The Influence of the Cholesterol Level in Cells on Endovanilloid Cytotoxicity.

The influence of the cellular cholesterol content on the cytotoxicity of endovanilloids acyldopamines was studied in MDA-MB-231 and MCF 10A cells. The activity of acyldopamines depends on the cellular cholesterol content, and a decrease in cholesterol content increases the cytotoxicity of acyldopamines.

Neuroprotective and Antioxidant Activity of Arachidonoyl Choline, Its Bis-Quaternized Analogues and Other Acylcholines.

The antioxidant activity and protective effect in the toxicity model of H2O2 were studied for arachidonic (AA-CHOL), docosahexaenoic (DHA-CHOL), linoleic (Ln-CHOL), and oleic (Ol-CHOL) fatty acids, as well as arachidonoyl dicholine (AA-diCHOL) and O-arachidonoyl bistetramethylaminoisopropanol (ABTAP). AA-CHOL, DHA-CHOL and Ln-CHOL provided a 20% increase in cell survival. AA-CHOL, AA-diCHOL, Ol-CHOL, and ABTAP had a radical-scavenging effect in the ABTS test, approximately equal to the activity of a standard radical scavenger Trolox.

Early-life N-arachidonoyl-dopamine exposure increases antioxidant capacity of the brain tissues and reduces functional deficits after neonatal hypoxia in rats.

Perinatal hypoxia-ischemia is one of the most common causes of perinatal brain injury and subsequent neurological disorders in children. The aim of this work was to evaluate the potential antioxidant and neuroprotective effects of N-arachidonoyl-dopamine (NADA) in the model of acute neonatal hypoxia (ANH) in rat pups. Male and female Wistar rats were exposed to a hypoxic condition (8% oxygen for 120 min) at postnatal day 2 (P2). Transcription factor HIF1-α and glutathione peroxidases GPx2 and GPx4 gene expression was increased in rat brains in the hypoxic group compared to control 1.5 h but not 4 days after ANH. There were no post-hypoxic changes in reduced (GSH) and oxidised (GSSG) glutathione levels in the brain of rat pups 1.5 h and 4 d after hypoxia. Hypoxic rats displayed retarded performance in the righting reflex and the negative geotaxis tests. ANH resulted in increased ambulation in Open field test and impaired retention in the Barnes maze task under stressful conditions as compared with the control group. Treatment with NADA significantly attenuated the delayed development of sensorimotor reflexes and stress-evoked disruption of memory retention in hypoxic rats but had no effect on the hypoxia-induced hyperactivity. In rats exposed to hypoxia, treatment with NADA decreased GPx2 gene expression and increased GSH/GSSG ratio in whole brains 1.5 h after ANH. These results suggest that the long-lasting beneficial effects of NADA on hypoxia-induced neurobehavioural deficits are mediated, at least in part, by its antioxidant properties.

Neuroprotective Action of Amidic Neurolipins in Models of Neurotoxicity on the Culture of Human Neural-Like Cells SH-SY5Y.

It was established that in neurodegeneration models in the human neuron-like cell line SH-SY5Y, amide derivatives of arachidonic and docosahexaenoic acids were inactive in experiments with MPP+ and CoCl2 but protected from H2O2. The protective activity of neurolipins decreased in the series DHA-DA > AA-SER ≥ AA-GLY > AA-GABA ≥ AA-EA and was manifested starting from a concentration of 0.5 nM.

Selective Action of N-Arachidonoyl Dopamine on Viability and Proliferation of Stromal Cells from Eutopic and Ectopic Endometrium.

We performed a comparative study of the cytotoxic effect of endocannabinoid N-arachidonoyl dopamine (AA-DA) on cultured stromal cells of ectopic and eutopic endometrium. It was found that AA-DA in the concentration range of 1-20 µM produces more selective cytotoxic effect on the stromal cells of the ectopic endometrium due to interaction with cannabinoid type 1 receptor. In concentrations below 1 µM, AA-DA stimulated the proliferation of stromal cells of the eutopic endometrium and did not affect the division of ectopic endometrium cells. This effect was realized due to its interaction with cannabinoid type 2 receptor.

Neuroprotective Properties of Endocannabinoids N-Arachidonoyl Dopamine and N-Docosahexaenoyl Dopamine Examined in Neuronal Precursors Derived from Human Pluripotent Stem Cells.

