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Augmentation of collagen synthesis by ascorbic acid in vitro
404
SHORT COMMUNICATIONS
dibromophenol with isolated mitochondria from houseflies as found by CHANCE AND SACKTOR1. The present results also complement the recent findings from BirCHER'S laboratory with mitochondria of locust flight muscle which showed that ADP produced a stimulation of eGP oxidation of 2. 5 times (see ref. 6) and caused an oxidation in the steady-state level of D P N H v. The observations of VA~ DEN BERGH AND SLATER8 on the ADP stimulation of respiration in a system containing mitochondria and supernatant fraction from thoracic muscle of houseflies are also consistent with the data found with teased muscle. This is as one would predict since the teased-muscle preparations contain mitochondria, the soluble cytoplasm (sarcoplasm) and myofibrils. It should be emphasized that with substrates other than ~GP the 2- to 5-fold increase in O~ uptake due to ADP is not of sufficient magnitude to satisfy completely the requirements for a physiological-control mechanism in the insect, just beginning flight. In an insect, such as the fly, oxygen uptake is increased 5O-lOO times on initiation of flight. Thus, this relatively small stimulation of respiration by ADP is of significance only when considered with other possible components of the respiratorycontrolmechanism for flies, including: availability and oxidation of a critical substrate (i.e., ~GP) ; inorganic phosphate; divalent cation ; as well as others, yet to be described. Detailed discussions of the respiratory-control problem in flight muscle were given elsewhere 9,10.
Physiological Chemistry Branch, Directorate of Medical Research, U.S. Army Chemical Research and Development Laboratories, Army Chcmical Ccnter, (U.S.A .) and Johnson Foundation/or Medical Physics, University of P ennsylvania,Philadelphia (U.S.A.)
BERTRAM SACKTOR LESTER PACKER*
]3. CHANCE AND B. SACKTOR, Arch. Biochem. Biophys., 76 (1958) 509 • 2 B. SACKTOR, Am. Chem. Soc., Abstr. of Papers, i 3 5 t h Meeting (1959) 3oC, 3 B. CHANCE, Rev. Sci. Instr., 22 (1951) 634. 4 B. CHANCE AND G. R. WILLIAMS, Advances in Enzymol., 17 (1956) 65. 5 g . SACKTOR, J. Biophys. Biochem. Cytol., i (1955) 29. e T. B/JCHER, M. KLINGENBERG AND E. ZEBE, Proc. 4th Intern. Congr. Biochem., Vienna, 12 (1959) 153. 7 M. I{LINGENBERG, W. SLENCZKA AND E. RITT, Biochem. Z., 332 (1959) 47. s S. G. VAN DEN BERGH AND E. C. SLATER, Biochim. Biophys. Acta, 4 ° (196o) 176. 9 B. SACI~TOR, Proc. Celebrazione Spallanzaniana, Reggio-Pavia, in the press. 10 B. SACKTOR, Ann. Rev. Entomol., 6 (1961) lO 3. 1
Received December 2ist, 196o • P r e s e n t address: D e p a r t m e n t of Microbiology, The University of Texas S o u t h w e s t e r n Medical School, Dallas, Texas.
