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CELLULAR AND MOLECULAR

Effects of Dinucleoside Polyphosphates on Trabecular Meshwork Cells and Aqueous Humor Outflow Facility

Laboratori de Neurofisiologia, Facultat de Medicina-Institut d'Investigacions Biomèdiques August Pi i Sunyer, Universitat de Barcelona, Barcelona, Spain (D.S., A.G., X.G.); and Departamentos de Bioquímica (J.P.) and Óptica (A.P.), Escuela Universitaria de Óptica, Universidad Complutense de Madrid, Spain

Received February 22, 2005; accepted June 7, 2005.

	Abstract

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The most important risk factor for the development of glaucoma is elevated intraocular pressure (IOP). Hypotensive drugs decrease IOP, preventing optic nerve damage and further vision loss. The balance between aqueous humor (AH) production and drainage determines IOP, and problems in AH outflow pathways are associated with open-angle glaucoma development. Previous studies have shown the presence of diadenosine tetraphosphate (Ap₄A) and pentaphosphate (Ap₅A) in the AH. Topic application of Ap₄A to the cornea decreased IOP, whereas Ap₅A increased it. Because dinucleoside polyphosphates stimulate P2Y purinergic receptors, we studied their presence in trabecular meshwork (TM) cells. Additionally, the effects of diadenosine polyphosphates (Ap_nAs; n = 3–5) and Up₄U (P¹,P⁴-(diuridine 5')-tetraphosphate; INS365) in outflow facility were tested. P2Y₁, P2Y₂, and P2Y₄ receptors were detected in TM cells by Western blot and immunocytochemistry. In TM cells, Ap₃A, Ap₄A, and Ap₅A induced discrete intracellular calcium concentration ([Ca²⁺]_i) mobilizations compared with higher and more sustained [Ca²⁺]_i mobilizations after Up₄U application. In bovine ocular anterior segments perfused at constant pressure, 1 µM Ap₃A or Ap₄A increased outflow facility, whereas Up₄U or Ap₅A did not modify it. 2-MeSADP, a selective P2Y₁ agonist, induced outflow facility increases similar to those obtained after Ap₃A and Ap₄A, and these were prevented by addition of the selective P2Y₁ receptor antagonist MRS-2179 (2'-deoxy-N₆-methyladenosine-3',5'-diphosphate). Our results demonstrate that the hypotensive effect of Ap₄A and other dinucleotides is mediated, at least in part, by increasing trabecular outflow facility through activation of P2Y₁ receptors. The latter would seem to be an interesting target in the development of antiglaucomatous drugs to selectively increase AH outflow.

Several compounds have been identified as present in the aqueous humor, and among these, diadenosine polyphosphates have recently been found in rabbit aqueous humor (Pintor et al., 2003b). Diadenosine polyphosphates are a group of dinucleotides formed by two adenosine moieties bridged by a variable number of phosphates oscillating from 2 to 7 (Ap_nA, n = 2–7). Diadenosine tetraphosphate (Ap₄A) and diadenosine pentaphosphate (Ap₅A), in particular, have been found in similar concentrations to ADP and ATP (Pintor et al., 2003b). Moreover, Ap₄A degradation by phosphodiesterases is a source of other nucleotides such as ATP (Pintor et al., 2003b). Diadenosine polyphosphates activate both P2X and P2Y purinergic receptors (Ralevic and Burnstock, 1998). The P2Y purinergic receptor subfamily comprises purine and pyrimidine nucleotide receptors coupled to G proteins. These receptors, cloned and characterized in different cell types, are known to activate PLC, leading to IP₃ formation and intracellular Ca²⁺ mobilization (for review, see Ralevic and Burnstock, 1998). However, Ca²⁺ release from intracellular stores after P2Y activation has also been reported to stimulate a variety of signaling pathways, including PKC, PLA₂, Ca²⁺-dependent K⁺ channels and nitric-oxide synthase. In the eye, P2Y receptors have been found in different tissues, including retina, ciliary body, cornea, conjunctiva, choroids, and optic nerve head (Cowlen et al., 2003; Pintor et al., 2003b). Moreover, it is known that TM cells release ATP upon stimulation with hypotonic stimuli (Cui et al., 2001; Fleischhauer et al., 2003; Soto et al., 2004) or shear stress (Cui et al., 2001), and recently the presence of purinergic P2Y receptors has also been found in a human TM cell line (Crosson et al., 2004). However, a detailed study of the effects of purinergic receptor activation on outflow facility has yet to be conducted.

Recent studies by Pintor et al. (2003b) have found that application of certain dinucleoside polyphosphates to the eye modify IOP. In particular, Ap₄A has a hypotensive effect in the rabbit eye, whereas Ap₂A, Ap₃A, and Ap₅A have the opposite effect. The ocular targets for dinucleoside polyphosphates have not been fully characterized. It is likely that some of these compounds modify aqueous humor production, but they may also affect outflow. From a physiological point of view, understanding how all these dinucleotides interact with ocular structures is important. Moreover, one of them, Ap₄A, presents interesting features from the therapeutic point of view, since it reduces IOP and therefore could be used in ocular hypertension and glaucoma treatment.

