Transepithelial Transport Processes Research Articles

Expanding Genotype-Phenotype Correlation of CLCNKA and CLCNKB Variants Linked to Hearing Loss.

The ClC-K channels CLCNKA and CLCNKB are crucial for the transepithelial transport processes required for sufficient urinary concentrations and sensory mechanoelectrical transduction in the cochlea. Loss-of-function alleles in these channels are associated with various clinical phenotypes, ranging from hypokalemic alkalosis to sensorineural hearing loss (SNHL) accompanied by severe renal conditions, i.e., Bartter's syndrome. Using a stepwise genetic approach encompassing whole-genome sequencing (WGS), we identified one family with compound heterozygous variants in the ClC-K channels, specifically a truncating variant in CLCNKA in trans with a contiguous deletion of CLCNKA and CLCNKB. Breakpoint PCR and Sanger sequencing elucidated the breakpoint junctions derived from WGS, and allele-specific droplet digital PCR confirmed one copy loss of the CLCNKA_CLCNKB contiguous deletion. The proband that harbors the CLCNKA_CLCNKB variants is characterized by SNHL without hypokalemic alkalosis and renal anomalies, suggesting a distinct phenotype in the ClC-K channels in whom SNHL predominantly occurs. These results expanded genotypes and phenotypes associated with ClC-K channels, including the disease entities associated with non-syndromic hearing loss. Repeated identification of deletions across various extents of CLCNKA_CLCNKB suggests a mutational hotspot allele, highlighting the need for an in-depth analysis of the CLCNKA_CLCNKB intergenic region, especially in undiagnosed SNHL patients with a single hit in CLCNKA.

Evaluation of Blood-CSF Barrier Transport by Quantitative Real Time Fluorescence Microscopy.

Transporters at the blood-cerebrospinal fluid (CSF) barrier (BCSFB) play active roles in removing drugs and toxins from the CSF. The goal of this study is to develop a fluorescence microscopy approach to quantitatively study the transepithelial transport processes at the murine BCSFB in real time. Choroid plexus (CP) tissues were isolated from mouse lateral ventricles and incubated with anionic (fluorescein-methotrexate, 8-fluorescein-cAMP) or cationic (IDT307) fluorescent probes. The CSF-to-blood transport was imaged and quantified using compartmental segmentation and digital image analysis. Real time images were captured and analyzed to obtain kinetic information and identify the rate-limiting step. The effect of transporter inhibitors was also evaluated. The transport processes of fluorescent probes can be captured and analyzed digitally. The intra- and inter- animal variability were 20.4% and 25.7%, respectively. Real time analysis showed distinct transport kinetics and rate-limiting step for anionic and cationic probes. A CP efflux index was proposed to distinguish between transepithelial flux and intracellular accumulation. Rifampin and MK571 decreased the overall transepithelial transport of anionic probes by more than 90%, indicating a possible involvement of organic anion transporting polypeptides (Oatps) and multidrug resistance-associated proteins (Mrps). A CP isolation method was described, and a quantitative fluorescence imaging approach was developed to evaluate CSF-to-blood transport in mouse CP. The method is consistent, reproducible, and capable of tracking real time transepithelial transport with temporal and spatial resolution. The approach can be used to evaluate transport mechanisms, assess tissue drug accumulation, and assay potential drug-drug interactions at the BCSFB.

Impact of Campylobacter spp. on the Integrity of the Porcine Gut.

