Life Sciences 336 (2024) 122286 Contents lists available at ScienceDirect Life Sciences journal homepage: www.elsevier.com/locate/lifescie Evidence for the involvement of TRPV2 channels in the modulation of vascular tone in the mouse aorta Alex Perálvarez-Marín a, b, Montse Solé b, c, d, Judith Serrano b, e, Alice Taddeucci b, e, Belén Pérez b, e, Clara Penas b, d, f, Gemma Manich b, g, Marcel Jiménez b, f, Pilar D'Ocon h, i, Francesc Jiménez-Altayó b, e, j, * a Biophysics Unit, Department of Biochemistry and Molecular Biology, School of Medicine, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain b Institute of Neurosciences, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain c Department of Biochemistry and Molecular Biology, School of Medicine, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain d Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, Madrid, Spain e Department of Pharmacology, Therapeutics and Toxicology, School of Medicine, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain f Department of Cell Biology, Physiology and Immunology, Universitat Autonoma de Barcelona, Cerdanyola del Vallès, Spain g Human Anatomy and Embriology Unit, Department of Morphological Sciences, School of Medicine, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain h Department of Pharmacology, School of Pharmacy Universidad de Valencia, Burjassot, Spain i Estructura de Recerca Interdisciplinar en Biotecnologia i Biomedicina (ERI BIOTECMED), Universidad de Valencia, Valencia, Spain j Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Instituto de Salud Carlos III, Madrid, Spain A R T I C L E I N F O A B S T R A C T Keywords: Aims: Transient receptor potential vanilloid 2 (TRPV2) channels are expressed in both smooth muscle and TRPV2 channels endothelial cells and participate in vascular mechanotransduction and sensing of high temperatures and lipids. Mouse aorta Nevertheless, the impact of TRPV2 channel activation by agonists on the coordinated and cell-type specific Endothelium-dependent and independent modulation of vasoreactivity is unknown. vasodilatation Potassium ion channels Main methods: Aorta from 2- to 4-months-old male Oncins France 1 mice was dissected and mounted in tissue Nitric oxide baths for isometric tension measurements. TRPV2 channel expression was assessed by immunofluorescence and KATP channels western blot in mice aortas and in cultured A7r5 rat aortic smooth muscle cells. Key findings: TRPV2 channels were expressed in all three mouse aorta layers. Activation of TRPV2 channels with probenecid evoked endothelium-dependent relaxations through a mechanism that involved activation of smooth muscle Kir and Kv channels. In addition, TRPV2 channel inhibition with tranilast increased endothelium- independent relaxations to probenecid and this effect was abrogated by the KATP channel blocker glibencla­ mide, revealing that smooth muscle TRPV2 channels induce negative feedback on probenecid relaxations mediated via KATP channel inhibition. Exposure to the NO donor sodium nitroprusside increased TRPV2 channel translocation to the plasma membrane in cultured smooth muscle cells and enhanced negative feedback on probenecid relaxations. Significance: In conclusion, we present the first evidence that TRPV2 channels may modulate vascular tone through a balance of opposed inputs from the endothelium and the smooth muscle leading to net vasodilation. The fact that TRPV2 channel-induced activity can be amplified by NO emphasizes the pathophysiological rele­ vance of these findings. 1. Introduction vanilloid (TRPV) channel subfamily (TRPV1–6) is located mostly in the plasma membrane and is expressed in different cell types and tissues [4]. Transient receptor potential (TRP) channels are a set of membrane Like other TRPV channels, TRPV2 are formed by a long cytoplasmic N- proteins that act as non-selective cation channels [1–3]. The TRP terminal domain, followed by six transmembrane segments containing a * Corresponding author at: Department of Pharmacology, Therapeutics and Toxicology, School of Medicine, Institut de Neurociències, Av. de Can Domènech, 737, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain. E-mail address: francesc.jimenez@uab.cat (F. Jiménez-Altayó). https://doi.org/10.1016/j.lfs.2023.122286 Received 20 October 2023; Received in revised form 16 November 2023; Accepted 20 November 2023 Available online 24 November 2023 0024-3205/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). A. Perálvarez-Marín et al. Life Sciences 336 (2024) 122286 loop that forms the pore, and the cytoplasmic C-terminal domain [5]. To the thoracic and abdominal aorta, the thoracic cavity of the mouse was date, it has been reported that these channels are activated by high opened, and the lungs and heart were removed. Immediately after temperatures (>52 ◦ C), mechanical stress, and various chemical signals dissection, the aorta was placed in a cold oxygenated (95 % O2, 5 % CO2) [5–8]. TRPV2 channels are ubiquitously expressed in the body and have Krebs Henseleit (KH) physiological solution. While the artery was been detected in smooth muscle cells [9–11], endothelial cells [12], and immersed in the cold KH solution, the connective and adipose tissue was in the heart [13]. This evidence suggests that these channels may have gently removed and the artery thoracic ascendent, arch, thoracic widespread roles in cardiovascular physiology [5,14]. descendent, and abdominal portions were dissected. TRPV2 channels are essential for the maintenance of cardiac struc­ ture and function [15,16] and play a critical role in dystrophic cardio­ 2.3. Evaluation of aortic functionality myopathy [17] and myocardial infarction [18]. Regarding the involvement of TRPV2 in physiological or pathological vascular re­ Aortic segments (1.5 mm) were mounted under isometric conditions sponses, there is insufficient knowledge. In the few studies that reported on a 4-channel wire myograph (Model 620 M; Danish Myo Technology, the participation of TRPV2 channels in vascular physiology, the authors Aarhus, Denmark) with cold KH solution [31]. Next, the aortic segments showed that these channels could act as sensors of membrane stretch were incubated for 30 min at 37 ◦ C in oxygenated (95 % O2, 5 % CO2) and cell swelling in mouse aortic smooth muscle cells [11]. Likewise, KH solution. Once stabilization was complete, the arteries were gradu­ other studies have proved that myogenic responses in retinal arterioles ally stretched until the basal ring tension reached the optimal tension of are initiated by pressure-induced stretching, which activates TRPV2 14.7 mN. After a 45 min stabilization period, tissue viability was channels [19]. Altogether, available evidence suggests that TRPV2 analyzed by exposing the segments twice to a K+-rich KH solution (100 channels play important roles in vascular physiology. Nevertheless, mM KCl). Subsequently, a series of washes were carried out and the there is scarce knowledge about the impact of these channels on the segments were again subjected to a stabilization period of 30 min. The coordinated and cell-type specific modulation of vasoreactivity. stable analogue of thromboxane A2, 9,11-Dideoxy-9α,11α-methane- Up to now, one of the main limitations of the study of TRPV2 channel epoxy prostaglandin F 2α (U46619; Merck KGaA, Darmstadt, Germany) functions is the lack of ligands that specifically interact with them. was added to the bath at a concentration to induce a pre-contraction of Certain molecules such as some growth factors, cannabinoids, lyso­ 70–100 % compared to the contraction produced by the 100 mM KCl phospholipids, insulin, 2-aminoethoxydiphenyl borate (2-APB), nitric solution. Once a plateau was reached, increasing concentrations of oxide (NO), and cytokines activate TRPV2 channels-derived responses acetylcholine (ACh; from 1 nM up to 100 μM) were added to assess [5–7,20]. Some of them induce translocation of TRPV2 channels from endothelium-dependent relaxations. Those aortic rings that showed the endoplasmic reticulum to the plasma membrane (e.g., NO), while <50 % relaxation were discarded, as they were considered to have others can act as direct modulators of channel gating. For instance, damaged endothelium. Subsequently, a series of washes were performed cannabidiol and 2-APB activate TRPV2 channels directly, but they can and after a stabilization period of 30 min aortic segments were pre­ also activate other channels and receptors [21]. In addition, this activity contracted with U46619 to then perform a concentration-response curve could be species-dependent since human TRPV2 channels are insensitive (from 1 nM to 300 μM) using the agonist of TRPV2 channels, probenecid. to 2-APB [22]. Interestingly, the uricosuric agent probenecid, which Probenecid relaxations in the presence of pre-contractions to phenyl­ shows antihypertensive effects in rats [23], has also been recognized as a ephrine (Phe), an α1-adrenergic receptor agonist, or 5-hydroxytrypta­ TRPV2 channel activator [24–27], whereas the antiallergic drug trani­ mine (5-HT), a 5-HT receptor agonist, were also studied. Preliminary last has been used as a TRPV2 channel antagonist [28–30]. Nevertheless, studies showed no sex-dependent differences in probenecid relaxations both drugs have also been considered to interact with other targets in aortas from C57BL/6 mice (unpublished results); consequently, only [5,7]. male mice were used. The involvement of smooth muscle and/or endothelial TRPV2 To analyze probenecid mechanisms of action we used the abdominal channel signaling in vascular responses is not explored. The present aorta, since relaxations were prominent in this aortic portion and four study was designed to investigate the putative participation of endo­ segments per animal could be regularly obtained. We evaluated the ef­ thelial and smooth muscle TRPV2 channels in regulating vascular fect produced by several inhibitors that were pre-incubated 30 min reactivity of the mouse aorta, using probenecid and tranilast as phar­ before the probenecid responses began. Tranilast (100 μM), a TRPV2 macological tools. channel inhibitor, glibenclamide (30 μM), an ATP-dependent K+ (KATP) channels blocker, 4-aminopyridine (1 mM), an inhibitor of voltage- 2. Materials and methods gated K+ (Kv) channels, apamin (100 nM) and charybdotoxin (100 nM), an inhibitor of Ca2+-activated K+ channels, barium chloride 2.1. Animals (BaCl2; 100 μM), an inhibitor of inwardly rectifying K+ (Kir) channels, prazosin (0.1 μM), a selective α1-adrenergic receptor blocker, and so­ Two- to four-months old male mice (n = 64) of the Oncins France 1 dium nitroprusside (NPS; 100 μM), a NO donor. (OF1) strain were used (Charles River, Sant Cugat del Vallès, Spain). Finally, with the aim of establishing the contribution of endothelium Preliminary data showed that probenecid responses were comparable in probenecid relaxations, the vessels were pre-incubated for 30 min within this range of age. The animals were housed in accordance with with NG-nitro-L-arginine methyl ester (L-NAME; 300 μM), a non- the regulations of the institutional guidelines: constant temperature and selective inhibitor of synthases of NO, and with indomethacin (10 humidity, light-dark cycle of 12–12 h and no limit of food or water. All μM), a non-selective inhibitor of cyclooxygenases. In addition, the procedures were carried out following the Spanish Legislation (RD35/ endothelium was removed mechanically by gently rubbing the interior 2013) and the Guide for the Care and Use of Laboratory Animals pub­ of the vessel with a plastic cannula. Arteries with ACh relaxations of lished by the US National Institutes of Health (NIH publication number <20 % were considered endothelium denuded. In endothelium-denuded 85–23, revised in 1996). Likewise, the procedure was approved by the aortas, probenecid relaxations were tested in the absence and presence Ethics Committee of the Universitat Autònoma de Barcelona (approval of BaCl2 (100 μM) plus 4-aminopyridine (1 mM) or tranilast (100 μM) in code: FJA-eut/01). different experimental conditions. 