apyrase and alpha-beta-methyleneadenosine-5--triphosphate

Eosinophils play an important role in the pathogenesis of asthma and can be activated by extracellular nucleotides released following cell damage or inflammation. For example, increased ATP concentrations were reported in bronchoalveolar lavage fluids of asthmatic patients. Although eosinophils are known to express several subtypes of P2 receptors for extracellular nucleotides, their function and contribution to asthma remain unclear. In this article, we show that transcripts for P2X1, P2X4, and P2X5 receptors were expressed in healthy and asthmatic eosinophils. The P2X receptor agonist α,β-methylene ATP (α,β-meATP; 10 μM) evoked rapidly activating and desensitizing inward currents (peak 18 ± 3 pA/pF at -60 mV) in healthy eosinophils, typical of P2X1 homomeric receptors, which were abolished by the selective P2X1 antagonist NF449 (1 μM) (3 ± 2 pA/pF). α,β-meATP-evoked currents were smaller in eosinophils from asthmatic patients (8 ± 2 versus 27 ± 5 pA/pF for healthy) but were enhanced following treatment with a high concentration of the nucleotidase apyrase (17 ± 5 pA/pF for 10 IU/ml and 11 ± 3 pA/pF for 0.32 IU/ml), indicating that the channels are partially desensitized by extracellular nucleotides. α,β-meATP (10 μM) increased the expression of CD11b activated form in eosinophils from healthy, but not asthmatic, donors (143 ± 21% and 108 ± 11% of control response, respectively). Furthermore, α,β-meATP increased healthy (18 ± 2% compared with control 10 ± 1%) but not asthmatic (13 ± 1% versus 10 ± 0% for control) eosinophil adhesion. Healthy human eosinophils express functional P2X1 receptors whose activation leads to eosinophil αMβ2 integrin-dependent adhesion. P2X1 responses are constitutively reduced in asthmatic compared with healthy eosinophils, probably as the result of an increase in extracellular nucleotide concentration.

Topics: Adenosine Triphosphate; Apyrase; Asthma; Benzenesulfonates; CD11b Antigen; Cell Adhesion; Eosinophils; Healthy Volunteers; Humans; Leukocyte Count; Purinergic P2X Receptor Agonists; Real-Time Polymerase Chain Reaction; Receptors, Purinergic P2X1; Receptors, Purinergic P2X4; Receptors, Purinergic P2X5; Reverse Transcriptase Polymerase Chain Reaction

Evidence from patients with inflammatory bowel disease (IBD) and animal models suggests that inflammation alters blood flow to the mucosa, which precipitates mucosal barrier dysfunction. Impaired purinergic sympathetic regulation of submucosal arterioles, the resistance vessels of the splanchnic vasculature, is one of the defects identified during IBD and in mouse models of IBD. We hypothesized that this may be a consequence of upregulated catabolism of ATP during colitis. In vivo and in vitro video microscopy techniques were employed to measure the effects of purinergic agonists and inhibitors of CD39, an enzyme responsible for extracellular ATP catabolism, on the diameter of colonic submucosal arterioles from control mice and mice with dextran sodium sulfate [DSS, 5% (wt/vol)] colitis. Using a luciferase-based ATP assay, we examined the degradation of ATP and utilized real-time PCR, Western blotting, and immunohistochemistry to examine the expression and localization of CD39 during colitis. Arterioles from mice with DSS colitis did not constrict in response to ATP (10 microM) but did constrict in the presence of its nonhydrolyzable analog alpha,beta-methylene ATP (1 microM). alpha,beta-Methylene ADP (100 microM), an inhibitor of CD39, restored ATP-induced vasoconstriction in arterioles from mice with DSS-induced colitis. CD39 protein and mRNA expression was markedly increased during colitis. Immunohistochemical analysis demonstrated that, in addition to vascular CD39, F4/80-immunoreactive macrophages accounted for a large proportion of submucosal CD39 staining during colitis. These data implicate upregulation of CD39 in impaired sympathetic regulation of gastrointestinal blood flow during colitis.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Antigens, CD; Antigens, Differentiation; Apyrase; Arterioles; Colitis; Colon; Dextran Sulfate; Disease Models, Animal; Electric Stimulation; Enzyme Inhibitors; Macrophages; Male; Mice; RNA, Messenger; Splanchnic Circulation; Submucous Plexus; Sympathetic Nervous System; Up-Regulation; Vasoconstriction

