PGE2, Ca2+, and cAMP mediate ATP activation of Clminus channels in pigmented ciliary epithelial cells

Johannes C. Fleischhauer1, Claire H. Mitchell1, Kim Peterson-Yantorno1, Miguel Coca-Prados2, and Mortimer M. Civan1,3

Departments of 1 Physiology and 3 Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; and 2 Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06510


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Purines regulate intraocular pressure. Adenosine activates Cl- channels of nonpigmented ciliary epithelial cells facing the aqueous humor, enhancing secretion. Tamoxifen and ATP synergistically activate Cl- channels of pigmented ciliary epithelial (PE) cells facing the stroma, potentially reducing net secretion. The actions of nucleotides alone on Cl- channel activity of bovine PE cells were studied by electronic cell sorting, patch clamping, and luciferin/luciferase ATP assay. Cl- channels were activated by ATP > UTP, ADP, and UDP, but not by 2-methylthio-ATP, all at 100 µM. UTP triggered ATP release. The second messengers Ca2+, prostaglandin (PG)E2, and cAMP activated Cl- channels without enhancing effects of 100 µM ATP. Buffering intracellular Ca2+ activity with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid or blocking PGE2 formation with indomethacin inhibited ATP-triggered channel activation. The Rp stereoisomer of 8-bromoadenosine 3',5'-cyclic monophosphothioate inhibited protein kinase A activity but mimicked 8-bromoadenosine 3',5'-cyclic monophosphate. We conclude that nucleotides can act at >1 P2Y receptor to trigger a sequential cascade involving Ca2+, PGE2, and cAMP. cAMP acts directly on Cl- channels of PE cells, increasing stromal release and potentially reducing net aqueous humor formation and intraocular pressure.

aqueous humor formation; P2Y receptors; adenosine 3',5'-cyclic monophosphate; prostaglandins; calcium


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AQUEOUS HUMOR SECRETION is a determinant of intraocular pressure, so that reducing the secretory rate is a major strategy in treating glaucomatous patients. The aqueous humor also delivers substrates, oxygen, and the antioxidant ascorbate to the avascular cornea, lens, and trabecular meshwork, removes metabolic waste products, and facilitates immune responses (20). The bilayered ciliary epithelium forms aqueous humor by transferring solute (and, secondarily, water) from the stroma of the ciliary processes to the contralateral posterior chamber of the eye. Solute is taken up from the stroma by the pigmented ciliary epithelial (PE) layer, passes through gap junctions to the nonpigmented ciliary epithelial (NPE) layer, and is then released into the aqueous humor.

Several lines of evidence suggest that Cl- channel activity limits the rate of secretion (7). Activating Cl- channels of the NPE cells is expected to increase secretion, whereas activating Cl- channels of the PE cells should favor reabsorption, thereby reducing net secretion. Purines may regulate activity of Cl- channels on both sides of the tissue. At the aqueous surface of the epithelium, A3-subtype adenosine agonists activate Cl- channels (5, 25). At the stromal surface, ATP and the estrogen receptor antagonist tamoxifen synergistically activate Cl- channels of bovine PE cells (26). The aim of the present study was to examine the effect of ATP itself on these cells.

The strategy of the study was to focus on ATP-triggered transfer of fluid out of PE cells with volumetric measurements and to verify the role of Cl- channels by patch clamping. These approaches were supplemented by luciferin/luciferase assay of ATP release in addressing the identity of the nucleotide receptor(s) involved.


    METHODS
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Cellular model: Bovine PE cells. We have extended our studies of an immortalized PE cell line developed by M. Coca-Prados from a primary culture of bovine PE and characterized by several investigators (10, 26, 34). Cells were grown in Dulbecco's modified Eagle's medium (DMEM; no. 11965-084, GIBCO-BRL, Grand Island, NY) with 10% fetal bovine serum (SH30071.03, HyClone Laboratories, Logan, UT) and 50 µg/ml gentamicin (no. 15750-060, GIBCO-BRL), at 37°C in 5% CO2 (36). The medium had an osmolality of 328 mosmol/kgH2O. Cells were passaged every 6-7 days.

Volumetric measurements and analysis. After reaching confluence, cells from a T-75 flask were harvested by trypsinization within 3-10 days after passage (8). A 0.5-ml aliquot of the cell suspension in DMEM was added to 20 ml of each test solution. Parallel aliquots of cells were studied on the same day. One or two aliquots served as control, and the others were exposed to different experimental conditions at the time of suspension. The same amount of solvent vehicle was always added to the control and experimental aliquots. The sequence of studying the suspensions was varied to preclude systematic time-dependent artifacts. Cell volumes of isosmotic suspensions were measured with a Coulter counter (model ZBI-Channelyzer II) with a 100-µm aperture. As previously described, the cell volume (vc) of the suspension was taken as the peak of the distribution function. The time course of cell shrinkage was fit to a monoexponential by nonlinear least-squares analysis, and the probability of the null hypothesis (that any 2 sets of observations were derived from the same population) was obtained from the F distribution (10).

