Abstract
This study reports the actions of enantiomer pairs of anesthetic steroids 3α5αP/ent-3α5αP and 3α5βP/ent-3α5βP as modulators of γ-aminobutyric acid (GABA)A receptors and as anesthetics. The enantiomers of structurally related 17-carbonitrile analogs also are examined. These studies were aimed at 1) determining whether the steroid recognition site could distinguish between molecules differing in shape, but not other physical properties (enantioselectivity); 2) providing further insight into the structure-activity relationships of anesthetic steroids; and 3) determining whether modulation of GABAA receptor function correlates with anesthetic potency for anesthetic steroid enantiomers. Stereoselective actions of the compounds were evaluated in four different bioassays: 1) noncompetitive displacement of [35S]t-butylbicyclophosphorothionate from the picrotoxin site of GABAA receptors present in rat brain membrane preparations; 2) modulation of GABA currents in cultured rat hippocampal neurons; 3) loss of righting reflex in tadpoles; and 4) loss of righting reflex in mice. The data indicate that 5α-reduced steroids, but not 5β-reduced steroids, show a high degree of enantioselectivity/enantiospecificity in their actions as modulators of GABAA receptors and as anesthetics. For all compounds studied, the effects on GABAA receptor function closely tracked with anesthetic effects. These data show that the anesthetic steroid recognition site is capable of distinguishing enantiomers, suggesting a protein-binding site of specific dimensions and shape. The results are compatible either with a structural model of the binding site that can accommodate 3α5αP, 3α5βP, andent-3α5βP, but not ent-3α5αP, or with two different binding sites for steroid anesthetics.
The steroids 3α5αP (allopregnan-3α-ol-20-one) and 3α5βP (pregnanolone) are potent anesthetics (Phillipps, 1974). A large amount of data supports the hypothesis that the anesthetic actions of these steroids are correlated with their enhancement of γ-aminobutyric acid (GABA)A receptor-mediated neuronal inhibition (Lambert et al., 1995). The number of binding sites for these steroids and their location on GABAA receptors are not known. Moreover, whether the steroids share a common site is not known. Molecular-modeling studies suggest that the anesthetic activities of these steroids result from binding at a common binding site (Purdy et al., 1990; Han et al., 1996). However, binding studies for steroid-induced noncompetitive displacement oft-butylbicyclophosphorothionate (TBPS) from the picrotoxin-binding site on GABAA receptors indicate that, although these steroids may share a common binding site, multiple binding sites are detectable for some steroids (Morrow et al., 1990; Hawkinson et al., 1994a,b, 1998).
Because 3α5αP and 3α5βP are molecules that each contain eight chiral centers (C-3, C-5, C-8, C-9, C-10, C-13, C-14, and C-17), their stereoselective modulation of GABAA receptor function can be studied by inverting each, some, or all of the chiral centers in the molecules. Inversion of one center in a molecule containing multiple chiral centers gives compounds called epimers. Epimers are a subset of compounds called diastereomers. Diastereomers are compounds with multiple chiral centers that differ in configuration at one or some, but not all, chiral centers. Studies of the C-3 (inversion of the 3-hydroxyl group) and C-17 (inversion of the acetyl group) epimers of 3α5αP and 3α5βP have been very informative in establishing the structure-activity relationships for anesthetic steroids (Phillipps, 1974). No doubt, studies of additional diastereomers of 3α5αP and 3α5βP will further define these structure-activity relationships. However, because diastereomers have different physical properties due to the relative positions of some atoms in each diastereomer being different, the membrane-perturbing effects as well as the direct effects of these steroids on GABAA receptor function will be different. Thus, C-3 epimers (3β-OH) of 3α5αP and 3α5βP not only fail to potentiate GABA effects at GABAA receptors but also interact differently with membrane phospholipids (Makriyannis et al., 1991).
To avoid the membrane-perturbing effects caused by studying anesthetic steroid diastereomers having different physical properties, we have studied anesthetic steroid enantiomers (Fig.1). Enantiomers are the stereoisomers of optically active compounds that are mirror images of each other (all chiral centers have the opposite absolute configuration). Enantiomers have identical physical properties because the relative positions of all atoms in each enantiomer are identical. For example, any group having an axial configuration in one enantiomer also has an axial configuration in the other enantiomer.
