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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.

The article presents an analysis of current literature covering general information, as well as clinical and experimental research on autism spectrum disorder. Autism is a complex mental disorder. A growing body of literature suggests the association of autism spectrum disorder with dysfunction of the autonomic nervous system, especially those affecting the gastrointestinal tract and bladder. In addition, there are problems with nutrition, metabolism, immune and endocrine systems, and microbiota. Prevalence of autism has increased significantly over the past 40 years. As more and more children with autism become adults, understanding this condition throughout life is of paramount importance. Although many research has focused on understanding how diagnosis and treatment can help little children, few are focused on adults with autism and how primary care groups can better help these people. Despite significant progress toward identifying the factors influencing the development of autism spectrum disorder, the etiology of the disease remains uncertain. In this regard, scientists are trying to obtain models of autism in rodents to continue further research. Based on the data obtained during clinical and experimental researches, a hypothesis about the possible role of the purinergic system in the pathogenesis of autism spectrum disorder is considered. The results are encouraging, but further research is required. Thus, somatic disorders can worsen the main symptoms of autism, which affect communication and behavior functioning. In this regard, further research is necessary, including in a rodent model of autism spectrum disorder to contribute to identifying the possible causes of the disorder.

The pre- and postsynaptic effects of ATP on thermal sensibility in the diaphragm of the rat have been studied. It has been found that reduction in the bath temperatute from 37 to 22°С provokes a decrease in contractile force of diaphragm caused both by electrical stimulation and carbachol. At 37°C adding ATP to the incubation medium increases contractive activity of the diaphragm caused both by electrical stimulation and carbachol. These effects of ATP persist at lower temperatures, but are more prominent in carbachol-induced contractions. It is assumed that the thermosensitive component of purine modulation is located mostly postsynaptically in the diaphragm of the rat.

—We have previously demonstrated that hypothermia significantly modulates the effect of ATP on the contractile activities of various types of rodent locomotor muscles. The aim of this study was to compare the effects of ATP on contraction of mouse locomotor (m. soleus) and respiratory (m. Diafragma) muscles at normal and low temperatures. It has been found that a decrease in the ambient temperature to 22°C is associated with a decrease in the force of muscle contraction (m. Diafragma) induced by electrical stimulation and carbachol, as well as with an increase in the contractile force of m. soleus. The potentiating effects of ATP in these muscles are preserved even in response to temperature reduction, but they are enhanced only in carbachol-induced contractions, thus indicating a postsynaptic localization of a thermal sensitive element in purine modulation. The characteristic features of ATP release modulation with the predominant postsynaptic thermal sensitive link in different motor units in hypothermia in mice may underlie the adaptive mechanisms of this small rodent.

Autism is a pressing global problem in a number of medical and related scientific disciplines. For autism, a polysystemic feature is typical, and neurological changes are usually accompanied by somatic ones, most often affecting the intestine, pancreas, and often lungs, pelvic organs, kidneys, adrenal glands and other organs. It is not surprising that the mortality from somatic causes in such children exceeds the mortality of healthy children of the same age groups by 3–10 or more times (depending on the severity of autism). Many studies report a high prevalence of gastrointestinal symptoms in autistic people. The most common of these is chronic constipation (22% on average). The functional interaction of the gastrointestinal tract and the central nervous system is due to the presence of various connections and includes the autonomic nervous, immune and neuroendocrine systems. Of particular importance in gastrointestinal disorders and the pathogenesis of autism is the intestinal microbiota, a complex bacterial community located in the gastrointestinal tract. Under the influence of external and internal factors, the microbiota changes the permeability of the intestinal and blood-brain barriers, and the metabolites produced by the altered microbiota can enter the bloodstream and the central nervous system, disrupting its functioning. It was proven that there are pronounced differences between the intestinal microbiota of healthy children and autistic children, and directed individual correction often leads to normalization or significant improvement in social and communicative behavior and other deviations typical of children with autism. Thus, violations in the somatic sphere can increase the severity of the clinical presentation of autism, causing various behavioral and communication disorders. Identification of the spectrum of these disorders, as well as the study of the mechanisms of their development and interrelationship, is an urgent problem, the solution of which may be important for determining the tactics of complex therapy of patients with autism spectrum disorders.

Randomized clinical trials (RCTs) are currently recognized as the gold standard for evaluating the efficacy and safety of medical interventions. However, often the presentation of the results of RCTs in reports and articles does not give a complete and reliable description of all the results of the test, which often leads to incorrect conclusions and guidelines. In order to comply with certain uniform requirements for reporting documents based on materials of the conducted RCT, a group of authors and editors of medical journals in the early 1990s came out with a proposal to the medical community about using common standards for presenting RCT results, which they called CONSORT (CONsolidated Standards Of Reporting). The first version of CONSORT, however, caused a lot of criticism from both authors and editors of journals. The accumulated critical comments were taken into account by the developers of the revised and published in 2010 version of CONSORT, which is currently accepted as a reference for presenting the results of RCT in the world's leading medical journals. CONSORT 2010 contains a statement, supplemented with a set of questions on the conducted RCT, requiring a clearly formalized answer, and a flowchart for visual presentation of the results. In addition, the CONSORT developers submitted a separate document to clarify the terms in order to clarify the cases of application of this methodology. It is important to understand that CONSORT does not include recommendations for developing, conducting and analyzing the trials. It contains only reports on what and how it was done, what was eventually established, and only indirectly affects the design and conduct of RCT. However, authors who are familiar with the requirements of CONSORT will strive to meet these requirements when planning and conducting RCTs. As an example, the article presents a case of assessing the quality of the presentation of one of the RCTs according to CONSORT 2010 standards.

