Simons Svirskis

The Evolutionary History and Tissue Mapping of Amino Acid Transporters Belonging to Solute Carrier Families SLC32, SLC36, and SLC38

Members of the solute carrier families (SLC) 32, 36, and 38, together also designated the β-group of SLCs, are known to transport neutral amino acids. In this paper, we show that these three families were present before the split of the animal lineage and that they are likely to share a common decent. We also show that the APF transporters found in plants are most likely homologous to the mammalian β-group, suggesting that this type of transporters arouse early in the evolution of eukaryotes. We performed detailed tissue expression analysis of all the members of the β-group in rat and found several examples of highly specific expression patterns, with SLC38A7 being exclusively found in liver, SLC38A5 in blood, and SLC38A4 in muscle and liver. Moreover, we found that SLC38A10 is expressed in several endocrine organs. We also found that SLC38A1 is highly up regulated in the cortex from rats treated with diazepam and that SLC38A2 is significantly down regulated in the same tissue. In addition, we performed a detailed expression analysis of SLC38A1 and SLC38A6 in mouse brain using in situ hybridization, which showed that both these transporters are widely expressed in the brain.

Evaluation of behavioural effects of neural melanocortin receptor antagonists injected ICV and in VTA in rats

The natural melanocortic peptides are known to exert a variety of effects after central administration. Recently, we discovered the first potent and selective substances for the MC4 receptor, i.e. HS964 and HS014. We found HS964 to be an antagonist for the MC1, MC3, MC4 and MC5 receptors in vitro. HS014 is an antagonist for the MC3 and MC4 receptors and a partial antagonist for the MC1 and MC5 receptors. We injected alpha-MSH and these substances, both intracerebroventricular (ICV) and in the ventral tegmental area (VTA) in rats and scored several behavioural effects. The results show that alpha-MSH caused intensive grooming which was antagonized by pre-treatment of both HS014 and HS964. The data give further support to the hypothesis that it is the MC4 receptor which mediates grooming in rodents. The grooming effects of alpha-MSH were more pronounced after intra-VTA administration compared to the ICV administration. Both alpha-MSH, HS014 and HS964 caused an increase in vertical activity of the rats after intra-VTA administration but not after ICV administration. Horizontal activity was virtually not affected by the administration of the peptides. The data indicate that the neural MC3 and MC4 receptors are not likely to be an important mediators of locomotor activity in rats.

The g2-MSH peptide mediates a central analgesic effect via a GABA-ergic mechanism that is independent from activation of melanocortin receptors

Using the latency for tail-flick after thermal stimulation we have assessed the effects of alpha-, gamma(1)- and gamma(2)-MSH on nociceptive threshold in the mice. Intracisternal injections of gamma(2)-MSH induced a distinct analgesia, while gamma(1)-MSH in the same doses gave only a minor analgesia. Intracisternal alpha-MSH instead gave a short-term hyperalgesia. The effect of gamma(2)-MSH was not blocked by any of the MC(4)/MC(3)receptor antagonist HS014, naloxone or by the prior intracisternal administrations of gamma(1)-MSH. However, the gamma(2)-MSH analgesic response was completely attenuated by treating animals with the GABA(A)antagonist bicuculline. The gamma(2)-MSH analgesic effect was moreover additive to the analgesia afforded by muscimol and ethanol, but not to that afforded by diazepam. In addition both gamma(1)- and gamma(2)-MSH induced moderate catalepsy, but could at the same time attenuate haloperidol induced catalepsia. We conclude that gamma(2)-MSH mediates a central analgesic effect via GABA-receptor dependent pathway that is distinct from melanocortic- and opioid-receptors. Moreover, the mechanism for gamma(2)-MSHs analgesic effect appears to be distinct from that causing moderate catalepsia by gamma-MSHs.

Neuroprotective properties of mildronate, a mitochondria-targeted small molecule.

