Expression of P2Y1, P2Y2, P2Y4, and P2Y6 Receptor Subtypes in the Rat Retina

Abstract

purpose. To elucidate the expression pattern of different types of metabotropic P2Y receptors in the adult rat retina.

methods. Qualitative RT-PCR was used to investigate the expression profile of different P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, and P2Y6), and in situ hybridization studies were performed to show their cellular localization within the retina. Immunohistochemical staining was used to detect the corresponding P2Y proteins (P2Y1, P2Y2, and P2Y4) and their cellular localization. Southern blot analysis and sequencing verified the identity of the P2Y PCR products.

results. RT-PCR revealed the presence of P2Y1, -2, -4, and -6 mRNA in the neural retina and the retinal pigment epithelium (RPE) and choroid. In situ hybridization showed labeling in the retinal ganglion cell layer for all four P2Y receptor subtypes, although the intensity varied. In addition, staining for P2Y1, -4, and -6 mRNA was shown in the inner nuclear layer, but was absent for the P2Y2 receptor subtype. Immunohistochemistry showed intense staining for P2Y1, -2, and -4 in the ganglion cell layer and the outer plexiform layer. There was also a specific subtype staining in the inner plexiform layer (P2Y2, -4), the inner (P2Y1, -4) and outer (P2Y1) nuclear layers and the inner segments of the photoreceptors (P2Y1, -2).

discussion. The data suggest that extracellular nucleotides may play complex roles as autocrine–paracrine mediators and may have neuromodulatory effects in the retina through metabotropic P2Y receptors.

Nucleotides play important roles in different physiological functions of the mammalian organism, such as cell growth, cell death, and the release of hormones. ¹ The cloning and characterization of multiple purine receptors in the past 15 years has identified adenosine triphosphate (ATP) and other nucleotides as essential extracellular signaling molecules. ² They have been shown to act as neurotransmitters in various brain areas of the mammalian nervous system as well as in non-neuronal tissues. ³  

Purine receptors of the P2 type constitute a new class of receptors that recognize these nucleotides and mediate the action of extracellular nucleotides in many cell types. They comprise two large families: ligand-gated (ionotropic) P2X receptors and metabotropic P2Y receptors. Receptors of the P2Y family are seven-transmembrane-domain receptors that couple to different intracellular signaling pathways through heterotrimeric G-proteins. However, the degree of homology with other members of the G-protein–coupled receptor family, such as the muscarinic receptors and the β-adrenergic receptors, is very low. ^{3 4 5 6} Currently, eight different P2Y-receptor subtypes are known in mammals: P2Y1, -2, -4, -6, -11, -12, -13, and -14; some other P2Y receptors have been cloned from non–mammalian species (P2Y3/chicken, P2Y8/Xenopus), and the subtypes P2Y5, -7, -9, and -10 were found not to be true members of the P2Y-family. ^{3 5 6 7 8 9 10} P2Y1, -2, -4 and -6 receptors show a wide distribution and are widely accepted as functional receptors in mammals. On the basis of their nucleotide selectivity, these mammalian P2Y receptors can be roughly divided into three categories: receptors activated solely by adenine nucleotides (P2Y1); receptors activated essentially by uridine nucleotides (P2Y6); and receptors activated by both adenine and uridine nucleotides (P2Y2, -4). ^{3 11 12} P2Y11, -12, -13, and -14 subtypes have been discovered only recently, and only limited information about their distribution and function is currently available. P2Y11 receptors have been found in the placenta, kidney, and spleen as well as in immune cells, particularly in lymphocytes. ^{7 11 13 14 15 16} P2Y12 receptors seem to play an important role in coagulation of platelets and have been also found in the brain. ^{8 17 18 19 20 21 22 23 24} P2Y13 receptors were found to be present in various inner organs and the brain, and they show numerous similarities with P2Y12 receptors. ^{9 25 26} P2Y14 receptors have uridine diphosphate (UDP)-glucose and UDP-galactose as ligands, ^{10 27} and they have been identified in various inner organs and the brain and seem to have a special function in the immune system. ^{27 28 29}  

