Increased Na(+)-H+ antiporter activity in apical membrane vesicles from mutant LLC-PK1 cells.

In whole cell experiments, the PKE20 mutant of the renal epithelial cell line LLC-PK1 displays a severalfold elevation of Na(+)-H+ antiporter activity at the apical surface (J.G. Haggerty, N. Agarwal, R.F. Reilly, E. A. Adelberg, and C.W. Slayman. Proc. Natl. Acad. Sci. USA 85: 6797-6801, 1988). The present study was undertaken to explore the properties of the mutant at the membrane level. Apical membrane vesicles were prepared by the magnesium-aggregation technique, with a similar enrichment (ca. 10-fold) of the marker enzyme gamma-glutamyltranspeptidase in vesicles from parent and mutant cell lines. In both cases, 22Na influx was stimulated by an inside-acid pH gradient, inhibited by ethylisopropylamiloride (EIPA), and unaffected by valinomycin, indicating that it was mediated by Na(+)-H+ antiport. Quantitatively, PKE20 vesicles showed a 4.2-fold increase in the maximal velocity of Na(+)-H+ antiporter activity compared with the parent, with only minor increases in the activity of two other Na(+)-dependent transporters (14-56% for alpha-methylglucoside and L-glutamate). Dose-response curves for EIPA indicated that the increased Na(+)-H+ antiport activity in PKE20 vesicles was due to an increased activity of the relatively amiloride-resistant form of the Na(+)-H+ antiporter with little or no change in the amiloride-sensitive form. No differences in polypeptide composition of the two vesicle preparations could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Taken together, the results indicate that the mutation in PKE20 is expressed at the membrane level and is specific for the relatively amiloride-resistant Na(+)-H+ antiporter.(ABSTRACT TRUNCATED AT 250 WORDS)

LLC-PK1 mutant with increased Na+-H+ exchange and decreased sensitivity to amiloride.

LLC-PK1 cells contain a well-characterized Na+-H+ antiporter that is sensitive to ethylisopropylamiloride (EIPA) in the submicromolar range. Using a modification of the method of Franchi et al. (J. Biol. Chem. 261: 14614-14620, 1986), we have selected mutants that can recover from an acid load in the presence of 100 microM EIPA. One such mutant, designated PKE20, has been studied in detail. The maximal velocity (Vmax) for the Na+-H+ antiporter, assayed as EIPA-sensitive 22Na+ uptake, has increased from 44 nmol.min-1.10(6) cells-1 in the parent cells to 106 nmol.min-1.10(6) cells-1 in PKE20. No detectable change has occurred in the Km for Na+ (118 mM in the parent, 111 mM in the mutant) or in the dependence of Na+ uptake on intracellular pH. However, the PKE20 antiporter exhibits a greatly decreased sensitivity to amiloride and its derivatives, with drops in inhibitory potency ranging from 25-fold (amiloride) to 100-fold (EIPA). The mutation is specific for the antiporter; measurements of Na+-K+ pump and Na+-dependent amino acid uptake show only small changes, which appear to result from minor antiporter-induced alterations in internal Na+ concentration. PKE20 cells should prove useful in experiments to identify and isolate the antiporter protein.

Pharmacologically different Na/H antiporters on the apical and basolateral surfaces of cultured porcine kidney cells (LLC-PK1).

Proximal tubule cells of the kidney contain, on their apical surface, an amiloride-sensitive Na/H antiporter that functions in Na reabsorption and proton secretion. We have investigated the localization of the antiporter in a cloned cell line of porcine renal origin, LLC-PK1/Cl4, which is often considered to be a useful model of the proximal tubule. Transport measurements were performed with differentiated monolayers grown on Nuclepore filters, permitting independent access to the apical and basolateral cell surfaces. In control experiments with LLC-PK1/Cl4 monolayers, three marker transport systems showed the expected polarity: 87% of ouabain-sensitive Rb uptake was at the basolateral surface, and 99% of Na-dependent alpha-methylglucoside transport and 93% of Na-dependent D-aspartate (L-glutamate) transport were at the apical surface. By contrast, the monolayers displayed significant Na/H antiporter activity (assayed as ethylisopropylamiloride-sensitive 22Na uptake) at both cell surfaces, with an apical uptake rate amounting to 44% and a basolateral rate amounting to 56% of the total. Significantly, the apical and basolateral antiporters could readily be distinguished from one another on the basis of ethylispropylamiloride sensitivity. The apical system had an IC50 of 13 microM, close to that reported for kidney brush border vesicle preparations, whereas the basolateral system had an IC50 of 44 nM, similar to values seen in undifferentiated LLC-PK1 cells and other cultured cell lines. The PKE20 mutant, previously selected from LLC-PK1/Cl4 on the basis of resistance to ethylisopropylamiloride, was found to overexpress the more resistant antiporter both during rapid growth and on its apical cell surface at confluence; normal amounts of the more sensitive antiporter were seen on the basolateral surface of confluent PKE20 cells. Taken together, these results suggest that there are two distinct forms of the Na/H antiporter, which are under separate genetic control.

