Ectonucleotidase Inhibitor ARL67156 Research Articles

Indoxyl sulfate decreases uridine adenosine tetraphosphate-induced contraction in rat renal artery.

The protein-bound uremic toxin indoxyl sulfate has negative effects on a variety of physiological activities including vascular function. Uridine adenosine tetraphosphate (Up4A), a new dinucleotide molecule affects vascular function including induction of vasocontraction, and aberrant responsiveness to Up4A is evident in arteries from disorders such as hypertension and diabetes. The link between indoxyl sulfate and the Up4A-mediated response is, however, unknown. We used Wistar rat's renal arteries to see if indoxyl sulfate will affect Up4A-mediated vascular contraction. In renal arteries of indoxyl sulfate, the contractile response generated by Up4A was dramatically reduced compared to the non-treated control group. Indoxyl sulfate increased endothelin-1-induced contraction but had no effect on phenylephrine, thromboxane analog, or isotonic K+-induced renal arterial contractions. UTP, ATP, UDP, and ADP-produced contractions were reduced by indoxyl sulfate. CH223191, an aryl hydrocarbon receptor (AhR) antagonist, did not reverse Up4A, and UTP contraction decreases caused by indoxyl sulfate. The ectonucleotidase inhibitor ARL67156 prevents indoxyl sulfate from reducing Up4A- and UTP-mediated contractions. In conclusion, we discovered for the first time that indoxyl sulfate inhibits Up4A-mediated contraction in the renal artery, possibly through activating ectonucleotidase but not AhR. Indoxyl sulfate is thought to play a function in the pathophysiology of purinergic signaling.

Delivery of an ectonucleotidase inhibitor with ROS-responsive nanoparticles overcomes adenosine-mediated cancer immunosuppression.

Tumor evasion of immune destruction is associated with the production of immunosuppressive adenosine in the tumor microenvironment (TME). Anticancer therapies can trigger adenosine triphosphate (ATP) release from tumor cells, causing rapid formation of adenosine by the ectonucleotidases CD39 and CD73, thereafter exacerbating immunosuppression in the TME. The goal of this study was to develop an approach to facilitate cancer therapy-induced immunogenic cell death including ATP release and to limit ATP degradation into adenosine, in order to achieve durable antitumor immune response. Our approach was to construct reactive oxygen species (ROS)-producing nanoparticles that carry an ectonucleotidase inhibitor ARL67156 by electronic interaction and phenylboronic ester. Upon near-infrared irradiation, nanoparticle-produced ROS induced ATP release from MOC1 cancer cells in vitro and triggered the cleavage of phenylboronic ester, facilitating the release of ARL67156 from the nanoparticles. ARL67156 prevented conversion of ATP to adenosine and enhanced anticancer immunity in an MOC1-based coculture model. We tested this approach in mouse tumor models. Nanoparticle-based ROS-responsive drug delivery reprogramed the immunogenic landscape in tumors, eliciting tumor-specific T cell responses and tumor regression, conferring long-term survival in mouse models. We demonstrated that TME reprograming sets the stage for response to anti-programmed cell death protein 1 (PD1) immunotherapy, and the combination resulted in tumor regression in a 4T1 breast cancer mouse model that was resistant to PD1 blockade. Furthermore, our approach also induced immunological effects in patient-derived organotypic tumor spheroid model, suggesting potential translation of our nanoparticle approach for treating human cancers.

Inhibition of the hypercapnic ventilatory response by adenosine in the retrotrapezoid nucleus in awake rats

The brain regulates breathing in response to changes in tissue CO2/H+ via a process called central chemoreception. Neurons and astrocytes in the retrotrapezoid nucleus (RTN) function as respiratory chemoreceptors. The role of astrocytes in this process appears to involve CO2/H+-dependent release of ATP to enhance activity of chemosensitive RTN neurons. Considering that in most brain regions extracellular ATP is rapidly broken down to adenosine by ectonucleotidase activity and since adenosine is a potent neuromodulator, we wondered whether adenosine signaling contributes to RTN chemoreceptor function. To explore this possibility, we pharmacologically manipulated activity of adenosine receptors in the RTN under control conditions and during inhalation of 7–10% CO2 (hypercapnia). In urethane-anesthetized or unrestrained conscious rats, bilateral injections of adenosine into the RTN blunted the hypercapnia ventilatory response. The inhibitory effect of adenosine on breathing was blunted by prior RTN injection of a broad spectrum adenosine receptor blocker (8-PT) or a selective A1-receptor blocker (DPCPX). Although RTN injections of 8PT, DPCPX or the ectonucleotidase inhibitor ARL67156 did not affected baseline breathing in either anesthetized or awake rats. We did find that RTN application of DPCPX or ARL67156 potentiated the respiratory frequency response to CO2, suggesting a portion of ATP released in the RTN during high CO2/H+ is converted to adenosine and serves to limit chemoreceptor function. These results identify adenosine as a novel purinergic regulator of RTN chemoreceptor function during hypercapnia.

