Active Anion Secretion Research Articles

Epithelial Barrier Dysfunction in Diarrhea-Predominant Irritable Bowel Syndrome (IBS-D) via Downregulation of Claudin-1.

In patients with diarrhea-predominant irritable bowel syndrome (IBS-D), the diarrheal mechanisms are largely unknown, and they were examined in this study on colon biopsies. Electrophysiological measurements were used for monitoring functional changes in the diarrheic colon specimens. In parallel, tight junction protein expression was analyzed by Western blot and confocal laser-scanning microscopy, and signaling pathway analysis was performed using RNA sequencing and bioinformatics. Epithelial resistance was decreased, indicating an epithelial leak flux diarrheal mechanism with a molecular correlate of decreased claudin-1 expression, while induction of active anion secretion and impairment of active sodium absorption via the epithelial sodium channel, ENaC, were not detected. The pathway analysis revealed activation of barrier-affecting cytokines TNF-α, IFN-γ, IL-1β and IL-4. Barrier dysfunction as a result of epithelial tight junction changes plays a role in IBS-D as a pathomechanism inducing a leak flux type of diarrhea.

Intestinal Barrier in Post-Campylobacter jejuni Irritable Bowel Syndrome.

Campylobacter jejuni (C. jejuni) is one of the most common causes of bacterial gastroenteritis worldwide. One sequela of this infection is the development of post-infectious irritable bowel syndrome (PI-IBS). It has been suggested that a dysfunctional intestinal barrier may promote IBS development. We aimed to test this hypothesis against the background of the leaky gut concept for low-grade inflammation in PI-IBS. We identified patients with persistent PI-IBS symptoms after C. jejuni infection. During sigmoidoscopy, forceps biopsies were obtained for electrophysiological measurements of epithelial transport and barrier function in miniaturized Ussing devices. C. jejuni absence was checked by PCR and cytokine production with immunohistochemistry. In PI-IBS, the epithelial resistance of the colon epithelium was unaltered, reflecting an intact paracellular pathway. In contrast, temperature-dependent horseradish peroxidase (HRP, 44 kDa) permeation increased. Short-circuit current (Isc) reflecting active anion secretion and ENaC-dependent electrogenic sodium absorption was unaffected. Early endosome antigen-1 (EEA1) and IL-4 levels increased. C. jejuni is not incorporated into the resident microbiota of the colon mucosa in PI-IBS. In PI-IBS after C. jejuni infection, macromolecule uptake via endocytosis was enhanced, leading to low-grade inflammation with pro-inflammatory cytokine release. The findings will allow C. jejuni-induced pathomechanisms to be targeted during infection and, thereafter to reduce sequelae such as PI-IBS.

Cholinergic-induced anion secretion in murine jejunal enteroids involves synergy between muscarinic and nicotinic pathways.

Acetylcholine induces robust electrogenic anion secretion in mammalian intestine and it has long been hypothesized that it mediates the epithelial response through the M3 and, to a lesser extent, the M1 muscarinic receptors in the mouse. However, nicotinic receptors have recently been identified in intestinal enterocytes by quantitative real-time (qRT)-PCR/RNAseq, although any direct influence on intestinal transport has not been identified. We tested the hypothesis that cholinergic-induced anion secretion in the intestine is a result of both muscarinic and nicotinic pathways that are intrinsic to the intestinal epithelia. We developed a method to generate mouse jejunal enteroid monolayers which were used to measure active electrogenic anion secretion by the Ussing chamber/voltage-clamp technique. Here, we show that the cholinergic agonist carbachol (CCh) and the muscarinic agonist bethanechol (BCh) stimulate short-lived, concentration-dependent anion secretion in the epithelial cell-only enteroid monolayers. The muscarinic antagonist atropine completely inhibited CCh- and BCh-induced secretion, while the nicotinic antagonist hexamethonium reduced the CCh response by ~45%. While nicotine alone did not alter anion secretion, it increased the BCh-induced increase in short-circuit current in a concentration-dependent manner; this synergy was prevented by pretreatment with hexamethonium. In addition to being sensitive to hexamethonium, monolayers express both classes of cholinergic receptor by qRT-PCR, including 13 of 16 nicotinic receptor subunits. Our findings indicate that an interaction between muscarinic and nicotinic agonists synergistically stimulates anion secretion in mouse jejunal epithelial cells and identify a role for epithelial nicotinic receptors in anion secretion.