Biochim. Biophys. Acta, 49 (1961) 4o2-4o4
Augmentation of collagen synthesis by ascorbic acid in vitro Ascorbic acid is essential for adequate synthesis of collagen in species for which ascorbic acid is a vitamin 1-3, but since pertinent experiments have been carried out iI~ intact animals we have not known whether vitamin C acts directly on the cells synthesizing collagen (fibroblasts) or whether its action is primarily systemic. High Biochim. Biophys. Acta, 49 (r96I) 404-4 °6
SHORT COMMUNICATIONS
405
concentrations of ascorbic acid in the adrenal cortex and other endocrine tissues 4, and demonstrable effects of hormones on collagen synthesis 5, as well as the finding that some collagen synthesis may occur in the absence of demonstrable ascorbic acid e have suggested that the vitamin may be primarily involved in the production or release of a hormone affecting collagen synthesis. A local action of ascorbic acid, on the other hand, is suggested by the accumulation of ascorbic acid at sit~s of active collagen synthesis in healing wounds ~, and by the direct relation of collagen concentration to ascorbic acid concentration in the carrageenan granuloma 8. More direct evidence for a local action of ascorbic acid has been presented by GOULI)9, who implanted vinyl sponges on either flank of guinea pigs, and then deprived them of ascorbic acid. After injections of small amounts of vitamin directly into one of the pair of sponges, the hydroxyproline (and presumably collagen) content of the injected sponge was several-fold higher than that of the contralateral, uninjected sponge. However, even these experiments do not distinguish between an effect on the whole tissue, such as restoration of blood capillaries, and a direct effect on the collagensynthesizing cells. The present experiments show that ascorbic acid added in vitro to suspensions of scorbutic granuloma cells increases collagen synthesis, thus demonstrating a direct effect on the collagen-synthesizing fibroblasts. GREEN AND LOWTI~ER1° have shown that labeled hydroxyproline can be isolated from collagen fractions from slices of non-scorbutic, carrageenan granulomas incubated with [l~C]proline. This is acceptable evidence for collagen synthesis even without rigorous purification of collagen, since collagen contains approx. 14 % hydroxyproline and this amino acid has not been found in other purified mammalian proteins. We have been able to show, using similar techniques, that suspensions of scorbutic granuloma cells are also able to synthesize collagen hydroxyproline. The data in Table I illustrate this synthesis of collagen by scorbutic granuloma in vitro and the stimulation obtained when ascorbic acid is added to the incubation media. The first 5 guinea pigs received no ascorbic acid for 14 days before being killed. The ascorbic acid concentrations were typical for scorbutic granulomas 8. The specific activity of collagen hydroxyproline was greater in all of these granulomas when I mg of ascorbic acid was added to the incubation media. The last four guinea pigs were deprived of ascorbic acid for 14 days but were injected intraperitoneally with 3oo mg sodium ascorbate 1-1.5 h before being killed. The ascorbic acid of these granulomas was, as expected, very high and addition of ascorbic acid to the incubation media did not lead to an increased specific activity of hydroxyproline. Indeed, in two tissues the specific activity was lower when the media were supplemented with ascorbic acid suggesting inhibition by excess ascorbic acid. A comparison of the effectiveness of injected ascorbic acid with ascorbic acid added in vitro is difficult because of variation among animals. However, the average specific activity of collagen hydroxyproline did not differ significantly when ascorbic acid was added in vitro to scorbutic granulomas and when it was inj ected before killing the animals. Although one can not exclude additional systemic effects of ascorbic acid, the data show that ascorbic acid added in vitro to a sugpension of scorbutic carrageenan granuloma cells stimulates collagen synthesis and does this as effectively as ascorbic acid which reaches the granuloma via the blood stream. Biochim. Biophys. Acta, 49 (1961) 404-406
406
SHORT COMMUNICATIONS TABLE I EFFECT OF ASCORBIG ACID ON COLLAGEN SYNTHESIS
E a c h flask contained a p p r o x . 2 g of tissue brei (obtained b y pressing I 4 - d a y scorbutic carrageenan g r a n u l o m a ~ t h r o u g h a plate h a v i n g o . 8 - m m holes), suspended in 8 ml of K r e b s - R i n g e r b i c a r b o n a t e solution to which h a d been added 8 #C [14C]proline (13/,C/#mole)*, 42.5 # m o l e s glycine, 12. 5 # m o l e s alanine, IO/zmoles glutamic acid, 7 . 5 / , m o l e s each of aspartic acid and arginine, 5 # m o l e s lysine, valine, serine a n d phenylalanine, 2. 5/~moles leucine, isoleucine, m e t h i o n i n e a n d threonine, t /~mole t y r o s i n e a n d t r y p t o p h a n . T h e p H w a s 7.2-7.4. i m g of ascorbic acid (o.i ml) w a s added to the designated flasks and to the u n i n c u b a t e d controls. After i n c u b a t i o n w i t h s h a k i n g a t 37 ° for 6 h in a 95 o/002 - 5 % COs a t m o s p h e r e the reactions were s t o p p e d b y addition of o. 75 ml trichloroacetic acid (I g/ml). H y d r o x y p r o l i n e w a s isolated f r o m t h e collagen fraction a n d its specific activity was d e t e r m i n e d as described in ref. 3. Ascorbic acid w a s determined on a l i q u o t s of tissue as described b y RoE AND KUETHER 11. All e x p e r i m e n t s and analyses were carried o u t in duplicate.
