Localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the hypothalamus-pituitary system in rats: light and electron microscopic immunocytochemical studies.

The localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the hypothalamus-pituitary system in rats was examined in light and electron microscopic immunocytochemistry using a specific antiserum to synthetic PACAP 1-38 (R0831). In light microscopic study, intensely PACAP-immunostained perikarya were observed in the supraoptic and paraventricular magnocellular nucleus in the hypothalamus. In the median eminence, many immunoreactive nerve fibers were observed in the internal layer, but a few immunoreactive terminals were noticed in the external layer. In the pituitary gland, numerous immunoreactive nerve fibers were observed in the posterior lobe. In the intermediate lobe, moderately immunostained cells were observed, but in the anterior lobe no immunostained cells were noticed. In electron microscopic study, PACAP-immunoreactivity was examined by avidin-biotin peroxidase complex method. In the perikarya of the supraoptic and paraventricular magnocellular nucleus, DAB-reaction products were distributed diffusely in the cytoplasmic matrix, frequently attaching to the rough-surfaced endoplasmic reticulum. In the nerve terminals of the posterior lobe, reaction products were observed among the secretory granules, but sometimes upon them. In the cells of the intermediate lobe, reaction products were also distributed in the cytoplasmic matrix.

Pituitary adenylate cyclase-activating polypeptide (PACAP)-like immunoreactive neuronal elements in rat hypothalamus and median eminence with special reference to morphological background of its effect on anterior pituitary--light and electron microscopic immunocytochemistry.

Pituitary adenylate cyclase-activating polypeptide-like immunoreactive (PACAP-LI) neuronal elements in the rat hypothalamus including the median eminence (ME) were investigated by light and electron microscopic immunocytochemistry. PACAP-LI neuronal perikarya with well-developed cell organelles and dense granules were distributed mainly in the magnocellualr portion of the paraventricular nucleus and throughout the entire supraoptic nucleus. In the ME, numerous PACAP-LI neuronal processes were found in the internal layer (IL), and immunoreactive terminals containing dense granules, vesicles and mitochondria were detected around portal capillaries which penetrated into the IL from the external layer. Thereafter, PACAP is released into the portal capillaries in the IL, transported to the anterior pituitary and plays a role in the stimulation of adenylate cyclase of anterior pituitary cells.

Co-existence of PACAP and nitric oxide synthase in the rat hypothalamus.

The possible co-existence of pituitary adenylate cyclase-activating polypeptide (PACAP)-38 and nitric oxide synthase (NOS) in the rat hypothalamus was examined by a combination of PACAP-immunocytochemistry and NADPH-diaphorase histochemistry. Virtually all PACAP-38-immunoreactive neurones in the paraventricular and supraoptic nuclei exhibited NADPH-diaphorase activity. Since NADPH-diaphorase activity was identical to NOS-immunoreactivity in the magnocellular neurosecretory neurones, this finding indicates that the PACAP neurones synthesize NO.

Immunohistochemical localization of gonadotropin-associated peptide (GAP) in the human hypothalamus.

GAP was immunohistochemically localized for the first time in the human hypothalamus. GAP-immunoreactivity was present in the cytoplasm of neuronal cells in the arcuate nucleus. GAP-immunoreactive nerve fibers were also present in the primary plexus around capillaries in the infundibular region. No GAP-immunoreactive neurons were detected in the paraventricular nuclei or supraoptic nuclei.

Characterization of a high-affinity galanin receptor in the rat anterior pituitary: absence of biological effect and reduced membrane binding of the antagonist M15 differentiate it from the brain/gut receptor.

