Diinosine Pentaphosphate Research Articles

Effect of diinosine polyphosphates on intraocular pressure in normotensive rabbits

The ability of diinosine polyphosphates, diinosine triphosphate (Ip3I), diinosine tetraphosphate (Ip4I) and diinosine pentaphosphate (Ip5I) to modify intraocular pressure in normotensive New Zealand white rabbits was tested. Ip5I produced increase in intraocular pressure, while Ip3I and Ip4I produced a decrease. Ip4I was the most effective reducing intraocular pressure inducing a maximal decrease of intraocular pressure to 74.2 ± 2.5% compared with the control value. Dose–response analysis demonstrated a concentration dependent pattern which presented a pD2 value of 6.19 ± 0.18, equivalent to an EC50 of 0.63 μM. Regarding the underlying mechanism used by Ip4I to reduce intraocular pressure, studies with agonists and antagonists revealed that Ip4I reduces intraocular pressure via P2Y receptors in the eye. We suggest that topical application of Ip4I to the cornea has therapeutic potential for lowering intraocular pressure, a major risk factor for glaucoma.

Uridine adenosine tetraphosphate induces contraction of airway smooth muscle

Contraction of airway smooth muscle (ASM) plays an important role in the regulation of air flow and is potentially involved in the pathophysiology of certain respiratory diseases. Extracellular nucleotides regulate ASM contraction via purinergic receptors, but the signaling mechanisms involved are not fully understood. Uridine adenosine tetraphosphate (Up(4)A) contains both pyrimidine and purine moieties, which are known to potentially activate P2X and P2Y receptors. Both P2X and P2Y receptors have been identified in the lung, including airway epithelial cells and ASM. We report here a study of purinergic signaling in the respiratory system, with a focus on the effect of Up(4)A on ASM contraction. Up(4)A induced contraction of rat isolated trachea and extrapulmonary bronchi as well as human intrapulmonary bronchioles. Up(4)A-induced contraction was blocked by di-inosine pentaphosphate, a P2X antagonist, but not by suramin, a nonselective P2 antagonist. Up(4)A-induced contraction was also attenuated by α,β-methylene-ATP-mediated P2X receptor desensitization. Several P2X receptors were detected at the mRNA level: P2X1, P2X4, P2X6, and P2X7, and to a lesser extent P2X3. Furthermore, the Up(4)A response was inhibited by removal of extracellular Ca(2+) and by the presence of the L-type Ca(2+) channel blocker, nifedipine, or the Rho-associated kinase inhibitor, H1152. We conclude that Up(4)A stimulates ASM contraction, and the underlying signaling mechanism appears to involve P2X (most likely P2X1) receptors, extracellular Ca(2+) entry via L-type Ca(2+) channels, and Ca(2+) sensitization through the RhoA/Rho-associated kinase pathway. This study will add to our understanding of the pathophysiological roles of extracellular nucleotides in the lung.

Uridine adenosine tetraphosphate-induced contraction is increased in renal but not pulmonary arteries from DOCA-salt hypertensive rats

Uridine adenosine tetraphosphate (Up(4)A) was reported as a novel endothelium-derived contracting factor. Up(4)A contains both purine and pyrimidine moieties, which activate purinergic (P2)X and P2Y receptors. However, alterations in the vasoconstrictor responses to Up(4)A in hypertensive states remain unclear. The present study examined the effects of Up(4)A on contraction of isolated renal arteries (RA) and pulmonary arteries (PA) from DOCA-salt rats using isometric tension recording. RA from DOCA-salt rats exhibited increased contraction to Up(4)A versus arteries from control uninephrectomized rats in the absence and presence of N(G)-nitro-l-arginine (nitric oxide synthase inhibitor). On the other hand, the Up(4)A-induced contraction in PA was similar between the two groups. Up(4)A-induced contraction was inhibited by suramin (nonselective P2 antagonist) but not by diinosine pentaphosphate pentasodium salt hydrate (Ip(5)I; P2X(1) antagonist) in RA from both groups. Furthermore, 2-thiouridine 5'-triphosphate tetrasodium salt (2-ThioUTP; P2Y(2) agonist)-, uridine-5'-(γ-thio)-triphosphate trisodium salt (UTPγS; P2Y(2)/P2Y(4) agonist)-, and 5-iodouridine-5'-O-diphosphate trisodium salt (MRS 2693; P2Y(6) agonist)-induced contractions were all increased in RA from DOCA-salt rats. Protein expression of P2Y(2)-, P2Y(4)-, and P2Y(6) receptors in RA was similar between the two groups. In DOCA-salt RA, the enhanced Up(4)A-induced contraction was reduced by PD98059, an ERK pathway inhibitor, and Up(4)A-stimulated ERK activation was increased. These data are the first to indicate that Up(4)A-induced contraction is enhanced in RA from DOCA-salt rats. Enhanced P2Y receptor signaling and activation of the ERK pathway together represent a likely mechanism mediating the enhanced Up(4)A-induced contraction. Up(4)A might be of relevance in the pathophysiology of vascular tone regulation and renal dysfunction in arterial hypertension.

