Pattern of polyamines and related monoacetyl derivatives in chick embryo retina during development

Int. J. Devl Neuroscience, Vol. 12.No. 5, pp. 423-429,1994

Pergamon

0736-5748(94)E0022-T

ElsevierScienceLtd Copyright© 1994ISDN Printedin GreatBritain.All rightsreserved 0736-5748/94$7.00+0.00

P A T T E R N OF POLYAMINES A N D R E L A T E D M O N O A C E T Y L D E R I V A T I V E S IN CHICK E M B R Y O R E T I N A D U R I N G DEVELOPMENT G. TAIBI,* M. R. SCHIAVO, G. CALVARUSO and G. TESORIERE Institute of BiologicalChemistry, Faculty of Medicine, Universityof Palermo, Palermo, Italy (Received 6 December

1993;in revised form 28 February 1994;accepted 3 March 1994)

Abstraet--Polyamines and related monoacetyl derivatives were studied in chick embryo retina during development (6th-19th day). Putrescine, which is high in the first phase of retinogenesis, is necessaryto sustain both tissueproliferationand via N-acetylputrescine,~-aminobutyricacid synthesis.A later increase in spermidine and particularlyspermine may play a role in the last phase of developmentwhen the retina reaches maturation. The presence of/Vl-acetylspermidinealready at the 8th day indicates that in chick embryo retina, putrescine synthesis can depend on two separate pathways. The first involvesornithine decarboxylase activity; the second, spermidine/spermine Nt-acetlytransferase and probably polyamine oxidase that converts spermidine to putrescine via Nl-acetylspermidine. chick embryo, retina, polyamines,monoacetylpolyamines,metabolism, GABA, proliferation, differentiation. Key words:

The polyamines putrescine, spermidine and spermine are polycationic molecules that are commonly present in significant amounts in both prokaryotic and eukaryotic cells 11,14-16,21-23 and are essential for cell proliferation and differentiation. Their biosynthesis greatly depends on ornithine decarboxylase (ODC), the key enzyme of the polyamine-biosynthetic pathway. 1,9-11,14 T h e r e is evidence to support the view that putrescine takes part mainly in the regulation of cell proliferation, whilst spermidine and, particularly, spermine are more related to differentiative events. 2,10,11,21-28,3° The presence of monoacetylpolyamines has also been documented in the cells; however, their biological function is still unclear. 42 It has been ascertained that the intracellular concentration of these acetylated forms varies greatly, according to the proliferative status of the cell and also in response to a wide range of stimuli. 4,6,37 Some authors have identified acetylated polyamines as an excretion product of cultured cells, 4° where acetylation seems to be a prerequisite for their excretion, 41,42 while others have hypothesized on their precise metabolic role.17,2°,28-31,33 In our previous studies, we have found that, during chick embryo retina development, the ontogenic pattern of O D C activity is closely related to D N A synthesis. 35 This activity, in fact, shows the highest levels in 7-day-old chick embryo retina, but decreases quickly, reaching very low values between the 12th and 14th day of development. Many authors have reported that putrescine, apart from ornithine decarboxylation, can be also synthesized by an alternative route that involves acetylation and oxidation of spermidine. 17,20,29,31-34 It has been observed by Grillo and co-workers 7 that, in chick embryo retina, spermidine/spermine Nl-acetyltransferase, which is low at the beginning of the embryonic life, markedly increases to its highest activity at the 14th day of development and subsequently decreases to a minimum after hatching. We have studied changes in natural polyamines and their acetylated forms during chick embryo retina development to evaluate the relation between these compounds and retinal growth and differentiation, and to get insight into the relationship between the alternative biosynthetic pathways. *To whom all correspondence should be addressed at: Istituto di Chimica Biologica,Policlinico"Paolo Giaccone", Via Del Vespro No. 129,90127 Palermo, Italy. Fax +39-91-6552463. Abbreviations: GABA, ~/-amino butyric acid; GAD, glutamate decarboxylase (glutamic acid decarboxylase); ODC, ornithine decarboxylase(EC 4.1.1.17);SAMDC, S-adenosylmethioninedecarboxylase(EC 4.1.1.50);spermidine synthase, S-methyl-adenosylhomocysteamine: putrescine aminopropyltransferase;spermine synthase, S-methyl-adenosylhomocysteamine : spermidineaminopropyltransferase;NLSAT, spermidine/spermine,Nt-acetyltransferase;PAO, FAD-dependent polyamine oxidase. 423