In this study, we identified the purinergic receptors present in TM cells and tested whether dinucleoside polyphosphates have any effects on intracellular calcium. Also, in a more physiological approach, we used a constant-pressure perfusion technique of ocular anterior segments to test the effects of dinucleotides on outflow facility. Our results show that Ap₄A and Ap₃A increase aqueous humor outflow, this not being the case of the others dinucleotides tested. Among the purinergic receptors identified in trabecular meshwork cells (P2Y₁, P2Y₂, and P2Y₄), the effects seem to be mediated by P2Y₁, since a selective P2Y₁ agonist (2-MeSADP) elicited similar effects.

	Materials and Methods

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Bovine Trabecular Meshwork Cell Culture. Bovine TM cells were cultured using a modification of the technique described by Stamer et al. (1995). As described previously (Llobet et al., 1999), bovine TM strips were digested with 2 mg/ml collagenase (Sigma, Madrid, Spain) and 0.5 mg/ml bovine serum albumin (BSA) (Sigma) at 37°C for 2 h. After mechanical digestion, the supernatant was collected, centrifuged, and resuspended. The resuspended solution was seeded in culture flasks containing Dulbecco's modified Eagle's medium (DMEM) (Cambrex Bio Science, Barcelona, Spain) plus 10% fetal bovine serum, 100 mg/ml L-glutamine (Sigma), 100 U.I./ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin-B (Cambrex Bio Science). Cell growth was observed 2 to 4 days after seeding, and cells reached confluence 12 to 15 days later. Cell passages were performed using Trypsin-EDTA (Cambrex Bio Science). Cells from passages 1 to 3 were used.

Immunolabeling and Western Blotting with P2Y Receptor Antibodies. For the immunocytochemical study, we used trabecular meshwork cells glued to coverslips pretreated with poly-L-lysine. Covers were then treated with 4% p-formaldehyde (w/v) for 15 min before being washed twice with phosphate-buffered saline (PBS) medium. Cells were incubated overnight at room temperature in PBS containing 1% BSA and anti-P2Y primary antibodies (Alomone Labs, Jerusalem, Israel). The dilutions of the primary antibodies were anti-P2Y₁, 1/200; anti-P2Y₂, 1/500; anti-P2Y₄, 1/500; anti P2Y₆, 1/200; and anti P2Y₁₁, 1/1000. The covers were washed three times in PBS in the presence of 3% BSA and then incubated for 1 h with the secondary antibody that was also diluted in PBS/BSA solution. The secondary antibody used was goat anti-rabbit IgG-TRITC from Sigma (40 µg/ml). The covers were washed three times with PBS and mounted following standard procedures. Controls were carried out by following the same procedures but substituting the primary antibody with the same volume of PBS/BSA solution. Cells were analyzed by confocal microscopy using a Zeiss Axiovert 200M microscope equipped with an LSM 5 Pa confocal module. Trabecular meshwork cells were observed with a Zeiss 63x oil immersion lens, numerical aperture 1.40. TRICT was monitored by exciting at a wavelength of 543 nm. Differential interference contrast (Nomarski) was performed with the same 63x lens bypassed through the corresponding polarizers and analyzers. All the images were managed using the LSM 5 Pa software.

For the Western blot analysis, trabecular meshwork cells were homogenized with a lysis buffer that contained 50 mM HEPES, pH 7.5, 2.5% Triton (w/v), 10 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 5 µg/ml leupeptin. After homogenization, proteins were quantified by the Bradford method. Protein samples (40 µg) were separated by SDS-polyacrylamide gel electrophoresis (10% acrylamide gel) using the Bio-Rad Mini-Protein 3 cell system (Bio-Rad, Hercules, CA). Proteins were transferred to nitrocellulose membranes. After transfer, the membranes were washed with PBS and blocked for 1 h at room temperature with 5% (w/v) skimmed milk powder in PBS. Blots were then incubated overnight at 4°C with primary antibodies in 5% (w/v) skimmed milk powder dissolved in PBS/Tween 20 (0.5% by volume). The dilutions of primary antibodies were as follows: anti-P2Y₁, 1/200; anti-P2Y₂, 1/500; anti-P2Y₄, 1/200; anti P2Y₆, 1/200; and anti P2Y₁₁, 1/1000. The primary antibodies were removed and the blots extensively washed with PBS/Tween 20. Blots were then incubated for 1 h at room temperature with the secondary antibody (mouse anti-rabbit IgG coupled to horseradish peroxidase; A-2074, Sigma) at 1/1000 dilution in 5% (w/v) skimmed milk powder dissolved in PBS/Tween 20. After removal of the secondary antibody, blots were extensively washed as described above and developed using the enhanced chemiluminescence detection system (Amersham Biosciences, Inc., Piscataway, NJ).