Vascular responses to adenine nucleotides in human corpora cavernosa from men with vasculogenic erectile dysfunction were investigated. We also evaluated the catabolism of extracellular adenine nucleotides to probe its relevance to vascular hemodynamics in impotent men. Human corpora cavernosa have high NTPDase1/CD39 activity, converting ATP directly into AMP, without significant ADP formation. Extracellular ATP hydrolysis is slower in impotent patients. Adenine nucleotides have dual roles on phenylephrine-contracted strips of corpora cavernosa operated by P2X-contractant and P2Y-relaxant receptors. Prolonged exposure to endogenous ATP related to decreased NTPDase1/CD39 activity leads to P2-purinoceptor desensitization in impotent men. Shutting down ATP signaling in vasculogenic impotent men may represent a defense mechanism for preventing purinergic overstimulation.

Topics: Adenosine A2 Receptor Agonists; Adenosine Triphosphate; Adolescent; Adult; Antigens, CD; Apyrase; Case-Control Studies; Dose-Response Relationship, Drug; Endothelium; Humans; Impotence, Vasculogenic; Male; Middle Aged; Muscle Contraction; Muscle Tonus; Penis; Receptor, Adenosine A2B; Receptors, Purinergic P2; Signal Transduction

The role of ATP-stimulated P2X1 receptors in human platelets is still unclear. They may act alone or in synergy with other pathways, such as P2Y1 or P2Y12 receptors, to accelerate and enhance calcium mobilisation, shape change and aggregation. To date very few pharmacological means of selectively inhibiting platelet P2X1 receptors have been described, although recent work has shown that suramin is a useful lead compound for the development of high-affinity P2X1 antagonists. We therefore investigated the effects of a series of bivalent and tetravalent suramin analogues on alphabeta meATP (P2X1 receptors)-induced or ADP (P2Y1 receptors)-induced intracellular calcium increases and shape change, as well as on ADP-induced aggregation (P2Y1 & P2Y12 receptors) in human platelets. Changes in intracellular calcium were measured using standard fluorescence techniques, while shape change and aggregation were determined by turbidimetry. The novel tetravalent compound NF864 (8,8',8'',8'''-(carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino)))tetrakis-naphthalene-1,3,5-trisulfonic acid-dodecasodium salt) proved to be the most potent platelet P2X1 antagonist reported to date, blocking alphabeta meATP-induced Ca2+ increases and shape change in a concentration-dependent manner, with a pA2 of 8.17 and 8.49, respectively. The ability to inhibit the platelet P2X1 receptor displayed the following order : NF864 > NF449 > or = NF110 > NF023 = MK-HU1 = suramin. A different antagonistic profile was observed for ADP-induced Ca2+ increases, shape change and aggregation; however, overall four compounds showed sufficient ability to selectively inhibit P2X1 responses, with the order NF110 > NF449 > or = NF864 > or = MK-HU1. Therefore, these compounds should prove useful tools for investigating the functional significance of platelet P2X1 receptors in thrombosis and haemostasis, NF864 being the most promising compound.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Apyrase; Blood Platelets; Calcium; Cell Shape; Dose-Response Relationship, Drug; Humans; Least-Squares Analysis; Linear Models; Phenylmercury Compounds; Platelet Aggregation Inhibitors; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2X; Receptors, Purinergic P2Y1; Suramin; Thionucleotides

The role of ATP on the modulation of noradrenaline release elicited by electrical stimulation (100 pulses/8 Hz) was studied in the prostatic portion of rat vas deferens preincubated with [3H]noradrenaline. In the presence of P1 antagonists, the nucleotides 2-methylthioadenosine-5'-triphosphate (2-MeSATP), 2-methylthioadenosine 5'-diphosphate (2-MeSADP), ADP, and ATP decreased electrically evoked tritium overflow up to 44%, with the following order of potency: 2-MeSATP > 2-MeSADP > ADP > or = ATP. The P2Y antagonists reactive blue 2 (RB2) and 2-methylthioadenosine 5'-monophosphate (2-MeSAMP) increased, whereas the P2X antagonist pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) (PPNDS) decreased evoked tritium overflow. The inhibitory effect of 2-MeSATP was antagonized by RB2 (10 microM) and by 2-MeSAMP (10 microM) but not by the selective P2Y1 receptor antagonist 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS 2179; 10 microM). When, besides P1 receptors, inhibitory P2Y receptors were blocked with RB2, alpha,beta-methyleneadenosine 5'-triphosphate (alpha,beta-meATP), beta,gamma-imidoadenosine 5'-triphosphate (beta,gamma-imidoATP), beta,gamma-methyleneadenosine 5'-triphosphate (beta,gamma-meATP), 2-MeSATP, and ATP enhanced tritium overflow up to 140%, with the following order of potency: alpha,beta-meATP > 2-MeSATP = ATP = beta,gamma-meATP > or = beta,gamma-imidoATP. The facilitatory effects of alpha,beta-MeATP and beta,gamma-imidoATP were prevented by PPNDS. Under the same conditions, apyrase attenuated, whereas the ectonucleotidase inhibitor 6-N,N-diethyl-D-beta,gamma-dibromomethylene 5'-triphosphate enhanced tritium overflow, an effect that was prevented by PPNDS. In the prostatic portion of the rat vas deferens, endogenous ATP exerts a dual and opposite modulation of noradrenaline release: an inhibition through activation of P2Y receptors with a pharmacological profile similar to that of the P2Y12 and P2Y13 receptors and a facilitation through activation of P2X receptors with a pharmacological profile similar to that of P2X1 and P2X3, or PX2/P2X3 receptors.

