Tetracaine, a local anesthetic, preferentially induces translational inhibition with processing body formation rather than phosphorylation of eIF2α in yeast

Abstract

It is unclear whether local anesthetics, such as tetracaine, and antipsychotics, such as phenothiazines, act on lipids or proteins. In Saccharomyces cerevisiae, these drugs inhibit growth, translation initiation, and actin polarization, and induce cell lysis at high concentrations. These activities are likely due to the cationic amphiphilic structure common to these agents. Although drug-induced translational inhibition is conserved in mammalian cells, other mechanisms, including the phosphorylation of eIF2α, a eukaryotic translational initiation factor, remain poorly understood. At a concentration of 10 mM, tetracaine rapidly inhibited translation initiation and lysed cells, whereas, at 2.5 mM, it slowly induced inhibition without lysis. The pat1 disruptant defective in mRNA decapping and the xrn1 disruptant defective in 5'-3' exoribonuclease were partially resistant to translational inhibition by tetracaine at each concentration, but the gcn2 disruptant defective in the eIF2α kinase was not. Phosphorylation of eIF2α was induced by 10 mM but not by 2.5 mM tetracaine, whereas processing bodies (P-bodies) were formed at 2.5 mM in Pat1-dependent and -independent manners. Therefore, administration of tetracaine inhibits translation initiation with P-body formation at both concentrations but acts via the Gcn2-eIF2α system only at the higher concentration. Because other local anesthetics and phenothiazines induced Pat1-dependent P-body formation, the mechanisms involved in translational inhibition by these cationic amphiphiles are similar. These results suggest that this dose-dependent biphasic translational inhibition by tetracaine results from an increase in membrane proteins that are indirectly inhibited by nonspecific interactions of cationic amphiphiles with membrane lipids.

Local Anesthetics and Antipsychotic Phenothiazines Interact Nonspecifically with Membranes and Inhibit Hexose Transporters in Yeast

“…However, administration of excessive amounts of auxotrophic amino acids and uracil did not alleviate the sensitivity to 2.5 mM TC in the presence of 2% glucose, which indicates that amino acid starvation and uracil starvation are not involved in the sensitivity to TC at low concentrations. This finding is in agreement with reports that TC induced eIF2a phosphorylation at 10 mM but not at 2.5 mM (Araki et al 2015). The addition of excessive amounts of sorbitol, a nonfermentable sugar alcohol, did not alleviate TC sensitivity, as observed with other polysaccharides, which indicates that alleviation of the effects of TC is not due to high osmolarity.…”

“…In contrast, actin polarization recovered to just 27% after omission of TC from cells treated with 10 mM for 30 min ( Figure 3C, right, black circle), which shows that 10 mM is severely toxic. Based on ALP activity, TC treatment led to rapid cell lysis at 10 mM, possibly via its surfactant activity, but not at 2.5 mM ( Figure 3C, bottom) (Araki et al 2015). This suggests that the cell membrane is damaged by 10 mM TC and that membrane damage would not be rescued by additional glucose or OMG.…”

“…This is so possibly because maltose transporters have structural similarity to Hxts (Cheng and Michels 1989). Because administration of TC stimulated the eIF2a phosphorylation that is induced by amino acid starvation (Araki et al 2015), TC also might induce amino acid starvation. However, administration of excessive amounts of auxotrophic amino acids and uracil did not alleviate the sensitivity to 2.5 mM TC in the presence of 2% glucose, which indicates that amino acid starvation and uracil starvation are not involved in the sensitivity to TC at low concentrations.…”

“…Polysome analysis was carried out as described previously (Araki et al 2015). Logarithmic cultures (1 3 10 7 cells/ml) grown in 40 ml of YPD (pH 6.0) were treated with the indicated drugs and used for polysome analysis.…”

“…This activity accounts for the common toxicity of these compounds at high concentrations. At low concentrations that do not cause cell lysis, these compounds induce similar responses, including the inhibition of both translation initiation and polarization of the actin cytoskeleton, nuclear localization of the stress-responsible transcription factor Msn2, and processingbody (P-body) formation for messenger RNA (mRNA) decay in a dose-dependent manner in yeast (Uesono et al 2008(Uesono et al , 2011Araki et al 2015). Volatile anesthetics (Bruce 1975;Heys et al 1989;Horber et al 1988), local anesthetics (Banerjee and Redman 1977;Wang et al 2011), and phenothiazines (Raghupathy et al 1970;Kumar et al 1991) are known to inhibit protein synthesis in animals at cellular, tissue, and whole-body levels.…”