Aim. Conduction of quantitative and qualitative analysis of registration parameters of medications of two groups - angiotensin converting enzyme inhibitors and angiotensin II receptor blockers in Russian Federation compared to those of leading drug regulators. Methods. The whole list of currently existing angiotensin converting enzyme inhibitors and angiotensin II receptor blockers according to international Anatomical Therapeutic Chemical Classification of drugs was analyzed by quantitative and qualitative characteristics in four national drug regulators: State drug register of the Russian Federation, American drug regulator - Food and Drug Administration, European Medicines Agency, and Australian drug regulator - Therapeutic Goods Administration. Results. It was found that by the number of registered international nonproprietary names and by the number of trade names in the group of angiotensin converting enzyme inhibitors the leading position is taken by State drug registry of the RF, and in the group of angiotensin II receptor blockers Therapeutic Goods Administration is leading. On the territory of Russian Federation among angiotensin converting enzyme inhibitors, lisinopril, captopril, perindopril and enalapril are registered in the highest numbers of trade names, and among angiotensin II receptor blockers - losartan and valsartan. The example of the best practice is presented by European Medicines Agency with the lowest number of registered international nonproprietary and trade names. The qualitative analysis of registration parameters of angiotensin converting enzyme inhibitors and angiotensin II receptor blockers (accepted indications) revealed that in RF the highest number of indications for both angiotensin converting enzyme inhibitors and angiotensin II receptor blockers is accepted compared to those of the leading drug regulators. The example of the best practice by this criterion is presented by the European regulator - European Medicines Agency. Based on the understanding that all international nonproprietary names in every analyzed pharmacological group are medications of the «me too» category, the decrease of the number of registration positions by national regulator and thorough analysis of evidence of effects are recommended when approving indications. Conclusion. In the official state drug registry of RF compared to three foreign drug regulators, the highest number of international nonproprietary names and trade names of angiotensin converting enzyme inhibitors are presented as well as indications for both angiotensin converting enzyme inhibitors and angiotensin II receptor blockers.

Purinergic P2 receptors, the basic endogenous agonist of which is adenosine triphosphoric acid (ATP), are widely spread in the organs and tissues of human and animals including urogenitary system. Physiologically, in the peripheral nervous system the role of P2 receptors in most cases is not leading, they only complement or modulate the action of main neuromediators (acetylcholine, norepinephrine). But in pathology the role of P2 receptors significantly increases and often takes the lead in the pathogenesis of one or another disease. In particular, it was determined that purinergic component of contractile bladder response increases from 2-5% in normal state to 40% in some pathological processes (such as interstitial cystitis, neurogenic bladder, urinary obstruction). In the bladder of experimental animals different subtypes of P2 receptors were revealed, their functional role was established in normal conditions and models of pathological processes. Certain subtypes of P2 receptors were also detected in the human bladder, including in some urinary tract diseases. The level of ATP in patients’ urine was established to significantly increase in lower urinary tract obstruction that holds certain promise for the diagnosis of these diseases. Variety and large representation of P2 receptors in lower urinary tract make them attractive as potential targets for novel drugs. On this evidence, evaluation of effect of P2 receptor agonists and antagonists as well as medications affecting the metabolism of endogenous nucleotides and nucleosides, is one of promising direction for the search for new urological drugs.

The review surveys the impact of “stress hormones”–glucocorticoids and catecholamines–on the functioning of the neuromuscular synapse. The review brings together the data on the influence of the main agents of stress–cortisol and norepinephrine–on the intensity and timing of the acetylcholine release, as well as signaling effect of its co-mediator ATP.

This review focuses on the effects of temperature on the functioning of the neuromuscular system. The changes in environmental temperature could affect the contractile acts in both ectotherms and endotherms by changing the amplitude and velocity of contractions and, accordingly, the mechanical work of skeletal muscles. In this study, we summarize the data on the effects of hypo- and hyperthermia on the supraspinal and peripheral components of regulation of the neuromuscular function.

P2 receptors, the main endogenous agonist of which is adenosine triphosphate (ATP), are widely distributed in mammalian tissues and organs, including the cardiovascular system. In human blood vessels, various types of the P2Y (metabotropic, G-protein coupled receptors) and P2X (ligand-gated ion channels) family of receptors are present. Several subtypes of P2X and P2Y receptors have been found on the surface of endothelial cells as well as smooth muscle cells of the vessels. Activation of various subtypes of P2 receptors located in different cells of the blood vessel can have multidirectional action on the tone of the vessel’s wall, thereby causing both vasoconstriction and vasodilatation. To date, two main physiologic mechanisms have been identified, via which Р2 receptors participate in controlling the vascular tone: (1) neuronal - ATP is released as a co-transmitter from perivascular sympathetic nerve terminals and activates P2 receptors located on vascular smooth muscle cells; (2) endothelial - ATP is released into the vessel’s lumen by endothelial cells and blood cells and activates P2 receptors located on the endothelial cells. In the first mechanism, simultaneous release of ATP and norepinephrine from sympathetic nerve terminals results in vasoconstriction caused by rapid depolarization, which is completely inhibited by P2X receptor antagonists, and slow depolarization, which is inhibited by alpha-adrenergic blockers. In the second mechanism, during shear stress and hypoxic conditions, ATP activates P2 receptors of endothelial cells causing vasodilatation. These differing effects, mediated via P2 receptors, make it very tempting to develop novel drugs that would regulate vascular tone via these receptors.

