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Am J Physiol Endocrinol Metab 290: E1267-E1275, 2006. First published January 24, 2006; doi:10.1152/ajpendo.00463.2005
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Inhibition of mammalian translation initiation by volatile anesthetics

Laura K. Palmer,1 Sharon L. Rannels,1 Scot R. Kimball,1 Leonard S. Jefferson,1 and Ralph L. Keil2

Departments of 1Cellular and Molecular Physiology and 2Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania

Submitted 22 September 2005 ; accepted in final form 18 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Volatile anesthetics are essential for modern medical practice, but sites and mechanisms of action for any of their numerous cellular effects remain largely unknown. Previous studies with yeast showed that volatile anesthetics induce nutrient-dependent inhibition of growth through mechanisms involving inhibition of mRNA translation. Studies herein show that the volatile anesthetic halothane inhibits protein synthesis in perfused rat liver at doses ranging from 2 to 6%. A marked disaggregation of polysomes occurs, indicating that inhibition of translation initiation plays a key role. Dose- and time-dependent alterations that decrease the function of a variety of translation initiation processes are observed. At 6% halothane, a rapid and persistent increase in phosphorylation of the {alpha}-subunit of eukaryotic translation initiation factor (eIF)2 occurs. This is accompanied by inhibition of activity of the guanine nucleotide exchange factor eIF2B that is responsible for GDP-GTP exchange on eIF2. At lower doses, neither eIF2{alpha} phosphorylation nor eIF2B activity is altered. After extended exposure to 6% halothane, alterations in two separate responses regulated by the target of rapamycin pathway occur: 1) redistribution of eIF4E from its translation-stimulatory association with eIF4G to its translation-inactive complex with eIF4E-binding protein-1; and 2) decreased phosphorylation of ribosomal protein S6 (rpS6) with a corresponding decrease in active forms of a kinase that phosphorylates rpS6 (p70S6K1). Changes in the association of eIF4E and eIF4G are observed only after extended exposure to low anesthetic doses. Thus dose- and time-dependent alterations in multiple processes permit liver cells to adapt translation to variable degrees and duration of stress imposed by anesthetic exposure.

halothane; eukaryotic translation initiation factor 2{alpha} phosphorylation; mammalian target of rapamycin pathway

ALTHOUGH VOLATILE ANESTHETICS revolutionized medical practice when introduced in 1846, the mechanisms of action responsible for the physiological effects of these drugs remain essentially unknown. In addition to affecting cells of the central nervous system, these drugs affect all cells, tissues, and organisms examined (4, 26, 36). Molecular genetic studies with the small, relatively simple eukaryote Saccharomyces cerevisiae (yeast) provide an opportunity for gaining insight regarding physiologically relevant effects of these drugs (28) and generating hypotheses that are testable in more complex eukaryotes. Extensive similarities exist between the activity of these drugs in mammals and simpler eukaryotes, such as yeast, suggesting conservation of cellular mechanisms responsible for the responses (28, 34, 45). These similarities include rapid and reversible induction of responses, a sharp dose-response curve, correlation between the lipophilicity of various anesthetics and their potency for inducing responses (termed the Meyer-Overton relationship), additivity of doses of different anesthetics in producing effects, and lack of effect in yeast of volatile lipophilic compounds that do not induce anesthesia in mammals (termed nonimmobilizers).

Results from yeast show that the volatile anesthetic isoflurane affects availability of certain critical amino acids, such as leucine and tryptophan (38), and is accompanied by inhibition of both cell division and translation initiation in strains auxotrophic for these nutrients (28, 37). Results from yeast suggest that a variety of volatile anesthetics, including halothane, enflurane, methoxyflurane, and sevoflurane, act by the same or very similar mechanisms of action being elucidated for isoflurane. Results supporting this statement include finding that all these drugs rapidly and reversibly arrest yeast growth (28) and that all mutants tested respond similarly to all five anesthetics (28, 46).

