Activation of the nociceptin/orphanin FQ system is unable to reverse CRF2 receptor mediated anorexia in the rat

Central injection of Nociceptin/Orphanin FQ (N/OFQ), inhibits the anorectic effect of corticotropin-relasing factor (CRF) and stress in rats. Recently, Urocortin II (Ucn II) and Urocortin III (Ucn III), two selective CRF₂ receptor agonists, have been identified. Here, we investigated the effect of intracerebroventricular (ICV) injection of 0.25, 0.75, 1.50 or 3 nmol/rat of Ucn II or Ucn III on food and water intake in food deprived rats. The effect of N/OFQ on Ucn II and UCNIII-induced anorexia was also studied. Results showed a greater inhibition of food consumption by Ucn II than Ucn III. Pretreatment with N/OFQ (0.25–2.0 nmol/rat) did not block the effects of Ucn II and UCNIII. Conversely, injection of N/OFQ (0.25–2.0 nmol/rat) blocked the anorectic effect of CRF (0.1 nmol/rat). These findings suggest that N/OFQ selectively prevent the anorectic effect mediated by activation of the CRF₁ receptor system.     

Differential regulation of gonadotropin-releasing hormone by corticotropin-releasing factor family peptides in hypothalamic N39 cells

Corticotropin-releasing factor (CRF) is involved in a variety of physiological functions including regulation of hypothalamo-pituitary-adrenal axis activity during stressful periods. Urocortins (Ucns) are known to be members of the CRF family peptides. CRF has a high affinity for CRF receptor type 1 (CRF(1) receptor). Both Ucn2 and Ucn3 have very high affinity for CRF receptor type 2 (CRF(2) receptor) with little or no binding affinity for the CRF(1) receptor. Gonadotropin-releasing hormone (GnRH) is known to be involved in the regulation of the stress response. Gonadotropin-inhibitory hormone (GnIH) neurons interact directly with GnRH neurons, and the action of GnIH is mediated by a novel G-protein coupled receptor, Gpr147. This study aimed to explore the possible function of CRF family peptides and the regulation of GnRH mRNA in hypothalamic GnRH cells. Both mRNA and protein expression of the CRF(1) receptor and CRF(2) receptor were found in hypothalamic GnRH N39 cells. CRF suppressed GnRH mRNA levels via the CRF(1) receptor, while Ucn2 increased the levels via the CRF(2) receptor. Both CRF and Ucn2 increased Gpr147 mRNA levels. The results indicate that CRF and Ucn2 can modulate GnRH mRNA levels via each specific CRF receptor subtype. Finally, CRF suppressed GnRH protein levels, while Ucn2 increased the levels. Differential regulation of GnRH by CRF family peptides may contribute to the stress response and homeostasis in GnRH cells.     

Modulation of T-type Ca2+ channels by corticotropin-releasing factor through protein kinase C pathway in MN9D dopaminergic cells

Corticotrophin-releasing factor (CRF) is the main regulator of the body's stress axis and its signal is translated through G-protein-coupled CRF receptors (CRF-R1, CRF-R2). Even though CRF receptors are present in the midbrain dopamine neurons, the cellular mechanism of CRF action is not clear yet. Since voltage-dependent Ca(2+) channels are highly expressed and important in dopamine neuronal functions, we tested the effect of CRF on voltage-dependent Ca(2+) channels in MN9D cells, a model of dopamine neurons. The application of CRF-related peptide, urocortin 1, reversibly inhibited T-type Ca(2+) currents, which was a major Ca(2+) channel in the cells. The effect of urocortin was abolished by specific CRF-R1 antagonist and was mimicked by protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate. PKC inhibitors abolished the effect of urocortin. These results suggest that urocortin modulates T-type Ca(2+) channel by interacting with CRF-R1 via the activation of PKC signal pathway in MN9D cells.     