Mildronate, a representative of the aza-butyrobetaine class of drugs with proven cardioprotective efficacy, was recently found to prevent dysfunction of complex I in rat liver mitochondria. The present study demonstrates that mildronate also acts as a neuroprotective agent. In a mouse model of azidothymidine (anti-HIV drug) neurotoxicity, mildronate reduced the azidothymidine-induced alterations in mouse brain tissue: it normalized the increase in caspase-3, cellular apoptosis susceptibility protein (CAS) and iNOS expression assessed by quantitative and semi-quantitative analysis. Mildronate also normalized the changes in cytochrome c oxidase (COX) expression, reduced the expression of glial fibrillary acidic protein (GFAP) and cellular infiltration. The present results show that the neuroprotective action of mildronate results at least partially from anti-neurodegenerative (anti-apoptotic) and anti-inflammatory mechanisms. It might be suggested that the molecular conformation of mildronate can facilitate its easy binding to mitochondria, and regulate the expression of different signal molecules, hence maintaining cellular signaling and survival.

Opposite effects of γ1- and γ2-melanocyte stimulating hormone on regulation of the dopaminergic mesolimbic system in rats

Opposite effects of gamma(1)- and gamma(2)-melanocyte stimulating hormone on regulation of the dopaminergic mesolimbic system in rats.

Neuroprotective Properties of Mildronate, a Small Molecule, in a Rat Model of Parkinson’s Disease

Previously, we have found that mildronate [3-(2,2,2-trimethylhydrazinium) propionate dihydrate], a small molecule with charged nitrogen and oxygen atoms, protects mitochondrial metabolism that is altered by inhibitors of complex I and has neuroprotective effects in an azidothymidine-neurotoxicity mouse model. In the present study, we investigated the effects of mildronate in a rat model of Parkinson’s disease (PD) that was generated via a unilateral intrastriatal injection of the neurotoxin 6-hydroxydopamine (6-OHDA). We assessed the expression of cell biomarkers that are involved in signaling cascades and provide neural and glial integration: the neuronal marker TH (tyrosine hydroxylase); ubiquitin (a regulatory peptide involved in the ubiquitin-proteasome degradation system); Notch-3 (a marker of progenitor cells); IBA-1 (a marker of microglial cells); glial fibrillary acidic protein, GFAP (a marker of astrocytes); and inducible nitric oxide synthase, iNOS (a marker of inflammation). The data show that in the 6-OHDA-lesioned striatum, mildronate completely prevented the loss of TH, stimulated Notch-3 expression and decreased the expression of ubiquitin, GFAP and iNOS. These results provide evidence for the ability of mildronate to control the expression of an array of cellular proteins and, thus, impart multi-faceted homeostatic mechanisms in neurons and glial cells in a rat model of PD. We suggest that the use of mildronate provides a protective effect during the early stages of PD that can delay or halt the progression of this neurodegenerative disease.

Modification of swelling–contraction–aggregation processes in rat muscle mitochondria by the 1,4‐dihydropyridines, cerebrocrast and glutapyrone, themselves and in the presence of azidothymidine

The influence of the 1,4‐dihydropyridines (DHPs), water‐soluble glutapyrone available as sodium, potassium and ammonium salts of 2‐(2,6‐dimethyl‐3,5‐diethoxycarbonyl‐1,4‐DHP‐4‐carboxamide)glutaric acid, from one side, and a lipophylic cerebrocrast, 2‐propoxyethyl 2,6‐dimethyl‐4‐(2‐difluoromethoxyphenyl)‐1,4‐DHP‐3,5‐dicarboxylate, from the other side, on partially damaged mitochondria of the Wistar rat hindlimb muscle was also studied. The following tests were made: (1) rates of endogenous respiration and substrate (succinate) oxidation and oxidative phosphorylation; (2) rates and amplitudes of high‐amplitude swelling and contraction after the addition of ATP, ADP and succinate to the previously swollen mitochondria and (3) rate of reversible self‐aggregation of mitochondria isolated in salt media after ATP‐induced contraction without and in the presence of azidothymidine (AZT). Cerebrocrast (10–100 μM) partially normalized the endogenous respiration rate and slightly augmented the respiration rate after the addition of succinate and to lesser extent ADP. Cerebrocrast in a concentration‐dependent manner (2·5–50 μM) increased (two‐fold at 20–50 μM) the active contraction amplitude of swollen mitochondria, induced by single or repeated additions of ATP. The influence of cerebrocrast on the ADP‐ and succinate‐induced contractions was less obvious. Unlike cerebrocrast glutapyrone caused a reduction of the ATP‐induced contraction amplitude (two‐fold at 0·5–5·0 mM), not impairing the mitochondrial contraction ability in response to ATP or succinate. Pre‐exposure to 2·5 mM glutapyrone resulted in at least a 10‐fold inhibition of the reversible aggregation rate in the presence of 99 and 198 μM AZT. The results suggest the usefulness of further study of cerebrocrast and glutapyrone in preventing AZT‐induced and some other mitochondrial myopathies.