Pharmacological properties of P2 receptors on rat otic parasympathetic ganglion neurons

To elucidate the pharmacological profile of P2X receptors and the probable expression of P2Y receptors in otic ganglion neurons from 17-day-old rats, single neurons were enzymatically isolated and maintained in tissue culture for up to 30 h. Whole-cell voltage-clamp recording was carried out at a holding potential of − 60 mV. Most otic ganglion neurons responded to adenosine 5′-triphosphate (ATP), 2-methylthio ATP (2-MeSATP) and α,β-methylene ATP (αβ-meATP) with sustained currents and EC 50 values of 19 µM, 47 µM and 94 µM, respectively. 2′,3′- O-trinitrophenyl-ATP (TNP-ATP) inhibited the response to αβ-meATP and ATP with an IC 50 values of 3.9 nM and 18.3 nM, respectively, which was closed to that observed in nodose neurons. The response to ATP was antagonized by suramin and cibacron blue. The dose–response curve of suramin against ATP response at a pH of 6.5 was shifted to the left compared to that at a pH of 7.4. Diinosine pentaphosphate (Ip 5I), which blocks P2X 3, but not P2X 2/3-mediated responses, had no effect on the currents evoked by ATP or αβ-meATP. In some neurons, uridine 5′-triphosphate (UTP) induced a tiny, but long-lasting current with a mean amplitude of 0.034 ± 0.011 nA. Reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed the expression of mRNAs for P2X 2, P2X 3, P2X 4, P2X 6 and P2X 7, but not for P2X 1 and P2X 5 receptors in otic ganglion. In conclusion, in rat otic ganglion neurons, P2X 2/3 heteromultimer receptors dominate, but P2X 7 and P2Y 2 or P2Y 4 receptors also play roles.

Existence of high and low affinity dinucleotides pentaphosphate-induced calcium responses in individual synaptic terminals and lack of correlation with the distribution of P2X 1–7 subunits

Individual analysis of synaptic terminals calcium responses, induced by dinucleotides pentaphosphate, Ap 5A or Gp 5G, demonstrates the presence of two main groups considering the concentration required for stimulation. The first group corresponds to those responding to Ap 5A or Gp 5G at nanomolar concentration, representing 16% and 12%, respectively, and the second one responds to micromolar concentration and represents, respectively, 17% and 14%, of the total functional synaptosomal population in rat midbrain. Dose–response curves in single terminals showed an Ap 5A EC 50 values of 0.9 ± 0.2 nM and 11.8 ± 0.9 μM, being the maximal intrasynaptosomal calcium increase of 200 ± 0.3 and 125 ± 0.2 nM for the high and low affinity responding terminals, respectively. Combination of microfluorimetric and immunocytochemical studies showed lack of correlation between dinucleotides pentaphosphate responses and P2X receptor subunits expression, in spite of the abundance of P2X 2, P2X 3 and P2X 7 at the presynaptic level in rat midbrain synaptosomes. Pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), a P2X receptors antagonist, showed no effect on low affinity dinucleotides receptors population, and partial inhibition on the high affinity one. On the other hand, diinosine pentaphosphate (Ip 5I) completely abolished the low affinity dinucleotides responses, and 60% inhibition of the high affinity ones.

