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.     

Urocortin 1 reduces food intake and ghrelin secretion via CRF(2) receptors

Although it is known that urocortin 1 (UCN) acts on both corticotropin-releasing factor receptors (CRF(1) and CRF(2)), the mechanisms underlying UCN-induced anorexia remain unclear. In contrast, ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, stimulates food intake. In the present study, we examined the effects of CRF(1) and CRF(2) receptor antagonists (CRF(1)a and CRF(2)a) on ghrelin secretion and synthesis, c-fos mRNA expression in the caudal brain stem, and food intake following intracerebroventricular administration of UCN. Eight-week-old, male Sprague-Dawley rats were used after 24-h food deprivation. Acylated and des-acylated ghrelin levels were measured by enzyme-linked immunosorbent assay. The mRNA expressions of preproghrelin and c-fos were measured by real-time RT-PCR. The present study provided the following important insights into the mechanisms underlying the anorectic effects of UCN: 1) UCN increased acylated and des-acylated ghrelin levels in the gastric body and decreased their levels in the plasma; 2) UCN decreased preproghrelin mRNA levels in the gastric body; 3) UCN-induced reduction of plasma ghrelin and food intake were restored by CRF(2)a but not CRF(1)a; 4) UCN-induced increase of c-fos mRNA levels in the caudal brain stem containing the nucleus of the solitary tract (NTS) was inhibited by CRF(2)a; and 5) UCN-induced reduction of food intake was restored by exogenous ghrelin and rikkunshito, an endogenous ghrelin secretion regulator. Thus, UCN increases neuronal activation in the caudal brain stem containing NTS via CRF(2) receptors, which may be related to UCN-induced inhibition of both ghrelin secretion and food intake.     

Effect of protein synthesis inhibition on brain corticotropin-releasing factor and plasma adrenocorticotropin

The effect of inhibiting protein synthesis on concentrations of corticotropin-releasing factor (CRF) in rat brain and plasma adrenocorticotropin (ACTH) was assessed following the administration of the general protein synthesis inhibitor anisomycin. Compared with vehicle-injected controls, protein synthesis inhibition resulted in significantly reduced CRF immunoreactivity (CRF-ir) in median eminence within 1 h (p less than 0.01), remained decreased after 4 h (p less than 0.025), and was nonsignificantly decreased after 24 h. Plasma ACTH levels were greatly increased within 1 h posttreatment (p less than 0.0005), continued elevated after 4 h (p less than 0.01), and returned to normal levels after 24 h. CRF-ir measured in other brain areas 24 h after anisomycin showed decreased levels in medulla-pons (p less than 0.025) and neurointermediate lobe of pituitary (p less than 0.05), with no change noted in frontal cortex, hippocampus, midbrain-thalamus, or cerebellum. Overall these data show that blockade of normal protein synthesis with anisomycin can elicit changes in CRF-ir and ACTH content.     

Heparin-binding angiogenesis factors: detection by immunological methods

Immunological methods for the detection of basic fibroblast growth (FGF) are described. Polyclonal antibodies directed against synthetic peptides representing amino-, internal, and carboxy-terminal regions of basic FGF, were raised in rabbits. Five techniques, enzyme-linked immunosorbent assay, immunoblot, electrophoretic transfer (Western) blot, immunoprecipitation and radioimmunoassay, were used to detect basic FGF. These techniques were used to demonstrate that a human hepatoma cell line synthesizes a growth factor structurally related to brain and pituitary basic FGF.     

