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Anesth Analg 2008; 106:838-845
© 2008 International Anesthesia Research Society
doi: 10.1213/ane.0b013e31816095bd
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ANESTHETIC PHARMACOLOGY

The Anesthetic-Like Effects of Diverse Compounds on Wild-Type and Mutant {gamma}-Aminobutyric Acid Type A and Glycine Receptors

Liya Yang, PhD, and James M. Sonner, MD

From the Department of Anesthesia and Perioperative Care, University of California, San Francisco, California.

Address correspondence to Dr. Sonner, Department of Anesthesia and Perioperative Care, Room S-455i, University of California, San Francisco, CA 94143-0464. Address e-mail to sonnerj{at}anesthesia.ucsf.edu.


   Abstract
Top
 Abstract
Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
INTRODUCTION: No theory of inhaled anesthetic action requires volatility of the anesthetic to accomplish the biophysical interaction of anesthetic with biological target. The identification of mutations that attenuate the effect of inhaled anesthetics on various receptors raises the possibility that nonvolatile compounds with anesthetic effects can be identified with the aid of these receptors. In previous studies, we identified compounds that were either charged or had an exceptionally low vapor pressure and which modulated anesthetic-sensitive receptors in a manner similar to inhaled anesthetics. We tested whether these, and another charged compound, shared a common mechanism with volatile anesthetics, by comparing their effect on wild-type {gamma}-aminobutyric acid type A (GABAA) or glycine receptors and mutant receptors that were engineered to be relatively resistant to inhaled anesthetics.

METHODS: The effect of β-hydroxybutyric acid, ammonium chloride, diethylhexyl phthalate, and GABA were tested on homomeric {alpha}1 and mutant {alpha}1 (S267I) glycine receptors. The effect of sodium dodecyl sulfate and glycine were tested on {alpha}1b2{gamma}2s and mutant {alpha}1(S270I)β2{gamma}2s GABAA receptors. Receptors were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping. For both GABAA and glycine receptors, isoflurane and ethanol were used as positive controls and propofol as a negative control (i.e., unaffected by the mutation).

RESULTS: β-hydroxybutyric acid, ammonium chloride, diethylhexyl phthalate, and GABA all enhanced glycine receptor function. This effect was reduced by the S267I mutations. Sodium dodecyl sulfate and glycine enhanced GABAA receptor function, and the S270I mutation attenuated this effect.

CONCLUSION: These findings support the hypothesis that the compounds studied modulate GABAA or glycine receptors by a mechanism similar to that of isoflurane and ethanol. Comparing the effect of drugs on anesthetic-sensitive wild-type receptors with relatively less sensitive mutant receptors may help identify compounds with anesthetic effects.


   Introduction
Top
 Abstract
 Introduction
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
For many years, the lipid bilayer was considered the major target of general inhaled anesthetics.1–3 More recently, studies have focused on neuronal proteins, particularly ligand-gated ion channels.4–6 However, neither theory has provided sufficient molecular insight into anesthetic action to develop new drugs, nor does either theory of anesthesia require volatility of the anesthetic to accomplish the biophysical interaction of anesthetic with biological target.

Over the past decade, point mutations have been made in anesthetic-sensitive receptors that diminish the effect of anesthetics on those receptors.7,8 This raises the possibility that a comparison of the effect of drugs on wild-type (WT) anesthetic-sensitive ion channels and mutant channels, which are less anesthetic-sensitive, can be used to help identify new anesthetic compounds, including nonvolatile compounds with anesthetic effects. In previous studies, we identified compounds that were either charged or had an exceptionally low vapor pressure and which modulated anesthetic-sensitive receptors in a manner similar to inhaled anesthetics.9–12 Here, we tested whether these, and another charged compound, shared a common mechanism with volatile anesthetics. We did this by comparing their anesthetic-like effects on WT {gamma}-aminobutyric acid type A (GABAA) or glycine (Gly) receptors and on mutant GABAA and Gly receptors engineered to be relatively resistant to inhaled anesthetics.7,13,14

We identified these compounds using a variety of physical and biological principles. First, we attempted a test of the interfacial theory of anesthesia.15 An interface is the boundary between two immiscible condensed phases, such as that between a membrane and water or protein and water. The interfacial theory of anesthesia states that anesthetics are interfacially active, that is, they accumulate at interfaces. We asked if interfacial activity was a sufficient condition for anesthetic-like modulation of receptor function. We studied a nonvolatile interfacially active amphiphile, sodium dodecyl sulfate (SDS), applying it to WT and mutant GABAA receptors. If interfacial activity suffices to produce anesthetic-like modulation of receptor function, then SDS should have the same modulatory effect as an inhaled anesthetic on these receptors, assuming that it mimics the effect of anesthetic on appropriate interfacial properties.

