Endogenous ATP release inhibits electrogenic Na + absorption and stimulates Cl − secretion in MDCK cells Our previous studies with a line of Madin-Darby canine kidney -LRB- MDCK -RRB- cells -LRB- FL-MDCK -RRB- transfected with FLAG-labeled α , β , and γ subunits of epithelial Na + channel -LRB- ENaC -RRB- showed that , although most of the short-circuit current -LRB- Isc -RRB- was amiloride sensitive -LRB- AS-Isc -RRB- , there was also an amiloride-insensitive component -LRB- NS-Isc -RRB- due to Cl − secretion -LRB- Morris and Schafer , J Gen Physiol 120:71 -- 85 , 2002 -RRB- . In the present studies , we observed a progressive increase in NS-Isc and a corresponding decrease in AS-Isc during experiments . There was a significant negative correlation between AS-Isc and NS-Isc both in the presence and absence of treatment with cyclic adenosine monophosphate -LRB- cAMP -RRB- . NS-Isc could be attributed to both cystic fibrosis transmembrane conductance regulator -LRB- CFTR -RRB- and a 4 , 4 ' - diisothiocyano-2 , 2 ' - disulfonic acid stilbene -LRB- DIDS -RRB- - sensitive Ca2 + - activated Cl − channel -LRB- CaCC -RRB- . Continuous perfusion of both sides of the Ussing chamber with fresh rather than recirculated bathing solutions , or addition of hexokinase -LRB- 6 U/ml -RRB- , prevented the time-dependent changes and increased AS-Isc by 40 -- 60 % , with a proportional decrease in NS-Isc . Addition of 100 μM adenosine triphosphate -LRB- ATP -RRB- in the presence of luminal amiloride produced a transient four-fold increase in NS-Isc that was followed by a sustained increase of 50 -- 60 % above the basal level . ATP release from the monolayers , measured by bioluminescence , was found to occur across the apical but not the basolateral membrane , and the apical release was tripled by cAMP treatment . These data show that constitutive apical ATP release , which occurs under both basal and cAMP-stimulated conditions , underlies the time-dependent rise in Cl − secretion and the proportional fall in ENaC-mediated Na + absorption in FL-MDCK cells . Thus , endogenous ATP release can introduce a significant confounding variable in experiments with this and similar epithelial cells , and it may underlie at least some of the observed interaction between Cl − secretion and Na + absorption . Introduction Madin-Darby canine kidney -LRB- MDCK , type I -RRB- cells grown as epithelial monolayers have been used extensively as a model of the mammalian collecting duct -LRB- CD -RRB- . These monolayers exhibit many characteristics of the CD , including a high transepithelial resistance and amiloride-sensitive Na + reabsorption that is mediated by the epithelial Na + channel -LRB- ENaC -RRB- and stimulated by agonists that increase intracellular cyclic adenosine monophosphate -LRB- cAMP -RRB- -LSB- 1 , 2 -RSB- . Similar cell lines that are derived from the CD , including mouse cortical CD -LRB- M-1 -RRB- and inner medullary CD -LRB- mIMCD-K2 -RRB- cells -LSB- 3 -- 5 -RSB- and A6 cells from the Xenopus distal nephron -LSB- 6 , 7 -RSB- share these characteristics . However , in contrast to the CD , all of these cell lines also have Cl − channels in their apical membranes , and , when studied under short-circuit conditions , exhibit Cl − secretion , which is observed as an amiloride-insensitive component of the short-circuit current -LRB- NS-Isc -RRB- that is also stimulated by cAMP -LSB- 2 , 3 , 5 -- 8 -RSB- . We previously developed a line of MDCK cells -LRB- FL-MDCK -RRB- that had been retrovirally transfected with rat ENaC subunits containing the FLAG epitope in their extracellular loops -LSB- 2 -RSB- . Monolayers of these cells had higher rates of ENaC-mediated Na + absorption , measured as the amiloride-sensitive short-circuit current -LRB- AS-Isc -RRB- , than did the original MDCK cell line . Our previous studies with FL-MDCK monolayers in Dulbecco 's modified Eagle 's medium -LRB- DMEM -RRB- showed that cAMP treatment produced a rapid transient peak in the total short-circuit current -LRB- Isc -RRB- within 5 min , followed by a broad peak that decayed over 20 min -LSB- 2 -RSB- . This biphasic response to cAMP treatment has also been reported in A6 and M-1 cultures and has been attributed to rapid stimulation of Cl − secretion mediated by cystic fibrosis transmembrane conductance regulator -LRB- CFTR -RRB- followed by a slower activation of ENaC -LSB- 6 -- 9 -RSB- . In FL-MDCK cells , the biphasic response to cAMP treatment was followed by a decay of Isc , which reflected a decrease in AS-Isc -LSB- 2 -RSB- . Morris and Schafer -LSB- 2 -RSB- found that this late fall in AS-Isc in FL-MDCK monolayer was prevented by the omission of Cl − from the bathing solution and attributed the effect to Cl − secretion via CFTR , which has subsequently been demonstrated to be present in this cell line -LSB- 10 -RSB- . A large body of evidence indicates that CFTR is a cAMP-regulated Cl − channel as well as a conductance regulator , which is colocalized with ENaC in airway , colonic , and other epithelial tissues -LSB- 11 , 12 -RSB- . It has been proposed that when cAMP activates CFTR , ENaC is inhibited , and this inhibitory effect of CFTR has been hypothesized to explain the pathophysiology of cystic fibrosis in airway epithelia -LSB- 13 , 14 -RSB- . In Xenopus oocytes expressing ENaC , coexpression of CFTR reduces the ENaC-mediated Na + current -LSB- 15 -RSB- , an effect that might be attributed to a direct effect of CFTR on ENaC activity . However , Kunzelman and his collaborators -LSB- 16 , 17 -RSB- have shown that the inhibitory effect of CFTR can be mimicked by coexpression of other anion channels or treatment with amphotericin B and is explained by an increase in intracellular Cl − -LRB- -LSB- Cl − -RSB- i -RRB- . We have shown that an increase in -LSB- Cl − -RSB- i also inhibits ENaC in FL-MDCK cells ; however , this effect can not explain the effect of Cl − secretion on Na + absorption with cAMP treatment , because stimulation of Cl − secretion by cAMP results in a fall rather than a rise in -LSB- Cl − -RSB- i -LSB- 10 -RSB- . Moreover , in the Xenopus oocyte expression system , CFTR activation inhibits ENaC at a continuous holding potential at which CFTR mediates inward currents corresponding to Cl − efflux -LSB- 18 -RSB- . Under these conditions , activation of CFTR will not result in an increase in -LSB- Cl − -RSB- i. Therefore , a change in -LSB- Cl − -RSB- i is unlikely to fully explain the observed reciprocal regulation of ENaC and CFTR . In the present studies , we show that adenosine triphosphate -LRB- ATP -RRB- accumulation in the apical solution produces a progressive stimulation of NS-Isc and a corresponding decrease in AS-Isc when FL-MDCK cells are studied in Ussing chambers . The possibility of constitutive ATP release raises an important issue when interpreting changes in ion transport in experiments such as these . More important in the present context , ATP release can explain at least part of the inverse relationship between Cl − secretion and Na + reabsorption , and , as suggested by Wilson et al. -LSB- 19 -RSB- , it may be involved in the conversion of CD cells from absorptive to secretory in polycystic kidney disease . Materials and methods Cell culture The FL-MDCK cells used were a clone of the type-1 MDCK line that had been retrovirally transfected with flagged rat α - , β - , and γENaC by Morris and Schafer -LSB- 2 -RSB- . The cells used were from passages 5 -- 35 and were cultured in T-75 flasks at 37 °C in DMEM -LRB- Life Technologies -RRB- supplemented with 10 % fetal bovine serum -LRB- FBS -RRB- , 50 mM N-2-hydroxyethylpiperazine-N ' -2-ethanesulfonic acid -LRB- HEPES -RRB- -LRB- pH 7.4 -RRB- , 1 % Pen/strep-fungizone , and the selection antibiotics G418 -LRB- 800 μg/ml -RRB- , hygromycin -LRB- 300 μg/ml -RRB- , and puromycin -LRB- 5 μg/ml -RRB- . For transepithelial transport studies , cells were seeded onto 24-mm Transwell inserts -LRB- Costar ; Catalog No. 3412 -RRB- at a density of ∼ 105 cells/cm2 . For bioluminescence assays , cells were seeded on 12-mm collagen-coated Millicell inserts -LRB- Millipore ; Catalog No . PHIP 012 50 -RRB- at the same density . After seeding , cells were fed daily with DMEM containing FBS and HEPES but without selection antibiotics for 5 -- 7 days . Before use in the experiments , the cell monolayers were induced with 1 μM dexamethasone plus 2 mM Na + butyrate in the culture medium overnight . Ussing chamber experiments FL-MDCK monolayers were carefully cut from the plastic insets and were mounted in