Molecular Basis and Differentiation-Associated Alterations of Anion Secretion in Human Duodenal Enteroid Monolayers

Background & AimsHuman enteroids present a novel tool to study human intestinal ion transport physiology and pathophysiology. The present study describes the contributions of Cl- and HCO3- secretion to total cyclic adenosine monophosphate (cAMP)-stimulated electrogenic anion secretion in human duodenal enteroid monolayers and the relevant changes after differentiation.MethodsHuman duodenal enteroids derived from 4 donors were grown as monolayers and differentiated by a protocol that includes the removal of Wnt3A, R-spondin1, and SB202190 for 5 days. The messenger RNA level and protein expression of selected ion transporters and carbonic anhydrase isoforms were determined by quantitative real-time polymerase chain reaction and immunoblotting, respectively. Undifferentiated and differentiated enteroid monolayers were mounted in the Ussing chamber/voltage-current clamp apparatus, using solutions that contained as well as lacked Cl- and HCO3-/CO2, to determine the magnitude of forskolin-induced short-circuit current change and its sensitivity to specific inhibitors that target selected ion transporters and carbonic anhydrase(s).ResultsDifferentiation resulted in a significant reduction in the messenger RNA level and protein expression of cystic fibrosis transmembrane conductance regulator, (CFTR) Na+/K+/2Cl- co-transporter 1 (NKCC1), and potassium channel, voltage gated, subfamily E, regulatory subunit 3 (KCNE3); and, conversely, increase of down-regulated-in-adenoma (DRA), electrogenic Na+/HCO3- co-transporter 1 (NBCe1), carbonic anhydrase 2 (CA2), and carbonic anhydrase 4 (CA4). Both undifferentiated and differentiated enteroids showed active cAMP-stimulated anion secretion that included both Cl- and HCO3- secretion as the magnitude of total active anion secretion was reduced after the removal of extracellular Cl- or HCO3-/CO2. The magnitude of total anion secretion in differentiated enteroids was approximately 33% of that in undifferentiated enteroids, primarily owing to the reduction in Cl- secretion with no significant change in HCO3- secretion. Anion secretion was consistently lower but detectable in differentiated enteroids compared with undifferentiated enteroids in the absence of extracellular Cl- or HCO3-/CO2. Inhibiting CFTR, NKCC1, carbonic anhydrase(s), cAMP-activated K+ channel(s), and Na+/K+-adenosine triphosphatase reduced cAMP-stimulated anion secretion in both undifferentiated and differentiated enteroids.ConclusionsHuman enteroids recapitulate anion secretion physiology of small intestinal epithelium. Enteroid differentiation is associated with significant alterations in the expression of several ion transporters and carbonic anhydrase isoforms, leading to a reduced but preserved anion secretory phenotype owing to markedly reduced Cl- secretion but no significant change in HCO3- secretion.

Campylobacter fetus impairs barrier function in HT-29/B6 cells through focal tight junction alterations and leaks.

