Ectonucleotidase Activity in Smooth Muscle Tissues of Rats with a Valproate Model of Autism.

Ectonucleotidases play an important role in regulating the level of extracellular nucleotides and nucleosides and are an important part of the regulation of the effects of adenosine and ATP on adenosine and P2 receptors, respectively. We have previously established the ambiguous effect of P2 receptor agonists on the contractile activity of smooth muscle tissue in rats with the valproate model of autism. In this work, HPLC was used to evaluate the activity of ectonucleotidases in the smooth muscle tissues of the internal organs of rats with a valproate model of autism. The activity of ectonucleotidases was significantly higher in the smooth muscle tissues of the duodenum, vas deferens, and bladder, but lower in the ileum and uterus. The results obtained make it possible to compare the activity of ectonucleotidases identified here with changes in P2 receptor-mediated contractility of smooth muscle tissues revealed in our previous experiments.

Comparative Assessment of Disturbances of Contractions of the Isolated Uterus in 3- and 9-Month-Old Rats with a Model of Autism.

We performed a comparative study of the effects of carbachol, α,ß-methylene-ATP, ß,γ-methylene-ATP, and electric field stimulation on the contractile activity of the isolated uterus from rats aged 3 and 9 months with valproic model of autism. The contractile responses of isolated rat uterine preparations induced by P2X-receptor agonists α,ß-methylene-ATP and ß,γ-methylene-ATP were significantly lower than in the control. In addition, the contractions of the isolated uterus of 9-month-old rats induced by carbachol were significantly lower than in controls. No significant differences in uterine smooth muscle contractions in both age groups of rats induced by electric field stimulation in comparison with the control were found. Thus, significant impairment of uterine contractile activity was revealed in rats with valproic model of autism, which persisted up to the age of 9 months. The absence of changes in the contractions induced by electric field stimulation suggests that the changes in the contractile activity of the uterus of the rats with modeled autism spectrum disorder are caused by the disorders occurring at the postsynaptic level.

[Involvement of p2 receptors in the contractile activity of the bladder in patients with benign prostatic hyperplasia].

AIM: to study the role of P2 receptors in impaired bladder contractility in patients with lower urinary tract obstruction. MATERIALS AND METHODS: in pharmacological studies, tissue samples from the bladder wall of 30 patients were used, obtained during planned surgical interventions for benign prostatic hyperplasia (transvesical simple prostatectomy without placement of cystostomy tube). Based on these tissue, isolated smooth muscle specimens were prepared. Their mechanical activity and the efficiency of ligands of purine P2 and other receptors were evaluated. With this aim, the following P2-receptor agonists were used: adenosine triphosphoric acid (ATP), adenosine diphosphoric acid (ADP), uridine-5'-triphosphoric acid (UTP), alpha, beta-methylene-ATP, 2-methylthio-ADP, as well as antagonists of P2-disulfonate receptors acid (PPADS), suramin, NF023, MRS2500. In addition, the efficiency of ligands of other receptors, including carbacholine, epinephrine, histamine, serotonin, atropine was evaluated. RESULTS: the most effective agonist was alpha-beta-methylene-ATP, while ATP and 2-methylthio-ADP were significantly less active. In our experiments, ADP and UTP did not show an effect on human bladder. The influence of P2 receptor agonists was inhibited by P2 receptor antagonists PPADS and suramin, as well as MRS2500, although to a lesser extent. Carbacholine caused a strong concentration-dependent contractile response of the bladder, which was inhibited by atropine. Histamine resulted in mild bladder contractions only at high concentrations. Epinephrine and serotonin did not cause significant changes in the contractile activity of the bladder. CONCLUSION: The main subtype of P2 receptors involved in the contractile activity of the human bladder is P2X1 receptors. P2Y1 receptors also have some influence on the contraction, while other subtypes of P2 receptors are not detected by pharmacological methods.

ATP-induced changes in rat skeletal muscle contractility.

