Secretion In Intestinal Epithelia Research Articles

Antisecretory effect of prescribed appetite stimulator drug cyproheptadine in rat intestine

Cyproheptadine (Cph) is an antiserotoninergic and antihistaminergic agent with alpha-blocking activity and central sedative effect. Cph has been found to be effective in stimulating appetite, but to our knowledge, its direct effects on the intestine have not been documented. We aimed to assess the antisecretory effects of Cph in rat proximal colon using Ussing chambers' technique. In basal and serotonin (5-HT)-stimulated conditions, Cph induced a dose-dependent reduction in short-circuit current (Isc). This effect was different in fed vs. fasted rats (EC50 = 1.9 × 10(-5 ) m and 4.9 × 10(-5 ) m, respectively). As expected, Cph induced a marked dose-dependent rightward shift of the concentration-response curve to 5-HT (pA2 = 5.4). The effect of Cph was found to be close to that of antisecretory agents in the following sequence: peptide YY > somatostatin > clonidine > Cph > C7-sorbin. To our knowledge, this is the first demonstration that Cph has a direct effect on the inhibition of electrogenic ionic secretion in intestinal epithelium in vitro. Our results indicate that Cph can modulate the intestinal transport of electrolytes and provide a new insight into the peripheral effects of this drug, which is frequently prescribed as appetite stimulator in developing countries.

Ceramide activates JNK to inhibit a cAMP-gated K+ conductance and Cl- secretion in intestinal epithelia.

Sphingomyelinases (SMases) hydrolyze membrane sphingomyelin to ceramide and are expressed by diverse host and microbial cell types populating mucosal surfaces. Exogenous bacterial SMase acts on the basolateral membrane of polarized human intestinal epithelial cells to repress the cAMP-induced Cl(-) secretory response, but how this occurs is unknown. We show here that SMase acts by down-regulating a cAMP-gated basolateral membrane K(+) conductance. Neither phosphocholine, ceramide-1-phosphate, nor sphingosine-1-phosphate recapitulates this effect, indicating that ceramide production is the decisive factor. Basolaterally applied SMase induced the phosphorylation of c-Jun NH(2)-terminal kinase (JNK), and inhibition of JNK rescued the effect of SMase on cAMP-dependant secretion. SMase secreted by normal human fibroblasts specifically recapitulated the effect on cAMP-induced Cl(-) secretion, indicating that cell types inhabiting the subepithelial space can provide such an activity to the basolateral membrane of intestinal enterocytes in trans. Thus, conversion of sphingomyelin to ceramide in basolateral membranes of intestinal cells rapidly activates JNK to inhibit a cAMP-gated K(+) conductance and thereby attenuates Cl(-) secretion. These results define a novel lipid-mediated pathway for regulation of salt and water homeostasis at mucosal surfaces.

Shark rectal gland vasoactive intestinal peptide receptor: cloning, functional expression, and regulation of CFTR chloride channels

Vasoactive intestinal peptide (VIP) is a secretagogue that mediates chloride secretion in intestinal epithelia. We determined the relative potency of VIP and related peptides in the rectal gland of the elasmobranch dogfish shark and cloned and expressed the VIP receptor (sVIP-R) from this species. In the perfused rectal gland, VIP (5 nM) stimulated chloride secretion from 250 +/- 66 to 2,604 +/- 286 microeq x h(-1) x g(-1); the relative potency of peptide agonists was VIP > PHI = GHRH > PACAP > secretin, where PHI is peptide histidine isoleucine amide, GHRH is growth hormone-releasing hormone, and PACAP is pituitary adenylate cylase activating peptide. The cloned sVIP-R from shark rectal gland (SRG) is only 61% identical to the human VIP-R1. It maintains a long, extracellular NH2 terminus with seven cysteine residues, and has three N-glycosylation sites and eight other residues implicated in VIP binding. Two amino acids considered important for peptide binding in mammals are not present in the shark orthologue. When sVIP-R and the CFTR chloride channel were coexpressed in Xenopus oocytes, VIP increased chloride conductance from 11.3 +/- 2 to 127 +/- 34 microS. The agonist affinity for activating chloride conductance by the cloned receptor was VIP > GHRH = PHI > PACAP > secretin, a profile mirroring that in the perfused gland. The receptor differs from previously cloned VIP-Rs in having a low affinity for PACAP. Expression of both sVIP-R and CFTR mRNA was detected by quantitative PCR in shark rectal gland, intestine, and brain. These studies characterize a unique G protein-coupled receptor from the shark rectal gland that is the oldest cloned VIP-R.

