Presence of diadenosine polyphosphates in human tears

Diadenosine polyphosphates are naturally occurring substances that facilitate tear secretion. The occurrence of these dinucleotides in human tears has been established and quantified by an HPLC technique and phosphodiesterase treatment. The concentration of these compounds found in tears was 2.0+/-2.2 nM for diadenosine triphosphate (Ap3A), 108.0+/-18.3 nM for diadenosine tetraphosphate (Ap4A) and 37.0+/-6.2 nM for diadenosine pentaphosphate (Ap5A). When subjects were treated with topical ocular anaesthesia, the concentrations of Ap3A, Ap4A and Ap5A changed to 1.5+/-1.7 nM, 189.3+/-19.5 nM and 112.6+/-12.3 nM, respectively. Ap4A and Ap5A increased tear secretion in rabbits, the effect presenting an EC50 value of 19.0+/-1.2 ng/microl and 11.4+/-1.3 ng/microl respectively. In conclusion, diadenosine polyphosphates are released from the corneal epithelium, they stimulate tear production and therefore they may be considered as physiological modulators of tear secretion.     

Possible role of diadenosine polyphosphates in glucose-stimulated insulin release: effects of NaF upon metabolic and functional variables in rat isolated islets

Diadenosine polyphosphates, such as diadenosine triphosphate (A2P3) and diadenosine tetraphosphate (A2P4), were recently proposed to participate in the stimulus-secretion coupling for nutrient-stimulated insulin release. Since NaF, an inhibitor of inorganic pyrophosphatase, was reported to lower A2P3 and A2P4 content in glucose-stimulated pancreatic islets, its effects upon metabolic, cationic, biosynthetic and secretory variables in rat pancreatic islets were investigated in the present study. Up to a concentration close to 0.1 mM, NaF failed to affect most of these variables, except for a decrease in 45Ca net uptake. Much higher concentrations of NaF (e.g. 5.0 mM) were required to cause inhibition of the metabolic, ionic, biosynthetic and secretory responses of the islets to nutrient secretagogues. Yet, even at this high concentration, NaF failed to lower the islet content in tritiated A2P3 and A2P4 in islets prelabelled with [2,8-3H]adenosine and failed to prevent the glucose-induced increase in such a content. It is concluded, therefore, that NaF may not represent a suitable tool to assess the participation of diadenosine polyphosphates in the process of nutrient-induced insulin secretion.     

Diadenosine pentaphosphate affects electrical activity in guinea pig atrium via activation of potassium acetylcholine-dependent inward rectifier

Diadenosine pentaphosphate (Ap5A) belongs to the family of diadenosine polyphosphates, endogenously produced compounds that affect vascular tone and cardiac performance when released from platelets. The previous findings indicate that Ap5A shortens action potentials (APs) in rat myocardium via activation of purine P2 receptors. The present study demonstrates alternative mechanism of Ap5A electrophysiological effects found in guinea pig myocardium. Ap5A (10^?4 M) shortens APs in guinea pig working atrial myocardium and slows down pacemaker activity in the sinoatrial node. P1 receptors antagonist DPCPX (10^?7 M) or selective GIRK channels blocker tertiapin (10^?6 M) completely abolished all Ap5A effects, while P2 blocker PPADS (10^?4 M) was ineffective. Patch-clamp experiments revealed potassium inward rectifier current activated by Ap5A in guinea pig atrial myocytes. The current was abolished by DPCPX or tertiapin and therefore was considered as potassium acetylcholine-dependent inward rectifier (I _KACh). Thus, unlike rat, in guinea pig atrium Ap5A produces activation of P1 receptors and subsequent opening of KACh channels leading to negative effects on cardiac electrical activity.     

Diadenosine polyphosphates induce intracellular Ca2+ mobilization in human neutrophils via a pertussis toxin sensitive G-protein.

