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(Investigative Ophthalmology and Visual Science. 2006;47:2065-2074.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0228
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Effect of cAMP on Porcine Ciliary Transepithelial Short-Circuit Current, Sodium Transport, and Chloride Transport

Ying Ni,1 Renyi Wu,1,2 Wen Xu,1,2 Helmut Maecke,3 Josef Flammer,1 and Ivan O. Haefliger1

1From the Laboratory of Ocular Pharmacology and Physiology, University Eye Hospital Basel, Basel, Switzerland; 2The Second Affiliated Hospital, Medical College of Zhejiang University, Hangzhou, China; and the 3Division of Radiological Chemistry, University Hospital Basel, Basel, Switzerland.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To investigate the effects of 8-bromo-cAMP (cAMP) on porcine ciliary transepithelial short-circuit current (Isc) and transport of chloride (Cl–) and sodium (Na+).

METHODS. With Ussing-type chambers, cAMP-induced changes in Isc, electrical resistance (ER), and transepithelial 36Cl– and 22Na+ fluxes were measured. Drugs were applied to the nonpigmented epithelium (NPE) and/or pigmented epithelium (PE) side(s). The effect of IBMX (1, 5, or 10 µM; 3-isobutyl-1-methylxanthine) on Isc-increase induced by 8-bromo-cAMP on the PE side was also tested.

RESULTS. On the NPE side, a single concentration (10 µM, 100 µM, or 1 mM) of 8-bromo-cAMP induced a biphasic (transient peak followed by sustained plateau) Isc increase. On the PE side, 8-bromo-cAMP induced a similar but delayed biphasic Isc increase at 1 mM, a slight plateau-Isc increase at 100 µM, and no Isc increase at 10 µM. In the concentration–response curve, the cAMP-induced peak-Isc increase became significant at a concentration 10,000 times lower on the NPE than on the PE side. At 10 µM, the cumulative cAMP-induced Isc-increase reached its maximum on the NPE side, but was virtually nonexistent on the PE side. IBMX (a phosphodiesterase inhibitor) but not 8-CPT-6-Phe-cAMP (higher permeability than 8-bromo-cAMP) significantly increased the peak-Isc concentration–response curve induced by 8-bromo-cAMP (10 nM–1 mM) on the PE side. On the NPE but not the PE side, 10 µM 8-bromo-cAMP induced a significant but transient increase in net PE-to-NPE 36Cl– flux (1.03 ± 0.18 µEq/min per square centimeter; P < 0.001). Neither ER nor transepithelial 22Na+ flux was changed after cAMP exposure.

CONCLUSIONS. In porcine ciliary processes, apparently on the NPE side, cAMP triggers a biphasic (transient peak followed by a sustained plateau) Isc increase. Only the peak-Isc increase involves an increase in net PE-to-NPE Cl– transport.

In the eye, the ciliary epithelium of the ciliary body is the main site of aqueous humor production. It consists of a nonpigmented epithelial (NPE) layer that faces the aqueous humor side and a pigmented epithelial (PE) layer that faces the stromal side. ß-Adrenergic antagonists and {alpha}2-adrenergic agonists are drugs that inhibit aqueous humor production, and therefore they are commonly used to reduce intraocular pressure.1 2 3 At the same time, these drugs also decrease intracellular concentration of cAMP in the ciliary epithelium.4 5 6 This observation suggests that cAMP could be a mediator involved in aqueous humor production through the modulation of ionic and fluid transport across the ciliary epithelium.

The Ussing-type chamber is a system that can be used to measure ionic currents7 8 (short-circuit current: Isc) or ionic fluxes9 10 (such as 22Na+ or 36Cl–) across the ciliary epithelium. Both Na+ and Cl– have been proposed to be involved in the process of aqueous humor production under baseline conditions.11 12 In the present study, we primarily investigated how 8-bromo-cAMP modulates Isc and 36Cl– and 22Na+ transepithelial transport in isolated porcine ciliary processes and/or ciliary epithelial bilayer.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Tissue Preparation
Porcine eyes were obtained from a local abattoir immediately after the animal’s death. Eyes were cut in half at the equator, and the anterior half was placed in a Petri dish with the cornea facing down. Under a dissecting microscope, the lens, the remnant retina, the choroid, and the vitreous were removed. Then, the ciliary body with its underlying stroma was dissected as a ring from the sclera and the iris. For experiments, only radial sections (of about one fifth the surface of the ciliary body’s ring) were used.

