Corticotropin-releasing Factor Antibody Research Articles

Role of CRF and the hypothalamic-pituitary-adrenal axis in stroke: revisiting temporal considerations and targeting a new generation of therapeutics.

Ischaemic neurovascular stroke represents a leading cause of death in the developed world. Preclinical and human epidemiological evidence implicates the corticotropin-releasing factor (CRF) family of neuropeptides as mediators of acute neurovascular injury pathology. Preclinical investigations of the role of CRF, CRF receptors and CRF-dependent activation of the hypothalamic-pituitary-adrenal (HPA) axis have pointed toward a tissue-specific and temporal relationship between activation of these pathways and physiological outcomes. Based on the literature, the major phases of ischaemic stroke aetiology may be separated into an acute phase in which CRF and anti-inflammatory stress signalling are beneficial and a chronic phase in which these contribute to neural degeneration, toxicity and apoptotic signalling. Significant gaps in knowledge remain regarding the pathway, temporality and systemic impact of CRF signalling and stress biology in neurovascular injury progression. Heterogeneity among experimental designs poses a challenge to defining the apparent reciprocal relationship between neurological injury and stress metabolism. Despite these challenges, it is our opinion that the elucidated temporality may be best matched with an antibody against CRF with a half-life of days to weeks as opposed to minutes to hours as with small-molecule CRF receptor antagonists. This state-of-the-art review will take a multipronged approach to explore the expected potential benefit of a CRF antibody by modulating CRF and corticotropin-releasing factor receptor 1 signalling, glucocorticoids and autonomic nervous system activity. Additionally, this review compares the modulation of CRF and HPA axis activity in neuropsychiatric diseases and their counterpart outcomes post-stroke and assess lessons learned from antibody therapies in neurodegenerative diseases.

Female-biased sexual dimorphism of corticotropin-releasing factor neurons in the bed nucleus of the stria terminalis

BackgroundThe bed nucleus of the stria terminalis (BNST) contains the highest density of corticotropin-releasing factor (CRF)-producing neurons in the brain. CRF-immunoreactive neurons show a female-biased sexual dimorphism in the dorsolateral BNST in the rat. Since CRF neurons cannot be immunostained clearly with available CRF antibodies in the mouse, we used a mouse line, in which modified yellow fluorescent protein (Venus) was inserted to the CRF gene, and the Neo cassette was removed, to examine the morphological characteristics of CRF neurons in the dorsolateral BNST. Developmental changes of CRF neurons were examined from postnatal stages to adulthood. Gonadectomy (GDX) was carried out in adult male and female mice to examine the effects of sex steroids on the number of CRF neurons in the dorsolateral BNST.MethodsThe number of Venus-expressing neurons, stained by immunofluorescence, was compared between male and female mice over the course of development. GDX was carried out in adult mice. Immunohistochemistry, in combination with Nissl staining, was carried out, and the effects of sex or gonadal steroids were examined by estimating the number of Venus-expressing neurons, as well as the total number of neurons or glial cells, in each BNST subnucleus, using a stereological method.ResultsMost Venus-expressing neurons co-expressed Crf mRNA in the dorsolateral BNST. They constitute a group of neurons without calbindin immunoreactivity, which makes a contrast to the principal nucleus of the BNST that is characterized by calbindin immunostaining. In the dorsolateral BNST, the number of Venus-expressing neurons increased across developmental stages until adulthood. Sexual difference in the number of Venus-expressing neurons was not evident by postnatal day 5. In adulthood, however, there was a significant female predominance in the number of Venus expressing neurons in two subnuclei of the dorsolateral BNST, i.e., the oval nucleus of the BNST (ovBNST) and the anterolateral BNST (alBNST). The number of Venus-expressing neurons was smaller significantly in ovariectomized females compared with proestrous females in either ovBNST or alBNST, and greater significantly in orchiectomized males compared with gonadally intact males in ovBNST. The total number of neurons was also greater significantly in females than in males in ovBNST and alBNST, but it was not affected by GDX.ConclusionVenus-expressing CRF neurons showed female-biased sexual dimorphism in ovBNST and alBNST of the mouse. Expression of Venus in these subnuclei was controlled by gonadal steroids.

