DBA/2J mouse strain has become a popular model for studying glaucoma, because it develops the pathology spontaneously. DBA/2J mice contain mutations in two genes, Tyrp1 and Gpnmb, encoding tyrosinase-related and glycosylated transmembrane proteins, respectively. These mutations lead to pigment dispersion, iris transillumination, iris atrophy, and anterior synechia (Anderson et al., 2002). Due to the blockade of aqueous outflow, DBA/2J mice suffer from ocular hypertension by the age of 9 months, which is accompanied by the canonical symptoms of a glaucoma-related death of retinal ganglion cells (RGCs), optic nerve atrophy and cupping, as well as visual deficits (Libby et al., 2005). The pathophysiology of glaucoma remains in part unknown, although there are several studies demonstrating that elevated intraocular pressure (IOP) affects disease development, leading to a progressive optic neuropathy characterized by functional and structural impairment of ocular tissues that may result in the loss of vision (Davson, 1993). This elevated IOP mainly leads to glaucoma as a result of impeded aqueous humor outflow (Tomarev, 2001). Aqueous humor is produced by the ciliary epithelium in the posterior chamber of the eye and circulates through the pupil to the anterior chamber, where it drains through the trabecular meshwork into Schlemm’s canal and episcleral veins (Morrison and Acott, 2003). Thus, the ciliary processes provide the pressure inside the eye, which is maintained as a balance between the production and the drainage of the aqueous humor throughout the trabecular meshwork.

It is well known that purinergic P2 receptors, which are activated by extracellular nucleotides, can be involved in aqueous humor production and drainage and are therefore involved in IOP control (Guzman-Aranguez et al., 2013). P2 receptors are classified into two subfamilies: G protein-coupled P2Y receptors and ligand-gated cation channels called P2X receptors. So far, seven P2X subunits (P2X1-7) and eight P2Y receptors (P2Y_{1,2,4,6,11,12,13,14}) have been cloned and characterized in humans, according to their agonist sensitivity, sequence identities, and signal transduction mechanisms (Burnstock, 2000; Abbracchio et al., 2006). P2Y receptors contain seven hydrophobic transmembrane domains connected by three extracellular loops and three intracellular loops. The extracellular amino-terminus presents sites for glycosylation and the intracellular domain contains potential sites for phosphorylation, which may participate in receptor desensitization and internalization. P2Y receptors can be characterized according to their responses to nucleotide agonists and subtype-preferring antagonists. Adenine nucleotides, such as ADP or its synthetic analogous 2-methylthio-ADP (2-MeSADP), can activate selectively P2Y₁ receptors (Waldo and Harden, 2004), whereas uracil nucleotides activate P2Y₆ receptors (Nicholas et al., 1996). Moreover, both adenine and uracil nucleotides (ATP and UTP) activate equipotently P2Y₂ receptors (Lazarowski et al., 1995).

P2Y₁ and P2Y₂ receptors are expressed in rabbit ciliary body epithelial cells, which may be responsible for the action of 2-MeSATP, ATPγS, as well as other P2Y agonists (Farahbakhsh and Cilluffo, 2002). The expression of P2Y₁, P2Y₂, and P2Y₄ receptors in bovine trabecular meshwork has also been reported (Soto et al., 2005) as well as the presence of P2Y₆ and P2Y₁₁ receptors in rat ocular structures (Pintor et al., 2004).

The dinucleotide diadenosine tetraphosphate (Ap₄A) (Guzman-Aranguez et al., 2011) shows a hypotensive effect when instilled topically in the eye (Fonseca et al., 2016), which is related to an increase in the outflow of the aqueous humor through activation of several purinergic receptors, mainly the P2Y₁ (Schachter et al., 1996; Soto et al., 2005).

Most of the experiments performed to analyze the modulation of aqueous humor production and drainage by purinergic receptors previously described in the scientific literature, have been carried out on normotensive mice models or on induced models of glaucoma. Here we characterize the presence of P2Y₁, P2Y₂, and P2Y₆ receptors in the ciliary processes of the eye of control versus glaucomatous mice and analyze the expression levels of these receptors during aging and the development of the pathology in these animals.

The existence of nucleotides in the aqueous humor (Mitchell et al., 1998; Pintor et al., 2003), as well as the participation of P2 purinergic receptors in the regulation of IOP, has already been described in the scientific literature (Crooke et al., 2008). Mono and dinucleotides, both in the μM range, are able to activate P2 receptors, which in turn modify both the synthesis and drainage of the aqueous humor. In normotensive animal models, P2Y₁ and P2Y₂ receptors are located in the trabecular meshwork and the ciliary body, respectively (Soto et al., 2005; Martin-Gil et al., 2012). When the activation of P2Y₁ receptors is carried out by Ap₄A in the trabecular meshwork, the observed effect is a reduction in IOP (Soto et al., 2005), whereas the activation of P2Y₂ receptors in the ciliary body produces a hypertensive effect (Martin-Gil et al., 2012). P2Y₂ receptors exert this effect by increasing the presence of Aquaporin-1 in the ciliary body epithelial cell membranes (Martin-Gil and Pintor, 2010; Pintor et al., 2011). Finally, the activation of P2Y₆ receptor by molecules such as uridine diphosphate (UDP), reduces IOP in the New Zealand normotensive model (Markovskaya et al., 2008).

