Molecular Vision 2006; 12:937-948 <http://www.molvis.org/molvis/v12/a106/> Received 10 August 2005 | Accepted 16 June 2006 | Published 16 August 2006

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Identification of the A₃ adenosine receptor in rat retinal ganglion cells

Mei Zhang,^1,2 Murat T. Budak,^3,4 Wennan Lu,¹ Tejvir S. Khurana,^4,5 Xiulan Zhang,^1,6 Alan M. Laties,¹ Claire H. Mitchell⁵
 
 

Departments of ¹Ophthalmology, ³Cell and Developmental Biology, ⁵Physiology, and ⁴Pennsylvania Muscle Institute, University of Pennsylvania School of Medicine, Philadelphia, PA; ²Eye Institute and Xiamen Eye Center, Xaimen University Medical School, Xiamen, People's Republic of China; ⁶Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, People's Republic of China

Correspondence to: Dr. Claire H. Mitchell, Department of Physiology, University of Pennsylvania, 3700 Hamilton Walk, Philadelphia, PA, 19104-6085; Phone: (215) 573-2176; FAX: (215) 573-5851; email:chm@mail.med.upenn.edu

Abstract

Purpose: Adenosine can protect retinal ganglion cells from the death that accompanies a general ischemic challenge as well as excitotoxic death. In other tissues, both A₁ and A₃ adenosine receptor subtypes can mediate protection. While a role for the A₁ adenosine receptor in ganglion cell protection has been established, a potential for the A₃ receptor has only recently been proposed. Although the pharmacology is promising, the molecular identity of the responsible receptor is unclear as previous studies were unable to detect message for the A₃ receptor in retinal ganglion cells. We combined laser capture microdisection (LCM) and immunopurification with traditional and real-time PCR to unequivocally demonstrate the presence of the A₃ receptor message in rat retinal ganglion cells.

Methods: Retinal ganglion cells of Long-Evans rat pups were retrograde labeled with aminostilbamidine. Eyeballs were enucleated, embedded, frozen, sectioned, and fluorescent cells in the ganglion cell layer were collected with LCM. Purified ganglion cells were also isolated with a two-step panning procedure. cDNA for the A₃ receptor obtained from the microdissected ganglion cell layer, immunopurified ganglion cells, whole retina and testis was amplified using RT-PCR, confirmed by DNA sequencing and compared with published sequences. A₃ receptor message was also amplified using real-time PCR. Ca²⁺ levels in immunopanned ganglion cells were measured ratiometrically with fura-2.

Results: RNA from immunopurified ganglion cells and from dye-loaded cells in the ganglion cell layer contained message for the A₃ receptor when amplified with either traditional RT-PCR or real-time PCR. The entire encoding region was sequenced and found to be 99% identical to the published code. The sequence closely resembled the consensus form of the gene, with other sequences deviating from this default code. Molecular identification was functionally confirmed in purified ganglion cells as the A₃ receptor agonist Cl-IB-MECA prevented the excessive Ca²⁺ rise triggered by P2X₇ agonist BzATP.

Conclusions: Retinal ganglion cells express A₃ adenosine receptor mRNA. Stimulation of this receptor can reduce the Ca²⁺ overload following excessive activation of P2X₇ receptors.

Introduction

Growing evidence indicates that adenosine is an important intracellular mediator in the retina and has considerable potential to protect retinal neurons [1-3]. Changes in retinal adenosine levels occur during ischemic events [4-6], with increased adenosine levels both enhancing the recovery of the electroretinogram (ERG) b-wave after ischemia, and preventing the ischemia-induced thinning of retinal cell layers [1]. Although the A₁, A_2A, A_2B, and A₃ subtypes of G-protein-coupled surface receptors are all stimulated by adenosine [7-9], protective effects on retinal ganglion cells have been primarily associated with the A₁ receptor. Stimulation with A₁ receptor agonists protects the ischemic retina [1-3], and attenuates the damaging effects of N-methyl-D-aspartate (NMDA) [10-12]. The A₁ and the A₃ receptors are both coupled to the G_i protein and their stimulation can decrease cytoplasmic levels of cAMP [13], although other downstream signaling pathways may differ [14-16]. In both neural and cardiac tissues, stimulation of the A₃ receptor with moderate levels of agonists can produce protective actions which equal or exceed those of A₁ receptor agonists, while excessive receptor stimulation is deleterious [17-19]. In studies of cerebral ischemia, postischemic and chronic preischemic administration of A₃ receptor agonists was neuroprotective, whereas acute administration of agonists during ischemia exacerbated the histological and functional damage [19-23]. These reports suggest that stimulation of the A₃ receptor could augment the protective effects of the A₁ receptor on retinal ganglion cells, if the conditions of agonist delivery were examined with care.

