The apical membrane glycocalyx of MDCK cells.

The microenvironment near the apical membrane of MDCK cells was studied by quantitation of the fluorescence of wheat germ agglutinin attached to fluorescein (WGA). WGA was shown to bind to sialic acid residues attached to galactose at the alpha-2,3 position in the glycocalyx on the apical membrane. Young MDCK cells (5-8 days after splitting) showed a patchy distribution of WGA at stable sites that returned to the same locations after removal of sialic acid residues by neuraminidase treatment. Other lectins also showed stable binding to patches on the apical membrane of young cells. The ratio of WGA fluorescence emission at two excitation wavelengths was used to measure near-membrane pH. The near-membrane pH was markedly acidic to the pH 7.4 bathing solution in both young and older cells (13-21 days after splitting). Patches on the apical membrane of young cells exhibited a range of near-membrane pH values with a mean +/- SEM of 6.86 +/- 0.04 (n = 121) while the near-membrane pH of older cells was 6.61 +/- 0.04 (n = 120) with a uniform WGA distribution. We conclude that the distribution of lectin binding sites in young cells reflects the underlying nonrandom location of membrane proteins in the apical membrane and that nonuniformities in the pH of patches may indicate regional differences in membrane acid-base transport as well as in the location of charged sugars in the glycocalyx.

Diffusion of small solutes in the lateral intercellular spaces of MDCK cell epithelium grown on permeable supports.

The diffusion coefficients of four solutes ranging in molecular weight from 238 to 10,000 in the lateral intercellular spaces (LIS) of cultured kidney cells (MDCK) grown on permeable supports were determined from the spread of fluorescence produced after the release of caged compounds by a pulse from a UV laser. Two types of experiments were performed: measurement of the rate of change of fluorescence after releasing a caged fluorophore, and measurement of the change in fluorescence of a relatively static fluorescent dye produced by the diffusion of an uncaged ligand for the dye. Fluorescence intensity was determined by photon-counting the outputs of a multichannel photomultiplier tube. Diffusion coefficients were determined in free solution as well as in the LIS of MDCK cells grown on permeable supports and the hindrance factor, theta, determined from the ratio of the free solution diffusivity to that in the LIS. The hindrance factors for 3000-MW dextran, 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, MW 524) and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, MW 238) were not significantly different from 1. The diffusion of 10,000-MW dextran was substantially reduced in the LIS with a theta of 5.6 +/- 0.3. Enzymatic digestion by neuraminidase of the sialic acid residues of the glycosylation groups in the LIS increased the diffusivity of the 10,000-MW dextran 1.8-fold indicating hindrance by the glycocalyx. We conclude that small solutes, such as Na(+) and Cl(-), would not be significantly restricted in their diffusion in the LIS and that solute concentration gradients could not develop along the LIS under physiologic conditions.

Diffusion coefficients in the lateral intercellular spaces of Madin-Darby canine kidney cell epithelium determined with caged compounds.

The diffusion coefficients of two caged fluorescent dyes were measured in free solution and in the lateral intercellular spaces (LIS) of cultured Madin-Darby canine kidney (MDCK) cells after photoactivation by illumination with a continuous or pulsed UV laser. Both quantitative video imaging and a new photometric method were utilized to determine the rates of diffusion of the caged fluorescent dyes: 8-((4,5-dimethoxy-2-nitrobenzyl)oxy)pyrene-1,3,6-trisulfonic acid (DMNB-HPTS) and (4,5-dimethoxy-2-nitrobenzyl) fluorescein dextran (10,000 MW) (DMNB-caged fluorescein dextran). The diffusion coefficients at 37 degrees C in free solution were 3.3 x 10(-6) cm2/s (HPTS) and 0.98 x 10(-6) cm2/s (10,000 MW dextran). Diffusion of HPTS within nominally linear stretches of the LIS of MDCK cells grown on glass coverslips was indistinguishable from that in free solution, whereas dextran showed a 1.6 +/- 0.5-fold reduction in diffusivity. Measurements of HPTS diffusion within the LIS of multicellular regions also exhibited a diffusivity comparable to the free solution value. The restriction to diffusion of the dextran within the LIS may be due to molecular hindrance.

Regulation of the MDCK cell tight junction.

