bluuebunny/crossref_metadata_2025 huggingface dataset:
Catecholamines and ascorbic acid as stimulators of bovine ovarian oxytocin secretion
Authors: Luck M. R. , Jungclas B.
Published: December 2008

The effects of catecholamines and ascorbic acid on cultured bovine granulosa cells have been examined to assess their possible role in the initiation and maintenance of luteal oxytocin secretion. The actions of these agents have also been compared with the previously reported ability of follicular theca tissue to enhance oxytocin secretion.

Using granulosa cells cultured in serum-supplemented medium, we observed a highly significant enhancement of oxytocin secretion in the presence of adrenaline and noradrenaline, particularly over the concentration range 1–10 μmol/l. This effect was accompanied by smaller and less consistent changes in progesterone secretion and did not involve any change in the time-course of oxytocin secretion. Acetylcholine was without effect. Ascorbic acid stimulated oxytocin secretion when used alone over a range of concentrations, but was also able to synergize with adrenaline. Lactic acid was ineffective. The stimulation of oxytocin secretion by adrenaline could be blocked by equimolar propranolol, but the stimulation of progesterone was not blocked. Propranolol had a variable effect on the ability of theca tissue to stimulate oxytocin secretion by granulosa cells but the results also suggested the presence of some β-agonistic activity in the culture medium.

We conclude, first, that catecholamines may be involved in the regulation of ovarian oxytocin secretion, secondly, that ascorbate may regulate oxytocin secretion through its involvement in the biosynthesis of oxytocin but also through interaction with catecholamines and, thirdly, that the stimulatory action of theca tissue probably does not involve the action of β-agonists.

J. Endocr. (1987) 114, 423–430
Passive immunization of pregnant goats against ovine LH
Authors: Malecki J. , Jenkin G. , Thorburn G. D.
Published: December 2008

Groups of three goats at 50, 90 and 130 days of gestation were passively immunized against ovine LH (oLH) by i.v. infusion of 8 ml serum equivalent of the immunoglobulin fraction of rabbit anti-oLH serum (LHAS). Goats at the same stages of gestation as above served as controls and received 8 ml serum equivalent of the immunoglobulin fraction of normal rabbit serum (NRS). Plasma concentrations of progesterone were determined by specific radioimmunoassay of blood collected at 20-min intervals from 6 h before infusion of LHAS or NRS to 12 h after infusion. Less frequent sampling was performed from 2 days before to 6 days after infusion. Plasma from all LHAS-immunized goats exhibited binding of oLH. Twelve hours after immunization, titres ranged from 1:135 to 1:215. All LHAS-treated goats had titres of less than 1:10 by 5 days after immunization, but a low level of oLH binding was still detectable. Treatment with LHAS or NRS did not shorten the length of gestation, with all goats delivering live offspring between 142 and 147 days after conception. Plasma concentrations of LH ranged from less than 0·15 μg/l to 4·8 μg/l and were greater than 0·15 μg/l in 181 of 255 samples (71%) for both the NRS-treated group, throughout the experiment, and the LHAS-treated groups before infusion of antiserum. Luteinizing hormone was not detectable in plasma samples obtained after LHAS infusion in goats at 50 or 130 days of pregnancy. Plasma concentrations of LH exceeded 0·15 μg/l in only five of 51 (10%) samples in 90-day-pregnant goats treated with LHAS, the maximum value reached being 0·80 μg/l.

Plasma concentrations of progesterone did not alter significantly after infusion of LHAS or NRS at 50, 90 or 130 days of gestation, nor were they significantly different between the groups treated with LHAS and NRS.

The role of LH in the maintenance of pregnancy in the goat after 50 days of gestation is questioned.

J. Endocr. (1987) 114, 413–436
Thyroid hormones regulate the amount of thyrotrophin-releasing hormone in the hypothalamic median eminence of the rat
Authors: Mori M. , Yamada M.
Published: December 2008

The negative-feedback action of thyroid hormones on TRH in the hypothalamic median eminence was examined. Thyroidectomy caused a progressive decrease in TRH content of the median eminence, but not of the whole hypothalamus. In contrast, it did not affect the LHRH content of the median eminence. Administration of thyroid hormone prevented the decrease in TRH content of the median eminence after thyroidectomy. Destruction of the hypothalamic paraventricular nucleus (PVN) led to a significant reduction in TRH content of the median eminence in normal and thyroidectomized rats. These data provide evidence that thyroid hormones regulate directly the amount of median eminence TRH which is derived from the hypothalamic PVN in the rat.

J. Endocr. (1987) 114, 443–448
Comparison of receptors for insulin-like growth factor II from various rat tissues
Authors: Taylor J. E. , Scott C. D. , Baxter R. C.
Published: December 2008

Receptors for insulin-like growth factor II (IGF-II) have been compared in solubilized microsomal membranes from rat lung, brain, kidney, heart, epididymal and subcutaneous fat, ovary, testis and adrenals. Highest binding/μg protein was seen with testicular membranes. Receptors from all tissues showed high affinity for human IGF-II (mean association constant = 65 litres/nmol) and a high degree of specificity (mean IGF-I cross-reactivity 0·3%; no cross-reactivity with insulin). Affinity labelling followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis showed binding was only to a type-II IGF receptor, with a major band seen at a molecular weight of about 230 000 in lung, brain, kidney and testis, and 240 000 in heart, fat and adrenal gland. All tissues showed broad or bimodal pH dependence of binding, with optima seen at about pH 6 and pH 9. Mild stimulation of IGF-II binding by low calcium concentrations (1–2 mmol/l) was seen in all tissues, although higher concentrations were inhibitory in the brain. It was concluded that IGF-II receptors from different rat tissues, when studied under uniform conditions, show similar binding affinities but differences in size and regulation which might be missed if receptors are examined in separate studies.