The diadenosine polyphosphates diadenosine 5',5"'-P1,P3-triphosphate (Ap3A), diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), diadenosine 5',5"'-P1,P5-pentaphosphate (Ap5A) and diadenosine 5',5"'-P1,P6-hexaphosphate (Ap6A) all stimulated increases in intracellular Ca2+ in human neutrophils. Maximal increases in intracellular Ca2+ of 650 nM were obtained at dinucleotide concentrations of 500-700 microM. These increases in intracellular, Ca2+ were completely abolished by pre-treatment of the neutrophils with pertussis toxin and were hardly affected when the extracellular buffer was devoid of Ca2+. On the other hand, adenosine triphosphate (ATP) could stimulate much greater increases in intracellular Ca2+ (up to 1.1 microM) at much lower concentrations (half maximal responses obtained at around 5 microM ATP). Receptor de-sensitization experiments indicate that human neutrophils may possess two types of P2-purinoceptors. The first of these may bind ATP (but not the dinucleotides) with high affinity whilst the second may bind the dinucleotides with lower affinity and also bind ATP.     

The enzymatic activity of the VEGFR2 receptor for the biosynthesis of dinucleoside polyphosphates

The group of dinucleoside polyphosphates encompasses a large number of molecules consisting of two nucleosides which are connected by a phosphate chain of variable length. While the receptors activated by dinucleoside polyphosphates as well as their degradation have been studied in detail, its biosynthesis has not been elucidated so far. Since endothelial cells released the dinucleoside polyphosphate uridine adenosine tetraphosphate (Up₄A), we tested cytosolic proteins of human endothelial cells obtained from dermal vessels elicited for enzymatic activity. When incubated with ADP and UDP, these cells showed increasing concentrations of Up₄A. The underlying enzyme was isolated by chromatography and the mass spectrometric analysis revealed that the enzymatic activity was caused by the vascular endothelial growth factor receptor 2 (VEGFR2). Since VEGFR2 but neither VEGFR1 nor VEGFR3 were capable to synthesise dinucleoside polyphosphates, Tyr-1175 of VEGFR2 is most likely essential for the enzymatic activity of interest. Further, VEGFR2-containing cells like HepG2, THP-1 and RAW264.7 were capable of synthesising dinucleoside polyphosphates. VEGFR2-transfected HEK 293T/17 but not native HEK 293T/17 cells synthesised dinucleoside polyphosphates in vivo too. The simultaneous biosynthesis of dinucleoside polyphosphates could amplify the response to VEGF, since dinucleoside polyphosphates induce cellular growth via P2Y purinergic receptors. Thus the biosynthesis of dinucleoside polyphosphates by VEGFR2 may enhance the proliferative response to VEGF. Given that VEGFR2 is primarily expressed in endothelial cells, the biosynthesis of dinucleoside polyphosphates is mainly located in the vascular system. Since the vasculature is also the main site of action of dinucleoside polyphosphates, activating vascular purinoceptors, blood vessels appear as an autocrine system with respect to dinucleoside polyphosphates. We conclude that VEGFR2 receptor is capable of synthesising dinucleoside polyphosphates. These mediators may modulate the effects of VEGFR2 due to their proliferative effects.     

Effect of diadenosine polyphosphates on Ca2+ ATPase activity

 Diadenosine tri-, tetra-, penta-, and hexaphosphate (Ap₃A, Ap₄A, Ap₅A and Ap₆A) have been described as having various effects on vascular tone depending on the number of phosphate groups. This study examined the effect of diadenosine polyphosphates on Ca²⁺ ATPase activity. The activity of the enzyme was measured spectrophotometrically as the difference in hydrolysis of ATP in the presence and absence of Ca²⁺ with various concentrations of ATP and diadenosine polyphosphates. The diadenosine polyphosphates increased the activity of the Ca²⁺ ATPase. The effect tended to be stronger with Ap₅A and Ap₆A than with Ap₃A and Ap₄A in the order of potency: Ap₃A≈AP₄A<Ap₅A≈AP₆A. The stimulatory effect of diadenosine polyphosphates was not competitive with that of ATP, suggesting an allosteric activation of Ca²⁺ ATPase by diadenosine polyphosphates. This effect may be physiologically relevant for limiting the increase in cytosolic free Ca²⁺ concentration elicited by diadenosine polyphosphates by receptor activation and modulating Ca²⁺ ATPase function under resting conditions. Received: 24 January 1997 / Accepted: 9 May 1997     

