Transmural distribution of biochemical markers of total protein and collagen synthesis, myocardial contraction speed and capillary density in the rat left ventricle in angiotensin II-induced hypertension.

The effect of angiotensin II-induced hypertension on selected biochemical parameters was studied in Sprague-Dawley rats. Angiotensin II infusion at rates of 41.7 micrograms h-1 kg-1 and 12.5 micrograms h-1 kg-1 for 2, 5, 10 and 15 days elevated the systolic blood pressure from 143 +/- 7 mmHg to 215-230 mmHg (P less than 0.001) and 185-195 mmHg (P less than 0.001), respectively. The left ventricular weight/body weight ratio increased 10-14% (P less than 0.05) and 23-32% (P less than 0.001) after 2-15 days in rats treated at the lower and higher infusion rates, respectively. Prolyl 4-hydroxylase (PH) activity, a marker of collagen synthesis, was evenly distributed in the left ventricle. PH activity increased by about 100% in both subendocardial and subepicardial layers of the left ventricular wall after angiotensin II infusion for 10 days at 41.7 micrograms h-1 kg-1, but remained unaltered at 12.5 micrograms h-1 kg-1. No change was observed in hydroxyproline concentration. Myosin isoenzymes (V1-V3), which reflect myocardial contractility, were unevenly distributed in the left ventricular wall: the proportion of the fast-turnover isoenzyme (V1) was smaller in the subendocardial layer than in the subepicardial layer. The proportion of V1 decreased after treatment in both layers. Alkaline phosphatase activity, a marker of capillary density, was evenly distributed transmurally in the left ventricular wall. Angiotensin II caused a slight decrease in this activity in both myocardial layers. The results suggest that the elevation of blood pressure leads to transmurally evenly distributed changes in biochemical parameters reflecting collagen synthesis, capillary density and contractile properties of the myocardium.

Prolonged exercise causes an increase in the activity of galactosylhydroxylysyl glucosyltransferase and in the concentration of type III procollagen aminopropeptide in human serum.

The effect of prolonged heavy physical exercise on serum galactosylhydroxylysyl glucosyltransferase activity (S-GGT) and serum type III procollagen aminoterminal propeptide (S-Pro(III)-N-P) concentration was studied in healthy male long-distance runners. S-GGT increased gradually by about 70% (p less than 0.01) during a competitive 24-h run, and a rising trend was also observed in S-Pro(III)-N-P. After the termination of the run S-GGT was normalized in two days, but the increase in S-Pro(III)-N-P continued up to one day after the race, reaching nearly 40% (p less than 0.005). The alterations in S-GGT and S-Pro(III)-N-P showed no significant correlation with S-CK, S-LDH or the distance run. The most likely explanation for the increases in S-GGT and S-Pro(III)-N-P is that prolonged heavy exercise injures the collagen-synthesizing cells of the connective tissue, leading to a short-term increase in type III procollagen production.

Two serum markers of collagen biosynthesis as possible indicators of irreversible pulmonary impairment in farmer's lung.

Serum galactosylhydroxylysyl glucosyltransferase activity (S-GGT) and serum procollagen type III aminoterminal propeptide concentration (S-PRO(III)-N-P) were measured in 40 patients with farmer's lung at the time of an acute attack of the disease and 6 months later in order to study the usefulness of these serum markers for predicting the development of interstitial lung fibrosis. These 2 serum parameters have previously been found to reflect tissue collagen synthesis, especially in fibrotic hepatic diseases. The mean S-GGT was significantly increased when compared with the reference mean both at the time of the acute attack (p less than 0.001) and after 6 months (p less than 0.001). Approximately half of the patients had a S-GGT value higher than the upper normal limit at the acute stage, after which the levels decreased significantly (p less than 0.01). S-PRO(III)-N-P remained at the control level in most patients at the acute stage of the disease, followed by a small but significant increase (p less than 0.01), so that by the end of 6 months of follow-up one third of the patients had a S-PRO(III)-N-P value slightly above the upper normal reference value. Double blind tests on the administration of corticosteroids to half of the patients suggested that corticosteroids might have some favorable effect on the disease, although no significant response was found. The patients with definitely abnormal pulmonary function tests 1 yr after the acute stage had significantly higher initial S-GGT values than the rest of the patients (p less than 0.001). Elevated S-GGT at the acute stage was observed in 90% of the patients with definitely abnormal lung function after 1 yr but in only 30% of the other patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention by zinc of rat lung collagen accumulation in carbon tetrachloride injury.