Structure-activity studies demonstrate that galanin fragments 1-15 and 2-29 are fully active, whereas fragment 3-29 has been reported to be inactive, in a number of different in vivo models. M15, a chimeric peptide comprising galanin 1-13 and substance P5-11, has recently been found to be a potent galanin antagonist. Direct effects of galanin at the level of the pituitary have been defined, yet, paradoxically, a number of studies have been unable to demonstrate galanin binding to an anterior pituitary receptor. Porcine galanin stimulated prolactin release from dispersed rat anterior pituitary cells up to 180% +/- 12% (mean +/- SEM) of control secretion. The addition of a specific galanin antiserum caused a profound inhibition of basal prolactin release, maximal inhibition being 12% +/- 0.5% of control secretion. Addition of M15 produced no effect on basal or galanin-stimulated prolactin release. Galanin fragment 3-29 was fully active when compared to galanin 1-29. Fragments 5-29 and 8-29 stimulated prolactin release to a lesser extent and galanin 1-15, 10-29, and 20-29 had no significant prolactin-releasing activity. Using [mono(125I)iodo-Tyr26]galanin or porcine 125I-labeled Bolton-Hunter [mono(125I)iodo-Lys25]galanin, no anterior pituitary membrane binding was observed. In contrast, 125I-labeled Bolton-Hunter N-terminally labeled galanin allowed characterization of a single high-affinity anterior pituitary galanin receptor with a Kd of 4.4 +/- 0.34 nM and a Bmax of 79 +/- 8.3 fmol/mg of protein. The IC50 for porcine galanin was 0.51 +/- 0.04 nM but for M15 was in excess of 10 microM. Galanin 3-29 fully displaced the label with an IC50 of 0.96 +/- 0.7 nM. The IC50 for galanin 5-29 was 200 nM, whereas 8-29 and 1-15 were > 1 microM. Galanin 10-29 and 20-29 failed to displace the label. These data suggest the presence of a high-affinity pituitary galanin receptor, designated GAL-R2, in which region 3-10 and amino acid 25 are crucial for membrane binding and biological activity, in contrast to the known gut/brain galanin receptor (designated GAL-R1). A number of tissues known to bind or respond to galanin were screened. GAL-R2 would appear to be expressed only in the anterior pituitary and hypothalamus.

Production of immunoreactive corticotropin-releasing hormone in various neuroendocrine tumors.

The concentrations of immunoreactive (IR) corticotropin-releasing hormone (CRH) in 218 neuroendocrine tumors were determined by CRH radioimmunoassay. The tumors examined were 86 pancreatic endocrine tumors (PET), 22 neuroblastic tumors (NBT), 26 carcinoid tumors (CA), 24 pheochromocytomas (PHEO), 40 small cell lung carcinomas (SCLC) and 20 medullary thyroid carcinomas (MTC). IR-CRH was detectable in 21 neuroendocrine tumors (10 PET, four NBT, three CA, two PHEO and two SCLC) at levels of 10-2,700 ng/g wet weight (9.6%). The 21 patients with these CRH-producing tumors showed no clinical symptoms suggestive of Cushing's syndrome. The levels of plasma IR-CRH extracted by immunoaffinity chromatography were < 7.5 pg/ml in five normal subjects and a patient with a neuroblastic tumor containing 55 ng/g wet weight IR-CRH, but in a patient with a thymic carcinoid tumor containing 1,000 ng/g wet weight IR-CRH, the plasma level was elevated to 180 pg/ml. This patient did not have Cushing's syndrome nor an elevated plasma adrenocorticotropic hormone (ACTH) level. The concentrations of nine peptides (growth hormone-releasing hormone, somatostatin, ACTH, calcitonin, gastrin-releasing peptide, glucagon, vasoactive intestinal peptide, neuropeptide tyrosine and pancreatic polypeptide) were determined in extracts of the 21 IR-CRH-producing tumors. Some of these peptides were frequently found to be produced concomitantly with CRH. The results indicate IR-CRH to be produced by various neuroendocrine tumors, but Cushing's syndrome, due to the CRH, to be very rare. The results also show that CRH-producing tumors produce multiple hormones.

A new brain glucosensor and its physiological significance.

The concentration of fibroblast growth factor (FGF), which is found in cerebrospinal fluid (CSF), markedly increases after the start of feeding. Food intake was dose-dependently suppressed by picomole doses of FGF and facilitated by anti-FGF antibody. This suppression was caused by activation of protein kinase C in glucose-sensitive neurons in the lateral hypothalamus. In situ hybridization by use of cDNA showed that acidic (a)FGF was produced in ependymal cells. The ependymal cells released aFGF by responding to glucose increase in CSF after feeding. Released aFGF diffused into the brain parenchyma and was taken by neurons. Passive avoidance was significantly more reliable after aFGF infusion into CSF. Clamping cerebral arteries in the gerbil induced ischemia, which damaged neurons in the CA1 layer of the hippocampus. Pretreatment with aFGF prevented this damage. Thus, aFGF is not only the most potent substance yet found for the suppression of feeding, but it is also extremely effective as a neurotrophic and memory facilitating substance.

