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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/jpet.112.202473
J Pharmacol Exp Ther 345:331–341, June 2013
Perspectives in Pharmacology
Nucleotides in the Eye: Focus on Functional Aspects
and Therapeutic Perspectives
Ana Guzman-Aranguez, Concepcion Santano, Alba Martin-Gil, Begoña Fonseca,
and Jesús Pintor
Received December 5, 2012; accepted March 14, 2013
ABSTRACT
The presence and activity of nucleotides and dinucleotides in
the physiology of most, if not all, organisms, from bacteria to
humans, have been recognized by the scientific community,
and the eye is no exception. Nucleotides in the dynamic fluids
interact with many ocular structures, such as the tears and
aqueous humor. Moreover, high concentrations of nucleotides
in these secretions may reflect disease states such as dry eye
and glaucoma. Apart from the nucleotide concentration in these
fluids, P2 purinergic receptors have been described on the
ocular surface (cornea and conjunctiva), anterior pole (ciliary
body, trabecular meshwork), and posterior pole (retina). P2X and
Introduction
The role of purine nucleotides and nucleosides as extracellular messengers was first proposed in the 1970s (Burnstock,
1972). Since then, the rapidly accelerating progress in this field
has helped determine the involvement of these compounds in
key biochemical and physiologic processes in different tissues.
Their therapeutic potential in a wide range of diseases is also
attracting increasing interest (Burnstock, 2012).
Extracellular nucleotides act by stimulating P2 purinergic
receptors on the cell surface. This group of receptors is divided into two families, the ionotropic P2X receptors and the
This study was supported by grants from the Spanish Ministry of Economy
and Competition [SAF2010-16024]; the Ministry of Health, Social Services and
Equality RETICS [RD07/0062/0004]; and the Universidad Complutense de
Madrid [GR35/10-A-920777].
A.G. and C.S. contributed equally to this work.
dx.doi.org/10.1124/jpet.112.202473.
P2Y purinergic receptors are essential in maintaining the homeostasis of ocular processes, such as tear secretion, aqueous
humor production, or retinal modulation. When they are functioning properly, they allow the eye to do its job (to see), but in
some cases, a lack or an excess of nucleotides or a malfunction in
the corresponding purinergic receptors leads to disease. This
Perspective is focused on the nucleotides and dinucleotides and
the P2 purinergic receptors in the eye and how they contribute
to normal and disease states. We also emphasize the action of
nucleotides and their receptors and antagonists as potential
therapeutic agents.
metabotropic P2Y receptors that are coupled to G proteins (von
Kugelgen and Harden, 2011).
P2X receptors are ligand-gated ion channels that allow
calcium influx from the extracellular space after nucleotide
binding. Seven mammalian purinergic receptor subunits,
denoted P2X1 through P2X7, and several spliced forms of
these subunits have been identified. Cloning of P2X receptors
revealed that all subunits have a large extracellular loop, two
transmembrane domains, and intracellular-located amino
and carboxyl termini of variable lengths. Three subunits are
necessary for forming a native functional channel, and these
channels are organized as homotrimers or heterotrimers
(Coddou et al., 2011).
P2Y receptors, as G protein-coupled receptors, contain seven
hydrophobic transmembrane domains connected by three extracellular loops and three intracellular loops (Jacobson and
Boeynaems, 2010). The P2Y family is composed of eight
members encoded by distinct genes that can be subdivided into
two groups based on their coupling to specific G proteins. The
ABBREVIATIONS: Ap3A, diadenosine triphosphate; Ap4A, diadenosine tetraphosphate; Ap5A, diadenosine pentaphosphate; ATPgS, adenosine59-O-(3-thiotriphosphate); BBG, Brilliant Blue G; BzATP, 29(39)-O-(4-benzoylbenzoyl)adenosine-59-triphosphate; ERK, extracellular-signal-regulated
kinases; IL, interleukin; INS365, diuridine tetraphosphate; INS37217, P(1)-(uridine 59)-P(4)-(29-deoxycytidine 59)tetraphosphate; IOP, intraocular
pressure; Ip4I, diinosine tetraphosphate; MRS2179, 29-deoxy-N6-methyl adenosine 39,59-diphosphate; MRS2578, 1,4-di[3-(3-isothiocyanatophenyl)
thioureido]butane; PVR, proliferative vitreoretinopathy; ROCK, Rho-associated protein kinase; ROS, reactive oxygen species; RPE, retinal pigment
epithelium; SRS, subretinal space; Up4U, diuridine tetraphosphate; Y27632, (R)-(1)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide;
YO-PRO-1, 4-[(3-methyl-1,3-benzoxazol-2(3H)-ylidene)methyl]-1-[3-(trimethylammonio)propyl]quinolinium diiodide.
331
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Department of Biochemistry and Molecular Biology, Faculty of Optics and Optometry, Universidad Complutense de Madrid,
Madrid, Spain
332
Guzman-Aranguez et al.
Nucleotides at the Ocular Surface
Identification of Nucleotides in Tears and Changes
in Pathologic Conditions. The tear film covering the ocular
surface consists of two distinct layers: an outer lipid layer
produced by meibomian glands and an inner layer containing
a mixture of mucins, glycoproteins, and different aqueous components such as electrolytes and glucose, together with other
proteins (lysozyme and lactoferrin, among others). The aqueous
phase is mainly derived from the main and accessory lacrimal
glands, but the conjunctiva can also contribute to secretion of
fluid into the tear film. Finally, the mucin component is provided by the conjunctival goblet cells as well as corneal and
conjunctival epithelial cells (Murube, 2012).
Mono- and dinucleotides have been detected in rabbit and
human tears using high-performance liquid chromatography
(Pintor et al., 2002a,b). Particularly, diadenosine polyphosphates, diadenosine tetraphosphate (Ap4A), and diadenosine
pentaphosphate (Ap5A) were found at micromolar concentrations in rabbit tears. Human tears also contain these
dinucleotides together with diadenosine triphosphate (Ap3A)
and Ap4A, the latter being the most abundant diadenosine
polyphosphate in human tears. Interestingly, the concentrations of diadenosine polyphosphates in rabbits were one order
of magnitude higher than that found in humans. Despite this
difference, the Ap4A/Ap5A ratio in rabbits and humans remained stable, at 2.97 and 2.91, respectively. (Pintor et al.,
2002a,b).
Nucleotides in tears can be released into the extracellular
medium by cell rupture or neural exocytosis, and it also has been
suggested that the release of nucleotides may be a consequence
of mechanical shear stress on the corneal epithelium (Srinivas
et al., 2002). In humans, there is evidence pointing to a mechanical release of nucleotides, in particular Ap4A and Ap5A, as
a consequence of the lid effect on the corneal surface. Normal
individuals instructed to blink at different frequencies release
more nucleotides the more often they blink (Peral et al., 2006)
(Fig. 2).
An increase in blinking frequency is a typical sign of eye
dryness, which occurs to compensate tear instability (Tsubota
et al., 1996). Therefore, the concentrations of these molecules
should be elevated in cases of dry eye, and indeed they are. In
symptomatic non-Sjögren dry eye patients with normal tear
production, Ap4A levels were increased by 5-fold and Ap5A levels
by 1.5-fold, and almost by 100- and 345-fold for Ap4A and Ap5A,
respectively, in symptomatic non-Sjögren dry eye patients with
low tear production (Peral et al., 2006). Interestingly, differences
were found between male and female tear samples. Samples
from symptomatic women apparently had higher levels of
diadenosine polyphosphates than those of men. This divergence gives rise to the possibility that there may be a hormonal
factor involved, which may deserve further investigation.
Additionally, Ap4A concentration increased 42-fold and Ap5A
concentration 595-fold in Sjögren dry eye patients, compared
with normal individuals (Carracedo et al., 2010). The association between the increase of diadenosine polyphosphates in
tears and dry eye pathology has been proposed as a potential
molecular biomarker for this disease (Pintor, 2007).
Effects on Tear Secretion and Tear Composition. The
physiology of the ocular surface of both mammalian animals
and humans may be modified by nucleotides in tears. One of the
physiologic processes regulated by extracellular nucleotides on
the ocular surface is tear production.
Topical instillation of the mononucleotides, UTP and ATP, in
an equipotent way, increased tear secretion in rabbits around
4-fold as determined by Schrimer tests (Murakami et al., 2000),
whereas UDP and ADP did not modify tear secretion (Pintor
et al., 2002b). Based on this response to nucleotides, the effect
seems to be mediated by P2Y2 receptors. This conclusion is
reinforced by the promotion of chloride secretion and net fluid
flux in the serosal-to-mucosal direction induced by P2Y2 receptor activation in conjunctival epithelium (Li et al., 2001b).
Dinucleotides can also stimulate tear fluid secretion. Diadenosine polyphosphate Ap4A markedly increased tear production
to around 60% higher than normal in New Zealand White
rabbits. Ap5A and Ap6A also significantly increased tear production by about 20% (Pintor et al., 2002b). Likewise, the application of diuridine tetraphosphate (Up4U, INS365), a
selective P2Y2 agonist, induced a 1.5-fold transient increase in
tear fluid secretion in a rat model (Fujihara et al., 2001).
Nucleotide application not only alters tear volume (water
and electrolytes) but also affects tear composition, modifying
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P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors couple to Gq
to activate phospholipase C b; the P2Y12, P2Y13, and P2Y14
receptors associate with Gi to inhibit adenylyl cyclase. The
P2Y11 receptor has the unique property of coupling through
both Gq and Gs (Jacobson et al., 2012).
