Hypothalamic Origin Research Articles

Hypothalamic-pituitary dysfunction in patients with craniopharyngioma.

Previous studies of preoperative pituitary function in patients with craniopharyngioma have been limited in scope and have focused on children. We have evaluated the impact of craniopharyngiomas and their surgical treatment on pituitary function in a large group of mostly adult patients. We performed a retrospective study of patients treated at our centre between 1980 and 1992. Twenty-two men and 13 women, most of them adults, treated surgically for craniopharyngioma during the above period. Serum glucose, GH, LH, FSH, TSH and cortisol were measured both before and after a combined insulin induced hypoglycaemia, GnRH and TRH test. Basal concentrations of thyroid hormones, PRL and gonadal steroids were also measured. Preoperative computed tomographic scan was performed in all patients, and a detailed study of visual function before and after surgery was available for 32 of them. Endocrine function was reevaluated post-operatively. In preoperative studies, 29 patients had some anterior pituitary deficit and 13 had diabetes insipidus. The most common abnormality was gonadotrophin deficiency, followed by GH deficiency. Dynamic studies suggested a hypothalamic origin for these deficits. In post-surgical evaluation, impaired pituitary function was observed in most patients. Panhypopituitarism was present in 28 cases and diabetes insipidus in 24. Our report illustrates the high incidence of endocrine deficits in patients with craniopharyngioma. Additional hypothalamic-pituitary dysfunction usually occurs following surgical treatment of these tumours.

Triiodo- L-thyronine enhances TRH-induced TSH release from perifused rat pituitaries and intracellular Ca 2+ levels from dispersed pituitary cells

There is now increasing evidence that Ca 2+ serves as the first messenger for the prompt and non-genomic effects of 3,5,3′ triiodo- L-thyronine (T 3) in several tissues. We have previously shown that the first phase of thyroid stimulating hormone (TSH) release in response to thyrotropin-releasing hormone (TRH) can be potentiated by messenngers of hypothalamic origin, by a Ca 2+-dependent phenomenon involving the activation of dihydropyridine-sensitive Ca 2+ channels. By perifusing rat pituitary fragments, we have investigated whether T 3 would modify TSH release when the hormone is applied for a short time (i.e. 30 min) before a 6 min pulse of physiological concentrations of TRH, thus excluding the genomic effect of T 3. We show that: (1) increasing concentrations of T 3 (100 nM−10 μM) in the perifused medium potentiates the TRH-induced TSH release in a dose-dependent manner; (2) the T 3 potentiation is not reproduced by diiodothyronine and T 3 does not potentiate the increase of TSH release induced by a depolarizing concentration of KCl; (3) the protein synthesis inhibitor cycloheximide, does not significantly modify the effect of T 3; (4) addition of Co 2+, nifedipine, verapamil, or ω-conotoxin in the medium, at a concentration which does not modify the TSH response to TRH, reverses the T 3 potentiation of that response. We also tested whether T 3 would change intracellular concentrations of Ca 2+, by measuring [Ca 2+] i with fura-2 imaging on primary cultures of dispersed pituitary cells, either in basal conditions or after stimulation by TRH or/and T 3. Both substances induced a fast increase of [Ca 2+] i, with a peak at 15 s, followed by a subsequent progressive decay with TRH and a rapid return with T 3. Our data suggest that T 3 enhances TRH-induced TSH release by a protein synthesis-independent and Ca 2+-dependent phenomenon, probably due to an increase in Ca 2+ entry through the activation of dihydropyridine- and ω-conotoxin-sensitive Ca 2+ channels. They also show that T 3 may acutely enhance [Ca 2+] i in pituitary cells. These findings support the idea of the occurence of a prompt and stimulatory role of T 3 at the plasma membrane level in normal rat pituitary gland.

Effect of 15-day treatment with growth-hormone-releasing hormone alone or combined with different doses of arginine on the reduced somatotrope responsiveness to the neurohormone in normal aging.

