Release of neuropeptide Y and hemodynamic changes during surgical removal of human pheochromocytomas

This study investigates the release of Neuropeptide Y from eight human pheochromocytomas. Profil immunoreactive Neuropeptide Y (Ir-NPY) levels during the management of surgery were compared with these of norepinephrine (NE) while hemodynamics were monitored. Plasma IrNPY and NE levels increased during tumor manipulation and returned to near normal one hour after operation. However, Ir-NPY levels remained high just after tumor resection while NE levels were significantly decreased. At tumor manipulation and just after tumor resection, plasma Ir-NPY levels were correlated with the systemic vascular resistances (SVR) (r = 0.74; P<0.04 and r = 0.86; P<0.006 respectively). No correlation was found either between plasma Ir-NPY and NE levels or between plasma NE levels and SVR. The release of Ir-NPY from tumor tissue, studied by a superfusion method, exhibited a significant correlation with the plasma Ir-NPY concentrations at the time of corresponding tumor resection (r = 0.95; P<0.007). Chromatographic analysis showed that Ir-NPY in plasma and outflow migrate as human NPY (1-36). These results confirmed that in pheochromocytoma, plasma NPY mainly originates from the tumor and argue for an important role of NPY in pheochromocytoma hypertension as indicated by the correlation between the Ir-NPY levels and the SVR.     

Radioimmunoassay of neuropeptide Y

The development of a radioimmunoassay to the newly isolated peptide, neuropeptide Y is described. Four separate antisera have been developed using different immunisation schedules. Two of these antisera (YNI and YNIO) are directed to the C-terminal region of the peptide and cross-react with the related peptide PYY, whereas YN7 is specific being directed to the N-terminal region of NPY, YN6 is similarly specific for NPY, but is unable to bind the available fragments. These four antisera provide similar results for determination of NPY immunoreactivity within porcine brain extracts, however YN6 consistently undervalues all extracts from the other species examined (human, rat, guinea pig, cat and mouse). Chromatographic analysis by means of reverse phase high pressure liquid chromatography (HPLC) shows that NPY immunoreactivity of human extracts elutes in an earlier position than the porcine standard. It seems likely therefore that human and porcine NPY differ in their amino acid sequences.     

Synthesis of neuropeptide Y

Neuropeptide Y (NPY), a hexatriacontapeptide amide, was synthesized on benzhydrylamine resin. The peptide product obtained by HF treatment contained 63% of the target peptide, NPY. A comparison of the chemical, immunochemical and biological properties of the synthetic peptide with natural NPY indicated that they were identical.     

Isolation and characterization of neuropeptide Y from porcine intestine

The isolation and primary structure of intestinal neuropeptide Y (NPY) is described. The peptide was purified from porcine intestinal extracts using a chemical assay and radioimmunoassay for NPY. The amino acid sequence of this peptide is: Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala- Arg-Tyr-Tyr- Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2. This the structure of intestinal NPY is identical to the NPY of brain origin.     

Ligands of the neuropeptide Y Y2 receptor

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian brain and exerts a variety of physiological processes in humans via four different receptor subtypes Y1, Y2, Y4 and Y5. Y2 receptor is the most abundant Y subtype receptor in the central nervous system and implicated with food intake, bone formation, affective disorders, alcohol and drugs of abuse, epilepsy, pain, and cancer. The lack of small molecule non-peptidic Y2 receptor modulators suitable as in vivo pharmacological tools hampered the progress to uncover the precise pharmacological role of Y2. Only in recent years, several potent, selective and non-peptidic Y2 antagonists have been discovered providing the tools to validate Y2 receptor as a therapeutic target. This Letter reviews Y2 receptor modulators mainly non-peptidic antagonists and their structure–activity relationships.     

Y1 and Y2 receptors for neuropeptide Y

By using monoiodinated radioligands of both intact neuropeptide Y (NPY) and of a long C-terminal fragment, NPY13-36, two subtypes of binding sites, which differ in affinity and specificity, have been characterized. The Y1 type of binding site, characterized on a human neuroblastoma cell line, MC-IXC, and a rat pheochromocytoma cell line, PC-12, binds NPY with a dissociation constant (Kd) of a few nanomolar but does not bind NPY13-36. The Y2 type of binding site, characterized on porcine hippocampal membranes and on another human neuroblastoma cell line, SMS-MSN, is of higher affinity and binds both NPY and NPY13-36. None of the binding sites distinguish between NPY and the homologous peptide YY (PYY). It is concluded that NPY/PYY-binding sites occur in two subtypes which may represent two types of physiological receptors.     

