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ABSTRACT: Die Mukoviszidose ist eine häufige, autosomal-rezessive Erbkrankheit mit deutlich reduzierter Lebenserwartung, die sich überwiegend
bereits im Kindesalter manifestiert. Der therapeutische Fortschritt hat dazu geführt, dass die Lebenserwartung dieser Patienten
in den letzten Jahren deutlich gestiegen ist: Mittlerweile ist etwa die Hälfte der Patienten im Erwachsenenalter. Daneben
gibt es atypische Verläufe, die sich erst im späten Erwachsenenalter manifestieren. Der zugrunde liegende genetische Defekt
betrifft den Cystic Fibrosis Transmembrane Conductance Regulator, einen Chloridionenkanal. Dieser Defekt führt über die Bildung
dehydrierter, abnorm visköser respiratorischer und gastrointestinaler Sekrete zu einer Multisystemerkrankung. Eine gestörte
mukoziliäre Clearance führt zur respiratorischen Besiedelung mit pathogenen Erregern (Pseudomonas aeruginosa) und einer chronischen pulmonalen Inflammation. Es kommt zu einer zunehmenden kombinierten Ventilationsstörung mit Ausbildung
von Bronchiektasen. Typische akute Komplikationen sind Infektexazerbationen, die auch die häufigste Todesursache bei Mukoviszidosepatienten
darstellen, daneben kommen die allergische bronchopulmonale Aspergillose, Hämoptysen und Pneumothoraces vor. Eine gastrointestinale
Beteiligung zeigt sich meist in einer exo- und im Verlauf auch endokrinen Pankreasinsuffizienz mit Diabetes mellitus, Malabsorption
und seltener einer biliären Zirrhose. Akute gastrointestinale Komplikationen sind Pankreatitis und Ileus. Der Artikel stellt
Epidemiologie und Pathophysiologie der Mukoviszidose dar und fokussiert auf Klinik, Diagnostik und die multimodale Therapie
beim erwachsenen Patienten.
Cystic fibrosis (CF) is a common autosomal-recessive inherited disease, which often results in premature death. Due to treatment
advances, life expectancy has however continuously improved in recent years. Currently about half of all patients are adults.
There are also “atypical” variants of CF with symptoms occurring in late adulthood. CF is caused by a mutation in the gene
coding for a chloride ion channel, known as the cystic fibrosis transmembrane conductance regulator (CFTR). This mutation
results in abnormally viscous mucosal secretions, leading to multi-organ disease with particular emphasis in the respiratory
and digestive tracts. Impaired mucociliary clearance results in bacterial colonization of the airways (e.g. Pseudomonas aeruginosa) and consequently in chronic pulmonary inflammation, inevitably leading to progressive bronchiectasis and combined ventilatory
disorders. Typical acute complications are infective exacerbations – the most frequent cause of death in cystic fibrosis –
along with allergic bronchopulmonary aspergillosis, haemoptyses and pneumothoraces. Involvement of the gastrointestinal tract
generally manifests as exo- and later endocrine pancreatic insufficiency with diabetes mellitus, malabsorption and sometimes
biliary liver cirrhosis. Typical acute complications are pancreatitis and ileus. The article describes epidemiology and pathophysiology
of CF and focuses on the signs and symptoms, as well as the diagnostic and multi-modal therapeutic strategies used in adult
patients.
