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(Investigative Ophthalmology and Visual Science. 2002;43:2317-2325.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

P2 Purinergic Receptor–Coupled Signaling in the Rabbit Ciliary Body Epithelium

Nasser A. Farahbakhsh and Marianne C. Cilluffo

From the Department of Physiological Science, University of California, Los Angeles, California.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To identify and characterize P2 purinergic receptors and their signaling pathways in the epithelial cells of the rabbit ciliary body.

METHODS. Real-time fluorescence ratio imaging of the intact fura-2-loaded nonpigmented ciliary body epithelial (NPE) cells of rabbit were used to record changes in the intracellular free calcium concentration ([Ca2+]i), in response to a number of purinergic agonists and antagonists. The effects of some of these drugs on the inositol phosphate (IP) levels in ciliary processes were also examined.

RESULTS. Adenosine diphosphate (ADP), adenosine triphosphate (ATP), and uridine triphosphate (UTP) dose dependently increased the [Ca2+]i and IP levels. The [Ca2+]i increases induced by ADP and UTP were distinguishable, both kinetically and pharmacologically. The effect of ADP on [Ca2+]i was mimicked by a number of P2Y1-selective agonists, and was blocked by three P2Y1-receptor-specific antagonists. The [Ca2+]i increases elicited by ADP (or its analogs) and UTP were additive.

CONCLUSIONS. Rabbit ciliary body epithelium possesses both P2Y1 and P2Y2 metabotropic purinergic receptor subtypes, which differentially use the IP3/Ca2+ second-messenger pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The presence of adenosine nucleotides in aqueous humor has been known for some time.1 Adenosine triphosphate (ATP) was first reported to be released from the sensory nerve endings in the ciliary body.2 However, a more recent report suggests that ATP may originate in the ciliary body’s epithelial cells as well,3 raising the possibility of an autocrine role for ATP and its metabolites. For example, it has been reported that adenosine is present in the aqueous humor,4 and its level is increased in response to topical application of epinephrine.5

It has been reported that all four subtypes of the P1 purinergic (adenosine) receptor are expressed, to varying degrees, in the mammalian ciliary body epithelium.6 7 8 It has been suggested that these receptors play a role in modulating aqueous humor secretion, and consequently the intraocular pressure (IOP).9 10 11 12 13 However, not much is known about the presence and role of the P2 purinergic receptor subtypes in this tissue. Of six cloned and characterized mammalian metabotropic P2Y receptors,14 15 only the P2Y2 subtype has been reported to be present in human16 and bovine 17 cultured ciliary body epithelial cells.

Recently, it was reported that topical application of ATP or some of its structurally related derivatives (2-methylene thioATP, {alpha}ß-methylene adenosine diphosphate [{alpha}ßmeADP], and ATP{gamma}S) increases IOP in rabbit, whereas others ({alpha}ßmeATP and ß{gamma}-methylene ATP) lower it. The hypotensive effect was inhibited by the P2 antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), but not by the A1 blocker 8-cyclopentyl-1,3-dipropylxanthine (DPCPX).18 Neither the P2 receptor subtypes that mediate these effects, nor their cellular locations are known. In another report, it has been shown that ATP, uridine triphosphate (UTP), adenosine diphosphate (ADP), and uridine diphosphate (UDP) activate a chloride channel in pigmented epithelial cells of the bovine ciliary body, suggesting that this may lead to a decrease in net formation of aqueous humor. It was concluded that more than one P2Y subtype may be involved.19

In the studies reported herein, we sought to determine whether rabbit ciliary body epithelial cells express receptors for purine and pyrimidine nucleotides, and if that is the case, to characterize the signal transduction pathways coupled to these receptors. The results of our experiments suggest that rabbit ciliary body epithelial cells possess at least two types of P2 purinergic receptors linked to phospholipase C (PLC) activation: the metabotropic P2Y1 and P2Y2 subtypes.

Part of this work has been published recently in abstract form.20


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue Isolation and Experimental Setup
Intact ciliary body epithelial processes were isolated from pigmented rabbits by procedures previously described.21 Briefly, rabbits weighing 2 to 3 kg were killed with a lethal dose of pentobarbital sodium. The procedure was in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The eyes were then rapidly enucleated, rinsed in HEPES-buffered Ringer’s solution (formulation described later) and hemisected. Single processes were sectioned from the ciliary body by cutting along the base of the process from the iridial margin to the pars plana. Individual processes were laid on their sides in Ringer’s solution in the center of 35-mm Petri dishes that had been modified by cementing a glass coverslip over a hole drilled into the bottom of the dish. The processes were covered and held down with a 2- to 3-mm2 piece of glass coverslip, to provide mechanical stability. A Plexiglas insert was placed in the chamber to reduce the chamber volume. The process was continually superfused with Ringer’s or a test solution at a rate of 10 mL/min.

