Introduction

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In recent years, a number of studies have shown that steroids can have a rapid action in the brain and can produce clinical anesthesia as well as affecting mood and performance in humans. These rapid actions are apparently mediated by binding to membrane proteins, including ligand-gated ion channels. A variety of steroids can enhance activation of the -aminobutyric acid type A (GABA_A) receptor by GABA and may cause direct gating of this receptor (reviewed in Lambert et al., 1995). Certain sulfated steroids block GABA_A receptors (Majewska and Schwartz, 1987) and can also enhance the activity of N-methyl-D-glutamate-type glutamate receptors (Park-Chung et al., 1997). Finally, steroids are known to inhibit the muscle (Gillo and Lass, 1984) and neuronal (Bertrand et al., 1991; Ke and Lukas, 1996) nicotinic receptor.

The most prevalent nicotinic receptor in the brain is composed of the 4 and 2 subunits, which form the high-affinity nicotine binding component. The physiological role of this particular subunit combination is not fully understood, but it is known that the numbers in the brain are altered by chronic nicotine exposure and in some conditions, including Alzheimer's disease (reviewed in Role and Berg, 1996). In general, nicotinic receptors in the brain may serve as presynaptic receptors that modulate transmitter release (reviewed in McGehee and Role, 1996). Steroids can inhibit the neuronal nicotinic 42 receptor, and it has been suggested that it may be a target for rapid steroid actions in the brain (Bertrand et al., 1991). Previous studies on neuronal nicotinic receptors have indicated that there is some specificity for steroid structure in producing inhibition and that steroids act from the external solution, because progesterone covalently coupled to bovine serum albumin can inhibit (Valera et al., 1992; Ke and Lukas, 1996). Because neuronal nicotinic receptors and GABA_A receptors are members of the same extended gene family, it is also of interest to compare the structural requirements for block by steroids, in one case, and potentiation, in the other. Finally, some volatile and i.v. anesthetic agents are extremely potent blockers of recombinant rat (Violet et al., 1997) and chicken (Flood et al., 1997) nicotinic 42 receptors expressed in Xenopus laevis oocytes. This raises the possibility that other anesthetic agents may also act on these receptors at clinically relevant concentrations.

We took advantage of a stably transfected cell line expressing rat 42 type neuronal nicotinic receptors (HN42 cells; Sabey et al., 1999) to examine the inhibitory effects of steroids on a major class of neuronal nicotinic receptor expressed in brain. One steroid, 3,5,17-3-hydroxyandrostane-17-carbonitrile (ACN), was chosen for detailed study because it has been shown to be a potent anesthetic agent in mice and tadpoles and to potentiate responses of GABA_A receptors (Wittmer et al., 1996). We explored the steroid structural requirements for activity to compare the requirements for action on the nicotinic receptor with those for action on the GABA_A receptor. We also examined the ability of these steroids to produce anesthesia in X. laevis tadpoles to determine whether action at nicotinic receptors was likely to be involved in producing anesthesia.

	Materials and Methods

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Unless otherwise noted, chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).

Most steroids were synthesized in the laboratory of D. Covey; these include ACN and its enantiomer, ent-ACN; (3,5,17)-17-hydroxyestrane-3-carbonitrile (ECN) and its enantiomer, ent-ECN; (3,5,17)-3-hydroxyandrostane-17-carbonitrile (B163); (3,5,17)-3-hydroxyandrostane-17-carbonitrile (B164); (3,5,17)-3-hydroxyandrostane-17-carbonitrile (B260); (3,5,17)-3-hydroxyandrostane-17-carbonitrile (B372) (see Fig. 6 for structures of steroids used).

HN42 cells were produced and maintained as described previously (Sabey et al., 1999). In brief, HEK 293 cells were transfected with expression constructs for the rat 4 and 2 subunits and selected for resistance to G418 (450 µg/ml; Life Technologies, Grand Island, NY). Drug-resistant cells were repeatedly immunoselected using monoclonal antibody 270, which binds to an epitope on the extracellular surface of the 2 subunit. The cells used in this study had been sequentially selected six times but had not been cloned. The cDNAs for the rat neuronal nicotinic subunits were kindly provided by Dr. J. Patrick (Baylor College of Medicine, Houston, TX).

