The addition of corticotropin-releasing hormone to 2-day low dose dexamethasone suppression test provides additional case detection.

ABSTARCT: PURPOSE: The diagnostic value of adding a Corticotropin-Releasing Hormone (CRH) Stimulation Test to the 2-day Low Dose Dexamethasone Suppression Test (Dex-CRH Test) has been debated in the literature. METHODS: We identified 65 patients with Cushing disease (CD) and 42 patients in whom a diagnosis of Cushing disease could not be confirmed (NCD) after a minimum follow-up of 14 months who underwent the Dex-CRH test. RESULTS: The female sex ratio, median (range) age, and BMI were similar between the two groups. The follow-up for patients with CD and NCD was 74 (4-233) and 52 (14-146) months, respectively. Among 65 patients with CD, 5 (7.7%) had a cortisol level ≤1.4 µg/dl after LDDST but were appropriately classified as CD with a cortisol level >1.4 µg/dL at 15-min post CRH stimulation. In contrast, 3/42 patients (7.1%) in NCD had an abnormal Dex-CRH test. In only one of three patients, the LDDST was marginally normal (cortisol was 1.4 µg/dL and increased to 3.1 µg/dL 15-min post CRH). A cortisol cutoff value of >1.4 µg/dL during the Dex-CRH test provided a sensitivity of 100%, specificity of 93%, and diagnostic accuracy of 97% to diagnose CD. When patients without a Dex level were excluded (n = 74), the sensitivity did not change, but the specificity and accuracy of the Dex-CRH test increased to 97 and 99%, respectively. CONCLUSION: The Dex-CRH Test provided additional case detection in 5/65 (7.7%) patients with CD. It resulted in one false-positive case compared to LDDST. Measurement of dexamethasone improved diagnostic accuracy of the test.

Corticotropin-Releasing Factor Family: A Stress Hormone-Receptor System's Emerging Role in Mediating Sex-Specific Signaling.

No organ in the body is impervious to the effects of stress, and a coordinated response from all organs is essential to deal with stressors. A dysregulated stress response that fails to bring systems back to homeostasis leads to compromised function and ultimately a diseased state. The components of the corticotropin-releasing factor (CRF) family, an ancient and evolutionarily conserved stress hormone-receptor system, helps both initiate stress responses and bring systems back to homeostasis once the stressors are removed. The mammalian CRF family comprises of four known agonists, CRF and urocortins (UCN1-3), and two known G protein-coupled receptors (GPCRs), CRF1 and CRF2. Evolutionarily, precursors of CRF- and urocortin-like peptides and their receptors were involved in osmoregulation/diuretic functions, in addition to nutrient sensing. Both CRF and UCN1 peptide hormones as well as their receptors appeared after a duplication event nearly 400 million years ago. All four agonists and both CRF receptors show sex-specific changes in expression and/or function, and single nucleotide polymorphisms are associated with a plethora of human diseases. CRF receptors harbor N-terminal cleavable peptide sequences, conferring biased ligand properties. CRF receptors have the ability to heteromerize with each other as well as with other GPCRs. Taken together, CRF receptors and their agonists due to their versatile functional adaptability mediate nuanced responses and are uniquely positioned to orchestrate sex-specific signaling and function in several tissues.

Bioelectronic sensor mimicking the human neuroendocrine system for the detection of hypothalamic-pituitary-adrenal axis hormones in human blood.

In the neuroendocrine system, corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) play important roles in the regulation of the hypothalamic-pituitary-adrenal (HPA) system. Disorders of the HPA system lead to physiological problems, such as Addison's disease and Cushing's syndrome. Therefore, detection of CRH and ACTH is essential for diagnosing disorders related to the HPA system. Herein, receptors of the HPA axis were used to construct a bioelectronic sensor system for the detection of CRH and ACTH. The CRH receptor, corticotropin-releasing hormone receptor 1 (CRHR1), and the ACTH receptor, melanocortin 2 receptor (MC2R), were produced using an Escherichia coli expression system, and were reconstituted using nanodisc (ND) technology. The receptor-embedded NDs were immobilized on a floating electrode of a carbon nanotube field-effect transistor (CNT-FET). The constructed sensors sensitively detected CRH and ACTH to a concentration of 1 fM with high selectivity in real time. Furthermore, the reliable detection of CRH and ACTH in human plasma by the developed sensors demonstrated their potential in clinical and practical applications. These results indicate that CRHR1 and MC2R-based bioelectronic sensors can be applied for rapid and efficient detection of CRH and ACTH.