Some information about the action of purines in the eye under normal and pathologic conditions is already available. In human lens cells, activation of P2Y receptors leads to calcium release, ³⁰ in bovine cornea cells, P2Y receptors participate in the transport of fluids ^{31 32} and in the iris of rats the release rate of noradrenalin is modulated by ATP. ³³ In contrast, only limited information is available about P2Y receptors in the retina. In the photoreceptors of the frog retina, ATP modulates sensitivity to light. ³⁴ In mice and ferrets, purine receptors play a role in the development and regeneration of retinal cells ³⁵ and ATP as well as uridine triphosphate (UTP) cause calcium release from intracellular stores in the embryonic chick retina. ³⁶ In a recent paper, ³⁷ the occurrence of P2Y2 receptor mRNA in various ocular tissues of macaques and rabbits is described. In situ hybridization has verified the presence of P2Y2 mRNA in different retinal cell layers, for instance in the ganglion cell layer and the inner nuclear layer (INL) of adult rhesus macaques and albino rabbits. ³⁷  

Nucleosides and nucleotides are interesting new pharmacological tools that may be suitable for the treatment of some ocular diseases. ³⁸ Investigation of the effects of ATP and UTP in human Müller cells indicates that P2 receptors may be altered in the retina of patients with vitreoretinopathy. ^{39 40}  

In view of the limited information about P2Y receptors in the mammalian retina, ⁴¹ the present study was aimed at elucidating their expression and cellular localization by means of RT-PCR, in situ hybridization ISH, and immunohistochemistry. 

Of the eight known P2Y-receptor subtypes in mammals, we confined ourselves to the subtypes P2Y1, -2, -4, and -6 because they are widely accepted as functional receptors in mammals and show a wide distribution in various tissues. In addition, antibodies against at least the subtypes P2Y1, -2, and -4 are available. 

Materials and Methods

All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Pigmented adult rats (Brown Norway; aged 50–70 days) were killed by CO₂ incubation, the eyes were dissected immediately after enucleation at room temperature (RT) by hemisection along the ora serrata, and the anterior segment was discarded. For RT-PCR, the retina was carefully removed from the underlying tissue, to avoid any contamination with retinal pigment epithelium (RPE). Then, the choroid with the adhering RPE was isolated. All tissues were frozen immediately in liquid nitrogen and stored at −80°C. For ISH, the eyecups were fixed in 4% paraformaldehyde (PFA) in phosphate buffer (PB; 0.2 M, pH 7.4) for 30 minutes at 4°C. Afterward, the eyecups were immersed for cryoprotection in 30% sucrose overnight at 4°C and embedded in optimal cutting temperature compound (OCT; Tissue Tek; Leica, Nussloch, Germany). Cryosections 10 μm thick were collected on coated (Vectabond; Vector Laboratories, Burlingame, CA) glass slides, dried at 60°C for 3 hours, and stored at −80°C until use. For immunohistochemistry the eyecups were fixed in 4% PFA and 0.5% glutaraldehyde in PB (PB; 0.2 M, pH 7.4) for 1 hour at 4°C. After they were washed with 0.1 M PB, the eyecups were immersed for cryoprotection in 30% sucrose overnight at 4°C and embedded in OCT (Tissue Tek; Leica). Cryosections 12 μm thick were collected on poly-l-lysine (Sigma-Aldrich, Deisenhofen, Germany)-coated glass slides, air dried for 2 hours, and stored at −20°C until use. 

