1. Volume 127, Issue 1, Pages 188-202 (July 2004)
Endogenous adenosine differentially modulates 5-hydroxytryptamine release from a human enterochromaffin cell model 
Fievos L. Christofi, Minsoo Kim, Jacqueline E. Wunderlich, Jianjing Xue, Zach Suntres, Arturo Cardounel, Najma H. Javed, Jun Ge Yu, Iveta Grants, Helen J. Cooke 
Gastroenterology 
Volume 127, Issue 1, Pages (July 2004)
DOI: /j.gastro

2. Figure 1
Expression of mRNA transcripts for adenosine receptors in human BON cells. (A) The PCR products in lanes 3, 5, 7, and 9 are predicted sizes of 294, 321, 325, and 361 bps, respectively. Positive controls are from human lung and human cell lines T98G, U373, and HT 29 cells. Each band was isolated and sequenced. (B) Western blots indicate the presence of A1, A2a, and A3R proteins: 74 kilodaltons (A1R), 48–50 kilodaltons (A2aR), and 23–24 kilodaltons or 52 kilodaltons (A3R).
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
Expression of adenosine receptors in human carcinoid BON cells. (A-C) A2aR immunoreactivity in the human BON cells is found predominately in the cytoplasm and cell membrane. (E-G) Polarized clustering of A2bR immunoreactivity is clearly evident in many cells (E and F, arrows); observed at one end of the cell (E), sometimes surrounding the cell (G), or at opposite poles (F, upper arrow). In other cells, strong staining is observed in membrane and cytoplasmic regions in clusters (F and G) or vesicular structures (G). Arrows (B and F) indicate putative cell membrane staining. (I-L) A3R immunoreactivity is distributed throughout (I-K), and occasionally dense immunoreactivity is present in the outer cell membranes (I-K, arrows). (M-O) A1R immunoreactivity has a punctate appearance and is distributed throughout the cell. Arrows (N) are putative outer membrane staining. Preabsorption of each antibody, A2a, A2b, A3, and A1R, with its immunogenic peptide prevents all immunoreactivity (D, H, L, and P, respectively). Scale bar = 30 μm.
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
Comparative immunohistochemical staining of adenosine receptors, 5-HT, and synaptophysin in 2 human gastrointestinal carcinoid tumors. Sections of ileal (A-H) and gastric (I-O) carcinoid tumors are illustrated. Primary A3R antibody preabsorbed with antigenic peptides (B and J) and primary antibody omitted (H) were used as negative controls. Counterstaining was performed with Mayer’s hematoxylin (blue). (A) Shows nests of tumor cells with strong A3R immunoreactivity in cell membranes and cytoplasm (arrows). Arrows in (C) and (K) indicate that granular staining with A1R antibody is mainly distributed in the peripheral cells in tumor nests. Arrows in (D) and (L) show tumor nests stained with an anti-A2aR antibody. In (E) and (M), a finely granular A2bR immunoreactivity is present in most tumor cells (arrows). 5-HT is distributed in the majority of carcinoid cells in terminal ileum (F) but in fewer cells in stomach (N). Synaptophysin, a marker for neuroendocrine cells, is present in most tumor cells as shown in (G) and (O). Scale bar = 30 μm.
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
Adenosine concentration in the extracellular medium of a monolayer of BON cells. (A) Representative chromatograms for adenosine analysis using the LC-MS/MS technique: exogenous adenosine with internal 13C standard beneath. (B) Chromatogram of supernatant from BON cells seeded at a density of 1 × 106 cells and incubated 20 minutes under resting conditions; the internal 13C standard is beneath. (C) Adenosine release in the extracellular medium during baseline (Base) conditions and during mechanical stimulation (MS) with or without the adenosine uptake inhibitor S-(p-nitrobenzyl)-6-thio-inosine (1 μmol/L NBTI). NBTI elevated mechanically evoked extracellular adenosine levels and baseline adenosine (n = 3). (D) In the same cells as C, NBTI blocked 5-HT release.
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
Evidence that endogenous adenosine release may not activate A1Rs to suppress baseline or stimulated 5-HT release or cAMP levels. (A) Concentration-dependent increase in baseline 5-HT release by adenosine deaminase, indicating that endogenous adenosine provides ongoing inhibition of baseline 5-HT release. In A, for 0.5 units/mL, P < 0.05 from control (BASE); 1 vs. 2, P < (B) The A1R antagonist CPT (100 nmol/L) did not mimic the response to adenosine deaminase nor did it alter baseline 5-HT levels. In B, P > 0.05 for BASE vs. CPT for 5-HT or cAMP. (C) Isoproterenol increased 5-HT and cAMP in a concentration-dependent manner. In C, for cAMP, BASE vs. 10−7 to 10−4 mol/L isoproterenol, P < 0.05; for 5-HT, BASE vs. 10−6 to 10−4 mol/L isoproterenol, P < (D) The A1R agonist, CCPA (100 nmol/L) had no significant effect on BASE or isoproterenol-stimulated 5-HT release (P > 0.05). Baseline 5-HT and cAMP with the number of experiments in parentheses are expressed as pmol/well/30 minutes.
Gastroenterology  , DOI: ( /j.gastro )