For some experiments, isolated ciliary epithelial bilayers were prepared. In brief and after the procedure initially described by Sears et al.,13 eyes were first perfused through the posterior ciliary arteries for approximately 2 minutes with oxygenated Dulbecco’s modified Eagle’s medium (DMEM), and then for 45 minutes with DMEM containing 0.1% collagenase and 0.05% hyaluronidase (37°C). After having been perfused, eyes were prepared as just described, except that the epithelial bilayer was obtained by further peeling the epithelium from the underlying stroma.

Electrical Parameters Measurements
As described previously,7 8 14 a self-modified Ussing-type chamber with an exposure area of 0.1 cm2 was used. Each of the two hemichambers of the system was filled with 5 mL physiological Krebs-Ringer (mM: NaCl 118.05, KCl 4.69, KH2PO4 1.20, MgSO4 1.20, NaHCO3 25.01, EDTA 0.026, CaCl2 2.51, glucose 11.1, HEPES 10 [pH 7.4], 95% O2 and 5% CO2, 37°C). Each hemichamber was connected to two Ag/AgCl electrodes, a voltage and a current electrode (World Precision Instruments, Sarasota, FL). Electrodes were bridged with 3% agar-3 M KCl and connected to a DVC-1000 voltage/current clamp apparatus (World Precision Instruments). Signals were amplified (Cyto 721; World Precision Instruments), analogue-digital converted (Maclab 2e; ADInstruments, Castle Hill, Australia), and stored in real-time on a computer (Macintosh Powerbook 1400; Apple Computers, Cupertino, CA). With this system, Isc measurements were performed under voltage clamped conditions and PD measurements under current clamped conditions. Before conducting any experiments, the system was calibrated (~20 minutes) by monitoring any voltage differences between the two voltage electrodes and adjusting any difference to zero.

The tissues were then mounted between the two hemichambers (that were filled again with modified Krebs-Ringer physiologic solution). The preparations were left quiescent for approximately 60 minutes until the electrophysiological parameters Isc or PD reached their baseline. Before starting any experimental protocol, the electrical resistance (ER) was determined from the current deflections induced by offsetting the short-circuited condition by 500 µV.

Transepithelial Cl– and Na+ Flux Measurement
Using radioactive tracers (5 µCi 36Cl–, 7.5 µCi 22Na+), transepithelial fluxes (influx: JPE-NPE, and outflux: JNPE-PE) of Cl– and Na+ were determined in Ussing-chambers under short-circuited conditions. For these experiments paired radial sections obtained from the same ciliary body of an eye were mounted in two Ussing-chamber systems as described and were set in parallel. Once the electrophysiological parameters Isc reached their baseline (after approximately 60 minutes), the preparations were exposed to the radioactive tracers. One of the two systems was used to measure influx JPE-NPE. This was done by loading the hemichamber facing the PE with radioactive tracers (loading hemi-chamber) and comparing the radioactivity in this hemichamber with the radioactivity in the other hemichamber facing the NPE (collecting hemichamber). The other Ussing-chamber system was used to measure JNPE-PE. Outflux was measured by loading the hemichamber facing the NPE side (loading hemichamber) with radioactive tracers and comparing the radioactivity in this hemichamber with the radioactivity in the other hemichamber facing the PE (collecting hemichamber). The net transport (net transport: Jnet) was then calculated as the difference between the paired unidirectional influx JPE-NPE and outflux JNPE-PE.

To measure radioactivity over time, 100 µL samples were collected simultaneously from each hemichamber and replaced by an equivalent volume (100 µL) of Krebs-Ringer solution. Each sample was mixed with 3 mL scintillation cocktail and radioactivity was measured using a liquid scintillation counter (1500 TriCab; PerkinElmer, Meriden, CT) for 20 minutes.

