Effects of neuropeptide Y (NPY) at the sympathetic neuroeffector junction. Can pre- and postjunctional receptors be distinguished?

Neuropeptide Y (NPY) is widely distributed in central and peripheral neurons. In sympathetic postganglionic neurons, NPY coexists with noradrenaline. NPY and its structural relative peptide YY (PYY) appear to exert three principally different effects at the sympathetic neuroeffector junction. Firstly, NPY has a direct postjunctional effect; this effect is manifested as a vasoconstriction when studied on the guinea pig iliac vein. Secondly, NPY has an indirect postjunctional effect in that it potentiates the response to various vasoconstrictors; this was studied on the rabbit femoral artery and vein, using noradrenaline and histamine, respectively, as vasoconstrictors. Thirdly, NPY acts prejunctionally in that it suppresses the release of noradrenaline from sympathetic nerve terminals; this was studied in the rat vas deferens. The aim of the investigation was to examine whether the three effects of NPY were mediated by the same type of receptor. For this purpose, we examined the effects of a series of NPY-related peptides, namely NPY, PYY, desamido-NPY, and five C-terminal fragments (NPY 19-36, NPY 24-36, PYY 13-36, PYY 24-36 and PYY 27-36). NPY and PYY were active in all three assay systems. The C-terminal amide appears to be crucial for maintaining the biological activity, since desamido-NPY was inactive in the three test systems. Interestingly, PYY 13-36 was almost as active as NPY and PYY in suppressing the electrically evoked contractions of the vas deferens; PYY 13-36 was inactive in the two other test systems. None of the shorter fragments had any biological activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presynaptic effects of neuropeptide Y on [3H]noradrenaline and [3H]acetylcholine release

The electrically evoked release of radioactivity from mouse vas deferens and rat hypothalamic slices preloaded with [3H]noradrenaline was measured. In addition the release of [3H]acetylcholine from longitudinal muscle strip of guinea-pig ileum was also measured. Neurochemical evidence has been obtained that neuropeptide Y (NPY), although it co-exists and is released with (-)-noradrenaline (NA), it behaves differently as far as its effect on presynaptic modulation of chemical neurotransmission is concerned. It exerts a frequency-dependent presynaptic inhibitory effect on noradrenaline release from mouse vas deferens but has no effect on the electrically evoked release of NA from rat hypothalamus. Unlike NA, NPY does not influence the release of [3H]acetylcholine from the longitudinal muscle strip of guinea-pig ileum and does not potentiate the presynaptic effect of NA. It seems very likely, that the inhibitory effect of NPY is mediated via receptors. Its action is concentration dependent. While exogenous noradrenaline inhibited the release of noradrenaline by 91%, the maximum inhibition reached with NPY was not higher than 60%, indicating that either the intrinsic activity of NPY is lower or much less axon terminals are equipped with NPY receptors. Peptide YY (PYY) also reduced the release of NA from mouse vas deferens.     

Synthesis of analogues of peptide YY with modified N-terminal regions: relationships of amphiphilic secondary structures and activity in rat vas deferens

Peptide YY (PYY) not only has distinct sequence homology with neuropeptide Y (NPY) and avian pancreatic polypeptide (APP), but it also exhibits both NPY- and APP-like biological activities. We synthesized two analogues of PYY, A1 and A2, with modified N-terminal regions, and compared their chemical and biological properties to those of PYY and the C-terminal fragment of PYY, (13-36)PYY. This study shows that there is a good correlation between the stability of amphiphilic alpha-helical structure and the biological activity of these peptides. A CD study of (13-36)PYY in mixed H2O and trifluoroethanol (TFE) solutions indicated a significant increase in alpha-helical segments (26-79%) with increasing TFE proportions. Since the fragment (13-36)PYY had potent activity in the rat vas deferens (RVD) assay, the secondary structure is possibly required on the RVD cell surface receptors. The analogues, A1 and A2, were designed to increase the stability of the alpha-helical structures by incorporation of modified N-terminal regions. The CD studies and the RVD assays of A1 and A2, suggest that the amphiphilic alpha-helical structures are stabilized by intramolecular hydrophobic interactions with the N-terminal regions and/or by intermolecular hydrophobic interactions in the self-association process, and subsequently potentiate the activities of the peptides compared to those of PYY and (13-36)PYY.     

