ABSTRACT

The hexapeptide His-d-Trp-Ala-Trp-d-Phe-Lys-NH₂ (GHRP-6) and GH-releasing factor (GHRH) produced a rapid release of GH upon perifusion of dispersed rat pituitary cells. In contrast to the native hormone GHRH, GHRP-6 elicited a response of short duration. When perifusion of each secretagogue was continued until the cells no longer released GH, a challenge by the alternative secretagogue immediately resulted in a secondary release of GH. These results are consistent with each secretagogue causing desensitization of discrete receptor-linked second messenger pathways. Cells which were perifused for 1 min with GHRP-6 required continued perifusion with culture medium alone for 60 min before they completely regained responsiveness to a subsequent challenge with GHRP-6. Somatostatin (SRIF) was able to inhibit the action of either secretagogue completely. However, when both GHRH and GHRP-6 were perifused together, SRIF attenuated but did not block GH secretion. These perifusion data add support to conclusions derived from static cell culture studies, that GHRH and GHRP-6 act through different receptor sites and that through discrete signalling pathways their individual effects on GH release are amplified

Journal of Endocrinology (1991) 129, 11–19

ABSTRACT

In order to obtain a phenotypically stable cell population of chondrocytes, high density primary monolayer cultures of bovine articular chondrocytes were established. Using these cultures, a specific insulin-like growth factor-I/somatomedin-C (IGF-I/SM-C) receptor was demonstrated and characterized. At 15 °C steady-state binding was attained by 5 h, and averaged 25% per 2·2 × 10⁶ cells. Fifty per cent displacement of ¹²⁵I-labelled IGF-I/SM-C by unlabelled IGF-I/SM-C occurred at concentrations of only 2·3 ng/ml, whereas IGF-II and porcine insulin were approximately 15-and 1000-fold less potent respectively. Scatchard analysis gave a linear plot, with a calculated association constant of 2·26 × 10⁹ l/mol and a receptor number of 15 400 sites per cell.

Preincubation of chondrocyte monolayers with either IGF-I/SM-C or porcine insulin at 37 °C for 20 h resulted in reduction of ¹²⁵I-labelled IGF-I/SM-C binding in a dose-dependent manner, although higher concentrations were required with insulin. More than 40% down-regulation of the receptor occurred with IGF-I/SM-C at concentrations of 10 nmol/l and nearly 70% reduction at 50 nmol/l. Interestingly, after preincubation with either human (h) or bovine (b)GH, 40% down-regulation of ¹²⁵I-labelled IGF-I/SM-C binding was observed at concentrations of 10 μmol/l. Local production of IGF-I/SM-C by chondrocytes in response to GH stimulation may have occurred, but, because only 120 pmol IGF-I/SM-C and < 30 pmol IGF-I/SM-C per litre were recovered from serum-free conditioned media preincubated with bGH and hGH respectively, this was not established.

These studies demonstrate that cultured bovine articular chondrocytes possess a highly specific IGF-I/SM-C receptor, and that this receptor population is regulated not only by IGF-I/SM-C and insulin but also by high concentrations of either hGH or bGH. These results are consistent with the growth-promoting action of IGF-I/SM-C on skeletal tissues, and suggest the possibility that GH itself may play its own role to modulate IGF-I/SM-C receptors on chondrocytes.

J. Endocr. (1985) 107, 275–283

ABSTRACT

The possible involvement of a deficit of GH and insulin-like growth factor-I (somatomedin C) (IGF-I/SMC) in mediating the effects of propylthiouracil (PTU)-induced hypothyroidism on body and skeletal growth and myelination was studied in the neonatal rat. Myelination (as assessed by 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) activity), skeletal growth (as assessed by tail length) and body weight of pups from PTU-treated mothers were significantly retarded compared with normal animals or euthyroid controls. At 20 days after birth, plasma GH in hypothyroid animals was undetectable (< 10 μg/l), pituitary GH content was 1 ·2% of control, and plasma, liver and kidney IGF-I/SMC concentrations were 63, 68 and 50% of control values respectively. CNP activity in hypothyroid brain was 52% of normal controls but the concentration of IGF-I/SMC was 113–154% of control. Treatment of hypothyroid animals from day 1 with GH (10 mg/kg body weight per day) restored liver and plasma IGF-I/SMC concentrations at 20 days to values above those of normal animals and euthyroid controls. The concentration of IGF-I/SMC was also significantly (P< 0·001) restored in hypothyroid kidney (79% of normal), but the concentration in brain was unaffected. These observations provide evidence that the GH treatment employed in the present experiments was adequate to restore the deficit. GH treatment had no significant effect on tail length or CNP activity, and only a small (4–24%) effect on body weight at 20 days. Only thyroxine was able fully to restore body weight and substantially restore tail length and CNP activity.

