scholarly journals The role of mechanical forces and adenosine in the regulation of intestinal enterochromaffin cell serotonin secretion

2012 ◽  
Vol 302 (3) ◽  
pp. G397-G405 ◽  
Author(s):  
A. Chin ◽  
B. Svejda ◽  
B. I. Gustafsson ◽  
A. B. Granlund ◽  
A. K. Sandvik ◽  
...  
Keyword(s):  
Mechanical Stimulation ◽  
Tryptophan Hydroxylase ◽  
Cell Function ◽  
Cell System ◽  
Receptor Expression ◽  
Intracellular Camp ◽  
Serotonin Secretion ◽  
A2b Receptor ◽  
Ec Cells ◽  
Inflammatory Bowel

Enterochromaffin (EC) cells of the diffuse neuroendocrine cell system secrete serotonin (5-HT) with activation of gut motility, secretion, and pain. These cells express adenosine (ADORA) receptors and are considered to function as mechanosensors. Physiological pathways mediating mechanosensitivity and adenosine responsiveness remain to be fully elucidated, as do their roles in inflammatory bowel disease (IBD) and neoplasia. Pure (98–99%) FACS-sorted normal and IBD human EC cells and neoplastic EC cells (KRJ-I) were studied. IBD-EC cells and KRJ-I overexpressed ADORA2B. NECA, a general ADORA receptor agonist, stimulated, whereas the A2B receptor antagonist MRS1754 inhibited, 5-HT 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 Tph1 (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 Tph1 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 ADORA2B, and 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 disease.

Isolation, functional characterization, and transcriptome of Mastomys ileal enterochromaffin cells

2006 ◽  
Vol 291 (5) ◽  
pp. G778-G791 ◽  
Author(s):  
M. Kidd ◽  
I. M. Modlin ◽  
G. N. Eick ◽  
M. C. Champaneria
Keyword(s):  
Tryptophan Hydroxylase ◽  
Somatostatin Receptor ◽  
Receptor Expression ◽  
Intestinal Cell ◽  
Marker Genes ◽  
Cell Marker ◽  
Serotonin Content ◽  
Serotonin Secretion ◽  
Ec Cells ◽  
Cell Transcriptome

Although the enterochromaffin (EC) cell is one of the primary neuroendocrine regulatory cells of the small intestine, the lack of a purified cell system has precluded characterization of the cell and limited precise physiological evaluation. We developed methodology to obtain a pure population of Mastomys ileal EC cells, evaluated their functional regulation, and defined the transcriptome. Mastomys ilea were everted, end ligated, pronase-collagenase digested, and Nycodenz gradient centrifuged, and EC cells were collected by fluorescence-activated cell sorting (FACS) of acridine orange-labeled cells. Enrichment was confirmed by immunostaining of tryptophan hydroxylase and chromogranin A, specific EC cell markers, serotonin content, EC cell marker gene expression, and electron microscopy. Pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin, and gastrin receptor expression was determined by real-time RT-PCR. Live post-FACS-sorted cells were cultured, and the effects of forskolin, isoproterenol, acetylcholine, GABAA, PACAP-38, and gastrin on serotonin secretion were measured by ELISA. GeneChip Affymetrix profiling of FACS-sorted cells was undertaken to obtain the EC cell transcriptome. FACS produced a >70-fold enrichment of EC cells with a serotonin content of 240 ± 22 ng/mg protein. Preparations were 99 ± 0.7% pure by immunostaining for tryptophan hydroxylase. Vasoactive intestinal peptide/PACAP receptor 1 (VPAC1) and somatostatin receptor 2 were present, whereas PACAP receptor 1 (PAC1) and CCK2 receptors were undetectable. Forskolin, isoproterenol, and PACAP-38 stimulated serotonin secretion at EC50 values of 5 × 10−10, 4.5 × 10−10, and 1.2 × 10−9 M, respectively. Isoproterenol stimulated cAMP levels by ∼3.5 ± 0.62-fold vs. unstimulated cells (EC50 of ∼10−9 M). Octreotide, acetylcholine, and GABAA inhibited serotonin secretion with IC50 values of 3 × 10−11, 3 × 10−10, and 2.9 × 10−10 M, respectively. Gastrin had no effect on serotonin secretion. The naive EC cell transcriptome revealed highly expressed EC cell marker genes, the absence of marker genes for other small intestinal cell types, and a receptor profile that included cholinergic, adrenergic, dopaminergic, serotoninergic, GABAergic, and prostaglandin receptors. We were able to isolate homogeneous preparations (>99%) of live ileal EC cells and demonstrated regulation of serotonin secretion as well as established the normal EC cell transcriptome. Application of this methodology to normal and diseased human ileum will facilitate the elucidation of the pathophysiology of EC cells.

