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M Paulmichl

Publications and source records attributed to M Paulmichl.

At least 19 recordsLinked to original sources
PubMed2005

S-CMC-Lys-dependent stimulation of electrogenic glutathione secretion by human respiratory epithelium.

Glutathione (GSH) is one of the most important defense mechanisms against oxidative stress in the respiratory epithelial lining fluid. Considering that GSH secretion in respiratory cells has been postulated to be at least partially electrogenic, and that the mucoregulator S-carbocysteine lysine salt monohydrate (S-CMC-Lys) can cause an activation of epithelial Cl(-) conductance, the purpose of this study was to verify whether S-CMC-Lys is able to stimulate GSH secretion. Experiments have been performed by patch-clamp technique, by high-performance liquid chromatography (HPLC) assay, and by Western blot analysis on cultured lines of human respiratory cells (WI-26VA4 and CFT1-C2). In whole-cell configuration, after cell exposure to 100 microM S-CMC-Lys, a current due to an outward GSH flux was observed, which was inhibitable by 5-nitro-2-(3-phenylpropylamino)-benzoate and glibenclamide. This current was not observed in CFT1-C2 cells, where a functional cystic fibrosis transmembrane conductance regulator (CFTR) is lacking. Inside-out patch-clamp experiments (GSH on the cytoplasm side, Cl(-) on the extracellular side) showed the activity of a channel, which was able to conduct current in both directions: the single channel conductance was 2-4 pS, and the open probability (P(o)) was low and voltage-independent. After preincubation with 100 microM S-CMC-Lys, there was an increase in P(o), in the number of active channels present in each patch, and in the relative permeability to GSH vs Cl(-). Outwardly directed efflux of GSH could also be increased by protein kinase A, adenosine 5'-triphosphate, and cyclic adenosine monophosphate (cAMP) added to the cytoplasmic side (whole-cell configuration). The increased secretion of GSH observed in the presence of S-CMC-Lys or 8-bromoadenosine-3',5'-cyclic monophosphate was also confirmed by HPLC assay of GSH on a confluent monolayer of respiratory cells. Western blot analysis confirmed the presence of CFTR in WI-26VA4 cells. This study suggests that S-CMC-Lys is able to stimulate a channel-mediated GSH secretion by human respiratory cells: electrophysiological and pharmacological characteristics of this channel are similar to those of the CFTR channel.

Carbocysteine↗
PubMed2005

Jejunal creatine absorption: what is the role of the basolateral membrane?

The mechanism of the intestinal creatine absorption is not well understood. Previous studies have established the involvement of a CT1 carrier system in jejunal apical membrane. The current research was aimed at completing the picture of creatine absorption. To investigate the process supporting creatine exit from enterocyte, basolateral membrane vesicles isolated from rat jejunum were used. The presence of various symport and antiport mechanisms was searched and a NaCl-dependent electrogenic transport system for creatine was evidenced, which shares some functional and kinetic features with the apical CT1. However, Western blot and immunohistochemical experiments ruled out the presence of a CT1 transporter in the basolateral membrane. Further studies are required to identify the basolateral transport mechanism. However, in the in vivo conditions, the NaCl gradient is inwardly directed, therefore such a mechanism cannot energetically mediate the exit of creatine from the cell into the blood during the absorptive process, but rather it may drive creatine into the enterocyte. To shed more light on the creatine absorption process, a possible creatine movement through the paracellular pathway has been examined using the jejunal tract everted and incubated in vitro. A linear relationship between creatine transport and concentration was apparent both in the mucosa-to-serosa and serosa-to-mucosa directions and the difference between the two slopes suggests that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption. As a matter of fact, when transepithelial water flux is reduced by means of a mucosal hypertonic solution, the opposite creatine fluxes tend to overlap. The findings of the present study suggest that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption.

Animals↗
PubMed2005

Ion transport across the gallbladder epithelium.

