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Home , Biopterin, Sepiapterin, Sepiapterin reductase, Tetrahydrobiopterin

Proc. Nati. Acad. Sci. USA Vol. 86, pp. 5864-5867, August 1989 Cell Biology , the for aromatic hydroxylases, is synthesized by and regulates proliferation of erythroid cells (differentiation/erythrogenesis) KEIKO TANAKA, SEYMOUR KAUFMAN, AND SHELDON MILSTIEN* Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, MD 20892 Contributed by Seymour Kaufman, May 18, 1989

ABSTRACT The only known role for 6(R)-5,6,7,8- mopoietic cell proliferation. As a model system to study tetrahydrobiopterin (BH4) is as the cofactor for the aromatic possible mechanisms of action of BH4 during erythrogenesis, amino acid hydroxylases. However, BH4 has been shown to be we have examined the correlation between BH4 synthesized by cells that do not contain any hydroxylase and the differentiation of murine erythroleukemia (MEL) activity, suggesting that it may have still undiscovered func- cells, a tissue culture model system for erythrogenesis. tions. Our fmding of much higher levels of BH4 and GTP Undifferentiated MEL cells have nuclei and are rapidly cyclohydrolase, the first of de novo BH4 biosynthesis, dividing, whereas treatment with the differentiation-inducing in rat reticulocytes compared to mature erythrocytes raised the agent dimethyl sulfoxide or hexamethylene bisacetamide possibility that BH4 might play a role in erythrocyte matura- (HMBA) inhibits proliferation and the cells begin to produce tion. We have now demonstrated, by using murine erythro- hemoglobin (7, 8). Differentiation of MEL cells has been leukemia (MEL) cells as a model for erythrogenesis, that BH4 shown to be a multistep process (9, 10). In the early stage, synthesis is required for proliferation of these cells. Inhibition within the first 8-10 hr that precede irreversible commitment of BH4 biosynthesis in rapidly dividing MEL cells with N- to differentiation, there are numerous alterations in cellular acetylserotonin, a potent inhibitor ofsepiapterin reductase, the processes (7). During the following committed phase, there is terminal enzyme in the BH4 biosynthetic pathway, results in a progressive expression of the differentiated phenotype as inhibition of DNA synthesis and mitogenesis without induction seen by hemoglobin accumulation and cessation of cell of hemoglobin synthesis. The inhibition of DNA synthesis is division. We have now demonstrated that there is a require- reversed by repletion of cellular BH4 levels with , a ment for intracellular BH4 for DNA synthesis in the prolif- that is readily taken up by the cells and converted to BH4 erative stage of these cells. In contrast, differentiation, as by the sequential reductions of and measured by the appearance of hemoglobin, occurs after the . Treatment of MEL cells with hexa- cessation of BH4 synthesis. The results presented here may methylene bisacetamide, an inducer of differentiation, results provide some clues as to the function of BH4 in the human in a decrease in BH4 synthesis accompanied by a cessation of erythrocyte. growth and concomitant hemoglobin synthesis. The inhibition ofproliferation induced by hexamethylene bisacetamide can be reversed by maintaining high intracellular levels ofBH4, which EXPERIMENTAL PROCEDURES also decreases the amount of hemoglobin. The mechanism of Rat Red Blood Cells. Reticulocytosis was induced in the BH4 effect has not yet been elucidated, but it appears as Sprague-Dawley rats by phenylhydrazine treatment (11). though BH4 synthesis is more intimately linked with cell Red blood cells were counted with a hemocytometer and proliferation than with the differentiation process. reticulocytes were determined after staining with methylene blue. Red blood cells were separated according to density It is well established that 6(R)-5,6,7,8-tetrahydrobiopterin (age) by density gradient centrifugation (12). (BH4) is the natural cofactor required for the Cell Cultures. Friend virus-transformed MEL cells, strain of the aromatic amino acids, , , and 745, provided by A. Razin (Department of Cellular Biochem- (1). The presence of significant pools of BH4 and istry, The Hebrew University-Hadassah Medical School), the required for