Éva M. Fekete

Physiology, pharmacology, and therapeutic relevance of urocortins in mammals: ancient CRF paralogs

Urocortins, three paralogs of the stress-related peptide corticotropin-releasing factor (CRF) found in bony fish, amphibians, birds, and mammals, have unique phylogenies, pharmacologies, and tissue distributions. As a result and despite a structural family resemblance, the natural functions of urocortins and CRF in mammalian homeostatic responses differ substantially. Endogenous urocortins are neither simply counterpoints nor mimics of endogenous CRF action. In their own right, urocortins may be clinically relevant molecules in the pathogenesis or management of many conditions, including congestive heart failure, hypertension, gastrointestinal and inflammatory disorders (irritable bowel syndrome, active gastritis, gastroparesis, and rheumatoid arthritis), atopic/allergic disorders (dermatitis, urticaria, and asthma), pregnancy and parturition (preeclampsia, spontaneous abortion, onset, and maintenance of effective labor), major depression and obesity. Safety trials for intravenous urocortin treatment have already begun for the treatment of congestive heart failure. Further understanding the unique functions of urocortin 1, urocortin 2, and urocortin 3 action may uncover other therapeutic opportunities.

CRF system recruitment mediates dark side of compulsive eating

Dieting to control body weight involves cycles of deprivation from palatable food that can promote compulsive eating. The present study shows that rats withdrawn from intermittent access to palatable food exhibit overeating of palatable food upon renewed access and an affective withdrawal-like state characterized by corticotropin-releasing factor-1 (CRF1) receptor antagonist-reversible behaviors, including hypophagia, motivational deficits to obtain less palatable food, and anxiogenic-like behavior. Withdrawal was accompanied by increased CRF expression and CRF1 electrophysiological responsiveness in the central nucleus of the amygdala. We propose that recruitment of anti-reward extrahypothalamic CRF-CRF1 systems during withdrawal from palatable food, analogous to abstinence from abused drugs, may promote compulsive selection of palatable food, undereating of healthier alternatives, and a negative emotional state when intake of palatable food is prevented.

MPZP: A novel small molecule corticotropin-releasing factor type 1 receptor (CRF1) antagonist

The extrahypothalamic stress peptide corticotropin-releasing factor (CRF) system is an important regulator of behavioral responses to stress. Dysregulation of CRF and the CRF type 1 receptor (CRF(1)) system is hypothesized to underlie many stress-related disorders. Modulation of the CRF(1) system by non-peptide antagonists currently is being explored as a therapeutic approach for anxiety disorders and alcohol dependence. Here, we describe a new, less hydrophilic (cLogP approximately 2.95), small molecule, non-peptide CRF(1) antagonist with high affinity (K(i)=4.9 nM) and specificity for CRF(1) receptors: N,N-bis(2-methoxyethyl)-3-(4-methoxy-2-methylphenyl)-2,5-dimethyl-pyrazolo[1,5-a] pyrimidin-7-amine (MPZP). The compound was systemically administered to adult male rats in two behavioral models dependent on the CRF(1) system: defensive burying (0, 5, 20 mg/kg, n=6-11 for each dose) and alcohol dependence (0, 5, 10, 20 mg/kg, n=8 for each self-administration group). Acute administration of MPZP reduced burying behavior in the defensive burying model of active anxiety-like behavior. MPZP also attenuated withdrawal-induced excessive drinking in the self-administration model of alcohol dependence without affecting nondependent alcohol drinking or water consumption. The present findings support the proposed significance of the CRF(1) system in anxiety and alcohol dependence and introduce a promising new compound for further development in the treatment of alcohol dependence and stress-related disorders.

Delayed satiety-like actions and altered feeding microstructure by a selective type 2 corticotropin-releasing factor agonist in rats: intra-hypothalamic urocortin 3 administration reduces food intake by prolonging the post-meal interval.

