Corticotrophin-releasing factor-like immunoreactivity and bioactivity of human fetal and adult hypothalami

ABSTRACT Corticotrophin releasing factor-like immunoreactivity (CRF-LI) and bioactivity, and arginine vasopressinlike immunoreactivity (AVP-LI) have been measured in extracts of human fetal and adult hypothalamic tissue and their development with the gestational age of the fetuses (12–27 weeks) studied. CRF-LI was measured by a radioimmunoassay developed for ovine corticotrophin-releasing factor (oCRF-41). Corticotrophin-releasing factor bioactivity was measured in a rat isolated anterior pituitary cell perfusion system. CRF-LI and bioactivity and AVP-LI were all detectable in fetal hypothalamic extracts from 12 to 13 weeks of gestational age. CRF-LI was also present in human fetal pituitary glands from 12 weeks of gestational age. The concentration of CRF-LI in the fetal hypothalamic extracts (9·2±11·4 ng/g, mean ± s.e.m., n = 33) showed no significant correlation with the gestational age of the fetuses. However the concentration of AVP-LI (25·0–36·8 ng/g, n = 17) did show a positive correlation (r = 0·508, P<0·05) with gestational age, as did the concentration of CRF bioactivity (471·3–556·3 ng ACTH released/g tissue, n = 13, r = 0·725, P < 0·01). The CRF bioactivity of all fetal hypothalamic extracts was potentiated by the addition of synthetic human (h)AVP, but the bioactivity of the adult hypothalamic extracts was not, presumably because of the higher levels of AVP-LI already present in the adult extracts. Pretreatment of tissue extracts with antisera to oCRF-41 and/or hAVP reduced the CRF bioactivity of all hypothalamic extracts. Sephadex chromatography of fractions which co-eluted with synthetic oCRF-41 or hAVP contained CRF bioactivity and this bioactivity was potentiated when synthetic hAVP or oCRF-41, respectively, were added to the fractions. However, a larger molecular weight form of CRF-LI (8000–10 000 daltons), which was observed only in fetuses of 20 weeks of gestational age or less, did not contain any significant CRF bioactivity. J. Endocr. (1986) 108, 171–180

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Production and utilization of monoclonal antibodies to human/rat corticotrophin-releasing factor-41

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0050159 ◽

1990 ◽

Vol 5 (2) ◽

pp. 159-166 ◽

Cited By ~ 6

Author(s):

N. G. N. Milton ◽

E. W. Hillhouse ◽

S. A. Nicholson ◽

C. H. Self ◽

A. M. McGregor

Keyword(s):

Monoclonal Antibodies ◽

Enzyme Linked Immunosorbent Assay ◽

Dependent Manner ◽

Corticotrophin Releasing Factor ◽

Tissue Extracts ◽

Rat Pituitary ◽

Hypothalamic Tissue ◽

Dose Dependent

ABSTRACT Murine monoclonal antibodies against human/rat corticotrophin-releasing factor-41 (CRF-41) were produced and characterized for use in the immunological and biological characterization of CRF-41. Spleen cells from BALB/c mice immunized with CRF-41 conjugated to bovine γ-globulin were fused with a BALB/c-derived non-secretor X-63 myeloma line. Hybridomas were selected for CRF antibody production by enzyme-linked immunosorbent assay, and positive hybridomas cloned twice. Three monoclonal antibodies were obtained (KCHMB001, KCHMB002 and KCHMB003) and characterized as IgG1, IgG1 and IgG2a isotypes respectively, with affinity constants for rat CRF-41 of 30, 53 and 34 nmol/l respectively. All three monoclonal antibodies recognize an epitope contained between residues 34 and 41 of the human/rat sequence. The antibodies were able to neutralize the ACTH-releasing activity of rat CRF-41, applied to rat pituitary fragments in vitro, in a dose-dependent manner. Isoelectric focusing showed that KCHMB 003 detected bands of synthetic rat CRF-41 and rat [Met(O)21,38]-CRF-41 at pH 7·1 and 6·8 respectively. Use of KCHMB003 in a two-site enzyme-amplified immunoassay showed that this antibody recognizes both synthetic rat CRF-41 and immunoreactive CRF-41 in rat hypothalamic tissue extracts.

