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V. P. MICHELANGELI
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N. H. HUNT
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T. J. MARTIN
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SUMMARY

Several aspects of the activation of adenylate cyclase by guanosine 5′-triphosphate (GTP), 5′-guanylylimidodiphosphate (Gpp(NH)p) and bovine parathyroid hormone (bPTH) have been studied in chick kidney plasma membrane preparations. GTP (10−4 mol/l), Gpp(NH)p (10−4 mol/l) and bPTH (10 i.u./ml) activated adenylate cyclase without any significant time lag. However a 2 min delay was observed before the activity of the enzyme increased after the addition of bPTH (−6 → + 34) to incubations.

The early (0–3 min) effects of GTP and Gpp(NH)p upon chick kidney adenylate cyclase activity were antagonized by the addition of the alternative guanyl nucleotide. After 5 min of incubation with kidney plasma membranes, Gpp(NH)p induced a stable state of activation of adenylate cyclase which was not reversible by subsequent addition of GTP. GTP did not induce an irreversible state of enzyme activation. In pre-incubation studies, GTP did not produce a persistent enzyme activation and did not modify the effect of Gpp(NH)p added subsequently at the incubation stage. Gpp(NH)p produced a stable state of activation of adenylate cyclase which was not inhibited by addition of GTP at the incubation stage.

Bovine PTH (2–34) inhibited the effect of bPTH upon adenylate cyclase activity when the native hormone (10 i.u./ml) had been incubated with plasma membranes for up to 8 min before addition of the analogue (5 μg/ml). Incubation of plasma membranes with bPTH (2–34) for as little as 10 s prevented activation of adenylate cyclase by subsequent addition of bPTH. This pattern was confirmed in pre-incubation studies. After pre-incubation of kidney membranes with bPTH and bPTH (2–34), followed by washing, an acid extract of the membranes contained immunoreactive bPTH. Gpp(NH)p produced a greater increase in adenylate cyclase activity in membranes pre-incubated with bPTH or bPTH (2–34) than in membranes pre-incubated with buffer alone, suggesting that the hormone and analogue facilitated the interaction of Gpp(NH)p with adenylate cyclase.

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A. E. ARGÜELLES
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H. MARTÍN
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M. CHEKHERDEMIAN
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I. MOLOCZNIK
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Metyrapone administered orally (Liddle, Estep, Kendall, Williams & Townes, 1959) or intravenously (Gold, Di Raimondo & Forsham, 1960) has been widely used as a test of corticotrophin (ACTH) reserve. Although both modes of administration are thought to be equally satisfactory (Gold, Kent & Forsham, 1961) a comparison in the same subjects has not been reported.

The subjects (10 men and 4 women) received first an infusion of 30 mg. metyrapone/kg. body weight (Metopirone, Ciba) in 0·9 % NaCl solution at the rate of 125 ml./hr. for 4 hr. starting between 8.00 and 9.00 hr. Urine collections were started at the same time. The oral test was performed 5–7 days later, eight doses of two 250 mg. capsules being taken every 2–3 hr. Total 17-oxogenic steroid (17-OGS) excretion was estimated by the method of Few (1961).

The results are shown in Table 1. After i.v. administration of metyrapone the rise in

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O. F. X. Almeida
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A. H. S. Hassan
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K. E. Nikolarakis
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G. B. Martin
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ABSTRACT

