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Biomedical subjects

F Lang

Publications and source records attributed to F Lang.

At least 289 records · Page 16Linked to original sources
PubMed1994

Increase of c-jun mRNA upon hypo-osmotic cell swelling of rat hepatoma cells.

c-jun mRNA levels were increased in rat hepatoma cells (H4-II-E-C3) when exposed to hypotonic medium (205 mosmol/l) with a maximal induction observed after 1 h of hypotonic exposure. At this time point an approximate 5-fold increase in c-jun expression could be detected in relation to normotonic control incubations (305 mosmol/l). Hypertonic exposure (405 mosmol/l) had only a slight effect on c-jun expression. In contrast to the increased c-jun mRNA levels under hypotonic conditions, expression of the c-fos proto-oncogene was unaffected by changes in the osmolarity. The hypotonicity-induced increase in c-jun expression was also detectable in the presence of a protein kinase C (PKC) inhibitor. This indicates that PKC is not involved in the signal transduction pathway leading to c-jun expression upon hypotonic cell swelling in these cells.

Animals↗
PubMed1994

Alkalinization of acidic cellular compartments following cell swelling.

Osmotic swelling of rat hepatocytes increases fluorescence of Acridine orange and of fluorescein isothiocyanate (FITC)-dextran, both indicative of alkalinization of acidic intracellular vesicles. Similar to osmotic cell swelling, insulin and glutamine lead to an increase in Acridine orange fluorescence, an effect virtually abolished upon osmotic reversal of glutamine-induced cell swelling. Barium, which blocks K+ channels in the plasma membrane, similarly leads to cell swelling and increase of Acridine orange fluorescence. Since proteolysis is governed by lysosomal pH, these observations indicate that the anti-proteolytic action of osmotic cell swelling is mediated by lysosomal alkalinization. Thereby, insulin, glutamine and barium probably exert their anti-proteolytic action by cell swelling and subsequent lysosomal alkalinization.

Acridine Orange↗
PubMed1994

Atomic force microscopy of peritoneal macrophages after particle phagocytosis.

The atomic force microscope was used to image peritoneal macrophages after phagocytosis of latex beads with 0.45 microns in diameter and of zymosan particles. The rigidity of the phagocytosed material allowed to image the live membrane at forces below 2 nN. Repeated scanning of the membrane unavoidably caused the protrusion of the beads and increased their virtual height. The influence of fixation by glutaraldehyde on the image and the corresponding force vs. distance curves were analyzed and compared. Short treatment with Triton X-100 enabled us to identify intracellular components, such as embedded latex beads, cell nucleus and cytoskeletal strands. The data demonstrate that it is possible to image living cells if they are bolstered by stiff material.

Animals↗
PubMed1994

Morphological alterations and cytoskeletal reorganization in opossum kidney (OK) cells during osmotic swelling and volume regulation.

Cells from a variety of tissues regulate their volume when exposed to anisotonic conditions. After exposure of cells to hypotonic conditions, the rapid phase of cell swelling is followed by a slower phase of cell shrinkage towards the initial volume. The present study investigates morphological alterations of adherent and fully spread cells after exposure to hypotonic conditions and the reorganization of cytoskeletal components such as F-actin, actin-binding proteins, microtubules and intermediate-sized filaments. We used cells of a continuous epithelial cell line from the opossum kidney (OK cells), which were exposed to hypotonic conditions for a period of 60 min at 25 degrees C. The osmolarity was reduced by 40% from 320 mosmol/l (isotonic conditions) to 192 mosmol/l (hypotonic conditions). The initial swelling after exposure of OK cells to hypotonic conditions caused enhanced ruffling membrane activity, formation of lamellipodia and an extended space between adjacent cells which was caused by a more rounded cell shape. Moreover, the height of cells located in the centre of cell clusters increased by 32 +/- 8% (mean value +/- SEM) as checked by morphometric analysis of the vertical distance between the apical and basolateral F-actin domain. Although the fluorescence intensity and organization of F-actin in a horizontal direction remained unaltered during cell swelling, we observed a loss of periodicity and irregular distribution of myosin aggregates and a partial rearrangement of vimentin filaments in the form of short fragments. In all experiments the organization of microtubles was observed to be unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1994

Effects of urea on K+ fluxes and cell volume in perfused rat liver.

