Introduction

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Neurosteroids have received recent wide attention because of their endogenous presence in the central nervous system at concentrations that may modulate GABAergic and/or glutamatergic synaptic communication (Baulieu, 1998; Concas et al., 1998). Synthetic analogs of endogenous neurosteroids may be clinically useful neuroprotectants, anesthetics, and anticonvulsants (Gasior et al., 1999; Zorumski et al., 2000). Generally, augmentation of GABAergic transmission and block of NMDA receptor-mediated transmission are associated with anesthetic, anticonvulsant, and antiexcitotoxic properties (Macdonald and Greenfield, 1997). Unfortunately, whereas many neuroactive steroids have activity at both of these receptor types, no known endogenous or synthetic steroid dampens NMDA receptor signaling without also inhibiting GABAergic signaling. These are opposing cellular effects with regard to the clinically desirable properties mentioned above.

Pregnane steroid derivatives with a sulfate or other negatively charged substituent at the carbon 3 (C3) position in the -configuration are negative modulators of NMDA receptors through noncompetitive, voltage-independent block (Park-Chung et al., 1994). The -configuration of the ring fusion at C5 is also important for blocking action, as  sulfate substitution at C3 retains NMDA receptor blocking activity if the steroid is 5-reduced (Park-Chung et al., 1994; Weaver et al., 2000). Hemisuccinate and other hemiester -substituents at C3 retain NMDA receptor blocking activity (Weaver et al., 1997, 2000). Unfortunately, the neuroprotective profile of each of these NMDA receptor antagonists is compromised by the fact that each of these derivatives also blocks GABA_A receptor activity (Park-Chung et al., 1999). Additionally, whereas (3,5)-3-hydroxypregnan-20-one hemisuccinate, the hemisuccinate analog of naturally occurring pregnanolone sulfate, has been shown to be neuroprotective (Weaver et al., 1997), this analog is subject to hydrolysis of the hemisuccinate group.

We report here the cellular characterization of a novel neurosteroid analog, (3,5)-20-oxo-pregnane-3-carboxylic acid (35PC), with both NMDA antagonist and GABA-potentiating actions. We synthesized this steroid as a nonhydrolyzable analog of (3,5)-3-hydroxypregnan-20-one hemisuccinate (Weaver et al., 1997). Like the hemisuccinate, at physiological pH the carboxylic acid group of 35PC should be largely deprotonated, making 35PC a likely NMDA receptor blocker. However, like GABA-potentiating steroids, which contain a 3-hydroxyl group as a hydrogen bond donor (Phillipps, 1975), the ---COOH of un-ionized 35PC is a similarly located hydrogen bond donating group, and therefore un-ionized 35PC might be expected to potentiate GABA receptors.

35PC exhibits complex actions on GABA_A receptors, with potentiation, voltage-dependent block, and direct gating of GABA_A receptors. At low concentrations of drug and at physiological pH, net potentiation of GABA_A receptor function and IPSCs are observed. At higher concentrations, the postsynaptic GABA_A potentiation is decreased by steroid-induced block of receptor function. Block shows little stereoselectivity and is relatively insensitive to placement of the carboxylate at either end of the steroid ring structure. The pH dependence of both block and potentiation by carboxylated steroids suggests a higher apparent pK than expected of these organic acids in water. This probably reflects the influence of membrane/protein constituents on the pK of the carboxylate group.

	Materials and Methods

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Hippocampal Cultures. Primary hippocampal microcultures were prepared from 1- to 3-day-old postnatal albino rats using established methods (Mennerick et al., 1995). Under halothane anesthesia, rats were decapitated, and the hippocampi were dissected and cut into 500-µm-thick transverse slices. The slices were dissociated with 1 mg/ml papain in oxygenated Leibovitz L-15 medium and mechanically triturated in modified Eagle's medium containing 5% horse serum, 5% fetal calf serum, 17 mM D-glucose, 400 µM glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. Isolated cells were plated (75 cells/mm²) onto plastic culture dishes coated first with 0.15% agarose then with atomized droplets of rat tail collagen. To halt glial proliferation, cultures were treated with 10 µM cytosine arabinoside after 3 days in vitro. Experiments were performed in cultures that were 4 to 14 days old.

