May 25, 1979 - cell population density there was a significant increase in the ability of CDR to inhibit cyclic AMP formation. Further, the intracellular levels of ...
Proc. Nati. Acad. Sci. USA Vol. 76, No. 8, pp. 3962-3966, August 1979
Cell Biology
Chinese hamster ovary cell population density affects intracellular concentrations of calcium-dependent regulator and ability of regulator to inhibit adenylate cyclase activity (calmodulin/cyclic AMP)
DANIELE EVAIN*, CLAUDE KLEEt, AND WAYNE B. ANDERSON* *Laboratory of Molecular Biology and tLaboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205
Communicated by E. R. Stadtman, May 25, 1979
The adenylate cyclase activity [ATP pyroABSTRACT phosphate-lyase (cyclizing), EC 4.6.1.1] of crude Chinese hamster ovary cell membranes was inhibited 30-40% by low concentrations (6-600 ng/ml) of calcium-dependent regulator (CDR). This inhibitory effect was lost at concentrations of CDR above 600 ng/ml. The adenylate cyclase activity of membranes prepared from low population density Chinese hamster ovary cells was not appreciably altered by CDR. However, with increasing cell population density there was a significant increase in the ability of CDR to inhibit cyclic AMP formation. Further, the intracellular levels of CDR determined in the 12,000 X g supernatant and particulate fractions varied inversely with increasing cell population density. As cell number increased from 2 X 106 to 10 X 106 cells per dish the CDR concentration present in the supernatant fraction increased from 0.4 to 0.8 ,ug of CDR per mg of protein, while the. amount of endogenous CDR associated with the particulate fraction decreased from 0.6 to 0.4 ,ug of CDR per mg of protein. This suggests that possible changes in the distribution of CDR between the supernatant and membrane fractions might serve as a regulatory mechanism for activities under CDR control. A low molecular weight Ca2+-binding protein that mediates some of the regulatory effects of Ca2+ has been isolated from various tissues (1-5). This polypeptide of molecular weight 16,500 binds 4 mol of Ca2+ per mol of protein (6-7) and is referred to as calmodulin or calcium-dependent regulator (CDR). At least one form of 3',5'-cyclic-nucleotide phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) (Ca2+-dependent) requires CDR for full activity (1-5). More recently, CDR has been shown to exhibit other Ca2+-dependent, regulatory functions, including activation of erythrocyte Ca2+,Mg2+-ATPase (8, 9), actomysin ATPase (10, 11), stimulation of myosin light chain kinase (12, 13), modulation of phosphorylase kinase (14), and depolymerization of microtubules (15). Other reports indicate that CDR also functions as an activator of adenylate cyclase tATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from brain (16, 17). A number of cellular-properties of Chinese hamster ovary (CHO) cells are influenced by the intracellular contentration of cyclic AMP (see ref. 18 for review). Thus, it is of interest to identify factors that may be involved in regulating cyclic nucleotide metabolism in these cells. Intracellular Ca2+ concentrations have been implicated in the regulation of cyclic nucleotide metabolism (1, 2).. In this communication we report on studies carried out to investigate the possible involvement of CDR in this regard. Contrary to the findings with brain tissue, CDR is inhibitory toward adenylate cyclase activity of crude CHO membranes. With increasing cell population density, it is observed that the intracellular concentration of
soluble CDR increases while there is a progressive decrease in the amount of CDR associated with the crude membrane fraction. Corresponding to this apparent change in intracellular CDR localization, membrane adenylate cyclase activity increases with increasing cell population density while the inhibitory effect of added CDR toward adenylate cyclase activity is potentiated. EXPERIMENTAL PROCEDURES Materials. The sources of materials used in the adenylate cyclase assay (19) and cyclic nucleotide phosphodiesterase assay (20) were as previously described. The cyclic AMP antigen was from Collaborative Research (Waltham, MA). The a modified Eagle's medium was from Flow Laboratories (Rockville, MD) and fetal bovine serum was purchased from Associated Biomedic Systems (Buffalo, NY). CDR was purified from bovine brain acetone powder as described (20). Growth of Cells. CHO cell line 10001 was a subclone of a CHO pro-line originally derived by Puck et al. (21) and kindly provided to us by M. Gottesman. Cells were grown in suspension at 370C in a modified Eagle's medium with Earle's salts with 10% fetal bovine serum. The cells were plated on 100-mm tissue culture dishes (Costar, Cambridge, MA) 3 days prior to each assay and grown in a 5% CO2 humidified atmosphere. The growth medium was changed 24 hr prior to assay. For the cell density studies, cells were planted at different densities and allowed to grow for 3 days .to reach the density indicated. Studies with cells at densities less than 2 X 106 cells per dish are not included because the adenylate cyclase activity of such cells was too low for accurate measurement. For cell counts, cells were removed from two dishes by trypsin treatment (0.1% trypsin in 25 mM Tris/5.5 mM dextrose/0.14 M NaCl/5 mM KCI/0.7 mM Na2HPO4) and counted with a Coulter Counter.