424

(i. l'aibi ~,ta/ EXPERIMENTAL P R O C E D U R E S

Materials Amino acids, polyamines and their monoacetylderivatives (hydrochloride forms) were purchased from Sigma Chimica (Milan, Italy). Fertilised Warren Breed chicken eggs were supplied by CISA (Cocconato D'Asti, Italy), All commercial reagents (A-grade or for chromatographic use) were from Farmitalia Carlo Erba (Milan, Italy).

Sample preparations Eggs of uniform size and weight were placed in an automatic incubator (Victoria) at 38°C and 60% relative humidity. Chick embryos at various days of development were killed by decapitation and their eye-balls bisected near the equatorial plane. The neural retinas were accurately separated from the pigment epithelium and placed into a polypropylene tube containing sterile saline at 4°C. After centrifugation at 500 g for 5 min the retinas were re-suspended in one volume of 0.2 M perchloric acid (20 mg wet weight/ml) containing 20 mM 1,7-diaminoheptane as internal standard. The samples were homogenized by sonication at 10-sec bursts (10 ~m amplitude), in a Soniprep 150 (MSE), centrifuged at 4°C (20,000 g) for 15 rain and the supernatants were collected and stored in a polypropylene tube at -80°C. Aliquots (100 ~l) of the homogenate were always taken for protein determination. 13

Polyamine analysis To obtain the polyamine derivatives, samples were neutralised with potassium hydroxide, centrifuged to remove potassium per chlorate and then submitted to benzoyl chloride derivatization. 38 The derivatized samples were filtered through a MiUex-FG13 filter (0.2 ~m pore size) and assayed by reverse-phase H.P.L.C. 38 Chromatographic separations were achieved by using a Spherisorb C18 $ 3 0 D S 2 column (15×0.46 cm I.D.; 3 txm particles) (Phase Separation Ltd, Deeside, U.K.). The polyamines and their acetylated derivatives were eluted with 62% methanol (v/v) (flow rate, 1.0 ml/min) at room temperature (approx. 20°C) and detected at 254 nm (detector sensitivity 0.01 a.u.f.s.). Individual compounds were estimated using calibration curves previously generated from standard solutions. Recovery was determined by the percent recovery of the internal standard (1,7-diaminoheptane). Each determination was performed in triplicate.

Amino acid analysis Samples were filtered through a Millex-FGt3 filter (0.2 Ixm pore size), mixed 1 : 1 with pH 2.2 samples diluting buffer and injected (50 IA) into Beckman System 6300 High Performance Autoanalyzer. RESULTS AND DISCUSSION Chick embryo retina development can be subdivided into three different phases. The first one is characterized by proliferative activity until the 7th day; the second shows a remarkable decrease of proliferation with a concurrent start of differentiative events (7th-12th day); the third phase leads to tissue maturation (12th hatching). In this paper we report on the concentration of both polyamines and their monoacetyl derivatives between the 6th and 9th day of retina development. For this purpose we have employed a rapid and sensitive assay capable of detecting simultaneously both polyamines and their conjugated formsY As shown in Fig. 1, polyamine concentrations, and in particular putrescine, were high at the 6th-8th day of development. From the 8th to 10th day, putrescine concentration markedly decreased and then declined more slowly until the 16th day. Spermidine and spermine were present at much lower concentrations than putrescine. Spermidine concentration changed during retina development similarly to that of putrescine, whereas changes in spermine were much less conspicuous. For the three polyamines the lowest value was found at the 16th day; afterwards their levels increased slowly until the 19th day. Cadaverine (1,5-diaminopentane) was also present in chick embryo retina from the 6th day on and its concentration showed a trend similar to that found for putrescine except between the 16 and 18th day (Fig. 2). Cadaverine is exclusively

425

Polwmaincsand monoacclyl dcrivalivcs during dcvclopmcnl 0,9 ]

0,6

0,3

E ¢:

0,0

6

8

10

12

14

16

18

19

Day of Development Fig. 1. Polyamine concentrations in chick embryo retina during development. Results, mean and S.D. values, are from triplicate determinations from one experiment representative of two done for every day of development.

