Abstract: The neuroprotective effect of neuropeptide Y (NPY) receptor activation was investigated in organotypic mouse hippocampal slice cultures exposed to the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Exposure of 2-week-old slice cultures, derived from 7-day-old C57BL/6 mice, to 8 microm AMPA, for 24 h, induced degeneration of CA1 and CA3 pyramidal cells, as measured by cellular uptake of propidium iodide (PI). A significant neuroprotection, with a reduction of PI uptake in CA1 and CA3 pyramidal cell layers, was observed after incubation with a Y(2) receptor agonist [NPY(13-36), 300 nm]. This effect was sensitive to the presence of the selective Y(2) receptor antagonist (BIIE0246, 1 microm), but was not affected by addition of TrkB-Fc or by a neutralizing antibody against brain-derived neurotrophic factor (BDNF). Moreover, addition of a Y(1) receptor antagonist (BIBP3226, 1 microm) or a NPY-neutralizing antibody helped to disclose a neuroprotective role of endogenous NPY in CA1 region. Cultures exposed to 8 microm AMPA for 24 h, displayed, as measured by an enzyme-linked immunosorbent assay, a significant increase in BDNF. In such cultures there was an up-regulation of neuronal TrkB immunoreactivity, as well as the presence of BDNF-immunoreactive microglial cells at sites of injury. Thus, an increase of AMPA-receptor mediated neurodegeneration, in the mouse hippocampus, was prevented by neuroprotective pathways activated by NPY receptors (Y(1) and Y(2)), which can be affected by BDNF released by microglia and neurons.

Abstract: Neuropeptide Y (NPY) is a 36 amino acid peptide widely present in the CNS, including the retina. Previous studies have demonstrated that NPY promotes cell proliferation of rat post-natal hippocampal and olfactory epithelium precursor cells. The aim of this work was to investigate the role of NPY on cell proliferation of rat retinal neural cells. For this purpose, primary retinal cell cultures expressing NPY, and NPY Y(1), Y(2), Y(4) and Y(5) receptors [Alvaro et al., (2007) Neurochem. Int., 50, 757] were used. NPY (10-1000 nM) stimulated cell proliferation through the activation of NPY Y(1), Y(2) and Y(5) receptors. NPY also increased the number of proliferating neuronal progenitor cells (BrdU(+)/nestin(+) cells). The intracellular mechanisms coupled to NPY receptors activation that mediate the increase in cell proliferation were also investigated. The stimulatory effect of NPY on cell proliferation was reduced by l-nitroarginine-methyl-esther (l-NAME; 500 muM), a nitric oxide synthase inhibitor, 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ; 20 muM), a soluble guanylyl cyclase inhibitor or U0126 (1 muM), an inhibitor of the extracellular signal-regulated kinase 1/2 (ERK 1/2). In conclusion, NPY stimulates retinal neural cell proliferation, and this effect is mediated through nitric oxide-cyclic GMP and ERK 1/2 pathways.

Abstract: Activation of the hypothalamic-pituitary-adrenal gland (HPA) axis can modulate the immune system. Cytokines and neuropeptide Y (NPY) are potent regulators of the HPA axis and are both produced by the adrenal medulla. The cytokine interleukin-1beta (IL-1beta) belongs to the interleukin-1 family along with interleukin-1alpha and the interleukin receptor antagonist (IL-1ra). The aim of the present study was to determine the interaction between NPY and IL-1beta in catecholamine (norepinephrine, NE and epinephrine, EP) release from mouse chromaffin cells in culture. We found that IL-1beta increased the constitutive release of NPY, NE and EP from mouse chromaffin cells. This IL-1beta stimulatory effect was blocked by IL-1ra. The immunoneutralization of NPY and the use of the NPY Y(1) receptor antagonist (BIBP 3226) inhibited the stimulatory effect of IL-1beta on catecholamine release from these cells. The present work shows that IL-1beta induces catecholamine release, and in turn this peptide will induce an additional increase in catecholamine release acting through the Y(1) receptor. This work suggests that NPY is involved in the regulatory loop between the immune and the adrenal system in some pathophysiological conditions where plasmatic IL-1beta increases, like in sepsis, rheumatoid arthritis, stress or hypertension.

