Selective inhibition of collagen synthesis by D-penicillamine in carrageenin-induced inflammation in rats.

The effect of d-penicillamine on syntheses of collagen and noncollagen protein in inflammed tissued was studied by incubating minced granulation tissue with [³H]proline and various amounts of d-penicillamine. d-Penicillamine selectively inhibited the incorporation of [³H]proline into collagen hydroxy-proline when 30 mM d-penicillamine was present in the incubation medium, and the accumulation of [³H]proline-labeled protocollagen was found in the minced granulation tissue incubated with 30 mM d-penicillamine. In addition, administration of d-penicillamine (800 mg/kg) into granuloma pouch every 12 hr for 4 days, selectively inhibited the collagen synthesis and increased the collagen solubility in granulation tissue. When partially purified prolyl hydroxylase was incubated with [3,4-³H]proline-labeled protocollagen substrate in the presence of d-penicillamine, the enzyme activity was decreased in proportion to the concentration of d-penicillamine. This inhibition was restored by increasing the amount of ferrous iron in the incubation medium. These results suggest that the selective inhibition of collagen synthesis in vivo and in vitro may be mainly due to the ability of d-penicillamine to chelate ferrous iron, a cofactor of prolyl hydroxylase.     

An experimental model of hepatic fibrosis induced by the administration of dibutyltin dichloride.

Dibutyltin dichloride (DBT) was investigated for its ability to induce hepatic fibrosis in rats. Since hydroxyproline does not appear in significant amounts in noncollagen protein and prolyl residues in collagen are not hydroxylated before they are in peptide-bound form, the conversion of isotopically labeled proline to hydroxyproline by prolyl hydroxylase was taken as one parameter reflecting the rate of collagen formation. In vitro collagen synthesis was determined by incubation of liver biopsies with labeled proline, after which incorporation of label into collagenase-digestible protein was compared to incorporation into collagenase-resistant protein. Hydroxyproline content served as a parameter of collagen accumulation. DBT, administered by oral intubation (10 and 20 mg/kg) daily for 4 days, produced inflammation in portal tracts and biliary damage at the end of this period. Prolyl hydroxylase activity was increased over control values in both treated groups, and in vitro collagen synthesis was increased in the higher dose group. DBT, administered by oral intubation (10 and 20 mg/kg) every other day for 12 days, produced extensive inflammation in portal tracts, biliary damage, fibrosis, necrosis, infarcted areas, and granulomatous lesions. In the higher dose group increases in hydroxyproline content, prolyl hydroxylase activity, and relative collagen synthesis in vitro were observed at the end of the 12-day period. DBT did not increase prolyl hydroxylase activity of L929 cells when added to culture media for 24 hr. These results suggest that DBT-induced fibrogenesis is a result of biliary damage and/or inflammatory processes rather than direct stimulation of fibroblasts.     

Effect of a single injection of betamethasone disodium phosphate on the synthesis of collagen and noncollagen protein of carrageenin granuloma in rats

The effect of a single injection of betamethasone disodium phosphate on the incorporation of [³H]proline into collagen and noncollagen protein of rat carrageenin granuloma was studied. In all series of experiments, both [³H]proline and the steroid were given intravenously and the animals were killed at 30 min after the labeled proline injection in order to investigate precisely the time course of the drug action. The incorporation was not influenced at all by the steroid when it was given 30 min before the injection of [³H]proline. The inhibitory effect of the steroid, for both the collagen and the noncollagen protein, became significant at 1 hr after its intravenous administration, increasing progressively with the passage of time. Inhibition of the labeled proline incorporation into collagen hydroxyproline was significantly greater than that into noncollagen protein. Neither accumulation of protocollagen nor any change of protocollagen proline hydroxylase activity was demonstrated in the granuloma obtained from the rats treated with the steroid. The results show that the steroid does not inhibit the process of hydroxylation of protocollagen, suggesting indirectly the inhibition of synthesis of protocollagen or of [³H]proline transport through cell membrane, or of both.     