Whole cell patch-clamp recording. Micropipettes were pulled from Corning no. 7052 glass, coated with Sylgard, and fire polished. The resistances of the micropipettes in the bath usually ranged from ~1.0 to 2.6 MOmega ; successful seals displayed gigaohm resistances. Unless otherwise stated, currents were recorded in the ruptured-patch mode. After rupture of the membrane patch, the series resistance was measured to be only 8.1 ± 0.7 MOmega and was therefore not usually compensated; whole cell capacitance was 10.4 ± 1.2 pS. The baseline whole cell currents were 83 ± 17 pA/pF. In a subset of experiments (n = 8), we measured whole cell currents in the perforated-patch mode. In those experiments, we back-filled the micropipettes with solution containing amphotericin (168 µg/ml) and filled the tips with amphotericin-free solution (1). The applied voltages were not corrected for the small junction potentials (approximately -2.8 mV; Ref. 6) arising from the present micropipette filling and external solutions.

Data were acquired at 2-5 kHz with either an Axopatch 1D (Axon Instruments, Foster City, CA) or a List L/M-EPC7 (Darmstadt, Germany) patch-clamp amplifier and filtered at 500 Hz. The membrane potential was held at -40 mV and stepped to test voltages from -100 to +80 mV in 20-mV increments at 1-s intervals. Each step lasted 300 ms with intervening periods of 1.7 s at the holding potential. Stimulatory responses were measured at peak levels and inhibitory responses at the nadirs.

ATP measurements. Bovine PE cells were grown for 4-48 h to confluence on glass coverslips. Cells were washed in control solution and mounted on an inverted microscope, and bath ATP levels were measured continuously by including 2 mg/ml of luciferin/luciferase assay mixture (33). After background levels were recorded, a solution containing luciferin/luciferase assay mixture and either control solution or UTP was carefully added to the cells. ATP released from cells into the extracellular bath reacted with the luciferase and led to the production of a photon. Light produced was captured with a ×20 objective, filtered at 520 nm, measured with a photomultiplier tube, and recorded on-line using the Felix software suite (Photon Technologies International, Princeton, NJ). The luminescence values were converted to concentrations of ATP using a standard curve; UTP did not alter luminescence in the presence of the luciferin/luciferase assay mixture alone. The control solution contained (in mM) 105 NaCl, 4.5 KCl, 2.8 Na-HEPES, 7.2 HEPES acid, 1.3 CaCl2, 0.5 MgCl2, 5 glucose, and 75 mannitol. The pH was adjusted to 7.4 with NaOH, and the solution had an osmolality of 304-312 mosmol/kgH2O.

Chemicals. All chemicals were reagent grade. Gramicidin D, ionomycin, tamoxifen, ATP, UTP, ADP, UDP, GTP, 2-methylthio-ATP, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), dibutyryl cAMP (DBcAMP), and indomethacin were purchased from Sigma (St. Louis, MO); 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), and N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) from Molecular Probes (Eugene, OR); and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) from Biomol Research Laboratories, (Plymouth Meeting, PA). 9-Phenylanthranilic acid (DPC) was obtained from Fluka (Ronkonkoma, NY), and the inhibitory (Rp) and stimulatory (Sp) diastereoisomers of 8-bromoadenosine 3',5'-cyclic monophosphothioate (8-Br-cAMPS) were from Biolog (La Jolla, CA).


    RESULTS
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
REFERENCES

Effect of nucleotides on PE cell volume. ATP triggered concentration-dependent shrinkage of PE cells at 10 and 100 µM but not at 3 µM (Fig. 1A). Test solutions in Fig. 1A contained the cation ionophore gramicidin (5 µM) to incorporate an exit port for K+ in the plasma membrane. Under these conditions, the observed shrinkage reflected activation of a Cl- release pathway (8). ATP also produced comparable shrinkage without gramicidin (Fig. 1B). As previously reported (26), ATP did not uniformly trigger shrinkage; no response was noted in ~20% of the present series of volumetric measurements. Averaging the results of 11 series of experiments (reflecting 43 experiments), ATP produced a magnitude of shrinkage (Delta vinfinity ) of 4.2 ± 0.3% with a time constant (tau ) of 5.2 ± 0.6 min. In controls (14 series, 50 experiments), Delta vinfinity  = 1.3 ± 0.3% and tau  = 9.5 ± 2.5 min (in 10 series of controls with significant shrinkage). In agreement with our previous study (26), tamoxifen uniformly enhanced the response to ATP (n = 10, Fig. 2B). In contrast, adenosine has no significant effect on the volume of these PE cells with or without tamoxifen (26).


