Stimulation of Ca(2+) influx through ATP receptors on rat brain synaptosomes: identification of functional P2X(7) receptor subtypes

Abstract

1. ATP receptors of the P2X class have previously been identified on autonomic nerve endings and on a limited population of CNS neurons. 2. In the present study P2X receptors on mammalian cortical synaptosomes have been identified by a variety of functional and biochemical studies. In choline buffer ATP analogues caused concentration/time dependent Ca(2+) influx. Relative to the effects caused by ATP, benzoylbenzoyl ATP (BzATP) was about seven times more active than ATP while 2-me-S-ATP and ATPgammaS were much less active. alpha,beta-me- ATP and beta,gamma-me-ATP were virtually inactive. In sucrose buffer, relative to choline buffer, the activity of BzATP was more than doubled while activity in sodium buffer was reduced. Moreover, the P2X antagonists PPADS or Brilliant Blue G both significantly attenuated influx. These observations suggest the presence of P2X receptors on synaptosomes which subserve Ca(2+) influx. This activity profile of the ATP analogues and the response to blocking agents are characteristic of responses of P2X(7) receptors. 3. Influx was unaffected by the VSCC inhibitors omega-CTx-MVIIC and (-) 202 - 791, indicating that ATP induced Ca(2+) influx occurred primarily through P2X receptors. 4. P2X(7) receptor protein was identified by Western blotting and immunohistochemical staining. Purified preparations were devoid of significant concentrations of GFAP or the microglial marker OX-42 but contained greatly enriched amounts of syntaxin and SNAP 25. 5. The various pharmacological and biochemical studies were all consistent with the presence of functional P2X(7) receptors.

Figure 1

Concentration and time dependence of ATP and BzATP-evoked Ca²⁺ influx in rat brain cortical synaptosomes carried out in choline buffer as outlined in Methods. (A) ATP (solid bars) or BzATP (open bars) were incubated with the synaptosomes for 60 s at the concentrations indicated and calcium influx was measured. (B) Time dependence of ATP and BzATP-evoked Ca²⁺ influx. Synaptosomes were incubated with maximally effective concentrations of ATP (1 mM) or BzATP (100 μM) for 10 – 90 s as indicated. Values shown are mean±s.e.mean, n=4.

Figure 2

K⁺ and adenine nucleotide-evoked Ca²⁺ influx in rat brain cortical synaptosomes. K⁺-evoked and adenine nucleotide induced influx was measured in the presence and absence of (ω-CTx-MVIIC, 1 μM) as indicated. ATP evoked influx (solid bar, third bar from left) is shown in the absence and presence (solid bar, far right side) of PPADS (30 μM) n=4. Synaptosomes were exposed to 25 mM K⁺ for 3 s and 1 mM of the nucleotides for 60 s. The influx induced by maximally effective concentrations of 1 mM ATP or BzATP were not significantly different from one another. Inset: shows lack of effect of ω-CTx-MVIIC on ATP-evoked Ca²⁺ influx (*P<0.05, n=4).

Figure 3

(A) Calcium influx was measured in synaptosomal preparations exposed to increasing concentrations of BzATP in three incubating solutions of different ionic composition. Each bar represents the mean±s.e.mean, obtained in three experiments carried out in triplicate. BzATP induced influx was significantly greater in sucrose buffer than in the high ionic strength sodium or choline based buffers (2-way ANOVA, P<0.001). (B) Calcium influx initiated by BzATP in synaptosomal preparations incubated in the presence of the selective P2X₇ antagonist Brilliant Blue G. The influx represented by the bars is the mean±s.e.mean of three experiments carried out in triplicate. The asterisk denotes a significant reduction in influx as compared to that caused by BzATP alone (P<0.01, Student's t-test).

Figure 4

Western blots carried out to detect the presence of P2X₂ and P2X₇ receptor protein in rat cortical homogenates and synaptosomal preparations. Cortical homogenates and purified synaptosomes were prepared and treated with antibodies to P2X₂ and P2X₇ receptors. (A) Shows intense staining for P2X₂ receptors in PC-12 cells which were used as a positive control, weak detection in brain homogenates (Br), but no evidence of P2X₂ subtypes were found in purified synaptosomes (Syn). (B) Shows P2X₇ protein in brain homogenates and purified synaptosomes, but not in rat vas deferens (Vas) where P2X₁ receptors have been located.

Figure 5

Immunoreactivity in cortical slices or in purified rat cortical synaptosomes treated with fluorescent antibodies to P2X₇ receptors or to the microglial marker OX-42. (A and B) show the background fluorescence in the absence of the primary antibodies to P2X₇ receptors or to OX-42 in cortical sections or synaptosomes respectively. Immunofluorescence demonstrated following treatment of cortical sections with fluorescent antibodies to P2X₇ receptors (C) or cytospun cortical synaptosomes (D). (E) reveals specific immunofluorescence in cortex treated with antibodies to the microglial marker OX-42. This fluorescence is absent from synaptosomal preparations (F).

Figure 6

Western blots showing the presence of the synaptic markers syntaxin and SNAP-25 and the virtual absence of glial fibrillary acid protein (GFAP) in purified synaptosomes (Syn). Top panel: GFAP staining in whole cortical homogenates and virtual absence of staining in purified synaptosomes is shown. The figure shows the enhanced concentration of synaptic proteins syntaxin (middle panel) and SNAP-25 (bottom panel) in purified synaptosomes (Syn).

Figure 7

Immunoreactivity of purified synaptosomes treated with antibodies to the synaptic markers syntaxin and SNAP-25. (A) non-specific staining; (B) SNAP 25 staining; and (C) syntaxin staining.