Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania
Submitted 26 April 2004 ; accepted in final form 26 August 2004
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ABSTRACT |
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cystic fibrosis transmembrane conductance regulator; recycling endosomes; brefeldin A; autostimulation; retinal detachment
This complex interaction between the RPE and the photoreceptors is dependent on close communication between the two cell types. Various neurochemicals have been implicated in this communication, including dopamine (15, 59), serotonin (39), and epinephrine (13, 24, 42). The purines ATP and adenosine also contribute to RPE-retina interaction. Stimulation of the RPE by adenosine can modify the rate of outer segment phagocytosis (17), while stimulation of apical P2Y2 receptors by ATP can trigger elevation of Ca2+ (40, 55). This elevation likely opens a basolateral Cl conductance and increases the flux of ions and fluid across the RPE (40). Triggering this fluid absorption by stimulating apical P2Y2 receptors with agonist INS37217can reduce the size of fluid blebs in the subretinal space (29, 31), emphasizing the potential of purinergic signaling in the treatment of retinal detachment.
The endogenous source of ATP capable of stimulating these P2 receptors is likely the RPE cells themselves. The existence of a physiological release of ATP from epithelial cells in many different cell types is now widely accepted (6, 48, 50), and RPE cells have been shown to release ATP in response to stimulation (14, 36). However, the mechanisms involved in this release remain to be determined. Previous work has shown that while the Ca2+ ionophore ionomycin is not sufficient to trigger release, the general Cl channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate inhibits it, suggesting anion channel involvement (36). RPE cells have been shown to express the anion channels ClC-2, ClC-3, ClC-5, the pCLCA1 Cl channel regulator, and the cystic fibrosis transmembrane conductance regulator (CFTR; Refs. 4, 26, 63, 64). Of these, ATP release has been associated most consistently with CFTR (7, 41, 52, 60). However, recent evidence suggests that the exocytotic pathway may also be important, because substances that interfere with vesicular transport, such as botulinum toxin, tetanus neurotoxin, and brefeldin A (BFA) can prevent stimulated ATP release (5, 30, 58). Consequently, we investigated the involvement of CFTR in ATP release from RPE cells and examined whether vesicular transport contributed to this release. Preliminary results were presented previously in abstract form (43, 44).
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MATERIAL AND METHODS |
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Solutions. The isotonic solution used as a basis for all solutions was composed of (in mM) 105 NaCl, 5 KCl, 6 HEPES acid, 4 Na-HEPES, 5 NaHCO3, 60 mannitol, 5 glucose, 0.5 MgCl2, and 1.3 CaCl2, pH 7.4. Hypotonic solution was obtained by adding the required amount of deionized distilled water, with "%hypotonicity" defined as the percentage of water added. Glibenclamide, forskolin, 3-isobutyl-1-methylxanthine (IBMX), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and CFTR-172 were mixed as stock solutions in dimethyl sulfoxide. The CFTR-172 was a kind gift from A. S. Verkman (Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA). The cAMP-stimulating mixture contained (in µM) 10 forskolin, 100 IBMX and 500 8-4-chlorophenylthio(cpt)-cAMP. Brefeldin A was dissolved in ethanol. All chemicals were from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.
ATP measurements from cultured cells. ATP release was detected through the chemiluminescent luciferin-luciferase reaction, and the light emitted was recorded using a microplate luminometer (Luminoskan Ascent; Labsystems, Franklin, MA). ARPE-19 cells were grown to confluence in 96-well white assay plates with clear bottoms for 47 days (Corning, Corning, NY). To minimize the ATP release that accompanies solution change (18), growth medium was removed from the cells 1 h before the experiment and replaced with 100 µl of isotonic solution. After 1 h, 50 µl of the isotonic solution was replaced by 50 µl of a solution containing twice the drug concentration or the amount of distilled water required to obtain the final concentration or hypotonicity. To produce a 60% hypotonic solution, 60 µl were removed. The plate was placed in the microplate luminometer 6090 s after addition of the drugs, and 10 µl of the luciferase working solution were injected in each well through the internal injector system. Measurements were taken every 30 s for each well for 30 min with an integration time of 100 ms/measurement. The luciferase solution was prepared in a stock solution from one vial of the ATP assay kit diluted in 450 µl of isotonic solution and 50 µl of distilled water. A working solution was made by diluting 40 µl of the stock solution in 1 ml of isotonic solution. The ATP released was calculated at the different time points indicated in the RESULTS with the use of a standard curve to transform the arbitrary units obtained through the luciferase reaction to an ATP concentration.
All the substances were tested for the effect on the luciferin-luciferase assay as shown in Fig. 1. Only hypotonic solutions and genistein were found to interfere with the assay itself. All of the data obtained using hypotonic solutions were corrected for this enhancement of the chemiluminescence reaction. A standard curve covering three orders of magnitude showed the relationship between luminescence and ATP concentration to be linear over the range of interest. The results from hypotonic experiments were thus scaled down by the factor shown to enhance the response to 10 nM ATP.
