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

[3H]Ouabain binding to intact MDCK (cultured monolayers of dog kidney) cells of 60 serial passages is dependent upon ouabain concentration, time and medium K+. By utilising high K+ incubations to estimate non-specific [3H]ouabain-binding, the concentration of ouabain giving half maximal specific binding was estimated to be 1.0 . 10(-7) M and the total maximum binding to be 2.33 . 10(5) sites/cell. Ouabain inhibition of (Na+, K+)-pump function was monitored by the cellular uptake of 86Rb over 5 min. The larger fraction of 86Rb uptake was ouabain sensitive and the ouabain concentration giving half-maximal inhibition was 2 . 10(-7) M. The cellular distribution of the (Na+ + K+)-ATPase was investigated using [3H]ouabain autoradiography of intact freeze-dried epithelial monolayers of MDCK cells grown upon millipore filter supports. Binding of [3H]ouabain is localised over the lateral cellular membranes. Autoradiographic silver grain density is close to background levels over both the apical and basal (attachment) membranes.     

Leucine transport in relation to the activities of Na+-H+ antiporter and Na+/K+ pump stimulated by serum and a tumor promoter

Fetal calf serum and 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the rate of leucine uptake by Chang liver cells in Na+-containing medium. Addition of monensin to the incubation medium also increased the leucine uptake. All these agents were capable of raising the cytoplasmic pH, which was blocked by a prior addition of amiloride or removing Na+ from assay medium, suggesting activation of Na+-H+ exchange across the cell membrane by fetal calf serum and TPA. The stimulation of leucine uptake by monensin and fetal calf serum was blocked completely or incompletely by addition of ouabain or amiloride. The basal and fetal-calf-serum- or TPA-stimulated leucine uptake was extensively depressed by the presence of an excess of 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid in the incubation medium. Based on these results it is proposed that the transport of leucine by the system L is stimulated by fetal calf serum and TPA with a high concentration of Na+ outside the cells as a result of alkalinization of the cytoplasm and coordinated activation of (Na+ + K+)-ATPase by these stimulators to maintain the transmembrane Na+ gradient and also hyperpolarize the cell membrane.     

Amiloride and amiloride analogs inhibit Na+/K+-transporting ATPase and Na+-coupled alanine transport in rat hepatocytes

Amiloride, a commonly used inhibitor of Na+-H+ exchange, has been shown to exhibit a variety of nonspecific effects. Recently, the more potent amiloride analogs, 5-(N,N-dimethyl)amiloride hydrochloride (DMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIA), have been used to control for the nonspecific effects of the parent compound. In the present study, we have explored the effects of these analogs on Na+/K+-transporting ATPase (Na+/K+-ATPase) and Na+-coupled alanine transport in primary rat hepatocyte cultures and rat liver plasma membranes, and we have compared the effects of these analogs with the effects of amiloride and ouabain. Amiloride, DMA, and EIA increased steady-state Na+ content and inhibited ouabain-sensitive 86Rb+ uptake in a reversible, concentration-dependent, ouabain-like manner, with estimated 50% inhibitory concentrations (IC50) of 3.0.10(-3) M, 5.2.10(-4) M, and 1.2.10(-4) M, respectively. Amiloride, DMA and EIA also inhibited ouabain-sensitive ATP hydrolysis in rat liver plasma membranes with similar potency (IC50 values of 2.2.10(-3) M, 2.2.10(-3) M, and 1.7.10(-4) M, respectively). In separate experiments, amiloride (5.10(-3) M), DMA (10(-3) M), and EIA (2.5.10(-4) M) decreased the uptake into hepatocytes of alanine by 20%, 61%, and 59%, respectively, and further studies with DMA (10(-3) M) demonstrated that this inhibition was largely due to a decrease in the Na+-dependent fraction of alanine uptake. These findings indicate that amiloride, DMA, and EIA inhibit hepatic Na+/K+-ATPase directly, reversibly, and with a relative rank order potency of EIA greater than DMA greater than amiloride. All three compounds also inhibit the hepatic uptake of alanine, and presumably could indirectly inhibit other Na+-coupled transport processes as well.     