The P2X receptors are more structurally restrictive than
P2Y receptors with regard to agonist selectivity. They respond
principally to ATP and their analogs 39-O-(4-benzoyl) benzoyl
ATP, a,b-methylene ATP, b,g-methylene ATP, and 2-methylthio
ATP as active ligands, while P2Y receptors, other than ATP
and its analogs, are also activated by nucleotides such as ADP,
UTP, or UDP. In particular, the ligand preferences [shown in
brackets] of the eight human P2Y receptors are as follows:
P2Y1 [ADP], P2Y2 [UTP 5 ATP], P2Y4 [UTP], P2Y6 [UDP],
P2Y11 [ATP, NAD1], P2Y12 [ADP], P2Y13 [ADP], and P2Y14
[UDP, UDP-glucose, and other nucleotide sugars] (Zimmermann
et al., 2012; Burnstock, 2013).
Dinucleoside polyphosphates, known as dinucleotides, also
act as agonists for P2X and P2Y receptors. They are composed
of two nucleoside moieties linked by their ribose 59-ends to a
variable number of phosphates (2–7), the diadenosine polyphosphates (containing two adenosine moieties) being the most
abundant and widely studied of these compounds (GuzmanAranguez et al., 2007).
In the eye, the identification of nucleotides and dinucleotides
in ocular secretions such as tears and aqueous humor (Mitchell
et al., 1998; Pintor et al., 2002a,b) and the expression of P2
receptors in different eye locations (see Table 1) suggests the
involvement of these compounds in the physiology of the eye. In
fact, during the last decade, the role of several nucleotides and
their receptors in ocular processes, including tear secretion,
corneal wound healing, regulation of intraocular pressure
(IOP), or retinal detachment, has been established (Pintor
et al., 2003a; Crooke et al., 2008).
Moreover, the expanding knowledge of the contribution of
nucleotides to ocular functions reveals that these compounds
can provide new opportunities for therapeutic interventions
in ocular diseases (Fig. 1).
In this article, we review the main biologic roles of nucleotides in the eye and the applications of these compounds
as therapeutic agents for the treatment of eye disease.
Function and Therapeutic Potential of Nucleotides in Eye
re-epithelialization, whereas Ap3A or Ap5A did the opposite,
delaying the wound-healing process (Mediero et al., 2006).
An increased re-epithelialization rate has been observed
with nucleotides such as Ap4A, UTP, and ATP. This agonistic
profile coincides with the observations of Lazarowski et al.
(1995) showing Ap4A and UTP to be the best agonists on the
cloned P2Y2 receptor.
Concerning the second messenger systems triggered by
the P2Y2 receptor to accelerate the rate of cell migration
during wound healing, two intracellular pathways are implicated: extracellular-signal-regulated kinases (ERK) and
Rho-associated protein kinase (ROCK-1). ROCK-1 is linked
to the actin cytoskeleton, modifying cellular contractility. The
involvement of this pathway has been demonstrated with
compounds such as Y27632 [(R)-(1)-trans-4-(1-aminoethyl)-N(4-pyridyl)cyclohexanecarboxamide] or (2)-blebbistatin, which
inhibit ROCK-1 and myosin light chain kinase (Mediero et al.,
2008). When these compounds were used, the ability of epithelial cells to migrate after Ap4A treatment disappeared.
This notion has been recently confirmed by specific silencing
of the P2Y2 receptor using small interfering RNA (Crooke
et al., 2009; Boucher et al., 2010).
The P2X7 receptor may also mediate later phases of wound
repair in the cornea. In vivo studies with P2X7 knockout mice
(P2X72/2) revealed that P2X7 is necessary for the timely
healing of abrasions and the normal structural organization of
the corneal stroma (Mayo et al., 2008).
Effects on Cell Proliferation. After the damaged area
has been covered with a cell monolayer, the final step in
corneal wound healing is to initiate the mitotic process for
restoring normal corneal epithelial thickness. In vitro experiments have shown that after treatment with Ap4A or Ap3A,
corneal epithelial cell proliferation increased similarly with
both dinucleotides at 24 and 36 hours (Mediero et al., 2010;
http://www.journalofemmetropia.org/2171-4703/jemmetropia.
2010.1.81.87.php). Thus, pretreatment with several inhibitors demonstrated that Ap4A is linked to the phospholipase
C/protein kinase C/RhoA/ROCK-1 and Erk1/2 pathways,
and Ap3A modulates proliferation by activating ERK1/2 and
p38 mitogen-activated protein kinase cascades (Mediero
et al., 2010). Pharmacologic experiments have demonstrated
the sequential involvement of P2Y2 and P2Y6 receptors in
the proliferative stage of wound healing. It is interesting
that corneal epithelial cell proliferation during the wound
healing process was found to be initiated by P2Y2 receptor
activation via the ERK1/2 pathway (Muscella et al., 2004).
Wound healing could not be completed without the participation of a P2Y6 receptor to maintain the mitotic process. In
the case of the P2Y6 receptor, the natural activator would
be Ap3A, which recruits the p38 mitogen-activated protein
kinase pathway to lead the whole proliferation process (Mediero
et al., 2010).
Anterior Chamber Nucleotides
Nucleotides in Aqueous Humor and Their Effect on
Intraocular Pressure. Aqueous humor, produced by ciliary
processes, is a transparent fluid that provides nutritional
support to avascular intraocular structures, mainly the cornea and the lens. Aqueous humor is drained through the trabecular meshwork, Schlemm’s canal, and episcleral veins (To
et al., 2002).
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its protein content. Activation of P2X7 receptors in rat
lacrimal gland by the nucleotide (benzoylbenzoyl)adenosine
59triphosphate (BzATP) increased intracellular calcium and
stimulated protein secretion (Hodges et al., 2009). Furthermore, the expression of specific tear proteins such as secreted
mucins can also be modified by nucleotides. UTP and ATP
stimulated mucin release from isolated rabbit and human
conjunctival tissues through P2Y2 activation (Jumblatt and
Jumblatt, 1998). Similarly, the mononucleotides ATP and
UTP and the dinucleotide Up4U increased mucin secretion
from conjunctival goblet cells in living animals (Fujihara
et al., 2001; Murakami et al., 2003)
In view of the simultaneous effect of nucleotides via P2Y2
receptors on fluid/mucin secretion, P2Y2 receptor agonists
have been tested in different dry eye animal models for
restoring moisture and rehydration of the ocular surface.
In fact, the dinucleotide diuridine tetraphosphate (INS365,
Diquafosol) has been under development as a new treatment
of dry eye (Nichols et al., 2004) and was recently launched in
Japan by Santen Pharmaceutical after completion of clinical
trials.
Interestingly, as mentioned above, non-Sjögren and Sjögren
dry eye patients showed higher Ap4A and Ap5A dinucleotide
levels than normal subjects (Peral et al., 2006; Carracedo
et al., 2010). These increased levels may represent a compensatory attempt by the ocular surface epithelium to preserve
a wet-surface phenotype by increasing tear fluid and mucin
secretion, although this hypothesis remains to be confirmed.
Finally, it has been observed that nucleotides can modify
the levels of not only secreted mucins, but also other tear
proteins. In particular, changes in lysozyme levels, the most
abundant protein in tears, have been detected after topical
application of nucleotides (Peral et al., 2008). UTP, Ap4A, and
Up4U increased lysozyme concentrations by 67, 93, and 119%,
respectively, compared with lysozyme basal levels. Because
this enzyme is one of the first defense mechanisms against
bacterial infection, increasing the levels of this natural bacteriologic agent with these nucleotides could provide additional protection against pathogen invasion.
Effects on Wound Healing and Cell Migration. The
cornea is a multilayered tissue of the eye characterized by its
transparency, avascularity, and its ability to refract light. Its
most superficial layer is the corneal epithelium, which is
susceptible to damage by several factors. After injury, normal
epithelium is regenerated by the wound healing process (Dupps
and Wilson, 2006).
Nucleotides have been shown to stimulate events associated with epithelial wound repair. The mononucleotides ATP
and UTP and the dinucleotide Ap4A induce cell migration and
stimulate corneal re-epithelialization, both in vitro and in
vivo (Pintor et al., 2004a; Mediero et al., 2008). In particular,
UTP increased the rate of healing by 168% and Ap4A by 130%
in experiments in New Zealand White rabbits (Pintor et al.,
2004a). Pharmacologic studies using different P2 receptor
antagonists showed that the wound healing effect triggered by
these nucleotides was mediated by P2Y2 receptor activation
(Pintor et al., 2004a; Mediero et al., 2006).
Experiments performed in vitro with immortalized rabbit
corneal epithelial cells indicate that diadenosine polyphosphates can produce several effects on corneal epithelium,
which differ depending on phosphate chain bridging between the two adenosines. Ap4A accelerated the rate of
333
334
Guzman-Aranguez et al.