Now it is recognized that extracellular adenosine triphosphate (ATP), along with certain other purine and pyrimidine compounds is capable to regulate many intracellular processes by affecting specific receptors - P2-receptors. These receptors are widely distributed in the organs and tissues of humans and animals. It has been shown that P2-receptors are involved in the vascular tone maintenance, nerve transmission modulation, hemostasis regulation and functions of many internal organs. P2-receptors wide variety and broad representation makes them very attractive as potential targets for new drugs with the original mechanism of action. About 20 years ago in the Kazan State Medical University laboratory was created and research group on studying the fundamental and applied aspects of the P2-receptors was formed. Professor A.U. Ziganshin, a disciple and colleague of professor G. Burnstock, leads this group. In this article, an overview of research carried out by the author or under his supervision over the past two decades to study the physiological and pathophysiological role of P2-receptors in humans and animals, as well as the evaluation of these receptors as potential targets for action of new drugs, is given. In particular, it describes the work to identify new and effective P2-receptors antagonists, the role and characteristics of ecto-ATPase activity in different tissues are described, an overview of studies to assess the unique hypersensitivity of P2-receptors at low temperatures is given. Also studies on assessing the presence and functional role of P2-receptors in the pregnant human uterus, inflamed fallopian tubes, various blood vessels are presented in summary form. Obviously, due to the growing interest of many pharmaceutical companies to this area, in the near future we can expect new drugs, which are P2-receptors agonists or antagonists and are effective in treatment of various human diseases.

Aim. To study the presence and localization of the P2X and P2Y receptor subtypes in the human cystic artery and great saphenous vein (with and without varicose disease).Methods. Segments of the human blood vessels were stained using a standard two-step immunohistochemical analysis using primary and secondary antibodies. In the experiments primary antibodies to the following receptors were used: Р2Х1, Р2Х2, Р2Х3, Р2Х4, Р2Y1, Р2Y2, Р2Y4. In order to determine the presence of a receptor in a vessel sample a comparison was made between staining of the experimental and the control samples, which were not treated with primary antibodies.Results. Immunohistochemical analysis of the cystic artery showed the presence of Р2Х1, Р2Х3, Р2Y1, Р2Y2 receptors. All receptor subtypes were found to be located in the muscular layer of the artery, whereas the P2Y1 receptor was also expressed on the surface of the endothelial cells. In the great saphenous vein without varicose disease Р2Х1, Р2Х2 и Р2Y1 receptor subtypes were identified, all of which were found to be located on the smooth muscle cells of the vein. Similarly to the cystic artery, the Р2Y1 receptor was also found within the endothelial layer of the vein. At the same time, only Р2Х2 и Р2Y1 receptor subtypes were expressed in the muscular layer of the great saphenous vein affected by varicose disease. No P2 receptor subtypes were identified on the endothelial layer of the varicose-diseased vein.Conclusion. Different P2 receptor subtypes were found to be present in the smooth muscle and endothelial layers of the human cystic artery and great saphenous vein. The identified differences in the receptor subtypes between samples of great saphenous veins with and without varicose disease are, most likely, explained by the restructuring of the receptor apparatus as a result of varicose disease progression.

The article reviews papers researching the biologic activity of peat and sapropel-derived humic substances, published by domestic and foreign authors. Peat is an accumulation of mainly partially decayed vegetation decomposed by microorganisms, found at mires. Sapropel is natural organic minerals that are derived from freshwater body sediment and formed by decayed vegetation and animal residues, minerals of biochemical and chemical origin and mineral components. Humic substances are a specific group of macromolecular substances derived from peat and sapropel as a result of vegetation and animal residues deterioration. It was found that drugs based on humic substances have the broadest spectrum of biological properties that are already widely used in veterinary medicine and can be used in various spheres of medicine. In particular, medications containing humic substances affect the nonspecific and specific resistance of the organism, show antioxidant, anti-inflammatory, antiviral, antibacterial, antifungal, membranotropic, hepatoprotective properties, have the ability to enhance the metabolic processes activity in the body. Sodium humate stimulates the tumor regression, helps to prevent serotonin ulcers, increases the body’s resistance to hypoxic conditions. Humic substances are do not have any toxic, teratogenic, embryotoxic and carcinogenic properties, while at the same time they are able to positively influence the health status of animals, increasing their productivity. Positive effect of humic substances on plant adaptation to ionizing radiation and pesticides was shown. Plenty of research and favorable results provides a basis for the development of a new direction in pharmacy and pharmacology related to the creation of new domestic products based on peat and sapropel.

The features of synaptic organization of tonic motor units in vertebrates, which are widely represented in amphibians and reptiles but preserved only as muscular systems serving sensory organs in mammals, are reviewed. Based on the previous classical works, the comparative description of innervation and neurotransmitter secretion of tonic muscular systems is given. The data on synaptic organization of tonic muscles obtained in the studies that were continued after a 20-year break and published in the past few years are discussed.

P2 receptors are detected in different tissues and organs, which makes them a potential target of pharmacological action. A number of studies confirming the maturity of purinergic theory are currently published. Literature review focuses on P2 receptors characteristics, their main current agonists and antagonists, as well as on the possibilities of pharmacological action on these receptors. The most important studies addressing new chemical compounds important for studying P2 receptors and also compounds with potential for medical use are analyzed. Data on current successes of P2 receptors pharmacology and introduction of P2Y receptors antagonists into clinical practice are presented. Over the last decades a certain progress was observed in studying P2 receptors agonists and antagonists. There is a growing interest to pathophysiology and therapeutic potential of purinergic neurotransmission. Nevertheless, there is a need for developing new compounds acting selectively to these receptors both in vivo and in vitro. Despite the large number of P2 receptors agonists and antagonists, most of them have certain drawbacks, including incomplete of non-selective antagonism, or significant effect on ecto-ATPase activity. Thus, studying P2 receptors to find novel compounds acting on these receptors is of significant clinical value. Apparently, this trend of developing novel drugs — P2 receptors agonists and antagonists — is particularly promising.

