A. A. Rubashkin

The Role of the Tight Junction in Paracellular Fluid Transport across Corneal Endothelium. Electro-osmosis as a Driving Force

The mechanism of epithelial fluid transport is controversial and remains unsolved. Experimental difficulties pose obstacles for work on a complex phenomenon in delicate tissues. However, the corneal endothelium is a relatively simple system to which powerful experimental tools can be applied. In recent years our laboratory has developed experimental evidence and theoretical insights that illuminate the mechanism of fluid transport across this leaky epithelium. Our evidence points to fluid being transported via the paracellular route by a mechanism requiring junctional integrity, which we attribute to electro-osmotic coupling at the junctions. Fluid movements can be produced by electrical currents. The direction of the movement can be reversed by current reversal or by changing junctional electrical charges by polylysine. Aquaporin 1 (AQP1) is the only AQP present in these cells, and its deletion in AQP1 null mice significantly affects cell osmotic permeability but not fluid transport, which militates against the presence of sizable water movements across the cell. By contrast, AQP1 null mice cells have reduced regulatory volume decrease (only 60% of control), which suggests a possible involvement of AQP1 in either the function or the expression of volume-sensitive membrane channels/transporters. A mathematical model of corneal endothelium predicts experimental results only when based on paracellular electro-osmosis, and not when transcellular local osmosis is assumed instead.Our experimental findings in corneal endothelium have allowed us to develop a novel paradigm for this preparation that includes: (1) paracellular fluid flow; (2) a crucial role for the junctions; (3) hypotonicity of the primary secretion; (4) an AQP role in regulation and not as a significant water pathway. These elements are remarkably similar to those proposed by the Hill laboratory for leaky epithelia.

Evidence for a central role for electro-osmosis in fluid transport by corneal endothelium.

The mechanism of transepithelial fluid transport remains unclear. The prevailing explanation is that transport of electrolytes across cell membranes results in local concentration gradients and transcellular osmosis. However, when transporting fluid, the corneal endothelium spontaneously generates a locally circulating current of approximately 25 microA cm(-2), and we report here that electrical currents (0 to +/-15 microA cm(-2)) imposed across this layer induce fluid movements linear with the currents. As the imposed currents must be approximately 98% paracellular, the direction of induced fluid movements and the rapidity with which they follow current imposition (rise time < or =3 sec) is consistent with electro-osmosis driven by sodium movement across the paracellular pathway. The value of the coupling coefficient between current and fluid movements found here (2.37 +/- 0.11 microm cm(2) hr(-1) microA (-1), suggests that: 1) the local endothelial current accounts for spontaneous transendothelial fluid transport; 2) the fluid transported becomes isotonically equilibrated. Ca(++)-free solutions or endothelial damage eliminate the coupling, pointing to the cells and particularly their intercellular junctions as a main site of electro-osmosis. The polycation polylysine, which is expected to affect surface charges, reverses the direction of current-induced fluid movements. Fluid transport is proportional to the electrical resistance of the ambient medium. Taken together, the results suggest that electro-osmosis through the intercellular junctions is the primary process in a sequence of events that results in fluid transport across this preparation.

Epithelial Fluid Transport: Protruding Macromolecules and Space Charges Can Bring about Electro-Osmotic Coupling at the Tight Junctions

The purpose of the present work is to investigate whether the idea of epithelial fluid transport based on electro-osmotic coupling at the level of the leaky tight junction (TJ) can be further supported by a plausible theoretical model. We develop a model for fluid transport across epithelial layers based on electro-osmotic coupling at leaky tight junctions (TJ) possessing protruding macromolecules and fixed electrical charges. The model embodies systems of electro-hydrodynamic equations for the intercellular pathway, namely the Brinkman and the Poisson-Boltzmann differential equations applied to the TJ. We obtain analytical solutions for a system of these two equations, and are able to derive expressions for the fluid velocity profile and the electrostatic potential. We illustrate the model by employing geometrical parameters and experimental data from the corneal endothelium, for which we have previously reported evidence for a central role for electro-osmosis in translayer fluid transport. Our results suggest that electro-osmotic coupling at the TJ can account for fluid transport by the corneal endothelium. We conclude that electro-osmotic coupling at the tight junctions could represent one of the basic mechanisms driving fluid transport across some leaky epithelia, a process that remains unexplained.

