Seven lessons from seven renin secreting tumors.
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Angiography↗
Biomedical subjects
M T Corvol
Publications and source records attributed to M T Corvol.
Evidence for a direct in vitro action of sex steroids on rabbit cartilage cells during skeletal growth: influence of age and sex.
A direct effect of sex steroid hormones on in vitro cartilage cell metabolism was demonstrated. Cells were derived from rabbit fetuses on day 20 of gestation, and from male and female rabbits aged from 2 to 80 days. Testosterone (T), dihydrotestosterone (DHT), or 17 beta-estradiol (E2) (10(-9) -10(-9) M) were added to primary culture of epiphyseal articular chondrocytes. They showed an age-dependent stimulatory effect on [35S]sulfate incorporation into newly synthesized proteoglycans. In cultured rabbit fetal cartilage cells, the maximum active concentration of T and DHT was 10(-9) M with a 40% stimulating effect over control values. E2 was even more active with 80% stimulating effect when added at 10(-8) M. Chondrocytes from animals aged up to 5 days responded poorly and those from animals aged 5-30 days not at all. The response of cells from older animals varied with animal age and sex. T and DHT stimulated chondrocytes from males aged 32-55 days and females aged 40-52 days to about the same extent. E2 stimulated cells from animals of the same ages, but the response of female-derived cells was twice that of male-derived cells. The stimulating effect was dose dependent from 10(-11) to 10(-8) M and maximal at 10(-9) M for T and DHT and at 10(-8) M for E2. Puromycin completely abolished the effect.
Aging↗
Isolation of renin-producing human cells by transfection with three simian virus 40 mutants.
A human juxtaglomerular cell (JGC) tumor was used for the immortalization of renin-secreting cells. The transfection of primary JGC with three different simian virus 40 (SV40) mutants resulted in the continuous production of renin-secreting cells. The most efficient renin-producing cells (producing about 400 pg of renin per 24 hr per ml of culture medium) were those transfected with the PAS SV40 mutant. The renin production was stable and the cell cultures have been maintained for greater than 1 year. Two types of cells were cultured together and could not be separated: round and birefringent cells, which exhibited features of mast cells, and elongated cells containing myofilaments and secretory granules. Immunocytochemical staining showed the presence of renin in this latter cell type. The renin produced by the transfected cells was not stored within the cells but was released rapidly into the medium. More than 95% of the renin produced was prorenin, which, after activation, had characteristics similar to those of pure human standard renin as to its enzymatic, immunologic, and biochemical properties, except that it was less glycosylated. These stable JGC tumoral cell lines provide a unique system for studying human renin biosynthesis and its regulation in vitro.
Antigens, Viral, Tumor↗
Cell multiplication and type II collagen production by rabbit articular chondrocytes cultivated in a defined medium.
The complexity and the variations in the efficiency of different batches of serum stimulated the preparation of a serum-free medium which could promote not only growth, but also the differentiation properties of rabbit articular chondrocytes in culture. The serum-free medium (SFM) developed in this study contained insulin, transferrin, Na-selenite, human fibronectin bovine serum albumin (BSA), brain growth factor (BGF) or fibroblast growth factor (FGF), hydrocortisone and multiplication stimulating activity (MSA). Primary or secondary cultures of chondrocytes in such a medium attained a proliferation rate equal to 70-80% of that obtained with chondrocytes grown in a serum control medium. The deletion of various factors from SFM indicates that BGF or FGF are the most stimulating of growth factors. Insulin was beneficial when used individually; when combined with BGF or FGF, they had a synergistic effect on cell proliferation. MSA seemed not to play any role in chondrocyte growth in culture. The SFM medium did not modify either the morphology or the progression of cells into the cell cycle. It moreover allowed the maintenance of the specific function of chondrocytes to synthesize type II collagen.
Animals↗
Testosterone metabolism in prepubertal rabbit cartilage.
Using thin-layer chromatography, celite column chromatography and recrystallization methods, articular (AR) and growth plate (GP) cartilage tissues and cells from prepubertal rabbits were shown to convert testosterone (T) into at least three main metabolites: dihydrotestosterone (DHT), delta 4-androstenedione and androstanediols. In tissue incubation experiments the amount of each newly formed metabolite per mg of tissue was always greater in AR than in GP cartilage. After a 24 h incubation with AR or GP cartilage tissues, T was mainly converted to DHT and delta 4-androstenedione in approximately equal amounts. The amount of androstanediol metabolites formed was much lower. In a time-course experiment, the conversion of T to DHT and delta 4-androstenedione was shown to increase in a linear fashion, while the conversion to androstanediols was more variable. Using cultured AR cartilage cell incubations, similar results were obtained. In addition, DHT was shown to be the sole metabolite which accumulated in the cellular pool during the first 3 h incubation, as well as during the 24 h incubation when maximum cellular uptake of radioactivity was observed. At this time, the intracellular amount of unmetabolized [3H]T (88 pmoles/100 micrograms DNA) was similar to the amount of [3H]DHT (70 pmoles/100 micrograms DNA) accumulated in the chondrocytes. For both delta 4-androstenedione and androstanediols, 99% of radioactivity was extracted from the incubation medium.
