Posprint of: Pharmacological Research Volume 63, Issue 5, May 2011, Pages 414–422
Smooth muscle neurokinin-2 receptors mediate contraction in human saphenous veins
HakimaMechiche (a), StanislasGrassin-Delyle (a), Francisco M. Pinto (b), Amparo Buenestado (a), Luz Candenas (b), PhilippeDevillier (a)
(a) Laboratory of PulmonaryPharmacology, UPRES EA 220, Faculté de Médecine Île-de-France Ouest, UniversityVersailles 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 mMKCl 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 NK2-receptor 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 NK1- or 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
NeurokininA (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 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.
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 transcription-polymerase 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 guanidiumisothiocyanate–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% agarose-formaldehyde 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 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 iCycleriQ 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 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. Emonds-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 t-test 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 NK1-receptors was observed on the endothelium (Fig. 2).
3.2. Vascular muscle responses of human saphenous veins to tachykinin receptor agonists
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 NK1-receptor 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 NK3-receptor 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 undistendedprecontracted 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 NK3-tachykinin 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 NK1- and 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.