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Effects of diadenosine polyphosphates (ApnAs) and adenosine polyphospho guanosines (ApnGs) on rat mesenteric artery P2X receptor ion channels
British Journal of Pharmacology
Abstract
Diadenosine polyphosphates (ApnAs, n=3–7) and adenosine polyphospho guanosines (ApnGs, n=3–6) are naturally occurring vasoconstrictor substances found in platelets. These vasoconstrictor actions are thought to be mediated through the activation of P2X receptors for ATP. The effects of ApnAs and ApnGs at P2X receptors on rat mesenteric arteries were determined in contraction studies and using the patch clamp technique on acutely dissociated artery smooth muscle cells.
P2X1 receptor immunoreactivity was detected in the smooth muscle layer of artery rings. The sensitivity to α,β-methylene ATP and desensitizing nature of rat mesenteric artery P2X receptors correspond closely to those of recombinant P2X1 receptors.
Ap4A, Ap5A and Ap6A evoked concentration dependent P2X receptor inward currents which desensitized during the application of higher concentrations of agonist. The agonist order of potency was Ap5AAp6AAp4A>>Ap3A. Ap2A and Ap7A were ineffective. Similar results were obtained in contraction studies except for Ap7A which evoked a substantial contraction.
ApnGs (n=2–6)(30 μM) evoked P2X receptor inward currents in mesenteric artery smooth muscle cells. ApnGs (n=4–6) were less effective than the corresponding ApnA.
This study shows that at physiologically relevant concentrations ApnAs and ApnGs can mediate contraction of rat mesenteric arteries through the activation of P2X1-like receptors. However the activity of the longer chain polyphosphates (n=6–7) may be overestimated in whole tissue studies due to metabolic breakdown to yield the P2X receptor agonists ATP and adenosine tetraphosphate.
British Journal of Pharmacology (2000) 129, 124–130; doi:10.1038/sj.bjp.0702993
... Other than ATP, P2X1 receptors are also stimulated by several related compounds that have been shown to be released into the bloodstream. These include a number of diadenosine polyphosphates (Ap n As) and adenosine polyphosphoguanosines (Ap n Gs) [44,[143][144][145][146][147][148] that are stored and secreted from platelet dense granules. Of these compounds, Ap 4 A and Ap 5 A have received most attention as they are stored at the highest concentrations, although at levels many fold lower than ATP [149][150][151]. ...
... Of these compounds, Ap 4 A and Ap 5 A have received most attention as they are stored at the highest concentrations, although at levels many fold lower than ATP [149][150][151]. Furthermore, most Ap n As and Ap n Gs active at P2X1 receptors in rat mesenteric arteries are partial agonists and 10-fold-less potent than α,β-meATP or ATP [146,152]. Since diadenosine polyphosphates are less rapidly degraded compared to ATP [153,154], they may act as longer range P2X1 receptor stimuli. Uridine adenosine tetraphosphate (Up 4 A) and adenosine tetraphosphate (Ap 4 ) are released from endothelial cells in response to agonists or mechanical shear and reach plasma concentrations that induce potent P2X1-dependent vasoconstriction in a rat perfused kidney model [155,156]. ...
... More work is required to confirm the true potency of these various compounds compared to ATP on platelet P2X1 receptors. One study reports an EC 50 of 0.1 μM for Ap 4 A at platelet P2X1 receptors [157], however this is much lower than concentrations reported previously to be effective for this receptor in human platelets (EC 50 23.5 μM) [44,111] or rat mesenteric smooth muscle (no maximal response, but ≈50 μM required for 50% of the response to 10 μM αβ-meATP [146]). Although earlier data suggested that ADP is a weak agonist of wild-type P2X1 receptors [28,31,91,92,147] or a preferential agonist at a naturally occurring shortened variant [158], it is now firmly established that ADP has no agonist activity at native or recombinant P2X1 receptors [64,94]. ...
Extracellular nucleotides are ubiquitous signalling molecules, acting via the P2 class of surface receptors. Platelets express three P2 receptor subtypes, ADP-dependent P2Y1 and P2Y12 G-protein-coupled receptors and the ATP-gated P2X1 non-selective cation channel. Platelet P2X1 receptors can generate significant increases in intracellular Ca(2+), leading to shape change, movement of secretory granules and low levels of α(IIb)β(3) integrin activation. P2X1 can also synergise with several other receptors to amplify signalling and functional events in the platelet. In particular, activation of P2X1 receptors by ATP released from dense granules amplifies the aggregation responses to low levels of the major agonists, collagen and thrombin. In vivo studies using transgenic murine models show that P2X1 receptors amplify localised thrombosis following damage of small arteries and arterioles and also contribute to thromboembolism induced by intravenous co-injection of collagen and adrenaline. In vitro, under flow conditions, P2X1 receptors contribute more to aggregate formation on collagen-coated surfaces as the shear rate is increased, which may explain their greater contribution to localised thrombosis in arterioles compared to venules within in vivo models. Since shear increases substantially near sites of stenosis, anti-P2X1 therapy represents a potential means of reducing thrombotic events at atherosclerotic plaques.
... Once at the extracellular space, this nucleotide can activate both ionotropic P2X and metabotropic P2Y receptors. In this sense, the effect of Ap 4 on the vascular system is to reduce blood pressure by means of the activation of a P2Y receptor; however, in stress situations, such as in an hemorrhage, it produces vasoconstriction by the activation of smooth muscle P2X receptors (Lee et al., 1995b;Lewis et al., 2000). Very recently, Ap 4 has been identified in human myocardial tissue. ...
... Other locations where Ap 4 stimulates P2X receptors are vas deferens, vascular smooth muscle, and mesenteric artery (Bailey and Hourani, 1995b;Lee et al., 1995a;Lewis et al., 2000). In these models, adenosine 5Ј-tetraphosphate produces vasoconstriction, with this nucleotide being especially active on P2X 1 receptors (Lewis et al., 2000). ...
... Other locations where Ap 4 stimulates P2X receptors are vas deferens, vascular smooth muscle, and mesenteric artery (Bailey and Hourani, 1995b;Lee et al., 1995a;Lewis et al., 2000). In these models, adenosine 5Ј-tetraphosphate produces vasoconstriction, with this nucleotide being especially active on P2X 1 receptors (Lewis et al., 2000). It is premature to elucidate which subunits form the P2X receptor involved in the reduction of intraocular pressure, according to the preliminary pharmacological studies presented in this work. ...
Adenosine 5' tetraphosphate, Ap(4), is a natural nucleotide present in many biological systems. This nucleotide has been found as a constituent of the nucleotide pool present in the aqueous humor of New Zealand rabbits. HPLC analysis confirmed its identity and calculated its concentration levels to be 197 +/- 21 nM. When applied topically to the rabbit eyes, this mononucleotide produced a reduction in the intraocular pressure, which was dose-dependent. The pD(2) value calculated from the dose-response curve was 7.28 +/- 0.47, which is equivalent to 52.48 nM. The time course of such intraocular pressure reduction presented a maximal decrease of IOP to 75.1 +/- 2.3% compared with the vehicle control value (100%), and the effect lasted for more than 2 h. Cross-desensitization studies demonstrated that Ap(4) effect was mediated via a P2X receptor in this system. P2 receptor antagonists suramin, pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS), and reactive blue 2 (RB-2) showed that only the latter was able to revert the effect of Ap(4). Antagonists of adrenoceptors and cholinoceptors were able to partially reverse the effect of this nucleotide; this might indicate a connection with the neural mechanisms that control the intraocular pressure.
... In contrast to Ap n As, the Ap n Gs and Gp n Gs have only been investigated in a few studies. In one study recently published by Lewis et al. (2000), the receptor-mediated vascular actions of Ap n Gs have been investigated in mesenteric arteries. However Ap n Gs and Gp n Gs have not been characterized so far with respect to their eects on kidney vasculature. ...
... Furthermore, most of the vasoconstrictive eects of Ap 4 G, Ap 5 G and Ap 6 G, which have never been investigated before with respect to their receptor activation in the rat kidney, appear to be mediated by P 2X -receptors as evidenced by the inhibition by both suramin and PPADS. It has been shown recently that Ap n G activate P 2X -receptors in isolated mesenteric arteries (Lewis et al., 2000). Gp 4 G, Gp 5 G and Gp 6 G only show very weak vasoconstriction at high dose applications. ...
The activation of P2x-receptors in the rat renal vasculature by dinucleoside polyphosphates with variable phosphate group chain length (XpnX; X=Adenin (A) /Guanin (G), n=4 – 6) was studied by measuring their effects on perfusion pressure of the isolated perfused rat kidney at constant flow in an open circuit.
Like Ap4A, Ap5A and Ap6A the dinucleoside polyphosphates Ap4G, Ap5G and Ap6G exerted a vasoconstriction which could be blocked by suramin and pyridoxal-phosphate-6-azophenyl-2; 4-disulphonic acid (PPADS).
Gp4G, Gp5G and Gp6G showed only very weak vasoconstriction at high doses.
Ap6A and α, β-meATP could not be blocked by the selective P2x1-receptor antagonisten NF023 (30 μM), whereas Ap4A, Ap4G, Ap5A, Ap5G and Ap6G were partially blocked by NF023.
Inhibition of endothelial NO-synthase by Nω-nitro-L-arginine methyl ester (L-NAME) did not affect vasoconstrictions induced by dinucleosidepolyphosphates.
P2x-receptor can only be activated if at least one adenosine moiety is present in the molecule.
ApnG show a weaker vasoconstrictive action than corresponding ApnA, concluding that two adenosine moieties enhance the P2x-receptor binding and activation.
XpnX containing five phosphate groups show the most pronounced vasoconstrictive effect whereas four phosphate groups show the less effect, therefore the number of phosphate groups critically changes receptor affinity.
Additional experiments using permanent perfusion with α, β-methylene ATP (α,β-meATP) and the selective P2x1-receptor antagonist NF023 showed that the newly discovered human dinucleoside polyphosphates activated the vascular P2x1-receptor and an recently identified new P2x-receptor subtype.
The differential effects of dinucleoside polyphosphates allow a fine tuning of local perfusion via composition of XpnXs.
British Journal of Pharmacology (2001) 132, 467–474; doi:10.1038/sj.bjp.0703817
... For instance, the decreasing effect described for Ap 4 on blood pressure is mediated by P2Y receptors present in endothelial cells, but under certain conditions such as haemorrhage, Ap 4 produces vasoconstriction probably by acting through smooth muscle P2X receptors, being more potent than ATP on this receptor (Lee et al., 1995b). Similar effects have been described in the vas deferens (Taylor et al., 1983;Bailey and Hourani, 1995) and in the mesenteric artery (Lewis et al., 2000), where Ap 4 can also stimulate P2X 1 receptors showing a higher potency than ATP in this respect. ...
... Moreover, the experiments performed with the antagonists showed that both Ap 4 and ATP responses were antagonised by the same antagonists and with similar potencies. These results suggest that Ap 4 was acting on a P2X receptor and would be consistent with the results obtained for this nucleotide on a P2X receptor present in the vas deferens (Bailey and Hourani, 1995), in vascular smooth muscle (Lee et al., 1995b) and in mesententeric artery (Lewis et al., 2000). In addition, responses were blocked with relatively high potency by PPADS, a P2 antagonist (Windscheif et al., 1994). ...
The aim of this study was to see whether the compound adenosine 5'-tetraphosphate (Ap(4)) is active in the central nervous system by examining its effect on isolated rat brain synaptic terminals. Ap(4) proved to be more resistant to ecto-enzymatic hydrolysis than adenosine triphosphate (ATP), showing only 2% hydrolysis after a 2-min incubation, compared to 75% for ATP. In addition, Ap(4) was able to produce concentration-dependent increases in intracellular Ca(2+) when applied extracellularly. This action was dependent upon the presence of extracellular calcium. Ap(4) acts through ionotropic ATP receptors (P2X receptors) and not through diadenosine polyphosphate receptors, since ATP abolished the response elicited by Ap(4) whereas Ap(5)A did not. Ap(4), ATP and ATP-gamma-S were of similar potency (EC(50) approximately 20 microM) while 2MeSATP, alpha,beta-meATP and ADP-beta-S possessed slightly lower potency (EC(50) approximately 50 microM). The P2-purinoceptor antagonists suramin and PPADS blocked the Ap(4) effect. The IC(50) values for these compounds were 35.5 and 7.8 microM respectively. Diinosine polyphosphates and inosine tetraphosphate inhibited the response elicited by Ap(4) with IC(50) values that varied between approximately 40 and 50 microM. These results show that Ap(4) is as good an agonist as ATP on synaptosomal P2X receptors, being more resistant to extracellular hydrolysis by ecto-nucleotidases.
... Up 4 A is the first dinucleotide containing one purine and one pyrimidine moiety found in living organisms (Jankowski et al., 2005). The potency and type of response of the dinucleotides are largely determined by the number of phosphates in the polyphosphate chain (Lewis et al., 1994;Lewis et al., 2000;Ralevic et al., 1995;Ralevic et al., 2001). In general, dinucleotides containing a phosphate chain of 2-3 compounds elicit vasorelaxation, whereas those containing a longer chain of 4 to 6 elicit vasoconstriction followed by vasodilation. ...
