Biomedical subjects

L Schild

Publications and source records attributed to L Schild.

At least 19 recordsLinked to original sources

PubMed2005

Targeted antioxidative and neuroprotective properties of the dopamine agonist pramipexole and its nondopaminergic enantiomer SND919CL2x [(+)2-amino-4,5,6,7-tetrahydro-6-Lpropylamino-benzathiazole dihydrochloride].

Pramipexole has been shown to possess neuroprotective properties in vitro that are partly independent of its dopaminergic agonism. The site of neuroprotective action is still unknown. Using [(3)H]pramipexole, we show that the drug enters and accumulates in cells and mitochondria. Detoxification of reactive oxygen species (ROS) by pramipexole is shown in vitro and in vivo by evaluating mitochondrial ROS release and aconitase-2 activity, respectively. Pramipexole and its (+)-enantiomer SND919CL2X [low-affinity dopamine agonist; (+)2-amino-4,5,6,7-tetrahydro-6-l-propylamino-benzathiazole dihydrochloride] possess equipotent efficacy toward hydrogen peroxide and nitric oxide generated in vitro and inhibit cell death in glutathione-depleted neuroblastoma cells. IC(50) values ranged from 15 to 1000 microM, consistent with the reactivity of the respective radical and the compartmentalization of ROS generation and ROS detoxification. Finally, both compounds were tested in superoxide dismutase 1-G93A mice, a model of familial amyotrophic lateral sclerosis. SND919CL2X (100 mg/kg) prolongs survival time and preserves motor function in contrast to pramipexole (3 mg/kg), which shows an increase in running wheel activity before disease onset, presumably caused by the dopaminergic agonism. We conclude that both enantiomers, in addition to their dopaminergic activity, are able to confer neuroprotective effects by their ability to accumulate in brain, cells, and mitochondria where they detoxify ROS. However, a clinical use of pramipexole as a mitochondria-targeted antioxidant is unlikely, because the high doses needed for antioxidative action in vitro are not accessible in vivo due to dopaminergic side effects. In contrast, SND919CL2X may represent the prototype of a mitochondria-targeted neuroprotectant because it has the same antioxidative properties without causing adverse effects.

Aconitate Hydratase↗

PubMed2004

The epithelial sodium channel: from molecule to disease.

Genetic analysis has demonstrated that Na absorption in the aldosterone-sensitive distal nephron (ASDN) critically determines extracellular blood volume and blood pressure variations. The epithelial sodium channel (ENaC) represents the main transport pathway for Na+ absorption in the ASDN, in particular in the connecting tubule (CNT), which shows the highest capacity for ENaC-mediated Na+ absorption. Gain-of-function mutations of ENaC causing hypertension target an intracellular proline-rich sequence involved in the control of ENaC activity at the cell surface. In animal models, these ENaC mutations exacerbate Na+ transport in response to aldosterone, an effect that likely plays an important role in the development of volume expansion and hypertension. Recent studies of the functional consequences of mutations in genes controlling Na+ absorption in the ASDN provide a new understanding of the molecular and cellular mechanisms underlying the pathogenesis of salt-sensitive hypertension.

Aldosterone↗

PubMed2001

Mitochondrial polarization in rat hippocampal astrocytes is resistant to cytosolic Ca(2+) loads.

