Pharmacological dissection of the cellular mechanisms associated to the spontaneous and the mechanically stimulated ATP release by mesentery endothelial cells: roles of thrombin and TRPV

Abstract

Endothelial cells participate in extracellular ATP release elicited by mechanosensors. To characterize the dynamic interactions between mechanical and chemical factors that modulate ATP secretion by the endothelium, we assessed and compared the mechanisms participating in the spontaneous (basal) and mechanically stimulated secretion using primary cultures of rat mesentery endothelial cells. ATP/metabolites were determined in the cell media prior to (basal) and after cell media displacement or a picospritzer buffer puff used as mechanical stimuli. Mechanical stimulation increased extracellular ATP that peaked within 1 min, and decayed to basal values in 10 min. Interruption of the vesicular transport route consistently blocked the spontaneous ATP secretion. Cells maintained in media lacking external Ca2+ elicited a spontaneous rise of extracellular ATP and adenosine, but failed to elicit a further extracellular ATP secretion following mechanical stimulation. 2-APB, a TRPV agonist, increased the spontaneous ATP secretion, but reduced the mechanical stimulation-induced nucleotide release. Pannexin1 or connexin blockers and gadolinium, a Piezo1 blocker, reduced the mechanically induced ATP release without altering spontaneous nucleotide levels. Moreover, thrombin or related agonists increased extracellular ATP secretion elicited by mechanical stimulation, without modifying spontaneous release. In sum, present results allow inferring that the spontaneous, extracellular nucleotide secretion is essentially mediated by ATP containing vesicles, while the mechanically induced secretion occurs essentially by connexin or pannexin1 hemichannel ATP transport, a finding fully supported by results from Panx1−/− rodents. Only the latter component is modulated by thrombin and related receptor agonists, highlighting a novel endothelium-smooth muscle signaling role of this anticoagulant.

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Abbreviations

ATP:

Adenosine 5′-triphosphate

ADP:

Adenosine 5′-diphosphate

AMP:

Adenosine 5′-monophosphate

ADO:

Adenosine

2-APB:

2-Aminoethoxydiphenylborane

CMD:

Cell medium displacement

DMSO:

Dimethyl sulfoxide

ECs:

Endothelial cells

Gd:

Gadolinium III

H-1152P:

(S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine

HC 067047:

2-Methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H–pyrrole-3-carboxamide

Panx1−/− :

Pannexin1 knockout

MβCD:

Methyl-β-cyclodextrin

NEM:

N-ethylmaleimide

PAR:

Protease-activated receptor

TRP:

Transient receptor potential

TRPV:

Transient receptor potential vanilloid

VNUT:

vesicular nucleotide transporter

Y-27632:

(R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide

References

  1. 1.

    Burnstock G (1980) Purinergic nerves and receptors. Prog Biochem Pharmacol 16:141–154

    CAS  PubMed  Google Scholar 

  2. 2.

    Burnstock G (2017) Purinergic signaling in the cardiovascular system. Circ Res 120(1):207–228. https://doi.org/10.1161/CIRCRESAHA.116.309726

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Coddou C, Stojilkovic SS, Huidobro-Toro JP (2011) Allosteric modulation of ATP-gated P2X receptor channels. Rev Neurosci 22(3):335–354. https://doi.org/10.1515/RNS.2011.014

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Ralevic V, Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50(3):413–492

    CAS  PubMed  Google Scholar 

  5. 5.

    Navarrete LC, Barrera NP, Huidobro-Toro JP (2014) Vas deferens neuro-effector junction: from kymographic tracings to structural biology principles. Auton Neurosc 185:8–28. https://doi.org/10.1016/j.autneu.2014.05.010

    CAS  Article  Google Scholar 

  6. 6.

    Buvinic S, Briones R, Huidobro-Toro JP (2002) P2Y1 and P2Y2 receptors are coupled to the NO/cGMP pathway to vasodilate the rat arterial mesenteric bed. Br J Pharmacol 136(6):847–856. https://doi.org/10.1038/sj.bjp.0704789

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Buvinic S, Poblete MI, Donoso MV, Delpiano AM, Briones R, Miranda R, Huidobro-Toro JP (2006) P2Y1 and P2Y2 receptor distribution varies along the human placental vascular tree: role of nucleotides in vascular tone regulation. J Physiol 573(Pt 2):427–443. https://doi.org/10.1113/jphysiol.2006.105882

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Coste B, Mathur J, Schmidt M, Earley TJ, Ranade S, Petrus MJ, Dubin AE, Patapoutian A (2010) Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science 330(6000):55–60. https://doi.org/10.1126/science.1193270

