Kozo Hashimoto

Corticotropin-releasing hormone and pituitary-adrenocortical responses in chronically stressed rats

Brain corticotropin-releasing hormone (CRH) concentration and pituitary adreno-cortical responses were examined in chronically stressed rats: body restraint stress (6 h/day) for 4 or 5 weeks. Stressed rats showed a reduction in weight gain. CRH concentration in the median eminence and the rest of the hypothalamus were not different between control and chronically immobilized rats. The anterior pituitary adenocorticotropic hormone (ACTH) concentration was elevated in chronically stressed rats, whereas plasma ACTH and corticosterone levels did not differ from the control values. The median eminence CRH concentration was reduced to the same extent at 5 min after onset of ether exposure (1 min) in chronically immobilized rats and controls. However, plasma ACTH and corticosterone showed greater responses to ether stress in chronically immobilized rats than in control rats. Plasma ACTH and corticosterone responses to exogenous CRH were not different between control and chronically immobilized rats, while the response to arginine vasopressin (AVP) was significantly greater in chronically immobilized rats. These results suggest that chronic stress caused an increase in the ACTH-secreting mechanism and that pituitary hypersensitivity to vasopressin might at least be partly responsible for this.

Starvation-induced changes in rat brain corticotropin-releasing factor (CRF) and putuitary-adrenocortical response

Starvation-induced changes in CRF concentration in major brain regions and abnormalities in the pituitary-adrenal axis were examined in rats using rat CRF radioimmunoassay. The CRF concentrations in the hypothalamus and cerebellum were significantly reduced in the completely starved rats, while those in the midbrain, thalamus and neurointermediate lobe of the pituitary were significantly increased in the semi-starved or completely starved rats. No significant changes in the CRF concentrations were found in the pons, medulla oblongata and cerebral cortex. In the completely starved rats, the serum ACTH level was significantly reduced, whereas the serum corticosterone level was markedly elevated. These observations suggest that starvation may stimulate the CRF-ACTH-corticosterone system and that not only hypothalamic CRF but also extrahypothalamic CRF may be discretely related to feeding behavior or starvation. The reduced serum ACTH level in starved rats may be ascribed to the negative feedback effect of the elevated serum corticosterone.

Corticotropin-releasing factor (CRF)-like immunoreactivity in the adrenal medulla

Bovine adrenal medulla extract prepared by acid-acetone or acid methanol extraction showed two peaks of CRF-like immunoreactivity on Sephadex G-50 chromatography. One eluted near the void volume and another (low molecular weight CRF-like immunoreactivity) eluted slightly before arginine vasopressin (AVP), while most of the immunoreactivity in bovine hypothalamus coeluted with synthetic ovine CRF. When low molecular weight CRF fractions were chromatographed by reversed phase high performance liquid chromatography, three CRF-like immunoreactive peaks appeared. The first peak appeared near TRH, the second one eluted near AVP and the last one eluted near somatostatin. These three peaks of immunoreactivity showed ACTH releasing bioactivity in rat pituitary cells cultures. Therefore, the adrenal medulla-CRF-like substances might be tissue-CRF which may play a role to stimulate ACTH release in the severe stress conditions.

Immunohistochemical demonstration of the localization of corticotropin releasing factor-containing neurons in the hypothalamus of mammals including primates

SummaryThe presence of the CRF-containing neurons in the hypothalamus was investigated in four different species (cats, dogs, pigs, and monkeys) by the peroxidase-antiperoxidase technique using specific anti-serum to CRF. In all animals examined, CRF-containing perikarya were found mainly in the paraventricular and supraoptic nuclei, and a small number of the immunoreactive cells were observed in the accessory supraoptic nucleus and the lateral hypothalamic area. The size of the CRF-containing perikarya ranged from 20–35 μm in diameter. These findings suggest that the magnocellular paraventricular and supraoptic nuclei are the center not only of the classical neurosecretory system for the production of the posterior lobe hormones, but also that of the CRF neuronal system.

Immunohistochemical identification of neurons containing corticotropin-releasing factor in the rat hypothalamus

SummaryA specific rabbit anti-CRF serum and the immunoperoxidase technique were used to show that CRF-containing neurons are mainly distributed in the paraventricular and supraoptic nuclei of the rat hypothalamus. In addition, immunoreactive neurons are scattered in other hypothalamic regions. These neurons are 20–30 μm in diameter. From the present and previous investigations it may be concluded that the hypothalamic magnocellular nuclei, i.e., paraventricular and supraoptic, and other hypothalamic accessory nuclei, are the producing sites not only for vasopressin and oxytocin, but also for corticotropin-releasing factor.

