Receptor Antagonist BIBP3226 Research Articles

Neuropeptide Y attenuates cardiac remodeling and deterioration of function following myocardial infarction

Plasma levels of neuropeptide Y (NPY) are elevated in patients with acute myocardial infarction (AMI), but its role in AMI remains unclear, which was examined here in NPY wild-type/knockout (WT/KO) mice treated with/without exogenous NPY and its Y1 receptor antagonist (Y1Ra) BIBP 3226. We found that AMI mice lacking NPY developed more severe AMI than WT mice with worse cardiac dysfunction, progressive cardiac inflammation and fibrosis, and excessive apoptosis but impairing angiogenesis. All of these changes were reversed when the NPY KO mice were treated with exogenous NPY in a dose-dependent manner. Interestingly, treatment with NPY also dose dependently attenuated AMI in WT mice, which was blocked by BIBP 3226. Phenotypically, cardiac NPY was de novo expressed by infiltrating macrophages during the repairing or fibrosing process in heart-failure patients and AMI mice. Mechanistically, NPY was induced by transforming growth factor (TGF)-β1 in bone marrow-derived macrophages and signaled through its Y1R to exert its pathophysiological activities by inhibiting p38/nuclear factor κB (NF-κB)-mediated M1 macrophage activation while promoting the reparative M2 phenotype invivo and invitro. In conclusion, NPY can attenuate AMI in mice. Inhibition of cardiac inflammation and fibrosis while enhancing angiogenesis but reducing apoptosis may be the underlying mechanisms through which NPY attenuates cardiac remodeling and deterioration of function following AMI.

Determination of neuropeptide Y Y1 receptor antagonist BIBP 3226 and evaluation of receptor expression based on liquid chromatography coupled with tandem mass spectrometry.

Neuropeptide Y (NPY) is a peptide widely distributed throughout the body that is involved in various physiological processes, including the regulation of feeding behavior and energy homeostasis. 5-Carbamimidamido-2-(2,2-diphenylacetamido)-N-[(4-hydroxyphenyl)methyl]pentanamide (BIBP 3226) is a selective NPY Y1 receptor antagonist with recognized application in bone regeneration studies, requiring quantification at picogram levels. Hence, BIBP 3226 determination is proposed here by a validated HPLC-MS/MS method, based on a reversed-phase Kinetex® core-shell C8 column (2.6μm, 150 × 2.1mm) at 30°C, elution in isocratic mode using a mixture of acetonitrile and water (30:70, v/v), containing 0.1% (v/v) formic acid, at 0.25mLmin-1, detection in positive ionization mode, and data acquisition in selected reaction monitoring mode. Calibration curves were linear for concentrations ranging from 0.25 to 30ngmL-1 with LOD and LOQ values as low as 0.1 and 0.3pg in cell extracts and 16 and 48pg in supernatant culture media, respectively. BIBP 3226 was successfully determined in cell extracts and supernatants obtained from internalization assays. Using similar exposure conditions, the amount of BIBP 3226 found in breast cancer cells (MCF7) was 72 to 657 times higher than that found in bone marrow cells (Wt C57BL/6 mice), providing an indirect indicator of NPY Y1 receptor expression.

QRFP administration into the medial hypothalamic nuclei improves memory in rats

Even though several of RFamide peptides have been shown to modify memory and learning processes in different species, almost nothing is known regarding cognitive effects of recently discovered neuropeptide QRFP. Considering multiple physiological functions of QRFP, localization of QRFP-synthesizing neurons in the hypothalamus and its' widely spread binding sites within the CNS, the present study was designed to investigate the possible role of QRFP in the consolidation of spatial memory.As target area for microinjection, the medial hypothalamic area, including dorsomedial (DMN) and ventromedial (VMN) nuclei, has been chosen. At first, the effects of two doses (200 ng and 400 ng) of QRFP were investigated in Morris water maze. After that receptor antagonist BIBP3226 (equimolar amount to the effective dose of neuropeptide) was applied to elucidate whether it can prevent effects of QRFP. To reveal possible changes in anxiety level, animals were tested in Elevated plus maze.The higher dose of QRFP (400 ng) improved short-term memory consolidation in Morris water maze. Pretreatment with antagonist BIBP3226 abolished cognitive effects of QRFP. The neuropeptide did not affect anxiety level of rats.This study provides unique evidence regarding the role of QRFP in the consolidation of memory and gives the basis for further investigations of neuropeptide's cognitive effects.