BH4 biosynthesis in various cell were grown at 37°C in suspension in Dulbecco's modified types (2) and in areas of the brain where there are negligible Eagle's medium (GIBCO) with 10%o fetal bovine serum (Flow amounts of aromatic amino acid hydroxylase activity (3) Laboratories), penicillin (100 units/ml), and streptomycin raised the possibility that BH4 may have other, as yet (100 ,g/ml) (Advanced Biotechnology, Silver Spring, MD). undiscovered, roles in physiological processes (3, 4). HT medium (Flow Laboratories) was also used in some It has also long been known that there are relatively high experiments described below. Cell viability was checked concentrations of BH4 in rat erythrocytes (2). Erythrocytes with trypan blue and was always >95%, except where are terminally differentiated blood cells whose major function indicated. is to carry hemoglobin; they have no known BH4-dependent Measurement of BH4 and Enzyme Activities. Total reactions. We have found that young rat red blood cells and BH4 content were measured by high-performance liquid contain and synthesize much more BH4 than do erythrocytes. chromatography with fluorescent detection after differential Based on the increase in cellular biopterin levels in prolifer- iodine oxidation (13). ating hemopoietic cells during bone marrow transplantation Red blood cells were lysed in 9 volumes of5 mM potassium in beagle dogs, Ziegler et al. (5, 6) proposed that there was an , pH 7.0/0.1 mM EDTA/0.25 mM phenylmethyl- essential connection between biopterin synthesis and he- Abbreviations: BH4, 6(R)-5,6,7,8-tetrahydrobiopterin; HMBA, hex- The publication costs of this article were defrayed in part by page charge amethylene bisacetamide; 5-HT, ; MEL, murine erythro- payment. This article must therefore be hereby marked "advertisement" leukemia; NAS, N-acetylserotonin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

5864 Downloaded by guest on September 25, 2021 Cell Biology: Tanaka et A Proc. Natl. Acad. Sci. USA 86 (1989) 5865 sulfonyl fluoride. Since hemoglobin interferes with the en- To determine BH4 levels in erythrocytes of an earlier age, zyme assays described below, it was removed from the rats were treated with phenylhydrazine to remove erythro- hemolysate by applying 3 ml to a DEAE-Sephacel (Pharma- cytes and stimulate production of erythrocyte precursor cia, 1 ml) column equilibrated with hemolysis buffer, washing cells. Four days after a 3-day course of phenylhydrazine with buffer until all of the hemoglobin was eluted, and then treatment, the reticulocyte content in rat blood reached a eluting bound proteins with 2 ml of0.01 M Tris HCl (pH 7.4) maximum of >80% of the total erythrocytes compared to containing 1 M NaCl. Control experiments indicated that normal rat blood, which contained only 1-2% reticulocytes. >90o of both dihydropteridine reductase and sepiapterin The biopterin concentration in hemolysates prepared from reductase activities were recovered from the hemolysate by the phenylhydrazine-treated rats was 17.5-fold higher than in this procedure. the control hemolysates [560 + 7 and 41.5 ± 7.6 pmol/mg, respectively (mean ± SEM)]. The activity ofthe rate-limiting MEL cells were washed and resuspended in phosphate- enzyme in the BH4 biosynthetic pathway, GTP cyclohydro- buffered saline at 4 x 107 cells per ml. After freeze-thawing lase, was 23-fold higher in the reticulocyte-rich blood [1.59 ± and sonication on ice (two times for 10 s with 1-min interval), 0.09 and 0.07 ± 0.01 pmol/min per mg, respectively (mean ± the suspensions were centrifuged at 10,000 X g for 10 min and SEM)]. In fact, the GTP cyclohydrolase activity of reticulo- the supernatants were used for the assays. GTP cyclohydro- cytes is higher than in rat liver (14), the organ that contains lase activity was measured essentially as described by Blau the largest amount of BH4 (2). Thus, the rate of BH4 biosyn- and Niederwieser (14). Sepiapterin reductase activity was thesis in erythrocyte precursors must be very high, and measured spectrophotometrically (15). Dihydropteridine re- during the maturation process in vivo, there is a large ductase activity was determined as described (16). decrease in BH4 content and the capacity to synthesize it. Measurement