7. Figure 6
Endogenous adenosine release activates excitatory A2R and inhibitory A3Rs to regulate 5-HT release in BON cells. (A) The A3R agonist IB-MECA (IB; 100 nmol/L) reduced baseline (BASE) 5-HT and cAMP levels. The A3R antagonist MRS 1191 (10 nmol/L) augmented 5-HT release but not cAMP levels (P < 0.05). The MRS HT response was sensitive to IB-MECA inhibition. (B) The A2R agonist MECA (ME; 100 nmol/L) increased 5-HT and cAMP levels. The A2R antagonist alloxazine (AL, 5 μmol/L) reduced both 5-HT and cAMP responses to MECA. (C) Alloxazine (AL) or the A2aR antagonist CSC (1 μmol/L) reduced the MECA effect; AL + CSC caused the biggest inhibition of the agonist response. (D) The A2aR agonist CGS21680 stimulated 5-HT release that could be antagonized by CSC (100–1000 nmol/L). (E) CGS elevated cAMP levels. (F) Mechanical activation by rotational shaking caused an increase in both cAMP and 5-HT levels. (G) Mechanically evoked 5-HT release was significantly reduced by the combined effect of CSC (75 nmol/L) and AL. Baseline values (pmol/well/30 minutes) of 5-HT and cAMP are at the top of each figure, with the number of experiments indicated in parentheses.
Gastroenterology  , DOI: ( /j.gastro )

8. Figure 7
Baseline and touch evoked changes in [Ca2+]i in BON cells: (A) Control touch response without drugs. LSM imaging reveals that touch activation of a BON cell leads to a time-dependent elevation in [Ca2+]i. (B) The A3R antagonist MRS 1191 (100 nmol/L) augments the touch Ca2+ response compared with A; corresponding Ca2+ transients (changes in pixel intensities) are shown on the right for both A and B: 1, before touch; 2, touch; 3, 15 seconds after touch; 4, 45 seconds after touch; the arrow (↑) indicates the touch. (C) Isoproterenol (10 μmol/L) caused a slow increase in baseline [Ca2+]i over 20 minutes. The Ca2+ transient is shown next to the images. (A–C). Images are pseudo-colored based on a gray scale of 0–255. A 10 × 10 pixel square area was used to calculate pixel intensities. (D). Isoproterenol caused an increase in baseline [Ca2+]i and reached a plateau after 20 minutes of perfusion before touch. Touch Ca2+ response during the plateau reached with isoproterenol in the bath was similar in amplitude to the control touch response, thus isoproterenol and touch gave additive responses. Values are mean ± SEM.
Gastroenterology  , DOI: ( /j.gastro )

9. Figure 8
Adenosine A1 and A3R inhibitory components of touch-evoked Ca2+ responses in BON cells. (A) The A3R antagonist MRS1191 augmented the touch Ca2+ transient (P < 0.05). (B) Chronic exposure to the A1 antagonist CPT for 24 hours caused a significant augmentation in the touch Ca2+ response (P < 0.05). (C) The stimulatory effects of CPT and MRS1191 were additive on the touch Ca2+ response. (D) The additive effects of CPT and MRS1191 are shown in a histogram. The first stacked bars were obtained in separate experiments with CPT or MRS1191. The single (nonshaded bar) was obtained in cells exposed to both CPT and MRS1191. The bars are nearly identical. (E) Adenosine deaminase (5 U/mL)-induced augmentation is reduced by the A3R agonist IB-MECA (50 nmol/L). (F) Histogram summarizing the peak Ca2+ responses for each condition in A–E. Values are mean ± SEM.
Gastroenterology  , DOI: ( /j.gastro )