Involvement of endogenous CRF in carbon tetrachloride-induced acute liver injury in rats

Central neuropeptides play important roles in many physiological and pathophysiological regulation mediated through the autonomic nervous system. In regard to the hepatobiliary system, several neuropeptides act in the brain to regulate bile secretion, hepatic blood flow, and hepatic proliferation. Central injection of corticotropin-releasing factor (CRF) aggravates carbon tetrachloride (CCl4)-induced acute liver injury through the sympathetic nervous pathway in rats. However, still nothing is known about a role of endogenous neuropeptides in the brain in hepatic pathophysiological regulations. Involvement of endogenous CRF in the brain in CCl4-induced acute liver injury was investigated by centrally injecting a CRF receptor antagonist in rats. Male fasted Wistar rats were injected with CRF receptor antagonist alpha-helical CRF-(9-41) (0.125-5 microg) intracisternally just before and 6 h after CCl4 (2 ml/kg) administration, and blood samples were obtained before and 24 h after CCl4 injection for measurement of hepatic enzymes. The liver sample was removed 24 h after CCl4 injection, and histological changes were examined. Intracisternal alpha-helical CRF-(9-41) dose dependently (0.25-2 microg) reduced the elevation of alanine aminotransferase and aspartate aminotransferase levels induced by CCl4. Intracisternal alpha-helical CRF-(9-41) reduced CCl4-induced liver histological changes, such as centrilobular necrosis. The effect of central CRF receptor antagonist on CCl4-induced liver injury was abolished by sympathectomy and 6-hydroxydopamine pretreatment but not by hepatic branch vagotomy or atropine pretreatment. These findings suggest the regulatory role of endogenous CRF in the brain in experimental liver injury in rats.     

Antibody changes in paragonimiasis patients after praziquantel treatment as observed by ELISA and immunoblot

To observe antibody changes after praziquantel treatment in paragonimiasis, a total of 46 serum samples from 13 serologically diagnosed patients was collected for 4-28 months. The specific antibody (IgG) levels were measured by enzyme-linked immunosorbent assay (ELISA). All but one patient who needed retreatment became symptom-free within a week. Antibody levels were dropped near to or below a cut-off absorbance (abs.) of 0.25 in varying intervals from 4 to 18 months. Of 9 patients who were retested within 3 months, 5 revealed temporary elevation of antibody level. After the elevation, the levels began to decline slowly to negative ranges. If treated earlier after symptoms developed, the temporary elevation did not occur and intervals to negative conversion were shorter. By sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)/immunoblot, antigen-antibody reactions in individual patient faded gradually without significant changes in reacting antigen bands.     

In vitro study on proopiomelanocortin messenger RNA levels in cultured rat anterior pituitary cells

The fundamental examination on the measurement of proopiomelanocortin (POMC) mRNA levels in cultured rat anterior pituitary (AP) cells was studied. In addition, the detailed study on time- and dose-related effects of corticotropin-releasing factor (CRF) and dexamethasone on the level of POMC mRNA in AP cells in vitro was examined. Basal levels of POMC mRNA in AP cells cultured with serum initially declined after 1-day culture, gradually increased and reached a peak after 3-day culture, and then slightly decreased after 4- and 5-day culture. These mRNA levels after 3-day culture did not change through subsequent 15-hr incubation without serum. CRF treatment caused a time- and dose-dependent increase in POMC mRNA levels. The minimum effective dose of CRF was 0.1 nM for 15-hr incubation. The significant increase in POMC mRNA levels was observed after 3 hrs of 1 nM CRF treatment with a 2-fold elevation seen after 15 hrs of exposure. Dexamethasone treatment caused a dose-dependent decrease in POMC mRNA levels in AP cells. The minimum effective dose was 0.1 microgram/ml and such mRNA levels did not decrease until 15 hrs of exposure.     

Immunoblot assay: a rapid and sensitive method for identification of salmonid fish viruses

An immunoblot assay was used to identify the viruses of infectious pancreatic necrosis, infectious hematopoietic necrosis, and viral hemorrhagic septicemia. Viral antigen in infected cell culture supernatant was adsorbed onto nitrocellulose membrane or Whatman 541 filter paper and detected by enzyme-linked immunosorbent assay techniques. The immunoblot assay took less than 4 hr to perform and required no special instrumentation. Assays using cell culture supernatant fluids showed immunoblot sensitivity was 10(5) - 10(6) PFU/ml. Assay sensitivity, determined using purified virus, is 0.85-4.0 ng of viral antigen. The immunoblot assay was used to detect and identify virus in cell culture fluids.     