Biochemical evidence for an ecto alkaline phosphodiesterase I in human airways

Because dinucleotides are signaling molecules that can interact with cell surface receptors and regulate the rate of mucociliary clearance in lungs, we studied their metabolism by using human airway epithelial cells. A membrane-bound enzyme was detected on the mucosal surface of polarized epithelia that metabolized dinucleotides with a broad substrate specificity (diadenosine polyphosphates and diuridine polyphosphates [Up(n)U], n = 2 to 6). The enzymatic reaction yielded nucleoside monophosphates (NMP) and Np(n)(-)(1) (N = A or U), and was inhibited by nucleoside 5'-triphosphates (alpha,betamet adenosine triphosphate [ATP] > ATP >/= uridine triphosphate > guanidine triphosphate > cytidine triphosphate). The apparent Michaelis constant (K(m,app)) and apparent maximal velocity (V(max,app)) for [(3)H]Up(4)U were 22 +/- 4 microM and 0.24 +/- 0.05 nmoles. min(-)(1). cm(-)(2), respectively. Thymidine 5'-monophosphate p-nitrophenyl ester and adenosine diphosphate (ADP)- ribose, substrates of ecto alkaline phosphodiesterase I (PDE I) activities, were also hydrolyzed by the apical surface of airway epithelia. ADP-ribose competed with [(3)H]Up(4)U, with a K(i) of 23 +/- 3 microM. The metabolism of ADP-ribose and Ap(4)A was not affected by inhibitors of cyclic nucleotide phosphodiesterases (3-isobutyl-1-methylxanthine, Ro 20-1724, and 1,3-dipropyl-8-p-sulfophenylxanthine), but similarly inhibited by fluoride and N-ethylmaleimide. These results suggest that a PDE I is responsible for the hydrolysis of extracellular dinucleotides in human airways. The wide substrate specificity of PDE I suggests that it may be involved in several signaling events on the luminal surface of airway epithelia, including purinoceptor activation and cell surface protein ribosylation.     

Diadenosine polyphosphate-induced inhibition of cardiac KATP channels: Operative state-dependent regulation by a nucleoside diphosphate

It has been proposed that the regulatory action of mononucleotides, such as ATP and UDP, on cardiac ATP-sensitive K⁺ (K_ATP) channels is determined by the state of the channel. Recently, dinucleotides, such as diadenosine tetraphosphate (Ap₄A) and diadenosine pentaphosphate (Ap₅A), have been recognized as novel intracellular ligands of cardiac K_ATP channels. However, it is not known whether the state of K_ATP channels also determines the response of the channel to dinucleotides. Therefore, we examined the action of diadenosine polyphosphates on K_ATP channel activity during different operative channel states, using the inside-out patch clamp technique applied to patches excised from guinea-pig ventricular myocytes. Spontaneous openings of K_ATP channels (or operative condition 1) were inhibited by Ap₄A and Ap₅A. Addition of UDP, which on its own did not affect spontaneous openings of K_ATP channels, prevented Ap₄A and Ap₅A to inhibit spontaneous K_ATP channel activity. In contrast, following run-down of spontaneous channel activity, UDP induced K_ATP channel openings (or operative condition 2), but could no longer antagonize the inhibitory effect of Ap₄A and Ap₅A. We conclude that the inhibitory action of diadenosine polyphosphates on K_ATP channels can be blocked by UDP only when K_ATP channels are in operative condition 1, but not in operative condition 2. Thus, the operative condition of KATp channels determines the UDP-mediated regulation of the diadenosine polyphosphate-dependent inhibitory channel gating. This finding further suggests that the operative state of the cardiac K_ATP channel protein is a critical determinant of the regulatory action of an intracellular ligand.     

