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J Fischbarg

Publications and source records attributed to J Fischbarg.

At least 73 records · Page 4Linked to original sources
PubMed1993

Cultured bovine corneal endothelial cells express CHIP28 water channels.

Cultured bovine corneal endothelial cells (CBCEC) transport fluid from the basal to the apical surface. In this study, we examined whether the plasma membranes of these cells have water channels. We cultured BCEC on glass plates and monitored the intensity of the light scattered (IS) by the cells. We determined the kinetic constant (k) of the change in IS on exposure to a 10% hypoosmotic challenge to calculate the osmotic permeability (Pf) of the plasma membrane. At 37 degrees C, we found values of k = 0.68 +/- 0.07 s-1 and Pf = 93.3 +/- 33 microns/S (n = 13). The sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS; 1 mM) reduced Pf by 75%; 5 mM dithiothreitol reversed such inhibition. The activation energy (Ea) of Pf in the range 10-37 degrees C was 4.7 +/- 0.7 kcal/mol (n = 5). The high Pf, values, the inhibition by pCMBS, and the low Ea strongly suggest the presence of water channels. Therefore, we tested whether the injection of poly(A)+ RNA prepared from CBCEC into Xenopus laevis oocytes results in the expression of water channels. Four days after injection, we measured oocyte Pf values from the rate of volume increase on exposure to hypoosmotic medium. In control oocytes injected with 50 nl of water, Pf was 13.4 +/- 0.3 microns/S (n = 63). In oocytes injected with poly(A)+ RNA (50 ng/oocyte in 50 nl water), Pf was 40.9 +/- 1.6 microns/S (n = 72).(ABSTRACT TRUNCATED AT 250 WORDS)

4-Chloromercuribenzenesulfonate↗
PubMed1993

Determination of volume and water permeability of plated cells from measurements of light scattering.

Measurements of cell membrane water osmotic permeabilities can be inaccurate because of the technical difficulties inherent to cell volume measurements and because of the presence of an unstirred water layer in contact with the cells. We detail here a method we have developed to quantify transient changes in cell volumes from the intensity of light scattered by cells. For this, we theorize how an unstirred layer originates in a perfusing chamber, and we calculate values for both cell membrane water osmotic permeability and unstirred layer thickness from time transient changes in scattered light. We apply a computer algorithm that finds the best correspondence between experimental data and estimated values. This is done by solving a differential equation governing cell volume changes by numerical integration (Runge-Kutta) and iterating the procedure varying the test values of osmotic permeability and unstirred layer thickness until the best fit is achieved. We exemplify this procedure with experimental results obtained in adherent cultured cells.

Animals↗
PubMed1992

Kinetic analysis of water transport through a single-file pore.

We apply the diagrammatic method developed by Hill (1977. Free Energy Transduction in Biology. Academic Press, New York) to analyze single-file water transport. We use this formalism to derive explicit expressions for the osmotic and diffusive permeabilities Pf and Pd of a pore. We first consider a vacancy mechanism of transport analogous to the one-vacancy pore model previously used by Kohler and Heckmann (1979. J. Theor. Biol. 79:381-401). (a) For the general one-vacancy case, we find that the permeability ratio can be expressed by Pf/Pd = (Pf/Pd)eqf(wA,wB), where the second factor is a function of the water activities in the two adjoining compartments A and B. As a consequence, the permeability ratio in general can effectively differ from its value at equilibrium. We also find that n - 1 less than or equal to (Pf/Pd)eq less than or equal to n, a result already proposed by Kohler and Heckmann (1979. J. Theor. Biol. 79:381-401). (b) When vacancy states are transient intermediates, the model can be reduced to a diagram consisting of only fully occupied states. Such a diagram resembles the one describing a no-vacancy mechanism of transport (c), but in spite of the similarity the expressions obtained for the permeability coefficients still retain the basic relationships of the original (a) nonreduced one-vacancy model. (c) We then propose a kinetic description of a no-vacancy mechanism of single-file water transport. In this case, the expressions derived for Pf and Pd are formally equivalent to those obtained by Finkelstein and Rosenberg (1979. Membrane Transport Processes. Vol. 3. C.F. Stevens and R.W. Tsien, editors, Raven Press, New York. 73-88.) A main difference with the vacancy mechanism is that here the permeability coefficients are independent of the water activities.