SchlüsselwörterZystische Fibrose-Diabetes mellitus-Hämoptysen-Aspergillose-Pankreasinsuffizienz
KeywordsCystic fibrosis-Diabetes mellitus-Haemoptysis-Aspergillosis-Pancreatic insufficiency
Der Internist 04/2012; 51:277-288. · 0.30 Impact Factor
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M Donowitz,
S Singh,
P Singh,
M Chakraborty,
Y Chen,
R Murtazina,
M Gucek,
R N Cole,
N C Zachos,
F F Salahuddin,
O Kovbasnjuk,
N Broere,
W G Smalley-Freed,
A B Reynolds,
A L Hubbard, U Seidler,
E Weinman,
H R de Jonge,
B M Hogema,
X Li
[show abstract]
[hide abstract]
ABSTRACT: To identify additional potential functions for the multi-PDZ domain containing protein Na+/H+ exchanger regulatory factor 2 (NHERF2), which is present in the apical domain of intestinal epithelial cells, proteomic studies of mouse jejunal villus epithelial cell brush border membrane vesicles compared wild-type to homozygous NHERF2 knockout FVB mice by a two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-iTRAQ approach. Jejunal architecture appeared normal in NHERF2 null in terms of villus length and crypt depth, Paneth cell number, and microvillus structure by electron microscopy. There was also no change in proliferative activity based on BrdU labeling. Four brush border membrane vesicles (BBMV) preparations from wild-type mouse jejunum were compared with four preparations from NHERF2 knockout mice. LC-MS/MS identified 450 proteins in both matched wild-type and NHERF2 null BBMV; 13 proteins were changed in two or more separate BBMV preparations (9 increased and 4 decreased in NHERF2 null mice), while an additional 92 proteins were changed in a single BBMV preparation (68 increased and 24 decreased in NHERF2 null mice). These proteins were categorized as 1) transport proteins (one increased and two decreased in NHERF2 null); 2) signaling molecules (2 increased in NHERF2 null); 3) cytoskeleton/junctional proteins (4 upregulated and 1 downregulated in NHERF2 null); and 4) metabolic proteins/intrinsic BB proteins) (2 upregulated and 1 downregulated in NHERF2 null). Immunoblotting of BBMV was used to validate or extend the findings, demonstrating increase in BBMV of NHERF2 null of MCT1, coronin 3, and ezrin. The proteome of the NHERF2 null mouse small intestinal BB demonstrates up- and downregulation of multiple transport proteins, signaling molecules, cytoskeletal proteins, tight junctional and adherens junction proteins, and proteins involved in metabolism, suggesting involvement of NHERF2 in multiple apical regulatory processes and interactions with luminal contents.
Physiological Genomics 03/2011; 43(11):674-84. · 2.73 Impact Factor
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[show abstract]
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ABSTRACT: Electrolyte transporters located in the basolateral membrane of the colonic epithelium are increasingly appreciated as elaborately regulated components of specific transport functions and cellular homeostasis: During electrolyte absorption, Na(+) /K(+) ATPase, Cl⁻ conductance, Cl⁻/HCO₃⁻ exchange, K(+) /Cl⁻ cotransport and K(+) channels are candidates for basolateral Na(+) , Cl⁻ and K(+) extrusion. The process of colonic anion secretion involves basolateral Na(+) /K(+) /2Cl⁻ , and probably also Na(+) /HCO₃⁻ cotransport, as well as Na(+) /K(+) ATPase and K(+) channels to supply substrate, stabilize the membrane potential and generate driving force respectively. Together with a multitude of additional transport systems, Na(+) /H(+) exchange and Na(+) /HCO₃⁻ cotransport have been implicated in colonocyte pH(i) and volume homeostasis. The purpose of this article is to summarize recently gathered information on the molecular identity, function and regulation of the involved basolateral transport systems in native tissue. Furthermore, we discuss how these findings can help to integrate these systems into the transport function and the cellular homoeostasis of colonic epithelial cells. Finally, disturbances of basolateral electrolyte transport during disease states such as mucosal inflammation will be reviewed.