Volatile anesthetics, including halothane, isoflurane, enflurane, sevoflurane, and desflurane, have also been shown to inhibit protein synthesis in larger eukaryotes. This has been reported in a number of different systems, including intact animals (23–25), perfused organs (10, 40, 41), tissue slices in culture (15–18), and cells in culture (3, 6, 8, 21, 35). Studies regarding effects of volatile anesthetics on protein synthesis in perfused rat liver, the system employed in the work reported here, demonstrate that halothane exposure produces a rapid and dose-dependent inhibition of translation initiation affecting synthesis of both secreted and retained proteins (10, 12). Cellular mechanisms responsible for inhibition of translation induced by volatile anesthetics in any of the larger eukaryotic systems remain unknown. Halothane, the anesthetic whose activity in inhibiting protein synthesis in perfused liver has been best characterized (10, 12), is a halogenated hydrocarbon. Clinically, concentrations of halothane up to 5% are used in children for induction of anesthesia (7, 19) or for endotracheal intubation without muscle relaxants (20), whereas concentrations up to 2% can be used to maintain anesthesia (9).

In the present study, halothane was found to induce posttranslational modification of translation initiation factors regulated by multiple signaling pathways. The alterations identified are consistent with the observed decrease in translation initiation. The various modifications occur in a time- and dose-dependent pattern, indicating that liver cells adapt their translational response to the degree and duration of stress imposed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male Sprague-Dawley rats (Charles River Breeding Laboratories) weighing ~125–145 g were maintained on a 12:12-h light-dark cycle and were provided food (Harlan-Teklad Rodent Chow) and water ad libitum. The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee of The Pennsylvania State University College of Medicine.

Liver perfusions. Animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 µg/g body wt; Abbott Laboratories) and were prepared for in situ liver perfusion as previously described (10), with the following modifications: halothane without thymol as a preservative (Halocarbon Laboratories) was vaporized in a mixture of 95% O2 and 5% CO2 with the use of a Fluotec 3 vaporizer and delivered to the perfusion apparatus at a flow rate of 5.5 l/min; the perfusate was preequilibrated with the halothane gas mixture for ~45 min before the start of the perfusion. In addition, all 20 amino acids were added to the medium at 2.5 times the normal plasma levels in rats (44). This level of amino acids is required to partially activate positive translation regulatory factors (e.g., p70S6K1), thus permitting examination of the effect of the translation inhibitory activity of halothane on these factors.

Measurement of liver protein synthesis. After an initial 45-min perfusion with nonradioactive medium, perfusate containing L-[3H]valine (1.25 x 10–4 µCi/ml; Amersham Biosciences) was delivered for 15 min. A portion of liver was then removed, immediately frozen between aluminum blocks precooled in liquid nitrogen, and stored at –80°C. Incorporation of L-[3H]valine into perchloric acid-precipitable protein was determined as described previously (32, 43).

Polysome profiles. Polysomes were isolated and analyzed by sucrose density gradient (SDG) centrifugation by slight modification of a previously described procedure (11). Briefly, a freshly excised liver sample was homogenized with a Dounce homogenizer in 3 vol of SDG buffer (20 mM HEPES, pH 7.5, 100 mM KCl, 5 mM MgCl2) per gram of liver. The homogenate was centrifuged at 3,000 g for 10 min at 4°C. One volume of detergent mix (10% Triton X-100 and 10% sodium deoxycholate) was added to 9 vol of supernatant. A 500-µl aliquot of this mixture was layered onto a 20–48% SDG formed by alternately adding and freezing (in liquid nitrogen) 1.45-ml aliquots of 20, 24, 28, 32, 36, 40, 44, and 48% sucrose in SDG buffer. Before use, gradients were thawed overnight at 4°C. Gradients were centrifuged at 40,000 rpm for 110 min at 4°C using a Beckman SW41 rotor. After centrifugation, gradients were fractionated, and the A254 was continuously recorded using an Isco gradient fractionator (Instrumentation Specialities).