The function of the gallbladder is not only to store bile, but also to concentrate it during the interdigestive phase by means of salt-dependent water reabsorption. On the contrary, secretions of water and salt take place during the digestive phase. Dysregulation of ion absorption or secretion are common in many gallbladder diseases, such as colelithiasis. Transepithelial absorptions are determined by the Na+/K+ pump on the basolateral membrane, and by several apical membrane Na(+)-coupled transporters. Among these, some isoforms of Na+/H+ and Cl-/HCO3(-) exchangers have been studied. The presence of a Na(+)-Cl(-) simport has been molecularly and functionally characterized in some animal species. The ion transepithelial secretion is mainly dependent on an apical chloride transport attributable to a CFTR-like cAMP-activated channel with high permeability to HCO3(-). The apical membrane electrical potential is one of the factors influencing anion secretion and is maintained by the activity of cAMP-dependent K+ channels. The regulation of the activity of these channels is complex, because of their sensitivity to voltage, and to intracellular calcium and pH. The coordinated interplay underlying the regulation of transporters and channels needs to be clarified yet, as well as the interactions between transporters, channels and aquaporins.

Animals↗
PubMed2001

Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells.

In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579-1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Fick's law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic Ca2+ concentration. Consistently, content release correlated with the occurrence of Ca2+ oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation.

Adenosine Triphosphate↗
PubMed2001

The presence of NHE1 and NHE3 Na+-H+ exchangers and an apical cAMP-independent Cl- channel indicate that both absorptive and secretory functions are present in calf gall bladder epithelium.

We investigated the transport systems that can sustain Na+ and Cl- movements across bovine gall bladder epithelium, focusing on the Na+-H+ exchanger (NHE) family and chloride conductive pathways. Experiments conducted using the fluorescent probe acridine orange (AO) with brush-border membrane vesicles (BBMV) or vesicles obtained from the total epithelium (EMV) demonstrated the presence of a Na+-H+ exchange in both preparations. The use of specific inhibitors indicated the presence of an apical NHE3 exchanger and a NHE1 isoform which should reside in the basolateral membrane. Using reverse transcriptase (RT) PCR, we identified cDNA fragments corresponding to the NHE1, NHE3, Cl--HCO3- (AE2a) transporters and to the CFTR channel. Using the patch-clamp technique, we investigated Cl- conductances on cultured epithelial cells. We found a 5 pS Cl- channel with a voltage-independent open probability, insensitive to stilbenes (SITS), Zn2+ and cAMP. The results suggest that absorption and secretion coexist in calf gall bladder epithelium. A Na+-H+-Cl--HCO3- double exchange may, at least partially, sustain the absorptive function, and a Cl- apical conductive pathway may be involved in secretion. The conductance we observed does not seem to be cAMP-regulated, unlike other mammalian gall bladders.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed2001

Na(+)/H(+)exchangers: linking osmotic dysequilibrium to modified cell function.

The Na(+)/H(+) exchangers (NHEs) are among the major ion transporters involved in cell volume regulation. NHE activation leads to a cellular influx of Na(+) ions and extrusion of H(+) ions, which are readily replenished from intracellular buffers. This will result in a net import of Na(+). In many systems NHE operates in parallel to Cl(-)/ HCO3(-) exchange, resulting in cellular uptake of NaCl. The influx of osmotically obliged water will consequently lead to cell swelling. This makes NHEs suitable to serve as powerful mechanisms for increasing cell volume (CV). The low volume threshold for NHE activation enables the cells to respond to very minute reductions of the CV. By the coupling to the export of H(+) ions cell volume regulatory NHE activation may lead to changes in intracellular pH. On the other hand NHEs are activated by a broad variety of ligands and by intracellular acidosis, which, in turn, may consequently lead to cell swelling. In addition, NHEs are linked to other intracellular proteins and structures, like e.g. the cytoskeleton, which themelves are involved in the regulation of numerous cellular processes. Therefore NHEs link CV regulation to a diversity of cellular functions, both in physiological and pathophysiological conditions. Six isoforms of the Na(+)/H(+) exchanger, termed NHE1--6, have been cloned so far. NHE 1--5 are located in the plasma membrane, whereas NHE6 is sorted to the mitochondrial membrane. NHE1 and NHE6 are the ubiquitously expressed isoforms. The expression of the isoforms NHE2 to NHE5 is restricted to specific tissues and the pattern of their expression, as well as their subcellular localization indicate that they fulfill specialized functions. Cell shrinkage induced activation has been shown for NHE1,2 and 4. In contrast, NHE3 is inhibited by cell shrinkage. In many cells several isoforms are present and assigned to specific membrane domains where they may serve a functional crosstalk between the different ion transporters.

