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SUMMARY
The maternal administration of meclofenamic acid (a prostaglandin synthetase inhibitor) to pregnant sheep prevented the dexamethasone-induced delivery of live lambs and delayed delivery after foetal death in utero. Administration of meclofenamic acid had no effect on the changes in the levels of progesterone and oestrogen in the plasma which occur before lambing in response to foetal glucocorticoid. Despite normal maternal endocrine changes, increased uterine activity did not occur at the expected time, although it could be elicited by vaginal distension or by administration of oxytocin. The rates of cervical ripening and dilatation were reduced by meclofenamic acid and lambing was frequently associated with some degree of cervical dystocia. Withdrawal of meclofenamic acid did not immediately result in an increase in the level of prostaglandin F in the plasma despite the appearance of co-ordinated uterine contractions; the concentration of prostaglandin in the plasma was not raised until vaginal passage of the lambs. It is concluded that the synthesis or release of prostaglandins mediates the effects of changes in the levels of steroids in the maternal plasma on uterine contractility in sheep.
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SUMMARY
A technique for the continuous superfusion of small tissue samples in vitro has been applied to the study of prostaglandin production by ovine intra-uterine tissues. Basal and oxytocin-stimulated production of prostaglandins was studied at 120–125 days of pregnancy and after dexamethasone-induced delivery. In general, the relative rate of prostaglandin production by tissues was: foetal cotyledon = maternal cotyledon>myometrium and in quantitative order the prostaglandins produced were prostaglandin E (PGE) > prostaglandin F (PGF) = 13,14-dihydro-15-oxo-prostaglandin F (PGFM). Considerable variation was found between the rates of prostaglandin production in individual sheep. Oxytocin had no effect on the production of prostaglandins by tissues obtained before labour but myometrium and maternal cotyledon obtained at delivery exhibited a significant increase in production of PGE and PGF (though not PGFM) in response to oxytocin. Administration of arachidonic acid increased the production of PGE and PGF by the foetal cotyledon.
Queensland Health Pathology Service, Royal Brisbane and Women’s Hospital, Brisbane, Queensland 4029, Australia
Department of Endocrinology, Royal Brisbane and Women’s Hospital, Brisbane, Queensland 4029, Australia
Department of Obstetrics and Gynaecology, University of Queensland, Brisbane, Queensland 4029, Australia
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Queensland Health Pathology Service, Royal Brisbane and Women’s Hospital, Brisbane, Queensland 4029, Australia
Department of Endocrinology, Royal Brisbane and Women’s Hospital, Brisbane, Queensland 4029, Australia
Department of Obstetrics and Gynaecology, University of Queensland, Brisbane, Queensland 4029, Australia
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Queensland Health Pathology Service, Royal Brisbane and Women’s Hospital, Brisbane, Queensland 4029, Australia
Department of Endocrinology, Royal Brisbane and Women’s Hospital, Brisbane, Queensland 4029, Australia
Department of Obstetrics and Gynaecology, University of Queensland, Brisbane, Queensland 4029, Australia
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Verapamil inhibits tri-iodothyronine (T3) efflux from several cell types, suggesting the involvement of multidrug resistance-associated (MDR) proteins in T3 transport. The direct involvement of P-glycoprotein (P-gp) has not, however, been investigated. We compared the transport of 125I-T3 in MDCKII cells that had been transfected with mdr1 cDNA (MDCKII-MDR) versus wild-type MDCKII cells (MDCKII), and examined the effect of conventional (verapamil and nitrendipine) and specific MDR inhibitors (VX 853 and VX 710) on 125I-T3 efflux. We confirmed by Western blotting the enhanced expression of P-gp in MDCKII-MDR cells. The calculated rate of 125I-T3 efflux from MDCKII-MDR cells (around 0.30/min) was increased twofold compared with MDCKII cells (around 0.15/min). Overall, cellular accumulation of 125I-T3 was reduced by 26% in MDCKII-MDR cells compared with MDCKII cells, probably reflecting enhanced export of T3 from MDCKII-MDR cells rather than reduced cellular uptake, as P-gp typically exports substances from cells. Verapamil lowered the rate of 125I-T3 efflux from both MDCKII and MDCKII-MDR cells by 42% and 66% respectively, while nitrendipine reduced 125I-T3 efflux rate by 36% and 48% respectively, suggesting that both substances inhibited other cellular T3 transporters in addition to P-gp. The specific MDR inhibitors VX 853 and VX 710 had no effect of 125I-T3 efflux rate from wild-type MDCKII cells but reduced 125I-T3 export in MDCKII-MDR cells by 50% and 53% respectively. These results have provided the first direct evidence that P-gp exports thyroid hormone from cells.
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ABSTRACT
The chicken pituitary gland contains a number of naturally occurring, developmentally regulated forms of GH which have identical molecular weights but differ in their isoelectric points. In order to characterize their biological properties, each must be separated from non-GH proteins and other forms of GH. Chicken GH (cGH) was separated from other pituitary proteins by immunoaffinity chromatography using an anti-GH monoclonal antibody covalently linked to Sepharose 4B. The cGH eluted from this column as a single peak and migrated as a single band during sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), but showed multiple bands on isoelectric focussing. This material was chromatographed on a high-performance cation exchange column, and separation of charge isomers was monitored by a combination of isoelectric focussing and immunoblotting. Chicken GH eluted from this column in two distinct peaks. The minor peak (cGH P1) contained an isomer with an isoelectric point of 6·86 and the major peak (cGH P2) an isomer with an isoelectric point of 7·52. Each isomer migrated as a single band during isoelectric focussing and SDS-PAGE (M r = 23 500), and as a single peak during high-performance gel permeation chromatography and reverse-phase high-performance liquid chromatography. Analysis of cGH P2 through 30 cycles in a gas-phase microsequencer gave an amino acid sequence identical to that predicted by translation of the GH complementary DNA nucleotide sequence.
