Abstract
Serotonin (5-HT) functions as a neurotransmitter and neuromodulator in both the central and enteric nervous systems of mammals. The dynamic degradation of 5-HT metabolites in 5-HT-containing nervous system tissues is monitored by capillary electrophoresis with wavelength-resolved laser-induced native fluorescence detection in an effort to investigate known and novel 5-HT catabolic pathways. Tissue samples from wild type mice, genetically altered mice, Long Evans rats, and cultured differentiated rat pheochromocytoma PC-12 cells, are analyzed before and after incubation with excess 5-HT. From these experiments, several new compounds are detected. One metabolite, identified as 5-hydroxyindole thiazoladine carboxylic acid (5-HITCA), has been selected for further study. In 5-HT-incubated central and enteric nervous system tissue samples and differentiated PC-12 cells, 5-HITCA forms at levels equivalent to 5-hydroxyindole acetic acid, via a condensation reaction between L-cysteine and 5-hydroxyindole acetaldehyde. In the enteric nervous system, 5-HITCA is detected without the addition of 5-HT. The levels of L-cysteine and homocysteine in rat brain mitochondria are measured between 80 and 140 microm and 1.9 and 3.4 microm, respectively, demonstrating that 5-HITCA can be formed using available, free L-cysteine in these tissues. The lack of significant accumulation of 5-HITCA in the central and enteric nervous systems, along with data showing the degradation of 5-HITCA into 5-hydroxyindole acetaldehyde, suggests that an equilibrium coupled to the enzyme, aldehyde dehydrogenase type 2, prevents the accumulation of 5-HITCA. Even so, the formation of 5-HITCA represents a catabolic pathway of 5-HT that can affect the levels of 5-HT-derived compounds in the body.
Highlights
Using capillary electrophoresis (CE) with a wavelength-resolved laser-induced native fluorescence (LINF) detection system and mass spectrometry (MS), we identify this particular component as 5-hydroxyindole thiazolidine carboxylic acid (5-HITCA) and explore the mechanism of its formation
We demonstrate here that in the presence of 5-HT, 5-HITCA is formed in different quantities dependent on the brain region being studied, the levels can be similar to the levels of 5-hydroxyindole acetic acid (5-HIAA) in these tissues, and that it forms without exogenously added L-Cys
A significant percentage (30 –50%, data not shown) of the 5-HT added in the incubation solutions is converted to something other than 5-HT, but is not detected as known 5-HT metabolites. Possible fates for this undetected 5-HT include conversion into unknown metabolites, formation of serotonin-protein complexes [30], or involvement in reactions relating to the opening of the ring structure, resulting in molecules that are undetectable via our native fluorescence spectroscopic detection system
Summary
All reagents were obtained from Sigma at analytical grade or higher, with no further purification. Modified Gray’s balanced salt solution (mGBSS) was previously described [16] and consisted of the following components (in mM): CaCl2 (1.5), KCl (4.9), KH2PO4 (0.2), MgCl2 [11], MgSO4 (0.3), NaCl [138], NaHCO3 (27.7), Na2HPO4 (0.8), HEPES [25], glucose [10], pH 7.2
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