Neuropeptide Y and neuropeptide Y18-36. Structural and biological characterization

Neuropeptide Y (NPY), a 36-residue peptide amide, has been shown by numerous studies to be a potent vasoconstrictor. In order to gain an appreciation of the structural requirements for this action, we have previously synthesized a number of fragments of NPY. It had been shown that sequential deletions from the N-terminus resulted in peptides with decreasing hypertensive activity. In the present study we present data supporting the unexpected finding of two fragments, NPY17-36 and NPT18-36 with substantial hypotensive action in vivo. This action was dose dependent (data not shown) and was also observed to a lesser extent with NPY19-36 but not NPY16-36 or NPY20-36. It was, however, slower in onset and of longer duration than the hypertensive action of NPY. These differing kinetics of action may suggest that NPY and NPY18-36 act through different mechanisms. Structural studies using circular dichroism were performed. While NPY was found to assume an ordered helical structure in both aqueous buffer and trifluoroethanol (TFE), 30% TFE in aqueous buffer was required to induce substantial helicity for NPY18-36. This structural investigation suggests that both NPY and NPY18-36 assume an ordered conformation upon reaching the lipid rich receptor environment.     

Neuropeptide Y microinjected into the suprachiasmatic region phase shifts circadian rhythms in constant darkness

The geniculohypothalamic tract (GHT) is a projection from the intergeniculate leaflet to the suprachiasmatic nucleus (SCN). The GHT exhibits neuropeptide Y (NPY) immunoreactivity and appears to communicate photic information to the SCN. Microinjection of NPY into the SCN has been found to phase shift circadian rhythms of hamsters housed in constant light in a manner similar to the phase shifts produced by pulses of darkness or triazolam injections. In the present study, NPY was injected into the SCN of Syrian hamsters housed in constant darkness and was found to produce phase shifts similar to those seen in hamsters housed in constant light. Microinjections were not followed by wheel running during the subjective day (the time when NPY microinjections are followed by significant phase advances). These data suggest that NPY produces phase shifts by some mechanism other than by inducing wheel running or by inhibiting the response of SCN neurons to light and supports a role for NPY in nonphotic shifting of the circadian clock.     

Control of the expression of human neuropeptide Y by leptin: in vitro studies

Neuropeptide Y (NPY) participates in the regulation of reproduction and food intake. The adipose-secreted hormone, leptin, has also been involved in these processes, and has been shown to exert its effects in part by controlling NPY synthesis and release at the hypothalamic level. In the present study, we utilized the SH-SY5Y human neuroblastoma cell line, to study the leptin-NPY interrelationships. SH-SY5Y cells were found to express leptin receptors (RT-PCR and Western blot analyses). A 24-h treatment with leptin at different concentrations did not affect NPY gene expression, but resulted in a stimulation of NPY release. This stimulated secretion was blocked by the combined treatment with leptin and the muscarinic agonist carbachol or the phorbol ester TPA. Leptin and carbachol also caused an increased intracellular content of NPY. In conclusion, the SH-SY5Y human neuroblastoma cell line appears to be a suitable in vitro model for studying the pharmacological effects of leptin on the biosynthesis and secretion of NPY.     

The ATPase domain of hsp70 possesses a unique binding specificity for 3'-sulfogalactolipids

The region(s) of hsp70 critical for sulfogalactolipid (SGL) recognition has been defined through deletion analysis and site-directed mutagenesis. Truncated polymerase chain reaction products of hsp70 generated N-terminal fragments of 43, 35, 29, and 22 kDa. The C terminus substrate-binding domain (28 kDa) was also expressed. The N-terminal ATPase domain (rP43) shared the binding specificity of hsp70, because only sulfogalactosyl ceramide and sulfogalactosyl glycerolipid were recognized by both TLC overlay and RELISA. The C-terminal domain showed no binding. SGL binding of rP29 and rP22 was severely reduced. The loss of SGL binding for rP35 by RELISA but not TLC overlay was considered as a function of receptor presentation. The truncation of rP43 to rP35 demonstrates that residues 318-387 (the base of the ATP binding cleft) are critical for high affinity SGL binding. Mutagenesis showed that Arg(342) and Phe(198) are crucial for this process. SGL binding, mediated by these conserved residues within the ATPase domain of hsp70, implies that this binding specificity is evolutionarily conserved.     

