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Autophagy, a process of degradation and recycling of macromolecules and organelles to maintain cellular homeostasis, has also been shown to help eliminate invading pathogens. Conversely, various pathogens including parasites have been shown to modulate/exploit host autophagy facilitating their intracellular infectious cycle. In this regard, Cryptosporidium parvum (CP), a protozoan parasite of small intestine is emerging as a major global health challenge. However, the pathophysiology of cryptosporidiosis is mostly unknown. We have recently demonstrated CP‐induced epithelial barrier disruption via decreasing the expression of specific tight junction (TJ) and adherens junction (AJ) proteins such as occludin, claudin‐4 and E‐cadherin. Therefore, we utilised confluent Caco‐2 cell monolayers as in vitro model of intestinal epithelial cells (IECs) to investigate the potential role of autophagy in the pathophysiology of cryptosporidiosis. Autophagy was assessed by increase in the ratio of LC3II (microtubule associated protein 1 light chain 3) to LC3I protein and decrease in p62/SQSTM1 protein levels. CP treatment of Caco‐2 cells for 24 hr induced autophagy with a maximum effect observed with 0.5 × 106 oocyst/well. CP decreased mTOR (mammalian target of rapamycin, a suppressor of autophagy) phosphorylation, suggesting autophagy induction via mTOR inactivation. Measurement of autophagic flux utilizing the lysosomal inhibitor chloroquine (CQ) showed more pronounced increase in LC3II level in cells co‐treated with CP + CQ as compared to CP or CQ alone, suggesting that CP‐induced increase in LC3II was due to enhanced autophagosome formation rather than impaired lysosomal clearance. CP infection did not alter ATG7, a key autophagy protein. However, the decrease in occludin, claudin‐4 and E‐cadherin by CP was partially blocked following siRNA silencing of ATG7, suggesting the role of autophagy in CP‐induced decrease in these TJ/AJ proteins. Our results provide novel evidence of autophagy induction by CP in host IECs that could alter important host cell processes contributing to the pathophysiology of cryptosporidiosis. [ABSTRACT FROM AUTHOR]

Background/Aims: Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter and hormone with important physiological functions in many organs, including the intestine. We have previously shown that 5-HT activates the aryl hydrocarbon receptor (AhR) in intestinal epithelial cells (IECs) via a serotonin transporter (SERT)-dependent mechanism. AhR is a nuclear receptor that binds a variety of molecules including tryptophan (TRP) metabolites to regulate physiological processes in the intestine including xenobiotic detoxification and immune modulation. We hypothesized that 5-HT activates AhR indirectly by interfering with metabolic clearance of AhR ligands by cytochrome P450 1A1 (CYP1A1). Methods: Inhibition of CYP1A1 activity by 5-HT was assessed in the human intestinal epithelial cell line Caco2 and recombinant CYP1A1 microsomes using both luciferase and LC-MS/MS. Degradation of 5-HT by recombinant CYP1A1 was measured by LC-MS/MS. For in vitro studies, CYP1A1 and CYP1B1 mRNA expression levels were measured by RT-PCR and CYP1A1 activity was measured by ethoxyresorufin-O-deethylase (EROD) assays. For in vivo studies, AhR ligands were administered to SERT KO mice and WT littermates and intestinal mucosa CYP1A1 mRNA was measured. Results: We show that 5-HT inhibits metabolism of both the pro-luciferin CYP1A1 substrate Luc-CEE as well as the high affinity AhR ligand 6-formylindolo[3,2-b] carbazole (FICZ). Recombinant CYP1A1 assays revealed that 5-HT is metabolized by CYP1A1 in an NADPH dependent manner. Treatment with 5-HT in TRP-free medium, which is devoid of trace AhR ligands, showed that 5-HT requires the presence of AhR ligands to activate AhR. Cotreatment with 5-HT and FICZ confirmed that 5-HT potentiates induction of AhR target genes by AhR ligands. However, this was only true for ligands which are CYP1A1 substrates such as FICZ. Administration of β-napthoflavone by gavage or indole-3-carbinol via diet to SERT KO mice revealed that lack of SERT impairs intestinal AhR activation. Conclusion: Our studies provide novel evidence of crosstalk between serotonergic and AhR signaling where 5-HT can influence the ability of AhR ligands to activate the receptor in the intestine. [ABSTRACT FROM AUTHOR]