Electrophysiology of Nicotinic Responses. Cells were plated onto 35- or 60-mm tissue culture dishes and used within 4 days after plating. Recordings were made in an extracellular saline composed of 140 mM NaCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, 5 mM KCl, and 10 mM glucose, pH 7.3. The internal solution was composed of 4 mM NaCl, 4 mM MgCl₂, 0.5 mM CaCl₂, 10 mM HEPES, 5 mM EGTA, and 140 mM CsCl, pH 7.3. Standard methods were used to record currents in the whole-cell configuration. Records were filtered with a four-pole Bessel filter (Frequency Devices, Haverhill, MA) and directly digitized by a PC clone computer using a Digidata interface (Axon Instruments, Foster City, CA). Drugs were dissolved in external solution and applied through a three-tube perfusion system (Sabey et al., 1999). The three-tube perfuser provided a solution change around cells attached to the culture substrate with a 10-to-90% time of about 50 ms (data not shown; from the time course of changes in holding current when bath solutions with different ion concentrations were applied to cells). The cell was perfused with extracellular solution continuously between applications of agonists or other drugs. To reduce loss of steroids as a result of sticking to plastics, the perfusion system used glass syringe reservoirs, Teflon tubing, Teflon valves, and quartz perfuser tips.

View this table: [in this window] [in a new window]   TABLE 1 Inhibition of acetylcholine responses by 3 µM concentrations of steroids The table is organized with the compound producing the greatest block (ACN) at the top and that producing the least (bath solution) at the bottom. The structures of the drugs used are shown in Fig. 6. The first column gives the name of the compound. The second column shows the mean residual response to 100 µM acetylcholine after 30-s preexposure to the steroid (all at 3 µM concentration) for N cells. The next three columns give the substituent and orientation at the 3, 5, and 17 positions, respectively. The next two columns show the measured IC50 values and Hill coefficient for inhibition or the calculated IC50 value (in parentheses). The calculated IC50 value was estimated from the mean residual current with 3 µM compound () and the mean value for the Hill coefficient for all the blocking curves determined (1.4), using the equation IC50 = 1.4 th root {(1  )/} × 3 µM.

Steroid Action on GABA_A Receptors. The ability of steroids to act on GABA_A receptors was examined as described (Wittmer et al., 1996). In brief, GABA (2 µM) or GABA (2 µM) plus steroid (10 µM) were applied to voltage-clamped cultured rat hippocampal neurons using matched puffer pipettes. Data on potentiation are presented as the ratio of the peak response in the presence of GABA plus steroid to the response to GABA alone (no potentiation is 1.0 and block is <1).

Tadpole Loss of Righting Reflex (LRR). The tadpole LRR assay was conducted as described (Wittmer et al., 1996). In brief, groups of 10 tadpoles were exposed to various concentrations of steroid in Ringer's solution. After equilibration at room temperature for 3 h, tadpoles were flipped onto their backs using a smooth glass rod. LRR was defined as failure of the tadpole to right itself within 5 s after being turned over. Reversibility was determined by returning the tadpoles to normal Ringer's solution and demonstrating that they recovered normal righting reflexes. The concentrations of anesthetic agents producing LRR in half the tadpoles correlate well with concentrations necessary to produce anesthesia in mammals (Tonner et al., 1992; Franks and Lieb, 1993).

Data Analysis. All values are presented and shown in graphs as the arithmetic mean ± 1 S.D., based on N observations. Concentration-effect relationships were analyzed in Excel (Microsoft, Redmond, WA) and SigmaPlot (SSPS, Chicago, IL). Comparisons of the relative ability of steroids to block responses were made using the unpaired t test assuming unequal variances.