Solutions
HEPES-buffered Ringer’s contained (in millimolar) 137 NaCl, 4.3 KCl, 1.7 CaCl2, 0.8 MgCl2, 10 sucrose, 7 glucose, 10 HEPES, 6 NaOH (pH 7.6, 293–298 mOsM). Low-calcium Ringer’s was prepared by substituting 10 mM EGTA for equiosmolar NaCl (extracellular free calcium concentration, [Ca2+]o < 3 nM). Adenosine-3'-phosphate-5'-phosphosulfate (A3P5PS), ADP, adenosine 5'-monophosphate (AMP), adenosine 5'-O-(2-thiodiphosphate) (ADPßS), adenosine 5'-O-(3-thiotriphosphate) (ATP{gamma}S), ATP, DPCPX, ionomycin, 2-methylthioadenosine diphosphate (2-meSADP), 2-methylthioadenosine triphosphate (2-meSATP), {alpha}ßmeADP, {alpha}ßmeATP, N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179), 3'-O-(4-benzoyl)benzoyl ATP (bzATP), PPADS, UDP, UTP, and Ringer’s salts were purchased from Sigma Chemical Co. (St. Louis, MO). All drugs were prepared as concentrated stocks and stored at -20°C.

Fura-2 Loading and Ca2+ Imaging
Ciliary processes were loaded with 10 µM fura 2-acetoxymethyl ester (fura-2/AM; Molecular Probes Inc., Eugene, OR) for 45 to 120 minutes at room temperature and then washed with Ringer’s. [Ca2+]i measurements were made as described previously.21 We used the method of Owen22 to estimate the error in calculating [Ca2+]i. Only the results of experiments in which relative error remained below 15% were used. Data are expressed either as peak [Ca2+]i increase over the resting level immediately before the drug application (peak minus base), in nanomolar, or as a percentage increase over the baseline: 100 · (peak –base)/base. The effects of inhibitors are presented as relative decrease in percentage increase in response to each agonist in the presence and absence of the blocker: 100 · (1 - percentage increase in the presence of inhibitor/percentage increase in the absence of inhibitor). Statistical analysis was made using an unpaired Student’s t-test. P < 0.05 was considered significant.

Inositol Phosphate Measurements
Total inositol phosphates (IPs) were measured from isolated ciliary processes, as described previously,23 with slight modification as follows.

Intact single or double processes were isolated from rabbit eyes, as described earlier for Ca2+ imaging experiments. All the processes from two to four eyes were collected into a 60-mm Petri dish containing balanced salt solution (BSS). The processes were then equally distributed among the wells of four-well culture plates containing BSS. In most experiments, each well contained between 12 and 20 processes. The BSS was removed, replaced with 0.5 mL of NCTC culture medium, and the processes were placed in a tissue culture incubator. After 30 minutes, 0.5 mL of NCTC containing 4 to 6 µCi of myo-3[H]-inositol was added to each well, and the tissues were incubated for 3 hours.

The labeled tissues were then washed and stimulated with the agonists for 3 minutes, after which the IPs were extracted with formic acid. Blockers were added 3 minutes before the agonists. After 1 to 2 hours on ice, the total IPs produced were separated from myo-inositol by ion-exchange chromatography. The counts from the total IP fraction were divided by the sum of the counts from the IP fraction plus the myo-inositol fraction. This technique normalizes the increase in IP formation resulting from different amounts of tissue in each well by comparing the counts in the IP fraction with the total counts present in the cytosolic portion of the tissue. In each experiment, each drug tested was run in either duplicate or triplicate (1 well of a multiwell culture dish per replicate). Data are expressed as the mean ± SE of the number of measurements indicated in the text. Statistical analysis was performed with the unpaired Student’s t-test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We recorded the time course of changes in the [Ca2+]i in the rabbit ciliary body nonpigmented epithelial (NPE) cells, elicited by a number of purine and pyrimidine nucleotides and their analogues. In the 317 ciliary processes used for these experiments, the baseline [Ca2+]i was 62.1 ± 2.4 nM (mean ± SE). Both the amplitude and kinetics of the responses appeared to be agonist dependent. Figure 1A shows a typical time course, during which the increases in the [Ca2+]i in response to UTP, ADP, and ATP (all at 100 µM) were recorded successively. The response to UTP was relatively small and round, usually composed of both fast- and slow-rising phases. It took more than 1 minute for the response to UTP to reach its peak, after which the [Ca2+]i declined very little. The response to ADP however, was biphasic, consisting of a fast-rising phase that reached its peak in less than 30 seconds, followed by a rapid decline to a lower, yet elevated level, within the next 2 to 5 minutes. Kinetics of the ATP-evoked response appeared to be intermediary between the responses to ADP and UTP, having both a significant transient phase and a relatively large sustained component. Table 1 summarizes the kinetic data of the [Ca2+]i increases induced by ADP, UTP, and ATP.



