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1.
Cell Mol Gastroenterol Hepatol ; 18(5): 101383, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39089626

RESUMEN

BACKGROUND & AIMS: Diarrhea occurs in up to 50% of cases of COVID-19. Nonetheless, the pathophysiologic mechanism(s) have not been determined. METHODS: This was examined using normal human enteroid monolayers exposed apically to live SARS-CoV-2 or non-replicating virus-like particles (VLPs) bearing the 4 SARS-CoV-2 structural proteins or irradiated virus, all of which bound and entered enterocytes. RESULTS: Live virus and VLPs incrieased secretion of multiple cytokines and reduced mRNAs of ACE2, NHE3, and DRA. Interleukin (IL)-6 plus IL-8 alone reduced NHE3 mRNA and protein and DRA mRNA and protein. Neither VLPs nor IL-6 plus IL-8 alone altered Cl- secretion, but together they caused Cl- secretion, which was Ca2+-dependent, CFTR-independent, blocked partially by a specific TMEM16A inhibitor, and entirely by a general TMEM16 family inhibitor. VLPs and irradiated virus, but not IL-6 plus IL-8, produced Ca2+ waves that began within minutes of VLP exposure, lasted for at least 60 minutes, and were prevented by pretreatment with apyrase, a P2Y1 receptor antagonist, and general TMEM16 family inhibitor but not by the specific TMEM16A inhibitor. CONCLUSIONS: The pathophysiology of COVID-19 diarrhea appears to be a unique example of a calcium-dependent inflammatory diarrhea that is caused by direct viral effects plus the virus-induced intestinal epithelial cytokine secretion.


Asunto(s)
COVID-19 , Citocinas , Diarrea , SARS-CoV-2 , Humanos , COVID-19/virología , COVID-19/inmunología , COVID-19/metabolismo , COVID-19/patología , SARS-CoV-2/fisiología , Diarrea/virología , Citocinas/metabolismo , Interleucina-6/metabolismo , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/patología , Enzima Convertidora de Angiotensina 2/metabolismo , Pandemias , Neumonía Viral/virología , Neumonía Viral/inmunología , Neumonía Viral/patología , Neumonía Viral/metabolismo , Betacoronavirus/fisiología , Enterocitos/virología , Enterocitos/metabolismo , Enterocitos/patología , Interleucina-8/metabolismo
2.
Gastroenterology ; 165(4): 986-998.e11, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37429363

RESUMEN

BACKGROUND & AIMS: 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. METHODS: 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 in vitro and in vivo. N3SP was delivered into cells via a hydrophobic fluorescent maleimide or nanoparticles. RESULTS: 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 in vitro mouse intestine. N3SP also stimulated intestinal fluid absorption in the mouse small intestine in vivo 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. CONCLUSIONS: These findings suggest pharmacologic stimulation of NHE3 activity as an efficacious approach for the treatment of moderate/severe diarrheal diseases.


Asunto(s)
Enterotoxinas , Intercambiadores de Sodio-Hidrógeno , Ratones , Animales , Humanos , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Enterotoxinas/farmacología , Enterotoxinas/metabolismo , Células CACO-2 , Intercambiadores de Sodio-Hidrógeno/metabolismo , Enterocitos/metabolismo , Sodio/metabolismo , Diarrea/tratamiento farmacológico , Diarrea/prevención & control , Diarrea/inducido químicamente , Péptidos/efectos adversos , Microvellosidades/metabolismo
3.
Am J Physiol Cell Physiol ; 324(6): C1263-C1273, 2023 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-37154494

RESUMEN

In polarized intestinal epithelial cells, downregulated in adenoma (DRA) is an apical Cl-/[Formula: see text] exchanger that is part of neutral NaCl absorption under baseline conditions, but in cyclic adenosine monophosphate (cAMP)-driven diarrheas, it is stimulated and contributes to increased anion secretion. To further understand the regulation of DRA in conditions mimicking some diarrheal diseases, Caco-2/BBE cells were exposed to forskolin (FSK) and adenosine 5'-triphosphate (ATP). FSK and ATP stimulated DRA in a concentration-dependent manner, with ATP acting via P2Y1 receptors. FSK at 1 µM and ATP at 0.25 µM had minimal to no effect on DRA given individually; however, together, they stimulated DRA to levels seen with maximum concentrations of FSK and ATP alone. In Caco-2/BBE cells expressing the Ca2+ indicator GCaMP6s, ATP increased intracellular Ca2+ (Ca2+i) in a concentration-dependent manner, whereas FSK (1 µM), which by itself did not significantly alter Ca2+i, followed by 0.25 µM ATP produced a large increase in Ca2+ that was approximately equal to the elevation caused by 1 µM ATP. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) pretreatment prevented the ATP and FSK/ATP synergistically increased the DRA activity and the increase in Ca2+i caused by FSK/ATP. FSK/ATP synergistic stimulation of DRA was similarly observed in human colonoids. In Caco-2/BBE cells, subthreshold concentrations of FSK (cAMP) and ATP (Ca2+) synergistically increased Ca2+i and stimulated DRA activity with both being blocked by BAPTA-AM pretreatment. Diarrheal diseases, such as bile acid diarrhea, in which both cAMP and Ca2+ are elevated, are likely to be associated with stimulated DRA activity contributing to increased anion secretion, whereas separation of DRA from Na+/H+ exchanger isoform-3 (NHE3) contributes to reduced NaCl absorption.NEW & NOTEWORTHY The BB Cl-/[Formula: see text] exchanger DRA takes part in both neutral NaCl absorption and stimulated anion secretion. Using intestinal cell line, Caco-2/BBE high concentrations of cAMP and Ca2+ individually stimulated DRA activity, whereas low concentrations, which had no/minimal effect, synergistically stimulated DRA activity that required a synergistic increase in intracellular Ca2+. This study increases understanding of diarrheal diseases, such as bile salt diarrhea, in which both cAMP and elevated Ca2+ are involved.


