ABSTRACT

Fragments of rat anterior pituitary glands incubated in vitro and challenged with either of two ACTH secretagogues were used to investigate the extent to which the acute, biphasic, feedback-like inhibitory effects on hormone secretion exerted by the synthetic glucocorticoid dexamethasone were related to alterations in second messenger responses. Addition of dexamethasone was shown to cause both an immediate inhibition (fast inhibition) of the release of ACTH-like immunoreactivity induced by arginine vasopressin (AVP) or corticotrophin-releasing factor (CRF-41), and also an inhibition that occurred after removal of the steroid and was maximal 90 min after its introduction (early delayed inhibition).

The accumulation of adenosine 3′,5′-monophosphate (cAMP) in the tissue was enhanced in a dose-related manner by CRF-41, as was that of phosphate esters of inositol (inositol phosphates) by AVP. The dose–response curve for the effect of CRF-41 on cAMP production was markedly shifted to the right by dexamethasone acting in the time-domain of fast inhibition (i.e. the response was attenuated, but not abolished). Application of the steroid during the same time-period reduced significantly the inositol phosphate response induced by the higher concentration of AVP tested (3000 mmol/l), but had no effect on the action of a lower concentration (30 mmol/l).

In contrast, the cAMP and inositol phosphate dose–response curves to CRF-41 and AVP respectively were unaffected by the glucocorticoid when it was applied at the time which generated early delayed inhibition of ACTH release.

It is concluded that part of the fast inhibitory action of dexamethasone on ACTH secretion in vitro (but not its delayed effects) involves a rapid alteration of second messenger responses to secretagogues, but the precise mechanism underlying this process remains to be established.

Journal of Endocrinology (1989) 122, 545–551

ABSTRACT

Rat adrenal gland slices, when incubated in vitro with [1,4-¹⁴C]putrescine, accumulate the radioactive diamine and convert it, in part, to a compound indistinguishable (in four separative systems) from [¹⁴C]γ-aminobutyrate (GABA). Adrenal glands taken from animals that had undergone adrenal enucleation 28 days previously, so that the cortex of the tissue had regenerated, likewise formed [¹⁴C]GABA from [1,4-¹⁴C]putrescine. Putrescine-derived GABA was released from adrenal slices in vitro by 48 mmol K⁺/l, the release being dependent upon the presence of Ca²⁺ in the incubation medium. ACTH(1–24) and 8-bromocyclic AMP both provoked a dose-related release of putrescine-derived GABA, although the dose–response curve for the latter differed somewhat from that for the release of corticosterone by this secretogogue.

The enzyme believed to be responsible for the first step in the metabolic transformation of putrescine into GABA, diamine oxidase (DAO), is present in extracts of adrenal tissue and its catalytic activity underwent a transient increase followed by a fall below resting levels upon stimulation of adrenal slices with ACTH(1–24). The conclusion that this enzyme initiates the formation of GABA by this pathway is indicated by the observation that adrenal slices pretreated with the DAO inhibitor, aminoguanidine, released significantly less [1,4-¹⁴C]putrescine-derived GABA in response to 48 mmol K⁺/l than did control tissues. The functional significance of these findings remains to be established.

Journal of Endocrinology (1989) 123, 227–232

The concentration of cytochrome P-450 in microsomes prepared from the livers of mature female Wistar-derived rats was significantly lower than in mature males. This sex difference was abolished after hypophysectomy, when the concentration of the cytochrome in males and females was not significantly different from that in the intact male. A concentration of cytochrome P-450 characteristic of females was restored by two anterior pituitary transplants under the kidney capsule of hypophysectomized females; a partial 'feminization' occurred in similarly treated hypophysectomized males. A partial 'feminization' was also achieved by the administration of rat or sheep prolactin to hypophysectomized females. Unexpectedly, the administration of l-dihydroxyphenylalanine to normal females was without effect on cytochrome P-450, whereas in intact males 'feminization' resulted.

Castration of adult rats resulted in the 'feminization' of cytochrome P-450, whereas ovariectomy was without effect. Administration of testosterone propionate for 10 days, either immediately after the operation or 14 weeks later to rats castrated when adult failed, however, to reverse the fall in cytochrome P-450. The establishment of a higher concentration of cytochrome P-450 in the liver of female rats could not be brought about by the administration of testosterone propionate, whether given as a single dose on the second day after birth or as a 10-day course of treatment after puberty or both.

