Neuropeptide Y: potential role in recurrent developmental seizures

Seizures induce profound plastic changes in the brain, including altered expression of neuropeptide Y (NPY) and its receptors. Here, I discuss a potential role of NPY plasticity in the developmental brain: in a rat model of febrile seizures (FS), the most common type of seizures in infants and young children, NPY expression was up-regulated in hippocampus after experimentally induced FS. Interestingly, NPY up-regulation was associated with an increased seizure threshold for additional (recurrent) FS, and this effect was abolished when an antagonist against NPY receptor type 2 was applied. These findings suggest that inhibitory actions of NPY, released after seizures, exert a protective effect that reduces the risk of seizure recurrence in the developing brain.     

Neuropeptide Y receptor in the rat brain

The specific binding of the chloramine-T iodinated neuropeptide Y (125I-NPY) to membranes from rat cerebral cortex was investigated using equilibrium binding and kinetic methods. The equilibrium binding of 125I-NPY at 37 degrees C was characterized by a Kd value of 0.38 nM. The receptor densities in the cerebral cortex, hypothalamus and cerebellum were 0.45 pmol/mg, 0.47 pmol/mg and 0.04 pmol/mg protein respectively. The binding site for 125I-NPY was sensitive to treatment with proteolytic enzymes and thiol reagents. The binding showed a sharp optimum at pH 7-7.7 and was inhibited by increasing concentrations of Mg2+.     

Neuropeptide Y in the human male genital tract

Neuropeptide Y (NPY) was found in high concentrations in the male genital tract. NPY levels were highest in the seminal vesicles, prostate, corpus cavernosum and vas deferens, where large numbers of immunoreactive nerve fibres were detected. Considerable quantities were also found in the epididymis and spongiosum. Lower concentrations were found in the glans penis, testis and foreskin. The presence of a large number of nerves containing NPY suggest that this active neuropeptide may play a role in control of genital function.     

Neuropeptide Y Y5 receptors inhibit kindling acquisition in rats

Neuropeptide Y inhibits neuronal excitability and seizures in various experimental models. This peptide delays kindling epileptogenesis but the receptors involved in this action are unknown. We have studied the role of Y5 receptors in kindling using the selective antagonist GW438014A (IC50=210 nM), a small heterocycle molecule that crosses the blood-brain barrier, and the selective peptide agonist Ala31Aib34 NPY (IC50=6.0 nM). Intraperitoneal injection of GW438014A (10 mg/kg), 30 min before the beginning of a rapid-kindling protocol, significantly accelerated the rate of kindling acquisition as compared to vehicle-injected rats. Thus, the number of electrical stimuli required to reach stages 3 and 4-5 of kindling were reduced by 50% and 25%, respectively. The average afterdischarge duration in the stimulated hippocampus was prolonged by 2-fold. Conversely, kindling rate was delayed by intracerebroventricular administration of 24 nmol Ala31Aib32 NPY. Thus, the number of stimuli necessary to reach stages 2 and 3 of kindling was increased by 3- and 4-fold, respectively. During the stimulation protocol (40 stimuli) none of the rats treated with the Y5 agonist showed stages 4-5 seizures. Twenty-four hours after the last kindling stimulation, thus during the re-test session, Y5 agonist- or antagonist-treated rats had stages 4-5 seizures as their controls. In rats treated with both the antagonist and the agonist, kindling rate was similar to vehicle-injected rats. These data indicate that Y5 receptors mediate inhibitory effects of NPY in kindling and display anticonvulsant rather then antiepileptogenic effects upon agonist stimulation.     

