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.     

In vitro biosynthesis of radioactive estradiol-17 beta, 17-glucosiduronate by rhesus monkey liver.

A method for the preparation of radioactive estradiol-17 beta, 17-glucosiduronate by incubating 3H-estradiol with rhesus monkey liver microsomal preparation in the presence of uridine diphosphoglucuronic acid is described. Small but significant amounts of the conjugate were also obtained from the 150,00 pellet and cytosol fractions. The addition of NADPH to the incubation media increased the yield of radioactive-estradiol-17 beta, 17-glucosiduronate perhaps by preserving the integrity of the C-17-hydroxyl group. As expected, the effect of the reduced nucleotide was more pronounced in the fractions other than the microsome. The biosynthesized conjugate was isolated and purified by multiple column chromatography and the structure was confirmed by derivative formation, enzyme hydrolysis and crystallization of the aglycone.     

Contrasting effects of estradiol-17 beta and 17 alpha-ethinyl estradiol-17 beta on cultured whole embryos

Estradiol-17 beta (E2) and 17 alpha-ethinyl estradiol-17 beta (EE) were compared in terms of their relative capacities to alter growth and developmental patterns of cultured whole embryos during the early stages of organogenesis. Embryos exhibited a notable differential susceptibility to the embryotoxic effects of parents E2 vs EE when these estrogens were added directly to the media at the onset of the culture period. At initial concentrations of 0.1 mM, E2 failed to produce statistically significant effects whereas EE elicited marked embryotoxicity. Inclusion of a P-450-dependent biotransformation system in the culture media resulted in a significant attenuation of the embryotoxic effects of parent E2 vs EE when these estrogens were added directly to the media at the onset of the culture period. At initial concentrations of 0.1 mM, E2 failed to produce statistically embryotoxicity by hepatic S9. The divergent results produced by the two steroids could not be attributed to differences in rates of catecholestrogen generation in the culture medium or by the conceptuses. The results demonstrate definitive dissimilarities between the effects of two steroidal estrogens on developmental parameters and document marked differences in the effects of biotransformation on their embryotoxic potential. The data strongly suggest that the embryotoxicity of these steroids is not mediated via interactions with estrogen receptors. Additionally, the data show that the differential capacity of these two steroids to produce embryotoxic effects is diametrically opposite to earlier reported patterns of their carcinogenic potential in the Syrian hamster kidney.     

Competitive protein-binding assay of estrone, estradiol-17 or estradiol-17 in human or ruminant plasma

A procedure for the assay of estrone, estradiol-17β or estradiol-17α in plasma is described. The technique also appears applicable to the assay of estriol in plasma. The procedure uses a semi-automatic extraction of plasma, rapid micro-alumina column chromatography and competitive binding of the estrogens to stable proteins of sheep uterine cytosol. The use of alumina column chromatography results in consistently low blanks. The assay has been evaluated for the measurement of estradiol-17β and estrone in human and sheep plasma, and for estradiol-17α and estrone in goat plasma. The change in binding affinity of estradiol-17α relative to estradiol-17β when incubated in sheep uterine cytosol at two different temperatures (25°C and 4°C), makes it possible to differentiate the two epimers of estradiol. Measurement of estradiol-17β down to 10 pg and of estrone and estradiol-17α to 25 pg are maintained in routine analyses. The specificity of the procedure was thoroughly checked by various methods, including comparison with spectrophotofluorimetric analysis.     

Effect of amino acids on collagen biosynthesis

Summary The effect of various amino acids on collagen biosynthesis was studied in organ cultures of chicken embryo tibiae. Competitive interrelationship between selected amino acids influences independently the uptake of proline and lysine, precursors of hydroxyproline and hydroxylysine, respectively, which are the two amino acids characteristics of collagen. On the basis of these influences, the possibility of biosynthesis of anomalous collagens is stressed. Parallel studies of biosynthesis of hydroxyproline and hydroxylysine are necessary.     

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.     

Comparative effects of estradiol-17beta and estriol on uterine RNA polymerases I, II, and III in vivo.

The early and later effects of estradiol-17β and estriol on the RNA polymerase activities of uterine nuclei obtained from ovariectomized rats were compared. At 4 hr of hormone action both estradiol-17β and estriol stimulated the activity of polymerase I, but not the activities of polymerases II and III. At 24 hr, however, the effect of estriol had disappeared, whereas estradiol-17β stimulated all three polymerase activities. These results indicate that estrogen-induced growth of the uterus occurs in two phases, initiation and maintenance. Estriol initiates uterine growth, but does not maintain the process. Estradiol-17β, in contrast, does both. The differences in the effects of the two estrogens may reside in their different binding affinities.     