Orally administered zinc was studied as a protective antifibrotic agent with respect to experimentally caused lung collagen accumulation in rats. Intraperitoneally injected carbon tetrachloride induced a diffuse alveolar damage with interstitial pulmonary fibrosis, and the morphologic findings suggested a primary toxic effect on the lungs. The carbon tetrachloride induction increased significantly the lung to body weight ratio, lung total protein and collagen content, lung total prolyl hydroxylase and galactosylhydroxylysyl glucosyltransferase activities, and daily urinary hydroxyproline excretion. Treatment with 114 mg/L of zinc in the animals' drinking water inhibited the lung prolyl hydroxylase activity and prevented the increases in lung collagen content and urinary hydroxyproline excretion but did not normalize any of the other above parameters. Enhanced lung prolyl hydroxylase activity was noted when a ferrous ion excess was included in the assay in order to reverse the competitive inhibition of the enzyme activity by zinc. It is suggested that zinc has a direct and selective preventive effect on rat lung collagen accumulation by inhibiting procollagen proline hydroxylation.

Elevated serum galactosylhydroxylysyl glucosyltransferase, a collagen synthesis marker, in fibrosing lung diseases.

The activity of galactosylhydroxylysyl glucosyltransferase, an enzyme catalyzing collagen biosynthesis, was measured in the sera of 101 patients with various pulmonary diseases to study whether detectable enzyme amounts are liberated into the serum from the lung tissue, and whether this is associated with the development of lung fibrosis. Increased serum galactosylhydroxylysyl glucosyltransferase activity was found in all the patients with progressive pulmonary fibrosis and in about half of the patients with acute stages of farmer's lung and infectious pneumonia. In one third of the patients with stage I sarcoidosis the serum enzyme activity was slightly increased, whereas in bronchial asthma and chronic bronchitis the values were mostly within the normal range. In conclusion, elevated serum enzyme activity was demonstrated in connection with those respiratory diseases in which pulmonary fibrosis was already verifiable or relatively often develops later. Measurements of serum galactosylhydroxylysyl glucosyltransferase may, thus, be useful in evaluating actual lung collagen synthesis in human pulmonary diseases.

Decrease in liver collagen accumulation in carbon tetrachloride-injured and normal growing rats upon administration of zinc.

Several attempts have been made to develop antifibrotic drugs for human use, but their success has been limited. The present data suggest that peroral zinc treatment has a direct and selective inhibitory effect on carbon tetrachloride-induced collagen accumulation in rat liver. Zinc did not normalize the carbon tetrachloride-induced increases in either liver relative weight, liver total protein content, fat accumulation, or the standard liver function tests, but it did efficiently inhibit liver collagen accumulation. It also reduced skin and liver collagen content and urinary hydroxyproline excretion in normal growing animals, indicating that the inhibition is not limited to the fibroproliferative inflammation associated with carbon tetrachloride injury. Neither inhibition of polysomal protein synthesis nor increased degradation of mature collagen fibers was found to play any major role in the effect of zinc. Instead, a plausible mechanism is inhibition of proline hydroxylation.

Increased activities of prolyl 4-hydroxylase and galactosylhydroxylysyl glucosyltransferase, enzymes of collagen biosynthesis, in skeletal muscle of endurance-trained mice.

