Pressor and tachycardic responses to intrathecal administration of neuropeptide FF in anesthetized rats

Article abstract of DOI:10.1016/j.peptides.2009.11.003

Neuropeptide FF (NPFF) belongs to a neuropeptide family including two precursors (pro-NPFF_A and pro-NPFF_B) and two receptors (NPFF₁ and NPFF₂). NPFF and NPFF receptor mRNAs have been reported to be highly expressed and localized in the rat and human spinal cord. In the present study, the i.t. action of NPFF system on blood pressure and heart rate were examined using NPFF and two related agonists, NPVF and dNPA, which exhibit highest selectivities for NPFF₁ and NPFF₂ receptors, respectively. In urethane-anesthetized rats, NPFF and related peptides (5-40 nmol, i.t.) produced significant pressor and tachycardic responses at the spinal cord level. These effects were dose-dependent and similar with respect to time-course for the three peptides. Furthermore, i.t. injection of RF9 (20 nmol), a selective NPFF antagonist, significantly antagonized the cardiovascular responses to 20 nmol NPFF and related peptides (i.t.). Moreover, pretreatment of the rats with α-adrenoceptor antagonist phentolamine (1 mg/kg, i.v.) significantly reduced the pressor effects of NPFF. Nevertheless, pretreatment with muscarinic receptor and adrenoceptor antagonists (i.v.) could block the tachycardic effects induced by NPFF. Collectively, our results suggested that i.t. administration of NPFF and related peptides increased MAP and HR which were possibly mediated by the activation of both NPFF₁ and NPFF₂ receptors in the rat spinal cord. In addition, our results showed that the muscarinic receptor and adrenoceptor participated in the tachycardic response to i.t. NPFF, while α-adrenoceptor played an important role in the regulation of pressor effect of NPFF.

Full text of DOI:10.1016/j.peptides.2009.11.003

Peptides 31 (2010) 683–688
Contents lists available at ScienceDirect
Peptides
journal homepage: www.elsevier.com/locate/peptides
Pressor and tachycardic responses to intrathecal administration of neuropeptide
FF in anesthetized rats
a,1
a,1
a
a
a
a,b,
Quan Fang , Ning Li , Tian-nan Jiang , Qian Liu , Yu-lin Li , Rui Wang
*
a
Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Medicine, and Institute of Biochemistry and Molecular Biology, School of Life Sciences, and State Key
Laboratory of Applied Organic Chemistry, Lanzhou University, 222 Tian Shui South Road, Lanzhou, 730000, PR China
b
State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong,
China
A R T I C L E I N F O
A B S T R A C T
Article history:
Neuropeptide FF (NPFF) belongs to a neuropeptide family including two precursors (pro-NPFFA and pro-
Received 12 September 2009
Received in revised form 9 November 2009
Accepted 9 November 2009
Available online 14 November 2009
B 1 2
NPFF ) and two receptors (NPFF and NPFF ). NPFF and NPFF receptor mRNAs have been reported to be
highly expressed and localized in the rat and human spinal cord. In the present study, the i.t. action of
NPFF system on blood pressure and heart rate were examined using NPFF and two related agonists, NPVF
1 2
and dNPA, which exhibit highest selectivities for NPFF and NPFF receptors, respectively. In urethane-
anesthetized rats, NPFF and related peptides (5–40 nmol, i.t.) produced significant pressor and
tachycardic responses at the spinal cord level. These effects were dose-dependent and similar with
respect to time-course for the three peptides. Furthermore, i.t. injection of RF9 (20 nmol), a selective
NPFF antagonist, significantly antagonized the cardiovascular responses to 20 nmol NPFF and related
Keywords:
Neuropeptide FF (NPFF)
MAP (mean arterial pressure)
HR (heart rate)
i.t. (intrathecal)
Rat
peptides (i.t.). Moreover, pretreatment of the rats with a-adrenoceptor antagonist phentolamine (1 mg/
kg, i.v.) significantly reduced the pressor effects of NPFF. Nevertheless, pretreatment with muscarinic
receptor and adrenoceptor antagonists (i.v.) could block the tachycardic effects induced by NPFF.