The transcripts of CRF and CRF receptors under fasting stress in Dabry's sturgeon (Acipenser dabryanus Dumeril).

Dabry's sturgeon (Acipenser dabryanus Dumeril, 1868) belongs to Sturgeon and is distributed throughout the mainstream of the upper Yangtze River. While there is little research onphysiological mechanism of Dabry's sturgeon, such as feeding regulation by the CRF system. At present, CRF is thought to regulate feeding via CRF receptors (CRF-Rs) in several mammals, but relatively few studies of CRF and feeding exist in teleosts. Herein, the transcripts of CRF and CRF-Rs under fasting stress in Dabry's sturgeon (Acipenser dabryanus Dumeril) have been explored. A full length Dabry's sturgeon CRF cDNA of 953 bp was identified, which contained a 447 bp open reading frame (ORF). A partial CRF-R1 cDNA of 1053 bp and CRF-R2 cDNA of 906 bp corresponding to the coding sequences (CDS) was obtained. In addition, analysis of the tissue distribution of CRF and CRF-Rs mRNAs revealed they were widely distributed in the central and peripheral nervous systems. Furthermore, periprandial (preprandial and postprandial), fasting, and re-feeding experiments revealed CRF mRNA was significantly increased 1 h and 3 h after feeding and CRF and CRF-Rs transcripts were significantly decreased after 10 days fasting, and significantly increased on re-feeding on day 10. These results suggest that CRF and CRF-Rs might regulate feeding by acting as satiety factors.

Development of an electrochemical impedimetric immunosensor for Corticotropin Releasing Hormone (CRH) using half-antibody fragments as elements of biorecognition.

An electrochemical impedimetric immunosensor was developed for the detection of the neuropeptide Corticotropin Releasing Hormone (CRH) based on the immobilization of half-antibody fragments on gold nanoparticles (AuNp). Then, the optimal conditions for the obtainment of AuNp through electroplating on a bare gold electrode were studied. The results showed that the obtainment of AuNp at a fixed potential of -0.2 V for 330 s, at 80 °C and 2·10-3 mol·L-1 of HAuCl4 generates an adequate nanostructured surface and is a highly reproducible method. Also, the optimal conditions for immobilizing the half-antibody on AuNp were studied. The interaction of the CRH with the recognition layer of the immobilized half-antibody on the nanostructured surface was carried out by incubation at 4 °C for 2 h. A dissolution of [Fe(CN)6]4-/[Fe(CN)6]3- as a redox probe was used to study the electrochemical responses of the nanostructured surface and the immobilization processes of the half-antibody and detection of CRH, using cyclic voltammetry and electrochemical impedance spectroscopy. An immunosensor was obtained for the specific detection of CRH, within a range of 10.0-80.0 µg mL-1, with a limit of detection of 2.7 µg mL-1 and a limit of quantification of 9.2 µg mL-1. Additionally, the association constant between the CRH and the immobilized half-antibody was calculated at 1.96·105 M-1.

In silico binding mechanism prediction of benzimidazole based corticotropin releasing factor-1 receptor antagonists by quantitative structure activity relationship, molecular docking and pharmacokinetic parameters calculation.

Despite the various research efforts toward the treatment of stress-related disorders, the drug has not yet launched last 20 years. Corticotropin releasing factor-1 receptor antagonists have been point of great interest in stress-related disorders. In the present study, we have selected benzazole scaffold-based compounds as corticotropin releasing factor-1 antagonists and performed 2D and 3D QSAR studies to identify the structural features to elucidating the binding mechanism prediction. The best 2D QSAR model was obtained through multiple linear regression method with r2 value of .7390, q2 value of .5136 and pred_r2 (predicted square correlation coefficient) value of .88. The contribution of 2D descriptor, T_2_C_1 was 60% (negative contribution) and 4pathClusterCount was 40.24% (positive contribution) in enhancing the activity. Also 3D QSAR model was statistically significant with q2 value of .9419 and q2_se (standard error of internal validation) value of .19. Statistical parameters results prove the robustness and significance of both models. Further, molecular docking and pharmacokinetic analysis was performed to explore the scope of investigation. Docking results revealed that the all benzazole compounds show hydrogen bonding with residue Asn283 and having same hydrophobic pocket (Phe286, Leu213, Ile290, Leu287, Phe207, Arg165, Leu323, Tyr327, Phe284, and Met206). Compound B14 has higher activity compare to reference molecules. Most of the compounds were found within acceptable range for pharmacokinetic parameters. This work provides the extremely useful leads for structural substituents essential for benzimidazole moiety to exhibit antagonistic activity against corticotropin releasing factor-1 receptors.