The present study provides strong evidence against an important involvement of GH or IGF-I/SMC in mediating the effects of thyroid hormone on myelination and body growth in the infant rat. It does not, however, rule out the possibility that thyroid hormone is required for the expression of the growth-promoting effects of IGF-I/SMC by other mechanisms such as the expression of the IGF-I/SMC receptor.

J. Endocr. (1988) 119, 117–125

Abstract

GH release is normally stimulated by the naturally occurring GH-releasing factor (GRF). However, smaller GH-releasing peptides (GHRPs) and non-peptide analogues have been described which stimulate GH release in animals and man. Although these compounds release GH in vitro, their in vivo activity in conscious animals has proved more difficult to study since the GH responses are variable, and prone to desensitization. We now compare the GH-releasing properties of GHRP-6 and a novel benzolactam GH secretagogue L-692,585 using chronically cannulated guinea pigs and automated blood microsampling to study the effects of repeated exposure to these secretagogues. L-692,585 was approximately tenfold less potent than GHRP-6 for GH release, but it synergized strongly with GRF. Serial injections of GRF, GHRP-6 or L-692,585 at intervals of 60 or 90 min produced variable GH release which followed a cyclic pattern of responsiveness. Prolonging the pulse interval to 3 h produced more regular responses to both GHRP-6 and L-692,585. Continuous i.v. infusion of low doses of either secretagogue elicited an initial GH release, and amplified the spontaneous GH secretory pattern over the next 6 h. We conclude that L-692,585 and GHRP-6 share similar in vivo properties, and that intermittent responsiveness to frequent injections is similar for all three secretagogues, and is a property of the conscious animal rather than of any secretagogue type. More consistent responses can be obtained with less frequent injections that more closely match the endogenous GH rhythm, whereas continuous exposure to these secretagogues leads to amplified endogenous secretion. Our results show that the interpretation of in vivo effects of these peptide and non-peptide secretagogues will need to take account of their interaction with the endogenous mechanisms governing GH release.

Journal of Endocrinology (1995) 145, 417–426

Abstract

The negative feedback regulation by ovarian steroids of luteinizing hormone secretion may be partially mediated by a hypothalamic endogenous opioid mechanism. This could be affected by ovarian steroid-regulated changes in hypothalamic opioid receptor binding mechanisms. In this report we show that in the presence of blocking concentrations of site-selective opioid analogues, [³H]diprenorphin homogeneously labelled μ, δ or κ receptor subtypes respectively. Using this receptor binding model, we characterized each opioid receptor subtype in the hypothalamic preoptic area and medio-basal hypothalamus of ovariectomized (OVX) and OVX plus progesterone- or oestradiol-17β (OE₂)-treated ewes. In the preoptic area, progesterone treatment did not influence the affinity or capacity of δ or κ receptor binding sites, but significantly reduced μ receptor subtype content (20% less than control) with no statistically significant change in affinity. There was no effect of OE₂ on either the affinity or capacity of each opioid receptor subtype in this area. In the mediobasal hypothalamus, progesterone treatment significantly decreased δ subtype receptor affinity (22 ± 11 nm vs control 7 ± 2 nm) and increased binding capacity (78 ± 9 fmol/mg protein vs control 37 ± 16 fmol/mg protein). OE₂ treatment had a similar, though more profound effect on affinity (51 ± 17 nm) and binding capacity (139 ± 26 fmol/mg protein) at the δ receptor binding site. There were no significant changes in the affinity or capacity of μ or κ binding sites in the medio-basal hypothalamus. These results indicate that steroid hormones modulate hypothalamic opioid receptors in the OVX ewe in a receptor subtype- and region-dependent manner. The precise role of these steroid-induced changes in opioid receptor characteristics remains to be determined.

Journal of Endocrinology (1995) 145, 559–567

ABSTRACT

The placenta has been shown to contain ACTH and β-endorphin but the roles of these peptides are unknown. To investigate whether they are released into the maternal circulation from the placenta in response to physiological stimuli the effects of hypoglycaemic stress were investigated. Plasma samples were collected from the femoral artery (FA) and uterovarian (UV) vein of nine pregnant sheep before and during hypoglycaemia induced by intravenous insulin (100U). Plasma concentrations of ovine β-endorphin (oβ-EP) were measured by radioimmunoassay. Concentrations of oβ-EP rose in both vessels by 60 min after insulin. The peak concentrations of oβ-EP (pmol/l) were 122 ± 29 (mean ± SEM, n=8) in the UV and 96±24 (n=9) fmol/ml in the FA 60 min after insulin injection. There was no difference between the concentrations of oβ-EP in the vessels before insulin injection but at 60 and 120 min after insulin the concentrations of oβ-EP were significantly higher in the UV than FA (P<0.02, analysis of variance). This indicates that the pregnant uterus or placenta can respond to hypoglycaemia by secreting β-EP into the maternal circulation. It is therefore possible that placental pro-opiomelanocortin (POMC) peptides may have a role in maternal endocrinology and metabolism.

