astressin and antalarmin

The characterization of the corticotropin-releasing factor (CRF) family of neuroendocrine regulatory peptides, the cloning and pharmacological characterization of two CRF receptor subtypes (CRF(1) and CRF(2)), and the development of selective CRF receptor antagonists provided new insight to unravel the mechanisms of stress and the potential involvement of the CRF system in different pathophysiological conditions, including functional gastrointestinal disorders, mainly irritable bowel syndrome (IBS), and psychopathologies such as anxiety/depression. Compelling pre-clinical data showed that brain CRF administration mimics acute stress-induced colonic responses and enhances colorectal distension-induced visceral pain in rats through CRF(1) receptors. Similarly, peripheral CRF reduced the pain threshold to colonic distension and increased colonic motility in humans and rodents. These observations mimic the manifestations of IBS, characterized by abdominal bloating/discomfort and altered bowel habits. Moreover, CRF-CRF(1) pathways have been implicated in the development of anxiety/depression. These psychopathologies, together with stressful life events, have high comorbidity with IBS, and are considered significant components of the disease. From these observations, CRF(1) receptors have been suggested as a target to treat IBS. Peripherally acting CRF(1) antagonists might directly improve IBS symptoms, as related to motility, secretion and immune response. On the other hand, central actions will be beneficial as to prevent the psychopathologies that co-exist with IBS and as a way to modulate the central processing of stress- and visceral pain-related signals. Here, we review the pre-clinical and clinical data supporting these assumptions, and address the efforts done at a pharmaceutical level to develop effective therapies targeting CRF(1) receptors for functional gastrointestinal disorders.

Topics: Animals; Brain; Corticotropin-Releasing Hormone; Gastrointestinal Motility; Gastrointestinal Tract; Hormone Antagonists; Humans; Hyperalgesia; Intestinal Mucosa; Irritable Bowel Syndrome; Peptide Fragments; Pyrimidines; Pyrroles; Receptors, Corticotropin-Releasing Hormone; Stress, Psychological; Urocortins

Corticosterone-releasing hormone (CRH) and arginine vasopressin (AVP) are crucial components of the hypothalamic-pituitary-adrenal axis that stimulates the release of adrenocorticotropic hormone from the pituitary and mediate the stress response. CRH binds to two subtypes of CRH receptors (CRH-R1 and CRH-R2) that are present in both central and peripheral tissues. We used the CRH-R1-specific antagonist, antalarmin (ANT), the CRH-R1 and CRH-R2 peptide antagonist, astressin (AST), and the CRH-R2-specific peptide antagonist, astressin2b (AST2b), to determine which CRH receptor is involved in the nicotine-stimulated secretion of corticosterone. Male C57BL/6 mice were administered ANT (20 mg/kg, i.p.), AST (0.3 mg/kg, i.p.), AST2b (0.3 mg/kg, i.p.) or vehicle prior to administration of nicotine (1.0 mg/kg, s.c.), CRH (10 μg/kg, s.c.), AVP (10 μg/kg, s.c.) or saline (s.c.), killed 15 min later and trunk blood collected and assayed for corticosterone plasma levels. We found that CRH enhanced corticosterone release, and this response was blocked by both AST and ANT. Nicotine also increased corticosterone secretion, but this effect persisted in the presence of either CRH antagonist. Furthermore, AST but not ANT or AST2b decreased corticosterone levels associated with stress of handling and injection. We also assessed the role of AVP V(1b) -specific receptor antagonist, SSR149415 alone and in combination with AST and AST2b. Although the AVP antagonist did not alter basal or nicotine-stimulated corticosterone secretion, it attenuated the AVP-induced stimulation of corticosterone and its combination with AST but not AST2b completely abolished nicotine-mediated stimulation of corticosterone secretion. Our results demonstrate that the nicotine-induced stimulation of the hypothalamic-pituitary-adrenal axis is mediated by both the CRH-R and the AVP V(1b) receptor and when the CRH receptor is blocked, nicotine may utilize the AVP V(1b) receptor to mediate secretion of corticosterone. These results argue in favor of the development of specific antagonists that block both AVP and CRH receptors to decrease the pleasurable component of nicotine, which may be mediated by corticosterone.

