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 mu M), the NOS substrate L-arginine (10-470 mu M) and the NO donor 3-morpholinylsydnoneimine (SIN-1, 1-10 mu M), concentration-dependently inhibited [H-3]-acetylcholine ([H-3]-ACh) release from rat motor nerve endings. Increasing stimulus frequency from 5 Hz-trains to 50 Hz-bursts enhanced [H-3]-ACh release inhibition by L-arginine (47 mu M) and L-citrulline (470 mu M), whereas the effect of SIN-1 (10 mu M) remained unchanged. NOS inhibition with N-omega-nitro-L-arginine (100 mu M) 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 mu M), 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 (10 nM), 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 mu M) increased basal adenosine outflow via the equilibrative nucleoside transport system sensitive to NBTI (10 mu M), without significantly (P > 0.05) changing the nucleoside release subsequent to nerve stimulation. Data indicate that NOS-derived L-citrulline negatively modulates [H-3]-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. (C) 2007 Elsevier Ltd. All rights reserved.