J. Biol. Macromol., 2(1), 3-10, 2002

Review

Pain transmission regulated by novel neuropeptides nocistatin andnociceptin/orphanin FQ
Emiko Okuda-Ashitaka1, Toshiaki Minami2 and Seiji Ito1
1

Department of Medical Chemistry, Kansai Medical University, Moriguchi 570-8506, Japan and2

Department of Anesthesiology, Osaka Medical College, Takatsuki 569-8686, Japan

Received December 30, 2001
A neuropeptide nociceptin or orphanin FQ (Noc/OFQ) was previously identified as an
endogenous ligand for the orphan opioid receptor-like receptor. Studies on the analysis of the
precursor of Noc/OFQ, we identified another novel neuropeptide and named it “nocistatin”.
Noc/OFQ is involved in a broad range of pharmacological actions in various tissues from thecentral nervous system to the periphery.

We found that intrathecal administration of Noc/OFQ

induced pain responses including touch-evoked pain (allodynia) and thermal hyperalgesia. Whileboth the allodynia and the hyperalgesia evoked by Noc/OFQ were transmitted bycapsaicin-sensitive primary afferent fibers and initially in the disinhibition of the inhibitory
glycinergic response, the allodynia was mediated by glutamate through the N-methyl-D-asparateate
receptor comprising the GluRε1 subunit and the thermal hyperalgesia was mediated by substance P.
Twobioactive

peptides,

Noc/OFQ

andnocistatin

exist

the

same

precursor

prepronociceptin/orphanin FQ. Simultaneous administration of nocistatin blocked the allodynia
and thermal hyperalgesia induced by Noc/OFQ, and anti-nocistatin antibody decreased thethreshold for the Noc/OFQ-induced allodynia.

The carboxy-terminal hexapeptide of nocistatin(Glu-Gln-Lys-Gln-Leu-Gln), which is conserved in bovine, human and murine species, possesses
the blocking activity. Nocistatin also attenuated the allodynia and thermal hyperalgesia evoked byprostaglandin E2 and the inflammatory pain induced by formalin or carrageenan/kaolin, andreversed morphine tolerance and the Noc/OFQ-induced inhibition of morphine analgesia.
Nocistatin and Noc/OFQ are novel bioactive peptides produced from the same precursor, and theyplay opposite roles in the regulation of pain transmission.

1, Introductionin the periphery.

In contrast to acute pain,

Under physiological conditions, nociceptive

pathophysiological conditions of chronic pain

signals evoked by noxious thermal, mechanical,

states, which are often associated with prolonged

and chemical stimuli are transmitted to the central

tissue damage or injury, give rise to hyperalgesia

nervous system (CNS) via activated nociceptors

and allodynia (2, 29). Hyperalgesia is defined asrunning title

4

an increased response to stimuli that are

Pharmacological studies have establishedphysiologically painful, whereas allodynia isthat there are at least three major types, µδ and κ

defined as pain due to innocuous stimuli that doof opioid receptors.

not

The

δ-opioid receptor was succeeded in 1992, andpathophysiology of pain involves a very complex

followed by the cloning of the µ and κ-opioid

interaction of many different peripheral andreceptors.

central structures. The dorsal horn of the spinal

cyclase, inactivate voltage-gated Ca2+ channels,

normally

provokepain.

cord is an important site for pain transmission,and it constitutes the first relay station forincoming somatosensory information.

PrimaryandMolecular cloning of the

These receptors inhibit adenylate

stimulateaninwardly

rectifying

K+conductance through a pertussis toxin-sensitive
G-protein (10, 25). cDNA cloning studies haveafferent nerve fibers including nociceptive Aδ, Aβ

revealed a novel G-protein-coupled receptor withand C-fibers terminate in spinal dorsal horn.

a high degree of amino acid sequence homology

Modification of synaptic strength between theses(approximately 60%) to the µ- δ-, and κ-opioid

afferents and second order neurons in spinal cord
is considered a cellular mechanism of some formsof hyperalgesia and allodynia.

In the spinal

dorsal horn, the excitatory neurotransmitters, e.g.
glutamate and substance P, released from theterminals induce the excitability of spinal neurons.
On the other hand, serotonergic and noradrenergic
descending pathways from the brainstem exert astronginhibitorytransmission.

effecton

dorsalhorn

Morphine and endogenous opioid

peptides such as endorphins, enkephalins, anddynorphins produce their analgesic effects bymodulating pain pathways through specificmembrane receptors. Using molecular screening
methods based on opioid receptor gene sequence,an orphan G protein-coupled receptor was
identified (12).