神经肽Y—Y1受体样免疫反应物质在大鼠中枢神经系统的分布

为探明神经肽Ｙ受体在中枢神经系统的分布，用免疫组织化学方法，研究了ＮＰＹ－Ｙ１受体亚型免疫反应神经细胞及纤维在大鼠中枢神经系统的分布。     

Contribution of P2Y(1) receptors to ADP signalling in mouse spinal cord cultures

Mixed neuronal and glial cell spinal cord cultures from neonates express ADP sensitive P2Y(1,12&13) receptors. ADP (10microM) evoked increases in intracellular calcium that were essentially abolished by the P2Y(1) receptor antagonist MRS2179 (10microM), responses were also absent in preparations from P2Y(1) receptor deficient mice however UTP (100microM) evoked calcium rises were unaffected. ADP also evoked a robust increase in extracellular signal-regulated protein kinase (ERK) phosphorylation that was of similar magnitude in the cultures from wild type and P2Y(1) receptor deficient mice. These results suggest that ADP acts through P2Y(1) receptors to mediate an increase in intracellular calcium but not to stimulate ERK phosphorylation in the spinal cord.     

Diadenosine tetraphosphate protects sympathetic terminals from 6-hydroxydopamine-induced degeneration in the eye

Aims: To examine diadenosine tetraphosphate (Ap₄A) for its ability to protect the eye from neurodegeneration induced by subconjunctival application of 6-hydroxydopamine (6-OHDA). Methods: Intraocular neurodegeneration of anterior structures was induced by subconjunctival injections of 6-OHDA. Animals were pre-treated with topical corneal applications of Ap₄A or saline. Results: 6-OHDA caused miosis, abnormal pupillary light reflexes, a precipitous drop in intraocular pressure and loss of VMAT2-labelled (vesicle monoamine transporter-2, a marker for sympathetic neurones) intraocular neurones. Pre-treatment with Ap₄A prevented all of these changes from being induced by 6-OHDA, demonstrably preserving the sympathetic innervation of the ciliary processes. This neuroprotective action of Ap₄A was not shared with the related compounds adenosine, ATP or diadenosine pentaphosphate. P2-receptor antagonists showed that the effects of Ap₄A were mediated via a P2-receptor. Conclusion: Ap4A is a natural component of tears and aqueous humour, and its neuroprotective effect indicates that one of its physiological roles is to maintain neurones within the eye. Ap₄A can prevent the degeneration of intraocular nerves, and it is suggested that this compound may provide the basis for a therapeutic intervention aimed at preventing or ameliorating the development of glaucoma associated with neurodegenerative diseases. Furthermore, subconjunctival application of 6-OHDA provides a useful model for studying diseases that cause ocular sympathetic dysautonomia.     

Localization and possible function of P2Y(4) receptors in the rodent retina

Extracellular ATP acts as a neurotransmitter in the retina, via the activation of ionotropic P2X receptors and metabotropic P2Y receptors. The expression of various P2X and P2Y receptor subtypes has been demonstrated in the retina, but the localization of P2Y receptors and their role in retinal signaling remains ill defined. In this study, we were interested in determining the localization of the P2Y(4) receptor subtype in the rat retina, and using the electroretinogram (ERG) to assess whether activation of these receptors modulated visual transmission. Using light and electron microscopy, we demonstrated that P2Y(4) receptors were expressed pre-synaptically in rod bipolar cells and in processes postsynaptic to cone bipolar cells. Furthermore, we show that the expression of P2Y(4) receptors on rod bipolar cell axon terminals is reduced following dark adaptation, suggesting receptor expression may be dependent on retinal activity. Finally, using the electroretinogram, we show that intravitreal injection of uridine triphosphate, a P2Y receptor agonist, decreases the amplitude of the rod PII, supporting a role for P2Y receptors in altering inner retinal function. Taken together, these results suggest a role for P2Y(4) receptors in the modulation of inner retinal signaling.     

