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Abstract
Microsome fractions prepared from the mammary glands of non-pregnant, pregnant and lactating sheep have been used to study binding of 125I-labelled transforming growth factor-α (TGF-α). Binding was dependent on microsomal protein concentration, time and temperature. It showed the characteristics of an epidermal growth factor (EGF) receptor, being displaced by TGF-α and EGF, but not by insulin or IGF-I. The non-linear curve fitting program LIGAND was used to determine affinity and number of binding sites. A single class of high-affinity binding sites was found. The apparent dissociation constant (K d) was similar in all physiological states (2·43±0·27 mol/l × 10−10, n=23). Numbers of binding sites were lower in late-pregnant (20 weeks) and lactating sheep (14·07± 2·45 fmol/mg protein, n=10) than in non-pregnant, 10-or 15-week pregnant sheep (43·04±5·93 fmol/mg protein, n=13).
DNA synthesis by mammary alveolar epithelial cells cultured on collagen gels was increased twofold by TGF-a (maximum response at 10 μg/l; 1·8 nmol/l) but not by EGF. Cells derived from 15- to 20-week pregnant sheep responded significantly to TGF-α on day 3 of culture, but the response was delayed to day 4–5 of culture in cells from other physiological states. Dose–response was not significantly affected. TGF-α and IGF-I produced an additive effect on DNA synthesis. Oestradiol (10−12 to 10−9 m), a potential stimulator of the TGF-α gene, did not stimulate DNA synthesis alone, or in combination with IGF-I. It is concluded that growth factors acting via the EGF receptor play a role in ruminant mammary development, but whether they mediate oestradiol effects remains unresolved.
Journal of Endocrinology (1995) 144, 165–171
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1. The ACTH concentration has been estimated in lyophilized pituitary tissue from five adults, seven infants and eight foetuses. Results are expressed in terms of the international standard. Adult pituitaries contain more ACTH than infantile glands, and infantile glands more than foetal ones.
2. ACTH activity was detected in foetal pituitaries at a period of gestation as early as 16 weeks.
3. The accuracy of the Sayers test was considerably improved when the time interval between hypophysectomy and assay was increased from 24 to 48 hr.
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SUMMARY
A sensitive and specific radioimmunoassay for α-melanocyte-stimulating hormone (α-MSH) was developed.
Extracts of the neurointermediate lobe of the rat produced displacement curves which were parallel to those obtained with synthetic α-MSH. The mean immunoreactive a-MSH concentration in neurointermediate lobes from normal adult rats was 2768 ± 200 (s.e.m.) ng/lobe. This accounted for approximately 78% of the MSH activity of the neurointermediate lobe as measured by bioassay. Much lower levels of immunoreactive α-MSH were found in the anterior lobe of the rat. Extracts of rat serum and plasma also contained immunoreactive α-MSH and the mean level was found to be 237 ± 20 pg/ml. This was slightly lower than the level measured in rat plasma by bioassay.
Increased levels of α-MSH were found in plasma of rats 1 and 3 h after a single injection of trifluoperazine and after 1·5 min of ether anaesthesia. These changes were reflected by decreases in the α-MSH content of the neurointermediate lobe.
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Search for other papers by J C Buckingham in
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Abstract
Glucocorticoids have been shown repeatedly to inhibit the secretion of TSH in experimental animals and in man but their site and mode of action are unknown. In the present study, we have used an in vitro model to examine the effects of dexamethasone on the resting and pharmacologically evoked secretion of TSH by the rat anterior pituitary gland, and to show how they are influenced by inhibitors of RNA/protein synthesis. In addition, we have investigated the potential role of lipocortin 1 (LC1), a protein shown previously to contribute to glucocorticoid action in several systems, as a mediator of the glucocorticoid-induced suppression of TSH release in our in vitro preparation.
The significant (P<0·01) increases in the release of immunoreactive (ir)TSH from rat anterior pituitary tissue initiated by submaximal concentrations of TRH (10 nmol/l), vasoactive intestinal polypeptide (VIP, 10 nmol/l) or the adenyl cyclase activator, forskolin (100 μmol/l) were reduced significantly (P<0·05) by preincubation of the tissue with dexamethasone (0·1 μmol/l). In contrast, irTSH secretion evoked by a submaximal concentration of the L-Ca2+ channel opener BAY K8644 (10 μmol/l) was unaffected by the steroid, although readily antagonised (P<0·01) by nifedipine (1–100 μmol/l). Inclusion of actinomycin D (1·78 μmol/l) or cycloheximide (0·8 μmol/l), inhibitors of RNA and protein synthesis respectively, in the medium effectively abrogated the inhibitory effects of dexamethasone (0·1 μmol/l) on the secretory responses to TRH (10 nmol/l), VIP (10 nmol/l) and forskolin (100 μmol/l).
