Table 1.
Mouse primers for RT-PCR.
Fig 1.
Expression of nAChR subunits, taste receptors, and BDNF in STC-1 cells.
Consensus primers to amplify mouse nAChR subunits, taste receptor (T2R38), downstream signaling intermediate (TRPM5), and BDNF were designed based on the published sequences and are shown in Table 1. (A) Based on the predicted sizes of the PCR products (Table 1), we detected the mRNAs for the nAChR subunits: chrna3, chrna4, chrna5, chrna6, chrna7, chrnb2, and chrnb4 in the STC-1 cell cDNA sample. In addition, we detected the mRNAs for T2R38, TRPM5, α-ENaC, and TRPV1. (B) Based on the predicted size of the PCR product (Table 1), we also detected the mRNA for BDNF in STC-1 cells. Brain tissue was used as a positive control.
Fig 2.
Immunofluorescence staining of nAChR subunits and BDNF in STC-1 cells.
(Panel A) Immunostaining of nAChR α3, α4, and α5. (Panel B) Immunostaining of nAChR α7, β2, and β4. (Panel C) Negative control (NC) without primary antibody and immunostaining of BDNF. Blue color represents staining of cell nuclei with DAPI. The panels show merged confocal images of DAPI and secondary antibody fluorescence.
Fig 3.
Co-localization of nAChR subunits with bitter taste receptors and downstream intracellular signaling intermediates in STC-1 cells.
(Panel A) Immunostaining of AChRα3 and AChRβ4. (Panel B) Immunostaining of AChRβ2 and T2R38. (Panel C) Negative control (NC) without primary antibody. (Panel D) Immunostaining of TRPM5 and T2R38. (Panel E) Negative control (NC) without primary antibody. Blue color represents staining of cell nuclei with DAPI. The panels show merged confocal images of DAPI and fluorescence images of Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate (green) and Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 594 (red) conjugate.
Fig 4.
Co-IP of nAChRs in STC-1 cell lysates.
In STC-1 cell lysates nAChRs α5 and β4 proteins were immunoprecipitated by AChRα3 antibody (A, B), and nAChR α4 protein was immunoprecipitated by AChRβ2 antibody (C). IP = immunoprecipitation; WB = Western blot; IgG = negative control.
Fig 5.
Effect of nicotine exposure on the nAChR mRNA expression level in STC-1 cells.
(A) After 24h treatment. Relative to control at 250 nM nicotine the p values for chrna5, chrna6, chrna7, and chrnb2 were 0.0125, 0.0001, 0.0219, and 0.0001, respectively. Relative to control at 500 nM nicotine the p values for chrna4, chrna5, chrna6, chrna7, and chrnb2 were 0.0001, 0.0001, 0.0001, 0.0019, and 0.0012, respectively. Relative to control at 1000 nM nicotine the p values for chrna3, chrna4, chrna5, chrna6, chrnb2, and chrnb4 were 0.0272, 0.0041, 0.0125, 0.0001, 0.0184, and 0.0284. Relative to 250 nM nicotine at 500 nM nicotine the p values for chrna4, chrna5, chrna6, and chrna7 were 0.0003, 0.0002, 0.0001, 0.0006, respectively. Relative to 500 nM nicotine at 1000 nM nicotine the p values for chrna4, chrna5, chrna6, and chrnb2 were 0.0025, 0.01, 0.0146, and 0.042, respectively. (B) After 4 day treatment. Relative to control at 250 nM nicotine the p values for chrna3, chrna4, chrna5, chrna6, chrna7, and chrnb4 were 0.0001, 0.0022, 0.002, 0.0001, 0.0001, and 0.0001, respectively. After 4 day treatment, relative to control the p values for chrna4, chrna5, chrna6, chrna7, and chrnb4 at 500 nM nicotine were 0.0001, 0.0001, 0.0001, 0.0001, and 0.0001, respectively. Increasing nicotine concentration from 250 nM to 500 nM only produced a significant increase in mRNA level of chrna4 (p = 0.029). No significant changes were observed in the mRNA levels of the other nAChR subunits. In contrast, increasing nicotine from 500 nM to 1000 nM produced significant increases in all nAChR subunits investigated. After 4 day treatment, relative to 500 nM nicotine at 1000 nM nicotine the p values for chrna3, chrna4, chrna5, chrna6, chrna7, and chrnb4 were 0.0003, 0.0001, 0.0004, 0.0021, 0.0001, and 0.0001, respectively.
Fig 6.
Effect of Mec on the nicotine induced increase in the nAChR mRNA expression level in STC-1 cells.
STC-1 cells were treated with 0 Nic (control), 500 nM nicotine, 500 nM nicotine + 10 μM Mec or 10 μM Mec for 24h. In control STC-1 cells, the mRNA levels of chrna4 and chran6 increased as before. The increase in mRNA of chrna4 and chran6 by nicotine was blocked in the presence of Mec. Mec by itself did not affect the mRNA level of chrna4 and chran6 (data not shown).