Neuroprotective properties of endocannabinoids N-arachidonoyl dopamine (NADA) and N-docosahexaenoyl dopamine (DHDA) were examined in neuronal precursor cells differentiated from human induced pluripotent stem cells and subjected to oxidative stress. Both compounds exerted neuroprotective activity, which was enhanced by elevating the concentration of the endocannabinoids within the 0.1-10 µM range. However, both agents at 10 µM concentration showed a marked toxic effect resulting in death of ~30% of the cells. Finally, antagonists of cannabinoid receptors as well as the receptor of the TRPV1 endovanilloid system did not hamper the neuroprotective effects of these endocannabinoids.

Synthesis of N-acyl derivatives of Pro-Gly-Pro-Leu peptide: Proteolytic stability in vitro and effects on mouse macrophage cells RAW264.7.

Acetyl, oleoyl, arachidonoyl, and docosahexaenoyl derivatives of the Pro-Gly-Pro-Leu peptide with a chemical purity of 99.8% were synthesized. The degradation kinetics of the Pro-Gly-Pro-Leu derivatives under the action of leucine aminopeptidase, nasal mucus, and microsomal fraction of the brain and blood of rats was studied. It was shown that the N-acyl derivatives of Pro-Gly-Pro-Leu proved to be more resistant to the action of leucine aminopeptidase and other enzyme systems. The study of the cytotoxic and anti-inflammatory activity of preparations on the mouse macrophage cell line RAW264.7 showed that acylation with oleic and arachidonic acid makes the peptide cytotoxic with LC50 in the range of 70-15 µM and gives it anti-inflammatory properties with EC50 of 32 and 36 µM, respectively.

N-acyl dopamines induce apoptosis in PC12 cell line via the GPR55 receptor activation.

Dopamine amides of arachidonic, docosahexaenoic, and oleic acids were found to induce apoptosis in PC12 cells, which was blocked exclusively by antagonists and preincubation agonists of the receptor GPR55, belonging to the group of non-CB1/CB2 receptors.

Expression of Type I Cannabinoid Receptors at Different Stages of Neuronal Differentiation of Human Fibroblasts.

Differential expression of type 1 cannabinoid receptors (CR1) was evaluated at different stages of human skin fibroblast transformation into terminally differentiated neurons. Immunocytochemical staining detected no CR1 on fibroblasts, but their transformation into induced pluripotent stem cells was accompanied by marked stimulation of CR1 expression. In neuronal precursors, the receptors were located mainly on cell bodies and at the base of their processes. This distribution was retained at the terminal stage of differentiation of induced pluripotent stem cells into neurons.

N-acyl dopamines induce cell death in PC12 cell line via induction of nitric oxide generation and oxidative stress.

It was shown that dopamine amides of arachidonic, oleic, and docosahexaenoic acids exhibit toxicity with respect to PC12 pheochromocytoma cell line. The mechanism of realization of the cytotoxic effect of acyl dopamines is the induction of oxidative stress. This event is preceded by triggering the synthesis of nitric oxide.

Binding assessment of two arachidonic-based synthetic derivatives of adrenalin with ß-lactoglobulin: Molecular modeling and chemometrics approach.

A computational approach to predict the main binding modes of two adrenalin derivatives, arachidonoyl adrenalin (AA-AD) and arachidonoyl noradrenalin (AA-NOR) with the ß-lactoglubuline (BLG) as a nano-milk protein carrier is presented and assessed by comparison to the UV-Vis absorption spectroscopic data using chemometric analysis. Analysis of the spectral data matrices by using the multivariate curve resolution-alternating least squares (MCR-ALS) algorithm led to the pure concentration calculation and spectral profiles resolution of the chemical constituents and the apparent equilibrium constants computation. The negative values of entropy and enthalpy changes for both compound indicated the essential role of hydrogen bonding and van der Waals interactions as main driving forces in stabilizing protein-ligand complex. Computational studies predicted that both derivatives are situated in the calyx pose and remained in that pose during the whole time of simulation with no any significant protein structural changes which pointed that the BLG could be considered as a suitable carrier for these catecholamine compounds.

[STUDYING NEUROPROTECTIVE ACTIVITY OF GABA CONJUGATE WITH ARACHIDONIC ACID UNDER CONDITIONS OF LOCAL PERMANENT BRAIN ISCHEMIA IN RATS].

