Publications by authors named "Amelia Mazzone"

26 Publications

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Expression of the regulated isoform of the electrogenic Na/HCO cotransporter, NBCe1, is enriched in pacemaker interstitial cells of Cajal.

Am J Physiol Gastrointest Liver Physiol 2021 01 28;320(1):G93-G107. Epub 2020 Oct 28.

Enteric NeuroScience Program, Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota.

Interstitial cells of Cajal (ICCs) generate electrical slow waves, which are required for normal gastrointestinal motility. The mechanisms for generation of normal pacemaking are not fully understood. Normal gastrointestinal contractility and electrical slow-wave activity depend on the presence of extracellular HCO. Previous transcriptional analysis identified enrichment of mRNA encoding the electrogenic Na/HCO cotransporter (NBCe1) gene () in pacemaker myenteric ICCs in mouse small intestine. We aimed to determine the distribution of NBCe1 protein in ICCs of the mouse gastrointestinal tract and to identify the transcripts of the gene in mouse and human small intestinal tunica muscularis. We determined the distribution of NBCe1 immunoreactivity (NBCe1-IR) by immunofluorescent labeling in mouse and human tissues. In mice, NBCe1-IR was restricted to Kit-positive myenteric ICCs of the stomach and small intestine and submuscular ICCs of the large intestine, that is, the slow wave generating subset of ICCs. Other subtypes of ICCs were NBCe1-negative. Quantitative real-time PCR identified >500-fold enrichment of and transcripts ["IP3-receptor-binding protein released by IP3" (IRBIT)-regulated isoforms] in Kit-expressing cells isolated from Kit, Rpl22 mice and from single GFP-positive ICCs from Kit mice. Human jejunal tunica muscularis ICCs were also NBCe1-positive, and and RNAs were >300-fold enriched relative to . In summary, NBCe1 protein expressed in ICCs with electrical pacemaker function is encoded by gene transcripts that generate IRBIT-regulated isoforms of NBCe1. In conclusion, Na/HCO cotransport through NBCe1 contributes to the generation of pacemaker activity in subsets of ICCs. In this study, we show that the electrogenic Na+/HCO cotransporter, NBCe1/Slc4a4, is expressed in subtypes of interstitial cells of Cajal (ICCs) responsible for electrical slow wave generation throughout the mouse gastrointestinal tract and is absent in other types of ICCs. The transcripts of expressed in mouse ICCs and human gastrointestinal smooth muscle are the regulated isoforms. This indicates a key role for HCO transport in generation of gastrointestinal motility patterns.
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http://dx.doi.org/10.1152/ajpgi.00255.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8112189PMC
January 2021

A Method for Multi-day Tracking of Gastrointestinal Smooth Muscle Contractile Patterns in Organotypic Culture.

Annu Int Conf IEEE Eng Med Biol Soc 2019 Jul;2019:4791-4794

Gastrointestinal (GI) motility is a key component of digestive health, and it is a complex both rhythmic and arrhythmic process governed by many physiological factors. The ability to accurately track the movements of the GI tissues in vitro over multiple days would provide valuable insights into GI tract physiology. A correlational analysis tracking algorithm was developed and applied to intestinal smooth muscle tissues that were maintained in organotypic culture. Physiologically relevant metrics, such as frequency, amplitude and motility index were independently assayed to quantify smooth muscle contractions. The results were validated by manually detected frequency determined using a standard software package. The algorithm was capable of tracking the changes in contractions of the same tissues over six days. This proof-of-concept study demonstrates the feasibility of long-term tracking of GI motility in organotypic cultures over multiple days. The approach allows study of the effects on GI smooth muscle contractility of direct controlled targeting of the cells and molecules in the GI tunica muscularis.
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http://dx.doi.org/10.1109/EMBC.2019.8857365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7138509PMC
July 2019

microRNA overexpression in slow transit constipation leads to reduced Na1.5 current and altered smooth muscle contractility.

Gut 2020 05 22;69(5):868-876. Epub 2019 Nov 22.

Enteric NeuroScience Program (ENSP), Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA

Objective: This study was designed to evaluate the roles of microRNAs (miRNAs) in slow transit constipation (STC).

Design: All human tissue samples were from the of the colon. Expression of 372 miRNAs was examined in a discovery cohort of four patients with STC versus three age/sex-matched controls by a quantitative PCR array. Upregulated miRNAs were examined by quantitative reverse transcription qPCR (RT-qPCR) in a validation cohort of seven patients with STC and age/sex-matched controls. The effect of a highly differentially expressed miRNA on a custom human smooth muscle cell line was examined by RT-qPCR, electrophysiology, traction force microscopy, and ex vivo by lentiviral transduction in rat organotypic cultures.

Results: The expression of 13 miRNAs was increased in STC samples. Of those miRNAs, four were predicted to target , the gene that encodes the Na channel Na1.5. The expression of mRNA was decreased in STC samples. Let-7f significantly decreased Na current density in vitro in human smooth muscle cells. In rat organotypic cultures, overexpression of let-7f resulted in reduced frequency and amplitude of contraction.

Conclusions: A small group of miRNAs is upregulated in STC, and many of these miRNAs target the SCN5A-encoded Na channel Na1.5. Within this set, a novel Na1.5 regulator, let-7f, resulted in decreased Na1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest Na1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.
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http://dx.doi.org/10.1136/gutjnl-2019-318747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7147984PMC
May 2020

mutation G615E results in Na1.5 voltage-gated sodium channels with normal voltage-dependent function yet loss of mechanosensitivity.

