Colonic Epithelial Cell Line T84 Research Articles

Intracellular Signaling Mechanisms Pharmacological Action of Jasminum amplexicaule Buch.-Ham. (Oleaceae) on Gastrointestinal Secretion.

Jasminum amplexicaule Buch-Ham. (Oleaceae) has been commonly used in the traditional medicine in dysentery, diarrhoea and bellyache in China. In the present work, the methanol extract of Jasminum amplexicaule (JME) was examined for pharmacology on human colonic epithelial cell line T84 by the short-circuit current technique. The results showed that pretreatment of T84 cells with JME produced a concentration-dependent (0-1000 μg/mL. EC50 = 0.055 mg/ mL) inhibition effect on adrenalin (Adr.)-induced Cl- secretion. The maximal response was observed at 200 μg/mL. It has been demonstrated that JME has a direct effect on the enterocyte. Our results also demonstrated that the JME exerted inhibitory effect on gastrointestinal Cl(-)secretion that effected by acting on basolateral β-adrenoreceptors. These results suggested that the Chinese traditional medicine of JME can be used for the treatment of acute diarrhea and bellyache.

Epithelial expression of interleukin-37b in inflammatory bowel disease

Interleukin (IL)-37 is a member of the IL-1 cytokine family. We investigated IL-37b expression in the inflamed mucosa of inflammatory bowel disease (IBD) patients. Furthermore, we analysed IL-37b expression in human colonic epithelial cells. The human colonic epithelial cell line T84 and human colonic subepithelial myofibroblasts (SEMFs) were used. IL-37b expression in the IBD mucosa was evaluated by immunohistochemistry. IL-37b mRNA and protein expression were determined by real time-polymerase chain reaction (PCR) and Western blotting, respectively. IL-37b was not detected in the normal colonic mucosa. In the inflamed mucosa of IBD patients, epithelial IL-37b expression was increased markedly. In ulcerative colitis (UC) and Crohn's disease (CD) patients, IL-37b expression was enhanced in the affected mucosa. In the intestinal epithelial cell line T84, the expression of IL-37b mRNA and protein was enhanced by tumour necrosis factor (TNF)-α. This IL-37b induction by TNF-α was mediated by nuclear factor (NF)-κB and activator protein (AP)-1 activation. Furthermore, IL-37b inhibited TNF-α-induced interferon-γ-inducible protein (IP)-10 expression significantly in human colonic SEMFs. Epithelial IL-37b expression was increased in IBD patients, especially UC patients. IL-37b may be involved in the pathophysiology of IBD as an anti-inflammatory cytokine and an inhibitor of both innate and acquired immune responses.

Lactose in Human Breast Milk an Inducer of Innate Immunity with Implications for a Role in Intestinal Homeostasis

Postpartum, infants have not yet established a fully functional adaptive immune system and are at risk of acquiring infections. Hence, newborns are dependent on the innate immune system with its antimicrobial peptides (AMPs) and proteins expressed at epithelial surfaces. Several factors in breast milk are known to confer immune protection, but which the decisive factors are and through which manner they work is unknown. Here, we isolated an AMP-inducing factor from human milk and identified it by electrospray mass spectrometry and NMR to be lactose. It induces the gene (CAMP) that encodes the only human cathelicidin LL-37 in colonic epithelial cells in a dose- and time-dependent manner. The induction was suppressed by two different p38 antagonists, indicating an effect via the p38-dependent pathway. Lactose also induced CAMP in the colonic epithelial cell line T84 and in THP-1 monocytes and macrophages. It further exhibited a synergistic effect with butyrate and phenylbutyrate on CAMP induction. Together, these results suggest an additional function of lactose in innate immunity by upregulating gastrointestinal AMPs that may lead to protection of the neonatal gut against pathogens and regulation of the microbiota of the infant.