Asunto(s)
Células Epiteliales , Cloruro de Sodio , Humanos , Células CACO-2 , Células Epiteliales/metabolismo , Aniones/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Diarrea/metabolismo , Adenosina Trifosfato/farmacología , Adenosina Trifosfato/metabolismo , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismo , Antiportadores de Cloruro-Bicarbonato/genética , Antiportadores de Cloruro-Bicarbonato/metabolismo
4.
Front Physiol ; 13: 892112, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35928564

RESUMEN

Use of human enteroids studied in the undifferentiated and differentiated state that mimic the intestinal crypt and villus, respectively, has allowed studies of multiple enterocyte populations, including a large population of enterocytes that are transitioning from the crypt to the villus. This population expresses NHE3, DRA, and CFTR, representing a combination of Na absorptive and anion secretory functions. In this cell population, these three transporters physically interact, which affects their baseline and regulated activities. A study of this cell population and differentiated Caco-2 cells transduced with NHE3 and endogenously expressing DRA and CFTR has allowed an understanding of previous studies in which cAMP seemed to stimulate and inhibit DRA at the same time. Understanding the contributions of these cells to overall intestinal transport function as part of the fasting and post-prandial state and their contribution to the pathophysiology of diarrheal diseases and some conditions with constipation will allow new approaches to drug development.

5.
Cell Physiol Biochem ; 56(1): 39-49, 2022 Jan 26.
Artículo en Inglés | MEDLINE | ID: mdl-35076190

RESUMEN

BACKGROUND/AIMS: NHE3 (Na+/H+ exchanger3) and SLC26A3 (Cl-/HCO3- exchanger, DRA) are the major components of the intestinal neutral NaCl absorptive process and based on the intestinal segment, contribute to HCO3- absorption and HCO3- secretion. NHE3 and DRA are highly regulated by changes in second messengers, cAMP, cGMP and Ca2+. Precise and convenient measurement of exchanger activity is necessary to allow rapid study of physiologic and pharmacologic functions. Some epithelial cells are difficult to load with AM ester dyes and loading may not be uniform. METHODS: The use of a genetically modified fluorescent protein, mOrange2 was explored as an intracellular pH sensor protein to measure exchange activity of NHE3 and DRA. The model used was FRT cells stably expressing NHE3 or DRA with intracellular pH measured by changes of mOrange2 fluorescence intensity. Intracellular pH was monitored using a) Isolated single clones of FRT/mOrange2/HA-NHE3 cells studied in a confocal microscope with time-lapse live cell imaging under basal conditions and when NHE3 was inhibited by exposure to forskolin and stimulated by dexamethasone, b) coverslip grown FRT/mOrange2 cells expressing NHE3 or DRA using a computerized fluorometer with a perfused cuvette with standardization of the mOrange2 absorption and emission signal using K+/Nigericin as an internal standard in each experiment. RESULTS: A similar rate of intracellular alkalization by Na+ addition in cells expressing NHE3 and by Cl- removal in cells expressing DRA was found in mOrange2 expressing cells compared to the same cells loaded with BCECF-AM,both using the same pH calibration with K+/Nigericin. Using mOrange2 as the pH sensor, NHE3 basal activity was quantitated and shown to be inhibited by forskolin and stimulated by dexamethasone, and DRA was oppositely shown to be stimulated by forskolin, responses similar to results found using BCECF-AM. CONCLUSION: This study demonstrates that mOrange2 protein can be an effective alternate to BCECF-AM in measuring intracellular pH (preferred setting Ex520nm, Em 563nm) as affected by NHE3 and DRA activity, with the advantage, compared to AM ester dyes, that genetic expression can provide uniform expression of the pH sensor.


