Colonic Cl Research Articles

LOCAL REGULATION OF ANION SECRETION BY PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE IN HUMAN COLONIC T84 CELLS

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel hypothalamic peptide, which has been shown to exert various functions in a number of tissues, including exocrine and endocrine tissues. The present study investigated the role of local PACAP in the control of anion secretion by the human colonic T84 cell. Both bioactive forms of PACAP-27 and PACAP-38 gave rise to a dose-dependent increase in the short-circuit current (ISC). However, there was a reversal in the order of potency observed at different concentration ranges for the two bioactive forms. PACAP-27 was greater than PACAP-38 when the peptide concentrations were below 10n m; PACAP-38 was greater than PACAP-27 in the range of 10–80n m. The effects of both PACAP forms were restricted to the apical aspect of the T84 cell. The ISCresponses to both PACAP-27 and PACAP-38 were suppressed respectively by the non-selective Cl−channel blocker, diphenylamine-dicarboxylic acid (DPC), by the Ca2+dependent Cl−channel blocker, diisothiocyanatostilbene-disulfonic acid (DIDS) and by the Ca2+chelator, BAPTA-AM, indicating the involvement of Ca2+. The expression of PACAP was demonstrated and localized specifically to the perinuclear cytoplasm of the T84 cell using immunocytochemistry, indicating its epithelial origin. Thus, the present data suggest that, in addition to the well-known cAMP-dependent pathway, PACAP may play a role in regulating colonic Cl−secretion via a Ca2+-dependent pathway, perhaps through two distinct PACAP receptor subtypes. Moreover, the regulation of anion secretion by T84 cells may be mediated by locally formed PACAP in an autocrine or paracrine fashion.

17beta-oestradiol acutely regulates Cl- secretion in rat distal colonic epithelium.

In this study we used the short circuit current (ISC) technique to measure the non-genomic effects of the female sex steroid 17beta-oestradiol (E2) on electrogenic transepithelial ion transport in rat distal colonic epithelium. Basal ISC was largely composed of a transepithelial Cl- secretory component with minimal electrogenic Na+ movement. E2 (1-100 nM) caused a significant decrease in basal ISC after 15 min. In addition, pre-treating colonic epithelial tissues with E2 (0.1-100 nM) for 10 min significantly reduced forskolin (20 microM)-induced Cl- secretion. E2 also down-regulated Cl- secretion which was pre-stimulated by forskolin. Cl- secretory responses to the Ca2+-dependent secretagogue carbachol (10 microM) were also significantly reduced in the presence of E2 (10- 100 nM). However, E2 had no effect on amiloride-sensitive Na+ absorption. The rapid anti-secretory effect of E2 was abolished in the presence of the intracellular Ca2+ chelator BAPTA (50 microM) or the protein kinase C (PKC) inhibitor chelerythrine chloride (1 microM). However, in the presence of the nuclear oestrogen receptor antagonist tamoxifen (10 microM), E2 still produced an inhibition of Cl- secretion. Testosterone, progesterone and 17alpha-oestradiol had no significant effect on colonic Cl- secretion. Also, E2 (100 nM) did not alter Cl- secretion in colonic epithelia isolated from male rats. We conclude that E2 inhibits colonic Cl- secretion via a non-genomic pathway that involves intracellular Ca2+ and PKC. It is possible that this gender-specific mechanism contributes to the salt and water retention associated with high E2 states.

Role of CFTR in the colon.