COX-2 as a novel target of CRF family peptides’ participating in inflammation

In mammals, corticotropin-releasing factor (CRF) family peptides include CRF, Urocortin (Ucn) 1, Ucn2, and Ucn3. In contrast to their systemic indirect immunosuppressive effects on the hypothalamic-pituitary adrenal axis, CRF family peptides act as locally expressed autocrine or paracrine pro-inflammatory factors in a series of inflammatory diseases. Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in metabolism of arachidonic acid, has been abundantly reported to take part in inflammatory diseases. Recently, reports indicate that CRF family peptides may play an important role in the regulation of COX-2 under inflammatory conditions. Moreover, CRF receptors are involved in this process. This review aims to highlight the current novel findings on regulation of COX-2 by CRF family peptides in inflammation. Furthermore, the relevant mechanisms are discussed.     

Cardioprotective action of urocortin in postconditioning involves recovery of intracellular calcium handling

Ischemia/reperfusion (I/R) damage in the heart occurs mainly during the first minutes of reperfusion. Urocortin (Ucn) is a member of the corticotrophin-releasing factor that has been identified as a potent endogenous cardioprotector peptide when used in pre- and postconditioning protocols. However, the underlying mechanisms are not completely elucidated. Here, we focused on intracellular calcium ([Ca²⁺]_i) handling by Ucn when applied in early reperfusion. We used Langendorff-perfused rat hearts to determine hemodynamic parameters, and confocal microscopy to study global [Ca²⁺]_i transients evoked by electrical stimulation in isolated cardiomyocytes loaded with fluorescence Ca²⁺ dye fluo-3AM. We found that the acute application of Ucn at the onset of reperfusion, in isolated hearts submitted to ischemia, fully recovered the hearts contractility and relaxation. In isolated cardiac myocytes, following ischemia we observed that the diastolic [Ca²⁺]_i was increased, the systolic [Ca²⁺]_i transients amplitude were depressed and sarcoplasmic reticulum (SR) Ca²⁺ load was reduced. These effects were correlated to a decrease in the Na⁺/Ca²⁺ exchanger (NCX) activity. Importantly, Ucn applied at reperfusion produced a complete recovery in diastolic [Ca²⁺]_i and global [Ca²⁺]_i transient amplitude, which were due to NCX activity improvement. In conclusion, we demonstrated that [Ca²⁺]_i handling play an essential role in postconditioning action of Ucn.     

Urocortin – From Parkinson's disease to the skeleton

Urocortin (Ucn 1), a 40 amino acid long peptide related to corticotropin releasing factor (CRF) was discovered 19 years ago, based on its sequence homology to the parent molecule. Its existence was inferred in the CNS because of anatomical and pharmacological discrepancies between CRF and its two receptor subtypes. Although originally found in the brain, where it has opposing actions to CRF and therefore confers stress-coping mechanisms, Ucn 1 has subsequently been found throughout the periphery including heart, lung, skin, and immune cells. It is now well established that this small peptide is involved in a multitude of physiological and pathophysiological processes, due to its receptor subtype distribution and promiscuity in second messenger signalling pathways. As a result of extensive studies in this field, there are now well over one thousand peer reviewed publications involving Ucn 1. In this review, we intend to highlight some of the less well known actions of Ucn 1 and in particular its role in neuronal cell protection and maintenance of the skeletal system, both by conventional methods of reviewing the literature and using bioinformatics, to highlight further associations between Ucn 1 and disease conditions. Understanding how Ucn 1 works in these tissues, will help to unravel its role in normal and pathophysiological processes. This would ultimately allow the generation of putative medical interventions for the alleviation of important diseases such as Parkinson's disease, arthritis, and osteoporosis.     