Furthermore, studies on the“orphan” receptor led to the discovery of a novel
neuropeptide nociceptin/orphanin FQ (Noc/OFQ)(11, 20, 24) and nocistatin (21).

This review

describes recent advances in studies on Noc/OFQ
and nocistatin, and particular attention will be
paid to pain transmission at the level of the spinal
dorsal horn.
2, Noc/OFQ and its receptor-

receptors.

This opioid receptor homologue,

designated as ORL1 in humans, as ROR-CandLC132 in rats, and as KOR-3 in mice, did notbind any of the known natural and synthetic
opioid ligands and remained an “orphan” (12).
A heptadecapeptide Noc/OFQ was isolated fromrat, porcine, and bovine brains as an endogenous
ligand for this “orphan” receptor on the basis ofits

inhibitionofforskolin-inducedcAMP

accumulation in cells expressing these receptors(11, 20, 24). Noc/OFQ is structurally similar todynorphin A, but structurally lacks the N-terminal
tyrosine essential for opioid peptides to activate
the receptor.
3, Pain transmission of Noc/OFQ
Noc/OFQ and its receptor are densely
expressed in the superficial dorsal horn of the
spinal cord. We found that the intrathecal (i.t.)
administration of Noc/OFQ was shown to induce
hyperalgesia to noxious stimuli by the hot plate
test (3) and allodynia to innocuous tactile stimuli(3, 20).

Noc/OFQ-induced allodynia wasblocked by glycine, an N-methly-D-asparateate(NMDA) receptor antagonist, a non-NMDA5

E. Okuda-Ashitaka et al.

receptor antagonist, and a soluble guanylate

Noc/OFQ involved in primary afferent C fibers

cyclasevia substance P release.

inhibitor.

Noc/OFQ-induced

hyperalgesia was inhibited by glycine only.

Noc/OFQ is reported to have different

Noc/OFQ-induced allodynia and hyperalgesia

effects including nociception, no effect, andmay involve a common neurochemical event

analgesia, depending on animal speicies tested,

beginning with the disinhibition of the inhibitory

doses, routes of administration, and so on.

glycinergic responses (3).

Noc/OFQ is also known to have many central

Activation of primary afferent C-fibers giveeffects including locomotion, neuroendocrine

rise to spinal release of substance P and glutamate,secretion, and memory and/or learning. Recent

and these mediators facilitate the cascade ofreviews provide details on central roles ofnociceptive

Noc/OFQ (8, 12).

processing.

We

designatedexperiments to elucidate the pathway andmediators of Noc/OFQ-evoked pain responses

4, Discovery of a novel bioactive peptide(14).

nocistatin

Neonatal capsaicin treatment eliminatedthe induction of hyperalgesia and allodynia by

Noc/OFQ is processed from its precursor

Whereas this treatment markedly

prepronociceptin/orphanin FQ (ppNoc/OFQ) (Fig.

reduced the content of substance P in the spinal1a), and its sequence is bounded by pairs of basic

cord, it did not affect the Noc/OFQ content or theamino acids Lys-Arg, a general cleavage site forexpression of Noc/OFQ receptors and GluRε andprecursor

maturation.

GluRζ subunits of NMDA receptor.

laboratories

have

Noc/OFQ.

The

We

and

severaldeterminedtheprimary

substance P antagonists CP96,345 and CP99,994

structures of bovine, human, mouse, and rat

blocked the Noc/OFQ-induced hyperalgesia, but

ppNoc/OFQ (6, 11, 19, 21, 23), and the amino

not allodynia. In contrast, the Noc/OFQ-evoked

acids sequence of ppNoc/OFQ are highly

allodynia, but not hyperalgesia, disapperared inconserved

NMDA receptor GluRε1 subunit knockout mice.

ppNoc/OFQ gene displays organizational and

Taken together, these results demonstrated thatstructural features that are very similar to thosecapsaicin-sensitive primary affenrent fibers are

encoding precursors of the endogenous opioid

involved not only in the thermal hyperalgesia but

peptides preproenkephalin, preprodynorphin, andalso in tactile allodynia induced by Noc/OFQ, but

preproopiomelanocortin, (15).

in different pathways; the former is mediated bypeptide precursors possess several bioactive

substance P and the latter is mediated bypeptides (Fig. 1a). The ppNoc/OFQ comprises

glutamateother putative cleavage sites.

throughthe

NMDA

receptoramong

species.