Some pharmacological effects of prodolic acid, a new anti-inflammatory compound, indicative of inhibition of prostaglandin synthesis.

Prodolic acid, a new non-steroidal anti-inflammatory compound, inhibited bradykinin-induced bronchoconstriction but did not affect histamine-induced bronchoconstriction in the guinea pig. Prodolic acid potentiated the responses of the isolated rabbit vas deferens and portal vein to electrical stimulation without altering the response to noradrenaline. The potentiating effects of prodolic acid on the vas deferens were reversible but the potentiating effects in the portal vein were frequency-dependent. It is concluded that these effects of prodolic acid are probably related to its ability to inhibit prostaglandin biosynthesis.     

Potent and selective tools to investigate neuropeptide Y receptors in the central and peripheral nervous systems: BIB03304 (Y1) and CGP71683A (Y5)

We have evaluated 3 newly developed neuropeptide Y receptor antagonists in various in vitro binding and bioassays: BIBO3304 (Y1), T4[NPY33-36]4 (Y2), and CGP71683A (Y5). In rat brain homogenates, BIBO3304 competes for the same population of [125I][Leu31,Pro34] peptide YY (PYY) binding sites (75%) as BIBP3226, but with a 10 fold greater affinity (IC50 of 0.2 +/- 0.04 nM for BIBO3304 vs. 2.4 +/- 0.07 nM for BIBP3226),while CGP71683A has high affinity for 25% of specific [125I][Leu31,Pro34]PYY binding sites. Both BIBO3304 and CGP71683A (at 1.0 microM) were unable to compete for a significant proportion of specific [125I]PYY3-36/Y2 sites. The purported Y2 antagonist T4[NPY33-36]4 competed against [125I]PYY3-36 binding sites with an affinity of 750 nM. These results were confirmed in HEK 293 cells transfected with either the rat Y1, Y2, Y4, or Y5 receptor cDNA. BIBO3304, but not CGP71683A, competed with high affinity for [125I][Leu31,Pro34]PYY binding sites in HEK 293 cells transfected with the rat Y1 receptor cDNA, whereas the reverse profile was observed upon transfection with the rat Y5 receptor cDNA. Additionally, both molecules were inactive at Y2 and Y4 receptor subtypes expressed in HEK 293 cells. Receptor autoradiographic studies revealed the presence of [125I][Leu31,Pro34]PYY/BIBO3304-insensitive sites in the rat brain as reported previously for BIBP3226. Finally, the selective antagonistic properties of BIBO3304 were demonstrated in a Y1 bioassay (rabbit saphenous vein; pA2 value of 9.04) while being inactive in Y2 (rat vas deferens) and Y4 (rat colon) bioassays. These results confirm the high affinity and selectivity of BIBO3304 and CGP71683A for the Y1 and Y5 receptor subtypes, respectively, while the purported Y2 antagonist, T4[NPY33-36]4 possesses rather low affinity for this receptor.     

Synthesis and biological evaluation of pNPY fragments

Peptide fragments of pNPY corresponding to the C-terminal segments (13-36) and (25-36), the N-terminal segments (1-12) and (1-24), the segments (6-14) and (7-20), which contain a putative beta-turn, and the internal segments (13-24) and (20-30) were synthesized using solid phase methodology. These fragments were assayed for NPY receptor binding activity in the rat hypothalamus membrane preparation, enhancement of food intake in the rat following ivt administration and inhibition of electrically stimulated muscle contraction in the rat vas deferens. Only the C-terminal fragment (13-36) retained some of the activities of pNPY, appearing to act as a weak agonist, having an additive effect with pNPY on the inhibition of muscle contraction and prolonging the duration of action of pNPY in the feeding assay. It also had considerable alpha-helical character, as did pNPY. None of the other peptide fragments had any agonist or antagonist activity. These results suggest that the expression of full biological NPY activity requires both the C- and the N-terminal segments as well as a putative amphiphilic alpha-helical segment (14-31).     

