JOURNAL OF CELLULAR PHYSIOLOGY 143555-562 119901

Cystine and Dibasic Amino Acid Uptake by Opossum Kidney Cells BEATRICE STATES*

AND

STANTON SEGAL

Division of Biochemical Development and Molecular Diseases, Children'$ Hospital of Philadelphia, Department of Pediatrics and Medicine, University of Pennsylvania School ot Medicine, Philadelphia, Pennsylvania 79 104 The characteristics of the uptake of L-cystine by the continuous opossum kidney cell line, OK, were examined. Uptake of cystine is rapid and, in contrast to other continuous cultured cell lines, these cells retain the cystine/dibasic amino acid transport system which i 5 found in vivo and in freshly isolated kidney tissue. Confluent monolayers of cells also fail to show the presence of the cystinei glutamate transport system [irebent in LLC-PK, cells, fibroblasts, and cultured hepatocytes. Uptake of cystine occurs via a high-affinity saturable process which is independent of medium sodium concentration.The predominant site of cystine transport is across the apical cell membrane. The intracellular concentration of GSH far exceeds that of cysteine with a ratio greater than 1OO:l for GSH:cysteine. Incubation of cells for 5 minutes with a physiological level of labelled cystine resulted in the labelling of 66% and 5% of the total intracellular cysteine and glutathione, respectively. The ability of these cells to reflect the shared cystine/ dibasic amino acid transport system makes them a suitable model for investigation of the cystine carrier which is altered in h u m a n cystinuria.

One of the more important disorders of proximal tu- phosphate transport by the parathyroid hormone bule transport is human cystinuria. This transport de- (PTH),a property not found in LLC-PK, and JTC-12P3 fect is characterized as a n alteration in the cystinel cells (Malmstrom and Murer, 19861, the role of PTH in lysine transporter, a system shared also with the Naf-H' anti-porter activity (Pollack et al., 1986), and dibasic amino acids, ornithine and arginine (Foreman for its transport of amino acids (Malmstrom et al., and Segal, 1985).Information on this system has come 1987; Scheinman, 1988; Schwegler et al., 1989). The mainly from studies using renal cortical tubule seg- data suggest that these OK cells retain several imporments (Forman et al., 1980), brushborder membrane tant functional properties found in vivo in proximal vesicles (Segal et al., 1977; McNamara et al., 19811, tubules and in renal cortex. We, therefore, examined the OK cell line to deterand primary dog proximal tubule cells (States et al., 1990). However, such model systems lack cell homoge- mine a ) whether the cystinetdibasic amino acid transneity. A continuous kidney epithelial cell line, on the port system was present and b) the kinetic properties of other hand, offers several advantages such as: a) a cystine and dibasic amino acid uptake. These results more homogeneous cell population, b) the ability to ma- form the basis of this report. nipulate the growth medium prior to transport studies, MATERIALS AND METHODS c) long-term storage so that the same type of experimental material may be used again, and d) the possiMaterials bility of developing mutants. Two continuous kidney epithelial cell lines retain The Opossum Kidney (OK) cell line, passage 73, was several characteristics in common with the proximal the generous gift of Dr. Joseph Handler (Laboratory of tubule. One, the LLC-PK, line, derived from juvenile Kidney and Electrolyte Metabolism, National Instipig kidney (Hull et al., 1976), has been studied exten- tutes of Health, Bethesda, MD). The following labelled sively for its Na+-dependent hexose (Mullin et al., compounds were obtained from Amersham Interna1980; Rabito and Ausiello, 1980), phosphate (Rabito, tional, Arlington, IL: (35S) L-cystine (332mCi/mmol), 1983), and amino acid transport (Rabito and Karish, 1983) capacities. Recently, however, these cells have been shown to lack the genetically determined cystinel lysine transport system (Foreman e t al., 1988). This Received December 12, 1989; accepted February 14, 1990. cell line, therefore, is a n inappropriate model for stud- *To whom reprint requests/correspondence should be addressed. ies of this specific renal tubule cell cystine transport Abbreviations used: a-MEM, a-minimal Eagle's Medium; CSH, system (Segal and Thier, 1989). The second continuous cysteine; D.R., distribution ratio; GSH, reduced glutathione; epithelial cell line, the OK line, derived from opossum HPLC, high performance liquid chromatography; OK, opossum kidney (Koyama et al., 1978), is of more recent vintage kidney; PBS, phosphate buffered saline; S.E.M., standard error and has been studied for its capacity for regulation of of the mean. D

