Collagenase activity in the normal rat myocardium

Summary Fibrillar collagen in the myocardium provides a supportive framework for myocytes and capillaries. Disruption of this organized framework has been observed in certain pathological states. Collagen degradation is primarily mediated by the specific enzyme collagenase, which has been found to exist in various tissues including the myocardium. In this report we describe a method that detects collagenase activity in sections of cardiac tissue. This method is on the basis of degradation of collagen by collagenase on one hand and the visualization of disrupted collagen fibers by immunofluorescence on the other. Frozen rat heart secctions were incubated under optimal conditions for collagenase activity (37°C in the presence of 0.1 M calcium at pH 7.4) for 24 h and 48 h. Subsequently, immunofluorescence staining with antibody to type I collagen was performed and the collagenous structures were visualized by immunofluorescence light microscopy. As control, untreated rat heart sections and sections incubated in the absence of calcium were similarly treated with antibody. After the 24 h of incubation, we found no change in the structural integrity of collagen fibers. Marked disruption of the type I collagen fibers was observed 48 h after incubation. No evidence of collagen fiber disruption was found in control sections. Experiments with exogenous collagenase resulted in similar collagen fiber disruption in the frozen rat heart sections. We conclude that the disruption of collagen type I fibers after 48 h of incubation, under optimal conditions for collagenolytic digestion, is. the result of collagen degradation by intrinsic collagenase of the myocardium.     

Collagenase activity in the normal rat myocardium. An immunohistochemical method

Fibrillar collagen in the myocardium provides a supportive framework for myocytes and capillaries. Disruption of this organized framework has been observed in certain pathological states. Collagen degradation is primarily mediated by the specific enzyme collagenase, which has been found to exist in various tissues including the myocardium. In this report we describe a method that detects collagenase activity in sections of cardiac tissue. This method is on the basis of degradation of collagen by collagenase on one hand and the visualization of disrupted collagen fibers by immunofluorescence on the other. Frozen rat heart sections were incubated under optimal conditions for collagenase activity (37 degrees C in the presence of 0.1 M calcium at pH 7.4) for 24 h and 48 h. Subsequently, immunofluorescence staining with antibody to type I collagen was performed and the collagenous structures were visualized by immunofluorescence light microscopy. As control, untreated rat heart sections and sections incubated in the absence of calcium were similarly treated with antibody. After the 24 h of incubation, we found no change in the structural integrity of collagen fibers. Marked disruption of the type I collagen fibers was observed 48 h after incubation. No evidence of collagen fiber disruption was found in control sections. Experiments with exogenous collagenase resulted in similar collagen fiber disruption in the frozen rat heart sections. We conclude that the disruption of collagen type I fibers after 48 h of incubation, under optimal conditions for collagenolytic digestion, is the result of collagen degradation by intrinsic collagenase of the myocardium.     

Collagenase in carcinoma cells

Collagenolytic enzyme activity has been found in high levels in homogenates of a tumor (ascitic V₂ carcinoma) implanted in rabbit muscle. Unlike other mammalian collagenases, this enzyme was not actively synthesized and released by primary cultures of the tumor. Most of the collagenolytic activity in tumor homogenates was found to sediment at 1000–4000 × g along with 5′-nucleotidase activity indicating that it might be bound to a membraneous component of the cell.     

Collagenase and gelatinase messenger ribonucleic acid expression and activity during follicular development in the rat ovary.

Metalloproteinases are members of a family of proteinases that remodel the extracellular matrix throughout the body. To test the hypothesis that metalloproteinases are regulated by gonadotropin-induced changes during follicular growth, rats were injected with eCG (20 IU, s.c.), and ovaries and serum were collected at the time of eCG administration (0 h) and at 6, 12, 24, 36, or 48 h later for analysis of metalloproteinase mRNA expression, metalloproteinase activity, and steroidogenesis. Serum estradiol levels increased from 18.9 pg/ml at 0 h to 503.8 pg/ml at 48 h. Analysis of mRNA expression was performed for collagenase-3, 72-kDa gelatinase, and 92-kDa gelatinase (n = 3-4). For collagenase-3, eCG stimulated a 32-fold increase in collagenase-3 mRNA at 48 h after eCG injection as compared to that in ovaries collected at the time of eCG administration (i.e., 0-h control). The mRNA levels for 72-kDa gelatinase were 2.8-fold compared to 0 h at 36 h after eCG treatment and returned to control levels by 48 h after gonadotropin treatment. Levels of the 92-kDa mRNA expression peaked at 24 h (4. 2-fold compared to 0 h) and returned to control levels by 36 h. Gel zymography revealed 3 gelatinolytic bands corresponding to the gelatinases of approximately 72 kDa, 92 kDa, and 105 kDa. Analysis of metalloproteinase activity as the degradation of collagen or gelatin per ovary showed an increase in gelatinolytic and collagenolytic activity between 12 and 48 h after eCG treatment. In summary, these findings demonstrate that the gonadotropin induction of folliculogenesis results in changes in the metalloproteinases that may be responsible for extracellular matrix remodeling associated with follicular growth.     