Second, we studied two endogenous compounds which, in millimolar concentrations, have anesthetic-like effects in animals.9,12 These compounds are metabolites that are increased in conditions producing a reversible state of coma resembling general anesthesia. We have shown that β hydroxybutyric acid in concentrations that are observed clinically in diabetic ketoacidosis has a large anesthetic-like positive modulatory effect on Gly receptors, and anesthetizes tadpoles.12 Ammonia, which is increased in fulminant hepatic failure and in uremia, also positively modulates Gly receptors, and reduces isoflurane requirement in rats.9 We hypothesized that both compounds would behave like volatile anesthetics on WT and mutant Gly receptors.

Third, we examined the effect of diethylhexylphthalate (DEHP) on Gly receptor function. DEHP is an uncharged aromatic compound which is a liquid with a low vapor pressure at room temperature. It also has a large positive modulatory effect on Gly receptors.11 This compound was of interest to us because it is much larger (molecular weight approximately 390, molecular volume approximately 450 Å3) than most volatile compounds that are anesthetics.16

Finally, we tested the modulatory effect of two neurotransmitters. It has been proposed that certain neurotransmitters might have anesthetic-like effects on their receptors.17 We previously tested a related prediction that GABA positively modulates Gly receptors, and that Gly positively modulates GABAA receptors, finding it to be correct.10 We sought to strengthen this finding with additional studies on these Gly receptors and on a different subtype of GABAA receptor to support the hypothesis of a common mechanism with volatile anesthetics.

We chose GABAA and Gly receptors for study. These receptors are members of the pentameric cysteine-loop ligand-gated ion channels superfamily, and are the major inhibitory neurotransmitter receptors in the central nervous system (CNS).18,19 A broad range of inhaled anesthetics, at clinically effective concentrations, can enhance their function.4,7,13,20,21

In the mutants we studied, serine-267 in transmembrane domain 2 (TM2) of the Gly receptor {alpha}1 subunit and the homologous residue of the GABAA {alpha}1 subunit (S270) were mutated to isoleucine. This serine is a key residue for anesthetic modulation.7,13,14,16,22 We used {alpha}1β2{gamma}2s GABAA receptors in this study since this receptor is the most common neuronal subunit combination in the mammalian CNS.23 We studied {alpha}1 Gly receptors because these have been the most widely studied isoform of that receptor for anesthetic mechanisms.7 We found that the mutant receptors attenuated the modulatory effect of all of the compounds under study compared with WT receptors, suggesting a common modulatory mechanism with alcohols and volatile anesthetics.


   METHODS
Top
 Abstract
 Introduction
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All studies on animals were approved by the institutional animal care and use committee of the University of California at San Francisco.

Materials
WT and mutant {alpha}1(S270I)β2{gamma}2s GABAA receptors and WT and mutant {alpha}1(S267I) Gly receptor clones were a gift of Professor R.A. Harris (University of TX, Austin). Xenopus laevis female frogs were purchased from Nasco (Modesto, CA). Isoflurane was a gift of Baxter PPD. GABA (99%), Gly (>99%), ethanol (>99.8%), ammonium chloride (99.5%), 3-hydroxybutyric acid (95%), DEHP (99%), SDS (>99%), and propofol (97%) were purchased from Sigma-Aldrich (St. Louis, MO).

