Biomedical subjects

W Nagel

Publications and source records attributed to W Nagel.

At least 37 records · Page 2Linked to original sources

PubMed1999

Trypsin inhibits voltage-activated chloride conductance of toad skin.

The effect of trypsin on the voltage-activated chloride conductance (GCl) of toad skin was investigated. Serosal application of > 0.1 mg ml-1 trypsin decreased the voltage-activated GCl without notable delay. The maximal inhibition to 38% of the control values, exerted within 15 min, was in some experiments partly or completely reversible. Chymotrypsin had much lower effect than trypsin. Mucosal application of trypsin did not have any effect. Trypsin did neither interfere with the conductive pathway opened by supramaximal concentrations of cAMP nor with the inhibitory effect of epinephrine on the voltage-activated GCl. The effect of trypsin required influx of Ca2+ from the extracellular space. It is concluded that protease-activated receptors or trypsin-sensitive proteins in the basolateral membrane of toad skin epithelial cells interfere with regulative steps involved in the voltage-activation of GCl. This may be harmful for the segregation of epithelial cells using this enzyme.

Animals↗

PubMed1998

Phosphoinositide 3-OH kinase activates the beta2 integrin adhesion pathway and induces membrane recruitment of cytohesin-1.

Signal transduction through phosphoinositide 3-OH kinase (PI 3-kinase) has been implicated in the regulation of lymphocyte adhesion mediated by integrin receptors. Cellular phosphorylation products of PI 3-kinases interact with a subset of pleckstrin homology (PH) domains, a module that has been shown to recruit proteins to cellular membranes. We have recently identified cytohesin-1, a cytoplasmic regulator of beta2 integrin adhesion to intercellular adhesion molecule 1. We describe here that expression of a constitutively active PI 3-kinase is sufficient for the activation of Jurkat cell adhesion to intercellular adhesion molecule 1, and for enhanced membrane association of cytohesin-1. Up-regulation of cell adhesion by PI 3-kinase and membrane association of endogenous cytohesin-1 is abrogated by overexpression of the isolated cytohesin-1 PH domain, but not by a mutant of the PH domain which fails to associate with the plasma membrane. The PH domain of Bruton's tyrosine kinase (Btk), although strongly associated with the plasma membrane, had no effect on either membrane recruitment of cytohesin-1 or on induction of adhesion by PI 3-kinase. Having delineated the critical steps of the beta2 integrin activation pathway by biochemical and functional analyses, we conclude that PI 3-kinase activates inside-out signaling of beta2 integrins at least partially through cytohesin-1.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1998

Mitochondria-rich cells and voltage-activated chloride current in toad skin epithelium: analysis with the scanning vibrating electrode technique.

The pathway for the voltage-activated chloride current across isolated toad skin was analyzed using a scanning 2D-vibrating voltage probe technique, which permits discrimination of local current peaks if their origins are more than 50 microns apart. The epithelium was separated from the corial connective tissue after enzymatic digestion with collagenase. Cl- current was activated by voltage clamping the transepithelial potential to 60-100 mV, serosa positive. Activated inward current was between 85 and 450 microA/cm2. In more than 25 tissue areas of 150 x 100 microns from 10 animals, which were automatically scanned with the vibrating probe, between 0 and 4 peaks of elevated local current (up to 800 microA/cm2) could be identified in individual fields. The density of current peaks, which were generally located at sites of mitochondria-rich (MR) cells, was less than 10% of the density of microscopically identified MR cells. The total current across individual sites of elevated conductance was 3.9 +/- 0.6 nA. Considering the density of peaks, they account for 17 +/- 2.5% of the applied transepithelial clamping current. The time course of current activation over previously identified conductive sites was in most cases unrelated to that of the total transepithelial current. Moreover, initially active sites could spontaneously inactivate. The results indicate that detection of elevated current above some MR cells is not sufficient to verify these cells as the pathway for transepithelial voltage-activated Cl- current. Since the major fraction of activated current is apparently not associated with a route through MR cells, channel-like structures in the tight junctions of the paracellular pathway must be considered as an alternative possibility. Current peaks over MR cells could be due to high density of such sites in tight junctions between MR and surrounding principal cells. Improvement of the spatial resolution of the vibrating probe is required to verify this view.