It was found in previous studies that the neurotransmitter control of the secretion of LHRH and LH differs between long-term castrated and ovariectomized rats. One interpretation of these data was that there was a reduced 'positive drive' in the male, and the question was raised 'how do the gonadotrophs of long-term castrated rats maintain a high level of LH secretion?'. In the present series of experiments, evidence for a reduced dependence of the gonadotrophs upon LHRH stimulation is provided. Although sensitivity to native LHRH was not completely lost in long-term castrated rats, two potent LHRH antagonists (d-pyroglu1,d-Phe2,d-Trp3,6)-LHRH and (N-acetyl-3,4-dehydro-Pro,p-fluoro-d-Phe2,d-Trp3,6)-LHRH, were found to inhibit LH secretion in short-term castrated and long-term ovariectomized rats, but not in long-term castrated rats. Neither blockade of axonal transport with colchicine nor immunoneutralization of LHRH with an antiserum against LHRH (both administered 48 h before blood sampling) produced reductions in serum concentrations of LH in long-term castrated rats, although these treatments significantly suppressed LH levels in short-term castrated animals. Chronic (6-day) infusions of the second LHRH antagonist (up to 450 μg/day) neither reduced LH secretion nor altered the morphology of the 'castration cells' in the pituitaries of long-term castrated rats. Chronic treatment with testosterone (15 days), however, reversed these parameters to some extent, and when the testosterone treatment was coupled with chronic infusions of the LHRH antagonist, significantly lower serum levels of LH and reductions in the size of the castration cells were observed. These data thus indicate that castration cells may function autonomously, without the need for LHRH, and that testosterone in some way restores the dependency on LHRH and/or the responsiveness to LHRH of these cells.

Journal of Endocrinology (1989) 123, 263–273

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A. CRAWFORD
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N. H. HUNT
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J. K. DAWBORN
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V. P. MICHELANGELI
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T. J. MARTIN
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SUMMARY

Adenylate cyclase activity in particulate fractions from a transplantable rat osteogenic sarcoma was stimulated in a dose-dependent manner by prostaglandins E1 and E2 (PGE1 and PGE2) and parathyroid hormone (PTH). Prostaglandin F was active at a high concentration (3 × 10− 4 mol/l). Pretreatment of membranes with collagenase plus hyaluronidase reduced the magnitude of the PTH effect but did not affect the size of the PGE1 effect.

Guanosine 5′-triphosphate and its synthetic analogue 5′-guanylylimidodiphosphate (Gpp(NH)p) activated adenylate cyclase in particulate preparations from the osteogenic sarcoma. The latter agent produced much larger effects, although the concentrations required for half-maximal enzyme activation were the same for both agonists (approximately 2 × 10−6 mol/l). The effects of PTH and Gpp(NH)p were supra-additive at some concentrations of hormone. The effects of PGE1 and Gpp(NH)p were supra-additive at all hormone concentrations tested. Pre-incubation of membrane particles for 6 min with PTH produced an enzyme activation which was not reversed by dilution through washing; pre-incubation with PGE1 did not produce this effect. The response of membrane adenylate cyclase to Gpp(NH)p (10−4 mol/l) was 75% greater in preparations pre-incubated with PTH than in membranes pre-incubated in buffer alone or in buffer containing PGE1. The basal rate of cyclic AMP production in the adenylate cyclase assay system decreased over a 35 min incubation period. This decrease was prevented by addition of PTH or PGE1. Addition of NaF or Gpp(NH)p produced a steady increase in the rate of production of cyclic AMP with time.

Membrane preparations did not reduce the biological activity of PTH and did not degrade 125I-labelled PTH.

The results demonstrate that the PTH- and PGE-responsive adenylate cyclases of the osteogenic sarcoma have distinctly different properties and that particulate preparations of the tumour do not metabolize PTH.

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CHRISTINE BECK
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R. G. LARKINS
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T. J. MARTIN
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H. G. BURGER
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SUMMARY

The paradoxical plasma growth hormone (GH) responses to oral glucose in certain patients with lung cancer prompted an examination of tumour extracts for GH releasing activity. Exposure of superfused rat pituitary to pulses (30 s) of aminophylline and to extracts from rat, sheep and human hypothalami resulted in a rapid and short-lived release of immunoreactive rat GH into the medium. Fresh extracts from five lung tumours, and from the surrounding lung tissue of four of these tumours significantly stimulated the release of GH, while extracts of a metastatic chondrosarcoma and normal rat lung were inactive. Gel filtration experiments suggested that the releasing activity in rat, sheep and human hypothalamic tissue and in human lung tumour extracts was present in at least two molecular species of different sizes.

