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Search for other papers by J. M. BASSETT in
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SUMMARY
A sensitive method for the determination of corticosteroids in 0·1 ml. or less of ovine plasma is described. The method uses the steroid-binding properties of corticosteroid-binding globulin (CBG) and gel filtration on small columns of Sephadex G-25 (fine) at 4° for separation of CBG-bound and free steroids. Cortisol was found to be the predominant corticosteroid in ovine plasma and accounts for about 90% of the value determined by this method. The corticosteroid concentration in peripheral plasma of unstressed sheep was in the range 0·1–1·0 μg./100 ml. In untrained animals, venipuncture increased corticosteroid concentration substantially; training reduced the effect. An infusion of cortisol sodium succinate (100 μg. cortisol/min.) increased the plasma corticosteroid level to 9·5 ± 0·49 μg./100 ml. Intravenous infusion of the synthetic adrenocorticotrophic preparation Synacthen at rates of 10 and 20 μg./hr. for 2 hr. increased peripheral corticosteroid concentrations to 8 μg./100 ml. Single i.v. injections of 0·2–0·8 μg. Synacthen also significantly increased peripheral corticosteroid concentrations 7–15 min. later. The injection of 0·05 and 0·1 μg. Synacthen significantly increased the corticosteroid concentration too, but the increase was not significantly greater than that produced by the injection of acidified saline diluent alone. Injection of insulin (0·25 unit/kg. body weight, i.v.) caused a fivefold increase in the corticosteroid concentration 30–60 min. later, in both adult sheep and lambs. Glucose (0·25 g./kg. body weight, i.v.) had no effect on corticosteroid concentration.
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SUMMARY
Incubation, with [4-14C]progesterone, of adrenocortical tissue (posterior cardinal vein preparations) from the eel yielded 14C-labelled cortisol and cortisone. These two hormones, containing both 14C and 3H, appeared when similar preparations were incubated simultaneously with [4-14C]progesterone and [16-3H]pregnenolone. In addition, the following intermediaries were isolated: [14C, 3H]progesterone; [14C, 3H]17α-hydroxyprogesterone; [3H]17α-hydroxypregnenolone and [14C, 3H]21-deoxycortisol. Analysis of the isotope content of the end products and isolated intermediaries seemed to indicate that the transformation of pregnenolone to corticosteroids proceeded to a large extent through intermediaries other than progesterone. In none of these experiments could the formation of aldosterone be demonstrated. Similarly, incubation of eel adrenocortical tissue with a mixture of [4-14C]-progesterone and [1,2-3H]corticosterone failed to yield detectable aldosterone.
A further search for aldosterone, using a large amount of eel adrenocortical tissue with [4-14C]progesterone and [16-3H]pregnenolone as substrates with added angiotensin, also gave negative results. In similar preparations, [1-14C]sodium acetate was not transformed to any recognizable corticosteroids.
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ABSTRACT
Previous studies utilizing steroid-binding assays have suggested that corticosteroid-binding globulin (CBG)-like glucocorticoid binding sites are present in various tissues of the rat. It is not known, however, whether such binding reflects the intracellular presence of CBG derived from serum or a special class (type III) of receptors. In order to elucidate this problem, immunocytochemical localization of rat CBG was carried out using a specific antiserum prepared against rat serum CBG and the peroxidase-antiperoxidase technique. Positive staining was found in certain cells of the liver, the distal and/or convoluted tubules of the kidney, the uterus, the follicular cells of the thyroid, and some cells of the anterior pituitary. Other tissues including heart, muscle, thymus, hypothalamus, supraoptic and paraventricular nuclei, and diaphragm were negative. The presence of immunoreactive CBG in specific cells of some glucocorticoid-responsive tissues and not others raises interesting questions concerning the transport of glucocorticoids and their mechanism of action.
J. Endocr. (1986) 108, 31–36
Search for other papers by JOAN D. FULLER in
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* Zoology Department, University of St Andrews, Scotland, KY16 9TS, and † Medical Research Council Blood Pressure Research Unit, Western Infirmary, Glasgow, G11 6NT
(Received 2 April 1976)
Although several corticosteroids have been reported to be present in teleost plasma (review by Idler, 1972) relatively few studies have taken adequate steps to ensure that these compounds have been reliably characterized. Methods of establishing the identity of steroids have been discussed by Brooks, Brooks, Fotherby, Grant, Klopper & Klyne (1970) and those related to teleost studies have been reviewed and assessed by Idler (1972). This report describes the analysis of a small number of plasma samples from Salmonidae caught in Loch Lomond, Scotland. Analysis was by a highly specific method based on gas–liquid chromatography (g.l.c.) which satisfies many of the criteria suggested by the above authors.