Specifi~ activity o] hydrozyproli~a Guinea pig No.
Ascorbicacid in gra•uloma #g/g
No addition
Added ascorblc
Unincubated control
counls/rain/#mole** I 2 3 4 5 Mean 6 7 8 9 Mean
15 3.8 7.3 7.3 2.8 680 480 58o 380
4,900 5,400 2,400 5,1oo 3,000 5,3oo 1,9oo 3,800 700 2,3oo 2,58o~_ 690*** 4 , 3 8 0 ± 590*** 7,200 7,000 4,4 °0 1,9oo 2,ooo 2,ooo 3,1oo 2,300 4, I8o ! 66o*** 3,3oo ± 1,24o***
7 o 16 6 38 20 8 3 3
* [14C]proline as obtained f r o m Nuclear-Chicago contained [14C]hydroxyproline. Unlabeled h y d r o x y p r o l i n e was added and t h e proline was purified b y c h r o m a t o g r a p h i n g twice on 4o-cm c o l u m n s of Dowex-5o. ** A b o u t i # m o l e of h y d r o x y p r o l i n e w a s counted for IO,OOO counts. * * * S t a n d a r d error.
We are indebted to the U.S. Public Health Service for a grant (A-36o) supporting this work and to Mr. L. STOLOFF, Marine Colloids, Rockport, Me., U.S.A., for a generous supply of purified carrageenan.
College of Medicine, University of Vermont, Burlington, Vt. (U.S.A.)
WILLIAM "CAN B . ROBERTSON* JEAN HEWITT
I S. B. WOLBACH A~D P. R. HOWE, Arch. o[ Pathol. Lab. Med., I (1926) I. 2 W. VAN B. ROBERTSON AND B. SCHWARTZ, J . Biol. Chem., 21o (1953) 689. * W. VAN B. ROEERTSON, J. HEWlTT AND C. HERMAN, J . Biol. Chem., 234 (1959) lO5. 4 M. E. REID, The V i t a m i n s , Vol. I, Academic Press, Inc., N e w York, 1954, p. 311. 6 W. VAN B. ROBERTSON AND E. C. SANBORN, Endocrinology, 63 (1958) 250. 6 j . F. WOESSNER AND B. S. GOULD, J . Biophys. Biochem. Cytol., 3 (I957) 685. v A. F. ABT, S. V. SCHUCHING AND J. H. R o E , J . Nutrition, 7 ° (196o) 427 . 8 W. VAN B. ROBERTSON, A n n . N . Y . Acad. Sci., in the press, 9 B. S. GOULD, J. Biol. Chem., 232 (1958) 637. 10 N. M. GREEN AND D. A. LOWTHER, Biochem. J . , 71 (1959) 55. xt j . H. RoE, Methods o[ Biochemical A n a l y s i s , Vol. I, N e w York, 1954, p. 127.
Received December 6th, 196o * P r e s e n t address: Dept. of Pediatrics, Stanford U n i v e r s i t y Medical School, Palo Alto, Calif. (U.S.A.).
