Co-localization of neuropeptide tyrosine (NPY) and its C-terminal flanking peptide (C-PON)

Neuropeptide tyrosine (NPY) is one of the most abundant and widespread peptides in the mammalian nervous system. Recent isolation and sequencing of the DNA encoding NPY has predicted the existence of a 97 amino acid precursor peptide. Proteolytic processing of this precursor could yield three separate peptide products, an N-terminal signal peptide, neuropeptide tyrosine and a 30 amino acid C-terminal flanking peptide (C-PON). Here, we present evidence that the predicted C-flanking peptide of NPY is widely distributed in both the central and peripheral nervous systems of several mammalian species including man, and has an identical distribution to NPY. It was also demonstrated, using correlative light microscopic immunostaining on serial sections and double electron microscopic immunocytochemistry, that C-PON and NPY immunoreactivities are co-localized in neuronal cell bodies of the brain cortex, sympathetic ganglion cells, norepinephrine-containing granules of the adrenal medulla and in human pheochromocytoma tumor cells.     

Neuropeptide Y and autonomic nervous system

Neuropeptide Y (NPY) containing 6 amino acid residues belongs to peptides widely spread in the central and peripheral nervous system. NPY and its receptors play an extremely diverse role in the nervous system, including regulation of satiety, of emotional state, of vascular tone, and of gastrointestinal secretion. In mammals, NPY has been revealed in the majority of sympathetic ganglion neurons, in a high number of neurons of parasympathetic cranial ganglia as well as of intramural ganglia of the metasympathetic nervous system. At present, six types of receptors to NPY (Y₁–Y₆) have been identified. All receptors to NPY belong to the family of G-bound proteins. Actions of NPY on peripheral organs-targets are predominantly realized through postsynaptic receptors Y₁, Y₃–Y₅, and presynaptic receptors of the Y₂ type. NPY is present in large electrondense vesicles and is released at high-frequency stimulation. NPY affects not only vascular tone, frequency and strength of heart contractions, motorics and secretion of the gastrointestinal tract, but also has trophic effect and produces proliferation of cells of organs-targets, specifically of vessels, myocardium, and adipose tissue. In early postnatal ontogenesis the percent of the NPY-containing neurons in ganglia of the autonomic nervous system increases. In senescent organisms, this parameter decreases. This seems to be connected with the trophic NPY effect on cell-targets as well as with regulation of their functional state.     

Neuropeptide Y: anatomical distribution and possible function in mammalian nervous system

Neuropeptide Y (NYP) is a 36 amino acid peptide which shares considerable sequence homology with pancreatic polypeptide and peptide YY. NPY is widely distributed within neurons of the central and peripheral nervous systems, and occurs in mammalian brain in higher concentrations than all other peptides studied to date. Radioimmunoassay studies demonstrated high concentrations of NPY immunoreactivity within many regions of the hypothalamus and within the paraventricular thalamic nucleus, nucleus accumbens, the septum and medial amygdala. These findings correspond with the distribution of NPY containing terminals. Numerous cell bodies containing NPY are located within the cerebral cortex, caudate-putamen, hippocampus, hypothalamus, and nucleus tractus solitarius. Central administration of NPY causes a marked increase in ingestive behaviors, possibly related to the release of NPY from neurons in the arcuate nucleus that innervate the paraventricular nucleus of the hypothalamus. NPY projections from the arcuate nucleus to the medial preoptic area may be related to the central effects of NPY on luteinizing hormone release and sexual behavior. NPY immunoreactive terminals heavily innervated neurons within the amygdala and hypothalamus that are connected to the dorsal vagal complex, suggesting a role of NPY in central autonomic regulation. NPY terminals form a dense plexus around cerebral vessels and are probably responsible for NPY's potent vasoconstrictor effects in the cerebral cortex. Coronary vessels are also innervated heavily by NPY terminals, indicating a role for NPY in the pathogenesis of coronary vasospasm. NPY is present in pheochromocytomas and circulating levels of NPY may prove useful in the diagnosis of pheochromocytoma. Thus, anatomical and physiological studies suggest a varied, but important, function for NPY in mammalian nervous system.     

