Regional distribution of immunoreactive prolactin-releasing peptide in the human brain.

Regional distribution of prolactin-releasing peptide (PrRP) in the human brain was studied by radioimmunoassay. The antiserum raised against human PrRP-31 in a rabbit was used in the assay, which showed 100% cross reaction with PrRP-20 and no significant cross reaction with other peptides. The highest concentrations of immunoreactive-PrRP were found in hypothalamus (912 +/- 519 fmol/g wet weight, n = 6, mean +/- SEM), followed by medulla oblongata (496 +/- 136 fmol/g wet weight) and thalamus (307 +/- 117 fmol/g wet weight). On the other hand, immunoreactive-PrRP was not detected in frontal lobe or temporal lobe (<50 fmol/g wet weight). Sephadex G50 column chromatography of the immunoreactive-PrRP in the hypothalamus and medulla oblongata showed three immunoreactive peaks; one peak eluting in the position of PrRP-20, one eluting in the position of PrRP-31 and one eluting earlier. Reverse phase high-performance liquid chromatography (HPLC) of these brain tissue extracts showed a peak eluting in the position of PrRP-20 and PrRP-31. The present study has shown for the first time the presence of immunoreactive-PrRP in the human brain. The immunoreactive-PrRP levels in the human hypothalamus were, however, lower than the levels of other neuropeptides with prolactin-releasing activity, such as thyrotropin-releasing hormone and vasoactive intestinal polypeptide.

Immunocytochemical localization of adrenomedullin-like immunoreactivity in the human hypothalamus and the adrenal gland.

Adrenomedullin is a potent vasodilator peptide that was isolated from pheochromocytoma. Localization of adrenomedullin-like immunoreactivity was studied by immunocytochemistry in the human hypothalamus and adrenal gland. Adrenomedullin-immunoreactive cell bodies were found in the paraventricular, supraoptic and infundibular nuclei of the hypothalamus. Both magnocellular and parvocellular cells of the paraventricular nucleus were positively immunostained. Adrenomedullin-like immunoreactivity was localized in the adrenal medulla. No positive immunostaining was observed in the vascular endothelium, vascular smooth muscle cell or adrenal cortex. The preabsorption of the antiserum with synthetic human adrenomedullin (1-52) abolished the immunostaining. These findings indicate that adrenomedullin-like immunoreactivity is localized in the paraventricular, supraoptic and infundibular nuclei as well as in the adrenal medulla, and suggest that adrenomedullin acts as a neurotransmitter, a neuromodulator or a neurohormone in the human hypothalamus.

Melanin-concentrating hormone in human and rat.

Melanin-concentrating hormone (MCH) is a neuropeptide originally isolated from chum salmon pituitaries. To explore physiological roles of MCH in mammals, we studied the regional distribution of immunoreactive MCH in the rat tissues and the presence of immunoreactive MCH in human adrenal glands, adrenal tumors and plasma by radioimmunoassay, and the expression of MCH mRNA in rat tissues and human brain tissues by Northern blot analysis. Immunoreactive MCH was present in every region of rat brain and neurointermediate lobe of the pituitary gland, with the highest concentrations found in the hypothalamus (48.3 +/- 6.6 pmol/g wet weight, mean +/- SEM, n = 6). The immunoreactive MCH in rat hypothalamus, frontal lobe, and pons and medulla oblongata was eluted in the position of synthetic human/rat MCH in reverse-phase high-performance liquid chromatography. No immunoreactive MCH was detected in the rat peripheral tissues. Northern blot analysis showed that a single species of MCH mRNA (approximately 1 kb) was expressed specifically in the rat and human hypothalamus, but not detectable in other regions of brain or rat peripheral tissues. Immunoreactive MCH was not detected in human adrenal glands (< 0.5 pmol/g wet weight, n = 9) or adrenal tumors including pheochromocytomas. Immunoreactive MCH were not detected in plasma obtained from human healthy subjects (< 0.25 pmol/l) and rat (< 0.25 pmol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

Pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity in human hypothalamus: co-localization with arginine vasopressin.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel hypothalamic peptide consisting of 38 amino acids (PACAP1-38) with a potent stimulatory action on adenylate-cyclase in rat pituitary. The presence of PACAP-like immunoreactivity in human brain was studied by radioimmunoassay. Co-localization of PACAP with arginine vasopressin and oxytocin was investigated by immunocytochemistry in the human hypothalamus. Immunoreactive PACAP was detected in all regions of human brain (cortex, thalamus, hypothalamus, pons and hemisphere of cerebellum) with the highest levels found in the hypothalamus (8.5 +/- 1.9 pmol/g wet weight, n = 4, mean +/- S.E.M.). High performance liquid chromatography of the human hypothalamic extract showed that approximately 50% of the immunoreactive PACAP was eluted in the position of PACAP1-38. Immunocytochemical studies showed the presence of PACAP immunoreactive neurons in the paraventricular and supraoptic nuclei of human hypothalamus. PACAP co-localized with arginine vasopressin in magnocellular cells of these nuclei. These findings suggest that PACAP1-38 plays important physiological roles in the human hypothalamus.