It is well known that both spontaneous and growth hormone-releasing hormone (GHRH)-stimulated GH secretion undergo an age-related decrease; in addition, there is supportive evidence that the GH hyposecretory state of aging is of hypothalamic origin. The aims of the study in 35 normal elderly subjects (20 males and 15 females aged 65-89 years) were to verify whether the low somatotrope responsiveness to GHRH (1 microgram/kg) can be primed by a daily GHRH treatment and whether the potentiating effect of both high intravenous (0.5 g/kg) and low oral (8 g) doses of arginine (ARG) on GH response to GHRH is maintained with time. In group A (N = 14) the GH response to GHRH on day 1 (AUC: 373.5 +/- 78.5 micrograms.l-1.h-1) was unchanged after 7 (3720 +/- 38 micrograms.l-1.h-1) and 15 days (377.9 +/- 63.8 micrograms.l-1.h-1) of daily GHRH administration. In group B (N = 6) the GH response to GHRH co-administered with iv ARG on day 1 (1614.2 +/- 146.2 micrograms.l-1.h-1) was higher (p < 0.05) than that of GHRH alone (group A) and persisted unchanged after 7 (1514.7 +/- 366.5 micrograms.l-1.h-1) and 15 days (1631.7 +/- 379.1 micrograms.l-1.h-1) of treatment. In group C (N = 15) the GH response to GHRH co-administered with oral ARG on day 1 (950.6 +/- 219.4 micrograms.l-1.h-1) was higher (p < 0.03) than that of GHRH alone (group A) but lower (p < 0.05) than that to GHRH plus iv ARG (group B).(ABSTRACT TRUNCATED AT 250 WORDS)

Cortisol levels alter the response to metoclopramide in patients with hypothalamic amenorrhea.

The reduction in frequency and/or amplitude of gonadotropin-releasing hormone (GnRH) pulses in patients with amenorrhea of hypothalamic origin has been attributed to increased dopamine activity. The objective of the present study was to determine the role of dopamine in the pathogenesis of hypothalamic amenorrhea. Fourteen patients with hypothalamic amenorrhea, nine of whom had psychogenic amenorrhea and five anorexia nervosa, were studied and compared with nine normal women during the early follicular phase. Metoclopramide (10 mg), a dopamine antagonist, was infused intravenously and blood samples were collected at 15-min intervals for 2 h for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) measurement by radioimmunoassay. Both the hypothalamic amenorrhea (psychogenic amenorrhea and anorexia nervosa) and control groups were unresponsive to FSH, suggesting that dopamine may have little or no effect on FSH secretion. Five patients of the psychogenic amenorrhea group responded to LH (responsive psychogenic amenorrhea) and four did not (non-responsive psychogenic amenorrhea). No anorexia nervosa or control patient responded to the stimulus. Responsive psychogenic amenorrhea patients showed decreased basal cortisol levels compared to the non-responsive psychogenic amenorrhea and anorexia nervosa groups. It is possible that patients with exclusive alterations in the dopaminergic system are those who respond to metoclopramide (responsive psychogenic amenorrhea group), whereas patients who also have involvement of the hypothalamic-adrenal axis like the women with anorexia nervosa, are not responsive to metoclopramide and tend to have elevated cortisol levels. The non-responsive psychogenic amenorrhea group, with elevated cortisol levels, probably represents an intermediate step between the responsive psychogenic amenorrhea and anorexia nervosa patients.

DNA regulatory sequences of the rat tyrosine hydroxylase gene direct correct catecholaminergic cell-type specificity of a human growth hormone reporter in the CNS of transgenic mice causing a dwarf phenotype

Transgenic mice bearing 4.8 kilobases (kb) of upstream rat tyrosine hydroxylase (TH) sequences linked to a human growth hormone gene (hGH) exhibited cell-specific expression of hGH in all the appropriate catecholaminergic neurons in the central nervous system (CNS), although with different penetrance in two different mouse lineages. No ectopic expression was observed in any brain or peripheral region in one founder and its progeny. In another founder there was some ectopic expression in addition to appropriate and high levels of tissue-specific expression in all catecholaminergic areas. These results identify regulatory sequences that are sufficient for targeting expression to all catecholaminergic CNS neurons. Also, expression of exogenous hGH in the hypothalamus caused a dwarf phenotype, generating a novel genetic model for GH deficiency of hypothalamic origin.

