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P2X receptors regulate adenosine diphosphate release from hepatic cells

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Abstract

Extracellular nucleotides act as paracrine regulators of cellular signaling and metabolic pathways. Adenosine polyphosphate (adenosine triphosphate (ATP) and adenosine diphosphate (ADP)) release and metabolism by human hepatic carcinoma cells was therefore evaluated. Hepatic cells maintain static nanomolar concentrations of extracellular ATP and ADP levels until stress or nutrient deprivation stimulates a rapid burst of nucleotide release. Reducing the levels of media serum or glucose has no effect on ATP levels, but stimulates ADP release by up to 10-fold. Extracellular ADP is then metabolized or degraded and media ADP levels fall to basal levels within 2–4 h. Nucleotide release from hepatic cells is stimulated by the Ca2+ ionophore, ionomycin, and by the P2 receptor agonist, 2′3′-O-(4-benzoyl-benzoyl)-adenosine 5′-triphosphate (BzATP). Ionomycin (10 μM) has a minimal effect on ATP release, but doubles media ADP levels at 5 min. In contrast, BzATP (10–100 μM) increases both ATP and ADP levels by over 100-fold at 5 min. Ion channel purinergic receptor P2X7 and P2X4 gene silencing with small interference RNA (siRNA) and treatment with the P2X inhibitor, A438079 (100 μM), decrease ADP release from hepatic cells, but have no effect on ATP. P2X inhibitors and siRNA have no effect on BzATP-stimulated nucleotide release. ADP release from human hepatic carcinoma cells is therefore regulated by P2X receptors and intracellular Ca2+ levels. Extracellular ADP levels increase as a consequence of a cellular stress response resulting from serum or glucose deprivation.

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Abbreviations

ADP:

Adenosine diphosphate

AK1:

Adenylate kinase

ATP:

Adenosine triphosphate

BzATP:

2′3′-O-(4-Benzoyl-benzoyl)-adenosine 5′-triphosphate

NTPDase:

Ectonucleotidase

P2X:

Ion channel purinergic receptor

P2Y:

G-protein-coupled purinergic receptor

VNUT:

Vesicular nucleotide transporter

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Authors and Affiliations

  1. Atherosclerosis, Genetics and Cell Biology Group, University of Ottawa Heart Institute, 40 Ruskin Street, Ottawa, Ontario, K1Y 4 W7, Canada

    Cynthia Chatterjee & Daniel L. Sparks

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  1. Cynthia Chatterjee
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  2. Daniel L. Sparks
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Correspondence to Daniel L. Sparks.

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Supplemental Figure 1

Adenylate kinase inactivation has no effect on nucleotide release. (A) Hepatic cells were transfected with either negative control (si-Ctrl) or adenylate kinase (AK1) siRNA and incubated for 48 h. Hepatic cells were then incubated in FBS-depleted media overnight, media was changed and media aliquots were sampled (A) or treated with 100 μM of the P2 receptor agonist, BzATP (B). Media ATP&ADP concentrations were determined by bioluminescence assay and values are expressed as mean ± SD of 3 independent experiments. (PDF 41 kb)

Supplemental Figure 2

P2X receptor inhibition blocks ADP release. (A) Hepatic cells were incubated in FBS-depleted media overnight and then media was changed. Cells were treated with the P2X receptor inhibitor, A438079 (100 μM) and media aliquots were sampled over 120 min. Media ATP and ADP concentrations were determined by bioluminescence assay and values are expressed as mean ± SD of 3 independent experiments. *P < 0.05 vs control (B) A438079 treated cells were treated with 10 μM of the P2 receptor agonist, BzATP and media ATP&ADP concentrations were determined by bioluminescence assay and values are expressed as mean ± SD of 3 independent experiments. (PDF 19 kb)

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Chatterjee, C., Sparks, D.L. P2X receptors regulate adenosine diphosphate release from hepatic cells. Purinergic Signalling 10, 587–593 (2014). https://doi.org/10.1007/s11302-014-9419-2

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  • DOI: https://doi.org/10.1007/s11302-014-9419-2

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