Stimulatory Effect Of Neuropeptide Y Research Articles

Abstract 3663: Neuropeptide Y stimulates neuroblastoma cell migration via Y5R/RhoA pathway

Abstract Neuroblastoma (NB) is a pediatric malignancy that originates from precursors of the sympathetic neurons and expresses neuronal proteins, such as neuropeptide Y (NPY). NPY is a neurotransmitter released from sympathetic nerves, acting via its Y1-Y5 receptors (Y1R-Y5R). NB cells constitutively express Y2R, while Y5R is induced in pro-apoptotic conditions. Our previous data indicated that Y2R is involved in NB cell proliferation and angiogenesis, while Y5R acts as a pro-survival factor. In NB patients, elevated serum NPY levels are associated with poor prognosis and metastasis. Interestingly, in NB tissues, tumor cells with angioinvasive phenotype have a strong expression of Y5R on the leading edge of the penetrating cell. Thus, the goal of our study was to determine the role of NPY and its receptors in NB dissemination. To this end, we assessed the effect of NPY system perturbations on NB cell motility (IncuCyte Live Cell Analysis system), cytoskeleton organization (phalloidin staining) and activity of a cytoskeleton regulator, RhoA (ICC, pull-down assay). The role of particular NPY receptors in these processes was validated by their overexpression in CHO-K1 cells. Treating NB cells with NPY stimulated NB cell migration in a dose-dependent manner, with a peak of activity at 10-8M and lower effects at higher and lower peptide concentrations. This effect was inhibited by Y5R antagonist, while in some cell lines further reduction was observed when combining Y5R and Y2R antagonists. The stimulatory effect of NPY on cell migration was associated with cytoskeleton remodeling, which included a higher number of filopodia positive for Y5R and co-localization of Y5R with an active form of RhoA at both leading and trailing edges of a migrating cell. In line with this role for Y5R in NB motility, expression of this receptor was up-regulated by hypoxia, a known pro-metastatic factor. Consistent with the results on native NB cells, overexpression of Y5R in CHO-K1 cells significantly increased their motility, while transfection with Y1R or Y2R had no such effect. Stimulation of CHO-K1/Y5R cells with NPY increased migration in a dose-dependent manner, while blocking Y5R inhibited cell motility. Moreover, NPY exerted a chemotactic effect for CHO-K1/Y5R cells. Like in NB cells, in CHO-K1 transfectants, Y5R co-localized with active RhoA in both leading and trailing edges of migrating cells. In line with this, CHO-K1/Y5R cells had elevated basal RhoA activity, while NPY stimulation further increased RhoA-GTP levels. Altogether, our data implicate Y5R as the main NPY receptor mediating its stimulatory effect on cell motility. In NB cells, Y2R may further enhance this effect. The Y5R-induced cell motility is associated with cytoskeleton remodeling driven by RhoA activation. The concentration-dependent activity of NPY and its chemotactic effect indicates that the peptide may spatially regulate NB cell migration within tumor tissue. Citation Format: Nouran Abualsaud, Lindsay Caprio, Abrar Bakr, Lamia Alamri, Ewa Krawczyk, Susana Galli, Sung Hyeok Hong, Joanna Kitlinska. Neuropeptide Y stimulates neuroblastoma cell migration via Y5R/RhoA pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3663.

Inhibitory effect of D3 dopamine receptors on neuropeptide Y‑induced migration in vascular smooth muscle cells.