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N. H. HUNT
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T. J. MARTIN
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V. P. MICHELANGELI
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J. A. EISMAN
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SUMMARY

Both guanosine 5′-triphosphate (GTP) and 5′-guanylylimidodiphosphate (Gpp(NH)p) activated adenylate cyclase (EC 4.6.1.1) in chick kidney plasma membranes. Half-maximal stimulation occurred at 3·1 × 10−6 m for both agents. The maximum increases in adenylate cyclase activity produced by GTP and Gpp(NH)p were respectively 130 and 720% over basal activity.

At the end of a 12 min incubation period GTP concentration was 85% of that originally added in the presence of an ATP-regenerating system but less than 20% in its absence.

GTP and guanosine 5′-diphosphate inhibited the activation of adenylate cyclase by Gpp(NH)p, suggesting that they all acted at a common site. Gpp(NH)p facilitated the stimulation of adenylate cyclase activity by bovine parathyroid hormone (BPTH) and by the synthetic amino terminal fragment BPTH (1–34), decreasing the concentrations required for half-maximal enzyme activation by a factor of approximately eight in both cases. This property was not shared by the native nucleotide GTP. Gpp(NH)p rendered active (at certain concentrations) a synthetic parathyroid hormone peptide fragment, BPTH (2–34), which was incapable of activating adenylate cyclase in the absence of the nucleotide analogue. This suggested that the GTP analogue, in addition to a direct effect upon adenylate cyclase activity, was capable of influencing hormone interaction with the enzyme complex.

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C. BIDDULPH
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M. SAN MARTIN
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S. C. BECKER
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W. H. McSHAN
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SUMMARY

The succinic dehydrogenase (SDHase) and adenosinetriphosphatase (ATPase) activities were determined in the corpora lutea, ovarian interstitial tissue and endometria of pseudopregnant (PP) rabbits and rabbits in which superovulation was produced by treatment with pituitary gonadotrophins (SO). Statistical analysis of the results showed that the weight, SDHase, ATPase and percentage solids of the corpora lutea, and the SDHase of the endometria of each of the two groups varied significantly (P<0·01) between days. The day values for the SDHase of the endometria, however, were particularly high in SO rabbits at the 2-day stage and low in the 12- to 16-day stages as compared with the PP group. Other differences found were of less statistical significance.

The results are considered in relation to the poor survival of embryos formed from eggs of SO rabbits. The lesser weight of the corpora lutea and the lesser SDHase activity of the endometria of the SO group, as compared with the PP group during the latter half of the experimental period, may be significant in relation to the poorer survival of embryos.

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Junny Chan
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Elizabeth H Rabbitt
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Barbara A Innes
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Judith N Bulmer
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Paul M Stewart
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Mark D Kilby
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Martin Hewison
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Glucocorticoids play a fundamental role in the endocrinology of pregnancy but excess glucocorticoids in utero may lead to abnormalities of fetal growth. Protection against fetal exposure to cortisol is provided by the enzyme 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) located in the human placental trophoblast. By contrast, relatively little is known concerning the function of glucocorticoid-activating 11β-HSD1, which is strongly expressed within human maternal decidua. To address this we have assessed: i) changes in decidual 11β-HSD1 expression across gestation and ii) the functional role of glucocorticoids in decidua. Human decidua was collected from women undergoing surgical termination of pregnancy in first (n = 32) and second (n = 10) trimesters, and elective caesarean sections in the third trimester (n = 9). Analysis of mRNA for 11β-HSD1 by real-time RT-PCR showed increased expression in second (9.3-fold, P < 0.01) and third (210-fold, P < 0.001) trimesters. Studies using primary cultures of decidual cells also revealed higher levels of cortisol generation in the third trimester. Changes in decidual 11β-HSD1 with gestation were paralleled by increased expression of the apoptosis markers caspase-3 and annexin-V, particularly in cluster designation (CD)10−VE non-stromal cells (20-fold in third trimester relative to first trimester). Apoptosis was also readily induced in primary cultures of third trimester decidual cells when treated with cortisol, cortisone, or dexamethasone (all 100 nM for 24 h). The effect of cortisone but not cortisol or dexamethasone was blocked by an 11β-HSD inhibitor confirming the functional significance of endogenous cortisol generation. These data show that autocrine metabolism of glucocorticoids is an important facet of the feto-placental unit in late gestation and we propose that a possible effect of this is to stimulate programmed cell death in human decidua.