Release and functional role of neuropeptide Y as a sympathetic modulator in human saphenous vein biopsies

Transmural electrical stimulation of the sympathetic nerve endings of human saphenous vein biopsies released two forms of NPY identified chromatographically as native and oxidized peptide. The release process is dependent on extracellular calcium, the frequency, and the duration of the stimuli. While guanethidine reduced the overflow of ir-NPY, phenoxybenzamine did not augment NPY release, but increased that of noradrenaline. Oxidized NPY, like native NPY, potentiated the noradrenaline and adenosine 5'-triphospahate-induced vasoconstriction, an effect blocked by BIBP 3226 and consonant with the RT-PCR detection of the mRNA encoding the NPY Y1 receptor. These results highlight the functional role of NPY in human vascular sympathetic reflexes.     

Novel non-peptidic neuropeptide Y Y2 receptor antagonists

Through SAR studies of a piperidinylindoline cinnamide HTS lead, the first potent, non-peptide, low molecular weight selective Neuropeptide Y Y2 (NPY Y2) antagonists have been synthesized. The SAR studies around the piperidinyl, the indolinyl, and the cinnamyl moieties are discussed.     

Modeling of neuropeptide receptors Y1, Y4, Y5, and docking studies with neuropeptide antagonist

Neuropeptide Y (NPY), receptors belong to the G-protein coupled receptor superfamily. NPY mediates several physiological responses, such as blood pressure, food intake, sedation. These actions of NPY are mediated by six receptor subtypes denoted as Y1-Y5 and y6. Modeling of receptor subtypes and binding site identification is an important step in developing new therapeutic agents. We have attempted to model the three NPY receptor types, Y1, Y4, and Y5 using homology modeling and threading methods. The models are consistent with previously reported experimental evidence. To understand the interaction and selectivity of NPY analogues with different neuropeptide receptors, docking studies of two neuropeptide analogues (BVD10 and BVD15) with receptors Y1 and Y4 were carried out. Results of the docking studies indicated that the interaction of ligands BVD10 and BVD15 with Y1 and Y4 receptors are different. These results were evaluated for selectivity of peptide analogues BVD10 and BVD15 towards the receptors.     

Solution structure of human neuropeptide Y

Summary The three-dimensional structure of synthetic human neuropeptide Y in aqueous solution at pH 3.2 and 37°C was determined from two-dimensional ¹H NMR data recorded at 600 MHz. A restraint set consisting of 440 interproton distance restraints inferred from NOEs and 11 backbone and 4 side-chain dihedral angle restraints derived from spin-spin coupling constants was used as input for distance geometry calculations in DIANA and simulated annealing and restrained energy minimisation in X-PLOR. The final set of 26 structures is well defined in the region of residues 11–36, with a mean pairwise rmsd of 0.51 Å for the backbone heavy atoms (N, C and C) and 1.34 Å for all heavy atoms. Residues 13–36 form an amphipathic -helix. The N-terminal 10 residues are poorly defined relative to the helical region, although some elements of local structure are apparent. At least one of the three prolines in this N-terminal region co-exists in both cis and trans conformations. An additional set of 24 distances was interpreted as intermolecular distances within a dimer. A combination of distance geometry and restrained simulated annealing yielded a model of the dimer having antiparallel packing of two helical units, whose hydrophobic faces form a well-defined core. Sedimentation equilibrium experiments confirm the observation that neuropeptide Y associates to form dimers and higher aggregates under the conditions of the NMR experiments. Our results therefore support the structural features reported for porcine neuropeptide Y [Cowley, D.J. et al. (1992) Eur. J. Biochem., 205, 1099–1106] rather than the aPP fold described previously for human neuropeptide Y [Darbon, H. et al. (1992) Eur. J. Biochem., 209, 765–771].Abbreviations NPY neuropeptide Y - PP pancreatic polypeptide - 1D, 2D one-, two-dimensional - NOE nuclear Overhauser enhancement - NOESY 2D NOE spectroscopy - TOCSY 2D total correlation spectroscopy - E.COSY exclusive correlation spectroscopy - HMQC heteronuclear multiple-quantum coherence - rmsd root-mean-square deviation     

