Biochemical Genetics, Vol. 15, Nos. 7/8, 1977
Membrane Function in Cystic Fibrosis. I. Putrescine Transport in Normal and Cystic Fibrosis Fibroblasts JoAnn C. Kelly 1 ,z and A. Gib D e B u s k 1,3
Received 11 Nov. 1976~Final 3 Jan. 1977
Putrescine transport was examined in normal and cystic fibrosis fibroblasts. No differences were observed in accumulation pattern, kinetics of uptake, or efftux between CF and normal cells. In both growing and growth-arrested CF and normal fibrobIasts, exogenously supplied putrescine remained unchanged for at least 60 min. Some differences were observed in the response of CF and normal cells to environmental (media) changes. KEY WORDS: cystic fibrosis; putrescine; transport.
INTRODUCTION Disturbances in polyamine metabolism in cystic fibrosis (CF) have been reported. Altered levels of polyamines have been observed in the blood (Arvanitakis et al., 1973; Lundgren et al., 1975; Cohen et aL, 1975) and in cell culture medimn (Rennert et al., 1973; Lindheimer, personal communication). It is possible that the altered spermidine/spermine ratio in CF is a response to the defect. What role polyamine metabolism plays in the pathogenesis of cystic fibrosis is not known at this time. It is known, however, that glycoprotein metabolism can be influenced by spermine concentration (Baker and Hellegrass, 1974). Also, polyamines are important in bacteria for their role of maintaining a salt balance across cellular membranes (Munro This research was supported by a grant from the Cystic Fibrosis Foundation and by a grant from the National Institutes of Health, Training Grant (GM01316 11 GNC). 1 Genetics Group, Department of Biological Science, Florida State University, Tallahassee, Florida. 2 Present address: Department of Pediatrics, Division of Medical Genetics, Washington University School of Medicine, St. Louis, Missouri. 3 Address correspondence to A. G. D. 695 © 1977 Plenum Publishing Corp., 227 West 17th Street, New York, N.Y. 10011. To promote freer access to published material in the spirit o f the 1976 Copyright Law, Plenum sells reprint articles from all its journals. This availability underlines the fact that no part o f this publication may be reproduced, stored in a retrieval system~ or transmitted, i n a n y form or by any means, electronic, mechanical, ohotocoovinm microfilmin~
Kelly and DeBusk
et al., 1972). Both of these aspects are of interest in CF, for it is still unsettled as to whether or how glycoprotein metabolism is altered, and high sweat salt concentration is the most consistent chemical finding in CF--in fact, it is diagnostic. Previous studies of transport phenomena demonstrated no abnormalities in the uptake of small metabolites by CF fibroblasts. Benke et al. (1972), Fletcher and Lin (1973), and Sullivan et al. (1976) found the uptake of several amino acids and sugars to be unaltered and concluded that the transport mechanisms of the plasma membrane was probably intact in CF fibroblasts. Brown et al. (1971) found that plasma from CF patients inhibited sugar transport. However, Taussig and Gardner (1972) demonstrated that CF saliva and plasma have no effect on ion, amino acid, or sugar transport. The intent of this investigation has been to determine whether putrescine transport is altered in CF fibroblasts.
MATERIALS AND METHODS Cells Human diploid fibroblasts were used throughout this study. Cells were used immediately after receipt from the supplier, or after two to ten subcultivations in this laboratory. All cells were of passage number 8-18. Cystic fibrosis fibroblasts were obtained from the American Type Culture Collection (1139, male, 3 months; 1142, male, 3 months; 1154, female, 8 years; 1137, female, 8 years). All CF fibroblasts were derived from skin biopsy. Normal fibroblasts were obtained from Dr. Leonard Hayflick (WI38, female, fetal lung), from Dr. James Regan (HSBP, male, foreskin), and from the American Type Culture Collection (1141, male, 9 years, skin biopsy; 1121, male, 3 years, skin biopsy; 1147, female, 9 years, skin biopsy). One of the normal strains, HSBP, was derived from a Black subject. Cultivation of Cells All fibroblasts were maintained in Dulbecco's modified minimal essential medium (Morton, 1970) supplemented with 10~ v/v active fetal calf serum, 100 #g of streptomycin, and 100 units of penicillin per milliliter and 15 mmoles HEPES buffer per liter. Generally, cells were subcultivated at 1:3 split ratios every 1-2 weeks. At each subcultivation, cells were removed with 0.05~o trypsin in Hanks' balanced salt solution supplemented with 100 #g of streptomycin and 100 units of penicillin per milliliter and 15 mmoles HEPES buffer per liter. Cells were routinely maintained in 75-cm 2 Falcon flasks or 490-cm 2 Corning tissue culture roller flasks.
