Synthesis and degradation rates of collagens in vivo in whole skin of rats, studied with 1802 labelling.

Rats of synthesis and degradation in vivo of collagens in 0.5 M-acetic acid-soluble and -insoluble extracts from skins of three growing rats were determined by using a labelling procedure involving exposure of the animals to an atmosphere of 18O2 for 36 h. For comparison, rats also received injections of [2H]proline. Serial skin biopsies were taken at frequent intervals over 392 days. Enrichment of 18O and 2H in the hydroxyproline of the collagen fractions was determined by gas chromatography-mass spectrometry. Changes in size of the soluble and insoluble collagen pools were considered in the evaluation of isotope kinetic data. The insoluble collagen fraction showed no degradation. The efflux (mean +/- S.D., expressed as mumol of hydroxyproline) from the soluble collagen pool was estimated to be 59.9 +/- 1.9 per day from the 18O data, and 25.5 +/- 7.5 per day from the 2H results. The finding indicates significant reutilization of 2H-radiolabelled proline for hydroxyproline synthesis. From these isotope data and estimates of size of the collagen pools it was determined that 55% of the collagen disappearing from the soluble pool was due to maturation into insoluble collagens and 45% of the disappearance was a result of actual degradation of soluble collagen. These results confirm the utility of 18O2 as a non-reutilizable label for studies of collagen turnover in vivo.     

Influence of bleomycin on the metabolism of dermal collagen in albino rats

The metabolism of collagen in male rats by treatment with bleomycin was studied following the injection of [3H]proline and the determination of specific and total activity of [3H]hydroxyproline in skin collagen fractions and urine. In the case of the bleomycin-treated animals, there was found to be an increase in the neutral salt soluble collagen content with no change in insoluble collagen content as compared to the control group. The specific and total radioactivity of [3H]hydroxyproline in soluble and insoluble collagen fractions was also increased. Examination of [3H]hydroxyproline activity in soluble and insoluble collagen showed that the conversion of soluble to insoluble collagen was improved by the bleomycin-treated group. It was found that this was accompanied by a decrease in urinary excretion of total hydroxyproline and [3H]hydroxyproline during the first 12 hr after the administration of [3H]proline. Therefore, the results of the present investigation clearly indicate that the maturation of soluble to insoluble collagen is promoted and accompanied by a decrease in the catabolism of soluble collagen in the bleomycin-treated animals. In addition, administration of bleomycin increased the synthesis of collagen.     

A study of the biosynthesis of collagenous and non-collagenous proteins and of the proline/hydroxyproline ratios of collagens isolated from embryonic tissues

1. After incubation of chick-embryo skin slices with [(14)C]proline for 2hr. the specific activities of [(14)C]proline and [(14)C]hydroxyproline in soluble and insoluble collagens and [(14)C]proline in non-collagenous proteins were determined as well as the total amounts of both imino acids in these proteins. On the basis of these results it was demonstrated that soluble collagens having a high proline/hydroxyproline ratio are contaminated with non-collagenous proteins. 2. It was found that, in the presence of a mixture of amino acids in the incubation medium, the rate of synthesis of soluble collagen is significantly decreased. 3. The metabolic activity of collagenous proteins is related to their solubility, but that of non-collagenous proteins is not.     

Free and small peptide-bound [14C]hydroxyproline synthesis in rat liver in vitro in CCl4-induced hepatic fibrosis

To clarify the process of free and small peptide-bound hydroxyproline synthesis in hepatic fibrogenesis, we measured the in vitro synthesis of [14C]hydroxyproline in the 67% ethanol soluble fraction in rat liver slices, together with hepatic protein-bound [14C]hydroxyproline synthesis. In control rat liver, the amount of free and small peptide-bound [14C]hydroxyproline synthesized was 13.1 +/- 2.6 10(-4) x dpm/g liver/3 hr. In the CCl4-treated rat liver, where the hepatic hydroxyproline content was increased 4.6-fold, the protein-bound [14C]hydroxyproline synthesis was significantly increased 1.5-fold, but free and small peptide-bound [14C]hydroxyproline synthesis was decreased into 70%. There was a significant inverse correlation between free and small peptide-bound [14C]hydroxyproline synthesis, and hepatic hydroxyproline content. These results suggest that the combination of an increase in collagen synthesis and a decrease in free and small peptide-bound [14C]hydroxyproline synthesis contributes to rapid accumulation of collagen in hepatic fibrosis.     

