Effects of bromosulfophthalein on structure and function of the plasma membrane of S37 ascites tumor cells

Bioelectrochemistry and Bioenergetics, 10 (1983) 75-85 A section of J. Electroanal. Chem., and constituting Vol. 155 (1983) Elsevier Sequoia S .A ., Lausanne - Printed in The Netherlands

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531-EFFECTS OF BROMOSULFOPHTHALEIN ON STRUCTURE AND FUNCTION OF THE PLASMA MEMBRANE OF S37 ASCITES TUMOR CELLS

RICHARD H. MATTHEWS and TERESA K . PICONE Department of Physiological Chemistry, The Ohio State University, Columbus, OH 43210 (U.S.A .) ABRAMO C. OTTOLENGHI Department of Medical Microbiology and Immunology, The Ohio State University, Columbus, OH 43210 (U.S.A.) (Revised manuscript received September 1st 1982)

SUMMARY Bromosulfophthalein had been reported to inhibit the y-glutamyl cycle . The 7-glutamyl cycle has been implicated in amino acid transport in mammalian cells . Bromosulfophthalein was therefore employed in an attempt to correlate function of the y-glutamyl cycle with amino acid transport by two well-defined ascites cell amino acid transport systems . Inhibition of both transport systems occurred in the presence of 1 mM bromosulfophthalein . However, further studies suggested that this inhibition was associated with a functional and morphological alteration of the plasma membrane. The S37 cells had an increased non-specific permeability demonstrated by sulfate . Steady state retention of amino acids was decreased and 51 Cr release was increased by bromosulfophthalein. Vital staining was also increased by bromosulfophthalein and electron micrographs revealed membrane solubilization in the presence of bromosulfophthalein .

ABBREVIATIONS

BSP = bromosulfophthalein ; co = extracellular concentration of a test solute ; v = initial velocity of uptake of a test solute ; NMeAIB = N-methyl-a-amino isobutyric acid ; EDTA = ethylene diamine tetraacetic acid . INTRODUCTION

Bromosulfophthalein (Fig. 1) has been used routinely in a liver function test [1] . This compound has been shown to be conjugated to glutathione by an enzyme present in rat liver [2]. It has also been suggested that bromosulfophthalein is an inhibitor of y-glutamyl transpeptidase [3,4]. This was of interest in that y-glutamyl transpeptidase is an enzyme in the y-glutamyl cycle, which has been postulated to be the energetic 0302-4598/83/$03.00

0 1983 Elsevier Sequoia S.A.

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support for amino acid transport in mammalian tissues, especially brain and kidney [5]. The postulated support of amino acid transport by y-glutamyl transpeptidase has been strengthened by demonstration of amino acid transport in reconstituted

10 OH Fig . 1 . The structure of bromosulfophthalein .

lecithin vesicles containing this enzyme [6] . This laboratory had previously reported that dihydroorotic acid, an inhibitor of 5-oxoprolinase, and L-methionine sulfoximine, an inhibitor of y-glutamyl cysteine synthetase, both inhibited function of the L and A transport systems for neutral amino acids in S37 ascites tumor cells . We also reported that glutathione reversed certain inhibitions of transport through the L and A transport systems [7] . The aforementioned data was consistent with the support of the L and A transport systems by the y-glutamyl cycle, and the object of the present study was to test further the proposed relationship between amino acid transport systems L and A and the y-glutamyl cycle by employing an inhibitor of the enzyme directly engaged in the transfer of incoming amino acids . Bromosulfophthalein did indeed inhibit amino acid uptake through transport systems L and A, but the basis of the effect was more general than inhibition of a specific enzyme . EXPERIMENTAL

General

Preparation of S37 cells, preparation of media and liquid scintillator, and general procedures for the execution of amino acid transport experiments have been described in detail previously [8] . Chemicals

Sulfobromophthalein (bromosulfophthalein) and L-histidine were obtained from Sigma Chemical Co. ; NMeAIB was obtained from Aldrich ; [ 3 H]-L-histidine and [ 14 C]-NMeAIB were purchased from New England Nuclear . [ 35 S]-sulfate was purchased from Amersham . Solutions were sterilized by ultrafiltration .