Enantioselectivity of 3α5αP-induced GABAAreceptor modulation and anesthesia in Xenopus laevistadpoles and mice has been reported previously (Wittmer et al., 1996). Herein, we report the corresponding actions of the 3α5βP enantiomers. Additionally, new information for the displacement of TBPS binding by both pairs of enantiomers is reported. Some comparative data for the corresponding enantiomers of the 17-carbonitrile analogs of 3α5αP and 3α5βP also are presented. The new data show that enantioselectivity [comparison of 3α5αP/ent-3α5αP with 3α5βP/ent-3α5βP) and diastereoselectivity (comparison of 3α5αP/3α5βP withent-3α5αP/ent-3α5βP) for modulation of GABAA receptor function by these anesthetic steroids are different. The enantioselective actions of 3α5αP are significantly greater than the enantioselective actions of 3α5βP. The diastereoselective actions of the 3α5αP/3α5βP steroid pair are not significant and those of theent-3α5αP/ent-3α5βP pair are significantly different.
Materials and Methods
Chemicals.
The ent-3α5βP,ent-3α5αP, 3α5αACN [(3α,5α,17β)-3-hydroxyandrostane-17-carbonitrile],ent-3α5αACN, and 3α5βACN [(3α,5β,17β)-3-hydroxyandrostane-17-carbonitrile] were prepared and characterized as described previously (Hu et al., 1993, 1997; Han et al., 1996; Nilsson et al., 1998). The ent-3α5βACN was prepared from ent-(3α,5β)-3-hydroxyandrostan-17-one with the methods described for the preparation of 3α5βACN (Han et al., 1996). The infrared, 1H NMR, and13C NMR spectra of the 3α5βACN andent-3α5βACN were identical. The 3α5αP and 3α5βP were purchased from either Sigma Chemical Co. (St. Louis, MO) or Steraloids (Newport, RI). The [35S]TBPS was purchased from NEN Life Science Products (Boston, MA) and TBPS was purchased from Research Biochemicals International (Natick, MA).
[35S]TBPS Binding.
Rat brain cortical membranes were prepared with minor modifications of the method previously reported (Hawkinson et al., 1994a). Briefly, frozen rat cerebral cortices (Pel-freez, Rogers, AK) were thawed and homogenized in 10 volumes of ice-cold 0.32 M sucrose with a glass/Teflon pestle. The homogenate was centrifuged at 1500g for 10 min at 4°C. The resultant supernatant was centrifuged at 10,000g for 30 min at 4°C. The pellet (P2) from this centrifugation was resuspended in 200 mM NaCl, 50 mM potassium phosphate buffer, pH 7.4, and centrifuged at 10,000g for 20 min at 4°C. This washing procedure was done three times, and then pellets were resuspended in buffer (∼4 ml/brain) with a glass/Teflon pestle. The membrane suspension was aliquoted, frozen in liquid nitrogen, and stored at −80°C before use. [35S]TBPS binding assays were done according to the procedure described previously (Hawkinson et al., 1994a) with modifications. Briefly, aliquots of membrane solution (0.5 mg/ml final protein concentration in assay) were incubated with 5 μM GABA, 2 nM [35S]TBPS (45–120 Ci/mmol), and 5 μl-aliquots of steroid in dimethyl sulfoxide (DMSO) solution (final assay concentrations ranged from 1 nM to 10 μM), and brought to a final volume of 1 ml with 200 mM NaCl, 50 mM potassium phosphate buffer, pH 7.4. Control binding was defined as binding observed in the presence of 0.5% DMSO and the absence of steroid. Nonspecific binding was defined as binding observed in the presence of 200 μM picrotoxin and ranged from 6.1 to 14.3% of total binding. Assay tubes were incubated for 2 h at room temperature. A Brandel (Gaithersburg, MD) cell harvester was used for filtration of the assay tubes through Whatman/GF/C glass filter paper. Filter paper was rinsed with 4 ml of ice-cold buffer three times. Radioactivity bound to the filters was read by liquid scintillation counter and data were fit with Sigma Plot version 3.0 to the Hill equation
Electrophysiological Recording.