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Fig. 1.   Effects of ATP on cell volume. A: ATP triggered a concentration-dependent shrinkage (n = 4). In this and subsequent figures presenting volumetric data, solid curves are least-square fits to monoexponentials. Otherwise, data points are connected by dotted lines. Fit values for steady-state magnitude (Delta vinfinity ) and time constant (tau ) of shrinkage: control (2.4 ± 0.2%, 8.6 ± 2.6 min), 3 µM ATP (2.6 ± 0.2%, 12.7 ± 2.4 min), 10 µM ATP (2.7 ± 0.3%, 6.8 ± 2.1 min; P < 0.01), 100 µM ATP (5.0 ± 0.2%, 8.0 ± 1.0 min; P < 0.01). Probabilities of the null hypothesis were obtained from the F distribution. B: 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 9-phenylanthranilic acid (DPC) inhibited ATP-triggered shrinkage. Fit values for Delta vinfinity and tau : control (0.7 ± 0.4%, 14.8 ± 18.8 min), 100 µM ATP (3.1 ± 0.2%, 2.5 ± 0.8 min; P < 0.01 vs. control), 1 mM DPC + 100 µM ATP [not significant (NS); P < 0.01 vs. ATP alone], 100 µM NPPB + 100 µM ATP (1.2 ± 1.0%, 16.2 ± 15.5 min; P < 0.01 vs. ATP alone). Iso, isotonic control; Gram, gramicidin D.



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Fig. 2.   Relative effects of nucleotides on cell volume. A: fit values for Delta vinfinity and tau  for UTP, UDP, and ATP (n = 4): control (1.9 ± 0.6%, 23.6 ± 15.5 min), 100 µM ATP (4.1 ± 0.2%, 4.8 ± 1.1 min; P < 0.01), 100 µM UTP (3.4 ± 1.2%, 31.6 ± 20.2 min; P < 0.01 vs. ATP, P > 0.05 vs. control), 100 µM UDP (2.7 ± 0.3%, 11.0 ± 2.8 min; P < 0.01 vs. both ATP and control). B: fit values for Delta vinfinity and tau  for ADP, tamoxifen, and ATP (n = 4): control (1.5 ± 0.2%, 1.0 ± 1.3 min), 100 µM ADP (2.4 ± 0.5%, 7.3 ± 1.9 min; P < 0.05 vs. control, P < 0.01 vs. ATP), 100 µM ATP (5.6 ± 0.5%, 7.3 ± 1.9 min; P < 0.01), 10 µM tamoxifen + 100 µM ATP (not fit by single exponential, but data points significantly different from control, ATP, and ADP at P = 0.01).

The ATP-triggered shrinkage was inhibited by Cl- channel blockers. When tested in parallel aliquots of cell suspensions, NPPB (100 µM) and DPC (1 mM) were similarly effective in blocking shrinkage (Fig. 1B).

UTP and UDP (Fig. 2A) and ADP (Fig. 2B) also triggered shrinkages. These effects were smaller than the ATP-triggered shrinkage, precluding a definitive ranking of the effects of ADP, UDP, and UTP.

Effect of nucleotides on PE whole cell currents. ATP altered whole cell currents in approximately one-third of the bovine PE cells (see Table 2). Two different electrophysiological effects were noted, sometimes in the same cells (Fig. 3). The stimulatory effect is exemplified by the increase in whole cell currents beginning ~1 min after initiation of perfusion with 10 µM ATP (Fig. 3A). Raising the external ATP concentration to 100 µM then triggered the second characteristic effect, a small inhibition of outward current at +80 and +60 mV within ~40s. The later rate of increase in whole cell currents was not detectably altered by this increase in ATP concentration. Application of the Cl- channel blocker NPPB (100 µM) subsequently inhibited the currents by ~70%. The time courses of ATP-difference currents after step changes in voltage are presented in Fig. 3B, and the current-voltage relationships of the ATP- and NPPB-difference currents are presented in Fig. 3C. Slight inactivation was noted at highly depolarizing potentials (Fig. 3B); the magnitude of depolarization-induced inactivation displayed by Cl- channels of many cells depends on the free intracellular Mg2+ concentration and other unidentified factors (29). The current-voltage relationship was outwardly rectifying (Fig. 3C).


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Fig. 3.   Effects of ATP on whole cell currents. A: time course of currents before, during, and after perfusion with ATP and NPPB initiated at the times indicated by the vertical lines. B: responses of 100 µM ATP-difference currents to step changes in voltage from the holding potential of -40 mV. In Figs. 4, 5, and 8, difference currents were calculated by subtracting means of 3-5 sets of measurements just before change in perfusion from a similar set of records at the peak stimulation or maximal inhibition. C: current-voltage relationships for the absolute baseline () and activated (black-down-triangle ) currents and 100 µM ATP ()- and 100 µM NPPB (black-lozenge )-difference currents.

The reversal potential for the ATP-activated currents (Fig. 3C) was -26.8 ± 1.8 mV. Taking into account a junction potential of approximately -2.8 mV estimated for similar filling and bath solutions (5), the corrected reversal potential was -29.6 mV. When this value and the known anionic concentrations inside and outside the cell (Table 1) are inserted in the Goldman equation, the relative permeability of aspartate/Cl- of the ATP-activated anion channels can be estimated (5) to be ~0.14.