Abnormal migration of vascular smooth muscle cells (VSMCs) serves an important role in hypertension, atherosclerosis and restenosis following angioplasty, which is regulated numerous hormonal and humoral factors, including neuropeptide Y (NPY) and dopamine. Dopamine and NPY are both sympathetic neurotransmitters, and a previous study reported that NPY increased VSMC proliferation, while dopamine receptor inhibited it. Therefore, the authors wondered whether or not there is an inhibitory effect of dopamine receptor on NPY‑mediated VSMC migration. The present study demonstrated that stimulation with NPY dose‑dependence (10‑10‑10‑7M, 24h) increased VSMC migration, the stimulatory effect of NPY was via the Y1 receptor. This is because, in the presence of the Y1 receptor antagonist, BIBP3226 (10‑7 M), the stimulatory effect of NPY on VSMC migration was blocked. Activation of the D3 receptor by PD128907 dose‑dependence (10‑11‑10‑8 M) reduced the stimulatory effect of NPY on VSMC migration. The effect of PD128907 was via the D3 receptor, because the inhibitory effect of PD128907 on NPY‑mediated migration was blocked by the D3 receptor antagonist, U99194. The authors' further study suggested that the inhibitory effect of the D3 receptor was via the PKA signaling pathway, in the presence of the PKA inhibitor, 14‑22 (10‑6 M), the inhibitory effect of PD128907 on VSMC migration was blocked. Moreover, the inhibitory effect of PD128907 was imitated by PKA activator, Sp‑cAMP [S], in the presence of Sp‑cAMP [S], the NPY‑mediated stimulatory effect on VSMC migration was abolished. The present study indicated that activation of the D3 receptor inhibits NPY Y1‑mediated migration on VSMCs, PKA is involved in the signaling pathway.

The stimulatory effect of neuropeptide Y on growth hormone expression, food intake, and growth in olive flounder (Paralichthys olivaceus).

Neuropeptide Y (NPY) is a 36-amino acid peptide known to be a strong orexigenic (appetite-stimulating) factor in many species. In this study, we investigated the effect of NPY on food intake and growth in the olive flounder (Paralichthys olivaceus). Recombinant full-length NPY was injected intraperitoneally into olive flounder at the dose of 1μg/g body weight; phosphate buffered saline was used as the negative control. In a long-term experiment, NPY and control groups were injected every fifth day over a period of 30days. In a short-term experiment, NPY and control groups were given intraperitoneal injections and maintained for 24h. Food intake and growth rates were significantly higher in fish injected with recombinant NPY than in the control fish (P<0.05). Higher growth hormone (GH) and NPY mRNA transcript levels were observed in both experiments, indicating a stimulatory effect of NPY on GH release. These findings demonstrate that NPY is an effective appetite-stimulating factor in olive flounder with the potential to improve the growth of domestic fish species and enhance efficiency in aquaculture.

Inhibitory effect of D1-like dopamine receptors on neuropeptide Y-induced proliferation in vascular smooth muscle cells

Proliferation of vascular smooth muscle cells (VSMCs) is thought to have a key role in the development of atherosclerotic lesions. Neuropeptide Y (NPY), norepinephrine and dopamine are sympathetic neurotransmitters. NPY has been particularly shown to stimulate proliferation of VSMCs. NPY, norepinephrine and dopamine are all sympathetic transmitters. In our previous study, we found that in the presence of the dopamine receptor, the α1-adrenergic receptor-mediated VSMC proliferation is reduced. We hypothesize that the activation of the D1-like receptor might inhibit the NPY-mediated VSMC proliferation. In our present study, we found that NPY, mainly via the Y1 receptor, increased VSMC proliferation. This was determined by [(3)H]-thymidine incorporation, in a concentration (10(-11) to 10(-8) M)-dependent manner. In the presence of the D1-like receptor agonist, fenoldopam (10(-12) to 10(-5) M), the stimulatory effect of NPY on VSMC proliferation was reduced. The involvement of the D1-like receptor was confirmed when the inhibitory effect of fenoldopam was reversed in the presence of the D1-like receptor antagonist SCH-23390 (10(-8) M). Moreover, the inhibitory effect of fenoldopam on NPY-mediated VSMC proliferation was also blocked in the presence of the PKA inhibitor 14-22 (10(-6) M). Protein kinase A activator 8-(4-chlorophenylthio) adenosine-3,5-cyclic monophosphorothioate, Sp-isomer sodium salt (10(-6) M) could simulate the stimulatory effect of fenoldopam. It indicated that the inhibitory effect of D1-like receptors on NPY-mediated VSMC proliferation may have an important role in the regulation of blood pressure or prevention of atherosclerosis.