Adenylate Cyclase Assay. Cells grown on dishes were washed, harvested, and homogenized, and crude membranes were isolated as described (19). In determinations with light and heavy cell population densities approximately the same numbers of cells were harvested in the same final volume of buffer to give similar protein concentrations for each experimental sample. Adenylate cyclase activity of crude membranes was determined by measuring the conversion 'of [a-32P]ATP to cyclic [32P]AMP as described (19). The standard incubation mixture (final volume 50 Ml) contained 25 mM Tris-HCl, pH 7.8, 10 mM phosphoenolpyruvate, 4 ,ug of pyruvate kinase, 5 mM MgCl2, 0.2 mM ATP, [a-32P]ATP (4-5 X 106 cpm), and 30-60 ug of membrane protein. CDR was added to the incu-
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Abbreviations: CDR, calcium-dependent regulator or calmodulin; CHO, Chinese hamster ovary cells; EGTA, ethylene glycol bis(f3aminoethyl ether)-N,N,N',N'-tetraacetic acid.
3962
Proc. Natl. Acad. Sci. USA 76 (1979)
Cell Biology: Evain et al. bation mixture just prior to initiation of the reaction with membranes. The stock solution of CDR contained 0.21 mg of CDR per ml of 30 mM Tris1HC1, pH 7.4/0.1 M NaCi, and was diluted with buffer containing 0.5 mg of bovine serum albumin per ml just prior to use. Each experiment has been- repeated at least two times and the results of duplicate and triplicate determinations within each experiment agreed within + 10%. While the extent of inhibition noted with a given concentration of CDR was somewhat variable from experiment to experiment (i.e., 15-50% inhibition with CDR at 60 ng/ml), inhibition was consistently observed: Cyclic Nucleotide Phosphodiesterase Assay. Cyclic AMP phosphodiesterase was assayed by measuring the formation of [3H]AMP from cyclic [3H]AMP as described by Klee (20). One unit is formation of lpmol per min. A crude homogenate was centrifuged at 12,000 X g for 10 min and the resulting supernatant was used as the source of enzyme. The assay was carried out with 10 ,gM cyclic AMP at 30 'C for 20 min. Determination of CDR Level. CDR was assayed by its ability to activate CDR-dependent cyclic AMP phosphodiesterase purified by affinity chromatography with CDR coupled to Sepharose(5). Cells were homogenized, and the particulate fraction was resuspended, in 0.33 M sucrose/1 mM MgCl2/50 mM Tris-HCl, pH 7.8, buffer. The specificity for activation of phosphodiesterase activity by CDR was determined by showing inhibition of this effect with ethylene glycol bis(3-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The incubation mixture contained 0.04 M Tris-HCI (pH 8.0), 3 mM MgCI2, 0.05 mM CaCl2, 0.2 M NH4CI, 0.5 mM dithioerythritol, 0.01 mg of bovine serum albumin, 0.002 unit of cyclic AMP phosphodiesterase (specific activity 22 units/A28o unit), 80,000 cpm of cyclic [3H]AMP, 1 mM cyclic AMP, and 1500 cpm of [14C]AMP in a final volume of 0.1 ml. Samples to be assayed were boiled for 3 min or not boiled, and 2- 30-,M1 aliquots were tested for their ability to activate cyclic AMP hydrolysis. Precipitates resulting from boiling from not removed prior to assay of CDR. The concentration of CDR present in the samples was determined by using a standard curve obtained simultaneously with purified CDR. The linear range was between 1 and 5 nM (n17-85 ng of CDR per ml). Determinations were carried out at two or three concentrations within this range. Protein Determination. Total cellular protein was determined in identically treated companion cultures and in membrane and supernatant preparations by the method of Lowry et al. (22) with bovine serum albumin as standard. RESULTS CDR Inhibition of Adenylate Cyclase. Fig. 1 shows the effect of increasing CDR concentration on the adenylate cyclase activity of crude CHO membranes. When added at low concentrations (6-600 ng/ml), CDR inhibited basal (Fig. )- and GTP-stimulated (unpublished data) adenylate cyclase activity. This inhibitory effect was lost at concentrations of CDR above 600 ng/ml. The addition of 10,uM Ca2+ did not appreciably enhance the inhibitory effect of CDR, presumably because the reaction mixture, as well as the CDR preparation itself, contained sufficient Ca2+ to support the CDR effect. It should be emphasized, however, that the amount of Ca?