Correlated Effects of External Magnesium on Cation Content and DNA Synthesis in Cultured Chicken Embryo Fibroblasts HISASHI SANUI AND HARRY RUBIN Department of Molecular Biology and Virus Laboratory, University of CaZifornia, Berkeley, California 94720
ABSTRACT Depletion of Mg2+in the growth medium for chicken embryo fibroblasts produces a large decrease in DNA synthesis as measured by 3H-thymidine incorporation, and concomitant decreases in cellular K + and Mg2+ and increases in Na+ and Ca2+.In cells grown in media containing 0.2 mM Ca2+, graded reduction of Mg2+from 0.8 mM (control) to 0.016 mM produced graded decreases in DNA synthesis to 10%of control a t 0.016 mM Mg2+.Concomitantly, cell cations showed graded changes, Na+ increasing t o 227%,K+ decreasing to 52.5%,Mg2+decreasing to 57.5%and Ca2+increasing to 153.5%of control. The effects of Mg2+depletion on DNA synthesis and cell cation content exhibited a dependence on Ca2+concentration, the effects being larger at low CaZ+concentration. Use of inorganic pyrophosphate in the growth medium a s a selective complexor of Mg2+caused a marked decrease in DNA synthesis which was accompanied by changes in cellular cation content similar to those produced by direct Mg2+depletion. The effects of Mg2+depletion on cell cation content are explainable in terms of changes in membrane permeability caused by rapid external surface exchange of bound divalent cations. Among the several interpretations of the data in terms of possible mechanisms by which changes in external Mg2+concentration may affect cell metabolism, the most consistent with known properties of the system is the concept of a central role for intracellular free Mgz+in the coordinate control of growth and metabolism in animal cells. The rates of metabolism and growth of cells grown in culture depend on a variety of environmental factors. Recent investigations (e.g., Rubin, '71; Ceccarini and Eagle, '71; Eagle, '73) have shown that pH variations in the range 6.6-8.0have a profound effect on cell growth. The role of divalent cations is of particular interest because of their involvement a s important structural and functional components of the plasma membrane and their requirement as cofactors in numerous enzymatically catalyzed intracellular reactions. Zinc is required for deoxyribonucleic acid (DNA) synthesis, and the availability of intracellular Zn2+may play a role in the regulation of cell multiplication (Rubin, '72; '73). Ca2+has been implicated a s a regulator of cell growth by several investigators (e.g., Balk, '71; Balk et al., '73; Dulbecco and Elkington, '75; Luckasen et al., '74). J. CELL. PHYSIOL.,92: 23-32.
Although a n extensive literature exists describing the importance of Ca2+to cell growth and physiology, much less has been said about the possible role of Mg2+.Nevertheless, Mg2+ is known to be essential for the growth of living cells (e.g., Walser, '671, and various investigations have shown t h a t cells grown in media deficient in Mg2+exhibit reduced rates of growth and metabolism (Gunther, '66; Gunther and Averdunk, '70; Lusk et al.,, '68; Whitfield e t al., '69; Rubin, '75). On the basis of the antagonistic action of Mg2+and Ca2+on common metabolic processes and the ability of the cells to regulate Ca2+levels via specific cellular and mitochondria1 membrane transport mechanisms, Bygrave ('76) suggested that modification of the MgZ+/Ca2+ ratio is a means of regulating intracellular metabolic reactions. However, Rubin and Koide ('76) reReceived July 26, 1976. Accepted Dec 8,1976.
23
24
HISASHI SANUI AND HARRY RUBIN
ported a mutual potentiation of cell growth by Mg2+and Ca2+and proposed that the major metabolic effects of varying Ca2+concentration in the growth medium ([Ca"],) are produced indirectly through a n effect on free intracellular Mg". In view of the importance of Mg2+ as a cofactor for a multitude of intracellular reactions and its role in preserving the structure of ribosomes (Heaton, '73) and in maintaining the permeability properties of plasma membranes, it is significant that the primary importance of Mg2+in the regulation of metabolic and physiological processes is now receiving serious consideration (Achs and Garfinkel, '73; Gerber e t al., '73; Peck and Ray, '71; Lostroh and Krahl, '74). On the basis of many studies of the effects of various environmental factors on growth and metabolism of cells grown in culture in this laboratory, Rubin ('75) proposed that free intracellular Mg2+is the central agent in the coordinate control of metabolism and growth in animal cells. The striking effects of external Mg2+depletion and other environmental stimuli on a variety of intracellular reactions are by now well established. However, the critical questions of the mechanism of action of the agents and the possible role of free intracellular Mg2+ still require elucidation. The plasma membrane regulates the passage of materials into and out of a cell and is the first site of action of many environmental agents which affect the activities of the cell. Mg2+and Ca2+bound to the membrane appear to be of importance in determining the permeability properties of this structure (e.g., Leive, '65; Tolberg and Macey, '711, and considerable evidence attests to its ion exchange properties (e.g., Sanui, '70; Sanui and Pace, '65, '67b). In investigations aimed a t understanding the mechanisms of action of the various external agents, therefore, it is important to determine changes in membrane divalent cation binding and the associated effects on active and passive solute transport. Alterations in the cation content of cells grown under different environmental conditions can be important indicators of changes in intracellular ion activities and steady state ratios of active to passive cation transport. As an initial step in determining the mode of action of external Mg2+in affecting cellular growth and metabolism, therefore, we investigated the effects of varying external Mg2+ concentration ([Mg2+l,) on cellular Na+, K +
Mg2+ and Ca2+ content and correlated the observed changes with modifications in cellular DNA synthesis as measured by 3H-thymidine incorporation (Sanui and Rubin, '76). Our results show that the decrease in rate of DNA synthesis produced by depletion of Mg2+ in the growth medium is accompanied by concomitant changes in the cellular concentration of the cations. The observed effects indicate a change in the plasma membrane cation transport properties and suggest several possibilities as the immediate source of regulation of cellular metabolism. Among these possibilities, the most consistent with known properties of the system is the concept of a central role for intracellular free Mg2+in the co0rdinat.e control of growth and metabolism in animal cells. METHODS AND MATERIALS
Cell cultures Primary cultures of chicken embryo fibroblasts (CEF) were prepared in plastic petri dishes (Falcon) from 10-day-old embryos according to well established procedures (Rein and Rubin, '68). Secondary cultures were prepared in 100mm dishes by seeding 2 X lo 6 cells per dish in a standard medium consisting of medium 199, 2% tryptose phosphate broth (TPB)and 1% chicken serum (designated 199 (2-0-1))and grown in an incubator a t 38°C and containing 5% CO, in air to maintain a constant pH of 7.4-7.5 in the bicarbonate buffered medium. Unless otherwise indicated, the standard medium contained 150 mM Na+, 5 mM K+, 0.8 mM Mg2+and 1.8 mM Ca2+. Experimental procedure In experiments designed t o determine the effect of varying [Mg2+l,on cell cation content and DNA synthesis, secondary cultures were grown in standard medium for three days to yield essentially confluent cultures. The old medium was then removed by aspiration, cells were rinsed twice with new standard or modified medium and grown in the new medium for 16 hours. Measurement of H-thymidine incorporation 3H-methyl thymidine (New England Nuclear) incorporation into trichloroacetic acid (TCA) insoluble fractions of the cell was used for determination of the rate of DNA synthesis (Rubin, '73). The increased incorporation of thymidine into DNA on stimulation of a cell results from an acceleration of cells through the G1 period into the S-period, and
Mgz+ CONTROL OF CELL CATIONS AND METABOLISM
represents an increase in the fraction of cells in the S-period rather than a n increase in the rate of synthesis of DNA in those cells already in the S-period (Rubin and Fodge, '74). Following 16-hour incubation in control or modified medium, the medium was replaced with 4 ml per 100 mm dish of 199 medium containing 0.25 pC 3H-thymidine per ml. After incubation for one hour, cells were rinsed three times with tris-saline buffer and the acid-soluble pools were removed by five minutes of exposure to ice cold 5% TCA followed by three rinses with this solution. The cells were then dissolved in 4 ml per dish of 0.1 N NaOH, and 1 ml of the extract was added to 10 ml of scintillation fluid (10 g of Omnifluor in 2 liters of toluene and 1 liter of Triton X-100) acidified with 0.1 ml of 100%(w/v) TCA for counting i n a liquid scintillation spectrometer (Packard model 3375) (Rubin and Koide, '73).
25
dish of pH 7,0.25 M sucrose solution dispensed from a Repipet. Advantage was taken of the normally strong adherence of the cells to the dish to quickly (5-10 seconds per wash) wash the cells in situ by carefully adding the wash solution and gently swirling before removing with a vacuum aspirator system. Samples were then harvested, sonicated and analyzed for protein and cations as described. Results (fig. 1) show that after the first wash, which removes protein contained in the medium, there is essentially no loss of total protein (cells) up to seven washes. There is also essentially no loss of any of the cations from two to seven washes. The washout data on a semilogarithmic plot can be resolved into two main components, a relatively steep component representing dilution of adhering medium and a second component of nearly zero slope indicating essentially no loss of cell cations in the time required for the washes. Dishes without cells processed in the same manner generally showed negligible carryover of cations.