The review covers the history and evolution of the WHO initiatives in pharmaceutical policy - for better and safer use of medicines, their scientifically based selection and ethical promotion. The WHO Concept of Essential Medicines, the WHO vision of the Rational Use of medicines and the WHO Ethical Criteria for Medicinal Drug Promotion are reviewed. Contribution of these WHO designed initiatives, definitions and documents to Global Health, particularly relating to modern Russian health system problems is discussed. The principles and methodology of drug selection are presented. The review describes health system benefits of the WHO Essential Medicines Concept implementation at a national level. Advantages of the WHO Model List of Essential Medicines use not only as a model product, but as a model process and an healthcare instrument are emphasized. The WHO principles of the Rational Use of Medicines, the history of its International development, the WHO key strategies towards the Rational Use of Medicines and the major barriers on the way are described. The history of the WHO Ethical Criteria for medicinal drug promotion development is presented in line with the basic principles and objectives of this unique international document. The core goal of the WHO Ethical Criteria is to improve health and healthcare through the rational use of medicines. The global implementation of the WHO Essential Medicines Concept and the ever growing barriers to the rational use of medicines and advances in public health, as well the advances and problems of the Russian national health system, are discussed in the historical perspective.

This literature review focuses on the role of purinergic P2-receptors in the physiology and pathophysiology of the eye, as well as on the possibilities of pharmacologic stimulation of these receptors. The most important studies from the clinical point of view on the involvement of purinergic neuronal transmission in the physiological processes have been analyzed, ranging from normal embryogenesis to cell apoptosis in age-related degenerative diseases of the eye. Data on the effect of agonists and antagonists of P2 receptors on corneal wound healing, lacrimal fluid production, regulation of intraocular pressure, neurotransmission, proliferation of glial tissue in the retina has been presented along with data on the possibilities of modulating these processes by using potential drugs acting via P2 receptors.

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.

The inhibitory effects of ATP and adenosine on the nerve-mediated contractile responses of isolated sartorius muscle of the frog, Rana ridibunda, evoked by electrical field stimulation (EFS) were studied using pharmacological organ-bath technique. The effects of hydrocortisone applied in vitro and in vivo on contractility of sartorius muscle were also examined. ATP (100 microM) significantly reduced the amplitude of contraction to EFS of sartorius muscle, while pyridoxalphosphate-6-azonphenyl-2',4'-disulfonic acid (PPADS; 10 microM), a P2 receptor antagonist, abolished inhibitory effect of ATP. A similar inhibitory effect of adenosine (100 microM) was fully antagonized by 8-(p-sulfophenyl)-theophylline (8-SPT, 100 microM), a P1 receptor antagonist. Incubation of the tissue with hydrocortisone (10 microM) caused a slight, but significant, decrease of muscle contractions. After incubation of muscle preparations with both hydrocortisone and ATP, no inhibition of muscle contractility was registered. A single injection of hydrocortisone (100 mg/kg) 12 h prior to experiments to frogs did not significantly change the nerve-mediated contractility of isolated sartorius muscle; however, it abolished the inhibitory action of ATP without changing inhibitory activity of adenosine. After treatment of frogs with hydrocortisone for 14 days (100 mg/kg/day), both ATP and adenosine retained their inhibitory action on EFS-induced contractions of the muscle, and their effects were antagonized by PPADS and 8-SPT, respectively. It is concluded that hydrocortisone has antagonistic actions against the inhibitory effects of ATP at the frog neuromuscular junction, although this effect is lost following long-term treatment with hydrocortisone.

Skeletal muscles play key roles in the development of various pathologies, including bronchial asthma and several types of auto-immune disorders, e.g. polymyositis. Since most of these maladies have an immunological/allergic element, this paper is devoted to assessing the impact of immunobiological reorganization on the functional properties of isolated skeletal muscles in mice. A combination of two methods (myography and electrophysiology) was used to evaluate extensor digitorum longus (EDL) and diaphragmatic muscle (DM) in this regard. Conventional myographic technique showed that ovalbumin-induced sensitization (OS) produced different changes in the contractile properties of EDL and DM. The amplitudes of carbachol (CCh)-induced contractions increased in DM but decreased in EDL. Those changes were inversely related to OS-mediated changes of non-quantal acetylcholine (ACh) release intensity within the muscle endplate, as shown by the electrophysiologically measured H-effect. These results clearly show that OS-mediated changes of non-quantal ACh release alter the functional properties of postjunctional ACh receptors and therefore contribute to the disturbance of CCh-induced contractility of skeletal muscles. Other mechanisms of OS-mediated changes of skeletal muscle contractility are also proposed and discussed.

Global biopharmaceutical industry is entering a new area. The first biopharmaceuticals, released 20–25 years ago are out of patent protection already. The top selling biopharmaceuticals with a market volume of tens of billions of US dollars will be out of patent by 2010. And the process will go on and on. Transnational corporations are sounding alarm: by producing bio-generics new biotechnologies, mostly of India and China, threaten multibillion profits of Big Pharma, the phrase often used to refer to companies with revenue in excess of $3 billion, and/or R&D expenditure in excess of $500 million. The largest transnational companies launched huge PR campaigns against bio-generic producers under the cover of safety concerns for the public. Though there is a scientific basis for some concern due to the fact that bio-generics are not absolute copies of originator brands, and thus differ from chemical generics, patient safety care is definitely not the sole reason for their anxiety. There are at least two major points among the other objectives on the agenda for biologics of Big Pharma. Firstly, they aim at not allowing bio-generics on established markets for as long as possible and specifically on the markets of those countries with weak biotechnological expertise. This PR campaign is aimed at the regulatory authorities of these countries. Thus limiting generic competition transnational companies keep for themselves sales at the desirable high level. For example, Johnson & Johnson received more than 25 billion dollars only for its erythropoietin over a period of less than 10 years. Sales of biopharmaceuticals of the so called “big four” consisting of erythropoietin, interferon alfa and beta, and filgrastim are still at the multibillion level.