Electrophysiological-anatomic correlates of ATP-triggered vagal reflex in the dog. V. Role of purinergic receptors

The mechanism of extracellular ATP-triggered vagal depressor reflex was further studied in a closed-chest canine model. Adenosine and ATP were administered individually in equimolar doses (0.01-1.0 mumol/kg) into the right coronary artery (RCA) and left circumflex coronary artery (LCA). When administered into the RCA, adenosine and ATP exerted an identical and relatively small negative chronotropic effect on sinus node automaticity; the time to peak negative chronotropic effect was >/=7 s. When administered into the LCA, adenosine had no effect on sinus node automaticity, whereas ATP markedly suppressed sinus node automaticity. This effect of ATP 1) reached its peak in <2 s after its administration, 2) was short lasting, and 3) was completely abolished by either intravenous administration of the muscarinic cholinergic blocker atropine (0.2 mg/kg) or intra-LCA administration of 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP), a potent P2X(2/3) purinergic receptor (P2X(2/3)R) antagonist, but not by diinosine pentaphosphate (Ip(5)I), a potent inhibitor of P2X(1)R and P2X(3)R. Repetitive administrations of ATP were not associated with reduced effects, indicative of receptor desensitization, thereby excluding the involvement of the rapidly desensitized P2X(1)R in the action of ATP. It was concluded that ATP triggers a cardio-cardiac vagal depressor reflex by activating P2X(2/3)R located on vagal sensory nerve terminals localized in the left ventricle. Because these terminals mediate vasovagal syncope, these data could suggest a mechanistic role of extracellular ATP in this syndrome and, in addition, give further support to the hypothesis that endogenous ATP released from ischemic myocytes is a mediator of atropine-sensitive bradyarrhythmias associated with left ventricular myocardial infarction.

Positive inotropic and sustained anti‐β‐adrenergic effect of diadenosine pentaphosphate in human and guinea pig hearts. Role of dinucleotide receptors and adenosine receptors

Diadenosine polyphosphates are present intracellularly and in extracellular fluid due to release from secretory vesicles in platelets, chromaffin cells and other cells. This study investigates effects of diadenosine pentaphosphate (AP5A) on heart muscle function. Contractile force amplitude and action potential duration at 90% repolarization (APD90) were measured after challenge with AP5A 50 microm or isoproterenol 50-70 nM in guinea pig papillary muscles. Isoproterenol was given immediately after AP5A-exposure or after 45 min washout. AP5A was combined with antagonists to the purinergic P2 receptor (suramin 100 microm), the dinucleotide receptor [diinosine pentaphosphate 30 microm (IP5I)] or adenosine receptors [8-(P-sulfophenyl) theophylline 50 microm (8-SPT)]. Results are %-change (mean +/- SEM) from value before exposure. AP5A increased contractile force by 22 +/- 3%* (*P <0.05), and IP5I abolished this. AP5A prolonged APD90 by 7 +/- 2%*. AP5A significantly reduced response to isoproterenol acutely from 31 +/- 4* (controls) to 9 +/- 4% and after 45 min washout from 61 +/- 14* (controls) to 16 +/- 5%. 8-SPT abolished the sustained effect. Increase in contractile force by AP5A was confirmed in human atria trabecula preparations. AP5A increased contractile force and prolonged APD90. Contractile force increased by stimulation of the dinucleotide receptor in guinea pig myocardium. The sustained anti-beta-adrenergic effect of AP5A was due to adenosine receptor stimulation.

Diadenosine Tetraphosphate Protects against Injuries Induced by Ischemia and 6-Hydroxydopamine in Rat Brain

Diadenosine tetraphosphate (AP4A), an endogenous diadenosine polyphosphate, reduces ischemic injury in the heart. In this study, we report the potent and protective effects of AP4A in rodent models of stroke and Parkinson's disease. AP4A, given intracerebroventricularly before middle cerebral artery (MCA) ligation, reduced cerebral infarction size and enhanced locomotor activity in adult rats. The intravenous administration of AP4A also induced protection when given early after MCA ligation. AP4A suppressed terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) induced by hypoxia/reperfusion in primary cortical cultures, and reduced both ischemia-induced translocation of mitochondrial cytochrome c and the increase in cytoplasmic caspase-3 activity in vivo. The purinergic P2/P4 antagonist di-inosine pentaphosphate or P1-receptor antagonist sulfonylphenyl theophylline, but not the P2-receptor antagonist suramin, antagonized the effect of AP4A, suggesting that the observed protection is mediated through an anti-apoptotic mechanism and the activation of P1- and P4-purinergic receptors. AP4A also afforded protection from toxicity induced by unilateral medial forebrain bundle injection of 6-hydroxydopamine (6-OHDA). One month after lesioning, vehicle-treated rats exhibited amphetamine-induced rotation. Minimal tyrosine hydroxylase immunoreactivity was detected in the lesioned nigra or striatum. No KCl-induced dopamine release was found in the lesioned striatum. All of these indices of dopaminergic degeneration were attenuated by pretreatment with AP4A. In addition, AP4A reduced TUNEL in the lesioned nigra 2 d after 6-OHDA administration. Collectively, our data suggest that AP4A is protective against neuronal injuries induced by ischemia or 6-OHDA through the inhibition of apoptosis. We propose that AP4A may be a potentially useful target molecule in the therapy of stroke and Parkinson's disease.