Simple SummaryCampylobacteriosis is recognised as a leading food-borne zoonotic enteric disease of humans, mainly caused by Campylobacter jejuni, and to a minor extent by C. coli. In general, pigs are symptomless carriers primarily of C. coli, but may also harbour C. jejuni. In a swine infection model, weaned piglets were intragastrically inoculated with C. coli (ST-5777; n = 8), with C. jejuni (ST-122; n = 10), or with both strains (n = 8) and 11 piglets served as control. The health status was monitored and the influence on the intestinal barrier was investigated using the Ussing chamber technique and histological examinations. After inoculation, no clinical manifestations were noted. No gross lesions were observed during dissection four weeks post inoculation, and no pathohistological changes were detected in the intestinal mucosal sections. On the other hand, in the caecum of C. jejuni mono-inoculated pigs, we recognized an impact on transepithelial transport processes. We observed an increased Cl− secretion by using the Ussing chamber technique.Campylobacter (C.) is the most common food-borne zoonosis in humans, which mainly manifests with watery to bloody diarrhoea. While C. jejuni is responsible for most cases of infection, C. coli is less frequently encountered. The object of the study was to prove the clinical impact of mono- and co-colonisation of C. coli and C. jejuni on weaned piglets in an infection model and to investigate the impact on transepithelial transport processes in the jejunum and caecum. At an age of eight weeks, eight pigs were infected with C. coli (ST-5777), 10 pigs with C. jejuni (ST-122), eight pigs with both strains, and 11 piglets served as control. During the four-week observation period, no clinical signs were observed. During dissection, both strains could be isolated from the jejunum and the caecum, but no alteration of the tissue could be determined histopathologically. Mono-infection with C. jejuni showed an impact on transepithelial ion transport processes of the caecum. An increase in the short circuit current (Isc) was observed in the Ussing chamber resulting from carbachol- and forskolin-mediated Cl− secretion. Therefore, we speculate that caecal colonisation of C. jejuni might affect the transport mechanisms of the intestinal mucosa without detectable inflammatory reaction.

The bitter taste receptor agonist denatonium influences mouse tracheal epithelial ion transport

In the airway epithelium, mucociliary clearance is strictly regulated by ciliary beat and transepithelial ion transport to provide homeostasis. During recent years it was shown that ciliated cells and specialized chemosensory cells in the airway epithelium express bitter taste receptors (Tas2R). We have previously shown that the Tas2R agonist denatonium stimulates particle transport speed in murine trachea via acetylcholine (ACh) release from brush cells. However, it remains to be elucidated if Tas2R stimulation also influences transepithelial ion transport processes. To address this question, we here performed measurements of the transepithelial ion current (ISC) of murine tracheas in Ussing chamber experiments. Apical application of denatonium changed ISC in a dose dependent manner (EC50 132.6 µM). Application of the Gai subunit inhibitor pertussis toxin (500 ng/ml) significantly reduced the denatonium-effect (1 mM), while the Gbg subunit inhibitor gallein (50 µM) had no effect. In the presence of TPPO (100 µM) an inhibitor of the transient receptor potential melastatin-5 (TRPM5) ion channel, a member of the canonical bitter signalling cascade, and in TRPM5 knock-out mice, the denatonium-effect was significantly reduced. Inhibition of an increase of intracellular Ca2+ with the phospholipase C inhibitor U-73122 (10 µM) did not change the denatonium-effect. Furthermore, the denatonium-effect was not dependent on cholinergic signalling, as blocking ACh receptors with mecamylamine (100 µM) and atropine (50 µM) had no influence. Interestingly application of the pannexin1 cannel inhibitor probenecid (1 mM) also reduced the denatonium-effect, pointing towards a release of ATP via pannexin channels. A decrease in intracellular ATP-levels leads to an activation of KATP potassium channels as shown with the inhibitor glibenclamide (100 µM). Additionally, blocking ATP-receptors with suramin (100 µM) significantly reduced the denatonium-effect, indicating that the released ATP acts in a paracrine way on the airway epithelial cells. Taken together we showed that activation of Tas2R in brush cells might represent important regulators of ion transport activity via ATP-dependent signalling. Selectively targeting Tas2R in the trachea might represent a useful target for stimulation of the mucociliary clearance.