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  		Figure 1. Purine and pyrimidine nucleotides elicited increases in concentration of [Ca2+]i in the rabbit ciliary body nonpigmented epithelial cells. (A) The change in [Ca2+]i, recorded from intact nonpigmented ciliary body epithelial cells loaded with fura-2, is shown as a function of time. The responses to UTP, ADP, and ATP, each applied at a 100-µM concentration, are shown. Solid bars: duration of exposure. (B) The dose–response curves for ADP, UTP, and ATP. For each concentration of the agonist, the difference between the peak response and the prestimulus baseline was calculated and normalized to the baseline level. Each data point represents the average ±SE of 3 to 48 measurements. (C) Relative [Ca2+]i increases in response to 12 nucleotides. All agonists were applied at a 30-µM concentration except {alpha}ßmeATP, which was used at 100 µM. Each bar represents the average ±SE of 3 to 25 measurements.

 

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  		Table 1. The Kinetics of Nucleotide-Induced Responses

 
Concentration Dependence of the ADP-, UTP-, and ATP-Induced Increase in [Ca2+]i
Figure 1B shows the dose–response data for ADP, UTP, and ATP. The dose–response data for UTP was modeled as a first-order reaction with a 50% effective concentration (EC50) at 42.8 ± 1.1 µM, and a Hill coefficient (nH) of 13.0 ± 2.8. However, the dose–response curves of ADP and ATP did not show any inflection point within the concentration range used (1–300 µM). Therefore, no reliable fit to these data could be made with the Michaelis-Menten equation. These results suggest that first, the ADP- and ATP-evoked [Ca2+]i increases may be mediated, at least in part, by a receptor subtype other than the purinergic receptor underlying the response to UTP, and second, overestimation of the EC50 and nH for these agonists is indicative of hydrolysis of ADP, UTP, and ATP by extracellular enzymes.24 Therefore, we investigated the responses elicited by nine additional ligands, some of which are considered non- or less-hydrolyzable.25 The data in Figure 1C compares the relative [Ca2+]i increases recorded in response to these 12 agonists. All agonists were applied at a 30-µM concentration, except {alpha}ßmeATP, which was tested at 100 µM. These agonists included those considered specific for the P2Y1 subtype (ADPßS, 2-meSADP, 2-meSATP, and ADP), the P2Y2 subtype (UTP and ATP), the P2Y4 subtype (UTP), the P2Y6 subtype (UDP), and the P2Y11 subtype (ATP{gamma}S).26 27 Agonists specific for ionotropic P2X receptors ({alpha}ßmeADP and {alpha}ßmeATP), and the P2X7-specific agonist bzATP28 were also tested.

These agonists were divided into three groups based on the size of the [Ca2+]i increases they elicited: those inducing relatively large responses (400%–600% [Ca2+]i increase)—ADP, ADPßS, and ATP; those producing moderate responses (100%–200% increase)—2-meSADP, 2-meSATP, ATP{gamma}S, AMP and UTP; and those for which the increase was less than 50%—{alpha}ßmeATP, {alpha}ßmeADP, bzATP, and UDP. The size of the [Ca2+]i increases induced by these ligands did not show a receptor-subtype–specific pattern. However, the weak responses to {alpha}ßmeATP, {alpha}ßmeADP, or bzATP, suggests that P2X receptors do not significantly contribute to the ATP-induced [Ca2+]i increase in the rabbit ciliary body NPE cells.

P2Y1 and P2Y2 Receptors in Rabbit Nonpigmented Epithelium
To determine whether both P2Y1 and P2Y2 receptors are present in this tissue, we measured the [Ca2+]i changes in response to ADP and UTP, individually or in combination. Figure 2A shows the result of one such experiment, in which the responses to 100 µM ADP, 100 µM UTP, and 100 µM ADP+100 µM UTP were recorded. ADP and UTP generated responses comparable to those shown in Figure 1A , even though the order of the agonist application was reversed. This indicates that the size and kinetics of these responses are agonist specific and independent of the order in which ADP and UTP were applied. In addition, the 10-minute washout between the drug applications appeared sufficient for minimizing any heterologous desensitization. Therefore, the simultaneous application of ADP and UTP was also preceded by a 10-minute washout. The response to the combination of ADP and UTP was additive. In three experiments, the response to ADP+UTP was 13% ± 27% larger than the sum of the responses to ADP and UTP (P > 0.31).