In contrast to the airways, the defects in colonic function in cystic fibrosis (CF) patients are closely related to the defect in CFTR. The gastrointestinal phenotype of CF transgenic mice closely resembles the phenotype in CF patients, which clearly indicates the crucial role of CFTR in colonic Cl- secretion and the absence of an effective compensation. In the colon, stimulation of CFTR Cl- channels involves cAMP- or cGMP-dependent phosphorylation. Exocytosis is not involved. Activation of CFTR leads to coactivation of basolateral KVLQT1-type K+ channels and inhibition of luminal Na+ channels (ENaC). In contrast to cultured cells, Ca2+ does not activate luminal Cl- channels in intact enterocytes. It activates basolateral SK4-type K+ channels and luminal K+ channels, which provide additional driving force for Cl- exit. The magnitude of Cl- secretion, however, completely depends on the presence of at least a residual CFTR function in the luminal membrane. These findings have been clearly demonstrated by Ussing chamber experiments in colon epithelium biopsies of CF and normal individuals: Colonic Cl- secretion in CF patients is variable and reflects the genotype; a complete defect of CFTR is paralleled by the absence of Cl- secretion and unmasks Ca(2+)-regulated K+ channels in the luminal membrane; overabsorption of Na+ in CF reflects the absence of ENaC inhibition by CFTR; and the functional status of CF colon can be mimicked by the complete suppression of cAMP stimulation in enterocytes of healthy individuals.

The very small-conductance K+ channel KvLQT1 and epithelial function.

KvLQT1 (KCNQ1) is a very small conductance K+ channel distributed widely in epithelial and non-epithelial tissues. Its specific biophysical and pharmacological properties are determined by the regulatory subunits IsK (KCNE1) and MiRP2 (KCNE3). In epithelial cells of the inner ear, pancreas, and airways it interacts with IsK to conduct a voltage-gated and slowly activating K+ current. In the colon it coassembles with KCNE3 to conduct an instantaneous and constitutively active K+ current. In Cl- secretory epithelia, such as the colon and pancreas, this K+ channel provides the driving force for Cl- exit and is located in the basolateral membrane. In the inner ear it enables luminal secretion of K+ into the endolymphatic space. The functional relevance of KvLQT1 to epithelial function is revealed by blocking it pharmacologically or by studying animals with a genetic defect for it, which result in the breakdown of colonic Cl- secretion and endolymph production, respectively. KvLQT1 K+ channels are activated via cAMP or Ca2+ and inhibited by the chromanol 293B. Interaction with as yet unknown regulatory subunits may determine the properties of KvLQT1 in the rectal gland and other epithelial tissues in which KvLQT1 is not inhibited by chromanols.

Effect of short-chain fatty acids on cyclic 3′,5′-guanosine monophosphate-mediated colonic secretion

Short chain fatty acids (SCFA) prevent and reverse cyclic 3′,5′-adenosine monophosphate (cAMP) but not Ca 2+-mediated Cl − secretion. Mucosal [HCO 3 −] i has an opposite effect on these secretagogues. We examined whether SCFA and [HCO 3 −] i affect cyclic 3′,5′-guanosine monophosphate (cGMP)-induced secretion. Stripped segments of male Sprague–Dawley rat ( Rattus norvegicus) proximal and distal colon, and cultured T84 cells were studied in Ussing chambers, and pH i and [HCO 3 −] i were determined. Mucosal [cGMP] was measured in proximal colon. In T84 cells, the increase in Cl − secretion (measured as I sc) induced by mucosal 0.25 μM Escherichia coli heat-stable enterotoxin (ST a) was prevented/reversed by bilateral 50 mM Na + butyrate (71%/73%), acetate (58%/76%), propionate (68%/73%) and (poorly metabolized) isobutyrate (80%/79%). In proximal colon in HCO 3 − Ringer, basal Cl − secretion was not affected by [HCO 3 −] i or 25 mM butyrate. Mucosal 0.25 μM ST a decreased net Na + and Cl − absorption. Bilateral but not mucosal 25 mM SCFA reversed ST a-induced effects on Na + absorption and Cl − secretion. Bilateral and mucosal 25 mM SCFA but not [HCO 3 −] i prevented ST a-induced Cl − secretion and increases in mucosal [cGMP]. ST a did not produce Cl − secretion in distal colon. It was concluded that SCFA but not [HCO 3 −] i can prevent and reverse cGMP-induced colonic Cl − secretion.

Bradykinin-stimulated Cl- secretion in T84 cells. Role of Ca2+-activated hSK4-like K+ channels.