SARS‐CoV‐2 May Hijack GPCR Signaling Pathways to Compromise Lung Ion and Fluid Transport

The tropism of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), a virus responsible for the ongoing coronavirus disease 2019 (COVID‐19) pandemic, towards the host cells is determined, at least in part, by the expression and distribution of its cell surface receptor, angiotensin‐converting enzyme 2 (ACE2). The virus further exploits the host cellular machinery to gain access into the cells; its spike protein is cleaved by a host cell surface transmembrane serine protease 2 (TMPRSS2) shortly after binding ACE2 followed by its proteolytic activation at a furin cleavage site. The virus primarily targets the epithelium of the respiratory tract which is covered by a tightly regulated airway surface liquid (ASL) layer that serves as a primary defense mechanism against respiratory pathogens. The volume and viscosity of this fluid layer is regulated and maintained by a coordinated function of different transport pathways in the respiratory epithelium. We argue that SARS‐CoV‐2 may potentially alter evolutionary conserved second‐messenger signaling cascades via activation of G‐protein coupled receptors (GPCRs) or by directly modulating G protein signaling. Such signaling may in turn adversely modulate transepithelial transport processes, especially those involving cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na+ channel (ENaC), thereby shifting the delicate balance between anion secretion and sodium absorption that controls homeostasis of this fluid layer. As a result, activation of the secretory pathways including CFTR‐mediated Cl‐ transport may overwhelm the absorptive pathways such as ENaC‐dependent Na+ uptake and initiate a pathophysiological cascade leading to lung edema, one of the most serious and potentially deadly clinical manifestations of COVID‐19.

SARS-CoV-2 may hijack GPCR signaling pathways to dysregulate lung ion and fluid transport.

The tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a virus responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic, toward the host cells is determined, at least in part, by the expression and distribution of its cell surface receptor, angiotensin-converting enzyme 2 (ACE2). The virus further exploits the host cellular machinery to gain access into the cells; its spike protein is cleaved by a host cell surface transmembrane serine protease 2 (TMPRSS2) shortly after binding ACE2, followed by its proteolytic activation at a furin cleavage site. The virus primarily targets the epithelium of the respiratory tract, which is covered by a tightly regulated airway surface liquid (ASL) layer that serves as a primary defense mechanism against respiratory pathogens. The volume and viscosity of this fluid layer is regulated and maintained by a coordinated function of different transport pathways in the respiratory epithelium. We argue that SARS-CoV-2 may potentially alter evolutionary conserved second-messenger signaling cascades via activation of G protein-coupled receptors (GPCRs) or by directly modulating G protein signaling. Such signaling may in turn adversely modulate transepithelial transport processes, especially those involving cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na+ channel (ENaC), thereby shifting the delicate balance between anion secretion and sodium absorption, which controls homeostasis of this fluid layer. As a result, activation of the secretory pathways including CFTR-mediated Cl− transport may overwhelm the absorptive pathways, such as ENaC-dependent Na+ uptake, and initiate a pathophysiological cascade leading to lung edema, one of the most serious and potentially deadly clinical manifestations of COVID-19.

The gasotransmitter hydrogen sulfide inhibits transepithelial anion secretion of pregnant mouse endometrial epithelium

Endometrial epithelium exhibits a robust ion transport activity required for dynamical regulation of uterine fluid environment and thus embryo implantation. However, there still lacks a thorough understanding of the ion transport processes and regulatory mechanism in peri-implantation endometrial epithelium. As a gaseous signaling molecule or gasotransmitter, hydrogen sulfide (H2S) regulates a myriad of cellular and physiological processes in various tissues, including the modulation of ion transport proteins in epithelium. This study aimed to investigate the effects of H2S on ion transport across mouse endometrial epithelium and its possible role in embryo implantation. The existence of endogenous H2S in pregnant mouse uterus was tested by the detection of two key H2S-generating enzymes and measurement of H2S production rate in tissue homogenates. Transepithelial ion transport processes were electrophysiologically assessed in Ussing chambers on early pregnant mouse endometrial epithelial layers, demonstrating that H2S suppressed the anion secretion by blocking cystic fibrosis transmembrane conductance regulator (CFTR). H2S increased intracellular Cl− concentration ([Cl−]i) in mouse endometrial epithelial cells, which was abolished by pretreatment with the CFTR selective inhibitor CFTRinh-172. The cAMP level in mouse endometrial epithelial cells was not affected by H2S, indicating that H2S blocked CFTR in a cAMP-independent way. In vivo study showed that interference with H2S synthesis impaired embryo implantation. In conclusion, our study demonstrated that H2S inhibits the transepithelial anion secretion of early pregnant mouse endometrial epithelium via blockade of CFTR, contributing to the preparation for embryo implantation.

