SUMMARY

Thyroidal uptake and release of 131I were used to study the response of pituitary autografts to locally infused synthetic thyrotrophin releasing factor (TRF) and porcine median eminence extract (MEE). Thyroidal release rates of 14% ¹³¹I/24h were maintained during continuous infusion of grafts with MEE (equivalent to 4 hypothalami/day) while 12·6% ¹³¹I/24 h was lost from thyroids of rats whose pituitary autografts were infused with 1000 ng synthetic TRF/day. Infusion of autografts with 5, 50 and 250 ng synthetic TRF/day resulted in lower, but dose-dependent, thyroidal responses suggesting that synthetic TRF could induce thyrotrophin (TSH) synthesis in the grafts.

At all doses of TRF, thyroidal ¹³¹I release was significantly increased by daily injection of cortisone (2 mg s.c.).

Local TRF stimulation of TSH release from pituitary grafts significantly increased the dose of thyroxine (T₄) needed to decrease thyroid activity, and while 1·0 μg T₄/100 g depressed activity in rats receiving saline infusions, 1·7–2·7 μg T₄/100 g was needed to inhibit ¹³¹I release in rats receiving 250 ng TRF/day, and 3 μg T₄/100 g was needed by rats receiving 1000 ng TRF/day.

Continuous TSH infusion (25–28 mu./day) into hypophysectomized rats induced thyroidal ¹³¹I release at rates similar to those in rats with TRF-stimulated pituitary autografts.

It is suggested that while synthetic TRF can enhance TSH synthesis in the pituitary its effects on TSH synthesis may normally be potentiated by other humoral substances.

SUMMARY

Thyroidal uptake and release of ¹³¹I were used to study the effects of prolonged infusion of porcine hypothalamic extract on the ability of rat pituitary autografts to secrete thyrotrophin (TSH). The extract used contained insufficient TSH to induce release of thyroidal ¹³¹I in hypophysectomized rats.

After infusion for 10 days, the time of maximal uptake of ¹³¹I and onset of ¹³¹I release was significantly shortened by infusion of hypothalamic, but not cerebral cortical extract, when compared with non-infused autografted controls. The rate of release of thyroidal ¹³¹I was significantly increased by the infusion of hypothalamic extract so that by 96–120 h after the administration of ¹³¹I the rate of release was not significantly different from that in intact controls.

Accelerated thyroidal release of ¹³¹I began 42–48 h after the application of hypothalamic extracts to pituitary autografts and fell rapidly after withdrawal of the extract. At the end of 14–17 days of infusion sections of the autografts contained aldehyde-fuchsin positive staining basophils.

Abstract

Although GnRH is believed to be the primary secretagogue for LH, oxytocin has also been shown to stimulate LH release from the anterior pituitary. We investigated the possibility that the two secretagogues interact in the modulation of LH release. Anterior pituitaries were removed from adult female rats at pro-oestrus, and tissue pieces were pre-incubated in oxytocin for 3 h prior to being stimulated with 15 min pulses of GnRH. LH output over the 1 h period from the beginning of the GnRH pulse was determined. Control incubations were carried out in the absence of oxytocin, and background secretory activities without GnRH stimulation were also determined.

Tissue which was pre-exposed to oxytocin (0·012–1·25 μm) had an increased LH response to GnRH (1·25 nm). The increase was larger than the sum of the LH outputs obtained with oxytocin and GnRH separately, revealing that oxytocin synergistically enhanced LH secretion elicited by GnRH (P<0·05; ANOVA). If stimulation by GnRH was delayed for 2 h after incubation with oxytocin, an increase in LH secretion was still observed, indicating that oxytocin-induced modulation did not rapidly disappear. Oxytocin pre-incubation was observed to result in an increase of maximal GnRH-induced LH output (P<0·001; t-test), as well as an increase of intermediate responses.

The LH response of the anterior pituitary to subsequent pulses of GnRH was modified by the self-priming process. The effect of oxytocin pretreatment on the response of primed tissue to GnRH was also investigated. It was found that pre-incubation in oxytocin also enhanced the LH response of primed tissue to GnRH.

The study has revealed that oxytocin increases the LH output of anterior pituitary tissue in response to GnRH. The effect occurs on both GnRH-primed and unprimed tissues. The results suggest that oxytocin has the potential to regulate the dynamics of the pro-oestrous LH surge.