of Hemoglobin. Hemoglobin content was BH4 Levels During Differentiation of MEL Cells. Cultured determined by measurement ofthe absorbance at 410 nm (17) MEL cells, induced to differentiate along the erythroid or by the colorimetric assay of Melloni et al. (18), with some pathway, have been extensively studied as a model for minor modifications. Hemoglobin-containing cells were cellular differentiation (20). In agreement with previous stud- scored according to Orkin et al. (19). ies (8), we found that treatment ofMEL cells with HMBA (5 Measurement of [3HJThymidine Incorporation. MEL cells mM) for 96 hr resulted in differentiation of >90o of the cells were seeded at a density of 105 cells per ml in 96-well culture to produce hemoglobin, as determined by counting benzi- plates and cultured for 24 hr prior to experiments. After dine-positive cells. As in the case of the reticulocyte-rich treatment with the various agents as described in table blood from phenylhydrazine-treated rats, the biopterin levels legends, [3H]thymidine (1 GCi/ml; 1 Ci = 37 GBq; ICN) was in continuously growing MEL cells prior to HMBA treatment added, and incubations were continued for 2 hr. Cells were are high relative to those ofthe terminally differentiated cells harvested with a cell harvester onto glass fiber filters, which (Table 2). As can be seen in Fig. 1, total intracellular BH4 were then dried; radioactivity was determined in a liquid content declines markedly during the period of HMBA treat- scintillation counter. ment and the GTP cyclohydrolase activity also decreases proportionally, suggesting that the decreased BH4 content results from a decrease in the rate ofbiosynthesis rather than RESULTS AND DISCUSSION from an increased turnover. From these data, it appears as Biopterin in Rat Erythrocytes. Since rat erythrocytes con- though there is an inverse relationship between intracellular tain relatively high concentrations ofbiopterin in the absence BH4 concentration and the appearance of hemoglobin, but of any known BH4-dependent hydroxylases, measurements these experiments do not establish whether the loss of BH4 of levels of BH4 and its relative rate of synthesis during the biosynthetic capacity is an epiphenomenon or is directly course of erythrogenesis might yield some clues as to its connected to the phenotypic expression of differentiation. function in the erythrocyte. During the process of erythro- Effect of Alterations of Intracellular BH4 Concentrations on cyte maturation, precursor cells lose many biochemical func- Proliferation and Differentiation of MEL Cells. To study the tions, including the capacity to proliferate. Therefore, it was relationship between the processes of erythroid proliferation of interest to examine the distribution of BH4 and GTP and differentiation and the intracellular concentration of cyclohydrolase activity in cells ofdiffering ages separated by BH4, the effects of agents that either increase or decrease density gradient centrifugation. As can be seen in Table 1, BH4 concentrations were studied in MEL cells with and older or more dense cells have a lower content of biopterin without HMBA treatment. BH4 is taken up from the medium and lower GTP cyclohydrolase activity than younger or less by MEL cells (data not shown). However, BH4 added to cell cultures was not stable and was rapidly oxidized (til2 1 hr) dense cells. This result suggests the possibility that BH4 may in the medium at 37°C. Prolonged incubations with BH4 also also be present at high levels in precursor cells. decreased the viability of MEL cells, probably due to reac- tive oxygen species produced during the autoxidation of BH4 Table 1. Biopterin content and GTP cyclohydrolase activities of (data not shown). The autoxidation could be prevented by the rat erythrocytes separated by density gradient centrifugation addition of an enzymatic tetrahydropterin reducing system GTP cyclohydrolase, Total biopterin, (21) or 1 mM ascorbic acid. However, these agents them- Fraction pmol/min per ml nmol/ml selves were found to have toxic effects on the viability of the Top 1 48.7 2.8 cultured cells (data not shown). Therefore, to increase intra- 2 12.0 2.0 cellular BH4 concentrations we utilized the endogenous 3 7.9 1.8 salvage