促肾上腺皮质激素释放激素及去甲肾上腺素对高原鼠兔体液免疫的抑制作用

采用第三脑室注入CRF 及N E 的方法观察对高原鼠兔(Ochotona curzoniae) 体液免疫的影响。结果表明: 第三脑室注入CRF 1 Lg 可抑制抗体生成, 比对照下降29.2%(P<0.01) , 而在第三脑室注入CRF 受体阻断剂α-helical CRF2 (9-41) 50 Lg 后再注入CRF 1 Lg 则可取消CRF 对抗体生成的抑制作用; 第三脑室注入5 nM NE, 与对照相比, 抗体水平下降38.85%(P < 0.01) , 而使用62OHDA 损毁脑内交感神经系统则使抗体水平升高24.31% (P <0.01)。这些结果表明, 高原鼠兔中枢CRF 升高对体液免疫有抑制作用, 中枢交感神经系统对体液免疫也具有紧张性抑制作用。     

Circulating human corticotropin-releasing factor-binding protein levels following cortisol infusions.

Corticotropin-releasing factor-binding protein (CRF-BP) is a 37 kDa protein present in the brain and plasma and is known to regulate the actions of CRF. It has been demonstrated that CRF-BP in the brain and the pituitary appears to be positively regulated by glucocorticoids. In this study, the effect of various doses of hydrocortisone infusions on plasma CRF-BP levels was assessed. Four groups of 10 age-matched males received a 100 min infusion of either placebo (saline), 40 microg/kg/h, 300 microg/kg/h or 600 microg/kg/h hydrocortisone. CRF-BP levels were measured via a LIRMA. In addition, levels of plasma ACTH and cortisol were measured by standard radioimmunoassay. As expected, plasma cortisol levels increased and plasma ACTH levels were suppressed following the infusion. When expressed as proportion of pre-infusion baseine, no significant changes in plasma CRF-BP levels were observed following the infusion for all hydrocortisone groups relative to the control group. However, a significant time-averaged positive correlation was found between CRF-BP and cortisol levels at low to moderate, but not high, cortisol levels. The data obtained in this study indicate that CRF binding protein levels within the time course examined may slightly appear to be affected in the peripheral circulation in response to pronounced, sustained hypercortisolemia.     

Social Opportunity Rapidly Regulates Expression of CRF and CRF Receptors in the Brain during Social Ascent of a Teleost Fish,Astatotilapia burtoni

In social animals, hierarchical rank governs food availability, territorial rights and breeding access. Rank order can change rapidly and typically depends on dynamic aggressive interactions. Since the neuromodulator corticotrophin releasing factor (CRF) integrates internal and external cues to regulate the hypothalamic-pituitary adrenal (HPA) axis, we analyzed the CRF system during social encounters related to status. We used a particularly suitable animal model, African cichlid fish, Astatotilapia burtoni, whose social status regulates reproduction. When presented with an opportunity to rise in rank, subordinate A. burtoni males rapidly change coloration, behavior, and their physiology to support a new role as dominant, reproductively active fish. Although changes in gonadotropin-releasing hormone (GnRH1), the key reproductive molecular actor, have been analyzed during social ascent, little is known about the roles of CRF and the HPA axis during transitions. Experimentally enabling males to ascend in social rank, we measured changes in plasma cortisol and the CRF system in specific brain regions 15 minutes after onset of social ascent. Plasma cortisol levels in ascending fish were lower than subordinate conspecifics, but similar to levels in dominant animals. In the preoptic area (POA), where GnRH1 cells are located, and in the pituitary gland, CRF and CRF₁ receptor mRNA levels are rapidly down regulated in ascending males compared to subordinates. In the Vc/Vl, a forebrain region where CRF cell bodies are located, mRNA coding for both CRFR₁ and CRFR₂ receptors is lower in ascending fish compared to stable subordinate conspecifics. The rapid time course of these changes (within minutes) suggests that the CRF system is involved in the physiological changes associated with shifts in social status. Since CRF typically has inhibitory effects on the neuroendocrine reproductive axis in vertebrates, this attenuation of CRF activity may allow rapid activation of the reproductive axis and facilitate the transition to dominance.     

