Three groups of workers (Heller, Hasan & Saifi, 1968; Vorherr, Bradbury, Hoghoughi & Kleeman, 1968; Unger & Schwarzberg, 1970) have found that stimuli which are known to release endogenous vasopressin (vagal stimulation, haemorrhage, barbiturate anaesthesia) raised the antidiuretic activity (ADA) in the cerebrospinal fluid (CSF) of experimental animals (rabbits, dogs). The nature of the stimuli, the fact that the ADA was abolished by thioglycollate and the recent finding of neurophysin in the CSF (Robinson & Zimmerman, 1973) indicate strongly that the ADA was exerted by vasopressin. However, it is not clear whether the hormone is directly secreted into the CSF by juxtaependymal processes of neurosecretory neurones as suggested by morphological evidence (for references see Heller et al. 1968; Heller & Zaidi, 1974) or whether it reaches the CSF from the blood.

Heller et al. (1968) and Vorherr et al. (1968) have tried to solve this problem by administering vasopressin intravenously

Eight highly purified human pituitary FSH preparations and purified preparations of the α-and β-subunits of FSH were fractionated by an electrofocusing technique in the pH range of 2·5–10·0 on a sucrose density gradient. The human (h) FSH activity in each of the eluted fractions was monitored by an in-vitro bioassay and a radioimmunoassay procedure.

After electrofocusing, the overall recovery of the biological activity of the eight preparations was between 80 and 94% (mean 88%). On the other hand, the recovery of immunoreactivity ranged between 30 and 84% (mean 71%). A loss of over 85% of hFSH immunoreactivity was observed when the α- and β-subunits of hFSH were fractionated by the same procedure.

The specific loss of varying amounts of immunoreactivity in all preparations during electrofocusing was also reflected by a proportional increase in the ratios of biological activity (B) to immunoreactivity (I); preparation A, which exhibited a loss of 70% of the immunoreactivity, had a threefold increase in its B/I ratio after electrofocusing.

Significant differences were observed in the electrofocusing profiles of the eight preparations both in terms of their pI values and of their spread.

The disparity in the relative distribution of hFSH activities in different pH regions suggested major differences in the carbohydrate moieties (sialic acid content) of the preparations studied, probably as a result of the chemical manipulations involved in the purification of the hormone. It is suggested that a combination of several (but certainly not all) of the preparations might serve as a provisional International Reference Preparation for hFSH radioimmunoassays.

Five highly purified human pituitary LH preparations (candidate preparations for a new International Reference Preparation (IRP) and coded A to E) and three highly purified commercial preparations (referred to as Kabi I, II and III) were fractionated by electrofocusing on a sucrose density gradient with ampholytes in the pH range of 3·5–10·0 The LH activity was monitored in each of the eluted fractions by an in-vitro bioassay and a radioimmunoassay procedure and the profiles of biological activity and immunoreactivity were compared with those of the first IRP of Human Pituitary Luteinizing Hormone for Immunoassay (code no. 68/40).

The overall recovery of the biological activity after electrofocusing of the nine preparations ranged between 75 and 92% (mean 86%) and was higher (P < 0·05) than that of the immunological reactivity which varied between 71 and 94% (mean 79%).

The profiles of the biological activity and immunological reactivity were in close agreement with each other in all preparations. Significant differences were, however, observed in the distribution of various molecular species between the currently used standard and the eight other highly purified preparations.

The ratios of biological activity (B) to immunoreactivity (I) of preparation B and of the three Kabi preparations were significantly higher than those of the other five preparations, both before and after electrofocusing. After electrofocusing, a slight but significant (P < 0·05) rise in the B/I ratios was observed, suggesting that some biologically inactive immunoreactive material was removed during fractionation. All purified preparations lacked the characteristic 'acidic' human LH species which accompanies FSH and which is abundant in the IRP (69/104) and is also present in aqueous extracts of postmenopausal pituitary glands.

A comparison of the electrofocusing profiles of the nine highly purified preparations with those of human LH from plasma and individual pituitary glands revealed marked differences, suggesting that the methods used for the purification of the hormone significantly altered its molecular composition.

ABSTRACT

The propensity of ionic lithium to interfere with the coupling of receptors to guanine nucleotide binding proteins (G-proteins) has only recently been investigated using rat cortical membranes. In the present study we have used intact isolated osteoclasts to investigate lithium-induced uncoupling of the receptor-mediated actions of calcitonin. All actions of calcitonin on the osteoclast were abolished by ionic lithium. We believe that the cation prevents signal transduction by inhibiting G protein-receptor interaction, the first step in intracellular signalling.