scholarly journals Activation of Type 1 CRH Receptor Isoforms Induces Serotonin Release from Human Carcinoid BON-1N Cells: An Enterochromaffin Cell Model

Endocrinology ◽  
2011 ◽  
Vol 152 (1) ◽  
pp. 126-137 ◽  
Author(s):  
S. Vincent Wu ◽  
Pu-Qing Yuan ◽  
Jim Lai ◽  
Kelvin Wong ◽  
Monica C. Chen ◽  
...  
Keyword(s):  
Tryptophan Hydroxylase ◽  
Cell Function ◽  
Splice Variants ◽  
Cell Model ◽  
Serotonin Release ◽  
Intracellular Camp ◽  
Mrna Levels ◽  
Maximal Increase ◽  
Ec Cells

Abstract CRH and 5-hydroxytryptamine (5-HT) are expressed in human colonic enterochromaffin (EC) cells, but their interactions at the cellular level remain largely unknown. The mechanistic and functional relationship between CRH and 5-HT systems in EC cells was investigated in a human carcinoid cloned BON cell line (BON-1N), widely used as an in vitro model of EC cell function. First, we identified multiple CRH1 splice variants, including CRH1a, CRH1c, CRH1f, and a novel form lacking exon 4, designated here as CRH1i, in the BON-1N cells. The expression of CRH1i was also confirmed in human brain cortex, pituitary gland, and ileum. Immunocytochemistry and immunoblot analysis confirmed that BON-1N cells were CRH1 and 5-HT positive. CRH, urocortin (Ucn)-1, and cortagine, a selective CRH1 agonist, all increased intracellular cAMP, and this concentration-dependent response was inhibited by CRH1-selective antagonist NBI-35965. CRH and Ucn-1, but not Ucn-2, stimulated significant ERK1/2 phosphorylation. In transfected human embryonic kidney-293 cells, CRH1i isoforms produced a significant increase in pERK1/2 in response to CRH1 agonists that was sensitive to NBI-35965. CRH and Ucn-1 stimulated 5-HT release that reached a maximal increase of 3.3- and 4-fold at 10−8m over the basal level, respectively. In addition, exposure to CRH for 24-h up-regulated tryptophan hydroxylase-1 mRNA levels in the BON-1N cells. These findings define the expression of EC cell-specific CRH1 isoforms and activation of CRH1-dependent pathways leading to 5-HT release and synthesis; thus, providing functional evidence of a link exists between CRH and 5-HT systems, which have implications in stress-induced CRH1 and 5-HT-mediated stimulation of lower intestinal function.

scholarly journals Further delineation of the continuous human neoplastic enterochromaffin cell line, KRJ-I, and the inhibitory effects of lanreotide and rapamycin

2007 ◽  
Vol 38 (1) ◽  
pp. 181-192 ◽  
Author(s):  
Mark Kidd ◽  
Geeta N Eick ◽  
Irvin M Modlin ◽  
Roswitha Pfragner ◽  
Manish C Champaneria ◽  
...  
Keyword(s):  
Substance P ◽  
Cell Line ◽  
Tryptophan Hydroxylase ◽  
Intracellular Camp ◽  
Cell Of Origin ◽  
Rt Pcr ◽  
Cytoplasmic Vesicles ◽  
Ec Cells ◽  
Gastrointestinal Carcinoid

