Long-term trophic effect of ACTH on rat adrenocortical cells

Summary The changes occurring in rat adrenocortical cells (zona fasciculata) during an 8 day period of treatment with ACTH, were investigated by morphometric and autoradiographic methods.The most important ultrastructural change consists in a conspicuous increase in the smooth endoplasmic reticulum, that accounts for about 50% of the total increase of cellular volume. Also the mitochondrial fraction shows a significant increase, which is found to be due both to the increment in the number of mitochondria per cell and to the increase in the mean volume of organelles themselves.The quantitative autoradiographic data, indicating an increment in the incorporation of 3H-orotate and 3H-leucine into adrenocortical cells of the treated animals, allow us to conclude that the ACTH-induced ultrastructural changes are the morphological expression of a stimulation of the cellular protein synthesis.Since mitochondria are largely autonomous in the synthesis of their enzymes and structural proteins, it is possible to hypothesize that ACTH also intervenes in the regulation of the mitochondrial protein synthesis.The authors wish to express their sincere appreciation to Mr. G. Gottardo for his excellent technical assistance.     

Biphasic effect of ACTH on growth of rat adrenocortical cells in primary culture

Summary The proliferation rate of differentiating fetal rat adrenocortical cells was studied in primary culture. In this system, stimulation with ACTH induces differentiation of zona glomerulosa-like cortical cells into zona fasciculata-like cells. Incorporation of bromodeoxyuridine (BrdU) was studied immunocytochemically by use of anti-BrdU antibody, and the proliferation rate was counted from the monolayer colonies of adrenocortical cells. After 21 days of cultivation in the absence of ACTH, the proliferation rate of zona glomerulosa-like cells was 10%. The rate slowly declined to 1% at the age of 100 days during continuous cultivation in the absence of ACTH. Stimulation with ACTH induced a strong inhibition in the proliferation rate (down to 2% during the first 24 h). Treatment with ACTH during the following 48 h led to an extremely intense proliferation of adrenocortical cells at a proliferation rate of 25%. Continuous treatment with ACTH up to 100 days led to a persistent growth of adrenocortical cells, and a proliferation rate over 2-fold higher than in control cells cultivated in the absence of ACTH. Thus, ACTH is the principal growth-promoting factor also in vitro, as has been found in in vivo studies. This growth effect is mediated by a biphasic course; at the beginning of differentiation the effect is inhibitory and is followed by a persistent stimulation of the growth of adrenocortical cells.     

Effect of ACTH on mitochondrial RNA synthesis of rat adrenocortical cells

Summary The incorporation of 3H-thymidine and 3H-uridine into adrenocortical cells of intact and ACTH-treated rats was investigated by high-resolution autoradiography. The quantitative analysis of autoradiographs shows no effect of ACTH on the incorporation of 3H-thymidine, at least in our experimental conditions. On the contrary, ACTH was found to enhance the incorporation of 3H-uridine into both adrenocortical nuclei and mitochondria. These findings are discussed in relation to numerous biochemical and morphological data, indicating that ACTH stimulates the synthesis of enzymes and structural proteins of adrenocortical cells.It is suggested that the mechanism of action of ACTH on adrenal cortex, consists in an integrated stimulation of both nuclear and mitochondrial DNA-dependent RNA synthesis.The authors wish to express their sincere appreciation to Mrs. L. Rebonato and Mr. G. Gottardo for skilled technical assistance.     

In vitro interaction of ACTH with rat brain muscarinic receptors

ACTH-(1-24) inhibits the in vitro binding of the muscarinic antagonist [3H]QNB to membranes from rat brain. The magnitude of inhibition is dependent on the concentration of ACTH-(1-24). Kinetic analysis indicates a pure competitive inhibition which is suggestive of a reversible interaction of ACTH with muscarinic receptors. A mechanism involving an interaction of ACTH-(1-24) with the phospholipid core of the receptors is suggested. Structure activity studies point to a relation with reported effects of intracerebroventricularly administered ACTH on the turnover rate of acetylcholine and the ACTH-induced stretching and yawning syndrome.     

