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B Püschel

Publications and source records attributed to B Püschel.

10 recordsLinked to original sources
PubMed1999

Expression of rat kidney anion exchanger 1 in type A intercalated cells in metabolic acidosis and alkalosis.

By enzyme-linked in situ hybridization (ISH), direct evidence is provided that acid-secreting intercalated cells (type A IC) of both the cortical and medullary collecting ducts of the rat kidney selectively express the mRNA of the kidney splice variant of anion exchanger 1 (kAE1) and no detectable levels of the erythrocyte AE1 (eAE1) mRNA. Using single-cell quantification by microphotometry of ISH enzyme reaction, medullary type A IC were found to contain twofold higher kAE1 mRNA levels compared with cortical type A IC. These differences correspond to the higher intensity of immunostaining in medullary versus cortical type A IC. Chronic changes of acid-base status induced by addition of NH(4)Cl (acidosis) or NaHCO3 (alkalosis) to the drinking water resulted in up to 35% changes of kAE1 mRNA levels in both cortical and medullary type A IC. These experiments provide direct evidence at the cellular level of kAE1 expression in type A IC and show moderate capacity of type A IC to respond to changes of acid-base status by modulation of kAE1 mRNA levels.

Acidosis↗
PubMed1997

Cellular and regional distribution of the glutamate transporter GLAST in the CNS of rats: nonradioactive in situ hybridization and comparative immunocytochemistry.

Oligonucleotide and cRNA probes were used for nonradioactive in situ hybridizations carried out to identify the neural cell types expressing the glutamate transporter GLAST mRNA in the rat CNS. Additionally, the regional distribution of GLAST mRNA-expressing cells was studied, and the results were complemented by immunocytochemical investigations using an antibody against a synthetic GLAST peptide. The findings documented that GLAST is expressed by Bergmann glia and by astrocytes throughout the CNS. The glial localization of GLAST mRNA was verified unequivocally by double-labeling with an astrocytic marker protein. Additionally, GLAST mRNA reactivity and GLAST immunoreactivity were found in ependymal cells. In other neural cell types of the CNS, GLAST expression was not detectable. A high level of astrocytic immunolabeling was observed in the entire gray matter of the brain, with variations in intensity in different regions. Those brain areas that are known to possess high glutamatergic activity and astrocytic glutamate metabolism stained intensely for both GLAST mRNA and GLAST protein. The latter observation suggests that the GLAST glutamate transporter participates in the regulation of extracellular glutamate concentrations, especially in brain areas receiving an intense glutamatergic innervation.

Amino Acid Transport System X-AG↗
PubMed1997

Role of cadherins and plakoglobin in interendothelial adhesion under resting conditions and shear stress.

The role of cadherins and the cadherin-binding cytosolic protein plakoglobin in intercellular adhesion was studied in cultured human umbilical venous endothelial cells exposed to fluid shear stress. Extracellular Ca2+ depletion (< 10(-7) M) caused the disappearance of both cadherins and plakoglobin from junctions, whereas the distribution of platelet endothelial cell adhesion molecule 1 (PECAM-1) remained unchanged. Cells stayed fully attached to each other for several hours in low Ca2+ but began to dissociate under flow conditions. At the time of recalcification, vascular endothelial (VE) cadherin and beta-catenin became first visible at junctions, followed by plakoglobin with a delay of approximately 20 min. Full fluid shear stress stability of the junctions correlated with the time course of the reappearance of plakoglobin. Inhibition of plakoglobin expression by microinjection of antisense oligonucleotides did not interfere with the junctional association of VE-cadherin, PECAM-1, and beta-catenin. The plakoglobin-deficient cells remained fully attached to each other under resting conditions but began to dissociate in response to flow. Shear stress-induced junctional dissociation was also observed in cultures of plakoglobin-depleted arterial endothelial cells of the porcine pulmonary trunk. These observations show that interendothelial adhesion under hydrodynamic but not resting conditions requires the junctional location of cadherins associated with plakoglobin. beta-Catenin cannot functionally compensate for the junctional loss of plakoglobin, and PECAM-1-mediated adhesion is not sufficient for monolayer integrity under flow.

Antigens, CD↗
PubMed1996

Taste receptor-like cells in the rat gut identified by expression of alpha-gustducin.