pathway that converts sepiapterin (7,8-dihydrolac- 4 6.4 1.3 toylpterin) to BH4 by the sequential action of sepiapterin Bottom 5 2.3 0.8 reductase and dihydrofolate reductase (22). DNA synthesis in rapidly dividing MEL cells, as measured by [3H]thymidine Washed erythrocytes from a control rat were separated on a density gradient. GTP cyclohydrolase activities and total biopterin incorporation into DNA, was increased significantly by pre- concentrations are expressed as activity or concentration per ml of loading the cells with 100 ,uM sepiapterin, which increased cells isolated from each fraction. Sixty-five percent of the erythro- intracellular BH4 by >70-fold. DNA synthesis was also cytes were recovered in fraction 5, which contained approximately markedly decreased after inhibition of BH4 synthesis with the same biopterin content and GTP cyclohydrolase activity as the N-acetylserotonin (NAS) (Table 3), a potent inhibitor of washed erythrocytes. sepiapterin reductase (23), the terminal enzyme of BH4 Downloaded by guest on September 25, 2021 5866 Cell Biology: Tanaka et al. Proc. Natl. Acad. Sci. USA 86 (1989) Table 2. Effect of increasing cellular levels of BH4 with sepiapterin on the HMBA-induced differentiation of MEL cells Hemoglobin, Biopterin, GTP cyclohydrolase, Addition ,ug per 106 cells nmol/mg pmol/min per mg None 105 0.25 0.55 HMBA 685 0.06 0.07 + sepiapterin (6 hr) 685 0.68 0.09 + sepiapterin (96 hr) 209 6.95 0.07 Cells were incubated in the presence or absence of HMBA (5 mM) for 96 hr. Sepiapterin (0.1 mM) was added at the start and either was washed offat 6 hr and fresh medium containing HMBA was added or was left in for the entire 96-hr period. The data are representative of results obtained from three separate experiments. biosynthesis. Pretreatment of MEL cells for 4 hr with 1 mM However, inhibition of proliferation has been shown to NAS resulted in an inhibition ofDNA synthesis of48.8% and usually accompany differentiation, although it is not suffi- a decrease in BH4 content of 54%. This was reflected by a cient to induce differentiation (25). Incubation of MEL cells decrease in cell number of 44.1%, measured 24 hr later (Fig. with NAS, in the absence of HMBA, results in reduced DNA 1). The degree ofreduction ofMEL cell proliferation was also synthesis and cellular proliferation but does not lead to an dependent on the concentration of NAS. Serotonin (5-HT) increase in differentiation, as measured by the appearance of and , in vitro inhibitors ofBH4 synthesis with lower hemoglobin-positive cells (data not shown). Maintenance of inhibitory potencies (23, 24), were also somewhat less potent high intracellular levels of BH4 with sepiapterin during the as inhibitors of DNA synthesis and proliferation (Fig. 2). entire 96-hr induction of differentiation with HMBA appears Furthermore, increasing the intracellular BH4 concentration to interfere with the differentiation process (Table 2). In effectively reversed the inhibition of DNA synthesis caused contrast, when the sepiapterin is washed out after the first 6 by NAS treatment, strongly indicating that the inhibition of hr, the amount of hemoglobin made is the same as in the cells DNA synthesis by NAS was indeed directly related to the treated with HMBA alone, even though the BH4 concentra- inhibition of BH4 synthesis (Table 3). tion remains above that of the rapidly growing cells for the It should be noted that the effects of exogenous sepiapterin entire 96 hr. In the absence of further data on globin that we have observed-namely, the small increase in DNA expression in these cells, we can only speculate that the synthesis when added to rapidly dividing MEL cells and the difference between the results of short- and long-term main- restoration of the rate of DNA synthesis to normal when tenance ofhigh BH4 concentrations on hemoglobin synthesis added to NAS-treated cells-are probably due to the sepi- may be due to the previously mentioned deleterious effects of apterin-mediated increase in intracellular BH4 rather than to BH4 on cell viability. The explanation for this difference and a different, more direct effect of sepiapterin itself. If sepiap- the mechanism by which BH4 interferes with the expression terin directly enhanced DNA synthesis, it would seem likely of hemoglobin have not yet been elucidated. that NAS, by inhibiting the intracellular conversion of sepi- Tetrahydrofolates and BH4 Synthesis in MEL Cells. Tet- apterin to BH4, would increase intracellular sepiapterin and rahydrofolates play a vital role in cell growth, providing the thereby increase DNA synthesis rather than, as we have methyl groups required for methionine, purine, and deoxy- observed, to decrease it. thymidylate synthesis. 