RT-PCR Analysis

Total RNA from the retina and RPE/choroid was isolated (RNeasy Total RNA Kit; Qiagen, Hilden, Germany). First-strand cDNA was synthesized from 1 μg total RNA, using an oligo-d(T)–primed, 20-μL reaction mixture (Superscript RNase H-Reverse transcriptase; Invitrogen-Life Technologies Inc., Gaithersburg, MD), according to standard procedures. PCR amplification of P2Y-receptor-subunit–specific fragments was performed with the following primer pairs (kind gift of Eric A. Barnard, Glaxo Institute of Applied Pharmacology, Cambridge, UK) ⁴² : P2Y1 sense primer 5′-TGG CGT GGT GCT GCA CCC TCT CAA GTC-3′ and antisense primer 5′-CGG GAC AGT CTC CTT CTG AAT GTA-3′, yielding a fragment of 561bp; P2Y2 sense primer 5′-CTG CCA GGC ACC CGT GCT CTA CTT-3′ and antisense primer 5′-CTG AGG TCA AGT GAT CGG AAG GAG-3′, yielding a fragment of 339 bp; P2Y4 sense primer 5′-CAC CGA TAC CTG GGT ATC TGC CAC-3′ antisense primer 5′-CAG ACA GCA AAG ACA GTC AGC ACC-3′, yielding a fragment of 377 bp; P2Y6 sense primer 5′-GGA GAC CTT GCC TGC CGC CTG GTA-3′ and antisense primer 5′-TAC CAC GAC AGC CAT ACG GGC CGC-3′, yielding a fragment of 410 bp. The PCR amplifications were performed on a thermal cycler (GeneAmp PCR System 2400; Applied Biosystems (ABI), Weiterstadt, Germany) using 5 μL of the first strand cDNA in a final volume of 50 μL 1 U Taq (AmpliTaq; ABI) and a primer concentration of 40 ng/μL. Reaction conditions were 40 cycles of 30 seconds at 94°C, 30 seconds at 65°C (for P2Y4) and 66°C (for P2Y1, -2, -6), and 30 seconds at 72°C. The reaction products were analyzed by gel electrophoresis. 

Southern Blot Analysis

Identity of the PCR products was tested by Southern blot analysis, using P2Y-subunit–specific oligonucleotides (P2Y1: 5′-AGT ATG TGC ACG ACT GTG GCC ATG TTC TGC-3′; P2Y2: 5′-TAT GGG ACC ACA GGT CTG CCT CGG GCC AAG-3′; P2Y4: 5′-CAT CCT GTG CCA TGA CAC TAC TCT GCC AGA-3′; and P2Y6: 5′-GCC GTG CTG CTT GGG TGG TAT GTG GAG TCG-3′) as hybridization probes, according to standard procedures. ⁴³ Labeling of the oligonucleotides with digoxigenin-dUTP was performed with a kit (DIG Oligonucleotide Tailing Kit; Roche, Mannheim, Germany). After hybridization to the target nucleic acids, oligonucleotides were detected by enzyme-linked immunoassay using an anti-digoxigenin alkaline phosphatase conjugate with subsequent enzyme-catalyzed color reaction (nitroblue tetrazolium/5-bromo-4-chloro-3-indoyl phosphate; NBP/BCIP; Roche, Mannheim, Germany). As an additional control, PCR products of the P2Y subtypes were cloned into the multiple cloning site of a vector (pCR-Script; Stratagene, Heidelberg, Germany) and dye-terminator cycle-sequenced with DNA polymerase (AmpliTaq) on a gene analyzer (Prism 310; ABI). 

In Vitro Transcription

The orientation of the different P2Y rat cDNA fragments (P2Y1: 561 bp, P2Y2: 339 bp, P2Y4: 377 bp, and P2Y6: 410 bp), subcloned in a vector (pCR-Script; Stratagene), were determined by sequencing the corresponding clones. Transcription from the T3 promoter yielded antisense riboprobes, and transcription from the T7 promotor yielded sense riboprobes. Before in vitro transcription, the plasmids were linearized by digestion with either BamHI or EcoRV, respectively. In vitro runoff transcripts thus contained only the corresponding P2Y sequences, and the RNA was nonradioactively labeled with digoxigenin-UTP (DIG RNA Labeling Kit; Roche, Mannheim, Germany), according to the manufacturer’s instructions. Antisense digoxigenin-labeled RNA probes were used as the positive control and sense digoxigenin-labeled RNA probes were used as the negative control. 