... In rat aortic smooth muscle cells, the presence of P2X1, P2X2, P2X4, P2Y2 and P2Y6 have been shown [for review: (Kunapuli and Daniel, 1998)]. In addition, a study reported that purine dinucleotides containing 4 to 6 phosphates in the polyphosphate chain cause contraction mediated by P2X1 receptor present on the smooth muscle (Lewis et al., 2000). Based on these data, our results indicate that dinucleotides containing both purine and pyrimidine moieties and a long polyphosphate chain induce contraction by partially activating P2X1 receptors on the smooth muscle. ...
Uridine adenosine tetraphosphate (Up(4)A) has been recently reported as an endothelium-derived vasoconstrictor and plasma levels of this dinucleotide are increased in juvenile hypertensive subjects. This study aimed to evaluate the vascular actions of Up(4)A, typify the putative purinergic receptors that might mediate these effects and characterize the intracellular signaling pathways that may govern Up(4)A responses. Up(4)A induced a modest endothelium-dependent relaxation of rat aortic rings contracted with phenylephrine. From baseline, Up(4)A induced concentration-dependent contractions that were significantly potentiated by endothelium removal or nitric oxide synthase inhibition. The contractile response induced by Up(4)A was not tachyphylactic and was significantly reduced in the presence of P1 or P2X receptor antagonists, L-type Ca(2+) channel blocker and Rho-kinase inhibitor. Up(4)A-induced contraction apparently involves superoxide anion formation since it was significantly reduced by treatment with apocynin or tempol. This study presents the unique findings that the endogenous compound Up(4)A is able to induce relaxation in addition to contraction of rat aorta. Up(4)A-induced contraction is modulated by nitric oxide production, mediated by P1 and P2X receptor activation, and involves L-type Ca(2+) channels, Rho-kinase pathway and superoxide formation.
... natural agonist, diadenosine pentaphosphate (Ap5A), is stored and released from platelets 17 and acting at P2X1Rs mediates arterial contraction 18,19 . The ATP analogue α ,β -methylene ATP (α β meATP) also has agonist activity at P2X1Rs. ...
Structural studies of P2X receptors show a novel U shaped ATP orientation following binding. We used voltage clamp fluorometry (VCF) and molecular dynamics (MD) simulations to investigate agonist action. For VCF the P2X1 receptor (P2X1R) K190C mutant (adjacent to the agonist binding pocket) was labelled with the fluorophore MTS-TAMRA and changes in fluorescence on agonist treatment provided a real time measure of conformational changes. Studies with heteromeric channels incorporating a key lysine mutation (K68A) in the ATP binding site demonstrate that normally three molecules of ATP activate the receptor. The time-course of VCF responses to ATP, 2′-deoxy ATP, 3′-deoxy ATP, Ap5A and αβmeATP were agonist dependent. Comparing the properties of the deoxy forms of ATP demonstrated the importance of the 2′ hydroxyl group on the ribose ring in determining agonist efficacy consistent with MD simulations showing that it forms a hydrogen bond with the γ-phosphate oxygen stabilizing the U-shaped conformation. Comparison of the recovery of fluorescence on agonist washout, with channel activation to a second agonist application for the partial agonists Ap5A and αβmeATP, showed a complex relationship between conformational change and desensitization. These results highlight that different agonists induce distinct conformational changes, kinetics and recovery from desensitization at P2X1Rs.
... While intracellular Ap(n)As are proposed as inhibitory ligands of the ATP-sensitive K+ channels (Jovanovic et al. 1996;, extracellular Ap(n)As are considered as agonists of purine receptors (Laubinger et al. 2003;Lewis et al. 2000;Nahum et al. 2006). Earlier studies provided the data supporting P1 or P2 purine receptors as a main target for extracellular Ap(n)As in various tissues including myocardium (Arvola et al. 2004;Brandts et al. 1998;2003;Conant et al. 2000;McDonald et al. 2002;Neumann et al. 1999;Vahlensieck et al. 1999). ...
Diadenosine polyphosphates are now considered a novel class of endogenous paracrine signal compounds. The putative role of these compounds in pathogenesis of myocardial infarction was proposed, since the concentration of diadenosine polyphosphates increases in the cardiac tissue following the ischemic lesion and myocardial necrosis. Therefore, possible effects of diadenosine polyphosphates on cardiac electrical activity and their ionic mechanisms are of considerable interest.
In the present study, we have investigated the effects of diadenosine pentaphosphate (Ap5A), diadenosine tetraphosphate (Ap4A), and NAD+ on transmembrane currents belonging to the family of potassium inward rectifiers: background inward rectifier (IKI), ATP-dependent potassium current (IKATP), and acetylcholinedependent current (IKACh). Experiments were performed using the whole-cell patch-clamp technique on isolated atrial and ventricular rat cardiomyocytes. We have demonstrated that none of the tested adenine compounds affects IKI and IKACh. Ap5A (10–5 M) induces considerable decrease of both inward and outward component of IKATP by 22.1 and 19% of control value, respectively. Higher concentration of Ap5A (3 × 10–5 M) induces stronger suppression of IKATP—by 47.5% and 37.8%, respectively. However, IKATP was found to be insensitive to Ap4A and NAD+.
... While intracellular Ap(n)As are proposed as inhibitory ligands of the ATP-sensitive K+ channels (Jovanovic et al. 1996;, extracellular Ap(n)As are considered as agonists of purine receptors (Laubinger et al. 2003;Lewis et al. 2000;Nahum et al. 2006). Earlier studies provided the data supporting P1 or P2 purine receptors as a main target for extracellular Ap(n)As in various tissues including myocardium (Arvola et al. 2004;Brandts et al. 1998;2003;Conant et al. 2000;McDonald et al. 2002;Neumann et al. 1999;Vahlensieck et al. 1999). ...
Diadenosine polyphosphates (Ap(n)As) are endogenously produced molecules which have been identified in various tissues of mammalian organism, including myocardium. Ap(n)As contribute to the blood clotting and are also widely accepted as regulators of blood vascular tone. Physiological role of Ap(n)As in cardiac muscle has not been completely elucidated. The present study aimed to investigate the effects of diadenosine tetra- (Ap4A) and penta- (Ap5A) polyphosphates on contractile function and action potential (AP) waveform in rat supraventricular and ventricular myocardium. We have also demonstrated the effects of A4pA and Ap5A in myocardial sleeves of pulmonary veins (PVs), which play a crucial role in genesis of atrial fibrillation. APs were recorded with glass microelectrodes in multicellular myocardial preparations. Contractile activity was measured in isolated Langendorff-perfused rat hearts. Both Ap4A and Ap5A significantly reduced contractility of isolated Langendorff-perfused heart and produced significant reduction of AP duration in left and right auricle, interatrial septum, and especially in right ventricular wall myocardium. Ap(n)As also shortened APs in rat pulmonary veins and therefore may be considered as potential proarrhythmic factors. Cardiotropic effects of Ap4A and Ap5A were strongly antagonized by selective blockers of P2 purine receptors suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), while P1 blocker DPCPX was not effective. We conclude that Ap(n)As may be considered as new class of endogenous cardioinhibitory compounds. P2 purine receptors play the central role in mediation of Ap4A and Ap5A inhibitory effects on electrical and contractile activity in different regions of the rat heart.
... In fact, studies suggest that the potency and type of response of Ap n A are determined by the number of phosphates (n) in the polyphosphate chain (van der Giet et al. 1999, Gabriels et al. 2000, Ralevic et al. 2001. In mesenteric arteries, Ap n A (n = 2-3) act as dilators via endothelial P2Y1-like receptors, while Ap n A (n = 4-6) constrict via P2X1 like (Lewis et al. 2000). In renal vasculature, vasoconstriction predominates. ...
INTRODUCTION AND AIMS: Recently, uridine adenosine tetraphosphate (Up4A) was described as a strong vasoconstrictor released from endothelial cells
after stimulation with mechanical stress. In this study, we isolated and identified Up4A from kidney tissue, and we characterized
the essential varying effects of Up4A on the afferent and efferent arterioles.
METHODS: Porcine and human kidney tissue was fractionated by size-exclusion-chromatography affinity-chromatography, anion-exchange-chromatography,
and reverse phase-chromatography. In fractions purified to homogeneity, Up4A was identified by matrix assisted laser desorption/ionisation
mass-spectrometry (MALDI-TOF-MS), MALDI-LIFT-fragment-massspectrometry (MALDI-TOF-TOF-MS), retention-time comparison, and
enzymatic cleavage analysis. We analysed the release of Up4A from cultivated renal proximal tubule cells after stimulation
of protein kinase C with OAG. Up4A was identified in renal tissue, and the effect of Up4A on the vascular tone of isolated
perfused afferent and efferent arterioles was tested.
RESULTS: Stimulation of tubule cells with OAG increased the release-rate of Up4A from tubule cells about ten fold. Up4A acts as a
strong vasoconstrictive mediator on afferent arterioles, but has no significant effect on the tone of efferent arterioles,
suggesting a functional role of Up4A a an autocrine hormone for glomerular perfusion. Because of the predominant effect of
the Up4A on afferent arterioles, we assume that Up4A may decrease glomerular perfusion, intraglomerula pressure, and hence
glomerular filtration rate. The release of Up4A from renal tubular cells may be an additional mechanism whereby tubular cells
could affect renal perfusion. Up4A release may further contribute to renal vascular autoregulation mechanisms.
CONCLUSIONS: As Up4A occurs in renal tissue and has marked effects on afferent but not efferen arterioles, Up4A may play a role in renal
hemodynamics and blood pressure regulation.
... Therefore, the mechanism by which Ap 4 A inhibits ADP-induced platelet activation and its possible effects on P2Y 1 and P2Y 12 are unknown. Diadenosine polyphosphates are potent agonists of P2X receptors expressed on a variety of human and rat cell types [16,17]. Although human platelets express P2X 1 receptors [18][19][20], whether Ap 4 A is an agonist via platelet P2X 1 is unknown. ...
INTRODUCTION: Diadenosine 5',5'''-P(1),P(4)- tetraphosphate (Ap(4)A) is stored in platelet dense granules, but its effects on platelet function are not well understood.
METHODS AND RESULTS: We examined the effects of Ap(4)A on platelet purinergic receptors P2Y(1), P2Y(12) and P2X(1). Flow cytometry was used to measure the effects of Ap(4)A in the presence or absence of ADP on: a) P2Y(12)-mediated decrease in intraplatelet phosphorylated vasodilator stimulated phosphoprotein (VASP), b) P2Y(1)-mediated increase in platelet cytosolic Ca(2+), and c) P2X(1)-mediated intraplatelet entry of extracellular Ca(2+). ADP-stimulated platelet shape change (P2Y(1)-mediated) and aggregation (P2Y(1)- and P2Y(12)-mediated) were measured optically. Ap(4)A inhibited 3 microM ADP-induced: a) platelet aggregation (IC(50) 9.8+/-2.8 microM), b) P2Y(1)-mediated shape change, c) P2Y(1)-mediated increase in platelet cytosolic Ca(2+) (IC(50) 40.8+/-12.3 microM), and d) P2Y(12)-mediated decrease in VASP phosphorylation (IC(50)>250 microM). In the absence of added ADP, Ap(4)A had agonist effects on platelet P2X(1) and P2Y(12), but not P2Y(1), receptors.
CONCLUSION: Ap(4)A, a constituent of platelet dense granules, is a) an antagonist of platelet P2Y(1) and P2Y(12) receptors, where it inhibits the effects of ADP, and b) an agonist of platelet P2X(1) and P2Y(12) receptors.
... In fact, studies suggest that the potency and type of response of Ap n A are determined by the number of phosphates (n) in the polyphosphate chain (van der Giet et al. 1999, Gabriels et al. 2000, Ralevic et al. 2001. In mesenteric arteries, Ap n A (n = 2-3) act as dilators via endothelial P2Y1-like receptors, while Ap n A (n = 4-6) constrict via P2X1 like (Lewis et al. 2000). In renal vasculature, vasoconstriction predominates. ...
IntroductionMechanisms and participating substances involved in the reduction of glomerular filtration (GFR) in contrast-induced acute kidney injury (CI-AKI) are still matter of debate. We hypothesized that diadenosine polyphosphates are released by the action of contrast media on tubular cells and may act on glomerular arterioles and reduce GFR.Methods
Freshly isolated rat tubules were treated with the contrast medium iodixanol (47 mg iodine/ml) at 37°C for 20 min. The content of ApnA (n=3-6) in the supernatant of treated tubules and in the plasma of healthy persons and patients with AKI was analyzed by using reversed phase chromatography, affinity chromatography and mass-spectrometry. GFR was obtained in conscious mice by inulin clearance. Concentration response curves for ApnA (n=3-6, 10-12-10-5 mol/l) were measured in isolated perfused glomerular arterioles.ResultsIodixanol treatment of tubules significantly increased the concentration of ApnA (n=3-5) in the supernatant. Ap6A was below the detection limit. AKI patient show higher concentrations of ApnA compared to healthy. Application of Ap5A significantly reduced the GFR in conscious mice. Ap5A reduced afferent arteriolar diameters, but did not influence efferent arterioles. The constrictor effect on afferent arterioles was strong immediately after application, but weakened with time. Then non-selective P2 inhibitor suramin blocked the Ap5A induced constriction.Conclusion
The data suggest that Ap5A plays a role in the pathophysiology of CI-AKI. We show a contrast media induced release of Ap5A from tubules, which might increase afferent arteriolar resistance and reduce the GFR.This article is protected by copyright. All rights reserved.