Neuropeptide Y Y1 receptor mediated mesenteric vasoconstriction in the pig in vivo

The object of the present study was to investigate the effects of the sympathetic cotransmitter neuropeptide Y (NPY), and the closely related gut hormone peptide YY (PYY), on splanchnic blood flow regulation in the anaesthetized pig in vivo. Systemic injections of NPY, PYY and the NPY Y(1) receptor agonist [Leu(31)Pro(34)]NPY (470 pmol kg(-1) each) evoked pressor and mesenteric vasoconstrictor responses that were largely abolished by the selective NPY Y(1) receptor antagonist H 409/22 (60 nmol kg(-1) min(-1)). In contrast, the NPY Y(2) receptor agonist N-acetyl[Leu(28)Leu(31)]NPY(24-36) (1.1 nmol kg(-1)), a dose of which potently evoked splenic NPY Y(2) receptor mediated (not affected by H 409/22) vasoconstriction, did not evoke any mesenteric vascular response. Mesenteric vascular responses to angiotensin II (10 pmol kg(-1)), alpha,beta-methylene ATP (10 nmol kg(-1)) and the alpha(1)-adrenoceptor agonist phenylephrine (15 nmol kg(-1)), were not inhibited by H 409/22. It is concluded that NPY and PYY evokes porcine mesenteric vasoconstriction mediated by the NPY Y(1) receptor subtype, as demonstrated by selective and specific inhibition exerted by the NPY Y(1) receptor antagonist H 409/22, in vivo.     

Regulation of Neuropeptide Y Release by Neuropeptide Y Receptor Ligands and Calcium Channel Antagonists in Hypothalamic Slices

Neuropeptide Y (NPY) is an important regulator of energy balance in mammals through its orexigenic, antithermogenic, and insulin secretagogue actions. We investigated the regulation of endogenous NPY release from rat hypothalamic slices by NPY receptor ligands and calcium channel antagonists. High-potassium stimulation (60 mM) of the slices produced a calcium-dependent threefold increase in NPY release above basal release. The Y2 receptor agonists NPY(13-36) and N-acetyl[Leu28,Leu31]NPY(24-36), the Y4 agonist rat pancreatic polypeptide (rPP), and the Y4/Y5 agonist human pancreatic polypeptide (hPP) significantly reduced both basal and stimulated NPY release. NPY(13-36)-induced reduction of NPY release could be partially prevented in the presence of the weak Y2 antagonist T4-[NPY(33-36)]4, whereas the hPP- and rPP-induced inhibition of release was not affected by the Y5 antagonist CGP71683A or the Y1 antagonist BIBP3226. The selective Y1, Y2, and Y5 antagonists had no effect on either basal or potassium-stimulated release when administered alone. The calcium channel inhibitors omega-conotoxin GVIA (N-type), omega-agatoxin TK (P/Q-type), and omega-conotoxin MVIIC (Q-type) all significantly inhibited potassium-stimulated NPY release, without any effect on basal release, whereas nifedipine had no effect on either basal or stimulated release. Addition of both omega-conotoxin GVIA and omega-agatoxin TK together completely inhibited the potassium-stimulated release. In conclusion, we have demonstrated that NPY release from hypothalamic slices is calcium-dependent, involving N-, P-, and Q-type calcium channels. NPY release is also inhibited by Y2 agonists and rPP/hPP, suggesting that Y2 and Y4 receptors may act as autoreceptors on NPY-containing nerve terminals.     

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.     

Reduced Neuropeptide Y Concentrations in Suicide Brain

Neuropeptide Y (NPY) was measured in postmortem brain tissue from victims of suicide and from individuals dying a sudden natural or accidental death (controls). Concentrations of NPY-immunoreactivity were measured by radioimmunoassay in frontal cortex (BA 10), temporal cortex (BA 22), caudate nucleus, and cerebellum. Concentrations of NPY-immunoreactivity were significantly lower in postmortem frontal cortex (-14%) and caudate nucleus (-27%) from suicide victims compared with age-matched controls. A subgroup of suicides with evidence of a history of depression revealed more robust reductions in concentrations of NPY-immunoreactivity in frontal cortex and caudate nucleus, as did four individuals who died from natural causes and also were described as having a possible history of depression. Concentrations of NPY-immunoreactivity in temporal cortex and cerebellum from victims of suicide or from the subgroup of subjects with a possible history of depression were not significantly different from those of age-matched controls. We suggest there is a deficit in the brain NPY system leading to region-specific reductions in peptide concentrations in subjects who have a history of depression.     