Efficient gene delivery to primary alveolar epithelial cells by nucleofection

Primary alveolar epithelial cells play a pivotal role in lung research, particularly when focusing on gas exchange, barrier function, and transepithelial transport processes. However, efficient transfection of primary alveolar epithelial cells continues to be a major challenge. In the present study, we applied nucleofection, a novel method of gene and oligonucleotide delivery to the nucleus of cells by electroporation, to achieve highly efficient transfection of primary alveolar epithelial type II (ATII) cells. To quantify the amount of ATII cells effectively transfected, we applied a plasmid expressing GFP and assessed the amount of GFP-expressing cells by flow cytometry. Analysis of the nucleofected ATII cells revealed a concentration-dependent transfection efficiency of up to 50% when using 3-8 μg plasmid DNA without affecting cell viability. Nucleofection of cultured A549 and H441 cells yielded similar transfection rates. Importantly, nucleofection of ATII cells did not interfere with the integrity of ATII monolayers even with use of relatively high concentrations of plasmid DNA. In subsequent studies, we also efficiently delivered small interfering RNAs to ATII cells by nucleofection, thereby silencing Akt and the multiligand receptor megalin, which has been recently shown to play a key role in removal of excess protein from the alveolar space, and effectively inhibited megalin-driven uptake and transcellular transport of albumin in ATII cells. Thus we report successful transfection of primary rat alveolar epithelial cells with both plasmids and oligonucleotides via nucleofection with high viability and consistently good transfection rates without impairing key physiological properties of the cells.

P02 The impact of hydrogen sulfide on ion channels and transporters in airway epithelial cells

Hydrogen sulfide (H2S) is emerging as an important regulator of numerous ion channels, particularly potassium channels. A growing body of evidence demonstrates the importance of potassium channel regulation by H2S for physiological processes such as smooth muscle relaxation. In the airways, H2S-induced smooth muscle relaxation leads to airway dilatation [1] . Aside from smooth muscle cells, epithelial cells are important for airway physiology. The ciliated airway epithelium is covered by a thin fluid layer (airway surface liquid, ASL) which allows effective ciliary beat and thereby mucociliary clearance. The volume and composition of the ASL is regulated by the vectorial transport of ions and, consequently, water across the epithelium. We questioned if H2S can be produced by airway epithelial cells and influences ion channels and transepithelial ion transport processes by those cells. RT-PCR and western blot experiments demonstrated the expression of H2S generating enzymes (cystathionine-γ-lyase and cystathionine-β-synthase) in human H441 airway epithelial cells. Furthermore, the measurement of H2S production by H441 lysates with the methylene blue method showed H2S generation which was inhibited by D,L-propargylglycine and aminooxyacetic acid. These data indicate that H441 airway epithelial cells have the capacity of H2S production. Transepithelial ion transport was electrophysiologically measured by Ussing chamber recordings on cultured human H441 airway epithelial cells at the air/liquid interface as well as tracheal preparations form pigs. In both models, exogenously applied H2S (by NaHS) decreased sodium absorption by ∼60% [2] . In H441 cells, this effect was dose-dependent (IC50 146 μM of NaHS) and fully reversible. There was no effect of H2S on amiloride-sensitive sodium channels which are located in the apical membrane of the epithelial cells. By contrast, there was a reduction in the activity of the basolaterally located Na+/K+-ATPase. The membrane abundance of the Na+/K+-ATPase, as determined by surface biotinylation and western blot, or cellular ATP concentrations were not affected by H2S. Furthermore, H2S also inhibited potassium channels in the basolateral membrane, the inhibition of which decreased Na+/K+-ATPase activity and overall sodium absorption by H441 cells. In sum, these data indicate that airway epithelial cells have the capacity of H2S production. Exogenously applied H2S decreases sodium absorption across airway epithelial cells by inhibiting the Na+/K+-ATPase as well as basolateral potassium channels. We conclude that H2S might represent a signaling molecule which affects ion transport processes across airway epithelial cells. However, the physiological regulation of ion transport by endogenously produced H2S remains to be determined. Supported by grants from the German Research Foundation AL1453/1–1 and AL1453/1–2.

