Desensitization of μOR signaling in enteric neurons.

<p>A: Single exposure to DAMGO (1 µM, 5 min) induced significant MAPK activation in naïve enteric neurons, whereas a second exposure to the same DAMGO dose following 2 hours DAMGO pretreatment abolished DAMGO-mediated MAPK response, indicating desensitization. B: Single exposure to DAMGO (1 µM) or morphine (10 µM) activated MAPK in chronic neurons. A second exposure to the same dose of DAMGO or morphine following 2 hours DAMGO or morphine pretreatment induced the same effect in chronic neurons as single exposures, indicating suppression of desensitization. (** p<0.01 vs. control in A and B). C and D: DAMGO and morphine inhibit forskolin-stimulated cAMP in naïve (C) and chronic (D) enteric neurons. This effect was not observed in naïve enteric neurons (C) with a second opioid stimulation following a prior 2 hour exposure, indicative of desensitization. D: Note the over 2 fold increase in cAMP in unstimulated chronic neurons (cAMP superactivation or “overshooting”) vs. naïve control; DAMGO and morphine inhibition of cAMP was not prevented by 2 hours DAMGO or morphine pretreatment in chronic neurons, indicating suppression of desensitization. **p<0.01 vs. controls. N = 5–7 experiments performed in duplicate per group.</p

Effect of opioids on CREB phosphorylation in enteric neurons.

<p>Naïve (A) and chronic (B) neurons were stimulated with 1 µM DAMGO, 10 µM morphine or medium (control) for 0–20 minutes. DAMGO and morphine induced a significant, transient CREB activation in chronic, but not naïve enteric neurons. CREB phosphorylation in chronic neurons was blocked by the MEK1/2 inhibitor (U0126) treatment. *p<0.05 and **p<0.01 vs. control; n = 4–7 experiments in triplicate per group. Representative gels of pCREB and CREB are shown at the bottom of the figure. Total CREB was used to verify that the treatment did not affect the total level of this protein and to confirm equal gel loading.</p

μOR immunoreactivity in naïve enteric neurons (A–C) and in neurons chronically treated with morphine (D–F).

<p>μOR immunoreactivity is at the cell surface in unstimulated and morphine-stimulated neurons (A, C arrows), and it is in the cytosol following stimulation with DAMGO (B) in naïve enteric neurons. μOR immunoreactivity is at the cell surface in unstimulated neurons (D, arrows), but in the cytosol following DAMGO or morphine stimulation (E, F) in chronic enteric neurons.</p

Opioid-induced MAPK activation in naïve (A) and chronically treated (B) enteric neurons.

<p>DAMGO (1 µM, black bars) induced a transient MAPK/ERK1/2 activation in naïve (A) and chronic (B) neurons at 5 and 10 minutes, whereas morphine (grey bars) induced MAPK/ERK1/2 activation only in chronic (B) neurons. **p<0.01 compared to controls (white bars). N = 4–7 experiments in triplicate. Representative gels of pERK1/2 and tERK are shown at the bottom of each graph. tERK was used to verify that the treatment did not affect the total level of this protein and to confirm equal gel loading.</p

Diet-Induced Regulation of Bitter Taste Receptor Subtypes in the Mouse Gastrointestinal Tract

<div><p>Bitter taste receptors and signaling molecules, which detect bitter taste in the mouth, are expressed in the gut mucosa. In this study, we tested whether two distinct bitter taste receptors, the bitter taste receptor 138 (T2R138), selectively activated by isothiocyanates, and the broadly tuned bitter taste receptor 108 (T2R108) are regulated by luminal content. Quantitative RT-PCR analysis showed that T2R138 transcript is more abundant in the colon than the small intestine and lowest in the stomach, whereas T2R108 mRNA is more abundant in the stomach compared to the intestine. Both transcripts in the stomach were markedly reduced by fasting and restored to normal levels after 4 hours re-feeding. A cholesterol-lowering diet, mimicking a diet naturally low in cholesterol and rich in bitter substances, increased T2R138 transcript, but not T2R108, in duodenum and jejunum, and not in ileum and colon. Long-term ingestion of high-fat diet increased T2R138 RNA, but not T2R108, in the colon. Similarly, α-gustducin, a bitter taste receptor signaling molecule, was reduced by fasting in the stomach and increased by lowering cholesterol in the small intestine and by high-fat diet in the colon. These data show that both short and long term changes in the luminal contents alter expression of bitter taste receptors and associated signaling molecules in the mucosa, supporting the proposed role of bitter taste receptors in luminal chemosensing in the gastrointestinal tract. Bitter taste receptors might serve as regulatory and defensive mechanism to control gut function and food intake and protect the body from the luminal environment.</p></div