Western blotting. Sample preparation for immunoblot analysis was performed as described previously (33). Briefly, freshly excised liver samples were homogenized in 7 vol of ice-cold homogenization buffer [20 mM HEPES, pH 7.4, 2 mM EGTA, 50 mM NaF, 100 mM KCl, 0.2 mM EDTA, 50 mM beta-glycerophosphate, 1 mM DTT, 1 mM benzamidine, 0.5 mM sodium vanadate, 1x protease inhibitor cocktail (Sigma)] per gram of liver using a Polytron homogenizer. Homogenates were centrifuged at 1,000 g for 3 min at 4°C. An aliquot of supernatant was diluted with an equal volume of SDS sample buffer and boiled for 5 min. Equal amounts of total protein were loaded in the various lanes on a gel. Samples were subjected to SDS-PAGE, the separated proteins transferred to polyvinylidene difluoride (PVDF) membranes (Pall), and the membranes blocked in 5% (wt/vol) nonfat dry milk in Tris-buffered saline plus Tween 20 (TBST; 25 mM Tris, pH 7.6, 154 mM NaCl, 0.01% Tween 20). Phosphorylation of eukaryotic initiation factor (eIF)2{alpha} on Ser51, p70S6K1, or ribosomal protein (rp)S6 on Ser235/236 and Ser240/244 was evaluated as described previously (2, 14, 33), using antibodies purchased from BioSource International, Bethyl Laboratories, or Cell Signaling, respectively. Blots were washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (Bethyl Laboratories) at room temperature for 1 h. Blots were washed again with TBST and developed using enhanced chemiluminescence reagents (GE Healthcare) according to the manufacturer's instructions. Blots were quantitated using a GeneGnome chemiluminescence documentation and analysis system with GeneTools software (Syngene).

Measurement of eIF2B activity. Guanine nucleotide exchange activity of eIF2B in liver homogenates was measured as described previously (2, 30) from homogenate of a sample of freshly excised liver. Briefly, homogenates were centrifuged at 10,000 g for 10 min at 4°C and the supernatants immediately assayed for guanine nucleotide exchange factor activity by the addition of preformed eIF2·[3H]GDP complex and assay buffer (62.5 mM MOPS, pH 7.4, 125 mM KCl, 1.25 mM DTT, 2.5 mM magnesium acetate, 250 µg/ml bovine serum albumin). After 0, 1, 2, and 3 min, aliquots were removed, chilled by the addition of ice-cold wash buffer (assay buffer lacking bovine serum albumin), and immediately filtered through a nitrocellulose disc. Discs were dissolved in 7 ml of Filtron X (National Diagnostics), and the amount of eIF2·[3H]GDP complex bound to the filters was determined by scintillation counting.

Quantitation of eIF4E-binding protein-1·eIF4E and eIF4G·eIF4E complexes. The amount of eIF4E bound to either eIF4E-binding protein (4E-BP)1 or eIF4G was determined by a microtiter plate-based assay, as described previously (29). Liver samples were prepared for the assay by homogenizing freshly excised liver in 7 vol of ice-cold homogenization buffer (20 mM HEPES, pH 7.4, 2 mM EGTA, 50 mM NaF, 100 mM KCl, 0.2 mM EDTA, 50 mM beta-glycerophosphate, 1 mM DTT, 1 mM benzamidine, 0.5 mM sodium vanadate, 1x protease inhibitor cocktail) per gram of liver with the use of a Polytron homogenizer. Homogenates were centrifuged at 1,000 g for 3 min at 4°C before conducting the assays.

Statistical analysis. Values are presented as means ± SE. The number of rat livers per group (n) is indicated in the figure legends. When appropriate, data were analyzed by a one-way analysis of variance followed by a Tukey-Kramer multiple comparisons test or Student's t-test using Graphpad Instat 3.0 software with statistical significance set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Initially, we established conditions that produced the previously reported inhibition of protein synthesis by halothane in perfused liver (10). On the basis of incorporation of radiolabeled valine, we found significant anesthetic-induced inhibition that was dose dependent, ranging from a reduction to 77% of control at 2% halothane up to a reduction to 36% of control at 6% halothane after 1 h of anesthetic exposure (Fig. 1A).


