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ABSTRACT
Submaxillary salivary gland tissue from large White, Göttingen miniature and Meishan (Chinese) breeds of pig, and European wild boars, was incubated with [35S]methionine. The radiolabelled amino acid was incorporated into protein in all incubations as demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Specifically [35S]methionine was predominantly incorporated into the α- and β-charge isomers of pheromaxein, a 16-androstene steroid-binding protein, as shown by SDS-PAGE in combination with vertical isoelectric focusing on polyacrylamide slab gels. The synthesis of pheromaxein occurred in submaxillary gland tissue from both sexes, including tissues stored frozen at −70 °C for long periods. There was little evidence for pheromaxein synthesis in parotid gland tissue or skeletal muscle. Total protein, pheromaxein and total 16-androstenes were determined in the submaxillary gland cytosols of six mature Göttingen miniature boars and a positive correlation was found between these glandular constituents. The amounts of endogenous pheromaxein relative to total protein in the submaxillary gland cytosols (range 10·3–18·0%), together with the predominant synthesis of this protein in vitro, indicate that pheromaxein is a major protein produced in porcine submaxillary glands, particularly in those of the male.
Journal of Endocrinology (1991) 128, 205–212
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ABSTRACT
The effects of chronic infusion of porcine relaxin on oxytocin release were studied in lactating rats. Infusion of relaxin (4·2 μg/h for either 4 or 6 days) suppressed reflex milk ejection and reduced litter weight gain for 48 h compared with saline-infused controls. After 2 days, neither the rate of growth nor the frequency of milk ejection were significantly different from controls. For 24 h after the infusion of relaxin ended, litters gained weight more quickly than controls but there was no difference seen in the frequency of milk ejection. The effects on oxytocin release of stopping an infusion of relaxin after 3 days were investigated. There was a significant (P <0·01) rise in plasma oxytocin (up to 90 pmol/l) 30 min after the infusion was stopped, followed by a sustained rise in intramammary pressure. Treatment of relaxin-infused rats with naloxone (0·1 mg/kg) when the infusion was halted caused a more rapid release of oxytocin (within 2 min), a greater release of oxytocin (up to 140 pmol/l) and a prolonged rise in intramammary pressure.
J. Endocr. (1987) 114, 241–246
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ABSTRACT
Hydrogen peroxide (H2O2) is an essential substrate for the peroxidase reaction in thyroid hormone biosynthesis. We demonstrated the production of H2O2 from porcine thyroid cells stimulated with extracellular ATP, using a scopoletin–horseradish peroxidase (HRP) system. Incubation of isolated cells for 1 day in the presence of 10% (v/v) newborn calf serum was necessary for the detection of induction by ATP of H2O2 production. The rate of H2O2 production induced by the addition of ATP increased in a dose-dependent manner, and the concentration of ATP required for half-maximum stimulation was about 10 μmol/l. ADP and GTP were also effective, but only at higher concentrations than ATP. In the absence of extracellular Ca2+, the production rate was very low.
Production of H2O2 from thyroid cells was also measured by a method which discriminated between H2O2 and superoxide anion (O2 −); in this, diacetyl-deuteroheme-substituted HRP was employed as the trapping agent for both O2 metabolites. The thyroid cells produced H2O2, but not O2 −, when the cells were stimulated by extracellular ATP.
Journal of Endocrinology (1990) 126, 283–287
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ABSTRACT
19-Hydroxytestosterone and 19-hydroxyandrostenedione have been identified as secretory products of the testes in the mature male domestic pig. Their isolation and identification were made by reverse-phase high-performance liquid chromatography and capillary gas chromatography-mass spectrometry (CGC-MS) of extracts from testicular vein blood and media of incubations with Leydig cells. Blood was collected from veins on the surface of the testes of anaesthetized boars. Collagenase-dispersed Percoll-purified cells (> 90% pure) were incubated (20 × 106 cells/flask) with androstenedione (8·75 μmol/l) or [3H]androstenedione (5 × 106 c.p.m.) for < 60 min. Steroids were recovered from plasma or media by solid-phase extraction and the unconjugated fractions chromatographed isocratically in two solvent systems (acetonitrile: water, 37:63 (v/v) and methanol: water, 70:30 (v/v)) before CGC-MS analysis. 19-Hydroxy-testosterone was present in greater quantities than 19-hydroxyandrostenedione in testicular vein blood; it was also seen as a quantitatively significant metabolite of unlabelled and radioactive androstenedione in the incubation studies. The demonstration of the secretion of 19-hydroxyandrogens from porcine testes thus raises questions concerning the physiological significance of a testicular, rather than an adrenal, secretion of these compounds.