2.2. Preparation of aortic rings 2.4. Cell culture Mice were euthanized by rapid decapitation under anesthesia (4 % The rat aortic smooth muscle cell line A7r5 was obtained from isoflurane mixed with 0.8 L/min O2). With the aim of gently dissecting ECACC (European Collection of Cell Cultures) and cultured in high 2 A. Perálvarez-Marín et al. Life Sciences 336 (2024) 122286 glucose Dulbecco's Modified Eagle Medium (DMEM; Sigma-Aldrich) times in lysis buffer and reconstituted in sample buffer, boiled for 5 min supplemented with 10 % fetal bovine serum (FBS). Cells were main­ at 95 ◦ C and centrifuged to obtain the membrane fraction. Samples were tained at 37 ◦ C in a humidified atmosphere containing 5 % CO2. Cells analyzed by western blot. were seeded at 40,000 cells/mL on collagen-coated (collagen 1 rat tail, BD Biosciences) coverslips in case of immunofluorescences, grown for 2.7. Western blot 48 h and then starved in 0.2 % FBS medium for 1 h before adding treatments. NPS was freshly prepared in 0.2 % FBS DMEM, protected The protein concentration of samples was determined by the Brad­ from light, and used at 10, 100 and 300 μM final concentrations for 1 h. ford method (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were loaded for the total and internal fractions (15 to 25 μg, depending 2.5. Immunofluorescence on the experiment), while equal volumes of the membrane fractions were loaded between treatments (40 to 50 μL). Proteins were separated Aortic segments were fixed in 4 % paraformaldehyde and embedded by SDS-polyacrylamide gel electrophoresis using the Bio-Rad Mini- in Tissue Tek OCT embedding medium (Sakura Finetek Europe, Zoe­ PROTEAN 3 system and transferred onto nitrocellulose membranes terwoude, the Netherlands), frozen in liquid nitrogen and kept at (Schleicher & Schüll). Membranes were blocked for 1 h with PBS con­ − 70 ◦ C. Sections were processed for immunofluorescence staining as taining 0.1 % Tween-20 and 10 % non-fat dry milk and then incubated previously described [32]. Briefly, 14 μm-thick frozen transverse sec­ overnight at 4 ◦ C with the indicated primary antibody diluted in tions were incubated for 1 h with anti-TRPV2 (1:100; #ACC-039, lot blocking buffer. After incubation with the corresponding HRP (horse­ ACC039AN0502, Alomone Labs, Jerusalem, Israel) primary polyclonal radish)-conjugated secondary antibodies, blots were developed using antibody in a humidified chamber at 37 ◦ C. Afterwards, sections were the EZ-ECL Enhanced Chemiluminescence Detection Kit for HRP (Bio­ incubated for 45 min with a donkey anti-rabbit IgG secondary antibody logical Industries, Kibbutz Beit Haemek, Israel) and captured with Fuji conjugated to Cyanine 3 (1:200; #711-165-152, lot 110351, Jackson X-ray films Super RX-N (Fujifilm, Tokyo, Japan). The antibodies used ImmunoResearch Laboratories, West Grove, PA, USA) in a humidified were: rabbit anti-TRPV2 (1:500; #ACC-039, lot ACC039AN0502, Alo­ chamber at 37 ◦ C. The specificity of the immunostaining was verified by mone Labs, Jerusalem, Israel); mouse anti-GAPDH (1:10000; Enzo Life omission of the primary antibody (negative control). Images were Sciences); rabbit anti-insulin-like growth factor 1 receptor b (IGFRecB; captured using a FV1000 confocal microscope (Olympus Iberia, Barce­ 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), HRP-conjugated lona, Spain). Average fluorescence signal was analyzed in at least two goat anti-rabbit IgG (1:2000; Sigma-Aldrich), HRP-conjugated rabbit rings of each animal with Image J software (National Institutes of anti-mouse IgG (1:2000; Sigma-Aldrich). Band intensities were quanti­ Health, Bethesda, MD, USA), and the results were expressed as arbitrary fied with ImageJ-FIJI software (National Institutes of Health, Bethesda, units. MD, USA), and normalized with the Ponceau staining corresponding to In the case of cultured smooth muscle cells, after the corresponding each lane. GAPDH and IGFRecB were used as controls for the correct treatments, they were washed once with phosphate-buffered saline fractionation protocol in internal and membrane fractions, respectively. (PBS) containing 0.5 mM Ca2+ chloride (CaCl2), 1 mM magnesium chloride (MgCl2) and 4 % sucrose (PBS-Ca-Mg-Suc). Cells were then 2.8. High-performance liquid chromatography (HPLC) incubated for 5 min at 37 ◦ C with anti-TRPV2 (1/60; #ACC-039, lot ACC039AN0502, Alomone Labs, Jerusalem, Israel) in PBS-Ca-Mg-Suc. The concentration of ATP in mice aortas was evaluated by HPLC with After a wash in ice-cold PBS-Ca-Mg, cells were fixed in PBS-Ca-Mg-Suc ultraviolet detection. A pool of thoracic plus abdominal aorta segments containing 2 % paraformaldehyde for 15 min on ice. Then, cells were from 2 animals was used until reaching n = 5 independent experiments washed twice in PBS-Ca-Mg and blocked in PBS containing 2 % normal per experimental condition (10 mice). The tissue was homogenized with goat serum (NGS) for 20 min at 37 ◦ C under slow agitation. After one 60 μL of modified RIPA buffer (50 mM Tris-HCl, 0.5 % sodium deoxy­ wash with PBS-Ca-Mg, cells were incubated with Cy3-conjugated cholate, 0.2 % SDS, 100 mM NaCl, 1 mM EDTA, 1 % Triton X-100) with a donkey anti-rabbit IgG secondary antibody (1:250 in PBS containing douncer. The method used for ATP extraction from biological samples 2 % NGS) for 1 h at 37 ◦ C under slow agitation. Finally, after two 10 min was adapted from Sudo et al. (2000) [34]. After incubating at 37 ◦ C for washes in PBS, coverslips were mounted using Vectashield plus con­ 20 min, samples were centrifuged, and 25 μL of supernatant was injected taining DAPI (#H-2000-2) to counterstain nuclei (Vector Laboratories, into the HPLC system (Perkin Elmer series 200 Pump, 717 plus Auto Burlingame, CA, USA) and images were captured using a FV1000 sampler, 2487 Dual λ absorbance detector). A reverse-phase silica-based confocal microscope (Olympus Iberia, Barcelona, Spain). Average fluo­ chromatography column (Kromasil 100-5-c18; 4.0 × 200 mm) was ob­ rescence signal per area was analyzed in at least two regions per tained from Teknokroma (Sant Cugat del Vallès, Spain). The mobile experimental condition with ImageJ-FIJI software (National Institutes of phase was methanol-10 mM tetrabutylammonium hydrogen sulfate in Health, Bethesda, MD, USA), and the results were expressed as arbitrary 20 mM ammonium dihydrogen phosphate-diammonium hydrogen units. phosphate (pH 6.5) (25:75), which was run with an isocratic regular flow rate of 1.0 mL/min and the wavelength UV detector was set at 260 2.6. Biotinylation and subcellular fractionation nm. To obtain surface-protein fractions, a biotinylation protocol was 2.9. Drugs and reagents