In Situ Hybridization

Sections were thawed at RT for 90 minutes, treated with proteinase K buffer (PKB: 0.1 M Tris-HCl [pH 8.0] 0.05 M EDTA [pH 8.0]) at 37°C for 5 minutes, and digested with 0.3 μg/mL proteinase K (Sigma-Aldrich) in PKB at 37°C for 8 minutes, washed two times in 0.1% diethylpyrocarbonate-treated water (DEPC-H₂O; Sigma-Aldrich), and postfixed in PFA 4% in 0.2 M PB for 15 minutes. After three rinses in DEPC-H₂O, slides were dried at RT for 15 minutes. No prehybridization procedure was performed. The P2Y-RNA probes (0.13 ng/μL) were added to the hybridization buffer (Amersham Pharmacia, Freiburg, Germany) at 37°C, and the mixture was heated to 68°C for 10 minutes. The probes were put on ice for 2 minutes, and hybridization was performed at 64°C overnight. The slices were washed twice in 0.1× SSC at 64°C, washed for 10 minutes in Tris-buffered saline (TBS; 0.15 M NaCl and 0.1 M Tris-HCl [pH 7.5]) at RT, and then treated for 30 minutes with blocking solution (10% blocking reagent in 0.1 M maleic acid and 0.15 M NaCl [pH 7.5]; Roche). Afterward, the slices were incubated with anti-digoxigenin-AP, Fab-fragment antibodies (1:500, in 10% blocking solution, 0.15% Triton X-100, TBS; Roche) for 45 minutes at 37°C. Samples were then washed twice in TBS for 15 minutes and 10 minutes in AP-buffer (0.1 M Tris-HCl [pH 9.5], 0.001M MgCl₂, and 10% tetramisolhydrochloride) at RT. Detection took place at 4°C overnight in detection buffer (AP-buffer with 400 μg/mL NBT/BCIP). The color reaction was stopped with 0.1 M Tris-HCl [pH 8.0], 0.01 M EDTA for 15 minutes at RT, and then slices were embedded (Karion F; Merck, Darmstadt, Germany). 

Immunohistochemistry

Sections were thawed at RT for 90 minutes, and endogenous peroxide activity was blocked by incubating the slides in 3% hydrogen peroxide and 40% methanol for 20 minutes at RT. Slides were rinsed with 0.05 M phospate-buffered saline (PBS) two times for 10 minutes each and preincubated with 10% normal goat serum (NGS) in PBST (PBS containing 0.03% Triton-X-100) for 1 hour at RT to block nonspecific binding. 

The antibodies against P2Y1 and -4 (Alamone Laboratories, Jerusalem, Israel) and P2Y2 (the generous gift of Mark Knepper, National Heart, Lung, and Blood Institute, Bethesda, MD) ⁴⁴ were used in a dilution of 1:250 in PBST, containing 10% NGS and incubated at 4°C overnight. After rinsing in PBS (3 × 10 minutes) the sections were incubated with biotinylated goat anti-rabbit IgG (Sigma-Aldrich) and diluted 1:200 in PBST, containing 5% NGS for 2 hours at RT. The slides were rinsed in PBS and incubated for 1 to 2 hours with an avidin-biotin peroxidase complex kit (Vectastain ABC Kit; Vector Laboratories, Inc.). After three washes in PB, the slides were incubated in 3,3′-diaminobenzidine (DAB)/nickel solution (1 mg/mL DAB, 0.2% glucose 0.004% NH₄Cl, 0.09% (NH₄)₂Ni(SO₄)₂, 1 μL/mL glucose oxidase in PB) for 7 to 10 minutes. After the reaction was terminated in PB, the slides were coverslipped with glycerol/PBS (9:1). Control experiments were performed by omitting the primary antibody or by replacing it with PBS. No antibodies directed against P2Y6 are currently available. 