Further,the

These opioidThis prompted us

Inoue et al.

to investigate the possibility that the Noc/OFQ

showed that intraplanter injection of Noc/OFQ

precursor may comprise not only Noc/OFQ but

evoked behavioral responses in a flexor-reflex

also an additional bioactive peptide(s) as aparadigm (7).

maturation product(s).

comprising the GluRε1 subunit.

This response was suppressed byneonatal capsaicin pretreatment, NK1 receptorThebovineppNoc/OFQ

proteinantagonist, or in tachykinin gene knockout mice.

consists of 176 amino acid residues. It contains

Consistent with our results of hyperalgesia,

three additional potential cleavage sites, which

6

b

RRR
KK

COOH

K

M

M

M

L

M

bPNP-3 (Nocistatin)bPNP-2

KR

KR

KR

M

KR
KR

M

KR
KR

SP

COOH

COOH

KR
KR

NH 2

DynA DynB

KR
KK

NE

KK
KR

ppENK

KR

β -Endorphin

ACTH

SP

NH 2

KK

ppDYN

Noc/OFQ bPNP-4 bPNP-5

KR

KKRR

KR

γ -MHS

SP

KR

NH 2

KR

ppOMC

bPNP-3KR

bPNP-2

SP

RK

NH 2

RR

ppNoc/OFQ

KR

KR

a

KR

running title

COOH

Nociceptin

BovineKRMPRVRSLFQRQ----------- KRTEPGLEEVGEIEQKQLQ KRFGGFTGARKSARKLANQ KR

Human

RR*********E*-----------EEP***M**A**M****** KR***************** KR

Mouse

KR*******V*VRDAEPGADAEPGADA***ADDAE*V****** KR***************** KR

Rat

KR******VV*ARDAEPEA------DA**VAD*AD*V****** KR***************** KR

Figure 1 (a) Schematic representation of the structure of opioidpeptide precoursors. ppNoc/OFQ, preproNoc/OFQ; ppMOC,
preproopiomelanocortin; ppDYN, preprodynorphin; ppENK, preproenkephalin; SP, signal peptide; γ-MSH, γ- melanocyte
stimulating hormon; ACTH, adrenocorticotropic hormone; NE, neoendorphin; DynA, dynorphin A; DynB, dynorphin B; M,
Met-enkepharin; L, Leu-enkepharin. bPNP-2, bPNP-3, bPNP-4 and bPNP-5 are putative peptides produced from bovine
ppNoc/OFQ. (b) Alignment of deduced amino acid sequences among bovine, human, mouse, and rat nocistatin and Noc/OFQ.
The conserved amino acids are shaded, and the putative proteolytic cleavage motifs are boxed.

woulddelineatefourprocessing

products

Noc/OFQ-induced allodynia (Fig. 2c).

The

(bPNP-2, bPNP-3, bPNP-4 and bPNP-5), as

dose-dependency of Noc/OFQ-induced allodynia

shown in Fig. 1a.

in

To clarify whether thesemice

without

pretreatment

showedaputative peptides produced from ppNoc/OFQ arebell-shaped pattern in the dose-range from 1 tobiologically active in vivo, we synthesized them

100 pg.

chemically and investigated i.t. injection of them

the left by 2.5 orders at the half-maximal effective

effcts on pain responses (Fig. 2a).

Althoughdose by i.t. pretreatment of mice with thebPNP-3 (500 pg/mouse) did not induce allodynia

antibody to nocistatin, but not with normal IgG.

by itself, the allodynia caused by i.t. injection ofThis result strongly suggests that the endogenous

Noc/OFQmPNP-3-immunoreactive peptide, possibly mouse(50

pg/mouse)

blocked by bPNP-3.

wassignificantly

The i.t. administration of

The dose-response curve was shifted tonocistatin,

plays

an

inhibitory

role

in

bPNP-3 (500 pg/mouse) also attenuated the

Noc/OFQ-evoked allodynia in the spinal cord.

hyperalgesia evoked by Noc/OFQ (50 pg/mouse)Inin the hot plate test (Fig, 2b). So, we termed theimmunoreactivities were most abundant in thebPNP-3 “nocistatin”, after its apparent analgesic

superficial laminae of mouse spinal dorsal horneffects on nociception induced by Noc/OFQ.