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Measurement of intracellular Ca2+.
ARPE-19 cells were plated onto coverslips and grown as described above until confluent. After washing, cells were loaded with 10 µM fura-2 and 0.2% pluronic acid at 37°C for 30 min. The dye was washed off, and the coverslips were mounted on a Nikon inverted microscope (Nikon Instruments, Melville, NY) and visualized with a x20 objective. The field of 20 cells was alternatively excited at 340 and 380 nm with a monochromator (Photon Technologies International, Lawrenceville, NJ), and the fluorescence emitted at 510 nm was detected with a photometer (Photon Technologies International). The ratio of light detected after excitation at 380 and 340 nm was converted into Ca2+ concentration using a calibration performed after each measurement as previously described (36).
Data analysis. Baseline levels of ATP varied considerably by experimental day for both fresh and cultured experiments. Efforts to remove this variability, such as limiting cell age and changing the solution 1 h before the measurements, were only partially effective, and the raw concentrations throughout the text reflect this variation. To allow comparison of experiments, values were normalized to the mean control value of each day. All data are expressed as means ± SE, and the unpaired Student's t-test was used for statistical analysis, with P < 0.05 defined as significantly different. The IC50 of CFTR-172 and the EC50 of hypotonicity were determined by fitting the data curve with first-order exponential curves using SigmaPlot graphing software (SPSS, Chicago, IL).
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RESULTS |
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ATP release from ARPE-19 cells reached a peak 10 min after the addition of a 50% hypotonic solution, after which levels slowly declined for the remainder of the 30-min measurement period (Fig. 2A). Peak levels rose more than eightfold in this series of experiments, from 1.7 ± 0.5 nM to 14.1 ± 1.6 nM (n = 8 for both). The amount of ATP present in the well rose with the degree of hypotonicity >30% and reached a peak at 50% hypotonicity. Increasing hypotonicity to 60% did not produce any additional increase, giving an EC50 of 42% (Fig. 2B).
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Release triggered by cAMP. To determine whether CFTR was involved in ATP release from RPE cells, the effect of intracellular cAMP was examined because activation by the cAMP-dependent protein kinase A is typically necessary for the opening of the CFTR conductance pathway (2). Intracellular cAMP was increased with a cAMP cocktail containing cell-permeant cpt-cAMP, forskolin, and IBMX. The cocktail triggered an immediate increase in ATP levels bathing ARPE-19 cells (Fig. 3A). While recording from the assay plate generally began within 6090 s after addition of stimuli, continuous monitoring during cocktail addition confirmed that ATP levels reached their peak within 30 s. Peak levels of ATP rose fourfold when cells were exposed to the cocktail (Fig. 3B). Although both forskolin and cpt-cAMP increased bath levels of ATP, levels were highest with the complete cocktail. Addition of the cocktail at the peak of the hypotonic response did not lead to a further increase. In fresh bovine RPE cells, the cAMP-stimulating cocktail increased ATP levels 2.5-fold, from 10.1 ± 2.1 nM ATP (n = 6) to 24.3 ± 9.3 nM ATP (n = 6, Fig. 3C). Forskolin itself led to a small but not significant increase in the fresh cells. Because the CFTR activator genistein significantly inhibited the luciferin-luciferase assay (Fig. 1), it was not possible to reliably quantify its enhancement of ATP release.
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BFA also blocked the cAMP-stimulated ATP release across the apical membrane of fresh bovine RPE cells. In these experiments, the cAMP cocktail increased the ATP levels 1.6-fold, to 64.7 ± 12.9 nM (n = 12) from control levels of 46.0 ± 8.8 nM (n = 12). A 30-min incubation with BFA produced a small but not significant reduction in ATP concentration to 52.4 ± 9.7 nM (n = 11) (Fig. 7E).
Because vesicular release is known to require Ca2+ (8), the effect of the Ca2+ chelator BAPTA on ATP release was examined. Incubation of ARPE-19 cells in cell-permeant BAPTA-AM for 30 min reduced the ATP release triggered by hypotonicity by >77% (Fig. 7F). Levels were increased from 0.55 ± 0.02 nM in control (n = 16) to 5.63 ± 0.82 nM in hypotonic conditions (n = 14; 10.2-fold increase) but fell to only 1.68 ± 0.29 nM in the presence of BAPTA and hypotonicity (n = 16; 1.7-fold increase from control). This indicates that ATP release requires intracellular Ca2+ and further supports a role for the vesicular process in the release of ATP from RPE cells.