... Sections were blocked for 10 min with 50 mM glycine in PBS, incubated for 10 min with immunobuffer (PBS with 50 mM glycine, 0.25% bovine serum albumin, 0.03% saponin, and 0.05% sodium azide) and labelled for 90 min with primary antibodies diluted in immunobuffer. The following primary antibodies were used, either alone or two together (rabbit+mouse): Rabbit polyclonal anti-P2X 1 (APR-001, Alomone Labs, Israel; 1:500; previously used in the following studies [6,8,[13][14][15][16][17][18][19]); mouse polyclonal anti-P2X 4 (H00005025-B01P; Abnova, Taiwan; 1:500); rabbit polyclonal anti-P2Y 1 (APR-009, Alomone Labs; 1:500; previously used in the following studies [18,[20][21][22]); polyclonal anti-P2Y 2 (APR-010, Alomone Labs; 1:500; previously used in the following studies [6,8,18,[22][23][24]); rabbit polyclonal anti-P2Y 4 (P6497, Sigma-Aldrich, Denmark; 1:250); rabbit polyclonal anti-P2Y 11 (APR-015, Alomone Labs; 1:500; previously used in the following studies [18,[25][26][27][28][29][30]); rabbit polyclonal anti-P2Y 12 (HPA013796, Sigma-Aldrich; 1:30; previously used in the following study [31]). The P2X 1 antibody was highly specific and directed against the epitope corresponding to amino acid residues 382-399 of rat P2X 1 (human 15/18 residues identical); the P2Y 11 antibody was corresponding to amino acid residues 357-373 of human P2Y 11 ; western blots for anti-P2X 1 and anti-P2Y 11 are shown on the Alomone homepage. ...
Purinergic receptors are present in most tissues and thought to be involved in various signalling pathways, including neural signalling, cell metabolism and local regulation of the microcirculation in skeletal muscles. The present study aims to determine the distribution and intracellular content of purinergic receptors in skeletal muscle fibres in patients with type 2 diabetes and age-matched controls. Muscle biopsies from vastus lateralis were obtained from six type 2 diabetic patients and seven age-matched controls. Purinergic receptors were analysed using light and confocal microscopy in immunolabelled transverse sections of muscle biopsies. The receptors P2Y(4), P2Y(11) and likely P2X(1) were present intracellularly or in the plasma membrane of muscle fibres and were thus selected for further detailed morphological analysis. P2X(1) receptors were expressed in intracellular vesicles and sarcolemma. P2Y(4) receptors were present in sarcolemma. P2Y(11) receptors were abundantly and diffusely expressed intracellularly and were more explicitly expressed in type I than in type II fibres, whereas P2X(1) and P2Y(4) showed no fibre-type specificity. Both diabetic patients and healthy controls showed similar distribution of receptors. The current study demonstrates that purinergic receptors are located intracellularly in human skeletal muscle fibres. The similar cellular localization of receptors in healthy and diabetic subjects suggests that diabetes is not associated with an altered distribution of purinergic receptors in skeletal muscle fibres. We speculate that the intracellular localization of purinergic receptors may reflect a role in regulation of muscle metabolism; further studies are nevertheless needed to determine the function of the purinergic system in skeletal muscle cells.
... Support that the region around the terminal phosphate is open at the protein surface and comes from ADP ribosylation (see above)(Adriouch et al., 2007). In addition, the ATP analogues adenosine tetraphosphate and diadenosine pentaphosphate (Ap5A) are agonists at some, but not all P2X receptors, showing that bulky substitutions, such as the addition of an ADP to ATP to make Ap5A, can be accommodated at the P2X1 receptor binding pocket (Ralevic et al., 1995; Lewis et al., 2000). These results suggest that the terminal phosphate of ATP faces out of the binding pocket, and the adenine and ribose components of ATP are bound deeper within the protein or further along the binding groove. ...
P2X receptors for ATP are ligand gated cation channels that form from the trimeric assembly of subunits with two transmembrane segments, a large extracellular ligand binding loop, and intracellular amino and carboxy termini. The receptors are expressed throughout the body, involved in functions ranging from blood clotting to inflammation, and may provide important targets for novel therapeutics. Mutagenesis based studies have been used to develop an understanding of the molecular basis of their pharmacology with the aim of developing models of the ligand binding site. A crystal structure for the zebra fish P2X4 receptor in the closed agonist unbound state has been published recently, which provides a major advance in our understanding of the receptors. This review gives an overview of mutagenesis studies that have led to the development of a model of the ATP binding site, as well as identifying residues contributing to allosteric regulation and antagonism. These studies are discussed with reference to the crystal to provide a structural interpretation of the molecular basis of drug action.
... Therefore, the mechanism by which Ap 4 A inhibits ADP-induced platelet activation and its possible effects on P2Y 1 and P2Y 12 are unknown. Diadenosine polyphosphates are potent agonists of P2X receptors expressed on a variety of human and rat cell types[16,17]. Although human platelets express P2X 1 receptors181920, whether Ap 4 A is an agonist via platelet P2X 1 is unknown. ...
Diadenosine 5',5'''-P(1),P(4)- tetraphosphate (Ap(4)A) is stored in platelet dense granules, but its effects on platelet function are not well understood.
We examined the effects of Ap(4)A on platelet purinergic receptors P2Y(1), P2Y(12) and P2X(1). Flow cytometry was used to measure the effects of Ap(4)A in the presence or absence of ADP on: a) P2Y(12)-mediated decrease in intraplatelet phosphorylated vasodilator stimulated phosphoprotein (VASP), b) P2Y(1)-mediated increase in platelet cytosolic Ca(2+), and c) P2X(1)-mediated intraplatelet entry of extracellular Ca(2+). ADP-stimulated platelet shape change (P2Y(1)-mediated) and aggregation (P2Y(1)- and P2Y(12)-mediated) were measured optically. Ap(4)A inhibited 3 microM ADP-induced: a) platelet aggregation (IC(50) 9.8+/-2.8 microM), b) P2Y(1)-mediated shape change, c) P2Y(1)-mediated increase in platelet cytosolic Ca(2+) (IC(50) 40.8+/-12.3 microM), and d) P2Y(12)-mediated decrease in VASP phosphorylation (IC(50)>250 microM). In the absence of added ADP, Ap(4)A had agonist effects on platelet P2X(1) and P2Y(12), but not P2Y(1), receptors.
Ap(4)A, a constituent of platelet dense granules, is a) an antagonist of platelet P2Y(1) and P2Y(12) receptors, where it inhibits the effects of ADP, and b) an agonist of platelet P2X(1) and P2Y(12) receptors.
... As ATP is an antagonist at platelet P2Y 1 receptors this infers a low receptor density. The diadenosine polyphosphates, Ap 4 A, Ap 5 A and Ap 6 A, are also P2X 1 receptor agonists (Sage et al., 1997; Wildman et al., 1999; Lewis et al., 2000) and are released from platelet dense granules (Flodgaard & Klenow, 1982; Jankowski et al., 1999), therefore may be important physiological activators of platelet ionotropic purinoceptors. The role of P2X 1 receptors during platelet activation following vascular injury is presently unclear. ...
ADP, an important agonist in thrombosis and haemostasis, has been reported to activate platelets via three receptors, P2X1, P2Y1 and P2TAC. Given the low potency of ADP at P2X1 receptors and recognized contamination of commercial samples of adenosine nucleotides, we have re-examined the activation of P2X1 receptors by ADP following HPLC and enzymatic purification.
Native P2X1 receptor currents in megakaryocytes were activated by α,β-meATP (10 μM) and commercial samples of ADP (10 μM), but not by purified ADP (10–100 μM).
Purified ADP (up to 1 mM) was also inactive at recombinant human P2X1 receptors expressed in Xenopus oocytes. Purification did not modify the ability of ADP to activate P2Y receptors coupled to Ca2+ mobilization in rat megakaryocytes.
In human platelets, P2X1 and P2Y receptor-mediated [Ca2+]i responses were distinguished by their different kinetics at 13°C. In 1 mM Ca2+ saline, α,β-meATP (10 μM) and commercial ADP (40 μM) activated a rapid [Ca2+]i increase (lag time 0.5 s) through the activation of P2X1 receptors. Hexokinase treatment of ADP shifted the lag time by ∼2 s, indicating loss of the P2X1 receptor-mediated response.
A revised scheme is proposed for physiological activation of P2 receptors in human platelets. ATP stimulates P2X1 receptors, whereas ADP is a selective agonist at metabotropic (P2Y1 and P2TAC) receptors.
British Journal of Pharmacology (2000) 131, 108–114; doi:10.1038/sj.bjp.0703517
... Transverse sections (12 mm) of mesenteric arteries were processed for immunochemistry as described previously (Lewis et al., 2000) using the following P2X receptor subtype selective antibodies; anti-P2X 1 , anti-P2X 2 , anti-P2X 4 and anti-P2X 7 +control antigen blocking peptide (Alomone Lab. Israel); anti-P2X 3 (gift from Prof Elde and Dr Vulchanova, University of Minnesota, U.S.A.), anti-P2X 5 and anti- P2X 6 +control antigen blocking peptide (gift from Roche Bioscience, Palo Alto, U.S.A.). ...
Immunoreactivity for P2X1, P2X4 and P2X5 receptor subtypes was detected in the smooth muscle cell layer of second and third order rat mesenteric arteries immunoreactivity, for P2X2, P2X3, P2X6 and P2X7 receptors was below the level of detection in the smooth muscle layer.
P2X receptor-mediated currents were recorded in patch clamp studies on acutely dissociated mesenteric artery smooth muscle cells. Purinergic agonists evoked transient inward currents that decayed rapidly in the continued presence of agonist (τ∼200 ms). Standard whole cell responses to repeated applications of agonist at 5 min intervals ran down. Run-down was unaffected by changes in extracellular calcium concentration, intracellular calcium buffering or the inclusion of ATP and GTP in the pipette solution.
Run-down was overcome and reproducible responses to purinergic agonists were recorded using the amphotericin permeabilized patch recording configuration.
The rank order of potency at the P2X receptor was ATP=2 methylthio ATP>α,β-methylene ATP>CTP=l-β,γ-methylene ATP. Only ATP and 2meSATP were full agonists. The P2 receptor antagonists suramin and PPADS inhibited P2X receptor-mediated currents with IC50s of 4 μM and 70 nM respectively.
These results provide further characterization of artery P2X receptors and demonstrate that the properties are dominated by a P2X1-like receptor phenotype. No evidence could be found for a phenotype corresponding to homomeric P2X4 or P2X5 receptors or to heteromeric P2X1/5 receptors and the functional role of these receptors in arteries remains unclear.
British Journal of Pharmacology (2000) 131, 1659–1666; doi:10.1038/sj.bjp.0703744
... However, P2X 5 like immunoreactivity has not been reported in renal vasculature (Chan et al., 1998), nor has it been observed in the vascular supply of the adjacent adrenal gland (Afework and Burnstock, 1999). P2X 5 transcripts have been found in rat mesenteric artery (Phillips and Hill, 1999), but Ap n A and Ap n G compounds do not elicit nondesensitizing responses in this arterial preparation (Lewis et al., 2000). Thus, it is unlikely that P2X 1/5 receptors could account for the sustained agonism by these dinucleotides in renal vasculature. ...
We have investigated the activity of naturally occurring high-performance liquid chromatography-purified diadenosine polyphosphates (Ap(n)A, n = 5-6), adenosine polyphospho guanosines (Ap(n)G, n = 5-6), and diguanosine polyphosphates (Gp(n)G, n = 5-6) under voltage-clamp conditions at recombinant rat P2X1-4 purinoceptor subtypes expressed in Xenopus laevis oocytes. At rP2X1 and rP2X3 receptors, Ap(n)As and Ap(n)Gs evoked concentration-dependent inward currents. Gp(n)Gs were not active at these receptors. At rP2X2 and rP2X4 receptors, dinucleotides did not show significant activity. For the rP2X1 receptor, Ap(n)As and Ap(n)Gs were partial agonists; for the P2X3 receptor, only Ap5G was full agonist, whereas the other tested substances were partial agonists. The rank order of potency at rP2X1 was ATP > or = Ap6A > or = Ap5A > or = Ap6G > or = Ap5G, and rank order of efficacy was ATP > or = Ap5A > or = Ap6A > Ap5G > Ap6G, whereas at rP2X3 the rank order of potency was ATP > Ap5G > or = Ap5A > or = Ap6A > or = Ap6G and the rank order of efficacy was ATP approximately Ap5G > or = Ap5A approximately Ap6A > or = Ap6G. For rP2X1 and rP2X3 it is evident that receptor agonism depended on the presence of at least one adenine moiety in the dinucleotide, while the presence of a guanine moiety had a significant impact and decreased agonist efficacy. The data suggest that naturally occurring Ap(n)As and Ap(n)Gs may play an important physiological role in different human tissues and systems by activating group I P2X receptors.
... These Wndings suggested that compared to the binding of the adenine ring or ribose the detection/co-ordination of the three phosphate residues plays a dominant role in determining the aYnity of drugs at the agonist binding site. It is interesting that longer chain phosphates also activate the channel with for example adenosine tetraphosphate and the diadenosine polyphosphate Ap 5 A acting as agonists (Lewis et al. 2000). This suggests that the structure of the binding site that recognises the phosphate tail of ATP is relatively open-ended and can accommodate additional substitutions i.e. in the case of Ap 5 A essentially an ADP molecule bound to the end of ATP. ...
