L-citrulline inhibits [3H]acetylcholine release from rat motor nerve terminals by increasing adenosine outflow and activation of A1 receptors

Abstract

Background and purpose: Nitric oxide (NO) production and depression of neuromuscular transmission are closely related, but little is known about the role of L-citrulline, a co-product of NO biosynthesis, on neurotransmitter release.

Experimental approach: Muscle tension recordings and outflow experiments were performed on rat phrenic nerve-hemidiaphragm preparations stimulated electrically.

Key results: L-citrulline concentration-dependently inhibited evoked [(3)H]ACh release from motor nerve terminals and depressed nerve-evoked muscle contractions. The NO synthase (NOS) substrate, L-arginine, and the NO donor, 3-morpholinosydnonimine chloride (SIN-1), also inhibited [(3)H]ACh release with a potency order of SIN-1>L-arginine>L-citrulline. Co-application of L-citrulline and SIN-1 caused additive effects. NOS inactivation with N(omega)-nitro-L-arginine prevented L-arginine inhibition, but not that of L-citrulline. The NO scavenger, haemoglobin, abolished inhibition of [(3)H]ACh release caused by SIN-1, but not that caused by L-arginine. Inactivation of guanylyl cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) fully blocked SIN-1 inhibition, but only partially attenuated the effects of L-arginine. Reduction of extracellular adenosine accumulation with adenosine deaminase or with the nucleoside transport inhibitor, S-(p-nitrobenzyl)-6-thioinosine, attenuated the effects of L-arginine and L-citrulline, while not affecting inhibition by SIN-1. Similar results were obtained with the selective adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine. L-citrulline increased the resting extracellular concentration of adenosine, without changing that of the adenine nucleotides.

Conclusions and implications: NOS catalyses the formation of two neuronally active products, NO and L-citrulline. While, NO may directly reduce transmitter release through stimulation of soluble guanylyl cyclase, the inhibitory action of L-citrulline may be indirect through increasing adenosine outflow and subsequently activating inhibitory A(1) receptors.

Figure 1

Concentration–response curves for the inhibitory effects of L-arginine (0.01–4.7 mM), L-citrulline (0.01–4.7 mM) and SIN-1 (1–100 μM) on [³H]ACh release from motor nerve terminals stimulated with 5 Hz-trains (750 pulses). Abscissa, log of the concentration (M) of L-arginine (0.01–4.7 mM, squares), L-citrulline (0.01–4.7 mM, circles) and SIN-1 (1–100 μM, triangles), applied 15 min before S₂. Ordinate, percentage change in S₂/S₁ ratio as compared with the S₂/S₁ ratio in control experiments. Zero per cent represents identity between the two ratios, negative values indicate inhibition of evoked [³H]ACh release. Each point represents the mean±s.e.m. of 4–11 experiments.

Figure 2

Effect of the extracellular NO scavenger, haemoglobin, on L-arginine- and SIN-1-induced inhibition of [³H]ACh release from stimulated motor nerve terminals. The time course of tritium outflow from phrenic nerve terminals shown here is taken from typical experiments in the absence (control, filled squares) and in the presence of (a) L-arginine (L-Arg, 47 μM) or (b) SIN-1 (10 μM), used either alone (filled circles) or in the presence (open circles) of haemoglobin (Hb, 10 μM). Tritium outflow (ordinates) is expressed as a percentage of the total radioactivity present in the tissue at the beginning of the collection period and was measured in samples collected every 3 min. [³H]ACh release was elicited by stimulating the phrenic nerve trunk with 750 pulses delivered with a frequency of 5 Hz at the indicated times (S₁ and S₂). L-arginine (47 μM) and SIN-1 (10 μM) were applied 15 min before S₂; haemoglobin (10 μM) was present throughout the assay, including S₁ and S₂ (horizontal bars). None of the drugs changed spontaneous tritium outflow.