EGF mediates aldosterone‐induced regulatory effects of myofibroblasts on the junctional permeability of cultured colonic epithelial cells

Aldosterone (ALDO) regulates the growth of pericryptal myofibroblasts and the permeability of the colonic epithelium in vivo. We have examined whether these effects of ALDO can be reproduced in vitro, using the myofibroblast CCD-18Co cell line to study proliferation and the colonic epithelial cell line T84 to study the expression of tight junction proteins. The possible role of the epidermal growth factor (EGF) as mediator of myofibroblasts effects on the expression of junctional proteins was also studied. Cell proliferation was quantified from bromodeoxyuridine incorporation, the expression of tight-junction proteins β-catenin and claudin IV by Western blot and EGF expression by RT-PCR. ALDO stimulated CCD-18Co proliferation by 70% (P<0.05) and increased EGF expression (P<0.05), and both effects were prevented by Spironolactone. ALDO had no direct effects on the expression of junctional proteins by the T84 cells; however, both β-catenin and claudin IV were increased when the T84 cells were incubated with medium from myofibroblasts stimulated by ALDO (conditionated medium, CM). The CM increased 40% (P<0.05) T84 proliferation and about 30% (P<0.05) the expression of β-catenin and claudin IV and these effects were prevented by anti-EGF antibodies. These results indicate that EGF is a candidate to mediate the ALDO effects on crypt epithelial permeability. Supported by BFU2006-08410, MEC, Spain.

Expression of integrin alphavbeta6 in the intestinal epithelial cells of patients with inflammatory bowel disease.

Background and aims:The prevalence of inflammatory bowel disease (IBD) is about 0.05% in industrialized countries. The pathogenesis of IBD remains to be further understood. The present study aims to elucidate the expression of integrin αvβ6 in the intestinal mucosa of patients with IBD.Materials and Methods:Colonic biopsy was obtained from a group of IBD patients. The expression of αvβ6 in the intestinal mucosa was detected by Western blotting. Human colonic epithelial cell line T84 cells were stimulated by microbial antigen flagellin. The expression of αvβ6 in T84 cells was evaluated by quantitative RT-PCR and Western blotting.Results:The levels of αvβ6 in the intestinal mucosa were much lower than it in normal control subjects. The serum levels of myeloperoxidase (MPO) were higher in IBD patients that were negatively correlated with the levels of αvβ6 in the intestinal mucosa. The expression of αvβ6 was detectable in T84 cells at naοve status that could be upregulated by exposure to microbial antigen flagellin. Pretreatment with MPO dramatically suppressed the expression of αvβ6 in T84 cells.Conclusions:We conclude that the expression of αvβ6 was suppressed in IBD intestinal mucosa, which could be resulted from the high levels of MPO.

Hydrogen Sulfide Is a Novel Prosecretory Neuromodulator in the Guinea-Pig and Human Colon

Hydrogen sulfide (H(2)S) has been suggested as a novel gasomediator. We explored its unknown neuromodulatory role in human and guinea-pig colon. We used immunohistochemistry to detect H(2)S-producing enzymes cystathionine gamma-lyase (CSE) and cystathionine beta-synthase (CBS) in enteric neurons, Ussing chambers to measure mucosal ion secretion, and neuroimaging with voltage- and Ca(++)-sensitive dyes to record H(2)S effects on guinea-pig and human enteric neurons. More than 90% of guinea-pig and human submucous and myenteric neurons were colabeled for CSE and CBS. Myenteric interstitial cells of Cajal were CSE-immunoreactive. The exogenous H(2)S donor NaHS (0.2-2.5 mmol/L) concentration-dependently increased chloride secretion in human and guinea-pig submucosa/mucosa preparations, but not in the colonic epithelial cell line T84. The secretory response was reduced significantly by tetrodotoxin (0.5 micromol/L), capsaicin desensitization (10 micromol/L), and the transient receptor potentials vanilloid receptor 1 antagonist capsazepine (10 micromol/L). The endogenous H(2)S donor L-cysteine also induced secretion that was diminished significantly by capsaicin desensitization, the CBS inhibitor amino-oxyacetic acid, and the CSE inhibitor propargylglycine. NaHS increased spike discharge in 23% of guinea-pig and 36% of human submucous neurons, but had no effect on Ca(++) mobilization in cultured guinea-pig enteric neurons. This excitatory response was reduced significantly by capsaicin desensitization and capsazepine, but not by glibenclamide (10 micromol/L). The presence of H(2)S-producing enzymes in human and guinea-pig enteric neurons, the excitatory action on enteric neurons, and the prosecretory effects of NaHS suggest H(2)S as a novel gut-signaling molecule. Its action mainly involves transient receptor potentials vanilloid receptor 1 receptors on extrinsic afferent terminals, which in turn activate enteric neurons.