P2X receptors for ATP comprise a distinct family of ligand gated ion channels with a range of properties. They have been shown to be involved in a variety of physiological processes including blood clotting, sensory perception, pain sensation, bone formation as well as inflammation and may provide a number of novel drug targets. In addition to the orthosteric site for ATP binding it has been suggested that there may be additional allosteric sites that regulate agonist action at the receptor. There is currently no crystal structure available for P2X receptors and the lack of sequence similarity to other ATP binding proteins has meant that a mutagenesis-based approach has been used primarily to investigate receptor structure-function. This review aims to provide an overview of recent work that gives an insight into residues involved in ATP action and allosteric regulation.
The activity of novel pharmacological agents at recombinant P2X receptors was studied to find agents that distinguish between P2X receptor subtypes, particularly P2X1 and P2X3. Adenine nucleotide derivatives and diadenosine polyphosphates (ApnA, n = 2-6) were investigated as P2X receptor agonists. PAPET and HT-AMP were agonists, to varying degrees, at P2X1-4 receptors. PAPET displayed higher affinity but lower efficacy than ATP at P2X1 and P2X3 receptors. HT-AMP showed higher affinity than ATP at P2X3 receptors yet acted as a partial agonist at P2X1-4 receptors. Diadenosine polyphosphates also showed selectivity in their actions at P2X1-4 receptors. Ap2A was inactive and Ap3-6A showed varying affinities and efficacies as agonists at P2X1-4. Ap3A was most effective at distinguishing between P2X1 and P2X3 receptors with over 100 fold difference between their respective EC50 values A series of PPADS derivatives, involving chemical manipulation of the phenylazo moiety and/or the pyridoxal phosphate moiety, showed nanomolar activity at P2X1 and P2X3 receptors with variable degrees of selectivity between these receptor subtypes. The most potent compounds were studied in detail and shown to be nonsurmountable antagonists. A comparison of like data for recombinant P2X1 receptors and native P2X1-like receptors in vas deferens revealed a number of pharmacological anomalies. Co-expression of P2X1 and P2X2 revealed a novel pH-sensitive phenotype although this heteromeric receptor is unlikely to account for the difference between rP2X1 and the native P2X subtype(s) in this tissue. A step forward has been made in the search of pharmacological agents that distinguish between P2X1 and P2X3 receptors. Antagonist-resistant ATP responses in the vas deferens lend weight for other contraction-mediating P2 receptors in this preparation. Greater diversity of purinergic signalling was revealed through co-expression of P2X receptors and underlines the need for further novel pharmacological tools.
Ligand-gated ion channels on the cell surface are directly activated by the binding of an agonist to their extracellular domain and often referred to as ionotropic receptors. P2X receptors are ligand-gated non-selective cation channels with significant permeability to Ca2+ whose principal physiological agonist is ATP. This chapter focuses on the mechanisms by which P2X1 receptors, a ubiquitously expressed member of the family of ATP-gated channels, can contribute to cellular responses in non-excitable cells. Much of the detailed information on the contribution of P2X1 to Ca2+ signalling and downstream functional events has been derived from the platelet. The underlying primary P2X1-generated signalling event in non-excitable cells is principally due to Ca2+ influx, although Na+ entry will also occur along with membrane depolarization. P2X1 receptor stimulation can lead to additional Ca2+ mobilization via a range of routes such as amplification of G-protein-coupled receptor-dependent Ca2+ responses. This chapter also considers the mechanism by which cells generate extracellular ATP for autocrine or paracrine activation of P2X1 receptors. For example cytosolic ATP efflux can result from opening of pannexin anion-permeable channels or following damage to the cell membrane. Alternatively, ATP stored in specialised secretory vesicles can undergo quantal release via the process of exocytosis. Examples of physiological or pathophysiological roles of P2X1-dependent signalling in non-excitable cells are also discussed, such as thrombosis and immune responses.
Vascular effects of diadenosine polyphosphates (ApnAs), adenosine polyphospho guanosines (ApnGs) and guanosine polyphospho guanosines (GpnGs), novel families of naturally-occurring signalling molecules, were investigated in methoxamine preconstricted rat isolated perfused mesenteric arterial beds.
Three different types of response were elicited by ApnAs and ApnGs. Those with a short polyphosphate chain (n=2 – 3) elicited vasorelaxation. Ap3A was more potent than Ap2A, and both were more potent than the corresponding ApnG. Relaxations to Ap3A and Ap3G, but not to Ap2A and Ap2G, were blocked by endothelium removal and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), a P2 receptor antagonist.
Longer polyphosphate chain ApnAs and ApnGs (n=4 – 6) elicited dose-dependent vasoconstriction followed by prolonged vasorelaxation, with a potency order for both types of response of Ap5AAp6A>Ap4A. A similar order and potency was observed for ApnGs. Contractions and prolonged relaxations were blocked by PPADS and P2X1 receptor desensitization with α,β-methylene ATP (α,β-meATP), and were largely endothelium-independent.
In the presence of α,β-meATP rapid relaxations to contractile ApnAs and ApnGs (n=4 – 6) were revealed.
GpnGs were virtually inactive, except for Gp2G which elicited vasoconstriction via PPADS- and α,β-meATP-sensitive smooth muscle P2X1-like receptors.
These data show that, as with ApnAs, the length of the polyphosphate chain (n) is an important determinant of the activity of ApnGs at P2 receptors in the rat mesenteric arterial bed. When the chain is short (n=2 – 3) the purines elicit rapid vasorelaxation, which for Ap3A and Ap3G is mediated via endothelial P2Y1-like receptors. When the chain is long (n=4 – 6) ApnAs and ApnGs elicit vasoconstriction via P2X1-like receptors, followed by prolonged endothelium-independent vasorelaxation. Rapid relaxation to contractile dinucleotides (n=4 – 6) is revealed by block of vasoconstriction. Regarding the purine moiety, one adenine is crucial and sufficient for vasoactivity as GpnGs were largely inactive, and ApnAs and ApnGs approximately equipotent.
British Journal of Pharmacology (2001) 134, 1073–1083; doi:10.1038/sj.bjp.0704341
Background:
Diadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap(4)A), a natural compound stored in platelet dense granules, inhibits ADP-induced platelet aggregation. Ap(4)A inhibits the platelet ADP receptors P2Y(1) and P2Y(12), is a partial agonist of P2Y(12), and is a full agonist of the platelet ATP-gated ion channel P2X1. Modification of the Ap(4)A tetraphosphate backbone enhances inhibition of ADP-induced platelet aggregation. However, the effects of these Ap(4)A analogs on human platelet P2Y(1), P2Y(12) and P2X1 are unclear.
Objective:
To determine the agonist and antagonist activities of diadenosine tetraphosphate analogs towards P2Y(1), P2Y(12), and P2X1.
Methods:
We synthesized the following Ap(4)A analogs: P(1),P(4)-dithiotetraphosphate; P(2),P(3)-chloromethylenetetraphosphate; P(1)-thio-P(2),P(3)-chloromethylenetetraphosphate; and P(1),P(4)-dithio-P(2),P(3)-chloromethylenetetraphosphate. We then measured the effects of these analogs on: (i) ADP-induced platelet aggregation; (ii) P2Y(1)-mediated changes in cytosolic Ca(2+); (iii) P2Y(12)-mediated changes in vasodilator-stimulated phosphoprotein phosphorylation; and (iv) P2X1-mediated entry of extracellular Ca(2+).
Results:
Ap(4)A analogs with modifications in the phosphate backbone inhibited both P2Y(1) and P2Y(12), and showed no agonist activity towards these receptors. The dithio modification increased inhibition of P2Y(1), P2Y(12), and platelet aggregation, whereas the chloromethylene modification increased inhibition of P2Y(12) and platelet aggregation, but decreased P2Y(1) inhibition. Combining the dithio and chloromethylene modifications increased P2Y(1) and P2Y(12) inhibition. As compared with Ap(4)A, each modification decreased agonist activity towards P2X1, and the dual modification completely eliminated P2X1 agonist activity.
Conclusions:
As compared with Ap(4)A, tetraphosphate backbone analogs of Ap(4)A have diminished activity towards P2X1 but inhibit both P2Y(1) and P2Y(12) and, with greater potency, inhibit ADP-induced platelet aggregation. Thus, diadenosine tetraphosphate analogs with dual receptor selectivity may have potential as antiplatelet drugs.
Purinergic signaling plays important roles in control of vascular tone and remodeling. There is dual control of vascular tone by ATP released as a cotransmitter with noradrenaline from perivascular sympathetic nerves to cause vasoconstriction via P2X1 receptors, whereas ATP released from endothelial cells in response to changes in blood flow (producing shear stress) or hypoxia acts on P2X and P2Y receptors on endothelial cells to produce nitric oxide and endothelium-derived hyperpolarizing factor, which dilates vessels. ATP is also released from sensory-motor nerves during antidromic reflex activity to produce relaxation of some blood vessels. In this review, we stress the differences in neural and endothelial factors in purinergic control of different blood vessels. The long-term (trophic) actions of purine and pyrimidine nucleosides and nucleotides in promoting migration and proliferation of both vascular smooth muscle and endothelial cells via P1 and P2Y receptors during angiogenesis and vessel remodeling during restenosis after angioplasty are described. The pathophysiology of blood vessels and therapeutic potential of purinergic agents in diseases, including hypertension, atherosclerosis, ischemia, thrombosis and stroke, diabetes, and migraine, is discussed.
Dinucleoside polyphosphates are an interesting group of signalling molecules that control numerous physiological functions. Diadenosine compounds, with a backbone of anything from two to seven phosphates, are known to occur naturally. Some of them have been isolated from cerebral nerve terminals and, acting via nucleoside (P1), nucleotide (P2), or dinucleotide receptors, can affect central nervous system function. Many of them have been isolated from human blood platelet secretory granules and are potentially involved in haemostatic mechanisms and peripheral control of vascular tone. Many visceral organs respond to the application of adenine dinucleotides and, although they act on receptors in the periphery that can be mainly defined as either P1 or P2, evidence is now accumulating for discrete dinucleotide receptors. In the periphery, adenine dinucleotides can be potent agonists, with diverse functions, causing contraction or relaxation of smooth muscle. Many P2X receptor proteins and P2Y receptors have been cloned and adenine dinucleotides have a variable pharmacological profile at these receptors and may be useful tools for characterising subtypes of P2X and P2Y receptors. This review provides a broad description of the many extracellular roles of diadenosine polyphosphates as emerging, yet increasingly important, natural ligands for a plethora of structurally diverse mononucleotide and dinucleotide receptors. Drug Dev. Res. 52:260–273, 2001. © 2001 Wiley-Liss, Inc.
Mammalian ATP-gated nonselective cation channels (P2XRs) can be composed of seven possible subunits, denoted P2X1 to P2X7. Each subunit contains a large ectodomain, two transmembrane domains, and intracellular N and C termini. Functional P2XRs are organized as homomeric and heteromeric trimers. This review focuses on the binding sites involved in the activation (orthosteric) and regulation (allosteric) of P2XRs. The ectodomains contain three ATP binding sites, presumably located between neighboring subunits and formed by highly conserved residues. The detection and coordination of three ATP phosphate residues by positively charged amino acids are likely to play a dominant role in determining agonist potency, whereas an AsnPheArg motif may contribute to binding by coordinating the adenine ring. Nonconserved ectodomain histidines provide the binding sites for trace metals, divalent cations, and protons. The transmembrane domains account not only for the formation of the channel pore but also for the binding of ivermectin (a specific P2X4R allosteric regulator) and alcohols. The N- and C- domains provide the structures that determine the kinetics of receptor desensitization and/or pore dilation and are critical for the regulation of receptor functions by intracellular messengers, kinases, reactive oxygen species and mercury. The recent publication of the crystal structure of the zebrafish P2X4.1R in a closed state provides a major advance in the understanding of this family of receptor channels. We will discuss data obtained from numerous site-directed mutagenesis experiments accumulated during the last 15 years with reference to the crystal structure, allowing a structural interpretation of the molecular basis of orthosteric and allosteric ligand actions.
in the anaesthetized rat, uridine adenosine tetraphosphate (Up(4) A) is a circulating, endothelium-derived vasoconstrictor presumably operating as such in un-anaesthetized animals. The present study investigated the in vivo effects of Up(4) A in conscious mice and rats, and its direct vascular effects in the mouse aorta in vitro.
in vivo, Up(4) A was given as step-up infusion at rates of 8-512 nmol min(-1) kg(-1) for 30 min periods in chronically catheterized rodents. In vitro, the effect of Up(4) A on rings of mouse aortae mounted in a myograph was tested.
high doses of Up(4) A (mice: 512 nmol min(-1) kg(-1) ; rats: 128 nmol min(-1) kg(-1) ) caused hypotension (99 (+/-)4 to 64 7(+/-) mmHg and 114 (+/-) 3 to 108 (+/-) 3 mmHg, respectively, both P < 0.01). In rats, Up(4) A significantly decreased sodium excretion by >75% and potassium excretion by approximately 60% without significant changes in urine flow. Exposure of phenylephrine-contracted rings to increasing concentrations of Up(4) A elicited contraction at 10(-7) and 10(-6) molL(-1) (18 ± 2% and 76 (+/-) 16% respectively); unexpectedly, 10(-5) molL(-1) caused a biphasic response with a contraction (19 6(+/-)2%) followed by a relaxation (-46 (+/-) 6%). No relaxation was observed when the concentration was increased further. Bolus exposure to 10(-5) molL(-1) of Up(4) A caused contraction (+80 (+/-) 2%). Added successively to untreated vessels, increasing concentrations of Up(4) A (10(-7) -10(-5) molL(-1) ) induced a biphasic response of contraction followed by relaxation.
up(4) A has direct biphasic effects on vascular smooth muscle of the mouse aorta but vasoconstriction dominates at low concentrations. In conscious rodents, step-up infusions of Up(4) A elicit hypotension and electrolyte retention.