Involvement of P2X2 and P2X3 receptors in neuropathic pain in a mouse model of chronic constriction injury

AbstractIn the present study, we examined the involvement of P2X receptors in neuropathic pain using the chronic constriction injury (CCI) model. The thermal and mechanical nociceptive thresholds were evaluated using the paw withdrawal test and a von Frey test, respectively. Also, the expression of P2X receptors in the dorsal root ganglia (DRG) was analyzed using quantitative real‐time RT‐PCR. In the acute stage of CCI (3–30 days after injury), thermal hyperalgesia and mechanical allodynia had developed and mRNA of P2X2 and P2X3 was upregulated in the ipsilateral DRG. The thermal hyperalgesia at 7 days was inhibited by intrathecal (i.t.) administration of P2X antagonists, pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonate (PPADS), 2′‐O‐(trinitrophenyl)adenosine 5′‐triphosphate, trisodium salt (TNP‐ATP) and diinosine pentaphosphate (IP5I). In contrast, mechanical allodynia (7 days) was inhibited by the i.t. administration of PPADS and TNP‐ATP but not IP5I. These pharmacological and gene expression data suggest that upregulated homomeric P2X3 and heteromeric P2X2 and P2X3 (P2X2/3) receptors augmented thermal hyperalgesia and mechanical allodynia, respectively, at the spinal level in acute stage of CCI. In the chronic stage (&gt;40 days), thermal hyperalgesia disappeared but the mechanical allodynia persisted. Only P2X3 mRNA was downregulated. PPADS and TNP‐ATP failed to restore the mechanical threshold to the normal level. These data suggest that the involvement of P2X receptors changed in the chronic stage of CCI. Drug Dev. Res. 59:104–111, 2003. © 2003 Wiley‐Liss, Inc.

P2Y receptor specific for diadenosine tetraphosphate in lung: selective inhibition by suramin, PPADS, Ip 5I, and not by MRS-2197

Extracellular dinucleotides, which act as signaling molecules in a variety of systems, may regulate fluid homeostasis in the human lung by activation of a specific P2Y receptor subtype. Previously, we presented evidence for a G protein-coupled P2Y receptor with high affinity for dinucleotides in both rat and human lung tissue. In a human bronchial epithelial cell line (HBE-1), diadenosine polyphosphates (Ap nA, n=2–6) increase intracellular Ca 2+. The aim of the present work was to find additional evidence that, in these cells, the receptors selectively activated by diadenosine polyphosphates are distinct from already known P2Y receptors, which are activated by the mononucleotides ATP or UTP. We tested antagonists suitable to classify P2Y receptor subtypes. The P2Y 1 receptor-selective antagonist 2′-deoxy- N 6-methyl adenosine 3′,5′-diphosphate (MRS-2197) did not affect Ca 2+ mobilization induced by diadenosine tetraphosphate (Ap 4A). However, suramin, pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS) and diinosine pentaphosphate (Ip 5I) inhibited the Ca 2+ response by 96%, 92% and 32%, respectively. Moreover, these results were confirmed by assessing the specific binding of [ 3H]Ap 4A to membranes from human and rat lung. Suramin (100 μM), PPADS (400 μM) and Ip 5I (200 μM), reduced [ 3H]Ap 4A binding in lung membrane preparations by 66%, 77% and 80%, respectively. The Ap 4A-induced Ca 2+ response in HBE-1 cells was inhibited to a much greater extent by these antagonists than the ATP- or UTP-evoked Ca 2+ rise. Thus, Ap 4A in lung epithelial cells also activates a still unidentified P2Y receptor that is specific for dinucleotides over mononucleotides.