New molecular evidence that Teladorsagia circumcincta (Nematoda: Trichostrongylidea) is a species complex

In the polymorphic Teladorsagia circumcincta (morphs circumcincta and trifurcata), a sheep and goat line (SGL) and a goat line (GL) have been previously described on the basis of the malate dehydrogenase allozyme polymorphism (MDH-2) and of the morphology of the dorsal ray. The GL were never found alone in 1 host, so the status of species was not given to these 2 lines. To investigate further whether there are other genetic markers that will delineate them, we collected T. circumcincta worms from goat and sheep at 8 farms in Touraine (west-central France). The worms were identified individually as being SGL or GL on the basis of MDH-2 polymorphism. This distinctiveness was corroborated by sequences of the beta-tubulin isotype I gene, the second internal transcribed spacer (ITS2) of their rDNA, and the nicotinamide dehydrogenase (ND4) gene of their mDNA. The extent of the divergence in the 3 additional genetic markers was such that SGL and GL may be considered as 2 species. A third putative species was found in the SGL line based exclusively on the ND4 gene. These findings suggest that T. circumcincta is a species complex and that further investigation is required on a wider geographic scale.     

Characterization of the ceramide moieties of sphingoglycolipids from mouse brain by ESI-MS/MS: identification of ceramides containing sphingadienine

Sphingoglycolipids (SGLs) are cell membrane constituents. As the ceramide structure influences the biological properties of the SGL, we characterized by electrospray ionization tandem mass spectrometry the molecular species of ceramides present in SGL of mouse brain. We report here for the first time the presence in mammalian brain of sphingadienine (d18:2). Sphingenine (d18:1) is present in all SGL species, in contrast to eicosasphingenine (d20:1), which is a constituent of only gangliosides. Sphingadienine is present in galactosylceramide and sulfatides. Free ceramides contain the three types of bases. Thus, there could be two separate pools of free ceramides (d18:1, d18:2 and d20:1, d18:1) as precursors of complex SGL.     

Leptin and neuropeptide Y (NPY) modulate nitric oxide synthase: further evidence for a role of nitric oxide in feeding.

Recent studies have suggested a role for nitric oxide in the regulation of food intake. Neuropeptide Y (NPY) is one of the most potent orexigenic agents. Chronic administration of leptin decreases food intake. This study examined the effects of NPY and leptin on nitric oxide synthase (NOS) in the hypothalamus. Previously it has been demonstrated that obese (ob/ob) mice have elevated NOS levels in the hypothalamus. In this study we demonstrated that the administration of leptin (6 microg/day) subcutaneously (SC) for 3 days decreased body weight (P < 0.001) and food intake P < 0.001) in obese (ob/ob) mice as expected. In addition, leptin decreased NOS in the hypothalamus nu 37% (P < 0.01) and in brown adipose tissue by 69% (P < 0.01) but not in white adipose tissue. NPY was administered intracerebroventricularly to CD-1 mice at doses of 0.25 and 0.50 microg. Mice were sacrificed 15 min after injection and NOS was measured in their hypothalami. NPY at the lower dose increased NOS in the hypothalamus by 147%. These results, taken together, with previously published studies support the concept that nitric oxide may play a role as a mediator of the effects of NPY and leptin on food intake. The alterations of NOS in brown adipose tissue following leptin administration could result in changes in blood flow or metabolism in the brown fat.     

