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M J Sanders

Publications and source records attributed to M J Sanders.

27 records · Page 2Linked to original sources
PubMed1982

Ouabain-sensitive 86Rb(K) influx is linked to transepithelial Na transport in pig kidney cell line.

The pig kidney cell line, LLC-PK1, exhibits rheogenic D-glucose coupled transepithelial Na+ transport that is inhibited by phlorizin. By measuring the difference in initial rates of influx of 86Rb+ with and without coupled Na+ transport, we can demonstrate an 86Rb+ uptake linked to Na+ transport, The simultaneous determination of phlorizin-inhibited Na coupled D-[3H] glucose uptake and 86Rb+ influx allows calculation of an Na+/Rb+ stoichiometry that is consistent with an electrogenic Na+ for Rb+ exchange.

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PubMed1982

Transepithelial transport in cell culture: stoichiometry of Na/phlorizin binding and Na/D-glucose cotransport. A two-step, two sodium model of binding and translocation.

The renal cell line LLC-PK1 cultured on a membrane filter forms a functional epithelial tissue. This homogeneous cell population exhibits rheogenic Na-dependent D-glucose coupled transport. The short-circuit current (Isc) was accounted for by net apical-to-basolateral D-glucose coupled Na flux, which was 0.53 +/- 0.09(8) mueq cm-2hr-1, and Isc, 0.50 +/- 0.50(8) mueq cm-2hr-1. A linear plot of concurrent net Na vs. net D-glucose apical-to-basolateral fluxes a gave a regression coefficient of 2.08. As support for a 2:1 transepithelial stoichiometry, sodium was added in the presence of D-glucose and the response of Isc analyzed by a Hill plot. A slope of 2.08 +/- 0.06(5) was obtained confirming a requirement of 2 Na for 1 D-glucose coupled transport. A Hill plot of Isc increase to added D-glucose in the presence of Na gave a slope of 1.02 +/- 0.02(5). A direct determination of the initial rates of Na and D-glucose translocation across the apical membrane using phlorizin, a nontransported glycoside competitive inhibitor to identify the specific coupled uptake, gave a stoichiometry of 2.2. A coupling ratio of 2 for Na, D-glucose uptake, doubles the potential energy available for Na-gradient coupled D-glucose transport. In contrast to coupled uptake, the stoichiometry for Na-dependent-phlorizin binding was 1.1 +/- 0.1(8) from Hill plot analyses of Na-dependent-phlorizin binding as a function of [Na].(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1982

Transepithelial transport by pulmonary alveolar type II cells in primary culture.

Fluid and electrolyte transport by epithelial cells in vitro can be recognized by the ability of cultured cells to form domes and by the electrical properties of monolayer cultures. Pulmonary alveolar epithelial cells are thought to be partially responsible for fluid movement in the fetal lung, but their role in electrolyte transport in the adult lung is not known. We isolated alveolar type II cells from adult rat lung and maintained them on plastic culture dishes alone, on plastic culture dishes coated with an extracellular matrix, and on collagen-coated Millipore filters. Numerous large domes were formed on culture dishes coated with the extracellular matrix; smaller domes were formed on uncoated plastic culture dishes. Sodium butyrate (3 mM) stimulated dome formation. Transmission electron microscopy showed that the epithelial cells had flattened but still retained lamellar inclusions and that the cells were polarized with microvilli on the apical surface facing the culture medium. The electrical properties of the monolayers maintained on collagen-coated Millipore filters were tested in two laboratories. The transepithelial potential differences were 0.7 +/- 0.1 mV (24 filters, seven experiments) and 1.3 +/- 0.1 mV (13 filters, two experiments) apical side negative, and the corresponding resistances were 217 +/- 11 ohm X cm2 and 233 +/- 12 ohm X cm2. Terbutaline (10 microM) produced a biphasic response with a transient decrease and then a sustained increase in potential difference. Amiloride (0.1 mM) completely abolished the potential difference when it was added to the apical side but not when it was added to the basal side, whereas 1 mM ouabain inhibited the potential difference more effectively from the basal side. Thus, type II cells form a polarized epithelium in culture, and these cells actively transport electrolytes in vitro.

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PubMed1982

Electrical effects of histamine on monolayers formed in culture from enriched canine gastric chief cells.

To develop techniques for studying transport properties and secretory function of selected cell types in the gastric mucosa, separated fractions of dispersed canine fundic mucosal cells were placed in short-term culture to form epithelial monolayers. Cell fractions enriched in either chief, parietal, or mucous cells were prepared by using counterflow centrifugation and were plated on type I collagen. An epithelial monolayer formed by approximately equal to 36 hr. Immunofluorescence with an antipepsinogen I antibody revealed pepsinogen-containing granules in greater than 95% of the cells, regardless of whether the monolayers were formed from the mucous, chief, or parietal cell-enriched fractions. Upon achieving confluency, chief cell monolayers were mounted in Ussing chambers to study their electrical properties. Under basal conditions, monolayers (n = 6) had a spontaneous potential difference (PD) (+/- SEM) of 26 +/- 4 mV (apical surface negative), a short-circuit current (Isc) (+/- SEM) of 16 +/- 2 microA/cm2, and a transepithelial resistance (R) (+/- SEM) of 1,480 +/- 210 omega X cm2. Histamine increased the short-circuit current, an effect blocked by an H2-receptor antagonist. Seventy percent of the spontaneous PD was amiloride sensitive, suggesting sodium absorption accounted for a major component of the PD. These preparative techniques yield highly enriched chief cell monolayers, which maintain morphological and functional cellular differentiation for greater than 48 hr in culture, thus allowing study of oriented functions of a selected cell type. The present studies indicate that an H2 receptor enhances electrogenic ion transport in chief cell monolayers, indicating that histamine can act on fundic mucosal cells other than just parietal cells.