Biological Transport↗
PubMed1992

Fluid transport across cultured bovine corneal endothelial cell monolayers.

The mammalian corneal endothelium is known to transport fluid from the stromal compartment to the aqueous humor, thereby maintaining corneal transparency. Corneal endothelial cells have been cultured for some years now, but whether they preserve their in vivo ability to actively transport fluid is not known. We have now grown bovine corneal endothelial cell monolayers (BCECM) on permeable substrates (Transwell) and report that, just like their counterparts in vivo, these cultured cells pump fluid from the basal to the apical compartment and display measurable electrical resistance and potential difference across the monolayer. BCECM were grown on collagen-treated permeable supports using Dulbecco's modified Eagle's medium (DMEM)/20% fetal bovine serum with antibiotics. Cells grew to confluence in 5-7 days and displayed polygonal shape. Only cells from passages 1-3 were utilized. Inserts were fitted directly into Lucite chambers specially built. The rate of fluid pumping by BCECM was 3.96 +/- 0.49 (SE) microliter.h-1.cm-2 (n = 13) and could be measured continuously for several hours; fluid pumping was inhibited by 0.2 mM amiloride. The specific electrical resistance of the monolayers was 180 +/- 22 omega.cm2 (n = 11). A mean electrical potential difference of 63.8 +/- 3.7 microV (n = 15, range 40-100 microV, apical side negative) was recorded across the monolayers in DMEM. The availability of the commercial inserts makes this procedure practical; as a consequence, the rate of fluid transport by cultured corneal endothelium has been quantitated for the first time. This method can now be extended to other cultured layers.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1990

Effects of ambient bicarbonate, phosphate and carbonic anhydrase inhibitors on fluid transport across rabbit corneal endothelium.

Bicarbonate has been long held to be indispensable for fluid pumping by the endothelium; however, such need has been disputed recently. We investigated this issue and found that: (1) the corneal endothelium pumps fluid equally well (at 6-8 microliters hr-1 cm-2) whether the bathing solution contains 43 mM bicarbonate or 10 mM phosphate, (2) if bicarbonate and most of the phosphate are absent, fluid pumping is noticeably lowered (2-4 microliters hr-1 cm-2), (3) carbonic anhydrase inhibitors (5 mM acetazolamide; 0.1, 0.2 and 0.3 mM ethoxzolamide) block this lowered fluid pumping, and (4) in the absence of external bicarbonate, 20 mM HEPES is insufficient to preserve adequate fluid pumping. These results are consistent with existing models for endothelial transport in which exogenous and endogenous CO2 are converted to HCO3- by carbonic anhydrase, with HCO3- fueling the transport mechanism and therefore the fluid pump.

Acetazolamide↗
PubMed1990

Glucose transporters serve as water channels.

Water traverses the plasma membranes of some eukaryotic cells faster than can be explained by the water permeability of their lipid bilayers. This has led to a search for a water channel. Our previous work identified glucose transporters as candidates for such a channel. We report here that Xenopus laevis oocytes injected with mRNA encoding the brain/Hep G2, adult skeletal muscle/adipocyte, or liver forms of the glucose transporter exhibit an osmotic water permeability of their plasma membranes larger than that of untreated oocytes. The osmotic water permeability component attributable to glucose transporters increased an average of 4.8-fold in the injected oocytes. These studies provide direct evidence that the facilitative, sodium-independent mammalian glucose transporters serve as membrane water channels.

Animals↗
PubMed1990

Effect of fluorescein on the electrical potential difference across isolated rabbit corneal endothelium.

The authors investigated whether fluorescein sodium affects the in vitro endothelial function of rabbit corneas. As an index of this function, the transendothelial electrical potential difference (TEPD) was used. The TEPD in a balanced salts and glucose (BSG) control solution increased for the first 30 min and then decayed slowly, reaching about 60% of its original value after 5 hr. When a BSG solution containing 5 micrograms/ml of fluorescein sodium was used, the TEPD time course was similar to the control solution. Since this fluorescein sodium concentration is about sevenfold higher than that seen in the anterior chamber of ocular patients, these results reassure users that no toxic effect of fluorescein is discernible at concentrations relevant to ophthalmic practice. With a fluorescein sodium concentration of 500 micrograms/ml, the TEPD decreased below control values after 4 hr of exposure, but such a concentration is approximately 5000-fold higher than that seen in the anterior chamber of patients. The adverse effect of fluorescein on TEPD is probably irrelevant for standard systemic clinical use.

