The evaluation of sodium valproate in the treatment of Nelson's syndrome.

It has previously been reported that sodium valproate (Epilim) lowers plasma ACTH levels in Nelson's syndrome. This report describes further experience with its use. Ten patients with Nelson's syndrome were treated with sodium valproate (600-1200 mg/day) for 5-32 weeks. Plasma ACTH was measured by cytochemical methods and RIA. Initial treatment for 5-12 weeks significantly (P less than 0.005) lowered plasma ACTH from a pretreatment mean of 2460 +/- 1870 ng/liter to 480 +/- 330 ng/liter, and the ACTH circadian rhythm was restored in two patients. On discontinuing treatment, plasma ACTH levels remained suppressed for 3 weeks and rose to pretreatment values in 5-12 weeks. Two patients' plasma ACTH levels failed to show a second response to treatment, while a third patient had a favorable second response to treatment over 32 weeks. In six patients, skin pigmentation lightened with treatment, and in one patient, a reduction in size of a pituitary microadenoma, demonstrated radiographically, occurred with treatment. gamma-Aminobutyric acid and sodium valproate were shown to be ineffective in inhibiting ACTH secretion from cultured pituitary tumor cells from a patient with Nelson's syndrome. The results show that sodium valproate is effective in some cases of Nelson's syndrome. We suggest that it reduces the hypersecretion of ACTH by enhancing gamma-aminobutyric acid function in the hypothalamus, thereby inhibiting the release of corticotropin-releasing factor.

Influence of prolonged glucocorticoid treatment on intracellular mechanisms involved in ACTH secretion in the rat.

Two chemically characterized peptides, arginine vasopressin (AVP) and corticotrophin-releasing factor-41 (CRF-41), known to stimulate ACTH secretion by interaction with their respective specific receptors on the corticotroph, were shown to cause the accumulation of phosphate esters of inositol (IP) and adenosine 3',5'-monophosphate (cAMP) respectively when added to rat anterior pituitary fragments incubated in vitro. The former 'second messenger' response (IP production) was unaffected in tissues removed from animals treated with prednisolone in the drinking water (1035 mumol/1) for 14 days. On the other hand, the cAMP response, whilst still present, was inhibited by some 50% in tissues taken from such animals. In contrast, pituitary glands from steroid-treated rats failed to respond to challenge with a variety of substances expected to cause the release of ACTH by mimicking or provoking the production of IP or cAMP. Indeed, of the wide range of ACTH secretagogues tested, only the phospholipase A2 activator melittin was able to cause attenuated ACTH release from tissues removed from treated rats. The failure to provoke ACTH release from tissues removed from steroid-treated animals was also seen when submaximal concentrations of CRF-41 or AVP, or hypothalamic extract or 48 mmol K+/1 were used as the stimuli. The staged recovery of the ACTH secretory response and IP and cAMP accumulation in vitro following the withdrawal of prednisolone treatment was also investigated. A cAMP response that did not differ significantly from that of control tissue and a normal ACTH response to K+ and to melittin were all recovered by 3 days after withdrawal, and the response to cholera toxin showed a partial recovery. Responses to all stimuli of ACTH secretion which cause their effect by entering the corticotrophs were normal by 5 days after withdrawal, when the response to CRF-41 was still significantly, and that to AVP still slightly, reduced compared with controls. Surprisingly, restoration of the ACTH response was most delayed when the expectedly most potent extracellular stimulus (hypothalamic extract) was used. In this case, release was still significantly impaired 7 days after steroid withdrawal. The results show that the glucocorticoid acts to compromise several distinct steps in the process whereby extracellular signals such as CRF-41 and AVP cause the secretion of ACTH. The only step that appears to be spared is the generation of IP by AVP.(ABSTRACT TRUNCATED AT 400 WORDS)

Glucocorticoids act rapidly in vitro to attenuate second messenger responses to ACTH secretagogues in rats.

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 nmol/l), but had no effect on the action of a lower concentration (30 nmol/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.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of releasable gamma-aminobutyrate from putrescine by rat adrenal slices in vitro.

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]gamma-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.

Stress responsiveness of hypothalamic corticotrophin-releasing factor and pituitary pro-opiomelanocortin mRNAs following high-dose glucocorticoid treatment and withdrawal in the rat.

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Recovery of the components of the hypothalamo-pituitary-adrenocortical axis in the rat after chronic treatment with prednisolone.

Male Wistar-derived rats (200-250 g) were treated for 14 days with prednisolone 21-sodium succinate at a concentration of 1035 mumol/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.(ABSTRACT TRUNCATED AT 250 WORDS)

Biphasic inhibition of bioactive hypothalamic corticotrophin releasing factor secretion in vitro by corticosterone and prevention of the second phase by various steroids.