Acta Physiologica 01/2011; 201(1):33-46. · 3.09 Impact Factor
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M Donowitz,
S Singh,
P Singh,
F F Salahuddin,
Y Chen,
M Chakraborty,
R Murtazina,
M Gucek,
R N Cole,
N C Zachos,
O Kovbasnjuk,
N Broere,
W G Smalley-Freed,
A B Reynolds,
A L Hubbard, U Seidler,
E Weinman,
H R de Jonge,
B M Hogema,
X Li
[show abstract]
[hide abstract]
ABSTRACT: Na/H exchanger regulatory factor 1 (NHERF1) is a scaffold protein made up of two PDZ domains and an ERM binding domain. It is in the brush border of multiple epithelial cells where it modulates 1) Na absorption by regulating NHE3 complexes and cytoskeletal association, 2) Cl secretion through trafficking of CFTR, and 3) Na-coupled phosphate absorption through membrane retention of NaPi2a. To further understand the role of NHERF1 in regulation of small intestinal Na absorptive cell function, with emphasis on apical membrane transport regulation, quantitative proteomic analysis was performed on brush border membrane vesicles (BBMV) prepared from wild-type (WT) and homozygous NHERF1 knockout mouse jejunal villus Na absorptive cells. Jejunal architecture appeared normal in NHERF1 null; however, there was increased proliferative activity, as indicated by increased crypt BrdU staining. LC-MS/MS analysis using iTRAQ to compare WT and NHERF1 null BBMV identified 463 proteins present in both WT and NHERF1 null BBMV of simultaneously prepared and studied samples. Seventeen proteins had an altered amount of expression between WT and NHERF1 null in two or more separate preparations, and 149 total proteins were altered in at least one BBMV preparation. The classes of the majority of proteins altered included transport proteins, signaling and trafficking proteins, and proteins involved in proliferation and cell division. Affected proteins also included tight junction and adherens junction proteins, cytoskeletal proteins, as well as metabolic and BB digestive enzymes. Changes in abundance of several proteins were confirmed by immunoblotting [increased CEACAM1, decreased ezrin (p-ezrin), NHERF3, PLCβ3, E-cadherin, p120, β-catenin]. The changes in the jejunal BBMV proteome of NHERF1 null mice are consistent with a more complex role of NHERF1 than just forming signaling complexes and anchoring proteins to the apical membrane and include at least alterations in proteins involved in transport, signaling, and proliferation.
Physiological Genomics 11/2010; 42A(3):200-10. · 2.73 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Cystic fibrosis (CF) is a common autosomal-recessive inherited disease, which often results in premature death. Due to treatment advances, life expectancy has however continuously improved in recent years. Currently about half of all patients are adults. There are also "atypical" variants of CF with symptoms occurring in late adulthood. CF is caused by a mutation in the gene coding for a chloride ion channel, known as the cystic fibrosis transmembrane conductance regulator (CFTR). This mutation results in abnormally viscous mucosal secretions, leading to multi-organ disease with particular emphasis in the respiratory and digestive tracts. Impaired mucociliary clearance results in bacterial colonization of the airways (e. g. Pseudomonas aeruginosa) and consequently in chronic pulmonary inflammation, inevitably leading to progressive bronchiectasis and combined ventilatory disorders. Typical acute complications are infective exacerbations - the most frequent cause of death in cystic fibrosis - along with allergic bronchopulmonary aspergillosis, haemoptyses and pneumothoraces. Involvement of the gastrointestinal tract generally manifests as exo- and later endocrine pancreatic insufficiency with diabetes mellitus, malabsorption and sometimes biliary liver cirrhosis. Typical acute complications are pancreatitis and ileus. The article describes epidemiology and pathophysiology of CF and focuses on the signs and symptoms, as well as the diagnostic and multi-modal therapeutic strategies used in adult patients.
Der Internist 03/2010; 51 Suppl 1:277-88. · 0.30 Impact Factor
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N Broere,
M Chen,
A Cinar,
A K Singh,
J Hillesheim,
B Riederer,
M Lünnemann,
I Rottinghaus,
A Krabbenhöft,
R Engelhardt,
B Rausch,
E J Weinman,
M Donowitz,
A Hubbard,
O Kocher,
H R de Jonge,
B M Hogema, U Seidler
[show abstract]
[hide abstract]
ABSTRACT: We investigated the role of the Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as an attenuated in vitro Na(+) absorption in isolated jejunal and colonic, but not of ileal, mucosa. However, cAMP-mediated inhibition of both parameters remained intact. Acid-activated NHE3 transport rate was reduced in surface colonocytes, while its inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the BBM of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated BBM from the small and large intestines. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adaptor proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates, and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides.
Pflügers Archiv - European Journal of Physiology 09/2008; 457(5):1079-91. · 4.46 Impact Factor
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ABSTRACT: We investigated the role of the recently discovered, villous-expressed anion exchanger Slc26a6 (PAT1) and the predominantly crypt-expressed cystic fibrosis transmembrane regulator (CFTR) in basal and acid-stimulated murine duodenal HCO(3)(-) secretion in vivo, and the influence of blood HCO(3)(-) concentration on both.
