Introduction

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The discovery of diadenosine 5'-polyphosphates (Ap_nA, n = 2-7) (Fig. 1) and their release from platelets and chromaffin cells has led to many studies of the biological activity and cellular processing of these intra- and extracellular signaling molecules (Pintor, 1999; Hoyle et al., 2001). Diadenosine polyphosphates have interesting pharmacological effects on nucleotide receptors; that is, depending on the chain length, they may be agonists or antagonists at P2X and P2Y receptors with varying selectivity. There is not a clear relationship between the phosphate chain length and the selective activity on P2X or P2Y receptors; however, from a physiological point of view, some of them can act as vasodilators (Ap₂A and Ap₃A), whereas others act as vasoconstrictors (Ap₄A, Ap₅A, and Ap₆A) (Ralevic and Burnstock, 1998).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Structures of diadenosine polyphosphates (ApnA, n = 2-5).

Interest in uridine-containing nucleotides as extracellular signaling molecules increased with the discovery that UTP was a full agonist at the P2Y₂ receptor, with potency comparable to that of the purine agonist ATP. Diadenosine polyphosphates were also found to be potent and full agonists in cells overexpressing the human P2Y₂ receptor (Lazarowski et al., 1995). Also, the avian P2Y₁ receptor is sensitive to Ap₄A, presenting EC₅₀ values in the nanomolar range (Pintor et al., 1996). Important differences have been observed on native P2Y₁ receptors, where diadenosine tetraphosphate behaved as an antagonist in clear contrast to the behavior of this dinucleotide in cloned P2Y₁ receptors (Vigne et al., 2000). On the other hand, other expressed P2Y receptors, such as P2Y₄, are also activated by Ap₃A-Ap₆A in the micromolar range (Communi et al., 1996; Janssens et al., 1997). Recently, several of the pyrimidine dinucleotides Up_nU (n = 2-7) have been prepared and their ability to stimulate P2Y receptors has been studied. Up₄U is the most potent P2Y₂ receptor agonist in the series (Pendergast et al., 2001). P2Y₂ agonists are known to have prosecretory effects on the surface of the eye, such as stimulating the secretion of ions, fluid, and mucin (Hosoya et al., 1999; Fujihara et al., 2000, 2001; Li et al., 2001).

Despite various studies on P2Y₂ agonists in ocular surface tissues, little is known about the role of Ap_nA signaling molecules on ocular physiology. Dinucleotides have been shown to affect intraocular pressure in rabbits: Ap₄A lowered intraocular pressure, whereas others raised it (Peral et al., 2000). Here we report for the first time the presence of diadenosine polyphosphates in rabbit tears and suggest that these dinucleotides, through interactions with the P2Y₂ receptor, are potent stimulators of tear secretion.

	Experimental Procedures

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Materials. UTP and ATP were purchased from Amersham Biosciences, Inc. (Piscataway, NJ); UDP, Ap₂A, Ap₃A, Ap₄A, Ap₅A, Ap₆A, and phosphodiesterase were purchased from Sigma Chemical (St. Louis, MO); 2MeSADP, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), suramin, and reactive blue-2 (RB-2) were purchased from Sigma/RBI (Natick, MA). Adrenoceptor antagonists (yohimbine and ICI 118,551) and cholinoceptor antagonists (hexamethonium and atropine) were obtained from Tocris (Bristol, UK). The purity of all nucleotide agonists was established by HPLC (95-99% purity). Schirmer strips were kindly provided by Allergan (Irvine, CA). Fluo-3/AM was obtained from Molecular Probes (Eugene, OR). Dulbecco's modified Eagle's medium, fetal bovine serum, G-418, and other cell culture reagents were obtained from the Tissue Culture Facility at the University of North Carolina (Chapel Hill, NC) or from Invitrogen (Carlsbad, CA). 1321N1 human astrocytoma cells stably expressing the P2Y₁, P2Y₂, P2Y₄, P2Y₆, or P2Y₁₁ receptors, and wild-type 1321N1 cells were obtained from the University of North Carolina (Chapel Hill, NC).

Animals. Male New Zealand White rabbits weighing 2.0 to 2.5 kg were placed in individual cages with free access to food and water and subjected to regular cycles of light/dark (12 h). All the experiments were performed according to Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and to the European Directive 86/609/EEC.

