Proc. Nat. Acad. Sci. USA Vol. 70, No. 12, Part I, pp. 3609-3612, December 1973
Role of Cyclic 3': 5 '-Adenosine Monophosphate in the Early Transport Changes Induced by Serum and Insulin in Quiescent Fibroblasts (uridine/phosphate/prostaglandins/tissue culture) ENRIQUE ROZENGURT* AND LUIS JIMENEZ DE ASUA* Department of Biological Sciences, Princeton University, Princeton, New Jersey 08540; and Imperial Cancer Research Fund Laboratories, Lincoln's Inn Fields, London, England Communicated by Arthur B. Pardee, July 23, 1973
ABSTRACT Serum or insulin added to quiescent Looking for a relationship between these early membrane mouse-embryo fibroblasts produced rapid increases in the changes, we found that the endogenous levels of cAMP rate of transport of uridine and phosphate and a decrease of the intracellular concentration of cyclic AMP. Incuba- dramatically affect the activation of uridine transport in- tion of the cells with prostaglandin E1, theophylline, or duced by serum or insulin. Addition or removal of prosta- both prevented the increase in uridine transport produced glandin E1 (PGE1) and theophylline brought about large and by serum or insulin. Prostaglandin E1 was more effective rapid transport changes that were associated with variations than prostaglandins E2 and B1. Kinetic experiments showed that the addition of prostaglandin E1 and theophylline in cAMP concentration. In contrast, phosphate uptake was causes the uridine transport rate to return to the basal not affected by the intracellular levels of cAMP. Thus, the level within 5 min; the rate rose rapidly after their removal. cyclic nucleotide is involved in the control of some specific Similar effects were obtained when insulin was used in- changes in membrane permeability. stead of serum. Associated with these transport changes were significant variations in the levels of cyclic AMP. MATERIALS AND METHODS An inverse correlation between the changes in uridine transport and those in cyclic AMP concentration was Secondary mouse-embryo fibroblasts were seeded in Dul- shown under various experimental conditions. In contrast becco's modified Eagle's medium supplemented with 0.2% to the effects observed with uridine transport, phosphate serum, 100 units/ml of penicillin, and 100 ug/ml of strepto- uptake was only slightly affected by changes in the endog- mycin in Nunc petri dishes. The cells were allowed to become enous level of cyclic AMP. We propose that at least two different sorts of membrane changes are rapidly initiated quiescent by incubating in this low-serum medium for at by serum, one under cyclic AMP control and the other not least 3 days before they were used. related to this nucleotide. Transport was measured with cells attached to 30-mm dishes. In most experiments, the medium was replaced by Quiescent cultures of untransformed fibroblasts recommence fresh serum-free medium with or without additions and after growth when serum is added. Selective increases in membrane 10 min, serum or insulin was added at a final concentration of permeability and changes in the intracellular concentration of 10% or 8 ug/ml, respectively. At the times indicated in each cyclic AMP (cAMP) are among the earliest events associated experiment the cells were exposed to labeled substrate for 5 with reinitiation of growth stimulated by addition of serum min and then washed four times with isotonic saline. Uptake (see ref. 1 for review). The rate of transport of uridine and into the acid-soluble fraction was measured by extracting the phosphae but not that of glucose and amino acids, is increased washed cells for 30 min at 40 with 5% trichloroacetic acid and several fold within 15 min after addition of serum to density- counting an aliquot in Triton-toluene scintillation fluid. inhibited 3T3 cells (2). cAMP, which is becoming implicated Cyclic AMP was determined by the binding competition increasingly in control of growth of fibroblasts (3-6), shows method described by Gilman (7), with cAMP binding protein opposite changes. A rapid fall in cAMP concentration was and protein kinase inhibitor from bovine muscle. The