                              
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Table 1.   Compositions of solutions

Whole cell responses to UTP are illustrated in Fig. 4. As shown in Fig. 4A, 10 µM UTP produced small initial inhibitions of outward currents followed by stimulation of both outward and inward currents. The UTP-difference currents displayed the same characteristics observed with ATP-difference currents (Fig. 3): slight inactivation at highly depolarizing potentials (Fig. 4B) and outward rectification with a comparable uncorrected reversal potential (-33.4 ± 1.2 mV; Fig. 4C). As for the ATP experiments, the magnitudes of the responses to UTP are displayed in Fig. 5 and the frequency of the responses is given in Table 2. ADP and UDP triggered similar changes, but the magnitudes were smaller (Fig. 5, Table 2).


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Fig. 4.   Effects of UTP on whole cell currents. The time courses during changes in perfusion (A), the responses of the UTP-difference currents to step changes in voltage (B), and the current-voltage relationships of the baseline (black-down-triangle ), activated (), and difference () currents (C) are presented. Iso Ctrl in A indicates onset of washouts with isotonic control solution.



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Fig. 5.   Relative effects of nucleotides on whole cell currents. The percent activations and percent blocks were calculated as mean ± SE values for the cells displaying responses. Approximately 25-45% of the cells responded to the nucleotides (see text for discussion). Nucleotide concentrations are in µM.


                              
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Table 2.   Number of responses (activation or block) per total number of experiments

To summarize the electrophysiological results obtained with all nucleotides, the means ± SE of stimulatory and inhibitory responses to 10 and 100 µM concentrations are presented in Fig. 5 and the frequency with which these effects were observed is presented in Table 2. No response to ATP was seen after 1 or 3 µM ATP. At 10 and 100 µM concentrations, stimulations were seen in ~25-45% of the cells studied. There was considerable variance in response, but the changes appeared generally larger after ATP than after UTP, ADP, and UDP (Fig. 5). Block was observed in a similar fraction of the cells studied, with UTP producing a larger effect than did the other three nucleotides (Fig. 5). For all nucleotides studied, the nonzero reversal potential, outward rectification, inactivation at highly depolarizing potentials, and sensitivity to NPPB established that the stimulated currents reflected activation of Cl- channels.

Because <50% of the cells responded to nucleotides, we wondered whether dialysis of important components out of the cell could have limited the frequency of response. However, the response rate to ATP and UTP was not significantly enhanced by using the perforated-patch mode of whole cell recording. Stimulatory and inhibitory responses were observed in 5 and 3 of 11 cells, respectively.

Second messenger cascade assayed by shrinkage. ATP increases intracellular Ca2+ activity of bovine PE cells (26). Raising intracellular Ca2+ levels by adding ionomycin triggered PE cell shrinkage similar to that produced by ATP, and the two effects were not additive (Fig. 6A).


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Fig. 6.   Effects of Ca2+, prostaglandin (PG)E2, and cAMP on cell volume. A: fit values for Delta vinfinity and tau  for the responses to raising intracellular Ca2+ with ionomycin (n = 3): control (NS), 2 µM ionomycin (4.6 ± 0.4%, 3.3 ± 1.1 min; P < 0.01), 100 µM ATP (4.9 ± 0.4%, 5.3 ± 1.3 min; P < 0.01), 2 µM ionomycin + 100 µM ATP (4.5 ± 0.5%, 3.5 ± 1.3 min; P < 0.01). The data obtained with ionomycin, ATP, or both together were not significantly different from one another. B: fit values for Delta vinfinity and tau  for the responses to PGE2 (n = 3): Control (1.6 ± 0.2%, 6.7 ± 2.9 min), 100 µM ATP (4.5 ± 0.2%, 5.2 ± 0.8 min; P < 0.01), 10 µM PGE2 (4.6 ± 0.4%, 6.1 ± 1.7 min; P < 0.01), 10 µM PGE2 + 100 µM ATP (6.5 ± 0.8%, 8.3 ± 2.6 min; P < 0.01 vs. control, P > 0.05 vs. PGE2 alone). C: 100 µM ATP and 500 µM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) produced comparable degrees of shrinkage (n = 4). Fit values for Delta vinfinity and tau : control (1.17 ± 0.09%, 1.4 ± 0.7 min), 8-BrcAMP (3.8 ± 0.4%, 4.0 ± 1.6 min; P < 0.05), 100 µM ATP (4.37 ± 0.07%, 5.0 ± 0.3 min; P < 0.01), 8-BrcAMP + ATP (4.1 ± 0.1%, 2.4 ± 0.3 min; P < 0.01). Adding 8-BrcAMP clearly increased the speed but not the magnitude of the shrinkage response.

