Synthesis of serotonin by neurons of the myenteric plexus in situ and in organotypic tissue culture

Previous studies have suggested that serotonin is a neurotransmitter in the mammalian myenteric plexus. The present study was done to test this hypothesis by determining whether neurons of the myenteric plexus synthesize 5-HT from L-trypophan. Isolated strips of longitudinal muscle with attached myenteric plexus took up L-tryptophan by a saturable transport mechanism K_m 4.7 × 10^-5 M; V_max 6.7 nmoles/g/min). These strips also synthesized tritiated serotonin ([³H]5-HT) and [³H]5-hydroxy-indoleacetic acid from L-[³H]tryptophan. This synthesis of [³H]5-HT was antagonized by prior treatment of guinea pigs with p-chlorophenylalanine. The drug treatment did not interfere with tryptophan uptake as did inclusion of p-chlorophenylalanine with L-[³H]tryptophan in vitro and thus probably decreased synthesis of [³H]5-HT by inhibiting tryptophan hydroxylase. Organotypic tissue cultures of intestine, lacking mucosa, argentaffin cells or mast cells also converted L-[³H]tryptophan to [³H]5-HT and [³H]5-hydroxyindoleacetic acid. Examination of the myenteric plexus from chemically sympathectomized (6-hydrpxydopamine) animals given L-tryptophan and a monoamine oxidase inhibitor for formaldehyde-induced histofluorescence revealed a diffuse yellow fluorophore in the myenteric plexus with the spectral characteristics of 5-HT. Pretreatment with p-chorophenylalanine prevented development of the diffuse fluorescence induced by L-tryptophan but revealed clusters of fluorescent neuronal cell bodies. The fluorescence of these cell bodies also had the spectral characteristics of the 5-HT fluorophore. Organotypic tissue cultures of intestine, grown for 3 weeks, and then dried and exposed to gaseous formaldehyde contained groups of fluorescent neuronal cell bodies. Their fluorescence was enhanced by adding L-tryptophan and an inhibitor of monoamine oxidase to the culture medium. This fluorescence also had the spectral characteristics of 5-HT. We conclude that the myenteric plexus synthesizes 5-HT from L-tryptophan. The responsible neurons survive in culture and are therefore intrinsic to the gut itself. These observations support the hypothesis that 5-HT is a transmitter of neurons in the mammalian myenteric plexus.