Pre- and postjunctional actions of neuropeptide Y and related peptides

The effects of neuropeptide Y (NPY) and related peptide fragments on blood pressure and vagal action at the heart were compared in the anaesthetized rat. A change in vagal action was taken as a measure of presynaptic activity and a change in blood pressure was taken as a measure of postsynaptic activity. NPY, NPY-(13-36), PYY-(13-36), des-Ser22-NPY-(13-36) and a stabilized 13-36 analogue of NPY (ANA NPY) all exerted pressor actions and attenuated vagal action at the heart. The maximum vagal inhibitory or presynaptic action in order of potency was NPY, ANA-NPY, PYY-(13-36) significantly greater than NPY-(13-36), des-Ser22-NPY-(13-36). The order of potency for the half time of this effect was NPY, ANA-NPY significantly longer than PYY-(13-36) and NPY-(13-36), which were significantly longer than des Ser22-NPY-(13-36). For the pressor or postsynaptic effects, NPY increased blood pressure significantly more and for a longer duration than all the 13-36 fragments, which were not demonstrably different in this respect. These results are consistent with the proposal that there are two populations of NPY receptors. The C-terminal flanking peptide of NPY (CPON) and desamido-NPY had no effect on either vagal action at the heart or on blood pressure.     

Evidence for different pre-and post-junctional receptors for neuropeptide Y and related peptides

The effects of neuropeptide Y (NPY), peptide YY (PYY), desamido-NPY and five C-terminal fragments of NPY or PYY were tested on different smooth muscle preparations in vitro. The fragments were NPY 19-36, NPY 24-36, PYY 13-36, PYY 24-36 and PYY 27-36. NPY and PYY appear to exert three principally different effects at the level of the sympathetic neuroeffector junction. Firstly, they have a direct post-junctional effect, leading to constriction of certain blood vessels; this was studied on the guinea-pig iliac vein. Secondly, they potentiate the response to various vasoconstrictors; this was studied on the rabbit femoral artery and vein, using noradrenaline and histamine, respectively, as agonists. Thirdly, NPY and PYY act prejunctionally in that they suppress the release of noradrenaline from sympathetic nerve endings upon stimulation; this was studied in the rat vas deferens. NPY and PYY were approximately equipotent in constricting the guinea-pig iliac vein, while desamido-NPY and the fragments were without effect. Desamido-NPY and the fragments were ineffective also in potentiating the response to noradrenaline in the rabbit femoral artery, nor did they potentiate the response to histamine in the rabbit femoral vein. NPY and PYY potentiated the response to noradrenaline in the artery, as well as the response to histamine in the vein. The NPY- and PYY-induced suppression of noradrenaline release from the prostatic portion of the rat vas deferens was reproduced by PYY 13-36 but not by the shorter fragments nor by desamido-NPY. In conclusion, a C-terminal portion seems to be sufficient for exerting the prejunctional effect of NPY and PYY, while the whole sequence seems to be required for post-junctional (direct and modulatory) effects. An amidated C-terminal is crucial for maintaining the biological activity of NPY. Desamido-NPY and the fragments that were inactive as agonists also seemed inactive as antagonists.     

Neuromodulation of the cardiac vagus: comparison of neuropeptide Y and related peptides

Neuropeptide Y (NPY), a putative co-transmitter in noradrenergic sympathetic nerves of the cardiovascular system, inhibits the negative chronotropic action of the cardiac vagus. In the present study, peptides related to NPY were tested for potency in producing this effect. In bilaterally vagotomized, anaesthetised dogs, the increase in pulse interval caused by electrical stimulation of the peripheral stump of the right vagus was measured before and after intravenous administration of peptide. The effects of doses of NPY were compared with those of equimolar doses of peptide YY (PYY), and of avian and human pancreatic polypeptides (APP and HPP). PYY inhibited the vagal action more effectively than did NPY. APP and HPP, however, caused no change in strength of vagal action at the doses used. The response to a second injection of NPY, given soon after the injection of APP or HPP, was not significantly different from the original. Thus no evidence was obtained for a competitive inhibition of the action of NPY by either pancreatic polypeptide. A C-terminal hexapeptide fragment of human pancreatic polypeptide was also tested. Like APP and HPP, it neither inhibited the cardiac vagus nor blocked the action of NPY. The order of potency obtained here (PYY greater than NPY much greater than APP, HPP, CFPP) can be expected to be of use in efforts to distinguish the active site(s) of the NPY molecule, and to characterise the receptors involved in these modulatory effects.     

