Nicotinic acetylcholine receptor (CHRN) expression and function in cultured human adult fungiform (HBO) taste cells

<div><p>In rodents, CHRNs are involved in bitter taste transduction of nicotine and ethanol. Currently, it is not clear if CHRNs are expressed in human taste cells and if they play a role in transducing the bitter taste of nicotine and ethanol or in the synthesis and release of neurohumoral peptides. Accordingly, we investigated the expression and functional role of CHRNs in HBO cells. Using molecular techniques, we demonstrate that a subset of HBO cells express CHRNs that also co-express TRPM5, T1R3 or T2R38. Exposing HBO cells to nicotine or ethanol acutely or to nicotine chronically induced a differential increase in the expression of CHRN mRNA and protein in a dose- and time-dependent manner. Acutely exposing HBO cells to a mixture containing nicotine plus ethanol induced a smaller increase in CHRN mRNAs relative to nicotine or ethanol treatment alone. A subset of HBO cells responded to nicotine, acetylcholine and ATP with a transient increase in [Ca²⁺]_i. Nicotine effects on [Ca²⁺]_i were mecamylamine sensitive. Brain-derived neurotrophic factor (BDNF) protein was detected in HBO cells using ELISA. Acute nicotine exposure decreased BDNF in HBO cells and increased BDNF release in the medium. CHRNs were also detected in HEK293 cells by RT-PCR. Unlike HBO cells, CHRNs were localized in most of HEK293 cells and majority of HEK293 cells responded to nicotine and ethanol stimulation with a transient increase in [Ca²⁺]_i. BDNF levels in HEK293 cells were significantly higher than in HBO cells but the nicotine induced release of BDNF in the media was a fraction of the BDNF cellular content. We conclude that CHRNs are expressed in TRPM5 positive HBO cells. CHRN mRNA expression is modulated by exposure to nicotine and ethanol in a dose- and time-dependent manner. Nicotine induces the synthesis and release of BDNF in HBO cells.</p></div

Co-localization of CHRNA and CHRNB subunits in HBO cells.

<p>Dual immunostaining was used to co-localize CHRNA and CHRNB subunits in individual HBO cells. <b>(A and B)</b> Show immunostaining of CHRNA3 <b>(α3)</b> with CHRNB4 <b>(β4)</b>. The panels show confocal images of α3 (green), β4 (red), DAPI (blue), and merged images of DAPI and dual fluorescence labels. <b>(C and D)</b> Show immunostaining of CHRNA5 <b>(α5)</b> with CHRNB2 <b>(β2)</b>. The panels show confocal images of α5 (green), β2 (red), DAPI (blue), and merged images of DAPI and dual fluorescence labels.</p

Immunofluorescence staining of CHRNA and CHRNB subunits in HEK293 cells.

<p><b>(A)</b> Shows immunostaining of CHRNA3 <b>(α3)</b>, CHRNA4 (α4), and CHRNA5 <b>(α5)</b> in HEK293 cells. <b>(B)</b> Shows immunostaining of CHRNB2 <b>(β2)</b> in HEK293 cells. The panels show merged confocal images of DAPI (blue) and secondary antibody fluorescence (green). The negative control <b>(NC)</b> without primary antibody is also shown. Dual immunostaining was used to co-localize CHRNA5 <b>(α5)</b> with CHRNB2 <b>(β2)</b>. <b>(B, α5/β2)</b> Shows immunostaining of CHRNA5 (α5; green) with CHRNB2 (β2; red). The panel shows merged confocal images of DAPI and dual fluorescence labels.</p

Effect of Nic, ETOH, and mecamylamine (Mec) on temporal changes in [Ca²⁺]_i in individual HBO cells.

<p><b>(A)</b> Shows a representative HBO cell that responded with a dose-dependent increase in FIR when stimulated with 0.01, 0.05 and 1.0 mM Nic. <b>(B)</b> Shows 1 out of 8 HBO cells that responded with a transient increase in FIR when stimulated with 50 mM ETOH. Two additional cells responded with a small but significant increase in FIR when stimulated with 50 mM ETOH. <b>(C)</b> Shows another representative HBO cell that responded with an increase in FIR in the presence of 1 mM Nic. Mec (50 μM) inhibited the Nic-induced increase in FIR. In each cell, the FIR was normalized to 1 with respect to its value under the control conditions.</p

Effect of Nic exposure on the CHRN subunit mRNA expression level in HBO cells.

