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Constitutive and evoked release of ATP in adult mouse olfactory epithelium

  • Sébastien Hayoz ORCID logo EMAIL logo , Cuihong Jia and Colleen Cosgrove Hegg
Published/Copyright: January 16, 2024
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Open Life Sciences
From the journal Open Life Sciences Volume 19 Issue 1

Abstract

In adult olfactory epithelium (OE), ATP plays a role in constant cell turnover and post-injury neuroregeneration. We previously demonstrated that constitutive and ATP-evoked ATP release are present in neonatal mouse OE and underlie continuous cell turn-over and post-injury neuroregeneration, and that activation of purinergic P2X7 receptors is involved in the evoked release. We hypothesized that both releases are present in adult mouse OE. To study the putative contribution of olfactory sensory neurons to ATP release, we used olfactory sensory neuronal-like OP6 cells derived from the embryonic olfactory placode cells. Calcium imaging showed that OP6 cells and primary adult OE cell cultures express functional purinergic receptors. We monitored ATP release from OP6 cells and whole adult OE turbinates using HEK cells as biosensors and luciferin–luciferase assays. Constitutive ATP release occurs in OP6 cells and whole adult mouse OE turbinates, and P2X7 receptors mediated evoked ATP release occurs only in turbinates. The mechanisms of ATP release described in the present study might underlie the constant cell turn-over and post-injury neuroregeneration present in adult OE and thus, further studies of these mechanisms are warranted as it will improve our knowledge of OE tissue homeostasis and post-injury regeneration.

Keywords: primary cell cultures; OP6 cells; confocal imaging; immunohistochemistry; luciferin–luciferase assay

1 Introduction

The olfactory epithelium (OE) comprises several distinct cell types [1]. The OE is pseudostratified: the cell bodies of the glia-like sustentacular cells are located in the apical layer, the cell bodies of the olfactory sensory neurons (OSN) that detect odorants are located in the middle layer, and progenitor/stem cells that allow neuroregeneration and constant cell turn-over are located in the basal layer. The OE also houses in the apical layer a population of microvillous cells whose exact function is still unclear. Cells of all OE layers express both P2X purinergic receptors and P2Y purinergic receptors [2–4]. P2X are ionotropic receptors and P2Y are metabotropic receptors; their activation by ATP plays an essential role in mechanisms involving ATP as a neurotransmitter [5]. Several subtypes of P2X and P2Y purinergic receptors are present in mouse OE [2–4]. Purinergic signaling has been well studied in the vertebrate olfactory system [6] and ATP has been shown to modulate responses to odorants [7,8]. Moreover, in vertebrate OE, purinergic signaling has been shown to be involved in communication between the different cell types [2,9,10]. Purinergic signaling also plays a very important injury-related role in mouse OE. Indeed, ATP has both protective and proliferative effects following injury [11,12]. One ATP mechanism of action is direct stimulation of the purinergic receptors located on the OE basal progenitor cells [2,13,14]. ATP also indirectly induces the upregulation and release of other neurotrophic factors such as neuropeptide Y and fibroblast growth factor 2 [15–18], leading to cell proliferation in adult mouse OE via activation of neuropeptide Y and fibroblast growth factor receptors [16–18]. Therefore, study of the mechanisms underlying ATP release is essential to fully understand how the OE maintains tissue homeostasis and recovers from injury.

ATP release has been well documented in several organ systems and sensory systems including sensory axons in the olfactory bulb [5,19]. ATP-induced ATP release has been demonstrated in endothelial cell culture [20,21], in taste bud cells [22] and in cultured astrocytes [23]. Moreover, we showed that both ATP-evoked ATP release and constitutive ATP release occur in neonatal mouse OE through numerous mechanisms [4]. The constitutive release of ATP likely occurs in physiological, i.e., injury-free conditions, to promote constant cell turnover and tissue homeostasis in the OE. The evoked ATP release, activated by purinergic receptor stimulation, is likely triggered by ATP leaking from damaged cells, inducing subsequent post-injury OE regeneration. When we previously investigated the mechanisms underlying the evoked ATP release in mouse neonatal OE, we became interested in one sub-type of P2X purinergic receptors, P2X7. This sub-type, encoded by the P2rx7 gene, has unique properties. Activated P2X7 receptors form large pores leading to either influx or efflux of ions and other molecules [24–26]. Moreover, it has been shown in several cell types that activated P2X7 receptors can also form complexes with pannexin1 channels, which can lead to efflux of ATP or other molecules [27–32]. We indeed showed that one of the mechanisms involved in ATP-evoked release of ATP in neonatal mouse OE is activation of P2X7 receptors and subsequent formation of P2X7 large pores and/or P2X7–pannexin complexes [4]. We also showed that stimulation of P2Y purinergic receptors also contribute to the evoked release of ATP. We hypothesized that similar mechanisms of constitutive and evoked ATP release are present in adult OE. To specifically investigate the putative role of OSN in ATP release, we used the OP6 cell line, a clonal temperature-sensitive cell line derived from the E10 mouse olfactory placode. Undifferentiated OP6 cells are similar to immature OSN and differentiated OP6 cells are similar to mature OSN, as described by Illing et al. [33]. We first used calcium imaging to show the presence of functional purinergic receptors in OP6 cells and adult OE cells, a requirement to study purinergic receptor-mediated ATP release in adult OE. Then, using luciferin–luciferase assays and human embryonic kidney (HEK-293) cells as ATP biosensors, we showed that constitutive and evoked ATP release both occur in whole adult OE turbinates, but that both immature and mature OP6 cells only have a constitutive release of ATP. We also showed that P2X7 purinergic receptors are involved in the evoked release of ATP. We conclude that (a) functional purinergic receptors are present in adult mouse OE and activation of the P2X7 receptor subtype is sufficient for evoked release of ATP, (b) constitutive ATP release is also present in adult mouse OE, and (c) OSN only constitutively release ATP, the evoked release of ATP thus originating most likely from other cell types. However, it does not exclude that the other OE cell types might constitutively liberate ATP as well, and that activation of other P2X and/or P2Y receptor subtypes might contribute to the evoked release of ATP.

2 Materials and methods

2.1 Chemicals

Fluo-4 AM was purchased from Invitrogen (Carlsbad, CA, USA). ATP determination kits were purchased from Molecular Probes (Eugene, OR, USA). All other chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA).

2.2 Solutions

Ringer’s solution contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2 6H2O, 2 CaCl2, 10 HEPES, and 10 glucose (pH 7.4). Calcium-free Ringer’s solution contained (in mM) 140 NaCl, 5 KCl, 10 HEPES, 4 EGTA, and 20 glucose (pH 7.4). Concentrated stock solutions of ATP, 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (Bz-ATP), and Clostridium difficile toxin A were made in Ringer’s solution, stored at −20°C and, on the day of the experiment, diluted to concentration values indicated in the text. Fresh stock solutions of carbenoxolone and probenecid dissolved in Ringer’s solution were made on the day of the experiment and diluted to concentration values indicated in the text.

2.3 Olfactory turbinates dissection

All animal procedures were approved by Michigan State University’s IACUC, and all applicable guidelines from NIH were followed. Swiss Webster mice around 3–8 weeks old (Charles River, Portage, MI) were anesthetized (i.p. ketamine/xylazine 65/5 mg/kg body weight, respectively) and sacrificed by decapitation. We used Swiss Webster mice to be consistent with our previous study that characterized constitutive and evoked ATP releases in neonatal OE, and that also used Swiss Webster mice [4]. Fur and skin were removed to expose the bone and the skull cut in half with a razor blade to expose the septum and turbinates. Fine forceps were used to remove the septum and extract the full set of turbinates, identified via the paper and online atlas by Barrios et al. [34]. Turbinates were immediately placed in Ringer’s solution (see Section 2.2) and used either for preparing cell cultures (see Section 2.4) or for luciferin–luciferase assays (see Section 2.6).

  1. Ethical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals, and has been approved by the Michigan State University’s IACUC.

2.4 Cell cultures

Primary cell cultures were prepared from olfactory turbinates collected from 3 weeks old Swiss Webster mice (see Section 2.3) and dissociated as described previously [35]. Primary cultures were grown on coverslips coated with concavalin A (10 mg/mL; Sigma type IV) and used at 7–8 days in vitro (DIV) for calcium imaging or immunohistochemistry. The OP6 cell line, a clonal temperature-sensitive cell line derived from the E10 mouse olfactory placode [33], was generously provided by Dr Mary Lucero from University of Utah. At a permissive temperature (33°C), OP6 cells are undifferentiated and behave like immature OSN. At a non-permissive temperature (39°C), all-trans retinoic acid induces the differentiation into a population of mature bipolar OSN-like cells. OP6 cells were plated onto coverslips coated with laminin (20 μg/mL), grown and/or differentiated as described previously [33], and used at 4–8 DIV for calcium imaging or luciferin–luciferase assays. P2X2-transfected HEK-293 cells used as biosensors were generously provided by Dr James Galligan from Michigan State University and were maintained as described in our previous study [4]. Briefly, they were grown in Dulbecco’s modified Eagle’s medium F-12 containing 10% fetal bovine serum, 10% GluMax (Invitrogen, Carlsbad, CA, USA), and penicillin and streptomycin (Invitrogen, Carlsbad, CA, USA; 100 units/mL each). HEK-293 cells were passaged once every 3 days when they reached 90% confluence. Afterward, HEK-293 cells were plated on 35 mm coverslips and maintained at 37°C (5% CO2) for 24 h before use in calcium imaging experiments.