TABLE 1
Expression of purinergic receptors in different localizations of the eye
P2 Receptor Subtypes
Species
Function
Ocular surface
Corneal epithelium
P2X5
Rat
P2X7
Rat, mouse, and human
P2Y1, P2Y4
P2Y2
P2Y6
P2Y11
Conjunctival
epithelium
P2X7
P2Y2
P2Y1, P2Y11,
P2Y13
Anterior and
posterior chamber
Ciliary processes
P2X2
P2X7
P2Y1
P2Y2
P2Y4
P2Y6
Trabecular
meshwork
P2X2
P2X7
P2Y1
P2Y2
P2Y4
P2Y11
Retina
Photoreceptors
P2X2
P2X7
P2Y1, P2Y2,
P2Y4, P2Y6
Bipolar cells
P2X3, P2X4,
P2X5
P2X7
P2Y1, P2Y2,
P2Y6
P2Y4
Horizontal cells
P2X7
Amacrine cells
P2X1, P2X3,
P2X4, P2X5
Not determined
Corneal epithelial migration and stromal
organization
Rabbit, rat, and human
Not determined
Macaque, rabbit, rat, and Modulation of corneal re-epithelialization
human
(acceleration)
Rabbit, rat, and human
Modulation of corneal re-epithelialization
(delay)
Rabbit and human
Not determined
Groschel-Stewart et al., 1999
Groschel-Stewart et al., 1999; Dutot et al.,
2008; Mayo et al., 2008
Klepeis et al., 2004; Pintor et al., 2004b
Cowlen et al., 2003; Klepeis et al., 2004; Pintor
et al., 2004b
Klepeis et al., 2004; Pintor et al., 2004b
Human
Dutot et al., 2008
Rabbit, macaque, and
human
Membrane permeabilization associated with
iatrogenic pathology
Stimulation of tear secretion
Klepeis et al., 2004
Jumblatt and Jumblatt, 1998; Cowlen et al.,
2003
Rat
Not determined
Hodges et al., 2011
Rat
Induction of intracellular calcium
concentration and protein secretion
Induction of intracellular calcium
concentration
Hodges et al., 2009, 2011
Rat
Cow
Cow
Rabbit
Rabbit, cow, monkey, rat,
and human
Not determined
Membrane permeabilization
Not determined
Increase intraocular pressure
Rat
Rabbit, rat
Not determined
Not determined
Rabbit and cow
Decrease IOP (increase aqueous humor
outflow)
Human
Membrane permeabilization, ATP release
Cow, human, pig, and rat Decrease IOP (increase aqueous humor
outflow).
Cell swelling
Cow and pig
Not determined
Cow, human, and pig
Not determined
Human
Not determined
Ohtomo et al., 2011
Pintor and Peral, 2001
Li et al., 2011
Farahbakhsh and Cilluffo, 2002
Shahidullah and Wilson, 1997; Cullinane et al.,
2001a,b; Farahbakhsh and Cilluffo, 2002;
Cowlen et al., 2003; Pintor et al., 2004b;
Martin-Gil et al., 2012
Pintor et al., 2004b
Pintor et al., 2004b; Markovskaya et al., 2008b
Pintor and Peral, 2001
Li et al., 2012
Crosson et al., 2004; Pintor et al., 2004b; Soto
et al., 2005; Chow et al., 2007
Soto et al., 2005; Chow et al., 2007
Crosson et al., 2004; Soto et al., 2005; Chow
et al., 2007
Crosson et al., 2004
Rat
Not determined
Mouse, rat, and
Modulation of synaptic transmission
marmoset
Rabbit, rat, and macaque Not determined
Greenwood et al., 1997
Franke et al., 2005; Puthussery et al., 2006;
Notomi et al., 2011
Cowlen et al., 2003; Fries et al., 2004a; Pintor
et al., 2004b
Rat
Not determined
Wheeler-Schilling et al., 2000
Mouse and rat
Rat
Modulation of synaptic transmission
Not determined
Vessey and Fletcher, 2012
Fries et al., 2004b
Rat
Modulation of synaptic transmission
Ward et al., 2008
Mouse and rat
Modulation of synaptic transmission
Vessey and Fletcher, 2012
Mouse and rat
Not determined
P2X2
Mouse
P2X7
Mouse
Wheeler-Schilling et al., 2001; Yazulla and
Studholme, 2004; Puthussery and Fletcher,
2007; Shigematsu et al., 2007
Inhibition of Ach release from OFF cholinergic Kaneda et al., 2004; Puthussery et al., 2006;
amacrine
Kaneda et al., 2008, 2010
Modulation of synaptic transmission
Vessey and Fletcher, 2012
(continued )
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Lacrimal gland
P2X1, P2X2,
P2X4, P2X6,
P2X3, P2X7
References
Function and Therapeutic Potential of Nucleotides in Eye
335
TABLE 1—Continued
P2 Receptor Subtypes
Ganglion cells
P2X2, P2X3,
P2X4, P2X5
P2X7
P2Y1, P2Y2,
P2Y4, P2Y6
Astrocytes and
Müller cells
P2X7
P2Y2
P2Y1, P2Y2,
P2Y4, P2Y6
P2Y1
Species
Rat
Not determined
Mouse
Increase intracellular calcium concentrations
Rabbit, rat, and macaque Not determined
Human
Guinea pig
Rat
Increase DNA synthesis
Increase DNA synthesis
Elicitation of Ca2+ responses
Pannicke et al., 2000; Bringmann et al., 2001
Moll et al., 2002; Milenkovic et al., 2003
Li et al., 2001a; Fries et al., 2004b, 2005
Mouse and rat
Homeostasis of the extracellular space volume Uckermann et al., 2006; Iandiev et al., 2007;
Wurm et al., 2008, 2010
Elicitation of Ca2+ responses
Fries et al., 2005
P2Y6
P2Y12
P2X7
Depolarization and contraction
Rat
Regulation of calcium-activated chloride
channel
Human
Rabbit, cow, rat, and
human
Not determined
Increase of fluid flow from apical to basolateral
membrane.
Increase IL-8 secretion
Increase IL-8 secretion
Not determined
Not determined
Human
Human
Human
Measurable amounts of nucleotides and dinucleotides,
such as ATP or Ap4A, have been detected in aqueous humor
(Mitchell et al., 1998; Pintor et al., 2003b). Purinergic receptors
P2X and P2Y are present in the main structures bathed by
the aqueous humor (the ciliary process and trabecular meshwork) (Table 1), suggesting that these compounds contribute
to physiologic functions in these areas, such as the control of
IOP. The mechanisms that control and regulate IOP are not
fully understood, but they result from the dynamic equilibrium between the production and drainage of aqueous humor. Disruption of this equilibrium results in raised IOP,
which is an important risk factor in the pathogenesis of
glaucoma, a multifactorial optic neuropathy characterized by
damage to the optic nerve head and irreversible field of vision
loss (Friedman et al., 2004). For this reason, most glaucoma
drug treatments involve lowering IOP, either by decreasing
the production of aqueous humor or by improving its outflow
(Gupta et al., 2008).
Interestingly, the levels of ATP and Ap4A in the aqueous
humor of glaucomatous patients were around 14 and 15 times
higher than in healthy individuals, respectively (Castany
et al., 2011; Li et al., 2011), suggesting the involvement of
these compounds in the pathology of glaucoma.
A factor connecting high nucleotide levels in glaucoma
patients and raised IOP is the presence of P2Y2 receptors in
the ciliary body. The activation of P2Y2 receptors in the ciliary
processes clearly has a hypertensive effect on the eye. Hypertensive nucleotides comprise compounds such as ATP and its
analogs 2-methylthio-ATP and ATPgS [adenosine-59-O-(3thiotriphosphate)] (Peral et al., 2009) or UTP (Markovskaya
et al., 2008a) and its analogs 2-thioUTP, uridine-59-O-(3-
Kawamura et al., 2003; Sugiyama et al., 2005,
2006
Kawamura et al., 2003; Sugiyama et al., 2005
Reigada et al., 2005
Peterson et al., 1997; Sullivan et al., 1997;
Maminishkis et al., 2002; Meyer et al., 2002;
Relvas et al., 2009
Relvas et al., 2009
Reigada et al., 2005
Dutot et al., 2008; Yang et al., 2011
thiotriphosphate) (Martin-Gil et al., 2012). Pharmacologic
studies using antagonists have suggested that the P2Y2
receptor is involved in the hypertensive effect induced by
these nucleotides. In fact, silencing the P2Y2 receptor by small
interfering RNA produced a robust decrease in IOP of 48% 6
5% compared with control (Martin-Gil et al., 2012). Therefore,
the use of small interfering RNA against the P2Y2 receptor
may be an interesting therapeutic approach in the reduction
of IOP in the treatment of the abnormally raised IOP observed
in many glaucoma patients.
It is worth pointing out that there are also nucleotides that,
when topically applied, produce a hypotensive effect. Among
the nucleotides with a hypotensive effect on IOP, there are
compounds capable of facilitating the aqueous humor outflow.
For instance, a,b-me ATP and b,g-me ATP produce a clear
and marked decrease in IOP (14.2% 6 2.2% and 33.2% 6 1.3%
decrease, respectively) (Peral et al., 2009). This effect seems
to be mediated by P2X2 receptors present in the cholinergic terminals that innervate the ciliary muscle. The P2X2
receptor triggers the release of acetylcholine, leading to the
widening of the iridocorneal angle with the corresponding
increased aqueous humor drainage and reduced IOP (Peral
et al., 2009).
Other nucleotides can also reduce IOP, improving aqueous
humor outflow by the direct activation of metabotropic P2Y
receptors on the trabecular meshwork. Thus, Ap4A induces
a hypotensive effect by increasing trabecular outflow through
activation of P2Y1 receptors (Soto et al., 2005).
When instilled topically, nucleotides can also stimulate
purinergic receptors in the ciliary processes responsible for
aqueous humor production. Thus, the nucleotide UDP
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Retinal pigment
epithelium
P2Y1
P2Y2
References
Greenwood et al., 1997; Brandle et al., 1998;
Wheeler-Schilling et al., 2001; Puthussery
et al., 2006
Brandle et al., 1998; Franke et al., 2005; Zhang
et al., 2006; Reigada et al., 2008; Mitchell
et al., 2009; Hu et al., 2010; Vessey and
Fletcher, 2012
Cowlen et al., 2003; Fries et al., 2004a
P2Y1, P2Y2, P2Y4, Human
P2Y6
Retinal pericytes
P2X7
Rabbit and rat
P2Y4
Function
336
Guzman-Aranguez et al.
stimulates P2Y6 receptors of the blood vessels of the ciliary
processes, inducing vasoconstriction and significantly reducing aqueous humor production (Markovskaya et al., 2008a)
with a concomitant decrease in IOP (around 20%).