Neuropeptide Y co-exists and co-operates with noradrenaline in perivascular nerve fibers

Neuropeptide Y (NPY)-immunoreactive nerve fibers were numerous around arteries and few around veins. NPY probably co-exists with noradrenaline in such fibers since chemical or surgical sympathectomy eliminated both NPY and noradrenaline from perivascular nerve fibers and since double staining demonstrated dopamine-beta-hydroxylase, the enzyme that catalyzes the conversion of dopamine to noradrenaline, and NPY in the same perivascular nerve fibers. Studies on isolated blood vessels indicated that NPY is not a particularly potent contractile agent in vitro. NPY greatly enhanced the adrenergically mediate contractile response to electrical stimulation and to application of adrenaline, noradrenaline or histamine, as studied in the isolated rabbit gastro-epiploic and femoral arteries. The potentiating effect of NPY on the response to electrical stimulation is probably not presynaptic since NPY affected neither the spontaneous nor the electrically evoked release of [3H]noradrenaline from perivascular sympathetic nerve fibers.     

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.     

Neuropeptide Y (NPY), an endogenous presynaptic modulator of adrenergic neurotransmission in the rat vas deferens: structural and functional studies

The role of neuropeptide tyrosine (NPY) on adrenergic neurotransmission was assessed in the rat vas deferens transmurally stimulated with square pulses of 0.15 or 15 Hz. Nanomoles of NPY inhibited the electrically-induced contractions on the prostatic half but not on the epididymal end of the ductus. NPY was at least 200-fold more potent than norepinephrine or adenosine to produce an equivalent inhibition. Complete amino acid sequence of NPY is required for full agonist activity; deletion of tyrosine at the amino terminus, i.e., NPY fragment 2-36 was 3-fold less potent than the native peptide. NPY fragment 5-36, 11-36 or 25-36 were proportionally less potent than NPY. Avian pancreatic polypeptide was inactive. The presynaptic nature of the NPY activity was established measuring the outflow of 3H-norepinephrine from the adrenergic varicosities of the vas deferens electrically stimulated. In this assay, NPY was more potent than NPY 2-36 or NPY fragment 5-36. No inhibitory action of NPY was detected in K+ depolarized tissues. The inhibitory effect of NPY on the rat vas deferens neurotransmission was not significantly modified by yohimbine, theophylline or naloxone, indicating that the effect of NPY is not due to the activation of alpha 2-adrenoceptors, adenosine receptors or opiate receptors respectively. Picrotoxin or apamin did not modify the inhibitory potency of NPY; verapamil or methoxyverapamil significantly reduced its potency. The inhibitory action of NPY is best explained through the activation of presynaptic NPY receptors that regulate norepinephrine release via a negative feedback mechanism. Structure activity studies give support to the notion of NPY receptors.     

Receptor binding profiles of NPY analogues and fragments in different tissues and cell lines

To investigate receptor selectivity and possible species selectivity of a number of NPY analogues and fragments, receptor binding studies were performed using cell lines and membranes of several species. NPY displays 4–25-fold higher affinity for the Y₂ receptor than for the Y₁ receptor. The affinity of [Leu³¹,Pro³⁴]NPY is 7–60-fold higher for the Y₁ receptor when compared with the Y₂ subtype. Species selectivity within the Y₂ receptors is demonstrated by PYY(3–36), NPY(2–36), NPY(22–36), and NPY(26–36). It is shown that NPY(22–36) is species selective for the human Y₂ subtype (K_i of 0.3 nM) compared with the rabbit and rat Y₂ receptor (K_i of 2 and 10 nM, respectively). PYY(3–36) displays highest affinity for the human and rabbit Y₂ subtype (K_i of 0.03 and 0.17 nM). The screening of NPY analogues and fragments revealed that highest affinity for the human Y₂ receptor is shown by NPY(2–36) and PYY(3–36). In addition, PYY(3–36) and NPY(2–36) are not only subtype selective, but also species selective.     