trichlo- found when serum or insulin was added to quiescent fibroblasts roacetic-acid extracts were purified by Dowex 50 chromatog- (4-6). Changes that occur earliest are of particular interest raphy as described (8). Treatment of samples prepared in in the search for triggering events and for cause and effect this way with cyclic nucleotide phosphodiesterase showed that connections between addition of serum and subsequent meta- the competing material was cAMP. Protein was determined bolic changes. by the method of Lowry et al. (9), after the attached cells The purpose of this paper is to investigate a possible role were washed five times with phosphate-buffered salt solution of cAMP in the early transport changes. The increases in up- (pH 7.2) take might be mediated by an indirect mechanism involving Nucleotides, theophylline, and cyclic nucleotide phospho- changes in the intracellular level of cAMP; conversely, the diesterase were obtained from Sigma. Crystalline insulin was transport changes might signal a modification of the mem- from Calbiochem. Prostaglandins were the generous gift of brane structure, that includes lower internal concentrations Dr. John Pike, Upjohn Co. Isotopically labeled compounds of cAMP; or the two phenomena could be unconnected. were obtained from The Radiochemical Center. Abbreviations: cAMP, cyclic 3': 5'-adenosine monophosphate; RESULTS PGE1, prostaglandin E1. Addition of serum to mouse-embryo fibroblasts made quies- * Present address: Imperial Cancer Research Fund, Lincoln's Inn cent by incubating in a low-serum medium brings about a 2- Fields, London, WC2, England. to 4-fold increase in uridine transport within 30 min. Pros- 3609 Downloaded by guest on October 2, 2021 3610 Biochemistry: Rozengurt and Jimenez de Asua Proc. Nat. Acad. Sci. USA 70 (1978)
or insulin stimulation of uridine uptake (Fig. 2C and D). Experiments with exogenous cyclic nucleotides including dy- butyryl cyclic AMP were of limited value in this system be- cause 5'-AMP itself was inhibitory in the concentration range tested (0.2-1 mM). The kinetics of the changes in uridine uptake produced by addition or removal of PGE1 and theophylline in the presence of serum or insulin are shown in Fig. 3. A control without inhibitors shows that the rate of transport reached a maximum in 30 min. The addition of PGE1 plus theophylline before serum completely prevented the stimulatory effect of the latter. Removal of these compounds after 40 min of exposure was followed by a rapid recovery in transport which, however, was not complete (Fig. 3A). Conversely, when PGE1 and theophylline were added at different times after serum the 10 20 30 Prostaglandin yg/ml transport rate returned to the basal level in 5 min (Fig. 3B). Similar effects were obtained when insulin was used instead of FIG. 1. Effect of different prostaglandins on the serum-induced serum (Fig. 3C and D). increase in uridine transport. Prostaglandin El (0), prostaglandin cAMP levels were determined under conditions parallel E2 (0), and prostaglandin B1 (0) at the concentrations indicated, to those described for the kinetics of transport changes (Fig. were added 10 min before the fresh serum-free medium was sup- 4). Addition of serum to quiescent cultures reduced the in- plemented with serum. Some dishes were incubated with prostag- tracellular level of cAMP, as was reported for other cell lines landin El but without serum (A). The concentration of [3H] uridine (4-6). PGE, plus theophylline added 10 min before serum was 2.5 ACi/ml (29 Ci/mmol) and the cells were labeled (for 5 min) 30 min after addition of serum. Uptake into acid-soluble pool 8 (A) (B) was measured. All points represent averages of duplicate samples. la 6 ZIA V taglandin El added 10 min before serum preferentially in- a'A .v hibits the rise and was more effective than prostaglandins 'A~ v E2 and B1 (Fig. 1). Since all these prostaglandins activated 4 , ,_\v _# the adenylate cyclase activity of mouse-embryo fibroblasts A A 2 (10), the results suggest that cAMP is involved in the early 0 transport changes induced by serum. This possibility is fur- ther substantiated by the experiments shown in Fig. 2. 