Channels (Austin) 2019 12;13(1):287-298

a Enteric NeuroScience Program, Division of Gastroenterology and Hepatology, Mayo Clinic , Rochester , MN , USA.

is expressed in cardiomyocytes and gastrointestinal (GI) smooth muscle cells (SMCs) as the voltage-gated mechanosensitive sodium channel Na1.5. The influx of Na through Na1.5 produces a fast depolarization in membrane potential, indispensable for electrical excitability in cardiomyocytes and important for electrical slow waves in GI smooth muscle. As such, abnormal Na1.5 voltage gating or mechanosensitivity may result in channelopathies. mutation G615E - found separately in cases of acquired long-QT syndrome, sudden cardiac death, and irritable bowel syndrome - has a relatively minor effect on Na1.5 voltage gating. The aim of this study was to test whether G615E impacts mechanosensitivity. Mechanosensitivity of wild-type (WT) or G615E-Na1.5 in HEK-293 cells was examined by shear stress on voltage- or current-clamped whole cells or pressure on macroscopic patches. Unlike WT, voltage-clamped G615E-Na1.5 showed a loss in shear- and pressure-sensitivity of peak current yet a normal leftward shift in the voltage-dependence of activation. In current-clamp, shear stress led to a significant increase in firing spike frequency with a decrease in firing threshold for WT but not G615E-Na1.5. Our results show that the G615E mutation leads to functionally abnormal Na1.5 channels, which cause disruptions in mechanosensitivity and mechano-electrical feedback and suggest a potential contribution to smooth muscle pathophysiology.
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http://dx.doi.org/10.1080/19336950.2019.1632670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629189PMC
December 2019

Direct repression of anoctamin 1 () gene transcription by Gli proteins.

FASEB J 2019 05 25;33(5):6632-6642. Epub 2019 Feb 25.

Enteric NeuroSciences, Mayo Clinic, Rochester, Minnesota, USA.

The Ca-activated Cl channel, anoctamin 1 (Ano1, also known as transmembrane protein 16A) contributes to intestinal pacemaking, fluid secretion, cellular excitability, and tissue development. The human promoter contains binding sites for the glioma-associated oncogene (Gli) proteins. We investigated regulation of transcription by Gli. promoter activity was determined using a luciferase reporter system. Binding and functional effects of Glis on transcription and expression were demonstrated by chromatin immunoprecipitation, small interfering RNA knockdown, PCR, immunolabeling, and recordings of Ca-activated Cl currents in human embryonic kidney 293 (HEK293) cells. Results from previous genome-wide association studies were used to test promoter polymorphisms for association with disease. Gli1 and Gli2 bound to the promoter and repressed transcription. Repression depended on Gli binding to a site close to the transcriptional start site. Mutation of this site prevented Gli binding and transcriptional repression. Knockdown of Gli expression and inhibition of Gli activity increased expression of RNA and Ca-activated Cl currents in HEK293 cells. A single-nucleotide polymorphism prevented Gli binding and showed association with irritable bowel syndrome. We conclude that Gli1 and Gli2 repress by a novel mechanism that is independent of Gli cleavage and that has a role in gastrointestinal function.-Mazzone, A., Gibbons, S. J., Eisenman, S. T., Strege, P. R., Zheng, T., D'Amato, M., Ordog, T., Fernandez-Zapico, M. E., Farrugia, G. Direct repression of anoctamin 1 () gene transcription by Gli proteins.
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http://dx.doi.org/10.1096/fj.201802373RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463924PMC
May 2019

Irritable bowel syndrome patients have SCN5A channelopathies that lead to decreased Na1.5 current and mechanosensitivity.

Am J Physiol Gastrointest Liver Physiol 2018 04 22;314(4):G494-G503. Epub 2017 Nov 22.

Enteric NeuroScience Program, Division of Gastroenterology and Hepatology, Department of Physiology and Biomedical Engineering, Mayo Clinic , Rochester, Minnesota.

The SCN5A-encoded voltage-gated mechanosensitive Na channel Na1.5 is expressed in human gastrointestinal smooth muscle cells and interstitial cells of Cajal. Na1.5 contributes to smooth muscle electrical slow waves and mechanical sensitivity. In predominantly Caucasian irritable bowel syndrome (IBS) patient cohorts, 2-3% of patients have SCN5A missense mutations that alter Na1.5 function and may contribute to IBS pathophysiology. In this study we examined a racially and ethnically diverse cohort of IBS patients for SCN5A missense mutations, compared them with IBS-negative controls, and determined the resulting Na1.5 voltage-dependent and mechanosensitive properties. All SCN5A exons were sequenced from somatic DNA of 252 Rome III IBS patients with diverse ethnic and racial backgrounds. Missense mutations were introduced into wild-type SCN5A by site-directed mutagenesis and cotransfected with green fluorescent protein into HEK-293 cells. Na1.5 voltage-dependent and mechanosensitive functions were studied by whole cell electrophysiology with and without shear force. Five of 252 (2.0%) IBS patients had six rare SCN5A mutations that were absent in 377 IBS-negative controls. Six of six (100%) IBS-associated Na1.5 mutations had voltage-dependent gating abnormalities [current density reduction (R225W, R433C, R986Q, and F1293S) and altered voltage dependence (R225W, R433C, R986Q, G1037V, and F1293S)], and at least one kinetic parameter was altered in all mutations. Four of six (67%) IBS-associated SCN5A mutations (R225W, R433C, R986Q, and F1293S) resulted in altered Na1.5 mechanosensitivity. In this racially and ethnically diverse cohort of IBS patients, we show that 2% of IBS patients harbor SCN5A mutations that are absent in IBS-negative controls and result in Na1.5 channels with abnormal voltage-dependent and mechanosensitive function. NEW & NOTEWORTHY The voltage-gated Na channel Na1.5 contributes to smooth muscle physiology and electrical slow waves. In a racially and ethnically mixed irritable bowel syndrome cohort, 2% had mutations in the Na1.5 gene SCN5A. These mutations were absent in irritable bowel syndrome-negative controls. Most mutant Na1.5 channels were loss of function in voltage dependence or mechanosensitivity.
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http://dx.doi.org/10.1152/ajpgi.00016.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5966747PMC
April 2018

EAVK segment "c" sequence confers Ca-dependent changes to the kinetics of full-length human Ano1.

Am J Physiol Gastrointest Liver Physiol 2017 Jun 23;312(6):G572-G579. Epub 2017 Mar 23.

Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota

Anoctamin1 (Ano1 and TMEM16A) is a calcium-activated chloride channel specifically expressed in the interstitial cells of Cajal (ICC) of the gastrointestinal tract muscularis propria. Ano1 is necessary for normal electrical slow waves and ICC proliferation. The full-length human Ano1 sequence includes an additional exon, exon "0," at the NH terminus. Ano1 with exon 0 [Ano1] had a lower EC for intracellular calcium ([Ca]) and faster chloride current () kinetics. The Ano1 alternative splice variant with segment "c" encoding exon 13 expresses on the first intracellular loop four additional amino acid residues, EAVK, which alter at low [Ca] Exon 13 is expressed in 75-100% of Ano1 transcripts in most human tissues but only 25% in the human stomach. Our aim was to determine the effect of EAVK deletion on Ano1 parameters. By voltage-clamp electrophysiology, we examined in HEK293 cells transiently expressing Ano1 with or without the EAVK sequence [Ano1ΔEAVK]. The EC values of activating and deactivating for [Ca] were 438 ± 7 and 493 ± 9 nM for Ano1 but higher for Ano1ΔEAVK at 746 ± 47 and 761 ± 26 nM, respectively. Meanwhile, the EC values for the ratio of instantaneous to steady-state were not different between variants. Congruently, the time constant of activation was slower for Ano1ΔEAVK than Ano1 currents at intermediate [Ca] These results suggest that EAVK decreases the calcium sensitivity of Ano1 current activation and deactivation by slowing activation kinetics. Differential expression of EAVK in the human stomach may function as a switch to increase sensitivity to [Ca] via faster gating of Ano1. Calcium-activated chloride channel anoctamin1 (Ano1) is necessary for normal slow waves in the gastrointestinal interstitial cells of Cajal. Exon 0 encodes the NH terminus of full-length human Ano1 [Ano1], while exon 13 encodes residues EAVK on its first intracellular loop. Splice variants lack EAVK more often in the stomach than other tissues. Ano1 without EAVK [Ano1ΔEAVK] has reduced sensitivity for intracellular calcium, attributable to slower kinetics. Differential expression of EAVK may function as a calcium-sensitive switch in the human stomach.
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http://dx.doi.org/10.1152/ajpgi.00429.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495914PMC
June 2017

Constipation-Predominant Irritable Bowel Syndrome Females Have Normal Colonic Barrier and Secretory Function.

Am J Gastroenterol 2017 Jun 21;112(6):913-923. Epub 2017 Mar 21.

Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota, USA.

Objectives: The objective of this study was to determine whether constipation-predominant irritable bowel syndrome (IBS-C) is associated with changes in intestinal barrier and secretory function.

Methods: A total of 19 IBS-C patients and 18 healthy volunteers (all females) underwent saccharide excretion assay (0.1 g C mannitol and 1 g lactulose), measurements of duodenal and colonic mucosal barrier (transmucosal resistance (TMR), macromolecular and Escherichia coli Bio-Particle translocation), mucosal secretion (basal and acetylcholine (Ach)-evoked short-circuit current (Isc)), in vivo duodenal mucosal impedance, circulating endotoxins, and colonic tight junction gene expression.

Results: There were no differences in the in vivo measurements of barrier function between IBS-C patients and healthy controls: cumulative excretion of C mannitol (0-2 h mean (s.e.m.); IBS-C: 12.1 (0.9) mg vs. healthy: 13.2 (0.8) mg) and lactulose (8-24 h; IBS-C: 0.9 (0.5) mg vs. healthy: 0.5 (0.2) mg); duodenal impedance IBS-C: 729 (65) Ω vs. healthy: 706 (43) Ω; plasma mean endotoxin activity level IBS-C: 0.36 (0.03) vs. healthy: 0.35 (0.02); and in colonic mRNA expression of occludin, zonula occludens (ZO) 1-3, and claudins 1-12 and 14-19. The ex vivo findings were consistent, with no group differences: duodenal TMR (IBS-C: 28.2 (1.9) Ω cm vs. healthy: 29.8 (1.9) Ω cm) and colonic TMR (IBS-C: 19.1 (1.1) Ω cm vs. healthy: 17.6 (1.7) Ω cm); fluorescein isothiocyanate (FITC)-dextran (4 kDa) and E. coli Bio-Particle flux. Colonic basal Isc was similar, but duodenal basal Isc was lower in IBS-C (43.5 (4.5) μA cm) vs. healthy (56.9 (4.9) μA cm), P=0.05. Ach-evoked ΔIsc was similar.

Conclusions: Females with IBS-C have normal colonic barrier and secretory function. Basal duodenal secretion is decreased in IBS-C.
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http://dx.doi.org/10.1038/ajg.2017.48DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5502210PMC
June 2017

A novel exon in the human Ca2+-activated Cl- channel Ano1 imparts greater sensitivity to intracellular Ca2.

Am J Physiol Gastrointest Liver Physiol 2015 Nov 10;309(9):G743-9. Epub 2015 Sep 10.

Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota

Anoctamin 1 (Ano1; TMEM16A) is a Ca(2+)-activated Cl(-) channel (CACC) expressed in interstitial cells of Cajal. The mechanisms by which Ca(2+) regulates Ano1 are incompletely understood. In the gastrointestinal tract, Ano1 is required for normal slow wave activity and is involved in regulating cell proliferation. Splice variants of Ano1 have varying electrophysiological properties and altered expression in disease states. Recently, we identified a transcript for human Ano1 containing a novel exon-"exon 0" upstream of and in frame with exon 1. The electrophysiological properties of this longer Ano1 isoform are unknown. Our aim was to determine the functional contribution of the newly identified exon to the Ca(2+) sensitivity and electrophysiological properties of Ano1. Constructs with [Ano1(+0)] or without [Ano1(-0)] the newly identified exon were transfected into human embryonic kidney-293 cells. Voltage-clamp electrophysiology was used to determine voltage- and time-dependent parameters of whole cell Cl(-) currents between isoforms with varying concentrations of intracellular Ca(2+), extracellular anions, or Cl(-) channel inhibitors. We found that exon 0 did not change voltage sensitivity and had no impact on the relative permeability of Ano1 to most anions. Ano1(+0) exhibited greater changes in current density but lesser changes in kinetics than Ano1(-0) in response to varying intracellular Ca(2+). The CACC inhibitor niflumic acid inhibited current with greater efficacy and higher potency against Ano1(+0) compared with Ano1(-0). Likewise, the Ano1 inhibitor T16Ainh-A01 reduced Ano1(+0) more than Ano1(-0). In conclusion, human Ano1 containing exon 0 imparts its Cl(-) current with greater sensitivity to intracellular Ca(2+) and CACC inhibitors.
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http://dx.doi.org/10.1152/ajpgi.00074.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4628966PMC
November 2015

Ranolazine inhibits voltage-gated mechanosensitive sodium channels in human colon circular smooth muscle cells.