<p><b>(A) After 24h Nic treatment</b>. Relative to control, at 0.25 μM Nic the *p values for CHRNA3, CHRNA5, CHRNA6, CHRNB2, and CHRNB4 mRNAs were 0.0111, 0.0306, 0.0017, 0.0239, and 0.0173, respectively. Relative to control, at 0.50 μM Nic the *p values for CHRNA3, CHRNA5, CHRNA6, CHRNB2, and CHRNB4 mRNAs were 0.0022, 0.0473, 0.0014, 0.0049, and 0.0141, respectively. Relative to control, at 1.0 μM Nic the *p values for CHRNA3, CHRNA5, CHRNA6, CHRNB2, and CHRNB4 mRNAs were 0.0194, 0.0055, 0.0013, 0.0236, and 0.0003, respectively. Relative to 0.25 μM Nic, at 0.50 μM Nic the *p values for CHRNA6 and CHRNB4 were 0.0039 and 0.0024. Relative to 0.50 μM Nic at 1.0 μM Nic the *p values for CHRNA6, CHRNB2, and CHRNB4 were 0.0192, 0.0144, and 0.0046, respectively. <b>(B) After 24h ETOH treatment</b>. Relative to control, at 50 mM ETOH the *p values for CHRNA3, CHRNA5, CHRNA6, and CHRNB2 mRNAs were 0.0048, 0.0207, 0.0020, and 0.0169, respectively. Relative to 50 mM ETOH, at 50 mM ETOH + 0.5 μM Nic the *p values for CHRNA3 and CHRNA6 mRNAs were 0.0049 and 0.0024. <b>(C) After 4 days treatment</b>. Relative to control, at 0.25 μM Nic the *p values for chrna6 and CHRNA7 mRNAs were 0.0003 and 0.024, respectively. After 4 days treatment, relative to control, at 0.50 μM Nic the *p values for CHRNA5, CHRNA6, CHRNA7, and CHRNB4 were 0.0402, 0.0001, 0.0049, and 0.0001, respectively. Relative to control, at 1.0 μM Nic, the *p values for CHRNA5, CHRNA6, CHRNA7, and CHRNB4 were 0.0009, 0.0005, 0.0001, and 0.0001, respectively. The values represent mean ± SEM of triplicate measurements.</p

Co-localization of CHRNA5 subunit with TRPM5 in HBO cells.

<p>Dual immunostaining was used to co-localize CHRNA5 <b>(α5)</b> subunit with <b>TRPM5</b> in individual HBO cells. <b>(A and B)</b> Show confocal images of α5 (green), TRPM5 (red), DAPI (blue), and merged images of DAPI and dual fluorescence labels.</p

Effect of Nic and ETOH on temporal changes in [Ca²⁺]_i in individual HEK293 cells.

<p><b>(A)</b> Shows changes in FIR in all 45 HEK293 cells in the visual field that were stimulated with 0.01 mM Nic. In each cell, the FIR was normalized to 1 with respect to its value under the control conditions. The values are mean ± SEM of the number of cells in the visual field. <b>(B)</b> Shows changes in FIR in all 43 HEK293 cells in the visual field that were stimulated with 10 mM ETOH. In each cell, the FIR was normalized to 1 with respect to its value under the control conditions. The values are mean ± SEM of the number of cells in the visual field.</p

RT-PCR primers for CHRN subunits.

<p>RT-PCR primers for CHRN subunits.</p

Co-localization of CHRNB2 with T2R38 in HBO cells.

<p>Dual immunostaining was used to co-localize CHRNB2 <b>(β2)</b> with <b>T2R38</b> in individual HBO cells. <b>(A and B)</b> Show confocal images of DIC, β2 (green), DAPI (blue), T2R38 (red), and merged images of DAPI and dual fluorescence labels.</p

Co-localization of CHRNA4 and CHRNB2 subunits with TRPM5 in individual HBO cells.

<p>Dual immunostaining was used to co-localize CHRNA4 <b>(α4)</b> or CHRNB2 <b>(β2)</b> subunit with <b>TRPM5</b> in individual HBO cells. <b>(A)</b> Show confocal images of DIC, α4 (green), DAPI (blue), TRPM5 (red), and merged images of DAPI and dual fluorescence labels. <b>(B)</b> Show confocal images of DIC, β2 (green), DAPI (blue), TRPM5 (red), and merged images of DAPI and dual fluorescence labels.</p