Figure 1
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  		Fig. 1. Halothane (Hal) rapidly inhibits translation in perfused liver. A: after perfusion of rat livers with medium exposed or not exposed to varying concentrations of Hal for 45 min, L-[3H]valine was added to the perfusate, and perfusion was continued for an additional 15 min. Liver samples were harvested and immediately frozen. Incorporation of L-[3H]valine into acid-precipitable complexes was determined, and the rate of protein synthesis was calculated (32, 43). At least 6 livers were used for each condition. Percentages indicate rate of synthesis at the different levels of Hal exposure vs. unexposed (0% Hal) controls. White bar, results from livers that were not exposed to Hal; light gray, medium gray, and black bars, results from perfusions conducted with 2, 4, or 6% Hal, respectively. B: perfusions were conducted for a total of 15, 30, or 60 min with perfusate exposed or not exposed to 6% Hal. During the last 15 min of each perfusion, L-[3H]valine was added, and rates of protein synthesis were determined as described above. At least 8 livers that were exposed to Hal and at least 5 livers that were not exposed to Hal were used at each time point. White bars, results obtained from control livers not exposed to Hal for each of the indicated lengths of time; light gray, medium gray, and black bars, results from livers exposed to 6% Hal for 15, 30, or 60 min, respectively. **P < 0.01 vs. unexposed controls.

 
A hallmark of anesthetic action is the rapidity of induced responses. Examination of the time course for inhibition of protein synthesis during exposure to 6% halothane showed that protein synthesis was significantly decreased to 73% of the control after only 15 min of exposure (Fig. 1B). During longer periods of exposure (30 or 60 min), the rate of synthesis was further reduced to 52 and 38% of control rates, respectively. Thus inhibition occurred rapidly, and the amount of inhibition increased as time of exposure was extended.

Alteration of translation initiation plays a major role in regulation of protein synthesis in response to numerous cellular stresses (27). To examine whether halothane exposure affected initiation, the distribution of ribosomal subunits within polysome profiles was examined. There was a dramatic redistribution from polysomes to monosomes and free ribosomal subunits (i.e., ribosomal subunits not associated with mRNA) after 60 min of exposure to 2, 4, or 6% halothane (Fig. 2, A and B, bottom). During exposure to 6% halothane, the shift was rapid, as it was observed after only a 15-min exposure (Fig. 2B). The extent of inhibition increased substantially with longer periods of exposure. These findings are consistent with the results obtained when incorporation of radiolabeled valine was examined.


Figure 2
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  		Fig. 2. Hal rapidly inhibits initiation of protein synthesis in perfused liver. A and B: perfusions with medium exposed to 0, 2, 4, or 6% Hal were conducted as described in the legend to Fig. 1 and MATERIALS AND METHODS. Samples of liver were homogenized, and aliquots of the 3,000-g supernatants were mixed with detergent and separated by ultracentrifugation on 20–48% sucrose density gradients. Location of ribosomal material in the gradient fractions was monitored at A254. Locations of subpolysomal and polysomal regions are indicated. B: perfusion with 6% Hal rapidly inhibits translation initiation. Perfusions conducted as described above were performed for 15, 30, or 60 min with perfusate exposed to 6% Hal. Typical results from a total of at least 5 gradients per condition are shown.

 
One mechanism that regulates translation initiation in response to environmental stimuli is phosphorylation of the {alpha}-subunit of eIF2. Phosphorylated eIF2 bound to GDP forms an inactive complex with its guanine nucleotide exchange factor, eIF2B, inhibiting regeneration of the eIF2·GTP complex required for translation initiation (39). To assess effects of halothane exposure on this translation initiation process, we examined alterations in both phosphorylation of eIF2{alpha} and activity of eIF2B. At 6% halothane, phosphorylation of eIF2{alpha} increased significantly after only a 15-min exposure. After 60 min of exposure, the level of eIF2{alpha}(P) increased to more than twofold above control levels (Fig. 3, A and B). Total eIF2{alpha} levels were not altered during halothane exposure (Fig. 3B). In contrast to the rapid increase in phosphorylation of eIF2{alpha} observed when perfusate was exposed to 6% halothane, there was no significant increase in eIF2{alpha} phosphorylation in rat livers after 60 min of exposure to 2 or 4% halothane (Fig. 3A).