Urocortins: Actions in health and heart failure.

The urocortins (Ucns), endogenous peptides belonging to the corticotropin-releasing factor (CRF) family, are increasingly recognized as having diverse and important multi-system functions, especially within the cardiovascular system. The biological actions of the three Ucns (Ucn1, Ucn2, Ucn3) are mediated via G-protein-coupled CRF receptors, with both peptides and receptors widely distributed throughout tissues and organs contributing to pressure/volume homeostasis including the heart, vasculature, kidneys and adrenals. The Ucns activate a variety of signaling cascades in cardiomyocytes, vascular smooth muscle cells and endothelial cells including, but not limited to, adenyl cyclase/cAMP and several kinase pathways, with downstream effects comprising vasodilation, augmented cardiac contractility, and protection against hypoxic injury. Increasing evidence suggests the Ucns may be clinically significant molecules in the pathogenesis, treatment and/or management of several conditions, with some of the most compelling data demonstrating a therapeutic potential for the peptides in the setting of heart failure. Circulating levels of the Ucns are elevated in this setting, and antagonism of the endogenous peptides exacerbates manifestations of the syndrome in animal models. All three Ucns exert salutary hemodynamic, neurohormonal and renal effects in experimental heart failure and recent clinical trials have demonstrated hemodynamic benefits of Ucn2 administration.

Prospective Clinical Applications of CRF Peptide Antagonists.

It is noteworthy that thirty three years of efforts in corticotropin releasing factor (CRF) research by academia and the pharmaceutical industry resulted in several thousand papers and patents, yet little progress has been made to identify and market diagnostic or therapeutic CRF peptides and small molecule ligands. We document the potential relevance of CRF peptide antagonists to reinvigorate stress/anxiety affected "anatomy systems" in order to boost their efficacy.

CRH peptide evolution occurred in three phases: Evidence from characterizing sea lamprey CRH system members.

The corticotropin releasing hormone (CRH) system, which includes the CRH family of peptides, their receptors (CRHRs) and a binding protein (CRHBP), has been strongly conserved throughout vertebrate evolution. The identification of invertebrate homologues suggests this system evolved over 500 million years ago. However, the early vertebrate evolution of the CRH system is not understood. Current theory indicates that agnathans (hagfishes and lampreys) are monophyletic with a conservative evolution over the past 500million years and occupy a position at the root of vertebrate phylogeny. We isolated the cDNAs for three CRH family members, two CRHRs and a CRHBP from the sea lamprey, Petromyzon marinus. Two of the CRH peptides are related to the CRH/urotensin-1 (UI) lineage, whereas the other is a urocortin (Ucn) 3 orthologue. The predicted amino acid identity of CRH and UI is 61% but they possess distinct motifs indicative of each peptide, suggesting an early divergence of the two genes. Based on our findings we propose the CRH peptides evolved in at least 3 distinct phases. The first occurring prior to the agnathans gave rise to the CRH/UI-like and Ucn2/3-like paralogous lineages. The second was a partial sub-genomic duplication of the ancestral CRH/UI-like gene, but not the Ucn2/3-like gene, giving rise to the CRH and UI (Ucn) lineages. The third event which resulted in the appearance of Ucn2 and Ucn3 must have occurred after the evolution of the cartilaginous fishes. Interestingly, unlike other vertebrate CRHRs, we were unable to classify our two P. marinus receptors (designated CRHRα and CRHRß) as either type 1 or type 2, indicating that this split evolved later in vertebrate evolution. A single CRHBP gene was found suggesting that either this gene has not been affected by the vertebrate genome duplications or there have been a series of paralogous gene deletions. This study suggests that P. marinus possess a functional CRH system that differs from that of the gnathostomes and may represent a model for the earliest functioning CRH system in vertebrates.

Combination of Thiol-Additive-Free Native Chemical Ligation/Desulfurization and Intentional Replacement of Alanine with Cysteine.

We report a novel strategy for native chemical ligation (NCL). Alanines not located at a ligation site are temporarily replaced with cysteines, and this enables efficient thiol-additive-free NCL, with subsequent desulfurization to regenerate the target peptide. We synthesized stresscopin-related peptide and neuroendocrine regulatory peptide-2 (NERP-2) by this method. We confirmed that both conventional alkyl thioester and thioester-equivalent N-acyl-N'-methyl-benzimidazolinone (MeNbz) can be adopted as thioester components for thiol-additive-free NCL of multi-Cys-containing peptides.