An increase in intracellular Ca2+ activity can in turn activate phospholipase A2 (4), enhancing formation of prostaglandin (PG)E2. Indeed, 10 µM PGE2 produced effects similar to those of Ca2+, replicating the actions of 100 µM ATP. Application of ATP together with PGE2 did not significantly enhance the action of PGE2 alone (Fig. 6B). The effects of ionomycin and PGE2 were also not additive (Fig. 7C).


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Fig. 7.   Effects of buffering intracellular Ca2+ and blocking PGE2 on ATP-triggered shrinkage and nonadditivity of cAMP and PGE2 in triggering shrinkage. A: buffering intracellular Ca2+ with acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), but not N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) blocked ATP-induced shrinkage (n = 3). Fit values for Delta vinfinity and tau : control (3.8 ± 1.8%, 20.6 ± 16.5 min), 100 µM ATP (4.8 ± 0.1%, 1.6 ± 0.2 min; P < 0.01), 20 µM BAPTA-AM + 100 µM ATP (1.9 ± 0.3%, 6.4 ± 2.5 min; P > 0.05 vs. control, P < 0.01 vs. ATP), 20 µM TPEN + 100 µM ATP (4.3 ± 1.3%, 1.3 ± 0.4 min; P < 0.01 vs. control and BAPTA, P > 0.05 vs. ATP alone). B: cyclooxygenase inhibition with indomethacin (Indo) also blocked ATP-triggered shrinkage (n = 5). Fit values for Delta vinfinity and tau : control (1.9 ± 0.2%, 8.8 ± 1.9 min), 100 µM ATP (3.8 ± 0.6%, 7.1 ± 3.3 min; P < 0.01), 1 µM Indo + 100 µM ATP (NS, P < 0.05 vs. ATP), 100 µM Indo + 100 µM ATP (2.1 ± 0.3%, 6.2 ± 2.6 min; P < 0.01 vs. ATP, P > 0.05 vs. both control and 1 µM Indo + ATP). C: adding cAMP did not enhance PGE2-triggered shrinkage (n = 3). Fit values for Delta vinfinity and tau : control (NS), 10 µM PGE2 (4.3 ± 0.5%, 4.6 ± 1.7 min; P < 0.01), 0.5 mM cAMP (3.9 ± 0.3%, 4.4 ± 1.2 min; P < 0.01), cAMP + PGE2 (3.6 ± 0.2%, 2.6 ± 0.7 min; P < 0.01). PGE2, cAMP, and both agents together triggered similar shrinkages (P > 0.05).

Production and release of PGE2 would in turn be expected to trigger cAMP formation by occupancy of EP2 receptors known to be functionally expressed in bovine PE cells (2), so we tested whether cAMP could replicate the responses to ATP. 8-BrcAMP (500 µM) and 100 µM ATP produced similar degrees of shrinkage (Fig. 6C).

The foregoing data suggested that ATP might activate Cl- channels, and thereby shrinkage, by stimulating increases in Ca2+, PGE2, and cAMP, but it was unclear whether these second messengers were acting in parallel or in tandem, as recently found in Madin-Darby canine kidney (MDCK) cells (30). We addressed this issue by attempting to block increases 1) in Ca2+ (with the Ca2+ buffer BAPTA), 2) in PGE2 (with the cyclooxygenase inhibitor indomethacin), and 3) in cAMP-activated protein kinase activity (with the inhibitory Rp stereoisomer of 8-BrcAMPS).

Preincubation with BAPTA-AM for 1 h to buffer intracellular Ca2+ (18) exerted no direct effect on cell volume but completely abolished ATP-triggered shrinkage (Fig. 7A). In contrast, similar treatment with TPEN, a chelator of heavy metals other than Ca2+ and Mg2+, did not alter the subsequent response to ATP (Fig. 7A). Even in the presence of BAPTA-AM, both 10 µM PGE2 and 500 µM 8-BrcAMP triggered shrinkage (data not shown; n = 4; P < 0.01).

We then examined whether blocking the cyclooxygenase pathway of arachidonic acid metabolism would also affect the response to ATP. As illustrated by Fig. 7B, both 1 and 100 µM indomethacin inhibited the ATP-triggered shrinkage. The indomethacin was not acting simply as a channel blocker. Suspending cells in solution containing 10 µM PGE2 together with 100 µM indomethacin and 100 µM ATP overcame the indomethacin inhibition (data not shown; n = 5; P < 0.01 compared with parallel aliquots suspended in indomethacin and ATP alone).

