Stimulation of insulin secretion by corticotropin-releasing factor (CRF) in anesthetized rats

Corticotropin-releasing factor-containing cells have been recently found in the endocrine pancreas of several vertebrate species by immunocytochemistry. In order to clarify the possible physiological significance of these findings, we have studied the effect of the administration of CRF on endocrine pancreatic function. Five minutes, after injection of ovine CRF 1-41 into the jugular vein, a dose-related increase in insulin levels in the hepatic-portal vein of anesthetized rats was found. This dose-dependent insulin increase was delayed to fifteen minutes after CRF injection into rats exposed to greater surgical stress and was partially blunted in adrenalectomized animals. Glucose and glucagon levels were not altered after CRF administration under these conditions. These results suggest that CRF may play a modulatory role in insulin secretion; however, whether CRF acts directly on the beta-cell or through some CRF-stimulated mediator remains to be established.     

Brain corticotropin-releasing factor (CRF) and catecholamine responses in acutely stressed rats

Ether-laparotomy stress produced a rapid increase in rat hypothalamic CRF concentration, followed by a rapid reduction and subsequent increase. Cold-restraint stress significantly reduced hypothalamic CRF concentration at 15 min after stress onset. Serum ACTH and corticosterone levels were significantly elevated at 15 min after the onset of both stresses. The CRF responses in the medulla oblongata were not similar to the hypothalamic CRF responses. Norepinephrine concentration in the hypothalamus was reduced, whereas dopamine concentration in the hypothalamus and medulla oblongata was significantly increased. Epinephrine concentrations in these tissues did not show any significant change throughout the stress period. The observations lead to the following conclusions: hypothalamic CRF plays a major role in stimulating ACTH secretion under acute stress; the reduction in hypothalamic CRF is due to an excess release in the early phase of acute stress; hypothalamic CRF and medulla oblongata CRF are controlled by different mechanisms; norepinephrine in the hypothalamus may not be involved in stimulating hypothalamic CRF secretion in the early phase of acute stress; and catecholamines are regulated differently in the hypothalamus and medulla oblongata.     

Reduction in brain immunoreactive corticotropin-releasing factor (CRF) in spontaneously hypertensive rats

The brain CRF concentration of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) was examined by rat CRF radioimmunoassay. Anti-CRF serum was developed by immunizing rabbits with synthetic rat CRF. Synthetic rat CRF was also used as tracer and standard. The displacement of 125I-rat CRF by serially diluted extracts of male Wistar rats hypothalamus, thalamus, midbrain, pons, medulla oblongata, cerebral cortex, cerebellum and neurointermediate lobe was parallel to the displacement of synthetic rat CRF. In both WKY and SHR the highest levels of CRF immunoreactivity were shown by the hypothalamus and neuro-intermediate lobe, and considerable CRF immunoreactivity was also detected in other brain regions. The CRF immunoreactivity in the hypothalamus, neurointermediate lobe, midbrain, medulla oblongata and cerebral cortex was significantly reduced in SHR and it may suggest that CRF abnormality may be implicated in the reported abnormalities in the pituitary-adrenal axis, autonomic response and behavior of SHR.     

Endocytosis of the vasopressin receptor by anterior pituitary cells is increased by corticotropin-releasing factor (CRF)

Endocytosis of certain receptors such as the transferrin receptor and the EGF-receptor appears to be influenced by second messengers. If second messengers are involved in modulation of endocytosis, not only endocytosis of the stimulated receptor itself but also of receptors for other ligands on the cell surface may be influenced by receptor occupancy. Corticotropin-releasing factor (CRF) and vasopressin act synergistically on secretion of ACTH from the anterior pituitary. The results presented here demonstrate that CRF increases retrieval of the vasopressin receptor in anterior pituitary cells in primary culture without influencing the surface binding of vasopressin. This is not a function of an increased membrane turnover since endocytosis of the transferrin receptor is not influenced by CRF.     

Chronic ethanol exposure potentiates the locomotor-activating effects of corticotropin-releasing factor (CRF) in rats

The effects of chronic exposure (21 days) to ethanol vapors on locomotor response to intracerebroventricular (i.c.v.) administration of corticotropin releasing factor (CRF) was investigated in male Wistar rats. Responses to CRF were tested during chronic exposure, 1 1/2 hours following removal of ethanol vapors, and two weeks after withdrawal of ethanol. A greater sensitivity to the locomotor-activating effects of CRF was found in ethanol-treated rats as compared to their controls during ethanol exposure (P less than 0.001) and 90 min following removal of ethanol vapors (P less than 0.001) but not two weeks following withdrawal. These results support clinical findings of a reversible activation in the hypothalamic-pituitary-adrenal (HPA) axis in alcoholism. In addition, it appears that chronic exposure to ethanol can also modify central neuronal systems specifically responsive to the locomotor activating effects of CRF.     

