SUMMARY

Cholesterol and cholesterol esters have been identified on the basis of thin-layer and gas—liquid chromatographic data and fluorimetric characteristics as major interfering fluorogens in a simple fluorimetric method for determining plasma corticosteroids.

In a small series of normal subjects the average concentration of fluorogen, which was not 11-hydroxysteroid, was 3·4 μg. apparent cortisol/100 ml. plasma, equivalent to 22·4% of the total fluorogen present.

Queen Elizabeth Hospital, Birmingham, B15 2TH, and *Department of Clinical Endocrinology, Women's Hospital, Birmingham, BU 4HL

(Received 19 January 1976)

Administration of the natural or synthetic decapeptide known as luteinizing hormone/follicle-stimulating hormone releasing hormone (LH-RH) causes prompt release of both luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The decapeptide has a short half-life of only a few minutes, so that its use as a therapeutic agent in the treatment of infertility is limited. Attempts have been made therefore to synthesize more potent and longer-acting analogues. Many of these have only weak activity in experimental animals, but some, notably those with modifications to structure at residues 5, 6 and 10, are more effective (Arimura, Vilchez-Martinez, Coy, Coy, Hirotsu & Schally, 1974; Kastin, Arimura, Gonzalez-Barcena, Coy, Clinton-Miller, Nishi, Lee, Duron-Huerta, Schalch & Schally, 1974a; Kastin, Schally, Gonzalez-Barcena, Coy, Clinton-Miller, Porias & Schalch, 1974b; Vilchez-Martinez, Coy, Arimura, Coy, Hirotsu & Schally, 1974; Zañartu,

ABSTRACT

The possible involvement of the lipoxygenase pathway of arachidonic acid metabolism in the events which take place during ACTH-induced stimulation of corticosterone secretion has been studied using an isolated rat adrenal cell system. Incubation with arachidonic acid resulted in an inhibition of ACTH-stimulated corticosterone production. The lipoxygenase pathway inhibitors nordihydroguaretic acid (NDGA), eicosatetraynoic acid (ETYA) and compound BW755C also produced inhibition of ACTH-stimulated corticosterone synthesis. The concentrations of the inhibitors at which 50% inhibition occurred were 15, 34 and 37 μmol/l respectively. The inhibitions produced by NDGA and ETYA were independent of cyclic AMP output. NDGA also inhibited corticosterone production induced by dibutyryl cyclic AMP but had no effect on corticosterone synthesis induced by pregnenolone.

Preincubation of adrenal cells with the lipoxygenase products 5, 12 and 15 hydroxyeicosatetraenoic acid (HETE) and with leukotrienes A₄, B₄, C₄, D₄ and E₄ resulted in significant inhibitions of corticosterone production in response to ACTH with leukotriene A₄ (LTA₄) and with 15HETE and 5HETE. Conversely, incubation with glutathione (GSH), which is known to reduce intracellular LTA₄ levels, produced stimulation (at 5 mmol GSH/1) and inhibition (at 50 mmol GSH/1) of corticosterone output. These studies suggest that the lipoxygenase pathway may be involved in ACTH-stimulated corticosterone synthesis.

J. Endocr. (1987) 112, 253–258

Abstract

Many studies have consistently shown that castration induces a prompt increase in serum levels and pituitary content of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as a concomitant rise in steady state levels of the messenger RNAs directing their synthesis. The reports of effects of castration on the overall physiology of hypothalamic luteinizing hormone-releasing hormone (LHRH) — steady state levels of LHRH mRNA, post-translational processing and secretion — have, however, not been consistent.

The goal of the studies reported here was to provide the first analysis of the effect of castration, at multiple post-operative time points, on steady state levels of LHRH mRNA and on the levels of hypothalamic proLHRH. All these data are correlated with hypothalamic levels of the mature LHRH decapeptide and with serum and pituitary levels of immunoreactive LH and FSH.

Adult male rats were either castrated or sham-castrated (controls) and then sacrificed at 1, 3, 5, 7, 14, 21 or 28 days postoperatively. As expected, there was a prompt and sustained rise in serum immunoreactive LH and FSH in castrates compared with sham-operated animals. Intrapituitary LH levels rose above levels in the sham-operated animals by 14 days post castration. Intra-pituitary FSH showed a biphasic response, first falling significantly below control levels, then rising above control levels at 21 days.

Steady state levels of LHRH mRNA in castrates, measured by reverse transcription/polymerase chain reaction, were increased about 2-fold above control levels by 1 day postoperatively, but were virtually identical to control levels at each of the other time points despite marked changes in the gonadotropins.

ProLHRH content in castrates was 1·8-times that seen in controls at 1 day post castration (P<0·05), concomitant with the rise in steady state levels of LHRH mRNA at that time point. However, proLHRH content in castrates was no different from that seen in controls at each of the later time points examined. LHRH content was unchanged through 7 days after castration, but then fell significantly to 57% of control levels in hypothalami from animals gonadectomized 14 to 21 days previously (P<0·001 vs control), and to 54% of sham-operated levels at 28 days postoperatively (P<0·001).

We conclude that: (1) changes in steady state levels of LHRH mRNA after castration are small and transient and (2) increased proLHRH coupled with unchanged LHRH levels at 1 day post castration, and castrate animal pro-LHRH at control levels coupled with falling LHRH at later post-castration time points indicate that the effect of gonadectomy on post-translational processing of pro-LHRH to LHRH is, likewise, small and transient.

In aggregate our data suggest that most of the increase in serum LH and FSH seen in male rats after castration is not mediated at the hypothalamic level.