Effect of a High Fat Diet on T2R138, T2R108 and α-Gustducin (Gust) Expression in the Ileum and Colon.

<p>qRT-PCR analysis shows that T2R138 mRNA and α-gustducin mRNA levels are significantly (*p<0.05; **p<0.01) up-regulated in the colon, but not ileum by a long term (8 weeks) high fat (45% and 60%) diet compared to low (10%) fat diet. By contrast, T2R108 mRNA was not affected by this diet.</p

Cells Expressing T2R138 in the Gut.

<p>Confocal images of mouse ileum showing colocalization of T2R138 (green) (A) with α-Gustducin (red) (B) immunoreactivity; C: shows overlay of both T2R138 and α-Gustducin immunoreactivity. D–I: Confocal images showing colocalization of T2R138 (green) (D, G) with chromogranin A, a marker of enteroendocrine cells (red) (E, H) and overlay of both immunoreactivities in the same cells (F,I) in the ileum (D–F) and distal colon (G–I). Calibration bar: 20 µm.</p

TR2138, T2R108 and α-Gustducin (Gust) Regulation by Fasting/Re-feeding in the Stomach.

<p>mRNA levels for each transcript were analyzed by qRT-PCR and normalized to β-actin. T2R138, T2R108 and α-gustducin mRNA levels were markedly decreased by fasting (82%, 53% and 37%, respectively compared to controls) and restored by re-feeding. *p<0.05, **p<0.01 vs. fasted. CTR, control; F, fasted; F/R, re-feeding following fasting.</p

Expression of mT2R138, mT2R108 and α-Gustducin (Gust) mRNA in the mouse Gastrointestinal Tract.

<p>mRNA levels were analyzed in the different regions of the GI tract with qRT-PCR and normalized to β-actin levels in each tissue (A–C). Relative quantities were determined using the comparative ΔΔCt method. Each cDNA sample was amplified in duplicate and all data are expressed as the mean ± S.E.M. T2R138 expression was very low in the stomach compared to the small and large intestine (A), whereas T2R108 is more abundant in the stomach compared to the intestine (B). α-Gustducin is more abundant in the stomach and colon compared to the other regions (C). D: Single bands of the predicted size were found for each primer, in all GI segments analyzed as well as in STC1 cells or the tongue, which served as positive controls. No signal was detected with any of the primer in 3T3 cells, which were used as negative control. A:antrum, C:corpus, D:duodenum, J: jejunum, I: ileum, PC: proximal colon, DC: distal colon, STC1: STC 1 cells, BA: β actin; CTR, control; * the tongue is shown as control for α-gustducin.</p

Distribution of T2R138 Immunoreactivity in the Gastrointestinal Tract.

<p>Confocal images. T2R138 immunostaining in cells (arrows) of the tongue (A) and in isolated cells (arrows) along the GI tract (B, D, E). B: T2R138 immunoreactive cell in the jejunum. C: Lack of specific staining in a section incubated with T2R138 antibody pre-adsorbed with the antigen against which the antibody was raised. D and E: T2R138 immunoreactive cells in the proximal (D) and distal (E) colon. F: high magnification of a T2R138 immunoreactive cell of the colon showing the granular staining concentrated toward the base of the cell. Calibration bar: 20 µm in A–E, 10 µm in F.</p