Asunto(s)
Antiportadores/metabolismo , Fluoresceínas/farmacología , Proteínas Luminiscentes/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Transportadores de Sulfato/metabolismo , Animales , Antiportadores/genética , Concentración de Iones de Hidrógeno , Proteínas Luminiscentes/genética , Ratas , Ratas Endogámicas F344 , Intercambiador 3 de Sodio-Hidrógeno/genética , Transportadores de Sulfato/genética
6.
bioRxiv ; 2021 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-33948596

RESUMEN

Diarrhea occurs in 2-50% of cases of COVID-19 (∼8% is average across series). The diarrhea does not appear to account for the disease mortality and its contribution to the morbidity has not been defined, even though it is a component of Long Covid or post-infectious aspects of the disease. Even less is known about the pathophysiologic mechanism of the diarrhea. To begin to understand the pathophysiology of COVID-19 diarrhea, we exposed human enteroid monolayers obtained from five healthy subjects and made from duodenum, jejunum, and proximal colon to live SARS-CoV-2 and virus like particles (VLPs) made from exosomes expressing SARS-CoV-2 structural proteins (Spike, Nucleocapsid, Membrane and Envelope). Results: 1) Live virus was exposed apically for 90 min, then washed out and studied 2 and 5 days later. SARS-Cov-2 was taken up by enteroids and live virus was present in lysates and in the apical>>basolateral media of polarized enteroids 48 h after exposure. This is the first demonstration of basolateral appearance of live virus after apical exposure. High vRNA concentration was detected in cell lysates and in the apical and basolateral media up to 5 days after exposure. 2) Two days after viral exposure, cytokine measurements of media showed significantly increased levels of IL-6, IL-8 and MCP-1. 3) Two days after viral exposure, mRNA levels of ACE2, NHE3 and DRA were reduced but there was no change in mRNA of CFTR. NHE3 protein was also decreased. 4) Live viral studies were mimicked by some studies with VLP exposure for 48 h. VLPs with Spike-D614G bound to the enteroid apical surface and was taken up; this resulted in decreased mRNA levels of ACE2, NHE3, DRA and CFTR. 4) VLP effects were determined on active anion secretion measured with the Ussing chamber/voltage clamp technique. S-D614G acutely exposed to apical surface of human ileal enteroids did not alter the short-circuit current (Isc). However, VLPS-D614G exposure to enteroids that were pretreated for ∼24 h with IL-6 plus IL-8 induced a concentration dependent increase in Isc indicating stimulated anion secretion, that was delayed in onset by ∼8 min. The anion secretion was inhibited by apical exposure to a specific calcium activated Cl channel (CaCC) inhibitor (AO1) but not by a specific CFTR inhibitor (BP027); was inhibited by basolateral exposure to the K channel inhibit clortimazole; and was prevented by pretreatment with the calcium buffer BAPTA-AM. 5) The calcium dependence of the VLP-induced increase in Isc was studied in Caco-2/BBe cells stably expressing the genetically encoded Ca2+ sensor GCaMP6s. 24 h pretreatment with IL-6/IL-8 did not alter intracellular Ca2+. However, in IL-6/IL-8 pretreated cells, VLP S-D614G caused appearance of Ca 2+ waves and an overall increase in intracellular Ca 2+ with a delay of ∼10 min after VLP addition. We conclude that the diarrhea of COVID-19 appears to an example of a calcium dependent inflammatory diarrhea that involves both acutely stimulated Ca2+ dependent anion secretion (stimulated Isc) that involves CaCC and likely inhibition of neutral NaCl absorption (decreased NHE3 protein and mRNA and decreased DRA mRNA).

7.
Am J Physiol Gastrointest Liver Physiol ; 320(3): G258-G271, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33074011

RESUMEN

There is increasing evidence that the study of normal human enteroids duplicates many known aspects of human intestinal physiology. However, this epithelial cell-only model lacks the many nonepithelial intestinal cells present in the gastrointestinal tract and exposure to the mechanical forces to which the intestine is exposed. We tested the hypothesis that physical shear forces produced by luminal and blood flow would provide an intestinal model more closely resembling normal human jejunum. Jejunal enteroid monolayers were studied in the Emulate, Inc. Intestine-Chip under conditions of constant luminal and basolateral flow that was designed to mimic normal intestinal fluid flow, with human umbilical vein endothelial cells (HUVECs) on the basolateral surface and with Wnt3A, R-spondin, and Noggin only on the luminal surface. The jejunal enteroids formed monolayers that remained confluent for 6-8 days, began differentiating at least as early as day 2 post plating, and demonstrated continuing differentiation over the entire time of the study, as shown by quantitative real-time polymerase chain reaction and Western blot analysis. Differentiation impacted villus genes and proteins differently with early expression of regenerating family member 1α (REG1A), early reduction to a low but constant level of expression of Na+-K+-2Cl- cotransporter 1 (NKCC1), and increasing expression of sucrase-isomaltase (SI) and downregulated in adenoma (DRA). These results were consistent with continual differentiation, as was shown to occur in mouse villus enterocytes. Compared with differentiated enteroid monolayers grown on Transwell inserts, enteroids exposed to flow were more differentiated but exhibited increased apoptosis and reduced carbohydrate metabolism, as shown by proteomic analysis. This study of human jejunal enteroids-on-chip suggests that luminal and basolateral flow produce a model of continual differentiation over time and NaCl absorption that mimics normal intestine and should provide new insights in intestinal physiology.NEW & NOTEWORTHY This study showed that polarized enteroid models in which there is no basolateral Wnt3a, are differentiated, regardless of the Wnt3a status of the apical media. The study supports the concept that in the human intestine villus differentiation is not an all or none phenomenon, demonstrating that at different days after lack of basolateral Wnt exposure, clusters of genes and proteins exist geographically along the villus with different domains having different functions.


