Pituitary stimulation by combined administration of four hypothalamic releasing hormones in normal men and patients

Ten normal young men (22-28 yr of age), within 10% of their ideal body weight, were given the four releasing hormones (TRH, 200 micrograms; GnRH, 100 micrograms; ovine corticotropin-releasing hormone, 50 micrograms; GH-releasing hormone, 80 micrograms) iv on separate days and then in combination on the same day. Plasma TSH, PRL, FSH, LH, cortisol, ACTH, and GH were measured by RIA in samples collected from 20 min before to 120 min after injection. There were no significant differences in responses to the separate and combined tests for FSH, LH, cortisol, ACTH, and GH. The plasma TSH (0.001 less than P less than 0.01) and PRL (P less than 0.001) responses were significantly higher after the combined test. The tolerance was identical to that of TRH alone. In eight patients studied after pituitary surgery, combined administration provided results comparable to those obtained after separate administration of TRH, GnRH, and insulin.     

Temporal relationships between the circadian rhythmicity in plasma levels of pituitary hormones and in hypothalamic concentrations of releasing factors

Ovariectomized female rats were implanted with estradiol containing silastic implants to induce constant circulating levels of the steroid, and sacrificed every 2 h in order to determine neuroendocrine rhythms. Under these conditions, we observed very marked circadian fluctuations in the hypothalamic concentrations of corticotropin-releasing factor (CRF), luteinizing hormone-releasing hormone (LHRH) and thyrotropin-releasing hormone (TRH), and in plasma levels of adrenocorticotropin (ACTH), corticosterone, luteinizing hormone (LH) and prolactin; the amplitude of the prolactin cycle was in particular much higher than in non-chronically estrogenized animals. The daily variation in CRF, ACTH and corticosterone showed significant rank correlations. Changes in hypothalamic content of LHRH and TRH were biphasic; the increase observed during the light period was abruptly interrupted by a depletion episode, coincident with the period of maximal LH and prolactin secretion, respectively. The initial phase of ACTH, LH and prolactin increments occurred between 11.00 and 15.00 h, and was relatively well synchronized. The steepest rise in ACTH and prolactin occurred at the same time, and preceded that of LH by a constant lag of about 2 h. After that initial period, secretion kinetics of the three hormones followed an independent pattern. The data suggest that increased secretion of several hormones results from activation of neural mechanisms occurring within a limited period of the 24-hour cycle.     

Responsiveness of irradiated rat anterior pituitary cells to hypothalamic releasing hormones is restored by treatment with growth hormone

Hypopituitarism is a common sequela of irradiation in cancer patients. Here we report that recombinant human growth hormone (r-hGH) prevents cell death and restores secretory capacity of irradiated rat pituitary cells in vitro. Dispersed rat pituitary cells from male Sprague-Dawley rats, irradiated with a 9-Gy sublethal dose, were incubated with r-hGH before, after, or before and after irradiation. Treatment with GH resulted in increased cell survival, which reached its maximum at the concentration of 5 nM, with an EC(50) of 3.5 nM. Protective effects of GH on pituitary cells were more pronounced in cultures treated before and after irradiation. Similarly, beneficial effects of GH were observed on the secretory capacity of surviving cells. In fact, irradiated pituitary cells treated with GH secreted substantial amounts of GH, luteinizing hormone, follicle-stimulating hormone, prolactin, thyroid-stimulating hormone and adrenocorticotropic hormone in response to specific releasing hormones. Such effects of GH were prevented in the presence of the specific GH receptor antagonists B2036 and G120K. Our results show that r-hGH exerts a specific protective effect on irradiated rat pituitary cells and suggest possible use of GH as an adjuvant agent for prevention of postirradiation hypopituitarism.     

Hypothalamic hormones a.k.a. hypothalamic releasing factors

Originally searched for and eventually isolated as factors of hypothalamic origin controlling anterior pituitary secretions, these hypophysiotropic peptides are now a chapter of physiology and medical endocrinology of their own. Defying the concept of 'neuropeptides' they and their receptors are now known to be ubiquitous and to have subtle as well as profound effects on a large number of functions of both soma and psyche. This review will be composed of brief essays on current knowledge of each of the original 'hypothalamic hormones', TRH, GnRH, somatostatin, GHRH and corticotropin releasing hormone and will close on possible and probable futures.     