Two groups of women were used to compare effects of P2 receptor agonists on the contractile activity of isolated human fallopian tubes with and without acute purulent inflammation. A control group included women with uterine tubes without inflammation ( n = 15). A study group included women, operated for unsuccessful conservative anti-inflammatory treatment of acute purulent tuboovarial formations (group with acute purulent salpingitis, n = 16). Division into two groups was done according to pathohistological diagnosis. Spontaneous contractions of the isolated tubes were registered isometrically with electromechanical transducer before and after incubation of the tissues with agonists of P2 receptors—ATP, UTP, 2-methylthio-ATP and α,β-methylene-ATP. In the control group neither of the agonists produced any significant effect on fallopian tube contractility. In the study group, ATP, 2-methylthio-ATP and α,β-methylene-ATP significantly increased the spontaneous contractility of isolated tubes. It is suggested that higher activity of P2 receptor agonists in the uterine tubes with acute purulent inflammation is due to expression of several subtypes of P2 receptors during inflammation.

Nucleotides can modify intraocular pressure (IOP). We have tested the ability of uridine-5'-diphosphate, UDP, for modulating IOP in New Zealand white rabbits. Uridine 5' diphosphate, UDP, reduced IOP by 82.9+/-2.6% compared to control. Dose-response analysis demonstrated a concentration dependent pattern which presented a pD(2) value of 7.57+/-1.45, equivalent to an EC(50) of 26.91 nM. Of all the tested P2 receptor antagonists, suramin, pyridoxalphosphate-6-azophenyl-2, 4-disulfonic acid (PPADS) and Reactive Blue 2 (RB-2), only the last two were able to reverse the action triggered by UDP. Altogether, UDP acting probably on P2Y(6) receptors present on the ciliary processes, can reduce intraocular pressure, indicating that this substance may be used for the treatment of ocular hypertension and glaucoma.

The ability of 12 new thiazole derivatives to influence the muscle contractility mediated by purine P2X receptors has been studied in vitro using isolated tissues of rats and guinea pigs. Most of the synthesized compounds did not cause significant effects, but two compounds exhibited pronounced antagonism with respect to P2X-mediated contractility response. These compounds offer a good starting point for the synthesis of new effective antagonists of P2 receptors.

The aim of the present study was to investigate in vitro the differences in P2 receptor mediated responses of human greater saphenous vein (GSV) taken from patients with varicose disease and obliterating atherosclerosis. Samples of the inguinal part of the GSV were taken from the patients who underwent phlebectomia operation due to varicose disease (n=9, VD group) or femoropoplitea bypass operation using auto-vein due to obliterating atherosclerosis of lower extremities (n=11, OA group). The mechanical responses of the isolated segments of GSV to P2 receptor agonists were tested using standard organ-bath technique. ATP (10(-6)-10(-4) M), ADP (10(-6)-10(-4) M) and alpha,betamethyleneATP (10(-8)-10(-5) M) caused concentration-dependent contractions of the veins of both groups, the latter agonist being approximately tenfold more active than first two. ATP at all concentrations tested, alpha,betamethyleneATP at concentrations of 10(-6) and 10(-5) M and ADP at a concentration of 10(-6) M produced significantly higher contractions of the GSV taken from OA group than from VD group. UTP (10(-6)-10(-4) M) caused concentration-dependent contractions of the veins taken from OA group, while in VD group this agonist was virtually without effect. Adenosine (10(-6)-10(-4) M) and 2-methylthio-ATP (10(-7)-10(-5) M) had no significant contractile activity in this tissue in both groups. It is concluded from this study that there are P2 receptor and adrenoceptor mediated contractions in human greater saphenous veins, which are impaired by varicose disease, in contrast to contractions produced by histamine and carbachol which are, if anything, enhanced.

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.

Experiments on rats showed that pulse therapy with prednisolone (100 mg/kg intraperitoneally for 3 days) stimulated urinary excretion of hydroxyproline, increased the content of inorganic phosphorus, promoted the increase in the content of dienic conjugates and catalase activity, and decreased serum levels of MDA and ceruloplasmin. Ten-day treatment with dimephosphone (208 mg/kg) or xydiphone (45 mg/kg) after pulse therapy with prednisolone normalized urinary excretion of hydroxyproline and reduced the levels of dienic conjugates. Dimephosphone did not change, while xydiphone normalized the level of MDA decreased by prednisolone.

Here we review the results of physiological, pharmacological, and pathophysiological studies of ATP receptors, P2 type receptors. Characteristics of two families of P2 receptors (P2X and P2Y) are presented and the possibility of using preparations affecting these receptors is discussed.

Experiments on isolated preparations of varicose human great saphenous vein revealed different sensitivity of the distal and proximal portions to P2X receptor agonist α,β-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.

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.

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.

1. The effects of NF023, the symmetrical 3'-urea of 8-(benzamido)naphthalene-1,3,5-trisulphonic acid), and its parent compound suramin were investigated on vasoconstrictor responses to alpha, beta-methylene ATP in rabbit isolated saphenous artery and vasodilator responses to ATP in noradrenaline-precontracted rabbit isolated thoracic aorta. 2. In rabbit isolated saphenous artery, alpha, beta-methylene ATP-induced vasoconstrictor responses via P2X-receptors were concentration-dependently and competitively antagonised by NF023 (30-300 microM; pA2 = 5.69 +/- 0.04). Suramin (100-1000 microM) also competitively blocked vasoconstrictor responses to alpha, beta-methylene ATP, albeit with lower potency (pA2 = 4.79 +/- 0.05). In contrast, NF023 (100 microM) did not significantly affect contractile responses to noradrenaline or histamine in the saphenous artery. 3. In noradrenaline-precontracted rabbit isolated thoracic aorta preparations, ATP (3-3000 microM) concentration-dependently induced relaxations via endothelium-dependent or smooth muscle P2Y-receptor subtypes. NF023 (30-300 microM) failed to block relaxant responses to ATP at endothelium-dependent P2Y-receptors, whereas suramin (100-1000 microM) did antagonise endothelium-dependent vasodilator responses to ATP. Neither NF023 (100 microM) nor suramin (300 microM) influenced vasorelaxant responses to ATP via endothelium-independent P2Y-receptors. 4. In conclusion, this study outlines the selectivity of NF023 as an effective P2X-receptor antagonist in rabbit isolated blood vessels without affecting endothelium-dependent or endothelium-independent P2Y-receptor subtypes, adrenoceptors or histamine receptors.

