Effects of prolonged treatment with cyanoketone on the zona fasciculata of rat adrenal cortex

Summary We have examined IgG Fc receptor (FcR) activity of human and rabbit arachnoid granulations and leptomeninges using antibody (IgG)-coated erythrocytes (EIgG), covalently crosslinked IgG dimers, trimers and oligomers, immune complexes, aggregated Fc fragments and a monoclonal anti-human neutrophil Fc receptor antibody, 3G8. EIgG bound specifically to cells of the leptomeninges and arachnoid granulations; uncoated erythrocytes, F(ab) ₂-coated, or IgM-coated erythrocytes failed to bind. The specificity of this interaction was demonstrated by inhibition studies. Monomeric IgG and Fc fragments blocked EIgG adherence, whereas bovine serum albumin (BSA), Fab fragments of IgG and the monoclonal anti-neutrophil FcR antibody failed to inhibit EIgG adherence. Monomeric IgG inhibited FcR function in a dose-dependent fashion; maximal inhibition was achieved at 1.7 × 10^-5M IgG, indicating a relatively low avidity receptor. Oligomers of IgG inhibited EIgG adherence more effectively and inhibition was directly related to oligomer size. Additionally, these tissues were positive for specific and non-specific esterases. These studies suggest that the CSF pathway from the perivascular spaces to the arachnoid granulations plays a protective role in the clearance of IgG and IgG immune complexes in infections and immune-mediated disorders.Work partially supported by PHS Grant #Ca 38055, National Cancer InstituteWork primarily supported by the Veterans Administration Merit Program     

Subcellular distribution of calcium in zona fasciculata cells of the rat adrenal cortex

Zona fasciculata cells from the adrenal cortex of female Sprague-Dawley rats were fixed by immersion in potassium pyroantimonate-osmium tetroxide and potassium pyroantimonate-glutaraldehyde to study the distribution of calcium. Potassium pyroantimonate-osmium tetroxide treatment gave reproducible patterns of electron-opaque precipitate, whereas inconsistent deposits of reaction product were seen after potassium pyroantimonate-glutaraldehyde fixation. Nuclei showed sparse precipitate over heterochromatin and dense aggregates over areas of nucleoli surrounded by portions of the nucleolar-dense component. Two major cytoplasmic sites of precipitate were identified: mitochondria and vesicles of smooth endoplasmic reticulum. Most of the intramitochondrial precipitate was localized to the intracristal space. Precipitate was also seen in vesicles of Golgi apparatus. The extracellular space was filled with closely packed electron-opaque particles. Observation of tissues treated with control fixative saturated with EGTA showed little if any reaction, confirming that calcium was the primary cation precipitated by potassium pyroantimonate. Our results provide a method suitable for accurate localization of calcium in adrenocortical cells.     

Effects of a prolonged treatment with aminoglutethimide on the zona fasciculata of rat adrenal cortex: A morphometric investigation

Summary The effects of a 7-day administration of aminoglutethimide (AG) on the adrenal zona fasciculata were examined in normal and dexamethasone/ACTH-treated rats. There was a 70–74% decrease in the concentration of corticosterone in blood, but no conspicuous qualitative changes suggesting cell degeneration occurred. Morphometry showed that AG induced a significant hypertrophy of the zona fasciculata and its parenchymal cells only in normal animals, which was due to an increase in the volume of the mitochondrial compartment and to proliferation of the smooth endoplasmic reticulum. This response to AG was considered to be non-specific and mediated by the enhanced secretion of ACTH following the decrease in the blood level of corticosterone. AG administration significantly increased the volume of the lipid-droplet compartment and the number of intramitochondrial lipid-like inclusions in both groups of animals. These changes were interpreted as the morphological counterpart of the AG-induced block of cholesterol utilization in steroid synthesis.     

The effects of ageing on the morphology and function of the zonae fasciculata and reticularis of the rat adrenal cortex

Summary The morphological counterpart of the well-known age-dependent marked impairment of glucocorticoid secretion of rat adrenals was investigated by use of morphometric techniques. For this purpose 4-, 8-, 16- and 24-month-old rats were studied. Despite the notable lowering of both basal and ACTH-stimulated production of corticosterone by collagenase-dispersed inner adrenocortical cells, ACTH and corticosterone plasma concentrations displayed significant increases with ageing. Zona fasciculata (ZF) and zona reticularis (ZR) showed a notable hypertrophy in aged rats, which was due to rises in both the average volume and number of their parenchymal cells. The hypertrophy of ZF and ZR cells was in turn associated with increase in the volume of the mitochondrial compartment and proliferation of smooth endoplasmic reticulum, i.e., the two organelles involved in steroid-hormone synthesis. All these morphologic changes, conceivably due to the chronic exposure to high levels of circulating ACTH, are interpreted as a response enabling ZF and ZR to compensate for their age-dependent lowering in glucocorticoid secretion. Stereology also demonstrated that ZF and ZR cells underwent a striking age-related lipid-droplet repletion. Lipid droplets are the intracellular stores of cholesterol esters, the obligate precursors of steroid hormones in rats. This finding is in keeping with the contention that the mechanism underlying the age-dependent decline in rat-adrenal glucocorticoid secretion mainly involves impairments of the utilization of intracellular cholesterol previous to its intramitochondrial transformation to pregnenolone.     