The influence of physiological Ca(2+)-inducing stimuli and agents mimicking ischemic conditions on mitochondrial potential was studied in postnatal (P1) hippocampal astrocytes. Cytosolic Ca(2+) loads with characteristic kinetics of rise and duration, detected by Fura-2, were provoked by extracellular Ca(2+) influx, release from InsP(3)-sensitive intracellular stores, or inhibition of the reloading of endoplasmic reticulum Ca(2+) stores. Inhibitors of mitochondrial respiration caused only moderate release of Ca(2+) from intracellular stores, inducing a rise of less than 60 nM. The maximal Ca(2+) rise was found with InsP(3)-mediated responses (500 nM; via ATP) or with ionophore (4-Br-A23187)-mediated Ca(2+) influx from extracellular medium (770 nM). Remarkably, all these agents causing significant rise of cytosolic Ca(2+), only minimally depolarized the mitochondria. Membrane potential of mitochondria was monitored by Rh123 or TMRE. Depolarization was only found with very high cytosolic Ca(2+) levels (above 60 microM; measured by fura FF). These were achieved with external Ca(2+) influx by ionophore in combination with inhibition of glycolysis. Thus, mitochondria in the astrocytes are obviously not sensitive to moderate cytosolic Ca(2+) loads, irrespective of the source of Ca(2+). Furthermore, isolated rat brain mitochondria display a low sensitivity of respiratory activity to Ca(2+), which is consistent with the data obtained with the astrocytes in vitro. The capacity of isolated mitochondria to build up a potential was gradually reduced at low micromolar Ca(2+) and totally compromised only at Ca(2+) concentrations in the 100 microM range.

Adenosine Triphosphate↗

PubMed2001

Micromachined impedance spectroscopy flow cytometer for cell analysis and particle sizing.

A new cytological tool, based on the microCoulter particle counter (microCPC) principle, aimed at diagnostic applications for cell counting and separation in haematology, oncology or toxicology is described. The device measures the spectral impedance of individual cells or particles and allows screening rates over 100 samples s(-1) on a single-cell basis. This analyzer is intended to drive a sorting actuator producing a subsequent cell separation. Size reduction and integration of functions are essential in achieving precise measurements and high throughput. 3D finite element simulations are presented to compare various electrode geometries and their influence on cell parameters estimation. The device is based on a glass-polyimide microfluidic chip with integrated channels and electrodes microfabricated at the length scale of the particles to be investigated (1-20 microm). A laminar liquid flow carries the suspended particles through the measurement area. Each particle's impedance signal is recorded by a differential pair of microelectrodes using the cell surrounding media as a reference. The micromachined chip and processing electronic circuit allow simultaneous impedance measurements at multiple frequencies, ranging from 100 kHz to 15 MHz. In this paper, we describe the microfabrication and characterisation of an on-chip flow-cytometer as the first building block of a complete cell-sorting device. We then discuss the signal conditioning technique and finally impedance measurements of cells and particles of different sizes and types to demonstrate the differentiation of subpopulations in a mixed sample.

Flow Cytometry↗

PubMed2001

Distinct Ca2+ thresholds determine cytochrome c release or permeability transition pore opening in brain mitochondria.

In diseases associated with neuronal degeneration, such as Alzheimer's or cerebral ischemia, the cytosolic Ca2+ concentration ([Ca2+]cyt) is pathologically elevated. It is still unclear, however, under which conditions Ca2+ induces either apoptotic or necrotic neuronal cell death. Studying respiration and morphology of rat brain mitochondria, we found that extramitochondrial [Ca2+] above 1 M causes reversible release of cytochrome c, a key trigger of apoptosis. This event was NO-independent but required Ca2+ influx into the mitochondrial matrix. The mitochondrial permeability transition pore (PTP), widely thought to underlie cytochrome c release, was not involved. In contrast to noncerebral tissue, only relatively high [Ca2+] (is approximately equal to 200 M) opened PTP and ruptured mitochondria. Our findings might reflect a fundamental mechanism to protect postmitotic neuronal tissue against necrotic devastation and inflammation.

Animals↗

PubMed2001

Structure function relationships of ENaC and its role in sodium handling.