Cytosolic Free Ca²⁺ Measurement. Measurement of [Ca²⁺]_i was performed as described in detail previously (Llobet et al., 1999). Briefly, bovine TM cells were plated on 25-mm-diameter glass coverslips (VWR Scientific Inc., Philadelphia, PA) and then loaded with 5 µM fura-2/acetoxymethyl ester (Calbiochem, San Diego, CA) for 25 min at 37°C in incubation buffer (121 mM NaCl, 4.7 mM KCl, 5 mM NaHCO₃, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 2 mM CaCl₂, 10 mM glucose, 10 mM HEPES, and 0.01% BSA, at pH 7.4 with NaOH) (287 ± 2 mOsM/kg; mean ± S.D.). Coverslips with fura-2-loaded cells were transferred into an open flow chamber (1 ml of incubation buffer) mounted on the heated stage of a Nikon Diaphot-300 inverted epifluorescence microscope. Fluorescence images were obtained by a charge-coupled device camera (CH250; Photometrics, Tucson, AZ) and were digitized, stored, and analyzed on an Apple Macintosh 840AV computer (Apple Computer, Cupertino, CA). After a stabilization period of 10 min, image pairs were obtained alternately every 4 s, and for a total of 8 min, at excitation wavelengths of 340 (1) and 380 nm (2; 10-nm bandwidth filters) to excite the Ca²⁺-bound and Ca²⁺-free forms of this ratiometric dye, respectively. The emission wavelength was 510 nm (120-nm bandwidth filter). Experiments were calibrated by measuring the minimum and maximum fluorescence ratio in fura-2-loaded cells in the absence of Ca²⁺ in the bath (R_min) and in the presence of Ca²⁺ + ionomycin (R_max). Calcium concentrations were calculated according to the formula [Ca²⁺] = K_d · Q · [(R – R_min)/(R_max – R)], where K_d is the Ca²⁺ dissociation constant of the dye, R is the fluorescence ratio F₁/F₂, and Q is the ratio of F_min to F_max at 2. Typically, 10 to 20 cells were present in a field, and [Ca²⁺]_i values were calculated and analyzed individually for each single cell from the 340- to 380-nm fluorescence ratios at each time point (Llobet et al., 1999). In both control and experimental groups, Ca²⁺ was recorded for 1 min before drug application and for 7 min thereafter. Cells were considered as responders when [Ca²⁺]_i increased by more than 100% above the resting value. Drug responses in each field were homogeneous, and several experiments with cells from different primary cultures were used to calculate dose-response curves. Dose-response curves for [Ca²⁺]_i were calculated using the maximum [Ca²⁺]_i increase in the first peak recorded after drug application.

Perfusion of Anterior Segments. Eyes from 3- to 6-month-old cows were obtained from the local abattoir 0.5 to 2 h after death and were kept in PBS at 4°C for not more than 1.5 h. Isolation of bovine anterior segments was performed as described previously (Gual et al., 1997). The perfusion technique has also been described previously (Gual et al., 1997; Llobet et al., 1999). Briefly, bovine anterior segments were placed in a specially designed perfusion chamber. The anterior segments, located in their respective chambers together with force transducers (Letica, Barcelona, Spain) and the tubing system, were placed in an incubator (Selecta, Barcelona, Spain) at 36°C and 5% CO₂. Perfusion was carried out with DMEM. The pressure of the artificial anterior chamber was monitored and recorded throughout the experiment with a pressure transducer (9162-0; Mallinckrodt, Northampton, UK) and was maintained with a suspended reservoir at 10 mm Hg in bovine eyes. Outflow facility (C) was averaged over periods of 15 min (mean of 450 values). Baseline facility (C₀) was calculated during the first 90-min period of stable recording. When a drug was added to the perfusion medium, the tubes and anterior chamber were flushed and replaced with the new medium. This change was made by rapidly replacing the contents of the artificial anterior chamber by opening the exit needle until 200% of the volume had been exchanged; this exchange was always made at a pressure below 10 mm Hg. Recording of outflow facility measurements started after stabilization of flow.

The perfusion procedure was carried out using a protocol with three periods: perfusion with control isotonic DMEM for 90 min to establish the C₀ (baseline), 90 min perfusion with a drug (C_d), and 90 min of perfusion with DMEM returning to the baseline conditions (C_ret). Outflow facility was calculated as the ratio between C_d/C₀ or C_ret/C₀.

Studies of the Ectoenzymatic Degradation of Diadenosine Polyphosphates. Trabecular meshwork cells were plated in six-well dishes at a density of 3 x 10⁶ cells/well. DMEM was removed, and cells were washed twice with PBS before commencing the degradation studies. To monitor the ectoenzymatic degradation of diadenosine polyphosphates, cells were incubated with 3 ml of 1 µM Ap₃A, Ap₄A, or Ap₅A at 37°C. Aliquots of 50 µl were taken at 1, 5, 15, 30, and 60 min and analyzed by HPLC. The HPLC system consisted of a Waters 1515 Isocratic HPLC pump, a 2487 dual wavelength absorbance detector, and a Reodyne injector, all controlled by the Breeze software from Waters (Milford, MA). The column used was a NovaPak C18 (15-cm length, 0.4-cm diameter), also from Waters. The mobile phase consisted of 10 mM KH₂PO₄, 2 mM tetrabutyl ammonium, and 17% acetonitrile, pH 7.5. Detection was monitored at a wavelength of 260 nm. To transform chromatographic peaks into concentrations, diadenosine polyphosphate peak areas were compared with external standards of known concentrations prepared from commercial dinucleotides.