Ussing-type chambers . In most experiments , both sides of the monolayers were bathed with Krebs-Ringer bicarbonate -LRB- KRB -RRB- solution , which contained -LRB- in mM -RRB- : 113 NaCl , 1.2 Na2HPO4 , 25 NaHCO3 , 1.1 CaCl2 , 1.2 MgCl2 , 4.5 KCl , and 10 glucose . The KRB solutions were continuously gassed with a mixture of 95 % O2-5 % CO2 to give a pH of 7.40 at 37 °C . As shown by the schematic diagram in Fig. 1a , transepithelial short-circuit currents -LRB- Isc , μA/cm2 -RRB- and voltages -LRB- Vte -RRB- were measured and conductances were evaluated every 30 s by measuring the current deflections induced by 4-s symmetrical voltage pulses of ± 2 mV , as described previously -LSB- 2 , 10 -RSB- . In most experiments , KRB was continuously recirculated on both sides of the epithelium from 10-ml reservoirs -LRB- Fig. 1b -RRB- ; however , in the experiments involving `` fresh perfusion , '' as shown in Fig. 1c , both sides of the monolayers were continuously perfused with fresh rather than recirculated KRB . In those experiments involving cAMP treatment , 100 μM 8 - -LRB- 4-chlorophenylthio -RRB- - cAMP -LRB- CPT-cAMP -RRB- plus 100 μM isobutylmethylxanthine -LRB- IBMX -RRB- were added to both the apical and basolateral solutions . AS-Isc was defined as the change in Isc produced by the addition of 10 μM amiloride to the apical solution , and the remaining Isc was defined as NS-Isc . Because we observed time-dependent changes in AS-Isc and NS-Isc in the voltage clamp experiments , we conducted additional experiments in which the monolayers were left under open-circuit conditions during the course of the experiment , and the open-circuit transepithelial voltage -LRB- Vte , mV -RRB- was measured continuously except for intermittent voltage clamping to 0 mV and ± 2 mV for a total of 4 s every 30 s to obtain Isc and conductance measurements . Fig. 1a Schematic diagram of the electrophysiological circuitry . The Ussing chamber is constructed of two lucite half-chambers , which are clamped together with the Madin-Darby canine kidney -LRB- MDCK -RRB- cell monolayer -LRB- on a Transwell membrane -RRB- between them . Voltage-sensing electrodes are positioned close to the center and on either side of the monolayer . Current passing electrodes , made from silver disks with a central hole to accommodate the voltage electrodes , are placed at the back of each half chamber and parallel to the monolayer . The transepithelial voltage -LRB- Vte -RRB- is measured by the voltage clamp/analog to digital converter -LRB- called `` the clamp '' -RRB- . When no current is being passed , the voltage recorded is called the `` open circuit '' Vte . To short-circuit the epithelial monolayer , the clamp passes a current -LRB- I -RRB- that is sufficient to drive Vte to zero . The current measured in this situation is called the short-circuit current -LRB- Isc -RRB- . b In experiments in which the Krebs-Ringer bicarbonate solutions -LRB- KRB -RRB- in the Ussing chambers were recirculated , 10-ml syringes were used as reservoirs , and a roller pump was used to continuously move the KRB from each half-chamber to its respective reservoir . c In experiments in which fresh KRB solutions were continuously perfused , large beakers served as the solution reservoirs from which KRB was constantly infused into each half-chamber by a roller pump and constantly removed by suction to a waste reservoir . In both the recirculation and fresh perfusion methods , the solutions in the reservoirs -LRB- 10-ml syringes or beakers , respectively -RRB- were constantly bubbled with a 95 % O2 -- 5 % CO2 gas mixture Bioluminescence detection of ATP release ATP released from the MDCK monolayers was measured as described by Taylor et al. -LSB- 20 -RSB- . Briefly , culture medium was removed from the inserts , and the cell monolayers were washed three times with phosphate-buffered saline -LRB- PBS -RRB- solution to remove any FBS present in the culture medium . Opti-minimal essential medium -LRB- MEM -RRB- with 2 mg/ml luciferin-luciferase reagent -LRB- Sigma -RRB- was added to the apical or basolateral side of the monolayer . Each insert with cells contained 200 μl of solution , and the average light signal was measured for 10-s , nonintegrated photon collection periods with a TD-20 / 20 luminometer -LRB- Turner Designs ; Promega -RRB- . Treatment with cAMP was applied by adding 1 μl of 20 mM CPT-cAMP and 20 mM IBMX stock solution to the 200 μl apical solution of the inserts -LRB- final concentration 100 μM for both -RRB- , and control experiments were done with addition of the same volume of vehicle alone . The light output was measured for at least 2 min , and the average was taken for the calculation of ATP release . Individual batches of luciferin-luciferase reagent were tested for relative sensitivity by measuring the light output response to serial dilutions of an ATP stock solution with 2 mg/ml luciferin-luciferase reagent in Opti-MEM . Data analysis and statistics StatView for Macintosh -LRB- SAS Institute Inc. -RRB- was used for statistical analyses . Statistical significance -LRB- P < 0.05 -RRB- was determined by analysis of variance -LRB- ANOVA -RRB- with Bonferroni/Dunn post hoc testing for multiple comparisons and by paired or nonpaired t-tests , as appropriate , for single comparisons . Time dependence of transport was evaluated by linear regression analysis ; the R value and significance of the slope are given . Correlation analysis was used to compare AS-Isc and NS-Isc and calculate a correlation coefficient , r. Materials All chemicals were obtained from Sigma-Aldrich -LRB- St. Louis , MO , USA -RRB- unless otherwise noted . Results Time-dependent changes in Isc The Isc in FL-MDCK cells was quite high and variable in the first 5 min after recording was begun using continuous short-circuiting . In six experiments , 10 μM amiloride added to the apical solution in the initial 5 min decreased Isc by 91 ± 2 % , indicating that most of the current was due to ENaC-mediated Na + absorption . The initial high Isc subsequently declined to a nadir at ∼ 15 min -LRB- Fig. 2a and b -RRB- . Addition of amiloride at this nadir -LRB- Fig. 2a -RRB- almost completely inhibited Isc ; however , when the experiment was continued beyond the nadir , amiloride was less effective in reducing Isc -LRB- Fig. 2b -RRB- . In 35 experiments such as those in Fig. 2a and b , the time interval between the nadir and amiloride addition to the apical solution was varied from 0 to 45 min . In these experiments , the average Isc at the nadir was 15.2 ± 0.5 μA/cm2 . Regression analysis showed that NS-Isc increased significantly -LRB- R = 0.76 , P < .001 -RRB- and AS-Isc decreased significantly -LRB- R = 0.53 , P < .002 -RRB- as a function of the time between the nadir and the addition of amiloride . The same high initial Isc and the time-dependent increase of NS-Isc were also observed in experiments using the intermittent short-circuit technique -LRB- see `` Materials and methods '' -RRB- , which indicated that neither effect is produced by prolonged short-circuiting of the epithelium -LRB- data not shown -RRB- . Fig. 2Time course of the short-circuit current -LRB- Isc -RRB- and response to amiloride . Isc was measured across monolayers of FL-Madin-Darby canine kidney -LRB- FL-MDCK -RRB- cells induced overnight with 2 mM Na butyrate and 1 μM dexamethasone . Amiloride -LRB- 10 μM -RRB- was added to the apical solution either early -LRB- A -RRB- or late -LRB- B -RRB- at the point indicated by the arrow . The transepithelial conductance -LRB- Gte -RRB- is proportional to the height of the deflections in Isc produced by transiently clamping the voltage to ± 2 mV , as described in `` Materials and methods '' As shown in Fig. 3 , the effect of cAMP on Isc also depended on whether the treatment occurred early -LRB- at the nadir of Isc , Fig. 3a -RRB- or late -LRB- ∼ 40 min after the nadir , Fig. 3b -RRB- . With both early and late cAMP addition , Isc increased immediately , reaching a transient peak within 5 min ; however , the magnitude of this initial peak was exaggerated with late cAMP treatment -LRB- Fig. 3b -RRB- . After the initial response , Isc showed a secondary broader increase that decayed more rapidly after late -LRB- Fig. 3b -RRB- than after early -LRB- Fig. 3a -RRB- cAMP treatment . In 29 experiments such as those in Fig. 3 , the time interval between the nadir in Isc and cAMP treatment was varied from 2 to 58 min , and 10 μM amiloride was added ∼ 30 min after cAMP treatment . Regression analysis showed a