Review

Neuropeptide Y and its receptors as potential

therapeutic drug targets

Antonio P. Silva, Claudia Cavadas1, Eric Grouzmann*

Division of Hypertension and Vascular Medicine, Centre Hospitalier Universitaire Vaudois (CHUV),

Av. Pierre Decker, 1011 Lausanne, Switzerland

Received 23 July 2002; received in revised form 14 August 2002; accepted 20 August 2002

Abstract

Neuropeptide Y (NPY) is a 36-amino-acid peptide that exhibits a large number of physiological activities in the central and

peripheral nervous systems. NPY mediates its effects through the activation of six G-protein-coupled receptor subtypes named

Y1, Y2, Y3, Y4, Y5, and y6. Evidence suggests that NPY is involved in the pathophysiology of several disorders, such as the

control of food intake, metabolic disorders, anxiety, seizures, memory, circadian rhythm, drug addiction, pain, cardiovascular

diseases, rhinitis, and endothelial cell dysfunctions. The synthesis of agonists and antagonists for these receptors could be useful

to treat several of these diseases.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Neuropeptide Y; NPY receptors; Agonist; Antagonist; Drug target

1. Introduction

Since its discovery 20 years ago, neuropeptide Y

(NPY) has been implicated in several pathophysiolog-

ical states. Initial studies proposed NPY as a tumoral

marker for pheochromocytomas. At the same time,

NPY was shown to be a long-lasting vasoconstrictor

and to induce feeding when injected centrally. Then,

pharmacological experiments with peptidic analogues

of NPY ascertained the existence of two receptors for

this peptide. The subsequent cloning of five NPY

receptors and the generation of mice lacking these

receptors facilitated the study into the involvement of

NPY in the pathophysiology of a number of diseases

including feeding disorders and metabolic diseases,

seizures, anxiety, hypertension, congestive heart fail-

ure, and airway diseases.

2. Structure and biosynthesis of NPY

NPY is a 36-amino-acid peptide first discovered in

the porcine brain [1]. Together with the other peptides

of the NPY family (peptide YY (PYY), pancreatic

polypeptide (PP)), NPY may exhibit a three-dimen-

sional structure called PP fold as suggested by compu-

ter modeling studies in comparison with the structure

of the avian PP determined by X-ray crystallography.

This tertiary structure is characterized by a hairpin-like

0009-8981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0009 -8981 (02 )00301 -7

* Corresponding author. Tel.: +41-21-314-07-50; fax: +41-21-

314-07-61.

E-mail address: Eric.Grouzmann@chuv.hospvd.ch

(E. Grouzmann).1 Present address: Laboratory of Pharmacology, Faculty of

Pharmacy, University of Coimbra, 3000 Coimbra, Portugal.

www.elsevier.com/locate/clinchim

Clinica Chimica Acta 326 (2002) 3–25

structure conferred by a type II h-turn, connecting a

polyproline-like type II helix (residues 1 to 9) and an

amphiphilic a-helix (residues 14 to 30), thus leading to

a close proximity of N- and C-terminal ends of the

peptide [2–5]. The NPY gene is located on human

chromosome 7 at the locus 7p15.1. It is composed of

four exons. The first one encoding the 5Vuntranslatedregion (5V-UTR). The second exon encodes the signal

peptide and the main part of the mature NPY sequence.

The third exon contains the sequences encoding the

tyrosine36 of NPY, the glycine37 amide donor site, the

dibasic K38–R39 site for the cleavage by prohormone

convertases (PC), and the main part of the C-terminal

peptide of NPY (CPON). Finally, the fourth exon

encodes the end of CPON and the 3V-UTR.cAMP was previously identified as a NPY-elevat-

ing agent in the arcuate nucleus and the paraventric-

ular nucleus of rat brain [6]. Phospholipase Cg was

also shown to mediate the induction of NPY expres-

sion by brain-derived neurotrophic factor [7]. NPY

mRNA expression is also up-regulated by glucocorti-

coids such as dexamethasone [8].

The pre-pro-NPY generated after translation is

directed into the endoplasmic reticulum where the

signal peptide is removed. The following processing

step is the cleavage of the precursor pro-NPY at a

dibasic site by prohormone convertases, which gen-

erates NPY1–39 and CPON. Two further sequential

truncations at the C-terminal end by a carboxypepti-

dase and the peptidylglycine a-amidating monooxy-

genase, respectively, lead to the biologically active

amidated NPY. The amide moiety is essential for the

activity of NPY and prevents degradation by carbox-

ypeptidases (Fig. 1). The mature NPY can be further

Fig. 1. Biosynthesis of NPY (bp: base pairs; mRNA: messenger ribonucleic acid; nt: nucleotides; UTR: untranslated region; CPON: C-flanking

peptide of NPY).

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–254

processed by two enzymes, the dipeptidyl peptidase

IV, and the aminopeptidase P. The resulting products

are NPY3–36 and NPY2–36, respectively.

3. Localization

NPY is mainly localized in the nervous system. In

the central nervous system (CNS), the most abundant

source of NPY is hypothalamus, particularly the para-

ventricular nucleus, arcuate nucleus, suprachiasmatic

nucleus, median eminence, and dorsomedial nucleus

[9,10]. High levels of NPY are also detected in the

cerebral cortex [4]. Peripherally, NPY is abundant in

the sympathetic nervous system, where it is co-stored

and co-released with norepinephrine. It is also

expressed in a subpopulation of parasympathetic neu-

rons [11]. In the periphery, adrenal medulla is also a

source of NPY [12] and its expression also occurs in

the endocrine pancreas of dexamethasone-treated rats

[13,14]. Other non-neuronal cells have been shown to

express NPY, e.g. in rat, megakaryocytes express

NPY [15]. NPY is also expressed in liver, heart,

spleen, and in endothelial cells of blood vessels [16].

4. NPY receptors

4.1. Nomenclature and pharmacology

NPY exerts its biological action through G-protein-

coupled receptors. These receptors have been charac-

terized through their implication in physiological

processes. The NPY receptor family includes the Y1

receptor, first characterized as a post-synaptic recep-

tor, the Y2 receptor, known as a pre-synaptic receptor,

the Y3 receptor that is a NPY-preferring receptor, the

Y4 receptor, first characterized as a PP receptor, the

Y5 which is involved in feeding, and the y6 that has

been cloned, although this receptor is not functional in

humans [17]. All these receptors, except for the Y3

receptor, have been cloned [18–22]. The pharmaco-

logical characteristics described below are summar-

ized in Table 1.

Table 1

Pharmacological characteristics of NPY receptors

Receptor Endogenous agonists

order of potency

Selective agonists Antagonists Signal transduction

Y1 NPY, [Pro34]NPY,

PYYHPYY, NPY

C-terminal fragments, PP

[Phe7,Pro34]NPY,

[Leu31,Pro34]NPY, [Pro34]NPY,

[Leu31,Pro34]PYY, [Pro34]PYY

N-terminally truncated analogsa,

GW1229b, BIBP3226, SR120819A,

BIBO3304, LY357897, J-115814,

H394/84c

Gi/o inhibition of

adenylate cyclase; Ca2 +

Y2 PYY, NPYzNPY2–36,

NPY3– 36H[Pro34]NPY,

PP

NPY3– 36, NPY13 – 36,

Ac-[Lys28,Glu32]-(25–36)-NPY,

TASP-V

T4-[NPY(33–36)]4, BIIE0246d Gi/o inhibition of

adenylate cyclase; Ca2 +

Y3 NPYz [Pro34]NPYzNPY13– 36HPYY, PP

– – Inhibition of

adenylate cyclase; Ca2 +

Y4 PPHPYYHPYY

fragments

PP, GW1229b – Gi inhibition of

adenylate cyclase; Ca2 +

Y5 NPY, PYY, [Pro34]NPY

NPY2–36, NPY3– 36>PP

[Ala31,Aib32]-NPY CGP71683A, N-acetylated

a-(3-pyridylmethyl)-h-aminotetralin,

L-152,804

Gi inhibition of

adenylate cyclase; Ca2 +

Y6 NPY=PYY>PP – – Inhibition of

adenylate cyclase

a Sequences of the N-terminally truncated analogs of NPY: INPIXRLRY, where X can be Phenylalanine (F), (4-Ph)-F, or (2,6-dichloro-

benzyl)-Y; INPXYRLRY, where X is Aib (aminoisobutyric acid); INXIYRLRY, where X is (3,4-dehydro)-P.b Also called GR231118 or 1229U91.c 1,4-Dihydro-4-[3-[[[[3-[spiro(indene-4,1V-piperidin-1-yl)]propyl]amino]carbonyl]amino]phenyl]-2,6-dimethyl-3,5-pyridine dicarboxylic

acid, dimethylester).d (S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6h)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclo-pentyl]acetyl]-N-[2-[1,2-dihydro-

3,5(4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamide.

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–25 5

4.1.1. Y1 receptor

Binding of NPY on the Y1 receptor is largely

impaired when the N-terminal part of the peptide is

removed. Indeed, truncation of NPY leading to

NPY2–36 or NPY3–36 or NPY13– 36 results in a

marked loss of affinity and biological activity [23].

Peptides modified on the C-terminal end (i.e.

[Pro34]NPY and [Leu31,Pro34]NPY) retain full activity

on the Y1 receptor [23], but lose their affinity for the

Y2 receptor [19]. This suggests that N-terminal part of

the peptide determines its binding and activity on the

Y1 receptor. Recently, a highly selective Y1 agonist

was synthesized, [Phe7,Pro34]NPY, which shows an

affinity constant below the nanomolar range [24].

Peptidic antagonists of the Y1 receptor are numerous,

mainly N-terminally truncated analogues of NPY.

Their sequences are INPIXRLRY, where X can be

Phenylalanine (F), (4-Ph)-F, or (2,6-dichloro-benzyl)-

Y, or INPXYRLRY, where X is Aib (aminoisobutyric

acid), or INXIYRLRY, where X is (3,4-dehydro)-P

[25]. Another N-terminally truncated analogue is

the GW1229, also called GR231118 or 1229U91

[26], which has an affinity in the picomolar range

for NPY Y1 receptor. However, this antagonist of Y1

was found to also be a potent agonist at the Y4

receptor [27,28]. Nonpeptidic antagonists for the Y1

receptor have been synthesized, e.g. BIBP3226 [29],

and SR120819A, an orally active antagonist [30].

Two recent more potent antagonists have been devel-

oped. The first one is an analogue of BIBP3226, the

BIBO3304 [31], and the second one is the LY357897,

a trisubstituted indole [32]. Furthermore, 1,3-disub-

stituted benzodiazepines have been characterized as

novel, potent, selective Y1 antagonists [33]. The Y1

receptor can also be blocked by J-115814, an antag-

onist developed by Kanatani et al. [34] in 2001,

as well as by a dihydropyridine-like substance, the

H394/84 [35]. Another way to characterize the bind-

ing of NPY to the Y1 receptor is the use of specific

antibodies directed against the receptor [36]. The Y1

receptor is mainly localized in blood vessels, in the

central nervous system (anterior thalamus, cerebral

cortex, medial geniculate) and amygdala. The two

main effects of NPY mediated by the Y1 receptor are

vasoconstriction [37–39]. It seems to also be impli-

cated in the regulation of feeding behaviour [40].

NPY Y1 receptor is stably expressed in a neuro-

blastoma cell line: SK-N-MC [41].

4.1.2. Y2 receptor

In contrast to the Y1 receptor, binding to the Y2

receptor does not require the N-terminal sequence,

since it binds N-terminally truncated fragments well,

(i.e. NPY2–36, NPY3–36, NPY13–36, NPY18–36, and

NPY22–36). On the other hand, the C-terminal part of

the peptide, is more important, as suggested by the

fact that [Pro34]NPY has much lower affinity for Y2

than for Y1 receptor. Cyclic analogs of NPY have

full agonist properties, this is the case for the Ac-

[Lys28,Glu32]-(25–36)-NPY [42]. Another potent

agonist at the Y2 receptor is the TASP-V [43], a

molecule composed of two C-terminal fragments of

NPY21–36 attached to a cyclic template. Inversely,

T4-[NPY33–36]4, another TASP molecule, has antag-

onist action [44]. A potent Y2 nonpeptidic receptor

antagonist was designed, the BIIE0246 [45]. This

antagonist was characterized in a rat vas deferens

assay, where it antagonizes NPY effects at the Y2

receptor [45]. The Y2 receptor, in contrast to the Y1,

is mainly a pre-synaptic receptor, and it is implicated

in the inhibition of neurotransmitter release [17]. It is

expressed in the central and peripheral nervous

system, intestine and certain blood vessels [19,46–

48]. The Y2 receptor is also expressed by a human

astrocytoma cell line, LN319 [49], as well as by two

neuroblastoma cell lines: SMS-KAN and CHP234

[50,51].

4.1.3. Y3 receptor

The Y3 receptor is characterized by at least its 10-

fold lower affinity for PYY than for NPY [17]. This

receptor subtype is localized in the human adrenal

medulla where it mediates the NPY-induced secretion

of catecholamines [12], in the rat nucleus tractus

solitarius [52], in rat cardiac membranes, and in

bovine chromaffin cells [52–57].

4.1.4. Y4 receptor

The Y4 receptor binds to variable extents the three

members of the NPY family in mammals. The best

affinity to human Y4 receptor is obtained with the PP

[17]. PYY is bound with a lower affinity, and finally,

NPY does not bind to human Y4 receptor [20]. The

rodent Y4 receptor is more selective, since both PYY

and NPY have low affinities for this receptor, com-

pared to PP which binds to rodent Y4 receptor with

high affinity [58]. The Y1 antagonist, GW1229, is a

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–256

potent agonist, but no antagonists have been reported

[27,28]. The Y4 receptor is widely distributed in the

whole body including brain and periphery, e.g. hypo-

thalamus, hippocampus, skeletal muscle, thyroid

gland, heart, prostate, stomach, small intestine, colon,

pancreas, adrenal medulla and cortex, and nasal mu-

cosa [20,59]. In the CNS, Y4 receptor is also found in

cerebellum, medulla, and spinal cord [5]. The Y4 re-

ceptor exhibits a higher affinity for the pancreatic

polypeptide (PP) than for NPY. PP is known to inhibit

exocrine pancreatic secretion [60], to induce gall

bladder relaxation [61], and to stimulate LH secretion

[62]. Due to its tissue distribution, the Y4 receptor

might be involved in these effects described for PP.

The Y4 receptor is stably expressed in the Y4-CHO

clone 29 cell line [63].