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  		Figure 2. In the rabbit NPE cells, [Ca2+]i increases induced by P2Y1 and P2Y2 agonists were additive. (A) Responses to ADP, UTP, and ADP+UTP recorded successively from the same tissue. (B) Time course of successive responses to ADPßS, UTP, and ADPßS+UTP, in a different ciliary process. (C) Responses to 2-meSATP, UTP, and 2-meSATP+UTP, recorded from yet another ciliary process. These responses are representative of three recordings in each case. All agonists were applied at a 100-µM concentration, alone or in combination.

 
Two other P2Y1 agonists, 2-meSATP and ADPßS, induced [Ca2+]i changes kinetically similar to the response to ADP. ADPßS (100 µM) induced a [Ca2+]i increase with kinetics similar to that generated by ADP, with a relatively large transient component (555 ± 37 nM, or 1444% ± 524% increase, n = 3). The response to the ADPßS+UTP application was also additive (Fig. 2B) , 15% ± 14% larger than the sum of the [Ca2+]i increases induced by ADPßS and UTP in the same tissues (all applied at 100 µM, P > 0.29, n = 3). 2-meSATP (100 µM) elicited a somewhat lesser [Ca2+]i increase (81 ± 24 nM, or 216% ± 86%, n = 4), which was composed mainly of a transient component (Fig. 2C) . However, 2-meSATP also generated an additive response when it was combined with UTP. The [Ca2+]i increase evoked by 100 µM 2-meSATP+100 µM UTP was 60% ± 46% higher than the sum of the responses to 100 µM 2-meSATP and 100 µM UTP (P > 0.17, n = 3).

The Effect of P2Y1-Selective Antagonists
To further characterize the P2-purinergic receptors present in the rabbit ciliary body epithelial cell, we tested the effects of three P2Y1-selective antagonists on the [Ca2+]i increase induced by purinergic agonists. MRS2179 is the most potent and selective P2Y1-receptor antagonist (IC50 = 330 nM).29 At 10 µM, MRS2179 reduced the [Ca2+]i increase in the rabbit NPE cells in response to 100 µM ADP from 314 ± 67 nM (peak minus baseline) to 19 ± 10 nM (95.6% ± 2.5% reduction, n = 5, Fig. 3A ). After a 10-minute washout, the effect of MRS2179 was partially reversible (104 ± 34 nM). Similarly, 10 µM MRS2179 reduced the [Ca2+]i increase generated by 100 µM ATP from 607 ± 195 to 128 ± 40 nM (76.3% ± 5.3%, n = 5, Fig. 3C ). However, the 100 µM UTP-evoked increase in the [Ca2+]i was inhibited by only 42.9% ± 6.7% (from 105 ± 15 to 59 ± 9 nM, n = 4, Fig. 3B ) by the same concentration of MRS2179. In the presence of 10 µM MRS2179, the [Ca2+]i increases evoked by 100 µM ATP and 100 µM UTP were not significantly different (P > 0.08), suggesting that in the presence of MRS2179, both responses to ATP and UTP were mediated by the P2Y2 receptor.



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  		Figure 3. The effects of the P2Y1-selective antagonist MRS2179 on the nucleotide-induced [Ca2+]i increase in NPE cells. (A) MRS2179 inhibited the ADP-elicited response. The [Ca2+]i increases induced by 100 µM ADP, before, during, and after exposure to 10 µM MRS2179 are shown. The inhibitory effect of MRS2179 was only partly reversible. (B) The UTP-evoked response was partially inhibited by MRS2179. The [Ca2+]i increases induced by 100 µM UTP, before, during, and after exposure to 10 µM MRS2179 are shown. (C) The ATP-evoked response is significantly reduced by MRS2179. The [Ca2+]i increases induced by 100 µM ATP, before, during, and after exposure to 10 µM MRS2179 are shown. A 10-minute washout did not appear sufficient for recovery of UTP- or ATP-induced responses. Each record is representative of three similar experiments.