Animals↗

PubMed1998

alpha1-adrenoceptors antagonize activated chloride conductance of amphibian skin epithelium.

The effects of adrenoceptor agonists on the transepithelial Cl- conductance (GCl) in the skin of several amphibian species, both toads and frogs, were studied. Epinephrine (Epi) from the serosal side selectively and reversibly inhibited the voltage-activated GCl in toad skin and the short-circuit GCl in frog skin. The main effects of activation of the adrenoceptors must reside in the skin epithelium and not in the glands, since measurements were made both from intact skins and split epithelia with essentially the same results. Effective concentrations of Epi were variable among individual tissues. GCl was reduced to 34+/-17% (n=46) with 1 micromol/l Epi, but in some tissues 0.1 micromol/l inhibited more than 80% of GCl, whereas some preparations were little influenced at >3 micromol/l Epi. The affected receptor type was identified by the use of the alpha1-agonist phenylephrine, which mimicked the response of Epi at concentrations above 30 micromol/l, whereas the alpha2-agonists xylazine and iodoclonidine had no effect at supramaximal concentrations. Prazosin, a specific alpha1-antagonist, reduced or eliminated the inhibition by Epi, but the response pattern suggests a low affinity. The alpha2-antagonist yohimbine, at concentrations </=0.3 micromol/l, had a minimal effect, but reduced the inhibition by Epi at concentrations of 1-10 micromol/l. This might indicate affinity to alpha1-adrenoceptors in amphibian skin. Activation of beta-adrenoceptors by isoproterenol (0.1-5 micromol/l) led to a transient increase of the baseline inactivated component of GCl with a slight reduction of the voltage-activated GCl at the higher concentrations, but the inhibitory effect of Epi was not altered. Epi, on the other hand, neither prevented nor reversed the induction of a voltage-insensitive GCl in toad skin caused by application of cAMP at supramaximal concentrations (>100 micromol/l CPT-cAMP). Preincubation of the serosal medium with Ca2+-free solution (in the presence of 2 mmol/l EGTA) for extended periods of time (>30 min) eliminated the response to Epi. It is concluded that alpha1-adrenoceptors participate in the physiological control of voltage-activated Cl- conductance in amphibian skin epithelium via modulation of intracellular Ca2+, presumably by efflux from intracellular stores.

1-Methyl-3-isobutylxanthine↗

PubMed1998

The PH domain and the polybasic c domain of cytohesin-1 cooperate specifically in plasma membrane association and cellular function.

Recruitment of intracellular proteins to the plasma membrane is a commonly found requirement for the initiation of signal transduction events. The recently discovered pleckstrin homology (PH) domain, a structurally conserved element found in approximately 100 signaling proteins, has been implicated in this function, because some PH domains have been described to be involved in plasma membrane association. Furthermore, several PH domains bind to the phosphoinositides phosphatidylinositol-(4,5)-bisphosphate and phosphatidylinositol-(3,4,5)-trisphosphate in vitro, however, mostly with low affinity. It is unclear how such weak interactions can be responsible for observed membrane binding in vivo as well as the resulting biological phenomena. Here, we investigate the structural and functional requirements for membrane association of cytohesin-1, a recently discovered regulatory protein of T cell adhesion. We demonstrate that both the PH domain and the adjacent carboxyl-terminal polybasic sequence of cytohesin-1 (c domain) are necessary for plasma membrane association and biological function, namely interference with Jurkat cell adhesion to intercellular adhesion molecule 1. Biosensor measurements revealed that phosphatidylinositol-(3,4,5)-trisphosphate binds to the PH domain and c domain together with high affinity (100 nM), whereas the isolated PH domain has a substantially lower affinity (2-3 microM). The cooperativity of both elements appears specific, because a chimeric protein, consisting of the c domain of cytohesin-1 and the PH domain of the beta-adrenergic receptor kinase does not associate with membranes, nor does it inhibit adhesion. Moreover, replacement of the c domain of cytohesin-1 with a palmitoylation-isoprenylation motif partially restored the biological function, but the specific targeting to the plasma membrane was not retained. Thus we conclude that two elements of cytohesin-1, the PH domain and the c domain, are required and sufficient for membrane association. This appears to be a common mechanism for plasma membrane targeting of PH domains, because we observed a similar functional cooperativity of the PH domain of Bruton's tyrosine kinase with the adjacent Bruton's tyrosine kinase motif, a novel zinc-containing fold.