Bradykinin (BK)-stimulated colonic Cl- secretion was studied in T84 colonic adenocarcinoma cells by measuring BK (50 nM)-evoked changes in cytosolic free [Ca2+] ([Ca2+]i), membrane conductance and transepithelial ion transport. In T84 cells grown on impermeable supports, BK stimulated a transient increase in [Ca2+]i as assessed by fura-2 ratio imaging. In cell-attached, patch-clamp recordings, BK transiently activated low-conductance K channels. These channels were activated/inactivated reversibly in inside-out patches by switching [Ca2+]i in the bath between 30 nM and 100 nM. Excised channels recorded with 160 mM [K+] in bath and pipette exhibited an inwardly rectifying current/voltage-relation, conductances of 10+/-1 pS and 34+/-4 pS (n=10) at positive and negative voltages, respectively, and a 15-fold lower permeability for Na+ than for K+. The mean open probability of these channels did not depend on voltage but increased with increasing [Ca2+]i with an apparent concentration for a half-maximal response (EC50) of 110 nM, resembling that of hSK4 K+ channels. Application of the reverse transcriptase-polymerase chain reaction technique showed hSK4 messenger ribonucleic acid (mRNA) to be expressed in T84 cells. Macroscopic currents in T84 cells showed a similar dependence on [Ca2+]i. Whole cell conductance (in nS/10pF) increased from 0.5+/-0. 1 (n=6) at 10 nM [Ca2+]i in the pipette solution to 1.5+/-0.2 (n=7) at 100 nM, and to 2.0+/-0.5 (n=7) at 1 microM due to activation of a K+ conductance. In Ussing-chambered T84 monolayers grown on filters, BK did not evoke a short-circuit current (Isc). When, however, the monolayers were pre-stimulated by forskolin (1 microM), BK further enhanced Cl-secretion (DeltaIsc=21+/-5 microA/cm2, n=10) transiently and biphasically. In conclusion, BK enhances cyclic adenosine monophosphate-stimulated Cl- secretion in T84 cells, probably via basolateral, Ca2+-liganded activation of low-conductance hSK4-type K+ channels.

Hydrogen peroxide inhibits cAMP-induced Cl- secretion across colonic epithelial cells.

We examined the effects of H2O2 on Cl- secretion across human colonic T84 cells grown on permeable supports and mounted in modified Ussing chambers. Forskolin-induced short-circuit current, a measure of Cl- secretion, was inhibited in a concentration-dependent fashion when monolayers were pretreated with H2O2 for 30 min (30-100% inhibition between 500 microM and 5 mM). Moreover, H2O2 inhibited 76% of the Cl- current across monolayers when the basolateral membranes were permeabilized with nystatin (200 micrograms/ml). When the apical membrane was permeabilized with amphotericin B, H2O2 inhibited the Na+ current (a measure of Na+-K+-ATPase activity) by 68% but increased the K+ current more than threefold. In addition to its effects on ion transport pathways, H2O2 also decreased intracellular ATP levels by 43%. We conclude that the principal effect of H2O2 on colonic Cl- secretion is inhibitory. This may be due to a decrease in ATP levels following H2O2 treatment, which subsequently results in an inhibition of the apical membrane Cl- conductance and basolateral membrane Na+-K+-ATPase activity. Alternatively, H2O2 may alter Cl- secretion by direct action on the transporters or alterations in signal transduction pathways.

