Brain Research, 117 (1976) 297-304 © Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
297
THE INFLUENCE OF METHYLXANTHINES ON PRECURSOR INCORPORATION INTO PROTEIN AND RNA OF MOUSE BRAIN
I. BOKSAY*, V. CSANYI**, J. GERVAI** and A. LAJTHA
New York State Research Institute for Neurochemistry and Drug Addiction, Ward's Island, New York, N. Y. 10035 (U.S.A.) (Accepted April 1st, 1976)
SUMMARY
Pentoxifylline at 1 mM had no effect on [14C]isoleucine incorporation into mouse brain tissue suspension. At 5-20 raM, this compound inhibited incorporation. The inhibition was prompt, and it was reversible. Aminophylline at 3-12 mM produced inhibition, but theophylline at 2-16 m M h a d no effect. Pentoxifylline inhibited the incorporation of uridine into brain RNA to the same extent and with a similar time course as its effect on protein synthesis.
INTRODUCTION
The effect on protein synthesis of pentoxifylline (3,7'-dimethyl-l-(5-oxohexyl)xanthine, BL191), a new methylxanthine derivative is,a4, was compared with that of aminophylline and theophylline. Previously, methylxanthines were reported to have beneficial effects in reducing cerebral edema induced by cold in cats 7,s. In addition, several reports of the effects of methylxanthine derivatives on protein synthesis have appeared 5,e,~. The model system that we used, studying [14C]isoleucine incorporation into a tissue suspension from brain, has the advantages of being a more easily controlled environment than that in vivo, and, at the same time, of containing living cells, which reflect the properties of the brain more closely than do cell-free preparations. In this respect, the tissue suspension technique provides an intermediate stage between tissue slices and homogenates. Of particular importance to the study reported here is that the specific activity of the precursor can be kept constant, and therefore drug effects on macromolecular metabolism can be studied without interference from drug effects on precursor penetration. Since previous work showed that rate of in* Hoechst-Roussel Pharmaceuticals Inc., Somerville, N. J. 08876, U.S.A. ** Experimental Station of L. Eotvos University H-2131 G6d, Javorka, S.u. 14, Hungary.
298 corporation into proteins in slices from young brain is approximately 80-901'.,, of that in vivo whereas the rate in slices from adult brain is less than 20 ~.~,of that in vivo 1.~,, we used tissue from young animals. MATERIALS AND METHODS Brain hemispheres removed from decapitated 3- or 4-day-old albino mice (inbred strain of the laboratory) were placed in a few milliliters of incubation medium at 0 °C. The composition of the medium was 119 m M NaC1, 5.0 m M KC1, 1.2 m M MgSO4 • 7HzO, 1.0 mM NaH2PO4 • 2H20, 1.0 m M NaHCO3, 0.7 m M CaCI2, 12.0 mM NaOH, 10.0mM glucose, and 2 5 . 0 m M N-2-hydroxyethylpiperazine-N'-2ethanesulphonic acid (HEPES); the pH was adjusted to 7.2. Before use, 100 #g/ml of penicillin and 50 ktg/ml of streptomycin were added. The hemispheres were pressed through a polyester bolting cloth (165 nm apertures) into the same medium gently with a spoon. The usual ratio of tissue to medium was one brain hemisphere to 5 ml medium. We used 0.4#Ci of [14C]isoleucine (272mCi/mmole; Calbiochem) or [2-14C]uridine (44 mCi/mmole; Calbiochem) in each 25 ml Erlenmeyer flask (except 0.15 #Ci in the experiments described in Fig. 1). Incubations of the suspensions were at 37 °C, in a water bath without shaking following ox.ygenation. Shaking did not affect incorporation in suspensions, but at times it resulted in increased tissue damage. To measure radioactivity, 0.8 ml samples of the suspension were added to 1.2 ml of ice-cold 20 % perchloric acid (PCA) that contained unlabeled isoleucine or uridine (1 mM). Determination of the radioactivity in protein. PCA-treated samples were heated to 85 °C for 20 min and centrifuged. The precipitate was washed twice with 2 ml 5 ~o PCA, then with 2 ml of ethanol-ether (2:3). After being dried at room temperature, it was dissolved in 1 ml of 1 N N a O H for counting; 0.5-0.8 ml portions of