RECYCLING OF L-CITRULLINE TO SUSTAIN NITRIC OXIDE-DEPENDENT ENTERIC NEUROTRANSMISSION

Neurons that synthesize nitric oxide from arginine produce stoichiomet ric amounts of citrulline. We investigated whether nitric oxide-releas ing enteric neurons have the capacity to recycle citrulline to arginin e and thereby sustain nitrergic neurotransmission. Argininosuccinate s ynthetase-like immunoreactivity and argininosuccinate lyase-like immun oreactivity, enzymes capable of citrulline to arginine conversion, wer e both localized in discrete populations of myenteric and submucosal n eurons in the canine proximal colon. Argininosuccinate synthetase-like immunoreactivity and argininosuccinate lyase-like immunoreactivity we re also found in varicose nerve fibers within the circular and longitu dinal muscle layers. Argininosuccinate synthetase-like immunoreactivit y and argininosuccinate lyase-like immunoreactivity co-localized with neuronal beta-nicotinamide adenine dinucleotide phosphate diaphorase s taining, a marker for nitric oxide synthase. The functional significan ce of argininosuccinate synthetase-like immunoreactivity and argininos uccinate lyase-like immunoreactivity was shown by testing the effects of exogenous citrulline on responses to enteric inhibitory nerve stimu lation, which were assessed by measuring contractions, inhibitory junc tion potentials and electrical slow waves. As shown previously, argini ne analogues (L-nitroarginine methyl ester or L-nitroarginine; 100 mu M) inhibited nitric oxide-dependent responses, and excess L-arginine r estored inhibitory responses. Citrulline alone (0.1-2 mM) had no effec t on nitrergic transmission under control conditions, but in the prese nce of L-nitroarginine methyl ester or L-nitroarginine, citrulline (0. 1-2 mM) restored nitrergic transmission in a concentration-dependent m anner. Other neutral amino acids (L-serine, L-leucine) did not mimic t he effects of citrulline. Taken together, these data suggest that ente ric nitrergic neurons have the enzymatic apparatus and functional capa bility of recycling citrulline to arginine.