A specific radioimmunoassay for Cortricotropin Releasing Factor (CRF) using synthetic ovine CRF

This newly developed specific radioimmunoassay for corticotropin releasing factor (CRF) had a sensitivity range of 25 pg/tube to 4 ng/tube. Intra and interassay coefficient of variation were 4.6% and 9.8%, respectively. Rat median eminence extracts showed a parallel dose response curve with synthetic ovine CRF and a significant cross reaction was not evident with other tested neuropeptides. The highest mean levels of CRF were found in the median eminence (6.61 ng/mg protein). Considerable amounts of CRF were found in the arcuate nucleus, paraventricular nucleus, dorsomedial nucleus, suprachiasmatic nucleus and ventromedial nucleus. The immunoreactive CRF of the rat medial basal hypothalamus coeluted with bioassayable CRF and with iodinated CRF on Sephadex G-75 chromatography. The results indicate that rat hypothalamus contains a CRF similar to ovine CRF.

Reduction in brain immunoreactive corticotropin-releasing factor (CRF) in spontaneously hypertensive rats

The brain CRF concentration of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) was examined by rat CRF radioimmunoassay. Anti-CRF serum was developed by immunizing rabbits with synthetic rat CRF. Synthetic rat CRF was also used as tracer and standard. The displacement of 125I-rat CRF by serially diluted extracts of male Wistar rats hypothalamus, thalamus, midbrain, pons, medulla oblongata, cerebral cortex, cerebellum and neurointermediate lobe was parallel to the displacement of synthetic rat CRF. In both WKY and SHR the highest levels of CRF immunoreactivity were shown by the hypothalamus and neuro-intermediate lobe, and considerable CRF immunoreactivity was also detected in other brain regions. The CRF immunoreactivity in the hypothalamus, neurointermediate lobe, midbrain, medulla oblongata and cerebral cortex was significantly reduced in SHR and it may suggest that CRF abnormality may be implicated in the reported abnormalities in the pituitary-adrenal axis, autonomic response and behavior of SHR.

Distribution and Characterization of Corticotropin-Releasing Factor and Arginine Vasopressin in Rat Hypothalamic Nuclei

Corticotropin-releasing factor (CFR) was bioassayed and arginine vasopressin (AVP) radioimmunoassayed from punched-out hypothalamic nuclei. The highest concentration of CFR was found in the median eminence (ME), followed by the paraventricular nucleus (PVN), supraoptic nucleus (SON), suprachiasmatic nucleus (SCN), arcuate nucleus (ARC), dorsomedial nucleus (DMN) and ventromedial nucleus (VMN). The AVP concentration was in the order of ME, SON, PVN, SCN, ARC, VMN and DMN. Sephadex G-25 gel filtration of the ME extracts showed one peak for AVP and two peaks for CRF. One CRF peak appeared on the void volume (big CRF) and the other (small CRF) was coeluted with AVP. Gel filtration of the PVN and SON extracts showed one peak for AVP but three or four peaks for CRF. The addition of anti-AVP serum (AVP-AS) to pituitary cell cultures reduced the CRF activities of AVP and ME extracts by approximately 80 and 40%, respectively. When the small CRF fraction of ME extracts was treated with AVP-AS on affinity chromatography, the unbound fraction (AVP-free) still showed significant CRF activity. Re-examination of CRF concentration using AVP-AS showed that it was still highest in ME, but was significantly higher in PVN than in SON, SCN and ARC. These results suggest that the PVN is an important nucleus for producing corticotropin-releasing hormone.

Effect of atrial natriuretic peptide on acethylcholine-induced release of corticotropin-releasing factor from rat hypothalamus in vitro

Effect of rat atrial natriuretic peptide (rANP) on acetylcholine-induced release of corticotropin-releasing factor (CRF) from the rat hypothalamus was studied in vitro using perifusion method. Perifused acetylcholine at 100 and 1000 ng/ml evoked significant CRF release, whereas norepinephrine at 10, 100 and 1000 ng/ml did not show a definite effect on CRF release. Continuous administration of alpha-rANP(1-28) (20ng/ml) inhibited the acetylcholine (100ng/ml)-induced CRF release. It is likely that ANP is involved in the regulation of CRF release.

Central catecholaminergic control of ACTH secretion

Plasma adrenocorticotropic hormone (ACTH) has been measured after an intra-third ventricular administration of noradrenaline, an adrenergic agonist or an adrenergic antagonist. Centrally administered noradrenaline caused a significant increase in ACTH secretion. The alpha-agonist phenylephrine also increased the ACTH level. However, neither the alpha-antagonist phentolamine nor beta-agonist isoproterenol affected the ACTH level. The beta-antagonist propranolol evoked a significant elevation in ACTH. Passive immunoneutralization was examined with anti-rat corticotropin-releasing factor (CRF) rabbit serum, anti-arginine vasopressin (AVP) rabbit serum and normal rabbit serum (NRS) on the intra-third ventricular noradrenaline-induced ACTH secretion to study the involvement of endogenous CRF. An intra-third ventricular administration of noradrenaline caused a significant increase of ACTH levels in NRS-injected rats and anti-AVP-injected rats, whereas an i.v. anti-rat CRF injection significantly reduced the intra-third ventricular noradrenaline-induced ACTH secretion. These results suggest that central catecholamine stimulated ACTH secretion via the alpha-adrenergic mechanism and that endogenous CRF is at least partly involved in the noradrenaline-induced ACTH secretion.