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.

Food intake inhibition and reduction in body weight gain in lean and obese rodents treated with GW438014A, a potent and selective NPY-Y5 receptor antagonist.

Numerous reports have implicated theY5 receptor as the 'feeding' receptor mediating the orexigenic action of neuropeptide Y (NPY). This notion is supported by the correlation between the in vitro functional and binding activities of different peptide agonists and their potent stimulation of food intake in rodents. We have discovered a series of small molecule heterocycles with high affinity, selectivity, and functional antagonism for Y5 receptors. Intraperitoneal (i.p.) administration of GW438014A into rodents, resulted in a potent reduction of NPY-induced and normal overnight food intake. Brain levels of GW438014A were detected well in excess of its binding IC(50) for up to 3 h post-dosing. Daily (i.p., BID, 10 mg/kg) administration of this compound to Zucker Fatty rats for a period of 4 days resulted in a marked decrease in the rate of weight gain and a reduction in fat mass. No effect on food intake was observed following oral administration of GW438014A (25-100 mg/kg), consistent with the poor oral bioavailability (<3%) and low brain levels observed.

Food intake inhibition and reduction in body weight gain in rats treated with GI264879A, a non-selective NPY-Y1 receptor antagonist.

Neuropeptide Y has been proposed to play a major role in the hypothalamic regulation of feeding behavior through the activation of specific, central NPY receptor(s). In an effort to design small molecule antagonists of NPY receptors, we have synthesized a series of substituted dipeptides based on defined pharmacophores, previously identified by us and others as essential for the interaction with the peptide receptors. GI264879A behaves as a functional antagonist of Y1 receptors while displaying no binding selectivity for the different NPY receptor subtypes. We demonstrate here that administration of GI264879A to rats causes a significant decrease in food intake and body weight partly through a mechanism dependent on the integrity of the vagus nerve.

Novel neuropeptide Y receptor antagonists block vasoconstriction in the hamster cheek pouch microcirculation.

We investigated the efficacy of novel neuropeptide Y (NPY) antagonists to inhibit the microcirculatory dynamics of NPY in the hamster cheek pouch microcirculation using intravital microscopy and computer-assisted image analysis. Changes in arteriolar diameter served as an index of vasomotor alterations. Fluorescein isothiocyanate-labeled Dextran 150 served as a tracer for measurements of macromolecular transport. GW 383 and GW 1229, two novel NPY receptor antagonists, were applied topically in separate experiments. Pretreatment with 10(-5), 10(-6) and 10(-7) M GW 383 and with 10(-6) and 10(-8) M GW 1229 attenuated the vasoconstriction induced by 10(-7) M NPY in a dose-dependent manner. Furthermore, pretreatment with 10(-7) and 10(-8) M GW 1229 significantly inhibited the 10(-9) M NPY-induced vasoconstriction. At these doses, the NPY antagonists did not alter microvascular permeability. Our results demonstrate that the novel NPY antagonists inhibit the vasoconstriction induced by NPY in the hamster check pouch microcirculation. We suggest that the inhibition is due to binding of antagonists to Y1-type NPY receptors.

GW1229, a novel neuropeptide Y Y1 receptor antagonist, inhibits the vasoconstrictor effect on neuropeptide Y in the hamster microcirculation.

We studied the effect of GW1229, a novel neuropeptide Y Y1 receptor antagonists, on the vasoconstriction induced by neuropeptide Y and structurally related analogs in the hamster cheek pouch microcirculation. Changes in arteriolar diameter and microvascular conductance were assessed by intravital microscopy and measurement of sodium22 clearance. GW1229 did not affect basal vascular conductance but inhibited, concentration dependently, the reduction in arteriolar diameter and vascular conductance induced by 100 nM neuropeptide Y. GW1229 also counteracted the vasoconstrictor effect of 100 nM [Leu31,Pro34]neuropeptide Y, and that of 300 nM neuropeptide Y-[(13-36). In contrast, GW1229 had no effect on the vasoconstriction induced by noradrenaline. We conclude that the vasoconstrictor effect on neuropeptide Y in the hamster cheek pouch is mediated by neuropeptide Y Y1 receptors. The maintenance of physiological tone in this vascular bed does not involve the participation of endogenous neuropeptide Y.

Hemodynamic profile of neuropeptide Y in dogs: effect of ganglionic blockade.