Development of a selective photoactivatable antagonist for corticotropin-releasing factor receptor, type 2 (CRF2).

A novel photoactivatable analog of antisauvagine-30 (aSvg-30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF2), has been synthesized and characterized. The N-terminal amino-acid d-Phe in aSvg-30 [d-Phe11,His12]Svg(11-40) was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl (ATB) residue. The photoactivatable aSvg-30 analog ATB-[His12]Svg was tested for its ability to displace [125I-Tyr0]oCRF or [125I-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 (rCRF1) or mouse CRF receptor, type 2beta (mCRF2beta). Furthermore, the ability of ATB-[His12]Svg(12-40) to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRF1 (HEK-rCRF1 cells) or mCRF2beta (HEK-mCRF2beta cells) was determined. Unlike astressin and photo astressin, ATB-[His12]Svg(12-40) showed high selective binding to mCRF2beta (Ki = 3.1 +/- 0.2 nm) but not the rCRF1 receptor (Ki = 142.5 +/- 22.3 nm) and decreased Svg-stimulated cAMP activity in mCRF2beta-expressing cells in a similar fashion as aSvg-30. A 66-kDa protein was identified by SDS/PAGE, when the radioactively iodinated analog of ATB-[His12]Svg(12-40) was covalently linked to mCRF2beta receptor. The specificity of the photoactivatable 125I-labeled CRF2beta antagonist was demonstrated with SDS/PAGE by the finding that this analog could be displaced from the receptor by antisauvagine-30, but not other unrelated peptides such as vasoactive intestinal peptide (VIP).     

Synthesis and biological properties of ovine corticotropin-releasing factor (CRF)

The 41-residue sequence of recently identified ovine corticotropin-releasing factor (CRF) was assembled on a benzhydrylamine resin support. Deprotection and cleavage from the resin were accomplished by HF treatment. The crude peptide was purified by gel filtration and reverse-phase, medium pressure, followed by high-performance liquid chromatography (HPLC). In addition to the usual criteria, the homogeneity of the final material, obtained in 7% yield, was assessed by the isolation and examination of cyanogen bromide cleavage and tryptic digestion fragments by HPLC and amino acid analysis. The synthetic 41 amino acid CRF stimulated the release of corticotropin (ACTH) in three in vitro systems: isolated rat pituitary quarters, monolayer cultures of dispersed pituitary cells, and superfused pituitary cells on a column, the responses being related to the log-dose of CRF in the range of 0.05-125 ng/ml. The synthetic peptide also augmented in vivo release of ACTH in rats pretreated with chlorpromazine, morphine, and Nembutal, as assessed by the measurement of serum corticosterone. The data indicates chemical purity and high biological activity of synthetic material.     

Adrenocortical atrophy of hypophysectomized rats can be reduced by corticotropin-releasing hormone (CRH)

Summary The effects of high dose injections of corticotropin-releasing hormone (CRH) on the adrenal cortex of hypophysectomized rats were studied at the light-and electron-microscopical levels. Adrenocortical atrophy induced by hypophysectomy could be reduced by daily i.p. injection of 10 g (3 nmol) CRH given for 3 days starting at day 5 after the operation. The cortex broadened, mostly because of hypertrophy of the zona fasciculata. Blood vessels were enlarged. Although the adrenocortical cells of hypophysectomized rats showed features of a functionally suppressed state, such as tubular mitochondria, the cells of CRH-treated animals showed characteristics of stimulated cells. The inner membrane of the mitochondria formed the typical densely packed vesicles of adrenocortical cells that are active in steroidogenesis. Lipid droplets were found to be reduced, and the cells developed filopodia at their surface. These morphological observations indicate that CRH influences the adrenal cortex via extrapituitary mechanisms.     