Effects of fibroblast growth factors and platelet-derived growth factor on food intake in rats.

In the present study, the relations between acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), platelet-derived growth factor (PDGF), and food intake were studied. When aFGF-, bFGF-, and PDGF-like activity in cerebrospinal fluid (CSF) was examined by bioassay, the activity of those factors significantly increased in postfeeding CSF, compared to prefeeding CSF. Injections of aFGF, bFGF, aFGF (synthetic amino-terminal peptide of aFGF), and PDGF into the third cerebral ventricle decreased food intake, and injections of anti-aFGF, anti-bFGF, and anti-aFGF antibodies into the lateral hypothalamus (LHA) increased food intake. The activity of LHA glucose-sensitive neurons was inhibited by electrophoretic application of aFGF. These results suggest that aFGF, bFGF and PDGF have in vivo physiological roles in the central nervous system, distinct from those as mitogens.

Development of the hypothalamic luteinizing hormone-releasing hormone-containing neuron system in the rat: in vivo and in transplantation studies.

The development of the hypothalamic LHRH-containing neuron system was immunohistochemically investigated in vivo and in tissue transplantation using rat embryos aged from 12.5 to 17.5 days of gestation. The sera used were generated against rat gonadotropic hormone-releasing hormone-associated peptide (28-56) (rGAP) and LHRH. Immunoreaction for rGAP was first found in cells migrated from and in the vomeronasal organ on Days 13.5 and 14.5 of gestation. Immunoreactive cells seem to ascend along the terminal nerves, reaching the medial surface of the forebrain vesicles. Subsequently the cells occurred in the septum and further into their final position in the septopreoptic-diagonal band area on Days 16.5-17.5 of gestation; during this traverse the cells become secretory neurons after changes in morphology and in behavior. Intraventricular transplantation revealed that nasal epithelia of Day 12.5 embryos raised only a few cells immunoreactive both for LHRH and rGAP, but a great number of immunoreactive cells and fibers in the presence of the medial basal hypothalamus (MBH). The fibers formed a median eminence-like structure together with dense capillary plexus that had grown in the cografted MBH. The same phenomenon was apparently observed in the grafts obtained from older embryos of gestation, but not in the combined grafts of the anterior septum and the nasal epithelium or the MBH. We conclude that hypothalamic LHRH neurons originate from the nasal placode and acquire secretory behavior in the presence of the MBH.

Gonadotropin-releasing hormone-associated peptide immunoreactivity in bovine colostrum.

Gonadotropin-releasing hormone(GnRH)-associated peptide (GAP) is a 56-amino acid peptide found on the C-terminal of the GnRH (also called luteinizing hormone-releasing hormone) precursor and is assumed to be co-produced with GnRH. The purpose of this report is to demonstrate the presence of GAP immunoreactivity in bovine colostrum. Radioimmunoassay of acidified methanolic extracts demonstrated a concentration of GAP immunoreactivity of approximately 1.5 +/- 0.1 pmol/g dry skim bovine colostrum. Gel filtration (Sephadex G-10) and high-performance liquid chromatography of extracts containing GAP immunoreactivity showed it to be of low molecular weight and a high hydrophobic character. The presence of GAP immunoreactivity in bovine colostrum suggests that the GnRH precursor is synthesized and processed in mammary tissue itself.

Galanin stimulates the release of vasoactive intestinal polypeptide from perifused hypothalamic fragments in vitro and from periventricular structures into the cerebrospinal fluid in vivo in the rat.

Intracerebroventricular injection of galanin (2 micrograms/rat) raised plasma prolactin (PRL) levels in the rat, which was accompanied by an increase in immunoreactive vasoactive intestinal polypeptide (VIP) in the cerebrospinal fluid (CSF). Immunoreactive VIP release from superfused rat hypothalamic fragments in vitro was dose-relatedly stimulated by galanin (10(-7) and 10(-8) M). PRL release from superfused rat anterior pituitary cells was stimulated by TRH (10(-8) M) but not affected by galanin (10(-7) to 10(-5) M). These findings indicate that central galanin has a stimulating role in the release of hypothalamic VIP, which results in pituitary PRL secretion in the rat.