Small intestinal carcinoids (SICs) are the most prevalent gastrointestinal carcinoid and characterized by local invasion metastasis and protean symptomatology. The proliferative and secretory regulation of the cell of origin, the enterochromaffin (EC) cell has not been characterized. The absence of either a pure preparation of normal EC cells or human EC carcinoid cell lines has hindered the development of therapeutic agents. We therefore further characterized the neoplastic SIC cell line, KRJ-I by assessing its secretory (serotonin (5-HT)) and proliferative responses and defining its log growth phase transcriptome. Electron microscopy demonstrated oval, lobulated nuclei and substance P, and 5-HT-positive cytoplasmic vesicles. RT-PCR detected transcripts for chromogranin A (CHGA), VMAT1 (SLC18A1), tryptophan hydroxylase (TPH1), substance P (TAC1), guanylin (GUCA2A), and SERT (SLC6A4). By immunohistochemistry, all cells were positive for CHGA, SERT, VMAT1, and TPH1. Transcriptome analysis (Affymetrix U133 Plus chips) identified somatostatin SSTR2/3, adrenergic α1C and β1, dopamine D2, nicotinic-type cholinergic A5, A6, B1, muscarinic acetylcholine M4, and 5-HT-2A receptors. The presence of transcripts for SSTR1, SSTR2, and SSTR3 receptors was confirmed by RT-PCR and sequencing. Isoproterenol (ISO) resulted in a dose-dependent increase in intracellular cAMP (EC50=340 nM) and 5-HT (EC50=81 nM) which was completely inhibited by the cAMP antagonist 2′,5′-dideoxyadenosine (10 μM). Preincubation with a SSTR agonist, lanreotide, inhibited Ip-stimulated 5-HT secretion (IC50=420 nM). Both lanreotide (10 nM) and rapamycin (50 nM) inhibited proliferation (20±12 and 35±5% respectively) in serum-free medium whereas gefitinib (1 nM–10 μM) inhibited proliferation at micromolar concentrations. KRJ-I is a neoplastic EC cell line that can be used as an in vitro model of SICs as it will allow elucidation and clarification of the secretory and proliferative mechanism(s) of neoplastic EC cells and the molecular signatures that characterize each of these responses.

scholarly journals Characterization of Serotonin Signaling Components in Patients with Inflammatory Bowel Disease

2018 ◽  
Vol 2 (3) ◽  
pp. 132-140 ◽  
Author(s):  
Md Sharif Shajib ◽  
Usha Chauhan ◽  
Salman Adeeb ◽  
Yeshale Chetty ◽  
David Armstrong ◽  
...  
Keyword(s):  
Inflammatory Bowel Disease ◽  
Bowel Disease ◽  
Tryptophan Hydroxylase ◽  
Intestinal Inflammation ◽  
Quantitative Polymerase Chain Reaction ◽  
Effective Strategies ◽  
Serotonin Signaling ◽  
Ec Cells ◽  
Inflammatory Bowel ◽  
Signaling Components

AbstractBackgroundTryptophan hydroxylase (TPH)1 catalyzes the biosynthesis of serotonin (5-hydroxytrptamine; 5-HT) in enterochromaffin (EC) cells, the predominant source of gut 5-HT. Secreted 5-HT regulates various gut functions through diverse 5-HT receptor (5-HTR) families, and 5-HT transporter (5-HTT) sequesters its activity via uptake into surrounding cells. In inflammatory bowel disease (IBD) mucosal 5-HT signaling is altered, including upregulated EC cell numbers and 5-HT levels. We examined key mucosal 5-HT signaling components and blood 5-HT levels and, as part of a pilot study, investigated the association between 5-HTT gene-linked polymorphic region (5HTTLPR) and Crohn’s disease (CD).MethodsIn the context of inflammation, colonic expressions of TPH1, 5-HTT and 5-HTRs were studied in CD patients (n=15) and healthy controls (HC; n=10) using quantitative polymerase chain reaction (qPCR). We also investigated 5HTTLPR in 40 CD patients and HC utilizing PCR and measured platelet-poor plasma (PPP) and plasma 5-HT concentrations.ResultsCompared with HC, inflammation in CD patients was associated with elevated TPH1, 5-HTR3, 5-HTR4, 5-HTR7 and downregulated 5-HTT expressions. In our second cohort of participants, significantly higher PPP and plasma 5-HT levels and higher S-genotype (L/S+S/S) than L/L genotype were observed in CD patients compared with HC.ConclusionOur results suggest that augmented mucosal 5-HT signaling and specific 5-HTTLPR genotype–associated decreased efficiency in 5-HT reuptake, the latter through increased 5-HT availability, may contribute to inflammation in CD patients. These findings revealed important information on various components of 5-HT signaling in intestinal inflammation which may ultimately lead to effective strategies targeting this pathway in IBD.