Synthesis and degradation of collagens in skin of healthy and protein-malnourished rats in vivo, studied by 18O2 labelling.

To explore the effects of growth retardation, caused by restricted protein intake, on collagen turnover in the whole skin, Sprague-Dawley rats (n = 20) were labelled with 18O2 and fed on either an adequate (18%) or a low (3%) lactalbumin diet. Skin biopsies were obtained at intervals during the following 6 months. Independent groups of animals (n = 186) were used to determine the size of the 0.5 M-acetic acid-soluble and -insoluble collagen pools in the entire skin of healthy and malnourished rats. Collagen was estimated by measurement of hydroxyproline. Soluble-collagen synthesis rates were equivalent to 99 +/- 8 mumol of hydroxyproline/day in healthy animals and 11 +/- 2 mumol/day in malnourished rats. Insoluble-collagen synthesis rates were 32 and 5 mumol of hydroxyproline/day in the healthy and protein-depleted rats respectively. The degradation of soluble collagen amounted to 37 +/- 8 and 6 +/- 2 mumol of hydroxyproline/day in the healthy and malnourished groups respectively. Efflux of collagen from the soluble collagen, defined as the sum of the rate of soluble collagen that is degraded plus that which matures into insoluble collagen, was 70 +/- 8 and 11 +/- 2 mumol of hydroxyproline/day in the healthy and malnourished groups respectively. Insoluble collagen was not degraded in either group. The fraction of soluble collagen leaving the pool that was converted into insoluble collagen was 0.46 in both diet groups. It is concluded that the turnover of soluble collagen is markedly decreased with malnutrition, but degradation and conversion into insoluble collagen account for the same proportions of efflux from the soluble-collagen pool as in rapidly growing rats.     

Quantitative and metabolic changes of hepatic collagens in rats after carbon tetrachloride poisoning

1. The collagen hydroxyproline in rat liver was composed of 3.5% neutral-soluble collagen, 4.9% acid-soluble collagen and 91.6% insoluble collagen. In labelling studies with [(14)C]proline in vitro, the specific radioactivities of neutral-soluble, acid-soluble and insoluble collagens in rat liver were found to be 233000, 69000 and 830d.p.m./mumol of hydroxyproline respectively after 1h. 2. During subacute carbon tetrachloride poisoning the hepatic content of insoluble collagen markedly increased, whereas those of soluble collagens did not change. During recovery from subacute poisoning hepatic contents of soluble collagens were markedly decreased. 3. After 8 weeks of carbon tetrachloride poisoning the specific radioactivities of hepatic soluble collagens increased, while that of insoluble collagen decreased. During recovery from subacute poisoning, the specific radioactivities of soluble collagens decreased to the normal range and that of insoluble collagen further decreased. 4. Hepatic collagenolytic activity solubilizing insoluble collagen, which differs from mammalian collagenase, decreased under the conditions of the subacute poisoning and also during recovery from subacute poisoning.     

The phosphorylation of 5′-oligoadenylic acids by adenylate kinase and adenosine triphosphate

1. This paper reports studies on the metabolism of bone from normal chicks and from chicks with vitamin D-deficiency rickets. Both in vitro and in vivo there was an increased incorporation of [(14)C]proline into collagen hydroxyproline by rachitic bone. The proportion of the collagen that was soluble in cold salt solutions was greater with the rachitic bone. These results show that in rickets there is an increased synthesis of bone collagen, but they do not provide any evidence of a defect in the maturation of collagen. 2. Rachitic bone incubated aerobically in vitro consumed more glucose and released more lactate than normal bone. Bone from rachitic chicks treated with vitamin D 48hr. previously had rates of glycolysis that were nearly normal. Though we were unable to show any direct action of vitamin D in vitro, we consider that vitamin D probably has a direct action on bone, possibly related to matrix biosynthesis.     