Interferon-γ Down-regulates Adenosine 2b Receptor-mediated Signaling and Short Circuit Current in the Intestinal Epithelia by Inhibiting the Expression of Adenylate Cyclase

Adenosine is an endogenous signaling molecule that is highly up-regulated in inflammatory states. Adenosine acts through the A2b receptor, a G protein-coupled receptor that couples positively to Galpha(s) and activates adenylate cyclase. This leads to cAMP-mediated electrogenic chloride secretion in intestinal epithelia. To better understand the regulation of the A2b receptor in intestinal epithelia, we studied the effects of interferon-gamma (IFN-gamma), a potent immunomodulatory cytokine, in the T84 cell line. Pretreatment of cells with 500 units/ml IFN-gamma for 12 h inhibited an adenosine-induced short circuit current (Isc) without affecting the transepithelial resistance. Under these conditions, IFN-gamma did not inhibit the protein expression or membrane recruitment of the A2b receptor, shown to be essential for its function. Interestingly, IFN-gamma inhibited cAMP levels as well as its downstream signaling pathway as shown by the inhibition of adenosine-induced phosphorylation of cAMP response element-binding protein and protein kinase A activity. Similar studies with forskolin, a direct activator of adenylate cyclase, also demonstrated inhibition of cAMP and its downstream response by IFN-gamma. However, IFN-gamma did not affect secretory responses to the calcium-dependent secretagogue carbachol or cAMP analog 8-bromo-cAMP, indicating that normal secretory responses to adequate second messengers in IFN-gamma-treated cells are achievable. Moreover, IFN-gamma inhibited the expression of adenylate cyclase isoforms 5 and 7. In conclusion, we demonstrate that IFN-gamma down-regulates adenosine-mediated signaling possibly through the direct inhibition of adenylate cyclase expression. We propose that IFN-gamma may acutely affect global cAMP-mediated responses in the intestinal epithelia, thereby decreasing secretory responses, which may consequently aggravate inflammatory processes.

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.

Probiotics inhibit TNF-alpha-induced interleukin-8 secretion of HT29 cells.

To study the effect of probiotics on interleukin-8 secretion in intestinal epithelia when stimulated by proinflammatory cytokines. Colonic adenocarcinoma HT29 cells were cultured and divided into four groups: control, TNF-alpha (group T in short), bifidobacterium (group B), lactobacillus (group L). B. Longum and L. bulgaricus were suspended in culture medium with a concentration of 1 x 10(8) cfu/ml and added into 24 wells respectively. One hour later TNF-alpha (10 ng/ml) was added into each well of groups T, B, L. The supernatants were collected and measured for IL-8 after 3 hours, nuclear factor-kappaB (NF-kappaB) p65 was also examined by Western blotting. There was less interleukin-8 secretion in HT29 cells when preincubated with B. Longum or L. bulgaricus compared with group T. Less p65 appeared in nuclei in groups B and L compared with group T, as detected by Western blot. Probiotics can suppress interleukin-8 secretion in intestinal epithelia when stimulated by proinflammatory cytokines, which is most likely mediated by NF-kappaB.