BACKGROUND: Extracellular purine compounds, adenosine triphosphate (ATP) and adenosine, are involved in regulation of many cell functions, engaging in rapid and long-term cellular processes. The nucleotides, including ATP, exert their extracellular effects by influencing membrane P2 receptors. ATP outside of the cell rapidly is metabolized by the ecto-enzyme system to produce adenosine, which acts on separate adenosine (P1) receptors. Since adenosine and ATP often are functional antagonists, ATP degradation not only limits its effect, but also brings new ligand with different, often opposing, properties. Great variety and widespread of P2 and adenosine receptors in the body emphasize the important physiological and pathophysiological significance of these receptors, and make them very attractive as targets for potential drug action.The existence of several subtypes of P2 and adenosine receptors has been shown in the skeletal muscles. ATP as a co-transmitter is densely packed together with classical neurotransmitters in the presynaptic vesicles of vertebral motor units but until recently ATP was refused to have its own functional role there and was recognized only as a source of adenosine. However, on the eve of the third millennium there appeared data that ATP, released from the nerve ending and acting on presynaptic P2 receptors, suppresses subsequent quantum release of acetylcholine. The final product of its degradation, adenosine, performs a similar inhibitory effect acting on presynaptic adenosine receptors.Despite the fact that the mechanisms of presynaptic inhibitory action of ATP and other purines were studied earlier, the object of those studies was usually neuromuscular synapse of cold-blooded animals. The few studies, in which experiments were carried out on preparations of warm-blooded animals, described the basic effects of purines. These often were guided by the convenience of preparation of the synapses of the diaphragm. We think that those results cannot be considered as typical effects of ATP and other purines on skeletal muscles and could not be extrapolated to all warm-blooded animals. Furthermore the role of ATP and its derivatives in the accumulation of vertebrate muscular effort has not been investigated.It is known that in physiological conditions vertebrates may mobilize only up to a third of the maximum muscle force. Why the two-thirds of muscular strength are not used normally but may be used at stress, remains unknown.It is known that the body's adaptive response to stress is a change in the activity of the endocrine system. The leading role in this is given to catechol amines and glucocorticoids, mobilized in significant quantities in blood under stress.We have found previously that incubation of frog sartorius muscle with hydrocortisone resulted in a decrease of contraction amplitude. However, when hydrocortisone was used in combination with ATP, its inhibitory effect on contractile responses disappeared. It is interesting that hydrocortisone had no effect on the inhibitory effect of adenosine. In the following experiments, assessing the effect of hydrocortisone on rat soleus muscle, it was established that hydrocortisone and purines had similar inhibitory effect. When ATP and hydrocortisone were given together the same oppression occurred. OBJECTIVE: To study the effects of ATP and adenosine on contraction parameters of rat skeletal muscle and assess the impact of the catechol amines on these processes. METHODS: Contractions of rat soleus muscles were recorded isometrically by mechanical sensor Linton FSG-01 (UK) according to standard procedures. The average of muscle parameters received within 30 seconds (30 responses) was treated as one result. Amplitude and time characteristics of the curve reductions were estimated. During all experiments standard Krebs solution flowed through the bath continuously to which agents were added at necessary concentrations. All experimental animals were maintained and prepared for dissection under the European Convention for the Protection of Vertebrate Animals used in scientific experiments. All agents used in the study were supplied by Sigma Chemical Company Ltd. (UK), Tocris Cookson and Research Biochemicals International (USA). RESULTS: The concentration of 100 µM for adenosine is close to saturation [1], and for its predecessor ATP this concentration is created after the passage of a pulse through the synapse [2]. We used this concentration of purines to study the mechanism of action of adenosine and ATP on neuromuscular synapse.The effect of adenosine was partially inhibited in the presence of 100 µM 8-SPT, an antagonist of adenosine receptors. The contraction force of "fast" and "slow" rat skeletal muscles was raised by half in the presence of norepinephrine. In the presence of norepinephrine adenosine exerted its effect fully, but ATP by half reduced its depressor effect on the contraction force of both muscles. CONCLUSIONS: 1. Norepinephrine increases half times of the reduction of "fast" and "slow" skeletal muscle.2. In the presence of norepinephrine, inhibitory effect of adenosine on contraction force is maintained.3. Inhibitory effect of ATP on contraction force of studied skeletal muscles becomes twice less pronounced in the presence of norepinephrine.We think that reduction of ATP depressive effect on the skeletal muscle by norepinephrine may be an adaptive response to acute stress.

Functional activity of varicose human great saphenous vein at different sites.

Experiments on isolated preparations of varicose human great saphenous vein revealed different sensitivity of the distal and proximal portions to P2X receptor agonist α,ß-methylene-ATP, but not to norepinephrine and histamine. It is suggested that restructuring of the P2 receptor system plays an important role in the pathogenesis of varicose veins, which affects the trunk of the great saphenous vein in varying degrees.

[Purinergic P2-receptors as potential targets of new pharmaceutical products].

