Enterochromaffin (EC) cells of the diffuse neuroendocrine cell system secrete serotonin

Enterochromaffin (EC) cells of the diffuse neuroendocrine cell system secrete serotonin (5-HT) with activation of gut motility secretion and pain. agonist stimulated whereas the A2B receptor antagonist MRS1754 inhibited 5 release (EC50 = 1.8 × 10?6 M; IC50 = 3.7 × 10?8 M) which was associated with corresponding alterations in intracellular cAMP levels and pCREB (Ser133). Mechanical stimulation using a rhythmic flex model induced transcription and activation of (tryptophan hydroxylase) and VMAT1 (vesicular monoamine transporter 1) and the release of 5-HT which could be inhibited by MRS1754 and amplified by NECA. Secretion was also inhibited by H-89 (PKA inhibitor) while and VMAT1 transcription was regulated by PKA/MAPK and PI3K-mediated signaling. Normal and IBD-EC cells also responded to NECA and mechanical stimulation with PKA activation cAMP production and 5-HT release effects reversible by MRS1754. EC cells express stimulatory MK-2461 ADORA2B and MK-2461 rhythmic stretch induces A2B activation PKA/MAPK/IP3-dependent transcription and PKA-dependent secretion of 5-HT synthesis and secretion. Receptor expression is amplified in IBD and neoplasia and 5-HT release is increased. Determination of factors that regulate EC cell function are necessary for understanding its role as a mechanosensory cell and to facilitate the development of agents that can selectively target cell function in EC cell-associated MK-2461 disease. = 8) was obtained from patients who had undergone hemi- or colectomies for Crohn’s ileitis (= 2) or colitis (= 6). Only grossly affected tissue was studied. Macroscopically “normal” tissue was obtained from patients undergoing surgery for diverticulitis (= 3) or hemicolectomies for colon cancer (colon = 4). MK-2461 All tissue was collected between 2008 and 2010 at the Yale University Department of Surgery. The institutional review board has designated this as nonhuman subjects research. Normal and IBD-EC cell isolation. Crohn’s disease (= 8) and normal colon (= 7) mucosa were used for the isolation and study of MK-2461 short-term cultured EC cells (28 31 EC cells were isolated from the colon following mucosal stripping and enzymatic digestion using a combination of Nycodenz gradient fractionation and acridine orange uptake and cell FACS were sorted as described (38). In previous studies we obtained preparations of >98% pure EC cells (28 31 38 Approximately 1 × 106 cells were obtained per mucosal sample a quantity sufficient for real-time PCR short-term culture and mechanical strain and secretion studies (28 31 38 KRJ-I cell culture. KRJ-I cells were cultured in Ham’s F-12 media supplemented with 10% Has2 fetal calf serum penicillin (100 U/ml) and streptomycin (100 mg/ml) (Sigma) at 37°c with 5% CO2. Cells were cultured for 24 h before any experiments. Basal 5-HT concentrations in the cell medium of untreated cells were 3-4 ng/ml. In initial experiments real-time PCR was undertaken to confirm ADORA expression. Cells were then incubated with NECA SCH442416 or MRS1754 (10?11-10?4 M) for 60 min to determine effects on basal-line (nonmechanically stimulated) 5-HT release and cAMP activation. Flexercell strain experiments. Normal and IBD-EC cells or KRI-I cells were plated on collagen-coated flexible-bottomed wells (Flex I plates; Flexercell International Hillsborough NC). Normal and IBD-EC cells were cultured for 8 h before experiments whereas KRJ-1cells were precultured for 24 h. Cells were subjected to cycles of stretch and relaxation by a computer-driven vacuum-operated MK-2461 Flexercell strain unit FX-4000 (Flexercell International). Rhythmic deformation was applied at a frequency of 10 cycles/min with 10% average radial elongation of cells for 0-6 h as previously described (3). In some experiments KRJ-1 cells were exposed to rhythmic deformation with or without preincubation with ADORA receptor modulating compounds (NECA 1 × 10?5 M; SCH442416 1 × 10?6 M; MRS1754 1 × 10?7 M; controls Ham’s F12 medium alone). PKA/cAMP signaling pathway analysis. Cultured cells were stimulated with NECA (1 × 10?5 M) or mechanically stressed (Flexercell) for 1 h. In some studies ADORA antagonists were used. PKA activity and cAMP were quantified using SuperArray ELISA kits (SA Biosciences) as.