0 I 0 1 2 3 0 1 2 3 Theophylline, a well-known inhibitor of phosphodiesterase CL Theophylline ( mM ) activity, also inhibits the serum stimulation of uridine trans- co port in a concentration-dependent manner but does not de- (D) press the uptake rate in the absence of serum. This makes a ._ direct competitive effect of this compound on the uridine transport system unlikely. Insulin, which decreases the in- D mKZZ2 5, and see below), stimulated tracellular level of cAMP (4, 11, 50 uridine transport in the absence of serum. This stimulation 0f~ was completely inhibited by theophylline (Fig. 2B). Similar results were obtained when density-inhibited 3T3 cells were 0~~~~~~~~~~~~~~~- stimulated by serum or insulin. Furthermore, theophylline increased the effectiveness of PGE1 in preventing the serum I o 0 10 20 30,10 10 20 30 Prostaglandin (,ug/mI) TABLE 1. Effect of prostaglandin E1, theophylline, and El insulin on the cAMP levels of mouse-embryo fibroblasts FIG. 2. (A and B) Effect of different concentrations of theo- phylline on the serum (A)- or insulin (B)-induced increase in cAMP uridine transport. Theophylline was added 10 min before serum or Additions* (pmol/mg of protein) insulin (8,g/ml). Some dishes were incubated with theophylline but without serum or insulin (closed symbols). Transport rates are 22.7± 0.9 expressed as cpm X 10-3 per dish. (C and D). Potentiation by Insulin 14.3 ± 4 theophylline of the inhibition of uridine transport produced by Insulin + theophylline 27.5 ± 2.3 PGE,. Cells were incubated for 10 min with PGE1 (open symbols) PGE, 550 ± 14 or PGEI + theophylline (closed symbols) and then serum (C) or Insulin + PGE, 347 ± 27 insulin (D) was added. Bars represent the basal rate of transport Insulin + PGE1 + theophylline 1093 + 120 determined in serum-free medium. The concentration of theophyl- line used in the presence of serum or insulin was 1 and 0.6 mM, * Quiescent mouse-embryo fibroblasts were incubated with respectively. For comparison, transport was expressed as per- PGE1 (25,g/ml), theophylline (1.5 mM), or both for 10 min. centage of the stimulated value. Transport was determined 30 min Then, insulin (5 mg/ml) was added as indicated and cAMP was after addition of serum or insulin, and each point represents the determined 30 min later. average of duplicate samples. Downloaded by guest on October 2, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) Transport and Cyclic AMP 3611
promoted a 100-fold increase in cAMP concentration. This high level of cAMP was not substantially modified by the subsequent addition of serum and may, therefore, account for the inhibitory effect of PGE1 and theophylline on the stimulation of uridine transport by serum. Removal of the PGE1 and theophylline was followed by a rapid drop in cAMP concentration which, however, had not returned to the basal value after 5 min. A similar effect of removal of PGE, was observed with L cells (8). Possibly after an acute eleva- tion of cAMP a fraction of the total nucleotide pool is bound E
and not readily available to hydrolysis by phosphodiesterase E (12). When PGE1 and theophylline were added 30 min after Z 102 serum they produced a dramatic increase in cAMP level !. I~~~~~~
(A) (B)
to 10 ,
-10 0 10 20 30 40 Minutes tIO FIG. 4. Kinetics of the changes in cAMP concentration. Mouse-embryo fibroblasts were seeded in 90-mm dishes (3 X 106 o ~ ~ ~ ~ ~ cells) and allowed to become quiescent. The cultures were incu- bated with (0) or without (0) PGE, (25 ,ug/ml) and theophylline (1 mM) for 10 min. Then, serum was added (0 time) and cAMP was determined 20 and 35 min later. At 30 min, the medium con- 0 taining PGEI and theophylline was removed from some dishes. The dishes were washed and incubated with fresh serum contain- 0 ing medium without the compounds. cAMP was measured 5 Inin
co 0 0 20 40 60 0 20 40 -60 later (0). Other dishes received PGE, and theophylline, and the incubation was terminated 5 min later (U). Bars indicate SD. *< (C)/