performed on cultured smooth muscle cells, as previously described [33]. Briefly, cells were rinsed in ice-cold PBS-Ca-Mg and then incubated U46619 (#D8174), ACh (#A6625), phenylephrine (#P6126), sero­ for 30 min at 4 ◦ C under slow agitation in 0.5 mg/mL of biotin diluted in tonin (#H9523), probenecid (#P8761), tranilast (#T0318), 4-amino­ the same buffer (EZ-Link Sulfo-NHS-SS-Biotin; Thermo Fisher Scienti­ pyridine (#A78403), glibenclamide (#60639), L-NAME (#N5751), fic). Then, cells were washed three times in PBS-Ca-Mg containing 100 indomethacin (#I7378), NPS (#71778), BaCl2 (#B0750), prazosin mM Glycine, once in PBS-Ca-Mg, and scrapped in lysis buffer (50 mM (#P7791), carbenoxolone (#C4790), and reagents used to make solu­ NaCl, 10 mM EDTA, 10 mM EGTA, 50 mM HEPES, 1 % Triton X-100, and tions were purchased from Merck (KGaA, Darmstadt, Germany). Apamin protease and phosphatase inhibitors) to obtain lysates. These were (#L8407) and charybdotoxin (#L8213) were acquired from Latoxan incubated overnight at 4 ◦ C on rotation with NeutrAvidin-agarose beads (Portes-lès-Valence, France). Acetylcholine, probenecid, 4-aminopyri­ (Thermo Fisher Scientific), reserving part of the lysate as Total Fraction. dine, BaCl2, apamin, charybdotoxin, and carbenoxolone were dis­ After centrifugation at 4000 ×g for 4 min at 4 ◦ C, the supernatant was solved in distilled water, while tranilast, and stock solutions (10 mM) of recovered and stored as internal fraction, and beads were washed four glibenclamide and U46619 were dissolved in DMSO. We ensured that 3 A. Perálvarez-Marín et al. Life Sciences 336 (2024) 122286 the highest concentration of DMSO in the bath did not exceed 0.005 %. different aortic regions previously pre-contracted with U46619. Re­ L-NAME and NPS were dissolved in KH solution. Mounting medium laxations were similar in the ascending aorta, the aortic arch, and the containing DAPI was obtained from Vectashield. Reagents used for abdominal aorta, whereas the thoracic descending aorta showed biotinylation assay were obtained from ThermoFisher Scientific. significantly lower responses. At this stage, we continued our study in the abdominal aorta, since it showed high probenecid relaxations, while 2.10. Statistical analysis allowing four segments per animal to be obtained consistently for comparative studies. Results are expressed as mean ± standard error of the mean (SEM) of Previous studies in Phe-precontracted rat [23] and human [35] ar­ the number of animals (n) in each group (shown in figure legends). teries suggested that probenecid-induced vasodilatations could be Normal distribution was checked before choosing the appropriate sta­ directly or indirectly related to inhibition of α-adrenergic receptors. In tistical analysis. Comparison between two groups was carried out with our experimental conditions, probenecid relaxations were not likely to the Mann-Whitney U or Student's t-test. Comparison between more than be mediated via blockade of α1-adrenergic receptor signaling, since they two groups (one single factor) was carried out with one-way ANOVA or were induced in aortas previously pre-contracted with Phe, but also with the Kruskal-Wallis test followed by Tukey's or Dunn's post-test, respec­ other agents. In fact, in the abdominal aortic portion, probenecid re­ tively. The vasodilator responses induced by ACh and probenecid were laxations were higher in U46619 and 5-HT pre-contracted vessels versus expressed as % of the relaxation obtained after precontraction with other contractile agents such as Phe and KCl (100 mM) (Fig. 2B). We U46619, Phe, 5-HT, or with a 100 mM KCl solution. Statistical analysis therefore studied probenecid relaxations in aortas pre-contracted with was performed using GraphPad Prism software version 9.1 (GraphPad U46619 in posterior mechanistic studies. Moreover, the selective α1- Software, San Diego, California, USA). Two-way/factor ANOVA tests adrenergic receptor blocker prazosin 0.1 μM did not relax mouse aortas were performed with repeated measures on the concentration factor and pre-contracted with U46619 (Supplementary Fig. 1), thus, adding using the Bonferroni or Tukey's post-test. In some graphs, the area under further evidence against participation of α1-adrenoceptors in mouse the curve was calculated and expressed as arbitrary units of the number aorta probenecid relaxations. of animals (n) in each group (shown in figure legends). In this case, the It is remarkable that the relaxant activity of probenecid was almost statistical analysis consisted of an ordinary two-way ANOVA test. Sta­ indetectable in aortic rings pre-contracted with KCl, suggesting an tistical significance was considered based on a value of P < 0.05. intervention of K+ channels in this activity, which will be analyzed later (section 3.3). 3. Results Compared to ACh relaxations, an endothelium-dependent musca­ rinic receptor agonist used as a reference, probenecid concentration- 3.1. TRPV2 channels are expressed in the different layers of the mouse responses curves shifted to the right in all aortic segments, indicating aortic wall lower relaxations (Supplementary Fig. 2). However, maximum re­ laxations evoked by probenecid and ACh were not statistically different, Immunofluorescent expression of TRPV2 channels was assessed in except for thoracic descending aorta, which showed lower relaxations mouse aortic cross sections (Fig. 1). TRPV2 channel signal was detected (Supplementary Fig. 2). in all three (adventitia, media, and endothelial) layers of the vessel wall. Participation of the endothelium in probenecid vasorelaxant activity Nevertheless, in the case of endothelial and smooth muscle cells only, was shown by comparing its activity in intact and endothelium-denuded the signal was widely distributed throughout the cell body, whereas vessels. Fig. 3A shows a significant decrease of probenecid-induced TRPV2 channel expression in adventitial cells was in the nucleus. relaxation in aortas without endothelium. However, after endothelium removal, remaining probenecid-induced relaxations (24.30 ± 4.21 %) 3.2. TRPV2 channels modulate vascular tone in the mouse aorta could still be observed. The non-selective inhibition of NO synthase (Fig. 3B) and COX (Fig. 3C) isoforms with