Fitting of Kinetic Models. Fitting of the time course of block by 10 µM ACN and recovery from block by 10 µM ACN was performed with SCOP (Simulation Resources, Berrien Springs, MI), using numerical solution of the kinetic models. The models were implemented in SCOP using the "kinetic" form of model entry, in which transition rates were entered and quality of fit was judged from ². As presented in Results, four simple kinetic models were used in the analysis. For each model, it was assumed that there were an integral number (M) of binding sites for steroid on each receptor. The sites were assumed to be identical and independent for binding steroid.

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	Results

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Time Course of Steroid Inhibition of Responses. The steroid ACN reversibly reduced the acetylcholine-elicited response of cells expressing nicotinic receptors composed of rat 4 and 2 subunits (Fig. 1; see also Sabey et al., 1999). The block developed and reversed slowly. The slow kinetics of inhibition by ACN was not due to perfusion artifacts, as the response to acetylcholine was much more rapid (see Fig. 1).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   The steroid ACN reversibly blocks responses to 100 µM acetylcholine. The traces show responses of a cell to 100 µM acetylcholine before (left) and 60 s after (right) a response elicited immediately after a 30-s exposure to 10 µM ACN (middle). Responses were recorded at 100 mV.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Time course of inhibition and recovery. A, fraction of control current recorded immediately after exposure to 1 µM ACN (, dotted line) or 10 µM ACN (, solid line) for the indicated time. Responses were assayed with 100 µM acetylcholine. Each point shows the arithmetic mean ± 1 S.D. for 2 to 19 cells, whereas the lines show the fits of single exponential functions declining from 1 to a constant residual current. B, fraction of control current recorded after a 30-s exposure to 10 µM ACN followed by a wash with normal bath solution for the indicated time. Responses assayed with 1 µM acetylcholine. Each point shows the arithmetic mean ± 1 S.D. for 9 to 39 cells, whereas the line shows a single exponential rising from a constant to a final steady level. Note that zero time is offset from the ordinate. We will analyze the time courses further later, but note here that the time for development of half-maximal inhibition was longer when 1 µM ACN was used than with 10 µM ACN. When the data were fit with a single exponential function declining to a constant level, the half-time with 1 µM ACN was 1 s (final level 0.67), whereas with 10 µM ACN, it was 0.6 s (final level, 0.05). When the data for recovery were fit with a single exponential, the time for half-recovery was 7 s (initial value, 0.05; final value, 0.95).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Time course of inhibition. The traces show the currents for two cells that were exposed to 1 µM acetylcholine plus 10 µM ACN for 2 s, normalized to the currents recorded in the same cells in response to 1 µM acetylcholine alone (one cell continuous line, the other dotted). Superimposed on the traces are the mean values shown in Fig. 2A for development of inhibition during preexposure to 10 µM ACN alone. The time courses match very well.

Concentration-Effect Relationship for ACN. The relationship between inhibition and the concentration of ACN is shown in Fig. 4. The inhibition data were fit with eq. 1:

(1)

where IC₅₀ is the concentration of steroid (S) producing half-reduction in the response and n is the Hill coefficient. The fit parameters for the data in Fig. 4 (responses elicited with 100 µM acetylcholine) were an IC₅₀ value of 1.65 µM and an n value of 1.8. We usually used 100 µM acetylcholine to test the responsiveness of cells. However, we also tested the ability of ACN to block responses elicited by 1 µM acetylcholine and estimated that the IC₅₀ was about 1.7 µM (results not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Inhibition by ACN and ent-ACN. The fraction of control current remaining after a 30-s application of ACN (, solid line) or ent-ACN (, dotted line) is shown. The lines show the fits of eq. 1 to the data; for data with ACN, parameter values are IC50 = 1.6 µM, n = 1.8, and with ent-ACN, they are IC50 = 4.6 µM and n = 1.4. Each point shows the arithmetic mean ± 1 S.D. for 3 to 19 cells. Responses assayed using 100 µM acetylcholine.