Drugs. P¹,P³-diadenosine triphosphate (Ap₃A), P¹,P⁴-diadenosine tetraphosphate (Ap₄A), P¹,P⁵-diadenosine pentaphosphate (Ap₅A), 2-(methylthio) adenosine 5'-diphosphate (2-MeSADP), 2'-deoxy-N₆-methyladenosine-3',5'-diphosphate (MRS-2179), pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS), and suramin sodium salt were obtained from Sigma. P¹,P⁴-(diuridine 5')-tetraphosphate (Up₄U; INS365) was kindly provided by Inspire Pharmaceuticals (Durham, NC).

Data Analysis. Results are given as mean ± S.E.M. Results were statistically analyzed using paired or unpaired Student's t test, Fisher's exact test, and ² test. Two-way ANOVA plus Bonferroni post tests were used to evaluate statistical differences between control and drug effects on outflow facility. p values less than 0.05 were considered significant.

	Results

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Localization of P2Y Receptors in Trabecular Meshwork Cells. Primary cultured trabecular meshwork cells were used 3 to 5 days after passage. Under the Nomarski interferential technique, the cells presented an elongated aspect as observed in Fig. 1. Trabecular meshwork cells were incubated with commercially available P2Y receptor antibodies. Among the antibodies tested only P2Y₁, P2Y₂, and P2Y₄ positively labeled the cells (Fig. 1A). The other P2 antibodies used (P2Y₆ and P2Y₁₁) produced no positive result in these cells (Fig. 1A). Western blot analysis performed as described under Materials and Methods gave similar results to those observed in the immunocytochemistry studies. Blots presented single bands for P2Y₁, P2Y₂, and P2Y₄ receptors as shown in Fig. 1B. No bands were detected when the antibodies for P2Y₆ and P2Y₁₁ were assayed in the Western blots (Fig. 1B), this being consistent with the immunocytochemical results described above. The band molecular masses of 70 kDa for P2Y₁, 50 kDa for P2Y₂, and 92 kDa for P2Y₄ are in line with those described in the literature (Pintor et al., 2004).

View larger version (89K): [in this window] [in a new window]   Fig. 1. Detection of P2Y purinergic receptors in trabecular meshwork cells. A, immunolabeling of primary cultured trabecular meshwork cells. Cells were analyzed by confocal microscopy and differential interference contrast (Nomarski) with 63x objective. Antibodies against P2Y receptors showed positive staining to P2Y1, P2Y2, and blotting P2Y4 receptors and negative to P2Y6 and P2Y11. Goat anti-rabbit IgG-TRITC (40 µg/ml) was used as the secondary antibody. B, Western showing the presence of P2Y1, P2Y2, and P2Y4 receptors in a trabecular meshwork cells. P2Y6 and P2Y11 provided negative results, as did the immunocytochemistry shown in A. The dilutions of primary antibodies were as follows: anti-P2Y1, 1/200; anti-P2Y2, 1/500; anti-P2Y4, 1/200; anti-P2Y6, 1/200; and anti-P2Y11, 1/1000. The secondary antibody (mouse anti-rabbit IgG coupled to horseradish peroxidase) was at 1/1000. Bands at expected molecular masses are shown: 70 kDa for P2Y1, 50 kDa for P2Y2, and 92 kDa for P2Y4.

As described under Materials and Methods, cells, when stimulated with dinucleotides, were considered as responders when [Ca²⁺]_i increased by more than 100% above the resting value. All the nucleotides tested (Ap₃A, Ap₄A, Ap₅A, and Up₄U) mobilized [Ca²⁺]_i in TM cells, but the response patterns, the peak characteristics, and the potencies of each agonist varied between them.

Ap₃A and Ap₄A elicited similar [Ca²⁺]_i responses (Fig. 2). Response percentages at each concentration are shown in Fig. 3A and Table 1. At 10^–5 M, Ap₃A induced one Ca²⁺ peak in 74% of responder cells (n = 85), whereas Ca²⁺ oscillations were observed in the remainder. Ap₄A (10^–5 M) elicited Ca²⁺ oscillations in 60% of responder cells (n = 60) and only one peak in the rest. At the same concentration as the previous drugs, Ap₅A induced only one Ca²⁺ peak in all the responder cells (100%; n = 14). Calculated EC₅₀ from dose-response curves (Fig. 3A) were 1.1 x 10^–5 M for Ap₃A, 6.1 x 10^–5 M for Ap₄A, and 4.5 x 10^–5 M for Ap₅A. Figure 3B and Table 1 show [Ca²⁺]_i increases induced by Ap₃A, Ap₄A, and Ap₅A at concentrations ranging from 10^–8 to 10^–3 M. To further characterize the calcium responses, we also calculated T₇₀,as the time from the calcium peak until the cell has recovered 70% of the calcium increase. Ap₃A and Ap₄A (at 10^–5 M) showed similar T₇₀ values of 33.7 ± 4.3 and 33.2 ± 1.6 s (mean ± S.E.M.), respectively. Calcium peaks for Ap₅A were brief and recovered faster, within 15.0 ± 2.1 s.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Intracellular calcium mobilizations induced by dinucleoside polyphosphates in trabecular meshwork cells. Representative experiments of TM cells stimulated (arrow) with Ap3A (A and B), Ap4A (C and D), Ap5A (E), and Up4U (INS365; F) at 10–5 M. A, C, E, and F show representative experiments in cells where these drugs only induced a single [Ca2+]i peak. B and D show examples of cells that elicited multiple calcium peaks or calcium oscillations. Observe the different pattern of Ca2+ responses elicited by Up4U (larger and more sustained Ca2+ increases) and diadenosine polyphosphates (smaller and briefer).

