Distinct P2Y Receptor Subtypes Regulate Calcium Signaling in Human Retinal Pigment Epithelial Cells

Abstract

purpose. Nucleotide signaling plays a role in retinal pigment epithelial (RPE) function, and receptors for nucleotides are potential therapeutic targets for various ocular diseases. The purpose of this study was to investigate the expression of P2Y receptor subtypes in native and cultured human RPE cells.

methods. Intracellular Ca²⁺ levels were monitored using real-time fluorescence imaging in cultured human RPE cells loaded with Fura-2. Expression of P2Y receptors in native and cultured RPE cells was determined by quantitative RT-PCR and Western blot analysis.

results. Adenosine triphosphate (ATP), uridine triphosphate (UTP), adenosine diphosphate (ADP), 2-methylthio ATP (2MeSATP), and uridine diphosphate (UDP) produced concentration-related increases in [Ca²⁺]_i in cultured RPE cells. However, differences between the magnitude and shape of agonist responses were observed. ATP and UTP showed similar response characteristics, including a distinct Ca²⁺ influx component. ATP and UTP were equipotent (EC₅₀, 6 μM) and maximum responses were equivalent, suggesting activation of a P2Y₂ receptor. Maximal responses to ADP and 2MeSATP were equivalent with EC₅₀s of 1 μM and 0.3 μM. The P2Y₁ antagonist MRS 2179 (10 μM) inhibited these responses, confirming functional expression of P2Y₁ receptors. The presence of a response to UDP suggested P2Y₆ expression. There was no influx component to P2Y₁- and P2Y₆-mediated responses. mRNA for P2Y₁, P2Y_2, P2Y₄, and P2Y₆ receptor subtypes was found in cultured RPE cells, and for P2Y₁, P2Y_2, P2Y_4, P2Y₆, and P2Y₁₂ it was found in native RPE cells. Expression of P2Y₁, P2Y₂, and P2Y₆ protein was found in native and cultured RPE cells.

conclusions. These data define the expression profile of P2Y receptors in human RPE and show that different P2Y subtypes control distinct calcium responses in these cells.

The retinal pigment epithelium (RPE) is a multifunctional monolayer of pigmented epithelial cells located at the posterior of the eye. It has close structural and functional relationships with the neighboring photoreceptors of the retina and forms a polarized barrier between the retina (apical) and choroid (basolateral). Interactions between the RPE and the retina are vital for retinal homeostasis and visual function. The RPE regulates the fluid and ion balance of the subretinal space by the transport of fluid and ions from the subretinal space to the choroid. ¹ This process is regulated by a variety of G protein-coupled receptors (GPRCs). ² Abnormal accumulation of fluid between the RPE and the retina, as occurs in macular edema and retinal detachment, causes the loss of physical contact between these two tissues and results in a loss of visual function. 

The purinoceptor system is implicated in RPE-retinal interactions. ³ For example, studies have shown that phagocytosis of rod outer segments can be reduced by the activation of A2 receptors in rat RPE cells. ^{4 5} Activation of P2Y receptors by adenosine triphosphate (ATP) and uridine triphosphate (UTP) has been reported to increase [Ca²⁺]_i, Cl⁻ and K⁺ membrane conductances, transepithelial ion transport, and fluid movement in rat and bovine RPE. ^{6 7 8} In addition, the activation of apical P2Y₂ receptors by the synthetic agonist INS37217 can stimulate the fluid absorption across freshly isolated human fetal RPE monolayers. ⁹  

There are several potential sources of nucleotides and nucleosides in the posterior of the eye. RPE cells themselves have been reported to release ATP when exposed to osmotic stress, growth factors, glutamate, UTP, and ATP, ^{10 11 12 13 14} and cultured retinal neurons and astrocytes have also been shown to release ATP. ^{15 16} The RPE also expresses multiple enzymes for the breakdown of ATP, products of which (specifically adenosine diphosphate [ADP] and adenosine) are also pharmacologically active. ^{3 17} The release of ATP from the RPE and the retina may therefore lead to the activation of multiple purinoceptor signaling pathways in the RPE. 

