Multiple effects of ouabain on BHK cells

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Experimental Cell Research 74 (1972) 201-206

MULTIPLE

EFFECTS OF OUABAIN

T. F. MCDONALD, Carnegie Institution

ON BHK CELLS

H. G. SACHS, C. W. M. ORR and J. D. EBERT

of Washington, Department of Embryology,

Baltimore,

Md 21210, USA

SUMMARY The effects of ouabain on BHK cells have been investigated. Ouabain (4 :: 1O-4 M) inhibited growth, reduced the incorporation of SH-thymidine and 14C-amino acids, promoted a loss of cell potassium and gain of sodium, and reduced the membrane potential. Ouabain (1 x lo--* M) had very little effect while 2 x 1O-4 M ouabain had an intermediate effect. ATP content was not reduced after 24 h ouabain treatment except at a dose of lOAs M. The recovery from ouabain treatment was dependent on the concentration of ouabain and the period of exposure.

During studies on the relationship between external potassium concentration and growth in baby hamster kidney (BHK) cells [I, 21, it was necessary to employ ouabain as a means of changing the transmembrane ionic balance and lowering the membrane potential. Despite the widespread use of ouabain as an inhibitor of the Na+-K+ATPase system, few studies have looked at other direct or indirect effects on cultured cells. The intent of this study was to examine the effects of ouabain on BHK cell growth, macromolecular synthesis, ATP content, intracellular concentrations of sodium and potassium, and the cell membrane potential. Since the dose of ouabain required to produce changes in the transmembrane ionic balance varies both among species [3, 41 and among cell types in any particular organism [5], we have examined the effects of a wide range of ouabain concentration upon the abovementioned parameters. In addition, data is presented on the time- and dose-dependent recovery of BHK cells from ouabain.

MATERIALS

AND

METHODS

Cells A line of BHK-21 fibroblasts has been maintained in this laboratory by serial stationary culture of stock originally obtained from Dr H. V. Aposhian.

Media The cell line is maintained in Eagle minimal essentia1 medium supplemented with 5% tryptose phosphate broth and 10% calf serum (MEMSIO) in an atmosphere of 5 % CO8 in air at 37°C. Ouabain media were made by the addition of ouabain (Schwarz/Mann) to MEM510.

Growth experiments Cells were trypsinized from dense cultures and seeded on plastic Falcon tissue culture dishes (60 mm diameter) at 4 x lo5 cells/plate. All cells were fed 24 h later. Cells were removed from the plates with 0.25 % trypsin (trypsin, 1 : 250, Nutritional Biochemicals) and the density determined by counting an appropriate dilution in a hemocytometer. All results are the means of two replicate plates.

Sodium and potassium determinations Complete details are provided elsewhere [2]. Briefly, cells were grown on 150 mm diameter plastic dishes (Falcon), removed by trypsinization, briefly centrifuged and resuspended at 2-3 x 10’ cells/ml. Aliquots (0.3 ml) were transferred to small-bore polyethylene centrifuge tubes, centrifuged for 3 min at 1 200 g. Exptl Cell Res 74 (1972)

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Fig. 1. Abscissa: time (h); ordinate: cell number (x 10-S). The effect of ouabain on BHK cell growth. Incubation in ouabain medium commenced 24 h after seeding (arrow). l , Control; o, 1 x 10-4M ouabain; n , 2 x 10-4M ouabain; q , 4 x 10-4M ouabain. and the supernatant removed. The tip of the tube was cut off and the cell pellet extruded into a low-alkali test tube. Cells were digested with cont. HN03, and sodium and potassium analysed on a flame photometer (IL moclel 143). Water content was expressed as the difference between wet and dry weights. Intracellular concentrations of sodium and potassium (mmoles/kg cell water) were calculated following the subtraction of extracellular ions and water from the total ion and water content of the cell pellet. The extracellular space of cell pellets, determined with 14C-inulin or 14C-sucrose,was 27.0 ml/100 g wet pellet [2].