Thesis submitted for the degree of Doctor of Philosophy at the University of Leicester, September 2008. Awarded 3 July 2009. The identification of five predicted proteins (dP2XA-E) with homology to vertebrate P2X receptors in Dictyostelium discoideum proved a unique opportunity to explore purinergic receptor function in a simple unicellular eukaryote from outside the animal kingdom. dP2XE was shown to be expressed as a trimer and trafficked to the cell surface in Xenopus oocytes. However, no currents were detected to extracellularly applied ligands. This lack of function was potentially due to inaccurate post-translational modifications since dP2XE expressed in oocytes and D. discoideum displayed different molecular weights. Unexpectedly for a potential ligand-gated ion channel, dP2XE-eGFP displayed a solely intracellular distribution in D. discoideum, localising to the endolysosomal and contractile vacuole systems. Ablation of the p2xE gene by homologous recombination revealed a role for dP2XE in axenic growth in suspension, associated with a modest defect in cytokinesis, and a potential involvement in the calcium signalling and homeostasis functions of the contractile vacuole. Both this distribution and disruption phenotype were mirrored for p2xA. The existence of purinergic signalling in D. discoideum was demonstrated by utilising an apoaequorin expressing strain to show that extracellular ATP and ADP evoked increases in intracellular Caˆ{2,+}. Indicative of P2X receptor activation, responses were rapid and transient, required extracellular Caˆ{2,+}, inhibited by Gdˆ{3,+}, modified by extracellular pH and remained unaffected by deletion of either the single heterotrimeric G\beta or iplA genes. ATP/ADP responses were unaffected by ablation of either the p2xA or p2xE genes leaving dP2XB-D as potential candidates. Inhibition of the large P2X-like response with Znˆ{2,+} revealed the presence of a much smaller response with a slower time course indicating that P2Y-like receptors may also be present. The work presented in this thesis demonstrates that D. discoideum possesses cell surface purinergic receptors for extracellular ATP/ADP and extends our knowledge of the intracellular role of purinergic signalling in this organism.
alpha beta meATP-evoked concentration-dependent, PPADS-sensitive, desensitising, P2X receptor-mediated, constrictions of mesenteric, basilar and septal artery rings with EC(50) values of 1, 1 and 30 microM, respectively. In patch clamp studies on acutely dissociated artery smooth cells alpha beta meATP-evoked transient inward currents (tau approximately 100 ms) with mean current densities of approximately 340, 175 and 120 pA/pF, respectively. P2X(1) receptor immunoreactivity was expressed in mesenteric and basilar arteries and this receptor subunit appears to dominate the P2X receptor phenotype in these vessels. In contrast P2X(1) receptor immunoreactivity was not detected in septal arteries and the alpha beta meATP sensitivity of constriction was not consistent with the involvement of P2X(1) receptors. These results suggest that not all arteries share a common P2X receptor phenotype.
P2X receptors are nucleotide-gated cation channels composed of homomeric or heteromeric assemblies of three subunits. In the past 7 years, an extended series (P2X1-7) of P2X subunits has been cloned from vertebrate tissues. In this rapidly expanding field, one of the main current challenges is to relate the cloned P2X receptor subtypes to the diverse physiological responses mediated by the native P2X receptors. However, the paucity of useful ligands, especially subtype-selective agonists and antagonists as well as radioligands, acts as a considerable impediment to progress. Most of the ligands available are highly limited in terms of their kinetics of action, receptor-affinity, subtype-selectivity and P2X receptor-specificity. Their suspected ability to be a substrate for ecto-nucleotidases or to inhibit these enzymes also complicates their use. A number of new antagonists at P2X receptors have recently been described which to some degree are more potent and more selective than earlier antagonists like suramin or pyridoxal-5'-phosphate-6-azophenyl 2',4'-disulfonate (PPADS). This work moves us closer to the ideal goal of classifying the recombinant and native P2X receptor subtypes on the basis of antagonist profiles. This review begins with a brief account of the current status of P2X receptors. It then focuses on the pharmacological properties of a series of key P2 receptor agonists and antagonists and will finish with the discussion of some related therapeutic possibilities.
Peroxynitrite is implicated in many diseases. Hence, there is considerable interest in potential therapeutic peroxynitrite scavengers. Diet-derived phenolics have been claimed to be powerful peroxynitrite scavengers. However, the reactivity of peroxynitrite can be significantly modified by bicarbonate and this has not been considered in evaluations of the scavenging activity of phenols. Bicarbonate (25 mM) significantly decreased the ability of several phenolic compounds (caffeic acid, o- and p-coumaric acid, gallic acid, ferulic acid) but not others (catechin and epicatechin) to inhibit peroxynitrite-mediated tyrosine nitration. Bicarbonate (25 mM) also decreased the ability of catechin, epicatechin, quercetin and ferulic acid but not chlorogenic acid, gallic acid, caffeic acid and o-coumaric acid to inhibit peroxynitrite-mediated alpha(1)-antiproteinase inactivation. These results show that physiological concentrations of bicarbonate substantially modify the ability of dietary phenolics to prevent peroxynitrite-mediated reactions. When assessing compounds for peroxynitrite scavenging, experiments should be conducted in the presence of bicarbonate to avoid misleading results.
One hypothesis of the pathophysiology of pre-eclampsia is that placentally derived, yet unidentified, vasoactive factors are released into the maternal circulation, causing hypertension.
To determine if diadenosine polyphosphates, new potent vasoconstrictors, are present in human placenta.
Human placental tissue was homogenated and fractionated by size-exclusion chromatography, affinity chromatography, anion-exchange chromatography and reversed-phase chromatography. In fractions purified to homogeneity, diadenosine diphosphate, diadenosine triphosphate, diadenosine tetraphosphate, diadenosine pentaphosphate, diadenosine hexaphosphate and diadenosine heptaphosphate were identified by matrix-assisted laser desorption/ionization mass spectrometry, retention-time comparison and enzymatic cleavage analysis.
The presence of diadenosine polyphosphates in human placenta makes them possible candidates for involvement in the pathophysiology of pre-eclampsia. However, their contribution to the pathophysiology of eclampsia requires substantiation in further studies.
ADP plays a crucial role in haemostasis and thrombosis and its receptors are potential targets for antithrombotic drugs. Two G-protein coupled P2 receptors contribute to platelet aggregation: the P2Y1 receptor initiates aggregation through mobilisation of calcium stores, while the more recently identified P2Y12 receptor coupled to adenylyl cyclase inhibition is essential for a full aggregation response to ADP and the stabilisation of aggregates. The latter is defective in certain patients with a selective congenital deficiency of aggregation to ADP. It is also the target of the antithrombotic drug clopidogrel and of ATP analogues and other compounds currently under evaluation. In addition, the P2X1 ionotropic receptor is present in platelets but its role is not yet completely known. Studies in P2Y1 knock-out mice and experimental thrombosis models using selective P2Y1 antagonists have shown that the P2Y1 receptor, like the P2Y12 receptor, is a potential target for new antithrombotic drugs.
In this study, the receptor desensitizing effects of diadenosine polyphosphates at recombinant human P2X3 (hP2X3) receptors were examined. Administration of Ap3A, Ap4A, Ap5A or Ap6A inhibited the hP2X3 receptor-mediated response to a subsequent application of 3 muM alphabeta-methyleneATP (alphabeta-meATP), in a concentration-dependent manner, with IC50 values 2707, 42, 59 and 46 nM, respectively. These agonists did not desensitize alphabeta-meATP responses mediated by the slowly desensitizing heteromeric human P2X2/3 receptor. hP2X3 receptor desensitization was reversible and was not observed following the increase in intracellular Ca2+ levels produced by carbachol. A similar pattern of desensitization evoked by Ap5A was also observed using electrophysiological recordings of Xenopus oocytes expressing hP2X3 receptors. These data demonstrate that diadenosine polyphosphates, found endogenously in the central nervous system, can readily desensitize hP2X3 receptors at nanomolar concentrations that are 10-fold lower than are required to produce agonist-induced receptor activation. Thus, P2X3 receptor desensitization by diadenosine polyphosphates may provide an important modulatory mechanism of P2X3 receptor activation in vivo.
The aim of the present study was to investigate the effects of Xp(5)X and Xp(6)X (X = guanosine (G) or adenosine (A); n = 5 and 6), which have been identified in human platelets, on coronary vascular tone. The activation of purinoceptors in rat coronary vasculature by Xp(5)X and Xp(6)X was evaluated by measuring their effects on perfusion pressure in the Langendorff perfused rat. Ap(5)X and Ap(6)X induced dose-dependent vasodilation that was due to P2Y(1) receptor activation, as evidenced by use of the selective P2Y(1) receptor antagonist 2'-deoxy-N(6)-methyl-adenosine 3',5'-diphosphate diammonium (MRS2179). Vasodilation was induced by NO release, as evidenced by inhibition of nitric oxide synthases (NO synthases) by N(G)-nitro-L-arginine methyl ester (L-NAME). The dose-dependent decrease in coronary perfusion pressure induced by Ap(5)X and Ap(6)X was converted to a dose-dependent increase in perfusion pressure after inhibition of NO synthases by L-NAME. After endothelium removal, the vasodilation elicited by Ap(5)X and Ap(6)X was converted to a vasoconstriction which could be inhibited by P2X receptor blockade. Ap(5)A, Ap(5)G, Ap(6)A and Ap(6)G are vasodilating or vasoconstricting nucleotides that activate P2Y(1) or P2X receptors depending on the status of the coronary vascular endothelium.
Diadenosine polyphosphates (APnA) are endogenous dinucleoside molecules consisting of two adenosine moieties linked via their 5'-ribose positions by a variable number of phosphate groups. APnA have been shown to be present in different cell types and to be released from platelets as well as co-released with catecholamines and ATP from bovine adrenal medulla. Candidate metabolites of APnA are ATP, ADP, AMP and adenosine. Vascular effects induced by APnA and their metabolites in several models have been reported to be mediated by A1- and A2-adenosine receptors as well as P2-purinoceptors. APnA have been demonstrated to differentially affect regional perfusion, to influence cardiac output and blood pressure as well as the reactivity of isolated blood vessels and vascular beds. Vascular effects of APnA vary with the number of phosphate groups linking the adenosine molecules. This review outlines the effects of APnA on mesenteric and renal circulation. The effects of the antagonists varying with the type of vascular bed and the heterogeneous and dynamic vascular effects of diadenosine polyphosphates indicate a regionally different distribution of P2X and of P2Y purinoceptors in resistance arteries from different vascular beds. Although APnA have vasoconstrictor effects on the local level, it was repeatedly confirmed that systemically applied APnA induce hypotensive effects. The vasoconstrictor effects of APnA in isolated vessels are most prominent under resting tone conditions. In vivo, the vasculature exhibits a vasotone which makes dilatory effects more likely. Information on effects of APnA in vivo is still limited despite the fact that these compounds already have been used in man.
G-protein-coupled P2Y1 and P2Y12 receptors play key roles in platelet activation, however the importance of ionotropic P2X1 receptors remains unclear. Platelet P2X1 responses are highly labile in vitro, but were greatly enhanced by increasing [Ca2+]o in the range 1-10 mM. The P2X1 agonist alpha,beta-MeATP stimulated a shape change which saturated at peak [Ca2+]i of > or = 400 nM, without evidence for aggregation. The maximal P2X1-evoked transmission decrease was 82% of that obtained via P2Y1 receptors. alpha,beta-MeATP caused a disc to sphere transformation in virtually all platelets, but lacked the long processes produced by ADP. Following block of P2Y1 receptors with A3P5PS, co-stimulation with alpha,beta,-MeATP and ADP failed to induce aggregation despite the generation of peak [Ca2+]i responses similar to those stimulated via P2Y1 receptors. Therefore early, transient Ca2+ influx via P2X1 receptors can contribute to platelet activation by stimulating a significant morphological change, but does not readily synergise with P2Y12 receptors to support aggregation.
The platelet surface membrane possesses three P2 receptors activated by extracellular adenosine nucleotides; one member of the ionotropic receptor family (P2X(1)) and two members of the G-protein-coupled receptor family (P2Y(1) and P2Y(12)). P2Y(1) and P2Y(12) receptors have firmly established roles in platelet activation during thrombosis and haemostasis, whereas the importance of the P2X(1) receptor has been more controversial. However, recent studies have demonstrated that P2X(1) receptors can generate significant functional platelet responses alone and in synergy with other receptor pathways. In addition, studies in transgenic animals indicate an important role for P2X(1) receptors in platelet activation, particularly under conditions of shear stress and thus during arterial thrombosis. This review discusses the background behind discovery of P2X(1) receptors in platelets and their precursor cell, the megakaryocyte, and how signalling via these ion channels may participate in platelet activation.
P2X1 receptors for ATP are ligand-gated cation channels expressed on a range of smooth muscle preparations and blood platelets.