Rat mesenteric small artery neurogenic dilatation is predominantly mediated by β1 -adrenoceptors in vivo.

The prevailing dogma about neurogenic regulation of vascular tone consists of major vasodilatation caused by CGRP (and possibly substance P) released from sensory-motor nerves and vasoconstriction caused by noradrenaline, ATP and neuropeptode Y release from sympathetic nerves. Most studies on perivascular nerve-mediated vasodilatation are made in vitro. In the present study, we provide evidence indicating that in vivo electrical perivascular nerve stimulation in rat mesenteric small arteries causes a large β1-adrenoceptor-mediated vasodilatation, which contrasts with a smaller vasodilatation caused by endogenous CGRP that is only visible after inhibition of Y1 NPY receptors. Mesenteric arteries are densely innervated and the nerves are important regulators of vascular tone and hence blood pressure and blood flow. Perivascular sensory-motor nerves have been shown to cause vasodilatation in vitro. However, less is known about their function in vivo. Male Wistar rats (10-12 weeks old; n=72) were anaesthetized with ketamine (3mg kg-1 ) and xylazine (0.75mg kg-1 ) or pentobarbital (60mg kg-1 ). After a laparotomy, a section of second-order mesenteric artery was visualized in an organ bath after minimal removal of perivascular adipose tissue. The effects of electrical field stimulation (EFS) and drugs on artery diameter and blood flow were recorded with intravital microscopy and laser speckle imaging. EFS caused vasodilatation in arteries constricted with 1μm U46619 in the presence of 140μm suramin and 1μm prazosin. The vasodilatation was inhibited by 1μm tetrodotoxin and 5μm guanethidine, although not by the 1μm of the CGRP receptor antagonist BIBN4096bs. In the presence of 0.3μm Y1 receptor antagonist BIBP3226, BIBN4096bs partly inhibited the vasodilatation. Atenolol at a concentration 1μm inhibited the vasodilatation, whereas 0.1μm of the β2 -adrenoceptor selective antagonist ICI-118,551 had no effect. Increasing the extracellular [K+ ] to 20mm caused vasodilatation but was converted to vasoconstriction in the presence of 1μm BIBN4096bs, and constriction to 30mm potassium was potentiated by BIBN4096bs. Atenolol but not BIBN4096bs increased contraction to EFS in the absence of suramin and prazosin. In mesenteric small arteries of anaesthetized rats, EFS failed to stimulate major dilatation via sensory-motor nerves but induced sympathetic β1 -adrenoceptor-mediated dilatation.

Neuropeptide Y impairs the acquisition of conditioned defeat in Syrian hamsters

Recent evidence indicates that Neuropeptide Y (NPY) may function as a potent anxiolytic as well as a resilience factor that can insulate the brain from the effects of stress. However, most of these studies have utilized physical stressors such as shock or restraint. In the present study, we use an ethologically-based model in Syrian hamsters (Mesocricetus auratus) called Conditioned Defeat (CD) to investigate whether NPY can ameliorate the effect of social defeat stress. In the CD model, a male Syrian hamster is socially defeated by a larger, more aggressive conspecific. Subsequently, when paired with a smaller, non-aggressive intruder (NAI) in its own home cage, changes in its behavioral repertoire occur, including a reduction in aggression and chemosensory (social) investigation, and a concomitant increase in submissive behaviors. In Experiment 1, hamsters were infused intracerebroventricularly (icv) with NPY prior to social defeat, and 24-hours later, hamsters were exposed to a NAI. Results indicate that NPY significantly reduced submissive/defensive behaviors in socially defeated hamsters compared to control animals. In Experiment 2, we examined whether this effect was mediated by the NPY Y1 receptor. Subjects were first pre-treated with the Y1 receptor antagonist BIBP 3226 or vehicle, followed by NPY and then socially defeated. Upon testing with a NAI 24-hours later, pretreatment with BIBP 3226 failed to block the NPY effect compared to controls. These results demonstrate that NPY may function as an important resilience factor in socially defeated hamsters, but that these effects are not mediated by the Y1 receptor.