格拉姆柱花草(Stylosanthes gracilis H.B.K.cv.Graham)凝集素的纯化与性质研究

为了探讨凝集素在豆科植物与根瘤菌的识别过程中的作用,用ＤＥＡＥ－３２离子交换层析和ＳｅｐｈａｄｅｘＧ－１５０凝胶过滤分离、纯化格拉姆柱花草种子凝集素（ＳＧＬ）,其分子量约为４５ｋＤ,由两个相同的亚基组成,等电点约ｐＨ５．８,它是一种糖蛋白,含糖量约为２．６％．ＳＧＬ的热稳定性强．ＳＧＬ的血凝活性能被甘露糖所抑制．ＳＧＬ对红细胞的凝集作用可能具有种属专一性;ＳＧＬ具有强的促有丝分裂作用;荧光标记实验显示：９株能与格拉姆柱花草植株结瘤的菌株有７株能与ＳＧＬ结合,６株不能与之结瘤的菌株,只有１株能与ＳＧＬ结合,这表明不同根瘤菌菌株对ＳＧＬ的结合能力,和它们在格拉姆柱花草上结瘤能力之间可能具有一定的生物学相关性．     

Organization of calbindin D28K-immunoreactive neurons in the dog superior colliculus

We localized calbindin D28K-immunoreactive (IR) neurons in the superior colliculus (SC) of the dog and studied the distribution and effect of enucleation on the distribution of this protein. We also compared this labeling to that of GABA. Calbindin D28K was localized with antibody immunocytochemistry. Calbindin D28K-IR neurons formed three laminar tiers in the SC, one within the lower superficial gray layer (SGL), the second within the upper intermediate gray layers (IGL), and the third within the deep gray layer (DGL). The third tier was not very distinctive when compared with the other two tiers. Calbindin D28K-IR neurons in the SC varied dramatically in morphology and size, and included round/oval, vertical fusiform, stellate, pyriform, and horizontal neurons. Neurons with varicose dendrite were also labeled in the IGL. Enucleation appeared to have no effect on the distribution of calbindin D28K-IR neurons in the contralateral SC. Two-color immunofluorescence revealed that a small percentage (11.20%) of calbindin D28K-IR neurons co-localized with GABA. The current results demonstrate that the patterned distribution of calbindin D28K-IR neurons in the intermediate and deep SC is comparable with other animals, but that the distribution of this protein in the superficial SC is strikingly different from that in previously studied animals. The results also suggest that retinal projection may not control the activity of the expression of calbindin D28K in the dog SC. These results will not only provide valuable knowledge of the basic neurochemical architecture of the dog visual system, but also provide clues for the understanding of the similarities and differences among species.     

NPY and stress 30 years later: the peripheral view

Almost 30 years ago, neuropeptide Y (NPY) was discovered as a sympathetic co-transmitter and one of the most evolutionarily conserved peptides abundantly present all over the body. Soon afterward, NPY's multiple receptors were characterized and cloned, and the peptide's role in stress was first documented. NPY has proven to be pivotal for maintaining many stress responses. Most notably, NPY is known for activating long-lasting vasoconstriction in many vascular beds, including coronary arteries. More recently, NPY was found to play a role in stress-induced accretion of adipose tissue which many times can lead to detrimental metabolic changes. It is however due to its prominent actions in the brain, one of which is its powerful ability to stimulate appetite as well as its anxiolytic activities that NPY became a peptide of importance in neuroscience. In contrast, its actions in the rest of the body, including its role as a stress mediator, remained, surprisingly underappreciated and not well understood. Our research has focused on that other, "peripheral" side of NPY. In this review, we will discuss those actions of NPY on the cardiovascular system and metabolism, as they relate to adaptation to stress, and attempt to both distinguish NPY's effects from and integrate them with the effects of the classical stress mediators, glucocorticoids, and catecholamines. To limit the bias of someone (ZZ) who has viewed the world of stress through the eyes of NPY for over 20 years, fresh insight (DH) has been solicited to more objectively assess NPY's contributions to stress-related diseases and the body's ability to adapt to stress.     