Characterization of the transporterB0AT3 (Slc6a17) in the rodent central nervous system

BackgroundThe vesicular B0AT3 transporter (SLC6A17), one of the members of the SLC6 family, is a transporter for neutral amino acids and is exclusively expressed in brain. Here we provide a comprehensive expression profile of B0AT3 in mouse brain using in situ hybridization and immunohistochemistry.ResultsWe confirmed previous expression data from rat brain and used a novel custom made antibody to obtain detailed co-labelling with several cell type specific markers. B0AT3 was highly expressed in both inhibitory and excitatory neurons. The B0AT3 expression was highly overlapping with those of vesicular glutamate transporter 2 (VGLUT2) and vesicular glutamate transporter 1 (VGLUT1). We also show here that Slc6a17mRNA is up-regulated in animals subjected to short term food deprivation as well as animals treated with the serotonin reuptake inhibitor fluoxetine and the dopamine/noradrenaline reuptake inhibitor bupropion.ConclusionsThis suggests that the B0AT3 transporter have a role in regulation of monoaminergic as well as glutamatergic synapses.

Comparative study of taurine and tauropyrone: GABA receptor binding, mitochondrial processes and behaviour

Objectives  Taurine, a sulfur‐containing amino acid, has high hydrophilicity and is poorly absorbed. Tauropyrone, a taurine‐containing 1,4‐dihydropyridine derivative, is suggested to have greater activity than taurine owing to improved physicochemical properties that facilitate delivery of the compound to target cells. The aim of this study was to determine whether the 1,4‐dihydropyridine moiety in tauropyrone improves the pharmacological efficacy of taurine in vitro and in vivo.

Distinct Influence of Atypical 1,4‐Dihydropyridine Compounds in Azidothymidine‐Induced Neuro‐ and Cardiotoxicity in Mice Ex Vivo

This study demonstrates the effective protection by compounds of atypical 1,4-dihydropyridine (DHP) series cerebrocrast, glutapyrone and tauropyrone against neuro- and cardiotoxicity caused by the model compound azidothymidine, a well-known mitochondria-compromising anti-HIV drug. In previous in vitro experiments, we have demonstrated distinct effects of these DHP compounds to influence mitochondrial functioning. In the present in vivo experiments, DHP compounds were administered intraperitoneally in mice daily for 2 weeks, per se and in combinations with azidothymidine at doses: azidothymidine 50 mg/kg; cerebrocrast 0.1 mg/kg; glutapyrone 1 mg/kg; and tauropyrone 1 mg/kg. At the end of the experiment, mice were killed, heart and brain tissues were removed and examined ex vivo histopathologically and immunohistochemically. NF-kappaBp65 and caspase-3 were used as the markers indicating inflammatory and apoptotic events, respectively. Cerebrocrast (dicyclic structure) was the most potent DHP, which effectively reduced azidothymidine-induced overexpression of NF-kappaBp65 and caspase-3 in mouse myocardium and brain cortex. Glutapyrone per se increased the number of caspase-3-positive cells in the brain, whereas it reduced NF-kappaBp65 and caspase-3 expression in cardiac tissue caused by azidothymidine. Tauropyrone showed dual action: per se it increased caspase-3 in the brain and NF-kappaBp65 expression in the heart, but it considerably reduced these activations in azidothymidine-treated mice. This study provides the first demonstration of a distinct pharmacological action for atypical DHP compounds in cardiac and brain tissues. The dicyclic structure of cerebrocrast is considered beneficial for neuro- and cardioprotection at least in part via mitochondrial targeting and consequent regulation of inflammatory and apoptotic processes.