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M Taub

Publications and source records attributed to M Taub.

At least 37 records · Page 2Linked to original sources
PubMed1990

Altered basement membrane protein biosynthesis by primary cultures of cpk/cpk mouse kidney.

Previously, kidneys from three-week-old cpk/cpk C57/B16 mice were found to contain elevated mRNA levels for the basement membrane components collagen IV and laminin [1]. Here primary cultures of kidney epithelial cells derived from cpk/cpk C57/B16 mice were established and the production of these proteins in culture was studied. Primary cultures of cpk/cpk mouse kidney epithelial cells were observed to have a more polygonal, flattened morphology than cells from unaffected littermate kidneys. The rate of collagen IV and laminin biosynthesis was determined by means of [35S] labelling studies followed by immunoprecipitation. Collagen IV and laminin biosynthesis are elevated by approximately twofold or more in primary cultures derived from 20-day-old cpk/cpk mice, as compared with parallel primary cultures derived from their unaffected littermates. Similarly, laminin B1 chain mRNA is elevated in primary cultures derived from 20-day-old cpk/cpk mice. In primary cultures derived from younger (day 11) mice, similar differences in the rates of both collagen and laminin biosynthesis were not observed between the two culture types. These observations are consistent with the previously reported age-dependent differences observed in laminin and in collagen IV gene expression in both cpk/cpk and wild-type mouse kidneys, and suggest that the regulation of overproduction of these proteins is due to an alteration in the kidney cells and not due to systemic factors.

Animals↗
PubMed1990

Epidermal growth factor or transforming growth factor alpha is required for kidney tubulogenesis in matrigel cultures in serum-free medium.

The ability of matrigel, a reconstituted basement membrane gel, to induce the differentiation of baby mouse kidney cells has been examined in a hormonally defined serum-free medium. Primary cultures of baby mouse kidney cells were observed to form tubules over a time interval of 1-2 weeks in matrigel. Electron microscopic studies showed that tubules with lumens were present, and the tubule morphology was similar to that of the collecting duct. When using matrigel from which the growth factors had been removed, tubule formation no longer occurred, unless the medium was further supplemented with epidermal growth factor (10 ng/ml). Transforming growth factor alpha stimulated tubule formation as effectively as epidermal growth factor, whereas transforming growth factor beta had an inhibitory effect on tubule formation. These data suggest that both an extracellular matrix and specific growth factors may regulate kidney differentiation during development.

Animals↗
PubMed1989

Retinoic acid modulates dome formation by MDCK cells in defined medium.

Retinoic acid dramatically increases the size of domes in confluent MDCK monolayers in a hormonally defined medium (medium K-1). After 4-5 days of retinoic acid treatment, enlarged domes began to appear in confluent MDCK monolayers. After 7 days with 3 x 10(-7) M retinoic acid, the majority of the domes in the monolayers were between 27 and 80 x 10(-3) microns 2 in area, whereas in control medium the majority of the domes were between 0 and 9 x 10(-3) microns 2 in area. The dependence of the retinoic acid effect on prostaglandin E1 (PGE1) was examined. In normal MDCK cells, the effects of retinoic acid on dome size were observed only in medium K-1 supplemented with PGE1. This observation indicated that retinoic acid did not elicit its effects simply by stimulating PGE production. In contrast, in monolayers of PGE1-independent MDCK cells, retinoic acid treatment resulted in an increase in dome frequency even in medium K-1 lacking PGE1. This observation can be explained by the elevated cyclic adenosine monophosphate (cAMP) levels in these PGE1-independent MDCK cells. Dibutyryl cAMP-resistant MDCK cells, which normally do not form domes in medium K-1, were also studied. Remarkably, the dibutyryl cAMP-resistant MDCK cells were observed to form domes at a significant frequency when medium K-1 was supplemented with retinoic acid. However in medium K-1 lacking PGE1, an effect of retinoic acid on dome formation by dibutyryl cAMP-resistant MDCK monolayers was not observed. The inability of dibutyryl cAMP-resistant MDCK cells to form domes in medium K-1 has previously been attributed to their decreased cAMP-dependent protein kinase activity. The stimulatory effects of retinoic acid on dome formation may possibly be due to an increase in the activity of a particular cAMP-dependent protein kinase or activation of a separate pathway.

Alprostadil↗
PubMed1988

Preparation of basolateral membranes that transport p-aminohippurate from primary cultures of rabbit kidney proximal tubule cells.