Figure 3
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  		Fig. 3. Exposure to high levels of Hal induces hyperphosphorylation of eIF2{alpha} and inhibition of eIF2B activity. Perfusions with the indicated concentrations of Hal were conducted for the designated exposure times as described in the legend to Fig. 1 and MATERIALS AND METHODS. Fresh samples of liver were homogenized. A and B: aliquots of 1,000-g supernatants were subjected to SDS-PAGE. After transfer to polyvinylidene difluoride membrane, the separated proteins were probed with an antibody that specifically recognizes eIF2{alpha} phosphorylated on Ser51 [designated eIF2{alpha}(P); B, top] or an antibody against total eIF2{alpha} (B, bottom). Relative amounts of phosphorylated and total eIF2{alpha} were determined. Representative blots from livers exposed or not exposed to 6% Hal for 60 min are shown (B). C: aliquots of 10,000-g supernatants were used to determine eIF2B guanine exchange factor activity. At least 8 livers were used for each Hal exposure, and at least 5 were used for each unexposed control. Bars are as described in the legend of Fig. 1B. *P < 0.05; **P < 0.01 vs. unexposed controls.

 
Consistent with the increased phosphorylation of eIF2{alpha} observed during exposure to 6% halothane, eIF2B activity rapidly decreased to 70–80% of control values in response to this treatment (Fig. 3C). Thus inhibition of formation of the translationally active eIF2·GTP complex likely plays a role in the reduced translation precipitated by 6% halothane. In contrast, eIF2B activity was not altered by exposure to either 2 or 4% halothane for 60 min (Fig. 3C). Although this lack of effect of lower halothane concentrations on eIF2B activity was different from the inhibition observed during exposure to 6% halothane, it is consistent with the unaltered levels of eIF2{alpha}(P) observed during exposure to these lower drug levels (Fig. 3A).

A separate mechanism regulating translation initiation is assembly of the active eIF4F complex (containing eIF4E, eIF4G, and eIF4A) required for several processes, including recognition of the 5' cap structure of mRNA (22, 42). The eIF4E component of this complex is required for recognition of the mRNA cap but is sequestered in an inactive form when bound to 4E-BP(s). This leads to decreased amounts of active eIF4F complex. In contrast to the alterations of eIF2{alpha} phosphorylation and eIF2B activity, which occurred rapidly, alterations in eIF4E association arose only after extended exposure. After 15 min of exposure there was no significant difference in the association of eIF4E with either 4E-BP1 or eIF4G (Fig. 4, A and B, respectively). After a 30-min exposure, the association of eIF4E with eIF4G was reduced to ~50% of the control, although there was not a corresponding increase in the level of eIF4E associated with 4E-BP1. Binding of eIF4E by another member of the 4E-BP family or an alternative mechanism that regulates the association of eIF4E with eIF4G may be responsible for the dissociation of eIF4E and eIF4G at this time. A 60-min exposure to 6% halothane led to increased levels of eIF4E complexed with 4E-BP1 and a corresponding decrease in eIF4E associated with eIF4G. During exposure to lower concentrations of halothane, there was a decrease in levels of translationally active eIF4E·eIF4G complex, although only at 4% halothane was this statistically significant (Fig. 4B). As observed after a 30-min exposure to 6% halothane, there was no change in the association of eIF4E with 4E-BP1 at these lower concentrations of anesthetic (Fig. 4A), again suggesting that another member of the 4E-BP family or an alternative mechanism plays a role in regulating the association of eIF4E with eIF4G under these conditions.


Figure 4
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  		Fig. 4. Extended exposure to Hal decreases translationally active eIF4E. A microtiter dish assay (29) was used to determine relative levels of eIF4E complexed with 4E-BP1 (A) or eIF4G (B) after perfusions with medium exposed to the specified concentrations of Hal. Perfusions for the indicated times were conducted as described in the legend to Fig. 1 and MATERIALS AND METHODS. At least 8 livers that were exposed to Hal and at least 5 livers that were not exposed to Hal were used at each time point. Bars are as described in the legend of Fig. 1B. **P < 0.01 vs. unexposed controls.