Hippocampal neurons were cultured from 1- to 2-day-old Sprague-Dawley albino rats with methods described previously (Thio et al., 1991). After 4 to 7 days in culture, neurons were voltage clamped at −60 mV with whole-cell patch-clamp recording techniques. The extracellular recording solution contained 140 mM NaCl, 4 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM glucose, pH 7.3. Recording pipettes were filled with a solution containing 140 mM CsCl, 4 mM NaCl, 0.5 mM CaCl2, 4 mM MgCl2, and 10 mM HEPES, pH 7.3. GABA and steroids were applied for 500 ms with a pressure (20 psi of air) ejection drug delivery system with patch pipettes positioned ∼5 μm from the recorded neuron. This system allowed reliable drug application while minimizing exposure of neurons to steroids. The concentrations reported are those in the pipette and are ∼2- to 3-fold higher than the concentrations bathing the cell. Steroids were prepared in a DMSO stock at 10 to 100 mM and diluted so that the final concentration of DMSO was <0.5%. DMSO at this concentration had no effect on GABA responses. Concentration-response data were fit to an equation of the form
Tadpole Loss of Righting Reflex (LRR) Anesthesia Assay.
Tadpole LRR was measured as described previously (Wittmer et al., 1996). Briefly, groups of 10 early prelimb-bud stage Xenopus laevis tadpoles (Nasco, Fort Atkinson, WI) were placed in 100 ml of oxygenated Ringer's stock solution containing various concentrations of compound. Compounds were added from a 10 mM DMSO stock (final concentration of DMSO in test solutions was 0.1%). After equilibrating at room temperature for 3 h, tadpoles were evaluated with the LRR behavioral endpoint. LRR was defined as failure of the tadpole to right itself within 5 s after being flipped by a smooth glass rod. In all cases, the tadpoles regained their righting reflex when placed in fresh oxygenated Ringer's solution. Control beakers containing up to 0.6% DMSO produced no LRR in tadpoles. Concentration-response curves were fit with Sigma Plot version 3.0 to the Hill equation
Mouse Anesthesia Assay.
BALB/c mice (Sprague-Dawley; Harlan Breeders, Indianapolis, IN) weighing 20 to 30 g each were placed under a heat lamp for 1 to 2 min. Compounds were injected i.v. through a tail vein with various doses of either 3α5βP orent-3α5βP in an 8% ethanol, 16% Cremaphor EL (Sigma Chemical Co.) solution at a rate of 50 to 250 μl/5 to 10 s. Sleep time was measured from the moment mice displayed LRR until they were able to right themselves. All mice recovered fully without observable neurological deficits. Control solutions without steroids were administered with no observable neurobehavioral effect.
Results
[35S]TBPS Binding.
Anesthetic steroids are known to be noncompetitive displacers of [35S]TBPS from the picrotoxin-binding site of GABAAreceptors (Majewska et al., 1986). In this study, the enantioselectivity for [35S]TBPS displacement by four pairs of anesthetic steroid enantiomers was examined (Fig.2). For the two 5β-reduced steroid enantiomer pairs, 3α5βP/ent-3α5βP and 3α5βACN/ent-3α5βACN, the natural enantiomers were each ∼4-fold more potent displacers of [35S]TBPS. For the two 5α-reduced steroid enantiomer pairs, 3α5αP/ent-3α5αP and 3α5αACN/ent-3α5αACN, the natural enantiomers were the more potent displacers by factors of 26 and 80, respectively. Thus, enantioselectivity for [35S]TBPS displacement was found for all enantiomer pairs, but the degree of enantioselectivity was much higher for the 5α-reduced steroids.
Steroid Effects on GABAA Currents.
The enantioselectivity found for modulation of GABAAreceptor function in cultured rat hippocampal neurons by the enantiomer pairs is shown in Figs. 3 and4. At a steroid concentration of 10 μM, there was no enantioselectivity for potentiation of 2 μM GABA-mediated currents by the 3α5βP/ent-3α5βP pair (Fig. 3). In contrast, an ∼4-fold enantioselectivity for the degree of potentiation under the same experimental conditions was found for the 3α5αP/ent-3α5αP pair (Fig. 3). This last result is in close agreement with results previously reported from a more extensive electrophysiological evaluation of GABAA receptor modulation in these cells by the 3α5αP/ent-3α5αP pair (Wittmer et al., 1996).