The aim of the present study was to examine the effects of a number of P2-purinoceptor antagonists on degradation of adenine nucleotides by Xenopus laevis oocyte ecto-nucleotidase. Folliculated oocytes readily metabolize all three naturally-occurring nucleotides, the order of preferential substrates being ATPADPAMP. The degradation of ATP and ADP was decreased significantly in the presence of several P2X- and P2Y-purinoceptor antagonists, including suramin, PPADS, Cibacron blue, Coomassie Brilliant blue, Evans blue, Trypan blue, Congo red, and PIT (each compound was used at 100 microM). All these compounds inhibited the degradation of ATP by up to 60%, whereas the hydrolysis of ADP was inhibited by Congo red and PIT by 75-80%. In addition, DIDS (100 microM) and TNP-ATP (100 microM) selectively inhibited the breakdown of ATP, and sodium azide (10 mM) selectively inhibited the breakdown of ADP. The enzymatic breakdown of either ATP or ADP was unaffected by 8-pSPT (100 microM), an antagonist of P1-purinoceptors, or by oxidized ATP (100 microM), an antagonist of P2Z-purinoceptors. The degradation of AMP was prevented completely by PIT (100 microM) and ingibited significantly by Congo red (100 microM). In conclusion, the present study shows that most of currently available antagonists of P2-purinoceptors inhibit the enzymatic breakdown of extracellular ATP and ADP. The inhibitory effect on ecto-nucleotidase activity should be taken into account when these antagonists are used in pharmacological experiments.

1. 2,2'-Pyridylisatogen tosylate (PIT) has been reported to be an irreversible antagonist of responses to adenosine 5'-triphosphate (ATP) at metabotropic purinoceptors (of the P2Y family) in some smooth muscles. When a recombinant P2Y1 purinoceptor (derived from chick brain) is expressed in Xenopus oocytes, ATP and 2-methylthioATP (2-MeSATP) evoke calcium-activated chloride currents (ICl,Ca) in a concentration-dependent manner. The effects of PIT on these agonist responses were examined at this cloned P2Y purinoceptor. 2. PIT (0.1-100 microM) failed to stimulate P2Y1 purinoceptors directly but, over a narrow concentration range (0.1-3 microM), caused a time-dependent potentiation (2-5 fold) of responses to ATP. The potentiation of ATP-responses by PIT was not caused by inhibition of oocyte ecto-ATPase. At high concentrations (3-100 microM), PIT irreversibly inhibited responses to ATP with a IC50 value of 13 +/- 9 microM (pKB = 4.88 +/- 0.22; n = 3). PIT failed to potentiate inward currents evoked by 2-MeSATP and only inhibited the responses to this agonist in an irreversible manner. 3. Known P2 purinoceptor antagonists were tested for their ability to potentiate ATP-responses at the chick P2Y1 purinoceptor. Suramin (IC50 = 230 +/- 80 nM; n = 5) and Reactive blue-2 (IC50 = 580 +/- 130 nM; n = 6) reversibly inhibited but did not potentiate ATP-responses. Coomassie brilliant blue-G (0.1-3 microM) potentiated ATP-responses in three experiments, while higher concentrations (3-100 microM) irreversibly inhibited ATP-responses. The results indicated that potentiation and receptor antagonism were dissociable and not a feature common to all known P2 purinoceptor antagonists. 4. In radioligand binding assays, PIT showed a low affinity (pKi < 5) for a range of membrane receptors, including: alpha 1, alpha 2-adrenoceptors, 5-HT1A, 5-HT1B, 5-HT2, 5-HT3, D1, D2, muscarinic, central benzodiazepine, H1, mu-opioid, dihydropyridine and batrachotoxin receptors. PIT showed some affinity (pKi = 5.3) for an adenosine (A1) receptor. 5. In guinea-pig isolated taenia caeci, PIT (12.5-50 microM) irreversibly antagonized relaxations to ATP (3-1000 microM); PIT also directly relaxed the smooth muscle and histamine was used to restore tone. Relaxations to nicotine (10-100 microM), evoked by stimulating intrinsic NANC nerves of taenia caeci preparations in the presence of hyoscine (0.3 microM) and guanethidine (17 microM), were not affected by PIT (50 microM, for 25-60 min). 6. These experiments indicate that PIT causes an irreversible antagonism of ATP receptors but, for recombinant chick P2Y1 purinoceptors, this effect is preceded by potentiation of ATP agonism. The initial potentiation by PIT (and by Coomassie brilliant blue-G) of ATP-responses raises the possibility of designing a new class of modulatory drugs to enhance purinergic transmission at metabotropic purinoceptors.