Analysis of Variance↗
PubMed1989

Kinetic model of the effects of electrogenic enzymes on the membrane potential.

Electrogenic enzymes contribute to the electrical field existing across biological membranes by using a source of free energy to generate an ionic current. The model introduced here permits one to evaluate this contribution. Since the model incorporates the electrogenic enzyme in the form of a sequential kinetic diagram, it permits one to study the kinetic effects of the concentration of the enzyme, the substrates and the different ligands on the membrane potential. Ionic electrodiffusion is expressed in terms of a chemical reaction; ionic permeabilities are thus treated as voltage-dependent rate constants. We use the condition of global electroneutrality to obtain an expression for the electrical potential difference across the membrane; such expression constitutes an extension of the Goldman-Hodgkin-Katz equation. The enzyme-related terms appear in the equation as functions of the rate constants and the diverse concentrations. The model is used to analyze the case of a cell membrane traversed by Na+ and K+ by simple diffusion, and by electrogenic transport mediated by a Na+-K+ ATPase. The enzyme reaction is represented by the six-step scheme proposed by Chapman et al. (1983, J. membr. Biol. 74, 139-153). The main results of the numerical calculations are that, within a certain interval, the membrane potential difference depends linearly on the enzyme density and hyperbolically on the ATP concentration. A similar behavior has been experimentally observed for the electrogenic proton pump of Neurospora crassa. Thus, the model here can be useful in the explanation and prediction of effects of electrogenic enzymes on the membrane potential.

Animals↗
PubMed1989

Evidence that the glucose transporter serves as a water channel in J774 macrophages.

Water transport across plasma membranes is a universal property of cells, but the route of such transport is unclear. In this study, volume changes of cells of the J774 murine macrophage-like cell line were monitored by recording the intensity of light scattered by the cells. We investigated the effects of several inhibitors of glucose transport on cell membrane osmotic water permeability as calculated from the rates of cell volume change. Cytochalasin B (2.5 micrograms/ml), phloretin (20 microM), and tomatine (3 microM) reversibly blocked glucose uptake into these cells. All three inhibitors reversibly decreased the osmotic water permeability of J774 cells from 89.6 +/- 3.2 to 27.2 +/- 1.4 microns/sec. We conclude that a major component of the osmotic water flow across the plasma membranes of these cells is accounted for by water traversing their glucose transporters.

3-O-Methylglucose↗
PubMed1989

Hydraulic conductivity of endothelial cell monolayers cultured on human amnion.

Hydraulic conductivity (Lp) of endothelial cell monolayers cultured on a supporting matrix of human amnion was measured. Bovine aortic (Baec), bovine microvascular (Bmec), and human umbilical vein endothelial cell (Huvec) monolayers were mounted in a water-jacketed chamber maintained at 37 degrees C. The lower compartment of the chamber was connected to an electronic sensor-aspiration system that served to volume clamp the lower compartment. The aspirated volume was displayed on a chart recorder, providing continuous measurements of volume flow per unit area (Jv). In 36 monolayers, Jv was linearly related to hydrostatic pressure. The average slope of this relationship (Lp) was 1.14 +/- 0.8 X 10(-6), 1.67 +/- 1.49 X 10(-6), and 2.9 +/- 0.85 X 10(-6) cm.s-1.cmH2O-1 for Baec, Bmec, and Huvec, respectively. The effect of increased luminal oncotic pressure was studied in seven monolayers. The oncotic pressure difference (delta pi) ranged from 3.1 to 7.1 cmH2O (measured with a Wescor oncometer). In all cases, there was an immediate fall in Jv and a displacement to the right of the x-intercept of the line Jv vs. change in pressure (delta P) without a change in the slope (Lp). These results indicate that the Lp of endothelial cell monolayers cultured on amnion is within the range of values previously reported for single capillaries in vivo and that these monolayers appear to sustain delta pi sufficient to generate an osmotically driven water flow.

Amnion↗
PubMed1989

Inhibition of the hydrosmotic response to antidiuretic hormone by 3,3'-diallyldiethylstilbestrol (DADES).