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Wang S, Chennupati R, Kaur H, Iring A, Wettschureck N, Offermanns S (2016) Endothelial cation channel PIEZO1 controls blood pressure by mediating flow-induced ATP release. J Clin Invest 126(12):4527–4453. https://doi.org/10.1172/JCI87343

    Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Yin J, Kuebler WM (2010) Mechanotransduction by TRP channels: general concepts and specific role in the vasculature. Cell Biochem Biophys 56(1):1–18. https://doi.org/10.1007/s12013-009-9067-2

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Pankratov Y, Lalo U, Verkhratsky A, North RA (2006) Vesicular release of ATP at central synapses. Pflugers Arch 452(5):589–597. https://doi.org/10.1007/s00424-006-0061-x

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Locovei S, Wang J, Dahl G (2006) Activation of pannexin 1 channels by ATP through P2Y receptors and by cytoplasmic calcium. FEBS Lett 580(1):239–244. https://doi.org/10.1016/j.febslet.2005.12.004

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Rondaij MG, Bierings R, Kragt A, van Mourik JA, Voorberg J (2006) Dynamics and plasticity of Weibel-Palade bodies in endothelial cells. Arterioscler Thromb Vasc Biol 26(5):1002–1007. https://doi.org/10.1161/01.ATV.0000209501.56852.6c

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Lim To WK, Kumar P, Marshall JM (2015) Hypoxia is an effective stimulus for vesicular release of ATP from human umbilical vein endothelial cells. Placenta 36(7):759–766. https://doi.org/10.1016/j.placenta.2015.04.005

    Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Sawada K, Echigo N, Juge N, Miyaji T, Otsuka M, Omote H, Yamamoto A, Moriyama Y (2008) Identification of a vesicular nucleotide transporter. Proc Natl Acad Sci U S A 105(15):5683–5686. https://doi.org/10.1073/pnas.0800141105

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Lohman AW, Isakson BE (2014) Differentiating connexin hemichannels and pannexin channels in cellular ATP release. FEBS Lett 588(8):1379–1388. https://doi.org/10.1016/j.febslet.2014.02.004

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Silverman W, Locovei S, Dahl G (2008) Probenecid, a gout remedy, inhibits pannexin 1 channels. Am J Physiol Cell Physiol 295(3):C761–C767. https://doi.org/10.1152/ajpcell.00227.2008

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Ballerio R, Brambilla M, Colnago D, Parolari A, Agrifoglio M, Camera M, Tremoli E, Mussoni L (2007) Distinct roles for PAR1- and PAR2-mediated vasomotor modulation in human arterial and venous conduits. J Thromb Haemost 5(1):174–180. https://doi.org/10.1111/j.1538-7836.2006.02265.x

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Lin H, Liu AP, Smith TH, Trejo J (2013) Cofactoring and dimerization of proteinase-activated receptors. Pharmacol Rev 65(4):1198–1213. https://doi.org/10.1124/pr.111.004747

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Penuela S, Gehi R, Laird DW (2013) The biochemistry and function of pannexin channels. Biochim Biophys Acta 828:15–22

    Article  Google Scholar 

  21. 21.

    Bargiotas P, Krenz A, Hormuzdi SG, Ridder DA, Herb A, Barakat W, Penuela S, von Engelhardt J, Monyer H, Schwaninger M (2011) Pannexins in ischemia-induced neurodegeneration. Proc Natl Acad Sci U S A 108(51):20772–20777. https://doi.org/10.1073/pnas.1018262108

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Shoji KF, Sáez PJ, Harcha PA, Aguila HL, Sáez JC (2014) Pannexin1 channels act downstream of P2X 7 receptors in ATP-induced murine T-cell death. Channels (Austin) 8(2):142–156. https://doi.org/10.4161/chan.28122

    CAS  Article  PubMed Central  Google Scholar 

  23. 23.