It is concluded that the sex difference in hepatic microsomal cytochrome P-450 is maintained by the release in the female of an anterior pituitary factor(s) that serves to depress its concentration. The factor(s) shows some of the characteristics of prolactin but the findings are not consistent with that hormone being responsible for all of the effects observed. The release of the factor(s) in the male may be inhibited by a compound of gonadal origin other than testosterone. A sex difference could not be 'imprinted' in the female by either neonatal and/or postpubertal testosterone treatment.

The concentration of hepatic microsomal cytochrome b₅ and the specific activity of NADPH-cytochrome c reductase were found not to be sex-dependent in the rats used. However, anterior pituitary factor(s) other than prolactin and growth hormone act to suppress partially the concentration of the former and to promote the specific activity of the latter in the endoplasmic reticulum of rat hepatocytes.

The effect of various steroids on the functional activity of the rat hypothalamus in vitro was investigated. The addition of corticosterone (10⁻⁷ mol/l) for 30 min to the incubation medium inhibited immediately the release of bioactive corticotrophin releasing factor (CRF) by tissue induced by serotonin (2·6×10⁻⁸ mol/l). This was followed by a period lasting from 30 min (coincident with removal of the steroid from the medium) to 60 min when no inhibition was seen. Finally a second period of suppression of hypothalamic CRF activity in vitro was shown to be fully established 120 min after addition of the steroid. In more detailed investigations the latter inhibition was shown to occur when the tissue was exposed to the steroid (3×10⁻⁷ mol/l) for 5 or 30 min, but not for 1 min, and it was dose-related. Of other steroids investigated, progesterone in high concentrations (3 × 10⁻⁶ mol/l) suppressed to a small extent the functional activity of the hypothalamus in vitro but 17α-hydroxyprogesterone, 11α-hydroxyprogesterone, 11α,17α-dihydroxyprogesterone and 11-epicortisol had no effect on the delayed inhibition. Progesterone (10⁻⁷ mol/l) potentiated the ability of corticosterone (10⁻⁸ mol/l) to induce the delayed suppression of hypothalamic CRF activity in vitro. In contrast, 17α-hydroxyprogesterone, 11α-hydroxyprogesterone, 1 1α,17α-dihydroxyprogesterone and 11-epicortisol competitively antagonized this inhibitory action of corticosterone (3 × 10⁻⁷ mol/l) in a dose-related manner (1·5 × 10⁻⁸–3 × 10⁻⁸ mol/l). The action of the antagonist 11-epicortisol was similar whether it was added to the tissue in vitro before corticosterone or antagonist and agonist were added together. The functional characterization of steroid action on the hypothalamus may lead to a clearer understanding of the mechanism by which the compounds influence hormone release.

ABSTRACT

In-situ hybridization with synthetic oligonucleotide probes was used to determine the mRNA content of corticotrophin-releasing factor (CRF) and proenkephalin A mRNA in the paraventricular nucleus, and of pro-opiomelanocortin (POMC) mRNA in the anterior pituitary gland of male rats immediately after, and during recovery from, chronic high-dose prednisolone treatment. Levels of transcripts for mRNA for both CRF and POMC were markedly reduced after the treatment, but there was a rapid return to control values for CRF mRNA within 18 h of steroid withdrawal.

In untreated animals, the stressful stimulus of i.p. hypertonic saline increased CRF and proenkephalin A mRNA within 4 h with no significant difference in response seen whether the tissues were removed at 13.00 or 20.00 h. The increase in POMC mRNA did not reach statistical significance in these animals. Although prednisolone resulted in a marked reduction of basal CRF mRNA, the stress-induced increment of CRF mRNA remained comparable with that found in untreated animals. On the day following cessation of prednisolone treatment at 09.00 h, basal and stress levels of CRF mRNA were significantly higher in rats killed at 20.00 h than at 13.00 h. Proenkephalin A mRNA transcripts were below quantifiable levels of detection in control or non-stressed prednisolone-treated animals at all the time-points studied. Stress, however, resulted in the accumulation of proenkephalin A mRNA in control animals. This response was inhibited by prednisolone treatment and only returned 18 h after withdrawal. Prednisolone treatment reduced POMC mRNA below the levels detected in untreated animals, with no detectable response to stress. However, 36 h after prednisolone withdrawal there was a significant increase in POMC mRNA in response to stress.