A recent study suggested that stimulation of the A₃ receptor was beneficial to retinal ganglion cells [24]. Agonists for the A₃ receptor prevented the Ca²⁺ rise and cell death which accompanied activation of the P2X₇ receptor. While this study implied a neuroprotective role for the A₃ receptor using pharmacologic tools, the molecular identification of the A₃ receptor in retinal ganglion cells has been elusive. An initial study was unable to detect any message for the A₃ receptor in the eye using in situ hybridization [25]. Subsequently, message for the A₃ receptor was identified in the whole retinal tissue of mice with the more sensitive conventional reverse transcription-polymerase chain reaction (RT-PCR) technique [26]. This study indicated expression of the A₃ receptor promoter in a few ganglion cells, but was also unable to directly identify message for the A₃ receptor in ganglion cells using in situ hybridization.

The discovery of the neuroprotective potential of the A₃ receptor on retinal ganglion cells makes the molecular identification of the receptor more imperative. It is possible that moderately low levels of A₃ mRNA precluded detection with the in situ technique, while difficulties in isolating ganglion cells prevented the use of more sensitive amplification tools. In the present study, several approaches were employed to overcome these difficulties and directly demonstrate the presence of mRNA for the A₃ adenosine receptor in rat retinal ganglion cells. Message for the A₃ receptor obtained from retrograde labeled cells in the ganglion cell layer using laser capture microdissection (LCM), and from ganglion cells purified using a two-step immunopanning technique, was amplified using both traditional and real-time PCR. The receptor was cloned and sequenced to verify its identity, while physiologic effects in purified cells supported functional confirmation. Together these results provide direct evidence for the A₃ adenosine receptor in retinal ganglion cells.

Methods

Retrograde-labeling of retinal ganglion cells

Pups at postnatal day (PD) 3-5 from untimed pregnant Long-Evans rats (Jackson Laboratory Inc., Bar Harbor, ME) were back retrograde labeled by the injection of FluoroGold derivative aminostilbamidine (Molecular Probes, Eugene, OR) based upon protocols described previously [27]. Pups were anesthetized with an intraperitonial injection of 50/5 mg/kg ketamine/xylazine. An incision exposed the skull, while a 1 mm hole drilled through the skull exposed the cortex overlying each superior colliculus. Using a Hamilton syringe affixed to a micromanipulator, a needle was inserted 0.8 mm lateral from the midline and 0.8 mm anterior to Bregma's line and a total of 2.5 μl saturated dye dissolved in 0.9% saline was delivered to each side at a depth of 2 mm and 1 mm. The needle was retracted after a delay of 2 min to allow dye absorption and the wound closed with 2-3 sutures. Preliminary examination of labeled retinal whole mounts confirmed an even distribution of dye, showing a high degree of staining 2 days after injection. Consequently, eyes were enucleated 4-5 days after injection, at PD 7-10. Another three unlabeled pups were used to obtain RNA from the whole retina. Animals were sacrificed by i.p. injection of 50/5 mg/kg ketamine/xylazine followed by an overdose, in accordance with University of Pennsylvania IACUC approved protocols and the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research.

Tissue preparation and laser-capture microdissection (LCM)

Eyeballs from three Fluorogold labeled pups were quickly enucleated, embedded with OCT tissue embedding medium (Tissue Tek, Sakura, Tokyo, Japan) and snap-frozen in liquid nitrogen. The eyeballs were sectioned transversely into 7-10 μm sections with a cryostat, mounted on Superfrost Plus electrostatically charged slides (Fischer Scientific, Pittsburgh, PA) and stored at -80 °C until use. Histogene LCM frozen-section staining reagents were used for tissue dehydration and staining as described by the manufacturer (Arcturus Engineering, Mountain View, CA). Briefly, sections were fixed and dehydrated sequentially in 75%, 95%, and 100% ethanol followed by 100% xylene. Sections were allowed to air dry and were stored in a desiccator at room temperature until utilized for LCM; care was taken to ensure that LCM was completed within 1 h of the slides being placed in the desiccator. Two to three sections from each sample were stained with hematoxylin and eosin (H&E) to verify the quality of the section. A PixCell II LCM System (Acturus Engineering, Mountain View, CA) was used for LCM. Fluorogold labeled retinal ganglion cells were visualized using fluorescent microscopy. Caps and thermoplastic film were placed above the frozen section. The fluorescent cells with large cell bodies characteristic of ganglion cells were then selectively captured from the retina of each section with the 7.5 μm laser setting. The melted thermoplastic membrane and small region of tissue directly underneath adhering to it were removed by lifting the plastic cap. Approximately 500-1,000 ganglion cells were obtained from each specimen (typically from 6-8 slides containing 24-32 sections), with each specimen from a pup derived from a separate breeding pair.