Infections by Campylobacter species are the most common foodborne zoonotic disease worldwide. Campylobacter jejuni and C. coli are isolated most frequently from human stool samples, but severe infections by C. fetus (Cf), which can cause gastroenteritis, septicemia, and abortion, are also found. This study aims at the characterization of pathological changes in Cf infection using an intestinal epithelial cell model. The Cf-induced epithelial barrier defects appeared earlier than those of avian Campylobacter species like C. jejuni/C. coli. Two-path impedance spectroscopy (2PI) distinguished transcellular and paracellular resistance contributions to the overall epithelial barrier impairment. Both transcellular and paracellular resistance of Cf-infected HT-29/B6 monolayers were reduced. The latter was attributed to activation of active anion secretion. Western blot analysis showed no decrease in tight junction (TJ) protein expression (claudin-1, -2, -3, and -4) but showed redistribution of claudin-1 off the TJ domain. In addition, Cf induced epithelial cell death, cell detachment, and lesions (focal leaks), as the result of which macromolecule flux (10-kDa dextran) was increased in Cf-invaded cell monolayers. In conclusion, barrier dysfunction from Cf infection was due to TJ protein redistribution, cell death induction, and leak formation, resulting in bacterial translocation, ion leak flux, and antigen uptake (leaky gut).

Mo2029 Differential Activity of NHE3 and NHE2 in the Small Intestine of Irradiated Mice

Tight junctions are formed by mature enterocytes and create an efficient barrier that has the necessary machinery for electrolyte and nutrient absorption. Radiotherapy causes epithelial barrier dysfunction leading to increased macromolecular translocation into the systemic compartment causing endotoxemia and inflammatory response. Recently, it was shown that glucose and some amino acids (AAs) activated active anion secretion and/ or increased paracellular permeability, whereas our mitigating amino acids mixture (MAAM) comprised of lysine, glycine, tryptophan, tyrosine, aspartic acid, isoleucine, threonine, valine, and serine increased electrolyte absorption and decreased paracellular permeability. The mechanism by which these amino acids tightened the mucosal barrier was not known. Therefore, studies were undertaken to determine the mechanism by which MAAM tightened the mucosal barrier. NIH Swiss mice were irradiated using a 137Cs source. Radiation dose-dependent and time-dependent (0, 6, 12, 24, 48, 72, and 144 hr after irradiation) changes in transepithelial electrical resistance (TEER) and dilution potential (DP) and the relative permeability of Cland Na+ (PCl/PNa) were measured. Transmission electron microscopy (TEM) images, Western blot analysis, and immunohistochemistry (IHC) for the cell junction protein complex (Claudin 1, 2, 5, Nectin, and E-cadherin) were performed at similar time points. Radiation resulted in a dose-dependent increase in conductance (43.3 ± 1.2, 29.5 ± 1.2, 48.2 ± 2.3, 48.9 ± 1.7, 38.7 ± 1.2 mS at 0, 1, 3, 5, 7 Gy, respectively). Similarly, time-dependent changes in conductance showed a maximal increase occurring in the first 6 hr (46.3 ± 1.5); there was no significant difference with increasing time after irradiation. Treatment with MAAM resulted in a significant decrease in conductance. DP studies showed that MAAM restored ion selectivity. TEM showed disruption of the cell junction complex and formation of clear space between the cells as early as 6 hr after irradiation. The changes persisted for the duration of the experiment (6 days). MAAM prevented cell junction disruption in mice; this was identifiable at 6 hr after irradiation. Western analysis and IHC showed a radiation dose-dependent increase in Claudin 1, 2, 5, Nectin, and E-cadherin except at 7 Gy. MAAM further increased Claudin 1, 2, 5, Nectin, and E-cadherin across all radiation doses. MAAM treatment decreased paracellular permeability. TEM showed disruption of the cell junction complex that peaked at 6 hours and was corrected using MAAM. Western analysis and IHC showed that radiation-induced alterations in the cell junction protein complex were further corrected in the MAAM group. We conclude that the disruption in the barrier results from changes in the cell junction complex and that MAAM restores the barrier function by correcting these changes.