Very recently, the topical application of diinosine polyphosphates has been seen to have profound effects on IOP in
normotensive rabbits. Diinosine pentaphosphate produced
an increase in IOP, while diinosine triphosphate and Ip4I
(diinosine tetraphosphate) produced a decrease, the latter
being the most effective in reducing IOP, with an EC50 of 0.63
mM (Guzman-Aranguez et al., 2012).
The same study suggested that diinosine tetraphosphate
behaved as both an agonist and an antagonist. This was the
first time that an agonistic profile for Ip4I was described (Pintor
et al., 1997). Studies performed with selective and nonselective P2 receptor antagonists demonstrated that it was
possible to reverse the hypotensive effect of Ip4I. The selective
P2Y1 antagonist MRS2179 (29-deoxy-N6-methyladenosine
39,59-diphosphate) and the selective P2Y6 antagonist MRS2578
(1,4-di[3-(3-isothiocyanatophenyl)thioureido]butane) significantly blocked the hypotensive effect induced by Ip4I on IOP,
indicating that Ip4I effect is mediated partially by P2Y1 and
P2Y6 receptors. Moreover, the use of other nonselective
antagonists also supported this finding (Guzman-Aranguez
et al., 2012).
Nucleotides in the Retina
The main function of the retina is to transform the photic
input into electrical messages that will be sent to the lateral
geniculate nucleus that projects to the visual cortex. It
contains various types of neurons, Müller cells (radial glia),
astrocytes, and microglial cells.
The most representative nucleotide in the mammalian
retina is ATP, which is released from Müller cells and broken
down into adenosine by enzymatic degradation resulting in
autocrine activation of the P1 receptors in these glial cells
(Newman, 2003). ATP may also activate P2 receptors of neighboring retinal neurons, such as photoreceptors, amacrine
cells, and ganglion cells (Greenwood et al., 1997; Puthussery
et al., 2006; Kaneda et al., 2008). The co-release of ATP together with acetylcholine from cholinergic amacrine cells has
also been reported (Neal and Cunningham, 1994), and ATP
can also be released from retinal pigmented epithelial cells
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 5, 2017
Fig. 1. Therapeutic potential of nucleotides in the eye. Schematic representation of eye (left) and transmitted light combined with DAPI (4’,6-diamidino2-phenylindole) immunofluorescence in vertical sections of different ocular parts/structures (central): ciliary processes (upper), cornea (middle), and
retina (lower). Potential therapeutic applications in these ocular parts/structures are summarized on the right. c, capillar; En, endothelium; Ep,
epithelium; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; NPCE, nonpigmented ciliary epithelium; ONL, outer nuclear
layer; OPL, outer plexiform layer; PCE, pigmented ciliary epithelium; PS, photoreceptor segments; RPE, retinal pigment epithelium; St, stroma. Scale
bar = 20 mm.
Function and Therapeutic Potential of Nucleotides in Eye
337
Fig. 2. Levels of ATP after different blinking frequencies. (A) High-performance liquid chromatography eluting profiles of tear samples after 0, 12, 30, and
60 blinks per minute. An increase in the concentration
of ATP was observed with the increase in blinking
frequency. (B) Quantification of ATP levels in individuals blinking at different frequencies. As occurred
in (A), there was a gradual increase in the concentration of ATP with the frequency of blinking. Data are
expressed as mean 6 S.D.
blocking P2X7 receptors with Brilliant Blue G or oxidized
ATP or degrading extracellular ATP with apyrase (Resta
et al., 2007).
Müller Cell Volume Regulation. P2 receptors are also
involved in Müller cell volume regulation. ATP is released
after glutamate receptor activation from these glial cells and
is catabolized to ADP, which activates P2Y1 receptors, triggering adenosine release (Uckermann et al., 2006; Wurm
et al., 2010). The stimulation of adenosine A1 receptors then
triggers the opening of potassium and chloride channels in the
plasma membrane of Müller cells and inhibits their osmotic
swelling (Iandiev et al., 2007; Wurm et al., 2010). Moreover,
hyposmotic exposure of retinal slices from P2Y1 receptordeficient mice induced Müller cell swelling (Wurm et al.,
2010).
Exogenous UDP and UTP reduced Müller cell volume in
murine retinal slices (Wurm et al., 2010), probably via the
activation of P2Y4 and P2Y6 receptors. Given that, Müller
cells exhibited impaired cell volume regulation in ischemic
(Uckermann et al., 2006) and diabetic rat retina (Wurm et al.,
2008). Autocrine P2Y1 signaling contributes to the homeostasis of the extracellular space volume, preventing the osmotic
swelling of Müller cells. Therefore, this receptor may be
interesting as a therapeutic target for treating retinal diseases accompanied by ischemic or hypoxic processes.
Müller Cell Proliferation. The mitogenic effect of ATP in
cultured guinea pig Müller cells has been demonstrated (Moll
et al., 2002; Milenkovic et al., 2003). The activation of P2Y
receptors led to membrane conductance alterations through
stimulation of large-conductance calcium-activated potassium
channels and phosphorylation of ERK1/2, inducing stimulation
of DNA synthesis in guinea pig Müller cells (Moll et al., 2002;
Milenkovic et al., 2003). In contrast, the mitogenic action of
ATP on DNA synthesis rate was mediated by P2X7 receptors in
cultured human Müller cells (Bringmann et al., 2001).
Müller cell proliferation occurs in the retina of patients with
proliferative vitreoretinopathy (PVR), causing the formation
of epiretinal membranes (glial scars) which can result in
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(RPE cells) and may activate P2 receptors on photoreceptor
membranes (Mitchell and Reigada, 2008).
Histologic, biochemical, and pharmacologic data support
the presence of different P2 receptors in most types of neurons
and glial cells in the mammalian retina (Table 1). Their role in
retinal function is not well determined, although there is
evidence that the activation of some P2 receptors, such as
P2X7 (Vessey and Fletcher, 2012), P2X2 (Kaneda et al., 2010),
and P2Y4 (Ward et al., 2008), is involved in the modulation
of outer and inner retinal processing. Recent studies have
suggested that altered purinergic signaling mediated by P2
receptors may be an important player in pathophysiologic
processes of the retina. Therefore, P2 receptors could be
interesting targets for the development of future treatments
for retinal diseases.
Neuronal Apoptosis. Inherited retinal diseases cause
increased extracellular ATP levels that may lead to photoreceptor apoptosis. Selective photoreceptor apoptosis mediated
by P2X7 receptors (Notomi et al., 2011) occurred after
intravitreal injection of ATP in murine retina (Puthussery
and Fletcher, 2009; Notomi et al., 2011). In contrast, intraocular injections of Brilliant Blue G, a selective P2X7 receptor
antagonist, reduced ATP-induced photoreceptor apoptosis
(Notomi et al., 2011). Interestingly, P2X7 receptor-mediated
photoreceptor apoptosis was reported in BALBCrd mice,
a retinitis pigmentosa model (Franke et al., 2005), as upregulated expression of P2X7 receptor, and peak photoreceptor apoptosis occurred at the same time between P20 and
P40. Injections of a purinergic receptor antagonist also decreased photoreceptor apoptosis in rd1 mice (Puthussery and
Fletcher, 2009).
In vitro studies have demonstrated that ATP triggered
ganglion cell apoptosis through P2X7 receptor activation
(Zhang et al., 2006). Moreover, hypoxia and acute increase of
hydrostatic pressure induced retinal ganglion cell death due
to the increase of extracellular ATP, which activates P2X7
receptors (Resta et al., 2007; Sugiyama et al., 2010). Acute
pressure-induced ganglion cell apoptosis also was reduced by
338
Guzman-Aranguez et al.
RPE Nucleotides
The RPE is a monolayer of cells located between the outer
segments of photoreceptors and the vessels of the choriocapillaris. The RPE plays an important role in the homeostasis of the
retina by forming the outer blood-retinal barrier regulating
the transport of ions, water, and metabolic end products from
the subretinal space (SRS) to the choroid (Strauss, 2005; Simo
et al., 2010). Moreover, RPE cells also phagocytize the shed
photoreceptor outer segments (Strauss, 2005; Simo et al., 2010),
which is an essential function in the maintenance of photoreceptor excitability. Additionally, RPE cells produce cytokines,
which are necessary for retinal development and survival
(Wenkel and Streilein, 2000; Bian et al., 2011).
RPE cells can release ATP into the SRS and act as an
autocrine or paracrine signal by stimulating P2 receptors
(Mitchell, 2001; Eldred et al., 2003; Reigada et al., 2005). The
effects of ATP, ADP, and UTP are mediated through different
P2 receptors in RPE cells (Table 1). Moreover, ATP and UTP
can trigger the release of ATP by RPE cells, thus acting as
signal amplifiers (Mitchell, 2001; Reigada et al., 2005).
Ectonucleotidase expression has been shown in human
ARPE-19 cells (Reigada et al., 2005; Mitchell and Reigada,
2008). These enzymes are involved in ATP degradation to
ADP, the effects of which are mediated by PY1 and P2Y12
receptors (Reigada et al., 2005). Furthermore, the apical
membrane localization of nucleotidases in ARPE-19 cells
suggests a role in the balance of nucleotides in SRS that would
affect purinergic signaling of RPE cells.
Retinal Detachment. Deregulation of the transepithelial
transport of nutrients, ions, and water contributes to fluid
accumulation in the SRS, reducing adhesion between the
retina and the RPE and producing the retinal detachment
that is characteristic of a wide variety of ocular diseases with
loss of vision (Machemer, 1984; Cox et al., 1988; Kent et al.,
2000).