Cell harvesting for protein and cation measurements Cultures not used for 3H-thymidine incorAddition of inorganic pyrophosphate poration measurements were carefully and The external supply of available Mg2+can quickly rinsed with the appropriate rinse fluid. Routinely, this consisted of five rinses be regulated either by simple reduction in with 10 ml per rinse of C0,-free 0.25 M sucrose solution. Rinsed cells were harvested by care50tfully scraping each dish with a well-rinsed rubber policeman. Generally, cells from approximately seven dishes were combined for one sample, and made to 10 ml volume in double distilled water. Cell suspensions were then sonicated and aliquots were used to measure protein by the Lowry procedure (Lowry et al., '51) and cations by atomic absorption spectrophotometry (Sanui and Pace, '66). Cation analyses of dilutions of the sonicated cells yielded results essentially identical to those from analyses of cells ashed by exposure to activated oxygen (Sanui, '711. All samples and standards for atomic absorption spectrophotometry contained 15 mM La3+,4 mM Cs+ and 100 mM HC1 which minimized chemical and ionization interferences (Sanui and Pace, '66). Duplicates of each cell sample were read twice each against suitable standards using the 100 average mode of the atomic absorpFig. 1 Effect of repeated washes on cation and protein tion spectrophotometer (Perkin-Elmer model content of cells grown in culture. Chicken embryo fibro403). blasts were grown in 100 mm petri dishes to confluency in RESULTS
Washout curve In order to test the efficiency of the cell culture washing procedure, cells grown in standard medium to confluency were washed from zero to seven times with 10 ml per wash per
medium 199 containing 2% tryptose phosphate broth, 1% chicken serum, 150 mM Na+, 6 mM K', 0.8 mM Mgz+and 1.7mM Caz+.Cells were washed zero to seven times in situ using 10 ml/wash/dish of pH 7,0.25M sucrose solution dispensed from a Repipet. Washed cells from five dishes were combined, sonicated and aliquots taken for cation measurement by atomic absorption spectrophotometry and protein measurement by the Lowry procedure.
26
HISASHI SANUI AND HARRY RUBIN
1
0.501
9 . "
NO PP,
2 3 m M PP,
- precipitate
? Z
I
~
09
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3 0
5 030
07 025
--.
2 0
0.6 3.
0
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+-
+-
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1.95mM Co*+ 0 . 8 3 m M Ma*+
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Y
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z
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: 010 u
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Fig. 2 Comparison of the cation content of cells grown in standard medium without PP, and with 2.3 mM PP, with or without precipitate present. After 16 hours growth in the particular medium, cells in some dishes were labeled with 3H-thymidine for one hour, and activity in the 5%TCA insoluble fraction was measured. The measured activities of cells grown in the presence of 0 PPi was 12.0 cpm/@gprotein, in 2.3 mM PPi with precipitate, 0.6 cpm/pg protein and in 2.3 mM PP, with precipitate removed, 1.4 cpm/pg protein. Cells not used for labeling studies were washed five times with pH 7, 0.25 M sucrose solution, harvested, sonicated and analyzed for cations and protein.
Fig. 3 Effect of inorganic pyrophosphate on Wthymidine incorporation and cation content of cells grown in culture. Cells were grown in standard medium 199 (201) containing added 2.00, 2.25 and 2.50 mM PP, with precipitates removed by centrifugation. 3H-thymidine incorporation was measured after 16 hours by exposing cells to 3H-thymidine for one hour and measuring the activity in the TCA insoluble fraction. Remaining cells were washed five times with neutral 0.25 M sucrose solution, harvested, sonicated and analyzed for cations and protein.
external concentration or by addition of an agent which preferentially complexes Mg2+. In earlier studies (Rubin, '75) adenosinetriphosphate (ATP), adenosinediphosphate (ADP) and inorganic pyrophosphate (PPi) were employed a s complexing agents t o reduce available Mg2+. All inhibited DNA synthesis when added a t 2-4 mM concentration. Of the three, PPi is the most selective for Mg2+over Ca2+(Sillen and Martell, '64)and causes no vacuole formation in cells as do the adenine nucleotides. This agent was also found to yield the most reproducible metabolic effects (Rubin, '75) and the precipitates which formed did not damage the cells and posed no obvious problems. In the present studies, however, the precipitate seriously affected cell cation measurements. Nevertheless, because of the marked inhibition of DNA synthesis produced by PPi in the earlier experiments (Rubin, '751, i t was considered essential t o test the effects of PP, on cell cation content. Experiments were therefore run using media containing no added PP,,
added 2.3 mM PP, with precipitate left, and added 2.3 mM PP, with precipitate removed by centrifugation. Cultures were grown and processed as described earlier. Compared with cells grown in standard medium with no added PP,, cells grown in media containing 2.3 mM PP, (with precipitate) incorporated 3H-thymidine a t about a 19-fold lower rate, whereas those grown in medium plus PP, with the precipitate removed had a rate only about 9-fold lower than controls. Cell cultures grown in the presence of the precipitate also retained much higher levels of Ca2+ and larger amounts of Na+ and Mg2+ than those grown in the absence of precipitate. This possibly reflects a n incomplete removal of precipitate (fig. 2). Cells grown in media containing added PPi but with the precipitate removed contained 170% more Na+, 42% less K+, 35% less Mgz+ and 330% more Ca2+than those grown in the absence of added PP,. These changes were qualitatively similar to those observed when [Mg2+l,was reduced by depletion as is discussed in a later section.
Inorganic pyrophosphate concentration. mM
27
Mg2+ CONTROL OF CELL CATIONS AND METABOLISM
Thus, meaningful cation data is obtained for PP, if the precipitate is removed before incubation of the cells. In the presence of 0.8 mM Mg2+,addition of 2-3 mM PP, was previously found to produce a significant and graded decrease in the rate of 3H-thymidine incorporation (Rubin, '751, whereas lower levels of PP, did not have a significant effect. Cells were therefore grown in media with added 2.00, 2.25 and 2.50 mM PP, with the precipitate removed, and analyzed for cations. Results (fig. 3) showed a graded large increase in Na+ and Caz+and a decrease in K+ and Mg2+accompanying the graded decrease in the rate of 3H-thymidine incorporation with increasing PP,. This pattern of correlated changes was qualitatively similar in different experiments, suggesting a general increase in permeability of the membrane and/or a decrease in active cation transport. Removal of Mg 2+ Changes qualitatively similar to but quantitatively smaller and more consistent than those obtained in media containing added PP, with the precipitate removed were produced by directly reducing the growth medium Mg2+ concentration by depletion (fig. 4). These Mg2+-depletedsystems were also not complicated by the possibility of precipitate formation. For this reason, all subsequent studies in this series were performed by directly changing the Mg2+concentration in the medium. Standard 199 medium with no added Mg2+ (generally containing 15-30 pM Mg2+as contaminants from other constituents) was used and various amounts of MgC12 were added to obtain the desired [Mg2+lo. The changes in rate of 3H-thymidine incorporation produced by changes in [Mg2+lohave been found to depend on [Ca2+l0,indicating some kind of interaction between these cations (Rubin and Koide, '76). In view of the known ion exchange nature of the plasma membrane (Sanui and Pace, '65, '67a,b; Sanui, '70) this is not surprising. Generally, the effects of reduced [Mg2+loon DNA synthesis were found to be larger a t low [Ca2+l,.For this reason, experiments were carried out a t approximately 0.2 mM ICa2+l,. Changes in cellular cation content at reduced [Mg2+lo were also found to be affected by [Ca2+l,. In cells grown in media containing 0.22 mM CaZ+and varied levels of [Mg2+l,
-g c
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Fig. 4 The cation content of cells grown in media containing low or high concentrations of Mg". Cells were grown in medium 199 (201) containing 0.26 mM Ca" and either 0.06 or 0.90 mM Mg2+.3H-thymidine incorporation was measured after 16 hours by exposing cells to W t h y m i dine containing medium for one hour and measuring the activity in the TCA insoluble fraction. Cells grown in 0.90 mM Mg2+medium had a n activity of 27.8 cpm/pg protein, whereas those grown in 0.06 mM Mg2+had 7.7 cpm/fig protein, a level about 28%of the control. Cells in the remaining dishes were washed five times with 0.25 M sucrose solution, harvested, sonicated and analyzed for cations and protein.
j4E
0 02
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'
I I 1 0.4 06 concentrotion. mM
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Fig. 5 Mg2+dependence of the cation content and rate of 3H-thymidine incorporation in cells grown in 199 (201) medium containing 0.22 mM Ca2+ and varied concentrations of Mg2+. 3H-thymidine incorporation was measured after 16 hours by exposing cells to Wthymidine medium for one hour and measuring the activity in the 5% TCA insoluble fraction. Cells in the remaining dishes were washed five times with 0.25M sucrose solution, harvested, sonicated and analyzed for cations and protein.
