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Vol. 52, Issue 6, 1113-1123, 1997
Laboratory of Molecular Neurobiology, Department of Pharmacology, Boston University School of Medicine, Boston, Massachusetts 02118 (M.P.-C., F.-S.W., A.A.M., T.T.G., D.H.F.), and Department of Psychiatry, University of California School of Medicine, San Diego, California 92161 (R.H.P.)
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Summary |
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Summary
Introduction
Procedures
Results
Discussion
References
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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 NR1100 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.
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Introduction |
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Summary
Introduction
Procedures
Results
Discussion
References
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Nervous tissue synthesizes numerous steroids from cholesterol. Such neurosteroids have been proposed to control neuronal excitability by modulating ligand and/or voltage-gated ion channels (1, 2). Increasing evidence suggests that certain steroids also play a critical role in important physiological processes such as learning, memory, and aging (3, 4). This hypothesis is consistent with the findings that the levels of the neurosteroids dehydroepiandrosterone and its sulfate, DHEAS, are decreased with age (5) and DHEAS improves memory in aging mice (6). In addition, the risk of Alzheimer's disease and related dementia has been shown to be decreased in women who have received estrogen replacement therapy (7).
PS, one of the most abundant neurosteroids, has diverse modulatory
effects on ligand-gated ion channels, acting as a negative modulator of
-aminobutyric acidA (8, 9), glycine (10), kainate, and AMPA receptors, while positively modulating the NMDA receptor (11). PS and some structurally related derivatives also
augment NMDA receptor-mediated increases in intracellular Ca2+ in cultured rat hippocampal neurons (12,
13). Behavioral studies have shown that PS increases the convulsant
potency of NMDA (14) and enhances memory retention in mice (3, 15) and
memory performance in the rat when injected directly into the nucleus
basalis magnocellularis (16). PS also prevents NMDA receptor
antagonist-induced deficits in a passive avoidance memory task (17) and
antagonizes dizocilpine-induced amnesia in rats (18). Chronic
inhibition of steroid sulfatase activity by estrone sulfamate enhances
passive avoidance memory (19). These observations suggest that the
behavior of animals after PS administration or after inhibition of
sulfatase activity is altered in a manner that is consistent with
positive modulation of NMDA receptor function.
In contrast, 3
5
S (pregnanolone sulfate) inhibits the NMDA
response by a voltage-independent, noncompetitive mechanism (20). However, little is known about the steroid modulatory site or sites of
the NMDA receptor. In particular, it is not known whether positive and
negative steroid modulators of the NMDA receptor act at the same site,
nor is it known whether the site or sites of steroid action correspond
to the sites of action of other known NMDA receptor modulators, such as
polyamines (21), redox agents (22), arachidonic acid (23), and MK-801
(24).
In the current study, we examined the effects of sulfated steroids on
the current induced by NMDA in primary cultures of voltage-clamped chick spinal cord, rat hippocampal neurons, and
NR1100 + NR2A subunits expressed in Xenopus
laevis oocytes. A variety of sulfated steroids modulate the NMDA
response in either a positive or negative direction with a high degree
of structural specificity. 35S (epipregnanolone sulfate), a PS
analog, inhibits the NMDA response. 35S and PS do not act through
the spermine, arachidonic acid, or redox sites of the NMDA receptor,
suggesting that these sulfated steroids act through a unique modulatory
site or sites associated with the NMDA receptor. Surprisingly, the
interaction between PS and 35S is not competitive, demonstrating
the presence of independent pathways for negative as well as positive
modulation of the NMDA response. Furthermore, the modulation of
spontaneous EPSCs by PS and 35S demonstrates that these steroids
have the capacity to exert powerful effects on excitatory synaptic
transmission and brain excitability.
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Experimental Procedures |
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Summary
Introduction
Procedures
Results
Discussion
References
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Tissue culture.
Cultures of spinal cord neurons were
prepared from 7-day chick embryos as described previously (10).
Cultures of hippocampal neurons were prepared as described by Brewer
(25) with some modifications. Dissociated hippocampal cells from 18-day
Sprague-Dawley rat embryos were plated onto 35-mm culture dishes in
Dulbecco's modified Eagle's medium supplemented with 2.4 mg/ml bovine
serum albumin, 26.5 mM sodium bicarbonate, 1 mM
sodium pyruvate, 20 mM HEPES, 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and a modification of
Brewers B16 defined components (with 250 nM vitamin B12 and
without catalase, glutathione, and superoxide dismutase). All cultures
were maintained in a humidified atmosphere of 5%
CO2/95% air at 37°. Non-neuronal cell division was inhibited by exposure to 10
6 M
ara-C. The ara-C was added to spinal cord cultures 36 hr after plating.
