The role of dinucleoside polyphosphates on the ocular surface and other eye structures
Introduction
Dinucleoside polyphosphates are a family of dinucleotides related to others such as nicotinamide adenine dinucleotides (NAD and NADP), but slightly different in their structure. Dinucleoside polyphosphates comprise all those dinucleotides formed by two nucleosides bridged by a variable number of phosphates. The two nucleosides moiety can be formed by ribose which may present the same or different bases, either purines or pyrimidines (Fig. 1). Although in theory the possibilities of dinucleotides are enormous, simply by combining the nitrogenised bases and the phosphate length, there are only a few which have been recognised as naturally occurring dinucleoside polyphosphates, nonetheless, other dinucleoside polyphosphates have been synthesised either enzymatically or chemically (Guranowski et al., 1995, Mizuno and Sasaki, 1965, Ortiz et al., 1993). Dinucleoside polyphosphates can be named in different ways, for instance, α, ω dinucleoside polyphosphates, dinucleoside 5′, 5‴-P1, Pn polyphosphates or NpnN. The latter is the most common one in which “N” is substituted by the bases (A, G, U, for example) and “n” by the number of phosphates.
Dinucleoside polyphosphates found in biological systems are those formed by two adenosines, which are termed diadenosine polyphosphates, ApnA, the ones formed by guanosines, diguanosine polyphosphates, GpnG, or a combination of both, one moiety with adenosine and the other with guanosine (GpnA). Concerning the phosphate length linking both nucleosides, this can vary from two to seven, although the most common are those dinucleotides with 3–5 phosphates. Common, naturally occurring dinucleoside polyphosphates, according to the number of phosphates, are Ap5A or Gp4G.
Synthetic dinucleoside polyphosphates are relevant since they depict remarkable actions on ocular physiology (Guzman-Aranguez et al., 2007, Guzman-Aranguez et al., 2011). Among these the ones with uridine, diuridine polyphosphates (like Up4U AKA diquafosol), with deoxycytidine and uridine (dCp4U, also termed denufosol), or those with hypoxanthine, diinosine polyphosphates (IpnI), have demonstrated interesting therapeutic properties in the eye as it will later be described (Fujihara et al., 2001, Guzman-Aranguez et al., 2012).
The history of dinucleoside polyphosphates started far away from the future physiological and pharmacological applications found in the eye. Indeed, the first news on these molecules occurred in the 1960's. Dinucleoside polyphosphates were discovered in the eggs of brine shrimps, mostly diguanosine polyphosphates (Finamore and Warner, 1963, Warner, 1964). These dinucleotides are the basis for the development of the eggs into proper shrimps since they will be used for DNA synthesis.
It was not until a few years later when it was possible to identify a biochemical reaction showing the synthesis of a dinucleoside polyphosphate. Scientists, when investigating the reaction between aminoacids and their corresponding tRNAs during the protein synthesis process discovered the presence of some diadenosine polyphosphates (Randerath et al., 1966, Zamecnik et al., 1966). Over the following years, most of the efforts were dedicated to describing their structural and chemical properties, mainly by spectrometric methods (Bush and Tinoco, 1967, Cheng, 1968, Warshaw and Tinoco, 1965). Changes in the molecules present were also studied when environmental properties (Lee et al., 1979) such as temperature or pH was modified (Chan and Nelson, 1969, Davis and Tinoco, 1968, Glaubiger et al., 1968, Hruska and Danyluk, 1968, Ikehara et al., 1970, Rabczenko and Shugar, 1971, Ts'o et al., 1969). The structural changes are especially relevant when the pH medium is altered. The structure of a NpnN, for example, Ap4A, under physiological pH condition (pH ≈ 7), depicts a stacked conformation in which the adenine rings are piled up, this being possible due to the phosphate chain folding. This is caused by the negatively charged phosphate groups and the partially positively charged adenine rings and the consequent electrostatic interactions. A different panorama is observed when the pH lowers (pH ≈ 4). Under this condition the phosphates are folded but the adenine rings are pointing to opposite directions. The maximal open possible conformation is reached at a pH below 3. Under this condition the protonation of the phosphates occurs and the interactions among the adenines and phosphates do not exist (Guzman-Aranguez et al., 2011).
The conformational changes observed at different pH, may explain why these dinucleotides can interact with different purinergic receptors, mainly when in some environmental conditions, such as acidic synapses (glutamatergic) the conditions can be far from neutral conditions (Gualix et al., 2003).
Concerning temperature, it seems that dinucleoside polyphosphates at low temperatures present their bases in a stacking conformation being so close to each other that there is no room for water between them. On the contrary, when temperature is high, even at denaturing conditions (above 40 °C), the bases are not stacked and they do not interact with each other and therefore can interact with the solvent and other molecules. In this case, the only limitation of the aromatic bases would be their covalent bonds to the respective riboses (Davis and Tinoco, 1968, Glaubiger et al., 1968).