Exposure of the perfused rat liver to a perfusate made hyperosmotic by the presence of 200 mmol l-1 glucose led, as expected, to marked, transient hepatocellular shrinkage followed by volume-regulatory net K+ uptake. However, even after this volume-regulatory K+ uptake had ceased, the liver cells are still slightly shrunken. Withdrawal of glucose from the perfusate resulted in marked transient cell swelling, net K+ release from the liver and restoration of cell volume. However, when the Krebs-Henseleit perfusate was made hyperosmotic by the presence of urea (20-300 mM), there was no immediate decrease in liver mass, yet a slight and persistent cell shrinkage developing 2 min after the onset of exposure to urea. Surprisingly, urea induced concentration-dependent net K+ efflux from the liver and removal of urea net K+ reuptake from the inflowing perfusate. The urea (200 mM)-induced net K+ release resembled that observed following a lowering of the influent [NaCl]: making the perfusate hypoosmotic (245 mosmol l-1, by reducing influent [NaCl] by 30 mM) gave roughly the same K+ response as hyperosmotic exposure (505 mosmol/l) resulting from the presence of 200 mM urea. The urea-induced K+ efflux was not inhibited in the presence of ouabain (1 mM), or in Ca(++)-free perfusion, but was modified in the presence of quinidine (1 mM) or Ba++ (1 mM). The direction in which the liver was perfused had no effect on the urea-induced net K+ release.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

H2O2 induced hyperpolarization of pancreatic B-cells.

Conventional electrophysiology and the whole-cell patch-clamp technique have been applied to elucidate the effects of H2O2 on pancreatic B-cells of the mouse. In these cells, addition of 15 mmol/l glucose leads to depolarization and oscillation of the cell membrane potential. Subsequent addition of H2O2 (1 mmol/l) in the presence of glucose was followed by a marked and rapid hyperpolarization of the cell membrane with suppression of the electrical activity. Accordingly, in slow whole-cell patch-clamp experiments (with nystatin in the pipette solution) H2O2 induced a marked increase of cell membrane conductance. Tolbutamide, a blocker of K+ ATP channels, only partially blocked the effect of H2O2 even at high concentrations. The H2O2-induced, tolbutamide-insensitive current component, however, was largely abolished by a high concentration of TEA+ (80 mmol/l) or BaCl2 (10 mmol/l). It is concluded that in B-cells H2O2 stimulates a K+ current and that this effect leads to marked hyperpolarization and reversal of glucose-induced oscillations of cell membrane potential.

Animals↗
PubMed1994

Regulation of cell function by the cellular hydration state.

Cellular hydration can change within minutes under the influence of hormones, nutrients, and oxidative stress. Such short-term modulation of cell volume within a narrow range acts per se as a potent signal which modifies cellular metabolism and gene expression. It appears that cell swelling and cell shrinkage lead to certain opposite patterns of cellular metabolic function. Apparently, hormones and amino acids can trigger those patterns simply by altering cell volume. Thus alterations of cellular hydration may represent another important mechanism for metabolic control and act as another second or third messenger linking cell function to hormonal and environmental alterations.

Amino Acids↗
PubMed1994

Regulation of proximal renal tubular K+ conductance by intracellular pH.

Conventional electrophysiology and 2', 7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein fluorescence have been applied to elucidate the effects of metabolic acidosis on straight proximal tubules of the mouse kidney. Reduction of extracellular bicarbonate concentration from 20 to 10 mmol/l leads to a decline of intracellular pH from 7.00 +/- 0.06 to 6.85 +/- 0.05, a depolarization of the cell membrane (PDbl) from -72 +/- 1 to -59 +/- 2 mV, a decrease of the basolateral transference number for potassium (tK) from 0.80 +/- 0.01 to 0.54 +/- 0.03, an increase of the basolateral transference number for bicarbonate (tb) from 0.16 +/- 0.02 to 0.42 +/- 0.03 and an increase of the fractional resistance of the basolateral over the luminal cell membrane (Rb/Ra) by 64 +/- 8%. Upon return to 20 mmol/l bicarbonate after a 5-min exposure to 10 mmol/l bicarbonate, the intracellular pH approached a more alkaline value (7.28 +/- 0.08) than before exposure to acidosis. Despite the intracellular alkalosis, PDbl (-67 +/- 1 mV) and tK (0.73 +/- 0.02) remained significantly below, and tb (0.26 +/- 0.02) and Rb/Ra (32 +/- 8%) significantly above the respective values before induction of acidosis. Even transient exposure of the tubules to 40 mmol/l extracellular bicarbonate did not restore the original electrophysiological properties of the tubule cells. It is concluded that both a rapidly reversible and a long-lasting decrease of proximal tubular K+ conductance follows cellular acidosis.