Chronic Alcohol Ingestion Changes the Landscape of the Alveolar Epithelium

Similar to effects of alcohol on the heart, liver, and brain, the effects of ethanol (EtOH) on lung injury are preventable. Unlike other vital organ systems, however, the lethal effects of alcohol on the lung are underappreciated, perhaps because there are no signs of overt pulmonary disorder until a secondary insult, such as a bacterial infection or injury, occurs in the lung. This paper provides overview of the complex changes in the alveolar environment known to occur following both chronic and acute alcohol exposures. Contemporary animal and cell culture models for alcohol-induced lung dysfunction are discussed, with emphasis on the effect of alcohol on transepithelial transport processes, namely, epithelial sodium channel activity (ENaC). The cascading effect of tissue and phagocytic Nadph oxidase (Nox) may be triggered by ethanol exposure, and as such, alcohol ingestion and exposure lead to a prooxidative environment; thus impacting alveolar macrophage (AM) function and oxidative stress. A better understanding of how alcohol changes the landscape of the alveolar epithelium can lead to improvements in treating acute respiratory distress syndrome (ARDS) for which hospitalized alcoholics are at an increased risk.

Nicotine-induced activation of soluble adenylyl cyclase participates in ion transport regulation in mouse tracheal epithelium

AimsFunctional nicotinic acetylcholine receptors (nAChR) have been identified in airway epithelia and their location in the apical and basolateral membrane makes them targets for acetylcholine released from neuronal and non-neuronal sources. One function of nAChR in airway epithelia is their involvement in the regulation of transepithelial ion transport by activation of chloride and potassium channels. However, the mechanisms underlying this nicotine-induced activation of ion transport are not fully elucidated. Thus, the aim of this study was to investigate the involvement of adenylyl cyclases in the nicotine-induced ion current in mouse tracheal epithelium. Main methodsTo evaluate the nicotine-mediated changes of transepithelial ion transport processes electrophysiological Ussing chamber measurements were applied and nicotine-induced ion currents were recorded in the absence and presence of adenylyl cyclase inhibitors. Key findingsThe ion current changes induced by nicotine (100μM, apical) were not altered in the presence of high doses of atropine (25μM, apical and basolateral), underlining the involvement of nAChR. Experiments with the transmembrane adenylyl cyclase inhibitor 2'5'-dideoxyadenosine (50μM, apical and basolateral) and the soluble adenylyl cyclase inhibitor KH7 (10μM, apical and basolateral) both reduced the nicotine-mediated ion current to a similar extent. Yet, a statistically significant reduction was obtained only in the experiments with KH7. SignificanceThis study indicates that nicotine binding to nAChR in mouse tracheal epithelium activates transepithelial ion transport involving adenylyl cyclase activity. This might be important for novel therapeutic strategies targeting epithelial ion transport mediated by the non-neuronal cholinergic system.

Protein kinase A dependent and independent activation of the V-ATPase in Malpighian tubules ofAedes aegypti.

Transepithelial ion transport in insect Malpighian tubules is energized by an apical V-ATPase. In hematophagous insects, a blood meal during which the animal ingests huge amounts of salt and water stimulates transepithelial transport processes linked to V-ATPase activation, but how this is accomplished is still unclear. Here we report that membrane-permeant derivatives of cAMP increase the bafilomycin-sensitive ATPase activity in Malpighian tubules of Aedes aegypti twofold and activate ATP-dependent transport processes. In parallel, membrane association of the V(1) subunits C and D increases, consistent with the assembly of the holoenzyme. The protein kinase A inhibitor H-89 abolishes all cAMP-induced effects, consistent with protein kinase A (PKA) being involved in V-ATPase activation. Metabolic inhibition induced by KCN, azide and 2,4-dinitrophenol, respectively, also induces assembly of functional V-ATPases at the membrane without PKA involvement, indicating a phosphorylation-independent activation mechanism.

