Table 1.
RT-PCR primers for CHRN subunits.
Table 2.
RT-PCR primers for GPCRs and signaling components.
Fig 1.
Expression of CHRN subunits, taste receptors, and intracellular signaling intermediates in HBO and HEK293 cells.
Consensus primers to amplify CHRN subunits, GPCRs, and downstream signaling intermediate were designed based on the published sequences and are shown in Tables 1 and 2. (A) Based on the predicted sizes of the PCR products (Table 1), mRNAs for the CHRNA3, CHRNA4, CHRNA5, CHRNA6, CHRNA7, CHRNB2, and CHRNB4 were detected in HBO cells. (B) In addition, mRNAs for T1R1, T1R3, T2R38, PLCβ2, and TRPM5 were detected in HBO cells. (C) In Western blot analysis using specific AChRα4 and AChRα5 antibodies, the expression of α4 and α5 proteins were detected in HBO and HEK293 cell lysates. (D) Based on the predicted sizes of the PCR products (Table 1), mRNAs for the CHRNA3, CHRNA4, CHRNA5, CHRNA6, CHRNB2 and CHRNB4 were detected in HEK293 cells. In addition, mRNAs for T2R38 and TRPM5 were detected.
Fig 2.
Immunofluorescence staining of CHRNA4 and CHRNA5 in HBO cells.
Shows immunostaining of CHRNA4 (A; α4) and CHRNA5 (B; α5) in HBO cells using 20x and 40x objectives (total 200x and 400x magnification). The panels show merged confocal images of DAPI (blue) and secondary antibody fluorescence (green). The negative control (NC) without primary antibody is also shown. The horizontal red lines represent 10 μm.
Fig 3.
Co-localization of CHRNA subunits in HBO cells.
Dual immunostaining was used to co-localize CHRNA subunits in individual HBO cells. (A and B) Show immunostaining of CHRNA5 (α5) with CHRNA4 (α4). The panels show confocal images of α5 (green), α4 (red), DAPI (blue), and merged images of DAPI and dual fluorescence labels. (C and D) Show immunostaining of CHRNA5 (α5) with CHRNA3 (α3). The panels show confocal images of α5 (green), α3 (red), DAPI (blue), and merged images of DAPI and dual fluorescence labels.
Fig 4.
Co-localization of CHRNA and CHRNB subunits in HBO cells.
Dual immunostaining was used to co-localize CHRNA and CHRNB subunits in individual HBO cells. (A and B) Show immunostaining of CHRNA3 (α3) with CHRNB4 (β4). The panels show confocal images of α3 (green), β4 (red), DAPI (blue), and merged images of DAPI and dual fluorescence labels. (C and D) Show immunostaining of CHRNA5 (α5) with CHRNB2 (β2). The panels show confocal images of α5 (green), β2 (red), DAPI (blue), and merged images of DAPI and dual fluorescence labels.
Fig 5.
Co-localization of CHRNA5 subunit with TRPM5 in HBO cells.
Dual immunostaining was used to co-localize CHRNA5 (α5) subunit with TRPM5 in individual HBO cells. (A and B) Show confocal images of α5 (green), TRPM5 (red), DAPI (blue), and merged images of DAPI and dual fluorescence labels.
Fig 6.
Co-localization of CHRNA4 and CHRNB2 subunits with TRPM5 in individual HBO cells.
Dual immunostaining was used to co-localize CHRNA4 (α4) or CHRNB2 (β2) subunit with TRPM5 in individual HBO cells. (A) Show confocal images of DIC, α4 (green), DAPI (blue), TRPM5 (red), and merged images of DAPI and dual fluorescence labels. (B) Show confocal images of DIC, β2 (green), DAPI (blue), TRPM5 (red), and merged images of DAPI and dual fluorescence labels.
Fig 7.
Co-localization of CHRNA6 subunit with TRPM5 in individual HBO cells.
Dual immunostaining was used to co-localize CHRNA6 (α6) subunit with TRPM5 in individual HBO cells. (A, B and C) Show confocal images of DIC, DAPI (blue), α6 (green), TRPM5 (red), and merged images of DAPI and dual fluorescence labels.
Fig 8.
Co-localization of CHRNA5 subunit with T1R3 in HBO cells.
Dual immunostaining was used to co-localize CHRNA5 (α5) subunit with T1R3 in individual HBO cells. (A and B) Show confocal images of α5 (green), T1R3 (red), DAPI (blue), and merged images of DAPI and dual fluorescence labels.
Fig 9.
Co-localization of CHRNB2 with T2R38 in HBO cells.
Dual immunostaining was used to co-localize CHRNB2 (β2) with T2R38 in individual HBO cells. (A and B) Show confocal images of DIC, β2 (green), DAPI (blue), T2R38 (red), and merged images of DAPI and dual fluorescence labels.