Amino Acid Sequence↗
PubMed2001

Determination of protein-protein interactions of ICIn by the yeast two-hybrid system.

ICln is a ubiquitously expressed eukaryotic protein. Expression of the protein in Xenopus laevis oocytes, the knocking-down of the protein in fibroblasts, or the reconstitution of the protein in lipid bilayer led to the assumption that this protein is an ionic channel or a significant part thereof. However, other possible roles for ICln in potential regulatory mechanisms have been postulated, as diverse as regulator of cell morphology by interacting with the Skb1 protein and/or interaction with core spliceosomal proteins. Here we show that ICln is able to interact with SnRNP core proteins SmD1, SmD2, SmD3, SmX5 and SmB/B'.

Amino Acid Sequence↗
PubMed2000

Swelling-induced taurine release without chloride channel activity in Xenopus laevis oocytes expressing anion channels and transporters.

Taurine is an important osmolyte involved in cell volume regulation. During regulatory volume decrease it is released via a volume-sensitive organic osmolyte/anion channel. Several molecules have been suggested as candidates for osmolyte release. In this study, we chose three of these, namely ClC-2, ClC-3 and ICln, because of their expression in rat astrocytes, a cell type which is known to release taurine under hypotonic stress, and their activation by hypotonic shock. As all three candidates were also suggested to be chloride channels, we investigated their permeability for both chloride and taurine under isotonic and hypotonic conditions using the Xenopus laevis oocyte expression system. We found a volume-sensitive increase of chloride permeability in ClC-2-expressing oocytes only. Yet, the taurine permeability was significantly increased under hypotonic conditions in oocytes expressing any of the tested candidates. Further experiments confirmed that the detected taurine efflux does not represent unspecific leakage. These results suggest that ClC-2, ClC-3 and ICln either participate in taurine transport themselves or upregulate an endogenous oocyte osmolyte channel. In either case, the taurine efflux of oocytes not being accompanied by an increased chloride flux suggests that taurine and chloride can be released via two separate pathways.

Animals↗
PubMed2000

Deranged transcriptional regulation of cell-volume-sensitive kinase hSGK in diabetic nephropathy.

Transforming growth factor beta (TGF-beta) has been shown to participate in the pathophysiology of diabetic complications. As shown most recently, TGF-beta stimulates the expression of a distinct serine/threonine kinase (hSGK) which had previously been cloned as an early gene transcriptionally regulated by cell volume alterations. The present study was performed to elucidate transcription and function of hSGK in diabetic nephropathy. As shown by Northern blotting, an increase of extracellular glucose concentration increased hSGK mRNA levels in cultured cells, an effect qualitatively mimicked by osmotic cell shrinkage or treatment with TGF-beta (2 microgram/liter), phorbol 12,13-didecanoate (1 microM), or the Ca(2+) ionophore ionomycin (1 microM) and blunted by high concentrations of nifedipine (10 and 100 microM). In situ hybridization revealed that hSGK transcription was markedly enhanced in diabetic nephropathy, with particularly high expression in mesangial cells, interstitial cells, and cells in thick ascending limbs of Henle's loop and distal tubules. According to voltage clamp and tracer flux studies in Xenopus oocytes expressing the renal epithelial Na(+) channel ENaC or the mouse thick ascending limb Na(+),K(+),2Cl(-) cotransporter BSC-1, coexpression with hSGK stimulated ENaC and BSC-1 11-fold and 6-fold, respectively, effects reversed by kinase inhibitors staurosporine (1 microM) and chelerythrine (1 microM) and not elicited by inactive hSGK. In conclusion, excessive extracellular glucose concentrations enhance hSGK transcription, which in turn stimulates renal tubular Na(+) transport. These observations disclose an additional element in the pathophysiology of diabetic nephropathy.

