1Departments of Physiology and 2Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Submitted 1 April 2004 ; accepted in final form 3 June 2004
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ABSTRACT |
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cell volume; chloride secretion; aqueous humor formation
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In the present study, we examine the effects of cAMP on excised single-channel and whole cell currents and on cell volume in native bovine PE cells. Our results suggest that cAMP activates maxi-Cl channels in PE cells, facilitating Cl release from the PE cells to the ciliary stroma. This Cl recycling process may potentially serve as a pathway to regulate net aqueous humor formation.
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MATERIALS AND METHODS |
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Patch clamp measurements.
Micropipettes for excised patches and whole cell configurations with resistances of 510 and 24 M, respectively, were prepared from Corning glass (cat. no. 7052; World Precision Instruments, Sarasota, FL) using a Flaming/Brown micropipette puller (model P-97; Sutter Instruments). Micropipettes were coated with Sylgard (World Precision Instruments) and fire-polished with a microforge (model MF-830; Narishige). Recordings were conducted in a perfusion chamber connected to a Ag/AgCl pellet via a 3 M KCl agar bridge at room temperature (2224°C). Single-channel and whole cell currents were measured by using either Axopatch 1B or 1D patch clamps (Axon Instruments, Foster City, CA) coupled to an external Bessel filter (model 990C; Frequency Devices, Haverhill, MA). Single-channel recordings were acquired at 10 kHz and filtered at 2 kHz; whole cell measurements were obtained at 2 kHz and filtered at 500 Hz. For excised patches, the holding potential (Vh) was 0 mV, the command potentials were from 80 to +80 mV in 20-mV steps, and the upward current deflections depicted inward currents, and vice versa. The duration of each command step was 4 s, with intervening periods of 1.2 s at Vh. For whole cell measurements (Vh was also 0 mV) the step pulses ranged either from 100 to +80 mV in 20-mV increments (the duration of each command potential was 300 ms) or from 80 to +80 mV in 40-mV steps (each command pulse lasted for 4 s). In both cases, the upward current deflections indicated outward currents and vice versa. Data were acquired with the Axon Digidata using pClamp 8.2 software (Axon Instruments, Foster City, CA). Analysis was conducted by using Axon Instruments Clampfit 8.2 software.
Cell volume measurements. Cell volume was monitored by measuring cell area using calcein fluorescence (37). Coverglasses were mounted in a chamber connected to a Nikon Diaphot microscope. Fresh PE cells were loaded with 4 µM calcein-AM and 0.02% Pluronic for 3040 min and then perfused with isotonic Tyrodes solution for 30 min before initiating the experiment. Calcein was excited every 20 s at 488 nm, and light emitted at 520 nm was detected with an IC-200 charge-coupled device camera (Photon Technology International, Princeton, NJ). Cell area was determined from the number of pixels detected above threshold within the cell region. Results were analyzed by using Imagemaster software (Photon Technology International). Experiments were conducted at room temperature.
Solutions and pharmacological agents. For single-channel recordings, the baseline solutions for the micropipette and for the bath were identical and contained (in mM): 130 NaCl, 20 sucrose, 10 HEPES, 0.674 CaCl2, 1.1 EGTA, and 0.1 GTP (290 mosmol/kgH2O, pH 7.4). Cl concentrations were reduced by replacing NaCl with isosmolal amounts of sucrose. For whole cell measurements, the standard bathing solution contained (in mM): 140 NMDGCl, 0.5 MgCl2, 10 HEPES and 1.2 CaCl2 (290 mosmol/kgH2O, pH 7.4). The micropipette solution contained (in mM): 140 NMDGCl, 1.2 MgCl2, 10 HEPES, 1 EGTA, 2 ATP and 0.5 GTP (290295 mosmol/kgH2O, pH 7.3). Where appropriate, the Cl concentration in the micropipette was reduced to 30 mM by equimolar substitution of aspartate.
For cell volume measurements, the isotonic Tyrodes bathing solution contained (in mM): 110 NaCl, 15 HEPES, 2.5 CaCl2, 1.2 MgCl2, 4.7 KCl, 1.2 KH2PO4, 30 NaHCO3, and 10 glucose (295305 mosmol/kgH2O, pH 7.35). Solutions were made hypotonic by reducing the concentration of NaCl from 110 to 55 mM (240250 mosmol/kgH2O).
All chemicals were reagent grade. cAMP, 8-bromo-cAMP (8-Br-cAMP), 5-nitro-2-(phenylpropylamino)benzoate (NPPB), and SITS were purchased from Sigma (St. Louis, MO). All solutions were filtered through a 0.22-µm Millipore filter before use.