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Sulfate exclusion space A pool of S37 cells was divided ; one-half was incubated in the absence and the other half in the presence of 1 mM bromosulfophthalein for 30 minutes at 20°C . After washing the cell preparations, each of the preparations was divided in fifths, aliquots being added to tared centrifuge tubes, supernates removed after centrifuging, and pellets suspended in 8 cm3 of chilled Krebs-Ringer buffer containing [ 35 S]-sulfate . Following 1-minute centrifugations at 1940 X g, 50 mm3 (µl) aliquots of supernates were taken for liquid scintillation counting and the excess supernatant liquid removed by siphoning. Cell pellets were packed by a second 1-minute spin at 1940 X g and wet weights determined . All pellets were then resuspended in 8 cm3 of chilled Krebs-Ringer buffer lacking any label . After centrifuging for 1 minute at 1940 x g, a 500 mm3 aliquot of each second supernate was counted . The sulfate space was computed as x = [8 x R 2 X 1 .1]/[10 X R 1 - 1 .1 X R 2 ] where R, was the net c.p .m . associated with the 50 mm3 aliquot of the first supernate, R 2 was the net c .p .m . associated with the 500 mm3 aliquot of the second supernate, and the factor 1 .1 was associated with the degree of quenching . The sulfate space for each sample was divided by the wet weight to obtain a fraction regarded as apparent extracellular space for each pellet . Labeling of cells with S1 Cr The methodology for the "Cr labeling of cells was adapted from that used by Berke and co-workers [9] . [ 51 Cr]-chromate was obtained from the nuclear pharmacy of the O .S .U. Hospitals . 10 7 S37 cells were labeled by incubation for 30 min at 37°C with 50 µ Ci of [5 1 Cr]-chromate in a medium volume of 2 cm3 . The incubation was terminated by addition of 40 cm3 of cold phosphate-buffered saline with 10% fetal calf serum . Cells were centrifuged 10 min at 800 X g and resuspended in 40 cm3 cold phosphate-buffered saline with 10% fetal calf serum . After a second wash under the same conditions, the cells were incubated in 40 cm3 phosphate-buffered saline with 10% fetal calf serum for 30 min at 4°C and again centrifuged as above. Vital staining (Trypan blue) was done to check for dead cells in the labeled preparation . Labeled S37 cells were then mixed with unlabeled S37 cells . S37 cell preparations were then incubated with the indicated concentrations of BSP at 37°C or at the indicated temperatures in 0 .5 mM BSP for 30 min . Cells were pelleted at the end of the incubation period and radiolabel in both supernates and pellets was determined by gamma counting to estimate the percent release of 51 Cr. Results were computed as per cent of total label . Membrane protein solubilization Ghosts of S37 cells were prepared by treating 1 .5 X 10 9 cells according to the procedure developed for red blood cells [10] . The ghost preparation was divided into 5 equal portions . Each portion was incubated 30 min at 37 ° C in the presence of 0.5

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mM BSP in 20 mM phosphate buffer adjusted to the indicated pH values in total volume of 10 ml . 2 cm3 aliquots from each of the media were taken for analysis of total protein. The remaining material was centrifuged 20 min at 32000 x g and the supernate analyzed to determine solubilized protein . Protein was assayed by the fluorescamine assay described by Udenfriend et al . [11] . Fluorescamine solution in acetone was prepared fresh (30 mg/l00 cm3 ). Bovine plasma albumin was used as a standard . The pH of the 0.4 M borate buffer was adjusted to 8 .3 to avoid quench problems. Two cm3 aqueous samples were mixed with 2 cm 3 of buffer. Two cm3 of fluorescamine solution was added to each sample and mixed immediately. Excitation was at 390 nm ; emission was read at 475 nm using an Aminco-Bowman spectrophotofluorometer . Preparation of samples for electron microscopy