Asunto(s)
Diferenciación Celular , Yeyuno/citología , Microfluídica/métodos , Cultivo Primario de Células/métodos , Estrés Mecánico , Adulto , Apoptosis , Proteínas Portadoras/metabolismo , Células Cultivadas , Enterocitos/citología , Enterocitos/metabolismo , Femenino , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Yeyuno/metabolismo , Litostatina/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Trombospondinas/metabolismo , Proteína Wnt3A/metabolismo
8.
Am J Physiol Cell Physiol ; 319(2): C321-C330, 2020 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-32551856

RESUMEN

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.


Asunto(s)
Agonistas Muscarínicos/farmacología , Sistema Colinérgico no Neuronal/genética , Receptores Muscarínicos/genética , Receptores Nicotínicos/genética , Acetilcolina/farmacología , Animales , Aniones/metabolismo , Atropina/farmacología , Agonistas Colinérgicos/farmacología , Enterocitos/efectos de los fármacos , Enterocitos/metabolismo , Hexametonio/farmacología , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Ratones , Sistema Colinérgico no Neuronal/efectos de los fármacos , Receptores Muscarínicos/metabolismo , Receptores Nicotínicos/metabolismo
9.
Am J Physiol Cell Physiol ; 317(4): C737-C748, 2019 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-31365292

RESUMEN

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood death from diarrhea and the leading cause of Traveler's diarrhea. E. coli heat-stable enterotoxin (ST) is a major virulence factor of ETEC and inhibits the brush border Na/H exchanger NHE3 in producing diarrhea. NHE3 regulation involves multiprotein signaling complexes that form on its COOH terminus. In this study, the hypothesis was tested that ST signals via members of the Na/H exchanger regulatory factor (NHERF) family of scaffolding proteins, NHERF2, which had been previously shown to have a role, and now with concentration on a role for NHERF3. Two models were used: mouse small intestine and Caco-2/BBe cells. In both models, ST rapidly increased intracellular cGMP, inhibited NHE3 activity, and caused a quantitatively similar decrease in apical expression of NHE3. The transport effects were NHERF3 and NHERF2 dependent. Also, mutation of the COOH-terminal amino acids of NHERF3 supported that NHERF3-NHERF2 heterodimerization was likely to account for this dual dependence. The ST increase in cGMP in both models was partially dependent on NHERF3. The intracellular signaling pathways by which ST-cGMP inhibits NHE3 were different in mouse jejunum (activation of cGMP kinase II, cGKII) and Caco-2 cells, which do not express cGKII (elevation of intracellular Ca2+ concentration [Ca2+]i). The ST elevation of [Ca2+]i was from intracellular stores and was dependent on NHERF3-NHERF2. This study shows that intracellular signaling in the same diarrheal model in multiple cell types may be different; this has implications for therapeutic strategies, which often assume that models have similar signaling mechanisms.


Asunto(s)
Toxinas Bacterianas/farmacología , Enterotoxinas/farmacología , Proteínas de Escherichia coli/farmacología , Proteínas de la Membrana/efectos de los fármacos , Intercambiador 3 de Sodio-Hidrógeno/efectos de los fármacos , Animales , Células CACO-2 , GMP Cíclico/metabolismo , Diarrea/inducido químicamente , Escherichia coli/efectos de los fármacos , Humanos , Ratones Transgénicos
10.
Exp Mol Med ; 51(8): 1-14, 2019 08 05.
Artículo en Inglés | MEDLINE | ID: mdl-31383845

RESUMEN

Calcium-activated chloride channels (CaCCs) mediate numerous physiological functions and are best known for the transport of electrolytes and water in epithelia. In the intestine, CaCC currents are considered necessary for the secretion of fluid to protect the intestinal epithelium. Although genetic ablation of ANO1/TMEM16A, a gene encoding a CaCC, reduces the carbachol-induced secretion of intestinal fluid, its mechanism of action is still unknown. Here, we confirm that ANO1 is essential for the secretion of intestinal fluid. Carbachol-induced transepithelial currents were reduced in the proximal colon of Ano1-deficient mice. Surprisingly, cholera toxin-induced and cAMP-induced fluid secretion, believed to be mediated by CFTR, were also significantly reduced in the intestine of Ano1-deficient mice. ANO1 is largely expressed in the apical membranes of intestines, as predicted for CaCCs. The Ano1-deficient colons became edematous under basal conditions and had a greater susceptibility to dextran sodium sulfate-induced colitis. However, Ano1 depletion failed to affect tumor development in a model of colorectal cancer. We thus conclude that ANO1 is necessary for cAMP- and carbachol-induced Cl- secretion in the intestine, which is essential for the protection of the intestinal epithelium from colitis.