this N a O H solution (neutralized with HCI) were added to 10 ml of a scintillation mixture containing 6.35 g PPO (2,5-diphenyloxazole), 115 mg dimethyl POPOP (1-bis-2,4methyl-5-phenyloxazolyl-benzene), 538 ml toluene, and 462 ml Triton X-100 per liter. An Intertechnique scintillation spectrometer with external standard quench correction was used. Protein content was assayed by the method of Lowry et al. 2~. Determination of radioactivity of RNA. The samples suspended in PCA were centrifuged, the pellet was washed 3 times with ice-cold 5 % PCA, and the supernatant plus washings was used for determination of the precursors. The pellet was suspended in 5 % PCA and heated at 85 °C for 20 min; after centrifugation this supernatant was used for measurement of RNA radioactivity, and for RNA content by measuring the absorption at 260 nm in a Beckman spectrophotometer. Results presented in the figures are the averages of 4-6 experiments as indicated in the legends. The standard deviation of mean values was within 5 %. RESULTS The tissue suspension contained particles of 150-200 nm diameter, with many
299
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Fig. 1. Effect of antibiotics on [14C]isoleucine incorporation into mouse brain tissue suspension. Brain suspensions were incubated in a standard medium containing 0.4/~Ci/ml [14C]isoleucine. Averages of 6 experiments are shown. ×, control; +, no cells; O, 100/~g/ml penicillin, 50 pg/ml streptomycin; I , 50/~g/ml cycloheximide; IS],50 pg/ml streptomycin, 100/zg/ml penicillin, 50/zg/ml cycloheximide. intact cells. We found that cell suspensions incorporated radioactive amino acids for at least 6 h (Fig. 1). The rate of incorporation was somewhat higher in the first hour, but it remained fairly constant thereafter. In these preparations, the antibiotics were necessary to prevent incorporation due to bacterial contamination. Cycloheximide inhibits protein synthesis in m a m m a lian cells but does not affect bacterial growth. Penicillin and streptomycin inhibit bacterial growth, but do not inhibit protein synthesis in mammalian cells al. The rate of incorporation in the presence of penicillin and streptomycin was less than in their absence, the difference depending on the time period used for incorporation. Bac60O
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Fig. 2. Effect o f pentoxifylline on [ 1aC]isoleucine incorporation into mouse brain tissue suspension.
Experimental conditions as in Fig. 1 except that the medium contained pentoxifylline as indicated. Averages of 5 experiments are shown. × control; ©, 1 mM pentoxifylline; A, 10 mM pentoxffylline; A, 5 mM pentoxifylline; O, 20 mM pentoxifylline.
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Fig. 3. The reversibility of the inhibitory effect. At 15 min pentoxifyllin¢ (to 20 m M ) was added to some suspensions. At 45 min all suspensions were put in fresh (pentoxifylline-free) media. Averages of 5 experiments are shown.
terial contamination (penicillin- and streptomycin-sensitive incorporation) could not be eliminated by using sterilized media. As expected, the 3 antibiotics together completely inhibited the incorporation of isoleucine (Fig. 1). Pentoxifylline at a concentration of 1 mM had no effect, but higher concentrations (5-20 mM) inhibited isoleucine incorporation in brain tissue suspensions. Inhibition was almost complete at 20 mM (Fig. 2). The onset of inhibition of incorporation was prompt, because incorporation stopped immediately (Fig. 3) when incorporation was allowed to proceed and pentoxifylline was added later. Inhibition of the amino acid incorporation was reversible. Addition of pentoxifylline inhibited x ° r.
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TIME - rain. Fig. 4. Effect of pentoxifylline on [l~C]isoleucine and [!4C]uridine incorporation into mouse brain tissue suspension. Pentoxifylline when added was 20 raM. A v e r a ~ of 4 experiments are shown. Protein: × ×,control; × . . . . ×,l~ntoxifyllinv. Nucl¢icacid: © ©,control; O - - : O, pentoxifyllinc.
