The purinergic P2X family is composed of ionotropic receptors; seven receptor subtypes have been identified (P2X1 to P2X7). The P2X7 receptor, formerly also known as P2Z, is ubiquitously expressed in a wide variety of cell types including cells of haematopoietic origin (mast cells, macrophages, fibroblasts, erythrocytes, granulocytes, erythroleukaemia cells, and lymphocytes), central and spinal cord neurons, brain glial cells (microglia, astrocytes, and Müller cells), bone cells (osteoblasts, osteoclasts, and osteocytes), and epithelial and endothelial cells [1,2,3,4,5,6,7,8,9,10]. The P2X7 receptor has also been detected in both anterior and posterior segments of the eyeball: cornea and conjunctiva were immunopositive [11,12,13,14], as were the different layers of the retina [11,15,16,17,18,19], lacrimal glands , and lens cells [11,21] (see Table 1 for more details). The P2X7 receptor is highly polymorphic and nine different human splice variants have been identified .
The P2X7 receptor is restrictively activated by ATP4−, requiring higher concentrations of ATP to be activated when compared with other P2X purinoreceptors. The P2X7 receptor is sensitive to a few nucleotides apart from BzATP (2′(3′)-O-(4-Benzoylbenzoyl)adenosine 5′-triphosphate triethylammonium salt), which is 10 to 100 times more potent than ATP [23,24].
P2X7 receptors show complex gating behavior: several seconds of ATP exposure induces P2X7 receptors’ dilatation from a channel that allows for the passage of small cations to a pore that allows for permeation of larger cations and dyes such as YO-PRO-1 (see YO-PRO-1 staining protocol in ). P2X7 receptor activation triggers numerous cellular effects from oxidative stress to apoptosis, including inflammation. As previously described, P2X7 receptors activate apoptotic caspases 3, 8, and 9 [26,27], and are involved in actin reorganization and plasma membrane blebs formation through p38, Mitogen-Activated Protein Kinases (MAPK), and Rho-GTPases . The activation of P2X7 receptors can also induce an inflammatory response through the formation of the inflammasome complex, which leads to proinflammatory cytokine release [26,29,30,31,32,33,34,35,36]. All these toxic cellular events occur during the pathogenesis of degenerative disorders, which highlights the pivotal role of P2X7 receptors in these diseases. It is worth mentioning that P2X7 receptors are also involved in life signals such as cell proliferation [37,38]. This effect on proliferation has been associated to wound healing when P2X7 receptor activation is induced  but also to cancer when P2X7 receptor expression is increased, allowing cancerous cell survival and proliferation [40,41]. The proliferative effects triggered by P2X7 receptor activation may be elicited at basal or low ATP concentrations , and/or depends on isoform expression .
P2X7 receptors are of great interest to toxicologists because as membrane receptors, they can be easily targeted by therapeutic formulations to block the toxic mechanisms they trigger. This review will be dedicated to the implication of P2X7 receptors in ocular toxic stresses and the existing modulators that are or could be used in ophthalmology.
3. Anti-P2X7 Strategies in Ophthalmology
The P2X7 receptor has received particular attention as a potential therapeutic target because of its widespread involvement in numerous ocular diseases. It appears to be a key regulatory element of apoptosis, inflammation, and cell death in general. Several P2X7 receptor antagonists have been evaluated in ophthalmology, and both topical and oral administrations have been considered.
3.1. Topical Administration
Topical administration, mostly in the form of eye drops, is employed to treat diseases of the anterior segment, usually the cornea and the conjunctiva. Hyaluronan is a natural polysaccharide used in ophthalmology in artificial tears for the treatment of dry eye syndrome [88,89,90,91], due to its lubricant and viscoelastic properties. Hyaluronan has also been considered as a potent P2X7 receptor modulator: a pretreatment with hyaluronan before BAC or SLS incubation was able to significantly decrease SLS-induced P2X7 activation in corneal and conjunctival cells [59,92,93]. One possible mechanism is that hyaluronan physically coats the cell membrane via strong links with CD44 receptors. At the same time, it masks P2X7 receptors, preventing their activation.