Effects of Different Variants in the ENPP1 Gene on the Functional Properties of Ectonucleotide Pyrophosphatase/Phosphodiesterase Family Member 1

Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (E‐NPP1), encoded by ENPP1, is a plasma membrane protein that generates inorganic pyrophosphate (PP_i), a physiologic inhibitor of hydroxyapatite formation. In humans, variants in ENPP1 are associated with generalized arterial calcification of infancy, an autosomal‐recessive condition causing premature onset of arterial calcification and intimal proliferation resulting in stenoses. ENPP1 variants also cause pseudoxanthoma elasticum characterized by ectopic calcification of soft connective tissues. To determine the functional impact of ENPP1 missense variants, we analyzed 13 putative pathogenic variants in vitro regarding their functional properties, that is, activity, localization, and PP_i generation. Transfection of eight of the 13 variants led to complete loss of NPP activity, whereas four mutants (c.1412A > G, p.Tyr471Cys; c.1510A > C, p.Ser504Arg; c.1976A > G, p.Tyr659Cys; c.2330A > G, p.His777Arg) showed residual activity compared with wild‐type E‐NPP1. One putative pathologic variant (c.2462 G > A, p.Arg821His) showed normal activity. The five mutants with normal or residual E‐NPP1 enzyme activity were still able to generate PP_i and localized in the plasma membrane. In this study, we identified a functional ENPP1 polymorphism, which was expected to be pathogenic till now. Furthermore, we identified four mutants (p.Tyr471Cys, p.Ser504Arg, p.Tyr659Cys, p.His777Arg) with residual E‐NPP1 function, which would be potential therapeutical targets for conformational‐stabilizing agents.     

Cortical and subcortical distribution of ionotropic purinergic receptor subunit type 1 (P2X(1)R) immunoreactive neurons in the rat forebrain

Ionotropic purinergic receptors (P2XR) are ATP-gated cationic channels composed of seven known subunits (P2X(1-7)R) and involved in different functions in neural tissue. Although their presence has been demonstrated in the brain, few studies have investigated their expression pattern. In particular, ionotropic purinergic receptor subunit type 1 (P2X(1)R) has been observed in the cerebellum and in brainstem nuclei. The present study investigates the P2X(1)R expression pattern in the rat forebrain using immunohistochemistry. The specificity of the immunolabeling has been verified by Western blotting and in situ hybridization methods. P2X(1)R immunoreactivity was specifically localized in neurons, dendrites and axons throughout the forebrain. Characteristic differences in the distribution of P2X(1)R were observed in different cortical areas. In prefrontal, cingulate and perirhinal cortices, very intense labeling was present in neuronal bodies. In frontal, parietal, temporal and occipital cortices, immunostaining was lighter and mainly found in dendrites and axons. The hippocampal formation was intensely labeled. Labeling was present almost exclusively in dendrites and axons and never in neuronal bodies. The diencephalon was devoid of P2X(1)R positive neurons or fibers except for the medial habenular nucleus, which showed very intense P2X(1)R immunostaining. Furthermore, two subcortical regions, namely, the nucleus centralis of the amygdala and the bed nucleus of the stria terminalis, showed intense P2X(1)R neuronal labeling. Present data indicate that P2X(1)R are prevalent in forebrain areas involved in the integration of cognitive, limbic and autonomic functions.     