Tear Collection and Extraction. The dinucleotide tear components were extracted from tears using Whatman no. 51 paper strips (Schirmer strips; Whatman, Maidstone, UK) placed in the inferior lid margin of the eye for 5 min. The strips were then placed in Eppendorf tubes containing 500 µl of ultrapure water and strongly vortexed for 5 min. The Schirmer strips were carefully rinsed and the liquid in the tube was heated in a 100°C bath for 20 min. Precipitated proteins were removed by centrifugation at 4000 rpm for 30 min. Diadenosine polyphosphates are stable to this treatment as previously reported; however, any mononucleotides present in the sample are destroyed by this sample workup procedure (Pintor et al., 1992). One hundred microliters of the supernatant was injected into the HPLC apparatus for analysis.

HPLC Procedures. Identification and quantification of adenine dinucleotides in rabbit tears were performed by HPLC as previously described (Pintor et al., 1992). Briefly, the chromatographic system consisted of a Waters Novapak C18 column (15 cm in length, 0.4 cm in diameter), a 1515 Isocratic HPLC pump, a 2487 dual absorbance detector, and a Reodyne injector, all managed by the software Breeze from Waters (Milford, MA). The system was equilibrated overnight with 0.1 M KH₂PO₄, 3% methanol, pH 6.0, and detection of nucleotides was performed under isocratic conditions with the mobile phase described above at a flow rate of 1.5 ml/min. After injection of 100 µl of sample, detection was monitored at 260 nm. Peaks identified as putative diadenosine polyphosphates, based on comparing their retention times with the ones of commercial standards, were collected and subjected to phosphodiesterase treatment. Phosphodiesterase from Crotalus durissus (EC 3.1.15.1) at a concentration of 0.3 U/ml was incubated for 10 min with the corresponding putative dinucleotide, and the digestion products were analyzed by HPLC under the same conditions described previously. This treatment resulted in the quantitative hydrolysis of each compound isolated from tears and resulted in the appearance of two new peaks identified as adenine mononucleotides. Quantification of the products of hydrolysis of the phosphodiesterase was performed by comparing the areas under the curves with those of known amounts of commercial standards.

Measurement of Tear Secretion. Tear secretion was measured according to the Schirmer test. Briefly, 10 µl of the test compound at the indicated concentrations was instilled via pipette in the eye. Thirty seconds later, a Schirmer strip was placed in the inferior lid margin of the eye for 5 min. Control experiments were performed by applying 10 µl of saline solution (0.9% NaCl). Tear secretion was measured as the length (millimeters) of the strip wetted by the tears.

Dosing. Single-dose experiments were carried out by applying 10 µl of the corresponding nucleotide or dinucleotide at a concentration of 10 µg/µl. Dose-response analysis was performed by instilling doses ranging from 10¹⁰ to 10⁴ g/µl, always in a volume of 10 µl. Concentration-response curves were done by applying different doses in a noncumulative manner in one of the rabbit eyes, with the contralateral eye receiving the same volume of saline solution (control). Transformation of grams per microliter units into molar concentrations was performed by factoring in the corresponding molecular weight of each dinucleotide for each data point. Antagonists of P2 receptors such as PPADS, suramin, or RB-2 were applied 5, 10, 15, and 30 min before the application of any agonist. Adrenoceptor and cholinoceptor antagonists, yohimbine, ICI 118,551, atropine, and hexamethonium were applied at a concentration of 10 µg/µl (final volume 10 µl), 5 min before the instillation of agonist.

Cell Culture. 1321N1 human astrocytoma cells stably expressing the human P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁ receptors were grown in Dulbecco's modified Eagle's medium containing 4.5 g/l glucose, 5% fetal bovine serum, and 600 µg/ml G-418. For intracellular Ca²⁺ measurements, cells were seeded in 96-well black wall/clear bottom culture plates (3904; Corning Glassworks, Corning, NY), at a density of 35,000 cells/well and assays were conducted 2 days later when the cells had reached confluence.

Intracellular Ca²⁺ Measurements. On the day of the assay, the growth medium in the culture plates was aspirated and replaced with 2.5 µM Fluo-3/AM in a final volume of 50 µl and incubated for 1 h at 25°C. Then the dye was replaced with assay buffer (10 mM KCl, 118 mM NaCl, 2.5 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, and 20 mM HEPES, pH 7.4) by using a Columbus Plate Washer (Tecan Inc., Research Triangle Park, NC). Intracellular Ca²⁺ levels in response to P2Y receptor agonists was monitored as changes in fluorescence intensity using a fluorescent light imaging plate reader (Pendergast et al., 2001) from Molecular Devices (Sunnyvale, CA). Average fluorescence units corresponding to peak height were captured on disk and exported for further analysis. Changes in fluorescence data corresponding to concentrations of intracellular Ca²⁺ were normalized to the response of the cognate agonists (2MeSADP for P2Y₁ receptor, ATP for P2Y₂ receptor, UTP for P2Y₄ receptor, UDP for P2Y₆ receptor, and ATP for P2Y₁₁ receptor).