The Role of K + Channels in Colonic Cl − Secretion

Cl − secretion in the rat colonic crypt base cell (bc) requires the coordinated (a) opening of Cl − channels in the luminal membrane; (b) activation of the Na +2Cl −K + cotransporter; (c) enhanced conductive K + exit from the cell; and (d) increased pumping by the (Na + + K +)-ATPase. In this study we focus on the importance of conductive K + exit. After stimulation with the cholinergic agonist carbachol (CCH, 0.1–10 μmol/l) bc respond with a marked increase in whole cell (wc) conductance and a hyperpolarization of the membrane voltage (V m). This is paralleled by a marked increase in the (Cl − secretory) short-circuit current (I sc) in Ussing chamber studies of the intact distal colon. Current evidence favors the view that CCH, via IP 3, enhances cytosolic Ca 2+ activity, and that Ca 2+ increases the open probability of Cl − channels indirectly and that of K + channels directly. After stimulation with PGE 2 bc also enhance the wc conductance, but this is paralleled by a marked depolarization of V m. Again these effects correspond to a marked increase in (Cl − secretory) I sc. The depolarization and enhanced wc conductance is partly due to the activation of Cl − channels. However, current evidence suggests that these effects on Cl − channels are paralleled by an activation of K + channels. The chromanol 293B, by inhibiting these K + channels specifically, abolishes PGE 2-induced Cl − secretion completely, but has no effect on basal K + conductance or on CCH-induced Cl − secretion. CCH apparently activates a Ca 2+-dependent K + channel with a conductance of 10–20 pS, whilst PGE 2 (or cAMP) activate a much smaller K + channel. Only the latter K + channel can be inhibited by 293B in excised patches. Noise analysis suggests that this K + channel has a conductance of <3 pS and fast kinetics. The complete 293B induced inhibition of Cl − secretion caused by PGE 2 can be explained by the fact that PGE 2 induces a marked depolarization and that this depolarization reduces the basal K + conductance. Current evidence suggests that this inhibition of the basal K + conductance is caused by a depolarization induced inhibition of Ca 2+ entry.

Chloride transport in primary cultures of rabbit colonocytes at different stages of development

BACKGROUND & AIMS: Ontogeny of colonic Cl- transport and its regulation has been characterized inadequately. The aim of this report was to study developmental changes in Cl- transport in primary cultures of rabbit distal colonocytes. METHODS: Colonocytes from newborn (7-9 days old), weanling (25-28 days old), and adult (6 months old) rabbits were cultured for 24 hours on a collagen IV matrix, and Cl- transport was measured using the fluoroprobe 6-methoxyquinolyl acetoethyl ester. RESULTS: Cl- permeabilities were dependent on [Cl-]o with maximal rates (in millimoles per liter per second) at [Cl-]o = 75 mmol/L (newborns; 0.15 +/- 0.04; weanlings; 0.2 +/- 0.02; and adults, 0.32 +/- 0.06). Influx was inhibited significantly by the Cl- channel (50 mumol/L diphenylamine-2-carboxylate) and the Na(+)-K(+)- 2Cl- cotransport (10 mumol/L furosemide) inhibitors. The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretagogues, prostaglandin E1 (1 mumol/L), forskolin (1 mumol/L), and 8-bromo-cAMP (100 mumol/L), and the protein kinase C activator, phorbol 12-13 dibutyrate (1 mumol/L), increased Cl- influx significantly in all groups with adults showing greatest stimulation. However, taurodeoxycholate (0.025-1 mmol/L) had an effect only in the adult and the guanosine 3',5'-cyclic monophosphate (cGMP) activators STa and 8-bromo-cGMP had no effect. CONCLUSIONS: Rabbit distal colonocytes possess inhibitor-sensitive Cl- permeabilities even in neonates. However, the ontogeny of their regulation depends on the secretagogue-signaling pathway. (Gastroenterology 1996 Dec;111(6):1541-50)

Modulation of Cl − secretion in rat distal colon by genistein, a protein tyrosine kinase inhibitor

The protein tyrosine kinase inhibitor, genistein, caused an increase of short-circuit current ( Isc) across the rat distal colon in forskolin-pretreated tissues, suggesting a synergistic interaction of the drug with cAMP-dependent secretion. In the absence of forskolin, genistein had a dual effect on Isc, it increased Isc in tissues with a low baseline, but decreased Isc in tissues with a high baseline Isc. The secretory effect of genistein was dependent on the presence of Cl − and was blocked by inhibitors of Cl − secretion like bumetanide, an inhibitor of the Na +-K +-Cl − cotransporter, or 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), a Cl − channel blocker. Unidirectional flux measurements revealed that genistein inhibited Na + and Cl − absorption and induced net Cl − secretion. The protein tyrosine phosphatase inhibitor vanadate suppressed the secretory effect of genistein. In contrast, genistein caused an inhibition of carbachol-induced, i.e. Ca 2+-mediated secretion. Whole-cell patch-clamp experiments confirmed the synergistic effect of genistein on cAMP-induced Cl − currents. In the presence of forskolin, genistein caused a depolarization concomitant with an increase in membrane inward current. In addition, genistein caused an inhibition of a basal K + conductance and inhibited the Ca 2+-dependent K + conductance stimulated by carbachol. These results suggest a complex role of the protein tyrosine kinase pathway in the control of colonic Cl − secretion, an antagonistic action on the cAMP pathway and a synergistic action on the Ca 2+ pathway as revealed by the opposing effects of genistein. The physiological importance of this regulation remains to be clarified.