J. Endocr. (1987) 115, 35–41
Endocrine control of plasma concentrations of calcium-binding protein in the pig
Authors: Maunder E. M. W. , Pillay A. V. , Care A. D.
Published: December 2008

The aetiology of the rise in plasma calbindin-D9k (vitamin D-induced calcium-binding protein; CaBP), following insulin-induced hypoglycaemia, was studied in the pig. ACTH led to a rise in plasma concentrations of both CaBP and cortisol. Metyrapone, which blocks cortisol synthesis, abolished the increases in plasma concentrations of CaBP and cortisol normally observed in response to insulin-induced hypoglycaemia. However, there was no significant rise in plasma concentrations of CaBP in response to pharmacological or physiological doses of cortisol.

Injection of clonidine, an α2-adrenergic agonist, led to a rise in plasma concentrations of CaBP, whereas phenylephrine, an α1-adrenergic agonist, tended to exert an inhibitory effect. Also, administration of phentolamine (an α-adrenergic blocker) before injection of insulin abolished the usual increase in plasma concentrations of CaBP, whereas propranolol (a β-adrenergic blocker) enhanced the normal increase in plasma concentrations of CaBP in response to insulin-induced hypoglycaemia. Isoproterenol, a β-adrenergic agonist, was without effect on plasma CaBP. Neither GH nor glucagon appear to be involved in the rise in plasma CaBP following insulin-induced hypoglycaemia. Although atropine abolished the effect of acute hypoglycaemia on plasma CaBP, carbamylcholine was without effect on plasma CaBP concentration. It is concluded that the increases in plasma CaBP induced by either ACTH or α2-adrenergic stimulation may be interrelated since the administration of ACTH can lead to raised plasma concentrations of catecholamines.

J. Endocr. (1987) 115, 121–128
Maintenance of neonatal rat B cells in glucose-depleted medium: a perifusion study
Authors: Wakabayashi S. , Kagawa S. , Nakao K. , Matsuoka A.
Published: December 2008

Two groups of monolayer cultures of pancreatic cells from the neonatal rat were maintained in glucosedepleted TCM 199 medium, supplemented with 5·5 mmol galactose/l, with or without 0·1 mmol 2-deoxyglucose/1. Another group was kept in medium with 5·5 mmol galactose/l alone, following exposure for 2 days to a medium with 5·5 mmol galactose/1 and 10 μmol iodoacetic acid/l to kill fibroblasts selectively. Each of these monolayers was cultured in a perifusion system for a total of 7 days so that phasic insulin secretion could be compared. On day 0, B cells responded in a monophasic fashion to acute challenge with 16·7 mmol glucose/l whereas, in the presence of 10 μmol forskolin/1 and 1 mmol 3-isobutyl-1-methyl-xanthine/l, the same dose of glucose stimulated a biphasic response of approximately the same magnitude. At a concentration of 10 mmol/l, leucine and 2-ketoisocaproate both produced only minimal increases in the second phase of secretion above the basal level. No response to secretagogues was seen under culture conditions without 2-deoxyglucose. In contrast, addition of 2-deoxyglucose to the galactose-supplemented medium stimulated B cells to secrete insulin in a biphasic fashion in response to a single dose of glucose, and the stimulatory effects of leucine and 2-ketoisocaproate were also remarkably increased. Moreover, when exposed to a linear concentration gradient of glucose, leucine or 2-ketoisocaproate, these B cells responded to secretagogues in a dose-dependent fashion. On the other hand, B cells which had been exposed briefly to iodoacetic acid and then cultured in medium with galactose alone, showed a biphasic response to glucose, leucine and 2-ketoisocaproate; however, quantitative relationships differed. Thus the response (total insulin secreted during a 30-min stimulation) of B cells grown in 2-deoxyglucose to glucose was 610%, to leucine 504% and to 2-ketoisocaproate 810% of that of cells exposed to iodoacetic acid. The present data suggest that cultivation of neonatal B cells in a glucose-depleted medium containing 2-deoxyglucose may cause functional maturation.

J. Endocr. (1987) 115,169–175
Studies of the parathyroid hormone gene in normal subjects, and in subjects with primary hyperparathyroidism and familial benign hypercalcaemia
Authors: Almahroos G. M. , Docherty K. , Fletcher J. A. , Webb T. , Heath D. A.
Published: December 2008

Familial benign hypercalcaemia (FBH) closely resembles primary hyperparathyroidism (PHPT) both clinically and biochemically. Using a cDNA probe for the parathyroid hormone (PTH) gene we have studied restriction fragment length polymorphisms in normal British subjects and have shown them to be similar to those found in previous studies in a German population. The pattern of inheritance of these restriction fragment length polymorphisms in a family with FBH shows that the PTH gene is not involved in the pathogenesis of the condition. Limited studies in PHPT indicate that it is unlikely that a major structural defect or rearrangement is responsible for the sporadic form of the disease.

J. Endocr. (1987) 115, 183–186
A decrease in FSH receptors of granulosa cells during follicular maturation in the domestic hen
Authors: Ritzhaupt L. K. , Bahr J. M.
Published: December 2008

The follicles of the ovary in the domestic hen are arranged in a hierarchy. Responsiveness of the adenylyl cyclase enzyme system of the granulosa cells to FSH decreases as follicles proceed towards ovulation. To test the hypothesis that this decline in FSH responsiveness could be the result of a decrease in FSH receptor numbers, an FSH receptor assay was characterized for chicken granulosa cells and used to measure receptor number and affinity of the largest (F1), third largest (F3) and fifth largest (F5) follicles removed 18 h before ovulation. The numbers of binding sites for F1, F3 and F5 follicles (n = 4) were 0·22 ± 0·05, 0·5 ± 0·14 and 1·22 ± 0·27 pmol hormone bound/mg protein respectively, and were significantly (P < 0·001) different among follicles. The apparent association constants for the F1, F3 and F5 follicles were not different and had a value of 23·4 ± 4·9 litres/nmol (n = 12). Our results indicate that FSH receptor numbers decrease in granulosa cells without a change in affinity as follicles approach ovulation. The decrease in FSH receptor numbers is associated with the reported decline in FSH-stimulated steroidogenesis and adenylyl cyclase activity which occurs during follicular maturation.