(21).

fact,

both

Noc/OFQ

andnocistatin

Furthermore, in order to examine whether
the endogenous nocistatin participates in paintransmission in the spinal cord, we examined theeffectofpretreatment

withanti-nocistatin

antibody (antibody against mPNP-3) on the-5, Structure-activity relationship of nocistatin
Althoughbovinenocistatin(Thr1-Glu-Pro-Gly-Leu-Glu-Glu-Val-Gly-Glu-Ile-Glu-Gln-Lys-

7

E. Okuda-Ashitaka et al.

% Max Possible Cumulativea

Allodynia (% of control)

b

125

c

Nociceptin

100

100
Score

bPNP-275

bPNP-3*50**

bPNP-4
**

**
**

0
13

12

50

bPNP-5

25

14

75

**

11

10

25

Saline90-log[i.t. Agent (g)]

10

0

20

15

Response Time (s)

14

13

12

11

10

9

8

-log[i.t. Noc/OFQ (g)]Figure 2 Effects of putative peptides deduced from bovine ppNoc/OFQ on allodynia (a) and hyperalgesia (b). Noc/OFQ (a, 50
pg/mouse) was simultaneously injected i.t. with various doses of bPNP-2 (z), bPNP-3 ( ), bPNP-4 (∆) or bPNP-5 ( ). b,
Noc/OFQ (50 pg) was injected without or with 500 pg of the indicated peptide. Each point represents the mean ± s.e.m. (n=6-10).
*p<0.05, **p<0.01, versus nociceptin-injected group. c, Effect of i.t. pretreatment with anti-nocistatin antibody on Noc/OFQ–induced allodynia. Mice were not pretreated (z) or pretreated i.t. with 10 µg of anti-mPNP-3 IgG ( ) or 10 µg of normal rabbit
IgG (∆) 1 h before i.t. injection of various doses of nociceptin. The allodynic score (mean ± s.e.m., n=6) is expressed as a percentof the maximum possible cumulative score over a 50-min period. Reprinted from Peptide, 21, Okuda-Ashitaka E. and Ito S.,
Nocistatin:a novel neuropeptide encoded by the gene for the nociceptin/orphanin FQ precursor., 1101-1109, 2000, with permission
from Elsevier Science.

Gln-Leu-Gln17) is flanked by pairs of Lys-Arg at

parts of the Noc/OFQ precursors showed similar

positions 109-110 and 128-129, the human, rat,

biological activities beyond the species and thatand mouse precursors are devoid of the former

thecleavage site, resulting in processed products ofimportant for exhibiting the activity. Although

30, 35, and 41 amino acid residues, respectively

the structural and conformational constraint for(Fig. 1b). While this appears to delineate amino

Noc/OFQ activation is most stringent in theacid sequences of variable length and of low

amino-terminal

conservation among species, the variation of

Phe-Gly-Gly-Phe-Thr) of the Noc/OFQ molecule,length among peptides is due to the repetition of athe carboxyl-terminal part (positions 12-17,

six-amino acid motif, Asp-Ala-Glu-Pro-Gly-Ala,

Glu-Gln-Lys-Gln-Leu-Gln) appears to be theand its divergent form: three times in mouse,minimal essential

twice in rat, and once in human, and thenocistatin sequence.

carboxyl-terminal

consensus

part

portion(positions

is

1-5,

core present within the

carboxyl-terminal half of these peptides is fairly
conserved among species. We identified mature

6, Pain transmission of nocistatin

forms of NST in bovine, human, rat, and mouse

Allodynia

brains (9).

All of rat, mouse, and humanputative

peptides

Noc/OFQ-induced

suppressed

allodynia.

pentapeptides

theAlthough

The

i.t.

administrationof

attenuated the allodynia caused by i.t. injection of
Noc/OFQ.

Furthermore, i.t. administration of

Glu-Gln-Lys-Gln-Leu

prostaglandin (PG) E2 induces allodynia in mice

(PNP-3-6P∆C)

andGln-Lys-Gln-Leu-Gln(13,(PNP-3-6P∆N)

did

not

PGE2-evoked allodynia in mice (21).

affectthe

Noc/OFQ-induced allodynia at 500 pg/mouse, ahexapeptide

nocistatin

Glu-Gln-Lys-Gln-Leu-Gln

27).