Purine signaling has been identified in most eye tissue, and there is much interest in developing purinoceptor drugs for the treatment of various ocular disorders. ¹⁸ Investigation of purinoceptor subtypes enables identification of specific targets for novel therapeutic interventions in eye disease. For example, Soto et al. ¹⁹ recently identified the expression of P2Y₁, P2Y₂, and P2Y₄ in trabecular meshwork cells and showed that P2Y₁ agonists could increase aqueous humor outflow. In addition, the secretion of tear film has been found to be regulated by the P2Y₂ receptor. ^{18 20} These receptors represent potential new targets for the treatment of glaucoma and dry eye, respectively. In the RPE, the stimulation of fluid transport by P2Y₂ activation has been shown to facilitate retinal reattachment in animal models of retinal detachment. ^{9 21} Purinoceptors are therefore also a target for the treatment of this disease. In this article, we show that multiple P2Y receptor subtypes are expressed in the native human RPE, and we demonstrate functional expression in human cultured RPE cells. 

Methods

Tissue Culture

RPE tissue was obtained from human donor eyes provided by the East Anglian Eye Bank (Norwich, UK). Because no donor details, apart from age, sex, and cause of death, were released, this research followed the tenets of the Declaration of Helsinki. The culture method has previously been described. ²² Briefly, less than 48 hours after death, globes were dissected in sterile 1:1 Dulbecco modified Eagle medium—Ham F-12 (DMEM/F-12) supplemented with 10% fetal calf serum, 2 mM l-glutamine, 26 mM sodium bicarbonate, and 50 mg/L gentamicin. The anterior portion of the sclera, followed by the ciliary body and vitreous humor, were dissected from the eye. The RPE and neural retina were dissected at the optic nerve. The RPE was separated from the retina and transferred to fresh medium, where it was cut into 25-mm² pieces. Each piece of RPE (Fig. 1A)was transferred to a 35-mm culture dish with 1.5 mL fresh medium and was pinned securely with entomological pins (Watkins and Doncaster Ltd., Kent, UK). Medium was replaced every 3 to 4 days, and primary RPE cells began to migrate and proliferate on the culture dish at approximately 1 to 2 weeks in culture. Although native cells remained pigmented, daughter cells did not contain any pigment (Figs. 1B 1C) . Once a sufficient number of cells had grown onto the culture dish, the RPE explant was removed and discarded. RPE cultures were examined for cells with a mesenchymal morphology that were then scraped away, leaving only cells with an epithelial morphology. These cells were trypsinized and seeded into cell culture flasks, grown, and passaged onto coverslips or culture dishes, depending on the experimental procedure (Fig. 1B) . Cultured cells left to grow to confluence displayed characteristics similar to the native tissue (Fig. 1C) , and they expressed the junctional proteins ZO-1 and β-catenin at the lateral cell-cell interfaces as well as cytokeratins 8 and 18 (data not shown). Cells between passages 0 and 3 were used in this study. 

Native RPE Preparations

Immediately after dissection and under a dissecting microscope in a sterile lamina flow hood, RPE explants were pinned into Petri dishes containing DMEM/F12, with the apical surface uppermost (Fig. 1A) . RPE cells were gently brushed away from the basement membrane with a pair of curved forceps. Cells lifted from the basement membrane in rafts and were clearly identifiable as a monolayer of RPE cells. Cells were collected and centrifuged for 5 minutes at 13,000 rpm. The supernatant was removed, and the pellet was frozen in liquid nitrogen. Pellets were stored at −80°C until they were used for protein or RNA extraction. 