Distribution of neurotensin-like immunoreactivity in the diencephalon of the Japanese monkey (Macaca fuscata).

The distribution of neurotensin-like immunoreactive (NT-LI) neurons was examined in the thalamus and hypothalamus of the Japanese monkey (Macaca fuscata) by using the peroxidase-antiperoxidase immunocytochemistry technique. In the thalamus, NT-LI neuronal perikarya were distributed mainly in the midline nuclear group and the dorsomedial nucleus, and partially in the intralaminar nucleus: Immunoreactive fibers were mainly distributed in the midline nucleus, particularly in the nucleus rhomboidalis. Numerous immunoreactive fibers were also detected in the regions that contain the pathways to extrathalamic areas such as the stratum zonale and inferior thalamic peduncle. In the hypothalamus, many immunoreactive neuronal perikarya were distributed in the lateral hypothalamic area and in the arcuate nucleus. Immunoreactive fibers were disseminated throughout the hypothalamus, but they were dense in the preoptic area and sparse in the ventromedial nucleus. An accumulation of dense immunoreactive endings was also observed in the external layer of the median eminence. NT-LI fibers in the external layer of the median eminence were considered to represent nerve endings near portal vessels. Functional roles of neurotensin in the thalamus and hypothalamus are discussed from the anatomical point of view.

Central galanin stimulates pituitary prolactin secretion in rats: possible involvement of hypothalamic vasoactive intestinal polypeptide.

The effect of galanin, a newly identified neuropeptide, on pituitary prolactin (PRL) secretion was examined in the rat. Intracerebroventricular (i.c.v.) injection of all 5 doses of galanin (0.4, 1, 2, 5 and 10 micrograms/rat) raised plasma PRL levels in urethane-anesthetized rats. Galanin injection (2 micrograms/rat, i.c.v.) also increased plasma PRL levels in conscious rats. The intermediate dose of galanin (2 micrograms/rat, i.c.v.) produced a greater response in plasma PRL levels than either smaller or larger doses of galanin. Intravenous injection of galanin did not affect plasma PRL levels. Passive immunization with specific anti-vasoactive intestinal polypeptide (VIP) rabbit serum suppressed plasma PRL response to galanin (2 micrograms/rat, i.c.v.) in anesthetized rats. These findings indicate that central galanin has a stimulatory role in pituitary PRL secretion via the hypothalamus in the rat and that VIP may be involved in rat PRL release induced by galanin.

Regional distribution of gastrin-releasing peptide- and somatostatin-like immunoreactivity in the rabbit hypothalamus.

Regional distribution of gastrin-releasing peptide- (GRP) and somatostatin (SRIF)-like immunoreactivity in the discrete nuclei of the hypothalamus was examined in the rabbit according to Palkovits' microdissection method. GRP-like immunoreactivity (LI) was detected abundantly in the hypothalamus as compared with the cerebral cortex when measured by radioimmunoassay using the antiserum recognizing the C-terminal portion of synthetic porcine GRP. On gel-filtration chromatography of the hypothalamic extracts, two major peaks of GRP-LI were eluted; the peak with larger molecular size corresponded to synthetic porcine GRP1-27 and the smaller size to porcine GRP14-27. A concentration of GRP-LI was highest in the infundibular nuclei (IFN) as well as the ventromedial nuclei (VMN), and next high in the paraventricular nuclei (PVN), suprachiasmatic nuclei (SCN) and periventricular nuclei (PEV). The content of GRP-LI in the median eminence was not so much when compared with them. On the other hand, SRIF was localized in the highest concentration in the ME, followed by the VMN and IFN, as well as the PEV. The findings indicate that porcine GRP-LI exists in the hypothalamus of rabbits with characteristic regional distribution. Concurrent localization of GRP-LI and SRIF in some parts of the hypothalamus may suggest the interaction of both peptides in these areas under various physiological and pathological status.

Vasoactive intestinal peptide- and peptide histidine isoleucine amide-like immunoreactivity colocalize with vasopressin-like immunoreactivity in the canine hypothalamo-neurohypophysial neuronal system.