The cells were incubated for 30 min at 37 °C in Krebs-Ringer buffer with no BSP or 0 .25 mM, 0.50 mM and 1 .0 mM with respect to BSP . The pH was adjusted to 7.4 . The samples were then suspended for 3 hours in 2% gluteraldehyde in cacodylate-sucrose buffer (0.1 M cacodylate, 3 .42 g/100 cm3 sucrose, pH 7 .2) and following centrifugation, resuspended for 1 hour in the cacodylate-sucrose buffer . After another change in buffer, the sample was allowed to stand overnight at 4°C . Following centrifugation, the pellet was suspended in a drop of buffer and one drop of 4% molten agar added . The sample was postfixed in 1 % OsO 4 in cacodylate-sucrose buffer . Following dehydration through graded alcohols and propylene oxide, the pellets were embedded in . Epon . Sections were viewed with a Hitachi Hu 12 electron microscope . Representative areas were photographed to include both apparently intact and non-viable cells . RESULTS

Inhibition of amino acid transport : the initial velocity experiment

It was previously demonstrated that uptake of histidine across a broad concentration range into S37 cells gave a biphasic double-reciprocal plot [12]. It was further shown that the system dominant in the high-concentration region, near the origin of the double-reciprocal plot, was transport system A for neutral amino acids and that the system dominant in the low-concentration region, more distant from the origin of the double-reciprocal plot, was transport system L for neutral amino acids [13] . Deflections of one or both limbs of this biphasic double-reciprocal plot have been used in the elucidation of characteristics of the two transport systems for neutral amino acids [12-141. In the present study, preincubation with 1 mM bromosulfophthalein caused a marked deflection, both in slope and elevation of the two limbs of the biphasic double-reciprocal plot (Fig. 2) . This established inhibition of apparent function of both transport systems L and A by bromosulfophthalein .

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Fig . 2. The effect of bromosulfophthalein upon amino acid transport in S37 cells . S37 cells were first incubated in the presence (o) or absence (0) of 1 mM bromosulfophthalein for 30 min at 20°C . After washing and resuspending the cells, they were incubated for 2 min at 20°C in the presence of varying concentrations, c° (mM), of 3 H-histidine. The initial velocity of uptake, v (mM/min), was estimated by dividing the intracellular concentration of 3 H-histidine determined subsequent to the 2-min incubations by 2 min. Repetition of the experiment yielded similar results .

Inhibition of amino acid transport : the steady-state experiment The steady-state experiment (Fig . 3) indicated that inhibition of transport and retention of amino acids was very significant at 0 .5 mM BSP and was virtually MM [ 3 H]-histidine reprecomplete at 1 .0 mM BSP. Uptake and retention of 0 .1 sented activity of system L predominantly, whereas uptake and retention of [ 14 C]-NMeAIB was specific to system A [14] . The inhibitory effects of BSP on retention by the two transport systems showed a similar dependence upon the concentration of BSP . Inhibition of amino acid retention : endogenous amino acids Treatment of washed S37 cells with increasing concentrations of BSP caused progressive increases in the levels of endogenous amino acids released to the medium, and corresponding decreases in the levels of amino acids subsequently found in lysates of the cells (Fig. 4) . Effects of BSP on non-specific tests of membrane function BSP caused a significant increase in the sulfate space of the cell pellet from 6% to 32% of the pellet. This increase indicates entry of sulfate into cells. BSP also



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Fig. 3. BSP inhibition of steady-state labeled amino acid retention . S37 cells were incubated for 30 min at 37°C in the concentrations of BSP indicated . 0 .1 MM 3 H-L-histidine (0) or 1 MM 14 C-NMeAIB (o) was also present in each medium. Individual points were means of 3 determinations and repetition of the experiment yielded a similar result.

[a 5 0.25

P] (mM) 1 .0

1 .5

2 .0

Fig . 4. BSP effect on endogenous amino acid retention . S37 cells were exposed to the indicated concentrations of BSP for 30 min . a t 37° . Cells were then separated from the media by centrifugation and lysed with 30 µM EDTA . Total amino acids were measured in the media (0) and in the cell lysates (n) by the ninhydrin procedure of Rosen [15] .