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Controls of corticotrophin-releasing factor output by hypothalamic tissue from fetal sheep in vitro

Journal of Endocrinology ◽

10.1677/joe.0.1220015 ◽

1989 ◽

Vol 122 (1) ◽

pp. 15-22 ◽

Cited By ~ 7

Author(s):

A. N. Brooks ◽

L. A. Power ◽

S. A. Jones ◽

K. P. Yang ◽

J. R. G. Challis

Keyword(s):

G Protein ◽

Gestational Age ◽

Potassium Depolarization ◽

Fetal Sheep ◽

Corticotrophin Releasing Factor ◽

Basal Release ◽

Hypothalamic Tissue ◽

Negative Feedback Control ◽

Perifusion System

ABSTRACT Corticotrophin-releasing factor (CRF) is thought to be an important physiological regulator of the pituitary-adrenal axis in fetal sheep and, as such, plays a fundamental role in the initiation of parturition in this species. However, little is known of the controls of CRF secretion from the fetal hypothalamus. We looked for the presence of CRF in fetal hypothalami, and examined whether the hypothalamic CRF concentration or molecular species changed in relation to gestational age. We established an in-vitro perifusion system to examine the release of CRF from perifused hypothalami taken from fetuses at day 100 and day 140 of pregnancy, under basal conditions and in response to potassium depolarization and/or dexamethasone administration. Immunoreactive CRF was present in fetal hypothalami as early as day 100 (2·42 ± 0·99 (s.e.m.) μg/g protein, n = 9) and in similar concentrations at day 140 (2·31 ± 0·69 μg/g protein, n = 9). There was a significant (P < 0·05) increase in hypothalamic CRF content to 14·79 ± 4·09 μg/g protein (n = 16) between day 122 and day 135 of gestation. Using Sephadex G-75 chromatography, hypothalamic extracts at day 100, days 122–135 and day 140 eluted with a single peak of immunoreactivity which corresponded to synthetic ovine CRF(1–41). The basal release of CRF from perifused hypothalami at day 140 (76·6 ± 10·4 pg/fraction, n = 8) was significantly (P < 0·05) greater than at day 100 (50·1 ± 10·2 pg/fraction, n = 11). Dexamethasone significantly inhibited basal CRF release at day 140 of gestation but not at day 100. Potassium depolarization caused a rapid release of CRF in all cases, a response which was independent of gestational age or treatment with dexamethasone. We conclude that the fetal hypothalamus contains immunoreactive CRF as early as day 100 of gestation and that this material may be released when perifused in vitro under basal conditions and in response to a depolarizing agent. The basal release of CRF from perifused hypothalami of day-140 fetuses was greater than at day 100 and was inhibited by dexamethasone, suggesting maturation of negative feedback control of CRF output between days 100 and 140. Since dexamethasone had no effect on potassium-stimulated release of CRF, we suggest that its effects are at sites other than the hypothalamic CRF nerve terminals. Journal of Endocrinology (1989) 122, 15–22

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Serotonin and acetylcholine affect the release of prolactin and growth hormone from pituitary glands of domestic fowl in vitro in the presence of hypothalamic tissue

Acta Endocrinologica ◽

10.1530/acta.0.1050455 ◽

1984 ◽

Vol 105 (4) ◽

pp. 455-462 ◽

Cited By ~ 16

Author(s):

T. R. Hall ◽

S. Harvey ◽

A. Chadwick

Keyword(s):

Growth Hormone ◽

Pituitary Gland ◽

Anterior Pituitary ◽

Domestic Fowl ◽

Hormone Secretion ◽

Pituitary Hormone ◽

Cholinergic Drugs ◽

Pituitary Glands ◽

Hypothalamic Tissue

Abstract. Anterior pituitary glands from broiler fowl were incubated alone or with hypothalamic tissue in medium containing either serotonin or serotoninergic drugs, acetylcholine or cholinergic drugs, and the release of prolactin (Prl) and growth hormone (GH) measured by homologous radioimmunoassays. The neurotransmitters and drugs affected the release of hormones from the pituitary gland only when hypothalamic tissue was also present. Serotonin and its agonist quipazine stimulated the release of Prl and inhibited release of GH in a concentration-related manner. The antagonist methysergide blocked the effects of serotonin and quipazine on Prl. Acetylcholine and its agonist pilocarpine also stimulated release of Prl and inhibited release of GH in a concentration-related manner. Atropine blocked these responses. The results show that serotonin and acetylcholine affect pituitary hormone secretion by acting on the hypothalamus. They may stimulate the secretion of a Prl releasing hormone and somatostatin.