The organic anion p-aminohippurate (PAH) is specifically secreted by the renal proximal tubule. The possibility was examined that the probenecid sensitive PAH transport system (which is involved in this secretory process in renal proximal tubule cells in vivo) is retained in primary cultures of rabbit kidney proximal tubule cells. Significant 3H-PAH uptake into primary cultures of proximal tubule cells was observed. After 10 min, 150 pmole PAH/mg protein had accumulated intracellularly. Given an intracellular fluid volume of 10 microliter/mg protein, the intracellular PAH concentration was estimated to be 15 microM. The initial rate of PAH uptake (when 50 microM PAH was in the uptake buffer) was inhibited 50% by 2 mM probenecid. Intact monolayers also exhibited Na+-dependent alpha methyl-D-glucoside uptake (an apical marker). Basolateral membranes were purified from primary rabbit kidney proximal tubule cell cultures. Probenecid sensitive PAH uptake into the membrane vesicles derived from the primary cultures was observed. The rate of PAH uptake was equivalent to that obtained with vesicles obtained from the rabbit renal cortex. No significant Na+-dependent D-glucose uptake into the vesicles was observed, indicating that primarily basolateral membrane vesicles had indeed been obtained.

Aminohippuric Acids↗
PubMed1986

Sodium entry pathways in renal epithelial cell lines.

Na+ entry into kidney epithelial cells occurs by a multiplicity of pathways. Established cell lines such as the A6 cells, derived from the collecting duct of the kidney of Xenopus laevis, MDCK cells, from the distal tubule of a dog kidney, and the LLC-PK1 cells, originating from the proximal tubule of a pig kidney, provide excellent model cell systems for the detailed characterization and isolation of the proteins which comprise these entry pathways. Major pathways of Na+ entry include the amiloride-sensitive Na+ channel, the amiloride-sensitive Na+/H+ antiporter, and the loop diuretic-sensitive NaCl/KCl symporter. While the former two systems have been shown to exhibit an apical location in epithelial cells so far examined, the last system may be localized to either the basolateral or apical surface, depending on the transport function of the cell. Nutrient/Na+ symporters such as the glucose, phosphate, and p-aminohippurate symporters may all be localized to the apical surfaces of proximal tubular cells, but other systems, including those specific for neutral amino acids, may predominate in the basolateral surface or be distributed between the two membranes. Studies concerned with the catalytic, structural, and regulatory properties of these transport systems serve not only to characterize the individual translocators in established cell lines, but also to suggest their physiological functions in intact kidney tissues.

Aldosterone↗
PubMed1985

Phosphate uptake by primary renal proximal tubule cell cultures grown in hormonally defined medium.

The uptake of labeled inorganic phosphate into primary rabbit kidney proximal tubule cells has been examined. Phosphate was accumulated into the primary proximal tubule cells against a concentration gradient. This accumulation was sensitive to inhibition by metabolic inhibitors. The dependence of phosphate uptake on the extracellular phosphate concentration was examined. Similarities were observed between primary proximal tubule cells and the LLC-PK1 cell line in these regards. These phosphate uptake data were then plotted on a Lineweaver-Burke plot. A nonlinear plot was obtained, which suggested that phosphate uptake occurs by means of a Na+ dependent, carrier mediated process, as well as by another Na+ independent mechanism. The pH dependence of phosphate uptake was also examined. Unlike previous observations with LLC-PK1 cells, optimal phosphate uptake occurred at pH 6.5. However, this difference between the two cell culture systems may possibly be explained by differences in uptake conditions. The dependence of phosphate uptake on the extracellular NaCl concentration was examined at three different pH values. The rate of phosphate uptake at pH 7.0 was observed to saturate at a lower NaCl concentration than at either pH 6.0 or pH 6.5. Furthermore, the optimal rate of phosphate uptake at pH 7.0 was observed to be higher than at the other two pH values studied when the NaCl concentration was below 120 mM. However, when the NaCl concentration was raised to 150 mM, optimal phosphate was observed to occur at pH 6.5 rather than at pH 7.0. These observations may be explained if the pH affects not only the rate of phosphate uptake but also the affinity of the phosphate uptake system for sodium. Phosphate uptake was also observed to be sensitive to several agents, Na2 X SO4 and NaSCN, which affect the membrane potential. As observed with phosphate uptake by LLC-PK1 (and renal brush border membrane vesicles), phosphate uptake was highly sensitive to inhibition by the phosphate analogue arsenate. Novel observations were that the phosphate analogue vanadate and its cellular metabolite vanadyl stimulated the initial rate of phosphate uptake.

