Posprint of: Pharmacological Research Volume 63, Issue 5, May 2011, Pages 414–422
Smooth muscle neurokinin-2 receptors mediate contraction in human saphenous veins
Hakima Mechiche (a), Stanislas Grassin-Delyle (a), Francisco M. Pinto (b), Amparo Buenestado
(a), Luz Candenas (b), Philippe Devillier (a)
(a) Laboratory of Pulmonary Pharmacology, UPRES EA 220, Faculté de Médecine Île-de-France
Ouest, University Versailles Saint Quentin, 11 rue Guillaume Lenoir, 92150 Suresnes, France
(b) Instituto de Investigaciones Quimicas, CSIC, Avda. Americo Vespucio 49, 41092 Sevilla,
Spain
Abstract
Substance P (SP) and neurokinin A (NKA) are members of the tachykinin peptides family. SP
causes endothelial-dependant relaxation but the contractile response to tachykinins in human
vessels remains unknown. The objective was to assess the expression and the contractile
effects of tachykinins and their receptors in human saphenous veins (SV). Tachykinin
expression was assessed with RT-PCR, tachykinin receptors expression with RT-PCR and
immunohistochemistry, and functional studies were performed in organ bath. Transcripts of all
tachykinin and tachykinin receptor genes were found in SV. NK1-receptors were localized in
both endothelial and smooth muscle layers of undistended SV, whereas they were only found
in smooth muscle layers of varicose SV. The expression of NK2- and NK3-receptors was limited
to the smooth muscle in both preparations. NKA induced concentration-dependent
contractions in about half the varicose SV. Maximum effect reached 27.6 ± 5.5% of 90 mM KCl
and the pD2 value was 7.3 ± 0.2. NKA also induced the contraction of undistended veins from
bypass and did not cause the relaxation of these vessels after precontraction. The NK2receptor antagonist SR48968 abolished the contraction induced by NKA, and a rapid
desensitization of the NK2-receptor was observed. In varicose SV, the agonists specific to NK1or NK3-receptors did not cause either contraction or relaxation. The stimulation of smooth
muscle NK2-receptors can induce the contraction of human SV. As SV is richly innervated,
tachykinins may participate in the regulation of the tone in this portion of the low pressure
vascular system.
Keywords
Tachykinin receptors; Vascular smooth muscle; Contraction; Saphenous veins
1. Introduction
Neurokinin A (NKA) belongs to a structurally related peptide family named tachykinins, which
also includes substance P (SP), neurokinin B (NKB), and hemokinin-1 (HK-1). Three genes
encode for the members of this family: TAC1 encodes for SP and NKA through alternative
1
splicing, TAC3 encodes for NKB, and TAC4 encodes for HK-1. Their biological effects are
mediated through three specific G-protein coupled receptors: NK1-, NK2- and NK3-receptors,
which are encoded by TACR1, TACR2 and TACR3, respectively. SP and HK-1 are preferential
agonists for NK1-receptors, NKA for NK2-receptors and NKB for NK3-receptors. These peptides
may undergo enzymatic degradation by neutral endopeptidase (NEP), which is encoded by the
MME gene. Tachykinins are mainly localized in the central nervous system, but they are also
distributed in the sensory nerves (mainly in the afferent C-fibers) and are widely distributed
within the mammalian peripheral tissues [1]. Sensory C-fibers have already been involved in
vascular tone regulation, by acting on tachykinin release [2]. Most of the studies on vascular
tissues have focused on SP-induced relaxation. Indeed, SP causes the relaxation of numerous
human vascular preparations: omental arteries and veins [3], gastroepiploic arteries [4],
mesenteric arteries and veins [5], internal mammary arteries [6], coronary arteries and veins
[7], [8] and [9], umbilical artery [10], pulmonary arteries and veins [11] and [12], and penile
deep dorsal vein [13]. The relaxant effect obtained with SP was endothelium dependent [4],
[6], [7], [10], [13] and [14], and involved NK1-receptors [11] and [12], as confirmed with the
specific NK1-receptor agonist [Met-OMe11]SP which induced vasorelaxation in human
pulmonary arteries [15]. Only one human study showed a weak contraction of the internal
thoracic artery in response to high concentrations of SP [6], whereas a few animal studies
showed a contraction of the rabbit pulmonary artery [16], the rat gastric vasculature [17] and
[18], or the canine cerebral arteries [19] in response to NKA. NKA has been shown to induce a
contraction of several human smooth muscles, including bronchus [20], uterus [21], and colon
[22], but the contractile effects of NKA in human vessels have not been explored yet. The
human great saphenous vein (SV) is richly innervated with the presence of SP-immunoreactive
nerves [23]. As NKA is a product of the same gene as SP (TAC1), the human saphenous vein
provides a good basis for further in depth studies on neurovascular regulation [23].