Extracellular pH in Restricted Domains as a Gating Signal for Ion Channels Involved in Transepithelial Transport

The importance of intracellular pH (pH(i)) in the regulation of diverse cellular activities ranging from cell proliferation and differentiation to cell cycle, migration and apoptosis has long been recognised. More recently, extracellular pH (pH₀), in particular that of relatively inaccessible compartments or domains that occur between cells in tissues, has begun to be acknowledged as a relevant signal in cell regulation. This should not be surprising given the abundant reports highlighting the pH₀-dependence of the activity of membrane proteins facing the extracellular space such as receptors, transporters, ion channels and enzymes. Changes in pH affect the ionisation state of proteins through the effect on their titratable groups. There are proteins, however, which respond to pH shifts with conformational changes that are crucial for catalysis or transport activity. In such cases protons act as signalling molecules capable of eliciting fast and localised responses. We provide examples of ion channels that appear fastidiously designed to respond to extracellular pH in a manner that suggests specific functions in transporting epithelia. We shall also present ideas as to how these channels participate in complex transepithelial transport processes and provide preliminary experiments illustrating a new way to gauge pH₀ in confined spaces of native epithelial tissue.

Basolateral Cl−uptake mechanisms in Xenopus laevis lung epithelium

A thin liquid layer covers the lungs of air-breathing vertebrates. Active ion transport processes via the pulmonary epithelial cells regulate the maintenance of this layer. This study focuses on basolateral Cl(-) uptake mechanisms in native lungs of Xenopus laevis and the involvement of the Na(+)/K(+)/2 Cl(-) cotransporter (NKCC) and HCO(3)(-)/Cl(-) anion exchanger (AE), in particular. Western blot analysis and immunofluorescence staining revealed the expression of the NKCC protein in the Xenopus lung. Ussing chamber experiments demonstrated that the NKCC inhibitors (bumetanide and furosemide) were ineffective at blocking the cotransporter under basal conditions, as well as under pharmacologically stimulated Cl(-)-secreting conditions (forskolin and chlorzoxazone application). However, functional evidence for the NKCC was detected by generating a transepithelial Cl(-) gradient. Further, we were interested in the involvement of the HCO(3)(-)/Cl(-) anion exchanger to transepithelial ion transport processes. Basolateral application of DIDS, an inhibitor of the AE, resulted in a significantly decreased the short-circuit current (I(SC)). The effect of DIDS was diminished by acetazolamide and reduced by increased external HCO(3)(-) concentrations. Cl(-) secretion induced by forskolin was decreased by DIDS, but this effect was abolished in the presence of HCO(3)(-). These experiments indicate that the AE at least partially contributes to Cl(-) secretion. Taken together, our data show that in Xenopus lung epithelia, the AE, rather than the NKCC, is involved in basolateral Cl(-) uptake, which contrasts with the common model for Cl(-) secretion in pulmonary epithelia.

Modular structure of sodium-coupled bicarbonate transporters

Mammalian genomes contain 10 SLC4 genes that, between them, encode three Cl-HCO(3) exchangers, five Na(+)-coupled HCO(3) transporters (NCBTs), one reported borate transporter, and what is reported to be a fourth Cl-HCO(3) exchanger. The NCBTs are expressed throughout the body and play important roles in maintaining intracellular and whole-body pH, as well as contributing to transepithelial transport processes. The importance of NCBTs is underscored by the genetic association of dysfunctional NCBT genes with blindness, deafness, epilepsy, hypertension and metal retardation. Key to understanding the action and regulation of NCBTs is an appreciation of the diversity of NCBT gene products. The transmembrane domains of human NCBT paralogs are 50-84% identical to each other at the amino acid level, and are capable of a diverse range of actions, including electrogenic Na/HCO(3) cotransport (i.e. NBCe1 and NBCe2) and electroneutral Na/HCO(3) cotransport (i.e. NBCn1 and NBCn2), as well as Na(+)-dependent Cl-HCO(3) exchange (i.e. NDCBE). Furthermore, by the use of alternative promoters and alternative-splicing events, individual SLC4 genes have the potential to generate multiple splice variants (as many as 16 in the case of NBCn1), each of which could have unique temporal and spatial patterns of distribution, unitary transporter activity (i.e. flux mediated by one molecule), array of protein-binding partners, and complement of regulatory stimuli. In the first section of this review, we summarize our present knowledge of the function and distribution of mammalian NCBTs and their multiple variants. In the second section of this review we consider the molecular consequences of NCBT variation.

