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. 2011 Aug 22:12:86.
doi: 10.1186/1471-2202-12-86.

Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium

Ruth Dooley  1 , Anastasia Mashukova, Bastian Toetter, Hanns Hatt, Eva M Neuhaus
Affiliations

Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium

Ruth Dooley et al. BMC Neurosci. .

Abstract

Background: Extracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation.

Results: Here we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells.

Conclusion: Our results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.

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Figures

Figure 1
Figure 1
Odorants induce MAPK and CREB phosphorylation in the olfactory epithelium. (A) Cryosections (12 μm) of mice expressing GFP in every mature OSN (OMP-GFP) were subjected to immunohistochemistry using an antibody specific for phosphorylated p42/44 MAPK in the olfactory epithelium. After 2 hours of odorant exposure a strong anti-phospho-p42/44 MAPK immunoreactivity was observed in the cytoplasm of OSNs. (B) Staining for phospho-CREB (Ser-133). Phospho-CREB was observed in the sustentacular cell layer, in the nuclei of OSNs, and in immature OSNs not expressing OMP, after exposure to a mixture of 100 different odorants for 2 h. Control panel with secondary antibody only or after using a specific blocking peptide shows very low background labeling intensity in the nuclei of the different cell types. (C) WB analysis of control cell lysates (SK-N-MC cells prepared with IBMX and forskolin treatment) and olfactory epithelium samples with CREB and phospho-CREB (Ser-133) specific antibodies showing labeling of the proteins (bands marked by stars), which is abolished when the specific blocking peptide was pre-incubated with the antibody.
Figure 2
Figure 2
Time-dependent odorant induced CREB phosphorylation. (A) Staining for phospho-CREB (Ser-133). Low levels of basal phospho-CREB were observed in the nuclei of OSNs and immature OSNs not expressing OMP, with little or no staining in the sustentacular cell layer. An increase in phospho-CREB in the sustentacular cell layer was observed after 15 min of exposure to a mixture of 100 different odorants, and after 2 hours positive staining for phospho-CREB was observed in the nuclei of OSNs, immature OSNs not expressing OMP and the sustentacular cells. Scale bar 20 μm. (B) High magnification image (2X) of a section of the images in A, clearly showing the increase in CREB phosphorylation in the sustentacular cell layer (arrowhead) and in the OSNs (arrow) after 2 h odorant treatment. Scale bar 20 μm. (C) Bar chart shows mean ± SD average fluorescence intensity along the nuclei of different cell types indicated, normalized to cytosolic background staining. Sustentacular cell nuclei layer, control 0.96 ± 0.33 n = 78, 15 min odorant 4.88 ± 0.68 n = 71, 2 h odorant 7.10 ± 0.59 n = 76. OSN nuclei layer, control 2.58 ± 1.47 n = 68, 15 min odorant 2.80 ± 0.81 n = 81 (p = 0.24), 2 h odorant 4.16 ± 3.11 n = 74. Immature cell nuclei layer, control 0.79 ± 0.69 n = 74, 15 min odorant 2.76 ± 0.24 n = 79, 2 h odorant 3.97 ± 0.86 n = 69. n represents one standard field of view at 40 × magnification and 2 × zoom, one-way ANOVA p*** < 0.001, p** < 0.01. Three mice were used for each condition and four cryosections with complete olfactory epithelium were quantified per mouse.
Figure 3
Figure 3