Central Role of P2Y 6 UDP Receptor in Arteriolar Myogenic Tone

Objective— Myogenic tone (MT) of resistance arteries ensures autoregulation of blood flow in organs and relies on the intrinsic property of smooth muscle to contract in response to stretch. Nucleotides released by mechanical strain on cells are responsible for pleiotropic vascular effects, including vasoconstriction. Here, we evaluated the contribution of extracellular nucleotides to MT. Approach and Results— We measured MT and the associated pathway in mouse mesenteric resistance arteries using arteriography for small arteries and molecular biology. Of the P2 receptors in mouse mesenteric resistance arteries, mRNA expression of P2X 1 and P2Y 6 was dominant. P2Y 6 fully sustained UDP/UTP-induced contraction (abrogated in P2ry6 −/− arteries). Preventing nucleotide hydrolysis with the ectonucleotidase inhibitor ARL67156 enhanced pressure-induced MT by 20%, whereas P2Y 6 receptor blockade blunted MT in mouse mesenteric resistance arteries and human subcutaneous arteries. Despite normal hemodynamic parameters, P2ry6 −/− mice were protected against MT elevation in myocardial infarction–induced heart failure. Although both P2Y 6 and P2Y 2 receptors contributed to calcium mobilization, P2Y 6 activation was mandatory for RhoA–GTP binding, myosin light chain, P42–P44, and c-Jun N-terminal kinase phosphorylation in arterial smooth muscle cells. In accordance with the opening of a nucleotide conduit in pressurized arteries, MT was altered by hemichannel pharmacological inhibitors and impaired in Cx43 +/− and P2rx7 −/− mesenteric resistance arteries. Conclusions— Signaling through P2 nucleotide receptors contributes to MT. This mechanism encompasses the release of nucleotides coupled to specific autocrine/paracrine activation of the uracil nucleotide P2Y 6 receptor and may contribute to impaired tissue perfusion in cardiovascular diseases.

Stimulation of Glia Reveals Modulation of Mammalian Spinal Motor Networks by Adenosine.

Despite considerable evidence that glia can release modulators to influence the excitability of neighbouring neurons, the importance of gliotransmission for the operation of neural networks and in shaping behaviour remains controversial. Here we characterise the contribution of glia to the modulation of the mammalian spinal central pattern generator for locomotion, the output of which is directly relatable to a defined behaviour. Glia were stimulated by specific activation of protease-activated receptor-1 (PAR1), an endogenous G-protein coupled receptor preferentially expressed by spinal glia during ongoing activity of the spinal central pattern generator for locomotion. Selective activation of PAR1 by the agonist TFLLR resulted in a reversible reduction in the frequency of locomotor-related bursting recorded from ventral roots of spinal cord preparations isolated from neonatal mice. In the presence of the gliotoxins methionine sulfoximine or fluoroacetate, TFLLR had no effect, confirming the specificity of PAR1 activation to glia. The modulation of burst frequency upon PAR1 activation was blocked by the non-selective adenosine-receptor antagonist theophylline and by the A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, but not by the A2A-receptor antagonist SCH5826, indicating production of extracellular adenosine upon glial stimulation, followed by A1-receptor mediated inhibition of neuronal activity. Modulation of network output following glial stimulation was also blocked by the ectonucleotidase inhibitor ARL67156, indicating glial release of ATP and its subsequent degradation to adenosine rather than direct release of adenosine. Glial stimulation had no effect on rhythmic activity recorded following blockade of inhibitory transmission, suggesting that glial cell-derived adenosine acts via inhibitory circuit components to modulate locomotor-related output. Finally, the modulation of network output by endogenous adenosine was found to scale with the frequency of network activity, implying activity-dependent release of adenosine. Together, these data indicate that glia play an active role in the modulation of mammalian locomotor networks, providing negative feedback control that may stabilise network activity.

