Cycloheximide: no ordinary bitter stimulus

Abstract

Cycloheximide (CyX), a toxic antibiotic with a unique chemical structure generated by the actinomycete, Streptomyces griseus, has emerged as a primary focus of studies on mammalian bitter taste. Rats and mice avoid it at concentrations well below the thresholds for most bitter stimuli and T2R G-protein-coupled receptors specific for CyX with appropriate sensitivity are identified for those species. Like mouse and rat, golden hamsters, Mesocricetus auratus, also detected and rejected micromolar levels of CyX, although 1mM CyX failed to activate the hamster chorda tympani nerve. Hamsters showed an initial tolerance for 500microM CyX, but after that, avoidance of CyX dramatically increased, plasticity not reported for rat or mouse. As the hamster lineage branches well before division of the mouse-rat lineage in evolutionary time, differences between hamster and mouse-rat reactions to CyX are not surprising. Furthermore, unlike hamster LiCl-induced learned aversions, the induced CyX aversion neither specifically nor robustly generalized to other non-ionic bitter stimuli; and unlike adverse reactions to other chemosensory stimuli, aversions to CyX were not mollified by adding a sweetener. Thus, CyX is unlike other bitter stimuli. The gene for the high-affinity CyX receptor is a member of a cluster of five orthologous T2R genes that are likely rodent-specific; this "CyX clade" is found in the mouse, rat and probably hamster, but not in the human or rabbit genome. The rodent CyX-T2R interaction may be one of multiple lineage-specific stimulus-receptor interactions reflecting a response to a particular environmental toxin. The combination of T2R multiplicity, species divergence and gene duplication results in diverse ligands for multiple species-specific T2R receptors, which confounds definition of 'bitter' stimuli across species.

Figure 1

Cycloheximide Decomposition in Alkaline Solution. The major route of decomposition is shown. In neutral aqueous solution, cycloheximide is initially non-odorous to humans. It develops a detectable ketone-like odor similar to its breakdown products within 24 hrs (unpublished observations).

Figure 2

Two-Day, 2-Bottle Mean Intake (±se) of Cycloheximide (CyX), Chlorhexidine, Dulcin and n-Propylthiouracil (PROP). CyX solutions include pure 0.3 - 30 μM CyX, as well as 30 μM CyX treated with NaOH (X), and 30 μM CyX + dulcin. The data for the pure CyX solutions are plotted in the order that they were presented; i.e., the leftmost 30 μM CyX bar represents the hamsters' initial access to CyX. Other solutions are 0.2 mM chlorhexidine, the 0.2 mM chlorhexidine + dulcin mixture, 5 mM dulcin, and 1 mM and 5 mM PROP. Means ± se are plotted. 2-day water and stimulus intake differed for all stimuli except alkali-treated 30 μM cycloheximide X.

Figure 3

Data Traces of Responses to 10 mM Solutions by Hamster Chorda Tympani Nerve. Recordings from 1 nerve to cycloheximide (CyX), propylthiouracil (PROP) and quinine·HCl: 5-sec of neural activity recorded before and 10 sec after stimulus application (onset indicated by vertical lines aligned for the 3 records) is shown in each trace. Major divisions on the ordinate are 0.01 volts. Numerals at major divisions on the abscissa indicate stimuli were applied between 13 and 16 seconds after starting the recording of the 3 separate epochs.

Figure 4

Chorda Tympani Nerve Responses to Cycloheximide, Quinine·HCl and n-Propylthiouracil Concentration Series. Responses, measured as 5-sec post-stimulus minus 5-sec pre-stimulus areas beneath integrated response curves, are given as mean ±se percentages of the standard response to 500 mM NH₄Cl.

Figure 5

Effect of Presenting 500 μM Cycloheximide (CyX) for 1 Hr on Subsequent CyX Intake. Mean ± se CyX/water intake ratios for the 1^st presentation, open point (N=28), and 2^nd presentation, filled points for separate subgroups of hamsters (N = 6-10), are plotted. No matter the time after 1^st exposure, CyX intake was much diminished on the 2^nd offering

Figure 6

Effect of One 1-Hr Exposure to 500 μM Cycloheximide (CyX) and Self-Administration of 0.75 μMoles of CyX on Intake of Solutions. Means (± se) are plotted. After a single CyX presentation, CyX intake was much reduced compared to the reduction in water intake (p<.0005); drinking of other bitter solutions was reduced to a lesser extent, while drinking of NaCl and sucrose was unaffected.

Figure 7

Mean (±se) Percent Suppression of Taste Stimulus Intake following One Exposure to 500 μM Cycloheximide (CyX). Like CyX (p = 0002), bitter stimuli [1 mM quinine, 1 mM SOA, 5 mM PROP, 30 mM caffeine] were suppressed (p = 05); non-bitter stimuli [.1 M NaCl, .1 M sucrose] were not.

Figure 8

Percent Amino Acid Identity (± se) of Orthologous Proteins of Rat and Mouse or Human and Mouse. The 4 groups of proteins (see Table 2) from left to right are: 2 T2R receptors, 3 T1R receptors, 2 cytochrome P450 enzymes and (combined) potassium channel protein KCNJ6, olfactory receptor I7 and metabotropic glutamate receptor mGluR1. Amino acid (AA) identity is greater for the rat-mouse comparison than the human-mouse comparison (p = .000001); and the T2R candidate bitter receptors have the lowest AA identity of the 4 groups of proteins (α = .05).