J. Endocr. (1987) 115, 303–310
Purification of ovine somatotrophs using a combination of density gradient centrifugation and short-term culture
Authors: Gomm J. J. , Ray K. P. , Wallis M.
Published: December 2008

Freshly dispersed or cultured (18 h) ovine anterior pituitary cells were fractionated on 55% sigmoidal Percoll gradients. This resulted in the separation of two sub-populations of cells at densities of 1·075 g/ml (peak I) and 1·055 g/ml (peak II). Radioimmunoassay of fractions from a gradient on which cells were separated before culture showed the profiles of GH and prolactin to be virtually superimposable. After culture, however, the lactotroph population exhibited an apparent shift in its density profile, the lighter population increasing at the expense of the denser one. Immunocytochemistry of the cells remaining in the denser peak (peak I) showed that it consisted of about 65% somatotrophs which was approximately a three-fold enrichment over the proportion of somatotrophs present in the original cell preparation (24%). Refractionation studies indicated that the change in the density profile of the lactotrophs was due to an actual reduction in their density rather than a loss of viability of the denser sub-population. Secretion data showed the purified somatotrophs to be more responsive than the crude cell preparation to the regulatory factors, GH-releasing hormone and somatostatin. This enriched cell population should prove useful in the detailed study of GH secretion.

J. Endocr. (1987) 115, 395–403
Opioidergic modulation of the oestradiol-induced LH surge in the rat: roles of ovarian steroids
Authors: Lustig R. H. , Pfaff D. W. , Fishman J.
Published: December 2008

Sex steroids convey information on the status of the reproductive system, which the brain is able to integrate to promote ovulation, in the form of the LH surge. The present studies examined the influence of alterations in central opioidergic tone to initiate the LH surge, and the roles of oestradiol and progesterone to effect changes in opioidergic tone, by antagonizing this activity using either naloxone or nalmefene (N-cyclopropylmethyl-6-desoxy-6-methylene-noroxymorphone), a long-acting μ- and κ-opiate antagonist. The timing and amplitude of the LH surge was examined in (1) cyclic rats in pro-oestrus and (2) ovariectomized rats with varying doses of oestradiol supplementation. Plasma was obtained hourly through an indwelling intra-atrial catheter between 13.00 and 19.00 h, and later assayed for LH and oestradiol concentrations by radioimmunoassay.

Rats treated with either nalmefene or progesterone on pro-oestrus demonstrated similar advances in the time of initiation of the LH surge by 1–2 h compared with control rats. The effects of nalmefene and progesterone were evident within 2 and 3–5 h of their administration respectively. Conversely, rats treated with progesterone on dioestrus demonstrated low prooestrous oestradiol levels and abolition of the prooestrous LH surge, but continuous naloxone infusion restored the pro-oestrous LH surge, with raised oestradiol concentrations. In ovariectomized rats without oestradiol supplentation, nalmefene alone was able to increase basal LH levels, but unable to facilitate a spontaneous rise in LH amplitude indicative of an LH surge. Supplementation with low doses of oestradiol was itself ineffective in facilitating a spontaneous rise in LH concentration, but nalmefene co-administration significantly potentiated the ability of low doses of oestradiol to induce augmented LH secretion, in addition to advancing the timing of the spontaneous LH rise. Similarly, progesterone co-administration to ovariectomized, oestradiol-primed rats significantly advanced and augmented LH hypersecretion.

The results of these experiments are consistent with the concept that central opioidergic systems normally restrain the initiation of the LH surge, and that blocking opiate receptors removes this inhibition. They advance the hypothesis that oestradiol, the essential signal for LH surge induction, has, as one consequence of its action, the time-specific inhibition of hypothalamic opioidergic tone in the afternoon, which would otherwise restrain the LH surge-generating mechanism. Finally, the biphasic effect of progesterone on the LH surge appears to be mediated through dichotomous actions on opioidergic tone; in the highoestrogen state, progesterone advances LH surge timing, mimicking the effect of an opiate antagonist, but in the low-oestrogen state, progesterone abolishes the subsequent LH surge, and its effect is negated by an opiate antagonist.

J. Endocr. (1988) 116, 55–69
Androgen metabolism and regulation of rat ventral prostate growth and acid phosphatase during sexual maturation
Authors: Orlowski J. , Bird C. E. , Clark A. F.
Published: December 2008

