Biomedical subjects

M Duszyk

Publications and source records attributed to M Duszyk.

At least 37 records · Page 2Linked to original sources

PubMed1997

Determination of cell surface charge by photometric titration.

A colloid titration method has been frequently used to determine the number of charged residues at the cell surface. Here we present a new version of this technique, based on photometric measurements of a metachromatic shift in the maximum absorption of toluidine blue as it binds to the cell surface. The major improvements are: (1) simplified methodology and (2) increased precision of equivalence point determination. The data are analyzed using Gran's theory, which allows measurements to be taken at regular intervals instead of being concentrated around the equivalence titration point. We used this method to characterize the cell surface charge of three populations of rat mast cells: (1) peritoneal mast cells (PMC), (2) bone marrow-derived mast cells (BMMC) and (3) a rat cultured mast cell line (RCMC). Our results indicate that PMC have (4.23 +/- 0.59) x 10(8), while BMMC (8.58 +/- 0.26) x 10(7) negatively charged residues per cell. The results for RCMC were similar to those for BMMC. Taking into account the size differences between PMC and BMMC, the average charge density of PMC was also significantly higher than that of BMMC. The differences in cell surface charge were analyzed in the light of different sensitivities of mast cells to polycationic secretagogues.

Animals↗

PubMed1997

Decreased cell surface charge in cystic fibrosis epithelia.

A colloid titration technique has been used to determine the surface charge of cystic fibrosis (CF) and corresponding non-CF epithelial cells. We have shown that the negative surface charge of CF epithelial cells is significantly reduced in comparison with non-CF cells. This fact may play an important role in CF, where the increased adherence of microorganisms is known to cause chronic lung infection. Neuraminidase treatment removed approximately the same amount of surface charge in both cell lines, indicating no differences in cell surface sialylation. Similar results were obtained by direct measurements of the amount of N-acetylneuraminic acid released by neuraminidase. Therefore, our results indicate that sialic acid residues are not involved in the reduction of the negative surface charge in CF. This conclusion does not support the hypothesis that undersialylation of cell-membrane molecules occurs in cystic fibrosis.

Cell Membrane↗

PubMed1997

Ins(3,4,5,6)P4 specifically inhibits a receptor-mediated Ca2+-dependent Cl- current in CFPAC-1 cells.

We have examined the role of inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P4] in the control of Cl- current in CFPAC-1 cells. Intracellular Ins(3,4,5,6)P4 had no effect on basal current, but it produced a five- to sevenfold reduction in the Cl- current stimulated by either 2 microM extracellular ATP or by 1 microM extracellular thapsigargin. The half-maximally effective dose of Ins(3,4,5,6)P4 was 2.9 microM, and 4 microM blocked >80% of the ATP-activated current. In contrast, 10 microM Ins(1,4,5,6)P4, Ins(1,3,4,5)P4, or Ins(1,3,4,6)P4 enhanced rather than inhibited the ATP-activated Cl- current, although Ins(1,4,5,6)P4 only acted transiently. These stimulatory effects were Ca2+ dependent and largely inhibited by coapplication of equimolar Ins(3,4,5,6)P4. Inositol 1,3,4,5,6-pentakisphosphate, the precursor of Ins(3,4,5,6)P4, did not affect Cl- current. These data consolidate and extend the hypothesis that Ins(3,4,5,6)P4 is an important intracellular regulator of Cl- current in epithelial cells.

Adenosine Triphosphate↗

PubMed1997

Nitric oxide activates chloride currents in human lung epithelial cells.