2.5 Confocal calcium imaging

Live cell confocal imaging of primary OE cell cultures, OP6 cells, and HEK-293 cells, was performed using an Olympus Fluoview 1,000 LSM system. Cells were loaded with fluo-4 AM (Invitrogen, Carlsbad, CA, USA) via incubation for 30 min in Ringer’s solution containing 18 µM of the fluo-4 AM dye. Time series experiments were performed by collecting 256 × 128 pixel images at ≥1 Hz. ATP and Bz-ATP were applied using bath exchange and a 200 µL-volume loop injector (designated ▲ in Figure 1). The fluorometric signals obtained are expressed as relative fluorescence (F) change, ΔF/F = (F − F 0)/F 0, where F 0 is the basal fluorescence level (mean F of first ten frames). To be considered a true ATP-elicited response, a fluorescence increase had to have a peak normalized value above a cutoff of three times the standard deviation of the baseline. The cutoff was calculated for each individual cell from the baseline points preceding ATP application (imaging of primary OE cell cultures, OP6 cells, or HEK-293 cells in the absence of an OE set of turbinates) or Bz-ATP application (imaging of HEK-293 cells in the presence of a set of turbinates). During each imaging session, at least 30% of the imaged cells responded with calcium increases high enough to be considered true responses. To prevent the fluorescence increase of one cell from being detected by a neighboring cell during analysis (false positive), and only have cells with clearly defined borders when drawing region of interest for data analysis, we purposely imaged regions of the coverslips where we had a low density of cells.

Figure 1 OP6 cells and adult mouse OE primary cell cultures have functional purinergic receptors. (a–c). Representative grayscale images of (a) undifferentiated OP6 cells, (b) differentiated OP6 cells, and (c) 7–8 DIV primary adult OE cell cultures. Scale bar = 50 µm. (d–f) Representative calcium responses; each black or gray trace shows a response recorded in a single cell. Thus, traces from two different cells were superimposed in panel (f). Trace in (d): undifferentiated OP6 cells, trace in (e): differentiated OP6 cells; traces in (f): adult OE primary cell cultures. ▲, application of 50 µM ATP. n = 3 coverslips for each experiment.
Figure 1

OP6 cells and adult mouse OE primary cell cultures have functional purinergic receptors. (a–c). Representative grayscale images of (a) undifferentiated OP6 cells, (b) differentiated OP6 cells, and (c) 7–8 DIV primary adult OE cell cultures. Scale bar = 50 µm. (d–f) Representative calcium responses; each black or gray trace shows a response recorded in a single cell. Thus, traces from two different cells were superimposed in panel (f). Trace in (d): undifferentiated OP6 cells, trace in (e): differentiated OP6 cells; traces in (f): adult OE primary cell cultures. ▲, application of 50 µM ATP. n = 3 coverslips for each experiment.

2.6 Bioluminescence detection of ATP

ATP was quantified using 0.5 mM d-luciferin and 4 μg/mL luciferase in Ringer’s solution obtained from an ATP determination kit as previously described [4]. We continuously recorded luminescence (in relative light units) with 1-s photon collection intervals, using a Turner TD20/20n luminometer. On the day of the experiment, we made standard curves of ATP (Mg salt) ranging between 10 nM and 5 μM from a 0.5 M ATP stock and recorded them in 200 μL of luciferin and luciferase solution. The ATP standard curves were linear in the range of 10 nM–5 μM (r 2: 0.93–0.99). We used either whole olfactory turbinates or coverslips plated with OP6 cells. Whole olfactory turbinates were collected from 6 to 8 weeks old Swiss Webster mice as described (see Section 2.3). OP6 cells were either undifferentiated or differentiated. For each individual luciferin–luciferase assay, one set of OE turbinates (either dissected from the left side or the side right of the nose) or one OP6 cell coverslip was placed in a 200 μL bolus of luciferin and luciferase solution in a 35 mm petri dish and ATP release rates were measured for 10 min in presence or absence of inhibitors. Bz-ATP was used to trigger purine-evoked ATP release during the luciferin–luciferase assays since it was the purinergic receptor agonist that elicited the lowest amount of luminescence when applied alone in a bolus of luciferin and luciferase solution, as previously described [4]. Pilot studies showed that the luminescence baseline was stabilized at t = 10 min after a sample was put into the luciferin and luciferase solution. Therefore, all data were analyzed at t = 10 min. Raw relative light unit values were converted to ATP concentration using the standard curve (relative light units vs ATP concentration [μM]) and interpolation from a linear regression by Prism Software version 9.1.0 (Graphpad Software, San Diego, CA). At the end of each experiment, as a positive control, a bolus of Triton X-100 was added (0.5% final concentration) to release ATP via lysis of the cells.

2.7 Immunocytochemistry

Primary cell cultures (7–8 DIV) were processed for immunoreactivity to rabbit anti-calnexin (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-nestin (1:1,000; Millipore, Billerica, MA), goat anti-olfactory marker protein (OMP) (1:2,000; Wako Chemicals, Richmond, VA), or rabbit anti-phospholipase Cβ2 (PLCβ2) (1:100; Santa Cruz Biotechnology, Santa Cruz, CA). Cells were fixed in 4% paraformaldehyde for 20 min, washed with 0.1 M phosphate buffered saline (PBS), permeated with 0.05% tween 20 in 0.1 M PBS (PBST) for 10 min and blocked with 2% normal donkey serum (NDS) in PBST for 30 min. Cells were exposed to primary antibodies made in 2% NDS in PBST for 2 h at room temperature. Cy3-conjugated donkey anti-goat, fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit, or tetramethylrhodamine (TRITC)-conjugated donkey anti-mouse (1:200; Jackson ImmunoResearch Labs, West Grove, PA) was applied for 30 min at room temperature. Sections were then washed and mounted in Vectashield mounting medium for fluorescence with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Labs, Burlingame, CA) and visualized on an Olympus Fluoview 1,000 system. Cy3 dye was excited at 450 nm and low pass filtered at 560–620 nm. FITC dye was excited at 488 nm and low pass filtered at 505–525 nm. TRITC dye was excited at 543 nm and low pass filtered at 560–620 nm. DAPI was excited at 405 nm and fluorescence was detected at 430–470 nm. Antibody specificity was tested by omitting the primary antibodies or by using a peptide neutralization protocol in which the antibody (0.04 mg/mL) was combined with a 10-fold excess of the immunizing peptide (0.4 mg/mL). No immunoreactivity was observed in any of these controls. For each antibody, immunoreactive cells were tabulated based on size and optical density using the automated integrated morphometric analysis module from Metamorph software (Molecular Devices, Sunnyvale, CA) and expressed as a percent of total (DAPI-positive) cells; these cells are indicated by arrows in Figure 2. Positively-stained cells that could be dead or dying cells, or cell debris, were excluded from the tabulation (positively-stained cells not indicated by arrows in Figure 2).

Figure 2 Immature OSN, mature OSN, sustentacular cells, and microvillous cells are present in 7–8 DIV adult OE primary epithelium cell cultures. (a–d). Representative grayscale pictures of cells present in adult primary olfactory cell cultures. Each panel corresponds to a separate immunocytochemistry experiment testing one antibody and showing cells immuno-positive for the tested antibody. (a) Use of antibody against neuronal precursor marker nestin, (b) use of antibody against sustentacular cell marker calnexin, (c) use of antibody against mature olfactory neuron marker OMP, and (d) use of antibody against microvillous cell marker PLCβ2. Scale bar = 20 µm. In each panel, each arrow indicates a single immunopositive cell; n = 3 coverslips for each tested antibody.
Figure 2

Immature OSN, mature OSN, sustentacular cells, and microvillous cells are present in 7–8 DIV adult OE primary epithelium cell cultures. (a–d). Representative grayscale pictures of cells present in adult primary olfactory cell cultures. Each panel corresponds to a separate immunocytochemistry experiment testing one antibody and showing cells immuno-positive for the tested antibody. (a) Use of antibody against neuronal precursor marker nestin, (b) use of antibody against sustentacular cell marker calnexin, (c) use of antibody against mature olfactory neuron marker OMP, and (d) use of antibody against microvillous cell marker PLCβ2. Scale bar = 20 µm. In each panel, each arrow indicates a single immunopositive cell; n = 3 coverslips for each tested antibody.

2.8 Statistical analysis

We compared data sets with Prism Software version 9.1.0 (Graphpad Software, San Diego, CA). We used Student’s unpaired t-test to compare two data sets. To compare three or more data sets, if the standard deviation of any data set was not twice the value of the standard deviation of another data set, we used ordinary one-way ANOVA followed by Dunnett’s multiple comparison test to compare each data set to a control data set, or Tukey’s multiple comparison test, to make comparisons between all groups. If the standard deviation of any data set was at least twice the value of the standard deviation of any other data set, we used either Kruskal–Wallis test (if data sets failed the D’Agostino, Anderson–Darling, Shapiro–Wilk, and Kolmogorov–Smirnov normality tests) followed by Dunn’s multiple comparison test, or Brown–Forsythe and Welch ANOVA tests (if data sets did not fail the D’Agostino, Anderson–Darling, Shapiro–Wilk, and Kolmogorov–Smirnov normality tests) followed by Dunnett T3 multiple comparison test, for multiple comparisons correction [36,37]. A p value <0.05 was considered statistically significant.