Acute changes in myo-inositol uptake and 22Na+ flux in murine neuroblastoma cells (N1E-115) following insulin

myo-Inositol uptake was investigated in a murine neuroblastoma clone (N1E-115) to determine the effect of altered Na+,K+-ATPase activity. The Na+ ionophore monensin, and veratridine, an alkaloid affecting voltage-dependent Na+ entry, increased acute 22Na+ uptake and 22Na+ efflux from pre-loaded cells, concomitant with enhanced myo-inositol uptake. This effect was also seen following insulin. Insulin-stimulated myo-inositol uptake was inhibited by amiloride, ouabain and pyrithiamine. Amiloride inhibition suggests that activation of Na+/H+ exchange preceding Na+,K+-ATPase activation is involved in insulin stimulation of myo-inositol uptake. Pyrithiamine inhibition is an indication of prior activation of the Na+,K+-ATPase alpha + catalytic subunit by insulin. The results provide evidence that insulin contributes to the maintenance of Na+,K+-ATPase in neuronal tissue.     

Na+-dependent transport of alpha-aminoisobutyrate in isolated basolateral membrane vesicles from rat parotid glands

Basolateral plasma membranes were prepared from rat parotid gland after centrifugation in a self-orienting Percoll gradient. K+-dependent phosphatase [Na+ + K+)-ATPase), a marker enzyme for basolateral membranes, was enriched 10-fold from tissue homogenates. Using this preparation, the transport of alpha-aminoisobutyrate was studied. The uptake of alpha-aminoisobutyrate was Na+-dependent, osmotically sensitive, and temperature-dependent. In the presence of a Na+ gradient between the extra- and intravesicular solutions, vesicles showed an 'overshoot' accumulation of alpha-aminoisobutyrate. Sodium-dependent alpha-aminoisobutyrate uptake was saturable, exhibiting an apparent Km of 1.28 +/- 0.35 mM and Vmax of 780 +/- 170 pmol/min per mg protein. alpha-Aminoisobutyrate transport was inhibited considerably by monensin, but incubating with ouabain was without effect. These results suggest that basolateral membrane vesicles, which possess an active amino acid transport system (system A), can be prepared from the rat parotid gland.     

Regulation of Na+ transport in brown adipose tissue.

In order to test the hypothesis that Na+, K+-ATPase (Na+,K+-dependent ATPase) is involved in the noradrenaline-mediated stimulation of respiration in brown adipose tissue, the effects of noradrenaline on Na+,K+-ATPase in isolated brown-fat-cell membrane vesicles, and on 22Na+ and K+ (86Rb+) fluxes across the membranes of intact isolated cells, were measured. The ouabain-sensitive fraction of the K+-dependent ATPase activity in the isolated membrane-vesicle preparation was small and was not affected by the presence of noradrenaline in the incubation media. The uptake of 86Rb+ into intact hormone-sensitive cells was inhibited by 80% by ouabain, but it was insensitive to the presence of noradrenaline. 22Na+ uptake and efflux measured in the intact cells were 8 times more rapid than the 86Rb+ fluxes and were unaffected by ouabain. This indicated the presence of a separate, more active, transport system for Na+ than the Na+,K+-ATPase. This is likely to be a Na+/Na+ exchange activity under normal aerobic conditions. However, under anaerobic conditions, or conditions simulating anaerobiosis (2 mM-NaCN), the unidirectional uptake of Na+ increased dramatically, while efflux was unaltered.     