LC1 was readily detectable by Western blotting in protein extracts of freshly excised anterior pituitary tissue. A small proportion of the protein was found to be attached to the outer surface of the cells where it was retained by a Ca2+-dependent mechanism. Exposure of the tissue to dexamethasone (0·1 μmol/l) caused a pronounced increase in the amount of cellular LC1 attached to the outer surface of the cells and a concomitant decrease in the intracellular LC1 pool. Progesterone (0·1 μmol/l) and aldosterone (0·1 μmol/l) were also weakly active in this regard, but thyroxine and tri-iodothyronine (0·1 μmol/l) were not. Addition of an N-terminal LC1 fragment, LC1(1–188) (0·05–0·53 pmol/l) to the incubation medium reduced significantly (P<0·01) the increases in irTSH release induced by TRH (10 nmol/l), VIP (10 nmol/l) and forskolin (100 μmol/l), but failed to influence (P<0·05) those initiated by BAY K8644 (10 μmol/l). Furthermore, the inhibitory actions of dexamethasone (0·1 μmol/l) on the release of irTSH provoked by TRH (10 nmol/l), VIP (10 nmol/l) and forskolin (100 μmol/l) were substantially reversed (P<0·01) by a specific monoclonal anti-LC1 antibody, while an isotype-matched control antibody was without effect.
The results show clearly that dexamethasone, a semisynthetic glucocorticoid, acts at the pituitary level to inhibit the neurochemically evoked release of irTSH. They also provide novel evidence that the inhibitory actions of the steroid are dependent upon de novo RNA/protein synthesis and that they involve an LC1 dependent mechanism.
Journal of Endocrinology (1995) 147, 533–544
St Vincent’s Clinical School, Faculty of Medicine, University of New South Wales, Victoria St, Darlinghurst, NSW 2010, Australia
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St Vincent’s Clinical School, Faculty of Medicine, University of New South Wales, Victoria St, Darlinghurst, NSW 2010, Australia
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St Vincent’s Clinical School, Faculty of Medicine, University of New South Wales, Victoria St, Darlinghurst, NSW 2010, Australia
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Increased lipid availability is associated with diminished insulin-stimulated glucose uptake and glycogen synthesis in muscle, but it is not clear whether alterations in glycogen synthase activity itself play a direct role. Because intracellular localization of this enzyme is involved in its regulation, we investigated whether fat oversupply causes an inhibitory redistribution. We examined the recovery of glycogen synthase in subcellular fractions from muscle of insulin-resistant, fat-fed rats and chow-fed controls, either maintained in the basal state or after a euglycaemic-hyperinsulinaemic clamp. Although glycogen synthase protein and activity were mostly recovered in an insoluble fraction, insulin caused translocation of activity from the smaller soluble pool to the insoluble fraction. Fat-feeding, which led to a reduction in glycogen synthesis during the clamp, was associated with a depletion in the soluble pool, consistent with an important role for this component. A similar depletion was also observed in cytosolic fractions of muscles from obese db/db mice, another model of lipid-induced insulin resistance. To investigate this in more detail, we employed lipid-pretreated L6 myotubes, which exhibited a reduction in insulin-stimulated glycogen synthesis independently of alterations in glucose flux or insulin signalling through protein kinase B. In control cells, insulin caused redistribution of a minor cytosolic pool of glycogen synthase to an insoluble fraction, which was again forestalled by lipid pretreatment. Glycogen synthase recovered in the insoluble fraction from pre-treated cells exhibited a low fractional velocity that was not increased in response to insulin. Our results suggest that the initial localization of glycogen synthase in a soluble pool plays an important role in glycogen synthesis, and that its sequestration in an insulin-resistant insoluble pool may explain in part the reduced glycogen synthesis caused by lipid oversupply.
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ABSTRACT
The binding of 125I-labelled bovine TSH to a human thyroid cell line (SGHTL-34) has been studied. Binding to hormonally responsive cells was time dependent, specific and reversible. Scatchard analysis of the binding data indicated the presence of a single binding site with high affinity (intrinsic dissociation constant (K d) = 0·25 ±0·08 nmol/l; mean ± s.e.m.; n = 4) and low capacity (maximum binding (Bmax) = 104±29 fmol/mg protein; mean ± s.e.m.; n = 4). Hill plots confirmed the presence of a single site. Kinetic data demonstrated close agreement between the K d and Bmax obtained from the competition data (K d = 0·23±0·35 nmol/l; Bmax = 161 ±83 fmol/mg protein; n = 6).
