Autoradiographic localisation of [3H]ouabain bound to cultured epithelial cell monolayers of MDCK cells

Lamb, J. F.; Ogden, P.; Simmons, Nicholas L.

doi:10.1016/0005-2736(81)90391-6

Cited by 46 publications

(25 citation statements)

References 20 publications

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“…By analogy, transepithelial K § secretion would occur providing that the electrochemical gradient across the apical membrane was directed towards the apical solution. For MDCK epithelium the absence of net K § secretion, despite the existence of a functional (Na + -K +)-ATPase located along the lateral interspace membranes (Simmons, 1981a;Lamb et al, 1981) implies a functional impermeability to K § for the apical cell membranes. Experimental data consistent with this are (i) the isotope ratio of 86Rb: 42K within the tissue after a 2-hr incubation and (ii) the measured K + influx and efflux across the apical cell membranes.…”

Section: Discussionmentioning

confidence: 99%

K+ transport in ‘Tight’ epithelial monolayers of MDCK cells

et al. 1982

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Bidirectional transepithelial K+ flux measurements across 'high-resistance' epithelial monolayers of MDCK cells grown upon millipore filters show no significant net K+ flux. Measurements of influx and efflux across the basal-lateral and apical cell membranes demonstrate that the apical membranes are effectively impermeable to K+. K+ influx across the basal-lateral cell membranes consists of an ouabain-sensitive component, an ouabain-insensitive component, an ouabain-insensitive but furosemide-sensitive component, and an ouabain- and furosemide-insensitive component. The action of furosemide upon K+ influx is independent of (Na+ - K+)-pump inhibition. The furosemide-sensitive component is markedly dependent upon the medium K+, Na+ and Cl- content. Acetate and nitrate are ineffective substitutes for Cl-, whereas Br- is partially effective. Partial Cl- replacement by NO3 gives a roughly linear increase in the furosemide-sensitive component. Na+ replacement by choline abolishes the furosemide-sensitive component, whereas Li+ is a partially effective replacement. Partial Na+ replacement by choline abolishes the furosemide-sensitive component, whereas Li+ is a partially effective replacement. Partial Na+ replacement with choline gives an apparent affinity of approximately 7 mM Na, whereas variation of the external K+ content gives an affinity of the furosemide-sensitive component of 1.0 mM. Furosemide inhibition is of high affinity (K1/2 = 3 micrometer). Piretanide, ethacrynic acid, and phloretin inhibit the same component of passive K+ influx as furosemide; amiloride, 4,-aminopyridine, and 2,4,6-triaminopyrimidine partially so. SITS was ineffective. Externally applied furosemide and Cl- replacement by NO3- inhibit K+ efflux across the basal-lateral membranes indicating that the furosemide-sensitive component consists primarily of K:K exchange.

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Section: Discussionmentioning

confidence: 99%

K+ transport in ‘Tight’ epithelial monolayers of MDCK cells

et al. 1982

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“…Also membrane amplification (via the presence of a brushborder or extensive interdigitations of the basolateral surface) can significantly alter the effective surface areas for drug diffusion into the cytosol. In the extensively studied MDCK dog kidney cell-line, careful morphometric data show that the basolateral surface area exceeds that of the apical membrane by 7-10-fold (von Bonsdorff et al, 1985;Lamb et al, 1981). For epithelial colonic adenocarcinoma cells grown upon plastic the basolateral surface will be inaccessible from the apical medium due to tight-junction formation.…”

Section: IXmentioning

confidence: 99%

Epithelial secretion of vinblastine by human intestinal adenocarcinoma cell (HCT-8 and T84) layers expressing P-glycoprotein