Neuropeptide Y effects on vasorelaxation and intestinal contraction in the Atlantic cod Gadus morhua

Neuropeptide Y (NPY) has prominent cardiovascular effects in mammals and sharks, but no such effect has previously been demonstrated in any teleost fish. In the Atlantic cod, we found that cod NPY (10(-10)-10(-6) M) relaxed celiac arteries precontracted with epinephrine, and weak contractions were elicited in intestinal ring preparations. A few NPY-immunoreactive nerve fibers were present along small gut arteries. The results suggest that cod NPY produces vasorelaxation both by a direct action on smooth muscle and by release of prostaglandins, but with no involvement of nitric oxide, leukotrienes, or endothelium-derived relaxing factors. An additional indirect effect involving another neurotransmitter may occur. Cod NPY (10(-7) M) and human NPY (10(-7) M) had identical effects on the vessels. Small differences only in the effects of porcine [Leu(31),Pro(34)]NPY, NPY-(13-36), and cod NPY suggest the presence of a Y(1) subfamily receptor, similar to the zebrafish Ya receptor. A physiological role for NPY in teleost vasculature is concluded, but surprisingly the effect, a vasodilation, is opposite to that in mammals and is mediated by prostaglandins.     

Plasma concentration of neuropeptide Y in patients with adrenal hypertension.

The mechanisms of hypertension during primary hyperaldosteronism and Cushing's syndrome are not completely understood. An enhanced vascular sensitivity to noradrenaline has been described in both situations. Neuropeptide Y (NPY) induces direct vasoconstriction and potentiates the action of noradrenaline. Sodium retention and dexamethasone have been shown to increase circulating NPY levels in animals and the expression of NPY in neuroendocrine cells. In order to determine if NPY could be involved in the enhanced vascular sensitivity to noradrenaline associated with adrenocortical hyperactivity, we measured plasma NPY in patients with Cushing's syndrome (n = 26) and primary hyperaldosteronism (n = 15) and compared it with that of hypertensive patients with pheochromocytomas (n = 13) or essential hypertension (n = 51) and with normotensive controls (n = 47). The concentration of NPY-Like immunoreactivity (NPY-Li) (mean +/- S.E.) in controls was 39.6 +/- 3.0 pg/ml. Elevated concentrations were found in 77% of the samples collected from pheochromocytoma patients (1180.4 +/- 394.0 pg/ml). NPY-Li levels in patients with essential hypertension (35.0 +/- 2.6 pg/ml), primary hyperaldosteronism (31.3 +/- 3.9 pg/ml) and Cushing's syndrome (33.1 +/- 4.8 pg/ml) were not different from that of controls. NPY-Li levels in hypertensive and normotensive patients with Cushing's syndrome were similar (38.5 +/- 7.5 vs 24.2 +/- 3.7 pg/ml). No correlation was found between the NPY-Li level and the mean blood pressure at the time of sampling. Our results suggest that NPY is unlikely to be involved in the pathogenesis of hypertension associated with primary hyperaldosteronism and Cushing's syndrome.     

Deciphering sulfoglycolipids of Mycobacterium tuberculosis

For 4 decades, in vivo and in vitro studies have suggested that sulfoglycolipids (SGLs) play a role in the virulence or pathogenesis of the tubercle bacilli. However, the SGL structure and biosynthesis pathway remain only partially elucidated. Using the modern tools of structural analysis, including MALDI-time-of-flight MS, MS/MS, and two-dimensional NMR, we reevaluated the structure of the different SGL acyl (di-, tri-, and tetra-acylated) forms of the reference strain Mycobacterium tuberculosis H37Rv, as well as those produced by the mmpL8 knockout strains previously described to intracellularly accumulate di-acylated SGL. We report here the identification of new acyl forms: di-acylated SGL esterified by simple fatty acids only, as well as mono-acylated SGL bearing a hydroxyphthioceranoic acid, which were characterized in the wild-type strain. In a clinical strain, a complete family of mono-acylated SGLs was characterized in high abundance for the first time. For the mmpL8 mutant, SGLs were found to be esterified i) by an oxophthioceranoic acid, never observed so far, and ii) at nonconventional positions in the case of the unexpected tri-acylated forms. Our results further confirm the requirement of MmpL8 for the complete assembly of the tetra-acylated forms of SGL and also provide, by the discovery of new intermediates, insights in terms of the possible SGL biosynthetic pathways.     

