Vasoactivity of diadenosine polyphosphates in human small mesenteric resistance arteries

Diadenosine polyphosphates (ApnA) (n = 3-6) induced vasoconstrictions in isolated human mesenteric resistance arteries (hMRAs) mounted in a microvessel myograph (rank order of potency: Ap5A > Ap6A > Ap4A > Ap3A). The contractile effects of ApnA in hMRA were similar to their effects in rat MRA investigated previously. ATP, ADP, AMP, and adenosine had less contractile potency than ApnA, suggesting that the observed effects were not induced by the degradation products of ApnA. Ap4A- and Ap5A-induced vasoconstriction was inhibited by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (P2X purinoceptor antagonist) but not by ADP3'5' (P2Y purinoceptor antagonist). Thus, this purinergic vasoconstriction of hMRA seems to be P2X but not P2Y purinoceptor-mediated. In precontracted hMRA all ApnA caused vasorelaxations but (in contrast to rat MRA) the potencies of the ApnA did not differ significantly from each other. The ApnA degradation products had less vasorelaxing potency than ApnA, demonstrating that the vasorelaxations can be ascribed to the ApnA themselves. Ap5A-induced vasorelaxation of hMRA could neither be inhibited with ADP3'5' nor with PPADS, which reveals a decisive difference to the rat MRA where the inhibitory profile demonstrated the importance of the P2Y purinoceptor for Ap5A-induced vasorelaxation. However, Ap4A-induced vasorelaxation in hMRA could be inhibited by ADP3'5'. These findings show that Ap4A-induced vasorelaxation in hMRA is due to P2Y purinoceptor activation, that Ap5A evokes vasorelaxation in hMRA via another mechanism than Ap4A, and that data derived from the animal model cannot be simply transferred to human conditions.     

Diadenosine polyphosphates cause contraction and relaxation in isolated rat resistance arteries

The effects of diadenosine polyphosphates (APnA; n = 3-6) and adenine nucleotides on contractile reactivity of isolated rat mesenteric resistance arteries (MrA) and superior epigastric arteries (SEA), which display a dense and sparse autonomic innervation, respectively, were evaluated. All agonists examined, except adenosine and AMP, induced contractions. The rank order of potency was similar in both arteries: alpha,beta-methylene ATP (alpha,beta-meATP) > AP5A > AP6A > AP4A > ATP > ADP > AP3A. Contractions were stable during several minutes in SEA but highly transient in MrA. They were reduced after exposure to 10 microM alpha,beta-meATP and by 10 microM of the P2X antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid. During phenylephrine (10 microM)-induced contractions, the agonists induced a further contraction in SEA. In MrA, however, further contraction was followed by marked relaxation. The rank order of relaxing potency was comparable to that of the contractile potency of agonists. Also, the relaxing effects of APnA were blunted by 10 microM pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid and after exposure to alpha,beta-meATP. In vitro and in vivo sympathectomy with 6-hydroxydopamine and removal of the endothelium did not modify the effects of APnA in MrA. Thus, the contractile effects of APnA in resistance arteries 1) are due to a P2X purinoceptor-mediated stimulation of the smooth muscle; 2) depend on the length of the phosphate chain; and 3) are followed by endothelium-independent relaxing effects in MrA but not SEA, which may involve receptors that are similar to those mediating contraction. The regional heterogeneity of APnA effects cannot be attributed to a direct neurogenic influence.     

Influence of purinoceptor antagonism on diadenosine pentaphosphate-induced hypotension in anesthetized rats

Diadenosine polyphosphates (ApnA; n = 3-6) are potent vasoactive agents in isolated vessels. Information on effects of ApnA in vivo is still limited despite the fact that these compounds are starting to be used in humans. This study was designed to compare the effects of ApnA and their possible metabolites on blood pressure in vivo and to functionally identify purinoceptors involved in their action. All four ApnA and their degradation products induced a sustained drop of mean arterial blood pressure during i.v. infusion, which was fully reversible. The rank order of potency was Ap4A > or = Ap6A > Ap5A = Ap3A = ATP = ADP > AMP > or = adenosine, suggesting that the hypotensive effect is predominantly evoked by the original dinucleotides and not by their degradation products. The hypotensive effect of Ap5A was reduced by the P2X and P2Y(1) purinoceptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, the A(1) purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, and the A(2) purinoceptor antagonist 3, 7-dimethyl-1-propargylxanthine. The hypertensive effect by the prototype P2X receptor agonist alphabeta-methylene ATP was inhibited by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, too. Purinoceptor antagonists reduced the maximal effects of the agonists indicating a noncompetitive inhibition. In summary, the reported vasocontractile effect of ApnA seems to be limited to isolated preparations under resting tone conditions; however, the systemic cardiovascular effects of all four ApnA are hypotensive, also making them candidates for blood pressure reduction in humans. These effects are fast in onset and easily reversible. Activation of different purinoceptors in the vasculature (most probably P2Y(1) and A(2) receptors) contributes to the Ap5A-induced decrease of mean arterial blood pressure.     