Adenosine 2b receptor (A2bR) signals through adenylate cyclase (AC) 6 isoform in the intestinal epithelial cells

Adenosine 2b receptor (A2bR), a G-protein coupled receptor positively coupled to adenylate cyclase, mediates key events such as chloride, IL-6 and fibronectin secretion in intestinal epithelial cells and is upregulated during intestinal inflammation. In order to gain insight into the overall mechanism of A2bR activation, in this study, we sought to characterize the AC isoform associated with A2bR signaling. The colonic epithelial cell line T84, expressing only the A2b subtype of adenosine receptor, and Chinese hamster ovary (CHO) cells, were used in these studies. cAMP was measured by luminometric assay and AC isoform expression was determined by Western blot, RT-PCR, isoform-specific stealth RNAi and Quantigene. T84 and CHO cells express all nine known AC isoforms. In order to characterize which AC isoform(s) are associated with A2bR, we used the differential inhibition of specific AC isoforms by calcium and nitric oxide. Pretreatment of cells with carbachol or nitric oxide donors such as S-Nitroso-N-acetylpencillamine (SNAP) and PAPANANOATE inhibited A2bR mediated increase in cAMP. Further, overexpression of AC-5 or AC-6 potentiated A2bR-mediated increases in cAMP levels. Finally, transfection with AC isoform-specific RNAi demonstrated that AC-6 but not AC-5 RNAi inhibited adenosine-induced cAMP levels. Taken together, these results suggest that A2bR mediates signaling through AC-6 isoform. Since pro-inflammatory cytokines such as interferon-gamma (IFN-γ) modulate the expression of specific AC isoforms in the intestinal epithelia, our observation may have therapeutic implications for intestinal inflammation or diarrhea wherein aA2bR is upregulated.

Lipopolysaccharide Activates Human Mast Cells To Induce Intestinal Epithelial Barrier Dysfunction

Background: To determine the pathway via which lipopolysaccharide (LPS) compromises the intestinal epithelial barrier function. Materials and Methods: The human mast cell line HMC-1 cells were stimulated with LPS. The tryptase levels were evaluated in the supernatant. The human colonic epithelial cell line T84 cells were cultured with the supernatant from the culture of the HMC-1 cells that have been activated by LPS. The T84 monolayers' barrier function was evaluated by recording transepithelial electric resistance (TER) and horseradish peroxidase (HRP) flux. Results: HMC-1 cells expressed the toll like receptor 4. LPS stimulation induced HMC-1 cells to release tryptase. The supernatant caused significantly decrease in TER and increase in HRP flux that could be inhibited by pretreatment with tryptase inhibitor BABIM. Conclusion: LPS can induce human mast cells to release chemical mediators such as tryptase that can compromise the intestinal epithelial barrier function.

Agonist-induced polarized trafficking and surface expression of the adenosine 2b receptor in intestinal epithelial cells: role of SNARE proteins.