L-NAME (300 μM) or indo­ Probenecid, an agonist of TRPV2 channels, shows antihypertensive methacin (10 μM), respectively, did not modify probenecid relaxations, excluding participation of these endothelial mediators in the vaso­ effects in rats, which have been associated with the blockade of aortic contraction [23]. However, the involvement of TRPV2 channels in this relaxant response of probenecid. These findings demonstrate that aortic relaxations to probenecid have both endothelium-dependent and inde­ vascular activity has not been previously analyzed. Fig. 2A shows pendent components. concentration-dependent relaxations to this TRPV2 channel agonist in Fig. 1. Expression of TRPV2 channels in the mouse aorta. Representative photomicrographs of TRPV2 channel immunofluorescence (red; left) and negative control (right) of confocal microscopic mouse abdominal aorta sections (n = 5). Natural autofluorescence of elastin (green) and nuclear staining with Hoechst 33342 (10 μg/ mL) (blue) are also shown. 4 A. Perálvarez-Marín et al. Life Sciences 336 (2024) 122286 Fig. 2. Effects of the aortic region or the pre-contractile agonist on probenecid relaxations. (A) Concentration-response curves to probenecid in U46619 pre- contracted aortas from different aortic regions. (B) Concentration-response curves to probenecid in U46619, phenylephrine (Phe), 5-hydroxytryptamine (5-HT), and KCl pre-contracted abdominal aorta. Results are the mean ± SEM from 4 to 5 (A) and 4–5 (B) mice. *P < 0.05, **P < 0.01 versus thoracic descendent (A); **P < 0.01, ***P < 0.001 versus U46619 pre-contraction (B) by two-way ANOVA. Fig. 3. Modulation of probenecid relaxations by the endothelium. (A) Concentration-response curves to probenecid in aortic rings with and without endothelium. (B) Effect of the non-selective NO synthase inhibitor, L-NAME (300 μM), on the concentration-response curves to probenecid in U46619 pre-contracted abdominal aortas. (C) Effect of the non-selective cyclooxygenase inhibitor, indomethacin (10 μM), on the concentration-response curves to probenecid in U46619 pre-contracted abdominal aortas. Results are the mean ± SEM from 6 to 7 (A), 5 (B), and 5–8 (C) mice. *P < 0.05 by two-way ANOVA. We then used the TRPV2 channel blocker tranilast (100 μM) [28–30] relaxations excludes an unspecific activity of TRPV2 channels as mod­ to determine the role of TRPV2 channels in probenecid responses. In­ ulators of vascular tone and ascribes this activity to a specific antagonist cubation with tranilast significantly increased probenecid (Fig. 4A) but activity of tranilast at the probenecid-activated smooth muscle TRPV2 not ACh (Fig. 4B) relaxations in endothelium-intact aortas. The poten­ channels. These results suggest the presence of negative feedback on tiation effect of tranilast on probenecid relaxations was also observed in probenecid relaxations induced by muscular TRPV2 channels. endothelium-denuded arteries (Fig. 4A) suggesting that, in our experi­ mental conditions, tranilast antagonizes TRPV2 channels located out of the endothelial layer. The fact that tranilast did not modify ACh-induced 5 A. Perálvarez-Marín et al. Life Sciences 336 (2024) 122286 Fig. 4. Impact of inhibition of TRPV2 channels on probenecid or acetylcholine relaxations. (A) Effect of the selective TRPV2 inhibitor, tranilast (100 μM), on the concentration-response curves to probenecid in U46619 pre-contracted abdominal aortas with or without endothelium. (B) Effect of tranilast (100 μM) on the concentration-response curves to acetylcholine in U46619 pre-contracted abdominal aortas. Results are the mean ± SEM from 5 to 9 (A) and 4 (B) mice. *P < 0.05 versus control, ***P < 0.001 versus without endothelium by two-way ANOVA. 3.3. Probenecid-induced endothelium-dependent relaxations are mediated in KCl (100 mM)-pre-contracted arteries, which suggest the involvement by KIR and KV channels of smooth muscle hyperpolarization due to K+ efflux through K+ chan­ nels. Thus, concentration-response curves to probenecid were per­ As reported above (Fig. 2B), probenecid relaxations were abolished formed in vessels incubated with different K+ channel inhibitors and Fig. 5. Effects of inhibition of K+ channels on probenecid relaxations. (A) Concentration-response curves to probenecid in U46619 pre-contracted abdominal aortas in the absence (control) or presence of the inwardly rectifying K+ channel blocker BaCl2 (100 μM). (B) Concentration-response curves to probenecid in U46619 pre- contracted abdominal aortas in the absence (control) or presence of the voltage-gated K+ channel blocker 4-aminopyridine (1 mM). (C) Concentration-response curves to probenecid in U46619 pre-contracted abdominal aortas without endothelium in the absence or presence of BaCl2 plus 4-aminopyridine. Results are mean ± SEM from 4 to 5 (A), 4–5 (B), and 6 (C) mice. *P < 0.05, **P < 0.01 by two-way ANOVA. 6 A. Perálvarez-Marín et al. Life Sciences 336 (2024) 122286 with an inhibitor of the Na+K+-ATPase. Neither inhibition of KCa 3.5. Nitric oxide induces the translocation of TRPV2 channels to the channels with charybdotoxin (100 nM) and apamin (100 nM), nor in­ plasma membrane and increases the tranilast-induced potentiation of hibition of KATP channels with glibenclamide (30 μM) modified pro­ probenecid relaxations benecid relaxations (Supplementary Fig. 3). In addition, blockade of Na+K+-ATPase-mediated hyperpolarization with ouabain (1 μM) did not Previous studies showed that TRPV2 channels translocate from the have an effect (Supplementary Fig. 3). In contrast, either inhibition of endoplasmic reticulum to the plasma membrane in response to NO KIR channels with BaCl2 (1 mM) (Fig. 5A) or KV channels blockade with stimulation [20]. To elucidate whether exposure to NO translocates 4-aminopiridine (1 mM) (Fig. 5B) significantly reduced probenecid re­ TRPV2 channels to the plasma membrane in vascular cells, we cultured laxations in aortic rings with endothelium. Probenecid relaxations ob­ A7r5 rat aortic smooth muscle cells in the absence and presence of the tained in aortas without endothelium were not affected by incubation NO donor NPS, and measured TRPV2 channel location to the membrane with BaCl2 (1 mM) plus 4-aminopiridine (1 mM) (Fig. 5C). Altogether, by immunofluorescence