Acta med. scand. Vol. 195, pp. 37-38, 1974

INHIBITORY EFFECT OF L-DOPA ON COLLAGEN BIOSYNTHESIS

Nelly Blumenkrantz and Gustav Asboe-Hansen

From the Department of Dermatology (with Connective-Tissue Research Laboratories), Rigshospitalet, University of Copenhagen, Copenhagen, Denmark

Abstract. The effect of L-dopa on connective tissue bio- synthesis by 10-day-old chicken embryos has been stud- ied. Inhibited collagen formation as expressed by a re- duced hydroxylation of (“C)proline and (“C)lysine, and a similarly reduced glycosylation of (14C)hydroxylysine was found. N o influence on the biosynthesis of glyco- and/or mucoproteins was observed.

In a previous paper the authors reported on the effect of L-tyrosine and L-phenylalanine on col- lagen biosynthesis (2). It was demonstrated that L-tyrosine inhibits the incorporation of (14C)pro- line (Pr) into proteins and the biosynthesis of (14C)hydroxyproline (Hypro). L-phenylalanine af- fected neither the incorporation of (14C)Pr and ( 14C)lysine (Lys) nor their hydroxylation to (14C)- Hypro and (14C)hydroxylysine (Hylys) in the un- dialysable material synthesized by 10-day-old chicken embryo tibiae. In this communication the effect of the related amino acid L-dopa (di- hydroxyphenylalanine) on collagen and glyco- and f or mucoproteins is reported. The parameters considered were the hydroxylation of the amino acids (14C)Pr and (14C)Lys, the glycosylation of (I4C)Hylys, and the incorporation of (14C)glucos- amine. The extensive use of L-dopa in long-term treatment of Parkinson’s disease stimulated this study of its effect on connective tissue.

MATERIAL AND METHODS Embryonated chicken eggs were purchased from the State Serum Institute of Copenhagen. Uniformly labeled (“C)L-Pr, 197 pC/pmole, (”C)L-Lys, 248 p C / pmole, and (“C)D-glucosamine, 5-10 pC/pmole, were obtained from New England Nuclear Corp. L-dopa was a product of Sankyo, Japan.

Ten-day-old chicken embryo tibiae were removed by microscopic dissection. The tissues were preincubated during 1 hour in a medium of buffer, glucose and in-

organic salts (8). Increasing doses of L-dopa were then added and the preincubation was continued for 30 min. The medium without L-dopa was used for control.

After preincubation 5 p C (“C)Lys or (“C)Pr or (14C)glucosamine were added for an incubation period of 2 hours. At the end of this period the tissues were homogenized and the homogenates dialysed against run- ning tapwater overnight. The undialysable (“C)Pr and (“C)glucosamine labeled materials were hydrolysed in 6 N HCI at 110°C for 16 and 4 hours, respectively. The material labeled with (“C)Lys was divided into 2 aliquots. One was hydrolysed as indicated above for the (14C)Pr labeled material and used for determina- tion of total (14C)Hyllys, while the unhydrolysed aliquot was used for determination of unglycosylated (“C)Hylys (3). By subtraction the value of glycosylated (“C)Hylys was obtained. The HC1 was eliminated from the acid hydrolysed samples by evaporation under vacuum at 65OC. The total (”C) incorporated was determined on an aliquot of the (14C)Pr, (“C)Lys and (14C)glucosamine labeled material and expressed as percentage of controls. Total and unglycosylated (‘4C)Hylys were assayed on corre- sponding hydrolysed and unhydrolysed aliquots of the (“C)Lys labeled material according to Blumenkrantz and Prockop (3). (“C)Hypro was assayed by the method of Juva and Prockop (7).

The radioactivity was measured in a liquid scintilla- tion counter, and the observed cpm were corrected to dpm by the use of internal standards. The results were calculated as dpm per bone per hour of incubation and expressed as percentage of controls.

RESULTS

When tibiae of chick embryos were incubated with (I4C)Pr in the presence of increasing con- centrations of L-dopa, decreasing amounts of (14C)Hypro were synthesized. Although less pro- nounced, a reduced incorporation of (I4C)Pr was also observed. When, in parallel experiments, tis- sues were incubated with (14C)Lys in the presence of increasing concentrations of L-dopa, decreas- ing amounts of (14C)Hylys were synthesized,

Acta med. scand. 195

38 N . Blumenkrantz and G. Asboe-Hansen

Table I. Effect of increasing concentrations of L-dopa on uptake of (14C) Pr and v4C) Lys, on their hydroxy- lation, and on the glycosylation of (“C)Hylys by IO- day-old chicken embryo tibiae Results are expressed as % of controls. Values represent average of two separate assays

(14C)Hylys

L-dopa (14C) (14C) (14C) Glyco- (mM) Pr Hypro Lys Total sylated

0.85 111 91 105 80 79 1.7 103 81 88 52 45 7 78 25 88 39 39

10 68 26 92 38 38 17 62 23 91 25 * 25

Table 11. Effect of L-dopa on the uptake of (14C)D- glucosamine by 10-day-old chicken embryo tibiae

Total uptake of (14C)- L-dopa D-glucosamine (mM) ( % of control)

0.85 89.8 10 81.7 17 94.7

while the incorporation of the precursor (14C)Lys was not inhibited.

Glycosylation of (14C)Hylys was decreased parallel to the hydroxylation of (14C)Lys (Table I). No significant effect of L-dopa on the in- corporation of (14C)glucosamine into glyco and/ or mucoproteins was observed at the dosages used (Table 11).

DISCUSSION

The decreased incorpoGation of (14C)Pr under the effect of L-dopa may be due to an inhibitory competition for a similar transport system as ob- served between the related amino acid L-tyrosine and (14C)Pr (2). (14C)Lys is not affected on the basis of its transport by the basic amino acid

pathway (6, 9). According to Blasberg (1) the transport system that carries L-dopa into brain cells is probably shared with other neutral amino acids including phenylalanine and tyrosine. Our finding is in agreement with the statement of Wurtman et al. (10) that, although exogenous L-dopa is apparently not incorporated into pro- teins, it may inhibit protein synthesis by sharing the transport system with related amino acids.

The finding that the biosynthesis of both (‘4C)- Hypro and (14C)Hylys is more vigorously de- creased than the incorporation of their precursor amino acids suggests that an underhydroxylated collagen is synthesized in the presence of L-dopa. Considering that some amino acids are known to chelate various metal ions (9, this effect could be due to a chelation of Fe++ ions, one of the cofactors required for the hydroxylation step.

Our findings of an unaltered incorporation of (14C)D-glucosamine into glyco- and/or mucopro- teins synthesized by chicken embryo tibiae under the effect of L-dopa are comparable to the non- affected glycoprotein biosynthesis by rat liver mitochondria observed by Bosman et al. (4).

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