General anesthetics inhibit the nitrous-oxide-induced activation of corticotropin releasing factor containing neurons in rats

The activation of intracerebral corticotropin releasing factor (CRF) system is involved in nitrous oxide analgesia. We evaluated the effect of general anesthetics on nitrous-oxide-induced CRF activation and antinociception. Male Sprague–Dawley rats inhaled isoflurane (0%, 0.6%, 1.0% and 1.5%) or were administered with intravenous propofol (0, 0.1 and 0.2 mg/kg/min), with or without 75% nitrous oxide inhalation, for 90 min. The brain was fixed with fixative, and brain sections, including the paraventricular nucleus of the hypothalamus, were double immunostained with c-Fos and CRF antibodies to assess the activation of CRF-containing neurons. In other groups of rats, the effect of propofol on nitrous oxide antinociception was evaluated with tail flick latency tests. Both inhaled isoflurane and intravenous propofol inhibited nitrous-oxide-induced activation of CRF neurons, suggesting that these general anesthetics may inhibit one of the analgesic mechanisms of nitrous oxide. Indeed, propofol inhibited the antinociceptive action of nitrous oxide, as evaluated with tail flick latencies (TFL).

Does IL-6 Release ACTH by Exerting a Direct Effect in the Rat Anterior Pituitary

Adult male rats were used to determine whether high circulating levels of the pro-inflammatory cytokine interleukin-6 (IL-6) were capable of releasing ACTH independently of endogenous corticotropin-releasing factor (CRF). On one hand, CRF antibodies or a potent CRF antagonist significantly decreased, but did not totally abolish the ACTH response to the intravenous (i.v.) injection of recombinant rat IL-6. These results suggest that this cytokine might act either directly on the pituitary, or can release ACTH through mechanisms that do not involve CRF. On the other hand, the CRF antagonist or antibodies significantly (but not totally) blocked ACTH secretion due to the i.v. injection of endotoxin (LPS) while enhancing the ability of this immune stimulus to increase serum IL-6 concentrations. These results indicate that during endotoxemia, even very elevated circulating IL-6 concentrations were not able to release large amounts of ACTH in the absence of CRF drive. These data also illustrate the ability of a CRF antagonist or CRF antibodies to significantly augment IL-6 secretion, which indicates an inhibitory influence of the endogenous peptide in the paradigm we used. As comparable findings were obtained in adrenal-intact and adrenalectomized rats, they suggest that endogenous CRF is involved in the IL-6 response to LPS independently of circulating corticosteroids or other adrenal factors.

Involvement of Endogenous Corticotropin-Releasing Factor in Mediation of Neuroendocrine and Behavioral Effects to Alpha-Melanocyte-Stimulating Hormone

The present work was to study if the α-melanocyte-stimulating hormone (α-MSH) was involved in activation of the pituitary-adrenal axis (PAA) in rats. The hormone increased plasma corticosterone (CORT) level, and induced an anxiogenic response as indicated by results from the elevated plus-maze test. Intracerebroventricular administration of corticotropin-releasing factor (CRF) antiserum (1:10, 1:20 and 1:100 dilutions in 1µl volume), overcame both the anxiogenic response and the PAA activating effect induced by α-MSH (50 µg s.c.) in a concentration-dependent manner. CRF antibody at the doses applied did not modify either the elevated plus-maze responses or CORT level by itself. Our results reveal that both the anxiogenic and the PAA activating effects of alpha-MSH are mediated by CRF.

Acute alcohol administration stimulates the activity of hypothalamic neurons that express corticotropin-releasing factor and vasopressin

Alcohol (EtOH) treatment increases plasma ACTH levels in the rat, a response thought to depend at least partially on endogenous corticotropin-releasing factor (CRF). However, the inability of CRF antibodies to completely abolish EtOH-induced activation of the pituitary has suggested the possible importance of other factors. Because vasopressin (VP) may be acutely released by alcohol in the rat, and because this peptide interacts with CRF to promote ACTH secretion, we investigated its role in the hypothalamic-pituitary (HP) axis' response to one acute injection of EtOH (3 g/kg, i.p.). As expected, blockade of pituitary CRF receptors significantly blunted EtOH-induced ACTH secretion. Administration of a VP antagonist also interfered with this response, though to a lesser extent, while concomitant administration of both CRF and VP antagonists totally abolished it. These results indicate that both CRF and VP participate in the ability of alcohol to stimulate corticotrophs' function. Another hypothesis still awaiting confirmation is whether alcohol stimulates the activity of hypothalamic neurons important for ACTH release, which are primarily found in the paraventricular nucleus (PVN) of the hypothalamus. We therefore determined whether EtOH upregulated transcripts of the immediate early gene (IEG) NGFI-B, as well as those of CRF and VP. In the parvicellular portion of the PVN (pPVN), NGFI-B mRNA and CRF heteronuclear (hn) RNA levels increased within 20 min of EtOH treatment. Despite this robust response, densitometric assessments of CRF mRNA levels failed to indicate a significant elevation over control levels at any of the time points studied, a phenomenon that might be at least in part due to the abundance of these transcripts under basal conditions. Constitutive levels of VP ImRNA levels were very low in the pPVN, but high in the magnocellular division (mPVN). These signals tended to be increased in rats injected with alcohol, but these responses did not reach statistical significance. However, the mRNA encoding VP became significantly more expressed 3 h post-alcohol exposure, suggesting that gene expression had increased. Collectively, these results indicate that alcohol stimulates the activity of neurons in both the parvo- and the magnocellular portions of the PVN, though transcriptional activation of the CRF and VP genes in the PVN exhibit distinct time courses. Whether the influence of the drug is direct or is exerted through afferents to the PVN, and whether increased hypothalamic neuronal activity plays a role in mediating the acute ACTH response to alcohol, are issues that have not yet been adequately resolved.