Interestingly, previous in vitro studies have shown P2Y2
receptor activation with ATP or UTP triggered upregulation of the membrane fluid flow in the apical-to-basolateral
direction in bovine (Peterson et al., 1997) and rat RPE
monolayers (Maminishkis et al., 2002). Moreover, intravitreal
injection of P2Y2 receptor agonist INS37217 (a synthetic
dinucleotide, deoxycytidine 59 tetraphospho 59 uridine) enhanced fluid reabsorption from experimental subretinal blebs
and retinal reattachment in rabbit (Meyer et al., 2002) and rat
(Maminishkis et al., 2002) retinal detachment models. Surprisingly, UTP intravitreal injection neither increased fluid
reabsorption nor retinal reattachment in a retinal detachment rat model (Maminishkis et al., 2002). These authors
suggested that UTP may be degraded in vivo by ectonucleotidases located in retinal outer segments that were not present
in cultured RPE cells.
In view of these results, the P2Y2 receptor may be a good
therapeutic target for resolving retinal detachment, although
it would be necessary evaluate the hydrolytic resistance of
P2Y2 receptor agonists.
RPE Apoptosis. ATP is released into SRS under multiple
pathologic conditions, such as chemical ischemia, induced
oxidative stress, osmotic stress, growth factors, cell swelling,
or glutamate (Eldred et al., 2003; Reigada et al., 2005, 2006;
Dutot et al., 2008), inducing apoptosis through P2X7 receptor
activation. However, little is known about the apoptotic role of
ATP in RPE cells. P2X7 receptor expression has been shown
in cultured human ARPE-19 cells (Dutot et al., 2008) and
human RPE cells isolated from donor eyes (Yang et al., 2011).
P2X7 activation is caused by exposing human ARPE-19 cells
to ATP and BzATP, triggering pore formation and YO-PRO1 (4-[(3-methyl-1,3-benzoxazol-2(3H)-ylidene)methyl]-1-[3(trimethylammonio)propyl]quinolinium diiodide) dye uptake
in these cells (Dutot et al., 2008). Based on these results,
Dutot and coworkers proposed that the P2X7 receptor could
induce RPE apoptosis. A recent paper supports this hypothesis, as increases in Ca21 levels are concomitant with apoptotic markers such as caspase-3 activation and nuclear
condensation in native RPE cells treated with ATP or BzATP,
and these events are impeded by preincubation with oxidized
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retinal detachment and finally blindness (Bringmann and
Wiedemann, 2009). Müller cells from patients with PVR exhibited upregulated currents through P2X7 receptors compared with normal donors, which led to increased activity of
calcium-activated large-conductance potassium channels
(Bringmann et al., 2001). Hence, the increased density of
P2X7 receptor currents may contribute to the activation of
some mitogen pathways involved in stimulation of Müller
cell proliferation.
It would be interesting to identify effective P2 receptor
agonists for reducing the proliferation of Müller cells and to
investigate their therapeutic application in the prevention of
retinal detachment in PVR.
Regulation the Physiology of Retinal Pericytes. ATP
acts as a vasoactive molecule regulating the function of the
pericytes located in rat (Kawamura et al., 2003) and rabbit
(Sugiyama et al., 2006) retinal microvessels. ATP modifies
ionic currents, increases Ca21 levels, and causes contraction
of pericytes (Kawamura et al., 2003). ATP effects were mediated by P2X7 receptor activation, which initially caused
a rise in the steady-state and transient inward current of
pericytes, narrowing the microvascular lumen (Kawamura
et al., 2003); thus, ATP acts as a vasoconstrictor through the
P2X7 receptor and decreases retinal blood velocity. UTPinduced P2Y4 receptor activation also regulates calciumactivated chloride channel activity and increases the transient
inward current but not the steady-state inward current
(Kawamura et al., 2003). Consequently, P2Y4 receptor stimulation can also induce retinal pericyte contractions.
Diabetic retinal microvessels exhibited more pericyte
vulnerability to the lethal effect of P2X7 receptor activation
(Kawamura et al., 2003; Sugiyama et al., 2006). Low BzATP
concentrations (#100 mM), nonlethal for normal retina,
triggered pericyte apoptosis through the P2X7 receptor in
diabetic retinal microvasculature (Sugiyama et al., 2004).
This diabetes-induced increase in sensitivity suggests that
extracellular nucleotide concentrations that are not lethal in
the normal retina may cause pores to open and, consequently,
microvascular cells to die in the diabetic retina. Thus, diabetes appears to facilitate the channel-to-pore transition
that occurs during activation of P2X7 receptors, although the
mechanism by which diabetes induces this effect remains
unclear. Interestingly, activation of P2Y4 receptors inhibited
P2X7 pore formation and microvascular cell death (Sugiyama
et al., 2005). These findings suggest that ATP vasotoxicity
may be improved by P2Y4-mediated inhibition of the lethal
effects of P2X7 on the diabetic retina.
Function and Therapeutic Potential of Nucleotides in Eye
Perspectives and Conclusions
Nucleotides and dinucleotides participate in several relevant physiologic processes in the eye, and they have also been
shown to be involved in disease. From a pharmacologic point
of view, these molecules may help in restoring the normal
conditions of eye physiology.
The possibilities, therefore, of using nucleotides and dinucleotides agonists or antagonists in the treatment of ocular
disease are wide ranging. It is important to bear in mind that
due to the widespread distribution of P2 receptors in ocular
structures, sometimes we may need agonists and other times
antagonists to achieve the desired effect. Moreover, there are
few studies on the presence of ectonucleotidases in the eye,
which may limit the effect of some nucleotides. Finally, some
ocular structures, such as the retina, need special delivery
systems because the application of purinergic compounds is
not easy.
All these, and probably many others, are the challenges
facing purinergic investigators in the search for effective
medicines for the treatment of eye diseases. To date, the
dinucleotide Up4U (diuridine tetraphosphate), marketed under the name Diquas (Inspire Pharmaceuticals, Raleigh, NC),
is the only nucleotide-based medicine to treat an eye disease (i.e., dry eye). This should encourage researchers to continue in the search for new compounds and strategies based
on nucleotides or dinucleotides for the treatment of ocular
diseases.
Authorship Contributions
Wrote or contributed to the writing of the manuscript: GuzmanAranguez, Santano, Martin-Gil, Fonseca, Pintor.
References
Bian ZM, Elner SG, Khanna H, Murga-Zamalloa CA, Patil S, and Elner VM (2011)
Expression and functional roles of caspase-5 in inflammatory responses of human
retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 52:8646–8656.
Boucher I, Rich C, Lee A, Marcincin M, and Trinkaus-Randall V (2010) The P2Y2
receptor mediates the epithelial injury response and cell migration. Am J Physiol
Cell Physiol 299:C411–C421.
Brändle U, Kohler K, and Wheeler-Schilling TH (1998) Expression of the P2X7receptor subunit in neurons of the rat retina. Brain Res Mol Brain Res 62:106–109.
Bringmann A, Pannicke T, Moll V, Milenkovic I, Faude F, Enzmann V, Wolf S,
and Reichenbach A (2001) Upregulation of P2X(7) receptor currents in Müller glial
cells during proliferative vitreoretinopathy. Invest Ophthalmol Vis Sci 42:860–867.
Bringmann A and Wiedemann P (2009) Involvement of Müller glial cells in epiretinal
membrane formation. Graefes Arch Clin Exp Ophthalmol 247:865–883.
Burnstock G (1972) Purinergic nerves. Pharmacol Rev 24:509–581.
Burnstock G (2012) Purinergic signalling: its unpopular beginning, its acceptance
and its exciting future. Bioessays 34:218–225.
Burnstock G (2013) Introduction to purinergic signalling in the brain. Adv Exp Med
Biol 986:1–12.
Cai J, Nelson KC, Wu M, Sternberg P, Jr, and Jones DP (2000) Oxidative damage
and protection of the RPE. Prog Retin Eye Res 19:205–221.
Carracedo G, Peral A, and Pintor J (2010) Diadenosine polyphosphates in tears of
Sjogren syndrome patients. Invest Ophthalmol Vis Sci 51:5452–5459.
Castany M, Jordi I, Catala J, Gual A, Morales M, Gasull X, and Pintor J (2011)
Glaucoma patients present increased levels of diadenosine tetraphosphate, Ap(4)A,
in the aqueous humour. Exp Eye Res 92:221–226.
Coddou C, Yan Z, Obsil T, Huidobro-Toro JP, and Stojilkovic SS (2011) Activation
and regulation of purinergic P2X receptor channels. Pharmacol Rev 63:641–683.
Cowlen MS, Zhang VZ, Warnock L, Moyer CF, Peterson WM, and Yerxa BR (2003)
Localization of ocular P2Y2 receptor gene expression by in situ hybridization. Exp
Eye Res 77:77–84.
Cox SN, Hay E, and Bird AC (1988) Treatment of chronic macular edema with
acetazolamide. Arch Ophthalmol 106:1190–1195.
Crooke A, Guzmán-Aranguez A, Peral A, Abdurrahman MKA, and Pintor J (2008)
Nucleotides in ocular secretions: their role in ocular physiology. Pharmacol Ther
119:55–73.
Crooke A, Mediero A, Guzmán-Aránguez A, and Pintor J (2009) Silencing of P2Y2
receptor delays Ap4A-corneal re-epithelialization process. Mol Vis 15:1169–1178.
Crosson CE, Yates PW, Bhat AN, Mukhin YV, and Husain S (2004) Evidence for
multiple P2Y receptors in trabecular meshwork cells. J Pharmacol Exp Ther 309:
484–489.
Cullinane AB, Coca-Prados M, and Harvey BJ (2001a) Chloride dependent intracellular pH effects of external ATP in cultured human non-pigmented ciliary
body epithelium. Curr Eye Res 23:443–447.