The epithelial sodium channel (ENaC) in the apical membrane of polarized epithelial cells is the rate-limiting step for Na entry into the cell; in series with the basolateral Na pump, it allows the vectorial transepithelial transport of Na ions. ENaC is expressed in different epithelia like the distal nephron or colon, and the airways epithelium. In the lung ENaC controls the composition and the amount of pulmonary fluid, whereas in the distal nephron ENaC under the control of aldosterone and vasopressin, is essential to adapt the amount of Na+ reabsorbed with the daily sodium intake. Activating mutations of ENaC cause severe disturbances of Na+ homeostasis leading to hypertension in human and in mouse models. Functional expression of ENaC in different cell systems allowed the identification of structural domains of the protein that are essential for channel function and/or modulation of channel activity. Site-directed mutations in specific domains of the channel protein lead to channel hyperactivity or channel loss of function. Knowledge about ENaC structure-function relationships opens new opportunities for development of pharmacological tools for controlling ENaC activity, such as channel activators of potential benefit in the treatment of pulmonary edema, or highly potent ENaC blockers with natriuretic effects.

Animals↗

PubMed2001

Permeability properties of ENaC selectivity filter mutants.

The epithelial Na(+) channel (ENaC), located in the apical membrane of tight epithelia, allows vectorial Na(+) absorption. The amiloride-sensitive ENaC is highly selective for Na(+) and Li(+) ions. There is growing evidence that the short stretch of amino acid residues (preM2) preceding the putative second transmembrane domain M2 forms the outer channel pore with the amiloride binding site and the narrow ion-selective region of the pore. We have shown previously that mutations of the alphaS589 residue in the preM2 segment change the ion selectivity, making the channel permeant to K(+) ions. To understand the molecular basis of this important change in ionic selectivity, we have substituted alphaS589 with amino acids of different sizes and physicochemical properties. Here, we show that the molecular cutoff of the channel pore for inorganic and organic cations increases with the size of the amino acid residue at position alpha589, indicating that alphaS589 mutations enlarge the pore at the selectivity filter. Mutants with an increased permeability to large cations show a decrease in the ENaC unitary conductance of small cations such as Na(+) and Li(+). These findings demonstrate the critical role of the pore size at the alphaS589 residue for the selectivity properties of ENaC. Our data are consistent with the main chain carbonyl oxygens of the alphaS589 residues lining the channel pore at the selectivity filter with their side chain pointing away from the pore lumen. We propose that the alphaS589 side chain is oriented toward the subunit-subunit interface and that substitution of alphaS589 by larger residues increases the pore diameter by adding extra volume at the subunit-subunit interface.

Amino Acids↗

PubMed2001

Trafficking and cell surface stability of ENaC.

The epithelial Na(+) channel (ENaC) plays a key role in the regulation of Na(+) and water absorption in several epithelia, including those of the distal nephron, distal colon, and lung. Accordingly, mutations in ENaC leading to reduced or increased channel activity cause human diseases such as pseudohypoaldosteronism type I or Liddle's syndrome, respectively. The gain of ENaC function in Liddle's syndrome is associated with increased activity and stability of the channel at the plasma membrane. Thus understanding the regulation of channel processing and trafficking to and stability at the cell surface is of fundamental importance. This review describes some of the recent advances in our understanding of ENaC trafficking, including the role of glycosylation, ENaC solubility in nonionic detergent, targeting signal(s) and hormones. It also describes the regulation of ENaC stability at the cell surface and the roles of the ubiquitin ligase Nedd4 (and ubiquitination) and clathrin-mediated endocytosis in that regulation.

Animals↗

PubMed2000

Adaptation of protein carbonyl detection to the requirements of proteome analysis demonstrated for hypoxia/reoxygenation in isolated rat liver mitochondria.

The key technique in proteome analysis is high-resolution two-dimensional (2D) electrophoretic separation of proteins from biological samples. This method combines isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Derivatization of protein carbonyls with 2, 4-dinitrophenylhydrazine (DNPH) and subsequent anti-dinitrophenyl (DNP) immunoblotting is widely used for the detection of oxidatively modified proteins. In previous studies on adapting this method to 2D electrophoresis the derivatization step was carried out before and after the 2D procedure, resulting in an altered spot pattern and high background staining, respectively. The aim of the present experiments was to develop a method for protein derivatization with DNPH between the IEF and the SDS-PAGE steps. Mitochondria were exposed to 10 min hypoxia and 5 min reoxygenation. After IEF using immobilized pH gradients the gel strips were incubated in DNPH/trifluoroacetic acid/SDS for 20 min and neutralized, and SDS-PAGE was performed. Proteins were either stained with Coomassie dye or subjected to Western blotting using anti-DNP IgG. Gels and blots were scanned and matched to a master gel, and the relative carbonyl content of each spot was calculated and compared for five experiments. Importantly, the spot patterns in DNPH-treated and untreated gels were not different. Protein carbonyls could be detected in 59 of the 125 matched spots. Although there was no significant increase in the total protein carbonyl content after hypoxia/reoxygenation, eighteen 2D spots exhibited an increase in carbonyl content. However, most protein spots did not show a change or even a decline (4 spots) in protein carbonyls.