The activities of prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT), and the concentration of 4-hydroxyproline were measured in red and white parts of quadriceps femoris muscle of mice after 3, 10, and 20 sessions of daily endurance training. The activities of PH and GGT increased in the red part of the muscle after training for 3 and 10 times and returned to the control level after 20 training sessions. In the white muscle the increase of PH activity was less than in the red muscle. No alteration in GGT activity was observed in the white muscle. The concentration of hydroxyproline was unchanged in the both types of skeletal muscle. The results suggest that collagen turnover in leg muscles may be enhanced during the early phase of adaptation to endurance training. The enhancement is more prominent in red than in white skeletal muscle.

Evidence for a relative excess of lysyl hydroxylase in chick embryo tendon and cartilage compared with bone and skin.

Various chick embryo tissues were incubated in vitro with a range of Zn2+ concentrations, and inhibition of the hydroxylations of collagen proline and lysine residues was studied in the intact tissues. At an constant inhibition level of proline hydroxylation, lysine hydroxylation proved to be inhibited more in the skin and bone than in the tendon and cartilage. The ratios of lysyl hydroxylase to prolyl hydroxylase activity were also distinctly lower in the former than in the latter. The variations observed in the reduction of lysine hydroxylation by Zn2+ thus correlate well with the differences seen in the enzyme activity ratios. No differences are found in the Ki values for Zn2+ between purified prolyl and lysyl hydroxylases or between the same enzymes from different tissues, and consequently the differences in lysine hydroxylation inhibition between the various tissues cannot be explained by differences in the kinetic constants. Recent studies also suggest that the existence of tissue-specific lysyl hydroxylase isoenzymes is improbable. The data thus suggest that there is a relative excess of lysyl hydroxylase activity in tissues such as tendon and cartilage, in which the lysine hydroxylation was less sensitive to Zn2+ inhibition, compared with skin and bone, where lysine hydroxylation was inhibited to a greater extent. These data are in a good agreement with the findings concerning variation in the reduction in lysine hydroxylation in different tissues with age or in the Ehlers-Danlos Syndrome Type VI.

Diagnostic imaging of focal nodular hyperplasia of the liver developing during nitrofurantoin therapy.

An asymptomatic palpable liver tumor developed in a six-year-old girl seven months after commencement of prophylactic nitrofurantoin therapy for recurrent urinary tract infections. The tumor was examined by 99mTc colloid radionuclide scan, compound ultrasonography and angiography. Ultrasonography demonstrated a large, solid tumor (5 x 5 x 8 cm) in the right lobe of the liver which had an echogenic central core surrounded by an area giving low-amplitude echoes. Angiography disclosed that the tumor was well demarcated and hypervascular, containing large tortuous arteries. The uptake of radionuclide in the tumor was normal. The tumor was resected and the pathological findings were typical for focal nodular hyperplasia (FNH) of the liver. The combination of the findings of these three diagnostic imaging methods is probably specific for uncomplicated FNH, a benign and innocuous tumor of the liver.

Serum galactosylhydroxylysyl glucosyltransferase in acute myocardial infarction and during subsequent collagen scar formation.

Changes in serum galactosylhydroxylysyl glucosyltransferase, an enzyme catalysing one of the intracellular post-translational modifications in collagen biosynthesis, were studied in twenty-four patients with acute myocardial infarction. The enzyme activity was monitored for 18 days from the onset of infarction, and at least a two-peaked pattern was observed. The first peak corresponded to the stage of acute myocardial injury, there being a highly significant correlation between the maximal values for serum glucosyltransferase and alpha-hydroxybutyrate dehydrogenase. An average decreasing in serum glucosyltransferase activity of 41%, was noted during the following 24 h. A new gradual rise in serum glucosyltransferase activity, interpreted as indicating myocardial collagen scar formation, was observed 5 days after the onset of infarction, when the serum enzyme activities indicating myocardial injury had already declined. The average daily values for serum glucosyltransferase between 6 and 18 days correlated highly significantly with the maximal value for serum alpha-hydroxybutyrate dehydrogenase, which serves as a relative estimate of the size of the original myocardial infarction area. The data further suggest that certain other factors including heart failure and/or various drug treatments may also affect the magnitude of this second peak.

Differences between proline and lysine hydroxylations in their inhibition by zinc or by ascorbate deficiency during collagen synthesis in various cell types.