In our third approach, we focused on cAMP in trying to interrupt the signaling pathways initiated by ATP. In a preliminary test, we found that the specific form of cAMP used to activate shrinkage was not critical. Effects similar to those elicited by 8-BrcAMP, albeit faster, were triggered by the more permeable analog DBcAMP (500 µM) (Fig. 8A). Also, simultaneous addition of the two analogs produced the same response as did DBcAMP alone (Fig. 8A), so that 500 µM is maximally effective. Because cAMP commonly modifies channel activity through protein kinase A (PKA), we applied the inhibitory diastereoisomer (Rp) of 8-BrcAMPS. Unexpectedly, Rp-8-BrcAMPS produced cell shrinkage comparable to that triggered by ATP, and adding Rp-8-BrcAMPS and ATP together had the same effect as adding ATP alone (Fig. 8B). Parallel additions of the inhibitory diastereoisomer, the stimulatory isomer Sp-8-BrcAMPS, and 8-BrcAMPS in the same series of experiments elicited similar shrinkages (Fig. 8C).


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Fig. 8.   Effects of cAMP on cell volume. A: 8-BrcAMP and dibutyryl cAMP (DBcAMP) triggered shrinkages of the same magnitude, with the more permeable dibutyryl form triggering a faster response (n = 4). The 500 µM concentration was evidently saturating, because no additive effect was observed by adding the 2 forms of cAMP. Fit values for Delta vinfinity and tau : Control (NS), 500 µM 8-BrcAMP (4.2 ± 0.4%, 12.2 ± 0.7 min; P < 0.01), 500 µM DBcAMP (4.3 ± 0.3%, 2.2 ± 0.7 min; P < 0.01), 8-BrcAMP + DBcAMP (4.3 ± 0.3%, 4.3 ± 1.2 min; P < 0.05 vs. control, P > 0.05 vs. DBcAMP). B: the Rp diastereoisomer of adenosine 3',5'-cyclic monophosphothioate (cAMPS) inhibits PKA activity but it stimulated shrinkage (n = 4). Fit values for Delta vinfinity and tau : control (NS), 100 µM ATP (2.9 ± 0.4%, 7.2 ± 2.7 min; P < 0.01), 100 µM Rp (1.9 ± 0.1%, 1.3 ± 0.6 min; P < 0.01), Rp + ATP (3.0 ± 0.6%, 12.8 ± 5.5 min; P < 0.01). The addition of Rp-cAMPS reduced the speed and slightly increased the magnitude of the shrinkage response to ATP. C: 8-BrcAMP, the inhibitory diastereoisomer Rp, and the stimulatory diastereoisomer Sp-cAMPS produced similar degrees of cell shrinkage (n = 6). Fit values for Delta vinfinity and tau : control (1.4 ± 0.2%, 5.3 ± 2.0 min), 500 µM 8-BrcAMP (4.0 ± 0.4%, 7.1 ± 2.0 min; P < 0.01), 100 µM Rp (3.2 ± 0.2%, 3.2 ± 0.7 min; P < 0.01), 100 µM Sp (4.4 ± 0.4%, 6.0 ± 1.8 min; P < 0.01).

Second messenger cascade assayed by whole cell currents. Perfusion with 10 µM PGE2 activated Cl- currents in five of five cells by 88 ± 27% (+80 mV; P < 0.01). In the experiment shown in Fig. 9A, perfusion with either 1 mM DPC or 100 µM NPPB reversibly inhibited the activated currents and isotonic washout was associated with decay of the currents. The time courses of the PGE2-difference currents after step changes in voltage are displayed in Fig. 9B, and the current-voltage relationships are presented in Fig. 9C. With 100 µM indomethacin present to block PGE2 production, 100 µM ATP produced no stimulatory response (n = 3), and, conversely in the presence of 100 µM ATP, indomethacin reduced current by 31 ± 8% (+80 mV; n = 4; P < 0.05); one-half of these cells displayed a stimulatory response to the ATP pretreatment.


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Fig. 9.   Effects of PGE2 on whole cell currents. The time courses during changes in perfusion (A), the responses of the PGE2-difference currents to step changes in voltage (B), and the current-voltage relationships of the baseline (), PGE2-difference (black-down-triangle ), NPPB-difference (black-lozenge ), and DPC-difference () currents (C) are presented.

The effects of 8-BrcAMP on Cl- channels were also examined with whole cell patch clamping. In the experiment shown in Fig. 10A, 100 µM 8-BrcAMP increased outward and inward currents. Raising the concentration to 500 µM further stimulated the currents, producing a cumulative stimulation of ~80% at +80 mV. NPPB subsequently inhibited the activated currents reversibly by >90%. The time courses of the difference currents after step changes in voltage and the current-voltage relationships are displayed in Fig. 10, B and C, respectively. At concentrations of 100 and 500 µM, 8-BrcAMP stimulated currents in four of nine experiments without exerting any blocking effect (Fig. 5).


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Fig. 10.   Effects of 8-BrcAMP on whole cell currents. The time courses of the absolute currents (A), the responses of the 8-BrcAMP-difference currents to step changes in voltage (B), and the current-voltage relationships of the baseline (, bottom) and activated (, top) currents and the 8-BrcAMP (black-down-triangle )- and NPPB ()-difference currents (C) are presented.

