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G. R. Williams
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G. A. Brent
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The retinoids, vitamin D3 and thyroid hormone exert diverse and complex tissue-specific actions by a common mechanism within the cell nucleus. These hormones, like the classical steroid hormones, glucocorticoid and oestrogen, bind to nuclear receptor proteins and modify transcriptional activity of target genes. The receptors are members of the steroid/thyroid hormone nuclear receptor superfamily of structurally homologous ligand-responsive transcription factors which activate or repress expression of hormone-responsive target genes (Evans, 1988; Green & Chambon, 1988; Moore, 1990; O'Malley, 1990; Moore & Brent, 1991).

The receptors for 3,5,3′-l-tri-iodothyronine (T3Rs), 1,25(OH)2-vitamin D3 (VDRs) and all-trans retinoic acid (RARs) form a subclass of homologous and functionally related proteins within the steroid superfamily. The receptors can bind to DNA in the absence of ligand (Brent, Dunn, Harney et al. 1989; Graupner, Wills, Tzukerman et al. 1989), they reside in the nucleus and their response elements possess

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R. H. Underwood
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A. I. Menachery
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G. H. Williams
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ABSTRACT

To assess the impact of sodium intake on the adrenal phosphoinositide system, rats were maintained on a low or normal salt diet for 5 days, and glomerulosa cell preparations (2×105 cells) were stimulated by angiotensin II (AII; 10 nmol/l), potassium (K+; 8·7 mmol/l) or ACTH (0·1 nmol/l) for 0, 2, 4, 6, 12, 15 and 60 s. Levels of phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns 4-P), phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) and inositol 1,4,5-trisphosphate (Ins 1,4,5-P3)+inositol3) + inositol 1,3,4-trisphosphate (Ins 1,3,4-P3) were assayed by a microspectrophotometric procedure. Non-stimulated levels of PtdIns, PtdIns 4-P, PtdIns 4,5-P2 and Ins 1,4,5-P3 (+Ins 1,3,4-P3) (means ± s.e.m.; n = 36) in cells from rats on the low Na+ intake were 580 ± 6·5, 187 ± 2·6, 82 ± 3 and 95 ± 1·2 pmoler incubate respectively, indistinguishable from those observed in rats on a normal Na+ intake, except for the significantly (P<0·025) greater Ptdlns 4,5-P2 level. In response to AII stimulation, all four compounds showed an earlier and greater peak response when cells were obtained from animals on a low rather than a high sodium intake. All values had returned to control levels by 12–15 s, regardless of the level of sodium intake. In contrast, with K+ stimulation there were no differences in the peak response of cells from rats on the two dietary intakes, but there was a shift of the peak to a longer time-interval (6 versus 8 s) in animals maintained on a low sodium intake. In addition, PtdIns 4,5-P2 did not return to control levels in cells obtained from animals on a low sodium intake. However, the most striking differences were observed in response to ACTH. In animals maintained on a normal sodium intake, there were no changes in any of the four compounds. In contrast, there was a sharp increase in all four substances in response to ACTH when the cells were obtained from animals on a low sodium intake, with peak levels occurring at 8 s, similar to that observed with potassium. Thus, changes in the response pattern of the phosphoinositides may mediate the altered adrenal responsiveness to AII and ACTH with sodium restriction.

Journal of Endocrinology (1989) 122, 371–377

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R Bland
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R L Sammons
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M C Sheppard
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G R Williams
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Abstract

3,5,3′-Tri-iodothyronine (T3), 1α,25(OH)2-vitamin D3 (D3) and retinoids activate related nuclear receptors which interact by heterodimerisation to regulate gene expression. Actions of each hormone are discrete and may be specified by changes in the relative concentrations of their receptors (T3R, vitamin D receptor (VDR), retinoic acid receptor (RAR), retinoid X receptor (RXR)). T3, D3 and retinoids are essential for skeletal development and maintenance and we have previously shown complex interactions amongst their signalling pathways in osteosarcoma cells. In these studies we demonstrate that similar T3R, VDR, RAR and RXR proteins are co-expressed in both osteoblast lineage cell primary cultures and osteosarcoma cells by Western blotting. We investigated whether hormone interactions in bone result from changes in receptor stoichiometry. Cells were treated with combinations of T3, D3, 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (RA) that are known from previous studies to produce complex cell specific responses. No alteration in expression of any receptor protein was seen in response to any hormone combination in three phenotypically distinct osteosarcoma cell lines. Thus, in contrast to studies of overexpressed receptors in vitro, changes in the physiological concentrations of endogenous T3R, VDR, RAR and RXR do not specify discrete hormone actions in osteoblastic cells. Other unidentified factors are likely to modulate hormone action in these bone cells.