2,4-Dinitrophenol↗
PubMed1985

Dibutyryl cyclic AMP resistant MDCK cells in serum free medium have reduced cyclic AMP dependent protein kinase activity and a diminished effect of PGE1 on differentiated function.

Prostaglandin E1 (PGE1) has a stimulatory effect both on the growth and the expression of differentiated function of Madin Darby Canine Kidney (MDCK) cells in a hormonally defined medium (Medium K-1). While the stimulatory effect of PGE1 on MDCK cell growth is observed in subconfluent cultures, the effect of PGE1 on differentiated function (i.e., dome formation) is observed at confluency. PGE1 may possibly affect growth and such differentiated functions by separate mechanisms. In order to examine this possibility, dibutyryl cyclic AMP resistant variants of MDCK were selected. All of the variants were partially resistant to the growth inhibitory effects of dibutyryl cyclic AMP and theophylline. The cyclic AMP dependent protein kinase activity of four of the five variant clones studied was significantly reduced as compared with normal MDCK cells. The dependence of the kinase activity of several of the dibutyryl cyclic AMP resistant variants (DBr2 and DBr3) on the cyclic AMP concentration in the reaction mixture was compared with that of normal MDCK cells. At all of the cyclic AMP concentrations tested DBr2 and DBr3 cells had reduced protein kinase activity as compared with normal MDCK cells. This reduced activity could be attributed to a decrease in the Vmax for kinase in the two variants, rather than to a change in the Km of kinase for cyclic AMP. The cyclic AMP phosphodiesterase activity of dibutyryl cyclic AMP resistant variants was also studied. Unlike PGE1 independent clone 1, DBr2 and DBr3 cells did not differ significantly from normal MDCK cells with regard to their ability to degrade cyclic AMP. The growth and functional responsiveness of DBr2 and DBr3 cells to PGE1 was also examined. DBr2 and DBr3 cells were shown to retain a normal growth response to PGE1. However the capacity of DBr2 and DBr3 cells to form domes in response to PGE1 was dramatically reduced as compared with normal MDCK cells. Nevertheless DBr3 cells were shown to still retain the capacity to form domes in response to other inducers. The effect of PGE1 on one of the functional parameters involved in dome formation (the activity of the Na+/K+ATPase) was examined. The rate of ouabain-sensitive Rb+ uptake was observed to be elevated in confluent monolayers of normal MDCK cells maintained in Medium K-1, as compared with monolayers maintained in Medium K-1 minus PGE1.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

PGE1-independent MDCK cells have elevated intracellular cyclic AMP but retain the growth stimulatory effects of glucagon and epidermal growth factor in serum-free medium.

Prostaglandin E1 (PGE1), a component in the hormone-supplemented, serum-free medium for the Madin Darby canine kidney (MDCK) cell line, has been proposed to increase MDCK cell growth by increasing intracellular cyclic AMP levels. The association between increased intracellular cyclic AMP and the growth stimulatory effect of PGE1 has been examined in normal MDCK cells and in PGE1-independent variants of MDCK. These variant cells have lost the PGE1 requirement for long term growth in defined medium. Normal MDCK cells had almost twofold higher intracellular cyclic AMP levels during growth in Medium K-1 (9.0 pmol/mg protein) than in Medium K-1 minus PGE1. Furthermore, PGE1-independent clone 1 had higher intracellular cyclic AMP levels in Medium K-1 minus PGE1 than normal MDCK cells in Medium K-1. This latter observation suggests that the PGE1 requirement for MDCK cell growth is associated with the low intracellular cyclic AMP levels of this cell line. An involvement of cyclic AMP in the growth response to PGE1 is supported by these observations, as well as by the growth stimulatory effects of other agents that affect cyclic AMP metabolism in MDCK cells. These agents include glucagon, isobutyl methylxanthine (IBMX), and dibutyryl cyclic AMP. The growth of PGE1-independent clone 1 was inhibited rather than stimulated by PGE1. Similarly, PGE1-independent cell growth was inhibited by IBMX and dibutyryl cyclic AMP. However, the growth response to one agent which increases cyclic AMP (glucagon) was retained in PGE1-independent clone 1. This result suggests that the effect of glucagon is not associated with increases in intracellular cyclic AMP. The growth stimulatory effect of epidermal growth factor (EGF) on normal MDCK cells was also studied. Although EGF does not act via a cyclic AMP-mediated mechanism, EGF increased normal MDCK cell growth and substituted for PGE1 in Medium K-1. Thus, EGF and PGE1 could possibly affect similar growth-related functions in MDCK cells, although by different pathways. This possibility was examined further, using PGE1-independent clone 1. EGF, like glucagon, was still growth stimulatory to the PGE1-independent cells. Consequently, the biochemical pathways by which EGF and PGE1 increase MDCK cell growth probably do not converge.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Transport and metabolism of glucose by renal proximal tubular cells in primary culture.