Asunto(s)
Anoctamina-1/fisiología , Carbacol/farmacología , Cloruros/metabolismo , Toxina del Cólera/farmacología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Animales , Anoctamina-1/genética , Calcio/metabolismo , Canales de Cloruro/genética , Canales de Cloruro/fisiología , Colitis/inducido químicamente , Colitis/genética , Colitis/metabolismo , Colitis/patología , Femenino , Intestinos/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Vías Secretoras/efectos de los fármacos , Vías Secretoras/genética , Regulación hacia Arriba/efectos de los fármacos
11.
Cell Mol Gastroenterol Hepatol ; 7(3): 641-653, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30659943

RESUMEN

BACKGROUND & AIMS: SLC26A3 (DRA) is an electroneutral Cl-/HCO3- exchanger that is present in the apical domain of multiple intestinal segments. An area that has continued to be poorly understood is related to DRA regulation in acute adenosine 3',5'-cyclic monophosphate (cAMP)-related diarrheas, in which DRA appears to be both inhibited as part of NaCl absorption and stimulated to contribute to increased HCO3- secretion. Different cell models expressing DRA have shown that cAMP inhibits, stimulates, or does not affect its activity. METHODS: This study re-evaluated cAMP regulation of DRA using new tools, including a successful knockout cell model, a specific DRA inhibitor (DRAinh-A250), specific antibodies, and a transport assay that did not rely on nonspecific inhibitors. The studies compared DRA regulation in colonoids made from normal human colon with regulation in the colon cancer cell line, Caco-2. RESULTS: DRA is an apical protein in human proximal colon, differentiated colonoid monolayers, and Caco-2 cells. It is glycosylated and appears as 2 bands. cAMP (forskolin) acutely stimulated DRA activity in human colonoids and Caco-2 cells. In these cells, DRA is the predominant apical Cl-/HCO3- exchanger and is inhibited by DRAinh-A250 with a median inhibitory concentration of 0.5 and 0.2 µmol/L, respectively. However, there was no effect of cAMP in HEK293/DRA cells that lacked a cystic fibrosis transmembrane conductance regulator (CFTR). When CFTR was expressed in HEK293/DRA cells, cAMP also stimulated DRA activity. In all cases, cAMP stimulation of DRA was not inhibited by CFTRinh-172. CONCLUSIONS: DRA is acutely stimulated by cAMP by a process that is CFTR-dependent, but appears to be one of multiple regulatory effects of CFTR that does not require CFTR activity.


Asunto(s)
Antiportadores de Cloruro-Bicarbonato/metabolismo , Colon/metabolismo , AMP Cíclico/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Transportadores de Sulfato/metabolismo , Células CACO-2 , Diferenciación Celular/efectos de los fármacos , Colforsina/farmacología , Células HEK293 , Humanos , Transporte Iónico , Organoides/efectos de los fármacos , Organoides/metabolismo , Reproducibilidad de los Resultados
12.
Nat Chem ; 11(3): 254-263, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30532015

RESUMEN

Rapamycin and FK506 are macrocyclic natural products with an extraordinary mode of action, in which they form binary complexes with FK506-binding protein (FKBP) through a shared FKBP-binding domain before forming ternary complexes with their respective targets, mechanistic target of rapamycin (mTOR) and calcineurin, respectively. Inspired by this, we sought to build a rapamycin-like macromolecule library to target new cellular proteins by replacing the effector domain of rapamycin with a combinatorial library of oligopeptides. We developed a robust macrocyclization method using ring-closing metathesis and synthesized a 45,000-compound library of hybrid macrocycles (named rapafucins) using optimized FKBP-binding domains. Screening of the rapafucin library in human cells led to the discovery of rapadocin, an inhibitor of nucleoside uptake. Rapadocin is a potent, isoform-specific and FKBP-dependent inhibitor of the equilibrative nucleoside transporter 1 and is efficacious in an animal model of kidney ischaemia reperfusion injury. Together, these results demonstrate that rapafucins are a new class of chemical probes and drug leads that can expand the repertoire of protein targets well beyond mTOR and calcineurin.


Asunto(s)
Descubrimiento de Drogas/métodos , Macrólidos/química , Macrólidos/metabolismo , Sustancias Protectoras/química , Sustancias Protectoras/metabolismo , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/prevención & control , Animales , Línea Celular , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Proteoma/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/prevención & control , Sirolimus/química , Sirolimus/metabolismo , Porcinos , Serina-Treonina Quinasas TOR/química , Serina-Treonina Quinasas TOR/metabolismo , Tacrolimus/química , Tacrolimus/metabolismo , Proteínas de Unión a Tacrolimus/química , Proteínas de Unión a Tacrolimus/metabolismo
13.
Cell Mol Gastroenterol Hepatol ; 5(4): 591-609, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29930980

RESUMEN

BACKGROUND & AIMS: Human 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. METHODS: Human 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). RESULTS: Differentiation 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. CONCLUSIONS: Human 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.

