Ecto-Nucleotidases and Nucleoside Transporters Mediate Activation of Adenosine Receptors on Hippocampal Mossy Fibers by P2X7Receptor Agonist 2′-3′-O-(4-Benzoylbenzoyl)-ATP

Abstract

The ionotropic and cytolytic P2X₇ receptor is typically found on immune cells, where it is involved in the release of cytokines. Recently, P2X₇ receptors were reported to be localized to presynaptic nerve terminals and to modulate transmitter release. In the present study, we reassessed this unexpected role of P2X₇ receptors at hippocampal mossy fiber-CA3 synapses. In agreement with previous findings, the widely used P2X₇ agonist 2′-3′-O-(4-benzoylbenzoyl)-adenosine-5′-triphosphate (BzATP) clearly depressed field potentials (fEPSPs); however, no evidence for an involvement of P2X₇ receptors could be obtained. First, depression of fEPSPs by BzATP was unchanged in P2X₇^-/- mice. Second, experiments using P2X₇^-/- mice, immunohistochemistry, and electron microscopy showed that the antigen detected by frequently used P2X₇ antibodies is not compatible with a plasmalemmal P2X₇ receptor. Third, BzATP did not alter Ca²⁺ levels in synaptic terminals. In contrast, the depression of fEPSPs by BzATP was fully blocked by adenosine (A₁) receptor antagonists. Furthermore, the application of BzATP also activated postsynaptic A₁ receptor-coupled K⁺ channels. This effect of BzATP was mimicked by ATP and adenosine and was completely prevented by enzymes specifically degrading adenosine. Activation of A₁-coupled K⁺ channels by BzATP was dependent on ecto-nucleotidases, extracellular enzymes that convert ATP to adenosine. Moreover, the opening of A₁-coupled K⁺ channels by BzATP was dependent on nucleoside transporters. Taken together, our results indicate that BzATP is extracellularly catabolized to Bz-adenosine and subsequently hetero-exchanged for intracellular adenosine and then depresses mossy fiber fEPSPs through presynaptic A₁ receptors rather than through P2X₇ receptors. Thus, the present study casts doubts on the neuronal localization of P2X₇ receptors in rodent hippocampus.