Antiamnesic properties of analogs and mimetics of the tripeptide human urocortin 3.

Amnesia is a deficit in memory caused by brain damage, disease, or trauma. Until now, there are no successful medications on the drug market available to treat amnesia. Short analogs and mimetics of human urocortin 3 (Ucn 3) tripeptide were synthetized and tested for their action against amnesia induced by eletroconvulsion in mice. Among the 16 investigated derivatives of Ucn 3 tripeptide, eight compounds displayed antiamnesic effect. Our results proved that the configuration of chiral center of glutamine does not affect the antiamnesic properties. Alkyl amide or isoleucyl amide at the C-terminus may lead to antiamnesic compounds. As concerned the N-terminus, acetyl, Boc, and alkyl ureido moieties were found among the active analogs, but the free amino function at the N-terminus usually led to an inactive derivatives. These observations may lead to the design and synthesis of small peptidomimetics and amino acid derivatives as antiamnesic drug candidates, although the elucidation of the mechanism of the action requires further investigations.

Corticotropin-releasing hormone family evolution: five ancestral genes remain in some lineages.

The evolution of the peptide family consisting of corticotropin-releasing hormone (CRH) and the three urocortins (UCN1-3) has been puzzling due to uneven evolutionary rates. Distinct gene duplication scenarios have been proposed in relation to the two basal rounds of vertebrate genome doubling (2R) and the teleost fish-specific genome doubling (3R). By analyses of sequences and chromosomal regions, including many neighboring gene families, we show here that the vertebrate progenitor had two peptide genes that served as the founders of separate subfamilies. Then, 2R resulted in a total of five members: one subfamily consists of CRH1, CRH2, and UCN1. The other subfamily contains UCN2 and UCN3. All five peptide genes are present in the slowly evolving genomes of the coelacanth Latimeria chalumnae (a lobe-finned fish), the spotted gar Lepisosteus oculatus (a basal ray-finned fish), and the elephant shark Callorhinchus milii (a cartilaginous fish). The CRH2 gene has been lost independently in placental mammals and in teleost fish, but is present in birds (except chicken), anole lizard, and the nonplacental mammals platypus and opossum. Teleost 3R resulted in an additional surviving duplicate only for crh1 in some teleosts including zebrafish (crh1a and crh1b). We have previously reported that the two vertebrate CRH/UCN receptors arose in 2R and that CRHR1 was duplicated in 3R. Thus, we can now conclude that this peptide-receptor system was quite complex in the ancestor of the jawed vertebrates with five CRH/UCN peptides and two receptors, and that crh and crhr1 were duplicated in the teleost fish tetraploidization.

Characterization of Multisubstituted Corticotropin Releasing Factor (CRF) Peptide Antagonists (Astressins).

CRF mediates numerous stress-related endocrine, autonomic, metabolic, and behavioral responses. We present the synthesis and chemical and biological properties of astressin B analogues {cyclo(30-33)[D-Phe(12),Nle(21,38),C(α)MeLeu(27,40),Glu(30),Lys(33)]-acetyl-h/r-CRF(9-41)}. Out of 37 novel peptides, 17 (2, 4, 6-8, 10, 11, 16, 17, 27, 29, 30, 32-36) and 16 (3, 5, 9, 12-15, 18, 19, 22-26, 28, 31) had k(i) to CRF receptors in the high picomolar and low nanomole ranges, respectively. Peptides 1, 2, and 11 inhibited h/rCRF and urocortin 1-induced cAMP release from AtT20 and A7r5 cells. When Astressin C 2 was administered to adrenalectomized rats at 1.0 mg subcutaneously, it inhibited ACTH release for >7 d. Additional rat data based on the inhibitory effect of (2) on h/rCRF-induced stimulation of colonic secretory motor activity and urocortin 2-induced delayed gastric emptying also indicate a safe and long-lasting antagonistic effect. The overall properties of selected analogues may fulfill the criteria expected from clinical candidates.

Maternal separation enhances conditioned fear and decreases the mRNA levels of the neurotensin receptor 1 gene with hypermethylation of this gene in the rat amygdala.

