Impact of hypoxia and AMPK on CFTR-mediated bicarbonate secretion in human cholangiocyte organoids.

2021 ◽  
Author(s):  
Floris J.M. Roos ◽  
Marcel J.C. Bijvelds ◽  
Monique M.A. Verstegen ◽  
Henk P. Roest ◽  
Herold J. Metselaar ◽  
...  
Keyword(s):  
Liver Transplantation ◽  
Ussing Chamber ◽  
Bicarbonate Secretion ◽  
Transmembrane Conductance Regulator ◽  
Anion Channels ◽  
Transmembrane Conductance ◽  
Human Bile ◽  
Anion Secretion ◽  
Drug Interventions ◽  
Cystic Fibrosis Transmembrane

Background and Aims: Cholangiocytes express cystic fibrosis transmembrane conductance regulator (CFTR) which is involved in bicarbonate secretion for the protection against bile toxicity. During liver transplantation prolonged hypoxia of the graft is associated with cholangiocyte loss and biliary complications. Hypoxia is known to diminish CFTR activity in the intestine, but whether it effects CFTR activity in cholangiocytes remains unknown. Thus, the aim of this study is to investigate the effect of hypoxia on CFTR activity in intrahepatic cholangiocyte organoids (ICOs) and test drug-interventions to restore bicarbonate secretion. Methods: Fifteen different human ICOs were cultured as monolayers and ion channel (CFTR and ANO1) activity was determined using an Ussing chamber assay with or without AMP kinase (AMPK)-inhibitor under hypoxic and oxygenated conditions. Bile-toxicity was tested by apical exposure of cells to fresh human bile. Results: Overall gene-expression analysis showed a high similarity between ICOs and primary cholangiocytes. Under oxygenated conditions, both CFTR and ANO1 channels were responsible for forskolin and UTP activated anion-secretion. Forskolin-stimulation in absence of intracellular chloride showed ion-transport, indicating that bicarbonate could be secreted by CFTR. During hypoxia, CFTR activity significantly decreased (p=0.01). Switching from oxygen to hypoxia during CFTR-measurements, reduced CFTR activity (p=0.03). Consequently, cell death increased when ICO-monolayers were exposed to bile during hypoxia compared to oxygen (p=0.04). Importantly, addition of AMPK-inhibitor restored CFTR-mediated anion-secretion during hypoxia. Conclusion: ICOs provide an excellent model to study cholangiocyte anion channels and drug-related interventions. Here, we demonstrate that hypoxia affects cholangiocyte ion-secretion, leaving cholangiocytes vulnerable to bile toxicity. The mechanistic insights from this model maybe relevant for hypoxia-related biliary injury during liver transplantation.

scholarly journals Huqi San-Evoked Rat Colonic Anion Secretion through Increasing CFTR Expression

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Xiaowei Xue ◽  
Zhengming Shi ◽  
Wen Wang ◽  
Xiaotong Yu ◽  
Ping Feng ◽  
...  
Keyword(s):  
Protein Content ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Anion Secretion ◽  
Mrna Transcripts ◽  
And Function ◽  
Cystic Fibrosis Transmembrane

Huqi San (HQS) is a Chinese herbal preparation of eight medicinal herbs that promote diuresis, detoxification, blood circulation, and cholestasis. Defects in transporter expression and function can cause cholestasis and jaundice. However, the mechanism of the cholestasis underlying HQS effects, especially on the gastrointestinal tract ion secretion, has not been elucidated. Real-time RT-PCR and Western blotting were used to study the expression and localization of cystic fibrosis transmembrane conductance regulator (CFTR) andα-ENaC in rat alimentary tract, and then the effect of HQS on the ion transport in rat distal colon mucosa was investigated using the short-circuit current (ISC) technique. The results showed that pretreatment with HQS significantly enhanced mRNA transcripts and protein content of CFTR in liver and distal colon but notα-ENaC in alimentary organs. HQS increasesISCand decreases the transepithelial resistance. Pretreatment with epithelial Na+channel blocker did not affect theISCresponses elicited by HQS, but removal of extracellular Cl−or pretreatment with Cl−channel or Na+-K+-2Cl−cotransporter blocker inhibited HQS-elicitedISCresponses. These findings demonstrated that HQS, RA, and RP can stimulate Cl−secretion in the distal colon by increasing the mRNA transcripts and protein content of CFTR in liver and distal colon.