CREB phosphorylation in sustentacular cells is dependent on ACIII-mediated olfactory signaling. (A) Cryosections (12 μm) of OMP-GFP mice treated for 2 h with a mixture of 100 odorants or pre-treated with an inhibitor of adenylyl cyclase III (SQ22536). (B) High magnification image (2X) showing the inhibition of CREB phosphorylation in the sustentacular cell layer (arrowhead) and in the OSN cell soma (arrow), by an inhibitor of ACIII. Scale bar 10 μm. (C) Mean ± SD fluorescence intensity across the nuclei was quantified as before. Sustentacular cells showed a significant decrease from (6.47 ± 0.90, n = 79) after 2 h odorant treatment to (0.95 ± 0.24, n = 72) with SQ22536 pre-treatment; OSN nuclei decreased from 4.36 ± 3.47 (n = 70) after 2 h to 0.80 ± 1.39 (n = 81) with SQ22536 pre-treatment; immature cell nuclei did not change (2.90 ± 0.89 after 2 h (n = 80), 2.95 ± 0.86 (n = 74) with SQ22536 pre-treatment); p** < 0.01, p*** < 0.001.
Figure 4
Figure 4
CREB phosphorylation in sustentacular cells after 24 hours. (A) Low magnification representative image of 12 μm cryosection of OMP-GFP mice treated with a mixture of 100 odorants for 24 h. Sustentacular cells showed marked CREB phosphorylation, while phosphorylation in immature OSNs not expressing OMP is only weak and in OSNs decreased. (B) Higher magnification images. Scale bars = 50 μm. (C) Fluorescence intensity was quantified as before. Bar chart shows mean ± SD (Sustentacular cell control 0.96 ± 0.33 n = 78, 24 h odorant 3.87 ± 0.13 n = 73; OSN control 2.58 ± 0.15 n = 72, 24 h odorant 1.13 ± 0.62 n = 70), p** < 0.01, p*** < 0.001.
Figure 5
Figure 5
CREB phosphorylation is dependent on purinergic receptor signaling. (A) Representative 12 μm cryosection stained with antibodies to pCREB (Ser-133) after pre-treatment with PPADS/Suramin and treatment with 2 h odorant, CREB phosphorylation is markedly reduced in the sustentacular cell layer. Control sections show epithelium without odorant treatment and epithelium without odorant treatment, but with PPADS/Suramin incubation. Scale bar 20 μm. (B) Fluorescence intensities were quantified as before. Bar chart shows mean ± SD. Sustentacular cells 2 h odorant (7.10 ± 0.59 n = 79), +PPADS/Suramin (1.55 ± 0.48 n = 80), OSNs 2 h odorant (4.16 ± 3.11 n = 75), +PPADS/Suramin (4.21 ± 2.76 n = 71, p = 0.48), immature cells 2 h odorant (4.03 ± 2.04 n = 79), +PPADS/Suramin (3.51 ± 1.02 n = 80, p = 0.11), p*** < 0.001. (C) Western blot of whole olfactory epithelia lysates of OMP-GFP mice (2 mice pooled per condition) treated for 2 h with a mixture of 100 odorants or pre-treated with PPADS/Suramin. Blot representative of 3 independent experiments. (D) Bar chart shows average densitometry values, mean ± SEM of phospho-CREB (Ser133), shown as percentage of total CREB. Control (3.70 ± 1.29), 2 hours odorant treatment (68.04 ± 4.98), PPADS/suramin pre-treatment (45.93 ± 6.06), p** < 0.01.
Figure 6
Figure 6
ATP-mediated sustentacular cell CREB phosphoryation is dependent on adenylyl cyclase III. (A) Olfactory epithelia were incubated with ATP to activate purinergic receptors, resulting in CREB phosphorylation in sustentacular cells, OSNs, and immature OSNs not expressing OMP. Using an inhibitor to ACIII blocked the phosphorylation of CREB in OSNs, immature OSNs not expressing OMP, and in sustentacular cells to different extents. (B) Fluorescence intensities were quantified as before. Bar chart shows mean ± SD. Sustentacular cells 2 h ATP (7.52 ± 0.47 n = 78), +SQ22536 (4.39 ± 0.24 n = 78), OSNs 2 h ATP (1.81 ± 2.27 n = 75), +SQ22536 (0.84 ± 0.19 n = 69, p = 0.18), immature cells 2 h ATP (3.05 ± 0.91 n = 79), +SQ22536 (1.01 ± 0.27 n = 80), p*** < 0.001. Scale bar 10 μm.
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