This medium was removed 1 day later and replaced with fresh medium.
Cultured chick spinal cord neurons were used in experiments 2-4 weeks
after plating. The ara-C was added to rat hippocampal cultures 48 hr
after plating. This medium was removed 48 hr later and replaced with
serum-free Dulbecco's modified Eagle's medium plus defined
components. Cultured rat hippocampal neurons were used for experiments
14-18 days after plating.
Whole-cell current recordings.
Whole-cell currents were
recorded by the whole-cell variant of the patch-clamp technique (26).
Patch electrodes were fabricated with a double pull from borosilicate
glass microcapillary pipettes (Fisher Scientific, Fair Lawn, NJ) on a
David Kopf vertical pipette puller (model 700D). Electrode resistance
was 4.5 ± 0.06 M
(57 microelectrodes) when filled
with intracellular solution. The electrode solution usually contained
150 mM CsCl, 3 mM NaCl, 11 mM EGTA,
and 10 mM HEPES (pH adjusted to 7.2 with CsOH). To measure spontaneous excitatory synaptic currents, a low
Cl concentration (10 mM) pipette
solution (10 mM KCl, 3 mM sodium gluconate, 140 mM potassium gluconate, 11 mM EGTA and 10 mM HEPES, pH adjusted to 7.2 with KOH) was used to inhibit
Cl currents. The bath solution contained 150 mM NaCl, 4 mM KCl, 1 mM
CaCl2, 1 mM
MgCl2, and 10 mM HEPES, pH adjusted
to 7.2 with NaOH. All experiments were performed at room temperature
(23-25°) and, except where otherwise noted, at a holding potential
of 
70 mV.
(57 neurons), was
compensated (>53%). Currents were filtered at 1 kHz using an
eight-pole Bessel filter and digitized (40 msec/point) using an on-line
data acquisition system (pClamp; Axon Instruments). To measure cell
capacitance, a hyperpolarizing step (5 mV, 8 msec) was applied, and the
area of the resultant capacitative current spike was calculated by numerical integration.
Pregnenolone hemisuccinate methyl ester was prepared by treatment of
the parent compound in methanol at 0° with an etherial solution of
diazomethane in the usual manner. PHS and PS (sodium salt) were
obtained from Steraloids (Wilton, NH). All other steroid sulfates were
synthesized as their trimethylammonium salts according to the procedure
of Dusza et al. (27). Their structures were established by
elemental analysis and NMR spectroscopy. Three-dimensional structural
models were constructed by energy minimization using RASMOL.
Stock solutions of steroids were prepared in dimethylsulfoxide (final
concentration, 0.5% v/v). All other drug solutions, including NMDA and
external buffer (in the pressure pipette), also contained 0.5%
dimethylsulfoxide, which by itself had little or no effect on the
NMDA-induced current. All other drugs were obtained from Sigma, with
the exception of spermine tetrahydrochloride (Aldrich).
Drug solutions were applied to single neurons by pressure ejection (15 p.s.i.) from seven-barrel pipettes (28, 29). The drug solution in the
pressure pipette rapidly and effectively replaces the solution
surrounding the target neuron, with <10% dilution. To measure
responses to exogenously applied agonists, neurons received a 10-sec
prepulse of either external buffer or drug solution, followed by a
10-sec application of agonist or agonist plus drug, followed by a
10-20-sec pulse of external buffer solution. A period of 2-3 min was
allowed between successive applications of agonist. For spontaneous
excitatory postsynaptic current experiment, drugs were applied to the
target neuron by pressure ejection for a total of 80 sec; spontaneous
activity from the second half of the application period was analyzed
for each neuron. For each experiment, two 40-sec traces were analyzed
for each neuron. The target neuron was washed by pressure application
of buffer for 2-3 min between drug applications.
