Mrinalini C. Rao

Mode of action of heat-stable Escherichia coli enterotoxin Tissue and subcellular specificities and role of cyclic GMP

Some enteric strains of Escherichia coli release a heat-stable enterotoxin which, in contrast to cholera and heat-labile E. coli enterotoxins, stimulates guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2). We have examined the tissue spcificity of its action and the relation of its action to those of the 8-bromo analogues of cyclic GMP and cyclic AMP. Heat-stable enterotoxin stimulated guanylate cyclase activity and increased cyclic GMP concentration throughout the small and large intestine. It increased transepithelial electric potential difference and short-circuit current in the jejunum, ileum and caecum but not in the duodenum or distal colon. This pattern of electrical responses was mimicked by 8-bromo-cyclic GMP. However, 8-bromo-cyclic AMP produced an electrical response in all intestinal segments. The enterotoxin failed to stimulate guanylate cyclase inliver, lung, pancreas or gastric antral mucosa. In the intestines, it stimulated only the particulate and not the soluble form of the enzyme. Preincubation of the toxin with intestinal membranes did not render it capable of stimulating pancreatic guanylate cyclase. Cytosol factors did not enhance the toxins stimulation of intestinal guanylate cyclase. This study supports the role of cyclic GMP as intracellular mediator for heat-stable enterotoxin and suggests that the toxin affects a membrane-mediated mechanism for guanylate cyclase activation that is unique to the intestines.

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.

Isolation, Characterization, and Attachment of Rabbit Distal Colon Epithelial Cells

The authors investigated various enzymatic digestion procedures for isolating epithelial cells from the distal colon of New Zealand White male rabbits. Rabbit mucosa was washed, diced, and digested for 90 minutes in one of five different solutions, including a new combination consisting of 0.03% collagenase IV and 0.1% pronase (solution V). Solution I (0.3% dispase) yielded 14.2 +/- 8.2 x 10(6) colonocytes/g mucosa, solution II (0.15% dispase and 0.03% collagenase) yielded 7.7 +/- 2.8 x 10(6) colonocytes/g mucosa, and solution III (0.03% collagenase IV) yielded 15.4 +/- 10(6) cells/g mucosa. Solutions I-III have previously been described for the isolation of colonocytes. Solution IV (0.1% pronase and 325 U/mL DNAase) was originally described for the isolation of nasal epithelial cells but yielded only 2.5 +/- 1.2 x 10(6) cells/g mucosa when applied to the isolation of colonocytes. The new combination of pronase and collagenase, solution V, yielded significantly more colonocytes, 34.5 +/- 3.0 x 10(6) cells/g mucosa, than previously described methods (P less than 0.01). Inclusion of 5 mmol/L ethylenediaminetetraacetic acid in any of the solutions enhanced neither viability nor yield. The digestion product of solution V could be enriched for crypts by serial low-speed centrifugations. The epithelial origin of the colonocytes was confirmed by immunofluorescent staining for cytokeratins. Functional viability was tested by determining the presence of a Na+/H+ exchanger, using the pH fluorescent dye bis(carboxymethyl)-5(6)-carboxyfluorescein acetoxymethyl ester to measure intracellular pH. The authors document that sodium-dependent restoration of intracellular pH in colonocytes acid-loaded to a pH of 6.30 occurred at a rate of 0.19 +/- 0.02 pH U/min. Amiloride at concentrations of 1 mmol/L completely inhibited operation of the exchanger, as did sodium substitution with choline or tetramethylammonium. Lineweaver-Burke analysis at this intracellular pH showed a Michaelis constant of 10.71 mmol/L Na+ and a maximum velocity of 0.12 pH U/min. Exposing the colonocytes to 100 nmol/L phorbol 12,13-dibutyrate increased antiporter activity by 62.0%. Finally, the authors describe the synthesis of a new biomatrix composed of the basement membrane of 3T3 NIH fibroblasts that permits significantly improved colonocyte attachment than to glass, plastic, collagen types I or IV, or matrigel.

