Vija Klusa

Betulin binds to γ-aminobutyric acid receptors and exerts anticonvulsant action in mice

The lupane type pentacyclic triterpenes: lupeol, betulin, and betulinic acid are widely distributed natural compounds. Recently, pharmaceutical compositions from plant extracts (family Marcgraviaceae) containing betulinic acid, have been patented as anxiolytic remedies. To extend our knowledge of the CNS effects of the triterpenes, we suggest here that the chemically related lupeol, betulin and betulinic acid may interact with the brain neurotransmitter gamma-aminobutyric acid (GABA) receptors in vitro and in vivo. Using radioligand receptor-binding assay, we showed that only betulin bound to the GABA(A)-receptor sites in mice brain in vitro and antagonised the GABA(A)-receptor antagonist bicuculline-induced seizures in mice after intracisternal and intraperitoneal administration. Neither betulinic acid nor lupeol bound to GABA(A) receptor nor did they inhibit bicuculline-induced seizures in vivo. These findings demonstrate for the first time the CNS effects of betulin in vivo, and they also show distinct GABA(A)-receptor-related properties of lupane type triterpenes. These findings may open new avenues in understanding the central effects of betulin, and they also indicate possibilities for novel drug design on the basis of betulin structure.

Mitochondria as the target for mildronate's protective effects in azidothymidine (AZT)-induced toxicity of isolated rat liver mitochondria.

Previously mildronate, an aza‐butyrobetaine derivative, was shown to be a cytoprotective drug, through its mechanism of action of inhibition of carnitine palmitoyltransferase‐1, thus protecting mitochondria from long‐chain fatty acid accumulation and subsequent damage. Recently in an azidothymidine (AZT)‐induced cardiotoxicity model in vivo (in mice), we have found mildronates ability of protecting heart tissue from nuclear factor κB abnormal expression. Preliminary data also demonstrate cerebro‐ and hepatoprotecting properties of mildronate in AZT‐toxicity models. We suggest that mildronate may target its action predominantly to mitochondria. The present study in isolated rat liver mitochondria was designed to clarify mitochondrial targets for mildronate by using AZT as a model compound. The aim of this study was to investigate: (1) whether mildronate may protect mitochondria from AZT‐induced toxicity; and (2) which is the most critical target in mitochondrial processes that is responsible for mildronates regulatory action. The results showed that mildronate protected mitochondria from AZT‐induced damage predominantly at the level of complex I, mainly by reducing hydrogen peroxide generation. Significant protection of AZT‐caused inhibition of uncoupled respiration, ADP to oxygen ratio, and transmembrane potential were also observed. Mildronate per se had no effect on the bioenergetics, oxidative stress, or permeability transition of rat liver mitochondria. Since mitochondrial complex I is the first enzyme of the respiratory electron transport chain and its damage is considered to be responsible for different mitochondrial diseases, we may account for mildronates effectiveness in the prevention of pathologies associated with mitochondrial dysfunctions. Copyright

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.

Anti-inflammatory effects of cerebrocrast in a model of rat paw edema and on mononuclear THP-1 cells

Cerebrocrast (IOS 1.1212; 4-[2-(difluoromethoxy)phenyl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid di(2-propoxyethyl) diester) is a novel derivative of 1,4-dihydropyridine, which does not antagonize Ca(2+) influx in neuronal tissues. Since several classical dihydropyridines possess anti-inflammatory properties, we first studied the effects of cerebrocrast in a model of rat paw edema induced by carrageenan. Cerebrocrast had a preventative effect in this model of inflammation, with maximal activity (32-45% inhibition) in the 0.1-0.25 mg kg(-1) range. It was ineffective when added after the injection of carrageenan. Subsequent in vitro experiments showed that cerebrocrast in the micromolar range inhibited secretion of interleukin-1 beta, interleukin-6 and neurotoxic products by cells of the human monocytic THP-1 line while failing to affect secretion of tumor necrosis factor (TNF-alpha). It also lacked any direct neuroprotective effect against toxic secretions from stimulated THP-1 cells. The data obtained suggest that cerebrocrast may be useful not only in various inflammatory disorders of peripheral tissues, but also in treating neurodegenerative diseases, where inflammatory mechanisms in general and microglial activation, in particular, are thought to play an important role.

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.

Potato (Solanum tuberosum) Juice Exerts an Anticonvulsant Effect in Mice through Binding to GABA Receptors

Naturally occurring benzodiazepines have been identified in regular food such as wheat and potato, but there is still no evidence that potato extracts can affect CNS responses in vivo. Here we found that undiluted potato juice and potato juice diluted with saline 1 : 2 administered 10 min intracisternally ( I. C.) and 30 min per os before bicuculline exerted significant anticonvulsant activity in the bicuculline-induced seizure threshold test in mice. In vitro, potato juice from different harvests at dilution series from 10 % to 0.000001 %, diluted 100,000-fold, displaced 50 % of gamma-aminobutyric acid (GABA) receptor ligand [ (3)H]GABA and diluted 40-fold displaced 50 % of [(3)H]flunitrazepam from binding sites in mice forebrain membranes. The low content of diazepam (0.04 +/- 0.01 mg/kg) determined by HPLC and mass spectrometry in the potato extracts could not sustain the anticonvulsant activity of potato juice in vivo; therefore we hypothesized that potato juice might contain GABA (A) receptor GABA-site active compounds. The findings of this study suggest that potato juice as well as potato taken as food may have the capacity of influencing brain GABA-ergic activity.

The differential influences of melanocortins on nociception in the formalin and tail flick tests

Melanocortins exert multiple physiological effects that include the modulation of immune responses, inflammation processes, and pain transmission. In the present study we investigated the peripheral activity of natural melanocortins - alpha-, beta-, gamma1- and gamma2-melanocyte stimulating hormone (MSH) - and melanocortin receptor subtypes 3 and 4 (MC3/4 receptor) antagonist HS014 in pain (formalin and tail flick) tests after peptide subcutaneous administration in mice. In the formalin test, among all substances tested only alpha-MSH (1 micromol/kg) statistically significantly inhibited the formalin-induced first phase pain response, however, all tested peptides (except gamma1-MSH) at the dose of 1 micromol/kg produced a pronounced inhibitory effect on nociceptive behavior in the second phase and this activity was comparable with that of indomethacin (reference drug, 5 mg/kg intraperitoneally); beta-MSH was also active at a dose 0.1 micromol/kg. In the tail flick test, alpha-MSH (1 micromol/kg) showed algesic, whereas HS014 (0.5 micromol/kg) and indomethacin (10 mg/kg) exerted analgesic activity. Other peptides did not exert any activity in the tail flick test. These data indicate that peripherally administered melanocortin receptor agonists alpha-MSH, beta-MSH and gamma2-MSH, as well as MC3/4 receptor antagonist HS014 induced antinociception on pain/inflammatory events caused by formalin suggesting a predominant anti-inflammatory role of these peptides.