High‐Resolution Analysis of Barrier Function

High-resolution analysis of epithelial barrier function adds substantial information to that provided by conventional transepithelial electrical resistance (TER) measurements. This chapter describes three high-resolution techniques. First, two variants of impedance spectroscopy are delineated. One-path impedance spectroscopy discriminates vertically between serial pathways, namely resistances of the epithelial cell layer and of subepithelial tissues. As a typical application, measurements on human sigmoid colon biopsies from patients suffering from Crohn's disease are reported. Two-path impedance spectroscopy allows to discriminate between trans- and paracellular resistance, and the general principle of this technique is outlined. Second, the conductance scanning technique is presented, which discriminates horizontally between optically distinct parallel pathways over a wide range of spatial resolutions. Using this technique, it was shown that occludin--in contrast to the then prevailing opinion--is not irreplaceable to barrier function. Third, three-dimensional confocal fluorescence imaging for depicting transepithelial transport processes is introduced. Using this method the transepithelial translocation of bacteria which generate focal leaks was discovered.

The Ussing Chamber and Measurement of Drug Actions on Mucosal Ion Transport

Ussing chamber technology can be used to investigate the pharmacodynamic and pharmacokinetic properties of drugs and natural substances on polarized epithelia. With this approach, epithelial sheets or confluent epithelial cell monolayers are mounted between two half-chambers, permitting compound delivery to either the apical or basolateral cell surface. Described in this unit are protocols for determining the effects of test compounds on transepithelial ion transport processes.

Evolutionarily conserved WNK and Ste20 kinases are essential for acute volume recovery and survival after hypertonic shrinkage in Caenorhabditis elegans

Members of the germinal center kinase (GCK)-VI subfamily of Ste20 kinases regulate a Caenorhabditis elegans ClC anion channel and vertebrate SLC12 cation-Cl(-) cotransporters. With no lysine (K) (WNK) protein kinases interact with and activate the mammalian GCK-VI kinases proline-alanine-rich Ste20-related kinase (PASK) and oxidative stress-responsive 1 (OSR1). We demonstrate here for the first time that GCK-VI kinases play an essential role in whole animal osmoregulation. RNA interference (RNAi) knockdown of the single C. elegans GCK-VI kinase, GCK-3, dramatically inhibits systemic volume recovery and survival after hypertonic shrinkage. Tissue-specific RNAi suggests that GCK-3 functions primarily in the hypodermis and intestine to mediate volume recovery. The single C. elegans WNK kinase, WNK-1, binds to GCK-3, and wnk-1 knockdown gives rise to a phenotype qualitatively similar to that of gck-3(RNAi) worms. Knockdown of the two kinases together has no additive effect, suggesting that WNK-1 and GCK-3 function in a common pathway. We postulate that WNK-1 functions upstream of GCK-3 in a manner similar to that postulated for its mammalian homologs. Phylogenetic analysis of kinase functional domains suggests that the interaction between GCK-VI and WNK kinases first occurred in an early metazoan and therefore likely coincided with the need of multicellular animals to tightly regulate transepithelial transport processes that mediate systemic osmotic homeostasis.