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 X 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 X 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 X 10(-6 mol/l) suppressed to a small extent the functional activity of the hypothalamus in vitro but 17 alpha-hydroxyprogesterone, 11 alpha-hydroxyprogesterone, 11 alpha, 17 alpha-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, 17 alpha-hydroxyprogesterone, 11 alpha-hydroxyprogesterone, 11 alpha, 17 alpha-dihydroxyprogesterone and 11-epicortisol competitively antagonized this inhibitory action of corticosterone (3 X 10(-7) mol/l) in a dose-related manner (1.5 X 10(-8)-3 X 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.

Effect of difluoromethylornithine on the LH surge and subsequent ovulation in the rat.

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of hypothalamic neuropeptides on corticotrophin release from quarters of rat anterior pituitary gland in vitro.

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/l, and to a greater extent by the higher dose. Except in the case of 10(-6) mol SRIF/l 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/l was not potentiated by its combination with either 5 X 10(-10) or 10(-8) mol SRIF/l; 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of corticosteroid inhibition of adrenocorticotrophin release from the anterior pituitary gland of the rat.

The occurrence and nature of corticosteroid inhibition of ACTH secretion at the rat anterior pituitary gland was investigated using three experimental models: animals bearing lesions of the basal hypothalamus, and two preparations of the gland incubated in vitro; these were tissue segments and collagenase-dispersed cells. Release of ACTH in the experiments was provoked using one of three distinct stimuli: acid extracts of whole hypothalami, corticotrophin releasing activity released by serotonin from hypothalami incubated in vitro and synthetic ovine corticotrophin releasing factor. Irrespective of whether ACTH was measured directly by radioimmunoassay (in the experiments in vitro) or indirectly in terms of corticosterone production (in the lesioned animals), its stimulated release from the anterior pituitary gland was inhibited by corticosterone. Two phases of inhibition were observed; these had some of the characteristics inferred previously from experiments with intact animals and designated fast feedback and delayed feedback. However, the fast feedback demonstrable in lesioned animals did not show the rate-sensitivity shown previously in intact animals. 11-Deoxycortisol (or 11-deoxycorticosterone) and prednisolone proved to be agonists of corticosterone in provoking fast feedback in lesioned animals, whereas they had been shown respectively to act as an antagonist or to have no effect in intact rats. Several steroids were able to cause delayed feedback in lesioned rats, but beclomethasone dipropionate (shown to be an agonist of corticosterone in intact rats) proved to have no inhibitory effect at the anterior pituitary gland of lesioned animals. It is concluded that the dynamics of corticosteroid feedback mechanisms at the anterior pituitary gland, as indicated by experiments in lesioned animals, differ from those operative in the intact animals. Other work suggests that a more important site for such inhibitory mechanisms in vivo is the hypothalamus.

Relationship between adrenocorticotrophin bioactivity in blood and cerebrospinal fluid of rhesus monkeys.

Adrenocorticotrophin levels, measured by a cytochemical bioassay, were determined in the plasma and cerebrospinal fluid (CSF) of adult female rhesus monkeys which were ovariectomized and receiving oestrogen replacement therapy. In control monkeys, ACTH bioactivity was found in both CSF (10.2 +/- 1.8 ng/l) and plasma (186 +/- 51 ng/l) in samples taken at 14.00 h (lights on: 07.00-19.00 h). Dexamethasone treatment (0.2 mg/kg) twice daily for 4 days suppressed plasma ACTH levels (52.8 +/- 25.2 ng/l) but had no effect on CSF levels (7.6 +/- 2.7 ng/l). Raising plasma ACTH, either by daily injections of a long-acting preparation of ACTH (1-24) for 6 days or by bilateral adrenalectomy (and subsequently with-drawing cortisol replacement therapy) also resulted in no detectable changes in ACTH levels in the CSF. A regression analysis between ACTH in the plasma and CSF from samples taken throughout the experiments showed no correlation. In contrast, measurement of ACTH by radioimmunoassay, whilst satisfactory for determination of this peptide in plasma, could not identify authentic ACTH in the CSF. It is concluded that bioactive ACTH does not enter the CSF in detectable quantities from either the peripheral vascular compartment or from the animal's own pituitary gland, and that reducing ACTH secretion from the pituitary also has no effect on levels of ACTH in the CSF. This is in marked contrast to other pituitary peptide hormones, including prolactin, which is secreted together with ACTH during 'stress' but which, unlike ACTH, enters the CSF relatively easily.

Glucocorticoid effects on the molecular forms of adrenocorticotrophic hormone secreted by cultures of rat anterior pituitary cell monolayers.