 
A3P5PS, an antecedent to MRS2179,30 has also been used as a P2Y1-selective antagonist (IC50 {cong} 10 µM).31 32 In the NPE cells, A3P5PS, at 50 µM concentration, reduced the [Ca2+]i increase in response to 100 µM ADP by 70.0% ± 2.3% (from 356 ± 114 to 114 ± 26 nM, n = 3, Fig. 4A ). Similarly, in cells expressing P2Y1 or P2Y2 receptors, PPADS, has been found to selectively inhibit PLC activity in response to the P2Y1-receptor activation.33 34 35 36 In the rabbit NPE cells, PPADS at 25 µM, inhibited the response to ADP (100 µM) by 75.8% ± 8.3% (from 764 ± 187 to 185 ± 101 nM, n = 5, Fig. 4B ). In these experiments, neither the inhibition by A3P5PS nor that of PPADS was reversed after a 10-minute washout (Fig. 4) .



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  		Figure 4. The effects of pretreatment of the NPE cells with the P2Y1-selective antagonists A3P5PS and PPADS on ADP-induced [Ca2+]i increases. (A) A3P5PS significantly inhibited the ADP-elicited response. The [Ca2+]i increases induced by 100 µM ADP (bar ADP), before, during, and after exposure to 50 µM A3P5PS, are shown. Similar results were recorded in three experiments. (B) The ADP-evoked response was also inhibited by PPADS. A representative of five similar experiments shows the [Ca2+]i increases induced by 100 µM ADP, before, during, and after exposure to 25 µM PPADS.

 
P2-Receptor–Mediated Formation of IP
To further establish that the [Ca2+]i changes in the ciliary body NPE cells in response to purine and pyrimidine nucleotides were mediated by metabotropic receptors, we also measured the change in total IP formation induced by ADP, UTP, or ATP in the ciliary body processes (Fig. 5A) . ADP, at 100 µM, increased the counts in the IP fraction from 12.8% ± 2.0% to 17.3% ± 2.4% of the total counts (sum of the IP fraction+ inositol fraction, n = 6), representing a 35.2% increase in the IP formation over the basal level. UTP, at 100 µM, caused a 36.6% increase (from 12.3% ± 1.5% to 16.8% ± 0.5%, n = 6), whereas 100 µM ATP increased IP formation by 41.5% (from 12.3% ± 1.5% to 17.4% ± 1.2%, n = 6).



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  		Figure 5. The effects of MRS2179 on formation of IPs and increases in [Ca2+]i elicited by ADP, UTP, and ATP. (A) ADP, UTP, and ATP significantly increased IP formation over basal levels. The ADP-induced IP formation was inhibited by MRS2179, whereas the response to UTP and ATP were not significantly affected. Total IPs, normalized to the sum of the myo-inositol and IP fractions, were measured after a 3-minute incubation period. The basal (solid) and agonist-induced (hatched) IP formation, in the absence (white bars), and presence (gray bars) of 10 µM MRS2179, are shown. Each bar represents the average ±SE of six to nine measurements. (B) MRS2179 inhibited the [Ca2+]i increase induced by all three nucleotides. The baseline [Ca2+]i level (solid) and agonist-induced peak response (hatched bars), in the absence (open bars) and presence (shaded bars) of 10 µM MRS2179, are shown. Each bar represents the average ±SE of 4 to 48 measurements. Agonists were applied at a 100-µM concentration.

 
Pretreatment of the cells with 10 µM MRS2179 lowered the basal level of IP formation and significantly reduced the effect of ADP, but responses to UTP and ATP remained unchanged (Fig. 5A) . The presence of MRS2179 nearly completely inhibited the ADP-induced increase (from 10.1% ± 1.2% to 10.4% ± 0.5%, n = 6), a 91.5% inhibition. However in the presence of 10 µM MRS2179, the formation of IP in response to 100 µM UTP was enhanced from 10.6% ± 0.7% to 15.4% ± 1.2% (a 45.3% increase, n = 6), and the response to ATP was increased from 10.6% ± 0.7% to 17.8% ± 0.7% (a 67.9% increase, n = 6). In the MRS2179-treated ciliary processes, the difference between the UTP- and ATP-induced increase in formation of IP was not statistically significant (P > 0.05). In addition, the ADP-evoked IP formation was significantly smaller than the total IP formed by either of the other two agonists (P < 0.01 in both cases).