Modulation of the rat hippocampal dinucleotide receptor by adenosine receptor activation.

Diadenosine pentaphosphate (Ap(5)A) and ATP stimulate an intracellular free calcium concentration ([Ca(2+)](I)) increase in rat hippocampal synaptosomes via different receptors as demonstrated by the lack of cross-desensitization between Ap(5)A and ATP responses. The ATP response was inhibited by P2 receptor antagonists and not by the dinucleotide receptor antagonist, diinosine pentaphosphate (Ip(5)I). In contrast, the Ap(5)A response was inhibited by Ip(5)I but not by P2 receptor antagonists. Studies in single hippocampal synaptic terminals showed that 31% of them responded to Ap(5)A by a [Ca(2+)](i) increase. Adenosine receptors (A(1), A(2A), and A(3)) were also present in isolated terminals as demonstrated by immunohistochemistry. The activation of A(1) or A(2A) receptors by specific agonists changed the sigmoid concentration-response curve for Ap(5)A (EC(50) = 33.5 +/- 4.5 microM) into biphasic curves. When the high-affinity adenosine receptors A(1) or A(2A) were activated, the Ap(5)A dose-response curves showed a high-affinity component with EC(50) values of 41.1 +/- 1.9 pM and 99.9 +/- 10.2 nM, respectively. The low-affinity component showed EC(50) values of 17.1 +/- 0.8 and 21.6 +/- 1.4 microM for A(1) and A(2A) receptor activation, respectively. However, the adenosine A(3) receptor activation induced a right shift of the dinucleotide concentration-response curve, showing an EC(50) value of 331.4 +/- 54.6 microM. In addition, in the presence of the A(2A) agonist, the Ap(5)A calcium influx responses were increased up to 300% of the control values. These results clearly demonstrate that the activation of presynaptic adenosine receptors is able to modulate the dinucleotide response in hippocampal nerve terminals.

TNP-ATP, a potent P2X 3 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 P2X 3 ligand-gated ion channel, as it is preferentially expressed on nociceptive C-fibers. In addition, P2X 3 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 μmol/kg and ED 50=70 μmol/kg, respectively). Furthermore, 2′-(or-3′)- O-(trinitrophenyl)adenosine 5′- tri-phosphate (TNP-ATP) potently ( IC 50=10 nM) blocked the functional activation of P2X 3 receptors in vitro and attenuated acetic acid-induced visceral pain. In the abdominal constriction assay, TNP-ATP ( ED 50=6.35 μmol/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) (ED 50=63.5 μmol/kg, i.p.) at reducing acetic acid-induced nociception. At the highest dose, TNP-ATP completely abolished nociceptive behavior, as did morphine ( ED 50=3 μmol/kg, i.p.). While TNP-ATP is also a potent antagonist of P2X 1 receptors, P2X 1 receptor mediated responses have not been shown in dorsal root ganglia and diinosine pentaphosphate, IP5I, a potent and selective P2X 1 receptor antagonist, was ineffective at reducing abdominal constrictions. Thus, the antinociceptive effects of TNP-ATP appear to be mediated through activation of homomeric P2X 3 and/or heteromeric P2X 2/3 receptors. Together, these results show that activation of P2X 3 containing receptors plays a role in the transmission of inflammatory visceral pain.

Changes in P2X receptor responses of sensory neurons from P2X3-deficient mice.

Dorsal root ganglion (DRG) neurons respond to ATP with transient, persistent or biphasic inward currents. In contrast, the ATP responses in nodose neurons are persistent. These sustained currents are also heterogeneous, with one component being accounted for by P2X2/3 receptors, and the residual response probably mediated by P2X2 receptors, although the direct evidence for this has been lacking. In the present study, we examined the P2X receptors on DRG and nodose neurons from P2X3-deficient (P2X3-/-) mice, using whole cell voltage-clamp recording and immunohistochemistry. We found that all P2X3-/- DRG neurons lacked rapidly desensitizing response to ATP, and both DRG and nodose neurons from P2X3-null mutant mice no longer responded to alpha,beta-methylene ATP (alphabetameATP). In contrast, ATP evoked persistent inward current in 12% of DRG neurons and 84% of nodose neurons from P2X3-/- mice. This retained persistent response to ATP on nodose neurons had an EC50 for ATP of 77 microm, was antagonized by Cibacron blue and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid, potentiated by Zn2+ and acidification, but not enhanced by ivermectin or diinosine pentaphosphate. 2',3'-O-Trinitrophenyl-ATP antagonized this response with an IC50 of 8 microm. All these properties are consistent with those of recombinant P2X2 homomeric receptors. Furthermore, specific P2X2 receptor immunoreactivity detected in wild-type sensory neurons was unaltered in null mutant mice. Therefore, the alphabetameATP-insensitive persistent responses on nodose neurons are likely to be mediated by P2X2 homomers, which contribute to 60% of currents evoked by 100 microm ATP in the wild type.