Adenosine, acting through the A2b receptor, induces vectorial chloride and IL-6 secretion in intestinal epithelia and may play an important role in intestinal inflammation. We have previously shown that apical or basolateral adenosine receptor stimulation results in the recruitment of the A2b receptor to the plasma membrane. In this study, we examined domain specificity of recruitment and the role of soluble N-ethylmaleimide (NEM) attachment receptor (SNARE) proteins in the agonist-mediated recruitment of the A2b receptor to the membrane. The colonic epithelial cell line T84 was used because it only expresses the A2b-subtype adenosine receptor. Cell fractionation, biotinylation, and electron microscopic studies showed that the A2b receptor is intracellular at rest and that apical or basolateral adenosine stimulation resulted in the recruitment of the receptor to the apical membrane. Upon agonist stimulation, the A2b receptor is enriched in the vesicle fraction containing vesicle-associated membrane protein (VAMP)-2. Furthermore, in cells stimulated with apical or basolateral adenosine, we demonstrate a complex consisting of VAMP-2, soluble NEM-sensitive factor attachment protein (SNAP)-23, and A2b receptor that is coimmunoprecipitated in cells stimulated with adenosine within 5 min and is no longer detected within 15 min. Inhibition of trafficking with NEM or nocodazole inhibits cAMP synthesis induced by apical or basolateral adenosine by 98 and 90%, respectively. cAMP synthesis induced by foskolin was not affected, suggesting that generalized signaling is not affected under these conditions. Collectively, our data suggest that 1) the A2b receptor is intracellular at rest; 2) apical or basolateral agonist stimulation induces recruitment of the A2b receptor to the apical membrane; 3) the SNARE proteins, VAMP-2 and SNAP-23, participate in the recruitment of the A2b receptor; and 4) the SNARE-mediated recruitment of the A2b receptor may be required for its signaling.

Inhibition of epithelial chloride secretion by butyrate: role of reduced adenylyl cyclase expression and activity.

Butyrate and other short-chain fatty acids (SCFAs) are found at high concentrations in the colonic lumen and affect multiple epithelial cell functions. To better understand how SCFAs regulate ion transport, we investigated the effects of SCFAs on Cl(-) secretion in human colonic epithelial cell line T(84). Butyrate inhibited Cl(-) secretory responses to prostaglandin E(2), forskolin, and cholera toxin. Other SCFAs were less effective or inactive. Reduced secretion was associated with decreased synthesis of the second messenger cAMP rather than increased degradation. Expression and activity of adenylyl cyclase were decreased by butyrate, whereas phosphodiesterase activity was unaffected and phosphodiesterase inhibition did not reverse the effects of butyrate on Cl(-) secretion. Furthermore, butyrate decreased expression of the basolateral Na-K-2Cl cotransporter, indicating that it might modulate the secretory capacity of the cells. However, butyrate did not affect secretory responses to the calcium-dependent secretagogue carbachol, cAMP analogs, or uroguanylin, indicating that normal secretory responses to adequate levels of second messengers in butyrate-treated T(84) cells are possible. These results show that butyrate affects several aspects of epithelial Cl(-) secretion, including second messenger generation and expression of key ion transporters. However, these effects may not all be equally important in determining Cl(-) secretion in response to physiologically relevant secretagogues.

Distinct Ca2+- and cAMP-dependent anion conductances in the apical membrane of polarized T84 cells

Monolayers of the human colonic epithelial cell line T84 exhibit electrogenic Cl- secretion in response to the Ca2+ agonist thapsigargin and to the cAMP agonist forskolin. To evaluate directly the regulation of apical Cl- conductance by these two agonists, we have utilized amphotericin B to permeabilize selectively the basolateral membranes of T84 cell monolayers. We find that apical anion conductance is stimulated by both forskolin and thapsigargin but that these conductances are differentially sensitive to the anion channel blocker DIDS. DIDS inhibits thapsigargin-stimulated responses completely but forskolin responses only partially. Furthermore, the apical membrane anion conductances elicited by these two agonists differ in anion selectivity (for thapsigargin, I- > Cl-; for forskolin, Cl- > I-). However, the DIDS-sensitive component of the forskolin-induced conductance response exhibits anion selectivity similar to that induced by thapsigargin (I- > Cl-). Thus forskolin-induced apical anion conductance comprises at least two components, one of which has features in common with that elicited by thapsigargin.