Growth hormone reduces chloride secretion in human colonic epithelial cells via EGF receptor and extracellular regulated kinase

Background & Aims : Growth hormone (GH) has been shown to alleviate symptoms in patients with Crohn’s disease. Chloride secretion is important in driving intestinal fluid secretion. We examined whether GH inhibits chloride secretion induced by carbachol (CCh, a calcium-dependent pathway), and the downstream effectors responsible. Methods : T 84 cells were pretreated with GH at various concentrations followed by CCh (100 μmol/L). Chloride secretion was assessed as changes in short circuit current (▵I sc) in Ussing chambers. Tyrphostins AG1478 (an epidermal growth factor receptor [EGFr] inhibitor) and AG490 (a Janus kinase 2 [JAK2] inhibitor), SB203580 (a p38 inhibitor), and PD98059 (a MEK1 inhibitor) were used. Results : GH inhibited CCh-induced chloride secretion at up to 10 nmol/L, but higher concentrations were less effective. GH caused tyrosine phosphorylation of JAK2 and EGFr. AG490 suppressed activation of JAK2 and EGFr in response to GH. AG1478 prevented GH activation of EGFr and reversed its inhibitory effect on chloride secretion. GH also induced activation of both p38 and ERK1/2. AG490 reversed GH-induced tyrosine phosphorylation of both ERK1/2 and p38, but AG1478 reversed that of ERK1/2 only. PD98059, but not SB203580, reversed the inhibitory effect of GH on chloride secretion. Conclusions : GH inhibits CCh-induced chloride secretion via a JAK2-dependent mechanism involving transactivation of EGFr and consequent recruitment of ERK1/2. Although activated, p38 does not contribute to the inhibitory effect of GH on secretion. These data elucidate mechanisms of GH inhibition of chloride secretion in intestinal epithelia, which may be relevant to therapeutic benefits of GH in Crohn’s disease or other diarrheal diseases.

Medicinal Chemistry and Pharmacology of A2B Adenosine Receptors

The low affinity A(2B) adenosine receptor, like any other adenosine receptor subtype, belongs to the super-family of seven transmembrane domain protein-coupled receptors (7TMs GPCR) and is classified by the GPCR database in the family of rhodopsin like receptors (Class A of GPCR). It has been cloned from various species, including rat and human, and its sequences are highly similar across species, ranging from 85% identity between human and mouse and 95% identity between rat and mouse. The A(2B)receptors show a ubiquitous distribution, the highest levels are present in cecum, colon and bladder, followed by blood vessels, lung, eye and mast cells. Through A(2B) receptors adenosine seems to cause mast cells degranulation, vasodilation, cardiac fibroblast proliferation, inhibition of Tumor Necrosis Factor (TNF-alpha), increased synthesis of interleukin-6 (IL-6), stimulation of Cl(-) secretion in intestinal epithelia and hepatic glucose production. Hence, A(2B) adenosine receptor agonists could be useful in the treatment of cardiac diseases like hypertension or myocardial infarction and in the management of septic shock, while antagonists may serve as novel drugs for asthma, Alzheimer's disease, cystic fibrosis and type-II diabetes. No potent and selective A(2B) agonists have been reported so far; 5'-N-ethylcarboxamidoadenosine (NECA) is one of the most active. The monosubstitution on N(6)-position of adenosine is well tolerated and that position appears to be a useful site for increasing A(2B) potency. Among substituents in 2-position of adenosine only 1-alkynyl chains are effective for A(2B) potency. In particular, the (S)-2-hydroxypropynyl substituents brought about the highest activity demonstrating that the A(2B) receptors discriminate between (R) and (S) diastereomers. Hence, (S)-2-phenylhydroxypropynylNECA (PHPNECA), with an EC(50) = 0.22 micro M, proved to be the most potent A(2B) agonist reported so far. Classical antagonists for adenosine receptors are alkylxanthines which show considerable potency at A(2B) receptors. Para substituted 1,3-dialkyl-8-phenylxanthines possess high affinity in binding assays; the 3-unsubstituted 1-alkyl analogues resulted more A(2B) selective with the 8-[4-[(N-(2-hydroxyethyl)carboxamidomethyl)oxy]phenyl]-1-propylxanthine (60) showing the highest affinity (K(i) = 1.2 nM) and selectivity (A(1)/A(2B) = 60, A(2A)/A(2B) = 1,790, A(3)/A(2B) = 360). Among non-xanthine derivatives very promising are substituted purines, in which combination of appropriate substituents in 2-, 8-, and 9-position could lead to very potent and selective A(2B) antagonists.