Acute alcohol administration stimulates the activity of hypothalamic neurons that express corticotropin-releasing factor and vasopressin

Alcohol (EtOH) treatment increases plasma ACTH levels in the rat, a response thought to depend at least partially on endogenous corticotropin-releasing factor (CRF). However, the inability of CRF antibodies to completely abolish EtOH-induced activation of the pituitary has suggested the possible importance of other factors. Because vasopressin (VP) may be acutely released by alcohol in the rat, and because this peptide interacts with CRF to promote ACTH secretion, we investigated its role in the hypothalamic-pituitary (HP) axis' response to one acute injection of EtOH (3 g/kg, i.p.). As expected, blockade of pituitary CRF receptors significantly blunted EtOH-induced ACTH secretion. Administration of a VP antagonist also interfered with this response, though to a lesser extent, while concomitant administration of both CRF and VP antagonists totally abolished it. These results indicate that both CRF and VP participate in the ability of alcohol to stimulate corticotrophs' function. Another hypothesis still awaiting confirmation is whether alcohol stimulates the activity of hypothalamic neurons important for ACTH release, which are primarily found in the paraventricular nucleus (PVN) of the hypothalamus. We therefore determined whether EtOH upregulated transcripts of the immediate early gene (IEG) NGFI-B, as well as those of CRF and VP. In the parvicellular portion of the PVN (pPVN), NGFI-B mRNA and CRF heteronuclear (hn) RNA levels increased within 20 min of EtOH treatment. Despite this robust response, densitometric assessments of CRF mRNA levels failed to indicate a significant elevation over control levels at any of the time points studied, a phenomenon that might be at least in part due to the abundance of these transcripts under basal conditions. Constitutive levels of VP ImRNA levels were very low in the pPVN, but high in the magnocellular division (mPVN). These signals tended to be increased in rats injected with alcohol, but these responses did not reach statistical significance. However, the mRNA encoding VP became significantly more expressed 3 h post-alcohol exposure, suggesting that gene expression had increased. Collectively, these results indicate that alcohol stimulates the activity of neurons in both the parvo- and the magnocellular portions of the PVN, though transcriptional activation of the CRF and VP genes in the PVN exhibit distinct time courses. Whether the influence of the drug is direct or is exerted through afferents to the PVN, and whether increased hypothalamic neuronal activity plays a role in mediating the acute ACTH response to alcohol, are issues that have not yet been adequately resolved.

Corticotropin releasing hormone is involved in exercise-induced elevation in core temperature

To determine the involvement of corticotropin releasing factor (CRF) in exercise-induced elevation in core temperature of female rats, CRF antibody or vehicle was injected intracerebroventricularly (ICV) into rats that had free access to exercise wheels for 6 weeks. On the day of injection, there were no differences in body temperature or activity following these injections. However, exercising animals had a significantly attenuated daytime temperature when compared to vehicle control animals at one day postinjection with the CRF antibody. Although these animals have significantly lower body temperature than the animals that received vehicle, the injection of CRF antibody had no effect on locomotor activity. Therefore, this decrease in temperature is not due to a reduction in activity. These results suggest that CRF is involved in the exercise-induced elevation in daytime body temperature. Since the antibody also attenuates fevers caused by IL-1β (and presumably other cytokines), it is possible that the daytime elevation in body temperature of exercising rats is mediated by IL-1β other cytokines.