Characterization of PKA isoforms and kinase-dependent activation of chloride secretion in T84 cells.

Chloride exit across the apical membranes of secretory epithelial cells is acutely regulated by the cAMP-mediated second messenger cascade. To better understand the regulation of transepithelial chloride secretion, we have characterized the complement of cAMP-dependent protein kinase (PKA) isoforms present in the human colonic epithelial cell line T84. Our results show that both type I and type II PKA are present in T84 cells. Immunoprecipitation of 8-azido-[32P]cAMP-labeled cell lysates revealed that the major regulatory subunits of PKA were RIalpha and RIIalpha. In addition, immunogold electron microscopy showed that RIIalpha labeling was found on membranes of the trans Golgi network and on apical plasma membrane. In contrast, RIalpha was randomly distributed throughout the cytoplasm, with no discernible membrane association. Northern blot analysis of T84 RNA revealed that Calpha was the predominantly expressed catalytic subunit. Short-circuit current measurements were performed in the presence of combinations of site-selective cAMP analog pairs to preferentially activate either PKA type I or PKA type II in intact T84 cell monolayers. Maximal levels of chloride secretion (approximately 100 microA/cm2) were observed for both type I and type II PKA-selective analog pairs. Subsequent addition of forskolin was unable to further increase chloride secretion. Thus activation of either type I or type II PKA is able to maximally stimulate chloride secretion in T84 colonic epithelial cells.

Interleukin-15 signals T84 colonic epithelial cells in the absence of the interleukin-2 receptor beta-chain.

Interleukin-15 (IL-15) shares many biological functions with interleukin-2 (IL-2) due to common receptor components. IL-15 binds to the IL-2 receptor (IL-2R) beta-chain and the common gamma-chain receptor in addition to one other IL-15 binding receptor protein (IL-15R alpha). Both IL-2R beta- and gamma-chains are required to promote cell growth in hematopoietic cells. The colonic cryptlike epithelial cell line T84 contains the common gamma-chain but lacks the IL-2R beta-chain. We report IL-15R alpha-chain mRNA in T84 cells with the use of reverse transcriptase-polymerase chain reaction. T84 and normal colonic epithelial cells bind a FLAG-IL-15 fusion protein in immunoperoxidase and flow cytometric experiments. In addition, IL-15, but not IL-2, accelerates and enhances the development of transepithelial resistance across T84 monolayers in a dose-dependent fashion. We conclude that normal and T84 colonic epithelial cells express IL-15R alpha and are able to bind IL-15. IL-15 can deliver a nonproliferative functional signal in the absence of IL-2R beta-chain in T84 cells.

Rapid effects of steroid hormones on free intracellular calcium in T84 colonic epithelial cells.

Studies from our laboratory have demonstrated rapid (< 1 min) nongenomic activation of K+ recycling and Na+/H+ exchange by mineralocorticoids in human colonic epithelium, and studies from other laboratories have demonstrated rapid effects of aldosterone on intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells. Here a rapid nongenomic effect of aldosterone on [Ca2+]i is demonstrated in the human colonic epithelial cell line T84. Aldosterone induced a rapid increase in [Ca2+]i within approximately 2 min. The rise in [Ca2+]i after aldosterone appears to result from the activation of a Ca2+ influx pathway, inasmuch as 1) no increase in [Ca2+]i was observed with aldosterone when cells were bathed in Ca(2+)-free Krebs solution and 2) emptying of the intracellular Ca2+ stores by thapsigargin was not enhanced by addition of aldosterone to extracellular Ca(2+)-free solution. In contrast, the Ca2+ response to aldosterone, in the presence of 2 mM Ca2+ in the external bathing solution, was not decreased after intracellular Ca2+ stores were emptied by thapsigargin. Other mineralocorticoid hormones increased [Ca2+]i, whereas the glucocorticoid hydrocortisone failed to increase [Ca2+]i. These results demonstrate the existence of a mineralocorticoid-specific Ca(2+)-signaling pathway in human colonic T84 (crypt) epithelial cells.