A concentration-response curve for potentiation of GABA-mediated currents by the 3α5βP/ent-3α5βP pair is shown in Fig. 4A. The 3α5βP enantiomer was 3-fold more potent than theent-3α5βP enantiomer (EC50 = 1.2 and 3.6 μM, respectively). In Fig. 4B, the concentration-response curve for gating of GABAA receptors in the absence of added GABA is shown. The ent-3α5βP gates a current at the same concentration as 3α5βP and does so to a very similar extent. This contrasts with the results previously obtained with ent-3α5αP and ent-3α5αACN, neither of which gates GABAA receptors at a concentration of up to 100 μM (Wittmer et al., 1996).
Tadpole LRR.
The concentration-response relationships for the loss of tadpole LRR were determined for the 3α5βACN/ent-3α5βACN and 3α5βP/ent-3α5βP pairs. Little, if any, enantioselectivity was observed for these enantiomer pairs (Fig.5). The results contrast with the significant degrees of enantioselectivity found previously (Wittmer et al., 1996) for tadpole LRR with the 3α5αACN/ent-3α5αACN and 3α5αP/ent-3α5αP pairs (10- and 2.8-fold, respectively).
Mouse LRR.
When injected into mice, both 3α5βP andent-3α5βP produced anesthesia (as indicated by LRR) in a dose-dependent manner (Fig. 6). The slopes of the lines from a regression analysis of the dose-response data for this enantiomer pair differ by a factor of 2. The 3α5βACN/ent-3α5βACN pair was not tested for anesthetic activity in mice because adequate amounts ofent-3α5βACN were not available. These 3α5βP/ent-3α5βP results for mouse anesthesia are different from those obtained previously for the 3α5αACN/ent-3α5αACN pair in this bioassay (Wittmer et al., 1996). In that study, the lowest dose of 3α5αACN shown to cause LRR was 4 mg/kg; whereas ent-3α5αACN did not cause LRR at a dose as high as 80 mg/kg.
Discussion
The results from this and our previous enantioselectivity study (Wittmer et al., 1996) provide new information about the enantioselective and diastereoselective interactions of anesthetic steroids with GABAA receptors. Enantioselective interactions can be examined by comparing the results obtained with the 3α5α/ent-3α5α and 3α5β/ent-3α5β steroid pairs. Diastereoselective interactions can be examined by comparing the results obtained for the 3α5α/3α5β andent-3α5α/ent-3α5β steroid pairs because only the chiral center at C-5 is different in each of these steroid pairs.
As summarized in Table 1, all pairs of anesthetic steroid enantiomers studied in this and our previous study (Wittmer et al., 1996) exhibit some degree of enantioselectivity in electrophysiology, [35S]TBPS binding, and when performed, the mouse anesthesia tests. Significant enantioselectivity was not found in the tadpole LRR experiments for the 5β-reduced enantiomer pairs. Notably, the degree of enantioselectivity is uniformly greater across the various assays for the steroids in the 5α-reduced series. The consistency of these results is remarkable considering that different species and preparations were used in the bioassays. Of course, differences in species and preparation also limit interpretation of the results. For example, the enantioselectivity for anesthetic steroid effects on TBPS binding may differ for different GABAA receptor subtypes. Additonal studies with different subtypes of GABAA receptors are needed to address this possibility.
In the electrophysiological experiments, the enantioselectivity is manifested in different ways for the 5α- and 5β-series of compounds. For potentiation of GABA-mediated currents, enantioselectivity is detected as a difference in maximal response for the steroids in the 5α-reduced series, whereas it is detected as a difference in the EC50 values for the 3α5βP/ent-3α5βP enantiomer pair in the 5β-reduced series. Based on prior studies (Wittmer et al., 1996; Zorumski et al., 1998), there are likely to be significant differences in the potencies of the 5α-reduced enantiomers for potentiating GABA responses, but poor solubility at concentrations ≥100 μM limits the ability to generate accurate concentration-response data for theent-steroids. There is also a marked enantioselectivity difference in the gating of GABAA receptors by the two series of steroids. The ent-steroids in the 5α-reduced series do not gate these ion channels at concentrations up to 100 μM, whereas ent-3α5βP was found to gate the ion channels at least as well as 3α5βP over the concentration range of 1 to 100 μM.
