[Biochemistry of adrenal cortex hyperfunction].
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Alkaline lipolytic activities in bovine liver and adrenal cortex have been investigated; each tissue has a salt-resistant, hepatic-type lipase activity of which we describe a partial purification. Properties of the partially purified enzymes have been compared directly with those of authentic hepatic lipase prepared from rat liver. Furthermore, a similar activity has been detected in bovine post-heparin plasma. These findings contrast with a previous report that bovine post-heparin plasma and liver extracts lack hepatic salt-resistant lipase.
In an attempt to elucidate the mechanism(s) through which the functional adrenal cortex is established, we analyzed immunohistochemically the expression of various markers for the adrenocortical zones, i.e. the zona glomerulosa (zG), the zona fasciculata (zF), and the zona reticularis (zR), as well as markers for the medulla, and further examined the distribution and behavior of DNA-synthesizing cells in rat adrenal glands during development. The results showed that 1) separation of the cortex and medulla, and the development of functional zonation in the cortex began at around the time of birth, 2) at fetal stages when cortical zonation was not established, DNA-synthesizing cells were found scattered throughout the gland, where they proliferated without significant migration, and 3) after birth in the adrenal cortex with established cortical zonation, DNA-synthesizing cells were localized near the undifferentiated zone between zG and zF, and then they migrated centripetally. Cell death appeared to occur in the innermost portion of the cortex, where many resident macrophages are present. These findings illustrate basic processes underlying adrenal development and suggest that the undifferentiated region is apparently the stem cell zone of the adrenal cortex that maintains the cortical zonation.
Histo- and ultrastructure investigation of adrenal cortex in young animals after the single administration of hypobaric hypoxia has been carried out. Hypobaric hypoxia was modeled in ventilated barocamera with pO(2) decreasing till 364-372 mm Hg on the 15 albino male rats 51-120 days of age and 90-120 gr of body mass. Duration of the experiment was 4+/-1 hours. The animals of the control group were placed in normobaric conditions for the same time. Adrenocorticocytes of z. fasciculate are more sensible to oxygen insufficiency. Hyperchromocitous and shrinking of nucle-decrease of lipids, mitochondrion swelling and diffuse cells edema are presented. Myelin like transformation of lipids drops are remarkable. Hypobaric hypoxia is a severe stress-factor, which induces remarkable structure failure in adrenal cortex, especially in z. facsiculata.
Beta-endorphin-like peptide immunorphin (SLTCLVKGFY), a selective agonist of nonopioid beta-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of nonopioid beta-endorphin receptors on rat adrenal cortex membranes (Kd = 31.6 +/- 0.2 nM, Bmax = 37.4 +/- 2.2 pmol/mg protein). Immunorphin at concentrations of 10(-9) to 10(-6) M was found to inhibit the adenylate cyclase activity in adrenal cortex membranes, while intramuscular injection of immunorphin at doses of 10-100 microg/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.
Percentage of lipid content of 22 adrenal cortex lesions and their water proton longitudinal relaxation time were measured in vitro at 60 MHz. Although water relaxation times obtained on benign and malignant samples overlap each other, measurements of the percentage of lipids performed on the same tissues allow us to distinguish the two pathologies. The specific tool of characterization should be possible to obtain through in vivo spectroscopic imaging techniques.
Electron-microscopic investigations on the human adrenal cortex have been carried out in order to elucidate ultrastructural differences in the various zones. These studies indicate that certain differences in the ultrastructure of the mitochondria may be particularly useful as criteria to differentiate the three cortical zones. In this regard, the appearance of two types of mitochondria in the zona glomerulosa as well as in the zona fasciculata is of obvious interest. The probability of a relationship between the activity of the different cortical zones and the predominant presence of one form of mitochondria has been suggested. In addition, ultrastructural transformations following stress situations as well as problems dealing with the so-called 'dark and light cells' and the basal membrane are discussed.