    Ashley RA, Dubuque SH, Dvorak B, Woodward SS, Williams SK, Kling PJ (2002) Erythropoietin stimulates vasculogenesis in neonatal rat mesenteric microvascular endothelial cells. Pediatr Res 51(4):472–478. https://doi.org/10.1203/00006450-200204000-00012

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Lazarowski ER, Homolya L, Boucher RC, Harden TK (1997) Direct demonstration of mechanically induced release of cellular UTP and its implication for uridine nucleotide receptor activation. J Biol Chem 272(39):24348–24354. https://doi.org/10.1074/jbc.272.39.24348

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Hovater MB, Olteanu D, Hanson EL, Cheng NL, Siroky B, Fintha A, Komlosi P, Liu W, Satlin LM, Bell PD, Yoder BK, Schwiebert EM (2008) Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and flow-induced calcium signals. Purinergic Signal 4(2):155–170. https://doi.org/10.1007/s11302-007-9072-0

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Romanello M, Pani B, Bicego M, D'Andrea P (2001) Mechanically induced ATP release from human osteoblastic cells. Biochem Biophys Res Commun 289(5):1275–1281. https://doi.org/10.1006/bbrc.2001.6124

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Knight GE, Bodin P, De Groat WC, Burnstock G (2002) ATP is released from guinea pig ureter epithelium on distension. Am J Physiol Renal Physiol 282(2):F281–F288. https://doi.org/10.1152/ajprenal.00293.2000

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Ren Y, Liu W, Jiang H, Jiang Q, Feng J (2005) Selective vulnerability of dopaminergic neurons to microtubule depolymerization. J Biol Chem 280(40):34105–34112. https://doi.org/10.1074/jbc.M503483200

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Gödecke S, Roderigo C, Rose CR, Rauch BH, Gödecke A, Schrader J (2012) Thrombin-induced ATP release from human umbilical vein endothelial cells. Am J Physiol Cell Physiol 302(6):C915–C923. https://doi.org/10.1152/ajpcell.00283.2010

    Article  PubMed  Google Scholar 

  30. 30.

    Arcuino G, Lin JH, Takano T, Liu C, Jiang L, Gao Q, Kang J, Nedergaard M (2002) Intercellular calcium signaling mediated by point-source burst release of ATP. Proc Natl Acad Sci U S A 99(15):9840–9845. https://doi.org/10.1073/pnas.152588599

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Braet K, Aspeslagh S, Vandamme W, Willecke K, Martin PE, Evans WH, Leybaert L (2003) Pharmacological sensitivity of ATP release triggered by photoliberation of inositol-1,4,5-trisphosphate and zero extracellular calcium in brain endothelial cells. J Cell Physiol 197(2):205–213. https://doi.org/10.1002/jcp.10365

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Zanotti S, Charles A (1997) Extracellular calcium sensing by glial cells: low extracellular calcium induces intracellular calcium release and intercellular signaling. J Neurochem 69(2):594–602

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Earley S, Brayden JE (2015) Transient receptor potential channels in the vasculature. Physiol Rev 95(2):645–690. https://doi.org/10.1152/physrev.00026.2014

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Seki T, Goto K, Kiyohara K, Kansui Y, Murakami N, Haga Y, Ohtsubo T, Matsumura K, Kitazono T (2017) Downregulation of endothelial transient receptor potential Vanilloid type 4 channel and small-conductance of Ca2+-activated K+ channels underpins impaired endothelium-dependent hyperpolarization in hypertension. Hypertension 69:143–153

  35. 35.

    Sullivan MN, Earley S (2013) TRP channel Ca(2+) sparklets: fundamental signals underlying endothelium-dependent hyperpolarization. Am J Physiol Cell Physiol 305(10):C999–C1008. https://doi.org/10.1152/ajpcell.00273.2013

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Vriens J, Appendino G, Nilius B (2009) Pharmacology of vanilloid transient receptor potential cation channels. Mol Pharmacol 75(6):1262–1279. https://doi.org/10.1124/mol.109.055624

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    St Pierre M, Reeh PW, Zimmermann K (2009) Differential effects of TRPV channel block on polymodal activation of rat cutaneous nociceptors in vitro. Exp Brain Res 196(1):31–44. https://doi.org/10.1007/s00221-009-1808-3

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Adding LC, Bannenberg GL, Gustafsson LE (2001) Basic experimental studies and clinical aspects of gadolinium salts and chelates. Cardiovasc Drug Rev 19(1):41–56

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Li J, Hou B, Tumova S, Muraki K, Bruns A, Ludlow MJ, Sedo A, Hyman AJ, McKeown L, Young RS, Yuldasheva NY, Majeed Y, Wilson LA, Rode B, Bailey MA, Kim HR, Fu Z, Carter DA, Bilton J, Imrie H, Ajuh P, Dear TN, Cubbon RM, Kearney MT, Prasad KR, Evans PC, Ainscough JF, Beech DJ (2014) Piezo1 integration of vascular architecture with physiological force. Nature 515(7526):279–282. https://doi.org/10.1038/nature13701