Nocistatin

also

blockedtheThermal hyperalgesia
The

i.t.

administrationofnocistatin(PNP-3-6P), which is 100 % conserved among

attenuated the thermaol hyperalgesia caused by i.t.

species inhibited the Noc/OFQ-induced allodynia.

injection of Noc/OFQ and PGE2 (21).

These results demonstrate that these processed

Inflammatory hyperalgesia

running title

8

The i.c.v. injection of nocistatin (0.05-500

The formalin test is widely used as a model
Subcutaneous (s.c.)ng/rat) also reversed the anti-morphine analgesia

injection of formalin produces biphasic painof Noc/OFQ (8 µg/rat) in the rat tail-flick test, butbehaviors: a first transient phase (0-5 min)nocistatin did not affect basal tail-flick latency orascribed to the direct effect of formalin on

morphine analgesia (32). Furthermore, the i.c.v.

nociceptors and a second prolonged phase (10-30

administration of Noc/OFQ antibody reversedmin) related to the development of inflammation

morphine tolerance, and the release of Noc/OFQ

and central sensitization.

was increased during morphine tolerance.

of clinical pain states.

The i.t. administration

Bothof nocistatin significantly inhibited the first phasechronic and acute morphine tolerance wereand second phase of 2% formalin-evoked pain

reversed by the i.c.v. injection of nocistatin (26).

behaviors at 1 pg and at 10-1000 pg, respectively,

Glutamate releasein mice (17). Consistent with the results in mice,

The inhibition of K+-evoked glutamate

i.t. administration of nocistatin (3-30 µg/rat)

release by Noc/OFQ was fully reversed byattenuated the 5% formalin-induced flinchingnocistatin in rat brain slices, while nocistatin had

behavior in the first phase (29). On the otherno effect on the K+-evoked glutamate release

hand, i.t. Noc/OFQ at high doses of 0.3-3µg/mouse significantly inhibited both phases ofalone (18).

In vivo, the i.c.v. administration of

Noc/OFQ shortened the step-down latency and

2% formalin-induced pain, but it aggravated the

impaired spontaneous alternation in the Y-maze

1% formalin-induced second phase at low dose of

test in mice. Although there was no significant

10 pg/mouse.

effectNoc/OFQ

The latter aggravating effect ofwascompletelyofnociatatinbyitself,nocistatin

blocked by i.t.

counteracted the impairment of learning andadministration of 10 pg nocistatin, but the former

memory induced by Noc/OFQ (4). Furthermore,

inhibitory effect of 1 µg/mouse Noc/OFQ was not

the impairment of learning and memory induced

reversed by 1 µg nocistatin in mice.

by scopolamine, a muscarinic cholinergic receptor

In thisconnection, administration of nocistatin (30g/rat) failed to block the analgesic effect ofantagonist,wasreversedbyadministration of nocistatin (5).

thei.c.v.

Nocistatin is

Noc/OFQ (30 µg/rat) in the rat formalin test (30),

involved in not only pain transmission but alsosuggesting that the action mechanisms oflearning and memory.

Noc/OFQ at low and high doses are different (8,22).

7, Cellular mechanisms of nocistatin andAlthoughthei.c.v.

administrationofnocistatin induced neither hyperalgesia nor

Noc/OFQ
Althoughnocistatin

inhibitedtheanalgesia by itself, nocistatin (0.5-50 pmol/rat)

pharmacological functions induced by Noc/OFQ,

dose-dependently

nocistatin

reducedtheinflammatory

neither

hyperalgesia induced by hindlimb intraplantar

binding

injection

forskolin-inducedofcarrageenan/kaolin

in

the

rat

nor

displacedattenuated
cAMP

the

[3H]Noc/OFQ
inhibitionofaccumulationbypaw-pressure test (16).

Noc/OFQ in cells transfected with the receptor.

Morphine analgesia and tolerance

At the level of the spinal cord dorsal horn neuron,

9

E. Okuda-Ashitaka et al.

nocistatin

selectivity

reduces

inhibitory

glycinergic and GABAergic synaptic transmission

Pharmaceutical

Research,

and

the

UeharaMemorial Foundation.

but leaves excitatory glutamatergic transmission
unaffected, whereas Noc/OFQ only interferes

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Communicated by Hiroshi Ueno