High concentrations of a novel peptide, neuropeptide Y, in the innervation of mouse and rat heart

A newly discovered bioactive peptide, neuropeptide Y (NPY), has been found in the innervation of the mouse and rat heart by immunocytochemistry, NPY-immuno-reactive nerves were very dense around the nodal tissues. They also surrounded the coronary arteries and arterioles and were found in close association with the cardiac muscle. The distribution of NPY-containing nerves paralleled that of noradrenergic fibers, demonstrated by the use of antibodies to the catecholamine-converting enzymes tyrosine hydroxylase and dopamine beta-hydroxylase. Furthermore, NPY was seen to be present in a proportion of intrinsic neurons mostly found in the atria and in close proximity to the nodal tissue. The concentrations of extractable NPY-immunoreactive material (about 150 pmol/g in whole mouse heart) by far surpasses those of the other peptides so far reported in the cardiac tissue. High performance liquid chromatography demonstrated the NPY immunoreactivity to elute in a single sharp peak, in an identical position to brain NPY.     

Stimulation of apical Cl⁻/HCO₃⁻(OH⁻) exchanger, SLC26A3 by neuropeptide Y is lipid raft dependent

Neuropeptide Y (NPY), an important proabsorptive hormone of the gastrointestinal tract has been shown to inhibit chloride secretion and stimulate NaCl absorption. However, mechanisms underlying the proabsorptive effects of NPY are not fully understood. The present studies were designed to examine the direct effects of NPY on apical Cl?/HCO??(OH?) exchange activity and the underlying mechanisms involved utilizing Caco2 cells. Our results showed that NPY (100 nM, 30 min) significantly increased Cl?/HCO??(OH?) exchange activity (～2-fold). Selective NPY/Y1 or Y2 receptor agonists mimicked the effects of NPY. NPY-mediated stimulation of Cl?/HCO??(OH?) exchange activity involved the ERK1/2 MAP kinase-dependent pathway. Cell surface biotinylation studies showed that NPY does not alter DRA (apical Cl?/HCO??(OH?) exchanger) surface expression, ruling out the involvement of membrane trafficking events. Interestingly, DRA was found to be predominantly expressed in the detergent-insoluble (DI) and low-density fractions (LDF) of human colonic apical membrane vesicles (AMVs) representing lipid rafts. Depletion of membrane cholesterol by methyl-β-cyclodextrin (MβCD, 10 mM, 1 h) remarkably decreased DRA expression in the DI fractions. Similar results were obtained in Triton-X 100-treated Caco2 plasma membranes. DRA association with lipid rafts in the DI and LDF fractions of Caco2 cells was significantly enhanced (～45%) by NPY compared with control. MβCD significantly decreased Cl?/HCO??(OH?) exchange activity in Caco2 cells as measured by DIDS- or niflumic acid-sensitive 3?Cl? uptake (～50%). Our results demonstrate that NPY modulates Cl?/HCO??(OH?) exchange activity by enhancing the association of DRA with lipid rafts, thereby resulting in an increase in Cl?/HCO??(OH?) exchange activity. Our findings suggest that the alteration in the association of DRA with lipid rafts may contribute to the proabsorptive effects of NPY in the human intestine.     