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.

Evidence for the presence of cGMP-dependent protein kinase-II in human distal colon and in T84, the colonic cell line.

Heat-stable enterotoxin (STa) stimulates intestinal Cl(-) secretion by activating guanylate cyclase C (GCC) to increase intracellular cyclic GMP (cGMP). In the colon, cGMP action could involve protein kinase (PK) G-II or PKA pathways, depending on the segment and species. In the human colon, both PKG and PKA pathways have been implicated, and, therefore, the present study examined the mechanism of cGMP-mediated Cl(-) transport in primary cultures of human distal colonocytes and in T84, the colonic cell line. Both cell preparations express mRNA for CFTR, Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), GCC and PKG-II as detected by RT-PCR. The effects of STa and the PKG-specific cGMP analogues, 8Br-cGMP and 8pCPT-cGMP, on Cl(-) transport were measured using a halide-sensitive probe. In primary human colonocytes and T84 cells, STa, the cGMP analogues and the cAMP-dependent secretagogue, prostaglandin E(1) (PGE(1)), enhanced Cl(-) transport. The effects of 8Br-cGMP and 8pCPT-cGMP suggested the involvement of PKG, and this was explored further in T84 cells. The effects of 8pCPT-cGMP were dose-dependent and sensitive to the PKG inhibitor, H8 (70 microM), but H8 had no effect on PGE(1)-induced Cl(-) secretion. In contrast, a PKA inhibitor, H7 (50 microM), blocked PGE(1)-mediated but not 8pCPT-cGMP-induced Cl(-) transport. 8pCPT-cGMP enhanced phosphorylation of the PKG-specific substrate, 2A3, by T84 membranes in vitro. This phosphorylation was inhibited by H8. These results strongly suggest that cGMP activates Cl(-) transport through a PKG-II pathway in primary cells and in the T84 cell line of the human colon.

Localization of the Na+/H+ exchanger isoform NHE-3 in rabbit and canine kidney.

The distribution and subcellular localization of Na+/H+ exchanger isoform NHE-3 was studied in rabbit and canine kidney using polyclonal antibodies to an NHE-3 fusion protein. Western blot analyses were performed against microsomal, brush-border, and basolateral membranes isolated from rabbit kidney cortex, outer medulla, and inner medulla. Immunoblots indicated that NHE-3 antibody recognized a strong band with 95-100 kDa molecular mass in cortical microsomes. Subcellular localization studies showed that NHE-3 was expressed in brush-border membranes of kidney cortex. Expression of NHE-3 in the medullary regions was studied by immunoblot analysis of NHE-3 antibody against the microsomal membranes from the outer and inner medulla. NHE-3 antibody specifically labelled a 95-100 kDa protein in outer but not inner medulla. Subcellular localization studies demonstrated that NHE-3 is localized to the brush-border membranes of the outer medulla. Immunoblot analysis against brush-border membranes from canine kidney cortex and outer medulla demonstrated the presence of an 83-90 kDa protein. The above experiments suggest that NHE-3 in rabbit kidney is a 95-100 kDa protein and is expressed in brush-border membranes of the cortex and outer medulla. The canine kidney NHE-3 is a 83-90 kDa protein and is expressed in brush-border membranes of the cortex and outer medulla. Based on its subcellular localization, we conclude that NHE-3 may be involved in vectorial Na+ and HCO3- transport and pHo regulation.