Biochim. Biophys. Acta, 49 (1961) 404-4 °6
SHORT COMMUNICATIONS
dibromophenol with isolated mitochondria from houseflies as found by CHANCE AND SACKTOR1. The present results also complement the recent findings from BirCHER'S laboratory with mitochondria of locust flight muscle which showed that ADP produced a stimulation of eGP oxidation of 2. 5 times (see ref. 6) and caused an oxidation in the steady-state level of D P N H v. The observations of VA~ DEN BERGH AND SLATER8 on the ADP stimulation of respiration in a system containing mitochondria and supernatant fraction from thoracic muscle of houseflies are also consistent with the data found with teased muscle. This is as one would predict since the teased-muscle preparations contain mitochondria, the soluble cytoplasm (sarcoplasm) and myofibrils. It should be emphasized that with substrates other than ~GP the 2- to 5-fold increase in O~ uptake due to ADP is not of sufficient magnitude to satisfy completely the requirements for a physiological-control mechanism in the insect, just beginning flight. In an insect, such as the fly, oxygen uptake is increased 5O-lOO times on initiation of flight. Thus, this relatively small stimulation of respiration by ADP is of significance only when considered with other possible components of the respiratorycontrolmechanism for flies, including: availability and oxidation of a critical substrate (i.e., ~GP) ; inorganic phosphate; divalent cation ; as well as others, yet to be described. Detailed discussions of the respiratory-control problem in flight muscle were given elsewhere 9,10.
Physiological Chemistry Branch, Directorate of Medical Research, U.S. Army Chemical Research and Development Laboratories, Army Chcmical Ccnter, (U.S.A .) and Johnson Foundation/or Medical Physics, University of P ennsylvania,Philadelphia (U.S.A.)
BERTRAM SACKTOR LESTER PACKER*
]3. CHANCE AND B. SACKTOR, Arch. Biochem. Biophys., 76 (1958) 509 • 2 B. SACKTOR, Am. Chem. Soc., Abstr. of Papers, i 3 5 t h Meeting (1959) 3oC, 3 B. CHANCE, Rev. Sci. Instr., 22 (1951) 634. 4 B. CHANCE AND G. R. WILLIAMS, Advances in Enzymol., 17 (1956) 65. 5 g . SACKTOR, J. Biophys. Biochem. Cytol., i (1955) 29. e T. B/JCHER, M. KLINGENBERG AND E. ZEBE, Proc. 4th Intern. Congr. Biochem., Vienna, 12 (1959) 153. 7 M. I{LINGENBERG, W. SLENCZKA AND E. RITT, Biochem. Z., 332 (1959) 47. s S. G. VAN DEN BERGH AND E. C. SLATER, Biochim. Biophys. Acta, 4 ° (196o) 176. 9 B. SACI~TOR, Proc. Celebrazione Spallanzaniana, Reggio-Pavia, in the press. 10 B. SACKTOR, Ann. Rev. Entomol., 6 (1961) lO 3. 1
Received December 2ist, 196o • P r e s e n t address: D e p a r t m e n t of Microbiology, The University of Texas S o u t h w e s t e r n Medical School, Dallas, Texas.