The alpha-subunit of the trimeric G-protein complex specific for taste receptor cells of the tongue, alpha-gustducin, is described here to be also expressed in the stomach and intestine. The alpha-gustducin-containing cells were identified as brush cells that are scattered throughout the surface epithelium of the gut and share structural features of taste receptor cells of the tongue. These findings provide clues to the long-sought molecular and cellular basis for chemoreception in the gut.

Animals↗
PubMed1996

Expression of the glutamate transporter GLT1 in neural cells of the rat central nervous system: non-radioactive in situ hybridization and comparative immunocytochemistry.

Non-radioactive in situ hybridization using complementary RNA and oligonucleotide probes was applied in order to clearly identify the cell types expressing GLT1 and to show their regional distribution in the central nervous system of the rat. The results were compared with immunocytochemical data achieved using an antibody against a synthetic GLT1 peptide. The study showed that GLT1 was expressed in astrocytes and Bergmann glia which were identified by the detection of an astrocytic marker protein. Additionally, subsets of neurons in different brain regions (e.g., CA3/4 pyramidal cells of the hippocampus, endopiriform nucleus) were labelled by in situ hybridization. In other cell types of the central nervous system (oligodendrocytes, ependymal cells, epithelal cells of the choroid plexus, tanycytes), GLT1 expression was not detectable. The generally dense astrocytic immunolabelling of the gray matter of the brain showed an even higher intensity in regions reported to show high glutamatergic activity and astrocytic glutamate metabolism (e.g., the termination field of the glutamatergic perforant path in the hippocampus). On the basis of the cellular regional distribution of the GLT1 messenger RNA and protein demonstrated in the present study, it is reasonable to assume that this high affinity transporter is of importance for the maintenance of adequate extraneuronal glutamate levels.

ATP-Binding Cassette Transporters↗
PubMed1995

Identification of a binding motif for ankyrin on the alpha-subunit of Na+,K(+)-ATPase.

Cytoskeleton membrane associations are important for a variety of cellular functions. The anion exchanger of erythrocytes (AE1) and Na+,K(+)-ATPase of polarized epithelial cells provide well studied examples of how integral membrane proteins are anchored via the linker molecule ankyrin to the spectrin-based membrane cytoskeleton. In the present study we have generated several recombinant fragments of the large (third) cytoplasmic domain (CD3) of Na+,K(+)-ATPase to define binding sites of ankyrin on CD3 at a molecular level. We provide evidence that a cluster of four amino acids, ALLK, is essential for binding of ankyrin to both recombinant CD3 and to native Na+,K(+)-ATPase. Once bound, conformational changes might uncover further binding sites for ankyrin on Na+,K(+)-ATPase. A motif related to the ALLK cluster is also present in the cytoplasmic domain of AE1 where this sequence (ALLLK) turned out to be also important for ankyrin binding. These motifs are highly conserved during evolution of both Na+,K(+)-ATPase and AE1, further underlining their potential role in cytoskeleton to membrane linkage.

Amino Acid Sequence↗
PubMed1993

Cloning of a membrane-associated protein which modifies activity and properties of the Na(+)-D-glucose cotransporter.

