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ANESTHETIC PHARMACOLOGY

Luciferase as a Model for the Site of Inhaled Anesthetic Action

Yi Zhang, MD^*, Caroline R. Stabernack, MD^*, Robert Dutton, MD^*, James Sonner, MD^*, James R. Trudell, PhD, S. John Mihic, PhD, Tomohiro Yamakura, PhD, R. Adron Harris, PhD, Diane Gong, BS^*, and Edmond I Eger, II, MD^*

*Department of Anesthesia and Perioperative Care, University of California, San Francisco; Department of Anesthesia and the Program for Molecular and Genetic Medicine, Stanford University, Stanford, California; and the Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas

Address correspondence and reprint requests to Dr. Eger, Department of Anesthesia, S-455, University of California, San Francisco, CA 94143-0464. Address e-mail to egere{at}anesthesia.ucsf.edu

	Abstract

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The in vivo potencies of anesthetics correlate with their capacity to suppress the reaction of luciferin with luciferase. In addition, luciferin has structural resemblances to etomidate. These observations raise the issues of whether luciferin, itself, might affect anesthetic requirement, and whether luciferase resembles the site of anesthetic action. Because the polar luciferin is unlikely to cross the blood-brain barrier (we found that the olive oil/water partition coefficient was 100 ± 36 x 10^-7), we studied these issues in rats by measuring the effect of infusion of luciferin in artificial cerebrospinal fluid into the lumbar subarachnoidal space and into the cerebral intraventricular space on the MAC (the minimum alveolar anesthetic concentration required to eliminate movement in response to a noxious stimulus in 50% of tested subjects) of isoflurane. MAC in rats given lumbar intrathecal doses of luciferin estimated to greatly exceed anesthetizing doses of etomidate, did not differ significantly from MAC in rats receiving only artificial cerebrospinal fluid into the lumbar intrathecal space. MAC slightly decreased when doses of luciferin estimated to greatly exceed anesthetizing doses of etomidate were infused intraventricularly (P < 0.05). In contrast to the absent or minimal effects of luciferin, intrathecal or intraventricular infusion of etomidate at similar or smaller doses significantly decreased isoflurane MAC. Luciferin did not affect +-aminobutyric acid type A or acetylcholine receptors expressed in Xenopus oocytes. These results suggest that luciferin has minimal or no anesthetic effects. It also suggests that luciferin/luciferase may not provide a good surrogate for the site at which anesthetics act, if this site is on the surface of neuronal cells.

IMPLICATIONS: In proportion to their potencies, anesthetics inhibit luciferin’s action on luciferase, and luciferin structurally resembles the anesthetic etomidate. However, in contrast to etomidate, luciferin given intrathecally or into the third cerebral ventricle does not have anesthetic actions, and it does not affect +-aminobutyric acid or acetylcholine receptors in vitro. Luciferase may not provide a good surrogate for the site at which anesthetics act.

	Introduction

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Concepts of mechanisms of anesthetic action began with the observation by Meyer and Overton (1,2) in 1900 that anesthetic potency correlates with lipophilicity. For approximately 80 yr, this observation focused studies of mechanisms of anesthetic action on the neural (lipid) membrane. In the 1980s, this focus shifted from lipids to proteins. Franks and Lieb (3) published one of the seminal studies that caused this shift in paradigms. The study demonstrated that anesthetic potency correlated with the capacity of anesthetics to depress the response of a protein, luciferase, to its agonist, luciferin. Specifically, Franks and Lieb showed that the in vivo potencies of anesthetics correlated with their capacity to suppress the reaction of luciferin with luciferase.

The finding by Franks and Lieb raises the question of how closely luciferase resembles the actual site of anesthetic action. That is, how close a surrogate to that site is luciferase? The question is emphasized by the resemblance of luciferin to etomidate (Fig. 1). They have similar shapes and molecular volumes. For example, the molecular volume of luciferase is 271 ų and that of etomidate is 286 ų. Both contain an aromatic ring as well as a pentameric ring that includes a nitrogen atom. However, etomidate is an ethyl ester, whereas luciferin is a carboxylic acid, a polar compound that likely will not cross the blood-brain barrier. In the present study, we explored how closely luciferase resembles the actual site of anesthetic action at two levels: 1) that of the whole animal, and 2) that of receptors. Complementing the whole animal studies, we also tested the effects of etomidate using similar models. Finally, we measured the oil/water partition coefficient of luciferin.