Epithelial Cl- channels are regulated by various physiological factors, including guanosine 3',5'-cyclic monophosphate (cGMP). Because cGMP mediates many of the physiological actions of nitric oxide (NO), we have studied both the presence of endogenous NO and the effects of exogenous NO on Cl- currents in A549 human lung epithelial cells. We have detected Ca(2+)-dependent NO synthase activity in A549 cells. Using the perforated patch-clamp technique, we have shown that inhibition of this enzyme by NG-monomethyl-L-arginine decreased Cl- current, an effect that was reversed by the NO donor S-nitrosoglutathione (GSNO). In addition, the NO donors GSNO and S-nitroso-N-acetyl-D,L-penicillamine increased whole-cell Cl- currents in A549 cells. This stimulatory effect of the NO donors was sensitive to inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, suggesting that channels other than the cystic fibrosis transmembrane conductance regulator (CFTR) are involved in the action of NO on A549 cells. In addition, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a selective inhibitor of soluble guanylyl cyclase, decreased NO-mediated stimulation of Cl- currents. Our results suggest that, in lung epithelial cells, NO regulates a non-CFTR Cl- conductance acting via a cGMP-dependent mechanism.

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

PubMed1995

Characterization and regulation of a chloride channel from bovine tracheal epithelium.

1. The patch-clamp technique was used to characterize chloride channels from the apical membranes of bovine tracheal epithelial cells. Application of GTP gamma S or NaF to excised patches revealed the existence of a novel type of Cl- channel regulated by G-proteins in a membrane-delimited manner. 2. The channel had a linear current-voltage relationship, with a conductance of 100-120 pS. Its open probability was independent of voltage. 3. The channel was highly anion selective (permeability ratio, PNa/PCl = 0.06 +/- 0.04) and had the halide permeability sequence: I- > Br- > or = Cl- > F-, corresponding to the Eisenman I sequence. This suggested that neither ionic size nor diffusion rate determined ion permeation through the channel. 4. The mole fraction behaviour was studied using fluoride and chloride ions. Mixtures of ions produced currents that would be expected from the linear combination of the two ions acting independently, indicating relatively simple permeation through the pore and compatible with a single ion binding site. 5. The channel was inhibited by the stilbene disulphonates SITS (4-acetamido-4'-isothiocyanatostilbene-2, 2'-disulphonic acid) and DNDS (4,4'-dinitrostilbene-2,2'-sulphonic acid). SITS introduced voltage dependence to channel gating and indicated the possible involvement of lysine residues in the channel permeation pathway. 6. NaF was unable to activate Cl- channels in the presence of the aluminum chelator, deferoxamine mesylate. This indicates that Al3+ ions play an important role in chloride channel activation by fluoride. NaF activation was not dependent on the presence of calcium ions. 7. The channel was insensitive to alkaline phosphatase and to the specific inhibitors of protein phosphatase types I and 2A, okadaic acid and calyculin A. 8. The channels could be activated by GTP gamma S or by NaF in the presence of the phospholipase A2 inhibitor quinacrine, indicating that this enzyme is not involved in channel regulation.

Animals↗

PubMed1995

Evidence that pH-titratable groups control the activity of a large epithelial chloride channel.

The effects of pH changes were examined on the properties of a large, voltage dependent, epithelial Cl- channel from bovine tracheal cells. Alkaline solutions in the range pH = 7.4-9.2 had no detectable effects on channel conductance or gating. However, acid solutions significantly reduced channel open probability, raising the voltage required to open the channel. Analysis of channel activity in the acidic pH range suggested that at least one charged group on the channel with an apparent pK = 6.09, is responsible for its voltage dependence. Neutralization of this charge does not eliminate the voltage dependence, but changes the energy difference between the closed and open states. The absence of any change in channel conductance over this wide pH range suggests that the protonation site is far removed from the channel permeation pathway.

Animals↗

PubMed1994

Evidence that channels below 1 pS cause the volume-sensitive chloride conductance in T84 cells.

The volume-activated chloride current of T84 human colonic cells was studied using the whole-cell patch clamp. The current appeared reliably with a mild osmotic gradient and in the absence of intracellular ATP. It reversed at the chloride equilibrium potential and was blocked by the chloride channel blocker DIDS. Development of the current was accompanied by an increase in the current noise variance, typical of increasing ion channel open probability. Noise variance was always well-fitted by a double Lorentzian relationship with corner frequencies at approximately 1.7 Hz and approximately 60 Hz. The increase in variance during development of the volume-sensitive current was mostly due to an increase in the high frequency component. The relationship between noise variance and membrane current was well-fitted by a relationship with a single channel conductance of approximately 0.2 pS.