The aim of the present study was to investigate the expression and the function of tachykinins
and NEP, as well as the location and expression of the different tachykinin receptors in human
saphenous veins. We showed for the first time the presence of tachykinin transcripts, together
with tachykinin receptor transcripts and proteins in human saphenous veins, and that
neurokinin A induces vascular contractions of human saphenous veins by stimulating smooth
muscle NK2-receptor. We also provide evidences of NKA-induced NK2-receptor
desensitization, and a pharmacological characterization of the pathways involved in the
response showed a role for L-type voltage-operated calcium channels.
2. Methods
2.1. Tissues
Ring segments of human saphenous veins were obtained from 40 patients with primary
varicosity, 13 males (age range 33–77) and 27 females (age range 26–65), undergoing
stripping. In addition, undistended saphenous vein segments were obtained from 10 patients
(age range 52–66) undergoing arterial reconstruction. The study was approved by the local
ethics committee and the subjects gave informed consent.
2
Immediately after surgical removal, the blood vessels were quickly dissected free of connective
tissue and placed either in cooled (4 °C) Krebs-Henseleit solution for organ bath studies or
frozen in liquid nitrogen, and stored at −80 °C for immunochemistry. For reverse transcriptionpolymerase chain reaction (RT-PCR), intact tissue pieces obtained from different patients were
immediately submerged in RNAlater (Ambion, Huntingdon, UK), and then stored at −80 °C.
Segments of saphenous veins were chosen for subsequent RT-PCR analysis on the basis of their
response to NKA in functional studies.
2.2. Reverse transcription-polymerase chain reaction (RT-PCR)
RNA extraction, reverse transcription and PCR were performed as previously described [12].
2.2.1. RNA extraction and reverse transcription
Total RNA from human saphenous veins was extracted using the acid guanidium
isothiocyanate–phenol–chloroform extraction method [24]. The RNA samples were treated
with FPLC pure DNase I (Amersham Biosciences, Essex, UK) in DNase buffer (40 mmol/L Tris–
HCl, pH 7.5, 6 mmol/L MgCl2) containing 10 units of RNasin (Promega Corp., Madison, USA) to
eliminate contaminating genomic DNA. The integrity of the purified RNA was confirmed by
visualizing ribosomal RNA bands after the electrophoresis of RNA through a 1% agaroseformaldehyde gel. The quantity of total RNA was determined by spectrophotometric
measurement at 260 nm. RNA samples (10 μg each) were stored at −80 °C until use. Total RNA
(5 μg) was reverse transcribed using a first-strand cDNA synthesis kit (Amersham Biosciences).
2.2.2. PCR primers
The sequences of the primers used to amplify the genes that encode human SP/NKA, NKB and
HK-1 (TAC1, TAC3 and TAC4, respectively); the genes that encode human tachykinin receptors
(TACR1, TACR2 and TACR3), neutral endopeptidase (MME) or β-actin, the size of the expected
fragments and appropriate references are shown in Table 1.
The primer sets used to amplify TAC1, TAC3 or TAC4 were designed against a sequence
common to all mRNA isoforms. Two different isoforms have been described for NK1 and NK2
receptors [25] and [26]. The primer pair designed to analyse the expression of TACR1 allows
the amplification of both short and long isoforms. Two different primer pairs were designed to
analyse the expression of the two known splice variants of the tachykinin NK2 receptor. The
first set enables the simultaneous visualization of both α and the truncated TACR2 β isoforms
while the second one allows the amplification of a sequence only present in the long TACR2 α
isoform. A dual-labelled probe (FAM-CCATCGTCCACCCCTTCCAGCC-Tamra) was also designed
to specifically detect α TACR2. All primers and the probe were synthesized by Sigma–Aldrich
(London, UK).
2.2.3. Endpoint PCR
An endpoint PCR assay was used to detect the mRNAs of tachykinin, their receptors and NEP,
and to establish the identity of the amplified products. Amplification of the human β-actin
gene transcript was used to control the efficiency of RT-PCR among the samples. An aliquot of
the resulting cDNA (corresponding to 100 ng of total RNA) was used as a template for PCR
3
amplification, using a DNA thermal cycler (MJ Research, Watertown, USA). Each reaction
contained 0.2 μmol/L primers, 1.5 U of Taq polymerase (Amersham Biosciences), the buffer
supplied, 2.5 mmol/L MgCl2, 200 μmol/L dNTP's and cDNA in 25 μL. After a hot start (2 min at
94 °C), the parameters used for PCR were 10 s at 94 °C, 20 s at 60 °C, 30 s at 72 °C. Cycle
numbers were 35 for tachykinin and their receptors, and 24 for β-actin. PCR products were
separated by agarose gel electrophoresis, stained with ethidium bromide and visualized under
UV transiluminator (Spectronics Corp., New York, USA). mRNA expression for tachykinin, the
three tachykinin receptors, NEP and β-actin was analysed on each tissue and the identity of
each PCR product was established by DNA sequence analysis, as previously described [27]. No
PCR product was detectable when the samples were amplified without the RT step, suggesting
that there was no genomic DNA contamination. Similarly, no products were detected when the
RT-PCR steps were carried out with no added RNA, indicating that all reagents were free of
target sequence contamination.