The cocaine-induced elevation of plasma corticosterone is mediated by endogenous corticotropin-releasing factor (CRF) in rats

The role of endogenous corticotropin-releasing factor (CRF) in the cocaine-induced corticosterone response was investigated by using the immunoncutralization and receptor blockade of endogenous CRF. Pretreatment with different dilutions (1:5, 1:10 and 1:20, i.c.v.) of CRF antibody and different doses of an antagonist for CRF receptors, α-helical CRF 9–41 (αh-CRF, 0.001–1.0 μg, i.c.v.), dose-dependently prevented the cocaine-induced increase in corticosterone level. These results support the hypothesis that the activation of the hypothalamo-pituitary-adrenal (HPA) axis by cocaine is mediated through the release of endogenous CRF.

Thermogenic actions of tryptophan in the rat are mediated independently of 5-HT

Serotonin (5-HT) has been implicated in the central control of energy balance, via inhibition of food intake and stimulation of thermogenesis. Its rate of synthesis in brain is dependent on the availability of its precursor amino acid, tryptophan. The objective of the present study was therefore to investigate the thermogenic actions of tryptophan and to determine whether these actions are mediated by 5-HT. Central or peripheral injections of 5-HT (i.c.v.; 0.5–40μg), 5-hydroxytryptophan (5-HTP) (i.c.v.; 20μg) or tryptophan (i.p.; 20 mg/kg, i.c.v.; 12–60 μg) significantly increased resting oxygen consumption (uc|dot|V O2 by approximately 15–20%) in conscious rats, without apparent effects on physical activity. Small increases (5–7%) in uc|dot|V O2 were also observed following peripheral injections of aspartate or glycine (20 mg/kg) but not taurine, whilst central injection of tyrosine or leucine (15–18 μg) significantly increased uc|dot|V o2 by 15%. We have previously reported that the thermogenic and anorexic actions of 5-HT are mediated by corticotropin-releasing factor (CRF) 16. In the present study, the thermogenic actions of 5-HTP, like those of 5-HT, were significantly reduced by pretreatment (5 min before) with the CRF antagonist α-helical CRF 9–41 (25 μg, i.c.v.) or a polyclonal antibody to CRF. However, the thermogenic actions of tryptophan were not significantly modified by pretreatment with either the 5-HT antagonist, methylsergide (20 μg, i.c.v.) or with the CRF antagonist or antibody and thus appear to act through different mechanisms to 5-HT. Despite the well-documented effects of tryptophan on serotonergic systems, tryptophan administration (i.c.v.) did not significantly affect the concentration of either 5-HTP, 5-HT or its metabolite 5-HIAA within the cerebrospinal fluid or in the whole hypothalamus, measured 1 h post-injection. Similarly, no changes in the concentrations of other amino acids were observed either in the plasma or whole hypothalamus. In summary, central thermogenic actions of tryptophan are described which appear to be independent of 5-HT.

Mechanisms of early and late hypermetabolism and fever after localized tissue injury in rats

Inflammation and localized tissue injury were induced in rats by intramuscular injection of turpentine. This caused rapid and sustained (2-24 h) increases in colonic temperature (Tc; up by 2.5 degrees C) and oxygen consumption (VO2; up by 30%) as well as hypophagia (57%). The early responses (0-2 h) were attenuated by anesthesia, C-fiber deafferentation, peripheral but not central injection of a cyclooxygenase inhibitor, or central injection of a neutralizing antibody to corticotropin-releasing factor (CRF). In contrast, the later (18-20 h) changes in Tc and VO2 were unaffected by anesthesia, C-fiber deafferentation, or central injection of a CRF antibody but were inhibited by either peripheral or central administration of a cyclooxygenase inhibitor. beta-Adrenoceptor blockade reduced both phases of the response, and brown adipose tissue activity was significantly elevated from 4 to 24 h after turpentine injection. These data suggest that different mechanisms mediate the metabolic response to localized tissue injury, the early phase involving neural mechanisms characteristic of stress and inflammation, whereas the later phase appears to depend on humoral factors that are commonly associated with infection.