View larger version (22K): [in this window] [in a new window]   Fig. 3. Dose-response effects of dinucleoside polyphosphates on [Ca2+]i. Sigmoid fittings for the percentage of trabecular meshwork cells responding to each agonist (A) and the dose-response increase in Ca2+ in the responding cells (B). Up4U () was the most potent agonist to mobilize [Ca2+]i with an EC50 of 0.68 µM about 2 orders of magnitude lower than the other dinucleotides tested. Ap3A (), Ap4A (), and Ap5A () each showed about the same potency in terms of stimulating [Ca2+]i release with EC50 of 11, 61, and 45 µM, respectively.

In contrast to the above-mentioned results, Ca²⁺ mobilizations induced by Up₄U were consistently different from the effects induced by diadenosine polyphosphates. At 10^–5 M, Up₄U increased [Ca²⁺]_i in 74% of cells (Table 1). Most of the responder cells (78%; n = 102) elicited only one calcium peak. Ca²⁺ oscillations were rarely observed. Examples of these responses are shown in Fig. 2. Sigmoid fitting (Fig. 3A) shows that Up₄U was the most potent agonist in mobilizing [Ca²⁺]_i, it having an EC₅₀ of 6.8 x 10^–7 M, about 2 orders of magnitude lower than the other dinucleotides tested. When comparing the amplitude of the calcium peaks (Fig. 3B), Up₄U had an EC₅₀ value of 1.3 x 10^–6 M but a reduced maximal response. Finally, Up₄U calcium peaks where longer in duration, with a T₇₀ of 123.4 ± 7.5 s, as shown in Fig. 2 and Table 1.

Together, these data and the individual Ca²⁺ responses shown in Fig. 2 reveal a clear difference between the responses induced by Ap₃A, Ap₄A, and Ap₅A and those elicited by Up₄U. These distinct response patterns suggest that Up₄U activates different intracellular mechanisms from the other drugs. We did not observe any correlation between cell morphology and responsiveness to the different dinucleotides or the calcium peaks elicited.

Effects of Dinucleoside Polyphosphates on Outflow Facility. We perfused ocular anterior segments at constant pressure, as described previously (Gual et al., 1997; Llobet et al., 1999), and evaluated the effects on trabecular outflow facility of the same dinucleotides tested in the calcium study. All the compounds were tested at 10^–6 M since the physiological concentrations of dinucleotides found in rabbit aqueous humor were in the micromolar range (Pintor et al., 2003b). Control experiments with perfusion medium alone (DMEM; n = 9) showed no significant variations during the 300-min perfusion protocol. In contrast, both Ap₃A (n = 8) and Ap₄A(n = 8) increased outflow facility significantly (p < 0.001; two-way ANOVA versus control perfusion; Fig. 4, A and B). Interestingly, Ap₄A increased outflow facility right after adding the drug to the perfusion medium, whereas Ap₃A effects were slower to develop. Nevertheless, by the end of the perfusion experiments both drugs had increased outflow facility by a similar amount. In contrast, neither Ap₅A (n = 8) nor Up₄U (n = 8) increased outflow facility significantly (Fig. 4, C and D). In fact, both drugs slightly decreased outflow facility, although no significant changes were found compared with the control perfusion or with their respective baseline perfusion periods. These results suggest that the reported hypotensive effects of Ap₄A in the eye (Pintor et al., 2003b) may be mediated, at least in part, by an increase in trabecular outflow facility.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Effects of dinucleoside polyphosphates on outflow facility. Outflow facility ratio (normalized with baseline outflow facility) is plotted against time. Anterior segments were perfused with control medium (DMEM) for 90 min (baseline), and then perfusion medium was then exchanged for DMEM + drug (1 µM) for a further 90 min. Finally, a return period of 90 min with DMEM was carried out (same conditions as the baseline). Control perfusion group was done with DMEM in the absence of drugs (; n = 9). Ap3A (A; ; n = 8) and Ap4A (B; ; n = 8) increased outflow facility significantly (p < 0.001; two-way ANOVA versus control perfusion). Neither Ap5A (C; ; n = 8) nor Up4U (D; ; n = 8) changed outflow facility significantly.