scholarly journals Efficient IL-2R signaling differentially affects the stability, function, and composition of the regulatory T-cell pool

2021 ◽  
Author(s):  
Marc Permanyer ◽  
Berislav Bošnjak ◽  
Silke Glage ◽  
Michaela Friedrichsen ◽  
Stefan Floess ◽  
...  
Keyword(s):  
T Cell ◽  
Treg Cell ◽  
Cell Function ◽  
Interleukin 2 ◽  
Foxp3 Expression ◽  
Treg Cells ◽  
Receptor Expression ◽  
Spontaneous Mutant ◽  
Cell Pool ◽  
And Function

AbstractSignaling via interleukin-2 receptor (IL-2R) is a requisite for regulatory T (Treg) cell identity and function. However, it is not completely understood to what degree IL-2R signaling is required for Treg cell homeostasis, lineage stability and function in both resting and inflammatory conditions. Here, we characterized a spontaneous mutant mouse strain endowed with a hypomorphic Tyr129His variant of CD25, the α-chain of IL-2R, which resulted in diminished receptor expression and reduced IL-2R signaling. Under noninflammatory conditions, Cd25Y129H mice harbored substantially lower numbers of peripheral Treg cells with stable Foxp3 expression that prevented the development of spontaneous autoimmune disease. In contrast, Cd25Y129H Treg cells failed to efficiently induce immune suppression and lost lineage commitment in a T-cell transfer colitis model, indicating that unimpaired IL-2R signaling is critical for Treg cell function in inflammatory environments. Moreover, single-cell RNA sequencing of Treg cells revealed that impaired IL-2R signaling profoundly affected the balance of central and effector Treg cell subsets. Thus, partial loss of IL-2R signaling differentially interferes with the maintenance, heterogeneity, and suppressive function of the Treg cell pool.

scholarly journals Peutz-Jeghers syndrome: Quantitative study on enterochromaffin cells in hamartomatous intestine polyps

2013 ◽  
Vol 141 (9-10) ◽  
pp. 602-607
Author(s):  
Miljan Krstic ◽  
Vuka Katic ◽  
Slavica Stojnev ◽  
Dragan Mihailovic ◽  
Marijola Mojsilovic ◽  
...  
Keyword(s):  
Clinical Manifestations ◽  
Enterochromaffin Cells ◽  
Ileal Mucosa ◽  
Cell Hyperplasia ◽  
Ki 67 ◽  
Intestinal Polyps ◽  
Serotonin Secretion ◽  
Formalin Fixed Paraffin Embedded ◽  
Ec Cells ◽  
Peutz Jeghers Syndrome

Introduction. Peutz-Jeghers (PJ) syndrome is a rare familial disorder with the autosomal transmission characterized by multiple intestinal polyps, mucocutaneous pigmentation and increased incidence of various malignancies. Some clinical manifestations of PJ syndrome may be associated with the serotonin secretion from the enterochromaffin cells (EC). Objective. Since no data have been reported so far regarding EC cells in PJ polyps, the aim of our study was to quantitatively investigate EC population in hamartomatous intestinal polyps in patients with the PJ syndrome. Methods. The samples of surgically removed PJ polyps from family members with the PJ syndrome were collected during 34-year follow-up period. Formalin-fixed paraffin-embedded specimens of twenty-one PJ polyps were stained with HE, AB-PAS, Van Gieson, Fontana-Masson, FIF and Grimelius. For immuno- histochemical analysis, the following antibodies were used: chromogranin A, serotonin, Ki-67, desmin, vimentin and cytokeratin in order to eliminate differential diagnostic possibilities and to confirm diagnosis of PJ polyps. Results. Strong EC cell hyperplasia was observed within the tissue of the investigated polyps. Statistical analysis demonstrated significantly higher content of EC cells in PJ polyps than in the normal ileal mucosa. Conclusion. Marked hyperplasia of EC cells within the PJ polyps may be the most important contributor to functional disorders in patients with the PJ syndrome.