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The presence of atrial natriuretic peptide in canine cerebrospinal fluid and its possible origin in the brain

Journal of Endocrinology ◽

10.1677/joe.0.1190127 ◽

1988 ◽

Vol 119 (1) ◽

pp. 127-131 ◽

Cited By ~ 8

Author(s):

F. Marumo ◽

T. Masuda ◽

Y. Masaki ◽

K. Ando

Keyword(s):

Molecular Weight ◽

Cerebrospinal Fluid ◽

Atrial Natriuretic Peptide ◽

Natriuretic Peptide ◽

Gel Permeation Chromatography ◽

Tissue Extracts ◽

Molecular Weight Peak ◽

Hypothalamic Tissue ◽

The Brain ◽

Atrial Natriuretic

ABSTRACT The presence of atrial natriuretic peptide (ANP) in canine cerebrospinal fluid (CSF) was clearly demonstrated and an attempt was made to determine its origin as either the brain or the atrium. The concentration of ANP in canine CSF was 0·78 ±0·37 pmol/l (n = 31) and showed no evident correlation with that in plasma (r =0·12). Physiological doses of human α-ANP (α-hANP) were continuously infused intravenously into nine dogs, and ANP concentrations in CSF and plasma were examined six to eight times within a 120-min period following this. The ANP level in CSF was not influenced by the systemic administration of α-hANP up to 180 min. Only one low molecular weight peak corresponding to α-hANP could be obtained from the CSF samples, while both low and high molecular weight peaks were observed for plasma ANP by gel permeation chromatography. In the atrial and hypothalamic tissue extracts the same kinds of peaks were also evident. These results prove the presence of ANP in canine CSF and that it does not come from blood that has seeped across the blood–CSF barriers, but suggest that it may originate from the brain. J. Endocr. (1988) 119, 127–131

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THE PITUITARY-ADRENAL SYSTEM OF THE GENETICALLY OBESE (ob/ob) MOUSE

Journal of Endocrinology ◽

10.1677/joe.0.0650099 ◽

1975 ◽

Vol 65 (1) ◽

pp. 99-107 ◽

Cited By ~ 46

Author(s):

J. A. EDWARDSON ◽

C. A. M. HOUGH

Keyword(s):

Normal Values ◽

Obese Mice ◽

Litter Mate ◽

Corticotrophin Releasing Factor ◽

Adrenal System ◽

Pituitary Glands ◽

Hypothalamic Tissue ◽

Maintain Body Weight ◽

Perifusion System

SUMMARY Adult genetically obese (ob/ob) mice which are characterized by adrenal hypertrophy and increased secretion of corticosteroids have considerably increased levels of ACTH in the pituitary gland. At 5 weeks of age there is no difference in the pituitary ACTH content of lean and obese animals and dietary restriction, sufficient to maintain body weight at normal values, reduces the pituitary ACTH content of adult obese mice from 14 times the level found in lean litter-mate controls to almost normal values. Using an in-vitro perifusion system, the release of ACTH from isolated pituitary glands was studied. Pituitaries from lean and obese mice responded similarly to stimulation with a crude extract of hypothalamic tissue containing corticotrophin releasing factor (CRF). The CRF content of the hypothalamus in both groups appears to be similar. In contrast with the high pituitary content, plasma values for ACTH in unstressed obese mice are not increased. The results are discussed in relation to other evidence for a hypothalamic disorder in ob/ob mice.

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Ontogeny of corticotrophin-releasing factor (CRF) in the ovine fetal hypothalamus: use of multiple CRF antibodies

Journal of Endocrinology ◽

10.1677/joe.0.1290335 ◽

1991 ◽

Vol 129 (3) ◽

pp. 335-NP ◽

Cited By ~ 11

Author(s):

T. Watabe ◽

M. L. Levidiotis ◽

B. Oldfield ◽

E. M. Wintour

Keyword(s):