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Yang XC, Sachs F (1989) Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions. Science 243(4894):1068–1071. https://doi.org/10.1126/science.2466333

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Hollenberg MD, Compton SJ (2002) International Union of Pharmacology. XXVIII. Proteinase-activated receptors. Pharmacol Rev 54(2):203–217. https://doi.org/10.1124/pr.54.2.203

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Ikenoya M, Hidaka H, Hosoya T, Suzuki M, Yamamoto N, Sasaki Y (2002) Inhibition of rho-kinase-induced myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation in human neuronal cells by H-1152, a novel and specific Rho-kinase inhibitor. J Neurochem 81(1):9–16. https://doi.org/10.1046/j.1471-4159.2002.00801.x

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Lingor P, Teusch N, Schwarz K, Mueller R, Mack H, Bähr M, Mueller BK (2007) Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo. J Neurochem 103(1):181–189. https://doi.org/10.1111/j.1471-4159.2007.04756.x

    CAS  PubMed  Google Scholar 

  44. 44.

    Romanenko VG, Fang Y, Byfield F, Travis AJ, Vandenberg CA, Rothblat GH, Levitan I (2004) Cholesterol sensitivity and lipid raft targeting of Kir2.1 channels. Biophys J 87(6):3850–3861. https://doi.org/10.1529/biophysj.104.043273

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Norambuena A, Poblete MI, Donoso MV, Espinoza CS, González A, Huidobro-Toro JP (2008) P2Y1 receptor activation elicits its partition out of membrane rafts and its rapid internalization from human blood vessels: implications for receptor signaling. Mol Pharmacol 74(6):1666–1677. https://doi.org/10.1124/mol.108.048496

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Buvinic S, Bravo-Zehnder M, Boyer JL, Huidobro-Toro JP, González A (2007) Nucleotide P2Y1 receptor regulates EGF receptor mitogenic signaling and expression in epithelial cells. J Cell Sci 120(24):4289–4301. https://doi.org/10.1242/jcs.03490

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Lazarowski ER, Tarran R, Grubb BR, van Heusden CA, Okada S, Boucher RC (2004) Nucleotide release provides a mechanism for airway surface liquid homeostasis. J Biol Chem 279(35):36855–36864. https://doi.org/10.1074/jbc.M405367200

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Ralevic V, Milner P, Kirkpatrick KA, Burnstock G (1992) Flow-induced release of adenosine 5′-triphosphate from endothelial cells of the rat mesenteric arterial bed. Experientia 48(1):31–34. https://doi.org/10.1007/BF01923600

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Raqeeb A, Sheng J, Ao N, Braun AP (2011) Purinergic P2Y2 receptors mediate rapid Ca(2+) mobilization, membrane hyperpolarization and nitric oxide production in human vascular endothelial cells. Cell Calcium 49(4):240–248. https://doi.org/10.1016/j.ceca.2011.02.008

    CAS  Article  PubMed  Google Scholar 

  50. 50.

    Lazarowski ER, Sesma JI, Seminario-Vidal L, Kreda SM (2011) Molecular mechanisms of purine and pyrimidine nucleotide release. Adv Pharmacol 61:221–261. https://doi.org/10.1016/B978-0-12-385526-8.00008-4

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    Glick BS, Rothman JE (1987) Possible role for fatty acyl-coenzyme A in intracellular protein transport. Nature 326(6110):309–312. https://doi.org/10.1038/326309a0

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Bodin P, Burnstock G (2001) Evidence that release of adenosine triphosphate from endothelial cells during increased shear stress is vesicular. J Cardiovasc Pharmacol 38(6):900–908. https://doi.org/10.1097/00005344-200112000-00012

    CAS  Article  PubMed  Google Scholar 

  53. 53.

    Ferraro F, Mafalda Lopes d S, Grimes W, Lee HK, Ketteler R, Kriston-Vizi J, Cutler DF (2016) Weibel-Palade body size modulates the adhesive activity of its von Willebrand factor cargo in cultured endothelial cells. Sci Rep 6(1):32473. https://doi.org/10.1038/srep32473

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. 54.

    HZ H, Gu Q, Wang C, Colton CK, Tang J, Kinoshita-Kawada M, Lee LY, Wood JD, Zhu MX (2004) 2-Aminoethoxydiphenyl borate is a common activator of TRPV1, TRPV2, and TRPV3. J Biol Chem 279:35741–35748

    Article  Google Scholar 

  55. 55.