Androstane-3,17-diol↗
Renin biosynthesis by human tumoral juxtaglomerular cells. Evidences for a renin precursor.
Renin biosynthesis was studied in a juxtaglomerular cell tumor. The tumoral tissue had a high renin content (180 Goldblatt Units/g of tissue), was heavily stained by immunofluorescence using human renin antiserum, and exhibited numerous characteristic secretory granules by electron microscopy. In one series of experiments, renin biosynthesis was studied in tissue slices, by following the incorporation of radiolabeled amino acids into specific immunoprecipitable renin. Time course studies showed that renin was first synthesized in a high molecular weight form, 55,000 mol wt, i.e., 10,000 mol wt higher than that of active renin, and was then converted into a 44,000-mol wt form. In a second series of experiments renin tumoral cells were cultured. Small, round, birefringent cells obtained after collagenase digestion produced renin in both primary culture and subculture media. After 5 d most of the renin found in the culture medium was inactive, but could be activated by trypsin treatment. The tumoral tissue exhibited a strong renin immunofluorescence and numerous secretory granules were observed by electron microscopy. In contrast, the renin-producing cells isolated from this tumor and grown in culture showed little renin immunofluorescence and no secretory granule could be observed. The renin-producing cells in primary culture and subculture were pulsed with radiolabeled amino acids, and immunoprecipitable radiolabeled renin was found in the culture media, thus demonstrating the actual biosynthesis of the enzyme. This renin was not stored inside cultured cells but was rapidly released into the medium and had a molecular weight of 55,000. No conversion of this inactive high molecular weight renin into the active, 44,000 mol wt form of renin was observed. We postulate the existence of two pathways for the processing, packaging, and secretion of renin in the tumoral cells: in juxtaglomerular cells of tumoral tissue renin is synthesized as a preprorenin and rapidly converted into prorenin (55,000 mol wt), which is in turn packaged in secretory granules where it is processed into active renin (44,000 mol wt) and finally secreted; in the cultured tumoral cells renin is still biosynthesized as a preprorenin molecule and then converted into prorenin, but is neither stored as granules nor processed into active renin. In this case the renin is released in an inactive form.
Adult↗
Receptors for insulin-like growth factors in rabbit articular and growth plate chondrocytes in culture.
Receptors for the insulin-like peptide ILAs have been identified in cultured rabbit chondrocytes. The cell-ILAs interaction is a time-dependent, reversible and saturable process. The cell-bound radioactive material appears as intact hormone. Insulin-like growth factor II (IGFII) is as potent as ILAs in competing for 125I-ILAs binding, whereas insulin-like growth factor I is somewhat less potent. Insulin does not affect ILAs binding. Our results suggest that the cultured chondrocytes possess a common receptor for the somatomedin peptides and that insulin, which is able to stimulate sulfate incorporation into proteoglycans, acts through an insulin receptor distinct from the somatomedin site. In growth plate chondrocytes the specific binding of 125I-ILAs is ten times lower than in articular cells in culture, whereas the sulfation activity of ILAs, at all concentrations studied, is 2.5-3 times lower in growth plate than in articular chondrocytes. The total binding of 125I-ILAs is higher to a particulate cell fraction than to intact cultured cells, by a factor of 10 for the growth plate cells, and by a factor of 2 for the articular cells. These findings suggest a poor accessibility of the hormone to the receptor sites of the growth plate chondrocytes in cultures, which are known to be surrounded by a thick matrix.
Animals↗
Lipid composition of two types of chondrocytes in primary culture.
Chondrocytes from articular and growth plate cartilage were grown in primary culture. The lipid content, distribution into different types, as well as the fatty acid patterns of these lipids were compared when the cells had reached stationary phase and were synthesizing maximal amounts of proteoglycans. Numerous significant differences were observed, depending on the origin of the chondrocytes. In particular, growth plate chondrocytes showed increased dry weight, increased lipid content (phosphatides and triglycerides), and decreased cholesterol to phosphatide ratio when compared to articular chondrocytes; they also incorporated more of C18:1 and less C16:0 into their major lipid types. Whether these differences arise from specific metabolic regulation or are a consequence of chondrocyte organization in primary culture remains unclear.
Animals↗
Radioreceptor assay of insulin-like peptides in human plasma: growth hormone dependence and correlation with sulfation activity by two bioassays.
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Acromegaly↗
Partial purification and characterization of a binding protein for insulin-like activity (ILAs) in human amniotic fluid: a possible inhibitor of insulin-like activity.