Brain corticotropin-releasing factor/urocortin (CRF/Ucn) systems are hypothesized to control feeding, with central administration of ‘type 2’ urocortins producing delayed anorexia. The present study sought to identify the receptor subtype, brain site, and behavioral mode of action through which Ucn 3 reduces nocturnal food intake in rats. Non-food-deprived male Wistar rats (n=176) were administered Ucn 3 into the lateral (LV) or fourth ventricle, or into the ventromedial or paraventricular nuclei of the hypothalamus (VMN, PVN) or the medial amygdala (MeA), regions in which Ucn 3 is expressed in proximity to CRF2 receptors. LV Ucn 3 suppressed ingestion during the third–fourth post-injection hours. LV Ucn 3 anorexia was reversed by cotreatment with astressin2-B, a selective CRF2 antagonist and not observed following equimole subcutaneous or fourth ventricle administration. Bilateral intra-VMN and intra-PVN infusion, more potently than LV infusion, reduced the quantity (57–73%) and duration of ingestion (32–68%) during the third–fourth post-infusion hours. LV, intra-PVN and intra-VMN infusion of Ucn 3 slowed the eating rate and reduced intake by prolonging the post-meal interval. Intra-VMN Ucn 3 reduced feeding bout size, and intra-PVN Ucn 3 reduced the regularity of eating from pellet to pellet. Ucn 3 effects were behaviorally specific, because minimal effective anorectic Ucn 3 doses did not alter drinking rate or promote a conditioned taste aversion, and site-specific, because intra-MeA Ucn 3 produced a nibbling pattern of more, but smaller meals without altering total intake. The results implicate the VMN and PVN of the hypothalamus as sites for Ucn 3-CRF2 control of food intake.

Subtype-Selective Corticotropin-Releasing Factor Receptor Agonists Exert Contrasting, but Not Opposite, Effects on Anxiety-Related Behavior in Rats

The corticotropin-releasing factor (CRF) system mediates stress responses. Extrahypothalamic CRF1 receptor activation has anxiogenic-like properties, but anxiety-related functions of CRF2 receptors remain unclear. The present study determined the effects of intracerebroventricular administration of a CRF2 agonist, urocortin 3, on behavior of male Wistar rats in the shock-probe, social interaction, and defensive withdrawal tests of anxiety-like behavior. Equimolar doses of stressin1-A, a novel CRF1 agonist, were administered to separate rats. The effects of pyrazolo[1,5-a]-1,3,5-triazin-4-amine,8-[4-(bromo)-2-chlorophenyl]-N, N-bis(2-methoxyethyl)-2,7-dimethyl-(9Cl) (MJL-1-109-2), a CRF1 antagonist, on behavior in the shock-probe test also were studied. Stressin1-A increased anxiety-like behavior in the social interaction and shock-probe tests. Stressin1-A elicited behavioral activation and defensive burying at lower doses (0.04 nmol), but it increased freezing, grooming, and mounting at 25-fold higher (1-nmol) doses. Conversely, systemic administration of MJL-1-109-2 (10 mg/kg) had anxiolytic-like effects in the shock-probe test. Unlike stressin1-A or MJL-1-109-2, i.c.v. urocortin 3 infusion did not alter anxiety-like behavior in the shock-probe test across a range of doses that reduced locomotion and rearing and increased grooming. Urocortin 3 also did not decrease social interaction, but it decreased anxiety-like behavior in the defensive withdrawal test at a 2-nmol dose. Thus, i.c.v. administration of CRF1 and CRF2 agonists produced differential, but not opposite, effects on anxiety-like behavior. Urocortin 3 (i.c.v.) did not consistently decrease or increase anxiety-like behavior, the latter unlike effects seen previously after local microinjection of CRF2 agonists into the septum or raphe. With increasing CRF1 activation, however, the behavioral expression of anxiety qualitatively changes from “coping” to “noncoping” and offensive, agonistic behaviors.

Systemic urocortin 2, but not urocortin 1 or stressin1-A, suppresses feeding via CRF2 receptors without malaise and stress

BACKGROUND AND PURPOSE Infusion of corticotropin‐releasing factor (CRF)/urocortin (Ucn) family peptides suppresses feeding in mice. We examined whether rats show peripheral CRF/Ucn‐induced anorexia and determined its behavioural and pharmacological bases.

Urocortin 2 protects against retinal degeneration following bilateral common carotid artery occlusion in the rat

Urocortin 2 (Ucn 2) is corticotropin-releasing factor (CRF) paralog that preferentially activates CRF(2) receptors. Ucns exert CRF(2)-mediated cytoprotective effects against ischemia-reperfusion injury in cardiomyocytes. However, little is known regarding potential retinoprotective effects of Ucns despite the known presence of CRF family peptides and their receptors (predominantly CRF(2 alpha)) in retina. Therefore, the present study investigated the effects of post-ischemic intravitreal Ucn 2 (2 nmol) administration on ischemia-induced retinal degeneration. Two-month-old rats were subjected to permanent bilateral common carotid artery occlusion, and their retinas were processed histologically after two weeks survival to determine the density of viable cells in the ganglion cell layer and the thickness of all retinal layers. In vehicle-treated subjects, carotid occlusion reduced retina thickness by approximately 60% as compared to sham-operated animals. In contrast, intraocular Ucn 2 treatment led to a marked amelioration of the retinal layers, and the thickness of all layers was significantly increased by 40% compared to ischemic vehicle-treated subjects. Ucn 2 treatment also increased the number of cells by 55% in the ganglion cell layer as compared to those from carotid-occluded retinas of vehicle-treated subjects. These findings suggest that intraocular Ucn 2 treatment may protect against ischemia-induced retinal degeneration, results with potential therapeutic implications for ophthalmic diseases.