Study on fibroblasts in Marfan's syndrome

Fibroblasts grown from the skin of patients with Marfan's syndrome (mother and son) were investigated cytogenetically and biochemically together with fibroblasts from healthy individuals.In Marfan's syndrome the cross-linking of the tropocollagen chains is disturbed already on the cellular level. The high ratio of soluble collagen to insoluble collagen (3:1), compared with controls, where the ratio is 1:1, supports this concept. The high free hydroxyproline content in normal fibroblasts and in those of Marfan's syndrome (in early passages) forming about 70% of total cell hydroxyproline speaks for the participation of fibroblasts not only in the synthesis but also in the catabolism of newly formed young forms of collagenous proteins. In contrast to controls the fibroblasts from the skin of both patients suffering from Marfan's syndrome changed in the sense of ageing depending on the duration of their cultivation in vitro. This stability is illustrated by the equalization of the ratio of soluble to insoluble collagenous proteins to 1:1.Presented at the Symposium on Genetics in Ophthalmology, June 10th 1965 in Brno, Czechoslovakia.     

Metabolic fate of rat and human lipoprotein apoproteins in the rat

The fate of (125)I-labeled apolipoproteins was studied in vivo in rats that had received intravenous injections of (125)I-labeled rat HDL and (125)I-labeled human HDL, LDL, and VLDL. Plasma decay curves of rat and human HDL were exponential with similar half-lives in the circulation (11-12 hr). After injection, low molecular weight apolipoproteins (apoLP-alanine of human HDL and fraction HS-3 of rat HDL) were found to redistribute to other lipoproteins, predominantly VLDL. Decay curves of individual HDL proteins were constructed after lipoprotein fractionation, delipidation, and polyacrylamide gel electrophoresis. It was found that the half-lives of the different HDL apoproteins were not identical. A major rat HDL protein (52% of total counts) had a circulating half-life (t((1/2))) of 12.5 hr. Two others had a t((1/2)) of 8-9 hr while the t((1/2)) of several others was 11-12 hr. The t((1/2)) of three well-characterized human HDL apoproteins, apoLP-glutamine I, apoLP-glutamine II, and apoLP-alanine, were 13.5, 9.0, and 15.0 hr, respectively. The fate of (125)I-labeled human VLDL and LDL apoproteins in rats was similar to that described previously in humans. After injection of (125)I-labeled human VLDL into rats, apoLP-glutamic acid and apoLP-alanine rapidly transferred to rat HDL and were lost thereafter from the circulation from both VLDL and HDL. The apoLDL moiety of human VLDL moved metabolically to the LDL density range (d = 1.019-1.063) through a lipoprotein of intermediate density (d = 1.006-1.019).     

In vivo labeling studies on the biosynthesis and degradation of collagen in experimental myocardial infarction.

The biosynthesis and degradation of myocardial collagen was studied in myocardium infarcted rats after a single intraperitoneal injection of 3H-proline. The incorporation of tritiated proline into collagen as 3H-hydroxyproline was regarded as a measure of collagen synthesis. The total content as well as total activity of hydroxyproline were measured in the whole homogenate, neutral salt soluble fraction, insoluble fraction and in urine collected at different time intervals and specific activities were calculated. Both collagen anabolism and catabolism were found to be affected in infarcted rat hearts. Degradation of existing collagen at the earlier stages of myocardial infarction and a simultaneous new collagen synthesis and deposition as insoluble form later took place.     

Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs. Evidence for the formation and degradation of a partially hydroxylated collagen

1. After the administration of l-[G-(3)H]proline to guinea pigs deprived of ascorbic acid for increasing periods of time, the specific radioactivities of proline and hydroxyproline in skin collagen and aortic elastin were determined at various time-intervals after administration of the labelled compound with a view to studying the formation and degradation of collagen and elastin both deficient in hydroxyproline. 2. As judged from the incorporation of radioactivity into elastin proline, elastin synthesis was not decreased in the ascorbic acid-deficient animals. There was however, a rapid decline in the specific radioactivity of elastin hydroxyproline. The proline/hydroxyproline specific-radioactivity ratio was approx. 1.5:1 after 6 days and 20:1 after 12 days of ascorbic acid deprivation, in contrast with the ratio of 1:1 in controls. The results suggested that the effect of ascorbic acid deficiency on elastin biosynthesis could be regarded as simply an elimination of hydroxylation of elastin proline with the formation and retention of a polymer increasingly deficient in hydroxyproline. 3. Collagen proline and hydroxyproline specific radioactivities were derived from material that was soluble in hot trichloroacetic acid, non-diffusible and collagenase-degradable. In contrast with elastin, there was a rapid decline in the specific radioactivity of proline as well as hydroxyproline in collagen from the ascorbic acid-deficient animals. However, the proline/hydroxyproline specific-radioactivity ratio in all samples from scorbutic animals was consistently slightly above 1:1. The results suggest the appearance in place of collagen, but in rapidly diminishing amounts, of a partially hydroxylated collagen in which the degree of hydroxylation may be decreased only by approx. 10%. 4. Incorporation of radioactivity into the diffusible hydroxyproline in skin remained relatively high despite the rapid decline in the incorporation of radioactivity into collagen. This observation is interpreted as indicative of an increasing degree of degradation of partially hydroxylated collagen to diffusible peptides. An alternative explanation might be that partially hydroxylated peptides are released to an increasing extent from ribosomes before they attain a length at least sufficient to render them non-diffusible. In either case it implies the accumulation in scurvy of low-molecular-weight peptides enriched in proline and deficient in hydroxyproline and could explain the failure to accumulate a high-molecular-weight collagen deficient in hydroxyproline. 5. It is thought, however, that, in addition, an inhibition of ribosomal amino acid incorporation leading to decreased synthesis of partially hydroxylated collagen may also occur, perhaps secondarily to impaired hydroxylation.     

The effect of ascorbic acid on the nature and production of collagen and elastin by rat smooth-muscle cells.

1. The effects of various concentrations of ascorbic acid on the quality and quantity of the insoluble extracellular matrices produced by two strains of cultured rat smooth-muscle cells were studied. 2. Ascorbic acid was necessary for the appearance of insoluble collagen in the extracellular matrix. 3. Secretion of soluble collagen continued in the absence of ascorbic acid, but this soluble collagen was markedly underhydroxylated. 4. The amount of insoluble collagen present in the matrix was directly related to the ascorbic acid concentration. 5. The insoluble collagen that appeared in the matrix under conditions where ascorbic acid was limiting was no more than 7% underhydroxylated. 6. In contrast, the amount of insoluble elastin produced was inversely proportional to the ascorbic acid concentration. 7. The elastin produced in the absence of ascorbic acid had the expected amino acid composition, but hydroxyproline was absent. 8. The hydroxyproline content of elastin was also directly dependent on the ascorbic acid concentration. 9. Ascorbic acid had variable effects on the quantity of glycoprotein(s) present in the matrix. 10. The appearance of insoluble collagen in the extracellular matrices produced by cultured human fibroblasts and calf endothelial cells was also completely dependent on the presence of ascorbic acid.     

The synthesis of collagen during the development of anal-fin processes in ethisterone-treated females of Oryzias latipes

The synthesis of collagen during the development of anal-fin processes induced by ethisterone was studied by the autoradiographies of tritiated proline (Pro-T) and hydroxyproline (HyPro-T). The rapid incorporation of Pro-T was initiated in the screloblast masses between 48 and 72 hr of ethisterone treatment. The increased incorporation of HyPro-T was also detected in the screloblast masses after 48 hr. Furthermore, the grains of both radioactive amino acids were distributed distinctly in the horny substance secreted by the screloblast masses at and after 72 hr. It is suggested that the rapid increase in incorporation of labelled amino acids is mainly due to the collagen synthesis, and that the synthesis of collagen is initiated in the screloblast masses between 48 and 72 hr of the treatment.     

