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PATRICIA MARÍN-GARCÍA Y COLS. AN. R. ACAD. NAC. FARM.

Once we knew that resorufin fluorescence was dose-dependent
in the range nano-micromolar of glutamate, we decided to investigate
if extracellular glutamate could increase in response to pore formation
after P2X7 receptor activation. For that purpose, granule neurons
(14 days old) were stimulated for 1 min with several concentrations
of BzATP (100 nM-200 µM), in absence of magnesium, since this ion
has been shown to inhibit P2X7 receptor (2). Next, medium was taken
and the glutamate content was measured using Amplex® Red Glutamic
Acid/Glutamic Oxidase Assay Kit. As it can be observed in Figure 3a,
BzATP induced an increase in resorufin fluorescence that was dose-
dependent. Initially, these data seems to indicate that BzATP induced
pore formation through which glutamate reached extracellular
medium since the profile of glutamate release did not fit to a
saturation kinetic. To verify this hypothesis, granule neurons were
stimulated with BzATP and ATP in presence of YO-PRO-1 as indicated
in methods section. The lack of variations in the YO-PRO-1
fluorescence after granule cell exposure to BzATP (100 µM) or ATP
(100 µM) for 5 minutes discarded the possibility that prolonged
activation of P2X7 could induce pore formation in the plasma
membrane of granule neurons (Figure 3b). Therefore, the non-
saturable profile of the dose-response curve was caused by the
presence of BzATP in the incubation medium and not by a lineal
glutamate release through pore.

In a recent work (9) we showed that BzATP, and other molecules
that contain 4-benzoylbenzoyl groups in their structure, interfere
in the AR oxidation reaction catalyzed by HRP. Thus, BzATP
enhances the AR oxidation mediated by HRP in the absence of
externally added H2O2. This effect is produced by benzophenone
group of BzATP molecule since other ATP agonists did not reproduce
BzATP interferences (9). Therefore, we decided to investigate if
resorufin fluorescence increase observed in Figure 3 could be a
consequence of BzATP interference on Amplex Red oxidation. For
that purpose, a working solution containing 50 µM AR, 0.125 U/ml
HRP, 0.04 U/ml L-glutamate oxidase, 0.25 U/ml L-glutamate pyruvate
transaminase and 100 µM L-alanine was prepared following the
protocol of Amplex® Red Glutamic Acid/Glutamic Oxidase Assay Kit.
This working solution was supplemented with 0.1 µM, 0.5 µM and
1 µM L-Glu respectively and incubated for 30 min at 37º C. Resorufin

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