Chloride and sodium transport across bovine tracheal epithelium: Effects of secretagogues and indomethacin

The regulation of ion transport in bovine tracheal epithelium was studied in vitro. In the absence of exogenous midifiers of ion transport, average values for transepithelial electrical potential difference (ψt), short-circuit-current (Isc) and tissue resistance (Rt) were 35.4 mV (lumen negative), 5.4 μEq·h−1·cm−2 and 187 Ω·cm2 respectively; net Cl secretion (3.2 μEq·h−1·cm−2) and net Na absorption (1.3 μEq·h−1·cm2) accounted for 82% of theIsc. Amiloride reduced (ψ1) andIsc, and increasedRt. The values of (ψt),Rt andIsc obtained following addition of theophylline, epinephrine or prostaglandin E1 (PGE1) were not different from control values. Theophylline aldo did not alter Na and Cl fluxes but it increased tissue cAMP content 3-fold. Indomethacin did not affect (ψt) but it increasedRt and net Na absorption, and decreasedIsc and net Cl secretion; it did not significantly reduce tissue cAMP. When added to indomethacin-treated tissues, epinephrine restoredIsc,Rt and Na and Cl fluxes to control levels and increased tissue cAMP 3-fold. Similary, when PGE1 was added to indomethacin-treated tissues,Isc andRt were restored to control levels.We conclude that: (1) bovine tracheal epithelium, like its canine counterpart, absorbs Na and secretes Cl; the two tissues differ, however, in two ways: the spontaneous rate of Na absorption is higher in bovine trachea and the spontaneous rate of Cl secretion cannot be further increased in bovine trachea by secretagogues; (2) Cl secretion and Na absorption in bovine trachea are normally regulated by endogenous prostaglandins; (3) although cAMP may mediate changes in ion transport, a strict correlation between tissue cAMP content and Na and Cl transport rates is not evident; and (4) Na absorptive and Cl secretory rates are reciprocally related suggesting that both processes are present in the same cells.

Calcium regulated chloride permeabilities in primary cultures of rabbit colonocytes

To determine if calcium‐dependent secretagogues directly act on epithelial cells to elicit CI− secretion, their effects on CI− transport and intracellular Ca2+ concentrations ([Ca2+]i) were determined in primary cultures of rabbit distal colonic crypt cells. The Cl− sensitive fluorescent probe, 6‐methoxyquinolyl acetoethyl ester, MQAE and the Ca2+‐sensitive fluorescent probe, fura‐2AM were used to assess Cl− transport and [Ca2+]i, respectively. Basal Cl− transport (0.274 ± 0.09 mM/sec) was inhibited significantly by the Cl− channel blocker diphenylamine‐2‐carboxylate (DPC, 50 μM, 0.068 ± 0.02 mM/sec; P < 0.001) and the Na+/K+/2Cl− cotransport inhibitor furosemide (1 μM, 0.137 ± 0.04 mM/sec; P < 0.01). Ion substitution studies using different halides revealed the basal influx to be I− > F− ≥ Cl− > Br−. DPC inhibited I− influx by ∼50%, F− influx by 80%, Cl− influx by 85%, and Br− influx by 90%. Furosemide significantly inhibited influx of Br− (84%) and Cl− (81%) but not of F− and I−. The effects of agents known to alter biological response by increasing [Ca2+]i in other epithelial systems were used to stimulate Cl− transport. Cl− influx in mM/second was stimulated by 1 μM histamine (0.58 ± 0.05), 10 μM neurotensin (2.07 ± 0.32), 1 μM serotonin (1.63 ± 0.28), and 0.1 μM of the Ca2+ ionophore A23187 (2.05 ± 0.40). The Cl− permeability stimulated by neurotensin, serotonin, and A23187 was partially blocked by DPC or furosemide added alone or in combination. Histamine‐induced Cl− influx was significantly inhibited by only furosemide. Indomethacin blocked histamine‐stimulated Cl− permeability but had no effect on the actions of the other agents. These studies, focusing on isolated colonocytes without the contribution of submucosal elements, reveal that (1) histamine stimulates Cl− transport by activating the Na+/K+/2Cl− cotransporter via a cyclooxygenase‐dependent pathway; (2) neurotensin, serotonin, and A23187 activate both Cl− channels and the cotransporter, and their actions are cyclooxygenase‐independent.