Journal of Endocrinology (1997) 154, 63–74

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R G P Denis
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C Bing
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S Brocklehurst
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J A Harrold
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R G Vernon
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G Williams
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Rats normally eat about 85% of their food at night. Lactation increases food intake 3- to 4-fold, but the diurnal pattern of food intake persists. The mechanisms responsible for the diurnal and lactation-induced changes in food intake are still unresolved, hence we have further investigated the possible roles of serum leptin and hypothalamic expression of neuropeptide Y (NPY), agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) in rats. Suppressor of cytokine signalling-3 (SOCS-3) acts as a feedback inhibitor of leptin signalling in the hypothalamus, hence changes in expression of SOCS-3 were also investigated.

Changes in expression of NPY, AgRP or POMC alone could not account for the diurnal changes in intake and their alteration by lactation. However, there were increased AgRP mRNA:POMC mRNA ratios at night and also during lactation, which were very similar to estimated changes in food intake. Such changes in expression may result in dominance of the orexigenic AgRP peptide over the appetite-suppressing POMC-derived peptides, and so could contribute to the hyperphagia in these states. Diurnal and lactation-related changes in the AgRP mRNA:POMC mRNA ratio and food intake are not due to changes in leptin alone. However, hypoleptinaemia, possibly through increased expression of NPY, may contribute to the hyperphagia of lactation.

In the dark, expression of SOCS-3 was decreased in non-lactating rats; lactation decreased SOCS-3 expression in both light and dark phases. However, such changes are likely to enhance the ability of leptin-responsive neurones to transmit the leptin signal, and so are unlikely to contribute to either the nocturnal increase in appetite or the hyperphagia of lactation.

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T. A. Bramley
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G. S. Menzies
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R. J. Williams
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O. S. Kinsman
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D. J. Adams
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ABSTRACT

We have shown previously that partially purified human chorionic gonadotrophin (hCG) preparations inhibited the specific binding of I-labelled hLH or hCG to Candida albicans membranes at much lower concentrations than did highly purified hLH or hCG preparations. We now describe the characterization and partial purification of a heat-labile glycoprotein from commercially available gonadotrophin preparations. The factor strongly inhibited LH binding to Candida membranes, but not to sheep or pig luteal LH receptors. This material had a molecular weight of 16 000–21 000 daltons, bound strongly to CM-Sepharose at physiological pH, and could be resolved completely from hCG and from epidermal growth factor-like factors present in commercial gonadotrophin preparations. Its activity was not attenuated by a range of inhibitors specific for the four major classes of proteolytic enzymes, nor did it inhibit hormone binding by causing degradation of 125 I-labelled hLH or hCG tracers. Factors which inhibited hLH binding to Candida membranes were also present in partially purified human urinary and equine serum gonadotrophin preparations and in placental extracts, but were not detected in highly purified CG of hLH preparations. The properties of this factor were similar to those described for β-core protein, a cleavage product of the β subunit of hCG which is a contaminant of commercial gonadotrophin preparations. Highly purified β-core protein inhibited 125I-labelled hLH binding to Candida membranes, but not to sheep luteal binding sites.

Preparations of hCG depleted of inhibitor activity could stimulate adenylate cyclase activity in Candida membranes almost five fold. In contrast, partially purified inhibitor preparations strongly inhibited basal adenylate cyclase activity (to 18% of control levels). These observations suggest that endogenous LH-like factors, perhaps similar to β-core proteins of hCG, may play a role in the regulation of morphogenesis in Candida species.

Journal of Endocrinology (1991) 128, 139–151

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R. J. Schiebinger
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L. M. Braley
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A. Menachery
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G. H. Williams
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ABSTRACT

This study compared the extracellular calcium dependency and the enzymatic locus of that dependency for N6,O2′-dibutyryl cyclic AMP (dbcAMP)-, angiotensin II- and potassium-stimulated aldosterone secretion in dispersed rat glomerulosa cells. The need for extracellular calcium, calcium influx, and specifically for calcium influx through the calcium channel was examined. dbcAMP, angiotensin II and potassium, in the presence of calcium (3·5 mmol/l), significantly (P < 0·01) increased aldosterone output by at least 1·5-fold. Yet in the absence of extracellular calcium or in the presence of lanthanum (an inhibitor of calcium influx by most mechanisms) all three stimuli failed to increase aldosterone secretion. Nifedipine, a dihydropyridine calcium channel antagonist, significantly (P < 0·01) reduced angiotensin II- and potassium-stimulated aldosterone secretion, but had no effect on dbcAMP-stimulated aldosterone secretion (100 ± 14 vs 105 ± 19 pmol/106 cells). Likewise nitrendipine failed to inhibit ACTH-stimulated aldosterone secretion.