For the H&E staining, sections were air dried for several minutes, stained with 0.1% Mayer's hematoxylin for 10 min, rinsed in tap water for 5 min, and stained with 0.5% eosin for 30-60 s followed by a further wash. Sections were dehydrated using consecutive exposure to 70%, 95%, and 100% ethanol followed by 100% xylene. Sections were then mounted and examined using brightfield microscopy.

RNA isolation and linear RNA amplification of microdissected retinal ganglion cells

Five independent total RNA preparations were produced from the five sets of retinal ganglion cells obtained using LCM using the Pico pure RNA isolation kit (Arcturus Engineering). Total RNA was extracted from the captured cells by incubating LCM caps in extraction buffer for 30 min at 42 °C. RNA was purified using preconditioned MiraCol (Arcturus Engineering) purification columns. Eluted RNA was directly used for linear RNA amplification.

Linear T7-based RNA amplification was carried out by using the RiboAmp OA kit as suggested by manufacturer (Arcturus Engineering, Mountain View, CA). Total RNA was incubated with hybrid primers containing oligo dT/T7 RNA polymerase binding site containing primers, the RNA was reverse-transcribed into double-stranded cDNA and this cDNA was purified using MiraCol columns. Next, in vitro transcription was performed using T7 RNA polymerase, and the amplified antisense RNA (aRNA) purified using MiraCol columns. The aRNA was quantified and verified using a GeneQuant Pro spectrophotometer (Amersham Pharmacia Biotech, Upsala, Sweden). The total aRNA yields from each preparation were 1.8-6.0 μg, and the 260/280 ratio between 1.9-2.1 suggested the sample was of high quality.

Retinal ganglion cell panning

Purification of ganglion cells using the immunopanning procedure was performed as described previously [27] based upon the method of Hartwick et al. [10]. Neonatal rat retinas were dissociated enzymatically for 30 min with 15 U/ml papain, 0.2 mg/ml DL-cysteine and 0.004% DNAse I (Worthington/Cooper, Lakewood, NJ). The cells were washed, centrifuged, resuspended, and filtered through a 20 μm nylon mesh. Cells were incubated with rabbit antimacrophage antibody (1:75, Accurate Chemical, Westbury, NY), then incubated in a 100 mm dish coated with goat anti-rabbit IgG antibody (1:400, Jackson ImmunoResearch Laboratories Inc., West Grove, PA). Non-adherent cells were removed to a second petri-dish coated with goat anti-mouse IgM (1:300, Jackson ImmunoResearch Laboratories Inc., West Grove, PA) and anti-Thy 1.1 antibody (from hybridoma T11D7e2; American Type Culture Collection, Rockville, MD). After 30 min, non-adherent cells were washed off and the remaining cells were incubated with 0.125% trypsin for 8 min at 37 °C. Digestion was stopped with fetal bovine serum (30%) in neural basal medium and cells were centrifuged and plated on coverslips coated with poly-L-lysine and laminin. Total RNA was extracted from immunopanned cells using the TRIzol LS reagent (Invitrogen, Carlsbad, CA). RNA was purified using bromochloropropane (BCP, Molecular Research Center, Inc., Cincinnati, OH), then re-purified using the RNeasy Micro kit (Qiagen Sciences Inc., Germantown, MD) and eluted with 15 μl of DEPC-treated water. RNA was extracted from five separate preparations of immunopanned cells. Images were taken with a Nikon Eclipse E600 microscope equipped for epifluorescence with an excitation filter of 360±40 nm and with emission >515 nm.

RNA extraction from whole retina and testis

Eyeballs from three PD8-9 Long-Evans pups were enucleated. Retinas were dissected quickly from both eyes under microscope. Testes were used as positive control for A₃ receptor detection. A small part of testis from three male adult rats was dissected quickly. Total RNA preparations were made from each specimen using Trizol reagent as above.