Topics: Analysis of Variance; Animals; Antidiuretic Hormone Receptor Antagonists; Arginine Vasopressin; Corticosterone; Corticotropin-Releasing Hormone; Drug Interactions; Indoles; Male; Mice; Mice, Inbred C57BL; Nicotine; Nicotinic Agonists; Peptide Fragments; Pyrimidines; Pyrroles; Pyrrolidines; Receptors, Corticotropin-Releasing Hormone; Receptors, Vasopressin

Glucose transport across the placenta is mediated by glucose transporters (GLUT), which is critical for normal development and survival of the fetus. Regulatory mechanisms of GLUT in placenta have not been elucidated. Placental CRH has been implicated to play a key role in the control of fetal growth and development. We hypothesized that CRH, produced locally in placenta, could act to modulate GLUT in placenta. To investigate this, we obtained human placentas from uncomplicated term pregnancies and isolated and cultured trophoblast cells. GLUT1 and GLUT3 expressions in placenta were determined, and effects of CRH on GLUT1 and GLUT3 were examined. GLUT1 and GLUT3 were identified in placental villous syncytiotrophoblasts and the endothelium of vessels. Treatment of cultured placental trophoblasts with CRH resulted in an increase in GLUT1 expression while a decrease in GLUT3 expression in a dose-dependent manner. Cells treated with either CRH antibody or nonselective CRH receptor (CRH-R) antagonist astressin showed a decrease in GLUT1 and an increase in GLUT3 expression. CRH-R1 antagonist antalarmin decreased GLUT1 expression while increased GLUT3 expression. CRH-R2 antagonist astressin2b increased the expression of both GLUT1 and GLUT3. Knockdown of CRH-R1 decreased GLUT1 expression while increased GLUT3 expression. CRH-R2 knockdown caused an increase in both GLUT1 and GLUT3 expression. Our data suggest that, in placenta, CRH produced locally regulates GLUT1 and GLUT3 expression, CRHR1 and CRHR2-mediated differential regulation of GLUT1 and GLUT3 expression. Placental CRH may regulate the growth of fetus and placenta by modulating the expression of GLUT in placenta during pregnancy.

Topics: Corticotropin-Releasing Hormone; Female; Gene Expression Regulation, Developmental; Glucose Transport Proteins, Facilitative; Glucose Transporter Type 1; Glucose Transporter Type 3; Humans; Models, Biological; Peptide Fragments; Placenta; Pregnancy; Pyrimidines; Pyrroles; Receptors, Corticotropin-Releasing Hormone; RNA Interference; Trophoblasts

Corticotropin-releasing hormone (CRH) has been shown to modulate dendritic development in hippocampus. Mitotic kinesin-like protein 1 (MKLP1) plays key roles in dendritic differentiation. In the present study, we examined the effects of CRH on MKLP1 expression in cultured hippocampal neurons and determine subsequent signaling pathways involved. CRH dose-dependently increased MKLP1 mRNA and protein expression. This effect can be reversed by CRHR1 antagonist but not by CRHR2 antagonist. CRHR1 knockdown impaired this effect of CRH. CRH stimulated GTP-bound Gαs protein and phosphorylated phospholipase C (PLC)-β3 expression, which were blocked by CRHR1 antagonist. Transfection of GP antagonist-2A, an inhibitory peptide of Gαq protein, blocked CRH-induced phosphorylated PLC-β3 expression. PLC and PKC inhibitors completely blocked whereas adenylyl cyclase (AC) and PKA inhibitors did not affect CRH-induced MKLP1 expression. Our results indicate that CRH act on CRHR1 to induce MKLP1 expression via PLC/PKC signaling pathway. CRH may regulate MKLP1 expression, thereby modulating dendritic development.

Topics: Animals; Carbazoles; Cells, Cultured; Corticotropin-Releasing Hormone; Estrenes; GTP-Binding Protein alpha Subunits, Gs; Hippocampus; Kinesins; Microtubule-Associated Proteins; Neurons; Peptide Fragments; Protein Kinase C; Pyrimidines; Pyrroles; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptors, Corticotropin-Releasing Hormone; Signal Transduction; Transcription, Genetic; Transcriptional Activation; Type C Phospholipases

Corticotropin releasing hormone (CRH) has been detected in the adrenal gland of many species and may be involved in regulation of glucocorticoid secretion. In cultured human fetal adrenal definitive/transitional zone cells, CRH upregulates the adrenocorticotropic hormone (ACTH) receptor and steroidogenic enzymes and is blocked by the selective CRH type 1 receptor (CRH(1)) antagonist, antalarmin. Based on these findings and evidence that antalarmin infusion into sheep suppressed prepartum increases in cortisol, we hypothesized that antalarmin would influence adrenal cortisol secretion. Antalarmin strongly attenuated ACTH and forskolin (FSK)-stimulated cortisol and cyclic adenosine monophosphate (cAMP) release from cultured ovine adrenocortical cells but did not prevent ACTH binding to cells or ACTH-induced proliferation in adult cells. Corticotropin releasing hormone was minimally effective as a secretagogue but increased the cortisol response to subsequent ACTH. These results suggest that antalarmin attenuates ACTH-induced cortisol secretion from cultured ovine adrenal cortical cells at a site distal to the ACTH receptor. Although CRH may modulate the secretory response to ACTH, it is probably not a direct cortisol secretagogue in the sheep.

