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A Schwab

Publications and source records attributed to A Schwab.

At least 37 records · Page 2Linked to original sources
PubMed1997

The atomic force microscope detects ATP-sensitive protein clusters in the plasma membrane of transformed MDCK cells.

Plasma membrane proteins are supposed to form clusters that allow 'functional cross-talk' between individual molecules within nanometre distance. However, such hypothetical protein clusters have not yet been shown directly in native plasma membranes. Therefore, we developed a technique to get access to the inner face of the plasma membrane of cultured transformed kidney (MDCK) cells. The authors applied atomic force microscopy (AFM) to visualize clusters of native proteins protruding from the cytoplasmic membrane surface. We used the K+ channel blocker iberiotoxin (IBTX), a positively charged toxin molecule, that binds with high affinity to plasma membrane potassium channels and to atomically flat mica. Thus, apical plasma membranes could be 'glued' with IBTX to the mica surface with the cytosolic side of the membrane accessible to the scanning AFM tip. The topography of these native inside-out membrane patches was imaged with AFM in electrolyte solution mimicking the cytosol. The plasma membrane could be clearly identified as a lipid bilayer with the characteristic height of 4.9 +/- 0.02 nm. Multiple proteins protruded from the lipid bilayer into the cytosolic space with molecule heights between 1 and 20 nm. Large protrusions were most likely protein clusters. Addition of the proteolytic enzyme pronase to the bath solution led to the disappearance of the proteins within minutes. The metabolic substrate ATP induced a shape-change of the protein clusters and smaller subunits became visible. ADP or the non-hydrolysable ATP analogue, ATP-gamma-S, could not exert similar effects. It is concluded that plasma membrane proteins (and/or membrane associated proteins) form 'functional clusters' in their native environment. The 'physiological' arrangement of the protein molecules within a cluster requires ATP.

Adenosine Triphosphate↗
PubMed1997

Intracellular Ca2+ distribution in migrating transformed epithelial cells.

Migration of transformed Madin-Darby canine kidney (MDCK-F) cells depends on the polarized activity of a Ca2+-sensitive K+ channel. We tested whether a gradient of intracellular Ca2+-concentration ([Ca2+]i) underlies the horizontal polarization of K+ channel activity. [Ca2+]i was measured with the fluorescent dye fura-2/AM. Spatial analysis of [Ca2+]i indicated that a horizontal gradient exists, with [Ca2+]i being higher in the cell body than in the lamellipodium. Resting and maximal levels during oscillations of [Ca2+]i in the cell body were found to be 135 +/- 34 and 405 +/- 59 nml/l, respectively, whereas they were 79 +/- 18 and 307 +/- 102 nmol/l in the lamellipodium. This gradient can partially explain the preferential activation of K+ channels in the plasma membrane of the cell body. We applied a local superfusion technique during migration experiments and measurements of [Ca2+]i to test whether its maintenance is due to an uneven distribution of Ca2+ influx into migrating MDCK-F cells. Locally superfusing the cell body of migrating MDCK-F cells with La3+ alone or together with charybdotoxin, a specific blocker of Ca2+-sensitive K+ channels, slowed migration to 47 +/- 10% and 9 +/- 5% of control, respectively. Local blockade of Ca2+ influx into the cell body and the lamellipodium with la3+ was followed by a decrease of [Ca2+]i at both cell poles. This points to Ca2+ influx occurring over the entire cell surface. This conclusion was confirmed by locally superfusing Mn2+ over the cell body and the lamellipodium. Fura-2 fluorescence was quenched in both areas, the decrease of fluorescence being two to three times faster in the cell body than in the lamellipodium. However, this difference is insufficient to account for the observed gradient of [Ca2+]i. We hypothesize that the polarized distribution of intracellular Ca2+ stores contributes significantly to the generation of a gradient of [Ca2+]i.

Animals↗
PubMed1997

Imaging excised apical plasma membrane patches of MDCK cells in physiological conditions with atomic force microscopy.