Differential localization of P2 receptor subtypes in mesenteric arteries and veins of normotensive and hypertensive rats

ATP acts at P2 receptors to contract blood vessels and reactivity to vasoconstrictor agents is often altered in hypertension. This study was designed to identify P2 receptors in mesenteric arteries and veins and to determine whether ATP reactivity is altered in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Computer-assisted video microscopy was used to measure vessel diameter in vitro. ATP was a more potent constrictor of veins (EC(50) = 2.7 microM) than arteries (EC(50) = 196 microM) from normotensive rats; there was no change in ATP reactivity in vessels from DOCA-salt rats. The P2X1 receptor agonist alpha,beta-methylene ATP (alpha,beta-MeATP, 0.03-3 microM) contracted arteries but not veins. ATP-induced contractions in arteries were blocked by alpha,beta-MeATP (3 microM) desensitization. 2-Methylthio-ATP (0.1-10 microM), an agonist that can act at P2Y1 receptors, did not contract arteries or veins, whereas UTP, an agonist at rat P2Y2/P2Y4 receptors, contracted veins (EC(50) = 15 microM) and arteries (EC(50) = 24 microM). UTP-induced contractions of veins cross-desensitized with ATP, whereas UTP-induced contractions in arteries were unaffected by alpha,beta-MeATP-desensitization. The P2X/P2Y1 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4-disulfonic acid blocked ATP-induced contractions of arteries (IC(50) = 4.8 microM) but not veins. Suramin, an antagonist that blocks P2Y2 receptors, partly inhibited ATP- and UTP-induced contractions of veins. Immunohistochemical studies revealed P2X1 receptor immunoreactivity in arteries but not veins. These data indicate that mesenteric vascular reactivity to ATP is not altered in DOCA-salt hypertension. ATP acts at P2X1 and P2Y2 receptors to contract mesenteric arteries and veins, respectively, whereas in arteries UTP acts at an unidentified P2 receptor.     

Diadenosine polyphosphate analog controls postsynaptic excitation in CA3-CA1 synapses via a nitric oxide-dependent mechanism

Previously, we have described the modulatory effect of diadenosine polyphosphates Ap4A and Ap5A on synaptic transmission in the rat hippocampal slices mediated by presynaptic receptors (Klishin et al., 1994). In contrast, we now describe how nonhydrolyzable Ap4A analog diadenosine-5',5'-P1,P4-[beta,beta'-methylene]tetraphosphate (AppCH2ppA) at low micromolar concentrations exerts strong nondesensitizing inhibition of orthodromically evoked field potentials (OFPs) without affecting the amplitude of excitatory postsynaptic currents and antidromically evoked field potentials, as recorded in hippocampal CA1 zone. The effects of AppCH2ppA on OFPs are eliminated by a P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) but not mimicked by purinoceptor agonists alpha,beta-methylene-ATP and adenosine 5'-O-(3-thio)-triphosphate, indicating that a P2-like receptor is involved but not one belonging to the conventional P2X/P2Y receptor classes. Diadenosine polyphosphate receptor (P4) antagonist Ip4I (diinosine tetraphosphate) was unable to modulate AppCH2ppA effects. Thus, the PPADS-sensitive P2-like receptor for AppCH2ppA seems to control selectively dendritic excitation of the CA1 neurons. The specific nitric oxide (NO)-scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide is shown to significantly attenuate AppCH2ppA-mediated inhibitory effects, indicating that NO is involved in the cascade of events initiated by AppCH2ppA. Further downstream mediation by adenosine A1 receptors is also demonstrated. Hence, AppCH2ppA-mediated effects involve PPADS-sensitive P2-like receptor activation leading to the production of NO that stimulates intracellular synthesis of adenosine, causing in turn postsynaptic A1 receptor activation and subsequent postsynaptic CA1 dendritic inhibition. Such spatially selective postsynaptic dendritic inhibition may influence dendritic electrogenesis in pyramidal neurons and consequently mediate control of neuronal network activity.     