A highly purified suspension of rabbit proximal tubules was cultured in a hormone-supplemented serum-free medium. This primary culture yielded a homogeneous population of cells that demonstrated functional and morphological polarity in mono-layers. The characteristics of the Na-dependent glucose transporter in the luminal membrane were studied by measuring the uptake of alpha-methylglucoside (AMG). The kinetics of Na-dependent AMG uptake were consistent with a single saturable system with an apparent Km of 0.8 mM and Jmax of 0.14 nmol X mg-1 X min-1. AMG permeability was 0.10 microliter X mg-1 X min-1. Uptake was inhibited 95% by 0.1 mM phlorizin and by removal of sodium. The stoichiometry of Na/glucose interaction with the carrier was 2:1. These characteristics are typical of the characteristics described for the late proximal tubule. To examine whether the glucose that enters the cell across the luminal membrane is incorporated into the metabolic pool of the cell, we studied the oxidation of [14C]glucose to 14CO2 in the absence and presence of phlorizin. Significant decarboxylation of [1-14C]glucose and [6-14C]glucose was observed, consistent with the existence of aerobic metabolism and a hexose monophosphate shunt. In the presence of 0.1 mM phlorizin, uptake and oxidation of D-glucose were inhibited to an identical degree, suggesting that luminal uptake is a rate-limiting step in the oxidation of glucose by these proximal tubular cells. These studies indicate that proximal tubular cells in primary culture utilize glucose as an energy source and that the glucose derived from transport across the luminal membrane is incorporated into the metabolic pool of the cell.

Animals↗
PubMed1982

Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium.

Primary cultures of rabbit-kidney epithelial cells derived from purified proximal tubules were maintained without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium RK-1). A hormone-deletion study indicated that the primary cultures derived from purified rabbit proximal tubules required all of the three supplements in Medium RK-1 (insulin, transferrin, and hydrocortisone) for optimal growth but did not grow in response to EGF and T3. In contrast, the epithelial cells in primary cultures derived from an unpurified preparation of rabbit kidney tubules and glomeruli grew in response to EGF and T3, as well as insulin, transferrin, and hydrocortisone. These observations suggest that kidney epithelial cells derived from different segments of the nephron grow differently in response to hormones and growth factors. Differentiated functions of the primary cultures derived from proximal tubules were examined. Multicellular domes were observed, indicative of transepithelial solute transport by the monolayers. The proximal tubule cultures also accumulated alpha-methylglucoside (alpha-MG) against a concentration gradient. However, little or no alpha-MG accumulation was observed in the absence of Na+. Metabolic inhibitor studies also indicated that alpha-MG uptake by the primaries is an energy-dependent process, and depends upon the activity of the Na+/K+ ATPase. Phlorizin at 0.1 mM significantly inhibited 1 mM alpha-MG uptake whereas 0.1 mM phloretin did not have a significant inhibitory effect. Similar observations have been made concerning the Na+-dependent sugar-transport system located on the lumenal side of the proximal tubule, whereas the Na+-independent sugar transporter on the peritubular side is more sensitive to inhibition by phloretin than phlorizin. The cultures also exhibited PTH-sensitive cyclic AMP synthesis and brush-border enzymes typical of proximal cells. However, the activities of the enzymes leucine aminopeptidase, alkaline phosphatase, and gamma-glutamyl-transpeptidase were lower in the cultures than in purified proximal-tubule preparations from which they are derived.

Animals↗
PubMed1982

The solitary thyroid nodule. Selection of patients for surgery.