    Chung MK, Lee H, Mizuno A, Suzuki M, Caterina MJ (2004) 2-Aminoethoxydiphenyl borate activates and sensitizes the heat-gated ion channel TRPV3. J Neurosci 24(22):5177–5182. https://doi.org/10.1523/JNEUROSCI.0934-04.2004

    CAS  Article  PubMed  Google Scholar 

  56. 56.

    Pires PW, Sullivan MN, Pritchard HA, Robinson JJ, Earley S (2015) Unitary TRPV3 channel Ca2+ influx events elicit endothelium-dependent dilation of cerebral parenchymal arterioles. Am J Physiol Heart Circ Physiol 309(12):H2031–H2041. https://doi.org/10.1152/ajpheart.00140.2015

    Article  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Bishara NB, Murphy TV, Hill MA (2002) Capacitative Ca(2+) entry in vascular endothelial cells is mediated via pathways sensitive to 2 aminoethoxydiphenyl borate and xestospongin C. Br J Pharmacol 135(1):119–128. https://doi.org/10.1038/sj.bjp.0704465

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Eguchi R, Akao S, Otsuguro K, Yamaguchi S, Ito S (2015) Different mechanisms of extracellular adenosine accumulation by reduction of the external Ca(2+) concentration and inhibition of adenosine metabolism in spinal astrocytes. J Pharmacol Sci 128(1):47–53. https://doi.org/10.1016/j.jphs.2015.04.008

    CAS  Article  PubMed  Google Scholar 

  59. 59.

    Hahn C, Schwartz MA (2009) Mechanotransduction in vascular physiology and atherogenesis. Nat Rev Mol Cell Biol 10(1):53–62. https://doi.org/10.1038/nrm2596

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Mihara K, Ramachandran R, Saifeddine M, Hansen KK, Renaux B, Polley D, Gibson S, Vanderboor C, Hollenberg MD (2016) Thrombin-mediated direct activation of proteinase-activated receptor-2: another target for thrombin signaling. Mol Pharmacol 89(5):606–614. https://doi.org/10.1124/mol.115.102723

    CAS  Article  PubMed  Google Scholar 

  61. 61.

    Thuet KM, Bowles EA, Ellsworth ML, Sprague RS, Stephenson AH (2011) The Rho kinase inhibitor Y-27632 increases erythrocyte deformability and low oxygen tension-induced ATP release. Am J Physiol Heart Circ Physiol 301(5):H1891–H1896. https://doi.org/10.1152/ajpheart.00603.2011

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Sasaki Y, Suzuki M, Hidaka H (2002) The novel and specific Rho-kinase inhibitor (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinoline)sulfonyl]-homopiperazine as a probing molecule for Rho-kinase-involved pathway. Pharmacol Ther 93(2-3):225–232. https://doi.org/10.1016/S0163-7258(02)00191-2

    CAS  Article  PubMed  Google Scholar 

  63. 63.

    Ossovskaya VS1, Bunnett NW (2004) Protease-activated receptors: contribution to physiology and disease. Physiol Rev 84:579–621

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Acknowledgments

We would like to thank Prof. E. Lazarowski who patiently assisted and advised us to conduct some of these protocols, Prof. E. Leiva for graphical abstract figure design, and Ms. G. Sánchez for Panx1−/− mice and WT husbandry.

Funding sources

This work was funded by FONDECYT grants 114-1132 and 117-0842 and the Center for the Development of NanoScience and Nanotechnology, CEDENNA (FB 0807) also contributed with partial funds.

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Correspondence to J. Pablo Huidobro-Toro.

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M. Verónica Donoso declares that she has no conflict of interest.

Felipe Hernández declares that he has no conflict of interest.

Tania Villalón declares that she has no conflict of interest.

Claudio Acuña-Castillo declares that he has no conflict of interest.

J. Pablo Huidobro-Toro declares that he has no conflict of interest.

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The Universidad de Santiago Ethical Committee for the use of animals in biological research approved the specific protocols designed and supervised our strict adherence to the subscribed guidelines through the local Ethical Committee of the Faculty of Chemistry and Biology.

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Verónica Donoso, M., Hernández, F., Villalón, T. et al. Pharmacological dissection of the cellular mechanisms associated to the spontaneous and the mechanically stimulated ATP release by mesentery endothelial cells: roles of thrombin and TRPV. Purinergic Signalling 14, 121–139 (2018). https://doi.org/10.1007/s11302-017-9599-7

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Keywords

  • ATP release
  • Mechanically evoked ATP release
  • Thrombin receptors
  • PAR agonist analogs
  • TRPV
  • Pannexin/connexin hemichannels
  • Vesicular release