Oocyte Expression
Stage V and VI Xenopus laevis oocytes isolated from a surgically removed portion of ovary were defolliculated by gentle rotation in 500 U/mL collagenase type 1 (Worthingtom Biochemical Corporation, Lakewood, NJ) for 1 h at room temperature. GABAA receptors comprised of human {alpha}1 (WT or S270I) and {gamma}2s and rat β2 subunits ({alpha}1:β2:{gamma}2s in a molar ratio 1:1:4), and homomeric human {alpha}1 (WT or S267I) Gly receptors were expressed by microinjection into each oocyte nucleus of about 1 µg total cDNA. The cDNAs encoding GABAA receptor {alpha}1 WT and mutant subunits were cloned into pBK-CMV vector, and cDNAs encoding GABAA receptor β2 and {gamma}2s and Gly receptor {alpha}1 WT and mutant were cloned into pCIS2 vector. The injected oocytes were singly placed in a 96-well tissue plate (Fisher Scientific) containing modified Barth's solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 20 mM HEPES, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, with 5 mM sodium pyruvate, 50 µg/mL gentamycin, 50 U/mL penicillin, and 50 µg/mL streptomycin, filtered and adjusted to pH = 7.4) and incubated at 15°C. One to three days after injection, the oocytes were used for electrophysiological recordings.

Two-Electrode Voltage Clamp Recording
Two-electrode voltage clamping (GeneClamp 500B; Molecular Devices, Axon Instruments, Foster City, CA) was performed on oocytes at room temperature using frog Ringer's solution as perfusate (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2,10 mM HEPES, filtered and adjusted to pH 7.4) as previously described.24

In brief, oocytes were voltage clamped at –80mV using two glass electrodes filled with 3 M KCl (0.5–3 M{Omega} resistance). We studied compounds on GABAA or Gly receptors if there was significant modulation of WT channel function9–12; significant modulation was necessary to determine whether mutant receptors attenuated that effect. We used agonist concentrations, which we had used in previous studies. To study isoflurane (0.3 mM), ethanol (200 mM), Gly (10 mM), SDS (5 µM), and propofol (1 µM) modulation of GABA currents, the GABA concentration equivalent to 20% of the maximal effective concentration (EC20) was used. This concentration was determined by comparison to maximal currents produced by 1 mM GABA. To study isoflurane (0.3 mM), ethanol (200 mM), GABA (1 mM), 3-hydroxybutyic acid (7.5 mM), NH4Cl (5 mM), DEHP (1 µM), and propofol (25 µM) modulation of Gly currents, the Gly concentration equivalent to EC5 in WT and mutant {alpha}1 Gly receptors was determined by comparison to maximal currents produced by 1 mM Gly. Gly 1 mM was confirmed to produce maximal current by comparison to 10 mM Gly. Sucrose was used as an isosmolar control in the testing of Gly, GABA, and β-hydroxybutyic acid on receptors. NaCl was used as an isosmolar control for NH4Cl studies. Osmolarity was checked using a vapor pressure osmometer (VAPRO 5520, Wescor, Logan, UT). Uninjected oocytes and oocytes injected with Barth's solution also served as controls.

For each oocyte studied, stable inward currents in response to application of agonist(s) were verified by application of agonist(s) for 20 s followed by a 5 to 6 min washout, which was repeated three times.

Because absolute currents varied from oocyte to oocyte and from day to day, depending on the amount of receptor expressed, enhancement of currents was calculated as the percent change in current in oocytes during drug and agonist coadministration versus agonist alone. In determining the maximal absolute currents produced by WT and mutant channels (Fig. 1), this variation had to be minimized because currents were not normalized. In that study, currents in oocytes were all measured 1 day after cDNA injection.


Figure 123
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  		Figure 1. Maximal currents in wild-type and mutant receptors. (A) {gamma}-aminobutyric acid (GABA) maximal responses in {alpha}1β2{gamma}2s and {alpha}1(S270I)β2{gamma}2s GABAA receptors at 1 day after injection. (B) Glycine (Gly) maximal responses in {alpha}1 and {alpha}1(S267I) Gly receptors at 1 day after injection, using 1 or 10 mM Gly as the saturating agonist concentration. All data are expressed as mean ± se.

 

All data are presented as mean ± se, with 4 to 7 oocytes typically tested. Statistical significance was determined using Student's t-test.