81 increased the release of [51Cr] from S37 cells (Fig . 5). This effect showed a concentration dependence similar to the previous studies on amino acid retention . There was a definite increase in flux at 0 .5 mM BSP and the effect was nearing completion at 1 .0 mM BSP. The release of [51Cr] from S37 cells in the presence of BSP was temperature-dependent increasing from 9% to 28% as temperature was raised from 6° to 37°C . Increasing BSP concentrations also increased the vital staining of S37 cells from 9% to 75% . The vital staining increase began when BSP concentration attained 0 .5 mM . These tests all suggested an increased general permeability of the plasma membrane when it was exposed to 0 .5 mM BSP. pH Modulation of BSP effects BSP (Fig . 1) possesses titratable phenolic moieties and has a pK of 8 .8 [1]. The possible modulation of BSP effects by pH was therefore examined . Steady-state retention of labeled amino acids was decreased at lower pH values (Fig . 6). However, the pH effect was slight on endogenous amino acid release (Fig . 7) and suggested that elevated pH was associated with more release of amino acids . This aspect of the study was further complicated-by the finding that release of membrane proteins underwent a minimum at neutral pH (Fig . 8) . No simple and consistent conclusion was directly suggested by the pH variations . Morphology Examination of the electron micrographs (Fig . 9) revealed that treatment of the cells at the three higher concentrations of BSP (0 .5 mM to 1 mM) caused visible 100

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0

0 .2

I I 1 0 .6 1 .0 1 .4 1 .8

Fig. 5, BSP effect on 51 Cr release from S37 cells . For experimental details, see the text .



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o 3 H-Histidine ,& 14C-NMe AIB

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1 .0

12 (m#) /~

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10

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/

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/

v //

0 .8

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/

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8

6

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0 .4

4

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5

6

7

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9

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Fig . 6 . Effect of pH on amino acid retention with BSP present . S37 cells were incubated with 0 .1 mM [ 3 H]-L-histidine (0) or 1 MM [ 14 C]-NMeAIB (n) present for 30 min at 37°C . 0 .5 mM BSP was present in all cases . Individual points were mean values from two experiments done in triplicate . 1.2

1 .0

0.8 -t o Supernate

n Lysate 0.6

0.4

0.2

pH 0 1 ' 1

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5 6 7 8 9 Fig. 7. BSP release of endogenous amino acids as a function of pH . S37 cells were incubated in media containing 0.5 mM BSP for 30 min at 37°C with the pH adjusted as indicated . Endogenous amino acids were determined in the media (0) and the lysates (n) as for Fig . 4.

83

80

v

N

C 4J_ ~. 7 ao

60

o

40

20

0

6

7

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Fig . 8. BSP release of membrane proteins as a function of pH. For experimental details, see the text .

Fig. 9 . Effect of BSP on membranes of S37 mouse ascites tumor . Cells incubated in Krebs-Ringer buffer for 30 min at pH 7.4 . BSP concentration : (A) 0, (B) 0.50, (C) 1 .0 mM. All bars represent 0 .1 µm.

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changes in the structure of the cell membrane . When the appearance of the control membrane (Fig . 7) was compared with that seen following BSP treatment (Fig . 9), the diffuseness of the latter was apparent . The cells obtained from the mice consisted of two morphological types, a mononuclear cell and a cell with a multilobulated nucleus and containing many granules. Treatment with BSP did not appear to disintegrate the cells with the multilobulated nuclei as readily as the cells with the large single lobule nucleus . DISCUSSION