Results

Detection of P2Y Subunits by RT-PCR

Qualitative RT-PCR was used to determine whether mRNA of P2Y1, -2, -4, or -6 was expressed in the retinas of adult rats. Figure 1 shows a representative agarose gel in which the four amplified P2Y receptor subunit RNAs were demonstrated. A single PCR product of the predicted size of 561 bp for P2Y1, 339 bp for P2Y2, 377 bp for P2Y4, and 410 bp for P2Y6 was seen in the rat retina. All amplified bands showed specific hybridization signals with the P2Y-specific oligonucleotides used as probes in the Southern blot analysis. For comparison and as positive controls, we amplified the mRNA from the retinal pigment epithelium (RPE)/choroid and other rat tissues (kidney, muscle, cortex, lung, and adrenal gland) for their expression of P2Y mRNA. All transcripts for P2Y1, -2, -4 and -6 mRNAs were detected in the RPE/choroid as well as in the other tissues (data not shown), in agreement with previous reports. ³  

Although these qualitative results do not permit a comparison of mRNA levels between subtypes P2Y1, -4 and -6, the differences seen in the case of the amplification of P2Y2 were large enough to indicate that P2Y2 was less prominently expressed in the adult retina than P2Y1, -4, and -6. This was in accord with the ISH experiments that were part of the study (described later). However, quantitative analysis is needed to verify this in detail. 

Retinal Localization of P2Y mRNAs

The question arising from the PCR-based detection of all four P2Y receptor subunits (P2Y1, -2, -4 and -6) was whether retinal neurons show a cell-specific pattern of P2Y expression. To obtain data on this question, we performed ISH studies that allow a resolution of mRNA expression at the single-cell level. Prominent signals were detected in the ganglion cell layer (GCL) of the rat retina when cRNA probes specific for P2Y1 (Fig. 2A) , -2 (Fig. 2B) , -4 (Fig. 2C) , and -6 (Fig. 2D) were used. According to their size and position, several cells can be regarded as ganglion cells (Fig. 2 , triangles). However ganglion cells represent only slightly more than 50% of the neurons in the GCL of the rat retina. Since clearly more than half of the GCL neurons were stained (Fig. 2) , some of the P2Y-expressing cells in that layer can only have been displaced amacrine cells. The ISH signal for P2Y2 in the GCL was clearly weaker than that for P2Y1, -4 and -6, as was also observed in the PCR experiments for this subunit. The specific riboprobes for P2Y1 and -6 revealed an additional and distinct labeling of cell bodies in the inner row of the inner nuclear layer (Figs. 2A 2D ; arrows), indicating P2Y1 and -6 expression in amacrine cells. The signals obtained for the P2Y4 receptor subunit in the INL showed a more diffuse staining (Fig. 2C) in the inner half of the INL (arrows), both in the inner row and in cells above this first row. Very occasionally, short processes were stained in the INL (Fig. 2C) but could not be assigned unequivocally to a specific cell type. In contrast to the other P2Y subtypes under investigation, no staining was be detected for P2Y2 in the INL (Fig. 2B) by ISH. 

Cellular Localization of the Proteins for P2Y1, P2Y2, and P2Y4

Antibodies against P2Y6 are not currently available, so that P2Y6 protein detection was not possible. 

A strong immunoreaction was found in GCL somata for P2Y1 (Fig. 3A , filled triangles). No P2Y1 immunolabeling was observed in the inner plexiform layer (IPL); however, somata displaced to the IPL were immunoreactive. Cells in the inner row of the INL were stained frequently (Fig. 3A , filled arrows), even though the intensity of their labeling was more variable and generally lighter than that in the GCL. In contrast to the IPL the outer plexiform layer (OPL) was clearly P2Y1 immunoreactive (Fig. 3A , open triangles). Faint staining was also present in the inner segments (IS) of the photoreceptors and in some somata of the outer nuclear layer (ONL), directly along the outer limiting membrane (OLM; Fig. 3A , open arrow). 

