Dinucleotide Receptors Research Articles

Dinucleoside polyphosphates and their interaction with other nucleotide signaling pathways

Dinucleoside polyphosphates or Ap(n)A are a family of dinucleotides formed by two adenosines joined by a variable number of phosphates. Ap(4)A, Ap(5)A, and Ap(6)A are stored together with other neurotransmitters into secretory vesicles and are co-released to the extracellular medium upon stimulation. These compounds can interact extracellularly with some ATP receptors, both metabotropic (P2Y) and ionotropic (P2X). However, specific receptors for these substances, other than ATP receptors, have been described in presynaptic terminals form rat midbrain. These specific dinucleotide receptors are of ionotropic nature and their activation induces calcium entry into the terminals and the subsequent neurotransmitter release. Calcium signals that cannot be attributable to the interaction of Ap(n)A with ATP receptors have also been described in cerebellar synaptosomes and granule cell neurons in culture, where Ap(5)A induces CaMKII activation. In addition, cerebellar astrocytes express a specific Ap(5)A receptor coupled to ERK activation. Ap(5)A engaged to MAPK cascade by a mechanism that was insensitive to pertussis toxin and required the involvement of src and ras proteins. Diadenosine polyphosphates, acting on their specific receptors and/or ATP receptors, can also interact with other neurotransmitter systems. This broad range of actions and interactions open a promising perspective for some relevant physiological roles for the dinucleotides. However, the physiological significance of these compounds in the CNS is still to be determined.

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.

GABA modulates presynaptic signalling mediated by dinucleotides on rat synaptic terminals.

Diadenosine pentaphosphate (Ap(5)A) elicits Ca(2+) transients in isolated rat midbrain synaptic terminals acting through specific ionotropic dinucleotide receptors. The activation of GABA(B) receptors by baclofen changes the sigmoidal concentration-response curve for Ap(5)A (EC(50) = 44 microM) into biphasic curves. Thus, when GABA(B) receptors are activated, the curve shows a high-affinity component in the picomolar range (EC(50) = 77 pM) and a low-affinity component in the micromolar range (EC(50) = 17 microM). In addition, in the presence of GABA or baclofen, Ap(5)A calcium responses are increased up to 50% over the control values. Saclofen, a specific antagonist of GABA(B) receptors, blocks the potentiatory effect of baclofen. As occurs with Ap(5)A, GABA(B) receptors are also capable to modulate diguanosine pentaphosphate (Gp(5)G)-induced calcium responses. The combination of immunocytochemical and microfluorimetric techniques carried out on single synaptic terminals have shown that in the presence of baclofen, 64% of the terminals responding to 100 microM Ap(5)A are also able to respond to 100 nM Ap(5)A. This value is close to the percentage of synaptic terminals responding to Ap(5)A and labeled with the anti-GABA(B) receptor antibody (69%). The activity of cyclic AMP-dependent protein kinase (PKA) seems to be involved in the potentiatory effect of GABA(B) receptors on Ap(5)A calcium responses, because PKA activation by forskolin or dibutiryl cyclic AMP blocks the potentiatory effect of baclofen, whereas PKA inhibition facilitates calcium signaling mediated by Ap(5)A. These results demonstrate that the activation of presynaptic GABA(B) receptors is able to modulate dinucleotide responses in synaptic terminals.

Presence of functional ATP and dinucleotide receptors in glutamatergic synaptic terminals from rat midbrain.