Properties of Inhibition by ACN. A hydrophobic molecule such as ACN could produce effects mediated by a number of mechanisms, including indirect actions produced by perturbations of the lipid bilayer. One test for the site of action is to determine whether the mirror image of the compound (the enantiomer) has identical effects. The two enantiomers will have identical physical properties, including lipid solubility, but will differ in stereospecific interactions with chiral sites (e.g., a binding site on a protein). The data shown in Fig. 4 demonstrate enantioselectivity in the inhibition of responses elicited by 100 µM acetylcholine. The data with ent-ACN were fit by eq. 1 with parameters of IC₅₀ = 4.6 µM and n = 1.4. The block with 3 µM ACN (0.27 ± 0.09 residual current, N = 11) was significantly larger than that with 3 µM ent-ACN (0.61 ± 0.18, N = 8; P = .001).

A Simple Kinetic Description of Block by ACN. Based on the results we have obtained, it seems likely that steroids interact with a specific site on a target protein. It also is likely that a second messenger system is not required for the actions of steroids. Accordingly, it appears that steroids interact directly with the nicotinic receptor. We undertook an initial analysis of our data to determine whether some kinetic mechanisms for block could be ruled out and to make a provisional estimate of the number of steroid binding sites on the receptor. The goal of the analysis was not to provide a definitive statement on the likely mechanism of block.

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View larger version (22K): [in this window] [in a new window]   Fig. 5.   Fits and predictions of simple models for ACN action. The ability of the occupancy model Occ1 (described in Results) to describe the data for inhibition by ACN is shown. A, time course for development of inhibition (replotted from Fig. 2A) for 1 µM ACN () and 10 µM ACN (). B, time course for recovery (replotted from Fig. 2B, with altered time base to emphasize the early times). C, dependence of inhibition on the concentration of ACN (pooled data for inhibition of responses measured with 1, 10, or 100 µM acetylcholine; fit parameters for eq. 1 are IC50 = 1.6 µM, n = 1.4). The superimposed curves show the predictions for the following models: Occ1 with M = 1 (dotted lines), Occ1 with M = 5 (solid lines), or ConM with M = 2 (dashed lines). Note that the assumption of only one site does not describe the data well. In each case the onset (A, solid symbols) and recovery (B) time courses for 10 µM ACN were fit with an assumed value of M, as described in the text. The parameters were then used to generate the time course of inhibition using 1 µM ACN (A, ) and the concentration dependence of steady-state block (C), with no free parameters.

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Structure Activity Relationship. A number of structurally related steroids were examined with three goals in mind. The first was to define regions of the steroid molecule that are important for activity at this nicotinic receptor and to examine the actions of some endogenous neurosteroids. The second was to compare structural requirements for action at the rat neuronal nicotinic 42 receptor with requirements for action at rat hippocampal GABA_A receptors. This might provide some insight into similarities and differences between the sites on these two related proteins. The final goal was to compare the ability to inhibit nicotinic 42 receptors with the ability of a compound to produce an LRR in X. laevis tadpoles. This comparison would indicate the likelihood that inhibition of this nicotinic receptor is critically involved in producing anesthesia.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Structures of the steroids tested. The structures of the various steroids tested are shown. The structures are arranged in increasing order of effectiveness at reducing acetylcholine responses (see Table 1), from ACN (top left) to 35P (bottom right). Full chemical names are given in the abbreviations footnote.

Role of 3, 5, and 17 Positions. Changing the orientation of the 3-OH group (see Fig. 6 for position; compare ACN with B372 in Table 1) or the stereochemistry at carbon 5 ring (part of the A, B-ring fusion; compare ACN with B164 in Fig. 6) had no effect on ability to inhibit acetylcholine elicited responses (compare ACN with B164 in Table 1). This differs from the requirements for potentiation or gating of the GABA_A receptor. For the GABA_A receptor, changing the 3-OH group from - to -orientation eliminates potentiation, whereas changing the A, B-ring fusion reduces potentiation. Change at both the 3 and 5 positions slightly but significantly reduced the ability to inhibit acetylcholine responses (compare ACN with B260), whereas B260 is a very weak potentiator of GABA_A responses. However, changing the orientation of the carbonitrile group at the 17 position from  to  resulted in a significant decrease in inhibition of acetylcholine responses and greatly reduced potentiation of GABA_A responses (compare B163 with B164; B163 produces more block, P = .013).