Ca²⁺ Imaging

Cultured RPE cells grown on coverslips (Fig. 1B)were loaded with the acetoxymethyl ester (AM) form of Fura-2 (3 μM) for 60 minutes at 35°C. The coverslip formed the base of a chamber placed on the stage of an inverted epifluorescence microscope (Nikon, Melville, NY) fitted with a ×20 objective. Cells were washed for 20 minutes with physiological saline solution (PSS; pH 7.25) of the following composition: 130 mM NaCl, 20 mM HEPES, 5 mM glucose, 5 mM KCl, 5 mM NaHCO₃ (all from Fisher Scientific Ltd., Loughborough, UK), 1 mM CaCl₂ (BDH Laboratory Supplies, Poole, UK), and 0.5 mM MgCl₂ (Sigma, Poole, UK). RPE cells were continually perfused with PSS at a constant rate (approximately 1 mL/min), and agonist solutions were administered by a two-way tap. Ratiometric imaging was achieved by exciting cells at 340 nm and 380 nm and collecting the resultant emission intensities at 510 nm. Fluorescent emissions were collected by a charged-coupled device (CCD) camera (Photon Technology International, Birmingham, NJ) driven by imaging software (Image Master; Photon Technology International). Cells selected for imaging were subconfluent (Fig. 1B) , and data were collected from regions of interest placed around individual cells and acquired as a running ratio average. Fluorescence emissions were also collected from a region of each coverslip that was devoid of cells. This served as a background and was subtracted from 340-nm and 380-nm values on a spreadsheet (Excel; Microsoft, Redmond, WA). The resultant fluorescence ratios were converted to calcium concentrations after calibration of the cells using methods previously described. ²²  

Western Blot Analysis

Protein was extracted from cultured RPE cells or native RPE cells using Daub lysis buffer ²³ (50 mM HEPES [pH 7.5], 150 mM NaCl [both Fisher Scientific Ltd.], 1% Triton X-100, 1 mM EDTA, 10% glycerol [BDH Laboratory Supplies], 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 μg/mL aprotinin. Cell lysates were centrifuged at 13,000 rpm for 10 minutes at 4°C, and the supernatant was collected and analyzed for protein content by BCA assay (Pierce, Rockford, IL). Samples were loaded for electrophoresis at 100 μg/lane onto precast 10% polyacrylamide mini-gels (Pierce). The proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA) by semidry blotting (Trans-Blot semidry transfer cell; Bio-Rad Laboratories). 

Transferred proteins were blocked in 5% fat-reduced milk powder in 0.5% Tween-20 phosphate-buffered saline (PBS-T; Sigma). Membranes were incubated with rabbit anti-P2Y₁, anti-P2Y₂, anti-P2Y₄, anti-P2Y₆, anti-P2Y₁₁, or anti-P2Y₁₂ (Alomone Laboratories, Jerusalem, Israel). These are polyclonal antibodies raised to short peptide sequences (13–17 amino acids) specific to each receptor to enable selectivity. Either they are raised to a peptide sequence corresponding to residues from the human receptor (P2Y₁, P2Y₁₁, P2Y₁₂) or they have been shown to cross-react with human receptors. ²⁴ Rat brain was used as a positive control. Antibodies were diluted at 1:200 in 5% Marvel PBS-T, incubated for 2 hours at room temperature, and subsequently washed (6 × 10 minutes) in 1% Marvel PBS-T. Control membranes were incubated in antibody solutions that had previously been incubated with their homologous peptide antigen for 1 hour at room temperature. Incubation in secondary antibody (Amersham Biosciences, Amersham, UK), diluted in 5% Marvel PBS-T (1:1200), was carried out at room temperature for 1 hour and was followed by washes. Detection was by enhanced chemiluminescence (ECL Plus; Amersham Biosciences, Amersham, UK). 