The distribution of vasoactive intestinal peptide (VIP) and peptide histidine isoleucine amide (PHI) was investigated in the canine hypothalamus by immunocytochemistry. VIP- and PHI-like immunoreactive neurons were detected in the magnocellular supraoptic and paraventricular nucleus. These magnocellular VIP- and PHI-producing neurons coexist with vasopressin-like immunoreactivity and send axons to the median eminence and neurohypophysis. These findings may serve as an anatomical basis for studying the function of VIP and PHI on pituitary hormone secretion.

Stimulation by gastrin-releasing peptide, neurotensin and DN1417, a novel TRH analog, of dopamine and norepinephrine release from perifused rat hypothalamic fragments in vitro.

Both dopamine (DA) and norepinephrine (NE) release from perifused rat hypothalamic fragments was increased by depolarizing concentrations of potassium (K+, 20 mM and 56 mM) in a dose-related and Ca2+-dependent manner. DA and NE release induced by high K+ (20 mM) was further enhanced by gastrin-releasing peptide (GRP, 10(-5)-10(-6) M) and DN 1417 (10(-5) M), a thyrotropin-releasing hormone (TRH) analog. GRP (10(-5) M), neurotensin (NT, 10(-5) M) and DN 1417 (10(-5) M) also stimulated spontaneous release of DA and NE from the hypothalamus. These results suggest that GRP, NT and DN 1417 act at the hypothalamic catecholamine nerve terminals and stimulate the release of DA and NE in the rat.

Stimulation by serotonin of immunoreactive peptide histidine isoleucine release from rat hypothalamus in vitro.

The effect of serotonin (5-HT) on the release of peptide histidine isoleucine-like immunoreactivity (PHI-LI) from rat hypothalamus was investigated in vitro with a perifusion system. A high potassium concentration (56 mM) stimulated PHI-LI release in a calcium-dependent manner. PHI-LI release was dose-relatedly stimulated by 5-HT (10(-6), 10(-5) and 10(-4) M). PHI-LI release induced by 5-HT (10(-5) M) was abolished by cyproheptadine (10(-4) M), a 5-HT antagonist. These results suggest that 5-HT has a stimulating effect on PHI release from the hypothalamus.

Glucagon-related peptides in the rat hypothalamus.

Immunohistochemically, nerve fibers and terminals reacting with anti-N-terminal-specific but not with anti-C-terminal-specific glucagon antiserum were observed in the following rat hypothalamic regions: paraventricular nucleus, supraoptic nucleus, anterior hypothalamus, arcuate nucleus, ventromedial hypothalamic nucleus and median eminence. Few fibers and terminals were demonstrated in the lateral hypothalamic area and dorsomedial hypothalamic nucleus. Radioimmunoassay data indicated that the concentration of gut glucagon-like immunoreactivity was higher in the ventromedial nucleus than in the lateral hypothalamic area. In food-deprived conditions, this concentration increased in both these parts. This was also verified in immunostained preparations in which a marked enhancement of gut glucagon-like immunoreactivity-containing fibers and terminals was observed in many hypothalamic regions. Several immunoreactive cell bodies were found in the ventromedial and arcuate nuclei of starved rats. Both biochemical and morphological data suggest that glucagon-related peptides may act as neurotransmitters or neuromodulators in the hypothalamus and may be involved in the central regulatory mechanism related to feeding behavior and energy metabolism.

Involvement of peptide histidine isoleucine (PHI) in prolactin secretion induced by serotonin in rats.

The possible role of hypothalamic peptide histidine isoleucine (PHI) in prolactin (PRL) secretion induced by serotoninergic mechanisms was investigated in male rats using a passive immunization technique. Intracerebroventricular injection of serotonin (5HT, 10 micrograms/rat) raised plasma PRL levels both in urethane-anesthetized rats and in conscious rats pretreated with normal rabbit serum (0.5 ml/rat, iv, 30 min before). Plasma PRL responses to 5HT were blunted in these animals when they were pretreated with rabbit antiserum specific for PHI (0.5 ml/rat, iv, 30 min before) (mean +/- SE peak plasma PRL: anesthetized rats 271.3 +/- 38.3 ng/ml vs 150.0 +/- 12.6 ng/ml, p less than 0.01, conscious rats 54.3 +/- 6.8 ng/ml vs 30.7 +/- 4.1 ng/ml, p less than 0.025). These results suggest that hypothalamic PHI is involved, at least in part, in PRL secretion induced by central serotoninergic stimulation in the rat.