Genes involved in fatty acid metabolism: Molecular characterization and hypothalamic mRNA response to energy status and neuropeptide Y treatment in the orange-spotted grouper Epinephelus coioides

As in mammals, fatty acid (FA) metabolism plays diverse and vital roles in regulating food intake in fish. Multiple lines of evidence suggest that the effect of FA metabolism on food intake is linked to changes in the level of neuropeptide Y (NPY) in the hypothalamus of the rainbow trout. In mammals, the evidence suggests that FA metabolism regulates feeding via hypothalamic NPY. NPY is therefore considered an important factor that mediates the modulation of food intake by FA metabolism in vertebrates. The stimulatory effect of NPY on food intake is well known. However, to the best of our knowledge, the effect of NPY on FA metabolism in the hypothalamus has not been examined. In this study, we cloned the cDNA of four key enzymes involved in FA metabolism and assessed the effect of energy status and NPY on their mRNA expression in the hypothalamus of grouper. The full-length cDNAs of UCP2 and CPT1a and the partial coding sequence (CDS) of ACC1 and FAS were isolated from the grouper hypothalamus. These genes are expressed in the hypothalamus and during the organogenetic stage of embryogenesis. A feeding rhythm study showed that the hypothalamic expression level of NPY and CPT1a was highly correlated with feeding rhythm. Long-term fasting was found to significantly induce the hypothalamic mRNA expression of NPY, CPT1a and UCP2. An in vitro study demonstrated that NPY strongly stimulated CPT1a and UCP2 mRNA expression in a time- and dose-dependent manner. Collectively, these results suggest that these four genes related to FA metabolism may play a role in regulating food intake in grouper and, that NPY modulates FA metabolism in the grouper hypothalamus. This study showed, for the first time in vertebrates, the effect of NPY on the gene expression of FA metabolism-related enzymes.

Intracellular mechanisms coupled to NPY Y2 and Y5 receptor activation and lipid accumulation in murine adipocytes

The formation of adipose tissue is a process that includes the pre-adipocyte proliferation and differentiation to adipocytes that are cells specialized in lipid accumulation. The adipocyte differentiation is a process driven by the coordinated expression of various transcription factors, such as peroxisome proliferator-activated receptor (PPAR-γ). Neuropeptide Y (NPY) induces adipocyte proliferation and differentiation but the NPY receptors and the intracellular pathways involved in these processes are still not clear. In the present work we studied the role of NPY receptors and the intracellular pathways involved in the stimulatory effect of NPY on lipid accumulation. The murine pre-adipocyte cell line, 3T3-L1, was used as a cell model. Adipogenesis was evaluated by quantifying lipid accumulation by Oil red-O assay and by analyzing PPAR-γ expression using the Western blotting assay. Adipocytes were incubated with NPY (100nM) and a decrease on lipid accumulation and PPAR-γ expression was observed in the presence of NPY Y(2) receptor antagonist (BIIE0246, 1μM) or NPY Y(5) antagonist. Furthermore, NPY Y(2) (NPY(3-36), 100nM) or NPY Y(5) (NPY(19-23)(GLY(1), Ser(3), Gln(4), Thr(6), Ala(31), Aib(32), Gln(34)) PP, 100nM) receptor agonists increased lipid accumulation and PPAR-γ expression. We further investigate the intracellular pathways associated with NPY Y(2) and NPY Y(5) receptor activation. Our results show NPY induces PPAR-γ expression and lipid accumulation through NPY Y(2) and NPY Y(5) receptors activation. PKC and PLC inhibitors inhibit lipid accumulation induced by NPY Y(5) receptor agonist. Moreover, our results suggest that lipid accumulation induced by NPY Y(2) receptor activation occurs through PKA, MAPK and PI3K pathways. In conclusion, this study contributes to a step forward on the knowledge of intracellular mechanisms associated with NPY receptors activation on adipocytes and contributes to a better understanding and the development of new therapeutic targets for obesity treatment.