Statistics. Data are presented as means ± SE, where n is the number of experiments. The statistical significance of the differences was evaluated by using either one-way ANOVA followed by the Student-Newman-Keuls test or Students paired t-test. P < 0.05 was considered statistically significant.
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RESULTS |
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Effects of cytoplasmic Cl concentrations on maxi-Cl channel activity.
As noted above, PE cells are primarily involved in Cl uptake, thereby increasing the intracellular Cl concentration. The increased Cl might either diffuse to the NPE cells for subsequent Cl secretion into the posterior chamber or return to the ciliary stroma through Cl efflux pathways. We tested whether the intracellular Cl concentration itself might modify the behavior of the cAMP-activated maxi-Cl channels. This was done by reducing cytoplasmic NaCl concentration of inside-out patches from 130 to either 65 or 30 mM while maintaining extracellular NaCl concentration constant at 130 mM. Decreasing cytoplasmic Cl concentration from 130 to either 65 or 30 mM caused a stepwise inhibition of the maxi-Cl currents in the presence of cytoplasmic 500 µM cAMP. The inhibition could be explained partly by a reduction of Po at all potentials. The inhibition was, however, more pronounced when Vm was negative. The plots of Po as a function of Vm under different cytoplasmic Cl concentrations (130, 65, and 30 mM) are summarized in Fig. 3. In addition to the reduction of Po, cytoplasmic Cl concentration reduced channel conductance. The decrease in Cl channel conductance was observed with both inward and outward currents, the reduction being greater for inward currents (Fig. 7). Reducing cytoplasmic Cl concentration from 130 to 30 mM lowered Cl conductance by 30% when Vm was positive, whereas the reduction was
70% when Vm was negative. Taken together, decreasing the cytoplasmic Cl concentration caused a significant inhibition of maxi-Cl channels in excised inside-out patches, which could be ascribed to reductions in both Po and channel conductance.
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Stimulation of whole cell Cl currents by 8-Br-cAMP. To learn whether cAMP-activated maxi-Cl channels contributed significantly to PE-cell macroscopic currents, we studied the effects of 8-Br-cAMP and of the Cl channel blocker NPPB on whole cell current measurements. In symmetrical 140 mM NMDGCl, the addition of 500 µM 8-Br-cAMP reversibly stimulated the whole cell currents at all potentials (Fig. 8A). It was noted that the cAMP-activated currents displayed less outward rectification than did the baseline currents (Fig. 8B), suggesting that the cAMP-activated conductances were different from the baseline Cl conductances. These measurements were obtained with our standard whole cell protocol involving step changes in voltage of relatively brief duration (300 ms). For comparison with the single-channel records, we also measured whole cell currents with voltage steps of longer duration. With the prolonged voltage pulses of 4 s, the cAMP-activated whole cell currents showed significant current inactivation at both +80 (P < 0.01) and 80 mV (P < 0.05) but not at 40 and +40 mV (P > 0.05, n = 12). A typical experiment showing the voltage-dependent whole cell current inactivations is shown in Fig. 9. This is consistent with the channel inactivation due to Vm-dependent closing of maxi-Cl channels as displayed in excised inside-out patches (Fig. 3). These results strongly suggest that the cAMP-activated Cl channels observed with excised patches contribute currents that can be detected by macroscopic measurements, as well.
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DISCUSSION |
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Maxi-Cl channels share many similarities with VDAC including unitary channel conductance, multiple subconductance states, linear current-voltage (I-V) relationship, voltage dependence, anion selectivity, and sensitivity to Cl channel blockers (6, 33, 42, 44). The molecular identity of maxi-Cl channels is likely porin-1 (VDAC-1, porin-31HL), which has been localized in the plasma membrane of cultured T-lymphoblastic leukemia CEM cells (2) and astrocytes (15). Although plasma-membrane maxi-Cl channels have been recognized in many cells (6), including PE cells (38) for two decades, their physiological roles have been uncertain (1, 44, 47, 50), primarily because of the voltage-dependence of their activity. Their maximum Po is displayed within a narrow voltage range (20 to +20 mV), far from the Vm of many cells.