Evidence for the involvement of endogenous ATP and P2X receptors in TMJ pain.

Evidence is accumulating which supports a role for ATP in the initiation of pain by acting on P2X receptors expressed on nociceptive afferent nerve terminals. To investigate whether these receptors play a role in temporomandibular (TMJ) pain, we studied the presence of functional P2X receptors in rat TMJ by examining the nociceptive behavioral response to the application of the selective P2X receptor agonist alpha,beta-methylene ATP (alpha,beta-meATP) into the TMJ region of rat. The involvement of endogenous ATP in the development of TMJ inflammatory hyperalgesia was also determined by evaluating the effect of the general P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on carrageenan-induced TMJ inflammatory hyperalgesia. Application of alpha,beta-meATP into the TMJ region of rats produced significant nociceptive responses that were significantly reduced by the co-application of lidocaine N-ethyl bromide quaternary salt, QX-314, (2%) or of the P2 receptor antagonist PPADS. Co-application of PPADS with carrageenan into the TMJ significantly reduced inflammatory hyperalgesia. The results indicate that functional P2X receptors are present in the TMJ and suggest that endogenous ATP may play a role in TMJ inflammatory pain mechanisms possibly by acting primarily in these receptors.     

P2X3 receptor mediates heat hyperalgesia in a rat model of trigeminal neuropathic pain.

The present study was undertaken to determine the role of P2X3 receptor (P2X3R) on heat hyperalgesia in a newly developed rat model of trigeminal neuropathic pain. The unilateral infraorbital nerve (IoN) was partially ligated by 6-0 silk. To assess heat sensitivity, a vibrissal pad (VP) was placed on a hot plate and the latency until the rats withdrew their head was measured. Mechanical sensitivity of VP was also assessed by the use of von Frey filament. Both heat and mechanical hyperalgesia were observed at the VP ipsilateral to the IoN ligation. The latency to heat stimuli was prolonged after subcutaneous administration of pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, P2X1,2,3,5,7,1/5,2/3R antagonist) and 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP, P2X1,3,2/3,1/5R antagonist). The latency was shortened after administration of alpha,beta-methylene ATP (alpha,beta-meATP, P2X1,3,2/3R agonist), although no changes appeared after administration of beta,gamma-methylene-L-ATP (beta,gamma-me-L-ATP, P2X1R agonist). The protein gene product-9.5 and calcitonin gene-related peptide immunoreactive nerve fibers significantly decreased in the VP skin of ipsilateral to the IoN ligation. In the ipsilateral trigeminal ganglion, the number of P2X3-immunoreactive neurons significantly increased in the small cell group. In this study, we developed an experimental model of trigeminal neuropathic pain by partial ligation of IoN, which produced heat and mechanical hyperalgesia in the VP. Pharmacological and immunohistochemical studies revealed that the P2X3R plays an important role in the heat hyperalgesia observed in this model. PERSPECTIVE: The study describes the development of a novel model of trigeminal neuropathic pain. Heat hyperalgesia in this model was inhibited by peripheral injection of P2XR antagonists. The results suggest that P2X3R is a potential target for development of a novel therapy for trigeminal neuropathic pain.     

ATP inhibits glutamate synaptic release by acting at P2Y receptors in pyramidal neurons of hippocampal slices