Non‐Adrenocorticotropin Mediated Effects of Endogenous Corticotropin‐Releasing Factor on the Adrenocortical Activity in the Rat

Diurnal changes in plasma corticosterone concentrations of rats are supposed to be controlled by low amplitude changes in plasma adrenocorticotropin (ACTH) that run in phase with marked changes in adrenal sensitivity to ACTH (1, 2). These sensitivity changes are probably under neuronal control (3), but the mechanisms remain to be elucidated. By the use of a highly specific monoclonal antibody to rat corticotropin-releasing factor (CRF) (4), we studied the role of endogenous CR- in these processes. Blockade of endogenous CRF prevented the evening rise in plasma ACTH and reduced corticosterone levels to less than 10%. The CRF antibody did not affect morning ACTH concentrations but nevertheless reduced morning corticosterone levels by 40% to 60%. In dexamethasone-treated rats, immunoneutralization of endogenous CRF caused a similar reduction in the plasma corticosterone response to exogenous ACTH. We conclude that endogenous CRF plays a physiological role in controlling the adrenal sensitivity to ACTH.

Receptor-mediated immunomodulation by corticotropin-releasing factor

In both normal and adrenalectomized rats, exogenous Corticotropin-releasing factor (CRF) suppresses immune function, and stress-induced immunosuppression can be partially reversed with either CRF antibody or CRF antagonist, suggesting a role for CRF in immunomodulation. We now report binding of CRF to human monocyte-macrophages and T-helper lymphocytes but not to T-suppresser or B lymphocytes. Bound CRF was displaced by synthetic CRF as well as CRF antagonist. CRF binding at these sites was accompanied by increases in the concentration of cAMP (but not cGMP) in the cells, with minimal and maximal effective CRF doses of 10 −13 and 10 −6 M for the monocyte-macrophage and 10 −12 M and 10 −8 M for the T-helper cell. Production of cAMP in response to CRF was effectively inhibited by CRF antagonist in both cell types. Moreover, rat splenocyte proliferation induced by interleukin 2 (IL-2; 110 IU) was blocked by CRF, half-maximally at a CRF dose of 2.2 × 10 −10 M and completely at 3.5 × 10 −9 M. Finally, when CRF was added together with a 50-fold molar excess of CRF antagonist the IL-2 effect was fully restored. This demonstration of specific, physiologically relevant CRF receptors on two key immunocytes, the monocyte-macrophage and the T-helper lymphocyte, along with in vitro immunosuppression concomitant with CRF binding reinforces the growing body of evidence for a prominent role for CRF in immunomodulation.

Cloning and characterization of the cDNAs for human and rat corticotropin releasing factor-binding proteins

Corticotropin-releasing factor (CRF), is a potent stimulator of synthesis and secretion of preopiomelanocortin-derived peptides. Although CRF concentrations in the human peripheral circulation are normally low, they increase throughout pregnancy and fall rapidly after parturition. Maternal plasma CRF probably originates from the placenta, which responds to the bioactive peptide and produces the peptide and its messenger RNA. Even though CRF concentrations in late gestational maternal plasma are similar to those in rat hypothalamic portal blood and to those that can stimulate release of adrenocorticotropic hormone (ACTH) in vitro, maternal plasma ACTH concentrations increase only slightly with advancing gestation and remain within the normal range. Several groups have now reported the existence of a CRF-binding protein in human plasma which inactivates CRF and which has been proposed to prevent inappropriate pituitary-adrenal stimulation in pregnancy. The binding protein was recently purified from human plasma. We have now isolated and partially sequenced the binding protein, allowing us to clone and characterize its complementary DNA from human liver and rat brain. Expression of the cDNAs for human and rat binding protein in COS7 cells showed that these proteins bind CRF with the same affinity as the native human protein. Both rat and human recombinant binding proteins inhibit CRF binding to a CRF antibody and inhibit CRF-induced ACTH release by pituitary cells in vitro.

Human luteinizing hormone-like material in the neurosecretory system of Periplaneta americana: A histochemical study with monoclonal antibodies

