Corticotropin Releasing Factor (CRF): Immunocytochemical localization and Radioimmunoassay (RIA)

Recent isolation, structural identification, and synthesis of ovine CRF has made possible the generation of specific antibodies against this hypothalamic peptide. Two fragments of the amino acid sequence corresponding to ovine CRF (CRF 37-41 and CRF 22-41), as well as the full sequence of 41 residues (CRF 1-41), synthesized in our laboratories by solid-phase methods, were coupled to bovine serum albumin (BSA) with glutaraldehyde. New Zealand white rabbits were immunized with the emulsified mixtures of peptide-BSA conjugates and Freund's adjuvant as immunogens. The specificity of the generated antibodies was studied by agar-gel diffusion, absorption tests in the immunohistochemical system, and with the aid of displacement curves in RIA. ¹²⁵I-Tyr(35)-CRF 36-41 and ¹²⁵I-Tyr(0)-CRF 1-41 were used as radioligands in the RIA. The minimum detectable dose was 20 pg. The linearity observed in RIA for immunoreactive CRF in extracts of rat hypothalami, together with the immunocytochemical findings in the rat brain, indicate the presence of substance(s) immunologically indistinguishable from CRF. Immunohistochemistry with the peroxidase-antiperoxidase (PAP) technique detected the following CRF-immunoreactive structures in vibratome sections of hypothalami of colchicine-treated rats: CRF-containing cell bodies were observed mainly in smaller neurons of the paraventricular nucleus. CRF-positive nerve fibers and/or terminals were present in the external zone of the median eminence, with some immunoreactive CRF also present in the internal zone. The CRF-positive terminals were localized in the central regions of the median eminence. These morphological data reinforce the view that this polypeptide plays a physiological role in the control of ACTH release.     

Production of antiserum to CRF associated with adrenal atrophy in a rabbit

Corticotropin releasing factor (CRF) was recently isolated from ovine hypothalami by its ability to stimulate adrenocorticotropin (ACTH) and β-endorphin release from dispersed rat pituitary cells. Intramuscular injection of synthetic ovine CRF conugated to bovine thyroglobulin with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide and emulsified with Freund's complete adjuvant into a random bred New Zealand white rabbit resulted in antiserum production to CRF associated with adrenal atrophy. A decrease in the level of plasma coticosteroids was associated with an increase in mean total binding of ¹²⁵I-N-Tyr-CRF. Upon sacrifice, a decrease in pituitary content of ACTH and a decrease in adrenal weight and content of corticosteroids was observed in the rabbit producing antiserum to CRF. Adrenal atrophy was histologically verified with an observed decrease in the adrenal cortical zone not reflected in the zona glomerulosa. Individual cells were relatively larger either with more abundant pale cytoplasm or with distinctly vacuolated cytoplasm. The results presented here are consistent with a physiologically necessary role for this CRF or peptides with similar structures in the hypothalamic-pituitary-adrenal axis.     

Lack of effect of serotonin and norepinephrine on CRF release from hypothalami in vitro

A number of investigations utilizing hypothalami from adrenalectomized animals have provided conflicting results regarding the role of serotonin (5HT) and norepinephrine (NE) in CRF regulation. In order to further investigate these neurotransmitters, we performed three sets of experiments with hypothalami obtained from intact rats. In experiment I, freshly obtained rat hypothalami were randomly grouped and incubated in control medium and medium in the presence of 10(-11) M, 10(-10) M, and 10(-9) M serotonin. Aliquots of 200 microliters of these incubates were bioassayed for CRF activity using a dispersed anterior pituitary cell system, and ACTH secretion from the cells was determined by radioimmunoassay. A preliminary experiment had determined that a 200 microliters aliquot from hypothalami incubated in control medium resulted in a significantly (p less than 0.0001) greater ACTH release than obtained from cells alone. No significant effect of serotonin on hypothalamic CRF release was obtained. In experiment III, individual hypothalami were bisected longitudinally, and one half served as control. The contralateral half was incubated in medium containing 10(-11) M, 10(-10) M, and 10(-9) M serotonin. CRF release in this experiment again revealed no significant effect of serotonin. In experiment II, hypothalami were again randomly grouped and incubated with control medium and in the presence of 10(-8) M and 10(-6) M norepinephrine. This experiment resulted in no significant effect of norepinephrine on CRF release. These results suggest that serotonin and norepinephrine at the concentrations studied have no effect on CRF release from hypothalami obtained from intact rats.     

