Tamotsu Shibasaki

Stress-induced activation of neuronal activity and corticotropin-releasing factor gene expression in the paraventricular nucleus is modulated by glucocorticoids in rats.

Intronic in situ hybridization methodology provides a means of determining the rate of gene transcription under basal and stimulated conditions. In the present study, we have used intronic in situ hybridization to the corticotropin-releasing factor (CRF) gene to measure hypothalamic CRF gene transcription after stress as well as its modulation by glucocorticoids. Using this and conventional exonic in situ hybridization we examined the time course of changes in c-fos mRNA, and CRF heteronuclear RNA (hnRNA) and mRNA concentrations in the paraventricular nucleus (PVN) of male Wistar rats after restraint stress. In addition, we determined the effects of adrenalectomy and dexamethasone administration on c-fos and CRF gene expression in the PVN. Restraint stress induced a rapid induction (within 5 min) of c-fos mRNA and CRF hnRNA expression in the PVN. Both RNA concentrations peaked at 30 min then decreased and were undetectable 2 h after stress onset. In contrast, the concentration of CRF mRNA increased gradually and a significant elevation was first detected 60 min after the beginning of stress. Adrenalectomy augmented and dexamethasone pretreatment inhibited c-fos mRNA, CRF hnRNA, and mRNA induction after stress. The data suggest that stress-induced activation of neurons, CRF gene transcription, and CRF synthesis in the PVN are modulated by glucocorticoids.

Chlordiazepoxide attenuates stress-induced activation of neurons, corticotropin-releasing factor (CRF) gene transcription and CRF biosynthesis in the paraventricular nucleus (PVN)

Corticotropin-releasing factor (CRF) plays a role in coordinating endocrine, autonomic, and behavioral responses to stressful stimuli. Benzodiazepines exert many effects which oppose those of CRF, including anxiolysis and suppression of the pituitary-adrenal axis. In the present study, we employed in situ analysis of CRF heteronucleous RNA (hnRNA) and c-fos mRNA to assess stimulus-induced CRF gene transcription rate following stress and its modulation by chlordiazepoxide (CDP). Male albino rats were exposed to restraint stress for 30 min and sacrificed 30 and 120 min after the onset of stress. Either CDP or vehicle was given intraperitoneally 60 min before stress. To determine plasma ACTH levels by immunoradiometric assay, another group of rats was decapitated 10 min after the onset of restraint stress. Restraint stress induced rapidly and significantly c-fos mRNA and CRF hnRNA expression in the PVN at the 30 min time point. Increases in both RNA copies were significantly inhibited by administration of CDP at doses of 5 and 10 mg/kg. CRF mRNA concentrations were increased significantly in the PVN 120 min after stress and again, CDP attenuated significantly these increases in the PVN. The plasma ACTH increase in response to stress was inhibited significantly by CDP administration at every dose tested. CDP did not change CRF mRNA levels in the non-stressed animal.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of α-melanocyte stimulating hormone (α-MSH) in the central nervous system of the rat: an immunohistochemical study. II: Lower brain stem

Abstract The distribution of immunoreactive α-melanocyte stimulating hormone (α-MSHI) in the rat lower brain stem was examined by indirect immunofluorescence or peroxidase-anti-peroxidase immunohistochemical method using an antiserum against synthetic α-MSH. The results confirmed the presence of α-MSHI fibers in the midbrain central gray matter and parabrachial area, and demonstrated a much more extensive distribution of these fibers in various parts of the lower brain stem areas previously thought not contain α-MSHI fibers. In addition, the commissural nucleus was identified as a new α-MSHI neurons-containing site. No α-MSHI neurons were seen in other regions of the rat lower brain stem.

