HIF-1α, adenosine signaling and HRE activation.

<p><b>3A)</b> Curcumin inhibited (022±0.04 fold) while NECA (1.24±0.06 fold) stimulated and MRS1754 (0.74±0.02) inhibited HIF-1α protein levels compared to 30 minutes hypoxia in KRJ-I cells. SCH442146 had no effect. <b>3B)</b> Transient transfection with <i>Renilla</i> luciferase-encoding constructs in KRJ-I cells. Under normoxic conditions, NECA amplified activation of luciferase (RLU) while MRS1754 inhibited this. Curcumin and SCH442416 had no effect. Hypoxic conditions activated RLU (1.81±0.12), which was inhibited by curcumin and MRS1754 and amplified by NECA. <b>3C)</b> In IBD-EC cells, RLU was elevated in normoxic conditions. This could be reduced by curcumin and MRS1754 and amplified by NECA. Mean±SEM, <i>n</i> = 3–7, *<i>p</i><0.05 vs. 30 min hypoxia or control, **<i>p</i><0.05 vs. normoxic cells, ^#<i>p</i><0.05 vs. 30 min hypoxia. NS = not significant.</p

Analysis of 5-HT release and signaling pathways in ADORA2B-deficient rat EC cells.

<p><b>5A)</b> A 12 hr antisense approach inhibited ADORA2B transcript (real-time PCR) and protein expression (membrane-bound, flow cytometry) in isolated rat EC cells. Membrane expression was reduced to an estimated 5% of non-targeted cells. <b>5B)</b> In non-targeted cells, 5-HT release was elevated by hypoxia and inhibited by curcumin. Adenosine signaling responses were similar to human EC cells. Antisense reduced hypoxic-mediated responses (compared to controls) and ameliorated cell responses to NECA and MRS1754. <b>5C)</b> Western blot identified decreased pMAPK (0.56±0.09), pCREB (0.75±0.11) and pTPH-1 (0.63±0.07) in antisense treated cells confirming down-regulation of these pathways with loss of ADORA2B expression under hypoxic conditions. Mean±SEM, <i>n</i> = 3. *<i>p</i><0.05 vs. control, **<i>p</i><0.05 vs hypoxia, ^#<i>p</i><0.05 vs. hypoxia (in non-antisense treated cells). NS = not significant.</p

<i>HIF-1α</i> transcripts and protein in normal mucosa, IBD mucosa, isolated normal and IBD-EC cells.

<p><b>1A)</b> Transcript of HIF-1α was significantly elevated in IBD-associated conditions (mucosa: 3.4±0.63 fold, cells: 2.4±0.41). <b>1B)</b> Protein levels were significantly elevated in IBD-associated conditions (mucosa: 12.2±3.4, cells: 2.6±0.36). <b>1C)</b> Immunohistochemical staining of HIF-1α and Chromogranin A (white arrows) in normal mucosa and Crohn’s mucosa identified co-staining (yellow arrows) predominantly in IBD mucosa. <b>1D</b>) Quantitation identified significantly more enteroendocrine cells to be HIF-1α positive in IBD mucosa. Mean±SEM, <i>n</i> = 4–7, *<i>p</i> = 0.03 vs. normal mucosa, ^#<i>p</i> = 0.04 vs. normal EC cells,^##<i>p</i><0.001 vs. normal EC cells. DAPI – nuclei (blue), FITC-CgA (green), Cy5-HIF-1α (red), co-localization (yellow). N–M = normal mucosa, IBD-M = IBD mucosa, N–C = normal EC cells, IBD-C = IBD EC cells.</p

Effect of adenosine on the hypoxia-activated 5-HT pathway in KRJ-I cells.

<p><b>2A)</b> 5-HT was increased by hypoxia between 15–120 min, with a maximal effect at 30 min (2.28±0.12 fold). Curcumin and MRS1754 inhibited while NECA augmented secretion at all time points up to 120 mins. SCH442146 had no significant effect. <b>2B)</b> Total TPH-1 protein levels were unchanged after 30 min of hypoxia and after NECA or MRS1754 stimulation. Phosphorylated TPH-1 was significantly increased under hypoxia (1.80±0.26), was amplified by NECA (2.66±0.28) and reduced by MRS1754 to baseline. <b>2C)</b> VMAT-1 protein levels were significantly increased (2.83±0.31) by NECA during hypoxia and reduced by MRS1754 (2±0.1). <b>2D)</b> Chromogranin A protein levels did not change significantly after hypoxia or with the addition of NECA or MRS 1754. Mean±SEM, <i>n</i> = 3–8, *<i>p</i><0.05 vs. control, **<i>p</i><0.05 vs. hypoxia. NS = not significant.</p

Analysis of HIF-1α and ADORA2B in mucosa from the animal TNBS-induced colitis model with or without 5-ASA-treatment.

<p>6A) TNBS was associated with a significant elevation in HIF-1α and ADORA2B transcripts. Treatment with 5-ASA reversed these values to those similar to untreated rats. 6B) Protein expression was elevated by TNBS (HIF-1α: 1.68±0.11; ADORA2B: 1.37±0.09) and reversed by 5-ASA in mucosa. Mean±SEM, <i>n</i> = 3. *<i>p</i><0.05 versus control, **<i>p</i><0.05 versus TNBS.</p