An insulin radioreceptor assay (INS-RRA) and an insulin-like activity radioreceptor assay (ILAs-RRA) have been utilized to partially purify and characterize a protein from human amniotic fluid with ILAs-RRA reactivity. An acid-ethanol soluble protein with an apparent molecular weight of 34,500 daltons by calibrated Sephadex chromatography and an isoelectric point (pI) of 4.7 accounts for all of the ILA'S-RRA reactivity present in human amniotic fluid. Since this protein has been found to be a binding protein for ILAs, but not insulin, it has been termed amniotic fluid binding protein or AFBP. AFBP is reactive in a non-parallel manner in the ILAs-RRA and totally inactive in the INS-RRA. The activity of AFBP in the ILAs-RRA is thus to the competition of AFBP with the placental membrane receptor for the [125I]ILAs tracer employed in the ILA'S-RRA. AFBP inhibits the activity of employed insulin, in the INS-RRA, presumably by binding ILAs, while being inactive itself. In two biological assays studied to date, the rat epididymal fat pad assay and the rabbit chondrocyte sulphation assay, AFBP also inhibits the activity of added ILAs. These observations raise the possibility that binding protein(s) for insulin-like peptides may function as inhibitors of their bioactivity in different physiologic and pathologic states. The relation of AFBP to binding protein(s) in human plasma remains to be clarified.
Amniotic Fluid↗
Vitamin D and cartilage. I. In vitro metabolism of 25-hydroxycholecalciferol by cartilage.
In the present work, the capacity of cartilage to metabolize 25-hydroxycholecalciferol was investigated. Cartilage preparations from growth plate, articular surface, rib, scapula, and ear were isolated from 3-week-old normal rabbits and chickens. Each tissue was separately incubated with tritiated 25-hydroxycholecalciferol (, x 10(-9) M) for 1-24 h. Incubations of kidney and muscle were performed simultaneously for comparison. Similarly, cultured chondrocytes isolated from rabbit growth plate and articular cartilage were incubated for 1 or 20 h in medium free of fetal calf serum. After methanol-chloroform extraction of tissues, cells, and their respective media, chloroform phases were chromatographed on Sephadex LH-20 columns. The results show that kidney and cartilage are able to convert 25-hydroxycholecalciferol into a derivative which migrates in the 24,25-dihydroxycholecalciferol region. Cartilage tissue previously boiled is unable to metabolize 25-hydroxycholecalciferol. The conversion of 25-hydroxycholecalciferol occurs with all types of cartilage and is also observed in incubations of cultured chondrocytes. In the latter, the polar 25-hydroxycholecalciferol derivative is detected as early as 1 h after addition of 25-hydroxycholecalciferol. Two findings suggest that the polar derivative of 25-hydroxycholecalciferol produced by cartilage is 24,25-dihydroxycholecalciferol: 1) the cartilage derivative and 24,25-dihydroxycholecalciferol (synthetic and biosynthetic) comigrate during Sephadex LH-20 and high liquid pressure chromatography; and 2) both the cartilage derivative and 24,25-dihydroxycholecalciferol are sensitive to periodate treatment.
Animals↗
Partial purification, characterization, and assay of a slightly acidic insulin-like peptide (ILAs) from human plasma.
An insulin radioreceptor assay (RRA) using human placental microsomal membranes was used to measure insulin-like activity (ILA) extracted from human plasma concentrates (Cohn fraction IV-4) by acid ethanol. The soluble activity (ILAs), chromatographed on Sephadex G-75 in 1 M acetic acid, migrated as a small molecule (fractional elution volume, 0.56) ahead of insulin (fractional elution volume, 0.70), whereas at neutral pH, ILAs migrated as a large molecular weight species. The ILAs peak from acid gel filtration on Sephadex was further purified by chromatography on carboxymethyl cellulose (CMC). The ILAs peak from both Sephadex and CMC diluted parallel to the porcine insulin standard in the insulin RRA and was totally unreactive in an insulin RIA. The CMC-purified material was iodinated and purified by binding to and elution from human placental membranes. The binding of [125I]ILAs to human placental membranes was inhibited only minimally by insulin and proinsulin and not at all by epidermal growth factor, nerve growth factor, glucagon, or lactogenic hormones, including human growth hormone. Multiplication-stimulating activity (MSA) inhibited in a manner parallel to ILAs. A Scatchard plot of the binding data was nonlinear. Sephadex ILAs was subjected to isoelectric focusing. The fractions assayed in both insulin and ILAs RRAs yielded comparable results. Peaks of ILA were observed at pHs 5.3, 6.6, and 8.4. When CMC-ILA was subjected to isoelectric focusing in polyacrylamide, a single peak of activity migrating between pH 6.2-6.8 was seen. [125I]ILAs focused at exactly the same pH. Electrophoresis of CMC-ILAs in acid-urea revealed a sharp peak of activity migrating with one of the five protein bands seen after staining. Again, [125I]ILAs comigrated with unlabeled ILAs. The molecular weight of ILAs, as determined on a calibrated Sephadex G-150 column at neutral pH, was 9,000-10,000 daltons. CMC-ILAs stimulated [14C]glucose incorporation into triglycerides of rat adipose tissue and augmented [3H]thymidine incorporation into human fibroblasts, chicken embryo fibroblasts, and BALB 3T3 cells as well as [35S]sulfate incorporation into macromolecules of rabbit chondrocyte culture medium. In summary, ILAs isolated on the basis of a RRA for insulin is a slightly acidic peptide with some of the biological activities expected of a somatomedin.
