Nasal mucus glutathione transferase activity and impact on olfactory perception and neonatal behavior

Abstract

In olfaction, to preserve the sensitivity of the response, the bioavailability of odor molecules is under the control of odorant-metabolizing enzymes (OMEs) expressed in the olfactory neuroepithelium. Although this enzymatic regulation has been shown to be involved in olfactory receptor activation and perceptual responses, it remains widely underestimated in vertebrates. In particular, the possible activity of OMEs in the nasal mucus, i.e. the aqueous layer that lined the nasal epithelium and forms the interface for airborne odorants to reach the olfactory sensory neurons, is poorly known. Here, we used the well-described model of the mammary pheromone (MP) and behavioral response in rabbit neonates to challenge the function of nasal mucus metabolism in an unprecedented way. First, we showed, in the olfactory epithelium, a rapid glutathione transferase activity toward the MP by ex vivo real-time mass spectrometry (PTR-MS) which supported an activity in the closest vicinity of both the odorants and olfactory receptors. Indeed and second, both the presence and activity of glutathione transferases were evidenced in the nasal mucus of neonates using proteomic and HPLC analysis respectively. Finally, we strikingly demonstrated that the deregulation of the MP metabolism by in vivo mucus washing modulates the newborn rabbit behavioral responsiveness to the MP. This is a step forward in the demonstration of the critical function of OMEs especially in the mucus, which is at the nasal front line of interaction with odorants and potentially subjected to physiopathological changes.

Figure 1

Schematic representation of the system developed to analyse the metabolic capacity of ex vivo newborn rabbit OM or nasal mucus by PTR-MS in real-time. A known concentration of gaseous odorants (gas bag B) can be delivered above a fresh explant of OM or nasal mucus placed into a hermetically closed glassware and the flow was monitored by the PTR-MS instrument allowing the real-time analysis of metabolic activities. Shut-off valve and three-way valve luer were implemented to allow delivery of humidified zero air passing through the glassware (gas bag A) or not (gas bag C) to realize analysis and controls in different conditions. The whole system is enclosed in a thermostated oven (30 °C).

Figure 2

Real-time ex vivo newborn rabbit OM metabolism of the MP by PTR-MS analysis with the continuous method. The signal of MP on gaseous form (10⁻⁹ g/ml in gas bag B) passing through the glassware without OM (in blue), with OM (in red) or in presence of heated OM to denaturate enzymes (in grey) was monitored in real-time by the PTR-MS using the continuous method. (A) Kinetics of the ex vivo MP metabolism was measured in continuous over 60 s. Data represent the mean ± SEM from 5 independent assays for each condition. The significant differences is noted ^**p < 0.01 (Student’s t-test) for a comparison with MP signal without OM. (B) Data represent the normalized CPS mean ± SEM during the last 20 s of the continuous supply signal of MP measured by the PTR-MS instrument. No significant difference is noted ns and significant difference is noted ^*p < 0.05 (Student’s t-test) for a comparison with MP signal without OM (n = 5).

Figure 3

MP metabolism and enzymatic glutathione conjugation activity by nasal newborn rabbit mucus. (A) The MP signal in gaseous form (10⁻¹⁰ g/ml in gas bag B) passing through the glassware without nasal mucus (in green) or with nasal mucus (in orange) was monitored by the PTR-MS using the trapping method. Data represent the normalized CPS mean ± SEM during the first 90 sec of the 2 min-purge of the glassware trap of the continuous supply signal of MP measured by PTR-MS. ^**p < 0.01 (Student’s t-test) for a comparison of the MP signal with vs. without nasal mucus (n ≥ 6). (B) Glutathione conjugation of MP by nasal mucus GST was determined by measurement with HPLC-CAD, after incubation (80 min at 37 °C) of MP + reduced glutathione in presence or absence of mucus. Bars represent the production of the glutathione-MP conjugate without nasal mucus (hatched green lines) or with nasal mucus (hatched orange lines). Data consist in the mean of 3 independent assays ± SEM. ^***p < 0.001 (Student’s t-test) for a comparison of the glutathione-MP conjugate signal with vs. without nasal mucus (n ≥ 6).

Figure 4

Perception and behavioral responsiveness of newborn rabbits to the MP after nasal mucus washing. Proportions of rabbit pups responding by orocephalic movements to the MP in the glass-rod test (n = 15 to 30 pups from 3–6 litters): (A) responsiveness to MP at 10⁻⁶ g/ml before and after nasal mucus washing, (B) responsiveness to MP at 10⁻⁹ g/ml before and after nasal mucus washing and subsequently presented at 10⁻⁶ g/ml as a control and (C) responsiveness to MP at 10⁻⁹ g/ml before and after pseudo washing and subsequently presented at 10⁻⁶ g/ml as a control. Within each graph, distinct digits indicate statistical differences (p ≤ 0.05): Cochran’s Q test followed by McNemar test for pairwise comparisons.