The paraventriculo-infundibular corticotropin releasing factor (CRF) pathway as revealed by immunocytochemistry in long-term hypophysectomized or adrenalectomized rats

The immunocytochemical localization of corticotropin releasing factor (CRF)-containing pathways projecting from the paraventricular nucleus (PVN) to the external layer of the median eminence (ME) in long-term hypophysectomized or adrenalectomized rats is described. Immunocytochemistry was followed by silver intensification of the diaminobenzidine end-product. In comparison with untreated control rats, both hypophysectomy and adrenalectomy resulted in a dramatic increase in immunostaining of the CRF-containing perikarya and fibers, particularly those originating from the PVN and terminating in the ME. The staining was more intense in adrenalectomized than in hypophysectomized rats. The CRF-positive fibers emerging from the PVN form a medial, an intermediate and a lateral fiber pathway. The lateral and intermediate CRF tracts leave the dorsolateral part of the PVN and course laterally and medially of the fornix, respectively, then ventrally toward the optic tract. Just dorsal to the optic tract they turn in caudal direction and run parallel with and very close to the basal surface of the hypothalamus; individual fibers then turn medially to terminate in the external layer of the ME. Only a few fibers originate from the medial-ventral part of the PVN (medial pathway). These fibers run in ventral direction along the walls of the 3rd ventricle and terminate in the ME. Thus the majority of CRF fibers, similarly to other peptidergic systems, reach the medial basal hypothalamus from the anterolateral direction.     

Immunocytochemical localization of corticotropin-releasing factor (CRF) in the rat brain

The immunocytochemical localization of neurons containing the 41 amino acid peptide corticotropin-releasing factor (CRF) in the rat brain is described. The detection of CRF-like immunoreactivity in neurons was facilitated by colchicine pretreatment of the rats and by silver intensification of the diaminobenzidine end-product. The presence of immunoreactive CRF in perikarya, neuronal processes, and terminals in all major subdivisions of the rat brain is demonstrated. Aggregates of CRF-immunoreactive perikarya are found in the paraventricular, supraoptic, medial and periventricular preoptic, and premammillary nuclei of the hypothalamus, the bed nuclei of the stria terminalis and of the anterior commissure, the medial septal nucleus, the nucleus accumbens, the central amygdaloid nucleus, the olfactory bulb, the locus ceruleus, the parabrachial nucleus, the superior and inferior colliculus, and the medial vestibular nucleus. A few scattered perikarya with CRF-like immunoreactivity are present along the paraventriculo-infundibular pathway, in the anterior hypothalamus, the cerebral cortex, the hippocampus, and the periaqueductal gray of the mesencephalon and pons. Processes with CRF-like immunoreactivity are present in all of the above areas as well as in the cerebellum. The densest accumulation of CRF-immunoreactive terminals is seen in the external zone of the median eminence, with some immunoreactive CRF also present in the internal zone. The widespread but selective distribution of neurons containing CRF-like immunoreactivity supports the neuroendocrine role of this peptide and suggests that CRF, similarly to other neuropeptides, may also function as a neuromodulator throughout the brain.     

Impact of N-terminal domains for corticotropin-releasing factor (CRF) receptor-ligand interactions

The large extracellular N-terminal domains (NTs) of class B G protein-coupled receptors serve as major ligand binding sites. However, little is known about the ligand requirements for interactions with these receptor domains. Recently, we have shown that the most potent CRF receptor agonist urocortin 1 (Ucn1) has two segregated receptor binding sites Ucn1(1-21) and Ucn1(32-40). For locating the receptor domains interacting with these two sites, we have investigated the binding of appropriate Ucn1 analogues to the receptor N-termini compared to the corresponding full-length receptors. For this purpose receptor NTs of CRF(rat) subtypes 1 and 2(alpha) without their signal sequences were overexpressed in Escherichia coli and folded in vitro. For CRF2(a)-rNT, which bears five cysteine residues (C2-C6), the disulfide arrangement C2-C5 and C4-C6 was found, leaving C3 free. This is consistent with the disulfide pattern of CRF1-rNT, which has six cysteines and in which C1 is paired with C3. Binding studies of N-terminally truncated or C-terminally modified Ucn1 analogues demonstrate that it is the C-terminal part, Ucn1(11-40), that binds to receptor NT, indicating a two-domain binding mechanism for Ucn binding to receptor NT. Since the binding of Ucn1 to the juxtamembrane domain has been shown to be segregated from binding to the receptor N-terminus [Hoare et al. (2004) Biochemistry 43, 3996-4011], a third binding domain should exist, probably comprising residues 8-10 of Ucn, which particularly contribute to a high-affinity binding to full-length receptors but not to receptor NT.     

Discovery of NBI-77860/GSK561679, a potent corticotropin-releasing factor (CRF1) receptor antagonist with improved pharmacokinetic properties

Antagonists of the corticotropin-releasing factor (CRF) neuropeptide may prove effective in treating stress and anxiety related disorders. In an effort to identify antagonists with improved physico-chemical properties a new series of CRF(1) antagonists were designed to substitute the propyl groups at the C7 position of the pyrazolo[1,5-a]pyrimidine core of 1 with heterocycles. Compound (S)-8d was identified as a high affinity ligand with a pK(i) value of 8.2 and a functional CRF(1) antagonist with pIC(50) value of 7.0 in the in vitro CRF ACTH production assay.     