Journal of Endocrinology (1990) 127, 351–356
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ABSTRACT
Binding of [3H]arginine vasopressin (AVP) and [3H]oxytocin to primary monolayer cultures of bovine adrenal chromaffin cells was time-dependent, and the binding sites for each peptide were specific and saturable. Studies with the V1 AVP antagonist d(CH2)5Tyr(Me)2-AVP, the V2 agonist 1-deamino-8-d-AVP and the V2 antagonist d(CH2)5 d-Leu2,Val4-AVP indicated that the AVP receptor was V1 in specificity. Scatchard plots showed that each ligand interacted with a single high-affinity, low-capacity binding site: oxytocin dissociation constant (K d) 0·29 ± 0·02 nmol/l, maximum binding capacity (Bmax) 7·6 ± 0·2 fmol/106 cells (or 4500 ± 102 sites/cell) (n = 3); AVP K d 0·09±0·02 nmol/l, Bmax 5·1±0·63 fmol/106 cells (or 3050 ± 318 sites/cell) (n = 3). Although forskolin in concentrations from 1 nmol/l to 1 mmol/l stimulated cyclic AMP (cAMP) production in isolated chromaffin cells, this did not result in detectable catecholamine release. Neither AVP nor oxytocin in concentrations between 10 pmol/l and 10 μmol/l stimulated cAMP production in these cells. Vasopressin in concentrations as low as 10 pmol/l stimulated a sixfold increase in total inositol phosphates; the dose–response curve was triphasic. Oxytocin had little effect on total inositol phosphate accumulation at low concentrations, but concentrations above micromolar stimulated total inositol phosphate production approximately fourfold. There was no measurable release of catecholamines in response to either peptide. The physiological consequences of these AVP-induced changes in inositol phosphate concentrations remain to be elucidated.
Journal of Endocrinology (1989) 121, 133–139
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ABSTRACT
Human somatomedin C has been purified from Cohn fraction IV paste by a simplified procedure using chromatofocusing, hydroxylapatite chromatography and reverse-phase high performance chromatography. The purified material has a specific activity by somatomedin C radioimmunoassay of 9160 units/mg (1 unit is defined as the amount of somatomedin present in 1 ml normal adult male human serum), representing a 650 000-fold purification, and possesses sulphation, mitogenic and insulin-like activities (specific activities of 3388 units/mg, 832 units insulin equivalents/mg and 1122 units/mg respectively). Somatomedin C is shown to be a potent stimulator of DNA synthesis (50% maximum stimulation at 150 fmol/ml) in isolated chondrocytes derived from costal cartilage, a major physiological target tissue.
J. Endocr. (1986) 110, 151–158
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Abstract
125I-Labelled ovine prolactin was infused for 15 min into a pudic artery supplying one mammary gland of lactating goats (n=17). Between 0 and 4·25 h significandy more total (P<0·01) and trichloroacetic acid (TCA)-precipitable (P<0·001) radioactivity appeared in the milk of the infused compared with the non-infused gland. Gel chromatography and antibody precipitation indicated the presence of undegraded 125I-labelled prolactin in milk whey. Maximum transfer occurred 60–80 min after the end of infusion suggesting passage via a transcellular route. High plasma prolactin concentrations, resulting from infusion of cold prolactin with labelled prolactin in late lactation or from seasonally elevated prolactin at peak lactation, reduced the specific activity of infused prolactin and depressed the difference in secretion of 125I-labelled prolactin into milk of infused and non-infused glands. This suggests the operation of a competitive and saturable mechanism. Together with the increase in the milk to blood ratio of prolactin in goats given long-term (3 week) bromocriptine treatment, the results suggest that the goat mammary gland has a high avidity for prolactin especially when circulating prolactin is low. There was also evidence from TCA precipitation that prolactin may be protected from degradation in these circumstances. These mechanisms may contribute to the resistance of ruminant lactation to reduction in plasma prolactin and protect lactation from seasonal prolactin fluctuations.
Journal of Endocrinology (1995) 146, 411–420
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ABSTRACT
Basic fibroblast growth factor (bFGF) was quantitated in human primary thyrocyte cultures and thyroid cell lines produced by transfection with pSV3neo. Immunoreactive-bFGF (ir-bFGF) bound to heparin–Sepharose affinity columns eluted with 1·8–2·0 mol NaCl/l and had a molecular weight of approximately 17 000. Recombinant human bFGF in the presence of 5% serum increased the growth of transfected human thyrocytes but not the growth of primary human thyrocytes. Preincubation of cells with up to 100 μg bFGF/l potentiated TSH-stimulated cAMP release from the transfected cells but inhibited release from primary human thyroid cultures. bFGF may be an important modulator of thyroid cell function and growth.
Journal of Endocrinology (1993) 136, 339–344
