Polyamines and enzymes of polyamines metabolism in the cartilage during embryonic development

Int. J. Biochem. Vol. 20, No. 3, pp. 317-319, 1988 Printed in Great Britain. All rights reserved

0020-711X/88 $3.00 + 0.00 Copyright © 1988 Pergamon Journals Ltd

POLYAMINES A N D ENZYMES OF POLYAMINES METABOLISM IN THE CARTILAGE D U R I N G EMBRYONIC DEVELOPMENT N. BARGONI* and O. TAZARTES Dipartimento di Medicina e Oncologia Sperimentale, Sezione di Biochimica, Universitfi di Torino, Via Michelangelo 27, 10126 Torino, Italy [Tel. (011) 6698655] (Received 15 M a y 1987)

Abstract--1. In chicken embryo cartilage putrescine levels, maximal at day 8, fall by day 16 to a four-fold lower value, which remains unchanged through hatching and in the 12-day-old chick. 2. Spermine and spermidine, initially higher than putrescine, are almost halved between days 8 and 11, and remain constant afterwards. 3. Ornithine decarboxylase is down to 20% of the day 8 value by day 16, and is further reduced in the newly hatched chick. 4. S-Adenosyl-methionine decarboxylase activity shows a 50% reduction between days 8 and 1I, and no further changes. 5. Spermidine acetyltransferase activity at day 11 is 30% lower than at day 8, goes back up to the initial level by day 16, and progressively decreases through hatching and the first 12 days of life.

predominant. Also analyzed were samples obtained from chicks at hatching, and 5 and 12 days old. The involvement of polyamines in cellular prolifCartilage was obtained from the distal tips of the limb eration has been studied in several experimental bones of 8 and 11 day embryos, or from the epiphysis of systems, including the chicken embryo. The level of these bones in animals at later developmental stages. To determine concentration of polyamines, the tissue was putrescine, spermidine and spermine, as well as the activity of two key enzymes of polyamine bio- quickly isolated and immediately homogenized in 8 vol. of synthesis, ornithine decarboxylase (ODC) and S- 0.2M HC104, using an Ultra-Turrax homogenizer. The homogenate was then centrifuged at 10,000g for 10 min and adenosyl-methionine decarboxylase ( S A M D C ) , have the clear extract was used to prepare the dansyl-derivatives been recently determined by Lrwkvist et al. (1985) in of polyamines. These were then fractionated by HPLC, whole chicken embryo from day 1 to hatching, and in using acetonitrile-H20 on a 150 x 3.9 mm Nova Pak C18 a number of tissues of the 14 day embryo. Cartilage, Column. The diameter of the packing material was 5 lain. a tissue which undergoes remarkable morphological To determine enzymatic activities, cartilage was homchanges throughout maturation and mineralization, ogenized in 9 vol. of 0.15 M KC1, using an Ultra-Turrax was not included in this study. Scanty data on homogenizer. The homogenate was then centrifuged at cartilage are available from previous work, carried 10,000g for 10min, and the supernatant was used for out on rats during growth and on 14 day chicken activity assays. Day 8 samples were obtained pooling cartilage from 10-12 embryos; day 11 samples required cartilage embryo (Conroy et al., 1977), or on condrocytes, from 6--8 embryos; for day 16 samples cartilage was obeither cultured (Takigawa et al., 1980) or obtained tained fro m 2 embryos. One animal yielded enough cartilage from grafts of demineralized bone matrix (Rath et al., for measurements at subsequent time-points. 1978, 1981). ODC activity was measured from the amount of 14CO2 This paper describes the results of an analysis of produced from ~4C-labelled ornithine, according to the polyamine concentration and O D C , S A M D C , and method of J/inne and Williams-Ashman (1971), with the spermidine acetyltransferase (SAT) activities in the modifications described by Grillo and Bedino (1977). cartilage of chicken embryo during development, as SAMDC activity was determined measuring the production well as in the newly hatched chick. The determination of ~4CO2 from ~4C-labelled SAM, according to the method of SAT activity was included in this study, given the of Sturrnan (1980), as described by Grillo et al. (1978). SAT activity was measured according to the method of Libby involvement of this enzyme in both polyamines inter- (1978), following the modifications of Grillo (1983). conversion and catabolism. Putrescine, spermidine, spermine, dithiothreitol, pyridoxal phosphate, and S-adenosyl-methionine were Sigma products. D,L-Ornithine-l-~4C and S-adenosyl-methionineMATERIALS AND METHODS 1-14C were obtained from New England Nuclear Co. Embryos and chicks were obtained from eggs of "Red- (USA). t4C-Acetyl-SCoA was purchased from Amersham grey" hens. Determinations of substrates and enzymes were (England). performed on cartilage obtained from embryos at day 8 of development, when organogenesis is complete; at day 11, RESULTS when tissues and organs are still undergoing differentiation; and at day 16, when growth and "maturation" of tissues are The highest concentration of putrescine, spermidine and spermine was found in the cartilage of *To whom all correspondence should be addressed. the day 8 embryo (Fig. 1). By day 16 the concenINTRODUCTION