4.1.5. Y5 receptor

The Y5 receptor binds the Y1 receptor agonist

[Leu31,Pro34]NPY, the Y2 agonists NPY2– 36 and

NPY3–36, and PP [64]. A selective agonist of the Y5

receptor was synthesized by Beck-Sickinger et al., the

[Ala31,Aib32]-NPY, which stimulates food intake in

rats [65]. Y5 receptor was initially characterized by the

use of the recently developed nonpeptidic antagonist,

CGP71683A, which was also useful for the investiga-

tion of the role of the Y5 receptor in NPY-induced

increase in food intake [66]. Unfortunately, this antag-

onist showed side effects that were not attributed to the

Y5 receptor [67]. Antagonists of the Y5 receptor, based

on acetylation of a-(3-pyridylmethyl)-h-aminotetra-

lins, were also synthesized [68]. Another antagonist,

the L-152,804, which was recently developed, was

shown to be a useful tool to investigate the role of the

NPY Y5 receptor in the regulation of food intake [69].

This receptor is localized in the hypothalamus where it

stimulates appetite [21]. At the peripheral level, Y5 is

present in the intestine, ovary, testis, prostate, spleen,

pancreas, kidney, skeletal muscle, liver, placenta, and

heart [70]. This receptor is also stably expressed in a

human endometrial cancer cell line transfected with

the Y5 receptor cDNA: Y5-HEC-1B [71].

5. Intracellular signaling events

NPY receptors use similar signal transduction path-

ways (Fig. 2). In most cases, they are coupled to

pertussis toxin-sensitive G-proteins (i.e. Gi and Go).

However, some responses to NPY do not seem to be

sensitive to pertussis toxin [72–74]. One of the typical

signaling responses of NPY receptors is the inhibition

of adenylyl cyclase that occurs in almost every tissue

and cell type investigated [75,76]. The second type of

response is the variation of intracellular calcium con-

centration, which can be increased by the mobilization

of intracellular stores via inositol phosphate dependent

and independent pathways [77,78]. NPY can also act

on calcium signaling by activating or blocking calcium

channels at the plasma membrane level [79–81].

Potassium channels are also a target for NPY recep-

tor-coupled G-proteins. Indeed, activation of a recep-

tor by NPY can lead to the activation or inhibition of

potassium channels [73,82].

Some studies also suggest that NPY could activate

phospholipase A2 [83]. Moreover, NPY was shown to

stimulate prostacyclin production in porcine vascular

endothelial cells [84]. Another pathway activated by

NPY is the signaling through the mitogen-activated

protein kinases, as shown in human erythroleukemia

cells [85]. Another well-known signaling molecule

involved in the intracellular events activated by NPY

is nitric oxide [86]. For example, it was shown that

NPY induces vasodilation of human subcutaneous

arteries via Y1 receptor by a nitric oxide dependent

pathway [87]. It was also reported that NPY stimu-

lates the expression of genes containing a cAMP

response element in human neuroblastoma cell lines

[88], suggesting that NPY can induce cAMP produc-

tion or inhibit cAMP degradation by phosphodies-

terases in these cells.

6. Central effects of NPY

6.1. NPY and feeding behavior

It is now well established that the expression of

appetite is chemically coded in the hypothalamus

[10,89–92].

The results obtained with the studies employing

discrete lesions in the hypothalamus or surgical trans-

ection of neural pathways show that some hypothala-

mic sites, such as the ventromedial nucleus (VMN),

paraventricular nucleus (PVN), dorsomedial nucleus

(DMN), arcuate nucleus (ARC), and lateral hypothal-

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–25 7

amus (LH), contain neural circuitries affecting inges-

tive behaviour [93,94]. Several lines of evidence

suggest that NPY plays a key role in the control of

appetite, body weight gain, and obesity [10,92,95].

NPY-producing neurons are located in several sites

in the brainstem (BS) including locus coeruleus, and in

the hypothalamus along the length of the ARC and in

the DMN [10,96]. These neurons innervate various

hypothalamic sites including ARC, VMN, DMN,

PVN, and surrounding regions [97, 98]. Intracerebro-

ventricular (i.c.v.) injection of NPY in rats or mice

strongly stimulates food intake while inhibiting ther-

mogenesis and lipolysis [40,95,99–103]. Moreover,

hypothalamic NPY and its mRNA are increased in the

obese Zucker rat and during poor metabolic condition

such as fasting [104–108]. Interestingly, chronic i.c.v.

administration of NPY leads to obesity in rodents fed

ad libitum [96,101,103,109,110], suggesting that no

satiety signals are able to counteract the effect of NPY.

Besides this direct stimulatory effect, endogenous

NPY may have a physiological role on food intake

[102,111,112]. Leptin and insulin are the most likely

molecules to control NPYexpression. Leptin-deficient

ob/ob mice as well as rats with insulin-deficient

diabetes are hyperphagic and show increased NPY

synthesis [113,114]. In addition, NPY knock-out in

leptin-deficient ob/ob mice resulted in reduced body

weight [115]. Moreover, leptin infusion decreases

the expression and secretion of NPY from the hypo-

thalamus [116]. Similarly, insulin replacement in strep-

tozotocin-treated rats normalizes the increase in

hypothalamic NPY expression and reduces hyperpha-

gia that develops secondary to insulinopenia [117].

These studies suggest that NPY could be a target of

choice to develop antiobesity drugs. During the last 5

years, many studies aimed to identify the NPY recep-

tor subtype involved in the orexigenic effects of this

peptide. The Y1 and Y5 receptors appear to be the most

likely candidates for the orexigenic effect of NPY.

However, despite the use of more or less specific NPY

Fig. 2. Main signalling pathways activated by NPY (AC: adenylate cyclase; ATP: adenosine triphosphate; cAMP: 3V,5V-cyclic adenosine

monophosphate; PLC: phospholipase C; PIP2: phosphatidyl inositol 4,5-diphosphate; IP3: inositol 1,4,5-triphospate; DAG: diacylglycerol;

ROCC: receptor-operated calcium channel).

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–258

agonists/antagonists or gene-deficient mice for these

receptors, it appears that both Y1 and Y5 receptors

could be involved. The initial studies used C-terminal

fragments or substituted peptides of the NPY family to

investigate which NPY receptor(s) subtype(s) medi-

ates the role of NPY on feeding behaviour. PYY, the

Y1/Y5 agonist [Leu31,Pro34]NPY, the Y2/Y5 agonist

NPY2–36, were all potent to stimulate food intake

[118–120] whereas the Y2 receptor agonist, NPY13–

36, was a weak orexigenic agent in rats [118,120].

These studies indicate that the NPY Y1 and/or Y5

receptor but not Y2 may be involved in mediating

NPY-induced feeding.

Several Y1 receptor antagonists were developed

and tested for their ability to inhibit food intake in

rodents. The i.c.v. injection of the Y1 antagonist

(1229U911 or GR231118) inhibits both NPY-induced

food intake and physiological feeding behaviour after

an overnight fast [121,122]. The effect of i.c.v. injec-

tion of 1229U911 was larger in the obese fa/fa

(Zucker) than in lean rats. In contrast, 1229U911 does

not inhibit food intake in Wistar rats with diet-induced

obesity. Of note, 1229U911 has no effect on NPY-

induced feeding in mice, suggesting the involvement

of another receptor in the orexigenic effect of NPY in

mice [123–125]. Additionally, this compound has

also agonistic properties for the Y4 receptor even

though it does not seem to antagonize other receptors

[126]. BIBP 3226, a Y1 receptor antagonist, reduced

food consumption induced by fasting or the i.c.v.

infusion of NPY [119]. However, adverse effects were

observed with BIBP3226 that preclude all these

studies [125,127]. The Y1 antagonists BIBO3304

and SR120562A also inhibited food intake induced

by the i.c.v. infusion of NPY or by fasting [31,128].

The i.c.v. injection of J-104870, a high selective Y1

antagonist, in satiated Sprague–Dawley rats was

reported to significantly attenuate spontaneous food

intake in Zucker fatty rats and to decrease 74% of the

NPY-induced feeding [129]. Similarly, the intraperi-

toneal (i.p.) injection of the Y1 receptor antagonist J-

115814 reduced spontaneous feeding in wild type

mice and inhibits feeding induced by i.c.v. injections

of NPY in satiated Sprague–Dawley. Moreover, J-

115814 had no effect on i.c.v. NPY-induced food

intake in NPY Y1 knockout (KO) mice but reduced

food intake in NPY Y5 KO mice [34], strongly

suggesting a role of the Y1 receptor in mediating

feeding behaviour. On the other hand, the stimulatory

and inhibitory effects of the Y5 receptor agonists and

the Y5 receptor antagonists, respectively, on food

intake also favor the involvement of the Y5 receptor

subtype in eating behaviour. The Y5 agonist [D-

Trp32]NPY produced a similar increase in food intake

as NPYadministration in satiated rats [130] and a light

effect in obese Zucker rats, suggesting a down-regu-

lation of the Y5 receptor in the obese rat [131].

However, in mice and in another study with rats, [D-

Trp32]NPY does not increase food intake [125,132].

Another Y5 receptor-selective analog, [Ala31,Aib32]

NPY, also stimulates feeding in rats [65]. The repeated

central administration of Y5 antisense oligodeoxynu-

cleotides significantly decreased spontaneous as well

as NPY-induced food intake [133,134]. Several stud-

ies were performed using Y5 antagonists. In lean

satiated rats, the i.p. injection of the Y5 antagonist

CGP71683A also decreased i.c.v. NPY-induced food

intake [66] as well as fasting-induced food intake in

lean and obese Zucker fa/fa rats, with the advantage

that it does not produce any anxiogenic-like effect

as the Y1 antagonist H409/22 [67,135]. However,

it should be noted that CGP71683A did not affect

daily food intake, suggesting that the Y5 receptor

could be limited to stimulated conditions. However,

CGP71683A has a certain degree of affinity for

muscarinic or serotonin sites in rat brains and produce

inflammatory response [67]. More recently, the selec-

tive Y5 receptor antagonist L-152,804 did not affect

the NPY-induced food intake [69], limiting the spe-

cific central role of the NPY Y5 receptor subtype in

food intake. Although, more recently, another selec-

tive and potent Y5 antagonist, GW438014A, was

tested by i.p. administration into rodents, and showed

a potent reduction of NPY-induced and normal over-

night food intake. Interestingly, daily i.p. administra-

tion of GW438014A to Zucker fatty rats during 4 days

decreased the rate of weight gain and results in a

reduction in fat mass [136]. In order to clarify the

importance of the NPY and Y1 receptor and/or the Y5

receptor on the feeding behaviour, the development of

KO mice appeared to be a valuable and important

model [137]. The NPY-deficient mice (NPY� /� )

developed in 1996 [115] grow and eat normally, and

their response to i.c.v. NPY administration is similar

to that of wild type animals [138]. A more recent

report shows that KO NPY mice eat less after fasting

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–25 9

[139]. To clarify which NPY receptor subtype is

involved on food intake, different KO mice were

developed, namely Y1 KO mice, Y5 KO mice, and,

more recently, the Y2 KO mice. All three KO animals

grow and eat normally, but they are heavier and

accumulate more fat [69,140,141]. In the Y5 KO, this

obesity is due to hyperphagia and in the Y1 KO, to a

lower metabolic rate associated to lower locomotor

activity [140,141]. NPY is less potent in increasing

food intake in Y1 KO and Y5 KO mice, but not in Y2

KO, compared to control groups [69,140,141]. These

results corroborate the hypothesis that the stimulatory

effect of NPY in food intake is mediated by Y1 and Y5

receptor subtypes and not by the Y2 receptor. In

humans, the reports measuring regulation of NPY

expression in the brain are conflicting. For instance,

patients with nervous anorexia have higher NPY

concentrations in cerebrospinal fluid [142,143]

whereas patients with nervous bulimia have similar

cerebrospinal fluid NPY concentrations to control

group [142,143]. In conclusion, it is too early to

establish whether a single subtype selective drug or

a combination of Y1 and Y5 receptor selective antag-

onists will be necessary to regulate appetite.

6.2. NPY and anxiety

Several studies demonstrated that low doses of NPY

produce an anxiolytic effect and higher doses cause a

sedative action [144–147]. These findings were con-

firmed by using transgenic mice that overexpressed

NPY and displayed an anxiety-like behaviour [148],

whereas NPY KO mice had an anxiogenic behaviour

profile [139]. This NPY anxiolytic-sedative effect

appears to be mediated by the Y1 receptor subtype,

since in Y1KOmice, central administration of NPY did

not potentiate the pentobarbital-induced sedation as in

wild type animals [149]. The anxiolytic effect of NPY

measured by the increased preference for the open arms

of the elevated plus maze is mimicked by the i.c.v.

administration of PYY or the Y1/Y5 receptor agonist

[Leu31,Pro34]NPY in rats, but not by the Y2 receptor

agonist NPY13–36 [150]. In addition, the injection of

Y1 receptor antisense oligodeoxynucleotides into rat

amygdala produced an anxiogenic behaviour in animal

models of anxiety [39,151]. Moreover, the NPY-

induced anxiolytic-like effect was blocked by the Y1

receptor antagonist BIBO3304, but remained unaf-

fected by the Y2 antagonist BIIE0246 [152]. Finally,

central administration of BIBP3226 also results in an

anxiogenic behaviour in rats [119,153]; however,

BIBP3226 exhibits toxicity due to its poor solubility.

The sedative effect of NPY was also investigated in

humans. In fact, repeated intravenous injections of

NPY to young men produced an increase in sleep

period time, a decrease in sleep latency and time

awake, and also modulated rapid eyes movement sleep

[154]. However, this finding should be taken with care

since NPY must cross the blood–brain barrier to

achieve this effect.

6.3. NPY and epilepsy

The role of NPY in regulating seizure activity and

its possible involvement in epilepsy has been assessed

during the last decade. After acute seizures, there is an

increase in NPY and pre-pro-NPY mRNA levels in

the neurons of some cortical and limbic areas, partic-

ularly in the frontal, pyriform and entorhinal cortices,

in the amygdala and hippocampus [155–160].

In vitro and in vivo studies support the role of NPY

as an endogenous anticonvulsivant. In fact, NPY

inhibits excitatory neurotransmission and reduces glu-

tamate release in human and rodent hippocampus

slices [161–166]. Then, NPY KO mice display spon-

taneous seizure and are more sensitive to convulsant

agents while this effect is reverted by i.c.v. injection of

NPY [115,167,168]. Finally, transgenic rats overex-

pressing NPY, especially in the CA1 area of hippo-

campus, were less susceptible to epileptogenesis since

they showed a significant reduction in the number and

duration of electroencephalographic seizures induced

by kainate [169].