Central Neuropeptide Y Plays an Important Role in Mediating the Adaptation Mechanism Against Chronic Stress in Male Rats.

Exposure to continuous life stress often causes gastrointestinal (GI) symptoms. Studies have shown that neuropeptide Y (NPY) counteracts the biological actions of corticotrophin-releasing factor (CRF) and is involved in the termination of the stress response. However, in chronic repeated restraint stress (CRS) conditions, the actions of NPY on GI motility remain controversial. To evaluate the role of NPY in mediation of the adaptation mechanism and GI motility in CRS conditions, a CRS rat model was set up. Central CRF and NPY expression levels were analyzed, serum corticosterone and NPY concentrations were measured, and GI motor function was evaluated. The NPY Y1 receptor antagonist BIBP-3226 was centrally administered before stress loading, and on days 1 through 5 of repeated stress, the central CRF and the serum corticosterone concentrations were measured. In addition, gastric and colonic motor functions were evaluated. The elevated central CRF expression and corticosterone concentration caused by acute stress began to fall after 3 days of stress loading, whereas central NPY expression and serum NPY began to increase. GI dysmotility also returned to a normal level. Pretreatment with BIBP-3226 abolished the adaptation mechanism and significantly increased CRF expression and the corticosterone concentration, which resulted in delayed gastric emptying and accelerated fecal pellet output. Inhibited gastric motility and enhanced distal colonic motility were also recorded. CRS-produced adaptation, overexpressed central CRF, and GI dysmotility observed in acute restraint stress were restored to normal levels. Central NPY via the Y1 receptor plays an important role in mediating the adaptation mechanism against chronic stress.

Evidence for the involvement of neuropeptide Y in the antidepressant effect of imipramine in type 2 diabetes

Depression is a major comorbidity factor of diabetes and the outcome of one disorder influences the other. Our aim is to scrutinize the link between the two, if any. Since neuropeptide Y (NPY) system plays an important role in regulating central glucose sensing mechanisms, and also depression-related behavior, we test the involvement of NPY in the modulation of depression in type 2 diabetic mice. The mice were fed on high-fat diet and administered with low dose of streptozotocin to induce type 2 diabetes. These animals showed augmented plasma glucose and increased immobility time in tail suspension test (TST) suggesting induction of diabetes and depression. Intracerebroventricular (icv) treatment with NPY or NPY Y1 receptor agonist [Leu31, Pro34]-NPY and intraperitoneal treatment with imipramine decreased immobility time. However, opposite effect was produced by NPY Y1 receptor antagonist BIBP3226 (icv). Moreover, reduced immobility time by imipramine was potentiated by NPY and [Leu31, Pro34]-NPY, but attenuated by BIBP3226. Immunohistochemical analysis of the different nuclei of the extended amygdala, the region primarily involved in affective disorders, was undertaken. A significant reduction in NPY immunoreactivity in the central nucleus of amygdala, nucleus accumbens shell and lateral division of bed nucleus of stria terminalis of the diabetic mice was noticed; the response was ameliorated in imipramine treated animals. The results suggest that decreased NPY expression in the extended amygdala might be causally linked with the depression induced following type 2 diabetes and that the antidepressant action of imipramine in diabetic mice might be mediated by NPY-NPY Y1 receptor system.