Apical plasma membrane vesicles formed from organ donor colon demonstrate Na+ and H+ conductances and Na+/H+ exchange

Apical plasma membrane vesicles were prepared from human organ donor colon mucosal scrapings. These vesicles were enriched 10-fold in cysteine-sensitive alkaline phosphatase activity compared to starting homogenates, and showed minimal contamination of microsomal, mitochondrial or basolateral membranes. Transport studies using [22Na] uptake into proximal colonic vesicles demonstrated Na+ and H+ conductances, Na+/H+ exchange and amiloride inhibition of Na+ uptake. The isolation of these apical vesicles will permit detailed study of human colonic transport processes.     

Na+-ATPase is a different entity from the (Na+ + K+)-ATPase in rat kidney basolateral plasma membranes

In this work, we present evidence in agreement with the hypothesis that there exist two Na+-stimulated ATPase activities in basolateral plasma membranes from rat kidney proximal tubular cells: (1) (Na+ + K+)-ATPase activity, which is inhibited by ouabain and by treating the membranes with trypsin, is insensitive to furosemide and reaches maximal activity upon treatment with SDS at an SDS/protein ratio of 1.6; (2) the Na+-ATPase activity, which is insensitive to ouabain and to trypsin treatment, is inhibited by furosemide and reaches maximal activity upon treatment with SDS at an SDS/protein ratio of 0.4.     

A bicarbonate-dependent process inhibitable by disulfonic stilbenes and a Na+/H+ exchange mediate 22Na+ uptake into cultured bovine corneal endothelium

22Na+ uptake into confluent monolayers of cultured bovine corneal endothelial cells was studied in the presence of ouabain (10(-4)M) to inhibit active sodium extrusion. In bicarbonate saline, uptake was reduced to a similar degree either by amiloride (10(-3)M) or by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) (10(-3)M). A further reduction was obtained with SITS-pretreated cells in the presence of amiloride. SITS-sensitive uptake was further characterized in saline containing both ouabain (10(-4)M) and amiloride (10(-3)M). It was absolutely dependent on bicarbonate, which could not be substituted by other plasma membrane permeable buffers (50 mM acetate or 25 mM glycodiazine). It was a saturable function of both bicarbonate and sodium concentration. Half-maximal fluxes occurred between 3 and 7 mM HCO3 (at 151 mM Na) and between 35 and 60 mM Na (at 28 mM HCO3). Uptake into sodium-depleted cells was reduced as opposed to sodium-rich cells, and SITS-sensitive 22Na+ efflux out of 22Na+-loaded cells into sodium-free medium was less than efflux into sodium saline, indicating trans-stimulation by sodium. The amiloride-sensitive pathway was studied in the absence of bicarbonate to inhibit uptake via the SITS-sensitive pathway. 22Na+ uptake into sodium-depleted cells increased steeply with extracellular pH in the range between pH 6 and 8 and could be largely blocked by 10(-3), but not by 10(-5) M amiloride. It is concluded that bovine corneal endothelial cells possess at least two distinct pathways for sodium uptake, amiloride sensitive 22Na+ fluxes being mediated by a Na+/H+ antiport, while the SITS-sensitive process is probably identical to a bicarbonate-sodium cotransport system postulated earlier from electrophysiological studies.     

Polarized distribution of the Na+/H+ exchange system in a renal cell line (LLC-PK1) with characteristics of proximal tubular cells

Studies of Na+ and H+ transport by confluent monolayers of the epithelial cell line LLC-PK1 were performed to verify the presence of a Na+/H+ exchange system. The presence of an outwardly directed H+ gradient produced a large stimulation of Na+ influx measured under net flux conditions. Amiloride (10(-3) M) completely inhibited Na+ influx stimulated by the H+ gradient and part of the Na+ influx measured in the absence of a pH gradient. Half-maximal inhibition of the Na+ influx stimulated by a pH gradient at 143 mM Na was observed at 5 microM amiloride. The presence of an inwardly oriented proton gradient also stimulated Na+ efflux from Na+-loaded cells. The stimulation was completely inhibited by the presence of 10(-3) M amiloride in the washout medium. These results indicate that this system could operate in the opposite direction depending on the orientation of the Na+ and H+ gradient. Incubation in Na+-free medium or in the presence of 10(-3) M ouabain resulted in a dramatic decrease of H+ release from LLC-PK1 cells. This H+ release was largely, although not completely, inhibited by 10(-4) M amiloride. Neither chloride substitution by the impermeable anion isethionate nor incubation in the presence of the ionophore valinomycin in high K+ medium affected Na+ influx by stimulated by a pH gradient. Inhibition of the Na+ influx by amiloride occurred only from the apical side of the monolayer. These results indicate that the Na+/H+ exchange system in LLC-PK1 monolayers is specifically localized in the apical membrane of the epithelial cells.     