The receptors appear to be clustered close to sympathetic nerve varicosities and mediate the underlying membrane potential
changes and constriction following nerve stimulation in a range of arteries and resistance arterioles. In this study we have
used discontinuous sucrose density gradients, Western blot analysis, and cholesterol measurements to show that recombinant
and smooth muscle (rat tail artery, vas deferens, and bladder) P2X1 receptors are present in cholesterol-rich lipid rafts and co-localize with the lipid raft markers flotillin-1 and -2. Lipid
rafts are specialized lipid membrane microdomains involved in signaling and trafficking. To determine whether lipid raft association
was essential for P2X1 receptor channel function we used the cholesterol-depleting agent methyl-β-cyclodextrin (10 mm for 1 h). This led to a redistribution of the P2X1 receptor throughout the sucrose gradient and reduced P2X1 receptor-mediated (α,β-methylene ATP, 10 μm) currents in HEK293 cells by >90% and contractions of the rat tail artery by ∼50%. However contractions evoked by potassium
chloride (60 mm) were unaffected by methyl-β-cyclodextrin and the inactive analogue α-cyclodextrin had no effect on P2X1 receptor-mediated currents or contractions. P2X1 receptors are subject to ongoing regulation by receptors and kinases, and the present results suggest that lipid rafts are
an essential component in the maintenance of these localized signaling domains and play an important role in P2X1 receptor-mediated control of arteries.
P2X receptors comprise a family of ATP-gated ion channels with the basic amino acids Lys-68, Arg-292, and Lys-309 (P2X1 receptor numbering) contributing to agonist potency. In many ATP-binding proteins aromatic amino acids coordinate the binding
of the adenine group. There are 20 conserved aromatic amino acids in the extracellular ligand binding loop of at least 6 of
the 7 P2X receptors. We used alanine replacement mutagenesis to determine the effects of individual conserved aromatic residues
on the properties of human P2X1 receptors expressed in Xenopus oocytes. ATP evoked concentration-dependent (EC50 ∼1 μm) desensitizing currents at wild-type receptors and for the majority of mutants there was no change (10 residues) or a <6-fold
decrease in ATP potency (6 mutants). Mutants F195A and W259A failed to form detectable channels at the cell surface. F185A
and F291A produced 10- and 160-fold decreases in ATP potency. The partial agonists 2′,3′-O-(4-benzoyl)-ATP (BzATP) and P1,P5-di(adenosine 5′)-pentaphosphate (Ap5A) were tested on a range of mutants that decreased ATP potency to determine whether this resulted predominantly from changes
in agonist binding or gating of the channel. At K68A and K309A receptors BzATP and Ap5A had essentially no agonist activity but antagonized, or for R292A potentiated, ATP responses. At F185A receptors BzATP was
an antagonist but Ap5A no longer showed affinity for the receptor. These results suggest that residues Lys-68, Phe-185, Phe-291, Arg-292, and Lys-309
contribute to ligand binding at P2X1 receptors, with Phe-185 and Phe-291 coordinating the binding of the adenine ring of ATP.
Diadenosine pentaphosphate and diadenosine hexaphosphate have been isolated in human platelets and have been postulated to
play an important role in the control of vascular tone. Here we describe the isolation and identification of diadenosine heptaphosphate
from human platelets. Dinucleoside polyphosphates were concentrated by affinity chromatography from a nucleotide-containing
fraction from deproteinated human platelets. Dinucleoside polyphosphates were purified by anion-exchange and reversed phase
high performance liquid chromatography to homogeneity. Analysis of one of these fractions with matrix-assisted laser desorption/ionization
mass spectrometry revealed a molecular mass of 1076.4 (1077.4 = [M + H]+) Da. UV spectroscopic analysis of this fraction showed the spectrum of an adenosine derivative. Comparison of the postsource
decay matrix-assisted laser desorption/ionization mass spectrum of the fraction minus that of diadenosine heptaphosphate (Ap7A) demonstrated that the isolated substance was identical to Ap7A. The identity of the retention times of the authentic and the isolated compound confirmed this result. Enzymatic analysis
demonstrated an interconnection of the phosphate groups with the adenosines in the 5′-positions of the riboses. With thrombin-induced
platelet aggregation, Ap7A is released from the platelets into the extracellular space. The vasoconstrictive action of Ap7A on the vasculature of the isolated perfused rat kidney Ap7A was slightly less than that of Ap6A. The threshold of the vasoconstrictive action of Ap7A was 10−5mol/liter. The vasoconstrictive effect was abolished by suramin and pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid,
suggesting an activation of P2x receptors. Furthermore, Ap7A inhibits ADP-induced platelet aggregation. Thus, the potent vasoconstrictor Ap7A derived from human platelets, like other diadenosine polyphosphates, may play a role in the regulation of vascular tone
and hemostasis.
Several P2X receptor subunits were recently cloned; of these, one was cloned from the rat vas deferens (P2X1) and another from pheochromocytoma (PC12) cells differentiated with nerve growth factor (P2X2). Peptides corresponding to the C-terminal portions of the predicted receptor proteins (P2X1 391-399 and P2X2 460-472) were used to generate antisera in rabbits. The specificities of antisera were determined by staining human embryonic kidney cells stably transfected with either P2X1 or P2X2 receptors and by absorption controls with the cognate peptides. In the vas deferens and the ileal submucosa, P2X1 immunoreactivity (ir) was restricted to smooth muscle, whereas P2X2-ir was restricted to neurons and their processes. Chromaffin cells of the adrenal medulla and PC12 cells contained both P2X1- and P2X2-ir. P2X1-ir was also found in smooth muscle cells of the bladder, cardiac myocytes, and nerve fibers and terminals in the superficial dorsal horn of the spinal cord. In contrast, P2X2-ir was observed in scattered cells of the anterior pituitary, neurons in the hypothalamic arcuate and paraventricular nuclei, and catecholaminergic neurons in the olfactory bulb, the substantia nigra, ventral tegmental area, and locus coeruleus. A plexus of nerve fibers and terminals in the nucleus of the solitary tract contained P2X2-ir. This staining disappeared after nodose ganglionectomy, consistent with a presynaptic function. The location of the P2X1 subunit in smooth muscle is consistent with its role as a postjunctional receptor in autonomic transmission, while in neurons, these receptors appear in both postsynaptic and presynaptic locations.
Two new P2X receptor cDNAs (P2X5 and P2X6) were isolated and expressed. All six proteins are 36-48 percent identical and seem to have two transmembrane segments with a large extracellular loop. Functionally, P2X5 and P2X6 receptors most resemble P2X2 and P2X4; they desensitize only slowly and do not respond to alpha beta methylene-ATP. P2X6 receptors, like P2X4, receptors, are not blocked by the antagonists suramin and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid. P2X6 and P2X5 receptors express at lower levels than P2X1-P2X4 receptors do, perhaps indicating that they do not normally form homomultimeric channels. P2X6 and P2X4 are the receptors expressed most heavily in brain, where their RNAs have a widespread and extensively overlapping distribution. The spinal cord expresses all receptors except P2X3. P2X2, P2X4, and P2X6, are the most abundant in the dorsal horn. Sensory neurons of the trigeminal, dorsal root, and nodose ganglia express all six RNAs; P2X3 is found only there. The functional properties and tissue distribution of these six P2X receptors indicate new roles for ATP-gated ion channels.
We isolated and identified nucleoside(5') oligophospho-(5') nucleosides containing adenosine and guanosine (ApnG; n = 3-6) as well as diguanosine polyphosphates (GpnG; n = 3-6) in human platelets. For identification, UV spectrometry, matrix-assisted laser desorption/ionization, postsource decay matrix-assisted laser desorption/ionization mass spectrometry, and enzymatic cleavage experiments were used. The adenosine(5') oligophospho-(5') guanosines act as vasoconstrictors and growth factors. The diguanosine polyphosphates are potent modulators of growth in vascular smooth muscle cells, but do not affect vascular tone.
ATP-gated ion channels (P2X receptors) are ubiquitously present in autonomic and sensory neurons as well as in smooth muscle; they mediate fast excitatory synaptic transmission at sympathetic neuromuscular junctions, at some neuro-neuronal synapses and may be involved in the generation and transmission of primary afferent information. Five subtypes of native P2X receptors can be distinguished by their kinetics and their agonist and antagonist profile. Six distinct P2X receptors have been cloned; all are present in sensory neurons and most are present in autonomic neurons; homomeric or heteromeric forms of these cloned receptors reproduce the five phenotypes observed in native cells.
The actions of exogenously applied ATP were investigated with the whole‐cell patch clamp method in single cells isolated from guinea‐pig urinary bladder with a modified concentration jump technique.
Rapid application of ATP (threshold ca. 100 n m ) depolarized the cell membrane with superimposition of action potentials which was followed by transient hyperpolarization. In the presence of D600, the amplitude of the ATP‐induced depolarization was a function of the ATP concentration (EC 50 :0.5‐1 μ m ).
ATP activated a dose‐dependent inward current with a short latency (18 ms with 10 μ m ATP; measured as the time between the start of application and 10% of the peak). The relationship of the peak current versus ATP concentration was well fitted by a Michaelis‐Menten equation with a Hill coefficient (n) of 1.7 and a dissociation constant ( K d ) of 2.3 μ m . The current desensitized rapidly and the time course of desensitization was a function of the ATP concentration and could be fitted by two exponentials.
The reversal potential of the ATP‐activated current was near 0 mV. Replacement of extracellular Na by other monovalent or divalent cations indicated that the current flows through nonselective cation channels.
α,β‐Methylene ATP also produced a dose‐dependent inward current but was less potent than ATP (n: 1.6, K d : 10.4 μ m ). α,β‐Methylene ATP blocked the response to ATP by desensitization of the receptor.
α,β‐Methylene ATP was 50–100 times more potent than ATP at eliciting a contractile response of strips of detrusor smooth muscle.
The relevance of the above results to the possible role of ATP as the fast excitatory transmitter is discussed.
Receptor-operated Ca2+ entry has been proposed as a signalling mechanism in many cells. Receptor-operated Ca2+ channels (ROCs) were first postulated in smooth muscle by Bolton, van Breemen and Somlyo and Somlyo, but recordings of directly ligand-gated Ca2+ current are lacking. Here we describe receptor-operated Ca2+ current evoked in arterial smooth muscle cells by ATP, a sympathetic neurotransmitter. ATP activates channels with approximately 3:1 selectivity for Ca2+ over Na+ at near-physiological concentrations and with a unitary conductance of approximately 5 pS in 110 mM Ca2+ or Ba2+. The channels can be opened even at very negative potentials and resist inhibition by cadmium or nifedipine, unlike voltage-gated Ca2+ channels; they are not blocked by Mg2+, unlike NMDA (N-methyl-D-aspartate)-activated channels; they are directly activated by ligand, without involvement of readily diffusible second messengers, unlike cation channels in neutrophils and T lymphocytes. Thus, the ATP-activated channels provide a distinct mechanism for excitatory synaptic current and Ca2+ entry in smooth muscle.
Dinucleotides diadenosine triphosphate (AP3A) and diadenosine tetraphosphate (AP4A) are released from platelet-dense granules upon agonist-induced platelet aggregation. Since most platelet-derived compounds simultaneously affect aggregation and vascular tone, we investigated whether AP3A and AP4A have vasoactive properties. Experiments were performed in isolated, saline-perfused segments of rabbit mesenteric arteries precontracted with norepinephrine. In segments with intact endothelium, both dinucleotides (1-10 microM) induced vasodilation, with AP3A responses significantly greater. Vasodilator responses to AP3A in endothelium-denuded segments were not significantly different from those in segments with intact endothelium but those to AP4A in endothelium-intact segments were reversed to a pronounced contraction after endothelium removal. Likewise, pretreatment of endothelium-intact segments with gossypol (3 microM) reversed dilator responses to acetylcholine and to AP4A into contractions, whereas AP3A-induced dilation was not affected. In segments with intact endothelium but pretreated with reactive blue (10 microM), AP4A also induced a contraction. Dilator response to AP3A was not affected. High-performance liquid chromatographic analysis of effluent from vascular segments showed that neither AP3A nor AP4A was degraded during passage through segments. These results indicate that in rabbit mesenteric arteries both nucleotides act directly, and not through their hydrolysis products, on endothelial (AP4A) and/or smooth muscle receptors. The endothelium-dependent dilator effect of AP4A, is probably mediated by endothelial P2y-purinoceptors.
The adenosine-5'-triphosphate (ATP) molecule is an extracellular messenger in neural and non-neural tissues, where it activates several cell-surface-receptor subtypes, including G-protein-coupled receptors and ligand-gated ion channels. ATP-gated channels (termed P2x receptors) have been characterized on smooth muscle cells and autonomic and sensory neurons, where they mediate membrane depolarization and, in some cases, Ca2+ entry. P2x receptors are functionally heterogeneous, but resemble acetylcholine- and serotonin-gated channels with respect to ion selectivity and kinetic parameters of channel gating. We report here that despite such close functional similarities, the deduced sequence of a cloned P2x receptor predicts an unusual subunit structure resembling voltage-insensitive cation channels. Thus, the P2x receptor provides a striking example of convergent evolution, whereby proteins have been fashioned with similar functional properties from subunits having very different structural characteristics. There is sequence similarity between the ATP receptor and RP-2, a gene activated in thymocytes undergoing programmed cell death. RP-2 may encode a receptor for ATP or another metabolite released during apoptosis.