We combined the patch-clamp technique with atomic force microscopy (AFM) to visualize plasma membrane proteins protruding from the extracellular surface of cultured kidney cells (MDCK cells). To achieve molecular resolution, patches were mechanically isolated from whole MDCK cells by applying the patch-clamp technique. The excised inside-out patches were transferred on freshly cleaved mica and imaged with the AFM in air and under physiological conditions (i. e. in fluid). Thus, the resolution could be increased considerably (lateral and vertical resolutions 5 and 0.1 nm, respectively) as compared to experiments on intact cells, where plasma membrane proteins were hardly detectable. The apical plasma membrane surface of the MDCK cells showed multiple protrusions which could be identified as membrane proteins through the use of pronase. These proteins had a density of about 90 per micron(2), with heights between 1 and 9 nm, and lateral dimensions of 20-60 nm. Their frequency distribution showed a peak value of 3 nm for the protein height. A simplified assumption - modelling plasma membrane proteins as spherical structures protruding from the lipid bilayer - allowed an estimation of the possible molecular weights of these proteins. They range from 50 kDa to 710 kDa with a peak value of 125 kDa. We conclude that AFM can be used to study the molecular structures of membranes which were isolated with the patch-clamp technique. Individual membrane proteins and protein clusters, and their arrangement and distribution in a native plasma membrane can be visualized under physiological conditions, which is a first step for their identification.

Animals↗
PubMed1996

Rapid activation of calcium-sensitive Na+/H+ exchange induced by 20-hydroxyecdysone in salivary gland cells of Drosophila melanogaster.

Ecdysteroids play an important role in the larval moulting process of insects. 20-Hydroxyecdysone (20E) causes the induction of specific 'puffs' in polytene chromosomes of Drosophila melanogaster salivary gland cells. Although it is known that inorganic ions control pretranscriptional processes in the cell nucleus, the intracellular mechanisms of gene activation are still unclear. Therefore, we examined the effects of 20E on plasma membrane ion transport of Drosophila melanogaster salivary gland cells. Isolated glands of the third larval stage were superfused with a solution mimicking the haemolymph. The relative K+ conductance of the cell membrane (tK+) was measured with microelectrodes by performing ion substitution experiments. Under control conditions tK+ averaged to 0.16 + 0.02 (n = 15). Addition of 5 x 10(-6) M 20E increased tK+ within 2 min by 19.1 +/- 4.2% (n = 15). This rapid response to 20E was elicited only in the presence of calcium. Moreover, starting from a steady-state intracellular pH of 7.20-7.60, 20E induced a rise in cytoplasmic pH by 0.27 +/- 0.06 (n = 6) within minutes. Amiloride (10(-3) M), a blocker of plasma membrane Na+/H+ exchange, prevented the 20E-induced intracellular alkalinization. We conclude that 20E activates a calcium-sensitive plasma membrane Na+/H+ exchange leading to a rise of plasma membrane K+ conductance and intracellular alkalinization both being prerequisites for steroid hormone induced gene activation.

Amiloride↗
PubMed1996

[Intranasal administration of midazolam (Dormicum) in young children in emergency medicine].

A method for sedation of preschool children in emergency medicine is presented. Midazolam (Dormicum) is frequently used in premedication in children. Various routes of administration have been described for this drug. After use of nasal administration of midazolam for premedication we also used this method for sedation of children in emergencies. This technique provides rapid onset and cardiovascular stability while maintaining spontaneous ventilation. In two case reports the first results are demonstrated; the method is evaluated and discussed.

Burns↗
PubMed1996

Pressure threshold for fluid loss from the peritoneal cavity.

Ascites or dialysis fluid in the peritoneal cavity causes fluid loss from the cavity to the body. Experiments in animals and in humans have shown that the fluid loss rate increases with large increments in the intraperitoneal hydrostatic pressure (Pip). We hypothesized that there is a low-threshold Pip above which this fluid loss occurs. Because the full Pip force is exerted across the abdominal wall (AW), we further hypothesized that fluid movement into the abdominal wall would vary directly with the Pip. To address these questions, we dialyzed rats for 3 h in the supine position at constant levels of Pip with isotonic and hypertonic dialysis solutions containing a protein marker of fluid movement. We measured total fluid loss, AW fluid-marker concentration, and lymph flow. With variation of Pip from 0 to 8 cmH2O, we found that 1) lymph flows (0.61 +/- 0.03 ml/h) were not dependent on Pip, 2) measured isotonic fluid loss rate varied from 0.29 +/- 0.06 ml/h at 0 cmH2O to 0.62 +/- 0.02 at 2 cmH2O and then rose in a linear fashion to 5.06 +/- 0.10 ml/h at 8 cmH2O, 3) fluid movement into the AW paralleled the measured fluid loss rate, and 4) protein clearance from the cavity overestimated the true fluid loss because of adsorption of the marker to the peritoneal surface. We conclude that, although peritoneal lymph flow is not dependent on intraperitoneal hydrostatic or osmotic pressure, fluid loss from the cavity and fluid loss to the abdominal wall are directly proportional to Pip > 2 cmH2O. We also note that protein markers of fluid movement require correction for tissue surface adsorption for accurate results.