It has been proposed that extracellular ATP inhibits synaptic release of glutamate from hippocampal CA1 synapses after its catabolism to adenosine. We investigated the possibility that at least part of this effect is mediated by ATP itself acting on P2Y receptors. ATP and various analogs decreased the amplitude and duration of glutamate-mediated excitatory postsynaptic potentials in all tested neurons. This effect was reversible and concentration-dependent and had the following rank order of agonist potency: AMP = ATP = adenosine-5'-O-(3-thio)triphosphate > adenosine = ADP. alpha,beta-Methylene ATP, beta,gamma-methylene ATP, 2-methylthioadenosine 5'-triphosphate, GTP, and UTP induced only a partial response. The depolarization induced by exogenous glutamate was not affected by ATP, indicating that this nucleotide acts presynaptically to inhibit glutamate-mediated excitatory postsynaptic potentials. Neither inhibition of ectonucleotidase activity with alpha,beta-methylene ADP, suramin, or pyridaxalphosphate-6-azophenyl-2',4'-disulfonic acid 4-sodium nor removal of extracellular adenosine (with adenosine deaminase) altered ATP effects. 8-Cyclopentyltheophylline competitively inhibited ATP effects, whereas P2 receptor antagonists (pyridaxalphosphate-6-azophenyl-2',4'-disulfonic acid 4-sodium, suramin, and reactive blue 2) were ineffective. ATP effects were by far more sensitive to pertussis toxin (PTX) than those of adenosine. After PTX, adenosine-5'-O-(3-thio)triphosphate induced only a partial response, and ATP concentration-response curve was biphasic. The second phase of this curve was blocked by adenosine deaminase, implying that it is mediated by adenosine as a result of ATP catabolism. Under control conditions, however, catabolism of ATP is not required to explain its actions. In conclusion, ATP inhibits synaptic release of glutamate by direct activation of P2Y receptors that are PTX- and 8-cyclopentyltheophylline-sensitive.     

TNP-ATP, a potent P2X3 receptor antagonist, blocks acetic acid-induced abdominal constriction in mice: comparison with reference analgesics

Exogenous ATP has been shown to be algogenic in both animal and humans. Research has focused on the P2X3 ligand-gated ion channel, as it is preferentially expressed on nociceptive C-fibers. In addition, P2X3 receptor gene disrupted mice show decreased responses to somatic painful stimuli. However, the potential role of P2X receptor activation in visceral pain has not yet been evaluated. In the present study, the systemic administration of suramin, and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, PPADS, both non-selective P2X receptor antagonists, dose-dependently reduced acetic acid-induced abdominal constrictions in mice (ED(50)=34.5 micromol/kg and ED50=70 micromol/kg, respectively). Furthermore, 2'-(or-3')-O-(trinitrophenyl)adenosine 5'- tri-phosphate (TNP-ATP) potently (IC50=10 nM) blocked the functional activation of P2X3 receptors in vitro and attenuated acetic acid-induced visceral pain. In the abdominal constriction assay, TNP-ATP (ED(50)=6.35 micromol/kg, i.p.) was 6-10 fold more potent than suramin and PPADS to reduce nociceptive behavior. In addition, TNP-ATP was 10 fold more potent than TNP-AMP (2'-(or-3')-O-(trinitrophenyl)adenosine 5'-mono-phosphate) (ED50=63.5 micromol/kg, i.p.) at reducing acetic acid-induced nociception. At the highest dose, TNP-ATP completely abolished nociceptive behavior, as did morphine (ED50=3 micromol/kg, i.p.). While TNP-ATP is also a potent antagonist of P2X1 receptors, P2X1 receptor mediated responses have not been shown in dorsal root ganglia and diinosine pentaphosphate, IP5I, a potent and selective P2X1 receptor antagonist, was ineffective at reducing abdominal constrictions. Thus, the antinociceptive effects of TNP-ATP appear to be mediated through activation of homomeric P2X3and/or heteromeric P2X2/3 receptors. Together, these results show that activation of P2X3 containing receptors plays a role in the transmission of inflammatory visceral pain.     

Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), a putative P2Y(1) receptor antagonist, blocks signaling at a site distal to the receptor in Madin-Darby canine kidney-D(1) cells

Substantial evidence documents the potential importance of P2Y receptor subtypes in the regulation of cellular responses, but few selective antagonists exist for these receptors. In the current study, we assessed the use of pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonate (PPADS) as a putative P2Y(1) receptor-selective blocker in Madin-Darby canine kidney (MDCK-D(1)) cells. We found that the key action of PPADS in MDCK-D(1) cells was blockade of signaling at a postreceptor site. PPADS blocked UTP (P2Y(2))-stimulated accumulation of cAMP [which is dependent on arachidonic acid (AA) metabolism by cyclooxygenase] but not that by 2-methyl thio-adenosine triphosphate (2MeSATP; which is independent of cyclooxygenase and has been attributed to P2Y(1) and P2Y(11) receptors). By contrast, PPADS inhibited AA release mediated by both 2MeSATP and UTP. PPADS displayed uncompetitive antagonism in blockade of AA release in response to 2MeSATP. PPADS also inhibited AA release stimulated by various nucleotides, phenylephrine, and bradykinin, implying that the effect does not involve the inhibition of a specific receptor. Because PPADS also inhibited ionomycin-, thapsigargin-, and phorbol-12-myristate-13-acetate-promoted AA release, it appears to act at a site distal to an increase in intracellular Ca(2+) transients or PKC activation. Inhibition of melittin-stimulated AA release by PPADS suggested that the target of PPADS action may either be a phospholipase A(2) (PLA(2)) or a site distal to PLA(2), but PPADS did not inhibit Ca(2+)-dependent PLA(2) activity in MDCK-D(1) cell homogenate. The data indicate that PPADS blocks AA release in response to multiple compounds and suggest caution in the use of this compound for distinguishing P2Y receptor subtypes.     