Topics: Adrenal Cortex; Adrenocorticotropic Hormone; Animals; Cells, Cultured; Corticotropin-Releasing Hormone; Female; Hydrocortisone; Peptide Fragments; Pregnancy; Pyrimidines; Pyrroles; Sheep

Rats exposed to 3 h of restraint stress on each of 3 days (RRS) lose weight on the days of RRS and gain weight at the same rate as controls after stress ends, but do not return to the weight of controls. RRS rats also show an exaggerated endocrine response to subsequent novel stressors. Studies described here tested the effects of corticotropin-releasing factor receptor (CRFR) antagonism on RRS-induced weight loss, hypophagia, and corticosterone release during mild stress in the postrestraint period. Weight loss was not prevented by either peripheral or third-ventricle administration of a CRFR1 antagonist, antalarmin, before each restraint. Antalarmin did, however, allow recovery of body weight in the poststress period. Third-ventricle administration of a CRFR2 antagonist, antisauvagine 30, had no effect in RRS rats but caused sustained weight loss in control animals. Surprisingly, third-ventricle administration of the nonselective CRFR antagonist, astressin, caused hypophagia and reversible weight loss in control rats. It had no effect in RRS rats. None of the antagonists modified the corticosterone response to RRS or to mild stress in the post-RRS period, but antalarmin suppressed corticosterone during the period of restraint in Control rats. These results suggest that CRFR1 activation is required for the initiation of events that lead to a prolonged down-regulation of body weight in RRS rats. The sustained reduction in body weight is independent of the severity of hypophagia on the days of restraint and of RRS-induced corticosterone release.

Topics: Animals; Corticosterone; Corticotropin-Releasing Hormone; Dose-Response Relationship, Drug; Eating; Feeding Behavior; Infusions, Parenteral; Injections, Subcutaneous; Male; Peptide Fragments; Prosencephalon; Pyrimidines; Pyrroles; Rats; Rats, Sprague-Dawley; Receptors, Corticotropin-Releasing Hormone; Restraint, Physical; Stress, Psychological; Time Factors; Weight Loss

Corticotropin-releasing factor (CRF) is a neurotransmitter in Barrington's nucleus neurons. These neurons can coregulate parasympathetic tone to the bladder (to modulate micturition) and brain noradrenergic activity (to affect arousal). To identify the role of CRF in the regulation of micturition, the effects of CRF agonists and antagonists on urodynamics in the unanesthetized rat were characterized. Rats were implanted with bladder and intrathecal or intraperitoneal catheters under isoflurane anesthesia. Cystometry was performed in the unanesthetized, unrestrained state at least 24 h later. In some cases, cortical electroencephalographic activity (EEG) was recorded simultaneously to assess arousal state. During cystometry, the state of arousal often shifted between waking and sleeping and urodynamic function changed depending on the state. Micturition threshold, bladder capacity, and micturition volume were all increased during sleep. The CRF1/CRF2 receptor agonists CRF and urocortin 2 increased bladder capacity and micturition volume in awake but not in sleeping rats. Conversely, the CRF1 receptor antagonists antalarmin and NBI-30775 increased urinary frequency and decreased bladder capacity in awake rats. The present results demonstrate a profound effect of the state of arousal on urodynamic function and suggest that simultaneous monitoring of EEG and cystometry may provide a useful model for studying nocturnal enuresis and other urinary disorders. In addition, the results provide evidence for an inhibitory influence of CRF in the spinal pathway on micturition. Targeting the CRF system in the spinal cord may provide a novel approach for treating urinary disorders.