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Naomi Fleury Telethon Kids Institute, University of Western Australia, Perth, Australia

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Martin Feelisch Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, UK

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Prue H Hart Telethon Kids Institute, University of Western Australia, Perth, Australia

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Richard B Weller University of Edinburgh, MRC Centre for Inflammation Research, Edinburgh, Scotland

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Jordan Smoothy Telethon Kids Institute, University of Western Australia, Perth, Australia

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Vance B Matthews School of Medicine and Pharmacology – Royal Perth Hospital Unit, The University of Western Australia, Perth, Australia

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Shelley Gorman Telethon Kids Institute, University of Western Australia, Perth, Australia

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Exposure to sunlight may limit cardiometabolic risk. In our previous studies, regular exposure to sub-erythemal (non-burning) ultraviolet radiation (UVR) reduced signs of adiposity and cardiometabolic dysfunction in mice fed a high-fat diet. Some of the observed effects were dependent on skin release of nitric oxide after UVR exposure. Here, we examine the effects of sub-erythemal UVR on signs of adiposity and metabolic dysfunction in already overweight mice, comparing the effects of two sunlamps with distinct emitted light spectra. Mice were fed a high-fat diet from 8 weeks of age, with UVR administered twice a week from 14 weeks of age until they were killed at 20 weeks of age. Mice were irradiated with the same dose of UVB radiation (1 kJ/m2) from either FS40 (65% UVB, 35% UVA) or CLEO (4% UVB, 96% UVA) sunlamps, but substantially more UVA from the latter. FS40 UVR (but not CLEO UVR) significantly reduced mouse weights and weight gain, compared to mice fed a high-fat diet (only). These effects were dependent on nitric oxide. Conversely, CLEO UVR (but not FS40 UVR) significantly reduced circulating LDL cholesterol. Both light sources reduced fasting insulin levels, and the extent of hepatic steatosis; the latter was reversed by topical application of cPTIO, suggesting an important role for skin release of nitric oxide in preventing hepatic lipid accumulation. These results suggest that there may be a number of benefits achieved by regular exposure to safe (non-burning) levels of sunlight or UV-containing phototherapy, with effects potentially dependent on the predominance of the wavelengths of UVR administered.

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F. M. K. Law
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P. J. Moate
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D. D. Leaver
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H. Diefenbach-Jagger
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V. Grill
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P. W. M. Ho
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T. J. Martin
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ABSTRACT

Parathyroid hormone-related protein (PTHrP) has been shown to be present in milk of various mammals. We have assayed PTHrP in milk of various species by radioimmunoassay and estimated the molecular weights by Western blot analysis. PTHrP concentrations in bovine, ovine and human milk were 59·2±18·5, 74·1±35·0 and 36·6±20·7 μg/l (means ± s.d.) respectively, in pooled samples collected at various stages of lactation. PTHrP in mammalian milk was found to exist in two forms with molecular weights of 17·5 kDa and 21·5 kDa approximating those of PTHrP(1-108) and (1–141) respectively. In comparison, marsupial milk PTHrP appeared as a single low molecular weight form of 14·4 kDa which approximated to that of PTHrP(1–84). We performed a longitudinal study measuring the concentration of PTHrP in milk throughout lactation in cows, and found it to increase with the duration of lactation (r= 0·669, n = 91). We further examined the relationship between the concentration of PTHrP and total calcium in bovine milk, and the differences between these constituents in milk from Friesian and Jersey cows. PTHrP concentrations correlated positively with total milk calcium (r = 0·346, n = 105). The mean milk concentration of PTHrP of the Jerseys was significantly higher than that of the Friesians (52·6±5·4 μg/l compared with 41·0±4·8 μg/l, P < 0·01), as was the mean milk calcium concentration (30·5±3·0 mmol/l compared with 26·7±2·7 mmol/l, P < 0·01). We therefore postulate that production of PTHrP by the mammary gland may be associated with calcium transport from blood to milk. Also PTHrP may play a role in the development of milk fever in Jerseys which are predisposed to this condition.

Journal of Endocrinology (1991) 128, 21–26

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