It took quite a long time from the moment these dinucleotides were identified until some biochemical and physiological actions for these compounds were detected. One of the first actions was the effect of diadenosine tetraphosphate, Ap4A, on DNA polymerase triggering DNA replication (Grummt, 1978, Grummt, 1979, Grummt et al., 1979). Indeed, it has been possible to correlate changes (increases) in Ap4A concentrations during the cell cycle and specially during cell proliferation and DNA synthesis (Bambara et al., 1985). These experiments were quite controversial, although other authors could, in part, confirm the relationship between Ap4A and DNA synthesis, it seemed that apparently the dinucleotide would prime DNA synthesis rather than activating the polymerase (Zamecnik et al., 1982). Also, some other studies suggested a close relation between the amino acid activation process and DNA replication in mammalian cells (Grummt, 1983, Rapaport et al., 1981).
Another intracellular action suggested for dinucleoside polyphosphates has been its possible role of “alarmone” (Lee et al., 1983, Varshavsky, 1983). An alarmone is a molecule that reflects and indicates a stress situation within a cell. A classical situation is that in which an elevation of temperature, apart from increasing the levels of heat shock proteins (HSPs), also produces a rise in dinucleoside polyphosphates such as Ap3A, Ap4A, Gp4A and Gp3A (Lee et al., 1983). The same dinucleotides are increased when cells are submitted to oxidative stress in a process independent from the dinucleotide synthesis by aminoacyl-tRNA synthetases.
Although the presence and actions of dinucleoside polyphosphates have been reported in several tissues as indicated (Andersson, 1989, Rapaport and Zamecnik, 1976, Ripoll et al., 1996), two sources are relevant concerning the eye physiology: the blood vessels and the nervous system.
Apart from the presence of dinucleoside polyphosphates in crustaceans as previously indicated, these molecules were found as putative active vasoactive compounds when they were discovered in platelet secretory granules (Flodgaard and Klenow, 1982, Luthje and Ogilvie, 1983). From the moment they were discovered the main interest of the scientific community was to identify those actions triggered by the dinucleotides in the blood stream. In this sense, one of the first roles was their involvement in platelet aggregation. Interestingly, the two dinucleotides contained in the platelets (in their dense granules), Ap3A and Ap4A, depicted opposite actions; while the first was inducing platelet aggregation, the second, showed disaggregating properties (Luthje et al., 1985). Nonetheless, the existence of dinucleoside polyphosphates in the plasma and blood (Luthje and Ogilvie, 1988), also suggested an effect on vasoconstriction or/and vasodilation. When analysing the effect of the vascular tone, dinucleoside polyphosphates such as Ap4U produces a biphasic effect although the predominant one at low concentrations is vasoconstriction, infusions of this dinucleotide elicit hypotension and electrolyte retention (Flores et al., 1999, Hansen et al., 2010).
The identification of dinucleoside polyphosphates and mainly diadenosine polyphosphates in neural secretory vesicles showed the storage of these dinucleotides with classical transmitters such as noradrenalin (Rodriguez del Castillo et al., 1988). This co-localization was further extended to cholinergic and monoaminergic synaptic vesicles (Miras-Portugal et al., 1998, Miras-Portugal et al., 1999, Pintor et al., 1992). Moreover, it was possible to demonstrate that these dinucleotides were releasable in a Ca2+ dependent manner from brain synaptic terminals (Pintor et al., 1991, Pintor et al., 1992) and also by the effect of drugs such as amphetamines (Pintor et al., 1993). The role of diadenosine polyphosphates, mainly Ap4A and Ap5A in the nervous system has been the modulation of the release of monoamines (Giraldez et al., 2001), glutamate (Gualix et al., 2003), or the inhibition of synaptic transmission in the hippocampus (Klishin et al., 1994).
Extracellular actions of dinucleoside polyphosphates occurred by means of receptors on the cell surface termed P2 purinergic receptors. These types of receptors are divided into two families, the P2X ionotropic (Habermacher et al., 2015) and the P2Y metabotropic receptors (von Kugelgen and Harden, 2011).
P2X receptors are activated by nucleotides and dinucleotides which mediate the rapid and non-selective cation transients (mainly Na+ and Ca2+) from the extracellular space (Samways et al., 2014), causing depolarization of the membrane ion channels and subsequent activation of voltage dependent calcium channels. Seven members of P2X receptors in mammalians have been cloned (P2X1-P2X7) and they combined various forms of these subunits (Saul et al., 2013). Cloning of P2X receptors has revealed that all the subunits have large extracellular loop with ten cysteine residues, two transmembrane domains and short intracellular C and N terminals (Roberts et al., 2006). Three subunits are necessary to form a functional native channel, and these channels are organized as homotrimers or heterotrimers (Coddou et al., 2011).
P2Y receptors are metabotropic receptors, members of the family of G protein-coupled receptors containing seven hydrophobic transmembrane domains connected by three extracellular loops and three intracellular loops, with an extracellular N-terminal end and another C-terminal oriented intracellularly (Jacobson and Boeynaems, 2010). These receptors are coupled, via G proteins to others, such as phospholipase C (PLC), adenylate cyclase or to ion channels (von Kugelgen and Hoffmann, 2015). To date, the P2Y family comprises eight members (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14) encoded by different genes. They can be organized into two groups based on their specific G protein coupling. In one group, that includes the P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11 receptors, are all coupled to Gq and the further activation of phospholipase C β, with the consequent formation of inositol triphosphate (IP3) and the mobilization of intracellular Ca2+. The other group includes the P2Y12, P2Y13 and P2Y14 receptors, associated with Gi proteins and inhibiting adenylate cyclase. The P2Y11 is the only thing that can be coupled to both Gs and Gq proteins (Jacobson et al., 2015).