Quantitative RT-PCR

Tissue samples from different regions of human RPE were collected as described. Total RNA was isolated from native RPE and human cultured RPE cells (RNeasy Mini Kit; Qiagen, Valencia, CA) according to the manufacturer’s instructions. This includes a step for removal of genomic DNA on the column provided using RNase-free DNase1 (Qiagen). The RNA was used as a template for first-strand cDNA synthesis using random primers (Promega, Madison, WI) and superscript II reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA) according to standard protocols. P2Y receptor gene expression was measured by quantitative (Q) RT-PCR using validated primers and probes (Assay-on-Demand; Applied Biosystems, Foster City, CA). Assay identification numbers are P2RY ₁ (Hs00704965_s1), P2RY ₂ (Hs00175732_m1), P2RY ₄ (Hs00267404_s1), P2RY ₆ (Hs00602547_m1), P2RY ₁₁ (Hs01038858_m1), and P2RY ₁₂ (Hs00224470_m1). 18S rRNA primers and probes (Applied Biosystems) were used as an endogenous control. Each reaction mixture consisted of 2.5 ng cDNA (1 ng for the 18S analysis), 33% 2 × Master Mix (TaqMan; Applied Biosystems), 100 nM of each forward and reverse primer, and 200 nM probe in a total volume of 25 μL. Standard curves were constructed for each gene (fourfold serial dilutions) using a sample that expressed the gene of interest and for 18S rRNA (eightfold dilutions). Real-time PCR reactions were performed (ABI PRISM 7700 Sequence Detection System; Applied Biosystems). Reaction conditions for the PCR amplification were 2 minutes at 50°C and 10 minutes at 95°C, followed by 40 cycles, each consisting of 15 seconds at 95°C and 1 minute at 60°C. Levels of mRNA in each sample were determined using standard curves and normalized to 18S rRNA. 

Results

Ca²⁺ Responses to Purinoceptor Agonists

Potency sequences of agonists at purinoceptors are indicative of receptor subtype. ²⁵ The calcium-signaling responses of human cultured RPE cells to a series of purinoceptor agonists were therefore investigated. ATP, UTP, ADP, 2-methylthio ATP (2MeSATP), and uridine diphosphate (UDP; 10 μM, 30-second exposure) all caused increases in intracellular calcium concentration ([Ca²⁺]_i) (Figs. 2A 2B) . ATP and UTP produced similar response characteristics, with a rapid initial increase in [Ca²⁺]_i followed by a sustained plateau lasting approximately 5 minutes (Figs. 2A 2B) . Most cells responded to these agonists, with both ATP and UTP producing responses in approximately 90% of cells (Fig. 3) . ADP and 2-MeSATP produced a more transient response with a slower initial increase in [Ca²⁺]_i than ATP and UTP (Figs. 2A 2B) . Responses to these agonists occurred in approximately 55% of cells (Fig. 3) . Responses to UDP, which occurred in approximately 10% of cells, were less frequent than in other purinoceptor agonists. In cells that did respond to UDP, the increase in [Ca²⁺]_i was consistently lower in magnitude than that observed for all other agonists. The response to agonists occurred in all responding cells simultaneously on initial exposure to the agonist, with no secondary responses observed in either adjacent or adjoining cells. 

It was important to investigate whether Ca²⁺ mobilization originated from intracellular stores or whether it was caused by influx from the extracellular medium. P2X receptors are ligand-gated ion channels, some of which are permeable to Ca²⁺. Therefore, extracellular Ca²⁺ contributes to the initial Ca²⁺ peak when the receptor is activated. P2Y receptors are G-protein-coupled receptors, and the initial increase in [Ca²⁺]_i results from release of Ca²⁺ from intracellular stores. Removing extracellular Ca²⁺ can, therefore, determine whether agonists act through P2X or P2Y receptor subtypes by indication of whether this initial increase in [Ca²⁺]_i is from the extracellular medium or is from an intracellular source. All agonists were capable of eliciting increases in [Ca²⁺]_i in the absence of extracellular Ca²⁺ to the same magnitude as responses observed in the presence of extracellular Ca²⁺ (Figs. 4A 4B 4C 4D 4E) . This indicated that the receptors mediating the [Ca²⁺]_i increase were P2Y receptors and that the initial peak originated from intracellular Ca²⁺ stores. In addition, these experiments showed differences in the response profiles by different agonists. The rate of decrease of [Ca²⁺]_i occurred more rapidly in response to ATP and UTP in the absence of extracellular Ca²⁺ than in the presence of extracellular Ca²⁺ (Figs. 4A 4B) ; however, no significant difference was noted when comparing ADP, 2MeSATP, and UDP responses in Ca²⁺-containing medium compared with Ca²⁺-free medium (Figs. 4C 4D 4E) . Hence, ATP and UTP have a secondary Ca²⁺ influx component to their response—leading to the observed biphasic Ca²⁺ response—whereas the other agonists do not. This suggests that ATP and UTP activate different receptor subtypes to ADP, 2MeSATP, and UDP. To enable a more detailed comparison of the agonist responses, dose-response characteristics were investigated. 