Stress during postnatal development is associated with an increased risk for depression, anxiety disorders, and substance abuse later in life, almost as if mental illness is able to be programed by early life stressors. Recent studies suggest that such "programmed" effects can be caused by epigenetic regulation. With respect to conditioned fear, previous studies have indicated that early life stress influences its development in adulthood, whereas no potential role of epigenetic regulation has been reported. Neurotensin (NTS) is an endogenous neuropeptide that has receptors densely located in the amygdala and hippocampus. Recently, NTS systems have constituted an emerging target for the treatment of anxiety. The aim of the present work is to clarify whether the NTS system is involved in the disturbance of conditioned fear in rats stressed by maternal separation (MS). The results showed that MS enhanced freezing behaviors in fear-conditioned stress and reduced the gene expression of NTS receptor (NTSR) 1 but not of NTS or NTSR2 in the amygdalas of adult rats. The microinjection of a NTSR1 antagonist into the amygdala increased the percentage of freezing in conditioned fear, whereas the microinjection of NTSR1 agonist decreased freezing. These results suggest that NTSR1 in the amygdala may play a role in the effects of MS on conditioned fear stress in adult rats. Moreover, MS increased DNA methylation in the promoter region of NTSR1 in the amygdala. Taken together, MS may leave epigenetic marks in the NTSR1 gene in the amygdala, which may enhance conditioned fear in adulthood. The MS-induced alternations of DNA methylation in the promoter region of NTSR1 in the amygdala may be associated with vulnerability to the development of anxiety disorders and depression in adulthood.

Cloning of corticotropin-releasing hormone (CRH) precursor cDNA and immunohistochemical detection of CRH peptide in the brain of the Japanese eel, paying special attention to gonadotropin-releasing hormone.

The stress-related corticotropin-releasing hormone (CRH) was first identified by isolation of its cDNA from the brain of the Japanese eel Anguilla japonica. CRH cDNA encodes a signal peptide, a cryptic peptide and CRH (41 amino acids). The sequence homology to mammalian CRH is high. Next, the distribution of CRH-immunoreactive (ir) cell bodies and fibers in the brain and pituitary were examined by immunohistochemistry. CRH-ir cell bodies were detected in several brain regions, e.g., nucleus preopticus pars magnocellularis, nucleus preopticus pars gigantocellularis and formatio reticularis superius. In the brain, CRH-ir fibers were distributed not only in the hypothalamus but also in various regions. Some CRH-ir fibers projected to adrenocorticotropic hormone (ACTH) cells in the rostral pars distalis of the pituitary and also the α-melanocyte-stimulating hormone (α-MSH) cells in the pars intermedia of the pituitary. Finally, the neuroanatomical relationship between the CRH neurons and gonadotropin-releasing hormone (GnRH) neurons was examined by dual-label immunohistochemistry. CRH-ir fibers were found to be in close contact with GnRH-ir cell bodies in the hypothalamus and in the midbrain tegmentum and GnRH-ir fibers were in close contact with CRH-ir cell bodies in the nucleus preopticus pars magnocellularis. These results suggest that CRH has some physiological functions other than the stimulation of ACTH and α-MSH secretion and that reciprocal connections may exist between the CRH neurons and GnRH neurons in the brain of the Japanese eel.

Regulation of a putative corticosteroid, 17,21-dihydroxypregn-4-ene,3,20-one, in sea lamprey, Petromyzon marinus.

In higher vertebrates, in response to stress, the hypothalamus produces corticotropin-releasing hormone (CRH), which stimulates cells in the anterior pituitary to produce adrenocorticotropic hormone (ACTH), which in turn stimulates production of either cortisol (F) or corticosterone (B) by the adrenal tissues. In lampreys, however, neither of these steroids is present. Instead, it has been proposed that the stress steroid is actually 17,21-dihydroxypregn-4-ene-3,20-dione (11-deoxycortisol; S). However, there have been no studies yet to determine its mechanism of regulation or site of production. Here we demonstrate that (1) intraperitoneal injections of lamprey-CRH increase plasma S in a dose dependent manner, (2) intraperitoneal injections of four lamprey-specific ACTH peptides at 100µg/kg, did not induce changes in plasma S concentrations in either males or females; (3) two lamprey-specific gonadotropin-releasing hormones (GnRH I and III) and arginine-vasotocin (AVT), all at single doses, stimulated S production as well as, or to an even greater extent than CRH; (4) sea lamprey mesonephric kidneys, in vitro, converted tritiated 17α-hydroxyprogesterone (17α-P) into a steroid that had the same chromatographic properties (on HPLC and TLC) as S; (5) kidney tissues released significantly more immunoassayable S into the incubation medium than gill, liver or gonad tissues. One interpretation of these results is that the corticosteroid production of the sea lamprey, one of the oldest extant vertebrates, is regulated through multiple pathways rather than the classical HPI-axis. However, the responsiveness of this steroid to the GnRH peptides means that a reproductive rather than a stress role for this steroid cannot yet be ruled out.