Adsorption↗
PubMed1996

Plasticity of renal epithelial cells: the way a potassium channel supports migration.

In this article we review aspects of plasticity of renal epithelial cells. We focus on one particular feature, namely on cell migration. For normal renal and other epithelial cells migration is a motif of plasticity which can be activated after disrupture of epithelial integrity. In the case of transformed renal epithelial cells, however, migration is "pathophysiological" as it is no longer regulated as in normal cells. We studied migration in a permanently transformed Madin-Darby canine kidney cell line, called MDCK-F. Locomotion of these cells strictly depends-in addition to the cytoskeletal "migration machinery"-upon the oscillatory activity of a Ca(2+)-sensitive plasma membrane K+ channel. We propose that K+ channel activity is linked to migration via changes of cell volume. We deduced from patch-clamp experiments in combination with high resolution 3D-images obtained by atomic force microscopy that periods of high K+ channel activity are parallelled by cell shrinkage. By locally superfusing either cell body (rear part) or lamellipodium (front part of the cell) with specific K+ channel blockers we disclosed a polar distribution of K+ channel activity in MDCK-F cells. K+ channels are preferentially active at the rear part of MDCK-F cells. We discuss how localized K+ channel activity, in concert with other migration-relevant phenomena such as "tail contraction" or asymmetric cell-matrix interactions, may result in localized changes of cell volume supporting migration. Finally, we define cell polarization for a migrating epithelial cell. Whereas normal epithelial cells are "vertically" polarized, transformed cells are "horizontally" polarized, i.e., in the plane of movement. Such a distinct view could be helpful for better understanding the transition from a normal differentiated epithelial cell to a tumorigenic migrating cell.

Animals↗
PubMed1995

Polarized ion transport during migration of transformed Madin-Darby canine kidney cells.

Epithelial cells lose their usual polarization during carcinogenesis. Although most malignant tumours are of epithelial origin little is known about ion channels in carcinoma cells. Previously, we observed that migration of transformed Madin-Darby canine kidney (MDCK-F) cells depended on oscillating K+ channel activity. In the present study we examined whether periodic K+ channel activity may cause changes of cell volume, and whether K+ channel activity is distributed in a uniform way in MDCK-F cells. After determining the average volume of MDCK-F cells (2013+/-270 microm3; n=8) by means of atomic force microscopy we deduced volume changes by calculating the K+ efflux during bursts of K+ channel activity. Therefore, we measured the membrane conductance of MDCK-F cells which periodically rose by 22.3+/-2.5 nS from a resting level of 6.5+/-1.4 nS (n=12), and we measured the membrane potential which hyperpolarized in parallel from -35.4+/-1.2 mV to -71.6+/-1.8 mV (n=11). The distribution of K+ channel activity was assessed by locally superfusing the front or rear end of migrating MDCK-F cells with the K+ channel blocker charybdotoxin (CTX). Only exposure of the rear end to CTX inhibited migration providing evidence for "horizontal" polarization of K+ channel activity in transformed MDCK-F cells. This is in contrast to the "vertical" polarization in parent MDCK cells. We propose that the asymmetrical distribution of K+ channel activity is a prerequisite for migration of MDCK-F cells.

Animals↗
PubMed1995

Xenopus laevis oocyte: using living cells to teach the theory of cell membrane potential.