The vasoconstrictor effect of neuropeptide Y and related peptides in the guinea pig isolated heart

Neuropeptide Y (NPY) infusions into isolated, perfused, spontaneously beating hearts of guinea pigs elicited concentration-dependent increases of myocardial perfusion pressure and decreases of myocardial tension, but no consistent changes of heart rate. The increase of perfusion pressure caused by NPY (attributed to a constrictor effect on coronary vessels) was not affected by atropine, prazosin, yohimbine, propranolol, cimetidine, diphenhydramine, indomethacin or a mixture of methysergide and morphine. However, it was reduced by verapamil, a Ca²⁺ antagonist. Deletion of the N-terminal amino acid Tyr¹ from the NPY molecule caused a 12-fold reduction of NPY potency as a coronary constrictor. Further shortening of the NPY molecule by removal of sequence Tyr¹ through Glu¹⁵ or Tyr¹ through Ala¹⁸ caused major losses of potency without detectable reduction of intrinsic activity. The results suggest that the constrictor effect of NPY on guinea pig coronary vessels results from a direct effect on vascular smooth muscle cells, is mediated by specific receptors and is likely to involve the participation of extracellular calcium ions. The results also suggest that the chemical groups responsible for the vasoconstrictor effect of NPY in guinea pig hearts might be scattered in the C-terminal end of the peptide.     

Role of atrial natriuretic peptides and neuropeptide Y in blood pressure regulation

Atrial natriuretic peptides (ANP) are released into the circulation in response to enhanced atrial stretching. These peptides not only have diuretic and natriuretic properties, but also exert a relaxing effect on the vasculature. Moreover, they antagonize the contractions induced by norepinephrine and angiotensin II. Neuropeptide Y (NPY) is also a vasoactive peptide. It is widely distributed throughout the central and peripheral nervous systems. NPY is coreleased with norepinephrine by perivascular nerve endings. At high concentrations, this peptide has a direct vasoconstrictor effect. In addition, it enhances the vascular effect of various agonists, including norepinephrine and angiotensin II. Both ANP and NPY have an inhibitory effect on renin secretion. This effect may have important implications for the role of these peptides in cardiovascular regulation.     

Structure-activity relationships of neuropeptide Y Y1 receptor antagonists related to BIBP 3226

Analogues of BIBP 3226, (R)-N(alpha)-diphenylacetyl-N-(4-hydroxybenzyl)argininamide, were synthesized and investigated for Y1 antagonism (Ca2+-assay, HEL cells) and binding on Y1, Y2 and Y5 receptors. Replacing the benzylamino by a tetrahydrobenzazepinyl group preserves most of the Y1 activity. Combination with a N(G)-phenylpropyl arginine and a N(alpha)-p-biphenylylacetyl moiety shifted the NPY receptor selectivity towards Y5.     

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.     

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.     

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.     