The P2X7 receptor is potently inhibited by divalent cations such as calcium, magnesium, zinc, and copper, that on the one hand alter the affinity of ATP binding to the P2X7 receptor in an allosteric manner, and on the other hand directly interact with the P2X7 receptor [94,95,96,97,98]. It has been demonstrated that some marine solutions containing Ca2+, Mg2+, and Zn2+ inhibited basal activation of P2X7 receptors in ocular surface cell lines . Such solutions that are rich in divalent cations could be easily used as P2X7 receptor modulators or to potentiate P2X7 receptor antagonists.
3.2. Oral Administration
The oral route of drug administration to target the eye may not be the most efficient delivery system due to absorption from the gastrointestinal tract and high systemic clearance rates. Nevertheless, oral antioxidants are prescribed to dry AMD patients since no truly effective treatment is currently available for patients with advanced disease. A proof-of-principle clinical trial in AMD patients confirmed the positive effects of antioxidant saffron administration in neurodegenerative diseases and its persistence over time [99,100]. Recent data showed that saffron may exert its protective role in neurodegeneration by reducing the intracellular calcium response evoked by P2X7 receptor stimulation .
Omega-3 polyunsaturated fatty acids were included in the formulation used in the Second Age-Related Eye Disease Study (AREDS2) to evaluate their effects on slowing the progression of AMD . The results of AREDS2 showed that omega-3 fatty acid supplementation did not yield a statistically significant reduction in the progression of AMD . However, previous observational studies suggested a link between high dietary consumption of omega-3 fatty acids and decreased risk of developing advanced AMD [104,105]. We demonstrated that eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) represent efficient modulators of amyloid β toxicity in retinal cells with the condition that these omega-3 fatty acids are brought as triglycerides in fish oils, and not as ethyl esters like in AREDS2 . The preventive effects of fish EPA and DHA against amyloid β-induced apoptosis relies on the inhibition of P2X7 receptors through lipid raft disruption. In combination with Brilliant Blue G (BBG), a specific P2X7 receptor inhibitor, they fully prevented amyloid β cytotoxic effects. It was then concluded that marine oils rich in EPA and DHA plus BBG in food supplements could be proposed to prevent AMD. Other studies determined the promising role of BBG as a therapeutic agent to inhibit AMD expansion, as it might prevent retinal pigmented epithelium and photoreceptor cell death [106,107,108].
Ca2+ ions play a critical role in the biochemical cascade of signal transduction pathways, leading to the activation of immune cells. In the present study, we show that the exposure of freshly isolated human monocytes to NAD+ results in a rapid concentration-dependent elevation of [Ca2+]i (intracellular free Ca2+ concentration) caused by the influx of extracellular Ca2+. NAD+ derivatives containing a modified adenine or nicotinamide ring failed to trigger a Ca2+ increase. Treating monocytes with ADPR (ADP-ribose), a major degradation product of NAD+, also resulted in a rise in [Ca2+]i. Selective inhibition of CD38, an NAD-glycohydrolase that generates free ADPR from NAD+, does not abolish the effect of NAD+, excluding the possibility that NAD+ might act via ADPR. The NAD+-induced Ca2+ response was prevented by the prior addition of ADPR and vice versa, indicating that both compounds share some mechanisms mediating the rise in [Ca2+]i. NAD+, as well as ADPR, were ineffective when applied following ATP, suggesting that ATP controls events that intersect with NAD+ and ADPR signalling.
Abbreviations: ADPR, ADP-ribose; cADPR, cyclic ADP-ribose; β-araF-NAD, 2′-deoxy-2′-fluoroarabinoside NAD; ART, mono-ADP-ribosyltransferase; [Ca2+]i, intracellular free Ca2+ concentration; fMLP, N-formylmethionyl-leucylphenylalanine; fura, 2/AM, fura 2 acetoxymethyl ester; NADase, NAD+-glycohydrolase; β-riboF-NAD, 2′-deoxy-2′-F-ribose-NAD; TRP, transient receptor potential
- The Biochemical Society, London