Gill RK, Pant N, Saksena S, Singla A, Nazir TM, Vohwinkel L, Turner JR, Goldstein J, Alrefai WA, Dudeja PK. Function, expression, and characterization of the serotonin transporter in the native human intestine. Am J Physiol Gastrointest Liver Physiol 294: G254-G262, 2008. First published November 8, 2007 doi:10.1152/ajpgi.00354.2007.-The enteric serotonin transporter (SERT) plays a critical role in modulating serotonin availability and thus has been implicated in the pathogenesis of various intestinal disorders. To date, SERT expression and function in the human intestine have not been investigated. Current studies were designed to characterize the function, expression, distribution, and membrane localization of SERT in the native human intestine. Real-time PCR studies showed relatively higher SERT mRNA expression in the human small intestine compared with colon (ileum ϾϾ duodenum ϾϾ jejunum). Northern blot analysis revealed three mRNA hybridizing species encoding SERT (3.0, 4.9, and 6.8 kb) in the human ileum. Consistent with SERT mRNA expression, SERT immunostaining was mainly detected in the epithelial cells of human duodenal and ileal resected tissues. Notably, SERT expression was localized predominantly to the apical and intracellular compartments and was distributed throughout the crypt-villus axis. Immunoblotting studies detected a prominent protein band (ϳ70 kDa) in the ileal apical plasma membrane vesicles (AMVs) isolated from mucosa obtained from organ-donor intestine. Functional studies showed that uptake of [ 3 H]serotonin (150 nM) in human ileal AMVs was 1) significantly increased in the presence of both Na ϩ and Cl Ϫ ; 2) inhibited (ϳ50%) by the neuronal SERT inhibitor, fluoxetine (10 M) and by unlabeled 5-HT; and 3) exhibited saturation kinetics indicating the presence of a carrier-mediated process. Our studies demonstrated differential expression of SERT across various regions of the human intestine and provide evidence for the existence of a functional SERT capable of removing intraluminal serotonin in human ileal epithelial cells. serotonin uptake; serotonin transporter expression SEROTONIN (5-hydroxytryptamine, 5-HT) is a neurotransmitter and hormone that influences diverse physiological functions. About 90% of the whole body content of 5-HT is present in the gastrointestinal (GI) tract, where it plays a critical role in modulation of gut motility and fluid and electrolyte transport (10,12,28,30). The majority of 5-HT in the GI tract is synthesized and stored in secretory granules of mucosal enterochromaffin (EC) cells. EC cells continuously release small amounts of 5-HT in response to a number of chemical and mechanical stimuli (13). Because 5-HT exerts its effects via specific 5-HT receptor subtypes, a mechanism for 5-HT deactivation is necessary to prevent the receptors from desensitization. The enzymes that catabolize 5-HT, including monoamine oxidases and glucuronyl transferases, are localized intracellularly and thus require transport of 5-HT across plasma membranes (5, 31). However, 5-...

Lipopolysaccharide (LPS) is recognized as an inducer of the inflammatory response associated with gram-negative sepsis and systemic inflammatory response syndrome. LPS induction proceeds through Toll-like receptor (TLR) in immune cells and intestinal epithelial cells (IEC). This report presents the first identification of Bcl10 (B-cell CLL/lymphoma 10) as a mediator of the LPS-induced activation of IL-8 in human IEC. Bcl10 is a caspase-recruitment domain-containing protein, associated with constitutive activation of NF-kappaB in MALT (mucosa-associated lymphoid tissue) lymphomas. The normal human IEC line NCM460, normal primary human colonocytes, and ex vivo human colonic tissue were exposed to 10 ng/ml of LPS for 2-6 h. Effects on Bcl10, phospho-IkappaBalpha, NF-kappaB, and IL-8 were determined by Western blot, ELISA, immunohistochemistry, and confocal microscopy. Effects of Bcl10 silencing by small-interfering RNA (siRNA), TLR4 blocking antibody, TLR4 silencing by siRNA, and an IL-1 receptor-associated kinase (IRAK)-1/4 inhibitor on LPS-induced activation were examined. Following Bcl10 silencing, LPS-induced increases in NF-kappaB, IkappaBalpha, and IL-8 were significantly reduced (P < 0.001). Increasing concentrations of LPS were associated with higher concentrations of Bcl10 protein when quantified by ELISA, and the association between LPS exposure and increased Bcl10 was also demonstrated by Western blot, immunohistochemistry, and confocal microscopy. Exposure to TLR4 antibody, TLR4 siRNA, or an IRAK-1/4 inhibitor eliminated the LPS-induced increases in Bcl10, NF-kappaB, and IL-8. Identification of Bcl10 as a mediator of LPS-induced activation of NF-kappaB and IL-8 in normal human IEC provides new insight into mechanisms of epithelial inflammation and new opportunities for therapeutic intervention.