We describe a student course in membrane physiology that includes demonstration of the cell membrane potential of a living cell, the Xenopus laevis oocyte. By use of K+ macroelectrodes and a computer simulation program, students are introduced to the membrane concept on the basis of ion gradients, diffusion potentials, and membrane conductances. Subsequently, some basic principles are demonstrated in living cells by measuring the cell membrane potential of a X. laevis oocyte. The dependence of the cell membrane potential on ion gradients is shown by stepwise elevation of the extracellular K+ concentration. The clinical relevance of disturbances of the K+ homeostasis is discussed in this context. The effect of changing the membrane conductance for particular ions on the cell membrane potential is demonstrated by applying a K+ channel blocker. The experiments are discussed in the context of the action of pharmaceuticals and toxins. This student course on membrane physiology has now been held for 2 yr for medical and dental students, and their response to the oocyte demonstration, as evaluated by a student questionnaire, was mainly positive.

Animals↗
PubMed1994

Imaging nuclear pores of aldosterone-sensitive kidney cells by atomic force microscopy.

In nuclei of renal target cells, aldosterone enhances transcriptional activity followed by the translocation of specific RNA molecules across the nuclear envelope. Trafficking between cell nucleus and cytoplasm occurs via nuclear pore complexes (NPCs) located in the double-layered nuclear envelope. We investigated the nucleocytoplasmic transport route by structure-function analysis at subcellular level in quiescent and aldosterone-stimulated cells. With atomic-force microscopy (AFM) we imaged individual pores of the nuclear surface of cultured kidney cells and related the number of pores per micron2 to nuclear envelope conductance (Gn, per micron2) evaluated electrically by current injection into the isolated nucleus. NPCs were equally distributed resembling "donut-like" structures with outer diameters of 134 +/- 12 nm (n = 50), each equipped with a central channel. Six hours of aldosterone exposure (0.1 microM) increased the number of NPCs per micron 2 of nuclear surface from 7.4 +/- 0.4 to 9.8 +/- 0.4 (n = 12; P < 0.01). At the same time Gn rose from 6900 +/- 520 to 9600 +/- 610 pS/micron2 paralleled by an increase of the intranuclear electrical potential from -2.8 +/- 0.2 to -6.2 +/- 0.4 mV (n = 18; P < 0.01). Assuming that NPCs represent the sole conductive pathway in the nuclear envelope, we calculate a mean single NPC conductance of 932 and 980 pS, in the absence and presence of aldosterone, respectively. We conclude that aldosterone facilitates nucleocytoplasmic transport by increasing the number of NPCs but not by modifying their biophysical properties. Possibly, aldosterone controls similar transport mechanisms in both plasma membrane and nuclear envelope.

Aldosterone↗
PubMed1994

Pharmacokinetics of torasemide and its metabolites in end-stage renal disease.

The pharmacokinetics of torasemide, a new loop diuretic, as well as its active metabolites M1 and M3, and its inactive main metabolite, M5, were studied in 12 patients with end-stage renal failure during single i.v. (n = 6) or single oral (n = 6) dosing of 200 mg torasemide, and during chronic oral treatment for 9 days (n = 12). The elimination half-life (t1/2) of torasemide was unchanged in renal failure, whereas t1/2 of the torasemide metabolites M1, M3, and M5 were markedly prolonged. However t1/2 as well as the area under the plasma level time curve of torasemide and its metabolites were unchanged during chronic compared to acute administration. The results of this study suggest that despite the increased half-life of torasemide metabolites M1, M3 and M5 in end-stage renal failure patients, no accumulation of the parent drug torasemide and its metabolites during chronic dosing is demonstrable.

Diuretics↗
PubMed1994

Extracellular pH determines the rate of Ca2+ entry into Madin-Darby canine kidney-focus cells.

