Intracellular acidification increases adenosine transport in human umbilical vein endothelial cells

Celis, Natalia, Araos, Joaquín, Sanhueza, Carlos, Toledo, Fernando, Beltran, Ana R., Pardo, Fabian, Leiva, Andrea, Ramirez, Marco A. and Sobrevia, Luis (2017) Intracellular acidification increases adenosine transport in human umbilical vein endothelial cells. Placenta, 51 10-17. doi:10.1016/j.placenta.2017.01.120

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Author Celis, Natalia
Araos, Joaquín
Sanhueza, Carlos
Toledo, Fernando
Beltran, Ana R.
Pardo, Fabian
Leiva, Andrea
Ramirez, Marco A.
Sobrevia, Luis
Title Intracellular acidification increases adenosine transport in human umbilical vein endothelial cells
Journal name Placenta   Check publisher's open access policy
ISSN 1532-3102
0143-4004
Publication date 2017-03-01
Sub-type Article (original research)
DOI 10.1016/j.placenta.2017.01.120
Open Access Status File (Author Post-print)
Volume 51
Start page 10
End page 17
Total pages 8
Place of publication London, United Kingdom
Publisher Elsevier
Language eng
Subject 2743 Reproductive Medicine
2729 Obstetrics and Gynaecology
1309 Developmental Biology
Abstract Introduction Adenosine is taken up via human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) at a physiological extracellular pH (pHo ∼7.4) in human umbilical vein endothelial cells (HUVECs). Acidic pHo increases the uptake of adenosine and 5-hydroxytryptamine (5HT) via hENT4 in this cell type. However, modulation of hENT1 and hENT2 transport activity by the pHi is unknown. We investigated whether hENT1 and hENT2-adenosine transport was regulated by acidic pHi. Methods HUVECs loaded with a pH sensitive probe were subjected to 0.1–20 mmol/L NHCl pulse assay to generate 6.9–6.2 pHi. Before pHi started to recover, adenosine transport kinetics (0–500 μmol/L, 37 °C) in the absence or presence 1 or 10 μmol/L S-(4-nitrobenzyl)-6-thio-inosine (NBTI), 2 mmol/L hypoxanthine, 2 mmol/L adenine, 100 μmol/L 5HT, or 500 μmol/L adenosine, was measured. Results Overall adenosine transport (i.e., hENT1+hENT2) was semisaturable and partially inhibited by 1 μmol/L, but abolished by 10 μmol/L NBTI in cells non-treated or treated with NHCl. The initial velocity and non-saturable, lineal component for overall transport were increased after NHCl pulse. hENT1 and hENT2-mediated adenosine transport maximal capacity was increased by acidic pHi. hENT1 activity was more sensitive than hENT2 activity to acidic pHi. Discussion hENT1 and hENT2-adenosine transport is differentially regulated by acidic pHi in HUVECs. These findings are important in pathologies associated with pHi alterations such as gestational diabetes mellitus.
Formatted abstract
Introduction: Adenosine is taken up via human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) at a physiological extracellular pH (pHo ∼7.4) in human umbilical vein endothelial cells (HUVECs). Acidic pHo increases the uptake of adenosine and 5-hydroxytryptamine (5HT) via hENT4 in this cell type. However, modulation of hENT1 and hENT2 transport activity by the pHi is unknown. We investigated whether hENT1 and hENT2-adenosine transport was regulated by acidic pHi.

Methods: HUVECs loaded with a pH sensitive probe were subjected to 0.1–20 mmol/L NH4Cl pulse assay to generate 6.9–6.2 pHi. Before pHi started to recover, adenosine transport kinetics (0–500 μmol/L, 37 °C) in the absence or presence 1 or 10 μmol/L S-(4-nitrobenzyl)-6-thio-inosine (NBTI), 2 mmol/L hypoxanthine, 2 mmol/L adenine, 100 μmol/L 5HT, or 500 μmol/L adenosine, was measured.

Results: Overall adenosine transport (i.e., hENT1+hENT2) was semisaturable and partially inhibited by 1 μmol/L, but abolished by 10 μmol/L NBTI in cells non-treated or treated with NH4Cl. The initial velocity and non-saturable, lineal component for overall transport were increased after NH4Cl pulse. hENT1 and hENT2-mediated adenosine transport maximal capacity was increased by acidic pHi. hENT1 activity was more sensitive than hENT2 activity to acidic pHi.

Discussion: hENT1 and hENT2-adenosine transport is differentially regulated by acidic pHi in HUVECs. These findings are important in pathologies associated with pHi alterations such as gestational diabetes mellitus.
Keyword Adenosine transport
Endothelium
ENT transporters
Foetus
Intracellular pH
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID 11150083
1150344
3140516
FONDECYT 1150377
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: UQ Centre for Clinical Research Publications
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