Histophysiology of the zona fasciculata of the adrenal cortex of albino rats

Three phases have been detected in the fascicular zone of the white rat adrenal cortex. During the first phase (normoemia) blood stream is rather moderate, lipids accumulate in adrenocorticocytes. This results in increase of their volume and in decrease of nuclear-cytoplasmic relations. The second phase (functional hyperemia) is characterized with an elevated blood stream and plethora, maximal parameters of nuclear volume and nuclear-cytoplasmic relations, decreasing content of lipids and volume of adrenocorticocytes. During the third phase (functional hypoemia) parameters characterising intensity of blood stream, nuclear volume and nuclear-cytoplasmic relations decrease.     

A comparative morphometric analysis of the effects of thyroxine and ACTH on the zona fasciculata of rat adrenal cortex.

The effects of thyroxine and ACTH on the ultrastructure of cells of the adrenal zona fasciculata were studied by morphometric analysis and compared by two-way analysis of variance. The results indicate that the effects of thyroxine and ACTH are different and independent. The statistically significant differences in effect of thyroxine and ACTH are: thyroxine causes an increase in the volume fraction of mitochondria and a decrease in the volume fraction of endoplasmic reticulum independent of ACTH; thyroxine causes a decrease in surface density of mitochondria and endoplasmic reticulum in the absence of ACTH, but in the presence of ACTH this effect is moderated by an interaction of throxine and ACTH. There were no statistically significant effects of either thyroxine or ACTH on volume fraction or surface density of liposomes.     

Evidence that long-term methionine-enkephalin administration stimulates rat adrenal zona fasciculata

Short-term methionine-enkephalin (DALA) treatment did not induce evident changes in the adrenal zona fasciculata and in the basal corticosterone output of dexamethasone-treated rats administered with maintenance doses of ACTH. Conversely, prolonged (5 days) DALA treatment caused a notable hypertrophy of zona fasciculata cells, along with a significant increase in the plasma concentration of corticosterone. It is suggested that enkephalins exert a trophic action on the rat zona fasciculata.     

Inhibited autophagic degradation during ACTH-stimulated growth of rat adrenal zona fasciculata

Autophagic vacuoles (AVs) in adrenal parenchymal cells of the outer part of the zona fasciculata were investigated by means of electron microscopic morphometry in 40 adult male Sprague Dawley rats. Four test groups and four control groups received twice daily subcutaneous injections of ACTH (12.5 U/kg body weight) or physiological saline, respectively, for 3, 6, 9 and 12 days. At 12 days the total dry weight of the adrenals was increased in ACTH-treated animals from 17.2 to 71.0 mg (about fourfold) and the average volume of zona fasciculata cells from 1,500 to 5,100 microns3 (about threefold). The relatively larger increase in total mass reflects mitotic activity in the growing adrenal tissue. The calculated growth rate of total tissue expressed as the percentage increase in dry weight per day was 12% at day 3 and decreased to 4.6% at day 12 of ACTH stimulation. During the time interval investigated the cytoplasmic volume fraction of autophagic vacuoles was significantly reduced from 10 X 10(-5) in controls to 1 X 10(-5) in ACTH-treated animals (p less than 0.001). Assuming a half-life for AVs of 7.9 min (Papadopoulos and Pfeifer 1986), autophagic degradation of cytoplasm can be calculated as 1.3%/day in controls, but only 0.15%/day in ACTH-treated animals. Thus growth of 1.15%/day can be attributed to inhibition of autophagic degradation. Although this is only part of total growth, it is evident from the present results that anticatabolic growth induction is utilized nearly to its maximum in adrenocortical fasciculata cells under the influence of ACTH.     

The involvement of coated vesicles in the secretion of corticosterone by the zona fasciculata of the rat adrenal cortex

Following exposure of rats to unpredictable stress there was a marked increase in the number of ‘coated’ vesicles in contact with or close to the cell membrane of the zona fasciculata cells. The close correlation between the vesicle numbers and the plasma levels of corticosterone led to the hypothesis that the coated vesicles were intimately involved in the secretory process. The use of horseradish peroxidase as a tracer protein confirmed that the coated vesicles were not involved in pinocytosis and the inward movement of materials, this function being performed by a much larger uncoated vesicle. The presence of microtubules associated with the coated vesicles and radiating through the Golgi body region, the site of formation of the vesicles, suggested that they may be involved in the transport of the secretory product to the cell membrane. The use of microtubule inhibitors, colchicine and vinblastine, were found to significantly reduce the plasma steroid response to stress. On the basis of these findings a new secretory mechanism was postulated.     