Androgen metabolism and the regulation of rat ventral prostate cell proliferation and secretory function were examined during sexual maturation. Changes in acid phosphatase (AP) characteristics were measured as a marker of androgen-dependent prostatic secretory function. In immature (21-day-old) rats, total AP activity per cell was low (14.2±1.3 mol p-nitrophenol phosphate hydrolysed/h per mg DNA); it increased threefold as the weight, protein and DNA contents of the prostate increased to adult (65-day) levels. This corresponded with significant (P<0.001) increases in the staining intensities of three of the four bands of secretory AP on isoelectric focusing gels. The extent of inhibition of AP by tartrate decreased at the same time. Secretory AP is known to be relatively tartrate-resistant. The changes in AP activity occurred after prostatic 5α-dihydrotestosterone (5α-DHT) levels increased from 4.6 ± 0.7 pmol/mg DNA (21 days) to reach a peak of 17.6±2.3 pmol/mg DNA at 58 days. Prostatic 5α-DHT concentrations were always higher than testosterone levels. Prostatic 5α-androstane-3α,17β-diol (3α-Adiol) levels were lower than 5α-DHT levels except on day 58 when levels peaked dramatically at 26.2±5.5 pmol/mg DNA. Changes in prostatic 5α-DHT and 3α-Adiol levels corresponded with changes in 5α-reductase and 3α-hydroxysteroid oxidoreductase (3α-HSOR) activities. The oxidative reaction of 3α-HSOR was approximately fourfold higher than the reductive reaction, indicating a preference for the formation of 5α-DHT. The plasma levels of testosterone, 5α-DHT and 3α-Adiol cannot account for their respective prostatic levels, indicating the importance of the steroid-metabolizing enzymes in regulating intracellular androgen levels. Changes in the AP characteristics could be correlated with the androgen status of the prostate.

J. Endocr. (1988) 116,81-90
Effect of hypothyroidism on the in-vivo and in-vitro uptake of sugar by rat thymocytes and on the stimulatory response to 3,5,3′-tri-l-iodothyronine
Authors: Segal J.
Published: December 2008

We have previously demonstrated, both in vivo and in vitro, that 3,5,3′-tri-l-iodothyronine (T3) increases the uptake of the glucose analogue 2-deoxy-d-glucose (2-DG) in rat thymocytes by acting at the level of the plasma membrane. In the present study, the effect of hypothyroidism on the basal uptake of 2-DG by rat thymocytes and their response to T3 was examined.

Rats were rendered hypothyroid by thyroidectomy at 21 days of age, and experiments performed with 31-and 56-day-old animals. Uptake of 2-DG by thymocytes from hypothyroid rats, both in vivo and in vitro, was greater in 31-day-old animals and less in 56-day-old animals than that in euthyroid rats. In both age-groups, hypothyroidism increased cellular responsiveness to T3, shifting the dose–response curve to the left. Similar results were obtained in experiments in which animals were rendered hypothyroid by KClO4. Injection of thyroid hormones into rats treated with KClO4 reversed the effects of hypothyroidism on uptake of [3H]2-DG by thymocytes and their response to T3. From these observations it was concluded that hypothyroidism produces a time-dependent change in basal sugar uptake by rat thymocytes, and increases cellular responsiveness to the effect of T3 at the level of the plasma membrane.

J. Endocr. (1988) 116, 107–114
Further evidence that N-terminal pro-opiomelanocortin peptides are involved in adrenal mitogenesis
Authors: Estivariz F. E. , Carino M. , Lowry P. J. , Jackson S.
Published: December 2008

In order to demonstrate the mitogenic effects of N-terminal pro-opiomelanocortin (N-POMC) peptides on the adrenal glands further, female rats with bilateral adrenal enucleation (hereafter referred to as enucleation) were hypophysectomized 11 days after enucleation and injected twice daily with 5 μg purified human N-POMC(1–28), ACTH(1–24) or 0·9% (w/v) NaCl. On day 14 after enucleation, rats were injected with colchicine and, after killing, their adrenal glands weighed, fixed and mitotic counts in histological sections assessed. Plasma corticosterone was measured fluorometrically. In other experiments, rats 7 days after enucleation were hypophysectomized and implanted with osmotic minipumps delivering 5 μg purified N-POMC(1–28) per day. On day 14 after enucleation, animals were treated as above. Collagenase-dispersed adrenal cells were incubated with purified or synthetic N-POMC(1–28) or synthetic N-POMC (1–36) (1–300 nmol/l) and [3H]thymidine incorporation into DNA was determined. Intact female rats were implanted with osmotic minipumps delivering 8 μg purified or synthetic N-POMC(1–28)/day, 8 μg synthetic N-POMC(1–36)/day or saline alone. Mitotic counts were performed on histological sections.

Both s.c. injection or continuous delivery from minipumps of purified N-POMC(1–28) partially prevented the atrophy of regenerating adrenal glands after hypophysectomy (s.c. injection of N-POMC: 2·29 ± 0·92 mitoses/section compared with 0·52 ± 0·39 for controls; minipump delivery: 5·02 ± 0·97 compared with 0·13 ± 0·05; P < 0·01 for both experiments). ACTH did not augment mitotic activity in enucleated– hypophysectomized rats but significantly increased plasma concentrations of corticosterone in s.c. injection experiments. Adrenal weights were not significantly changed. Both purified and synthetic N-POMC(1–28) increased thymidine incorporation in incubated adrenal cells, the synthetic peptide being less potent than the purified peptide. Whilst synthetic and purified N-POMC(1–28) significantly increased mitotic counts in histological sections of intact adrenal glands after chronic delivery from minipumps, N-POMC(1–36) had no significant effect.

It was concluded that N-POMC(1–28) can partially reverse the adrenal atrophy in enucleated rats after hypophysectomy, whereas ACTH is mitotically inactive in the same situation. The low potency of the synthetic peptides compared with purified N-POMC(1–28) preparations reported in this study could be due to differences in secondary structure.

J. Endocr. (1988) 116, 201–206
The time-course of oxytocin secretion from cultured bovine granulosa cells, stimulated by ascorbate and catecholamines
Authors: Luck M. R. , Jungclas B.
Published: December 2008

Bovine granulosa cells secrete oxytocin when cultured in a serum-supplemented medium. The time-course of secretion is similar to that in the early corpus luteum in vivo, with a delay of 1 to 2 days followed by a peak and decline over the first 5 days of culture. We have investigated the basis of this time-course in vitro and studied the temporal characteristics of the stimulatory actions of ascorbic acid and adrenaline on this process.