Comparison of the biologic actions of corticotropin-releasing factor and sauvagine

Corticotropin-releasing factor (CRF), a peptide isolated from ovine hypothalamus, and sauvagine, a peptide isolated from frog skin, share significant structural homology and elicit a number of similar biological responses. CRF is more potent than sauvagine in stimulating pituitary ACTH secretion. Sauvagine, however, is 5–10 times more potent than CRF to act within the brain to increase plasma levels of catecholamines and glucose and to elevate mean arterial pressure. Sauvagine is likewise more potent than CRF to act outside the brain to increase superior mesenteric artery flow and plasma glucose concentrations and to decrease mean arterial pressure. CRF and sauvagine produce important effects representative of biologically active peptides.     

Interaction of arginine vasopressin and corticotropin releasing factor demonstrated with an improved bioassay

We developed an improved in vivo bioassay for corticotropin releasing factor (CRF) by modifying the injection schedule in the standard chlorpromazine-morphine-pentobarbital assay procedure. A combined injection of chlorpromazine and morphine followed 75 min later by injection of pentobarbital produced low basal levels of corticosterone and rendered the animals highly sensitive to synthetic CRF but insensitive to the stress of ether or histamine. The lowest dose of CRF that significantly elevated plasma corticosterone levels was 0.01 micrograms/kg. Using this assay, we studied CRF-arginine vasopressin (AVP) interactions at doses that were expected to raise systemic peptide concentrations to levels measured in hypophysial portal blood. The threshold for a significant corticosterone response was found to be at least 250-fold lower for CRF-41 than for AVP. The order in which CRF and AVP are injected was also found to be important, potentiation being greater if CRF was given first. In addition, rats deprived of water for 24 hr were more sensitive to CRF than normally hydrated animals.     

Mosquitofish (Gambusia affinis) vitellogenin: identification, purification, and immunoassay

Vitellogenin is a phospholipoglycoprotein precursor of egg yolk. In mature female fish, vitellogenin is synthesized and secreted by the liver in response to circulating estrogens. Vitellogenin is normally undetectable in the blood of male fish, but can be induced by exposure to compounds possessing estrogenic activity. Thus, the presence of vitellogenin in blood of male fish can serve as a useful biomarker for assessing previous exposure to estrogenic compounds. In the present study, we report identification and purification of vitellogenin in the mosquitofish (Gambusia affinis). Anti-vitellogenin immune serum was generated and used to develop an immunoblot assay for detection of vitellogenin. A combination of immunoblotting and densitometric scanning was used to assess the time- and dose-dependent effects of 17alpha-ethynylestradiol on vitellogenesis in male G. affinis. The results indicate that changes in the level of vitellogenin in mosquitofish blood can be reliably detected by the immunoblot assay, and that the mosquitofish may be a useful bioindicator organism for detecting estrogenic contamination of the aquatic environment.     

CRF receptors in the nucleus accumbens modulate partner preference in prairie voles

Recent evidence suggests a role for corticotropin-releasing factor (CRF) in the regulation of pair bonding in prairie voles. We have previously shown that monogamous and non-monogamous vole species have dramatically different distributions of CRF receptor type 1 (CRF(1)) and CRF receptor type 2 (CRF(2)) in the brain and that CRF(1) and CRF(2) receptor densities in the nucleus accumbens (NAcc) are correlated with social organization. Monogamous prairie and pine voles have significantly lower levels of CRF receptor type 1 (CRF(1)), and significantly higher levels of type 2 (CRF(2)) binding, in NAcc than non-monogamous meadow and montane voles. Here, we report that microinjections of CRF directly into the NAcc accelerate partner preference formation in male prairie voles. Control injections of CSF into NAcc, and CRF into caudate-putamen, did not facilitate partner preference. Likewise, CRF injections into NAcc of non-monogamous meadow voles also did not facilitate partner preference. In prairie voles, this CRF facilitation effect was blocked by co-injection of either CRF(1) or CRF(2) receptor antagonists into NAcc. Immunocytochemical staining for CRF and Urocortin-1 (Ucn-1), two endogenous ligands for CRF(1) and CRF(2) receptors in the brain, revealed that CRF, but not Ucn-1, immunoreactive fibers were present in NAcc. This supports the hypothesis that local CRF release into NAcc could activate CRF(1) or CRF(2) receptors in the region. Taken together, our results reveal a novel role for accumbal CRF systems in social behavior.     