Electrophysiology

RESULTS The effect of ouabain on cell multiplication was investigated over the range lo-’ M to 4 x lop* M. At concentrations below 2 X 1O-4 M there was little or no inhibition of growth during the 72 h observation period (fig. 1). At 4 x 10~~ M ouabain there was no increase in cell number while at 2 x 1O-4 M the doubling time was lengthened from 20 to 28 h. The incorporation of 3H-thymidine and 14Camino acids into the acid-insoluble fraction was measured in BHK cells treated with ouabain. Cells were pulsed for 15 min at various times during a 24 h incubation. The results are shown in fig. 2. At 2 x 10d4 M ouabain, the incorporation of both labels was within 15 Y0 of control. At 4 X 10h4 M ouabain, the incorporation of both labels declined to about 30 % of control within 12 h. It may be noted that the incorporation of 14C-amino acids declined rapidly during the first 3 h in comparison with the slow decline of 3H-thymidine incorporation. Distinct morphological changes were associated with ouabain treatment. These changes became more pronounced with increased

Full details are provided elsewhere [2, 61. Membrane potentials were measured in cells on 35 mm plastic dishes (Falcon) at 37°C under an atmosphere of 10 % OJ80 % N&O % CO,. The intracellular electrode was filled with 2 M KC1 by the method of Tasaki et al. [7]; the indifferent electrode, was filled with an isotonic balanced salt solution using the same technique.

ATP determination The ATP content of cells was determined with luciferin-luciferase using the procedure of Chapman et al. [8]. Determinations were made on extracts of approx. 5 x lo6 cells [2].

Pulse labeling experiments A measure of the rates of DNA synthesis and protein synthesis was obtained by incubating the cells with 0.2-0.5&i/ml SH-thymidine (spec. act. 16.4Ci/mmole, SchwarziMann) or with 0.1 &i/ml 14C-amino acids (spec. act. 5i mCi/mAtok &bon, Amersham/ Searle). Full details are nrovided elsewhere Il. 21. The TkA-insoluble fraction was counted in a P&a& liquid scintillation counter, and was usually 1O5-lOa cpm/plate. Exptl Cell Res 74 (1972)

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Fi... 2. Abscissa: time (h); ordinate: % control (acidinsoluble cpm/cell). The effect of ouabain on the incorporation of (a) SH-thymidine and (b) 14C-amino acids in BHK cells. Cells were seeded at 4 x lo5 cells/plate and ouabain incubation commenced 24 h later. Control cells and ouabain cells were pulsed for 15 min at the times indicated, and values are the means of duplicate plates. 0, 2 x 10-4M ouabain; l , 4 x 10-4M ouabain.

Ouabain and BHK cells

F!p. 3. Photomicrographs of BHK cells: (a), normal; (b), after 24 h in medium containing 4

duration of exposure and concentration of the drug. A comparison of BHK cells in normal medium and after 24 h in 4 x 1O-4 M ouabain medium is presented in fig. 3. The ouabaintreated cells show signs of cytoplasmic vacuolation as well as a marked increase in filapodial extensions. An electron microscopic examination of ouabain-treated cells (kindly provided by Dr J. Rash) indicated that the cytoplasm and nucleus were essentially normal. However, these cells appeared to have an increased number of mitochondria and lipid deposits. It was possible that the effect of ouabain on BHK cell growth and macromoleclular synthesis was related to a disturbance in energy metabolism. As an indication of cell metabolic state, the ATP content of BHK cells was determined following incubation with I ji 10d4 M to 1 x 1O-3 M ouabain. The

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results are summarized in table 1. Control cells had an ATP content of 2.63 j-O.21 nmoles,’ lo6 cells (mean j.S.E., n =4). At doses of 1, 2 and 4 x 10~” M ouabain there was no decline in ATP content. ATP content uas reduced by about one-third when the dose was raised to 10 3 M. BHK cells incubated for 24 h in medium containing 1, 2 or 4 X 1O-4 M ouabain were analysed for water content and intracellular concentrations of sodium and potassium. The results are shown in table 2. The water content of ouabain-treated cells was not significantly different from control at any dose. After 24 h in 1 x lo-” M ouabain there was only a slight increase in intracellular sodium and a slight decrease in intracellular potassium. BHK cells incubated with 4 i 10 -J M ouabain gained 118.2 mM sodium and lost 120.7 mM potassium. At the intermediate Exptl Cell Res 74 (1972)

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Table 1. The effect of ouabain on the ATP content of BHK cells ATP content was measured after 24 h incubation in ouabain medium Values for control and 4 x lo-* M ouabain are mean +S.E. (n =4). All other values are means from 2 experiments Ouabain concentration

ATP content (nmoles/106 cells)