View larger version (33K): [in this window] [in a new window]   Figure 1. Molecular models of luciferin and etomidate illustrate their similarities in shape and molecular volume. The molecular volume of luciferase is 271 Å3 and that of etomidate is 286 Å3. Both contain an aromatic ring as well as a pentameric ring that includes a nitrogen atom. However, etomidate is an ethyl ester, whereas luciferin is a carboxylic acid.

	Materials and Methods

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Effect of Intrathecal Luciferin and Etomidate
In 12 rats anesthetized with isoflurane, a 32-gauge polyurethane catheter (Micor Inc., Allison Park, PA) was placed through the atlantooccipital membrane according to the method described by Yaksh and Rudy (4), and threaded caudally 6 to 8 cm to the lumbar sac. At the neck, sutures fixed the catheter to adjacent muscle and skin. Rats recovered from anesthesia and surgery for 24 h before study.

Each study consisted of two parts. First, we infused artificial cerebrospinal fluid (aCSF) made up daily from stock solutions. Stock solution #1 was made by adding NaCl 3.6963 g, NaHCO₃ 1.1551 g, KCl 0.0895 g, KH₂PO₄ 0.0340 g, and Na₂SO₄ 0.0355 g in deionized, distilled water to a volume of 500 mL. Stock solution #2 was made from CaCl₂ · 2H₂O 0.8086 g and MgCl₂ · 6H₂O 0.8437 g in deionized, distilled water to a volume of 10 mL. To make aCSF, 25 mL stock solution #1 was added to 0.0266 g glucose, adjusted to pH 7.4 with bubbles of CO₂ for 1–2 min, and added to 50 µL stock solution #2, giving a final composition of 154.7 mM Na⁺, 0.82 mM Mg²⁺, 2.9 mM K⁺, 132.49 mM Cl^-, 1.1 mM Ca²⁺, 5.9 mM glucose, at a pH of 7.4. In the second study, we infused aCSF to which we added the luciferin (Sigma Chemical Co., St. Louis, MO) at a concentration of 4 mg/mL.

Each rat was placed in a gas-tight clear plastic cylinder. A rectal temperature probe was inserted, and the temperature probe and the tail of the rat were separately drawn through holes in the rubber stopper used to seal one end of the cylinder. Ports through rubber stoppers in each end of the cylinder allowed delivery-exit of gases. Ports were added for sampling gases. A fresh gas inflow containing 1.1%–1.2% isoflurane in oxygen (produced using a conventional variable bypass vaporizer) at a total inflow rate of 8 L/min entered at the head end of the cylinders and exited at the tail end. The concentrations of the isoflurane were monitored with an infrared analyzer (RGM; Datex-Ohmeda, Madison, WI) and analyzed at the end of each concentration step with gas chromatography. The chromatograph reading was accepted as the value for the exposure concentration. The chromatograph was calibrated with secondary standards from tanks. Four to eight rats were studied concurrently.

Animals were equilibrated with the 1.1%–1.2% isoflurane partial pressure for 30 min during infusion of aCSF at 1 µL/min. After 30 min, a tail clamp was applied, and all rats moved. The isoflurane partial pressure was then increased by 0.2%–0.3% atmospheres. After equilibration for 30 min, the tail clamp was applied again and isoflurane partial pressure measured. This procedure was repeated until a partial pressure at which the animals did not move was achieved.

After obtaining this control value for MAC for each rat, the inspired isoflurane concentration was decreased to 0.8%, and the rats were reequilibrated at this concentration for 50 min. All rats moved in response to tail clamp at this concentration. In 6 of the 12 rats, the infusion of aCSF then was changed to an infusion of aCSF containing luciferin 4.0 mg/mL at an infusion rate of 4 µL/min. The remaining 6 rats continued to receive aCSF alone at an infusion rate of 1 µL/min. After 1–2 h, the tail clamp was applied, revealing movement in all rats (isoflurane concentration 0.8% to 1.1%). We then increased the isoflurane concentration in steps of 0.2% to 0.3%, holding the concentration constant for 30 min at each step, and testing for movement in response to the tail clamp at the end of each period of equilibration. This process continued until no rat moved in response to the tail clamp. MAC was calculated as the mean of the concentrations just permitting and just preventing movement in response to the tail clamp.

