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 P2X 7 receptor is typically found on immune cells, where it is involved in the release of cytokines. Recently, P2X 7 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 P2X 7 receptors at hippocampal mossy fiber-CA3 synapses. In agreement with previous findings, the widely used P2X 7 agonist 2Ј-3Ј-O-(4-benzoylbenzoyl)-adenosine-5Ј-triphosphate (BzATP) clearly depressed field potentials (fEPSPs); however, no evidence for an involvement of P2X 7 receptors could be obtained. First, depression of fEPSPs by BzATP was unchanged in P2X 7 Ϫ/Ϫ mice. Second, experiments using P2X 7 Ϫ/Ϫ mice, immunohistochemistry, and electron microscopy showed that the antigen detected by frequently used P2X 7 antibodies is not compatible with a plasmalemmal P2X 7 receptor. Third, BzATP did not alter Ca 2ϩ levels in synaptic terminals. In contrast, the depression of fEPSPs by BzATP was fully blocked by adenosine (A 1 ) receptor antagonists. Furthermore, the application of BzATP also activated postsynaptic A 1 receptor-coupled K ϩ channels. This effect of BzATP was mimicked by ATP and adenosine and was completely prevented by enzymes specifically degrading adenosine. Activation of A 1 -coupled K ϩ channels by BzATP was dependent on ecto-nucleotidases, extracellular enzymes that convert ATP to adenosine. Moreover, the opening of A 1 -coupled K ϩ 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 A 1 receptors rather than through P2X 7 receptors. Thus, the present study casts doubts on the neuronal localization of P2X 7 receptors in rodent hippocampus.

p38 Mitogen-Activated Protein Kinase Contributes to Adenosine A1Receptor-Mediated Synaptic Depression in Area CA1 of the Rat Hippocampus

“…It is unlikely that SB203580 exerts its effects on mossy fiber-CA3 synaptic depression by blocking adenosine transporters as previously suggested (Kukley et al, 2004). The inactive analog SB202474 (Sweeney et al, 1999) has a similar effect on nucleoside transporters as SB203580 (Huang et al, 2002).…”

Dual Presynaptic Control by ATP of Glutamate Release via Facilitatory P2X1, P2X2/3, and P2X3and Inhibitory P2Y1, P2Y2, and/or P2Y4Receptors in the Rat Hippocampus

“…When studying other synapses, it was found that P2X 2 receptors controlled glutamatergic transmission onto interneurons of the CA1 region (Khakh et al, 2003), P2Y 1 receptors controlled interneuron excitability (Kawamura et al, 2004), whereas in mossy fiber/CA3 pyramid synapses, a mixed P2/P1 modulation by exogenously added ATP was concluded (Kukley et al, 2004). Thus, it primarily remains to be tested whether P2 receptors might exert a more important control of glutamate release in other glutamatergic synapses in the hippocampus.…”

“…P2X 7 receptor does not presynaptically control glutamate release It was described recently that the P2X 7 receptor is targeted to glutamatergic nerve terminals in different brain regions (Deuchars et al, 2001;MirasPortugal et al, 2003), including the hippocampus (Armstrong et al, 2002;Sperlágh et al, 2002). However, some studies cast doubts on the selectivity of the most commonly used antibodies against P2X 7 receptors (Sim et al, 2004), and it has been shown that purported P2X 7 receptor agonists might also indirectly activate adenosine A 1 receptors (Kukley et al, 2004). We report that Bz-ATP (5 M; a concentration selective for P2X 7 or P2X 1 receptor activation) (North, 2002) facilitated by 48.5 Ϯ 11.2% (n ϭ 4) the evoked release of glutamate.…”

Physiology of Microglia

“…They include an agonist 2=,3=-(benzoyl-4-benzoyl)-ATP (BzATP) which is ϳ30 times more potent than ATP at P2X 7 receptors (although BzATP can activate other P2X receptors and can be degraded to Bz adenosine, which stimulates P1 receptors; Ref. 481), and several antagonists such as Brilliant Blue G (BBG) which blocks P2X 7 receptors at 10 -200 nM concentrations and oxidized ATP (oxATP). The latter, however, also inhibits P2X 1 and P2X 2 receptors (660).…”