14.
Mol Biol Cell ; 28(13): 1754-1767, 2017 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-28495796

RESUMEN

Casein kinase 2 (CK2) binds to the NHE3 C-terminus and constitutively phosphorylates a downstream site (S719) that accounts for 40% of basal NHE3 activity. The role of CK2 in regulation of NHE3 activity in polarized Caco-2/bbe cells was further examined by mutation of NHE3-S719 to A (not phosphorylated) or D (phosphomimetic). NHE3-S719A but not -S719D had multiple changes in NHE3 activity: 1) reduced basal NHE3 activity-specifically, inhibition of the PI3K/AKT-dependent component; 2) reduced acute stimulation of NHE3 activity by LPA/LPA5R stimulation; and 3) reduced acute inhibition of NHE3 activity-specifically, elevated Ca2+ related (carbachol/Ca2+ ionophore), but there was normal inhibition by forskolin and hyperosmolarity. The S719A mutant had reduced NHE3 complex size, reduced expression in lipid rafts, increased BB mobile fraction, and reduced binding to multiple proteins that bind throughout the NHE3 intracellular C-terminus, including calcineurin homologous protein, the NHERF family and SNX27 (related PDZ domains). These studies show that phosphorylation of the NHE3 at a single amino acid in the distal part of the C-terminus affects multiple aspects of NHE3 complex formation and changes the NHE3 lipid raft distribution, which cause changes in specific aspects of basal as well as acutely stimulated and inhibited Na+/H+ exchange activity.


Asunto(s)
Quinasa de la Caseína II/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Células CACO-2 , Calcio/metabolismo , Carbacol/metabolismo , Células Epiteliales/metabolismo , Exocitosis , Humanos , Lisofosfolípidos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas , Fosforilación , Unión Proteica , Dominios Proteicos , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Sodio/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno
15.
Am J Physiol Gastrointest Liver Physiol ; 313(2): G129-G137, 2017 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-28495802

RESUMEN

Na+/H+ exchanger NHE3 mediates the majority of intestinal and renal electroneutral sodium absorption. Dysfunction of NHE3 is associated with a variety of diarrheal diseases. We previously reported that the NHE3 gene (SLC9A3) has more than 400 single-nucleotide polymorphisms (SNPs) but few nonsynonymous polymorphisms. Among the latter, one polymorphism (rs2247114-G>A), which causes a substitution from arginine to cysteine at amino acid position 799 (p.R799C), is common in Asian populations. To improve our understanding of the population distribution and potential clinical significance of the NHE3-799C variant, we investigated the frequency of this polymorphism in different ethnic groups using bioinformatics analyses and in a cohort of Japanese patients with cardiovascular or renal disease. We also characterized the function of human NHE3-799C and its sensitivity to regulatory ligands in an in vitro model. NHE3-799C had an allele frequency of 29.5-57.6% in Asian populations, 11.1-23.6% in European populations, and 10.2-22.7% in African populations. PS120/FLAG-NHERF2 fibroblasts stably expressing NHE3-799C had lower total protein expression but a higher percentage of surface expression than those expressing NHE3-799R. NHE3-799C had similar basal activity to NHE3-799R and was similarly stimulated or inhibited, by serum or forskolin, respectively. Tenapanor, a small-molecule NHE3 inhibitor, dose-dependently inhibited NHE3-799R and NHE3-799C activities. The IC50 values of tenapanor for NHE3-799C and NHE3-799R were significantly different, but both were in the nanomolar range. These results suggest that NHE3-799C is a common variant enriched in Asian populations, is not associated with compromised function or abnormal regulation, and is unlikely to contribute to clinical disease.NEW & NOTEWORTHY This study reports results on the functional significance of human NHE3-799C under basal conditions and in response to regulatory ligands, including a novel NHE3 inhibitor called tenapanor. We demonstrate that NHE3-799C is a common variant of NHE3 that is enriched in Asian populations; however, in contrast to our previous studies using rabbit NHE3, its presence seems to have limited clinical significance in humans and is not associated with compromised function or abnormal transport regulation.


Asunto(s)
Alelos , Frecuencia de los Genes , Polimorfismo de Nucleótido Simple , Intercambiadores de Sodio-Hidrógeno/genética , Pueblo Asiatico/genética , Enfermedades Cardiovasculares/genética , Biología Computacional , Genotipo , Humanos , Enfermedades Renales/genética , Mutación , Intercambiador 3 de Sodio-Hidrógeno , Población Blanca/genética
16.
J Biol Chem ; 292(20): 8279-8290, 2017 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-28283572