Control 1 x 1O-4 M 2 x 1O-4 M 4 x 10m4M 1 x 1O-3M

2.63 +0.21 2.13 2.55 2.47 kO.25 1.74

concentration of 2 x 1O-4 M, cells gained 60.6 mM sodium and lost 66.9 mM potassium. From these results it may be concluded that the loss of potassium and gain of sodium in ouabain treated BHK cells proceeds on a 1 : 1 basis. The recovery of BHK cell growth from ouabain treatment is illustrated in fig. 4. Two parameters were investigated: the recovery after exposure to 2, 3 or 4 x 10~~M ouabain for 24 h (fig. 4a), and the recovery after 6, 12 or 24 h at 4 x 1O-4 M ouabain (4b). After 2 x 10d4 M ouabain for 24 h, cell growth resumed at control rates. A further increase in concentration to 3 or 4 x 1O-4M for 24 h resulted in a decreasedrate of growth after washout of the drug (three changes of medium). When cells were incubated with 4 X 10e4 M ouabain for 6 or 12 h, growth resumed at a normal rate. The incorporation of 3H-thymidine and 14C-amino acids was measured in BHK cells incubated for 24 h in 4 x 1O-4M ouabain and subsequently for 6 h in control medium. Cells were pulsed for 15 min at the times indicated in fig. 5. As shown previously, the incorporation of both labels declined to about 25 % control within 12 h and thereafter stabilized at this level. Following washout of the drug (arrow), label incorporation recovered to 5&60 y0 control after 6 h. Exptl Cell Res 74 (1972)

The intracellular potassium concentration and the membrane potential of BHK cells were measured during 24 h in 4 x lop4 M ouabain and during the subsequent 6 h recovery (fig. 6). Control values prior to ouabain were 157.1 mM potassium and -49.9 mV membrane potential. After 6 h of recovery, intracellular potassium had recovered to 126.0 mM and the membrane potential to -46.8 mV. Further measurements of potassium following 24 h of recovery indicated almost complete recovery (149.2? 6.8 mM, mean iS.E., n=4). It is apparent from fig. 6 that changes in the intracellular potassium concentration were matched by changes in the membrane potential. Further experiments involving manipulation of the external and internal potassium concentrations indicate that the membrane potential of BHK cells is mainly determined by the potassium distribution acrossthe cell membrane (Sachs&McDonald. In prep.). DISCUSSION The effect of ouabain on BHK cells was markedly dependent on the dose employed. At 1 X 1O-4 M, ouabain had little effect on growth, macromolecular synthesis and intracellular ion concentrations. At 4 x 1O-4 M, growth was almost completely inhibited, the Table 2. The effect of ouabain on the water content and intracellular sodium andpotassium concentrations of BHK cells Determinations were made following a 24 h incubation in ouabain medium Values are mean + S.E. (n = 6) Water Ouabain content concentration ( % wet wt) Control 1 x 1O-4 M 2 x lo-’ M 4 x 1O-4M

Intracellular ions (mM) Na

84.6 +0.45 25.3 +2.3 84.OkO.49 35.1 i2.9 84.7kO.42 85.9k4.1 83.9kO.50 143.5+9.0

K 161.7k5.9 148.9f4.8 94.8 +4.1 41.Ok3.8

Ouabain and BHK

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Fig. 5. Abscissa: time (h); ordinate:

96 control (acidinsoluble cpm/cell). The incorporation of l , aH-thymidine; and 0, Y-amino acids in BHK cells during 24 h incubation in the 4 x 10-4M ouabain medium and subsequent 6 h recovery. Cells were seeded at 4 x 105 cells/plate, ouabain treatment commenced 24 h later, and cells were washed 3 times (arrow) and re-incubated in control (MEM) medium. Cells were pulsed for 15 min and values are the means of duplicate plates.