Comparing the IC50 values from the [35S]TBPS binding experiments and the EC50 values from tadpole LRR and electrophysiological experiments shows that the effects of the compounds occur in the same concentration range throughout these bioassays. However, the enantioselectivity for [35S]TBPS displacement by the 3α5αACN and 3α5αP enantiomer pairs is much greater than the enantioselectivity found for the anesthetic effects of these compounds in the tadpole LRR test. Additionally, whereas a small degree of enantioselectivity is found for the 5β-reduced steroids in the [35S]TBPS-binding experiments, significant enantioselectivity for these compounds was not found in the tadpole LRR test. The reasons for the failure of the tadpole LRR results to more closely correlate with those of the [35S]TBPS binding experiment are not clear, although the complexity of factors involved in behavioral responses is likely to contribute.
Overall the results show that 3α5α-, 3α5β- andent-3α5β-steroids potently modulate GABAA receptors, whereasent-3α5α-steroids do not effectively modulate these receptors. Enantioselectivity for GABAA receptor modulation/anesthesia by anesthetic steroids in the 5α- and 5β-reduced steroid series is different (5α > 5β). Because of this enantioselectivity difference, the diastereoselective interactions of the ent-3α5α/ent-3α5β and 3α5α/3α5β steroid pairs are also different (ent-pairs > natural pairs). These stereoselectivity results raise interesting questions. Do these stereoselectivity differences imply different mechanisms (direct versus indirect) for modulation of GABAA receptor function and the correlated anesthetic actions of the two series of anesthetic steroids? Do these stereoselectivity differences suggest different binding sites for the two series of anesthetic steroids on GABAA receptors? These questions are addressed in the following discussion.
Our studies of the enantioselectivity of anesthetic steroid effects on GABAA receptor function were initiated as a way to distinguish between direct (a steroid-binding site on the receptor) and indirect (membrane perturbation) effects of these compounds on receptor function. We define direct effects as those arising from molecular interactions involving the steroid and amino acids of the receptor. We define indirect effects as those arising from molecular interactions involving the steroid and other nonprotein membrane constituents such as cholesterol and phospholipids. The degree to which enantioselectivity can be used to make the distinction between the two types of interactions is dependent on how large a difference is expected for the direct and indirect actions of the steroid on receptor function.
Binding interactions of ligands with receptors are expected to be highly enantioselective because receptor proteins are made of onlyl-amino acids. For substrates binding to enzymes, where both the initial binding and then a subsequent chemical reaction must occur, the expected result is complete enantioselectivity (i.e., the enzymatic transformation is expected to be enantiospecific). However, there are exceptions to the expected results for these types of interactions. For example, the binding of nornicotine to nicotinic acetylcholine receptors is not enantioselective. Both (+)- and (−)-nornicotine displace (−)-[3H]nicotine with equal potency and efficacy (Zhang and Nordberg, 1993; Badio and Daly, 1994; Abreo et al., 1996).
Many examples of esterases that demonstrate varying degrees of enantioselectivity for the hydrolysis of racemic esters are reported in the literature. Indeed, the effective separation of racemic esters by enzymes (one ester enantiomer is preferentially hydrolyzed and readily separated from the unreacted ester enantiomer as the corresponding alcohol enantiomer) as a method for the resolution of the optically active components may require the screening of different esterases to identify the most enantioselective esterase for a particular separation (Chen et al., 1982 and references therein). The implication of these examples for this study is that the varying degrees of enantioselectivity found for the anesthetic steroids are all consistent with a direct steroid-receptor interaction. Indeed, even if enantioselectivity had not been observed in this study, the possibility of a direct steroid-receptor interaction could not be unequivocally ruled out.
Based on information currently available, no enantioselectivity would be expected for the indirect modulation of GABAAreceptor function by anesthetic steroid enantiomers. This conclusion is based on the following reasoning and supporting evidence. Because enantiomers have identical physical properties, they alter the physical properties of a membrane (e.g., fluidity) in an identical manner unless the physical properties of the membrane are already dependent on preexisting enantiospecific interactions between membrane constituents. Most obviously, because the cholesterol and phospholipids found in cell membranes occur in enantiomerically pure form, interactions between these molecules could fulfill the preexisting enantiospecificity requirement. The extent to which the different anesthetic steroid enantiomers altered these preexisting enantiospecific cholesterol-phospholipid interactions would then determine the anesthetic steroid enantioselectivity for indirect modulation of channel function.