N(ω)-Carbamoylation of the Argininamide Moiety: An Avenue to Insurmountable NPY Y1 Receptor Antagonists and a Radiolabeled Selective High-Affinity Molecular Tool ([(3)H]UR-MK299) with Extended Residence Time.

Analogues of the argininamide-type NPY Y1 receptor (Y1R) antagonist BIBP3226, bearing carbamoyl moieties at the guanidine group, revealed subnanomolar Ki values and caused depression of the maximal response to NPY (calcium assay) by up to 90% in a concentration- and time-dependent manner, suggesting insurmountable antagonism. To gain insight into the mechanism of binding of the synthesized compounds, a tritiated antagonist, (R)-N(α)-diphenylacetyl-N(ω)-[2-([2,3-(3)H]propionylamino)ethyl]aminocarbonyl-(4-hydroxybenzyl)arginin-amide ([(3)H]UR-MK299, [(3)H]38), was prepared. [(3)H]38 revealed a dissociation constant in the picomolar range (Kd 0.044 nM, SK-N-MC cells) and very high Y1R selectivity. Apart from superior affinity, a considerably lower target off-rate (t1/2 95 min) was characteristic of [(3)H]38 compared to that of the higher homologue containing a tetramethylene instead of an ethylene spacer (t1/2 3 min, Kd 2.0 nM). Y1R binding of [(3)H]38 was fully reversible and fully displaceable by nonpeptide antagonists and the agonist pNPY. Therefore, the insurmountable antagonism observed in the functional assay has to be attributed to the extended target-residence time, a phenomenon of relevance in drug research beyond the NPY receptor field.

Effects of direct QRFP-26 administration into the medial hypothalamic area on food intake in rats.

The RFamide peptide family comprises a number of biologically active peptides sharing RF motif at their C-terminal end. These peptides are involved in the control of multiple physiological functions including regulation of metabolism and feeding behavior. QRFP-43 as well as its 26-aminoacid residue QRFP-26 are able to cause orexigenic effect when administered to the rodents' cerebral ventricles. QRFPs have been suggested as the endogenous ligands of the previously orphan GPR103 receptors. GPR103 receptors share amino acid identity with other receptors of neuropeptides involved in feeding (NPY, NPFF, galanin). QRFP-26 expressing neurons and binding sites are densely present in the rat medial hypothalamus (MHA), an area directly responsible for the regulation of feeding. QRFP-26 was delivered to the target area by direct intrahypothalamic microinjection, and the consumption of liquid food was measured over a 60 min period. Both doses (100 and 200 ng) significantly increased food intake. Non-specific receptor antagonist BIBP3226 eliminated the orexigenic effect caused by QRFP-26 administration. Effective doses of QRFP-26 did not modify general locomotor activity and behavioral patterns examined in the open-field test. This study is the first reporting feeding modulating effects following direct intrahypothalamic QRFP-26 administration.

Neuropeptide FF receptors as novel targets for limbic seizure attenuation

Neuropeptide Y (NPY) is a well established anticonvulsant and first-in-class antiepileptic neuropeptide. In this study, the controversial role of NPY1 receptors in epilepsy was reassessed by testing two highly selective NPY1 receptor ligands and a mixed NPY1/NPFF receptor antagonist BIBP3226 in a rat model for limbic seizures. While BIBP3226 significantly attenuated the pilocarpine-induced seizures, neither of the highly selective NPY1 receptor ligands altered the seizure severity. Administration of the NPFF1/NPFF2 receptor antagonist RF9 also significantly attenuated limbic seizure activity. To further prove the involvement of NPFF receptors in these seizure-modulating effects, low and high affinity antagonists for the NPFF receptors were tested. We observed that the low affinity ligand failed to exhibit anticonvulsant properties while the two high affinity ligands significantly attenuated the seizures. Continuous NPFF1 receptor agonist administration also inhibited limbic seizures whereas bolus administration of the NPFF1 receptor agonist was without effect. This suggests that continuous agonist perfusion could result in NPFF1 receptor desensitization and mimic NPFF1 receptor antagonist administration. Our data unveil for the first time the involvement of the NPFF system in the management of limbic seizures.