The seizure-related increase in NPY expression is

accompanied by modified levels of NPY receptor

subtypes Y1, Y2, and Y5 in the hippocampus: Y1

receptor and Y5 receptor levels are decreased and Y2

receptors are increased in animal models [170–174].

Also, in hippocampal specimens obtained from

patients with temporal epilepsy, Y2 receptor binding

was increased and Y1 receptor binding was reduced,

compared to controls obtained at autopsy. In the same

patients, NPY-immunoreactive fibers and NPY mRNA

were also increased [175].

Several investigations suggest that the anticonvul-

sive action of NPY is mediated by both Y2 and Y5

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–2510

receptors. In fact, in hippocampal slice, NPY sup-

presses glutamate release through presynaptic activa-

tion of Y2 receptors [115,162,164,172,176–178].

Therefore, the development of nonpeptidergic Y2

receptor agonists would generate valuable compounds

for anticonvulsive treatment.

Other studies showed that Y5 receptor subtype also

mediates the anticonvulsant properties of NPY.

Indeed, the rank order of NPY agonists in inhibiting

seizures induced by kainic acid was similar to phar-

macological profile of Y5 receptor [179]. Moreover,

although Y5 receptor KO mice do not exhibit sponta-

neous seizure-like activity, they are more sensitive to

exposure to kainate and the antiepileptic effect of

exogenous NPY was suppressed in this model [180].

However, application of Y5 agonists ([6-aminohexa-

noic acid8-20][Pro34]NPY or [D-Trp32]NPY) on normal

rat hipoccampal slices does not suppress epileptiform

activity in hippocampal CA3 area, despite reducing

synaptic excitation [181]. Taken together, these results

suggest that Y5 receptor partially mediates the anti-

epileptic activity of NPY and support a modulating

role for Y5 receptor of limbic seizures, while Y5

receptor does not appear to be required for normal

hippocampal function.

Although many of the effects of NPY in limbic

regions appear to be inhibitory, this peptide may

facilitate seizures by acting on the Y1 receptor. The

intrahippocampal injection of BIBP3226, but not its

inactive enantiomer BIBP3435, reduced electroence-

phalographic seizures induced by kainate in rats. This

anticonvulsant effect was reversed by the Y1/Y5

agonist [Leu31,Pro34]NPY [182]. It is therefore possi-

ble that the loss of Y1 receptors on the dendrites of

granule cells in the epileptic tissue might represent an

adaptative change aimed at counteracting hyperexcit-

ability [155]. Thus, it is still unclear whether a Y1

receptor antagonist or Y2 and Y5 agonists may be an

alternative to treating seizures.

6.4. NPY and circadian rhythms

Mammalian circadian rhythms are generated and

regulated by the hypothalamic suprachiasmatic nuclei

(SCN) [183–187]. These fibers arise from the thala-

mic intergeniculate leaflet and ventral lateral genicu-

late nucleus [188,189]. NPY has been implicated in

the circadian rhythm [190]. In fact, during the sub-

jective day, NPY can change the phase of the clock by

itself, and during the subjective night, NPY can

inhibit the phase shifting effect of light [190–196].

NPY has also a direct inhibitory effect in the SCN

[196]. The effects of NPY on SCN may be mediated

through different NPY receptor subtypes. In the

hamster, in vivo and in vitro, NPY, Y1/Y5 and Y2

receptor agonists produce a shift in circadian rhythm

in the SCN [194,195]. In SCN slices taken from rat, a

Y2 agonist advanced the peak of the circadian rhythm

but did not inhibit cell firing, while the application of

the Y5 agonist [D-Trp32]NPY produced only direct

neuronal inhibition [196].

Therefore, NPY may act by altering levels of some

circadian clock-related genes. In fact NPY, Y1/Y5 and

Y2 agonists reduced the mRNA levels for the genes

acutely sensitive to light Period 1 and Period 2 in

hamster and mice [197,198]. If the effect of NPY on

circadian rhythm observed in hamsters [194,195] is

also verified in humans, the ability of NPY to mod-

ulate the circadian-clock responses to light may be of

clinical importance.

6.5. NPY and memory processing

Several studies indicate that NPY could have a role

in learning and memory. Post-training i.c.v. adminis-

tration of NPY in mice improves memory retention

and reverse amnesia induced by scopolamine

[199,200].

Depending on the brain site of injection, NPY has

differential effects on memory retention. NPY injec-

tion into the rostral hippocampus and septum

enhanced memory retention, while administration into

the amygdala or caudal hippocampus induced amne-

sia. No effects were observed after injection in the

caudate/putamen, thalamus, or cerebral cortex [201].

In addition, the immunoneutralization of NPY into the

same brain sites prevents the effects normally pro-

duced by NPY, suggesting that NPY has a physio-

logical role in memory processing. The Y2 receptor

subtype agonist appears to be implicated since the Y2

agonist, NPY20–36, also improved memory retention

[201].

The implication of endogenous NPY on memory

was observed in transgenic rats with hippocampal

NPY overexpression. These animals showed an im-

pairment of spatial memory acquisition and retention

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–25 11

in the Morris water maze [202]. However, NPY KO

mice have a normal performance in an inhibitory

avoidance test, a model designed to measure memory

(i.e. 24 h retention) [139]. These observations strongly

suggest a physiological role of NPY in learning and

memory, but further studies using new pharmacolog-

ical tools are warranted to confirm the NPY receptor

subtypes involved.

6.6. NPY and pain

Different studies support an important role for

NPY in mediating analgesia and hyperalgesia by

distinct central and peripheral mechanisms [203].

In the spinal cord, NPY has an antinociceptive

effect; NPY produces analgesia after thermal noxious

stimuli, but has no effect upon mechanical noxious

stimuli [204]. NPY could exert antinociceptive actions

by inhibiting substance P and other ‘‘pain neurotrans-

mitters’’ released in the spinal cord dorsal horn [203–

205]. This effect may be mediated by the Y1 or Y2

receptors [206,207]. In a neuropathic rat model (rats

whose sciatic nerves had been partially transected), the

local administration of NPYand Y2 receptor agonist N-

acetyl[Leu28,31]NPY24–36 in the injured paw increased

mechanical and thermal hyperalgesia. Similar admin-

istration of the Y1/Y5 agonist [Leu31,Pro34]NPY

increased mechanical hyperalgesia but decreased ther-

mal hyperalgesia [206]. However, in another study

using a similar model, intrathecal administration of

NPY also exacerbated nerve injury-induced hyperal-

gesia [208] but [Leu31,Pro34]NPY enhanced hyper-

algesia and N-acetyl[Leu28,31]NPY24–36 has no effect.

Interestingly, Y1 KO mice exhibit a hyperalgesic

phenotype in thermal tests (hot plate and tail-flick

test), mechanical tests (Von Frey hairs), chemical tests

(first phase of formalin test, acetic-acid- and magne-

sium-sulphate-induced writhings), and neuropathic

nociception experiments (partial nerve injury) [207].

In addition, the analgesic response to tail-flick follow-

ing spinal cord injection of NPY was completely

abolished in Y1 KO mice, suggesting that the Y1

receptor is exclusively responsible for the antinoci-

ceptive effects of NPY [207].

In the brain, the effect of NPY on nociceptive

modulation is unclear, but is predominantly antinoci-

ceptive. I.c.v. administration of low doses of NPY in

mice decreased pain threshold in both the hot plate

test and the electrical tail stimulation, while in the

formalin test, NPY induced a decrease in phase I of

the licking response, demonstrating an antinociceptive

effect [209]. Using another behaviour test in mice, the

writhing test induced by acetic acid, NPYexhibits also

an antinociceptive action [210]. The antinociceptive

effect of NPY was also corroborated in the nucleus

raphe magnus, in the periqueductal grey, or in the

nucleus accumbens of rats [211–213]. Nevertheless,

this antinociceptive effect of NPY was reduced by

subsequent intranucleus accumbens administration of

kappa- and sigma-opioid receptor antagonists, but not

by the delta-opioid receptor antagonist, suggesting a

cross talk between opiates and NPY [213]. Several

results suggest that the effect of NPYon nociception is

modulated by the Y1 and/or Y5 receptor since NPY,

PYY, the Y2/Y5 agonist NPY2–36or the Y1/Y5 agonist

[Leu31, Pro34]NPY, but not the Y2 agonist NPY13–36,

produced antinociceptive effect [210]. However, ano-

ther study shows that the Y2 agonist NPY13– 36

enhanced analgesia in spontaneously hypertensive rats,

but was not effective in normotensive rats [214]. In

conclusion, NPY exert a role in nociception but the

specific NPY receptor(s) subtype(s) involved in this

action is still unclear. Furthermore, the physiological

significance and potential therapeutic value of NPY in

pain disorders in man remains to be solved.

6.7. NPY and drug addiction

Recent data obtained on adult Sprague–Dawley

rats suggest that a dysfunction of the NPY system in

the central nervous system, i.e. the perifornical hypo-

thalamus and the nucleus accumbens, could give rise

to both eating disorders and substance abuse [215].

Many studies report a correlation between NPY

expression and alcohol consumption. Indeed, it was

shown that in alcohol-preferring rats NPY expression

was higher than in alcohol-avoiding rats [216]. Fur-

thermore, an acute alcohol administration induces a

decrease of NPY mRNA in the arcuate nucleus of

adults male Sprague–Dawley rats [217]. It was also

reported that mice lacking NPY expression show

increased consumption of alcohol and are less sensi-

tive to alcohol effects compared to wild type mice.

Conversely, NPY overexpressing mice show less al-

cohol consumption and are more sensitive to alcohol

sedative effects [218]. In humans, a correlation has

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–2512

been found between alcohol consumption and Leu(7)

to Pro(7) polymorphism of the NPY gene [219].

In regard to NPY receptors, there is paucity of data

reporting the involvement of NPY receptor subtypes

in the regulation of alcohol consumption. First, a low

Y2 receptor expression in the medial amygdala of rats

has been associated with the alcohol-preferring phe-

notype [216]. More information about the involve-

ment of NPY receptors in the regulation of alcohol

consumption and effects comes from the use of trans-

genic mice models. Y5� /� mice show a higher

ethanol-induced sedation, and have higher plasma

ethanol levels 1 and 3 h after ethanol intraperitoneal

injection [220]. Y1� /� mice show an increased

voluntary consumption of alcohol and are less sensi-

tive to the sedative effect of alcohol, compared to the

wild type mice. These effects are not related to a

difference in the ethanol plasma levels [221].

At present, it is difficult to conclude the role of each

receptor subtype in the regulation of alcohol con-

sumption, even if it seems clear that the Y1 receptor

negatively controls the voluntary consumption of al-

cohol in a mouse model. There are conflicting results

about the effect of NPYon ethanol self-administration.

Indeed, some studies report a positive effect of NPYon

alcohol ingestion [222], while other investigations

found no effect [223,224] and even a negative effect

of NPY on alcohol consumption [225]. These differ-

ences may be due to varying sites of NPY i.c.v.

injection resulting in no effect of NPY, and injection

in the paraventricular nucleus leading to an increased

alcohol consumption.

For the therapeutic use of NPY receptor agonists or

antagonists in the treatment of alcohol abuse, it would

be of great interest to determine if one or more NPY

receptor subtype over- or down-regulation is associ-

ated with this behavior. On the other hand, the Leu(7)

to Pro(7) polymorphism that has been associated with

increased alcohol consumption in humans has also

been associated with an increased pro-NPYprocessing

[226], which suggests that increased NPY synthesis

might be associated with higher alcohol consumption

in humans. The negative effect of NPY on alcohol

might be related to its anxiolytic action. Chronic

opioid treatment in adult male rats with codeine or

morphine has previously been demonstrated to reduce

NPY levels in striatum and hypothalamus. [227,228],

suggesting that reduced NPY gene transcription could

be involved in basic mechanisms of opioid tolerance

or withdrawal. However, another study does not sup-

port this hypothesis since no changes were observed

in NPY mRNA expression in the locus coeruleus of

rats after both chronic morphine treatment and nalox-

one-precipitated withdrawal [229]. Nevertheless, the

i.c.v. application of exogenous NPYand various NPY-

related peptides were studied on naloxone-precipitated

withdrawal from morphine in rats. NPY mediates an

antiwithdrawal effect through a Y5-like pharmacolog-

ical profile [230]; however, this matter still remains to

be demonstrated conclusively using specific Y5 recep-

tor antagonists. Therefore, NPY could be a therapeutic

target in opioid dependence and withdrawal.

7. Peripheral effects of NPY

7.1. Cardiovascular effects

NPYis present in sympathetic nerve endings around

the blood vessels. Several studies demonstrated that

NPY is a potent and long-acting vasoconstrictor and

could play a role in modulating the sympathetic nerv-

ous activity to control blood pressure. Indeed, NPY can

elicit a long-lasting vasoconstriction insensitive to

alpha-adrenergic blockers [231]. The vasoconstrictive

effect of NPY is mediated by the Y1 receptor present on

smooth muscle cells. The physiological role of this

receptor subtype was confirmed in mice lacking NPY

Y1 receptor expression, which show no blood pressure

response to NPY but a normal response to NE [140]. In

addition, NPY does not potentiate NE-induced vaso-

constriction of blood vessel isolated from NPY Y1-

deficient animals. Interestingly, NPY Y1 KO have a

normal blood pressure, suggesting that the NPY Y1

receptor does not play a crucial role in maintaining

blood pressure homeostasis in unstimulated conditions

[140]. A role for NPY on the control of basal blood

pressure is also not supported by pharmacological

studies. Indeed, BIBP3226, a specific Y1 antagonist,

does not affect blood pressure in normotensive and

in spontaneous hypertensive rats despite increased

circulating NPY concentrations in the latter [232].