3,3'-diallyldiethylstilbestrol (DADES), a blocker of the facilitated diffusion of glucose, was found to interfere markedly with the hydrosmotic response to antidiuretic hormone and its related agonists. Frog urinary bladders were isolated and monitored for transmural net water flow. DADES was added either to the serosal or to the apical medium at concentrations ranging from 10(-4) M to 10(-6) M. Pretreatment for 30 min with apical 10(-4) M DADES drastically reduced the subsequent hydrosmotic response: (a) to oxytocin (4.4 x 10(-8) M) by 91.7 +/- 17.6% versus 6.2 +/- 7.8 in control; (b) to 8-bromo 3',5'-cyclic AMP by 93.5 +/- 19.4% versus 19.4 +/- 11.4%; (c) to serosal hyperosmolarity (mannitol 220 mOsm) by 99.3 +/- 0.5% versus 12.3 +/- 18.2%. This effect was dose-dependent. Inhibitory action of DADES was more effective on the apical side than on the serosal side (97.0 +/- 1.5 versus 45.8 +/- 10.8). Freeze-fracture studies revealed a modified distribution of the particles and unusual endocytotic pits and vesicles in the apical membrane of both granular and mitochondria-rich epithelial cells. These observations point to multiple and complex effects of the drug. Thus, it seems that DADES has numerous effects on urinary epithelium, which makes it a nonspecific inhibitor of water permeation. Conclusions on its use should therefore be drawn with suitable caution.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Effects of human neutrophil chemotaxis across human endothelial cell monolayers on the permeability of these monolayers to ions and macromolecules.

We have developed a method for studying the permeability properties of human endothelia in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured on a substrate of human amnion. Confluent monolayers of these cells demonstrated 6-12 delta.cm2 of electrical resistance (a measure of their permeability to ions) and restricted the transendothelial passage of albumin from their apical to their basal surface. To determine whether leukocyte emigration alters endothelial permeability in this model, we examined the effects of migrating human polymorphonuclear leukocytes (PMN) on these two parameters. Few PMN migrated across the HUVEC monolayers in the absence of chemoattractants. In response to chemoattractants, PMN migration through HUVEC monolayers was virtually complete within 10 minutes and occurred at random locations throughout the monolayer. PMN migrated across the monolayer via the paracellular pathway. Although one PMN migrated across the monolayer for each HUVEC, PMN migration induced no change in electrical resistance or albumin permeability of these monolayers. At this PMN:HUVEC ratio, these permeability findings were correlated morphologically to measurements that HUVEC paracellular pathway size increases by less than 0.22% with PMN migration. This increase is insufficient to effect a measurable change in the electrical resistance of the endothelial cell monolayer. These findings demonstrate that increased permeability of cultured endothelial cell monolayers is not a necessary consequence of PMN emigration.

Albumins↗
PubMed1988

Extracellular ATP induces a large nonselective conductance in macrophage plasma membranes.

Extracellular ATP in its tetra-anionic form (ATP4-) induces ion fluxes and membrane depolarization in the mouse macrophage-like cell line J774.2 and in resident mouse macrophages. We analyzed the effects of extracellular ATP4- by both patch-clamp and intracellular microelectrode techniques. Whole-cell patch-configuration membrane potential measurements on J774.2 cells revealed that ATP4- -induced depolarization occurred within 40 ms of pulsed application of ATP and was completely reversible. The depolarizations were accompanied by a dramatic increase in membrane conductance and showed no sign of adaptation to ATP over a period of 30 min. At 5 mM total ATP (ATPt) the whole-cell conductance was approximately 10 nS, and an upper limit of 20 pS for a single-channel conductance has been established. The reversal potential associated with the ATP-induced depolarization at asymmetric K+, Na+, Ca2+, and Cl- concentrations across the membrane was 0 mV. In patch-clamped cells depolarization was complete at 20 microM ATP4-, and repolarization from full depolarization occurred in approximately 5 s. In contrast, in intact cells measured by microelectrode impalement, complete depolarization occurred at approximately 2 mM ATP4- and repolarization was much slower (approximately 100 min). These findings indicate that the changes in intracellular ionic composition that occur after ATP treatment affect the rate of cell repolarization. At lower concentrations of ATP, potassium conductances modulated the depolarizing effect of ATP. ATP also depolarized mouse peritoneal macrophages, but a variant cell line (ATPR B2), derived from J774.2 cells by prolonged exposure to ATP, was insensitive to ATP. Our results provide a membrane electrophysiological description and analysis of a large nonselective plasma membrane conductance of macrophages induced by extracellular ATP.