The sodium flux across individual tight junctions (TJ) of low-resistance MDCK cell monolayers grown on glass coverslips was determined as a measure of paracellular permeability. Increases in perfusate glucose concentration from 5 to 25 mM decreased tight junction Na permeability. This permeability decrease was not specific as nonmetabolizable analogues of glucose caused similar diminutions in TJ Na permeability. Stimulation of protein kinase A increased TJ Na permeability, and inhibition of protein kinase A decreased TJ Na permeability. Transepithelial electrical resistance of monolayers grown on permeable supports did not change as predicted from the observed alterations in TJ Na permeability of monolayers grown on glass coverslips. Fluorescent labeling of cell F-actin showed that increased F-actin in the perijunctional ring correlated with higher TJ Na permeability. Although a low dose of cytochalasin D did not change TJ Na permeability, it disrupted the cytoskeleton and blocked the decrease in TJ Na permeability caused by glucose. Cytochalasin D failed to block the effects of protein kinase A stimulation or inhibition on TJ Na permeability. We conclude that tight junction sodium permeability is regulated both by protein kinase A activity and by other processes involving the actin cytoskeleton.

Effect of the alkyl chain length of monocarboxylic acid on the permeation through bilayer lipid membranes.

Electrically silent hydrogen ion fluxes across a planar bilayer lipid membrane (BLM) induced by an addition of monocarboxylic acid at one side of BLM were studied by measuring pH changes in the unstirred layers near the BLM surface. The pH changes were assayed by recording protonophore-dependent potentials as well as by direct measurements of pH shifts in he unstirred layers close to the membrane by the pH microelectrode. It was shown that the mechanism of the acid transport changed qualitatively upon the increase of the hydrophobic chain length of the acid. In the case of short-chain acids at pH < pKa, the total transport was limited by diffusion of the anionic form of the acid across the unstirred layers, while at the alkaline pH (pH>>pKa) the transport was limited by diffusion of the neutral form across the membrane. In the alkaline pH range the pH shifts induced by short-chain acids were sensitive to the presence of cholesterol in the BLM as well as to the stirring conditions in the cell. However, in the case of long chain acids (more than 8 carbonic atoms) the transport was limited by diffusion of the anionic form of the acid in the whole range of pH studied. In the latter case, pH changes in the unstirred layers did not depend on the presence of cholesterol in the membrane, and moreover pH shifts were not dependent on the thickness of the unstirred layer. It was proposed that the peculiarities of the long-chain acid-induced proton transport were associated with the formation of micelles of the acid in bathing solutions.

Evidence in favor of the existence of a kinetic barrier for proton transfer from a surface of bilayer phospholipid membrane to bulk water.

When the hydrogen-ion flux is induced by nigericin across the planar bilayer lipid membrane (BLM) with bulk pH values being equal at the opposite sides of the BLM, formation of a difference in boundary potentials (delta phi b) on the membrane is observed by the method of inner membrane field compensation. pH gradients are titrated routinely by the addition of sodium acetate at one side of the membrane. The increase in buffer concentration (citrate, phosphate, Mes) leads to a decrease in delta phi b. delta phi b forms in the presence of phosphatidylserine in the membrane-forming solution only. It is concluded that the steady-state difference of the hydrogen ion binding to the opposite surfaces of the membrane (HIBD) is created under the conditions of equal pH values near surfaces of the BLM. The model of the processes implies that nigericin transfers proton predominantly from interface to interface while acetate transfers the proton from bulk phase to bulk phase. In the other series of experiments the monensin-mediated formation of the HIBD leads to the formation of an potassium-ion gradient in the presence of nigericin. Thus, a possibility of performing a work due to the formation of HIBD is demonstrated. Owing to these properties the hydrogen-ion binding difference can be interpreted in a first approximation as a difference of surface hydrogen-ion concentration at the opposite sides of the membrane, arising due to the existence of a kinetic barrier for the proton transfer at the membrane interfaces. These findings can be significant for the mechanism of energy transduction in membrane phosphorylation in mitochondria and chloroplasts.

Proton dissociation from nigericin at the membrane-water interface, the rate-limiting step of K+/H+ exchange on the bilayer lipid membrane.

The rate of K+/H+ exchange through bilayer lipid membranes (BLM) induced by nigericin was measured by the method of pH gradient offset according to Antonenko, Yu.N. and Yaguzhinsky L.S. [(1990) Biochim. Biophys. Acta 1026, 236-240]. It was shown that under the conditions of high potassium ion concentration the rate of nigericin-mediated K+/H+ exchange increased with an increase in the concentrations of such buffer compounds as citric acid and MES. The concentration dependence was different for citrate and MES. The buffer concentration effect was absent at low potassium ion concentrations. Citrate increased the rate of K+/H+ exchange being added to the side of BLM where the K+ concentration was higher and had no effect at the opposite side. At high KCl and citrate concentrations, the rate of K+/H+ exchange was about 6 times lower in D2O when compared to H2O solutions. It is concluded that under certain experimental conditions the overall rate of the K+/H+ exchange induced by nigericin is determined by the rate of proton dissociation from nigericin at the membrane-water interface.