The proximal duodenum of anaesthetized mice was perfused in situ, and HCO(3)(-) secretion was determined by back-titration. Duodenal mucosal permeability was assessed by determining (51)Cr-EDTA leakage from blood to lumen.
Compared with wild type (WT) littermates basal duodenal HCO(3)(-) secretory rates were slightly reduced in Slc26-deficient mice at low ( approximately 21 mm), and markedly reduced at high blood HCO(3)(-) concentration ( approximately 29 mm). In contrast, basal HCO(3)(-) secretion was markedly reduced in CFTR-deficient mice compared with WT littermates both at high and low blood HCO(3)(-) concentration. A short-term application of luminal acid increased duodenal HCO(3)(-) secretory rate in Slc26a6-deficient and WT mice to the same degree, but had no stimulatory effect in the absence of CFTR. Luminal acidification to pH 2.5 did not alter duodenal permeability.
The involvement of Slc26a6 in basal HCO(3)(-) secretion in murine duodenum in vivo is critically dependent on the systemic acid/base status, and this transporter is not involved in acid-stimulated HCO(3)(-) secretion. The presence of CFTR is essential for basal and acid-induced HCO(3)(-) secretion irrespective of acid/base status. This suggests a coupled action of Slc26a6 with CFTR for murine basal duodenal HCO(3)(-) secretion, but not acid-stimulated secretion, in vivo.
Acta Physiologica 05/2008; 193(4):357-65. · 3.09 Impact Factor
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ABSTRACT: In the gastrointestinal tract, CFTR, in conjunction with one or several members of the SLC26 anion exchanger family, mediates electrogenic Cl- and HCO3- secretion. Na+/H+ exchanger isoform NHE3, on the other hand, coupled to one or several of the SLC26 isoforms, mediates electroneutral NaCl absorption. The agonist-induced activation of anion secretion and inhibition of salt absorption causes secretory diarrhea. Current dogma sees the formation of a multiprotein complex of transport proteins, postsynaptic density-95/discs large/zonula occludens-1 (PDZ) adapter proteins, anchoring proteins, the cytoskeleton, and the involved protein kinases as one crucial step in the regulation of these transport processes. Data obtained in heterologous expression studies suggest an important role of these PDZ adapter proteins in trafficking, endocytic recycling, and membrane retention of the respective transmembrane proteins. This article reviews recent advances in our understanding of the role of the PDZ adapter proteins NHERF, E3KARP, PDZK1, IKEPP (NHERF-1 to NHERF-4), CAL, and Shank-2 that bind to CFTR, NHE3, and the intestinal SLC26 members in the regulation of intestinal fluid transport. Current concepts are mostly derived from heterologous expression studies and studies on their role in organ physiology are still in infancy. Recently, however, PDZ adapter protein-deficient mice and organ-specific cell lines have become available, and the first results suggest a more cell-type and possibly signal-specific role of these adapter proteins. This opens the potential for drug development targeted to PDZ domain interactions, which is, in theory, one of the most efficient antidiarrheal strategies.
AJP Gastrointestinal and Liver Physiology 12/2006; 291(5):G766-77. · 3.43 Impact Factor
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ABSTRACT: The Na(+)-HCO(3)(-) cotransporter (NBC) mediates HCO(3)(-) import into the colonocyte via its pNBC1 isoform. Whereas renal kNBC1 is inhibited by increased cAMP levels, pNBC1 is stimulated. Cholinergic stimulation activates renal NBC, but the effect on intestinal NBC is unknown. Therefore, crypts were isolated from the murine proximal colon by Ca(2+) chelation and loaded with the pH-sensitive dye 2',7'-bis-carboxyethyl-5,6-carboxyfluorescein. Na(+)-HCO(3)(-) cotransport activity was calculated from the dimethylamiloride-insensitive (500 microM) intracellular pH recovery from an acid load in the presence of CO(2)-HCO(3)(-) and the intracellular buffering capacity. Carbachol strongly increased Na(+)-HCO(3)(-) cotransport activity compared with control rates. Ca(2+) chelation with BAPTA-AM, blockade of the M(3) subtype of muscarinergic receptors with 4-diphenylacetoxy-N-methylpiperidine methiodide, and inhibition of Ca(2+)/calmodulin kinase II with KN-62 all caused significant inhibition of the carbachol-induced NBC activity increase. Furthermore, PKC inhibition with Gö-6976 and Gö-6850 significantly reduced the carbachol effect, which may be related to the unique NH(2)-terminal consensus site for PKC-dependent phosphorylation of pNBC1. We conclude that NBC in the murine colon is thus activated by carbachol, consistent with its presumed function as an anion uptake pathway during intestinal anion secretion, but that the signal transductions pathways are distinct from those involved in the cholinergic activation of renal NBC1.