However, NPY appears to tune sympathetic system

activity in several vascular beds. First of all, in vitro, the

long-lasting vasoconstriction induced by high fre-

quency sympathetic stimulation of the guinea pig vena

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–25 13

cava can be significantly blunted in the presence of

SR120107 or BIBP3226, two NPY Y1 antagonists

[233–235]. In vivo, BIBP3226 can also inhibit the

vasoconstrictive response caused by high frequency

stimulation of the sympathetic nerves in pigs, indicat-

ing that endogenous NPYvia the NPYY1 receptor may

play a role in evoking the long-lasting vasoconstriction

seen in nasal mucosa, hind limb, and skin [235]. In

addition, stress-induced hypertension in rats was

reversed by BIBP3226, suggesting that NPY could

be involved in mediating physiological responses to

stress [236]. These findings are substantiated by

observations showing an increase in adrenal NPY

expression during chronic stress. Together, these ob-

servations suggest that enhanced NPY secretion could

be important in modulating the blood pressure res-

ponse to chronic stress [236]. NPY is also able to

potentiate the noradrenaline-induced vasoconstriction

through the Y1 receptor, suggesting a cross talk

between catecholamines and NPY [237]. This coop-

erative effect is strengthened by the fact that NPY and

NE are co-stored in sympathetic nerve endings. NPY

is also reported to increase the hemodynamic effects

of histamine, vasopressin, ATP, and angiotensin II

[4,238,239]. Therefore, NPY Y1 antagonists could

be useful for treating hyperadrenergic reactive sub-

jects with an increased secretion of NPY associated to

hypertensive crises.

The sympathetic vascular control is also modulated

at the prejunctional level by NPY. Indeed, by action

on the prejunctional Y2 receptors, NPY inhibits nor-

epinephrine release from sympathetic nerve terminals

[37]. In the kidney and the spleen of the pig, the Y2

receptor was also shown to assume an autoinhibitory

function, since it mediates the down-regulation of

NPY release from sympathetic nerve endings in these

organs. These effects were demonstrated by the use of

the Y2 receptor antagonist BIIE0246 [240]. It was also

suggested in this study that basal splenic vascular tone

was in part due to the Y2 receptor activation. The

vasopressive effect of NPY as well as its capacity to

potentiate adrenergic responsiveness may be benefi-

cial in endotoxic shock, since NPY is able to restore

the vasoconstriction in response to NE in LPS-in-

duced hypotensive rats [241,242]. Indeed, in humans

experiencing sepsis, plasma levels of NPY are signifi-

cantly increased [243,244]. Then, NPY-mediated vas-

oconstriction was shown to be preserved during

endotoxemia [242]. NPY infusion is able to prevent

the development of hypotension and restore the sen-

sitivity to pressor agents in endotoxemic rats at doses

that do not affect the blood pressure [242]. The fact

that the Y1 antagonist BIBP3226 significantly exac-

erbates the detrimental effects of both endotoxemia

and hemorrhage strongly suggests that NPY acts

through Y1 receptors to maintain blood pressure

during septic and hemorrhagic shock [245]. It is also

indicative of a role of endogenous NPY in controlling

blood pressure during shock.

7.2. NPY and catecholamine secretion by adrenal

chromaffin cells

Adrenal medulla is a target organ during stress by

mediating catecholamine release. Catecholamine by

increasing heart rate and blood pressure and glucose

consumption help our body to respond to stress. NPY

is produced by the chromaffin cells of human adrenal

gland [246] and human pheochromocytomas [247–

249]. Recent studies have shown that NPY causes the

release of catecholamine by intact rat adrenal capsular

tissue [250] while other investigations report an inhib-

itory effect of NPY on catecholamine secretion in rat

adrenomedullary primary cell cultures [251].A weak

inhibitory effect of NPY on NE and epinephrine (E)

release from bovine chromaffin cells evoked by a

cholinergic agonist [250,252] has been observed.

However, depending on the experimental conditions,

conflicting results were obtained with perfused bovine

adrenal gland, where NPY was reported to stimulate

the secretion of catecholamine in the presence of

cholinergic agents [54]. The exact subtype(s) of

NPY receptor(s) involved in the modulation of bovine

catecholamine secretion remain(s) undefined although

the presence of NPY Y1 and the Y3 receptors has been

reported in bovine chromaffin cells [57,253–255].

More recently, we demonstrated that human chormaf-

fin cells in culture expressed the mRNA for the Y1,

Y2, Y4, and Y5 receptors [12]. These receptors are

functional, as various receptor-specific agonists elicit

an increase in intracellular calcium. [12]. Moreover,

the stimulatory effect of NPY on catecholamine

release from human chromaffin cells was also ob-

tained by hPP, NPY13–36, and NPY3–36, but PYY did

not produced any effect, suggesting that the Y3

receptor modulates NPY effect.

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–2514

Another important aspect observed in human

chromaffin cells in culture is the fact that NPY

is constitutively released from these cells [12].

Moreover, no receptor-specific antagonists (not Y1,

nor Y2, nor Y5 antagonists) were able to reduce

constitutive catecholamine release; however, an

NPY-immunoneutralizing antibody markedly reduced

constitutive catecholamine release. Since NPY is co-

released with NE from nerve endings and acts as an

important modulator of the sympathetic function by

potentiating the catecholamine vasoconstrictor activ-

ity through the Y1 receptor, we speculate that NPY

could behave as an amplifier of catecholamine

release by acting on the Y3 receptor (Fig. 3). In this

context, a better knowledge of the Y3 receptor could

allow the development of specific receptor antago-

nists useful for treating hyperadrenergic reactive

subjects, as well as patients having pheochromocy-

toma with an increased secretion of NPY associated

to hypertensive crises.

7.3. Endothelial cells

At the vascular level, NPYaction is not restricted to

smooth muscle cells (SMCs), it also acts on vascular

endothelial cells (EC). In porcine aortic endothelial

cells, NPY can stimulate prostacyclin production [84],

indicating that NPY plays a role in endothelium-

dependent regulation of vascular motility. Further

evidence on this point was given by the potentiating

effect of NPY on noradrenaline-induced vasoconstric-

tion, which was shown to be mediated by endothelium

on human saphenous veins [256]. On rat microvascu-

lar coronary EC, NPY was shown to reduce macro-

molecule permeability, as well as cAMP intracellular

content, via a pertussis toxin-sensitive pathway, thus

Fig. 3. Schematic drawing representing the involvement of NPY originating from the sympathetic nerve systems and the adrenal gland during

stress. NPY is co-released with NE from nerve endings, and it acts as an important modulator of the sympathetic function by potentiating the

catecholamine vasoconstrictor activity through the Y1 receptor. Furthermore, NPY secreted from chromaffin cells stimulates catecholamine

release by an autocrine/paracrine mechanism. Therefore, NPY could play an important role on sustaining the catecholamine plasmatic levels

during stress situations.

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–25 15

indicating the involvement of a Gi protein. In this

model, NPY could antagonize the effect of isoproter-

enol, which elevated macromolecule permeability and

cAMP intracellular content, suggesting an antiadrener-

gic effect of NPY [257]. This effect of NPY is in

opposition to the potentiating effects described before,

but this can be explained by the difference in NPY

concentrations used and by the high heterogeneity of

endothelial cells over the vascular tree. Moreover,

NPY is able to promote endothelial cell proliferation,

migration and adhesion to the extracellular matrix. It

also stimulates capillary tube formation in vitro and

angiogenesis in vivo [47]. More recently, NPY was

shown to activate ECs migration in response to

wounding. This effect was produced by the processing

product NPY3–36, resulting from the cleavage of the

two N-terminal amino acids of NPY by dipeptidyl

peptidase IV [258]. Thus, the use of Y2 selective

agonists could be beneficial in wound healing.

7.4. NPY and rhinitis

NPY reduces blood flow and NO levels in the

human nasal mucosa. This effect appears to be medi-

ated by the Y1 receptor subtype, as suggested by RT-

PCR expression analysis [259]. In addition, local

treatment with exogenous NPY reduces nasal obstruc-

tion and mucus secretion evoked by allergen chal-

lenge in allergic patients [260]. This effect could be

due to the vasoconstrictor effect of NPY in the nasal

mucosa [261].

TASP V, a potent and selective NPY Y2 receptor

agonist, is able to attenuate the histamine-induced

nasal airway resistance increase and minimal cross-

section area decrease [43] in humans. Although the

presence of Y2 receptor has not been determined in the

human nasal mucosa by RT-PCR [259], the effect of

TASP V suggests the presence of the Y2 receptor in the

human nasal mucosa. It was previously observed that,

in anaesthetized dogs, sympathetic nerve stimulation

attenuates parasympathetic vasodilatation via NPY

release acting on pre-junctional Y2 receptors [262]. It

is possible, therefore, that TASP V could act via this

mechanism. Thus, it would be interesting to analyse

the effect of NPY and TASP V on parasympathetic

peptide secretion. The development of Y2 specific

agonists would be valuable in the treatment of nasal

congestion occurring in chronic rhinosinusitis.

8. Conclusion

In summary, numerous investigations to date sug-

gest that NPY is implicated in the pathophysiology of

a number of diseases including feeding and metabolic

disorders, anxiety, seizures, memory, circadian rhythm,

drug addiction, pain, cardiovascular diseases, rhinitis,

and endothelial cell dysfunctions. Thus, the design of

selective antagonists or agonists of NPY receptors

could be useful compounds for the treatment of all

these diseases. However, one should keep in mind that

the large tissue distribution of NPY receptors and their

stimulation or blockade by insufficiently selective

drugs could produce untoward effects.

Acknowledgements

We are thankful to Dr. Mary Holdom for careful

reading of the manuscript. This work was supported

by the Swiss National Foundation (31-065068.01).

References

[1] Tatemoto K, Carlquist M, Mutt V. Neuropeptide Y: a novel

brain peptide with structural similarities to peptide YY and

pancreatic polypeptide. Nature 1982;296:659–60.

[2] Blundell TL, Pitts JE, Tickle IJ, Wood SP, Wu C-W. X-ray

analysis (1.4A resolution) of avian pancreatic polypeptide:

small globular protein hormone. Proc Natl Acad Sci U S A

1981;78:4175–9.

[3] Schwartz TW, Fuhlendorff J, Kjems LL, Kristensen MS,

Vervelde M, O’Hare M, et al. Signal epitopes in the three-

dimensional structure of neuropeptide Y. Ann NY Acad Sci

1990;611:35–47.

[4] Dumont Y, Martel JC, Fournier A, St-Pierre S, Quirion R.

Neuropeptide Yand neuropeptide Y receptor subtypes in brain

and peripheral tissues. Prog Neurobiol 1992;38:125–67.

[5] Cerda-Reverter JM, Larhammar D. Neuropeptide Y family of

peptides: structure, anatomical expression, function, and mo-

lecular evolution. Biochem Cell Biol 2000;78:371–92.

[6] Akabayashi A, Zaia CT, Gabriel SM, Silva I, Cheung WK,

Leibowitz SF. Intracerebroventricular injection of dibutyryl

cyclic adenosine 3V,5V-monophosphate increases hypothala-

mic levels of neuropeptide Y. Brain Res 1994;660:323–8.

[7] Williams AG, Hargreaves AC, Gunn-Moore FJ, Tavare JM.

Stimulation of neuropeptide Y gene expression by brain-de-

rived neurotrophic factor requires both the phospholipase

Cgamma and Shc binding sites on its receptor, TrkB. Bio-

chem J 1998;333:505–9.

[8] Higuchi H, Yang HY, Sabol SL. Rat neuropeptide Y precur-

sor gene expression. mRNA structure, tissue distribution, and

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–2516

regulation by glucocorticoids, cyclic AMP, and phorbol ester.

J Biol Chem 1988;263:6288–95.

[9] Palkovits M. Peptidergic neurotransmitters in the endocrine

hypothalamus. In: Ciba Foundation Symposia, editor, Func-

tional anatomy of the neuroendocrine hypothalamus. Chiches-

ter: Wiley; 1992. p. 3–15.

[10] Kalra SP, Dube MG, Pu S, Xu B, Horvath TL, Kalra PS.

Interacting appetite-regulating pathways in the hypothalamic

regulation of body weight. Endocr Rev 1999;20:68–100.

[11] Sundler F, Bottcher G, Ekblad E, Hakanson R. PP, PYY, and

NPY. Occurrence and distribution in the periphery. New Jer-

sey: Colmers W.F. and Wahlestedt C.; 1993. p. 157–93.

[12] Cavadas C, Silva AP, Mosiman F, Cotrim MD, Fontes Ri-

beiro CA, Brunner HR, et al. NPY regulates catecholamine

secretion from human adrenal chromaffin cells. J Clin

Endocrinol Metab 2001;86:5956–63.

[13] Myrsen U, Ahren B, Sundler F. Neuropeptide Y is expressed

in subpopulations of insulin- and non-insulin-producing islet

cells in the rat after dexamethasone treatment: a combined

immunocytochemical and in situ hybridisation study. Regul

Pept 1995;60:19–31.

[14] Myrsen U, Ahren B, Sundler F. Dexamethasone-induced

neuropeptide Y expression in rat islet endocrine cells. Rapid

reversibility and partial prevention by insulin. Diabetes

1996;60:1306–16.

[15] Ericsson A, Schalling M, McIntyre KR, Lundberg JM, Lar-

hammar D, Seroogy K, et al. Detection of neuropeptide Y

and its mRNA in megakaryocytes: enhanced levels in certain

autoimmune mice. Proc Natl Acad Sci U S A 1987;84:

5585–9.

[16] Strand FL. Neuropeptides: regulators of physiological pro-

cesses. London: Charles F. Stevens; 1999.

[17] Michel MC, Beck-Sickinger A, Cox H, Doods HN, Herzog

H, Larhammar D, et al. XVI International Union of pharma-

cology recommendations for the nomenclature of neuropep-

tide Y, peptide YY, and pancreatic polypeptide receptors.

Pharmacol Rev 1998;50:143–50.

[18] Larhammar D, Blomqvist AG, Yee F, Jazin E, Yoo H, Wah-

lested C. Cloning and functional expression of a human neu-

ropeptide Y/peptide YY receptor of the Y1 type. J Biol Chem

1992;267:10935–8.

[19] Rose PM, Fernandes P, Lynch JS, Frazier ST, Fisher SM,

Kodukula K, et al. Cloning and functional expression of a

cDNA encoding a human type 2 neuropeptide Y receptor. J

Biol Chem 1995;270:22661–4.

[20] Lundell I, Blomqvist AG, Berglund MM, Schober DA,

Johnson D, Statnick MA, et al. Cloning of a human receptor

of the NPY receptor family with high affinity for pancreatic

polypeptide andpeptideYY. JBiolChem1995;270:29123–8.

[21] Gerald C, Walker MW, Criscione L, Gustafson EL, Batzl-

Hartmann C, Smith KE, et al. A receptor subtype involved

in neuropeptide Y-induced food intake. Nature 1996;382:

168–71.

[22] Weinberg DH, Sirinathsinghji DJ, Tan CP, Shiao LL, Morin

N, Rigby MR, et al. Cloning and expression of a novel neuro-

peptide Y receptor. J Biol Chem 1996;271:16435–8.