Figure 5B shows a summary of the results of our measurements of the ADP-, UTP- and ATP-induced [Ca2+]i increases for comparison with the IP data. As we described before (Fig. 3) , MRS2179 treatment reduced the response to ADP and ATP to a much larger extent than it reduced the response to UTP. It is noteworthy that in the absence of MRS2179 the [Ca2+]i increases elicited by ADP and ATP (both at 100 µM), were not statistically different (P > 0.05) and were significantly larger than the response to UTP (P < 0.001 in both cases, see also Fig. 1B ). In the presence of 10 µM MRS2179, UTP- and ATP-induced [Ca2+]i increases (such as those in Figures 3B 3C ), were not significantly different (P > 0.08), and both were significantly larger than the response to ADP (P < 0.03 in both cases). The data summarized in Figure 5 show that MRS2179 had different effects on ADP- and UTP-induced responses: In its absence, the formation of IP by the three P2 agonists was not significantly different (Fig. 5A) . MRS2179 significantly reduced ADP-induced IP formation, and ADP- and ATP-generated [Ca2+]i increases, moderately reduced the [Ca2+]i increase elicited by UTP, and left the UTP- and ATP-evoked formation of IP unchanged.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Six mammalian P2Y subtypes have been cloned. Of these, the P2Y1, P2Y11, and P2Y12 receptors are selective for purinergic nucleotides,37 P2Y4 and P2Y6 are pyrimidine nucleotide receptors,38 and P2Y2 is sensitive to both ATP and UTP, but not to ADP or UDP.38 In this study, we used a functional assay—the [Ca2+]i measurement—both to provide evidence for the presence in the rabbit ciliary body NPE cells of at least two P2-purinergic receptors, possibly metabotropic P2Y1 and P2Y2 subtypes, and to characterize the signal transduction pathways linked to these receptors.

P2Y1 Receptor Subtype
Our evidence for the presence of the P2Y1 receptor in the rabbit ciliary body NPE cells follows. First, ADP elicited significant formation of IPs and increases in [Ca2+]i in rabbit NPE cells (Figs. 1 2A 3A 4 5) . The ADP-induced [Ca2+]i increase was dose dependent (Fig. 1B) . ADP is primarily a P2Y1 agonist, with an EC50 of 10 nM to 1 µM in different tissues.35 39 It is approximately 500 times less potent than ATP and UTP in the cloned mouse P2Y2 receptor.40 It is not active at the P2Y4 receptor, and is only a weak and partial agonist at the P2Y6 receptor.38

Second, ADPßS, 2-meSADP, and 2-meSATP also induced significant increases in [Ca2+]i (Figs. 1C 2B 2C) . ADPßS, 2-meSADP, and 2-meSATP are considered P2Y1-specific ligands, with the order of potency, 2-meSADP > ADP > ADPßS > 2-meSATP > ATP.39 Except for the P2Y1, P2Y11, and P2Y12 subtypes, ADPßS, 2-meSADP, and 2-meSATP are not considered to be active agonists in any other cloned P2Y receptors.38 40 In the case of bovine and human cultured ciliary epithelial cells, which have been reported to express the P2Y2 purinergic receptor subtype, 2-meSATP was found to be ineffective in increasing formation of IP16 or in increasing [Ca2+]i.17 However, 2-meSATP is a full agonist for cloned human P2Y11-receptor-mediated formation of IP (EC50, 210 µM).41 It is also a full agonist for P2Y12-mediated adenylyl cyclase inhibition in C6-2B rat glioma cells (IC50, 3.9 nM).26

Third, the kinetics of the response to ADP, ADPßS, or 2-meSADP were unlike that of the UTP-induced [Ca2+]i increase (Table 1 ; Figs 1A 2 ). Similar observations have been made in a number of other tissues that express both P2Y1 and P2Y2 subtypes. For example, in endothelial cells of the bovine pulmonary artery,42 human umbilical vein,43 and rat glioma C6 cells,44 the [Ca2+]i increase elicited by ADP or 2-meSATP is more transient than the response to UTP.

Fourth, the ADP-evoked [Ca2+]i increase in rabbit NPE cells was strongly inhibited by three P2Y1-selective antagonists: MRS2179,29 A3P5PS,30 31 32 and PPADS.33 34 35 36 MRS2179 reduced the ADP-induced [Ca2+]i increase by 95.6% (Fig. 3A) , and IP formation by 91.5% (Fig. 5A) . These results are in agreement with the effects of MRS2179 on the ADP-induced Ca2+ mobilization in rat platelets,45 the 2-meSATP-induced IP formation in turkey erythrocytes,29 and the [Ca2+]i increase in the rat cerebellar astrocytes.46 In the NPE cells, A3P5PS inhibited the response to ADP by 70% (Fig. 4A) , in agreement with its reported effect on human platelets.31 Another putative P2Y1 receptor antagonist, PPADS also inhibited the ADP-induced [Ca2+]i increase in the NPE cells by 75.6% (Fig. 4B) . PPADS was originally considered a P2X-selective inhibitor.47 However, the P2Y1 receptor–coupled IP formation and [Ca2+]i mobilization in a number of tissues have also been reported to be inhibited by PPADS.33 34 35 36

P2Y2 Receptor Subtype
Ciliary body epithelial cells possess P2Y2 (formerly classified as P2U) receptors, as previously reported in simian virus (SV)-40–transformed human NPE and bovine pigmented epithelial cells16 and nontransformed bovine ciliary epithelial cells.17 Our results suggest that a purine-pyrimidine nucleotide receptor with pharmacologic characteristics of the P2Y2 subtype is also present in the intact rabbit ciliary body epithelial cells. The evidence for a P2Y2 receptor in the rabbit NPE cells follows.