Discordances in the temporal pattern of the ACTH/beta-endorphin releasing effects of synthetic CRFs and partially purified bovine CRF in a superfusion system

Temporal characteristics of ACTH and beta-endorphin secretion induced by bovine hypothalamic CRF-A (void volume) and CRF-B (Kav = 0.583) separated by Sephadex G-100 were compared to those of synthetic ovine or rat CRF, sauvagine and vasopressin. Dispersed cells or minced fragments of rat adenohypophyses perifused in a column were exposed to various secretagogues, and ACTH and/or beta-endorphin concentrations of the effluent were measured by radioimmunoassays. CRF-A or CRF-B induced an immediate brisk rise of ACTH and beta-endorphin within 1 min after initiation of CRF perifusion. The maximum rate of ACTH or beta-endorphin secretion was reached 1-2 min later. Hormone secretion persisted throughout a 10-min exposure to these secretagogues. More than 80% of the total ACTH or beta-endorphin secretion induced by 10-min perifusion with bovine CRF occurred during exposure to CRF. With 10-min perifusion with 300 ng/ml ovine or rat CRF, the onset of the major CRF-stimulated ACTH or beta-endorphin secretion was markedly delayed compared to that following bovine CRF. During perifusion with ovine or rat CRF, a modest slow increase in ACTH or beta-endorphin secretion was observed. More than 60-70% of the total ACTH or beta-endorphin secretion induced by 10-min perifusion with rat or ovine CRF occurred after CRF withdrawal. The ACTH secretory patterns for sauvagine were very similar to those for synthetic rat or ovine CRF. These results suggest some qualitative differences between partially purified bovine CRF and synthetic ovine or rat CRF.     

Distribution of corticotropin releasing factor-like immunoreactivity in the rat brain by immunohistochemistry and radioimmunoassay: comparison and characterization of ovine and rat/human CRF antisera

The distribution of corticotropin releasing factor (CRF)-like immunoreactivity in the rat brain has been demonstrated by immunohistochemistry and radioimmunoassay using 4 different antisera. Two antisera were directed against synthetic ovine CRF, two antisera were directed against synthetic rat/human CRF. Immunohistochemistry revealed that there are discrete regions where CRF immunoreactive cell bodies are seen with all 4 antisera (e.g., the paraventricular nucleus, the dorsolateral tegmental nucleus) whereas there are cells observed only with one rat CRF antiserum (e.g., in the cortex) or terminal fields observed only with ovine CRF antisera (e.g., the spinal trigeminal tract, the substantia gelatinosa, the spinal cord). Radioimmunoassay showed different cross reactivity of the antisera with synthetic ovine or rat/human CRF and sauvagine, however, there was no cross reactivity with a variety of other peptides. Tissue values of CRF obtained by RIA of micropunched brain nuclei with the 4 antisera were frequently dissimilar suggesting that different antisera recognize different substances. High performance liquid chromatography and radioimmunoassay of brain tissue samples, revealed that there is more than one form of CRF-like immunoreactivity present. There is indirect evidence that there exists at least one peptide in the rat brain, prominent in the medulla and the spinal cord, which cross reacts with antisera directed to ovine CRF only.     

Adrenalectomy does not change CRF secretion induced by interleukin-1 from rat perifused hypothalami.

Interleukin-1 (IL-1) is a potent hypothalamic-pituitary-adrenal (H-P-A) axis activator. The hypothalamus is considered one of the main sites of action of IL-1 on the H-P-A axis, inducing CRF secretion, which is modulated by glucocorticoids. Glucocorticoids, which modulate CRF release by a negative feedback inhibition, have been postulated to exert a permissive action on the IL-1 effect on CRF secretion. Using a continuous perifusion system of rat hypothalami, the results of the present study indicate that at the same concentrations, IL-1 beta exerted a more potent effect than IL-1 alpha stimulating CRF secretion. The increase in hypothalamic CRF release induced by IL-1 was rapidly inhibited by both dexamethasone and corticosterone. However, adrenalectomy 2 or 8 days before did not modify CRF secretion induced by IL-1 from the in vitro perifused hypothalami. These data indicate that IL-1 does not seem to induce CRF secretion by interfering with an impeding action of glucocorticoids, although the cytokine effect is negatively modulated by corticosteroids.     

In vitro study of immunoreactive corticotropin-releasing factor release from the rat hypothalamus

Immunoreactive corticotropin-releasing factor (I-CRF) release from rat hypothalami was studied in vitro utilizing a perifusion of rat hypothalami and a rat CRF RIA. Basal release of I-CRF from the hypothalamus of adrenalectomized or hypophysectomized rats was higher than in that of normal rats. K+-induced I-CRF release was completely suppressed by omission of Ca++ from the medium. Dexamethasone suppressed I-CRF release from hypothalami, but not from median eminence (ME). C-AMP and angiotensin II had mild stimulatory effects on I-CRF release. These results suggest that 1) the feedback mechanism acts mainly on a higher level than ME, and 2) c-AMP and angiotensin II may be involved in CRF-releasing mechanism(s).     