Glutamatergic terminals from rat midbrain were characterized by immunolocalization of synaptophysin and the vesicular glutamate transporters, either VGLUT1 or VGLUT2. Terminals containing these markers represent about 31% (VGLUT1) and 16% (VGLUT2) of the total synaptosomal population. VGLUT1-positive glutamatergic terminals responded to ATP or P1,P 5-di(adenosine-5') pentaphosphate (Ap5A) with an increase in the intrasynaptosomal calcium concentration as measured by a microfluorimetric technique in single synaptosomes. Roughly 20% of the VGLUT1-positive terminals responded to ATP, 13% to Ap5A and 11% to both agonists. Finally 56% of the terminals labeled with the anti-VGLUT1 antibody did not show any calcium increase in response to ATP or Ap5A. A similar response distribution was also observed in the VGLUT2-positive terminals. The Ca2+ responses induced by ATP and Ap5A in the glutamatergic terminals could be selectively inhibited by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 80 micro m) and P1,P 5-di(inosine-5') pentaphosphate (Ip5I, 100 nm), respectively. Both ATP and Ap5A, once assayed in the presence of extrasynaptosomal calcium, were able to induce a concentration-dependent glutamate release from synaptosomal populations, EC50 values being 21 micro m and 38 micro m for ATP and Ap5A, respectively. Specific inhibition of glutamate release was obtained with PPADS on the ATP effect and with Ip5I on the dinucleotide response, indicating that separate receptors mediate the secretory effects of both compounds.

Ca2+ signalling in brain synaptosomes activated by dinucleotides.

Diadenosine polyphosphates are a family of dinucleotides formed by two adenosines joined by a variable number of phosphates. Diadenosine tetraphosphate, Ap4A, diadenosine pentaphosphate Ap5A, and diadenosine hexaphosphate, Ap6A, are stored in synaptic vesicles and are released upon nerve terminal depolarization. At the extracellular level, diadenosine polyphosphates can stimulate presynaptic dinucleotide receptors. Responses to diadenosine polyphosphates have been described in isolated synaptic terminals (synaptosomes) from several brain areas in different animal species, including man. Dinucleotide receptors are ligand-operated ion channels that allow the influx of cations into the terminals. These cations reach a threshold for N- and P/Q-type voltage-dependent calcium channels, which become activated. The activation of the dinucleotide receptor together with the activation of these calcium channels triggers the release of neurotransmitters. The ability of Ap5A to promote glutamate, GABA or acetylcholine release has been recently described by the present authors in rat midbrain synaptosomes.

Independent receptors for diadenosine pentaphosphate and ATP in rat midbrain single synaptic terminals.

Diadenosine pentaphosphate (Ap5A) and adenosine 5'-triphosphate (ATP) stimulate a intrasynaptosomal calcium concentration [Ca(2+)](i) increase via specific purinergic receptors in rat midbrain synaptosomes, although nothing is known about their distribution in presynaptic terminals. A microfluorimetric technique to measure [Ca(2+)](i) increase using the dye FURA-2AM, has permitted study of the presence of dinucleotide and P2X receptors in independent isolated synaptic terminals. Our results demonstrate the existence of three populations of synaptosomes: one with dinucleotide receptors (12%), another with P2X receptors (20%) and a third with both (14%). It has been possible to demonstrate that the activation of these receptors occurs only in the presence of extracellular Ca(2+) and that it is also coupled with voltage-dependent Ca(2+) channels. Finally 54% of the synaptosomes that responded to K(+) did not present any calcium increase mediated by the nucleotides used. In summary, ATP and dinucleotides exhibit specific ionotropic receptors that can coexist or not on the same synaptic terminal.

Single GABAergic synaptic terminals from rat midbrain exhibit functional P2X and dinucleotide receptors, able to induce GABA secretion

GABAergic terminals from rat midbrain characterized by immunolocalization of glutamic acid decarboxylase and/or the vesicular inhibitory amino acid transporter respond to ATP or P1,P5‐di(adenosine‐5′) pentaphosphate (Ap5A) with an increase in the intrasynaptosomal calcium concentration measured by a microfluorimetric technique in single synaptic terminals. The ATP response is mediated through the activation of P2X receptors with an abundant presence of P2X3 subunits. Ap5A, however, exerts its effects by acting through a different receptor termed the dinucleotide receptor. Both receptors, once activated in the presence of extrasynaptosomal calcium, induce a concentration‐dependent GABA release from synaptosomal populations with EC50 values of 16 and 20 µm for ATP and Ap5A, respectively. Specific inhibition of GABA release is obtained with pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (80 µm) on the ATP effect and with P1,P5‐di(inosine‐5′) pentaphosphate (100 nm) on the dinucleotide receptor.

Single GABAergic synaptic terminals from rat midbrain exhibit functional P2X and dinucleotide receptors, able to induce GABA secretion.