Journal of Endocrinology (1996) 148, 509–515

SUMMARY

Renal iodide clearance in rats was reduced rapidly when the animals were fed a protein-depletion diet. If the iodine content of the protein-depletion diet is high, this results in an increase in the serum iodide levels to concentrations in excess of 150 μg./100 ml. Protein-bound iodine was elevated due to the accumulation of iodinated serum albumin. Thyroid hormonal iodoamino acid content was transiently depressed, presumably by the mechanism described by Wolff & Chaikoff (1948). Thyroxine metabolism was not affected except for a change in the partition of thyroxine between liver and serum. There was no evidence for pituitary involvement in the effects of protein depletion on thyroid function.

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

ABSTRACT

When bovine adrenocortical cells from the zona fasciculata/reticularis (zfr) are maintained in primary culture, cortisol secretion in response to acute stimulation with ACTH and adrenaline (which activate adenylate cyclase) is seen to increase steadily over the first 48 h, while secretion in response to angiotensin II and acetylcholine (which activate phosphoinositidase C) shows an initial decline in the first 24 h and a recovery to maximum after 48 h. We have investigated whether these discrepant changes in cortisol secretory response to the different agonists are due to changes in formation of the associated second messengers (cAMP or inositol phosphates), or altered coupling of these second messenger signals to steroid secretion.

Increases in steroid secretion in response to ACTH and adrenaline were paralleled by increased cAMP. Steroid secretion in response to exogenous 8-bromoadenosine 3′:5′-cyclic monophosphate also increased steadily during this 48-h period. Thus increased responsiveness was due to both increased second messenger formation and increased coupling to the steroid secretory response.

The decreased steroid secretory response to angiotensin and acetylcholine after 24 h, and subsequent recovery after 48 h in culture, were accompanied by an increased formation of phosphoinositols after 24 h and a further increase by 48 h. However, the steroid secretory response to a combination of calcium ionophore and the protein kinase C activator, phorbol 12-myristate 13-acetate, was reduced after 24 h and recovered by 48 h of culture. Fura-2-loaded cells also showed an increase in intracellular [Ca²⁺] after 24 h in culture. Thus the impaired steroid secretory response to angiotensin II and acetylcholine after 24 h of culture was not due to reduced formation of second messengers but to a failure of Ca²⁺ and diacylglycerol so formed to activate the steroid secretory process.

Reversible uncoupling of the steroid secretory response from the Ca²⁺- and diacylglycerol-based but not the cAMP-based second messengers observed in bovine zfr cells suggests that differential control of steroid secretion and other cell functions may be possible in vivo for activators of phosphoinositidase C, and may explain apparently discrepant results from studies on other in-vitro adrenocortical cell preparations.

Journal of Endocrinology (1992) 133, 21–28

Abstract

Jurkat cells were used to study the immunomodulatory role of luteinizing hormone-releasing hormone (LHRH) in immune cells. The Jurkat cell, a human mature leukemic cell line, phenotypically resembles resting human T lymphocytes and has been widely used to study T cell physiology. The data from this study demonstrate that the Jurkat cell concentration of immunoreactive LHRH was 210 ± 36 pg/10⁶ cells and that of proLHRH was 188 ± 27 pg/10⁶ cells (means ± s.e.m.). The authenticity of this LHRH immunoreactivity is documented in two ways. First, both Jurkat LHRH and proLHRH immunoreactivity demonstrate dilutional parallelism with hypothalamic LHRH and proLHRH. Second, Jurkat lysates show LHRH bioactivity by releasing luteinizing hormone from rat anterior pituitary cells in culture. The presence of substantial amounts of LHRH in medium in which Jurkat cells were cultured for 72 h indicated that LHRH can be released from the cells. Using specific primers to exons 2 and 4 of the LHRH gene, we have found that Jurkat cells (like human T cells) express LHRH mRNA.

The LHRH agonist, des-Gly¹⁰,d-Trp⁶-LHRH ethylamide, significantly increases the proliferative activity of Jurkat cells, as assessed by tritiated thymidine incorporation, from 15 980 ± 1491 c.p.m. in controls to 28 934 ± 3395, 30 457 ± 3861 (P=0·05 vs control) or 35 299 ± 5586 c.p.m. (P<0·01 vs control) with 10⁻¹¹, 10⁻⁹ or 10⁻⁷ m agonist respectively. LHRH antagonist, [d-pGlu¹,d-Phe²,d-Trp^3,6]-LHRH, at a concentration of 10⁻⁸ m decreases Jurkat cell proliferative activity from 17 145 ± 526 c.p.m. in control medium to 10 653 ± 1323 c.p.m. (P=0·05). Co-incubation with the LHRH antagonist completely inhibits the proliferative stimulation induced by the LHRH agonist. Furthermore, applying monoclonal LHRH antibody to Jurkat cells inhibits the cell proliferative activity assessed by tritiated thymidine incorporation from 19 900 ± 2675 c.p.m. in controls to 15 680 ± 2254, 15 792 ± 1854 and 9700 ± 908 c.p.m. in media with 1:40, 1:20 and 1:10 dilution of purified antibody respectively (P<0·01, 1:10 dilution compared with control). In addition, the cAMP level in LHRH-stimulated Jurkat cells is decreased to 74, 27 and 57% of control levels after 15, 30 and 45 min respectively of exposure to 10⁻⁷ m LHRH agonist.

In summary, Jurkat cells produce, process and release immunoreactive and bioactive LHRH, as do normal human T cells. Endogenous and exogenous LHRH increase Jurkat cell proliferative activity, and cAMP may be involved in LHRH-induced Jurkat cell proliferation. The Jurkat cell may be a useful model with which to study the role of LHRH in human T cell function.

Journal of Endocrinology (1997) 153, 241–24