Advances in induction of ovulation

The emphasis of this review is placed on the treatment of clomiphene-resistant women with polycystic ovary syndrome (PCOS) or with hypogonadotropic hypogonadism. There has been an increasing awareness of the need to avoid the consequences of multiple folliculogenesis, and this is reflected in the more widespread use of low-dose regimens for induction of ovulation, particularly in PCOS. The past 12 months have seen the first reports of the clinical applications of recombinant human follicle-stimulating hormone (FSH), and there is an intriguing suggestion that long-acting opiate agonists may have a part to play in the management of anovulation of hypothalamic origin.

Olfactory function in patients with hypogonadotropic hypogonadism: an all-or-none phenomenon?

Hypogonadotropic hypogonadism (HH) refers to an endocrine defect of hypothalamic origin resulting in gonadal hypoplasia and frequently associated with anosmia or severely impaired olfactory function (Kallmann's syndrome). This apparently results from a disruption in the migration of neurons from the olfactory placode to the bulb and hypothalamus early in development, and so provides a unique opportunity to investigate olfactory function in human subjects with congenitally incomplete peripheral systems. Olfactory performance in 37 HH patients and 37 age-matched controls was compared using a modified version of the Munich Olfaction Test. This test is based on the sniff-bottle method and includes tests of (i) odor quality discrimination, (ii) intensity discrimination, (iii) detection thresholds, and (iv) recognition, hedonic evaluation and identification ability. The patients could be divided into two distinct groups differing significantly on all four subtests and showing no overlap in performance: 20 anosmics, conforming to Kallmann's syndrome, and 17 apparent normosmics whose performance was slightly poorer, but not significantly different to that of the controls. The unexpected failure to find a continuum of olfactory dysfunction now raises the question whether HH with or without anosmia represents two syndromes with distinct etiologies, or rather reflects the ability of the olfactory system to function well despite morphological impairment.

Increased luteinizing hormone and alpha-subunit secretion in women with hyperandrogenic anovulation.

Women with hyperandrogenic anovulation (HAA) have increased circulating levels of LH relative to those of FSH. The cause of this disturbance in gonadotropin secretion is uncertain. Previous investigations have sought to determine if increased GnRH drive is responsible for the excessive LH concentrations. Because previous results have conflicted, we addressed this question by comparing the 24-h secretory patterns of alpha-subunit and LH in women with HAA (n = 9) to those in eumenorrheic women in the midfollicular phase (n = 9). The mean (+/- SEM) pulse frequency was increased in women with HAA compared to that in eumenorrheic women of comparable age and percent ideal body weight for both LH (23.0 +/- 0.7 pulses/24 h vs. 3 17.1 +/- 1.7; P = 0.002) and alpha-subunit (23.0 +/- 0.8 vs. 19.1 +/- 1.2; P = 0.02). LH and alpha-subunit, but not FSH, responses to a submaximal dose of exogenous GnRH were increased in HAA, as were basal LH and alpha-subunit levels (P < 0.01). The present observations provide evidence for increased GnRH drive, including pulse frequency, in HAA. Although the results confirm the presence of a disturbance in gonadotropin secretion and suggest that its proximate cause may be of hypothalamic origin, they do not exclude the possibility that other factors, perhaps of ovarian origin, play a role in the establishment and/or maintenance of the altered gonadotropin secretory patterns and the chronic anovulation characteristic of HAA.

Measurement of cAMP accumulation in Chinese hamster ovary cells transfected with the recombinant human TSH receptor (CHO-R): a new bioassay for human thyrotropin.

