Adenosine stimulates Clminus channels of nonpigmented ciliary epithelial cells

David A. Carré1, Claire H. Mitchell1, Kim Peterson-Yantorno1, Miguel Coca-Prados2, and Mortimer M. Civan1,3

Departments of 1 Physiology and 3 Medicine, The University of Pennsylvania, Philadelphia, Pennsylvania 19104-6085; and 2 Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06510

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Ciliary epithelial cells possess multiple purinergic receptors, and occupancy of A1 and A2 adenosine receptors is associated with opposing effects on intraocular pressure. Aqueous adenosine produced increases in short-circuit current across rabbit ciliary epithelium, blocked by removing Cl- and enhanced by aqueous Ba2+. Adenosine's actions were further studied with nonpigmented ciliary epithelial (NPE) cells from continuous human HCE and ODM lines and freshly dissected bovine cells. With gramicidin present, adenosine (>= 3 µM) triggered isosmotic shrinkage of the human NPE cells, which was inhibited by the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) and niflumic acid. At 10 µM, the nonmetabolizable analog 2-chloroadenosine and AMP also produced shrinkage, but not inosine, UTP, or ATP. 2-Chloroadenosine (>= 1 µM) triggered increases of whole cell currents in HCE cells, which were partially reversible, Cl- dependent, and reversibly inhibited by NPPB. Adenosine (>= 10 µM) also stimulated whole cell currents in bovine NPE cells. We conclude that occupancy of adenosine receptors stimulates Cl- secretion in mammalian NPE cells.

cell volume; whole cell recording; short-circuit current; 2-chloroadenosine; adenosine 5'-triphosphate; purines

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE CILIARY EPITHELIUM secretes the aqueous humor from the underlying stroma into the posterior chamber of the eye (10). This epithelium is structurally unique in consisting of two layers of cells whose apical faces are apposed. The basolateral surfaces of the pigmented ciliary epithelial and nonpigmented ciliary epithelial (NPE) cells abut the stroma and aqueous humor, respectively. The cells within each layer and between the two layers intercommunicate through gap junctions (9, 20), forming a functional syncytium. After uptake at the stromal-syncytial interface, ions diffuse through the gap junctions and are released across the basolateral membranes into the aqueous humor. The small negative transepithelial potential provides a driving force for Na+ transfer from the stroma into the aqueous humor through the paracellular pathway in parallel with the transcellular pathway.

The circadian rhythm of aqueous humor formation is the most striking indication that this secretion is regulated. The rate of secretion in humans has been reported to be two- to threefold higher during the day than during the early morning hours (1). The basis for the phenomenon remains unknown, although attention has been addressed to the potential roles of many hormones and of sympathetic nerve activity (1). Among the mediators considered to be possible triggers of circadian rhythms has been adenosine (5). The potential role of purinergic agonists is of particular interest, since activation of adenosine A2 receptors has been associated with ocular hypertension (12), while occupancy of adenosine A1 receptors has been associated with ocular hypotension (11).

Despite the potential importance of purinergic regulation of aqueous humor formation, very little information is available concerning the effects of adenosine on isolated tissue preparations. Adenosine increases the adenosine 3',5'-cyclic monophosphate (cAMP) level of the rabbit iris-ciliary body (11) and acts synergistically with acetylcholine to increase the Ca2+ activity of NPE cells in rabbit ciliary processes (14). The purpose of the present study was to determine whether adenosine exerts effects on transport by the ciliary epithelial cells. For this purpose, we have studied the intact rabbit iris-ciliary body, immortalized lines of human NPE cells, and freshly dissected bovine NPE cells. The immortalized human NPE cells are known to possess several forms of adenosine and ATP receptors (23, 24).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Transepithelial measurements. Adult male Dutch belted rabbits weighing 1.8-2.4 kg (Ace Animals, Boyertown, PA) were anesthetized with pentobarbital and killed (2). Treatment and death were in accordance with the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Research. After enucleation, the iris-ciliary body was isolated, as previously described (2).