Journal of Endocrinology (1995) 145, 113–119

ABSTRACT

Human genital skin fibroblasts were used to study aromatase activity by analysing the [³H]H₂O released as [1β-³H]androstenedione is converted to oestrone. 4-Hydroxyandrostenedione was shown to be a competitive inhibitor of this aromatase activity, the concentration of inhibitor producing 50% inhibition being 1·29 nmol/l. Dexamethasone stimulated the enzyme complex in a dose-dependent manner with half-maximal stimulation at 11·5 nmol/l. A peak of induction occurred after 16 h of preincubation. Measurement of aromatase activity in normal cell strains provided a normal range for the Michaelis–Menten constant (K _m) and the maximum velocity (V _max) of 6·72±0·54 nmol/l and 215·3±33·9 fmol/mg protein per h (means ± s.e.m., n = 20) respectively. K _m values obtained for partial and complete androgen insensitivity syndrome (PAIS and CAIS) patient cell strains were all within the normal range. The mean V_max±s.e.m. in cell strains from patients with PAIS (n = 13) and CAIS (n = 11) were 127·4±19·2 and 54·8±19·3 fmol/mg protein per h respectively. V _max values for patients with CAIS were significantly (P < 0·001) lower than normal subjects. This suggests that aromatase expression in genital skin fibroblasts is androgen-dependent.

Journal of Endocrinology (1990) 127, 177–183

The release of prolactin may be related to that of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (Bern & Nicoll, 1968). The present study was designed to determine whether there is any relationship between the short-term variation in blood LH and FSH levels (Naftolin, Yen & Tsai, 1972; Nankin & Troen, 1972), and that of prolactin levels in the blood.

A total of 230 blood samples were obtained from five normal men (age 24–29) at 15-min intervals between 09.00 and 16.30 h. All subjects continued their normal daily routine during the experiment. Samples were collected through an indwelling Polythene cannula, and the plasma was separated and stored at −20 °C. Prolactin levels were determined by radioimmunoassay (McNeilly, 1973). The results are expressed as ng standard human pituitary prolactin (kindly provided by Dr H. Friesen)/ml. Serum LH and FSH were measured by double-antibody radioimmunoassay as described by McNeilly & Chard (1974).

Abstract

Isoflavonoids and lignans, constituents of many plant foods, have been proposed as protective agents in those populations with a low incidence of hormone-dependent cancers. They may act by their inhibition of the metabolism of growth-promoting steroid hormones. This report describes the inhibition of 5α-reductase and 17β-hydroxysteroid dehydrogenase by six isoflavonoids and two lignans in human genital skin fibroblast monolayers and homogenates, and in benign prostatic hyperplasia tissue homogenates. In genital skin fibroblasts, genistein, biochanin A and equol were the most potent inhibitors of 5α-reductase activity, each resulting in greater than 80% inhibition at a concentration of 100 μm. The IC₅₀ values for genistein and a seven-compound mixture were approximately 35 μm and 20 μm (2·9 μm of each compound) respectively. Of the lignans, enterolactone was the most potent inhibitor. Inhibition by biochanin A was shown to be reversible. When genital skin fibroblast homogenates were used, biochanin A was found to inhibit 5α-reductase isozymes 1 and 2 to differing extents (30% and 75% respectively). Genistein was shown to inhibit 5α-reductase 2 in a non-competitive nature (V^max and K^m values without and with inhibitor were 30 and 20 pmol/mg protein per h and 177 and 170 nm respectively). All of the compounds tested inhibited 17β-hydroxysteroid dehydrogenase activity in genital skin fibroblast monolayers. When prostate tissue homogenates were used, the compounds tested were better inhibitors of 5α-reductase 1 than 2. It is possible that a life-long dietary exposure to these lignans and isoflavonoids may have a significant influence on the development of hormone-dependent tumours.

Journal of Endocrinology (1995) 147, 295–302

Abstract

Perifused equine anterior pituitary cells were used to investigate the relationships between the secretion of ACTH and substances known to either stimulate (corticotrophin-releasing hormone (CRH), and arginine vasopressin (AVP)) or inhibit (cortisol) ACTH secretion. The experiments were designed to mimic the hormone milieu present in vivo in the horse, with cortisol (0 or 100 nmol/l) and CRH (0 or 0·02 nmol/l) perifused continuously, and pulses of AVP (10 nmol/l) applied for 5 min at 30-min intervals.

In columns perifused with 0·02 nmol CRH/1 there was no significant overall effect of 100 nmol cortisol/l on the ACTH responses to pulses of AVP, although there was a significant interaction between AVP pulse number and cortisol showing that ACTH total area (pmol ACTH proportional to area under response curve) in response to AVP pulses 1 and 2 was significantly (P<0·05) decreased in columns perifused with 100 nmol cortisol/l. However ACTH incremental area (pmol ACTH proportional to the area above the CRH-induced baseline) was not affected by cortisol at any AVP pulse.

