Glaucoma patients present increased levels of diadenosine tetraphosphate, Ap4A, in the aqueous humour
Research highlights
► Diadenosine polyphosphates are present in the human aqueous humour. ► This molecules can activate P2 receptors in the ciliary body and trabecular meshwork. ► Diadenosine polyphosphates are increased 15 times in primary open-angle glaucoma patients. ► Diadenosine tetraphosphate might be considered as a possible biomarker for glaucoma condition.
Introduction
There is an increasing interest in the physiological role of diadenosine polyphosphates in the ocular structures (Guzman-Aranguez et al., 2007). These natural dinucleotides consist of two adenosine nucleosides joined by a phosphate chain whose length can vary from 2 to 7 phosphates (abbreviated ApnA, where n = 2–7) (Miras-Portugal et al., 1999). Diadenosine polyphosphates are synthesized as secondary products of aminoacid activation by t-aminoacyl synthestases during the assembly of aminoacids to the corresponding t-RNA (Zamecnik et al., 1966). After their synthesis these molecules are stored in secretory systems such as chromaffin cells or synaptic terminals (Pintor et al., 1992, Rodriguez del Castillo et al., 1988). Diadenosine polyphosphates are released in a Ca2+-dependent manner from brain nerve endings and chromaffin cells after stimulation by depolarizing agents such as 4-aminopyridine, veratridine or high K+ (Pintor et al., 1992). Moreover, their release can be directly induced in some neural models by cholinergic agonists and indirectly by dopaminergic agents (Pintor et al., 1991, Pintor et al., 1992, Pintor et al., 1995). The release of diadenosine polyphosphate has been demonstrated as a consequence of mechanical shear stress in various locations including the eye, in structures such as the ocular surface (Carracedo et al., in press, Peral et al., 2006). In the extracellular space, they can activate different types of purinergic receptors (Miras-Portugal et al., 1999) to be finally degraded by extracellular ectonucleotidases (Vollmayer et al., 2003).
Their presence in the New Zealand rabbit aqueous humour or in animal tears, together with the discovery of their receptors and effects on ocular physiology after application of these dinucleotides, indicates that these molecules may be physiological regulators of some relevant ocular processes. For instance, diadenosine polyphosphates have shown to increase tear production in animal models, hence suggesting potential clinical utility for the treatment of eye dryness (Pintor et al., 2002b). Also, some of them can modify intraocular pressure behaving as putative regulators of IOP. In this sense, diadenosine tetraphosphate, Ap4A, reduce IOP by facilitating the drainage of the aqueous humour through the trabecular meshwork (Soto et al., 2005), while others such as Ap3A or Ap5A can increase IOP (Pintor et al., 2003). On the ocular surface Ap4A can speed up corneal wound healing, whereas Ap3A or Ap5A have been shown to delay this process (Mediero et al., 2006, Mediero et al., 2008, Pintor et al., 2004).
These substances have been also found in human tears (Pintor et al., 2002a) and, for instance, Ap4A levels have a positive correlation with different states of dry eye. Thus, Ap4A is increased by 5-fold in symptomatic normal tear production patients and almost by 100-fold in symptomatic dry eye patients with low tear production (Peral et al., 2006). More recently, the concentration of some dinucleotides has been demonstrated to correlate with the progression of Sjögren syndrome pathology. In these patients, diadenosine tetraphosphate concentration was increased by 42-fold when compared to normal individuals (Carracedo et al., 2010). Altogether, it seems that patients with dry eye and related pathologies such as Sjögren syndrome exhibit abnormally elevated concentrations of diadenosine tetraphosphate.
Although the presence of diadenosine polyphosphates in the aqueous humour of New Zealand rabbits has been demonstrated (Pintor et al., 2003), nothing is known about the existence of diadenosine polyphosphates in human aqueous humour. Nevertheless, an interesting study demonstrated the presence of ATP in the aqueous humour of both, normal and patients with acute increase of intraocular pressure (Zhang et al., 2007), indicating that nucleotides may have a pathophysiological role in the modulation of IOP. In that study, all the cases investigated by Zhang and coworkers were primary acute angle-closure glaucoma patients. These authors demonstrated that increased IOP levels in these patients lead to increased levels of extracellular ATP in the anterior chamber which favours the hypothesis that nucleotides could be released by mechanical stress (Gomes et al., 2005, Pedersen et al., 1999).
In this study, we investigated the presence of diadenosine polyphosphates in the aqueous humour of normal individuals as well as in patients suffering from primary open-angle glaucoma. We demonstrated higher levels of diadenosine polyphosphates in glaucoma patients when compared to normal individuals, thus indicating that nucleotides and dinucleotides are likely involved in glaucoma pathology.
Section snippets
Subject recruitment and aqueous humour collection
26 eyes of 26 patients who underwent cataract surgery were included in the study. The study protocol was approved by the clinical research ethics committee of the Hospital Sagrat Cor-ICR, Barcelona. Signed informed consent to the aqueous humour contribution was obtained from all patients in accordance with the Declaration of Helsinki. They underwent an ophthalmologic examination that included measurement of best-corrected visual acuity, intraocular pressure measured by Goldmann tonometer,
Presence of diadenosine polyphosphates in the human aqueous humour
The chromatographic analysis of the aqueous humour samples taken from non-glaucomatous individuals (control group) showed several nucleotide peaks, one of which was tentatively identified as diadenosine tetraphosphate (Ap4A), after comparing its retention time with the ones displayed by commercial samples (Fig. 1A, upper trace). Another peak, that eluted later, was also tentatively identified as diadenosine pentaphosphate (Ap5A) (Fig. 1A, upper trace).