Effect of chronic ACTH treatment on the physical state of lipid droplets in rat adrenocortical cells

A histophysical method has been adapted to determine the thermotropic phase transitions of adrenocortical lipid droplets using a polarizing microscope equipped with a cold/hot stage. Cryosections of freshly-removed, unfixed adrenals, derived from control (untreated), and 14 days ACTH-treated rats were examined. The lipid droplets in the zona fasciculata and zona reticularis of the untreated rats were birefringent at room temperature (22°C). The birefringence of zona glomerulosa lipids selectively increased in the temperature range from −10 to −15°C. In cryosections prepared from ACTH-treated rats, thermotropic phase transitions of the lipid droplets appeared at a temperature range between −30 and −40°C in each cortical zone. The chemical analysis of the isolated lipids revealed that the relative amount of triglycerides in the zona fasciculata lipids increased, while that of free and esterified cholesterol decreased after chronic ACTH treatment. Present data suggest that the increased fluidity of lipid droplets promotes lipid mobilization in response to the enhanced demand of the chronically stimulated adrenocortical cells. Viscosity-dependent mobilization of free cholesterol from lipid droplets is not a rate-limiting process in adrenal steroidogenesis, but rather may represent an important control of the availability of precursor from lipid stores.     

Stage-dependent variation in the rat Sertoli cells nuclear volume

A morphometric study was carried out to investigate stage-dependent variation in sertoli cell nuclear volume in the rat testis. sertoli cell nuclei had the largest volumes in stages IX to X of the seminiferous epithelium cycle (746 microns3), and the smallest volumes in stage XIV (624 microns3). In the remaining stages, the nuclei presented intermediate values, without significant differences. The results were discussed in terms of a possible functional cyclic variation in the sertoli cell reflecting changes in their nuclear size.     

Influences of heparin on ACTH distribution and immunoreactivity in plasma of the rat. In vivo and in vitro studies

1. Blood samples from non-pregnant female rats were incubated in vitro with porcine 125I-ACTH, and the corresponding plasmas were chromatographed on fine Sephadex G 50. When heparin was added in vivo or in vitro, almost all the radioactivity appeared in the void volume of the columns; the same was observed when labelled ACTH was added to heparin-containing saline. In contrast, when NaCl instead of heparin was added to the blood in vivo as well as in vitro, almost all the plasma radioactivity was eluted later, with 125I-ACTH. 2. When labelled ACTH was i.v. administered to pregnant females, it was eluted in the void volume in the presence of heparin, and further down in its absence. 3. The same plasma samples from non-stressed or ether-stressed females were radioimmunoassayed for ACTH, with and without heparin. The degradation of ACTH was greater in the presence of heparin, and plasma ACTH concentration was understimated for low blood levels of heparin (5 UI/ml or less) and in contrast overestimated for high ones (25 or 50 UI/ml). 4. In conclusion, the reported data clearly demonstrated firstly that heparin added to rat blood traps ACTH molecules, promoting the formation of aggregates with apparent height molecular weight; secondly that heparin interferes with the direct radioimmunoassay of ACTH in the plasma.     

In vitro studies of cells in histiocytosis X

In a female patient with histiologically ensured histiocytosis X, cell cultures modified according to NEZELOF were performed in methylcellulosis and Eagle's medium. From the 5th to the 15th day of incubation the cells were daily observed natively through a normal microscope and taken for histochemical examinations and for checking their capacity of phagocytosis. Two cell types could be distinguished: spindle-shaped fibroblasts as well as round cells of changing form and size, the alpha-naphthylacetate esterase response of which proved to be positive. The round cells can partly be regarded as HK cells, whereas giant cells could be predominantly observed during the second week. A phagocytosis of iron chips and human erythrocytes loaded with antibodies could be revealed in both types. HK cells represented mature macrophages, from which giant cells developed under in vitro conditions. There is still not explanation as to the local and immunological factors characterizing the clinical picture of histiocytosis X.     