Isolation and characterization of a glycine transport mutant in an established mammalian cell line, CHO(PEOT/1).

Glycine uptake in CHO(PEOT/1) cells is mediated by at least three systems, of which two have been identified and partially characterized in this study: (1) a low affinity "A" system that transports a number of small neutral amino acids including glycine and methylaminoisobutyric acid (MeAIB), and (2) a high-affinity system, specific for glycine and sarcosine. By a combination of tritium suicide and replica plating, we have isolated a mutant (CHY-3) with a 47% decrease in glycine transport at the standard test concentration of 2.5 microM. Uptake studies with radioactive glycine, MeAIB, and sarcosine revealed that the mutant lacks the glycine-sarcosine system, but has undergone a compensatory 30-50% increase in the A system. Thus, there appears to be a regulatory interaction between these two systems for glycine uptake by CHO cells.

Stimulation by serum of the Na+/H+ antiporter in quiescent pig kidney epithelial (LLC-PK1) cells and role of the antiporter in the reinitiation of DNA synthesis.

LLC-PK1 cells can be brought into a classical quiescent state by depriving them of serum for 6 days. At this time, pulse-labeling with [3H]-thymidine shows that only 3% of the cells are synthesizing DNA, but the quiescent cells can be stimulated with serum to re-enter the cell cycle at a point early in G1. The rate of amiloride-sensitive 22Na+ uptake (as a measure of the Na+/H+ antiporter) is relatively low during quiescence; it rises 2- to 3-fold within 4 h after serum addition. This increase in antiporter activity appears to be required for the resumption of DNA synthesis in the absence of bicarbonate, because ethylisopropylamiloride (EIPA) blocks [3H]-thymidine incorporation when serum is added to cells in bicarbonate-free medium. In the presence of bicarbonate, however, EIPA has no effect on [3H]-thymidine incorporation, indicating that another (bicarbonate-dependent) transport system can substitute for the antiporter under these conditions.

Isolation and characterization of a Na-H antiporter-deficient mutant of LLC-PK1 cells.

LLC-PK1 is an established cell line derived from pig kidney epithelium; it differentiates in vitro to form a polarized epithelial sheet, capable of the vectorial transport of solutes and water. We have used a modification of the "proton-suicide" method of Pouyssegur et al. (Proc. Natl. Acad. Sci. USA. 81:4833-4837, 1984) to isolate a mutant of LLC-PK1 cells that is deficient in Na-H antiporter activity. The mutant grows normally at pH 7.0 and above in the presence and absence of bicarbonate; at pH 6.5, however, it requires bicarbonate for growth. Alkalinization of the cytoplasm by the Na-H antiporter thus appears to be essential for growth at acidic pH in the absence of bicarbonate. The antiporter is also essential for the formation of domes (a consequence of vectorial water transport) in the absence of bicarbonate.

Stimulation of bumetanide-sensitive K+ transport in Swiss 3T3 fibroblasts by serum and mitogenic hormones.

Rapidly growing Swiss 3T3 fibroblasts possess a bumetanide-sensitive K+ transport system that is dependent on both Na+ and Cl- ions; a smaller bumetanide-insensitive component of K+ transport is also present. In cells brought to the quiescent state by 8-11 days of incubation without a medium change, the bumetanide-sensitive rate of transport was reduced by 63%; the bumetanide-insensitive rate did not change. Removal of dialyzed fetal calf serum from the uptake medium resulted in a substantial reduction in bumetanide-sensitive uptake in both rapidly growing cells (33% reduction) and quiescent cells (68% reduction) but had no effect on bumetanide-insensitive uptake. Insulin was almost as effective as dialyzed fetal calf serum in stimulating bumetanide-sensitive uptake; insulin was maximally stimulatory at 2.5 micrograms/ml. The combination of insulin, epidermal growth factor, and arginine-vasopressin was maximally effective in stimulating both bumetanide-sensitive K+ uptake and 3H-thymidine incorporation in quiescent cells; bumetanide, however, did not interfere with the hormonal stimulation of DNA synthesis. Thus, the bumetanide-sensitive K+ transport system is not necessary for such stimulation to occur. Furthermore, concentrations of hormones which stimulated significant levels of DNA synthesis produced no elevation in the intracellular concentration of K+. We conclude that the bumetanide-sensitive pathway of K+ transport is modulated by serum and by mitogenic hormones, but does not play a role in the stimulation of DNA synthesis by these factors.