ABSTRACT

The switch from γ (fetal) to β (adult) globin production was studied by the analysis of globin synthesis in chronically cannulated ovine fetuses and newborn lambs. The γ/α globin synthesis ratio decreased from 0·98 ± 0·11 (s.d.) (n = 4 samples) at 100–120 days of gestation to 0·15± 0·07 (n = 4) in lambs of 150–156 days post-conception, and the β/α synthesis ratio increased from 0·04 ± 0·06 (n = 4) to 1·13 ± 0·21 (n = 4) over the same period. In bilaterally adrenalectomized fetuses, which survived in utero until 151–156 days, the γ/α and β/α synthesis ratios were 0·64 ± 0·14 (n = 3) and 0·25 ± 0·07 (n = 3) respectively in the 150- to 156-day period. Bilateral adrenalectomy did not affect the time of onset of β globin synthesis, but significantly decreased the rate. In one bilaterally adrenalectomized fetus the infusion of increasing concentrations of cortisol restored the rate of β globin synthesis to normal. Treatment of three intact fetuses with 100 μg cortisol/h for 3 weeks, from 100 to 121 days, did not affect the timing or rate of switch from γ to β globin synthesis. Thus fetal adrenal secretions, probably cortisol, affected the rate of change of γ to β globin synthesis but other factors must have been involved in the initiation of the switch.

J. Endocr. (1985) 104, 165–170

SUMMARY

In the male rat injections of CaCl₂ and MgCl₂ stimulated mitosis in bone marrow and thymus tissue. The magnesium salt was also mitogenic in the normal female, but calcium only exerted its mitogenic effect after ovariectomy. Oestradiol, but not progesterone replacement therapy abolished calcium-induced mitosis in the ovariectomized rat. The inability of calcium to stimulate cell division was also apparent in the thyroparathyroidectomized female rat, suggesting the oestradiol blockade did not operate via some indirect action on the calcium homeostatic hormones calcitonin or parathyroid hormone.

When thymic lymphocytes derived from male or female rats were isolated and maintained in suspension, increased calcium or magnesium concentrations in the culture medium stimulated the entry of cells into mitosis. Addition of oestradiol to the culture medium abolished the mitogenic effect of increased calcium levels, but had no effect on magnesium-induced proliferation. These experiments suggested that oestradiol might act at the cell surface to prevent the influx of calcium but not magnesium ions into the interior of the cell and thus to block the sequence of biochemical events which lead to the initiation of DNA synthesis and culminate in mitosis.

Treatment of type 1 diabetes by islet transplantation is currently limited by loss of functional β-cell mass after transplantation. We investigated here whether adenovirus-mediated changes in AMP-activated protein kinase (AMPK) activity, previously shown to affect insulin secretion in vitro, might affect islet graft function in vivo. In isolated mouse and rat islets, insulin secretion stimulated by 17 (vs 3) mmol/l glucose was inhibited by 36.5% (P<0.01) and 43% (P<0.02) respectively after over-expression of constitutively-active AMPK- (AMPK CA) versus null (eGFP-expressing) viruses, and glucose oxidation was decreased by 38% (P<0.05) and 26.6% (P<0.05) respectively. Increases in apoptotic index (terminal deoxynucleotide transferase-mediated deoxyuridine trisphosphate biotin nick end-labelling) (TUNEL)) were also observed in AMPK CA- (22.8 ± 3.6% TUNEL-positive cells, P<0.001), but not AMPK DN- (2.72 ± 3.9%, positive cells, P=0.05) infected islets, versus null adenovirus-treated islets (0.68 ± 0.36% positive cells). Correspondingly, transplantation of islets expressing AMPK CA into streptozotocin-diabetic C57 BL/6 mice improved glycaemic control less effectively than transplantation with either null (P<0.02) or AMPK-DN-infected (P<0.01) islets. We conclude that activation of AMPK inhibits β-cell function in vivo and may represent a target for therapeutic intervention during islet transplantation.

SUMMARY

C₁₉-steroids, testosterone and oestrogen production rates have been measured simultaneously by urinary isotope dilution in a group of 18 patients with polycystic ovaries and in two normal women.

The production of total C₁₉-steroids remained high in seven patients under dexamethasone suppression, suggesting a major ovarian contribution. In eight it did not remain high, suggesting a major adrenal contribution.

The rate of testosterone production was usually normal under dexamethasone. The rate of oestrogen production was not subnormal and even occasionally raised.

Ovarian wedge resection produced a good clinical response in 75% of the observations, and C₁₉-steroid production fell. The latter was not so evident if there was no clinical response. Testosterone production was usually reduced. Oestrogen production rate changed irregularly.

Administration of human pituitary follicle-stimulating hormone caused a concurrent increase in C₁₉-steroids, testosterone and oestrogen production in four out of five patients.

The inadequacy of the urinary isotope dilution technique, in attempting to measure testosterone secretion and interconversion of testosterone and androstenedione in these patients, has been stressed and discussed.

Some correlations have been established with direct observations on ovarian steroid metabolism in certain patients reported elsewhere.