Adipose Tissue↗
The effect of a slightly acidic somatomedin peptide (ILAs) on the sulphation of proteoglycans from articular and growth plate chondrocytes in culture.
Chondrocyte cultures were prepared from rabbit growth plate (GPC) and articular (ARC) chondrocytes. These two cell types have distinct morphological characteristics. The cells reached maximum numbers by days 10 and 21 for ARC and GPC, respectively. The proteoglycans (PG) contained in the cellular pool were extracted and purified by DEAE cellulose chromatography. The effect of a partially purified somatomedin peptide with insulin-like activity on [35S]sulphate incorporation into PG was evaluated. In both ARC and GPC a significant stimulation of [35S]sulphate uptake into PG subunits was obtained with 1 ng Eq./ml of somatomedin peptide. In order to obtain the same stimulatory effect with porcine insulin, a 1000-fold greater concentration was required. The electrophoretic patterns of the PG subunits on acrylamide-agarose electrophoresis were identical on control incubations and after stimulation with the somatomedin peptide. These data demonstrate in vitro biological activity of this peptide on well differentiated articular and epiphyseal growth plate chondrocytes in culture. These cultures appear to provide a sensitive biological assay for somatomedin peptides.
Animals↗
The in vitro production and activity of 24, 25-dihydroxycholecalciferol in cartilage and calvarium.
Previously reported results from our laboratory have indicated that 24, 25-dihydroxycholecalciferol can be formed in vitro during incubations of cartilage tissue or cartilage cells with 25-hydroxycholecalcified. They have also demonstrated the high potency of this dihydroxymetabolite of vitamin D3 in stimulating in vitro the sulfate incorporation into proteoglycans of cartilage cells in culture and in decreasing in vitro the parathyroid hormone action on rat calvarium phosphatases activities. The present report shows that 24, 25-)OH)2 D3 can also be produced during rat calvarium incubations with 25-hydroxycholecalciferol and that therefore calvarium as well as cartilage might be both a site of formation and a site of action for 24, 25-(OH)2 D3. The review of recent experimental and clinical investigations strongly suggests that this metabolite has a physiological significance and may be specifically active on some parameters of bone mineralization. Further studies on the cartilage and calvarium abilities to convert 25-hydroxycholecalciferol into 24, 25-(OH)2 D3 shows that this transformation occurs in the mitochondrial fraction but that it does not seem modified by factors known to control the 25-hydroxycholecalciferol metabolism in the kidney. Finally the analysis of experimental and clinical results published so far does not yet bring enough information to understand the significance of this extrarenal metabolism of 25-hydroxycholecalciferol.
25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
[Culture of normal and pathological human growth cartilage. Action of vitamin D derivatives and somatomedin].
Chondrocytes from normal cartilage of a 2 month old child have been cultured. In vitro the cells maintained their morphological identity and synthesised proteoglycans with electrophoretic properties similar to those synthesised by cartilage in vivo. Sulphation was stimulated by vitamin D metabolites and by somatomedin. This technique has been used to study biopsies of abnormal cartilage.
Cartilage, Articular↗
Culture of chondrocytes from the proliferative zone of epiphyseal growth plate cartilage from prepubertal rabbits.
Chondrocytes from epihyseal growth plate cartilage of prepubertal rabbits were cultured. Cells from the proliferative zone showed a very different adhesiveness: they did not stick to the flasks until the 8th to 10th days. They need more oxygen tension to develop and grew in small separated colonies. Each colony was composed by several layers of round or polygonal cells, metachromatic with toluidine blue and they were surrounded by one layer of fibroblastic-like cells. At the end of primary culture the colonies looked like "craters". Cells from the resting zone multiplied as monolayer culture identical to articular chondrocytes. The stage of differentiation of the two different kinds of cells is discussed.
Animals↗
[Comparison of the electrophoretic properties of the proteoglycans secreted by the chondrocytes of rabbit growth cartilage and articular cartilage in culture].
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Animals↗
Proceedings: Joint involvement in spondylo-epiphyseal dysplasias.
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Adult↗