AJP Gastrointestinal and Liver Physiology 11/2006; 291(4):G650-7. · 3.43 Impact Factor
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ABSTRACT: Cell migration is crucial for immune defence, wound healing or formation of tumour metastases. It has been shown that the activity of the Na(+)-H(+) exchanger (NHE1) plays an important role in cell migration. However, so far it is unknown whether Na(+)- HCO(3)(-) cotransport (NBC), which has similar functions in the regulation of intracellular pH (pH(i)) as NHE1, is also involved in cell migration. We therefore isolated NHE-deficient Madin-Darby canine kidney (MDCK-F) cells and tested whether NBC compensates for NHE in pH(i) and cell volume regulation as well as in migration. Intracellular pH was measured with the fluorescent pH indicator 2'7'-bis(carboxyethyl)-5-carboxyfluorescein (BCECF). The expression of NBC isoforms was determined with semiquantitative PCR. Migration was monitored with time-lapse video microscopy and quantified as the displacement of the cell centre. We found that MDCK-F cells express the isoform NBC1 (SLCA4A gene product) at a much higher level than the isoform kNBC3 (SLCA4A8 gene product). This difference is even more pronounced in NHE-deficient cells so that NBC1 is likely to be the major acid extruder in these cells and the major mediator of propionate-induced cell volume increase. NHE-deficient MDCK-F cells migrate more slowly than normal MDCK-F cells. NBC activity promotes migration during an acute intracellular acid load and increases migratory speed and displacement on a short timescale (< 30 min) whereas it has no effect on the long-term behaviour of migrating MDCK-F cells. Taken together, our results show that NBC actvity, despite many functional similarities, does not have the same importance for cell migration as NHE1 activity.
The Journal of Physiology 10/2005; 568(Pt 2):445-58. · 4.72 Impact Factor
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The Journal of Physiology 09/2004; 505(2):411 - 423. · 4.72 Impact Factor
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ABSTRACT: The Na(+)/H(+) exchanger isoform NHE2 is highly expressed in the intestinal tract, but its physiological role has remained obscure. The aim of this study was to define its expression, location, and regulatory properties in murine colon and to look for the compensatory changes in NHE2 (-/-) colon that allow normal histology and absorptive function. To this end, we measured murine proximal colonic surface and crypt cell NHE1, NHE2, and NHE3 expression levels, transport rates in response to acid, hyperosmolarity and cAMP in murine proximal colonic crypts, as well as changes in transcript levels and acid-activated NHE activity in NHE2 (-/-) crypts. We found that NHE2 was expressed most abundantly in crypts, NHE1 equally in crypts and surface cells, and NHE3 much stronger in surface cells. NHE2, like NHE1, was activated by low intracellular pH (pH(i)), hyperosmolarity, and cAMP, whereas NHE3 was activated only by low pH(i). Crypts isolated from NHE2 (-/-) mice displayed increased acid-activated NHE1- and NHE3-attributable Na(+)/H(+) exchange activity, no change in NHE1 expression, and NHE3 expression levels twice as high as in normal littermates. No change in cellular ultrastructure was found in NHE2 (-/-) colon. Our results demonstrate high NHE2 expression in the crypts and suggest a role for NHE2 in cryptal pH(i) and volume homeostasis.