Immunoreactivity to the P2Y2 antibody was observed in the GCL, IPL, OPL, and IS of the photoreceptors. P2Y2 staining in the GCL was mainly found in cells with a cell body size larger than 12 μm (Fig. 3B , filled triangles), implying an expression of P2Y2 on ganglion cells rather than on displaced amacrine cells. ⁴⁵ Immunoreaction in the IPL was strong and homogeneously distributed over the entire layer, with a small lighter band occasionally visible in the middle of the layer (Fig. 3B) . Similar homogeneous but lighter staining were visible in the OPL (Fig. 3B , open triangles). No labeled cells were present in the INL or in the ONL. However, the IS of the photoreceptors were again immunoreactive for the P2Y2 antibody (Fig. 3B , open arrows). 

Neurons of all soma sizes showed strong P2Y4 immunoreactivity in the GCL (Fig. 3C , filled triangles). For the P2Y4 receptor three broadly stained sublayers, roughly corresponding to sublayers 1, 3, and 5 can be distinguished in the IPL (Fig. 3C , white stars). The immunoreaction in the IPL was generally pronounced, and sublayer 5 in particular showed massive, dotlike staining in close proximity to the cells in the GCL, so that it was occasionally unclear whether the immunoreaction was exclusively in the IPL or partly on the dendritic side of the stained somata in the GCL as well. Numerous stained somata were found in cells in the inner row of the INL (Fig. 3C , filled arrows) also showing dots of enhanced P2Y4 immunoreaction on the side facing the IPL. Other than the clear layers in the IPL, however, no processes originating from cells in the INL or GCL and running into the IPL were found. At the distal border of the INL, lightly stained, vertically elongated cells were present, bearing immunoreactive dots at their distal ends facing the OPL (Fig. 3C , open arrow). These hot spots of immunoreactivity were seen along the entire border between the INL and OPL (Fig. 3C , open triangles); the more distal parts of the OPL were also immunoreactive, but lighter. No specific labeling was observed distal to the OPL. 

Discussion

Despite considerable evidence of signaling by extracellular nucleotides in other sensory systems, few studies have been undertaken in the eye. Several recent reports have suggested that nucleotides may play complex roles as autocrine or paracrine mediators in ocular tissues. Several studies relating to P2Y receptors have focused on the P2Y2 subtype in the anterior segment of the eye. P2U (P2Y2) receptors were described in lens epithelium by one study, ⁴⁶ but another group reported that P2Y1 and -2 receptor transcripts were found in lens fiber cells but not in epithelial cells. ⁴⁷ In addition, it has been demonstrated that rabbit and human conjunctival cells contain functional P2Y2 receptors that govern mucin secretion, ⁴⁸ and P2Y2 receptors have been found in ciliary epithelial cells, ⁴⁹ although their function has not yet been established. P2Y2 receptors in the retinal pigment epithelium probably have a remedial function in retinal detachment, because they take part in the reabsorption of subretinal liquids. ^{50 51} Cowlen et al. ³⁷ recently described P2Y2 receptors found by ISH in various ocular tissues of macaques and rabbits—for instance, in the conjunctival, corneal, and ciliary body epithelium and parts of the lens, the cornea, and the choroid. Other studies have indicated the occurrence of different P2Y-subtypes in human lens cells, ³⁰ bovine corneal cells, ^{31 32} and iris cells of rats. ³³  

In contrast to all other ocular tissues, the retina is part of the central nervous system (CNS). Numerous studies in the past 15 years have verified that the CNS is especially rich in purine receptors. ^{52 53} In the visual cortex of rats, purine receptors seem to mediate changes in calcium signaling during neuronal development. ⁵⁴ P2 agonists act as neurotrophic factors in neuronal signaling pathways, where they support the growth and survival of neurons together with, for example, nerve growth factor (NGF). ⁵⁵ P2Y-receptor activation in cortical astrocytes through ATP has been shown to mediate the induction of neurodegenerative diseases and inflammation in the rat, ⁵⁶ and purine receptor involvement has also been suggested in such human neurodegenerative diseases as Alzheimer and schizophrenia. ^{57 58}  