Animals↗

PubMed2000

Regulation of the epithelial Na+ channel by Nedd4 and ubiquitination.

The epithelial Na+ channel (ENaC) is comprised of three subunits, alpha, beta and gamma, and plays an essential role in Na+ and fluid absorption in the kidney, colon and lung. We had identified proline-rich sequences at the C termini of alpha beta gamma ENaC, which include the sequence PPxY, the PY motif. This sequence in beta or gamma ENaC is deleted or mutated in Liddle's syndrome, a hereditary form of arterial hypertension. Our previous work demonstrated that these PY motifs bind to the WW domains of Nedd4, a ubiquitin protein ligase containing a C2 domain, three or four WW domains and a ubiquitin protein ligase Hect domain. Accordingly, we have recently demonstrated that Nedd4 regulates ENaC function by controlling the number of channels at the cell surface, that this regulation is impaired in ENaC bearing Liddle's syndrome mutations, and that ENaC stability and function are regulated by ubiquitination. The C2 domain is responsible for localizing Nedd4 to the plasma membrane in a Ca(2+)-dependent manner, and in polarized epithelial MDCK cells this localization is primarily apical. In accordance, electrophysiological characterization of ENaC expressed in MDCK cells revealed inhibition of channel activity by elevated intracellular Ca2+ levels. Thus, in response to Ca2+, Nedd4 may be mobilized to the apical membrane via its C2 domain, where it binds ENaC via Nedd4-WW:ENaC-PY motifs' interactions, leading to ubiquitination of the channel by the Nedd4-Hect domain and subsequent channel endocytosis and lysosomal degradation. This process may be at least partially impaired in Liddle's syndrome due to reduced Nedd4 binding, leading to increased retention of ENaC at the cell surface.

Animals↗

PubMed1999

Effect of adenine nucleotide pool size in mitochondria on intramitochondrial ATP levels.

Net adenine nucleotide transport into and out of the mitochondrial matrix via the ATP-Mg/Pi carrier is activated by micromolar calcium concentrations in rat liver mitochondria. The purpose of this study was to induce net adenine nucleotide transport by varying the substrate supply and/or extramitochondrial ATP consumption in order to evaluate the effect of the mitochondrial adenine nucleotide pool size on intramitochondrial adenine nucleotide patterns under phosphorylating conditions. Above 12 nmol/mg protein, intramitochondrial ATP/ADP increased with an increase in the mitochondrial adenine nucleotide pool. The relationship between the rate of respiration and the mitochondrial ADP concentration did not depend on the mitochondrial adenine nucleotide pool size up to 9 nmol ADP/mg mitochondrial protein. The results are compatible with the notion that net uptake of adenine nucleotides at low energy states supports intramitochondrial ATP consuming processes and energized mitochondria may lose adenine nucleotides. The decrease of the mitochondrial adenine nucleotide content below 9 nmol/mg protein inhibits oxidative phosphorylation. In particular, this could be the case within the postischemic phase which is characterized by low cytosolic adenine nucleotide concentrations and energized mitochondria.

Adenine Nucleotides↗

PubMed1999

A single point mutation in the pore region of the epithelial Na+ channel changes ion selectivity by modifying molecular sieving.