Balance of unidirectional monovalent ion fluxes in cells undergoing apoptosis: why does Na+/K+ pump suppression not cause cell swelling?

Non‐technical summary  Apoptosis is a crucial mechanism for tissue maintenance and deregulation of apoptosis may lead to catastrophic consequences in humans (e.g. cancer). The present work is a first attempt to quantitatively characterize rearrangement of the monovalent ion fluxes in cells during apoptosis. An established model of apoptosis induced by staurosporine in lymphoid U937 cells is used to experimentally measure cellular Cl− content and fluxes, K+, Na+ and water content as well as ouabain‐sensitive and ‐resistant Rb+ fluxes. A mathematical model is developed to account for the unidirectional ion fluxes and water balance in a cell as a whole. A decrease in the channel permeability of the plasma membrane for Na+ proved to be crucial for preventing cell swelling due to the decrease in Na+/K+ pump activity in cells undergoing apoptosis whereas opening of the K+ and Cl− channels is not required. Supplemental Table S1 is given for easy calculating flux balance under specified conditions.

Analysis of the monovalent ion fluxes in U937 cells under the balanced ion distribution : Recognition of ion transporters responsible for changes in cell ion and water balance during apoptosis

Unidirectional 22Na, Li+ and Rb+ fluxes and net fluxes of Na+ and K+ were measured in U937 human leukemic cells before and after induction of apoptosis by staurosporine (1 μM, 4 h) to answer the question which ion transporter(s) are responsible for changes in cell ion and water balance at apoptosis. The original version of the mathematical model of cell ion and water balance was used for analysis of the unidirectional ion fluxes under the balanced distribution of major monovalent ions across the cell membrane. The values of all major components of the Na+ and K+ efflux and influx, i.e. fluxes via the Na+,K+‐ATPase pump, Na+ channels, K+ channels, Na/Na exchanger and Na‐Cl symport were determined. It is concluded that apoptotic cell shrinkage and changes in Na+ and K+ fluxes typical of apoptosis in U937 cells induced by staurosporine are caused by a complex decrease in the pump activity, Na‐Cl symport and integral Na+ channel permeability.

POTENTIAL DISTRIBUTION ACROSS THE ELECTROACTIVE-POLYMER FILM BETWEEN THE METAL AND SOLUTION AS A FUNCTION OF THE FILM CHARGING LEVEL

Abstract The distribution of the electrostatic potential across the electroactive-polymer film between the metal electrode and the solution has been analysed theoretically at various charging levels. The content of the film is determined by the electronic equilibrium with the metal and the exchange by co- and counterions with the solution. The electronic and ionic concentrations inside the film are assumed to be well below their saturation values. Two limiting shapes of the potential profile have been found, with a plateau region inside the film separating two space-charge layers near the interfaces, and with the potential drop distributed within the whole film, like that in an insulating film. In its turn, profiles with the bulk-film region have been separated into three different types, referred to as the “membrane”, “electroncounterion” and “semiconductor” forms. Depending on the electronic and ionic parameters of the system, the profile may show various scenarios of evolution among these states upon sweeping the electrode polarization. Available ways to distinguish between these variants are discussed.

The role of potassium, potassium channels, and symporters in the apoptotic cell volume decrease: experiment and theory.