Membrane Function in Cystic Fibrosis
Fibroblasts were checked for mycoplasma contamination by the procedure of Schneider et al. (1974), which is based on the differential rates of incorporation of uridine and uracil by human cells and mycoplasma. Materials
Putrescine was obtained in radiolabeled form from New England Nuclear Corp. as [1,4-14C] putrescine dihydrochloride. The radiolabeled putrescine was checked for purity by paper chromatography and found to be free of contamination by other compounds. The [2-14C]uracil and [2-a4C]uridine were obtained from Schwarz/Mann. Fetal bovine serum, growth medium, and antibiotics were obtained from Grand Island Biological Company. Twice-crystallized type II bovine pancreatic trypsin was obtained from Sigma Chemical Company. All other chemicals, which in general were of the highest grade available, were obtained from Sigma Chemical Company, Calbiochem, Fisher Scientific Company, and Mallinckrodt Chemical Works. Transport Assay
The transport assay was a modification of a method used by several workers in other cell culture systems (Bose and Zlotnick, 1973; Kletzien and Perdue, 1974). With minor variations, the following protocol was used throughout. All assays were carried out at 37 C unless otherwise specified. Cells were planted in 6-cm Falcon dishes at a density of approximately 1 × l0 s cells per dish and allowed to incubate 24 hr in complete growth medium at 37 C. The dishes were rinsed with 5 x 2.0 ml tris-buffered saline (TBS) at pH 7.4 immediately prior to the initiation of transport. The buffered saline contained, per liter, 116 mEq Na +, 2.7 mEq K +, 2 mEq Mg 2+, 2 mEq Ca 2÷ (all cations added with C1-), and buffered at pH 7.4 with 20 mM tris-HC1. Transport was initiated by removing the buffered saline and adding 2.0 ml radioisotope in buffered saline. The reaction was stopped after removal of the radioisotope by rinsing with 5 × 2.0 ml TBS at 0 C. With the use of a repeating syringe and aspirator, these rinsings can be made very rapidly, in less than 0.3 rain. The washed cells were extracted with 1.0 ml and then 2x0.5 ml ice-cold 5% trichloroacetic acid (TCA). Radioactivity in the combined extracts was determined by liquid scintillation counting using internal standardization. The extracted cells were then dissolved in 1 N NaOH. Cell protein was measured by the method of Lowry et al. (1951) against a bovine serum albumin standard. Background for both protein and radioactivity measurements was taken from dishes without cells incubated in growth medium and serum and processed in the same manner as those with cells. After the G
Kelly a n d D e B u s k
appropriate conversion, uptake was expressed as nmol/mg/min. Determination of the counts remaining in the TCA-precipitated material was made by dissolving in 1.0 ml 1 N NaOH. Exactly half was taken for protein determination and half was counted in 60 min. Ninety-eight percent of the counts entering as putrescine in TBS remained in the TCA-soluble fraction.
Efliux Assay In the efltux experiments, cells were labeled for 30 min before the etttux was initiated. The efflux reaction was started after the removal of the radioisotope-containing TBS by the addition of 2.0 ml TBS. The etttux was stopped by 5 x 2.0 ml rinses of TBS at 0 C. The cells were then processed as in the transport assay.
Chromatography For the fate-of-label studies, cells labeled with [t4C]putrescine were extracted with cold 5 ~ TCA. The TCA was removed with three extractions of equal volumes of ether. After reduction of the volume, aliquots were mixed with unlabeled putrescine and assayed using ascending chromatography, as with
10 laJ I-" 0
MINUTES OF UPTAKE Fig. 1. Time course of putrescine accumulation in CF and normal fibroblasts. Uptake of [14C]putrescine in TBS, p H 7.4, at 37 C was measured at a substrate concentration of 10/tM. The cells were rinsed with TBS immediately prior to uptake. Each point represents the mean of triplicate determinations. ©, 1141 (normal); c3, HSBP (normal); lb, 1139 (CF); m, 1142 (CF); , , 1154 (CF).