Extracellular ATP exerts its effects through P2 purinoceptors: these are ligand-gated ion channels (P2x) or G-protein-coupled receptors (P2Y, P2U). ATP at P2x receptors mediates synaptic transmission between neurons and from neurons to smooth muscle, being responsible, for example, for sympathetic vasoconstriction in small arteries and arterioles. We have now cloned a complementary DNA encoding the P2x receptor from rat vas deferens and expressed it in Xenopus oocytes and mammalian cells. ATP activates a cation-selective ion channel with relatively high calcium permeability. Structural predictions suggest that the protein (399 amino acids long) is mostly extracellular and contains only two transmembrane domains plus a pore-forming motif which resembles that of potassium channels. The P2x receptor thus defines a new family of ligand-gated ion channels.
cDNAs encoding P2x purinoceptors from human bladder smooth muscle and from rat PC-12 cells were expressed in oocytes and human embryonic kidney 293 cells. Agonist potencies of 2-methylthio-ATP = 2-chloro-ATP = ATP > = 2'- and 3'-O-(4-benzoylbenzoyl)-ATP > or = adenosine-5'-O-(3-thio)-triphosphate > or = P1,P5-di(adenosine-5') pentaphosphate > ADP prevailed for both P2x purinoceptors. There were two main differences in agonist sensitivity between the two receptors. First, ATP was 10 times more potent at the receptor from bladder (EC50, 0.8 microM) than at the receptor from PC-12 cells (EC50, 8.2 microM). Second, alpha,beta-methylene-ATP and L- and D-beta,gamma-methylene-ATP were agonists in cells expressing the bladder smooth muscle receptor (EC50, 1-3 microM) but were ineffective in cells expressing the PC-12 receptor. The P2 purinoceptor antagonists suramin, pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, and pyridoxal-5-phosphate acted similarly at both receptor forms, producing noncompetitive inhibition, with IC50 values of 1-5 microM for suramin and pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid and 10-20 microM for pyridoxal-5-phosphate. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid distinguished receptor subtypes, producing potent inhibition of the bladder smooth muscle P2x-mediated response, with an IC50 value of 3 microM; it inhibited the PC-12 form by < 40% at 100 or 300 microM. This study thus defines the pharmacological properties of homo-oligomeric forms of these two types of cloned P2x receptor channels.
1. The isolated perfused rat mesenteric arterial bed was used to examine the activity of the adenine dinucleotides: beta-nicotinamide adenine dinucleotide (NAD); beta-nicotinamide adenine dinucleotide phosphate (NADP); flavin adenine dinucleotide (FAD); and of the alpha,omega-diadenosine polyphosphates: adenylyl adenosine (AP1A); P1,P2-diadenosine pyrophosphate (AP2A); P1,P3-diadenosine triphosphate (AP3A); P1,P4-diadenosine tetraphosphate (AP4A); P1,P5-diadenosine pentaphosphate (AP5A); P1,P6-diadenosine hexaphosphate (AP6A). Responses were compared with those of ADP, ATP, 2-methylthio-ATP (2-meSATP) and alpha,beta-methylene ATP (alpha,beta-meATP). 2. In basal tone preparations mono- and dinucleotides elicited vasoconstriction with the order of potency: alpha,beta-meATP > or = AP5A > or = AP6A > or = AP4A > or = 2-meSATP > ATP > ADP. The dinucleotides NAD, NADP, FAD, AP1A, AP2A and AP3A had no effect. 3. The P2X-purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (30 microM) virtually abolished vasoconstrictor responses to AP4A, AP5A and AP6A. 4. Auto- and cross-desensitization of vasoconstrictor responses to AP4A, AP5A, AP6A, ATP and alpha,beta-meATP were observed. 5. In raised tone preparations nucleotides elicited endothelium-dependent vasodilatation with the order of potency: 2-meSATP = ADP > ATP > AP3A > AP2A > AP1A = NADP = FAD > NAD. The nucleotides AP4A, AP5A, AP6A and alpha,beta-meATP had no vasodilator effects. 6. It is concluded that the alpha,omega-adenine dinucleotides AP4A, AP5A and AP6A elicit vasoconstriction, but not vasodilatation, in the rat mesenteric arterial bed via P2x-purinoceptors. In contrast, the dinucleotides NADP, FAD, AP1A, AP2A and AP3A elicit vasodilatation, but not vasoconstriction, via endothelial P2Y-purinoceptors. 7. It is suggested that there is a crucial relationship between the structure of the alpha,omega-diadenosine polyphosphates and their activity at P2X- and P2Y-purinoceptors with a pivotal role played by the polyphosphate chain. Molecules with four or more phosphates are vasoconstrictors, while those with three or less phosphates are vasodilators.
1. By use of a 'grease-gap' technique, the depolarizing effects of adenosine 5'-triphosphate (ATP) and ATP analogues on the rat isolated vagus nerve were determined in normal and in Ca2+/Mg(2+)-free (+ 1 x 10(-3) M ethylenediamine tetraacetic acid) physiological salt solution (PSS). 2. In normal PSS, ATP produced concentration-dependent depolarization responses but the concentration-effect curve to ATP was incomplete and a maximum effect was not achieved. The threshold concentration for depolarization was 1 x 10(-5) M and at the highest concentration tested (1 x 10(-3) M) the peak amplitude of the response to ATP only amounted to 71% of the depolarization produced by a near maximal response to 5-hydroxytryptamine (5-HT, 1 x 10(-5) M). 3. In Ca2+/Mg(2+)-free PSS, ATP produced depolarization responses at much lower concentrations and of markedly larger amplitude. Under these conditions, the threshold concentration for depolarization was 1-3 x 10(-7) M and the maximal response to ATP amounted to 526% of the response to 5-HT (1 x 10(-5) M) in normal PSS. The concentration-effect curve to ATP was sigmoid, with a defined maximum effect and a mean EC50 value of 1.2 x 10(-6) M. 4. In contrast to the effects on responses to ATP, the absence of divalent cations in the PSS did not modify the effective concentrations of either alpha, beta-methylene ATP or 5-HT. However, the maximum responses to both alpha, beta-methylene ATP and 5-HT were significantly increased in Ca2+/Mg(2+)-free PSS.(ABSTRACT TRUNCATED AT 250 WORDS)
The electrophysiological actions of the P 2 ‐purinoceptor agonists, adenosine 5′‐triphosphate (ATP), 2‐methylthioATP (2‐meSATP), α,β‐methyleneATP (α,β‐meATP) and uridine 5′‐triphosphate (UTP) were studied under concentration and voltage‐clamp conditions in acutely dissociated rat tail artery smooth muscle cells. For comparison, their actions as vasoconstrictors were studied in intact ring preparations.
Rapid application of ATP (100 n m ‐1 μ m ) via a U‐tube supervision system activated concentration‐dependent inward currents with a latency to onset of less than 3 ms. The inward current decayed by more than 95% during a 2 s application of 300 n m and 1 μ m ATP.
2‐meSATP (100nM‐1 μ m ) and α,β‐meATP (100 n m ‐1 μ m ) also evoked transient inward currents. The agonist order of potency was ATP = 2‐meSATP ≥ α,β‐meATP. UTP (300nM‐1μ m ) did not produce a change in the holding current.
A second application of ATP (300 n m and 1 μ m ) 10 min after the first, evoked currents which were one third of the initial amplitude. This decline was dependent upon activation of the P 2 ‐purinoceptor. Similar results were seen with 2‐meSATP and α,β‐meATP (both 300 n m and 1 μ m ). Cross‐desensitization was seen between ATP and 2‐meSATP or α,β‐meATP.
Inward currents evoked by ATP, 2‐meSATP and α,β‐meATP (all 1 μ m ) were abolished by the P 2 ‐purinoceptor antagonist suramin (100 μ m ).
αβ‐meATP (100 n m ‐30 μ m ), 2‐meSATP (3 μ m ‐100 μ m ), ATP (3 μ m ‐ 1 μ m ) and UTP (3 μ m ‐ 1 μ m ) produced concentration‐dependent contractions of rat tail artery rings. When measured at a level equal to 50% of the maximum response to noradrenaline, the rank order of agonist potency was α,β,‐meATP > > 2‐meSATP >UTP >ATP.
This study shows that the rank order of agonist potency at the P 2X ‐purinoceptor which mediates contractions of the rat isolated tail artery is very different from the potency order for evoking the inward current which initiates the contractions. It is concluded that this difference may be due to the relative absence of breakdown of some of the agonists in the single cell system compared with artery rings.
Our understanding of the regulation of vascular tone has been extended since the identification of vasoactive agents such as the atrial natriuretic peptides, endothelial-derived relaxing factor and endothelin. Unidentified vasopressive agents have been found in platelets. Here we isolate these vasopressors and identify them as diadenosine pentaphosphate (AP5A) and diadenosine hexaphosphate (AP6A) by chromatography, mass spectrometry, ultraviolet spectroscopy and enzymatic cleavage. In the vasculature of isolated perfused rat kidney, both diadenosine phosphates were active at a concentration of 10(-9) M; in aortic rings, contractions were elicited at 10(-8) M. Intra-aortic injection in the rat caused a prolonged increase in blood pressure. We conclude that AP5A and AP6A may play a part in local vasoregulation and possibly in the regulation of blood pressure.
The P2Z receptor is responsible for adenosine triphosphate (ATP)-dependent lysis of macrophages through the formation of membrane
pores permeable to large molecules. Other ATP-gated channels, the P2X receptors, are permeable only to small cations. Here, an ATP receptor, the P2X7 receptor, was cloned from rat brain and exhibited both these properties. This protein is homologous to other P2X receptors but has a unique carboxyl-terminal domain that was required for the lytic actions of ATP. Thus, the P2X7 (or P2Z) receptor is a bifunctional molecule that could function in both fast synaptic transmission and the ATP-mediated lysis of
antigen-presenting cells.
1. The relationship between the stimulation of ATP receptors, the increase in intracellular free calcium concentration ([Ca2+]i; measured using the fluorescent indicator fura-2), contraction and the subtypes of purinoceptors involved were investigated in the small mesenteric artery of the rat. 2. In normal physiological solution, ATP (0.001-3 mM) caused concentration-dependent increases in both [Ca2+]i and contraction. Both responses produced by ATP (1 mM) were inhibited by 50% in the presence of nitrendipine (1 microM) and were abolished in the presence of nitrendipine plus SK&F 96365 (30 microM). 3. In Ca(2+)-free medium, ATP (3 mM) elicited a transient increase in both [Ca2+]i and tension which were abolished by caffeine and decreased by 65% by thapsigargin (1 microM). Moreover, ATP (1 and 3 mM) produced increases in the [3H]D-myo-inositol 1,4,5-trisphosphate ([3H]IP3) content of vessels in a concentration-dependent manner. 4. Treatment of the vessels with Bordetella pertussis toxin (PTX) inhibited contractions to ATP linked to the influx of calcium through nitrendipine-sensitive mechanisms, but not those linked to the release of Ca2+ from intracellular stores nor the capacity of ATP in increasing IP3 content of the vessels. 5. The order of potency of ATP and its analogues in eliciting contraction was alpha, beta-methylene-ATP (alpha, beta-MeATP) > 2-methylthio-ATP (2-MeSATP) > ATP = ADP. The response to ATP was inhibited by suramin. Reactive Blue 2 (up to 100 microM) did not affect the contractile response to ATP. Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium (PPADS) and alpha, beta-MeATP abolished the response to low concentrations of ATP and reduced contractions elicited by high concentrations of ATP. 6. After blockade of P2X-purinoceptors with PPADS, the order of potency of ATP and its analogues was 2-MeSATP > ATP = ADP. UTP produced concentration-dependent contractions which were not affected by suramin, Reactive Blue 2, PPADS or alpha, beta-MeATP, suggesting the presence of P2U-purinoceptors. 7. The results suggest that low concentrations of ATP activate P2X-purinoceptors and produce an influx of calcium through both voltage-dependent calcium channels sensitive to nitrendipine and through receptor-operated calcium channels sensitive to SK&F 96365. High concentrations of ATP activate P2Y-purinoceptors which promote firstly a nitrendipine-sensitive calcium influx via a PTX-sensitive G protein and secondly a release of Ca2+ from an internal source via the production of IP3.
1. Whole-cell potassium currents were studied in single smooth muscle cells enzymatically isolated from pig coronary arteries. 2. In cells isolated from small diameter branches of the left anterior descending coronary artery (LAD), an inward rectifier potassium current (IK(IR)) was identified, which was inhibited by extracellular barium ions, suggesting the presence of inward rectifier potassium (KIR) channels. 3. The conductance for IK(IR) measured in 6, 12, 60 and 140 mM extracellular potassium was a function of membrane potential and the extracellular potassium concentration. 4. On hyperpolarization, IK(IR) activated along an exponential time course with a time constant that was voltage dependent. 5. Inward rectifier current was compared in cells isolated from coronary vessels taken from different points along the vascular tree. Current density was greater in cells isolated from small diameter coronary arteries; at -140 mV it was -20.5 +/- 4.4 pA pF-1 (n = 23) in 4th order branches of the LAD, but -0.8 +/- 0.2 pA pF-1 (n = 11) in the LAD itself. 6. In contrast to IK(IR), there was little effect of arterial diameter on the density of voltage-dependent potassium current; densities at +30 mV were 12.8 +/- 1.3 pA pF-1 (n = 19) in 4th order branches and 17.4 +/- 3.1 pA pF-1 (n = 11) in the LAD. 7. We conclude that KIR channels are present in pig coronary arteries, and that they are expressed at a higher density in small diameter arteries. The presence of an enhanced IK(IR) may have functional consequences for the regulation of cell membrane potential and tone in small coronary arteries.