Effect of morphine on hypothalamic corticotropin-releasing factor (CRF) and pituitary-adrenocortical activity

The effects of intraperitoneal and intra-third ventricular administration of morphine on the hypothalamic corticotropin-releasing factor (CRF) and the pituitary-adrenocortical activity were examined in unanesthetized, freely moving rats. Hypothalamic CRF was measured by rat CRF radioimmunoassay. Intraperitoneal or intra-third ventricular administration of morphine increased blood concentrations of ACTH and corticosterone while intraperitoneal administration tended to increase CRF concentration in the whole hypothalamus including the median eminence and intra-third ventricular administration increased CRF concentration in the hypothalamus excluding the median eminence. However, morphine seemed to inhibit the increase in CRF concentration in the hypothalamus induced by the ether-laparotomy stress. The main site of morphine action on the hypothalamo-pituitary-adrenocortical system seemed to be in the hypothalamic area.     

Corticotropin-releasing factor (CRF) stimulates locomotor activity in intact and hypophysectomized newts (Amphibia)

Locomotor activity of rough-skinned newts (Taricha granulosa) was significantly higher in intact and hypophysectomized males injected intracranially with 100 ng CRF (ovine corticotropin-releasing factor) than in those injected with 10 ng CRF or saline. In addition, an injection of corticosterone or dexamethasone failed to stimulate newt locomotor activity. These results provide evidence that CRF can act independently of pituitary hormones to stimulate locomotor activity in a nonmammalian vertebrate.     

Starvation-induced changes in rat brain corticotropin-releasing factor (CRF) and pituitary-adrenocortical response

Starvation-induced changes in CRF concentration in major brain regions and abnormalities in the pituitary-adrenal axis were examined in rats using rat CRF radioimmunoassay. The CRF concentrations in the hypothalamus and cerebellum were significantly reduced in the completely starved rats, while those in the midbrain, thalamus and neurointermediate lobe of the pituitary were significantly increased in the semi-starved or completely starved rats. No significant changes in the CRF concentrations were found in the pons, medulla oblongata and cerebral cortex. In the completely starved rats, the serum ACTH level was significantly reduced, whereas the serum corticosterone level was markedly elevated. These observations suggest that starvation may stimulate the CRF-ACTH-corticosterone system and that not only hypothalamic CRF but also extrahypothalamic CRF may be discretely related to feeding behavior or starvation. The reduced serum ACTH level in starved rats may be ascribed to the negative feedback effect of the elevated serum corticosterone.     

N-methyl-D-aspartate (NMDA)-mediated corticotropin-releasing factor (CRF) release in cultured rat amygdala neurons

Corticotropin-releasing factor (CRF) plays an important role in the activation of centrally mediated responses to stress. The amygdala, a limbic structure involved in the stress response, has a significant number of CRF cell bodies and CRF receptors. Activation of glutamatergic projections to the amygdala has been implicated in the stress response. Few studies have evaluated neurotransmitter-stimulated CRF release in the amygdala. We measured the effects of glutamate (0.1-1000 microM) and N-methyl-D-aspartate (NMDA, 0.1-1000 microM) on CRF release from the amygdala using primary neuronal cultures from embryonic rat brains (E18-19). Experiments were performed after the cultures grew for 17-20 days. CRF was measured using radioimmunoassay. The excitatory amino acid neurotransmitters, glutamate and NMDA, stimulated CRF release in a concentration-dependent manner. The apparent EC50 values for glutamate and NMDA were 17.5 microM and 12 microM, respectively. Consistent with a NMDA receptor-driven event, glutamate-stimulated CRF release was blocked by the NMDA antagonist, 2-amino-5-phosphonovaleric acid (AP-5, 1-100 microM) and antagonized by the addition of 1.2 mM MgCl2 to the incubation medium. These results implicate an inhibition of CRF release in the amygdala as a possible mechanism for the reported anxiolytic effects of NMDA antagonists.     