+ added with the CDR (;15 nM Ca2+ with CDR at 60 ng/ml) did not alter cyclase activity. CDR did not significantly alter fluoride-stimulated cyclase activity (unpublished data). In order to establish the specificity of the CDR response it was of interest to determine if troponin C, a protein structurally similar to CDR (23, 24), or another acidic protein, lactalbumin, would also alter adenylate cyclase activity. As shown in Table 1, lactalbumin and troponin C both inhibited adenylate cyclase
3963
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CDR, ng/mI FIG. 1. Effect of increasing CDR concentration on the basal adenylate cyclase activity of crude membranes prepared from confluent CHO cells (10 X 106 cells per dish), measured in the presence
(A)
or
absence (0) of
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Ca2~
activity. However, the presence of EGTA did not alter the inhibitory effect of these two proteins, whereas it did negate the inhibition noted with CDR. This indicates that the CDR effect on adenylate cyclase activity is a Ca2+-specific function. In addition, the CDR inhibitory protein (25) did not modify the inhibition observed with lactalbumin and troponin C, whereas it did abolish the inhibitory effect of CDR toward adenylate cyclase (unpublished data). Calcium alone did inhibit adenylate cyclase activity in a concentration-dependent manner, with 50% inhibition observed at f0.2-0.5 mM Ca2+ (see Fig. 2). The addition of EGTA to the cyclase
reaction mixture
increased the activity of
adenylate
cyclase, apparently by removal of Ca2+ or other heavy metal inhibitors. As shown -in Fig. 2, no Ca2+ stimulation or inhibition was observed with concentrations of added Ca2+ that were less than the concentration of EGTA (0.1 mM) present. These results differ from those reported for the enzyme from C-6 glioma cells, in which stimulation of cyclic AMP formation was noted with concentrations of Ca2+ added below the concentration of EGTA (26). Influence of Cell Population Density on CDR Effects. Considerable variability in the ability of CDR to inhibit adenylate cyclase activity was initially observed. This led us to determine the effect of CDR on adenylate cyclase at different cell densities. As shown in Fig. 3, CDR had little effect on the adenylate cyclase activity of membranes prepared from low population density CHO cells. However, with increasing cell population density there was a significant increase in the ability of CDR to inhibit cyclic AMP formation. With increasing cell population density there was also a progressive increase of the ability of 0.1 mM EGTA to activate adenylate cyclase activity (see Fig. 3). However, it is not apTable 1. Effect of acidic proteins and CDR on basal adenylate cyclase activity in the presence and absence of EGTA Adenylate cyclase activity, pmol cyclic AMP formed/15 min per mg protein CDR Condition Control Lactalbumin. Troponin.C
17±0.01 18 0.06 19+0.05 25±0.1 24±0.05 35±0.08 23 0.06 38±0.6 The assay was carried out with acidic proteins or CDR at 60 ng/ml in the presence or absence of 0.1 mM EGTA. The values are the average of triplicate samples ± SEM. -EGTA
+EGTA
Cell Biology: Evain et al.