GABAergic terminals from rat midbrain characterized by immunolocalization of glutamic acid decarboxylase and/or the vesicular inhibitory amino acid transporter respond to ATP or P(1),P(5)-di(adenosine-5') pentaphosphate (Ap(5)A) with an increase in the intrasynaptosomal calcium concentration measured by a microfluorimetric technique in single synaptic terminals. The ATP response is mediated through the activation of P2X receptors with an abundant presence of P2X(3) subunits. Ap(5)A, however, exerts its effects by acting through a different receptor termed the dinucleotide receptor. Both receptors, once activated in the presence of extrasynaptosomal calcium, induce a concentration-dependent GABA release from synaptosomal populations with EC(50) values of 16 and 20 microM for ATP and Ap(5)A, respectively. Specific inhibition of GABA release is obtained with pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (80 microM) on the ATP effect and with P(1),P(5)-di(inosine-5') pentaphosphate (100 nM) on the dinucleotide receptor.

Adenosine triphosphate and diadenosine pentaphosphate induce [Ca(2+)](i) increase in rat basal ganglia aminergic terminals.

Synaptosomal preparations from rat midbrain exhibit specific responses to both ATP and Ap(5)A, which stimulate a [Ca(2+)](i) increase in the presynaptic terminals via specific ionotropic receptors, termed P2X, and diadenosine polyphosphate receptors. Aminergic terminals from rat brain basal ganglia were characterized by immunocolocalization of synaptophysin and the vesicular monoamine transporter VMAT2 and represent 29% of the total. These aminergic terminals respond to ATP and/or Ap(5)A with an increase in the intrasynaptosomal calcium concentration as measured by a microfluorimetric technique. This technique, which allows single synaptic terminals to be studied, showed that roughly 8.2% +/- 1.6% of the aminergic terminals respond to ATP, 16.9% +/- 1.3% respond to Ap(5)A, 32.6% +/- 0.8% to both, and 42.3% +/- 1.5% of them have no response. Immunological studies performed with antibodies against ionotropic ATP receptor subunits showed positive labelling with anti-P2X(3) antibodies in 39% of the terminals. However, colocalization studies of VMAT and P2X(3) receptor subunit indicate that only 25% of the aminergic terminals also contain this receptor subtype. These results demonstrate that the aminergic terminals from the rat brain basal ganglia are to a large extent under the modulation of presynaptic nucleotide and dinucleotide receptors.

Presynaptic diadenosine polyphosphate receptors: Interaction with other neurotransmitter systems

AbstractDiadenosine polyphosphates (ApnA n = 2–6) are natural compounds that can play a neurotransmitter role in the synaptic terminals of the central nervous system. Microfluorimetric studies of [Ca2+]i in single synaptic terminals have shown the presence of specific ionotropic receptors for nucleotides and dinucleotides. These dinucleotide receptors may or may not coexist at the same terminal. Aminergic terminals from rat basal ganglia have been immunologically characterised by the presence of the vesicular monoamine transporter 2 after the functional studies. Fifty‐eight percent of these terminals respond to nucleotides, and of these, 17% respond only to Ap5A. Cholinergic terminals from rat midbrain were immunologically characterised by the vesicular acetylcholine transporter. Sixty‐three percent of these terminals responded to nucleotides, and of these, 22% responded only to Ap5A. The presynaptic ionotropic dinucleotide receptors can coexist not only with the ATP receptors, but also with various subtypes of nicotinic receptors. GABAergic terminals from rat midbrain were immunologically characterised by the vesicular inhibitory amino acid transporter. Fifty‐nine percent of these terminals responded to nucleotides, and of these, 17% responded only to Ap5A. The presynaptic dinucleotide receptors, when stimulated, are able to induce the GABA release from synaptosomal preparations. These data clearly show the broad interaction of nucleotides and dinucleotides with other neurotransmitter systems. Drug Dev. Res. 52:239–248, 2001. © 2001 Wiley‐Liss, Inc.

Diadenosine polyphosphates as extracellular signal molecules

AbstractDinucleoside 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.