significant increase in NS-Isc with the time between the nadir and cAMP treatment -LRB- R = 0.60 , P < .002 -RRB- . Fig. 3Time course of Isc response to cyclic adenosine monophosphate -LRB- cAMP -RRB- treatment . A mixture of 100 μM 8 - -LRB- 4-chlorophenylthio -RRB- -LRB- CPT -RRB- - cAMP plus 100 μM isobutylmethylxanthine -LRB- IBMX -RRB- was added as indicated by the lower bar , either early -LRB- a -RRB- or late -LRB- b -RRB- to both sides of the monolayers , followed by 10 μM amiloride addition to the apical solution only -LRB- upper bar -RRB- Further examination of the data from these experiments -LRB- see Fig. 4 -RRB- revealed a significant negative correlation between AS-Isc and NS-Isc , both with and without cAMP treatment . In other words , the time-dependent increase in NS-Isc observed in both sets of experiments was correlated with a corresponding decrease in AS-Isc . As expected , the average AS-Isc was significantly greater in those experiments with cAMP treatment -LRB- 20.2 ± 1.1 μA/cm2 , n = 29 -RRB- than in those without -LRB- 12.8 ± 0.6 , n = 35 -RRB- , but the correlation with NS-Isc was not significantly different between the two groups . Fig. 4Negative correlation between the amiloride-sensitive component of the short-circuit current -LRB- Isc -RRB- -LRB- AS-Isc -RRB- and the nonamiloride-sensitive component -LRB- NS-Isc -RRB- in the absence and presence of cyclic adenosine monophosphate -LRB- cAMP -RRB- . The 35 experiments in the first group -LRB- open circles , solid line -RRB- were conducted using the same protocol as the representative experiments in Fig. 1 with no cAMP addition . The 29 experiments in the second group -LRB- solid dots , dashed line -RRB- followed the protocol of the representative experiments in Fig. 2 , with the addition of 100 μM 8 - -LRB- 4-chlorophenylthio -RRB- -LRB- CPT -RRB- - cAMP plus 100 μM isobutylmethylxanthine -LRB- IBMX -RRB- ∼ 30 min before the addition of amiloride . In both groups , there was a significant correlation between AS-Isc and NS-Isc : − cAMP group , r = 0.79 , P < .001 ; + cAMP group , r = 0.65 , P < .001 Effect of cAMP and intracellular Ca2 + on NS-Isc In the experiments shown in Fig. 5 , NS-Isc was measured throughout the experiments in the continuous presence of 10 μM amiloride in the apical solution . Treatment with cAMP produced an initial rapid peak in NS-Isc , followed by a sustained plateau . We then tested the effects of two inhibitors of Cl − transport pathways : glibenclamide , which is a well-established inhibitor of CFTR ; and 4 , 4 ' - diisothiocyano-2 , 2 ' - disulfonic acid stilbene -LRB- DIDS -RRB- , which is a less selective inhibitor of Cl − channels including the Ca2 + - activated chloride channel -LRB- CaCC -RRB- and the chloride channel -LRB- CLC -RRB- family of Cl − channels but not CFTR . -LRB- See the Discussion for more detail about these inhibitors and references to their specificity . -RRB- Addition of 200 μM glibenclamide followed by 300 μM DIDS to the apical solution ∼ 20 min after cAMP treatment significantly decreased NS-Isc to or below the magnitude prior to cAMP treatment -LRB- Fig. 5a & b -RRB- . In other experiments , addition of apical DIDS before cAMP treatment reduced the late plateau but not the initial peak of NS-Isc -LRB- Fig. 5c and e -RRB- , whereas apical glibenclamide significantly decreased the initial peak , with no effect on the late plateau -LRB- Fig. 5d & e -RRB- . These data suggest that at least two types of Cl − channels are stimulated by cAMP . One is sensitive to glibenclamide and responds to cAMP quickly while the other is sensitive to DIDS and responds to cAMP slowly . Fig. 5Effects of glibenclamide and 4 , 4 ' - diisothiocyano-2 , 2 ' - disulfonic acid stilbene -LRB- DIDS -RRB- on the nonamiloride-sensitive component -LRB- NS-Isc -RRB- . In these experiments , 10 μM amiloride was continuously present in the apical solution . A Effect of cyclic adenosine monophosphate -LRB- cAMP -RRB- treatment -LSB- 100 μM cAMP plus 100 μM isobutylmethylxanthine -LRB- IBMX -RRB- , lowest bar -RSB- on NS-Isc . The middle and upper bars indicate the time of addition of 300 μM DIDS and 200 μM glibenclamide -LRB- Gliben . -RRB- to the apical solution . B Summary of 14 experiments such as that in A . The mean NS-Isc values immediately before cAMP treatment -LRB- point 1 in A -RRB- , 3 -- 5 min -LRB- `` initial '' , point 2 -RRB- and 15 -- 20 min -LRB- `` late '' , point 3 , just before DIDS or glibenclamide addition -RRB- after cAMP treatment , and after DIDS plus glibenclamide -LRB- point 4 -RRB- . * Significantly different from value before cAMP treatment , P < .001 ; † significantly different from the initial and late NS-Isc , P < .001 . C Effect of 300 μM apical DIDS added before cAMP treatment . D Effect of 200 μM apical glibenclamide added before cAMP treatment . E Mean values of the change in NS-Isc -LRB- the cAMP-stimulated NS-Isc -RRB- produced by cAMP treatment in the initial -LRB- point 2 in A , C , and D -RRB- or late -LRB- point 3 -RRB- response period in three sets of experiments : with no inhibitor -LRB- control , from summary of 15 experiments in B -RRB- , DIDS before cAMP -LRB- as in C , n = 6 -RRB- , and glibenclamide before cAMP -LRB- as in D , n = 5 -RRB- . * Significant difference compared with the corresponding control value -LRB- calculated from the data in B -RRB- , and between initial and late response to cAMP , P < .001 The Cl − channel that was quickly activated by cAMP and inhibited by glibenclamide has been attributed to CFTR , the expression and function of which has been identified by us in this same cell line -LSB- 10 -RSB- . Because cAMP increases intracellular Ca2 + -LRB- -LSB- Ca2 + -RSB- i -RRB- in MDCK cells -LSB- 21 -RSB- , we examined the hypothesis that CaCC contributes to the fraction of NS-Isc that is inhibited by DIDS by using thapsigargin to produce a modest but sustained elevation of -LSB- Ca2 + -RSB- I -LSB- 22 , 23 -RSB- . -LRB- Thapsigargin is known to elevate intracellular Ca2 + by releasing it from the endoplasmic reticulum -LSB- 22 -RSB- . -RRB- In the experiments shown in Fig. 6 , NS-Isc was measured in the continuous presence of luminal amiloride . The addition of 1 μM thapsigargin to both sides of the monolayer produced a transient spike followed by a sustained increase in NS-Isc , most of which was sensitive to DIDS -LRB- Fig. 6a -RRB- . When 300 μM DIDS was added to apical solution before thapsigargin , it almost completely blocked the response of NS-Isc to thapsigargin -LRB- Fig. 6b -RRB- . Fig. 6Effect of thapsigargin on the nonamiloride-sensitive component -LRB- NS-Isc -RRB- and prevention of that effect by 4 , 4 ' - diisothiocyano-2 , 2 ' - disulfonic acid stilbene -LRB- DIDS -RRB- . All experiments were performed in the presence of 10 μM luminal amiloride . A Mean NS-Isc values for seven experiments at four time points : just before adding thapsigargin to the apical and basolateral solutions , 3 -- 5 min after addition of 1 μM thapsigargin to the apical and basolateral solutions -LRB- TG-initial -RRB- , 15 -- 20 min after thapsigargin addition -LRB- TG-late -RRB- , and 3 -- 5 min after the addition of 300 μM DIDS to the apical solution . * Significantly different compared with NS-Isc before thapsigargin treatment , P < .001 ; † significantly different compared with both the initial and late NS-Isc after thapsigargin , P < .001 . B Mean values of the change in NS-Isc produced by thapsigargin : the initial peak -LRB- gray bars -RRB- and the late response -LRB- open bars -RRB- for control experiments -LRB- as in A -RRB- and in three experiments in which 300 μM DIDS was added before thapsigargin . * Significantly different compared with the corresponding control values , P < .001 Effect of Ussing chamber perfusion on time-dependent changes in Isc In all of the experiments presented up to this point , the KRB solutions that bathed the MDCK monolayers were recirculated as shown in Fig. 1b . To examine whether the time-dependent changes in AS-Isc and NS-Isc might be caused by the accumulation of a secretagogue or metabolite in these recirculated solutions , we conducted paired experiments in which one Ussing chamber was perfused with recirculated KRB solutions and the other chamber was continuously perfused with fresh KRB solutions , as shown in Fig. 1c . The representative control experiment shown in Fig. 7a was conducted in the usual way : the apical and basolateral KRB solutions were recirculated through both sides of the Ussing chamber from 10-ml reservoirs . In the