Neuropeptide Y is crucial for nutritional state-dependent regulation of maternal behavior.

Lactation is indispensable for the survival of mammalian pups. However, any excess of energy expenditure for lactation over energy intake threatens the mother's survival. Here, we report that an orexigenic molecule, neuropeptide Y (NPY), mediates nutritional state-dependent regulation of maternal behavior. After 9h of fasting, dams showed a dramatic decrease in the expression of maternal behavior. Intracerebroventricular or direct dorsal raphe nucleus (DRN) injection of NPY inhibited the expression of maternal behavior in non-fasted dams. In contrast, injection of the NPY Y1 receptor antagonist BIBP-3226 into the DRN, in which the expression of the Y1 receptor was confirmed in serotonergic (5-HT) and GABAergic interneurons, recovered the expression of maternal behavior in fasted dams. When the pups were presented, the increase in the number of c-Fos-positive GABAergic, but not serotonergic, neurons was smaller in fasted than in non-fasted dams. These results suggest that NPY may inhibit pup-induced activation of GABAergic neurons via the Y1 receptor. Injection of a 5-HT1A agonist, GABAA receptor antagonist, or GABAB receptor antagonist into the DRN induced incomplete maternal behavior in non-fasted dams. In contrast, each of a 5-HT2A receptor agonist or a GABAB receptor agonist, but not a GABAA receptor agonist, recovered separate components of maternal behavior in fasted dams. These results suggest that NPY inhibits both 5-HT neuronal activity and its modulation via the GABA receptor in the DRN, resulting in the suppression of maternal behavior under food-restricted conditions.

Involvement of amygdaloid neuropeptide Y in the anxiolytic effects of acupuncture during ethanol withdrawal in rats

The role of neuropeptide Y (NPY) in the central nucleus of amygdala (CeA) in the preventive effects of acupuncture against ethanol withdrawal-induced anxiety was investigated. Rats were treated with 3g/kg/day of ethanol for 28 days, followed by 3 days of withdrawal. Bilateral acupuncture treatment at HT7 (Shen-Men), PC6 (Nei-Guan) or a non-acupoint was respectively added to the rats during the withdrawal once a day for three days. Enzyme-linked immunosorbent assays and real-time polymerase chain reaction analyses showed there was a significant decrease in NPY protein and mRNA expression in the CeA during ethanol withdrawal, which was reversed by acupuncture at HT7 but neither at PC6 nor at a non-acupoint. Acupuncture at HT7 also greatly inhibited the decrease in cAMP response element-binding protein (CREB) phosphorylation in the CeA. In elevated plus maze tests, a selective NPY Y1 receptor antagonist BIBP 3226 into the CeA before the acupuncture abolished almost completely the anxiolytic effect of acupuncture at HT7. These results suggest that acupuncture at HT7 rescues the depletion of amygdaloid NPY and reverses the decrease in CREB phosphorylation to produce anxiolytic effects during ethanol withdrawal.

Neuropeptide Y protects cerebral cortical neurons by regulating microglial immune function.

Neuropeptide Y has been shown to inhibit the immunological activity of reactive microglia in the rat cerebral cortex, to reduce N-methyl-D-aspartate current (INMDA) in cortical neurons, and protect neurons. In this study, after primary cultured microglia from the cerebral cortex of rats were treated with lipopolysaccharide, interleukin-1β and tumor necrosis factor-α levels in the cell culture medium increased, and mRNA expression of these cytokines also increased. After primary cultured cortical neurons were incubated with the lipopolysaccharide-treated microglial conditioned medium, peak INMDA in neurons increased. These effects of lipopolysaccharide were suppressed by neuropeptide Y. After addition of the neuropeptide Y Y1 receptor antagonist BIBP3226, the effects of neuropeptide Y completely disappeared. These results suggest that neuropeptide Y prevents excessive production of interleukin-1β and tumor necrosis factor-α by inhibiting microglial reactivity. This reduces INMDA in rat cortical neurons, preventing excitotoxicity, thereby protecting neurons.