Circulating TSH bioactivity may vary in several clinical and experimental conditions. Since the reliability of the current methods for the measurement of TSH bioactivity is limited, a new bioassay based on cAMP accumulation in Chinese Hamster Ovary cells transfected with recombinant human TSH receptor (CHO-R) was set up. The sensitivity was 0.3 +/- 0.1, 0.4 +/- 0.1 and 0.01 +/- 0.01 micrograms/L for TSH IRP 80/558, recombinant human TSH and bovine TSH, respectively. Standard curves were parallel, and the intra- and inter-assay coefficients of variation were 13 +/- 1.1% and 22 +/- 1.9%, respectively. LH, FSH, CG and TSH subunits did not stimulate cAMP accumulation up to high concentrations. Circulating TSH was partially purified by immunoaffinity separation and concentrated before being bioassayed. However, plain sera with high TSH levels, such as those from primary hypothyroid patients (PH), could be directly tested in CHO-R bioassay, provided that sera were added at concentrations lower than 10%. TSH from 6 normal subjects had biological to immunological ratio (B/I) ranging from 0.6 to 2.1 (mean +/- SD = 1.4 +/- 0.5). TSH from 6 patients with PH showed bioactivity significantly lower than in normals (B/I = 0.6 +/- 0.3; p < 0.001; range = 0.3-1.1). TSH from 5 patients with central hypothyroidism of hypothalamic origin (CH) had undetectable basal bioactivity (B/I < 0.2), which normalized in only one patient after acute TRH and in all patients after chronic TRH administration. In conclusion, CHO-R cells provide an excellent tool for evaluating TSH bioactivity, owing to high sensitivity, specificity, reproducibility and feasibility of the assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoreactive gonadotropin-releasing hormone in porcine reproductive tissues

Studies were aimed to identify and characterize IR-GnRH in the porcine endometrium, ovary, oviduct, and compare that with GnRH of hypothalamic and placental origin. RP-HPLC profiles revealed that all tissue extracts contained three peaks of IR-GnRH. Extraction with radioiodinated 125I-GnRH also resulted in three similar peaks, indicating these two extra peaks as an extraction artifact. Radioreceptor assay showed endometrial and ovarian ultrafiltrates displaced binding of 125I-GnRH analog to pituitary membrane. Secretion of uterine IR-GnRH was increased in ovariectomized, progesterone-treated gilts, as compared to the control. Treatment with estradiol + progesterone in ovariectomized gilts further increased uterine secretion of IR-GnRH. These results demonstrate that IR-GnRH identified in the porcine reproductive tissues possesses hydrophobicity similar to those in hypothalamus and placenta; endometrial IR-GnRH binds to pituitary GnRH receptors; and uterine secretion of IR-GnRH is modulated by ovarian steroids.

ENDOCRINE DISORDERS IN SEPTO-OPTIC-DYSPLASIA (DE MORSIER SYNDROME)

The syndrome of septo-optic-dysplasia (SOD) is characterized by hypoplasia of the optic nerve, various types of forebrain defects and hormonal deficiencies. To better define the endocrine abnormalities in children with SOD we have studied the clinical and endocrinological characteristics of 16 such patients retrospectively. The children were seen in the pediatric endocrinology service at the Children's Hospital of the University of Munich from 1976 through 1992 (7 boys, 9 girls; age at initial presentation: 1 day to 13 years of age, mean 1.9 years). Mothers' age at delivery was between 16 and 32 years, mean 21.6. Seven of the 16 patients had neonatal hyperbilirubinaemia. Seven patients had perinatal problems with cyanosis and apnea which necessitated mechanical ventilation. Three neonates suffered from hypoglycaemia. Unilateral hypoplasia of the optic nerve was found in 5 cases, bilateral hypoplasia in 10. Sonographic, CT or NMR imaging yielded the following results: Five of the patients had a cavum septi pellucidi. Three patients had hypoplasia of the cerebellum, one aplasia of the corpus callosum and one aplasia of the fornix. An empty sella with or without an ectoptc pituitary was seen in four cases. Height at time of diagnosis was -3.96 to +2.09 SDS, mean -2,38. Endocrine deficiencies were present in all 11 patients who had undergone endocrinological investigations. Seven patients suffered from GH deficiency (GH below 10 ng/ml in two provocative tests). One had diabetes insipidus centralis, one had hypogonadotropic hypogonadism, one had hypothyroidism and two adrenal insufficiency. Hypothalamic testing was performed in a subset of patients: in five of eight children tested a TRH test, in two out of eight a LHRH test, and in three out of five a GHRH test yielded abnormal results. High prolactin levels were measured in two out of five patients. In conclusion, (1) SOD is characterized by optic nerve hypoplasia and a variety of endocrine deficiencies. In addition, forebrain malformations are present in some SOD patients. (2) The putative hypothalamic origin of the hormonal disorders in some SOD patients needs to be investigated systematically. (3) Pediatric endocrinotogists need to participate in the care of aH patients with SOD.