Am J Physiol Gastrointest Liver Physiol 2015 Sep 16;309(6):G506-12. Epub 2015 Jul 16.

Enteric NeuroScience Program, Mayo Clinic College of Medicine, Rochester, Minnesota; Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, Minnesota;

Human jejunum smooth muscle cells (SMCs) and interstitial cells of Cajal (ICCs) express the SCN5A-encoded voltage-gated, mechanosensitive sodium channel NaV1.5. NaV1.5 contributes to small bowel excitability, and NaV1.5 inhibitor ranolazine produces constipation by an unknown mechanism. We aimed to determine the presence and molecular identity of Na(+) current in the human colon smooth muscle and to examine the effects of ranolazine on Na(+) current, mechanosensitivity, and smooth muscle contractility. Inward currents were recorded by whole cell voltage clamp from freshly dissociated human colon SMCs at rest and with shear stress. SCN5A mRNA and NaV1.5 protein were examined by RT-PCR and Western blots, respectively. Ascending human colon strip contractility was examined in a muscle bath preparation. SCN5A mRNA and NaV1.5 protein were identified in human colon circular muscle. Freshly dissociated human colon SMCs had Na(+) currents (-1.36 ± 0.36 pA/pF), shear stress increased Na(+) peaks by 17.8 ± 1.8% and accelerated the time to peak activation by 0.7 ± 0.3 ms. Ranolazine (50 μM) blocked peak Na(+) current by 43.2 ± 9.3% and inhibited shear sensitivity by 25.2 ± 3.2%. In human ascending colon strips, ranolazine decreased resting tension (31%), reduced the frequency of spontaneous events (68%), and decreased the response to smooth muscle electrical field stimulation (61%). In conclusion, SCN5A-encoded NaV1.5 is found in human colonic circular smooth muscle. Ranolazine blocks both peak amplitude and mechanosensitivity of Na(+) current in human colon SMCs and decreases contractility of human colon muscle strips. Our data provide a likely mechanistic explanation for constipation induced by ranolazine.
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http://dx.doi.org/10.1152/ajpgi.00051.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4572410PMC
September 2015

Identification and characterization of a novel promoter for the human ANO1 gene regulated by the transcription factor signal transducer and activator of transcription 6 (STAT6).

FASEB J 2015 Jan 28;29(1):152-63. Epub 2014 Oct 28.

Enteric NeuroScience Program,

Anoctamin-1 (Ano1) is a widely expressed protein responsible for endogenous Ca(2+)-activated Cl(-) currents. Ano1 is overexpressed in cancer. Differential expression of transcriptional variants is also found in other diseases. However, the mechanisms underlying regulation of Ano1 are unknown. This study identifies the Ano1 promoter and defines a mechanism for regulating its expression. Next-generation RNA sequencing (RNA-seq) analysis in human gastric muscle found a new exon upstream of the reported exon 1 and identified a promoter proximal to this new exon. Reporter assays in human embryonic kidney 293 cells showed a 6.7 ± 2.1-fold increase in activity over empty vector. Treatment with a known regulator of Ano1 expression, IL-4, increased promoter activity by 1.6 ± 0.02-fold over untreated cells. The promoter region contained putative binding sites for multiple transcription factors including signal transducer and activator of transcription 6 (STAT6), a downstream effector of IL-4. Chromatin immunoprecipitation (ChIP) experiments on T84 cells, which endogenously express Ano1, showed a 2.1 ± 0.12-fold increase in binding of STAT6 to P0 after IL-4 treatment. These results were confirmed by mutagenesis, expression, and RNA interference techniques. This work allows deeper understanding of the regulation of Ano1 in physiology and as a potential therapeutic target in a variety of diseases.
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http://dx.doi.org/10.1096/fj.14-258541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4285536PMC
January 2015

Ano1, a Ca2+-activated Cl- channel, coordinates contractility in mouse intestine by Ca2+ transient coordination between interstitial cells of Cajal.

J Physiol 2014 Sep 25;592(18):4051-68. Epub 2014 Jul 25.

Department of Physiology and Biomedical Engineering Enteric NeuroScience Program, Mayo Clinic, Rochester, MN, USA

Interstitial cells of Cajal (ICC) are pacemaker cells that generate electrical activity to drive contractility in the gastrointestinal tract via ion channels. Ano1 (Tmem16a), a Ca(2+)-activated Cl(-) channel, is an ion channel expressed in ICC. Genetic deletion of Ano1 in mice resulted in loss of slow waves in smooth muscle of small intestine. In this study, we show that Ano1 is required to maintain coordinated Ca(2+) transients between myenteric ICC (ICC-MY) of small intestine. First, we found spontaneous Ca(2+) transients in ICC-MY in both Ano1 WT and knockout (KO) mice. However, Ca(2+) transients within the ICC-MY network in Ano1 KO mice were uncoordinated, while ICC-MY Ca(2+) transients in Ano1 WT mice were rhythmic and coordinated. To confirm the role of Ano1 in the loss of Ca(2+) transient coordination, we used pharmacological inhibitors of Ano1 activity and shRNA-mediated knock down of Ano1 expression in organotypic cultures of Ano1 WT small intestine. Coordinated Ca(2+) transients became uncoordinated using both these approaches, supporting the conclusion that Ano1 is required to maintain coordination/rhythmicity of Ca(2+) transients. We next determined the effect on smooth muscle contractility using spatiotemporal maps of contractile activity in Ano1 KO and WT tissues. Significantly decreased contractility that appeared to be non-rhythmic and uncoordinated was observed in Ano1 KO jejunum. In conclusion, Ano1 has a previously unidentified role in the regulation of coordinated gastrointestinal smooth muscle function through coordination of Ca(2+) transients in ICC-MY.
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http://dx.doi.org/10.1113/jphysiol.2014.277152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4198014PMC
September 2014

Loss-of-function of the voltage-gated sodium channel NaV1.5 (channelopathies) in patients with irritable bowel syndrome.