Membrane Function in Cystic Fibrosis
the radiolabeled putrescine. Appropriate standards were chromatographed beside the cellular extracts. RESULTS
Some of the aspects of putrescine transport were studied in CF and normal human fibroblasts. The putrescine accumulation patterns are shown in Fig. 1. For the strains studied here, the range of the CF values was contained within the range of observed normal values. Lineweaver-Burk plots of initial rates of transport of putrescine show some degree of individual differences (Fig. 2). However, no large or obvious differences appeared which could be attributed to the mutant gene directly. Examination of the Michaelis-Menten constants (Table I) derived from Hofstee transformations supported this
Fig. 2. Lineweaver-Burk plot of initial rates of putrescine transport. Double reciprocal plots of initial rates of putrescine transport as a function of substrate concentration. Initial rates of [l~Clputrescine transport were measured in TBS, p H 7.4, at 37 C. Cells were rinsed with TBS immediately prior to uptake. Lines shown were fitted by linear regression. Each point is the mean of triplicate measurements. V is in nmol/mg/min; S is M. A, 1141 (normal); ©, 1121 (normal); A, W138 (normal); , HSBP (normal); v , 1154 (CF); 0 , 1142 (CF).
Kelly and DeBusk Table I. Initial Rate Kinetic Constants for Putrescine Transport in CF and Normal Fibroblasts"
Apparent/2,, ( x 10- s M)
Apparent Vmax (nmol/mg/min)
1141 (normal) 1121 (normal) WI38 (normal) HSBP (normal) 1154 (CF) 1142 (CF)
0.185 0.168 0.217 0.128 0.167 0.151
0.202 0.368 0.181 0.204 0.285 0.438
" Michaelia-Menten constants were derived using the Hofstee transformation.
Fig. 3. Effect of time after planting on initial rates of putrescine transport as a function of substrate concentration. Initial rates of [14C]putrescine transport were measured in TBS, p H 7.4, at 37 C at substrate concentrations of less than or equal to 10/ZM. Cells were rinsed with TBS immediately prior to uptake. Double reciprocal plots were drawn and lines shown were fitted by linear regression. V is in nmol/mg/min; S is M. A, 1141, normal, planted 22 hr; ©, 1141, normal, planted 4 days; A, 1121, CF, planted 22 hr; O, 1121, CF, planted 4 days.
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observation. The a p p a r e n t Km's o f the n o r m a l strains r a n g e d f r o m 0.128 × 10 - 5 to 0.217 × 10 -5 M, whereas the C F values were 0.151-0.167 × 10 - 5 M. The a p p a r e n t Vmax'S o f the n o r m a l strains ranged f r o m 0.181 to 0.368 n m o l / m g / m i n ; the C F values, which o v e r l a p p e d this range, were 0.285-0.438 nmol/mg/min. Initial rates o f putrescine t r a n s p o r t as a function o f substrate conc e n t r a t i o n were m e a s u r e d in C F a n d n o r m a l fibroblasts which h a d been p l a n t e d in the 6-cm F a l c o n dishes for longer t h a n the usual lengths o f time (Fig. 3). By 4 days, these cells (1) were at a higher density t h a n w h a t was s t a n d a r d a n d (2) h a d been in the same m e d i u m m u c h longer (there were no m e d i a changes over this time). Analysis o f the initial rates o f t r a n s p o r t for the kinetic constants in the n o r m a l strain at 4 days showed that the a p p a r e n t
~" 5 0 0
Fig. 4. Metabolic fate of putrescine in growing CF and normal fibroblasts. Cultures were split 1:2 into 25cm 2 Falcon flasks 12 hr prior to the experiment.~The ceils were labeled with 1/tM [14C]putrescine in growth medium for 60 rain. The cells were then washed with TBS and extracted with 5 ~ ice-cold TCA. Following ether extraction and volume reduction, an aliquot was mixed with unlabeled putrescine and chromatographed ascending on Whatman No. 1 chromatography paper in methanol-pyridine-acetic acid-water (6:6:1:4). After development, the chromatograms were cut into 1-cm strips and counted. Unlabeled spermidine, spermine, and putrescine standards were chrornatographed beside the samples. Arrow 1, spermine; arrow 2, spermidine; arrow 3, putrescine; , 1141 (normal); A, 1121 (normal); 0, 1154 (CF); ©, 1142 (CF).