Transepithelial bioelectrical properties of rabbit acinar cell monolayers on polyester membrane scaffolds

In our quest to develop a tissue-engineered tear secretory system, we have tried to demonstrate active transepithelial ion fluxes across rabbit lacrimal acinar cell monolayers on polyester membrane scaffolds to evaluate the bioelectrical properties of the cultured cells. Purified lacrimal gland acinar cells were seeded onto polyester membrane inserts and cultured to confluency. Morphological properties of the cell monolayers were evaluated by transmission electron microscopy and immunofluorescence staining for Na(+),K(+)-ATPase and the tight junction-associated protein occludin. Sections revealed cell monolayers with well-maintained epithelial cell polarity, i.e., presence of apical (AP) secretory granules, microvilli, and junctional complexes. Na(+),K(+)-ATPase was localized on both the basal-lateral and apical plasma membranes. The presence of tight cell junctions was demonstrated by a positive circumferential stain for occludin. Bioelectrical properties of the cell monolayers were studied in Ussing chambers under short-circuit conditions. Active ion fluxes were evaluated by inhibiting the short-circuit current (I(sc)) with a Na(+),K(+)-ATPase inhibitor, ouabain (100 microM; basal-lateral, BL), and under Cl(-)-free buffer conditions after carbachol stimulation (CCh; 100 microM). The directional apical secretion of Cl(-) was demonstrated through pharmacological analysis, using amiloride (1 mM; BL) and bumetanide (0.1 mM; BL), respectively. Regulated protein secretion was evaluated by measuring the beta-hexosaminidase catalytic activity in the AP culture medium in response to 100 microM basal CCh. In summary, rabbit lacrimal acinar cell monolayers generate a Cl(-)-dependent, ouabain-sensitive AP --> BL I(sc) in response to CCh, consistent with current models for Na(+)-dependent Cl(-) secretion.     

Effect of inhibition of Na+/K(+)-ATPase on the vasopressin-induced increase in intracellular Na+ concentration in vascular smooth muscle cells.

The effect of inhibition of Na+/K(+)-ATPase by ouabain on the arginine vasopressin (AVP)-induced increase in intracellular Na+ concentration [( Na+]i) was examined in cultured rat vascular smooth muscle cells (VSMC) by the direct measurement of [Na+]i using a fluorescent indicator dye. AVP at a concentration of 1 x 10(-9) M or higher increased [Na+]i in a dose-dependent manner in cultured rat VSMC. The preincubation of cells with 1 x 10(-4) M ouabain for 1 hr at 37 degrees C did not affect the basal [Na+]i but enhanced the 1 x 10(-6) M AVP-induced increase in [Na+]i. The preincubation was not necessary because similar results were obtained after the simultaneous administration of AVP and ouabain. The treatment with ouabain did not affect the intracellular pH changes induced by AVP. These results therefore indicate that the inhibition of Na+/K(+)-ATPase enhances the AVP-induced increase in [Na+]i by decreasing cellular Na+ efflux in cultured rat VSMC.     