Evolution of neuropeptide Y and its related peptides

1. The neuropeptide Y (NPY) family of peptides includes also the gut endocrine peptide YY (PYY), tetrapod pancreatic polypeptide (PP), and fish pancreatic peptide-tyrosine (PY). All peptides are 36 amino acids long.2. Sequences from many types of vertebrates show that NPY has remained extremely well conserved throughout vertebrate evolution with 92% identity between mammals and cartilaginous fishes.3. PYY has 97–100% identity between cartilaginous fishes and bony fishes, but is less conserved in amphibians and mammals (83% identity between amphibians and sharks and 75% identity between mammals and sharks).4. NPY and PYY share 70–80% identity in most species.5. Both NPY and PYY were present in the early vertebrate ancestor because both peptides have been found in lampreys.6. The tissue distribution appears to have been largely conserved between phyla, except that PYY has more widespread neuronal expression in lower vertebrates.7. Pancreatic polypeptide has diverged considerably among tetrapods leaving only 50% identity between mammals, birdsJreptiles and frogs.8. Several lines of evidence suggest that the PP gene arose by duplication of the PYY gene, probably in the early evolution of the tetrapods.9. The pancreatic peptide PY found in anglerfish and daddy sculpin may have resulted from an independent duplication of the PYY gene.10. The relationships of the recently described mollusc and worm peptides NPF and PYF with the NPY family still appear unclear.     

Immunocytochemical localization of neuropeptide Y-like immunoreactivity in adrenergic and non-adrenergic neurons of the rat gastrointestinal tract

The immunocytochemical location of neuropeptide Y (NPY)-like immunoreactivity (LI) within the neuronal structures of the rat gastrointestinal (GI) tract was investigated with the indirect immunofluorescence method. NPY immunoreactive neurons were found throughout all regions of the GI tract with the largest number in the duodenum. NPY immunoreactive perikarya were mainly located in the submucosal ganglia. NPY labeled processes were extensively seen in the submucosal and myenteric plexuses, smooth muscles, muscularis mucosa, mucosa and surrounding blood vessels. Following 6-hydroxydopamine (6-OHDA) treatment, NPY immunoreactive nerve fibers around blood vessels disappeared completely and the reactive fibers in other regions were reduced in number. NPY immunoreactive nerve cell bodies in the ganglionic plexuses, however, were not affected by 6-OHDA treatment. Serial sections of the coeliac ganglion showed that NPY-LI was present in cell bodies which also displayed tyrosine hydroxylase (TH) immunoreactivity. Our results suggest that NPY is abundantly contained in both adrenergic and non-adrenergic neurons of the gut and may play an important role in the regulation of the GI tract.     

Neuropeptide Y immunoreactivity in human cerebrospinal fluid

Neuropeptide Y (NPY) immunoreactivity has been determined in human cerebrospinal fluid (CSF). High pressure liquid chromatography revealed that the NPY immunoreactivity co-eluted with authentic NPY. The range of NPY levels was 108 +/- 18 pg/ml (mean +/- S.D.) in a group of 28 normal subjects. In five additional subjects NPY immunoreactivity was measured in 4 sequential 8 ml aliquots of CSF to determine whether a rostro-caudal gradient was present. No significant differences in NPY levels were detected between any of the 4 aliquots.     

Neuropeptide Y potentiates beta-adrenergic stimulation of lipolysis in 3T3-L1 adipocytes

Recently, we have shown that neuropeptide Y (NPY) is produced and upregulated in visceral adipose tissue of an early-life programmed rat model of central obesity. Moreover, we have demonstrated that NPY promotes proliferation of adipocyte precursor cells and contributes to the pathogenesis of obesity. However, the role of NPY in regulating adipocyte metabolism is poorly understood. The present study was designed to examine the effects of NPY on adipocyte metabolic function using 3T3-L1 adipocytes as an in vitro cell model system. We found that although it did not affect basal lipolysis, NPY potentiated isoproterenol (a β-adrenergic receptor agonist) stimulated lipolysis. Furthermore, this potentiation occurred upstream of adenylyl cyclase, since NPY did not enhance forskolin (an activator of adenylyl cyclase) stimulated lipolysis. In addition, NPY also augmented isoproterenol-stimulated phosphorylation of hormone sensitive lipase. In contrast, NPY did not alter the expression of several key lipolytic and lipogenic enzymes/proteins. Taken together, our results revealed a novel cross talk between the NPY and β-adrenergic signaling pathways in regulating lipolysis. Thus, the present findings add a new dimension to the dynamic role NPY plays in regulating energy balance.