Collectively, our results suggested that i.t. administration of NPFF and related peptides increased MAP
and HR which were possibly mediated by the activation of both NPFF
1 2
and NPFF receptors in the rat
spinal cord. In addition, our results showed that the muscarinic receptor and adrenoceptor participated
in the tachycardic response to i.t. NPFF, while
of pressor effect of NPFF.
a
-adrenoceptor played an important role in the regulation
ß 2009 Elsevier Inc. All rights reserved.
NPFF) and pro-NPFF
B
peptides (NPVF) are the preferred ligands
1
receptors, respectively. Since the isolation
1
. Introduction
for NPFF and NPFF
2
of NPFF in 1985, the biological functions suggested for this
neuropeptide including pain modulation, food intake, gastro-
intestinal and hormonal modulation, modulation of opiate
tolerance and abstinence and cardiovascular actions.
Immunoreactive NPFF has been found to be present in the
central nervous system including the spinal cord and the pituitary
gland [4,16,19,21,25]. The highest NPFF transcripts were also
detected in the spinal cord and medulla oblongata [28,32].
Additionally, the binding assays and autoradiographic studies
have revealed the presence of NPFF bindings sites in rat spinal cord
Neuropeptide FF (NPFF, FLFQPQRFamide) was originally iso-
lated from bovine brain through its cross-reaction with antibodies
to the molluscan cardioexcitory peptide FMRF-NH which
2
,
possessed the similar C-terminal sequence [31]. Recent reports
have shown that NPFF belongs to a neuropeptide family including
two precursors (pro-NPFF
coupled receptors (NPFF and NPFF
it has been suggested that the pro-NPFF
A
and pro-NPFF
) [6,7,13,18,22,28]. In addition,
peptides (NPFF and NPA-
B
) and two G-protein
1
2
A
[
1,2]. Subsequently, several studies have shown that NPFF
receptor mRNA is highly localized in superficial layer of dorsal
spinal cord while NPFF receptor is expressed abundantly in
2
Abbreviations: 1DMe, D.YL(N-Me)FQPQRFamide; HR, heart rate; MAP, mean arterial
pressure; dNPA, D.NP(N-Me)AFLFQPQRFamide; i.t., intrathecal; NPA-NPFF,
NPAFLFQPQRFamide; NTS, the nucleus tractus solitarius; NPVF, VPNLPQRFamide;
PAP, pulsatile arterial pressure.
1
human spinal cord [6,32]. Moreover, the pharmacological studies
have demonstrated involvement of spinal NPFF system in pain
processing [33]. A summary of findings reported to date indicated
that: NPFF induced analgesic and morphine modulating activities
at the spinal level [21,25]; the spinal NPFF system is up-regulated
*
Corresponding author at: Institute of Biochemistry and Molecular Biology,
School of Life Science, Lanzhou University, 222 Tian Shui South Road, Lanzhou,
7
30000, PR China. Tel.: +86 931 8912567; fax: +86 931 8912567.
E-mail address: wangrui@lzu.edu.cn (R. Wang).
1
Both authors contributed equally to this work.
0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2009.11.003

6
84
Q. Fang et al. / Peptides 31 (2010) 683–688
by peripheral inflammation in the rat [32]; i.t. infusion of NPFF
analogue 1DMe produced a long lasting increase in spinal outflow
of met-enkephalin-like immunoreactive materials in rats [5].
Taken together, these data strongly suggested that the spinal cord
should be one of the important sites to explore the bioactivities of
NPFF system.