Maternal Western diet increases adiposity even in male offspring of obesity-resistant rat dams: early endocrine risk markers

Maternal overnutrition or associated complications putatively mediate the obesogenic effects of perinatal high-fat diet on developing offspring. Here, we tested the hypothesis that a Western diet developmental environment increases adiposity not only in male offspring from obesity-prone (DIO) mothers, but also in those from obesity-resistant (DR) dams, implicating a deleterious role for the Western diet per se. Selectively bred DIO and DR female rats were fed chow (17% kcal fat) or Western diet (32%) for 54 days before mating and, thereafter, through weaning. As intended, despite chow-like caloric intake, Western diet increased prepregnancy weight gain and circulating leptin levels in DIO, but not DR, dams. Yet, in both genotypes, maternal Western diet increased the weight and adiposity of preweanlings, as early as in DR offspring, and increased plasma leptin, insulin, and adiponectin of weanlings. Although body weight normalized with chow feeding during adolescence, young adult Western diet offspring subsequently showed decreased energy expenditure and, in DR offspring, decreased lipid utilization as a fuel substrate. By mid-adulthood, maternal Western diet DR offspring ate more chow, weighed more, and were fatter than controls. Thus, maternal Western diet covertly programmed increased adiposity in childhood and adulthood, disrupted relations of energy regulatory hormones with body fat, and decreased energy expenditure in offspring of lean, genetically obesity-resistant mothers. Maternal Western diet exposure alone, without maternal obesity or overnutrition, can promote offspring weight gain.

Enteric neuromuscular junctions: Comparison of ultrastructural features in different phylogenetic groups

The enteric neuromuscular junctions of snail (Helix pomatia), locust (Locusta migratoria migratorioides), cockroach (Periplaneta americana), carp (Cyprinus carpio) and tench (Tinca tinca) were studied by means of different light and electron microscopic methods. The nitroblue tetrazolium staining revealed that the myenteric plexuses of the above species are composed of nerve cells, a network of varicose nerves and nerve bundles. Instead of highly organized ganglia, single neurons or small groups of 2-4 cells are characteristic of the invertebrates and fish studied. Catecholaminergic fluorescence induced by glyoxylic acid was detected in the muscular layer of the entire alimentary tract in snail and the hindgut of tench. Fluorescent nerves and perikarya were frequent in the snail gut, while only nerves and no perikarya were found in tench. A close contact between enteric muscles and nerves is the most common form of enteric neuromuscular junction in both the smooth (i.e. the molluscan and fish gut) and the striated (i.e. the insect gut) musculature. The striated musculature (i.e. the insect gut, the oesophagus of carp, and the oesophagus, stomach and the midgut of tench) also receives a synaptic input. Cytochemical evidence is provided of the cholinergic character of fish motor endplates. The ultrastructural appearance and vesicle population of certain nerve terminals suggest a universal role of aminergic and peptidergic control in gut motility.

Relationship between appearance of GABA, fluorogenic monoamines and cytochrome oxidase activity during prenatal morphogenesis of chick myenteric plexus

SummaryThe basic histology of the developing embryonic gut wall of the chick was examined on haematein and eosin-stained paraffin sections. In parallel with this, the ontogenic sequence of myenteric plexus formation was followed on whole mounts after NADH diaphorase histochemistry. The presence of nerve elements was verified also by electron microscopy. The appearance of enteric γ-aminobutyric acid-containing neurons, as an example of an intrinsic inhibitory neuronal system, was studied by using an antiserum against the γ-aminobutyric acid glutaraldehyde bovine serum albumin conjugate. The development of noradrenergic innervation as an extrinsic inhibitory supply was followed by means of a glyoxylic acid-induced fluorescence method. Cytochrome oxidase activity was detected histochemically. Three consecutive steps of the morphogenesis of the myenteric plexus were revealed; first the appearance of a cellular crest at the mesenteric border on embryonic day 9; second the migration and clustering of nerve cells between embryonic days 10 and 16; then the elongation of neurites on embryonic days 16 and 21. Immunoreaclive and also fluorescent fibres were first detected on the 14th day of incubation, while immunopositive cell bodies appeared only after hatching. In the early stages the cytochrome oxidase activity was restricted to the perikarya, while at the end of embryonic development the activity also appeared in the ganglionic neuropile. On the basis of these observations, we concluded that there is a close time relation between the morphogenesis and the biochemical and functional maturation of the myenteric plexus.