The influence of indomethacin on collagen synthesis during tendon healing in the rabbit

The influence of indomethacin on collagen synthesis in intact and healing plantaris longus tendons in the rabbit was investigated. Forty-four male New Zealand White rabbits were subjected to a standardized trauma (tenetomy + repair) on the left hindlimb. Half of the animals were subsequently treated with indomethacin, 10 mg/kg per day orally, and the other half with placebo. After 2 and 4 weeks the rabbits were injected intravenously with 3H-proline and killed 18 h later. Indomethacin affected the collagen metabolism differently depending on whether the tendons were involved in wound healing or not. In intact tendons the drug caused a small general inhibition of collagen synthesis. In the healing tendon there was a shift towards the synthesis of more insoluble collagen with little effect on the total synthesis. After 4 weeks there was also a slight but significant decrease in the amount of hydroxyproline in the most soluble collagen fraction from the tenotomized, indomethacin treated tendons.     

The influence of indomethacin on collagen synthesis during tendon healing in the rabbit

The influence of indomethacin on collagen synthesis in intact and healing plantaris longus tendons in the rabbit was investigated. Forty-four male New Zealand White rabbits were subjected to a standardized trauma (tenetomy + repair) on the left hindlimb. Half of the animals were subsequently treated with indomethacin, 10 mg/kg per day orally, and the other half with placebo. After 2 and 4 weeks the rabbits were injected intravenously with ³H-proline and killed 18 h later. Indomethacin affected the collagen metabolism differently depending on whether the tendons were involved in wound healing or not. In intact tendons the drug caused a small general inhibition of collagen synthesis. In the healing tendon there was a shift towards the synthesis of more insoluble collagen with little effect on the total synthesis. After 4 weeks there was also a slight but significant decrease in the amount of hydroxyproline in the most soluble collagen fraction from the tenotomized, indomethacin treated tendons.     

Effect of hyperbarism on collagenous protein metabolism in granulation tissue.

Rats with subcutaneously implanted polyurethane sponges were exposed 6 hours daily for 7 days to high ambient atmospheric pressures (1.5, 2, 2.5 and 3 ATA). Another group was exposed 4 hours daily for 4 weeks to 3 ATA before inducing granulation tissue formation. 14C-proline was administered 16 hours before terminating the experiment. Free hydroxyproline, soluble and insoluble collagen and total noncollagenous protein were isolated from the 7-day granuloma and the amount and radioactivity of 14C-hydroxyproline and 14C-proline were determined. Seven days' graduated hyperbarism did not affect collagen synthesis; the maturation of collagen to insoluble forms was inhibited at 2 and 2.5 ATA, but not at 3 ATA. Stimulated degradation of collagen (free hydroxyproline) was observed at 2, 2.5 and 3 ATA. In animals subjected to long-term exposure at 3 ATA pressure, the collagen in the granuloma matured to insoluble forms more quickly. Biochemical changes were correlated with changes in the fine structure of the granulation tissue. The appearance of the fibroblast proteosynthetic apparatus was not influenced by hyperbarism. Progressive spherical transformation, fusion of mitochondria and lysosomal activation in the pericapillary fibroblasts occurred at 2, 2.5 and 3 ATA. In short-term experiment, the formation of cytosegresomes and cellular necrosis also contributed to the effect at 3 ATA, which is thus already a toxic pressure for granulation tissue.     