Involvement of the P2Y₁ Receptor in Outflow Facility Modulation. Ap₃A and Ap₄A can activate both P2Y₁ and P2Y₂ purinergic receptors (Ralevic and Burnstock, 1998; Hoyle et al., 2001). In contrast, Up₄U is only active on P2Y₂. Since Up₄U did not induce significant changes in outflow facility, we tested the hypothesis that Ap₃A and Ap₄A effects on outflow facility may be mediated by P2Y₁ receptor activation. Stimulation of TM cells with the selective P2Y₁ agonist 2-MeSADP (1 µM) increased [Ca²⁺]_i in 80.3 ± 8.2% (mean ± S.E.M.; n = 234 cells) of cells (p < 0.001 versus control; Fig. 5, A and B). The mean [Ca²⁺]_i increase was 384.5 ± 10 nM (n = 234), whereas T₇₀ was 24.9 ± 1.0 s, these values being similar to those observed for Ap₃A and Ap₄A. To further characterize the effects of P2Y₁ activation, we tested [Ca²⁺]_i mobilizations induced by 2-MeSADP after preincubation of TM cells with two P2Y₁ antagonists, MRS-2179 and PPADS. At 10 µM, both compounds reduced significantly the percentage of cells responding to 2-MeSADP (p < 0.01 and p < 0.001, respectively; Fig. 5A). In contrast, suramin (100 µM), a nonselective P2Y₂ antagonist, did not significantly reduce 2-MeSADP effects on [Ca²⁺]_i (Fig. 5A). When 2-MeSADP (1 µM; n = 10) was tested on trabecular outflow facility, it increased outflow facility significantly (p < 0.001; two-way ANOVA versus control perfusion; Fig. 5C), thus providing evidence of P2Y₁ receptor involvement in outflow facility modulation. In agreement with this result, 2-MeSADP (1 µM) was unable to increase outflow facility in the presence of the selective P2Y₁ receptor antagonist MRS2179 (10 µM; n = 5; Fig. 5C). Finally, we tested whether the effect of Ap₄A on outflow facility was also blocked by the P2Y₁ receptor antagonist. MRS-2179 (10 µM) partially blocked an Ap₄A-induced increase in outflow facility (p < 0.001; two-way ANOVA versus Ap₄A alone; Fig. 5D; n = 8), showing that at least part of the effect of this compound is mediated by activation of P2Y₁ receptors.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Involvement of P2Y1 receptor in outflow facility modulation. A, stimulation of TM cells with the selective P2Y1 agonist 2-MeSADP (1 µM) increased [Ca2+]i in 80.3 ± 8.2% of cells. Preincubation of TM cells with two selective P2Y1 antagonists, MRS-2179 (10 µM) and PPADS (10 µM), reduced significantly the percentage of cells responding to 2-MeSADP (**, p < 0.01 and ***, p < 0.001, respectively; Fisher's exact test). Suramin (100 µM), a nonselective P2Y antagonist, did not significantly reduce 2-MeSADP effects on [Ca2+]i (results are mean ± S.E.M.). B, representative [Ca2+]i response to 2-MeSADP at 1 µM. C, 2-MeSADP (1 µM; ; n = 10) increased outflow facility significantly (p < 0.001; two-way ANOVA) versus control (; n = 11) in anterior segment perfusion experiments. When MRS-2179 (10 µM; ; n = 5) was present in the perfusion medium, it selectively blocked the effect of 2-MeSADP (1 µM) on aqueous humor outflow. D, Ap4A (1 µM; ; n = 8) increased outflow facility significantly (p < 0.001; two-way ANOVA) versus control (; n = 11) in anterior segment perfusion experiments. When MRS-2179 (10 µM; ; n = 8) was present in the perfusion medium, it partially blocked the effect of Ap4A (1 µM) on aqueous humor outflow (p < 0.001; two-way ANOVA versus Ap4A alone).

View larger version (20K): [in this window] [in a new window]   Fig. 6. Ectoenzymatic degradation of diadenosine polyphosphates. TM cells plated in multiwells in a number of 3 x 106 cells/well were studied in terms of their ability to cleave diadenosine polyphosphates. Incubation of single concentrations of 1 µM demonstrated that both dinucleotides were slowly degraded, although Ap4A was more resistant to cleavage than Ap5A. A, chromatographic composition of the corresponding profiles of Ap4A and Ap5A areas after the incubation with TM cells for 0, 1, 5, 15, 30, and 60 min (indicated by the labels on the corresponding peaks). The peaks occurred at the same retention time, but the starting point has been adjusted in the figure for each peak to allow comparison of peak magnitude. B, time-dependent degradation of Ap4A and Ap5A. After 1 h in the presence of the corresponding dinucleotide, TM cells were able to degrade 18% of Ap4A and 44% of Ap5A.

	Discussion

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Diadenosine polyphosphates are natural compounds that control numerous physiological functions (Hoyle et al., 2001). For example, they have been found in tears (Pintor et al., 2002a), where they increase tear secretion (Pintor et al., 2002b). Thus, synthesized pharmacological compounds with related structures (e.g., Up₄U) have been proposed for dry eye treatment, and topical application of Ap₄A accelerates the rate of reepithelialization after corneal wound healing (Pintor et al., 2003a). In the retina, where P2X and P2Y purinergic receptors have been found, synthetic nucleotidic compounds such as INS37217increase fluid absorption in rat models of retinal detachment (Maminishkis et al., 2002). Interestingly, micromolar concentrations of dinucleotides have been detected in rabbit AH, together with other mononucleotides (Pintor et al., 2003b). When tested in rabbit eyes, Ap₂A, Ap₃A, and Ap₅A induced a dose-dependent increase in IOP, whereas Ap₄A produced the opposite effect (Pintor et al., 2003b). Here, the finding that Ap₃A and Ap₄A increase trabecular outflow facility leads us to hypothesize that, at least in part, the hypotensive effect of Ap₄A in rabbit eyes is mediated by an increase in aqueous outflow. When instilled topically, dinucleotides are likely to stimulate purinergic receptors present in the TM to increase AH outflow as well as other purinergic receptors present in the eye, such as those in the ciliary processes (Farahbakhsh and Cilluffo, 2002), regulating inflow simultaneously. Since the effects of Ap₄A on IOP were tested in rabbits and our study has been performed in bovine eyes, the results cannot be fully extrapolated. Nevertheless, the hypotensive effect of Ap₄A seems to be well correlated with the increase in outflow facility found here. In contrast, Ap₃A, which also increased outflow facility, had a hypertensive effect in rabbit eyes. This effect may be due to the different selectivity of each agonist for the purinergic receptors stimulated and the receptor population present in the inflow and outflow pathways of the rabbit.