P2-receptors are widespread in animal and human organs and tissues. Therefore, they attract much attention as potential targets of new drugs. This article presents in brief characteristics of P2 receptors and reviews the author's search for new antagonists of P2-receptors as well as his studies on the role of ectonucleotidases and unique temperature-dependence of P2 receptor activity. In addition, the presence and functional activity of P2 receptors in human uterus, fallopian tubes, heart and blood vessels are described. The possible physiological and pathophysiological role of P2 receptors in these tissues, as well as prospects for the development of drugs acting via P2 receptors is discussed.

Expression of P2X receptor subtypes on CD34+ cells and c-kit+ cells of human umbilical blood.

The presence of several subtypes of P2X receptors on early hemopoietic precursors (CD34+) from human umbilical blood was detected by flow cytometry. The expression of P2X receptors on umbilical blood lymphocytes was an order of magnitude higher than that on adult human blood cells. Our results attest to early involvement of P2X receptors in differentiation of human hemopoietic cells.

Opposite effect of ATP on contraction force of tonic and phasic skeletal muscles in frogs.

Experiments in vitro showed that ATP and adenosine equally suppressed contractions of frog m. sartorius, which belongs to the phasic type muscles. Adenosine receptors antagonist 8-SPT abolished the effect of adenosine, but did not change the effect of ATP. This fact proves the independence of signaling pathways of these purines. ATP produced an opposite effect on the tonic muscle m. cruralis and increased the force of its contraction. Adenosine produced an inhibitory effect on the force of m. cruralis contration. In this case, 8-SPT also eliminated the effect of adenosine, but did not change the effect of ATP. The potentiating effect of ATP was blocked by suramin, a nonselective antagonist of P2 receptors, which attests to their involvement into the effects of this purine. The opposite effects of purinergic regulation reflect fundamental differences in functional organization of phasic and tonic muscular systems. It was hypothesized that the increase in contraction force under the effect of ATP is a mechanism providing maitenance of the contracted state of tonic muscle without appreciable metabolic costs.

New specific marker of cytochrome P450 1A2 activity.

Various methods were proposed for phenotyping of patients by activity of cytochrome P450 1A2, each has some advantages and disadvantages. However, no reference parameters were developed for measuring CYP1A2 activity that could be used as a unified standard for phenotyping of patients. We propose a mathematic model of caffeine metabolism allowing calculation of rate constants for the formation of its primary metabolites. First-order rate constant of paraxanthine formation was tested as a new specific marker of isoenzyme 1A2 in healthy volunteers.

[On the possibility of patient phenotyping on the basis of cytochrome p-450 1A2 isoenzyme activity using caffeine as the test substrate].

Reactions of caffeine biotransformation and enzymes participating in the caffeine metabolism via the paraxanthine pathway are reviewed. Data on the drugs metabolized mainly in cytochrome P-450 1A2 isoenzyme (CYP 1A2) are analyzed. Information on the phenotypes of CYP 1A2 activity in smoking healthy volunteers, women using oral contraceptives, diabetes patients, those with liver disorders, etc. is summarized. Methodological approaches to patient phenotyping with respect to CYP 1A2 activity as proposed in the available literature are analyzed.

[Hexestrol inhibits ATP-induced contractile response of isolated preparations of pregnant human uterus].

The results of in vitro experiments showed that hexestrol, a synthetic analog of estrogen hormones, at high concentrations inhibited the contractile response of isolated preparations of pregnant human uterus induced by ATP. It is suggested that estrogen hormones can interact with P2 receptors in human uterus during pregnancy.

[Features of the metabolism of various drugs involving cytochrome P-450 isoenzymes].

Substrates of cytochrome P450 isoenzymes have been analyzed and systematized, and a database is created in which the substrates are classified in accordance with the chemical structure of drugs. Each substrate is characterized by the oxidation reaction and the resulting metabolites, with allowance for the Michaelis - Menten pharmacokinetic parameters. Each isoenzyme metabolises certain preferred substrates, depending on the presence of active structural groups or moieties susceptible to oxidation and on the stereoselectivity of drugs. The reaction direction also depends on the specific interactions between the active groups of a substrate and the given isoenzyme.

[Effects of sodium humate from peat of Tomsk region on allergic reactions].

Sodium humate from peat of Tomsk region was tested on three animal models of allergic reaction. It was found that sodium humate suppresses the development and reduces the intensity of anaphylactic shock in guinea pigs and decreases the intensity of the delayed hypersensitivity reaction to goat erythrocytes. It is suggested that sodium humate can be a promising substance for the treatment of allergic states.

Role of protein kinase C in the effect of ATP on contractile function of the isolated strip from mouse diaphragm.