Xenopus laevis Oocytes. Stage V-VI oocytes were harvested from sexually mature female X. laevis (Xenopus One, Northland, MI) under 0.1% tricaine (3-aminobenzoic acid ethyl ester) anesthesia. Oocytes were defolliculated by shaking for 20 min at 37°C in collagenase (2 mg/ml) dissolved in calcium-free solution containing: 96 mM NaCl, 2 mM KCl, 1 mM MgCl₂ and 5 mM HEPES at pH 7.4. Capped mRNA, transcribed in vitro (mMessage mMachine; Ambion, Austin, TX) from linearized plasmids containing receptor-coding regions, were injected into oocytes 24 h after defolliculation. Oocytes were incubated for up to 2 weeks at 18°C in ND96 medium containing 96 mM NaCl, 1 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, and 10 mM HEPES at pH 7.4 supplemented with 5 mM pyruvate and the above-mentioned antibiotics. The cDNAs for the GABA_A receptor subunits were provided by A. Tobin [University of California, Los Angeles (1)], P. Malherbe [Hoffman-La Roche, Switzerland (2)], and C. Fraser [National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD (2L)].

Electrophysiology. For synaptic studies, the growth medium was exchanged for a solution containing 138 mM NaCl, 4 mM KCl, 10 mM HEPES, 10 mM D-glucose, 2 mM CaCl₂, and 1 mM MgCl₂, pH 7.25. Whole-cell voltage-clamp recordings of autaptic currents were performed from neurons on single-neuron islands using recording pipettes with open tip resistances of 2 to 5 M. The pipette solution contained 140 mM KCl, 4 mM NaCl, 5 mM EGTA, 0.5 mM CaCl₂, 10 mM HEPES, pH 7.25. Neurons were recorded using an Axopatch 1-D patch-clamp amplifier (Axon Instruments, Foster City, CA), and series resistance (<10 M) was compensated 90 to 100% during experiments. Synaptic transmission was activated by stimulating neurons with 1.5-ms voltage steps from 70 to +30 mV at intervals of 20 to 30 s. During experiments, microislands were continuously superfused locally using a gravity-driven multibarrel pipette with a common exit port. The tip of this local perfusion system was placed ~400 µm from the microisland being recorded, and solution flowed at a rate of 0.1 to 0.5 ml/min. All recordings were done at room temperature (~22°C) on the stage of a Nikon inverted microscope equipped with phase-contrast optics. Studies examining exogenous applications of agonists were performed using whole-cell recordings as described above, except the pipette solution contained 140 mM CsCl in place of KCl. Experiments examining low-Mg²⁺-induced action potentials (Fig. 1F) were performed in the current-clamp mode of the patch amplifier on islands containing small networks of neurons.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Synaptic effects of 35PC. A, structure of 35PC showing the pregnane core and stereochemistry of the C3 and C5 substituents. B and C, 35PC selectively depressed the slow NMDA receptor component of EPSCs. B, autaptic EPSCs were elicited from solitary glutamatergic neurons in microcultures with a 2-ms voltage pulse to 0 mV (from a holding potential of 70 mV). The extracellular bath solution contained no added Mg2+ and 10 µM glycine to unmask NMDA receptors. The traces represent average responses obtained in the absence (thin trace) and presence (thick trace) of 50 µM 35PC. Note that the peak EPSC was not affected by the drug, but the slow NMDA component was selectively diminished. Transient currents associated with stimulation have been blanked in this and subsequent figures for clarity. C, summary of the effects of 50 µM 35PC on peak EPSC and the NMDA component (measured 30 ms after stimulation) in nine neurons. D and E, concentration-response effects of 35PC on hippocampal IPSCs. D, top to bottom, represent the effect of 35PC on an IPSC from a solitary GABAergic cell (all from the same cell). In all cases, the thick trace represents the IPSC in the presence of drug, whereas the thin trace represents the IPSC in the absence of drug. As noted in the text, the effect on peak amplitude seen with 50 µM 35PC was variable from cell to cell. E, summary data representing the concentration dependence of 35PC on IPSC 10 to 90% decay time. Asterisks denote significantly different than zero (p < 0.05, two-tailed t test). The values for the 10 to 90% decay times from which the normalized data in Fig. 1E were derived were baseline, 114 ± 13 ms; 1 µM 35PC, 105 ± 10 ms; 5 µM 35PC, 143 ± 10 ms; 10 µM 35PC, 132 ± 7 ms; and 50 µM 35PC, 101 ± 16 ms. F, in a current-clamp recording from a small network of neurons grown in a microculture, action potential activity was increased by removing extracellular Mg2+ and adding glycine to potentiate NMDA receptor activity (low Mg2+). 35PC (5 µM) reversibly eliminated the firing. The apparent increase in firing upon removal of drug in this cell was not consistently observed in other neurons.