Cells cultured on stirred microcarriers showed a similar pattern of secretion of oxytocin to those cultured on conventional flat plates, despite continuing and rapid mitosis. This indicated that the secretion profile in conventional culture was not an artifact related to the cessation of mitosis. Furthermore, secretion of oxytocin and progesterone by cells on microcarriers was stimulated without a corresponding change in mitotic rate, showing that the secretion per cell had been increased.

In conventional culture, addition of ascorbic acid to culture media (0·5 mmol/l) increased the secretion of oxytocin (up to 4·5-fold) but only if ascorbic acid was present during the first day of culture. The cells showed a progressive refractoriness to stimulation after 12 h. Since the time-course of secretion was unaltered by treatment, this resulted in a delay of 1 to 2 days before the action of the ascorbate was seen. The secretion of progesterone was similarly affected but with less stimulation and less consistency. In contrast, cells treated with adrenaline (10 μmol/l) secreted more oxytocin on the day of treatment and did so at any time during culture provided that there was sufficient basal secretion of hormone. Adrenaline also failed to alter the time-course of secretion but treated cells showed a persistent response, maintaining enhanced secretion for up to 3 days after the adrenaline had been removed.

Ascorbate and adrenaline were highly synergistic in their effects, provided that the ascorbate was present from the start of culture; the response to adrenaline strongly reflected the degree of ascorbate stimulation. We conclude that granulosa cells secrete oxytocin according to an inherent time-schedule and that there is a limited period during which they can respond to ascorbate. Since ascorbate is required for the biosynthesis of oxytocin, this suggests that the availability of ascorbate during corpus luteum formation may determine the amount of oxytocin which can be released subsequently in response to catecholamines.

J. Endocr. (1988) 116, 247–258
Calcitonin inhibition of prolactin secretion in isolated rat pituitary cells
Authors: Shah G. V. , Epand R. M. , Orlowski R. C.
Published: December 2008

Salmon calcitonin inhibited TRH-stimulated release of prolactin in isolated pituitary cells from untreated female rats. These cells were still capable of responding to the fresh addition of TRH after the removal of calcitonin. Calcitonin gene-related peptide had only a weak effect in inhibiting prolactin release in these cells.

Pituitary cells isolated from female rats which had been treated with weekly s.c. injections of 1 mg oestradiol dipropionate for 4 weeks, exhibited a marked increase in the magnitude of the inhibition of prolactin release by salmon calcitonin. Both basal and TRH-stimulated release of prolactin were inhibited by concentrations of 0·1 nmol salmon calcitonin/l or higher. Prolactin release from these cells was also inhibited at somewhat higher concentrations by calcitonin gene-related peptide. Our results demonstrate that calcitonin can directly inhibit basal as well as TRH-stimulated prolactin release by acting directly at the pituitary. The results strongly suggest that the peptide may be involved in the regulation of prolactin release in certain physiological conditions.

J. Endocr. (1988) 116, 279–286
Analysis of release of porcine relaxin by reverse haemolytic plaque assay: evidence for autoregulation
Authors: Taylor M. J. , Clark C. L.
Published: December 2008

Relaxin release from monodispersed luteal cells derived from pregnant pigs (days 100–110 of gestation; n = 3) was detected by a reverse haemolytic plaque assay. In this technique, luteal cells were co-cultured in monolayers with ovine erythrocytes coupled to protein-A. In the presence of porcine relaxin antiserum and complement, a zone of haemolysis–a plaque–developed around relaxin-releasing luteal cells. Luteal cells were preincubated with porcine relaxin (up to 1000 ng/ml) or progesterone (up to 0·5 μg/ml) for 18 h before the plaque assay. Relaxin (but not progesterone) pretreatment inhibited subsequent relaxin release. The inhibitory effect of relaxin on its own release (autoregulation) has the potential to be a significant part of the local mechanisms which contribute to the overall control of ovarian secretion of protein hormones.

J. Endocr. (1988) 116, 287–291
Possible mechanisms responsible for the rise in plasma vasopressin associated with diabetic ketoacidosis in the rat
Authors: Charlton J. A. , Thompson C. J. , Baylis P. H.
Published: December 2008

The plasma concentration of arginine vasopressin (AVP) is increased in diabetic ketoacidosis (DKA) in man and the rat. Although haemodynamic changes and nausea/emesis may account for the increased secretion of AVP in severe human DKA, they appear not to be responsible in moderate DKA. Streptozotocin-treated rats were studied to investigate other factors possibly involved in the secretion of AVP in DKA.

Wistar rats were injected i.p. with streptozotocin (150 mg/kg body weight). Diabetic rats were maintained on 3–4 units protamine–zinc insulin (PZI)/day for 11 days, after which PZI was withdrawn for 3 days in half the rats. The plasma concentration of AVP was greater in rats with DKA than in normal controls (mean 11·4 pmol/l compared with 1·6 pmol/l; P <0·05). Rats with DKA had higher plasma osmolality and concentrations of blood glucose, β-hydroxybutyrate and acetoacetate, but lower plasma carbon dioxide content than diabetic and normal controls (P <0·05). There were no differences in plasma levels of sodium, urea or haematocrit between rats with DKA and controls. In a separate study involving the same procedures, daily systolic blood pressure was measured using a tail cuff to occlude arterial inflow to the tail, and subsequent detection of the cuff pressure at which the first arterial pulsation appeared. No significant differences were detected between normal and diabetic rats and rats with DKA. Exponential relationships between plasma osmolality and plasma AVP (correlation coefficient, r = +0·75; P <0·01), and plasma ketone bodies and plasma AVP (r= +0·60; P <0·05) were obtained. The increase in plasma AVP in the DKA group was not due to haemodynamic changes or nausea/emesis. The results suggest that in early DKA in the rat, increases in the plasma concentration of AVP are related to the metabolic consequences of insulin deficiency and not to hypovolaemia or hypotension.