Thiamine, a water-soluble vitamin, is essential for normal cellular functions, growth and development. Thiamine deficiency leads to significant clinical problems and occurs under a variety of conditions. To date, however, little is known about the mechanism of thiamine absorption in the native human small intestine. The objective of this study was, therefore, to characterize the mechanism of thiamine transport across the brush-border membrane (BBM) of human small intestine. With the use of purified BBM vesicles (BBMV) isolated from the jejunum of organ donors, thiamine uptake was found to be 1) independent of [Na.sup.+] but markedly stimulated by an outwardly directed [H.sup.+] gradient (pH [5.5.sub.in]/pH [7.5.sub.out]); 2) competitively inhibited by the cation transport inhibitor amiloride (inhibitor constant of 0.12 mM); 3) sensitive to temperature and osmolarity of the incubation medium; 4) significantly inhibited by thiamine structural analogs (amprolium, oxythiamine, and pyrithiamine), but not by unrelated organic cations (tetraethylammonium, N-methylnicotinamide, or choline); 5) not affected by the addition of ATP to the inside and outside of the BBMV; 6) potential insensitive; and 7) saturable as a function of thiamine concentration with an apparent Michaelis-Menten constant of 0.61 [+ or -] 0.08 [micro]M and a maximal velocity of 1.00 [+ or -] 0.47 pmol [multiplied by] mg [protein.sup.-1] [multiplied by] 10 [s.sup.-1]. Carrier-mediated thiamine uptake was also found in BBMV of human ileum. These data demonstrate the existence of a [Na.sup.+]-independent, pH-dependent, amiloride-sensitive, electroneutral carrier-mediated mechanism for thiamine absorption in native human small intestinal BBMV. thiamine transporter; human small intestine; brush-border membranes

Mechanism of thiamine uptake by human colonocytes: studies with cultured colonic epithelial cell line NCM460. Am J Physiol Gastrointest Liver Physiol 281: G144-G150, 2001.-- Thiamine (vitamin [B.sub.1]) is essential for normal cellular functions and growth. Mammals cannot synthesize thiamine and thus must obtain the vitamin via intestinal absorption. The intestine is exposed to a dietary thiamine source and a bacterial source in which the vitamin is synthesized by the normal microflora of the large intestine. Very little is known about thiamine uptake in the large intestine. The aim of this study was, therefore, to address this issue. Our results with human-derived colonic epithelial NCM460 cells as a model system showed thiamine uptake to be 1) temperature- and energy dependent, 2) [Na.sup.+] independent, 3) increased with increasing buffer pH from 5 to 8 and after cell acidification but inhibited by amiloride, 4) saturable as a function of concentration, 5) inhibited by thiamine structural analogs but not by unrelated organic cations, and 6) inhibited by modulators of a [Ca.sup.2+]/calmodulin-mediated pathway. NCM460 cells and native human colonic mucosa expressed the recently cloned human thiamine transporter THTR-1 (product of the SLC19A2 gene) at both mRNA and protein levels. These results demonstrate for the first time that human NCM460 colonocytes possess a specific carrier-mediated system for thiamine uptake that appears to be under the regulation of an intracellular [Ca.sup.2+]/calmodulin-mediated pathway. It is suggested that bacterially synthesized thiamine in the large intestine may contribute to thiamine nutrition of the host, especially toward cellular nutrition of the local colonocytes. colonic thiamine transport

