Intestinal Fluid Secretion Research Articles

Partially Differentiated Ileal and Rectal Human CFTR-F508del Enteroids Secrete Fluid in Response to cAMP and cGMP: Targeted Preclinical Studies of Pharmaco-Therapy of CF Constipation

Introduction: Treatment of Cystic Fibrosis (CF) patients with Trikafta is prolonging life. This has allowed the importance of other causes of morbidity in these patients to become clearer and to emphasize the need to improve their quality of life by developing additional treatments. Chronic constipation is one such continuing unsolved medical issue for patients with CF even on Trikafta. In general, approaches to treat chronic constipation include increasing intestinal fluid secretion that involves inhibiting intestinal NaCl absorption and/or stimulating intestinal Cl secretion, with inhibition of the latter being the primary defect in CF. Drug development efforts have mostly aimed to improve CFTR function and control pancreatic insuffciency. We have recently reported an enterocyte population in the human ileal and colon that is present between the crypt Cl− secretory cells and the mature small intestinal villus or colonic surface Na+ absorptive cells that contain both DRA and CFTR. This population is called partially differentiated enterocytes. This study was undertaken to derive a strategy to stimulate intestinal fluid secretion from partially differentiated enterocytes in the F508del-CF-patient population by enhancing residual CFTR F508del activity using drugs that stimulate cAMP and cGMP. Methods: Ileal and rectal enteroids from F508del CF patients and healthy subjects (HS/normal) were studied. Enteroids were maintained in growth media containing Wnt3A, R-spondin, and Noggin (undifferentiated/UD); induced to partially differentiate (3d-DF) or fully differentiate (6d-DF) by removal of growth factors (Wnt3A and R-spondin) for 3 or 6 days, respectively. Forskolin-induced swelling (FIS) assay and live cell microscopy were used to determine fluid secretion in enteroids. A relative increase in organoid surface area over a time series of 60 min was measured to quantify FIS response. mRNA expression was determined using qRT-PCR. Results: Compared to HS enteroids, F508del enteroids had significantly higher mRNA expression of LGR5, and Ki67 in the UD state. Expression of goblet cell marker MUC2 and enteroendocrine (EEC) marker ChgA increased with differentiation in both HS and F508del, while 6d-DF F508del enteroids had a significantly higher ChgA compared to 6d-DF enteroids from HS. UD F508del enteroids had significantly higher expression of phosphodiesterase-3a (PDE3a) than HS enteroids, which further increased with differentiation, but did not change in HS. While UD and 6d-DF F508del rectal and ileal enteroids did not swell when exposed to cAMP (forskolin) and cGMP (linaclotide), 3d-DF F508del enteroids showed a swelling response when exposed to cAMP (forskolin) and cGMP (linaclotide). Fluid secretion from 3d-DF F508del enteroids was ~50% (cAMP) and ~67% (cGMP) of the response of intestinal enteroids from HS enteroids. Combinations of CFTR correctors and potentiators mimicking the makeup of Trikafta only partially enhanced fluid secretion in F508del CF enteroids as compared to HS. However, cGMP (linaclotide) additively enhanced Trikafta-induced fluid secretion from 3d-DF F508del enteroids. Conclusions: CFTR F508del can be stimulated by cAMP and cGMP in partially differentiated enterocyte populations. Our findings identify a novel role of the partially differentiated ileal and rectal enterocyte populations which behave in a way that offers the potential to improve treatment for CF constipation. Funding: This study is partly supported by Cystic Fibrosis Foundation grant CF-SINGH20G0, National Institutes of Health grants R01-DK-116352 and P30-DK-89502 (the Hopkins Basic and Translational Research Digestive Diseases Research Core Center). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abatement of Forskolin-Induced Intestinal Fluid Secretion with Penicillin

Abatement of Forskolin-Induced Intestinal Fluid Secretion with Penicillin

Crucial role of carbon monoxide as a regulator of diarrhea caused by cholera toxin: Evidence of direct interaction with toxin

The present study evaluated the role of heme oxygenase 1 (HO-1)/carbon monoxide (CO) pathway in the cholera toxin-induced diarrhea and its possible action mechanism. The pharmacological modulation with CORM-2 (a CO donor) or Hemin (a HO-1 inducer) decreased the intestinal fluid secretion and Cl− efflux, altered by cholera toxin. In contrast, ZnPP (a HO-1 inhibitor) reversed the antisecretory effect of Hemin and potentiated cholera toxin-induced intestinal secretion. Moreover, CORM-2 also prevented the alteration of intestinal epithelial architecture and local vascular permeability promoted by cholera toxin. The intestinal absorption was not altered by any of the pharmacological modulators. Cholera toxin inoculation also increased HO-1 immunoreactivity and bilirubin levels, a possible protective physiological response. Finally, using fluorometric technique, ELISA assay and molecular docking simulations, we show evidence that CO directly interacts with cholera toxin, forming a complex that affects its binding to GM1 receptor, which help explain the antisecretory effect. Thus, CO is an essential molecule for protection against choleric diarrhea and suggests its use as a possible therapeutic tool.