	Results

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Presence of Diadenosine Polyphosphates in Rabbit Tears. Diadenosine polyphosphates were isolated from rabbit tears and quantified by HPLC as indicated under Experimental Procedures. Chromatographic separation of tear extracts resulted in two well defined peaks that were identified as Ap₄A and Ap₅A by comparing their retention times with those of authentic external standards (Fig. 2, A and B). To confirm the identity of these two dinucleotides, samples were rechromatographed and enriched with 25 pmol of commercial Ap₄A and Ap₅A. The subsequent chromatographic analysis demonstrated the coelution of the standards and the peaks found in the sample, thus suggesting that both naturally occurring peaks and commercial diadenosine polyphosphates are the same (data not shown).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Representative HPLC chromatograms and retention times for Ap5A and Ap4A in rabbit tear fluid (A) and standard solution (B).

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Phosphodiesterase (PDE) analysis of putative diadenosine polyphosphates. Diadenosine polyphosphates were treated with phosphodiesterase from C. durissus (EC 3.1.15.1) as described under Experimental Procedures. HPLC elution profile of putative Ap4A before and after the treatment of PDE. The appearance of AMP and ATP is concomitant with the disappearance of Ap4A (A). HPLC elution profile of Ap5A before and after the PDE treatment. The disappearance of Ap5A was concomitant with the appearance of AMP and adenosine tetraphosphate (B).

Effect of Mono- and Dinucleotides on Tear Secretion. To determine whether mononucleotides were able to modify rabbit tear secretion, single doses of ATP, UTP, ADP, and UDP were applied at 10 µg/µl (final volume 10 µl). As shown in Fig. 4A, among the tested mononucleotides, UTP and ATP significantly increased Schirmer scores to 160 ± 8% (n = 16) (P < 0.001) and 131 ± 6%, respectively (n = 12), compared with control. UDP and ADP, on the other hand, did not significantly increase tear secretion, their values being 105 ± 2% for UDP and 107 ± 1% for ADP (n = 10).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Effect of mononucleotides (A) and ApnA (B) on tear secretion in rabbits as measured by the Schirmer test. Ten microliters of the test compound solution (10 µg/µl) was instilled via pipette in the eye. Thirty seconds later, a Schirmer strip was placed in the inferior lid margin of the eye for 5 min. Control experiments were performed by applying 10 µl of saline solution (0.9% NaCl). Tear secretion was measured as the length (mm) of the strip wetted by the tears and values are expressed as percentage of saline control (***P < 0.001, **P < 0.05) compared with saline solution (Student's t test).

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View larger version (17K): [in this window] [in a new window]   Fig. 5.   Dose-effect curve for ApnA stimulation of tear secretion by using the Schirmer test. Diadenosine polyphosphates Ap2A-Ap6A were assayed at concentrations ranging from 1010 to 104 g/µl. Tranformation of grams per microliter into molar concentrations was performed by taking into account the corresponding molecular weight of each dinucleotide. Doses were applied in a noncumulative manner in one of the rabbit eyes, with the contralateral eye receiving the same volume of saline solution. Values are the mean ± S.E.M. of eight independent experiments. , Ap6A; , Ap5A; , Ap4A; , Ap3A; , Ap2A.

Effects of Antagonists on Tear Secretion. The activity of diadenosine polyphosphates on tear secretion suggests the activation of P2 nucleotide receptors. To confirm this, the effects of three nonselective, nucleotide receptor antagonists on tear secretion were studied: PPADS, suramin, and RB-2. The pretreatment with these antagonists for 5 min before the administration of nucleotides did not modify tear secretion induced by either Ap₄A or UTP (10 µg/µl, n = 10; data not shown). Only RB-2 modified very slightly the Schirmer scores of both compounds (n = 12), reducing tear secretion as observed in Fig. 6. Similar effects were observed when the rabbits were pretreated with antagonists for 10, 15, or 30 min. In rabbits, RB-2 reduced tear secretion from 155 to 132% for UTP and from 150 to 125% for Ap₄A. Although PPADS and suramin have been shown to antagonize the P2Y₁ receptor, their effects on other P2Y receptor subtypes are less known (Charlton et al., 1996; Schachter et al., 1996). In our hands, addition of varying concentrations of the nonselective P2 receptor antagonists PPADS, suramin, and RB-2 to cells transfected with P2Y₂, P2Y₄, or P2Y₆ receptors revealed no meaningful antagonism of the calcium responses (data not shown).