We studied the effects of adenosine and ATP on contractile function of the isolated strip from mouse diaphragm. ATP significantly increased the strength of muscle contraction induced by carbachol. Adenosine had no effect on carbachol-induced muscle contraction. P2 receptor antagonist suramin abolished the effect of ATP. The positive chronotropic effect of ATP was not observed after treatment with specific protein kinase C inhibitor chelerythrine. Our results indicate that the effect of ATP on contractile function of mouse diaphragm is realized via protein kinase C.

[Studying the efficacy of a series of quinoxaline and azolo[a(b)]quinoxaline derivatives as P2X receptor antagonists].

The influence of a series of 14 quinoxaline and azoloquinoxaline derivatives on the P2X and P2Y receptor mediated response was studied in vitro on isolated rat and guinea pig tissues. Most of the compounds studied did not affect the response, while paratolylhydrazone-3-benzoyl-1,2-dihydro-2-oxoquinoxaline exhibited antagonism with respect to the P2X receptor mediated response, while not influencing the P2Y mediated relaxation. It is suggested that this compound can be used for the synthesis of new effective P2 receptor antagonists.

Stimulation of vagus nerve modifies negative chronotropic and hypotensive effects of adenosine.

The effects of electrical stimulation of the vagus nerve on the negative chronotropic and hypotensive effects of adenosine were studied on adult rats. Intravenous injection of adenosine to control rats significantly reduced the heart rate and induced a transient blood pressure drop followed its increase. Preliminary electrical stimulation of the right vagus nerve reduced the magnitude and duration of adenosine-induced bradycardia and changed the dynamics of adenosine-induced arterial pressure perturbations.

Pharmacological characterization of P2-receptors in human fallopian tubes.

In women with various gynecological disorders, ATP, ADP, and 2-methylthio-ATP dose-dependently potentiated spontaneous contractility of isolated fallopian tubes, while alpha,beta-methylene-ATP and uridine triphosphate were little efficient. Pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid, a P2-receptor antagonist, inhibited responses to 2-methylthio-ATP, produced no effect on responses to ATP, and potentiated ADP-induced responses in fallopian tubes. During inflammation, sensitivity of fallopian tubes to P2 agonists and antagonists decreased. The data attest to the presence of functionally active P2 receptors in human fallopian tubes probably involved in the regulation of their mechanical activity.

Effect of PPADS on P2X receptor-mediated responses of human blood vessels.

In vitro experiments showed that pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid almost completely suppressed contractile responses of the gallbladder artery to alpha,beta-methylene-ATP, while alpha,beta-methylene-ATP-induced contractions of the major subcutaneous vein of patients with varicose disease did not change under the effect of the antagonist. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid significantly reduced contractions of the major subcutaneous veins induced by alpha,beta-methylene-ATP (in two highest concentrations) in patients without varicosity. These results indicate different sensitivity of human blood vessels to the studied P2 receptor agonist and antagonist.

Effects of dimephosphone, xydiphone, and ionol on the content and activities of rat liver cytochromes P-450 during long-term treatment with phenobarbital.

Effects of dimephosphone, xydiphone, and ionol administered in parallel with phenobarbital on the content of cytochromes P-450 in rat liver and on the rate of C-hydroxylation of diazepam, haloperidol, and prednisolone by rat liver microsomal enzymes were studied in vitro. Dimephosphone, xydiphone, and ionol exhibited similar inductive effects on C-hydroxylation reactions in the CYP P-450 system during treatment with phenobarbital. Xydiphone and ionol in a dose of 1 mmol/kg canceled phenobarbital-induced increase in P-450 cytochrome content in rat liver. Sex-dependent cytochromes P-450 are involved in the prednisolone and haloperidol C-hydroxylation reactions in rats.

[Types of biochemical response to acute pharmacological indomethacin probe in healthy volunteers]. / Tipy biokhimicheskogo reagirovaniia zdorovykh dobrovol'tsev na ostruiu farmakologicheskuiu probu s indometatsinom.

By the type of biochemical response to acute pharmacological indomethacin probe, a group of both male and female healthy volunteers can be subdivided into two parts. The first part includes volunteers with a stability index reduced as a result of accumulation of the oxidized products and a decrease in the content of reduced glutathione (GSH). The second part includes volunteers with the stability index increased as a result of decrease in the amount of lipid peroxidation products and an increase in the GSH content. A difference between males and females with respect to the response type consisted in that females features changes in the gutathione buffer components, and males, in the activity of antioxidant enzymes (catalase and peroxidase).