Escherichia coli heat-stable enterotoxin-mediated colonic Cl− secretion is absent in cystic fibrosis

Background/Aims: Calcium- and adenosine 3′,5′-cyclic monophosphate (cAMP)-mediated Cl− secretions in the human colon are abnormal in cystic fibrosis, but the effect of guanosine 3′,5′-cyclic monophosphate (cGMP) is unknown. This study examined the effects of the cGMP activator Escherichia coli heat-stable enterotoxin (STa) on rectal ion transport of controls and subjects with cystic fibrosis. Methods: In vivo rectal potential difference (PD) was measured in response to 10−7 mol/L STa in adult cystic fibrosis (n = 6) and control subjects (n = 7). Cl− transport was also evaluated in 24-hour primary cultures of human colonocytes using 6-methoxy-quinolyl-acetoethyl ester in response to STa (1 μmol/L) and 8-bromo-cGMP (100 μmol/L) with or without Cl− transport inhibitors. Results: Whereas STa increased rectal potential difference in controls, there was no effect in cystic fibrosis subjects. STa stimulated the cGMP concentration in rectal biopsy specimens from both control and cystic fibrosis subjects approximately twofold. In vitro Cl− transport in non-cystic fibrosis colonocytes increased threefold and fivefold with STa and 8-bromo-cGMP, respectively. These transport increases were inhibited by furosemide and the Cl− channel blocker diphenylamine-2-carboxylate. Conclusions: Human colonocytes secrete Cl− in response to STa and cGMP in normal subjects, but this response is absent in cystic fibrosis.

The gene for congenital chloride diarrhea maps close to but is distinct from the gene for cystic fibrosis transmembrane conductance regulator.

Congenital chloride diarrhea (CLD) is characterized by watery stools with high chloride content beginning prenatally and is inherited as an autosomal recessive trait. Perfusion studies have established a basic defect in ileal and colonic Cl-/HCO3- transport, resulting in defective chloride absorption. The protein and its gene defects have, however, remained uncharacterized. We attempted to exclude candidate genes by considering linkage disequilibrium as well as genetic linkage in a small number of Finnish families. Initial results were suggestive of linkage between CLD and the cystic fibrosis transmembrane regulator gene (CFTR). Extended analysis in eight families confirmed close linkage to chromosome 7 markers proximal of CFTR, with maximum logarithm of odds scores of 5.11 and 5.06 for D7S501 and D7S496, respectively, at zero recombination. Allelic associations were observed that were striking between CLD and D7S496 and weaker between CLD and D7S501. Multipoint analyses mapped CLD unequivocally at D7S496 with a maximum logarithm of odds score of 9.33. We conclude that the CLD gene maps close to, but is distinct from, CFTR.

Basolateral mechanisms of intracellular pH regulation in the colonic epithelial cell line HT29 clone 19A.

The intracellular pH (pHi) of the colonic tumour cell line HT29 cl.19A was studied by microspectrofluorometry using the pH-sensitive dye BCECF. Single cells within a confluent monolayer, grown in a polarized manner on permeable supports, were examined. An amiloride-sensitive Na+/H+ exchange and a stilbene-insensitive Cl-/HCO3- exchange mechanism have been identified in the basolateral membrane. Removal of Na+ from the basolateral solution caused a decrease of pHi by 0.50 +/- 0.09 unit (n = 4). Amiloride or Na(+)-free solution at the apical side had no effect on pHi. Cl- removal at the basolateral side led to an increase of pHi by 0.20 +/- 0.03 unit (n = 4) whereas apical removal had no influence on pHi. This effect was independent of Na+ and was insensitive to 0.2 mM 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulphonic acid. A basolateral Cl-/HCO3- exchanger is the most likely explanation for this observation. The Na+/H+ exchange mechanism in the basolateral membrane is an acid extruder, whereas the Cl-/HCO3- exchanger is an acid loader. Both of these mechanisms are important for the maintenance of intracellular pH in HT29 cl.19A cells.