Protective role of the neuropeptide urocortin II against experimental sepsis and leishmaniasis by direct killing of pathogens.

We currently face an alarming resurgence in infectious diseases characterized by antimicrobial resistance and therapeutic failure. This has generated the urgent need of developing new therapeutic approaches that include agents with nontraditional modes of action. A recent interest focused on approaches based on our natural immune defenses, especially on peptides that combine innate antimicrobial activity against diverse pathogens and immunoregulatory functions. In this study, to our knowledge, we describe for the first time the antimicrobial activity of the neuropeptide urocortin II (UCNII) against a panel of Gram-positive and Gram-negative bacteria and tropical parasites of the genus Leishmania. Importantly, this cytotoxicity was selective for pathogens, because UCNII did not affect mammalian cell viability. Structurally, UCNII has a cationic and amphipathic design that resembles antimicrobial peptides. Using mutants and UCNII fragments, we determined the structural requirements for the interaction between the peptide and the surface of pathogen. Following its binding to pathogen, UCNII caused cell death through different membrane-disrupting mechanisms that involve aggregation and membrane depolarization in bacteria and pore formation in Leishmania. Noteworthily, UCNII killed the infective form of Leishmania major even inside the infected macrophages. Consequently, UCNII prevented mortality caused by polymicrobial sepsis and ameliorated pathological signs of cutaneous leishmaniasis. Besides its presence in body physical and mucosal barriers, we found that innate immune cells produce UCNII in response to infections. Therefore, UCNII could be considered as an ancient highly-conserved host peptide involved in the natural antimicrobial defense and emerge as an attractive alternative to current treatments for microbial disorders with associated drug resistances.

Sex-specific cell signaling: the corticotropin-releasing factor receptor model.

Elucidating the biological basis for sex differences in diseases can reveal their pathophysiology and guide the development of individualized treatments. Here, we review evidence for the novel concept that receptor signaling can be sex biased such that the specific pathways engaged by ligand binding are determined by sex. As an example, this review focuses on the receptor for corticotropin-releasing factor (CRF), a stress-related peptide implicated in diverse psychiatric and medical disorders that are more prevalent in females. There is evidence for sex biases in CRF receptor coupling to G proteins and ß-arrestin that render females more sensitive to acute stress and less able to adapt to chronic stress. Taken with evidence for sex biased signaling in other receptor systems, the studies demonstrate the broad potential impact of this characteristic in determining sex differences in disease and therapeutic efficacy and underscore the importance of studying females in medical and pharmacological research.

Posttranslational processing of human and mouse urocortin 2: characterization and bioactivity of gene products.

Mouse (m) and human (h) urocortin 2 (Ucn 2) were identified by molecular cloning strategies and the primary sequence of their mature forms postulated by analogy to closely related members of the corticotropin-releasing factor (CRF) neuropeptide family. Because of the paucity of Ucn 2 proteins in native tissues, skin, muscle, and pancreatic cell lines were transduced with lentiviral constructs and secretion media were used to isolate and characterize Ucn 2 products and study processing. Primary structures were assigned using a combination of Edman degradation sequencing and mass spectrometry. For mUcn 2, transduced cells secreted a 39 amino acid peptide and the glycosylated prohormone lacking signal peptide; both forms were C-terminally amidated and highly potent to activate the type 2 CRF receptor. Chromatographic profiles of murine tissue extracts were consistent with cleavage of mUcn 2 prohormone to a peptidic form. By contrast to mUcn 2, mammalian cell lines transduced with hUcn 2 constructs secreted significant amounts of an 88 amino acid glycosylated hUcn 2 prohormone but were unable to further process this molecule. Similarly, WM-266-4 melanoma cells that express endogenous hUcn 2 secreted only the glycosylated prohormone lacking the signal peptide and unmodified at the C terminus. Although not amidated, hUcn 2 prohormone purified from overexpressing lines activated CRF receptor 2. Hypoxia and glycosylation, paradigms that might influence secretion or processing of gene products, did not significantly impact hUcn 2 prohormone cleavage. Our findings identify probable Ucn 2 processing products and should expedite the characterization of these proteins in mammalian tissues.