The P2X3 antagonist P1, P5-di[inosine-5'] pentaphosphate binds to the desensitized state of the receptor in rat dorsal root ganglion neurons

P2X3 purinergic receptors are predominantly expressed in dorsal root ganglion (DRG) neurons and play an important role in pain sensation. P2X3-specific antagonists are currently being sought to ameliorate pain in several indications. Understanding how antagonists interact with the P2X3 receptor can aid in the discovery and development of P2X3-specific antagonists. We studied the activity of the noncompetitive antagonist P1, P5-di[inosine-5'] pentaphosphate (IP5I) at the P2X3 receptor, compared with the well studied competitive antagonist TNP-ATP, using a whole-cell voltage-clamp technique in dissociated rat DRG neurons. IP5I blocked alphabeta-methylene ATP (alphabeta-meATP)-evoked P2X3 responses in a concentration-dependent manner (IC50 = 0.6 +/- 0.1 microM). IP5I effectively inhibited P2X3 currents when pre-exposed to desensitized but not unbound receptors. Furthermore, IP5I equally blocked 1 and 10 microM alphabeta-meATP-evoked currents and had no effect on the desensitization rate constant of these currents. This supports the action of IP5I as a noncompetitive antagonist that interacts with the desensitized state of the P2X3 receptor. In contrast, TNP-ATP inhibited the current evoked by 1 microM alphabeta-meATP significantly more than the one evoked by 10 microM alphabeta-meATP. It also significantly slowed down the desensitization rate constant of the current. These results suggest that TNP-ATP acts as a competitive antagonist and competes with alphabeta-meATP at the P2X3 agonist binding site. These findings may help to explain why IP5I acts selectively at the fast-desensitizing P2X1 and P2X3 subtypes of the P2X purinoceptor, while having much less potency at slow-desensitizing P2X2 and P2X(2/3) subtypes that lack the fast desensitized conformational state.     

Adenosine 5'-(2-fluorodiphosphate) is not a selective P2Y purinoceptor agonist in the rabbit jugular vein.

The relaxant properties of the putative selective P2Y agonist adenosine 5'-(2-fluorodiphosphate) (ADP-beta-F) and its structural analog adenosine 5'-(2-thiodiphosphate) have been investigated in the rabbit precontracted jugular vein preparation. In tissues with intact endothelium, ADP-beta-F produced a multiphasic agonist concentration/effect curve made up of two vasorelaxant components which were kinetically and pharmacologically distinct. The higher potency phase (p[A50] 5.58 +/- 0.13), characterized by slow, tonic responses, was retained after endothelial denudation and blocked by the selective P1 purinoceptor antagonist 8-sulphophenyltheophylline. The lower potency phase (p[A50] 3.98 +/- 0.07), characterized by fast, phasic responses, was abolished by endothelial denudation and is presumed to be mediated at P2Y purinoceptors. By contrast, the agonist concentration/effect curve to adenosine 5'-(2-dithiophosphate) in endothelium-intact tissues appear monophasic and was unaffected by 8-sulphophenyltheophylline (p[A50] 6.86 +/- 0.12), although endothelial denudation revealed a secondary P1-induced relaxant component (p[A50] 5.73 +/- 0.20). This study demonstrates that in the rabbit jugular vein, relaxant responses to ADP-beta-F are mediated primarily by activation of P1 purinoceptors, and it is, therefore, invalid to regard ADP-beta-F as a selective probe for P2Y purinoceptors, whereas adenosine 5'-(2-thiodiphosphate) does show some selectivity for this receptor.     