Gastroenterology 2014 Jun 5;146(7):1659-1668. Epub 2014 Mar 5.

Enteric Neuroscience Program, Division of Gastroenterology &Hepatology, Department of Physiology & Biomedical Engineering, Mayo Clinic, Rochester, Minnesota.

Background & Aims: SCN5A encodes the α-subunit of the voltage-gated sodium channel NaV1.5. Many patients with cardiac arrhythmias caused by mutations in SCN5A also have symptoms of irritable bowel syndrome (IBS). We investigated whether patients with IBS have SCN5A variants that affect the function of NaV1.5.

Methods: We performed genotype analysis of SCN5A in 584 persons with IBS and 1380 without IBS (controls). Mutant forms of SCN5A were expressed in human embryonic kidney-293 cells, and functions were assessed by voltage clamp analysis. A genome-wide association study was analyzed for an association signal for the SCN5A gene, and replicated in 1745 patients in 4 independent cohorts of IBS patients and controls.

Results: Missense mutations were found in SCN5A in 13 of 584 patients (2.2%, probands). Diarrhea-predominant IBS was the most prevalent form of IBS in the overall study population (25%). However, a greater percentage of individuals with SCN5A mutations had constipation-predominant IBS (31%) than diarrhea-predominant IBS (10%; P < .05). Electrophysiologic analysis showed that 10 of 13 detected mutations disrupted NaV1.5 function (9 loss-of-function and 1 gain-of-function function). The p. A997T-NaV1.5 had the greatest effect in reducing NaV1.5 function. Incubation of cells that expressed this variant with mexiletine restored their sodium current and administration of mexiletine to 1 carrier of this mutation (who had constipation-predominant IBS) normalized their bowel habits. In the genome-wide association study and 4 replicated studies, the SCN5A locus was strongly associated with IBS.

Conclusions: About 2% of patients with IBS carry mutations in SCN5A. Most of these are loss-of-function mutations that disrupt NaV1.5 channel function. These findings provide a new pathogenic mechanism for IBS and possible treatment options.
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http://dx.doi.org/10.1053/j.gastro.2014.02.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4096335PMC
June 2014

Non-canonical translation start sites in the TMEM16A chloride channel.

Biochim Biophys Acta 2014 Jan 28;1838(1 Pt B):89-97. Epub 2013 Aug 28.

U.O.C. Genetica Medica, Istituto Giannina Gaslini, Genova, Italy.

TMEM16A is a plasma membrane protein with voltage- and calcium-dependent chloride channel activity. The role of the various TMEM16A domains in expression and function is poorly known. In a previous study, we found that replacing the first ATG of the TMEM16A coding sequence with a nonsense codon (M1X mutation), to force translation from the second ATG localized at position 117, only had minor functional consequences. Therefore, we concluded that this region is dispensable for TMEM16A processing and channel activity. We have now removed the first 116 codons from the TMEM16A coding sequence. Surprisingly, the expression of the resulting mutant, Δ(1-116), resulted in complete loss of activity. We hypothesized that, in the mutant M1X, translation may start at a position before the second ATG, using a non-canonical start codon. Therefore, we placed an HA-epitope at position 89 in the M1X mutant. We found, by western blot analysis, that the HA-epitope can be detected, thus demonstrating that translation starts from an upstream non-ATG codon. We truncated the N-terminus of TMEM16A at different sites while keeping the HA-epitope. We found that stepwise shortening of TMEM16A caused an in parallel stepwise decrease in TMEM16A expression and function. Our results indicate that indeed the N-terminus of TMEM16A is important for its activity. The use of an alternative start codon appears to occur in a naturally-occurring TMEM16A isoform that is particularly expressed in human testis. Future experiments will need to address the role of normal and alternative amino-terminus in TMEM16A structure and function.
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http://dx.doi.org/10.1016/j.bbamem.2013.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898931PMC
January 2014

Inhibition of cell proliferation by a selective inhibitor of the Ca(2+)-activated Cl(-) channel, Ano1.

Biochem Biophys Res Commun 2012 Oct 17;427(2):248-53. Epub 2012 Sep 17.

Enteric NeuroScience Program, Mayo Clinic, Rochester, MN 55905, USA.

Background: Ion channels play important roles in regulation of cellular proliferation. Ano1 (TMEM16A) is a Ca(2+)-activated Cl(-) channel expressed in several tumors and cell types. In the muscle layers of the gastrointestinal tract Ano1 is selectively expressed in interstitial cells of Cajal (ICC) and appears to be required for normal gastrointestinal slow wave electrical activity. However, Ano1 is expressed in all classes of ICC, including those that do not generate slow waves suggesting that Ano1 may have other functions. Indeed, a role for Ano1 in regulating proliferation of tumors and ICC has been recently suggested. Recently, a high-throughput screen identified a small molecule, T16A(inh)-A01 as a specific inhibitor of Ano1.

Aim: To investigate the effect of the T16A(inh)-A01 inhibitor on proliferation in ICC and in the Ano1-expressing human pancreatic cancer cell line CFPAC-1.

Methods: Inhibition of Ano1 was demonstrated by whole cell voltage clamp recordings of currents in cells transfected with full-length human Ano1. The effect of T16A(inh)-A01 on ICC proliferation was examined in situ in organotypic cultures of intact mouse small intestinal smooth muscle strips and in primary cell cultures prepared from these tissues. ICC were identified by Kit immunoreactivity. Proliferating ICC and CFPAC-1 cells were identified by immunoreactivity for the nuclear antigen Ki67 or EdU incorporation, respectively.