Ovine corticotropin-releasing factor stimulates the concomitant secretion of corticotropin, β-lipotropin, β-endorphin and γ-melanotropin by the bovine adenohypophysis invitro

Bovine anterior pituitary cells in culture respond to synthetic ovine corticotropin-releasing factor (CRF) with concomitant release of immunoreactive adrenocorticotropin, β-endorphin and γ-melanotropin. Gel permeation chromatography shows that the major molecular forms of these peptides released into the culture medium are ACTH1–39 β-lipotropin and two large forms of γ3-MSH (Mr >8.000). The effect of CRF is dose dependent and the maximal stimulation (5 × 10−9M CRF) gives increases in immunoreactive ACTH (5 fold), β-endorphin (2 fold) and γ3-MSH (6 fold) that are not significantly different from those obtained with 10 mM isobutyrylmethyl xanthine (IBMX). The ED50 for the ACTH, β-LPH and γ3-MSH dose-response curves to CRF are 3 × 10−10, 3 × 10−10 and 2 × 10−10 molar respectively. CRF has no such effect on bovine intermediate lobe cells. The results suggest that CRF acts to release the post-translational products of pro-opiomelanocortin processing in the anterior pituitary without discrimination or selectivity.

Discovery of atrial natriuretic factor in the brain: its characterization and cardiovascular implication.

Summary1.We have devised a radioimmunoassay for atrial natriueretic factor (ANF). Its application to rat brain extract led to the discovery of ANF in the brain. In addition to the hypothalamus and the pontine medullary region, it was widely distributed.2.ANF in the brain is stored in a low molecular weight form, in contrast to pro-ANF in the atria. Thus, the processing of pro-ANF in the bran neuronal cells is different from that in the atria.3.ANF was found in the anterior and posterior lobes of the pituitary, the peripheral ganglia, adrenergic neurons, and the adrenal medulla.4.Brain ANF suppressed stimulated dipsogenesis, basal and stimulated vasopressin release, and angiotensin II-stimulated pressor effects.5.ANF in the peripheral neuronal system inhibits catecholamine synthesis and release. Thus, central ANF functions to reduce the peripheral fluid volume and vascular tone in concert with the peripheral ANF.

Coronary hemodynamics and cardiac beating modulate atrial natriuretic factor release from isolated Langendorff-perfused rat hearts

The role of coronary hemodynamics and cardiac beating on atrial natriuretic factor (ANF) release was studied in the isolated Langendorff-perfused rat heart. ANF release was measured by radioimmunoassay. When the coronary flow rate was changed, ANF release decreased or increased in a flow-dependent manner. When the perfusion pressure was changed, ANF release also increased or decreased, respectively, with concomitant changes in coronary flow rate. Furthermore, perfusion with 50 mM potassium chloride showed immediate cardiac arrest and a decrease of ANF release to an undetectable level with a significant decrease in coronary flow. However, low but readily detectable amounts of ANF were released when coronary flow rate was maintained. These results may suggest that coronary hemodynamics and cardiac beating could be factors modulating ANF secretion from the atrium.

Effects of Cyproheptadine, Reserpine, and Synthetic Corticotropin-releasing Factor on Pituitary Glands from Patients with Cushingʼs Disease

Direct effects of cyproheptadine, reserpine, synthetic ovine corticotropin-releasing factor (CRF), dexamethasone, and lysine-8-vasopressin (LVP) on the secretion of immunoreactive ACTH and beta-endorphin from the adenoma and the nonadenomatous tissue of patients with Cushings disease were examined using a superfusion system. Cyproheptadine and reserpine (10(-9)-10(-7) M of each) suppressed immunoreactive ACTH and beta-endorphin secretion from both tissues. CRF (10(10)-10(7) M) stimulated the secretion of both peptides from the nonadenomatous tissue, but only a high dose of CRF could stimulate the secretion of these peptides from some adenomas. Such CRF-induced secretion was partially suppressed by dexamethasone. LVP (10(-9)-10(-7) M) stimulated peptide secretion from both types of tissue. These results suggest direct inhibitory effects of cyproheptadine and reserpine on the secretion of these peptides from the pituitary of patients with Cushings disease, a different stimulatory mechanism of LVP from that of CRF in these tissues, and low sensitivity of the adenoma to CRF.