Angiotension II and potassium activation of both the early aldosterone biosynthetic pathway (as reflected by pregnenolone production in the presence of cyanoketone) and also its late pathway (as reflected by the conversion of exogenous corticosterone to aldosterone in the presence of cyanoketone) were significantly (P < 0·01) inhibited by lanthanum, nifedipine and by reducing the extracellular calcium concentration. However, with dbcAMP stimulation, none of these manipulations modified pregnenolone production. Late pathway activation by dbcAMP was inhibited by lanthanum and a reduction in extracellular calcium, but not by nifedipine.

These observations suggest that: (1) the extracellular calcium dependency of dbcAMP-, angiotensin II- and potassium-stimulated aldosterone secretion reflects a need for calcium influx; (2) with dbcAMP stimulation, activation of the late pathway is dependent on calcium influx by a calcium channel-independent mechanism, whereas activation of the early pathway is not dependent on extracellular calcium or calcium influx and (3) activation of both the early and late pathway by angiotensin II and potassium is dependent on calcium influx by a calcium channel-dependent mechanism. Therefore, we conclude that the mechanism of activation of the early aldosterone biosynthetic pathway by dbcAMP is different from angiotensin II or potassium and early pathway activation is distinct from that of late pathway activation with dbcAMP stimulation.

J. Endocr. (1986) 110, 315–325

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T. A. Bramley
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G. S. Menzies
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R. J. Williams
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O. S. Kinsman
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D. J. Adams
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ABSTRACT

We have described recently the presence of binding sites for human LH (hLH) and human chorionic gonadotrophin (hCG) in microsomal and cytosol fractions prepared from the dimorphic, pathogenic fungus, Candida albicans. We have now compared the properties ofCandida LH/hCG-binding sites with those of the ovine luteal LH receptor. Sheep luteal LH-binding sites were associated with luteal membranes, and little or no binding activity was present in cytosol fractions. In contrast, significant LH/hCG-binding activity was present in Candida cytosol. Moreover, there were marked differences in sensitivity to inhibition by metal ions, association and dissociation rates, and affinity constants between sheep and Candida LH-binding sites. Scatchard plots of 125I-labelled hLH binding to sheep luteal receptors demonstrated a single high-affinity component (association constant (K a) 0·3 litres/pmol) which was displaceable by hCG. In contrast, Scatchard plots of binding to Candida microsomes and cytosol fractions demonstrated two components, one with high affinity (K a 0·18 litres/pmol) and low capacity and a second site with lower affinity (K a 4 litres/nmol) and high capacity, both of which were displaceable by unlabelled hCG. Gel permeation chromatography of cytosol demonstrated two distinct peaks of LHbinding activity with approximate molecular weights of > 1 000 000 and 30 000–50 000. Scatchard plots of 125I-labelled hLH binding to the higher molecular weight peak demonstrated a single, high-affinity LH-binding site (K a 0·18 litres/pmol), whereas the lower molecular weight fraction contained both high- (K a 0·17 litres/pmol) and low-affinity (K a 4 litres/nmol) LH-binding sites.

Both partially purified and highly purified hCG and hLH preparations displaced binding of 125I-labelled hLH and hCG to sheep luteal LH receptors at similar concentrations. 125I-Labelled hLH/hCG binding to Candida membranes was also displaceable by low levels (ng) of partially purified hCG preparations, but much higher levels (μg) of highly purified hLH and hCG were required. This paradoxical observation suggested the presence of radiolabelled contaminants, or damaged forms induced during radioiodination of hormone tracers, which can bind more strongly to Candida membranes than unlabelled hCG and hLH but which do not bind to sheep LH receptors. However, no evidence for hLH tracer contaminants with differential binding to Candida and sheep luteal receptors was obtained following gel exclusion chromatography or fractionation on Concanavalin A–Sepharose. (Although three distinct 125I-labelled hLH fractions were resolved on Concanavalin A–Sepharose, presumably reflecting differences in their carbohydrate compositions, all three tracer peaks bound equivalently to both Candida membranes and ovine luteal LH receptors.) Moreover, iodination of highly purified hLH and hCG with 127I failed to generate substances with differential binding to sheep luteal and Candida LH-binding sites. Furthermore, preincubation of 125I-labelled hLH tracer with Candida membranes at different temperatures with or without protease inhibitors did not affect the ability of the hormone tracer to bind to fresh ovine luteal or Candida LH-binding sites. Thus, differences between ovine luteal and Candida LH-binding sites cannot be attributed to differential binding of contaminants present in the hormone tracer preparations, nor to (proteolytic) modification of tracer by Candida membranes during incubation.