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PubMed1981

Transepithelial transport in cell culture: D-glucose transport by a pig kidney cell line (LLC-PK1).

The pig kidney cell line LLC-PK1 cultured on a collagen coated membrane filter formed a continuous sheet of oriented asymmetrical epithelial cells joined by occluding junctions. A transepithelial electrical potential (PD) and short-circuit current (SCC) were dependent on the presence of Na and sugar in the apical bathing solution. In the presence of 5.5 mM D-glucose, a PD of 2.8 mV. apical surface negative a SCC of 13 microA cm-2 and transepithelial resistance of 211 ohm.cm2 were recorded. The SCC was promptly reduced by the addition of phlorizin to the apical bath but unaffected when placed in the basolateral bath. The effect on SCC of various sugars was compared by the concentrations required for half-maximal SCC: 0.13 mM beta-methyl-D-glucoside, 0.28 mM D-glucose, 0.65 mM alpha-methyl-D-glucoside, 0.77 mM 6-deoxy-D-glucose, 4.8 mM D-galactose, and 29 mM 3-O-methyl-glucose. When [Na] was reduced, the concentration of D-glucose required for half-maximal SCC increase. Isotopically labeled 3H and 14C D-glucose were used to simultaneously determine bidirectional fluxes; a resultant net apical-to-basolateral transport was present and abolished by phlorizin. The transported isotope cochromatographed with labeled D-glucose, indicating negligible metabolism of transported glucose. The pig kidney cell line, LLC-PK1, provides a cell culture model for the investigation of mechanisms of transepithelial glucose transport.

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PubMed1981

Transepithelial glucose transport in cell culture.

Pig kidney cell line LLC-PK1 cultured on a collagen-coated membrane filter formed a continuous sheet of oriented asymmetrical epithelial cells joined by circumferential occluding junctions. In the presence of 5.5 mM D-glucose, a potential difference (PD) of 2.8 mV, apical bath negative, short-circuit current Isc of 13.2 microA . cm-2, and transepithelial resistance of 211 omega . cm2 were recorded. Isc and PD were reduced by phlorizin added to the apical bath but were unaffected when phlorizin was placed in the basolateral bath. Ouabain or the replacement of Na by tris-(hydroxymethyl)aminomethane or choline abolished the Isc. The sugar concentrations required to produce the half-maximal Isc were 0.13 mM beta-methyl-D-glucoside, 0.28 mM D-glucose, 0.65 mM alpha-methyl-D-glucoside, 0.77 mM 6-deoxy-D-glucose, 4.8 mM D-galactose, and 29 mM 3-O-methylglucose. When [Na] was reduced, the D-glucose required for half-maximal SCC increased. Isotopically 3H- and 14C-labeled D-glucose were used to determine simultaneous bidirectional fluxes; a resultant net apical-to-basolateral flux was present and could be abolished by phlorizin. The transported isotope cochromatographed with labeled D-glucose, indicating negligible metabolism. The cell culture model provides advantages for investigation of mechanisms of transepithelial glucose transport.

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PubMedDate not supplied

The apical surface of canine chief cell monolayers resists H+ back-diffusion.

The resistance of the gastric mucosa to acid and peptic injury is reflected by a resistance to the back-diffusion of H+ from gastric lumen to blood. The nature of this 'barrier', however, remains undefined. Using Ussing chambers, we have now studied the acid-barrier function of monolayers prepared from dispersed canine fundic chief cells. These monolayers secrete pepsinogen in response to stimulation. We found that, on acidification of the apical solution to pH 2, transepithelial resistance (R) increased 2.6-fold and the monolayers maintained this 1:100,000 H+ concentration gradient for more than 4 h. The addition of aspirin to the acidified apical solution caused a rapid decay in R, as did acidification of the basolateral solution to a pH less than 5.5. Ouabain-treated monolayers displayed the rise in R expected with apical acidification, while potential difference (V) and short-circuit current (Isc) decreased essentially to zero, indicating impermeability to H+. However, if the integrity of the ouabain-treated monolayers was disrupted by low apical pH, H+ permeation occurred, reflected by an Isc that was dependent on the H+ gradient across monolayers. These data indicate that the apical surface of chief cells is a very tight barrier to H+ diffusion and may be an important element resisting acid-peptic injury.

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