Expression of parathyroid hormone-related peptide (PTHrP) and PTH/PTHrP receptor in newborn human calvaria osteoblastic cells

1997 ◽  
Vol 136 (6) ◽  
pp. 640-648 ◽  
Author(s):  
Abderrahim Lomri ◽  
Cindy de Pollak ◽  
Michael Sebag ◽  
David Goltzman ◽  
Richard Kremer ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Parathyroid Hormone ◽  
Culture Media ◽  
Receptor Expression ◽  
Osteoblastic Cells ◽  
Pcr Analysis ◽  
Intracellular Camp ◽  
Related Peptide ◽  
Parathyroid Hormone Related Peptide ◽  
Pthrp Receptor

Abstract We examined the expression of parathyroid hormone-related peptide (PTHrP) and its receptor in normal newborn human calvaria osteoblastic (NHCO) cells. Northern blot analysis showed that NHCO cells express a single 1·6 kb transcript of PTHrP, which was increased within 1 h (2x) and peaked at 6 h (7x) after serum treatment. In the culture media, the release of PTHrP peptide was maximally increased (4x) 24 h after the addition of serum, as determined by immunoradiometric assay. NHCO cells exhibited a cytoplasmic immunostaining for PTHrP in the presence of serum, and most PTHrP-positive cells were alkaline phosphatase-negative, suggesting that PTHrP was expressed in undifferentiated cells. Furthermore, RT-PCR analysis showed that both PTHrP and PTH/PTHrP receptor were expressed in NHCO cells in basal conditions or after stimulation with serum. The maximal PTHrP expression induced by serum suppressed PTH/PTHrP receptor expression, suggesting that PTHrP down-regulated its receptor in NHCO cells. Treatment with 10 nm human PTH(1–34—which binds to PTH/PTHrP receptors, increased intracellular cAMP levels and alkaline phosphatase activity, and decreased cell growth, indicating that ligand binding to PTH/PTHrP receptors regulates NHCO cell proliferation and differentiation. The expression and synthesis of PTHrP and the presence of functional PTH/PTHrP receptors suggest a possible paracrine mechanism of action of PTHrP in normal human calvaria osteoblastic cells. European Journal of Endocrinology 136 640–648

Sensitization to Mechanical Stimulation by Inflammatory Mediators and by Mild Burn in Canine Visceral Nociceptors In Vitro

2002 ◽  
Vol 87 (4) ◽  
pp. 2043-2051 ◽  
Author(s):  
Hisashi Koda ◽  
Kazue Mizumura
Keyword(s):  
Mechanical Stimulation ◽  
Inflammatory Mediators ◽  
Mechanical Response ◽  
Second Messengers ◽  
Intracellular Camp ◽  
Solution Ph ◽  
In Vitro Investigation ◽  
Anesthetized Dogs ◽  
C 30

Hyperalgesia to mechanical stimulation and heat is commonly observed in inflamed conditions. Although sensitization to heat is well documented and its mechanism has also been well studied, it remains unclear whether and how nociceptors are sensitized to mechanical stimulation. Therefore we conducted in vitro investigation of which inflammatory mediators (bradykinin, histamine, prostaglandin E2, and protons) sensitize nociceptors to suprathreshold mechanical stimulation and at what concentrations. In addition, we studied the effects of possible second messengers for these mediators downstream of the receptors and also the effects of mild burn. Single polymodal receptor activities were recorded in canine testis-spermatic nerve preparations excised from deeply anesthetized dogs. Mechanical stimulation was applied to the identified receptive field for 10 s with a servo-controlled mechanical stimulator. Bradykinin at 0.001 μM induced neither excitation nor facilitation of the mechanical response; however, it facilitated the mechanical response at 0.01 μM and higher, levels at which significant excitation was also induced by bradykinin alone. Histamine excited the nociceptor and sensitized it to mechanical stimulation at 10 μM and higher. PG E2 also sensitized the mechanical response, but starting at 1 μM, without inducing excitation by itself. The effects of two possible intracellular messengers for these mediators were studied using forskolin (10 μM), which increases intracellular cAMP, and a protein-kinase-C-stimulating phorbol ester, phorbol 12,13-dibutyrate (0.1 μM). Both substances reversibly facilitated the mechanical response of testicular polymodal receptors. In contrast, low-pH solution (pH: 6.6–4.5) seldom induced excitation and failed to facilitate the mechanical response. After 55°C, 30-s heat stimulation, testicular polymodal receptors were sensitized to mechanical stimulation. These results demonstrated that inflammatory mediators and burn sensitized nociceptor responses to mechanical stimulation and provide support for the idea that peripheral nociceptor sensitization is a mechanism involved in hyperalgesia to mechanical stimulation in inflamed tissues.

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