Reverse transcriptase polymerase chain reaction and sequencing

RNA from the immunopanned ganglion cells, microdissected cells of the ganglion cell layer, whole retina and testis was reverse transcribed to cDNA using Superscript II (Invitrogen Carlsbad, CA) reverse transcriptase (RT) following the protocol provided by the manufacturer. Polymerase chain reaction (PCR) was performed using the following conditions: PCR was carried out in a total volume of 25 μl, containing 1X PCR buffer, 2.5 mM MgCl₂, 200 μM dNTP, 0.5 μM each of the two primers used and 1 U of Taq DNA Polymerase (Promega, Fitchburg, WI). All primers were produced by Invitrogen. Two sets of overlapping primers were used for A₃ adenosine receptor message amplification (Table 1) [28]. Samples were denatured at 94 °C for 45 s (for 641 bp and 724 bp), annealed to primers at 65 °C (for 641 bp) or 56 °C (for 724 bp) for 45 s, and elongated at 72 °C for 60 s (for 641 bp and 724 bp). This was repeated for 30 to 35 cycles, followed by extension at 72 °C for 10 min, using a T gradient 96 instrument (Biometra, Goettingen, Germany). RT-PCR products were separated on a 1.0% agarose gel containing ethidium bromide and visualized under UV light. As the RT-PCR product was present as a single band on agarose gel, the products of RT-PCR were purified with QIAquick PCR purification kit (Qiagen Sciences, Germantown, MD) and underwent sequencing by the dideoxy termination method at the University of Pennsylvania School of Medicine sequencing facilities. Sequence data was analyzed using the MacVector software (version 7.2; Accelrys, Inc., San Diego, CA). The putative phosphorylation sites in the predicted protein sequences were assessed using Scansite [29].

Real-time polymerase chain reaction

Gene expression analysis was performed on an ABI Prism 7900HT machine (Applied Biosystems) using SYBR Green PCR master kit (PE Applied Biosystems, Foster City, CA). PCR primers for A₃ receptor were summarized in Table 1 [30]. PCR primers for real-time amplification are summarized in Table 1 [30]. As an internal control for efficiency of transcription, we simultaneously amplified the housekeeping gene GAPDH [31]. All real-time PCR reactions were done in triplicate. PCR conditions were identical for both genes. Cycle conditions were: 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s (denaturation) and 60 °C for 90 s (annealing and extension). At the end of the amplification cycles, samples were heated to 95 °C with a ramp time of 20 min to construct dissociation curves to check that single PCR products were obtained, and verify the lack of product in the absence of template. Real-time PCR product was verified using conventional PCR and sequencing as described above. PCR (35 cycles) conditions were: denaturing at 94 °C for 60 s, annealing to primers at 59 °C for 60 s, and elongating at 72 °C for 60 s.

Intracellular Ca²⁺ measurements

Intracellular Ca²⁺ levels from immunopanned ganglion cells were measured as previously reported [27]. In brief, panned RGCs grown on coverslips for 24 h were loaded with 10 μM fura-2 and 2% pLuoronic F-127 (Molecular Probes, Eugene, OR) for 60-90 min at 25 °C, then rinsed for 30 min. Coverslips were mounted on a Nikon Diaphot inverted microscope and visualized with a 40x objective. The field was alternatively excited at 340 nm and 380 nm with a scanning monochromator and the light emitted at >520 nm from a region of interest surrounding individual retinal ganglion cells was imaged with a CCD camera and analyzed (Photon Technologies International, Inc., Lawrenceville, NJ). Cells were perfused with a control solution at the start of Ca²⁺ imaging experiments containing (in mM) 105 NaCl, 4.5 KCl, 2.8 NaHepes, 7.2 Hepes acid, 1.3 CaCl₂, 5 glucose, 75 mannitol, pH 7.4. Drugs were dissolved in the control solution. Calibration was performed separately on each cell after the experiment by perfusing cells in the presence of 5 μM ionomycin and control solution (with 1.3 mM Ca²⁺) followed by ionomycin in the base solution without Ca²⁺ and with the addition of 5 mM EGTA (pH 8.0). The 340/380 ratio was converted to Ca²⁺ concentration as previously described [27]. All experiments were performed at 25 °C. All materials were from the Sigma-Aldrich Corp. (St. Louis. MO) unless otherwise noted.

Results

Expression of A₃ adenosine receptor in cells isolated by LCM

Retinal ganglion cells comprise less than 5% of the total retinal cell population. As ganglion cells are sandwiched among the inner retinal neurons, glial cells, and the inner limiting membrane, obtaining samples of sufficient purity for molecular analysis is difficult using standard dissection techniques. In present study we used both LCM and immunopanning to concentrate genetic material from retinal ganglion cells in order to search for the A₃ adenosine receptor.