2.3. Cardiovascular measurement
Experiments were performed, as described earlier [8,9]. Male
Wistar rats weighing 200–250 g (n = 53) were anesthetized with
urethane (l.2 g/kg, i.p.). Supplemental doses of urethane were
given as needed to maintain a uniform level of anesthesia. The
trachea was incised to get rid of mucus. The animals spontaneously
breathed room air. Polyethylene catheter was inserted into the left
external jugular vein for i.v. administration of drugs. The right
carotid artery was cannulated with polyethylene catheter and
connected to a PT100 pressure transducer with its output
In previous studies, both intracerebroventricular (i.c.v.) and
intravenous (i.v.) administration of NPFF produced increases in
blood pressure and heart rate in rats [3,14,24]. Additionally,
bilateral microinjection of NPFF into the commissural NTS caused
pressor and bradycardia effects [17]. It was notable that NPFF
exerted different modulatory roles in nociceptive activities at the
supraspinal and spinal level [21,25]. However, little if anything is
known about the cardiovascular effects of NPFF system at the
spinal level. Therefore, the present study was undertaken to
investigate the cardiovascular responses to NPFF system follow-
ing i.t. administration in urethane-anesthetized rats. Further-
more, selective agonists and antagonist for the NPFF receptors
were used in an attempt to characterize the NPFF receptors
subtype which mediates the spinal action of NPFF on the
cardiovascular system. In addition, the effects of muscarinic
receptor and adrenoreceptors in mediating cardiovascular
responses to NPFF were also examined.
connected to
a recorder system (model BL-420F, Taimeng
Technology Corporation of Chengdu, China). MAP and HR were
measured directly from the pre-calibrated BL-420F recorder
system. For i.t. administration, a third catheter (PE-10) was passed
through a slit in the dura at the atlanto-occipital junction and
positioned so that the inner tip lay at the selected level, at the T12-
L1 vertebral level as described by the previous studies [29,30]. The
animals were then allowed to recover for 30–40 min. The position
of the i.t. catheter was verified at necropsy after each experiment.
2.4. Administration of peptides
Fori.v. administration, drugswereinjectedin200-mlvolumeover
2
. Materials and methods
a period of 30 s in a random sequence. For i.t. administration, drugs
were injected according to the method of Yaksh and Rudy [30] and
2.1. Animals
wereadministeredintrathecallyoveraperiodof30 sin20-
mlvolume
of saline or drugs. The catheter was flushed with 5 l of saline. PAP,
m
Male Wistar rats were obtained from the Experimental Animal
MAP and HR were simultaneously measured with a pressure
transducerplugged intoa BL-420Frecordersystemand continuously
recorded throughout the experiments on a recorder system.
Center of Lanzhou University. All animals were cared for and
experiments were carried out in accordance with the European
Community guidelines for the use of experimental animals (86/
Incontrolexperiments, 20
mlofsalinereplaced the drugsolution.
6
09/EEC). All the protocols in this study were approved by the
To examine the cardiovascular responses of NPFF and related
peptides, after control experiments, increasing doses of NPFF (5, 10,
Ethics Committee of Lanzhou University, China.
20, and 40 nmol) were i.t. injected in the same animals in the
2
.2. Chemicals
experiments at 35-min intervals (n = 6). Similarly, dose–response
curves were obtained for the effect of 5, 10, 20, and 40 nmol of NPVF
(n = 6) and dNPA (n = 7) on cardiovascular responses.
NPFF, NPVF, dNPA and RF9 were synthesized on a solid support
following the previous report [11]. Peptides were prepared by
manual solid-phase synthesis using standard N-fluorenylmethox-
ycarbonyl (Fmoc) chemistry. Fmoc-protected amino acids (GL
Biochem (Shanghai) Ltd.) were coupled to a Rink Amide MBHA
resin (Tianjin Nankai Hecheng Science & Technology Co., Ltd, China).
The following schedule was employed: (1) DMF wash (3Â); (2) 20%
In experiments in which the effects of NPFF antagonist RF9 on
responses to NPFF and related peptides (n = 16) were investigated,
maximal changes in MAP and HR were compared before
administration of RF9 and 10 min after administration, in a dose
of 20 nmol, i.t. Furthermore, the roles of muscarinic receptor,
a-
and -adrenoreceptors in mediating cardiovascular responses to
b
a
piperidine/DMF (3Â, 4 min); (3) DMF wash (3Â); (4) N -Fmoc-
NPFF were also investigated. As to muscarinic receptor antagonist
atropine sulfate (2 mg/kg, i.v.), the effects of atropine (n = 6) on the
cardiovascular responses to NPFF were tested before administra-
Amino Acid (2.5 eq.)/HBTU (2.5 eq.)/HOBt (2.5 eq.)/DIPEA (5 eq.) in
DMF (1Â), 1 h; (5) DMF wash (3Â); (6) Kaiser Test. RF9 was obtained
after acylation of the N-terminus with1-adamantanecarboxylic acid
tion and 10 min after administration.