J. Endocr. (1988) 116, 343–348
Refractoriness in quail leads to a reduction in the photoperiodic drive on LH secretion
Authors: Follett B. K.
Published: December 2008

The hypothesis was advanced that the long-day castration response in quail should become less pronounced in birds made refractory by exposure to long photoperiods. This was tested by pretreating two groups of castrated quail with testosterone for 9 weeks to suppress LH secretion whilst exposing them either to short days or to long days. The castration response was then measured on long days by withdrawing the testosterone and following the subsequent rate of increase in LH secretion. In both groups LH concentrations increased steadily for 7 weeks after removing the testosterone but the rate of increase in the group previously exposed to long days was only 50% of that in the group previously held on short days (P<0·025). The long-day castration response in quail not pretreated with testosterone was not altered by retaining the birds for 9 weeks on short days before transfer to long days.

J. Endocr. (1988) 116, 363–366
Glucocorticoid regulates secretion of epidermal growth factor in the human salivary gland adenocarcinoma cell line
Authors: Kurokawa R. , Kyakumoto S. , Ota M.
Published: December 2008

The treatment of a human submandibular gland adenocarcinoma cell line (HSG cell line) for 48 h with triamcinolone acetonide (TA; 1–100 nmol/l) reduced the secretion of epidermal growth factor (EGF) in a closely related manner to a maximum of 66%. The reduction in the level of EGF secreted resulted in the suppression of DNA synthesis in the HSG cells to a similar extent. When the cells were incubated with TA and exogenous human EGF (hEGF), DNA synthesis was 1·7-fold higher than that without added hEGF. The removal of EGF by the addition of hEGF antibody reduced DNA synthesis in HSG cell cultures to the same extent as did TA. These results suggest that the growth inhibition of HSG cells by TA is due to the reduction in the amount of EGF secreted.

J. Endocr. (1988) 116, 451–455
Reduction of testicular blood flow and focal degeneration of tissue in the rat after administration of human chorionic gonadotrophin
Authors: van Vliet J. , Rommerts F. F. G. , de Rooij D. G. , Buwalda G. , Wensing C. J. G.
Published: December 2008

The effect of 100IU human chorionic gonadotrophin (hCG) on testicular capillary blood flow was studied in adult male rats using a 133Xe clearance method and a radioactive microsphere technique. To investigate the role of Leydig cells in regulation of testicular blood flow after treatment with hCG, rats were pre-treated with ethane dimethylsulphonate (EDS) which selectively destroys mature Leydig cells. Six hours after treatment with hCG, testicular blood flow decreased in control and hypophysectomized rats to 25–50% of normal values, but not in EDS-pretreated animals. Prostaglandin E2 levels were also determined 6 h after an injection of hCG. A 300-fold increase in the concentration of prostaglandin E2 occurred in normal testis tissue. This rise was markedly inhibited if EDS was given 3 days before administration of hCG. Furthermore, 6 h after administration of hCG, the filling of the testicular capillary bed with methylacrylate was decreased, while in control rats and rats treated with EDS and hCG, complete filling of the capillaries was seen. Cell degeneration in some subcapsular seminiferous tubules was observed 6–10 days after treatment with hCG.

The results suggest that the hCG-induced precapillary vasoconstriction, probably mediated (in part) by prostaglandins, causes reduction in testicular blood flow 6 h after administration of hCG, and may result in cell damage.

J. Endocr. (1988) 117, 51–57
Only tissue-type plasminogen activator is secreted by immature bovine Sertoli cell-enriched cultures
Authors: Coombs R. J. , Ellison J. , Woods A. , Jenkins N.
Published: December 2008

Two molecular variants of plasminogen activator (PA): urokinase (uPA) and tissue-type plasminogen activator (tPA), have been reported to be synthesized in the rat testis. Data obtained in this study using monospecific antibodies raised against uPA and tPA in immunoblotting and bioimmunoassay protocols consistently demonstrate that only tPA (and not uPA) is synthesized by bovine Sertoli cell-enriched cultures, and is induced by bovine FSH. Zymographic analysis of conditioned medium on gels containing plasminogen and casein showed a dominant PA proteolytic band (72 kDa) which co-migrated with human tPA. A proteolytic band (43 kDa), which was also secreted by FSH-stimulated cells, was not present when protection was afforded from auto-proteolysis by aprotinin, and was therefore concluded to be a proteolytic fragment of tPA, and not uPA.

J. Endocr. (1988) 117, 63–67
Estimating the rate of externalization of gonadotrophin-releasing hormone receptors in ovine anterior pituitary cells in vitro
Authors: Starling L. , McIntosh J. E. A. , McIntosh R. P.
Published: December 2008

We report an estimate of the rate of externalization of unstimulated receptors for gonadotrophin-releasing hormone (GnRH), and derive from this the turnover time of the unstimulated receptor. The binding of the GnRH antagonist acetyl-d-pCl-Phe1,2,d-Trp3,d-Lys6,d-Ala10]-GnRH to dispersed sheep anterior pituitary cells was non-saturable at 37 °C. Further experiments showed that the binding had two distinct phases. We suggest that these phases correspond to the initial, saturable binding to existing plasma membrane receptors, followed by binding to receptors as they are inserted into the surface membrane. The two processes are temporally distinct, and can be inhibited independently by pharmacological manipulations. The initial phase was inhibited by treatments that could be expected to reduce the number of active receptors on the cell surface (preincubation of the cells for 30 min with 100 μg neuraminidase/ml or 50 μmol GnRH/ml), and was complete in less than 30 min after the addition of the antagonist tracer. The second phase occurred continuously in the presence of tracer, and was reduced or abolished by inhibitors of microtubule function (100 μmol vinblastine/l), protein synthesis (25 μg cycloheximide/ml), or energy metabolism (0·25 mmol 2,4-dinitrophenol/l). The rate of insertion of receptors into the plasma membrane was calculated from the rate of increase of the second phase of binding. The calculated rate implies a 100% turnover of unstimulated receptors every 150 min. In contrast, previously published estimates of the rate of internalization of the GnRH–receptor complex in the rat pituitary suggest that the stimulated receptor is turned over much faster.