-10

01Ft

Putrescine

--

.lg

8

"~

0~6 '

01U m

E

0,4 E

E

4 ..

0,2 2

I.

~

0,0 ;

" 8

10

1'2

14

1'6

18

19

Day of Development Fig. 2. Putrescine (11) and cadaverine (D) patterns in chick embryo retina during retinogenesis. Points represent the mean values_+S.D, from triplicate determinations of one experiment representative of two done for every day of development.

produced through the low affinity decarboxylation of lysine by ODC, in mammalian cells,25,26 and its concentration reflects only the rate of ODC activity.28 Consequently the increase of cadaverine concentration observed at the 16th day seems to indicate that the increment of spermidine and spermine, in the last phase of development, is due probably to an activation of the normal biosynthetic pathway and, in particular, of S-adenosylmethionine decarboxylase (SAMDC), spermidine and spermine synthase, as demonstrated by the low concentration of putrescine.

2,0!

426

G. Taibi eta/.

• []

Spd/Pt Spm/Pt

1,5 t O o~

1,0

eL

0,5

0,0 6

8

10

12

14

16

18

19

Day of Development Fig. 3. Molar ratios of spermidine putrescine and spermine/putrescine during chick embryo retinogenesis. 0,8'

800

0,6"

600

O

iiilil

0 Q

""

X

E 0 .400

0,4 '

Z

E E C

,200

0,2'

• i

0,0 6

8

10

Day

12

of

14

16

18

E E

Q. o

0,0

19

Development

Fig. 4. Relation between potyamine concentrations (putrescine, &; spermidine. ©: spermine, U3) and rate of DNA synthesis (bars) during chick embryo retina development. The DNA data are derived from our previous paper and represent the mean+S.E. (bars) values from six separate experiments. 39 Points represent the mean values_+S.D, from triplicate determinations of one experiment representative of two done for every day of development.

This is more evident if spermidine and spermine to putrescine ratios are evaluated (Fig. 3). As can be seen, both ratios markedly increased during retinal development, particularly in the last phase when differentiative events become predominant. Results reported in this paper support the view that putrescine is mainly related to cellular proliferation. Its level declines in parallel to the decrement of proliferative events and to the decrease in ODC activity and DNA synthesis. 39 On the contrary, spermidine and, especially, spermine appear to be more related than putrescine to the last phase of development when neural retina reaches maturation (Fig. 4). As shown in Fig. 5, N-acetylputrescine is present in high concentrations during all embryonic life. Because its level is always much higher than Nl-acetylspermidine, we conclude that

Polvamincs and monoacclvl dcrivalivcs during development

427

0,010

[] N-Acetylputrescine

] 1,0

N-Acetylspermidine ["7] N-Acetylspermine I--]

--o- Putrescin¢

0,008

0,8

¢= ,,¢ ~o

~gJ 0~ 0,006

0 , 6 ._~

m 0,004

~s

E 0,002

[]