Specimens of four salmonid species were collected by means of seine or gill-nets. Coregonus lavaretus,
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Search for other papers by E. W. HILLHOUSE in
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Search for other papers by J. L. BURDEN in
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SUMMARY
Structure–activity studies on the corticosteroid fast and delayed feedback receptor mechanisms controlling the secretion of corticotrophin releasing factor (CRF) were carried out with the rat hypothalamus in vitro. The secretion of CRF was induced by acetylcholine (3 pg/ml).
The fast feedback receptor appears highly specific, and the structure essential for efficacy involves an 11β-hydroxyl group and an unblocked 21-hydroxyl group. Several steroids showed antagonism and so the binding site is not very specific. 18-Hydroxy,11-deoxycorticosterone, progesterone, 17α-hydroxyprogesterone and 11-deoxycorticosterone were antagonists of fast feedback.
The delayed feedback receptor required either an 11β- or a 21-hydroxyl group for efficacy. The binding site required a 17-hydroxyl group when the 11β- or 21-hydroxyl groups were absent. Binding also involved the 3-oxo,4,5-ene structure since steroids in which these are absent were inactive.
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Search for other papers by M Orchinik in
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Stressors elicit a complex but variable suite of endocrine events. Comparative studies of the stress response have focused primarily on the adrenocortical response to stress, in particular the measurement of plasma levels of glucocorticoids. However, a number of other factors contribute to and modify cellular and organismal responses to glucocorticoids. Notably, plasma corticosteroid binding globulins (CBGs) can regulate the general availability of steroid to tissues, and/or direct the delivery of hormones to specific sites. In this paper, we discuss possible functions of CBG and mechanisms of CBG action, review CBG characteristics among vertebrates, and discuss our recent studies indicating that CBG may indeed modulate responses to stressors. For example, in house sparrows, we found that basal and stress-induced concentrations of total corticosteroid (cortisol or corticosterone) (CORT) vary seasonally, but CBG concentrations change proportionally, so that free CORT concentrations appear static year-round. In contrast, in white-crowned sparrows and tree lizards, CBG concentrations change under conditions when total CORT levels do not, resulting in significant changes in circulating free CORT. These differences in free CORT are masked if CBG is not accounted for. We have also found that the binding properties of CBG vary considerably between species and need to be determined empirically. Such studies led to the observation that CBG in several species may also serve as a functional androgen binding protein; this is especially important for birds, because previous studies had concluded that birds lack androgen binding globulins. We propose that consideration of CBG is paramount to understanding the role of glucocorticoids in mediating behavioral and physiological responses to stress.
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Chronic corticosteroid treatment is known to induce bone loss and osteoporosis. Osteoclasts are specialised bone-resorbing cells that are formed from mononuclear phagocyte precursors that circulate in the monocyte fraction. In this study we have examined the effect of the synthetic glucocorticoid, dexamethasone, on human osteoclast formation and bone-resorbing activity. Human monocytes were cultured for up to 21 days on glass coverslips and dentine slices, with soluble receptor activator for nuclear factor kappaB ligand (RANKL; 30 ng/ml) and human macrophage-colony stimulating factor (M-CSF; 25 ng/ml) in the presence and absence of dexamethasone (10(-8) M). The addition of dexamethasone over a period of 7 and 14 days of culture of monocytes (during which cell proliferation and differentiation predominantly occurred) resulted in a marked increase in the formation of tartrate-resistant acid phosphatase-positive multinucleated cells and an increase in lacunar resorption. The addition of dexamethasone to monocyte cultures after 14 days (when resorptive activity of osteoclasts had commenced) reduced the extent of lacunar resorption compared with cultures to which no dexamethasone had been added. The addition of dexamethasone to osteoclasts isolated from giant cell tumours of bone significantly inhibited resorption pit formation. Our findings indicate that dexamethasone has a direct effect on osteoclast formation and activity, stimulating the proliferation and differentiation of human osteoclast precursors and inhibiting the bone-resorbing activity of mature osteoclasts.
