Mei Ao

Specific Immunomodulatory and Secretory Activities of Stevioside and Steviol in Intestinal Cells

Stevioside, isolated from Stevia rebaudiana, is a commercial sweetener. It was previously demonstrated that stevioside attenuates NF-kappaB-dependent TNF-alpha and IL-1beta synthesis in LPS-stimulated monocytes. The present study examined the effects of stevioside and its metabolite, steviol, on human colon carcinoma cell lines. High concentrations of stevioside (2-5 mM) and steviol (0.2-0.8 mM) decreased cell viability in T84, Caco-2, and HT29 cells. Stevioside (2 mM) potentiated TNF-alpha-mediated IL-8 release in T84 cells. However, steviol (0.01-0.2 mM) significantly suppressed TNF-alpha-induced IL-8 release in all three cell lines. In T84 cells, steviol attenuated TNF-alpha-stimulated IkappaB --> NF-kappaB signaling. Chloride transport was stimulated by steviol (0.1 mM) > stevioside (1 mM) at 30 min. Two biological effects of steviol in the colon are demonstrated for the first time: stimulation of Cl(-) secretion and attenuation of TNF-alpha-stimulated IL-8 production. The immunomodulatory effects of steviol appear to involve NF-kappaB signaling. In contrast, at nontoxic concentrations stevioside affects only Cl(-) secretion.

Lubiprostone Activates Cl− Secretion via cAMP Signaling and Increases Membrane CFTR in the Human Colon Carcinoma Cell Line, T84

BackgroundLubiprostone, used clinically (b.i.d.) to treat constipation, has been reported to increase transepithelial Cl− transport in T84 cells by activating ClC-2 channels.AimTo identify the underlying signaling pathway, we explored the effects of short-term and overnight lubiprostone treatment on second messenger signaling and Cl− transport.MethodsCl− transport was assessed either as Isc across T84 monolayers grown on Transwells and mounted in Ussing chambers or by the iodide efflux assay. [cAMP]i was measured by enzyme immunoassay, and [Ca2+]i by Fluo-3 fluorescence. Quantitation of apical cell surface CFTR protein levels was assessed by Western blotting and biotinylation with the EZ-Link Sulfo-NHS-LC-LC-Biotin. ClC-2 mRNA level was studied by RT-PCR.ResultsLubiprostone and the cAMP stimulator, forskolin, caused comparable and maximal increases of Isc in T84 cells. The Isc effects of lubiprostone and forskolin were each suppressed if the tissue had previously been treated with the other agent. These responses were unaltered even if the monolayers were treated with lubiprostone overnight. Lubiprostone-induced increases in iodide efflux were ~80% of those obtained with forskolin. Lubiprostone increased [cAMP]i. H89, bumetanide, or CFTRinh-172 greatly attenuated lubiprostone-stimulated Cl− secretion, whereas the ClC-2 inhibitor CdCl2 did not. Compared to controls, FSK-treatment increased membrane-associated CFTR by 1.9 fold, and lubiprostone caused a 2.6-fold increase in apical membrane CFTR as seen by immunoblotting following cell surface biotinylation.ConclusionsLubiprostone activates Cl− secretion in T84 cells via cAMP, protein kinase A, and by increasing apical membrane CFTR protein.

Ion transport in the small intestine.

Purpose of review The 2009 review on small intestinal ion transport, in this series, focused on recent advances in duodenal bicarbonate secretion, the importance of scaffolding proteins and the pathophysiology of inflammation-associated diarrhea. The current review focuses on advances in ion-coupled solute transport, the dynamic role of the paracellular pathway in transepithelial-fluid transport and of elucidating the cellular basis of diarrheas associated with enteric infections. Recent findings In understanding the cellular pathophysiology underlying diarrheal diseases, there is increased focus on the role of altering Na+ absorptive mechanisms as well as the role of the paracellular pathway. This is not to minimize the role of Cl−-secretory pathways, especially cystic fibrosis transmembrane conductance regulator (CFTR), which continues to have pleiotropic roles in modulating other transporters. The Na+-glucose cotransporter (SGLT) was the first transporter ever to be cloned. Twenty-one years later, with another first, the crystal structure of the related Na+-galactose transporter has been described and opens new avenues to understand structure–function relationships and intelligent drug design for transporters. Summary Progress continues to be made on integrating information obtained from reductionist models into more complex in-vivo animal models and where possible in human studies. Recognition of the coordinated regulation of cellular Na+ absorptive and Cl−-secretory pathways together with the paracellular route in health and disease will help develop a more holistic picture of the multifaceted nature of small intestinal ion transport.