3964
Proc. Natl. Acad. Sci. USA 76 (1979)
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parent if there is an interrelationship between the increased response to EGTA and the increased sensitivity of the cyclase activity to CDR. Effect of Cell Population Density on the Intracellular CDR Level. Because the CDR inhibitory effect toward adenylate cyclase is influenced by cell population density, it was of interest to establish the intracellular concentrations of CDR I
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present in the 12,000 X g supernatant and particulate fractions at various cell densities. The results presented-in Fig. 4 show that 0.4 jig of CDR per mg of supernatant protein was present in the supernatant prepared from low population density cells. As the cell number increased to 9 X 106 cells per dish, there was a corresponding increase in the concentration of supernatant CDR (to 0.8 jug of CDR per mg of protein). As the population density continued to increase, the level of supernatant CDR remained constant or decreased slightly. In another study, values of 0.37 and 1.2 tig of CDR per mg of protein were observed in the supernatants from low and high cell densities, respectively. On the other hand, when CDR levels were determined in the 12,000 X g particulate fraction, there was a progressive decrease in the amount of CDR present with increasing cell population density. At low cell population density (2 X 106 cells per dish) there was 0.6 Mg of CDR per mg of particulate protein, and this decreased to ;0.4 jig of CDR per mg of particulate protein at high cell densities. Influence of Cell Population Density on Cyclic AMP Metabolism in CHO Cells. Because the intracellular CDR levels and the ability of CDR to inhibit adenylate cyclase are modulated with increasing cell population density, it was of interest to determine any changes that might occur in cyclic AMP metabolism as a function of cell density. As has been observed in other fibroblastic cells (27, 28), the activities of adenylate cyclase and cyclic AMP phosphodiesterase both, increased with increasing cell population- density (Fig. 5). Basal adenylate cyclase activity increased with increasing cell population until a density of 10-11 X 106 cells per dish was reached. At cell densities above this level, when the cells began to grow in multilayers, there was a sharp fall in cyclase activity. Cyclic AMP phosphodiesterase activity also increased with increasing cell density, and it continued to increase even at population densities above 10-11 X 106 cells per dish. Such an increase in cyclic AMP phosphodiesterase activity would correlate well with an increase in CDR levels to poten-
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FIG. 3. Effect of CDR (0) and EGTA (-) on adenylate cyclase activity as a function of increasing cell population density. The effect of CDR on cyclase activity was determined in the presence or absence of CDR at 60 ng/ml with 10 ,M GTP and 10 ,uM Ca2+ and membranes prepared from cells at the indicated population density. Results with CDR are expressed as the percent inhibition of cyclase activity relative to controls measured in the absence of CDR. The effect of EGTA on basal cyclase activity was determined in the presence or absence of 0.1 mM EGTA with membranes prepared from cells at the indicated population density. Results with EGTA are given as relative activation, which is the specific activity of adenylate cyclase determined in the presence-of EGTA divided by the specific activity of adenylate cyclase determined in the absence of EGTA.
0.4 -
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FIG. 4. Effect of increasing cell population density on supernatant (0) and particulate (0) levels of CDR. The concentration of CDR was determined in the 12,000 X g supernatant and particulate fractions prepared from cells at the indicated population densities. The results presented are from two separate experiments. Values are expressed as the mean I SEM of triplicate determinations of each
sample.
Proc. Natl. Acad. Sci. USA 76 (1979)
Cell Biology: Evain et al.
7
11
9
Cells X 10O
per
1:
dish
FIG. 5. Basal adenylate cyclase activity (0) and cyclic AMP phosphodiesterase activity (A) in CHO cells as a function of increasing cell population density.
have been unable to demonor Ca2+ plus CDR on the cyclic AMP phosphodiesterase activity (determined with either 1 AuM or 1 mM cyclic AMP) of crude supernatants prepared from either low or high cell population density CHO cells (unpublished results). The apparent inability of CDR to modulate the supernatant phosphodiesterase activity of CHO cells is similar to the results obtained with the cyclic AMP phosphodiesterase of smooth muscle (29). However, it does not rule out the possibilities that a more highly purified enzyme preparation would be activated by CDR or that in crude extracts proteolytic activities or inhibitors mask the CDR effect. tiate this activity. However,
we
strate any effect of EGTA, Ca2
,
DISCUSSION The adenylate cyclase activity of brain tissue (14, 15) and of C-6 glioma cells (26) shows a biphasic response to changes in Ca2+ and CDR concentration; i.e., activity is enhanced by low concentrations of CDR or free Ca2+ (