Malakooti, Jaleh, Refka Y. Dahdal, Pradeep K. Dudeja, Thomas J. Layden, and Krishnamurthy Ramaswamy. The human [Na.sup.+]/[H.sup.+] exchanger NHE2 gene: genomic organization and promoter characterization. Am J Physiol Gastrointest Liver Physiol 280: G763-G773, 2001.--The [Na.sup.+]/[H.sup.+] exchanger (NHE) 2 belongs to a family of plasma membrane transporters involved in intracellular pH and cell volume regulation. We recently reported cloning of human NHE2 (hNHE2) from a colonic cDNA library. Northern blot analysis has identified NHE2 mRNA only in small intestine, prostate, kidney, colon, and skeletal muscle. In this study, we describe the structure and 5'-regulatory region of the hNHE2 gene. The hNHE2 gene spans [is greater than] 90 kb and is organized in 12 exons intervened by 11 introns. All introns contain the conserved GT and AG dinucleotides at the donor and acceptor sites, respectively. The hNHE2 gene was mapped to chromosome 2q11.2. Primer extension analysis revealed a single transcription initiation site in human colonic adenocarcinoma cell lines. Analysis of the DNA nucleotide sequences of a 1.4-kb fragment of the 5'-flanking region shows no canonical TATA or CAAT boxes. However, the promoter region contains several potential cis-regulatory elements such as Sp1, early growth response-1, activator protein-2, MyoD, p300, nuclear factor-[Kappa]B, myeloid zinc finger protein-1, caudal-related homeobox (Cdx) gene A, and Cdx protein-2 binding sites. In transient transfection studies, a reporter construct containing the 1.4-kb promoter region exhibited low luciferase activity levels. However, after deletion upstream of -664, its activity increased approximately threefold. Thus our data suggest that an inhibitory element may exist in the NHE2 promoter 5'-upstream region. exon-intron junction; chromosome 2q11.2; transcription factor; C2BBe1; transfection

Hadjiagapiou, Christos, Larry Schmidt, Pradeep K. Dudeja, Thomas J. Layden, and Krishnamurthy Ramaswamy. Mechanism(s) of butyrate transport in Caco-2 cells: role of monocarboxylate transporter 1. Am J Physiol Gastrointest Liver Physiol 279: G775-G780, 2000.--The short-chain fatty acid butyrate was readily taken up by Caco-2 cells. Transport exhibited saturation kinetics, was enhanced by low extracellular pH, and was [Na.sup.+] independent. Butyrate uptake was unaffected by DIDS; however, [Alpha]-cyano-4-hydroxycinnamate and the butyrate analogs propionate and L-lactate significantly inhibited uptake. These results suggest that butyrate transport by Caco-2 cells is mediated by a transporter belonging to the monocarboxylate transporter family. We identified five isoforms of this transporter, MCT1, MCT3, MCT4, MCT5, and MCT6, in Caco-2 cells by PCR, and MCT1 was found to be the most abundant isoform by RNase protection assay. Transient transfection of MCT1, in the antisense orientation, resulted in significant inhibition of butyrate uptake. The cells fully recovered from this inhibition by 5 days after transfection. In conclusion, our data showed that the MCT1 transporter may play a major role in the transport of butyrate into Caco-2 cells. short-chain fatty acids; intestinal absorption; antisense cDNA

Said, Hamid M., Alvaro Ortiz, Chandira K. Kumar, Nabendu Chatterjee, Pradeep K. Dudeja, and Stanley Rubin. Transport of thiamine in human intestine: mechanism and regulation in intestinal epithelial cell model Caco-2. Am. J. Physiol. 277 (Cell Physiol. 46): C645-C651, 1999.-- The present study examined the intestinal uptake of thiamine (vitamin [B.sub.1]) using the human-derived intestinal epithelial cells Caco-2 as an in vitro model system. Thiamine uptake was found to be 1) temperature and energy dependent and occurred with minimal metabolic alteration; 2) pH sensitive; 3) [Na.sup.+] independent; 4) saturable as a function of concentration with an apparent Michaelis-Menten constant of 3.18 [+ or -] 0.56 [Micro] m and maximal velocity of 13.37 + 0.94 pmol.mg [protein.sup.-1] [multiplied by] 3 [min.sup.-1]; 5) inhibited by the thiamine structural analogs amprolium and oxythiamine, but not by unrelated organic cations tetraethylammonium, N-methylnicotinamide, and choline; and 6) inhibited in a competitive manner by amiloride with an inhibition constant of 0.2 mM. The role of specific protein kinase-mediated pathways in the regulation of thiamine uptake by Caco-2 cells was also examined using specific modulators of these pathways. The results showed possible involvement of a [Ca.sup.2+]/calmodulin (CaM)-mediated pathway in the regulation of thiamine uptake. No role for protein kinase C- and protein tyrosine kinase-mediated pathways in the regulation of thiamine uptake was evident. These results demonstrate the involvement of a carrier-mediated system for thiamine uptake by Caco-2 intestinal epithelial cells. This system is [Na.sup.+] independent and is different from the transport systems of organic cations. Furthermore, a CaM-mediated pathway appears to play a role in regulating thiamine uptake in these cells. thiamine transport; human intestinal cells; transport mechanism; transport regulation