Oocyte electrophysiology: X. laevis expression system. mRNA was prepared through in vitro transcription of NR1100 and NR2A cDNAs using the mMessage mMachine kit (Ambion, TX). NR1100 and NR2A clones were kindly provided by Dr. R. S. Zukin (Albert Einstein College of Medicine, New York, NY) and Dr. S. Nakanishi (Faculty of Medicine, Kyoto University, Kyoto, Japan), respectively. The oocytes were removed from X. laevis frogs. After defolliculation, isolated oocytes were transferred into glass petri dishes containing ND96 solution [96 mM NaCl, 1 mM MgCl2, 2 mM KCl, 1.8 mM CaCl2, 5 mM HEPES, 2.5 mM pyruvate (pH adjusted to 7.4 with NaOH)] and were maintained in an incubator at 18°. On the following day, batches of 20-40 selected oocytes were injected with 50 nl of prepared RNA solution (0.5 ng of NR1 and 12 ng of NR2A mRNAs/oocyte). The injected oocytes were used for electrophysiological experiments after a 4-10-day incubation at 18°.
Electrical recordings and drug application.
Recordings from
X. laevis oocytes were obtained in two electrode
voltage-clamp mode using an Axoclamp-2A amplifier (Axon Instruments). The microelectrodes were fabricated from glass capillaries and were
filled with 3 M KCl solution. The resistance of filled
microelectrodes was 2.5-3.5 M. The oocytes were clamped
at a holding potential of 70 mV. The membrane current was filtered at
500 Hz and sampled at 100 Hz frequency. The drugs were applied using a
gravity-driven external perfusion system. The data acquisition and
external perfusion control were done using custom-written software
implemented in the SuperScopeII programming language (GW Instruments,
Somerville, MA).
/I 1) × 100%,
where I and I are, respectively, the agonist-induced currents in the
absence and presence of steroid. Throughout, results are expressed as
mean ± standard error; statistical comparison of groups was
carried out using Student's t test.
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Results |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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Bidirectional modulation of the NMDA receptor by PS and
35S.
Currents elicited by 30 µM NMDA were
recorded in primary cultures of chick spinal cord neurons by whole-cell
recording. At a holding potential of 70 mV, 100 µM PS
increases the amplitude of the NMDA-induced whole-cell current by
~2.5 fold (150 ± 14% potentiation, 14 neurons; Fig.
1B). In contrast, 35S, an analog of
PS, inhibits (50 ± 4%, 12 neurons) the NMDA response (Fig. 1C).
35S (100 µM) has a similar inhibitory effect
(66 ± 3%, 5 neurons; Park-Chung et al., 1994),
whereas 35S (100 µM) potentiates (39 ± 6%, 7 neurons) the NMDA response, demonstrating that the interaction of
35S with the NMDA receptor is stereospecific about C5.
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5S and recovery from inhibition are rapid, with no evidence of
use dependence (Fig. 2C), in contrast to
MK-801 (24), which in a similar experiment exhibits clear use
dependence (data not shown). To determine whether inhibition of the
NMDA response by 35S is voltage dependent, we constructed
current/voltage curves for the NMDA response in the presence and
absence of 35S. As shown in Fig. 1, D and E, the percentage
inhibition of the NMDA response is independent of holding potential,
indicating the absence of voltage-dependence.
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PS and 35S act through extracellularly directed sites.
When PS (100 µM) is applied intracellularly by inclusion
in the electrode buffer, the average NMDA response does not differ significantly from control and remains stable throughout the recording period (3-5 min; Fig. 2B). The same result is obtained when NMDA responses are normalized to the cell capacitance (pF) to adjust for
cell-to-cell variability in the membrane area, demonstrating that
intracellular PS cannot gain access to the steroid modulatory site of
the NMDA receptor. In addition, the ability of PS to potentiate the
NMDA response is unaltered in the presence of a high intracellular concentration of PS (Fig. 2, A and B), indicating that PS acts at the
extracellular surface of the membrane. Similarly, the addition of
35S (200 µM) to the intracellular buffer does not
inhibit NMDA-induced currents or prevent inhibition of the NMDA
response by extracellular 35S (100 µM, Fig. 2, C
and D), indicating that 35S also acts at a site associated with
the extracellular surface of the membrane.
Bidirectional modulation is not exerted through the polyamine
site.
In agreement with previous results (21), spermine (10-250
µM) potentiates the NMDA-induced current. Potentiation is
maximal (136 ± 33%, four neurons) at a spermine concentration of
100 µM (Fig. 3A). When the
concentration of spermine is further increased to 250 µM,
it is less effective in potentiating the NMDA response (67 ± 21%, three neurons; not shown). To determine whether PS, 35S,
and spermine act through a common modulatory site on the NMDA receptor,
we examined the effect of PS (100 µM) on the NMDA response in the presence of a maximally potentiating concentration of
spermine (100 µM). In the absence of spermine, PS
potentiates the NMDA response by 150 ± 14% (14 neurons), whereas
in the presence of spermine, PS potentiates the NMDA induced current by
178 ± 12% over the response in the presence of spermine alone
(six neurons; Fig. 3B).