The intriguing mission of neuropeptide Y in the immune system

For many years, the central nervous system and the immune system were considered two autonomous entities. However, extensive research in the field of neuroimmunomodulation during the past decades has demonstrated the presence of different neuropeptides and their respective receptors in the immune cells. More importantly, it has provided evidence for the direct effects of neuropeptides on the immune cell functions. Neuropeptide Y (NPY) is generally considered the most abundant peptide in the central and peripheral nervous system. However, it is also distinguished by exhibiting pleiotropic functions in many other physiological systems, including the immune system. NPY affects the functions of the cells of the adaptive and innate immunity. In this respect, NPY is known to modulate immune cell trafficking, T helper cell differentiation, cytokine secretion, natural killer cell activity, phagocytosis and the production of reactive oxygen species. The specific Y receptors have been found in immune cells, and their expression is amplified upon immune stimulation. Different Y receptor subtypes may mediate an opposite effect of NPY on the particular function, thus underlining its regulatory role. Since the immune cells are capable of producing NPY upon appropriate stimulation, this peptide can regulate immune cell functions in an autocrine/paracrine manner. NPY also has important implications in several immune-mediated disorders, which affirms the clear need for further investigation of its role in either the mechanisms of the disease development or its possible therapeutic capacity. This review summarises the key points of NPY’s mission throughout the immune system.     

NPY L7P polymorphism and metabolic diseases

Neuropeptide Y (NPY) is an abundant and widespread peptide in mammalian nervous system, both in the central and peripheral nervous systems. NPY is a multifunctional neurotransmitter with multiple modulator effects in the regulation of physiological functions and responses in the body. NPY is a potent orexigenic peptide, which has effects on energy balance at the level of energy intake, expenditure, and partition. There are many association studies between the NPY gene variants and cardiovascular and metabolic disease. Most of them are done by using p.L7P substitution as a marker. At the moment it seems that the p.L7P substitution of preproNPY protein causes altered NPY secretion, which leads to haemodynamic disturbances caused by sympathetic hyperactivity and to various effects caused by altered local signalling by NPY. SNP association studies using p.L7P polymorphism suggest that this functional substitution may be a strong independent risk factor for various metabolic and cardiovascular diseases.     

Neuropeptide Y, peptide YY and aluminum in Alzheimer's disease: is there an etiological relationship?

Neuropeptide Y (NPY) and peptide YY (PYY) are members of the pancreatic polypeptide family which have a high degree of primary and tertiary structural homology. They function as neurotransmitters and humoral agents in central nervous system and gastrointestinal function. During the last two decades, NPY body fluid concentrations and NPY/PYY brain receptor numbers have been demonstrated to be altered during the course of Alzheimer's disease. Recent research has shown that both NPY and PYY may be involved in aluminum metabolism in animal models. A brief discussion of the structure, biological activity and possible involvement of these peptides in aluminum metabolism and Alzheimer's disease is contained herein.     

Attenuation of hypercholesterolemia and hyperglycemia in ob/ob mice by NPY Y2 receptor ablation

Neuropeptide Y (NPY) is a 36 amino acid peptide well known for its role in regulating food intake and energy homeostasis. It has previously been shown that the NPY Y2 receptor is required for a full biological response to leptin in the central nervous system. We have examined the impact of this receptor on plasma levels of lipid and cholesterol in wild type and obese (ob/ob) mice. The results show that an absence of Y2 in female mice has no effect on cholesterol level in normal lean mice but profoundly decreases serum cholesterol and glucose levels in ob/ob mice. We conclude that NPY, interacting with the Y2 receptor, participates in cholesterol and glucose homeostasis of obese mice.     

Molecular evolution of the neuropeptide Y (NPY) family of peptides: cloning of three NPY-related peptides from the sea bass (Dicentrarchus labrax)

Neuropeptide Y (NPY) is a 36-amino-acid peptide that is widely and abundantly expressed in the central nervous system of all vertebrates investigated. Related peptides have been found in various vertebrate groups: peptide YY (PYY) is present in gut endocrine cells of many species and pancreatic polypeptide (PP) is made in the pancreas of all tetrapods. In addition, a fish pancreatic peptide called PY has been reported in three species of fishes. The evolutionary relationships of fish PY have been unclear and it has been proposed to be the orthologue (species homologue) of each of the three tetrapod peptides. We demonstrate here with molecular cloning techniques that the sea bass (Dicentrarchus labrax), an acanthomorph fish, has orthologues of both NPY and PYY as well as a separate PY peptide. Sequence comparisons suggest that PY arose as a copy of the PYY gene, presumably in a duplication event separate from the one that generated PP from PYY in tetrapods. PY sequences from four species of fish indicate that, similar to PP, PY evolves much more rapidly than NPY and PYY. The physiological role of PY is unknown, but we demonstrate here that sea bass PY, like NPY and PYY but in contrast to the tetrapod PP, is expressed in brain.     