Cullinane AB, Coca-Prados M, and Harvey BJ (2001b) Extracellular ATP effects on
calcium signaling in cultured human non-pigmented ciliary body epithelium. Curr
Eye Res 23:448–454.
Chow J, Liton PB, Luna C, Wong F, and Gonzalez P (2007) Effect of cellular senescence on the P2Y-receptor mediated calcium response in trabecular meshwork
cells. Mol Vis 13:1926–1933.
Dupps WJ, Jr and Wilson SE (2006) Biomechanics and wound healing in the cornea.
Exp Eye Res 83:709–720.
Dutot M, Liang H, Pauloin T, Brignole-Baudouin F, Baudouin C, Warnet JM,
and Rat P (2008) Effects of toxic cellular stresses and divalent cations on the
human P2X7 cell death receptor. Mol Vis 14:889–897.
Eldred JA, Sanderson J, Wormstone M, Reddan JR, and Duncan G (2003) Stressinduced ATP release from and growth modulation of human lens and retinal pigment epithelial cells. Biochem Soc Trans 31:1213–1215.
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 5, 2017
ATP, an irreversible inhibitor of the P2X7 receptor (Yang
et al., 2011).
Oxidative stress is also an important contributing factor to
RPE apoptosis during the development of retinal diseases
such as age-related macular degeneration or diabetic retinopathy (Cai et al., 2000; Hao et al., 2012; Sreekumar et al.,
2012). Interestingly, overproduction of reactive oxygen species (ROS) and increased P2X7-mediated permeabilization
of YO-PRO-1 were reported in cultured human RPE cells
treated with tert-butyl hydroperoxide (Dutot et al., 2008),
a chemical oxidant. Further studies are required to determine
the implication of ROS overproduction in RPE apoptosis
induced by P2X7 receptor activation and the breakdown of the
outer blood-retina barrier in age-related macular degeneration or diabetic retinopathy. Furthermore, it would be very
interesting to investigate whether treatment with P2X7
receptor antagonists can reduce RPE apoptosis and improve
the damaged visual function and homeostasis of the retina in
these diseases.
Immune Responses. Monocyte-derived proinflammatory
cytokines such as tumor necrosis factor a, interleukin-1b (IL1b), or IL-18, among others, can be released to the SRS and
act as danger signals, stimulating inflammatory cytokine
production in human RPE cells (Bian et al., 2011). Interestingly, activation of IL-8 secretion through an ERK1/
2–dependent pathway was demonstrated in stimulated
human ARPE-19 cells with ATPgS, UTP and UDP (Relvas
et al., 2009). In addition, these nucleotides and tumor necrosis
factor a had a synergic effect on induced IL-8 secretion by
human ARPE-19 cells (Relvas et al., 2009).
Relvas et al. (2009) proposed that ATPgS and UTP effects
on IL-8 production may be mediated through the P2Y2 receptor. UDP action can be mediated by P2Y6 or P2Y4 receptors, but considering that P2Y4 receptors are not expressed in
ARPE-19 cells, it is clear that the increase of IL-8 secretion is
mediated through the P2Y6 receptor.
Proinflammatory cytokines induce ROS overproduction in
human RPE apoptosis (Yang et al., 2007). Moreover, increases
in Ca21 levels, ROS, and caspase activation have been involved in activated monocyte-induced mouse (Yang et al.,
2011) and human (Elner et al., 2003) RPE cell apoptosis. In
addition, IL-8 attracts and activates neutrophils, and this also
involves the breakdown of the outer blood-retina barrier and
monocyte infiltration into the SRS, the main hallmarks of
immune response in a variety of retinal pathologic processes
(Penfold et al., 2001). Moreover, abnormal IL-8 production
and immune cell infiltration into SRS occur in these eye
diseases, so it would be very interesting to investigate whether
the therapeutic application of P2Y2 and P2Y6 receptor antagonists can contribute to the repair of RPE breakdowns
during retinal inflammation.
339
340
Guzman-Aranguez et al.
Machemer R (1984) The importance of fluid absorption, traction, intraocular currents, and chorioretinal scars in the therapy of rhegmatogenous retinal detachments. XLI Edward Jackson memorial lecture. Am J Ophthalmol 98:681–693.
Maminishkis A, Jalickee S, Blaug SA, Rymer J, Yerxa BR, Peterson WM, and Miller
SS (2002) The P2Y2 receptor agonist INS37217 stimulates RPE fluid transport in
vitro and retinal reattachment in rat. Invest Ophthalmol Vis Sci 43:3555–3566.
Markovskaya A, Crooke A, Guzmán-Aranguez AI, Peral A, Ziganshin AU, and Pintor
J (2008a) Hypotensive effect of UDP on intraocular pressure in rabbits. Eur J
Pharmacol 579:93–97.
Markovskaya A, Crooke A, Guzmán-Aranguez AI, Peral A, Ziganshin AU, and Pintor
J (2008b) Hypotensive effect of UDP on intraocular pressure in rabbits. Eur J
Pharmacol 579:93–97.
Martin-Gil A, de Lara MJ, Crooke A, Santano C, Peral A, and Pintor J (2012) Silencing of P2Y2 receptors reduces intraocular pressure in New Zealand rabbits. Br
J Pharmacol 165 (4b):1163–1172.
Mayo C, Ren R, Rich C, Stepp MA, and Trinkaus-Randall V (2008) Regulation by
P2X7: epithelial migration and stromal organization in the cornea. Invest Ophthalmol Vis Sci 49:4384–4391.
Mediero A, Crooke A, and Pintor J (2010) Diadenosine polyphosphates, Ap4A and Ap3A,
increase epithelial cell proliferation during corneal wound healing. J Emmetropia 1:
81–87.
Mediero A, Guzmán-Aranguez A, Crooke A, Peral A, and Pintor J (2008) Corneal reepithelialization stimulated by diadenosine polyphosphates recruits RhoA/ROCK
and ERK1/2 pathways. Invest Ophthalmol Vis Sci 49:4982–4992.
Mediero A, Peral A, and Pintor J (2006) Dual roles of diadenosine polyphosphates in
corneal epithelial cell migration. Invest Ophthalmol Vis Sci 47:4500–4506.
Meyer CH, Hotta K, Peterson WM, Toth CA, and Jaffe GJ (2002) Effect of INS37217,
a P2Y2 receptor agonist, on experimental retinal detachment and electroretinogram in adult rabbits. Invest Ophthalmol Vis Sci 43:3567–3574.
Milenkovic I, Weick M, Wiedemann P, Reichenbach A, and Bringmann A (2003) P2Y
receptor-mediated stimulation of Müller glial cell DNA synthesis: dependence on
EGF and PDGF receptor transactivation. Invest Ophthalmol Vis Sci 44:1211–1220.
Mitchell CH (2001) Release of ATP by a human retinal pigment epithelial cell line:
potential for autocrine stimulation through subretinal space. J Physiol 534:193–202.
Mitchell CH, Carré DA, McGlinn AM, Stone RA, and Civan MM (1998) A release
mechanism for stored ATP in ocular ciliary epithelial cells. Proc Natl Acad Sci USA
95:7174–7178.
Mitchell CH, Lu W, Hu H, Zhang X, Reigada D, and Zhang M (2009) The P2X7
receptor in retinal ganglion cells: a neuronal model of pressure-induced damage
and protection by a shifting purinergic balance. Purinergic Signal 5:241–249.
Mitchell CH and Reigada D (2008) Purinergic signalling in the subretinal space:
a role in the communication between the retina and the RPE. Purinergic Signal 4:
101–107.
Moll V, Weick M, Milenkovic I, Kodal H, Reichenbach A, and Bringmann A (2002)
P2Y receptor-mediated stimulation of Müller glial DNA synthesis. Invest Ophthalmol Vis Sci 43:766–773.
Murakami T, Fujihara T, Nakamura M, and Nakata K (2000) P2Y2 receptor stimulation increases tear fluid secretion in rabbits. Curr Eye Res 21:782–787.
Murakami T, Fujihara T, Nakamura M, and Nakata K (2003) P2Y2 receptor elicits
PAS-positive glycoprotein secretion from rabbit conjunctival goblet cells in vivo.
J Ocul Pharmacol Ther 19:345–352.
Murube J (2012) The origin of tears. II. The mucinic component in the XIX and XX
centuries. Ocul Surf 10:126–136.
Muscella A, Greco S, Elia MG, Storelli C, and Marsigliante S (2004) Differential
signalling of purinoceptors in HeLa cells through the extracellular signal-regulated
kinase and protein kinase C pathways. J Cell Physiol 200:428–439.
Neal M and Cunningham J (1994) Modulation by endogenous ATP of the light-evoked
release of ACh from retinal cholinergic neurones. Br J Pharmacol 113:1085–1087.
Newman EA (2003) Glial cell inhibition of neurons by release of ATP. J Neurosci 23:
1659–1666.
Nichols KK, Yerxa B, and Kellerman DJ (2004) Diquafosol tetrasodium: a novel dry
eye therapy. Expert Opin Investig Drugs 13:47–54.
Notomi S, Hisatomi T, Kanemaru T, Takeda A, Ikeda Y, Enaida H, Kroemer G,
and Ishibashi T (2011) Critical involvement of extracellular ATP acting on P2RX7
purinergic receptors in photoreceptor cell death. Am J Pathol 179:2798–2809.
Ohtomo K, Shatos MA, Vrouvlianis J, Li D, Hodges RR, and Dartt DA (2011) Increase
of intracellular Ca21 by purinergic receptors in cultured rat lacrimal gland
myoepithelial cells. Invest Ophthalmol Vis Sci 52:9503–9515.