Several days after the dye aminostilbamidine was injected into the superior colliculus, the spherical cell bodies of retinal ganglion cells fluoresced yellow-green, with retinal whole mounts indicating dense and even staining. As illustrated in Figure 1A, this yellow staining of ganglion cell bodies was evident within the laminar organization of the retina. Only large, fluorescent cell bodies were "captured" or collected with the laser, and those in close proximity to unlabeled material were left behind to avoid obtaining material from unlabeled cells. However, the 7.5 μm width of the laser made it difficult to ensure that captured material was exclusively from ganglion cells. Figure 1B shows the same retinal section after removal of material from the ganglion cell layer, while Figure 1C shows the selected cells adherent to the underside of the capturing cap. Corresponding retinal anatomy from an adjacent section after HE staining is illustrated in Figure 1D.

Expression of A₃ adenosine receptor mRNA in retinal ganglion cells isolated by LCM was detected using traditional RT-PCR. Two sets of primers, A₃U (upper) and A₃L (lower), with a 173 bp overlapping sequence, were used to amplify the entire encoding region (Figure 2). Bands of the predicted product sizes were detected; 641 bp for A₃U and 724 bp for A₃L (Figure 3A,B). Analogous bands were observed in three replicates. No product was detected when RT was omitted from the reaction as a negative control.

Amplification of mRNA obtained from the total retina of rat pups and from the testes of adult rat also revealed bands of 641 bp and 724 bp using primer pairs A₃U and A₃L. When sequenced, the products from the total retinal and testes material maintained the distinct sequence found in retinal ganglion cells, with complete identity in samples from all three sources.

Confirmation of the A₃ message in captured cells from the ganglion cell layer was obtained using real-time PCR. Product was detected from 5 captured preparations using A₃Q (quantitative) primers described in Table 1. The dissociation curve run after the reaction indicated the products of the GAPDH and A₃ primers were not contaminated, and that no product was detected in the absence of template. The specificity of these primers was confirmed by using them for traditional PCR (Figure 3C) and sequencing the product. Qualitative analysis suggested the A₃ message was present in relatively low abundance, with product crossing threshold 7.0±0.8 (n=5) cycles after that detected with GAPDH primers. While this analysis does not enable predictions of relative copy number, it is consistent with relatively small amounts of mRNA for the A₃ receptor.

Expression of A₃ adenosine receptor in immunopurified ganglion cells

While the large, fluorescently labeled cell bodies in the ganglion cell layer were most likely ganglion cells, the imprecision of the LCM laser, combined with the presence of amacrine, glial, and endothelial cells in the ganglion cell layer made it possible that material from these cells contributed to the mRNA used above. To confirm that mRNA for the A₃ receptor was present in ganglion cells, a two-step immunopanning technique was used to obtain a purified population of ganglion cells. When retinas containing labeled ganglion cells were used, >98% of the immunopanned cells were fluorescent, indicating a high degree of purity (Figure 4A,B). RNA extracted from immunopanned cells displayed bands of appropriate size using A₃U, A₃L, and A₃Q primers (Figure 4C-E). Sequencing found the product 100% identical to the message from microdissected cells. Product was amplified from cDNA obtained from each of 5 independent preparations of panned cells using real-time PCR (Figure 4F). The amplification product crossed threshold at 13.0±0.2 (n=5) cycles after that detected with GAPDH primers. Again, the dissociation curve verified the purity of the product found with GAPDH and A₃ primers, and that no product was found in the absence of template.

Comparison of A₃ sequence

The sequence of the encoding region from ganglion cell layer and purified ganglion cells was over 99% identical to sequences for the rat A₃ receptor with accession numbers M94152 [32] and X59249 [33], although several single nucleotide alterations and a few more extensive changes were present. The unique nucleotide sequence obtained in the present study is found in Figure 5A, and was entered into the Genbank database with accession number DQ075463. Most of the single substitutions were conservative, but G128C resulted in the substitution of a basic arginine to a nonpolar proline in the first intracellular loop. The sequence differed from X59249 at base pairs 219-249, and differed from M94152 at base pairs 859-883. The A₃ receptor in retinal ganglion cells shares both these regions with the splice variant of this gene expressed in brain cells (accession number X93219) [34], although the spliced insertion was not present in our material. The resulting comparison of these four amino acid sequences for the A₃ receptor is illustrated in Figure 5B.