1990 WILEY-LISS, INC.

556

STATES AND SEGAL

(U-14C)a-methyl-D-glucose (279 mCi/mmol), ('H)3-0methylglucose (8.5Ci/mmol), (14C) polyethyleneglycol (15 mCi/gm), (U-14C)L-lysine(348 mCi/mmol), (U- 4C) L-arginine (342 mCi/mmol), and (1-l4C)L-ornithine (59 mCi/mmol). ("S)L-cystine was greater than 95% pure as determined by thin layer chromatography (States and Segal, 1969) and HPLC (States et al., 1987). L( l-3H (N)-glucose (10.7 Ci/mmol) was purchased from New England Nuclear, Boston, MA. a-Minimal Eagle's Medium was obtained from Flow Laboratories, Inc., McLean, VA. Fetal calf serum was purchased from Whittaker M.A. Bioproducts, Walkersville, MD. Plastic flasks were obtained from Fisher Scientific, King of Prussia, PA. All reagents were of the highest available grade. Cell culture All these studies were performed with OK cells between passages 77 and 82. Cells were grown routinely in T25 and T75 flasks with a-minimal Eagle's Medium supplemented with 4 mM glutamine and 3% fetal calf serum (complete u-MEM) at 37°C under compressed air:CO, (95:5). At confluency, cells were transferred to new T25 flasks a s follows: the culture medium was removed, the cell monolayer was washed with 4 ml Puck's Versene solution B, and the cells were unsheeted by incubation for 10 minutes with 2 ml Puck's solution C containing 0.04% trypsin and 0.02% Versene. Once cells were free of the plastic surface, they were transferred a t a 1:4 split. When cells were grown and subcultured in T75 flasks, twice the amounts of P u c k s solutions B and C were required. Five or 15 milliliters complete a-MEM media then was added to each T25 or T75 flask, respectively. Media from representative flasks of OK cells were taken to the Human Genetic Cell Science Center of the University of Pennsylvania for monitoring for mycoplasma contamination. Only cells free of mycoplasma were used in experiments. Also, periodically, the cells were examined by electron microscopy for intracellular contaminants. Quantitation of cysteine (CSH) and reduced glutathione (GSH) CSH and GSH levels in OK cells were determined by HPLC (States et al., 1987). Confluent monolayers of cells in T25 flasks were washed twice with 2 ml of cold phosphate-buffered saline (Ca and Mg free). The cells were ruptured by addition of 0.5 ml 0.02M EDTA. The material was scraped with a rubber policeman and transferred to a 1.5 ml polypropylene conical centrifuge tube for deproteinization by addition of 0.05 ml of either cold 50% trichloroacetic acid (TCA) or cold 30% metaphosphoric acid. The mixture was vortexed and placed at 4°C for 1 hour before centrifugation at 12,OOOg for 5 minutes. The supernatant was removed and either used immediately or stored a t -40°C for no longer than 48 hours before assay of intracellular CSH and GSH. The pellet was dissolved in 1 ml of either 0.1N NaOH for protein determination a s described below or solubilized by addition of 1 ml 0.2% Triton X100:1N NH,OH followed by freezing and thawing three times for determination of both DNA (Fiszer-Szafarz et al., 1981) and protein by using the Bio-Rad protein assay kit with bovine y-globulin as the standard. The