RESUMEN

NHE3 directly binds Na+/H+ exchanger regulatory factor (NHERF) family scaffolding proteins that are required for many aspects of NHE3 regulation. The NHERFs bind both to an internal region (amino acids 586-660) of the NHE3 C terminus and to the NHE3 C-terminal four amino acids. The internal NHERF-binding region contains both putative Class I (-592SAV-) and Class II (-595CLDM-) PDZ-binding motifs (PBMs). Point mutagenesis showed that only the Class II motif contributes to NHERF binding. In this study, the roles in regulation of NHE3 activity of these two PBMs were investigated, revealing the following findings. 1) Interaction occurred between these binding sites because mutation of either removed nearly all NHERF binding. 2) Mutations in either significantly reduced basal NHE3 activity. Total and percent plasma membrane (PM) NHE3 protein expression was reduced in the C-terminal but not in the internal PBD mutation. 3) cGMP- and Ca2+-mediated inhibition of NHE3 was impaired in both the internal and the C-terminal PBM mutations. 4) There was a significant reduction in half-life of the PM pool of NHE3 in only the internal PBM mutation but no change in total NHE3 half-life in either. 5) There were some differences in NHE3-associating proteins in the two PBM mutations. In conclusion, NHE3 binds to NHERF proteins via both an internal Class II PBM and C-terminal Class I PBM, which interact. The former determines NHE3 stability in the PM, and the latter determines total expression and percent PM expression.


Asunto(s)
Calcio/metabolismo , Membrana Celular/metabolismo , GMP Cíclico/metabolismo , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Secuencias de Aminoácidos , Línea Celular , Membrana Celular/genética , GMP Cíclico/genética , Humanos , Mutación , Dominios PDZ , Fosfoproteínas/genética , Unión Proteica/fisiología , Estabilidad Proteica , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genética
17.
Eur J Pharmacol ; 791: 544-551, 2016 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-27388143

RESUMEN

Equilibrative nucleoside transporters (ENTs) play a crucial role in the transport of nucleoside and nucleoside analogues, which are important for nucleotide synthesis and chemotherapy. In addition, ENTs regulate extracellular adenosine levels in the vicinity of its receptors and hence influence adenosine-related functions. The clinical applications of ENT inhibitors in the treatment of cardiovascular diseases and cancer therapy have been explored in numerous studies. However, all ENT inhibitors to date are selective for ENT1 but not ENT2. In the present study, we investigated the novel compound 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) as an inhibitor of ENT1 and ENT2. Nucleoside transporter-deficient PK15NTD cells stably expressing ENT1 and ENT2 showed that FPMINT inhibited [3H]uridine and [3H]adenosine transport through both ENT1 and ENT2 in a concentration-dependent manner. The IC50 value of FPMINT for ENT2 was 5-10-fold less than for ENT1, and FPMINT could not be displaced with excess washing. Kinetic studies revealed that FPMINT reduced Vmax of [3H]uridine transport in ENT1 and ENT2 without affecting KM. Therefore, we conclude that FPMINT inhibits ENTs in an irreversible and non-competitive manner. Although already selective for ENT2 over ENT1, further modification of the chemical structure of FPMINT may lead to even better ENT2-selective inhibitors of potential clinical, physiological and pharmacological importance.


Asunto(s)
Tranportador Equilibrativo 1 de Nucleósido/antagonistas & inhibidores , Transportador Equilibrativo 2 de Nucleósido/antagonistas & inhibidores , Naftalenos/farmacología , Piperazinas/farmacología , Triazinas/farmacología , Transporte Biológico/efectos de los fármacos , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Transportador Equilibrativo 2 de Nucleósido/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Cinética , Naftalenos/química , Piperazinas/química , Triazinas/química
18.
Cell Physiol Biochem ; 36(2): 670-82, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25998606

RESUMEN

BACKGROUND: Vasopressin induced trafficking of aquaporin-2 (AQP2) containing vesicles has been studied in kidney cell lines using conventional fluorescent proteins as tags. However, trafficking of fluorescent tagged AQP2, which resembles the vectorial translocation of native AQP2 from cytoplasm to apical membrane has not been demonstrated at real time. Using a photoconvertible fluorescent protein tag on AQP2 might allow the simultaneous tracking of two separate populations of AQP2 vesicle after subcellular local photoconversion. METHODS: A spacer was used to link a photoconvertible fluorescent protein (mEos2) to the amino-terminus of AQP2. The DNA constructs were expressed in mpkCCD cells. The trafficking of chimeric protein was visualized with high speed confocal microscopy in 4 dimensions. RESULTS: Chimeric AQP2 expressed in mpkCCD cell conferred osmotic water permeability to the cells. Subcellular photoconversion with a 405 nm laser pulse converted green chimeras to red chimeras locally. Forskolin stimulation triggered chimeric AQP2 to translocate from acidic organelles to apical plasma membrane. By serendipity, the rate of apical accumulation was found to increase when mEos2 was tagged to the carboxyl-terminus in at least one of the AQP2 molecules within the tetramer. CONCLUSION: Functional photoconvertible chimeric AQP2 was successfully expressed in mpkCCD cells, in which forskolin induced apical trafficking and accumulation of chimeric AQP2. The proof-of-concept to monitor two populations of AQP2 vesicle simultaneously was demonstrated.


