PERSONAL DE APOYO
VEGGETTI Mariela Iris
artículos
Título:
Presynaptic inhibition of spontaneous acetylcholine release mediated by P2Y receptors at the mouse neuromuscular junction
Autor/es:
S. DE LORENZO, M. VEGGETTI, S. MUCHNIK AND A. LOSAVIO
Revista:
NEUROSCIENCE
Editorial:
PERGAMON-ELSEVIER SCIENCE LTD
Referencias:
Lugar: Amsterdam; Año: 2006 vol. 142 p. 71 - 85
ISSN:
0306-4522
Resumen:
At the neuromuscular
junction, ATP is co-released with the neurotransmitter acetylcholine (ACh) and
once in the synaptic space, it is degraded to the presynaptically active metabolite
adenosine. Intracellular recordings were performed on diaphragm fibers of CF1
mice to determine the action of extracellular ATP (100 µM) and the
slowly hydrolysable ATP analog 5=-adenylylimidodiphosphate
lithium (βg-imido ATP) (30 µM) on miniature
end-plate potential (MEPP) frequency. We found that application of ATP and βg-imido ATP decreased
spontaneous secretion by 45.3% and 55.9% respectively. 8-Cyclopentyl-1,3-dipropylxanthine
(DPCPX), a selective A1 adenosine receptor antagonist and αβ-methylene ADP
sodium salt (αβ-MeADP), which is an inhibitor of ecto-5?-nucleotidase, did not prevent the inhibitory effect of ATP,
demonstrating that the nucleotide is able to modulate spontaneous ACh release through
a mechanism independent of the action of adenosine. Blockade of Ca2+channels by both, Cd2+or the combined application of nitrendipine
and
w-conotoxin GVIA (w-CgTx) (L-type and N-type Ca2+ channel antagonists, respectively) prevented
the effect of βg-imido ATP, indicating that
the nucleotide modulates Ca2+ influx through
the voltage-dependent Ca2+ channels related to spontaneous secretion. βg-Imido ATP-induced
modulation
was antagonized by the non-specific
P2 receptor antagonist suramin and the P2Y receptor antagonist
1-amino-4-[[4-[[4-chloro-6-[[3(or4)-sulfophenyl]
amino]-1,3,5-triazin-2-yl]amino]-3-sulfophenyl] amino]-9,10-dihydro-9,10-dioxo-2-anthracenesulfonic
acid (reactive blue-2), but not by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic
acid) tetrasodium salt (PPADS), which has a preferential antagonist effect on
P2X receptors. Pertussis toxin and N-ethylmaleimide (NEM), which
are blockers of Gi/o proteins,
prevented the action of the nucleotide, suggesting that the effect is mediated
by P2Y receptors coupled to Gi/o proteins. The protein kinase C (PKC)
antagonist chelerythrine and the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide
hydrochloride (W-7) occluded the effect of βg-imido ATP, while the
protein kinase A (PKA) antagonist KT-5720 and the inhibitor of the calcium/calmodulin-dependent
protein kinase II (CAMKII) KN-62 failed to do so. βg-Imido ATP did not affect 10, 15 and 20 mM
K+-evoked release and application of reactive blue-2
before incubation in high K+ induced a
higher asynchronous secretion Thus, our results show that at mammalian
neuromuscular junctions, ATP induces presynaptic inhibition of spontaneous ACh
release due to the modulation of Ca2+ channels
related to tonic secretion
through the activation of P2Y receptors coupled to Gi/o proteins. We also
demonstrated that at increasing degrees of membrane depolarization evoked by K+, endogenously released ATP induces presynaptic
inhibition as a means of preventing excessive neurotransmitter secretion.