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Inhibition by EST, PROG, and 35P. The fraction of control current after a 10- or 30-s application of the various steroids is shown, assayed using 100 µM acetylcholine. Data are shown for EST (, solid line), PROG (, dotted line), and 35P (, dashed line). The lines show the fits of eq. 1 to the data with parameters IC50 = 3.0 µM, n = 1.2 (PROG), IC50 = 10 µM, n = 1.2 (EST), and IC50 = 12.3 µM and n = 1.55 (35P). The fits were made assuming that complete block occurred at high steroid concentrations. Each point shows the arithmetic mean ± 1 S.D. for data from the following numbers of cells: PROG, 3 µM 6, 10 µM 4, 30 µM 3; EST, 3 µM 15, 10 µM 6, 30 µM 4; and 35P, 3 µM 4, 10 µM 4, 20 µM 8.

Correlation with Anesthetic Potency in Tadpoles. We obtained data on the concentration of steroid producing an LRR in half of the X. laevis tadpoles exposed to steroid. Because we did not have full nicotinic receptor blocking curves for all compounds, we initially converted the data into ranks for examining correlations. The rank 1 was assigned to the compound producing the most block when used at a steroid concentration of 3 µM and correspondingly to the compound that caused LRR at the lowest concentration. The ranks were correlated significantly, with a correlation coefficient of 0.71 and the probability that the coefficient differs from zero of P = .007 (Fig. 8A).

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Correlations between inhibition of nicotinic receptors and LRR in tadpoles. A, scatterplot of the rank value for the ability of a compound to block acetylcholine elicited currents (smallest residual current in the presence of 3 µM compound is given rank 1) and the rank for ability to cause LRR in tadpoles (smallest IC50 is given rank 1). The linear regression coefficient is 0.71 (P = .007), indicating that compounds that produce a greater amount of block are likely to have a greater potency at producing LRR. The line shows the predicted regression relationship (for data values, see Table 1). B, scatterplot of IC50 values (filled symbols measured, open symbols calculated) for the ability of a compound to block acetylcholine-elicited currents and measured IC50 values for ability to cause LRR. Note that both ordinates are logarithmically scaled. The solid line shows the regression relationship for logarithmically transformed data, and the dotted line shows a slope of 1. The linear regression coefficient for logarithmically transformed data is 0.2 (P = .035). When only the experimentally determined IC50 values for block of acetylcholine elicited currents are used (filled symbols), the linear regression coefficient for logarithmically transformed data is 0.5 (P = .08).

	Discussion

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Mechanism of Action of ACN. These data indicate that the actions of ACN are not mediated by a second messenger pathway, at least one requiring high-energy phosphate compounds or the involvement of GTP-binding proteins. The observed enantioselectivity between ACN and ent-ACN indicates that the steroid interacts with a chiral site, rather than intercalating into a bulk hydrophobic milieu to produce the block. Furthermore, the structure-activity data indicate some specificity in interactions between steroid and site. These data suggest that steroids interact with specific sites on the 42 neuronal nicotinic receptor. Previous work has found that ACN does not affect the binding of radiolabeled cytisine to homogenates prepared from these cells (Sabey et al., 1999), which indicates that is not a competitive inhibitor of agonist binding.

Comparison of Steroid Structure Important for Actions at Neuronal Nicotinic and GABA_A Receptors. There is a clear effect of the stereochemical orientation of the substituent attached at the 17 position, with a -orientation of either a hydroxyl or carbonitrile group producing more potent nicotinic block than an -orientation. This stereoselectivity is qualitatively similar to that for potentiation of responses from GABA_A receptors. In contrast, the stereochemistry of the A, B-ring fusion or the orientation of the hydroxy group at position 3 has much less of an effect on inhibition of the rat 42 nicotinic receptor than on potentiation of GABA_A receptors. These comparisons demonstrate that the interactions between the steroids and the binding site have different structural requirements for neuronal nicotinic receptors and GABA_A receptors. There are undoubtedly additional features of steroid structure that play a role in the inhibition of rat 42 nicotinic receptors, which may involve both the A and D rings. These are suggested by comparisons between the structure and activity for drugs such as ACN, 35P, and progesterone (see Fig. 6 and Table 1).