U ZU 4U t>U 0U 1W UZ 40 60 80 100 Minutes TABLE 2. Effect of prostaglandin E1 and theophylline on FIG. 3. Kinetics of the uridine transport changes induced by phosphate and uridine transport induced by serum and insulin PGEI and theophylline in the presence of serum (A and B) or in- sulin (C and D). (A) Cells were incubated in serum-free medium PGE, + [3H]- [32P]_ with (A) or without (A) PGEI (25 ,g/ml) and theophylline (1 Conditions theophylline Uridine Phosphate mM). After 10 min, serum was added (0 time). Other dishes did not receive serum and served to determine the basal transport - 100 100 level (X). At 43 min, the medium containing PGEI and theophyl- Serum - 254 339 line was removed from some dishes. The dishes were washed and Serum + 132 274 incubated with fresh serum without the compounds (0). (B) Insulin - 200 140 PGE1 and theophylline were added 23 (0) and 53 (o) min after Insulin + 78 138 addition of serum. (X) Basal transport level. (C) Cultures were incubated in serum-free medium with (U) or without (0) PGE, The figures represent percentages of the transport rates ob- and theophylline. 10 min later insulin (8 pg/ml) was added (0 tained in serum-free medium. PGE, (20 /Ag/ml) + theophylline time). (X) Basal transport level. At 35 min, the reversibility of (1 mM) were added 10 min before serum or insulin. Transport the transport inhibition was determined (V) as in (A) for serum. was determined 30 min after addition of serum or insulin. The (D) PGE, and theophylline were added 20 (V) and 50 (V) mi specific activity of phosphate was 50 Ci/mol and 5,oCi/ml were after insulin. Transport was measured at different times, as in- added. Uridine transport rates were measured in the same experi- dicated. ( X ) Basal transport level. ment for comparison. Downloaded by guest on October 2, 2021 3612 Biochemistry: Rozengurt and Jimenez de Asua Proc. Nat. Acad. Sci. USA 70 (1973) changes in uridine transportand the endogenous concentration indicates that these transport changes may be a direct effect of cAMP. of cAMP. In contrast to the results obtained with uridine transport, In contrast to the effects observed with uridine transport, PGEI and theophylline inhibit only slightly the increase in phosphate uptake is not sensitive to the endogenous level of phosphate uptake observed shortly after addition of serum cAMP. Thus it seems that at least two sorts of membrane (Table 2). Furthermore, insulin had only a small stimulatory changes are rapidly stimulated by serum; one cAMP-depen- effect on phosphate transport. These results suggest that the dent and the other not related to this nucleotide. Whether increase in phosphate transport is controlled by a mechanism this second mechanism depends on alteration of membrane that is not dependent on cAMP. state directly or by influence of another intracellular mediator such as cGMP is unknown. DISCUSSION Selective changes in transport have been proposed as an The present results indicate that the endogenous levels of important regulatory point for modulating growth (20, 21). cAMP may be involved in regulation of early transport Although the uptake of nucleosides constitutes a salvage changes induced by serum and insulin in quiescent fibro- metabolic pathway that is unlikely to control growth, the blasts. Support for this notion comes from the following lines possibility that some of the effects on growth produced by of evidence: (1) both serum and insulin decrease cAMP and cAMP agents are mediated by functional alterations of the stimulate uridine transport; (2) prostaglandins El, E2, and cell surface remains attractive. B1, which activate adenylate cyclase activity from mouse- embryo fibroblasts (10), inhibit stimulation of transport; (3) E.R. is indebted to Dr. A. B. Pardee for advice and support blocks the rise in uridine transport and (Grant AI-CA 04409 from the U.S. Public Health Service). theophylline, which L.J.deA. was a Fellow of the John Simon Guggenheim Memorial increases the effectiveness of prostaglandin, prevents the drop Foundation. We thank Drs. R. Dulbecco and R. R. Burk for in cAMP by insulin and potentiates the accumulation of critical discussions during this