Structural insight into guanylyl cyclase receptor hijacking of the kinase–Hsp90 regulatory mechanism

Membrane receptor guanylyl cyclases play a role in many important facets of human physiology, from regulating blood pressure to intestinal fluid secretion. The structural mechanisms which influence these important physiological processes have yet to be explored. We present the 3.9 Å resolution cryo-EM structure of the human membrane receptor guanylyl cyclase GC-C in complex with Hsp90 and its co-chaperone Cdc37, providing insight into the mechanism of Cdc37 mediated binding of GC-C to the Hsp90 regulatory complex. As a membrane protein and non-kinase client of Hsp90–Cdc37, this work shows the remarkable plasticity of Cdc37 to interact with a broad array of clients with significant sequence variation. Furthermore, this work shows how membrane receptor guanylyl cyclases hijack the regulatory mechanisms used for active kinases to facilitate their regulation. Given the known druggability of Hsp90, these insights can guide the further development of membrane receptor guanylyl cyclase-targeted therapeutics and lead to new avenues to treat hypertension, inflammatory bowel disease, and other membrane receptor guanylyl cyclase-related conditions.

Structural insight into guanylyl cyclase receptor hijacking of the kinase-Hsp90 regulatory mechanism.

Membrane receptor guanylyl cyclases play a role in many important facets of human physiology, from regulating blood pressure to intestinal fluid secretion. The structural mechanisms which influence these important physiological processes have yet to be explored. We present the 3.9 Å resolution cryo-EM structure of the human membrane receptor guanylyl cyclase GC-C in complex with Hsp90 and its co-chaperone Cdc37, providing insight into the mechanism of Cdc37 mediated binding of GC-C to the Hsp90 regulatory complex. As a membrane protein and non-kinase client of Hsp90-Cdc37, this work shows the remarkable plasticity of Cdc37 to interact with a broad array of clients with significant sequence variation. Furthermore, this work shows how membrane receptor guanylyl cyclases hijack the regulatory mechanisms used for active kinases to facilitate their regulation. Given the known druggability of Hsp90, these insights can guide the further development of membrane receptor guanylyl cyclase-targeted therapeutics and lead to new avenues to treat hypertension, inflammatory bowel disease, and other membrane receptor guanylyl cyclase-related conditions.

Moringa oleifera Lam. Leaf improves constipation of rats induced by low-fiber-diet: A proteomics study

Ethnopharmacological relevanceThe leaf of Moringa oleifera Lam., a medicinal and edible herb for thousands of years in Ayurveda, is used as Pancha (na) karma (purgative) during and after the body cleansing process, to treat constipation, reduce cholesterol and body weight. Aim of studyThe aim was to investigate the diarrhea effects and possible mechanism of M. oleifera leaves in constipation rats. Materials and methodsThe hot water extract of M. oleifera leaves (WEMOL) was prepared and analyzed using LC-20AT HPLC system. The constipated rat model was induced by feeding with low fiber diet for 21 days. After oral administration of WEMOL for 7 days, the excretion parameter analysis, gastro-intestinal propulsion, histological analysis by HE and Alcian blue staining, and gastrointestinal hormone in rat's digestive tract through ELISA were used to evaluate the laxative effect of WEMOL. Label-free quantitation (LFQ) with LC-MS/MS, bioinformatics and Western blot were used to discover and verify the signal pathways and key proteins of WEMOL related to diarrhea. ResultsThe contents of isoquercitrin and astragalin were 2.7 mg/g and 1.7 mg/g, respectively in WEMOL. The stool number, weight, and water content of constipation rats were significantly reduced, indicating model had been established successfully. WEMOL (1.25 and 2.5 g/kg) increased water content of feces and the levels of Gas, MTL, NPY in gastric antrum and VIP, SP in colon of constipation rats, improved the muscle layer thickness and mucin secretion of colon. The proteomics revealed a total of 1731 differential proteins and 9 signaling pathways, WEMOL increased the expression of Vamp2, Gnai3, and Prkacb. ConclusionsThe laxative mechanism of WEMOL maybe modulate the signaling pathways mediated by 5-HT and Ach receptors, related to gastrointestinal motility and intestinal fluid secretion. It can be considered as the scientific connotation of Pancha (na) karma of M. oleifera leaves in ayurveda.