Fetal Brain ◽

Molecular Size ◽

Immunohistochemical Localization ◽

Nerve Fibres ◽

Adult Sheep ◽

Corticotrophin Releasing Factor ◽

Hypothalamic Tissue ◽

Ovine Fetal ◽

Newcastle Upon Tyne ◽

Molecular Weight Form

ABSTRACT In previous studies, using one particular antibody, immunohistochemical localization of corticotrophin-releasing factor (CRF) in ovine fetal brain was not possible before 90 days of gestation (term is approximately 150 days), although radioimmunoassay of hypothalamic extracts, using a different antibody, had shown CRF to be present from 63 days. The purpose of this study was to use a variety of CRF antibodies in both immunohistochemistry and radioimmunoassay to determine the presence and concentration of the CRF peptide as early in gestation as possible, and to determine whether more than one molecular size of CRF is detectable at any time in gestation. Seven different antibodies were used on hypothalamic tissue or extracts from seven adult sheep and 37 fetuses from 48 to 140 days of gestation. With one ovine CRF antibody (provided by Dr W. Vale, Salk Institute) immunohistochemical detection of CRF-labelled neurones and nerve fibres of the paraventricular nucleus and median eminence was possible from 49 days. The antibody with the greatest sensitivity in radioimmunoassay was one raised against human CRF, Ab-code R1 (provided by Dr E. Hillhouse, University of Newcastle upon Tyne). The hypothalamic contents of CRF (ng/whole hypothalamus) were 0·28±0·06 (mean ± s.e.m.) (n = 4), 9·0±0·6 (n = 5), 14·3±0·6 (n = 5), 30·0±3·4 (n = 4) in fetuses at 48–50, 100–109 and 139–140 days of gestation and in adult sheep respectively. At all ages only one peak of CRF-like activity, which co-eluted with synthetic ovine CRF, was observed after chromatography of hypothalamic extracts, and assays performed with three different antibodies. Thus there are small amounts of CRF in the ovine fetal pituitary from very early in gestation (50 days), and there is no evidence for a larger molecular weight form, as seen in the immature human fetus. Journal of Endocrinology (1991) 129, 335–341

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Involvement of rat corticotrophin-releasing factor-41-related peptide and vasopressin in adrenocorticotrophin-releasing activity from superfused rat hypothalami in vitro

Journal of Endocrinology ◽

10.1677/joe.0.1030025 ◽

1984 ◽

Vol 103 (1) ◽

pp. 25-29 ◽

Cited By ~ 13

Author(s):

G. Gillies ◽

A. Puri ◽

S. Hodgkinson ◽

P. J. Lowry

Keyword(s):

Anterior Pituitary ◽

Pituitary Cell ◽

Cell Column ◽

Anterior Pituitary Cell ◽

Related Peptide ◽

Corticotrophin Releasing Factor ◽

High K ◽

Specific Antisera

ABSTRACT Superfused rat hypothalamic pieces stimulated with a high K+ medium released corticotrophin-releasing factor (CRF) as detected in the rat isolated anterior pituitary cell column bioassay. This activity has components related to ovine CRF-41 (oCRF-41) and vasopressin as assessed by the effects of specific antisera on the ACTH-releasing activity of the hypothalamic column effluent. Release of immunoactive vasopressin paralleled that of CRF bioactivity. Immunoactive oCRF-41 could not be detected, as it is likely to be below the sensitivity of present radioimmunoassays. J. Endocr. (1984) 103, 25–29

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EFFECT OF CRF ON MAINTENANCE OF THE ADRENOCORTICOTROPHIC FUNCTION OF ANTERIOR PITUITARY GRAFTS

Journal of Endocrinology ◽

10.1677/joe.0.0250465 ◽

1963 ◽

Vol 25 (4) ◽

pp. 465-472 ◽

Cited By ~ 9

Author(s):

V. CRITCHLOW ◽

H. S. LIPSCOMB ◽

R. GUILLEMIN

Keyword(s):

Anterior Pituitary ◽

Chronic Administration ◽

Plasma Corticosterone ◽

Challenge Dose ◽

Prolonged Administration ◽

Kidney Capsule ◽

Melanocyte Stimulating Hormone ◽

Corticotrophin Releasing Factor ◽

Pituitary Glands ◽

Adrenal Corticosterone

SUMMARY Rats were hypophysectomized and their own anterior pituitary glands transplanted under the kidney capsule. Effects of chronic administration of corticotrophin-releasing factor (CRF), lysin-vasopressin and α-melanocyte stimulating hormone (α-MSH) on the adrenocorticotrophic activity of the pituitary grafts were studied using as criteria the concentration of ACTH in the grafts, resting levels of circulating plasma corticosterone, adrenal weight and also response of the graft to a challenge dose of CRF measuring plasma and adrenal corticosterone levels 12 min. later. The results show that prolonged administration of CRF has maintained synthesis of ACTH by the transplanted pituitary as well as its ability to release ACTH when acutely stimulated by CRF. These results were not duplicated by the administration of lysin-vasopressin or α-MSH in conditions identical to the ones used for studying CRF, and using doses equivalent to the vasopressor and melanophoretic activities of this particular preparation of CRF.

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