Inositol 3,4,5,6-Tetrakisphosphate Inhibits the Calmodulin-dependent Protein Kinase II-activated Chloride Conductance in T84 Colonic Epithelial Cells

The mechanism by which inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5, 6)P4) regulates chloride (Cl-) secretion was evaluated in the colonic epithelial cell line T84 using whole cell voltage clamp techniques. Our studies focused on the calcium-dependent chloride conductance (gClCa) that was activated either by mobilizing intracellular calcium (Cai) stores with thapsigargin or by introduction of the autonomous, autophosphorylated calmodulin-dependent protein kinase II (CaMKII) into the cell via the patch pipette. Basal concentrations of Ins(3,4,5,6)P4 (1 microM) present in the pipette solution had no significant effect on Cl- current; however, as the concentration of the polyphosphate was increased there was a corresponding reduction in anion current, with near complete inhibition at 8-10 microM Ins(3,4,5,6)P4. Corresponding levels are found in cells after sustained receptor-dependent activation of phospholipase C. The Ins(3,4,5, 6)P4-induced inhibition of gClCa was isomer specific; neither Ins(1, 3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, nor Ins(1,3,4,5,6)P5 induced current inhibition at concentrations of up to 100 microM. Annexin IV also plays an inhibitory role in modulating gClCa in T84 cells. When 2 microM annexin IV was present in the pipette solution, a concentration that by itself has no effect on gClCa, the potency of Ins(3,4,5,6)P4 was approximately doubled. The combination of Ins(3,4,5,6)P4 and annexin IV did not alter the in vitro activity of CaMKII. These data demonstrate that Ins(3,4,5,6)P4 is an additional cellular signal that participates in the control of salt and fluid secretion, pH balance, osmoregulation, and other physiological activities that depend upon gClCa activation. Ins(3,4,5,6)P4 metabolism and action should also be taken into account when designing treatment strategies for cystic fibrosis.

Forskolin- but not ionomycin-evoked Cl- secretion in colonic epithelia depends on intact microtubules

Several transport proteins are known to be trafficked to the cell membrane in response to appropriate secretagogues. In several cases, the response has been shown to be dependent on the cytoskeleton. We tested the hypothesis that the forskolin- and/or ionomycin-sensitive Cl- secretory response in colonic epithelia is dependent on an intact cytoskeleton. Using 125I- efflux as an assay for Cl- transport in the colonic epithelial cell line T84, we found that preincubation of the tissue for 3 h with either of two inhibitors of microtubule polymerization, nocodazole or colchicine, disrupted the cellular tubulin architecture and also reduced the forskolin- but not the ionomycin-evoked I- efflux. In contrast, brief exposure (4 min) to nocodazole was without effect on the forskolin-sensitive efflux, suggesting that the drug is not acting to block the stimulus-response pathway. An inactive structural analogue of colchicine, beta-lumicolchicine, had no inhibitory effect on either the forskolin-sensitive efflux or on microtubular structure. In a second model of Cl- secretion, the stripped rat colon, both colchicine and nocodazole reduced the forskolin-dependent short-circuit current by an average of 30-40%, suggesting a similar mechanism for insertion of Cl- channels into the plasma membrane. These findings suggest that the Cl- secretory response is dependent on microtubules and has a physiological role in the adenosine 3',5'-cyclic monophosphate-dependent, but not the Ca(2+)-dependent, Cl- secretion in colonic epithelia.

Regulated endocytosis in a chloride secretory epithelial cell line.