Structural and functional characterization of neuropeptide Y in a primitive teleost, the Japanese eel (Anguilla japonica)

In the present study, the first full-length cDNA encoding Neuropeptide Y (NPY) was cloned from the brain of Japanese eel (Anguilla japonica). The open reading frame of Japanese eel NPY gene is 294bp in length, encoding a precursor protein of 97 amino acids, which contains a 36-amino-acid mature peptide. Sequence analysis showed that the Japanese eel NPY peptide is similar to that of other species. Real-time PCR revealed that NPY in Japanese eel is mainly expressed in the brain, especially in the hypothalamus and the optic tectum thalamus. The effect of a negative energy balance on NPY gene expression was examined subsequently. The mRNA level of NPY in the hypothalamus and the optic tectum thalamus showed a pronounced increase after 4days of food deprivation. The biological activities of Japanese eel NPY were further investigated in vivo and in vitro. Intraperitoneal injection of the NPY peptide into Japanese eel could potently elevate the expression of the mammalian gonadotropin-releasing hormone (mGnRH) in hypothalamus and the follicle-stimulating hormone beta (FSHβ), the luteinizing hormone beta (LHβ) and growth hormone (GH) in pituitary. In static incubation studies, the stimulatory effects of NPY on mGnRH expression in hypothalamic fragments and on FSHβ, LHβ and GH expression in pituitary cells were also observed. However, in vivo and in vitro studies showed that NPY exhibits an inhibitory action on the expression of thyroid-stimulating hormone beta (TSHβ) in pituitary. The results indicate that NPY is involved in the regulation of multiple physiological processes in Japanese eel.

Neuropeptide Y stimulates retinal neural cell proliferation – involvement of nitric oxide

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 microM), a nitric oxide synthase inhibitor, 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ; 20 microM), a soluble guanylyl cyclase inhibitor or U0126 (1 microM), 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.

Leptin attenuates the acute effects of centrally administered neuropeptide Y on somatotropin but not gonadotropin secretion in ovariectomized cows

We tested the hypothesis that recombinant ovine leptin would attenuate the acute effects of neuropeptide Y (NPY) on secretion of GH and gonadotropins (LH and FSH) in cows. Ovariectomized cows ( n=6) fitted with third ventricle guide cannulas were assigned randomly to each of three groups in a Latin square arrangement: (1) control; saline treatment only, (2) NPY; saline followed by NPY, and (3) L-NPY; leptin pretreatment followed by NPY. Treatments were: s.c. injection of saline or leptin (30 μg/kg BW) at time 0, i.v. injection of saline or leptin (30 μg/kg BW) at 70 min, and intracerebroventricular (i.c.v.) injection of saline or NPY (500 μg) at 90 min. Plasma leptin was elevated ( P<0.01) at least four-fold throughout the experiment in the L-NPY group. Mean plasma concentrations of LH declined within 1 h and were lower ( P<0.03) than controls in both the NPY and L-NPY groups beginning 2 h after NPY injection. An acute increase in plasma concentrations of GH was observed within 1 h after NPY in the NPY group and mean values were greater ( P<0.01) than controls. However, in the L-NPY group, leptin pretreatment attenuated the NPY effect on GH. Treatments had no effect on FSH secretion. Results confirm suppressive and stimulatory effects of NPY on LH and GH secretion, respectively, and indicate that leptin can attenuate the acute effects of NPY on GH secretion in cattle.