ATP-induced inflammation was investigated using subplantar injection in the mouse hind paw. The order of efficacy of purinoceptor agonists for inducing paw oedema (30 nmol per paw) was ATP = alpha, beta-methylene ATP = 2-methylthio ATPadenosineUTPADPAMP. Diadenosine polyphosphates effectively induced paw oedema formation with an order of efficacy of: P1,P4-di(adenosine-5')tetraphosphate = P1,P5-di(adenosine-5')-pentaphosphate = P1,P6-di(adenosine-5')hexaphosphateATP = P1,P3-di(adenosine-5')triphosphateP1,P2-di(adenosine-5')pyrophosphate. Systemic administration of P2-purinoceptor antagonists (30-100 mu mol/kg), suramin, 4,4'-diisothiocyanatostilbene-2,2'-disulphonate, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid and cibacron blue, reduced the intensity of ATP-induced oedema. At 30 mu mol/kg 8-(p-sulfophenyl)theophylline (non-selective adenosine receptor antagonist), 3,7-dimethyl-1,1-propargylxanthine (adenosine A2 receptor antagonist), triprolidine (histamine H1 receptor antagonist), ranitidine (histamine H2 receptor antagonist) and ketanserin (5-hydroxytryptamine 5-HT2 receptor antagonist), but neither 8-cyclopentyl-1,3-dipropylxanthine (adenosine A1 receptor antagonist), nor indomethacin (cyclooxygenase inhibitor) inhibited the ATP-induced swelling. Topical (100 nmol per paw), but not systemic (100 mu mol/kg) administration of NG-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor) reduced the intensity of the ATP-induced paw oedema. These results show that ATP can induce an inflammatory oedematous reaction and contribute to our understanding of the underlying mechanisms.

1. The effect of pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on the relaxant response to adenine nucleotides was examined in the carbachol-contracted guinea-pig taenia coli and rat duodenum, two tissues possessing P2y-purinoceptors. In addition, in the taenia coli PPADS was investigated for its effect on relaxations evoked by adenosine, noradrenaline and electrical field stimulation. In order to assess the selectivity of PPADS between P2-purinoceptor blockade and ectonucleotidase activity, its influence on ATP degradation was studied in guinea-pig taenia coli. 2. The resulting rank order of potency for the adenine nucleotides in guinea-pig taenia coli was: 2-methylthio ATPATPalpha,beta-methylene ATP with the respective pD2-values 7.96 +/- 0.08 (n = 23), 6.27 +/- 0.12 (n = 21) and 5.88 +/- 0.04 (n = 24). 3. In guinea-pig taenia coli, PPADS (10-100 microM) caused a consistent dextral shift of the concentration-response curve (CRC) of 2-methylthio ATP and ATP resulting in a biphasic Schild plot. A substantial shift was only observed at 100 microM PPADS, the respective pA2-values at this particular concentration were 5.26 +/- 0.16 (n = 5) and 5.15 +/- 0.13 (n = 6). Lower concentrations of PPADS (3-30 microM) antagonized the relaxant effects to alpha,beta-methylene ATP in a surmountable manner. An extensive shift of the CRC was produced only by 30 microM PPADS (pA2 = 5.97 +/- 0.08, n = 6), and the Schild plot was again biphasic. 4. The relaxant responses to electrical field stimulation (80 V, 0.3 ms, 5 s, 0.5-16 Hz) in guinea-pigtaenia coli were concentration-dependently inhibited by PPADS (10-100 microM).5. In guinea-pig taenia coli, the potency of ATP in inducing relaxation appeared to be independent of its rate of degradation by ecto-nucleotidases, since the Km-value (366 microM) obtained in the enzyme assay was much higher than the functional EC50-value (0.45 microM) of ATP. PPADS (3-100 microM) was only weakly active in inhibiting ecto-nucleotidase activity leaving a residual activity of 81.8 +/- 5.1% at 100 microM.Enzyme inhibition by PPADS was concentration-independent and non-competitive.6. In rat duodenum, the rank order of potency was: 2-methylthio ATPATPalpha,beta-methylene ATP,the respective pD2-values being 6.98 +/- 0.04 (n = 76), 6.26 +/- 0.02 (n = 6) and 4.83 +/- 0.02 (n = 6). Among these agonists, 2-methylthio ATP displayed the lowest apparent efficacy.7. The CRC of 2-methylthio ATP in rat duodenum was shifted to the right by PPADS (10-100 microM) ina concentration-dependent manner, and Schild analysis gave a pA2-value of 5.09 +/- 0.06 (slope = 1.02,n=14).8 PPADS was without any effect on the carbachol-induced contraction in guinea-pig taenia coli or rat duodenum and on the relaxation to noradrenaline or adenosine in guinea-pig taenia coli.9 In conclusion, the antagonistic properties of PPADS at the taenia coli and rat duodenum P2y-purinoceptors were different from those recently described at the P2x-subtype: inhibition of P2y-purinoceptor-mediated responses was observed at higher concentrations (3-100 microM vs. 1-10 (30) microM).Furthermore, we conclude that in addition to the classical P2y-subtype, which is largely PPADS-resistant,the guinea-pig taenia coli may be endowed with a distinct relaxation-mediating P2-purinoceptor subtype which is sensitive to PPADS.

1. 1. In the guinea-pig urinary bladder and vas deferens, several α,β-unsaturated sulphones and phosphonium salts that were tested inhibited ecto-ATPase activity. The sulphones were more active in the bladder but the phosphonium salts were more effective in the vas deferens. 2. 2. These compounds either potentiated or inhibited purinergic contractile responses in the guinea-pig urinary bladder and vas deferens. 3. 3. α,β-Unsaturated sulphones and phosphonium salts represent a new promising class of compounds, capable of modulating purinergic neurotransmission.