ATP, UTP, ADP, 2MeSATP, and UDP all produced concentration-related increases in [Ca²⁺]_i in cultured human RPE cells (Fig. 5A) . The EC₅₀ for both ATP and UTP was 6 μM, with maximal responses occurring at approximately 100 μM. Maximal responses induced by ADP and 2MeSATP were significantly lower (approximately 50%) than ATP and UTP maximal responses (Fig. 5B) . No significant difference in maximal response amplitude was found between ATP and UTP or ADP and 2MeSATP (Fig. 5B) . ADP and 2MeSATP were more potent than the other agonists; EC₅₀ values were 1 μM and 0.3 μM, respectively. UDP was the least potent of the agonists investigated; the EC₅₀ value was 20 μM, and the maximal response was at 50 μM. The maximal response for UDP was approximately 30% that for ATP and UTP. 

P2Y₁ Receptor Antagonism

Responses to ADP and 2MeS-ATP suggest the presence of a P2Y₁ receptor; therefore, studies using the P2Y₁-selective antagonist MRS 2179 ²⁶ were carried out. Example traces from individual cells are shown (Fig. 6) , together with data compiled from approximately 90 cells (Fig. 7) . MRS 2179 (10 μM) had no effect on submaximal responses induced by 10-μM applications of ATP, UTP, or UDP (Figs. 6C 6D 6E 7) . The antagonist, however, almost completely abolished maximal concentrations of 2MeSATP and ADP (both at 10 μM; Figs. 6A 6B ). The antagonism was fully reversible. 

Gene and Protein Expression of P2Y Receptor Subtypes

To augment the pharmacologic data, P2Y receptor expression was determined at the molecular level in cultured RPE cells and freshly isolated human native RPE cells. Gene expression for P2Y receptor subtypes was investigated using QPCR methods. P2Y_1, P2Y_2, P2Y₄, and P2Y₆ mRNA was expressed in human cultured RPE cells (Fig. 8) . In addition to these, native RPE cells also showed expression of the P2Y₁₂ receptor subtype (Fig. 8) . 

At the protein level, immunoblots showed the presence of P2Y₁, P2Y₂, and P2Y₆ in cultured cells and native RPE (Fig. 9) . The P2Y₁ receptor antibody revealed a major band at 105 kDa (Fig. 9A)in native and cultured RPE cells. Binding was abolished in the control membrane incubated with the preabsorbed antibody, indicating that binding was specific. A P2Y₂ receptor band was visible at 38 kDa in cultured RPE cells and was not present in the control membrane (Fig. 9B) . In native samples, a major band was also seen at 38 kDa, with minor bands at 35 and 40 kDa. Again, the control peptide abolished binding, indicating that each of these bands represented specific binding to the receptor. P2Y₆ receptor expression was also detected in native and cultured RPE cells and showed a major band at 95 kDa in cultured samples and at 140 kDa in native samples (Fig. 9C) . Expression of P2Y₄, P2Y₁₁, and P2Y₁₂ receptor subtypes was not found in cultured or native RPE cells but was evident in the positive controls (Figs. 9D 9E 9F) . 