Topics: Animals; Arousal; Corticotropin-Releasing Hormone; Electroencephalography; Hormone Antagonists; Injections, Spinal; Male; Movement; Neuropeptides; Peptide Fragments; Pyrimidines; Pyrroles; Rats; Rats, Sprague-Dawley; Receptors, Corticotropin-Releasing Hormone; Sleep; Urocortins; Urodynamics; Wakefulness

Peptide ligands bind the CRF(1) receptor by a two-domain mechanism: the ligand's carboxyl-terminal portion binds the receptor's extracellular N-terminal domain (N-domain) and the ligand's amino-terminal portion binds the receptor's juxtamembrane domain (J-domain). Little quantitative information is available regarding this mechanism. Specifically, the microaffinity of the two interactions and their contribution to overall ligand affinity are largely undetermined. Here we measured ligand interaction with N- and J-domains expressed independently, the former (residues 1-118) fused to the activin IIB receptor's membrane-spanning alpha-helix (CRF(1)-N) and the latter comprising residues 110-415 (CRF(1)-J). We also investigated the effect of nonpeptide antagonist and G-protein on ligand affinity for N- and J-domains. Peptide agonist affinity for CRF(1)-N was only 1.1-3.5-fold lower than affinity for the whole receptor (CRF(1)-R), suggesting the N-domain predominantly contributes to peptide agonist affinity. Agonist interaction with CRF(1)-J (potency for stimulating cAMP accumulation) was 12000-1500000-fold weaker than with CRF(1)-R, indicating very weak direct agonist interaction with the J-domain. Nonpeptide antagonist affinity for CRF(1)-J and CRF(1)-R was indistinguishable, indicating the compounds bind predominantly the J-domain. Agonist activation of CRF(1)-J was fully blocked by nonpeptide antagonist, suggesting antagonism results from inhibition of agonist-J-domain interaction. G-protein coupling with CRF(1)-R (forming RG) increased peptide agonist affinity 92-1300-fold, likely resulting from enhanced agonist interaction with the J-domain rather than the N-domain. Nonpeptide antagonists, which bind the J-domain, blocked peptide agonist binding to RG, and binding of peptide antagonists, predominantly to the N-domain, was unaffected by R-G coupling. These findings extend the two-domain model quantitatively and are consistent with a simple equilibrium model of the two-domain mechanism: (1) The N-domain binds peptide agonist with moderate-to-high microaffinity, substantially increasing the local concentration of agonist and so allowing weak agonist-J-domain interaction. (2) Agonist-J-domain interaction is allosterically enhanced by receptor-G-protein interaction and inhibited by nonpeptide antagonist.

Topics: Amphibian Proteins; Aniline Compounds; Animals; Binding, Competitive; Cell Line; Corticotropin-Releasing Hormone; Extracellular Space; GTP-Binding Proteins; Humans; Ligands; Models, Chemical; Peptide Fragments; Peptide Hormones; Peptides; Protein Binding; Protein Structure, Tertiary; Pyrazoles; Pyrimidines; Pyrroles; Rats; Receptors, Corticotropin-Releasing Hormone; Triazines; Urocortins

Urocortin, a new mammalian member of the corticotropin-releasing factor (CRF) family has been proposed to be the endogenous ligand for CRF receptor 2 (CRF-R2). We studied the influence of intravenous urocortin on gastric emptying and the role of CRF-R2 in peptide action and postoperative gastric ileus in conscious rats. The intravenous doses of rat CRF and rat urocortin producing 50% inhibition of gastric emptying were 2.5 and 1.1 microgram/kg, respectively. At these intravenous doses, CRF and urocortin have their actions fully reversed by the CRF-R1/CRF-R2 antagonist astressin at antagonist/agonist ratios of 5:1 and 67:1, respectively. Astressin (12 microgram/kg iv) completely prevented abdominal surgery-induced 54% inhibition of gastric emptying 3 h after surgery while having no effect on basal gastric emptying. The selective nonpeptide CRF-R1 antagonists antalarmin (20 mg/kg ip) and NBI-27914 (400 microgram/kg iv) did not influence intravenous CRF-, urocortin- or surgery-induced gastric stasis. These results as well as earlier ones showing that alpha-helical CRF9-41 (a CRF-R2 more selective antagonist) partly prevented postoperative ileus indicate that peripheral CRF-R2 may be primarily involved in intravenous urocortin-, CRF-, and abdominal surgery-induced gastric stasis.

Topics: Animals; Corticotropin-Releasing Hormone; Gastric Emptying; Humans; Injections, Intravenous; Intestinal Obstruction; Male; Peptide Fragments; Pyrimidines; Pyrroles; Rats; Rats, Sprague-Dawley; Receptors, Corticotropin-Releasing Hormone; Recombinant Proteins; Urocortins