paired experiment in Fig. 7b , we continuously perfused fresh KRB on both sides of the Ussing chamber . In both the recirculation and fresh perfusion experiments , cAMP treatment produced an initial transient spike of Isc lasting less than 6 min , followed by a broader peak ; however , Isc decayed more rapidly in the recirculation experiment than it did in the fresh perfusion experiment . In four sets of paired experiments summarized in Fig. 7c , AS-Isc was greater -LRB- 39.8 ± 1.1 μA/cm2 vs. 25.2 ± 3.3 μA/cm2 , P < 0.01 -RRB- and NS-Isc was less -LRB- 2.8 ± 0.2 μA/cm2 vs. 7.7 ± 1.6 μA/cm2 , P < 0.01 -RRB- with fresh perfusion than in the recirculation group , suggesting that some substance , which inhibited AS-Isc and stimulated NS-Isc , was accumulating in the recirculated bathing solution . Fig. 7Time course of the short-circuit current -LRB- Isc -RRB- in experiments with and without recirculation of the bathing solutions . The sequence of treatments was the same in these paired experiments : 100 μM cyclic adenosine monophosphate -LRB- cAMP -RRB- plus 100 μM isobutylmethylxanthine -LRB- IBMX -RRB- were added to both apical and basolateral solutions , followed by 10 μM amiloride to the apical solution . A Apical and basolateral Krebs-Ringer bicarbonate -LRB- KRB -RRB- solutions were recirculated through the two sides of the Ussing chamber from 10-ml reservoirs . 4 , 4 ' - diisothiocyano-2 , 2 ' - disulfonic acid stilbene -LRB- DIDS -RRB- -LRB- 300 μM -RRB- was added to the apical solution near the end of the experiment . B In this paired experiment , fresh KRB solution was perfused through both sides of the Ussing chamber at the same rate -LRB- 1 ml/min -RRB- as for the recirculated solutions in A. C Summary results for four sets of paired experiments such as those in A and B. * Significantly different from control -LRB- with recirculation as in A -RRB- , P < .001 Effect of extracellular ATP on time-dependent changes in Isc We hypothesized that ATP was released by the FL-MDCK cells and accumulated in the recirculated solutions . Extracellular nucleotides are known to act as autocrine and paracrine agents that affect Na + absorption and Cl − secretion , and ATP has been shown to increase intracellular -LSB- Ca2 + -RSB- -LSB- 24 , 25 -RSB- , which might augment the Ca2 + - activated component of NS-Isc as well as inhibit ENaC activity . As shown in Fig. 8a , in the presence of amiloride , the addition of 100 μM ATP to the apical solution increased NS-Isc , and , as shown in Fig. 8b , this effect was almost completely abolished when the ATP was added together with hexokinase as an ATP scavenger -LSB- 26 -RSB- . -LRB- These experiments were conducted using the recirculated KRB method . -RRB- In five experiments such as that in Fig. 8a , basal NS-Isc -LRB- just before ATP addition -RRB- averaged 4.1 ± 0.1 μA/cm2 . With the addition of ATP , NS-Isc reached a peak value of 15.4 ± 2.2 μA/cm2 -LRB- P < 0.004 -RRB- within 2 -- 3 min , and then declined gradually to a plateau of 5.9 ± 0.3 μA/cm2 , which was still significantly larger than the basal level -LRB- P < 0.01 -RRB- . Addition of 300 μM DIDS to the apical solution before the addition of ATP had no effect on the peak amplitude -LRB- 14.6 ± 0.7 μA/cm2 , n = 3 -RRB- , but decreased the plateau value to 4.5 ± 0.2 μA/cm2 -LRB- n = 3 -RRB- , which was not significantly different from the basal value . Fig. 8Effect of adenosine triphosphate -LRB- ATP -RRB- on the nonamiloride-sensitive component -LRB- NS-Isc -RRB- and prevention of that effect by hexokinase . All experiments were performed in the presence of 10 μM apical amiloride . A Effect of 100 μM ATP added to the apical solution on NS-Isc . Mean values of five such experiments are given in the text . B Effect of 100 μM ATP on NS-Isc . In these experiments , 6 U/ml hexokinase was continuously present in the bathing solution . The same results were obtained in three such experiments To test whether endogenous ATP production by the epithelium might be involved in the time-dependent increase of NS-Isc and decrease of AS-Isc , we examined the effect of hydrolyzing extracellular ATP with hexokinase on the time course of NS-Isc and AS-Isc . Paired experiments without or with hexokinase were conducted as shown , respectively , in Fig. 9a and b , both using the recirculated KRB method . . With hexokinase in the bathing solutions , the response of Isc to cAMP treatment was exaggerated , and the broader peak was more sustained -LRB- Fig. 9b -RRB- . As shown by the summary of four such sets of paired experiments -LRB- Fig. 9c -RRB- , hexokinase had no effect on the basal Isc measured just before cAMP addition ; however , after cAMP , AS-Isc was significantly higher -LRB- 40.4 ± 2.9 μA/cm2 vs. 29.7 ± 2.8 μA/cm2 , P < 0.05 -RRB- with hexokinase , whereas the DIDS-sensitive NS-Isc was not significantly different from zero and significantly less than in the control group -LRB- − 0.3 ± 0.2 μA/cm2 vs. 3.6 ± 0.7 μA/cm2 , P < 0.01 -RRB- . Thus , all of the above data were consistent with the hypothesis that the accumulation of extracellular ATP causes the time-dependent increase of NS-Isc and decrease of AS-Isc that were observed in those experiments in which the bathing solutions were recirculated . Fig. 9Time course of the short-circuit current -LRB- Isc -RRB- response to cyclic adenosine monophosphate -LRB- cAMP -RRB- treatment in the absence and presence of hexokinase . Both types of experiments followed the same general protocol : A mixture of 100 μM -LRB- 4-chlorophenylthio -RRB- -LRB- CPT -RRB- - cAMP plus 100 μM isobutylmethylxanthine -LRB- IBMX -RRB- was added as indicated , followed by 10 μM apical amiloride and 300 μM apical 4 , 4 ' - diisothiocyano-2 , 2 ' - disulfonic acid stilbene -LRB- DIDS -RRB- . A In this experiment , the Krebs-Ringer bicarbonate -LRB- KRB -RRB- solution had no hexokinase added . B A paired experiment in which 6 U/ml hexokinase was continuously present in apical and basolateral bathing solution . C Average data for four paired experiments : the mean values of Isc immediately before cAMP was added -LRB- Basal-Isc -RRB- , amiloride-sensitive -LRB- AS-Isc -RRB- at 30 min after cAMP addition -LRB- AS-Isc -RRB- , and DIDS-sensitive Isc -LRB- DS-Isc -RRB- . * Significantly different compared with the corresponding control values Measurement of ATP released from MDCK cells ATP released from polarized MDCK cells was determined by a bioluminescence assay , in which a luminometer was used to measure light production from ATP consumption in the luciferin-luciferase reaction -LSB- 20 -RSB- . MDCK cells were grown to confluence in 12-mm permeable supports -LRB- Millicell -RRB- , and the ATP was measured in the apical or basolateral solution by adding the reagents only to that chamber . As shown in Fig. 10 , under basal conditions , apically directed ATP release produced a light emission of 16.4 ± 2.1 ALU -LRB- artificial light units -RRB- , whereas basolaterally directed ATP release was nearly zero -LRB- 0.7 ± 0.2 ALU -RRB- . In ten paired experiments , addition of 100 μM cAMP and 100 μM IBMX to the apical solution significantly increased the apically directed ATP release from 16.0 ± 2.1 ALU -LRB- basal -RRB- to 51.1 ± 9.4 ALU -LRB- n = 10 , P < 0.005 -RRB- , whereas addition of the same volume of solution without cAMP and IBMX -LRB- sham control for the possible effects of manipulating the monolayers -RRB- had no significant effect -LRB- 17.4 ± 3.5 ALU basal vs. 15.1 ± 2.3 ALU sham ; n = 4 , P = 0.29 -RRB- . Treatment with cAMP had no significant effect on basolaterally directed ATP release -LRB- 0.8 ± 0.3 , n = 8 , P > 0.5 -RRB- . Fig. 10Adenosine triphosphate -LRB- ATP -RRB- release across the apical and basolateral membranes of FL-Madin-Darby canine kidney -LRB- MDCK -RRB- monolayers . ATP release into the apical and basolateral solutions was measured for FL-MDCK monolayers in 12-mm culture inserts using the luciferin-luciferase assay . To measure apical ATP release , these reagents were present only in the apical solution -LRB- n = 14 -RRB- , and they were present only in the basolateral solution to measure basolateral release -LRB- n = 11 -RRB- . The monolayers were first incubated with 200 μl of standard Opti-minimal essential medium -LRB- MEM -RRB- solution on each side to measure basal ATP production . The monolayers were then removed from the instrument and either 1 μl of a stock solution of 8 - -LRB- 4-chlorophenylthio -RRB- -LRB- CPT -RRB- - cAMP + isobutylmethylxanthine -LRB- IBMX -RRB- stock solution -LRB- cAMP , n = 10 ; final concentrations : 100 μM cAMP and 100 μM IBMX -RRB- or 1 μl of plain Opti-MEM medium -LRB- Sham , n = 4 -RRB- were added . Average values of the light emission in arbitrary light units -LRB- ALU -RRB- ± standard error of the mean -LRB- SEM -RRB- are given . * Significantly different from basal and sham , P < 0.005 Discussion Previous work -LSB- 10 -RSB- has shown that the triply transfected FL-MDCK cells used in these experiments -LRB- a subclone of αFβ FγF MDCK cells used in the experiments of Morris et al. -LSB- 2 -RSB- -RRB- expressed all three rat-ENaC subunits and CFTR , and that it was a very useful model epithelium for the study of ENaC regulation . In the current study , we examined in greater detail the relationship between Cl − secretion and Na + absorption in this epithelium . Time-dependent changes in NS-Isc and AS-Isc FL-MDCK cells uniformly exhibited a high initial Isc , which was almost completely eliminated by 10 μM amiloride added to the apical solution . The initial Isc fell to a nadir within 15 min and then increased slowly -LRB- Figs. 2b and 3b -RRB- due to an increase in NS-Isc . This progressive rise of NS-Isc occurred consistently regardless of whether amiloride was present or not and whether the epithelium was continuously short-circuited or was left in the open-circuit condition with only intermittent short-circuiting to measure Isc . Moreover , the time-dependent increase of NS-Isc could not be inhibited by the further addition of amiloride but was partially blocked by apical DIDS , which is consistent with anion secretion -LRB- see below -RRB- . The time course of the Isc response to cAMP using these FL-MDCK cells was like that observed in the previous studies of Morris and Schafer -LSB- 2 -RSB- and in other studies of similar epithelia -LSB- 6 , 9 , 27 -RSB- . There was an initial transient increase lasting less than 6 min , followed by a broader peak , which decayed over the next 30 min -LRB- Fig. 3 -RRB- . Morris and Schafer -LSB- 2 -RSB- have shown that when these experiments are conducted in a Cl − - free solution , the initial transient spike was absent and Isc was stable for at least 30 min . The initial transient and the broader peak of Isc were attributed to , respectively , stimulation of Cl − secretion and Na + absorption by cAMP -LSB- 2 -RSB- . Interestingly , the effect of cAMP on Isc was also time dependent . With late-cAMP treatment -LRB- Fig. 3b -RRB- , the decay of Isc was faster and the magnitude of NS-Isc was larger . As shown in Fig. 4 , there was a significant inverse correlation between AS-Isc and NS-Isc both in the absence and in the presence of cAMP . This correlation does not indicate a causal relationship between the rise in NS-Isc and the fall in AS-Isc , but suggests that whatever process is responsible for the time dependence produces reciprocal changes in NS-Isc and AS-Isc . Cl − channels associated with NS-Isc As discussed previously -LSB- 2 -RSB- , we attributed NS-Isc to Cl − secretion because in the presence of apical amiloride , Cl − was the only ion present in sufficient concentration to account for it . Furthermore , glibenclamide blocked the initial rise in NS-Isc in response to subsequent cAMP treatment , whereas DIDS blocked the broader late transport response but not the early peak -LRB- Fig. 5 -RRB- . Glibenclamide is a well-established inhibitor of CFTR at the concentration -LRB- 200 μM -RRB- used in these experiments . Although glibenclamide also inhibits ATP-activated K + channels -LSB- 28 , 29 -RSB- , previous studies have shown that the apical membrane of MDCK cells has no measurable K + conductance -LSB- 10 , 30 -RSB- . Although DIDS effectively inhibits Cl − channels such as CaCC as well as the outwardly rectifying Cl − channel -LRB- ORCC -RRB- , and the CLC family of Cl − channels , it does not inhibit CFTR -LSB- 31 , 32 -RSB- . These results indicate that at least two types of Cl − channels contribute to NS-Isc and that they respond differently to cAMP . Based on these data , we have attributed the early , glibenclamide-sensitive , increase in NS-Isc after cAMP treatment to CFTR , which we have demonstrated is expressed in the apical membrane of these FL-MDCK cells -LSB- 10 -RSB- . CaCC appears to be responsible for the later increase in NS-Isc after cAMP treatment . As shown in Fig. 6 , we found that thapsigargin , which moderately increases -LSB- Ca2 + -RSB- i -LSB- 22 -RSB- , produced a transient spike and a sustained increase in NS-Isc that could be completely blocked by apical DIDS as expected for a CaCC -LSB- 33 -RSB- . Furthermore , cAMP increases -LSB- Ca2 + -RSB- i in MDCK cells -LSB- 21 -RSB- , and thus a CaCC may contribute to the DIDS-sensitive component of the NS-Isc response to cAMP . A CaCC has also been described in the M-1 and mIMCD-K2 cell lines -LSB- 34 , 35 -RSB- and in primary cultures of rabbit proximal and distal tubule cells -LSB- 36 -RSB- . It should also be noted , however , that the molecular composition of CaCC is not as yet know . Thus , we were unable to provide unequivocal evidence for its existence in the FL-MDCK cell line . Endogenous ATP release inhibits amiloride-sensitive Na + absorption When the MDCK monolayers were continuously perfused with fresh apical and basolateral KRB solutions rather than recirculated solutions , the time-dependent increase of NS-Isc and decrease of total Isc was prevented -LRB- Fig. 7 -RRB- . AS-Isc was also significantly increased during the continuous perfusion , suggesting that inhibition of ENaC was prevented by washing out accumulated metabolites -LRB- Fig. 7c -RRB- . Furthermore , our experiments showed that hexokinase , a scavenger of extracellular ATP , produced the same effects on AS-Isc and NS-Isc as the perfusion of fresh KRB solutions -LRB- Fig. 9 -RRB- . In the presence of hexokinase , AS-Isc was significantly higher but the DIDS-sensitive NS-Isc was significantly lower than in control experiments , indicating that extracellular ATP caused the inhibition of ENaC and activation of NS-Isc , the later possibly via a CaCC . Extracellular ATP is a well-established agonist for purinergic receptors , leading to an increase of intracellular -LSB- Ca2 + -RSB- and activation Ca2 + - activated Cl - channels -LSB- 24 , 37 , 38 -RSB- . In parallel , ATP attenuates amiloride-sensitive Na + absorption in a variety of tissues including airway -LSB- 39 -- 41 -RSB- and renal epithelia -LSB- 42 -- 44 -RSB- . Similarly , activation of CFTR also increases Cl − secretion and inhibits ENaC in native epithelial tissues -LSB- 14 , 45 -RSB- . Because CFTR has been suggested to potentiate ATP release -LSB- 46 , 47 -RSB- and it has been shown to be expressed in this MDCK cell line -LSB- 10 -RSB- , we examined a possible contribution of ATP release to the time-dependent transport in FL-MDCK cells . As shown in Fig. 8a , addition of 100 μM ATP stimulated anion secretion , possibly via a Ca2 + - activated Cl − channel , which was inhibited by DIDS , and this effect was prevented in the presence of hexokinase -LRB- Fig. 8b -RRB- . Endogenous release of ATP by the FL-MDCK monolayers was demonstrated by the bioluminescence assay experiments shown in Fig. 10 . Consistent with measurements in cortical CD -LRB- see -LSB- 48 -RSB- -RRB- , ATP was shown to be released into the apical solution under basal conditions , and this release was augmented more than three-fold by cAMP treatment . In contrast , there was no significant release of ATP into the basolateral solution either without or with cAMP treatment . ATP has been shown to inhibit AS-Isc and stimulate NS-Isc in other epithelia of distal nephron origin , including M-1 -LSB- 43 -RSB- , mIMCK-K2 -LSB- 42 -RSB- , and A6 cells -LSB- 49 -RSB- . Release of endogenous ATP into the extracellular medium has been proposed as a general response to hypotonicity and mechanical stimulation in many types of cells , including MDCK and A6 cells -LSB- 49 , 50 -RSB- . Ostrom et al. -LSB- 51 -RSB- demonstrated that mechanical stimulation increases ATP release from MDCK cells as well as COS-7 and HEK-293 cells , and they proposed that ATP acts to alter the set point for a variety of signal transduction pathways . Praetorius et al. -LSB- 50 -RSB- also observed that mechanical stimulation caused ATP release in MDCK cells and that this release was associated with a transient increase in -LSB- Ca2 + -RSB- i. Because of the opposite actions of ATP in the extracellular apical membrane , it can serve to shift the balance between active NaCl absorption and active NaCl secretion