28
HISASHI SANUI AND HARRY RUBIN 0 . 9 6 m M Co+'
r~ /= Total cations
,
DISCUSSION 1.2
r-
Y
u
0.2 Mg"
0.4 0.6 c o n c e n t r a t i o n , mM
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Fig. 6 Mg'+ dependence of the cation content and rate of 3H-thymidine incorporation in cells grown in 199 (201) media containing 0.96 mM Ca'+ and varied concentrations of Mg". W-thymidine incorporation was measured after 16 hours by exposing cells to Wthymidine medium for one hour and measuring the activity in the 5% TCA insoluble fraction. Cells in the remaining dishes were washed five times with 0.25 M sucrose solution, harvested, sonicated and analyzed for cations and protein.
from 0.016-0.80 mM, 3H-thymidine incorporation decreased to about 10%of control a t 0.016 mM [Mg2+lo(fig. 5). Concomitantly, cell Na+ increased to 227%, K+ decreased to 52.5%, Mg2+decreased to 57.5%and Ca2+increased to 153.5% of control. Variation of [Mg2+l,over the same range a t 0.96 mM [Ca2+loproduced similar but generally smaller changes (fig. 6). 3H-thymidine incorporation decreased to 23% of control a t 0.017 mM [Mg2+loand a t the same time, cellular Na+ increased to 116%,K+ decreased to 82%,Mg2+decreased to 62% and Ca2+decreased to 83% of control. I t is interesting t o note that cell Ca2+levels remained essentially the same at 0.96 mM and 0.22 mM LCa2+l,and showed a small apparent decrease relative to the 0.8 mM [Mg2+locontrol level at 0.96 mM [Ca2+l0only because of a slight increase in Ca2+a t that [Mg2+lo. In figures 5 and 6, i t is to be noted that K' levels are plotted on a different scale than that for the other three cations. The cellular levels for K + are a t least an order of magnitude larger than for the other cations, and the molar changes in K+ are the largest cation changes produced.
Extensive previous investigations (Rubin, '75; Rubin and Koide, '76) as well as those discussed here clearly show that depletion of Mg2+ in the growth medium produces large changes in the rate of DNA synthesis a s measured by 3H-thymidine incorporation. Results of the studies presented here show that these changes, observed after 16 hours incubation, are accompanied by significant concomitant changes in cellular cation content, K+ and Mg2+generally changing in parallel with 3Hthymidine incorporation, and Na+ and Ca2+ changing in a n opposite direction. Use of PP, in the growth medium as a selective complexor of Mg2+ had previously been observed to cause a marked decrease in the rate of 3H-thymidine incorporation into DNA (Rubin, '75) but measurements of the cell cation content under those conditions were complicated by formation of a precipitate which may be incompletely removed by rinsing. However, in experiments with PP, in which the precipitate was removed by centrifugation prior to application of the medium to the cells, the change in 3H-thymidine incorporation was accompanied by changes in cellular cation content which were qualitatively similar to those produced by direct Mg2+depletion. Although the rate of DNA synthesis was used as an indicator of the general metabolic state of the cell, i t has previously been shown (Rubin, '75) that limiting the external supply of Mg2+also causes reductions in the rates of protein synthesis, 3H-uridine incorporation into ribonucleic acid (RNA), glycolysis, and the transport of 2-deoxy-D-glucose. These changes are similar to those observed after removing serum or increasing population density. Most of these studies utilized a 16-hour incubation time with the specified medium and the present studies therefore correlate changes in cell cation content with changes in DNA synthesis after this period of incubation. Necessary correlations after various times of incubation are underway. Metabolic changes similar to those observed in this laboratory were reported by Gunther and Averdunk ('70) for Yoshida-Ascites Tumor cells grown in a Mg2+-deficientmedium. The reduced rates of synthesis of DNA, RNA and protein were accompanied by a decreased concentration of cellular K', an increased Na+, a decreased Mgz+and a n increased Cat+. The change in intracellular K+ was presum-
Mg2+ CONTROL OF CELL CATIONS AND METABOLISM
ably accounted for by a n increased K' efflux. In the present study, the observed changes in cellular cations also suggest a change in membrane permeability, with a more rapid movement of cations down concentration gradients at the low [Mg2'1,. No data are available at present as to whether changes in passive fluxes alone are involved or whether active transport processes are also affected. Preliminary studies of cation binding to the external surface of the plasma membrane measured in situ show changes in membrane bound cations with changing [Mg2+l,.In view of the known role of divalent cations in regulating membrane permeability, i t is of interest to note that with decreasing [Mg2'l, a decrease in externally bound Mg2' was accompanied by an increase in bound Ca2+.These changes would be expected on the basis of the known ion exchange nature of cell membranes. Increased rates of efflux of Na+and K + occur in cells grown in Mg2+-deficientmedia so that the changes in cell cation content are attributable at least in part to changes in membrane permeability. We suggest that changes in cellular cation (particularly Mg2+) content result in changes in intracellular free Mg2+concentration, which affect the rate of cell metabolism and growth. Other interpretations of the present results are possible. For example, in the experiments with Yoshida-Ascites tumor cells depleted of Mg2+,Gunther and Averdunk ('70)suggested t h a t the changed intracellular K'-Na' concentrations are responsible for the decrease in rates of RNA, DNA and protein synthesis. Shank and Smith ('76) postulated that cellular growth in mouse lymphoblasts was regulated by sodium pump activity primarily because of the Na+-dependency of amino acid transport. However, in chicken embryo cells, no significant changes in transport of amino acids have been found associated with changes in growth rate (Fodge and Rubin, '75). In the case of Ca2+,which has been suggested by several groups as a regulator of cell growth (e.g., Balk, '71; Balk, Whitfield et al., '73; Duibecco and Elkington, '75; Luckasen et al., '74) it seems likely that the effects, at least in chicken embryo fibroblasts, a r e primarily a t the membrane level related to permeability changes. Our results with Mg2+depletion show only a small increase in total cell Ca2+ under conditions involving large decreases in rates of DNA synthesis. Most of the
29
changes in Ca2+can be accounted for by a n increase in external membrane surface binding. Although the possibility does exist that release of a pool of Caz+as in the sarcoplasmic reticulum in muscle or from mitochondria could occur without a change in total cell Ca2+,no evidence for this exists a t present in chicken embryo fibroblasts. Recent experiments concerning the specificity of t h e requirements for Mg2+and CaZ+provide further evidence that Mg2+is the direct intracellular effector and that Ca2' (and Sr2+)act indirectly by making Mg2+available for metabolic work (Rubin and Koide, '76; Rubin, '77). Several important lines of evidence favor a key role for Mg2+ as a primary regulator of cell metabolism and growth: (1) the known requirement of Mg2' as a cofactor for a multitude of enzymatically catalyzed intracellular reactions, especially those involving transphosphorylations, and protein syntheis. Mg2+is required by various aminoacyl-tRNA synthetases and is also involved in maintaining the conformation of ribosomes and nucleic acids (Heaton, '73); (2) strong evidence that [Mg2+l,is in the range required for significant effects on key reactions with small changes in free Mg2+.For example, experimental (England e t al., '67; Rose, '68; Palaty, '71) and computer calculations (Kerson, e t al., '67) both indicate that free [Mg2'l in mammalian tissue is 1mM or less. This is below the concentration necessary for maximum activity of pyruvate kinase in rat heart (Kerson et al., '67) and hexokinase in ox brain (Bachelard and Goldfarb, '69) so that small changes in free Mg2' are potentially capable of exerting a considerable degree of metabolic control; (3) the recent observation that the coordinate inhibition of 2-deoxy-D-glucose and uridine uptake and the incorporation of uridine and thymidine into acid insoluble material by omission of serum or addition of cortisol is reproduced quantitatively by lowering the concentration of Mg2' in the growth medium (Rubin, '76) ; (4) the closely related graded decreases in intracellular [Mg2+lparalleling decreases in rates of DNA synthesis as demonstrated in the present investigations. Similar relationships between [Mg2+l,and DNA synthesis have been observed in subsequent studies with insulin as the stimulating agent (Sanui and Rubin, '77). Additional investigations now underway are necessary to clarify the role inorganic cations play in the regulation of cell metabo-
30
HISASHI SANUI AND HARRY RUBIN
lism. Those concerning the relative rates of change in cell cations and metabolism, the levels of free intracellular cations, and changes in membrane cation binding, pump activity and permeability are of primary importance. The information derived from these studies should elucidate further the key role that intracellular Mg2+may play in the coordinate control of metabolism and growth in animal cells.