phentolamine hydrochloride (1 mg/kg, i.v.) and b
a
-Adrenoceptor antagonist
-adrenoceptor
(
3 eq.)/HBTU (3 eq.)/HOBt (3 eq.)/DIPEA (6 eq.) in DMF (1Â), 1 h. The
protected peptide-resin was treated with reagent K (TFA/H O/
2
antagonist propranolol hydrochloride (2 mg/kg, i.v.), as well as
atropine sulfate, were tested individually in six rats.
phenol/ethanedithiol/thioanisole, 82.5:5:5:2.5:5) for 2 h at room
temperature. Gelfiltration (Sephadex G-10) was performed todesalt
the crudepeptides. Thedesalted peptide was purified bypreparative
reversed-phase HPLC using a Waters Delta 600 system coupled to a
UV detector. Fractions containing the purified peptides were pooled
and lyophilized. The purity of the peptide was established by
analytical HPLC. The molecular weight of the peptide was confirmed
by an electrospray ionization mass spectrometer (Mariner ESI-TOF
MS, Applied Biosystems, CA).
2.5. Statistical analysis
Data were given as means Æ S.E.M. The cardiovascular responses
of the peptides alone were analyzed with one-way ANOVA followed
by the Dunnett’s post hoc test, and paired Student’s t test was used to
establish the differences between the groups. A probability level of
0.05 or less was considered as statistically significant.
In addition, propranolol hydrochloride and phentolamine
hydrochloride were obtained from Sigma Chemical Company
3. Results
(USA); atropine sulfate was obtained from Dongting Lake Drug
Factory of Hunan, China. All drugs were dissolved in sterilized
saline, and the solutions were divided into aliquots and stored in
3.1. Effects of i.t. administration of NPFF on MAP and HR
2
ml tubes at À20 8C. The aliquots were thawed and used on the
In the present study, the cardiovascular responses to i.t.
administration of NPFF on MAP and HR have been investigated
day of the experiment.

Q. Fang et al. / Peptides 31 (2010) 683–688
685
Fig. 1. Dose–response curves of i.t. NPFF and related peptides on MAP and HR in
anesthetized rats. (A) Dose–response curves of NPFF (5, 10, 20 and 40 nmol, i.t.,
n = 6), NPVF (5, 10, 20 and 40 nmol, i.t., n = 6) and dNPA (5, 10, 20 and 40 nmol, i.t.,
n = 7) on MAP; (B) dose–response curves of NPFF (5, 10, 20 and 40 nmol, i.t., n = 6),
NPVF (5, 10, 20 and 40 nmol, i.t., n = 6) and dNPA (5, 10, 20 and 40 nmol, i.t., n = 7) on
HR. Data points represent means Æ S.E.M. To establish statistical significance, the data
were statistically analyzed by one-way ANOVA followed by the Dunnett’s post hoc test
Fig. 2. Typical pressor responses to the maximum dose of NPFF (40 nmol, i.t., A),
NPVF (40 nmol, i.t., B) and dNPA (40 nmol, i.t., C). Top and bottom tracings represent
PAP and MAP, respectively. Arrows indicate time of injection.