J. Endocr. (1988) 117, 97–107
Regulation of preproparathyroid hormone messenger RNA and hormone synthesis in human parathyroid adenomata
Authors: Farrow S. M. , Karmali R. , Gleed J. H. , Hendy G. N. , O'Riordan J. L. H.
Published: December 2008

Preproparathyroid hormone (preproPTH) mRNA and PTH secretion were measured in human parathyroid adenomata (n = 8) cultured in 1·0 or 3·0 mmol calcium/l and compared with changes in bovine parathyroid glands (n = 3) as a control. Incubation of bovine glands in 3·0 mmol calcium/l for 24 h resulted in a fall in mRNA levels to 47·3±21·7% (mean ± s.d.) compared with cells incubated in 1·0 mmol calcium/l, with a concomitant decrease in secretion to 62·6± 10·8%. These values fell further to 30·1 ± 15·5% and 42·1 ±18·7% respectively after 48-h incubation. One human adenoma responded to high levels of calcium in a similar manner with mRNA levels falling to 44·6± 11·9% and secretion to 30·3±17·3% within 24 h. However, in the majority of adenomata (seven out of eight), after 24-h incubation in 3·0 mmol calcium/l, mRNA levels fell to 54·1 ± 14·6% but there was no change in secretion. In two of these adenomata which were cultured for 48 h, there was no suppression of secretion despite mRNA levels having fallen to approximately 60% of control.

Incorporation of [35S]methionine into PTH secreted from human adenomatous cells was quantified by densitometry. There was no difference in the amount of radiolabelled PTH secreted from cells incubated in high levels of calcium compared with those in normal levels of calcium. In similar experiments, the effects of high calcium on the synthesis of PTH in bovine cells was assessed by determination of radiolabelled intracellular PTH. After 24 h in high calcium, there was a fall of 59·0± 10·0% in the incorporation of [35S]methionine into PTH compared with cells incubated in normal calcium.

These results suggest the existence of two or more sites of regulation of synthesis and secretion by calcium. There appears to be control at the level of transcription with suppression of mRNA levels in human adenomata and bovine glands. This may be due to a decreased rate of transcription, or an increased rate of degradation of mRNA or both. There is also evidence for a post-transcriptional site which is less sensitive in human adenomata to control by calcium than in bovine glands.

J. Endocr. (1988) 117,133–138
Food intake alters the serum growth hormone response to bovine growth hormone-releasing factor in meal-fed Holstein steers
Authors: Moseley W. M. , Alaniz G. R. , Claflin W. H. , Krabill L. F.
Published: December 2008

The hypothesis that endocrine or nutritional factors related to feeding may affect pituitary responsiveness to an acute challenge with bovine GH-releasing factor (1–44)-NH2 (GRF) was examined in steers. In these experiments, either steers were trained to consume their total daily food allotment in a 2-h period (meal-fed) or food was withheld at the normal time of feeding (sham-fed).

In the first of three experiments, the serum GH pattern was determined around the time of feeding in meal-fed and sham-fed steers. The temporal GH rhythm in both groups appeared to be synchronized to the time of feeding, with limited pulsatile GH activity occurring 2–3 h after feeding. Baseline secretion of GH and total area under the GH response curve were lower (P<0·01) in meal-fed compared with sham-fed steers. In the second experiment, 50 μg GRF was injected i.v. in meal-fed steers at −4, −2, 0, + 2, +4, +6 and + 8 h relative to the time of feeding. The number of steers responding to GRF (53%), the amplitude of the GH peak (15·8 μg/l) and the area under the GH response curve (0·6 arbitrary units) were lower (P< 0·001) after than before feeding (90 ± 6 (s.e.m.)%, 61·3 ± 3·2 μg/l and 2·0 ± 0·3 units respectively). Of those steers responding to GRF, the GH response was significantly reduced following feeding compared with before feeding. In the third experiment, 50 μg GRF was injected i.v. in sham-fed steers at −4, −2, 0, +4 and +6 h relative to the time of sham-feeding. The amplitude of the GRF-induced GH peak (55·4 ± 9·3 μg/l), time to the GH peak (12 ± 2 min) and area under the GH response curve (0·8 ± 4 units) were not significantly different (P>0·1) among the different times of injection of GRF before or after sham-feeding. Apparently factors associated with the ingestion of feed can modulate the release of GH from the pituitary as evidenced by (a) the synchronization of the GH pattern to the time of feeding, and (b) the reduction of the serum GH response to i.v. injection of GRF following feeding.