The hemodynamic effects of neuropeptide Y (NPY) were examined over a dose range of 0.03-30 nmol/kg, i.v., in anesthetized open-chest, ventilated dogs with and without ganglionic blockade. In normal (non-ganglion-blocked) animals, NPY produced significant, dose-dependent, and sustained (lasting 15-45 min) increases in mean arterial blood pressure and systemic vascular resistance (SVR) with a threshold dose of 0.3 nmol/kg and a maximum effective dose of 10 nmol/kg. Cardiac index (CI) decreased at doses > 1 nmol/kg, but stroke volume was not altered; heart rate (HR) decreased significantly at and above the 3 nmol/kg dose. No significant changes were observed in the left ventricular dP/dt (LVdP/dt) or the contractility index (LVdP/dt divided by systolic pressure). In ganglion-blocked animals, pressor and SVR responses to NPY were similar to those seen in normal animals but HR was not affected and a small but significant decrease in CI was seen only at the 30 nmol/kg. Furthermore, whereas LVdP/dt of ganglion-blocked dogs increased significantly at and above the 1 nmol/kg dose, the contractility index increased slightly only with the 10 and 30 nmol/kg doses. These data indicate that NPY produces sustained hypertension in dogs secondary to peripheral vasoconstriction, has a weak, direct positive inotropic action on the heart, and lacks chronotropic effects.

Pharmacological characterization and selectivity of the NPY antagonist GR231118 (1229U91) for different NPY receptors.

Neuropeptide Y (NPY) is widely distributed throughout the central and peripheral nervous system and exerts a wide range of physiological responses by activating specific receptors. In this study we have characterized the potency of the high affinity peptide dimer antagonist, GR231118, to displace radiolabeled NPY/PYY from different tissues and cell lines expressing Y1 or Y2 receptors and from CHO cells stably transfected with human cDNA encoding for Y1, Y2 and Y4 receptors. GR231118 displays high affinity for Y1 and Y4 receptors, equal or better than that of NPY itself, while its activity is several fold weaker for Y2 receptors. Displacement of radiolabeled PYY from rat hypothalamic membranes by GR231118, reveals the existence of high and low affinity binding sites which may be equated to Y1 and Y2 receptors respectively suggesting that the compound maybe used as a tool to dissect central NPY receptors.

Hemodynamic characterization of a novel neuropeptide Y receptor antagonist.

Defining the roles of the vasoconstrictor peptide neuropeptide Y (NPY) in the cardiovascular system is difficult due to lack of availability of specific NPY receptor antagonists. We report the in vivo NPY receptor blocking actions of a novel nonapeptide dimer, 1229U91 {(IleGluProDprTyrArgLeuArgTyrNH(2)(2)}, and describe its hemodynamic effects. In anesthetized normotensive rats, 1229U91 produced significant and dose-dependent reductions in NPY-reduced hemodynamic responses. 1229U91 (3-30 nmol/kg intravenously, i.v.) attenuated the pressor response (34 +/- 6-84 +/- 1%) and the increases in renal vascular resistance (RVR, 56 +/- 9-94 +/- 2%) produced by NPY (1 nmol/kg i.v.). Intravenous norepinephrine (NE)-induced hemodynamic responses were not altered by 1229U91. 1229U91 also produced dose-dependent inhibition of NPYinduced vasoconstrictor responses in anesthetized dogs and spontaneously hypertensive rats (SHR). These data demonstrate that 1229U91 is a selective NPY receptor antagonist. 1229U91 had no effect on resting hemodynamic variables in these preparations. In conscious SHR, 1229U91 did not produce significant changes in blood pressure (BP) or heart rate (HR) over a wide dose-range (15-1,500 nmol/kg i.v.). Lack of effect of the NPY receptor antagonist in SHR suggests that NPY does not contribute to the maintenance of BP in this hypertension model.

Synthesis, structure and stability of novel dimeric peptide-disulfides.

Oxidation of nonapeptide dithiol (2) with K3Fe(CN)6 leads to either monomeric disulfide (4) or antiparallel and parallel dimeric disulfides (3a and 3b) depending upon reaction conditions. When exposed to small amounts of thiols or cyanide in aqueous solution, these three species interconvert to an equilibrium mixture of 2:1:8 (3a:3b:4).

Structure-activity relationship of novel pentapeptide neuropeptide Y receptor antagonists is consistent with a noncontinuous epitope for ligand-receptor binding.

We report the first systematic study on short peptide structure affinity and activity for the neuropeptide Y (NPY) receptor. A series of linear pentapeptides has been synthesized that display affinities in the low micromolar range toward rat brain NPY receptors. Furthermore, some of these compounds competitively antagonize the Y1-type NPY receptor-mediated increase in cytosolic Ca2+ in human erythroleukemic (HEL) cells. The inactive NPY carboxyl-terminal pentapeptide (Thr-Arg-Gln-Arg-Tyr-NH2; IC50 > 100 microM) was modified by replacing threonine with an aromatic amino acid and glutamine with leucine. This resulted in a series of pentapeptides with dramatically improved affinity (IC50 = 0.5-4 microM) for the rat brain receptor. The structure-affinity data suggest that these peptides may represent a noncontinuous epitope containing the amino-terminal tyrosine and the carboxyl-terminal residues Arg-35 and Tyr-36 of NPY.