Only a few studies so far have addressed the potential role of P2Y receptors in the retina or RPE. In the embryonic chick retina, ATP resulted in an increase in [Ca²⁺]_i caused by the release of Ca²⁺ from intracellular stores. In light of the pharmacological profile, it was suggested that this response to ATP is mediated by P2U (P2Y2) purine receptors. The developmental profile of the Ca²⁺ response to ATP was studied from embryonic days E3 to E13. The Ca²⁺ response to ATP was largest at E3, drastically declined toward E8, and decreased farther until E11 to E13, indicating a special function of P2Y2 in the development of the chicken retina. ³⁶ Other studies on embryonic chick retina have shown that activation of P2Y2 receptors by ATP and UTP resulted in an activation of the second messenger inositol-triphosphate (IP₃) and an increase of intracellular calcium levels. ^{59 60} Human RPE-cells in culture have been reported to be sensitive to extracellular application of ATP, adenosine diphosphate (ADP), or UTP, resulting in a transient increase in [Ca²⁺]_i. The characteristics of calcium mobilization, together with RT-PCR experiments, indicate an activation of P2Y2 receptors. ⁶¹ P2Y2 receptors were recently described in various ocular tissues ³⁷ of macaques and rabbits (e.g., in the retina and the retinal pigment epithelium). In situ hybridization has verified the presence of P2Y2 mRNA in the GCL, INL, and RPE and in the astrocytes of the optic nerve head of adult rhesus macaques and albino rabbits. P2Y2 staining in the inner segments of the photoreceptors was seen only in the rabbit retina. ³⁷ Activation of P2Y receptors in Müller cells of guinea pigs resulted in an influx of calcium ions from the extracellular space, an increase in DNA synthesis, and cellular proliferation. ⁶²  

In addition, extracellularly applied ATP has been shown to evoke calcium waves that propagate through glial cells in the isolated rat retina. ⁶³ These waves travel synchronously in astrocytes and Müller cells and functionally link the two retinal glia cell types. The calcium wave arise from the release of Ca²⁺ from internal stores, mediated by inositol 1,4,5-triposphate, rather than from an influx of external Ca²⁺. In view of the ATP evocation of calcium waves, this suggests the involvement of a metabotropic P2Y-receptor in this extraneuronal signaling pathway of the retina. Our own data on cellular localization of the P2Y receptor subtypes do not indicate a clear P2Y expression in Müller cells of the rat retina. However, this may be due to the limited sensitivity of the techniques used, because initial results from single-cell RT-PCR studies performed in our laboratory on isolated Müller cells revealed the presence of P2Y receptors in the Müller glia as well. 

P2Y receptors may play an important role in neuron–glia signal transfer, as suggested from studies of cultured cells of an oligodendrocyte lineage obtained from the rabbit retina. ⁶⁴ ATP is released there as a neurotransmitter and/or cotransmitter, but also under pathologic conditions from damaged cells. In glial cells of the optic nerve in rats P2Y1, -2 and -4 receptors seem to take part in the mediation of the glial reaction on injuries. ^{65 66 67} Their reaction to ATP and UTP is much stronger in degenerated tissue than in healthy tissue. P2Y6 receptors in cultured human astrocytes proved a protective character. Cell death induced by tumor necrosis factor (TNF)-α was decreased by UDP-mediated activation of P2Y6 receptors. ⁶⁸  

Our results provide direct evidence for the expression of P2Y1, -2, -4, and -6 receptor subtypes in different cell types in the mammalian retina. RT-PCR analysis from the retina and RPE/choroid of the rat led to the identification of these four distinct P2Y receptor subtype transcripts (P2Y1, -2, -4, and -6), thus confirming the P2Y2 data from rabbit and monkey ³⁷ whereas the expressions of P2Y1, -4, and -6 mRNAs are novel findings in these eye tissues. In situ hybridization experiments revealed the cell-specific pattern of P2Y mRNA expression (P2Y1, -2, -4, and -6) and immunohistochemical staining shows the cell-specific pattern of at least P2Y1, -2, and -4 proteins in the cell bodies and synaptic layers in the adult rat retina. 