:], 2

1

0,000 6

8

O, 0 10

12

14

16

18

19

Day of Development

Fig.5. Polyaminemonoacetylderivatives(bars) and putrescine (open circle)contentin retina during chick embryo retinogenesis. Results, mean and S.D. values from triplicate determinations, are from one experiment representative of two done for everyday of development.ND, non-detectable. N-acetylputrescine arises from a direct acetylation of putrescine. Considering that putrescine is scarcely utilised by N1-SAT activity,7 it is possible to hypothesize that also in chick embryo retina, just as in human and rat brain 24,32 putrescine acetylation depends on acetyl-CoA: 1,4diaminebutane N-acetyltransferase activity. As shown in the same Fig. 5, Nl-acetylspermidine that was detected in chick embryo retina on the 8th day, increased until day 14 and then decreased. Acetylspermine was not detectable until the 14th day and appeared only at days 16-18. This is peculiar in the light of the known higher affinity of N1-SAT for spermine than to spermidine. 7 It might be explained by different sub-cellular localisation of these polycations; in particular, spermidine and N1-SAT may be both prevalently localized in the cytosol,7 whereas spermine may be predominant in the particulate. This conclusion is in agreement with data reported in literature about a possible role of this compound which involves membrane function. 11,19,20,35,43 The relatively high levels of putrescine observed between the 12th and 14th day, when ODC reached its lowest level, may suggest that in chick embryo retina, putrescine pool can also depend on an alternative route involving spermidine and spermine metabolism.17,20,31-34,4° This reverse pathway consists of a two-step mechanism. The first step is the conversion of spermine or spermidine into their NLacetyl derivatives, by spermidine/spermine Nl-acetyltrans ferase (N1-SAT activity); the second is the oxidation step of the monoacetylpolyamine catalysed by FAD-dependent polyamine oxidase (PAO), which splits off the acetylated aminopropyl group as acetylamidopropanale, thus converting Nl-acetylspermidine into putrescine and N 1acetylspermine into spermidine. N1-SAT, which is highly inducible by various agents as specific polyamines and derivatives 3,29 and is capable of inhibiting ODC activity,8:2, 44 has been found in chick embryo retina already at the 10th day of development. 7 The observation, reported in this paper, that Nl-acetylspermidine is at the highest level at the 12-14th day makes it very probable that the reverse pathway can contribute to the production of putrescine at this stage of development. G A B A synthesis is an important event in the ripening process of neural retina; in fact, several lines of investigation have suggested trophic actions for GABA. 18,36 Our recent observations indicate that G A B A is already present in the retina at the 6th day of development and its concentration increases rapidly until the 10th day. Afterwards its concentration declines until the 14th day and then again increases reaching the maximum value at hatching (Fig. 6). G A B A can be synthesized in chick embryo retina in at least two ways. The first is based upon the conversion of glutamic acid to GABA, catalysed by glutamic acid decarboxylase (GAD), 5 an

428

(i. Taibi et al.

0~5"

m

0,4

m

m

,4= ~JD e~

m

0,3

~0

E

0,2 m

[]

0,1

0,0 6

8

10

12

14

16

18

19

Day of Development

Fig. 6. GABA levels in chick embryo retina during development. Results, mean and S.D. values from quadruplicate determinations, are from one experiment representative of two done for every day of development. activity which is lacking in the early stage o f d e v e l o p m e n t and m a r k e d l y increases after the 14th day. 5 T h e second, by the conversion of putrescine to G A B A via the intermediates m o n o a c e t y l - p u trescine, N-acetyl-butyraldehyde, and N - a c e t y l - G A B A . D e Mello et al. 5 suggested that the latter biosynthetic p a t h w a y that depends on putrescine, can be active in retinal tissues in the first phase of retinogenesis when G A D activity is absent in the retina, while in the second phase only a small p e r c e n t a g e of G A B A was derived from putrescine. O u r data indicating that N-acetylputrescine is present in the retina in high c o n c e n t r a t i o n f r o m the 6th day of d e v e l o p m e n t supports strongly this conclusion according to the observation 31 that the acetylated f o r m is a necessary intermediate for the conversion of putrescine into G A B A . A f t e r the t 4 t h day (see Fig. 5), when G A D b e c o m e s active in chick e m b r y o retina, 5 N-acetyl-putrescine m a y not be necessary for G A B A synthesis. Therefore, in the last phase of dev e l o p m e n t the acetylated f o r m m a y be considered only as a catabolic p r o d u c t of putrescine, which is synthesized to permit its excretion. H o w e v e r , one should not exclude the possibility that also during terminal differentiation, s o m e a m o u n t of G A B A m a y arise f r o m N-acetylputrescine. In fact, the higher c o n c e n t r a t i o n o f N-acetylputrescine at this stage of d e v e l o p m e n t correlates with the increased conversion of putrescine into G A B A during the 14-18-day period. Acknowledgements--This work as supported by grants from Assessorato Regionale BB. CC. AA. and P.I. We thank M.