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5S
inhibits the NMDA response by 50 ± 5% (five neurons) in the
presence of spermine, which is not significantly different from the
percentage inhibition in the absence of spermine. Because this
concentration of 35S is close to its EC50
(see Fig. 6C), the percentage inhibition by 35S should be reduced
if 35S and spermine compete for a common site. Therefore, the
inhibitory steroid modulatory site is also distinct from the spermine
modulatory site.
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Bidirectional modulation is not exerted through the arachidonic
acid site.
Arachidonic acid potentiates the NMDA response by
acting directly at the NMDA receptor (23). Because PS and
35S have amphiphilic properties, we asked whether these sulfated
steroids and arachidonic acid might act through a common site. As shown
in Fig. 4A, a maximal concentration of
arachidonic acid (10 µM) potentiates the NMDA response by
120 ± 35% (five neurons) and 158 ± 22% (four neurons) in
the absence and presence of 100 µM PS (Fig. 4B).
Conversely, PS potentiates the NMDA response by 182 ± 25% (four
neurons) in the presence of arachidonic acid (Fig. 4C), which does not
differ significantly from potentiation in the absence of arachidonic acid. Similarly, arachidonic acid does not affect the percentage inhibition by 35S (49 ± 5%, five neurons; Fig. 4D). Thus,
these observations demonstrate that the potentiating and inhibitory steroid site or sites are also distinct from the arachidonic acid modulatory site.
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Bidirectional modulation is not exerted through the redox
site.
There is a gradual "run-up" of the NMDA response in the
presence of 4 mM DTT, producing a 173 ± 19% (six
neurons) enhancement of the NMDA current after 180 sec of DTT exposure
(Fig. 5A). To examine whether PS and/or
35S interacts with the redox modulatory site of the NMDA
receptor, potentiation of the NMDA response by PS was measured after
prolonged exposure (1 hr) to DTT (4 mM). Under these
conditions, the NMDA response is increased by 169% (326 ± 82 pA,
13 neurons with DTT versus 121 ± 16 pA, 25 neurons without DTT),
but PS potentiation of the NMDA response remains unchanged (165 ± 26%, four neurons; Fig. 5B). Similarly, PS potentiation of the NMDA
response is not significantly changed (161 ± 12%, four neurons)
after exposure to 10 mM DTT for 1 hr (not shown). As an
additional test, we treated cells with the sulfhydryl alkylating agent
NEM. Cultures were treated for 5 min with 4 mM DTT,
followed by 2-min exposure to 4 mM DTT plus 300 µM NEM. Cultures were then washed four times, and
potentiation of the 30 µM NMDA response by PS or
inhibition by 35S was measured. Alkylation of thiols with NEM
increases the NMDA response (367 ± 82 pA, 25 neurons), compared
with control cultures (121 ± 16 pA, 25 neurons), but PS
potentiation or 35S inhibition of the NMDA response remains unchanged (Fig. 5, C and D).
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PS and 35S act through distinct sites.
To investigate
whether the structurally similar steroids 35S and PS act through
the same site to modulate the NMDA response, we examined the effects of
35S and PS in combination. In the presence of PS, 35S
inhibits the peak NMDA response by 50 ± 4% (four neurons, Fig.
6A), which is not significantly different from the inhibition of 59 ± 3% (four neurons) measured in the absence of PS. Conversely, potentiation of the NMDA response by PS (100 µM) is still evident in the presence of 35S (Fig.
6B). Moreover, the EC50 for inhibition of the
NMDA response by 35S is similar in the presence and absence of PS
(Fig. 6C). Taking the PS EC50 of 57 µM for potentiation of the NMDA response (11) as an
approximation of its Kd, competitive
inhibition would predict that the IC50 for
35S measured in the presence of PS would be increased by a factor
of 4.5 (Fig. 6C, gray curve). These results indicate that
35S and PS modulate the NMDA response through distinct sites. In
contrast, the enhancing effects of PHS and PS are not additive. PS (200 µM) reduces the ability of PHS (100 µM) to increase the NMDA response (PHS,
204 ± 18%, four neurons versus PHS + PS, 14 ± 6%, five
neurons, not shown).