First, UTP dose dependently increased the [Ca2+]i (Figs.1 2 3B 5B) . However, the calculated EC50 of the UTP-induced increase in [Ca2+]i (42.8 µM, Fig. 1B ), is much larger than those reported for the cloned P2Y2 receptor (0.1–1.1 µM)38 40 and is close to the EC50 of UTP in cells that express the P2Y6 receptor.38 The P2Y6 is much more sensitive to UDP (EC50, ~0.1 µM) than to UTP. However, in the rabbit ciliary body NPE cells, the UDP-evoked response was less than that induced by UTP (Fig. 1C) , thus making P2Y6’s presence less likely. A possible explanation for the overestimated EC50 for the NPE cells is the ectonucleotidase activity.24 According to this interpretation, the concentration of UTP in the vicinity of the receptor may be much lower than that in the bulk solution.

Second, UTP increased formation of IP in the ciliary process (Fig. 5A) . As metabotropic receptors, all three pyrimidine-sensitive subtypes are coupled through G-proteins, to the enzyme PLC, which, when activated, hydrolyzes phosphatidylinositol 4,5-bisphosphate to form inositol 1,4,5-trisphosphate (IP3) and diacylglycerol.14 48 49

Third, the P2Y1-selective antagonist MRS2179 had a much smaller inhibitory effect on the 100-µM UTP-induced [Ca2+]i increase, than on the response to the same concentration of ADP (Figs. 3A 3B 5B) . Furthermore, MRS2179 did not inhibit formation of IP induced by UTP (Fig. 5A) . The latter observation is similar to that made in 1321N1 human astrocytoma cells expressing the P2Y2 receptor.50

Fourth, in the presence of MRS2179, ATP and UTP were equipotent (Figs. 3B 3C 5) . Equipotency of ATP and UTP is a hallmark of the P2Y2 receptor.40 49 Thus, when the contribution of the P2Y1 receptor was minimized with MRS2179, neither the UTP- or ATP-induced IP formation nor the UTP- or ATP-elicited [Ca2+]i increases in the ciliary body NPE cells, were significantly different (Fig. 5) .

A puzzling aspect of these results is the discrepancy between the observed effects of MRS2179 on the formation of IP (Fig. 5A) , and on the increases in [Ca2+]i (Fig. 5B) generated by ATP and UTP. MRS2179 did not inhibit IP formation induced by these agonists. However, it significantly reduced the [Ca2+]i increase induced by ATP and to a lesser degree lowered the response to UTP (Figs. 3B 3C 5B) . We do not know the reason for these differences. However, we can offer the following possibilities: We have measured the global average of the total IPs in the ciliary body’s epithelial cells, as opposed to the local concentration of IP3 in the vicinity of the IP3 receptors responsible for calcium release. As such, our measurements may have underestimated local variations in the IP3 level.23 Alternatively, MRS2179 may have additional effects in stages beyond formation of IP. Such a possibility has been suggested for the effects of another P2Y1-antagonist, PPADS.51

Other P2Y subtypes
Are there other receptors for purine or pyrimidine nucleotides in the rabbit ciliary body epithelium? Of the remaining four known mammalian P2Y subtypes, P2Y11 is reported to be equally sensitive to bzATP (EC50, 10.5 µM) and ATP{gamma}S (EC50, 13.5 µM).41 Because at 30 µM, bzATP produced a very small [Ca2+]i increase in the NPE cells (Fig. 1C) , we do not believe P2Y11 contributed significantly to the responses we have recorded.