It has been shown that Vm of both PE and NPE cells is approximately 60 mV in rabbit and shark ciliary epithelium (9, 26, 53). A membrane-permeant form of cAMP, 8-Br-cAMP, was reported to depolarize porcine ciliary epithelium by 10 mV (23). That action could reflect activation of Cl channels of either the NPE (10, 11, 20) or of PE cells (24). Consistent with the observation in transformed cultured cells (24), we have now documented that 8-Br-cAMP activates Cl channels of native bovine PE cells, facilitating Cl release and thereby reducing the cell volume. At least one of the channel targets is the maxi-Cl channel and the activation is likely to be direct, because perfusion with cAMP activates the channels in excised patches. Interestingly, cAMP has been recently reported (16) to have no effect on baseline whole cell currents of C1300 mouse neuroblastoma cells but to block the antiestrogen-induced activation of maxi-Cl channels; the inhibitory effect of cAMP was prevented by staurosporine. The basis for the diverse effects of cAMP on the two cell types is not apparent but might reflect differential expression of two distinct pathways for cAMP to modulate maxi-Cl channels, either by direct activation or by indirect inhibition through protein kinases (49).
Several lines of evidence suggest that the cAMP-activated maxi-Cl channels observed with excised patches contribute significantly to PE whole cell currents. First, both unitary maxi-Cl and whole cell currents are stimulated by the addition of cAMP, and the effects are prevented by NPPB. Second, measured as the mean value of initial 300 ms, cAMP-stimulated whole cell currents displayed a more linear I-V relationship than did baseline currents, consistent with the linear relationship displayed by cAMP-activated maxi-Cl channels. Third, the cAMP-activated unitary maxi-Cl and whole cell currents are both dependent on cytoplasmic Cl concentration, displaying an increase of currents at higher intracellular Cl concentration. Fourth, time- and voltage-dependent current inactivations are observed at Vm of ± 80 mV and not at ± 40 mV in both excised single-channel and whole cell measurements, consistent with the typical characteristics of maxi-Cl channels. In addition to maxi-Cl channels, it is possible that cAMP enhances whole cell currents by activating other Cl channels in PE cells. However, if an additional channel is targeted, it is unlikely to be CFTR, because Northern blot analysis has not detected mRNA for CFTR in human ciliary body (13). Moreover, patients with cystic fibrosis demonstrate normal aqueous flow rate compared with healthy subjects (34).
The cAMP-activated maxi-Cl channels observed in the present study were strongly influenced by cytoplasmic Cl concentration. Raising intracellular Cl concentration enhanced both inward and outward currents by a dual effect on Po and channel conductance. Under physiological conditions, the PE-cell cytoplasmic Cl concentration is determined by the rate of uptake from the stroma by electroneutral transporters and the rate of Cl diffusion through gap junctions to the NPE cells and final release into the aqueous humor. Thus the Cl concentration is expected to rise when the rate of PE-cell Cl uptake from the stroma exceeds the NPE-cell transport capacity to secrete Cl into the aqueous humor. The rise in PE-cell Cl concentration, accompanied by a rise in the counter-ion Na+, is predicted to present an osmotic gradient for water and cell swelling. Hypotonic swelling has been found to trigger NPPB-sensitive ATP release from cultured bovine PE cells (36). The ATP can occupy P2Y2 receptors, initiating sequential increases in free Ca2+ concentration, phospholipase A2 activity, PGE2 formation and release, occupancy of PGE2 receptors, formation of cAMP in Madin-Darby canine kidney epithelial cells (40), and transformed bovine PE cells (24). In other words, Cl-overloaded PE cells would be expected to swell, triggering a cascade of events leading to cAMP formation, and thereby activating the maxi-Cl channels directly. In agreement with the electrophysiological measurements, cAMP triggers a reduction of cell volume, which can be prevented by pretreatment with NPPB. Increased fluid transfer from the PE cells to the ciliary stroma is expected to reduce net aqueous humor secretion.
Although Cl overload could provide one source of delivery of cAMP to the PE cells through an ATP-release-triggered cascade, other mechanisms may prove of even greater physiological importance. For example, PE cells express 2-adrenergic receptors (21) so that sympathetic nerve endings located close to the ciliary epithelium in the ciliary processes could stimulate cAMP production in these cells. Multiple biologically active peptides are also thought to be released from those nerve endings (48). Included among the neuropeptides are VIP and substance P, both of which trigger increased production of cAMP (e.g., see Refs. 31 and 43).
The focus of the current work has been on the cAMP activation of the PE cell maxi-Cl channels and their potential physiological role in modifying net Cl secretion by the ciliary epithelium. However, strong published evidence indicates that maxi-Cl channels also mediate swelling-triggered ATP release by mouse mammary C127 cells (19, 44). Consistent with their findings, and in contrast to the cAMP insensitivity of release by transformed bovine PE cells (36), we have recently found that 8-Br-cAMP (500 µM) enhances isotonic ATP release by native bovine PE cells by 50% (Do CW, Reigada D, Mitchell CH, and Civan MM, unpublished data). The potential role of maxi-Cl-mediated ATP release in regulating aqueous humor formation is currently under study.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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