Na+/H+ exchanger activity in the pig kidney epithelial cell line, LLC-PK1: inhibition by amiloride and its derivatives.

Rapidly growing pig-kidney-derived epithelial cells, LLC-PK1, lack detectable amiloride-sensitive Na+/H+ exchange activity when assayed directly. A large 22Na uptake is induced when the cells are acid-loaded prior to assay by incubation with buffer containing ammonium chloride or nigericin. The acid-stimulated sodium uptake is sensitive to amiloride, with half-maximal inhibition at 3.5-4.5 microM in buffer containing 15 mM sodium ion. There is simple competitive interaction between amiloride and sodium ion when the amiloride concentration is below 25 microM and the sodium ion concentration is above 20 mM. Derivatives of amiloride which carry substituents on the 5-amino group are 35- to 175-fold more inhibitory than amiloride itself.

Rapid changes in bidirectional K+ fluxes preceding DMSO-induced granulocytic differentiation of HL-60 human leukemic cells.

When grown in medium containing 5 mM potassium and 140 mM sodium, HL-60, a human promyelocytic cell line, maintained a steady-state intracellular K+ concentration of 145 mmol/L cells and a steady-state intracellular Na+ concentration of 30 mmol/L cells. Nearly 90% of the unidirectional 42K+ influx could be inhibited by the cardiac glycoside ouabain with a Ki of 5 X 10(-8) M. This ouabain-sensitive component of influx rose as a saturating function of the extracellular K+ concentration with a K1/2 of 0.85 mM. The component of 42K+ influx resistant to ouabain inhibition was a linear function of the extracellular K+ concentration and was insensitive to inhibition by the diuretic furosemide. Unidirectional K+ efflux followed first order kinetics with a half-time of 55 min. Addition of 1.5% dimethyl sulfoxide (DMSO) to a culture of HL-60 cells allowed two population doublings followed by the cessation of growth without an impairment of cell viability. Beginning 2 to 3 days after DMSO addition, the cells underwent a dramatic reduction in volume (from 925 microns 3 to 500 microns 3) and began to take on the morphological features of mature granulocytes. Throughout this process of differentiation there was no change in the intracellular sodium or potassium concentration. However, immediately following the addition of DMSO to a culture of cells, there began an immediate, coordinated reduction in bidirectional K+ flux. The initial rate of the ouabain-sensitive component of K+ influx fell with a half-time of 11 h to a final rate, at 6 days induction, equal to one ninth that of the uninduced control, and over the same period, the rate constant for K+ efflux fell with a half-time of 14 h to a final value one fourth that of the uninduced control. The rapidity with which these flux changes occur raises the possibility that they play some role in the control of subsequent events in the process of differentiation.

Furosemide-sensitive potassium efflux in cultured mouse fibroblasts.

Transfer of LM(TK-) cells from normal growth medium to medium lacking K+ leads to a rapid loss of intracellular K+, which is 50-70% inhibited by furosemide or bumetanide. The diuretic-sensitive component of K+ efflux requires both Na+ and Cl-, and is presumably mediated by a K+, Na+, Cl- cotransport system of the kind described in avian erythrocytes and Ehrlich ascites cells. It can be calculated that such a system should be near equilibrium under normal growth conditions but should mediate net efflux (as observed) when the driving force is altered by reducing extracellular K+. The diuretic-sensitive component of net K+ efflux is also sensitive to amiloride. This effect is probably indirect, however, with amiloride acting to block the Na+ influx that supplies Na+ to the cotransport system. At the low extracellular K+ concentrations employed in these studies, the diuretic-sensitive system is a physiologically important pathway of K+ loss. The rate of growth in low-K+ medium can be increased (or the rate of cell lysis decreased) by adding diuretic or by reducing external Na+ or Cl-.

Isolation and characterization of a CHO amino acid transport mutant resistant to melphalan (L-phenylalanine mustard).

A mutant of Chinese hamster ovary cells, CHY-2, was isolated on the basis of its reduced ability to grow on a limiting concentration of leucine and was found to be defective in uptake of leucine via the sodium-independent L system. Consistent with published reports that the L system can mediate melphalan uptake, the D10 of the mutant for melphalan was increased threefold under conditions designed to limit drug uptake to the L system (brief exposure in sodium-free medium). Unlike a previously described melphalan-resistant CHO mutant (CHr), CHY-2 displays no cross-resistance to colchicine or puromycin. It differs from a second melphalan-resistant CHO mutant, melr, in its sensitivity to melphalan in the presence of high Na+, and from a melphalan-resistant mouse leukemic cell in possessing normal levels of intracellular glutathione. Thus, CHY-2 represents a new melphalan-resistant mutant class. The effect of the CHY-2 mutation is pleiotropic, involving significant reductions in amino acid uptake via the L, A and Ly+ (but not ASC) systems. The primary defect is unknown; however, the mutant possesses normal intracellular concentrations of Na+ and K+ and normal membrane fluidity. The growth rate of the mutant in standard medium is greatly reduced (generation time of 60 h vs. 24 h), although it can be improved by the addition of a supplement containing high concentrations of leucine, proline, and peptides.