Mo2028 A Functional Approach to Improve Barrier Function in Irradiated Mice

Tight junctions are formed by mature enterocytes and create an efficient barrier that has the necessary machinery for electrolyte and nutrient absorption. Radiotherapy causes epithelial barrier dysfunction leading to increased macromolecular translocation into the systemic compartment causing endotoxemia and inflammatory response. Recently, it was shown that glucose and some amino acids (AAs) activated active anion secretion and/ or increased paracellular permeability, whereas our mitigating amino acids mixture (MAAM) comprised of lysine, glycine, tryptophan, tyrosine, aspartic acid, isoleucine, threonine, valine, and serine increased electrolyte absorption and decreased paracellular permeability. The mechanism by which these amino acids tightened the mucosal barrier was not known. Therefore, studies were undertaken to determine the mechanism by which MAAM tightened the mucosal barrier. NIH Swiss mice were irradiated using a 137Cs source. Radiation dose-dependent and time-dependent (0, 6, 12, 24, 48, 72, and 144 hr after irradiation) changes in transepithelial electrical resistance (TEER) and dilution potential (DP) and the relative permeability of Cland Na+ (PCl/PNa) were measured. Transmission electron microscopy (TEM) images, Western blot analysis, and immunohistochemistry (IHC) for the cell junction protein complex (Claudin 1, 2, 5, Nectin, and E-cadherin) were performed at similar time points. Radiation resulted in a dose-dependent increase in conductance (43.3 ± 1.2, 29.5 ± 1.2, 48.2 ± 2.3, 48.9 ± 1.7, 38.7 ± 1.2 mS at 0, 1, 3, 5, 7 Gy, respectively). Similarly, time-dependent changes in conductance showed a maximal increase occurring in the first 6 hr (46.3 ± 1.5); there was no significant difference with increasing time after irradiation. Treatment with MAAM resulted in a significant decrease in conductance. DP studies showed that MAAM restored ion selectivity. TEM showed disruption of the cell junction complex and formation of clear space between the cells as early as 6 hr after irradiation. The changes persisted for the duration of the experiment (6 days). MAAM prevented cell junction disruption in mice; this was identifiable at 6 hr after irradiation. Western analysis and IHC showed a radiation dose-dependent increase in Claudin 1, 2, 5, Nectin, and E-cadherin except at 7 Gy. MAAM further increased Claudin 1, 2, 5, Nectin, and E-cadherin across all radiation doses. MAAM treatment decreased paracellular permeability. TEM showed disruption of the cell junction complex that peaked at 6 hours and was corrected using MAAM. Western analysis and IHC showed that radiation-induced alterations in the cell junction protein complex were further corrected in the MAAM group. We conclude that the disruption in the barrier results from changes in the cell junction complex and that MAAM restores the barrier function by correcting these changes.

Chloride secretion by cultures of pig tracheal gland cells

Malfunction of airway submucosal glands contributes to the pathology of cystic fibrosis (CF), and cell cultures of CF human airway glands show defects in Cl(-) and water transport. Recently, a transgenic pig model of CF (the CF pig) has been developed. Accordingly, we have developed cell cultures of pig airway gland epithelium for use in investigating alterations in gland function in CF. Our cultures form tight junctions (as evidenced by high transepithelial electrical resistance) and show high levels of active anion secretion (measured as amiloride-insensitive short-circuit current). In agreement with recent results on human airway glands, neurohumoral agents that elevate intracellular Ca(2+) potently stimulated anion secretion, while elevation of cAMP was comparatively ineffective. Our cultures express lactoferrin and lysozyme (serous gland cell markers) and MUC5B (the main mucin of airway glands). They are, therefore, potentially useful in determining if CF-related alterations in anion transport result in altered secretion of serous cell antimicrobial agents or mucus.

Neural involvement in 5-hydroxytryptamine-induced net electrogenic ion secretion in the rat intestine in-vivo.

