Fig. 3
Role of 5-HIAA precursors on NEP protein expression. a Tryptophan is the precursor of both serotonin and kynurenine pathways. IDO (indolamine dioxygenase 2,3 dioxygenase) induction by inflammatory cytokines favorizes the production of kynurenine pathway intermediates, including kynurenic acid. Tryptophan, 5-hydroxytryptophan (5-HTP) and serotonin (5-HT) are precursors of 5-HIAA. 5-HTP degradation is prevented by carbidopa while serotonin catabolism is blocked by IMAO (tranylcypromine + clorgyline). b Dose-response curves for the induction of NEP protein in SH-SY5Y cells. Effect of tryptophan, 5-HIAA, 5HTP + carbidopa (100 μM), Serotonin + tranylcypromine (30 μM) and clorgyline (20 μM) on NEP protein level in SH-SY5Y cells after 30-min treatment. In the absence of inhibitor, 5-HIAA and L-tryptophan exhibit an EC50 of 8 ± 3 μM and 19 ± 2 μM for NEP induction, respectively. In the presence of carbidopa, 5-HTP shows an intrinsic activity on NEP induction with an EC50 of 5 ± 1 μM but the maximum activity is 20%. Incubation of SH-SY5Y cells with increasing doses of serotonin in the presence of tranylcypromine showed no effect on NEP induction. c Effects of 24-h pharmacological inhibition of 5-HIAA synthesis from precursors (Tryptophan, 5-HTP, or 5-HT) on NEP protein expression in SH-SY5Y cells. In the presence of Tranylcypromine, NEP induction by tryptophan is reduced from 87 +/− 9 to 30 +/− 1%. In the presence of either carbidopa (inhibitor of 5-HTP decarboxylation into 5-HT), or tranylcypromine (blocker of 5-HTP conversion into 5-HIAA), NEP induction by 5-HTP was significantly reduced compared to 5-HTP tested without inhibitors (respectively 35 +/− 1 and 36 +/− 1% vs 57 +/− 3%). 5-HT at 100 μM exhibited some effect on NEP (51 +/− 7%) because of its role as a precursor of 5-HIAA. Consistently, the effect of 5-HT was inhibited by tranylcypromine (13 +/− 1%). The statistical analysis of triplicatesamples was done by an ANOVA completed with a Bonferroni’s Multiple Comparison Test. ## p < 0.005, ### or *** p < 0.0001. * by reference to non-treated (NT) cells, or # by reference to treatments with specific enzyme’s inhibitor