Similar to the ATP- and UTP-triggered responses, the PGE2 (Fig. 9B)- and 8-BrcAMP (Fig. 10B)-difference currents displayed slight inactivation at highly depolarizing potentials (Fig. 8B) and outward rectification (Figs. 9C, 10C) with similar uncorrected reversal potentials (-31.2 ± 3.3 mV in Fig. 9C and -32.7 ± 2.0 mV in Fig. 10C).

P2 receptors. If a single P2 receptor modulated Cl- channel activity in PE cells, the data of Figs. 2 and 5 might point to P2Y11 (see DISCUSSION). Because the bovine sequence for this receptor is unknown, we conducted a functional test for its presence. The EC50 values of 2-methylthio-ATP at turkey P2Y1 and human P2Y11 receptors are 6 nM and 50 µM, respectively (17). However, neither 100 nM nor 100 µM concentrations of 2-methylthio-ATP triggered the shrinkage produced by 100 µM ATP (n = 4; data not shown).

ATP release by PE cells. Multiple complexities limit the identification of P2 receptors affecting cell functions (see DISCUSSION). One such complexity is potential UTP-triggered ATP release, which has been reported in other cells (9, 23, 30). This possibility was examined by perfusing PE cells with 100 µM UTP. As illustrated by Fig. 11, the mechanical perturbation associated with solution change triggered a transient release in ATP even in the control solution. However, UTP triggered a sustained release of ATP. At the peak, 7 min after solution change, ATP levels in control solution were 41.1 ± 9.4 nM whereas those in UTP were 206.8 ± 73.5 nM (P < 0.05; n = 10). The subsequent decline in detected external ATP concentration likely reflects the activity of ecto-ATPase/apyrase and the utilization of substrate (23, 24).


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Fig. 11.   UTP triggers release of ATP. After ~4 min in control solution, the bath was changed to a control solution containing luciferin/luciferase. After a further ~10 min, the solution was replaced with UTP containing luciferin/luciferase, which triggered a clear increase in extracellular ATP concentration. The vertical lines at ~5 and ~15 min represent room light reaching the chamber during solution changes. Solid data lines represent mean responses, and dotted lines show associated SE (n = 10).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Initial observation. External nucleotides activated Cl- channels and triggered release of intracellular solute and water from bovine PE cells. On average, ATP produced larger stimulations of Cl- currents than did UTP, ADP, and UDP at the same concentration (Fig. 5). However, the observation that more than half the cells did not respond and the ranges in magnitude of the responses made it difficult to rank the nucleotide responses from the patch-clamp data. In this respect, electronic cell sorting provided a more feasible approach, directly measuring the parameter of central interest (the transfer of fluid out of the cells) and sampling 50,000 cells or more for each data point. By this measure and at a concentration of 100 µM, the ranking is ATP > UTP, ADP, UDP (Fig. 2).

To place the nonuniformity of response to ATP in perspective, we note that previous investigators reported a nonuniform expression for other channels in studies of PE cells. For example, only ~15% of fresh and cultured bovine PE cells exhibit T-type Ca2+ channels (16) and 22% of rabbit PE cells display L-type Ca2+ channels (13). Fain and Farahbakhsh (12) found voltage-gated Na+ channels in ~25% of the primary cultures of rabbit PE cells they studied. Stelling and Jacob (32) reported that carbachol increased K+ conductances in only ~30% and Cl- conductance in 49% of the freshly dissected bovine PE cells they studied. Mitchell et al. (27) also found that GTPgamma S activated two-thirds of large-conductance Cl- channels in patches taken from freshly dissected bovine PE cells ultimately shown to possess such a channel. Given the syncytial nature of the bilayered ciliary epithelium (7), activation of the Cl- channels in 25-45% of the PE cells should provide a physiologically significant pathway for release of solute to the stroma.

Nucleotide receptors. Identification of functionally important receptor(s) is generally even more complex for P2 than for P1 (adenosine) receptors because (11, 21) 1) specific agonists and antagonists are not available; 2) the ectoenzymes apyrase, ecto-ATPase, ecto-ADPase, 5'-nucleotidase, and ectonucleoside diphosphokinase not only metabolize adenine nucleotides to adenosine but also interconvert purine and pyrimidine nucleotides; 3) cells frequently possess multiple P2 and P1 receptors, which can exert opposing effects; and 4) the final functional effects can be highly dependent on the specific cell studied because of synergistic interactions of the second messenger cascades. An additional caveat is illustrated by Fig. 11. Consistent with observations in other cells (9, 23, 30), application of one triphosphate nucleotide can trigger release of another nucleotide from intracellular stores. The concentrations of ATP measured directly in this study, although probably an underestimate, should be sufficient to stimulate the P2Y2 receptor with an EC50 of 200 nM (22).