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Km was unchanged from that of the controls (0.221 x 10 .5 M) and that the apparent Vmax was slightly increased (0.766 nmol/mg/min) as compared to 0.202 nmol/mg/min for 22 hr. In the CF strain (1154) after 4 days, again there was no change in the apparent Km (0.203 x 10 -s M). There was a very slight change in the apparent Vm,x (0.593 nmol/mg/min as compared to 0.285 nmol/mg/min for 22 hr). It appeared that the normal cells had a slightly greater response to the media conditions than the CF cells. Chromatography of extracts of normal and CF fibroblasts labeled for 60 rain with [1,4-~4C]putrescine dihydrochloride was performed to determine to what extent externally supplied putrescine was metabolized. These extracts were made from cells which either were growing (12 hr after a 1:2 split) (see Fig. 4) or were growth-arrested (confluent) (see Fig. 5). In both the growing and growth-arrested cultures, for 60 rain most of the
Fig. 5. Metabolic rate of putrescine in n0ngrowing CF and normal fibroblasts. Confluent cultures in 25-cm2 Falcon flasks were labeled with 1 pM [~4C]putrescine in growth medium for 60 min. The cells were then washed with TBS and extracted with 5 ~ icecold TCA. Following ether extraction and volume reduction, an aliquot was mixed with unlabeled putrescine and chromatographed ascending on Whatman No. 1 chromatography paper in methanolpyridine-acetic acid-water (6:6:1:4). After development, the chromatograms were cut into 1-cm strips and counted. Unlabeled spermidine, spermine, and putrescine standards were chromatographed beside the samples. Arrow 1, spermine; arrow 2, spermidine; arrow 3, putrescine; , 1141 (normal); A, 1121 (normal); O, 1154 (CF); ©, 1142 (CF).
Membrane Function in Cystic Fibrosis
Fig. 6. Putrescine efflux in CF and normal fibroblasts. Efflux of [14C]putrescine into TBS, pH 7.4, was measured at 37 C. The cells were labeled for 30 min with 10/tM putrescine in TBS, pH 7.4, immediately prior to efftux. Each point is the mean of triplicate determinations. I1, 1144 (normal), 1141 (normal), 1139 (CF), 1154 (CF), 1142 (CF); ©, HSBP (normal).
externally supplied putrescine (95%) remained unchanged in both CF and normal fibroblasts. Uptake measurements in this study all involved labeling times of less than 60 min; therefore, no corrections for metabolic conversion were necessary. The rate of exit of exogenously supplied putrescine was examined in CF and normal fibroblasts. The etttux for putrescine in CF and normal fibroblasts are shown in Fig. 6. There was no loss of putrescine in 15 min by any of the cells which had been derived from skin biopsies, both CF and normal. Interestingly, the one strain (HSBP) which did show some slight efflux (10% in 15 rain) was derived from foreskin from the only known Black in this study. This difference could have been due either to the effect of the site of culture origin or to the genetic background. DISCUSSION Although there has been a considerable amount of work on the polyamines in various mammalian systems, there have been very few studies on the uptake of the polyamines by mammalian cells (Kano and Oka, 1976; Pohjanpelto, 1976). Studies in bacteria and lower organisms have shown the existence of active transport systems for the polyamines (Munro et al., 1974; Johnson and Bach, 1968; Tabor and Tabor, 1966). This present study demonstrated a similar transport system in human fibroblasts. At plasma concentrations, uptake was probably mediated by the active transport system. Polyamine concentrations have been examined in many animal tissues. Marked variation has been found in various tissues in total polyamine concentration as well as relative concentration of the different
Kelly and DeBusk
polyamines (Tabor and Tabor, 1964, 1972). The highest concentration of polyamines in humans was found in semen, 12-15/~mol/ml (Rosenthal and Tabor, 1956; Williams-Ashman and Lockwood, 1970). The apparent Km of the active transport system correlated well with plasma concentrations reported in the literature (McEvoy and Hartley, 1975). Putrescine transport was examined in several CF and normal strains. There were no significant differences in the accumulation patterns, kinetic analysis, etttux, or fate of exogenously supplied putrescine. Therefore, it appears that the mechanism of putrescine transport is intact in cystic fibrosis cells. The transport of putrescine was examined in growing and nongrowing cells. Under both of these conditions, it was observed that the metabolic fate of exogenously supplied putrescine was the same; i.e., 95% remained as putrescine for 1 hr. However, when cells were allowed to remain in unchanged medium until a high density was obtained, kinetic analysis showed an increase in the apparent Vmax.Thus it was demonstrated that it was possible for media components to alter the rate of transport, but not the K,, or metabolic fate. Such a system may prove useful in the study of some diseases where certain metabolites are produced (or overproduced). Cystic fibrosis is such a disease. Indeed, the CF cells, in this case, appeared to be less sensitive to the environment (media). Although performed with only one CF and one normal strain, this experiment suggested that it may be useful to further examine the effect of media components and factors produced into the media by CF fibroblasts On polyamine transport. Such follow-up studies are currently under way in this laboratory. Polyamine transport may eventually prove a useful tool in the furthering of our understanding of the action of the CF factors on cell membranes. ACKNOWLEDGMENTS
The authors would like to thank Dr. Ben Andrean and Mrs. Carmen Chaviano for their help in the preparation of the manuscript.