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.

Modulation of the dinucleotide receptor present in rat midbrain synaptosomes by adenosine and ATP.

Diadenosine polyphosphates activate dinucleotide receptors in rat midbrain synaptic terminals. The agonist with highest affinity at this receptor, diadenosine pentaphosphate (Ap(5)A), elicits Ca(2+) transients at concentrations ranging from 10(-7) to 10(-3) M with a single-phase curve and an EC(50) value of 56.21+/-1.82 microM. Treatment of synaptosomal preparations with alkaline phosphatase (AP) changes the dose-response control curve into a biphasic one presenting two EC(50) values of 6.47+/-1.25 nM and 11.16+/-0.83 microM respectively. The adenosine A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX) reversed the biphasic concentration-response for Ap(5)A curve in the presence of AP, to a monophasic one with an EC(50) value of 76.05+/-7.51 microM. The application of adenosine deaminase produced the same effect as DPCPX, the EC(50) value for Ap(5)A, in the presence of AP being 18.62+/-4.03 microM. Activation of the adenosine A(1) receptor by means of cyclohexyladenosine (CHA) shifted the dose response curve for Ap(5)A to the left, resulting in a monophasic curve with an EC(50) of 5. 01+/-0.02 pM. The destruction of extrasynaptosomal nucleotides by AP or the addition of pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), a broad P2 antagonist compound, enhance maximal effect of the Ap(5)A up to 55.6% on the dose response curve, thus suggesting a negative modulation by P2 receptors. In a summary, ATP and adenosine present at the extra-synaptosomal space, are relevant natural modulators of the dinucleotide receptor, via P2 and adenosine A(1) receptors respectively.

Chapter 32 Diadenosine polyphosphates, extracellular function and catabolism

Publisher Summary Diadenosine polyphosphates (Ap n A n = 2-6) are natural compounds that play an important role inside the cell, in both the nucleus and at the cytosol. The transport of Ap n A to cytoplasmatic storage granules provides both the end of their intracellular actions and at the same time the way to be released by controlled exocytosis. Diadenosine polyphosphates are co-stored and co-released from a large variety of synaptic terminals. Their physiological extracellular concentration range can be expected to be between the nM and low μM. Binding studies have detected the presence of high affinity sites, correlating with the physiological concentrations . From a functional point of view, specific receptors, called “dinucleotide receptors” or “P4 receptors,” have been described in presynaptic terminals and elicit calcium entrance through ionotropic receptors that are not susceptible to inhibition by voltage dependent calcium channel toxins. The extracellular actions of diadenosine polyphosphates are terminated by the enzymatic hydrolysis by an ecto-diadenosine polyphosphate hydrolase.

Diadenosine polyphosphates evoke Ca2+ transients in guinea-pig brain via receptors distinct from those for ATP.

1. The ability of diadenosine polyphosphates, namely P1,P2-di(adenosine) pyrophosphate (Ap2A), P1,P3-di(adenosine) triphosphate (Ap3A), P1,P4-di(adenosine) tetraphosphate (Ap4A), P1,P5-di(adenosine) pentaphosphate (Ap5A) and P1,P6-di(adenosine) hexaphosphate (Ap6A) to evoke Ca2+ signals in synaptosomes prepared from three different regions of the guinea-pig brain was examined. 2. In synaptosomal preparations from the paleocortex (cortex), diencephalon/brainstem (midbrain) and cerebellum all the dinucleotides evoked Ca2+ signals that were concentration dependent over the range 1-300 microM. ATP and its synthetic analogues, alpha,beta-methylene ATP, 2-methylthio ATP and adenosine 5'-O-(2-thio)diphosphate (all 100 microM) also evoked Ca2+ signals in these preparations. 3. In the midbrain and cerebellum preparations, responses to ATP and its analogues were attenuated or abolished by the P2 receptor antagonist suramin (100 microM) but responses to the dinucleotides were not. Also, desensitization by a dinucleotide blocked responses to dinucleotides but not mononucleotides, and desensitization by a mononucleotide blocked responses to mononucleotides but not dinucleotides. 4. In cortical preparations, suramin (100 microM) blocked responses to both classes of nucleotides. Furthermore, there was mutual cross-desensitization between the mono- and dinucleotides. 5. The adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine, did not affect responses evoked by the dinucleotides, nor did the pyrimidine UTP. 6. It is concluded that there are specific dinucleotide receptors, activated by diadenosine polyphosphates, but not ATP or UTP, on synaptic terminals in guinea-pig diencephalon/ brainstem and cerebellum. These receptors bear a similarity to the dinucleotide receptor (P4 receptor) in rat brain. In guinea-pig cerebral cortex synaptosomes, diadenosine polyphosphates appear to act via the same receptor as ATP.