Results: T16A(inh)-A01 inhibited Ca(2+)-activated Cl(-) currents by 60% at 10μM in a voltage-independent fashion. Proliferation of ICC was significantly reduced in primary cultures from BALB/c mice following treatment with T16A(inh)-A01. Proliferation of the CFPAC-1 human cell-line was also reduced by T16A(inh)-A01. In organotypic cultures of smooth muscle strips from mouse jejunum, the proliferation of ICC was reduced but the total number of proliferating cells/confocal stack was not affected, suggesting that the inhibitory effect was specific for ICC.

Conclusions: The selective Ano1 inhibitor T16A(inh)-A01 inhibited Ca(2+)-activated Cl(-) currents, reduced the number of proliferating ICC in culture and inhibited proliferation in the pancreatic cancer cell line CFPAC-1. These data support the notion that chloride channels in general and Ano1 in particular are involved in the regulation of proliferation.
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http://dx.doi.org/10.1016/j.bbrc.2012.09.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3479349PMC
October 2012

Hydrogen sulfide is a partially redox-independent activator of the human jejunum Na+ channel, Nav1.5.

Am J Physiol Gastrointest Liver Physiol 2011 Jun 10;300(6):G1105-14. Epub 2011 Mar 10.

Enteric Neuroscience Program, Division of Gastroenterology and Hepatology, Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55905, USA.

Hydrogen sulfide (H(2)S) is produced endogenously by L-cysteine metabolism. H(2)S modulates several ion channels with an unclear mechanism of action. A possible mechanism is through reduction-oxidation reactions attributable to the redox potential of the sulfur moiety. The aims of this study were to determine the effects of the H(2)S donor NaHS on Na(V)1.5, a voltage-dependent sodium channel expressed in the gastrointestinal tract in human jejunum smooth muscle cells and interstitial cells of Cajal, and to elucidate whether H(2)S acts on Na(V)1.5 by redox reactions. Whole cell Na(+) currents were recorded in freshly dissociated human jejunum circular myocytes and Na(V)1.5-transfected human embryonic kidney-293 cells. RT-PCR amplified mRNA for H(2)S enzymes cystathionine β-synthase and cystathionine γ-lyase from the human jejunum. NaHS increased native Na(+) peak currents and shifted the half-point (V(1/2)) of steady-state activation and inactivation by +21 ± 2 mV and +15 ± 3 mV, respectively. Similar effects were seen on the heterologously expressed Na(V)1.5 α subunit with EC(50)s in the 10(-4) to 10(-3) M range. The reducing agent dithiothreitol (DTT) mimicked in part the effects of NaHS by increasing peak current and positively shifting steady-state activation. DTT together with NaHS had an additive effect on steady-state activation but not on peak current, suggesting that the latter may be altered via reduction. Pretreatment with the Hg(2+)-conjugated oxidizer thimerosal or the alkylating agent N-ethylmaleimide inhibited or decreased NaHS induction of Na(V)1.5 peak current. These studies show that H(2)S activates the gastrointestinal Na(+) channel, and the mechanism of action of H(2)S is partially redox independent.
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http://dx.doi.org/10.1152/ajpgi.00556.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3119119PMC
June 2011

Altered expression of Ano1 variants in human diabetic gastroparesis.

J Biol Chem 2011 Apr 24;286(15):13393-403. Epub 2011 Feb 24.

Enteric Neuroscience Program, Division of Gastroenterology and Hepatology, Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.

Diabetes affects many organs including the stomach. Altered number and function of interstitial cells of Cajal (ICC), the gastrointestinal pacemaker cells, underlie a number of gastrointestinal motility disorders, including diabetic gastroparesis. In the muscle layers, ICC selectively express Ano1, thought to underlie classical Ca(2+)-activated Cl(-) currents. Mice homozygous for Ano1 knock-out exhibit abnormal ICC function and motility. Several transcripts for Ano1 are generated by alternative splicing of four exons. Here, we report expression levels of transcripts encoded by alternative splicing of Ano1 gene in gastric muscles of patients with diabetic gastroparesis and nondiabetic control tissues. Expression of mRNA from two alternatively transcribed exons are significantly different between patients and controls. Furthermore, patients with diabetic gastroparesis express mRNA for a previously unknown variant of Ano1. The 5' end of this novel variant lacks exons 1 and 2 and part of exon 3. Expression of this variant in HEK cells produces a decreased density of Ca(2+)-activated Cl(-) currents that exhibit slower kinetics compared with the full-length Ano1. These results identify important changes in expression and splicing of Ano1 in patients with diabetic gastroparesis that alter the electrophysiological properties of the channel. Changes in Ano1 expression in ICC may directly contribute to diabetic gastroparesis.
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http://dx.doi.org/10.1074/jbc.M110.196089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3075685PMC
April 2011

The alpha1H Ca2+ channel subunit is expressed in mouse jejunal interstitial cells of Cajal and myocytes.

J Cell Mol Med 2009 Nov-Dec;13(11-12):4422-31. Epub 2008 Dec 24.

Enteric Neuroscience Program, Mayo Clinic College of Medicine, Rochester, MN, USA.

T-type Ca(2+) currents have been detected in cells from the external muscular layers of gastrointestinal smooth muscles and appear to contribute to the generation of pacemaker potentials in interstitial cells of Cajal from those tissues. However, the Ca(2+) channel alpha subunit responsible for these currents has not been determined. We established that the alpha subunit of the alpha(1H) Ca(2+) channel is expressed in single myocytes and interstitial cells of Cajal using reverse transcription and polymerase chain reaction from whole tissue, laser capture microdissected tissue and single cells isolated from the mouse jejunum. Whole-cell voltage clamp recordings demonstrated that a nifedipine and Cd(2+) resistant, mibefradil-sensitive current is present in myocytes dissociated from the jejunum. Electrical recordings from the circular muscle layer demonstrated that mibefradil reduced the frequency and initial rate of rise of the electrical slow wave. Gene targeted knockout of both alleles of the cacna1h gene, which encodes the alpha(1H) Ca(2+) channel subunit, resulted in embryonic lethality because of death of the homozygous knockouts prior to E13.5 days in utero. We conclude that a channel with the pharmacological and molecular characteristics of the alpha(1H) Ca(2+) channel subunit is expressed in interstitial cells of Cajal and myocytes from the mouse jejunum, and that ionic conductances through the alpha(1H) Ca(2+) channel contribute to the upstroke of the pacemaker potential. Furthermore, the survival of mice that do not express the alpha(1H) Ca(2+) channel protein is dependent on the genetic background and targeting approach used to generate the knockout mice.
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http://dx.doi.org/10.1111/j.1582-4934.2008.00623.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855776PMC
July 2010

Production of the gaseous signal molecule hydrogen sulfide in mouse tissues.