Effects of prolonged infusion with endothelin-1 on the function and morphology of rat adrenal cortex

A week of SC infusion with endothelin-1 (ET-1) (0.2 microgram.kg-1.hr-1) lowered PRA and raised plasma aldosterone (A) concentration in rats. Kalaemia and the plasma levels of ACTH and corticosterone (B) were not affected. Prolonged ET-1 administration caused a notable hypertrophy of zona glomerulosa (ZG) and its parenchymal cells, without inducing any apparent change in zona fasciculata. Stereology showed that ZG cell hypertrophy was mainly due to the increase in the volume of the mitochondrial compartment and to the proliferation of smooth endoplasmic reticulum (i.e., the two organelles in which the enzymes of steroid synthesis are contained). Isolated ZG cells from ET-1-infused animals evidenced a notable enhancement in their basal production of A and B. The secretory responses of ZG cells to the maximal effective concentrations of their three main stimulators (ACTH, angiotensin-II and K+) displayed comparable increases. These findings indicate that ET-1, when chronically administered, is able to specifically enhance the growth and steroidogenic capacity of rat ZG, and suggest that the mechanism underlying this ET-1 effect involves stimulation of the de novo synthesis of both the steroidogenic enzymes and the membrane framework in which they are located.     

Effects of streptozotocin-induced long-term diabetes on parietal cell function and morphology in rats

Gastric pathology is a common complication in diabetes mellitus. The aim of the study was to evaluate the functions and morphological changes of the parietal cells of the rat stomach after streptozotocin-induced diabetes. Diabetes mellitus was induced in Wistar rats by a single intraperitoneal injection of streptozotocin (60 mg/kg body weight). The rats were weighed weekly and sacrificed after 6 months. The glandular portion of the stomach was removed and processed for H⁺-K⁺-ATPase immunohistochemistry and light and electron microscopy studies. Acid secretion was measured in vivo. After 6 months of diabetes, the mean weight of the rats was significantly lower (P < 0.001) compared to control. The mean weight of the stomach to body weight percentage increased significantly (P < 0.001) compared to control. The blood glucose level in diabetic rats was significantly higher (P < 0.001) than in normal control. Diabetic rats showed significant (P < 0.001) decrease in basal and stimulated acid secretion when compared to control. Electron micrographs of the parietal cells of glandular stomach of diabetic rats revealed significant (P < 0.0002) reduction in the number of mitochondria and a small though not significant increase in the number of canaliculi in the parietal cells compared with normal. Immunohistochemistry showed reduced H⁺-K⁺-ATPase (P < 0.00001) compared to control. Long-term diabetes induces morphological as well as functional changes in gastric parietal cells. The decrease in the number of mitochondria accompanied by reduced in H⁺-K⁺-ATPase in parietal cells may explain the reduced acid secretion observed in diabetics.     

肾上腺皮质束状带特异性表达Cre重组酶转基因小鼠的构建

肾上腺是人体重要的内分泌器官。由于缺乏肾上腺皮质束状带特异性表达Cre酶的工具鼠,目前对肾上腺皮质束状带细胞中特异表达基因的功能缺乏深入的解析。CYP11B1基因编码类固醇11β-羟化酶,该酶是糖皮质激素合成的关键酶,在肾上腺皮质束状带中特异性表达。本研究旨在利用CYP11B1基因在束状带特异性表达的特点,构建在肾上腺皮质束状带中特异性表达Cre重组酶的转基因动物。采用CRISPR/Cas9技术在CYP11B1基因终止密码子位点定点敲入2A-GfpCre表达框,获得CYP11B1-2A-GfpCre同源重组载体,进而构建CYP11B1Cre小鼠,并通过mTmG和LacZ染色确定Cre酶主要表达在小鼠肾上腺皮质束状带。在此基础上,本研究还用该工具鼠与胱硫醚-γ-裂解酶(cystathionineγ-lyase, CTH)条件性敲除鼠交配,获得了肾上腺皮质束状带CTH特异性敲除的小鼠,并证实了该动物肾上腺皮质束状带中CTH表达缺失。以上结果充分说明肾上腺皮质束状带特异性表达Cre重组酶小鼠构建成功。该工具鼠的成功构建,为深入研究肾上腺皮质束状带相关功能提供了有力工具。