Measurement of liver collagen synthesis by heavy water labeling: effects of profibrotic toxicants and antifibrotic interventions

Enhanced production of collagen is central to fibrotic disorders such as hepatic cirrhosis and pulmonary fibrosis. We describe a sensitive, quantitative, and high-throughput technique for measuring hepatic collagen synthesis in vivo through metabolic labeling with heavy water ((2)H(2)O). Rats and mice received (2)H(2)O in drinking water for up to 35 days. Deuterium incorporation into collagen-bound amino acids (AA) alanine and hydroxyproline (OHP) was measured by gas chromatography-mass spectrometry. A threefold stimulation of collagen fractional synthesis was observed under the maximum dosage of carbon tetrachloride (CCl(4); 1.67 ml/kg). Deuterium enrichment was systematically 20% higher in AA from monomeric collagen relative to dimeric collagen, consistent with slower turnover of the latter. Administration of 1% griseofulvin to mice resulted in a significant, threefold increase in liver collagen synthesis, observable within 12 days and consistent with predicted interstrain differences (C57/Bl6J > BALB/c). Deuterium enrichments of OHP from total liver proteins correlated well with alanine or OHP from isolated collagen. Fibrogenesis subsided after withdrawal of CCl(4) exposure and was reduced to various degrees by coadministration of interferon-gamma, rosiglitazone, atorvastatin, or enalapril. Changes in isotopically measured collagen synthesis correlated with, but were more sensitive and reproducible than, standard histological staining (trichrome) for fibrosis. In summary, liver collagen synthesis can be measured sensitively and with high precision over a short time period, without radioactivity, thereby providing a relatively high-throughput in vivo strategy for rapidly measuring profibrotic activities of suspected hepatotoxicants and antifibrotic activities of drug candidates.     

Investigation of total, free, peptide-bound, protein-bound, soluble and insoluble collagen hydroxyproline content in tissues from the Arabian camel (Camelus dromedarius)

This study was conducted to determine the concentration of total, free, peptide-bound, protein-bound, soluble and insoluble collagen hydroxyproline (Hyp) in tissues from the Arabian camel (Camelus dromedarius). Results indicated that there were significant differences in the concentration of total, free, peptide-bound, protein-bound, soluble and insoluble collagen Hyp in various tissues (P < 0.01). Camel kidney showed a significantly high concentration of total, free, peptide-bound and protein-bound Hyp and collagen content as compared to other tissues examined (P < 0.01). Kidney also showed a significantly high concentration of soluble collagen Hyp as compared to other tissues examined (P < 0.01). However, the concentration of insoluble collagen Hyp was significantly high in liver when compared to other tissues (P < 0.01). These variations may result from differences in the collagen structure and/or composition in this species.     

Effect of beta-D-xylosides on collagen synthesis in cultured fibroblasts.

Cultured normal human skin fibroblasts were incubated with [¹⁴C]proline in the presence and absence of 1.0 mM p-nitrophenyl-β-D-xylose. Formation of non-dialyzable hydroxyproline was used as a measure of collagen synthesis. Although total [¹⁴C]proline incorporation was similar in the two cultures, [¹⁴C]hydroxyproline formation was significantly decreased in the β-xyloside-treated cultures. Increasing the period of incubation increased the radioactivity of the insoluble collagen fraction in untreated fibroblasts, however, in β-xyloside-treated cultures no such increase was observed. In contrast to the decreased production of collagen, growth of cells in the presence of the β-xyloside induced the synthesis of high levels of soluble glycosaminoglycans as measured by ³⁵SO₄ incorporation into isolated polysaccharide.     

Changes in synthesis of types-I and -III collagen during matrix-induced endochondral bone differentiation in rat.

The changes in rates of hydroxyproline formation and biosynthesis of types-I and -III collagen during bone matrix-induced sequential differentiation of cartilage, bone and bone marrow in rat were investigated. Biosynthesis of types-I and -III collagen at different stages of this sequence was studied by labelling in vivo and in vitro with [2,3-3H]proline. Pepsin-solubilized collagens were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis. The results revealed that maximal amounts of type-III collagen were synthesized on day 3 during mesenchymal-cell proliferation. Thereafter, there was a gradual decline in type-III collagen synthesis. On days 9--20 during bone formation predominantly type-I collagen was synthesized. Similar results were obtained by the use of labelling techniques both in vivo and in vitro.