3 Results

3.1 Cells in adult OE and OP6 cells express functional purinergic receptors

To conduct our study on ATP release in mouse adult OE, we decided to use adult OE tissue to characterize overall ATP release mechanisms regardless of the cell types involved, and OP6 cells to specifically characterize the contribution of OSN to ATP release. We used both undifferentiated OP6 cells that behave like immature OSN and differentiated OP6 cells that have the properties of mature OSN [33]. Part of our study requires us to determine whether an ATP-evoked ATP release mediated by stimulation of purinergic receptors, a mechanism that we characterized in neonatal mouse OE [4], is also present in adult OE. Therefore, we first verified that all preparations we were planning to use do express functional purinergic receptors, using calcium imaging. We chose adult OE primary cell cultures to determine whether adult OE as a whole does express functional purinergic receptors, since we had an established method of preparing and maintaining these cell cultures and using them for calcium imaging [38]. Application of 50 µM of non-specific purinergic receptor agonist ATP induced robust calcium responses in adult OE primary cell cultures and also in undifferentiated and differentiated OP6 cells (Figure 1; n = 3 coverslips and n = ≥7 cells for each preparation). ATP triggered an oscillatory calcium response in undifferentiated OP6 cells (Figures 1a–d) and a transient calcium response in differentiated OP6 cells (Figure 1b–e). In primary adult OE cell cultures (Figure 1c–f), ATP triggered either an oscillatory response (Figure 1f; black trace) or a transient response (Figure 1f; grey trace).

These data show that primary cell cultures from adult mouse OE tissue, undifferentiated OP6 cells, and differentiated OP6 cells all express functional purinergic receptors. While they might differ from cells present in whole intact adult OE, these preparations are suitable models to investigate what adult OE cell types express functional purinergic receptors and what cell types might contribute to purinergic receptor-mediated ATP release mechanisms in adult mouse OE. Furthermore, these results are in accordance with our previous report that both non-neuronal and neuronal cells cultured from the OE are responsive to purines [38]. However, the fact that our primary OE cell cultures responded to ATP application during calcium imaging does not show per se they contain all three cell types present in adult OE: OSN, sustentacular cells, and microvillous cells. We needed to confirm our cell cultures had all three cell types so that we could confidently conclude that OSN, sustentacular cells, and microvillous cells in adult OE express functional purinergic receptors, and that whole adult OE turbinates preparation can thus be used to study purine-evoked ATP release in subsequent experiments. To validate that our primary adult OE cell cultures had OSN, sustentacular cells, and microvillous cells, we used immunohistochemistry and tested four antibodies (one experiment for each tested antibody, and n = 3 coverslips for each tested antibody). We used antibodies against four markers: nestin, a neuronal precursor marker that would thus show the presence of the stem cells like basal cells; OMP, a marker of mature OSN; calnexin, a sustentacular cell marker; and PLCβ2, a microvillous cell marker. For each tested antibody, a fraction of the cells was immuno-positive (Table 1 and Figure 2).

Table 1

Percentage of immuno-positive cells in adult mouse OE primary cell cultures for each tested cell marker

Percentage of immuno-positive cells in OE primary cell cultures (mean ± SEM) for each tested antibody
Nestin 8.16 ± 1.37
Calnexin 33.93 ± 9.42
OMP 49.43 ± 3.04
PLCβ2 40.94 ± 11.8

These positive immunocytochemistry results indicate that cells that responded to ATP application during calcium imaging of OE primary cell cultures and thus, expressed functional purinergic receptors, included all cell types present in the OE (basal cells, OSN, sustentacular cells, and microvillous cells). Thus, functional purinergic receptors are likely present in all cell types of adult OE tissue as well. Therefore, we validated that the whole adult OE turbinates are an adequate model to study mechanisms of purinergic receptor-mediated ATP release in adult mouse OE.

3.2 Purinergic receptor-mediated ATP release is present in adult OE but not in OP6 cells

Characterization of ATP release mechanisms in adult mouse OE requires to investigate whether the evoked purinergic receptor-mediated ATP release we described in mouse neonatal OE [4] is also present in mouse adult OE cells. We quantified ATP release from whole adult OE turbinates using luciferin–luciferase assays, with 50 µM Bz-ATP as the purinergic receptor agonist. We also used both undifferentiated and differentiated OP6 cells to investigate whether immature and/or mature OSN could contribute to the evoked release of ATP. Bz-ATP was used to trigger an evoked ATP release since we previously showed that the amount of luminescence induced by application of 50 µM Bz-ATP alone is lower than the total luminescence produced by the evoked release of ATP triggered by this agonist [4]. Moreover, Bz-ATP is known to activate not only P2X7 receptors, but also several sub-types of P2Y receptors [39–42]. Statistical analysis of our data using one-way ANOVA followed by Dunnett’s multiple comparison showed the amount of bioluminescence measured when the agonist was applied in absence of cells or tissue was not significantly different compared to conditions where undifferentiated or differentiated OP6 were present (Figure 3a; p > 0.05). In contrast, the amount of bioluminescence detected when Bz-ATP was applied in presence of a set of turbinates was significantly higher compared to Bz-ATP alone (Figure 3a; p < 0.05), compared to Bz-ATP in presence of undifferentiated OP6 cells (Figure 3a; p < 0.05), and compared to Bz-ATP in presence of differentiated OP6 cells (Figure 3a; p < 0.05). For these experiments, the n value is ≥10 luciferin–luciferase assays for each group. To confirm that Bz-ATP can elicit an evoked release of ATP in adult OE turbinates, we ran experiments where we recorded calcium responses from transfected human embryonic kidney cells stably expressing P2X2 and used as ATP biosensors (n = 3 coverslips of biosensors and n ≥ 16 biosensors for each set of experiments). To show that the biosensors respond to the presence of ATP, calcium responses were recorded when they were perfused with varying ATP concentrations (50 nM, 500 nM, 1 µM, and 5 µM). The biosensors responded to all tested ATP concentrations (Figure 3b). Statistical analysis with one-way ANOVA followed by Tukey’s multiple comparison test showed the mean normalized fluorescence in presence of 5 µM ATP was significantly higher compared to 50 nM ATP (Figure 3b; F (33,37) = 1.750; p < 0.05), compared to 500 nM ATP (Figure 3b; F (33,60) = 1.630; p < 0.05), and compared to 1 µM ATP (Figure 3b; F (33,16) = 2.178; P < 0.05). We then ran experiments where we imaged one coverslip of biosensors after application of 50 µM Bz-ATP alone, application of 50 µM Bz-ATP in presence of adult OE turbinates, application of 50 µM Bz-ATP in presence of one coverslip of undifferentiated OP6 cells, and application of 50 µM Bz-ATP in presence of one coverslip of differentiated OP6 cells. No calcium responses were triggered in the biosensors when Bz-ATP was applied alone, or in presence of undifferentiated or differentiated OP6 cells (Figure 3b). However, when Bz-ATP was applied in presence of turbinates, the biosensors responded with calcium increases, indicating that they detected ATP released by the turbinates following activation of the purinergic receptors located on the OE cells. Overall, the mean normalized fluorescence recorded in presence of the purinergic receptor-mediated ATP release was only significantly different when compared to the mean normalized fluorescence in presence of 5 µM of ATP (Figure 3b; F (33,87) = 1.570; p < 0.05). Thus, the amount of ATP released by the adult OE turbinates when purinergic receptors were activated by Bz-ATP ranges from 50 nM to 1 µM.

Figure 3 ATP-evoked release of ATP is present in adult OE but not in OP6 cells. (a) The luciferin and luciferase assay was used to quantify the amount of ATP (in nM; mean + SEM) released by undifferentiated OP6 cells (n = 18 assays; green triangles), differentiated OP6 cells (n = 19 assays; green squares), and adult OE turbinates (n = 10 assays; green circles) in presence of 50 µM purinergic receptor agonist Bz-ATP. Application of 50 µM Bz-ATP alone was used as a control (n = 19 assays; red circles). *: p < 0.05 vs 50 µM Bz-ATP (one-way ANOVA followed by Dunnett’s multiple comparison test). (b) Normalized fluorescence increase (mean + SEM) recorded during calcium imaging of transfected human embryonic kidney cells stably expressing P2X2 and used as ATP biosensors. Calcium responses were triggered in the biosensors by application of several ATP concentrations (red circles) and also by application of 50 µM Bz-ATP in presence of adult OE turbinates (n = 3 turbinates; green circles). Application of 50 µM Bz-ATP in absence of turbinates or in presence of undifferentiated or differentiated OP6 cells (n = 3 coverslips) did not trigger calcium responses in the biosensors. *: p < 0.05 vs any other mean (one-way ANOVA followed by Tukey’s multiple comparison test).
Figure 3

ATP-evoked release of ATP is present in adult OE but not in OP6 cells. (a) The luciferin and luciferase assay was used to quantify the amount of ATP (in nM; mean + SEM) released by undifferentiated OP6 cells (n = 18 assays; green triangles), differentiated OP6 cells (n = 19 assays; green squares), and adult OE turbinates (n = 10 assays; green circles) in presence of 50 µM purinergic receptor agonist Bz-ATP. Application of 50 µM Bz-ATP alone was used as a control (n = 19 assays; red circles). *: p < 0.05 vs 50 µM Bz-ATP (one-way ANOVA followed by Dunnett’s multiple comparison test). (b) Normalized fluorescence increase (mean + SEM) recorded during calcium imaging of transfected human embryonic kidney cells stably expressing P2X2 and used as ATP biosensors. Calcium responses were triggered in the biosensors by application of several ATP concentrations (red circles) and also by application of 50 µM Bz-ATP in presence of adult OE turbinates (n = 3 turbinates; green circles). Application of 50 µM Bz-ATP in absence of turbinates or in presence of undifferentiated or differentiated OP6 cells (n = 3 coverslips) did not trigger calcium responses in the biosensors. *: p < 0.05 vs any other mean (one-way ANOVA followed by Tukey’s multiple comparison test).