Chenodeoxycholic acid stimulates Cl− secretion via cAMP signaling and increases cystic fibrosis transmembrane conductance regulator phosphorylation in T84 cells

High levels of chenodeoxycholic acid (CDCA) and deoxycholic acid stimulate Cl(-) secretion in mammalian colonic epithelia. While different second messengers have been implicated in this action, the specific signaling pathway has not been fully delineated. Using human colon carcinoma T84 cells, we elucidated this cascade assessing Cl(-) transport by measuring I(-) efflux and short-circuit current (Isc). CDCA (500 μM) rapidly increases I(-) efflux, and we confirmed by Isc that it elicits a larger response when added to the basolateral vs. apical surface. However, preincubation with cytokines increases the monolayer responsiveness to apical addition by 55%. Nystatin permeabilization studies demonstrate that CDCA stimulates an eletrogenic apical Cl(-) but not a basolateral K(+) current. Furthermore, CDCA-induced Isc was inhibited (≥67%) by bumetanide, BaCl2, and the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh-172. CDCA-stimulated Isc was decreased 43% by the adenylate cyclase inhibitor MDL12330A and CDCA increases intracellular cAMP concentration. The protein kinase A inhibitor H89 and the microtubule disrupting agent nocodazole, respectively, cause 94 and 47% reductions in CDCA-stimulated Isc. Immunoprecipitation with CFTR antibodies, followed by sequential immunoblotting with Pan-phospho and CFTR antibodies, shows that CDCA increases CFTR phosphorylation by approximately twofold. The rapidity and side specificity of the response to CDCA imply a membrane-mediated process. While CDCA effects are not blocked by the muscarinic receptor antagonist atropine, T84 cells possess transcript and protein for the bile acid G protein-coupled receptor TGR5. These results demonstrate for the first time that CDCA activates CFTR via a cAMP-PKA pathway involving microtubules and imply that this occurs via a basolateral membrane receptor.

Calcitonin receptor-mediated CFTR activation in human intestinal epithelial cells.

High levels of calcitonin (CT) observed in medullary thyroid carcinoma and other CT‐secreting tumours cause severe diarrhoea. Previous studies have suggested that CT induces active chloride secretion. However, the involvement of CT receptor (CTR) and the molecular mechanisms underlying the modulation of intestinal electrolyte secreting intestinal epithelial cells have not been investigated. Therefore, current studies were undertaken to investigate the direct effects of CT on ion transport in intestinal epithelial cells. Real time quantitative RT‐PCR and Western blot analysis demonstrated the expression of CTR in intestinal epithelial T84 cells. Exposure of T84 cells to CT from the basolateral but not from apical side significantly increased short circuit current (ISC) in a dose‐dependent manner that was blocked by 1 μM of CTR antagonist, CT8–32. CT‐induced ISC was blocked by replacing chloride in the bath solutions with equimolar gluconate and was significantly inhibited by the specific cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor, CFTR127inh. Further, biotinylation studies showed that CT increased CFTR levels on the apical membrane. The presence of either the Ca2+ chelator, bis(2‐aminophenoxy)ethane tetraacetic acid‐acetoxymethyl (BAPTA‐AM) ester or the protein kinase A (PKA) inhibitor, H89, significantly inhibited ISC induced by CT (∼32–58% reduction). Response to CT was retained after permeabilization of the basolateral or the apical membranes of T84 cells with nystatin. In conclusion, the activation of CTR by CT induced chloride secretion across T84 monolayers via CFTR channel and the involvement of PKA‐ and Ca2+‐dependent signalling pathways. These data elucidate the molecular mechanisms underlying CT‐induced diarrhoea.

Chenodeoxycholic Acid Requires Activation of EGFR, EPAC and Ca2+ to Stimulate CFTR-dependent Cl- Secretion in Human Colonic T84 Cells.