Pannicke T, Fischer W, Biedermann B, Schädlich H, Grosche J, Faude F, Wiedemann
P, Allgaier C, Illes P, and Burnstock G, et al. (2000) P2X7 receptors in Müller glial
cells from the human retina. J Neurosci 20:5965–5972.
Penfold PL, Madigan MC, Gillies MC, and Provis JM (2001) Immunological and
aetiological aspects of macular degeneration. Prog Retin Eye Res 20:385–414.
Peral A, Carracedo G, Acosta MC, Gallar J, and Pintor J (2006) Increased levels of
diadenosine polyphosphates in dry eye. Invest Ophthalmol Vis Sci 47:4053–4058.
Peral A, Gallar J, and Pintor J (2009) Adenine nucleotide effect on intraocular
pressure: Involvement of the parasympathetic nervous system. Exp Eye Res 89:
63–70.
Peral A, Loma P, Yerxa B, and Pintor J (2008) Topical application of nucleotides
increase lysozyme levels in tears. Clin Ophthalmol 2:261–267.
Peterson WM, Meggyesy C, Yu K, and Miller SS (1997) Extracellular ATP activates
calcium signaling, ion, and fluid transport in retinal pigment epithelium. J Neurosci 17:2324–2337.
Pintor J (2007) A molecular marker for dry eye [article in Spanish]. Arch Soc Esp
Oftalmol 82:129–130.
Pintor J, Bautista A, Carracedo G, and Peral A (2004a) UTP and diadenosine tetraphosphate accelerate wound healing in the rabbit cornea. Ophthalmic Physiol
Opt 24:186–193.
Pintor J, Carracedo G, Alonso MC, Bautista A, and Peral A (2002a) Presence of
diadenosine polyphosphates in human tears. Pflugers Arch 443:432–436.
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 5, 2017
Elner SG, Yoshida A, Bian ZM, Kindezelskii AL, Petty HR, and Elner VM (2003)
Human RPE cell apoptosis induced by activated monocytes is mediated by caspase3 activation. Trans Am Ophthalmol Soc 101:77–89, discussion 89–91.
Farahbakhsh NA and Cilluffo MC (2002) P2 purinergic receptor-coupled signaling in
the rabbit ciliary body epithelium. Invest Ophthalmol Vis Sci 43:2317–2325.
Franke H, Klimke K, Brinckmann U, Grosche J, Francke M, Sperlagh B, Reichenbach
A, Liebert UG, and Illes P (2005) P2X(7) receptor-mRNA and -protein in the mouse
retina; changes during retinal degeneration in BALBCrds mice. Neurochem Int 47:
235–242.
Friedman DS, Wilson MR, Liebmann JM, Fechtner RD, and Weinreb RN (2004) An
evidence-based assessment of risk factors for the progression of ocular hypertension and glaucoma. Am J Ophthalmol 138(3, Suppl)S19–S31.
Fries JE, Goczalik IM, Wheeler-Schilling TH, Kohler K, Guenther E, Wolf S, Wiedemann P, Bringmann A, Reichenbach A, and Francke M, et al. (2005) Identification of
P2Y receptor subtypes in human Müller glial cells by physiology, single cell RT-PCR,
and immunohistochemistry. Invest Ophthalmol Vis Sci 46:3000–3007.
Fries JE, Wheeler-Schilling TH, Guenther E, and Kohler K (2004a) Expression of
P2Y1, P2Y2, P2Y4, and P2Y6 receptor subtypes in the rat retina. Invest Ophthalmol Vis Sci 45:3410–3417.
Fries JE, Wheeler-Schilling TH, Kohler K, and Guenther E (2004b) Distribution of
metabotropic P2Y receptors in the rat retina: a single-cell RT-PCR study. Brain
Res Mol Brain Res 130:1–6.
Fujihara T, Murakami T, Fujita H, Nakamura M, and Nakata K (2001) Improvement
of corneal barrier function by the P2Y(2) agonist INS365 in a rat dry eye model.
Invest Ophthalmol Vis Sci 42:96–100.
Greenwood D, Yao WP, and Housley GD (1997) Expression of the P2X2 receptor
subunit of the ATP-gated ion channel in the retina. Neuroreport 8:1083–1088.
Gröschel-Stewart U, Bardini M, Robson T, and Burnstock G (1999) Localisation of
P2X5 and P2X7 receptors by immunohistochemistry in rat stratified squamous
epithelia. Cell Tissue Res 296:599–605.
Gupta SK, Niranjan D G, Agrawal SS, Srivastava S, and Saxena R (2008) Recent
advances in pharmacotherapy of glaucoma. Indian J Pharmacol 40:197–208.
Guzmán-Aranguez A, Crooke A, Peral A, Hoyle CH, and Pintor J (2007) Dinucleoside
polyphosphates in the eye: from physiology to therapeutics. Prog Retin Eye Res 26:
674–687.
Guzman-Aranguez A, Díez LM, Martín-Gil A, Gualix J, Miras-Portugal MT,
and Pintor J (2012) Effect of diinosine polyphosphates on intraocular pressure in
normotensive rabbits. Exp Eye Res 101:49–55.
Hao LN, Wang M, Ma JL, and Yang T (2012) Puerarin decreases apoptosis of retinal
pigment epithelial cells in diabetic rats by reducing peroxynitrite level and iNOS
expression. Sheng Li Xue Bao 64:199–206.
Hodges RR, Vrouvlianis J, Scott R, and Dartt DA (2011) Identification of P2X3 and
P2X7 purinergic receptors activated by ATP in rat lacrimal gland. Invest Ophthalmol Vis Sci 52:3254–3263.
Hodges RR, Vrouvlianis J, Shatos MA, and Dartt DA (2009) Characterization of P2X7
purinergic receptors and their function in rat lacrimal gland. Invest Ophthalmol
Vis Sci 50:5681–5689.
Hu H, Lu W, Zhang M, Zhang X, Argall AJ, Patel S, Lee GE, Kim YC, Jacobson KA,
and Laties AM, et al. (2010) Stimulation of the P2X7 receptor kills rat retinal
ganglion cells in vivo. Exp Eye Res 91:425–432.
Iandiev I, Wurm A, Pannicke T, Wiedemann P, Reichenbach A, Robson SC,
Zimmermann H, and Bringmann A (2007) Ectonucleotidases in Müller glial
cells of the rodent retina: involvement in inhibition of osmotic cell swelling.
Purinergic Signal 3:423–433.
Jacobson KA, Balasubramanian R, Deflorian F, and Gao ZG (2012) G protein-coupled
adenosine (P1) and P2Y receptors: ligand design and receptor interactions.
Purinergic Signal 8:419–436.
Jacobson KA and Boeynaems JM (2010) P2Y nucleotide receptors: promise of therapeutic applications. Drug Discov Today 15:570–578.
Jumblatt JE and Jumblatt MM (1998) Regulation of ocular mucin secretion by P2Y2
nucleotide receptors in rabbit and human conjunctiva. Exp Eye Res 67:341–346.
Kaneda M, Ishii K, Morishima Y, Akagi T, Yamazaki Y, Nakanishi S, and Hashikawa
T (2004) OFF-cholinergic-pathway-selective localization of P2X2 purinoceptors in
the mouse retina. J Comp Neurol 476:103–111.
Kaneda M, Ishii T, and Hosoya T (2008) Pathway-dependent modulation by P2purinoceptors in the mouse retina. Eur J Neurosci 28:128–136.
Kaneda M, Ito K, Shigematsu Y, and Shimoda Y (2010) The OFF-pathway dominance
of P2X2-purinoceptors is formed without visual experience. Neurosci Res 66:86–91.
Kawamura H, Sugiyama T, Wu DM, Kobayashi M, Yamanishi S, Katsumura K,
and Puro DG (2003) ATP: a vasoactive signal in the pericyte-containing microvasculature of the rat retina. J Physiol 551:787–799.
Kent D, Vinores SA, and Campochiaro PA (2000) Macular oedema: the role of soluble
mediators. Br J Ophthalmol 84:542–545.
Klepeis VE, Weinger I, Kaczmarek E, and Trinkaus-Randall V (2004) P2Y receptors
play a critical role in epithelial cell communication and migration. J Cell Biochem
93:1115–1133.
Lazarowski ER, Watt WC, Stutts MJ, Boucher RC, and Harden TK (1995) Pharmacological selectivity of the cloned human P2U-purinoceptor: potent activation by
diadenosine tetraphosphate. Br J Pharmacol 116:1619–1627.
Li A, Banerjee J, Peterson-Yantorno K, Stamer WD, Leung CT, and Civan MM (2012)
Effects of cardiotonic steroids on trabecular meshwork cells: search for mediator of
ouabain-enhanced outflow facility. Exp Eye Res 96:4–12.
Li A, Zhang XL, Zheng DY, Ge J, Laties AM, and Mitchell CH (2011) Sustained
elevation of extracellular ATP in aqueous humor from humans with primary
chronic angle-closure glaucoma. Exp Eye Res 93:528–533.
Li Y, Holtzclaw LA, and Russell JT (2001a) Müller cell Ca21 waves evoked by
purinergic receptor agonists in slices of rat retina. J Neurophysiol 85:986–994.
Li Y, Kuang K, Yerxa B, Wen Q, Rosskothen H, and Fischbarg J (2001b) Rabbit
conjunctival epithelium transports fluid, and P2Y22 receptor agonists stimulate
Cl2 and fluid secretion. Am J Physiol Cell Physiol 281:C595–C602.
Function and Therapeutic Potential of Nucleotides in Eye
Sugiyama T, Kobayashi M, Kawamura H, Li Q, and Puro DG (2004) Enhancement of
P2X7-induced pore formation and apoptosis: an early effect of diabetes on the
retinal microvasculature. Invest Ophthalmol Vis Sci 45:1026–1032.