Dinucleotide receptor modulation by protein kinases (protein kinases A and C) and protein phosphatases in rat brain synaptic terminals.

The diadenosine polyphosphates, diadenosine tetraphosphate and diadenosine pentaphosphate (Ap5A), can activate an ionotropic dinucleotide receptor that induces Ca2+ transients into synaptosomes prepared from rat brain. This receptor, also termed the P4 purinoceptor, is sensitive only to adenine dinucleotides and is insensitive to ATP. Studies on the modulatory role of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatases on the response of diadenosine polyphosphate receptors were performed by measuring the changes in the intracellular Ca2+ levels with fura-2. Activation and inhibition of PKA were carried out by means of forskolin and the PKA inhibitory peptide (PKA-IP), respectively. The Ap5A response was inhibited by forksolin to 35% of control values, but PKA-IP induced an increase of 37%. The effect of PKC activation was similar to that observed for PKA. PKC stimulation with phorbol 12,13-dibutyrate produced an inhibition of 67%, whereas the PKC inhibitors staurosporine and PKC inhibitory peptide enhanced the responses elicited by Ap5A to 40% in both cases. Protein phosphatase inhibitors diminished the responses elicited by Ap5A to 17% in the case of okadaic acid, to 50% for microcystin, and to 45% in the case of cyclosporin A. Thus, the activity of dinucleotide receptors in rat brain synaptosomes appears to be modulated by phosphorylation/dephosphorylation. These processes could be of physiological significance in the control of transmitter release from neurons that are postsynaptic to nerves that release diadenosine polyphosphates.

Adenine dinucleotides: a novel class of signalling molecules.

1. Adenine dinucleotides (Ap3A, Ap4A, Ap5A, Ap6A) are stored in secretory granules of thrombocytes, chromaffin cells and neuronal cells. After release into the extracellular space, the dinucleotides exhibit divergent biological effects on a variety of target cells and organs. The dinucleotides are metabolized by soluble enzymes in the blood plasma as well as by membrane-bound ectoenzymes of endothelial cells, smooth muscle cells, and other cell types. 2. The enzymatic cleavage of the dinucleotides plays a dual role for their biological function: (a) termination of the signal; and (b) generation of purinergically active products such as ATP, ADP and finally adenosine. In contrast to ATP the dinucleotides are long-lived purine nucleotides in the blood. 3. The potential role of the dinucleotides as signalling molecules has been demonstrated in several systems. The adenosine polyphosphates have autocrine function for thrombocytes. Ap3A at low concentration reversibly activates isolated platelets. The mechanism of activation has been elucidated by showing a continuous cleavage of Ap3A, leading to the formation of ADP which is a known agonist of the P2T receptor on thrombocytes. Ap4A and other dinucleotides act as antagonists and inhibit platelet activation. 4. The vasotone of perfused isolated arteries as well as of resistance vessels in the beating heart is differentially influenced by adenine dinucleotides. While Ap3A and Ap4A exhibit relaxing effects at micromolar concentrations, Ap5A and Ap6A elicit vasoconstriction in these vessels. 5. In rat kidney mesangial cells adenine dinucleotides efficiently promote growth. Stimulation of DNA synthesis by various growth factors is enhanced synergistically. ApnA significantly increase the expression of the early growth response gene Egr-1. 6. The specificity and, in some tissues, the uniqueness of effects evoked by dinucleotides may be mediated by genuine dinucleotide receptors (P4) or by specialized P2 receptors (P2D).