Ample evidences indicate an essential role of ion channels in apoptosis [3, 4]. The channels permeable for K + ions are of primary interest because the efflux of K + via channels is believed to be responsible for cell volume decrease, known as a hallmark of apoptosis. It has been shown that at least 14 species of K + channels are involved in apoptosis of various cells [5, 6]. The role of K + channels in the regulation of apoptosis is assumed to be related to the influence of intracellular K + concentration on apoptotic caspases and nucleases [6–8]. However, this assumption is based mostly on the study of the shift in intracellular K + concentration with use of a fluorescent probe and measurement of the forward light scattering. These methods are not adequate for the quantitative analysis of the ion and water balance of cells. We studied changes in the intracellular content and concentration of K + during apoptotic decrease in cell volume using other methods. Cell water was assayed by measuring the buoyant density of cells in a continuous Percoll gradient; cell K + and Na + , by flame emission analysis. Well-known models of apoptosis were studied, namely, the apoptosis of rat thymocytes induced by dexamethasone (1 μ M, 4–6 h) and the apoptosis of human lymphoma cell line U937 treated with staurosporine (1 μ M, 4 h). Apoptosis was examined using confocal microscopy and flow cytometry. The methods were described in details earlier [9, 10].

Regulatory volume increase (RVI) and apoptotic volume decrease (AVD) in U937 cells in hypertonic medium

Changes in intracellular water, K+ and Na+ of U937 cells incubated in hyperosmolar medium supplemented with 200 mM sucrose have been studied. Cells were stained with acrydine orange, ethydium bromide, APOPercentage dye, which marks the phosphatidyl serine distribution on the plasma membrane; and FLICA polycaspase fluorescent dye. It was found that cell shrinkage produced by direct osmotic effect induced both a regulatory volume increase and apoptotic volume decrease. The regulatory volume increase dominated at the early stage, whereas apoptotic volume decrease prevailed at the later stage. Therefore, U937 cells were capable of triggering apoptosis and apoptotic volume decrease, despite the unimpaired regulatory volume increase response, and the current opinion that the dysfunction of the regulatory volume increase is a prerequisite for apoptosis and apoptotic volume decrease (Subramanyam et al., 2010) should be revised. It is concluded that the apoptotic volume decrease plays a significant role in preventing osmotic lysis in apoptotic cells, rather than in initiating apoptosis.

Changes in K+, Na+, Cl− balance and K+, Cl− fluxes in U937 cells induced to apoptosis by staurosporine: On cell dehydration in apoptosis

The K+, Na+, and Cl− balance and K+ (Rb+) and 36Cl− fluxes in U937 cells induced to apoptosis by 0.2 or 1 μM staurosporine were studied using flame emission and radioisotope techniques. It is found that two-thirds of the total decrease in the amount of intracellular osmolytes in apoptotic cells is accounted for by monovalent ions and one-third consists of other intracellular osmolytes. A decrease in the amount of monovalent ions results from a decrease in the amount of K+ and Cl− and an increase in the Na+ content. The rate of 36Cl−, Rb+ (K+), and 22Na+ equilibration between cells and the medium was found to significantly exceed the rate of apoptotic change in the cellular ion content, which indicates that unidirectional influxes and effluxes during apoptosis may be considered as being in near balance. The drift of the ion flux balance in apoptosis caused by 0.2 μM staurosporine was found to be associated with the increased ouabain-resistant Rb+ (K+) channel influx and insignificantly altered the ouabain-sensitive pump influx. Severe apoptosis induced by 1 μM staurosporine is associated with reduced pump fluxes and slightly changed channel Rb+ (K+) fluxes. In apoptotic cells, the 1.4–1.8-fold decreased Cl− level is accompanied by a 1.2–1.6-fold decreased flux.

Electrostatic contribution to the ion solvation energy: cavity effects

ABSTRACT Ion solvation process has been analysed for the spherically symmetrical system where an ion is located inside a cavity surrounded by an isotropic nonlocal dielectric medium. It has been proven that for any dielectric properties of the medium, the electric field outside the cavity as well as the ion solvation energy depend only on the total ion charge but not of the particular distribution of the ion charge density inside the cavity. These characteristics remain unchanged if the charge is displaced from the external boundary of the cavity into it. Analytical formulas for them have been derived for a particular model of the nonlocal dielectric function. Comparison of results for the solvation energy on the basis of this new theory and of the conventional approach (disregarding the existence of the cavity) shows a significant difference between their predictions if the ion charge is displaced inside the ion cavity.