The epithelial Na+ channel (ENaC) belongs to a new class of channel proteins called the ENaC/DEG superfamily involved in epithelial Na+ transport, mechanotransduction, and neurotransmission. The role of ENaC in Na+ homeostasis and in the control of blood pressure has been demonstrated recently by the identification of mutations in ENaC beta and gamma subunits causing hypertension. The function of ENaC in Na+ reabsorption depends critically on its ability to discriminate between Na+ and other ions like K+ or Ca2+. ENaC is virtually impermeant to K+ ions, and the molecular basis for its high ionic selectivity is largely unknown. We have identified a conserved Ser residue in the second transmembrane domain of the ENaC alpha subunit (alphaS589), which when mutated allows larger ions such as K+, Rb+, Cs+, and divalent cations to pass through the channel. The relative ion permeability of each of the alphaS589 mutants is related inversely to the ionic radius of the permeant ion, indicating that alphaS589 mutations increase the molecular cutoff of the channel by modifying the pore geometry at the selectivity filter. Proper geometry of the pore is required to tightly accommodate Na+ and Li+ ions and to exclude larger cations. We provide evidence that ENaC discriminates between cations mainly on the basis of their size and the energy of dehydration.

Amino Acid Sequence↗

PubMed1999

Mutational analysis of cysteine-rich domains of the epithelium sodium channel (ENaC). Identification of cysteines essential for channel expression at the cell surface.

One of the characteristic features of the structure of the epithelial sodium channel family (ENaC) is the presence of two highly conserved cysteine-rich domains (CRD1 and CRD2) in the large extracellular loops of the proteins. We have studied the role of CRDs in the functional expression of rat alphabetagamma ENaC subunits by systematically mutating cysteine residues (singly or in combinations) into either serine or alanine. In the Xenopus oocyte expression system, mutations of two cysteines in CRD1 of alpha, beta, or gamma ENaC subunits led to a temperature-dependent inactivation of the channel. In CRD1, one of the cysteines of the rat alphaENaC subunit (Cys158) is homologous to Cys133 of the corresponding human subunit causing, when mutated to tyrosine (C133Y), pseudohypoaldosteronism type 1, a severe salt-loosing syndrome in neonates. In CRD2, mutation of two cysteines in alpha and beta but not in the gamma subunit also produced a temperature-dependent inactivation of the channel. The main features of the mutant cysteine channels are: (i) a decrease in cell surface expression of channel molecules that parallels the decrease in channel activity and (ii) a normal assembly or rate of degradation as assessed by nondenaturing co-immunoprecipitation of [35S]methionine-labeled channel protein. These data indicate that the two cysteines in CRD1 and CRD2 are not a prerequisite for subunit assembly and/or intrinsic channel activity. We propose that they play an essential role in the efficient transport of assembled channels to the plasma membrane.

Animals↗

PubMed1999

Identification of a highly conserved sequence at the N-terminus of the epithelial Na+ channel alpha subunit involved in gating.

The epithelial Na+ channel (ENaC) is responsible for Na+ reabsorption in aldosterone target tissues such as distal nephron and colon. ENaC is a heterotetramer composed of three homologous subunits, alpha, beta, and gammaENaC. Mutations leading to loss of function or reduced channel activity have been identified in all three subunits in patients with pseudohypoaldosteronism type-1. One missense mutation substituting a glycine (G95S) which is completely conserved throughout the gene family reduced ENaC open probability, Po. In this study we have performed systematic alanine substitutions of 28 residues of alphaENaC encompassing the glycine (G95). This screen identified a stretch of ten consecutive amino acids (alphaT92-alphaC101) which, when mutated, lead to a decrease in Na+ current (I(Na)) expressed with no significant changes in channel surface expression. This inhibitory effect was strongest for G95 and for two additional highly conserved amino acids--H94 and R98. The R98A mutant led to an important reduction in channel Po with no change in single-channel conductance, indicating that the segment encompassing H94, G95 and R98 is involved in modulation of channel gating kinetics.

Alanine↗

PubMed1999

Induction of permeability transition in pancreatic mitochondria by cerulein in rats.