   RESULTS
Top
 Abstract
 Introduction
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Comparison of Maximal Currents Between WT and Mutant Receptors
We wanted to know the maximal current in WT and mutant receptors in order to determine the EC5 and EC20 agonist concentration for these receptors. {alpha}1(S270I)β2{gamma}2s GABAA receptors had 62% of the maximum current of WT receptors (Fig. 1A). The maximal current was 51% and 53% in the mutant {alpha}1(S267I) Gly receptors compared with the WT receptors with 1 and 10 mM Gly, respectively (Fig. 1B). There was no significant difference in the current produced by 1 or 10 mM Gly.

The S267I Point Mutation in {alpha}1 Gly Receptors Abolished or Significantly Reduced the Enhancing Effects of Isoflurane, Ethanol, GABA, β-Hydroxybutyric Acid, NH4Cl, and DEHP but not the Negative Control, Propofol
Isoflurane (0.3 mM) and ethanol (200 mM) enhancements were significantly decreased in {alpha}1(S267I)-containing Glyreceptors compared to WT controls, whereas modulation by 25 µM propofol was not affected by the S267I mutation (Fig. 2). GABA, β-hydroxybutyric acid, NH4Cl, and DEHP positively modulated Gly receptor function (Fig. 2). GABA 1 mM and 5 mM NH4Cl enhanced the WT gly receptor function (with the application of EC5 Gly) by 47% ± 14% and 40% ± 11%, respectively, and the S267I mutation abolished this effect (Fig. 2). β-hydroxybutyric acid 7.5 mM and 1 µM DEHP enhanced the EC5 Gly response by 130% ± 23% and 192% ± 34% in WT receptors, respectively, and the point mutation significantly reduced the effects of these compounds (Fig. 2). NH4Cl induced an inward current (100 ± 12 nA, n = 17 oocytes) in the absence of agonist that was not a function of the Gly-gated chloride current. This current was indistinguishable from that observed in oocytes injected with only Barth's solution (80 ± 15 nA, n = 4 oocytes). Uninjected oocytes did not show this current. In calculating enhancements for NH4Cl, this current was subtracted from the total current.


Figure 223
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  		Figure 2. The mutant {alpha}1 S267I glycine (Gly) receptors expressed in Xenopus oocytes showed significantly reduced enhancement of the Gly responses by all test compounds compared to wild-type (WT) receptors. Isoflurane and ethanol were positive controls (i.e., affected by the mutation); propofol was a negative control (unaffected by the mutation). Summary bar graphs show the Gly-induced responses, at EC5 agonist concentrations, from multiple oocytes expressing WT or mutant {alpha}1 subunits in the presence of test compounds. Bars indicate means ± se (n = 4–7). *P < 0.05, **P < 0.01, compared with WT Gly receptor responses using Student's t-test. Iso = isoflurane, EtOH = ethanol, GABA = {gamma} aminobutyric acid, βHB = β hydroxybutyric acid, NH4Cl = ammonium chloride, DEHP = diethylhexylphthalate.

 

The S270I Point Mutation in {alpha}1 GABAA Receptors Abolished or Significantly Reduced the Enhancing Effects of Isoflurane, Ethanol, Gly, and SDS but not the Negative Control, Propofol
Responses to isoflurane and ethanol were significantly decreased in {alpha}1(S270I)-containing GABAA receptors compared to WT controls. Isoflurane (0.3 mM) enhancement of the EC20 GABA response in {alpha}1(S270I) β2{gamma}2s GABAARs was <25% of the enhancement observed in WT receptors (42% ± 5% enhancement), and 200 mM ethanol induced 9% ± 3% inhibition of the GABA responses in the mutants compared with 20% ± 7% enhancement in WT controls (Fig. 3). Gly 10 mM and 5 µM SDS enhanced GABAAR functions (20% ± 2% and 60% ± 12% enhancements, respectively) and this modulation was also affected by the S270I point mutation (Fig. 3). The S270I mutation almost abolished the enhancing effects of Gly and SDS. This TM2 serine point mutation did not affect enhancement of 1 µM propofol (77% ± 12% in WT and 75% ± 11% in the mutant) (Fig. 3).