The colonic epithelial cell line T84 has been shown to be a good model to investigate the regulation of Cl- secretion by the adenosine 3',5'-cyclic monophosphate (cAMP)-mediated second messenger cascade. Regulated exocytic insertion and endocytic retrieval of transport proteins, or proteins that regulate transport proteins, is one mechanism proposed to regulate plasma membrane solute permeabilities. The aims of our studies were to characterize endocytic processes in T84 cells and to investigate their regulation by known activators of Cl- secretion that are mediated by the cAMP second messenger cascade. Forskolin, an activator of adenylate cyclase, caused a marked inhibition of endocytic uptake of the fluid-phase marker horseradish peroxidase (HRP) and the adsorptive marker wheat germ agglutinin conjugated to HRP. Similar inhibition was obtained with vasoactive intestinal peptide, a secretagogue whose receptor is coupled to adenylate cyclase, and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, a membrane-permeable cAMP analogue. 1,9-Dideoxy-forskolin, a forskolin analogue that fails to activate adenylate cyclase, was without effect on endocytosis. Our data show that the net rate of endocytosis, as measured by fluid-phase uptake, is decreased by a cAMP-mediated mechanism. Because the number of Cl- channels or associated regulatory proteins in the plasma membrane reflects a balance between their exocytic insertion and endocytic retrieval, we propose that the cAMP-mediated decrease in endocytosis could contribute to the concomitant increase in plasma membrane Cl- permeability.

Enhancement of kallikrein production and kinin sensitivity in T84 cells by growth in the nude mouse

The production of tissue kallikrein and the short-circuit current (SCC) response in the human colonic epithelial cell line T84 to bradykinin have been studied. These cells produce true tissue kallikrein and secrete it into the growth medium, and addition of bradykinin results in a small increase in SCC. After growth of T84 cells as a xenograft in athymic (nude) mice (NuT84), however, the cellular concentration of the enzyme increases 38-fold, and the maximal change in SCC (delta SCC) induced by bradykinin increases 35-fold. The SCC response to prostaglandin E1 is not changed, and the response to forskolin increases eightfold. The bradykinin-induced delta SCC was inhibited by piretanide, suggesting that a secretory movement of chloride was responsible for the change, and the effect was attenuated by an antagonist to kinin receptors. We conclude that both the production of true tissue kallikrein and the chloride secretory response to bradykinin can be regulated in T84 cells by changes in the cell environment.

Separate Cl- conductances activated by cAMP and Ca2+ in Cl(-)-secreting epithelial cells.

We studied the cAMP- and Ca2(+)-activated secretory Cl- conductances in the Cl(-)-secreting colonic epithelial cell line T84 using the whole-cell patch-clamp technique. Cl- and K+ currents were measured under voltage clamp. Forskolin or cAMP increased Cl- current 2-15 times with no change in K+ current. The current-voltage relation for cAMP-activated Cl- current was linear from -100 to +100 mV and showed no time-dependent changes in current during voltage pulses. Ca2+ ionophores or increased pipette Ca2+ increased both Cl- and K+ currents 2-30 times. The Ca2(+)-activated Cl- current was outwardly rectified, activated during depolarizing voltage pulses, and inactivated during hyperpolarizing voltage pulses. Addition of ionophore after forskolin further increased Cl- conductance 1.5-5 times, and the current took on the time-dependent characteristics of that stimulated by Ca2+. Thus, cAMP and Ca2+ activate Cl- conductances with different properties, implying that these second messengers activate different Cl- channels or that they induce different conductive and kinetic states in the same Cl- channel.

Differing effects of apical and basolateral adenosine on colonic epithelial cell line T84

Adenosine and its agonists, 5'-(N-ethylcarboxamido)adenosine and N6-(D-2-phenylisopropyl)-adenosine, induced a sustained increase in chloride secretion when added to either the apical or basolateral aspect of monolayers of the human colonic epithelial cell line T84. Secretion was induced with identical kinetics by addition to both sides, but apical addition was less potent. The rank order of potency of the agonists on either side was consistent with the presence of an adenosine A2-receptor, but the apical and basolateral receptors differed in both their ability to stimulate increases in adenosine 3',5'-cyclic monophosphate, and their susceptibility to down-modulation by chronic exposure to 5'-(N-ethylcarboxamido) adenosine in culture.