Tumour necrosis factor alpha potentiates ion secretion induced by histamine in a human intestinal epithelial cell line and in mouse colon: involvement of the phospholipase D pathway.

Patients suffering from inflammatory bowel disease show increased levels of the mast cell products histamine and tumour necrosis factor alpha (TNF-alpha). Treating these patients with antibodies against TNF-alpha diminishes the symptoms of diarrhoea. In this study, the effect of TNF-alpha on ion secretion induced by the mast cell mediator histamine in HT29cl.19A cells and mouse distal colon was investigated and the possible second messengers involved were studied. Electrophysiology of filter grown HT29cl.19A cells and isolated mouse distal colon was used to monitor the secretory response to histamine with and without prior exposure to TNF-alpha for 3-24 hours. Phospholipase D (PLD) activity and phosphatidic acid levels were analysed by 32P(i) labelling of HT29cl.19A cells. In both experimental systems TNF-alpha was found to potentiate ion secretion induced by histamine. Phospholipid analysis of HT29cl.19A cells revealed that histamine activates the PLD pathway. Furthermore, TNF-alpha pretreated cells were found to have decreased phosphatidic acid levels, the intermediate product of the PLD pathway, which indicates upregulation of the enzyme phosphatidic acid phosphatase. The mast cell products TNF-alpha and histamine synergistically stimulate ion secretion in intestinal epithelium via upregulation of the PLD pathway.

The impact of different flavonoid classes on colonic CI − secretion in rats

The plant polyphenol quercetin was shown to induce a significant CI − secretion in intestinal epithelium. In order to elucidate the structural requirements of quercetin and related flavonoids for this activity, we tested the ability of further flavonols and other flavonoids found in edible plants to induce CI − secretion which was measured as an increase in short-circuit current ( I sc ) in rat colon. Whereas several flavonols and the flavon luteolin increased I sc , other flavonoids such as flavanones, flavans, flavanols, and anthocyanidins failed to do so. Two glycosides of quercetin, spiraeosid, and isoquercitrin, as well as two methoxylated quercetin metabolites, isorhamnetin and tamarixetin, were also able to increase I sc . We conclude that a 2,3-double bond in conjunction with the 4-oxo group in the C ring and a hydroxylated B ring are necessary for the secretory activity of flavonoids. This activity requires different structural features than those mandatory for the antioxidative properties of flavonoids. Glucosidation and methoxylation of several hydroxyl groups does not necessarily abolish the secretory potential.

Fatty acids inhibit anion secretion in rat colon: apical and basolateral action sites.

Ca2+-dependent secretagogues evoke only a transient Cl- secretion in intestinal epithelia, although they induce a prolonged increase in the intracellular Ca2+ concentration, suggesting that they may exert an additional antisecretory action. In order to study the mechanism of this antisecretory effect, Cl- secretion, measured as the increase in short-circuit current (Isc), was evoked by carbachol in the absence and presence of different inhibitors. Neither a calmodulin antagonist, calmidazolium, nor different inhibitors of the nitric oxide (NO) pathway, i.e. Nomega-nitro-L-arginine (L-NNA) and Nomega-nitro-L-arginine methylester (L-NAME), affected the carbachol-induced Isc. However, inhibition of phospholipases A2 (PLA2) by quinacrine or arachidonyltrifluoromethyl ketone (AACOCF3) enhanced the Isc response evoked by carbachol, suggesting a role of fatty acids in the downregulation of anion secretion. Neither econazole, a cytochrome P450 inhibitor, nor nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenases, mimicked the action of the PLA2 blockers. Conversely, short- or medium-chain fatty acids inhibited the carbachol- and forskolin-induced Isc with caprate (C10:0) being the most efficient water-soluble fatty acid. This fatty acid inhibited a Cl- current, which was driven across the apical membrane by a serosally to mucosally directed Cl- gradient after depolarization of the basolateral membrane. A second action site of fatty acids seems to be the basolateral membrane. After permeabilization of the apical membrane with the ionophore nystatin, a mucosally to serosally directed K+ gradient induced a K+ current, which was also inhibited by caprate. These results indicate that carbachol not only acts as a secretagogue but at the same time initializes downregulation by increasing the intracellular concentration of fatty acids, a mechanism limiting the resulting Cl- secretion.