A novel cyclic analog of neuropeptide Y specific for the Y2 receptor.

The low-molecular-mass, cyclic analog of neuropeptide Y, [Ahx5-24, gamma-Glu2-epsilon-Lys30] NPY (YESK-Ahx-RHYINKITRQRY; Ahx, 6-aminohexanoic acid; NPY, neuropeptide Y), was synthesized and investigated for receptor binding, inhibition of forskolin-stimulated cAMP accumulation, inhibition of electrically stimulated rat vas deferens contractions and ability to increase blood pressure. Like the linear peptide [Ahx5-24] NPY (YPSK-Ahx-RHYINLITRQRY), the more rigid, cyclic analog showed good correlation between receptor binding to rabbit kidney membranes and biological activity in the vas deferens assay. Binding of this peptide to a new Y2-receptor-expressing cell line was slightly reduced, compared to the linear peptide [Ahx5-24] NPY, however inhibition of cAMP accumulation was even more efficient. Unlike the linear peptide [Ahx5-24] NPY, the cyclic analog did not induce a blood pressure increase in rats. Reduced binding to Y1 receptor-expressing SK-N-MC cells, as well as the loss of capability of signal transduction, suggest that only Y2-mediated activity is preserved after cyclization. The selectivity of the cyclic compound for Y2 subtypes of NPY receptors with respect to inhibition of cAMP accumulation is more than fortyfold increased, as compared to the linear NPY-(13-36) peptide, which has been used to determine Y2 selectivity so far.     

Multiple receptor interactions in the electrically stimulated ductus deferens of the guinea-pig

The evidence that ATP is released with noradrenaline as a cotransmitter from sympathetic nerve endings in the isolated guinea-pig vas deferens, had led to investigate the potential interactive effects of P2 and alpha 1 receptors, extending the observations to potentially synergistic interactions of histamine-, acetylcholine- and serotonin-receptors. Ineffective concentrations of histamine, carbachol and serotonin increase the neurogenic response to field stimulation; this effect is prevented by the specific antagonist. The responses to the exogenously applied ATP and noradrenaline are increased as well, suggesting that this synergistic interaction of carbachol is less specific. It can be envisaged that receptor complex is a multicomponent system with subunits specific for the primary neurotransmitter and accessory subunits which may interact to increase the responsiveness of the target cell to simultaneous afferent stimuli.     

The effect of caesium on adrenergic transmission in rabbit vas deferens.

The effect of caesium on the responses of rabbit vas deferens to transmural stimulation was investigated. The tissue responded to transmural stimulation with a phasic spike contraction followed bya sustained contractile response. The sustained response was inhibited by phentolamine and guanethidine and thus apparently results from noradrenaline release from adrenergic nerves. Addition of 2-5mM Cs+ greatly potentiated this secondary response without altering the sensitivity of the tissue to added (minus)-noradrenaline. This potentiation was not due to Cs+ decreasing the neuronal uptake of noradrenaline, or by Cs+ altering prostaglandin synthesis. Addition of 2mM Cs+ significantly increased the amount of (plus or minus)-[3-H] metaraminol released from tissues in response to transmural stimulation (5 Hz). It is suggested that caesium potentiated responses of rabbit vas deferens to transmural stimulation by increasing the amount of transmitter released per nerve impulse, possibly as a result of prolongation of the action potential.     