Management of the solitary thyroid nodule, especially the nodule which is hypofunctional on radionuclide scan, is still controversial. The major controversy centers around the selection of patients for surgery. A review of 50 patients who were recently operated on for suspected thyroid cancer at the Naval Regional Medical Center, Portsmouth, Virginia, was undertaken by us in an effort to determine which factors suggest the presence of malignancy preoperatively. Not surprisingly, carcinoma was most frequently found in the solitary, hypofunctional, solid thyroid nodule, although there were exceptions to each of these clinical criteria.

Adenocarcinoma↗
PubMed1981

Amiloride-resistant Madin-Darby canine kidney (MDCK) cells exhibit decreased cation transport.

Cation transport systems were investigated in mutant Madin-Darby Canine Kidney (MDCK) cells resistant to the diuretic drug amiloride. The mutants were isolated previously as clones resistant to the cytotoxic effects of 3 X 10(-4) M amiloride. Decreased amiloride transport by the Na+ channel was implicated as the basis of the resistance (Taub, '78). Consistent with this hypothesis, Na+ accumulation was lower in amiloride-resistant cells than in normal sensitive MDCK cells. Kinetic studies indicated that Na+ uptake in MDCK cells occurs by a single ATP independent transport system--the Na+ channel. In several amiloride-resistant clones, including clone Amr2, the decreased Na+ uptake was associated with a decrease in both the Km and Vmax for Na+ uptake by the Na+ channel. In Amr2 cells no significant alteration in the inhibitory effect of amiloride on Na+ uptake was observed. As the Na+ channel may actually be a general uptake system for monovalent cations (a number of cations inhibit Na+ uptake), the uptake of these inhibitory cations was examined in Amr2 cells. Both Ca++ and ouabain-insensitive Rb+ uptake occurred at decreased rates in Amr2 cells as compared with normal MDCK cells. However, further uptake studies suggested that Na+, Ca++ and ouabain-insensitive Rb+ uptake all occur by different systems. Thus several transport systems may be defective in Amr2 cells. Amr2 cells were also resistant to the inhibitory effects of amiloride on CO2 evolution from pyruvate. These observations indicate that alterations at a number of molecular sites may be associated with defective Na+ transport via the Na+ channel in amiloride-resistant cells. Thus the amiloride-resistant cells are potentially valuable in examining the interrelationships between Na+ transport and other cellular functions.

Amiloride↗
PubMed1981

Alterations in growth requirements of kidney epithelial cells in defined medium associated with malignant transformation.

The possibility has been investigated that 1) the supplements required for the growth of the Madin Darby Canine Kidney (MDCK) cell line in serum-free Medium K-1 are indeed requirements for the growth of normal kidney cells in vitro, and 2) that alterations in these growth requirements are associated with malignant transformation. Consistent with the hypothesis that MDCK cells resemble normal kidney cells in culture, primary cultures of baby mouse kidney epithelial cells grow in Medium K-1 and respond to the 5 components in the medium. The growth properties of Moloney sarcoma virus (MSV)-transformed MDCK cells in defined media have been examined. Unlike MDCK cells, MSV-transformed MDCK cells form tumors in adult nude mice. Although they still respond to the 5 factors in Medium K1, the optimal dosage for insulin is lower for the MSV transformants than for MDCK cells. The MSV transformants also have an additional requirement for growth in Medium K-1-fibronectin. Variants of MDCK cells have been isolated that have lost the PGE1 requirement for growth in defined medium. These variant cells have acquired 1) the ability to form tumors in adult nude mice and 2) an alteration affecting cAMP metabolism, in addition to PGE1 independence.

Animals↗
PubMed1981

Treatment of choledocholithiasis with oral chenodeoxycholic acid.

The aim of this study was to conduct a controlled trial of oral chenodeoxycholic acid in the management of radiolucent choledocholithiasis. Thirteen patients were randomized in double-blind fashion to receive either 750 mg/day of chenodeoxycholic acid (CDCA) or a placebo. After 4 months, those who had the placebo were administered CDCA; those who had received CDCA and showed a 25% or more decrease in the size of stones (evaluated blindly) received CDCA for an additional 4 months. Five of the 13 patients did not complete the study; four (one initially placebo and three CDCA) because acute biliary symptoms mandated operative intervention and one (initially placebo, then CDCA) because of asymptomatic elevations of the serum transaminase levels. Patients who were withdrawn from the study had significantly larger stones (P less than 0.02) (mean largest diameter, 11.4 mm +/- 1.6 SEM) than those who completed the study (6.5 +/- 0.5). Of the eight patients who completed the study, two of the three who received CDCA initially for 6 to 8 months experienced complete disappearance of stones; all five patients who took the placebo failed to show dissolution, and one of these subsequently had dissolution of stones after 8 months of CDCA. Biliary lipid analyses during treatment showed bile unsaturated with respect to cholesterol in the three patients whose stones dissolved with CDCA therapy. In conclusion, a patient with partial dissolution of stones and unsaturated bile after 4 months of CDCA probably will have complete dissolution of stones after 6 to 8 months of CDCA.