3T3 Cells↗
PubMed2000

The promoter for constitutive expression of the human ICln gene CLNS1A.

The ICln protein is expressed ubiquitously in mammals. Experiments designed to knock down the ICln protein in NIH 3T3 fibroblasts as well as in epithelial cells led to the conclusion that this protein is crucially involved in volume regulation after cytoplasmic swelling. Reconstitution of the ICln protein in lipid bilayers revealed the ion channel nature of ICln. Here we describe a new human promoter sequence, composed of 89 nucleotides, which is responsible for a highly constitutive expression of the ICln protein. The promoter sequence lacks a TATA box, and the transcription can be effected at multiple sites. In addition to the starting sites, upstream sequence elements are mandatory for an efficient transcription of the ICln gene (CLNS1A). These new nucleotide elements were defined by site-directed mutagenesis.

Animals↗
PubMed2000

Functional reconstitution of ICln in lipid bilayers.

Reconstitution of purified ICln in lipid bilayer leads to functional ion channels showing varying rectification. The reconstituted single channels have a conductance of approximately equal to 3 pS and their open probability is sensitive to nucleoside analogues. Mutation of a putative nucleotide binding site identified at the predicted extracellular mouth of the ICln channel protein leads to the reduction of the nucleoside-analogue sensitivity. Reconstituted ICln channels can be permeated both by cations and anions. The relative permeability of cations over anions depends on the presence of calcium. In the presence of calcium reconstituted ICln channels are more permeable to bromide than chloride, and more permeable to potassium than sodium. Similarly in NIH3T3 fibroblasts, the relative permeability of cations over anions of swelling-dependent chloride channels depends on extracellular calcium. Site-directed mutagenesis revealed the calcium-binding site responsible for the shift of the selectivity from cations towards anions of reconstituted ICln channels. Additional indirect structural information has been obtained by mutating a histidine in the predicted pore region of ICln. This histidine seems to have access to the ion-conducting tunnel of the pore. Our experiments show that ICln can act as an ionic channel, which does not exclude additional functions of the protein in regulatory mechanisms of the cell. Since knocking down the ICln protein in fibroblasts and epithelial cells leads to an impaired regulatory volume decrease (RVD) after cytoplasmic swelling and reconstituted ICln channels show several biophysical features of ion channels activated after swelling, ICln is a molecular candidate for these channels.

3T3 Cells↗
PubMed2000

The gastric H,K-ATPase blocker lansoprazole is an inhibitor of chloride channels.

1. It was postulated that swelling dependent chloride channels are involved in the proton secretion of parietal cells. Since omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are structurally related to phenol derivatives known to block swelling dependent chloride channels, we set out to test, whether these substances--which are known to block the H,K-ATPase--could also lead to an inhibition of swelling-dependent chloride channels. Swelling-dependent chloride channels--characterized in many different cell types--show highly conserved biophysical and pharmacological features, therefore we investigated the effect of omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 on swelling-dependent chloride channels elicited in fibroblasts, after the reduction of the extracellular osmolarity. 2. Omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are able to block swelling-dependent chloride channels (IClswell). 3. Lansoprazole and its protonated metabolite AG2000 act on at least two different sites of the IClswell protein: on an extracellular site which seems to be in a functional proximity to the nucleotide binding site, and on an intracellular site which allows the formation of disulfide-bridges. 4. The inhibition of the proton pump and the simultaneous blocking of chloride channels by omeprazole, lansoprazole and its acid activated sulphenamide form AG2000, as described here could be an effective mode to restrict proton secretion in parietal cells.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed2000

Structure and function of the ion channel ICln.

Normal function of organs and cells is tightly linked to the cytoarchitecture. Control of the cell volume is therefore vital for the organism. A widely established strategy of cells to counteract swelling is the activation of chloride and potassium channels, which leads to a net efflux of salt followed by water - a process termed regulatory volume decrease. Since there is evidence for swelling-dependent chloride channels (IClswell) being activated also during pathological processes, the identification of the molecular entity underlying IClswell is of utmost importance. Several proteins are discussed as the channel forming IClswell, i.e. phospholemman, p-glycoprotein, CLC-3 and ICln. In this review we would like to focus on the properties of ICln, a protein cloned from a Madin Darby canine kidney (MDCK) cell library whose expression in Xenopus laevis oocytes resulted in a nucleotide sensitive outwardly rectifying chloride current closely resembling the biophysical properties of IClswell.