Corticotropin releasing factor (CRF)-like immunoreactivity in the hypothalamus and posterior pituitary of the goat, bovine, rat, monkey and human

Goat hypothalamic extract prepared by HCl extraction and chromatographed on a Sephadex G-50 column showed two immunoreactive CRF peaks. Most of the immunoreactivity coeluted with synthetic ovine CRF, and a small peak eluted near the void volume. Bovine, monkey, rat and human hypothalamic extracts prepared by acid-acetone or acid-methanol extraction showed three immunoreactive peaks. Most of the immunoreactivity coeluted with ovine CRF, and other smaller peaks eluted near the void volume and slightly before arginine vasopressin. Goat hypothalamic extract showed the highest cross-reactivity with anti-ovine CRF serum, followed by bovine hypothalamic extract. Less cross-reactivity was found in human, rat and monkey hypothalamic extracts. CRF immunoreactivity in goat hypothalamic extract coeluted with ovine CRF on reversed phase high performance liquid chromatography (HPLC) and main CRF immunoreactivity in human and rat hypothalamic extracts eluted slightly later than ovine CRF. These results suggest that there is a heterogeneity among the CRF molecules in these species and that goat CRF may be more similar to that of sheep CRF and the amino acid sequence or molecular weight of other animals CRF may be different from that of sheep CRF. The monkey posterior pituitary and rat neurointermediate lobe showed similar elution patterns of CRF immunoreactivity to their hypothalamic extracts on Sephadex gel filtration and HPLC. These results indicate that the posterior pituitary contains a similar CRF to hypothalamic CRF.     

Identification of Two Corticotropin-Releasing Factor Receptors from Xenopus laevis with High Ligand Selectivity: Unusual Pharmacology of the Type 1 Receptor

Abstract: Two cDNA clones encoding distinct members of the corticotropin-releasing factor (CRF) receptor family have been isolated from Xenopus laevis with PCR-based approaches. The first full-length cDNA amplified from Xenopus brain encoded a 415-amino acid protein with ∼80% identity to mammalian CRF receptor type 1 (CRF-R1). The second full-length cDNA isolated from Xenopus brain and heart encoded a 413-amino acid protein with ∼81% identity to the α-variant of mammalian CRF receptor, type 2 (CRF-R2). No evidence could be obtained that the β-variant of CRF-R2 existed in Xenopus laevis . Binding studies using human embryonic kidney 293 (HEK 293) cells stably transfected with xenopus CRF-R2 showed that the CRF analogues urotensin I, urocortin, and sauvagine were bound with higher affinities than human/rat CRF, xenopus CRF, and ovine CRF. In contrast to human CRF-R1, xenopus CRF-R1 (xCRF-R1) was very selective for different CRF ligands. Urotensin I, urocortin, human/rat CRF, and xenopus CRF were bound with significantly (10–22-fold) higher affinities than ovine CRF ( K _D = 31.7 n M ) and sauvagine ( K _D = 51.4 n M ). In agreement with these binding data, EC₅₀ values of 39.7 and 1.1 n M were found for sauvagine and for human/rat CRF or xenopus CRF, respectively, when the cyclic AMP production in HEK 293 cells stably transfected with xCRF-R1 was determined.     

Structural characterization and localization of corticotropin-releasing factor in testis

To sequence and thereby definitively characterize corticotropin-releasing factor (CRF)-like material from a representative peripheral tissue, CRF was obtained from 76 ovine testes. The novel extraction procedure involved use of an immunoaffinity column to which a high-affinity CRF monoclonal antibody was attached as well as fast protein liquid chromatography. The complete sequence was elucidated by gas-phase sequencing, carboxyamidopeptidase digestion and cyanogen bromide cleavage. Aside from microheterogeneity at position 39, all the other amino acids were identical to ovine hypothalamic CRF. Additionally, in immunohistochemical studies in the rat, CRF was localized to the Leydig cell. These findings along with related observations by ourselves and others are compatible with the hypothesis that CRF plays a significant local role, possibly by paracrine or autocrine mechanisms.     

