Article Figures & Data
Figures
- Figure 1.
GC/MS analysis for the enzymatic conversion of benzaldehyde and acetyl isoeugenol in nasal mucus. A , Total ion chromatograms of the organic extracts from Ringer's solution (black line, top panel), nasal mucus (red line, middle panel), and heated nasal mucus (blue line, bottom panel), each incubated with benzaldehyde. The extracted ion chromatogram of the molecular ion peak of benzoic acid (m/z 122) is shown as the inset. Peaks for benzaldehyde, benzyl alcohol, and benzoic acid are shown by arrowheads. B , Total ion chromatograms of the organic extracts from Ringer's solution (black line, top panel), nasal mucus (red line, middle panel), and heated nasal mucus (blue line, bottom panel), each incubated with acetyl isoeugenol (AIEG). Peaks for AIEG and isoeugenol (IEG) are shown by arrowheads.
- Figure 2.
Structure–activity relationships of the enzyme reactions in nasal mucus of odorants with an aldehyde or acetate group. A , The percentage of converted odorants with an aldehyde group (conversion ratio) was plotted as the mean ± SEM (n = 3). B , The percentage of converted odorants with an acetate group (conversion ratio) was plotted as the mean ± SEM (n = 3). C , The compounds not converted.
- Figure 3.
The in vivo enzymatic conversions of benzaldehyde and acetyl isoeugenol in the nasal cavity. A , Total ion chromatogram of the benzaldehyde sample recovered from the nasal cavity. Upper panel, Control benzaldehyde; lower panel, the sample retrieved from the nasal cavity after perfusion. Extracted ion chromatogram of the molecular ion peak of benzoic acid (m/z 122) is shown as the inset. Peaks for benzaldehyde, benzyl alcohol, and benzoic acid are shown by arrowheads. B , Total ion chromatogram of the acetyl isoeugenol (AIEG) sample recovered from the nasal cavity. Upper panel, Control AIEG; lower panel, the sample retrieved from the nasal cavity after perfusion. Peaks for AIEG and isoeugenol (IEG) are shown by arrowheads. C , Extracted ion chromatogram of the molecular ion peaks m/z 122, 108, 106, 105, 94, and 79 of the nasal mucus sample obtained from mice that naturally inhaled benzaldehyde vapor. D , Extracted ion chromatogram of the molecular ion peak m/z 164 of the nasal mucus sample obtained from mice that naturally inhaled AIEG vapor. The samples in C and D were analyzed by GC/MS-SIM.
- Figure 4.
Effects of inhibitors for carboxylesterase and carbonic anhydrase on acetyl isoeugenol–isoeugenol conversion in nasal mucus. A , Dose-dependent inhibition by carboxylesterase inhibitor BNPP, but not by carbonic anhydrase inhibitor AZ. The mean conversion ratio was shown as percentage ± SEM (n = 3). Statistics, One-way ANOVA and post hoc test (Dunnett's test), *p < 0.05; ***p < 0.001. B , Analysis of the enzymatic activity of nasal mucus collected 1 d after application of Ringer's solution (left panel), AZ (middle panel), or BNPP (right panel) in the nasal cavity. C , The amount of nonconverted acetyl isoeugenol (AIEG) remaining 1 d after application of AZ or BNPP upon odorant perfusion into the nasal cavity. Statistics, One-way ANOVA and post hoc test (Dunnett's test), **p < 0.01.
- Figure 5.
Comparison of odorant-evoked glomerular activation patterns stimulated with acetyl isoeugenol before and after the application of inhibitor. A , Glomerular Ca2+ responses to acetyl isoeugenol (AIEG) before/after the application of BNPP shown as pseudocolored images. Left, Fluorescent image of the olfactory bulb. Middle images, Pseudocolored images of the olfactory bulb before and after the application of BNPP showing the glomerular activation patterns; red corresponds to the greatest response. The data from three animals (#1, #2, #3) are shown. Right, Schematic illustrations of glomerular activation patterns in pseudocolored images. The filled circles indicate glomeruli activated by AIEG, while the glomeruli with open circles are the ones that responded to AIEG only after BNPP application. Some extra glomeruli are activated by AIEG after BNPP treatment. B , Glomerular Ca2+ responses to acetyl isoeugenol (AIEG) before/after the application of AZ shown as pseudocolored images. Left, Fluorescent image of the olfactory bulb. Middle images, Pseudocolored images of the olfactory bulb before and after the application of AZ showing the glomerular activation patterns. The data from two animals (#4, #5) are shown. Right, Schematic illustrations of glomerular activation patterns in pseudocolored images. The filled circles indicate glomeruli activated by AIEG. There was no effect of AZ treatment on glomerular activation patterns.
- Figure 6.
Effects of enzymatic conversion on the recognition of acetyl isoeugenol (AIEG) in a behavioral context. Mice were trained for 4 d to associate a reward (sugar grains) with either of two odorants. After 4 d of training, BNPP or AZ was applied to the nasal cavities of the mice. On day 5, investigation times were measured for each pair of odorants without the sugar reward. Mean investigation times (in seconds) ± SEM during the 2 min test period are shown as bar graphs for odorants paired with the sugar reward [odor (+), gray bars], and unpaired odorants [odor (−), white bars]. A , Eugenol (EG) versus pentanol; B-1 , EG versus AIEG; B-2 , pentanol versus AIEG; B-3 , 2-heptanone versus AIEG; C , IEG versus AIEG. Statistics, Paired t test was used for two related samples (pair of odorants in each group); *p < 0.05, **p < 0.01 (black asterisk). Non-repeated-measures ANOVA for three independent groups (control, AZ, BNPP group) and post hoc test (Dunnett's test), *p < 0.05, **p < 0.01 (gray asterisk).
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