Amino Acid Sequence↗

PubMed1998

Effects of aminoperimidine on electrolyte transport across amphibian skin.

The effect of aminoperimidine (AP) on transepithelial Na+ transport and Cl- conductance (G(Cl)) of isolated amphibian skin (Bufo viridis and Rana esculenta) was analyzed using transepithelial and intracellular electrophysiological techniques. AP, applied at concentrations between 30 and 100 microM from the mucosal side, stimulated Na+ transport rapidly and reversibly by more than 30% of the control value due to an increase in apical membrane Na+ permeability. Influence of AP on basolateral membrane conductance and effective driving force for Na+ were negligible. Voltage-activated G(Cl) of toad skin, but not the resting, deactivated conductance, as well as spontaneously high G(Cl) in frog skin was rapidly inhibited by AP in a concentration-dependent manner. The half-maximal inhibitory concentration of 20 microM is the highest hitherto reported inhibitory power for G(Cl) in amphibian skin. The effect of AP on G(Cl) was slowly and incompletely reversible even after brief exposure to the agent. Serosal application of AP had similar, albeit delayed effects on both Na+ and Cl- transport. AP did not interfere with the Cl- pathway after it was opened by 100-300 microM CPT-cAMP, a membrane-permeable, nonhydrolyzed analogue of cAMP. Inhibition of the voltage-activated G(Cl) by AP was attenuated or missing when AP was applied during voltage perturbation to serosa-positive potentials. Since AP is positively charged at physiological pH, it suggests that the affected site is located inside the Cl- pathway at a certain distance from the external surface. AP affects then the Na+ and Cl- transport pathways independent of each other. The nature of chemical interference with AP, which is responsible for the influence on the transport of Na+ and Cl-, remains to be elucidated.

Amiloride↗

PubMed1998

Localizing transepithelial conductive pathways using a vibrating voltage probe.

A model simulation of the electrical field distribution for the voltage-activated C1- current across amphibian skin has been carried out for two potential pathways, i.e. transcellularly, through the mitochondria-rich cells (MRCs), or paracellularly, across the tight junctions (TJs) between the outermost living cell layer. The calculations are based on the mean density of MRCs and the typical dimensions of stratum granulosum cells of amphibian skin. It is demonstrated that current flow across MRCs would be detectable by scanning with the vibrating voltage probe in the extracellular space above the epithelium, whereas accurate representation of current flow through the TJs cannot be obtained using a probe of the present design. The experimental data indicate that field patterns corresponding to an MRC origin for the C1- current are never observed. It is concluded that the voltage-activated C1- conductance is localized to ion-selective structures in the paracellular pathway which may be regulated by the TJs.

Animals↗

PubMed1997

Effects of NEM on voltage-activated chloride conductance in toad skin.

The regulation of the voltage-activated chloride current conductance (GCl) in toad skin was investigated by the use of the SH reagents N-ethylmaleimide (NEM) and p-chloro-mercuricbenzenesulfonic acid PCMBS. This anion pathway is controlled by a voltage-sensitive gating regulator. Mucosal application of NEM decreased the voltage-activation in a time and concentration dependent manner, half-maximal inhibition being exerted at a concentration of 30 microM within 20 min. At concentrations higher than 100 microM, the voltage-activated GCl was near-completely and irreversibly inhibited in less than 10 min. Resting, deactivated conductance was essentially unaffected. NEM had no effect on active sodium transport (measured as Isc) under conditions, which fully dissipated the voltage-activated GCl. After complete inhibition of the voltage-activated GCl with NEM, chloride conductance could still be stimulated by CPT-cAMP as in control tissues. Under these conditions, NEM at concentrations above 1 mm decreased GCl reversibly. Mucosal application of PCMBS at 500 microM inhibited the activated conductance by 35%, which was slightly reversible. Inhibition of voltage-activated GCl, which was observed after mucosal addition of the membrane-impermeable NEM analogue, eosin-5-maleimide, was completely reversible after washout. This suggests that the binding site for the maleimide is not accessible from the external face of the apical membrane. Brief application of NEM at lower concentrations (1-3 min, </= 100 microM) led to partial inhibition of GCl, followed by occasionally complete recovery upon washout of NEM. Recovery of voltage-activated GCl was progressively attenuated and eventually disappeared after subsequent brief applications of NEM. This could reflect recruitment of permeation/control sites from a finite pool. The data are discussed in the frame of a working model for the voltage-activated Cl--pathway, that contains two principle components, i.e., an anion-selective permeation path which is controlled by regulatory protein(s).