Bile acids are known to initiate intricate signaling events in a variety of tissues, primarily in the liver and gastrointestinal tract. Of the known bile acids, only the 7α-dihydroxy species, deoxycholic acid and chenodeoxycholic acid (CDCA), and their conjugates, activate processes that stimulate epithelial Cl- secretion. We have previously published that CDCA acts in a rapid manner to stimulate colonic ion secretion via protein kinase A (PKA)-mediated activation of the dominant Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR) (Ao M, Sarathy J, Domingue J, Alrefai WA, and Rao MC. Am J Physiol Cell Physiol 305: C447-C456, 2013); however, PKA signaling did not account for the entire CDCA response. Here we show that in human colonic T84 cells, CDCAs induction of CFTR activity, measured as changes in short-circuit current (Isc), is dependent on epidermal growth factor receptor (EGFR) activation and does not involve the bile acid receptors TGR5 or farnesoid X receptor. CDCA activation of Cl- secretion does not require Src, mitogen-activated protein kinases, or phosphoinositide 3-kinase downstream of EGFR but does require an increase in cytosolic Ca2+ In addition to PKA signaling, we found that the CDCA response requires the novel involvement of the exchange protein directly activated by cAMP (EPAC). EPAC is a known hub for cAMP and Ca2+ cross talk. Downstream of EPAC, CDCA activates Rap2, and changes in free cytosolic Ca2+ were dependent on both EPAC and EGFR activation. This study establishes the complexity of CDCA signaling in the colonic epithelium and shows the contribution of EGFR, EPAC, and Ca2+ in CDCA-induced activation of CFTR-dependent Cl- secretion.

Epidermal Growth Factor Stimulates Chloride Transport in Primary Cultures of Weanling and Adult Rabbit Colonocytes

Objectives: We have shown that Ca2+-dependent regulation of Cl− secretion in the mammalian colon exhibits age dependence. Because epidermal growth factor (EGF) has a well-established role in growth and can increase intracellular calcium [Ca2+]i, it is conceivable that its developmental influence may extend to the regulation of intestinal ion transport. In this study, we examined the role of EGF in the regulation of Cl− transport in the developing rabbit distal colon. Materials and Methods: Because serum contains growth factors, which could have confounded our studies, we first established an optimal milieu for testing EGF in primary cultures of adult rabbit distal colonocytes by culturing them for 24 h in media containing 0%, 1%, 5%, and 20% serum. Chloride transport (millimoles per second) and [Ca2+]i were measured with use of the fluorescent indicator N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) and Fura-2AM, respectively. Results: Serum depletion had no effect on cell number, DNA content, or basal Cl− transport, but it significantly affected cell viability. In media with 0%, 1%, or 20% serum, bethanechol, 8BrcAMP, taurodeoxycholate, and EGF stimulated Cl− transport to a similar extent. EGF maximally stimulated Cl− transport at 16.3 nmol/L and 20 minutes. Bethanechol, but not EGF, increased [Ca2+]i. EGF did not alter bethanechol-stimulated Cl− transport or [Ca2+]i. EGF acts via an EGF-receptor and mitogen activated protein kinase (MAPK) signaling pathway, since stimulation of Cl− transport was abolished by genistein, AG1478, and PD98059. Weanling and adult colonocytes, cultured in 1% serum, showed similar basal and EGF-stimulated Cl− transport. Conclusions: EGF stimulates rabbit colonic Cl− transport via a Ca2+-independent, tyrosine kinase- and MAPK-dependent pathway, and its effects are not age dependent.

HEK-293 cells expressing the cystic fibrosis transmembrane conductance regulator (CFTR): a model for studying regulation of Cl- transport.

The Human Embryonic Kidney 293 cell line (HEK‐293) readily lends itself to genetic manipulation and is a common tool for biologists to overexpress proteins of interest and study their function and molecular regulation. Although these cells have some limitations, such as an inability to form resistive monolayers necessary for studying transepithelial ion transport, they are nevertheless valuable in studying individual epithelial ion transporters. We report the use of HEK‐293 cells to study the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. While HEK‐293 cells endogenously express mRNA for the Cl− channels, ClC‐2 and TMEM16A, they neither express CFTR mRNA nor protein. Therefore, we stably transfected HEK‐293 cells with EGFP‐CFTR (HEK‐CFTR) and demonstrated CFTR function by measuring forskolin‐stimulated iodide efflux. This efflux was inhibited by CFTRinh172, and the protein kinase A inhibitor H89, but not by Ca2+ chelation. In contrast to intestinal epithelia, forskolin stimulation does not increase surface CFTR expression and does not require intact microtubules in HEK‐CFTR. To investigate the role of an endogenous GαS‐coupled receptor, we examined the bile acid receptor, TGR5. Although HEK‐CFTR cells express TGR5, the potent TGR5 agonist lithocholic acid (LCA; 5–500 μmol/L) did not activate CFTR. Furthermore, forskolin, but not LCA, increased [cAMP]i in HEK‐CFTR suggesting that endogenous TGR5 may not be functionally linked to GαS. However, LCA did increase [Ca2+]i and interestingly, abolished forskolin‐stimulated iodide efflux. Thus, we propose that the stable HEK‐CFTR cell line is a useful model to study the multiple signaling pathways that regulate CFTR.