PS and 35S act through distinct sites on NR1100/
NR2A recombinant NMDA receptors expressed in X.
laevis oocytes.
To investigate the possibility that
PS and 35S might be acting on separate receptor populations
differing in subunit composition, the modulatory effects of steroids
were assessed using oocytes expressing NR1100 and
NR2A subunits. Control oocytes failed to respond to NMDA, whereas
oocytes injected with NR1100 and NR2A subunit
mRNA exhibited robust responses to NMDA (30), with an EC50 value of 62 ± 4 µM and a
Hill coefficient of 1.5 ± 0.1 (four neurons, not shown).
Consistent with reports that potentiation by polyamines is absent with
NR1 splice variants bearing the amino-terminal insert (which is present
in NR1100) or when the NR2A subunit is present
(31, 32), NMDA responses of oocytes expressing
NR1100 and NR2A subunits are not potentiated by
spermine (0.5-50 µM), whereas higher concentrations of
spermine are inhibitory (17 ± 2% inhibition at 100 µM spermine). In contrast, oocyte NMDA responses are
robustly potentiated by PS (257 ± 44%, five neurons at 100 µM PS), providing additional evidence that PS
potentiation is not mediated by the polyamine site.
5S decreases the maximal NMDA response in oocytes but produces
only a slight shift in the NMDA EC50 (48 ± 4 µM, four neurons; not shown), suggesting that 35S
inhibits the NMDA response noncompetitively. 35S inhibits the
peak NMDA response by an equal amount whether in the presence (54 ± 5%; five neurons) or absence of PS (46 ± 8%; four neurons).
Moreover, there is no significant change in the
EC50 for 35S inhibition in the presence or
absence of 200 µM PS (Fig. 6D). These results are
consistent with the view that 35S inhibits the NMDA response via
a site distinct from the PS modulatory site.
PS or 35S potentiates or inhibits spontaneously occurring
EPSCs in cultures of rat hippocampal formation.
Next, we
considered whether steroids could modulate ongoing synaptic
transmission, where exposure to the endogenous neurotransmitter is
brief and localized to synaptic receptors. To approach this question,
we examined the effects of PS and 35S on spontaneously occurring
EPSCs in primary cultures of rat hippocampal formation. In the absence
of added Mg2+, cultured hippocampal neurons
exhibit frequent spontaneous synaptic activity. By measuring this
spontaneous activity, we were able to investigate the effects of
steroids on glutamate-mediated synaptic responses. To distinguish EPSCs
from IPSCs, the membrane potential was clamped at 70 mV, which is
very close to the Cl equilibrium potential.
Thus, IPSCs are not expected to be observed and spontaneous activity
should consist only of EPSCs. Spontaneously occurring EPSCs are blocked
completely by coapplication of the NMDA receptor antagonist APV and the
non-NMDA receptor antagonist DNQX (10 µM), indicating
that the observed spontaneous activity is glutamate receptor mediated
(data not shown). Fig. 7 shows that PS
enhances spontaneous EPSCs of neurons in culture. The EC50 for potentiation of EPSCs by PS is 11.9 µM. PS augments DNQX-resistant but not APV-resistant
EPSCs, which is consistent with the observation that PS positively
modulates the NMDA receptor but not AMPA/kainate receptors (11, 33).
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5S, a negative modulator of the NMDA, AMPA,
and kainate receptors, inhibits EPSCs (Fig. 7E). 35S, another
steroid inhibitor of glutamate responses, also inhibits EPSCs (data not
shown).
Structure-activity relationships for positive and negative
modulation by steroids.
Like PS, PHS strongly potentiates the NMDA
response, indicating that the 3-sulfate group is not essential for
potentiation (Table 1). In
contrast, pregnenolone hemisuccinate methyl ester is
without activity, suggesting that a negatively charged group at C3
is essential for positive modulation of the NMDA receptor. 11-Keto PS,
which differs from PS by the presence of a ketone group at C11, is
virtually without effect, whereas 11-hydroxy PS, with a hydroxyl
group at C11, is weakly inhibitory. 7-Keto PS is also inactive. In
contrast, compounds with modifications to the C17 side chain, such as
20-dihydro PS and 21-acetoxy-PS, still potentiate the NMDA response.
17-Hydroxy-PS, which differs from PS by the presence of a hydroxyl
group at C17, has activity similar to that of PS. Thus, addition of a
C17 hydroxyl to the PS structure does not change its effect on the NMDA
response, whereas addition of a keto group or a hydroxyl group at
either C7 or C11 results in loss of potentiating activity.