Acidosis↗
PubMed1994

Positive regulation by chloride channel blockers of IsK channels expressed in Xenopus oocytes.

cRNA encoding the human IsK protein was injected into Xenopus oocytes and the induced IsK channels were investigated using the two-microelectrode voltage-clamp method. Niflumic acid, mefenamic acid, flufenamic acid, and 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid, which are commonly used in Xenopus oocytes to suppress endogenous Ca(2+)-activated Cl- channels, were tested for their effects on IsK channels. At low concentrations (10 microM) all compounds increased IsK amplitude and decreased the rate of IsK deactivation. At 100 microM these compounds further decreased the rate of IsK deactivation, resulting in persistent activation of IsK, similar to what has been previously described for the action of organic cross-linkers on IsK. However, at 100 microM niflumic acid and flufenamic acid decreased the time-dependent outward current, whereas 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and mefenamic acid caused an additional increase. When Cl- was completely substituted with gluconate, IsK had somewhat altered activation properties, but niflumic acid produced similar positive regulatory effects on IsK and shifted the voltage needed to evoke half-maximal IsK activation (V1/2) by about -20 mV. In summary, these compounds positively regulate IsK, presumably by stabilizing open IsK channels.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Cytoskeletal reorganization in NIH 3T3 fibroblasts expressing the ras oncogene.

Expression of the Ha-ras oncogene in NIH 3T3 fibroblasts leads to a set point shift of cell volume regulation and causes an increase in cell volume by activation of Na+/H+ exchange and Na+, K+, 2Cl- cotransport. Since both ion transport systems are thought to be governed by the cytoskeleton, the aim of this study was to examine the alterations in growth characteristics and cytoskeletal organization due to the expression of the oncogene. The experiments were performed on NIH 3T3 fibroblasts transfected with a transforming Ha-ras MMTV-LTR construct and expressing the oncogene after treatment with low serum medium and 1 mumol/l dexamethasone (+ras cells). Transfected cells not expressing the oncogene (-ras cells) and treated with low serum medium, but without the addition of dexamethasone, served as controls. The growth characteristics were examined and the cytoskeletal architecture was visualized by indirect immunofluorescence microscopy using specific antibodies and fluorescent dyes. Expression of the ras oncogene was accompanied by a significant and serum-independent increase in proliferative activity irrespective from the coating of the dishes with attachment factors (poly-L-lysine, collagen type I). Both, -ras and +ras cells, proliferated slower on substrates coated with poly-L-lysine than on tissue culture plastic or collagen type I. Expression of the ras oncogene also resulted in a significant increase in cell volume which was independent from the substrate. +ras Cells became more elongated, exhibited long cytoplasmic protrusions and tended to detach when compared with -ras cells. Examination of the cytoskeletal architecture in +ras and -ras cells revealed marked differences such as a depolymerization of the stress fiber network to strongly fluorescent "focals" as well as the absence of vinculin-containing attachment plaques (focal contacts), a disorganization of non-muscle myosin and of cell surface fibronectin in +ras cells. In addition, a retraction of microtubules and vimentin filaments to the perinuclear region was also observed in +ras cells. For comparison, NIH 3T3 fibroblasts which were not transfected with the ras oncogene (0ras cells) and which were also subjected to the experimental conditions described above (low serum medium +/- dexamethasone), did not exhibit the cytoskeletal alterations as observed for +ras cells. The results demonstrate that the expression of ras oncogene causes not only profound alterations in the proliferative activity, cell volume and cell morphology, but also a marked reorganization of cytoskeletal architecture, which may participate in the altered regulation of volume-regulatory ion transporters in the cell membrane.

3T3 Cells↗
PubMed1993

Time dependent changes in biophysical properties of minK channels expressed in Xenopus oocytes.

Slowly activating, voltage-dependent minK channels cloned from rat kidney were expressed in Xenopus oocytes. The maximal conductance (gmax) through these channels increased from 11 microS at day two after mRNA injection to 22 microS after one week. This increase of gmax is presumably the result of an increase in the number of functional channels through protein synthesis of the oocyte. Concurrent with the increase of gmax the voltage needed to evoke a half-maximal conductance (V1/2) was shifted to more negative potentials, while the activation of minK channels was accelerated. These results suggest a relation between protein density and activation of minK channels. Twofold changes of gmax were also observed in oocytes expressing the Shaker related rat potassium channel RBK1; however, there was no relation between gmax, V1/2 and the activation kinetics of RBK1. These results are consistent with the hypothesis that minK channel-formation and activation might involve subunit assembly.