Dissociation of pituitary glycoprotein response to releasing hormones in chronic renal failure

The LH, FSH and TSH response to LRH and TRH has been evaluated in patients with chronic renal failure. Basal gonadotrophins were elevated in 3 out of 6 males; one of 4 pre-menopausal females had increased basal LH. Exaggerated LH responses to LRH were noted in 4 out of 6 males and one of 4 females; FSH reponses were increased in 3 of these males. One male and one female had attenuated LH and FSH responses to LRH. Both testosterone and oestradiol levels were reduced. In 5 out of 6 subjects tested both pre- and post-dialysis there was a greater LH and FSH response to LRH following dialysis. This suggests the presence of a dialysable toxin which is inhibiting the gonadotrophin response to LRH. Gonadotrophin levels remained elevated during 4 h of dialysis suggesting prolongation of the metabolic clearance rate. Despite low T3 levels, TSH response to TRH (200 microgram) was only elicited in 2 of 6 cases. However, all 3 responded to 500 microgram and 2 out of 3 to 1000 microgram TRH, the third showing an attenuated response. TSH levels also remained persistently elevated in the responders. Dialysis however, failed to improve the relative TSH non-responsiveness to TRH. In conclusion the data has shown that there is a dissociation in glycoprotein hormone responses to releasing hormones in ureamia. Whereas the gonadotrophs retain their responsiveness to LRH, the thyrotrophs appear to be more effected by the uraemic process and demonstrate an impaired response to TRH.     

Multiple stimulation of the adenohypophysis by combinations of hypothalamic releasing factors

We have investigated the in vitro and in vivo interactions of the four hypothalamic releasing factors, LHRH, corticotropin-releasing factor, TRH, and GH-releasing factor on anterior pituitary hormone secretions, using a 2 X 2 X 2 X 2 factorial experimental design. This experimental design allows for the evaluation of both the main treatment effects of the hypothalamic releasing factors as well as all of the possible interactions between them. Significant main treatment effects were: LHRH on LH and FSH, corticotropin-releasing factor on ACTH and beta-endorphin, TRH on TSH, and GH-releasing factor on GH. These results confirm the specificity of the four releasing factors on their respective target cells. There were no significant interactions between any of the releasing factors on anterior pituitary hormone secretions. These results suggest that the changes in pituitary secretion that are observed under physiological conditions are not due to interactions between the hypothalamic releasing factors at the level of the pituitary, but rather to other secondary interactions that modify pituitary activation or response. These results also indicate that the clinical pituitary reserve tests can be expanded to include all four hypothalamic releasing factors, since any lack of response will reflect a specific pituitary defect and not a failure to respond owing to interaction of the secretagogues administered.     

Corticotropin releasing hormone stimulation test: diagnostic aspects in Cushing's syndrome

The effect of exogenous ovine Corticotropin-Releasing Hormone (oCRH) on plasma ACTH and cortisol levels was investigated in 10 normal volunteers and in 37 patients with Cushing's syndrome (26 with pituitary-dependent disease, 5 with an adrenal adenoma, 2 with an adrenal carcinoma and 4 with bilateral nodular hyperplasia). In all normal subjects and in patients with Cushing's disease, oCRH 100 micrograms as a bolus produced an increase in both plasma ACTH and cortisol. The peak of ACTH occurred after 15-30 min, while plasma cortisol showed highest levels between 30 and 60 min after oCRH administration. The hormonal response in Cushing's disease showed great variability with a clear hyperresponsiveness at least in 6 out of 26 patients with Cushing's disease. A slight and delayed response occurred in 3 cases of bilateral nodular adrenal hyperplasia, while a fourth case showed hyperresponsiveness similar to that found in pituitary-dependent Cushing's disease. No response was observed in patients with an adrenal tumor. Eleven patients with Cushing's disease were tested before and 1 month after pituitary microadenomectomy. After surgery basal cortisol levels were reduced in 10 and became unresponsive or less responsive to oCRH. ACTH patterns were variable with a normal response only in few cases. Although this test seems of limited value in the diagnosis of hypercortisolism, it is a useful tool to differentiate some types of Cushing's syndrome (adrenal tumor from pituitary-dependent Cushing's disease). Variable patterns of response in cases with bilateral nodular adrenal hyperplasia limit the usefulness of this test in recognizing this rare form of hypercortisolism.     

Development of radioimmunoassay for bullfrog thyroid-stimulating hormone (TSH): effects of hypothalamic releasing hormones on the release of TSH from the pituitary in vitro