Neuropeptide Y: Behavioral effects in the golden hamster

Neuropeptide Y (NPY) is found abundantly in nervous tissues of vertebrate species including the golden hamster. Centrally-administered NPY has been reported to elicit ingestive behaviors in the rat, squirrel, pig, mouse, and chick. To assess NPY's behavioral effects in a New World rodent that does not increase food intake after deprivation, NPY was injected intracerebroventricularly (10.0-0.04 μg/5 μl) in home-caged golden hamsters with ad lib access to food, water and 5% w/v ethanol solution. Food and fluid intakes, and behavior displays were monitored after NPY injection. NPY promptly increased short-term food intake and observed feeding behaviors at 10.0, 3.3, 1.1, and 0.37 μg NPY, but there was no effect on 24 hr food intake. Water and ethanol intakes were increased only at 10.0 and 0.37 μg NPY, respectively. Resting behaviors decreased at NPY doses that increased feeding, but there were no consistent effects of NPY on any other category of behavior. Results demonstrate that NPY potently stimulates short-term food intake and decreases resting behavior in the golden hamster. The lack of compensatory food intake in deprived hamsters cannot be explained as an insensitivity to the putative orexigenic function of endogenous neuropeptide Y.     

Neuropeptide Y reduces ovarian blood flow in the rabbit

Neuropeptide Y-containing nerve fibers have previously been demonstrated to innervate the mammalian ovary. These nerve fibers innervate primarily the vasculature. In this study we have developed a method for in vivo measurement of the ovary blood flow rate by means of the 133Xe method. Using this technique we measured the ovary blood flow rate and investigated the dose-response relationship between close intraarterial-injected NPY and the ovary blood flow rate. A monoexponential washout curve for 133Xe was found for the whole washout process, ensuring that the blood flow rate at any time could be calculated from the curve. We found a mean blood flow rate in the nonpregnant rabbit ovary at 43.6 +/- 4.4 ml.(100 g)-1.min-1 (mean +/- SEM). Injection of NPY (20, 200, 2000 pM) in the aorta close to a. ovarica resulted in a dose-dependent decrease in the ovarian blood flow rate with a maximum reduction to 40.7 +/- 6.3% (mean +/- SEM) of the control blood flow rate. These findings make it likely that receptors able to interact with NPY are present in the vasculature of the rabbit ovary.     

Neuropeptide Y1 receptors in the rat genital tract

Using in situ hybridization and immunohistochemistry, the expression of type 1 neuropeptide Y (NPY) receptors (Y1-Rs) has been demonstrated in the rat genital tract. In the male Y1-R mRNA and Y1-R-like immunoreactivity (LI) were found in smooth muscles of predominantly arterioles and small arteries inside testis. Fibers showing NPY-LI could not be detected within testis but only in the tunica albuginea. These Y1-Rs are suggested to mediate vasoconstriction, possibly activated by NPY released from nerves in the tunica albuginea. In the female rat Y1-R mRNA, but not Y1-R-LI was found in vascular smooth muscles of arteries in the ovary and oviduct. In the oviduct Y1-R mRNA was also detected in the non-vascular smooth muscle layer. Fibers showing NPY-LI were found around blood vessels both in the ovary and oviduct. In the female genital tract also Y1-Rs may thus be involved in regulatory mechanisms mediating, for example, vasoconstriction.     

Neuropeptide Y: a putative neurotransmitter

Since its isolation in 1982, neuropeptide Y (NPY) has received considerable interest. This 36 amino acid peptide has been identified widely throughout the central and peripheral nervous systems, and within the autonomic system it appears in close association but not exclusively within catecholamine containing nerves. NPY begins to meet some of the criteria required to be established as a neurotransmitter. Thus, the peptide has been localised exclusively within nerves, and electron microscopy has shown NPY within nerve terminals. High affinity, saturable binding sites for NPY have been demonstrated in rat brain membranes, and the peptide has been reported to be released into the circulation during sympathetic nerve stimulation. The peptide is pharmacologically active both within the central nervous system by altering blood pressure, feeding and anterior pituitary function and in the periphery where NPY acts as a vasoconstrictor.     