Gunther, Th. 1966 Uber die Rolle des Mg bei der Regulation des Zellstoffwechsels. Z. fur Naturforsch., 21 b: 1174-1177. Gunther, Th., and R. Averdunk 1970 K-transport und Stoffwechsel von Mg-arm gewachsenen Yoshida-Ascitestumorzellen. 2. Klin. Chim. u. Klin. Biochem., 8:621-625. Heaton, F. W. 1973 Magnesium requirement for enzymes and hormones. Biochem. SOC.Trans., 1: 67-70. Kerson, L. A,, D. Garfinkel and A. S. Mildvan 1967 Computer simulation studies of mammalian pyruvate kinase. J. Biol. Chem., 242: 2124-2133. Leive, L. 1965 A nonspecific increase in permeability in Escherichia coti produced by EDTA. Proc. Nat. Acad. Sci., 53: 745-750. ACKNOWLEDGMENTS Lostroh, A. J., and M. E. Krahl 1974 Magnesium, a second We thank Toshiko Koide, Virginia Rimers messenger for insulin: ion translocation coupled to transport activity. Adv. in Enzyme Regulation. G. Weber, ed. and Joyce Walton for their capable assistance Pergamon Press, New York, 22: 73-81. during the course of this investigation. Lowry, 0.H., N. J. Rosebrough, A. L. Farr and R. J. Randall This investigation was supported by NIH 1951 Protein measurement with the Folin phenol Research Grant CA 15744 from the National reagent. J. Biol. Chem., 193: 265-275. Cancer Institute. Luckasen, J. R., J. G. White and J. H. Kersey 1974 Mitogenic properties of a calcium ionophore, A23187. LITERATURE CITED Proc. Nat. Acad. Sci., 71: 5088-5090. Achs, M., and D. Garfinkel 1973 Available magnesium ion Lusk, J. E., R. J. P. Williams and E. P. Kennedy 1968 Magnesium and the growth of Escherichia coli. J. Biol. Chem., as an effective metabolic switch. In: Regulation and Con243: 2618-2624. trol in Physiological Systems. A. Iherall and A. Guyton, eds. International Federation of Automatic Control Sym- Palaty, V. 1971 Distribution of magnesium in the arterial wall. J. Physiol. (London), 218: 353-368. posium, pp. 19-21. Bachelard, H. S., and P. S. G. Goldfarb 1969 Adenine nu- Peck, E. J.,Jr., and W. J. Ray, Jr. 1971 Metal complexes of phosphoglucomutase in uiuo. Alterations induced by cleotides and magnesium ions in relation to control of insulin. J. Biol. Chem. 246: 1160-1167. mammalian cerebral-cortex hexokinase. Biochem. J.. Rein, A., and H. Rubin 1968 Effects of local cell concentra112 579-586 tion upon the growth of chick embryo cells in tissue culBalk, S D 1971 Calcium as a regulator of the prohferatlon ture. Expt. Cell Res., 49: 666-678. of normal but not of transformed, chicken fibroblasts in a plasma-containing medium. Proc. Nat. Acad. Sci., 68: Rose, I. 1968 The state of magnesium in cells as estimated from the adenylate kinase equilibrium. Proc. Nat. 271-275. Acad. Sci. 61: 1079-1086. Balk, S. D., J. F. Whitfield, T. Youdale and A. C. Braun 1973 Roles of calcium, serum, plasma and folic acid in Rubin, H. 1971 pH and population density in the regulation of animal cell multiplication. J. Cell. Biol., 51: 686th e control of proliferation of normal and Rous sarcoma 702. virus-infected chicken fibroblasts. Proc. Nat. Acad. Sci., 1972 Inhibition of DNA synthesis in animal 70: 675-679. cells by ethylene diamine tetraacetate, and its reversal Bygrave, F. L. 1976 Cellular calcium and magnesium by zinc. Proc. Nat. Acad. Sci., 69: 712-716. metabolism. I n : An Introduction to Bio-Inorganic 1973 pH. serum and Zn++in the regulation of Chemistry. D. R. Williams, ed. Charles C. Thomas, PuhDNA synthesis in cultures of chick embryo cells. J. Cell. lishers, Springfield, Illinois, pp. 171-184. Physiol., 82: 231-238. Ceccarini, C., and H. Eagle 1971 pH as a determinant of 1975 A central role for magnesium in coordicellular growth and contact inhibition. Proc. Nat. Acad. nate control of metabolism and growth in animal cells. Sci., 68: 229-233. Proc. Nat. Acad. Sci., 72: 3551-3555. Duibecco, R., and J. Elkington 1975 Induction of growth in 1976 Magnesium deprivation reproduces the resting fibroblastic cell Eultures by Ca". Proc. Nat. Acad. coordinate effects of serum removal or cortisol addition Sci., 72: 1584-1588. on transport and metabolism in chick embryo fibroblasts. Eagle, H. 1973 The effect of environmental pH on the J . Cell. Physiol., 89: 613-625. growth of normal and malignant cells. J. Cell. Physiol., 1977 Specificity of the requirements for mag82: 1-8. nesium and calcium in the growth and metabolism of England, P. J.,R. M. Denton and P. J. Randle 1967 The inchick embryo fibroblasts. J. Cell. Physiol., 91: 449fluence of magnesium ions and other bivalent metal ions 458. on the aconitase equilihrium and its hearing on the binding of magnesium ions by citrate in rat heart. Biochem. Rubin, H., and D. Fodge 1974 Interrelationships of glycolysis, sugar transport and initiation of DNA synthesis in J., 105: 32C-33C. chick embryo cells. In: Control of Proliferation of Animal Fodge, D., and H. Rubin 1975 Stimulation of lactic acid Cells. Vol. 1.B. Clarkson and R. Baserga, eds. Cold Spring production in chick embryo fibroblasts hy serum and high Harbor Laboratory, New York, pp. 801-816. pH in the absence of external glucose. J. Cell. Physiol.. Rubin, H., and T. Koide 1973 Inhibition of DNA synthesis 86: 453-457. in chick embryo cultures by deprivation of either serum Gerber, G., H. Berger, G-R. Janig and S. M. Rapoport 1973 or zinc. J. Cell Biol. 56: 777-786. Interaction of haemoglobin with ions. Quantitative description of the state of magnesium, adenosine 5'-triphos- _ _ 1976 Mutual potentiation by magnesium and calcium of growth in animal cells. Proc. Nat. Acad. Sci., and human haemoglobin phate, 2,3-b1sphosphoglycerate, 73: 168-172. under simulated intracellular conditions. Eur. J. BioSanui, H. 1970 pH dependence of the effect of adenosine chem., 38: 563-571.
Mg2+ CONTROL OF CELL CATIONS AND METABOLISM triphosphate and ethylene-diaminetetraacetate on sodium and magnesium binding by cellular membrane fragments. J. Cell. Physiol., 75: 361-368. 1971 Activated oxygen ashing of biological specimens for t h e microdetermination of Na, K, Mg and Ca by atomic absorption spectrophotometry. Anal. Biochem., 42: 21-28. Sanui, H., and N. Pace 1965 Mass law effects of adenosine triphosphate on Na, K, Mg and Ca binding by rat liver microsomes. J. Cell. Comp. Physiol., 65: 27-30. 1966 Application of atomic absorption in the study of Na, K, Mg and Ca binding by cellular membranes. Appl. Spectry., 20: 135-144. 1967a The Mg, Ca, EDTA and ATP dependence of Na binding by rat liver microsomes. J. Cell. Physiol., 69: 3-10. 1967b Effect of ATP, EDTA and EGTA on t he simultaneous binding of Na, K, Mg and Ca by rat liver microsomes. J. Cell. Physiol., 69: 11-20. Sanui, H., and H. Rubin 1976 Role of magnesium in the
31
regulation of cellular cation concentration and growth. Fed. Proc., 35: 500. 1977 Insulin effects on cation content and DNA synthesis in cultured cells. Fed. Proc. 36: 338. Shank, B. B., and N. E. Smith 1976 Regulation of cellular growth by sodium pump activity. J. Cell. Physiol., 87: 377-388. Sillen, L. G., and A. E. Martell 1964 Stability constants of metal-ion complexes. The Chemical Society, Special Publication No. 17. Burlington House, London, pp. 190-191. Tolberg, A. B., and R. I. Macey 1972 The release of membrane-bound calcium by radiation and sulfhydryl reagents. J. Cell. Physiol., 79: 43-51. Walser, M. 1967 Magnesium metabolism. Ergebnisse der Physiologie Biologischen Chemie und Experimentellen Pharmakologie, 59: 185-341. Whitfield, J. F., A. D. Perris and R. H. Rixon 1969 Stimulation of mitotic activity and the initiation of deoxyribonucleic acid synthesis in populations of rat thymic lymphocytes by magnesium. J. Cell. Physiol. 74: 1-8.
