Search Results

SUMMARY

Specific binding of radio-iodinated ovine prolactin to subcellular tissue fractions of the tammar wallaby (Macropus eugenii) was investigated. Specific binding was found, in order of decreasing binding activity, in the lactating mammary gland, corpus luteum, corpus albicans, adrenal gland and ovary. Specific binding was absent in kidney, liver, brain and inactive mammary gland.

The mean association constant (K_a at 23 °C) was determined as 0·90 × 10⁹, 2·20 × 10⁹, 2·44 × 10⁹, 3·38 × 10⁹ and 10·98 × 10⁹1/mol for mammary gland, adrenal, corpus albicans, corpus luteum and ovary respectively. The mean receptor concentration (N) varied from 92·87 × 10⁻¹⁴ mol/mg protein for the mammary gland to 1·03 × 10⁻¹⁴ mol/mg protein for the ovary. The concentration in the corpus luteum varied between tissue pools collected at different times of the annual breeding cycle.

The specificity for prolactin was shown in the mammary gland and corpus luteum by the failure of ovine FSH, LH, GH and TSH to displace ¹²⁵I-labelled ovine prolactin, whereas it was displaced readily by both ovine and bovine prolactin.

Abstract

In this study oxytocin (OT) receptors have been characterized and localized in the testis of the rat using the radioiodinated OT receptor antagonist ¹²⁵I-labelled d(CH₂)⁵ [Tyr(Me)²,Thr⁴,Tyr⁹-NH₂]-vasotocin (OTA). Receptor density and localization have been compared with the rat testis arginine vasopressin (AVP) receptor using the radioiodinated AVP V_1a receptor antagonist ¹²⁵I-labelled d(CH₂)₅Sar⁷-AVP and the radioiodinated linear AVP V_1a antagonist ¹²⁵I-labelled [(C₆H₅-CH₂CO)-O-methyl-d-Tyr-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH₂]. ¹²⁵I-labelled OTA bound with high affinity to membrane fractions of the rat testis (K _a = 13·8 ± 1·25 litres/nmol), mammary tissue (K _a=20·3± 4·36 litres/nmol) and uterus (K _a=27·8±0·74 litres/nmol). Competition studies with various OT and AVP receptor agonists and antagonists confirmed that the binding was to OT receptors. AVP receptors in the testis were found to be identical to AVP V_1a receptors in the liver. The AVP receptor density in the testis was much higher than the OT receptor density (109 ±12·3 vs 5·2 ±0·79 (mean ± s.e.m.) fmol/mg protein). Autoradiographical localization showed that both OT and AVP receptors were present in the interstitial spaces in the testis consistent with binding to Leydig cells. AVP receptors were also localized on the epithelial surfaces of the seminiferous tubules and on testicular blood vessels. This study has, for the first time, found OT receptors in the testis of the rat which have similar ligand-binding characteristics to mammary and uterine OT receptors. The receptor localizations are consistent with binding to Leydig cells.

Journal of Endocrinology (1994) 141, 343–352

Abstract

In this study arginine vasopressin (AVP) and oxytocin (OT) receptors have been characterized in the brushtail possum. AVP receptors were characterized using [³H]AVP and the radioiodinated AVP V_1a receptor antagonist ¹²⁵I-labelled [(C₆H₅-CH₂CO)-O-methyl-d-Tyr-Phe-Gln-Asn-Arg-Pro-Arg-Tyr- NH₂] while OT receptors were characterized using the radioiodinated OT receptor antagonist ¹²⁵I-labelled d(CH₂)₅[Tyr(Me)²,Thr⁴,Orn⁸, Tyr-NH₂ ⁹]-vasotocin. The receptor affinities and densities have been compared with the rat AVP and OT receptors. Low densities of OT receptors were present in the possum ovary and kidney. High densities of AVP-binding sites were found in the possum adrenal, testis, mesenteric artery, ovary and renal medulla and lower densities in the possum liver. The AVP-binding sites showed marked differences in ligand-binding characteristics from the rat AVP V_1a and V₂ receptors. Receptor affinities were similar between tissues, except for a distinctly lower value in the renal medulla. It is concluded that the brushtail possum expresses AVP receptors with distinct ligand specificities from those of the rat AVP V_1a and V₂ receptors.