We have studied the effect of a GABA conjugate with arachidonic acid (AA) on the morphological state of rat brain tissues after left median cerebral artery occlusion. The results showed that a 6- and 12-day course administration of the GABA - AA conjugate at dose of 2 mg/kg (i.p.) in rats with this model of local permanent brain ischemia led to significant recovery processes in brain tissues. The tissue morphology pattern in the group of animals treated with the GABSA - AA conjugate for 12 days was almost identical to that in intact tissues.

[The optimization of the nitric oxide quantitative analysis for its determination in the cultural medium of mammalian cell culture].

The protocol for the quantitative analysis of nitric oxide as nitrite-ion suitable for determination of its production by a mammalian cell culture was developed. The optimal results were obtained using microvolume-adjusted Griess method after the preliminary reduction of NO3- to NO2- with non-activated cadmium. The protocol was verified on a rat glioma C6 cell culture. The developed method may be used for the nitric oxide determination in 96-well and 48-well microplates; the detection limit is 2.1 ± 0.1 µM for NO2- and 2.9 ± 0.1 µM for NO3-.

[GABA-ergic mechanism of cerebrovascular and antiischemic effects of docosahexaenoic acid].

In experiments on rats, measurements of the local blood flow in the cortex of cerebrum with the aid of a laser Doppler flow meter showed that docosahexaenoic acid (DHA) enhanced the local cerebral circulation in animals with global transient cerebral ischemia, while not influencing that in intact animals. This vasodilatory effect of DHA in ischemized rats is blocked by bicuculline (specific GABA(A) receptor blocker), which is indicative of a GABA-ergic mechanisms of the vascular tone regulation. The results of radioligand binding assay in vitro showed the possibility of direct DHA interaction with cerebrovascular GABA(A) receptors.

[The influence of docosahexaenoic acid moiety on cytotoxic activity of 1,2,4-thiadiazole derivatives].

Among 3-(2-aminopropyl)-1,2,4-thiadiazole derivatives contatining substitution-ready secondary amino group and exhibiting cytotoxic towards rat C 6 glioma cells three compounds with LD 50 values ranged from 6 to 48 мM were chosen. For these compounds amides with docosahexaenoic acid were synthetised and their cytotoxic activity was studied. It was shown that, although docosahexaenoic acid itself was not toxic for C 6 glioma cells, its addition to the amino derivatives of 1,2,4-thiadiazole increased or decreased resultant cytotoxicity. The effect depended on the structure of 1,2,4-thiadiazole substituents. The obtained data show that the acylation of cytotoxic compounds with docosahexaenoic acid does not necessarily lead to the increase of their activity, but sometimes can inactivate a compound. This fact should be taken into account, especially in the case of anti-cancer drug development.

[Antiaggregation activity of arachidonic acid conjugates with neurotropic peptides proglyprol and semax].

The influence two original derivatives of a therapeutically important peptide, bearing arachidonic acid residue with semax and proglyprol, upon platelet aggregation have been studied in vitro. It is established that both derivatives, in contrast to the parent peptide, possess moderate anti-aggregant properties and produce a dose-dependent decrease in the interplatelet interaction induced by ADP, epinephrine, and arachidonic acid within the concentration range of 0.018 - 1.8 mM. This activity was more pronounced for arachidonoylsemax in comparison with arachidonoylproglyprol.

Effect of N-arachidonoyl dopamine on activity of neuronal network in primary hippocampus culture upon hypoxia modelling.

We studied the effect of endocannabinoid N-arachidonoyl dopamine on spontaneous bioelectric activity of cultured hippocampal neurons in a model of hypoxia/reoxygenation. Incubation under hypoxic conditions induced irreversible decrease in spontaneous bioelectric activity of neurons and their death. Application of N-arachidonoyl dopamine during hypoxia and in the post-hypoxic period preserved bioelectric activity and viability of neurons. The protective effect of N-arachidonoyl dopamine was primarily mediated by type I cannabinoid receptors.

[A new fluorescent analogue for the investigation of anandamide transport in cell cultures].

For the first time a new fluorescent analogue of anadamide incorporating BODIPY®-FL-fluorophore, attached to arachidonic acid via 2,2'-(ethylenedioxy)-bis(ethylenediamine), was prepared. Using rat glioma C6 cells it was demonstrated that the fluorescent analogue is a substrate of the cellular anandamide uptake system (Km 4.5 ± 0.9 µM, Vmax 20 ± 1 amol/(min x cell)).