Uridine adenosine tetraphosphate is a novel vasodilator in the coronary microcirculation which acts through purinergic P1 but not P2 receptors

Uridine adenosine tetraphosphate (Up4A) has been identified as an endothelium-derived contracting factor, which acts through purinergic P2X and P2Y receptors. Since the coronary vascular actions of Up4A are unknown, we investigated the vasoactive profile of Up4A in coronary microvessels, and studied the involvement of purinergic receptor subtypes. Studies were performed in isolated porcine coronary small arteries (diameter∼250μm), with and without endothelial denudation, mounted on a Mulvany wire myograph. Purinergic receptor expression was assessed by real-time PCR. Up4A (10−9–10−5M) failed to induce contraction at basal tone, but produced concentration-dependent vasorelaxation in precontracted microvessels. Up4A was slightly less potent than adenosine, ATP, and ADP in producing vasorelaxation, but significantly more potent than UTP and UDP. mRNA expression of P2X4, P2Y1, P2Y2, P2Y4, P2Y6 and A2A, but not P2X1, receptors was observed. Up4A-induced vasodilation was unaffected by non-selective P2 receptor antagonist PPADS, P2X1 antagonist MRS2159, P2Y1 antagonist MRS2179 and P2Y6 antagonist MRS2578, but was markedly attenuated by non-selective P1 receptor antagonist 8PT and A2A antagonist SCH58261. Up4A-induced vasodilation was not affected by ectonucleotidase inhibitor ARL67156, suggesting that A2A stimulation was not the result of Up4A breakdown to adenosine. Up4A-induced vasodilation was blunted in denuded vessels; additional A2A receptor blockade further attenuated Up4A-induced vasodilation, suggesting that A2A receptor-mediated vasodilation is only partly endothelium-dependent. In conclusion, Up4A exerts a vasodilator rather than a vasoconstrictor influence in coronary microvessels, which is mediated via A2A receptors and is partly endothelium-dependent.

Autocrine regulation of γ-irradiation-induced DNA damage response via extracellular nucleotides-mediated activation of P2Y6 and P2Y12 receptors

A key component of the response to DNA damage caused by ionizing radiation is DNA repair. Release of extracellular nucleotides, such as ATP, from cells plays a role in signaling via P2 receptors. We show here that release of ATP, followed by activation of P2Y receptors, is involved in the response to γ-irradiation-induced DNA damage. Formation of phosphorylated histone variant H2AX (γH2AX) foci, which are induced in nuclei by DNA damage and contribute to accumulation of DNA-repair factors, was increased at 1–3h after γ-ray irradiation (2.0Gy) of human lung cancer A549 cells. Focus formation was suppressed by pre-treatment with the ecto-nucleotidase apyrase. Pre-treatment with ecto-nucleotidase inhibitor ARL67156 or post-treatment with ATP or UTP facilitated induction of γH2AX, indicating that extracellular nucleotides play a role in induction of γH2AX foci. Next, we examined the effect of P2 receptor inhibitors on activation of ataxia telangiectasia mutated (ATM; a protein kinase) and accumulation of 53BP1 (a DNA repair factor), both of which are important for DNA repair, at DNA damage sites. P2Y6 receptor antagonist MRS2578, P2Y12 receptor antagonist clopidogrel, and P2X7 receptor antagonists A438079 and oxATP significantly inhibited these processes. Release of ATP was detected within 2.5min after irradiation, but was blocked by A438079. Activation of ATM and accumulation of 53BP1 were decreased in P2Y6 or P2Y12 receptor-knockdown cells. We conclude that autocrine/paracrine signaling through P2X7-dependent ATP release and activation of P2Y6 and P2Y12 receptors serves to amplify the cellular response to DNA damage caused by γ-irradiation.