 
A third mechanism that affects translation initiation is altered phosphorylation of rpS6 (13). Halothane administered at 6% in the perfusate induced a significant decrease to 60% of control levels of phosphorylated rpS6 after 60 min of perfusion (Fig. 5, A and B). The levels of phosphorylated rpS6 were relatively stable during shorter periods of drug exposure. The levels of phosphorylated rpS6 were not affected by exposure to 2 or 4% halothane for 60 min (Fig. 5A).


Figure 5
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  		Fig. 5. A and B: decreased phosphorylation of rpS6 occurs during extended exposure to 6% Hal. Perfusions with medium exposed to the indicated concentrations of Hal were conducted for the specified times as described in the legend to Fig. 1 and MATERIALS AND METHODS. Aliquots of 1,000-g supernatants were subjected to SDS-PAGE, and the separated proteins were probed with an antibody that specifically recognizes phosphorylated rpS6 after transfer to membrane. Relative levels of phosphorylated rpS6 were determined from at least 8 livers that were exposed to Hal and at least 5 livers that were not exposed to Hal at each time point. Representative blots from livers exposed or not exposed to 6% halothane for 60 min are shown (B). C and D: extended exposure to high levels of Hal induces accumulation of hypophosphorylated forms of p70S6K1. Perfusions were conducted, and samples to examine p70S6K1 were prepared as described above. Differentially phosphorylated forms of this enzyme were detected by an antibody against p70S6K1. Representative blots from livers exposed or not exposed to 6% Hal for 60 min are shown (D). Relative levels of hyperphosphorylated forms of this enzyme were determined by quantitation of the levels of chemiluminescence in the beta- and {gamma}-bands vs. total chemiluminescence in the {alpha}-, beta-, and {gamma}-bands. Bars are as described in the legend of Fig. 1B. *P < 0.05; **P < 0.01 vs. unexposed controls.

 
Activity of the 70-kDa kinase that phosphorylates rpS6, termed p70S6K1, is regulated by a series of phosphorylations (13). Dephosphorylation of p70S6K1 decreases its kinase activity. Consistent with the decreased levels of phosphorylated rpS6 observed only after extended exposure to high levels of halothane, we found that levels of phosphorylated p70S6K1 also decreased only under these conditions (Fig. 5, C and D).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Perfused rat liver provides advantages for studying direct effects of volatile anesthetics on regulation of protein synthesis in mammals. These include maintenance of the intact liver in a relatively normal physiological state during extended exposure, the ability to harvest sufficient tissue for a variety of assays to examine the extent and mechanisms of translational alteration, the ability to administer a wide range of anesthetic doses, the ability to precisely regulate the level of amino acids and other metabolites present in the perfusate and isolate the liver from anesthetic-induced alterations in hormones and metabolites, and well-characterized translational responses of the liver to a variety of stresses.

Consistent with findings in yeast (37) and mammalian systems (3, 6, 8, 10, 15–18, 21, 23–25, 35, 40, 41), the volatile anesthetic halothane inhibits protein synthesis in perfused rat liver. Analysis of polysome profiles showed that translation initiation is dramatically inhibited by this drug. Both inhibition of incorporation of radiolabeled amino acid and disaggregation of polysomes are dose- and time-dependent. It is possible that the pentobarbital sodium used to anesthetize the animals (50 µg/g body wt) before the surgery, independently or in combination with halothane, affects protein synthesis. Two lines of reasoning suggest that pentobarbital sodium does not significantly affect protein synthesis in these studies. First, pentobarbital sodium included in perfusate at up to 100 µg/ml does not affect protein synthesis in perfused lung (41). Second, the concentration of pentobarbital sodium in the liver is reduced during perfusion when perfusate void of pentobarbital sodium replaces the blood containing pentobarbital sodium. The injected pentobarbital sodium would have to continue to affect protein synthesis despite this reduced concentration. Regardless of whether pentobarbital sodium affects protein synthesis, it is clear that halothane is essential for the observed effects because both control and experimental animals received equivalent doses of pentobarbital sodium.