A Novel Peptide Prevents Enterotoxin- and Inflammation-Induced Intestinal Fluid Secretion by Stimulating Sodium-Hydrogen Exchanger 3 Activity

Acute diarrheal diseases are the second most common cause of infant mortality in developing countries. This is contributed to by lack of effective drug therapy that shortens the duration or lessens the volume of diarrhea. The epithelial brush border sodium (Na+)/hydrogen (H+) exchanger 3 (NHE3) accounts for a major component of intestinal Na+ absorption and is inhibited in most diarrheas. Because increased intestinal Na+ absorption can rehydrate patients with diarrhea, NHE3 has been suggested as a potential druggable target for drug therapy for diarrhea. A peptide (sodium-hydrogen exchanger 3 stimulatory peptide [N3SP]) was synthesized to mimic the part of the NHE3 C-terminus that forms a multiprotein complex that inhibits NHE3 activity. The effect of N3SP on NHE3 activity was evaluated in NHE3-transfected fibroblasts null for other plasma membrane NHEs, a human colon cancer cell line that models intestinal absorptive enterocytes (Caco-2/BBe), human enteroids, and mouse intestine invitro and invivo. N3SP was delivered into cells via a hydrophobic fluorescent maleimide or nanoparticles. N3SP uptake stimulated NHE3 activity at nmol/L concentrations under basal conditions and partially reversed the reduced NHE3 activity caused by elevated adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, and Ca2+ in cell lines and in invitro mouse intestine. N3SP also stimulated intestinal fluid absorption in the mouse small intestine invivo and prevented cholera toxin-, Escherichia coli heat-stable enterotoxin-, and cluster of differentiation 3 inflammation-induced fluid secretion in a live mouse intestinal loop model. These findings suggest pharmacologic stimulation of NHE3 activity as an efficacious approach for the treatment of moderate/severe diarrheal diseases.

Efficacy and safety of lubiprostone for the treatment of chronic idiopathic constipation: A phase 3, randomized, placebo-controlled study

IntroductionChronic idiopathic constipation (CIC) negatively impacts quality of life and increases healthcare costs. Lubiprostone stimulates the secretion of intestinal fluid, in turn facilitating the passage of stools and alleviating associated symptoms. Lubiprostone has been available in Mexico since 2018, but its clinical efficacy has not been studied in a Mexican population. AimTo evaluate the efficacy of lubiprostone, assessed by changes in spontaneous bowel movement (SBM) frequency after one week of treatment with 24 μg oral lubiprostone (b.i.d.), as well as its safety, over four weeks of treatment. StudyRandomized, double-blind, placebo-controlled study on 211 adults with CIC in Mexico. ResultsThe increase in SBM frequency, after one week of treatment, was significantly higher in the lubiprostone group than in the placebo group (mean: 4.9 [SD: 4.45] vs. 3.0 [3.14], p = 0.020). Secondary efficacy endpoints revealed a significantly higher proportion of SBM frequency/week in the lubiprostone group at weeks 2, 3, and 4. There was a better response within 24 h after the first dose with lubiprostone vs. placebo (60.0% vs. 41.5%; OR: 2.08, CI95%: [1.19, 3.62], p = 0.009) and the lubiprostone group also had significant improvement, with respect to straining, stool consistency, abdominal bloating, and Satisfaction Index. The main adverse events were gastrointestinal disorders in 13 (12.4%) lubiprostone-treated subjects and 4 (3.8%) control subjects. ConclusionsOur data confirm the efficacy and safety of lubiprostone for the treatment of CIC in a Mexican population. Lubiprostone treatment induces relief from the most bothersome symptoms associated with constipation.

Eficacia y seguridad de la lubiprostona para el tratamiento de estreñimiento crónico idiopático: un ensayo fase 3 aleatorizado controlado con placebo