Drugs. Unless otherwise stated, drugs were from Sigma (St. Louis, MO). Pregnenolone sulfate and (3,5)-pregnan-20-one sulfate (35PS) were obtained from Steraloids (Newport, RI) and from Sigma. The 35PS was prepared as described elsewhere (Park-Chung et al., 1997) and was the generous gift of Dr. Robert H. Purdy (Scripps Research Institute, La Jolla, CA). A preliminary account of the synthesis of 35PC and 35PC has been published (Zeng et al., 1999). Full synthetic details will be published elsewhere. The methyl ester of 35PC was prepared by reacting the acid with diazomethane dissolved in ether. The (3,5)-20-oxo-pregnane-3-carboxamide (35 PA) was prepared from 35PC by converting this carboxylic acid to the acid chloride and then reacting this intermediate with ammonia dissolved in methylene chloride.

	Results

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Alteration of Synaptic Activity by 35PC. Figure 1A shows the structure of 35PC. 35PC selectively depressed the slow NMDA receptor component of EPSCs in hippocampal neurons, consistent with the effect of other neuroactive steroids with a charged moiety in the -configuration at C3 (Park-Chung et al., 1997). 35PC (20 µM) produced only 8 ± 5% depression of the NMDA component (N = 3; data not shown), whereas 50 µM 35PC produced 20 ± 4% depression (N = 9; Fig. 1, B and C). Effects on the fast -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor component of EPSCs were negligible with 8 ± 5% potentiation produced at 50 µM (Fig. 1, B and C).

35PC Potentiates and Blocks GABA_A Receptors. In subsequent experiments we sought to explain the complicated actions of 35PC on IPSC decays using a combination of biophysical and pharmacological approaches. Prolongation of the decay phase of IPSCs is a common effect of GABA potentiators, including neuroactive steroids, benzodiazepines, and barbiturates, and probably underlies at least part of the clinical utility of these drugs. The reversal of IPSC prolongation at high steroid concentrations thus presumably undermines a potentially important property of the drug, especially because these high concentrations of steroid overlap with the concentrations that affect NMDA receptor-mediated EPSCs. We therefore probed structural and functional mechanisms of IPSC block by 35PC in more detail.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Postsynaptic effects of 35PC reveals block of NMDA receptors and both potentiation and block of GABAA receptors. A, responses to the nondesensitizing AMPA receptor agonist kainic acid (KA, 100 µM) at two membrane potentials are represented. There was no effect of 35PC (50 µM) at either potential. B, responses to 100 µM NMDA in another neuron. Extracellular Mg2+ was omitted and 10 µM glycine was added to all solutions. In addition, extracellular calcium concentration was lowered to 0.2 mM to reduce Ca2+-dependent desensitization of NMDA receptors (Clark et al., 1990; Legendre et al., 1993). 35PC (50 µM) inhibited responses to NMDA with little voltage dependence. C, responses to GABA (2 µM) in the absence and presence of 50 µM 35PC. Note the potentiation of GABA responses at 70 mV but block of responsiveness at + 40 mV. D, responses to 1 mM GABA at 70 mV in the absence (left) and presence (right) of 10 µM 35PC.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Potentiation is stereoselective about C3 but block is not. A, structure of 35PC with emphasis (arrow) on the stereochemistry of the C3 substituent. B, leak subtracted GABA currents from a hippocampal neuron. A voltage pulse from 70 to +50 mV (top trace) was delivered in the absence and presence of 2 µM GABA. The current in the absence of GABA was digitally subtracted to reveal the GABA-gated current (bottom trace). Note the outward voltage-dependent relaxation of the GABA current. C, same protocol was applied to the same cell, except that 35PC (50 µM) was also present in the GABA solution. Note the steady-state potentiation of GABA current apparent at 70 mV (before the voltage pulse) and the potentiation of the instantaneous GABA current observed at +50 mV, which gives rise to an inward relaxation and block of current relative to B at steady state. D-F, I/V relationships obtained from pulses to various membrane potentials. D, I/V relationship for GABA in the absence of modulator. Instantaneous current was measured between 3 and 5 ms after the voltage pulse. Steady-state current was measured at the end of the 200-ms voltage pulse. Note the nearly linear instantaneous I/V and outward rectification of the steady-state I/V. E, instantaneous I/V for GABA alone is replotted from D with the instantaneous current I/V relationship obtained in the presence of GABA plus 50 µM 35PC. F, steady-state current I/V relationship for GABA alone is replotted from D along with the steady-state I/V relationship measured in the presence of GABA and 50 µM 35PC. G-L, same protocols as in A-F were applied to a different cell using the diastereomer 50 µM 35PC. Note that potentiation is absent from both the instantaneous and steady-state currents at all potentials, but block is similar to that observed with 35PC.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Voltage and [GABA] dependence of 35PC block. A1, I/V relationship obtained from steady-state responses to 2 µM GABA at various membrane potentials from an X. laevis oocyte expressing recombinant GABAA receptors (122L subunit combination). A voltage-pulse subtraction protocol similar to that used in hippocampal neurons (Fig. 3) was used. , GABA alone. , currents in response to GABA plus 10 µM 35PC. A2, data in the presence of 35PC were replotted on an expanded y-axis to highlight the shape of the I/V curve. B1 and B2, same protocols applied to the same oocyte in A, except that 100 µM GABA was used as the agonist (plus 10 µM 35PC for open circles). Note the linearity of the 100 µM GABA I/V curve and the persistence of voltage-dependent block.