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-reduced derivatives of pregnenolone sulfate
(35S and 35S) exhibit strong inhibitory effects, whereas 35S potentiates the NMDA response.
To examine structural differences of steroids, we constructed
three-dimensional structural models by energy minimization using RASMOL. The presence of a C5-C6 double bond results in significant stereochemical differences between PS and 35S at the ring A/ring B junction and at the sulfate moiety (Fig.
1A).
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Discussion |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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Sulfated steroids can selectively modulate the actions of NMDA receptors by enhancing or inhibiting NMDA-induced conductance increases (11, 20), but the mechanism by which modulation occurs remains unknown. Previous reports have suggested that an intracellularly directed site may modulate PS action (33, 34), and it is not known whether positive and negative modulatory steroids work at one or more sites on the NMDA receptor. Whether steroids can modulate ongoing synaptic transmission also remains an important question in determining whether such compounds have the potential to act as endogenous neuromodulators. To approach these questions, we used the power of electrophysiology by combining studies of a series of extracellularly and intracellularly applied steroids on vertebrate neurons expressing native NMDA receptors with X. laevis oocytes expressing recombinant NR1100 and NR2A receptor subunits. The results show that there are at least two novel extracellularly directed modulatory sites on the NMDA receptor, one for potentiation and the other for inhibition, that can also enhance or inhibit ongoing synaptic transmission.
Sulfated steroids modulate the NMDA receptor at a novel site facing
the extracellular surface of the membrane.
Previous reports
suggest that PS might be able to diffuse within or across the membrane
to access its site of action (33, 34). However, we do not observe any
significant changes in the average amplitude of the NMDA response when
steroids are added intracellularly. Responses to repetitive application
of NMDA remain stable for up to 5 min with PS or 35S present in
the intracellular (electrode) buffer, suggesting that intracellular
steroids do not modulate the NMDA response even on this extended time
scale. The effects of extracellularly applied PS or 35S persist
when the corresponding steroid is present in the intracellular buffer. These results argue that the sites of action of PS and 35S are associated with the extracellular surface of the membrane.
5S is charged, voltage-dependence of inhibition would be
expected if access to its binding site requires entry into the channel.
However, inhibition of the NMDA response by 35S is not voltage
dependent, nor is there any indication of use-dependent inhibition or
recovery, such as is seen with MK-801. Considering that 35S has a
greater molecular weight than MK-801 and would be traveling
"upstream" in the electric field of the channel, it is hard to
imagine that 35S would move into the channel much faster than
MK-801 does. These results argue that inhibition by 35S is not
mediated by the Mg2+ or MK-801 binding sites.
Potentiation or inhibition of the NMDA response by PS or 35S are
independent of the glycine modulatory site (11, 20, 33). Similarly,
35S inhibition of the NMDA response is not reduced in the
presence of saturating glycine (data not shown), indicating that its
inhibitory effect is not due to competition with endogenous glycine.
A variety of other modulatory sites associated with the NMDA receptor
have been proposed, including sites for polyamines, arachidonic acid,
and redox agents. Polyamines such as spermine or spermidine at
micromolar concentrations increase the NMDA response (21). Arachidonic
acid has amphiphilic properties similar to PS and has been proposed to
act at the putative fatty acid binding domain of the NMDA receptor
(35). In addition, reducing agents enhance NMDA-induced currents,
whereas oxidation has the opposite effect (22). Both potentiation of
the NMDA response by PS and inhibition by 35S persist in the
presence of high concentrations of spermine or arachidonic acid.
Additional evidence against the involvement of the polyamine site is
provided by the observation that PS potentiates NMDA responses of
X. laevis oocytes expressing NR1100
and NR2A subunits, whereas spermine does not. Taken together, these
results argue that the modulatory effects of these steroids are not
mediated by either the polyamine or arachidonic acid sites.
Similarly, potentiation by PS and inhibition by 35S persist
following prolonged incubation with DTT or after alkylation with NEM,
indicating that excitatory and inhibitory steroids also do not interact
with redox modulatory sites. Taken together, these results provide
strong support for the existence of a novel extracellularly directed
steroid modulatory site(s) on or associated with the NMDA receptor.
Distinct sites for positive and negative modulation of the NMDA
receptor by sulfated steroids.
