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.

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.

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.

[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.

[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.

[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.

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.

Identification of potent, selective P2Y-purinoceptor agonists: structure-activity relationships for 2-thioether derivatives of adenosine 5'-triphosphate.

Study of P2-purinoceptor subtypes has been difficult due to the lack of potent and selective ligands. With the goal of developing high affinity P2-purinoceptor-selective agonist, we have synthesized a series of analogues of adenine nucleotides modified on the purine ring as chain-extended 2-thioethers or as N6-methyl-substituted compounds. Chemical functionality incorporated in the thioether moiety included cyanoalkyl, nitroaromatic, amino, thiol, cycloalkyl, n-alkyl, and olefinic groups. Apparent affinity of the compounds for P2Y-purinoceptors was established by measurement of P2Y-purinoceptor-promoted phospholipase C activity in turkey erythrocyte membranes and relaxation of carbachol-contracted smooth muscle in three different preparations (guinea pig taenia coli, rabbit aorta, and rabbit mesenteric artery). Activity at P2X-purinoceptors was established by measurement of contraction of rabbit saphenous artery and of the guinea pig vas deferens and urinary bladder. All 11 of the 2-thioethers of ATP stimulated the production of inositol phosphates with K0.5 values of 1.5-770 nM, with an (aminophenyl)ethyl derivative being most potent. Two adenosine diphosphate analogues were equipotent to the corresponding ATP analogues. Adenosine monophosphate analogues were full agonists, although generally 4 orders of magnitude less potent. ATP 2-thioethers displayed pD2 values in the range of 6-8 in smooth muscle assay systems for activity at P2Y-receptors. There was a significant correlation for the 2-thioether compounds between the pK0.5 values for inositol phosphate production and the pD2 values for relaxation mediated via the P2Y-purinoceptors in the guinea pig taenia coli, but not for the vascular P2Y-receptors or for the P2X-receptors. At P2X-receptors, no activity was observed in the rabbit saphenous artery, but variable degrees of activity were observed in the guinea pig vas deferens and bladder depending on distal substituents of the thioether moiety. N6-Methyl-ATP was inactive at P2X-receptors, and approximately equipotent to ATP at taenia coil P2Y-receptors. This suggested that hybrid N6-methyl and 2-thioether ATP derivatives might be potent and selective for certain P2Y-receptors, as was shown for one such derivative, N6-methyl-2-(5-hexenylthio)-ATP.

Contractility of urinary bladder and vas deferens after sensory denervation by capsaicin treatment of newborn rats.

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.

Effects of divalent cations and La3+ on contractility and ecto-ATPase activity in the guinea-pig urinary bladder.

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.

The activation of P1- and P2-purinoceptors in the guinea-pig left atrium by diadenosine polyphosphates.

1. The effects of P1, P2-di(adenosine) pyrophosphate (AP2A), P1, P3-di(adenosine) triphosphate (AP3A), P1,P4-di(adenosine) tetraphosphate (AP4A), P1,P5-di(adenosine) pentaphosphate (AP5A), ATP, alpha, beta-methylene ADP and 2-chloroadenosine (2-ClAd) were examined in the guinea-pig driven left atrium. 2. All these purine compounds except alpha, beta-methylene ADP produced a negative inotropic response with a rank order of potency of: 2-ClAd > > AP2A > or = ATP > or = AP4A = AP3A = AP5A. The EC50 value for 2-ClAd was approximately 1 microM, while those for the remaining compounds were in the range 10 microM-100 microM, alpha, beta-Methylene ADP (10-300 microM), a selective P2Y-purinoceptor agonist, produced a small positive inotropism. 3. The P1-purinoceptor antagonist, 8-para-sulphophenyltheophylline (8-pSPT, 20 microM) caused a right-ward shift in the concentration-response curves for 2-ClAd, ATP and AP2A, but converted the responses of AP3A, AP4A, and AP5A into positive inotropisms. 4. The non-selective P2-purinoceptor antagonist, suramin (300 microM), had no significant effect on the concentration-response curves for 2-ClAd, ATP or AP2A, but significantly antagonized inhibitory responses to AP3A, AP4A and AP5A, and excitatory responses to alpha, beta-methylene ADP. 5. In the presence of 8-pSPT (20 microM), suramin (300 microM) abolished the positive inotropic responses evoked by the dinucleotides. 6. ATP was degraded far more rapidly than any of the dinucleotides, and AP3A was the least stable of the diadenosine compounds. The relative order of stability was AP2A > AP4A = AP5A > AP3A > > ATP. Suramin (300 microM) reduced the rate of degradation of ATP and AP3A by approximately 30%. Suramin had no significant effect on the degradation of AP2A, AP4A or AP5A. 7. It is concluded that the diadenosine polyphosphates cause negative inotropic responses via P1-purinoceptors and a hitherto undefined suramin-sensitive P2-purinoceptor, and that they appear to have positive inotropic effects mediated via another suramin-sensitive P2-purinoceptor.

Effects of cyclopiazonic acid on contractility and ecto-ATPase activity in guinea-pig urinary bladder and vas deferens.

1. Cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic ATPase, was tested on guinea-pig urinary bladder and vas deferens for its ability: (1) to modify contractile responses to electrical field stimulation (EFS), exogenous ATP, alpha,beta-methylene ATP (alpha,beta-MeATP), carbachol, noradrenaline (NA), histamine, and KCl; (2) to affect ecto-ATPase activity; (3) to modify the release of ATP evoked by EFS. 2. In the urinary bladder, CPA (10 microM) potentiated contractile responses to EFS, exogenous ATP (100 microM), alpha,beta-meATP (1 microM), carbachol (0.5 microM), histamine (30 microM) and KCl (30 mM). In the vas deferens, CPA (10 microM) potentiated responses to EFS, ATP, alpha,beta-meATP, NA (100 microM) and KCl. CPA at a concentration of 1 microM had no effect on ATP-induced relaxation of carbachol-precontracted guinea-pig taenia coli, and at a concentration of 10 microM it markedly increased spontaneous contractile activity of taenia. 3. Ecto-ATPase was estimated to have Vmax and Km values of 0.98 nmol Pi 30 min-1 mg-1 wet tissue and 881 microM ATP in the urinary bladder, and 0.75 nmol Pi 30 min-1 mg-1 wet tissue and 914 microM ATP in the vas deferens, respectively. CPA at a concentration of 10 microM significantly inhibited ecto-ATPase activity by 18% in the urinary bladder and by 24% in the vas deferens. 4. In the guinea-pig vas deferens, CPA significantly potentiated ATP release evoked by EFS from 2.2 +/- 0.8 (6) pmol ATP min-1 g-1 wet tissue to 35.2 +/- 4.8 (6) pmol ATP min-1 g-1 wet tissue (P < 0.01). 5. In conclusion, the potentiation of contractile responses of the guinea-pig urinary bladder and vas deferens by CPA has a non-specific character. CPA inhibited ecto-ATPase activity and increased ATP release, but these effects do not appear to contribute to the potentiation of Pu-purinoceptor-mediated responses since the contractile actions of all the agonists studied were potentiated to the same extent.

Selective antagonism by PPADS at P2X-purinoceptors in rabbit isolated blood vessels.

1. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), a P2-purinoceptor antagonist, was investigated for its ability to antagonize: (1) P2X-purinoceptor-mediated contractions of the rabbit central ear artery and saphenous artery evoked by either alpha,beta-methylene ATP (alpha,beta-MeATP) or electrical field stimulation (EFS); (2) P2Y-purinoceptor-mediated relaxations of the rabbit mesenteric artery; (3) endothelium-dependent and endothelium-independent, P2Y-purinoceptor-mediated relaxations of the rabbit aorta. 2. alpha,beta-MeATP (0.1-100 microM) caused concentration-dependent contractions of the rabbit ear and saphenous arteries. The negative log[alpha,beta-MeATP] that produced a contraction equivalent to the EC25 for noradrenaline (ear artery) or histamine (saphenous artery) in the absence of PPADS was 6.60 +/- 0.18 (9) and 6.18 +/- 0.17 (9) in the ear artery and saphenous artery, respectively. These effects of exogenous alpha,beta-MeATP were concentration-dependently inhibited by PPADS (1-30 microM). In the ear artery, the negative log[alpha,beta-MeATP] producing a contractile response equivalent to the EC25 of noradrenaline, in the presence of PPADS at 1, 3 and 10 microM was 6.16 +/- 0.18 (8), 5.90 +/- 0.18 (8) and 4.72 +/- 0.36 (8), respectively (P < 0.01). In the saphenous artery, the negative log[alpha,beta-MeATP] values equivalent to the EC25 for histamine in the presence of PPADS at concentrations of 1, 3, 10 and 30 microM were 5.90 +/- 0.19 (8), 5.73 +/- 0.16 (8), 4.99 +/- 0.14 (8) and 4.51 +/- 0.13 (8), respectively (P < 0.01). 3. PPADS at a concentration of 1 microM had no effect on contractions of the ear artery evoked by EFS (4-64 Hz; 1 microM phentolamine present). At higher concentrations (3-30 MicroM) it caused concentration dependent inhibition of neurogenic contractions. In the saphenous artery, PPADS (1-30 MicroM) concentration-dependently inhibited contractions evoked by EFS at frequencies of 4, 8 and 16 Hz. Contractions evoked by EFS at frequencies of 32 and 64 Hz were significantly inhibited by PPADS only at concentrations of 10 and 30 MicroM.4. PPADS (30 MicroM) had no effect on relaxations to 2-methylthio ATP (3 nM-3 MicroM) in rabbit mesenteric artery and to ATP (1 MicroM-I mM) in rabbit aorta (with endothelium intact or removed). In addition,PPADS (30 MicroM) had no significant influence on the contractile potency of noradrenaline and histamine in rabbit ear and saphenous artery, respectively.5. In conclusion, these results support the evidence that PPADS is a selective antagonist of P2X-purinoceptor-mediated responses.