Impaired Response of Free Alpha-Subunits after Luteinizing Hormone-Releasing Hormone and Thyrotropin-Releasing Hormone Stimulations in Beta-Thalassemia Major

In order to clarify whether the damage in gonadotropin secretion due to iron overload in patients with beta-thalassemia is of pituitary or hypothalamic origin, 14 euthyroid patients (8 females and 6 males, age 15-24 years) affected by beta-thalassemia major with hypogonadotropic hypogonadism were studied. Luteinizing-hormone (LH), follicle-stimulating hormone (FSH) and free alpha-subunit (FAS) were measured during LH-releasing hormone (LH-RH) stimulation test, and thyroid-stimulating hormone (TSH), prolactin (PRL) and FAS during thyrotropin-releasing hormone (TRH) stimulation test. During LH-RH stimulation, the mean basal LH, FSH and FAS levels were similar to those found in normal prepubertal children, but the peak values were lower than those found in such children. Also during TRH stimulation, the mean peak values of FAS were lower than those of normal prepubertal children, but the TSH response was normal. The lack of response of gonadotropins and FAS to LH-RH cannot exclude hypothalamic failure; however, the normal response of TSH to TRH, in spite of the poor response of FAS, indicates that the origin of hypogonadotropic hypogonadism is the pituitary damage concerning not only the gonadotroph but also the thyrotroph cells.

Presence of somatostatin or neurotensin in lateral septal dopaminergic axon terminals of distinct hypothalamic and midbrain origins: convergence on the somatospiny neurons.

In the lateral septal area (LSA), both inhibitory and excitatory dopamine (DA) actions, as well as hypothalamic and midbrain DA efferents, have been described. Some neurons of the hypothalamic and midbrain DA systems contain somatostatin (SOM) or neurotensin (NT), and, in the LSA, the distribution of fibers containing these peptides overlaps with DA fibers. These data prompted us to test for the presence of SOM and NT in LSA dopaminergic axon terminals of hypothalamic and midbrain origins. To verify the origins of SOM and NT innervation of the LSA, the retrograde tracer horseradish peroxidase conjugated with wheat germ agglutinin (HRP-WGA) was injected into the LSA, and alternate brain sections were immunostained for SOM, NT, or tyrosine hydroxylase (TH) in group 1 rats. Numerous retrogradely labeled neurons were found immunopositive for SOM in the periventricular and basolateral hypothalamus, many HRP-WGA labeled cells contained NT immunoreactivity in the ventral tegmental area, and TH-immunoreactive retrogradely labeled neurons were observed in both brain areas. In a new approach, the presence of these peptides in dopaminergic boutons was assessed by combining peptide immunocytochemistry with acute 6-hydroxydopamine (6-OHDA) induced lesioning of DA cell groups. These groups of rats were treated with desipramine to protect the noradrenergic fibers, and 45 min later 1 microgram 6-OHDA (in 0.5 microliter saline) was unilaterally injected into the periventricular hypothalamus (group 2) or the ventral tegmental area (group 3). After 48 h the rats were killed and alternate septal sections of both groups were immunostained for TH, SOM, or NT. On the operated side of the LSA in both groups, electron microscopy revealed numerous axon terminals that were immunopositive for TH and contained autophagous cytolysosomes, an early sign of catecholamine fiber degeneration induced by 6-OHDA. In group 2, phagosome-containing boutons were found immunopositive for SOM, but not for NT; vice versa, in group 3, only NT-positive degenerated boutons were detected. SOM- and NT-positive degenerated axon terminals in both groups formed synaptic contacts with LSA neurons, in particular with somatospiny cells. On the contralateral side of the LSA, all of the axon terminals were intact. It has been shown that SOM exerts an inhibitory action, whereas NT has an excitatory effect on limbic area neurons. Thus, the results implicate that the differential peptide content of dopamine fibers marks their functional differences. It appears that LSA neurons receive double innervation from an inhibitory "somatostatinergic" DA system of the hypothalamus, and from an excitatory "neurotensinergic" DA system of the midbrain.