The age-related discrepancy in the effect of neuropeptide Y on select catecholamine biosynthetic enzymes in the adrenal medulla and hypothalamus in rats

The elevated levels of circulating catecholamines (CAs) with age may be related to the increased expression of CA biosynthetic enzymes, tyrosine hydroxylase (TH) and dopamine β hydroxylase (DβH) in the adrenal medulla of senescent compared with younger animals. Neuropeptide Y (NPY) is co-synthesized and co-released with CAs in the adrenal medulla. NPY inhibits the stimulated secretion of CAs, however, its role in regulation of the genes encoding CA biosynthetic enzymes is not clear. We hypothesized that NPY up-regulates TH, DβH and NPY expression in the adrenal medullae of young and old Fischer-344 rats. NPY increased mRNA expression of TH, DβH, NPY and also enhanced TH protein level in the adrenal medullae of young rats by 50%, 35%, 45% and by 20%, respectively. We also examined the effect of NPY on TH and NPY mRNA in the hypothalamus. Basal expression of TH mRNA was decreased in the hypothalamus with age. DNA binding activities of activator protein-1 and cAMP response element binding protein were also augmented only in the young by 140% and 125%, respectively. We conclude that NPY stimulates the CA biosynthetic pathway in the adrenal medulla and positive auto-regulation of NPY might be involved in this process. The stimulatory effect of NPY on adrenomedullary CA biosynthetic pathway is blunted with age.

Novel neuropeptide Y1 and Y5 receptor gene variants: associations with serum triglyceride and high‐density lipoprotein cholesterol levels

Neuropeptide Y (NPY) appears to play a critical role in the integration of appetite and energy expenditure through NPY Y1 and Y5 receptor subtypes. Moreover, the NPY Y1 receptor is highly expressed on human adipocytes, where it inhibits lipolysis. The genes encoding these receptors are transcribed co-ordinately in opposite directions from a common promoter in a region of chromosome 4 that has been previously linked to triglyceride and small low-density lipoprotein (LDL) particle concentration. Therefore, the purpose of this investigation was to examine the relationship between polymorphisms in the genes encoding NPY Y1 and Y5 and the development of obesity and dyslipidemia. We screened the promoter and coding regions and identified four polymorphic variants. One of these, a cytosine to thymine (C-->T) substitution in the untranslated region between the genes for NPY Y1 and Y5 (allele frequency 0.11), was significantly associated with both lower fasting triglyceride level (152 vs 125 mg/dl), and higher high-density lipoprotein (HDL) concentrations (49 vs 45 mg/dl) (p < 0.01) in 306 obese subjects. Given the stimulatory effect of NPY on adipocyte lipoprotein lipase (LPL) activity, and the lack of association of other polymorphisms with serum lipid levels, we hypothesize that this is a gain-in-function polymorphism.

Regulation of hypothalamic neuropeptide Y Y1 receptor gene expression during the estrous cycle: role of progesterone receptors.

Neuropeptide Y (NPY) stimulates the release of GnRH in an estrogen (E2)-dependent manner, which is important in generating preovulatory GnRH surges. We tested the hypothesis that E2 up-regulates NPY's actions by stimulating NPY Y1 receptor (Y1r) gene expression through a mechanism mediated by E2's ability to induce progesterone (P) receptors (PRs). In initial experiments, a specific Y1r antagonist BIBP3226 was used to confirm the involvement of Y1r in the stimulatory effects of NPY on in vivo GnRH release. Hypothalamic Y1r messenger RNA (mRNA) levels were then measured using competitive RT-PCR and were found to be significantly increased at 1000, 1200, and 1400 h on proestrus compared with other times of the day or cycle stage. Ovariectomy eliminated these increases, and E2 treatment restored them. Additional P treatment produced even larger increases in Y1r mRNA levels. To assess the role of PRs in stimulating Y1r expression, proestrous rats were treated with PR antagonist or oil vehicle and killed at 1200 h. Treatment with PR antagonist completely blocked the proestrous rise in Y1r gene expression. In parallel experiments, the same in vivo PR antagonist treatments also blocked NPY stimulation of GnRH release in vitro. Together our findings reveal that 1) Y1r mRNA levels are increased during the late morning and afternoon of proestrus; 2) Y1r mRNA levels are similarly increased by E2, and to an even greater extent by additional P; and 3) PR antagonism blocks both increased Y1r mRNA and induction of GnRH responsiveness to NPY. These observations support the idea that E2 up-regulates GnRH neuronal responses to NPY through stimulation of Y1r gene expression, and that E2's actions are mediated by the induction and subsequent activation of PRs.

