Isatin binding proteins in rat brain: in situ imaging, quantitative characterization of specific [3H]isatin binding, and proteomic profiling.

Isatin (indole-2,3-dione) is an endogenous indole that has a distinct and discontinuous distribution in the brain and in other mammalian tissues and body fluids. Its output is increased under conditions of stress and anxiety. Its biological targets remain poorly characterized, although [(3)H]isatin binding sites have been demonstrated in various brain structures. In this study, by using a real-time beta-imager, [(3)H]isatin radioligand binding analysis, and proteomic identification of proteins specifically bound to the affinity sorbent 5-aminocaproyl-isatin-Sepharose, we have investigated the distribution of [(3)H]isatin specific binding sites in the rat brain, characterized their K(d) and B(max), and identified some individual brain isatin binding proteins. The binding of [(3)H]isatin to rat brain sections was saturable and characterized by K(d) values (of 0.2-0.3 microM) consistent with physiological concentrations. The highest B(max) was found in the hypothalamus, consistent with a role in stress. In most brain regions, the homologous inhibition of [(3)H]isatin binding by increasing concentrations of cold isatin demonstrated complex behavior suggesting involvement of various binding proteins characterized by different affinity to isatin. Affinity chromatography of Triton X-100 lysates of whole-brain homogenates on 5-aminocaproyl-isatin-Sepharose followed by subsequent proteomic analysis resulted in identification of 25 individual proteins, including glyceraldehyde-3-phosphate dehydrogenase, one of few previously reported isatin binding proteins, and a group of cytoskeleton-related proteins. These binding sites may be related to the known antiproliferative and proapoptotic activities of isatin.

5-hydroxyoxindole, an indole metabolite, is present at high concentrations in brain.

5-Hydroxyoxindole has been identified as a urinary metabolite of indole, which is produced from tryptophane via the tryptophanase activity of gut bacteria. We have demonstrated recently that 5-hydroxyoxindole is an endogenous compound in blood and tissues of mammals, including humans. To date, 5-hydroxyoxindole's role is unknown. The aim of this study was to compare 5-hydroxyoxindole levels in plasma and cerebrospinal fluid (CSF) during day-night and seasonal changes, as a common approach to pilot physiological characterization of any compound. Simultaneous blood and CSF sampling was performed in the ewe, because its size allows collection in quantities suitable for 5-hydroxyoxindole assay (HPLC-ED) in awake animals, without obvious physiological or behavioral disturbance. 5-Hydroxyoxindole concentration was quite stable in plasma (2-6 nM range), whereas, in CSF, it displayed marked day-night and photoperiodic variations (4-116 nM range). 5-Hydroxyoxindole levels in CSF were twofold higher at night than during the day and at least one order of magnitude higher during the long compared with the short photoperiod. These day/night and photoperiodic variations persisted after pinealectomy, indicating that 5-hydroxyoxindole rhythms in CSF are independent of melatonin formation. In conclusion, high levels of 5-hydroxyoxindole in the CSF during long photoperiod and its daily modulation suggest physiological involvement of 5-hydroxyoxindole in rhythmic adjustments in the brain, independently of the pineal gland.

Inhibition of brain mitochondrial monoamine oxidases by the endogenous compound 5-hydroxyoxindole.

5-Hydroxyoxindole is a recently identified endogenous compound. Its physiological role remains unclear but certain evidence exists, that it may share some regulatory properties with isatin, a known endogenous inhibitor of monoamine oxidase (MAO) type B (MAO-B). In this study several oxidized indoles were tested for their in vitro inhibition of MAO type A (MAO-A) and B of rat brain non-synaptic mitochondria. 5-Hydroxyoxindole was less potent MAO-A inhibitor (IC50 56.8 microM) than isatin (31.8 microM) and especially 5-hydroxyisatin (6.5 microM), but it was the only highly selective MAO-A inhibitor among the all compounds studied (IC50 MAO-A:IC50 MAO-B = 0.044). Thus, the in vitro data suggest that MAO-A may represent potential target for 5-hydroxyoxindole.

Detection and quantification of 5-hydroxyoxindole in mammalian sera and tissues by high performance liquid chromatography with multi-electrode electrochemical detection.

OBJECTIVE: Since 5-hydroxyoxindole structurally related indole metabolites play different roles in some hepatic and neurologic disorders we found necessary to develop an assay to further investigate the physiologic relevance of this compound. METHODS: We have designed a convenient assay to determine 5-hydroxyoxindole in serum using solid phase extraction and a highly selective High Performance Liquid Chromatography system with multi-Electro Chemical Detection (HPLC-ECD). RESULTS: We have identified and quantified 5-hydroxyoxindole in various mammalian species. Its distribution in tissues showed that the molecule is also present in brain, liver, kidney and spleen, but not in skeletal muscle. CONCLUSIONS: 5-hydroxyoxindole is an endogenous tryptophan metabolite present in circulating blood and in some tissues at the nmol level, its determination using HPLC-ECD will be useful for elucidating the role of this molecule in normal and disease conditions.