The effects of glucocorticoids and corticotrophin-releasing factor (CRF) on the release of various molecular forms of adrenocorticotrophic hormone (ACTH) have been investigated in primary cultures of rat anterior pituitary. The rat cells responded to a 30 min challenge with CRF by secreting increased amounts of ACTH, as assessed both by bioassay, using rat adrenocortical cells, and by radioimmunoassay. Inclusion of a synthetic glucocorticoid, such as dexamethasone (DEX), in the incubation for 5 min prior to, and during the CRF challenge, had no effect on the response as measured by radioimmunoassay. Bioassay, however, indicated profound suppression of the response to CRF. This discrepancy between ACTH immuno- and bioactivity was investigated by fractionating the immunoreactive ACTH species using high-performance liquid chromatography. The lower molecular weight (<15kd) forms (ACTH(1-39) , phosphorylated ACTH(1-39) and glycosylated ACTH(1±39) ) were separated from higher molecular weight (>15kd) forms (i.e. ACTH biosynthetic intermediate and proopiomelanocortin) using C-18 Sep-Pak. The lower molecular weight molecules were further resolved into glycosylated and non-glycosylated ACTH, using an acetonitrile gradient high-performance liquid chromatography with trifluoroacetic acid as an ion-pairer. Neither the proportion of low molecular weight forms of ACTH, nor that of glycosylated ACTH(1-39) secreted in response to CRF, were affected by DEX. Further fractionation of non-glycosylated ACTH, also using acetonitrile gradient high-performance liquid chromatography but with heptafluorobutyric acid as the ion-pairer, yielded peaks corresponding to phosphorylated and non-phosphorylated ACTH(1-39) . DEX significantly increased the proportion of phosphorylated ACTH secreted in response to CRF by 18%. An additional effect of DEX was revealed when Sep-Pak extracts were treated with alkaline phosphatase, prior to analysis. After dephosphorylation, it became clear that the peptides released by CRF-stimulated cells were different if DEX was present in the medium. The peptide released in the presence of DEX (ACTH-S) had a slightly, but consistently, different retention time on high-performance liquid chromatography and very little biological activity. Antibody cross-reactivity studies suggested that ACTH-S was modified in the 1-24 region of the peptide. It is concluded that challenge of anterior pituitary cells in primary culture with CRF, following 5 min previous exposure to DEX, results in a molecular change. The consequence of this is that ACTH immunoreactivity is released, but the molecule has reduced biological activity. This may be part of the mechanism by which fast feedback inhibition of ACTH secretion is effected.

24-hour variation in content and release of hypothalamic neuropeptides in the rat.

The tissue content of up to eight neuropeptides, viz bombesin (BOM), cholecystokinin (CCK-8), neurotensin (NT), neuropeptide Y (NPY), peptide histidine isoleucine amide (PHI), somatostatin (SRIF), substance P (SP) and vasoactive intestinal polypeptide (VIP), in rat hypothalami removed at various times of the day, was measured using specific radioimmunoassays. There was significant variation in the content of BOM, CCK-8, NT, PHI, SP and VIP across a 24-h period. The levels of BOM, CCK-8 and NT were lowest around the onset of darkness (1900 h) and rose throughout the night to reach a peak around the time of lights on. Hypothalamic content of all eight peptides fell between 0700 h and 1300 h by an average of 45 +/- 4%. Basal release of these peptides, as well as that in the presence of 48 mM potassium (K+), was measured from hypothalami removed between 0700 and 1900 h and incubated in vitro in a CSF-like medium. Basal secretion of NT significantly increased, whilst that of CCK-8 significantly decreased over the same period. There was no significant change in the basal release of the other neuropeptides. The release in the presence of 48 mM K+ of SP decreased significantly during the day, whilst that of VIP significantly increased. There was also a significant change in the stimulated release of BOM, levels falling during the morning and rising again at 1900 h. 48 mM K+ caused a significant increase in the release of SRIF and SP at all times tested. Whilst 48 mM K+ induced a significantly higher release of CCK-8 and NT in the morning, this stimulus was ineffective in the evening. The contrary was true in the case of BOM, NPY and VIP, where a significant stimulation was induced only at 1900 h. The possible implications of these findings are discussed.

Unemployment rates, single parent density, and indices of child poverty: their relationship to different categories of child abuse and neglect.

OBJECTIVE: There were two main research questions: First, is there a relationship between rates of child physical abuse, child sexual abuse and child neglect and levels of female and male unemployment, single-parent density and child poverty in the immediately local area; and second, is this relationship different for different categories of abuse and neglect and different categories of deprivation? METHOD: Using archival data--registered cases of abuse and neglect and official data on child population, social worker ratio, unemployment rates, single-parent density, means-tested clothing grants and free school meals for children--a multiple correlational analysis was carried out of the 5,551 referrals and 1,450 registered cases of abuse and neglect in Glasgow, Scotland for the years 1991 through to 1993. RESULTS: Substantial correlations were found with all indices of deprivation but particularly physical abuse with rates of male unemployment. Lower and more variable correlations were found with female unemployment rates. Sexual abuse and neglect rates showed a less consistent relationship with the indices of deprivation. In general male unemployment rates alone accounted for two-thirds of the variance in total abuse and neglect rates, other factors adding little or nothing to this. CONCLUSIONS: The results demonstrate the importance of selecting small and relatively homogeneous areas for this kind of analysis to achieve ecological validity. Male unemployment rates at this level allow for the ranking of areas in terms of priority need.