Is there any evidence that enantiospecific cholesterol-phospholipid interactions are determinants of membrane physical properties? Because of the difficulty involved in addressing this question (the unnatural enantiomers of cholesterol and/or a phospholipid are required for the studies), few experiments have been conducted to examine this question. However, the few available experiments (lipid monolayer and NMR studies) provide no evidence that enantiospecific cholesterol-phospholipid interactions have any effect on the physical properties of the membrane (Ghosh et al., 1971; Arnett and Gold, 1982). Thus, we have referred previously to the membrane as a nonchiral environment and we have concluded that no enantioselectivity for anesthetic steroid action is expected if the effects of these compounds are indirectly caused by membrane perturbation (Wittmer et al., 1996). Realizing that this conclusion is based on very limited experimental data, we recently reported a new method for preparing the unnatural enantiomer of cholesterol (Kumar and Covey, 1999) and we plan to address the biological significance of enantiospecific interactions between cholesterol and different classes of phospholipids in future studies. Because, in this study, we find some degree of enantioselectivity for both series of anesthetic steroids, we find no compelling reasons to conclude that either series of anesthetic steroids modulates GABAA receptor function by an indirect mechanism.
Whether the different degrees of enantioselectivity imply different receptor-binding sites for the two series of anesthetic steroids is the final question to address. Although there are data from previous studies suggesting the existence of more than one class of binding sites for some steroids on GABAA receptors (Morrow et al., 1990; Hawkinson et al., 1994a,b, 1998), the enantioselectivity results we have obtained thus far present no new evidence for this phenomenon. Previous data from studies of the diastereoselective modulation of GABAA receptor function by 3α5αP and 3α5βP have been reasonably explained by a common binding-site hypothesis developed from molecular modeling studies (Purdy et al., 1990; Han et al., 1996). The results from this and the previous enantioselectivity study of 5α-reduced anesthetic steroids (Wittmer et al., 1996) establish new criteria that must be satisfied by molecular models of a common binding site for 5α- and 5β-reduced anesthetic steroids. Molecular models of a common binding site for both classes of anesthetic steroids also must be able to explain the greater enantioselectivity of 5α-reduced anesthetic steroids and the greater diastereoselectivity observed for theent-3α5αP/ent-3α5βP steroid pair versus the 3α5αP/3α5βP steroid pair.
Footnotes
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Send reprint requests to: Douglas F. Covey, Ph.D., Department of Molecular Biology and Pharmacology, Box 8103, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. E-mail: dcovey{at}molecool.wustl.edu
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↵1 This study was supported by U.S. Public Health Service Grant GM47969, Research Scientist Development Award MH00964, and the Bantly Foundation.
- Abbreviations:
- 3α5αP
- (3α,5α)-3-hydroxypregnan-20-one
- 3α5βP
- (3α,5β)-3-hydroxypregnan-20-one
- GABA
- γ-aminobutyric acid
- TBPS
- t-butylbicyclophosphorothionate
- ent-3α5αP
- (3β,5β,8α,9β,10α,13β,14α,17α)-3-hydroxypregnan-20-one
- ent-3α5βP
- (3β,5α,8α,9β,10α,13β, 14α,17α)-3-hydroxypregnan-20-one
- 3α5αACN
- (3α,5α,17β)-3-hydroxyandrostane-17-carbonitrile
- 3α5βACN
- (3α,5β,17β)-3-hydroxyandrostane-17-carbonitrile
- ent-3α5αACN
- (3β,5β,8α,9β,10α,13β,14α,17α)-3-hydroxyandrostane-17-carbonitrile
- ent-3α5βACN
- (3β,5α,8α,9β, 10α,13β,14α,17α)-3-hydroxyandrostane-17-carbonitrile
- DMSO
- dimethylsulfoxide
- LRR
- loss of righting reflex
- Received November 17, 1999.
- Accepted February 22, 2000.
- The American Society for Pharmacology and Experimental Therapeutics