Prednisolone concentrations in the plasma on the morning of the day after the final administration of the steroid were very high (880 nmol/l), but these were halved by the evening of that day and had fallen to 60 nmol/l 72 h after withdrawal.

These data show that both CRF and POMC mRNA were profoundly reduced by long-term prednisolone treatment. Stress-induced accumulation of CRF mRNA, however, persisted although the proenkephalin A mRNA responses to this stress were inhibited. No increase in pituitary POMC mRNA in response to stress was apparent until 36 h after withdrawal of prednisolone.

Journal of Endocrinology (1990) 127, 407–415

ABSTRACT

Male Wistar-derived rats (200–250 g) were treated for 14 days with prednisolone 21-sodium succinate at a concentration of 1035 μmol/l in their drinking water. The drug was then replaced with normal tap water and groups of animals were killed at various times during recovery, trunk blood being collected after decapitation. At the same time, hypothalamic slices, anterior pituitary gland fragments and adrenals were removed and their responsiveness assessed by exposure to appropriate stimuli in vitro. Tissues were also extracted to measure changes in content of hormones during recovery. Treatment with prednisolone produced marked reductions in body weight gain, adrenal weight and pituitary ACTH content, but no significant change in hypothalamic corticotrophin-releasing factor (CRF) bio- or immunoreactivity. The ACTH content was restored by 5 days after withdrawal but adrenal weight remained significantly reduced after 9 days of recovery. The responsiveness of the hypothalamus to acetylcholine in vitro was markedly inhibited and was still significantly reduced 7 days after withdrawal. The responsiveness of the anterior pituitary gland to synthetic CRF or arginine vasopressin and that of the adrenal gland to ACTH added in vitro were restored simultaneously after 7 days of withdrawal. In vivo, recovery was assessed by measurement of the response to laparotomy stress. Treatment with prednisolone prevented the increase in the plasma concentrations of ACTH and corticosterone produced by stress, and these responses recovered by 5 days (corticosterone) and 7 days (ACTH) after withdrawal. The abolition of the circadian rhythms of ACTH and corticosterone by treatment was also reversed by 5 days after withdrawal. This pattern of recovery is different from that which we observed after long-term treatment with dexamethasone, where the responsiveness of the hypothalamus and adrenal gland in vitro recovered before that of the anterior pituitary gland.

J. Endocr. (1987) 113, 239–247

ABSTRACT

Female Wistar-derived rats with regular oestrous cycles were injected s.c. at 15.00 h on pro-oestrus with difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase. The drug (10–100 mg/rat) caused a dose-related reduction in the concentration of LH in plasma taken at 19.00 h (the time of the peak of the LH surge in this colony). There was also a dose-related reduction in the pituitary content of total polyamines. The reduction in the plasma concentration of LH was not due to the shifting of the time of the peak of the surge, as concentrations were significantly lower than control from 17.00 to 21.00 h, the overall reduction in total LH release being approximately 50%. The number of ova in the oviducts at 06.00 h next morning was significantly reduced by treatment with 50 mg DFMO/rat, by an average of 70%.

Injection of DFMO enhanced the fall in plasma oestradiol concentrations seen between 15.00 and 19.00 h, in a dose-related manner. It also prevented the rise in progesterone concentrations seen in control animals during this period. The ability of DFMO to prevent the rise in plasma concentrations of LH was not secondary to the effects of the drug on ovarian steroid production because DFMO also significantly reduced the LH surge in animals ovariectomized on dioestrus and given appropriate replacement injections of oestradiol and progesterone.

It seems possible that part of the action of DFMO is exercised at the hypothalamus, since when 50 mg DFMO/rat was given either 2 or 4 h before the expected peak of the LH surge, the LHRH content of the hypothalamus was significantly reduced at that time. These results suggest that activation of ornithine decarboxylase is a necessary prerequisite for a normal LH surge, and that this activation is steroid-dependent. This conclusion is borne out by results from direct observations on the activity of the enzyme in pituitary tissue incubated in vitro.