317

318

N. BARGONIand O. TAZARTES

7O(; 40

500

% ~ (4)

T(6) [(6) ..M,--o--'-e4-...

• (.3~,,{...-.-

'-

...... I00

I

......

......

~ ~ ( 6 )

o

1

I

8

T(7)

I

16 Embryos

f

12

5

Hatchlng

~ (6)

i

N ( 7 )

T ~ i

C7)

Pu

I

21

-- e.(3)

=~

.... ;;-'!

.16)

I

II

O 30

"-...LC6l

Chicks

o

__1

8

I

II

[

16

I

2J

I

5

I

12

Ctlicks Doys Fig. 1. Concentration of polyamines in cartilage of chicken embryo during development, and in cartilage of the newly Fig. 3. Activity of spermidine acetyltransferase (SAT) in Ernl~ryos

Hotching

Days

hatched chick. Polyamine concentration is expressed as nmol/g of wet tissue. Putrescine (Pu); Spermidine (Sd); Spermine (Sp). Values at each time point are the mean of several determinations (number in parenthesis)___SD.

tration of putrescine drops to 25% of the original level, while the value measured at hatching and in the chick is only 15% of that found at day 8 of embryonal development. Conversely, the concentrations of spermidine and spermine, after decreasing to values 30 and 20% lower than day 8 values by day 11, remain about the same up to hatching. After hatching both values go down slightly: spermidine diminishes progressively to 60% of the original value by day 12, while spermine is already at a minimum by day 5 (60% of day 8 value). ODC appears to be very active in the cartilage of the day 8 embryo (Fig. 2). This activity shows a sharp decline (50% decrease) between days 8 and 11, and

80

-

cartilage of chicken embryo during development and in the cartilage of the newly hatched chick. Enzyme activity is expressed as nmol acetyl-'4C-spermidine/g wet tissue per hr. Number in parenthesis indicates the number of separate determinations at each time point. The values given represent the mean + SD. is down to 20% of the initial value by day 16 of development all throughout hatching. After hatching, ODC activity further declines to values which are undetectable by our method at day 12 of age. SAMDC activity of embryo cartilage also shows a 50% drop between days 8 and I1, but the day 11 value remains constant throughout the rest of embryonal development, as well as at hatching and in the chick (Fig. 2). SAT shows a 30% decline between days 8 and 11, but goes back to day 8 level by day 16 of development. The enzyme activity subsequently declines sharply to 50% of the starting value by hatching, and goes further down to 30% by 12 days of age (Fig. 3).

• (3) DISCUSSION

~

6o

:p

~ 5o ~ 4o 0

~

3o

\ '\,., \l cSl

",?'

2o ,o

o

t 8

:I6l

S.MOC

~,lgl .....

~"~'~ T k 'i .','- - ..,

r(7)

I II

t6

I

~ ~ ( 6 )

Emb3yos

I'-

21

Hotching

T(7) -t

12

5

Chicks

Days Fig. 2. Activity of ornithine decarboxylase (ODC) and S-adenosyl-methionine decarboxylase (SAMDC) in cartilage of chicken embryo during development and in cartilage of the newly hatched chick. Enzyme activity is expressed as nmol '4CO2/g wet tissue per hr. Number in parenthesis indicates the number of separate determinations at each time point. The values given represent the mean + SD.