Hyperstimulation with cholecystokinin analogue cerulein induces a mild edematous pancreatitis in rats. There is evidence for a diminished energy metabolism of acinar cells in this experimental model. The aim of this study was to demonstrate permeability transition of the mitochondrial inner membrane as an early change in mitochondrial function and morphology. As functional parameters, the respiration and membrane potential of mitochondria isolated from control and cerulein-treated animals were measured, and changes in volume and morphology were investigated by swelling experiments and electron microscopy. Five hours after the first injection of cerulein, the leak respiration was nearly doubled and the resting membrane potential was decreased by about 17 mV. These alterations were reversed by extramitochondrial ADP or did not occur when cyclosporin A was added to the mitochondrial incubation. A considerable portion of the mitochondria isolated from cerulein-treated animals was swollen and showed dramatic changes in morphology such as a wrinkled outer membrane and the loss of a distinct cristae structure. These data provide evidence for the opening of the mitochondrial permeability transition pore at an early stage of cerulein induced pancreatitis. This suggests that the permeability transition is an initiating event for lysis of individual mitochondria and the initiation of apoptosis and/or necrosis, as had been shown to occur in this experimental model.

Animals↗

PubMed1999

Interaction of nitric oxide donors and ascorbic acid on D-[3H] aspartate efflux from rat striatal slices.

There are conflicting reports in the literature concerning the neuroprotective effect of ascorbic acid on excitotoxic processes in which excessive glutamate release and nitric oxide are supposed to be major factors. To study the influence of ascorbate on the nitric oxide modulated glutamate release rat striatal slices, preloaded with the tritiated glutamate analog D-aspartate, were used. The high potassium-induced efflux of D-[3H]aspartate was concentration dependently stimulated by the nitric oxide donors sodium nitroprusside, S-nitroso-N-acetylpenicillamine (SNAP) or 5-amino 3-morpholinyl-1,2,3-oxadiazolium chloride (SIN-1), as well as by solutions of gaseous nitric oxide and, interestingly, by cyanide. Only the stimulation of D-[3H]aspartate release by SNAP and nitroprusside was affected by ascorbate in terms of a highly significant potentiation. Ascorbate was shown to exert its effect primarily by influencing the decomposition of these nitric oxide donors rather than by a direct interaction of ascorbate with nitric monoxide on glutamate release.

Animals↗

PubMed1999

On the molecular basis of ion permeation in the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.

Amiloride↗

PubMed1999

Defective regulation of the epithelial Na+ channel by Nedd4 in Liddle's syndrome.

Liddle's syndrome is an inherited form of hypertension linked to mutations in the epithelial Na+ channel (ENaC). ENaC is composed of three subunits (alpha, beta, gamma), each containing a COOH-terminal PY motif (xPPxY). Mutations causing Liddle's syndrome alter or delete the PY motifs of beta- or gamma-ENaC. We recently demonstrated that the ubiquitin-protein ligase Nedd4 binds these PY motifs and that ENaC is regulated by ubiquitination. Here, we investigate, using the Xenopus oocyte system, whether Nedd4 affects ENaC function. Overexpression of wild-type Nedd4, together with ENaC, inhibited channel activity, whereas a catalytically inactive Nedd4 stimulated it, likely by acting as a competitive antagonist to endogenous Nedd4. These effects were dependant on the PY motifs, because no Nedd4-mediated changes in channel activity were observed in ENaC lacking them. The effect of Nedd4 on ENaC missing only one PY motif (of beta-ENaC), as originally described in patients with Liddle's syndrome, was intermediate. Changes were due entirely to alterations in ENaC numbers at the plasma membrane, as determined by surface binding and immunofluorescence. Our results demonstrate that Nedd4 is a negative regulator of ENaC and suggest that the loss of Nedd4 binding sites in ENaC observed in Liddle's syndrome may explain the increase in channel number at the cell surface, increased Na+ reabsorption by the distal nephron, and hence the hypertension.

Animals↗