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ABSTRACT
A radioimmunoassay (RIA) for human corticosteroid binding globulin (CBG) has been developed using 125I-labelled CBG and a monospecific solid-phase CBG-antiserum (CBG-Ab-cellulose). In an RIA of serum CBG concentrations, pure CBG standards (1–100 ng protein) or samples (1 : 200) were incubated (16 h at 20 °C) with 125I-labelled CBG and CBG-Ab-cellulose. After addition of 2 ml 0·9% NaCl, the tubes were centrifuged, supernatants were aspirated and the 125I-labelled CBG bound to the CBG-Ab-cellulose pellet was counted. The specificity of the RIA was confirmed by parallel displacement curves for serial dilutions of male, female and pregnancy sera, as well as pure CBG standards. The mean ± s.d. recovery (99±8%) of pure CBG (1·6–25·0 ng) added to a diluted serum sample verified the accuracy of the method, and a good correlation (r = 0·97; n = 43) existed between serum CBG cortisol binding capacity (nmol/l) measurements and CBG concentrations (mg protein/l) measured by RIA. Intra- and interassay precisions (C.V.) at low to high serum CBG concentrations were <5% and <9% respectively. The mean ± s.d. serum CBG concentrations (mg protein/l) measured by the RIA were: 21·8±4·6 in boys (n = 12), 20·0±4·2 in girls (n = 9), 20·7±2·7 in men (n = 6), 20·5±2·9 in women (n = 6) and 47·1 ±10·5 in pregnant women (n = 5). The sensitivity of the standard curve used in the routine RIA of serum CBG was 1·0 ng CBG/assay tube, but this could be increased to 0·2 ng/assay tube by reducing the amount of CBG-Ab-cellulose used. The RIA is suitable for both clinical and research purposes, and will aid the identification of abnormal forms of CBG and facilitate studies of the regulation of CBG production in vitro.
J. Endocr. (1985) 104, 259–267
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Corticosteroids (CS) can modulate gene expression and are often used to treat a range of immunological and inflammatory diseases such as asthma, inflammatory bowel disease and rheumatoid arthritis. However, a proportion of patients fail to show an adequate response. On this basis patients have been subdivided into CS-sensitive (SS) and -resistant (SR) subgroups. The ability of CS to inhibit peripheral blood T cell proliferation in vitro has also been used similarly. In rheumatoid arthritis (RA), the in vitro-defined SS and SR subgroups correlate with the clinical responses to CS therapy. The mechanisms responsible for this observation are unknown but they appear to involve a number of known molecular events related to the described mechanisms of action of CS. These include alterations in the functional status of CS receptor-alpha, perturbations of the cytokine and hormonal milieu and intracellular signalling pathways. Peripheral blood mononuclear cells (MNCs) from SR significantly overexpress activated NF-kappaB. In vitro, CS fail to significantly inhibit concanavalin A (conA)-induced NF-kappaB activation in MNCs from SR RA patients. The alterations in the intracellular signalling pathways may explain in part our observations seen in SR RA subjects, CS fail to significantly inhibit conA-induced interleukin (IL)-2 and IL-4 secretion and lipopolysaccharide-induced IL-8 and IL-1beta secretion in vitro. CS therapy fails to reduce the circulating levels of IL-8 and IL-1beta in RA patients. In asthma, CS fail to induce L10 in SR asthma patients. Other molecular mechanisms such as enhanced AP-1 expression and alterations in the MAP kinase pathway are most likely to be involved too and we are currently investigating such possibilities. A full understanding of the molecular basis of SR will lead to the development of more rational therapeutic strategies.
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Search for other papers by W. C. DICK in
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SUMMARY
The response of plasma 11-hydroxycorticosteroids (11-OHCS) to parenteral lysine-vasopressin (LVP) was studied in 47 patients with rheumatoid arthritis, 25 of whom had received long-term oral corticosteroid therapy; the remaining 22 had received no oral corticosteroids at any time, and served as controls. Both groups of patients showed excellent reproducibility of the plasma 11-OHCS response to LVP, when administered intravenously or intramuscularly. Comparable plasma 11-OHCS levels were reached in control patients when the intravenous LVP test was carried out in the morning and in the evening. Criteria of a normal response to both intramuscular and intravenous LVP were defined. In the control group, intravenous infusion of LVP led to a more sustained plasma 11-OHCS response than did intramuscular injection of the same dose. No such differences were observed in the corticosteroid-treated group, but the intramuscular test was subnormal more frequently than the intravenous test.
In the control patients, insulin-induced hypoglycaemia and intravenous LVP led to comparable plasma 11-OHCS response, although a higher mean level at 60 min. was observed in the former. This difference was not observed in the corticosteroid-treated patients.
Direct comparison of these three tests lead to the conclusion that the intramuscular LVP test is more sensitive than the intravenous LVP test, but less sensitive than the insulin hypoglycaemia test, in detecting corticosteroid-induced suppression of hypothalamo-pituitary-adrenal function. The intramuscular LVP test is therefore to be preferred to the intravenous test because of its greater sensitivity, its relative lack of side-effects, and its ease of performance.