These data confirm that a purinergic receptor-mediated evoked release of ATP does occur in adult OE. Undifferentiated and differentiated OP6 cells might differ from immature and mature OSN present in whole OE. Still, our data combining use of biosensors and OP6 cells still raise the possibility that immature and mature OSN do not contribute to the purinergic receptor-mediated ATP release present in adult mouse OE.

3.3 Characterization of the constitutive ATP release present in OP6 cells

Lack of purinergic receptor-mediated ATP release in OP6 cells does not mean absence of ATP release from these cells. Indeed, we showed the presence of a constitutive ATP release occurring in neonatal OE and driven by constitutive calcium-independent exocytosis, connexin and/or pannexin1 hemichannels, and ATP binding cassette (ABC) transporters [4]. To investigate whether similar mechanisms are present in OP6 cells, we used luciferin–luciferase assays. During these assays, coverslips of either undifferentiated or differentiated OP6 cells were placed in control Ringer’s solution, Ringer’s solution containing 0.5 nM vesicular fusion inhibitor Clostridium difficile toxin A, Ringer’s solution containing 100 µM of non-specific connexin and pannexin inhibitor carbenoxolone, or Ringer’s solution containing 500 µM probenecid, an inhibitor of pannexin1 hemichannels and of ABC transporters (n ≥ 3 coverslips for each group of experiments). Statistical analysis using Brown–Forsythe and Welch ANOVA tests followed by Dunnett T3 multiple comparison showed carbenoxolone was the only treatment to significantly impact the amount of bioluminescence, inducing a decrease compared to control Ringer’s conditions (Figure 4a; p < 0.05). Similar results were observed when we used differentiated OP6 cells (Figure 4b; p < 0.05). Overall, these data obtained from OP6 cells raises the possibility that constitutive ATP release does occur in immature and mature OSN in adult OE, via ATP efflux through connexin hemichannels. Additionally, the amount of ATP released in control conditions from undifferentiated OP6 cells was significantly lower than the amount of ATP released in control conditions from differentiated cells (2.3 ± 0.25 vs 13.26 ± 1.35; p < 0.05, unpaired two-tailed Student’s t-test) indicating that OSN release more ATP as they mature. The difference in the responsiveness to carbenoxolone and probenecid treatments between panels a and b could be explained by the difference in OP6 cell maturation.

Figure 4 Constitutive ATP release characteristics in undifferentiated and differentiated OP6 cells. The luciferin and luciferase assay was used to quantify the amount of ATP (in nM; mean + SEM) released by undifferentiated OP6 cells (n = 3 assays for each group; a) and differentiated OP6 cells (n = 3–6 assays for each group; b) in absence of any inhibitors (control; red circles), in presence of 0.5 nM vesicular fusion inhibitor Clostridium difficile toxin A (green circles), 100 µM carbenoxolone, an inhibitor of connexin hemichannels and pannexin channels (green triangles), or 500 µM ABC transporter inhibitor probenecid (green squares). *: p < 0.05 vs control (Brown–Forsythe and Welch ANOVA tests followed by Dunnett T3 multiple comparison test).
Figure 4

Constitutive ATP release characteristics in undifferentiated and differentiated OP6 cells. The luciferin and luciferase assay was used to quantify the amount of ATP (in nM; mean + SEM) released by undifferentiated OP6 cells (n = 3 assays for each group; a) and differentiated OP6 cells (n = 3–6 assays for each group; b) in absence of any inhibitors (control; red circles), in presence of 0.5 nM vesicular fusion inhibitor Clostridium difficile toxin A (green circles), 100 µM carbenoxolone, an inhibitor of connexin hemichannels and pannexin channels (green triangles), or 500 µM ABC transporter inhibitor probenecid (green squares). *: p < 0.05 vs control (Brown–Forsythe and Welch ANOVA tests followed by Dunnett T3 multiple comparison test).

3.4 Characterization of the constitutive and evoked ATP release present in adult mouse OE turbinates

Results obtained from luciferin–luciferase assays and use of biosensors showed that Bz-ATP elicited a purinergic receptor-mediated evoked release of ATP in adult mouse OE turbinates. However, constitutive release of ATP might also be present in adult mouse OE, similar to what occurs in neonatal mouse OE [4], and could be detected via luciferin–luciferase assays. We used the same experimental conditions as the one used for the OP6 cells study (Figure 5a), with one set of turbinates per assay, and n ≥ 5 assays for each group. Statistical analysis using Kruskal–Wallis test showed that 500 µM probenecid was the only treatment to significantly impact the mean bioluminescence, significantly decreasing it compared to control Ringer’s condition (Figure 5a; p < 0.05). Therefore, the overall constitutive release of ATP occurring in adult mouse OE through all involved cell types is mediated by ABC transporters. We then aimed to characterize the precise mechanisms of the evoked release of ATP that we revealed in adult mouse OE turbinates. The ATP-evoked ATP release we characterized in mouse neonatal OE occurs via several mechanisms: (a) calcium influx through activated P2X7 receptors and followed by calcium-dependent exocytosis (b) through activated P2X7 receptor–pannexin1 complexes, and (c) via calcium-dependent exocytosis following P2Y purinergic receptor stimulation [4]. We used luciferin–luciferase assays to test whether similar mechanisms underlie the evoked release of ATP triggered by Bz-ATP in adult mouse OE. We hypothesized that the evoked release of ATP would be inhibited by use of vesicular fusion inhibitor Clostridium difficile toxin A, use of calcium-free Ringer’s solution, use of Ringer’s solution containing non-specific connexin and pannexin inhibitor carbenoxolone, or Ringer’s solution containing probenecid, a specific inhibitor of pannexin1 hemichannels. For each assay, a single set of turbinates was put into Ringer’s solution and stimulated with 50 µM Bz-ATP in presence or absence of one of the inhibitors, and application of 50 µM Bz-ATP in absence of turbinates was used as control assays (n ≥ 5 assays for each group). Statistical analysis using Brown–Forsythe and Welch ANOVA tests followed by Dunnett T3 multiple comparison test showed stimulation of turbinates with Bz-ATP in absence of any inhibitor significantly increased the amount of bioluminescence compared to control (Figure 5b; p < 0.05). Use of 0.5 nM Clostridium difficile toxin A significantly decreased the amount of bioluminescence compared to application of Bz-ATP in presence of tissue (Figure 5b; p < 0.05). Use of calcium-free Ringer’s had a similar effect (Figure 5b; p < 0.05), as well as use of 100 µM carbenoxolone (Figure 5b; p < 0.05), or 500 µM probenecid (Figure 5b; p < 0.05).

Figure 5 Constitutive and ATP-evoked ATP release characteristics in adult whole OE turbinates. (a) The luciferin and luciferase assay was used to quantify the amount of ATP (in nM; mean + SEM) released from adult turbinates by the constitutive release of ATP in absence of any inhibitors (control red circles), in presence of 0.5 nM vesicular fusion inhibitor Clostridium difficile toxin A (green circles), 100 µM carbenoxolone, an inhibitor of connexin hemichannels and pannexin channels (green triangles), or 500 µM ABC transporter inhibitor probenecid (green squares). n = 5–6 assays for each group. *: p < 0.05 vs control (Kruskal–Wallis test). (b) The luciferin and luciferase assay was used to quantify the amount of ATP (in nM; mean + SEM) released from adult turbinates by the evoked release of ATP triggered by application of 50 µM Bz-ATP (green symbols). Essays were done in Ringer’s solution alone (50 µM Bz-ATP; green circles), in presence of 0.5 nM vesicular fusion inhibitor Clostridium difficile toxin A (green triangles), calcium-free Ringer’s solution (green squares), 100 µM carbenoxolone, an inhibitor of non-specific pannexin and activated P2X7 receptor inhibitor (green inverted triangles), or 500 µM selective pannexin 1 channel and ABC transporter inhibitor probenecid (green diamonds). Application of 50 µM Bz-ATP in Ringer’s solution in absence of adult turbinates was used as a control (red circles). n = 5–11 assays for each group. *: p < 0.05 vs control (Brown–Forsythe and Welch ANOVA tests followed by Dunnett T3 multiple comparison test).
Figure 5

