Short-Term Administration of Common Anesthetics Does Not Dramatically Change the Endogenous Peptide Profile in the Rat Pituitary

Abstract

Cell−cell signaling peptides (e.g., peptide hormones, neuropeptides) are among the largest class of cellular transmitters and regulate a variety of physiological processes. To identify and quantify the relative abundances of cell−cell signaling peptides in different physiological states, liquid chromatography−mass spectrometry-based peptidomics workflows are commonly utilized on freshly dissected tissues. In such animal experiments, the administration of general anesthetics is an important step for many research projects. However, acute anesthetic administration may rapidly change the measured abundance of transmitter molecules and metabolites, especially in the brain and endocrine system, which would confound experimental results. The aim of this study was to evaluate the effect of short-term (<5 min) anesthetic administration on the measured abundance of cell−cell signaling peptides, as evaluated by a typical peptidomics workflow. To accomplish this goal, we compared endogenous peptide abundances in the rat pituitary following administration of 5% isoflurane, 200 mg/kg sodium pentobarbital, or no anesthetic administration. Label-free peptidomics analysis demonstrated that acute use of isoflurane changed the levels of a small number of peptides, primarily degradation products of the hormone somatotropin, but did not influence the levels of most other peptide hormones. Acute use of sodium pentobarbital had negligible impact on the relative abundance of all measured peptides. Overall, our results suggest that anesthetics used in pituitary peptidomics studies do not dramatically confound observed results.

Effects of Anesthetic Administration on Rat Hypothalamus and Cerebral Cortex Peptidome

“…This result suggests that neither sodium pentobarbital nor isoflurane have dramatic effects on the overall peptide profile in the hypothalamus and cerebral cortex, which parallels our previous results from the pituitary. 26 We also did not detect any major sex-specific differences in the peptide profile (Figure S5). Within each tissue, pairwise comparisons for each experimental group were then performed to identify any individual peptides that significantly differ on the basis of treatment effect (Figures 2 and 3, Tables 1 and 2, and Tables S1−S4).…”

“…We previously demonstrated that the short-term (<5 min) use of isoflurane or sodium pentobarbital had minimal impact on the peptide profile in rat pituitary, thereby suggesting that acute anesthetic administration did not confound peptide profile in this endocrine organ. 26 In the present study, we aimed to extend our method to investigate the effect of anesthetic administration on two regions of the brain: the hypothalamus and the cerebral cortex. Both of these brain regions are relatively rich in endogenous peptides 4,27 and have previously been shown to be influenced by anesthetic administration.…”

“…After the indicated treatment, the animals were sacrificed by decapitation, and the tissues were rapidly dissected and flash-frozen in liquid nitrogen (Figures S1 and S2). Protocols were kept as consistent as possible with our prior study on the rat pituitary 26 to allow meaningful comparisons between the two studies. Peptides were extracted from tissues and analyzed by LC-MS and LC−tandem mass spectrometry (LC-MS/MS) using label-free quantification.…”

Peptidomic Analysis Reveals Seasonal Neuropeptide and Peptide Hormone Changes in the Hypothalamus and Pituitary of a Hibernating Mammal

“…Endogenous peptides were extracted from tissues using both organic and aqueous extraction steps and enriched using molecular weight-based centrifugation filtration devices and solid-phase extraction. Extracted peptides were analyzed by a LC–MS and LC–MS/MS-based label-free peptidomics workflow, , which includes steps for data filtering, batch correction, imputation, and normalization (Figures S2–S5). Following data processing and normalization, relative peptide abundances were compared between groups using multivariate and univariate statistical analyses.…”

“…We hypothesized that changes in cell–cell signaling peptides from the hypothalamus and pituitary may be responsible for regulating critical physiological changes during the hibernation season. To test this hypothesis, we applied a non-targeted liquid chromatography–mass spectrometry (LC–MS) and LC-tandem mass spectrometry (LC–MS/MS) “peptidomics” approach ,− to identify and quantify endogenous neuropeptides and peptide hormones as a function of season and hibernation stage in the thirteen-lined ground squirrel. LC–MS and LC–MS/MS-based peptidomics allow the detection, identification, and quantification of peptides without preselection, allowing for findings not possible with targeted antibody-based methods.…”