Taurodeoxycholate activates potassium and chloride conductances via an IP3-mediated release of calcium from intracellular stores in a colonic cell line (T84)

Whole-cell patch-clamp techniques and fluorescence measurements of intracellular Ca2+ concentration, (Ca2+)i, were used to investigate the mechanism of taurodeoxycholate (TDC) stimulation of Cl- secretion in the T84 colonic cell line. During perforated whole-cell recordings, the cell membrane voltage was alternately clamped to EK and ECl. Initially, TDC (0.75 mM) stimulated inward nonselective cation currents that were composed of discrete large conductance single-channel events. This initial response was followed by activation of K+ and Cl- currents with peak values of 385 +/- 41 pA and 98 +/- 28 pA, respectively (n = 12). The K+ and Cl- currents oscillated while TDC was present and returned to baseline levels upon its removal. The threshold for activation of the oscillatory currents was 0.1 mM TDC. Taurocholate, a bile acid that does not stimulate colonic Cl- secretion, induced no current response. The TDC-induced currents could be activated in Ca(2+)-free bathing solutions. Preincubation of cells with the Ca2+ chelator, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethy)-ester (20 microM), (BAPTA-AM), eliminated the K+ and Cl- current responses, although the nonselective cation channel events were still present. Replacement of bath Na+ with NMDG+ inhibited the TDC-induced nonselective cation current but did not affect the K+ or Cl- currents. TDC induced a transient (Ca2+)i rise of 575 +/- 70 nM from a baseline of 71 +/- 5 nM (n = 15); thereafter, (Ca2+)i either plateaued or oscillated. TDC-induced (Ca2+)i oscillations were observed in the absence of bath Ca2+; however, removal of bath Ca2+ during the TDC response caused (Ca2+)i to return to near baseline values. Simultaneous K+ current and (Ca2+)i measurements confirmed that the initial nonselective cation current was independent of (Ca2+)i, while K+ current oscillations were in phase with the (Ca2+)i oscillations. TDC induced inositol monophosphate (IP) accumulation, reflecting production of inositol 1,4,5-trisphosphate (IP3) during TDC stimulation. The response to TDC during standard whole-cell patch-clamp was similar to that observed with perforated whole-cell recordings, except the nonselective cation current was prolonged. When heparin (1 mg/ml) was added to the pipette under these conditions, the Ca(2+)-activated currents were inhibited, but the nonselective cation currents were unaffected. These data suggest that TDC induces a Ca(2+)-independent nonselective cation conductance, perhaps by directly permeabilizing the plasma membrane. TDC stimulates Cl- secretion by activating K+ and Cl- conductances via an IP3-mediated release of Ca2+ from intracellular stores.

Trinitrophenyl-ATP blocks colonic Cl- channels in planar phospholipid bilayers. Evidence for two nucleotide binding sites.