[23] Krause J, Eva C, Seeburg P, Sprengel R. Neuropeptide Y1

subtype pharmacology of a recombinantly expressed neuro-

peptide receptor. Mol Pharmacol 1992;41:817–21.

[24] Soll RM, Dinger MC, Lundell I, Larhammer D, Beck-

Sickinger AG. Novel analogues of neuropeptide Y with a

preference for the Y1-receptor. Eur J Biochem 2001;268:

2828–37.

[25] Cabrele C, Beck-Sickinger AG. Molecular characterization

of the ligand– receptor interaction of the neuropeptide Y

family. J Pept Sci 2000;6:97–122.

[26] Daniels AJ, Matthews JE, Slepetis RJ, Jansen M, Viveros

OH, Tadepalli A, et al. High affinity neuropeptide Y receptor

antagonists. Proc Natl Acad Sci U S A 1995;92:9067–71.

[27] Schober DA, Van Abbema AM, Smiley DL, Bruns RF,

Gehlert DR. The neuropeptide Y Y1 antagonist, 1229U91,

a potent agonist gor the human pancreatic polypeptide-pre-

ferring (NPY Y4) receptor. Peptides 1998;19:537–42.

[28] Parker EM, Babij CK, Balasubramaniam A, Burrier RE,

Guzzi M, Hamud F, et al. GR231118 (1229U91) and other

analogues of the C-terminus of neuropeptide Y are potent

neuropeptide Y Y1 receptor antagonists and neuropeptide

Y Y4 receptor agonists. Eur J Pharmacol 1998;349:97–105.

[29] Rudolf K, Eberlein W, Engel W, Wieland HA, Willim KD,

Entzeroth M, et al. The first highly potent and selective non-

peptide neuropeptide Y Y1 antagonist: BIBP3226. Eur J

Pharmacol 1994;271:R11–3.

[30] Serradeil-Le G, Valette G, Douby PE, Pellet A, Oury-Donat

F, Brossard G, et al. SR120819A, an orally-active and selec-

tive neuropeptide Y Y1 receptor antagonist. FEBS Lett 1995;

362:192–6.

[31] Wieland HA, Engel W, Eberlein W, Rudolf K, Doods HN.

Subtype selectivity of the novel nonpeptide neuropeptide Y

Y1 receptor antagonist BIBO3304 and its effect on feeding in

rodents. Br J Pharmacol 1998;125:549–55.

[32] Hipskind PA, Lobb KL, Nixon JA, Britton TC, Bruns RF,

Catlow J, et al. Potent and selective 1,2,3-trisubstituted in-

dole NPY Y-1 antagonists. J Med Chem 1997;40:3712–4.

[33] Murakami Y, Hara H, Okada T, Hashizume H, Kii M, Ishihara

Y, et al. 1,3-Disubstituted benzodiazepines as novel, potent,

selective neuropeptide Y Y1 receptor antagonists. J Med

Chem 1999;42:2621–32.

[34] Kanatani A, Hata M, Mashiko S, Ishihara A, Okamoto O,

Haga Y, et al. A typical Y1 receptor regulates feeding beha-

viors: effects of a potent and selective Y1 antagonist, J-

115814. Mol Pharmacol 2001;59:501–5.

[35] Malmstrom RE, Balmer KC, Weilitz J, Nordlander M,

Sjolander M. Pharmacology of H 394/84, a dihydropyri-

dine neuropeptide Y Y(1) receptor antagonist, in vivo. Eur

J Pharmacol 2001;418:95–104.

[36] Eckard CP, Beck-Sickinger AG, Wieland HA. Comparison of

antibodies directed against receptor segments of NPY-recep-

tors. J Recept Signal Transduct Res 1999;19:379–94.

[37] Wahlestedt C, Hakanson R. Effects of neuropeptide Y (NPY)

at the synaptic neuroeffector junction. Can pre- and postjunc-

tional receptors be distinguished? Med Biol 1986;64:85–8.

[38] Grundemar L, Jonas SE, Morner N, Hogestatt ED, Wahles-

tedt C, Hakanson R. Characterization of vascular neuropep-

tide Y receptors. Br J Pharmacol 1992;105:45–50.

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–25 17

[39] Wahlestedt C, Pich EM, Koob GF, Yee F, Heilig M. Modu-

lation of anxiety and neuropeptide Y-Y1 receptors by anti-

sense oligonucleotides. Science 1993;259:528–31.

[40] Inui A. Feeding and body-weight regulation by hypothalamic

neuropeptides—mediation of the actions of leptin. Trends

Neurosci 1999;22:62–7.

[41] Aakerlund L, Gether U, Fuhlendorff J, Schwartz TW,

Thastrup O. Y1 receptors for neuropeptide Y are coupled to

mobilization of intracellular calcium and inhibition of adeny-

late cyclase. FEBS Lett 1990;15:73–8.

[42] Rist B, Zerbe O, Ingenhoven N, Scapozza L, Peers C,

Vaughan PF, et al. Modified, cyclic dodecapeptide analog

of neuropeptide Y is the smallest full agonist at the human

Y2 receptor. FEBS Lett 1996;394:169–73.

[43] Malis DD, Grouzmann E, Morel DR, Mutter M, Lacroix JS.

Influence of TASP-V, a novel neuropeptide Y/NPY Y2 ago-

nist, on nasal and bronchial responses evoked by histamine

in anaesthetized pigs and in humans. Br J Pharmacol 1999;

126:989–96.

[44] Grouzmann E, Buclin T, Martire M, Canizzaro C, Dorner B,

Razaname A, et al. Characterization of a selective antagonist

of neuropeptide Y at the Y2 receptor. Synthesis and pharma-

cological evaluation of a Y2 antagonist. [published erratum

appears in J. Biol. Chem. 1998;273:27033] J Biol Chem

1997;272:7699–706.

[45] Doods H, Gaida W, Wieland HA, Dollinger H, Schnorren-

berg G, Esser F, et al. BIIE0246: a selective and high affinity

neuropeptide Y Y2 receptor antagonist. Eur J Pharmacol

1999;384:R3–5.

[46] Goumain M, Voisin T, Lorinet AM, Laburthe M. Identifica-

tion and distribution of mRNA encoding the Y1, Y2, Y4, and

Y5 receptors for peptides of the PP-fold family in the rat

intestine and colon. Biochem Biophys Res Commun 1998;

247:52–6.

[47] Zukowska-Grojec Z, Karwatowska-Prokopczuk E, Rose W,

Rone J, Movafagh S, Ji H, et al. Neuropeptide Y: a novel

angiogenic factor from the sympathetic nerves and endothe-

lium. Circ Res 1998;83:187–95.

[48] Pheng LH, Fournier A, Dumont Y, Quirion R, Regoli D. The

dog saphenous vein: a sensitive and selective preparation for

the Y2 receptor of neuropeptide Y. Eur J Pharmacol 1997;

327:163–7.

[49] Beck-Sickinger AG, Grouzmann E, Hoffmann E, Gaida W,

Van Meir EG, Waeber B, et al. A novel cyclic analog of

neuropeptide Y specific for the Y2 receptor. Eur J Biochem

1992;206:957–64.

[50] Lynch JW, Lemos VS, Bucher B, Stoclet JC, Takeda K. A

pertussis toxin-insensitive calcium influx mediated by neuro-

peptide Y2 receptors in a human neuroblastoma cell line. J

Biol Chem 1994;269:8226–33.

[51] Shigeri Y, Fujimoto M. Y2 receptors for neuropeptide Y are

coupled to three intracellular signal transduction pathways in

a human neuroblastoma cell line. J Biol Chem 1994;269:

8842–8.

[52] Lee CC, Miller RJ. Is there really an NPY Y3 receptor?

Regul Pept 1998;75/76:71–8.

[53] Balasubramaniam A, Sheriff S, Rigel DF, Fischer JE. Char-

acterization of neuropeptide Y binding sites in rat cardiac

ventricular membranes. Peptides 1990;11:545–50.

[54] Wahlestedt C, Regunathan S, Reis DJ. Identification of

cultured cells selectively expressing Y1-, Y2-, or Y3-type

receptors for neuropeptide Y/peptide YY. Life Sci 1992;50:

PL7–PL12.

[55] Dumont Y, Satoh H, Cadieux A, Taoudi-Benchekroun M,

Pheng LH, St-Pierre S, et al. Evaluation of truncated neuro-

peptide Y analogues with modifications of the tyrosine res-

idue in position 1 on Y1, Y2 and Y3 receptor sub-types. Eur J

Pharmacol 1993;238:37–45.

[56] Dumont Y, Cadieux A, Pheng LH, Fournier A, St-Pierre S,

Quirion R. Peptide YY derivatives as selective neuropeptide

Y/peptide YY Y1 and Y2 agonists devoided of activity for the

Y3 receptor sub-type. Brain Res Mol Brain Res 1994;26:

320–4.

[57] Norenberg W, Bek M, Limberger N, Takeda K, Illes P.

Inhibition of nicotinic acetylcholine receptor channels in

bovine adrenal chromaffin cells by Y3-type neuropeptide

Y receptors via the adenylate cyclase/protein kinase A sys-

tem. Naunyn-Schmiedeberg’s Arch Pharmacol 1995;351:

337–47.

[58] Lundell I, Statnick MA, Johnson D, Schober DA, Starback P,

Gehlert DR, et al. The cloned rat pancreatic polypeptide

receptor exhibits profound differences to the orthologous

receptor. Proc Natl Acad Sci U S A 1996;93:5111–5.

[59] Bard JA, Walker MW, Branchek TA, Weinshank RL. Clon-

ing and functional expression of a human Y4 subtype recep-

tor for pancreatic polypeptide, neuropeptide Y, and peptide

YY. J Biol Chem 1995;270:26762–5.

[60] Schwartz TW. Pancreatic polypeptide: a hormone under va-

gal control. Gastroenterology 1983;85:1411–25.

[61] Ledeboer M, Masclee AA, Biemond I, Lamers CB. Gallblad-

der motility and cholecystokinin secretion during continuous

enteral nutrition. Am J Gastroenterol 1997;92:2274–9.

[62] Jain MR, Pu S, Kalra PS, Kalra SP. Evidence that stimulation

of two modalities of pituitary luteinizing hormone release in

ovarian steroid-primed ovariectomized rats may involve neu-

ropeptide Y Y1 and Y4 receptors. Endocrinology 1999;140:

5171–7.

[63] Voisin T, Goumain M, Lorinet AM, Maoret JJ, Laburthe M.

Functional and molecular properties of the human recombi-

nant Y4 receptor: resistance to agonist-promoted desensitiza-

tion. J Pharmacol Exp Ther 2000;292:638–46.

[64] Borowsky B, Walker MW, Bard J, Weinshank RL, Laz TM,

Vaysse P, et al. Molecular biology and pharmacology of

multiple NPY Y5 receptor species homologs. Regul Pept

1998;75–76:45–53.

[65] Cabrele C, Langer M, Bader R, Wieland HA, Doods HN,

Zerbe O, et al. The first selective agonist for the neuropeptide

YY5 receptor increases food intake in rats. J Biol Chem 2000;

275:36043–8.

[66] Criscione L, Rigollier P, Batzl-Hartmann C, Rueger H,

Stricker-Krongrad A, Wyss P, et al. Food intake in free-feed-

ing and energy-deprived lean rats is mediated by the neuro-

peptide Y5 receptor. J Clin Invest 1998;102:2136–45.

[67] Della ZuanaO, SadloM,GermainM, FeletouM, Chamorro S,

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–2518

Tisserand F, et al. Reduced food intake in response to CGP

71683Amay be due to mechanisms other than NPYY5 recep-

tor blockade. Int J Obes Relat Metab Disord 2001;25:84–94.

[68] McNally JJ, Youngman MA, Lovenberg TW, Nepomuceno

D, Wilson S, Dax SL. N-acetylated alpha-(3-pyridylmethyl)-

beta-aminotetralin antagonists of the human neuropeptide Y

Y5 receptor. Bioorg Med Chem Lett 2000;10:1641–3.

[69] Kanatani A, Ishihara A, Iwaasa H, Nakamura K, Okamoto O,

Hidaka M, et al. L-152,804: orally active and selective neu-

ropeptide Y Y5 receptor antagonist. Biochem Biophys Res

Commun 2000;272:169–73.

[70] Gerald C, Walker MW, Criscione L, Gustafson EL, Batzl-

Hartmann C, Smith KE, et al. A receptor subtype involved

in neuropeptide-Y-induced food intake. Nature 1996;382:

168–71.

[71] Moser C, Bernhardt G, Michel J, Schwartz H, Buschauer A.

Cloning and functional expression of the hNPY Y5 receptor

in human endometrial cancer (HEC-1B) cells. Can J Physiol

Pharm 2000;78:134–42.

[72] Colmers WF, Pittman QJ. Presynaptic inhibition by neuro-

peptide Y and baclofen in hippocampus: insensitivity to per-

tussis toxin treatment. Brain Res 1989;498:99–104.

[73] Millar BC, Weis T, Piper HM, Weber M, Borchard U,

McDermott BJ, et al. Positive and negative contractile effects

of neuropeptide Y on ventricular cardiomyocytes. Am J

Physiol 1991;261:H1727–33.

[74] Lemos VS, Takeda K. Neuropeptide Y2-type receptor-medi-

ated activation of large-conductance Ca(2+)-sensitive K+

channels in a human neuroblastoma cell line. Pflugers Arch

1995;430:534–40.

[75] Olasmaa M, Terenius L. Neuropeptide Y receptor interaction

with beta-adrenoceptor coupling to adenylate cyclase. Prog

Brain Res 1986;68:337–41.

[76] Michel MC. Receptors for neuropeptideY: multiple subtypes

and multiple second messengers. [Erratum appears in Trends

Pharmacol. Sci. 1991;12:448] Trends Pharmacol Sci 1991;

12:389–94.

[77] Perney TM, Miller RJ. Two different G-proteins mediate

neuropeptide Y and bradykinin-stimulated phospholipid

breakdown in cultured rat sensory neurons. J Biol Chem

1989;264:7317–27.

[78] Motulsky HJ, Michel MC. Neuropeptide Y mobilizes Ca2+

and inhibits adenylate cyclase in human erythroleukemia

cells. Am J Physiol 1988;255:E880–5.

[79] Ewald DA, Sternweis PC, Miller RJ. Guanine nucleotide-

binding protein Go-induced coupling of neuropeptide Y re-

ceptors to Ca2+ channels in sensory neurons. Proc Natl Acad

Sci U S A 1988;85:3633–7.