We found the highest levels of putrescine, spermidine and spermine in the cartilage of chicken embryo at day 8 of development. This value significantly decreases by day 11 for spermidine and spermine, and by day 16 for putrescine, but appears to remain stable throughout subsequent stages of embryonic development, at hatching, and in the young chick. Our data are in agreement with those obtained by others on the whole embryo, as far as putrescine is concerned, but not with respect to spermidine and spermine. The sharp drop in ODC activity that we observe throughout embryonic development in cartilage, appears to be a common feature of a number of different tissues in chicken embryo, as observed by Russell and Lombardini (1971). On the other hand, the reduction of SAMDC activity that we see in embryo cartilage between days 8 and 11, is more drastic than that observed in whole embryo by Snyder and Russell (1970). It is not possible to directly compare the activity of the enzymes that we described in cartilage with that

Polyamines in cartilage of the same enzymes measured in other tissues of the 14 day chicken embryo by L6wkvist et al. (1985) because of the different way of expressing the activity (nmol/mg proteins vs nmol/g wet tissue). Our data on the activity of ODC in chicken embryo cartilage throughout development do confirm the hypothesis of Raina et al. (1980) that a high ODC activity is characteristic of embryonic tissues. The observed low level of putrescine could be due to the action of diamino-oxydase (DAO), which is also known to be elevated in actively proliferating tissues (Andersson et al., 1980). The concentration of spermidine and spermine, on the other hand, correlates well with the level of activity of SAMDC and SAT, which concur with their metabolism. The interplay of all these enzymes results in the sustained production of an adequate amount of polyamines throughout development in cartilage. wish to thank Mr Antonio Baldassarre for the valuable technical help provided by determining the polyamines concentration. This work was supported by a grant from the Ministero per la Pubblica Istruzione. Acknowledgements--We

REFERENCES

Andersson A. C., Henningsson S. and Rosengren E. (1980) In Polyamines in Biomedical Research, pp. 273-283. Wiley, New York. Conroy P. D., Simms D. M. and Pointon J. J. (1977) Occurrence of ornithine decarboxylase and polyamines in cartilage. Biochem. J. 162, 347-350. Grillo M. A. (1983) Acetylation of spermidine in chicken tissues: effect of starvation, insulin, and glucagon. Adv. Polyamines Res. 4, 321-330. Grillo M. A. and Bedino S. (1977) Stimulation by insulin of chicken liver ornithine decarboxylase. Int. J. Biochem. 8, 711-713.

319

Grillo M. A., Bedino S., Testore G. (1978) Regulation of polyamine synthesis in chicken liver. Int. J. Biochem. 9, 185-189. J/inne and WiUiams-Ashman H. G. (1971) On the purification of L-ornithine decarboxylase from rat prostate and effects of the thiol compounds on the enzyme. J. biol. Chem. 246, 1725-1732. Libby P. R. (1978) Calf liver nuclear N-acetyltransferases. Purification and properties of two enzymes with both spermidine acetyltransferase and histone acetyltransferase activities. J. biol. Chem. 253, 233-237. L6wkvist B., Emanuelsson H. and Heby O. (1985) Changes in polyamine synthesis and concentration during chick embryo development. J. exp. Zool. 234, 375-382. Raina A., Eloranta T., Pajula R. L., M/intyj/irvi R. and Tuomi K. (1980) In Polyamines in Biomedical Research (Edited by Gawgas J. M.), pp. 35-49. Wiley, New York. Rath N. C. and Reddi A. H. (1978) Changes in ornithine decarboxylase activity during matrix-induced cartilage, bone, and bone marrow differentiation. Biochem. biophys. Res. Commun. 81, 106-113. Rath N. C. and Reddi A. H. (1981) Changes in polyamines, RNA synthesis and cell proliferation during matrixinduced cartilage, bone, and bone marrow development. Dev. Biol. 82, 211-216. Russell D. H. and Lombardini J. B. (1971) Polyamines: (1) enhanced S-adenosyl-L-methioninedecarboxylase in rapid growth systems, and (2) the relationships between polyamine concentration and RNA accumulation. Biochim. biophys. Acta 240, 273-286. Snyder S. H. and Russell D. H. (1970) Polyamine synthesis in rapidly growing tissues. Fedn Proc. Fedn Am. Socs exp. Biol. 29, 1575-1582. Sturman J. A. (1976) Subcellular distribution of Sadenosylmethionine decarboxylase in rat liver. Evidence of decarboxylation of S-adenosylmethionine separated from synthesis of spermidine. Biochim. biophys. Acta 428, 70-77. Takigawa M., Hishida H., Takano T. and Suzuki F. (1980) Polyamine and differentiation: induction of ornithine decarboxylase by parathyroid hormone is a good marker of differentiated condrocytes. Proc. natn. Acad. Sci. U.S.A. 77, 1481-1485.