We describe the presence of P2Y₁, P2Y₂, and P2Y₄ in bovine TM cells. In contrast, the immunocytochemistry and Western blot results seem to clearly indicate the absence of P2Y₆ and P2Y₁₁ purinoceptors. Using the same antibodies, it was possible to identify these receptors in rat ocular structures such as the corneal epithelium (P2Y₆) and the retinal pigmented epithelium (P2Y₁₁) and to confirm the existence of P2Y₁ and P2Y₂ in sections containing the TM (Pintor et al., 2004). Other studies have also reported the presence of P2Y₁ and P2Y₂ receptors in bovine TM cells (Cui et al., 2001) and P2Y₁, P2Y₄, and P2Y₁₁ in a human TM cell line (Crosson et al., 2004). Ap_nAs are known to activate several different purinergic receptors: Ap₃A and Ap₄A both activate P2Y₁ receptors with different selectivity, whereas Ap₅A is, in general, less effective at this receptor (Schachter et al., 1996). Furthermore, Ap₄A is a good agonist at P2Y₂ and P2Y₄ receptors, and although Ap₃A and Ap₅A can also activate these receptors, they do so with less affinity. Finally, Up₄U exhibits comparable potency with UTP as an agonist for P2Y₂ and P2Y₄ receptors, being inactive at P2Y₁ (Pendergast et al., 2001). P2Y receptors act via a G_q/11 protein coupling to activate PLC, IP₃ formation, and mobilization of [Ca²⁺]_i, although coupling to adenylyl cyclase, PLA₂, PKC, NO synthase, or BK_Ca channels activation has also been described (Ralevic and Burnstock, 1998).

In TM cells, all the Ap_nAs tested increased [Ca²⁺]_i in a dose-dependent manner (Figs. 2 and 3) with similar EC₅₀. Compared with Up₄U, Ap_nAs exhibited a different pattern of [Ca²⁺]_i mobilization, with faster transients and frequent induction of Ca²⁺ oscillations. In contrast, Up₄U induced Ca²⁺ transients with longer durations, similar to the Ca²⁺ increases triggered by ATP (Crosson et al., 2004; Soto et al., 2004), bradykinin (Llobet et al., 1999), and carbachol (Shade et al., 1996). Interestingly, drugs that induce large and sustained Ca²⁺ mobilizations either decrease outflow facility (bradykinin, carbachol, and endothelin-1) (Wiederholt et al., 1995; Llobet et al., 1999) or do not modify it (Up₄U and ATP; this study and D. Soto and X. Gasull, unpublished observations). Large and long-lasting Ca²⁺ increases may lead to cellular contraction or activation of different intracellular pathways, producing a decrease in TM permeability and thus reducing outflow facility. In fact, TM contractions have been described after application of carbachol or endothelin-1, known also to decrease outflow facility (Wiederholt et al., 1995, 2000).

Our study suggests that Ca²⁺ mobilizations produced by Ap₃A and Ap₄A are not correlated with the observed outflow facility increase. Three observations support this hypothesis: 1) Up₄U increased intracellular Ca²⁺ but did not modify outflow facility; 2) Ap₅A mobilized intracellular Ca²⁺ with similar response patterns to those of Ap₃A and Ap₄A, but again, without modifying outflow facility; and 3) Ap₃A and Ap₄A increased outflow facility at a concentration (1 µM) at which only about 15% of the cells elicited Ca²⁺ peaks. Although we cannot rule out the possibility that even in a small population of cells these fast Ca²⁺ transients and Ca²⁺ oscillations could lead to rhythmic activation of certain enzymes such as Ca²⁺/calmodulin-dependent protein kinase II (De Koninck and Schulman, 1998) or PKC (Oancea and Meyer, 1998) that may be involved in outflow facility regulation by TM cells, this explanation seems less plausible.

This and other studies (Wiederholt et al., 2000) suggest that contractile effects (e.g., cell contraction and PKC activation) would predominate after large Ca²⁺ mobilizations, whereas relaxing effects may be preferentially activated by discrete Ca²⁺ transients or by other signaling pathways (NO synthase, adenylyl cyclase, and prostaglandin release). The balance between the relaxing (likely to increase outflow facility) and contractile effects (which would lead to outflow facility reduction) would determine the permeability of the TM to the passage of aqueous humor (Wiederholt et al., 2000). In this regard, Ap₃A and Ap₄A seem to activate some of these relaxing mechanisms.