Presynaptic signalling mediated by mono- and dinucleotides in the central nervous system

Synaptosomal preparations from rat midbrain exhibit specific responses to both ATP and Ap 5A, which elicit a Ca 2+ entrance to the presynaptic terminals. Studies of isolated single terminals showed that not all the terminals contain ionotropic receptors for nucleotides, in fact only 46% of them do. Of these, 12% responded only to the dinucleotide Ap 5A, and 20% to the mononucleotide ATP. At the presynaptic level, diinosine pentaphosphate, Ip 5I, is a good tool to specifically block dinucleotide responses, which are inhibited at low nM concentration, versus the high μM concentrations required to block ATP responses. There is evidence for a presynaptic control of mononucleotide and dinucleotide responses, mediated by metabotropic and ionotropic receptors. Stimulation of adenosine A1 receptors increases the affinity of dinucleotide receptors by five orders of magnitude, from 30 μM to 680 pM for control and in the presence of A1 agonist, respectively.

Diadenosine Polyphosphates Activate a Ca2+-Dependent K+-Conductance in Porcine Aortic Smooth Muscle Cells via P2-Purinoceptors

Effects of the diadenosine polyphosphates P¹,P³-diadenosine triphosphate (Ap3A), P¹,P⁴-diadenosine tetraphosphate (Ap4A), P¹,P⁵-diadenosine pentaphosphate (Ap5A) and P¹,P⁶-diadenosine hexaphosphate (Ap6A) and of adenosine, ATP, ADP, AMP, UTP on smooth muscle cells from porcine aorta were examined. Membrane voltages and cellular conductances were measured in the slow whole cell configuration of the patch clamp technique. All four diadenosine polyphosphates, adenosine, AMP and ADP predominantly hyperpolarized membrane voltages with only occasional transient initial depolarizations whereas ATP and UTP led to sustained depolarizations. All four diadenosine polyphosphates increased cellular conductances. The effects of Ap5A on membrane voltages were almost completely inhibited by the putative P2-purinoceptor antagonist pyridoxal-phosphate-6-azophenyl-2’,4’-disulphonic acid (PPADS, 10 μmol/l) and only partially reduced by the putative A₂-purinoceptor antagonist 3,7-dimethyl-1-propragyl-xanthine (DMPX, 10 μmol/l) or the Ap4A-receptor antagonist diinosine pentaphosphate (Ip5I, 10 μmol/l). The adenosine-induced hyperpolarization was partially reduced by the putative A₁-purinoceptor antagonist 8-cyclopentyl-1,3-dipropargylxanthine (DPCPX, 0.1 μmol/l) or by DMPX while PPADS or Ip5I were without effects. Ap5A-induced hyperpolarizations were inhibited by Ba²⁺ and clotrimazole but not by glibenclamide. We conclude that diadenosine polyphosphates activatepredominantly a Ca²⁺-dependent K⁺-conductance in smooth muscle cells obtained from porcine aorta most likely mediated via P2Y-purinoceptors and possibly partially also by Ap4A receptors.

P2X 2 characteristics of the ATP receptor coupled to [Ca 2+] i increases in cultured Purkinje neurons from neonatal rat cerebellum

