Synaptic terminals from mice midbrain exhibit functional P2X7 receptor.
P2X(7) receptor has been recently localized in mice cerebellar granule neuron fibers. Here, the expression of this subunit has been detected in wild type mice midbrain, by quantitative real time-polymerase chain reaction, immunocytochemistry and Western blot assays. The functionality of this P2X(7) subunit has been confirmed using microfluorimetric experiments in isolated synaptic terminals from mice midbrain. 2'-3'-O-(4-benzoylbenzoyl)-ATP (BzATP) was 30-fold more potent than ATP and EC(50) values were 20 microM and 630 microM respectively. Brilliant Blue G (BBG) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) produced an inhibition in the responses induced by BzATP, with IC(50) values of 0.027 nM and 2.23 nM, respectively. In addition, P2X(7) inhibitors as ZnSO(4), BBG and suramin abolished partially or totally the responses induced by the physiological agonist ATP. According to immunochemical and PCR assays the presence of a "P2X(7)-like" protein in synaptosomes from validated P2X(7) knockout (KO) model have been detected. In KO animals, BzATP was sixfold more potent than ATP and the EC(50) values were 87 microM and 590 microM respectively. BBG and KN-62 also produced an inhibition in the responses induced by BzATP, with IC(50) value of 0.61 nM and 118 nM respectively, both of them higher than in wild type mice. Moreover, the calcium mobilization ability of native P2X(7) receptors was higher in control compared with KO mice. These biochemical and pharmacological experiments are consistent with the presence of a functional P2X(7) receptor in wild type mice midbrain, and the existence of a less efficient "P2X(7)-like" receptor in the KO model.
Marín-García
P
P
Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madrid, Av. Puerta de Hierro s/n, 28040 Madrid, Spain.
Sánchez-Nogueiro
J
J
Gómez-Villafuertes
R
R
León
D
D
Miras-Portugal
M T
MT
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D005470:Fluorometry; D007150:Immunohistochemistry; D008297:Male; D008636:Mesencephalon / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017729:Presynaptic Terminals / Q000502:physiology*; D058919:Purinergic P2 Receptor Antagonists; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000502:physiology*; D058486:Receptors, Purinergic P2X7; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism
C546469:P2rx7 protein, mouse; D058919:Purinergic P2 Receptor Antagonists; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D000255:Adenosine Triphosphate