Metabolism and functions of neuropeptide Y

Neuropeptide Y is one of the most abundant neuropeptides in the central and peripheral nervous systems and its sequence is highly conserved among species. A number of key physiological roles for NPY are now emerging, especially in the control of feeding and energy homeostasis. Other physiological actions of NPY are also reviewed. The metabolism of NPY has been examined by employing certain purified ectopeptidases and by using different membrane preparations. These approaches reveal that NPY is processed at its N-terminus by two proline-preferring aminopeptidases: aminopeptidase P and dipeptidyl peptidase IV. The action of the latter enzyme generates NPY (3−36) which has previously been shown to be a selective agonist at the Y2 class of NPY receptor. Thus, post-secretory processing of NPY can modify receptor selectivity. NPY is found to be resistant to the action of two other membrane aminopeptidases (N and W), and to the action of angiotensin converting enzyme. However, it is a substrate for endopeptidase-24.11 (K _m=15.4 μM) which can cleave the Tyr²⁰−Tyr2¹ and Leu³⁰−Ile³¹ bonds consistent with the known specificity of the enzyme. In striatal synaptic and renal brush border membranes, NEP is shown to be the major NPY hydrolysing activity but plays a lesser role in intestinal brush border membranes. Knowledge of the proteolytic processing of NPY should aid in the design of stable analogues of this neuropeptide. Special issue dedicated to Dr. Herman Bachelard.     

Solubilization and characterization of active neuropeptide Y receptors from rabbit kidney

Active neuropeptide Y receptors were solubilized from rabbit kidney membranes using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). In membrane fragments and soluble extracts neuropeptide Y binding was time dependent, saturable, reversible, and of high affinity. Scatchard analysis of equilibrium binding data indicated a single class of binding sites with respective KD and Bmax values of 0.09 nM and 530 fmol/mg of protein for the membrane-bound receptors and 0.10 nM and 1585 fmol/mg of protein for the soluble receptors. Neuropeptide Y binding was specifically inhibited by the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) in a concentration-dependent manner, with IC50 values of 28 and 0.14 microM for membrane-bound and soluble receptors, respectively, suggesting that neuropeptide Y receptors are functionally coupled to GTP-binding regulatory proteins. Cross-linking studies were performed with the heterobifunctional N-hydroxysuccinimidyl-4-azidobenzoate and the monofunctional neuropeptide Y derivative, azidobenzoyl and led to the identification of a 100 kDa peptide that should represent the covalently labeled neuropeptide Y receptor.     

Cardiac function in neuropeptide Y Y4 receptor-knockout mice

Autonomic control of cardiovascular function in neuropeptide Y (NPY) Y4 receptor-knockout mice was investigated using pancreatic polypeptide (PP), NPY and specific agonists and antagonists for other NPY receptors well characterised in cardiovascular function. Y4 receptor-knockout mice, anaesthetised with sodium pentobarbitone, displayed slower heart rate, indicated by a higher pulse interval and lower blood pressure compared to control mice. After vagus nerves were cut heart rate increased but was still significantly slower than in control mice. PP had no effect on blood pressure or cardiac vagal activity in either group of mice, which was consistent with earlier studies in other species. Injection of NPY evoked an increase in blood pressure but the response was significantly reduced in Y4 receptor-knockout mice compared to the controls. The reduction in pressor activity was not Y1 mediated as the selective Y1 antagonist, BIBP 3226, was effective in blocking NPY pressor activity in knockout mice. In addition, cardiac vagal inhibitory activity evoked by low doses of NPY was also reduced when compared to control responses. As N-acetyl [Leu(28, 31)] NPY 24-36 inhibited vagal activity dose dependently in both groups of mice with no difference in response at any dose, it is unlikely that this effect also is receptor mediated. We propose that the reduced vasoconstrictor and vagal inhibitory activity evoked by NPY in Y4 receptor-knockout mice is due to a lack of adrenergic tone bought about by a proposed reduction in sympathetic activity, possibly resulting from altered NPY activity secondarily affecting adrenergic transmission. We conclude that Y4 receptor deletion disrupts autonomic balance within the cardiovascular system.     