Outwardly rectifying 30-50-pS Cl- channels mediate cell volume regulation and transepithelial transport. Several recent reports indicate that rectifying Cl- channels are blocked after addition of ATP to the extracellular bath (Alton, E. W. F. W., S. D. Manning, P. J. Schlatter, D. M. Geddes, and A. J. Williams. 1991. Journal of Physiology. 443:137-159; Paulmichl, M., Y. Li, K. Wickman, M. Ackerman, E. Peralta, and D. Clapham. 1992. Nature. 356:238-241). Therefore, we decided to conduct a more detailed study of the ATP binding site using a higher affinity probe. We tested the ATP derivative, 2',3',O-(2,4,6-trinitrocyclohexadienylidene) adenosine 5'-triphosphate (TNP-ATP), which has a high affinity for certain nucleotide binding sites. Here we report that TNP-ATP blocked colonic Cl- channels when added to either bath and that blockade was consistent with the closed-open-blocked kinetic model. The TNP-ATP concentration required for a 50% decrease in open probability was 0.27 microM from the extracellular (cis) side and 20 microM from the cytoplasmic (trans) side. Comparison of the off rate constants revealed that TNP-ATP remained bound 28 times longer when added to the extracellular side compared with the cytoplasmic side. We performed competition studies to determine if TNP-ATP binds to the same sites as ATP. Addition of ATP to the same bath containing TNP-ATP reduced channel amplitude and increased the time the channel spent in the open and fast-blocked states (i.e., burst duration). This is the result expected if TNP-ATP and ATP compete for block, presumably by binding to common sites. In contrast, addition of ATP to the bath opposite to the side containing TNP-ATP reduced amplitude but did not alter burst duration. This is the result expected if opposite-sided TNP-ATP and ATP bind to different sites. In summary, we have identified an ATP derivative that has a nearly 10-fold higher affinity for reconstituted rectifying colonic Cl- channels than any previously reported blocker (Singh, A. K., G. B. Afink, C. J. Venglarik, R. Wang, and R. J. Bridges. 1991. American Journal of Physiology. 260 [Cell Physiology. 30]:C51-C63). Thus, TNP-ATP should be useful in future studies of ion channel nucleotide binding sites and possibly in preliminary steps of ion channel protein purification. In addition, we have obtained good evidence that there are at least two nucleotide binding sites located on opposite sides of the colonic Cl- channel and that occupancy of either site produces a blocked state.

Synergistic opioid inhibition of colonic Cl − secretion by κ-opioid receptor agonism plus μ-opioid receptor antagonism

On the basis of the novel concept of a dual prosecretory/antisecretory intestinal opioid system, a voltage-clamp experiment was performed with guinea-pig colonic mucosa, and net anion (Cl −) secretion was measured after stimulation with prostaglandin E 1 (PGE 1) plus theophylline. Synergism between κ-opioid receptor agonism by U69593 and μ-opioid receptor antagonism by CTOPNH 2 ▪ was observed.

Oligosaccharide structures of mucins secreted by the human colonic cancer cell line CL.16E.

Cl.16E, a stably differentiated clonal derivative of the human colonic cancer cell line HT29, was used to investigate the structure of oligosaccharide chains of mucins in colonic cancer. Secretory mucins were purified by equilibrium density gradient centrifugation in CsCl. Oligosaccharide side chains were isolated after beta-elimination. Compositional analysis of oligosaccharide-alditols performed after purification by gel filtration on a Bio-gel P-6 column showed 1) that GalNAc residues were located exclusively at the reducing ends of the chains, and 2) that fucose was absent from the preparation. Oligosaccharide-alditols were separated by high performance liquid chromatography (HPLC) on quaternary amine packings into a minor neutral fraction representing about 6.5% by weight of released oligosaccharides and four acidic fractions. Two acidic fractions, namely FI and FII encompassing mono- and disialylated structures, respectively, and containing 78% of total oligosaccharide alditols, were separated by HPLC. Structural determinations were carried out using methylation analysis, 1H NMR spectroscopy, and fast atom bombardment-mass spectrometry. Twelve oligosaccharide structures were determined which ranged in size from 3 to 8 residues. These oligosaccharides were based on core types 1, 2, and 4. Elongation of oligosaccharide chains was terminated by addition of sialic acid in alpha 2-3 linkage to Gal beta 1-3R and to Gal beta 1-4R residues. The predominant structure was a hexasaccharide (fraction FII-4). This contrasts with normal colonic mucins whose oligosaccharides were previously found to be based on core 3 structures and carry sialic acids in alpha (2-6) linkage to Gal beta 1-3R, to Gal beta 1-4R, and to GalNAc alpha-R (Podolsky, D.K. (1985) J. Biol. Chem. 260, 8262-8271; Podolsky, D.K. (1985) J. Biol. Chem. 260, 15510-15515). Collectively our findings suggest that Cl.16E colon cancer cells are able to synthesize mucin oligosaccharides of gastric type whose elongation is truncated by premature sialylation.