1 2
agonists for NPFF and NPFF receptors, respectively, were also
(*P < 0.05, **P < 0.01, ***P < 0.001 compared with the vehicle control group).
measured [12,23,26]. The selectivity and the affinity to NPFF
receptors of NPFF and related peptides used in this study are
presented in Table 1 [12]. Similar to NPFF, both NPVF and dNPA
significantly caused pressor and tachycardic effects when injected
in doses of 5–40 nmol, i.t. (Fig. 1A and B). Interestingly, the dose–
response curves of NPFF and related peptides indicated that the
order of potency of these peptides to induce changes in MAP and
HR were NPFF = NPVF ꢀ dNPA and dNPA > NPVF ꢀ NPFF, respec-
tively (Fig. 1). Fig. 2B and C showed typical recordings of PAP and
MAP before and after injections of NPVF and dNPA. The pressor
responses to i.t. administration of NPFF and related peptides
(40 nmol) were similar with respect to the time-course (Fig. 2).
in anesthetized rats. As shown in Fig. 1A, i.t. injection of NPFF (5,
0, 20 and 40 nmol) induced dose-dependent increases in MAP and
HR. MAP increased 4.76 Æ 1.66, 12.12 Æ 0.97, 18.14 Æ 2.01,
4.56 Æ 4.68 mmHg from the baseline at the 5, 10, 20 and 40 nmol
dose, respectively, of NPFF (n = 6). In addition, HR increased
.79 Æ 5.69, 23.93 Æ 5.63, 25.68 Æ 2.29, 50.30 Æ 8.13 bpm from the
1
3
3
baseline in the same dose range. Fig. 2A displays the records from
experiments illustrating the time-course of effects of NPFF (40 nmol,
i.t.) on PAP and MAP. The peak of the pressor effects was reached at
approximately 1 min after i.t. injection of NPFF. The blood pressure
parameter returned to the pretreatment levels within 10 min.
Furthermore, control experiments with injections of the vehicle
control (saline, i.t.) had no consistent effects on baseline MAP and HR
in urethane-anesthetized rats (from 95.06 Æ 3.46 mmHg and
3.3. Effects of NPFF antagonist RF9 (i.t.) on the cardiovascular
responses to NPFF and related peptides (i.t.)
It is well known that the selective antagonists are required to
explore the mechanism of the varied pharmacological effects
induced by the G-protein coupled receptors. The effects of NPFF
receptors selective antagonist RF9 (20 nmol, i.t.) on the cardio-
vascular responses induced by NPFF and related peptides are
shown in Fig. 3 [27]. Baseline MAP has no significant changes when
compared before and after i.t. administration of RF9 (from
1.29 Æ 0.86 to 5.17 Æ 1.73 mmHg, n = 16, P > 0.05), but a small
increase in baseline HR was noted in several animals after i.t.
3
96.09 Æ 10.27 bpm to 94.87 Æ 3.90 mmHg and 395.56 Æ 9.95 bpm,
respectively; n = 6, P > 0.05).
3.2. Effects of i.t. administration of NPVF and dNPA on MAP and HR
In order to characterize the NPFF receptors subtype which
mediates the spinal action of NPFF on the cardiovascular system,
the cardiovascular responses to NPVF and dNPA, the selective

6
86
Q. Fang et al. / Peptides 31 (2010) 683–688
Table 1
Affinities (K
i
) of neuropeptide FF agonists (NPFF, NPVF and dNPA) and antagonist (RF9) on human NPFF
1
2
and NPFF receptors.
a
b
NPFF
1
K
i
(nM)
NPFF
2
K
i
(nM)
S
1/2
Reference
NPFF, FLFQPQRF-NH
NPVF, VPNLPQRF-NH
2
2.82 Æ 0.06
0.59 Æ 0.07
2.9 Æ 0.5
75 Æ 9
0.21 Æ 0.03
23.0 Æ 2.1
13.4
0.026
107.4
1.29
[12]
[12]
[12]
[27]
2
dNPA, D.NP(N-Me)AFLFQPQRF-NH
2
0.027 Æ 0.001
58 Æ 5
RF9,
Data are cited from the previous reports [12,27].
a
K
i
value are expressed as means Æ S.E.M.
b
S1/2 = K (NPFF )/K (NPFF ) for the selectivity index.
i
1
i
2
administration of RF9 in a dose of 20 nmol (from 1.26 Æ 1.59 to
of RF9 in a dose of 20 nmol, the increases in MAP and HR in
response to i.t. administration of NPFF and related peptides were
almost completely antagonized (Fig. 3).