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DISCUSSION |
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CFTR-172 was recently identified as a high-affinity inhibitor of CFTR that does not block volume-sensitive Cl channels, Ca2+-sensitive Cl channels, KATP channels, AQP1, AE1, NHE3, or MDR-1 (28). It produces a voltage-independent block and does not affect cAMP production, phosphatase activity, or single-channel conductance, but leads to a reduction in the mean closed time of the channel and may effect channel gating by binding to NBD-1 (28, 56). Inhibition of anion transport by CFTR was maximal 10 min after application of CFTR-172, consistent with the enhanced inhibition of ATP release from RPE cells after preincubation. While the IC50 for ATP release was slightly higher than that found for anion influx or measurements of short-circuit current (28, 56), this is consistent with a more negative membrane potential in RPE cells. In addition, 10 µM CFTR-172 produced maximal block both of ATP release in the present study and of anion flux from thyroid epithelial cells in previous work (28). The effect of CFTR-172 on ATP release provides strong support for a role of CFTR in RPE physiology and is consistent with the idea that the Cl and ATP efflux pathways share a common gating mechanism.
The block of ATP release with CFTR-172 also strengthens the link between ATP release and CFTR. CFTR has been associated with ATP efflux since Reisin et al. (45) indicated overexpression of CFTR led to increased ATP efflux. While additional conduits for ATP efflux are likely (11, 18, 47) and release from some cells can clearly occur in the absence of CFTR (18, 19, 37, 61), the activity of CFTR is correlated with the augmentation of both baseline and stimulated ATP release in many cell types (33, 49), and the presence of immunopurified CFTR in lipid bilayers is linked to ATP conductance (9). ATP was initially thought to permeate the CFTR pore used by Cl, but the precise pathway of ATP movement is now a matter of some debate. Selective elimination of Cl and ATP conductance with different site mutations suggests that both CFTR and a separate cofactor are necessary for ATP efflux (23). Anion channel blockers diphenylamine carboxylate and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid were found to have differential effects on the Cl and ATP conductance at the single-channel level, although the two pathways shared a gating mechanism (54). The ability of CFTR-172 to prevent the release of ATP provides further evidence that one gating mechanism controls the passage of both anions (56) and identifies CFTR-172 as an important tool to explore the role of CFTR in release. However, the effects of CFTR-172 on Cl and ATP currents must be examined at the single-channel level to provide further insight into the relationship between CFTR and ATP release.
Brefeldin A and CFTR-172 both blocked >70% of the hypotonically triggered release of ATP. This overlap implies that both processes apply to the same population of released ATP and act in series. Freshly synthesized CFTR has a half-life of 16 h (51), but CFTR on the cell surface can be internalized at 5%/min (27). Because a 2-h preincubation with BFA completely inhibited the ATP release from our preparation, this process is unlikely to be dependent on synthesis of new protein but may instead involve recycled material. BFA has traditionally been associated with vesicular transport in the Golgi, but it can also interfere with a range of trafficking events, including transport of recycling endosomes to the plasma membrane. BFA prevented the formation of clathrin-coated buds at endosomes with <30-min incubation and reduced the clathrin-dependent endosomal recycling of transferrin within 10 min (53, 57). The ability of cAMP to stimulate the insertion of CFTR-containing vesicles into the membrane has been hotly debated, with the process occurring in some cells but not in others (3, 38, 62). At least part of the inhibition of ATP release by BFA in RPE cells indicated above is likely to be on the number of CFTR proteins already present in the membrane, because the block was time dependent and was observed in association with baseline levels of ATP released from unstimulated cells. Whether the larger increase in extracellular ATP levels after hypotonic stimulation reflects the insertion of CFTR-containing vesicles into the membrane as seen with other channel types (16, 58) awaits biochemical assessment of CFTR localization in stimulated and unstimulated cells. Regardless of the reason for this phenomenon, the ability of BFA to prevent ATP release by reducing the amount of CFTR reaching the membrane may reconcile opposing "channel" and "vesicular" theories about the release of ATP. Although the vesicular transport pathway has previously been implicated in the release of ATP from non-neuronal cells, the vesicles were assumed to contain transmitters (1, 5, 30, 58). The hybrid mechanism illustrated in Fig. 8 may underlie ATP release from cells in which roles for both anion channels and vesicular transport have been identified.
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It is tempting to assume that the increase in subretinal ATP after the stimulation of apical CFTR would autostimulate P2Y2 receptors and elevate Ca2+ in vivo as shown in Fig. 6. This rise in Ca2+ may open basolateral Ca+-sensitive Cl channels and increase retinal to basolateral fluid absorption if the released ATP acts like the P2Y2 agonists (29, 31). The released ATP may also stimulate P2X receptors present on the RPE (46), but because their activation opens a cation channel, stimulation would further increase intracellular Ca2+. The effects of cAMP on the RPE are complex, possibly reflecting species differences or the action of CFTR on the apical and basolateral membranes in addition to other mechanisms (4, 22). The activation of basolateral Cl conductance after apical autostimulation with ATP would add an additional level of complexity that may underlie some of the variation in effects attributed directly to cAMP. However, the ability of CFTR-172 to alter Ca2+ physiology shows a definitive role for the transporter, and in patients with cystic fibrosis, the fast oscillation of the electrooculogram was significantly reduced (33), suggesting that CFTR function in wild types is regulated by light. It will be interesting to see whether subretinal ATP levels are also affected by light.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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