Regulation of citrulline recycling in nitric oxide-dependent neurotransmission in the murine proximal colon

摘要

class="enumerated" style="list-style-type:decimal">We investigated the contribution of nitric oxide (NO) to inhibitory neuromuscular transmission in murine proximal colon and the possibility that citrulline is recycled to arginine to maintain the supply of substrate for NO synthesis.Intracellular microelectrode recordings were made from circular smooth muscle cells in the presence of nifedipine and atropine (both 1 μM). Electrical field stimulation (EFS, 0.3–20 Hz) produced inhibitory junction potentials (i.j.ps) composed of an initial transient hyperpolarization (fast component) followed by a slow recovery to resting potential (slow component).L-Nitro-arginine-methyl ester (L-NAME, 100 μM) selectively abolished the slow component of i.j.ps. The effects of L-NAME were reversed by L-arginine (0.2–2 mM) but not by D-arginine (2 mM). Sodium nitroprusside (an NO donor, 1 μM) reversibly hyperpolarized muscle cells. This suggests that NO mediates the slow component of i.j.ps.L-Citrulline (0.2 mM) also reversed the effects of L-NAME, and this action was maintained during sustained exposures to L-citrulline (0.2 mM). This may reflect intraneuronal recycling of L-citrulline to class="small-caps">L-arginine.Higher concentrations of class="small-caps">L-citrulline (e.g. 2 m class="small-caps">M) had time-dependent effects. Brief exposure (15 min) reversed the effects of class="small-caps">L-NAME, but during longer exposures (30 min) the effects of class="small-caps">L-NAME gradually returned. In the continued presence of class="small-caps">L-citrulline, class="small-caps">L-arginine (2 m class="small-caps">M) readily restored nitrergic transmission, suggesting that during long exposures to high concentrations of class="small-caps">L-citrulline, the ability to generate arginine from citrulline was reduced.Aspartate (2 m class="small-caps">M) had no effect on i.j.ps, the effects of class="small-caps">L-NAME, or the actions of class="small-caps">L-citrulline in the presence of class="small-caps">L-NAME. class="small-caps">L-Citrulline (0.2–2 m class="small-caps">M) alone had no effect on i.j.ps under control conditions.>S-methyl- class="small-caps">L-thiocitrulline (10 μ class="small-caps">M), a novel NOS inhibitor, blocked the slow component of i.j.ps. The effects of this inhibitor were reversed by class="small-caps">L-arginine (2 m class="small-caps">M), but not by class="small-caps">L-citrulline (2 m class="small-caps">M).These results suggest that i.j.ps in the murine colon result from release of multiple inhibitory neurotransmitters. NO mediates a slow component of enteric inhibitory neurotransmission. Recycling of class="small-caps">L-citrulline to class="small-caps">L-arginine may sustain substrate concentrations in support of NO synthesis and this pathway may be inhibited when concentrations of class="small-caps">L-citrulline are elevated.