View larger version (10K): [in this window] [in a new window]   Fig. 6.   Partial reduction of the UTP and Ap4A-stimulated tear secretion in rabbits with the nonselective P2 receptor antagonist RB-2. Rabbits pretreated with RB-2 showed a slight reduction in Schirmer scores after Ap4A and UTP (10 µg/µl) treatment, reducing tear secretion from 155 to 132% for UTP and from 150 to 125% for Ap4A (*P < 0.1 versus control).

View larger version (35K): [in this window] [in a new window]   Fig. 7.   Effects of cholinoceptor and adrenoceptor blockers on UTP and Ap4A-stimulated tear secretion. Blockade of muscarinic and nicotinic receptors with atropine 10 µg/µl (10 µl) and hexamethonium 10 µg/µl (10 µl) produced a decrease in basal tear secretion by 51.3%, whereas the blockade of 1- and 2-adrenoceptors decreased basal tear secretion by 18%. No effect of either cholinoceptor antagonists or adrenoceptor antagonists was observed for UTP- and Ap4A-stimulated tear secretion (**, P < 0.05, *, P < 0.1 versus control).

Pharmacological Profile of Diadenosine Polyphosphates on Transfected P2Y Receptors. Adenine dinucleotides were tested for their ability to activate human P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁ receptors as measured by the mobilization of intracellular Ca²⁺. P2Y₄, P2Y₆, and P2Y₁₁ were insensitive to diadenosine polyphosphates and were only stimulated by different mononucleotides (data not shown). Two receptors, P2Y₁ and P2Y₂, were fully activated by diadenosine polyphosphates with different pharmacological patterns (Fig. 8, A and B). The P2Y₁ and P2Y₂ receptors presented EC₅₀ and corresponding pD₂ values as shown in Table 1. The P2Y₁ receptor was fully activated by submicromolar concentrations of Ap₃A, whereas the P2Y₂ receptor was activated by Ap₄A at this low concentration. The diadenosine polyphosphates were all inactive at the other P2Y receptors, and, as stated above, no meaningful antagonism of the P2Y receptors was observed with PPADS, suramin, or RB-2 (data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Dose-effect curves for ApnA-stimulated intracellular calcium mobilization in 1321N1 astrocytoma cells expressing human P2Y1 (A) and P2Y2 (B) receptors. Curves are normalized to positive controls (2MeSADP for P2Y1 and UTP for P2Y2). EC50 and pD2 values are given in Table 1.

	Discussion

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The results show for the first time that New Zealand White rabbit tears contain diadenosine polyphosphates and that these compounds have effects on tear secretion. The concentrations of diadenosine polyphosphates in rabbit tears, as demonstrated by HPLC, were in the micromolar range, which is sufficient to allow them to activate nucleotide receptors present on the ocular surface. Applied dinucleotides altered the rate of tear production: dinucleotides with four, five, and six phosphates increased tear secretion, whereas Ap₂A and Ap₃A were inactive. Ap₄A was the most efficacious at increasing tear secretion, giving values similar to that obtained with UTP.

Diadenosine di- and triphosphates were unable to change tear secretion even when they were assayed at the highest concentrations. The concentrations necessary to increase tear secretion (EC₅₀ for Ap₄A, 2.27 µM) correspond well with the basal concentration of Ap₄A determined under normal conditions (2.92 µM). This indicates that Ap₄A is in tears at concentrations high enough to stimulate tear secretion, and therefore it may be a naturally occurring compound that promotes a normal tear secretion rate.