AJP Gastrointestinal and Liver Physiology 08/2004; 287(1):G125-33. · 3.43 Impact Factor
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ABSTRACT: Reduced gastrointestinal HCO3- secretion contributes to malabsorption and obstructive syndromes in cystic fibrosis. The apical HCO3- transport pathways in these organs have not been defined. We therefore assessed the involvement of apical Cl-/HCO3- exchangers and anion conductances in basal and cAMP-stimulated duodenal HCO3- secretion. Muscle-stripped rat and rabbit proximal duodena were mounted in Ussing chambers, and electrical parameters, HCO3- secretion rates, and 36Cl-, 22Na+, and 3H+ mannitol fluxes were assessed. mRNA expression levels were measured by a quantitative PCR technique. Removal of Cl- from or addition of 1 mM DIDS to the luminal perfusate markedly decreased basal HCO3- secretion but did not influence the HCO3- secretory response to 8-bromo-cAMP, which was inhibited by luminal 5-nitro-2-(3-phenylpropylamino)-benzoate. Bidirectional 22Na+ and 36Cl- flux measurements demonstrated an inhibition rather than a stimulation of apical anion exchange during cAMP-stimulated HCO3- secretion. The ratio of Cl- to HCO3- in the anion secretory response was compatible with both Cl- and HCO3- being secreted via the CFTR anion channel. CFTR expression was very high in the duodenal mucosa of both species. We conclude that in rat and rabbit duodena, an apical Cl-/HCO3- exchanger mediates a significant part of basal HCO3- secretion but is not involved in the HCO3- secretory response to cAMP analogs. The inhibitor profile, the strong predominance of Cl- over HCO3- in the anion secretory response, and the high duodenal CFTR expression levels suggest that a major portion of cAMP-stimulated duodenal HCO3- secretion is directly mediated by CFTR.
AJP Gastrointestinal and Liver Physiology 12/2003; 285(5):G887-97. · 3.43 Impact Factor
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ABSTRACT: Defective regulation and/or reduced expression of the Na+-K+-2Cl- cotransporter NKCC1 may contribute to the severe secretory defect that is observed in cystic fibrosis, but data concerning the expression and function of NKCC1 in cystic fibrosis transmembrane conductance regulator (CFTR)-deficient cells are equivocal. We therefore investigated NKCC1 mRNA expression, Na+-K+-2Cl- cotransport activity and regulation by cAMP in crypts isolated from the proximal colon of CFTR-containing (CFTR (+/+)) and CFTR-deficient (CFTR (-/-)) mice. mRNA expression levels were determined by semiquantitative PCR, transport rates were measured fluorometrically in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetomethylester (BCECF)-loaded crypts, cytoplasmic volume changes were assessed by confocal microscopy, and [Cl-]i changes were examined by N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) quenching. NKCC1 mRNA expression levels were not significantly reduced in CFTR (-/-) crypts compared to controls. Azosemide-sensitive NH4+ influx (used as a measure of Na+-K+-2Cl- cotransport) was 2.23 +/- 0.72 vs. 1.56 +/- 0.16 mM min-1, and increased by 63.6 % in (+/+) and 87.3 % in (-/-) crypts upon stimulation for 5 min with forskolin. After 20 min of stimulation with forskolin, the Na+-K+-2Cl- cotransport rates in (-/-) and (+/+) crypts were identical. Crypt cross-sectional area and [Cl-]i decreased only in (+/+) crypts upon stimulation. In conclusion, normal NKCC1 expression levels, somewhat reduced Na+-K+-2Cl- cotransport rates, but preserved activation by cAMP were found in colonic crypts from CFTR (-/-) mice, ruling out a severe dysfunction of the Na+-K+-2Cl- cotransporter in the CF intestine. Furthermore, these studies establish the existence of a direct, cell-volume- and [Cl-]i-independent activation of colonic NKCC1 by an increase in intracellular cAMP.