2.2.4. Real-time PCR
Real-time PCR was used to quantify the expression of TAC1, TAC3, TAC4, TACR1, TACR2, TACR3
and MME, using the iCycler iQ real-time detection system (Bio-Rad, CA, USA) and SYBR green
(Molecular Probes, Leiden, The Netherlands). β-actin was used as endogenous control for
variations in cDNA amounts. The PCR reaction mixture was identical to the one used in the
endpoint PCR assay, adding SYBR green I (1:75,000 dilution of the 10,000× stock solution) and
fluorescein (1:100,000 dilution) used as a reference dye for the normalization of the reactions.
Thermal cycling conditions were the same as those described for endpoint assays. Following
the final cycle of the PCR, the reactions were subjected to a heat dissociation protocol.
2.3. Immunohistochemistry
Cryostat sections (5 μm) of saphenous vein segments were immunostained with antibodies
against NK1, NK2 or NK3 receptors through the streptavidin–biotin-complex/peroxydase
method. The slides were fixed for 10 min with fresh aceton at room temperature. After
rehydrating the slides in phosphate buffered saline for 5 min, non specific binding was
eliminated by incubating the slides for 10 min in blocked serum (Clinisciences, Trappes,
France). The slides were then incubated overnight at 4 °C with the primary antibody raised
against NK1- (Sigma, St. Quentin Fallavier, France), NK2- (antibody kindly provided by Dr. P.
Geppetti, University of Ferrara, Italy) or NK3- (Calbiochem, Nottingham, UK) receptors.
Negative controls were produced by substituting the primary antibody with phosphate
buffered saline. After washing in phosphate buffered saline, the slides were incubated for 30
min with multilink biotinylated anti-IgG (Biogenex, Chevilly Larue, France). All slides were then
washed and incubated for 30 min with streptavidin–biotin complex reagent (Biogenex, Chevilly
Larue, France). Immunoreactivity was visualized with amino-3-ethyl-9-carbazol (AEC). Slides
were dehydrated and mounted in a hydrophobic mounting medium (Glycergel, Dako,
Montrouge, France).
2.4. Functional experiments
Functional experiments were performed essentially as previously described [12]. Saphenous
veins were cut into segments, about 4–5 mm long, and suspended in a 10 mL-organ bath
4
containing Krebs solution (composition in mmol/L: NaCl 118, KCl 5.4, CaCl2, 2.5, MgSO4 0.6,
KH2PO4 1.2, NaHCO3 25.0 and glucose 11.7, pH 7.4), continuously gassed with 5% CO2 in O2
and maintained at 37 °C. They were suspended on wires; the lower wire was fixed to a
micrometer (Mitutoyo, Japan) and the upper wire was attached to an isometric force
displacement transducer (UF-1, Pioden). Changes in force were recorded on two-channel
recorders (Linseis E200, Polylabo, France). At the beginning of the experiments, rings were
stretched to an initial tension of 2.5 g and left to equilibrate for an hour in the bath medium
which was changed every 15 min. The saphenous vein rings were then challenged twice with
90 mmol/L KCl to stabilize the preparations. The preparations were left to equilibrate again for
an hour, with the Krebs solution changed every 15 min. Concentration–response curves were
generated for [Sar9Met(O2)11]SP, NKA, [Nle10]NKA(4-10) or [MePhe7]NKB. The concentration
of the different tachykinin agonists was increased by 0.5 log-increments, each concentration
being added when the maximal effect had been produced by the previous concentration, or
every 5 min when no response occurred. The effect of a NK1- or a NK2-receptor selective
antagonist (SR140333 or SR48968 respectively, 0.01 μmol/L each) was examined by adding the
compounds to the tissue bath 40 min before the addition of TK receptor agonists [28]. In some
experiments, stimulation with TK receptor agonists was performed in preparations contracted
with phenylephrine (30 μmol/L).
Endothelial dependence of NKA-induced contraction was assessed in experiments in which the
endothelium of one of the pairs of adjacent saphenous vein rings was removed. Endothelium
was mechanically removed by inserting a smooth-edged arm of a dissecting forceps into the
lumen of the vessel ring and gently rolling the moistened preparation between the surface of a
forefinger and the forceps for about 10 s without undue stretch. The second ring of the pair, in
which the endothelium was left intact, served as control. Cumulative concentration–response
curves for NKA were generated in endothelial-denuded and -intact preparations as detailed
above. The removal of endothelium was confirmed by the loss of the relaxation response to
acetylcholine (100 μmol/L) in phenylephrine-contracted rings assessed at the end of the
experimental protocol.