Discussion

Identification of purinoceptor subtypes and their function is important in gaining a wider knowledge of the mechanisms involved in the homeostasis of the RPE and its surrounding tissues. Sullivan et al. ²⁷ have previously identified and characterized a P2Y₂ receptor in cultured human RPE cells, and localization of P2Y₂ mRNA has also been shown in rabbit, rat, and monkey RPE cells. ^{28 29} It is this receptor that has been targeted for the treatment of retinal detachment because an increase in intracellular Ca²⁺ through this receptor has been linked to fluid transport across the RPE. ^{9 21} In addition to the P2Y₂ receptor, other subtypes have been reported. This is significant because if several purinoceptor subtypes are expressed, they could work synergistically in control of downstream physiological responses. Expression of mRNA has been demonstrated for the P2Y₁ and P2Y₁₂ receptor subtypes in the ARPE-19 cell line, and Ca²⁺ imaging data suggest functional expression of the P2Y₁ receptor in these cells. ¹⁷ P2Y₁, P2Y₂, P2Y₄, and P2Y₆ mRNA expression has been reported in rat RPE. ³⁰ Pintor et al. ²⁹ also reported expression of P2Y₂, P2Y₄, and P2Y₁₁ in rat RPE. There is good evidence, therefore, that purine signaling has a role to play in RPE function. Some discrepancies, however, clearly exist. These may be methodological, or they could be accounted for by species or strain differences or by differences between cell lines and native material. It was important, therefore, to use several methodologies to determine the expression profile in human RPE. 

Our results indicated the presence of multiple P2Y receptor subtypes in human RPE. All the purinoceptor agonists investigated showed an initial release of Ca²⁺ from intracellular stores, indicating that ionotropic P2X receptor subtypes did not contribute to the responses observed. These agonists, therefore, acted through the P2Y family of receptors. After the initial release from intracellular stores, ATP and UTP responses showed a second phase that was dependent on the presence of extracellular Ca²⁺. This influx phase of Ca²⁺ signaling, produced by ATP and UTP, has been seen previously in human RPE cells. ^{7 10 27} Responses to ADP, 2MeSATP, and UDP, with much more transient responses to these agonists, did not show this influx phase. The difference in response characteristics induced by ATP and UTP compared with ADP, 2MeSATP, and UDP suggested the activation of distinct receptor subtypes with different downstream signaling mechanisms. In addition, there were clear divisions when the number of cells responding was analyzed (Fig. 2) . ATP and UTP gave responses in 90% of cells, ADP and 2MeSATP gave responses in 55% of cells, and UDP gave responses in 10% of cells. These divisions were recapitulated in the dose-response relationships. ATP and UTP showed equivalent EC₅₀ values and maximal responses. ADP and 2MeSATP had equivalent maximal responses of approximately 50% of the ATP/UTP responses, and UDP reached a maximal response at approximately 30% that of ATP/UTP. Evidence from the agonist profiling data therefore suggested expression of three receptor subtypes. 

The equipotent ATP/UTP response is most characteristic of the P2Y₂ receptor subtype. ²⁵ In addition, the dynamics of the Ca²⁺ response, with the initial release from intracellular Ca²⁺ stores followed by Ca²⁺ influx through the plasma membrane, is consistent with the response profile shown by the P2Y₂ agonist INS37217. ⁹ However, the presence of the P2Y₁₁ receptor subtype cannot be ruled out by the pharmacologic data. ³¹ In addition, it should be noted that UTP could also be signaling through the P2Y₄ receptor, which is selectively active for this uridine nucleotide. ^{32 33} Cells responded to ADP and 2MeSATP, with potency orders consistent with those reported in other systems for the P2Y₁ receptor subtype. ³⁴ The P2Y₁ receptor antagonist MRS 2179 inhibited the ADP and 2MeSATP-induced responses but had no effect on submaximal concentrations of ATP, UTP, and UDP. The functional expression of P2Y₁ receptors can, therefore, be confirmed by these data. UDP is an agonist at P2Y₆ receptors, suggesting that this subtype may also be functionally active in these cells. 

Identification of the functionally expressed receptor subtypes was facilitated by determination of gene and protein expression in both native and cultured RPE cells. Native samples showed gene expression for the P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₂ receptor subtypes. The expression of P2Y₁, P2Y₂, and P2Y₆ receptors was confirmed by Western blot analysis. However, no protein expression was found for the P2Y₄ and P2Y₁₂ receptor subtypes. Neither mRNA nor protein was found for P2Y₁₁. In cultured RPE cells, mRNA expression was found for the P2Y₁, P2Y₂, P2Y₄, and P2Y₆ receptor subtypes. P2Y₁, P2Y₂, and P2Y₆ receptors were found at the protein level, with no protein expression of the P2Y₄ receptor subtype. 