P2Y12 (formerly known as P2YAC, P2Ycyc, or P2TAC) is reported to be negatively coupled to the enzyme adenylyl cyclase through the pertussis-toxin–sensitive G-protein Gi.15 In this regard, P2Y12 resembles a number of receptors in the ciliary body epithelium (e.g., {alpha}2-adrenergic, A1-adenosinergic, and somatostatinergic receptors), whose activation we have previously reported to enhance synergistically the formation of IP and the increase in [Ca2+]i elicited by muscarinic receptor agonists.21 52 53 It is conceivable that if present, P2Y12-receptor–mediated signaling can contribute to the purine-nucleotide–induced [Ca2+]i increase through a synergistic interaction with the PLC/IP3/Ca2+ cascade. Such a synergistic interaction between the P2Y12-coupled pathway and the Gq-linked PLC activity in platelets has been suggested.54 However, P2Y12 has a very limited distribution. It is expressed only in platelets and possibly in brain glial cells.15 There is currently no evidence for its expression in ocular tissues. Furthermore, the P2Y12 receptor is sensitive to block by ARL 6609655 and 2-meSAMP,56 but not to that by MRS217945 or PPADS.57 58 59 Our preliminary test of the effects of the P2Y12 antagonist 2-meSAMP, showed that this agent, at a 50-µM concentration, enhanced the UTP-induced [Ca2+]i increase by 29% ± 6% (n = 3), suggesting that P2Y12 does not contribute to the purinergic responses we have been recording.

The presence of any pyrimidine-selective receptor also remains unclear. UDP was found to induce a small but significant increase in [Ca2+]i (Fig. 1C) . This [Ca2+]i increase could be due to the contamination of commercially supplied UDP with UTP or other nucleotides,38 ectonucleoside diphosphokinase activity,60 or, indeed, UDP itself. Considering these possibilities, we are not able to rule out the presence of either P2Y6 or P2Y4 receptor subtypes.

Finally, we find it difficult to explain the response to AMP (Fig. 1C) . This response, again, may be produced by a contaminant or might be mediated by an as yet unknown receptor.43

Ectonucleotidase Activity
The pharmacological characterization we have presented so far is further complicated by the presence of P1 receptors in this tissue, which can be activated by degradation products of some of these agonists. These products could arise from natural degradation of the nucleotide or from the activity of an ectonucleotidase.61 62 63 This could explain why the [Ca2+]i increase in response to 30 µM ADP, ADPßS, or ATP was significantly larger than the response to the same concentration of 2-meSADP, 2-meSATP, ATP{gamma}S, or UTP (Fig. 1C) . Metabolism of 2-meSADP, 2-meSATP, and UTP generates nucleosides (2-methylthio adenosine, and uridine), which are not A1-adenosinergic agonists (Farahbakhsh NA, Cilluffo MC, unpublished observation, 2001). However, adenosine formed as a result of the hydrolysis of ATP, ATP{gamma}S, ADP, or ADPßS, activates A1 receptors and synergistically increases the response to the P2 agonists. Even though both ADPßS and ATP{gamma}S are metabolized to adenosine, they elicited different [Ca2+]i increases (Fig. 1C) . This discrepancy can be explained on the basis of the difference between their respective rates of hydrolysis by the ectonucleotidase.

Two other observations, the dose–response curves of ADP, ATP, and UTP (Fig. 1B) , and the superadditive nature of some of the responses to the combined application of P2Y1 and P2Y2 agonists (Fig. 2) can be expected if an exofacial nucleotidase with finite capacity24 were present in this tissue. The limited capacity of the ectonucleotidase would generate a nonlinear relationship between local concentration of the nucleotide in the vicinity of the receptor and that in the bulk solution. At low concentration of nucleotides, a large part of the agonist is hydrolyzed, lowering the effective concentration, leading to a relatively small response. At high concentrations of nucleotide(s), the enzyme becomes saturated, allowing the local concentration to approach that of the bulk solution, eliciting maximal response.

In this report, we provide evidence for the presence of the metabotropic purinergic receptor subtypes P2Y1 and P2Y2 in rabbit ciliary body epithelial cells. Several P1 receptor subtypes have been identified in this tissue.6 7 8 10 11 12 52 Furthermore, the presence of ATP, ADP, adenosine, and inosine in the aqueous humor has been reported.1 4 5 These results, along with those reported recently by Fleischhauer et al.19 and Mitchell et al.64 provide strong evidence for the regulatory role of the purinergic system in aqueous humor secretion. The results reported in this study also suggest that ectonucleotidases may have a role in shaping purinergic signaling in the ciliary body epithelium. However, the identity and role of specific ectonucleotidases in the ciliary body epithelial cells remain to be investigated.


   Acknowledgements
 
The authors thank Gordon L. Fain for valuable comments on the manuscript and Urvi Patel and Maya Novgorodsky for helping with the tissue dissections.


   Footnotes
 
Supported by National Eye Institute Grant EY06969.

Submitted for publication February 1, 2002; accepted March 6, 2002.

Commercial relationships policy: N.

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

Corresponding author: Nasser A. Farahbakhsh, Department of Physiological Science, 3817 Life Sciences, University of California, Los Angeles, CA 90095-1606; nasserf{at}lifesci.ucla.edu.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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