5-Hydroxytryptamine (5-HT) induces active electrogenic anion secretion by both the small intestine and the colon, responses that can be detected from measurements of transmural electrical activity. This approach was adopted to examine the involvement of neural mechanisms in 5-HT-induced secretion in rat proximal jejunum, distal ileum and proximal colon in-vivo. Under control conditions, 5-HT caused maximum rises in transintestinal potential difference of 4.7 +/- 0.3, 3.8 +/- 0.4 and 7.6 +/- 0.3 mV, respectively, with corresponding ED50 values of 28 +/- 3, 38 +/- 4 and 41 +/- 4 nmol kg-1 (n = 12). In each region examined a neural component in the secretory response to 5-HT was identified. Hexamethonium (22 mumol kg-1) reduced the 5-HT response in each region: in the jejunum and colon, it also attenuated the responses to the 5-HT3 agonist, phenylbiguanide and to 5-methoxytryptamine (5-MeOT), an agonist at all 5-HT receptors except 5-HT3, indicating that in these regions the nicotinic pathway can be activated by more than one 5-HT receptor subtype. Atropine (0.27 and 2.7 mumol kg-1) was found to have regional effects on the intestinal responses to 5-HT receptor agonists. In the jejunum, evidence for a pro-secretory muscarinic pathway which could be activated by more than one 5-HT receptor subtype was found. In the ileum and colon no muscarinic pro-secretory pathway was identified, indeed in the colon, an anti-secretory pathway may be present. This muscarinic anti-secretory pathway was observed with phenylbiguanide and 5-MeOT, but not 5-HT. Substance P release does not appear to be involved in mediating the intestinal secretory response to 5-HT. 5-HT-induced intestinal anion secretion may involve a direct secretory action on the enterocyte which can be modified by neurally-mediated pro-secretory and anti-secretory pathways, the balance between these processes varying down the length of the gut.

Organic Anion Secretion across the Ciliary Body Epithelium of the Eye

Several organic anions have been shown to accumulate in the ciliary body following intraocular injection. Additionally, clearance of organic anions from the eye is slowed by the organic anion transport inhibitor probenecid. Here, we hypothesized that the ciliary body epithelium would support organic anion secretion in an aqueous humor (AH)‐to‐blood direction. Bovine ciliary body epithelium was mounted in Ussing chambers for determination of unidirectional 3H‐p‐aminohippurate (PAH) fluxes under short‐circuited conditions (at 0.01 mM PAH). The unidirectional AH‐to‐blood flux was 6‐fold greater than the blood‐to‐AH flux (0.6 ± 0.15 vs. 0.1 ± 0.03 nmoles x cm−2 x hr−1), indicating net active secretion. Net active PAH secretion was strongly inhibited by 0.25 mM probenecid. Consistent with the transport data, mRNA (RT‐PCR) and protein (Western blot) analysis revealed several organic anion transporters, including OAT1, MRP2 and MRP4 in the ciliary body. Active organic anion secretion (AH‐to‐blood) by the ciliary body epithelium may influence the effectiveness of thereaputic drugs, such as antivirals, antibiotics and chemotherapeutics, to treat ocular diseases. Supported by NIH (EY006915).

Structural and functional changes of the duodenum in human norovirus infection

Background:Norovirus infection is the most frequent cause of infectious diarrhoea in the western world. This study aimed to characterise functionally and histomorphologically the diseased duodenum in human biopsies.Methods:Norovirus infection was...

T cell activation altersintestinal structure and function

Treatment with anti-CD3 antibody (anti-CD3) causes transient diarrhea. In this issue of the JCI, Clayburgh et al. show that, in jejunum of mice injected with anti-CD3 or with TNF, fluid accumulation and changes in epithelial phenotype develop, the latter including an increase in the passive permeability to proteins, smaller solutes, and water and the endocytosis of the brush border Na+/H+ exchanger, thereby inhibiting Na+ absorption (a second cytokine, LIGHT, has the former effect, but not the latter) (see the related article beginning on page 2682). These phenotypic changes, by themselves, do not, however, explain increased fluid secretion. Since active anion secretion is not stimulated (in fact it is inhibited), a non-epithelial cell-mediated driving force must be present--most likely an increase in interstitial pressure due to an effect of TNF on capillary permeability, smooth muscle contractility, or both.