4-Chloromercuribenzenesulfonate↗

PubMed1997

[Carotid endarterectomy--5-year results in St. Gallen canton hospital].

The 5-year results following carotid endarterectomy at our institution were analysed retrospectively. The aim of this study was to compare our results with those published in the ECST and NASCET studies as a measure of quality control. We studied the outcome of 101 carotid endarterectomies which were performed between 1989 and 1993 in 90 patients. 74 operations were carried out in "symptomatic" and 27 in "asymptomatic" patients. The standard anaesthetic procedure was a cervical plexus block, which was completed in 87% of all operations. The 84 surviving patients on December the 31th 1993 were all followed up with a clinical and directional Doppler examination in our angiologic outpatient clinic during the first half of 1994. The perioperative stroke rate was 1.98% for the whole group and 2.7% for the "symptomatic" group. The cumulative stroke rate for a period of three years (measured by the Kapplan-Meier method) amounted to 3% (1% per year); the cumulative stroke rate for the "symptomatic" group was 4% in a two year period (2% per year). The results and outcome of carotid endarterectomy at our institution are comparable to the ECST and NASCET studies regarding perioperative mortality, perioperative stroke rate and rate of ipsilateral stroke during the first 2 years after operation.

Aged↗

PubMed1996

Alpha L beta 2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a cytoplasmic regulatory molecule.

The avidity of integrin adhesion receptors for extracellular ligands is subject to dynamic regulation by intracellular programs that have yet to be elucidated. We describe here a protein, cytohesin-1, which specifically interacts with the intracellular portion of the integrin beta 2 chain (CD18). The molecule shows homology to the yeast SEC7 gene product and bears a pleckstrin homology (PH) domain. Overexpression of either the full-length cytohesin-1 or the SEC7 domain induces beta 2 integrin-dependent binding of Jurkat cells to ICAM-1, whereas expression of the isolated cytohesin-1 PH domain inhibits T cell receptor-stimulated adhesion. Similar inhibition is not exhibited by PH domains taken from other proteins, showing that the interaction is specific and that individual PH domains are capable of discriminating between alternative targets.

Amino Acid Sequence↗

PubMed1995

Effects of cyclic AMP and theophylline on chloride conductance across toad skin.

1. The effects of the phosphodiesterase inhibitors theophylline and isobutylmethylxanthine (IBMX) on baseline and voltage-activated Cl- conductance (gCl) of toad skin were compared with those of the potent 2-chlorophenylthio analogue of cAMP (CPT-cAMP). 2. Using intact and split skins of Bufo viridis we confirmed that theophylline and IBMX raised the voltage-activated gCl with a pattern identical to that seen under control conditions. This effect was small or missing if gCl was already high in the control. 3. CPT-cAMP, in contrast, increased the Cl(-)-specific conductance by up to 6 mS cm-2 at short circuit. The characteristic time-dependent, slow activation of gCl by serosa-positive clamp potentials was completely lost under these conditions. 4. Coinciding with the loss of voltage activation of gCl the plateau value of the Lorentzian component of fluctuation in current at serosa-positive clamp potentials decreased by almost 50%. The corner frequencies were not notably different. 5. After CPT-cAMP, the sigmoidal voltage-conductance relation that is characteristic of control conditions or after theophylline disappeared; the patterns were variable and incompatible with voltage activation. 6. The voltage-activated gCl under control conditions and with theophylline was blocked by mucosal NO3-, I- or SCN-, the last two being almost equally effective. In the presence of CPT-cAMP, mucosal NO3- had minimal influence on tissue conductance, whereas the effects of I- and SCN- were essentially unchanged. Br- on the mucosal side could substitute for Cl- under all conditions. 7. The results suggest that protein phosphorylation by supramaximal concentrations of cAMP induces maximal conductance through anion-specific routes, while the voltage sensitivity of this pathway is lost. The effects of theophylline and IBMX on the voltage-activated Cl-conductance of toad skin cannot be explained solely by inhibition of the phosphodiesterase.