Cytokines in reproduction.

A growing body of evidence indicates that, in addition to the hormones of hypothalamic, pituitary and gonadal origin, autocrine/paracrine regulators play important roles in the reproductive functions. Cytokines, originally known as immunoregulatory proteins, may affect the neuroendocrine events of reproduction, ovarian/testis function, endometrium, the developing embryo, placenta and parturition. Cytokines can modulate and mediate the actions of hormones at their target cells and, in the opposite way, hormones may regulate the production and action of cytokines at three different levels: cytokine secretion, cytokine receptor expression and cellular responses. Cytokines may also function in an endocrine manner affecting distant targets. As many of the cyclic changes that occur in the ovary and endometrium during the normal menstrual cycle are similar to those associated with the inflammatory and regenerative processes, it is likely that cytokines are involved in these reactions. Furthermore, cytokines secreted by endometrial white blood cells may influence embryo development and trophoblast growth and may play a fundamental role in the mechanisms of immunological reproductive failure. Cytokines have also been implicated in the mechanisms responsible for the onset of parturition.

Thyrotropin‐Releasing Hormone Mediates Growth Hormone Release Induced by Milk and Nursing in Neonatal Rats

Previous evidence from this laboratory suggested that growth hormone (GH) release induced by milk in vitro and by nursing in vivo from neonatal rat pituitary glands is mediated by an alternative GH-releasing factor(s) (GRF) distinct from GH-releasing hormone (GHRH(1-43) ). In the present experiments we tested whether thyrotropin-releasing hormone (TRH) could fulfil the criteria of this alternative GRF in neonatal rats. The water-soluble fraction of rat milk (infranatant, prepared by ultracentrifugation) and its methanol/acetic acid extract (milk-borne peptides) stimulated GH release from perifused pituitary glands obtained from 2-day-old rats. Dialysis of the infranatant (mol wt cut-off: 2,000) against 500 volumes of culture medium at 4°C eliminated its GH-releasing activity in the perifusion system, while the infranatant retained its full GRF-like activity when incubated at 4°C without dialysis. The milk-borne GRF eluted as a single peak and coeluted with TRH in a combined gel permeation chromatography (Sephadex G-10) and perifusion set-up. Prolactin secretion was also stimulated simultaneously with the release of GH induced either by milk or by TRH. In a stepwise C(18) reversed-phase chromatography, milk-borne GRF was highly hydrophilic and coeluted with synthetic TRH. The in vitro GH-releasing bioactivities of synthetic TRH and a milk extract purified in C(18) reversed-phase chromatography were abolished by proline-specific endopeptidase. Thus, TRH and milk-borne GRF displayed similar molecular weights, hydropathic characteristics and proteolytic enzyme resistance. In vivo, nursing (which has been reported as a potent stimulus of GH secretion even in the absence of milk-intake) increased serum GH levels in 2-day-old pups. A supramaximal dose of TRH (10 ng/g intraperitoneally) stimulated GH release in 2-day-old pups separated from their mothers for 6 h to a similar extent as nursing. Nursing-induced levels of serum GH were not further elevated by TRH. This failure of TRH to further increase serum GH levels was not due to a maximal GH output by the neonatal pituitary gland, since the GH release induced by the serotonin precursor 5-hydroxy-L-tryptophan was augmented either by TRH or by nursing. These data provide evidence that the milk-borne GRF-like activity in vitro is indistinguishable from TRH, and suggest that TRH (probably of hypothalamic origin) might be the mediator of the nursing-induced release of GH in vivo as a physiological GRF in neonatal rats.

Effects of loperamide on the human hypothalamo-pituitary-adrenal axis in vivo and in vitro.