Peptide S is a novel potent inhibitor of voluntary and fast-induced food intake in rats

Peptide S (NPS or PEPS) and its cognate receptor have been recently identified both in the central nervous system and in the periphery. NPS/PEPS promotes arousal and has potent anxiolytic-like effects when it is injected centrally in mice. In the present experiment, we tested by different approaches its central effects on feeding behaviour in Long-Evans rats. PEPS at doses of 1 and 10 microg injected in the lateral brain ventricle strongly inhibited by more than 50% chow intake in overnight fasted rats with effects of longer duration with the highest dose (P<0.0001). A similar decrease was observed for the spontaneous intake of a high-energy palatable diet (-48%; P<0.0001). This anorexigenic effect was comparable to that induced by corticotropin-releasing hormone in fasted rats at equimolar doses. However, peptide S did not modify food intake stimulated by neuropeptide Y (NPY) at equimolar doses. It also did not affect the fasting concentrations of important modulators of food intake like leptin, ghrelin, and insulin in circulation. This study therefore showed that peptide S is a new potent anorexigenic agent when centrally injected. Its inhibitory action appears to be independent of the NPY, ghrelin, and leptin pathways. Development of peptide S agonists could constitute a new approach for the treatment of obesity.     

Neuropeptide Y (NPY) and depression: from animal studies to the human condition

Neuropeptide Y (NPY) is widely distributed throughout the central nervous system (CNS) and is one of the most conserved peptides in evolution, suggesting an important role in the regulation of basic physiological functions. In addition, both pre-clinical and clinical evidence have suggested that NPY, together with its receptors, may have a direct implication in several psychiatric disorders, including depression and related illnesses. NPY-like immunoreactivity and NPY receptors are expressed throughout the brain, with varying concentrations being found throughout the limbic system. Such brain structures have been repeatedly implicated in the modulation of emotional processing, as well as in the pathogenesis of depressive disorders. This review will concentrate on the distribution of NPY, its receptors, and the putative role played by this peptide in depressive illness based on both pre-clinical and clinical evidence.     

Receptor autoradiography as mean to explore the possible functional relevance of neuropeptides: focus on new agonists and antagonists to study natriuretic peptides, neuropeptide Y and calcitonin gene-related peptides

Over the past 20 years, receptor autoradiography has proven most useful to provide clues as to the role of various families of peptides expressed in the brain. Early on, we used this method to investigate the possible roles of various brain peptides. Natriuretic peptide (NP), neuropeptide Y (NPY) and calcitonin (CT) peptide families are widely distributed in the peripheral and central nervous system and induced multiple biological effects by activating plasma membrane receptor proteins. The NP family includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). The NPY family is composed of at least three peptides NPY, peptide YY (PYY) and the pancreatic polypeptides (PPs). The CT family includes CT, calcitonin gene-related peptide (CGRP), amylin (AMY), adrenomedullin (AM) and two newly isolated peptides, intermedin and calcitonin receptor-stimulating peptide (CRSP). Using quantitative receptor autoradiography as well as selective agonists and antagonists for each peptide family, in vivo and in vitro assays revealed complex pharmacological responses and radioligand binding profile. The existence of heterogeneous populations of NP, NPY and CT/CGRP receptors has been confirmed by cloning. Three NP receptors have been cloned. One is a single-transmembrane clearance receptor (NPR-C) while the other two known as CG-A (or NPR-A) and CG-B (or NPR-B) are coupled to guanylate cyclase. Five NPY receptors have been cloned designated as Y(1), Y(2), Y(4), Y(5) and y(6). All NPY receptors belong to the seven-transmembrane G-protein coupled receptors family (GPCRs; subfamily type I). CGRP, AMY and AM receptors are complexes which include a GPCR (the CT receptor or CTR and calcitonin receptor-like receptor or CRLR) and a single-transmembrane domain protein known as receptor-activity-modifying-proteins (RAMPs) as well as an intracellular protein named receptor-component-protein (RCP). We review here tools that are currently available in order to target each NP, NPY and CT/CGRP receptor subtype and establish their respective pathophysiological relevance.     

The Effects of NPY and Insulin on Food Intake Regulation in Fish

Recent abundant studies report that in rodents starvation inducesincreased neuropeptide Y (NPY) mRNA expression and peptide secretionin the hypothalamus which reduces autonomic nervous activityand promotes food intake, and intracerebroventricular (ICV)injection of NPY has potent orexigenic effects. Conversely,the effect of insulin in the central nervous system is to inhibitfood intake and NPY biosynthesis and secretion. In mammals bodyfatness is regulated and insulin acts as one intake inhibitorysignal related to fatness. In salmon (Oncorhynchus sp.) we havedemonstrated a rise in NPY-like mRNA expression and a coincidentdecrease in plasma insulin levels during 2 to 3 weeks of starvation.Additionally, experimentally manipulating body fatness withhigh and low fat diets has demonstrated that body fatness affectsfood intake in teleost fishes, raising the possibility thatNPY and insulin act to regulate their food intake. Therefore,we hypothesized that as in rodents, ICV treatment with NPY wouldstimulate food intake while ICV insulin would reduce food intake.Preliminary results suggest that ICV NPY administration doesstimulate food intake in channel catfish (Ictalurus punctatus),but central injection of insulin has no effect. Results of treatmentswith the sulfated octapeptide of cholecystokinin and the recombinantfragment of rat leptin 22–56 are also discussed.     