Intracellular K+ and the mitogenic response of 3T3 cells to peptide factors in serum-free medium.

The stimulation of DNA synthesis in cultures of Swiss 3T3 cells by vasopressin, epidermal growth factor, and insulin added in serum-free medium is strikingly dependent on the intracellular K+ content or concentration. The relationship between these parameters is sigmoid; DNA synthesis commences only when the intracellular K+ increases above a certain threshold level (0.56 mumol/mg of protein; 90 mM). Addition of K+ to K+- depleted cultures reverses the block on DNA synthesis after a lag period of at least 8 hr. The sigmoid dependence of DNA synthesis on intracellular K+ is generated in early G1 phase rather than at the G1/S boundary. The effects of K+ on the G1-S transition are, at least in part, exerted through its control of protein synthesis. In serum-free medium, the K+ content is close to the threshold required for allowing a mitogenic response. The findings suggest that a small change in the intracellular K+ level can influence the ability of these cells to initiate DNA synthesis in serum-free medium.

Isolation of a spontaneous CHO amino acid transport mutant by a combination of tritium suicide and replica plating.

A spontaneous transport mutant of Chinese hamster ovary cells, CHY-1, was isolated by a combination of [3H]proline suicide and replica plating. The mutant took up less tritium than the parent, resulting in a lower killing rate during storage. Transport by four separate amino acid transport systems (A, ASC, L, Ly+) was examined. The CHY-1 mutant exhibited normal uptake via the ASC, L, and Ly+ systems. By contrast, uptake of the most specific substrate of the A system, 2-(methylamino)-isobutyric acid, was significantly reduced at low, but not high, concentrations, due to a 3.5-fold increase in Km and a 1.5-fold increase in Vmax. Taken together, these data suggest that the CHY-1 mutation may be in the structural gene coding for the A transport protein. The tritium suicide procedure is discussed, and general equations are derived to predict the maximum storage time for the survival of one mutant cell and the optimum size of the cell population for maximum mutant enrichment.

Reduction of K+ efflux in cultured mouse fibroblasts, by mutation or by diuretics, permits growth in K+-deficient medium.

The mouse fibroblastic cell line LM(TK-) is unable to grow at external K+ concentrations below a threshold value of 0.4 mM. At subthreshold K+ concentrations, LM(TK-) cells rapidly lose intracellular K+ and eventually lyse. We have analyzed the pathway primarily responsible for K+ efflux under these experimental conditions and reports its specific inhibition by two diuretics, furosemide and bumetanide. Bumetanide, an analog of furosemide, was a more potent inhibitor (by several orders of magnitude) than was furosemide itself. The effects of ouabain and bumetanide were additive, suggesting independence of diuretic-sensitive K+ efflux from Na+/K+ pump-mediated fluxes. Characterization of K+ efflux in LTK-5, a mutant derived from LM(TK-) and selected for its ability to grow at 0.2 mM K+ indicated that the mutant had lost the diuretic-sensitive K+ efflux pathway. Net cation fluxes, steady-state intracellular cation concentrations, and growth at reduced K+ concentrations were comparable for LM(TK-) cells maximally inhibited by diuretics and for the LTK-5 mutant grown either in the presence or absence of diuretics. Thus, reduction in K+ efflux, either by diuretic addition diuretics. Thus, reduction in K+ efflux, either by diuretic addition or by genetic alteration, can permit the cell to maintain normal cation gradients and to grow at otherwise subthreshold external K+ concentrations.

Genetic alterations in potassium transport in L cells.

Starting with mutagenized cultures of the mouse fibroblastic cell line LM(TK-), we have selected mutant clones by their ability to grow at 0.2 micrometer K+, a concentration unable to support the growth of the parent cell. The mutants fall into two classes on the basis of their potassium transport properties. Both classes maintain a high intracellular K+ concentration when growing in low-potassium medium, and both are unaltered in the ouabain-sensitive Na/K pump. One class shows an increased activity of a ouabain-resistant, furosemide-sensitive K+ transport system; the other class shows a decreased activity of a specific component of K+ efflux.