Steroids, intracellular sodium levels, and Na+/K+-ATPase regulation

In outer medullary kidney tubules, both specific mineralocorticoid, and specific glucocorticoid Na+/K+-ATPase activation in vitro were inhibitable by amiloride, an inhibitor of a number of Na+-transporting mechanisms (Bentley, P.J. (1968) J. Physiol. (Lond.) 195, 317-330; Kinsella, J. L., and Aronson, P. S. (1980) Am. J. Physiol. 238, F461-F469). In addition, dexamethasone raised, whereas amiloride reduced, intracellular Na+ levels. These observations are consistent with the possibility that the steroidal responses are mediated by changes in intracellular Na+ ion activity. However, when intracellular Na+ levels were increased by the incubation of tubule segments in medium containing ouabain (10(-4) M), no Na+/K+-ATPase activation was observed, over incubation periods of up to 6 h. As mineralocorticoid and glucocorticoid effects are maximal within 2 h (Rayson, B.M., and Lowther, S.O. (1984) Am. J. Physiol. 246, F656-F662), these results suggest that the Na+ ion per se does not mediate the steroidal effects observed, directly. Incubation of tubule segments in medium containing 10(-4) M ouabain, at 37 degrees C, for longer periods (18 h), however, did indeed increase Na+/K+-ATPase activity, markedly. Thus, a potential homeostatic mechanism was demonstrable, where a chronic increase in intracellular Na+ level, measured after 2-4 h of treatment, resulted in an increase in Na+/K+-ATPase activity, such that the intracellular Na+ level was restored after 18-20 h of incubation to one not significantly different from the control value. This mechanism, however, appears to be clearly distinguishable from that which mediates steroidal Na+/K+-ATPase activation.     

Intracellular Na+ and K+ activities and membrane conductances in the collecting tubule of Amphiuma

Membrane potentials and conductances, and intracellular ionic activities were studied in isolated perfused collecting tubules of K+-adapted Amphiuma. Intracellular Na+ (aNai) and K+ (aKi) activities were measured, using liquid ion-exchanger double-barreled microelectrodes. Apical and basolateral membrane conductances were estimated by cable analysis. The effects of inhibition of the apical conductance by amiloride (10(-5) M) and of inhibition of the basolateral Na-K pump by either a low K+ (0.1 mM) bath or by ouabain (10(-4) M) were studied. Under control conditions, aNai was 8.4 +/- 1.9 mM and aKi 56 +/- 3 mM. With luminal amiloride, aNai decreased to 2.2 +/- 0.4 mM and aKi increased to 66 +/- 3 mM. Ouabain produced an increase of aNai to 44 +/- 4 mM, and a decrease of aKi to 22 +/- 6, and similar changes were observed when the tubule was exposed to a low K+ bath solution. During pump inhibition, there was a progressive decrease of the K+-selective basolateral membrane conductance and of the Na+ permeability of the apical membrane. A similar inhibition of both membrane conductances was observed after pump inhibition by low K+ solution. Upon reintroduction of K+, a basolateral membrane hyperpolarization of -23 +/- 4 mV was observed, indicating an immediate reactivation of the electrogenic Na-K pump. However, the recovery of the membrane conductances occurred over a slower time course. These data imply that both membrane conductances are regulated according to the intracellular ionic composition, but that the basolateral K+ conductance is not directly linked to the pump activity.     

Two classes of ouabain receptors in chick ventricular cardiac cells and their relation to (Na+,K+)-ATPase inhibition, intracellular Na+ accumulation, Ca2+ influx, and cardiotonic effect