Figure 323
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  		Figure 3. The mutant {alpha}1(S270I)β2{gamma}2s {gamma}-aminobutyric acide (GABA)A receptors expressed in Xenopus oocytes showed significantly reduced enhancement of the GABA responses by all test compounds compared to {alpha}1β2{gamma}2s GABAA receptors. Isoflurane and ethanol served as positive controls (i.e., affected by the mutation). Propofol was the negative control (unaffected by the mutation). Summary bar graphs show the GABA-induced responses, at agonist EC20 concentrations, from multiple oocytes expressing wild-type (WT) or mutant {alpha}1 subunits in the presence of test compounds. Bars indicate means ± se (n = 4–7). **P < 0.01 compared with WT GABAA receptor responses using Student's t-test. Iso = isoflurane, EtOH = ethanol, SDS = sodium dodecyl sulfate.

 

Current tracings are shown in Figures 4 and 5. Peak currents on application of agonist before and after study drug are reproducible for each oocyte. Peak currents vary between oocytes because of differences in expression of receptors from oocyte to oocyte.


Figure 423
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  		Figure 4. Traces show the responses of the wild-type (WT) or mutant receptors to a submaximal concentration of Gly (EC5) before, during, and after application of study drug. The enhancement of the Gly response by the test compounds was reversible. The duration of application of agonist or study drug is shown above the tracings; current and time scales are below and to the left. The first tracing of each group shows the response to agonist before application of the test compound. The second tracing shows the response to test compound and agonist. The third tracing shows the response to agonist after washout of the study drug. GABA = {gamma}-amino butyric acid, βHB = β hydroxybutyric acid, NH4Cl = ammonium chloride, DEHP = diethylhexyl phthalate.

 


Figure 523
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  		Figure 5. Traces show the responses of the wild-type (WT) or mutant {alpha}1(S270I)β2{gamma}2s {gamma}-aminobutyric acid (GABA)A receptors to submaximal concentration of GABA (EC20) before, during, and after application of study drug. The enhancement of the GABA response by the test compounds was reversible. The duration of application of agonist or study drug is shown above the tracings; current and time scales are below and to the left. The first tracing shows the response to agonist before application of the test compound. The second tracing shows the response to test compound and agonist. The third tracing shows the response to agonist after washout of the study drug. SDS = sodium dodecyl sulfate.

 

   DISCUSSION
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We studied a diverse set of compounds which we thought would have anesthetic-like modulatory effects on GABAA or Gly receptors. We determined their effect on GABAA receptors or Gly receptors harboring point mutations in the TM2 domain of the {alpha}1 subunit. Previous studies have shown that S270 in GABAA receptors and the homologous S267 in Gly receptors are important residues for alcohol and anesthetic modulation because these point mutations selectively abolished or significantly reduced the enhancing effects of alcohol and inhaled anesthetics.7,14,16,22,25 We found that these mutations abolished or significantly reduced the effect of our study compounds on Gly receptor function as well, but did not affect the negative control, propofol.13 We saw similar effects of study compounds on GABAA receptors, but again no effect on the negative (propofol) control. Propofol appears to modulate GABAA receptors via a different mechanism than alcohols and inhaled anesthetics, as shown by the different point mutations in the GABAA receptor which affect propofol's action.26 These findings support the hypothesis that the compounds investigated here modulate GABAA or Gly receptors by a mechanism similar to that of isoflurane and ethanol but different than propofol.

Two hypotheses have been formulated to explain the effect of this mutation1: that these residues line a binding pocket for alcohol and inhaled anesthetics,7,14,16,25 and that they are part of a transduction site required for anesthetic and alcohol actions.19 To the extent that our compounds have effects similar to alcohol and inhaled anesthetics on the WT and mutant receptors, our study places constraints on these two possibilities. Because we studied an uncharged compound (DEHP), a cation (ammonium), two anions (β hydroxybutyrate and SDS), and two zwitterions (GABA and Gly), a common binding site must be able to accommodate compounds with this range of charges. In addition, these compounds span more than an order of magnitude in molecular weight, from 18 for ammonium ion (NH4+) to 390 for DEHP (C24H38O4), with the latter molecule having a calculated volume of approximately 450 Å3. A binding site16 would have to accommodate compounds of these sizes.