This suggests that it could be misleading simply to use mRNA expression to indicate the functional presence of a receptor. Certainly, detection of the mRNA for a specific receptor did not necessarily give detectable levels of receptor protein or pharmacologic activity. Protein expression, however, was consistent with pharmacologic data, which together confirm the presence of P2Y₁, P2Y₂, and P2Y₆ receptors in human RPE. 

It is interesting that both the PCR and the blot data show a difference in expression level of the P2Y₂ receptor between native tissue and cultured cells. The data suggest that P2Y₂ expression is upregulated in cultured RPE cells. This has also been shown to be the case in rat salivary gland cells, where P2Y₂ upregulation in short-term culture has been described ³⁵ and again highlights the importance of comparing cultured cell data with native tissue. It should be stated, however, as validation of the cultured RPE cells used here, that the same receptor profile was found in native and cultured cells. 

Purinoceptors have been implicated in the pathophysiology of a number of diseases, including diabetes, thrombosis, cancer, bone disorders, and diseases of the ear and eye. ³⁶ In the eye, there is much interest in the development of drugs acting at purinoceptors for several ocular abnormalities, including dry eye, glaucoma, corneal wound healing, and retinal detachment. ¹⁸ We have shown here that human RPE expresses P2Y₁, P2Y₂, and P2Y₆ receptors. It now remains to investigate the physiological consequences of stimulation of these receptors, to determine how they contribute individually and collectively to fluid transport. Of specific interest here will be whether the different Ca²⁺ response profiles relate to differences in the regulation of fluid transport. It will also be important to determine how the P2Y receptors integrate with other GPRCs on the RPE. For example, we have previously shown that the Ca²⁺ response to ATP can be potentiated by stimulation of A₁ and A₂ adenosine receptors, ²² which again could have important consequences for fluid transport. In addition, it will be important to understand the mechanisms that regulate purine signaling under normal and pathologic conditions. For example, it has recently been demonstrated that stimulation of P2Y₁ receptors increases the expression of the ecto-ATPase NTPDase1, ³⁷ whereas ecto-5′ nucleotidase (CD73) activity is downregulated by stimulation of α1-adrenergic receptors. ³⁸ An understanding of the role of purine signaling in the RPE may lead to novel therapeutic strategies for retinal conditions. 

 Supported by The Humane Research Trust and the School of Chemical Sciences and Pharmacy.

 Submitted for publication August 10, 2007; revised September 23, 2007; accepted November 21, 2007.

 Disclosure: V.E. Tovell, None; J. Sanderson, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Phase-contrast images of human native and cultured RPE cells. (A) Apical view of human native RPE explant tissue. (B) Cultured RPE cells grown on coverslips for calcium-imaging experiments. (C) Cultured RPE cells grown to confluence. Cells exhibit the same hexagonal pattern and phase bright borders seen in native tissue.

Figure 2.

 

Calcium increases in response to P2 receptor agonists in human RPE cells. (A) Typical experimental trace showing changes in [Ca²⁺]_i in a single RPE cell in response to ATP, 2MeSATP, UTP, ADP, and UDP. Each agonist was administered at 10 μM as a 30-second pulse, with 15-minute intervals between the administrations of agonist. Differences in size and duration of responses can be seen between agonists. (B) Expanded overlay of typical responses to P2 receptor agonists. ATP and UTP show Ca²⁺ increases of similar magnitude and a sustained Ca²⁺ increase compared with ADP, 2MeSATP, and UDP. Ca²⁺ responses to ADP and 2MeSATP are also of similar magnitude. Scale bar, 30-second pulse of each agonist.

Figure 3.

 

Percentage of human RPE cells giving an increase in calcium in response to P2 receptor agonists. Cells were exposed to ATP, UTP, ADP, 2MeSATP, and UDP (10 μM, 30-second pulse). Percentages of cells responding to ATP and UTP in the field of view was 91% ± 1% and 92% ± 3%, respectively; 59% ± 5% and 54% ± 10% cells responded to ADP and 2MeSATP, and 8% ± 4% cells responded to UDP. Numbers responding to ATP and UTP were significantly higher than those responding to ADP and 2MeSATP (***P < 0.001, Student’s t-test), which, in turn, was significantly higher than the number of cells responding to UDP (^### P < 0.001, Student’s t-test). Data, expressed as mean ± SEM, were taken from four independent experiments and 120 cells.