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Fig. 5. Effect of aminophylline on amino acid incorporation into mouse brain tissue suspension. Averages of 4 experiments are shown. ×, control; O, 3 mM aminophylline; • 12 mM aminophylline; • , 6 mM aminophylline.
the incorporation immediately; after the removal of the BL191 the tissue suspension resumed isoleucine incorporation, although at a rate somewhat lower than that of the untreated control (Fig. 3). The incorporation of uridine into the brain cell suspension of mice was also inhibited. Pentoxifylline inhibited incorporation into protein and RNA to a similar extent, and with a similar time course (Fig. 4). Theophylline in 4 different concentrations (2, 4, 8, and 16 mM) did not affect the [laC]isoleucine incorporation. Aminophylline (tested in concentrations of 3, 6, or 12 mM) showed a stronger inhibition of amino acid incorporation in brain tissue suspensions than did pentoxifylline (Fig. 5) DISCUSSION
The exact mechanism of the observed inhibition of amino acid and nucleotide incorporation is not clear. The effect of aminophylline in cell suspensions is contrary to that reported in cell-free systems. Aminophylline enhanced polyphenylalanine synthesis in ribosomal systems from reticulocytes. Theophylline had similar effects; stimulation by caffeine was smaller; and theobromine had no effect5. Aminophylline stimulated amino acid incorporation into protein of rat liver and brain, in a microsomal and ribosomal system. Neither theophylline nor ethylenediamine, alone or together, had any stimulatory effect in these systems. The incorporation of [14C] glucosamine into acid precipitable glycoprotein was enhanced in intestinal slices of rats, but the uptake of glucosamine into acid soluble compartments of intestinal slices was not significantly affected by theophylline 6. Theophylline caused slight stimulation of [laC]leucine incorporation into protein in a microsomal system from bovine thyroid glands sa, but in isolated rat adrenal cells had no significant effect on protein synthesis 10. In rat adipocytes theophylline inhibited protein synthesis is.
302 Therefore, methylxanthines seem to stimulate protein synthesis in cell-free systems. although they inhibit incorporation in cellular systems. Both enhancement and suppression of RNA synthesis by 10-3-10 -9 M theophylline were demonstrated in human lymphocytes following the addition of varying doses of phytohemaglutinin34. Our observation that pentoxifylline inhibits [2-14C] uridine incorporation into mouse brain suspension RNA is in agreement with these findings. In irradiated lymphoma and fibroblast cells, caffeine or theophylline inhibited the rate of DNA synthesis. In non-irradiated cells higher methylxanthine doses were necessary for inhibition. Although the integrity and the repair of pre-existing DNA were unaffected, the DNA strands that were synthesized in the presence of the methylxanthines were smaller than those made in their absence 16,17. Many of the pharmacological and biochemical effects of methylxanthines could be explained by changes of cyclic AMP (cAMP) following the inhibition of cAMP phosphodiesterase. Pentoxifylline also produces an inhibition of this enzyme11,3°. The precise role of cAMP in the control of macromolecular synthesis is not known. In rat liver slices, cAMP caused inhibition of protein synthesis in cellular and cell-free preparations. Microsomes incubated with cAMP did not accelerate protein degradation 1,~,29. Both stimulation and inhibition of protein synthesis by cAMP were reported in cell-free systems isolated from thyroid glands4,19,zo,3L A progressive increase in cAMP produced by theophylline, ACTH, and epinephrine was coupled to inhibition of protein synthesis in rat fat cells~3. Inhibition of protein synthesis by cAMP was also demonstrated in spleen 9, cancer cells TM, muscle 3n, and adrenal glands1L In contrast to the observations of inhibition, in other systems cAMP is capable of stimulating protein ~6 and nucleic acid synthesis. In the chick embryo fibroblast, cAMP stimulated RNA biosynthesis, increasing the incorporation of [3H]uridine14. In monkey kidney cells, the rate of [ZH]thymidine and [3H]uridine incorporation was increased by cAMP, although there was no increase in RNA or DNA content27,2s. The incorporation of radioactive uridine into horse lymphocyte RNA was stimulated by cAMP without stimulation of the incorporation of thymidine into DNAL cAMP in the regenerating lens of the newt decreased prior to enhancement of RNA and protein synthesis, and increased immediately before initiation of DNA synthesis3L Low concentrations of cAMP itself were held to be responsible for initiating DNA synthesis, but there was an increase of intracellular cAMP levels immediately before the DNA synthesis began in liver remnants after hepatectomy in rats 22. Inhibition of amino acid and nucleotide uptake or transport by methylxanthines might be involved in the action of the methylxanthine3~. Although a causal relationship may exist between cAMP elevation and inhibition of protein, RNA, and DNA synthesis, no direct correlation has been established between inhibition of phosphodiesterase by methylxanthines, alteration of cAMP levels, and corresponding changes in macromolecular synthesis. The fact that protein and nucleic acid metabolism are inhibited by BL191 would indicate that methylxanthines act through a mechanism such as energy, or influence protein metabolism indirectly.