DNA standard was based on construction of a standard curve after reading a t 260 m p on a Zeiss Spectrophotometer PM2 DL. Under our culture conditions and assay method, OK cells were found to contain 70 -t 5.4 pg DNA/mg protein (mean t S.E.M., 20 determinations). Total intracellular cyst(e)ine was determined on a Beckman 6300 Amino Acid Analyzer with a n IBM F'C System Gold integrator. ' appearing The percentages of total intracellular S in CSH and GSH and the intracellular levels of CSH and GSH after 5 minutes incubation a t 37°C with ("S)L-cystine were determined by HPLC a s described above. Fractions of 0.8mUmin were collected from the column in a Pharmacia Frac-100 fraction collector (Pharmacia, Piscataway, NJ, USA) attached to the collector valve of the Waters Model 6000A HPLC (Waters, Milford, MA, USA). Each fraction was added to 10 ml of ACSII scintillation fluid and counted in a Packard TriCarb Liquid Scintillation Spectrometer (Packard Instrument Co., Downers Grove, IL, USA).

Transport studies For transport experiments, OK cells were suspended in 1 ml of complete u-MEM medium at approximately 0.1 x lo6 celldm1 medium. One milliliter of this suspension was added to each 35 x 10 mm well of a sixcluster well set. Four milliliters of complete a-MEM media were added immediately to each cluster well. Cells were refed every other day. At the point of confluency (i.e., in 5 to 6 days) and 24 hours prior to onset of the experiment, the cells were refed with 5 ml of complete a-MEM. To initiate transport studies, the growth media were removed and each cluster well was washed two times with 2 ml of 37°C phosphate buffered saline (PBS) supplemented with 0.1% glucose (pH 7.4). To begin a n experiment, 2 ml of 37°C PBS + 0.1% glucose containing approximately 0.2 pCi/ml of radioactive substrate w,as added to each cluster well. The wells were incubated a t 37°C with shaking at 100 rpm on a n Orbital Shaker. The incubation was terminated by aspirating the buffer media and immediately washing the cells twice with 2 ml of cold Mg-Ca-free phosphate-buffered saline. Tissue radioactivity was extracted with 1 ml of cold 10% TCA (Gazzola e t al., 1981). The entire TCA extract was added to scintillation cocktail, ACSII (Amersham), and assayed for radioactivity in a Packard Tri-Carb Liquid Scintillation Spectrometer. Intracellular fluid space (ICF) and extracellular fluid space (ECF) were determined by measuring the uptake of trace amounts of (3H)3-O-methylglucose (1 mCi/ml), a non-metabolizable substrate (Mullin et al., 19801, and (14C)polyethylene glycol (50 pCi/ml), a substance which does not enter the cell. These substrates, added to glucose-free media at a 20:l ratio of 3H:14C, were measured at the corresponding time period(s) in each experiment to correct for trapping or diffusion of transport substrates. Calculations were as described previously (States et al., 1974) with distribution ratios (D.R.) for cystine uptake calculated as cpm S ' in (ICF/ nlMECF/nl). The TCA-precipitated protein in each well was solubilized in 1ml of 0.1N NaOH and quantitated by using the Bio-Rad protein assay kit with bovine y-globulin a s the standard.

557

CYSTINE AND DIBASIC AMINO ACID UPTAKE BY OK CELLS

To determine the concentration dependence of uptake of several amino acids, cells in cluster well dishes were incubated at 37°C with shaking at 100 rpm for 5 minutes. One-tenth to 0.2 pCi/ml of labelled substrate plus unlabelled substrate was added to PBS supplemented with 0.1% glucose (pH 7.4) to give the desired final concentrations over the ranges examined. Transport kinetic parameters were determined from Lineweaver-Burk plots of the transport data by using the Enzfitter program, a non-linear regression data analysis program for the IBM PC by Robin J. Leatherbarrow, published in 1987 by Elsevier Science Publishers, Amsterdam, The Netherlands. All analyses of data for statistical significance were performed by using Student’s t-test. All studies were performed with at least six replicate cluster wells per data point.