An expression library from porcine kidney cortex was screened with a monoclonal antibody (R4A6) which stimulates high-affinity phlorizin binding in kidney and intestine but does not react with the membrane protein (SGLT1) which mediates Na(+)-coupled transport of D-glucose (Hediger, M.A., Coady, M.J., Ikeda, T.S., and Wright, E.M. (1987) Nature 330, 379-381). A cDNA (RS1) was obtained which codes for a hydrophilic M(r) 66,832 polypeptide and contains a predicted hydrophobic alpha-helix at the COOH terminus. After expression in Xenopus oocytes RS1 protein was found associated with the plasma membrane. RS1-homologous mRNAs were detected in renal outer cortex and outer medulla, small intestine, liver, and LLCPK1 cells, but not in skeletal muscle, heart muscle, Madin-Darby canine kidney (MDCK) cells, renal inner medulla, and Xenopus oocytes. After nondenaturing gel electrophoresis of renal brush-border membranes comigration of RS1- and SGLT1-homologous proteins as a high molecular weight complex was demonstrated. RS1 altered the expression of Na(+)-glucose cotransport by SGLT1 in Xenopus oocytes. There was no effect on the expression of the nonhomologous transporters for Na(+)-gamma-aminobutyric acid cotransport and for Na(+)-independent glucose transport. However, RS1 also changed the expression of the SGLT1-homologous Na(+)-myo-inositol cotransporter from MDCK cells. The Vmax of methyl-alpha-D-glucopyranoside (AMG) transport expressed after injection of a small amount of SGLT1-cRNA was increased 40-fold when a stoichiometric amount of RS1-cRNA was coinjected. In addition the voltage and glucose dependence of expressed AMG uptake and the concentration dependence of transport inhibition by phlorizin were changed when stoichiometric amounts of RS1-cRNA were coinjected with SGLT1-cRNA. Thus with SGLT1 one apparent transport site (K0.5 about 100 microM) and one apparent phlorizin inhibition site (Ki about 5 microM) was observed whereas with SGLT1 plus RS1 two apparent transport sites (K0.5(1) about 20 microM, K0.5(2) about 1 mM) and two apparent phlorizin inhibition sites (Ki(1) about 0.3 microM, Ki(2) about 30 microM) were found as has been described in brush-border membrane vesicles of kidney and intestine (see e.g. Koepsell, H., Fritzsch, G., Korn, K., and Madrala, A. (1990) J. Membr. Biol. 114, 113-132). The data suggest that the Na(+)-D-glucose cotransporter and possibly also other SGLT1-type Na(+)-cotransporters contain RS1-type regulatory subunits.

Amino Acid Sequence↗
PubMed1993

Role of H+ ions in volume and voltage of epithelial cell nuclei.

Condensation of chromatin depends upon the ion composition in the cell nucleus. We tested in isolated nuclei of Madin-Darby canine kidney cells the influence of various ions on nuclear volume (i. e. DNA packing) and intranuclear voltage. After isolation, nuclei were superfused with cytosolic solutions in which Na+, K+, Ca2+ and H+ ions were varied. With video-imaging and microelectrode techniques nuclear volume and intranuclear potential were measured in response to the various ions. In control cytosolic solution, isolated nuclei exhibited an intranuclear electrical potential of -6.5 +/- 0.5 mV (relative to a reference electrode in the cytosolic solution) corresponding to a nuclear volume of 250 +/- 10 fl (n = 104). Changing the Na+, K+ or free Ca2+ concentration in the superfusate in the physiological range resulted in minor changes of volume and intranuclear potential whereas pH altered both parameters dramatically. Nuclear swelling and intranuclear negative voltage increased with alkalinization and decreased when pH was reduced. An intact nuclear envelope was found to be no prerequisite for maintaining intranuclear negativity, indicating that the composition and functional state of nuclear chromatin rather than specific ion permeabilities of the nuclear envelope determine nuclear electrical potential. We present a model that explains nuclear volume and voltage on the basis of interaction between negatively charged DNA and positively charged histones of the nuclear chromatin.

Animals↗
PubMed1991

Comparison of a Na+/D-glucose cotransporter from rat intestine expressed in oocytes of Xenopus laevis with the endogenous cotransporter.

Epithelial Na+/D-glucose cotransport was incorporated into the plasma membrane of Xenopus oocytes after microinjection of poly(A)(+)-mRNA from rat intestine tissue and was detected by measurements of uptake of [14C]AMG (methyl alpha-D-glucopyranoside). In mRNA-injected oocytes, the rate of AMG uptake exceeds the rate of endogenous Na+/AMG cotransport by a factor of up to 30. It is demonstrated that the additionally expressed transport differs qualitatively from the endogenous transport with respect to several parameters which is a prerequisite for the demonstration of expression of a foreign transporter: (1) The expressed system is more sensitive to external glucose or AMG and to the specific inhibitor phlorizin, (2) it is less sensitive to external Na+ and to changes in membrane potential, and (3) it is susceptible to inhibition by monoclonal antibodies, known to bind specifically to Na+/glucose cotransporters and to modulate the cotransport in kidney and intestine. The use of the antibodies allows one to distinguish between endogenous Na+/AMG cotransport and foreign cotransport expressed by injection of foreign mRNA. The expression of the foreign transport leads to transport rates that are high enough to detect the electrical current generated by the Na+/glucose cotransport. This allows future characterization of the cotransport system under voltage-clamp conditions by analyzing membrane current.

Animals↗