Functional identification of the A₃ receptor in immunopurified cells

A₃ receptor agonists can protect ganglion cells present in a preparation of mixed retinal cells from stimulation of the P2X₇ receptor agonist [24]. However, it is not known whether these agonists can act directly on the ganglion cells. The protective effect of the specific A₃ receptor agonist 2-chloro-N⁶-(3-iodobenzyl)-adenosine-5'-N-methyluronimide (Cl-IB-MECA) [8] was thus examined on immunopurified retinal ganglion cells with the Ca²⁺ sensitive dye fura-2. Repetitive 15 s applications of 50 μM 3'-O-(4-benzoylbenzoyl)ATP (BzATP) led to large, repeatable elevations in the Ca²⁺ levels of isolated ganglion cells (Figure 6A). While BzATP can activate several P2 receptors, previous characterization has demonstrated that its actions are mediated by the P2X₇ receptor in retinal ganglion cells [27]. The rise in Ca²⁺ triggered by BzATP was prevented by 10 μM adenosine (Figure 6B), with a mean decrease of 65.8±3.8% (n=12; Figure 6C). Of particular note, the A₃ receptor agonist Cl-IB-MECA (1 μM) also produced a substantial inhibition of the Ca²⁺ rise in isolated ganglion cells (Figure 6D). The block was reversible, repeatable, and significant, with a mean decrease of 69.3±4.8% (n=7; Figure 6E). Approximately 80% of purified ganglion cells responded to Cl-IB-MECA with some degree of block.

Discussion

This study has demonstrated the presence mRNA for the A₃ adenosine receptor in genetic material isolated from the retinal ganglion cell layer using LCM microdissection and from immunopurified retinal ganglion cells. mRNA was detected using conventional PCR and confirmed with sequencing and real-time amplification. The message was distinct from previously reported rat A₃ sequences, with small differences throughout the reading frame. Receptor identification was supported pharmacologically with the A₃ receptor agonist Cl-IB-MECA. These data confirm the A₃ receptor in rat retinal ganglion cells on a molecular level and emphasize the protective opportunities of receptor activation.

In the past, detection of A₃ mRNA from retinal ganglion cells has been hampered by both the low copy number and the difficulty in obtaining a pure population of ganglion cells. The combination of LCM, immunopanning, and real-time PCR are well suited to address both concerns and provide direct information about gene expression in these cells. The application of LCM to labeled ganglion cells in rapidly frozen tissue sections provided genetic material from the ganglion cell layer. While the presence of additional cell types in this layer may have diluted the purity of the captured sample somewhat, the ability to select large, fluorescent cell bodies with the laser will have increased selectivity. The detection of mRNA for the A₃ receptor in immunopanned cells provides an additional level of specificity. However, the 4-6 h required to isolate ganglion cells with the immunopanning technique is not ideal, and may have been sufficient to enable mRNA degradation. Although prediction of copy number is not possible without standard curves constructed from known quantities of mRNA, the relative expression of the A₃ mRNA with respect to GAPDH suggests the captured material had more mRNA than panned cells. As the captured material was quick frozen, it is possible that the A₃ mRNA was particularly sensitive to degradation.

The relatively low expression of the mRNA for the A₃ receptor in both preparations is consistent with two previous studies that failed to detect A₃ mRNA in the entire eye [25] or in the retina itself using in situ hybridization [26]. The presence of the A₃ receptor was subsequently confirmed in the ciliary epithelium using functional and molecular tools [35,36], implying the in situ technique was not sufficiently sensitive to detect message present in low copy number in other ocular tissues. This observation in the eye agrees with that found elsewhere, with high levels of expression primarily confined to the testes [9,26,32]. Previous attempts to overcome this difficulty in the retina examined the activity of the A₃ receptor promoter linked to a β galactosidase reporter gene in transgenic animals. β galactosidase staining revealed promoter activity in the cerebrospinal vasculature and in a subset of ganglion cells [26]. Although staining was only observed in <10% of the total ganglion cells, this could underestimate of the proportion of cells expressing the A₃ gene if the turnover rate of the mature mRNA is low or if transgene expression was variable. Pharmacologic analysis indicates that A₃ agonist Cl-IB-MECA was effective on most immunopanned ganglion cells, suggesting the receptor itself is widely expressed.

The A₃ receptor genes obtained from ganglion cells, whole retina and testis were identical to each other, but unique from other published sequences for the rat A₃ receptor. Of the four sequences examined, ours appeared to reflect the baseline sequence, with each of the three larger changes occurring in just one of the other three sequences. The insertion of an additional 51 base pairs in an alternatively spliced variant cloned from rat brain (A3i; X93219) was associated with reduced coupling to G_i proteins [34]. This insertion was not present in our samples, implying activation of the A₃ receptor will lead to more robust activation of G_i in our preparation. According to the predicted transmembrane topology of the A₃ receptor [34], the differences between our sequence and X59249 affect the entire first extracellular loop, while those between our sequence and M94152 affect the cytoplasmic region just after the seventh transmembrane domain [32,33]. Although functional contributions of these regions, or these corresponding changes, are not presently understood, the shift from X59249 to our sequence removed a putative PKC δ phosphorylation site and this may have functional consequences. On a broader scale, the sequence changes illustrate the presence of different sequences in different rat strains, and emphasize caution when comparing pharmacologic or physiologic data from different laboratories.