An array of findings indicate that inhibition of translation in yeast exposed to volatile anesthetics results from deprivation for nutrients that the cells must obtain from their external environment (growth medium): strains prototrophic for all amino acids are highly resistant to the growth inhibitory effects of anesthetics (37); auxotrophic strains that contain extra copies of genes encoding permeases for critical amino acids that must be obtained from the medium are anesthetic resistant, whereas strains containing deletions of these genes are hypersensitive (38); increasing or decreasing the concentration of critical amino acids in the medium leads to resistance or hypersensitivity, respectively, to these drugs in appropriately auxotrophic strains (38); and uptake of leucine and tryptophan from medium is inhibited by isoflurane (38). The alterations of translation components observed during halothane exposure of rat liver are consistent with characterized changes known to occur during amino acid deprivation in mammalian systems (Fig. 6) (27), suggesting that limitation of nutrients may also play a critical role in the anesthetic response of liver.


Figure 6
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  		Fig. 6. Volatile anesthetics may affect amino acid availability. Amino acid limitation activates several signaling pathways that affect modification of various translation initiation factors inducing decreased protein synthesis. Modifications observed during Hal exposure are comparable to these. Dashed line ending with a cross hatch and labeled with a question mark indicates the possibility that volatile anesthetic exposure alters amino acid availability. Dashed line ending with an arrowhead and leading to the mammalian target of rapamycin (mTOR) pathway indicates unknown factors involved in transmitting signals regarding amino acid availability to mTOR. Dashed lines ending with arrowheads and labeled with question marks leading to "mRNA translation" indicate the possibility that additional pathways and/or modifications regulating translation initiation are affected by anesthetic exposure, as discussed in the text. eIF, eukaryotic translation initiation factor; 4E-BP1, eIF4E-binding protein-1; rpS6, ribosomal protein S6; p70S6K1, kinase that phosphorylates rpS6.

 
Studies in yeast show that inhibition of translation initiation by volatile anesthetics involves two processes: a rapidly induced general amino acid control (GCN)-independent pathway accompanied by a marked decrease in the level of eIF2{alpha}(P) and a GCN-dependent phase required to maintain inhibition that occurs only after more than 60 min of exposure and is accompanied by hyperphosphorylation of eIF2{alpha} (37). In contrast to our findings in yeast, increased phosphorylation of eIF2{alpha} in rat liver during exposure to 6% halothane occurs rapidly (within the first 15 min). Results from both systems indicate that response to anesthetic exposure is multifaceted and complex, involving multiple pathways operating at different times. It is intriguing to speculate that these mechanisms play roles in the activity of these drugs as general anesthetics due to alterations in transport of amino acids and related compounds that act as neurotransmitters (38).

Flaim et al. (10) previously reported a slight inhibition of initiation in liver when perfusate was exposed to 4% halothane for 45 min. The inhibition we observed at 4% halothane after 60 min of exposure is much more dramatic. This difference may be due to the fact that the perfusate in the previous studies (10) contained amino acids at 5 times rat arterial plasma concentrations, whereas the perfusate used in our studies contained amino acids at only 2.5 times these concentrations. Finding less inhibition of translation initiation when amino acid concentrations are higher is consistent with findings in yeast that increased levels of amino acids reverse the growth inhibitory effects of volatile anesthetics (38). The increased exposure time used in our studies is another potential factor in the observed difference.

The dissimilar alterations of translation initiation factors observed at 2, 4, or 6% halothane suggest that various mechanisms mediate inhibition at these different concentrations. For example, at 6% halothane the rapid and persistent increase in phosphorylation of eIF2{alpha} and the concurrent decrease in eIF2B activity suggest that these alterations are critical for reduced initiation at this anesthetic concentration. However, at 2 or 4% halothane, neither eIF2{alpha} phosphorylation nor eIF2B activity is affected even after extended exposure. One possible explanation for the failure to observe effects on eIF2{alpha} or eIF2B is that the extent of alterations induced by 2 or 4% halothane is low and fails to reach statistical significance under the experimental conditions. On the basis of our findings with 6% halothane, this seems unlikely. Significant changes in both eIF2{alpha} phosphorylation and eIF2B activity are observed when there are modest effects on protein synthesis during exposure to 6% halothane (e.g., 73% of control after 15 min). The level of inhibition of translation observed after a 60-min exposure to 4% halothane (61% of control) is greater than that observed after 15 min at 6% (Fig. 1, A and B, respectively). Thus relatively low levels of inhibition of translation by 6% halothane are accompanied by significant changes in both eIF2{alpha} and eIF2B, whereas neither factor is altered during exposure to 2 or 4% halothane even when inhibition of translation is greater. These results suggest that altered activity of eIF2 does not play a role in reducing translation at low halothane concentrations. This dramatic difference produced by a small increase in halothane concentration is similar to the sharp dose-response curve observed for other effects of volatile anesthetics.