The pupil and central area between pupil and ciliary processes were occluded with a Lucite disk, and the iris-ciliary body was mounted between the two halves of a Lucite chamber (2). The annulus of exposed tissue was bounded by outer and inner diameters of 12 and 5 mm, respectively, exposing a projected surface area of 0.93 cm2. Preparations were bathed with a Ringer solution (T1; Table 1) that was continuously bubbled with 95% O2-5% CO2 for maintenance of pH 7.4. Where appropriate, tissues were also bathed with a Cl--free Ringer solution (T2; Table 1). The transepithelial potential was fixed at 0 mV and corrected for solution series resistance, and the short-circuit current (Isc) was monitored on a chart recorder.

                              
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Table 1.   Compositions of solutions used in transepithelial and volumetric measurements

Cellular models. The ODM (17) and HCE immortalized NPE cell lines were developed by M. Coca-Prados from primary cultures of human epithelium. Cells were grown in Dulbecco's modified Eagle's medium (DMEM; no. 11965-027, GIBCO BRL, Grand Island, NY; and no. 51-43150, JRH Biosciences, Lenexa, KS) with 10% fetal bovine serum (A-1115-L; HyClone Laboratories, Logan, UT) and 50 µg/ml gentamicin (no. 15750-011; GIBCO BRL) at 37°C in 5% CO2 (26). The medium had an osmolality of 328 mosmol/kgH2O. Cells were passaged every 6-7 days and, after reaching confluence, were studied within 6-10 days after passage.

NPE cells were also obtained from freshly dissected bovine ciliary processes by the method of Jacob (16). The tips of the ciliary processes were excised and washed three times in a small volume of Ca2+- and Mg2+-free phosphate-buffered saline (PBS) solution. The tips were then transferred to 20 ml of a PBS solution containing 0.25% trypsin and 0.02% EDTA and digested at 37°C for 20-30 min with agitation. Thereafter, 10 ml of DMEM containing 10% fetal calf serum and gentamicin (100 µg/ml) were added to halt enzyme activity, and the residual tips were discarded. The preparation was next triturated to further dissociate the single cells and was centrifuged. The pellet was resuspended in PBS and spun again to remove blood cells and detritus. The cells were occasionally studied within 4 h of isolation. More commonly, the pellets were resuspended in medium containing 10% fetal calf serum, added to coverslips, and incubated overnight.

Measurement of cell volume. After harvesting of a single T-75 flask by a widely used trypsinization approach (26), a 0.5-ml aliquot of the cell suspension (usually in saline solution V; Table 1) was added to 20 ml of the test solution. The isosmotic solution V (Table 1) was used at a pH of 7.4. Four aliquots of cells were studied on the same day. One aliquot usually served as a control, with the other three aliquots being exposed to different experimental conditions. Comparisons were drawn only between experimental and control aliquots harvested from the same flask on the same day. The sequence of studying the suspensions was always varied to preclude systematic time-dependent artifacts. The stability of the control measurements was found to be enhanced by preincubating all cells for ~30 min in the control suspending solution rather than directly transferring the cells from DMEM to the test solutions.

Cell volumes of isosmotic suspensions were measured with a Coulter counter (model ZBI-Channelyzer II), using a 100-µm aperture (7). As previously described (26), the cell volume (vc) of the suspension was taken as the peak of the distribution function. The time course of cell shrinkage in the test solution was fit with the monoexponential function
v<SUB>c</SUB> = (v<SUB><IT>t</IT>o</SUB> − v<SUB>∞</SUB>) ⋅ <IT>e</IT><SUP>−(<IT>t</IT>/&tgr;)</SUP> + v<SUB>∞</SUB> (1)
where vto is the initial volume, vinfinity is the steady-state volume, t is time, and tau  is the time constant. The data were fit to Eq. 1 by a two-step approach. First, nonlinear least-squares regression analysis (Sigmaplot) was conducted, permitting both vinfinity and tau  to vary. Similar values of vinfinity were estimated for the parallel aliquots of the present study. To maximize the accuracy of the calculated value of vinfinity , the average for the parallel measurements was taken of all estimates of vinfinity whose coefficient of variation was <= 10%. This mean value of vinfinity was then inserted in Eq. 1 to obtain the best estimate of tau  by nonlinear regression analysis. This two-step approach provided satisfactory fits of the isosmotic data to monoexponentials (see Figs. 2-4).