Both ISH and immunohistochemistry clearly show labeling in ganglion cells either identified by a size criterion ^{45 69} or because the total amount of stained cells in the GCL outnumber the amount of displaced amacrine cells in the GCL. Furthermore, mRNA and/or protein staining revealed that P2Y1, -4, and -6 but not -2 receptors are expressed in amacrine cells along the inner border of the INL. The P2Y4 subtype, at least, is also present in displaced amacrine cells, shown by the sheer number of stained cells in the GCL. 

P2Y1, -2, and -4 receptor proteins were found in the OPL, indicating purinergic signal transmission at this very distal level of visual processing, either between the photoreceptors and their second order neurons or between the processes of the second order neurons themselves. The restriction of P2Y4 labeling to the proximal OPL indicates that this purinergic receptor subtype exists on the processes of the second order neurons but not on the synapses of the photoreceptors. 

The inner segments of the photoreceptors were stained for P2Y1 and -2 receptor proteins, but ISH showed no corresponding P2Y1 and -2 staining. Presumably, the available mRNA amount in the inner segments was too low to facilitate detection with this technique; however, protein accumulation was high enough that detection with P2Y1- and -2–specific antibodies was feasible. 

Our data show expression of purinergic receptors on amacrine and ganglion cells in the INL and GCL and on cell processes in both plexiform layers of the rat retina, indicating that extracellular ATP, acting as a neurotransmitter or neuromodulator, plays a crucial role in retinal signal processing. 

 Supported by Deutsche Forschungsgemeinschaft Grant GU 259/11.

 Submitted for publication February 11, 2004; revised April 22, 2004; accepted May 5, 2004.

 Disclosure: J.E. Fries, None; T.H. Wheeler-Schilling, None; E. Guenther, None; K. Kohler, 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.

 

Detection of amplification products for P2Y receptor subunits in the retina of adult pigmented rats (Brown Norway). Shown is expression of mRNA for the various P2Y subunits. Lane 1: P2Y1 (561 bp); lane 3: P2Y2 (339 bp); lane 5: P2Y4 (377 bp); lane 7: P2Y6 (410 bp). Lanes 2, 4, 6 and 8: the corresponding negative controls. Lane M: 100-bp ladder. The 1.5% agarose gel was stained with ethidium bromide.

Figure 2.

 

In situ hybridization with cRNA probes for P2Y1, -2, -4, and -6 receptor subtypes in the retina of adult pigmented rats (Brown Norway). (A) Expression of P2Y1 mRNA. Note the clear signal in the GCL (triangles) as well as in the innermost row of the INL (arrows). (B) Expression of P2Y2 mRNA. The labeling is restricted to the ganglion cell layer (triangles). (C) Micrograph with P2Y4-prominent labeling of virtually all cells in the GCL (triangles). A more diffuse signal was seen in the INL (arrows); very occasionally short ascending processes are stained (★). (D) Expression of P2Y6 mRNA. The staining pattern is comparable to that seen for the P2Y1 subunit. Hybridization with the sense (control) probes (to the left of the respective antisense probe) did not show any detectable signal. PHR: photoreceptors.

Figure 3.

 

Right: Immunohistochemical localization of P2Y in the rat retina; left: the control. (A) P2Y1, (B) P2Y2, and (C) P2Y4 immunoreactivity in the different retinal layers. Open triangles: staining of the OPL; filled arrows: staining of cells in the inner row of the INL (presumably amacrine cells); filled triangles: staining of the GCL. P2Y1 and -2 antibodies weakly labeled the inner segments of the photoreceptors (open arrows). The P2Y4 antibody occasionally labeled cells in the distal row of the INL (small open arrow, C), and three distinct sublayers in the IPL (☆). Bar, 10 μm.

The authors thank Gudrun Haerer for excellent technical assistance.