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Oxidized ATP. An irreversible inhibitor of the macrophage purinergic P2Z receptor
1National Research Council Unit for the Study of the Physiology of Mitochondria, Padova, Italy.
Abstract:
The effects of oxidized ATP (oATP) on responses triggered by extracellular adenosine 5'-triphosphate (ATPe) were investigated in the mouse macrophage-like cell line J774. ATPe induced in this cell line two kinds of responses mediated by two different P2 purinergic receptors: 1) an early permeabilization of the plasma membrane to extracellular hydrophilic markers of M(r) up to 900 mediated by P2Z receptors; and 2) a fast mobilization of Ca2+ from intracellular stores mediated by P2Y receptors. Low oATP concentrations (100 microM) completely blocked the first response without affecting the second. ATPe-dependent cell swelling, vacuolization, and lysis were also inhibited. Antagonism developed slowly, as an incubation at 37 degrees C for at least 2 h in the presence of oATP was needed and was irreversible, thus suggesting that the inhibitory action was due to covalent modification of the receptor. The rate of hydrolysis of exogenous ATP was slightly decreased by oATP, indicating a minor blocking effect of this compound on plasma membrane ecto-ATPases in the concentration range tested. These observations suggest that oATP may be a potentially very useful tool for isolation and characterization of the P2Z purinergic receptor.
Insights
Oxidized ATP (oATP) selectively inhibits P2Z purinergic receptor responses to extracellular ATP (ATPe) in J774 cells. This irreversible antagonism suggests oATP is a valuable tool for P2Z receptor research.
Area of Science:
- Immunology
- Cell Biology
- Pharmacology
Background:
- Extracellular adenosine 5'-triphosphate (ATPe) triggers cellular responses via P2 purinergic receptors.
- Two distinct ATPe-induced responses exist in J774 cells: plasma membrane permeabilization (P2Z receptors) and calcium mobilization (P2Y receptors).
Purpose of the Study:
- To investigate the effects of oxidized ATP (oATP) on ATPe-mediated responses in the J774 cell line.
- To determine if oATP can selectively modulate P2 purinergic receptor activity.
Main Methods:
- Incubation of J774 cells with oATP and ATPe.
- Assessment of plasma membrane permeability using hydrophilic markers.
- Measurement of intracellular calcium (Ca2+) mobilization.
- Evaluation of cell swelling, vacuolization, and lysis.
- Analysis of ATP hydrolysis by ecto-ATPases.
Main Results:
- Low concentrations of oATP (100 microM) completely blocked ATPe-induced plasma membrane permeabilization via P2Z receptors.
- oATP did not affect ATPe-induced calcium mobilization mediated by P2Y receptors.
- ATPe-dependent cell swelling, vacuolization, and lysis were inhibited by oATP.
- The inhibitory effect of oATP was slow to develop, irreversible, and suggested covalent modification of the P2Z receptor.
- oATP showed a minor inhibitory effect on plasma membrane ecto-ATPases.
Conclusions:
- Oxidized ATP (oATP) acts as a potent and selective antagonist of P2Z purinergic receptors.
- The irreversible nature of oATP's action suggests covalent modification of the P2Z receptor.
- oATP is a promising tool for the isolation and characterization of the P2Z purinergic receptor.