Opposing effects of ATP and adenosine on barrier function of rat coronary microvasculature

ATP can differentially affect the micro- and macrovascular endothelial barrier. It has been shown that it can both increase and/or decrease macromolecule permeability of microvascular endothelial cells and microvessels, in vivo. We hypothesised that the barrier stabilising effect is mediated by ATP itself via P2 receptors, while barrier-disrupting effect is mediated by its metabolite adenosine via adenosine receptors. The effects of ATP, ADP, AMP and adenosine on barrier function were studied in cultured rat coronary microvascular endothelial monolayers (RCEC) in vitro, as well as in rat mesentery vessels, and in rat hearts in vivo. ATP and ADP showed a biphasic effect on permeability of RCEC monolayers with a reduction followed by a later increase in albumin permeability. The permeability decreasing effect of ATP was enhanced by ecto-nucleotidase inhibitor ARL67156 while permeability increasing effect was enhanced by apyrase, an extracellular ecto-nucleotidase. Moreover, the permeability increasing effect was abrogated by adenosine receptor antagonists, 8-phenyltheophylline (8-PT) and DMPX. Adenosine and adenosine receptor agonists 5'-(N-ethylcarboxamido)-adenosine (NECA), CGS21680, and R-PIA enhanced albumin permeability which was antagonised by 8-PT, A(1), and A(2) but not by A(3) receptor antagonists. Likewise, immunofluorescence microscopy of VE-cadherin and actin showed that NECA induces a disturbance of intercellular junctions. Pre-incubation of ATP antagonised the effects of NECA on permeability, actin cytoskeleton and intercellular junctions. Similar effects of the applied substances were observed in rat mesentery artery by determining the vascular leakage using intravital microscopy as well as in rat hearts by assessing myocardial water contents in vivo. In conclusion, the study demonstrates that in RCEC, ATP, ADP, and its metabolite adenosine play opposing roles on endothelial barrier function.

Glial-derived adenosine modulates spinal motor networks in mice

The activation of purinergic receptors modulates central pattern generators controlling rhythmic motor behaviors, including respiration in rodents and swimming in frog tadpoles. The present study aimed to determine whether purinergic signaling also modulates the mammalian locomotor central pattern generator. This was investigated by using isolated spinal cord preparations obtained from neonatal mice in which locomotor-related activity can be induced pharmacologically. The application of either ATP or adenosine led to a reduction in the frequency of locomotor activity recorded from ventral roots. ATP had no effect when applied in the presence of both the adenosine receptor antagonist theophylline and the ectonucleotidase inhibitor ARL67156, demonstrating that the effects of ATP application result from the breakdown of ATP to adenosine and subsequent activation of adenosine receptors. The application of theophylline or the A1-specific antagonist cyclopentyl dipropylxanthine, but not the A2A-receptor antagonist SCH58261, caused an increase in locomotor burst frequency, demonstrating that endogenously derived adenosine activates A1 receptors during locomotor network activity. Furthermore, theophylline had no effect in the presence of the ectonucleotidase inhibitor ARL67156 or the glial toxins methionine sulfoximine or ethyl fluoracetate, suggesting that endogenous adenosine is derived from ATP, which is released from glia. Finally, adenosine had no effect on slow rhythmic activity recorded upon blockade of all inhibitory transmission, suggesting that adenosine may act via the modulation of inhibitory transmission. Together, these data highlight endogenous purinergic gliotransmission, involving activation of A1 receptors, as an important intrinsic modulatory system controlling the frequency of activity generated by spinal locomotor circuitry in mammals.

Astrocyte cultures exhibit P2X7 receptor channel opening in the absence of exogenous ligands

P2X7 receptors (P2X7Rs) gate the opening of large channels when activated by ATP or other ligands. P2X7Rs are expressed by astrocytes in culture and by reactive astrocytes in vivo, and astrocytes in culture have been shown to release glutamate and ATP in response to P2X7R activation. However, P2X7Rs are activated by ATP only at concentrations greater than 1 mM. The conditions under which astrocyte P2X7Rs would be activated in vivo are, thus, unclear. Here we show that astrocytes in culture exhibit basal P2X7R activity. Primary mouse astrocytes were found to take up the P2X7R permeant dyes YO-PRO-1 (YP) and propidium iodide in absence of any added ligands. By contrast, cultured rat astrocytes took up very little YP, consistent with their much lower level of P2X7R expression. The uptake by mouse astrocytes was inhibited by oxATP, suramin, KN-62 and brilliant blue G, and by siRNA knock-down of P2X7R. Astrocyte uptake of YP was also inhibited by phenol red at concentrations above 50 muM, suggesting that phenol red present in standard cell culture media may influence P2X7R channel activity. Treatment with apyrase, an enzyme that degrades extracellular ATP, partially decreased YP uptake in astrocytes. Conversely, exposure to the ectonucleotidase inhibitor ARL67156 enhanced YP uptake and astrocytes plated without contiguous neighboring astrocytes showed reduced basal YP uptake. These results suggest that the basal uptake of YP may be due to activation of P2X7R by release of ATP by astrocytes themselves into intercellular spaces.