study. The technical assistance of cAMP by PGEI; (4) the uptake changes induced by addition Mrs. C. A. Knight is also acknowledged. or removal of PGE1 and theophylline are observed in minutes; 1. Pardee, A. B. & Rozengurt, E. (1974) in Biochemistry of Cell associated with these transport changes are significant varia- Walls and Membranes, ed. Fox, C. F. (Medical & Technical tions in the levels of cAMP; and (6) the effect of PGE1 and Publ. Co., London), in press. theophylline on nucleoside transport is not simply due to a 2. Cunningham, D. D. & Pardee, A. B. (1969) Proc. Nat. Acad. general change in permeability; phosphate transport, which is USA 64,1049-1056. 3. Otten, J., Johnson, G. S. & Pastan, I. (1971) Biochem. also stimulated by serum, is not affected by the intracellular Biophys. Res. Commun. 44, 1192-1198. cAMP concentration. In addition, theophylline or PGE1 does 4. Otten, J., Johnson, G. S. & Pastan, I. (1972) J. Biol. Chem. not inhibit by a competitive effect since these compounds 247, 7082-7087. had little effect on the basal uridine transport rate. 5. Sheppard, J. R. (1972) Nature New Biol. 236, 14-16. Furthermore, the rate of uridine transport is sharply re- 6. Burger, M. M., Bombik, B.M., Breckenridge, B. M. & Sheppard, J. R. (1972) Nature New Biol. 239, 161-163. duced when the cells approach confluence (2, 13) whereas 7. Gilman, A. G. (1970) Proc. Nat. Acad. Sci. USA 67, 305- cAMP changes in the opposite direction (3, 4, 14). Conversely, 312. transformed cells do not show the reductions in uridine trans- 8. Manganiello, V. & Vaughan, M. (1972) Proc. Nat. Acad. Sci. port at confluence (2) nor do they display any change in USA 69, 269-273. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, cAMP (3, 14). R. J. (1951) J. Biol. Chem. 193, 265-273. All these results indicate that cAMP may control selective 10. Peery, C. V., Johnson, G. S. & Pastan, I. (1971) J. Biol. changes in cell permeability. The molecular basis of this con- Chem. 246, 5785-5790. trol is not known but the recent demonstration of cAMP- 11. Jimenez de Asua, L., Surian, E. S., Flawia, M. M. & Torres, endogenous phosphorylation of specific mem- H. N. (1973) Proc. Nat. Acad. Sci. USA 70, 1388-1392. dependent 12. O'Dea, R. F., Haddox, M. K. & Goldberg, N. D. (1971) brane proteins, a phenomenon that might be related to cell J. Biol. Chem. 246, 6183-6190. permeability regulation, provides a possible mechanism (15, 13. Weber, M. J. & Rubin, H. (1971) J. Cell. Physiol. 77, 157- 16). 168. Other studies have implicated cAMP in the regulation of 14. D'Armiento, M., Johnson, G. S. & Pastan, I. (1973) Nature Treatment of New Biol. 242, 78-80. membrane transport in cultured fibroblasts. 15. Rubin, C. S. & Rosen, 0. M. (1973) Biochem. Biophys. kes. CHO cells with dibutyryl cAMP and testosterone reduced Comm. 50, 421-429. transport rates for some amino acids (17), and exposure of 16. De Lorenzo, R. F., Walton, K. G., Curran, P. J. & Green- Py3T3 cells to dibutyryl cAMP and theophylline decreased gard, P. (1973) Proc. Nat. Acad. Sci. USA 70, 880-884. to that of 3T3 cells Re- 17. Rozengurt, E. & Pardee, A. B. (1972) J. Cell Physiol. 80, the transport rate of glucose (18). 273-279. cently, Kram et al. (19) have also implicated cAMP in the 18. Grimes, W. J. & Schroeder, J. L. (1973) J. Cell Biol. 56, 487- control of several membrane-transport systems of untrans- 491. formed 3T3 cells. However, in these studies the cells were ex- 19. Kram, R., Mamont, P. & Tomkins, G. M. (1973) Proc. Nat. posed to the compounds for hours or days and, therefore, the Acad. Sci. USA 70, 1432-1436. trans- 20. Pardee, A. B., Jiminez de Asua, L. & Rozengurt, E. (1974) important question of whether the effects observed on in Control of Proliferation in Animal Cells7 ed. Clarkson, B. port were a consequence of reduced growth rates or other & Baserga, R. (Cold Spring Harbor Laboratory), in press. metabolic alteration remained unanswered. The rapidity of 21. Holley, R. W. (1972) Proc. Nat. Acad. Sci. USA 69, 2840- the effects shown in the present paper for uridine transport 2841. Downloaded by guest on October 2, 2021