In vitro decrease of lyase activity in rat ovarian cells during incubation: effect of hCG

From PMSG-pretreated immature rats, dispersed ovarian cells were prepared with collagenase and DNase and incubated at 37 degrees C in McCoy's 5a medium under 95% air-5% CO2 atmosphere for 4 h. The activities of C17-C20 lyase measured in the 10,000 x g supernatant fluid of the cell homogenates decreased spontaneously with the lapse of time of the incubation. N,N'-Diphenyl-p-phenylenediamine (DPPD, an antioxidant) and actinomycin D inhibited the decrease most effectively. Cycloheximide was also an effective protector. Accordingly, the spontaneous decrease of the lyase activity was caused partly by an oxygen radical-mediated process and partly by a mechanism involving de novo synthesis of RNA and protein. Addition of hCG to the cells further decreased the lyase activity to about half of the control group at 4 h. DPPD itself did not affect the hCG-induced decrease of the lyase activity. However, actinomycin D and cycloheximide prevented the effect of hCG. These results indicate that de novo synthesis of RNA and protein is involved in the latter mechanism, while oxygen radical is not concerned in this process. The decrease of the enzyme activity by hCG during incubation is in agreement with the in vivo effect of hCG upon the lyase activity. On the contrary, at the end of incubation the activity of delta 5-3 beta-hydroxysteroid dehydrogenase (coupled with delta 5-delta 4 isomerase) was more than 89% of that before incubation, and the change of the enzyme activity according to the various treatments was less than 16%.     

Cytochemical and functional evaluation of ACTH responsive isolated rat adrenocortical cells. The maintenance of sex differences

The critical evaluation of isolation methods for obtaining the adrenocortical cell suspension due to trypsin or collagenase digestion was done. Some collagenase advantages were indicated by morphological observations on the staining smears as well as by ACTH stimulation test. The cytochemical reactions for enzyme activities had the limited applications for those purposes. It also appeared that commonly applied dye exclusion tests were inadequate for characterization of cell suspension. The possible role of the adrenocortical cell debris in the basal corticosterone production was pointed out. The maintenance of the sex dimorphism and the functional differences in the adrenocortical cells isolated from male and female rats have been observed.     

Transitory increase of run-off single ribosomes in rat adrenocortical cells, following ACTH administration, in vivo

Adrenal polysomes from control and adrenocorticotropin (ACTH)-treated rats were prepared. Sucrose gradient sedimentation analysis of the free polysome preparation showed an increased yield of monomeric and dimeric ribosomes from ACTH-treated animals. This effect was present at 7 minutes after ACTH injection and disappeared between 60 and 120 minutes of treatment. Similar change was observed in hypophysectomized rats following simultaneous injection of ACTH plus aminoglutethimide (an inhibitor of steroidogenesis) but not after dexamethasone. These increased particles are free of mRNA and peptidyl-tRNA for they dissociate completely by centrifugation through high ionic strength buffer. These results suggest that, in rat adrenal cells, ACTH could act at the level of the protein synthesizing system, most likely at the translational level.     

Proliferation and distribution of adrenocortical cells in ACTH treated female hamsters

The aim of the study was the analysis, by means of combined stereologic methods and metaphase arrest technique of the adrenocortical growth of ACTH treated hamster. Adult female hamsters were treated daily with 50 micrograms ACTH (Synacthen Depot, Ciba) for 3, 6 and 9 days. Between days 3 and 9 nearly linear increase in adrenal gland weight was observed, mainly due to the enlargement of the fasciculata and reticularis zones. Throughout the experiment, there was no marked change in the number of the zona glomerulosa cells while from the day 3 a notable increase in the number of fasciculata and reticularis cells occurred. Prolonged ACTH treatment resulted in a significant increase in 3H-thymidine incorporation with the highest value on day 6 and subsequent drop on day 9. Also metaphase index of adrenocortical cells notably increased due to ACTH administration and again the highest value was found on day 6 of experiment. From stereologic data and from metaphase index, adrenocortical cell birth-rates in individual adrenocortical zones and in the entire cortex were calculated. There were great differences between the values calculated from metaphase index and those obtained from stereologic data, with the former significantly lower. ACTH enhances centripetal migration of parenchymal cells in the hamster adrenal cortex and causes their accumulation in the zona reticularis.     