Abstract: The adrenal chromaffin cells synthesize and release catecholamine (mostly epinephrine and norepinephrine) and different peptides, such as the neuropeptide Y (NPY). NPY stimulates catecholamine release through NPY Y1 receptor in mouse chromaffin cells. The aim of our study was to determine the intracellular signaling events coupled to NPY Y1 receptor activation that lead to stimulation of catecholamine release from mouse chromaffin cells. The stimulatory effect of NPY mediated by NPY Y1 receptor activation was lost in the absence of extracellular Ca2+. On the other hand, inhibition of nitric oxide synthase and guanylyl cyclase also decreased the stimulatory effect of NPY. Moreover, catecholamine release stimulated by NPY or by the nitric oxide donor (NOC-18) was inhibited by mitogen-activated protein kinase (MAPK) and protein kinase C inhibitors. In summary, in mouse chromaffin cells, NPY evokes catecholamine release by the activation the NPY Y1 receptor, in a Ca2+-dependent manner, by activating mitogen-activated protein kinase and promoting nitric oxide production, which in turn regulates protein kinase C and guanylyl cyclase activation.

Abstract: NPY is present in the retina of different species but its role is not elucidated yet. In this work, using different rat retina in vitro models (whole retina, retinal cells in culture, microglial cell cultures, rat Müller cell line and retina endothelial cell line), we demonstrated that NPY staining is present in the retina in different cell types: neurons, macroglial, microglial and endothelial cells. Retinal cells in culture express NPY Y(1), Y(2), Y(4) and Y(5) receptors. Retina endothelial cells express all NPY receptors except NPY Y(5) receptor. Moreover, NPY is released from retinal cells in culture upon depolarization. In this study we showed for the first time that NPY is present in rat retina microglial cells and also in rat Müller cells. These in vitro models may open new perspectives to study the physiology and the potential pathophysiological role of NPY in the retina.

Abstract: BACKGROUND & AIMS: Glial-derived neurotrophic factor (GDNF) promotes the survival and proliferation of enteric neurons. Neuropeptide Y (NPY) is an important peptide regulating gastrointestinal motility. The role of NPY on the survival and proliferation of enteric neurons is not known. We examined the effects of GDNF on the expression and release of NPY from enteric neurons and the role of NPY in promoting enteric neuronal proliferation and survival. METHODS: Studies were performed in primary enteric neuronal cultures and NPY knockout mice (NPY(-/-)). GDNF-induced expression of NPY was assessed by reverse-transcription polymerase chain reaction (RT-PCR), immunocytochemistry, and enzyme-linked immunosorbent assay. Using NPY-siRNA and NPY-Y1 receptor antagonist, we examined the role of NPY in mediating the survival and proliferation effects of GDNF. Gastrointestinal motility was assessed by measuring gastric emptying, intestinal transit, and isometric muscle recording from intestinal muscle strips. RESULTS: GDNF induced a significant increase in NPY messenger RNA and protein expression in primary enteric neurons and the release of NPY into the culture medium. NPY (1 mumol/L) significantly increased proliferation of neurons and reduced apoptosis. In the presence of NPY-siRNA and NPY-Y1 receptor antagonist or in enteric neurons cultured from NPY(-/-) mice, GDNF-mediated neuronal proliferation and survival was reduced. NPY increased the phosphorylation of Akt, a downstream target of the PI-3-kinase pathway. In NPY(-/-) mice, there were significantly fewer nNOS-containing enteric neurons compared with wild-type (WT) mice. NPY(-/-) mice had accelerated gastric emptying and delayed intestinal transit compared with WT mice. CONCLUSIONS: We demonstrate that NPY acts as an autocrine neurotrophic factor for enteric neurons.

Abstract: The contribution of neuropeptide Y (NPY), deriving from adrenal medulla, to the adrenosympathetic tone is unknown. We found that in response to NPY, primary cultures of mouse adrenal chromaffin cells secreted catecholamine, and that this effect was abolished in cultures from NPY Y(1) receptor knockout mice (Y(1)-/-). Compared with wild-type mice (Y(1)+/+), the adrenal content and constitutive release of catecholamine were increased in chromaffin cells from Y(1)-/- mice. In resting animals, catecholamine plasma concentrations were higher in Y(1)-/- mice. Comparing the adrenal glands of both genotypes, no differences were observed in the area of the medulla, cortex, and X zone. The high turnover of adrenal catecholamine in Y(1)-/- mice was explained by the enhancement of tyrosine hydroxylase (TH) activity, although no change in the affinity of the enzyme was observed. The molecular interaction between the Y(1) receptor and TH was demonstrated by the fact that NPY markedly inhibited the forskolin-induced luciferin activity in Y(1) receptor-expressing SK-N-MC cells transfected with a TH promoter sequence. We propose that NPY controls the release and synthesis of catecholamine from the adrenal medulla and consequently contributes to the sympathoadrenal tone.