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-...

. Upregulation of P-glycoprotein by probiotics in intestinal epithelial cells and in the dextran sulfate sodium model of colitis in mice. Am J Physiol Gastrointest Liver Physiol 300: G1115-G1123, 2011. First published February 24, 2011 doi:10.1152/ajpgi.00027.2011.-P-glycoprotein (P-gp) mediates efflux of xenobiotics and bacterial toxins from the intestinal mucosa into the lumen. Dysregulation of P-gp has been implicated in inflammatory bowel disease. Certain probiotics have been shown to be effective in treating inflammatory bowel disease. However, direct effects of probiotics on P-gp are not known. Current studies examined the effects of Lactobacilli on P-gp function and expression in intestinal epithelial cells. Caco-2 monolayers and a mouse model of dextran sulfate sodium-induced colitis were utilized. P-gp activity was measured as verapamil-sensitive [ 3 H]digoxin transepithelial flux. Multidrug resistant 1 (MDR1)/P-gp expression was measured by real-time quantitative PCR and immunoblotting. Culture supernatant (CS; 1:10 or 1:50, 24 h) of Lactobacillus acidophilus or Lactobacillus rhamnosus treatment of differentiated Caco-2 monolayers (21 days postplating) increased (ϳ3-fold) MDR1/P-gp mRNA and protein levels. L. acidophilus or L. rhamnosus CS stimulated P-gp activity (ϳ2-fold, P Ͻ 0.05) via phosphoinositide 3-kinase and ERK1/2 MAPK pathways. In mice, L. acidophilus or L. rhamnosus treatment (3 ϫ 10 9 colonyforming units) increased mdr1a/P-gp mRNA and protein expression in the ileum and colon (2-to 3-fold). In the dextran sulfate sodium (DSS)-induced colitis model (3% DSS in drinking water for 7 days), the degree of colitis as judged by histological damage and myeloperoxidase activity was reduced by L. acidophilus. L. acidophilus treatment to DSS-treated mice blocked the reduced expression of mdr1a/ P-gp mRNA and protein in the distal colon. These findings suggest that Lactobacilli or their soluble factors stimulate P-gp expression and function under normal and inflammatory conditions. These data provide insights into a novel mechanism involving P-gp upregulation in beneficial effects of probiotics in intestinal inflammatory disorders. multidrug resistance 1; Lactobacillus species; phosphoinositide 3-kinase; ERK1/2 kinase; dextran sulfate sodium-induced colitis; intestinal inflammation

A family of anion exchangers (AEs) including AE1, AE2 and AE3 has been described. AE3 gene has been shown to encode two alternatively spliced isoforms termed as bAE3 (brain subtype) and cAE3 (cardiac subtype). The identity of the AE(s) involved in the human intestinal NaCl absorption is not fully understood. Current studies were undertaken to identify the AE isoforms expressed in the human intestine, to define their regional and vertical axis (crypt vs. surface cells) distribution, and to elucidate their membrane localization in the epithelial cells along the entire length of the human intestine. Our studies utilizing reverse transcription (RT)-PCR with total RNA extracted from pinch biopsies from various regions of the human intestine demonstrate that AE2 and bAE3 but not AE1 or cAE3 were expressed in all the regions of the human intestine. Utilizing in situ RT-PCR, we demonstrated that the message of AE2 was expressed throughout the vertical surface--crypt axis of the colon. Our Western blotting studies demonstrated that AE2 and bAE3 are localized to the basolateral but not the apical membranes of the intestinal epithelial cells from the human ileum and colon. In conclusion, our results demonstrated that in the human intestine, AE2 and bAE3, but not AE1 or cAE3, are expressed throughout the tract with the highest expression in the colon compared to the ileum and jejunum. Both the isoforms were found to be localized to the basolateral but not the apical membranes of the epithelial cells. We speculate that, in the human intestine, AE2 and bAE3 may be the 'housekeeping' isoforms, and the apical AE, the potential candidate for chloride absorption, remains to be identified.

Recent studies have indicated the presence of Na+/H+and Cl−/[Formula: see text]exchange activities in lung alveolar and tracheal tissues of various species. To date, the identity of the Na+/H+(NHE) and Cl−/[Formula: see text](AE) exchanger isoforms and their regional distribution in human airways are not known. Molecular species of the NHE and AE gene families and their relative abundance in the human airway regions were assessed utilizing RT-PCR and the RNase protection assay, respectively. Organ donor lung epithelia from various bronchial regions (small, medium, and large bronchi and trachea) were harvested for RNA extraction. Gene-specific primers for the human NHE and AE isoforms were utilized for RT-PCR. Our results demonstrated that NHE1, AE2, and brain AE3 isoforms were expressed in all regions of the human airways, whereas NHE2, NHE3, AE1, and cardiac AE3 were not detected. RNase protection studies for NHE1 and AE2, utilizing glyceraldehyde-3-phosphate dehydrogenase as an internal standard, demonstrated that there were regional differences in the NHE1 mRNA levels in human airways. In contrast, the levels of AE2 mRNA remained unchanged. Differential expression of these isoforms in the human airways may have functional significance related to the airway absorption and secretion of electrolytes.

Extensive loss of MLSN in PM correlated with aggressive metastatic melanoma. Ancillary testing for MLSN mRNA expression by CISH could offer a means to more accurately identify AJCC stage I and II patients at risk for metastatic disease, who could benefit from adjuvant therapy.