NSP4 PLAYS A MAJOR ROLE IN THE EARLY SECRETORY DIARRHEA INDUCED BY ROTAVIRUS IN A CACO-2 EXPERIMENTAL MODEL

Aim: Among Rotavirus' (RV) multiple mechanisms of diarrhea a major role is played by viral enterotoxin NSP4. RV infection induces an early anion secretion and subsequent cell damage in a Caco-2 cell model. Aim of the study was to address the role of NSP4 in a model of RV infection in human-derived intestinal cell line. Methods: Caco-2 cells were infected with RV strain SA-11 (5PFU/cell) and mounted in Ussing chambers at different times after infection. Production of NSP4 was assessed by western blotting in immunoprecipitates of supernatants and cell lysates. Short circuit current (Isc) and trans-epithelial resistance (TEER), reflecting transepithelial ion transport and tissue integrity were recorded. In parallel experiments, non-specific human immunoglobulins (Ig) preparations for parenteral use and specific anti-NSP4 Ig were added to enterocyte monolayers. Results: RV induced a biphasic response with a significant increase in Isc after 1-3 hours of infection, consistent with active anion secretion. TEER started to fall progressively 24 hours after infection, indicating monolayer loss of integrity. Starting from 1 hour after infection, NSP4 was detected in the supernatant of infected cells at increasing concentrations up to 48 hours post-infection. Addition of anti-NSP4 Ig, as well as no-specific human Ig, blocked the RV-induced secretion but not TEER modifications. Discussion: RV induces a dual, chronologically distinct, pathogenic effect in human enterocytes: 1) an early anion secretion which is NSP4-dependent and inhibited by specific Ig and by non-specific human Ig; 2) a subsequent cytopathic damage which is not prevented by anti-NSP4 Ig but blocked by human Ig.

T cell activation causes diarrhea by increasing intestinal permeability and inhibiting epithelial Na+/K+-ATPase.

Inflammatory bowel disease (IBD) is associated with mucosal T cell activation and diarrhea. We found that T cell activation with anti-CD3 mAb induces profound diarrhea in mice. Diarrhea was quantified by intestinal weight-to-length (wt/l) ratios, mucosal Na(+)/K(+)-ATPase activity was determined and ion transport changes were measured in Ussing chambers. Anti-CD3 mAb increased jejunal wt/l ratios by more than 50% at 3 hours, returning to base line after 6 hours. Fluid accumulation was significantly reduced in TNF receptor-1 (TNFR-1(-/-)), but not IFN-gamma knockout mice. Anti-CD3 mAb decreased mucosal Na(+)/K(+)-ATPase activity, which was blocked by anti-TNF mAb and occurred to a lesser degree in TNFR-1(-/-) mice. Neither alpha nor beta subunits of Na(+)/K(+)-ATPase decreased in abundance at 3 hours. Intestinal tissue from anti-CD3-treated mice exhibited increased permeability to mannitol at 1 hour and decreases in electroneutral Na(+) absorption, Na(+)-dependent glucose absorption, and cAMP-stimulated anion secretion at 3 hours. Furthermore, enteral fluid accumulation was observed in CFTR(-/-) mice, indicating a minor role of active anion secretion. These data suggest that diarrhea in IBD is due to TNF-mediated malabsorption rather than to secretory processes. T cell activation induces luminal fluid accumulation by increasing mucosal permeability and reducing epithelial Na(+)/K(+)-ATPase activity leading to decreased intestinal Na(+) and water absorption.

T cell activation causes diarrhea by increasing intestinal permeability and inhibiting epithelial Na+/K+-ATPase