1. Adenine dinucleotides (Ap3A, x = 2-6) are naturally-occurring polyphosphated nucleotidic substances which are found in the CNS and are known to be released in a calcium-dependent manner from storage vesicles in brain synaptosomes. The selectivity and activity of adenine dinucleotides for neuronally-derived recombinant P2 purinoceptors were studied using P2X2 and P2Y1 subtypes expressed in Xenopus oocytes. 2. For the P2Y1 subtype derived from chick brain, Ap3A was equipotent and as active as ATP (EC50 values: 375 +/- 86 nM and 334 +/- 25 nM, respectively). Ap4A was a weak partial agonist and other dinucleotides were inactive as agonists. None of the inactive dinucleotides were antagonists nor modulated the activity of Ap3A and ATP. 3. For the P2X2 subtype derived from rat PC12 cells, Ap4A was as active as ATP but less potent (EC50 values: 15.2 +/- 1 microM and 3.7 +/- 0.7 microM, respectively). Other adenosine dinucleotides were inactive as either agonists or antagonists. 4. Ap5A (1-100 nM) potentiated ATP-responses at the P2X2 subtype, showing an EC50 of 2.95 +/- 0.7 nM for this modulatory effect. Ap5A (10 nM) shifted the concentration-response curves for ATP to the left by one-half log10 unit but did not alter the Hill co-efficient for ATP (nH = 2.1 +/- 0.1). Ap5A (10 nM) failed to potentiate Ap4A-responses but did enhance the efficacy of the P2 purinoceptor antagonist, suramin, by 12 fold at the P2X2 subtype. 5. In conclusion, the results show that ionotropic (P2X2) and metabotropic (P2Y1) ATP receptors which occur in the CNS are activated selectively by naturally-occurring adenine dinucleotides which are known to be released with nucleotides from storage vesicles. The observed potentiation of P2X2-responses by Ap5A, where co-released with ATP by brain synaptosomes, may have a functional bearing in purinergic signalling in the CNS.
The activation of various purinoceptors in rat renal vasculature by P1,P2-diadenosine pyrophosphate (Ap2A), P1,P3-diadenosine triphosphate (Ap3A), P1,P4-diadenosine tetraphosphate (Ap4A), P1,P5-diadenosine pentaphosphate (Ap5A), P1,P6-diadenosine hexaphosphate (Ap6A) was studied by measuring their effects of perfusion pressure of a rat isolated perfused kidney.
The vasoconstrictive response to Ap5A was completely due to P2X purinoceptor activation, that to Ap4A and Ap6 was P2X purinoceptor mediated to a large extent, as evidenced by the inhibitory effects of suramin and pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid tetrasodium (PPADS).
The vasoconstrictive effects of Ap2A and Ap3A were mostly due to stimulation of A1-receptors, as shown by the inhibitory effect of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX).
The vasoconstrictive response to Ap6A was partially insensitive to A1 and P2X purinoceptor blockers.
In raised tone preparations Ap2A and Ap3A evoked vasodilatation, which was blocked by the A2 receptor blocker, 3,7-dimethyl-1-propargylxanthine (DMPX).
In raised tone preparations Ap4A evoked vasodilatation when the P2-purinoceptors were blocked by suramin.
The activation of different purinoceptor subtypes by diadenosine phosphates critically depends on the number of phosphate groups.
British Journal of Pharmacology (1997) 120, 1453–1460; doi:10.1038/sj.bjp.0701074
The site(s) at which diadenosine 5′,5′′′-P1, P4-tetraphosphate (AP4A) and diadenosine 5′, 5′′′-P1, P5-pentaphosphate (AP5A) act to evoke contraction of the guinea-pig isolated vas deferens was studied by use of a series of P2-receptor antagonists and the ecto-ATPase inhibitor 6-N, N-diethyl-D-β,γ-dibromomethyleneATP (ARL 67156).
Pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (300 nM–30 μM), suramin (3–100 μM) and pyridoxal-5′-phosphate (P-5-P) (3–1000 μM) inhibited contractions evoked by equi-effective concentrations of AP5A (3 μM), AP4A (30 μM) and α,β-methyleneATP (α,β-meATP) (1 μM), in a concentration-dependent manner and abolished them at the highest concentrations used.
PPADS was more potent than suramin, which in turn was more potent than P-5-P. PPADS inhibited AP5A, AP4A and α,β-meATP with similar IC50 values. No significant difference was found between IC50 values for suramin against α,β-meATP and AP5A or α,β-meATP and AP4A, but suramin was more than 2.5 times more potent against AP4A than AP5A. P-5-P showed the same pattern of antagonism.
Desensitization of the P2X1-receptor by α,β-meATP abolished contractions evoked by AP5A (3 μM) and AP4A (30 μM), but had no effect on those elicited by noradrenaline (100 μM).
ARL 67156 (100 μM) reversibly potentiated contractions evoked by AP4A (30 μM) by 61%, but caused a small, significant decrease in the mean response to AP5A (3 μM).
It is concluded that AP4A and AP5A act at the P2X1-receptor, or a site similar to the P2X1-receptor, to evoke contraction of the guinea-pig isolated vas deferens. Furthermore, the potency of AP4A, but not AP5A, appears to be inhibited by an ecto-enzyme which is sensitive to ARL 67156.
British Journal of Pharmacology (1997) 121, 57–62; doi:10.1038/sj.bjp.0701099
We investigated the extracellular degradation of diadenosine polyphosphates (ApnA) by cultured adrenomedullary endothelial cells using fluorogenic analogs of ApnA, the di(1,N6-ethenoadenosine) 5',5"'-P1,Pn-polyphosphates [epsilon-(ApnA)]. Kinetic parameters of epsilon-(ApnA) cleavage and effects of pH, ions, and inhibitors were determined by continuous fluorometric assays, using suspensions of endothelial cells grown on Cytodex-1 microspheres. Ecto-enzyme kinetic parameters for epsilon-(Ap3A), epsilon-(Ap4A), and epsilon-(Ap5A) hydrolysis are as follows: Michaelis-Menten constants of 0.39 +/- 0.07, 0.42 +/- 0.09, and 0.37 +/- 0.05 microM respectively, and maximal velocities of 26.1 +/- 6.8, 74.2 +/- 16.4, and 24.4 +/- 3.4 pmol.min-1.10(6) cells-1, respectively. ApnA and guanosine 5',5"'-P1,P4-tetraphosphate behave as competitor substrates of epsilon-(Ap4A) hydrolysis. The ectoenzyme is activated by Mg2+ and Mn2+ and inhibited by Ca2+, F-, adenosine 5'-tetraphosphate, adenosine 5'-O-(3-thiotriphosphate), and suramin. Optimum pH is around 9.0. High-performance liquid chromatography analysis reveals that the ecto-enzyme hydrolyzes epsilon-(ApnA) to give epsilon-adenosine-5'(n-1)-phosphate and epsilon-AMP, which are then further catabolized up to epsilon-adenosine via the membrane-bound nucleotidase system ecto-ATPase, ecto-ADPase (or apyrase), and ecto-5'-nucleotidase. The endothelial ecto-diadenosine polyphosphate hydrolase studied here exhibits different kinetic parameters and sensitivity to ions with respect to the enzyme from the tissue-related neurochromaffin cells. These different properties may be important in the extracellular signaling by ApnA.
The actions of ATP and uridine 5'-triphosphate (UTP) were compared at P2X1 receptors in acutely dissociated smooth muscles cells of the rat tail artery. ATP (30 nM-100 microM) and UTP (1 microM-1 mM) elicited concentration-dependent inward currents. ATP was approximately 100-fold more potent than UTP. In both cases, currents were activated within 3 ms of agonist application and had similar time-courses of activation and inactivation. The decay of responses for both agonists was concentration-dependent and in most cells could be fitted by two exponentials. The P2X receptor antagonists suramin (100 microM) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 5 microM) inhibited responses to both ATP and UTP. An action of UTP at P2X1 receptors has not previously been reported. However, since the responses to ATP and UTP had similar time-courses and as PPADS and suramin inhibited both agonists, it is concluded that ATP and UTP are acting at the same site in these cells, the P2X1 receptor.
1. P2X receptor activation by alpha,beta-meATP evoked inward currents in acutely dissociated rat mesenteric artery smooth muscle cells and contractions of whole artery rings. 2. The selective P2X1 and P2X3 receptor antagonist TNP-ATP inhibited P2X receptor mediated inward currents in response to 3 microM alpha,beta-meATP (an approximately EC90 concentration) with an IC50 of approximately 2 nM. This provides further evidence that the P2X receptor underlying membrane depolarisation associated with P2X receptor activation can be accounted for by the expression of P2X1 receptors. 3. TNP-ATP inhibited alpha,beta-meATP induced contractions with an IC50 of approximately 30 microM and had non-specific effects on smooth muscle contraction. 4. The reduced potency of TNP-ATP in whole tissue experiments probably reflects the breakdown of TNP-ATP by nucleotidases. Thus, TNP-ATP is of limited use in whole tissue experiments as a P2X receptor antagonist.
We have developed a method for measuring the local concentration of ATP at the extracellular surface of live cells. This method relies on the specific attachment to the cell surface of a chimeric protein that consists of the IgG-binding domain of Staphylococcus aureus protein A fused in-frame with the complete sequence for firefly luciferase (proA-luc). Expression of proA-luc in Escherichia coli and its one-step affinity purification are straightforward. Attachment to cells is demonstrated to be specific and antibody dependent using several suspended and adherent cell types. Light production by cell surface-attached luciferase is continuous, linearly related to ATP concentration, and sufficient to provide nanomolar sensitivity. The spatial resolution of this method enables the observation of strictly local changes in extracellular ATP during its secretion from activated platelets. Furthermore, the activity of cell-attached luciferase is relatively refractory to the inclusion of nucleotidases in the medium, arguing for its effectiveness in cell systems possessing potent ecto-ATPases.
The pharmacological activity of diadenosine polyphosphates was investigated at three recombinant P2X receptors (rat P2X1, rat P2X3, rat P2X4) expressed in Xenopus oocytes and studied under voltage-clamp conditions. For the rat P2X1 receptor, only P1,P6-diadenosine hexaphosphate (Ap6A) was a full agonist yet 2-3 folds less potent than ATP. At rat P2X3, P1,p4-diadenosine tetraphosphate (Ap4A), P1,P5-diadenosine pentaphosphate (Ap5A) and Ap6A were full agonists and more potent than ATP. Ap4A alone was equipotent with ATP at rat P2X4, but only as a partial agonist. Compared to known data for rat P2X2 and human P2X1 receptors, our findings contrast with rat P2X2 where only Ap4A is a full agonist although four folds less potent than ATP. At rat and human orthologues of P2X1, Ap5A was a partial agonist with similar potency. These data provide a useful basis for selective agonists of P2X receptor subunits.
ATP functions as a fast neurotransmitter through the specific activation of a family of ligand-gated ion channels termed P2X receptors. In this report, six distinct recombinant P2X receptor subtypes were pharmacologically characterized in a heterologous expression system devoid of endogenous P2 receptor activity. cDNAs encoding four human P2X receptor subtypes (hP2X1, hP2X3, hP2X4, and hP2X7), and two rat P2X receptor subtypes (rP2X2 and rP2X3), were stably expressed in 1321N1 human astrocytoma cells. Furthermore, the rP2X2 and rP2X3 receptor subtypes were co-expressed in these same cells to form heteromultimeric receptors. Pharmacological profiles were determined for each receptor subtype, based on the activity of putative P2 ligands to stimulate Ca2+ influx. The observed potency and kinetics of each response was receptor subtype-specific and correlated with their respective electrophysiological properties. Each receptor subtype exhibited a distinct pharmacological profile, based on its respective sensitivity to nucleotide analogs, diadenosine polyphosphates and putative P2 receptor antagonists. Alphabeta-methylene ATP (alphabeta-meATP), a putative P2X receptor-selective agonist, was found to exhibit potent agonist activity only at the hP2X1, hP2X3 and rP2X3 receptor subtypes. Benzoylbenzoic ATP (BzATP, 2' and 3' mixed isomers), which has been reported to act as a P2X7 receptor-selective agonist, was least active at the rat and human P2X7 receptors, but was a potent (nM) agonist at hP2X1, rP2X3 and hP2X3 receptors. These data comprise a systematic examination of the functional pharmacology of P2X receptor activation.
Pharmacological characterization of recombinant human and rat P2X receptor subtypes New structural motif for ligand-gated ion channels de®ned by an ionotropic ATP receptor
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Detection of local ATP release from activated platelets using cell surface-attached ®re¯y luciferase A novel receptor-operated Ca 2+ -permeable channel activated by ATP in smooth muscle
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Vasomotor activity of diadenosine triphosphate and diadenosine tetraphosphate in isolated arteries Cloning of P2X 5 and P2X 6 receptors and the distribution and properties of an extended family of ATP-gated ion channels
- R Busse
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Receptors for purines and pyrimidines
- V Burnstock
Receptors for purines and pyrimidines
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