in the MDCK cell , as shown by the hypothetical model in Fig. 11 . It is also quite important to note that the evidence we have presented for ATP accumulation in the extracellular fluid with longer incubation periods in Ussing chambers raises a caution for all such experiments , and it suggests that recirculation of the bathing solution , which has been in common use , should be avoided . Fig. 11Model for the role of adenosine triphosphate -LRB- ATP -RRB- in regulating Na + absorption and Cl − secretion in Madin-Darby canine kidney -LRB- MDCK -RRB- epithelia . The epithelial Na + channel -LRB- ENaC -RRB- mediates active Na + absorption and produces the amiloride-sensitive component of the short-circuit current -LRB- AS-Isc -RRB- . Because of the increased Na + conductance produced by ENaC , it also depolarizes the apical membrane , producing a negative transepithelial voltage -LRB- Vte -RRB- of − 30 mV . The basolateral membrane contains the ubiquitous Na , K-ATPase and , putatively , an Na-K-2Cl cotransporter , which actively accumulates Cl − in the cell . Cl − can then move across the apical membrane down its electrochemical potential gradient through Cl − channels , which include the glibenclamide-sensitive cystic fibrosis transmembrane conductance regulator -LRB- CFTR -RRB- channel , and a 4 , 4 ' - diisothiocyano-2 , 2 ' - disulfonic acid stilbene -LRB- DIDS -RRB- - sensitive , calcium-activated Cl - channel -LRB- CaCC -RRB- . The net secretory movement of Cl − gives rise to the amiloride-insensitive component of the short-circuit current -LRB- NS-Isc -RRB- . Our results show that ATP is secreted across the apical membrane . The pathway for ATP secretion is not known , but it has been proposed that it may also occur via CFTR -LSB- 46 -RSB- . When ATP accumulates in the apical extracellular space -LRB- e.g. , when the solution bathing this space is restricted in volume or is recirculated -RRB- , ATP inhibits ENaC and stimulates CaCC -LRB- and possibly CFTR or other Cl − channels -RRB- , resulting in a fall in AS-Isc and an increase in NS-Isc Implications for renal pathophysiology It must be recognized that the FL-MDCK cell line suffers from the limitations of any cell culture system when attempting to apply these results to an understanding of normal renal physiology or pathophysiology . Furthermore , even the type-1 MDCK cells used in these studies are heterologous , and their origin can not be ascribed definitively to any nephron segment . Nevertheless , the ability of ATP to inhibit ENaC-mediated Na + absorption and stimulate Cl − secretion , as shown in these experiments with FL-MDCK cells and in similar epithelia -LSB- 42 , 43 , 49 -RSB- , suggests a role for such signaling in the nephron . Significant amounts of ATP are released from epithelial cells originating from all regions of the nephron , and multiple P2Y and P2X receptors have been identified in all segments of the nephron -LSB- 48 -RSB- . Thus , changes in the luminal ATP concentration in the cortical CD and other aldosterone-sensitive segments of the distal nephron , in which ENaC mediates Na + transport , may be a physiologic signal for diminished Na + reabsorption . Also , Wilson et al. -LSB- 19 -RSB- demonstrated significant accumulation of ATP in cyst fluid obtained from patients with autosomal dominant polycystic kidney disease . Thus , based on our observations in MDCK cells , it seems reasonable to speculate that ATP might favor cyst enlargement and consequent exacerbation of polycystic disease by inhibiting Na + reabsorption and stimulating Cl − secretion -LSB- 19 , 52 -RSB- .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O

calcium-activated cl− channel
purinergic effects


Purinergic signalling in the subretinal space : a role in the communication between the retina and the RPE The retinal pigment epithelium -LRB- RPE -RRB- is separated from the photoreceptor outer segments by the subretinal space . While the actual volume of this space is minimal , the communication that occurs across this microenvironment is important to the visual process , and accumulating evidence suggests the purines ATP and adenosine contribute to this communication . P1 and P2 receptors are localized to membranes on both the photoreceptor outer segments and on the apical membrane of the RPE which border subretinal space . ATP is released across the apical membrane of the RPE into this space in response to various triggers including glutamate and chemical ischemia . This ATP is dephosphorylated into adenosine by a series of ectoenzymes on the RPE apical membrane . Regulation of release and ectoenzyme activity in response to light-sensitive signals can alter the balance of purines in subretinal space , and thus coordinate communication across subretinal space with the visual process . Introduction The retinal pigment epithelium -LRB- RPE -RRB- lies between the outer segments of the photoreceptors and the choroidal blood supply -LRB- Fig. 1 -RRB- . The RPE combines the functions of epithelial and glial cells , providing a barrier while also supporting the neural photoreceptors and modulating their function . Tight communication between photoreceptors and the RPE is critical to coordinate the multiple levels of interaction , and the purinergic contribution to this communication is becoming apparent . The relevance of this purinergic input is emphasized by the many functional effects of P1 and P2 receptor stimulation and by the multiple mechanisms in place to regulate subretinal levels of purine agonists . As the dynamics of ATP release and extracellular conversion into adenosine will modify agonist availability , the modulation of these processes can exert a temporal control on purinergic signaling . The following review will outline the main interactions between the RPE and photoreceptors , describe the effects of stimulating purinergic receptors on both sides of subretinal space , and summarize how levels of ATP , ADP , and adenosine are manipulated in this microenvironment . Fig. 1Schematic illustration of the key components of purinergic signaling in the subretinal microenvironment . Stimulation of P2 receptors on the RPE can enhance transepithelial fluid absorption while P1 receptors can modulate phagocytosis . ATP released through CFTR and other Cl − channels can stimulate P2 receptors or be converted to ADP , AMP , and adenosine -LRB- Ado -RRB- by a series of ectonucleotidases present on the apical membrane of the RPE . By controlling the balance of extracellular purines available to stimulate these receptors these mechanisms can control levels of endogenous purines available to activate the receptors . While theoretically possible , it remains to be determined whether these subretinal purines can actually stimulate photoreceptors Purines and subretinal space RPE-photoreceptor interactions across the subretinal space The outer segments of the rods and cones are responsible for the initial stages of vision , converting photon energy into a series of enzymatic reactions that close the light-sensitive channels on the photoreceptor plasma membrane , hyperpolarize the cells , and reduce the release of glutamate from the synaptic terminals -LSB- 1 , 2 -RSB- . Efficient photoreceptor function depends upon both short-term and long-term support from the RPE . The critical nature of these interactions is evident from the rapid degeneration of photoreceptors in the absence of a healthy RPE layer and by the RPE localization of defective gene product in some forms of hereditary photoreceptor degeneration -LSB- 3 -RSB- . The apical membrane of the RPE is separated from the plasma membrane of the outer segments by an extracellular space of only 10 -- 20 nm -LSB- 4 -RSB- . Although small , this subretinal space contains a highly structured matrix which ensheathes the outer segments and extends to the RPE -LSB- 5 , 6 -RSB- . The presence of enzymes within this interphotoreceptor matrix emphasizes that this extracellular space itself is functionally active -LSB- 7 , 8 -RSB- . This intimate anatomical relationship between photoreceptors and the RPE reflects multiple functional interactions . For example , the RPE accepts , recycles , and exports central components of the phototransduction process -LSB- 9 -RSB- . The outer segments are continuously resynthesized , and the phagocytosis , degradation , and processing of shed outer segment tips by the RPE cells is central to this renewal -LSB- 10 -RSB- . The ion channels and transporters on the apical membrane of the RPE help regulate the