Ruptured-patch whole cell recording. Cells harvested by trypsinization were resuspended and permitted to settle and attach either to glass coverslips, which were then mounted in a perfusion chamber, or to the bases of plastic petri dishes. External bath (EB) solutions were adjusted to pH 7.4 (Table 2). Micropipettes were double pulled from Corning no. 7052 glass, coated with Sylgard, and were fire polished. The micropipette (MP) filling solutions used were MP1, MP2, or MP3 at pH 7.2 or 7.4, as indicated in Table 2. Sufficient CaCl2 was included to generate a free Ca2+ concentration of either 10 or 100 nM. With these solutions, micropipettes displayed resistances of 1.5-4.0 MOmega . The seals formed commonly had resistances of ~10-20 GOmega . After rupturing the membrane patch, the series resistance was measured to be 10 ± 2 MOmega (n = 10).

                              
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Table 2.   Compositions of solutions used for patch clamping

Data were acquired at 2 kHz using Axopatch-1B electronics and associated headstage (Axon Instruments, Foster City, CA). In the experiments conducted with cultured human NPE cells (see Figs. 5-8), series resistance was compensated by >= 80%. In a series of experiments conducted with freshly dissected bovine NPE cells, series resistance compensation was not used, and the reduced current-voltage (I-V) relationships were corrected a posteriori for the calculated voltage drops across the series resistances.

The time courses of the whole cell currents (see Figs. 5 and 6) are presented as the raw measurements. The reduced I-V relations (see Figs. 7 and 8) have been corrected (19) for the junction potentials measured with reference to a flowing 3 M KCl junction, whose values were (in mV, filling solution with respect to perfusate) 1.50 ± 0.08 (MP1/EB1H), -3.43 ± 0.05 (MP2/EB1H), -1.64 ± 0.02 (MP2/EB2H), 6.69 ± 0.09 (MP1/EB1L), 3.73 ± 0.03 (MP2/EB1L), and 6.39 ± 0.04 (MP2/EB2L). The compositions of the pairs of solutions (MP/EB) are presented in Table 2.

The basic voltage protocol was to hold the potential at 0 mV, with cycles of step changes to ±40 and ±80 mV every 10 s. Each step usually lasted 1.3 s, with intervening periods of 0.7 or 1.3 s at the holding potential. In one-half the experiments, the protocol was modified to also include a voltage step of -20 mV (see Figs. 5-8).

Chemicals. All chemicals were reagent grade. Niflumic acid, adenosine, 2-chloroadenosine, inosine, AMP, ATP, UTP, gramicidin, and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) were purchased from Sigma Chemical (St. Louis, MO), and staurosporine and 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) were bought from Biomol Research Laboratories (Plymouth Meeting, PA).

Data presentation. Values are means ± SE, where n is the number of experiments. The probability (P) of the null hypothesis was tested with Student's two-tailed t-test.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Transepithelial measurements. The results of a series of eight experiments are presented in Fig. 1. Each rabbit iris-ciliary body served as its own series control. After a period of stable recording, 100 µM adenosine was added to the aqueous side solution bathing each tissue and then removed by perfusion with adenosine-free solution. In one-half of the eight experiments, tissues were next bathed in methylsulfonate (Cl--free) solution and then exposed to a repeat application of 100 µM adenosine. In the other four experiments, tissues were exposed to 5 mM BaCl2 on both surfaces (by isosmotic substitution for NaCl) and subsequently to adenosine. The absolute means ± SE for the baseline open-circuit transepithelial potential and Isc of the eight preparations were -0.96 ± 0.08 mV (with reference to stroma) and 6.93 ± 0.96 µA, respectively.


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Fig. 1.   Change in short-circuit current (Delta Isc) produced by adding adenosine to aqueous solution bathing isolated rabbit iris-ciliary bodies. Response to 100 µM adenosine was inhibited by substituting methylsulfonate (solution T2, Table 1) for Cl- (solution T1, Table 1) on both surfaces but was not inhibited by 5 mM Ba2+ added isosmotically to both surfaces.

After a lag time of 2-3 min, adenosine produced a stimulation of Isc that reached a peak change of 0.29 ± 0.04 µA within 20 min and decayed very slowly to 0.20 ± 0.03 µA by 45 min after addition of purine. The stimulation elicited by 100 µM nucleoside was abolished by Cl- removal. In contrast, the K+ channel blocker Ba2+ enhanced the magnitude of the adenosine-induced stimulation, presumably by blocking concurrent adenosine-triggered K+ secretion.