Topics: Adenosine Triphosphate; Animals; Apyrase; Male; Norepinephrine; Rats; Rats, Wistar; Receptors, Purinergic P2; Receptors, Purinergic P2X; Receptors, Purinergic P2Y1; Tritium; Vas Deferens

Adenosine triphosphate (ATP) and its stable analog, alpha,beta-methylene ATP, activate the platelet P2X(1) ion channel, causing a rapid Ca(++) influx. Here, we show that, in washed apyrase-treated platelets, alpha,beta-methylene ATP elicits reversible extracellular signal-regulated kinase 2 (ERK2) phosphorylation through a Ca(++)- and protein kinase C-dependent pathway. In contrast, high-performance liquid chromatography-purified adenosine diphosphate (ADP) did not trigger ERK2 phosphorylation. alpha,beta-Methylene ATP also activated the ERK2 pathway in P2X(1)-transfected HEK293 cells but not in cells expressing mutated P2X(1)delL nonfunctional channels. Because ATP released from the dense granules during platelet activation contributes to platelet aggregation elicited by low doses of collagen, and because collagen causes ERK2 phosphorylation, we have investigated the role of P2X(1)-mediated ERK2 activation in these platelet responses. We found that the antagonism of P2X(1) with ADP or desensitization of this ion channel with alpha,beta-methylene ATP both resulted in impaired ERK2 phosphorylation, ATP secretion, and platelet aggregation induced by low concentrations of collagen (< or = 1 microg/mL) without affecting the minor early dense granule release. Selective MEK1/2 inhibition by U-0126 and Ca(++) chelation with EGTA (ethyleneglycoltetraacetic acid) behaved similarly, whereas the PKC inhibitor GF109203-X totally prevented collagen-induced secretion and ERK2 activation. In contrast, when elicited by high collagen concentrations (2 microg/mL), platelet aggregation and secretion no longer depended on P2X(1) or ERK2 activation, as shown by the lack of their inhibition by alpha,beta-methylene ATP or U-0126. We thus conclude that mild platelet stimulation with collagen rapidly releases ATP, which activates the P2X(1)-PKC-ERK2 pathway. This process enhances further degranulation of the collagen-primed granules allowing platelet aggregation to be completed.

Topics: Adenosine Triphosphate; Apyrase; Blood Platelets; Calcium; Collagen; Egtazic Acid; Humans; In Vitro Techniques; Kinetics; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Models, Cardiovascular; Platelet Aggregation; Protein Kinase C; Receptors, Purinergic P2; Receptors, Purinergic P2X

The possibility was tested that endogenous ATP released upon alpha(1)-adrenoceptor activation causes relaxation of the rat vas deferens smooth muscle. ATP, 2-methylthio ATP and adenosine relaxed the vas deferens precontracted with 80 mM K(+). The metabolically stable P2 receptor agonists alpha,beta-methylene ATP (alpha,beta-MeATP) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) had little or no effect. The adenosine P1 receptor antagonist 8-(para-sulfophenyl)theophylline did not significantly affect the response to ATP. The P2 receptor antagonist reactive blue 2 markedly reduced the relaxation (by up to 73%); suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and acid blue 129 caused no change. ATP, but not alpha,beta-MeATP, also attenuated contractions elicited by noradrenaline at resting tension; reactive blue 2 blocked the inhibitory effect of ATP. Reactive blue 2, by itself, enhanced the response to noradrenaline (by up to 36%); suramin, PPADS and acid blue 129 caused no change. In the presence of the ATP-degrading enzymes apyrase and nucleotide pyrophosphatase, the facilitatory effect of reactive blue 2 was lost. Apyrase, by itself, enhanced the response to noradrenaline (by 13%). The results indicate that endogenous ATP, released from rat vas deferens smooth muscle upon alpha(1)-adrenoceptor stimulation, causes relaxation. The site of action of ATP is not a typical smooth muscle P2Y receptor.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Apyrase; Dose-Response Relationship, Drug; In Vitro Techniques; Male; Muscle Relaxation; Norepinephrine; Nucleotides; Potassium; Purinergic P1 Receptor Antagonists; Pyridoxal Phosphate; Pyrophosphatases; Rats; Rats, Wistar; Receptors, Adrenergic, alpha-1; Suramin; Theophylline; Thionucleotides; Triazines; Vas Deferens; Vasoconstrictor Agents