Other mechanisms proposed as playing important roles in TM function include changes in extracellular matrix (Tian et al., 2000), up-regulation/down-regulation of genes (Borras, 2003), and changes in cell shape (Gills et al., 1998) or cell volume (Al-Aswad et al., 1999; Soto et al., 2004). It is possible that dinucleotides could modify some of these parameters such as cell volume, as proposed for other compounds (Fleischhauer et al., 2003; Srinivas et al., 2004). Indeed, stimulation of adenosine A₁, A_2A, and A₃ receptors reduces TM cell volume (Fleischhauer et al., 2003) and their agonists reduce IOP by increasing outflow facility and decreasing inflow (Crosson, 2001). This effect on outflow facility could be mediated by TM cell shrinkage (Fleischhauer et al., 2003). As seen in other tissues (Sumiyoshi et al., 1997), it is possible that dinucleotides raise cAMP concentration, which has also been linked to outflow facility increase (Erickson-Lamy and Nathanson, 1992; Gilabert et al., 1997). As previously shown, cAMP-mediated reduction in TM cell volume (Srinivas et al., 2004) could increase outflow facility. Finally, since activation of the NO/cGMP pathway has also been involved in aqueous outflow facilitation (Kee et al., 1994), we cannot rule out the possibility that Ap_nAs may induce NO release in TM cells as reported in other cell types (Hilderman and Christensen, 1998).

The finding that selective (2-MeSADP) and nonselective (Ap₃A and Ap₄A) P2Y₁ agonists increase outflow facility together with the lack of effect observed by the P2Y₂/P2Y₄ agonist (Up₄U) leads us to propose P2Y₁ as a specific receptor linked to an increase in TM permeability. This seems reinforced by the fact that the P2Y₁ antagonist MRS-2179 blocks the increase induced by 2-MeSADP and significantly reduces the effect of Ap₄A on outflow facility. Therefore, P2Y₁ receptors seem to be a suitable target for antiglaucoma therapy to improve aqueous humor outflow through the trabecular meshwork.

Independently of the receptor subtype activated by diadenosine polyphosphates, the existence of ectonucleotidases with the ability to cleave these molecules into mononucleotides has been preliminarily demonstrated in the present study. However, we can only speculate which of the cloned ectonucleotide pyrophosphatase/phosphodiesterases is involved in the differential rate of hydrolysis of Ap₄A and Ap₅A (Vollmayer et al., 2003). This cleavage is slow and does not allow the accurate measurement of the mononucleotides generated after dinucleotide cleavage. This reflects the fact that the rate of transformation of the generated mononucleotides by means of ecto-ATPase, ecto-ADPase, ecto-apyrase, and ecto-5'nucleotidase needs to be higher than that which degrades diadenosine polyphosphates. Indeed, in chromaffin cells, the V_max for ATP degradation is almost 1000-fold higher than that for Ap₄A (Torres et al., 1990). It is important to note that either mononucleotides or adenosine, both of which are products of the dinucleotide cleavage, may contribute to the total facilitatory effect triggered by Ap₄A and Ap₃A. ATP, due to its fast degradation in comparison with the time necessary to measure the dinucleotide action in the facilitation studies, does not seem to be so relevant. In fact, we have not observed significant effects of ATP on outflow facility (D. Soto and X. Gasull, unpublished observations).

It will be interesting in the future to study the cross talk between dinucleotides, mononucleotides, and adenosine in TM cells to understand the importance of the purinergic system regulating aqueous humor physiology.

	Acknowledgements

 
We thank Dr. Miguel Morales, Dr. Elisa Ferrer, and Núria Comes for support during the study and helpful comments on the manuscript as well as Dan Forsblade for help with English grammar.

	Footnotes

 
This work was supported by BFI2002-01202, BFI2003-01190, PI031495, SAF2003-00338, and FEDER UCOM03-33-024, Spain.

doi:10.1124/jpet.105.085274.

ABBREVIATIONS: IOP, intraocular pressure; AH, aqueous humor; TM, trabecular meshwork; Ap₄A, P¹,P⁴-diadenosine tetraphosphate; Ap₅A, P¹,P⁵-diadenosine pentaphosphate; PL, phospholipase; IP₃, inositol 1,4,5-trisphosphate; PKC, protein kinase C; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine B isothiocyanate; HPLC, high-performance liquid chromatography; Ap₃A, P¹,P³-diadenosine triphosphate; 2-MeSADP, 2-(methylthio) adenosine 5'-diphosphate; MRS-2179, 2'-deoxy-N₆-methyladenosine-3',5'-diphosphate; PPADS, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid; Up₄U, P¹,P⁴-(diuridine 5')-tetraphosphate; ANOVA, analysis of variance; [Ca²⁺]_i, intracellular calcium concentration; INS37217 P¹-(uridine 5')-P⁴-(2'-deoxycytidine-5')-tetraphosphate.

Address correspondence to: Dr. Xavier Gasull, Laboratori de Neurofisiologia, Facultat de Medicina-U.B., Casanova 143, E-08036 Barcelona, Spain. E-mail: xgasull{at}ub.edu

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