A case of ruptured dissecting aneurysm 5 years after pituitary microsurgical treatment of Cushing's disease: autopsy findings in the hypothalamic-pituitary-adrenal axis.

The patient was a 26-year-old man with Cushing's disease who underwent transsphenoidal microscopic surgery for a pituitary microadenoma. His postoperative course was uneventful, but he died suddenly five years after the operation. At autopsy, a ruptured dissecting aneurysm with marked atherosclerosis was observed in the aorta. In the pituitary, a small focus of adrenocorticotropic hormone (ACTH) producing adenoma, possibly residual adenoma, was detected and Crooke's degeneration was observed in the non-tumorous pituitary gland. But immunohistochemical patterns of pituitary hormones in the non-tumorous pituitary gland were normal and the adrenal cortex was unremarkable. In the hypothalamus, corticotropin-releasing hormone immunoreactivity was not detected and arginine vasopressin was sporadically positive. Considering these findings, this patient may have developed subclinical hypercortisolism due to the residual adenoma at the time of autopsy, despite clinical remission. Cushing's syndrome is considered to be a risk factor dissecting aneurysm, and in this case the metabolic changes in Cushing's disease may have influenced the development of the dissecting aneurysm. Periodic cardiovascular re-evaluations should therefore be performed when there is clinical remission of Cushing's syndrome.

Calcitonin gene-related peptide in the human hypothalamus.

Calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) in the human hypothalamus was investigated by radioimmunoassay and by immunocytochemistry. CGRP-LI was detected from two hypothalami obtained at autopsy (2.1 and 7.0 ng/g wet tissue) by radioimmunoassay. Reverse phase high performance liquid chromatography revealed that most of the CGRP-LI in the human hypothalamus was eluted in an identical position with synthetic human CGRP. For immunocytochemistry, human hypothalami obtained at autopsy were fixed and cryostat-sectioned at 40 microns. Free floating sections were immunostained with antibody to CGRP. CGRP-immunoreactive cell bodies were found in the supraoptic nucleus, paraventricular nucleus and infundibular nucleus. These findings indicate that CGRP exists in the cell bodies of the supraoptic nucleus, paraventricular nucleus and infundibular nucleus in the human hypothalamus and CGRP may play some roles in the endocrine and other functions of the human hypothalamus.

Corticotropin-releasing hormone in the human hypothalamus. Free-floating immunostaining method.

We have clearly demonstrated corticotropin-releasing hormone (CRH) immunoreactive cell bodies and nerve fibers in the human hypothalamus by immunocytochemistry using free-floating sections instead of paraffin-embedded sections. Human hypothalami were obtained at autopsy, fixed and cryostat-sectioned at 40 microns. Free-floating sections were immunostained with antibody to CRH using the Vector ABC system. Most of CRH immunoreactive nerve fibers from the paraventricular nucleus pass under the fornix, while some CRH immunoreactive nerve fibers pass beyond the fornix and some through the fornix. Then the CRH immunoreactive nerve fibers run downward, medially to the supraoptic nucleus and toward the pituitary stalk. This method of immunocytochemistry is a very sensitive and suitable means for immunocytochemical studies of neuropeptides in the human brain.

Peptide histidine methionine may be a prolactin-releasing hormone in humans.

Peptide histidine isoleucine (PHI) was initially isolated from the porcine gastrointestinal tract and may be present in the brain. It has been suggested that PHI may be PRL-releasing hormone (PRH) because of its potent PRL-releasing activity and its existence in hypophysial portal plasma in rats. Vasoactive intestinal peptide and PHI are coded by the same gene, and human PHI has a C-terminal methionine instead of isoleucine [peptide histidine methionine (PHM)]. To investigate the possibility that PHM is a physiological PRH in humans, we measured the immunoreactive PHM concentration in human hypothalamic tissue and cerebrospinal fluid (CSF) using a specific RIA. We also examined in vivo the PRH activity of synthetic PHM. The human hypothalamus contained 19.3 +/- 6.2 (+/- SD; n = 5) pmol/hypothalamus, very similar to the content of GHRH or CRH. Immunoreactive PHM was also present in CSF; its levels in CSF were significantly lower in patients with prolactinomas than in control subjects. The CSF PHM levels in such patients increased after correction of hyperprolactinemia by long term bromocriptine therapy. The CSF PHM levels also were low in pregnant women. There was a significant negative correlation between plasma PRL and CSF PHM levels in all of these subjects. Gel filtration profiles of CSF extracts from normal subjects revealed two peaks of immunoreactive PHM: a high mol wt peak and one at the elution position of synthetic PHM. This profile resembled that of hyppothalamic extract. In contrast, only high mol wt material was detected in CSF from hyperprolactinemic subjects. Intravenous administration of synthetic PHM elicited a significant increase in plasma PRL in normal subjects; the responses to PHM were higher in women than in men. The presence of large amounts of immunoreactive PHM in the human hypothalamus suggests that PHM may participate in the regulation of anterior pituitary hormone secretion. Its specific PRL-releasing activity in vivo and the low CSF PHM levels of hyperprolactinemic subjects suggest that PHM may be a physiological PRH in humans.