1

’5S-CYSTlNE

-PBS + I % GLUCOSE -LOW NO PBS+ I % GLUCOSE

D ,

Studies with cells on Cellagent” Filters Cells were trypsinized as described above and diluted to approximately 0.6 x lo6 celldm1 of complete a-MEM. A disc was placed in the center of a 35 x 10 mm (sixcluster well dish and washed with 1 ml of complete medium. After removal of this medium, 2 ml of complete a-MEM was added to the underside of the filter and 2 ml of the cell suspension added carefully onto the filter. The media were replaced 24 hours after seeding to remove unattached nonviable cells. Thereafter, cells were refed 2 ml of fresh complete a-MEM media every other day and were monitored under light microscopy for degree of confluency. After a complete cellular monolayer appeared on the filter, the cells were refed and 20 hours later were treated a s follows: The growth media were removed from both sides of the filter. The cell monolayer and underside of the filter were washed three times with 37°C complete PBS supplemented with 0.1% glucose (pH 7.4). To start the experiment, 2 ml of complete PBS containing 0.1% glucose and 0.025 mM (35S)L-cystinewere added either to the chamber containing the monolayer of cells for studies of apical cellular uptake or to the underside of the filter to determine basolateral cellular transport. Two milliliters of complete PBS containing 0.1% glucose without label were added to the other side of the filter. Incubations were for 5 minutes. To stop cystine uptake, the media were aspirated and each surface was washed carefully three times with cold Ca-Mg-free PBS, the complete operation being accomplished within a 1 minute period. The cell supernatant was extracted from the cell monolayer by addition of 1 ml 10%TCA to the filter surface. The filter chambers were left a t room temperature for 1 hour after which the cell extract was placed in 10 ml ACSII for counting radioactivity in a Packard Scintillation Spectrophotometer. D.R. was calculated as described above under “Transport Studies.” A trace amount of (’HI 3-0-methylglucose was used to measure the total (ICF + ECF) space from the apical side of the cell. In a similar manner, a trace amount of (3H) Lglucose measured total (ICF + ECF) space from the basolateral side of the cell. ECF spaces were determined by measuring the uptake of trace amounts of (14C)polyethylene glycol across the apical and basolateral sides. These ECF values were subtracted from the total (ICF + ECF) spaces on the corresponding sides to determine the ICF spaces on each side of the cell. Calcula-

0

5

10 TIMEhn)

15

20

Fig. 1. Time- and sodium-dependent uptake of 0.025 mM (:’‘Ss)Lcystine by confluent monolayers of OK cells. Each point represents the average f S.E.M. of six-cluster-well dishes. Labelled cystine and to low sodium I8 mM) P B S + added to P B S + 0.1% glucose, (0) 0.1% glucose, ( 0 ) .

tions were a s described by States e t al. (1974). To determine DNA and cellular protein, 0.5 mlO.2% Triton X-100:lN NH40H was added to each filter, the cells were scraped with a small disposable plastic scraper and the contents transferred to a 1.5 ml conical centrifuge tube. DNA and protein were determined as described above. RESULTS Cystine uptake Effect of sodium on uptake. Other model systems, such as isolated rat renal tubules (Foreman et al., 1980), dog kidney epithelial cells in primary culture (States et al., 1990), and cultured human kidney epithelial cells (States e t al., 1986, 1987) show that sodium is a n important ion for cystine handling. We, therefore, determined the effect of reduced medium sodium on cystine uptake in experiments conducted in parallel with cystine uptake in PBS. The only difference was in the substitution of a n equimolar concentration of choline chloride for NaC1. The data, plotted in Figure 1, show that OK cells rapidly take up 0.025 mM L-cystine and that the cystine uptake in the low sodium medium mimicks the uptake curve with complete PBS. Except for the 5 minute point, where there is a significant difference between distribution ratios of approximately 30% (P