Tuning adenosine A 1 and A 2A receptors activation mediates l-citrulline-induced inhibition of [ 3H]-acetylcholine release depending on nerve stimulation pattern

Abstract

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