Tuning adenosine A1 and A2A receptors activation mediates L-citrulline-induced inhibition of [3H]-acetylcholine release depending on nerve stimulation pattern.
The influence of nerve stimulation pattern on transmitter release inhibition by L-citrulline, the co-product of NO biosynthesis by nitric oxide synthase (NOS), was studied in the rat phrenic nerve-hemidiaphragm. We also investigated the putative interactions between NOS pathway and the adenosine system. L-citrulline (10-470 microM), the NOS substrate L-arginine (10-470 microM) and the NO donor 3-morpholinylsydnoneimine (SIN-1, 1-10 microM), concentration-dependently inhibited [(3)H]-acetylcholine ([(3)H]-ACh) release from rat motor nerve endings. Increasing stimulus frequency from 5 Hz-trains to 50 Hz-bursts enhanced [(3)H]-ACh release inhibition by l-arginine (47 microM) and L-citrulline (470 microM), whereas the effect of SIN-1 (10 microM) remained unchanged. NOS inhibition with N(omega)-nitro-L-arginine (100 microM) prevented the effect of L-arginine, but not that of L-citrulline. Adenosine deaminase (2.5 U/ml) and the adenosine transport inhibitor, S-(p-nitrobenzyl)-6-thioinosine (10 microM), attenuated release inhibition by L-arginine and L-citrulline. With 5 Hz-trains, blockade of A(1) receptors with 1,3-dipropyl-8-cyclopentyl xanthine (2.5 nM), but not of A(2A) receptors with ZM241385 (10nM), reduced the inhibitory action of l-arginine and L-citrulline; the opposite was verified with 50 Hz-bursts. Blockade of muscarinic M(2) autoreceptors with AF-DX116 (10 nM) also attenuated the effects of L-arginine and L-citrulline with 50 Hz-bursts. L-citrulline (470 microM) increased basal adenosine outflow via the equilibrative nucleoside transport system sensitive to NBTI (10 microM), without significantly (P>0.05) changing the nucleoside release subsequent to nerve stimulation. Data indicate that NOS-derived L-citrulline negatively modulates [(3)H]-ACh release by increasing adenosine outflow channelling to A(1) and A(2A) receptors activation depending on the stimulus paradigm. While adenosine acts predominantly at inhibitory A(1) receptors during 5 Hz-trains, inhibition of ACh release by L-citrulline at 50 Hz-bursts depends on the interplay between adenosine A(2A) and muscarinic M(2) receptors.
Timóteo
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Laboratório de Farmacologia e Neurobiologia/UMIB, Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, L. Prof. Abel Salazar, 2, 4099-003 Porto, Portugal.
Oliveira
L
L
Campesatto-Mella
E
E
Barroso
A
A
Silva
C
C
Magalhães-Cardoso
M T
MT
Alves-do-Prado
W
W
Correia-de-Sá
P
P
D000109:Acetylcholine / Q000378:metabolism*; D000241:Adenosine / Q000378:metabolism / Q000502:physiology; D000818:Animals; D001120:Arginine / Q000494:pharmacology; D002956:Citrulline / Q000494:pharmacology*; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006454:Hemoglobins / Q000378:metabolism; D008297:Male; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009045:Motor Endplate / Q000187:drug effects / Q000502:physiology; D009046:Motor Neurons / Q000187:drug effects / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000037:antagonists & inhibitors / Q000378:metabolism; D017729:Presynaptic Terminals / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D043682:Receptor, Adenosine A1 / Q000187:drug effects*; D043705:Receptor, Adenosine A2A / Q000187:drug effects*; D015398:Signal Transduction / Q000502:physiology
D004791:Enzyme Inhibitors; D006454:Hemoglobins; D043682:Receptor, Adenosine A1; D043705:Receptor, Adenosine A2A; D002956:Citrulline; C002385:linsidomine; D001120:Arginine; D008981:Molsidomine; D052248:Nitric Oxide Synthase Type I; D000241:Adenosine; D000109:Acetylcholine