Inflammatory bowel disease (IBD) is associated with mucosal T cell activation and diarrhea. We found that T cell activation with anti-CD3 mAb induces profound diarrhea in mice. Diarrhea was quantified by intestinal weight-to-length (wt/l) ratios, mucosal Na+/K+-ATPase activity was determined and ion transport changes were measured in Ussing chambers. Anti-CD3 mAb increased jejunal wt/l ratios by more than 50% at 3 hours, returning to base line after 6 hours. Fluid accumulation was significantly reduced in TNF receptor-1 (TNFR-1–/–), but not IFN-γ knockout mice. Anti-CD3 mAb decreased mucosal Na+/K+-ATPase activity, which was blocked by anti-TNF mAb and occurred to a lesser degree in TNFR-1–/– mice. Neither α nor β subunits of Na+/K+-ATPase decreased in abundance at 3 hours. Intestinal tissue from anti-CD3–treated mice exhibited increased permeability to mannitol at 1 hour and decreases in electroneutral Na+ absorption, Na+-dependent glucose absorption, and cAMP-stimulated anion secretion at 3 hours. Furthermore, enteral fluid accumulation was observed in CFTR–/– mice, indicating a minor role of active anion secretion. These data suggest that diarrhea in IBD is due to TNF-mediated malabsorption rather than to secretory processes. T cell activation induces luminal fluid accumulation by increasing mucosal permeability and reducing epithelial Na+/K+-ATPase activity leading to decreased intestinal Na+ and water absorption.

δ-opioid receptors inhibit neurogenic intestinal secretion evoked by mast cell degranulation and type I hypersensitivity

Histamine and the mast cell degranulator, compound 48/80 produced elevations in short-circuit current, an electrical measure of active anion secretion, across porcine ileal mucosa sheets mounted in Ussing chambers. Luminally-applied β-lactoglobulin produced similar effects in mucosal sheets from cow’s milk-sensitized pigs. Their secretory effects were attenuated by blockers of H 1-histamine receptors, neuronal conduction or epithelial Na +/K +/Cl − cotransport. The δ-opioid agonist [ d-Pen 2, d-Pen 5]enkephalin suppressed mucosal responses to these substances in a naltrindole-reversible manner. Furthermore, submucosal mast cells and δ-opioid receptor-immunoreactive nerve fibers were observed in close juxtaposition. Intestinal neural pathways linking immediate hypersensitivity to secretory host defense appear to express inhibitory δ-opioid receptors.

Regulation of Ion Transport in the Porcine Intestinal Tract by Enteric Neurotransmitters and Hormones

In the present paper, the mechanisms underlying the neural and hormonal regulation of mucosal ion transport in the pig intestinal tract are reviewed. The active transport of NaCl by isolated sheets of porcine intestinal mucosa is modulated by cholinergic and non-cholinergic neurons of undetermined neurochemical identity that lie in the submucosa. The application of electrical field stimulation to mucosa-submucosa preparations from porcine jejunum, ileum, or colon produces rapid elevations in short-circuit current which are inhibited by tetrodotoxin or omega-conotoxin GVIA, blockers of neuronal Na + and Ca 2+ channels, respectively. In porcine ileum, these elevations in current are mimicked in large part by cholinergic agonists and have been attributed to anion secretion. The majority of classical neurotransmitters and gut peptides that have been examined to date increase active transepithelial anion secretion through interactions with G protein-coupled receptors associated with submucosal neurons or situated on the basolateral membranes of epithelial cells. A small number of neuropeptides interact with neuronal receptors to augment NaCl absorption or decrease anion secretion. Noradrenergic control of intestinal transport differs in the porcine small and large intestines, and displays considerable inter-species variability in its cellular underpinnings. Transport regulation by bombesin-like peptides may be mediated by receptors distributed in both the apical and basolateral membrane domains of epithelial cells in porcine colon. The transport process affected by these peptides may be linked to epithelial growth and differentiation. The pig intestinal tract appears to be a useful biological model for resolving the cellular mechanisms by which gut neurotransmitters and hormones act in regulating transepithelial ion fluxes. Its general relevance to human intestinal function is discussed.

Separate regulation of Na+ and anion transport by IMCD: location, aldosterone, hypertonicity, TGF-beta 1, and cAMP.