Cytokine effects in a human colonic goblet cell line. Cellular damage and its partial prevention by 5 aminosalicylic acid.

The aim of the present study was to investigate the effects of cytokines on intestinal goblet cells in vitro. For this purpose, we examined the effects of recombinant interferon gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on the human colonic goblet cell line Cl.16E by morphological and kinetic studies, and by the assessment of mucus production during IFN-gamma/TNF-alpha treatment. Control cultures of Cl.16E cells grown on nitrocellulose filters formed monolayers of polarized goblet cells, which had kinetic characteristics similar to those of a differentiated epithelium in steady state. The combined action of IFN-gamma and TNF-alpha caused a dose-related cellular exfoliation, leading to the formation of a mucoid cap made of mucus and cellular debris. The remaining viable cells underlying the mucoid cap were cuboidal and devoid of mucus granules. A dose-related increase in cellular incorporation of [3H]thymidine was reactive to the cytokine-induced cell loss. The synergistic effects of IFN-gamm and TNF-alpha were found to be reversible when the cells were reincubated in a culture medium without cytokines. Furthermore, 5-aminosalicylic acid partially protected Cl.16E cells against cellular injury caused by IFN-gamma and TNF-alpha. On the whole, these morphological and kinetic findings argue that the changes induced in Cl.16E cells by IFN-gamma and TNF-alpha closely parallel those observed during the acute phase of ulcerative colitis, and show that these cytokines can regulate intestinal mucus production by modulating cellular exfoliation, thus leading probably to a reinforced protection of the damaged mucosa.

Vanadium-induced Cl −-secretion in rabbit descending colon is mediated by prostaglandins

Vanadium in the 4+ (vanadyl-ion) and 5+ (vanadate-ion) oxidation state stimulates furosemide-sensitive electrogenic Cl − secretion in isolated epithelia of rabbit descending colon. This effect is associated with an increased release of prostaglandin E 2 from the tissue. Inhibitors of phospholipase A 2 or cyclooxygenase abolish both vanadium-induced release of prostaglandin E 2 and Cl − secretion. Neuronal mechanisms are not likely to be involved, as tetrodotoxin does not affect the vanadate induced Cl − secretion. Although vanadate is known to inhibit Na +, K +-ATPase activity, no inhibition of active Na + transport was observed in intact colonic epithelia suggesting a rapid intracellular reduction of vanadate ions to vanadyl ions which have no inhibitory effect on the Na +, K +-ATPase. The present findings therefore indicate that vanadate stimulated colonic Cl − secretion involves intracellular conversion of vanadate to vanadyl and release of prostaglandin E 2.

Colonic Cl channel blockade by three classes of compounds

We compared the potency and inhibitory actions of three different classes of organic acids on a Cl channel derived from colonic enterocyte plasma membrane vesicles. Chloride channels were incorporated into planar lipid bilayer membranes to examine the effects of the anthranilic acids, diphenylamine 2-carboxylic acid (DPC) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), the indanyl alkanoic acids, 2-[(2-cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H-inden -5-yl)oxy] acetic acid (IAA-94) and its stereoenantiomer IAA-95, and the disulfonic stilbene, 4,4'-dinitro-stilbene-2,2'-disulfonic acid (DNDS). Except for DNDS, each of the blockers was equipotent from both the outer membrane and the cytoplasmic side of the channel protein. The potency order from the outmembrane side was DNDS greater than IAA-94 = IAA-95 greater than NPPB much greater than DPC. In contrast, the potency order from the cytoplasmic side was IAA-94 = IAA-95 greater than NPPB greater than DNDS much greater than DPC. DPC and NPPB caused a concentration-dependent decrease in the single-channel conductance (fast block). DNDS, IAA-94, and IAA-95 caused a flickery-type block and a concentration-dependent decrease in open-channel probability. Kinetic analysis revealed that blockade could be explained by a linear closed-opened-blocked kinetic scheme. Similarities in the electrostatic potential maps of these open-channel blockers suggest they may bind to a single shared binding site within the channel protein.