Three different murine monoclonal antibodies to human luteinizing hormone were applied in immunoperoxidase histochemistry on the adult cockroach brain. They all very selectively labelled a constant number of neurosecretory neurone-type cells localized within the pars intercerebralis. Immunoreactive processes of these neurones enter the corpora cardiaca, suggesting that the material might indeed be involved in neurosecretion. Since the very same staining pattern had earlier been obtained by use of rabbit polyclonal human luteinizing hormone antibodies (Verhaert and De Loof, 1986), the present data strongly support the idea that the immunolabelling observed is specific in terms of antigenic similarities between human luteinizing hormone and the insect molecule(s). With two of the antibodies a clear and relatively intense staining reaction of the protocerebral neurones was obtained. Both antibodies bound to precisely identical cells, as could be revealed by double labellings. The third antibody to human luteinizing hormone only very weakly marked these cells. A fourth monoclonal antibody, which was primed against the structurally closely related glycoprotein hormone, human chorionic gonadotropin, but which was found to be fully (100%) cross-reactive to human luteinizing hormone, gave an equally faint immunostaining of the pars intercerebralis neurones and their corpora cardiacal ramifications. Another human chorionic gonadotropin monoclonal antibody, having only a 90% cross-reactivity to human luteinizing hormone, as well as two monoclonal antibodies to human follicle cell stimulating hormone, did not result in a conclusively immunopositive stain of cockroach cerebral structures under the applied conditions. The results are discussed in the light of the characteristics of the antibodies, as determined by Dr K. Siddle (Cambridge U.K.), who actually produced them. They all favour the conclusion that the neurosecretory chemical(s) demonstrated in this study is (are) very similar, but probably not identical, to human luteinizing hormone. Finally, we could demonstrate, by means of double immunostaining experiments, that the histological structures marked with the monoclonal luteinizing hormone antibodies are different and clearly separated from the cells and axonal pathways detected by a polyclonal corticotropin releasing factor antiserum (see Verhaert et al., 1984), although the distribution of the immunoreactivity throughout the cockroach brain is very similar for both the luteinizing hormone and corticotropin releasing factor antibodies.

Effects of intravenous and intraventricular injection of antisera directed against corticotropin-releasing factor on the secretion of anterior pituitary hormones.

To determine the physiological significance of corticotropin-releasing factor (CRF) in the control of pituitary hormone secretion, highly specific antibodies directed against the peptide were injected either intravenously or intraventricularly (third ventricle) and the effect on plasma levels of pituitary hormones was determined before and after application of ether stress for 1 min. The intravenous injection of CRF antiserum (0.5 ml) did not significantly alter basal corticotropin (ACTH) levels in freely moving ovariectomized rats but largely blocked the increase in plasma ACTH resulting from ether stress. These antibodies had no effect on the ether-induced decline in plasma growth hormone (GH), and they failed to modify plasma luteinizing hormone levels. In a second experiment, CRF antiserum (3 microliter) or normal rabbit serum was injected into the third ventricle. A blood sample was drawn 24 hr later and immediately thereafter another injection of CRF antiserum or normal rabbit serum was made. There was no modification in the level of any of the hormones 24 hr after the first injections, and they were similar in CRF antiserum and normal rabbit serum-injected animals. After imposition of ether stress, the response of plasma ACTH was nearly completely blocked by the intraventricular CRF antiserum, but the degree of blockade was slightly less than that obtained by intravenous injection. The decline in plasma GH after ether stress was blocked by the intraventricular CRF antiserum. There was no effect of the intraventricular injection of the antiserum on the levels of the other pituitary hormones. The results with intravenous injection of the antisera indicate that CRF plays an extremely important but probably not completely indispensable role in the release of ACTH after ether stress. The results of the intraventricular injection of the antiserum suggest strongly that endogenous CRF may also modify its own release in response to stress, augmenting it by a positive ultrashort loop feedback, and that the antisera against the peptide blocked this action; however, an action at the pituitary of these intraventricularly injected antibodies cannot be completely ruled out. The blockade of the stress-induced suppression of GH release by the CRF antibodies suggests that CRF released intrahypothalamically during ether stress brings about an alteration in the hypothalamic control of GH secretion such that the stress-induced inhibition of GH release is blocked.

Decreased corticotropin-releasing factor-like immunoreactivity in rat intermediate and posterior pituitary after stalk section.

We determined the corticotropin-releasing factor(CRF)-like immunoreactivity by radioimmunoassay in intermediate and posterior pituitary lobes of stalk-sectioned and sham-operated control rats. The antigenic determinant read by the CRF antibody used was contained within the region of amino acids 26 (Gln) to 37 (Leu) of the molecule. Intermediate and posterior lobes of control rats contained similar amounts of CRF (591 +/- 78 and 487 +/- 34 pg/mg protein, respectively). The section of the pituitary stalk produced a marked decrease in CRF-like immunoreactivity in both structures. The CRF content on the intermediate lobe after stalk section was 62 +/- 17 pg/mg protein, a decrease of 90%, and that of the posterior lobe was 90 +/- 13 pg/mg protein, an 83% decrease. Our results suggest that most of the CRF-like immunoreactivity in the rat intermediate and posterior pituitary lobes is contained in nerve fibers of brain origin and support the hypothesis of a role of CRF in the release of intermediate and posterior lobe peptides.