Structural Requirements for Block and Potentiation. Stereoselectivity of 35PC effects on GABA receptors was examined to gain insight into the stereochemical requirements for potentiation and block. We synthesized the 3-diastereomer (35PC; Fig. 3G) and characterized 35PC in the same voltage-pulse protocol used to examine 35PC. Interestingly, 35PC exhibited voltage-dependent block similar to that exhibited by the 3-diastereomer, but the 3-compound elicited no potentiation (Fig. 3, H-L). These results suggest that potentiation is highly stereoselective whereas block is not.

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Effect of lithocholic acid on GABA responses. A, structure of lithocholic acid with emphasis on placement of the carboxylate group (arrow) at C24. B, effect of lithocholic acid on responses to 20 µM GABA in an oocyte expressing the 122 subunit combination. C, summary of the GABA-dependence of lithocholic acid. Average block (10 µM lithocholic acid) of responses to 2 and 100 µM GABA is shown.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   35PC truncated the decay of IPSCs. A, thin trace represents control IPSCs. The thick trace represents averaged sweeps in the presence of 50 µM 35PC. B, summary of effects of 50 µM 35PC on the peak amplitude, 10 to 90% decay time, and total charge transfer from five neurons.

Dependence of Potentiation and Block on pH. Studies of the structural requirements of neuroactive steroid potentiation and block have previously suggested that a hydrogen bond donor at C3 is necessary for potentiation, whereas a negative charge at C3 is important for block (Phillipps, 1975). The predicted pK of the carboxylate group in 35PC and 35PC is ~5.0 (Fini et al., 1987; Loudon, 1995). However, the pK of organic acids is dramatically altered by changing the solvent dielectric constant or hydrogen bonding ability (Fini et al., 1987; Smejtek et al., 1987). To explore the role of the ionization state of 35PC in the potentiating and blocking action at GABA_A receptors, we examined the effect of altered extracellular pH on 35PC potentiation and block. For these studies, we examined the effect of steroids on X. laevis oocytes expressing the GABA_A receptor subunit combination 122L, because these cells tolerate large and repeated shifts in pH (Fig. 7). As in hippocampal neurons, 50 µM 35PC potentiated oocyte responses to 2 µM GABA at 70 mV and physiological pH (Fig. 7, A1 and A3). Consistent with previous results, low pH usually diminished responsiveness to GABA (Fig. 7A2; Zhai et al., 1998). Importantly, lowering the pH to 5.8 increased the net potentiation of 35PC measured at 70 mV. Examination of steady-state I/V curves at pH 7.4 showed evidence for potentiation at negative membrane potentials and block at positive membrane potentials, again nearly identical to data from hippocampal neurons (Fig. 7A3). At pH 5.8, steady-state GABA responses were potentiated at all membrane potentials (Fig. 7A4). This result suggests that un-ionized 35PC may relieve block, augment potentiation, or both.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Potentiating effect of 35PC was pH-dependent. A1 and A2, traces from an X. laevis oocyte expressing an 122L subunit combination. A1, at pH 7.4 and a membrane potential of 70 mV, 50 µM 35PC potentiated the response to 2 µM GABA. A2, at the same membrane potential but pH 5.8, the response to GABA alone was slightly reduced but the potentiation is much larger than at the higher pH. A3 and A4, from the same cell, steady-state I/V curves showed loss of block and apparent enhanced potentiation at low pH. A3, steady-state I/V curve at pH 7.4. The potentiation and block at negative and positive membrane potentials at pH 7.4 are similar to results in hippocampal neurons (Fig. 3F). A4, steady-state I/V curve at pH 5.8. Note the potentiation of steady-state GABA responses at all membrane potentials. B, titration of the potentiating effect of 35PC. Potentiation, independent of block, was isolated by using low concentrations of GABA (2 µM) and 35PC (5 µM) and by examining negative membrane potentials 70 mV. Potentiation was calculated relative to the steady-state GABA response at each pH value. The solid line represents a titration curve with an apparent pK of 6.4. C1 and C2, potentiation of the amide derivative of 35PC is pH-independent. Protocol was similar to that used for A1 and A2. The inset shows the structure of 35PA. Holding potential was 70 mV.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   A1-A4, low pH diminished block by 35PC. Voltage pulses in the presence and absence of GABA were applied in 20-mV increments to potentials between 90 and +90 mV. The traces shown are subtractions of traces in the absence of GABA from those in the presence of GABA. A1, at pH 7.4 responses to 20 µM GABA showed typical outward rectification. Twenty µM GABA was used to enhance the effect of 35PC. A2, 35PC (50 µM) depressed GABA responsiveness at all membrane potentials. A3, GABA responsiveness was depressed slightly by pH 5.8. A4, 35PC depression was nearly eliminated at low pH. B and C, pH dependence of block by 35PC (B, N = 4) and by lithocholic acid (C, N = 5). Apparent pK values for the blockers were 6.9 and 6.4, respectively. D, low pH only slightly affected the block of GABA responses by 35PS (2 µM). Because the block by 35PS, like that of 35PC, is [GABA]-dependent, the small amount of reduction in effectiveness at low pH could be due to the decreased GABA responsiveness at pH 5.8. The summary reflects fractional block of steady-state response to 20 µM GABA at +90 mV for 50 µM 35PC (N = 5; different sample than Fig. 8B) and 2 µM 35PS (N = 4).