We investigated the relationship between intracellular Ca2+ and pH homeostasis in Madin-Darby canine kidney-focus (MDCK-F) cells, a cell line exhibiting spontaneous oscillations of intracellular Ca2+ concentration (Ca2+i). Ca2+i and intracellular pH (pHi) were measured with the fluorescent dyes Fura-2 and BCECF by means of video imaging techniques. Ca2+ influx from the extracellular space into the cell was determined with the Mn2+ quenching technique. Cells were superfused with HEPES-buffered solutions. Under control conditions (pH 7.2), spontaneous Ca2+i oscillations were observed in virtually all cells investigated. Successive alkalinization and acidification of the cytoplasm induced by an ammonia ion prepulse had no apparent effect on Ca2+i oscillations. On the contrary, changes of extracellular pH value strongly affected Ca2+i oscillations. Extracellular alkalinization to pH 7.6 completely suppressed oscillations, whereas extracellular acidification to pH 6.8 decreased their frequency by 40%. Under the same conditions, the respective pHi changes were less than 0.1 pH units. However, experiments with the Mn2+ quenching technique revealed that extracellular alkalinization significantly reduced Ca2+ entry from the extracellular space. Large increases of Ca2+i triggered by the blocker of the cytoplasmic Ca(2+)-ATPase, thapsigargin, had no effect on pHi. We conclude: intracellular Ca2+ homeostasis in MDCK-F cells is pH dependent. pH controls Ca2+ homeostasis mainly by effects on the level of Ca2+ entry across the plasma membrane. On the contrary, the intracellular pH value seems to be insensitive to rap changes of Ca2+i.

Animals↗
PubMed1994

Cell transformation induces a cytoplasmic Ca2+ oscillator in Madin-Darby canine kidney cells.

Alkaline stress transforms Madin-Darby canine kidney (MDCK) cells as indicated by loss of epithelial structure, multilayer cell growth and formation of foci. In the present study we report that transformed MDCK cells (MDCK-F cells) exhibit spontaneous and lasting oscillations of intracellular Ca2+ concentration ([Ca2+]i), which are absent in non-transformed cells. Oscillations, as revealed by Fura-2 video imaging, were due to the activity of an inositol 1,4,5-trisphosphate-(InsP3)-sensitive Ca2+ store since their frequency was dependent on bradykinin concentration and they were abolished by the phosphoinositidase C inhibitor U73122. Moreover, blockers of the cytoplasmic Ca(2+)-ATPase, thapsigargin and 2,5-di-(tetr-butyl)-1,4-benzohydroquinone inhibited oscillatory activity. In contrast, neither injection of ruthenium red, ryanodine nor caffeine had any effect on oscillations. Analysis of the spatial distribution of [Ca2+]i showed that Ca2+ transients originated from an initiation site constant for a given cell and spread through the cell as an advancing Ca2+ wave. Oscillations started in a random manner from single cells and spread over neighbouring cells, suggesting a kind of intercellular communication. We conclude that MDCK-F cells have acquired the ability for endogenous Ca2+ release through transformation. Oscillations are primarily due to the activity of an InsP3-sensitive cytosolic Ca2+ oscillator.

Animals↗
PubMed1994

Cytoplasmic Ca2+ determines the rate of Ca2+ entry into Mardin-Darby canine kidney-focus (MDCK-F) cells.

Transformed Mardin-Darby canine kidney-focus (MDCK-F) cells exhibit spontaneous Ca2+ oscillations from an inositol 1,4,5-triphosphate-sensitive cytoplasmic Ca2+ store. In this study, Ca2+ entry from the extracellular space and its role in generation of oscillations were investigated by means of Ca2+ video imaging and the Fura-2/Mn2+ quenching technique. Oscillations were dependent on extracellular Ca2+ concentration and were inhibited by extracellularly applied La3+, Co2+ and Ni2+. Depolarization of the cell membrane with high K+ concentrations and the L-type Ca2+ channel blocker nifedipine had no effect on oscillations, indicating the lack of involvement of voltage-gated Ca2+ channels. Mn2+ quenching experiments disclosed significant Ca2+ influx into MDCK-F cells. The rate of this influx was constant between Ca2+ spikes, but markedly increased during the spontaneous Ca2+ spikes. Similar transient increases in Ca2+ entry could be mimicked by agents triggering intracellular Ca2+ release such as bradykinin and thapsigargin. We conclude that the plasma membrane of MDCK-F cells exhibits a marked voltage-independent Ca2+ permeability permitting Ca2+ entry into the cytoplasm. The rate of Ca2+ entry which determines the frequency of oscillations is most likely to be regulated by the cytoplasmic Ca2+ concentration.

Animals↗
PubMed1994

Comparative pharmacokinetics and pharmacodynamics of loop diuretics in renal failure.