Acknowledgements

This work was supported by grants from the NIH (R01EY015537 and R01EY013434 to CHM and R01EY010009 to AML, R01EY013862, and R01AR051696 to TSK, and Vision Research Core Grant P30EY001583), by the Jody Sack Fund to M. Zhang and X. Zhang, and by grant 30471850 from the Chinese National Scientific Research Fund to X. Zhang. The authors would like to thank Dr. Richard A. Stone and Mortimer M. Civan for useful discussions. A preliminary report of some of these data was presented at the 2005 meeting of the Association of Vision in Research and Ophthalmology [37].

References

1. Larsen AK, Osborne NN. Involvement of adenosine in retinal ischemia. Studies on the rat. Invest Ophthalmol Vis Sci 1996; 37:2603-11.

2. Ghiardi GJ, Gidday JM, Roth S. The purine nucleoside adenosine in retinal ischemia-reperfusion injury. Vision Res 1999; 39:2519-35.

3. Li B, Roth S. Retinal ischemic preconditioning in the rat: requirement for adenosine and repetitive induction. Invest Ophthalmol Vis Sci 1999; 40:1200-16.

4. Roth S, Osinski JV, Park SS, ostwald P, Moshfeghi AA. Measurement of purine nucleoside concentration in the intact rat retina. J Neurosci Methods 1996; 68:87-90.

5. Roth S, Park SS, Sikorski CW, Osinski J, Chan R, Loomis K. Concentrations of adenosine and its metabolites in the rat retina/choroid during reperfusion after ischemia. Curr Eye Res 1997; 16:875-85.

6. Roth S, Rosenbaum PS, Osinski J, Park SS, Toledano AY, Li B, Moshfeghi AA. Ischemia induces significant changes in purine nucleoside concentration in the retina-choroid in rats. Exp Eye Res 1997; 65:771-9.

7. Linden J. Cloned adenosine A3 receptors: pharmacological properties, species differences and receptor functions. Trends Pharmacol Sci 1994; 15:298-306.

8. Fredholm BB, Abbracchio MP, Burnstock G, Daly JW, Harden TK, Jacobson KA, Leff P, Williams M. Nomenclature and classification of purinoceptors. Pharmacol Rev 1994; 46:143-56.

9. Dixon AK, Gubitz AK, Sirinathsinghji DJ, Richardson PJ, Freeman TC. Tissue distribution of adenosine receptor mRNAs in the rat. Br J Pharmacol 1996; 118:1461-8.

10. Hartwick AT, Lalonde MR, Barnes S, Baldridge WH. Adenosine A1-receptor modulation of glutamate-induced calcium influx in rat retinal ganglion cells. Invest Ophthalmol Vis Sci 2004; 45:3740-8.

11. Zhang C, Schmidt JT. Adenosine A1 and class II metabotropic glutamate receptors mediate shared presynaptic inhibition of retinotectal transmission. J Neurophysiol 1999; 82:2947-55.

12. Sun X, Barnes S, Baldridge WH. Adenosine inhibits calcium channel currents via A1 receptors on salamander retinal ganglion cells in a mini-slice preparation. J Neurochem 2002; 81:550-6.

13. Fredholm BB, IJzerman AP, Jacobson KA, Klotz KN, Linden J. International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev 2001; 53:527-52.

14. Schulte G, Fredholm BB. Signaling pathway from the human adenosine A(3) receptor expressed in Chinese hamster ovary cells to the extracellular signal-regulated kinase 1/2. Mol Pharmacol 2002; 62:1137-46.

15. Lee JE, Bokoch G, Liang BT. A novel cardioprotective role of RhoA: new signaling mechanism for adenosine. FASEB J 2001; 15:1886-94.

16. Englert M, Quitterer U, Klotz KN. Effector coupling of stably transfected human A3 adenosine receptors in CHO cells. Biochem Pharmacol 2002; 64:61-5.

17. Abbracchio MP, Cattabeni F. Brain adenosine receptors as targets for therapeutic intervention in neurodegenerative diseases. Ann N Y Acad Sci 1999; 890:79-92.