Biochemical and ultrastructural study of the effects of proline analogues on collagen synthesis in 3T6 fibroblasts

The effects of proline analogues, L-3,4-dehydroproline and L-azetidine-2-carboxylic acid, on collagen synthesis by cultured 3T6 fibroblasts have been studied. Prolyl hydroxylase activity was partially inhibited in cells cultured with dehydroproline for 24 h, resulting in the synthesis of collagen in which the proline was underhydroxylated. Azetidine had no effect on prolyl hydroxylase and less effect on the degree of hydroxylation of proline. Fibroblasts grown in the presence of either analogue and fixed in-situ contained greatly distended cisternae of the rough endoplasmic reticulum. Proline analogues otherwise caused few ultrastructural changes in the cells. Treated cells which had been handled more roughly during preparation for electron microscopy contained many large cytoplasmic vacuoles in addition to dilated cisternae. Our results indicate that the major effect of the proline analogues was the inhibition of prolyl hydroxylation. However, electron microscopy of the treated cells revealed hitherto unreported cytoplasmic damage.     

Effect of betamethasone-17-valerate on synthesis of collagen and prolyl hydroxylase activity in chick embryo tendon cells.

The effect of betamethasone-17-valerate on the biosynthesis of collagen was studied in matrix-free chick embryo tendon cells in vitro and the activity of prolyl hydroxylase was assayed in the cells after incubation with the steroid in vitro and after injection of the steroid on to chorioallantoic membrane of embryonated eggs. Tendon cells synthesized collagen at a rapid rate and the synthesis was essentially linear for up to about 7 hr when studied by labelling with [¹⁴C]proline. The secretion of collagen [¹⁴C]hydroxyproline was also almost linear for up to 7 hr. Betamethasone-17-valerate decreased the total incorporation of [¹⁴C]proline and the decrease in the synthesis of [¹⁴C]hydroxyproline was larger than the decrease in the total incorporation, indicating that the collagen synthesis was affected more than other protein synthesis. Betamethasone-17-valerate had no effect on the activity of prolyl hydroxylase, when the cells were incubated with this steroid in vitro or, when the activity of the purified enzyme was assayed in the presence of this steroid. When the steroid was injected on to chorioallantoic membrane of embryonated eggs, the activity of prolyl hydroxylase decreased markedly in the tendon cells, indicating that in the system studied the amount of prolyl hydroxylase is decreased or that the enzyme is inactivated by this steroid in vivo.     

Effect of (+)-catechin on purified prolyl hydroxylase and on collagen synthesis in skin fibroblasts in culture.

The flavonoid, (+)-catechin. inhibited the activity of purified rabbit prolyl hydroxylase, or the crude enzyme present in liver homogenate. (+)-Catechin at a concentration of 10^?4 M inhibited both forms of enzyme 50 percent. The inhibition was partially reversed when excessive amounts of Fe²⁺ (10^?3 M) or ascorbate (10^?2 M) were added to the reaction mixture. A higher recovery of the original activity was obtained when Fe²⁺ and ascorbate were added together to the same inhibited system. In addition, (+)-catechin revealed a competitive type of inhibition with respect to varying concentrations of enzyme. These results suggest that the drug and the enzyme compete for the free-radicals produced by the interaction of Fe²⁺, ascorbate and molecular oxygen. The effects of (+)-catechin on collagen synthesis and on proline hydroxylation were examined in confluent cultures of human skin fibroblasts. The activity of prolyl hydroxylase was significantly (P < 0.01) reduced (50 percent of the control) when the cells were incubated for 7 hrs in the presence of 2 × 10^?4 M (+)-catechin. When exposed to the same inhibitor concentration for 24 hr, collagen, and non-collagen, protein synthesis, as measured by the collagenase digestion method, were also significantly reduced (P < 0.01). However, the significant (P < 0.01) decrease in the proportion of collagen synthesized to total protein showed that the drug is more effective on the pathway of collagen production. The ratio of hydroxyproline/proline in total protein was also significantly decreased (P < 0.01) in the cultures exposed to (+)-catechin (2 × 10^?4 M) for 24 hr.     

The mechanism involved in the inhibitory action of tranilast on collagen biosynthesis of keloid fibroblasts.