IntroductionChronic idiopathic constipation (CIC) negatively impacts quality of life and increases healthcare costs. Lubiprostone stimulates the secretion of intestinal fluid, in turn facilitating the passage of stools and alleviating associated symptoms. Lubiprostone has been available in Mexico since 2018, but its clinical efficacy has not been studied in a Mexican population. AimTo evaluate the efficacy of lubiprostone, assessed by changes in spontaneous bowel movement (SBM) frequency after one week of treatment with 24μg oral lubiprostone (b.i.d.), as well as its safety, over four weeks of treatment. StudyRandomized, double-blind, placebo-controlled study on 211 adults with CIC in Mexico. ResultsThe increase in SBM frequency, after one week of treatment, was significantly higher in the lubiprostone group than in the placebo group (mean: 4.9 [SD: 4.45] vs. 3.0 [3.14], P=.020). Secondary efficacy endpoints revealed a significantly higher proportion of SBM frequency/week in the lubiprostone group at weeks 2, 3, and 4. There was a better response within 24hours after the first dose with lubiprostone vs. placebo (60.0% vs. 41.5%; OR: 2.08, [95%CI: 1.19, 3.62], P=.009) and the lubiprostone group also had significant improvement, with respect to straining, stool consistency, abdominal bloating, and satisfaction index. The main adverse events were gastrointestinal disorders in 13 (12.4%) lubiprostone-treated subjects and 4 (3.8%) control subjects. ConclusionsOur data confirm the efficacy and safety of lubiprostone for the treatment of CIC in a Mexican population. Lubiprostone treatment induces relief from the most bothersome symptoms associated with constipation.

Analysis of the causes of long-standing pelvic anterior sacral space infection and discussion of management techniques

Objective: To investigate the causes and management of long-term persistent pelvic presacral space infection. Methods: Clinical data of 10 patients with persistent presacral infection admitted to the Cancer Hospital of Zhengzhou University from October 2015 to October 2020 were collected. Different surgical approaches were used to treat the presacral infection according to the patients' initial surgical procedures. Results: Among the 10 patients, there were 2 cases of presacral recurrent infection due to rectal leak after radiotherapy for cervical cancer, 3 cases of presacral recurrent infection due to rectal leak after radiotherapy for rectal cancer Dixons, and 5 cases of presacral recurrent infection of sinus tract after adjuvant radiotherapy for rectal cancer Miles. Of the 5 patients with leaky bowel, 4 had complete resection of the ruptured nonfunctional bowel and complete debridement of the presacral infection using an anterior transverse sacral incision with a large tipped omentum filling the presacral space; 1 had continuous drainage of the anal canal and complete debridement of the presacral infection using an anterior transverse sacral incision. 5 post-Miles patients all had debridement of the presacral infection using an anterior transverse sacral incision combined with an abdominal incision. The nine patients with healed presacral infection recovered from surgery in 26 to 210 days, with a median time of 55 days. Conclusions: Anterior sacral infections in patients with leaky gut are caused by residual bowel secretion of intestinal fluid into the anterior sacral space, and in post-Miles patients by residual anterior sacral foreign bodies. An anterior sacral caudal transverse arc incision combined with an abdominal incision is an effective surgical approach for complete debridement of anterior sacral recalcitrant infections.

Natural statin derivatives as potential therapy to reduce intestinal fluid loss in cholera.

As a leading cause of death in children under 5 years old, secretory diarrheas including cholera are characterized by excessive intestinal fluid secretion driven by enterotoxin-induced cAMP-dependent intestinal chloride transport. This study aimed to identify fungal bioactive metabolites possessing anti-secretory effects against cAMP-dependent chloride secretion in intestinal epithelial cells. Using electrophysiological analyses in human intestinal epithelial (T84) cells, five fungus-derived statin derivatives including α,β-dehydrolovastatin (DHLV), α,β-dehydrodihydromonacolin K, lovastatin, mevastatin and simvastatin were found to inhibit the cAMP-dependent chloride secretion with IC50 values of 1.8, 8.9, 11.9, 11.4 and 5 μM, respectively. Being the most potent statin derivatives, DHLV was evaluated for its pharmacological properties including cellular toxicity, mechanism of action, target specificity and in vivo efficacy. DHLV at concentrations up to 20 μM did not affect cell viability and barrier integrity of T84 cells. Electrophysiological analyses indicated that DHLV inhibited cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent apical chloride channel, via mechanisms not involving alteration of intracellular cAMP levels or its negative regulators including AMP-activated protein kinases and protein phosphatases. DHLV had no effect on Na+-K+ ATPase activities but inhibited Ca2+-dependent chloride secretion without affecting intracellular Ca2+ levels. Importantly, intraperitoneal (2 mg/kg) and intraluminal (20 μM) injections of DHLV reduced cholera toxin-induced intestinal fluid secretion in mice by 59% and 65%, respectively without affecting baseline intestinal fluid transport. This study identifies natural statin derivatives as novel natural product-derived CFTR inhibitors, which may be beneficial in the treatment of enterotoxin-induced secretory diarrheas including cholera.