D'Ancona for her excellent technical assistance. REFERENCES l. Bachrach U. (1973) Function o f Naturally Occurring Po(vamines. Academic Press, New York. 2. Bolander F. F. Jr and Topper Y. J. (1979) Relationships between spermidine, glucocorticoids, and milk proteins in different mammalian species. Biochem. Biophys. Res. Commun. 90, 1131-1136. 3. Casero R. A., Celano P., Ervin S. J., Porter C. W., Bergeron R. J. and Libby P. R. (1989) Cancer Res. 49, 63%643. 4. Della Ragione F. and Pegg A. E. (1984) Advances in Polyamines in BioMedical Research (eds. Caldarera C. M. and Bacharach U.), pp. 97-104. CLUEB, Bologna. 5. De Mello F. G., Bachrach U. and Nirenberg M. (1976) Ornithine and glutamic acid decarboxylase activities in the developing chick retina. J. Neurochem. 27, 84%851. 6. Fuller D. J. M., Carper S. W., Clay L.. Chen J. and Gerner E. W. (1990) Polyamine regulation of heat-shock-induced spermidine NLacetyltransferase activity. Biochem. J. 267, 601-605. 7. G r i ~ M. A.~ Dianzani U. and Pezza~i D. C. ( ~984 ) A cety~ati~n ~f p~yamines in chicken brain and retina. ~nt. J. Bi~chem.

16, 1349-1352.