Effect of estradiol-17 beta and prostaglandins on rat myometrial gap junctions

The effects of estradiol-17 beta and indomethacin on myometrial gap junction development, plasma estradiol levels and uterine PGF2 alpha content were evaluated in immature and/or ovariectomized, mature rats. High doses of estradiol stimulated the development of gap junctions in the myometrium of animals from both groups. Concomitant injections of estradiol and indomethacin to ovariectomized rats potentiated the estradiol stimulation of gap junctions. Plasma estradiol levels were lower in ovariectomized rats treated with both estradiol and indomethacin than in animals treated with estradiol alone. Indomethacin also enhanced the uptake and retention of 3H-estradiol into uterine tissues. Uterine PGF2 alpha content of ovarectomized rats was stimulated with the initial injection of estradiol but thereafter, the PGF2 alpha content declined with repeated injections to values lower than that observed in controls. Prostaglandin F2 alpha content in tissues from rats treated with estradiol plus indomethacin were also higher than that observed in rats treated with indomethacin alone, however, the values obtained in both groups were significantly lower compared to those from control animals. These results are consistent with the hypothesis that steroid hormones and prostaglandins regulate myometrial gap junction formation. Regulation of myometrial gap junctions by prostaglandins is discussed with respect to a down regulation of the steroid-receptor mechanism and effects on cyclo-oxygenase or lipoxygenase products.     

Effect of estradiol-17ß and prostaglandins on rat myometrial gap junctions

The effects of estradiol-17ß and indomethacin on myometrial gap junction development, plasma estradiol levels and uterine PGF_2α content were evaluated in immature and/or ovariectomized, mature rats. High doses of estradiol stimulated the development of gap junctions in the myometrium of animals from both groups. Concomitant injections of estradiol and indomethacin to ovariectomized rats potentiated the estradiol stimulation of gap junctions. Plama estradiol levels were lower in ovariectomized rats treated with both estradiol and indomethacin than in animals treated with estradiol alone. Indomethacin also enhanced the uptake and retention of ³H-estradiol into uterine tissues. Uterine PGF_2α content of ovarectomized rats was stimulated with the initial injection of estradiol but thereafter, the PGF_2α content declined with repeated injections to values lower than that observed in controls. Prostaglandin F_2α content in tissues from rats treated with estradiol plus indomethacin were also higher than that observed in rats treated with indomethacin alone, however, the values obtained in both groups were significantly lower compared to those from control animals. These results are consistent with the hypothesis that steroid hormones and prostaglandins regulate myometrial gap junction formation. Regulation of myometrial gap junctions by prostaglandins is discussed with respect to down regulation of the steroid-receptor mechanism and effects on cyclo-oxygenase or lipoxygenase products.     

Effect of thyroid state on estradiol-17β metabolism in the rabbit

It is well known that the metabolic clearance rate (MCR) of a hormone is Influenced highly by the level of its specific binding protein. It was interesting, therefore, to study the metabolism of estradiol-17β (E₂) in an animal model such as the rabbit where there is a lack for a highly specific binding protein for the steroid. The kinetics of the hormone was studied in relation to the thyroid state, namely in rabbits receiving thyroxin or propylthiouracil.In the absence of any significant decrease of the level of the rabbit androgen binding protein (R-ABP), the accelerated MCR^E2 and the elevated conversion ratio of estradiol to estrone (CR ^E₂→E₁) observed in hyperthyroid rabbits were attributed to the important role of metabolizing enzymes in the liver and/or extrahepatic tissues. In hypothyroid rabbits, while the CR ^E₂→E₁ decreased significantly the MCR^E2 was not altered.     

Effect of streptozotocin-diabetes on rat liver asialoglycoprotein receptor turnover: in vivo degradation and in vitro biosynthesis

The metabolic turnover of the Hepatic Binding Protein (HBP) was investigated in streptozotocin-diabetic rats. We have already shown that diabetes induced a decreased ligand binding capacity while the immunoreactive HBP was normal. To explore the eventual modifications due to diabetic state upon the turnover of HBP, we followed the in vivo degradation of HBP and its biosynthesis in vitro. After in vivo labelling with L-[3H] leucine and purification of HBP from rat livers, we found a 20% decrease in diabetic HBP half-life. By in vitro incubations of freshly isolated hepatocytes and a 2 h-pulse in the presence of L-[35S] methionine, we showed that diabetes provokes an increased uptake of L-[35S] methionine in hepatocytes allowing an augmented synthesis of HBP although the L-[35S] methionine incorporation into total proteins was less efficient.     

In vivo studies on the metabolism of estrone and estradiol-17beta by the brain

³H-Estrone and H-estradiol-17β were infused in separate experiments into the jugular veins of each of 4 ewes. During the infusions blood samples were obtained from the ipsilateral jugular vein and common carotid artery. The blood samples were analyzed for radioactivity as free estrone and estradiol-17β and the conversion of infused precursor to product steroid by brain tissue, the transtissue conversion (ρ^PRE-PRO_AV) and the extraction by brain tissue of infused precursor, the transtissue extraction (1-ρ^PRE-PRE_AV) and the metabolic clearance rates were calculated.The mean ± SE for ρ_AV^1,2 (precursor, estrone = 1; product, estradiol = 2) was 0.09 ±0.03 and the mean ± SE for ρ_AV^2,1 (precursor, estradiol = 2; product, estrone = 1) was 0.08 ±0.02. The mean trans-tissue extraction of estrone was 0.13 ± 0.02 and of estradiol-17β was 0.14- ± 0.02. The transtissue extractions of estrone and estradiol-17β were greater than ρ_AV^1,2 ρ_AV^2,1 respectively in 2 of the 4 ewes.Brain metabolism of estrogens can account for only 2–4% of the total metabolism of these free estrogens from the blood pool.