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

PubMed1993

Halide permeation through three types of epithelial anion channels after reconstitution into giant liposomes.

Anion-selective channels from apical membranes of cultured CFPAC-1 cells were isolated and incorporated into giant liposomes for patch clamp recording. Liposomes were formed from L-alpha-lecithin by a dehydration-hydration method. Ion channels were characterized using the excised inside-out patch clamp configuration. The most commonly observed anion channels were similar to those observed in native epithelial tissues. The linear 20 pS Cl- channel had the halide permeability sequence Cl- > I- > or = Br- > F-, and showed anomalous mole-fraction behavior in solutions containing different proportions of Cl- and F- ions. The autwardly rectifying Cl- channel had the halide permeability sequence I- > Br- > Cl- > F-, and also showed anomalous mole-fraction behavior, indicating that both these channels probably contain multi-ion pores. The third, voltage-dependent anion channel showed at least five different substrates, had a conductance of 390 pS in the main state, and showed two types of kinetics, fast (openings and closings < 1 ms), and slow (openings and closings > 1 s). The channel was seen more frequently after reconstitution into giant liposomes than in intact cells. It was not selective amongst the halides, and there was no deviation from a linear dependence of relative current on molar fractions, indicating relatively simple permeation through the pore. Differences in halide permeabilities suggest that different anion channels may be related to different membrane proteins. Comparison with the chloride channel proteins isolated biochemically from epithelial cell membranes is discussed.

Cell Membrane Permeability↗

PubMed1992

Noise analysis and single-channel observations of 4 pS chloride channels in human airway epithelia.

Apical membranes of human airway epithelial cells have significant chloride permeability, which is reduced in cystic fibrosis (CF), causing abnormal electrochemistry and impaired mucociliary clearance. At least four types of chloride channels have been identified in these cells, but their relative roles in total permeability and CF are unclear. Noise analysis was used to measure the conductance of chloride channels in human nasal epithelial cells. The data indicate that channels with a mean conductance of 4.5 pS carry most of the chloride current, and that the mean number of such channels per cell is approximately 4,000. Chloride channels in this conductance range were also seen in single-channel recordings.

Biophysical Phenomena↗

PubMed1991

An analytical model of ionic movements in airway epithelial cells.

A new mathematical model of ion movements in airway epithelia is presented, which allows predictions of ion fluxes, membrane potentials and ion concentrations. The model includes sodium and chloride channels in the apical membrane, a Na/K pump and a cotransport system for Cl- with stoichiometry Na+:K+:2Cl- in the basolateral membrane. Potassium channels in the basolateral membrane are used to regulate cell volume. Membrane potentials, ion fluxes and intracellular ion concentration are calculated as functions of apical ion permeabilities, the maximum pump current and the cotransport parameters. The major predictions of the model are: (1) Cl- concentration in the cell is determined entirely by the intracellular concentration of negatively charged impermeable ions and the osmotic conditions; (2) changes in intracellular Na+ and K+ concentrations are inversely related; (3) cotransport provides the major driving force for Cl- flux, increases intracellular Na+ concentration, decreases intracellular K+ concentration and hyperpolarizes the cell interior; (4) the maximum rate of the Na/K pump, by contrast, has little effect on Na+ or Cl- transepithelial fluxes and a much less pronounced effect on cell membrane polarization; (5) an increase in apical Na+ permeability causes an increase in intracellular Na+ concentration and a significant increase in Na+ flux; (6) an increase in apical Cl- permeability decreases intracellular Na+ concentration and Na+ flux; (7) assuming Na+ and Cl- permeabilities equal to those measured in human nasal epithelia, the model predicts that under short circuit conditions, Na+ absorption is much higher than Cl- secretion, in agreement with experimental measurements.