Potentiation of peptide histidine methionine (PHM) on corticotropin-releasing hormone (CRH) induced ACTH secretion in humans.

The effects of rat corticotropin-releasing hormone (CRH) and of synthetic peptide histidine methionine (PHM) on ACTH release were studied in vivo in humans. PHM alone did not alter the basal plasma ACTH or cortisol levels, but it strongly potentiated CRH-induced ACTH secretion. These results, together with our previous observation that PHM immunoreactivity is present in human hypothalamus and pituitary stalk, suggest that PHM may play a physiological role in the regulation of ACTH secretion in humans.

Immunoreactive corticotropin-releasing hormone present in human plasma may be derived from both hypothalamic and extrahypothalamic sources.

Immunoreactive CRH concentrations were determined in human plasma using an immunoaffinity chromatographic extraction procedure and sensitive RIA. Immunoreactive CRH was detectable in the plasma of all normal subjects (mean +/- SD, 6.2 +/- 2.4 pg/mL; n = 15). Basal (0800-1000 h) plasma immunoreactive CRH levels were significantly lower in patients with Cushing's syndrome due to adrenal (2.8 +/- 1.1 pg/mL; n = 4) or pituitary adenomas (2.9 +/- 0.8 pg/mL; n = 5), in patients with hypothalamic hypopituitarism (3.2 +/- 0.9 pg/mL; n = 5), and in glucocorticoid-treated patients (3.9 +/- 1.9 pg/mL, n = 8). Basal plasma CRH levels were also low in patients with acromegaly (2.8 +/- 0.8 pg/mL; n = 14) and insulin-treated diabetic patients whose pituitary-adrenal function was normal (3.6 +/- 1.0 pg/mL; n = 12). In normal subjects plasma CRH levels increased after insulin-induced hypoglycemia; this response was abolished by the prior administration of dexamethasone. In contrast, basal plasma CRH levels were not affected by prior administration of metyrapone or dexamethasone. No evidence for diurnal variation in plasma immunoreactive CRH was found in normal subjects. In addition, in normal subjects oral glucose administration elicited a significant increase in plasma CRH (basal, 7.3 +/- 0.9 pg/mL; peak 30 min after glucose, 16.7 +/- 5.8 pg/mL; n = 5; P less than 0.05) without concomitant changes in ACTH. Gel filtration of extracts of pooled plasma from normal subjects revealed a major component of immunoreactive CRH in the position of synthetic rat CRH. Immunoreactive CRH-sized material had the same retention time as authentic rat CRH in a reverse phase high pressure liquid chromatography system. The content of immunoreactive CRH in human placenta, pancreas, and adrenal gland was much larger than that in hypothalamus. These findings suggest that immunoreactive CRH is present in peripheral plasma; the increase in plasma immunoreactive CRH after insulin-induced hypoglycemia may reflect stimulation of hypothalamic CRH release; the increase in plasma immunoreactive CRH after glucose administration may reflect extrahypothalamic CRH release; and the lack of diurnal variation in plasma immunoreactive CRH together with the lack of suppression of CRH by dexamethasone suggest that basal plasma CRH is of extrahypothalamic origin.

Immunocytochemical identification of CRF in the human hypothalamus.

Immunoreactive ovine corticotropin-releasing factor (CRF) was revealed in the cell bodies of parvocellular neurons in the paraventricular nucleus of the human hypothalamus by an immunocytochemical technique. The immunoreactivity was negative in magnocellular neurons. Immunoreactive nerve fibers were found in the subependymal layers of the third ventricle and in the perivascular space of the primary plexus in the lower hypothalamus. No CRF immunoreactive cell bodies were found in the supraoptic nucleus.

Role of bovine albumin in a serum-free suspension cell culture medium.

A serum-free culture medium, supplemented with 1% bovine serum albumin, supported the growth of both primary and continuous suspension-type cultures of various mammalian tumor cells. The role of albumin added to the medium was also studied. Defatted albumin failed to support cell growth, unless reconstituted with its lipid extract. Similarly, defatted albumin when combined with oleic and linoleic acids, also supported cell growth. Therefore, albumin-bound fatty acids play an important growth-promoting role in serum-free medium.