We concluded that adenosine was probably binding to purinergic receptors at the aqueous surface of the NPE cells, thereby triggering effects on ion transport across the epithelium. One of these effects was likely to have been stimulation of Cl- release through Cl- channels of the NPE cells, although other interpretations were possible. Given the complexity of the intact ciliary epithelium, we pursued this hypothesis by studying NPE cells in isolation by measuring both cell volume and whole cell currents.

Isotonic shrinkage of human NPE cells. Figure 2, A and B, demonstrates that adenosine produced a significant graded stimulation of isotonic shrinkage. A similar protocol was followed in the experiments of both panels. The control aliquot of cells received no adenosine, and another aliquot was exposed to the maximal concentration (10 mM) of adenosine at the time of initial suspension. The intermediate concentrations were 1 and 3 µM (Fig. 2A) and 10 and 100 µM (Fig. 2B). Adenosine triggered a shrinkage at 1 µM that was not significant at the 0.5 probability level but was highly significant (P < 0.001) at all higher concentrations. The half-maximal concentration (EC50) in Fig. 2A was <3 µM. However, biological variance was detectable among experiments conducted in different weeks, largely as a function of passage number. The EC50 in Fig. 2B was ~10 µM. The solid lines are the nonlinear least-squares regression fits to Eq. 1. Details concerning the values of the fits to the monoexponentials are provided in the legends to Figs. 2-4.


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Fig. 2.   Effects of adenosine on volume of HCE human nonpigmented ciliary epithelial (NPE) cells in isosmotic (Iso) suspension. A (n = 4) and B (n = 4) present data from 2 sets of experiments. Gramicidin (Gram; 5 µM) was always included in suspending solution (solution V, Table 1) to provide a constant pathway for cell K+ release. Both series contained aliquots without adenosine (as control) and with 10 mM adenosine (for maximal stimulation). Intermediate concentrations of 1 and 3 µM (A) and 10 and 100 µM (B) were added. Data are means ± SE. Solid lines are fits obtained by nonlinear least-squares regression analysis, using monoexponential equation vc = (vto - vinfinity ) · <IT>e</IT><SUP>−(<IT>t</IT>/&tgr;)</SUP> + vinfinity , where t is time, vto is initial volume (taken to be 100%), vinfinity is steady-state volume, and tau  is the time constant. Values of vinfinity (calculated as in MATERIALS AND METHODS) were 92.7 ± 0.9% (A) and 91.2 ± 0.4% (B). Calculated values of tau  (in min) for A: 85.4 ± 13.7 (control), 57.5 ± 8.2 (1 µM), 13.2 ± 1.1 (3 µM), and 7.8 ± 0.6 (10 mM); and for B: 234.8 ± 49.0 (control), 31.8 ± 0.8 (10 µM), 25.4 ± 1.1 (100 µM), and 16.5 ± 1.6 (10 mM). Adenosine triggered shrinkage at all concentrations. Stimulation at 1 µM was not significant at 0.05 probability level but was highly significant (P < 0.001) at all other concentrations.


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Fig. 3.   Effects of 10 µM concentrations of adenosine, AMP, ATP, UTP, 2-chloroadenosine, and inosine on volumes of HCE human NPE cells in isosmotic suspension. Symbols and fits are as in Fig. 2. Values of vinfinity (calculated as in MATERIALS AND METHODS) were 95.0 ± 0.6% (A) and 93.9 ± 0.8% (B). Calculated time constants (in min) for A (n = 3) were: 104.6 ± 12.6 (control), 10.8 ± 1.3 (adenosine), 141.7 ± 29.5 (ATP), and 164.0 ± 30.9 (UTP); and for B (n = 4): 54.6 ± 5.4 (control), 6.6 ± 0.4 (AMP), 13.3 ± 1.7 (2-chloroadenosine), and 47.6 ± 3.7 (inosine). Shrinkage was significantly produced by adenosine (P < 0.001), AMP (P < 0.001), and 2-chloroadenosine (P < 0.001).