Administration, Oral↗
PubMed1980

Growth of functional primary cultures of kidney epithelial cells in defined medium.

Primary cultures of baby mouse kidney epithelial cells can grow without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium K-1) designed for an established kidney epithelial cell line, MDCK. The five supplements in Medium K-1 are insulin, transferrin, PGE1, T3, and hydrocortisone. Medium K-1 also supports the growth of kidney epithelial cell cultures from a number of animals, including man, without fibroblast overgrowth. Outgrowth of kidney epithelial cells from kidney explants was also observed with Medium K-1. Thus, the medium appears to be selective for epithelial cell growth. The physiological properties of primary cultures of baby mouse kidney epithelial cells were studied in detail. Baby mouse kidney epithelial cells grew at equal rates (0.5 doublings/day) in Medium K-1 and serum-supplemented medium. Medium K-1 also supported the formation of baby mouse kidney epithelial colonies at low cell densities. The dependence of baby mouse kidney epithelial colony formation upon the five factors in Medium K-1 was examined. These studies indicated that the formation of baby mouse kidney epithelial colonies in defined medium depended upon all the five supplements in Medium K-1, in a manner similar, although not identical, to MDCK colonies. Primary cultures of baby mouse kidney epithelial cells grown in Medium K-1 retained kidney cell-associated properties, including the ability to form multicellular domes, a phenomenon associated with transepithelial salt transport. Amiloride-sensitive Na+ uptake and the mucosal surface enzyme leucine aminopeptidase were also observed in baby mouse kidney cultures. Similar functions were observed in MDCK monolayers.

Animals↗
PubMed1979

Regulation of 22Na+ transport by calcium in an established kidney epithelial cell line.

The role of calcium in regulating the Na+ channel in an established kidney epithelial cell line has been examined. Extracellular calcium was inhibitory to Na+ uptake, and a Dixon plot of the initial Na+ uptake rate in the presence of Ca2+ was nonlinear, suggesting a mixed pattern of inhibition. Similar patterns of inhibition were also observed for other divalent cations, including Ba2+, Mg2+, and Mn2+. In contrast elevated concentrations of intracellular calcium resulted in a stimulation of Na+ entry. This intracellular effect was specific to calcium, with Mg2+ and Mn2+ appearing much less effective. Lineweaver-Burk plots of Na+ influx in calcium-loaded and unloaded cells were linear, suggesting that under both conditions a single system transported Na+. Although Na+ entry was stimulated by intracellular Ca2+, the cells did not exhibit other counter transport phenomena reported with cell types in which a Na+/Ca2+ exchange system is operative. Thus, the results indicate that calcium acts as an allosteric regulator of Na+ transport by the Na+ channel.

Animals↗
PubMed1979

Uptake of 22Na+ by cultured dog kidney cells (MDCK).

22Na+ uptake measurements were conducted on the dog kidney cell line, MDCK, to determine the mechanism of ouabain-insensitive sodium transport. The radioisotope was found to be taken up into monolayer cultures via an ATP-independent, saturable process (Km = 40 mM). The presence of sodium on the opposite side of the membrane gave rise to a transstimulation of the 22Na+ flux. Studies utilizing potassium and valinomycin suggested that the transport system was insensitive to changes in the membrane potential. Replacement of chloride in the assay buffer with other anions did not decrease the rate of 22Na+ uptake at 14 mMNa+, but bicarbonate and acetate were stimulatory. Potassium and rubidium increased the rate of 22Na+ influx (Ka = 13mM with 14 mM NaCL in the medium). Lithium (Ki = 7.5mM) and amiloride (Ki = 1.7 x 10(-5) M) were competitive and partially (or mixed) competitive inhibitors, respectively. The data are consistent with a mechanism of sodium uptake that includes a carrier(s) capable of catalyzing net sodium uptake and sodium-sodium exchange.

Amiloride↗