The effect of corticotropin-releasing factor (CRF) on autonomic and behavioral responses during shock-prod burying test in rats

When administered intracerebroventricularly (ICV) in rats, corticotropin-releasing factor (CRF) possesses arousing and anxiogenic properties, which may be found reflected in autonomic and behavioral activation. As these responses are dependent on dose and situation, ICV-injected CRF may affect behavioral responses to a defined stimulus in a different fashion than autonomic concomitants. Two experiments were conducted in order to test this hypothesis. In both experiments, rats were treated ICV with CRF or an artificial cerebrospinal fluid (aCSF) 5 min prior to a 15-min exposure to an electrified prod (shock-prod burying test, SPB test) in their home cages. In the first experiment, 0.3 ng CRF injected ICV in unhandled rats significantly reduced the prod-burying response to electric shock, in favor of immobility, whereas following 300 ng CRF ICV, the predominant behavioral response was grooming behavior. In contrast, habituated rats, implanted with telemetric devices to measure heart rate, core temperature, and gross activity in the second experiment, showed a significant increase of burying behavior after 0.3 ng CRF ICV, in comparison to vehicle-treated controls. However, simultaneous cardiac acceleration was of the same magnitude and duration in both groups. In addition, whereas similar rises in CT were observed in both groups during the SPB test, CRF-treated rats showed more marked rise in core temperature during the first 15 min of the posttest period. At the 24-h retention test, rats belonging to the CRF group showed burying behavior and HR responses, in onset, magnitude, and duration similar to day 1, whereas extinction of the burying response and tachycardia was found in controls. Changes in CT, although less marked, showed the same pattern as on day 1 in both groups. These results show a differential effect of central CRF on behavioral and autonomic activation induced by a well-defined stressful stimulus. The response to CRF seems to be not only situation related, but also dependent on the pretest experience of the animal.     

Iontophoretic mapping of corticotropin-releasing factor (CRF) sensitive neurons in the rat forebrain

Iontophoretic application of corticotropin-releasing factor (CRF) onto the membrane of individual brain neurons produced changes in the spontaneous occurrence of their extracellular action potentials. Neurons in the cortex and hypothalamus tended to be excited by the application of this 41-residue peptide, while those in the thalamus and lateral septal area were inhibited. In general, neurons excited by CRF were also inhibited by the local application of dopamine (DA) and morphine (MOR), while those which were inhibited by CRF were excited by DA and MOR. Glutamate excited the majority of cells tested independent of the other peptide responses. The results suggest that CRF activates several CNS regions with some specificity, and may be involved in neuronal modulation of pituitary as well as extrapituitary events.     

Modulation of Ca2+ influx by corticotropin-releasing factor (CRF) family of peptides via CRF receptors in rat pancreatic beta-cells

The actions of the corticotropin-releasing factor (CRF) family of peptides are mediated by the seven transmembrane-domain G-protein-coupled receptors, the CRF receptors type 1 (CRF1 receptor) and type 2 (CRF2 receptor). In a previous study, we reported that CRF, an endogenous ligand for CRF1 receptor, modulated Ca2+ influx in rat pancreatic beta-cells. In addition to CRF, other additional members of the family, urocortins, have been identified in mammals. Urocortin 1 (UCN 1), a peptide of the CRF family, binds both CRF1 receptor and CRF2 receptor with equal affinities. Urocortin 3 (UCN 3), a highly selective ligand for CRF2 receptor with little affinity for CRF1 receptor, has been shown in rat pancreatic beta-cells. The present study focused on the effects of the CRF family peptides on intracellular Ca2+ ([Ca2+]i) concentration via CRF receptors in rat pancreatic beta-cells. Microfluorimetric experiments showed that CRF (0.2 nM) and UCN 1 (0.2 nM) elevated [Ca2+]i levels. Both CRF and UCN 1 effects were attenuated by astressin, a non-selective CRF receptor antagonist. Antisauvagine-30, a selective CRF2 receptor antagonist, appeared to enhance the UCN 1 effect on the elevation of [Ca2+]i. The CRF effect on the elevation of [Ca2+]i was inhibited by the addition of UCN 3. Taken together, the activation of CRF2 receptor antagonizes the CRF1 receptor-stimulated Ca2+ influx.     

Influence of corticotropin-releasing factor fragment CRF4-6 on metabolism in rats

Influence of corticotropin-releasing factor fragment CRF4-6 on metabolism in the rat was investigated in this study. Tripeptide (10 mkg/rat) after intracerebroventricular infusion to anesthetized animals increased metabolic rate, not affecting the respiratory ratio, induced hyperglycemia and hyperthermia. Synchronously with that heart rate and arterial pressure did also increase. Changes in energy metabolism induced by CRF4-6 closely resemble the effect of untruncated corticotropin-releasing factor molecule. Results of this study and the range of tripeptide effects described previously evidence that CRF4-6 is a physiologically active regulatory molecule, a derivative of corticotropin-releasing factor, that expands and prolongs effects of the parent molecule.