Characterization of neuropeptide Y-mediated corticotropin-releasing factor synthesis and release from human placental trophoblasts.

Neuropeptide Y (NPY) is a CRF secretagogue for human placental cells in culture. We have studied the involvement of intracellular calcium and calcium-dependent signaling in the NPY-induced CRF release in trophoblastic cells. The incubation of trophoblasts with NPY for 3 and 8 h led to a dose-dependent increase in CRF secretion. Also, NPY stimulated synthesis of this peptide hormone upon an 8-h incubation period. BIBP3226, a selective Y1 receptor antagonist, and pertussis toxin (PTX) eliminated these effects. NPY-stimulated CRF secretion was mostly prevented by loading cells with BAPTA-AM, suggesting that elevation of intracellular calcium is responsible for the increase of CRF secretion. However, this calcium chelator had no effect on CRF synthesis. Furthermore, U-73122, a phospholipase C-betas (PLC) inhibitor or xestospongin C, an inositol triphosphate receptor (InsP3-R) blocker, have partially prevented the effect of NPY on CRF synthesis and secretion. Therefore, the increase in CRF synthesis and secretion rely in part on the release of calcium from intracellular store. Interestingly, SKF 96365, an inhibitor of store operated calcium (SOC) influx, also partially blocked the NPY stimulatory effect on CRF release but not its synthesis, suggesting that calcium influx is also involved in this stimulation. In the syncytiotrophoblast, known to possess a NPY-activated protein kinase C (PKCs) activity, NPY also stimulated calcium calmodulin kinase II (CaMKII) and extracellular regulated kinase (ERK1/2) activities. In the present study, we observed that bisindolylmaleimide (BIM), a nonspecific PKCs inhibitor partially prevented the NPY-induced CRF release. On the other hand, autocamtide-2 related inhibitory peptide (AIP), a CaMKII inhibitor, prevented most of the stimulatory effect of NPY on both CRF synthesis and release. Go6976, an inhibitor of the conventional and mu PKCs and PD 098059, an inhibitor of the ERK cascade, had no effect on neither CRF synthesis nor release. Altogether, these results support a Y1 receptor-mediated PTX-sensitive induction on CRF synthesis and release by NPY from human placental trophoblasts. The stimulation of CRF synthesis by NPY seems to depend mainly on a PLC-beta to InsP3-R axis and on CaMKII activity. Also, the release of CRF depends on the PLC-beta to InsP3-R axis and CaMKII activity but also entails the participation of a calcium-independent PKCs.

Modulation of innate immune functions by intracerebroventricularly applied neuropeptide Y: Dose and time dependent effects

Centrally applied neuropeptide Y (NPY) interacts with the autonomic nervous system and the hypothalamo-pituitay-adrenal (HPA) axis activity. Since these physiological systems have been shown to modulate innate immune functions, the effects of intracerebroventricular (i.c.v.) NPY administration on leukocyte subsets in the blood, spleen and intravascular pool of the lung, blood granulocyte chemiluminescence response, and splenic natural killer (NK) cell-mediated lysis were studied in Lewis rats. Concentration-dependent NPY effects were tested at 15min and 24h post I.C.V. injection at dosages of 10 −6M, 10 −9M, and 10 −12M. Time dependent effects were investigated at 15min, 1h and 24h after i.c.v. administration of 10 −9M NPY. Compared to saline controls, an increased number of granulocytes and NK cells in the blood, associated with a decreased granulocyte function and NK cytotoxicity was observed 15min following NPY infusion. This initial immunosuppression was followed by long lasting stimulatory effects of NPY on the functional capacity of both cell populations when tested at 1h and 24h. The dosage of i.c.v. 10 −6M NPY produced no changes, whilst 10 −9M produced maximal, and 10 −12M still significant effects. Results provide evidence that centrally applied NPY influences innate immunity in a dose and time dependent fashion. Cell mobilization from the vascular marginal pool is likely to be an underlying mechanism for the initial immunosuppression.