Sugiyama T, Oku H, Komori A, and Ikeda T (2006) Effect of P2X7 receptor activation
on the retinal blood velocity of diabetic rabbits. Arch Ophthalmol 124:1143–1149.
Sugiyama T, Oku H, Shibata M, Fukuhara M, Yoshida H, and Ikeda T (2010) Involvement of P2X7 receptors in the hypoxia-induced death of rat retinal neurons.
Invest Ophthalmol Vis Sci 51:3236–3243.
Sullivan DM, Erb L, Anglade E, Weisman GA, Turner JT, and Csaky KG (1997)
Identification and characterization of P2Y2 nucleotide receptors in human retinal
pigment epithelial cells. J Neurosci Res 49:43–52.
To CH, Kong CW, Chan CY, Shahidullah M, and Do CW (2002) The mechanism of
aqueous humour formation. Clin Exp Optom 85:335–349.
Tsubota K, Hata S, Okusawa Y, Egami F, Ohtsuki T, and Nakamori K (1996)
Quantitative videographic analysis of blinking in normal subjects and patients
with dry eye. Arch Ophthalmol 114:715–720.
Uckermann O, Wolf A, Kutzera F, Kalisch F, Beck-Sickinger AG, Wiedemann P,
Reichenbach A, and Bringmann A (2006) Glutamate release by neurons evokes
a purinergic inhibitory mechanism of osmotic glial cell swelling in the rat retina:
activation by neuropeptide Y. J Neurosci Res 83:538–550.
Vessey KA and Fletcher EL (2012) Rod and cone pathway signalling is altered in the
P2X7 receptor knock out mouse. PLoS ONE 7:e29990.
von Kügelgen I and Harden TK (2011) Molecular pharmacology, physiology, and
structure of the P2Y receptors. Adv Pharmacol 61:373–415.
Ward MM, Puthussery T, and Fletcher EL (2008) Localization and possible function
of P2Y4 receptors in the rodent retina. Neuroscience 155:1262–1274.
Wenkel H and Streilein JW (2000) Evidence that retinal pigment epithelium functions as an immune-privileged tissue. Invest Ophthalmol Vis Sci 41:3467–3473.
Wheeler-Schilling TH, Marquordt K, Kohler K, Guenther E, and Jabs R (2001)
Identification of purinergic receptors in retinal ganglion cells. Brain Res Mol Brain
Res 92:177–180.
Wheeler-Schilling TH, Marquordt K, Kohler K, Jabs R, and Guenther E (2000) Expression of purinergic receptors in bipolar cells of the rat retina. Brain Res Mol
Brain Res 76:415–418.
Wurm A, Iandiev I, Hollborn M, Wiedemann P, Reichenbach A, Zimmermann H,
Bringmann A, and Pannicke T (2008) Purinergic receptor activation inhibits osmotic glial cell swelling in the diabetic rat retina. Exp Eye Res 87:385–393.
Wurm A, Lipp S, Pannicke T, Linnertz R, Krügel U, Schulz A, Färber K, Zahn D,
Grosse J, and Wiedemann P, et al. (2010) Endogenous purinergic signaling is required for osmotic volume regulation of retinal glial cells. J Neurochem 112:
1261–1272.
Yang D, Elner SG, Bian ZM, Till GO, Petty HR, and Elner VM (2007) Proinflammatory cytokines increase reactive oxygen species through mitochondria
and NADPH oxidase in cultured RPE cells. Exp Eye Res 85:462–472.
Yang D, Elner SG, Clark AJ, Hughes BA, Petty HR, and Elner VM (2011) Activation
of P2X receptors induces apoptosis in human retinal pigment epithelium. Invest
Ophthalmol Vis Sci 52:1522–1530.
Yazulla S and Studholme KM (2004) Vanilloid receptor like 1 (VRL1) immunoreactivity in mammalian retina: colocalization with somatostatin and purinergic P2X1
receptors. J Comp Neurol 474:407–418.
Zhang M, Budak MT, Lu W, Khurana TS, Zhang X, Laties AM, and Mitchell CH
(2006) Identification of the A3 adenosine receptor in rat retinal ganglion cells. Mol
Vis 12:937–948.
Zimmermann H, Zebisch M, and Sträter N (2012) Cellular function and molecular
structure of ecto-nucleotidases. Purinergic Signal 8:437–502.
Address correspondence to: Dr. Jesús Pintor, Department of Biochemistry
and Molecular Biology, Faculty of Optics and Optometry, Universidad
Complutense Madrid, C/Arcos de Jalón 118, 28037 Madrid, Spain. E-mail:
[email protected]
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 5, 2017
Pintor J, Gualix J, and Miras-Portugal MT (1997) Diinosine polyphosphates, a group
of dinucleotides with antagonistic effects on diadenosine polyphosphate receptor.
Mol Pharmacol 51:277–284.
Pintor J and Peral A (2001) Therapeutic potential of nucleotides in the eye. Drug Dev
Res 52:190–195.
Pintor J, Peral A, Hoyle CH, Redick C, Douglass J, Sims I, and Yerxa B (2002b)
Effects of diadenosine polyphosphates on tear secretion in New Zealand white
rabbits. J Pharmacol Exp Ther 300:291–297.
Pintor J, Peral A, Pelaez T, Carracedo G, Bautista A, and Hoyle CHV (2003a)
Nucleotides and dinucleotides in ocular physiology: new possibilities of nucleotides
as therapeutic agents in the eye. Drug Dev Res 59:136–145.
Pintor J, Peral A, Peláez T, Martín S, and Hoyle CHV (2003b) Presence of diadenosine polyphosphates in the aqueous humor: their effect on intraocular pressure. J Pharmacol Exp Ther 304:342–348.
Pintor J, Sánchez-Nogueiro J, Irazu M, Mediero A, Peláez T, and Peral A (2004b)
Immunolocalisation of P2Y receptors in the rat eye. Purinergic Signal 1:83–90.
Puthussery T and Fletcher EL (2009) Extracellular ATP induces retinal photoreceptor apoptosis through activation of purinoceptors in rodents. J Comp Neurol
513:430–440.
Puthussery T and Fletcher EL (2007) Neuronal expression of P2X3 purinoceptors in
the rat retina. Neuroscience 146:403–414.
Puthussery T, Yee P, Vingrys AJ, and Fletcher EL (2006) Evidence for the involvement
of purinergic P2X receptors in outer retinal processing. Eur J Neurosci 24:7–19.
Reigada D, Lu W, and Mitchell CH (2006) Glutamate acts at NMDA receptors on
fresh bovine and on cultured human retinal pigment epithelial cells to trigger
release of ATP. J Physiol 575:707–720.
Reigada D, Lu W, Zhang M, and Mitchell CH (2008) Elevated pressure triggers
a physiological release of ATP from the retina: possible role for pannexin hemichannels. Neuroscience 157:396–404.
Reigada D, Lu W, Zhang X, Friedman C, Pendrak K, McGlinn A, Stone RA, Laties
AM, and Mitchell CH (2005) Degradation of extracellular ATP by the retinal pigment epithelium. Am J Physiol Cell Physiol 289:C617–C624.
Relvas LJ, Bouffioux C, Marcet B, Communi D, Makhoul M, Horckmans M, Blero D,
Bruyns C, Caspers L, and Boeynaems JM, et al. (2009) Extracellular nucleotides
and interleukin-8 production by ARPE cells: potential role of danger signals in
blood-retinal barrier activation. Invest Ophthalmol Vis Sci 50:1241–1246.
Resta V, Novelli E, Vozzi G, Scarpa C, Caleo M, Ahluwalia A, Solini A, Santini E,
Parisi V, and Di Virgilio F, et al. (2007) Acute retinal ganglion cell injury caused by
intraocular pressure spikes is mediated by endogenous extracellular ATP. Eur J
Neurosci 25:2741–2754.
Shahidullah M and Wilson WS (1997) Mobilisation of intracellular calcium by P2Y2
receptors in cultured, non-transformed bovine ciliary epithelial cells. Curr Eye Res
16:1006–1016.
Shigematsu Y, Shimoda Y, and Kaneda M (2007) Distribution of immunoreactivity
for P2X3, P2X5, and P2X6-purinoceptors in mouse retina. J Mol Histol 38:369–371.
Simó R, Villarroel M, Corraliza L, Hernández C, and Garcia-Ramírez M (2010) The
retinal pigment epithelium: something more than a constituent of the blood-retinal
barrier—implications for the pathogenesis of diabetic retinopathy. J Biomed Biotechnol 2010:190724.
Soto D, Pintor J, Peral A, Gual A, and Gasull X (2005) Effects of dinucleoside polyphosphates on trabecular meshwork cells and aqueous humor outflow facility.
J Pharmacol Exp Ther 314:1042–1051.
Sreekumar PG, Spee C, Ryan SJ, Cole SP, Kannan R, and Hinton DR (2012)
Mechanism of RPE cell death in a-crystallin deficient mice: a novel and critical role
for MRP1-mediated GSH efflux. PLoS ONE 7:e33420.
Srinivas SP, Mutharasan R, and Fleiszig S (2002) Shear-induced ATP release by
cultured rabbit corneal epithelial cells. Adv Exp Med Biol 506 (Pt A):677–685.
Strauss O (2005) The retinal pigment epithelium in visual function. Physiol Rev 85:
845–881.
Sugiyama T, Kawamura H, Yamanishi S, Kobayashi M, Katsumura K, and Puro DG
(2005) Regulation of P2X7-induced pore formation and cell death in pericytecontaining retinal microvessels. Am J Physiol Cell Physiol 288:C568–C576.
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