Figure 3

Inhibitory effect of L-citrulline (0.01–47 mM) on diaphragm twitch tension induced by phrenic nerve stimulation (indirect stimulation, filled circles) or by direct muscle depolarization (direct stimulation, open circles) in conditions where the safety factor of neuromuscular transmission was reduced (high Mg²⁺, 6 mM). Twitch responses were induced alternating stimulus application to the phrenic nerve trunk or to muscle fibres at a frequency of 0.1 Hz. L-citrulline (0.01–47 mM) was applied in a cumulative manner; each concentration contacted the preparation at least 12 min before solution changeover. Ordinate, percentage change of the maximal twitch tension obtained in control conditions. Each point represents the mean±s.e.m. of six experiments. ^*P<0.05 (one-way ANOVA followed by Dunnett's modified t-test) compared with the effect of L-citrulline on twitch tension induced by direct muscle stimulation.

Figure 4

Influence of inhibition of soluble guanylyl cyclase (with ODQ) and NOS (with L-NOARG) on the reduction of evoked [³H]ACh release caused by L-arginine, L-citrulline and SIN-1. L-arginine (47 μM), L-citrulline (470 μM) and SIN-1 (10 μM) were applied 15 min before S₂ in concentrations that caused about 30% inhibition of [³H]ACh release from stimulated motor nerve terminals. ODQ (10 μM) and L-NOARG (100 μM) were present throughout the assay, including S₁ and S₂; the S₂/S₁ ratios obtained under these conditions were not statistically different from the ratio obtained in control experiments (without any drug during S₁ and S₂) (dashed horizontal line, see ‘Methods' section). The ordinates represent evoked tritium outflow expressed by S₂/S₁ ratios. Each column represents pooled data from 4 to 11 experiments. The vertical bars represent s.e.m. ^*P<0.05 (one-way ANOVA followed by Dunnett's modified t-test) when compared with the effects of L-arginine, L-citrulline and SIN-1 applied alone, respectively.

Figure 5

Role of endogenous adenosine on the inhibitory effect of L-arginine, L-citrulline and SIN-1 on evoked [³H]ACh release from motor nerve terminals. L-arginine (47 μM), L-citrulline (470 μM) and SIN-1 (10 μM) were applied 15 min before S₂ in concentrations that caused about 30% inhibition of [³H]ACh release from stimulated motor nerve terminals. (a) ADA (0.5 U ml⁻¹), the nucleoside transport inhibitor (NBTI, 10 μM) and (b) the two adenosine antagonists exhibiting high-subtype selectivity for A₁ (DPCPX, 2.5 nM) and A_2A (ZM 241385, 10 nM) receptors were present throughout the assay, including S₁ and S₂. The S₂/S₁ ratios obtained under these conditions were not statistically different from the ratio obtained in control experiments (without any drug during S₁ and S₂) (dashed horizontal line, see ‘Methods' section). The ordinates represent evoked tritium outflow expressed by S₂/S₁ ratios. Each column represents pooled data from (a) 4–11 and (b) 5–11 experiments. The vertical bars represent s.e.m. ^*P<0.05 (one-way ANOVA followed by Dunnett's modified t-test) when compared with the effects of L-arginine, L-citrulline and SIN-1 applied alone, respectively.

Figure 6

Effect of L-citrulline (470 μM) on the resting outflow of (a) adenosine and (b) ATP (and related adenine nucleotides) from the rat innervated hemidiaphragm in the absence and in the presence of the nucleoside transport inhibitor, NBTI (10 μM). L-citrulline (470 μM) was applied 15 min before S₂; NBTI (10 μM) was present throughout the assay, including S₁ and S₂. The ordinates represent the resting outflow of (a) adenosine and (b) ATP (and related adenine nucleotides) expressed by the B_2−n/B_1−n ratios; B_1−n and B_2−n correspond to the adenosine or adenine nucleotide content in bath samples collected n minutes before the first (without L-citrulline) and the second (in the presence of L-citrulline) stimulation periods, respectively. Each column represents pooled data from four experiments. The vertical bars represent s.e.m. ^*P<0.05 (one-way ANOVA followed by Dunnett's modified t-test), significant differences from the control.