Radioimmunoassay of CRF-like material in rat plasma: validation of the method

The availability of antibodies against the ovine corticotropin releasing factor (CRF), which cross-react with a CRF-like immunoreactivity (CRF-LI) in the rat, has enabled us to develop a radioimmunoassay (RIA) for rat CRF-LI in plasma and crude hypothalamic extracts. 125I-Tyr CRF 1-41 was used as the tracer, and synthetic ovine CRF as the reference hormone. The precision profile of the assay indicates a high degree of reproducibility except for the lower dose range. The minimum detectable dose was 20 pg/tube. This assay can detect differences in plasma CRF-LI levels after various manipulations that simultaneously alter the ACTH levels in plasma. A wide range of CRF concentrations has been found in plasma of normal rats. Caution should be exercised in the interpretation of the values obtained since an ovine RIA system was used.     

Immunohistochemical study on the development of CRF-containing neurons in the hypothalamus of the rat

Summary Appearance of immunoreactive corticotropin-releasing factor (CRF)-containing neurons was studied in developing hypothalamus of the rat by use of antisera against rat- and ovine CRF. These neurons were first recognized in the lateral and paraventricular nuclei on days 15.5 and 16.5 of gestation, respectively, when antiserum against rat CRF was employed. Antiserum against ovine CRF revealed the cells two days later exclusively in the latter nucleus. In both nuclei, the neurons increased in number with development. The neurons in the paraventricular nucleus appeared to project their immunoreactive processes to the median eminence via the periventricular and lateral pathways. In the median eminence, the immunoreaction with antiserum to rat CRF was first recognized in its anterior portion in the form of dots on day 16.5 of gestation but as beaded fibers in the external layer on day 17.5; these structures increased in amount with development in rostro-caudal direction. Although antiserum to ovine CRF was less potent in immunostainability than antiserum to rat CRF, it also revealed the beaded fibers in the median eminence on day 17.5 of gestation. Since evidence is available that the paraventricular nucleus is involved in corticotropin release, it is concluded that, in rats, the hypothalamic regulatory mechanism controlling the release of corticotropin initially appears on days 16.5–17.5 of gestation.     

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.     

The brain corticotropin-releasing factor (CRF) receptor is of lower apparent molecular weight than the CRF receptor in anterior pituitary. Evidence from chemical cross-linking studies

The ligand binding subunits of the corticotropin-releasing factor (CRF) receptors in brain and anterior pituitary of a number of species have been identified by chemical affinity cross-linking using the homobifunctional cross-linking agent disuccinimidyl suberate and 125I-Tyr0-oCRF (ovine CRF). In homogenates of rat, monkey, and human cerebral cortex, 125I-Tyr0-oCRF was covalently incorporated into a protein of Mr = 58,000. Under identical conditions in the anterior pituitary of rat, monkey, cow, and pig, 125I-Tyr0-oCRF was incorporated into a protein of apparent Mr = 75,000. The specificity of the labeling was typical of the CRF binding site since both the cerebral cortex- and pituitary-labeled proteins exhibited the appropriate pharmacological rank order profile characteristic of the CRF receptor (Nle21,Tyr32-oCRF approximately equal to rat/human CRF approximately equal to ovine CRF approximately equal to alpha-helical CRF(6-41) greater than alpha-helical oCRF(9-41) greater than or equal to oCRF(7-41) greater than rat/human CRF(1-20) approximately equal to vasoactive intestinal peptide). In addition to the major labeled proteins, 125I-Tyr0-oCRF was incorporated into higher molecular weight peptides which may represent precursors and into lower molecular weight components which may represent fragments of the major labeled proteins or altered forms of the CRF binding subunit. In summary, these data indicate a heterogeneity between brain and pituitary CRF receptors with the ligand binding subunit of the brain CRF receptor residing on a Mr = 58,000 protein, while in the anterior pituitary, the identical binding subunit resides on a protein of apparent Mr = 75,000.     

A novel peptide which stimulates adenylate cyclase: molecular cloning and characterization of the ovine and human cDNAs

A novel neuropeptide which remarkably stimulates adenylate cyclase in rat anterior pituitary cell cultures has been recently isolated from ovine hypothalami by A. Arimura and his collaborators (Biochem.Biophys.Res.Commun.164, 567-574(1989)). This peptide was designated as PACAP38(Pituitary Adenylate Cyclase Activating Polypeptide with 38 residues). In an attempt to investigate physiological implications of PACAP38, we have succeeded in cloning the cDNAs encoding the precursor of PACAP38 from ovine hypothalamus and human testis. An ovine cDNA encodes a protein of 176 amino acids in which PACAP38 is proceeded by a putative signal peptide and a "pro"-region (107 amino acids), and followed by a Gly-Arg-Arg sequence for proteolytic processing and amidation. Deduced amino-acid sequence of human PACAP38 was completely identical to that of the ovine isolated peptide. Cloning of PACAP38 cDNAs confirms the expression of the corresponding mRNAs and the presence of this neuropeptide in ovine hypothalamus and also in human testis.     