scholarly journals Infection by Toxoplasma gondii, a severe parasite in neonates and AIDS patients, causes impaired anion secretion in airway epithelia

2015 ◽  
Vol 112 (14) ◽  
pp. 4435-4440 ◽  
Author(s):  
Hong-Mei Guo ◽  
Jiang-Mei Gao ◽  
Yu-Li Luo ◽  
Yan-Zi Wen ◽  
Yi-Lin Zhang ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Toxoplasma Gondii ◽  
Infected Mouse ◽  
Inflammatory Responses ◽  
Short Circuit ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Airway Epithelia ◽  
Anion Secretion ◽  
Cystic Fibrosis Transmembrane

The airway epithelia initiate and modulate the inflammatory responses to various pathogens. The cystic fibrosis transmembrane conductance regulator-mediated Cl− secretion system plays a key role in mucociliary clearance of inhaled pathogens. We have explored the effects of Toxoplasma gondii, an opportunistic intracellular protozoan parasite, on Cl− secretion of the mouse tracheal epithelia. In this study, ATP-induced Cl− secretion indicated the presence of a biphasic short-circuit current (Isc) response, which was mediated by a Ca2+-activated Cl− channel (CaCC) and the cystic fibrosis transmembrane conductance regulator. However, the ATP-evoked Cl− secretion in T. gondii-infected mouse tracheal epithelia and the elevation of [Ca2+]i in T. gondii-infected human airway epithelial cells were suppressed. Quantitative reverse transcription–PCR revealed that the mRNA expression level of the P2Y2 receptor (P2Y2-R) increased significantly in T. gondii-infected mouse tracheal cells. This revealed the influence that pathological changes in P2Y2-R had on the downstream signal, suggesting that P2Y2-R was involved in the mechanism underlying T. gondii infection in airways. These results link T. gondii infection as well as other pathogen infections to Cl− secretion, via P2Y2-R, which may provide new insights for the treatment of pneumonia caused by pathogens including T. gondii.

cAMP regulation of Cl− and HCO 3 − secretion across rat fetal distal lung epithelial cells

2002 ◽  
Vol 282 (4) ◽  
pp. L650-L658 ◽  
Author(s):  
Ahmed Lazrak ◽  
Ulrich Thome ◽  
Carpantanto Myles ◽  
Janice Ware ◽  
Lan Chen ◽  
...  
Keyword(s):  
Short Circuit ◽  
Lung Epithelial Cells ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Anion Secretion ◽  
Ussing Chambers ◽  
Lung Epithelial ◽  
Cftr Protein ◽  
Distal Lung ◽  
Cystic Fibrosis Transmembrane

We isolated and cultured fetal distal lung epithelial (FDLE) cells from 17- to 19-day rat fetuses and assayed for anion secretion in Ussing chambers. With symmetrical Ringer solutions, basal short-circuit currents ( I sc) and transepithelial resistances were 7.9 ± 0.5 μA/cm2 and 1,018 ± 73 Ω · cm2, respectively (means ± SE; n = 12). Apical amiloride (10 μM) inhibited basal I sc by ∼50%. Subsequent addition of forskolin (10 μM) increased I sc from 3.9 ± 0.63 μA/cm2 to 7.51 ± 0.2 μA/cm2( n = 12). Basolateral bumetanide (100 μM) decreased forskolin-stimulated I sc from 7.51 ± 0.2 μA/cm2 to 5.62 ± 0.53, whereas basolateral 4,4′-dinitrostilbene-2,2′-disulfonate (5 mM), an inhibitor of HCO[Formula: see text] secretion, blocked the remaining I sc. Forskolin addition evoked currents of similar fractional magnitudes in symmetrical Cl−- or HCO[Formula: see text]-free solutions; however, no response was seen using HCO[Formula: see text]- and Cl−-free solutions. The forskolin-stimulated I sc was inhibited by glibenclamide but not apical DIDS. Glibenclamide also blocked forskolin-induced I sc across monolayers having nystatin-permeablized basolateral membranes. Immunolocalization studies were consistent with the expression of cystic fibrosis transmembrane conductance regulator (CFTR) protein in FDLE cells. In aggregate, these findings indicate the presence of cAMP-activated Cl− and HCO[Formula: see text] secretion across rat FDLE cells mediated via CFTR.

scholarly journals Rab11b Regulates the Apical Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator in Polarized Intestinal Epithelial Cells

2009 ◽  
Vol 20 (8) ◽  
pp. 2337-2350 ◽  
Author(s):  
Mark R. Silvis ◽  
Carol A. Bertrand ◽  
Nadia Ameen ◽  
Franca Golin-Bisello ◽  
Michael B. Butterworth ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Apical Membrane ◽  
Expression System ◽  
Anion Channel ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
T84 Cells ◽  
Anion Secretion ◽  
Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, ΔF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.