ABSTRACT Depletion of Mg2+in the growth medium for chicken embryo fibroblasts produces a large decrease in DNA synthesis as measured by 3H-thymidine incorporation, and concomitant decreases in cellular K + and Mg2+ and increases in Na+ and Ca2+.In cells grown in media containing 0.2 mM Ca2+, graded reduction of Mg2+from 0.8 mM (control) to 0.016 mM produced graded decreases in DNA synthesis to 10%of control a t 0.016 mM Mg2+.Concomitantly, cell cations showed graded changes, Na+ increasing t o 227%,K+ decreasing to 52.5%,Mg2+decreasing to 57.5%and Ca2+increasing to 153.5%of control. The effects of Mg2+depletion on DNA synthesis and cell cation content exhibited a dependence on Ca2+concentration, the effects being larger at low CaZ+concentration. Use of inorganic pyrophosphate in the growth medium a s a selective complexor of Mg2+caused a marked decrease in DNA synthesis which was accompanied by changes in cellular cation content similar to those produced by direct Mg2+depletion. The effects of Mg2+depletion on cell cation content are explainable in terms of changes in membrane permeability caused by rapid external surface exchange of bound divalent cations. Among the several interpretations of the data in terms of possible mechanisms by which changes in external Mg2+concentration may affect cell metabolism, the most consistent with known properties of the system is the concept of a central role for intracellular free Mgz+in the coordinate control of growth and metabolism in animal cells. The rates of metabolism and growth of cells grown in culture depend on a variety of environmental factors. Recent investigations (e.g., Rubin, '71; Ceccarini and Eagle, '71; Eagle, '73) have shown that pH variations in the range 6.6-8.0have a profound effect on cell growth. The role of divalent cations is of particular interest because of their involvement a s important structural and functional components of the plasma membrane and their requirement as cofactors in numerous enzymatically catalyzed intracellular reactions. Zinc is required for deoxyribonucleic acid (DNA) synthesis, and the availability of intracellular Zn2+may play a role in the regulation of cell multiplication (Rubin, '72; '73). Ca2+has been implicated a s a regulator of cell growth by several investigators (e.g., Balk, '71; Balk et al., '73; Dulbecco and Elkington, '75; Luckasen et al., '74). J. CELL. PHYSIOL.,92: 23-32.
Although a n extensive literature exists describing the importance of Ca2+to cell growth and physiology, much less has been said about the possible role of Mg2+.Nevertheless, Mg2+ is known to be essential for the growth of living cells (e.g., Walser, '671, and various investigations have shown t h a t cells grown in media deficient in Mg2+exhibit reduced rates of growth and metabolism (Gunther, '66; Gunther and Averdunk, '70; Lusk et al.,, '68; Whitfield e t al., '69; Rubin, '75). On the basis of the antagonistic action of Mg2+and Ca2+on common metabolic processes and the ability of the cells to regulate Ca2+levels via specific cellular and mitochondria1 membrane transport mechanisms, Bygrave ('76) suggested that modification of the MgZ+/Ca2+ ratio is a means of regulating intracellular metabolic reactions. However, Rubin and Koide ('76) reReceived July 26, 1976. Accepted Dec 8,1976.
23
24
HISASHI SANUI AND HARRY RUBIN
ported a mutual potentiation of cell growth by Mg2+and Ca2+and proposed that the major metabolic effects of varying Ca2+concentration in the growth medium ([Ca"],) are produced indirectly through a n effect on free intracellular Mg". In view of the importance of Mg2+ as a cofactor for a multitude of intracellular reactions and its role in preserving the structure of ribosomes (Heaton, '73) and in maintaining the permeability properties of plasma membranes, it is significant that the primary importance of Mg2+in the regulation of metabolic and physiological processes is now receiving serious consideration (Achs and Garfinkel, '73; Gerber e t al., '73; Peck and Ray, '71; Lostroh and Krahl, '74). On the basis of many studies of the effects of various environmental factors on growth and metabolism of cells grown in culture in this laboratory, Rubin ('75) proposed that free intracellular Mg2+is the central agent in the coordinate control of metabolism and growth in animal cells. The striking effects of external Mg2+depletion and other environmental stimuli on a variety of intracellular reactions are by now well established. However, the critical questions of the mechanism of action of the agents and the possible role of free intracellular Mg2+ still require elucidation. The plasma membrane regulates the passage of materials into and out of a cell and is the first site of action of many environmental agents which affect the activities of the cell. Mg2+and Ca2+bound to the membrane appear to be of importance in determining the permeability properties of this structure (e.g., Leive, '65; Tolberg and Macey, '711, and considerable evidence attests to its ion exchange properties (e.g., Sanui, '70; Sanui and Pace, '65, '67b). In investigations aimed a t understanding the mechanisms of action of the various external agents, therefore, it is important to determine changes in membrane divalent cation binding and the associated effects on active and passive solute transport. Alterations in the cation content of cells grown under different environmental conditions can be important indicators of changes in intracellular ion activities and steady state ratios of active to passive cation transport. As an initial step in determining the mode of action of external Mg2+in affecting cellular growth and metabolism, therefore, we investigated the effects of varying external Mg2+ concentration ([Mg2+l,) on cellular Na+, K +
Mg2+ and Ca2+ content and correlated the observed changes with modifications in cellular DNA synthesis as measured by 3H-thymidine incorporation (Sanui and Rubin, '76). Our results show that the decrease in rate of DNA synthesis produced by depletion of Mg2+ in the growth medium is accompanied by concomitant changes in the cellular concentration of the cations. The observed effects indicate a change in the plasma membrane cation transport properties and suggest several possibilities as the immediate source of regulation of cellular metabolism. Among these possibilities, the most consistent with known properties of the system is the concept of a central role for intracellular free Mg2+in the co0rdinat.e control of growth and metabolism in animal cells. METHODS AND MATERIALS
Cell cultures Primary cultures of chicken embryo fibroblasts (CEF) were prepared in plastic petri dishes (Falcon) from 10-day-old embryos according to well established procedures (Rein and Rubin, '68). Secondary cultures were prepared in 100mm dishes by seeding 2 X lo 6 cells per dish in a standard medium consisting of medium 199, 2% tryptose phosphate broth (TPB)and 1% chicken serum (designated 199 (2-0-1))and grown in an incubator a t 38°C and containing 5% CO, in air to maintain a constant pH of 7.4-7.5 in the bicarbonate buffered medium. Unless otherwise indicated, the standard medium contained 150 mM Na+, 5 mM K+, 0.8 mM Mg2+and 1.8 mM Ca2+. Experimental procedure In experiments designed t o determine the effect of varying [Mg2+l,on cell cation content and DNA synthesis, secondary cultures were grown in standard medium for three days to yield essentially confluent cultures. The old medium was then removed by aspiration, cells were rinsed twice with new standard or modified medium and grown in the new medium for 16 hours. Measurement of H-thymidine incorporation 3H-methyl thymidine (New England Nuclear) incorporation into trichloroacetic acid (TCA) insoluble fractions of the cell was used for determination of the rate of DNA synthesis (Rubin, '73). The increased incorporation of thymidine into DNA on stimulation of a cell results from an acceleration of cells through the G1 period into the S-period, and
Mgz+ CONTROL OF CELL CATIONS AND METABOLISM
represents an increase in the fraction of cells in the S-period rather than a n increase in the rate of synthesis of DNA in those cells already in the S-period (Rubin and Fodge, '74). Following 16-hour incubation in control or modified medium, the medium was replaced with 4 ml per 100 mm dish of 199 medium containing 0.25 pC 3H-thymidine per ml. After incubation for one hour, cells were rinsed three times with tris-saline buffer and the acid-soluble pools were removed by five minutes of exposure to ice cold 5% TCA followed by three rinses with this solution. The cells were then dissolved in 4 ml per dish of 0.1 N NaOH, and 1 ml of the extract was added to 10 ml of scintillation fluid (10 g of Omnifluor in 2 liters of toluene and 1 liter of Triton X-100) acidified with 0.1 ml of 100%(w/v) TCA for counting i n a liquid scintillation spectrometer (Packard model 3375) (Rubin and Koide, '73).
25
dish of pH 7,0.25 M sucrose solution dispensed from a Repipet. Advantage was taken of the normally strong adherence of the cells to the dish to quickly (5-10 seconds per wash) wash the cells in situ by carefully adding the wash solution and gently swirling before removing with a vacuum aspirator system. Samples were then harvested, sonicated and analyzed for protein and cations as described. Results (fig. 1) show that after the first wash, which removes protein contained in the medium, there is essentially no loss of total protein (cells) up to seven washes. There is also essentially no loss of any of the cations from two to seven washes. The washout data on a semilogarithmic plot can be resolved into two main components, a relatively steep component representing dilution of adhering medium and a second component of nearly zero slope indicating essentially no loss of cell cations in the time required for the washes. Dishes without cells processed in the same manner generally showed negligible carryover of cations.