5-Methylenetetrahydrofolate reduc- The process of differentiation of MEL cells is closely tase is a key enzyme in these pathways (26), which normally correlated with a limitation of cellular proliferation (25). catalyzes the NADPH-dependent reduction of 5,10-methyl- enetetrahydrofolate to 5-methyltetrahydrofolate. It has been v 120 shown that methylenetetrahydrofolate reductase catalyzes W Biopterin C GTP Cyclohydrolase Hemoglobin the NADPH-dependent reduction of quinonoid dihydrobiop- > terin to BH4 as well as the transfer of electrons directly from BH4 to 5,10-methylenetetrahydrofolate (27). If BH4 were ~80- being utilized by MEL cells in this fashion, inhibition of BH4

<60 biosynthesis would lead to decreases in purine nucleotide synthesis and inhibition of proliferation as is seen with 0 Table 3. Effect of sepiapterin and NAS pretreatment on DNA z 20 synthesis and biopterin content of rapidly growing MEL cells U Addition [3H]Thymidine 0 X START 1 2 3 4 incorporation,* BH4, 0-2 hr 2-4 hr % of control DAYS nmol/mg None None 100 0.12 FIG. 1. Effect of differentiation of MEL cells with HMBA on Sepiapterin (10 ,M) None 111.8 ± 3.9t 3.01 biopterin levels and GTP cyclohydrolase activity. MEL cells were Sepiapterin (100 uM) None 129.3 ± 4.9* 8.59 treated with 5 mM HMBA, and total biopterin, GTP cyclohydrolase, None NAS 50.5 ± 4.8t 0.05 and hemoglobin were determined at 24-hr intervals. The biopterin Sepiapterin (10 /LM) NAS 88.6 ± 9.8§ 0.20 concentrations and GTP cyclohydrolase activities are expressed Sepiapterin (100 ,uM) NAS 103.0 ± 4.8§ 0.60 relative to control cultures (99.4 pmol/mg and 0.35 pmol/min per mg, respectively). Due to spontaneous differentiation, hemoglobin con- The NAS concentration was 1 mM. Cells were labeled with centrations of about 15% of the maximum were always present. This [3H]thymidine during the 2- to 4-hr period. value was subtracted to obtain the maximum percent increase (final *The data are mean ± SD of eight separate experiments, which hemoglobin concentration = 540 ,ug per 106 cells). It should be noted yielded similar results. In a typical experiment, the control value that control cells are rapidly dividing and become growth limited was 27.3 ± 3.9 X 103 dpm per well. after 2-3 days. Total biopterin levels in confluent untreated cells after tAverages of duplicate samples. 96 hr actually increase by about 45%, whereas GTP cyclohydrolase tP 0.01 relative to the control. activity remains nearly constant. §P 0.01 relative to the cultures treated with NAS alone. Downloaded by guest on September 25, 2021 Cell Biology: Tanaka et al. Proc. Natl. Acad. Sci. USA 86 (1989) 5867

D 120 1. Kaufman, S. & Fisher, D. B. (1974) in Molecular Mechanisms MnCell Number C DNA Synthesis ofOxygen Activation, ed. Hayaishi, 0. (Academic, New York), 100 pp. 285-369. > 2. Fukushima, T. & Nixon, J. C. (1980) Anal. Biochem. 102, -j 0 176-188. ° 80 3. Milstien, S. (1987) in Unconjugated and Related Bio- z genic Amines, eds. Curtius, H. Ch., Blau, N. & Levine, R. A. 0 60 (de Gruyter, Berlin), pp. 49-65. U- 4. Kaufman, S. (1986) in and Folic Acid Derivatives, 0 40 I eds. Cooper, B. A. & Whitehead, V. M. (de Gruyter, Berlin), z pp. 185-200. 20 5. Ziegler, I., Fink, M. & Wilmann, W. (1982) Blut 44, 231-240. 6. Ziegler, I., Kolb, H. J., Bodenberger, U. & Wilmann, W. (1982) C 10MM NAS 100MM NAS 1 mM NAS 1 mM 5HT 1 mM Melatonin Blut 44, 261-270. 7. Friend, C., Scher, W., Holland, J. G. & Sato, T. (1971) Proc. FIG. 2. Effect of inhibitors of BH4 biosynthesis on the prolifer- Natl. Acad. Sci. USA 68, 378-382. ation ofMEL cells. MEL cells were seeded and cultured. After a 2-hr 8. Reuben, R. C., Wife, R. L., Breslow, R., Rifkind, R. A. & treatment with the indicated agents, the cells either were pulsed with Marks, P. A. (1976) Proc. Natl. Acad. Sci. USA 73, 862-866. [3H]thymidine for 2 hr for the measurement of DNA synthesis or 9. Chen, Z., Banks, J., Rifkind, R. A. & Marks, P. A. (1982) Proc. were cultured for a further 22 hr and cell numbers were determined. Natl. Acad. Sci. USA 79, 471-475. The results are expressed relative to control cultures. 