Similar Time Course Changes in Striatal Levels of Glutamic Acid Decarboxylase and Proenkephalin mRNA Following Dopaminergic Deafferentation in the Rat

An immunoblot procedure was developed to quantify the amount of tyrosine hydroxylase protein in homogenate of small brain regions. With the use of this method we have studied the variations in tyrosine hydroxylase activity and protein levels in some catecholaminergic neurons at different times following a single reserpine injection (10 mg/kg s.c.) and reevaluated the anatomical specificity of tyrosine hydroxylase induction by this drug. Reserpine administration provoked a long-lasting increase in both tyrosine hydroxylase activity and protein levels within locus ceruleus neurons. This effect culminated at day 4 after injection. At this time, the enzyme activity and protein levels in treated animals were respectively 2.7 and 2.6 times that measured in vehicle-treated animals. Both parameters varied in parallel so that tyrosine hydroxylase specific activity did not change over time. In contrast, reserpine did not cause any changes in tyrosine hydroxylase activity in the dopaminergic neurons of the substantia nigra, but provoked a moderate increase in tyrosine hydroxylase protein level. This latter effect was maximal (1.5 times) 4 days after treatment. In the adjacent dopaminergic area, i.e., the ventral tegmental area, a small decrease in the enzyme activity was recorded at day 2 without any significant change in the level of the protein. In conclusion, first, our data show the capacity of our method to assay tyrosine hydroxylase protein amounts in small brain catecholaminergic nuclei. Second, our results confirm and extend previous studies on the effect of reserpine on the regulation of tyrosine hydroxylase level within brain noradrenergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracerebroventricular injections of noradrenaline affect brain energy metabolism of rainbow trout

To assess the role of noradrenaline (NA) as a possible regulator of brain energy metabolism in teleost fish, the impact of increased noradrenaline levels within the brain on several parameters of energy metabolism was assessed in rainbow trout brain. Accordingly, two different doses of noradrenaline, producing increases in brain NA levels comparable to those occurring in several physiological processes in nature, were selected. In a subsequent set of three different experiments, fish were intracerebroventricularly injected with 1 microL 100 g(-1) body weight of Cortland saline alone (control) or containing NA (5 nmol NA and 10 nmol NA); after 30 min, brain and plasma samples were taken to assess changes in parameters of energy metabolism due to NA treatment. The results obtained clearly show dose-dependent changes in NA-treated fish in several parameters, including decreased glycogen and ATP levels, increased lactate and pyruvate levels, decreased fructose 1,6-bisphosphatase activity, and increased pyruvate kinase and lactate dehydrogenase activities. Altogether, the present experiments show for the first time in a teleost fish evidence supporting that increased noradrenaline levels in the brain elicit metabolic changes in the brain (enhanced glycogenolysis and glycolysis), resulting in an increased energy demand. These metabolic changes may be related to those occurring under several physiological conditions in nature such as hypoxia, in which increased energy demand and increased noradrenaline levels occur in the brain simultaneously.     