Biochim. Biophys. Acta, 49 (1961) 4o2-4o4
Augmentation of collagen synthesis by ascorbic acid in vitro Ascorbic acid is essential for adequate synthesis of collagen in species for which ascorbic acid is a vitamin 1-3, but since pertinent experiments have been carried out iI~ intact animals we have not known whether vitamin C acts directly on the cells synthesizing collagen (fibroblasts) or whether its action is primarily systemic. High Biochim. Biophys. Acta, 49 (r96I) 404-4 °6
SHORT COMMUNICATIONS
405
concentrations of ascorbic acid in the adrenal cortex and other endocrine tissues 4, and demonstrable effects of hormones on collagen synthesis 5, as well as the finding that some collagen synthesis may occur in the absence of demonstrable ascorbic acid e have suggested that the vitamin may be primarily involved in the production or release of a hormone affecting collagen synthesis. A local action of ascorbic acid, on the other hand, is suggested by the accumulation of ascorbic acid at sit~s of active collagen synthesis in healing wounds ~, and by the direct relation of collagen concentration to ascorbic acid concentration in the carrageenan granuloma 8. More direct evidence for a local action of ascorbic acid has been presented by GOULI)9, who implanted vinyl sponges on either flank of guinea pigs, and then deprived them of ascorbic acid. After injections of small amounts of vitamin directly into one of the pair of sponges, the hydroxyproline (and presumably collagen) content of the injected sponge was several-fold higher than that of the contralateral, uninjected sponge. However, even these experiments do not distinguish between an effect on the whole tissue, such as restoration of blood capillaries, and a direct effect on the collagensynthesizing cells. The present experiments show that ascorbic acid added in vitro to suspensions of scorbutic granuloma cells increases collagen synthesis, thus demonstrating a direct effect on the collagen-synthesizing fibroblasts. GREEN AND LOWTI~ER1° have shown that labeled hydroxyproline can be isolated from collagen fractions from slices of non-scorbutic, carrageenan granulomas incubated with [l~C]proline. This is acceptable evidence for collagen synthesis even without rigorous purification of collagen, since collagen contains approx. 14 % hydroxyproline and this amino acid has not been found in other purified mammalian proteins. We have been able to show, using similar techniques, that suspensions of scorbutic granuloma cells are also able to synthesize collagen hydroxyproline. The data in Table I illustrate this synthesis of collagen by scorbutic granuloma in vitro and the stimulation obtained when ascorbic acid is added to the incubation media. The first 5 guinea pigs received no ascorbic acid for 14 days before being killed. The ascorbic acid concentrations were typical for scorbutic granulomas 8. The specific activity of collagen hydroxyproline was greater in all of these granulomas when I mg of ascorbic acid was added to the incubation media. The last four guinea pigs were deprived of ascorbic acid for 14 days but were injected intraperitoneally with 3oo mg sodium ascorbate 1-1.5 h before being killed. The ascorbic acid of these granulomas was, as expected, very high and addition of ascorbic acid to the incubation media did not lead to an increased specific activity of hydroxyproline. Indeed, in two tissues the specific activity was lower when the media were supplemented with ascorbic acid suggesting inhibition by excess ascorbic acid. A comparison of the effectiveness of injected ascorbic acid with ascorbic acid added in vitro is difficult because of variation among animals. However, the average specific activity of collagen hydroxyproline did not differ significantly when ascorbic acid was added in vitro to scorbutic granulomas and when it was inj ected before killing the animals. Although one can not exclude additional systemic effects of ascorbic acid, the data show that ascorbic acid added in vitro to a sugpension of scorbutic carrageenan granuloma cells stimulates collagen synthesis and does this as effectively as ascorbic acid which reaches the granuloma via the blood stream. Biochim. Biophys. Acta, 49 (1961) 404-406
406
SHORT COMMUNICATIONS TABLE I EFFECT OF ASCORBIG ACID ON COLLAGEN SYNTHESIS
E a c h flask contained a p p r o x . 2 g of tissue brei (obtained b y pressing I 4 - d a y scorbutic carrageenan g r a n u l o m a ~ t h r o u g h a plate h a v i n g o . 8 - m m holes), suspended in 8 ml of K r e b s - R i n g e r b i c a r b o n a t e solution to which h a d been added 8 #C [14C]proline (13/,C/#mole)*, 42.5 # m o l e s glycine, 12. 5 # m o l e s alanine, IO/zmoles glutamic acid, 7 . 5 / , m o l e s each of aspartic acid and arginine, 5 # m o l e s lysine, valine, serine a n d phenylalanine, 2. 5/~moles leucine, isoleucine, m e t h i o n i n e a n d threonine, t /~mole t y r o s i n e a n d t r y p t o p h a n . T h e p H w a s 7.2-7.4. i m g of ascorbic acid (o.i ml) w a s added to the designated flasks and to the u n i n c u b a t e d controls. After i n c u b a t i o n w i t h s h a k i n g a t 37 ° for 6 h in a 95 o/002 - 5 % COs a t m o s p h e r e the reactions were s t o p p e d b y addition of o. 75 ml trichloroacetic acid (I g/ml). H y d r o x y p r o l i n e w a s isolated f r o m t h e collagen fraction a n d its specific activity was d e t e r m i n e d as described in ref. 3. Ascorbic acid w a s determined on a l i q u o t s of tissue as described b y RoE AND KUETHER 11. All e x p e r i m e n t s and analyses were carried o u t in duplicate.