Amino Acid Sequence↗
PubMed1999

ICln, an ion channel-forming protein associated with cell volume regulation.

It is not resolved whether the anionic channel involved in volume regulation after cell swelling comprises one or more subunits. Moreover, it remains to be determined which of the different proteins cloned so far, for which an involvement in cell volume regulation has been postulated, is the ideal candidate. In this review, we consider the role of the ICln protein, cloned from MDCK cells, in cell volume regulation.

Animals↗
PubMed1998

Genomic organization and chromosomal localization of the human SGK protein kinase gene.

The SGK protein kinase is a novel member of the serine/threonine protein kinase family. Its corresponding gene belongs to the group of immediate-early genes. SGK transcription is controlled by cell volume alterations in different cell lines. To analyze the genomic structure and chromosomal location of the SGK gene, a human P1 clone was isolated by screening a human genomic library with a SGK cDNA probe. This clone was confirmed to encode the authentic SGK gene by the detection of exon-intron structures and the correspondence between the nucleotide sequences of exons and human cDNA. Using this P1 clone as a probe for fluorescence in situ hybridization, a single chromosomal locus for SGK was assigned to band 6q23, a region frequently affected by deletion in various human neoplasms.

Cell Line↗
PubMed1998

Characterization of the human gene coding for the swelling-dependent chloride channel ICln at position 11q13.5-14.1 (CLNS1A) and further characterization of the chromosome 6 (CLNS1B) localization.

Expression cloning revealed a chloride channel (ICln) that we found to be fundamental for the regulatory volume decrease in a variety of cells. The chromosomal localization of the human ICln-gene showed two loci, one at chromosome 11 in position q13.5-q14.1, termed CLNS1A, and a second one at chromosome 6 at position p12.1-q13, termed CLNS1B. In this study, we offer a detailed characterization of the CLNS1A gene and provide the exact position (6p12) and sequence data of CLNS1B, an intronless gene 91.3% homologous to the coding region of CLNS1A.

Amino Acid Sequence↗
PubMed1998

Effect of inhibitors of Na+/H+-exchange and gastric H+/K+ ATPase on cell volume, intracellular pH and migration of human polymorphonuclear leucocytes.

1. Stimulation of chemotaxis of human polymorphonuclear leucocytes (PMNs) with the chemoattractive peptide fMLP (N-formyl-Met-Leu-Phe) is paralleled by profound morphological and metabolic alterations like changes of intracellular pH (pHi) and cell shape. The present study was performed to investigate the interrelation of cell volume (CV) regulatory ion transport, pHi and migration of fMLP stimulated PMNs. 2. Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling. 3. Inhibition of the Na+/H+ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP-induced cell swelling. 4. Step omission of extracellular Na+ caused intracellular acidification, which was accelerated by subsequent addition of gastric H+/K+ ATPase inhibitor SCH 28080, or by omission of extracellular K+ ions. In addition Na+ removal caused cell swelling, which was further enhanced by fMLP. 5. H+/K+ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP-induced increase in CV. 6. Increasing extracellular osmolarity by addition of mannitol to the extracellular solution caused cell shrinkage followed by regulatory volume increase, partially due to activation of the Na+/H+ exchanger. In fMLP-stimulated cells the CV increase was counteracted by simultaneous addition of mannitol. Under these conditions the fMLP stimulated migration was inhibited. 7. The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole. 8. Western analysis with a monoclonal anti gastric H+/K+ ATPase beta-subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs. 9. The results indicate that fMLP leads to cell swelling of PMNs due to activation of the Na+/H+ exchanger and a K+-dependent H+-extruding mechanism, presumably an H+/K+ ATPase. Inhibition of these ion transporters suppresses the increase in CV and precludes PMNs from stimulated migration.

Animals↗