NPY- and C-PON-immunoreactive substances in the central nervous system of Helix pomatia

The distribution of neuropeptide-tyrosin (NPY)- and C-flanking peptide of neuropeptide-tyrosine (C-PON)-immunoreactivities in the central nervous system of the pulmonate gastropod, Helix pomatia, was investigated. NPY- and C-PON-like substances were localized in neuronal somata and neuntes, but were not co-localized within the same cells. NPY-immunoreactive substances were also found in endocrine/paracrine like cells located in the epineurium. C-PON and NPY, both reduced serotonin activated isometric contractions of Helix aorta, suggesting that they may act as modulators in the control of the vascular system.     

NPY样免疫反应在蝾螈发育过程中神经系统的定位研究

By immuno-cytochemical method the localization of neuropeptide Y (NPY) in the nervous system during embryonic development of Cynops orientalis was studied. The results revealed that NPY was first localized in the peripheral nervous system (late tail-bud stage), and later appeared in the central nervous system (larval stage) where it appeared with the appearance of glial cells. Very probably with the migration of neural crest cells NPY appeared first in the peripheral nervous system and then distributed to the central nervous system.     

NPY样免疫反应在蝾螈发育过程中神经系统的定位研究

用免疫组化方法研究在蝾螈胚胎发育中神经肽Y(NPY)在神经系统中的定位.结果表明NPY最早分布在周边神经系统(尾芽晚期),之后才在中枢神经系统出现(幼虫期),而且是随着胶质细胞的出现而出现的.我们认为NPY是随着神经嵴细胞的迁移而进入周边神经系统,再分布到中枢神经系统的.     

Neuropeptide-Y stimulation of insulin secretion is mediated via the nucleus tractus solitarius.

Neuropeptide-Y (NPY) is widely distributed in nervous tissue. In the central nervous system, NPY has been shown to be densely located in specific brain regions wherein it may mediate specific functions. Previous data have indicated that NPY may act at a selective site in the brain to modulate insulin secretion. In this study, we investigated the effect of NPY on NTS-mediated insulin secretion. A limited occipital craniotomy was performed on anesthetized rats to expose the caudal medulla in the region of the obex. NPY was microinjected into the NTS and blood samples were subsequently collected from the femoral vein. NPY microinjection resulted in a significant increase in insulin secretion within 5 minutes that returned to baseline at 30 minutes. However, microinjections of NPY did not significantly alter the plasma glucose in this model system. We conclude that NPY can act directly on the NTS to increase circulating insulin levels. Thus, the NTS may be a major brainstem site that directly mediates the central action of NPY on nutrient homeostasis.     

Electron paramagnetic resonance backbone dynamics studies on spin-labelled neuropeptide Y analogues.

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammalians. NPY acts by binding to at least five G-protein coupled receptors (GPCRs) which have been named Y1, Y2, Y4, Y5 and Y6. Three spin-labelled NPY analogues containing the nitroxide group of the amino acid TOAC (2.2.6.6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) as a paramagnetic probe were synthesized by solid-phase peptide synthesis. Synthetic problems owing to the sensitivity of nitroxide towards acidic and reducing conditions have been overcome by using a cleavage cocktail that contains anisole and cresol scavengers. Concerning the receptor binding preferences, the analogues [TOAC34]-pNPY and [Ala31, TOAC32]-pNPY showed a marked selectivity for the Y5 receptor, while [TOAC2]-pNPY maintained a significant binding also to the Y2 receptor subtype. The modifications of the native peptide structure caused by the introduction of TOAC were examined by circular dichroism. In order to determine the rotational correlation time of the spin probes, electron paramagnetic resonance measurements were performed in solution and in the presence of liposomes. This allowed us to evaluate the backbone dynamics of the different parts of the NPY molecule in the free and membrane bound states. The results of these studies showed that NPY Interacts with liposomes by using the C-terminal alpha-helix while the N-terminal tail retains a flexibility that is comparable to that of the peptide in solution as already shown by NMR studies on DPC micelles. Furthermore, we demonstrated that TOAC-labelllng is a valuable tool to investigate changes in the backbone conformation and dynamics. This may be of major importance for peptides and small proteins when they bind to cell membranes.     