A bullfrog (Rana catesbeiana) thyroid-stimulating hormone (TSH) beta-subunit (TSHbeta) antiserum was produced by employing a C-terminal peptide synthesized on the basis of the amino acid sequence deduced from bullfrog TSHbeta cDNA. Immunohistochemical studies revealed that the bullfrog adenohypophyseal cells that immunologically reacted with the anti-bullfrog TSHbeta corresponded to those positively stained with an antiserum against human (h) TSHbeta. The antiserum was used for the development of a specific and sensitive radioimmunoassay (RIA) for the measurement of bullfrog TSH. The sensitivity of the RIA was 0.75+/-0.07ng TSH/100microl assay buffer. The interassay and intraassay coefficients of variation were 7.6 and 5.3%, respectively. Several dilutions of pituitary homogenates of larval and adult bullfrogs, or medium in which bullfrog pituitary cells were cultured, yielded dose-response curves that were parallel to the standard curve. Bullfrog prolactin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and alpha-subunit derived from glycoprotein hormones did not react in this assay. Immunoassayable TSH in the pituitary culture medium was confirmed to exist in the form of TSHbeta coupled with the alpha-subunit by an immunoprecipitation experiment using the TSHbeta antiserum and an alpha-subunit antiserum. TSH released from pituitary cells into the medium was also confirmed to possess a considerable activity in stimulating the release of thyroxine from the thyroid glands of larval bullfrogs in vitro.The effects of hypothalamic hormones such as mammalian gonadotropin-releasing hormone (mGnRH), ovine corticotropin-releasing hormone (oCRH), and thyrotropin-releasing hormone (TRH) on the release of TSH by dispersed anterior pituitary cells of the bullfrog larvae and adults were also studied. CRH markedly stimulated the release of TSH from both adult and larval pituitary cells. Both TRH and GnRH moderately stimulated the release of TSH from adult pituitary cells but not from the larval cells. This is the first report on the development of an RIA for amphibian TSH, which has provided the direct evidence that the release of TSH from the amphibian pituitary is enhanced by the hypothalamic releasing hormones such as CRH, TRH, and GnRH.     

Modulation of pancreatic islets-stress axis by hypothalamic releasing hormones and 11beta-hydroxysteroid dehydrogenase

Corticotropin-releasing hormone (CRH) and growth hormone-releasing hormone (GHRH), primarily characterized as neuroregulators of the hypothalamic-pituitary-adrenal axis, directly influence tissue-specific receptor-systems for CRH and GHRH in the endocrine pancreas. Here, we demonstrate the expression of mRNA for CRH and CRH-receptor type 1 (CRHR1) and of protein for CRHR1 in rat and human pancreatic islets and rat insulinoma cells. Activation of CRHR1 and GHRH-receptor significantly increased cell proliferation and reduced cell apoptosis. CRH stimulated both cellular content and release of insulin in rat islet and insulinoma cells. At the ultrastructural level, CRHR1 stimulation revealed a more active metabolic state with enlarged mitochondria. Moreover, glucocorticoids that promote glucose production are balanced by both 11b-hydroxysteroid dehydrogenase (11β-HSD) isoforms; 11β-HSD-type-1 and 11β-HSD-type-2. We demonstrated expression of mRNA for 11β-HSD-1 and 11β-HSD-2 and protein for 11β-HSD-1 in rat and human pancreatic islets and insulinoma cells. Quantitative real-time PCR revealed that stimulation of CRHR1 and GHRH-receptor affects the metabolism of insulinoma cells by down-regulating 11β-HSD-1 and up-regulating 11β-HSD-2. The 11β-HSD enzyme activity was analyzed by measuring the production of cortisol from cortisone. Similarly, activation of CRHR1 resulted in reduced cortisol levels, indicating either decreased 11β-HSD-1 enzyme activity or increased 11β-HSD-2 enzyme activity; thus, activation of CRHR1 alters the glucocorticoid balance toward the inactive form. These data indicate that functional receptor systems for hypothalamic-releasing hormone agonists exist within the endocrine pancreas and influence synthesis of insulin and the pancreatic glucocorticoid shuttle. Agonists of CRHR1 and GHRH-receptor, therefore, may play an important role as novel therapeutic tools in the treatment of diabetes mellitus.     

Dopaminergic stimulation of pituitary but not hypothalamic estrogen receptors in ovariectomized rats

This study was designed to examine the role of catecholamines and serotonin in the regulation of estrogen receptors (ER) in the medial basal hypothalamus (MBH) and anterior pituitary gland (AP) of adult ovariectomized rats. Plasma PRL and LH were also determined. Injection of alpha-methyl tyrosine (alpha-MT) resulted in the significant reduction of norepinephrine and dopamine (DA) content in the MBH, and of plasma LH levels as well as in the significant elevation of plasma PRL levels. This treatment also resulted in the significant reduction of ER concentration in the AP. The elevation in plasma PRL by alpha-MT was reversed by the simultaneous injection of bromocriptine (BC), a DA agonist, which also partially reversed the alpha-MT reduction of ER concentrations in the AP. BC had no effect on plasma LH levels. The ER concentration in the MBH was not significantly changed by any of these treatments. The reduction of serotonin content in the MBH by the injection of p-chlorophenylalanine had no effect on the ER concentration in either the MBH or AP, nor did it have any effect on plasma PRL levels. However, p-chlorophenylalanine treatment did decrease plasma LH levels. Neonatal treatment of female rats with monosodium glutamate which has been reported to destroy part of the MBH, resulted in a significant reduction in body and pituitary weight and in a significant elevation of plasma PRL levels in adults (2 months old). This treatment also resulted in a significant reduction in the AP and MBH ER concentration. Injection of BC to adults reversed the effects of neonatal monosodium glutamate treatment on plasma PRL levels and on the pituitary ER concentration, but had no effect on the ER concentration in the MBH. BC had no effect on the AP or MBH ER concentration in control rats, although it did as expected reduce the plasma PRL levels in these animals. Plasma LH levels were not significantly changed by any of these treatments. Injection of the DA antagonist, haloperidol, to adult rats resulted in a significant elevation of plasma PRL and in a significant reduction of ER concentration in the AP. Haloperidol treatment did not affect the binding affinity of these receptors. Overall, these data suggest that DA is involved with the regulation of ER in the AP.     