Cell harvesting for protein and cation measurements Cultures not used for 3H-thymidine incorAddition of inorganic pyrophosphate poration measurements were carefully and The external supply of available Mg2+can quickly rinsed with the appropriate rinse fluid. Routinely, this consisted of five rinses be regulated either by simple reduction in with 10 ml per rinse of C0,-free 0.25 M sucrose solution. Rinsed cells were harvested by care50tfully scraping each dish with a well-rinsed rubber policeman. Generally, cells from approximately seven dishes were combined for one sample, and made to 10 ml volume in double distilled water. Cell suspensions were then sonicated and aliquots were used to measure protein by the Lowry procedure (Lowry et al., '51) and cations by atomic absorption spectrophotometry (Sanui and Pace, '66). Cation analyses of dilutions of the sonicated cells yielded results essentially identical to those from analyses of cells ashed by exposure to activated oxygen (Sanui, '711. All samples and standards for atomic absorption spectrophotometry contained 15 mM La3+,4 mM Cs+ and 100 mM HC1 which minimized chemical and ionization interferences (Sanui and Pace, '66). Duplicates of each cell sample were read twice each against suitable standards using the 100 average mode of the atomic absorpFig. 1 Effect of repeated washes on cation and protein tion spectrophotometer (Perkin-Elmer model content of cells grown in culture. Chicken embryo fibro403). blasts were grown in 100 mm petri dishes to confluency in RESULTS
Washout curve In order to test the efficiency of the cell culture washing procedure, cells grown in standard medium to confluency were washed from zero to seven times with 10 ml per wash per
medium 199 containing 2% tryptose phosphate broth, 1% chicken serum, 150 mM Na+, 6 mM K', 0.8 mM Mgz+and 1.7mM Caz+.Cells were washed zero to seven times in situ using 10 ml/wash/dish of pH 7,0.25M sucrose solution dispensed from a Repipet. Washed cells from five dishes were combined, sonicated and aliquots taken for cation measurement by atomic absorption spectrophotometry and protein measurement by the Lowry procedure.
26
HISASHI SANUI AND HARRY RUBIN
1
0.501
9 . "
NO PP,
2 3 m M PP,
- precipitate
? Z
I
~
09
m 035
3 0
5 030
07 025
--.
2 0
0.6 3.
0
\
020
$
0.10
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cn 2
-
i:
1.0
08
+-
+-
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1.95mM Co*+ 0 . 8 3 m M Ma*+
04,-
0.15
E
Y
0
z
03
: 010 u
% U
0 2
0 05
01
n
n
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Mq**
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Fig. 2 Comparison of the cation content of cells grown in standard medium without PP, and with 2.3 mM PP, with or without precipitate present. After 16 hours growth in the particular medium, cells in some dishes were labeled with 3H-thymidine for one hour, and activity in the 5%TCA insoluble fraction was measured. The measured activities of cells grown in the presence of 0 PPi was 12.0 cpm/@gprotein, in 2.3 mM PPi with precipitate, 0.6 cpm/pg protein and in 2.3 mM PP, with precipitate removed, 1.4 cpm/pg protein. Cells not used for labeling studies were washed five times with pH 7, 0.25 M sucrose solution, harvested, sonicated and analyzed for cations and protein.
Fig. 3 Effect of inorganic pyrophosphate on Wthymidine incorporation and cation content of cells grown in culture. Cells were grown in standard medium 199 (201) containing added 2.00, 2.25 and 2.50 mM PP, with precipitates removed by centrifugation. 3H-thymidine incorporation was measured after 16 hours by exposing cells to 3H-thymidine for one hour and measuring the activity in the TCA insoluble fraction. Remaining cells were washed five times with neutral 0.25 M sucrose solution, harvested, sonicated and analyzed for cations and protein.
external concentration or by addition of an agent which preferentially complexes Mg2+. In earlier studies (Rubin, '75) adenosinetriphosphate (ATP), adenosinediphosphate (ADP) and inorganic pyrophosphate (PPi) were employed a s complexing agents t o reduce available Mg2+. All inhibited DNA synthesis when added a t 2-4 mM concentration. Of the three, PPi is the most selective for Mg2+over Ca2+(Sillen and Martell, '64)and causes no vacuole formation in cells as do the adenine nucleotides. This agent was also found to yield the most reproducible metabolic effects (Rubin, '75) and the precipitates which formed did not damage the cells and posed no obvious problems. In the present studies, however, the precipitate seriously affected cell cation measurements. Nevertheless, because of the marked inhibition of DNA synthesis produced by PPi in the earlier experiments (Rubin, '751, i t was considered essential t o test the effects of PP, on cell cation content. Experiments were therefore run using media containing no added PP,,
added 2.3 mM PP, with precipitate left, and added 2.3 mM PP, with precipitate removed by centrifugation. Cultures were grown and processed as described earlier. Compared with cells grown in standard medium with no added PP,, cells grown in media containing 2.3 mM PP, (with precipitate) incorporated 3H-thymidine a t about a 19-fold lower rate, whereas those grown in medium plus PP, with the precipitate removed had a rate only about 9-fold lower than controls. Cell cultures grown in the presence of the precipitate also retained much higher levels of Ca2+ and larger amounts of Na+ and Mg2+ than those grown in the absence of precipitate. This possibly reflects a n incomplete removal of precipitate (fig. 2). Cells grown in media containing added PPi but with the precipitate removed contained 170% more Na+, 42% less K+, 35% less Mgz+ and 330% more Ca2+than those grown in the absence of added PP,. These changes were qualitatively similar to those observed when [Mg2+l,was reduced by depletion as is discussed in a later section.
Inorganic pyrophosphate concentration. mM
27
Mg2+ CONTROL OF CELL CATIONS AND METABOLISM
Thus, meaningful cation data is obtained for PP, if the precipitate is removed before incubation of the cells. In the presence of 0.8 mM Mg2+,addition of 2-3 mM PP, was previously found to produce a significant and graded decrease in the rate of 3H-thymidine incorporation (Rubin, '751, whereas lower levels of PP, did not have a significant effect. Cells were therefore grown in media with added 2.00, 2.25 and 2.50 mM PP, with the precipitate removed, and analyzed for cations. Results (fig. 3) showed a graded large increase in Na+ and Caz+and a decrease in K+ and Mg2+accompanying the graded decrease in the rate of 3H-thymidine incorporation with increasing PP,. This pattern of correlated changes was qualitatively similar in different experiments, suggesting a general increase in permeability of the membrane and/or a decrease in active cation transport. Removal of Mg 2+ Changes qualitatively similar to but quantitatively smaller and more consistent than those obtained in media containing added PP, with the precipitate removed were produced by directly reducing the growth medium Mg2+ concentration by depletion (fig. 4). These Mg2+-depletedsystems were also not complicated by the possibility of precipitate formation. For this reason, all subsequent studies in this series were performed by directly changing the Mg2+concentration in the medium. Standard 199 medium with no added Mg2+ (generally containing 15-30 pM Mg2+as contaminants from other constituents) was used and various amounts of MgC12 were added to obtain the desired [Mg2+lo. The changes in rate of 3H-thymidine incorporation produced by changes in [Mg2+lohave been found to depend on [Ca2+l0,indicating some kind of interaction between these cations (Rubin and Koide, '76). In view of the known ion exchange nature of the plasma membrane (Sanui and Pace, '65, '67a,b; Sanui, '70) this is not surprising. Generally, the effects of reduced [Mg2+loon DNA synthesis were found to be larger a t low [Ca2+l,.For this reason, experiments were carried out a t approximately 0.2 mM ICa2+l,. Changes in cellular cation content at reduced [Mg2+lo were also found to be affected by [Ca2+l,. In cells grown in media containing 0.22 mM CaZ+and varied levels of [Mg2+l,
-g c
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0 . 0 6 m M Mg++ 0.90mM Mg"
0
016
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,
+-
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6
"
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Fig. 4 The cation content of cells grown in media containing low or high concentrations of Mg". Cells were grown in medium 199 (201) containing 0.26 mM Ca" and either 0.06 or 0.90 mM Mg2+.3H-thymidine incorporation was measured after 16 hours by exposing cells to W t h y m i dine containing medium for one hour and measuring the activity in the TCA insoluble fraction. Cells grown in 0.90 mM Mg2+medium had a n activity of 27.8 cpm/pg protein, whereas those grown in 0.06 mM Mg2+had 7.7 cpm/fig protein, a level about 28%of the control. Cells in the remaining dishes were washed five times with 0.25 M sucrose solution, harvested, sonicated and analyzed for cations and protein.
j4E
0 02
O L '
I
1
02 Mg'*
1
'
I I 1 0.4 06 concentrotion. mM
Y
1
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0.8
Fig. 5 Mg2+dependence of the cation content and rate of 3H-thymidine incorporation in cells grown in 199 (201) medium containing 0.22 mM Ca2+ and varied concentrations of Mg2+. 3H-thymidine incorporation was measured after 16 hours by exposing cells to Wthymidine medium for one hour and measuring the activity in the 5% TCA insoluble fraction. Cells in the remaining dishes were washed five times with 0.25M sucrose solution, harvested, sonicated and analyzed for cations and protein.