Aqueous humour samples from glaucoma
Discussion
The present study describes for the first time the existence of the dinucleotides Ap4A and Ap5A as soluble components of the human aqueous humour. Moreover, when comparing these molecules in glaucoma patients compared to normal individuals, it has been possible to prove that the concentrations of one of the dinucleotides, Ap4A, are significantly higher in glaucomatous patients than in normal individuals.
Among the different components present in the aqueous humour, nucleotides are important
Acknowledgements
This work was supported by grants from Ministerio de Ciencia e Innovación and Ministerio de Sanidad of Spain: BFU2005-01572; FIS 08/0014, SAF2007-60835, SAF2010-16024 and by RETIC Red de Patología ocular del envejecimiento, calidad visual y calidad de vida (RD07/0062/0004; RD07/0062/0006 and RD07/0062/0010), NEUROTRANS CM S-SAL 0253-2006 and BSCHUCM (GR58/08). The authors thank E. Ferrer for manuscript revision and commenting.
References (38)
- et al.
Dinucleoside polyphosphates in the eye: from physiology to therapeutics
Prog. Retin. Eye Res.
(2007) - et al.
Cloning and tissue distribution of the human P2Y1 receptor
Biochem. Biophys. Res. Commun.
(1996) - et al.
Diadenosine polyphosphates, extracellular function and catabolism
Prog. Brain Res.
(1999) - et al.
Mechanical stress induces release of ATP from Ehrlich ascites tumor cells
Biochim. Biophys. Acta
(1999) - et al.
Carbachol induced release of diadenosine polyphosphates – Ap4A and Ap5A – from perfused bovine adrenal medulla and isolated chromaffin cells
Life Sci.
(1991) - et al.
Presence of diadenosine polyphosphates – Ap4A and Ap5A – in rat brain synaptic terminals. Ca2+ dependent release evoked by 4-aminopyridine and veratridine
Neurosci. Lett.
(1992) - et al.
Elevated pressure triggers a physiological release of ATP from the retina: possible role for pannexin hemichannels
Neuroscience
(2008) - et al.
Endogenous nucleosides in the guinea-pig eye: analysis of transport and metabolites
Exp. Eye Res.
(1998) - et al.
Reversed-phase high-performance liquid chromatography of dinucleoside polyphosphates
J. Chromatogr. A
(1991) - et al.
Enzymatic synthesis of diadenosine tetraphosphate and diadenosine triphosphate with a purified lysyl-sRNA synthetase
Biochem. Biophys. Res. Commun.
(1966)
Acute increase of intraocular pressure releases ATP into the anterior chamber
Exp. Eye. Res.
Intraocular pressure responses to the adenosine agonist cyclohexyladenosine: evidence for a dual mechanism of action
Invest. Ophthalmol. Vis. Sci.
Characterization of ocular hypertension induced by adenosine agonists
Invest. Ophthalmol. Vis. Sci.
Purinergic signaling in the rabbit ciliary body epithelium
J. Exp. Zool. Part A, Comp. Exp. Biol.
P2 purinergic receptor-coupled signaling in the rabbit ciliary body epithelium
Invest. Ophthalmol. Vis. Sci.
ATP release through connexin hemichannels in corneal endothelial cells
Invest. Ophthalmol. Vis. Sci.
Diadenosine tetraphosphate protects sympathetic terminals from 6-hydroxxydopamine-induced degeneration in the eye
Acta Physiol. (Oxf.)
ATP is released from guinea pig ureter epithelium on distension
Am. J. Physiol. Ren. Physiol.
Cited by (25)
Ap<inf>4</inf>A Regulates Directional Mobility and Antigen Presentation in Dendritic Cells
2019, iScienceCitation Excerpt :Our group has previously demonstrated that non-canonical LysRS activity can drive increased intracellular Ap4A and control USF2 transcriptional activity, which up-regulates transforming growth factor-β2 in FcepsilonRI-activated mast cells (Lee and Razin, 2005). Ap4A can enhance phorbol myristate acetate (PMA)-stimulated reactive oxygen species production in lymphocytes (Schepers et al., 2010) and has been implicated in key immunological responses (Carracedo et al., 2013; Castany et al., 2011; Chang et al., 2014; Louie et al., 1988). Another pathway that is driven by the increase of intracellular Ap4A is the activation of microphthalmia-associated transcription factor (MITF), a master regulator in melanocyte development (Levy et al., 2006).
The role of dinucleoside polyphosphates on the ocular surface and other eye structures
2016, Progress in Retinal and Eye ResearchCitation Excerpt :These compounds are also present in the aqueous humour of human, with levels of 0.020 mM (for Ap4A) and 0.021 mM (for Ap5A) (Castany et al., 2011). Moreover, in glaucoma patients, Ap4A concentration is 15 times higher than in normal individuals (Castany et al., 2011). The origin of the dinucleotides in aqueous humour has only recently been elucidated as a mechanism dependent of the ciliary body epithelium although some clues about their extracellular origin were suggested some years ago (Pintor et al., 2003).
Evaluation of presumptive biomarkers of oxidative stress, immune response and apoptosis in primary open-angle glaucoma
2013, Current Opinion in PharmacologyCitation Excerpt :Das et al. reported that Ca2+ overload activates Cas cascades inducing RGC apoptosis [53]. Elevated molecules, such as homocysteine or diadenosine tetraphosphate, Ap(4)A in POAG patients evokes multiple cytotoxic mechanisms, involving increase of intracellular Ca2+ levels, ROS formation, and receptor-mediated activation of cell-death cascades [54]. Furthermore, augmented prostaglandin H2D isomerase (PGDS), Cas-14 and transtyretin (TTR), were detected by AH proteomic analyses in relation to apoptosis in POAG patients [55].
Metabolomics of the aqueous humor in patients with primary congenital glaucoma
2019, Molecular Vision