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Biochimica et Biophysica Acta, 442 (1976} 432--436 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98680

ARTERIAL ELASTIN SYNTHESIS IN THE YOUNG CHICK

IRENE LEE, M. CHEN YAU and R.B. RUCKER

Department of Nutrition, College of Agriculture and Environmental Sciences, University of California, Davis, Calif. 95616 (U.S.A.)

(Received February 18th, 1976)

Summary

In the growing chick a marked stimulation in soluble and mature arterial elastin synthesis occurs 2 and 5 weeks after hatching. Measurement of [3H]- valine and [3H]proline incorporation into arterial soluble protein during this period indicated that most of the label is found in a 70 000 dalton protein sub- unit. The labeled soluble subunit had the characteristics of native soluble elas- tin or tropoelastin. During the period in which the greatest percentage increase in mature elastin occurs, the highest specific activities of soluble [3H]valine- labeled protein were observed. These changes were striking and suggest a devel- opmental period for the growing chick in which factors related to elastin metabolism may be more easily studied.

Over the past few years significant advances have been made related to the metabolism and structure of arterial elastin [1]. We have previously described some of the properties and metabolism of native soluble elastin from the aortas of copper-deficient and normal chicks [2--6]. In collaborative studies with Dr. Kerry Yasunobu and coworkers, data regarding the N-terminal amino acid se- quences of arterial soluble elastin from copper-deficient chicks have been re- ported [6]. Foster et al. [7] have also reported similar data for soluble elastin from lathyritic chicks.

Some previous investigations on elastin metabolism have utilized embryonic chick aortas in culture (cf. refs. 3 and 8 and references cited}. In this communi- cation are described features pertaining to the apparent synthesis of arterial soluble elastin in the growing chick. Chicks from 1--6 weeks of age were used. The primary purpose of the study was to identify a period in early growth in which arterial elastin synthesis is significantly altered.

The birds were housed in a conventional brooder and fed a commercial ra- tion from the day of hatching. At 1, 2, 4 or 6 weeks of age, groups of birds were killed and the thoracic aorta removed. The aortas were cleansed of ad-

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hering tissue and quickly minced into I mm 3 segments. The minced segments were then used for studies pertaining to the apparent synthesis of elastin.

The segments were first incubated in Medium 199 with Hank's salts con- taining 20 pg/ml ~-aminopropionitrile for 60 rain (41°C, O2/CO2 (95 : 5, v/v)). This was done in order to allow the tissue to adjust to the medium. Approx. 200 mg of the tissue were then transferred to 5 ml of the same medium, but with 10 #Ci of L-[G-3H]valine (spec. act. 10 Ci/mmol) added. The tissue was incubated further for 120 min after which the tissue was removed and washed with chilled distilled water using a filtering flask. Following the wash the tissue was finely homogenized in 0.5 M acetic acid (5 ml) and centrifuged (25 000 × g, 60 min). The amount of protein extracted was estimated [9]. Portions of the supernatant fraction (2 ml) were also either dialyzed against 0.5 M acetic acid and lyophilized or trichloroacetic acid was added to 10% (w/v) in order to pre- cipitate soluble protein. The trichloroacetic acid was added to chilled solutions and the precipitated protein was collected using an ultramembrane filtration device with Millipore filters (Type HA, 0.45 pm). Radioactively labeled protein on the Millipore filters was determined [ 10].

The nature of the radioactively labeled products was assessed by means of polyacrylamide gel electrophoresis techniques in which sodium dodecyl sulfate [11] or 6 M urea at pH 4.3 [12] were employed using the dialyzed and lyo- philized samples. Approx. 150 pg of protein were applied to the gels. Purified nature soluble elastin was used as a standard [6]. The gels cut into contiguous 1 mm segments and the radioactivity determined [ 14].

In addition, minced cardiac and skeletal muscle were also incubated as de- scribed for the arterial segments. Furthermore, in one experiment L-[U-14C] - proline was added (2 pCi/ml) in addition to L-[G-3H]valine (2 pCi/ml) to the incubation medium. In this case radioactivity as 3H and 14C in the gel slices was measured simultaneously. The portion of the gel containing the major peak of radioactivity as [ ~4C]proline was also hydrolyzed and [14C] proline as L-proline and L-hydroxyproline was then estimated as described by Narayanan and Page [8].

Fig. 1 shows the distribution of radioactively labeled products from these ex- periments for birds at 2 weeks of age. Using the minced aorta segments the dis° tribution of radioactivity on polyacrylamide gels indicated essentially one major peak of labeled protein. When [3H]valine was present in the assays greater than 80% of the total counts were found under this peak (Fig. 1). Like- wise, essentially the same distribution of label was observed when [~4C]proline was used, except for a small peak of protein corresponding to the mobility of u-collagen. Separation of the radioactivity as [14C]proline and hydroxy[14C] - proline in the portion of the gel containing the major peak indicated that only 6% of the total radioactivity was hydroxyproline. This observation and the fact that the major peak of radioactivity always comigrated with a purified soluble elastin standard provided strong indirect evidence that the peak was not col- lagen and probably native soluble elastin. The observation that cardiac and ske- letal muscle minces did not synthesis a significant percentage of radioactively labeled acetic acid-soluble protein near 70 000 daltons also provided supporting evidence regarding the uniqueness of the arterial product (Fig. 1).

The data given in Fig. 2 were interpreted largely on the basis that valine was

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incorporated primarily into what appeared to be soluble elastin. It was ob- served that a marked stimulation in valine incorporation into arterial soluble protein occurs between 2 and 5 weeks, the period when there is greatest in- crease in the percentage of mature elastin in the vessel. The percentage of ma- ture or insoluble elastin was estimated using homogenized arterial tissue by a modification of the Lansing procedure [13]. The values for the DNA content

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F i g . 2. Indirect m e a s u r e m e n t o f so luble and m a t u r e elast in synthes i s in the aortas f r o m growing chicks . The upper figure corres p on d s to the change in the percentage o f thorac ic aorta m a t u r e e last in w i t h t ime. The b o t t o m figure c o r r e s p o n d s to changes in the D N A c o n t e n t ($ e ) and spec i f i c act iv i ty ( c p m / m g ) o f e x t r a c t e d so luble p r o t e i n per #g D N A (o o ) af ter i n c u b a t i o n o f aortas for 1 2 0 rain in a m e d i u m conta in ing 2 # C i / m l [ 3 H ] v a l i n e and ~ -aminopropr ion i tr i l e at 2 0 # g / m l .

of the tissue are also shown and were obtained using a method described by Ceriotti [14] . It was during the period of 2--5 weeks that there also appeared to be a slight increase and then decrease in the DNA content of tissue.

It is perhaps important to note that in nutritional copper deficiency the first s i ~ of aortic aneurysm and hemorrhage in the chick occur at 2--3 weeks [2]. Miller et al. [15] has also reported that it is about this time that greatest per- centage increase in the desmosine content of chick elastin occurs. Apparently this represents a period of rapid changes in the cardiovascular physiology of the growing chick. It would be of considerable interest to know to what degree these observations are directly related or correlated with specific developmental alternations in cardiovascular physiology. The chick as a model for elastin studies should prove to be very useful along these lines.

Acknowledgments

This work was supported in part by National Institutes of Health Grant No. HL-15965 and a grant from the Riverside, California Branch of the American Heart Association.

436

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