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Fig. 4.   Interactive effects of adenosine with staurosporine or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) on volume of ODM and HCE human NPE cells in isosmotic suspension. Symbols and fits are described in legend for Fig. 2. In presence of 5 µM gramicidin, values of vinfinity (calculated as in MATERIALS AND METHODS) were 93.6 ± 0.6% (A) and 91.2 ± 0.7% (B). A: significant shrinkage of HCE human NPE cells was initiated by 0.3 µM staurosporine (P < 0.005), 10 mM adenosine (P < 0.001), or both agents together (P < 0.001; n = 4). Calculated time constants (in min) for A: 83.6 ± 6.0 (control), 25.2 ± 3.7 (staurosporine), 8.8 ± 0.7 (adenosine), and 9.7 ± 1.0 (staurosporine + adenosine). Adding adenosine significantly enhanced staurosporine-triggered shrinkage (P < 0.001), but adding staurosporine did not significantly alter adenosine-elicited shrinkage (P > 0.4). B: either alone or together, 10 mM adenosine and 1 mM DBcAMP stimulated shrinkage of both HCE (n = 4) and ODM (n = 6) human NPE cells (P < 0.001 for all). Similar results were obtained with both cell lines, so that the data have been analyzed together. Calculated time constants (in min) for B: 49.6 ± 3.6 (control), 21.2 ± 1.6 (adenosine), 23.2 ± 1.3 (cAMP), and 9.5 ± 0.9 (adenosine + cAMP). Rate of shrinkage produced by both agents together was significantly faster than that produced by either agent alone (P < 0.001).

The experiments of Figs. 2, 3, and 4A were conducted with 5 µM gramicidin included in the control and experimental suspending solutions. Gramicidin distributes to the plasma membrane, providing an exit port for K+, so that Cl- channel activity is the rate-limiting factor for release of solute (KCl) and water (6). As expected, adenosine stimulated a similar shrinkage in the absence of gramicidin (P < 0.001; Fig. 4B) at all concentrations applied [100 µM (n = 4), 1 mM (n = 4), and 10 mM (n = 10)]. The putative role of Cl- channels in the isosmotic shrinkage of Fig. 2, A and B, was supported by data obtained with Cl- channel blockers. Both 100 µM NPPB (n = 4, P < 0.001) and 500 µM niflumic acid (n = 4, P < 0.001) abolished the shrinkage produced by 10 mM adenosine (data not shown).

Adenosine receptors and second messengers. Figure 3, A and B, indicates that the transport effects of adenosine were mediated by occupancy of adenosine (P1-purinergic) receptors. Consistent with the data of Fig. 2, 10 µM adenosine produced an isotonic shrinkage of the human NPE cells (n = 3; P < 0.001; Fig. 3A). In contrast, neither ATP nor UTP exerted significant effects on parallel aliquots at the same concentration, an observation incompatible with mediation through ATP (P2-purinergic) receptors (13, 15). Nevertheless, the responses to adenosine might possibly have been mediated by inosine, a deaminase product of adenosine, or by some later metabolite of inosine. This possibility was addressed by comparing the effects of 10 µM concentrations of inosine, 2-chloroadenosine (a nonmetabolizable analog of adenosine), and another purine agonist of adenosine receptors (AMP) (15) on the rate of isosmotic cell shrinkage (n = 4, Fig. 3B). Inosine had no effect, but both 2-chloroadenosine and AMP significantly accelerated shrinkage (P < 0.001), consistent with the effects of these purines on adenosine receptors in other tissues (13).

The results of Fig. 4, A and B, provide preliminary information concerning the possible second-messenger cascades involved in adenosine's actions on NPE transport. Some effects of stimulating adenosine receptors are mediated by changes in the intracellular cAMP level (13). The possible interaction of cAMP and adenosine was examined by adding 1 mM DBcAMP, 10 mM adenosine, and both agents together to parallel aliquots of cells (n = 10, Fig. 4B). Each agent alone produced shrinkage (P < 0.001), and the combined effect was at least additive (P < 0.001). The time constant was significantly smaller for cells exposed to cAMP and adenosine together than to either agonist separately (P < 0.001), suggesting that cAMP does not mediate the adenosine-triggered shrinkage.

Protein kinase C (PKC) has also been implicated in the transport effects of adenosine. Inhibiting baseline PKC activity blocks adenosine activation of a Cl- channel in RCCT-28A cells (22). Actually, blocking PKC activity with 0.3 µM staurosporine stimulated shrinkage (n = 4, P < 0.005; Fig. 4A), as previously reported (6). This shrinkage was significantly accelerated by adding 10 mM adenosine together with the staurosporine (P < 0.001). The converse was not true. Adding the two agents together did not further affect the rate of shrinkage triggered by 10 mM adenosine alone (P > 0.4). Thus the actions of adenosine cannot be mediated solely through an inhibition of endogenous PKC activity.