Hericium caput-medusae (Bull.:Fr.) Pers. fermentation concentrate polysaccharides improves intestinal bacteria by activating chloride channels and mucus secretion

Ethnopharmacological relevanceAs a traditional edible fungus in China and many other Asian countries, Hericium caput-medusae (Bull. Fr.) Pers. is widely used to improve the health of the gastrointestinal tract. For example, the drug “Weilexin Granules” is mainly composed of H. caput-medusae (Bull. Fr.) Pers. fermentation concentrate. However, the mechanism of action remains to be elucidated. Aims of the studyThe purpose of this study was to assess whether polysaccharides from H. caput-medusae (Bull. Fr.) Pers. fermentation concentrate (HFP) exerts a gut protective effect and a regulatory effect on the intestinal microbiota through the chloride channels and mucus secretion. Materials and methodsHFP was extracted, characterized and different concentrations of HFP (100, 200, 400 mg/kg) were administered to mice for 14 days. The changes in gut microbiota were observed via 16S high throughput sequencing. Short-chain fatty acids (SCFAs) was detected by GC-MS. AB-PAS staining was used to observe the secretion of mucus. The chloride channel activity and protein expression were verified by short-circuit current measurement and Western blot. ResultsHFP regulated the abundance of gut microbiota in mice, with increased levels of Ruminococcaceae and Lachnospiraceae and reduced proportions of Staphylococcus and Enterobacter. HFP enhanced mucus volume as well as increased intestinal fluid secretion by activating the chloride channels. In addition, short-circuit current experiments also proved that HFP activates Cl⁻ currents targeting cystic fibrosis transmembrane conductance regulator (CFTR) and Anoamin1 (ANO1). ConclusionIn conclusion, HFP might increase intestinal fluid secretion by promoting Cl⁻ secretion, which in turn advanced mucus hydration as well as regulated gut microbiota to improve intestinal health. Therefore, H. caput-medusae (Bull. Fr.) Pers. could be potentially used in the regulation of intestinal secretion and microbes.

Lactoferrin Decreases Enterotoxigenic Escherichia coli-Induced Fluid Secretion and Bacterial Adhesion in the Porcine Small Intestine.

Enterotoxigenic Escherichia coli (ETEC) infections are one of the most prevalent causes of post-weaning diarrhea in piglets, resulting in morbidity, mortality and elevated use of antibiotics. The emergence and further spread of antimicrobial resistance together with the growing demand for high quality animal protein requires the identification of novel alternatives for antimicrobials. A promising alternative is lactoferrin, as we previously showed that it can both inhibit the growth and degrade bacterial virulence factors of porcine ETEC strains in vitro. Aiming to confirm these findings in vivo, we performed a small intestinal segment perfusion experiment in piglets. Here, we showed that lactoferrin could not only decrease ETEC-induced fluid secretion, but also their ability to colonize the small intestinal epithelium. Furthermore, while ETEC infection induced pro-inflammatory cytokine mRNA expression in this experiment, lactoferrin was not able to counteract these responses. In addition, a bacterial motility assay showed that lactoferrin can reduce the motility of ETEC. Our findings further support the use of lactoferrin as an alternative for antimicrobials and also show its potential for the prevention of ETEC infections in pigs.

SUCCESSFUL MANAGEMENT OF REFRACTORY CONSTIPATION USING KAMPO MEDICINE MASHININGAN IN A PATIENT WITH WILD-TYPE ATTR CARDIAC AMYLOIDOSIS

TYPE: Case Report TOPIC: Cardiovascular Disease INTRODUCTION: A wild-type ATTR amyloidosis is a systemic disease with multi-organ dysfunction, involving heart, kidney, skin, and gastrointestinal tract, due to deposition of wild-type transthyretin in each organ. CASE PRESENTATION: We had a 76-year-old man diagnosed with wild-type ATTR cardiac amyloidosis, whose heart failure symptom improved by anti-heart failure medications but constipation refractory to multiple conventional medications persisted. Following the conversion from lubiprostone to Kampo medicine mashiningan, his average days per one evacuation decreased from around 7.0 days down to 1.6 days. DISCUSSION: Amyloid-related gastrointestinal comorbidities are often refractory to the conventional medications. Mashiningan might increase the proportion of soft feces via increasing intestinal fluid secretion through cystic fibrosis transmembrane conductance regulator chloride channel activation. CONCLUSIONS: Mashininigan might be alternative option to improve refractory constipation in patients with cardiac amyloidosis. DISCLOSURE: Nothing to declare. KEYWORD: heart failure

Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by the Nuclear Bile Acid Receptor, Farnesoid X Receptor