The involvement of nitric oxide and prostanoids in the vascular contraction produced by NKA
in human saphenous veins was tested by examining the effect of the nitric oxide synthase
inhibitor (NG-nitro-l-arginine, l-NOARG, 100 μmol/L) and of the cyclo-oxygenase inhibitor
(indomethacin, 10 μmol/L) on the contraction response to this agonist. At 40 min, cumulative
concentration–response curves were generated for the agonist as described above. The effect
of a pretreatment with the inhibitor of receptor-mediated calcium entry SKF96365 (30
μmol/L), the voltage-dependant calcium channel blocker nicardipine (3 μmol/L) or the inhibitor
of p38 mitogen-activated protein kinase (MAPK) SB203580 (10 μmol/L) on NKA-induced
response was also assessed. Paired control tissues received vehicles. In all experimental
protocols, only one cumulative concentration–effect curve was obtained for each vascular ring,
excepted where otherwise stated (tachyphylaxis studies).
2.5. Drugs and solutions for functional studies
[Sar9Met(O2)11]SP, NKA, [Nle10]NKA(4-10) and [MePhe7]NKB were obtained from Bachem
(Voisins-le-Bretonneux, France). SR140333 and SR48968 were kindly provided by Dr. Emonds5
Alt (Sanofi-Aventis, Montpellier, France). KCl, indomethacin, l-NOARG, SKF96365, SB203580
and nicardipine were obtained from Sigma (St. Quentin Fallavier, France). Stock solutions of
SR140333 (10 mmol/L) and SR48968 (10 mmol/L) were prepared in ethanol; SKF96365 (10
mmol/L) in DMSO; SB203580 (10 mmol/L) and TK-receptor agonists (1 mmol/L) in water. They
were diluted to final concentration in Krebs buffer solution. KCl, l-NOARG and indomethacin
were dissolved in distilled water.
2.6. Expression of the results and statistical analysis
All numerical data are expressed as arithmetic means ± standard error of the mean (S.E.M.). In
studies carried out on isolated human saphenous veins, pD2 values were determined for each
concentration–response curve as the negative logarithm of the molar EC50 value (the
concentration of agonist inducing a contraction which represented 50% of the maximal
contraction). The contractions produced by the NK2-receptor agonists were expressed as a
percent of KCl-induced contraction. Emax represents the maximal effect obtained with the
maximal concentrations of applied peptides. The potency (pD2) of agonists was defined as the
negative log10 of the agonist concentration achieving 50% of the maximal response (EC50). For
SR48968 antagonist studies, pKB was defined as the negative log10 of the dissociation
constant (KB) of antagonist NK2-receptors, which was estimated using the following equation:
KB = [B]/[DR − 1], where DR is the dose ratio (EC50 of the agonist in the presence of the
antagonist divided by the EC50 of the same agonist in the absence of the antagonist) and [B] is
the molar antagonist concentration [29]. Differences between concentration–response curves
were tested using analysis of variance (ANOVA) for repeated measures, followed by Bonferroni
post-test if necessary. For PCR data, statistical analysis was carried out using the Student's ttest for unpaired data, one-way ANOVA followed by Tukey's multiple comparison test
(GraphPad Prism 4.0, California, USA). p-values lower than 0.05 were considered to be
significant.
3. Results
3.1. Expression of tachykinins, tachykinin receptors and neutral endopeptidase
To investigate if tachykinins, tachykinin receptors and NEP were expressed in saphenous vein
preparations, an analysis of tachykinins, tachykinin receptors, NEP transcripts and tachykinin
receptor proteins was performed. By using endpoint RT-PCR, we detected the presence of
transcripts of TAC1 (encoding for SP and NKA), TAC3 (encoding for NKB), TAC4 (encoding for
HK-1), TACR1, TACR2, TACR3 (encoding for NK1-, NK2- and NK3-receptors, respectively) and
MME (encoding for NEP) in all fragments of assayed stripped veins (n = 23) (Fig. 1). In addition
to transcript expression, immunohistochemical studies were performed to assess the
localization of different receptor subtypes. In varicose SV from stripping (n = 4),
immunostainings for the three types of tachykinin receptors were positive in the smooth
muscle layers. In undistended SV from bypass, immunostainings for the three tachykinin
receptors were also positive in the smooth muscle layers and an immunostaining for NK1receptors was observed on the endothelium (Fig. 2).