[80] Gammon CM, Lyons SA, Morel P. Modulation by neuropep-

tides of bradykinin-stimulated second messenger release in

dorsal root ganglion neurons. Brain Res 1990;518:159–65.

[81] Michel MC, Rascher W. Neuropeptide Y: a possible role in

hypertension? J Hypertens 1995;13:385–95.

[82] Xiong Z, Cheung DW. ATP-dependent inhibition of Ca2+-

activated K+ channels in vascular smooth muscle cells by

neuropeptide Y. Pflugers Arch 1995;431:110–6.

[83] Martin SE, Patterson RE. Coronary constriction due to neuro-

peptide Y: alleviation with cyclooxygenase blockers. Am J

Physiol 1989;257:H927–34.

[84] Kawamura K, Smith TL, Zhou Q, Kummerow FA. Neuro-

peptide Y stimulates prostacyclin production in porcine vas-

cular endothelial cells. Biochem Biophys Res Comm 1991;

179:309–13.

[85] Keffel S, Schmidt M, Bischoff A, Michel MC. Neuropeptide

Y stimulation of extracellular signal-regulated kinases in hu-

man erythroleukemia cells. J Pharmacol Exp Ther 1999;291:

1172–8.

[86] You J, Zhang W, Jansen-Olesen I, Edvinsson L. Relation

between cyclic GMP generation and cerebrovascular reactiv-

ity: modulation by NPY and a-trinositol. Pharmacol Toxicol

1995;77:48–56.

[87] Nilsson T, Lind H, Brunkvall J, Edvinsson L. Vasodilation in

human subcutaneous arteries induced by neuropeptide Y is

mediated by neuropeptide Y Y1 receptors and is nitric oxide

dependent. Can J Physiol Pharm 2000;78:251–5.

[88] Sheriff S, Dayal R, Kasckow J, Regmi A, Chance W, Fischer

J, et al. NPY upregulates genes containing cyclic AMP re-

sponse element in human neuroblastoma cell lines bearing

Y1 and Y2 receptors: involvement of CREB. Regul Pept 1998;

75–76:309–18.

[89] Rohner-Jeanrenaud F. A neuroendocrine reappraisal of the

dual-centre hypothesis: its implications for obesity and insulin

resistance. Int J Obes Relat Metab Disord 1995;19:517–34.

[90] Elmquist JK, Maratos-Flier E, Saper CB, Flier JS. Unra-

veling the central nervous system pathways underlying re-

sponses to leptin. Nat Neurosci 1998;1:445–50.

[91] Elmquist JK, Elias CF, Saper CB. From lesions to leptin:

hypothalamic control of food intake and body weight. Neu-

ron 1999;22:221–32.

[92] Inui A. Neuropeptide Y feeding receptors: are multiple sub-

types involved? Trends Pharmacol Sci 1999;20:43–6.

[93] Luiten PG, ter Horst GJ, Steffens AB. The hypothalamus,

intrinsic connections and outflow pathways to the endocrine

system in relation to the control of feeding and metabolism.

Prog Neurobiol 1987;28:1–54.

[94] Bray GA. Integration of energy intake and expenditure in

animals and man: the autonomic and adrenal hypothesis. Clin

Endocrinol Metab 1984;13:521–46.

[95] Wieland HA, Hamilton BS, Krist B, Doods HN. The role of

NPY in metabolic homeostasis: implications for obesity ther-

apy. Expert Opin Investig Drugs 2000;9:1327–46.

[96] Kalra SP. Appetite and body weight regulation: is it all in the

brain? Neuron 1997;19:227–30.

[97] Sahu A, Kalra SP, Crowley WR, Kalra PS. Evidence that

NPY-containing neurons in the brainstem project into se-

lected hypothalamic nuclei: implication in feeding behavior.

Brain Res 1988;9(457):376–8.

[98] Sahu A, Dube MG, Kalra SP, Kalra PS. Bilateral neural trans-

ections at the level of mesencephalon increase food intake

and reduce latency to onset of feeding in response to neuro-

peptide Y. Peptides 1988;9:1269–73.

[99] Clark JT, Kalra PS, Crowley WR, Kalra SP. Neuropeptide Y

and human pancreatic polypeptide stimulate feeding behav-

ior in rats. Endocrinology 1984;115:427–9.

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–25 19

[100] Stanley BG, Kyrkouli SE, Lampert S, Leibowitz SF. Neuro-

peptide Y chronically injected into the hypothalamus: a

powerful neurochemical inducer of hyperphagia and obesity.

Peptides 1986;7:1189–92.

[101] Stanley BG, Magdalin W, Seirafi A, Nguyen MM, Leibowitz

SF. Evidence for neuropeptide Y mediation of eating pro-

duced by food deprivation and for a variant of the Y1 receptor

mediating this peptide’s effect. Peptides 1992;13:581–7.

[102] Larsen PJ, Tang-Christensen M, Stidsen CE, Madsen K,

Smith MS, Cameron JL. Activation of central neuropeptide

Y Y1 receptors potently stimulates food intake in male rhe-

sus monkeys. J Clin Endocrinol Metab 1999;84:3781–91.

[103] Raposinho PD, Pierroz DD, Broqua P, White RB, Pedrazzini

T, Aubert ML. Chronic administration of neuropeptide Y into

the lateral ventricle of C57BL/6J male mice produces an

obesity syndrome including hyperphagia, hyperleptinemia,

insulin resistance, and hypogonadism. Mol Cell Endocrinol

2001;185:195–204.

[104] Sahu A, Kalra PS, Kalra SP. Food deprivation and ingestion

induce reciprocal changes in neuropeptide Y concentrations

in the paraventricular nucleus. Peptides 1988;9:83–6.

[105] McCarthy HD, McKibbin PE, Holloway B, Mayers R, Wil-

liams G. Hypothalamic neuropeptide Y receptor character-

istics and NPY-induced feeding responses in lean and obese

Zucker rats. Life Sci 1991;49:1491–7.

[106] Dryden S, Pickavance L, Frankish HM, Williams G. In-

creased neuropeptide Y secretion in the hypothalamic para-

ventricular nucleus of obese (fa/fa) Zucker rats. Brain Res

1995;690:185–8.

[107] Stephens TW, Basinski M, Bristow PK, Bue-Valleskey JM,

Burgett SG, Craft L, et al. The role of neuropeptide Y in the

antiobesity action of the obese gene product. Nature 1995;

377:530–2.

[108] Widdowson PS. Regionally-selective down-regulation of

NPY receptor subtypes in the obese Zucker rat. Relationship

to the Y5 ‘feeding’ receptor. Brain Res 1997;758:17–25.

[109] Vettor R, Zarjevski N, Cusin I, Rohner-Jeanrenaud F, Jean-

renaud B. Induction and reversibility of an obesity syndrome

by intracerebroventricular neuropeptide Y administration to

normal rats. Diabetologia 1994;37:1202–8.

[110] Sainsbury A, Rohner-Jeanrenaud F, Grouzmann E, Jeanre-

naud B. Acute intracerebroventricular administration of neu-

ropeptide Y stimulates corticosterone output and feeding but

not insulin output in normal rats. Neuroendocrinology 1996;

63:318–26.

[111] Dube MG, Xu B, Crowley WR, Kalra PS, Kalra SP. Evi-

dence that neuropeptide Y is a physiological signal for nor-

mal food intake. Brain Res 1994;646:341–4.

[112] Sahu A, Sninsky CA, Kalra SP. Evidence that hypothalamic

neuropeptide Y gene expression and NPY levels in the para-

ventricular nucleus increase before the onset of hyperphagia

in experimental diabetes. Brain Res 1997;755:339–42.

[113] Schwartz MW, Baskin DG, Bukowski TR, Kuijper JL, Foster

D, Lasser G, et al. Specificity of leptin action on elevated

blood glucose levels and hypothalamic neuropeptide Y gene

expression in ob/ob mice. Diabetes 1996;45:531–5.

[114] White JD, Olchovsky D, Kershaw M, Berelowitz M. In-

creased hypothalamic content of preproneuropeptide-Y mes-

senger ribonucleic acid in streptozotocin-diabetic rats. Endo-

crinology 1990;126:765–72.

[115] Erickson JC, Clegg KE, Palmiter RD. Sensitivity to leptin

and susceptibility to seizures of mice lacking neuropeptide Y.

Nature 1996;381:415–21.

[116] Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin

DG. Identification of targets of leptin action in rat hypothal-

amus. J Clin Invest 1996;98:1101–6.

[117] McKibbin PE, McCarthy HD, Shaw P, Williams G. Insulin

deficiency is a specific stimulus to hypothalamic neuropep-

tide Y: a comparison of the effects of insulin replacement and

food restriction in streptozocin-diabetic rats. Peptides 1992;

13:721–7.

[118] Haynes AC, Arch JR, Wilson S, McClue S, Buckingham RE.

Characterisation of the neuropeptide Y receptor that mediates

feeding in the rat: a role for the Y5 receptor? Regul Pept

1998;75–76:355–61.

[119] Kask A, Rago L, Harro J. Evidence for involvement of neuro-

peptide Y receptors in the regulation of food intake: studies

with Y1-selective antagonist BIBP3226. Br J Pharmacol

1998;124:1507–15.

[120] Corpa ES, McQuade J, Krasnicki S, Conze DB. Feeding after

fourth ventricular administration of neuropeptide Y receptor

agonists in rats. Peptides 2001;22:493–9.

[121] Kanatani A, Ishihara A, Asahi S, Tanaka T, Ozaki S, Ihara M.

Potent neuropeptide Y Y1 receptor antagonist, 1229U91:

blockade of neuropeptide Y-induced and physiological food

intake. Endocrinology 1996;137:3177–82.

[122] Kanatani A, Ito J, Ishihara A, Iwaasa H, Fukuroda T, Fukami

T, et al. NPY-induced feeding involves the action of a Y1-like

receptor in rodents. Regul Pept 1998;75–76:409–15.

[123] Ishihara A, Tanaka T, Kanatani A, Fukami T, Ihara M,

Fukuroda T. A potent neuropeptide Y antagonist, 1229U91,

suppressed spontaneous food intake in Zucker fatty rats. Am

J Physiol 1998;274:R1500–4.

[124] Widdowson PS, Henderson L, Pickavance L, Buckingham R,

Tadayyon M, Arch JR, et al. Hypothalamic NPY status dur-

ing positive energy balance and the effects of the NPY antag-

onist, BW 1229U91, on the consumption of highly palatable

energy-rich diet. Peptides 1999;20:367–72.

[125] Iyengar S, Li DL, Simmons RM. Characterization of neuro-

peptide Y-induced feeding in mice: do Y1–y6 receptor. J

Pharmacol Exp Ther 1999;289:1031–40.

[126] Matthews JE, Jansen M, Lyerly D, Cox R, Chen WJ, Koller

KJ, et al. Pharmacological characterization and selectivity of

the NPY antagonist GR231118 (1229U91) for different NPY

receptors. Regul Pept 1997;72:113–9.

[127] Morgan DG, Small CJ, Abusnana S, Turton M, Gunn I,

Heath M, et al. The NPY Y1 receptor antagonist BIBP

3226 blocks NPY induced feeding via a non-specific mech-

anism. Regul Pept 1998;75–76:377–82.

[128] Polidori C, Ciccocioppo R, Regoli D, Massi M. Neuropeptide

Y receptor(s) mediating feeding in the rat: characterization

with antagonists. Peptides 2000;21:29–35.

[129] Kanatani A, Kanno T, Ishihara A, Hata M, Sakuraba A,

Tanaka T, et al. The novel neuropeptide Y Y(1) receptor

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–2520

antagonist J-104870: a potent feeding suppressant with oral

bioavailability. Biochem Biophys Res Commun 1999;266:

88–91.

[130] Hwa JJ, Witten MB, Williams P, Ghibaudi L, Gao J, Salis-

bury BG, et al. Activation of the NPY Y5 receptor regulates

both feeding and energy expenditure. Am J Physiol 1999;277:

R1428–34.

[131] Wyss P, Levens N, Stricker-Krongrad A. Stimulation of feed-

ing in lean but not in obese Zucker rats by a selective neuro-

peptide Y Y5 receptor agonist. NeuroReport 1998;9:2675–7.

[132] Parker EM, Balasubramaniam A, Guzzi M, Mullins DE,

Salisbury BG, Sheriff S, et al. [D-Trp(34)] neuropeptide Y

is a potent and selective neuropeptide Y Y(5) receptor ago-

nist with dramatic effects on food intake. Peptides 2000;21:

393–9.

[133] Schaffhauser AO, Stricker-Krongrad A, Brunner L, Cumin F,

Gerald C, Whitebread S, et al. Inhibition of food intake by

neuropeptide Y Y5 receptor antisense oligodeoxynucleotides.

Diabetes 1997;46:1792–8.

[134] Tang-Christensen M, Kristensen P, Stidsen CE, Brand CL,

Larsen PJ. Entral administration of Y5 receptor antisense

decreases spontaneous food intake and attenuates feeding in

response to exogenous neuropeptide Y. J Endocrinol 1998;

159:307–12.

[135] Kask A, Vasar E, Heidmets LT, Allikmets L, Wikberg JE.

Neuropeptide Y Y(5) receptor antagonist CGP71683A: the

effects on food intake and anxiety-related behavior in the rat.

Eur J Pharmacol 2001;414:215–24.

[136] Daniels AJ, Grizzle MK, Wiard RP, Matthews JE, Heyer D.

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. Regul Pept 2002;

106:47–54.

[137] Beck B. KO’s and organisation of peptidergic feeding behav-

ior mechanisms. Neurosci Biobehav Rev 2001;25:143–58.

[138] Hollopeter G, Erickson JC, Seeley RJ, Marsh DJ, Palmiter

RD. Response of neuropeptide Y-deficient mice to feeding

effectors. Regul Pept 1998;75–76:383–9.

[139] Bannon AW, Seda J, Carmouche M, Francis JM, Norman

MH, Karbon B, et al. Behavioral characterization of neuro-

peptide Y knockout mice. Brain Res 2000;868:79–87.

[140] Pedrazzini T, Seydoux J, Kunstner P, Aubert JF, Grouzmann

E, Beermann F, et al. Cardiovascular response, feeding be-

havior and locomotor activity in mice lacking the NPY Y1

receptor. Nat Med 1998;4:722–6.

[141] Marsh DJ, Hollopeter G, Kafer KE, Palmiter RD. Role of the

Y5 neuropeptide Y receptor in feeding and obesity. Nat Med

1998;4:718–21.