Regarding the nucleotide receptor subtype that may be involved in this physiological action, the lack of effect of UDP (P2Y₆ agonist) and ADP (P2Y₁ agonist), and, moreover, the full effect of Ap₄A, strongly suggested the involvement of a P2Y₂ receptor (Ralevic and Burnstock, 1998). This is consistent with P2Y receptors expressed in 1321N1 human astrocytoma cells. Among the receptors studied, only P2Y₁ and P2Y₂ were able to generate changes in the intracellular Ca²⁺ when challenged with diadenosine polyphosphates. When the agonist profile of both P2Y₁ and P2Y₂ receptors expressed in astrocytoma cells is compared with that obtained for tear secretion in the rabbits, it seems clear that the rabbit P2Y receptor matches better with the P2Y₂ receptor than with the P2Y₁ receptor. Although the expressed P2Y₄ receptor was not activated by diadenosine polyphosphates, previous studies have shown that in addition to being activated by UTP, UDP, and ATP, it is also activated by Ap_nA (Communi et al., 1996; Bogdanov et al., 1998; Kennedy et al., 2000; Patel et al., 2000). However, the activity of ATP is very low and there is no discrimination among the diadenosine polyphosphates (Communi et al., 1996). In the rabbit eye, only RB-2 was able to partially inhibit the induction of tear secretion by Ap₄A or UTP.

Blockade of adrenoceptors and cholinoceptors with the use of the corresponding antagonists did not alter the effect produced by adenine dinucleotides, in contrast to the partial reduction in basal tear secretion, as has been previously described (Salvatore et al., 1999). These results are consistent with the observation that P2Y₂ receptor gene expression is observed throughout the corneal and conjunctival epithelium, but not found in nerve terminals innervating the meibomian gland (Yerxa et al., 2000). Thus, the dinucleotides are likely acting at postjunctional rather than prejunctional sites.

Agonists of P2Y₂ receptors such as dinucleoside polyphosphates may prove to be useful in the development of compounds for ameliorating conditions in which enhanced tear secretion would be beneficial, such as dry eye. INS365, a diuridine polyphosphate P2Y₂ receptor agonist, is more efficacious at stimulating tear secretion than Ap₄A, showing a maximal 2-fold increase in tear secretion in the same rabbit model versus saline control (Yerxa et al., 1999). Positive safety and efficacy results of INS365 in dry eye patients were recently reported, and this compound is currently undergoing definitive evaluation in phase III clinical trials (Foulks et al., 2001). Additionally, Ap_nAs appear to be compounds with potential therapeutic value as in the case of ocular hypertension (Peral et al., 2000).

The presence of diadenosine polyphosphates in tears and their effect in stimulating tear secretion introduce new physiological elements in the regulation of tear secretion. The control of tear secretion is partially performed by the nervous system. Although it has been demonstrated that denervation may partially affect tear secretion, it has been shown that this process does not suppress tear secretion (Meneray et al., 1998). An explanation for this may be the presence of diadenosine polyphosphates such as Ap₄A, which would stimulate tear secretion in the absence of neural inputs. This would indicate that diadenosine polyphosphates, as occurs with ATP, may be released upon mechanical stimulation of corneal epithelial cells (Jensen, 2000; Srinivas and Fleiszig, 2000).

Activation of P2Y₂ receptors by diadenosine polyphosphates appears to be one of the chief actions of these signaling molecules, causing an increase in intracellular calcium, chloride, and fluid secretion; mucin secretion; and activation of downstream signaling events via the mitogen-activated protein kinase cascade (Fujita et al., 2001). With the isolation and characterization of nucleotides on the ocular surface now more clearly defined, it is possible to begin to construct an overall scheme of nucleotide release, action, metabolism, and reuptake. Nucleotides and dinucleotides are released onto the ocular surface in concentrations relevant to activate P2X and P2Y receptors. Their subsequent metabolism by ectonucleotidases (Gukasyan et al., 2002) can then generate different P2 receptor ligands with further physiological actions, although the presence and roles of these other receptors on the ocular surface remain to be elucidated. Finally, the resulting nucleoside base, e.g., adenosine, is then reabsorbed via nucleoside transporters (Hosoya et al., 1998). Although more experiments are necessary to confirm this hypothesis, nucleotides, dinucleotides, and their associated receptors and proteins are important to ocular surface signaling.

In summary, we present here evidence that Ap₄A and Ap₅A are present in rabbit tears and that exogenous application of diadenosine polyphosphates can facilitate tear secretion. This effect seems to be mediated by P2Y₂ receptors, according to the pharmacology obtained in rabbits when the effect of diadenosine polyphosphates is compared with expressed P2Y receptors. These findings suggest that these important signaling molecules may be involved in the regulation of basal tear secretion. Moreover, stimulation of this alternate, nonadrenergic, noncholinergic secretion mechanism may be useful for the treatment of dry eye syndrome.