Bcl10 (B-cell CLL/lymphoma 10) is a 233 amino acid CARD (caspase recruitment domain)-containing cellular protein, increasingly recognized as a mediator of NFkappaB activation in non-immune, as well as immune cells. Due to the importance of Bcl10 in diverse cell types, we developed a solid-phase, enzyme-linked immunosorbent (ELISA) assay to precisely measure Bcl10 in small volume cell lysates, using recombinant Bcl10 to standardize the assay. Standard curve measures Bcl10 from 0.25 ng/mL to 16 ng/mL, with very low intra- and inter-assay variation. Sample dilution and exogenous Bcl10 recovery experiments, comparisons with Western blot, and linear response to increasing doses of known Bcl10 activators confirm the specificity and precision of the ELISA.

We previously demonstrated that 5-HT treatment of Caco-2 cells inhibited Na ϩ /H ϩ exchangers (NHE) and Cl Ϫ /OH Ϫ exchange activities via distinct signaling mechanisms. Since regulation of several ion transporters such as NHE3 is influenced by intact cytoskeleton, we hypothesized that 5-HT modifies actin cytoskeleton and/or brush-border membrane architecture via involvement of signaling pathways. Ultrastructural analysis showed that 5-HT (0.1 M, 1 h) treatment of Caco-2 cells caused the apical membrane to assume a convex dome shape that was associated with shortening of microvilli. To examine whether these cellular architecture changes are cytoskeleton driven, we analyzed actin cytoskeleton by fluorescence microscopy. 5-HT induced basal stress fibers with prominent cortical actin filaments via 5-HT3 and 5-HT4 receptor subtypes. This induction was partially attenuated by chelation of intracellular Ca 2ϩ and PKC␣ inhibition (Go6976). In vitro assays revealed that PKC␣ interacted with actin and this association was increased by 5-HT. Our data provide novel evidence that 5-HT-induced signaling via 5-HT3/4 receptor subtypes to cause Ca 2ϩ and PKC␣-dependent regulation of actin cytoskeleton may play an important role in modulation of ion transporters that contribute to pathophysiology of diarrheal conditions associated with elevated levels of 5-HT. microvilli; serotonin and actin; 5-HT and curvature; stress fibers; ion transporter; cytoskeleton SEROTONIN (5-HT) is an important hormone and neurotransmitter that influences diverse functions in the gastrointestinal tract. High levels of 5-HT have been implicated in several diarrheal conditions associated with inflammatory bowel diseases or carcinoid syndrome (3,5,23,37). We have previously shown that 5-HT inhibits Na -dependent PKC␣ (16). In contrast, 5-HT inhibited Cl Ϫ /OH Ϫ exchange activity via a Ca 2ϩ -independent pathway involving Src-kinase induced activation of PKC␦ (31). Furthermore, recent studies have shown that 5-HT causes cytoskeletal reorganization via 5-HT4 receptors to induce pulmonary artery smooth muscle cell migration (10). However, nothing is known regarding the regulation of major cytoskeletal elements such as actin by 5-HT in intestinal epithelial cells.A number of ion transporters such as members of NHE gene family have been shown to interact with the cytoskeleton (40). Microfilaments have been suggested to maintain basal activity of NHE3 and have also been implicated in transducing regulatory signals from receptors or hormones. For example, binding of NHE3 to the cytoskeletal binding proteins is an important factor for its inhibition by cAMP/protein kinase A-, Ca 2ϩ -, or cGMP/PKG-mediated pathways (4,22,24,36,39). Increasing evidence suggests that distinct signaling molecules interact with cytoskeleton to mediate physiological functions (14). In addition, a role for the actin cytoskeleton has emerged during clathrin-and caveolin-mediated endocytosis and exocytosis (29, 34). Our previous studies suggested that 5-HT induced inhibition of NHE activ...