Fig 10.
Immunofluorescence staining of CHRNA and CHRNB subunits in HEK293 cells.
(A) Shows immunostaining of CHRNA3 (α3), CHRNA4 (α4), and CHRNA5 (α5) in HEK293 cells. (B) Shows immunostaining of CHRNB2 (β2) in HEK293 cells. The panels show merged confocal images of DAPI (blue) and secondary antibody fluorescence (green). The negative control (NC) without primary antibody is also shown. Dual immunostaining was used to co-localize CHRNA5 (α5) with CHRNB2 (β2). (B, α5/β2) Shows immunostaining of CHRNA5 (α5; green) with CHRNB2 (β2; red). The panel shows merged confocal images of DAPI and dual fluorescence labels.
Fig 11.
Expression analysis of CHRN subunits using single cell PCR.
(A, B, C) Single cell real-time RT-PCR technique was used to perform expression analysis of CHRN subunits using the Single Cell-to-CTTM Kit in 32 individual HBO cells. The final real-time PCR products were separated by electrophoresis on a 2% agarose gel containing 1 μg/ml ethidium bromide. In (A) T2R38 was not detected in cell number 1–11.
Fig 12.
Co-IP of CHRNs in HBO cell lysates.
In HBO cell lysates, CHRNA5 and CHRNB4 proteins were immune-precipitated by AChRα3 antibody. WB = Western blot; IgG = negative control.
Fig 13.
Effect of Nic and ETOH treatment on CHRNA5 and CHRNA6 protein expression in HBO cells.
HBO cells were treated with Nic (0.25–1.0 μM), ETOH (50 mM), and 0.5 μM Nic + 50 mM ETOH for 24h. Western blots were developed using specific AChRα5 or AChRα6 antibodies (A) and analyzed (B and C). The fold change in protein expression was calculated with respect to β-actin. The values are means of triplicate measurements.
Fig 14.
Effect of Nic exposure on the CHRN subunit mRNA expression level in HBO cells.
(A) After 24h Nic treatment. 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) After 24h ETOH treatment. 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. (C) After 4 days treatment. 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.
Fig 15.
Effect of Nic on BDNF synthesis and release in HBO cells and HEK293 cells.
(A) HBO cells were treated with 0.25, 0.50, and 1.0 μM Nic for 30 min. Following that BDNF concentration was measured in cell lysate and the media. Relative to control BDNF concentration in cell lysate increased at 0.25 μM Nic (p = 0.0274) and decreased at 0.5 μM Nic (p = 0.0055). No significant increase in BDNF concentration was observed at 0.25 μM Nic, however, at 0.5 μM Nic, BDNF concentration was significantly increased relative to control (p = 0.0001). Relative to control at 1.0 μM Nic, a small but significant increase in the BDNF concentration was observed in the media (p = 0.0004). The p values for the changes in total BDNF content for 0.25, 0.5 and 1.0 μM Nic were 0.0289, 0.0002, and 0.0014, respectively. The values are mean ± SEM of triplicate measurements. (B) In HEK293 cells, BDNF concentration in cell lysate was about 6 times greater than in HBO cells. Relative to control, at 0.25 μM Nic, BDNF concentration in cell lysate was significantly increased (p = 0.0001), and remained elevated at 0.5 μM (p = 0.0331) and 1.0 μM Nic (p = 0.0041). Relative to control at 0.25 μM, 0.5 μM, and 1.0 μM Nic, a small but significant increase in the BDNF concentration was observed in the media (p = 0.0001). The values are mean ± SEM of triplicate measurements.
Fig 16.
Effect of Nic, acetylcholine (ACh), and other taste stimuli on temporal changes in [Ca2+]i in individual HBO cells.
Temporal changes in [Ca2+]i were monitored as changes in Florescence Intensity Ratio (FIR; F340/F380) in Fura-2 loaded single HBO cells in response to stimulation with different taste stimuli. (A and B) Show two representative HBO cells that responded with a transient increase in FIR when stimulated with 200 μM ACh, 200 μM ATP, and 200 μM Nic. (C) Shows another HBO cell that responded with a transient increase in FIR when stimulated with 200 μM ACh and 200 μM glutamine. (D) Shows mean ± SEM changes in FIR from 5 individual HBO cells that were stimulated with 200 μM ACh and 200 μM ATP. In each cell, the FIR was normalized to 1 with respect to its value under the control conditions.
Fig 17.
Effect of Nic, ETOH, and mecamylamine (Mec) on temporal changes in [Ca2+]i in individual HBO cells.
(A) 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) 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. (C) 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.
Fig 18.
Effect of Nic and ETOH on temporal changes in [Ca2+]i in individual HEK293 cells.
(A) 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) 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.