J Neurochem 2008 Aug 29;106(4):1577-85. Epub 2008 May 29.

Enteric NeuroScience Program, and Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota, USA.

The gaseous molecule hydrogen sulfide (H(2)S) has been proposed as an endogenous signal molecule and neuromodulator in mammals. Using a newly developed method, we report here for the first time the ability of intact and living brain and colonic tissue in the mouse to generate and release H(2)S. This production occurs through the activity of two enzymes, cystathionine-gamma-lyase and cystathionine-beta-synthase. The quantitative expression of messenger RNA and protein localization for both enzymes are described in the liver, brain, and colon. Expression levels of the enzymes vary between tissues and are differentially distributed. The observation that, tissues that respond to exogenously applied H(2)S can endogenously generate the gas, strongly supports its role as an endogenous signal molecule.
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http://dx.doi.org/10.1111/j.1471-4159.2008.05502.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2836856PMC
August 2008

A mutation in telethonin alters Nav1.5 function.

J Biol Chem 2008 Jun 11;283(24):16537-44. Epub 2008 Apr 11.

Enteric Neuroscience Program, Mayo Clinic, Rochester, Minnesota 55905, USA.

Excitable cells express a variety of ion channels that allow rapid exchange of ions with the extracellular space. Opening of Na(+) channels in excitable cells results in influx of Na(+) and cellular depolarization. The function of Na(v)1.5, an Na(+) channel expressed in the heart, brain, and gastrointestinal tract, is altered by interacting proteins. The pore-forming alpha-subunit of this channel is encoded by SCN5A. Genetic perturbations in SCN5A cause type 3 long QT syndrome and type 1 Brugada syndrome, two distinct heritable arrhythmia syndromes. Mutations in SCN5A are also associated with increased prevalence of gastrointestinal symptoms, suggesting that the Na(+) channel plays a role in normal gastrointestinal physiology and that alterations in its function may cause disease. We collected blood from patients with intestinal pseudo-obstruction (a disease associated with abnormal motility in the gut) and screened for mutations in SCN5A and ion channel-interacting proteins. A 42-year-old male patient was found to have a mutation in the gene TCAP, encoding for the small protein telethonin. Telethonin was found to be expressed in the human gastrointestinal smooth muscle, co-localized with Na(v)1.5, and co-immunoprecipitated with sodium channels. Expression of mutated telethonin, when co-expressed with SCN5A in HEK 293 cells, altered steady state activation kinetics of SCN5A, resulting in a doubling of the window current. These results suggest a new role for telethonin, namely that telethonin is a sodium channel-interacting protein. Also, mutations in telethonin can alter Na(v)1.5 kinetics and may play a role in intestinal pseudo-obstruction.
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http://dx.doi.org/10.1074/jbc.M801744200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2423252PMC
June 2008

Evolving concepts in the cellular control of gastrointestinal motility: neurogastroenterology and enteric sciences.

Gastroenterol Clin North Am 2007 Sep;36(3):499-513, vii

Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA.

The enteric nervous system is an independent nervous system with a complexity comparable with the central nervous system. This complex system is integrated into several other complex systems, such as interstitial cells of Cajal networks and immune cells. The result of these interactions is effective coordination of motility, secretion, and blood flow in the gastrointestinal tract. Loss of subsets of enteric nerves, of interstitial cells of Cajal, malfunction of smooth muscle, and alteration in immune cells have been identified as the basis of many motility disorders. The initial factors triggering these changes and how to intervene to prevent, halt, and reverse them needs to be understood.
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http://dx.doi.org/10.1016/j.gtc.2007.07.003DOI Listing
September 2007

Isolation and characterization of lipid microdomains from apical and basolateral plasma membranes of rat hepatocytes.

Hepatology 2006 Feb;43(2):287-96

Center for Basic Research in Digestive Diseases, Mayo Medical School, Clinic and Foundation, Rochester, MN 55905, USA.

Canalicular bile is formed by the osmotic filtration of water in response to osmotic gradients generated by active transport at the apical and basolateral plasma membrane domains of hepatocytes. We recently demonstrated that mixed plasma membrane fractions isolated from rat hepatocyte couplets contain lipid microdomains ("rafts") enriched in cholesterol and sphingolipids and AQP8 and 9. We isolated lipid microdomains from hepatocyte apical and basolateral plasma membrane domains using Triton X-100 as detergent, and characterized their lipid and protein composition. A Triton-insoluble band ("raft fraction") at the 5%/30% sucrose interface in both apical and basolateral fractions was enriched for alkaline phosphatase (apical) and Na/K ATPase (basolateral) and was negative for amino peptidase-N. This detergent-insoluble band was also positive for caveolin-1 (a "raft" associated protein) and negative for clathrin (a "raft" negative protein). Lipid analysis showed that, the Triton-insoluble fraction was highly enriched in cholesterol and sphingolipids. Immunofluorescence staining on hepatocyte couplets for both caveolin-1 and cholera toxin B showed a punctate distribution on both the apical and basolateral plasma membranes, consistent with localized membrane microdomains. Dot blot analysis showed that the "raft" associated ganglioside GM1 was enriched in the detergent-insoluble fraction both domains. Furthermore, exposure of isolated hepatocytes to glucagon, a choleretic agonist, significantly increased the expression of AQP8 associated with the apical microdomain fractions but had no effect on AQP9 expression in the basolateral microdomain fractions. In conclusion, "rafts" represent target microdomains for exocytic insertion and retrieval of "flux proteins", including AQPs, involved in canalicular bile secretion.
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http://dx.doi.org/10.1002/hep.21039DOI Listing
February 2006

Expression and subcellular localization of the AQP8 and AQP1 water channels in the mouse gall-bladder epithelium.