NORADRENALINE METABOLIZING ENZYMES IN NORMAL AND SYMPATHETICALLY DENERVATED VAS DEFERENS

—A surgical technique for sympathetically denervating the vas deferens has been evaluated biochemically. A slight fall in soluble muscle protein content and no significant change in DNA content of the operated vas deferens were found. This indicates that the surgical procedure causes only a slight degree of tissue damage and may be useful for investigating the cellular localization and properties of noradrenaline metabolizing enzymes. In three species examined (rat, guinea pig and rabbit), monoamine oxidase activity of the vas deferens fell by approximately 50 per cent after denervation. The time course of the fall in monoamine oxidase activity of rat vas deferens was parallel to that of the disappearance of noradrenaline suggesting that this proportion of the total enzyme activity had a neuronal localization. The remaining enzyme activity is presumably located extraneuronally. Significant falls in catechol-O-methyl transferase activity were found in rat and rabbit vas deferens after denervation but not in guinea pig. The rabbit and rat vas deferens had respectively approximately 60 and 30 per cent of the catechol-O-methyl transferase activity associated with the sympathetic nerves. A complete loss of DOPA decarboxylase and tyrosine hydroxylase activities occurred in rat vas deferens after denervation, suggesting that these noradrenaline synthesizing enzymes have an entirely neuronal localization.     

A pharmacological investigation of the biphasic nature of the contractile response of rabbit and rat vas deferens to field stimulation

It has been demonstrated previously with the vas deferens of the guinea-pig that the first and second phases of the contractile response to motor nerve stimulation are preferentially antagonized by the P₂-purinoceptor antagonist arylazido aminopropionyl ATP (ANAPP₃), and the α₁-adrenoceptor antagonist prazosin, respectively. We have now investigated the effect of the two antagonists on the biphasic contraction in the vas deferens of two other species; rabbit and rat. ANAPP₃, in a concentration which antagonized responses to exogenously applied ATP but not those to exogenous norepinephrine, preferentially reduced the initial phasic response of the rabbit vas deferens to motor nerve stimulation without significantly reducing the secondary, tonic phase of the response. Prazosin had the opposite effect; antagonizing the response to norepinephrine but not to ATP and reducing the tonic response to motor nerve stimulation without significantly reducing the initial phasic response. Results obtained with the rat vas deferens were similar. The present results combined with previous findings suggest that ATP and norepinephrine act as cotransmitters in the vas deferens of several species.     

Neuropeptide Y (NPY) binding sites in rat brain labeled with 125I-Bolton-Hunter NPY: comparative potencies of various polypeptides on brain NPY binding and biological responses in the rat vas deferens

The binding of biologically active 125I-Bolton-Hunter (BH)-NPY to rat brain membranes was saturable and reversible and regulated by inorganic cations and guanyl nucleotides consistent with other neurotransmitter receptor systems. The concentration of specific 125I-NPY binding differed in various brain regions, being highest in the hippocampus and lowest in the cerebellum. Scatchard analysis of 125I-NPY binding showed a single class of receptor sites with a Kd = 0.1 nM and Bmax of 3 pmole/g tissue in hippocampus. Peptide YY, porcine and human NPY inhibited the specific 125I-BH-NPY binding with IC50 values of 50-120 pM. In contrast, human NPY free acid and pancreatic polypeptides from human (HPP), rat (RPP) and avian (APP) sources were much weaker (IC50 greater than or equal to 300 nM). The rank order of potencies for NPY analogs and the inactivity of APP and HPP fragment (31-36) on brain binding appeared to correlate with their relative activities in inhibiting contractions of the field-stimulated rat vas deferens. However, PYY, HPP and RPP exhibited activity in the field-stimulated rat vas deferens indicative of a possible action upon sites distinct from the brain NPY binding site.     

Pharmacological responses by different portions of guinea pig vas deferens circular muscle preparation