Journal of Endocrinology (1993) 137, 281–289
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ABSTRACT
The effects of thyrotrophin, cholera toxin and 8-chloro-cyclic AMP on thyroglobulin gene expression in cultured porcine thyroid cells were compared. Cells organized either into monolayers in culture chambers with porous bases or into inside-out follicles in suspension were used. Thyroglobulin mRNA content was measured by dot-blot hybridization, and thyroglobulin synthesis rate was determined by immunoprecipitation of [35S]thyroglobulin after 2 h labelling with [35S]methionine. Cholera toxin and 8-chlorocyclic AMP increased the thyroglobulin mRNA content and thyroglobulin synthesis rate in the same ratio (∼3) as did thyrotrophin, showing that cyclic AMP mediates the effect of thyrotrophin on thyroglobulin gene expression. The culture chamber system provides for contact of the effectors with either the apical or the basolateral membrane.
The addition of 0·02–0·1 mU thyrotrophin/ml on the basolateral side of the cell layer gave a maximal response whereas this response was not reached on the apical side even with the addition of 5 mU/ml. In contrast, cholera toxin and 8-chloro-cyclic AMP stimulated thyroid cells equally on both sides.
J. Endocr. (1988) 116, 25–33
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ABSTRACT
Cultured porcine thyroid cells exhibit a resting membrane potential of about − 73 mV and depolarize to about − 54 mV on exposure to TSH. The depolarizing response to TSH was preserved in a medium consisting only of inorganic salts and buffers, but was abolished in sodium-free medium, demonstrating dependence on an inward sodium current. Increasing the potassium concentration of the medium resulted in a reduction in the resting membrane potential of 60 mV per tenfold change in potassium concentration, and a diminished TSH response. A hyperpolarizing TSH response was observed in a sodium- and bicarbonate-free medium, indicating that a hyperpolarizing ion current (probably carried by potassium) was also enhanced in the presence of TSH. Tetrodotoxin blocked the TSH response. We conclude that the response of the thyroid cell membrane to TSH involves increases in permeability to sodium and potassium, and that the thyroid membrane ion channels bear some similarity to the voltage-dependent sodium channels of excitable tissues, despite the absence of action potentials in the thyroid.
J. Endocr. (1986) 108, 225–230
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ABSTRACT
The effect of relaxin on electrically evoked release of oxytocin from the posterior pituitary was examined by monitoring changes in intramammary pressure in the anaesthetized lactating rat.
The amount of oxytocin released by electrical stimulation of the neurohypophysis in vivo was dramatically reduced following i.v. injection of highly purified porcine relaxin (2·5–10 μg/rat). Relaxin inhibited oxytocin release in a dose-dependent manner and the onset of inhibition occurred within 6–10 min and lasted for 10–60 min. No effect on the sensitivity of the mammary gland to exogenous oxytocin was observed after relaxin treatment. During the period of inhibition, i.v. injection of the opioid antagonist naloxone chloride (1 mg/kg) completely and immediately restored electrically evoked oxytocin release. The neurohypophysis is known to contain endogenous opioid peptides, therefore the effect of relaxin on electrically stimulated release of oxytocin from the rat isolated neural lobe in vitro was examined. Relaxin (500–2000 ng/ml) failed to inhibit oxytocin release in vitro.
The results suggest that relaxin can inhibit the release of oxytocin from terminals in the neurohypophysis, but by an indirect mechanism. This action appears to be mediated through endogenous opioid peptides whose source is not clear. They are unlikely to be of neurohypophysial origin and may probably come from the adrenal medulla, since acute adrenalectomy negated the inhibitory effect of relaxin on oxytocin release.
