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
- 278 Downloads
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.
KeywordsATP release Mechanically evoked ATP release Thrombin receptors PAR agonist analogs TRPV Pannexin/connexin hemichannels Vesicular release
Cell medium displacement
- HC 067047
Transient receptor potential
Transient receptor potential vanilloid
vesicular nucleotide transporter
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.
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.
Compliance with ethical standards
Conflicts of interest
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.
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.
- 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 CrossRefPubMedPubMedCentralGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedPubMedCentralGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 69:143–153Google Scholar
- 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 CrossRefPubMedPubMedCentralGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 PubMedGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedPubMedCentralGoogle Scholar
- 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 CrossRefPubMedPubMedCentralGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedPubMedCentralGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 63.Ossovskaya VS1, Bunnett NW (2004) Protease-activated receptors: contribution to physiology and disease. Physiol Rev 84:579–621Google Scholar