This contrasts with the effect of cortisol in columns perifused with 0 nmol CRH/l, where 100 nmol cortisol/l significantly decreased ACTH total area (P=0·0075) and incremental area (P=0·049) at all AVP pulses compared with the responses in columns receiving 0 nmol cortisol/l.

There was a fall off in ACTH responsiveness with time during the experiment which, in the presence of 0·02 nmol CRH/1, was significantly (P<0·001) greater with 0 nmol cortisol/l than with 100 nmol cortisol/l and if 6 (rather than 3) pulses of AVP were given, whereas with 0 nmol CRH/l there was no difference in the fall off with time between columns receiving 0 and 100 nmol cortisol/l.

These results show that the control of ACTH secretion is influenced not only by independent action of secretagogues such as CRH and AVP, or inhibitors such as cortisol, but by a complex interaction of these factors with one another. CRH may have a role in 'protecting' the ACTH response to pulses of AVP in the presence of cortisol. It follows that, in vivo, 'background' CRH could allow an increase in ACTH in response to AVP released by a new stress, despite the presence of elevated cortisol.

Journal of Endocrinology (1996) 148, 475–483

ABSTRACT

The nature of circulating pro-opiomelanocortin (POMC)-related peptides was investigated in patients with a diagnosis of septic shock. Also, changes following administration of methylprednisolone were monitored using established chromatographic techniques and three radioimmunoassays directed towards the N-terminal, mid-portion and C-terminal regions of the precursor.

Adrenocorticotrophin and β-endorphin-like peptides were identified in the circulation. By 60 min after a pharmacological dose of methylprednisolone (30 mg/kg) concentrations of these peptides were reduced and continued to fall up to 180 min. β-Lipotrophin and N-terminal POMC(1–76) were also detected by the β-endorphin and γ₃-MSH assays respectively. The concentrations of these peptides were noticeably reduced only after 180 min.

The 31 000 Da MSH/ACTH/β-endorphin (POMC) and the 22 000 Da MSH/ACTH precursors were also present in the circulation in septic shock and their concentrations increased following steroid treatment.

J. Endocr. (1988) 119, 159–165

Abstract

Antagonists are useful for probing hormone action and receptor characteristics. In this study we have investigated the inhibitory effects of analogues of arginine vasopressin (AVP) and corticotrophin-releasing hormone (CRH) on stimulated release of immunoreactive ACTH from perifused equine anterior pituitary cells in vitro. Our aims were to gain some insight into the characteristics of the CRH and AVP receptors of the horse pituitary and to establish whether the response induced by AVP and CRH together could be blocked by combining antagonists. Experimental design included 5-min pulses of AVP (12·5 nmol/l), CRH (0·3 nmol/l) or CRH plus AVP given every 40 min alternately with pulses of secretagogue(s) plus appropriate antagonist(s). The effect of combined antagonists on the response to lower secretagogue concentrations (CRH, 0·03 nmol/l plus AVP, 2·5 nmol/l) was also tested. Response in the presence of an antagonist was compared with the mean response to secretagogue in the immediately preceding and following pulse and was expressed as per cent expected ACTH.

The ACTH response to AVP was inhibited over the dose range 0·4–50 μmol/l by Phaa-d-Tyr(Et)²Lys⁶Arg³VP (P<0·002; ANOVA) and by d(CH₂)₅[Tyr(Me)²]AVP (P<0·001). Suppression of the expected ACTH response to AVP by these two antagonists was most effectively achieved by antagonist concentrations of 10 μmol/l (to 28±2·1%) and 25 μmol/l (to 22±5·1%) respectively. Inhibition was not improved by preinfusion compared with a bolus pulse. Aaa-d-Tyr(Et)²Val⁴Abu⁶Arg^8·9VP and the non-peptide antagonist OPC-21268 had no inhibitory effect. Two α-helical (α-h) analogues of CRH, (α-hCRH(12–41) and α-hCRH(9–41) tested over the dose range 0·5–5 μmol/l, suppressed CRH-induced ACTH secretion (P<0·001) but CRH(23–41) had no significant effect. The α-hCRH(12–41) achieved greater suppression of ACTH release than the (9–41) derivative (8·7±4·2% compared with 19·3±3·5% of the expected ACTH response). Combination of d(CH₂)₅[Tyr(Me)²]AVP (25 μmol/l) plus α-hCRH (12–41) (5·0 μmol/l) achieved suppression to −0·5±1·3% and 0·8±1·5% of the expected response to CRH+AVP at 0·3+12·5 nmol/l and 0·03+2·5 nmol/l respectively. These effects were greater than seen by the individual antagonists alone.