Whole cell patch clamping. NPE cells were also patch clamped to examine the effects of purines under controlled conditions of intracellular composition and membrane potential. Figures 5-8 present results obtained by perfusing HCE human NPE cells with 2-chloroadenosine, the nonmetabolizable analog of adenosine. At a concentration of 10 µM, 2-chloroadenosine increased the whole cell currents of each of the 10 cells examined (P < 0.001; Figs. 5-8). The increase in current at the most depolarized potential (80 mV) was 1,420 ± 371 pA, corresponding to a stimulation of 136 ± 45% over baseline current. The effect was at least partially reversible in cells after prolonged washout with fresh perfusate (~10-15 min, e.g., Fig. 5), but this point was not systematically examined. The stimulation could be repeated in those cells, using the same 10 µM concentration of adenosine (Figs. 5 and 6). Stimulation of whole cell currents was also detectable in each of four experiments at a concentration of 1 µM (Fig. 6). The measured increase was 190 ± 92 pA, a stimulation of 36 ± 21%. For purposes of comparison, 10 µM adenosine increased the current by 1,519 ± 965 pA (199 ± 54%, P < 0.05) in the same set of four experiments.


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Fig. 5.   Time course of effects of 10 µM 2-chloroadenosine on ruptured-patch whole cell currents of an HCE human NPE cell. Each point of this time trace (and for Fig. 6) represents mean of 15 current measurements at 4-ms intervals, beginning 840 ms after onset of the voltage step. Positive outward current is presented upward. Command voltages were ±80, ±40, and -20 mV; holding potential was 0 mV. Micropipette and bath solutions were MP2 and EB2H and EB2L, respectively (Table 2). Raising external Cl- concentration from 28.5 (L) to 122.1 mM produced a small increase in outward currents. Perfusion with 10 µM 2-chloroadenosine (A) increased currents by ~200% at ±80 mV. Increased currents were highly dependent on external Cl- concentration and could be partially reversed during 6.4 min of washout. Reperfusion with 2-chloroadenosine again increased whole cell currents, which were reversibly reduced by 100 µM 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB; N) and by reducing external Cl- concentration (L).


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Fig. 6.   Effects of 1 and 10 µM 2-chloroadenosine on whole cell currents of an HCE human NPE cell. Perfusion with 1 µM 2-chloroadenosine (a) increased currents at ±80 mV by ~35%. This increase was reversed by one-third over a 2.8-min period of washout. Subsequent perfusion with 10 µM 2-chloroadenosine (A) produced a Cl--dependent increase in whole cell currents of ~135% at ±80 mV. Washout over the next 4.3 min reversed the stimulation by ~20%. Reperfusion with 10 µM 2-chloroadenosine elicited another stimulation, after which 100 µM NPPB (N) inhibited the whole cell currents. L, low-Cl- bath medium.


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Fig. 7.   2-Chloroadenosine difference currents as functions of time and external Cl- concentration. Difference currents measured in high (122.1 mM; A and C) and low (28.5 mM; B and D) Cl--containing solutions for experiment of Fig. 5. A large inactivating component is evident at +80 mV and a much smaller activating component at -80 mV. Lines are linear regressions fit over [-80, 40 mV] (high Cl-; C) and [-40, 80 mV] (low Cl-; D). Lowering Cl- concentration shifted reversal potential (Erev) by 37.3 ± 5.0 mV.


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Fig. 8.   2-Chloroadenosine difference currents as function of external Cl- concentration ([Cl-]o). Difference currents measured in high (122.1 mM) and low (28.5 mM) Cl--containing bicarbonate-free solutions for normalized data (n = 4). Lines are linear regressions fit over [-80, 40 mV] (high Cl-) and [-40, 80 mV] (low Cl-). Lowering the Cl- concentration shifted Erev from -24.9 ± 1.8 to 2.8 ± 1.5 mV; averaging the 4 values obtained by paired analysis led to a Delta Erev of 31.1 ± 3.6.