H(2)O(2) can contract many arteries, however the underlying mechanisms are not fully understood. This study aims to test whether H(2)O(2)-induced vasoconstriction could be functionally attributed to the activation of P(2)-purinoceptors in rat aorta and to explore its possible signaling mechanisms.. Isometric tension recording of H(2)O(2) and ATP-induced contractions of rat aortic rings were compared in the absence or presence of various pharmacological tools to identify their possible common signaling pathways.. Both H(2)O(2) and ATP induced transient phasic contractions in a concentration-dependent manner (1-1000 microM). Removal of endothelium potentiated the contractile responses to H(2)O(2) and to ATP. H(2)O(2) (30 microM)-induced phasic contraction could be abolished by catalase (800 U/ml), but not affected by SOD (150 U/ml), DMSO (5 mM) and apyrase (5 U/ml), suggesting no involvement of O(2)(-), hydroxyl free radicals and ATP release. Also, several receptor antagonists including phentolamine, atropine, methysergide and chlorpheniramine (each 3 microM) were without effect on H(2)O(2) (30 microM)-induced phasic contraction, suggesting no involvement of typical neurotransmitter release. However, both H(2)O(2) (30 microM) and ATP (1 mM)-induced phasic contractions not only presented homologous desensitization, but also showed heterogeneous desensitization. Furthermore, the phasic contractions in response to H(2)O(2) (30 microM) or ATP (100 microM) could be inhibited or abolished in a concentration dependent manner by RB-2 and suramin (10-100 microM), two widely used P(2)-purinoceptor antagonists, with only partial inhibition by Evans blue (300 microM), a moderately selective P(2x) receptor blocker, or by alpha-beta-methylene-ATP (100 microM), a selective P(2x) receptor desensitizer. On the other hand, both H(2)O(2) (30 microM) and ATP (100 microM)-induced phasic contractions were also attenuated, to different degree, by inhibitors of several enzymes including PLC, PKC, PLA(2) and cyclooxygenase. Lastly, removal of extracellular Ca(2+) or pretreatment with procaine (10 mM) and dantrolene (30 microM), two putative intracellular Ca(2+) release blockers, or with Ni(2+) (100 microM) and tetrandrine (5 microM), two Ca(2+) channel blockers, all significantly inhibited H(2)O(2) and ATP-induced contractions. However, nifedipine (1 microM), a voltage-dependent L-type Ca(2+) channel blocker, was without effect.. Our results demonstrate that H(2)O(2)-induced phasic contraction of rat aorta involves, at least in part, the activation of P(2)-purinoceptors in the aortic smooth muscle cells

Topics: Adenosine Triphosphate; Alkaloids; Analysis of Variance; Animals; Aorta; Apyrase; Benzylisoquinolines; Calcium; Calcium Channel Blockers; Catalase; Dantrolene; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Evans Blue; Hydrogen Peroxide; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Nickel; Nifedipine; Procaine; Purinergic P2 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Signal Transduction; Superoxide Dismutase; Suramin; Triazines; Vasoconstrictor Agents