Animals↗

PubMed1994

[Results in surgery of bronchus carcinoma].

During a 6-year period from 1986 to 1991, 119 patients with lung cancer underwent thoracotomy. 112 underwent surgical resection with lymphadenectomy. The mean age of the 97 male patients was 62.7 years, and of the 15 female patients was 62.7 years, and of the 15 female patients 57.1 years. 54 patients were in stage I, 23 stage II, 21 stage IIIa, 8 stage IIIb and 6 stage IV. Pneumonectomy was performed in 20 cases, bilobectomy in 2, lobectomy in 81, segmentectomy in 3 and sleeve lobectomy in 6. Perioperative death occurred in 2.7% of all patients, 5% of patients who underwent pneumonectomy and 2.2% of patients who underwent lobectomy. Serious complications occurred in 19% of all thoracotomy patients. The re-thoracotomy rate was 6% and mortality 0.8%. The cumulative 5-year survival rate in all 112 patients with surgical resection was 54.6%, and for the 54 patients with radical resection 60.3%.

Aged↗

PubMed1994

Electron microprobe analysis of electrolytes in whole cultured epithelial cells.

Microprobe analysis was used to determine electrolyte contents in whole epithelial sheets of A6 cells and to investigate the most critical points of this method. Analysis of dextran standard sections of different thickness revealed that low accelerating voltages of about 10 kV are best suited for whole freeze-dried cells on thick supports, since 5 microM thick sections are not penetrated by 10 kV electrons. Washing of A6 cells for 10 sec with distilled water led to cell swelling of about 40%, but the molar concentration ratios and the concentrations per dry weight (dw) were not altered. Washing for 60 sec with distilled water caused a further increase in cell volume (120%) and loss of cellular K and Cl (90 mmol/kg dw). Washing with isotonic NH4- acetate led to a loss of cell Cl already after 10 sec. To characterize the Na transport compartment, A6 cells cultured on permeable supports were washed for 5 sec with distilled water, freeze-dried, and analyzed. Inhibition of transepithelial Na transport by ouabain increased Na/P from 0.15 +/- 0.07 to 0.75 +/- 0.03 and Cl/P from 0.21 +/- 0.001 to 0.38 +/- 0.003 while K/P decreased from 0.83 +/- 0.08 to 0.32 +/- 0.03. The changes in cell Na and K contents can be explained by K/Na exchange; the increase in Cl content indicates some cell swelling. Since the ouabain-induced changes could be prevented by apical amiloride, the apical membrane provides the most important pathway for Na entry in A6 cells.

Animals↗

PubMed1993

Intestinal administration of copper and its transient release into venous rat blood serum concomitantly with metallothionein.

The molecular side of copper transport in biological systems is unknown. It was attempted to examine the copper and metallothionein (MT) release into the portal blood in rats in vivo. After direct administration of Cu(II) into the jejunum the copper and MT levels were distinctively higher in the portal venous serum compared with that of the vena cava inferior. MT in gel filtrated serum samples was analyzed immunologically employing ELISA and a monoclonal antibody to rat MT-I. Affinity chromatography on Protein A-Sepharose resulted in a higher immunoreactivity in the portal compartment as deduced from an elevated MT-antibody complex. It is assumed that MT serves as a genuine transport system for cuprous copper during the mucosal-to-serosal flux of this biologically important transition metal.

Animals↗

PubMed1993

K+ recirculation in A6 cells at increased Na+ transport rates.