Systemic treatment with neuropeptide Y receptor Y1-antagonist enhances atherosclerosis and stimulates IL-12 expression in ApoE deficient mice

AimsNeuropeptide Y (NPY) and Y1 receptors are involved in the mechanisms related to the development of atherosclerosis. We investigated the effects of systemically given NPY and its receptor Y1-antagonist on the development of atherosclerosis and associated inflammatory molecules in ApoE−/− mice during high-fat diet. MethodsFive weeks old ApoE−/− were fed atherogenic high cholesterol diet for 8weeks. The mice were injected with two doses of NPY (50 or 100μg/kg) or Y1 receptor antagonist BIBP3226 (100μg/kg) or vehicle intraperitoneally for 8weeks. Atherosclerosis lesion areas in aortic arch and descending aortas were determined, inflammatory molecules and NPY were determined in aortic wall, spleen, liver or in serum. ResultsNeuropeptide Y1 receptor antagonist, BIBP3226 (100μg/kg) increased atherosclerotic lesion areas compared to vehicle in descending aortas in ApoE−/− mice (p=0.021). The expression levels of macrophage-derived cytokine, interleukin-12 (IL-12) in spleens and livers were 8-fold increased with BIBP3226 (p=0.006 and p=0.003, respectively) as determined by RT-qPCR. Cholesterol levels in serum correlated positively with VCAM-1 expression (p=0.003) and negatively with NPY expression (p=0.044) in aortic wall in mice treated with BIBP 3226. ConclusionsThe results indicate that systemic treatment with Y1-antagonist enhances atherosclerosis development in ApoE deficient mice by triggering an overwhelming IL-12 production. The findings are highly valuable for evaluation of the development potential of Y1 ligands for therapeutics to treat or prevent atherosclerosis.

Central hypotensive effects of neuropeptide Y are modulated by endothelial nitric oxide synthase after activation by ribosomal protein S6 kinase

Neuropeptide Y (NPY) is a 36-amino acid polypeptide found abundantly in the central and peripheral nervous systems. NPY exerts a potent depressor effect via the activation of both Y(1) and Y(2) receptors in the nucleus tractus solitarii (NTS) of rats. However, the precise mechanisms involved in this NPY-mediated action remained unclear. Effects of a selective antagonist of Y(1) receptors, a PKC inhibitor, a PI3 kinase inhibitor, a NOS inhibitor, an endothelial NOS (eNOS)-selective inhibitor, a neuronal NOS (nNOS)-specific inhibitor or a MAPK inhibitor, on responses to microinjection of NPY into the NTS of Wistar-Kyoto rats were studied to determine the underlying mechanisms. Blood pressure and heart rate were measured and, in NTS, protein phosphorylation assessed by immunohistochemical techniques. Unilateral microinjection of exogenous NPY (4.65pmol/60nL) into the NTS of urethane-anesthetized Wistar-Kyoto rats markedly decreased blood pressure and heart rate. Microinjection of the Y(1) receptor antagonist BIBP3226 or the G(i) /G(o) -protein inhibitor, Pertussis toxin, into the NTS attenuated these NPY-induced hypotensive effects. A selective Y(1) receptor agonist increased expression of ERK1/2, ribosomal protein S6 kinase (RSK) and the phosphorylation of eNOS. RSK also bound directly to eNOS and induced its phosphorylation at Ser(1177) . Pretreatment of the NTS with an eNOS inhibitor, but not a nNOS inhibitor, attenuated the NPY-induced hypotensive effects. Together, these results suggested that NPY-induced depressor effects were mediated by activating NPY Y(1) receptor-PKC-ERK-RSK-eNOS and Ca(2+) -eNOS signalling pathways, which are involved in regulation of blood pressure in the NTS.