28
HISASHI SANUI AND HARRY RUBIN 0 . 9 6 m M Co+'
r~ /= Total cations
,
DISCUSSION 1.2
r-
Y
u
0.2 Mg"
0.4 0.6 c o n c e n t r a t i o n , mM
0.8
Fig. 6 Mg'+ dependence of the cation content and rate of 3H-thymidine incorporation in cells grown in 199 (201) media containing 0.96 mM Ca'+ and varied concentrations of Mg". W-thymidine incorporation was measured after 16 hours by exposing cells to Wthymidine medium for one hour and measuring the activity in the 5% TCA insoluble fraction. Cells in the remaining dishes were washed five times with 0.25 M sucrose solution, harvested, sonicated and analyzed for cations and protein.
from 0.016-0.80 mM, 3H-thymidine incorporation decreased to about 10%of control a t 0.016 mM [Mg2+lo(fig. 5). Concomitantly, cell Na+ increased to 227%, K+ decreased to 52.5%, Mg2+decreased to 57.5%and Ca2+increased to 153.5% of control. Variation of [Mg2+l,over the same range a t 0.96 mM [Ca2+loproduced similar but generally smaller changes (fig. 6). 3H-thymidine incorporation decreased to 23% of control a t 0.017 mM [Mg2+loand a t the same time, cellular Na+ increased to 116%,K+ decreased to 82%,Mg2+decreased to 62% and Ca2+decreased to 83% of control. I t is interesting t o note that cell Ca2+levels remained essentially the same at 0.96 mM and 0.22 mM LCa2+l,and showed a small apparent decrease relative to the 0.8 mM [Mg2+locontrol level at 0.96 mM [Ca2+l0only because of a slight increase in Ca2+a t that [Mg2+lo. In figures 5 and 6, i t is to be noted that K' levels are plotted on a different scale than that for the other three cations. The cellular levels for K + are a t least an order of magnitude larger than for the other cations, and the molar changes in K+ are the largest cation changes produced.
Extensive previous investigations (Rubin, '75; Rubin and Koide, '76) as well as those discussed here clearly show that depletion of Mg2+ in the growth medium produces large changes in the rate of DNA synthesis a s measured by 3H-thymidine incorporation. Results of the studies presented here show that these changes, observed after 16 hours incubation, are accompanied by significant concomitant changes in cellular cation content, K+ and Mg2+generally changing in parallel with 3Hthymidine incorporation, and Na+ and Ca2+ changing in a n opposite direction. Use of PP, in the growth medium as a selective complexor of Mg2+ had previously been observed to cause a marked decrease in the rate of 3H-thymidine incorporation into DNA (Rubin, '75) but measurements of the cell cation content under those conditions were complicated by formation of a precipitate which may be incompletely removed by rinsing. However, in experiments with PP, in which the precipitate was removed by centrifugation prior to application of the medium to the cells, the change in 3H-thymidine incorporation was accompanied by changes in cellular cation content which were qualitatively similar to those produced by direct Mg2+depletion. Although the rate of DNA synthesis was used as an indicator of the general metabolic state of the cell, i t has previously been shown (Rubin, '75) that limiting the external supply of Mg2+also causes reductions in the rates of protein synthesis, 3H-uridine incorporation into ribonucleic acid (RNA), glycolysis, and the transport of 2-deoxy-D-glucose. These changes are similar to those observed after removing serum or increasing population density. Most of these studies utilized a 16-hour incubation time with the specified medium and the present studies therefore correlate changes in cell cation content with changes in DNA synthesis after this period of incubation. Necessary correlations after various times of incubation are underway. Metabolic changes similar to those observed in this laboratory were reported by Gunther and Averdunk ('70) for Yoshida-Ascites Tumor cells grown in a Mg2+-deficientmedium. The reduced rates of synthesis of DNA, RNA and protein were accompanied by a decreased concentration of cellular K', an increased Na+, a decreased Mgz+and a n increased Cat+. The change in intracellular K+ was presum-
Mg2+ CONTROL OF CELL CATIONS AND METABOLISM
ably accounted for by a n increased K' efflux. In the present study, the observed changes in cellular cations also suggest a change in membrane permeability, with a more rapid movement of cations down concentration gradients at the low [Mg2'1,. No data are available at present as to whether changes in passive fluxes alone are involved or whether active transport processes are also affected. Preliminary studies of cation binding to the external surface of the plasma membrane measured in situ show changes in membrane bound cations with changing [Mg2+l,.In view of the known role of divalent cations in regulating membrane permeability, i t is of interest to note that with decreasing [Mg2'l, a decrease in externally bound Mg2' was accompanied by an increase in bound Ca2+.These changes would be expected on the basis of the known ion exchange nature of cell membranes. Increased rates of efflux of Na+and K + occur in cells grown in Mg2+-deficientmedia so that the changes in cell cation content are attributable at least in part to changes in membrane permeability. We suggest that changes in cellular cation (particularly Mg2+) content result in changes in intracellular free Mg2+concentration, which affect the rate of cell metabolism and growth. Other interpretations of the present results are possible. For example, in the experiments with Yoshida-Ascites tumor cells depleted of Mg2+,Gunther and Averdunk ('70)suggested t h a t the changed intracellular K'-Na' concentrations are responsible for the decrease in rates of RNA, DNA and protein synthesis. Shank and Smith ('76) postulated that cellular growth in mouse lymphoblasts was regulated by sodium pump activity primarily because of the Na+-dependency of amino acid transport. However, in chicken embryo cells, no significant changes in transport of amino acids have been found associated with changes in growth rate (Fodge and Rubin, '75). In the case of Ca2+,which has been suggested by several groups as a regulator of cell growth (e.g., Balk, '71; Balk, Whitfield et al., '73; Duibecco and Elkington, '75; Luckasen et al., '74) it seems likely that the effects, at least in chicken embryo fibroblasts, a r e primarily a t the membrane level related to permeability changes. Our results with Mg2+depletion show only a small increase in total cell Ca2+ under conditions involving large decreases in rates of DNA synthesis. Most of the
29
changes in Ca2+can be accounted for by a n increase in external membrane surface binding. Although the possibility does exist that release of a pool of Caz+as in the sarcoplasmic reticulum in muscle or from mitochondria could occur without a change in total cell Ca2+,no evidence for this exists a t present in chicken embryo fibroblasts. Recent experiments concerning the specificity of t h e requirements for Mg2+and CaZ+provide further evidence that Mg2+is the direct intracellular effector and that Ca2' (and Sr2+)act indirectly by making Mg2+available for metabolic work (Rubin and Koide, '76; Rubin, '77). Several important lines of evidence favor a key role for Mg2+ as a primary regulator of cell metabolism and growth: (1) the known requirement of Mg2' as a cofactor for a multitude of enzymatically catalyzed intracellular reactions, especially those involving transphosphorylations, and protein syntheis. Mg2+is required by various aminoacyl-tRNA synthetases and is also involved in maintaining the conformation of ribosomes and nucleic acids (Heaton, '73); (2) strong evidence that [Mg2+l,is in the range required for significant effects on key reactions with small changes in free Mg2+.For example, experimental (England e t al., '67; Rose, '68; Palaty, '71) and computer calculations (Kerson, e t al., '67) both indicate that free [Mg2'l in mammalian tissue is 1mM or less. This is below the concentration necessary for maximum activity of pyruvate kinase in rat heart (Kerson et al., '67) and hexokinase in ox brain (Bachelard and Goldfarb, '69) so that small changes in free Mg2' are potentially capable of exerting a considerable degree of metabolic control; (3) the recent observation that the coordinate inhibition of 2-deoxy-D-glucose and uridine uptake and the incorporation of uridine and thymidine into acid insoluble material by omission of serum or addition of cortisol is reproduced quantitatively by lowering the concentration of Mg2' in the growth medium (Rubin, '76) ; (4) the closely related graded decreases in intracellular [Mg2+lparalleling decreases in rates of DNA synthesis as demonstrated in the present investigations. Similar relationships between [Mg2+l,and DNA synthesis have been observed in subsequent studies with insulin as the stimulating agent (Sanui and Rubin, '77). Additional investigations now underway are necessary to clarify the role inorganic cations play in the regulation of cell metabo-
30
HISASHI SANUI AND HARRY RUBIN
lism. Those concerning the relative rates of change in cell cations and metabolism, the levels of free intracellular cations, and changes in membrane cation binding, pump activity and permeability are of primary importance. The information derived from these studies should elucidate further the key role that intracellular Mg2+may play in the coordinate control of metabolism and growth in animal cells.