The antagonist effects suggest that the CRH and AVP receptors of the equine pituitary have similar properties to those from other species and are consistent with the pituitary AVP receptor being unlike the V₂ receptor and resembling but not being identical to the V₁ type. We also conclude that α-hCRH(12–41) and d(CH₂)₅[Tyr(Me)²]AVP can together block the ACTH response to CRH plus AVP and suggest that these antagonists should provide a means of investigating additional secretagogues involved in ACTH release in the horse.

Journal of Endocrinology (1994) 143, 85–93

Oestrogen inhibits bone resorption, at least in part, by regulating the production of several cytokines, including interleukin-6 (IL-6), IL-1, receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) by cells of the osteoblastic lineage. The selective oestrogen receptor modulator raloxifene (RAL) acts on bone in a similar manner to oestrogen, although the mechanisms of action of RAL on osteoblasts still remain unclear. We investigated and compared the effects of 17-beta oestradiol (E(2)) and RAL on the regulation of IL-6, IL-1, RANKL and OPG in vitro in primary human osteoblastic (HOB) cells and in an immortalised clonal human bone marrow stromal cell line (HCC1) with osteoblastic characteristics. We tested E(2) and RAL at concentrations ranging from 10(-12) to 10(-6) M. IL-6, IL-1alpha and IL-1beta, OPG and RANKL were measured by ELISA. RANKL and OPG mRNA steady state level was assessed by quantitative PCR analysis. Both E(2) and RAL led to a significant reduction in IL-6 production in the HOB cells, although the effect was more marked with E(2) (P<0.05). IL-1alpha and IL-1beta also decreased significantly following treatment with E(2) and RAL in the HCC1 cells (E(2) (10(-8), 10(-7) and 10(-6) M), % reduction (means+/-S.E.M.) compared with vehicle-treated cells - IL-1alpha: 84+/-7.4, 70.8+/-2.9*, 78.2+/-4.8*; IL-1beta: 79+/-10, 72.8+/-8.2*, 66.6+/-2.8*; RAL (10(-8), 10(-7) and 10(-6) M) - IL-1alpha: 72.4+/-5*, 79+/- 5.2*, 102+/-7.7; IL-1beta: 67.9+/-3.2*, 69+/-2.5*, 73.8+/- 6.2*; *P<0.05). OPG protein concentration decreased significantly in a dose-dependent manner following treatment with E(2) and RAL (% reduction E(2) (10(-8), 10(-7) and 10(-6) M) - HOB: 72.5+/-8.4*, 80+/-6.7*, 62.8+/-8.9*; HCC1: 109+/-4, 98.8+/-6, 54.5+/-3.4*; RAL (10(-8), 10(-7) and 10(-6) M) - HOB: 81.5+/-5.5*, 62.7+/-7.4*, 55.2+/-10.9*; HCC1: 92.7+/-7.4, 67+/-12.2*, 39+/-4.5*; *P<0.05). In the HCC1 cells, RANKL protein did not change significantly following E(2). In contrast, a significant reduction in RANKL was seen with RAL at 10(-7) and 10(-6) M (66+/-6.4% and 74+/-3% respectively). There was no change in OPG mRNA expression following E(2) or RAL in the HCC1 cells, although in the HOB cells we observed a significant reduction in OPG mRNA. RANKL mRNA decreased significantly in the HCC1 cells following RAL (10(-8), 10(-7)and 10(-6) M) treatment (% change from controls: 52+/-2*, 62+/-1*, 53+/-5.8*; *P<0.05). Similar results were seen in the HOB cells with RAL at 10(-6) M (RANKL mRNA: 72+/-5.5, P<0.05). In addition, there was a significant decrease in the RANKL/OPG ratio after RAL at 10(-6) M (HOB: 65.6+/-5*, HCC1: 56.9+/-20*; *P<0.05). RANKL/OPG ratio did not change significantly in the HCC1 cells following E(2). However, in contrast to RAL, we observed an increase in the RANKL/OPG ratio in the HOB cells following treatment with E(2). In conclusion, the study shows that RAL and E(2) have divergent cell-specific effects on the regulation of cytokines. The data also suggest that, in contrast to E(2), RAL may exert its anti-resorptive actions, at least in part, via the RANKL/OPG pathway. Further in vivo studies are required to confirm this.