Myosin regulation of NKCC1: effects on cAMP-mediated Cl− secretion in intestinal epithelia

The basally located actin cytoskeleton has been demonstrated previously to regulate Cl- secretion from intestinal epithelia via its effects on the Na+-K+-2Cl- cotransporter (NKCC1). In nontransporting epithelia, inhibition of myosin light chain kinase (MLCK) prevents cell-shrinkage-induced activation of NKCC1. The aim of this study was to investigate the role of myosin in the regulation of secretagogue-stimulated Cl- secretion in intestinal epithelia. The human intestinal epithelial cell line T84 was used for these studies. Prevention of myosin light chain phosphorylation with the MLCK inhibitor ML-9 or ML-7 and inhibition of myosin ATPase with butanedione monoxime (BDM) attenuated cAMP but not Ca2+-mediated Cl- secretion. Both ML-9 and BDM diminished cAMP activation of NKCC1. Neither apical Cl- channel activity, basolateral K+ channel activity, nor Na+-K+-ATPase were affected by these agents. Cytochalasin D prevented such attenuation. cAMP-induced rearrangement of basal actin microfilaments was prevented by both ML-9 and BDM. The phosphorylation of mosin light chain and subsequent contraction of basal actin-myosin bundles are crucial to the cAMP-driven activation of NKCC1 and subsequent apical Cl- efflux.

Effect of Saccharomyces boulardii on cAMP- and Ca2+ -dependent Cl- secretion in T84 cells.

Several reports have confirmed that the cooperative interaction between cAMP- and Ca2+-mediated transduction pathways may contribute to the stimulatory or inhibitory regulation of Cl- secretion in intestinal epithelium. Saccharomyces boulardii has been shown to inhibit cholera toxin-induced secretion in rat jejunum. We have identified a 120-kDa protein in medium conditioned by Saccharomyces boulardii that reduces cholera toxin-induced cAMP in intestinal cells. The present study evaluated the effect of medium conditioned by Saccharomyces boulardii on cAMP- and Ca2+-mediated Cl- secretion in T84 cells. Experiments performed with cAMP agonists revealed that 1 hr of preincubation of cells with medium conditioned by Saccharomyces boulardii was necessary to elicit a 40-50% reduction in receptor (cholera toxin, prostaglandin E2, and vasoactive intestinal polypeptide) and nonreceptor (forskolin) mediated cAMP synthesis and 125I efflux. Secretion induced by carbachol was inhibited when cells were pretreated for 1 hr with medium conditioned by Saccharomyces boulardii despite the absence of inhibition of Ins (1,4,5)P3. From this study we conclude that Saccharomyces boulardii exerts an inhibitory effect in vitro on Cl- secretion mediated through both cAMP- and Ca2+-mediated signaling pathways.

Neuropeptide Y inhibits ion secretion in intestinal epithelium by reducing chloride and potassium conductance.