High-affinity neuropeptide Y receptor antagonists.

Neuropeptide Y (NPY) is one of the most abundant peptide transmitters in the mammalian brain. In the periphery it is costored and coreleased with norepinephrine from sympathetic nerve terminals. However, the physiological functions of this peptide remain unclear because of the absence of specific high-affinity receptor antagonists. Three potent NPY receptor antagonists were synthesized and tested for their biological activity in in vitro, ex vivo, and in vivo functional assays. We describe here the effects of these antagonists inhibiting specific radiolabeled NPY binding at Y1 and Y2 receptors and antagonizing the effects of NPY in human erythroleukemia cell intracellular calcium mobilization perfusion pressure in the isolated rat kidney, and mean arterial blood pressure in anesthetized rats.

Novel modified carboxy terminal fragments of neuropeptide Y with high affinity for Y2-type receptors and potent functional antagonism at a Y1-type receptor.

Peptide analogs of neuropeptide Y (NPY) with a Tyr-32 and Leu-34 replacement resulted in the decapeptide TyrIleAsnLeuIleTyrArgLeuArgTyr-NH2 (9; Table 1) and a 3700-fold improvement in affinity at Y2 (rat brain; IC50 = 8.2 +/- 3 nM) receptors when compared to the native NPY(27-36) C-terminal fragment. In addition, compound 9 was an agonist at Y1 (human erythroleukemia (HEL) cell; ED50 = 8.8 +/- 0.5 nM) receptors with potency comparable to that of NPY(1-36) (ED50 = 5 nM). Molecular dynamics and 1H-NMR were used to propose a solution structure of decapeptide 9 and for subsequent analog design. The replacement of Leu with Pro at position 4 of decapeptide 9 afforded an antagonist of NPY in HEL cells (18, TyrIleAsnProIleTyrArgLeuArgTyr-NH2; IC50 = 100 +/- 5 nM). Deletion of the N-terminal tyrosine of 18 resulted in a 10-fold improvement in antagonistic activity with a parallel 4-fold decrease in Y2 affinity. This potent antagonist may provide further insight into the physiological role(s) for NPY in the mammalian and peripheral nervous system.

The role of extracellular calcium in the neuropeptide-Y-induced increase in cytosolic calcium in human erythroleukemic (HEL) cells.

Cytosolic calcium changes were followed in human erythroleukemic (HEL) cells loaded with the fluorescent probe fura-2. Peak increases in cytosolic calcium were reduced by two-thirds in cells suspended in Ca(2+)-free medium, suggesting that calcium entry significantly contributes to the increases in cytosolic calcium after NPY receptor stimulation. To establish if Ca2+ entry was a direct consequence of receptor stimulation or indirectly via depletion of Ca2+ stores, the latter were totally or partially depleted by treatment with cyclopiazonic acid or alpha-thrombin, respectively, in Ca(2+)-free medium. Partial depletion markedly diminished and full depletion suppressed the NPY-induced response in Ca(2+)-free medium. After full depletion, the recovery of the NPY-induced increase in cytosolic calcium was dependent on the length of [Ca2+]e reexposure, suggesting a direct entry of Ca2+ to the storage sites followed by release to the cytosol. After partial depletion, transient reexposure to [Ca2+]e did not by itself increase cytosolic calcium levels or refill the stores as NPY stimulation did not increase cytosolic calcium if [Ca2+]e was chelated prior to stimulation. However, if partially depleted cells were exposed to NPY in the presence of readded [Ca2+]3, the peak calcium response was similar to that of control cells, indicating that partially depleted calcium stores can be refilled from extracellular sources only if NPY receptors are stimulated. Analysis of the data suggests that in HEL cells the entry of calcium and mobilization from intracellular stores are in series processes and that entry is triggered by intracellular levels only under extreme depletion, while under physiological conditions calcium entry is coupled to receptor stimulation.

Microcirculatory dynamics of neuropeptide Y.