J. Endocr. (1988) 117, 253–259
Circulating plasma cfactor-likeg immunoreactivity
Authors: Ellis M. J. , Livesey J. H. , Donald A.
Published: December 2008

Normal human peripheral plasma, examined by direct radioimmunoassay corticotrophin-releasing factor (CRF) using seven different antisera, showed marked inhibition of tracer binding to antiserum. This apparently high Cimmunoreactiv(CRF-LI) dilute in parallel with synthetic human CRF-41.parison, most equine and ovine plasma samples had low Cconcentrations.ractionation of both normal and late pregnancy human plasma on SG-75 neutral pH indicated the majority of the CRF-LI of large molecular size. The molecular weight was estimated to be approximately 29 000 following chromatography human plasma on Sephacryl S-200.igh CRF-LIrmal plasma was reduced to barely detectable levels by methanol treatment despite the recovery of added human CRF-41 of >80%. Methanol extracts of late pregnancy human plasma, however, retained high CRF-LI which exhibited the immunoreactive and Sephadex elution characteristics of synthetic human CRF-41. 125I-Labelled human CRF-41 added to human plasma showed a reversible, time-dependent alteration in molecular size and reduction in binding to excess antiserum. These findings indicate that direct RIA of human CRF in unextracted plasma leads to spurious results and suggest the presence in human plasma of an interfering factor and/or binding substance that is not generally apparent in equine or ovine plasma under the conditions described.

J. Endocr. (1988) 117, 299–307
Hydrolysis of phosphatidylinositol by human endometrium: modulating effects of steroids on arachidonic acid and 1,2-diacylglycerol release
Authors: Bonney R. C. , Franks S.
Published: December 2008

Phospholipase C and 1,2-diacylglycerol lipase activities were demonstrated in human endometrium using 1-stearoyl-2-[1-14C]arachidonyl phosphatidylinositol as substrate. Phosphatidylinositol is hydrolysed by phospholipase C to inositol phosphates and to 1,2-diacylglycerol which is then further metabolized by 1,2-diacylglycerol lipase to release free arachidonic acid. In the present study the radiolabelled products formed (1,2-diacylglycerol and arachidonic acid) were measured following chloroform/methanol extraction and thin-layer chromatography. Phospholipase C activity was calcium dependent and optimal at pH 5·0–5·5 and 7·5; 1,2-diacylglycerol lipase activity was also calcium dependent, with an optimum pH of 5·5. A significant increase in 1,2-diacylglycerol production was stimulated by steroid sulphates. Pregnenolone sulphate, oestrone sulphate, testosterone sulphate and dehydroepiandrosterone sulphate stimulated 4, 3·2-1·8- and 2·6-fold increases in release respectively. Oestradiol sulphate stimulated a 25% increase in diacylglycerol release which was not significantly different from the control value. Progesterone stimulated a fourfold increase but other free steroids had no effect. Arachidonic acid release was increased in the presence of oestradiol sulphate, oestrone and oestradiol but reduced by oestrone sulphate, dehydroepiandrosterone sulphate, progesterone, dehydroepiandrosterone and, to a lesser extent, by pregnenolone sulphate and testosterone sulphate. 5-Androstene-3β, 17β-diol had no effect on the liberation of either product.

This study demonstrates a potential route for the liberation of arachidonic acid from phosphatidylinositol in human endometrium. The opposing effects of steroids on phospholipase C and 1,2-diacylglycerol lipase activity could be important in regulating the release of arachidonic acid by this pathway.

J. Endocr. (1988) 117, 309–314
Effect of cortisol on the plasma and lymphoid tissue distributions of tritiated glucocorticoids in C57BL/6 mice
Authors: Durant S. , Duval D. , Homo-Delarche F.
Published: December 2008

The mechanism of action of very high doses of corticosteroids, such as those administered as bolus doses in the treatment of inflammatory and immune diseases or those currently used in rodents to isolate the small proportion of medullary thymocytes considered to be corticoresistant, is still undefined. The possible existence of selective local concentration by some tissues, particularly lymphoid organs, cannot be excluded. Therefore, using C57BL/6 mice, the kinetics of lymphoid tissue and plasma radioactivities after i.p. injection of steroids, either alone or with an excess of non-radioactive cortisol hemisuccinate (up to 10 mg/animal, i.e. 500 mg/kg ) were studied. There was a rapid and dose-dependent retention of [3H]corticosterone and [3H]cortisol in the thymuses of cortisol-treated compared with control animals. The spleen also appeared to be capable of accumulating steroids. However, when the tissue/plasma ratio of [3H]steroid concentration and the change in extracellular space in the presence of an excess of non-radioactive cortisol were taken into consideration, only the thymus was able to concentrate steroids above concentrations in the plasma. Moreover, this effect did not appear to be specific for glucocorticoids, since tracer accumulation was also observed when sex steroids were used as tracers. The cells of the reticuloendothelial system may, in part, be responsible for this phenomenon of steroid concentration in lymphoid organs.

J. Endocr. (1988) 117, 373–378
Pertussis toxin-catalysed ADP-ribosylation of endometrial proteins in sheep
Authors: Flint A. P. F.
Published: December 2008

Membrane fractions prepared from the uterine endometrium of untreated ovariectomized sheep contained a 41 × 103 Mr protein that was [32P]ADP-ribosylated by pertussis toxin in the presence of [32P]NAD. Progestin and progestin plus oestrogen treatment in vivo increased the concentration of this protein 2·7- and 3·6-fold respectively. Endometrial extracts from untreated or progestin-treated sheep also contained proteins of Mr 69 × 103 and 120 × 103 which were ADP-ribosylated in the absence of pertussis toxin; these proteins were not ADP-ribosylated in sheep receiving oestrogen. Incubation of endometrial slices from progestin plus oestrogen-treated sheep with oxytocin in vitro increased phosphoinositide hydrolysis 11-fold. This effect was not altered by prior incubation with pertussis toxin, although toxin treatment reduced by 64% subsequent labelling of the 41 × 103 Mr protein when membrane fractions prepared from pretreated slices were incubated with pertussis toxin and [32P]NAD. Thus the endometrium contains a pertussis toxin-sensitive protein which is induced by steroid treatment, but this protein is not involved in the phosphoinositide response to oxytocin.

J. Endocr. (1988) 117, 403–407