	Discussion

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Novel Combination of Cellular Actions for 35PC. The unique attribute of the neuroactive steroid 35PC is the ability of this compound to potentiate GABA_A receptor function and inhibit NMDA receptor function. This combination of cellular effects may enhance the clinical profile over previously characterized anesthetic neuroactive steroids. While block of NMDA receptors is perhaps sufficient to protect against various forms of glutamate-mediated toxicity (Michaelis, 1998), maximum block of NMDA receptors by neuroactive steroids is typically less than 100%. Therefore, the GABA-blocking actions of these same steroids may promote an increase in synaptic activity that at least partially undermines the direct effects on NMDA receptors. Likewise, GABA-potentiating actions should enhance the anesthetic properties of NMDA receptor blockers, because GABA_A receptor potentiation and NMDA receptor block are the cellular actions most closely correlated with anesthetic properties (Franks and Lieb, 1994).

Action at GABA_A Receptors: Site(s) of Action. Given that 35PC both potentiates and blocks GABA receptors, it is worth considering current evidence for possible sites on the GABA_A receptor that may mediate these actions. Potentiation by 35PC exhibits clear stereoselectivity, whereas block does not. Likewise, concentration-response data at synapses (Fig. 1E) suggest that potentiation is most prominent at low micromolar concentrations of steroid, whereas block becomes apparent at concentrations >10 µM. Potentiation and block by 35PC can also be temporally separated during voltage jumps to positive potentials (Fig. 3, A-F). Finally, low pH has opposite effects on block and potentiation, nearly eliminating block whereas enhancing potentiation. These results are all consistent with the hypothesis that potentiation and block occur at separate sites on the GABA_A receptor. Previous work also suggests that positive modulators of GABA_A receptors, such as (3,5)-3-hydroxypregnan-20-one, act at a different GABA_A receptor-associated site than blocking steroids such as pregnenolone sulfate (Zaman et al., 1992; Park-Chung et al., 1999). Unfortunately, specific potentiating and blocking sites on the GABA_A receptor have thus far eluded identification, although a point mutation in transmembrane domain 2 dramatically reduces block by pregnenolone sulfate, perhaps by altering the transduction mechanism of block (Akk et al., 2001). Interestingly, it was previously shown that 35PS and pregnenolone sulfate lack enantioselectivity for block of GABA_A receptors, possibly suggesting the lack of a conventional chiral protein-ligand recognition site for pregnane and pregnene series blockers (Nilsson et al., 1998).