Biological Transport, Active↗

PubMed1991

An inwardly rectifying chloride channel in ragweed-sensitized canine tracheal epithelial cells.

The single channel inside-out patch clamp technique was used to characterize ion channels in the apical membranes of ragweed-sensitized and control canine tracheal epithelial cells maintained in primary culture. Patches were obtained from single isolated cells or from cells at the edges of confluent sheets. A new type of chloride channel was seen in sensitized cells but not in control cells. The channel showed inward rectification in symmetric chloride solutions with conductance varying from 95 pS to 52 pS over the range of -60 mV to 60 mV membrane potential. Channel gating was voltage dependent with maximal opening at about -30 mV. Kinetic analysis showed that distributions of closed and open times could both be well fitted by the sums of three exponential components. Rate constants for transitions between the states of a linear kinetic model were calculated, with only one rate being significantly voltage dependent. The possible significance of this channel is discussed.

Animals↗

PubMed1990

The 20-pS chloride channel of the human airway epithelium.

The single-channel inside-out patch clamp technique was used to characterize chloride channels in the apical membranes of human airway epithelial cells maintained in primary culture. Patches were obtained from single isolated cells or from cells at the edges of confluent groups. The channel seen most often, in 24% of all patches, had a conductance of approximately 20 pS and had a linear current-voltage relationship in symmetric chloride solutions. The anion selectivity sequence for the channel was NO3- greater than Cl- greater than HCO3-, and it was impermeable to gluconate ions, indicating that the channel diameter lies between 4.7 and 5.5 A. Current through the channel saturated at high chloride concentrations, and the relationship between channel current and chloride concentration could be approximated by the Michaelis-Menten equation. Analysis of the channel's anion permeability and its current vs. concentration relationship indicates that it can be described by the one-ion channel theory, with a relatively weak binding site inside the channel. Histograms of channel open and closed durations were constructed using the log binning technique and could be well fitted by triple exponential distributions, suggesting that the channel has at least three open and three closed states.

Calorimetry↗

PubMed1989

Cell poking: quantitative analysis of indentation of thick viscoelastic layers.

A recently introduced device, the cell poker, measures the force required to indent the exposed surface of a cell adherent to a rigid substratum. The cell poker has provided phenomenological information about the viscoelastic properties of several different types of cells, about mechanical changes triggered by external stimuli, and about the role of the cytoskeleton in these mechanical functions. Except in special cases, however, it has not been possible to extract quantitative estimates of viscosity and elasticity moduli from cell poker measurements. This paper presents cell poker measurements of well characterized viscoelastic polymeric materials, polydimethylsiloxanes of different degrees of polymerization, in a simple shape, a flat, thick layer, which for our purposes can be treated as a half space. Analysis of the measurements in terms of a linear viscoelasticity theory yields viscosity values for three polymer samples in agreement with those determined by measurements on a macroscopic scale. Theoretical analysis further indicates that the measured limiting static elasticity of the layers may result from the tension generated at the interface between the polymer and water. This work demonstrates the possibility of obtaining quantitative viscoelastic material properties from cell poker measurements and represents the first step in extending these quantitative studies to more complicated structures including cells.

Cell Adhesion↗

PubMed1989

Cystic fibrosis affects chloride and sodium channels in human airway epithelia.

Abnormalities of epithelial function in cystic fibrosis (CF) have been linked to defects in cell membrane permeability to chloride or sodium ions. Recently, a class of chloride channels in airway epithelial cells have been reported to lack their usual sensitivity to phosphorylation via cAMP-dependent protein kinase, suggesting that CF could be due to a single genetic defect in these channels. We have examined single chloride and sodium channels in control and CF human nasal epithelia using the patch-clamp technique. The most common chloride channel was not the one previously associated with CF, but it was also abnormal in CF cells. In addition, the number of sodium channels was unusually high in CF. These findings suggest a wider disturbance of ion channel properties in CF than would be produced by a defect in a single type of channel.

Chlorides↗

PubMed1986

Poiseuille flow method for measuring cell-to-cell adhesion.