Role of Central Leptin Signaling in the Starvation-Induced Alteration of B-Cell Development

Nutritional deprivation or malnutrition suppresses immune function in humans and animals, thereby conferring higher susceptibility to infectious diseases. Indeed, nutritional deprivation induces atrophy of lymphoid tissues such as thymus and spleen and decreases the number of circulating lymphocytes. Leptin, a major adipocytokine, is exclusively produced in the adipose tissue in response to the nutritional status and acts on the hypothalamus, thereby regulating energy homeostasis. Although leptin plays a critical role in the starvation-induced T-cell-mediated immunosuppression, little is known about its role in B-cell homeostasis under starvation conditions. Here we show the alteration of B-cell development in the bone marrow of fasted mice, characterized by decrease in pro-B, pre-B, and immature B cells and increase in mature B cells. Interestingly, intracerebroventricular leptin injection was sufficient to prevent the alteration of B-cell development of fasted mice. The alteration of B lineage cells in the bone marrow of fasted mice was markedly prevented by oral administration of glucocorticoid receptor antagonist RU486 (11β-[p-(dimethylamino)phenyl]-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one). It was also effectively prevented by intracerebroventricular injection of neuropeptide Y Y(1) receptor antagonist BIBP3226 [(2R)-5-(diaminomethylideneamino)-2-[(2,2-diphenylacetyl)amino]-N-[(4-hydroxyphenyl)methyl]pentanamide], along with suppression of the otherwise increased serum corticosterone concentrations. This study provides the first in vivo evidence for the role of central leptin signaling in the starvation-induced alteration of B-cell development. The data of this study suggest that the CNS, which is inherent to integrate information from throughout the organism, is able to control immune function.

Influence of Neuropeptide Y and antidepressants upon cerebral monoamines involved in depression: An in vivo electrochemical study

Neuropeptide Y (NPY) and its receptors are present in the peripheral as well as the central nervous system (CNS). In vitro data have indicated NPY as an important mediator in the regulation of different diseases e.g. related to obesity, anxiety, depression, pain, memory loss and sleep disorders. In particular, studies of NPY levels in the cerebral spinal fluid (CSF) of depressed patients have shown a significant reduction of NPY levels when compared to control subjects. In addition, decreased concentrations of NPY were measured in the brain of suicide victims. These studies suggest that a reduction in cerebral NPY function may be associated with depressive symptoms. In the present work, a putative interaction between NPY, the catecholaminergic and serotonergic systems has been analysed by means of in vivo Differential Pulse Voltammetry (DPV) with treated carbon fibre micro electrodes (mCFE). It appeared that DPV with mCFE is an efficacious tool to monitor in vivo basal levels of catechols (Peak 2) and indoles (Peak 3) in discrete brain regions of rodents. Furthermore, it is shown that the peptidergic signal (Peak 5) simultaneously recorded with Peaks 2 and 3 in the amygdala could correspond to the oxidation of basal endogenous NPY. In addition, pharmacological treatments performed in vivo with exogenous NPY and with Y1 receptor antagonist BIBP3226 have indicated that these compounds interact positively with endogenous catecholaminergic and serotoninergic systems, in a way similar to that of the antidepressants imipramine and fluoxetine. In addition, the observed decrease of endogenous Peak 5 after treatment with imipramine or fluoxetine could be related to the concomitant stimulation of the catecholaminergic system with consequent reduced need for endogenous NPY. This would imply that NPY could be one of the endogenous chemicals acting on the maintenance of the mood. Thus, an antidepressant therapeutic potential of NPY and related compounds (e.g. BIBP3226) could be proposed.