Fig 7.
Effect of nicotine on the protein levels of α4 and β2 nAChR subunits in STC-1 cells.
(A) STC-1 cells in culture were treated for 24h with varying concentrations of nicotine (0–500 nM). Samples containing 30 μg total protein were resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes. Membranes were immune-blotted with primary antibodies against nAChRβ2, nAChRα4, and β-actin followed by HRP-conjugated secondary antibodies. Beta-actin was used as a protein loading control. Relative to control no significant changes were seen in β2 nAChR subunit protein level at nicotine concentrations tested. Relative to control, an increase in α4 nAChR subunit protein level was observed at 250 and 500 nM nicotine. The molecular weights of α4, β2, and β-actin are 75, 50 and 43 Kd. (B) Shows the ratio of the intensity of α4 and β2 bands relative to the intensity of the β-actin normalized to control (zero nicotine).
Fig 8.
Effect of nicotine on STC-1 cell Ca2+.
Changes in [Ca2+]i were measured as temporal changes in FIR (F340/F380) in individual STC-1 cells loaded with Fura-2 as a response to 100 μM or 250 μM nicotine exposure. (A) In the representative experiment shown, 41 out of 112 cells (36.6% of the cells) responded with a rapid but transient increase in FIR when exposed to 100 μM nicotine. (B) In the representative experiment shown, 40 out of 66 cells (60.6% of the cells) responded with a rapid increase in FIR when exposed to 250 μM nicotine. Relative to 100 μM nicotine, at 250 μM nicotine, STC-1 cells responded with a significantly higher (p <0.0001, unpaired) mean maximum increase in FIR (1.70 ± 0.09 versus 2.45 ± 0.12). (C) Exposing STC-1 cells to 100 μM nicotine + 10 μM Mec produced no change in FIR in all 66 cells investigated. The values are presented as mean ± SEM of FIR of the number of cells (N).
Fig 9.
Effect of denatonium on STC-1 cell Ca2+.
Changes in [Ca2+]i were measured as temporal changes in FIR (F340/F380) in individual STC-1 cells loaded with Fura-2 as a response to 5 mM denatonium or 250 μM nicotine exposure. (A) Exposing STC-1 cells to 5 mM denatonium induced a transient increase in FIR in 45 out of 72 cells investigated (62.5% of the cells). (B) In separate experiments, cells were first treated with 250 μM nicotine (Nic). The cells were than washed with control Ringer’s solution for 5 min. After the wash the same STC-1 cells were treated with 5 mM denatonium. All 50 STC-1 cells that responded with an increase in FIR to 250 μM Nic also responded with an increase in FIR when treated with 5 mM denatonium. The values are presented as mean ± SEM of FIR of the number of cells (N). (C) STC-1 cells were first treated with 5 mM denatonium (Den) and then with Den + 10 μM DHβE or Den + 10 μM Mec. A different batch of STC-1 cells was first treated with 1 mM nicotine (Nic) and then with Nic + 10 μM DHβE. The values are presented as mean ± SEM of FIR of the number of cells (N). The peak change in FIR in the presence of Nic or Den in each cell was normalized to 100%. DHβE inhibited the nicotine-induced changes in FIR to near zero. Mec or DHβE produced no significant (p >0.05, unpaired) changes in FIR induced by Den. In each case FIR values were measured from 13–20 STC-1 cells.
Fig 10.
Effect of nicotine on STC-1 cell BDNF.
STC-1 cells were treated with 1 or 100 μM nicotine (Nic) for 30 min in the absence and presence of 50 μM mecamylamine (Mec). BDNF was measured in cell lysates using ELISA. Nicotine decreased the BDNF content in STC-1 cells in a dose-dependent manner. The nicotine-induced decrease in BDNF was not observed in the presence of Mec. *BDNF value at 1 μM and 100 μM nicotine were significantly lower than the basal level (0 Nic) with p values of 0.0094 and 0.0044, respectively (paired). The BDNF values in the presence of Nic (1 or 100 μM) + 10 μM Mec were not different from the basal level (0 Nic) (p>0.05).
Fig 11.
Localization of nAChRs in gut tissue.
(A) Based on the predicted sizes of the PCR products (Table 1), we detected the mRNAs for chrna3 and chrnb4 in the intestinal mucosal cell cDNA sample. (B and C) Immunostaining of nAChR α3 in enteroendocrine cells in the gut (red arrows). We examined 3 different sections of the small intestine. In each of the 15 slides examined 2 or 3 enteroendocrine cells were positive for α3 nAChR in the crypts. (D and C) Negative control (NC) without primary antibody. Blue color represents staining of cell nuclei with DAPI. The panels C and E show merged confocal images of DAPI and secondary antibody fluorescence (Alexa Fluor® 488, green).