Outer and inner layer cells of bovine adrenal cortex were cultured separately to compare cellular structural characteristics and functional differences. Outer layer cells were polygonal in shape with radially distributed lipid droplets in the cytoplasm, and produced mainly aldosterone and cortisol. The aldosterone production increased upon stimulation with angiotensin II or dibutyryl-cAMP. In contrast, inner layer cells were spindle-shaped and had fine diffused lipid droplets. They produced four times as much cortisol as outer layer cells but no aldosterone. Cortisol production increased with ACTH or dibutyryl-cAMP stimulation. When stimulated by ACTH or by dibutyryl-cAMP, both types of adrenocortical cells showed "cellular retraction" whereby the number of cytoplasmic lipid droplets decreased and microvilli on the cellular surface increased. At the same time, the transverse distribution of actin fibers disappeared and the microtubules changed their distribution pattern from circular to radial. Stimulation by angiotensin II, on the other hand, brought no marked structural changes. These results indicate that, in functional terms, the outer layer cells and the inner layer cells in this culture system reflect zona glomerulosa and zona fasciculata-reticularis, respectively.
The various protein kinase activities characterized in adrenal cortex from different mammalian species do not differ in their molecular and catalytic properties from similar types of enzymes described in other tissues. The cyclic AMP-dependent system has been the most thoroughly investigated in adrenocortical tissue and is generally accepted as an obligatory intermediate in the intracellular response to ACTH action. In addition, adrenocortical cells contain two types of messenger independent protein kinases, one of which being selectively activated by polyamines in vitro and possibly related to the control of adrenocortical growth processes. The definition of intracellular phosphorylatable targets related to specific adrenocortical functions might have been obscured by the pleiotypic effects displayed by ACTH, the major effector of these cell activities. Cholesterol esterase appears one of these targets and 11 beta-hydroxylase cytochrome P-450 has been found activated in vitro by a cAMP dependent process. Future research should provide a better understanding of the role of protein phosphorylation-dephosphorylation processes as a regulatory device in adrenocortical cell functions. This includes possible complex modulation by multisite phosphorylations of a given target protein, involving several different protein kinase systems under the dependence of separate intracellular effectors.
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We have reported previously that a protein, ib, is produced in adrenal cortex and other steroidogenic cells with the same tissue-specific peptide hormone or cAMP dose-response and the same kinetics as the increase in steroid hormone biosynthesis. In this study, we have fractionated adrenal cortex cells into subcellular components and used two-dimensional electrophoresis to characterize the proteins in these fractions. We have demonstrated previously that inhibition of cytosolic translation, e.g. by cycloheximide, prevents the production of protein ib. We also report that the production of this protein is not affected by inhibition of mitochondrial translation by chloramphenicol.
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In order to establish the nature of the aldosterone synthetase activity in the adrenal cortex, we have used porcine adrenal, bovine adrenal cortex, highly purified bovine and porcine 11 beta-/18-hydroxylase, and antibodies raised against the latter enzyme. Mitochondria from two zones (glomerulosa and fasciculata) of the bovine cortex synthesize aldosterone, but those from glomerulosa are much more active than those from fasciculata. Partially purified (cholate-extracted plus ammonium sulfate-precipitated) extracts of mitochondria from the two zones are equally active in catalyzing all three steps in the conversion of 11-deoxycorticosterone to aldosterone. 18-Hydroxylase and aldehyde synthetase activities (18-hydroxycorticosterone----aldosterone) were completely precipitated from cholate extracts of mitochondria from bovine adrenal by antibodies to the pure porcine enzyme. No activity corresponding to any of the three steps in the conversion of 11-deoxycorticosterone to aldosterone was found in extramitochondrial fractions of the bovine cortex. Synthesis of aldosterone by the pure porcine enzyme was inhibited by antibodies to this enzyme and by metyrapone (an inhibitor of 11 beta-/18-hydroxylase). When fractions of porcine adrenal, resulting from purification of the enzyme from mitochondria, were exhaustively tested for any of the enzyme activities required for the synthesis of aldosterone, activity was found only in those fractions containing the 11 beta-/18-hydroxylase, i.e. no additional enzyme was discarded during the purification procedure. It is concluded that the only adrenocortical enzyme capable of synthesizing aldosterone in bovine and porcine adrenal is the well known 11 beta-hydroxylase, that the conversion of 18-hydroxycorticosterone to aldosterone is catalyzed by this cytochrome P-450, and that this step (aldehyde synthetase) requires the heme of the P-450 as demonstrated by the photochemical action spectrum.
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