Evidence that secreted dense granule adenine nucleotides mediate part of the agonist-induced cytosolic calcium ([Ca2+]i) responses in human platelets was obtained from comparisons of fura-2-loaded platelets from normal subjects and from patients with a form of platelet storage pool deficiency (SPD) in which the secretory dense granules and their contents are virtually absent. SPD platelets had normal initial [Ca2+]i increases induced by thrombin and the endoperoxide analog U46619, but a significantly enhanced decay of elevated [Ca2+]i levels following the initial increases. With thrombin, this enhanced [Ca2+]i decay was associated with decreased Ca2+ influx, as measured by Mn2+ quench of fura-2 fluorescence. Addition of micromolar concentrations of ADP, alone or together with ATP, after stimulation reversed the enhanced [Ca2+]i decay and increased Mn2+ quench in SPD platelets, but had no effect on these responses in normal platelets, while addition of 100-fold higher concentrations of ATP or apyrase before stimulation increased [Ca2+]i decay and decreased Mn2+ quench in normal platelets, but had little effect in SPD platelets. ATP and alpha,beta-methylene ATP, a specific agonist for P2X1 receptors, at micromolar concentrations also increased Mn2+ quench, but to lesser extents than did ADP, in SPD platelets isolated and loaded with fura-2 in the presence of apyrase. Similar effects of ADP and excess ATP were seen in U46619-stimulated platelets, but decreased Ca2+ influx could not be measured directly in SPD platelets, presumably due to the very transient influx response seen with U46619. These results suggest that secreted dense granule ADP and ATP contribute to the maintenance of elevated [Ca2+]i levels, but not to the initial [Ca2+]i increases, in stimulated human platelets, most likely via a nucleotide-specific component of Ca2+ influx which may be mediated by interactions with both P2X1 and P2Y1 purinoceptors.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenine Nucleotides; Adenosine Diphosphate; Adenosine Triphosphate; Apyrase; Blood Platelets; Calcium; Cytoplasmic Granules; Cytosol; Drug Interactions; Fura-2; Humans; Ion Transport; Platelet Activation; Platelet Storage Pool Deficiency; Purinergic P2 Receptor Agonists; Receptors, Purinergic P2X; Thrombin

We have investigated the purinoceptor subtypes responsible for calcium signaling in human platelets, which previous studies have shown to involve both Ca2+ influx via receptor-operated cation channels and release of Ca2+ from intracellular stores. Fura-2 measurements of [Ca2+]i in stirred platelet suspensions showed that both ADP (40 microM) and the non-hydrolyzable ATP analogue alphabeta-meATP (alpha, beta-methyleneadenosine 5-triphosphate, 10 microM) activated a rapid Ca2+ influx whereas only ADP mobilized Ca2+ from internal stores. In "nystatin" whole-cell patch clamp recordings, ATP, ADP, and the non-hydrolyzable ATP analogues, alpha, beta-meATP and ATPgammaS (adenosine 5 -O-(3-thiotriphosphate), all activated a cation channel permeable to both monovalent and divalent cations with a single-channel conductance of 11 picosiemens in NaCl saline. The current response to ATP (40 microM) was activated within 20 ms and desensitized with a time constant of 47-107 ms in the continued presence of agonist, which are characteristics of P2X1 receptors in other tissues. We conclude that human platelets possess a P2X1 purinoceptor, which mediates a rapid phase of ADP- or ATP-evoked Ca2+ entry via a cation channel, whereas one or more separate ADP-selective P2 purinoceptors evoke release of calcium from intracellular stores.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Apyrase; Aspirin; Blood Platelets; Calcium; Calcium Channels; Fluorescent Dyes; Fura-2; Humans; In Vitro Techniques; Membrane Potentials; Membrane Proteins; Patch-Clamp Techniques; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2X; Receptors, Purinergic P2Y12

Somatic sensation requires the conversion of physical stimuli into the depolarization of distal nerve endings. A single cRNA derived from sensory neurons renders Xenopus laevis oocytes mechanosensitive and is found to encode a P2Y1 purinergic receptor. P2Y1 mRNA is concentrated in large-fiber dorsal root ganglion neurons. In contrast, P2X3 mRNA is localized to small-fiber sensory neurons and produces less mechanosensitivity in oocytes. The frequency of touch-induced action potentials from frog sensory nerve fibers is increased by the presence of P2 receptor agonists at the peripheral nerve ending and is decreased by the presence of P2 antagonists. P2X-selective agents do not have these effects. The release of ATP into the extracellular space and the activation of peripheral P2Y1 receptors appear to participate in the generation of sensory action potentials by light touch.

Topics: Adenosine Triphosphate; Animals; Apyrase; Chickens; Female; Ganglia, Spinal; Membrane Potentials; Molecular Sequence Data; Nerve Fibers; Neurons, Afferent; Oocytes; Physical Stimulation; Purinergic P2 Receptor Agonists; Pyridoxal Phosphate; Rats; Receptors, Purinergic P2; Receptors, Purinergic P2X3; Receptors, Purinergic P2Y1; RNA, Messenger; Sciatic Nerve; Skin; Suramin; Time Factors; Transcription, Genetic; Xenopus laevis