Animals↗
PubMed1993

Endogenous hydroperoxide formation, cell volume and cellular K+ balance in perfused rat liver.

Addition of benzylamine (0.5 mM) to isolated perfused rat liver led to a net release of K+ of 10.5 +/- 0.3 mumol/g, which was accompanied by a decrease in liver mass by 9.3 +/- 0.4% and a decrease of the intracellular water space by 13.7 +/- 0.6%, suggestive of hepatocellular shrinkage. Benzylamine had no effect on the perfusion pressure, and there was a close relationship between benzylamine-induced net K+ release and the accompanying decrease in liver mass. Benzylamine-induced net K+ release was sensitive to inhibition of monoamine oxidase by pargyline and increased with benzylamine flux through monoamine oxidase, suggesting its dependence on intracellular H2O2 formation. In line with this, infusion of H2O2 (but not of benzaldehyde, the other product of benzylamine metabolism) stimulated net K+ release from the liver. However, at a given H2O2 load K+ release was about 2-3-fold higher when H2O2 was generated intracellularly during the oxidation of benzylamine, as compared with exogenously delivered H2O2. Inhibition of catalase by 3-amino-1,2,4-triazole (0.2 mM) significantly increased the benzylamine-induced net K+ release as well as the benzylamine-induced release of GSSG into bile, but had no effect on benzylamine oxidation at monoamine oxidase. In the presence of Ba2+ (1 mM) or in Ca(2+)-free perfusions, the benzylamine-induced net K+ efflux was diminished by 60-70% or about 30%, respectively. This was not explained by the 20-30% decrease in flux through monoamine oxidase observed under these conditions. The results suggest that metabolic generation of H2O2 inside the liver leads to a net K+ efflux and subsequent hepatocellular shrinkage. Net K+ efflux under these conditions is enhanced when catalase is inhibited, suggesting that the rate of both intracellular H2O2 generation and degradation can modulate cellular K+ balance and cellular volume. The data support the idea that oxidative stress may affect hepatocellular functions also by lowering the hepatocellular hydration state.

Animals↗
PubMed1993

Effects of [Ca2+]i and temperature on minK channels expressed in Xenopus oocytes.

Slowly activating, voltage-dependent minK channels cloned from rat kidney were expressed in Xenopus oocytes. Increase in the bath temperature from 22 to 32 degrees C resulted in a dramatic acceleration of minK channel activation. The extraordinarily high Q10 of minK channel activation was voltage-dependent, being higher at more negative potentials (Q10 at -20 mV; 7.02; at 20 mV: 4.0). While activation of minK channels was highly voltage-dependent at 22 degrees C, voltage had only little effect on minK channel activation at 32 degrees C. Increase in [Ca2+]i which has recently been shown to increase the maximal conductance gmax at room temperature, did not affect gmax at 32 degrees C. However, increase of [Ca2+]i caused acceleration of minK channel activation at both temperatures. The interaction of [Ca2+]i and temperature on gmax and activation rate of minK channels described here is very similar to recent findings on Ca- and temperature-effects on the slowly activating potassium conductance IKs in guinea pig cardiac myocytes.

Animals↗
PubMed1993

Effect of cell volume on Acridine Orange fluorescence in hepatocytes.

Hepatic proteolysis is inhibited by cell swelling following a variety of experimental manoeuvres, such as reduction of extracellular osmolarity, concentrative uptake of amino acids, or blockade of K+ channels by barium. On the other hand, proteolysis is known to be accomplished by pH-sensitive lysosomal proteases. Accordingly, NH3/NH4+ inhibits proteolysis by intralysosomal alkalinization. The present study has been performed to test for an effect of cell volume on the pH of acidic intracellular compartments, as assessed by Acridine Orange fluorescence at > 520 nm (F > 520). F > 520 is enhanced by NH3/NH4+ (2 and 20 mmol/l respectively), by glutamine (2 mmol/l), by the K(+)-channel blocker barium (10 mmol/l) and by reduction of extracellular osmolarity (by 20 and 80 mosmol/l respectively). The observations point to release of Acridine Orange from acidic cellular compartments, which is indicative of alkalinization of these compartments during cell swelling. This effect may contribute to the regulation of proteolysis.

Acridine Orange↗