Loop diuretics increase the fractional excretion of volume, sodium, potassium, chloride and calcium in all stages of renal failure, and their potency is directly correlated with these excretory activities. Tubular secretion of loop diuretics in renal failure is impaired both by reduced renal blood flow and by reduced activity of the tubular carrier system. For these reasons, high concentrations of diuretics in the peritubular capillaries are necessary to guarantee delivery of sufficient drug to their site of action in the ascending limb of the loop of Henle. Piretanide and furosemide have a constant extrarenal elimination and thus accumulate in renal failure. Decreased renal excretion of bumetanide is compensated by hepatic elimination and hence bumetanide does not accumulate. Elimination of torasemide is also independent of its renal excretion. Thus in renal failure, torasemide is the only loop diuretic in which the plasma concentration is strictly dose dependent. Loop diuretics follow a number of different metabolic pathways, but this may not be clinically relevant.

Diuretics↗
PubMed1994

Oscillating activity of a Ca(2+)-sensitive K+ channel. A prerequisite for migration of transformed Madin-Darby canine kidney focus cells.

Migration plays an important role in the formation of tumor metastases. Nonetheless, little is known about electrophysiological phenomena accompanying or underlying migration. Previously, we had shown that in migrating alkali-transformed Madin-Darby canine kidney focus (MDCK-F) cells a Ca(2+)-sensitive 53-pS K+ channel underlies oscillations of the cell membrane potential. The present study defines the role this channel plays in migration of MDCK-F cells. We monitored migration of individual MDCK-F cells by video imaging techniques. Under control conditions, MDCK-F cells migrated at a rate of 0.90 +/- 0.03 microns/min (n = 201). Application of K+ channel blockers (1 and 5 mmol/liter Ba2+, 5 mmol/liter tetraethylammonium, 100 mumol/liter 4-aminopyridine, 5 nmol/liter charybdotoxin) caused marked inhibition of migration, pointing to the importance of K+ channels in migration. Using patch-clamp techniques, we demonstrated the sensitivity of the Ca(2+)-sensitive 53-pS K+ channel to these blockers. Blockade of this K+ channel and inhibition of migration were closely correlated, indicating the necessity of oscillating K+ channel activity for migration. Migration of MDCK-F cells was also inhibited by furosemide or bumetanide, blockers of the Na+/K+/2Cl- cotransporter. We present a model for migration in which oscillations of cell volume play a central role. Whenever they are impaired, migration is inhibited.

Animals↗
PubMed1993

Mechanism of activation of K+ channels by minoxidil-sulfate in Madin-Darby canine kidney cells.

We studied the mechanism of K+ channel activation by minoxidil-sulfate (MxSO4) in fused Madin-Darby canine kidney (MDCK) cells. Patch-clamp techniques were used to assess single channel activity, and fluorescent dye techniques to monitor cell calcium. A Ca(2+)-dependent inward-rectifying K+ channel with slope conductances of 53 +/- 3 (negative potential range) and 20 +/- 3 pS (positive potential range) was identified. Channel activity is minimal in cell-attached patches. MxSO4 initiated both transient channel activation and an increase of intracellular Ca2+ (from 94.2 +/- 9.1 to 475 +/- 12.6 nmol/liter). The observation that K+ channel activity of excised inside-out patches was detected only at Ca2+ concentrations in excess of 10 mumol/liter suggests the involvement of additional mechanisms during channel activation by MxSO4. Transient K+ channel activity was also induced in cell-attached patches by 10 mumol/liter of the protein kinase C activator 1-oleoyl-2-acetyl-glycerol (OAG). OAG (10 mumol/liter in the presence of 1.6 mmol/liter ATP) increased the Ca2+ sensitivity of the K+ channel in inside-out patches significantly by lowering the Km for Ca2+ from 100 mumol/liter to 100 nmol/liter. The channel activation by OAG was reversed by the protein kinase inhibitor H8. Staurosporine, a PKC inhibitor, blocked the effect of MxSO4 on K+ channel activation. We conclude that MxSO4-induced K+ channel activity is mediated by the synergistic effects of an increase in intracellular Ca2+ and a PKC-mediated enhancement of the K+ channel's sensitivity to Ca2+.

Alkaloids↗