Ouabain has previously been shown to reduce protein synthesis in both intact tissue [lo] and cultured cells [ll]. Quastel & Kaplan [ 121reported that ouabain decreased b both DNA synthesis and protein synthesis I I 1 I I in human lymphocytes. They found that 0 24 48 72 96 protein synthesis was inhibited more rapidly Fig. 4. Abscissa: time (h); ordinate: cell number (X 10-S). than nucleic acid synthesis and this also (a), Recovery of BHK cell growth after 24 h inappears to be the case in ouabain-treated cubation in ouabain medium. Ouabain treatment commenced 24 h after seeding (first arrow). All BHK cells. plates were washed 3 times at 48 h (second arrow) The recovery of BHK cells from ouabain and reincubated in control (MEM) medium. l , Control; n , 2 x 10-4M ouabain; 0, 3 x 10-4M ouatreatment was dependent on the concentrabain; u, 4 x 10-4M ouabain. (b), effect of incubation time in 4 x 10-4M ouabain medium on the subse- tion of ouabain used and on the duration of quent growth of BHK cells. Ouabain treatment comtreatment. When the incubation period was menced 24 h after seeding. Plates were washed 3 24 h, cells resumed normal growth rates times at times indicated by arrows. Control plates washed 3 times at 36 h. l , Control; 0, 6 h in 4 k 10e4 after washing, provided the dose of ouabain M ouabain; q , 12 h in 4 x 10-4M ouabain; W, 24 h did not exceed 2 x 1O-4 M. Normal growth in 4 x lo-“M ouabain. also resumed when the incubation period in incorporation of 3H-thymidine and 14C- 4 x 1O-4 M ouabain did not exceed 12 h. amino acids was reduced by 70 %, and there The lack of recovery following 24 h in 4 :s’ were large changes in intracellular ion 10~” M ouabain medium does not appear to concentrations and the membrane potential. be due to a lingering presence of ouabain, for three reasons. First, after 12 h of incubaThese changes were not due to an inhibition of cellular energy metabolism since the tion with this concentration of ouabain, ATP content of these cells was not signifi- complete recovery followed drug washout (three changes of medium). Secondly, even cantly reduced. Exptl

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Fig. 6. Abscissa: time (h); ordinate: left-intracellular potassium concentration (mmole/kg cell water); right-membrane potential (mV). The intracellular potassium concentration (0) and membrane potential ( 0) of BHK cells during 24 h incubation in 4 x 10-4M ouabain medium and subsequent 6 h recovery. For potassium determinations, cells were seeded at 1 x 10’ cells/plate (150 mm diam.) and for membrane potential measurements at 2 x lo5 cells/plate (35 mm diam.). Ouabain treatment was begun 24 h later and after a further 24 h cells were washed 3 times (arrow) and reincubated in control (MEM) medium. Values are mean +S.E., n =4 - 6 for potassium determinations, n = 12 - 14 for membrane potential determinations.

[13, 141. However, since ATPase activity appeared to recover, as judged by potassium re-accumulation and the restoration of the membrane potential, it is apparent that prolonged exposure to high concentrations of ouabain irreversibly damages processes involved in macromolecular synthesis. We thank Dr R. L. DeHaan and Dr P. J. Stambrook for a critical review of this manuscript. Delores Somerville and Bessie Smith provided excellent technical assistance.

REFERENCES 1. Orr. C W M. Yoshikawa-Fukada. M & Ebert, J Di Proc natl acad sci US 69 (1972) 243. 2. McDonald. T F. Sachs. H G. Orr. C W M & ’ Ebert, J D; Dev biol. In’press.’ 3. Repke, K, New aspects of cardiac glycosides (ed W Wilbrandt) p. 47. Pergamon Press, London (1963). 4. Straub. W. Handbuch der experimentellen Pharmakologie. (ed A Hiffter) Bd II 2, p. 1355. Springer, Berlin (1924). 5. Williams, J A, Withrow, C D & Woodbury, D M, J physiol 212 (1971) 101. 6. DeHaan, R L & Gottlieb, S H, J gen physio152 (1968) 643. 7. Tasaki, K, Tsukahara, Y, Ito, S, Wayner, M G & Yu. W Y. Phvsiol behav 3 (1968) lCO9. 8. Chapman, J D,- Webb, R G ‘& Bbrsa, J, J cell biol 49 (1971) 229. 9. Lamb, J F & MacKinnon, M G A, J physiol213 (1971) 665. 10. Kypson, J & Hait, G, J pharmacol exptl ther 177 (1971) 398. 11. Lubin, M, Nature 213 (1967) 451. 12. Quastel, M R & Kaplan, J G, Exptl cell res 62 (1970) 407. 13. Winter, G G & Christensen, H N, J biol them 239 (1964) 872. 14. Yunis, A A, Arinmura, G K & Kipnis, D M, J lab clin med 62 (1963) 465.

when cells which had been exposed to this dose for 24 h were washed three times every 3 h for 12 h, growth did not resume at control level. Third, there did not appear to be any lingering effect on Na+-K+-ATPase activity, since cells re-accumulated potassium and the membrane potential recovered. The lack of recovery in growth was parallelled by a lack of recovery of DNA synthesis and protein synthesis. A relationship between Na+-K+-ATPase activity and amino acid transport has been suggested Received January 20, 1972

Exptl Cell Res 74 (1972)