Polvamincs and m o n o a c e t y l derivatives d u r i n g d e v e l o p m e n t

429

8. Guarneri C., Lugaresi A., Flamigni F., Muscari C. and Caldarera C. M. (1982) Effect of oxygen radicals and hyperoxia on rat heart ornithine decarboxylase activity. Biochim. Biophys. Acta 718, 157-164. 9. Heby O. (1981) Role of polyamines in the control of cell proliferation and differentiation. Differentiation 19, 1-20. 10. Ientile R., Macaione S. and Di Giorgio R. M. (1985a) Polyamine metabolic defects in inherited disorder of rd mice. In Recent Progress in Polyamine Research (eds Selmeci L., Brosnan M. E. and Seiler N.), pp. 401--409. Akad6midi Kiadb, Budapest. 11. Ientile R., Russo P. and Macaione S. (1986) Polyamine localisation and biosynthesis in chemically fractionated rat retina. J. Neurochem. 47, 1356-1360. 12. J~inne J. and Williams-Ashman H. G. (1971) On the purification of L-ornithine decarboxylase from rat prostate and effects of thiol compounds on the enzyme. J. biol. Chem. 246, 1725-1732. 13. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. 14. Macaione S. and Calatroni A. (1978) Polyamines and ornithine decarboxylase activity in the developing rat retina. Life Sci. 23, 683-690. 15. Macaione S. (1982) Polyamine metabolism in rat retina during postnatal development. In Proceedings of the Fourth Meeting of the European Society for Neurochemistry, September 12-18, Catania, p. 278. 16. Macaione S., Di Giorgio R. M., Nicotina P. and Ientile R. (1984) Retina maturation following administration of thyroxine in developing rats: effects on polyamine metabolism and glutamate decarboxylase. J. Neurochem. 43~ 303-315. 17. Matsui I., Wiegand L. and Pegg A. E. (1981) Properties of spermidine N-acetyltransferase from livers of rats treated with carbon tetrachloride and its role in the conversion of spermidine into putrescine. J. biol. Chem. 256, 2454-2459. 18. Meier M., Hertz L. and Schousboe A. (1991) Neurotransmitters as developmental signals. Neurochem. Int. 19, 1-15. 19. Missiaen L., Wuytack F., Raeymaekers L., De Smedt H. and Casteels R. (1989) Polyamines and neomycin inhibit the purified plasma-membrane Ca 2÷ pump by interacting with associated polyphosphoinositides. Biochem. J. 26L 10551058. 20. Morgan D. M. L. (1990) Polyamines and cellular regulation: perspectives. Biochem. Soc. Trans. 18, 1080-1084. 21. Oka T. and Perry J. W. (1974) Spermidine as a possible mediator of glucocorticoids effect on milk protein synthesis in mouse mammary epithelium in vitro. J. biol. Chem. 249, 7647-7652. 22. Pegg A. E. (1986) Recent advances in the biochemistry of polyamines in eukaryotes. Biochem. J. 234, 24%262. 23. Pegg A. E, and MacCann P. P. (1988) Polyamine metabolism and function in mammalian cells and protozoans. 1SIAtlas o f Science: Biochemistry, pp. 11-18. Institute of Science Information, Philadelphia. 24. Pegg A. E. (1988) Polyamine metabolism and its importance in neoplastic growth and as a target for chemotherapy. Cancer Res. 48, 75%774. 25. Pegg A. E. and McGill S. (1979) Decarboxylation of ornithine and lysine in rat tissues. Biochim. Biophys. Acta 568, 416-427. 26. Pegg A. E., Secrist J. A. III and Madhubala R. (1988) Properties of L1210 cells resistant to a-Difluoromethylornithine. Cancer Res. 48, 2678-2682. 27. Perry T. L., Hensen S. and MacDougall L. (1%7) Amines of human whole brain. J. Neurochem. 14, 775-782. 28. Poulin R., Coward J. K., Lakanen J. R. and Pegg A. E. (1993) Enhancement of the spermidine uptake system and lethal effects of spermidine over accumulation in ornithine decarboxylase-overproducing L1210 cells under hyposmotic stress. J. biol. Chem. 228, 4690-4698. 29. Seiler N. (1987) Functions of polyamine acetylation. Can. J. Physiol. Pharmac. 65, 2024-2035. 30. Seiler N. and Lamberty U. (1975) Interrelations between polyamines and nucleic acids: changes of polyamines and nucleic acid concentrations in the developing rat brain. J. Neurochem. 24, 5-13. 31. Seiler N. and A1-Therib M. J. (1974) Putrescine catabolism in mammalian brain. Biochem. J. 144, 2%35. 32. Seiler N. and AI-Therib M. J. (1974) Acetyl-CoA: 1,4-diaminobutane N-acetyltransferase. Occurrence in vertebrate organs and sub cellular localization. Biochim. Biophys. Acta 354, 206-212. 33. Sei•er N.• B••kenius F. N. and Kn•dgen B. ( •98•) Acety•ati•n •f spermidine in p••yamine catab••ism. Bi•chim. Biophys. Acta 663, 181-190. 34. Seiler N., Bolkenius F. N., Bey P., Mamont P. S. and Danzin C. (1985) Recent Progress in Polyamine Research (eds Selmeci L, Brosnan M. E. and Seiler N.), pp. 305-319. Akad6miai Kiadb, Budapest. 35. Smith C. D. and Snyderman R. (1988) M•dulati•n •f in•sit•l ph•sph•lipid metab•lism by polyamines. Bi•chem. J. 256, 125-130. 36. Spoerri P. E. (1988) Neurotrophic effects of G A B A in cultures of embryonic chick brain and retina. Synapse 2, 11-2Z 37. Stefanelli C., Carati D., Rossoni C., Flamigni F. and Caldarera C. M. (1986) Accumulation of Nl-acetylspermidine in heart and spleen of isoprenaline-treated rats. Biochem. J. 237, 931-934. 38. Taibi G. and Schiavo M. R. (1993) Simple high-performance liquid chromatographic assay for polyamines and their monoacetyl derivatives. J. Chromatogr. 614, 153-158. 39. Tesoriere G., Vento R., Taibi G., Calvaruso G. and Schiavo M. R. (1989) Biochemical aspects of chick embryo retina development: the effects of glucocorticoids. J. Neurochem. 52, 1487-1494. 40. Wallace H. M., MacGowan S. H. and Keir H. M. (1985) Polyamine metabolism in mammalian cells in culture. Biochem. Soc. Trans. 13, 32%330. 41. Wallace H. M. and Keir H. M. (1981) Uptake and excretion of polyamines from baby hamster kidney cells (BHK21/C13). The effect of serum on confluent cell cultures. Biochim. Biophys. Acta 676, 25-30. 42. Wallace H. M. and Coleman C. S. (1990) Changes in polyamine acetylation in human cancer cells. Biochem. Soc. Trans. 18, 1091-1094. 43. Wojcikiewicz R. J. H. and Fain J. N. (1988) Polyamines inhibit phospholipase C-catalysed polyphosphoinositide hydrolysis. Biochem. J. 255, 1015-1021. 44. Zuretti M. F. and Gravela E. (1983) Studies on the mechanisms of ornithine decarboxylase in vitro inactivation. Biochim. Biophys. Acta 742, 269-277.