1. Several cations (Ba2+, Cd2+, Co2+, Cu2+, Mn2+, Ni2+, Zn2+ and La3+, all as chloride salts, 1-1000 microM) were tested in the guinea-pig urinary bladder for their ability to: (i) modify contractile responses to electrical field stimulation (EFS), ATP, alpha,beta-methylene ATP (alpha,beta-meATP), carbachol (CCh), and KCl; (ii) affect ecto-ATPase activity. 2. Ba2+ (10-1000 microM) concentration-dependently potentiated contractile responses evoked by EFS (4-16 Hz), ATP (100 microM), alpha,beta-meATP (1 microM), CCh (0.5 microM), and KCl (30 mM). Ni2+ at concentrations of 1-100 microM also potentiated contractility of the urinary bladder, but at concentrations tested its effect was not concentration-dependent. Cu2+ at a concentration of 10 microM and Cd2+ at a concentration of 1 microM potentiated responses to all stimuli, except KCl. Ni2+ at a concentration of 1000 microM and Cd2+ at a concentration of 100 microM inhibited contractions evoked by all stimuli, and at a concentration of 1000 microM Cd2+ abolished any contractions. Responses to ATP and alpha,beta-meATP were selectively inhibited by Cu2+, Zn2+ or La3+, each at a concentration of 1 mM. 3. Cu2+, Ni2+, Zn2+ and La3+ (100-1000 microM) concentration-dependently inhibited ecto-ATPase activity in the urinary bladder smooth muscle preparations, while Ba2+ and Mn2+ were without effect, and Cd2+ and Co2+ caused significant inhibition only at a concentration of 1000 microM. 4. There was no correlation between the extent of ecto-ATPase inhibition and the effect on contractile activity of any of the cations. 5. In conclusion, the ability of some divalent cations to inhibit ecto-ATPase activity and to potentiate or inhibit contractile responses in the guinea-pig urinary bladder appear to be independent effects.

1. Capsaicin, a selective sensory neurotoxin, was given to newborn rats and at the age of 3 months the contractile activity of the urinary bladder detrusor muscle and vas deferens evoked by either electrical field stimulation (EFS) or exogenous adenosine 5'-triphosphate (ATP) and carbachol (urinary bladder), or ATP and noradrenaline (vas deferens) were tested. 2. EFS of the urinary bladder evoked contractions which consisted of cholinergic and purinergic components, since they could be partially blocked by either the muscarinic cholinoceptor antagonist, atropine (0.3 microM) or by desensitization of P2x-purinoceptors with alpha,beta-methylene ATP (10 microM). In capsaicin-treated rats, contractions of the urinary bladder evoked by EFS were significantly larger than those of control (vehicle-treated) animals, and this difference remained after the purinergic component of the contractions was blocked by desensitization of P2x-purinoceptors with alpha,beta-methylene ATP. However, when the cholinergic component of the contractions was blocked with atropine, the difference between the groups at 8 Hz and 16 Hz was abolished; EFS caused significantly larger contractions of the capsaicin-treated rat bladder only at frequencies of 2 Hz and 4 Hz. 3. EFS evoked contractions of the vas deferens consisted of adrenergic and purinergic components since they could be partially blocked by either the alpha-adrenoceptor antagonist, phentolamine (3 microM) or by alpha,beta-methylene ATP (10 microM). The contractions of the vas deferens were significantly larger than in the capsaicin-treated rats only at a frequency of 16 Hz. There were no differences between vas deferens contractions of the two groups either after desensitization of P2X-purinoceptors by alpha,beta-methylene ATP or in the presence of phentolamine.4. Contractions of the capsaicin-treated rat urinary bladder evoked by exogenous carbachol (0.1-100 microM) were not significantly different from those of controls, the pD2 values being 1.78 +/- 0.23 micro M and 1.90 +/- 0.20 micro M respectively. There was also no significant difference between the groups in contractions of the bladder evoked by ATP (10 micro M-3 mM).5. Contractions of the vas deferens evoked by either ATP (10 micro M-3 mM) or noradrenaline (1-1000 micro M) in the capsaicin-treated group showed no significant difference between control and capsaicin treated rats.6. In conclusion, the present results indicate that chronic capsaicin treatment increases the amplitude of contractions of the rat urinary bladder, an effect which preferentially involves the cholinergic component of the response; since the response to carbachol is unaffected, the change involves prejunctional mechanisms. In contrast, both the purinergic and adrenergic components of contraction in the vas deferens are unaffected by capsaicin. It is suggested that sensory nerves have a trophic influence on the development of parasympathetic nerves in the rat bladder; removal of sensory nerves shortly after birth results in an increase mainly in the cholinergic, and to a lesser extent purinergic component.

In vitro experiments were carried out to measure the contractile responses to P2-receptor agonists in the greater saphenous vein isolated from patients with obliterating vascular atherosclerosis and varicose veins of the legs. In patients with varicose veins, the contractile responses of the greater saphenous vein to ATP, α,β-methylene-ATP, and UTP were significantly lower than in patients with obliterating atherosclerosis, while the responses to ADP, adenosine, and 2-methylthio-ATP were similar in both groups. These data attest to the presence of P2-receptor-mediated contraction component in the greater saphenous vein, which are pronouncedly weakened during varicose disease.

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.

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.

Extracellular ATP can produce various effects acting via P2-purinoceptors. ATP is rapidly broken down by ecto-ATPase and other ecto-enzymes that limit its effect. Further, adenosine, a metabolite of ATP breakdown, can produce its own effect acting via P1-purinoceptors, sometimes masking the effects of ATP. An inhibitor of ATP degradation would be a useful pharmacological tool to discriminate between effects of ATP and its metabolites, as well as to potentiate its actions. Diverse compounds that have been claimed to be inhibitors of ATP-metabolising ectoenzymes are evaluated, but specific and selective Ca2+ /Mg2+ -dependent ecto-ATPase inhibitors still appear to be lacking.

We studied contractile responses of isolated smooth muscles from human uterus induced by P2 receptor agonists. In preparations from pregnant uterus all tested P2 receptor agonists caused smooth muscle contractions. The relative activity of P2 receptor agonists decreased in the following order: a,b-methylene-ATP-uridine triphosphate-ATP. Responses induced by ATP and ADP were similar. The amplitude of contractions induced by alpha,beta-methylene-ATP significantly decreased in the presence of P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid. None of tested P2 receptor agonists induced contractions of isolated myometrium from nonpregnant women. Our results indicate that pregnant human uterus contains P2 receptors mediating the contractile response.