Arvanitakis, S. N., Mangos, J. A., McSherry, N. R., Rennert, O. M., and LaPointe, D. (1973). Role of polyamines in cystic fibrosis. Pediat. Res. 7:336. Baker, A. P., and Hellegrass, L. M. (1974). Effects of polyamines on glycosyltransferases. Fed. Proe. 33:1300. Benke, P. J., Erbstoeszer, M., and Pitot, H. C. (1972). Transport of labelled compounds in control and cystic fibrosis cells in vitro. Lancet 1:182. Bose, S. K., and Zlotnick, B. J. (1973). Growth and density-dependent inhibition of deoxyglucose transport in Balb 3T3 cells and its absence in cells transformed by murine sarcoma virus. Proc. Natl. Acad. Sci. 70:2374.
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Brown, G. H., Oshin, A., Goodchild, M. C., and Anderson, C. M. (1971). Inhibition of sugar transport by plasma from cystic fibrosis patients. Lancet 2:639. Cohen, L. F., Farrell, P. M., Willison, J. W., and Lundgren, D. W. (1975). Localization of spermidine (Spd) and spermine (Spin) in blood of cystic fibrosis (CF) and control subjects. Pediat. Res. 9:312. Fletcher, D. S., and Lin, T. Y. (1973). Incorporation of L-leucine and D-glucosamine into skin fibroblasts derived from CF and normal individuals. Clin. Chim. Acta 44"5. Johnson, H. G., and Bach, M. K. (1968). Uptake and subcellular localization of tritiated spermine in Eseherichia coll. Arch. Biochem. Biophys. 128:113. Kano, K., and Oka, T. (1976). Polyamine transport and metabolism in mouse mammary gland: General properties and hormonal regulation. J. Biol. Chem. 251:2795. Kletzien, R. F., and Perdue, J. F. (1974). Sugar transport in chick embryo fibroblasts. I. Functional change in the plasma membrane associated with the rate of cell growth. J. Biol. Chem. 249:3366. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265. Lundgren, D. W., Farrell, P. M., and diSant'Agnese, P. A. (1975). Polyamine alterations in blood of male homozygotes and heterozygotes for cystic fibrosis. Clin. Chim. Acta 62:357. McEvoy, F. A., and Hartley, C. B. (1975). Polyamines in cystic fibrosis. Pediat. Res. 9:721. Morton, H. J. (1970). A survey of commercially available tissue culture media. In Vitro 6:89. Munro, G. F., Hercules, K., Morgan, J., and Sauerbier, W. (1972). Dependence of the putrescine content of Escherichia coli on the osmotic strength of the medium. J. Biol. Chem. 247:1272. Munro, G. F., Bell, C. A., and Lederman, M. (1974). Multiple transport components for putrescine in Escherichia coli. J. Bacteriol. 118:952. Pohjanpelto, P. (1976). Putrescine transport is greatly increased in human fibroblasts initiated to proliferate. J. Cell Biol. 68:512. Rennert, O. M., Frias, J., and La Pointe, D. (1973). Methylation of RNA and polyamine metabolism in cystic fibrosis. In Mangos, J. A., and Talamo, R. C. (eds.), Fundamental Problems o f Cystic Fibrosis and Related Diseases, Intercontinental Medical Book Corp., New York. Rosenthal, S. M., and Tabor, C. W. (1956). The pharmacology of spermine and spermidine distribution and excretion. J. Pharmacol. Exp. Ther. 116:131. Schneider, E. L., Stanbridge, E. J., and Epstein, C. J. (1974). Incorporation of 3H-uridine and all-uracil for the detection of mycoplasma contamination of cultured cells. Exp. Cell Res. 84:311. Sullivan, J. L., Kelly, J. C., Roess, W. B., and DeBusk, A. G. (1976). Methionine transport in fibroblasts from cystic fibrosis patients. In preparation. Tabor, C. W., and Tabor, H. (1966). Transport systems for 1,4-diaminobutane, spermidine and spermine in Escherichia coli. J. Biol. Chem. 241:3714. Tabor, H., and Tabor, C. W. (1964). Spermidine, spermine and related amines. Pharmacol. Rev. 16:245. Tabor, H., and Tabor, C. W. (1972). Biosynthesis and metabolism of 1,4-diaminobutane, spermidine, spermine and related amines. Adv. Enzymol. 36:203. Taussig, L. M., and Gardner, J. D. (1972). Effects of saliva and plasma of CF patients on membrane transport. Lancet 1:1367. Williams-Ashman, H. G., and Lockwood, D. H. (1970). Role of polyamines in reproductive physiology and sex hormone action. Ann. N. Y. Acad. Sci. 171:882.