303 REFERENCES
1 Akhtar, M. and Bloxham, D. P., The co-ordinated inhibition of protein and lipid biosynthesis by adenosine Y,5'-cyclic monophosphate, Biochem. J., 120 (1970) 11P. 2 Averner, M. J., Brock, M. L. and Jost, J.-P., Stimulation of ribonucleic acid synthesis in horse lymphocytes by exogenous cyclic adenosine Y,5'-monophosphate, J. biol. Chem., 217 (1972) 413-417. 3 Bloxham, D. P. and Akhtar, M., An anti-anabolic role of adenosine 3'-5'-cyclic monophosphate in the control of liver metabolism. A hypothetical mechanism for gluconeogenesis, Int. J. Biochem., 3 (1972) 294-308. 4 DeNayer, P. and De Visscher, M., Effect of 3',5'-cyclic AMP in a thyroid cell-free system, Arch. int. Physiol., 79 (1971) 1021-1022. 5 Fernandez Puentes, C., Carrasco, L. and Vazquez, D., The enhancement of polypeptide synthesis in mammalian systems by methylxanthines, FEBS Left., 45 (1974) 132-135. 6 Forstner, G., Shin, M. and Lukie, C., Cyclic AMP and intestinal glycoprotein synthesis: the effect of beta-adrenergic agents, theophyUine, and dibutyryl cyclic AMP, Canad. J. Physiol. Pharmacol., 51 (1973) 122-129. 7 Ganser, V. and Boksay, I., The influence of methylxanthines on experimental cerebral edema in cats, Naunyn-Schmeideberg's Arch. exp. Path. Pharmak., 277 (1973) R20. 8 Ganser, V. and Boksay, I., The effect of pentoxifylline on cerebral edema in cats, Neurology (Minneap.), 24 (1974) 487-493. 9 Gericke, D., Chandra, P., Haenzel, I. and Wacker, A., Studies on the effect of nucleoside cyclic Y,5'-monophosphates on antibody synthesis by spleen cells, Hoppe-Seylers Z. physiol. Chem., 351 (1970) 305-308. 10 Gericke, D. and Chandra, P., Inhibition of tumor growth by nucleoside cyclic Y,5-monophosphates, Hoppe-Seylers Z. physiol. Chem., 350 (1969) 1469-1471. 11 Hayashi, S. and Ozawa, H., Studies on 3,7-dimethyl-l-(5-oxo-hexyl)-xanthine (BL 191) 1. Cyclic Y,5'-nucleotide phosphodiesteras¢ (PDE),and the inhibitory effect of BL 191 on PDE in rat brain and heart, Chem. pharm. Bull., 22 (1974) 587-593. 12 Hechter, O. and Halkertson, I. D. K., The Hormones, Vol. 5, Academic Press, New York, 1964, pp. 397. 13 Jarett, L., Steiner, A. L., Smith, R. M. and Kipnis, D. M., The involvement of cyclic AMP in the hormonal regulation of protein synthesis in rat adipocytes, Endocrinology, 90 (1972) 1277-1284. 14 Koblet, H., Kohler, U. and Wyler, R., Stimulation of ribonucleic-acid synthesis in chick-embryo fibroblasts by exogenous adenosine Y,5'-monophosphate, Europ. J. Biochem., 37 (1973) 134-142. 15 Lajtha, A. and Dunlop, D., Alterations of protein metabolism during development of the brain. In S. Bogoch (Ed.), Biological Diagnosis of Brain Disorders, Spectrum-Wiley, New York, 1974, pp. 362-367. 16 Lehmann, A. R., Effect of caffeine on DNA synthesis in mammalian cells, Biophys. J., 12 (1972) 1316-1325. 17 Lehmann, A. R. and Kirk-Bell, S., Effects of caffeine and theophylline on DNA synthesis in unirradiated and UV-irradiated mammalian cells, Mutation Res., 26 (1974) 73-82. 18 Lehrach, F. and Muller, R., Results of a clinical investigation of the vasodilator 3,7-dimethyl-1(5-oxo-hexyl)-xanthine (BL 191), ArzneimitteI-Forsch., 21 (1971) 1160-1171. 19 Leung, B. S., Means, A. R. and O'Malley, B. W., Effect of cyclic adenosine Y,5'-monophosphate on the incorporation of aH-leucine into polypeptides by beef thyroid polysomes in vitro, Endocrinology, 89 (1971) 70-78. 20 Lissitzky, S., Mante, S., Attali, J.-C. and Cartouzou, G., Action of Y,5'-cyclic adenosine monophosphate on the protein synthesizing capacity of thyroid polyribosomes in vitro, Biochem. biophys. Res. Commun., 35 (1969) 437--443. 21 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 22 MacManus, J. P., Braceland, B. M., Youdale, T. and Whitfield, J. F., Adrenergic antagonists, and a possible link between the increase in cyclic adenosine Y,5', monophosphate and DNA synthesis during liver regeneration, J. Cell Physiol., 82 (1973) 157-164. 23 Mackie, C. and Schulster, D., Phosphodiesterase activity and the potentiation by theophylline of adrenocorticotrophin stimulated steroidgenesis and adenosine Y,5'-monophosphate levels in isolated rat adrenal cells, Biochem. biophys. Res. Commun., 53 (1973) 545-551.