3.2. Vascular muscle responses of human saphenous veins to tachykinin receptor agonists
6
As NK1-, NK2-, and NK3-receptors were found to be expressed, their relative contribution to
vascular smooth muscle response was examined in saphenous veins from stripping and bypass
surgery. Concentration–response curves were generated with specific agonists of each
receptor to determine the effects mediated by their stimulation. In saphenous vein
preparations from stripping, pre-contracted with 30 μmol/L phenylephrine, neither the NK1receptor agonist ([Sar9Met(O2)11]SP), the NK2-receptor agonists (NKA and [Nle10]NKA(4-10))
nor the NK3-receptor agonist ([MePhe7]NKB), applied up to 1 μmol/L, induced a relaxation of
the vessel rings (n = 4 for each). However, NKA and the selective NK2-receptor agonist
[Nle10]NKA(4-10) induced concentration-dependent contractions on basal tone in about half
the preparations (Fig. 3), whereas neither the NK1-receptor selective agonist nor the NK3receptor selective agonist caused contractions at concentrations up to 1 μmol/L (n = 6 for
these two agonists) on NKA-responsive veins. In the NKA-unresponsive SV, phenylephrine
induced similar contractions than in responsive preparations. In undistended precontracted SV
from bypass surgery, the presence of a functional endothelium was confirmed by the
observation of a relaxation (60 ± 12%, n = 9) to acetylcholine (1 μmol/L). In these preparations,
SP also caused a relaxation (45 ± 13%, n = 5). Similarly to what had been observed in SV from
stripping, no relaxation occurred in precontracted veins from bypass when NK2- or NK3tachykinin receptor agonists were applied. However, NKA also induced a contractile response
on basal tone (Table 2), and no contraction was observed with the agonists specific to NK1and NK3-receptors. The maximal contraction involved by NK2-receptor agonists in saphenous
vein preparations showed a large inter-individual variability (range: 8–95% of 90 mmol/L KCl),
and no correlation was found between the maximal level of contraction to KCl and to NKA.
3.3. Involvement and desensitization of smooth muscle NK2-receptors in the NKA-induced
contraction
In saphenous veins from stripping, the selective and potent NK2-receptor antagonist SR48968
(0.01 μmol/L) markedly inhibited the contraction to NKA with a pKB value of 8.7 ± 0.3 (n = 6)
(data not shown). In control experiments (n = 5), the NK1- and NK3-receptor antagonists,
SR140333 and SR142801, respectively (0.1 μmol/L each), did not alter the contraction induced
by NKA. In addition, the removal of endothelium did not alter the contractile response to NKA
(n = 6) (Fig. 4).
There were two patterns of concentration–response curves to NKA on these saphenous veins
from stripping. In the first case, in about 75% of the preparations, NKA induced an initial phase
of concentration-dependent contraction up to 3 × 10−7 mol/L, and for higher concentrations,
the contraction became transient and the addition of a consecutive incremental dose of
agonists did not prevent the preparations from relaxing progressively. This result suggested a
desensitization to NK2-receptor agonist-induced contractions. This desensitization was
confirmed by the much weaker response to a second cumulative addition of NKA (Fig. 5A, type
A, and Table 2) performed after an extensive washing of the preparation with Krebs solution
and a re-equilibration period of 45 min with bath fluid changes every 15 min. In the second
case, for the remaining 25% of the preparations, NKA-induced response increased dosedependently up to the highest applied concentration. In these preparations, no desensitization
was observed after a second cumulative addition of NKA, the contraction obtained with the
highest concentration applied being in the same range as the first application (Fig. 5B, type B,
7
and Table 2). No differences were observed in terms of relaxant response to Ach between the
two types of preparations. In all undistended SV from bypass surgery, an inverted U-shaped
concentration–response curve was observed after the cumulative addition of NKA (Emax and
pD2 values of 25.5 ± 8.9% and 7.5 ± 0.3, respectively). The rapid desensitization of NKAinduced contraction was confirmed by the absence of response to a second cumulative
addition of NKA (Emax of 3.1 ± 2.9%). Here again, the removal of endothelium did not alter the
contractile response to NKA (n = 4).
3.4. Pharmacological characterization of pathways involved in NK2-receptor-induced
contraction of human saphenous veins from stripping
The hypothesis that NK2-receptor-induced contraction could involve nitric oxide, prostanoids,
calcium channels and p38 MAPK were tested on concentration–response curves to NKA (10−10
to 10−6 mol/L) in the presence of the pharmacological modulators of these pathways. Results
are presented in Table 3. The addition of a NO synthase inhibitor (l-NOARG (100 μmol/L), n =
6), a cyclo-oxygenase inhibitor (indomethacin (10 μmol/L), n = 6), an inhibitor of receptormediated calcium-entry channel (SKF96365 (30 μmol/L), n = 3) or an inhibitor of p38 MAPK
(SB203580 (10 μmol/L), n = 8) did not alter the contraction induced by NKA in varicose SV. In
contrast, nicardipine (3 μmol/L, n = 16), an inhibitor of voltage-dependent calcium channels,
significantly reduced the maximal contraction induced by NKA (Fig. 6).
3.5. Quantitative determination of transcript expressions
Real-time RT-PCR showed that the relative abundance of the mRNA of all target genes was
similar in 12 selected human saphenous veins with high or weak responses to NKA (not
shown). No significant differences were found between the expression of TACR2 mRNA,
neither with primers that detect the two known TACR2 splicing variants nor when examining
exclusively the expression of TACR2 α isoform encoding the functionally active NK2-receptor,
and the maximal effect or the tachyphylaxis observed in the NKA-induced contraction of
human saphenous veins. No connection was observed between the tachykinin degrading
enzyme NEP mRNA levels and the magnitude of the maximal response to NKA.