Specifi~ activity o] hydrozyproli~a Guinea pig No.
Ascorbicacid in gra•uloma #g/g
No addition
Added ascorblc
Unincubated control
counls/rain/#mole** I 2 3 4 5 Mean 6 7 8 9 Mean
15 3.8 7.3 7.3 2.8 680 480 58o 380
4,900 5,400 2,400 5,1oo 3,000 5,3oo 1,9oo 3,800 700 2,3oo 2,58o~_ 690*** 4 , 3 8 0 ± 590*** 7,200 7,000 4,4 °0 1,9oo 2,ooo 2,ooo 3,1oo 2,300 4, I8o ! 66o*** 3,3oo ± 1,24o***
7 o 16 6 38 20 8 3 3
* [14C]proline as obtained f r o m Nuclear-Chicago contained [14C]hydroxyproline. Unlabeled h y d r o x y p r o l i n e was added and t h e proline was purified b y c h r o m a t o g r a p h i n g twice on 4o-cm c o l u m n s of Dowex-5o. ** A b o u t i # m o l e of h y d r o x y p r o l i n e w a s counted for IO,OOO counts. * * * S t a n d a r d error.
We are indebted to the U.S. Public Health Service for a grant (A-36o) supporting this work and to Mr. L. STOLOFF, Marine Colloids, Rockport, Me., U.S.A., for a generous supply of purified carrageenan.
College of Medicine, University of Vermont, Burlington, Vt. (U.S.A.)
WILLIAM "CAN B . ROBERTSON* JEAN HEWITT
I S. B. WOLBACH A~D P. R. HOWE, Arch. o[ Pathol. Lab. Med., I (1926) I. 2 W. VAN B. ROBERTSON AND B. SCHWARTZ, J . Biol. Chem., 21o (1953) 689. * W. VAN B. ROEERTSON, J. HEWlTT AND C. HERMAN, J . Biol. Chem., 234 (1959) lO5. 4 M. E. REID, The V i t a m i n s , Vol. I, Academic Press, Inc., N e w York, 1954, p. 311. 6 W. VAN B. ROBERTSON AND E. C. SANBORN, Endocrinology, 63 (1958) 250. 6 j . F. WOESSNER AND B. S. GOULD, J . Biophys. Biochem. Cytol., 3 (I957) 685. v A. F. ABT, S. V. SCHUCHING AND J. H. R o E , J . Nutrition, 7 ° (196o) 427 . 8 W. VAN B. ROBERTSON, A n n . N . Y . Acad. Sci., in the press, 9 B. S. GOULD, J. Biol. Chem., 232 (1958) 637. 10 N. M. GREEN AND D. A. LOWTHER, Biochem. J . , 71 (1959) 55. xt j . H. RoE, Methods o[ Biochemical A n a l y s i s , Vol. I, N e w York, 1954, p. 127.
Received December 6th, 196o * P r e s e n t address: Dept. of Pediatrics, Stanford U n i v e r s i t y Medical School, Palo Alto, Calif. (U.S.A.).
Biochim. Biophys. Acta, 49 (1961) 404-4 °6