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  • Author: S. M. Holland x
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S. L. JEFFCOATE, H. M. FRASER, A. GUNN and DIANE T. HOLLAND

The decapeptide luteinizing hormone releasing factor (LH-RF) has recently been isolated, sequenced and synthesized (Schally, Arimura, Kastin, Matsuo, Baba, Redding, Nair, Debeljuk & White, 1971). This has made possible the development of radioimmunoassays for this factor enabling it to be measured in biological fluids in vivo and in vitro.

Two milligrammes of the synthetic decapeptide (Hoechst) were conjugated to 2 mg bovine serum albumin (BSA) using 75 mg 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in 0·25 ml water (Goodfriend, Levine & Fasman, 1964). After dialysis overnight, 1 mg of the conjugate in 2 ml water, emulsified in Freund's complete adjuvant, was injected into 20 intradermal sites in a white New Zealand rabbit. A blood sample was obtained 8 weeks after this primary immunization and the assay developed using this antiserum.

LH-RF (0·1–1 μg) was iodinated with 125I (0·5–1 mCi) by the chloramine-T technique (5 μg of chloramine-T), specific activities between 100 and 500 μCi/μg being obtained.

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J. A. Millett, S. M. Holland, J. Alaghband-Zadeh and H. E. de Wardener

ABSTRACT

The plasma of normal man and the rat, and an acetone extract of hypothalamus from the rat, have an ability to inhibit Na-K-ATPase which is related directly to salt intake. The ability of the plasma to inhibit Na-K-ATPase is raised in essential hypertension.

The ability of plasma and of an acetone extract of hypothalamus from six spontaneously hypertensive (SHR) rats and six normotensive control (WKY) rats to inhibit Na-K-ATPase of fresh guinea-pig kidney was studied using cytochemical bioassay techniques. With a validated assay, which measures the capacity of biological samples to stimulate glucose-6-phosphate dehydrogenase (G6PD) as an index of their capacity to inhibit Na-K-ATPase, the mean G6PD-stimulating ability of the plasma from the SHR and the WKY rat was 772·3 ± 48·1 units/ml and 12·5 ± 2·6 units/ml respectively (P < 0·01) and of the hypothalamic extracts it was 2·2 ± 1·7 × 108 and 4·5 ± 1·8 × 104 units/hypothalamus (P < 0·01). With a semi-quantitative cytochemical assay, which measures Na-K-ATPase activity directly, plasma and an acetone extract of hypothalamus from the spontaneously hypertensive rat had much greater capacities to inhibit Na-K-ATPase than plasma and extract from the WKY rat.

These raised levels of Na-K-ATPase inhibitory activity in the plasma of the SHR rat are similar to the highest values found in the plasma of patients with essential hypertension. The results suggest that the substance responsible for the increased capacity of the plasma to inhibit Na-K-ATPase may originate from the hypothalamus and that it may, in part, be involved in the mechanisms which induce the rise of arterial pressure in inherited forms of hypertension.

J. Endocr. (1986) 108, 69–73

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H. M. FRASER, A. GUNN, S. L. JEFFCOATE and DIANE T. HOLLAND

SUMMARY

Autoimmunity to luteinizing hormone releasing hormone (LH-RH) in adult male rats, induced by immunization with LH-RH conjugated to bovine serum albumin, resulted in atrophy of the testes and secondary sex organs and aspermatogenesis. Both immunoreactive luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in serum and the pituitary were reduced to low levels compared with those of control animals. It is suggested that antibodies to LH-RH can inhibit the action of endogenous hormone and that LH-RH is, in fact, the gonadotrophin-releasing hormone in the rat, required for the release of both LH and FSH.

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H. M. FRASER, S. L. JEFFCOATE, DIANE T. HOLLAND and A. GUNN

The purpose of this study was to determine if luteinizing hormone releasing hormone (LH-RH) could be detected in the peripheral blood of the rat on the afternoon of pro-oestrus by the radioimmunoassay described elsewhere (Jeffcoate, Fraser, Gunn & Holland, 1973a; Jeffcoate, Fraser, Holland & Gunn, 1973b).