Introduction and AimsCFTR, a Cl‐ channel important in regulating intestinal fluid and electrolyte secretion, is implicated in the pathogenesis of a number of intestinal disorders. We have previously shown that bile acids, acting via the nuclear receptor, farnesoid X receptor (FXR), inhibit colonic epithelial CFTR expression. Dietary phytochemicals have been reported to have the capacity to modulate FXR signalling. Here, we set out to investigate mechanisms underlying FXR regulation of CFTR expression, and the potential for therapeutically targeting the receptor with dietary phytochemicals.MethodsMonolayers of T84 colonic epithelial cells were treated with the FXR agonist, GW4064 (5 µM) in the absence or presence of a plant phytochemical, designated here as KFS1 (5 µM). mRNA and protein expression were measured by qPCR and Western blotting. FXR binding to the CFTR promoter was investigated by chromatin immunoprecipitation (ChIP). Electrophysiological studies of T84cells were conducted in Ussing chambers. Studies on human and murine enteroids were carried out with ethical approval from Johns Hopkins University School of Medicine Institutional Review Board and the Institutional Review Board of the University of California San Diego, respectively.ResultsTreatment of T84 monolayers with GW4064 downregulated CFTR mRNA to 0.51 ± 0.06 fold after 12 hrs (n = 12; p < 0.001) and protein levels to 0.28 ± 0.06 fold after 48 hrs, compared to controls (n = 8; p < 0.001). Studies in Ussing chambers showed that GW4064 treatment for 48 hrs inhibited Cl‐ secretory responses to the Ca2+‐ and cAMP‐dependent agonists, carbachol (CCh; 100mM) and forskolin (FSK; 10mM), by 79.9 ± 7.5 % and 74.2 ± 8.9 %, respectively. Transcriptomic analysis of human colonic enteroids revealed FXR to be robustly expressed in secretory (crypt‐like) cells and that its activation also induced CFTR downregulation. FXR activation did not alter expression or phosphorylation of the p65 subunit of NF‐κB, or inhibit its translocation to the nucleus. ChIP‐qPCR analysis appears to show enhanced binding of FXR to the CFTR promoter upon GW4064 treatment. The phytochemical, KFS1 (5 mM; 24hrs), upregulated FXR mRNA and protein expression in T84 cells and enhanced GW4064‐induced downregulation of CFTR mRNA by 0.28 ± 0.05 fold (n = 8; p <0.01) and protein by 0.25 ± 0.11 fold (n = 4) after 24 hours. Similarly, KFS1 significantly upregulated FXR mRNA expression in murine colonic epithelial enteroids (2.3 ± 0.2; n = 4; p < 0.01) and enhanced GW4064‐induced downregulation of CFTR mRNA by 0.5 ± 0.1 fold (n = 4; p < 0.05) compared to GW4064 alone. Finally, KFS1 enhanced FXR inhibition of agonist‐induced Cl‐ secretory responses across T84 cells mounted in Ussing chambers.ConclusionFXR regulates colonic epithelial CFTR expression and function by a mechanism which appears to involve direct binding of FXR to the CFTR promoter. By virtue of their ability to upregulate FXR expression, and thereby enhance its antisecretory actions, plant extracts containing KFS1 have excellent potential to be developed as FXR‐targeted nutraceuticals for the treatment and prevention of intestinal disease.

Identification of Fungus‐derived Compounds as a Novel CFTR inhibitor: Mechanism and Potential Anti‐diarrheal Application

Secretory diarrhea, which has been considered as a health burden worldwide, could be alleviated by inhibiting fluid secretion. CFTR is considered to be a drug target for development of anti‐secretory agent. However, there is no currently FDA‐approval drug targeting CFTR. This study aimed to identify CFTR inhibitors from a library of fungal bioactive metabolites found in Thailand. Electrophysiological analyses were used for identifying the inhibitory effect of 40 compounds. Three active compounds with the best one being DHLV, which effectively inhibited CFTR‐mediated Clˉ secretion with low micromolar potency (IC50 ~1.8 μM) in T84 cells without effect on Na+‐K+ ATPase activity. In addition, DHLV inhibited Ca2+‐activated Clˉ channels stimulated by ATP. In vivo experiments showed that administration of DHLV by intraperitoneal route (2 mg/kg) and intraluminal (22 µg/kg or 20 µM) injections reduced cholera toxin‐induced intestinal fluid secretion in mice by ∼46.7% and ∼42.8%, respectively without affecting intestinal fluid absorption. This study indicates that the novel CFTR inhibitor, DHLV, is of potential utility in the treatment of secretory diarrhea.

