The stimulatory adenosine receptor ADORA2B regulates serotonin (5-HT) synthesis and release in oxygen-depleted EC cells in inflammatory bowel disease

PLoS One. 2013 Apr 26;8(4):e62607. doi: 10.1371/journal.pone.0062607. Print 2013.

Abstract

Objective: We recently demonstrated that hypoxia, a key feature of IBD, increases enterochromaffin (EC) cell 5-HT secretion, which is also physiologically regulated by the ADORA2B mechanoreceptor. Since hypoxia is associated with increased extracellular adenosine, we wanted to examine whether this nucleotide amplifies HIF-1α-mediated 5-HT secretion.

Design: The effects of hypoxia were studied on IBD mucosa, isolated IBD-EC cells, isolated normal EC cells and the EC cell tumor derived cell line KRJ-1. Hypoxia (0.5% O2) was compared to NECA (adenosine agonist), MRS1754 (ADORA2B receptor antagonist) and SCH442146 (ADORA2A antagonist) on HIF signaling and 5-HT secretion. Antisense approaches were used to mechanistically evaluate EC cells in vitro. PCR and western blot were used to analyze transcript and protein levels of HIF-1α signaling and neuroendocrine cell function. An animal model of colitis was evaluated to confirm hypoxia:adenosine signaling in vivo.

Results: HIF-1α is upregulated in IBD mucosa and IBD-EC cells, the majority (~90%) of which express an activated phenotype in situ. Hypoxia stimulated 5-HT release maximally at 30 mins, an effect amplified by NECA and selectively inhibited by MRS1754, through phosphorylation of TPH-1 and activation of VMAT-1. Transient transfection with Renilla luciferase under hypoxia transcriptional response element (HRE) control identified that ADORA2B activated HIF-1α signaling under hypoxic conditions. Additional signaling pathways associated with hypoxia:adenosine included MAP kinase and CREB. Antisense approaches mechanistically confirmed that ADORA2B signaling was linked to these pathways and 5-HT release under hypoxic conditions. Hypoxia:adenosine activation which could be reversed by 5'-ASA treatment was confirmed in a TNBS-model.

Conclusion: Hypoxia induced 5-HT synthesis and secretion is amplified by ADORA2B signaling via MAPK/CREB and TPH-1 activation. Targeting ADORA2s may decrease EC cell 5-HT production and secretion in IBD.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenosine / pharmacology
  • Adult
  • Aged
  • Animals
  • Cell Hypoxia / drug effects
  • Cell Hypoxia / genetics
  • Colitis / metabolism
  • Colitis / pathology
  • Disease Models, Animal
  • Enterochromaffin Cells / drug effects
  • Enterochromaffin Cells / metabolism*
  • Enterochromaffin Cells / pathology
  • Female
  • Humans
  • Hypoxia-Inducible Factor 1, alpha Subunit / genetics
  • Hypoxia-Inducible Factor 1, alpha Subunit / metabolism
  • Inflammatory Bowel Diseases / metabolism*
  • Inflammatory Bowel Diseases / pathology
  • Intestinal Mucosa / drug effects
  • Intestinal Mucosa / metabolism
  • Intestinal Mucosa / pathology
  • Male
  • Middle Aged
  • Oxygen / pharmacology*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Receptor, Adenosine A2B / metabolism*
  • Response Elements / genetics
  • Serotonin / biosynthesis*
  • Serotonin / metabolism*
  • Signal Transduction / drug effects
  • Signal Transduction / genetics
  • Trinitrobenzenesulfonic Acid

Substances

  • Hypoxia-Inducible Factor 1, alpha Subunit
  • RNA, Messenger
  • Receptor, Adenosine A2B
  • Serotonin
  • Trinitrobenzenesulfonic Acid
  • Adenosine
  • Oxygen