Although PS and 35S are
structurally similar, they do not interact competitively, arguing that
their respective positive and negative modulatory effects are exerted
through different sites on or associated with NMDA receptors. This
conclusion is based on the following observations: (i) the percentage
inhibition of the NMDA response by 35S does not change with the
addition of a near-maximal concentration of PS; (ii) potentiation by PS of the NMDA response is still evident in the presence of a high concentration of 35S; (iii) there is no significant change in the
IC50 for 35S in the presence of PS; and
(i.v.) the results cannot be explained by actions of PS and 35S
on subpopulations of NMDA receptors of different subunit composition,
as bidirectional modulation of the NMDA response by PS and 35S is
also observed in oocytes expressing only NR1100
and NR2A subunits, and as with neuronal NMDA receptors, the interaction
between these two modulators is not competitive.
5S is nearly complete, arguing against the
presence of a subpopulation of NMDA receptors resistant to 35S
but sensitive to PS. Moreover, it is possible to achieve nearly
complete inhibition of the NMDA response at high concentrations of
35S, arguing against the existence of a population of receptors
resistant to 35S but sensitive to PS.
Thus, there must be at least two distinct steroid modulatory sites with
the capacity to modulate NMDA receptor function. Although PS and
35S are structurally similar, energy minimization reveals significant differences in their three-dimensional structures (Fig.
1A). This finding further supports our conclusion that PS and 35S
act at two different sites.
In contrast, potentiation of the NMDA response by PHS is reduced in the
presence of PS, suggesting that PS and PHS act at a common site.
Because 11-keto PS is structurally similar to PS but has little effect
on the NMDA response (Table 1), we tested whether 11-keto PS could act
as a steroid site antagonist. In combination studies, 11-keto PS did
not antagonize potentiation by PS or inhibition by 35S (data not
shown), indicating that its inactivity reflects a lack of affinity for
the steroid modulatory sites.
Modulation of EPSCs by steroids.
As it does with chick spinal
cord neurons (11), PS enhances by up to 200% the NMDA-induced current
of neurons dissociated from embryonic rat hippocampal formation and
grown in culture (33). However, the effects of neurosteroids on
excitatory synaptic transmission have not been examined previously. Our
results demonstrate that PS potentiates spontaneous EPSCs in rat
hippocampal neurons, indicating that PS is able to enhance the response
of postsynaptic receptors to synaptically released glutamate. No
potentiation is observed in the presence of the NMDA antagonist APV,
confirming that the potentiation of EPSCs by PS is mediated by NMDA
receptors. The EC50 value for PS modulation of
spontaneously occurring EPSCs is about 12 µM, which is
lower than the EC50 of 57.4 µM for
PS modulation of the NMDA response in chick spinal cord neurons (11), suggesting that synaptic NMDA receptors of rat hippocampal neurons may
have greater sensitivity to modulation by sulfated steroids. 35S
and 35S inhibited EPSCs, consistent with their inhibitory effect
on the NMDA receptor. It seems unlikely that such bidirectional effects
are mediated through a presynaptic mechanism of action. The results
strongly suggest that sulfated steroids such as PS and 35S have
direct neuromodulatory effects on excitatory synaptic transmission in
CNS cultures by enhancing or inhibiting postsynaptic NMDA type
glutamate receptors.
Structure-activity relationships for steroid modulators of the NMDA
response.
The sulfate group of PS is not essential for
potentiation of the NMDA response, but a negatively charged group at C3
is required as potentiating activity is retained in the hemisuccinate
but not in the hemisuccinate methyl ester. The addition of a ketone group at C7 or C11 results in complete loss of activity, suggesting that structural requirements for potentiation of the NMDA response are
stringent. Of the four possible reduced derivatives of PS, 35S,
35S, and 35S are inhibitory, whereas 35S potentiates the NMDA response. The opposite effects of the stereoisomers 35S and 35S demonstrates that the interaction of these steroids with
the NMDA receptor is stereospecific about C5. This stereoselectivity of
action argues that 35S does not inhibit the NMDA receptor through
a nonspecific mechanism, such as perturbation of the lipid bilayer, as
proposed for short-chain alcohols (36), but rather acts through a
specific modulatory site.
Physiological and pharmacological significance. It remains to be determined whether certain NMDA receptor subtypes or spliced variants exhibit a higher affinity for sulfated steroids or whether local concentrations are adequate for modulation to occur under normal conditions.