Loperamide, an opiate agonist of high specificity for mu-receptors, was recently reported to suppress ACTH and cortisol levels in normal subjects, but not in patients with proven ACTH-dependent Cushing's disease. However, there is little information on the site of action of loperamide in the hypothalamo-pituitary-adrenal axis of man. We investigated the effect of loperamide on pituitary hormone secretion in vivo and in vitro. In seven normal subjects, basal ACTH plasma levels were significantly suppressed 3 h after loperamide administration (16 mg, orally) from 5 +/- 1 to 2 +/- 0 pmol/L (P less than 0.0001). After the combined pituitary stimulation test (100 micrograms human CRH, 100 micrograms GnRH, 100 micrograms GH-releasing hormone, and 200 micrograms TRH), the ACTH peak (maximum increase at 30 min) was significantly blunted by loperamide from 9 +/- 1 to 4 +/- 1 pmol/L (P less than 0.001) and the area under the curve of ACTH from 0-120 min was reduced from 35 +/- 5 to 23 +/- 4 pmol/L.2 h (P less than 0.05). In the insulin-hypoglycemia test (0.15 IU/kg BW), neither the ACTH peak nor the area under the curve of ACTH was affected by loperamide. In six patients with Cushing's disease and one patient with secondary adrenal insufficiency due to hypothalamic failure, neither basal ACTH and cortisol levels nor CRH-stimulated levels were influenced by loperamide. In four cultured human corticotropic adenomas, loperamide was not able to reduce basal and CRH-induced ACTH secretion. In summary, loperamide is able to reduce basal and CRH-induced ACTH and cortisol levels in normal subjects, but not in patients with Cushing's disease or secondary adrenal failure of hypothalamic origin. Loperamide has no significant effect on insulin-hypoglycemia-induced ACTH and cortisol levels and, therefore, no effect on stress-induced elevation of cortisol levels. Loperamide might act at a suprapituitary site in man in vivo, but, nevertheless, a pituitary site cannot be excluded.

Activity of vasoactive intestinal peptide and serotonin in the paraventricular nucleus reflects the periodicity of the endogenous stimulatory rhythm regulating prolactin secretion.

PRL secretion in the female rat is regulated by an endogenous stimulatory rhythm (ESR) of prolactin-releasing factors of hypothalamic origin which has a bimodal periodicity with distinct nocturnal (N) and diurnal (D) phases. The N phase reaches peak magnitude by 0300 h and the D phase reaches peak magnitude by 1700 h. This rhythm was first unmasked in ovariectomized rats by correctly timed injection of a dopamine antagonist. OT, vasoactive intestinal peptide (VIP), and serotonin (5-HT) are differentially involved in generating the ESR. Pharmacological studies suggest that OT is the neurohormone and VIP and 5-HT are neuromodulators which act to stimulate OT release. Recently, we reported that activity of OTergic neurons in the paraventricular nucleus (PVN) and OT concentrations in the anterior pituitary mirror the periodicity of the ESR. The present experiments were conducted to determine if VIP and 5-HT activity in the hypothalamus also mirrors the periodicity of the ESR. Push-pull cannulae were surgically implanted in the PVN of ovariectomized female rats. Following recovery, push-pull perfusion was conducted from either 0600-1400 h, 1400-2200 h, or 2200-0600 h. VIP was measured in perfusates by RIA. There was no difference in VIP pulse frequency between rats perfused during the three periods studied. However, animals perfused from 2200-0600 h had significantly greater pulse amplitude as compared to rats at either 0600-1400 h or 1400-2200 h. Activity of 5-HTergic neurons in the hypothalamus was studied by estimating the turnover of 5-HT 10 min following the injection of pargyline. Hypothalamic nuclei were dissected using Palkovits' punch technique and 5-HT concentration assayed by HPLC in conjunction with electrochemical detection. Turnover of 5-HT was estimated by calculating the slope of the accumulation of 5-HT over 10 min at differing times of day using least squares regression analysis. There was a distinct diurnal rhythm of 5-HT accumulation in the PVN. Rats killed at 1700 h had significantly greater slopes of 5-HT accumulation in the PVN than rats killed at either 0300 or 1200 h. Similarly, there was a diurnal rhythm of 5-HT turnover in the suprachiasmatic nucleus. Rats sampled at either 1200 or 1700 h had significantly greater slopes of 5-HT accumulation in the suprachiasmatic nucleus than rats sampled at 0300 h. There was no diurnal rhythm of 5-HT turnover evident in either the median eminence or the supraoptic nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)

Morphological and electrophysiological evidence for postsynaptic localization of functional oxytocin receptors in the rat dorsal motor nucleus of the vagus nerve