Polyamines in rat hepatocyte cultures.

In rat hepatocytes cultured for 120 h polyamine content was markedly modified. Putrescine concentration reached a maximum at 48 h, spermidine increased for 48 h and then remained constant, spermine after a decrease returned to its initial values. Total polyamine amount was increased by 75%. Both ornithine decarboxylase and the retroconversion pathway were responsible for these modifications. The possible correlation between polyamine metabolism and retrodifferentiation process was investigated by studying them in conditions which are known to preserve differentiated functions.     

Testosterone stimulation of mitochondrial aspartate aminotransferase in organ cultures of rat ventral prostate

Stimulation of mitochondrial aspartate aminotransferase (mAAT) activity by testosterone was determined in organ cultures of rat ventral prostate. The effect of testosterone on citrate accumulation in the culture medium was also determined. Testosterone stimulation of citrate accumulation and mAAT occurred in a dose dependent manner. Stimulation of mAAT activity occurred after a 1–3 h lag period and appeared to involve the synthesis of specific RNA since the response was inhibited by actinomycin D. Studies utilizing [³H]l-leucine indicated that unlike the total tissue, testosterone stimulated the incorporation of [³H]leucine into proteins of the mitochondrial fraction. The results suggested that mitochondrial proteins may be more sensitive to testosterone stimulation than cytosol proteins. The response was specific for mAAT since testosterone had no effect on mitochondrial malic dehydrogenase activity. The data suggested that testosterone may regulate prostate citrate content by the induction of mAAT in prostate mitochondria, which results in a source oxalacetic acid for citrate synthesis through transamination of aspartate by alpha ketoglutarate.     

Unusual increase in mitochondrial glycerolphosphate dehydrogenase activity in primary cultures of rat liver cells.

Primary cultures of neonatal rat liver cells show an increase in the activity of mitochondrial glycerolphosphate dehydrogenase between the second and tenth day of cultivation. At the end of cultivation the activity level exceeded that of liver tissue in vivo. Replacement of normal serum by hypothyroid serum or addition of triiodothyronine to the medium did not influence significantly the enzyme activity in vitro, in contrast to the very marked effects of thyroid hormones observed in vivo.     

Spectrophotometric measurement of flavin-containing monooxygenase activity in freshly isolated rat hepatocytes and their cultures.

The determination of the mixed function flavin-containing monooxygenase activity in rat liver and in hepatocytes and their cultures by spectrophotometric measurement of the oxygenation of methimazole is complicated by an inhibition caused by some of the reagents used during this method. Optimal conditions were determined for measuring this enzyme activity in microsomal preparations of rat liver and its hepatocytes. Optimal flavin-containing monooxygenase activities were obtained for measurements performed in a 0.25 M N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine-EDTA buffer at pH 8.7 and at a methimazole concentration of 2 mM. Data are also presented which show that no interferences caused by either cytochrome P450-dependent enzymes or by the reduction of methimazole disulfide by glutathione have to be taken into account when determining methimazole oxygenation. Finally, the above assay was also used to study flavin-containing monooxygenase activity in primary monolayer cultures of hepatocytes for 6 days.     