The Yin and Yang of bile acid action on tight junctions in a model colonic epithelium

Gastrointestinal epithelial barrier loss due to tight junction (TJ) dysfunction and bile acid‐induced diarrhea are common in patients with inflammatory diseases. Although excess colonic bile acids are known to alter mucosal permeability, few studies have compared the effects of specific bile acids on TJ function. We report that the primary bile acid, chenodeoxycholic acid (CDCA), and its 7α‐dehydroxylated derivative, lithocholic acid (LCA) have opposite effects on epithelial integrity in human colonic T84 cells. CDCA decreased transepithelial barrier resistance (pore) and increased paracellular 10 kDa dextran permeability (leak), effects that were enhanced by proinflammatory cytokines (PiC [ng/mL]: TNFα[10] + IL‐1ß[10] + IFNγ[30]). CDCA reversed the cation selectivity of the monolayer and decreased intercellular adhesion. In contrast, LCA alone did not alter any of these parameters, but attenuated the effects of CDCA ± PiC on paracellular permeability. CDCA, but not PiC, decreased occludin and not claudin‐2 protein expression; CDCA also decreased occludin localization. LCA ± CDCA had no effects on occludin or claudin expression/localization. While PiC and CDCA increased IL‐8 production, LCA reduced both basal and PiC ± CDCA‐induced IL‐8 production. TNFα + IL1ß increased IFNγ, which was enhanced by CDCA and attenuated by LCA. CDCA±PiC increased production of reactive oxygen species (ROS) that was attenuated by LCA. Finally, scavenging ROS attenuated CDCAs leak, but not pore actions, and LCA enhanced this effect. Thus, in T84 cells, CDCA plays a role in the inflammatory response causing barrier dysfunction, while LCA restores barrier integrity. Understanding the interplay of LCA, CDCA, and PiC could lead to innovative therapeutic strategies for inflammatory and diarrheal diseases.

Lithocholic acid attenuates cAMP-dependent Cl− secretion in human colonic epithelial T84 cells

Bile acids (BAs) play a complex role in colonic fluid secretion. We showed that dihydroxy BAs, but not the monohydroxy BA lithocholic acid (LCA), stimulate Cl(-) secretion in human colonic T84 cells (Ao M, Sarathy J, Domingue J, Alrefai WA, Rao MC. Am J Physiol Cell Physiol 305: C447-C456, 2013). In this study, we explored the effect of LCA on the action of other secretagogues in T84 cells. While LCA (50 μM, 15 min) drastically (>90%) inhibited FSK-stimulated short-circuit current (Isc), it did not alter carbachol-stimulated Isc LCA did not alter basal Isc, transepithelial resistance, cell viability, or cytotoxicity. LCAs inhibitory effect was dose dependent, acted faster from the apical membrane, rapid, and not immediately reversible. LCA also prevented the Isc stimulated by the cAMP-dependent secretagogues 8-bromo-cAMP, lubiprostone, or chenodeoxycholic acid (CDCA). The LCA inhibitory effect was BA specific, since CDCA, cholic acid, or taurodeoxycholic acid did not alter FSK or carbachol action. While LCA alone had no effect on intracellular cAMP concentration ([cAMP]i), it decreased FSK-stimulated [cAMP]i by 90%. Although LCA caused a small increase in intracellular Ca(2+) concentration ([Ca(2+)]i), chelation by BAPTA-AM did not reverse LCAs effect on Isc LCA action does not appear to involve known BA receptors, farnesoid X receptor, vitamin D receptor, muscarinic acetylcholine receptor M3, or bile acid-specific transmembrane G protein-coupled receptor 5. LCA significantly increased ERK1/2 phosphorylation, which was completely abolished by the MEK inhibitor PD-98059. Surprisingly PD-98059 did not reverse LCAs effect on Isc Finally, although LCA had no effect on basal Isc, nystatin permeabilization studies showed that LCA both stimulates an apical cystic fibrosis transmembrane conductance regulator Cl(-) current and inhibits a basolateral K(+) current. In summary, 50 μM LCA greatly inhibits cAMP-stimulated Cl(-) secretion, making low doses of LCA of potential therapeutic interest for diarrheal diseases.