Tranilast, an anti-allergic drug inhibiting the release of substances such as histamine and prostaglandins from mast cells, was previously reported to suppress collagen synthesis of fibroblasts derived from keloid tissues. However, the inhibitory mechanism on collagen synthesis is unknown. We studied its inhibitory mechanism on collagen synthesis by culturing fibroblasts from keloid and hypertrophic scar tissues of humans. Collagen synthesis of fibroblasts from keloid and hypertrophic scar tissue is greater than that from healthy human skin. Tranilast (3-100 microM) did not inhibit prolyl hydroxylase (the rate-limiting enzyme in collagen synthesis) activity. Tranilast (3-300 microM) suppressed the collagen synthesis of fibroblasts from keloid and hypertrophic scar tissue but not healthy skin fibroblasts. Tranilast (30-300 microM) inhibited the release of transforming growth factor (TGF)-beta 1 from keloid fibroblasts, which enhances the collagen synthesis of keloid fibroblasts. Anti-TGF-beta 1 antibody (50 microliter/ml) inhibited the collagen synthesis, although diphenhydramine (10 microM) and indomethacin (10 microM) did not show any inhibition. These results suggest that tranilast inhibits collagen synthesis of fibroblasts from keloid and hypertrophic scar tissue through suppressing the release of TGF-beta 1 from the fibroblasts themselves.     

Selective inhibition of collagen accumulation by N-(3,4-dimethoxycinnamoyl)anthranilic acid (N-5') in granulation tissue

The effect of topically applied N-5', an inhibitor of chemical mediator release from mast cells, on the carrageenin-air-pouch inflammation was studied. The formation of granulation tissue, the accumulation of exudate and the number of infiltrating cells were significantly reduced by the treatment with N-5' (100 mg/kg). The collagen content in granulation tissue was dose-dependently reduced without affecting the noncollagen protein and DNA content by treatment with N-5'. At a dose of 100 mg/kg of N-5', prolyl hydroxylase activity in the tissue was significantly decreased. The selective inhibition of collagen accumulation in granulation tissue resulted from reduction of collagen biosynthesis in vivo. N-5' did not directly inhibit collagen synthesis by diploid fibroblasts, but inhibited fibroblast proliferation in culture. Such results indicate that one of the inhibitory mechanisms of collagen accumulation by N-5' in inflamed sites may involve the inhibition of fibroblast proliferation.     

Effects of glucocorticosteroids on cultured human skin fibroblasts--IV. Specific decrease in the synthesis of collagen but no effect on its hydroxylation.

Confluent cultures of normal baby foreskin fibroblasts were exposed for 6 days to hydrocortisone-17-butyrate (5 μg/ml)or to clobetasol-17-propionate (1 or μg/ml). On day 5 [³H]proline was added to the cultures and on day 6 both medium and cell layer were analyzed for [³H]protein and protein-bound [³H]hydroxyproline. The synthesis of labeled protein was little affected, while that of labeled collagenous protein was greatly depressed, as compared to that of the control cells. This depression occurred for the collagenous protein present in the cell layer as well as for that released into the growth medium. This effect was not accompanied by a decrease in cellular prolyl hydroxylase activity or in collagen proline hydroxylation.     

Effect of proline analogs on oxygen toxicity-induced pulmonary fibrosis in the rat

Proline analogs inhibit collagen biosynthesis and prevent accumulation of collagen in tissues. The antifibrotic effects of three proline analogs, cis-hydroxyproline, L-azetidine-2-carboxylic acid, and L-3,4-dehydroproline, were compared in a rat oxygen toxicity model. The specificity of these agents for collagen was examined by measuring their effects on noncollagen protein and elastin accumulation in the lung. Increased lung collagen was produced by exposing rats to 95% O2 for 60 hr followed by a 2-week recovery period. Animals were treated with the proline analogs for the 2-week period. Oxygen exposure in untreated animals increased lung collagen 26% above air-breathing controls, and this increase was prevented by all three analogs. Increased noncollagen protein was also prevented by these agents, suggesting they were not entirely specific for collagen. Elastin accumulation, however, was not inhibited by cis-hydroxyproline. It was concluded that proline analogs were antifibrotic, but affected the metabolism of noncollagen protein.     