ABSTRACT

The Leydig cell cytotoxic ethylene dimethanesulphonate (EDS) was administered s.c. daily (50 mg/kg) to male rats aged 5–16 days. Apart from loss of weight and that the eyelids unfused earlier, no gross toxicity was observed during treatment. On day 17 testis weights, serum testosterone concentrations, testicular serum testosterone content and ¹²⁵I-labelled human chorionic gonadotrophin (hCG) binding to testicular homogenates were reduced. Serum LH and FSH concentrations were elevated.

The testes did not recover from EDS treatment and at 63 and 120 days were minute (<2% of control), and the prostate and seminal vesicles were small although not completely atrophied. In addition, body weights were substantially reduced. Serum and testicular testosterone and ¹²⁵I-labelled hCG binding to testicular homogenates were reduced but not absent. Serum LH and FSH concentrations were increased. Light microscopy of the adult testes showed that EDS treatment inhibited the development of the seminiferous tubules. Most of the tubules were devoid of germ cells and Sertoli cells were rare. Occasionally tubules also contained spermatogonia and spermatocytes but no signs of spermiogenesis. The testes were composed mainly of closely packed interstitial tissue with no lymphatic space. The interstitial cells resembled Leydig cells and stained for 3β-hydroxysteroid dehydrogenase. Histochemically identified Leydig cells were absent during treatment but reappeared when treatment was withdrawn. Testicular Leydig cell numbers were only 7% of control values in the 63-day-old EDS-treated rat. The effect on the testis of EDS treatment administered at a crucial time of testicular development may be explained by withdrawal of androgen; however, the systemic effects indicate non-specific toxicity so any explanation of these changes must be viewed with caution.

J. Endocr. (1988) 119, 467–474

Abstract

Prostaglandins exert marked but transient inhibitory effects on bone resorption. The present study examines the effects of prostacyclin (0·15 to 25 μm) on the morphology of freshly disaggregated rat osteoclasts. An area descriptor, p, represented changes in total cell spread area, and a motility descriptor, μ, represented overall changes in cell motility. The application of prostacyclin intercepted the trend of an increasing cell spread area with time and produced a transient reduction of ρ, an R effect. Its magnitude depended upon concentration and was marked at 25 μm prostacyclin. The subsequent recovery (+0·8/min) of ρ at this concentration resembled the persistent spreading seen in the absence of the agonist. There was also a sustained decrease in μ to approximately 60% of its pretreatment value (a Q effect) following the application of 25 μm prostacyclin. The extracellular application of 20 mm [Ca²⁺] produced a similarly transient cell retraction preceded by a rise of cytosolic [Ca²⁺], but without a corresponding decrease in μ. In contrast, prostacyclin did not elevate cytosolic [Ca²⁺], suggesting the triggering of an alternative transduction pathway. A fully reversible retraction together with incomplete quiescence may explain the transience characteristic of the antiresorptive action of prostacyclin.

Journal of Endocrinology (1994) 143, 375–381

The FSH potencies of 12 preparations of highly purified human pituitary FSH, originating from six different laboratories, were determined by in-vivo and in-vitro bioassays and by immunoassay in terms of the First International Reference Preparation of Human Pituitary Gonadotrophins (FSH and LH) for Bioassay (IRP; coded 69/104). The contamination of these FSH preparations with LH was also determined. Estimates of protein content were based on the absorbance at 280 nm of solutions of the preparations, assuming that A^1% _{1 cm 280} = 10.

The FSH potencies varied between different preparations from 827 i.u./mg to 13 100 i.u./mg by in-vivo bioassay; from 2930 to 14 600 i.u./mg by in-vitro bioassay and from 1680 to 5690 i.u./mg by immunoassay. The ratios of in-vivo biological activity relative to in-vitro biological activity and to immunoreactivity respectively varied between preparations from 0·06 to 2·3 and from 0·15 to 4·1, and there was a significant positive correlation between each of these ratios and the in-vivo biological potency of the preparations; such differences could be due to varying degrees of sialylation between preparations. On the other hand the ratios of in-vitro biological activity to immunoreactivity between preparations were fairly constant (approx. 2). The excess biological activity relative to immunoreactivity observed, in terms of the IRP, in all these materials is consistent with recent findings of some immunoreactive FSH in the IRP unassociated with biological activity. These data did not demonstrate any significant advantage, in terms of FSH in-vivo biological potency, from the use of fresh-frozen rather than acetone-dried pituitary glands for the isolation of FSH.

Contamination of all these preparations with LH appeared to be less than 3% (w/w), as determined by in-vitro bioassay and by immunoassay.

The results of this study are discussed in relation to the selection of material for an international reference preparation for immunoassay and attention is drawn to the value of high in-vivo biological FSH potency as a criterion of the identity of a preparation as well as of its freedom from contaminants without FSH biological activity.