Neuropeptide Y Regulation of LHRH Release in the Median Eminence: Immunocytochemical and Physiological Evidence in Hens

It has been suggested that hypothalamic median eminence (ME) might be a control site for luteinizing hormone-releasing hormone (LHRH) release. Thus, stimulatory and/or inhibitory inputs acting at this site might be involved in regulating LHRH release from the ME and, therefore, luteinizing hormone (LH) release from the anterior pituitary. Since a role for neuropeptide Y (NPY) on LH release has been suggested, we have hypothesized that NPY might act in the ME to control preovulatory LHRH release in hens. To examine this possibility we have determined: (a) the immunocytochemical distribution of LHRH and NPY in the ME of the hen, (b) the basal and NPY-stimulated release of LHRH in vitro from the ME of hens undergoing a natural or a premature preovulatory surge of LH, and (c) the tissue content of LHRH and NPY in microdissected MEs, at various times before and during a natural or a premature preovulatory surge of LH. A potential role for NPY on LHRH release in the ME is suggested for the following reasons. (a) There are opportunities for synaptic interactions between NPY and LHRH-containing axons at this site. LHRH-containing cell bodies localized in the anterior hypothalamus/medial preoptic area project to the ME. NPY-containing perikarya, concentrated in the ventromedial aspect of the arcuate nucleus, might contact LHRH processes going to the ME and/or might themselves send axons to the ME, (b) Addition of NPY to the incubation media increases LHRH release from microdissected ME tissue of hens killed at the time of the natural preovulatory surge of LH, but not in hens killed 7 h before the occurrence of this surge. However, the stimulatory effect of NPY on LHRH release can be induced at this latter time when a premature LH surge is elicited. While the natural preovulatory surge of LH occurs 4 h before the second ovulation in a sequence (C2 ovulation), administration of progesterone (P4) 10-14 h before the expected natural C2 ovulation advances the natural LH surge by 7-8 h. Thus, NPY might act as a physiological stimulus of LHRH release at the ME during the preovulatory surge of LH. This is suggested since in vitro basal LHRH release from denervated ME tissue does not change before and during the natural or the premature LH surge. Therefore, preovulatory release of LHRH in vivo might be under a continuous drive from stimulatory inputs to the LHRH neuron and NPY might be one of these stimulating factors.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibitory and stimulatory effects of neuropeptide Y(17-36) on rat cardiac adenylate cyclase activity. Structure-function studies.