Monoamine biosynthesis in hypothalamic regions of dwarf mice: effect of replacement of deficient anterior pituitary hormones

Female Ames dwarf and phenotypically normal female mice were killed 30 min after treatment with NSD-1015, an aromatic L-amino acid decarboxylase inhibitor. The accumulation of dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan were measured by high-performance liquid chromatography with electrochemical detection and provided estimates of the endogenous biosynthesis of dopamine (DA) in the median eminence (ME) and serotonin biosynthesis (5-HT) in all brain regions which were examined. Dopamine synthesis was markedly suppressed in the ME while 5-HT synthesis was enhanced in both the ME and mediobasal hypothalamus (MBH) of dwarfs as compared to phenotypically normal mice. Overall, catecholamine biosynthesis (DOPA accumulation) was suppressed in the MBH of the dwarf mice but was not different from that observed in normal mice in the preoptic area anterior hypothalamus (POA-AH). The biosynthesis of 5-HT was not different in the POA-AH of dwarf mice as compared to normal mice. In the second experiment dwarf mice received saline vehicle, ovine prolactin (PRL), growth hormone (GH) or thyroxin (T4) daily for 14 days. Normal mice received saline only. Replacement with PRL significantly enhanced DA synthesis in the ME and was the only hormone to suppress significantly the elevation of 5-HT synthesis normally observed in the ME and the MBH of the dwarfs. Both GH and T4 only partially reduced 5-HT synthesis in the ME and MBH so that this parameter was no longer statistically different from either saline-treated dwarfs or normal mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of adrenal function in patients with hypothalamic and pituitary disorders: comparison of serum cortisol, urinary free cortisol and the human-corticotrophin releasing hormone test with the insulin tolerance test

OBJECTIVE: This study aimed to evaluate the performance of screening tests (serum cortisol and 24-h urinary free cortisol) and the human-corticotrophin releasing hormone (h-CRH) test in the assessment of adrenal function in patients with hypothalamic-pituitary disorders. DESIGN: Summary receiver operating characteristics (SROC) curve analysis was applied with the insulin tolerance test (ITT) as reference test. A peak serum cortisol response to ITT > or = 500 nmol/l indicated adrenal sufficiency. The sensitivity at the intersect of the diagonal between sensitivity = 1 and (1-specificity) = 1 with the SROC curve, where sensitivity and specificity are equal, and the corresponding weighted kappa, an estimate of agreement with the ITT, served as parameters of test performance. The diagnostic yield, representing the proportion of tests obviating the need for an ITT, was also calculated. MEASUREMENTS: Serum cortisol at 0800 h (n = 122), at 1600 h (n = 116), 24-h urinary free cortisol (n = 115) and the peak serum cortisol to h-CRH (n = 129) were compared with the peak serum cortisol to ITT. PATIENTS: Eighty patients with hypothalamic-pituitary disorders in whom 75 ITT's were performed pre- and 57 post-operatively. RESULTS: Sensitivity at the intersect and weighted kappa were higher for 0800 h serum cortisol (0.873 and 0.763 respectively) than for 1600 h serum cortisol (0.769 and 0.561) and 24-h urinary free cortisol (0.777 and 0.576). These parameters were 0.868 and 0.756 for the h-CRH test. The diagnostic yield was 63.9% for 0800 h serum cortisol compared to 25.9% for 1600 h serum cortisol (P < 10(-8)), 23.5% for 24-h urinary free cortisol (P < 10(-8)) and 60.5% for the h-CRH test (NS). CONCLUSIONS: Serum cortisol measurement at 0800 h is better than 1600 h and 24-h urinary free cortisol to evaluate adrenal function in this patient category. The diagnostic applicability of the h-CRH test is not superior to 0800 h serum cortisol measurement.