In vitro decidualization of rat endometrial stromal cells

The induction of the decidualization of endometrial stromal cells is possible in an in vitro cell culture system. However, thus far, methods differ according to species or cell type, and a more stable or universal system has not yet been developed. The purpose of the present study has been to establish an in vitro decidualization system in primary cultured rat endometrial stromal cells (RES). The RES were treated with medroxyprogesterone acetate and dibutyryl-cyclic adenosine monophosphate (MPA treatment), estradiol and progesterone, or arachidonic acid. After 24 h of treatment, cells responded to all of the stimulations by expressing desmin mRNA. However, decidual/trophoblast prolactin-related protein (dPRP) mRNA was only expressed in the MPA-treated cells. Desmin and dPRP mRNA were not expressed after MPA treatment of the RES derived from immature rat uteri. However, mRNA from both desmin and dPRP were expressed in RES derived from gonadotrophin-injected immature rats. The expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 mRNA did not change after the decidual treatment of RES examined by real-time polymerase chain reaction. However, the results of gelatin zymography showed that the active forms of MMP-2 and MMP-9 significantly increased after in vitro decidualization (P < 0.05). We conclude that MPA treatment is the most effective method for stimulating decidualization in RES. Use of this system has revealed that sexual maturation and gonadotrophins are important for RES with regard to decidualization. Furthermore, the activity of MMP-2 and MMP-9 might increase during decidualization without a corresponding increase of the expression of these genes. This research was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (JSPS; no. 18580282, to N. Yamauchi).     

Tenascin gene expression in rat liver and in rat liver cells In vivo and in vitro studies

Tenascin is a major glycoprotein constituent of the extracellular matrix with a strong affinity to fibronectin; its distribution is believed to be temporarily and spatially limited. Tenascin gene expression is increased during wound healing processes. As repair mechanisms in chronic liver diseases resemble wound healing we studied tenascin gene expression in rat liver and in isolated rat liver cells. In normal rat liver a tenascin specific antiserum stains sinusoidal cells with fiber-like prolongations, which at the same time are desmin-positive (ITO-cells). In the CCl₄-acutely-damaged liver a strong tenascin staining is detected in cells located among the mononuclear cells of the inflammatory infiltrates in the areas of necrosis and in cells of the sinusoids. In CG⁴-chronically-damaged liver a strong tenascin staining is demonstrable in the connective tissue septa. In both cases, many of the tenascin-positive cells can be identified as desmin-positive by means of the double-staining fluorescence technique. The wall of larger vessels is always tensacin-negative. The staining pattern obtained with a fibronectin-specific antiserum is somewhat comparable with that of tenascin but the vessel wall was positive. Hepatocytes, Kupffer cells, ITO-cells and endothelial cells were isolated from rat liver and studied for their capacity to express the tenascin gene. Biosynthetically labeled tenascin was immunoprecipated from supernatants and cell lysates obtained from cultured ITO-cells and to a much lesser extent from intracellular lysates obtained from endothelial cells; its synthesis in ITO-cells increased during the time in culture. Tenascin was also identified immuno-cytochemically in increasing amount in ITO-cells in culture. We conclude that ITO-cells may play a major role in tenascin synthesis during liver fibrogenesis. Some of these results were presented at the Annual Meeting of the American Association Study of the Liver, Chicago, USA, 1990. G.R. holds a Hermann and Lilly Schilling professorship