Hunter

Hirst²,

Simmons³

1991

Br J Cancer

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Summary P-glycoprotein expression was demonstrated in two human intestinal adenocarcinoma cell-lines ileocaecal and T84, colonic) by immunoprecipitation of a 170-180 kDa protein with monoclonal antibody JSB-1. Both HCT-8 and T84 formed functional epithelial cell layers of high transepithelial electrical resistance (>700 Q cm2) when grown on permeable matrices. These epithelial layers demonstrated vectorial secretion (net vinblastine fluxes in the basal-to-apical direction of 0.135 and 0.452 pmol h-' cm-2 in HCT-8 and T84 cell layers, respectively, from bathing solutions containing 10 nM vinblastine). These vectorial vinblastine secretions were sensitive to inhibition by verapamil. Passive transepithelial vinblastine permeation was limited by the presence of intercellular (tight) junctions, as demonstrated by the high transepithelial electrical resistance, and verapamil increased this passive vinblastine permeation concomitant with a reduction in the electrical resistance. Cellular vinblastine loading was significantly greater from the basal side, and this was also susceptible to inhibition by basal verapamil. The demonstration of vectorial transport of vinblastine in human intestinal colonic adenocarcinoma cell layers is direct evidence in favour of the hypothesis that the function of mdrl in epithelia from the gastrointestinal tract is to promote detoxification by a process of epithelial secretion. This study also highlights that cellular vinblastine accumulation depends not only upon P-glycoprotein function, but also upon differential apparent membrane permeabilities and the presence of intercellular (tight) junctions that may restrict drug permeation and cellular accumulation to apical or basal membrane domains.

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“…The total magnitude ofuptake is compatible with measurements made on cells grown upon plastic Petri dishes (Rugg, Simmons & Tivey, 1986 Four separate membrane transport processes are thought to function in transepithelial Clsecretion; a Na+-K+-ATPase sensitive to cardiac glycoside inhibition, a Na+-K+-Clcotransport sensitive to loop diuretic inhibition, a Ca2+-activated K+ conductance (all present at the basal-lateral cell surfaces) and an apical Cl-conductance activated by intracellular cyclic AMP and inhibited by 9-anthracene carboxylic acid (Shorofsky et al 1982;Petersen & Maruyama, 1984;Welsh, 1984). For MDCK epithelial cells evidence for the existence of all four of these separate membrane transport processes has been provided (Lamb, Ogden & Simmons, 1981 ;Aiton et al 1982;Kolb, Brown & Murer, 1985). Evidence for the participation of a cotransport process in intact epithelium in Cl-secretion is given by the sidedness and potency of loop-diuretic inhibition of the adrenaline-stimulated short-circuit current.…”

Section: Transepithelial [3h]bumetanide Fluxes and Cellular Uptakementioning

confidence: 99%

“…The component of [3H]bumetanide insensitive to unlabelled loop-diuretic competition comprises < 50% of the total cellular uptake from both cell surfaces. The magnitude of this 'non-specific" component precludes sub-cellular localization as is possible for the Na+-K+-ATPase using [3H]ouabain autoradiography (Lamb et al 1981), so that the determination of the distribution of cotransport in epithelial and non-epithelial tissues using autoradiography requires ligands of higher specificity.…”

Section: Transepithelial [3h]bumetanide Fluxes and Cellular Uptakementioning

confidence: 99%

LOOP‐DIURETIC INHIBITION OF ADRENALINE‐STIMULATED Cl‐ SECRETION IN A CULTURED EPITHELIUM OF RENAL ORIGIN (MDCK)

1986

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SUMMARYLoop diuretics (furosemide, bumetanide, piretanide) inhibit the adrenaline-stimulated shortcircuit current due to transepithelial Cl-secretion in cultured renal epithelial layers (MDCK). The inhibition of Cl-secretion by loop diuretics is consistent with the presence of basal-lateral 'cotransport' since inhibition is observed only with the basal applications of loop diuretics, is of high potency (half-maximal bumetanide inhibition being observed at 0 8 /LM, bumetanide being more potent than furosemide) and is without effect upon the adrenaline-stimulated increase in tissue conductance. Loop diuretics are also shown to inhibit a component of K+ efflux across the basal-lateral surfaces. Cellular uptake of [3H]bumetanide across both apical and basal surfaces of intact epithelial layers was measured in order to localize the cotransport system. A component of cellular [3H]bumetanide uptake sensitive to competition by 0 1 mM unlabelled loop diuretic is only observed from the basal-lateral cell surfaces. There is no evidence for transepithelial bumetanide secretion as is seen in renal cortex.

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Autoradiographic localisation of [3H]ouabain bound to cultured epithelial cell monolayers of MDCK cells

Cited by 46 publications

References 20 publications

K+ transport in ‘Tight’ epithelial monolayers of MDCK cells

K+ transport in ‘Tight’ epithelial monolayers of MDCK cells

Epithelial secretion of vinblastine by human intestinal adenocarcinoma cell (HCT-8 and T84) layers expressing P-glycoprotein

LOOP‐DIURETIC INHIBITION OF ADRENALINE‐STIMULATED Cl‐ SECRETION IN A CULTURED EPITHELIUM OF RENAL ORIGIN (MDCK)

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