The addition of Zn2+ inhibited lysine hydroxylation markedly less effectively than it did proline hydroxylation in chick embryo tendon cells, 3T6 fibroblasts and lysyl hydroxylase-deficient Ehlers-Danlos Syndrome Type VI fibroblasts. With low Zn2+ concentrations, a similar difference was also seen in chick embryo cartilage cells, whereas with high concentrations both hydroxylations were affected to the same extent in this cell type. Ascorbate deficiency likewise had a much less effect on lysine than proline hydroxylation when studied with 3T6 fibroblasts. As these two effectors involve quite different mechanisms, it is suggested that relative insensitivity to inhibition may be a property of lysine hydroxylation seen in many cell types with a number of agents. Studies on the mechanism of the difference in the inhibition indicates that the phenomenon is probably not due to differences in the kinetic constants of Zn2+ and ascorbate for the two enzymes. Neither is it probably to any major extent due to delayed procollagen triple helix formation nor a difference in the location of the two hydroxylases within the cisternae of the rough endoplasmic reticulum. The difference similarly cannot be explained solely by an excess of lysyl hydroxylase in the cell. It may thus be due either to some other intracellular property or to the combined effect of several factors.

Regulation of the glycosylations of collagen hydroxylysine in chick embryo tendon and cartilage cells.

The regulation of the glycosylations of hydroxylysine was studied in isolated chick-embryo cells by labelling with a [14C]lysine pulse. The course of the procollagen lysyl modifications was compared in tendon and cartilage cells, and the effect on the gycosylations of the degree of lysyl hydroxylation and the concentration of Mn2+ and Fe2+ were also studied, in tendon cells. Procollagen triple helix formation was inhibited in most experiments in order to eliminate the effect of this process on the continuation of the reactions. Both in the tendon and cartilage cells the intracellular lysyl modifications proceeded in a biphasic fashion. After an initial sharp linear increase, the reactions did not cease but were protracted at a slower but constant rate. Lysyl hydroxylation was followed by rapid galactosylation in both cell types and this was followed almost immediately by rapid glucosylation, suggesting a close association of the corresponding enzymes. The data further suggest that other factors must also exist, in addition to the differences in the timing of triple helix formation and the actual hydroxylysine content, which are responsible for the different amounts of galactose in the collagens synthesized by these cell types. The amount of glucosylgalactosylhydroxylysine nevertheless seemed to be determined by the available acceptor sites, i.e., the amount of galactosylhydroxylysine. In further experiments with tendon cells the oxygen participating in lysyl hydroxylation was displaced by nitrogen at various points in time. When the degree of lysyl hydroxylation was reduced to less than one-third of the original, the total amounts of glycosylated residues decreased correspondingly, but their proportion relative to total hydroxylysine remained unchanged. Extra Mn2+ increased the proportion of galactosylated hydroxylysine, suggesting that the activity of hydrosylysyl galactosyltransferase is not saturating in respect of the catalyzed reaction. Experiments on the addition of Fe2+ or its chelation by alpha, alpha'-dipyridyl gave indications that the presence of this co-factor is not required for either glycosylation reaction in isolated tendon cells.

Effects of divalent cations on collagen biosynthesis in isolated chick embryo tendon cells.

Isolated chick embryo tendon cells were used in [14C]proline and [14C]lysine labelling experiments to investigate the effect of divalent cations on collagen biosynthesis with a special reference to prolyl hydroxylation and lysyl modifications. The following metals were studied by adding them to the incubation medium of the cells: Ca2+, Cd2+, Co2+, Hg2+, Mg2+, Mn2+, Ni2+, Pb2+ and Zn2+. Zn2+ caused a potent reductin in collagen prolyl hydroxylation with a concomitant increased cellular retention of collagenase-digestible material. These effects were detectable even at physiological concentrations. At the same concentrations of Zn2+, lysyl hydroxylation was considerably less inhibited than prolyl hydroxylation, and the extent of hydroxylysyl glycosylation was even increased. Co2+ was also an efficient inhibitor of collagen prolyl hydroxylation, but at concentrations ten times higher than those of Zn2+. In the presence of other metal ions, no or only up to 10% inhibition of prolyl hydroxylation was noted even at those concentrations at which [14C]proline incorporation into the protein was decreased. However, an increased cellular retention of collagen was detected in the presence of some metal ions. No reduction in lysyl hydroxylation was found in the presence of Ca2+ or Mg2+.