ionic composition of the subretinal space -LSB- 11 -RSB- . As extracellular levels of ions can modify the ionic driving forces across the photoreceptor plasma membrane , these RPE transporters can influence the state of neural activity . The transport of fluid and ions from the apical membrane to basolateral membrane of the RPE is also one of the main forces keeping the retina attached -LSB- 12 -RSB- . The control of photoreceptor activity by light gives a rapid temporal dependence to some interactions between the photoreceptors and the RPE . The volume of subretinal space increases in response to light -LSB- 13 -RSB- , linking small changes in the ionic composition of the subretinal space with activity of the RPE transport mechanisms which maintain this volume -LSB- 14 , 15 -RSB- . Other processes are controlled on a diurnal cycle . The shed tips of the outer segments are phagocytosed by the RPE soon after the onset of light -LSB- 16 , 17 -RSB- . These processes can both be modulated by purine levels in subretinal space , indicating purinergic regulation is important over multiple time scales . Purinergic receptors on photoreceptors A2 adenosine receptors were localized to both the inner and outer segments of photoreceptor outer segments over a decade ago by Blazynski and colleagues -LSB- 18 -RSB- , with more recent reports emphasizing their functional role . A2 agonists inhibit the L-type Ca2 + channel on rod outer segments -LSB- 19 -RSB- and can inhibit the synaptic release of glutamate from rods , suggesting changes in adenosine levels in subretinal space could modulate light sensitivity -LSB- 20 -RSB- . The A2 agonist DPMA and the adenosine deaminase inhibitor EHNA reduce mRNA for opsin in rods , suggesting that endogenous levels of adenosine can downregulate opsin message at night -LSB- 21 -RSB- . EHNA and the A2A receptor agonist CGS21680 also increase the survival of chick embryonic photoreceptors in culture -LSB- 22 -RSB- , indicating a long-term neuroprotective role for adenosine . P2 receptors are also present in the photoreceptors . mRNA for the P2X2 receptor is expressed in the photoreceptor cell bodies , with immunohistochemical localization of the protein to outer segments -LSB- 23 -RSB- . In situ hybridization indicates the photoreceptor layer has the highest level of P2Y2 receptor of any region in the rabbit retina , although staining was not pronounced in monkey -LSB- 24 -RSB- . P2X7 receptors have recently been localized to photoreceptor synaptic terminals , with evidence for ecto-ATPase activity in the synapse , and functional evidence suggesting ATP augments transmission of the light response by rods -LSB- 25 -RSB- . It was suggested that ATP might be co-released from photoreceptors with glutamate , although this remains to be tested directly . Purinergic receptors on the RPE Stimulation of P1 receptors can have a considerable impact on RPE cells . A2 receptors have been recognized on cultured and fresh RPE cells for some time -LSB- 26 , 27 -RSB- , with in situ hybridization confirming the presence of A2A receptors in rat RPE -LSB- 28 -RSB- . Stimulation of A2 receptors reduces the rate of rod outer segment phagocytosis by RPE cells -LSB- 29 -RSB- , while application of adenosine to the apical membrane of chick RPE cells increases the basolateral Cl - conductance , the transepithelial potential , and the c-wave , and decreases the hyperpolarization of the basal membrane in response to light -LSB- 30 -RSB- . Although adenosine alone does not increase intracellular Ca2 + levels -LSB- 31 -RSB- , adenosine acts synergistically with ATP to elevate Ca2 + levels in human RPE cells by stimulating both A1 and A2A receptors -LSB- 32 , 33 -RSB- . Stimulation of A1 receptors with high doses of NECA increases the active transport of fluorescein across the RPE , while activation of A2A receptors decreases this transport , and by extension , transport of the ions that underlie fluid movement -LSB- 34 -RSB- . Stimulation of A1 and A2A receptors produces analogous increases and decreases , respectively , in the absorption of subretinal fluid blebs . This is consistent with the negative coupling of the A1 receptor and the positive coupling of the A2 receptors to adenylate cyclase , as increasing cAMP inhibits the transport of fluid across the RPE towards the choroid -LSB- 35 -- 37 -RSB- . The agonist 2-Cl adenosine reverses the deficit in phosphoinositide metabolism found in diabetic RPE cells -LSB- 38 -RSB- , suggesting effects on metabolism in addition to transport and phagocytosis . Multiple P2 receptors have been localized to the RPE . The P2Y2 receptor was initially characterized in cultured human RPE -LSB- 31 -RSB- , with subsequent reports localizing transcript for P2Y1 , P2Y2 , P2Y4 , and P2Y6 in the rat RPE/choroid -LSB- 39 -RSB- and for P2Y1 and P2Y12 receptors in ARPE-19 cells -LSB- 40 -RSB- , and functionally identifying a P2X receptor in rat RPE cells -LSB- 41 -RSB- . ATP , ADP , and UTP induce numerous effects on RPE physiology -LSB- 32 , 33 , 42 , 43 -RSB- . While these effects likely involve multiple different receptor types , the contributions of the P2Y2 receptor have been explored in most detail to date . The P2Y2 receptor has been specifically localized to the apical membrane of fresh bovine RPE cells , and addition of ATP to this membrane transiently elevates Ca2 + , activates a basolateral Cl - conductance , inhibits an apical K + conductance , and increases the apical to basolateral flow of fluid -LSB- 43 -RSB- . This increased absorption of fluid from the subretinal space suggests P2Y2 receptor stimulation could reduce the excess fluid that accumulates in retinal edemas , and several reports have supported this theory . ATP , UTP , and the P2Y2 receptor agonist INS37217 decrease the size of subretinal fluid blebs when injected into subretinal space of rats -LSB- 44 -RSB- . In both normal and rds + / - mice with experimentally induced detachment , INS31217 improves the ERG recovery and decreased cell death -LSB- 45 -RSB- . INS37217 also reduces subretinal blebs in rabbits -LSB- 46 -RSB- . Injection of another P2Y2 agonist , INS542 , increases the active transport of fluorescein across the RPE , consistent with this upregulation of ion and fluid transport across the tissue -LSB- 47 -RSB- . Together these experiments emphasize the clinical potential of treating retinal edema with P2Y2 agonists . Regulation of purine levels in subretinal space While synthesized purinergic agonists may prove useful in treating some ocular disorders , the endogenous activation of P1 and P2 receptors in the subretinal microenvironment will ultimately be determined by availability of agonists . These levels are largely controlled by the release of ATP into the subretinal space , its conversion into other purines including adenosine , and the manipulation of adenosine by enzymes or transporters . Recent work has increased our understanding of both the stimuli that initiate changes in subretinal purine levels and the mechanisms that mediate these changes . Release of ATP by the RPE At least some of the ATP capable of stimulating the purinergic receptors on RPE cells is released from the RPE itself . The resulting autocrine stimulation ensures local delivery , and control , of purines to initiate the physiologic changes in the RPE . The release of ATP by RPE cells is triggered by multiple stimuli including osmotic stress , bFGF , UTP , NMDA , glutamate , and ATP -LSB- 39 , 40 , 48 -- 51 -RSB- . The ATP release following activation of NMDA receptors by glutamate may have the most interesting implications for communication across subretinal space , given that glutamate confers the light signal from photoreceptors to the rest of the visual system . Glutamate and the specific receptor agonist NMDA triggers ATP release from ARPE-19 cells , with the release inhibited by NMDA antagonist MK-801 , and by DCKA , which inhibits the glycine B binding site on NMDA receptors -LSB- 51 , 52 -RSB- . Although NMDA raises intracellular Ca2 + levels , this increase is prevented by eliminating ATP with apyrase , indicating autostimulation through released ATP is responsible for this Ca2 + signal . NMDA also triggers a release of ATP when applied to the intact bovine RPE eyecup -LSB- 51 -RSB- . The NMDA receptors and the ATP release sites have been functionally identified to the apical membrane of the bovine RPE , suggesting the neurotransmitter interactions could amplify the signal from any glutamate reaching subretinal space . The ability of both UTP