Glaucoma patients present increased levels of diadenosine tetraphosphate, Ap4A, in the aqueous humour

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Abstract

Previous studies have shown the presence of diadenosine tetraphosphate (Ap4A) and pentaphosphate (Ap5A) in the aqueous humour (AH) of different species. When topically applied to the rabbit cornea, Ap4A decreased IOP while Ap5A increased it. Here we study the presence of dinucleoside polyphosphates in the AH from human patients with or without glaucoma. AH was obtained at the time of cataract surgery from patients with (n = 16) or without (n = 10) primary open-angle glaucoma. AH (0.1–0.2 ml) was collected at the beginning of surgery through a corneal paracentesis and immediately cooled in liquid nitrogen, kept frozen and protected from light. AH aliquots were analyzed by HPLC for the presence of Ap4A and Ap5A. Both, Ap4A and Ap5A were detected in the AH of both experimental groups. No significant differences were found for Ap5A. In contrast, Ap4A levels were increased by ∼15-fold in the AH from glaucomatous eyes ranging from 19.5 ± 9.2 nM in normal individuals to 286.03 ± 30.9 nM in glaucomatous patients. In conclusion, both Ap4A and Ap5A were detected for the first time in human AH. Interestingly, glaucomatous eyes presented elevated concentrations of Ap4A compared to controls. The role of Ap4A needs to be elucidated but it may help to protect the autonomic innervation in the ciliary body/trabecular meshwork. Also, because of its higher levels in glaucoma patients it may be considered as a possible glaucoma biomarker.

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)

  • X. Zhang et al.

    Acute increase of intraocular pressure releases ATP into the anterior chamber

    Exp. Eye. Res.

    (2007)
  • Carracedo, G., Peral, A., Pintor, J., 2010. Diadenosine polyphosphates in tears of Sjogren syndrome patients. Invest....
  • C.E. Crosson

    Intraocular pressure responses to the adenosine agonist cyclohexyladenosine: evidence for a dual mechanism of action

    Invest. Ophthalmol. Vis. Sci.

    (2001)
  • C.E. Crosson et al.

    Characterization of ocular hypertension induced by adenosine agonists

    Invest. Ophthalmol. Vis. Sci.

    (1996)
  • N.A. Farahbakhsh

    Purinergic signaling in the rabbit ciliary body epithelium

    J. Exp. Zool. Part A, Comp. Exp. Biol.

    (2003)
  • N.A. Farahbakhsh et al.

    P2 purinergic receptor-coupled signaling in the rabbit ciliary body epithelium

    Invest. Ophthalmol. Vis. Sci.

    (2002)
  • P. Gomes et al.

    ATP release through connexin hemichannels in corneal endothelial cells

    Invest. Ophthalmol. Vis. Sci.

    (2005)
  • C.H.V. Hoyle et al.

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

    Acta Physiol. (Oxf.)

    (2010)
  • G.E. Knight et al.

    ATP is released from guinea pig ureter epithelium on distension

    Am. J. Physiol. Ren. Physiol.

    (2002)
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