NPY mediates ATP-induced neuroproliferation in adult mouse olfactory epithelium

In the CNS, ATP is released upon injury and promotes neuroproliferation via purinergic receptors. In the olfactory epithelium, ATP promotes the synthesis and release of neurotrophic factor NPY in neonates and induces neuroproliferation in neonatal and adult mice. We tested the hypothesis that NPY is involved in ATP-induced neuroproliferation in adult mice olfactory epithelium. Intranasal instillation of ATP significantly increased protein levels and number of NPY + cells. Pre-intranasal instillation of purinergic receptor antagonist PPADS significantly reduced ATP-induced upregulation of NPY. Intranasal instillation of NPY-Y1 receptor antagonist BIBP3226 following ATP instillation significantly inhibited the ATP-induced increase in BrdU incorporation, suggesting that NPY is released after ATP instillation and activates Y1 receptors to promote neuroproliferation. These data indicate that ATP initiates neuroproliferation via NPY upregulation, NPY release, and Y1 receptor activation, and suggests that the olfactory epithelium is good model to study neuroregenerative mechanisms in the CNS.

Tolerance to ethanol sedation and withdrawal hyper-excitability is mediated via neuropeptide Y Y1 and Y5 receptors

Aims Neuropeptide Y (NPY) is widely distributed throughout the brain and has been implicated in some of the actions of ethanol. The aim of the present study was to characterize the subtypes of NPY receptors in ethanol induced sedation, tolerance and withdrawal hyper-excitability. Main methods The loss of righting reflex paradigm was used to record the sleep duration in mice. Key findings The acute administration of ethanol (3–4 g per kg, i.p., 20% v/v) resulted in marked sedation. While prolonged ethanol consumption led to the development of tolerance, the mice showed hyper-excitability following ethanol withdrawal. Prior acute intracerebroventricular (i.c.v.) injection of NPY (5–20 ng per mouse) or NPY Y1 and Y5 receptors agonist [Leu 31, Pro 34]-NPY (0.02–0.2 ng per mouse) potentiated ethanol induced sedation. On the other hand, administration of selective NPY Y1 receptor antagonist BIBP3226 (5 ng per mouse, i.c.v.) inhibited ethanol induced sedation. Chronic concomitant treatment of NPY (20 ng per mouse, i.c.v.) or [Leu 31, Pro 34]-NPY (0.2 ng per mouse, i.c.v.) to ethanol-fed groups prevented the development of tolerance and attenuated withdrawal hyper-excitability. Moreover, acute treatment of NPY (5 ng per mouse, i.c.v.) or [Leu 31, Pro 34]-NPY (0.02 ng per mouse, i.c.v.) reversed the peak ethanol withdrawal hyper-excitability. Significance The results underscore a role for NPY Y1 and Y5 receptors in the ethanol induced sedation, tolerance and withdrawal hyper-excitability. We suggest that modulation of NPY Y1 and Y5 receptors may be a strategy to address the ethanol withdrawal conditions.

Neuropeptide Y induces gonadotropin-releasing hormone gene expression directly and through conditioned medium from mHypoE-38 NPY neurons

Neuropeptide Y (NPY) regulates reproductive function at the level of the hypothalamus through control of GnRH secretion. However, the direct control of GnRH gene expression by NPY has not yet been studied. GT1-7 neurons were treated with 100 nM of NPY over a 36 h time course. GnRH mRNA levels were significantly increased by NPY up to 12 h. We determined that GT1-7 neurons expressed Y1, Y2, and Y4 NPY receptors, but not Y5. Functional analysis of NPY receptor activation indicated that the Y1/Y4/Y5 receptor agonist [Leu31, Pro34] significantly induced cAMP accumulation in the GT1-7 neurons. Western blot studies demonstrated changes in the phosphorylation status of AKT, ERK1/2, CREB and ATF-1 after NPY exposure. Pharmacological inhibitors of the MAPK and PKA signal transduction pathways attenuated the NPY-mediated increase in GnRH transcription. This NPY-mediated increase in GnRH mRNA was also inhibited with the Y1-receptor specific antagonist BIBP-3226. The mHypoE-38 neurons secrete detectable levels of NPY and can be used as an endogenous source of NPY. Conditioned medium from mHypoE-38 neurons induced an increase in GnRH mRNA, which was inhibited by the Y1 receptor antagonist BIBP-3226. Together, these studies strengthen the evidence for the importance of NPY in the regulation of reproductive function.