and biotinylation of surface proteins followed these results suggest that endothelium-dependent probenecid re­ by subcellular fractionation and western blot. Immunofluorescence was laxations are mediated by KIR and KV channels. performed with primary antibodies incubated in alive cultured cells, allowing only the extracellularly exposed TRPV2 channels to bind. In 3.4. Activation of muscular TRPV2 channels by probenecid favors ATP non-treated cells, TRPV2 channel fluorescence was distributed in het­ accumulation with blockade of KATP channels erogeneous clusters located at the plasma membrane (Fig. 7A). Incu­ bation with NPS (300 μM) tended (P = 0.070) to increase the amount of We then investigated the mechanism involved in the TRPV2 channel- TRPV2 channel-derived clusters of fluorescence (Fig. 7A), suggesting an mediated modulation of endothelium-independent probenecid re­ elevated presence of the receptor in the cellular membrane. To confirm laxations, which was revealed after the blockade of TRPV2 channels that this phenomenon was a result of the translocation of TRPV2 with tranilast. The experiments were performed in aortas without channels to the plasma membrane, biotinylation of cell surface proteins endothelium to facilitate the interpretation of results. followed by subcellular fractionation was performed and then analyzed In the presence of the TRPV2 channel antagonist tranilast, neither by western blot. In biotinylated fractions of smooth muscle cells, cor­ incubation of BaCl2 (1 mM), nor charybdotoxin (100 nM) and apamin responding to proteins facing outside the plasma membrane, exposure to (100 nM), nor 4-aminopiridine (1 mM), nor ouabain (1 μM) modified NPS (100 μM) promoted an increased presence of TRPV2 channels in the probenecid responses (Supplementary Fig. 4). In contrast, incubation membrane fraction of the cells (Fig. 7B). In endothelium-denuded with the KATP channel inhibitor glibenclamide (30 μM) prevented aortas, incubation with NPS (100 μM) did not modify probenecid re­ endothelium-independent probenecid-induced relaxations evidenced by laxations, but significantly increased the tranilast-induced potentiation TRPV2 channel blockade with tranilast (Fig. 6A). Taken together, these of these responses (Fig. 8). results suggest that KATP channels are involved in the potentiation effect of tranilast on probenecid endothelium-independent relaxations. We 4. Discussion hypothesized that activation of muscular TRPV2 channels by probenecid favors ATP accumulation with blockade of KATP channels, depolariza­ TRPV2 channels are expressed in smooth muscle and endothelial tion, opening of voltage-dependent Ca2+ channels and increase in the cells of some vascular beds and participate in vascular mechano­ contractile tone, which opposes probenecid relaxations. Consistently, transduction and sensing of high temperatures and lipids [5–8]. aortic intracellular ATP concentrations in the presence of probenecid Nevertheless, the mechanisms associated with TRPV2 channel activa­ were significantly decreased by TRPV2 channel blockade with tranilast tion by agonists on the fine-tune modulation of vascular tone are un­ (Fig. 6B). However, carbenoxolone, an inhibitor of pannexin-1 channels, known. Here, for the first time, we demonstrate that the TRPV2 channel did not modify the decrease in ATP concentrations elicited by proben­ agonist probenecid causes endothelium-dependent and independent ecid (Fig. 6B), excluding the participation of these channels in this relaxations, which are counteracted by TRPV2 channels located at the vascular response. muscular layer. Endothelium-dependent relaxations induced by pro­ benecid are mediated through activation of Kir and Kv channels. Fig. 6. Impact of inhibition of KATP channels on the effect of tranilast on probenecid relaxations in abdominal aortas without endothelium. (A) Concentration- response curves to probenecid in U46619 pre-contracted abdominal aortas in the absence (control) or presence of tranilast (100 μM) and the KATP channel blocker glibenclamide (30 μM). (B) Intracellular concentrations of ATP measured by HPLC in abdominal aortas exposed to probenecid (300 μM) and treated with tranilast, or tranilast plus carbenoxolone (100 μM). Results are mean ± SEM from 5 to 10 (A) mice and 5 independent experiments (10 mice) (B). ***P < 0.001 versus control by two-way ANOVA (A); *P < 0.05; **P < 0.01 versus Probenecid by one-way ANOVA (B). 7 A. Perálvarez-Marín et al. Life Sciences 336 (2024) 122286 Fig. 7. Impact of NO exposure on TRPV2 channel location in the plasma membrane. (A) Representative photomicrographs of TRPV2 channel immunofluorescence (red; left) and quantification (right) of fluorescence (area x intensity) in non-treated (NT) and treated (sodium nitroprusside, NPS, 10–300 μM) A7r5 rat aortic smooth muscle cells. Nuclear staining with DAPI (blue) is also shown. (B) Western blot detection of TRPV2 channels in total, internal and membrane fractions obtained after cell surface biotinylation and subsequent subcellular fractionation in NT and treated (NPS, 10–300 μM) A7r5 rat aortic smooth muscle cells (above), with the corresponding signal quantification (below). Controls of membrane and internal fractions are represented by IGFRecB and GAPDH, respectively. Ponceau staining was used as a loading control. Results are the mean ± SEM from 5 (A) and 3 (B) independent experiments. The colored dots indicate values of each independent experiment. P = 0.070 by one-way ANOVA; *P < 0.05 by paired Student's t-test. Endothelium-independent relaxations are more evident when TRPV2 removal. It is worth noting that after the removal of this layer, pro­ channels are blocked by tranilast, demonstrating that TRPV2 channels benecid still evoked endothelium-independent relaxations, as discussed located out of the endothelium exert negative feedback on probenecid later. relaxations. We propose that smooth muscle TRPV2 channels activated In a previous study in rat thoracic aortas pre-contracted with the α1- by probenecid trigger an increase in ATP levels with the consequent adrenergic receptor agonist Phe, the authors reported that endothelium KATP channel inhibition (Fig. 9). Furthermore, we show that NO aug­ removal was insufficient to block