Ap₄A is a molecule present in the aqueous humor of animal models such as rabbits or mice (Pintor et al., 2003), but is also present in normal and glaucomatous human patients (Castany et al., 2011). Therefore, this dinucleotide seems to be involved in the pathophysiology of glaucoma, mainly by acting on P2 purinergic receptors. In this sense, the release of Ap₄A is produced by the activation of a pressure sensor, a TRPV4 channel, which is activated by the abnormal IOP often associated to glaucoma (Pintor et al., 2011). The TRPV4-induced Ap₄A release will stimulate mainly the P2Y₂ receptor present in the ciliary body, which mobilizes Aquaporin-1 from intracellular reservoirs to the plasma membrane of the ciliary epithelium (Martin-Gil and Pintor, 2010). This increase in the aquaporins facilitates the production of aqueous humor contributing to the elevation of IOP and therefore priming the described process.

In this current study, using a glaucoma animal model, we have analyzed the expression levels of three P2Y metabotropic receptors (P2Y₁, P2Y₂, and P2Y₆), which are known to be activated by Ap₄A (Guzman-Aranguez et al., 2007), in the ciliary body and iris of control (C57BL/6) and glaucomatous (DBA/2J) mice. Our results demonstrate that control animals express constant levels of P2Y₁, P2Y₂, and P2Y₆ receptors throughout their lives, in clear contrast to the glaucomatous strain, where more remarkable changes are observed. In this sense, P2Y₁ receptor expression is significantly reduced at both mRNA and protein levels in aged DBA/2J mice. On the contrary, however, P2Y₂ and P2Y₆ receptor expressions are enhanced in aged DBA/2J mice, correlating in time with the pathology development. Similar differences between control and glaucomatous mice have also been found when analyzing the retinal electrophysiology in both strains, observing a gradual ganglion cell death in DBA/2J model (Perez de Lara et al., 2014). Moreover, the retinal ATP released increases in the glaucomatous mouse, but not in the control one when the pathology is fully established (12 months of age). In addition, the expression of the vesicular nucleotide transporter (VNUT) is significantly increased during the development of glaucoma in DBA/2J mice, reaching maximal levels at 12 months of age (Perez de Lara et al., 2015). Altogether, this evidence suggests that the alteration in the purinergic system observed in the DBA/2J is related to the pathology development and not to mice aging.

We have also established that micromolar concentrations of Ap₄A significantly reduce IOP in DBA/2J mice, once the pathology is fully developed. Moreover, a chronic treatment with the dinucleotide for 3 months is able to ameliorate the elevation in IOP in the glaucomatous mice in a very significant manner (Fonseca et al., 2016). These results indicate that the predominant physiological effect of Ap₄A is the reduction of IOP, although the expression of both P2Y₂ and P2Y₆ in DBA/2J mice is increased and both receptors mediate opposite effects on IOP (Fonseca et al., 2016).

Previous studies reported that P2Y₂ receptor activation in the ciliary body exerted a hypertensive effect on IOP, which could be inhibited by antagonism or silencing of that receptor (Martin-Gil et al., 2012). The hypertensive role of the P2Y₂ receptor in the ciliary body correlated well with the abnormally elevated concentration of Ap₄A found in glaucoma patients, as previously commented (Castany et al., 2011). However, the topical application of this dinucleotide reduces IOP, so its use in the treatment of glaucoma cannot be discarded (Fonseca et al., 2016).

In our experimental model, the reduction in the expression of the P2Y₁ receptor in glaucomatous mice could produce an elevation in IOP, since the treatment with the specific antagonist MRS2179 prevented the hypotensive effect of Ap₄A (Fonseca et al., 2016). An increase P2Y₂ expression could have similar consequences, since its activation exerted a hypertensive effect (Fonseca et al., 2016). Thus, the pathology-related increased in P2Y₂ receptor expression and its activation by endogenous Ap₄A could justify high IOP values measured in the glaucomatous mice. Interestingly, exogenous applications of high doses of Ap₄A have hypotensive effects, probably by activating the P2Y₆ receptor, which is elevated in the glaucomatous mice. Noteworthy is that the high Ap₄A concentration (about 300 nM) found in the aqueous humor of glaucoma patients is unable to reduce IOP, probably because this concentration is enough to stimulate the hypertensive P2Y₂ receptor, but insufficient to activate the hypotensive P2Y₆, which requires micromolar concentrations (Guzman-Aranguez et al., 2007). Further studies will be necessary to clarify this refined equilibrium.

The pathogenesis of glaucoma is not fully understood, but most of the studies indicate that the level of IOP is linked to RGC’s death (Hollands et al., 2013). The rise in IOP causes mechanical stress and strain affecting eye posterior structures such as the lamina cribrosa and adjacent tissues, thus causing the deformation by compression of the lamina cribrosa. Consequently, the optic nerve axonal damage reduces the retrograde axonal transport (Burgoyne et al., 2005). This process produces the RGC’s death and the subsequent blindness. Slowing disease progression, by reducing IOP, can be suggested according to the changes in the expression of P2Y receptors sensitive to the dinucleotide Ap₄A presented in this manuscript. A previous study has already proved the hypotensive effect of this compound on the DBA/2J glaucoma model (Fonseca et al., 2016). Therefore, the application of Ap₄A may help to reduce IOP in humans if the changes in the P2Y receptors are similar to the ones described in the glaucomatous mice.

In summary, we have demonstrated that the expression of some P2Y receptors, P2Y₁, P2Y₂, and P2Y₆, changes during the development of the glaucomatous pathology in a mouse model. The prevalence of the P2Y₂ receptor between 9 and 12 months of age, together with the rise in Ap₄A concentration, may be a contributing factor that helps explain why the pressure is abnormally elevated when the pathology is fully established.

BF: Contributed in PCR, immunohistochemistry. AM-Á: Contributed in PCR, immunohistochemistry. MPdL: Contributed in IOP measurements. MTM-P: Contributed in paper organization and writing. RG-V: Contributed in PCR, statistical analysis, and writing. JP: Contributed in paper design, organization, and writing.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.