This single charge isomer increased the rate of lipolysis in chicken adipose tissue explants by about fourfold and was able to displace 125I-labelled cGH from binding sites in liver membranes with a dissociation constant of about 4 nmol/l. The output of insulin-like growth factor-I by hepatocytes in culture was increased from a basal rate of 50·4±11·6 (mean ± s.e.m.) to 787·9 ± 98·6 pg/6 × 106 cells per 48 h by two separate pulses of 1 μg cGH P2/ml. An i.v. injection of cGH P2 (15 μg/kg body weight) decreased the thyroxine:tri-iodothyronine ratio in serum of adult hens from 15·71 to 4·44, indicating an increase in 5′-monodeiodinase activity. These results demonstrate that the single most abundant charge isomer of chicken pituitary GH is likely to contain all the biological activity ascribed to the hormone.
Journal of Endocrinology (1990) 125, 207–215
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ABSTRACT
We investigated the uptake of l-tri-iodothyronine (T3) by cultured human trophoblast cells. Uptake was time-dependent, initially linear and approaching equilibrium after 60 min with an approximate half-time of 13 ± 4·5 min (mean ± s.e.m., n = 4). It had a non-saturable component accounting for about 50% of total uptake. We demonstrated a single saturable T3 uptake mechanism with a calculated Michaelis constant (K m) of 755 ± 145 nmol/l (n = 11–13) and a corresponding maximum velocity of 28·8 ± 5·3 pmol/min per mg protein (n = 11–13). The K m value was similar to those reported in other tissues.
Journal of Endocrinology (1992) 133, 483–486
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ABSTRACT
The ability of LHRH to induce Ca2+ mobilization and production of inositol phosphates in rat anterior pituitary tissue in vitro was investigated in relation to the self-priming effect of LHRH. Prior exposure to LHRH (which caused a characteristic potentiation of subsequent secretory responses) specifically enhanced LHRH-induced inositol phosphate production and mobilization of intracellular Ca2+ stores. LHRH-induced influx of Ca2+ through dihydropyridinesensitive Ca2+ channels was unaltered, as was ligand binding to LHRH receptors. These data suggest that a novel facilitation of signalling may occur in the phospho-inositide-Ca2+ mobilization response mechanism during LHRH priming, and that this may represent an important means of regulating cellular responsiveness in gonadotrophs.
J. Endocr. (1988) 119, 293–301
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Abstract
We have studied the uptake of 125I-thyroxine (125I-T4) in the human choriocarcinoma cell line JAR. Uptake of 125I-T4 was time-dependent, stereospecific and reversible, with a saturable component of 33% after 120 min of incubation. Kinetic analysis of the initial specific uptake rates indicated the presence of a single uptake process with a Michaelis constant of 59·4 ± 13·9 nm (n=12) and maximum velocity of 0·29 ± 0·06 pmol/min per mg protein. Uptake was dependent on intracellular energy as, in the presence of 2 mm potassium cyanide, saturable uptake was reduced to 60·6 ± 8·5% (n=4) of control uptake. Uptake was also temperature-dependent. Saturable 125I-T4 uptake after 60 min of incubation was 26·1 ± 3·0% at 25 °C (n=6) and 27·3 ± 5·7% at 4 °C of control uptake at 37 °C. Ouabain did not inhibit 125I-T4 uptake indicating that the uptake was independent of the Na gradient across the cell membrane. Although T4 uptake was stereospecific, as d-T4 failed to inhibit 125I-l-T4 uptake, it was not specific for T4, as tri-iodothyronine (T3) and reverse T3 also inhibited 125I-T4 uptake. We conclude that JAR cells have a saturable, stereospecific and reversible membrane transport mechanism for T4 which is dependent on intracellular energy, but independent of the Na+ gradient across the cell membrane.
Journal of Endocrinology (1995) 146, 233–238
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A specific radioimmunoassay for oxytocin has been established with a sensitivity of 0·8 pg/tube. This assay has been applied to the measurement of oxytocin in serial samples of peripheral plasma and amniotic fluid from pregnant rhesus monkeys, collected at weekly intervals by venepuncture and amniocentesis. Concentrations of oxytocin in both fluids were generally low and showed no trends throughout the latter half of gestation.
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The concentrations of 6-oxo-prostaglandin F1α (6-oxo-PGF1α) have been determined in maternal and foetal plasma from nine chronically catheterized sheep during late pregnancy and parturition. Labour occurred either spontaneously (three sheep) or was induced by continuous intrafoetal infusion of Synacthen (ACTH 1–24; 0·24 mg/24 h; three sheep) or dexamethasone (1 mg/24 h; three sheep). During spontaneous and Synacthen-induced parturition, concentrations of 6-oxo-PGF1α in maternal and foetal plasma remained at basal levels until 24 h before delivery. At varying times during the 24 h before delivery, levels of 6-oxo-PGF1α in maternal and foetal plasma were generally increased. When parturition was induced with dexamethasone, however, no increase was observed in the foetal plasma although the concentration of 6-oxo-PGF1α in maternal plasma was raised close to delivery.
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The concentration of thromboxane B2 has been measured in the plasma of women during late pregnancy, during term and pre-term labour, in women with pre-eclampsia and in umbilical cord arterial and venous plasma. In addition, the rates of production of thromboxane B2 in vitro were determined for placental tissues obtained after spontaneous vaginal delivery or elective Caesarean section. The results obtained indicate significant differences during parturition between the sources and controlling mechanisms of thromboxane and prostaglandin production.