10. Marks, P. A., Sheffery, M. & Rifkind, R. A. (1987) Res. 47, 659-666. antifolate drugs. However, attempts to reinitiate DNA syn- 11. Speiser, S. & Ettinger, J. D. (1982) J. Biol. Chem. 257, 14122- thesis of cells in which BH4 synthesis was inhibited by 14127. culturing them in hypoxanthine/thymine-supplemented me- 12. Limberd, L. E., Gill, D. M., Stadel, J. M., Hickey, A. R. & or addition of acid Lefkowitz, R. J. (1979) J. Biol. Chem. 255, 1854-1861. dium by direct 5-methyltetrahydrofolic 13. Milstien, S., Kaufman, S. & Summer, G. K. (1980) Pediatrics were not successful (data not shown). Furthermore, there 65, 806-810. were no significant changes in nucleoside triphosphate pools 14. Blau, N. & Niederwieser, A. (1983) Anal. Biochem. 128, in NAS-treated cells (data not shown). However, as shown 446-452. for a number of cell lines, the toxicity of agents that interfere 15. Sueoka, T. & Katoh, S. (1982) Biochem. Biophys. Acta 717, with thymidine synthesis and the reversal of the toxic effects 265-271. by exogenous thymidine administration are highly dependent 16. Craine, J. E., Hall, E. S. & Kaufman, S. (1972) J. Biol. Chem. on the timing and sequence of the treatments (28). 247, 6082-6091. 17. Rutherford, T. R. & Weatherall, D. J. (1979) Cell 16, 415-423. 18. Melloni, E., Pontremoli, S., Michetti, M., Sacco, O., Ca- CONCLUSIONS kiroglu, A. G., Jackson, J. F., Rifkind, R. A. & Marks, P. A. The results presented here demonstrate that BH4 plays a role (1987) Proc. Natl. Acad. Sci. USA 84, 5282-5286. in and differentiation of murine erythroid cells. 19. Orkin, S., Harosi, F. & Leder, P. (1975) Proc. Natl. Acad. Sci. proliferation USA 72, 98-102. The stimulation of DNA synthesis by increasing intracellular 20. Marks, P. A. & Rifkind, R. A. (1978) Annu. Rev. Biochem. 47, BH4 levels (with exogenous sepiapterin), the inhibition of 419-448. proliferation resulting from a block of de novo BH4 biosyn- 21. Kaufman, S. (1970) Methods Enzymol. 17, 603-609. thesis, and the decrease in BH4 accompanying terminal 22. Nichol, C. A., Lee, C. L., Edelstein, M. P., Chao, J. Y. & differentiation indicate that BH4 acts as a positive growth Duch, D. S. (1983) Proc. Natl. Acad. Sci. USA 80, 1546-1550. modulator when these cells are rapidly dividing. The mech- 23. Katoh, S., Sueoka, T. & Yamada, S. (1982) Biochem. Biophys. anism by which BH4 regulates cell growth remains elusive but Res. Commun. 105, 75-81. has important potential implications for human erythropoie- 24. Milstien, S. & Kaufman, S. (1985) Biochim. Biophys. Res. sis. Although it has not yet been demonstrated that matura- Commun. 128, 1099-1107. tion of the human erythrocyte responds to BH4 in the same 25. Tsiftsoglou, A. S. & Sartorelli, A. C. (1981) Biochim. Biophys. manner as does the rodent erythrocyte, it has been shown Acta 653, 226-235. that the enzymes of BH4 biosynthesis are present in human 26. Green, J. M., Ballou, D. P. & Matthews, R. G. (1988) FASEB 42-47. are greater levels J. 2, erythrocytes (29, 30) and that there much 27. Matthews, R. G. & Kaufman, S. (1980) J. Biol. Chem. 255, of GTP cyclohydrolase in human reticulocytes than in eryth- 6014-6017. rocytes (31). Furthermore, the present findings raise the 28. Jackson, R. C. (1980) Mol. Pharmacol. 18, 281-286. possibility that the growth of other BH4-containing, non- 29. Yoshioka, S., Masada, M., Yoshida, T., Mizokami, T., Akino, erythroid cell types may be regulated by BH4 in a similar M. & Matsuo, N. (1984) Zool. Soc. 1, 74-81. manner. 30. Niederwieser, A., Shintaku, H., Hasler, T., Curtius, H. Ch., Lehmann, H., Guardamagna, 0. & Schmidt, H. (1986) Eur. J. We thank Dr. Sarah Spiegel for advice and helpful comments. K.T. Pediatr. 145, 176-178. was supported by a grant from the American Health Assistance 31. Schoedon, G., Curtius, H. Ch. & Niederwieser, A. (1987) Foundation. Biochem. Biophys. Res. Commun. 148, 1232-1236. Downloaded by guest on September 25, 2021