The different segments of the guinea pig vas deferens circular muscle exhibit differential response patterns upon pharmacological stimulation. Namely, apart from barium chloride, the affinity and intrinsic activity of certain agonists and the strength of maximum contractions they induce appear to decrease along the path from the epididymis toward the prostate. If one subdivides the vas deferens into 3 parts of equal length such as epididymal, medial and prostatic portions, then adrenaline, acetylcholine, acetyl-beta-methylcholine, dopamine, histamine and bradykinin induce contractions on each of the 3 parts; whereas tyramine, ephedrine elicit responses in the epididymal and medial portions; amphetamine, DMPP, serotonin and PGF2 alpha in turn provoking contractions exclusively on the epididymal portion. The effects of adrenaline and noradrenaline are blocked by phentolamine and tolazoline; the responses to acetylcholine, acetyl-beta-methylcholine and carbamyl-beta-methylcholine are antagonized by atropine over a specific concentration range. The effects of tyramine, ephedrine and amphetamine are inhibited by phentolamine in an remarkably low dose range (pA2 = 13.51 +/- 0.09; 14.54 +/- 0.31; 14.35 +/- 0.12). The situation was the same when tyramine-dibenamine and tyramine-phenoxybenzamine combinations were tested (pD'2 = 14.03 +/- 0.37; 13.26 +/- 0.03). Based on these findings the presence of a peculiar alpha adrenergic receptor is suggested on the sympathetic postganglionic fibres. In addition to the already identified alpha adrenergic, muscarinic cholinergic and histamine H1 receptors, we could show the presence of dopaminergic receptors too in the vas deferens circular muscle.     

Interaction between norepinephrine, NPY and VIP in the ovarian artery.

The in vitro effect and the interaction between norepinephrine (NE), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) were studied in dissected segments of the rabbit ovarian artery. In addition, the structural requirement of the NPY receptor was investigated using NPY peptide analogs. NE induced a dose-dependent vasoconstriction with an Emax of 131.4 +/- 2.9% of K(+)-induced constriction. The vasoconstrictor effect of NPY was less than 5% of K(+)-induced vasoconstriction. Incubation of the artery with 10(-7) M NPY for 4 min induced a significant potentiation of NE-induced contractions. The selective NPY Y1 receptor agonist [Leu31, Pro34]NPY was also able to potentiate the NE response at the half-maximum contraction level, but not NPY(11-36), an NPY peptide fragment predominantly stimulating the NPY Y2 receptor. NPY exerted a dose-dependent vasoconstrictor effect on vessels contracted for 20 min with 10(-6) M NE. VIP induced a dose-dependent relaxation of vessels contracted with 10(-6) M NE. The VIP-induced relaxation could be reversed by NPY. In conclusion, receptors capable of interacting with NPY, presumably of the Y1 type, and VIP are present in the rabbit ovarian artery, and activation of these receptors may profoundly influence the response of the artery to norepinephrine.     

Melanotropin release inhibiting activity of neuropeptide Y: structure-activity relationships

We have recently shown that the release of alpha-MSH by the intermediate lobe of the frog pituitary is inhibited by neuropeptide Y (NPY). Using the perifusion technique, we have compared in the present study, the alpha-MSH release inhibiting activities of NPY, various NPY short chain analogues and two other members of the pancreatic polypeptide family, peptide YY (PYY) and avian pancreatic polypeptide (APP). The order of biological potency was NPY greater than NPY[2-36] greater than NPY[16-36] greater than NPY[25-36] greater than NPY[1-15]. Among the two pancreatic polypeptides tested, PYY appeared to be almost as potent as NPY while APP was 6 times less active than NPY. Neither NPY[1-15] nor NPY[16-36] could antagonize the inhibitory effect of NPY on alpha-MSH release. The structure-activity relationship study suggests that the bioactive determinant of NPY is located in the C-terminal part of the molecule.     

Specific [3H]propionyl-neuropeptide Y (NPY) binding in rabbit aortic membranes: comparisons with binding in rat brain and biological responses in rat vas deferens

The binding of biologically active [3H]propionyl-NPY to rabbit aortic membranes was specific and saturable. Scatchard analysis indicated a single class of binding sites with a Kd of 1.1 nM. The rank order of potencies for displacement of [3H]propionyl-NPY binding by NPY analogs in the aorta correlated with their potencies in displacing binding in brain and their activity in inhibiting contractions of the field-stimulated rat vas deferens. However, differences were noted in the absolute or relative potencies of other related polypeptides both in regards to aorta compared to brain NPY binding and NPY binding compared to activity in the vas deferens. Collectively, the results support proposals for heterogeneity of NPY receptors.