Homocellular regulation of K+ at increased transcellular Na+ transport implies an increase in K+ exit to match the intracellular K+ load. Increased K+ conductance, gK, was suggested to account for this gain. We tested whether such a mechanism is operational in A6 monolayers. Na+ transport was increased from 5.1 +/- 1.0 microA/cm2 to 20.7 +/- 1.3 microA/cm2 by preincubation with 0.1 mumol/l dexamethasone for 24 h. Basolateral K+ conductances were derived from transference numbers of K+, tK, and basolateral membrane conductances, gb, using conventional microelectrodes and circuit analysis with application of amiloride. Activation of Na+ transport induced an increase in gb from 0.333 +/- 0.067 mS/cm2 to 1.160 +/- 0.196 mS/cm2 and tK was reduced to 0.22 +/- 0.01 from a value of 0.70 +/- 0.05 in untreated control tissues. As a result, gK remained virtually unchanged at increased Na+ transport rates. The increase in gb after dexamethasone was due to activation of a conductive leak pathway presumably for Cl-. Increased K+ efflux, IK, was a consequence of the larger driving force for K+ exit due to depolarization at an elevated Na+ transport rate. The relationship between calculated K+ fluxes and Na+ transport rate, measured as the Isc, is described by the linear function IK = 0.624 x INa -0.079, which conforms with a stoichiometry 2:3 for the fluxes of K+ and Na+ in the Na+/K(+)-ATPase pathway. Our data show that homocellular regulation of K+ in A6 cells is not due to up-regulation of gK.

Amiloride↗

PubMed1992

Osmotic swelling and membrane conductances in A6 cells.

Hyposmotic basolateral perturbations (-30 mosmol/kg) in cultured renal layers (A6) increased basolateral membrane conductance more than 2-fold within 10 min; the increase was partly due to upregulation of K+ conductance, but other conductive pathways were also activated. The raise in apical membrane amiloride-sensitive Na+ conductance was less pronounced; it appears to be due to secondary effects.

Animals↗

PubMed1992

Basolateral membrane conductance in A6 cells: effect of high sodium transport rate.

Conductance of apical and basolateral membranes in short-circuited cultured renal distal cells (A6) was determined using microelectrodes. Epithelia were pre-incubated with 0.1 mumol/l dexamethasone in the presence of 4 mumol/l amiloride to prevent increase in apical Na+ entry. Omission of amiloride increased the Isc from 5.7 to 27.6 microA/cm2 due to the rise in apical membrane conductance from 21 to 595 microS/cm2. Apical fractional resistance decreased from 0.89 to 0.40 and cells depolarized from -52 to -4 mV. Basolateral membrane conductance, which was 320 microS/cm2 at partially inhibited transport, was not significantly altered during the first 2 min following establishment of high transport activity; it started to increase thereafter reaching a more than threefold higher value of 1324 microS/cm2 within 12 min. The gain cannot be explained by increase in partial K+ conductance. Disappearance of the conductance after reduction of basolateral Cl- or in the presence of the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate indicates a Cl- conductance, which appears to be activated by depolarization.

Amiloride↗

PubMed1992

Effect of forskolin on conductive anion pathways of toad skin.

The effect of the diterpene, forskolin, on pathways for conductive Cl- transport was analyzed using isolated skins of Bufo viridis. Forskolin did not stimulate the voltage-activated Cl- movement from mucosa to serosa; the Lorentzian component in the power density spectrum, which was present at serosa positive clamp potentials under control conditions, decreased significantly. The observation that stimulation of cytosolic adenosine 3'-5'-cyclic monophosphate (cAMP) by forskolin has no effect on the voltage-activated Cl- transport argues against control of this pathway by cAMP. Our data further demonstrate that the forskolin-activated Cl- conductive pathway is also permeable for NO3-. This pathway was studied in absence of mucosal Cl-, which eliminates Cl- movement through the voltage-activated pathway. With SO4(2-) and Cl- on the mucosal and serosal sides, respectively, this forskolin-induced pathway displayed a linear current-voltage relationship. The associated Lorentzians increased at serosa negative clamp potentials. Transepithelial current and plateau value of the Lorentzian were related by a quadratic function, which suggests voltage-independence of number and open-close probability of these conductance sites. Morphological sites for voltage-activated and forskolin-induced conductive Cl- transport remain to be identified.

Animals↗