Extrahypothalamic distribution of CRF-like immunoreactivity in the rat brain

CRF-like immunoreactivity was measured by radioimmunoassay in the brains of normal adult rats and found to be widely distributed in extrahypothalamic areas (e.g., thalamus, amygdala, hippocampus, frontal cerbral cortex, striatum, midbrain, pons-medulla and cerebellum) at levels approximately 10% of the hypothalamus. Sephadex G-50 gel filtration reveals that CRF-like immunoreactivity in the hypothalamus coelutes with synthetic ovine CRF and is also present in the void volume. However, in the extrahypothalamic areas of the rat brain, only CRF-like immunoreactivity that coelutes with synthetic ovine CRF was detected. High performance liquid chromatography revealed equal amounts of immunoreactivity coeluting with CRF and methionine sulfoxide CRF in hypothalamic extracts.     

Isolation, structure and synthesis of a heptapeptide with in vitro ACTH-releasing activity from porcine hypothalamus

Significant CRF activity was found in a fraction with Rf = 0.82-0.7 or VE/VT = 0.41-0.48 obtained by gel filtration of acid extracts of pig hypothalami on Sephadex G-25. The activity of this fraction decreased markedly during subsequent purification, particularly in the last two steps. From this fraction, a heptapeptide with significant ACTH releasing activity in vitro, was isolated in pure state, and its amino acid sequence was established as H-Phe-Ile-Tyr-His-Ser-Tyr-Lys-OH. This heptapeptide was synthesized by solid phase methods. The CRF activity of synthetic heptapeptide in vitro was low but could be potentiated by a cofactor fraction from rat hypothalamic extract.     

In vitro release of hypothalamic corticoliberine in the rat: incubation of slices from the whole hypothalamus

An experimental system allowing both the incubation and rapid transfert of rat hypothalamic slices has been developed in order to approach the regulation of CRF secretion. The release of CRF has been quantified by a specific radioimmunoassay. Under basal conditions, immunoreactive CRF release reached an optimum of 96.2 +/- 10.4 pg/3 hypothalami/20 min. A depolarizing concentration of KCl (56 mM) or veratridine (50 microM) applied for 20 min. induced a 222 and 257% increase, respectively, in CRF release. The in vitro CRF values released under basal and stimulated conditions are comparable to those of other hypothalamic neuropeptides. Furthermore, in vitro CRF release from the hypothalamus is in the same order of magnitude as in vivo CRF secretion estimated by hypophysial portal blood collection or median eminence push-pull cannulation.     

A priming effect of naloxone on in vitro LHRH release from the hypothalamus of mid-luteal ewes

The possible involvement of endogenous opioid peptides (EOPs) in LHRH release from hypothalami of ewes during the breeding season was investigated using an in vitro perifusion system. Hypothalami were procured in December from ovariectomized (OVX; 62-65 days before the experiment; n = 6) and mid-luteal (ML; n = 7) Western White-Face ewes. Hypothalami were mid-sagitally sectioned into halves containing the preoptic area, mediobasal hypothalamus, and infundibulum (median eminence). The left half (treated) received two 30-min challenges (beginning at 130 and 250 min, respectively, after onset of perifusion) of 500 microM naloxone (NAL) followed by a 30-min 60-mM potassium (K) challenge (at 370 min after onset of perifusion). The right half served as the control, receiving only K at the same time as the treated tissue. Both NAL challenges elicited (p less than 0.05) LHRH release from tissues of both ML and OVX ewes. Release of LHRH by hypothalami from ML, but not from OVX, ewes was greater (p less than 0.01) after the second than after the first NAL challenge. These results are consistent with the view that an inhibitory opioid influence exists on LHRH release from ovine hypothalami. The release of LHRH in response to NAL was dependent on the ovarian status in vivo since the priming effect of NAL on subsequent NAL-induced LHRH release occurred only from the hypothalami of ML ewes. We suggest from these results that EOPs may modulate LHRH release from ovine hypothalami in an ovarian steroid-dependent and independent manner.     

Synthetic corticotropin-releasing factor stimulates secretion of immunoreactive beta-endorphin/beta-lipotropin and ACTH by human fetal pituitaries in vitro

Synthetic ovine CRF, in an amount approximating that found in pituitary portal plasma of the rat, induced a significant increase in the secretion of both ACTH and immunoreactive beta-endorphin/beta-LPH (i beta-END/LPH) by human fetal hemipituitaries in an in vitro superfusion system. This finding suggests that a molecule similar to synthetic ovine CRF may be a physiologic hypothalamic releasing factor in man.