304 24 Popendiker, K., Boksay, I. and Bollmann, V., On the pharmacology of the new peripheral vasodilator 3,7-dimethyl-1-(5-oxo-hexyl)-xanthine, Arzneimittel-Forseh., 21 (1971) 1160-1171. 25 Raghupathy, E., Peterson, N. A. and McKean, C. M., Stimulatory effects of aminopbylline on amino acid incorporation into protein by cell-free systems, Biochem. Pharmacol., 20 (1971) 1901 1915. 26 Robison, A. G., Butcher, R. W. and Sutberland, E. W., Cyclic AMP, Academic Press, New York, 1971, 138 pp. 27 Roller, B., Hirai, K. and Defendi, V., Effect of cAMP on nucleoside metabolism I. Effect on thymidine transport and incorporation in monkey cells (CV-1), J. Cell PhysioL, 83 (1974) 163-176. 28 Roller, B., Hirai, K. and Defendi, V., Effect of cyclic AMP on nucleoside metabolism Ill. Effect on uridine incorporation in monkey (CV-1) cells, Biochim. biophys. Acta (Amst.), 366 (1974) 402-410. 29 Sellers, A., Bloxham, D. P., Munday, K. A. and Akhtar, M., Anti-anabolic effects of adenosine 3',5'-cyclic minophosphate. Inhibition of protein synthesis, Biochem. J., 138 (1974) 335-340. 30 Stefanovich, V., Concerning specificity of the influence of pentoxifylline on various cyclic phosphodiesterases, Res. Commun. Chem. Path. PharmacoL, 8 (1974) 673-680. 31 Strominger, J. L., In T. Bucher and H. Sies (Eds.), lnhibitors: Tools in Research, Springer, Berlin, 1969, p. 187. 32 Thorpe, C. W., Bond, J. S. and Collins, J. M., Early events in lens regeneration: changes in cyclic AMP concentrations during initiation of RNA and DNA synthesis, Biochim. biophys. Aeta (Amst.), 340 (1974) 413-418. 33 Wagar, G., Action of cyclic adenosine 3',5'-monophosphate on protein synthesis in a thyroidal cell-free system. Relation to ATP and GTP, Acta Endocr. (Kbh.), 75 (1974) 398-409. 34 Webb, D. R., Stities, D. P. and Fudenberg, H. H., Effects of cyclic AMP and theophylline on mitogen-induced RNA synthesis in human peripheral blood lymphocytes, lmmunok Commun., 2 (1973) 353-360. 35 Woo, Y.-T., Manery, J. F. and Dryden, E. E., Theophylline inhibition of nucleoside transport and its relation to cyclic AMP in skeletal muscle, C(mad. J. PhysioL PharmacoL, 52 (1974) 10631073. 36 Wool, I. G., Stirewalt, W. S., Kuihara, K., Low, R. B., Bailey, P, and Oyer, D., Mode of action of insulin in the regulation of protein biosysnthesis in muscle, Recent Progr. Hormone Res., 24 (1968) 139-213.