Regulation of S-adenosylmethionine decarboxylase activity in rat liver and prostate

Two methods were used for the quantitation of S-adenosylmethionine decarboxylase protein. The first involved titrating the active site of the enzyme by reduction of the Schiff base between 3H-decarboxylated S-adenosylmethionine and the pyruvate prosthetic group with sodium cyanoborohydride. The second method was radioimmunoassay with rabbit antiserum which was used to determine the total immunoreactive enzyme protein. It was found that the increased S-adenosylmethionine decarboxylase activity produced in rat prostate by treatment with alpha-difluoromethylornithine and in both prostate and liver by methylglyoxal bis(guanylhydrazone) were due entirely to increases in the amount of enzyme protein. The ratio of enzyme activity to protein (measured by either method) remained constant in rats treated with the drugs. Treatment with 2% alpha-difluoromethylornithine in the drinking water for 3 days increased prostatic S-adenosylmethionine decarboxylase protein by 5-fold. A substantial part, but not all, of this increase could be accounted for by a slowing of the rate of degradation of the enzyme. The half-life for loss of activity and titratable protein after inhibition of protein synthesis by cycloheximide was increased from 35 to 108 min by treatment with alpha-difluoromethylornithine. However, the half-life for loss of immunoreactive protein which was considerably longer was only increased from 139 to 213 min. The molecular weight of the S-adenosylmethionine decarboxylase subunit determined by immunoblotting was 32,000, and no smaller immunoreactive fragments were detected. These results indicate that spermidine depletion produced by alpha-difluoromethylornithine affects the degradation of S-adenosylmethionine decarboxylase at an early step involving the loss of the active site without substantial breakdown of the protein.     

Increased activity of plasminogen activators during involution of the rat ventral prostate.

Plasminogen activator was measured in the ventral prostates of non-castrated, castrated, and androgen-treated rats to determine whether changes in this activity correlated with the process of glandular involution. While the activity was very low in cytosolic extracts from the prostates of non-castrated rats, 2 days following castration the plasminogen activator activity increased in a near-linear fashion such that by day 7 it was 10-fold higher in terms of specific activity (per mg of protein) and cellular concentration (per mg of DNA). During this interval there was a rapid decrease in the cell population of the prostates. Treatment of the 7-day castrated rats with the potent androgen, dihydrotestosterone, both reduced the plasminogen activator activity and restored the cell number in a dose-related manner. Gel electrophoretic analysis revealed two major bands of plasminogen activator activity in the cytosolic extracts from 4- and 7-day castrated rats, plus additional minor bands in samples from 10- and 14-day castrated rats. Approx. 10% of the cellular concentration of plasminogen activator activity was recovered in association with an 18000g pellet fraction from the prostates; this fraction showed less heterogeneity of the plasminogen activator forms as observed by gel electrophoresis. Inhibitor studies indicated that the 18000g pellet fraction from the prostates of non-castrated rats possessed some plasminogen activator inhibitor activity, but the relative concentration of the inhibitor activity was small. We conclude that the involution of the prostate is probably associated with increased synthesis of plasminogen activators through a de-repression process which may involve loss of androgen receptors.     

Androgen metabolism and actions in rat ventral prostate epithelial and stromal cell cultures

The rat ventral prostate requires androgens for normal development, growth, and function. To investigate the relationship between androgen metabolism and its effects in the prostate and to examine differences between the epithelial and stromal cells, we have established a system of primary cell cultures of immature rat ventral prostate cells. Cultures of both cell types after reaching confluency (6-7 days) actively metabolized 3H-labelled testosterone (T), 5 alpha-dihydrotestosterone (5 alpha-DHT), 5 alpha-androstane-3 alpha,17 beta-diol, and 5 alpha-androstane-3 beta,17 beta-diol. The epithelial cells actively reduced T to 5 alpha-DHT and formed significant amounts of 5 alpha-androstane-3,17-dione from T, 5 alpha-DHT, and 5 alpha-androstane-3 alpha,17 beta-diol. All substrates were converted to significant amounts of C19O3 metabolites. The stromal cells also metabolized all substrates, but very little 5 alpha-androstane-3,17-dione was formed. The metabolism studies indicate that both cell types have delta 4-5 alpha-reductase, 3 alpha- and 3 beta-hydroxysteroid oxidoreductase and hydroxylase activities. The epithelial cells have significant 17 beta-hydroxysteroid oxidoreductase activity. The epithelial cells cultures grown in the presence of T have higher acid phosphatase (AP) contents (demonstrated histochemically and by biochemical assay). Tartrate inhibition studies indicate that the epithelial cells grown in the presence of T are making secretory AP. Stromal cell AP is not influenced by T. The results indicate that the cultured cells maintain differentiated prostatic functions: ability to metabolize androgens and, in the case of the epithelial cells, synthesize secretory AP.