Adenosine Triphosphate↗
PubMed1988

Use of transendothelial electrical potential difference to assess the chondroitin sulfate effect in corneal preservation media.

Corneal preservation time can be prolonged using chondroitin sulfate (CS) in preservation media and recently a great deal of attention has been focused on evaluating the effectiveness of CS. So far evaluations of the effectiveness of this and other additives have been based on determining the state of the cornea at the end of the preservation period. A more informative determination of the viability of stored corneas can be made by monitoring their physiological parameters throughout the storage period. We have accomplished this by monitoring in vitro the transendothelial electrical potential difference across deepithelialized rabbit corneas. We found that corneas stored in solutions containing basal salts, glucose and CS maintained higher transendothelial potential differences than corneas stored in the same solutions without CS, thus confirming the benefits of using CS for medium-term corneal preservation. The beneficial effects of CS were optimal at the 1% concentration, and were reduced at higher and lower concentrations.

Animals↗
PubMed1987

Inhibition of transepithelial osmotic water flow by blockers of the glucose transporter.

On the basis of evidence derived mostly from human erythrocytes, it has been suggested that water traverses cell membranes through membrane-spanning proteins such as the anion channel or the glucose transporter acting as water pores. However, specific inhibitors of such permeation processes have not been found to block water transport, and hence a precise identification of the water route has not been possible so far. We have investigated this issue by characterizing the osmotic flows across a fluid-transporting epithelium, the rabbit corneal endothelium. The rate of such flows was monitored continuously as a function of time. We confirmed prior findings of an inhibition by PCMBS on osmotic water flow, and lack of inhibition by DTNB and DIDS. On the other hand, we have found for the first time that several blockers of glucose facilitated diffusion, namely, phloretin (2 mM), phloridzin (2 mM), diallyldiethylstilbestrol (0.1 mM), cytochalasin B (20 micrograms/ml), and ethylidene-D-glucose (200 mM), all clearly inhibit osmotic flow. Our evidence is consistent with the hypothesis that both water and glucose may traverse these cell membranes through the same channel-like pathway contained in the glucose transporter membrane-spanning protein.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1987

Fractal model of ion-channel kinetics.

Markov models with discrete states, such as closed in equilibrium with closed in equilibrium with open have been widely used to model the kinetics of ion channels in the cell membrane. In these models the transition probabilities per unit time (the kinetic rate constants) are independent of the time scale on which they are measured. However, in many physical systems, a property, L, depends on the scale, epsilon, at which it is measured such that L(epsilon) alpha epsilon 1-D where D is the fractal dimension. Such systems are said to be 'fractal'. Based on the assumption that the kinetic rates are given by k(t) alpha t1-D we derive a fractal model of ion-channel kinetics. This fractal model has fewer adjustable parameters, is more consistent with the dynamics of protein conformations, and fits the single-channel recordings from the corneal endothelium better than the discrete-state Markov model.

Animals↗
PubMed1986

Membrane pores: a computer simulation of interacting pores analyzed by g1(tau) and g2(tau) correlation functions.

Ion channels in a cell membrane were modeled by a computer simulation of fluctuating pores distributed in a spatial array, a cellular automata. The sum of the currents through such a set of pores models a noise analysis experiment. These currents were analyzed by using the optical correlation functions gn(tau) = (fn(t)fn(t + tau))/(f2(t))n, where f(t) are the current deviations around the mean current and () denotes the time average. These functions can be easily used to determine if the noise is Gaussian. If the noise is not Gaussian, they provide additional information not already contained in the power spectrum. When the pores do not interact with each other, the noise is Gaussian and the power spectrum a Lorentzian. When the pores interact in a strongly cooperative way the noise was still Gaussian and the power spectrum still a Lorentzian, but the usual analysis applied to such a case would over-estimate the single channel conductance. If the kinetics of the pore opening and closing vary on the time scale of the experiment then the relationship g2(tau) = 1 + 2[g1(tau)]2 is no longer satisfied.

Cell Membrane↗