1
0.21 Æ 3.23 bpm, n = 16, P < 0.05). The change in HR in response to
RF9 returned to pre-injection values within 3 min.
At a dose of 20 nmol, NPFF and related peptides significantly
induced cardiovascular responses. Subsequently, this dose was
selected for evaluating the effects of RF9 and other pharmacolo-
gical antagonists (atropine, phentolamine and propranolol) on the
cardiovascular responses evoked by NPFF. After i.t. administration
3.4. Effects of atropine (i.v.), phentolamine (i.v.) and propranolol (i.v.)
on the cardiovascular responses to NPFF (i.t.)
Furthermore, the roles of muscarinic receptor,
a- and b-
adrenoreceptors in mediating cardiovascular responses to NPFF
were also determined. i.v. administration of atropine induced a
significant decrease in the baseline MAP and a slight increase in
baseline HR (À22.10 Æ 3.41 mmHg versus 1.61 Æ 0.61 mmHg for
saline, P < 0.001; 5.81 Æ 7.74 bpm versus 0.22 Æ 2.71 bpm for saline,
P > 0.05, respectively; n = 6), which returned to pre-injection values
within 3 min. Phentolamine, an a-adrenoreceptor antagonist, at a dose
of 1 mg/kg caused a significant decrease in baseline MAP (À39.29 Æ
5
.53 mmHgversusÀ1.64 Æ 1.15 mmHgforsaline,P < 0.01;n = 6),buta
slight change in baseline HR (À6.62 Æ 10.83 bpm versus 2.02 Æ
.28 bpm for saline, P > 0.05; n = 6), which was consistent with the
1
previousstudies[29].Similartoourrecentreport[10],basalMAPandHR
were reduced considerably after i.v. administration of 2 mg/kg
propranolol (À25.43 Æ 3.85 mmHg versus À0.15 Æ 2.00 mmHg for
saline,P < 0.01;À72.84 Æ 10.59 bpmversus0.14 Æ 0.61 bpmforsaline,
P < 0.001, respectively; n = 6), which returned to the pretreatment
levels about 10 min after injection of this antagonist.
In our study, pretreatment of the rats with
a-adrenoceptor
antagonist phentolamine (1 mg/kg, i.v.) significantly reduced the
cardiovascular effects of NPFF (20 nmol, i.t.). Nevertheless,
pretreatment with muscarinic receptor antagonist atropine
(
2 mg/kg, i.v.) and
b-adrenoceptor antagonist propranolol
(
2 mg/kg, i.v.) could only antagonized the tachycardic effects
induced by NPFF (20 nmol, i.t.), but had no significant effects on
pressor responses to i.t. NPFF (Table 2).
Table 2
Changes in MAP and HR caused by i.t. injection of NPFF (20 nmol) before and after
i.v. administration of muscarinic and adrenergic receptor antagonists in rats.
Treatments
DMAP (mm Hg)
DHR (bpm)
NPFF (i.t.)
19.0 Æ 2.5 (n = 6)
13.6 Æ 1.3 (n = 6)
26.2 Æ 5.1 (n = 6)
a
Atropine (2 mg/kg, i.v.) +NPFF (i.t.)
7.7 Æ 1.2 (n = 6)
NPFF (i.t.)
18.1 Æ 2.5 (n = 6)
29.6 Æ 9.6 (n = 6)
Phentolamine
b
a
(1 mg/kg, i.v.) + NPFF (i.t.)
4.7 Æ 2.1 (n = 6)
7.3 Æ 5.5 (n = 6)
NPFF (i.t.)
20.0 Æ 2.0 (n = 6)
21.8 Æ 3.2 (n = 6)
21.9 Æ 6.4 (n = 6)
Fig. 3. Changes in MAP and HR caused by i.t. injection of NPFF and related peptides
before and after the NPFF receptors antagonist RF9 (i.t., 20 nmol) in anesthetized
rats. (A) NPFF, NPVF and dNPA (i.t., 20 nmol) in MAP. (B) NPFF, NPVF and dNPA (i.t.,
a
Propranolol (2 mg/kg, i.v.) + NPFF (i.t.)