Constitutive and ATP-evoked ATP release characteristics in adult whole OE turbinates. (a) The luciferin and luciferase assay was used to quantify the amount of ATP (in nM; mean + SEM) released from adult turbinates by the constitutive release of ATP in absence of any inhibitors (control red circles), in presence of 0.5 nM vesicular fusion inhibitor Clostridium difficile toxin A (green circles), 100 µM carbenoxolone, an inhibitor of connexin hemichannels and pannexin channels (green triangles), or 500 µM ABC transporter inhibitor probenecid (green squares). n = 5–6 assays for each group. *: p < 0.05 vs control (Kruskal–Wallis test). (b) The luciferin and luciferase assay was used to quantify the amount of ATP (in nM; mean + SEM) released from adult turbinates by the evoked release of ATP triggered by application of 50 µM Bz-ATP (green symbols). Essays were done in Ringer’s solution alone (50 µM Bz-ATP; green circles), in presence of 0.5 nM vesicular fusion inhibitor Clostridium difficile toxin A (green triangles), calcium-free Ringer’s solution (green squares), 100 µM carbenoxolone, an inhibitor of non-specific pannexin and activated P2X7 receptor inhibitor (green inverted triangles), or 500 µM selective pannexin 1 channel and ABC transporter inhibitor probenecid (green diamonds). Application of 50 µM Bz-ATP in Ringer’s solution in absence of adult turbinates was used as a control (red circles). n = 5–11 assays for each group. *: p < 0.05 vs control (Brown–Forsythe and Welch ANOVA tests followed by Dunnett T3 multiple comparison test).

Our results show that the purinergic receptor-mediated evoked release of ATP in adult mouse OE occurs via mechanisms similar to part of the evoked release of ATP occurring in neonatal mouse OE. Namely, calcium influx through activated P2X7 receptors followed by calcium-dependent exocytosis of ATP, and ATP efflux through activated P2X7 receptor/pannexin1 complexes [4].

4 Discussion

Constitutive release of ATP occurs under physiological conditions in many organ systems [43]. Exogenous ATP, a simulation of injury, evokes the release of ATP in many cell types [20–23]. We previously revealed the presence of both constitutive and ATP-evoked ATP release in neonatal mouse OE [4] and extensively demonstrated how both types of ATP release play a role in constant mouse OE homeostasis and post-injury neuroregeneration, including ATP-induced release of other neurotrophic factors [2,4,11,12,14–18]. The evoked release of ATP in neonatal mouse OE includes pathways downstream of P2X7 and P2Y purinergic receptors stimulation [4]. We hypothesized that the adult mouse OE has mechanisms of ATP release similar to the ones present in neonatal mouse OE (e.g., constitutive and activated purinergic receptor-evoked ATP release) and tested this hypothesis using luciferin–luciferase assays and ATP biosensors. We did reveal the presence of both constitutive and activated purinergic receptor-mediated ATP release in adult mouse OE whole turbinate preparations. This is in accordance with our calcium imaging data showing the presence of functional purinergic receptors in adult OE primary cell cultures. This preparation is adequate to study calcium responses in adult mouse OE, since immunostaining showed all OE cell types in our cell cultures. We also investigated the contribution of a specific OE cell type, the OSN, to both releases of ATP, by using the OP6 cell line. Undifferentiated OP6 cells are similar to immature OSN, and differentiated OP6 cells are similar to mature OSN. Both immature and mature OSN displayed constitutive ATP release, but lacked a purinergic receptor-mediated one during our experiments; this absence is not due to absence of functional purinergic receptors in OP6 cells. Indeed, calcium imaging showed that both undifferentiated and differentiated OP6 cells do respond to ATP application, like our adult primary OE cell cultures. Therefore, either OSN only constitutively release ATP, or their putative evoked release of ATP does not involve receptors that can be activated by Bz-ATP.

When we investigated the precise mechanisms underlying both types of ATP release, we showed that the constitutive release of ATP in immature and mature OSN occurs via connexin and/or pannexin hemichannels. It is in accordance with the presence of both connexin and pannexin hemichannels in the central nervous system [44]. Furthermore, hemichannels are present in the OE [45] and it has been shown that both mature and immature OSN express the connexin 43 subunit [46]. Surprisingly, the constitutive release of ATP in our adult OE turbinate preparations was not inhibited by carbenoxolone, a non-specific connexin and pannexin inhibitor. An explanation is that the ATP released by OSN in adult mouse OE represents a minimal amount of the total ATP being released by the whole OE. Thus, the carbenoxolone treatment would not inhibit enough ATP release to have a significant effect. Our data support this explanation, since probenecid, which inhibits ABC transporters, significantly inhibited the constitutive release of ATP in the OE turbinate preparations. It shows that the main mechanism underlying the constitutive release of ATP in adult OE is ATP efflux through ABC transporters. It is coherent with the fact that we showed the same mechanism in neonatal mouse OE [4]. It was surprising that vesicular fusion inhibitor Clostridium difficile toxin A did not inhibit the constitutive ATP release in adult mouse OE. Indeed, we showed this inhibitor blocked part of the constitutive ATP release in neonatal mouse OE happening via constitutive calcium-independent exocytosis [4], a type of exocytosis that has been documented in other models as well [47–49]. This discrepancy can be explained by changes in mechanisms of ATP release when the OE matures from neonatal to adult. Indeed, it has been shown that exocytosis might be affected by age-related changes [50,51]. Moreover, our data from OP6 cells showed that OSN release more ATP as they mature, further supporting this explanation. Our results showed the evoked release of ATP occurring after purinergic receptor stimulation in adult OE shares mechanisms with the one present in neonatal mouse OE [4]: influx of calcium ions through activated P2X7 receptors followed by calcium-dependent exocytosis, and efflux of ATP through activated P2X7–pannexin1 complexes. Indeed, it was inhibited by use of (a) vesicular fusion inhibitor Clostridium difficile toxin A and calcium-free Ringer’s, conditions that inhibit calcium influx and calcium-dependent exocytosis and (b) use of non-specific connexin and pannexin inhibitor carbenoxolone and use of selective pannexin1 inhibitor probenecid, conditions that inhibit activated P2X7–pannexin1 complexes. While Bz-ATP can also activate some P2Y receptors besides P2X7 receptors, the fact that use of calcium-free Ringer’s totally abolished the evoked release of ATP suggests that this release occurs in adult OE solely via P2X7 receptors and does not involve P2Y receptors. Bz-ATP would still have induced a significant release of ATP in calcium-free Ringer’s if part of the evoked release of ATP in adult OE was mediated by calcium-dependent exocytosis of ATP following P2Y purinergic receptor stimulation. This is surprising since we showed P2Y receptor stimulation is involved in the evoked release of ATP occurring in neonatal mouse OE [4]. An explanation is that the mechanisms underlying evoked ATP release change during maturation of the OE. Another explanation is that the P2Y receptors involved in the evoked ATP release in adult OE are not activated by Bz-ATP. Further studies using P2Y receptor agonists besides Bz-ATP are required to explain this discrepancy between neonatal and adult evoked ATP release. Moreover, calcium influx through other types of activated P2X receptor subtypes besides P2X7 might also contribute to the evoked ATP release in adult mouse OE, since multiple P2X and P2Y receptor subtypes have been reported in mouse OE [2–4].

Now, a burning question is: what OE cell types are involved in constitutive and/or evoked ATP release in adult mouse OE? As shown in Figure 6, glial-like sustentacular cells are located in the apical layer of the OE; the OSN are located in the OE middle layer; and the basal layer of the OE houses the horizontal and the basal cells of the OE. The OE middle layer also houses microvillous cells. While their exact function is still unknown, we previously showed that, in neonatal OE, these cells express both IP3R3 receptors and neurotrophic factor NPY and that they are involved in tissue homeostasis and regeneration [18].

Figure 6 Schematic of the organization of the OE. The cell bodies of the glial-like sustentacular cells make the apical layer of the OE; the cell bodies of the OSN make the middle layer of the OE; and the stem cells-like horizontal and basal cells make the basal layer of the OE. All cell types express P2X and P2Y purinergic receptors.
Figure 6

Schematic of the organization of the OE. The cell bodies of the glial-like sustentacular cells make the apical layer of the OE; the cell bodies of the OSN make the middle layer of the OE; and the stem cells-like horizontal and basal cells make the basal layer of the OE. All cell types express P2X and P2Y purinergic receptors.

Could sustentacular cells, microvillous cells, and OSN all be involved in the ATP release mechanisms we describe in the present study and summarize in Figure 7?

Figure 7 Schematic of mechanisms underlying the evoked ATP release and the constitutive ATP release. Extracellular ATP, leaking from damaged cells or released constitutively, activates P2X7 purinergic receptors, leading to Ca2+ influx into the cell via activated P2X7 receptors and subsequent Ca2+-dependent exocytosis of ATP. Activated P2X7 receptors also form a complex with pannexin hemichannels through which ATP can efflux. The constitutive ATP release is mediated by ABC transporters. Inhibitors (−) and stimulators (+) are indicated in the figure.
Figure 7

Schematic of mechanisms underlying the evoked ATP release and the constitutive ATP release. Extracellular ATP, leaking from damaged cells or released constitutively, activates P2X7 purinergic receptors, leading to Ca2+ influx into the cell via activated P2X7 receptors and subsequent Ca2+-dependent exocytosis of ATP. Activated P2X7 receptors also form a complex with pannexin hemichannels through which ATP can efflux. The constitutive ATP release is mediated by ABC transporters. Inhibitors (−) and stimulators (+) are indicated in the figure.