probenecid-induced relaxations, ments the translocation of TRPV2 channels to the plasma membrane of which were attributed to α-adrenergic receptor blockade [23]. In our aortic smooth muscle cells, an effect that is associated with an increase conditions, the classic α1-adrenoceptor antagonist prazosin was unable in the negative feedback on probenecid relaxations (Fig. 10). to relax U44619 pre-contracted mouse aortas, suggesting that this Previous studies showed that TRPV2 channels are expressed at the signaling pathway is not likely to participate in our experimental con­ transcriptional level in rat aortas without endothelium [9]. In addition, ditions. Although we do not know the reasons for the discrepancy in the these channels were also detected in human pulmonary artery endo­ mechanisms involved in probenecid relaxations between our study and thelial cells [12]. In the present study, we show that TRPV2 channels are previous evidence [23], we speculate that the use of a different pre- expressed in endothelial, smooth muscle and adventitial cells of the contractile agonist (U46619 versus Phe), lower concentrations of pro­ mouse aorta, and then we study their potential functional role in benecid in our study (300 μM versus 1 mM), or different species (mice modulating aortic tone. We demonstrate that the TRPV2 channel agonist versus rats) might be involved in these differences. Altogether, the pre­ probenecid evokes aortic relaxations, largely attenuated by endothelium sent study does not exclude the possibility that probenecid may inhibit 8 A. Perálvarez-Marín et al. Life Sciences 336 (2024) 122286 Fig. 8. Impact of NO exposure on the tranilast-induced potentiation of probenecid relaxations. (A) Impact of incubation with the NO donor, sodium nitroprusside (NPS, 100 μM), on the effect of the selective TRPV2 inhibitor, tranilast (100 μM), on the concentration-response curves to probenecid in U46619 pre-contracted abdominal aortas without endothelium. (B) Quantification of the area under curve of concentration-response curves to probenecid. Results are the mean ± SEM from 5 to 6 mice. *P < 0.05, **P < 0.01 by two-way ANOVA. α-adrenergic receptors in the mouse aorta. However, α-adrenergic re­ tranilast did not decrease but largely increased probenecid relaxations ceptor signaling is not involved in mouse aorta probenecid relaxations in regardless of the presence of endothelium, suggesting that muscular U46619 pre-contracted vessels, which we hypothesized could be TRPV2 channel activity exerts negative feedback on probenecid re­ dependent on TRPV2 channel signaling. To our knowledge, this is the laxations. In a previous study, it was shown that tranilast increases ACh- first evidence of the possible involvement of TRPV2 channels in induced endothelium-dependent relaxations in rat mesenteric arteries, endothelial-dependent relaxations. but not in the rat aorta [42]. Likewise, tranilast was not able to modify Different members of the TRPV channel family have been associated ACh relaxations in the mouse aorta, which excludes the participation of with endothelium-dependent relaxations. Thus, endothelial TRPV1 TRPV2 channels in these responses and, in turn, confirms the specific channels participate in NO release leading to rat mesenteric and pig impact of TRPV2 channel inhibition on probenecid-induced relaxations. coronary artery relaxations [36,37]. In addition, activation of TRPV4 Notably, although the enhancement of relaxations after TRPV2 channel channels induces NO- and EDHF-dependent vasodilation in rat and inhibition may be a consequence of the blockade of TRPV2 channel- mouse conduit and resistance arteries [38–41]. A previous work induced smooth muscle Ca2+ entry leading to a decrease in smooth revealed that TRPV2 channel activation evokes relaxation in mouse muscle tone, we propose that these findings might also reveal the intestine through NO release from inhibitory motor neurons [30]. presence of a direct effect of probenecid on an endothelium-independent However, in the mouse aorta, inhibition of NO production by L-NAME relaxing mechanism, which would be counteracted by activation of did not significantly affect probenecid relaxations, which were only TRPV2 channels located in smooth muscle cells. attenuated after either Kir or Kv channel blockade. These results partly In endothelium-denuded aortic rings, the tranilast-induced blockade agree with those obtained in different TRPV2 channel-expressing cell of TRPV2 channels and the subsequent increase in probenecid re­ lines in which Kv channel blockade led to TRPV2 channel inhibition laxations was abrogated by KATP channel blockade, which suggests that [22]. The blockade of Kir or Kv channels did not have an effect on the increased probenecid relaxant activity after TRPV2 channel inhibi­ endothelium-denuded vessels, which suggests the essential role of this tion could be related to these channels. Nonetheless, at this stage, vascular layer. At this stage, the connection between Kir and Kv channels neither the present work nor previous evidence completely supports that and endothelium-dependent relaxations to probenecid is not clear, but probenecid may directly activate KATP channels (Fig. 9). KATP channels we propose that endothelial TRPV2 channel activation may release a couple cellular metabolism with membrane excitability, Ca2+ entry, and mediator that would diffuse into the smooth muscle layer to activate Kv cell functions, and KATP channel blockade results in vasoconstriction and and Kir channels (Fig. 9). Nevertheless, further studies are needed to depolarization of vascular smooth muscle cells [43]. In the present corroborate this hypothesis. study, antagonism of muscular TRPV2 channels with tranilast signifi­ The antiallergic drug tranilast has been used as a TRPV2 channel cantly decreased intracellular ATP concentrations in the mouse aorta, an antagonist [28–30], because it is one of the most specific, commercially environment that permits activation of KATP channels leading to smooth available TRPV2 channel inhibitors. Likewise, in the present study, we muscle hyperpolarization and relaxation. Besides, probenecid is re­ used tranilast to corroborate the role of TRPV2 channel activation on ported to block pannexin-1 