Further characterization of galactosylhydroxylysyl glucosyltransferase from chick embryos. Amino acid composition and acceptor specificity.

A modified purification procedure, consisting of affinity chromatographies on concanavalin A-agarose, collagen-agarose and UDP-glucose-derivative-agarose and one gel filtration, is reported for galactosylhydroxylysyl glucosyltransferase. The enzyme obtained is entirely pure when studied by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme protein was rich in glutamic acid + glutamine, aspartic acid + asparagine, glycine and alanine. The enzyme catalysed no significant glucose transfer to any of the glycoproteins tested, except for collagens. This included all the glycoproteins that have previously served as glucosyl acceptors for impure enzyme preparations, thus indicating a high degree of specificity of the enzyme for galactosylhydroxylysine. Galactosylsphingosine would act as a glucosyl acceptor, however. This compound has a close structural similarity to galactosylhydroxylysine in that they both have an unsubstituted amino group next to the hydroxy group to which the galactose is attached.

Further studies on the effect of the collagen triple-helix formation on the hydroxylation of lysine and the glycosylations of hydroxylysine in chick-embryo tendon and cartilage cells.

The hydroxylation of lysine and glycosylations of hydroxylysine were studied in isolated chick-embryo tendon and cartilage cells under conditions in which collagen triple-helix formation was either inhibited or accelerated. The former situation was obtained by incubating the tendon cells with 0.6mm-dithiothreitol, thus decreasing their proline hydroxylase activity by about 99%. After labelling with [(14)C]proline, the formation of hydroxy[(14)C]proline was found to have declined by about 95%. Since the hydroxylation of a relatively large number of proline residues is required for triple-helix formation at 37 degrees C, the pro-alpha-chains synthesized under these conditions apparently cannot form triple-helical molecules. Labelling experiments with [(14)C]lysine indicated that the degree of hydroxylation of the lysine residues in the collagen synthesized was slightly increased and the degree of the glycosylations of the hydroxylysine residues more than doubled, the largest increase being in the content of glucosylgalactosylhydroxylysine. Recovery of chick-embryo cartilage cells from temporary anoxia was used to obtain accelerated triple-helix formation. A marked decrease was found in the extent of hydroxylation of the lysine residues in the collagen synthesized under these conditions, and an even larger decrease occurred in the glycosylations of the hydroxylysine residues. The results support the previous suggestion that the triple-helix formation of the pro-alpha-chains prevents further hydroxylation of lysine residues and glycosylations of hydroxylysine residues during collagen biosynthesis.

Intracellular enzymes of collagen biosynthesis in human platelets.

Activities of four intracellular enzymes of collagen biosynthesis--prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase, and collagen glucosyltransferase--were demonstrated in human platelets, and the presence of prolyl hydroxylase protein was further demonstrated by direct radioimmunoassay. The ratio of the specific activities of the four enzymes in the four enzymes in the human platelet extract to those in human adult skin extract varied from about 0.1 to 1, the lowest relative activity being found with prolyl hydroxylase and the highest with collagen glucosyltransferase. Only a very small amount of prolyl hydroxylase protein, probably 1%, was in the form of the active enzyme tetramer. The collagen glucosyltransferase from human platelets readily glucosylated galactosylhydroxylysine in denatured collagen, but did not glucosylate native collagen. Also, native collagen did not act as an inhibitor of the glucosylation reaction. Therefore, platelet collagen glucosyltransferase cannot form either an enzyme--substrate complex or an enzyme--inhibitor complex with native collagen. The results thus argue against the theory which maintains that platelet collagen glucosyltransferase is involved in collagen--platelet adhesion.