BSP is a potential detergent in that it contains both hydrophobic (the tetrabromobutyrolactone) and polar phenolic and sulfonic acid functions . That BSP interacts with the S37 membrane is evidenced by the observation that when S37 ghost membranes are incubated with BSP, extensively washed and the pH adjusted to 9, the membranes assume the coloration characteristic of BSP at that pH . The observed release of "Cr and endogenous amino acids, as well as the increased dye uptake indicate that modifications are occurring in the general permeability of the cell membranes when S37 cells are subjected to concentrations of BSP in excess of 0 .25 mM. Further evidence of the probable role of BSP as a detergent is the increased release of 51 Cr which results from increasing the incubation temperature of the BSP treated cells . Increased temperature increases the fluidity of membrane bilayers and thus facilitates detergent action . The electron microscopy results also indicate that the membranes of S37 cells became altered as a result of treatment with BSP . The pH experiments do not provide a clear-cut and simple interpretation . This could be due to multiple effects of pH variation . Release of endogenous amino acids (Fig. 7) appeared slightly greater at high pH, whereas inhibition of labeled amino acid transport appeared greater at low pH (Fig. 6). The release of endogenous amino acids at high pH could be associated with a more effective action of the high pH form of BSP. The rationalization of its low pH effect on labeled amino acid transport would be to suggest that superimposed on the titration of BSP are inhibitions of system A and of histidine uptake through system L at low pH [16]. The evidence presented suggests that general functional damage to the plasma membrane occurs at a BSP concentration above 0 .25 mM. Impairment of amino acid transport and retention through both systems A and L follows similar concentration dependencies on BSP . This suggests that the observed inhibition of amino acid transport and retention could be associated with a general deleterious effect on the plasma membrane rather than with inhibition of a specific enzyme . It had been suggested that BSP inhibited y-glutamyl transpeptidase and also amino acid transport in intestinal epithelial cells . The effective concentration range was 0 .25-2 .0 mM [ 17]. The uptake of BSP by liver parenchymal cells is a specific process which is saturable at a concentration of 0.025 mM, an order of magnitude below the concentrations eliciting effects on S37 cells or on epithelial cells [181 . We conclude

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that BSP acts as a detergent and suggest that it would be hazardous to employ it in testing an amino acid transport mechanism . ACKNOWLEDGEMENTS

We appreciate the conscientious technical assistance of Misses Cheryll Buzby, Shelly Hall, Barbara Larger, and Rebecca McClaren . This work was supported in part by Grant CA 17925 from the National Institute of Health . REFERENCES I R.J. Henry, D.C . Cannon and J.W . Winkelman, Clinical Chemistry . Principles and Technics, 2nd ed ., Harper and Row, New York, 1974, pp . 1015-1019 . 2 R.F. Troxler, R. Greco and R. Lester, Clin. Chim. Acta, 49 (1973) 201 . 3 F. Binkley, J . Biol . Chem ., 236 (1961) 1075. 4 K. Tanaka and J . Carpenter, Fed . Proc . Fed . Am. Soc . Exp . Biol ., 34 (1975) 557 . 5 A. Meister, Science, 180 (1973) 33 . 6 S.C . Sikka and V .K. Kabra, J. Biol. Chem., 255 (1980) 4399 . 7 R.H. Matthews and M . Sardovia, Biophys. J., 15 (1975) 31 la . 8 R.H. Matthews and R . Zand, Biochemistry, 16 (1977) 3820 . 9 G. Berke, K .A. Sullivan and B. Amos, J. Exptl . Med., 135 (1972) 334 . 10 A.C . Ottolenghi and M .H. Bowman, J . Membr. Biol ., 2 (1972) 180 . 11 S. Undenfriend, S . Stein, P . Bohlen, W . Dairman, W. Leimgruber and M . Weigele, Science, 178 (1972) 871 . 12 R.H . Matthews, C .A . Leslie and P .G. Scholefield, Biochim . Biophys . Acta, 203 (1970) 457 . 13 R.H . Matthews, Biochim . Biophys. Acta, 282 (1972) 374 . 14 R.H. Matthews, M . Sardovia, N .J. Lewis and R. Zand, Biochim . Biophys. Acta, 394 (1975) 182 . 15 H. Rosen, Arch . Biochim. Biophys., 67 (1957) 10. 16 W .B. Im and H .N . Christensen, Biochim . Biophys. Acta, 455 (1976) 144. 17 T.Q. Garvey, P.E. Hyman and K.J . Isselbacher, Gastroenterology 71 (1976) 778 . 18 C .F .A . Van Bezooijen, T . Grell and D .L . Knock, Biochim. Biophys . Res. Commun ., 69 (1976) 354.