Corticotropin-releasing factor receptors and actions in rat Leydig cells

Rat Leydig cells possess functional high affinity receptors for corticotropin-releasing factor (CRF). CRF inhibited human chorionic gonadotropin (hCG)-induced androgen production in cultured fetal and adult Leydig cells in a dose-dependent manner, but it had no effect on basal testosterone secretion. Comparable inhibitory effects of CRF were observed in the presence or absence of 3-isobutyl-1-methylxanthine. CRF treatment caused a marked reduction of steroid precursors of the androgen pathway (from pregnenolone to testosterone) during gonadotropin stimulation, but it did not influence their basal levels. The inhibitory action of CRF on hCG-induced steroidogenesis was fully reversed by 8-bromo-cAMP but was not affected by pertussis toxin. The action of CRF was rapid; and it was blocked by coincubation with anti-CRF antibody. CRF caused no changes in hCG binding to Leydig cells, and in contrast to other target tissues, CRF did not stimulate cAMP production, indicating that CRF receptors are not coupled to Gs in Leydig cells. These studies have demonstrated that CRF-induced inhibition of the acute steroidogenic action of hCG is exerted at sites related to receptor/cyclase coupling or cAMP formation. The inhibitory effects of CRF in the Leydig cell do not occur through the Gi unit of adenylate cyclase, but could involve pertussis toxin-insensitive G protein(s). These observations demonstrate that CRF has a novel and potent antireproductive effect at the testicular level. Since CRF is synthesized in the testis and is present in Leydig cells, it is likely that locally produced CRF could exert negative autocrine modulation on the stimulatory action of luteinizing hormone on Leydig cell function.     

ACTH secretion in mouse pituitary tumor cells in culture: Inhibition of CRF-stimulated hormone release by somatostatin

Synthetic corticotropin-releasing factor (CRF) stimulates ACTH secretion in the clonal mouse pituitary cell strain AtT20/D16v (D16) in a dose-dependent manner with a half-maximal effect at 2×10^?9M. A single dose of 5×10^?9M CRF maximally stimulates the rate of ACTH secretion during the initial two hrs of treatment. During the period of maximal CRF stimulation intracellular hormone concentration declines progressively to a nadir at 4 hrs. During the ensuing 24 hrs of incubation intracellular hormone levels in CRF-stimulated cells increase gradually toward control values. Somatostatin (SRIF) inhibits the secretory response to CRF. This action of SRIF is dose-dependent with a half-maximal effect at 1×10^?9M and results in decreased maximal ACTH secretion with little effect on the ED₅₀ for CRF.     

Corticotropin-releasing factor (CRF)-induced behaviors are modulated by intravenous administration of teneurin C-terminal associated peptide-1 (TCAP-1)

The teneurin C-terminal associated peptides (TCAP) are a recently discovered family of bioactive peptides that can attenuate aspects of the behavioral stress responses of rats. Because TCAP has some structural similarity to the corticotropin-releasing factor (CRF) family of peptides, and modulates elements of the stress response, TCAP may act to modulate CRF actions in vivo. This hypothesis was tested by investigating anxiety-related behaviors in male rats following repeated intravenous (IV) TCAP-1 administration with either an acute intracerebroventricular (ICV) or IV CRF challenge. TCAP-1 alone did not affect behavioral responses significantly, however did significantly affect CRF-regulated behaviors depending on CRF's mode of injection. In both the elevated plus-maze and the open field tests, TCAP-1 had an anxiolytic effect on ICV CRF responses as indicated by decreased stretched-attend postures in the elevated plus maze (p<0.05), and increased center time and center entries in the open field (p<0.05). However, prior TCAP-1 treatment has an anxiogenic effect on the IV CRF-induced behaviors (decreased center entries and total distance in the open field (p<0.05)). TCAP-1's actions are not mediated through acute changes in glucocorticoid levels and may occur via a central action in the brain. A fluorescently (FITC)-labeled TCAP-1 analog was IV-administered to investigate whether IV TCAP-1 has the potential to regulate central mechanisms by crossing the blood-brain barrier. FITC-TCAP-1 was detected in blood vessels and fibers in the brain indicating that uptake into the brain is a possible route for its interaction with CRF and its receptors. Thus, TCAP may modulate CRF-associated behaviors by a direct action in the CNS.