4. Discussion
The present study demonstrates for the first time (i) the presence in human saphenous veins
of tachykinin and NEP transcripts, together with tachykinin receptor transcripts and proteins,
and (ii) that NKA induces vascular contractions of human saphenous veins by stimulating
smooth muscle NK2-receptor. We also provide evidences of NKA-induced NK2-receptor
desensitization in a majority of preparations.
We first found that the three known human genes encoding tachykinins (TAC1, TAC3 and
TAC4) and the gene encoding NEP (MME) were expressed in the human saphenous vein, which
is in accordance with the transcript expression observed in human pulmonary veins and
arteries [1]. A few studies focused on tachykinin receptors in human vascular tissue and
showed expressions of NK1-, NK2- but not NK3-receptor transcripts in umbilical vein
endothelial cells [30], expressions of NK1-, NK2- and NK3-receptor transcripts in pulmonary
artery and veins [1], and expressions of SP receptors in colon submucosal veins and arteries
8
[31]. Results of the present study revealed transcript expressions of the three tachykinin
receptors in human saphenous veins, supported by the immunohistochemical localization of
the three receptor subtypes in smooth muscle layer, and the presence of NK1-receptors in the
endothelium of saphenous veins from bypass, which confirm the body of evidence indicating
expression of tachykinin receptors in the human vasculature. The wide expression of
tachykinins, tachykinin receptors and NEP in the SV suggested that the tachykinin system may
have both physiologic and pathophysiologic interests at this richly innervated portion of the
vascular system [23], which we further investigated with functional studies. SP is known to
induce an endothelium-dependent relaxation in numerous human vascular preparations [3],
[4], [5], [6], [7], [8], [11], [12] and [13], and endothelial NK1-receptors have been shown to
mediate the relaxation of human pulmonary arteries and veins [11] and [12]. In pre-constricted
SV segments from patients undergoing coronary artery bypass surgery, SP produced a
relaxation which was markedly attenuated after the removal of the endothelium [32]. In
undistended SV, we have shown the presence of endothelial NK1-receptors, which therefore
explains the endothelium-dependent relaxation to a NK1-receptor agonist. No endothelial
NK2- or NK3-receptors were found on the endothelium of undistended SV, explaining the
absence of relaxation to NKA, [Nle10]NKA(4-10) and to the selective agonist for the NK3receptors [MePhe7]NKB in pre-constricted undistended SV (present study). In varicose SV
obtained from patients undergoing stripping, the endothelial function is impaired [33] as
illustrated by the lack of relaxation or the weak relaxation in response to acetylcholine
(present study). In pre-constricted varicose SV, the agonists for NK1-, NK2- and NK3-receptors,
[Sar9,Met(O2)11]SP, NKA and [MePhe7]NKB, did not induce relaxation. These results indicate
that in the absence of a functional endothelium, no relaxation occurs in response to
tachykinins.
NKA-mediated contraction has already been described in the rabbit pulmonary artery [16], in
the rat gastric vasculature [17] and [18], in the canine cerebral artery [19], but not in humans.
In the present study, in both varicose and undistended human SV, NKA induced a
concentration-dependent contraction and NK2-receptors were localized on the smooth muscle
layers. In varicose SV, the selective NK2-receptor agonist [Nle10]NKA(4-10) also induced a
concentration-dependent contraction but was less potent than NKA. This selective NK2receptor agonist has previously shown to be about 5-fold less potent than NKA to contract the
rabbit pulmonary artery, a NK2-receptor preparation [34]. Moreover, the NKA-induced
contraction was inhibited by the selective and potent NK2-receptor antagonist, SR48968, with
a calculated pKB value close to those previously found for this antagonist on NK2-receptors
[35] and [36]. All these results strongly suggest that NKA contracts human SV through the
activation of smooth muscle NK2-receptors. In agreement with the absence of endothelial
NK2-receptors, the mechanical removal of the endothelium did not alter the contractile
response to NKA in undistended SV. In addition to NK2-receptors, NK1- and NK3-receptors
were found on the smooth muscle layers of both undistended and varicose SV. However, in
contrast with observations of the rat gastric or mesenteric vasculature [17], [18] and [37], NK1and NK3-receptor agonists were not able to cause contractions in both varicose and
undistended human SV. The smooth muscle NK1- or NK3-receptors have therefore no
contractile or relaxant function in human SV, but may be involved in the smooth muscle
9
proliferation as previously shown for NK1-receptors in the rabbit airway [38] or rat aortic
smooth muscle cells [39].
In the present study, we also show a rapid development of tachyphylaxis in a majority of
preparations which was characterized by a bell-shaped response curve to the first cumulative
addition of NKA, or at least, by a transient response to the maximal concentration of NKA
added to the organ bath. NKA-induced desensitization of rat, bovine and human NK2-receptors
has been previously shown in transfected cells [40], [41] and [42], but to our knowledge, this is
the first demonstration of NK2-receptor desensitization in human smooth muscle
preparations. Desensitization to a second application of NKA in some preparations may
correspond to tissues with a relatively weak number of NK2 spare receptors. However, using
quantitative real-time PCR, we found no difference in the NK2-receptor transcript expression
in saphenous veins selected for their response to NKA and in a few preparations with or
without rapid tachyphylaxis, or in the expression level of the short (non-functional) or long
(functional) isoforms of NK2-receptors. Quantitative real-time PCR also showed that NK1-,
NK3-receptors and NEP mRNA levels were similar in highly responsive and weakly responsive
varicose SV.