Induction of novel agonist selectivity for the ADP-activated P2Y1 receptor versus the ADP-activated P2Y12 and P2Y13 receptors by conformational constraint of an ADP analog

ADP is the cognate agonist of the P2Y1, P2Y12, and P2Y13 receptors. With the goal of identifying a high potency agonist that selectively activates the P2Y1 receptor, we examined the pharmacological selectivity of the conformationally constrained non-nucleotide analog (N)-methanocarba-2MeSADP [(1'S,2'R, 3'S,4'R,5'S)-4-[(6-amino-2-methylthio-9H-purin-9-yl)-1-diphosphoryloxymethyl]bicyclo[3.1.0]hexane-2,3-diol] among the three ADP-activated receptors. Each P2Y receptor was expressed transiently in COS-7 cells, and inositol lipid hydrolysis was quantified as a measure of receptor activity. In the case of the Gi-linked P2Y12 and P2Y13 receptors, a chimeric G protein, Galphaq/i, was coexpressed to confer a capacity of these Gi-linked receptors to activate phospholipase C. 2MeSADP (2-methylthio-ADP) was a potent agonist at all three receptors exhibiting EC50 values in the sub to low nanomolar range. In contrast, whereas (N)-methanocarba-2MeSADP was an extremely potent (EC50=1.2 +/- 0.2 nM) agonist at the P2Y1 receptor, this non-nucleotide analog exhibited no agonist activity at the P2Y12 receptor and very low activity at the P2Y13 receptor. (N)-Methanocarba-2MeSADP also failed to block the action of 2MeSADP at the P2Y12 and P2Y13 receptors, indicating that the (N)-methanocarba analog is not an antagonist at these receptors. The P2Y1 receptor selectivity of (N)-methanocarba-2MeSADP was confirmed in human platelets where it induced the shape change promoted by P2Y1 receptor activation without inducing the sustained platelet aggregation that requires simultaneous activation of the P2Y12 receptor. These results provide the first demonstration of a high-affinity agonist that discriminates among the three ADP-activated P2Y receptors, and therefore, introduce a potentially important new pharmacological tool for delineation of the relative biological action of these three signaling proteins.     

Allosteric modulation and accelerated resensitization of human P2X(3) receptors by cibacron blue

The activity of ATP as a fast neurotransmitter is mediated by the P2X family of ligand-gated ion channels. P2X receptor subtypes are subject to functional modulation by a diverse set of factors, including pH, divalent cations, and temperature. The human P2X(3) (hP2X(3)) receptor subunit is expressed primarily in sensory ganglia where it exists as either a homomultimeric receptor or, in combination with P2X(2), as a heteromultimeric receptor. This article describes the allosteric modulatory effect of the putative P2X receptor antagonist cibacron blue on the activity of recombinant hP2X(3) receptors. In 1321N1 cells expressing the hP2X(3) receptor, cibacron blue mediated a 3- to 7-fold increase in both the magnitude and the potency of ATP-activated Ca(2+) influx and transmembrane currents. The half-maximal concentration of cibacron blue required to mediate maximal potentiation (EC(50) = 1.4 microM) was independent of the agonist used to activate the hP2X(3) receptor. The nonselective P2 receptor antagonist PPADS (pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid) caused a rightward shift of the cibacron blue concentration-effect curve, whereas increasing concentrations of cibacron blue attenuated PPADS antagonism. In addition to potentiating the effects of ATP at the hP2X(3) receptor, cibacron blue also produced a 6-fold increase in the rate of hP2X(3) receptor recovery from desensitization (from T(1/2) = 15.9 to 2.6 min), as evidenced by its ability to restore ATP responsiveness to acutely desensitized receptors. Consistent with the properties of other ligand-gated ion channels, these results suggest that hP2X(3) receptor activity can be allosterically modulated by a ligand distinct from the endogenous agonist.     

A differential role of the platelet ADP receptors P2Y1 and P2Y12 in Rac activation

The dynamics of the actin cytoskeleton, largely controlled by the Rho family of small GTPases (Rho, Rac and Cdc42), is critical for the regulation of platelet responses such as shape change, adhesion, spreading and aggregation. Here, we investigated the role of adenosine diphosphate (ADP), a major co-activator of platelets, on the activation of Rac. ADP rapidly activated Rac in a dose-dependent manner and independently of GPIIb/IIIa and phosphoinositide 3-kinase. ADP alone, used as a primary agonist, activated Rac and its effector PAK via its P2Y1 receptor, through a G(q)-dependent pathway and independently of P2Y12. The P2Y12 receptor appeared unable to activate the GTPase per se as also observed for the adenosine triphosphate receptor P2X1. Conversely, secreted ADP strongly potentiated Rac activation induced by FcgammaRIIa clustering or TRAP via its P2Y12 receptor, the target of antithrombotic thienopyridines. Stimulation of the alpha(2A)-adrenergic receptor/G(z) pathway by epinephrine was able to replace the P2Y12/G(i)-mediated pathway to amplify Rac activation by FcgammaRIIa or by the thrombin receptor PAR-1. This co-activation appeared necessary to reach a full stimulation of Rac as well as PAK activation and actin polymerization and was blocked by a G-protein betagamma subunits scavenger peptide.     