Protein/Protein Interactions (PDZ) in Proximal Tubules

IntroductionEssentially all cellular functions rely on the correctspatial organization of proteins. In epithelial cells,this is manifested by a separation of the plasmamembrane into an apical and a basolateral area,which is required for vectorial transport of solutes,and by the formation of junctional complexes toguarantee barrier function. In this article, we sum-marize the current knowledge of PDZ protein-basedmultimeric protein complexes in renal proximaltubular (PT) cells (for definition of the PDZ domain,seebelow), which are involved in the organization ofthe brush border membrane, the basolateral mem-brane and the tight junction of PT cells. In addition,we also consider a fourth subcellular structure, thesubapical compartment (SAC). This compartmenthas been ascribed to the recycling of endocytosedproteins and also to the routing of endocytosedmaterial to the lysosomes.In renal proximal tubules, the majority of filteredsolutes and water are reabsorbed by transepithelialtransport processes and by paracellular pathways[68]. In addition, PT-cells exhibit an exceptionallyhigh rate of endocytosis to recover small peptides andhormones. On a morphological basis, three differentdistinct epithelial cells can be distinguished along theproximal tubules. In the S1 segment, brush bordersare formed by long microvilli, whereas the appear-ance of the microvilli in the S2 and S3 segments is lesspronounced and varies among different species [37].Although this longitudinal morphological heteroge-neity of PT cells is also reflected by functional dif-ferences, in this article, we will not distinguish amongproximal tubular segments, i.e., the PDZ-based pro-tein complexes discussed below are assumed to be ofuniform nature along the entire proximal tubule.ThePDZmoduleNumerous protein domains have been described thatare implicated in protein-protein interactions (see e.g.,www.mshri.on.ca). Based on sequence similarities be-tween the post-synaptic density protein PSD-95, theDrosophilajunctional protein Disc-large and the tightjunctional protein ZO1, one such domain was definedas PDZ. PDZ proteins, among other functions, areprimarily thought to organize large functional unitssuch as synapses [55] or tight junctions [19]. Thecharacteristics of PDZ domains have been reviewedrecently[14,15,28,55]andaresummarizedasfollows:In mammals, over 400 different PDZ proteinshave been identified and grouped into three majorfamilies according to their domain organization:Afirst family encompasses proteins, which only containPDZ domains. The second, MAGUK family (mem-brane-associated guanylate kinases), contains one ormore PDZ domains besides a GuK domain (gua-nylate kinase domain) and a SH3 domain. A thirdfamily was defined containing proteins of multiplePDZ domains together with a variable number ofother protein domains.The PDZ domain comprises between 80 and 90amino-acid residues of which the three-dimensionalstructure has been basically resolved (see http://smart.enbl-heidelberg.de). The building principle is asandwich structure of 6 b-strands and two a-helicesthat form a hydrophobic cleft into which a short pep-tide can be accommodated. As an example, the struc-tural aspect of the CFTR-NHERF interaction hasbeen discussed in detail [38]. In most cases, PDZdomainsbindtoaC-terminalamino-acidmotif.Threedifferent classes of PDZ binding motifs have beenrecognized. They all include the last four C-terminalamino acids, whose characteristics of interaction canbe modulated by more upstream amino acids [55].Besides the classical canonical determinants, addi-tional PDZ binding motifs have been described and

Normal kinetics of intestinal glucose absorption in the absence of GLUT2: evidence for a transport pathway requiring glucose phosphorylation and transfer into the endoplasmic reticulum.

Glucose is absorbed through the intestine by a transepithelial transport system initiated at the apical membrane by the cotransporter SGLT-1; intracellular glucose is then assumed to diffuse across the basolateral membrane through GLUT2. Here, we evaluated the impact of GLUT2 gene inactivation on this transepithelial transport process. We report that the kinetics of transepithelial glucose transport, as assessed in oral glucose tolerance tests, was identical in the presence or absence of GLUT2; that the transport was transcellular because it could be inhibited by the SGLT-1 inhibitor phlorizin, and that it could not be explained by overexpression of another known glucose transporter. By using an isolated intestine perfusion system, we demonstrated that the rate of transepithelial transport was similar in control and GLUT2(-/-) intestine and that it was increased to the same extent by cAMP in both situations. However, in the absence, but not in the presence, of GLUT2, the transport was inhibited dose-dependently by the glucose-6-phosphate translocase inhibitor S4048. Furthermore, whereas transport of [(14)C]glucose proceeded with the same kinetics in control and GLUT2(-/-) intestine, [(14)C]3-O-methylglucose was transported in intestine of control but not of mutant mice. Together our data demonstrate the existence of a transepithelial glucose transport system in GLUT2(-/-) intestine that requires glucose phosphorylation and transfer of glucose-6-phosphate into the endoplasmic reticulum. Glucose may then be released out of the cells by a membrane traffic-based pathway similar to the one we previously described in GLUT2-null hepatocytes.