Changes in the control of gastric motor activity during metamorphosis in the amphibian Xenopus laevis, with special emphasis on purinergic mechanisms

The stomach of the amphibian Xenopus laevis is subject to extensive remodelling during metamorphosis. We investigated the changes in gastric activity control during this period using in vitro circular smooth muscle preparations mounted in organ baths. The nitric oxide synthase inhibitor L-NAME increased mean force in metamorphic and juvenile frogs but not in prometamorphic tadpoles. Serotonin (5-HT) relaxed stomach muscle prior to metamorphosis but elicited a biphasic response in juveniles consisting of contraction at low concentrations and relaxation at high concentrations. The effects of 5-HT were blocked by methysergide. In the prometamorphic tadpole, ATP elicited relaxation that was blocked by the ectonucleotidase inhibitor ARL67156 and the adenosine A(1) receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), suggesting adenosine as the mediator. Exogenous adenosine and the A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) induced relaxation at all stages. After metamorphosis, the potency of ATP decreased and neither DPCPX nor ARL67156 could block ATP-induced relaxation. Uridine 5'-triphosphate (UTP) induced relaxation prior to metamorphosis, but caused contraction of muscle strips from metamorphosing tadpoles. Single doses of UTP blocked phasic contractions in juveniles in a tetrodotoxin (TTX)-sensitive manner while the simultaneous increase in muscle tension was TTX insensitive. The P2X(1)/P2X(3) receptor agonist alpha-beta-MeATP elicited pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS)-sensitive contractions at all stages investigated. These results indicate the development of an inhibitory nitrergic tonus during metamorphosis and a 5-HT receptor involved in muscle contraction. Also, the development of UTP receptors mediating increased tension and neural UTP receptors decreasing contraction frequency in juveniles is indicated. An adenosine A(1)-like receptor mediating relaxation and a P2X-like receptor mediating contraction is demonstrated at all stages.

Inhibitory purinergic P2 receptor characterisation in rat distal colon

The aim of this study was to characterise the P2 receptors involved in purinergic relaxant responses in rat distal colon circular muscle. Concentration–response curves for purinergic agonists were constructed on methacholine-precontracted circular muscle strips of rat distal colon in the absence and presence of the nerve blocker TTX and the ecto-nucleotidase inhibitor ARL67156. The effects of the P2 receptor antagonists RB2, PPADS, suramin, MRS2179 and NF279, the NO-synthase inhibitor L-NAME and the small conductance K + channel blocker apamin were investigated. The localisation of the different P2 receptors was examined immunocytochemically. Immunocytochemistry demonstrated the expression of P2Y 1, P2Y 6 and P2X 1 receptors on smooth muscle cells and P2Y 2, P2Y 12, P2X 2 and P2X 3 receptors in the myenteric plexus; almost a quarter of the P2Y 2-immunopositive neurons co-expressed nNOS. The P2X-selective agonist αβmeATP and the P2Y-selective agonist ADPβS were the most potent relaxants; their effects were abolished by apamin. The effect of ADPβS was antagonised by the P2Y 1-selective antagonist MRS2179 pointing to interaction with the muscular P2Y 1-receptors. The relaxant effect of αβmeATP was partially reduced by TTX and concentration-dependently antagonised by PPADS, suramin, RB2 and the P2X 1-selective antagonist NF279; this correlates with an interaction with neuronal P2X 3 and muscular P2X 1 receptors. UTP was the least potent agonist; its effect was markedly increased by ARL67156, nearly abolished by TTX and reduced by L-NAME. This points to interaction with the neuronal P2Y 2-receptors inducing relaxation, at least partially, by NO release.