18. Liang BT, Jacobson KA. A physiological role of the adenosine A3 receptor: sustained cardioprotection. Proc Natl Acad Sci U S A 1998; 95:6995-9.

19. Von Lubitz DK, Simpson KL, Lin RC. Right thing at a wrong time? Adenosine A3 receptors and cerebroprotection in stroke. Ann N Y Acad Sci 2001; 939:85-96.

20. Von Lubitz DK, Lin RC, Popik P, Carter MF, Jacobson KA. Adenosine A3 receptor stimulation and cerebral ischemia. Eur J Pharmacol 1994; 263:59-67.

21. von Lubitz DK, Carter MF, Beenhakker M, Lin RC, Jacobson KA. Adenosine: a prototherapeutic concept in neurodegeneration. Ann N Y Acad Sci 1995; 765:163-78; discussion 196-7.

22. Von Lubitz DK, Lin RC, Boyd M, Bischofberger N, Jacobson KA. Chronic administration of adenosine A3 receptor agonist and cerebral ischemia: neuronal and glial effects. Eur J Pharmacol 1999; 367:157-63.

23. Baraldi PG, Cacciari B, Romagnoli R, Merighi S, Varani K, Borea PA, Spalluto G. A(3) adenosine receptor ligands: history and perspectives. Med Res Rev 2000; 20:103-28.

24. Zhang X, Zhang M, Laties AM, Mitchell CH. Balance of purines may determine life or death of retinal ganglion cells as A adenosine receptors prevent loss following P2X receptor stimulation. J Neurochem 2006; 98:566-75.

25. Kvanta A, Seregard S, Sejersen S, Kull B, Fredholm BB. Localization of adenosine receptor messenger RNAs in the rat eye. Exp Eye Res 1997; 65:595-602.

26. Yaar R, Lamperti ED, Toselli PA, Ravid K. Activity of the A3 adenosine receptor gene promoter in transgenic mice: characterization of previously unidentified sites of expression. FEBS Lett 2002; 532:267-72.

27. Zhang X, Zhang M, Laties AM, Mitchell CH. Stimulation of P2X7 receptors elevates Ca2+ and kills retinal ganglion cells. Invest Ophthalmol Vis Sci 2005; 46:2183-91.

28. Jackson EK, Zhu C, Tofovic SP. Expression of adenosine receptors in the preglomerular microcirculation. Am J Physiol Renal Physiol 2002; 283:F41-51.

29. Obenauer JC, Cantley LC, Yaffe MB. Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res 2003; 31:3635-41.

30. Rose'Meyer RB, Mellick AS, Garnham BG, Harrison GJ, Massa HM, Griffiths LR. The measurement of adenosine and estrogen receptor expression in rat brains following ovariectomy using quantitative PCR analysis. Brain Res Brain Res Protoc 2003; 11:9-18.

31. Fischer MD, Budak MT, Bakay M, Gorospe JR, Kjellgren D, Pedrosa-Domellof F, Hoffman EP, Khurana TS. Definition of the unique human extraocular muscle allotype by expression profiling. Physiol Genomics 2005; 22:283-91.

32. Zhou QY, Li C, Olah ME, Johnson RA, Stiles GL, Civelli O. Molecular cloning and characterization of an adenosine receptor: the A3 adenosine receptor. Proc Natl Acad Sci U S A 1992; 89:7432-6.

33. Meyerhof W, Muller-Brechlin R, Richter D. Molecular cloning of a novel putative G-protein coupled receptor expressed during rat spermiogenesis. FEBS Lett 1991; 284:155-60.

34. Sajjadi FG, Boyle DL, Domingo RC, Firestein GS. cDNA cloning and characterization of A3i, an alternatively spliced rat A3 adenosine receptor variant. FEBS Lett 1996; 382:125-9.

35. Mitchell CH, Peterson-Yantorno K, Carre DA, McGlinn AM, Coca-Prados M, Stone RA, Civan MM. A3 adenosine receptors regulate Cl- channels of nonpigmented ciliary epithelial cells. Am J Physiol 1999; 276:C659-66.

36. Carre DA, Mitchell CH, Peterson-Yantorno K, Coca-Prados M, Civan MM. Similarity of A(3)-adenosine and swelling-activated Cl(-) channels in nonpigmented ciliary epithelial cells. Am J Physiol Cell Physiol 2000; 279:C440-51.

37. Zhang M, Budak MT, Lu W, Khurana TS, Zhang X, Laties AM, Mitchell CH. Identification of the A3 Adenosine Receptor in Rat Retinal Ganglion Cells. ARVO Annual Meeting; 2005 May 1-5; Fort Lauderdale (FL).