Contribution of sensitized P2X receptors in inflamed tissue to the mechanical hypersensitivity revealed by phosphorylated ERK in DRG neurons

The mechanism of mechanical hyperalgesia in inflammation might involve a 'mechanochemical' process whereby stretch evokes the release of adenosine 5'-triphosphate (ATP) from the damaged tissue that then excites nearby primary sensory nerve terminals. In the present study, phosphorylated extracellular signal-regulated protein kinase (pERK) immunoreactivity was used as a marker indicating functional activation of primary afferent neurons to examine the P2X receptor-mediated noxious response in DRG neurons in a rat model of peripheral inflammation. We found that very few pERK-labeled DRG neurons were detected in normal rats after alpha, beta methylene-ATP (alphabetame-ATP) intraplantar injection. However, a number of DRG neurons were labeled for pERK after alphabetame-ATP injection to the complete Freund's adjuvant (CFA) induced inflamed paw. Seventy-three percent of pERK-labeled DRG neurons co-expressed the P2X3 receptor. After mechanical noxious stimulation to the hind paw of CFA-inflamed rats, we found many more pERK-labeled neurons compared to those in the normal rats. Administration of the P2X3 receptor antagonists, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid or 2'- (or 3')-O-(trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), significantly decreased the mechanical stimulation-evoked pERK labeling in CFA-inflamed rats, but not in normal rats. We also found the recruitment of neurons with myelinated A fibers labeled for pERK in CFA-inflamed rats, which was reversed by P2X3 receptor antagonists. Moreover, TNP-ATP dose dependently reduced the mechanical hypersensitivity of CFA rats. These data suggest that the P2X receptors in primary afferent neurons increase their activity with enhanced sensitivity of the intracellular ERK signaling pathway during inflammation and then contribute to the hypersensitivity to mechanical noxious stimulation in the inflammatory state.     

(N)-methanocarba-2MeSADP (MRS2365) is a subtype-specific agonist that induces rapid desensitization of the P2Y1 receptor of human platelets

Adenosine diphosphate (ADP) initiates and maintains sustained aggregation of platelets through simultaneous activation of both the Gq-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor. We recently described the synthesis and P2Y1 receptor-specific agonist activity of (N)-methanocarba-2MeSADP (MRS2365). Consequences of selective activation of the P2Y1 receptor by MRS2365 have been further examined in human platelets. Whereas MRS2365 alone only induced shape change, addition of MRS2365 following epinephrine treatment, which activates the Gi/z-linked, alpha2A-adrenergic receptor, resulted in sustained aggregation that was indistinguishable from that observed with ADP. Conversely, the platelet shape change promoted by ADP in the presence of the GPIIb/IIIa antagonist eptifibatide was similar to that promoted by MRS2365. Preaddition of the high affinity P2Y1 receptor antagonist MRS2500 inhibited the effect of MRS2365, whereas addition of MRS2500 subsequent to MRS2365 reversed the MRS2365-induced shape change. Preactivation of the P2Y1 receptor with MRS2365 for 2 min resulted in marked loss of capacity of ADP to induce aggregation as evidenced by a greater than 20-fold rightward shift in the concentration effect curve of ADP. This inhibitory effect of P2Y1 receptor activation was dependent on the concentration of MRS2365 (EC50 = 34 nm). The inhibitory effect of preincubation with MRS2365 was circumvented by activation of the Gq-coupled 5-HT2A receptor suggesting that MRS2365 induces loss of the ADP response as a consequence of desensitization of the Gq-coupled P2Y1 receptor. The time course of MRS2365-induced loss of aggregation response to epinephrine was similar to that observed with ADP. These results further demonstrate the P2Y1 receptor selectivity of MRS2365 and illustrate the occurrence of agonist-induced desensitization of the P2Y1 receptor of human platelets studied in the absence of P2Y12 receptor activation .