Journal of Endocrinology (1995) 144, 19–29

ABSTRACT

Previous studies on the hormonal regulation of hepatic angiotensinogen relied on in-vitro liver preparations and on the measurement of changes in plasma concentration. In this study ¹²⁵I-labelled angiotensinogen was used to measure simultaneously the production rate (PR) and metabolic clearance rate (MCR) in conscious rats by the constant-rate infusion and single-injection methods. Male rats received daily s.c. injections of isotonic saline (as control), 1 mg corticosterone acetate (CA), 25 μg 17β-oestradiol benzoate (OB) or 20 μg thyroxine (T₄) per 100 g body weight. On day 7 of treatment ¹²⁵I-labelled angiotensinogen was infused into a jugular vein at a rate of 1 μl/h by osmotic minipumps and blood samples taken 4, 5 and 6 days later. The PR of angiotensinogen increased from 576 ± 28 (s.e.m.; n = 9) to 954 ± 63 (n = 9), 1010±84 (n = 9) and 2359±150 (n = 10) μg/h per kg following treatment with CA, OB and T₄ respectively. In contrast, the PR of rat albumin did not change significantly from 218 ± 8 (n = 7) mg/h per kg. All three hormones increased MCR from 13 ± 1 (n = 17) ml/h per kg to 17± 1 (n = 9), 18 ± 2 (n = 9) and 27 ± 2 (n = 9) ml/h per kg for CA, OB and T₄ respectively. Single-injection experiments on five rats showed angiotensinogen to be distributed into three compartments with a half-time of disappearance of 4·4 ± 1 min, 116±11 min and 13·1 ±2·6 h. It was concluded that the production of angiotensinogen in vivo is at least tenfold higher than the reported in-vitro rates, that the clearance of angiotensinogen is under hormonal regulation and that angiotensinogen is distributed between at least three compartments.

J. Endocr. (1987) 112, 391–397

The effects of injections of cortisol, corticosterone and ACTH on indices of carbohydrate, fat and protein metabolism were investigated in the conscious echidna, Tachyglossus aculeatus.

Intravenous infusion of cortisol and corticosterone for 2 h at rates of 3 and 30 μg/kg/h respectively did not cause significant changes in the plasma concentrations of glucose, urea or amino acids during a 12·5 h observation period. In contrast, a dose-related increase in plasma free fatty acid (FFA) concentration was observed. Infusion of synthetic ACTH at 2 i.u./kg/h for 2 h caused a minor, short-lived increase in FFA concentration.

Daily i.m. injections of 0·2 mg cortisol or corticosterone acetates/kg, which raised plasma total corticosteroid concentrations to levels characteristic of maximal ACTH stimulation, did not cause glycosuria nor was there any change in body weight, nitrogen intake or urinary nitrogen excretion. However, there was a minor, but significant, increase in plasma glucose concentration. The liver glycogen content of 24 h fasted, corticosteroid-treated animals was similar to that of fasted control animals.

It is concluded that cortisol, corticosterone and ACTH have only minor effects on carbohydrate and protein metabolism and that the main action of these hormones may be to mobilize fat reserves.

SUMMARY

The peripheral plasma concentrations and production rates of corticosterone and cortisol were measured in the conscious, unrestrained echidna (Tachyglossus aculeatus) under basal conditions and during maximal ACTH stimulation.

Using Sephadex LH-20 column chromatography and radioligand assay, only cortisol and corticosterone could be detected in the peripheral blood plasma at very low concentrations of 0·07 ± 0·03 (s.e.m.) μg/100 ml and 0·14 ± 0·07 μg/100 ml respectively. Two-hourly sampling over periods of 36–48 h disclosed a diurnal periodicity in the combined plasma concentration of these corticosteroids, the high concentrations corresponding to periods of behavioural activity. Marked, short-term fluctuations in plasma corticosteroid concentration were also observed during periods of more frequent (20 min) sampling. Constant rate i.v. infusion of synthetic ACTH increased the plasma concentrations of both steroids to maximal values of 0·42 ± 0·23 μg cortisol/100 ml and 1·06 ± 0·56 μg corticosterone/100 ml at infusion rates of 1 i.u. ACTH/kg/h. This is approximately 1/160 of the potency of this ACTH in man.

The production rates of corticosterone and cortisol, measured by isotope dilution during constant rate i.v. infusion of ³H-labelled tracers, were only 0·35 ± 0·21 and 0·56 ± 0·26 μg/kg/h respectively during saline infusion, and increased to 2·86 ± 3·47 and 2·74 ± 2·07 μg/kg/h during the infusion of 1 i.u. ACTH/kg/h. The metabolic clearance rate of cortisol was greater than that of corticosterone and both were depressed by ACTH.