Asunto(s)
Acuaporina 2/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Agua/metabolismo , Animales , Acuaporina 2/análisis , Acuaporina 2/genética , Línea Celular , Colforsina/farmacología , Expresión Génica , Riñón/citología , Riñón/efectos de los fármacos , Riñón/metabolismo , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Presión Osmótica , Permeabilidad , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección
19.
Am J Physiol Gastrointest Liver Physiol ; 308(6): G482-8, 2015 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-25591867

RESUMEN

Transient receptor potential A1 (TRPA1) is a newly defined cationic ion channel, which selectively expresses in primary sensory afferent nerve, and is essential in mediating inflammatory nociception. Our previous study demonstrated that TRPA1 plays an important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in a guinea pig model of eosinophilic esophagitis (EoE). Antigen challenge-induced responses in esophageal mucosa were first assessed by histological stains and Ussing chamber studies. TRPA1 function in vagal sensory neurons was then studied by calcium imaging and by whole cell patch-clamp recordings in 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled esophageal vagal nodose and jugular neurons. Extracellular single-unit recordings were performed in vagal nodose and jugular C-fiber neuron subtypes using ex vivo esophageal-vagal preparations with intact nerve endings in the esophagus. Antigen challenge significantly increased infiltrations of eosinophils and mast cells in the esophagus. TRPA1 agonist allyl isothiocyanate (AITC)-induced calcium influx in nodose and jugular neurons was significantly increased, and current densities in esophageal DiI-labeled nodose and jugular neurons were also significantly increased in antigen-challenged animals. Prolonged antigen challenge decreased esophageal epithelial barrier resistance, which allowed intraesophageal-infused AITC-activating nodose and jugular C fibers at their nerve endings. Collectively, these results demonstrated that prolonged antigen challenge sensitized TRPA1 in esophageal sensory neurons and afferent C fibers. This novel finding will help us to better understand the molecular mechanism underlying esophageal sensory and motor dysfunctions in EoE.


Asunto(s)
Alérgenos , Esofagitis Eosinofílica/metabolismo , Esófago/inervación , Fibras Nerviosas Amielínicas/metabolismo , Ovalbúmina , Células Receptoras Sensoriales/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Nervio Vago/metabolismo , Potenciales de Acción , Animales , Señalización del Calcio , Modelos Animales de Enfermedad , Esofagitis Eosinofílica/inmunología , Esofagitis Eosinofílica/fisiopatología , Eosinófilos/inmunología , Eosinófilos/metabolismo , Cobayas , Isotiocianatos/farmacología , Masculino , Mastocitos/inmunología , Mastocitos/metabolismo , Fibras Nerviosas Amielínicas/efectos de los fármacos , Fibras Nerviosas Amielínicas/inmunología , Ganglio Nudoso/efectos de los fármacos , Ganglio Nudoso/inmunología , Ganglio Nudoso/metabolismo , Sensación , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/inmunología , Factores de Tiempo , Canales de Potencial de Receptor Transitorio/agonistas , Canales de Potencial de Receptor Transitorio/inmunología , Nervio Vago/efectos de los fármacos , Nervio Vago/inmunología , Nervio Vago/fisiopatología
20.
J Biol Chem ; 289(9): 5449-61, 2014 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-24398676

RESUMEN

Basal activity of the BB Na(+)/H(+) exchanger NHE3 requires multiprotein complexes that form on its C terminus. One complex stimulates basal NHE3 activity and contains ezrin and phosphoinositides as major components; how it stimulates NHE3 activity is not known. This study tested the hypothesis that ezrin dynamically associates with this complex, which sets ezrin binding. NHE3 activity was reduced by an Akti. This effect was eliminated if ezrin binding to NHE3 was inhibited by a point mutant. Recombinant AKT phosphorylated NHE3 C terminus in the domain ezrin directly binds. This domain (amino acids 475-589) is predicted to be α-helical and contains a conserved cluster of three serines (Ser(515), Ser(522), and Ser(526)). Point mutations of two of these (S515A, S515D, or S526A) reduced basal NHE3 activity and surface expression and had no Akti inhibition. S526D had NHE3 activity equal to wild type with normal Akti inhibition. Ezrin binding to NHE3 was regulated by Akt, being eliminated by Akti. NHE3-S515A and -S526D did not bind ezrin; NHE3-S515D had reduced ezrin binding; NHE3-S526D bound ezrin normally. NHE3-Ser(526) is predicted to be a GSK-3 kinase phosphorylation site. A GSK-3 inhibitor reduced basal NHE3 activity as well as ezrin-NHE3 binding, and this effect was eliminated in NHE3-S526A and -S526D mutants. The conclusions were: 1) NHE3 basal activity is regulated by a signaling complex that is controlled by sequential effects of two kinases, Akt and GSK-3, which act on a Ser cluster in the same NHE3 C-terminal domain that binds ezrin; and 2) these kinases regulate the dynamic association of ezrin with NHE3 to affect basal NHE3 activity.


Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Proteínas del Citoesqueleto/genética , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Células HEK293 , Humanos , Mutación Puntual , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Conejos , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genética
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