M. Yu. Bobrov

New aspects of biosynthesis and metabolism of N-acyldopamines in rat tissues

Possible biosynthetic pathways of N-acyldopamines in rat tissues were compared. It was shown that an insignificant amount of the conjugation products was formed during the incubation of arachidonic acid and dopamine, whereas the substitution of tyrosine for dopamine resulted in the productive biosynthesis of N-arachidonoyldopamine. The biosynthesis presumably involves several closely conjugated enzymatic stages, and free fatty acids rather than their CoA esters served as the starting substrates. The decarboxylation stage probably precedes the stage of catechol system formation, because N-acetyltyramine (a probable intermediate) was easily oxidized by monophenol monooxygenase to N-acyldopamine, whereas N-acyltyrosine is hydrolyzed under these conditions. Biosynthesis of N-acyldopamines in a cell-free medium was accompanied by their methylation. The possibility of oxidative metabolism of N-acyldopamines, which could serve as co-substrates or inhibitors of different oxidoreductases, was shown for the first time.

[Arachidonoyl amino acids and arachidonoyl peptides: synthesis and properties].

N-Arachidonoyl (AA) derivatives of amino acids (glycine, phenylalanine, proline, valine, γ-aminobutyric acid (GABA), dihydroxyphenylalanine, tyrosine, tryptophan, and alanine) and peptides (Semax, MEHFPGP, and PGP) were synthesized in order to study the biological properties of acylamino acids. The mass spectra of all the compounds at atmospheric pressure electrospray ionization display the most intense peaks of protonated molecular ions; the detection limits for these compounds are 10 fmol per sample. AA-Gly showed the highest inhibitory activity toward fatty acid amide hydrolase from rat brain (IC50 6.5 μM) among all the acylamino acids studied. AA-Phe, AA-Tyr, and AA-GABA exhibited a weak but detectable inhibitory effect (IC50 55, 60, and 50 μM, respectively). The acylated amino acids themselves, except for AA-Glu, were stable to the hydrolysis by this enzyme. All the arachidonoylamino acids inhibited cabbage phospholipase D to various degrees; AA-GABA and AA-Phe proved to be the most active (IC50 20 and 27 μM, respectively). Attempts to detect the biosynthesis of AA-Tyr in homogenates of rat liver and nerve tissue in vitro were unsuccessful; however, AA-dopamine and AA-Phe, the products of its metabolism, were found. The highest contents of these metabolites were detected in liver homogenate and in the brain homogenate, respectively. Acylamino acids exert no cytotoxic effect toward the glioma C6 cells. It was shown that N-acylation of Semax with arachidonic acid results in enhancement of its hydrolytic stability and increases its affinity for the sites of specific binding in rat cerebellum membranes.

Characteristic features of specific binding of pentapeptide HFPGP labeled at the C-terminal proline residue to rat forebrain plasma membranes.

101 The pentapeptide HFPGP is a major intermediate metabolite of N terminal degradation of the nootro pic peptide semax (MEHFPGP) in the brain [1]. It has its own biological activity, somewhat different from the effects of semax, and is sufficiently resistant to endogenous proteolytic enzymes [2, 3]. However, the question as to whether the biological effect of the pentapeptide is realized through the interaction with the receptor systems of nerve cells remains unan swered.

Nanocomplexes of recombinant proteins and polysialic acid: Preparation, characteristics, and biological activity

A preparation of nanocomplexes containing recombinant proteins (interferons α2b and β1b, insulin, and human granulocyte colony stimulating factor) and natural polysialic acid (PSA) has been described. The incorporation of protein into the complex changes its electrophoretic mobility. Atomic force microscopy reveals the average size of 23-kD insulin complexes with PSA of 10–20 nm and demonstrates that more than 60% of glycopolymer molecules carry a single protein molecule. Experiments with cultured cells show that cytokines bound to polysialic acid retain their ability to regulate cell proliferation. Insulin bound to PSA has a prolonged hypoglycemic effect in vivo.

Studies of peculiarities of binding of the Semax neuropeptide, with a labeled terminal proline residue to plasma membranes of rat brain

The peculiarities and characteristics of the binding of the Semax neuropeptide (Met-Glu-His-Phe-Pro-Gly-Pro) with a 3H-labeled terminal Pro to plasma membranes of the rat forebrain were studied. Specific binding sites were found that were characterized by a dissociation constant of the ligand-receptor complex of Kd = 100.4 ± 19.5 nM at 0–4°C. Significant biodegradation of Semax in the course of its interaction with plasma membranes was shown by HPLC. The rate of this degradation was considerably decreased at 0–4°C.

Specific Binding of Semax in Different Regions of the Rat Brain

Highly specific binding of Semax had been found in hippocampus and cerebellum ( K A is 104.6 and 106.7 nM, respectively), whereas in the basal ganlia and cortex, we have found only nonspecific binding. Semax, as a nootropic drug, can increase the acuity of vision, enhance attention, regulate processes of learning and memory formation [1‐3]. It is known that only specific regions of the brain are involved in these processes. Therefore, information on the amount of Semax and the rate of its metabolism in various brain parts is of special interest. Such information may help to compare activity of proteolytic enzymes in the various brain parts, assess link between the Semax concentration and specific binding of Semax with the plasma membranes of cells in various brain regions, and correlate these data with physiological functions controlled by these brain regions. In this work, we determined the binding properties of Semax labeled with tritium at the C-terminus proline in the cerebellum, basal ganglia, cortex, and hippocampus of the rat brain.

Antioxidant and neuroprotective properties of N-docosahexaenoyl dopamine.

N-Docosahexaenoyl dopamine exhibited antioxidant activity in the test with a stable oxygen radical galvinoxyl. This compound produced a dose-dependent protective effect on cultured granular cells from rat cerebellum under conditions of oxidative stress. N-Docosahexaenoyl dopamine decelerated the development of symptoms of Parkinson’s disease in mice receiving neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

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.

In vitro effects of anandamide and prostamide e2 on normal and transformed nerve cells

We studied the effects of endocannabinoid anandamide and its cyclooxygenase derivative prostamide E2 on cultured cerebellar granular cells and C6 glioma cells from rats. Prostamide E2 prevented apoptosis in cerebellar neurons induced by potassium deprivation of cultures, while anandamide had no neuroprotective properties. Prostamide E2 did not modulate the survival rate of glioma cells, while anandamide produced a cytotoxic effect. Our results indicate that cyclooxygenase transformation of anandamide is followed by the loss of antitumor activity of this agent. By contrast, prostamide E2 exhibited strong neuroprotective properties.

Introduction of hydrogen isotopes into Win 55212 and CP 55940, selective agonists of cannabinoid receptors

Selective agonists of cannabinoid receptors Win 55 212 and CP 55 940, labeled with hydrogen isotopes, were prepared. The content of isotopomers in deuterium-labeled Win 55 212 and CP 55 940, and also the deuterium distribution in fragments of the [2H]Win 55212 molecule were determined by mass spectrometry. [3H]Win 55 212 and [3H]CP 55 940 with molar radioactivities of 55 and 70 Ci mmol−1, respectively, were prepared by the reaction with gaseous tritium. The efficiency of isotope exchange with activated hydrogen species under the conditions of primary and secondary hydrogen spillover is discussed.