Our data rule out the presence of ATP efflux via ABC transporters in OSN but also shows that the main amount of constitutively released ATP from whole OE turbinates comes from ABC transporters action. Thus, the ABC transporter-mediated mechanism of constitutive ATP release has to occur in sustentacular and/or microvillous cells. The lack of ATP release mediated by Bz-ATP in OP6 cells means that sustentacular cells and/or microvillous are most likely responsible of the evoked release of ATP present in adult mouse OE. The fact that RNAseq databases showed P2rx7 is a non-OSN gene [52] supports this theory. It would make sense since astrocytes are known to release ATP via exocytosis [53,54] and sustentacular cells are the glia-like cells of the OE. Moreover, it has been well documented that vesicles, dense bodies, and secretory granules are present in the cytosol of sustentacular cells [55–59]. Sustentacular cells also display calcium store-mediated signaling [60]. The microvillous cells might also contribute to the evoked release of ATP via calcium-dependent exocytosis. They express IP3R3 receptors, a component of calcium-dependent exocytosis, and release neurotrophic factor NPY upon stimulation with ATP and UTP, two purinergic receptor agonists that lead to intracellular calcium increase, a requirement for calcium-dependent exocytosis [15,18]. Use of transgenic mice where specific reporters are used to label a specific OE cell type, like the IP3R3-tauGFP mouse we used in a previous study [18], are tools that could allow to precisely identify what OE cell type is involved in the constitutive and/or the evoked ATP release.

Our results suggest that sustentacular and/or microvillous cells are mostly responsible for both the constitutive and evoked release of ATP, OSN only minimally contributing to the constitutive release of ATP. Our results show that P2X7 receptors are definitely involved in the evoked release of ATP present in adult OE. Other purinergic receptors, including P2Y receptors might be involved as well. We are well aware of the limitations of our study. Our OP6 cells are a model to study OSN but are not OSN per se. Likewise, our primary OE cell cultures are not undissociated adult OE tissue per se. Also, probenecid inhibits both ABC transporters and pannexin hemichannels. If, in our characterization of the constitutive ATP release, probenecid and carbenoxolone would have inhibited the same target, the data sets for carbenoxolone and probenecid would have been similar in both panels of Figure 4 and in panel a of Figure 5. However, our luciferin–luciferase assays showed the effects of carbenoxolone and probenecid on the constitutive ATP release differ, enabling us to propose connexin hemichannels activity underlie the constitutive ATP release in OP6 cells, while ABC transporters activity is mainly responsible for the constitutive ATP release in whole adult OE. Also, the fact both probenecid and carbenoxolone target and inhibit the activated P2X7 receptor/pannexin hemichannel complex during the evoked release of ATP was not detrimental to our study, since it enabled us to confirm this complex is indeed involved in the evoked ATP release in adult mouse OE. We did calcium imaging of primary OE cell cultures because we do not have expertise in calcium imaging of whole intact adult OE turbinates, a method that has been successfully used by other laboratories [61]. Despite these limitations, our study revealed the presence of constitutive and purinergic receptor-mediated ATP release mechanisms in adult mouse OE. We thus paved the way for future projects aiming to characterize even more the mechanisms of ATP release in adult mouse OE, which could involve intact adult OE turbinates calcium imaging. Olfactory ensheathing glia (OEG) is another cell type that might also contribute to ATP release and the cell turnover and post-injury neuroregeneration mechanisms present in adult mouse OE. Indeed, these cells are found in association with the olfactory nerve [62]. Moreover, these cells contribute to regeneration of the primary olfactory nervous system and OEG transplantation promotes regeneration in the spinal cord, the visual system, and the central nervous system [63–66]. Thus, the putative contribution of OEG to the constitutive and/or evoked release of ATP present in adult mouse OE should be investigated. These future studies are required to determine what adult mouse OE cell type is involved in each kind of release, what purinergic receptor subtypes activate the evoked release of ATP, and how the two ATP releases are involved in OE homeostasis and post-injury neuroregeneration.

tel: +1-786 637-8121

Acknowledgements

P2X2-transfected human embryonic kidney cells used as biosensors were generously provided by Dr James Galligan from Michigan State University. The OP6 cell line was generously provided by Dr Mary Lucero from University of Utah.

  1. Funding information: This work was supported by National Institute on Deafness and Other Communication Disorders (NIDCD) (grant RO1DC006897).

  2. Author contributions: S.H., C.J., and C.C.H. designed the study, performed the experiments, and analyzed the data. S.H. made the final version of the figures and prepared the manuscript for publication. All authors approved the final version of the manuscript.

  3. Conflict of interest: Authors state no conflict of interest.

  4. Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Received: 2023-09-06
Revised: 2023-10-31
Accepted: 2023-11-28
Published Online: 2024-01-16

© 2024 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/biol-2022-0811
Keywords for this article
primary cell cultures; OP6 cells; confocal imaging; immunohistochemistry; luciferin–luciferase assay
Creative Commons
BY 4.0