Neuropeptide Y (NPY) inhibits cardiac adenylate cyclase activity by interacting with specific receptors coupled to a pertussis toxin-sensitive G protein. Structure-activity studies revealed that only C-terminal fragments can exhibit an NPY-like inhibitory effect on 125I-NPY binding and adenylate cyclase activity of rat cardiac ventricular membranes. Although NPY(17-36) inhibited 125I-NPY binding with high potency, it produced a biphasic effect on basal (GTP, 10 and 100 microM or guanosine 5'-gamma-O-(thio)triphosphate (GTP gamma S, 10 microM) adenylate cyclase activity. Low concentrations (less than 1 nM) of NPY(17-36) inhibited the adenylate cyclase activity whereas high concentrations (greater than 1 nM) reversed this action. GTP gamma S (100 microM) reversed the biphasic effect of NPY(17-36). NPY(17-36) exhibited only a stimulatory effect in the membranes from pertussis toxin-treated rats and an inhibitory effect with membranes from cholera toxin-treated rats. Low concentrations (less than 1 nM) of NPY(17-36) inhibited isoproterenol-stimulated adenylate cyclase activity whereas high doses (greater than 1 nM) reversed this activity. The cardiac NPY receptor antagonist, NPY(18-36) (1 microM), completely blocked the biphasic effect of NPY(17-36) on isoproterenol-stimulated activity. The inhibitory dose-response curve of NPY on isoproterenol-stimulated adenylate cyclase activity was shifted parallel to the right by NPY(17-36) (1 microM), suggesting that it is an antagonist of NPY at high concentrations. N-alpha-acetylated and C-terminally deamidated analogs of NPY(17-36) had no effect on the adenylate cyclase activity. [im-DNP-His26] NPY exhibited a more pronounced biphasic effect whereas N-alpha-myristoyl-NPY(17-36) elicited only a stimulatory effect. These investigations suggest that: 1) the inhibitory and stimulatory effects of NPY(17-36) are mediated by high affinity NPY receptors coupled to a pertussis toxin-sensitive G protein and a distinct population of low affinity receptors coupled to a cholera toxin-sensitive G protein, respectively; and 2) the stimulatory effect of NPY(17-36) is dissociable.

Neuropeptide Y (NPY) modulatesin vitro gonadotropin in release from rainbow trout pituitary glands

This work investigated the action of neuropeptide Y (NPY) on thein vitro pituitary release of the maturing gonadotropic hormone (GtH) of the rainbow trout using a perifusion system employing trout balanced salt solution (pH 7.5) at 15°C and a 12.5 ml/h flow rate. In vitellogenic females a 20 minutes NPY application (10(-7) M) induced a 20-30% decrease in GtH secretion. Removal of NPY was followed by a rebound in GTH secretion. On the contrary, in ovulated females, NPY (15 minutes, 10(-7) M) directly stimulated GTH secretion. The greatest stimulation was obtained the day of ovulation where the stimulatory effect of NPY was similar to those induced by s.GnRH in the same conditions, reaching 400% of the basal GTH level. In vitellogenic females treated with 1-4-6 androstadien 3-7 dione, an inhibitor of aromatase activity, the pituitary response to NPY was similar to that obtained in ovulated females. Thus thein vitro action of NPY might depend on thein vivo steroidogenic environment.

Studies on the physiological role and mechanism of action of neuropeptide Y in the regulation of luteinizing hormone secretion in the rat

It has been recently shown that intraventricular or systemic injection of neuropeptide Y (NPY) can produce a decrease in plasma luteinizing hormone (LH) levels in castrated rats of both sexes. In order to evaluate the physiological role of NPY in the regulation of LH secretion in the female rat, we proceeded to immunoneutralization experiments using specific antibodies to NPY. Injection of 0.5 ml antiserum to NPY produce a 20-fold increase of LH plasma levels, whereas injection of preimmune serum did not modify the plasma concentrations of LH. To investigate the possibility that catecholamines or serotonin might be involved in the effect of NPY in LH secretion, castrated rats were treated with α-methylparatyrosine (α-MPT), an inhibitor of catecholamine biosynthesis, or received an i.c.v. injection of the neurotoxin 5–7-dihydroxytryptamine (5,7-DHT) prior to the intraventricular injections of NPY. The pretreatment with α-MPT could not prevent the decrease of plasma LH induced by NPY injection whereas the pretreatment with 5,7-DHT reversed the effect of NPY injection. The anatomical connection between LH-releasing hormone (LHRH) and NPY neuronal systems were also investigated using double immunostaining technique. It appeared that NPY endings are in apposition to LHRH cell bodies in the preoptic area in proximity to the organum vasculosum of the lamina terminalis (OVLT). These studies strongly suggest that NPY is of physiological importance in the regulation of LH secretion in the rat and that the action of NPY could be exerted by at least two different ways: (1) a stimulatory effect of NPY on serotoninergic systems which in turn inhibit LHRH release and (2) a direct stimulatory effect of the peptide on LHRH neuronal system.