Neuropeptide Y, ubiquitous and elusive

This paper reviews aspects of NPY research that were emerging in 1985, shortly after the isolation and characterization of the peptide. NPY had become known for its widespread distribution especially in the central and peripheral nervous systems, but also in the gastro-intestinal and respiratory tracts and in fibers innervating smooth muscle around blood vessels. Consistent with its distribution, it was determined that NPY is a potent vasoconstrictor, affects neuroendocrine systems and is involved in appetite regulation--areas of research still relevant today. Through advances in technology knowledge about NPY's role in these and newly discovered physiological functions has deepened considerably. Successful cloning of a series of NPY receptors has opened up new and complex research vistas. Lately, the creation of mice genetically modified for NPY as well as for several receptor subtypes has brought many puzzling observations--followed by questions yet to be answered.     

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: identification of the binding site

Based on the hypothetical 3D structure of neuropeptide Y (NPY), NPY 1-4-Aca-25-36, a 17 amino acid analogue, has been synthesized replacing the sequence NPY 5-24 by epsilon-aminocaproic acid (Aca). This low-molecular weight deletion analogue showed nearly comparable receptor affinity to NPY. In order to elucidate the structural requirements for receptor recognition each amino acid of 1-4-Aca-25-36 was exchanged by its D-enantiomer, glycine and L-alanine. In addition distinct amino acids were replaced by closely related residues. Multiple peptide synthesis was applied using Fmoc-strategy and BOP activation. Binding assay was performed on rabbit kidney membrane preparations. The results of structure affinity studies suggest that the C-terminal tetrapeptide NPY 33-36 is essential for receptor recognition.     

Neuropeptide Y cotransmission with norepinephrine in the sympathetic nerve-macrophage interplay

The CNS modulates immune cells by direct synaptic-like contacts in the brain and at peripheral sites, such as lymphoid organs. To study the nerve-macrophage communication, a superfusion method was used to investigate cotransmission of neuropeptide Y (NPY) with norepinephrine (NE), with interleukin (IL)-6 secretion used as the macrophage read-out parameter. Spleen tissue slices spontaneously released NE, NPY, and IL-6 leading to a superfusate concentration at 3-4 h of 1 nM:, 10 pM:, and 120 pg/ml, respectively. Under these conditions, NPY dose-dependently inhibited IL-6 secretion with a maximum effect at 10(-10) M: (p = 0.012) and 10(-9) M: (p < 0.001). Simultaneous addition of NPY at 10(-9) M: and the alpha-2-adrenergic agonist p-aminoclonidine further inhibited IL-6 secretion (p < 0.05). However, simultaneous administration of NPY at 10(-9) M: and the beta-adrenergic agonist isoproterenol at 10(-6) M: or NE at 10(-6) M: significantly increased IL-6 secretion (p < 0.005). To objectify these differential effects of NPY, electrical field stimulation of spleen slices was applied to release endogenous NPY and NE. Electrical field stimulation markedly reduced IL-6 secretion, which was attenuated by the NPY Y1 receptor antagonist BIBP 3226 (10(-7) M, p = 0.039; 10(-8) M, p = 0.035). This indicates that NPY increases the inhibitory effect of endogenous NE, which is mediated at low NE concentrations via alpha-adrenoceptors. Blockade of alpha-adrenoceptors attenuated electrically induced inhibition of IL-6 secretion (p < 0.001), which was dose-dependently abrogated by BIBP 3226. This indicates that under blockade of alpha-adrenoceptors endogenous NPY supports the stimulating effect of endogenous NE via beta-adrenoceptors. These experiments demonstrate the ambiguity of NPY, which functions as a cotransmitter of NE in the nerve-macrophage interplay.