Presence of functional ATP and dinucleotide receptors in glutamatergic synaptic terminals from rat midbrain

Abstract

Glutamatergic terminals from rat midbrain were characterized by immunolocalization of synaptophysin and the vesicular glutamate transporters, either VGLUT1 or VGLUT2. Terminals containing these markers represent about 31% (VGLUT1) and 16% (VGLUT2) of the total synaptosomal population. VGLUT1-positive glutamatergic terminals responded to ATP or P1, P 5-di(adenosine-5′) pentaphosphate (Ap5A) with an increase in the intrasynaptosomal calcium concentration as measured by a microfluorimetric technique in single synaptosomes. Roughly 20% of the VGLUT1-positive terminals responded to ATP, 13% to Ap5A and 11% to both agonists. Finally 56% of the terminals labeled with the anti-VGLUT1 antibody did not show any calcium increase in response to ATP or Ap 5A. A similar response distribution was also observed in the VGLUT2-positive terminals. The Ca2+ responses induced by ATP and Ap5A in the glutamatergic terminals could be selectively inhibited by pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS, 80 μM) and P1, P5-di(inosine-5′) pentaphosphate (Ip5I, 100 nM), respectively. Both ATP and Ap5A, once assayed in the presence of extrasynaptosomal calcium, were able to induce a concentration-dependent glutamate release from synaptosomal populations, EC 50 values being 21 μM and 38 μM for ATP and Ap5A, respectively. Specific inhibition of glutamate release was obtained with PPADS on the ATP effect and with Ip5I on the dinucleotide response, indicating that separate receptors mediate the secretory effects of both compounds.