The Journal of Physiology 07/2003; 549(Pt 2):525-36. · 4.72 Impact Factor
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ABSTRACT: Basolateral Na(+)-HCO(3)(-) cotransport is essential for intestinal anion secretion, and indirect evidence suggests that it may be stimulated by a rise of intracellular cAMP. We therefore investigated the expression, activity, and regulation by cAMP of the Na(+)-HCO(3)(-) cotransporter isoforms NBC1 and NBCn1 in isolated murine colonic crypts. Na(+)-HCO(3)(-) transport rates were measured fluorometrically in BCECF-loaded crypts, and mRNA expression levels and localization were determined by semiquantitative PCR and in situ hybridization. Acid-activated Na(+)-HCO(3)(-) cotransport rates were 5.07 +/- 0.7 mM/min and increased by 62% after forskolin stimulation. NBC1 mRNA was more abundant in colonic crypts than in surface cells, and crypts expressed far more NBC1 than NBCn1. To investigate whether the cAMP-induced Na(+)-HCO(3)(-) cotransport activation was secondary to secretion-associated changes in HCO(3)(-) or cell volume, we measured potential forskolin-induced changes in intracellular pH and assessed Na(+)-HCO(3)(-) transport activity in CFTR -/- crypts (in which no forskolin-induced cell shrinkage occurs). We found 30% reduced Na(+)-HCO(3)(-) transport rates in CFTR -/- compared with CFTR +/+ crypts but similar Na(+)-HCO(3)(-) cotransport activation by forskolin. These studies establish the existence of an intracellular HCO(3)(-) concentration- and cell volume-independent activation of colonic NBC by an increase in intracellular cAMP.
AJP Gastrointestinal and Liver Physiology 02/2003; 284(1):G37-45. · 3.43 Impact Factor
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Pflügers Archiv - European Journal of Physiology 01/2002; 443(Supplement 1):S283-S284. · 4.46 Impact Factor
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ABSTRACT: 1. The anion exchanger isoform 2 (AE2) gene encodes three subtypes (AE2a, b and c), which have different N-termini and tissue distributions. AE2 is expressed at high levels in the stomach, where it is thought to mediate basolateral base exit during acid production. The present study investigated if the three AE2 subtypes are differentially expressed and regulated in different cell types within the gastric mucosa. 2. The cloning strategy to obtain rabbit AE2a, b and c cDNAs combined genomic PCR and RT-PCR based on primers deduced from the rat sequences. Semiquantitative RT-PCR using homologous primers revealed much higher AE2 mRNA expression in rabbit parietal cells (PCs) than in mucous cells (MCs). The subtype expression pattern was AE2b > AE2c > or = AE2a in PCs and AE2a >AE2b > AE2c in MCs. Sequence analysis revealed the presence of a highly conserved protein kinase C (PKC) consensus sequence in the AE2a alternative N-terminus. 3. Maximal Cl(-)-HCO(3)(-) exchange rates, measured fluorometrically in BCECF-loaded cultured gastric cells, were much higher in PCs than MCs. PKC activation by phorbol ester stimulated maximal Cl(-)-HCO(3)(-) exchange rates in MCs but not in PCs, whereas forskolin had no effect in each cell type. 4. In summary, rabbit PCs and MCs, which originate from the same gastric stem cell population, display a completely different AE2 subtype expression pattern. Therefore, AE2 subtype expression is not organ specific but cell type specific. The different regulation of anion exchange in parietal and mucous cells suggests that AE2 subtypes may be differentially regulated.
The Journal of Physiology 09/2001; 534(Pt 3):837-48. · 4.72 Impact Factor
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ABSTRACT: Several Na(+)/H(+) exchanger (NHE) isoforms are expressed in the stomach, and NHE1 and NHE2 knockout mice display gastric mucosal atrophy. This study investigated the cellular distribution of the NHE isoforms NHE1, NHE2, NHE3, and NHE4 in rabbit gastric epithelial cells and their regulation by intracellular pH (pH(i)), hyperosmolarity, and an increase in cAMP. Semiquantitative RT-PCR and Northern blot experiments showed high NHE1 and NHE2 mRNA levels in mucous cells and high NHE4 mRNA levels in parietal and chief cells. Fluorescence optical measurements in cultured rabbit parietal and mucous cells using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and NHE isoform-specific inhibitors demonstrated that in both cell types, intracellular acidification activates NHE1 and NHE2, whereas hyperosmolarity activates NHE1 and NHE4. The relative contribution of the different isoforms to pH(i)- and hyperosmolarity-activated Na(+)/H(+) exchange in the different cell types paralleled their relative expression levels. cAMP elevation also stimulated NHE4, whereas an increase in osmolarity above a certain threshold further increased NHE1 and not NHE4 activity. We conclude that in rabbit gastric epithelium, NHE1 and NHE4 regulate cell volume and NHE1 and NHE2 regulate pH(i). The high NHE1 and NHE2 expression levels in mucous cells may reflect their special need for pH(i) regulation during high gastric acidity. NHE4 is likely involved in volume regulation during acid secretion.