and ATP to stimulate release of ATP from the RPE supports the theory that the system acts to amplify signals . When applied at greater than 1 μM , ATP triggers a secondary release of ATP peaking 10 min after the initial stimuli -LSB- 40 -RSB- . UTP also initiates a release in extracellular ATP with a similar delay -LSB- 48 -RSB- . The rise in ATP triggered by UTP is inhibited by the Cl - channel blocker NPPB , and UDP is much less effective at triggering release than UTP ; both observations suggest the P2Y2 receptor contributes to the increase in ATP more than diphosphokinase , although influence from the enzyme can not be ruled out -LSB- 53 -RSB- . Recent evidence suggests that ischemia may lead to the release of ATP from RPE cells . Chemical ischemia triggers a substantial ATP release from cardiac myocytes -LSB- 54 -RSB- , while changes in oxygen levels trigger ATP release in central chemoreceptors -LSB- 55 -RSB- . We found that exposure to sodium cyanide led to a rapid release of ATP from ARPE-19 cells -LRB- Fig. 2 -RRB- . As hypoxic and/or ischemic challenge may lead to changes in the expression of growth factors in RPE cells during certain ocular disorders such as macular degeneration -LSB- 56 -RSB- , and as purines can induce expression of VEGF in other cells -LSB- 57 -RSB- , this ATP release may contribute to growth factor signaling by the diseased RPE . Fig. 2Chemical ischemia triggers ATP release from ARPE-19 cells . ATP release was measured in the bath directly from cells plated in 96-well plates to which the luciferin - luciferase reaction mixture was added -LSB- 51 -RSB- . Left Levels of ATP in the bath after addition of 5 mM NaCN to the cells . Measurement began 1 min after addition of NaCN or control solution to wells . Right Levels of ATP measured at the peak , 3 min after addition of NaCN -LRB- n = 12 -RRB- . Levels were normalized to concurrent levels in control -LRB- n = 14 -RRB- . Symbols and bars represent mean ± SE , * p < 0.001 The particular conduit for ATP release varies with the stimuli . The release in response to hypotonic challenge is largely dependent upon CFTR , as it was prevented by the specific CFTR inhibitor CFTR172 in addition to the more general blocker glybenclamide -LSB- 50 -RSB- . While the precise mechanisms by which CFTR contributes to this release are not yet known , a role for CFTR in ATP release into subretinal space is consistent with the reduction of certain ERG components in cftr - / - mice -LSB- 58 -RSB- and with the ability of apical ATP to activate conductances associated with these ERG components -LSB- 43 -RSB- . The release of ATP is also largely blocked by the vesicular transport inhibitor brefeldin A , suggesting the two processes occurred in series whereby ATP efflux follows the insertion of vesicles containing CFTR into the plasma membrane . Although the Ca2 + chelator BAPTA blocks this ATP release -LSB- 50 -RSB- , raising Ca2 + alone with ionophore ionomycin does not itself initiate release -LSB- 48 -RSB- . This necessary but not sufficient contribution of Ca2 + also supports a role for vesicular insertion . In contrast to the release following hypotonic challenge , the ATP release in response to NMDA does not involve CFTR -LSB- 51 -RSB- . Release is blocked by NPPB , however , suggesting another type of anion channel could serve as a conduit for ATP release . The presence of parallel mechanisms coexisting on the same cell for ATP release triggered by either agonists or by cell swelling has also been reported in astrocytes -LSB- 59 -RSB- and may reflect the multiple roles of purinergic signaling within a given tissue . As both stimuli lead to release across the apical membrane into subretinal space , both are expected to influence signaling in the microenvironment . Interconversion of purines in subretinal space The interconversion of nucleotides and nucleosides each capable of stimulating distinct receptors makes the purinergic signaling system of particular interest in a confined region such as the subretinal space . The main enzymes responsible for dephosphorylating extracellular ATP on the RPE cells have been analyzed and a basic understanding of their regulation has begun . This section first describes the enzymes that act on ATP and ADP , followed by enzymes which convert AMP into adenosine . The dephosphorylation of extracellular ATP by RPE cells involves enzymes from multiple families -LSB- 40 -RSB- , as found in airway epithelial cells -LSB- 60 -RSB- . Degradation of ATP by the apical membrane of the fresh bovine eyecup and by ARPE-19 cells is inhibited by ARL67156 or βγmATP . Message for eNPP1 , eNPP2 , and eNPP3 is present in ARPE-19 cells , consistent with the preference of βγmATP for members of the eNPP family -LSB- 61 -RSB- . The cells also express NTPDase2 , and NTPDase3 , although the intermittent presence of NTPDase1 likely reflects a regulated process -LSB- 40 -RSB- . Ecto-alkaline phosphatase has no effect on ATP degradation in RPE cells , in contrast to its considerable contribution in airway epithelium -LSB- 62 -RSB- . The putative contribution from diphosphokinases to interconversion of subretinal purines is presently unknown . Extracellular AMP is rapidly dephosphorylated into adenosine in subretinal space . The production of adenosine from ATP at the apical membrane of the bovine RPE eyecup is inhibited by the ecto-5 ′ - nucleotidase inhibitor αβmADP , confirming a role for this enzyme -LSB- 63 -RSB- . The enzyme is localized to rat RPE and ARPE-19 cells immunohistochemically . Degradation of 5 ′ AMP is highest near the subretinal space of rat retina -LSB- 63 -RSB- , although localization in mouse indicated larger amounts of ecto-5 ′ - nucleotidase at the tips of adjacent Müller cells -LSB- 64 -RSB- . Levamisole does not inhibit the dephosphorylation of 5 ′ AMP by the RPE , consistent with the absence of substantial ecto-alkaline phosphatase in subretinal space . The presence of light may alter the levels of adenosine in subretinal space . Epinephrine is released at the onset of light -LSB- 65 -RSB- and stimulation of the RPE with epinephrine can decrease activity of ecto-5 ′ - nucleotidase -LSB- 63 -RSB- . While norepinephrine and phenylephrine lead to similar decreases in enzyme activity , prazosin and corynanthine block the effects of norepinephrine , implicating the α1 epinephrine receptor in the inhibition of ecto-5 ′ - nucleotidase -LSB- 63 -RSB- . The kinetics of inhibition are consistent with cleavage of the nucleotidase from its GPI anchor . The phagocytosis of rod outer segments is maximal shortly after light onset -LSB- 16 -RSB- , and this phagocytosis is inhibited by adenosine -LSB- 29 -RSB- . The ability of epinephrine released by the illuminated retina to reduce ecto-5 ′ - nucleotidase activity and consequently adenosine levels may relieve this inhibition and enhance the rate of phagocytosis at light onset . Physiologic effects of subretinal purines on the RPE and photoreceptors The number of purinergic receptors on both photoreceptor and RPE membranes suggests purines make multiple contributions to the physiology of the outer retina . Our increased understanding of how agonist levels in subretinal space are controlled has begun to indicate how and when this contribution may occur . Future research will involve applying these findings from isolated systems to intact RPE-photoreceptor models , and pursuing the role of defective purinergic regulation in ocular disease . While it is unlikely that ATP released across the apical membrane of the RPE can diffuse to these P2 receptors in the outer plexiform layer given the ecto-ATPase activity in the synaptic clef -LSB- 25 -RSB- , stimulation of receptors elsewhere on the photoreceptor membrane is possible . It would be interesting to determine whether ATP released from the RPE and converted to adenosine by ecto-nucleotidases can actually modulate the response to light by stimulating the A2A receptors on photoreceptor outer segments . The impact of purinergic signaling on chronic ocular diseases is also of interest , such as the role of ischemia-driven ATP release in VEGF production . While the small size of subretinal space can complicate pharmacologic manipulation within the intact RPE-photoreceptor complex , molecular approaches may provide new insight into how endogenous purines in subretinal space affect the physiology , and pathophysiology , of both RPE and photoreceptors .

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atp release
cftr
glutamate
microenvironment
photoreceptors
rpe