ATP Release through Connexin Hemichannels in Corneal Endothelial Cells

Intercellular Ca(2+) wave propagation is a distinct form of cell-cell communication. In corneal endothelial cells, intercellular Ca(2+) wave propagation evoked by a point mechanical stimulus (PMS) is partially mediated by adenosine triphosphate (ATP) release and subsequent activation of P2Y receptors. This study was conducted to investigate the possibility that extrajunctional connexons (hemichannels) play a role in ATP release during PMS-induced Ca(2+) wave propagation in bovine corneal endothelial cells (BCECs). A Ca(2+) wave was evoked by a PMS applied to a single cell in a monolayer of cultured BCECs. Changes in [Ca(2+)](i) in the mechanically stimulated cell (MS cell) and in the neighboring (NB) cells were visualized by fluorescence imaging using the Ca(2+)-sensitive dye Fluo-4. From these images, the maximum normalized fluorescence (NF), the percentage of responsive cells (%RC), and the total area of cells reached by the Ca(2+) wave (active area [AA], in square micrometers) were calculated. Intercellular dye transfer, generally attributed to gap junctional coupling, was assessed by fluorescence recovery after photobleaching (FRAP) using 6-carboxyfluorescein diacetate. Opening of hemichannels was investigated by measuring cellular uptake of the fluorescent dye Lucifer yellow, which is known to permeate hemichannels. ATP release was measured by luciferin-luciferase bioluminescence. Flufenamic acid (FFA; 50 microM) and the connexin mimetic peptide Gap26 (300 microM), known blockers of hemichannels, significantly reduced AA in confluent monolayers as well as in contact-free cells. Neither FFA nor Gap26 affected the FRAP, indicating that reduction in AA of the PMS-induced wave by these agents is not due to a block of gap junction channels. FFA as well as Gap26 inhibited the increase in AA of the wave that was observed when cells were pretreated with the ectonucleotidase inhibitor ARL-67156 (100 microM). These findings suggest that the hemichannel blockers reduce the Ca(2+) wave propagation by inhibiting ATP release. Consistent with this finding, PMS or exposure to Ca(2+)-free solution (a maneuver known to induce the opening of hemichannels) led to ATP release; moreover, the release was inhibited by the hemichannel blockers. The extracellular ATP levels in response to both PMS and extracellular Ca(2+) removal were strongly enhanced by ARL-67156, and this effect was inhibited by FFA as well as by Gap26. Moreover, pretreatment of subconfluent BCEC monolayers with FFA or Gap26 inhibited the uptake of Lucifer yellow induced by removal of extracellular Ca(2+). Hemichannels contribute to ATP release on mechanical stimulation in BCECs. The released ATP contributes to propagation of the Ca(2+) wave.

Extracellular ATP facilitates flow-induced vasodilatation in rat small mesenteric arteries.

ATP can be released from endothelial cells, and this release is increased by intraluminal flow in blood vessels. In the present study, the effect of extracellular ATP (1 microM) on flow-induced vasodilatation was investigated in isolated and pressurized rat small mesenteric arteries. In the absence of extracellular ATP, only 46% of arteries developed dilatation in response to flow, and this response was both transient and unstable. In marked contrast, with ATP present, all vessels developed a prolonged and stable dilatation in response to flow. Even in the vessels that failed to respond to flow in the absence of ATP, dilatation could be stimulated once ATP was present. The ability of ATP to facilitate flow-induced vasodilatation was mimicked by UTP (1 microM), a P2Y agonist, or 3'-O-(4-benzoyl)benzoyl ATP (BzATP; 10 microM), an agonist for P2X1, P2X7, and P2Y11 purinoceptors. The involvement of P2X7 purinoceptors was further supported by the inhibitory effect of KN-62 (1 microM), a P2X7 antagonist, on the action of BzATP. P2X1 and P2X3 purinoceptors were not involved because their receptor agonist alpha,beta-methylene ATP had no effect. The facilitating effect of ATP on flow dilatation was also attenuated by the combined application of reactive blue 2 (100 microM), a P2Y antagonist, and suramin (100 microM), a nonselective P2X and P2Y antagonist. Furthermore, flow-induced dilatation obtained in the presence of ATP was reproducible. In contrast, in the additional presence of the ectonucleotidase inhibitor ARL-67156 (10 microM), although the first dilatation was normal, the responses to the second and later exposures to flow were greatly attenuated. The nonhydrolyzable ATP analogs adenosine-5'-(3-thiotriphosphate)trilithium salt (1 microM) and adenosine 5'-(beta,gamma-imido) triphosphate tetralithium salt hydrate (10 microM) had similar effects to those of ARL-67156. These data suggest that ATP acts through both P2X and P2Y purinoceptors to facilitate flow-induced vasodilatation and that ectonucleotidases prevent this effect by degrading ATP on the endothelial cell surface.