channels, which are master regulators of probenecid-induced vascular responses. Paradoxically, incubation with intracellular ATP concentrations [27,44]. However, in our study, the 9 A. Perálvarez-Marín et al. Life Sciences 336 (2024) 122286 Fig. 9. Proposed mechanism of modulation of aortic tone by TRPV2 channels. (A) Probenecid by binding to TRPV2 channels located in endothelial cells induces the release of an unknown mediator, which activates voltage-gated and inwardly rectifying K+ channels in smooth muscle cells, leading to membrane hyperpolarization and subsequent aortic relaxation. On the other hand, probenecid binding to smooth muscle TRPV2 channels increases intracellular ATP concentrations, which inhibit KATP channel activation inducing negative feedback on probenecid relaxations. (B) Tranilast-induced blockade of TRPV2 channels decreases intracellular ATP concentrations, which activates KATP channels, leading to increased endothelium-independent relaxations. A potential direct effect of probenecid on activating KATP channels could not be discarded. pannexin-1 channel inhibitor carbenoxolone did not affect intracellular studies are needed to define the precise mechanisms underlying the ATP concentrations in the presence of tranilast, suggesting that another contribution of ATP in this novel signaling pathway. mechanism or mechanisms may be involved in this effect. Taken Prior studies have shown that in response to NO exposure, TRPV2 together, we suggest that smooth muscle KATP channel opening is channels located in the endoplasmic reticulum membrane translocate to silenced by smooth muscle TRPV2 channel activation, though further the plasma membrane in a phosphoinositide 3-kinase-dependent 10 A. Perálvarez-Marín et al. Life Sciences 336 (2024) 122286 Fig. 10. Influence of nitric oxide on the proposed mechanism of modulation of aortic tone by TRPV2 channels. (A) Nitric oxide augments TRPV2 channel trans­ location to the plasma membrane and may also directly activate and/or overexpress KATP channels leading to increased channel activity, an effect that counteracts each other. (B) In the presence of tranilast, the blockade of KATP channels by TRPV2 channels is removed and this effect, in conjunction with a potential direct effect of nitric oxide on KATP channel activation and/or overexpression, jointly contributes to the enhancement of probenecid relaxations. manner [28]. To find out whether NO increases plasma membrane probenecid relaxations induced by tranilast and, thus, of the negative translocation of TRPV2 channels in vascular cells, we exposed cultured feedback exerted by TRPV2 channels, which was dependent on KATP aortic smooth muscle cells to the NO donor NPS. Incubation with this channel activation. In fact, KATP channels are activated and over- compound led to augmented TRPV2 channel translocation to the plasma expressed by NO in the rat aorta during septic shock [45], and addi­ membrane. This effect was associated with further potentiation of tional evidence showed that NO activates these channels in rabbit 11 A. Perálvarez-Marín et al. Life Sciences 336 (2024) 122286 mesenteric artery vascular smooth muscle cells [46]. However, this ac­ paper. AP-M, MS, JS, AT, BP, CP, GM, MJ, PD'O and FJ-A participated in tivity was not observed in other vessels, such as rabbit mesenteric data interpretation and manuscript drafting and editing. All authors arterial [47] and pig coronary arterial [48] smooth muscle cells. Overall, approved the final version of the manuscript; agree to be accountable for these results argue in favor of a direct activation of KATP channels by all aspects of the work in ensuring that questions related to the accuracy probenecid but as mentioned above, this should be confirmed in sub­ or integrity of any part of the work are appropriately investigated and sequent studies (Fig. 10). In addition, these results suggest that NO is a resolved; and qualify for authorship, while all those who qualify for modulator of smooth muscle cell TRPV2 channel function, which authorship are listed. highlights the pathophysiological relevance of vascular TRPV2 signaling identified in the present study. A limitation of the present study is that Funding we did not evaluate whether NO increases plasma membrane trans­ location of TRPV2 channels in endothelial cells and the potential func­ This work was supported by the Spanish Government PID2020- tional impact on aortic relaxations. Future studies should investigate 113634RB-C22/AEI/10.13039/501100011033 grant to FJ-A and this potential modulation as a priority. PID2020-120222GB-I00/AEI/10.13039/501100011033 grant to AP-M, The pathophysiological importance of the relationship between NO, and from the Generalitat de Catalunya 2021 SGR 00969 grant to FJ-A. TRPV2 channels, and KATP channels could be better interpreted considering that increased iNOS-derived NO contributes to the patho­ Declaration of competing interest genesis of multiple vascular disorders, including hypertension [49]. This is mostly ascribed to nitrosative stress due to excessive NO production The authors declare that the research was conducted in the absence by iNOS, which after reaction with superoxide anion forms peroxyni­ of any commercial or financial relationships that could be interpreted as trite. This harmful molecule subsequently leads to significantly a potential conflict of interest. increased oxidative/nitrosative stress and cellular damage in a vicious cycle. According to our hypothesis, a surge in iNOS-derived NO may Data availability increase the contribution of TRPV2 channels to vascular contractile re­ sponses by its translocation to the membrane, but may also induce a Data will be made available on request. potential direct activation of KATP channels by NO. Given that reactive oxygen species have been reported to activate TRPV2 channel function Acknowledgments and expression [50,51], we speculate that iNOS-derived NO may in­ crease the negative feedback of TRPV2 channels on relaxations, which The authors thank Jennifer Enrich-Bengoa for her technical support. may constitute a novel mechanism involved in vasodilatory dysfunction that needs further attention. Appendix A. Supplementary data 5. 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