Journal of Endocrinology (1991) 130, 177–190

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D. St.J. O'Reilly
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W. D. Fraser
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M. D. Penney
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F. C. Logue
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R. A. Cowan
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B. C. Williams
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G. Walters
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ABSTRACT

Six male volunteers were infused with arginine (0·5 g/kg body weight) over 30 min, after an overnight fast and water deprivation. There was a significant decrease in renal phosphate clearance (P<0·025) and urinary cyclic adenosine monophosphate (cAMP) output (P<0·025) during the 60- to 90-min period after the beginning of the infusion; both returned to the preinfusion basal levels within 150 min. The plasma levels of parathyroid hormone (PTH) were not affected by the infusion and remained unchanged during the subsequent 150 min. Plasma levels of arginine vasopressin (AVP) were also not significantly affected although plasma osmolality increased by 6–9 mmol/kg in all subjects. The infusion resulted in a diuresis, and a fall in urine osmolality but a decrease in free-water clearance; creatinine clearance was not affected. Six other subjects were given a bolus of 230 i.u. PTH intravenously, and 20 days later this was repeated during an infusion of arginine (0·5 g/kg body weight). There was a significant decrease in urinary phosphate (P< 0·025) and cAMP excretion (P<0·05) when PTH was given with arginine. It is suggested that arginine blocks the action of PTH on the proximal renal tubule but not that of vasopressin on the distal nephron and collecting ducts.

J. Endocr. (1986) 111, 501–506

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V. N. Anyaoku
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D. F. Wood
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R. Pavlou
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P. Williams
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K. Tan
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S. Fidler
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D. G. Johnston
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ABSTRACT

An enzyme-linked immunoadsorbent assay (ELISA) for measuring human pituitary glycoprotein free α-subunit is described. Pituitary tumours may secrete glycoprotein hormones, their free subunits (α, β) or a combination of these. Non-functioning adenomas often secrete free α-subunit. Assays for free α-subunit have previously used radioimmunoassay or immunoradiometric principles. Some of these methods are time-consuming and lack specificity and sensitivity. In order to overcome these problems, we have developed a two-site ELISA for α-subunit which uses a monoclonal antibody to α-subunit as the capture antibody to provide greater specificity. An affinity-purified polyclonal anti α-subunit conjugated to alkaline phosphatase was the signal antibody. Detection and quantification were based on phenolphthalein monophosphate conversion to phenolphthalein. The ELISA specifically measured glycoprotein free α-subunit in serum or plasma, discriminating between α-subunit and the intact glycoprotein hormones. The assay can be completed in 4 h. The assay sensitivity was 0·03 μg/l, and a normal range of 0·05 to 0·22 μg/l was established. In a retrospective study, elevated circulating glycoprotein α-subunit was detected in 22% of patients with non-functioning pituitary tumours previously treated with surgery and/or radiotherapy.

Journal of Endocrinology (1993) 136, 511–516

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Vincent Ricchiuti
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Christine G Lian
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Eveline M Oestreicher
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Loc Tran
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James R Stone Division of Endocrinology, Department of Pathology, Diabetes and Hypertension, Department of Medicine, Harvard Medical School, Brigham and Women's Hospital, 221 Longwood Avenue, Boston, Massachusetts 02115, USA

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Tham Yao
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Ellen W Seely
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Gordon H Williams
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Gail K Adler
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We tested the hypothesis that 17β-estradiol (E2) has dual effects on the heart, increasing levels of proteins thought to have beneficial cardiovascular effects (e.g. endothelial nitric oxide (NO) synthase (eNOS)) as well as those thought to have detrimental cardiovascular effects (e.g. type 1 angiotensin II (AngII) receptor (AT1R)). Ovariectomized Wistar rats consuming a high-sodium diet received one of four treatments (n=7 per group): group 1, placebo pellets; group 2, E2 (0.5 mg/pellet, 21-day release); group 3, NOS inhibitor, N ω-nitro-l-arginine-methyl-ester (l-NAME; 40 mg/kg per day for 14 days) plus Ang II (0.225 mg/kg per day on days 11–14); group 4, E2 plus l-NAME/Ang II. E2 increased cardiac levels of estrogen receptors ESR1 and ESR2, an ESR-associated membrane protein caveolin-3, eNOS, and phosphorylated (p)eNOS, thus, exerting potentially beneficial cardiovascular effects on NO. However, E2 also increased cardiac levels of proteins associated with cardiovascular injury and inflammation including, AT1R, protein kinase C delta (PRKCD), phosphorylated PRKC, and phosphorylated extracellular signal regulated kinase (pMAPK)3/1, plasminogen activator inhibitor-1 (PAI-1), osteopontin and ED-1, a monocyte/macrophage-specific protein. E2 treatment led to similar protein changes in the hearts of l-NAME/Ang II-treated rats except that the increase in peNOS was prevented, and l-NAME/Ang II and E2 had additive effects in increasing cardiac PRKCD and PAI-1. Thus, the highest levels of cardiac PAI-1 and PRKCD occurred in l-NAME/Ang II-treated rats receiving E2. In summary, E2 treatment increased cardiac expression of AT1R as well as the expression of pro-inflammatory and prothrombotic factors.

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