AJP Gastrointestinal and Liver Physiology 09/2001; 281(2):G447-58. · 3.43 Impact Factor
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ABSTRACT: In a search for the HCO(3)(-) supply mechanisms to the enterocyte we cloned and sequenced an intestinal subtype of the Na(+)HCO(3)(-) cotransporter isoform I (dNBC1), which turned out to be identical to the pancreatic NBC1 subtype (pNBC1). Within the intestine, we found particularly high NBC1 expression levels in the duodenum and proximal colon. Experiments with stripped rabbit duodenum in Ussing-chambers revealed that Na(+)HCO(3)(-) cotransport (NBC) and CO(2) hydration/Na(+)/H(+) exchange were equally important duodenal HCO(3)(-) supply pathways and were both upregulated during cAMP-mediated secretion. In the proximal colon, however, HCO(3)(-) secretion was low but NBC1 expression even higher than in the duodenum. Ussing-chamber experiments with an NBC-specific inhibitor revealed that NBC, coupled to basolateral Cl(-)/HCO(3)(-) exchange, was an important alternative Cl(-) supply pathway to Na(+)K(+)2Cl(-) cotransport (NKCC) during cAMP-stimulated colonic Cl(-) secretion. To investigate the functional integrity of anion uptake pathways in the absence of cystic fibrosis transmembrane conductance regulator (CFTR), we fluorometrically assessed NBC and NKCC transport rates and cell volume before and during forskolin-stimulation in isolated colonic crypts from normal and CFTR (-/-) mice. Although forskolin stimulation decreased cell volume only in normal, not in CFTR (-/-) crypts, it activated NBC and NKCC to a similar degree in both normal and CFTR (-/-) crypts. We conclude that, depending on the intestinal segment, NBC1 plays an important role in basolateral HCO(3)(-) or Cl(-) uptake. Expression and activation by cAMP is preserved in CFTR (-/-) intestine.
JOP: Journal of the pancreas 08/2001; 2(4 Suppl):247-56.
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ABSTRACT: HCO(3)(-) supply to the enterocyte is rate limiting for duodenal HCO(3)(-) secretion (J(HCO3-)). This study defines the molecular nature of the major HCO(3)(-) uptake pathways in rabbit duodenocytes and investigates their physiologic significance and regulation during basal and stimulated J(HCO3-).
pH gradient-driven (22)Na(+) uptake into duodenal basolateral membrane vesicles was partly HCO(3)(-) dependent, stilbene sensitive, and therefore mediated by Na(+)HCO(3)(-) cotransport, and partly HCO(3)(-) independent, Hoechst 642 sensitive, and therefore mediated by the Na(+)/H(+) exchanger isoform NHE1. Semiquantitative polymerase chain reaction (PCR) revealed high duodenal expression levels for the NBC1 isoform of the Na(+)HCO(3)(-) cotransporter gene family and NHE1. Cloning and comparison of full-length rabbit with human gastrointestinal and kidney NBC1 subtype revealed a conserved protein kinase A consensus sequence in the cytoplasmic N-terminus of the gastrointestinal NBC1. Inhibition of either Na(+)HCO(3)(-) cotransport or carbonic anhydrase reduced ouabain-sensitive J(HCO3-) in in vitro rabbit duodenal mucosae by approximately 50%, but did not affect 8-Br-cAMP-induced DeltaJ(HCO3-), suggesting cAMP-mediated up-regulation of the alternative pathway. However, inhibition of both Na(+)HCO(3)(-) cotransport and either carbonic anhydrase or NHE1 strongly reduced DeltaJ(HCO3-).
NBC1 and NHE1 are the major base importers in rabbit duodenocytes. Na(+)HCO(3)(-) cotransport and CO(2) hydration/Na(+)/H(+) exchange are equally important pathways for duodenal HCO(3)(-) supply and are up-regulated during cAMP-mediated stimulation.
Gastroenterology 08/2000; 119(2):406-19. · 11.68 Impact Factor