Central administration of neuropeptide FF and related peptides attenuate systemic morphine analgesia in mice

Neuropeptide FF (NPFF) belongs to an opioid-modulating peptide family. NPFF has been reported to play important roles in the control of pain and analgesia through interactions with the opioid system. However, very few studies examined the effect of supraspinal NPFF system on analgesia induced by opiates administered at the peripheral level. In the present study, intracerebroventricular (i.c.v.) injection of NPFF (1, 3 and 10 nmol) dose-dependently inhibited systemic morphine (0.12 mg, i.p.) analgesia in the mouse tail flick test. Similarly, i.c.v. administration of dNPA and NPVF, two agonists highly selective for NPFF(2) and NPFF(1) receptors, respectively, decreased analgesia induced by i.p. morphine in mice. Furthermore, these anti-opioid activities of NPFF and related peptides were blocked by pretreatment with the NPFF receptors selective antagonist RF9 (10 nmol, i.c.v.). These results demonstrate that activation of central NPFF(1) and NPFF(2) receptors has the similar anti-opioid actions on the antinociceptive effect of systemic morphine.     

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.     

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.     

Identification of a Drosophila brain-gut peptide related to the neuropeptide Y family.

A neuropeptide F (NPF) was isolated from the fruit fly, Drosophila mellanogaster, based on a radioimmunoassay for a gut peptide from the corn earworm, Helicoverpa zea. A partial sequence was obtained from the fly peptide, and a genomic sequence coding for NPF was cloned after inverse polymerase chain reaction and shown to exist as a single genomic copy. The encoded, putative prepropeptide can be processed into an amidated NPF with 36 residues that is related to invertebrate NPF's and the neuropeptide Y family of vertebrates. In situ hybridization and immunocytochemistry showed that Drosophila NPF was expressed in the brain and midgut of fly larvae and adults.     

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.     

Role of neuropeptide Y and its receptors in the progression of endocrine-related cancer

The neuropeptide Y (NPY) family of peptides, in addition to its many physiological actions, has also been involved in the modulation of tumor progression, with specific reference to endocrine-related cancers such as neuroendocrine tumors, breast and prostate cancers. These have been found either to express NPY receptors, or to secrete NPY-related peptides, or both. The study of the role of the NPY family of peptides in the biology of endocrine-related tumors, specifically concerning cell proliferation, angiogenesis, invasion and metastatization, may help to clarify some aspects of tumor pathophysiology, as well as to indicate novel diagnostic markers and therapeutical approaches.     

ProSAAS-derived peptides are colocalized with neuropeptide Y and function as neuropeptides in the regulation of food intake

ProSAAS is the precursor of a number of peptides that have been proposed to function as neuropeptides. Because proSAAS mRNA is highly expressed in the arcuate nucleus of the hypothalamus, we examined the cellular localization of several proSAAS-derived peptides in the mouse hypothalamus and found that they generally colocalized with neuropeptide Y (NPY), but not α-melanocyte stimulating hormone. However, unlike proNPY mRNA, which is upregulated by food deprivation in the mediobasal hypothalamus, neither proSAAS mRNA nor proSAAS-derived peptides were significantly altered by 1-2 days of food deprivation in wild-type mice. Furthermore, while proSAAS mRNA levels in the mediobasal hypothalamus were significantly lower in Cpe(fat/fat) mice as compared to wild-type littermates, proNPY mRNA levels in the mediobasal hypothalamus and in other subregions of the hypothalamus were not significantly different between wild-type and Cpe(fat/fat) mice. Intracerebroventricular injections of antibodies to two proSAAS-derived peptides (big LEN and PEN) significantly reduced food intake in fasted mice, while injections of antibodies to two other proSAAS-derived peptides (little LEN and little SAAS) did not. Whole-cell patch clamp recordings of parvocellular neurons in the hypothalamic paraventricular nucleus, a target of arcuate NPY projections, showed that big LEN produced a rapid and reversible inhibition of synaptic glutamate release that was spike independent and abolished by blocking postsynaptic G protein activity, suggesting the involvement of a postsynaptic G protein-coupled receptor and the release of a retrograde synaptic messenger. Taken together with previous studies, these findings support a role for proSAAS-derived peptides such as big LEN as neuropeptides regulating food intake.     

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.     

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.