Because the concentrations of neurosteroids rise in several physiological circumstances such as sexual activity, stress, and during the estrous cycle (1, 37), it is conceivable that PS or one of its sulfated metabolites may contribute to some of the physiological and behavioral changes known to occur in these conditions. Sulfated progesterone metabolites such as 35S, 35S, and 35S
are present at concentrations as high as 2 µM in the peripheral circulation (38) and the concentration of DHEAS is 8.9 µM in the blood of 40-year-old men (5), but it remains unknown whether sulfated steroids play a role in the functioning of the
intact nervous system.
Behavioral studies demonstrate that PS has memory-enhancing effects in
mice and rats (3, 16) and prevents NMDA receptor antagonist-induced
amnesia (17, 18) and that inhibition of pregnenolone sulfatase activity
enhances learning in rats (19). These findings raise the prospect that
PS or related sulfated neurosteroids may be active physiologically and
useful as a cognitive enhancers.
The inhibitory steroid sulfates such as 35S, 35S, or
related compounds may be useful as neuroprotective agents. Release of
glycine (39) and arachidonic acid (40) is increased in ischemia, and
probably contributes to NMDA receptor-mediated excitotoxicity. Because
35S can inhibit the NMDA response in the presence of glycine,
polyamines or arachidonic acid, inhibitory steroid sulfates may
effectively inhibit excessive NMDA receptor activation during pathophysiological conditions such as stroke.
Our results suggest that the sulfated steroids PS and 35S act at
unique sites to modulate the NMDA response and that they exert their
positive and negative modulation through independent pathways. These
results support the view that these sulfated steroids constitute a
novel class of endogenous functional modulators of the NMDA receptor.
It will be important to determine whether these steroids can be
released from nervous tissue in amounts sufficient to modulate
excitatory synaptic transmission and to develop pharmacological antagonists of the steroid modulatory sites to test the hypothesis that
sulfated steroids act as physiological neuromodulators in vivo. Further characterization of molecular interactions between steroids at specific sites on the NMDA receptor will be of extreme interest due to the critical role of this receptor in many
physiological and pathological processes in the central nervous system.
Moreover, the development of ligands that act specifically at one or
both of these sites should provide a useful strategy for the design of
therapeutic agents with neuroprotective, sedative-hypnotic, analgesic,
or anesthetic properties.
| |
Acknowledgments |
|---|
We would like to thank Dr. Kosta Steliou (Boston University, Boston, MA) for three-dimensional modeling of steroid structures. We would also like to thank N. Yaghoubi (Boston University School of Medicine, Boston, MA), who developed the automated system for oocyte electrophysiology and first demonstration of steroid modulation of recombinant NMDA receptors. We would particularly like to thank Dr. Suzanne Zukin for the NR1100 and Dr. S. Nakanishi for the NR2A clone.
| |
Footnotes |
|---|
Received March 3, 1997; Accepted August 12, 1997
1 Current affiliation: Gene Therapy Unit, Laboratory of Molecular Cell Biology, Institute of Bioscience and Biotechnology, Yasong, Taejon, Korea.
2 Current affiliation: Department of Physiology, College of Medicine, National Cheng Kung University, Tainan, Taiwan 70101, Republic of China
This work was supported by National Institute of Mental Health Grant MH49469.
Send reprint requests to: Dr. David H. Farb, Department of Pharmacology, Boston University, School of Medicine, 80 East Concord Street, Boston, MA 02118.
| |
Abbreviations |
|---|
NMDA, N-methyl-D-aspartate;
DHEAS, dehydroepiandrosterone sulfate;
PS, pregnenolone sulfate;
35S, 3-hydroxy-5-pregnan-20-one sulfate;
AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid;
EPSC, spontaneous excitatory postsynaptic current;
ara-C, 1--D-arabinofuranosylcytosine;
PHS, pregnenolone
hemisuccinate;
EGTA, ethylene glycol bis(-aminoethyl
ether)-N,N,N,N-tetraacetic
acid;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
DTT, dithiothreitol;
NEM, N-ethylmaleimide;
APV, (±)-2-amino-5-phosphonopentanoic acid;
35S, 3-hydroxy-5-pregnan-20-one sulfate;
35S, 3-hydroxy-5-pregnan-20-one sulfate;
35S, 3-hydroxy-5-pregnan-20-one sulfate.
| |
References |
|---|
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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) and presence
(
) of 200 µM PS. Results are mean values of pooled
data from four to seven neurons. Error bars, standard
errors. 3