Prevention of extracellular ADP-induced ATP accumulation of the cultured rat spinal astrocytes via P2Y(1)-mediated inhibition of AMPK

P2Y(1) is probably an important subtype of purinergic receptors (P2Rs) in modulation of the astrocyte activation in spinal cord. The aim of this study was to observe the effect of P2Y(1) receptor on the abnormal energy metabolism of the cultured rat spinal astrocyte induced by extracellular adenosine diphosphate (ADP). The results showed that adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP) in the astrocytes were up-regulated in the presence of ADP, which could be enhanced by MRS2179, a specific antagonist for P2Y(1) receptor. A higher level of expression of the AMP-activated protein kinase (AMPK) was found in the presence of MRS2179 and ADP together than that ADP alone. Blocking of AMPK with Compound C could effectively inhibit the enhancing effect of MRS2179 on ADP-induced astrocyte proliferation and ATP accumulation. Our results suggested that the P2Y(1) receptor mediated inhibition of AMPK may help to prevent the astrocytes from over activation induced by extracellular ADP.     

Adenosine 5'-triphosphate: a P2-purinergic agonist in the myocardium

ATP, besides an intracellular energy source, is an agonist when applied to a variety of different cells including cardiomyocytes. Sources of ATP in the extracellular milieu are multiple. Extracellular ATP is rapidly degraded by ectonucleotidases. Today ionotropic P2X(1--7) receptors and metabotropic P2Y(1,2,4,6,11) receptors have been cloned and their mRNA found in cardiomyocytes. On a single cardiomyocyte, micromolar ATP induces nonspecific cationic and Cl(-) currents that depolarize the cells. ATP both increases directly via a G(s) protein and decreases Ca(2+) current. ATP activates the inward-rectifying currents (ACh- and ATP-activated K(+) currents) and outward K(+) currents. P2-purinergic stimulation increases cAMP by activating adenylyl cyclase isoform V. It also involves tyrosine kinases to activate phospholipase C-gamma to produce inositol 1,4,5-trisphosphate and Cl(-)/HCO(3)(-) exchange to induce a large transient acidosis. No clear correlation is presently possible between an effect and the activation of a given P2-receptor subtype in cardiomyocytes. ATP itself is generally a positive inotropic agent. Upon rapid application to cells, ATP induces various forms of arrhythmia. At the tissue level, arrhythmia could be due to slowing of electrical spread after both Na(+) current decrease and cell-to-cell uncoupling as well as cell depolarization and Ca(2+) current increase. In as much as the information is available, this review also reports analog effects of UTP and diadenosine polyphosphates.     

Long-chain acyl-CoA esters and phosphatidylinositol phosphates modulate ATP inhibition of Katp channels by the same mechanism

Phosphatidylinositol phosphates (PIPs, e.g. PIP₂) and long-chain acyl-CoA esters (e.g. oleoyl-CoA) are potent activators of K _atp channels that are thought to link K _atp channel activity to the cellular metabolism of PIPs and fatty acids. Here we show that the two types of lipid act by the same mechanism: oleoyl-CoA potently reduced the ATP sensitivity of cardiac (Kir6.2/SUR2A) and pancreatic (Kir6.2/SUR1) K _atp channels in a way very similar to PIP₂. Mutations (R54Q, R176A) in the C- and N-terminus of Kir6.2 that greatly reduced the PIP₂ modulation of ATP sensitivity likewise reduced the modulation by oleoyl-CoA, indicating that the two lipids interact with the same site. Polyvalent cations reduced the effect of oleoyl-CoA and PIP₂ on the ATP sensitivity with similar potency suggesting that electrostatic interactions are of similar importance. However, experiments with differently charged inhibitory adenosine phosphates (ATP^4-, ADP^3- and 2'(3')- O -(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP^2-)) and diadenosine tetraphosphate (Ap₄A^5-) ruled out a mechanism where oleoyl-CoA or PIP₂ attenuate ATP inhibition by reducing ATP binding through electrostatic repulsion. Surprisingly, CoA (the head group of oleoyl-CoA) did not activate but inhibited K _atp channels (IC₅₀= 265 ± 33 μM). We provide evidence that CoA and diadenosine polyphosphates (e.g. Ap₄A) are ligands of the inhibitory ATP-binding site on Kir6.2.