14, 2000.
Received for publication December 21, 1999.
1 This study was supported by National Institute of Mental Health MH-49469. C.E.W. was supported in part by a Pharmacology-Clinical Pharmacology Research Fellowship Award from the Pharmaceutical Research and Manufacturers of America Foundation and a Medical Student Research Fellowship Award from the American Heart Association.
2 C.E.W. and M.B.L. contributed equally to this study.
3 Currrent address: Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037.
Send reprint requests to: David H. Farb, Department of Pharmacology, Boston University School of Medicine, 715 Albany St., Boston, MA 02118.
| |
Abbreviations |
|---|
CNS, central nervous system;
AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid;
NMDA, N-methyl-D-aspartate;
PS, pregnenolone
sulfate;
35S, 3-hydroxy-5-pregnan-20-one sulfate;
35S, 3-hydroxy-5-pregnan-20-one sulfate;
35HS, pregnanolone hemisuccinate;
DMSO, dimethyl sulfoxide;
DMEM, Dulbecco's
modified Eagle's medium;
35S, 3-hydroxy-5-pregnan-20-one
sulfate;
35S, 3-hydroxy-5-pregnan-20-one sulfate;
35HO, pregnanolone hemioxylate;
35HG, pregnanolone
hemiglutarate;
35F, pregnanolone formate;
PF, pregnenolone
formate;
PHO, pregnenolone hemioxylate;
PHS, pregnenolone
hemisuccinate;
PHG, pregnenolone hemiglutarate.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Their role in long-term potentiation.
Trends Neurosci
10:
288-293.-Hydroxy-5-pregnan-20-one sulfate: A negative modulator of the NMDA-induced current in cultured neurons.
Mol Pharmacol
46:
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; DMSO control) and 4 (
; 3
P < .05, significantly less protection than 3