GUCY2C‐Enriched Intestinal Neuropod Cells Modulate Visceral Pain

Chronic visceral pain (CVP) is a prevalent and debilitating syndrome with limited treatments. Guanylyl cyclase C (GUCY2C) is the receptor for uroguanylin (GUCA2B) in small intestine and guanylin (GUCA2A) in colorectum. This hormone‐receptor axis produces cyclic (c)GMP accumulation, inducing intestinal fluid secretion. This forms the basis for use of GUCY2C agonists linaclotide and plecanatide to treat constipation‐type irritable bowel syndrome (IBS‐C) and chronic idiopathic constipation (CIC). Unexpectedly, GUCY2C agonists relieve CVP in these patients, and in mouse models of visceral pain. Interestingly, these patients are deficient in uroguanylin, suggesting that constipation and CVP may reflect hormone insufficiency silencing GUCY2C. Indeed, eliminating GUCY2C in mice (GUCY2C‐/‐) produced CVP, evoked by cyclic rectal distension, quantified by the abdominal withdrawal reflex and by phospho‐ERK signaling in the spinal cord dorsal horn. CVP severity in GUCY2C‐/‐ mice recapitulated TNBS‐induced inflammatory bowel disease in wild type (GUCY2+/+) mice. Oral linaclotide relieved TNBS‐induced CVP in wild type mice, but was without effect on CVP in GUCY2C‐/‐ mice. The mechanistic basis of GUCY2C‐dependent visceral analgesia remains obscure. While GUCY2C is expressed by all epithelial cells in intestine, a GUCY2C promoter‐driven GFP reporter revealed a novel rare population of cells enriched in GUCY2C mRNA, protein, and activity. GUCY2CHigh cells, concentrated in duodenum but rare in rectum, morphologically resemble enteroendocrine cells with a basal neuropod potentially synapsing on visceral afferents in the lamina propria, offering a cellular substrate for GUCY2C visceral nociceptive signaling. RNAseq analysis revealed that GUCY2CHigh neuropod cells were deficient in gene products canonically associated with GUCY2C‐driven secretion, including GUCA2A, GUCA2B, CFTR and NHE3. Rather, these cells were enriched in gene sets characteristic of neurons. Importantly, dorsal root ganglia (DRG) cells formed functional connections with GUCY2CHigh neuropod cells in co‐culture. Thus, DRG cells with GUCY2CHigh neuropod cells alone (GUCA2B‐deficient, GUCY2C silenced) were hyperexcitable following current injection, with a reduced rheobase and repetitive action potentials (APs). In contrast, adding linaclotide (hormone replete, GUCY2C activated) silenced DRG neuron excitability, raising the rheobase and eliminating repetitive APs. DRG neuron excitability was not affected by linaclotide in co‐cultures with neuropod cells from GUCY2C‐/‐ mice. Moreover, the effects of linaclotide on DRG neuron excitability was not recapitulated by extracellular cGMP. These observations suggest that GUCY2CHigh neuropod cells synapse with DRG visceral afferents and modulate their excitability. They support a model in which GUCA2B sufficiency activates GUCY2C to suppress DRG neuron excitability, while GUCA2B insufficiency silences GUCY2C to produce DRG hyperexcitability and CVP. Finally, they suggest that linaclotide relieves CVP by stimulating GUCY2CHigh neuropod cells to inhibit DRG excitability and nociceptive signaling.

Vitamin D Receptor Activation Exerts Anti‐Secretory Actions In Colonic Epithelial Cells