Articles in the same Issue

  1. Biomedical Sciences
  2. Constitutive and evoked release of ATP in adult mouse olfactory epithelium
  3. LARP1 knockdown inhibits cultured gastric carcinoma cell cycle progression and metastatic behavior
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  12. Effectiveness and safety of a mumps containing vaccine in preventing laboratory-confirmed mumps cases from 2002 to 2017: A meta-analysis
  13. Low levels of sex hormone-binding globulin predict an increased breast cancer risk and its underlying molecular mechanisms
  14. A case of Trousseau syndrome: Screening, detection and complication
  15. Application of the integrated airway humidification device enhances the humidification effect of the rabbit tracheotomy model
  16. Preparation of Cu2+/TA/HAP composite coating with anti-bacterial and osteogenic potential on 3D-printed porous Ti alloy scaffolds for orthopedic applications
  17. Aquaporin-8 promotes human dermal fibroblasts to counteract hydrogen peroxide-induced oxidative damage: A novel target for management of skin aging
  18. Current research and evidence gaps on placental development in iron deficiency anemia
  19. Single-nucleotide polymorphism rs2910829 in PDE4D is related to stroke susceptibility in Chinese populations: The results of a meta-analysis
  20. Pheochromocytoma-induced myocardial infarction: A case report
  21. Kaempferol regulates apoptosis and migration of neural stem cells to attenuate cerebral infarction by O‐GlcNAcylation of β-catenin
  22. Sirtuin 5 regulates acute myeloid leukemia cell viability and apoptosis by succinylation modification of glycine decarboxylase
  23. Apigenin 7-glucoside impedes hypoxia-induced malignant phenotypes of cervical cancer cells in a p16-dependent manner
  24. KAT2A changes the function of endometrial stromal cells via regulating the succinylation of ENO1
  25. Current state of research on copper complexes in the treatment of breast cancer
  26. Exploring antioxidant strategies in the pathogenesis of ALS
  27. Helicobacter pylori causes gastric dysbacteriosis in chronic gastritis patients
  28. IL-33/soluble ST2 axis is associated with radiation-induced cardiac injury
  29. The predictive value of serum NLR, SII, and OPNI for lymph node metastasis in breast cancer patients with internal mammary lymph nodes after thoracoscopic surgery
  30. Carrying SNP rs17506395 (T > G) in TP63 gene and CCR5Δ32 mutation associated with the occurrence of breast cancer in Burkina Faso
  31. P2X7 receptor: A receptor closely linked with sepsis-associated encephalopathy
  32. Probiotics for inflammatory bowel disease: Is there sufficient evidence?
  33. Identification of KDM4C as a gene conferring drug resistance in multiple myeloma
  34. Microbial perspective on the skin–gut axis and atopic dermatitis
  35. Thymosin α1 combined with XELOX improves immune function and reduces serum tumor markers in colorectal cancer patients after radical surgery
  36. Highly specific vaginal microbiome signature for gynecological cancers
  37. Sample size estimation for AQP4-IgG seropositive optic neuritis: Retinal damage detection by optical coherence tomography
  38. The effects of SDF-1 combined application with VEGF on femoral distraction osteogenesis in rats
  39. Fabrication and characterization of gold nanoparticles using alginate: In vitro and in vivo assessment of its administration effects with swimming exercise on diabetic rats
  40. Mitigating digestive disorders: Action mechanisms of Mediterranean herbal active compounds
  41. Distribution of CYP2D6 and CYP2C19 gene polymorphisms in Han and Uygur populations with breast cancer in Xinjiang, China
  42. VSP-2 attenuates secretion of inflammatory cytokines induced by LPS in BV2 cells by mediating the PPARγ/NF-κB signaling pathway
  43. Factors influencing spontaneous hypothermia after emergency trauma and the construction of a predictive model
  44. Long-term administration of morphine specifically alters the level of protein expression in different brain regions and affects the redox state
  45. Application of metagenomic next-generation sequencing technology in the etiological diagnosis of peritoneal dialysis-associated peritonitis
  46. Clinical diagnosis, prevention, and treatment of neurodyspepsia syndrome using intelligent medicine
  47. Case report: Successful bronchoscopic interventional treatment of endobronchial leiomyomas
  48. Preliminary investigation into the genetic etiology of short stature in children through whole exon sequencing of the core family
  49. Cystic adenomyoma of the uterus: Case report and literature review
  50. Mesoporous silica nanoparticles as a drug delivery mechanism
  51. Dynamic changes in autophagy activity in different degrees of pulmonary fibrosis in mice
  52. Vitamin D deficiency and inflammatory markers in type 2 diabetes: Big data insights
  53. Lactate-induced IGF1R protein lactylation promotes proliferation and metabolic reprogramming of lung cancer cells
  54. Meta-analysis on the efficacy of allogeneic hematopoietic stem cell transplantation to treat malignant lymphoma
  55. Mitochondrial DNA drives neuroinflammation through the cGAS-IFN signaling pathway in the spinal cord of neuropathic pain mice
  56. Application value of artificial intelligence algorithm-based magnetic resonance multi-sequence imaging in staging diagnosis of cervical cancer
  57. Embedded monitoring system and teaching of artificial intelligence online drug component recognition
  58. Investigation into the association of FNDC1 and ADAMTS12 gene expression with plumage coloration in Muscovy ducks
  59. Yak meat content in feed and its impact on the growth of rats
  60. A rare case of Richter transformation with breast involvement: A case report and literature review
  61. First report of Nocardia wallacei infection in an immunocompetent patient in Zhejiang province
  62. Rhodococcus equi and Brucella pulmonary mass in immunocompetent: A case report and literature review
  63. Downregulation of RIP3 ameliorates the left ventricular mechanics and function after myocardial infarction via modulating NF-κB/NLRP3 pathway
  64. Evaluation of the role of some non-enzymatic antioxidants among Iraqi patients with non-alcoholic fatty liver disease
  65. The role of Phafin proteins in cell signaling pathways and diseases
  66. Ten-year anemia as initial manifestation of Castleman disease in the abdominal cavity: A case report
  67. Coexistence of hereditary spherocytosis with SPTB P.Trp1150 gene variant and Gilbert syndrome: A case report and literature review
  68. Utilization of convolutional neural networks to analyze microscopic images for high-throughput screening of mesenchymal stem cells
  69. Exploratory evaluation supported by experimental and modeling approaches of Inula viscosa root extract as a potent corrosion inhibitor for mild steel in a 1 M HCl solution
  70. Imaging manifestations of ductal adenoma of the breast: A case report
  71. Gut microbiota and sleep: Interaction mechanisms and therapeutic prospects
  72. Isomangiferin promotes the migration and osteogenic differentiation of rat bone marrow mesenchymal stem cells
  73. Prognostic value and microenvironmental crosstalk of exosome-related signatures in human epidermal growth factor receptor 2 positive breast cancer
  74. Circular RNAs as potential biomarkers for male severe sepsis
  75. Knockdown of Stanniocalcin-1 inhibits growth and glycolysis in oral squamous cell carcinoma cells
  76. The expression and biological role of complement C1s in esophageal squamous cell carcinoma
  77. A novel GNAS mutation in pseudohypoparathyroidism type 1a with articular flexion deformity: A case report
  78. Predictive value of serum magnesium levels for prognosis in patients with non-small cell lung cancer undergoing EGFR-TKI therapy
  79. HSPB1 alleviates acute-on-chronic liver failure via the P53/Bax pathway
  80. IgG4-related disease complicated by PLA2R-associated membranous nephropathy: A case report
  81. Baculovirus-mediated endostatin and angiostatin activation of autophagy through the AMPK/AKT/mTOR pathway inhibits angiogenesis in hepatocellular carcinoma
  82. Metformin mitigates osteoarthritis progression by modulating the PI3K/AKT/mTOR signaling pathway and enhancing chondrocyte autophagy
  83. Evaluation of the activity of antimicrobial peptides against bacterial vaginosis
  84. Atypical presentation of γ/δ mycosis fungoides with an unusual phenotype and SOCS1 mutation
  85. Analysis of the microecological mechanism of diabetic kidney disease based on the theory of “gut–kidney axis”: A systematic review
  86. Omega-3 fatty acids prevent gestational diabetes mellitus via modulation of lipid metabolism
  87. Refractory hypertension complicated with Turner syndrome: A case report
  88. Interaction of ncRNAs and the PI3K/AKT/mTOR pathway: Implications for osteosarcoma
  89. Association of low attenuation area scores with pulmonary function and clinical prognosis in patients with chronic obstructive pulmonary disease
  90. Long non-coding RNAs in bone formation: Key regulators and therapeutic prospects
  91. The deubiquitinating enzyme USP35 regulates the stability of NRF2 protein
  92. Neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio as potential diagnostic markers for rebleeding in patients with esophagogastric variceal bleeding
  93. G protein-coupled receptor 1 participating in the mechanism of mediating gestational diabetes mellitus by phosphorylating the AKT pathway
  94. LL37-mtDNA regulates viability, apoptosis, inflammation, and autophagy in lipopolysaccharide-treated RLE-6TN cells by targeting Hsp90aa1
  95. The analgesic effect of paeoniflorin: A focused review
  96. Chemical composition’s effect on Solanum nigrum Linn.’s antioxidant capacity and erythrocyte protection: Bioactive components and molecular docking analysis
  97. Knockdown of HCK promotes HREC cell viability and inner blood–retinal barrier integrity by regulating the AMPK signaling pathway
  98. The role of rapamycin in the PINK1/Parkin signaling pathway in mitophagy in podocytes
  99. Laryngeal non-Hodgkin lymphoma: Report of four cases and review of the literature
  100. Clinical value of macrogenome next-generation sequencing on infections
  101. Overview of dendritic cells and related pathways in autoimmune uveitis
  102. TAK-242 alleviates diabetic cardiomyopathy via inhibiting pyroptosis and TLR4/CaMKII/NLRP3 pathway
  103. Hypomethylation in promoters of PGC-1α involved in exercise-driven skeletal muscular alterations in old age
  104. Profile and antimicrobial susceptibility patterns of bacteria isolated from effluents of Kolladiba and Debark hospitals
  105. The expression and clinical significance of syncytin-1 in serum exosomes of hepatocellular carcinoma patients
  106. A histomorphometric study to evaluate the therapeutic effects of biosynthesized silver nanoparticles on the kidneys infected with Plasmodium chabaudi
  107. PGRMC1 and PAQR4 are promising molecular targets for a rare subtype of ovarian cancer
  108. Analysis of MDA, SOD, TAOC, MNCV, SNCV, and TSS scores in patients with diabetes peripheral neuropathy
  109. SLIT3 deficiency promotes non-small cell lung cancer progression by modulating UBE2C/WNT signaling
  110. The relationship between TMCO1 and CALR in the pathological characteristics of prostate cancer and its effect on the metastasis of prostate cancer cells
  111. Heterogeneous nuclear ribonucleoprotein K is a potential target for enhancing the chemosensitivity of nasopharyngeal carcinoma
  112. PHB2 alleviates retinal pigment epithelium cell fibrosis by suppressing the AGE–RAGE pathway
  113. Anti-γ-aminobutyric acid-B receptor autoimmune encephalitis with syncope as the initial symptom: Case report and literature review