Biol Cell 2005 Jun;97(6):415-23

Department of General and Environmental Physiology, University of Bari, via Amendola, 165/A-I-70126 Bari, Italy.

Background Information: Transepithelial transport of water is one of the most distinctive functions by which the gall-bladder rearranges its bile content. Water is reabsorbed from the gall-bladder lumen during fasting, whereas it is secreted into the lumen following meal ingestion. Nevertheless, the molecular mechanism by which water is transported across the gall-bladder epithelium remains mostly unclear.

Results: In the present study, we investigate the presence and subcellular localization of AQP (aquaporin) water channels in the mouse gall-bladder epithelium. Considerable AQP8 mRNA was detected in the gall-bladder epithelium of mouse, calf, rabbit, guinea pig and man. Studies of subcellular localization were then addressed to the mouse gall-bladder where the transcript of a second AQP, AQP1, was also detected. Immunoblotting experiments confirmed the presence of AQP8 and AQP1 at a protein level. Immunohistochemistry showed intense expression of AQP8 and AQP1 in the gall-bladder epithelial cells where AQP8 was localized in the apical membrane, whereas AQP1 was seen both in the apical and basolateral membranes, and in vesicles located in the subapical cytoplasm.

Conclusions: The pattern of subcellular distribution of AQP8 and AQP1 strongly corroborates the hypothesis of a transcellular route for the movement of water across the gall-bladder epithelium. Osmotic water would cross the apical membrane through AQP8 and AQP1, although AQP1 would be the facilitated pathway for the movement of water across the basolateral membrane. The presence of two distinct AQPs in the apical membrane is an unusual finding and may relate to the membrane's ability both to absorb and secrete fluid. It is tempting to hypothesize that AQP1 is hormonally translocated to the gall-bladder apical membrane to secrete water as in the bile duct epithelium, a functional homologue of the gall-bladder epithelium, whereas apical AQP8 may account for the absorption of water from gall-bladder bile.
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http://dx.doi.org/10.1042/BC20040137DOI Listing
June 2005

Water handling and aquaporins in bile formation: recent advances and research trends.

J Hepatol 2003 Nov;39(5):864-74

Section of Internal Medicine, Department of Internal Medicine and Public Medicine, Medical School, University of Bari, Bari, Italy.

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http://dx.doi.org/10.1016/s0168-8278(03)00294-0DOI Listing
November 2003

Ontogeny, distribution, and possible functional implications of an unusual aquaporin, AQP8, in mouse liver.

Hepatology 2003 Oct;38(4):947-57

Department of Zoology, Laboratory of Histology and Comparative Anatomy, University of Bari, via Amendola 165/A, I-70126 Bari, Italy.

Aquaporins are channel proteins widely expressed in nature and known to facilitate the rapid movement of water across numerous cell membranes. A mammalian aquaporin, AQP8, was recently discovered and found to have a very distinct evolutionary pathway. To understand the reason for this divergence, here we define the ontogeny and exact subcellular localization of AQP8 in mouse liver, a representative organ transporting large volumes of water for secretion of bile. Northern blotting showed strong AQP8 expression between fetal day 17 and birth as well as at weaning and thereafter. Interestingly, this pattern was confirmed by immunohistochemistry and coincided both temporally and spatially with that of hepatic glycogen accumulation. As seen by reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, fasting was accompanied by remarkable down-regulation of hepatic AQP8 that paralleled the expected depletion of glycogen content. The level of hepatic AQP8 returned to be considerable after refeeding. Immunoelectron microscopy confirmed AQP8 in hepatocytes where labeling was over smooth endoplasmic reticulum (SER) membranes adjacent to glycogen granules and in canalicular membranes, subapical vesicles, and some mitochondria. In conclusion, in addition to supporting a role for AQP8 in canalicular water secretion, these findings also suggest an intracellular involvement of AQP8 in preserving cytoplasmic osmolality during glycogen metabolism and in maintaining mitochondrial volume. AQP8 may have evolved separately to feature these intracellular roles as no other known aquaporin shows this specialization.
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http://dx.doi.org/10.1053/jhep.2003.50397DOI Listing
October 2003

Rat hepatocyte aquaporin-8 water channels are down-regulated in extrahepatic cholestasis.

Hepatology 2003 May;37(5):1026-33

Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Santa Fe, Argentina.

Hepatocytes express the water channel aquaporin-8 (AQP8), which is mainly localized in intracellular vesicles, and its adenosine 3',5'-cyclic monophosphate (cAMP)-induced translocation to the plasma membrane facilitates osmotic water movement during canalicular bile secretion. Thus, defective expression of AQP8 may be associated with secretory dysfunction of hepatocytes caused by extrahepatic cholestasis. We studied the effect of 1, 3, and 7 days of bile duct ligation (BDL) on protein expression, subcellular localization, and messenger RNA (mRNA) levels of AQP8; this was determined in rat livers by immunoblotting in subcellular membranes, light immunohistochemistry, immunogold electron microscopy, and Northern blotting. One day of BDL did not affect expression or subcellular localization of AQP8. Three days of BDL reduced the amount of intracellular AQP8 (75%; P <.001) without affecting its plasma membrane expression. Seven days after BDL, AQP8 was markedly decreased in intracellular (67%; P <.05) and plasma (56%; P <.05) membranes. Dibutyryl cAMP failed to increase AQP8 in plasma membranes from liver slices, suggesting a defective translocation of AQP8 in 7-day BDL rats. Immunohistochemistry and immunoelectron microscopy in liver sections confirmed the BDL-induced decreased expression of hepatocyte AQP8 in intracellular vesicles and canalicular membranes. AQP8 mRNA expression was unaffected by 1-day BDL but was significantly increased by about 200% in 3- and 7-day BDL rats, indicating a posttranscriptional mechanism for protein level reduction. In conclusion, BDL-induced extrahepatic cholestasis caused posttranscriptional down-regulation of hepatocyte AQP8 protein expression. Defective expression of AQP8 water channels may contribute to bile secretory dysfunction of cholestatic hepatocytes.
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http://dx.doi.org/10.1053/jhep.2003.50170DOI Listing
May 2003