We used the hamster cheek pouch microcirculation to investigate by intravital microscopy the effects of neuropeptide Y (NPY) on arteriolar diameter, leukocyte adhesion to microvascular endothelium, and postcapillary venular permeability. We applied NPY topically for 3 min at concentrations of 10(-7), 10(-9), and 10(-11) M. We quantified arteriolar diameter and permeability changes by digital image analysis. We used the mass of fluorescein isothiocyanate-Dextran 150 accumulated around postcapillary venules (10-30 microns) to calculate extravasation rates of macromolecules. We also measured the number of adhering white cells per 100-microns length of postcapillary venules using acridine orange to label white blood cells. At the applied doses, NPY did not alter either microvascular permeability to macromolecules or leukocyte adhesion to microvascular walls. NPY, in a dose-dependent manner, constricted arterioles ranging in control diameter from 10 to 60 microns. Vasoconstriction was strongest in arterioles ranging in diameter from 30 to 39 microns at a concentration of NPY of 10(-7) M. The Y1-type NPY receptor agonist, Leu31, Pro34-NPY, was as potent as NPY, whereas the carboxy-terminal fragment NPY 13-36 had no activity, indicating that the hamster cheek pouch microvasculature expresses the Y1 type of NPY receptor. We also blocked alpha-adrenergic receptors to test whether norepinephrine is required for NPY-induced vasoconstriction. This blockade did not inhibit the vasoconstriction caused by exogenous NPY. Our results demonstrate that (1) NPY modulates microvascular hemodynamics by changes in arteriolar diameter, (2) the NPY receptor on the hamster cheek pouch microvasculature is of the Y1 type, and (3) exogenous NPY-induced vasoconstriction is independent of the activity of endogenous norepinephrine.

Differences in the reserpine-sensitive storage in vivo of 1-methyl-4-phenylpyridinium in rats and mice may explain differences in catecholamine toxicity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Administration of reserpine, an inhibitor of vesicular catecholamine storage, differentially reduced the accumulation of MPP+ formed from MPTP in rats and mice. The effects were most pronounced in the adrenal gland for either species. In rats, reserpine decreased striatal and hippocampal MPP+ levels while in mice reserpine did not affect the disposition of MPP+ in the striatum but decreased hippocampal MPP+. The data suggest that mice may be more sensitive to the toxicant because less striatal MPP+ appears to be stored in the reserpine-sensitive storage vesicle.

Characterization of the neuropeptide Y-induced intracellular calcium release in human erythroleukemic cells.

Human erythroleukemic (HEL) cells, loaded with fura-2, respond to neuropeptide Y (NPY) with a fast and transient increase in intracellular calcium. The Y1 receptor-specific agonist (Leu-31,Pro-34)-NPY is 4-fold more potent and the carboxyl-terminal fragment NPY13-36 is 150-fold less potent than NPY. Thus, it is concluded that the response is mediated through the activation of a Y1 type of NPY receptor. HEL cells do not respond to a second addition of NPY but do respond to a further addition of alpha-thrombin (alpha-T). However, in a calcium-free medium, prior stimulation with NPY largely inhibits a subsequent response to alpha-T. Moreover, prior stimulation with alpha-T in the absence of external calcium completely prevents the response to the addition of NPY, indicating a common effector pathway. The latter is further reinforced by using thapsigargin (TG), which has been shown to deplete the Inositol 1,4,5-trisphosphate-dependent calcium pool in other systems. HEL cells preincubated with TG in calcium-free medium fail to respond to either NPY or alpha-T. Likewise, prior stimulation with NPY or alpha-T in calcium-free medium significantly inhibits the response to TG. Preincubation of cells with phorbol esters strongly inhibits the NPY-induced release of intracellular Ca2+ in HEL cells, an effect that is partially prevented by preincubation of the cells with H7, a protein kinase C inhibitor. However, neither the homologous nor the apparent heterologous desensitization of the NPY receptor can be prevented by H7. It is concluded that NPY releases intracellular Ca2+ from an inositol 1,4,5-trisphosphate-sensitive calcium pool, which is restored by external calcium, and that NPY receptor desensitization is protein kinase C independent.

Multicompartmental secretion of ascorbate and its dual role in dopamine beta-hydroxylation.

The neurobiological functions of ascorbate have both intra- and extracellular sites of action. Intracellularly, it participates predominantly in enzymic and transport reactions for neurotransmitter and hormone biosynthesis. Ascorbate is the cofactor for the dopamine beta-hydroxylase and peptidylglycine alpha-amidating monooxygenase systems, which catalyze the synthesis of norepinephrine and a variety of alpha-amidated peptides, respectively. The localization of these enzymes within the neurotransmitter- or hormone-containing storage vesicle requires a system for the constant regeneration of ascorbate to the reduced form. In fact, ascorbate participates in its own regeneration as a component of the vesicular electron-transport system. In addition to the roles of ascorbate in messenger synthesis, it is secreted from cells from different subcellular compartments. The extracellular role(s) of ascorbate are still unknown, although its interaction with and modification of plasma membrane proteins suggests some modulatory function.