Neuropeptide Y (NPY) is probably the most abundant neuropeptide, with a plethora of central as well as peripheral effects, including its proabsorptive action in the gastro-intestinal tract. The effects of NPY on electrical parameters related to three different pathways stimulating ion secretion were investigated using the human intestinal cell line HT29cl.19A. Transepithelial potential and resistance were measured with the preparation maintained in a horizontal Ussing chamber, allowing simultaneous measurement of the membrane potential and determination of the fractional resistance of the apical cell membrane. It was found that application of NPY, after the adenylyl-cyclase-activating drug forskolin, resulted in complete inhibition of forskolin-induced effects within approximately 20 min. The secretion stimulated by adenosine appeared to be insensitive to NPY. The acetylcholine analogue carbachol stimulates ion secretion by increasing intracellular free calcium concentrations ([Ca2+]i) which activates the basolateral potassium (K+) conductance. NPY caused 50% inhibition of the effect of carbachol. Measurements of [Ca2+]i showed that NPY inhibited the carbachol-induced rise in [Ca2+]i, which correlates with the reduced activation of basolateral K+ channels. From this study we conclude that NPY inhibits cAMP-stimulated as well as Ca2+-stimulated secretion via a reduction in the apical Cl- and basolateral K+ conductance. This double effect makes NPY an effective proabsorptive peptide.

The A Adenosine Receptor Mediates cAMP Responses to Adenosine Receptor Agonists in Human Intestinal Epithelia

Adenosine is thought to be a major effector in immunological stimulation of Cl- secretion in intestinal epithelia. Previous studies indicate that both apical and basolateral domains of intestinal epithelial cells possess functionally defined adenosine receptors. However, it is unclear whether the same receptor subclass is expressed, what the receptor subclass(es) is, or how the receptors signal the Cl- secretory response. We now characterize the intestinal epithelial adenosine receptor subtype using the model epithelium, T84. Both apical and basolateral adenosine receptor agonist response profiles revealed a hierarchy (ED50) of 5'-(N-ethylcarboxamido)adenosine > adenosine > CGS-21680. Similarly inhibition studies revealed identical ID50 hierarchies for apical and basolateral antagonism by xanthine amine congener > 1,3-diethyl-8-phenylxanthine > aminophylline. Analyses of both agonist and antagonist pharmacological hierarchies in Chinese hamster ovary cells stably expressing the A2b receptor revealed these same hierarchies. Northern blots performed on RNA extracted from polarized T84 monolayers demonstrated no detectable message for A1 or A2a adenosine receptor, but strong hybridization was detected for the A2b adenosine receptor. Subsequent Northern blots of RNA prepared from human alimentary tract revealed that A2b adenosine receptor message was heavily expressed throughout the colon, in the appendix, and more modestly expressed in the small intestine (ileum). Analyses of cAMP generation in T84 cells in response to adenosine indicated that the basolateral A2b receptor elicits Cl- secretion through this signaling pathway. Stimulation of Cl- secretion through the apical A2b receptor exhibited relatively small but significant increases in cAMP compared with basolateral stimulation. The protein kinase A inhibitor H-89, used at concentrations that did not affect short circuit current responses to the Ca(2+)-mediated agonist carbachol, effectively inhibited short circuit current elicited by either apical or basolateral adenosine. These data suggest that the major intestinal epithelial adenosine receptor is the A2b subclass, which is positively coupled to adenylate cyclase. Such observations have potentially important implications for the treatment of diarrheal diseases.

Diversity of kinin effects on transporting epithelia.

Over the last five years it has become clear that kinins can have major effects on the transepithelial transport of ions. From the first descriptions of effects upon electrogenic chloride secretion in intestinal epi-thelia it has been shown that kinins can affect the transport of ions other than chloride. Indeed not only can transport of other anions be influenced, but also the transepithelial movement of cations is affected by kinins. Even when transport of the same ion, say chloride is stimulated by kinins the mechanisms involved in different epithelial systems may be quite distinct. Finally the disposition of receptors for kinins in epithelia is also variable, in some they are located apically, in others basolaterally and yet in others in both these membrane domains.KeywordsChloride SecretionKinin EffectRapid Structural ChangeCanine Tracheal EpitheliumElectrogenic Chloride SecretionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.