  114. Comparative analysis of chloroplast genome of Lonicera japonica cv. Damaohua
  115. Human umbilical cord mesenchymal stem cells regulate glutathione metabolism depending on the ERK–Nrf2–HO-1 signal pathway to repair phosphoramide mustard-induced ovarian cancer cells
  116. Electroacupuncture on GB acupoints improves osteoporosis via the estradiol–PI3K–Akt signaling pathway
  117. Renalase protects against podocyte injury by inhibiting oxidative stress and apoptosis in diabetic nephropathy
  118. Review: Dicranostigma leptopodum: A peculiar plant of Papaveraceae
  119. Combination effect of flavonoids attenuates lung cancer cell proliferation by inhibiting the STAT3 and FAK signaling pathway
  120. Renal microangiopathy and immune complex glomerulonephritis induced by anti-tumour agents: A case report
  121. Correlation analysis of AVPR1a and AVPR2 with abnormal water and sodium and potassium metabolism in rats
  122. Gastrointestinal health anti-diarrheal mixture relieves spleen deficiency-induced diarrhea through regulating gut microbiota
  123. Myriad factors and pathways influencing tumor radiotherapy resistance
  124. Exploring the effects of culture conditions on Yapsin (YPS) gene expression in Nakaseomyces glabratus
  125. Screening of prognostic core genes based on cell–cell interaction in the peripheral blood of patients with sepsis
  126. Coagulation factor II thrombin receptor as a promising biomarker in breast cancer management
  127. Ileocecal mucinous carcinoma misdiagnosed as incarcerated hernia: A case report
  128. Methyltransferase like 13 promotes malignant behaviors of bladder cancer cells through targeting PI3K/ATK signaling pathway
  129. The debate between electricity and heat, efficacy and safety of irreversible electroporation and radiofrequency ablation in the treatment of liver cancer: A meta-analysis
  130. ZAG promotes colorectal cancer cell proliferation and epithelial–mesenchymal transition by promoting lipid synthesis
  131. Baicalein inhibits NLRP3 inflammasome activation and mitigates placental inflammation and oxidative stress in gestational diabetes mellitus
  132. Impact of SWCNT-conjugated senna leaf extract on breast cancer cells: A potential apoptotic therapeutic strategy
  133. MFAP5 inhibits the malignant progression of endometrial cancer cells in vitro
  134. Major ozonated autohemotherapy promoted functional recovery following spinal cord injury in adult rats via the inhibition of oxidative stress and inflammation
  135. Axodendritic targeting of TAU and MAP2 and microtubule polarization in iPSC-derived versus SH-SY5Y-derived human neurons
  136. Differential expression of phosphoinositide 3-kinase/protein kinase B and Toll-like receptor/nuclear factor kappa B signaling pathways in experimental obesity Wistar rat model
  137. The therapeutic potential of targeting Oncostatin M and the interleukin-6 family in retinal diseases: A comprehensive review
  138. BA inhibits LPS-stimulated inflammatory response and apoptosis in human middle ear epithelial cells by regulating the Nf-Kb/Iκbα axis
  139. Role of circRMRP and circRPL27 in chronic obstructive pulmonary disease
  140. Investigating the role of hyperexpressed HCN1 in inducing myocardial infarction through activation of the NF-κB signaling pathway
  141. Characterization of phenolic compounds and evaluation of anti-diabetic potential in Cannabis sativa L. seeds: In vivo, in vitro, and in silico studies
  142. Quantitative immunohistochemistry analysis of breast Ki67 based on artificial intelligence
  143. Ecology and Environmental Science
  144. Screening of different growth conditions of Bacillus subtilis isolated from membrane-less microbial fuel cell toward antimicrobial activity profiling
  145. Degradation of a mixture of 13 polycyclic aromatic hydrocarbons by commercial effective microorganisms
  146. Evaluation of the impact of two citrus plants on the variation of Panonychus citri (Acari: Tetranychidae) and beneficial phytoseiid mites
  147. Prediction of present and future distribution areas of Juniperus drupacea Labill and determination of ethnobotany properties in Antalya Province, Türkiye
  148. Population genetics of Todarodes pacificus (Cephalopoda: Ommastrephidae) in the northwest Pacific Ocean via GBS sequencing
  149. A comparative analysis of dendrometric, macromorphological, and micromorphological characteristics of Pistacia atlantica subsp. atlantica and Pistacia terebinthus in the middle Atlas region of Morocco
  150. Macrofungal sporocarp community in the lichen Scots pine forests
  151. Assessing the proximate compositions of indigenous forage species in Yemen’s pastoral rangelands
  152. Food Science
  153. Gut microbiota changes associated with low-carbohydrate diet intervention for obesity
  154. Reexamination of Aspergillus cristatus phylogeny in dark tea: Characteristics of the mitochondrial genome
  155. Differences in the flavonoid composition of the leaves, fruits, and branches of mulberry are distinguished based on a plant metabolomics approach
  156. Investigating the impact of wet rendering (solventless method) on PUFA-rich oil from catfish (Clarias magur) viscera
  157. Non-linear associations between cardiovascular metabolic indices and metabolic-associated fatty liver disease: A cross-sectional study in the US population (2017–2020)
  158. Knockdown of USP7 alleviates atherosclerosis in ApoE-deficient mice by regulating EZH2 expression
  159. Utility of dairy microbiome as a tool for authentication and traceability
  160. Agriculture
  161. Enhancing faba bean (Vicia faba L.) productivity through establishing the area-specific fertilizer rate recommendation in southwest Ethiopia
  162. Impact of novel herbicide based on synthetic auxins and ALS inhibitor on weed control
  163. Perspectives of pteridophytes microbiome for bioremediation in agricultural applications
  164. Fertilizer application parameters for drip-irrigated peanut based on the fertilizer effect function established from a “3414” field trial
  165. Improving the productivity and profitability of maize (Zea mays L.) using optimum blended inorganic fertilization
  166. Application of leaf multispectral analyzer in comparison to hyperspectral device to assess the diversity of spectral reflectance indices in wheat genotypes
  167. Animal Sciences
  168. Knockdown of ANP32E inhibits colorectal cancer cell growth and glycolysis by regulating the AKT/mTOR pathway
  169. Development of a detection chip for major pathogenic drug-resistant genes and drug targets in bovine respiratory system diseases
  170. Exploration of the genetic influence of MYOT and MB genes on the plumage coloration of Muscovy ducks
  171. Transcriptome analysis of adipose tissue in grazing cattle: Identifying key regulators of fat metabolism
  172. Comparison of nutritional value of the wild and cultivated spiny loaches at three growth stages
  173. Transcriptomic analysis of liver immune response in Chinese spiny frog (Quasipaa spinosa) infected with Proteus mirabilis
  174. Disruption of BCAA degradation is a critical characteristic of diabetic cardiomyopathy revealed by integrated transcriptome and metabolome analysis
  175. Plant Sciences
  176. Effect of long-term in-row branch covering on soil microorganisms in pear orchards
  177. Photosynthetic physiological characteristics, growth performance, and element concentrations reveal the calcicole–calcifuge behaviors of three Camellia species
  178. Transcriptome analysis reveals the mechanism of NaHCO3 promoting tobacco leaf maturation
  179. Bioinformatics, expression analysis, and functional verification of allene oxide synthase gene HvnAOS1 and HvnAOS2 in qingke
  180. Water, nitrogen, and phosphorus coupling improves gray jujube fruit quality and yield
  181. Improving grape fruit quality through soil conditioner: Insights from RNA-seq analysis of Cabernet Sauvignon roots
  182. Role of Embinin in the reabsorption of nucleus pulposus in lumbar disc herniation: Promotion of nucleus pulposus neovascularization and apoptosis of nucleus pulposus cells
  183. Revealing the effects of amino acid, organic acid, and phytohormones on the germination of tomato seeds under salinity stress
  184. Combined effects of nitrogen fertilizer and biochar on the growth, yield, and quality of pepper
  185. Comprehensive phytochemical and toxicological analysis of Chenopodium ambrosioides (L.) fractions
  186. Impact of “3414” fertilization on the yield and quality of greenhouse tomatoes
  187. Exploring the coupling mode of water and fertilizer for improving growth, fruit quality, and yield of the pear in the arid region
  188. Metagenomic analysis of endophytic bacteria in seed potato (Solanum tuberosum)
  189. Antibacterial, antifungal, and phytochemical properties of Salsola kali ethanolic extract
  190. Exploring the hepatoprotective properties of citronellol: In vitro and in silico studies on ethanol-induced damage in HepG2 cells
  191. Enhanced osmotic dehydration of watermelon rind using honey–sucrose solutions: A study on pre-treatment efficacy and mass transfer kinetics
  192. Effects of exogenous 2,4-epibrassinolide on photosynthetic traits of 53 cowpea varieties under NaCl stress
  193. Comparative transcriptome analysis of maize (Zea mays L.) seedlings in response to copper stress
  194. An optimization method for measuring the stomata in cassava (Manihot esculenta Crantz) under multiple abiotic stresses
  195. Fosinopril inhibits Ang II-induced VSMC proliferation, phenotype transformation, migration, and oxidative stress through the TGF-β1/Smad signaling pathway
  196. Antioxidant and antimicrobial activities of Salsola imbricata methanolic extract and its phytochemical characterization
  197. Bioengineering and Biotechnology
  198. Absorbable calcium and phosphorus bioactive membranes promote bone marrow mesenchymal stem cells osteogenic differentiation for bone regeneration
  199. New advances in protein engineering for industrial applications: Key takeaways
  200. An overview of the production and use of Bacillus thuringiensis toxin
  201. Research progress of nanoparticles in diagnosis and treatment of hepatocellular carcinoma
  202. Bioelectrochemical biosensors for water quality assessment and wastewater monitoring
  203. PEI/MMNs@LNA-542 nanoparticles alleviate ICU-acquired weakness through targeted autophagy inhibition and mitochondrial protection
  204. Unleashing of cytotoxic effects of thymoquinone-bovine serum albumin nanoparticles on A549 lung cancer cells
  205. Erratum
  206. Erratum to “Investigating the association between dietary patterns and glycemic control among children and adolescents with T1DM”
  207. Erratum to “Activation of hypermethylated P2RY1 mitigates gastric cancer by promoting apoptosis and inhibiting proliferation”
  208. Retraction
  209. Retraction to “MiR-223-3p regulates cell viability, migration, invasion, and apoptosis of non-small cell lung cancer cells by targeting RHOB”
  210. Retraction to “A data mining technique for detecting malignant mesothelioma cancer using multiple regression analysis”
  211. Special Issue on Advances in Neurodegenerative Disease Research and Treatment
  212. Transplantation of human neural stem cell prevents symptomatic motor behavior disability in a rat model of Parkinson’s disease
  213. Special Issue on Multi-omics
  214. Inflammasome complex genes with clinical relevance suggest potential as therapeutic targets for anti-tumor drugs in clear cell renal cell carcinoma
  215. Gastroesophageal varices in primary biliary cholangitis with anti-centromere antibody positivity: Early onset?
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