2.4 Æ 1.4 (n = 6)
Data were expressed as means Æ S.E.M. from experiments conducted on 6 rats,
respectively. To establish statistical significance, the data were statistically analyzed by
paired Student’s t test.
2
0 nmol) in HR. Data were expressed as means Æ S.E.M. ‘‘n’’ indicates the number of
experiments. To establish statistical significance, the data were statistically analyzed
by paired Student’s t test (P < 0.05 and P < 0.01 indicating significant differences from
the actions of agonists alone).
a
P < 0.05.
b
P < 0.01 (compared with the vehicle control group).

Q. Fang et al. / Peptides 31 (2010) 683–688
687
4
. Discussion
deduction that the pressor and tachycardia responses to i.t.
administration of NPFF agonists are mediated by different
mechanisms. Moreover, our results showed that the muscarinic
receptor and adrenoceptor participated in the cardiovascular
effects induced by i.t. administration of NPFF.
In summary, the results of the present study indicated that i.t.
administration of NPFF and related peptides induced significant
increases in MAP and HR which were possibly mediated by the
Unlike other neuropeptides, the distribution of NPFF was
found to be localized within the nervous and neuroendocrine
systems with the highest levels in dorsal spinal cord and the
posterior pituitary [4,16,19,21,25,33]. Results of the present
study confirmed that i.t. administration of NPFF to the rat spinal
cord evoked dose-related increases in MAP and HR. In addition,
NPVF and dNPA, two NPFF agonists exhibiting different
activation of NPFF
results also showed that the muscarinic receptor and adrenoceptor
participated in the tachycardic response to i.t. NPFF, while a-
1 2
and NPFF receptors in the rat spinal cord. Our
1 2
selectivities towards NPFF and NPFF receptors, respectively,
caused pressor and tachycardic responses at the spinal level. The
cardiovascular effects induced by NPFF and related peptides
were almost completely antagonized by the NPFF receptors
selective antagonist RF9 (i.t.) [11,15,27], indicating that the
increases in MAP and HR caused by these three peptides might
be linked to specific activation of NPFF receptors. Moreover, the
results caused by RF9 could be explained by the activation of
NPFF receptors by NPFF agonists in the spinal cord, which seem
to be consistent with the fact that the mammalian spinal cord
adrenoceptor played an important role in the regulation of pressor
effect of NPFF. Collectively, these data might be helpful to further
deduce the mechanisms of NPFF system in cardiovascular
responses. In addition, this in vivo bioassay may be applied as a
parameter to characterize the potential NPFF agonists and
antagonists.
Acknowledgements
expresses abundant NPFF receptors, especially NPFF
1,2,6,32].
The previous studies using cells expressing NPFF receptors have
been revealed that both NPFF and NPFF receptors have high
affinities for NPFF [6,12]. To further explore the roles of these two
types NPFF receptors in cardiovascular regulation, NPVF and dNPA,
2
receptor
[
This study was supported by the grants from the National
Natural Science Foundation of China (Nos. 90813012, 20902041
and 20932003), the Specialized Research Fund for the Doctoral
Program in Higher Education Institutions (Nos. 20060730017 and
200807301028) of the Ministry of Education of China, and
Interdisciplinary Innovation Research Fund for Young Scholars,
Lanzhou University (LZUJC2007006).
1
2
two selective agonists for NPFF
were used. dNPA, a stable analogue of pro-NPFF
NPFF, has at least 100 times higher affinity for the NPFF
the NPFF receptor [12,26], whereas the pro-NPFF peptide NPVF
has 30 times more affinity for the NPFF than for the NPFF receptor
12,20]. Interestingly, in our study, both NPVF and dNPA dose-
1
and NPFF
2
receptors, respectively,
peptide NPA-
than for
A
2
1
B
References
1
2
[
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1 2
both NPFF and NPFF receptors of the rat spinal cord could
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