297
THE INFLUENCE OF METHYLXANTHINES ON PRECURSOR INCORPORATION INTO PROTEIN AND RNA OF MOUSE BRAIN
I. BOKSAY*, V. CSANYI**, J. GERVAI** and A. LAJTHA
New York State Research Institute for Neurochemistry and Drug Addiction, Ward's Island, New York, N. Y. 10035 (U.S.A.) (Accepted April 1st, 1976)
SUMMARY
Pentoxifylline at 1 mM had no effect on [14C]isoleucine incorporation into mouse brain tissue suspension. At 5-20 raM, this compound inhibited incorporation. The inhibition was prompt, and it was reversible. Aminophylline at 3-12 mM produced inhibition, but theophylline at 2-16 m M h a d no effect. Pentoxifylline inhibited the incorporation of uridine into brain RNA to the same extent and with a similar time course as its effect on protein synthesis.
INTRODUCTION
The effect on protein synthesis of pentoxifylline (3,7'-dimethyl-l-(5-oxohexyl)xanthine, BL191), a new methylxanthine derivative is,a4, was compared with that of aminophylline and theophylline. Previously, methylxanthines were reported to have beneficial effects in reducing cerebral edema induced by cold in cats 7,s. In addition, several reports of the effects of methylxanthine derivatives on protein synthesis have appeared 5,e,~. The model system that we used, studying [14C]isoleucine incorporation into a tissue suspension from brain, has the advantages of being a more easily controlled environment than that in vivo, and, at the same time, of containing living cells, which reflect the properties of the brain more closely than do cell-free preparations. In this respect, the tissue suspension technique provides an intermediate stage between tissue slices and homogenates. Of particular importance to the study reported here is that the specific activity of the precursor can be kept constant, and therefore drug effects on macromolecular metabolism can be studied without interference from drug effects on precursor penetration. Since previous work showed that rate of in* Hoechst-Roussel Pharmaceuticals Inc., Somerville, N. J. 08876, U.S.A. ** Experimental Station of L. Eotvos University H-2131 G6d, Javorka, S.u. 14, Hungary.
298 corporation into proteins in slices from young brain is approximately 80-901'.,, of that in vivo whereas the rate in slices from adult brain is less than 20 ~.~,of that in vivo 1.~,, we used tissue from young animals. MATERIALS AND METHODS Brain hemispheres removed from decapitated 3- or 4-day-old albino mice (inbred strain of the laboratory) were placed in a few milliliters of incubation medium at 0 °C. The composition of the medium was 119 m M NaC1, 5.0 m M KC1, 1.2 m M MgSO4 • 7HzO, 1.0 mM NaH2PO4 • 2H20, 1.0 m M NaHCO3, 0.7 m M CaCI2, 12.0 mM NaOH, 10.0mM glucose, and 2 5 . 0 m M N-2-hydroxyethylpiperazine-N'-2ethanesulphonic acid (HEPES); the pH was adjusted to 7.2. Before use, 100 #g/ml of penicillin and 50 ktg/ml of streptomycin were added. The hemispheres were pressed through a polyester bolting cloth (165 nm apertures) into the same medium gently with a spoon. The usual ratio of tissue to medium was one brain hemisphere to 5 ml medium. We used 0.4#Ci of [14C]isoleucine (272mCi/mmole; Calbiochem) or [2-14C]uridine (44 mCi/mmole; Calbiochem) in each 25 ml Erlenmeyer flask (except 0.15 #Ci in the experiments described in Fig. 1). Incubations of the suspensions were at 37 °C, in a water bath without shaking following ox.ygenation. Shaking did not affect incorporation in suspensions, but at times it resulted in increased tissue damage. To measure radioactivity, 0.8 ml samples of the suspension were added to 1.2 ml of ice-cold 20 % perchloric acid (PCA) that contained unlabeled isoleucine or uridine (1 mM). Determination of the radioactivity in protein. PCA-treated samples were heated to 85 °C for 20 min and centrifuged. The precipitate was washed twice with 2 ml 5 ~o PCA, then with 2 ml of ethanol-ether (2:3). After being dried at room temperature, it was dissolved in 1 ml of 1 N N a O H for counting; 0.5-0.8 ml portions of this N a O H solution (neutralized with HCI) were added to 10 ml of a scintillation mixture containing 6.35 g PPO (2,5-diphenyloxazole), 115 mg dimethyl POPOP (1-bis-2,4methyl-5-phenyloxazolyl-benzene), 538 ml toluene, and 462 ml Triton X-100 per liter. An Intertechnique scintillation spectrometer with external standard quench correction was used. Protein content was assayed by the method of Lowry et al. 2~. Determination of radioactivity of RNA. The samples suspended in PCA were centrifuged, the pellet was washed 3 times with ice-cold 5 % PCA, and the supernatant plus washings was used for determination of the precursors. The pellet was suspended in 5 % PCA and heated at 85 °C for 20 min; after centrifugation this supernatant was used for measurement of RNA radioactivity, and for RNA content by measuring the absorption at 260 nm in a Beckman spectrophotometer. Results presented in the figures are the averages of 4-6 experiments as indicated in the legends. The standard deviation of mean values was within 5 %. RESULTS The tissue suspension contained particles of 150-200 nm diameter, with many
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Fig. 1. Effect of antibiotics on [14C]isoleucine incorporation into mouse brain tissue suspension. Brain suspensions were incubated in a standard medium containing 0.4/~Ci/ml [14C]isoleucine. Averages of 6 experiments are shown. ×, control; +, no cells; O, 100/~g/ml penicillin, 50 pg/ml streptomycin; I , 50/~g/ml cycloheximide; IS],50 pg/ml streptomycin, 100/zg/ml penicillin, 50/zg/ml cycloheximide. intact cells. We found that cell suspensions incorporated radioactive amino acids for at least 6 h (Fig. 1). The rate of incorporation was somewhat higher in the first hour, but it remained fairly constant thereafter. In these preparations, the antibiotics were necessary to prevent incorporation due to bacterial contamination. Cycloheximide inhibits protein synthesis in m a m m a lian cells but does not affect bacterial growth. Penicillin and streptomycin inhibit bacterial growth, but do not inhibit protein synthesis in mammalian cells al. The rate of incorporation in the presence of penicillin and streptomycin was less than in their absence, the difference depending on the time period used for incorporation. Bac60O
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Fig. 2. Effect o f pentoxifylline on [ 1aC]isoleucine incorporation into mouse brain tissue suspension.
Experimental conditions as in Fig. 1 except that the medium contained pentoxifylline as indicated. Averages of 5 experiments are shown. × control; ©, 1 mM pentoxifylline; A, 10 mM pentoxffylline; A, 5 mM pentoxifylline; O, 20 mM pentoxifylline.
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Fig. 3. The reversibility of the inhibitory effect. At 15 min pentoxifyllin¢ (to 20 m M ) was added to some suspensions. At 45 min all suspensions were put in fresh (pentoxifylline-free) media. Averages of 5 experiments are shown.
terial contamination (penicillin- and streptomycin-sensitive incorporation) could not be eliminated by using sterilized media. As expected, the 3 antibiotics together completely inhibited the incorporation of isoleucine (Fig. 1). Pentoxifylline at a concentration of 1 mM had no effect, but higher concentrations (5-20 mM) inhibited isoleucine incorporation in brain tissue suspensions. Inhibition was almost complete at 20 mM (Fig. 2). The onset of inhibition of incorporation was prompt, because incorporation stopped immediately (Fig. 3) when incorporation was allowed to proceed and pentoxifylline was added later. Inhibition of the amino acid incorporation was reversible. Addition of pentoxifylline inhibited x ° r.
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TIME - rain. Fig. 4. Effect of pentoxifylline on [l~C]isoleucine and [!4C]uridine incorporation into mouse brain tissue suspension. Pentoxifylline when added was 20 raM. A v e r a ~ of 4 experiments are shown. Protein: × ×,control; × . . . . ×,l~ntoxifyllinv. Nucl¢icacid: © ©,control; O - - : O, pentoxifyllinc.
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120
Fig. 5. Effect of aminophylline on amino acid incorporation into mouse brain tissue suspension. Averages of 4 experiments are shown. ×, control; O, 3 mM aminophylline; • 12 mM aminophylline; • , 6 mM aminophylline.