Female Sprague—Dawley rats, weighing 220–270 g, were maintained in a room illuminated from 05.00 to 19.00 h. The animals were observed for at least two consecutive 4-day oestrous cycles as judged by daily vaginal smears. At various times on the afternoon of pro-oestrus the animals were lightly anaesthetized with ether and rapidly desanguinated by cardiac puncture. Plasma luteinizing hormone (LH) was determined using the radioimmunoassay described by Daane & Parlow (1971) with minor modifications. This utilized purified rat LH for radio-iodination (NIAMD-Rat LH-1–3), NIAMD-Anti-Rat LH serum-1 and a rat LH preparation (NIAMD-Rat LH-RP-1) was used as standard. Serum (1–2 ml) samples were

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H. M. FRASER, S. L. JEFFCOATE, A. GUNN and D. T. HOLLAND

*Department of Surgery, University of Dundee, Dundee, DD1 4HN and †Department of Chemical Pathology, St Thomas's Hospital, London, SE1 7EH

(Received 23 August 1974)

Short-term inhibition of luteinizing hormone releasing hormone (LH-RH) in vivo can be conveniently studied by injection of antisera (Fraser & Gunn, 1973), and in the ovariectomized rat this was found to reduce both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels when measured 7 and 24 h after injection (Koch, Chobsieng, Zor, Fridkin & Lindner, 1973). Long-term inhibition of LH-RH cannot be satisfactorily achieved by injection of antisera but is induced by active immunization. In a previous study we have shown in the male rat that this results in low levels of LH and FSH in serum and pituitary, suggesting that LH-RH is required for synthesis as well as release of both LH and FSH (Fraser, Gunn, Jeffcoate & Holland, 1974). In this study, the work

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J. A. Millett, S. M. Holland, J. Alaghband-Zadeh and H. E. de Wardener

ABSTRACT

Some physicochemical properties of partially purified hypothalamic material from the spontaneously hypertensive rat, and of plasma from man and the rat, have been characterized using a validated cytochemical bioassay which measures the ability of biological fluids to stimulate fresh guinea-pig kidney glucose-6-phosphate dehydrogenase (G6PD) after 2 min of exposure to the test substance, as an indication of their ability to inhibit Na+/K+ adenosine triphosphatase (Na+/K+-ATPase) after 4–6 min of exposure.

The G6PD-stimulating activity of both hypothalamic extract and plasma is soluble in water and insoluble in chloroform. During electrophoresis the activity from both sites appears in the same fractions and travels considerably further than lysine. After high-pressure liquid chromatography the activity of hypothalamic extract appears in a discreet fraction which does not absorb u.v. light. The activity of both the hypothalamic extract and plasma survives boiling and acid hydrolysis, but is substantially inhibited by prior incubation with digoxin antibody. From ultrafiltration studies, the substance responsible for the ability to stimulate G6PD appears to have a molecular weight of less than 500. The G6PD-stimulating activity of hypothalamic extracts was destroyed by ashing and by base hydrolysis. The ability of plasma of high activity to stimulate G6PD is considerably increased by incubating at 37 °C for 15 min and destroyed by incubation for 45 min.

It is concluded that these and several other previously noted similarities suggest that the cytochemically assayable Na+/K+-ATPase-inhibiting/G6PD-stimulating activity in the plasma and hypothalamus may be due to the same ouabain-like substance.

J. Endocr. (1987) 112, 299–303

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S. L. JEFFCOATE, P. J. SHARP, H. M. FRASER, D. T. HOLLAND and A. GUNN

SUMMARY

Luteinizing hormone releasing hormone (LH-RH) was detected in hypothalamic extracts of rats, rabbits and chickens using a radioimmunoassay for synthetic LH-RH decapeptide. The mobilities of the immunologically active fraction and of synthetic LH-RH were the same in various chromatographic systems (gel filtration on Sephadex, thin-layer chromatography on silica gel and ion-exchange chromatography on carboxymethylcellulose) suggesting that mammalian, avian and synthetic LH-RH's are closely related.

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R. J. L. HOOPER, R. E. SILMAN, R. M. LEONE, M. D. A. FINNIE, S. J. CARTER, J. G. GRUDZINSKAS, Y. B. GORDON, DIANE T. HOLLAND, T. CHARD, P. E. MULLEN and I. SMITH

SUMMARY

The pineal indole 5-methoxytryptophol (ML) has been shown to have an antigonadal activity when administered to experimental animals, but data on its normal pattern of secretion have been lacking. Using a new gas chromatography–mass spectrometry assay, the concentration of ML at various phases of the human menstrual cycle has been studied. Daily samples were obtained throughout the month from five women with a normal cycle and two women taking an oral contraceptive. In women with a normal cycle levels of ML were found to be significantly lower in the last third of their cycle; this change was not seen in women taking an oral contraceptive who had low levels throughout the month. The changes in concentration of ML did not correlate with the changes in concentration of gonadotrophins.