J. Endocr. (1986) 109, 393–397
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ABSTRACT
Experiments were carried out to investigate the effects of porcine relaxin on the course of gestation and delivery in the rat. Plasma relaxin was maintained at approximately 600 nmol/l from day 19 to day 23 of gestation by i.v. infusion from chronically implanted minipumps. Relaxin significantly (P<0·001) prolonged the length of gestation in 17 rats compared with controls, without causing dystocia or affecting the number of live births. Six rats gave birth during relaxin infusion. In these animals there was a significant (P<0·001) increase in the interval between successive deliveries compared with control animals, resulting in prolonged labour. The remaining 11 rats gave birth after the infusion was completed, when the interval between successive deliveries was significantly (P< 0·025) shorter than controls. The results are consistent with the hypothesis that relaxin has a central action suppressing the release of oxytocin as well as a peripheral action on the myometrium and cervix.
J. Endocr. (1986) 109, 85–88
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ABSTRACT
Primary cultures of porcine thyroid cells, grown as monolayers, showed saturable binding of epidermal growth factor (EGF). Scatchard analysis resolved the binding to a high-affinity/low-capacity site (dissociation constant = 0·17 nmol/l, maximal binding capacity = 1·67 pmol/106 cells) and a low-affinity/high-capacity site. Preincubation of thyroid monolayers with TSH for 3 days caused an increase in binding of 125I-labelled EGF due to an increase in the number of receptors, with the binding affinity unchanged. This effect was dose-dependent within the range of TSH concentrations 0·01–100 mu/l. The same effect was seen with dibutyryl cyclic AMP (10–1000 μmol/l). When the protein synthesis inhibitor cycloheximide was included in the TSH preincubation, the increase in EGF binding was abolished. The TSH effect on EGF binding was not mediated by thyroid hormones, since neither thyroxine (T4) nor tri-iodothyronine (T3) at 01 nmol/l–10 μmol/l could mimic the effect of TSH, nor could antisera to T3 or T4 neutralize the effect of TSH. The concentration of extracellular iodide (10 nmol/l–10 mmol/l) had no effect on the binding of 125I-labelled EGF. The results demonstrate that TSH increases the number of receptor sites for binding of EGF to thyroid monolayers in vitro. This effect is dependent upon protein synthesis and is mediated by cyclic AMP but not by thyroid hormones or iodide. This effect on binding of EGF may contribute to the trophic action of TSH.
J. Endocr. (1987) 114, 179–184
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Abstract
An effort was made to evaluate the potential usefulness of a peptide vaccine in improving the growth performance of farm animals. It was previously reported that a murine PS-7·6 monoclonal antibody (mAb) which was specific to porcine GH (pGH) enhanced the growth-promoting activity of pGH in an experimental hypophysectomized rat model. Additional data from a epitope mapping study suggested that PS-7·6 mAb recognized a pGH fragment corresponding to an amino acid sequence 54–95. In this report, therefore, a peptide pGH(54–95) was synthesized in an attempt to induce PS-7·6-like antibodies in swine. It was demonstrated that the peptide pGH(54–95) competed with PS-7·6 mAb for the binding to radioactive pGH in a competition radioimmunoassay and also caused pigs to elicit polyclonal antibodies immunoreactive to pGH protein. The association and dissociation rate constants of the swine antibody to pGH were 1·9 × 102 m −1 s −1 and 3·2×10−4 s −1 respectively, thus producing an overall binding affinity of K d=1·6 × 10−6 m. The swine anti-body partially competed with murine PS-7·6 mAb for the binding to pGH, suggesting that the pGH-recognizing sites for both antibodies might be closely related. The biological effect of the swine antibody was examined in hypophysectomized rats and shown to significantly augment pGH activity in promoting the growth of these GH-deficient animals. The present findings suggest that a synthetic peptide may be developed as a potential growth vaccine for swine to generate antibodies capable of enhancing the effectiveness of endogenous pGH.
Journal of Endocrinology (1995) 145, 163–167