Each rat was allowed to awaken. It was determined that none had obvious neural injury (i.e., no impairment of mobility and movement in response to mild stimulation). The rats then were killed by immersion in 100% CO₂. The spinal cord was exposed, and placement of the tip of the catheter 5–10 mm from the end of the spinal cord demonstrated.

These studies were repeated, using commercial etomidate rather than luciferin as the test compound. In separate studies of 4 rats each, 2 mg/mL etomidate was infused at 1, 2, and 4 µL/min (i.e., doses of 2, 4, and 8 µg/min). Because etomidate contains 35% propylene glycol, we also tested the effect of intrathecal infusion of this vehicle at 1, 2, and 8 µL/min.

Effect of Luciferin and Etomidate Injected into a Cerebral Ventricle
Eight rats were anesthetized with xylazine and ketamine, intraperitoneally. The skull was exposed, and a hole drilled 0.5-mm posterior to the bregma and 1.5-mm lateral from the midline. A 25-g stainless steel guide cannula was placed through the hole to a depth of 4.2 mm from the surface of the skull. The cannula was secured in place with dental acrylic, plugged, and the wound sutured. Rats were allowed to recover for a minimum of 24 h.

On the day of study, each rat was prepared as for the previous study of MAC. MAC was determined first without infusion through the cannula. After completion of the determination, the isoflurane concentration was decreased to 1.3% to 1.4% (the largest concentration at which the rats had previously moved) where it was sustained for 30 min. All rats moved in response to tail clamp at this concentration. A 32-g stainless steel cannula was placed through and to the end of the guide cannula. PE10 tubing filled either with aCSF or aCSF containing 20 mg/mL luciferin was attached to the 32-g cannula and an infusion begun at 1 µL/min. The infusion continued for 2 h without changing the isoflurane concentration. All rats moved in response to tail clamp at the end of 2 h. The isoflurane concentration then was increased in 0.2% steps, each step was held for 30 min, and the tail clamp applied. This process continued until no rat moved in response to the tail clamp. Each rat was allowed to awaken after study to document that no obvious neural injury had occurred.

Each rat then was killed, and the catheter injected with 0.15 mL of India ink. The skull and cannula were removed and the brain was exposed. Sagittal sections through the brain were made to confirm that the India ink had lodged in the ventricles, including the third ventricle.

These studies were repeated, using etomidate rather than luciferin as the test compound. In separate studies of 4 to 7 rats, 2 mg/mL etomidate was infused at 1, 2, and 4 µL/min (i.e., doses of 2, 4, and 8 µg/min). Because etomidate contains 35% propylene glycol, we also tested the effect of intraventricular infusion of this vehicle at 1, 2, and 4 µL/min.

Effect of IV Etomidate
To assess the possibility that the effects seen with intrathecal and intraventricular etomidate did not result primarily from absorption of the etomidate and subsequent delivery to the brain, we also tested the effect of IV administered etomidate (with 35% propylene glycol) at 16, 32, and 64 µg/min.