P2X receptors present in cerebellar Purkinje cells have been studied by recording ATP-elicited [Ca 2+] i signals from immuno-identified (calbindin +) cells in culture using fura-2 microfluorescence. The [Ca 2+] i increases evoked by ATP were mimicked by 2MeSATP but not by α,β-meATP and other purinoceptor agonists. The selective P2X 1 antagonist diinosine pentaphosphate failed to inhibit ATP-elicited [Ca 2+] i transients, but suramin and PPADS rapidly and reversibly blocked the [Ca 2+] i responses to ATP and 2MeSATP. The IC 50 values for suramin and PPADS inhibition were 48.7±4.4 and 5.9±0.3 μM, respectively. Both antagonists blocked completely the signal elicited by ATP, revealing that there was not a separate antagonist-insensitive P2X receptor population in Purkinje cells. The effect of ATP was potentiated by Zn 2+ and H + ions. A one unit acidification from pH 7.4 to 6.4 enhanced by 172% the [Ca 2+] i transient elicited by an intermediate concentration of ATP. Conversely, alkalinization of the medium to pH 8.4 reduced the ATP response by 88%. This combination of pharmacological and modulatory properties indicates that endogenous P2X receptors present in Purkinje neurons are formed by P2X 2 subunits, rather than the more abundantly expressed P2X 4 purinoceptor subunits.

Presence of dinucleotide and ATP receptors in human cerebrocortical synaptic terminals

Human cerebrocortical synaptic terminals elicited concentration-dependent Ca 2+ transients after Ap 5A (diadenosine pentaphosphate) and ATP stimulation, with EC 50 values of 23.44±3.70 μM and 11.48±2.12 μM, respectively. The lack of cross-desensitisation and the selective antagonism by Ip 5I (diinosine pentaphosphate), suggests the activation of a dinucleotide receptor by Ap 5A, and a P2X receptor by ATP. Ap 5A Ca 2+ transients were partially abolished by ω-conotoxin GVI-A (53%), suggesting the participation of a N-type Ca 2+ channel in the dinucleotide response. ATP effect on Ca 2+ entry was abolished by nicardipine (44%) and by ω-conotoxin GVI-A (52%), suggesting the participation of L- and N-type Ca 2+ channels. These data suggest that Ap 5A and ATP activate dinucleotide and P2X receptors, respectively, in human brain synaptic terminals.

Antagonism of P2X receptors in guinea-pig vas deferens by diinosine pentaphosphate

Diinosine pentaphosphate (Ip 5I) antagonized contractions, mediated via P2X receptors, evoked by diadenosine pentaphosphate (Ap 5A) and ATP in the guinea-pig isolated vas deferens with pA 2 values of 6.4±0.17 (10 d.f.) and 6.5±0.10 (10 d.f.), respectively. Ip 5I (30 μM) did not affect contractile responses evoked by noradrenaline. Ip 5I (up to 100 μM) did not antagonize P2Y receptors in the guinea-pig taenia coli, nor P1 or P2 receptors in the guinea-pig left atrium.

Diinosine polyphosphates, a group of dinucleotides with antagonistic effects on diadenosine polyphosphate receptor.

A new family of dinucleotide derivatives, diinosine polyphosphates, has been synthesized through the use of the enzyme 5' adenylic acid deaminase from Aspergillus sp., starting from the corresponding diadenosine polyphosphates. Functional studies were performed on rat brain synaptic terminals in which a dinucleotide receptor has been described that is specific for adenine dinucleotides. The results demonstrated that diinosine polyphosphates did not behave as agonists on the diadenosine polyphosphate receptor (also know as P4 purinoceptor), but they were very efficient as antagonists in abolishing the Ca2+ responses elicited by diadenosine pentaphosphate. The IC50 values for diinosine triphosphate, diinosine tetraphosphate, and diinosine pentaphosphate were 4.90 +/- 0.10 microM, 8.33 +/- 0.22 microM, and 4.23 +/- 0.12 nM, respectively. The diinosine polyphosphates also antagonized the ATP receptors present in synaptic terminals, showing IC50 values of 100.08 +/- 5.72 microM for diinosine triphosphate, 29.51 +/- 1.40 microM for diinosine tetraphosphate and 27.75 +/- 1.65 microM for diinosine pentaphosphate. The antagonistic ability of these diinosine nucleotides was studied in comparison with other P1 and P2 purinoceptor antagonists, such as suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, and 8-cyclopentyl-1,3-dipropylxanthine. These purinergic antagonists did not inhibit the response of the P4 purinoceptor; only the diinosine polyphosphates were able to act as antagonists on the dinucleotide receptor. Suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid attenuated the responses elicited by ATP, as did the diinosine polyphosphate compounds. The most antagonistic diinosine polyphosphate for the dinucleotide and ATP receptors was diinosine pentaphosphate, which was 6000 times more selective for the P4 purinoceptor than it was for the ATP receptor.