A. E. Medvedev

Isatin: A link between natriuretic peptides and monoamines?

Isatin is an endogenous indole with a distinctive distribution in brain and tissues. In the brain, the highest levels have been found in the hippocampus (0.1 microgram/g), and an immunocytochemical stain has shown specific localization within particular cells. In vitro, its most potent known actions are as an inhibitor of monoamine oxidase B (IC50 approximately 3 microM), and of atrial natriuretic peptide (ANP) receptor binding and ANP-induced guanylate cyclase (both with an IC50 approximately 0.4 microM). In vivo, isatin administration (10-200 mg/kg) causes an increase of monoamine neurotransmitter levels in the brain. Isatin is anxiogenic in animal models at doses of 10-20 mg/kg and sedative at higher doses. Its anxiogenic effects are unlikely to be due to inhibition of monoamine oxidase, but may possibly stem from interaction with the ANP system. Isatin may mediate a link between monoamines and the natriuretic peptide system, and its analogues may provide new pharmacological tools.

Isatin : Role in stress and anxiety

(Indoledione 2,3) Isatin is an endogenous indole found both in mammalian brain and peripheral tissues. Isatin concentration in blood can exceed 1 μM and tissue concentrations vary from < 0.1 to 10 μM. Its level in the brain and periphery is increased by stress. Isatin has a wide spectrum of behavioural and metabolic effects. It is anxiogenic at lower doses and sedative at higher doses. Its most potent known in vitro actions are as an antagonist of atrial natriuretic peptide (ANP) function and NO signaling. In this review, we discuss isatin and stress in animal models, the few human studies, and also what it is known to date about the molecular mechanisms of its action. We suggest the possibility that isatin and its analogues may be interesting new pharmacological agents; Isatin antagonists may be anxiolytic, and isatin agonists may activate the HPA axis.

Isatin is a potent endogenous antagonist of guanylate cyclase-coupled atrial natriuretic peptide receptors

Isatin (indole-2,3-dione) is an endogenous compound with anxiogenic properties. In the brain, highest levels (0.1 microgram/g) have been found in the rat hippocampus. In the present study, we show that isatin has little effect on a wide range of neurotransmitter and hormonal receptors but that it acts as an inhibitor of atrial natriuretic peptide (ANP) binding, with an IC50 of 4x 10(-7) M. It also inhibits ANP-activated particulate guanylate cyclase from rat kidney, heart and brain membranes in dose-dependent fashion, varying also with ANP concentration. These findings suggest that isatin is a new endogenous regulator of mammalian ANP activity, with potential implications for the control of both anxiety and natriuresis.

Chromosome 18 Transcriptome Profiling and Targeted Proteome Mapping in Depleted Plasma, Liver Tissue and HepG2 Cells

The final goal of the Russian part of the Chromosome-centric Human Proteome Project (C-HPP) was established as the analysis of the chromosome 18 (Chr 18) protein complement in plasma, liver tissue and HepG2 cells with the sensitivity of 10(-18) M. Using SRM, we have recently targeted 277 Chr 18 proteins in plasma, liver, and HepG2 cells. On the basis of the results of the survey, the SRM assays were drafted for 250 proteins: 41 proteins were found only in the liver tissue, 82 proteins were specifically detected in depleted plasma, and 127 proteins were mapped in both samples. The targeted analysis of HepG2 cells was carried out for 49 proteins; 41 of them were successfully registered using ordinary SRM and 5 additional proteins were registered using a combination of irreversible binding of proteins on CN-Br Sepharose 4B with SRM. Transcriptome profiling of HepG2 cells performed by RNAseq and RT-PCR has shown a significant correlation (r = 0.78) for 42 gene transcripts. A pilot affinity-based interactome analysis was performed for cytochrome b5 using analytical and preparative optical biosensor fishing followed by MS analysis of the fished proteins. All of the data on the proteome complement of the Chr 18 have been integrated into our gene-centric knowledgebase ( www.kb18.ru ).

Effect of isatin on nitric oxide-stimulated soluble guanylate cyclase from human platelets

Isatin, an endogenous indole, has previously been shown to inhibit atrial natriuretic peptide (ANP)-stimulated particulate guanylate cyclase activity. Here, it was shown that it can be transported to human platelets where it inhibited nitric oxide (NO)-stimulated soluble guanylate cyclase activity obtained from human platelets. The effect was most pronounced at 10(-8)M isatin and is the most potent effect of isatin yet observed. The dose response curve was bell shaped with higher doses becoming less effective. The maximal inhibition observed was of 40%. Isatin had no effect on protoporphyrin IX-stimulated guanylate cyclase. Isatin-dependent regulation of ligand-stimulated guanylate cyclases is suggested to promote a stress-induced switch in metabolism.