The biochemical and pharmacological properties of the (Na+,K+)-ATPase have been studied at different stages of chick embryonic heart development in ovo and under cell culture conditions. The results show the existence of two families of ouabain binding sites: a low affinity binding site with a dissociation constant (Kd) of 2-6 microM for the ouabain-receptor complex and a high affinity binding site with a Kd of 26-48 nM. Levels of high affinity sites gradually decrease during cardiac ontogenesis to reach a plateau near 14 days of development. Conversely the number of low affinity binding sites is essentially invariant between 5 days and hatching. Cultured cardiac cells display the same binding characteristics as those found in intact ventricles. Inhibition of 86Rb+ uptake in cultured cardiac cells and an increase in intracellular Na+ concentration, due to (Na+,K+)-ATPase blockade, occur in a ouabain concentration range corresponding to the saturation of the low affinity ouabain site. Ouabain-stimulated 45Ca2+ uptake increases in parallel with the increase in the intracellular Na+ concentration. It is suppressed in Na+-free medium or when Na+ is replaced by Li+ suggesting that the increase is due to the indirect activation of the Na+/Ca2+ exchange system in the plasma membrane. Dose-response curves for the inotropic effects of ouabain on papillary muscle and on ventricular cells in culture indicate that the development of the cardiotonic properties is parallel to the saturation of the low affinity binding site for ouabain. Therefore, inhibition of the cardiac (Na+,K+)-ATPase corresponding to low affinity ouabain binding sites seems to be responsible for both the cardiotonic and cardiotoxic effects of the drug.     

Transport functions of the liver. Lack of correlation between hepatocellular ouabain uptake and binding to (Na+ + K+)-ATPase

Ouabain uptake was studied on isolated rat hepatocytes. Hepatocellular uptake of the glycoside is saturable (Km = 348 mumol/l, Vmax = 1.4 nmol/mg cell protein per min), energy dependent and accumulative. Concentrative ouabain uptake is not present on permeable hepatocytes, Ehrlich ascites tumor cells and AS-30D ascites hepatoma cells. There is no correlation between ouabain binding to rat liver (Na+ + K+)ATPase and ouabain uptake into isolated rat hepatocytes. While ouabain uptake is competitively inhibited by cevadine, binding to (Na+ + K+)-ATPase is not affected by the alkaloid. Although the affinities of digitoxin and ouabain to (Na+ + K+)-ATPase are similar, digitoxin is 10000-times more potent in inhibiting [3H]ouabain uptake as compared to ouabain. That binding to (Na+ + K+)-ATPase appears to be no precondition for ouabain uptake was also found in experiments with plasmamembranes derived from Ehrlich ascites tumor cells and AS-30D hepatoma cells. While tumor cell (Na+ + K+)-ATPase is ouabain sensitive, the intact cells are transport deficient. Hepatic ouabain uptake might be related to bile acid transport. Several inhibitors of the bile acid uptake system also inhibit ouabain uptake.     

Density-related changes of potassium (86Rb) uptake by amphibian endothelial cells

Potassium influx has been investigated in XTH-2 cells, a line derived from tadpole heart endothelia. In this line, the density at which the cultures become confluent is clearly separated from the density at which growth arrest takes place. Density-related changes in K+ influx were monitored by determining the uptake of 86Rb into well adhering cells kept in culture medium. The main observations were 1) 86Rb uptake is highest in single cells, and on confluency it reaches a low level, which is kept constant at higher cell density regardless of whether the cultures are stationary or still in logarithmic growth phase; 2) the relative amount of 86Rb taken up via the Na+ -K+ -2Cl- cotransport pathway and via the Na+/K+ pump changes from low cell density to confluent cultures; 86Rb uptake of single cells is nearly insensitive to ouabain, a maximum of ouabain sensitivity is reached around confluency, whereas piretanide-sensitive 86Rb uptake is highest in single cells and seems to reach a minimum at the onset of confluency; 3) the variations in Na+/K+ pumping rate reflect neither differences in the amount of enzyme present nor changes in enzyme repartition between apical and basolateral plasma membranes; they seem to result from either "masking" or "unmasking" of the enzyme; 4) no alterations in K+ uptake occur that would be characteristic of the "stationary growth phase." The only changes that seem to be related to arrest of proliferation are concerned with the Na+/K+-ATPase, which achieves an extraordinary susceptibility to stimulation by monensin and exhibits an increase in PNPPase activity.