Dihydropyridine neuropeptide Y Y(1) receptor antagonists

Dihydropyridine 5a was found to be an inhibitor of neuropeptide Y(1) binding in a high throughput (125)I-PYY screening assay. Structure-activity studies around certain portions of the dihydropyridine chemotype identified BMS-193885 (6e) as a potent and selective Y(1) receptor antagonist. In a forskolin-stimulated c-AMP production assay using CHO cells expressing the human Y(1) receptor, 6e demonstrated full functional antagonism (K(b)=4.5 nM). Compound 6e inhibited NPY-induced feeding in satiated rats when dosed at 3.0 and 10.0 mg/kg (ip), and also decreased spontaneous overnight food consumption in rats at doses of 10 and 20 mg/kg (ip).     

Naloxone blocks 'anxiolytic' effects of neuropeptide Y

Intracerebroventricular injection of neuropeptide Y (NPY) produces potent 'anxiolytic' effects in animal models of anxiety. Administration of opioid receptor antagonists suppresses NPY-induced food intake and thermogenesis. The present study examined whether the opiate antagonist naloxone would also suppress the 'anxiolytic' effects of neuropeptide Y. Following training and stabilization of responding in an operant conflict model of anxiety, rats were injected with either NPY or diazepam. Both NPY (veh., 2, 4, 6 microg, i.c.v.) and chlordiazepoxide (veh., 2, 4, 6 mg/kg, i.p.) produced a dose-dependent increase in punished responding in the conflict test. The 'anxiolytic' effects of NPY were not blocked by the administration of flumazenil (3, 6, 12 mg/kg, i.p.). The administration of naloxone (0.25-2.0 mg/kg, s.c.) antagonized the effects of NPY. Central administration of the selective mu opiate antagonist CTAP (1 microg, i.c.v.) partially blocked NPY-induced conflict responding. These results support the hypothesis that NPY may play an important role in experimental anxiety independent of the benzodiazepine receptor and further implicate the opioid system in the behavioral expression of anxiety.     

Pharmacological characterization of cloned chicken neuropeptide Y receptors Y1 and Y5

The neuropeptide Y (NPY) receptor subtypes Y1 and Y5 are involved in the regulation of feeding and several other physiological functions in mammals. To increase our understanding of the origin and mechanisms of the complex NPY system, we report here the cloning and pharmacological characterization of receptors Y1 and Y5 in the first non-mammal, chicken (Gallus gallus). The receptors display 80-83% and 64-72% amino acid sequence identity, respectively, with their mammalian orthologues. The three endogenous ligands NPY, peptide YY (PYY) and pancreatic polypeptide (PP) have similar affinities as in mammals, i.e. NPY and PYY have subnanomolar affinity for both receptors whereas chicken PP bound with nanomolar affinity to Y5 but not to Y1. A notable difference to mammalian receptor subtypes is that the Y1 antagonist SR120819A does not bind chicken Y1, whereas BIBP3226 does. The Y5 antagonist CGP71863A binds to the chicken Y5 receptor. Anatomically, both Y1 and Y5 have high mRNA expression levels in the infundibular nucleus which is the homologous structure of the hypothalamic arcuate nucleus in mammals. These results suggest that some of the selective Y1 and Y5 antagonists developed in mammals can be used to study appetite regulation in chicken.     

Endocrine function of neuropeptide Y knockout mice

Among its many proposed functions, neuropeptide Y (NPY) is thought to modulate the hypothalamic-pituitary axis. Specifically, increased hypothalamic NPY signaling may be critical in mediating the neuroendocrine response to fasting. To determine the consequences of NPY deficiency on endocrine physiology, multiple hormones were quantitated in wildtype and NPY-knockout mice under fed and fasted conditions. Serum concentrations of leptin, corticosterone, thyroxine, and testosterone were normal in NPY-knockout males fed ad libitum. A 48-hour fast resulted in a 50% reduction in leptin, a 60% reduction in thyroxine, a 75% reduction in testosterone, and a 12-fold increase in corticosterone in both wildtype and NPY-knockout mice. Fasting also increased the estrous cycle length by 3 days in both wildtype and NPY-deficient female mice. We conclude that NPY is not essential for appropriate function of the gonadotropic, thyrotropic, or corticotropic axes under ad lib fed conditions or in response to acute fasting.