Effect of Luciferin on Receptors
We have extensively characterized the effects of anesthetics and other modulators on +-aminobutyric acid type A (GABA_A) and nicotinic receptors by using the oocyte expression system (5,6), and these may be consulted for detailed descriptions of the methods. The following description summarizes those methods: Adult female Xenopus laevis were obtained from Xenopus I (Ann Arbor, MI) or Xenopus Express (Homosassa, FL). Acetylcholine was obtained from Bio-Rad Laboratories (Hercules, CA), and GABA from Sigma. Rat nicotinic acetylcholine (nACh) receptor subunit cDNAs were subcloned into several vectors: 2 and ß2 in pSP65, 3 and 4 in pSP64, and ß4 in pBluescript SK-. Human GABA_A receptor 1, ß2, and 2L subunits were in the pCDM8 vector. Oocytes were isolated and cDNA and cRNA microinjections performed as described elsewhere (7). Before injection, isolated oocytes were placed in modified Barth’s saline (MBS) containing (in mM) NaCl 88, KCl 1, HEPES 10, MgSO₄ 0.82, NaHCO₃ 2.4, CaCl₂ 0.91, and Ca(NO₃)₂ 0.33 adjusted to pH 7.5. GABA_A receptor subunit cDNAs (1.5 ng/30 nL) were injected into the nuclei of oocytes by the "blind" method of Colman (8). Acetylcholine receptor and ß cRNAs were combined in a 1:1 molar ratio before being injected (10–50 ng/40 nL) into the cytoplasm of oocytes. The injected oocytes were cultured at 15°–19°C in sterile MBS supplemented with 10 mg/L streptomycin, 10,000 U/L penicillin, 50 mg/L gentamycin, 90 mg/L theophylline, and 220 mg/L pyruvate.

GABA_A and acetylcholine receptor function was assayed electrophysiologically 1 to 4 days after cDNA or cRNA injection. Each oocyte was placed in a rectangular chamber (approximately 0.1 mL volume) and perfused at a rate of 2 mL/min via a pump (Cole-Palmer Instrument Co., Chicago, IL) using 18-gauge polyethylene tubing (Clay Adams Co., Parsippany, NJ) that delivered drug solutions to the recording chamber. Oocytes expressing GABA_A receptors were perfused with MBS whereas oocytes expressing nACh receptors were perfused with Ba²⁺-Ringer’s solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM BaCl₂, and 10 mM HEPES; pH 7.4) containing 1 µM atropine sulfate. The animal poles of oocytes were impaled with two glass electrodes (0.5–1.0 M) filled with 3 M KCl and voltage clamped at -70 mV using a Warner Oocyte Clamp OC-725C (Warner Instruments, Hamden, CT). In all cases, peak current was used as a response measure and sufficient time was allowed between drug applications to avoid desensitization or run-down. GABA or acetylcholine was applied before and after each drug application to assure that the baseline was stable. Luciferin (0 [control], 10 to 100 µM) was preapplied for 30 s before being coapplied with acetylcholine for 20 s. For the nAChR studies, an unpaired or paired t-test was used to determine whether a given point differed from control, accepting P < 0.05 as significant. In experiments on 1ß22L GABA_A receptors, GABA was coapplied with 10 to 200 µM luciferin for 30 s. Expression of all three types of subunits in a single receptor complex was verified by testing the action of flunitrazepam. A concentration of GABA producing 10% of a maximal effect (5–15 µM) was used in these studies, and a one-way analysis of variance was used in analyzing the effects of luciferin on GABA_A receptor function.

Solubility Studies
We measured the oil/water partition coefficient of luciferin. Preliminary studies indicated that the partition coefficient was small, dictating an approach that enhanced sensitivity. Eight milligrams of luciferin was dissolved in 3 mL of distilled water. This was equilibrated at 37°C with 250 mL of olive oil that previously had been washed with an equal volume of water (water discarded). Similarly, 3 mL of distilled water was equilibrated with 250 mL of washed olive oil at 37°C. The oil and aqueous phases were separated, and the oil was centrifuged at 10,000 rpm for 15 min. Forty-seven-milliliter aliquots of the centrifuged oil from each of the 250 mL of olive oil were placed in 50 mL syringes to which we added 3 mL of distilled water. The mixture was equilibrated in a rotator at 37°C for 30–60 min. The aqueous phase was removed and placed in cuvettes for analysis with a Bio-Rad spectrophotometer at 350 nM wavelength. Four such measurements were made for the oil equilibrated with luciferin (A values) and 4 for the oil equilibrated with distilled water (B values). Four cuvettes containing distilled water were used as (zero) blanks. The aqueous solution of luciferin was retrieved and centrifuged as above. The solution was diluted by half repeatedly, and each dilution was read with the spectrophotometer. The initial (larger) concentrations gave an alinear correlation with absorbance, but further dilutions provided a linear correlation that continued to the readings for values A and B. To determine the partition coefficient, the B values were averaged and the average value was subtracted from each of the A values to give C values. The C values were converted to C', the fraction of the original luciferin concentration, using the calibration curve obtained from the dilutions of the luciferin as described above. We assigned a value of 1.0 for the original value for the luciferin. The oil/water partition coefficient was calculated as 3C'/47.