Correlation with Production of LRR in Tadpoles. There is a strong correlation in the rank orders of the abilities of these compounds to produce anesthesia in X. laevis tadpoles (as assayed by the LRR) and the amount of block produced. The correlation suggests that the neuronal nicotinic receptor is involved in production of anesthesia. This simple interpretation is likely inappropriate, because the required concentrations are not linearly related. Three alternative explanations seem more likely. The first is that the rank correlation is simply fortuitous. This seems unlikely, given the statistical significance of the difference of the correlation coefficient from zero. The second is that a single alternative target with similar structural requirements is critical for anesthetic actions. The third possibility is that there are several targets that can result in anesthesia. For example, potentiation of GABA_A receptor activation might be a primary mechanism. However, for some agents that act weakly at GABA_A receptors, alternative targets might serve as the molecular substrate for effects leading to LRR (e.g., neuronal nicotinic receptors or voltage-gated calcium channels; Todorovic et al., 1998). Some of the compounds tested in the present study were chosen because they produced LRR with minimal apparent actions on GABA_A responses (see Table 2), and so the choice of drugs might have emphasized the role of targets other than GABA_A receptors. In sum, the data indicate that the neuronal nicotinic receptor is not likely to be a primary target by which steroid anesthetic agents produce their behavioral effects. However, block of nicotinic receptors may play a role in the actions of some anesthetic steroids.

Responses to Neuroactive Steroids Produced In Vivo. We tested several neuroactive steroids that are present in the rat brain. The compound with the strongest blocking activity is progesterone, with an apparent IC₅₀ value of about 3 µM. The concentration in human cerebrospinal fluid has been estimated to be much lower, about 0.1 nM (Backstrom et al., 1976; Uzunova et al., 1998). There are no comparable data for rat cerebrospinal fluid, but the amount of progesterone in rat brain tissue is about 20 nmol/kg wet wt. (Purdy et al., 1991; Corpechot et al., 1993), whereas the value for human brain is about 100 nmol/kg (Bixo et al., 1997). The concentration of -estradiol may be even lower: human cerebrospinal fluid concentration is about 0.01 nM (Backstrom et al., 1976), whereas the total brain amount is about 0.2 nmol/kg (female human brain, Bixo et al., 1995; female rat brain, Bixo et al., 1986). It is not certain what the relevant concentration of steroid would be. For example, the free concentration in the cleft might be critical or the concentration in the membrane, and there may be locally increased concentrations of some steroids. However, the bulk concentration in the brain is low compared with the IC₅₀ values we have measured, which suggests that the rat neuronal nicotinic 42 receptor is unlikely to be significantly modulated by endogenously produced steroids. It is possible that local high concentrations of steroids may appear in specific brain regions or that longer exposures to steroid might reveal an action on these receptors.

Other Studies of Steroid Action on Nicotinic Receptors. Previous studies of neuronal nicotinic receptors have found that chicken 42 receptors, expressed in X. laevis oocytes, are inhibited by progesterone with a similar potency as that seen in our studies (Bertrand et al., 1991; Valera et al., 1992), although the Hill coefficient is less than 1. They found that progesterone does not alter desensitization or compete for the agonist binding site, as our results indicate. 35P could produce only a partial block of responses from these chicken receptors even at maximally effective doses (Bertrand et al., 1991). In our hands, 35P could produce full block of rat 42 receptors, although with lower potency than progesterone. Ion fluxes in SH-SY5Y cells (likely expressing neuronal nicotinic receptors containing 3 and 4 subunits) also are inhibited by progesterone with an IC₅₀ value of 11 µM (Ke and Lukas, 1996). In both of these studies, progesterone covalently coupled to bovine serum albumin was effective at inhibition, indicating that the steroid can reach its site directly from the bathing solution.