J. Endocr. (1988) 117, 447–453

ABSTRACT

The effect of six hypothalamic peptides on the basal release of ACTH and that induced by arginine vasopressin (AVP) or by ovine corticotrophin releasing factor (oCRF) from fragments of the rat anterior pituitary gland incubated in vitro was investigated. Dose–response curves to AVP and to oCRF were obtained, and the response to a low dose of oCRF was potentiated by a low dose of AVP. Basal release of ACTH was not affected by any of the peptides in concentrations in the range 10⁻¹² to 10⁻⁶ mol/l, and only substance P (SP) and somatostatin (SRIF) inhibited significantly the response to oCRF in a dose-related manner. The responses to a range of doses of oCRF or AVP were reduced by 10⁻⁸ and 10 ^{− 6} mol SP or SRIF/1, and to a greater extent by the higher dose. Except in the case of 10⁻⁶ mol SRIF/1 on the response to AVP, the response was not further diminished by preincubation of the tissue with the peptide before the stimulating agent was added. The inhibition of the responses to AVP or oCRF by 10⁻⁹ mol SP/1 was not potentiated by its combination with either 5 × 10⁻¹⁰ or 10⁻⁸ mol SRIF/1; the inhibitory effects were merely additive. The results suggest that although SRIF and SP are able to modulate the release of ACTH from the anterior pituitary gland, they do so only at a high concentration. In the case of SRIF these concentrations are several orders of magnitude higher than those reported to be present in the hypophysial portal blood and therefore a physiological role for this peptide in the control of ACTH secretion is unlikely.

J. Endocr. (1984) 100, 219–226

Sherrington School of Physiology and Biochemistry Department, St Thomas's Hospital Medical School, London, SEI and * Department of Pharmacology, Royal Free Hospital School of Medicine, London, WC1

(Received 26 August 1977)

Substance P is present in the hypothalamus in relatively high concentrations. Its release is K⁺-evoked and Ca²⁺-dependent (Iversen, Jessell & Kanazawa, 1976) and it may be a neurotransmitter substance in the hypothalamus. We have tested its effect on secretion of corticotrophin releasing factor (CRF) by the hypothalamus in vitro and on production of corticotrophin (ACTH) by the anterior pituitary gland of the rat both in vivo and in vitro.

Production of CRF by rat hypothalami in vitro was stimulated by 5-hydroxytryptamine (5-HT) or acetylcholine (Gillham, Jones, Hillhouse & Burden, 1975; Buckingham & Hodges, 1977a) in the presence and absence of substance P. The activity of CRF was estimated by measuring cytochemically (Alghband-Zadeh, Daly, Bitensky & Chayen, 1974) the ACTH which

The status of serotonin as a putative neurotransmitter involved in the control of the secretion of corticotrophin-releasing activity from the rat hypothalamus has been further investigated. The experimental model used for the investigation was the rat hypothalamus incubated in vitro. It was confirmed that serotonin causes a dose-related release of corticotrophin-releasing activity from the tissue and it was shown that this effect was mimicked by the addition to the tissue of chlorimipramine or d-fenfluramine, two drugs expected to cause an increase in the concentration of serotonin in the synaptic cleft. The effect of the latter drug was greatly reduced by the simultaneous addition of either methysergide or metergoline.

Destruction of serotonin-containing nerve terminals in the hypothalamus was caused by the intraventricular administration of the neurotoxic drug 5,7-dihydroxytryptamine. This treatment resulted in an 84% reduction of the serotonin concentration in the hypothalamus 12 days later. Hypothalami taken from animals 12 days after treatment with the drug secreted corticotrophin-releasing activity in basal amounts equal to those found in tissues taken from control rats, but showed supersensitivity in response to added serotonin. No such supersensitivity was seen in response to d-fenfluramine and a diminished response to chlorimipramine was noted. Despite its intraventricular route of administration, 5,7dihydroxytryptamine was found to increase the sensitivity of segments of anterior pituitary gland in vitro to low doses of preparations containing corticotrophin releasing factor.

These results are consistent with the view that endogenous serotonin can act as a stimulator of the secretion of corticotrophin-releasing activity from the rat hypothalamus. They also suggest that conclusions about the control of the release of this trophic material inferred from measurements of corticotrophin or corticosterone in the circulation must be viewed with caution when the drug 5,7-dihydroxytryptamine has been used, because of the development of supersensitivity both in the hypothalamus and in the anterior pituitary gland.