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Purinergic Signalling

, Volume 14, Issue 2, pp 121–139 | Cite as

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

  • M. Verónica Donoso
  • Felipe Hernández
  • Tania Villalón
  • Claudio Acuña-Castillo
  • J. Pablo Huidobro-ToroEmail author
Original Article

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.

Keywords

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

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

Notes

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.

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.

Ethical approval

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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© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • M. Verónica Donoso
    • 1
  • Felipe Hernández
    • 1
  • Tania Villalón
    • 1
  • Claudio Acuña-Castillo
    • 1
  • J. Pablo Huidobro-Toro
    • 1
    Email author
  1. 1.Centro Desarrollo de NanoCiencia y Nanotecnología, CEDENNA y Laboratorio de Farmacología, Departamento de Biología, Facultad de Química y BiologíaUniversidad de SantiagoSantiagoChile

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