Three observations indicated that the stimulated currents largely reflected Cl- channels. First, purine-stimulated outward whole cell currents were strongly and reversibly dependent on the external Cl- concentration (Figs. 5-8). Second, the Cl- channel blocker NPPB (100 µM) produced a large reversible inhibition (57 ± 15%, n = 4, P < 0.05) of both outward and inward currents (Figs. 5-6). NPPB is known to block K+ as well as Cl- channels (21), but K+ was omitted from the micropipette filling solution for just this reason (Table 2). Third, the reversal potential (Erev) of the adenosine difference currents (Figs. 7-8) was shifted to more positive values by reducing the external Cl- concentration.

The difference in Erev (Delta Erev) in the high and low Cl--containing perfusates was less than the expected value of -37.3 mV for perfectly Cl--selective channels. This issue was most readily addressed in the set of four experiments conducted with the bicarbonate-free solutions (MP2, EB2H, and EB2L; Table 2). The ratio of the permeability of the anion (A) aspartate to that of Cl- (PA/PCl) through the 2-chloroadenosine-stimulated channels was estimated from the Goldman equation in the form
<IT>P</IT><SUB>A</SUB>/<IT>P</IT><SUB>Cl</SUB> = [(c<SUB>Cl</SUB>)<SUB>2</SUB> − (c<SUB>Cl</SUB>)<SUB>1</SUB> ⋅ (10)<SUP>&Dgr;E<SUB>rev</SUB>/59</SUP>]/
[(c<SUB>A</SUB>)<SUB>1</SUB> ⋅ (10)<SUP>&Dgr;E<SUB>rev</SUB>/59</SUP> − (c<SUB>A</SUB>)<SUB>2</SUB>] (2)
where c signifies the concentration of Cl- or aspartate anion, the subscripts 2 and 1 refer to the low and high Cl--containing perfusates, respectively, and Delta Erev is the reversal potential in high minus low Cl--containing perfusates. From Eq. 2, the mean measured value of Delta Erev (-32.5 ± 4.5 mV) is consistent with 2-chloroadenosine-stimulated channels whose permeability for aspartate relative to Cl- is very low [0.05 ± 0.05 (Fig. 8)].

The kinetics of the adenosine difference currents of Fig. 5 are presented in Fig. 7. Significant inactivation was noted only at 80 mV, with time constants of 284 ± 3 and 202 ± 3 ms in high (Fig. 7A) and low (Fig. 7B) external Cl- concentrations, respectively. Figure 7, C and D, presents the voltage dependence of the adenosine-stimulated currents measured early (4-24 ms) and late (1.17-1.19 s) after initiating each voltage step. Outward rectification was observed with external and internal Cl- concentrations of 122.1 and 22.9 mM, respectively. The rectification was far more prominent for the peak I-V relation, given the inactivating component at large depolarizing potentials (Fig. 7, A and B).

Freshly dissociated bovine NPE cells were also patch clamped to determine whether adenosine exerted similar electrophysiological effects on NPE cells other than simian virus 40-immortalized cell lines. In this series, the filling solution of the micropipette was MP3 and the perfusate was solution EB3 (Table 2), so that the Nernst potentials were (in mV) 0 (Cl-), -42 (K+), 78 (Na+), and 51 (for cation-nonselective channels). Adenosine (10 µM to 1 mM) stimulated whole cell currents of each of 13 cells with an Erev equal to the Cl- Nernst potential. In an initial series of nine experiments, adenosine increased the current at the most depolarized command voltage (80 mV) by 85 ± 24 pA (P < 0.01), a stimulation of 32 ± 10% over baseline current. In view of the known coupling of adenosine receptors to G proteins (15), GTP was included in the micropipette filling solution in four additional experiments [10 µM (n = 1); 100 µM (n = 3)]. For the entire series of 13 experiments, the mean increase in current at a command potential of 80 mV was 73 ± 18 pA (P < 0.005), corresponding to a stimulation of 28 ± 7% over baseline current. The percentage stimulations at the three adenosine concentrations applied were 12 ± 4% (10 µM, n = 4), 28 ± 6% (100 µM, n = 1), and 35 ± 10% (1 mM, n = 4).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Using Isc, cell volume, and whole cell currents as indexes of transport, the results demonstrate that adenosine and 2-chloroadenosine modulate the transport properties of NPE cells. Several observations indicate that the effects were triggered by occupancy of adenosine (purinergic P1 type) receptors. First, adenosine and AMP triggered isosmotic cell shrinkage, but ATP and UTP were ineffective at the same concentration (Fig. 3A), a ranking characteristic of P1 receptors and incompatible with ATP (purinergic P2 type) receptors (13, 15). Second, inosine (the deaminase product of adenosine) did not affect cell volume, whereas the nonmetabolizable analog 2-chloroadenosine stimulated both isosmotic shrinkage (Fig. 3B) and whole cell currents of HCE human NPE cells (Figs. 5-8). Third, the concentrations (1-3 µM) at which adenosine and 2-chloroadenosine stimulated shrinkage (Fig. 2A) and whole cell currents (Fig. 5) are consistent with the EC50 values at which these agonists stimulate both the A2b subtype (5-20 µM) and the A3 subtype (>1 µM) of P1 purinoceptors to alter intracellular cAMP (15). In this respect, it should be noted that Wax et al. (24) have reported that adenosine increased the cAMP content of both NPE and pigmented epithelial cells, but only at concentrations >100 µM (see p. 91 of Ref. 24). Lower concentrations have also been reported to have effects on the ciliary epithelium, but only in synergism with another agonist (14). Administered together with 10 µM acetylcholine, adenosine produced a marked synergistic increase in Ca2+ activity of rabbit ciliary processes with an EC50 of 0.25 µM. However, the change in Ca2+ activity produced by applying 1 µM adenosine alone was only 30 ± 17%, an effect of uncertain statistical significance (14).

