Tuning adenosine A sub(1) and A sub(2A) receptors activation mediates l- citrulline-induced inhibition of [ super(3)H]-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 [ super(3)H]-acetylcholine ([ super(3)H]-ACh) release from rat motor nerve endings. Increasing stimulus frequency from 5 Hz-trains to 50 Hz- bursts enhanced [ super(3)H]-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 super([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 sub(1) receptors with 1,3-dipropyl-8-cyclopentyl xanthine (2.5 nM), but not of A sub(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 sub(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 [ super(3)H]-ACh release by increasing adenosine outflow channelling to A sub(1) and A sub(2A) receptors activation depending on the stimulus paradigm. While adenosine acts predominantly at inhibitory A sub(1) receptors during 5 Hz-trains, inhibition of ACh release by l-citrulline at 50 Hz-bursts depends on the interplay between adenosine A sub(2A) and muscarinic M sub(2) receptors.