the incorporation immediately; after the removal of the BL191 the tissue suspension resumed isoleucine incorporation, although at a rate somewhat lower than that of the untreated control (Fig. 3). The incorporation of uridine into the brain cell suspension of mice was also inhibited. Pentoxifylline inhibited incorporation into protein and RNA to a similar extent, and with a similar time course (Fig. 4). Theophylline in 4 different concentrations (2, 4, 8, and 16 mM) did not affect the [laC]isoleucine incorporation. Aminophylline (tested in concentrations of 3, 6, or 12 mM) showed a stronger inhibition of amino acid incorporation in brain tissue suspensions than did pentoxifylline (Fig. 5) DISCUSSION
The exact mechanism of the observed inhibition of amino acid and nucleotide incorporation is not clear. The effect of aminophylline in cell suspensions is contrary to that reported in cell-free systems. Aminophylline enhanced polyphenylalanine synthesis in ribosomal systems from reticulocytes. Theophylline had similar effects; stimulation by caffeine was smaller; and theobromine had no effect5. Aminophylline stimulated amino acid incorporation into protein of rat liver and brain, in a microsomal and ribosomal system. Neither theophylline nor ethylenediamine, alone or together, had any stimulatory effect in these systems. The incorporation of [14C] glucosamine into acid precipitable glycoprotein was enhanced in intestinal slices of rats, but the uptake of glucosamine into acid soluble compartments of intestinal slices was not significantly affected by theophylline 6. Theophylline caused slight stimulation of [laC]leucine incorporation into protein in a microsomal system from bovine thyroid glands sa, but in isolated rat adrenal cells had no significant effect on protein synthesis 10. In rat adipocytes theophylline inhibited protein synthesis is.
302 Therefore, methylxanthines seem to stimulate protein synthesis in cell-free systems. although they inhibit incorporation in cellular systems. Both enhancement and suppression of RNA synthesis by 10-3-10 -9 M theophylline were demonstrated in human lymphocytes following the addition of varying doses of phytohemaglutinin34. Our observation that pentoxifylline inhibits [2-14C] uridine incorporation into mouse brain suspension RNA is in agreement with these findings. In irradiated lymphoma and fibroblast cells, caffeine or theophylline inhibited the rate of DNA synthesis. In non-irradiated cells higher methylxanthine doses were necessary for inhibition. Although the integrity and the repair of pre-existing DNA were unaffected, the DNA strands that were synthesized in the presence of the methylxanthines were smaller than those made in their absence 16,17. Many of the pharmacological and biochemical effects of methylxanthines could be explained by changes of cyclic AMP (cAMP) following the inhibition of cAMP phosphodiesterase. Pentoxifylline also produces an inhibition of this enzyme11,3°. The precise role of cAMP in the control of macromolecular synthesis is not known. In rat liver slices, cAMP caused inhibition of protein synthesis in cellular and cell-free preparations. Microsomes incubated with cAMP did not accelerate protein degradation 1,~,29. Both stimulation and inhibition of protein synthesis by cAMP were reported in cell-free systems isolated from thyroid glands4,19,zo,3L A progressive increase in cAMP produced by theophylline, ACTH, and epinephrine was coupled to inhibition of protein synthesis in rat fat cells~3. Inhibition of protein synthesis by cAMP was also demonstrated in spleen 9, cancer cells TM, muscle 3n, and adrenal glands1L In contrast to the observations of inhibition, in other systems cAMP is capable of stimulating protein ~6 and nucleic acid synthesis. In the chick embryo fibroblast, cAMP stimulated RNA biosynthesis, increasing the incorporation of [3H]uridine14. In monkey kidney cells, the rate of [ZH]thymidine and [3H]uridine incorporation was increased by cAMP, although there was no increase in RNA or DNA content27,2s. The incorporation of radioactive uridine into horse lymphocyte RNA was stimulated by cAMP without stimulation of the incorporation of thymidine into DNAL cAMP in the regenerating lens of the newt decreased prior to enhancement of RNA and protein synthesis, and increased immediately before initiation of DNA synthesis3L Low concentrations of cAMP itself were held to be responsible for initiating DNA synthesis, but there was an increase of intracellular cAMP levels immediately before the DNA synthesis began in liver remnants after hepatectomy in rats 22. Inhibition of amino acid and nucleotide uptake or transport by methylxanthines might be involved in the action of the methylxanthine3~. Although a causal relationship may exist between cAMP elevation and inhibition of protein, RNA, and DNA synthesis, no direct correlation has been established between inhibition of phosphodiesterase by methylxanthines, alteration of cAMP levels, and corresponding changes in macromolecular synthesis. The fact that protein and nucleic acid metabolism are inhibited by BL191 would indicate that methylxanthines act through a mechanism such as energy, or influence protein metabolism indirectly.
303 REFERENCES
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