From our results obtained with ion substitutions and channel inhibitors, at least one of the targets of the P1 purinergic receptors must be Cl- channels of the NPE cells. This conclusion is based on the observations that 1) the stimulation of Isc triggered by aqueous adenosine was entirely abolished by substituting methylsulfonate for Cl- (Fig. 1); 2) adenosine produced shrinkage of cultured human NPE cells, even when release of intracellular K+ was not rate limiting (Fig. 2), and this effect was inhibited by the Cl- channel blockers NPPB and niflumic acid; and 3) the currents activated by 2-chloroadenosine displayed a Cl--dependent Erev and were reversibly inhibited by the Cl- channel blocker NPPB (Figs. 5-8). Cl- channel activity of NPE cells has been posited to limit the rate of secretion (8), so that the stimulation of Cl- channels by adenosine should accelerate the rate of aqueous humor formation.

How P1 purinergic receptors are linked to Cl- channels is unclear. Occupancy of adenosine receptors A1, A2a, A2b, and A3 is thought to act at least partly through decreasing or increasing the intracellular cAMP concentration (15). However, the effects of very large concentrations of adenosine (10 mM) and DBcAMP (0.5 mM) appeared to be additive (Fig. 4B), presumably reflecting independent actions. Changes in PKC activity have also been thought to mediate some of the effects of adenosine (22). However, blocking PKC activity with even a large concentration of staurosporine (0.3 µM) was insufficient to replicate the adenosine-triggered stimulation (P < 0.001, Fig. 4A), whereas addition of the two agents was no more effective than the adenosine alone (P > 0.4). We conclude that some of the actions of adenosine may be mediated through an inhibition of endogenous PKC activity, but some must be mediated through a different signaling pathway and/or by targeting a different population of Cl- channels. Alternative possible mediators include intracellular Ca2+ (14) and G proteins (15). The latter possibility is particularly intriguing since all the known mammalian adenosine receptors (A1, A2a, A2b, and A3) are coupled to G proteins (15), and G proteins have been implicated in modulating Cl- channel activity of pigmented ciliary epithelial cells (18). These possibilities are currently under study.

    ACKNOWLEDGEMENTS

We thank Dr. Martin Pring for helpful discussions concerning statistical analysis of the data.

    FOOTNOTES

This work was supported in part by National Eye Institute Grants EY-10691 and EY-00785 for core facilities. C. H. Mitchell was partially supported by Visual Sciences Training Grant 5-T32-EY-07035-17 and Respiratory Training Grant 2-T32-HL-07027-21 in successive years.

Some of these results have been presented in preliminary form (3, 4).

Address for reprint requests: M. M. Civan, Dept. of Physiology, Univ. of Pennsylvania, Richards Bldg., Philadelphia, PA 19104-6085.

Received 3 September 1996; accepted in final form 25 June 1997.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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