Cytochemical demonstration of cholinergic, serotoninergic and peptidergic nerve elements in Gorgoderina vitelliloba (Trematoda: Digenea)

Standard enzyme cytochemical and indirect immunocytochemical techniques have been used in conjunction with light and confocal scanning laser microscopy (CSLM) to visualize cholinergic, serotoninergic and peptidergic nerve elements in whole-mount preparations of the amphibian urinary-bladder fluke, Gorgoderina vitelliloba. Cholinesterase (ChE) activity was localized in paired anterior ganglia, a connecting dorsal commissure and in the origins of the ventral nerve cords. Cholinergic ganglia were also evident in shelled embryos in the uterus. Serotonin-immunoreactivity (IR) was more extensive than ChE activity and was identified in both the central and peripheral nervous systems. Serotoninergic nerve fibres were associated with the somatic musculature and female reproductive ducts. Antisera to nine mammalian peptides and one invertebrate (FMRFamide) peptide have been used to investigate the peptidergic nervous system in the parasite. Immunoreactivity was obtained to five peptides, namely pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), substance P (SP) and FMRFamide. Peptidergic nerve fibres were found to be more abundant than demonstrable cholinergic or serotoninergic nerve fibres. NPY-IR was identified only in the main components of the central nervous system. However, PP- and PYY-IR occurred in the anterior ganglia, dorsal commissure, main nerve cords and in numerous small varicose fibres that ramified throughout the worm. Additionally, PP-immunoreactive nerve fibres were found to innervate the musculature of the female reproductive tracts. Six sites of IR were found in the acetabulum, using antisera directed towards the C-terminal end of PP and PYY, and these matched with the distribution of six non-ciliated rosette-like papillae observed by scanning electron microscopy. SP- and FMRFamide-IR were identified in the CNS, and FMRFamide-immunopositive nerve fibres were also evident in association with the gonopore cirrus region and with the terminal excretory pore. Results are discussed with respect to possible roles for each of the neurochemical types.     

Duplication of neuropeptide Y and peptide YY in Nile tilapia Oreochromis niloticus and their roles in food intake regulation

In vertebrates, the neuropeptide Y (NPY) family peptides have been recognized as key players in food intake regulation. NPY centrally promotes feeding, while peptide YY (PYY) and pancreatic polypeptide (PP) mediate satiety. The teleost tetraploidization is well-known to generate duplicates of both NPY and PYY; however, the functional diversification between the duplicate genes, especially in the regulation of food intake, remains unknown. In this study, we identified the two duplicates of NPY and PYY in Nile tilapia (Oreochromis niloticus). Both NPYa and NPYb were primarily expressed in the central nervous system (CNS), but the mRNA levels of NPYb were markedly lower than those of NPYa. Hypothalamic mRNA expression of NPYa, but not NPYb, decreased after feeding and increased after 7-days of fasting. However, both NPYa and NPYb caused a significant increase in food intake after an intracranial injection of 50 ng/g body weight dose. PYYb, one of the duplicates of PYY, had an extremely high expression in the foregut and midgut, whereas another form of duplicate PYYa showed only moderate expression in the CNS. Both hypothalamic PYYa and foregut PYYb mRNA expression increased after feeding and decreased after 7-days of fasting. Furthermore, the intracranial injection of PYYb decreased food intake, but PYYa had no significant effect. Our results suggested that although the mature peptides of NPYa and NPYb can both stimulate food intake, NPYa is the main endogenous functional NPY for feeding regulation. A functional division has been identified in the duplicates of PYY, which deems PYYb as a gut-derived anorexigenic peptide and PYYa as a CNS-specific PYY in Nile tilapia.     

Role of atrial natriuretic peptides and neuropeptide Y in blood pressure regulation

Atrial natriuretic peptides (ANP) are released into the circulation in response to enhanced atrial stretching. These peptides not only have diuretic and natriuretic properties, but also exert a relaxing effect on the vasculature. Moreover, they antagonize the contractions induced by norepinephrine and angiotensin II. Neuropeptide Y (NPY) is also a vasoactive peptide. It is widely distributed throughout the central and peripheral nervous systems. NPY is coreleased with norepinephrine by perivascular nerve endings. At high concentrations, this peptide has a direct vasoconstrictor effect. In addition, it enhances the vascular effect of various agonists, including norepinephrine and angiotensin II. Both ANP and NPY have an inhibitory effect on renin secretion. This effect may have important implications for the role of these peptides in cardiovascular regulation.