Despite these caveats, two general conclusions can be drawn from the data. First, the dominant functional receptors must be P2Y metabolotropic, rather than P2X ionotropic receptors. Otherwise, ATP activation of P2X cation-nonselective channels would have triggered influx of cation, leading to swelling of the PE cells, contrary to observation. Second, it is unlikely that the nucleotide-triggered shrinkage was mediated by occupancy of a single population of P2Y receptors. Of the six cloned functional P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y12; Refs. 11 and 14), only P2Y11 might conform to the nucleotide ranking displayed by Fig. 2. Our inability to detect a response to the P2Y11 agonist 2-methylthio-ATP at 100 µM suggests that this receptor is not playing a dominant role in mediating the responses to ATP. We conclude that the transport effects of the nucleotides likely reflect occupancy of multiple P2 receptors of the PE cells. This conclusion is consistent with the reported detection of at least two different P2Y receptors in bovine PE cells (31, 34).

Second messengers. The present data suggest a plausible signaling cascade. We have found that elevation of intracellular Ca2+ activity and separate application of PGE2 and 8-BrcAMP all mimic ATP in reducing cell volume, and the effects of these agents are not additive with those of ATP. In principle, all three second messengers could act in parallel to mediate the ATP-triggered activation of Cl- channels (Fig. 12A). Alternatively, ATP could trigger a cascade involving the sequential activation of the three second messengers (Fig. 12B). More complex pathways involving both parallel and series activations are also possible.


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Fig. 12.   Possible pathways of intracellular signaling mediating ATP-triggered Cl- channel (gCl-) activation. The data indicate that Ca2+, PGE2, and cAMP participate in mediating activation. In a purely parallel model (A), inhibition of cyclooxgenase with Indo should only partly inhibit activation, leaving the cAMP- and Ca2+-mediated paths operative. In a purely series model (B), block of PGE2 production should completely inhibit ATP-triggered Cl--channel activation, consistent with observation. Combinations of the 2 models may also account for the observations.

The current observations indicate that the purely parallel model of Fig. 10A cannot be correct. If ATP were to act independently through Ca2+, PGE2, and cAMP, blocking either the rise in intracellular Ca2+ or the formation of PGE2 should only partially inhibit the transport effects of ATP. This prediction is contrary to the observation that ATP-triggered shrinkage was completely blocked by either buffering intracellular Ca2+ levels or preventing PGE2 formation (Fig. 7, A and B).

In contrast, the series hypothesis (Fig. 12B) is consistent not only with the current results but also with a series cascade recently identified in MDCK cells (30). ATP-triggered elevation in cell Ca2+ activates phospholipase A2, stimulating cyclooxygenase-catalyzed PG synthesis and release. PGE2 occupancy of EP2 receptors is known to stimulate adenylyl cyclase-mediated cAMP production specifically by bovine PE cells (2). In turn, cAMP activates Cl- channels (Fig. 10), permitting release of K+ and Cl- through parallel ionic channels and, secondarily, release of water (Figs. 6C, 7C, 8A, and 8C). The data of Fig. 8, B and C, obtained with the PKA-inhibitory analog of 8-BrcAMP, Rp-8-BrcAMPS, suggest that cAMP acts directly on the PE Cl- channel, not through activation of PKA. This concept is consistent with reports that Rp-cAMPS mimics cAMP-triggered activation of hyperpolarization-activated currents (3, 15) and Rp-cGMPS activates the photoreceptor (but not the olfactory) cyclic nucleotide-gated channel (19).

Potential physiological implications. The Cl- channels of PE and NPE cells differ in their unitary conductances (27, 37) and pharmacological profiles (28, 35). In principle, activation of the NPE Cl- channels facing the aqueous humor is expected to increase net secretion, whereas activation of PE channels facing the stroma is expected to reduce net secretion. Release of ATP from both NPE and PE cells (24) provides the basis for autocrine regulation of secretion. Ectoenzyme metabolism of ATP provides a source of adenosine, which activates NPE Cl- channels at ~3 µM concentration (5, 25). The results of the present work demonstrate that ATP itself activates PE Cl- channels at ~10 µM concentration (Figs. 1 and 3). Which effect predominates should depend on local purine concentrations, on ectoenzyme activities and receptor densities at both surfaces, and possibly on activities of additional modulators. In particular, occupancy of plasma membrane estrogen receptors is thought to trigger synergistic enhancement of ATP activation of PE cell Cl- channels (35). The basis for the synergism between the estrogen-triggered cascade and the Ca2+-PGE2-cAMP cascade triggered by ATP remains to be determined.


    ACKNOWLEDGEMENTS

We thank Dr. Martin Pring for suggestions concerning statistical analysis of the data and Dr. Jeffrey W. Karpen for helpful information.


    FOOTNOTES

This work was supported in part by National Eye Institute Grants EY-12213, EY-08343, and EY-01583 (for core facilities). J. C. Fleischhauer received support from Swiss National Science Foundation Fellowship No. 1037.

Address for reprint requests and other correspondence: M. M. Civan, Dept. of Physiology, Univ. of Pennsylvania, 3700 Hamilton Walk, Philadelphia, PA 19104-6085.

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

Received 26 February 2001; accepted in final form 26 June 2001.


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