Plasma corticosteroid concentrations were increased after surgery during ether anaesthesia and in sick animals with heavy worm infestation. It is concluded that the adrenal cortex of echidnas responds to ACTH stimulation and stress in a similar way to eutherians, but the level of activity is much lower.

Abstract

While there have been reports on changes in the renin–angiotensin system and angiotensin II (AT) receptors in diabetes, there is no agreement on the nature of these changes. This study has characterised specific AT receptors in the heart, kidney, liver and adrenal glands of the streptozotocin (STZ)-diabetic rat using radioligand binding studies with the radioligand ¹²⁵I-[Sar¹,Ile⁸]-angiotensin II. Left ventricular AT receptor density increased by 135% 4 weeks after treatment and by 206% 12 weeks after treatment; in the liver, AT receptor density increased by 476% (4 weeks) and 263% (12 weeks) and in the adrenal gland by 236% (4 weeks) and 109% (12 weeks). In contrast, renal AT receptor density decreased by 49% (4 weeks) and 36% (12 weeks). Competition-displacement assays with losartan, an AT1-selective ligand, showed that the proportion of AT receptor subtypes remained unchanged. STZ treatment decreased plasma angiotensinogen by 72% (4 weeks) and 67% (12 weeks) and increased plasma renin concentration after 12 weeks; plasma renin activity and aldosterone concentrations remained unchanged. Treatment with human insulin (5 U/day) attenuated changes in plasma angiotensinogen and AT receptor density except in the left ventricle. We conclude that there are major changes in AT receptors in the STZ-diabetic rat that are tissue-specific and time-dependent. Plasma angiotensinogen and renin secretion change in directions that result in the maintenance of plasma renin activity and aldosterone concentration.

Journal of Endocrinology (1997) 154, 355–362

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are known to stimulate adenylate cyclase activity in rat pituitary cells but no direct effects have been reported on sheep pituitary cells. In this study we determined whether either peptide could stimulate intracellular cAMP accumulation in dispersed sheep pituitary cells in primary culture. Time course studies with PACAP showed that tachyphylaxis developed rapidly and so a short incubation time (5 min) was used to define the dose–response relationship. PACAP dose-dependently stimulated intracellular cAMP levels with a half-maximum response at 2·9 ± 0·2 nmol/l (n=4). In contrast, VIP only caused a small increase in intracellular cAMP levels at the highest dose tested (1 μmol/l). The VIP antagonist [4C1-d-Phe⁶,Leu¹⁷]VIP had no effect on the cAMP response to either PACAP or VIP while the peptide PACAP(6–38), a putative PACAP antagonist, blocked the cAMP response to PACAP. The desensitisation to PACAP was further investigated by pretreating cells with PACAP for 30 min. After a further 15 min in culture medium alone, these cells showed no cAMP response to subsequent treatment with PACAP but could respond to forskolin. When a longer incubation period of 240 min was used between the first and second treatment with PACAP, a partial return in responsiveness to PACAP was observed. In summary, these results show that PACAP activates adenylate cyclase in sheep pituitary cells but that there is rapid development of tachyphylaxis. Experiments with the antagonists suggest that the response to PACAP is via the PACAP type I receptor. In contrast, physiological doses of VIP do not stimulate cAMP accumulation in sheep pituitary cells.

Journal of Endocrinology (1996) 148, 545–552

ABSTRACT

There is inconclusive evidence that oxytocin acts directly on the corpus luteum and affects steroidogenesis. Since any such action would probably be mediated by oxytocin receptors, these should be present in luteal tissue. In this study, homogenates of corpora lutea from both pregnant and non-pregnant ewes were examined for oxytocin receptors by radio-receptor assay. Specific oxytocin binding was not observed in luteal tissue during the oestrous cycle. However specific binding was found in the corpora lutea of pregnant ewes; appearing at a fetal head length of approximately 0·65 cm (about 30 days of pregnancy) and persisting to a head size of 11 cm, the largest size examined in this study. The affinity (K _d) of the receptor was calculated as 2·9 ± 0·3 nmol/l (s.e.m.; n = 9), a value similar to that obtained for the uterus. The receptor number ranged from a low of 8·7± 3·2 fmol/mg protein (n = 6) at a head size of <0·65 cm, to a maximum of 40·1 ± 6·5 fmol/mg protein (n = 25) at a head size of 2·5–3·75 cm. These values were lower than our estimate of 588 ± 39 fmol/mg protein (n = 5) for the uterus. It is concluded that a direct action of oxytocin on the corpus luteum is possible but only after the first month of pregnancy and not in the corpus luteum of the oestrous cycle.

Journal of Endocrinology (1989) 121, 117–123