Localization of the cystic fibrosis transmembrane conductance regulator in human bile duct epithelial cells

1993 ◽  
Vol 105 (6) ◽  
pp. 1857-1864 ◽  
Author(s):  
Jonathan A. Cohn ◽  
Theresa V. Strong ◽  
Marina R. Picciotto ◽  
Angus C. Nairn ◽  
Francis S. Collins ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Bile Duct ◽  
Epithelial Cells ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Human Bile ◽  
Cystic Fibrosis Transmembrane

scholarly journals Gender-Specific Protection of Estrogen against Gastric Acid-Induced Duodenal Injury: Stimulation of Duodenal Mucosal Bicarbonate Secretion

Endocrinology ◽  
2008 ◽  
Vol 149 (9) ◽  
pp. 4554-4566 ◽  
Author(s):  
Anders Smith ◽  
Cheyanne Contreras ◽  
Kwang Hyun Ko ◽  
Jimmy Chow ◽  
Xiao Dong ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Duodenal Ulcer ◽  
Lower Incidence ◽  
Premenopausal Women ◽  
Bicarbonate Secretion ◽  
Transmembrane Conductance Regulator ◽  
Male Mice ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane

Because human duodenal mucosal bicarbonate secretion (DMBS) protects duodenum against acid-peptic injury, we hypothesize that estrogen stimulates DMBS, thereby attributing to the clinically observed lower incidence of duodenal ulcer in premenopausal women than the age-matched men. We found that basal and acid-stimulated DMBS responses were 1.5 and 2.4-fold higher in female than male mice in vivo, respectively. Acid-stimulated DMBS in both genders was abolished by ICI 182,780 and tamoxifen. Estradiol-17β (E2) and the selective estrogen receptor (ER) agonists of ERα [1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole] and ERβ [2,3-bis(4-hydroxyphenyl) propionitrile], but not progesterone, rapidly stimulated ER-dependent murine DMBS in vivo. E2 dose dependently stimulated murine DMBS, which was attenuated by a Cl−/HCO3− anion exchanger inhibitor 4,4′-didsothio- cyanostilbene-2, 2′-disulfonic acid, removal of extracellular Cl−, and in cystic fibrosis transmembrane conductance regulator knockout female mice. E2 stimulated murine DMBS in vitro in both genders with significantly greater response in female than male mice (female to male ratio = 4.3). ERα and ERβ mRNAs and proteins were detected in murine duodenal epithelium of both genders; however, neither ERα nor ERβ mRNA and protein expression levels differed according to gender. E2 rapidly mobilized intracellular calcium in a duodenal epithelial SCBN cell line that expresses ERα and ERβ, whereas BAPTA-AM abolished E2-stimulated murine DMBS. Thus, our data show that E2 stimulates DMBS via ER dependent mechanisms linked to intracellular calcium, cystic fibrosis transmembrane conductance regulator, and Cl−/HCO3− anion exchanger. Gender-associated differences in basal, acid- and E2-stimulated DMBS may have offered a reasonable explanation for the clinically observed lower incidence of duodenal ulcer in premenopausal women than age-matched men.

CFTR and Bicarbonate Secretion to Epithelial Cells

Physiology ◽  
2003 ◽  
Vol 18 (1) ◽  
pp. 38-42 ◽  
Author(s):  
Martin J. Hug ◽  
Tsutomu Tamada ◽  
Robert J. Bridges
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Fluid Secretion ◽  
Bicarbonate Secretion ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane

Defective HCO3– and fluid secretion are hallmarks of the pathophysiology of the pancreas of cystic fibrosis patients. Recently, impaired HCO3– secretion has been shown in most tissues known to express the cystic fibrosis transmembrane conductance regulator (CFTR). New results suggest that CFTR plays an important role in the transcellular secretion of HCO3–.

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