An increase of collagen biosynthesis precedes other symptoms of ethanol-induced liver damage in rats

Treatment of rats with 10% ethanol for 6 months induced a 3-4-fold increase of [3H]proline incorporation into liver collagen and enhanced the level of prolyl hydroxylase activity in liver and serum of intoxicated rats. Several diagnostic tests used to assess liver disease failed to demonstrate any symptom of liver damage. Therefore, increased collagen biosynthesis appears to precede other symptoms of liver damage induced by ethanol.     

Inhibitory effect of cadmium on collagenous peptide synthesis of embryonic chick bone in tissue culture

The effect of cadmium (Cd) on bone collagen synthesis was assessed in organ cultures of embryonic femur by measuring the incorporation of [³H]-proline(Pro) into collagenous-digestible protein (CDP) using purified bacterial collagenase. Cd produced a decrease when [³H] Pro was incorporated in CDP. There was little alteration in the hydroxylation of [³H] Pro to [³H]hydroxyproline(Hyp) in CDP of Cd-treated bone. An accumulation of underhydroxylated collagen and a decrease in the activity of prolyl hydroxylase (EC 1.14.11.2) in Cd-treated bone was not observed. These results indicated that the inhibitory effect of Cd on collagen synthesis was largely due to inhibition of collagenous peptide synthesis without inhibition of its hydroxylation.     

Increase in lipoperoxides and prolyl hydroxylase activity in rat liver following chronic ethanol feeding

The effect of lipid peroxidation on hepatic collagen synthesis was investigated in male Wistar strain rats after 7 weeks of ethanol feeding. Compared with control rats, the ethanol-fed rats had a significantly higher lipoperoxide content and a significantly lower reduced glutathione content al all times following ethanol treatment. Except for the earliest time (2 days), hepatic prolyl hydroxylase activity was also significantly increased and finally reached up to 214% of the control level. Hepatic hydroxyproline content was slightly increased, but not statistically significant. The lipoperoxides content was significantly correlated with prolyl hydroxylase activity and inversely correlated with reduced glutathione content. These findings were also confirmed in ethanol-pyrazole-treated rats. These results suggest that elevated lipoperoxides mediate an acceleration of collagen synthesis, even at an early stage, in ethanol-induced hepatic injury.     

Effect of 1,10-phenanthroline and desferrioxamine in vivo on prolyl hydroxylase and hydroxylation of collagen in various tissues of rats

We studied prolyl hydroxylase (PH) activity in various tissues of rats after systemic and local administrations of 1,10-phenanthroline and Desferrioxamine. We assayed PH in tissue extracts without adding iron to the incubation medium; we ascertained hydroxylation of collagen by measuring ³H release from biosynthetically 3,4-³H-proline-labeled collagen substrate. We studied the effect of both Fe²⁺ and Fe³⁺ chelating agents in tissues with a relatively high rate of collagen synthesis such as fetal skin and skin of newborn rats, 17-β-estradiol-stimulated uterus of immature rats, and carrageenan granuloma tissue. In addition, in some organs we studied collagen hydroxylation by measuring the ratio of proline to hydroxyproline either in highly purified samples or in the digest from samples treated with protease-free collagenase. Finally, the extent of underhydroxylation of collagen from some tissues of rats treated with either one or both chelating agents was determined using a 3,4,-³H-proline-labeled collagen as a substrate to partially purified PH. Neither systemic nor local injections of 1,10-phenanthroline or Desferrioxamine alone inhibited PH in any of the models. But simultaneous administration of both agents inhibited PH and hydroxylation of collagen in some models. Although local injections of 1,10-phenanthroline into granuloma tissue did not inhibit PH activity, hydroxylation of collagen synthesized in the granuloma from 4 to 16 hr after injection was reduced significantly. This seeming discrepancy is explained by the finding that PH is active even without the addition of Fe²⁺ in the assay medium; a strong reducing environment (α-ketoglutarate, ascorbic acid) reduces Fe³⁺, thus providing ferrous ions essential for PH activity. Both forms of iron, therefore, must be chelated simultaneously to affect PH activity in the assay system. In some experiments, within 8 hr after systemic administration of 1,10-phenanthroline, the activity of PH in skin almost doubled and remained significantly elevated for 40 hr. The mechanism responsible for this phenomenon is not known. The results of this study indicate that previously reported inhibitory effects of some Fe²⁺ chelating agents on collagen synthesis in various models of fibrosis may not be related to inhibition of PH or to collagen hydroxylation.     