SDS was predicted to modulate receptor function through its effects on interfacial properties. SDS might act on GABAA and Gly receptors at the protein/water interface, where it could adsorb to protein, or at the membrane/water interface where it might perturb bilayer properties which influence channel function. Our study does not allow us to distinguish these possibilities. Both possibilities would, however, suggest that, in this case, the mutations we studied affected transduction of a nonspecific effect of SDS on interfacial properties. This is also consistent with mutation of amino acid 270 of the GABAA receptor to other residues, which have been shown to affect signal transduction which causes mutation of the serine in position 270 of the GABAA receptor to tryptophan affects channel gating, but also reduces the effect of other positive modulators.27

The prediction that GABA would positively modulate Gly receptor function, and that Gly would modulate GABAA receptor function, was based on a theory of anesthesia that proposes that anesthetics modulate bilayer properties that are coupled to receptor function.17 This combination of neurotransmitters was chosen because at certain central synapses, GABA and Gly are packaged into the same vesicles and coreleased into the synaptic cleft.28 Our results confirm, over a broader range of neurotransmitter concentrations than previously studied and at a different GABAA receptor, our previous findings10 that these neurotransmitters can positively modulate each other's receptor. This effect is distinct from the agonist actions of these neurotransmitters.10 If this modulation occurs in vivo, then this anesthetic-like mechanism may be a means of modulating synaptic function.

Beta hydroxybutyric acid and ammonia modulate Gly receptor function in pathophysiological concentrations.9,12 Why would receptors respond to endogenous compounds in concentrations which have a deleterious effect? One might expect selection against such a response. That ion channels respond to these metabolites in an anesthetic-like manner may occur as a consequence of selection for some other beneficial effect, such as the hypothesized adaptation of receptors to indirect effects of neurotransmitters discussed above,17 in the same way that sickle cell trait is present in the population owing to selection for minimizing the deleterious impact of the malaria parasite, even though it leads to a harmful blood disorder. Alternately, the site at which these metabolites act (whether on the receptor itself or the bilayer) may have a beneficial effect that is selected for, independent of any endogenous compound. However, there is no function attached to the putative anesthetic binding site, or evidence of conserved membrane properties.

Because several of the compounds studied here are charged and of negligible vapor pressure (for example, millimolar concentrations of ammonium chloride in solution is undetectable by gas chromatography at the parts per million level (unpublished data)), our study suggests that volatility, per se, is not essential to anesthetic-like modulation of GABAA and Gly receptors. What then might be the role of volatility to inhaled anesthetic action? We suggest that because volatile anesthetics are uncharged, they cross the blood–brain barrier readily and enter the CNS in much the same manner as they pass from alveoli to blood, but that volatility itself has otherwise no mechanistic significance (Fig. 6).


Figure 623
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  		Figure 6. Venn diagram showing the hypothesized relation between compounds that have anesthetic effects on ion channels (circle on left), compounds that cross the blood–brain barrier (circle on right), those with anesthetic effects in animals (in the intersection of both circles), and those that are not volatile (B and D) and volatile (C and E). For example, A is a compound such as sodium dodecyl sulfate (SDS) that modulates receptor function like an anesthetic but does not cross the blood/brain barrier. B and C are anesthetics. C are volatile anesthetics, such as isoflurane and alcohols. B are nonvolatile compounds such as β hydroxybutyric acid, which acts as an anesthetic in animals and modulates receptors in a manner similar to volatile anesthetics.12 E are volatile compounds that cross the blood brain barrier but do not have anesthetic effects, such as the nonimmobilizers. D are nonvolatile compounds that cross the blood-brain barrier but are not anesthetics, such as glucose.

 

If this is correct, it has implications for studies of anesthetic mechanisms. Theoretical structure-activity studies (e.g., models of anesthetic interactions with GABAA and Gly receptors) aimed at determining properties of anesthetics should include both volatile and nonvolatile compounds that modulate channel function in an inhaled anesthetic-like manner. Experimental studies of a broad range of molecular structures, on expressed receptors of varying sensitivity to anesthetics, such as that reported here, might provide a method for identifying new anesthetics.


   Footnotes
 
Accepted for publication October 29, 2007.

Supported in part by NIGMS R01 GM069379.

Reprints will not be available from the author.


   REFERENCES
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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