ABSTRACT

Amylin-amide has been implicated in the pathogenesis of type II diabetes due to its proposed inhibitory effect on insulin release from β cells of the pancreatic islets, and on glucose uptake by the skeletal muscle. In experiments with rats and rabbits we failed to demonstrate these anti-insulin actions of amylin and amylin-amide. A single bolus dose of the two peptides (500 pmol) administered i.v. failed to supress plasma insulin levels or to elevate blood glucose levels. The continuous infusion of amylin-amide into rabbits also failed to supress the release of insulin in response to hyperglycaemia produced by an i.v. bolus injection of glucose. These in vivo observations imply that the amylin peptides may not have a primary physiological role in carbohydrate metabolism, but in view of our previous findings, we speculate that the peptide has a more prominent role in calcium homeostasis.

ABSTRACT

It is now established that calcium is a second messenger mediating the action of calcitonin on the osteoclast. We have demonstrated that an increase in the concentration of intracellular free calcium ([Ca²⁺]_i) is associated with (and possibly mediates) the functional effects of calcitonin, including an acute reduction of cell spread area (the R effect) and, in the longer term, a reduction in enzyme release. The present study addresses questions relating to mechanisms of calcitonin action on osteoclast [Ca²⁺]_i. We have used asusuberic(1–7) eel and human calcitonin as agonists, and an indo-1-based dual-emission microspectrofluorimetric method for the measurement of [Ca²⁺]_i in single osteoclasts. Whilst asusuberic(1–7) eel calcitonin caused a biphasic increase in [Ca²⁺]_i, human calcitonin produced only a monophasic [Ca²⁺]_i response of a much lower magnitude. Each biphasic response consisted of a rapid initial transient increase, occurring within seconds of exposure, followed by a sustained increase in [Ca²⁺]_i. The magnitude of the latter response was more variable, but was consistently below the peak value of [Ca²⁺]_i. The sustained phase of the calcitonin effect was abolished in extracellular Ca²⁺-free medium. This phase is therefore dependent on extracellular [Ca²⁺] ([Ca²⁺]_e) whilst the rapid transient increase appeared to be dependent on Ca²⁺ _i redistribution. The effects of calcitonin on [Ca²⁺]_i were concentration-dependent, with neither latency nor oscillations. Repetitive 30-s exposures to calcitonin failed to produce subsequent responses. There was a marked concentration-dependent correlation between changes in osteoclast [Ca²⁺]_i and the magnitude of the R effect. Thus the likely components of the biphasic [Ca²⁺]_i response are a rapid redistribution followed by the transmembrane flux of Ca²⁺. We suggest that the increase in [Ca²⁺]_i may mediate, in part, the inhibitory effect of calcitonin.

Journal of Endocrinology (1992) 132, 241–249

The effects of the related cytokines interleukin-6 (IL-6), leukemia inhibitory factor (LIF) and oncostatin-M on bone resorption and cytosolic Ca(2+) signaling were compared in isolated rat osteoclasts. In the traditional disaggregated osteoclast (pit) assay, IL-6 and LIF, but not oncostatin-M, conserved the bone resorption otherwise inhibited by high extracellular [Ca(2+)] (15 mM). It produced a paradoxical, concentration-dependent stimulation of resorption by elevated extracellular Ca(2+). In the micro-isolated single osteoclast resorption assay, IL-6, high [Ca(2+)] or IL-6 plus high [Ca(2+)] all increased pit formation. In contrast, the IL-6 receptor (IL-6R)-specific agonist antibody MT-18 inhibited bone resorption in a concentration-dependent manner (1:500 to 1:500 000). MT-18 triggered cytosolic Ca(2+) signals in fura 2-loaded osteoclasts within approximately 10 min of application. Each cytosolic Ca(2+) transient began with a peak deflection that persisted in Ca(2+)-free, EGTA-containing extracellular medium, consistent with a release of intracellularly stored Ca(2+). This was followed by a sustained elevation of cytosolic [Ca(2+)] that was abolished in Ca(2+)-free medium, as expected from an entry of extracellular Ca(2+), and by the Ca(2+) channel antagonist Ni(2+). The inclusion of either IL-6 or soluble human (sh) IL-6R specifically reversed both the above effects of MT-18, confirming that both effects were specific for the IL-6R. The findings suggest that IL-6R activation by IL-6 stimulates osteoclastic bone resorption either by reversing the inhibitory effect of high extracellular Ca(2+) in stromal-containing systems or itself stimulating bone resorption along with Ca(2+) by micro-isolated osteoclasts. In contrast, activation of the IL-6R by an agonist antibody produces an inhibition of bone resorption and an associated triggering of the cytosolic Ca(2+) signals previously associated with regulation of bone resorptive function in other situations.