[142] Kaye WH, Berrettini W, Gwirtsman H, George DT. Altered

cerebrospinal fluid neuropeptide Y and peptide YY immu-

noreactivity in anorexia and bulimia nervosa. Arch Gen Psy-

chiatry 1990;47:548–56.

[143] Baranowska B, Wolinska-Witort E, Wasilewska-Dziubinska

E, Roguski K, Chmielowska M. Plasma leptin, neuropeptide

Y (NPY) and galanin concentrations in bulimia nervosa and

in anorexia nervosa. Neuro endocrinol Lett 2001;22:356–8.

[144] Fuxe K, Agnati LF, Harfstrand A, Zini I, Tatemoto K, Pich

EM, et al. Central administration of neuropeptide Y induces

hypotension bradypnea and EEG synchronization in the rat.

Acta Physiol Scand 1983;118:189–92.

[145] Heilig M, Murison R. Intracerebroventricular neuropeptide Y

suppresses open field and home cage activity in the rat.

Regul Pept 1987;19:221–31.

[146] Heilig M, McLeod S, Brot M, Heinrichs SC, Menzaghi F,

Koob GF, et al. Anxiolytic-like action of neuropeptide Y:

mediation by Y1 receptors in amygdala, and dissociation

from food intake effects. Neuropsychopharmacology 1993;

8:357–63.

[147] Heilig M, Thorsell A. Brain neuropeptide Y (NPY) in stress

and alcohol dependence. Rev Neurosci 2002;13:85–94.

[148] Inui A, Okita M, Nakajima M, Momose K, Ueno N, Tera-

nishi A, et al. Anxiety-like behavior in transgenic mice with

brain expression of neuropeptide Y. Proc Assoc Am Physi-

cians 1998;11:171–82.

[149] Naveilhan P, Canals JM, Valjakka A, Vartiainen J, Arenas E,

Ernfors P. Neuropeptide Y alters sedation through a hypo-

thalamic Y1-mediated mechanism. Eur J Neurosci 2001;13:

2241–6.

[150] Broqua P, Wettstein JG, Rocher MN, Gauthier-Martin B,

Junien JL. Behavioral effects of neuropeptide Y receptor

agonists in the elevated plus-maze and fear-potentiated star-

tle procedures. Behav Pharmacol 1995;6:215–22.

[151] Heilig M. Antisense inhibition of neuropeptide Y (NPY)-Y1

receptor expression blocks the anxiolytic-like action of NPY

in amygdala and paradoxically increases feeding. Regul Pept

1995;59:201–5.

[152] Kask A, Nguyen HP, Pabst R, Von Horsten S. Neuropeptide

Y Y1 receptor-mediated anxiolysis in the dorsocaudal lateral

septum: functional antagonism of corticotropin-releasing hor-

mone-induced anxiety. Neuroscience 2001;104:799–806.

[153] Sajdyk TJ, Vandergriff MG, Gehlert DR. Amygdalar neuro-

peptide Y Y1 receptors mediate the anxiolytic-like actions of

neuropeptide Y in the social interaction test. Eur J Pharmacol

1999;368:143–7.

[154] Antonijevic IA, Murck H, Bohlhalter S, Frieboes RM, Hols-

boer F, Steiger A. Neuropeptide Y promotes sleep and inhib-

its ACTH and cortisol release in young men. Neurophar-

macology 2000;39:1474–81.

[155] Vezzani A, Sperk G, Colmers WF. Neuropeptide Y: emerg-

ing evidence for a functional role in seizure modulation.

Trends Neurosci 1999;22:25–30.

[156] Sperk G, Marksteiner J, Gruber B, Bellmann R, Mahata M,

Ortler M. Functional changes in neuropeptide Y- and soma-

tostatin-containing neurons induced by limbic seizures in the

rat. Neuroscience 1992;50:831–46.

[157] Bellmann R, Widmann R, Olenik C, Meyer DK, Maas D,

Marksteiner J, et al. Enhanced rate of expression and biosyn-

thesis of neuropeptide Y after kainic acid-induced seizures. J

Neurochem 1991;56:525–30.

[158] Rizzi M, Monno A, Samanin R, Sperk G, Vezzani A. Elec-

trical kindling of the hippocampus is associated with func-

tional activation of neuropeptide Y-containing neurons. Eur J

Neurosci 1993;5:1534–8.

[159] Gruber B, Greber S, Rupp E, Sperk G. Differential NPY

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–25 21

mRNA expression in granule cells and interneurons of the rat

dentate gyrus after kainic acid injection. Hippocampus

1994;4:474–82.

[160] Schwarzer C, Sperk G, Samanin R, Rizzi M, Gariboldi M,

Vezzani A. Neuropeptides-immunoreactivity and their

mRNA expression in kindling: functional implications for

limbic epileptogenesis. Brain Res Brain Res Rev 1996;22:

27–50.

[161] Colmers WF, Lukowiak K, Pittman QJ. Presynaptic action of

neuropeptide Y in area CA1 of the rat hippocampal slice. J

Physiol 1987;383:285–99.

[162] Colmers WF, Klapstein GJ, Fournier A, St-Pierre S, Treherne

KA. Presynaptic inhibition by neuropeptide Y in rat hippo-

campal slice in vitro is mediated by a Y2 receptor. Br J

Pharmacol 1991;102:41–4.

[163] Haas HL, Hermann A, Greene RW, Chan-Palay V. Action

and location of neuropeptide tyrosine (Y) on hippocampal

neurons of the rat in slice preparations. J Comp Neurol

1987;257:208–15.

[164] Greber S, Schwarzer C, Sperk G. Neuropeptide Y inhibits

potassium-stimulated glutamate release through Y2 receptors

in rat hippocampal slices in vitro. Br J Pharmacol 1994;113:

737–40.

[165] Patrylo PR, van den Pol AN, Spencer DD, Williamson A.

NPY inhibits glutamatergic excitation in the epileptic human

dentate gyrus. J Neurophysiol 1999;82:478–83.

[166] Reibel S, Nadi S, Benmaamar R, Larmet Y, Carnahan J,

Marescaux C, et al. Neuropeptide Y and epilepsy: varying

effects according to seizure type and receptor activation.

Peptides 2001;22:529–39.

[167] Baraban SC, Hollopeter G, Erickson JC, Schwartzkroin PA,

Palmiter RD. Knock-out mice reveal a critical antiepileptic

role for neuropeptide Y. J Neurosci 1997;17:8927–36.

[168] DePrato Primeaux S, Holmes PV, Martin RJ, Dean RG, Ed-

wards GL. Experimentally induced attenuation of neuropep-

tide-Y gene expression in transgenic mice increases mortality

rate following seizures. Neurosci Lett 2000;287:61–4.

[169] Vezzani A, Michalkiewicz M, Michalkiewicz T, Moneta D,

Ravizza T, Richichi C, et al. Seizure susceptibility and epi-

leptogenesis are decreased in transgenic rats overexpressing

neuropeptide Y. Neuroscience 2002;110:237–43.

[170] Kofler N, Kirchmair E, Schwarzer C, Sperk G. Altered ex-

pression of NPY-Y1 receptors in kainic acid induced epilepsy

in rats. Neurosci Lett 1997;230:129–32.

[171] Greisen MH, Sheikh SP, Bolwig TG, Mikkelsen JD. Reduc-

tion of neuropeptide Y binding sites in the rat hippocampus

after electroconvulsive stimulations. Brain Res 1997;776:

105–10.

[172] Schwarzer C, Sperk G. Glutamate-stimulated neuropeptide Y

mRNA expression in the rat dentate gyrus: a prominent role

of metabotropic glutamate receptors. Hippocampus 1998;8:

274–88.

[173] Madsen TM, Greisen MH, Nielsen SM, Bolwig TG, Mik-

kelsen JD. Electroconvulsive stimuli enhance both neuropep-

tide Y receptor Y1 and Y2 messenger RNA expression and

levels of binding in the rat hippocampus. Neuroscience 2000;

98:33–9.

[174] Bregola G, Dumont Y, Fournier A, Zucchini S, Quirion R,

Simonato M. Decreased levels of neuropeptide Y(5) receptor

binding sites in two experimental models of epilepsy. Neuro-

science 2000;98:697–703.

[175] Furtinger S, Pirker S, Czech T, Baumgartner C, Ransmayr G,

Sperk G. Plasticity of Y1 and Y2 receptors and neuropeptide

Y fibers in patients with temporal lobe epilepsy. J Neurosci

2001;21:5804–12.

[176] Klapstein GJ, Colmers WF. Neuropeptide Y suppresses epi-

leptiform activity in rat hippocampus in vitro. J Neurophysiol

1997;78:1651–61.

[177] Silva AP, Carvalho AP, Carvalho CM, Malva JO. Modula-

tion of intracellular calcium changes and glutamate release

by neuropeptide Y1 and Y2 receptors in the rat hippocampus:

differential effects in CA1, CA3 and dentate gyrus. J Neuro-

chem 2001;79:286–96.

[178] Woldbye DP. Antiepileptic effects of NPY on pentylenetetra-

zole seizures. Regul Pept 1998;75–76:279–82.

[179] Woldbye DP, Larsen PJ, Mikkelsen JD, Klemp K, Madsen

TG, Bolwig TG. Powerful inhibition of kainic acid seizures

by neuropeptide Y via Y5-like receptors. Nat Med

1997;3:761–4.

[180] Marsh DJ, Baraban SC, Hollopeter G, Palmiter RD. Role of

the Y5 neuropeptide Y receptor in limbic seizures. Proc Natl

Acad Sci U S A 1999;96:13518–23.

[181] Ho MW, Beck-Sickinger AG, Colmers WF. Neuropeptide

Y(5) receptors reduce synaptic excitation in proximal subic-

ulum, but not epileptiform activity in rat hippocampal slices.

J Neurophysiol 2000;83:723–34.

[182] Gariboldi M, Conti M, Cavaleri D, Samanin R, Vezzani A.

Anticonvulsant properties of BIBP3226, a non-peptide selec-

tive antagonist at neuropeptide Y Y1 receptors. Eur J Neuro-

sci 1998;10:757–9.

[183] Rusak B, Zucker I. Neural regulation of circadian rhythms.

Physiol Rev 1979;59:449–526.

[184] Ralph MR, Foster RG, Davis FC, Menaker M. Transplanted

suprachiasmatic nucleus determines circadian period. Sci-

ence 1990;247:975–8.

[185] Card JP, Moore RY. Neuropeptide Y localization in the rat

suprachiasmatic nucleus and periventricular hypothalamus.

Neurosci Lett 1988;88:241–6.

[186] Pelletier G. Ultrastructural localization of neuropeptide Y in

the hypothalamus. Ann NY Acad Sci 1990;611:232–46.

[187] Botchkina GI, Morin LP. Organization of permanent and

transient neuropeptide Y-immunoreactive neuron groups

and fiber systems in the developing hamster diencephalon.

Comp Neurol 1995;357:573–602.

[188] Moore E, Merali Z, Beninger RJ. Neuropeptide Y: intrastria-

tal injections produce contralateral circling that is blocked by

a dopamine antagonist in rats. Pharmacol Biochem Behav

1994;48:681–8.

[189] Harrington ME, Nance DM, Rusak B. Neuropeptide Y im-

munoreactivity in the hamster geniculo-suprachiasmatic

tract. Brain Res Bull 1985;15:465–72.

[190] Yannielli PC, Harrington ME. Neuropeptide Y in the mam-

malian circadian system: effects on light-induced circadian

responses. Peptides 2001;22:547–56.

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–2522

[191] Shibata S, Moore RY. Neuropeptide Y and optic chiasm sti-

mulation affect suprachiasmatic nucleus circadian function in

vitro. Brain Res 1993;615:95–100.

[192] Biello SM, Janik D, Mrosovsky N. Neuropeptide Y and

behaviorally induced phase shifts. Neuroscience 1994;62:

273–9.

[193] Huhman KL, Albers HE. Neuropeptide Y microinjected into

the suprachiasmatic region phase shifts circadian rhythms in

constant darkness. Peptides 1994;15:1475–8.

[194] Huhman KL, Gillespie CF, Marvel CL, Albers HE. Neuro-

peptide Y phase shifts circadian rhythms in vivo via a Y2

receptor. NeuroReport 1996;7:1249–52.

[195] Golombek DA, Biello SM, Rendon RA, Harrington ME.

Neuropeptide Y phase shifts the circadian clock in vitro via

a Y2 receptor. NeuroReport 1996;7:1315–9.

[196] Gribkoff VK, Pieschl RL, Wisialowski TA, van den Pol AN,

Yocca FD. Phase shifting of circadian rhythms and depres-

sion of neuronal activity in the rat suprachiasmatic nucleus

by neuropeptide Y: mediation by different receptor subtypes.

J Neurosci 1998;18:3014–22.

[197] Maywood ES, Mrosovsky N, Field MD, Hastings MH. Rap-

id down-regulation of mammalian period genes during be-

havioral resetting of the circadian clock. Proc Natl Acad Sci

U S A 1999;96:15211–6.

[198] Fukuhara C, Brewer JM, Dirden JC, Bittman EL, Tosini G,

Harrington ME. Neuropeptide Y rapidly reduces Period 1

and Period 2 mRNA levels in the hamster suprachiasmatic

nucleus. Neurosci Lett 2001;314:119–22.

[199] Flood JF, Hernandez EN, Morley JE. Modulation of memory

processing by neuropeptide Y. Brain Res 1987;421:280–90.

[200] Nakajima M, Inui A, Teranishi A, Miura M, Hirosue Y, Okita

M, et al. Effects of pancreatic polypeptide family peptides on

feeding and learning behavior in mice. J Pharmacol Exp Ther

1994;268:1010–4.

[201] Flood JF, Morley JE. Dissociation of the effects of neuro-

peptide Y on feeding and memory: evidence for pre- and

postsynaptic mediation. Peptides 1989;10:963–6.

[202] Thorsell A, Michalkiewicz M, Dumont Y, Quirion R, Caber-

lotto L, Rimondini R, et al. Behavioral insensitivity to re-

straint stress, absent fear suppression of behavior and im-

paired spatial learning in transgenic rats with hippocampal

neuropeptide Y overexpression. Proc Natl Acad Sci U S A

2000;97:12852–7.

[203] Munglani R, Hudspith MJ, Hunt SP. The therapeutic poten-

tial of neuropeptide Y. Analgesic, anxiolytic and antihyper-

tensive. Drugs 1996;52:371–89.