Figure 4.

 

Effects of extracellular Ca²⁺ on responses induced by P2 receptor agonists in human RPE cells. (A) ATP-, (B) UTP-, (C) ADP-, (D) 2MeSATP-, and (E) UDP-induced calcium responses in the presence and absence of extracellular calcium. Peak responses for all agonists were the same in the presence and absence of extracellular calcium, indicating that responses were mediated through P2Y receptors and not P2X receptors. ATP and UTP had a significant component of Ca²⁺ influx, which was not evident for the other agonists. Scale bars, 30-second pulse of the agonist at 10 μM. The experiment was repeated four times. Typical responses are shown.

Figure 5.

 

Concentration-response curves (A) and maximal responses (B) for P2Y receptor agonists in human RPE cells. (A) All agonists produced concentration-dependent increases in intracellular calcium. ATP (▪) EC₅₀ 6 μM, UTP (□) EC₅₀ 6 μM, ADP (•) EC₅₀ 1 μM, 2MeSATP (○) EC₅₀ 0.3 μM, and UDP (⋄) EC₅₀ 20 μM. Data for each curve were from four separate experiments performed on approximately 60 cells. (B) Maximal responses for ATP and UTP were significantly different from those of ADP and 2MeSATP (***P < 0.001, Student’s t-test), which, in turn, were significantly different from UDP (^### P < 0.001, Student’s t-test). No significant differences were found between ATP and UTP or ADP and 2MeSATP. All data (mean ± SEM) are expressed as the percentage increases in Ca²⁺ above baseline (control) Ca²⁺ levels.

Figure 6.

 

P2Y₁ receptor antagonist MRS 2179 inhibited the ADP and 2MeSATP responses. Typical experimental traces are shown. RPE cells were preincubated with the antagonist for 2 minutes before cells were exposed to a 30-second pulse of agonist. (A, B) MRS 2179 (10 μM) abolished responses induced by ADP and 2MeSATP (10 μM) in these cells. This effect was fully reversible. (C–E) MRS 2179 (10 μM) had no noticeable effect on responses induced by ATP, UTP, and UDP (10 μM).

Figure 7.

 

The P2Y₁ receptor subtype mediated the effects of ADP and 2MeSATP in human RPE cells. MRS 2179 (10 μM) significantly inhibited responses induced by ADP and 2MeSATP (10 μM). ATP, UTP, and UDP responses were unaffected. Results are expressed as mean ± SEM and for each agonist; four independent experiments were performed on approximately 90 cells. Asterisks: significant differences (P < 0.001) between responses induced by agonist alone and responses induced by agonist after pretreatment with MRS 2179.

Figure 8.

 

QPCR analysis of P2Y receptor mRNA expression in native and cultured RPE cells. Expression of P2Y₁, P2Y₂, P2Y₄, and P2Y₆ is shown in human cultured RPE cells, whereas human native RPE expressed P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₂. Expression of P2Y₂ was significantly lower in native than in cultured cells (P < 0.05, Student’s t-test). Gene expression data (mean ± SEM) are expressed relative to 18S ribosomal RNA expression.

Figure 9.

 

Protein expression of P2Y receptor subtypes in native and cultured human RPE cells. Expression of P2Y₁, P2Y₂, and P2Y₆ is demonstrated. (A–C) Western blotting of cultured RPE cells (C) and native RPE cells (N). Cultured and native samples were also preincubated with the control peptide antigen (P). (D–F) Western blotting of cultured RPE cells (C), native RPE cells, (N) and rat brain (B).

The authors thank Pamela Keely and Deborah Busby from the Norfolk and Norwich University Hospital Eye Bank for providing donor material; Caroline Pennington and Dylan Edwards for use of the PCR system; Sarah Burroughes for her contribution to the calcium imaging data; and The Humane Research Trust for its support.