We have investigated some of the factors known or suspected to influence ion transport by the rat inner medullary collecting duct and have analyzed their actions on active Na+ absorption and active anion secretion by primary cultures. Cells from the terminal 1-2 mm (tip) of the papilla had a lower basal rate of Na+ absorption (2.0 microA/cm2) than cells from the more proximal portions (6.5 microA/cm2). Aldosterone increased Na+ transport approximately sevenfold in the tip cells and approximately threefold in the proximal cells. The magnitude of anion secretion in response to adenosine 3',5'-cyclic monophosphate (cAMP) agonists was similar in the two regions and was unaffected by aldosterone. The morphology of monolayers from both regions was also similar. In monolayers cultured from the entire inner medulla, hypertonic (100 mosM) urea, NaCl, or sucrose reduced Na+ transport but had no significant effect on anion secretion. Transforming growth factor-beta 1, known to blunt the effect of steroids on Na+ transport, had no effect on anion secretion. Finally, cAMP had no effect on Na+ transport, a result that contrasts with its effect on Na+ transport by other epithelial cells demonstrating steroid-responsive, electrogenic Na+ transport. These results demonstrate some potential differences in the magnitude of Na+ transport by position along the inner medulla. They further demonstrate separate regulation of Na+ and anion transport.

Mucosal protection through active intestinal secretion: neural and paracrine modulation by 5-hydroxytryptamine

5-Hydroxytryptamine (5-HT) is widely distributed within enteroendocrine cells and neurons of the digestive tract. It stimulates active anion secretion in the intestinal epithelium, an effect which promotes the dilution and elimination of luminal pathogens. The intestinal secretory effects of 5-HT appear to be mediated in part by epithelial 5-HT 2 like receptors that are linked to phosphatidylinositol turnover. In addition, 5-HT appears to interact with 5-HT 3 or 5-HT 4 receptors on submucosal neurons to elicit neuronal depolarization and an associated increase in intestinal secretion. Although the precise cellular mechanisms by which 5-HT modulates mucosal ion transport are incompletely understood, it is clear that 5-HT plays an important role as a intestinal secretagogue in certain diarrheal states, in laxation, and in intestinal hypersensitivity reactions.

Cholinergic neurons and muscarinic receptors regulate anion secretion in pig distal jejunum

The neurotransmitter acetylcholine is thought to modulate epithelial ion transport in the mammalian small intestine. In this study, the non-selective acetylcholine agonist, carbachol, produced rapid changes in short-circuit current (Isc), an electrical measure of active anion secretion, across isolated sheets of the distal jejunal mucosa-submucosa from swine. The potency of carbachol in elevating Isc was decreased 26-fold in the presence of 10 nM atropine, but remained unchanged by 1–100 μM hexamethonium or 0.1 μM tetrodotoxin. The acetylcholine antagonists produced little or no decrease in spontaneous Isc, whereas tetrodotoxin decreased Isc by 54 μA/cm 2. [ 3H]Quinuclidinyl benzilate (QNB), a muscarinic acetylcholine receptor blocker, bound to a single species of sites within the mucosa-submucosa with a K D = 38 pM and B max = 94 fmol/mg protein. Selective muscarinic acetylcholine receptor blockers competed with [ 3H]QNB for this site with a rank order of affinity indicative of an interaction with a M 3-muscarinic acetylcholine receptor. Specific [ 3H]QNB binding sites were autoradiographically localized in the jejunal wall to the epithelium, submucosa and muscularis propria. Transmural electrical stimulation (3–600 pulses/10 s, 0.5 ms, 60 V) of the mucosa-submucosa produced increases in Isc which were dependent upon the number of impulses delivered and did not undergo tachyphylaxis upon repeated stimulation. Responses to electrical transmural stimulation were inhibited by atropine and hexamethonium, as well as the respective neuronal Na + and Ca 2+ channel blockers tetrodotoxin and ω-conotoxin GVIA, suggesting that electrical transmural stimulation depolarizes submucosal cholinergic neurons which terminate on ion-transporting epithelial cells. Thus acetylcholine, released upon electrical depolarization of intrinsic submucosal neurons, appears to stimulate anion secretion through interactions with M 3-muscarinic acetylcholine receptors in the mucosa of the porcine distal jejunum.