BackgroundThe vitamin D receptor (VDR) is a nuclear receptor that is expressed in many tissues throughout the body, but is particularly abundant within the colon. VDR is classically known to be activated by the active form of vitamin D, calcitriol, but is appreciated to also serve as a receptor for luminal bile acids. VDR has previously been shown to have anti‐inflammatory and barrier promoting actions in the large intestine. However, there is little known of the role VDR plays in regulating colonic epithelial transport function. Interestingly, previous studies from our laboratory have shown the nuclear bile acid receptor, farnesoid X receptor (FXR), to exert anti‐secretory actions in colonic epithelial cells. The current study aims to investigate a potential role for VDR in regulating colonic Cl‐ secretion and whether it may be a target for the treatment of diarrhoeal diseases.AimTo investigate the effects of VDR activation by calcitriol on colonic epithelial Cl‐ secretion.MethodsPolarised monolayers of T84 cells, grown on permeable supports, were used as a model of the colonic epithelium. Protein expression of VDR and CFTR was measured by western blotting. Calcitriol (1 ‐ 20 nM) was used to activate VDR and the VDR target protein, CYP24A1, was used to verify activation of the receptor by qRT‐PCR. The specificity of calcitriol as a VDR agonist was determined by investigating FXR activation using the FXR target protein, FGF19, and a FXR‐luciferase reporter cell line. The effects of VDR activation by calcitriol (10 nM) on chloride secretion over a timecourse (6 – 48 hr) was determined using the Ussing chamber technique. Changes in short‐circuit current (Isc) induced by the Ca2+‐dependent agonist, carbachol (CCh; 100 µM) were assessed.ResultsVDR was found to be expressed in T84 cell monolayers as determined by western blotting. Treatment of T84 cells with calcitriol (5 ‐ 20 nM) significantly (n = 3, *p ≤ 0.05) increased expression of CYP24A1, indicating activation of VDR by the agonist. Calcitriol did not alter the activity of FXR, either in an FXR‐luciferase reporter cell line or in T84 cells, indicating specificity of the agonist for VDR. In Ussing chambers, the FXR agonist, GW4064 (5 µM; 24 hrs), inhibited Cl‐ secretory responses to CCh, as previously reported (n = 5, **p ≤ 0.01). Calcitriol (10 nM) was also found to significantly inhibit CCh‐stimulated Cl‐ secretory responses by 39.9 ± 5.1% (6 hr), 40.4 ± 4.3% (18 hr) and 62.0 ± 6.1% (24 hr) (n = 5, **p ≤ 0.01). In addition to their effects on CCh‐induced chloride secretion, GW4064 (5 µM; 24 hrs) downregulated the protein expression of CFTR by 0.5 ± 0.1 (n = 8, ***p ≤ 0.001) (as previously reported) and calcitriol (10 nM) downregulated CFTR by 0.35 ± 0.1 (n=8, *p≤ 0.05).ConclusionSimilar to the bile acid receptor, FXR, VDR is expressed in colonic epithelial cells where activation by its natural ligand, calcitriol, downregulates CFTR expression and inhibits agonist‐induced Cl‐ secretory responses. Since Cl‐ secretion is the primary driving force for intestinal fluid secretion, our data suggest that VDR may serve as a target for the development of new anti‐diarrhoeal agents. Future studies will aim to determine the molecular mechanisms involved and investigate a possible role for VDR in mediating anti‐secretory actions of luminal bile acids.

Role of the Endocannabinoid System in the Regulation of Intestinal Homeostasis.

The maintenance of intestinal homeostasis is fundamentally important to health. Intestinal barrier function and immune regulation are key determinants of intestinal homeostasis and are therefore tightly regulated by a variety of signaling mechanisms. The endocannabinoid system is a lipid mediator signaling system widely expressed in the gastrointestinal tract. Accumulating evidence suggests the endocannabinoid system is a critical nexus involved in the physiological processes that underlie the control of intestinal homeostasis. In this review we will illustrate how the endocannabinoid system is involved in regulation of intestinal permeability, fluid secretion, and immune regulation. We will also demonstrate a reciprocal regulation between the endocannabinoid system and the gut microbiome. The role of the endocannabinoid system is complex and multifaceted, responding to both internal and external factors while also serving as an effector system for the maintenance of intestinal homeostasis.

The Glycemic Index, Postprandial Hypotension and Cardiovascular Disease

High glycemic index diets have been associated with an increased risk of cardiovascular disease events and all-cause mortality. We suggest that part of the reason for this association is through the effect of the rapidly digested high glycemic index carbohydrate diets in promoting the effects of postprandial hypotension in vulnerable individuals. Postprandial hypotension has been recognized as a problem especially affecting the frail elderly. The phenomenon occurs earlier in the day and includes syncope and falls acutely and more serious cardiovascular events and increased all-cause mortality in the longer-term. The mechanism appears to relate to the rapid digestion of carbohydrates foods. Strategies that reduce the amount of meal carbohydrates and their rate of absorption by enzyme inhibition or by delaying gastric emptying and have proved helpful as has increased fluid intake, presumably due to dilution of small intestinal contents and a reduction in the tonicity, so reducing the need for intestinal fluid secretion to dilute osmotically active sugars and products of digestion. In this way the need for increased blood flow to the gut can be reduced, that would otherwise steal blood from the systemic circulation resulting in a drop in blood pressure and an increase in heart rate. Slowly absorbed or low glycemic index carbohydrates would therefore appear potentially useful as part of the dietary strategy for the treatment of postprandial hypotension and conversely postprandial hypotension could be one of the reasons why high glycemic index diets have been associated with increased CVD risk.