In order to further characterize the NK2-receptor agonist-mediated contraction of human
saphenous veins, we have examined the effects of several pathway inhibitors on NKA-induced
contraction. Prostanoids generated following NK2-receptor activation have been shown to
amplify the direct contractile effect of NK2-receptor agonists in the hamster urinary bladder
[43]. However, the inhibition of prostanoid synthesis with indomethacin did not alter the
response to NKA in both human varicose and undistended SV. NKA can increase p38 MAPK
phosphorylation in canine smooth muscles [44]. The activation of p38 MAPK may be linked to
several functions, including the activation of transcription factors, cell motility and smooth
muscle contraction [44]. A selective inhibitor of p38 MAPK (SB203580) has been shown to
inhibit the contraction of vascular smooth muscles in response to thromboxane A2,
angiotensin II or endothelin-1 [45], [46] and [47]. SB203580, when used at a concentration
which is high enough to efficiently inhibit the activity of p38 MAPK [48], did not alter the NKAinduced contractions of human SV, therefore suggesting that the p38 MAPK pathway is not
involved in the contractile response to NKA in this human vessel. In addition, the activation of
L-type voltage calcium channels has been shown to be largely involved in the contractile
response to NKA in the hamster urinary bladder [43], in the rat myometrium [49], and in the
human colonic smooth muscle [50], whereas it has a minor role in the responses of guinea pig
trachea or human bronchus [51]. The inhibition of L-type calcium channels with nicardipine
reduced the contraction elicited by NKA in human varicose SV (present study). However, the
inhibition of receptor-operated calcium channels with SKF96365 [50], [52] and [53] did not
alter the NKA-induced contraction. These results suggest that NKA caused the contraction of
human saphenous veins mainly through the activation of L-type voltage-operated calcium
channels. Finally, the inhibition of NO synthesis with l-NOARG did not potentiate the
contraction to NKA.
10
In conclusion, the present study shows that the human genes encoding tachykinins, tachykinin
receptors and NEP were all expressed in the human saphenous vein. The results demonstrate
for the first time, in a human vessel, that NKA can induce contractions by stimulating smooth
muscle NK2-receptors, at least in part through the activation of voltage-dependent calcium
channels, and that SV NK2-receptors are subjected to rapid tachyphylaxis. These results may
have both physiologic and pathophysiologic interests since the saphenous veins are a richly
innervated portion of the low pressure vascular system [23]. In addition, sensitive nerve fibers
which form a network around the human temporal artery and coronary arteries [54], [55] and
[56] have also previously been involved in vascular tone regulation [2]. As the saphenous vein
is innervated in situ with peptidergic fibers [23], tachykinins may thus play a role in the control
of vascular smooth muscle tone, in particular since the localization of NK2-receptors to smooth
muscle cells would favor vasoconstriction by the closer proximity of these cells to the sensorymotor nerve endings. We also conclude that the vasodilatator properties of L-type calcium
channel blockers might involve the blockade of NKA-mediated vascular smooth muscle
contraction.
Conflict of interest
None.
Acknowledgments
C. Clement, Department of Vascular Surgery, University Hospital, Reims, France. The work by
FMP and LC was supported by a grant from the Ministerio de Ciencia e Innovación (CTQ200761024/BQU), Spain.
11
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19
Figure captions
Figure 1.
Expression of tachykinin, tachykinin receptors and NEP transcripts in human
saphenous veins. Agarose gel showing expression of TAC1, TAC3, TAC4, TACR1, TACR2, TACR3
and MME mRNAs in human saphenous veins. The figure is representative of typical results
observed in SV from 23 patients (12 with a high functional response to NKA and 11 with a
weak functional response to NKA). M = molecular weight standard.
Figure 2. Tachykinin receptor expression in human saphenous veins from bypass and from
stripping. Microphotographs showing NK1-, NK2- and NK3-receptor immunostainings in
endothelial (E) and smooth muscle (SM) layers of saphenous veins. Negative controls were
performed by omission of the anti-NK1-, NK2- or NK3-receptor antibody. Arrows are showing
the positive staining. L = Lumen.
Figure 3. Cumulative concentration–response curves of NKA (●, n = 39) and [Nle10]NKA(4-10)
(▴, n = 8) on human saphenous veins from stripping. The contraction is expressed as a
percentage of maximum contraction obtained with KCl (90 mmol/L). Values are expressed as
mean ± S.E.M.
Figure 4. Cumulative concentration–response curves of NKA on human saphenous veins from
stripping (n = 6) in the presence (●) or in the absence (○) of endothelium. The contraction is
expressed as a percentage of maximum contraction obtained with KCl (90 mmol/L). Values are
expressed as mean ± S.E.M.