Another feature of the effects of halothane is the time-dependent occurrence of alterations to translation factors. For example, phosphorylation of eIF2{alpha} increases rapidly after halothane administration, whereas altered phosphorylation of rpS6 and p70S6K1 occurs only after extended exposure. It is intriguing to speculate that rapid inhibition of translation at 6% is initially dependent on altered eIF2{alpha} phosphorylation, whereas the more dramatic inhibition of translation after extended exposure requires additional pathways, including decreased levels of phosphorylated rpS6 and redistribution of eIF4E out of its translationally active association with eIF4G. Other mechanisms affecting initiation may also participate in this anesthetic-induced inhibition of protein synthesis. Examination of effects on translation in perfused livers of mice mutant for various eIF2{alpha} kinases or 4E-BPs will provide additional insight regarding this possibility.

The dissociation of eIF4E and eIF4G observed at all concentrations of halothane appears to occur partly or wholly independently of eIF4E binding to 4E-BP1 regardless of the concentration of anesthetic. At 6% halothane, the decreased association of eIF4G with eIF4E is initially observed after 30 min of exposure, although increased association of eIF4E with 4E-BP1 is not observed until after 60 min. At both 2 and 4% halothane, the observed decrease in eIF4E·eIF4G complex is not accompanied by increased levels of eIF4E bound to 4E-BP1. In addition, this dissociation appears to initially occur independently of mTOR signaling, because alterations of the eIF4E·eIF4G complex occur in the absence of altered phosphorylation of p70S6K1 or rpS6 as well as in the absence of increased association of eIF4E with 4E-BP1. mTOR-independent regulation of the association of eIF4E with eIF4G has been observed in skeletal muscle (1, 31) but not in liver (31).

Further mechanisms responsible for inhibition of translation at 2 and 4% halothane may remain to be identified. It is not clear whether the decreased association of eIF4G with eIF4E, the only statistically significant change observed at 4% halothane, is sufficient to induce the substantial decline in translation observed (to 61% of control). Additional mechanisms that regulate translation initiation that were not uncovered in these experiments may be necessary for this inhibition. Inhibition of translation elongation may also play a role, although phosphorylation of elongation factor 2, which is regulated by mTOR (for a review see Ref. 5), is not altered after exposure to 6% halothane for 60 min (data not shown). Studies with mice containing mutations in various translation factors will provide additional insight.

In conclusion, the results of these studies indicate that the volatile anesthetic halothane inhibits translation initiation in mammalian liver in a dose- and time-dependent manner. Regulation of both eIF2{alpha} kinase activity and the mTOR response pathway plays a role in this inhibition. Volatile anesthetics may provide new tools for investigating cellular responses to altered amino acid nutrition in mammalian cells as well as in yeast.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from a Tobacco Cross-Campus Grant of The Pennsylvania State University (L. S. Jefferson, S. R. Kimball, and R. L. Keil) and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-13499 (L. S. Jefferson).


   ACKNOWLEDGMENTS
 
We thank Lynne Hugendubler, Jamie Crispino, and Courtney Bradley for expert technical assistance and Nikki Keasey for comments and suggestions regarding the manuscript.

Present address of L. K. Palmer: Division of Mathematics and Natural Sciences, The Pennsylvania State University, Altoona Campus, Altoona, PA 16601.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. L. Keil, Dept. of Biochemistry and Molecular Biology H171, The Pennsylvania State University College of Medicine, 500 University Dr., Hershey, PA 17033 (e-mail: rkeil{at}psu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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