Inhibition of mammalian translation initiation by volatile anesthetics

Abstract

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 ␣-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␣ 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 (p70 S6K1 ). 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␣ 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 effec...

Sex-dependent differences in the regulation of myocardial protein synthesis following long-term ethanol consumption

“…The homogenate was diluted with one-third volume of blocking solution (5% nonfat dry milk in phosphate-buffered saline). An aliquot was layered into wells of a microtiter plate previously coated with 1 g of mouse monoclonal anti-eIF4E antibody (20,37). After incubation overnight, the sample was removed, the wells were washed, and, subsequently, either polyclonal anti-eIF4E (Cell Signaling Technology, Beverly, MA), anti-4EBP1, or anti-eIF4G (Bethyl Laboratories, Montgomery, TN) in blocking solution was added to the wells.…”

“…Nonspecific binding of eIF4E, eIF4G ⅐ eIF4E complex or 4EBP1 ⅐ eIF4E complex is minimal. Values are expressed as the abundance of 4EBP1 or eIF4G divided by the abundance of eIF4E (20,37).…”

Rapid Turnover of the mTOR Complex 1 (mTORC1) Repressor REDD1 and Activation of mTORC1 Signaling following Inhibition of Protein Synthesis

“…The homogenate was centrifuged at 3,000 ϫ g for 15 min at 4°C centrifugation, and the supernatant was subjected to sucrose density gradient centrifugation as described previously (36). Briefly, discontinuous 9-step 20% (10 mM HEPES, pH 7.4; 250 mM KCl; 5 mM MgCl 2 ; 0.5 mM EDTA; 20% w/w sucrose) to 47% (10 mM HEPES, pH 7.4; 250 mM KCl; 5 mM MgCl 2 ; 0.5 mM EDTA; 47% w/w sucrose) sucrose density gradients were formed as previously described (36), except that the KCl concentration was changed to 75 mM. An equal amount of protein was loaded onto each gradient and centrifuged at 288,200 ϫ g at 4°C for 1 h 50 min.…”

Structural analysis of compounds with actions similar to local anesthetics and antipsychotic phenothiazines in yeast

“…Although translational inhibition is considered to be an indicator for an anesthetics effect (Palmer et al, 2005(Palmer et al, , 2006, it is not easy to evaluate quantitatively the translational inhibition of many samples under several conditions. At the drug concentrations that lead to shutdown of translation initiation, the actin cytoskeleton is also depolarized simultaneously but independently (Uesono et al, 2004(Uesono et al, , 2008.…”