A theoretical description of the cell aggregation process in the Poisseuille flow is presented, the capture efficiency being used as the measure of cell adhesiveness. The value of this coefficient is determined both for nonspecific and specific (i.e., mediated by molecules) cell-to-cell interactions. Regarding the cell adhesion described by the DLVO theory, it is possible to determine the conditions under which cell adhesion is maximal. The dependence of capture efficiency on parameters such as shear rate, size of cell, surface potential of cell, the Hamaker constant of substances constituting the cell membrane, and composition of the medium is analyzed. In the case of specific cell adhesion during flow, the change of suspension concentration is related to the number of free and occupied receptor sites, the diffusion constant of the receptors in the cell membrane, the force of a single bond, and the conditions of flow. It is possible to determine the diffusion constant from cell aggregation measurement when the force of a single bond and the number of free and occupied receptor sites are known.

Cell Adhesion↗

PubMed1986

Specific cell-to-cell adhesion under flow conditions.

An experimental method of measuring intercellular adhesion based on cell interactions in Poiseuille flow is presented. Hydrodynamic conditions of suspension flow, concentration of the suspension, and the size of cells allows determination of the capture efficiency, which is a measure of cell adhesiveness. The measurements of cell aggregation were performed with thymus cells and lectin from Ricinus communis. On the ground of theoretical description of the process of cell aggregation, it was possible to estimate the diffusion constant of the receptors in the cell membrane, which is equal to 5.6 X 10(-11) cm2/s. This value is in good agreement with the results of direct measurements of the diffusion constant of the lectin receptor. In the case of formalin-treated cells, the diffusion constant of the lectin receptors is equal to 6.8 X 10(-12) cm2/s. The total number of the receptor sites on the thymus cell surface for lectin R. communis and the affinity constant were also estimated.

Animals↗

PubMed1982

Surface interactions of leukemia and red cells passively moving in a quiescent fluid.

Adhesion to glass and silicone-coated surface of leukemia cells (L1210 and L5222) and their motion on an inclined plane have been investigated. The cells in quiescent fluid are subjected to the action of gravitational force; its component parallel to the surface ranges from 4.0 x 10(-14) N to 4.5 x 10(-13) N. The prolonged action of the gravitational force at 45 degrees (3.3 x 10(-13) N) causes the separation from the substratum of numerous cells which have adhered on a horizontal plane. When the cells passively move on the slope they are frequently arrested by adhesion which is partly short-lived. The analysis of the leukemia cell velocity reveals good agreement with the theory describing the motion of a sphere in quiescent fluid. The translation movement of nucleated cells on an inclined plane is not, however, accompanied by rotation. On the other hand, the cells flowing in mid-stream rotate with an angular velocity predicted by the theory.

Animals↗

PubMed1981

Contact of migrating L5222 leukemia cells with the substrate as studied under the action of gravitational force.

The interaction of actively migrating L5222 cells with the serum-coated glass surface was studied under the influence of an external force. In order to generate such force, the plane on which the cells were migrating, was inclined at different angles. The analysis of the L5222 cell migration pattern reveals that the modification of the cell movement direction relative to the surface appears when the plane is inclined at 1 degree. The increase of the slope to 2 degrees makes this effect more evident. When the slope is equal to 3 degrees the active motion of L5222 cells seems to disappear. The component of the gravitational force parallel to the plane on which the cells migrate ranges from 6.3 X 10(-15) N to 1.9 X 10(-14) N for the slopes from 1 degree to 3 degrees, respectively. The results of the study indicate that the force of interaction of migrating L5222 cells with the substrate is very small in comparison with the force of adhesion (not accompanied by locomotion) and seems to be comparable to that which operates in the near-substrate region of non-locomotive cells (e. g. L1210). Thus, the cell-substrate contact related to active movement and the non-locomotory adhesion of L5222 cells are probably different phenomena. As only L5222 cells were studied in this work, the above conclusions should be restricted to this cell type only.

Animals↗