Gunther, Th. 1966 Uber die Rolle des Mg bei der Regulation des Zellstoffwechsels. Z. fur Naturforsch., 21 b: 1174-1177. Gunther, Th., and R. Averdunk 1970 K-transport und Stoffwechsel von Mg-arm gewachsenen Yoshida-Ascitestumorzellen. 2. Klin. Chim. u. Klin. Biochem., 8:621-625. Heaton, F. W. 1973 Magnesium requirement for enzymes and hormones. Biochem. SOC.Trans., 1: 67-70. Kerson, L. A,, D. Garfinkel and A. S. Mildvan 1967 Computer simulation studies of mammalian pyruvate kinase. J. Biol. Chem., 242: 2124-2133. Leive, L. 1965 A nonspecific increase in permeability in Escherichia coti produced by EDTA. Proc. Nat. Acad. Sci., 53: 745-750. ACKNOWLEDGMENTS Lostroh, A. J., and M. E. Krahl 1974 Magnesium, a second We thank Toshiko Koide, Virginia Rimers messenger for insulin: ion translocation coupled to transport activity. Adv. in Enzyme Regulation. G. Weber, ed. and Joyce Walton for their capable assistance Pergamon Press, New York, 22: 73-81. during the course of this investigation. Lowry, 0.H., N. J. Rosebrough, A. L. Farr and R. J. Randall This investigation was supported by NIH 1951 Protein measurement with the Folin phenol Research Grant CA 15744 from the National reagent. J. Biol. Chem., 193: 265-275. Cancer Institute. Luckasen, J. R., J. G. White and J. H. Kersey 1974 Mitogenic properties of a calcium ionophore, A23187. LITERATURE CITED Proc. Nat. Acad. Sci., 71: 5088-5090. Achs, M., and D. Garfinkel 1973 Available magnesium ion Lusk, J. E., R. J. P. Williams and E. P. Kennedy 1968 Magnesium and the growth of Escherichia coli. J. Biol. Chem., as an effective metabolic switch. In: Regulation and Con243: 2618-2624. trol in Physiological Systems. A. Iherall and A. Guyton, eds. International Federation of Automatic Control Sym- Palaty, V. 1971 Distribution of magnesium in the arterial wall. J. Physiol. (London), 218: 353-368. posium, pp. 19-21. Bachelard, H. S., and P. S. G. Goldfarb 1969 Adenine nu- Peck, E. J.,Jr., and W. J. Ray, Jr. 1971 Metal complexes of phosphoglucomutase in uiuo. Alterations induced by cleotides and magnesium ions in relation to control of insulin. J. Biol. Chem. 246: 1160-1167. mammalian cerebral-cortex hexokinase. Biochem. J.. Rein, A., and H. Rubin 1968 Effects of local cell concentra112 579-586 tion upon the growth of chick embryo cells in tissue culBalk, S D 1971 Calcium as a regulator of the prohferatlon ture. Expt. Cell Res., 49: 666-678. of normal but not of transformed, chicken fibroblasts in a plasma-containing medium. Proc. Nat. Acad. Sci., 68: Rose, I. 1968 The state of magnesium in cells as estimated from the adenylate kinase equilibrium. Proc. Nat. 271-275. Acad. Sci. 61: 1079-1086. Balk, S. D., J. F. Whitfield, T. Youdale and A. C. Braun 1973 Roles of calcium, serum, plasma and folic acid in Rubin, H. 1971 pH and population density in the regulation of animal cell multiplication. J. Cell. Biol., 51: 686th e control of proliferation of normal and Rous sarcoma 702. virus-infected chicken fibroblasts. Proc. Nat. Acad. Sci., 1972 Inhibition of DNA synthesis in animal 70: 675-679. cells by ethylene diamine tetraacetate, and its reversal Bygrave, F. L. 1976 Cellular calcium and magnesium by zinc. Proc. Nat. Acad. Sci., 69: 712-716. metabolism. I n : An Introduction to Bio-Inorganic 1973 pH. serum and Zn++in the regulation of Chemistry. D. R. Williams, ed. Charles C. Thomas, PuhDNA synthesis in cultures of chick embryo cells. J. Cell. lishers, Springfield, Illinois, pp. 171-184. Physiol., 82: 231-238. Ceccarini, C., and H. Eagle 1971 pH as a determinant of 1975 A central role for magnesium in coordicellular growth and contact inhibition. Proc. Nat. Acad. nate control of metabolism and growth in animal cells. Sci., 68: 229-233. Proc. Nat. Acad. Sci., 72: 3551-3555. Duibecco, R., and J. Elkington 1975 Induction of growth in 1976 Magnesium deprivation reproduces the resting fibroblastic cell Eultures by Ca". Proc. Nat. Acad. coordinate effects of serum removal or cortisol addition Sci., 72: 1584-1588. on transport and metabolism in chick embryo fibroblasts. Eagle, H. 1973 The effect of environmental pH on the J . Cell. Physiol., 89: 613-625. growth of normal and malignant cells. J. Cell. Physiol., 1977 Specificity of the requirements for mag82: 1-8. nesium and calcium in the growth and metabolism of England, P. J.,R. M. Denton and P. J. Randle 1967 The inchick embryo fibroblasts. J. Cell. Physiol., 91: 449fluence of magnesium ions and other bivalent metal ions 458. on the aconitase equilihrium and its hearing on the binding of magnesium ions by citrate in rat heart. Biochem. Rubin, H., and D. Fodge 1974 Interrelationships of glycolysis, sugar transport and initiation of DNA synthesis in J., 105: 32C-33C. chick embryo cells. In: Control of Proliferation of Animal Fodge, D., and H. Rubin 1975 Stimulation of lactic acid Cells. Vol. 1.B. Clarkson and R. Baserga, eds. Cold Spring production in chick embryo fibroblasts hy serum and high Harbor Laboratory, New York, pp. 801-816. pH in the absence of external glucose. J. Cell. Physiol.. Rubin, H., and T. Koide 1973 Inhibition of DNA synthesis 86: 453-457. in chick embryo cultures by deprivation of either serum Gerber, G., H. Berger, G-R. Janig and S. M. Rapoport 1973 or zinc. J. Cell Biol. 56: 777-786. Interaction of haemoglobin with ions. Quantitative description of the state of magnesium, adenosine 5'-triphos- _ _ 1976 Mutual potentiation by magnesium and calcium of growth in animal cells. Proc. Nat. Acad. Sci., and human haemoglobin phate, 2,3-b1sphosphoglycerate, 73: 168-172. under simulated intracellular conditions. Eur. J. BioSanui, H. 1970 pH dependence of the effect of adenosine chem., 38: 563-571.
Mg2+ CONTROL OF CELL CATIONS AND METABOLISM triphosphate and ethylene-diaminetetraacetate on sodium and magnesium binding by cellular membrane fragments. J. Cell. Physiol., 75: 361-368. 1971 Activated oxygen ashing of biological specimens for t h e microdetermination of Na, K, Mg and Ca by atomic absorption spectrophotometry. Anal. Biochem., 42: 21-28. Sanui, H., and N. Pace 1965 Mass law effects of adenosine triphosphate on Na, K, Mg and Ca binding by rat liver microsomes. J. Cell. Comp. Physiol., 65: 27-30. 1966 Application of atomic absorption in the study of Na, K, Mg and Ca binding by cellular membranes. Appl. Spectry., 20: 135-144. 1967a The Mg, Ca, EDTA and ATP dependence of Na binding by rat liver microsomes. J. Cell. Physiol., 69: 3-10. 1967b Effect of ATP, EDTA and EGTA on t he simultaneous binding of Na, K, Mg and Ca by rat liver microsomes. J. Cell. Physiol., 69: 11-20. Sanui, H., and H. Rubin 1976 Role of magnesium in the
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regulation of cellular cation concentration and growth. Fed. Proc., 35: 500. 1977 Insulin effects on cation content and DNA synthesis in cultured cells. Fed. Proc. 36: 338. Shank, B. B., and N. E. Smith 1976 Regulation of cellular growth by sodium pump activity. J. Cell. Physiol., 87: 377-388. Sillen, L. G., and A. E. Martell 1964 Stability constants of metal-ion complexes. The Chemical Society, Special Publication No. 17. Burlington House, London, pp. 190-191. Tolberg, A. B., and R. I. Macey 1972 The release of membrane-bound calcium by radiation and sulfhydryl reagents. J. Cell. Physiol., 79: 43-51. Walser, M. 1967 Magnesium metabolism. Ergebnisse der Physiologie Biologischen Chemie und Experimentellen Pharmakologie, 59: 185-341. Whitfield, J. F., A. D. Perris and R. H. Rixon 1969 Stimulation of mitotic activity and the initiation of deoxyribonucleic acid synthesis in populations of rat thymic lymphocytes by magnesium. J. Cell. Physiol. 74: 1-8.