The vagal complex is innervated by oxytocin immunoreactive axons of hypothalamic origin. The presence of oxytocin binding sites in the dorsal motor nucleus of the vagus nerve of the rat was evidenced by autoradiography with a radioiodinated oxytocin antagonist as ligand. Two weeks following a unilateral vagotomy, distal to the nodose ganglion, binding sites were reduced below the level of detection in the ipsilateral dorsal motor nucleus of the vagus nerve. Choline acetyltransferase immunoreactivity was also markedly reduced in the vagal motoneurons whose axons had been transected. Electrophysiological studies were performed in vitro in brainstem slices from control rats. In antidromically identified vagal motoneurones, oxytocin applied at 0.1–1.0 μM either caused a reversible depolarization or generated, under voltage-clamp conditions, a transient inward current. These responses persisted under the condition of synaptic uncoupling. Taken together these observations favour the notion that oxytocin of hypothalamic origin acts directly on rat vagal motoneurones.

Is thyrotropin-releasing hormone immunoreactivity in peripheral blood an estimate for hypothalamic thyrotropin-releasing hormone release?

The significance of TRH for pituitary function is still unresolved mainly due to limitations in determining in vivo hypothalamic TRH release. We therefore examined whether TRH immunoreactivity (TRH-IR) in peripheral blood is an index for hypothalamic TRH release. Peripheral TRH-IR varied between 10 and 55 pmol/l and was similar in euthyroid and hypothyroid rats, but lower in hyperthyroid rats. Destruction of the hypothalamic paraventricular area reduced peripheral TRH-IR, while stimulation of this area increased it. Clearance of TRH during continuous TRH infusion was 1.9 +/- 0.2, 3.5 +/- 0.3 and 5.9 +/- 0.8 ml/min in hypothyroid, euthyroid and hyperthyroid rats, respectively. These and previous data on TRH in hypophysial portal blood indicate that 5-25 pmol TRH/l peripheral blood is of hypothalamic origin. Chromatography revealed that TRH-IR from hypothalamus and portal blood co-eluted with TRH, but in peripheral blood two peaks were found, one of which was authentic TRH. Thus, peripheral TRH-IR alters in experimental conditions and part of it seems to be of hypothalamic origin. However, the presence of TRH-like material in peripheral blood not identical to TRH and the fact that experimental conditions alter TRH clearance indicate that peripheral TRH-IR is not an index for hypothalamic TRH release.

Investigation and Characterization of Receptors for Pituitary Adenylate Cyclase-Activating Polypeptide in Human Brain by Radioligand Binding and Chemical Cross-Linking

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel peptide of hypothalamic origin which increases adenylate cyclase activity in rat anterior pituitary cell cultures. The 38-amino acid peptide shows a close sequence homology to vasoactive intestinal peptide (VIP). Binding sites for PACAP in membranes from postmortem human brain tissue were studied using [125I]PACAP27 as the radioligand. High specific binding sites (amount of specific binding measured at 0.25 nM [125I]PACAP27 in femtomoles per mg protein +/- SEM; n = 4) were present in hypothalamus (344.5 +/- 13.0), brain stem (343.0 +/- 29.3), cerebellum (292.0 +/- 21.1), cortex (259.6 +/- 19.8), and basal ganglia (259.2 +/- 50.3). Specific binding sites in pituitary, although present, were less abundant (35.0 +/- 8.9). Binding of [125I]PACAP27 was reversible and time, pH, and temperature dependent. Despite the homology with VIP, VIP was a poor inhibitor of [125I]PACAP27 binding (IC50, greater than 1 microM) compared with PACAP27 (IC50, 0.5-1.3 nM) and PACAP38 (IC50, 0.2-1.3 nM). Scatchard plots of [125I]PACAP27 binding showed the presence of both high and lower affinity sites. Chemical cross-linking of PACAP-binding sites revealed that [125I]PACAP27 was bound to polypeptide chains of 67,000 and 48,000 mol wt. Thus, we have demonstrated the presence of PACAP-specific receptors in human brain which are not VIP receptors. This opens the possibility of PACAP functioning as a novel neurotransmitter/neuromodulator in human brain.