[204] Hua XY, Boublik JH, Spicer MA, Rivier JE, Brown MR,

YakshTL.The antinociceptive effects of spinally administered

neuropeptide Y in the rat: systematic studies on structure–

activity relationship. J Pharmacol Exp Ther 1991;258:243–8.

[205] Duggan AW, Hope PJ, Lang CW. Microinjection of neuro-

peptide Y into the superficial dorsal horn reduces stimulus-

evoked release of immunoreactive substance P in the anaes-

thetized cat. Neuroscience 1991;4:733–40.

[206] Tracey DJ, Romm MA, Yao NN. Peripheral hyperalgesia in

experimental neuropathy: exacerbation by neuropeptide Y.

Brain Res 1995;669:245–54.

[207] Naveilhan P, Hassani H, Lucas G, Blakeman KH, Hao JX,

Xu XJ, et al. Reduced antinociception and plasma extrava-

sation in mice lacking a neuropeptide Y receptor. Nature

2001;409:513–7.

[208] White DM. Intrathecal neuropeptide Y exacerbates nerve in-

jury-induced mechanical hyperalgesia. Brain Res 1997;750:

141–6.

[209] Mellado ML, Gibert-Rahola J, Chover AJ, Mico JA. Effect

on nociception of intracerebroventricular administration of

low doses of neuropeptide Y in mice. Life Sci 1996;58:

2409–14.

[210] Broqua P, Wettstein JG, Rocher MN, Gauthier-Martin B,

Riviere PJ, Junien JL, et al. Antinociceptive effects of neuro-

peptide Y and related peptides in mice. Brain Res 1996;724:

25–32.

[211] Zhang Y, Lundeberg T, Yu L. Involvement of neuropeptide Y

and Y1 receptor in antinociception in nucleus raphe magnus

of rats. Regul Pept 2000;95:109–13.

[212] Wang JZ, Lundeberg T, Yu L. Antinociceptive effects in-

duced by intra-periaqueductal grey administration of neuro-

peptide Y in rats. Brain Res 2000;859:361–3.

[213] Li Y, Li JJ, Yu LC. Anti-nociceptive effect of neuropeptide Y

in the nucleus accumbens of rats: an involvement of opioid

receptors in the effect. Brain Res 2002;940:69–78.

[214] Pich EM, Solfrini V, Marrama P, Tiengo M, Agnati LF,

Carani C. Centrally administered neuropeptide Y fails to

increase food intake but enhances hypoalgesia in spontane-

ously hypertensive rats. Neurosci Lett 1993;149:209–12.

[215] Brown CM, Coscina DV, Fletcher PJ. The rewarding proper-

ties of neuropeptide Y in perifornical hypothalamus vs. nu-

cleus accumbens. Peptides 2001;21:1279–87.

[216] Caberlotto L, Thorsell A, Rimondini R, Sommer W, Hyytia

P, Heilig M. Differential expression of NPY and its receptors

in alcohol-preferring AA and alcohol-avoiding ANA rats.

Alcohol, Clin Exp Res 2001;25:1564–9.

[217] Kinoshita H, Jessop DS, Finn DP, Coventry TL, Roberts DJ,

Ameno K, et al. Acute ethanol decreases NPY mRNA but

not POMCmRNA in the arcuate nucleus. NeuroReport 2000;

11:3517–9.

[218] Thiele TE, Marsh DJ, Ste Marie L, Bernstein IL, Palmiter

RD. Ethanol consumption and resistance are inversely related

to neuropeptide Y levels. Nature 1998;396:366–9.

[219] Kauhanen J, Karvonen MK, Pesonen U, Koulu M, Tuomai-

nen TP, Uusitupa MI, et al. Neuropeptide Y polymorphism

and alcohol consumption in middle-aged men. Am J Med

Genet 2000;93:117–21.

[220] Thiele TE, Miura GI, Marsh DJ, Bernstein IL, Palmiter RD.

Neurobiological responses to ethanol in mutant mice lacking

neuropeptide Y or the Y5 receptor. Pharmacol Biochem Be-

hav 2000;67:683–91.

[221] Thiele TE, Koh MT, Pedrazzini T. Voluntary alcohol con-

sumption is controlled via the neuropeptide Y Y1 receptor. J

Neurosci 2002;22(RC208):1–6.

[222] Kelley SP, Nannini MA, Bratt AM, Hodge CW. Neuropep-

tide-Y in the paraventricular nucleus increases ethanol self-

administration. Peptides 2001;22:515–22.

[223] Slawecki CJ, Betancourt M, Walpole T, Ehlers CL. Increases

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–25 23

in sucrose consumption, but not ethanol consumption, fol-

lowing ICV NPY administration. Pharmacol Biochem Behav

2000;66:591–4.

[224] Katner SN, Slawecki CJ, Ehlers CL. Neuropeptide Y admin-

istration into the third ventricle does not increase sucrose or

ethanol self-administration but does affect the cortical EEG

and increases food intake. Psychopharmacology 2002;160:

146–54.

[225] Badia-Elder NE, Stewart RB, Powrozek TA, Roy KF, Mur-

phy JM, Li TK. Effect of neuropeptide Y (NPY) on oral

ethanol intake in Wistar, alcohol-preferring (P), and -nonpre-

ferring (NP) rats. Alcohol, Clin Exp Res 2001;25:386–90.

[226] Kallio J, Pesonen U, Kaipio K, Karvonen MK, Jaakkola U,

Heinonen OJ, et al. Altered intracellular processing and re-

lease of neuropeptide Y due to leucine 7 to proline 7 poly-

morphism in the signal peptide of preproneuropeptide Y in

humans. FASEB J 2001;15:1242–4.

[227] Pages N, Orosco M, Fournier G, Rouch C, Hafi A, Gourch

A, et al. The effects of chronic administration of morphine on

the levels of brain and adrenal catecholamines and neuro-

peptide Y. Gen Pharmacol 1991;22:943–7.

[228] Pages N, Orosco M, Rouch C, Fournier G, Comoy E, Bo-

huon C. Brain and adrenal monoamines and neuropeptide Y

in codeine tolerant rats. Gen Pharmacol 1992;23:159–63.

[229] Holmes PV, Bartolomeis A, Koprivica V, Crawley JN. Lack

of effect of chronic morphine treatment and naloxone-pre-

cipitated withdrawal on tyrosine hydroxylase, galanin, and

neuropeptide Y mRNA levels in the rat locus coeruleus.

Synapse 1995;19:197–205.

[230] Woldbye DPD, Klemp K, Madsen TM. Neuropeptide Y at-

tenuates naloxone-precipitated morphine withdrawal via Y5-

like receptors. J Pharmacol Exp Ther 1998;284:633–6.

[231] Lundberg JM, Tatemoto K. Pancreatic polypeptide family

(APP, BPP, NPY and PYY) in relation to sympathetic vaso-

constriction resistant to alpha-adrenoceptor blockade. Acta

Physiol Scand 1982;116:393–402.

[232] Grouzmann E, Aubert JF, Waeber B, Brunner HR. A sensi-

tive and specific two-site, sandwich-amplified enzyme immu-

noassay for neuropeptide Y. Peptides 1992;13:1049–54.

[233] Malmstrom RE, Lundberg JM. Endogenous NPY acting on

the Y1 receptor accounts for the long-lasting part of the

sympathetic contraction in guinea-pig vena cava: evidence

using SR 120107A. Acta Physiol Scand 1995;155:329–30.

[234] Malmstrom RE, Lundberg JM. Neuropeptide Y accounts for

sympathetic vasoconstriction in guinea-pig vena cava: evi-

dence using BIBP 3226 and 3435. Eur J Pharmacol 1995;

294:661–8.

[235] Lundberg JM, Modin A. Inhibition of sympathetic vasocon-

striction in pigs in vivo by the neuropeptide Y-Y1 receptor

antagonist BIBP 3226. Br J Pharmacol 1995;116:2971–82.

[236] Han S, Chen X, Cox B, Yang CL, Wu YM, Naes L, et al.

Role of neuropeptide Y in cold stress-induced hypertension.

Peptides 1998;19:351–8.

[237] Wahlestedt C, Edvinsson L, Ekblad E, Hakanson R. Neuro-

peptide Y potentiates noradrenaline-evoked vasoconstriction:

mode of action. J Pharmacol Exp Ther 1985;234:735–41.

[238] Franco-Cereceda A, Liska J. Neuropeptide Y Y1 receptors in

vascular pharmacology. Eur J Pharmacol 1998;349:1–14.

[239] Schuerch LV, Linder LM, Grouzmann E, Haefeli WE. Hu-

man neuropeptide Y potentiates alpha1-adrenergic blood

pressure responses in vivo. Am J Physiol 1998;275:H760–6.

[240] Malmstrom RE. Vascular pharmacology of BIIE0246, the

first selective non-peptide neuropeptide Y Y2 receptor antag-

onist, in vivo. Br J Pharmacol 2001;133:1073–80.

[241] Evequoz D, Waeber B, Corder R, Nussberger J, Gaillard R,

Brunner HR. Markedly reduced blood pressure responsive-

ness in endotoxemic rats: reversal by neuropeptide Y. Life

Sci 1987;41:2573–80.

[242] Hauser GJ, Myers AK, Dayao EK, Zukowska-Grojec Z. Neu-

ropeptide Y infusion improves hemodynamics and survival in

rat endotoxic shock. Am J Physiol 1993;265:H1416–23.

[243] Watson JD, Sury MR, Corder R, Carson R, Bouloux PM,

Lowry PJ, et al. Plasma levels of neuropeptide tyrosine Y

(NPY) are increased in human sepsis but are unchanged

during canine endotoxin shock despite raised catecholamine

concentrations. J Endocrinol 1988;116:421–6.

[244] Arnalich F, Sanchez JF, Martinez M, Jimenez M, Lopez J,

Vazquez JJ, et al. Changes in plasma concentrations of vaso-

active neuropeptides in patients with sepsis and septic shock.

Life Sci 1995;56:75–81.

[245] Qureshi NU, Dayao EK, Shirali S, Zukowska-Grojec Z,

Hauser GJ. Endogenous neuropeptide Y mediates vasocon-

striction during endotoxic and hemorrhagic shock. Regul

Pept 1998;75–76:215–20.

[246] Takiyyuddin MA, Brown MR, Dinh TQ, Cervenka JH, Braun

RJ, Parmer RJ, et al. Sympatho-adrenal secretion in humans:

factors governing catecholamine and storage vesicle peptide

co-release. J Auton Pharmacol 1994;14:187–92.

[247] Gulbenkian S, Wharton J, Hacker GW, Varndell IM, Bloom

SR, Polak JM. Co-localization of neuropeptide tyrosine

(NPY) and its C-terminal flanking peptide (C-PON). Pepti-

des 1985;6:1237–43.

[248] Lundberg JM, Hokfelt T, Hemsen A, Theodorsson-Norheim

E, Pernow J, Hamberger B, et al. Neuropeptide Y-like im-

munoreactivity in adrenaline cells of adrenal medulla and in

tumors and plasma of pheochromocytoma patients. Regul

Pept 1986;13:169–82.

[249] Osamura RY, Tsutsumi Y, Yanaihara N, Imura H, Watanabe

K. Immunohistochemical studies for multiple peptide-im-

munoreactivities and co-localization of Met-enkephalin-

Arg6-Gly7-Leu8, neuropeptide Y and somatostatin in human

adrenal medulla and pheochromocytomas. Peptides 1987;8:

77–87.

[250] Higuchi H, Costa E, Yang HY. Neuropeptide Y inhibits the

nicotine-mediated release of catecholamines from bovine

adrenal chromaffin cells. J Pharmacol Exp Ther 1988;244:

468–74.

[251] Shimoda K, Shen GH, Pfeiffer RF, McComb RD, Yang HY.

Antiserum against neuropeptide Y enhances the nicotine-

mediated release of catecholamines from cultured rat adrenal

chromaffin cells. Neurochem Int 1993;23:71–7.

[252] Hexum TD, Russett LR. Stimulation of cholinergic receptor

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–2524

mediated secretion from the bovine adrenal medulla by neu-

ropeptide Y. Neuropeptides 1989;13:35–41.

[253] Zheng J, Zhang P, Hexum TD. Neuropeptide Y inhibits chro-

maffin cell nicotinic receptor-stimulated tyrosine hydroxy-

lase activity through a receptor-linked G protein-mediated

process. Mol Pharmacol 1997;52:1027–33.

[254] Zhang P, Zheng J, Vorce RL, Hexum TD. Identification of an

NPY-Y1 receptor subtype in bovine chromaffin cells. Regul

Pept 2000;87:9–13.

[255] Lobaugh LA, Blackshear PJ. Neuropeptide Y binding and

inhibition of cAMP accumulation in human neuroepithelioma

cells. Am J Physiol 1990;258:C913–22.

[256] Fabi F, Argiolas L, Ruvolo G, del Basso P. Neuropeptide Y-

induced potentiation of noradrenergic vasoconstriction in the

human saphenous vein: involvement of endothelium gener-

ated thromboxane. Br J Pharmacol 1998;124:101–10.

[257] Noll T, Hempel A, Piper HM. Neuropeptide Y reduces mac-

romolecule permeability of coronary endothelial monolayers.

Am J Physiol 1996;271:H1878–83.

[258] Ghersi G, Chen W, Lee EW, Zukowska Z. Critical role of

dipeptidyl peptidase IV in neuropeptide Y-mediated endothe-

lial cell migration in response to wounding. Peptides 2001;

22:453–8.

[259] Cervin A, Onnerfalt J, Edvinsson L, Grundemar L. Func-

tional effects of neuropeptide Y receptors on blood flow

and nitric oxide levels in the human nose. Am J Respir Crit

Care Med 1999;160:1724–8.

[260] Lacroix JS, Mosimann BL. Attenuation of allergen-evoked

nasal responses by local pretreatment with exogenous neuro-

peptide Y in atopic patients. J Allergy Clin Immunol 1996;

98:611–6.

[261] Baraniuk JN, Silver PB, Kaliner MA, Barnes PJ. Neuropep-

tide Y is a vasoconstrictor in human nasal mucosa. J Appl

Physiol 1992;73:1867–72.

[262] Lacroix JS, Ulman LG, Potter EK. Modulation by neuropep-

tide Y of parasympathetic nerve-evoked nasal vasodilatation

via Y2 prejunctional receptor. Br J Pharmacol 1994;113:

479–84.

A.P. Silva et al. / Clinica Chimica Acta 326 (2002) 3–25 25