Molecular models of luciferin and etomidate were built with the Insight II software suite and were optimized by using Discover 98 with the CFF91 potential energy functions (MSI, San Diego, CA). Molecular volumes were calculated with Spartan 5.0 (Wavefunction Inc., San Diego, CA). Because luciferin has two fused rings, it is a more rigid molecule than etomidate. Therefore, luciferin was used as the template molecule and the single bonds in etomidate were manually rotated to maximize the overlap between the two molecules. All bond lengths and angles were constrained to their optimum values.

For the determinations of the effect of an infusion of luciferin on MAC, the average values were calculated for the control period and for the period during the fasted rate of luciferin infusion. We also compared the difference in MAC for a given infusion of etomidate with 35% propylene glycol (the vehicle) versus 35% propylene glycol. All differences were compared with a two-tailed t-test. We accepted P < 0.05 as indicative of a significant difference.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The structures for luciferin and etomidate (Fig. 1) have similar shapes and molecular volumes. Both structures contain an aromatic ring as well as a pentameric ring that includes a nitrogen atom. Luciferin is more polar because it is a carboxylic acid whereas etomidate is an ethyl ester.

MAC in rats receiving 4 µL/min of 4.0 mg/mL (16 µg/min) of luciferin in aCSF into the lumbar subarachnoid space did not differ from MAC in rats receiving only aCSF at the same inflow rate (Fig. 2). MAC in rats receiving 1 µL/min of 20 mg/mL (20 µg/min) of luciferin in aCSF in the cerebral ventricles had MAC values that did not differ from those of control rats (Fig. 2), but for those that were given luciferin, there was a slight decrease, whereas in those only given aCSF, there was a slight increase. A comparison of the changes in MAC was significant (P < 0.05). Autopsy examination showed that the infusions were confined to the lumbar and lower thoracic portions of the intrathecal space during intrathecal infusion, and to the third and fourth ventricle and medullary area during intraventricular infusion.

View larger version (25K): [in this window] [in a new window]   Figure 2. In six rats into which artificial cerebrospinal fluid (aCSF) was infused into the lumbar intrathecal space or four into which aCSF was infused into the cerebral ventricles, minimum alveolar anesthetic concentration (MAC) did not change with repeated measurement. Similarly, MAC did not change when 4 µL/min aCSF with 4.0 µg/µL luciferin was substituted for 1 µL/min aCSF alone, but MAC decreased slightly when 20 µg/µL was infused into the ventricles. Values are given as the mean, SD.

View larger version (26K): [in this window] [in a new window]   Figure 3. Intrathecal and intracerebroventricular infusion of 2 µg/µL of etomidate in 35% propylene glycol decreased isoflurane minimum alveolar anesthetic concentration (MAC) by 35% and 42%, respectively, at the fastest infusion rate (8 µg/min). Although intrathecal and intracerebroventricular infusion of 35% propylene glycol also decreased isoflurane MAC, the decrease was far less than that found during the infusion of etomidate (a maximum of 12% to 16%.) Etomidate (in propylene glycol) infused IV was approximately a quarter as potent as either intrathecal or intraventricular infusions.

View larger version (48K): [in this window] [in a new window]   Figure 4. Luciferin (10 or 100 µM) applied to rat neuronal nicotinic acetylcholine (nACh) receptors caused no change in nACh receptor response to acetylcholine. Luciferin was preapplied for 30 s before being coapplied with acetylcholine for 20 s. Acetylcholine concentrations were 60%–80% of a maximal effect for each receptor: 10 µM for 2ß2, 4ß2, and 4ß4 receptors, and 30 µM for 3ß2 receptors. Values for each bar are expressed as mean ± SD of measurements on four oocytes.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our results indicate that intrathecal or intraventricular luciferin does not affect, or minimally affects, isoflurane MAC (Fig. 2). In contrast, similar doses of etomidate given intrathecally or intraventricularly decrease isoflurane MAC (Fig. 3). The effects of etomidate are not explained by the concurrent administration of the vehicle for etomidate, 35% propylene glycol (Fig. 3). The far smaller potency of IV etomidate indicates that the effects of intrathecal and intraventricular etomidate are not explained by local absorption of etomidate and/or propylene glycol and their redistribution to the central nervous system. Consistent with an absence of effect of luciferin in vivo, we found no in vitroeffect of luciferin on GABA_A or acetylcholine receptors (Fig. 4).