The effect of ethanol on enzyme synthesis and secretion in isolated rat pancreatic lobules

This study investigates the effect of ethanol on enzyme synthesis and secretion in rat pancreatic lobules. Ethanol caused a dose-dependent inhibition of 3H-leucine incorporation into total protein. Examination of the time dependence showed that ethanol inhibited protein synthesis at each time point. Removal of ethanol partially reversed this inhibition. An autoradiograph of the newly synthesized proteins separated on SDS-PAGE showed that ethanol inhibited synthesis of all proteins. 14C-cycloleucine uptake was not altered by ethanol, excluding inhibition of amino acid uptake as the mechanism for the decreased protein synthesis induced by ethanol. Electron microscopy revealed no ultrastructural damage. Ethanol had no effect on the stimulated release of (i) amylase from zymogen granules nor (ii) newly synthesized pulse labelled enzymes. Acetaldehyde had no inhibitory effect on enzyme synthesis or secretion indicating that ethanol per se and not its metabolite is inhibitory. The decreased synthesis after acute exposure to ethanol with preservation of exocytosis would limit the autodigestive potential of pancreatic tissue. This may explain why isolated toxic doses of ethanol are rarely if ever associated with pancreatitis.     

Effects of prolyl hydroxylase inhibition on arterial collagen synthesis and blood pressure in hypertensive rats

Changes in arterial collagen synthesis and the effects of P-1894B, a potent inhibitor of prolyl hydroxylase, were investigated on rat hypertension, induced by deoxycorticosterone acetate (DOCA) and salt. In DOCA-salt hypertensive rats, prolyl hydroxylase activity increased significantly in the abdominal aorta and in the mesenteric artery. Incorporation of 14C-proline into the thoracic aorta, abdominal aorta and mesenteric artery was higher and the hydroxyproline content of the abdominal aorta and mesenteric artery was higher than in the control rats. Treatment with P-1894B significantly inhibited prolyl hydroxylase activity, reduced arterial collagen synthesis, but did not prevent or reverse hypertension.     

Effect of ethanol metabolism on initiation of protein synthesis in rat hepatocytes

Rat liver parenchymal cells were incubated in the presence and absence of ethanol (80 mM). Polysomes were isolated and analysed on sucrose gradients. Ethanol was shown to (1) inhibit the incorporation of ¹⁴C-valine into proteins, (2) result in a shift in the distribution of polysomes towards smaller sizes, (3) inhibit the formation of 40S initiation complexes, and (4) diminish the concentration of glucose-6-phosphate in the hepatocytes. Addition of 4-methylpyrazole (0.5 mM) partially prevented the inhibition of protein synthesis and completely restored the polysomal distribution. It is concluded that ethanol inhibits protein synthesis partly by a mechanism linked to ethanol metabolism. This effect takes place at the level of initiation and may be mediated by a reduced gluconeogenesis.     

Fate of aclacinomycin-A and its metabolites effect on cell growth and macromolecular synthesis

The relationship between the structure and activity of aclacinomycin-A (ACM) metabolites was investigated in vitro in Friend leukaemia cells (FLC). The cytotoxic effect was related to the ease with which ACM and its metabolites accumulate in the nucleus. Cellular uptake and nuclear incorporation are influenced by the hexopyranoses linked to aklavinone (AKV) and by the two methyls linked to the l-rhodosamine amino groups. The effect of ACM and its metabolites on macromolecular synthesis depended on the drug concentrations and the exposure time. ACM was the most active in the inhibition of nucleic acid synthesis whereas it had no direct effect on protein synthesis even at high drug concentrations. When cells were treated for a short time with low drug concentrations (1 μM), RNA synthesis was inhibited to a greater extent than DNA synthesis. But when incubated for longer periods, inhibition of DNA synthesis increased further. RNA and DNA syntheses were both inhibited to about the same extent only when cells were exposed to the higher drug concentrations (10 μM). We conclude therefore that at low drug concentrations the effect on DNA synthesis is probably a consequence of RNA synthesis inhibition. The early DNA synthesis inhibition which occurs at higher drug concentrations may result from the direct action on the cellular genome.