Corticotropin-Releasing Factor (CRF), CRF-Binding Protein (CRF-BP), and CRF/CRF-BP Complex in Alzheimer's Disease and Control Postmortem Human Brain

Abstract: In Alzheimer's disease (AD) there are dramatic reductions in human corticotropin-releasing factor (hCRF) concentration and reciprocal increases in CRF receptor density in the cortex. hCRF-binding protein (hCRF-BP), hCRF/hCRF-BP complex, and "free" hCRF were measured in 10 brain regions from control and AD postmortem human tissue. In the control brains hCRF-BP was heterogenously distributed and levels were at least 10-fold higher on a molar basis than total hCRF levels, suggesting that one major role of the binding protein is to limit the actions of hCRF at the hCRF receptors. Concordant with this hypothesis, the percentage of total hCRF that was in the bound inactive form ranged from 65 to 90% in most areas examined, with the exception of the caudate and globus pallidus where only 15 and 40% were complexed, respectively. hCRF-BP concentrations were similar in the control and AD groups except for Brodmann area (BA) 39 where there was a small but significant decrease in the AD group. Complexed hCRF levels were significantly decreased in BA 8/BA 9, BA 22, BA 39, nucleus basalis, and globus pallidus in the Alzheimer's group and free hCRF levels were significantly decreased only in three brain areas, BA 4, BA 39, and caudate; substantial (40%) but nonsignificant decreases were also noted in BA 8/BA 9 and BA 22. These data demonstrate that (1) a large proportion of the total hCRF in human brain is complexed to hCRF-BP and thus unavailable for hCRF receptor activation, (2) reductions in total hCRF alone do not necessarily predict reductions in bioactive free hCRF, and (3) total hCRF levels and hCRF-BP levels appear to be the main factors determining the quantity of bound and free hCRF in human brain.     

Discovery of Substituted Human Urocortin 1 Analogs with Improved CRF2 Receptor Selectivity and Increased Efficacy in Preventing Skeletal Muscle Atrophy

We have identified specific amino acid modifications of human Urocortin 1 (hUCN1) that lead to highly potent and selective Corticotropin Releasing Factor Receptor 2 (CRF2R) agonists that are efficacious in preventing skeletal muscle atrophy in animal models. We have demonstrated that the CRF2R versus CRF1R selectivity can be increased by modifying the 40 amino acid hUCN1 at amino acid positions 11, 12, 13, 35 and 39. Further improvement in drug properties, including reduced binding to the CRF binding protein, improved solubility, and improved in vivo potency, were achieved by modifying amino acids at positions 22, 23, 36, and 40. In vivo investigations of selected optimized hUCN1 analogs demonstrated significant anti-atrophy efficacy in a mouse casting model of hind leg muscle disuse atrophy.     

Chronic disruption of body weight but not of stress peptides or receptors in rats exposed to repeated restraint stress

Rats exposed to restraint stress for 3 h on each of 3 days lose weight and do not return to the weight of their non-stressed controls for extended periods of time. Studies described here demonstrate that the initial weight loss is associated with increased energy expenditure and reduced food intake on the days of restraint but that there is no difference between stressed and control rats once stress ends. The failure to compensate for this energy deficit accounts for the sustained reduction in weight which lasts for up to 80 days after the end of restraint. In an additional experiment, in situ hybridization was used to measure mRNA expression of corticotrophin releasing factor (CRF) and CRF receptors in hypothalamic nuclei, of urocortin (UCN) in the Edinger Westphal nucleus and of UCN III in the rostral perifornical area and medial amygdaloidal nucleus. Immediately after the second 3 h bout of restraint stress, there was a significant increase in expression of UCN in the Edinger Westphal nucleus and of CRF-R1 in the paraventricular nucleus of the hypothalamus and a less pronounced decrease in CRF-R2 expression in the ventromedial nucleus of the hypothalamus. There were no differences in expression of stress-related peptides or their receptors 40 days after the end of repeated restraint. These results suggest that the sustained reduction in body weight and increased responsiveness to subsequent stressors in rats that have been exposed to repeated restraint are not associated with prolonged changes in mRNA expression of CRF receptors or their ligands.