Figure 5. Cumulative concentration–response curves of NKA in “Type A” (A) (n = 11) or “Type
B” (B) (n = 29) human saphenous veins from stripping. The initial curve (●) and the second
curve (▴) obtained after a 45-min wash with Krebs solution are presented. The contraction is
expressed as a percentage of maximum contraction obtained with KCl (90 mmol/L). Values are
expressed as mean ± S.E.M. *p ≤ 0.05.
Figure 6. Cumulative concentration–response curves of NKA on human saphenous veins from
stripping following pre-treatment (▴) or not (●) with 3 μmol/L nicardipine (n = 16). The
contraction is expressed as a percentage of maximum contraction obtained with KCl (90
mmol/L). Values are expressed as mean ± S.E.M. *p ≤ 0.05.
20
Table 1
Table 1. Sequences of primers used in RT-PCR.
Gene
Forward primer
Reverse primer
Amplicon
size (bp)
References
TAC1
5′-ACTGTCCGTCGCAAAATCC-3′
5′-ACTGCTGAGGCTTGGGTCTC-3′
212
[57]
TAC3
5′-CCCCCGAGAGCAGAATAGGT-3′
5′-CCAGGGTCAGGTAGAAAAGATGG171
3′
[58]
TAC4
5′5′TCTCTTCTCTGTGTCTCCTGTCCTC- CATTTATTGAGTGCCTACTGTGTGCT- 246
3′
3′
[59]
TACR1
5′-ATGCCCAGCAGAGTCGTGT-3′
5′-TCGTGGTAGCGGTCAGAGG-3′
194
[60]
TACR2(α + β)
5′-GCCCTACCACCTCTACTTCATCC5′-AGCAAACCATACCCAAACCA-3′
3′
375
[61]
TACR2(α)
5′5′-GACGGTGGAGTAGAAGCACTGA-3′ 235
CAGCCACAACATCTGGTACTTTG-3′
[61]
TACR3
5′-GCCAGAAGGTCCCAAACAAC-3′ 5′-CAGCCAGCAGATAGCAAATGTC-3′ 229
[62]
MME
5′-AGCCTCTCGGTCCTTGTCCT-3′
[63]
β-actin
5′-TCCCTGGAGAAGAGCTACGA-3′ 5′-ATCTGCTGGAAGGTGGACAG-3′
5′-GGAGCTGGTCTCGGGAATG-3′
219
362
21
Table 2
Table 2. Emax and pD2 values for NKA-induced contractions of human
saphenous veins from stripping and from bypass, for the first and second
concentration–response curves applied to the same preparations.
Type
Stripping A
B
Bypass A
Emax (%)
1st curve
27.6 ± 5.5
24.5 ± 6.4a
25.5 ± 8.9
pD2
2nd curve
4.0 ± 1.5
20.9 ± 8.6a
3.1 ± 2.9
p
<0.001
NS
<0.001
1st curve
7.3 ± 0.2
NA
7.5 ± 0.3
n
2nd curve
NA
11
NA
29
NA
6
Emax values are presented as mean ± SEM and pD2 values as mean ± SD of n
independent experiments. NA = not applicable.
a Maximal effect when 1 μmol/L NKA was applied: an asymptote was not
reached and pD2 values were thus not calculated.
22
Table 3
Table 3. Emax and pD2 values for NKA-induced (10−10 to 10−6 mol/L)
contractions of human saphenous veins from stripping, in the absence (paired
control) or in the presence of cyclo-oxygenase inhibitor indomethacin
(10 μmol/L), NO synthase inhibitor l-NOARG (100 μmol/L), receptor-mediated
calcium-entry channel inhibitor SKF96365 (30 μmol/L), p38 mitogen-activated
protein kinases inhibitor SB203580 (10 μmol/L) or voltage-dependent calcium
channels inhibitor nicardipine (3 μmol/L).
Emax (%)
Paired
control
Indomethacin 20.1 ± 4.4
l-NOARG
15.2 ± 7.9
SKF96365
22.0 ± 3.6
SB203580
30.7 ± 12.1
Nicardipine 35.5 ± 5.4
pD2
Paired
control
NS
7.3 ± 0.2
NS
7.1 ± 0.4
NS
6.6 ± 0.2
NS
7.3 ± 0.4
<0.001 7.7 ± 0.2
n
Condition p
Condition p
17.9 ± 2.6
15.8 ± 8.7
19.4 ± 14.0
35.2 ± 13.0
19.5 ± 3.5
7.2 ± 0.2
7.2 ± 0.5
6.9 ± 0.6
6.7 ± 0.4
7.6 ± 0.2
NS
NS
NS
NS
NS
11
6
4
8
16
Emax values are presented as mean ± SEM and pD2 values as mean ± SD of n
independent experiments.
23
Figure 1
24
Figure 2
25
Figure 3
26
Figure 4
27
Figure 5
28
Figure 6
29