Interaction of isatin with type-A natriuretic peptide receptor: Possible mechanism

The effect of isatin on rat brain particulate guanylate cyclase (GC) was investigated. The enzyme was stimulated by atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and urodilatin, but not by C-type natriuretic peptide (CNP). Their effects were not additive, pointing to action via the GC-A receptor. Isatin, in dose-dependent manner, abolished this stimulation. The non-hydrolysable ATP analogue, adenylylimidodiphosphate, potentiated the effects of submaximal doses of ANP, BNP and urodilatin on this particulate GC-A, and attenuated or abolished sensitivity to isatin. These results suggest that isatin antagonises the generation of second messenger by GC-A; this sensitivity might be regulated at an ATP binding site, possibly a protein kinase-like domain.

Inhibitory potency of some isatin analogues on human monoamine oxidase A and B

Isatin is an endogenous compound which acts as a selective inhibitor of monoamine oxidase (MAO) B. In this study a range of isatin analogues were tested for their in vitro inhibition of human MAO A and B. Most of the analogues were less potent than isatin. Hydroxylation of the aromatic ring changed the inhibitory potency in favour of MAO A, with 5-hydroxyisatin being a potent and selective MAO A inhibitor (IC50 8 microM). Isatinic acid, which is formed reversibly from isatin at alkaline pH, showed no inhibition.

Isatin-binding proteins of rat and mouse brain: Proteomic identification and optical biosensor validation

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. Isatin itself and its analogues exhibit a wide range of pharmacological activities but its specific biological targets still are not well characterized. Affinity chromatography of Triton X‐100 lysates of soluble and particulate fractions of mouse and rat whole brain homogenates on 5‐aminocaproyl‐isatin‐Sepharose followed by subsequent proteomic analysis resulted in identification of 65 and 64 individual proteins, respectively. Isatin‐binding capacity of some of the identified proteins has been validated in an optical biosensor study using a Biacore 3000 optical biosensor, 5‐aminocarproyl‐isatin, and 5‐aminoisatin as the affinity ligands. The Kd values (of 0.1–20 μM) obtained during the optical biosensor experiments were consistent with the range of Kd values recently reported for [3H]isatin binding to brain sections. Although the number of isatin‐binding proteins identified in the mouse and rat brain was similar, only 21 proteins (about one‐third) were identical in the two species. This may be one reason for the differences in isatin effects in rats and mice reported in the literature.

Natriuretic peptide interaction with [3H]isatin binding sites in rat brain.

Isatin is an endogenous indole, which has a distinct and discontinuous distribution in the brain and exhibits a wide range of physiological and pharmacological effects. In the present study, we have demonstrated that atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) inhibited [3H]isatin binding to rat brain sections and isolated membrane fractions. Isatin itself antagonised not only natriuretic peptide receptor type A (NPR-A) (ANP-stimulation of guanylyl cyclase) but also NPR-C (ANP and CNP mediated inhibition of adenylyl cyclase) signalling. These results suggest that some [3H]isatin binding in the brain may be to NPR-A and NPR-C. Competitive interactions between isatin and natriuretic peptides and their receptors give a possible explanation of the known anxiogenic effect of low doses of isatin, interacting at NPR-A, and the sedative effects of higher doses, antagonising respectively the anxiolytic effect of ANP and the anxiogenic effect of CNP.

Tribulin and Endogenous MAO-Inhibitory Regulation In Vivo

Tribulin is the name given to a family of endogenous nonpeptide substances which inhibit monoamine oxidase (MAO) and benzodiazepine binding. It is widely distributed in mammalian tissues and body fluids, and exhibit some species and tissue variations. Several components selectively inhibiting MAO A, MAO B, central and peripheral benzodiazepine binding (tribulins A, B, BZc and BZp, respectively) have been recognised. Tribulin A represents some tissue-specific metabolites of trace amines, whereas isatin is the major component of tribulin B. Tribulin content increases in brain under conditions of stress and anxiety and is reduced under sedation. Changes in tribulin content in the brain are accompanied by corresponding changes in the content of monoamines and their acidic metabolites, and also by altered susceptibility of MAO to specific mechanism-based inhibitors. This suggests that tribulin is involved in MAO inhibitory regulation in vivo.