As noted in the Introduction, luciferin bears some structural resemblance to etomidate (Fig. 1), and if it acted like etomidate, we would have expected a decrease in MAC. Conversely, anesthetics decrease the capacity of luciferin to activate luciferase. If luciferin similarly stimulated the anesthetic site of action in vivo, we would have expected an increase in MAC. The results for intrathecal and cerebral ventricular infusions do not show either a material increase or decrease and thus support neither expectation.

We chose to infuse luciferin intraventricularly and in the lumbar intrathecal space rather than IV. An IV injection would not have been anticipated to have an effect on the brain because luciferin is ionized (consistent with the very low oil/water partition coefficient) and thus would not be expected to cross the blood-brain barrier. However, this also means that the intrathecal infusions would have resulted in large local concentrations of luciferin because the drug would be held within the brain and spinal cord by the same barrier. That is, luciferin injected intrathecally or intraventricularly would not readily leave the spinal cord or brain because of the blood-brain barrier. This differs from etomidate which should readily depart the spinal cord and brain during infusion of the fluid surrounding these structures.

Some further observations support the notion that we gave a dose of luciferin that should have had an effect if an effect was to be had. A bolus injection of 3.5 mg/kg of etomidate produces burst suppression in the rat (9). If we assume that the luciferin infused into the intrathecal space is confined to that space and the cord by the blood-brain barrier, and if we assume a weight of the spinal cord of 0.5 g (measured separately by ourselves), then the total dose of luciferin would be approximately 2100 mg/kg. Similarly, the dose for a 2-g rat brain (measured separately by ourselves) would be approximately 1200 mg/kg. Thus, in the context of luciferin as similar to etomidate, the doses injected would seem to be adequate.

The ionization of luciferin also would limit it to the outside of cells unless some active transport mechanism carried it into the cells. It would not have access to the internal, nonpolar aspects of proteins. This limitation also may explain the absence of material anesthetic effects of luciferin.

Another explanation for the absence of an anesthetic effect of luciferin might be that the effect of anesthetics on luciferase results from an interaction with luciferase at a site different from that at which luciferin acts. However, the information available suggests that anesthetics act competitively with luciferin. That is, they bind at the same site as the luciferin substrate (3,10,11). As noted in the preceding paragraph, we are not able to exclude the possibility that the site of action that luciferase represents is within a membrane and is not accessible to polar molecules like luciferin.

Studies in GABA_A and acetylcholine receptors did not show an effect of luciferin (Fig. 4). These results are consistent with our in vivo data in that potential molecular targets of anesthetic action would not be expected to be affected by compounds that could not induce anesthesia.

We conclude that luciferin has minimal or no anesthetic effects, either in vivo or at GABA_A or acetylcholine receptors. These results suggest that luciferase may not provide a good model of the site at which anesthetics act if that site is on the surface of neuronal cells.

	Acknowledgments

 
This work was supported by National Institutes of Health Grant 1P01GM47818-05.

The authors thank Drs. P. J. Whiting (Merck Sharpe & Dohme) and C. W. Luetje (University of Miami School of Medicine) for providing the GABA_A and nACh receptor subunit cDNAs, respectively.

	Footnotes

 
EIG is a paid consultant to Baxter, PPI, who donated the isoflurane used in these studies.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (4) Citing Articles via Google Scholar Google Scholar Articles by Zhang, Y. Articles by Eger, E. I Search for Related Content PubMed PubMed Citation Articles by Zhang, Y. Articles by Eger, E. I, II Related Collections Mechanisms Pharmacology