Concerning the action of dinucleoside polyphosphates and the activation of P2 receptors, most of the research has been carried out for ApnA compounds (Hoyle et al., 2001). In this sense, diadenosine polyphosphates can activate all the P2X receptors (Wildman et al., 1999), including the heteromeric P2X2/3. It is noteworthy to indicate that the most active dinucleotide has been Ap4A in most cases. (Pintor and Miras-Portugal, 1995b). Also, the P2X7 was only sensitive to these dinucleotides at concentrations above 1 mM, this effect being far from the physiological concentrations of these compounds.
Regarding the P2Y receptors, P2Y1 and P2Y2 are the ones that are stimulated by diadenosine polyphosphates (Patel et al., 2001). The tandem Ap4A-P2Y2 is of special interest since it participates in many physiological processes in many ocular structures (Fig. 2 see below).
The concentration of dinucleoside polyphosphates is tightly regulated by a variety of enzymes, mainly hydrolases that are present in all organisms and generally act on the polyphosphates chain of these molecules. The enzymes involved in the catabolism of dinucleoside polyphosphates have been reviewed by Guranowski (Guranowski, 2000).
Extracellular dinucleotides, which regulate physiological processes via P2 receptors activation, are degraded principally by ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) (Asensio et al., 2007, Vollmayer et al., 2003). This family of enzymes are hydrolases located on cell surface and are a type of the so called ecto-nucleotidases (Zimmermann et al., 2012). In vertebrates, NPPs have seven members (NPP1–NPP7) but only three (NPP1–NPP3) can degrade nucleotides (dinucleotides but also nucleoside triphosphates and diphosphates) (Goding et al., 1998, Jin-Hua et al., 1997, Stefan et al., 1999). Dinucleotides degradation by NPPs produce nucleoside monophosphates (NMP) plus nucleoside polyphosphates (NPn−1). Therefore, NPPs can generate metabolites biologically active such as ADP and ATP that are obtained from the naturally occurring diadenosine polyphosphates (Vollmayer et al., 2003, Zimmermann, 2000). These nucleotides can also be degraded by NPPs and essentially through ecto-nucleoside triphosphate diphosphohydrolases (NTPDases), other family of ecto-enzymes that can degrade nucleoside triphosphates and diphosphates (Zimmermann, 1999).
On the other hand, the degradation of diadenosine polyphosphates and their metabolites ATP as well as ADP produce AMP. This nucleoside monophosphate can also be catabolized through ecto-5′-nucleotidase (eN) as well as alkaline phosphatase enzymes (this last ecto-nucleotidase may also act on ATP and ADP molecules) (Zimmermann et al., 2012). Consequently, AMP is converted into the nucleoside adenosine, which mediates purinergic effects via P1 receptors interaction (Jacobson and Gao, 2006).
Finally, extracellular adenosine can be eliminated from the synaptic cleft through cell surface adenosine deaminases that convert adenosine to inosine and/or nucleoside transporters (Bonan, 2012, Franco et al., 1997, Pastor-Anglada and Perez-Torras, 2015). These transporters, equilibrative and concentrative nucleoside transporters, are integral membrane proteins implicated in the cellular uptake of adenosine (Bonan, 2012, Pastor-Anglada and Perez-Torras, 2015).
Section snippets
Ocular surface
The ocular surface is the outer zone of the eye. It is a functional unit composed of different structures such as corneal and conjunctival epithelium, lacrymal glands, eyelids and tear film. All components of this functional unit are linked through the innervation and vascular immunologic and endocrine systems. The main functions of the ocular surface are to maintain the transparency of the cornea (optic quality issues) and to hydrate and protect the corneal and conjunctival epithelium to the
Conclusions and future directions
The participation of dinucleoside polyphosphates in ocular physiology opens the possibility of using these compounds for the treatment of relevant eye pathologies. Beginning with the ocular surface to the retina, the application of these dinucleotides may help to improve several processes, which may be altered in the pathology.
Dry eye is treated in some countries such as Japan and South Korea by means of the dinucleotides Up4U. This nucleotide can clearly increase tear secretion, both the
Disclosure
All authors have no financial interest in the subject matter or materials discussed in this manuscript. The authors alone are responsible for the content and writing of the paper.
Acknowledgments
This work was supported by grants from Ministerio de Ciencia e Innovación (SAF2010-16024 and SAF2013-44416-R) and RETICS (RD12/0034/0003).
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Percentage of work contributed by each author in the production of the manuscript is as follows: Gonzalo Carracedo 35%; Almudena Crooke 10%; Ana Guzman-Aranguez 10%; M.J. Perez de Lara 10%; Alba Martin-Gil 10%; Jesus Pintor 25%.