The transepithelial unidirectional fluxes of Cl– (J'Cl) and Na+ (J'Na) were calculated using the following formula:

Formula
where J' is the unidirectional Cl– or Na+ flux through the area of ciliary processes studied (µEq/min per square centimeter) between two consecutive collections of samples n and n – 1 at time n (tn; in minutes) and at time n – 1 (tn–1; in minutes). In this formula, Cn and Cn–1 are the radioactivity (counts per minute: cpm) measured in the collecting hemichamber at time n and time n – 1, whereas Ln and Ln–1 the radioactivity measured in the loading hemichamber at time tn and at time tn–1. In this formula, [i] is the total concentration of ions Cl– or Na+ (micromolar per milliliter), V the volume (in milliliters) of the solution in each hemichamber, and A the area (in square centimeters) of tissue studied.

Experiment Protocols
In the first set of experiments, the changes in Isc induced by applying a single concentration (10 µM, 100 µM, or 1 mM) of 8-bromo-cAMP on the NPE side and/or the PE side(s) was measured over time in ciliary process preparations. In these set of experiments, the ER was measured every 5 minutes for 10 minutes before drug application and every 2 minutes for 10 minutes after 8-bromo-cAMP exposure, and then every 5 minutes for an additional 20 minutes.

In the second set of experiments, paired ciliary process preparations obtained from the same eye were studied in parallel and the changes in peak-Isc or -PD were measured after applying increasing cumulative concentrations of 8-bromo-cAMP either on the NPE (10 nM–30 µM) or on the PE (10 nM–1 mM) side. In the experiments where Isc was assessed, ER was measured after each drug application.

In the third set of experiments, the effect of different cAMP analogues that had, according to the providing manufacturer (Biolog-life Science Institute, Bremen, Germany; www.biolog.de/logkw.html), different relative lipophilicity (cell-membrane permeability) in comparison to cAMP (8-CPT-6-Phe-cAMP: 400; 8-bromo-cAMP: ~2, or 8-OH-cAMP: ~0.7) were studied. In this set of experiments, the drugs were applied in increasing cumulative concentrations (10 nM–30 µM) either on the NPE or on the PE side of ciliary processes or epithelial bilayer preparations. ER was measured like always before drug application and then at the end of the experiment.

In the fourth set of experiments, with and without preincubating the ciliary process preparations on both sides with one single concentration (1, 5, or 10 µM) of IBMX (3-isobutyl-1-methylxanthine, a nonspecific phosphodiesterase inhibitor) for approximately 30 minutes, the peak-Isc increase induced by applying 8-bromo-cAMP (10 nM–1 mM) on the PE side in an increasing cumulative manner were repeated. ER was measured like always before drug application and then repeated after IBMX application as well as at the end of the experiment.

In the fifth set of experiments, unidirectional ionic fluxes (J'Cl and J'Na) were assessed before and after application of a single concentration (10 µM) of 8-bromo-cAMP either on the NPE or on the PE side of ciliary processes. Samples were simultaneously collected from each hemichamber. The sampling rate was every 5 minutes for 10 minutes before 8-bromo-cAMP exposure. After 8-bromo-cAMP exposure, the sampling rate was every 2 minutes for another 10 minutes and then again every 5 minutes for an additional 20 minutes.

Drugs and Statistical Analysis
8-Bromo-cAMP was purchased from Sigma-Aldrich (Buchs, Switzerland). 8-OH-cAMP and 8-CPT-6-Phe-cAMP were bought from Biolog-life Science Institute. All cAMP analogues were prepared in physiological Krebs-Ringer solution. The radiolabeled isotopes 36Cl– and 22Na+ were purchased from GE Healthcare (Europe GmbH, Freiberg, Germany) and the scintillation cocktail from Perkin Life and Analytical Sciences (Wellesley, MA).

The ion fluxes (J'Cl, J'Na) are expressed in µEq/min per square centimeter. Results are given as mean ± SEM with n corresponding to the number of experiments (one eye per experiment) conducted. Data were analyzed using one-way ANOVA multiple comparison followed by Bonferroni correction with a two-tailed P < 0.05 considered to be statistically significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Baseline Isc and ER
Once tissues were mounted in the Ussing-type chambers electrophysiological parameters Isc and ER reached, after approximately 60 minutes, baseline values that remained stable for at least for 3 to 4 hours. The values of these electrophysiological parameters measured in baseline conditions in ciliary processes and ciliary epithelial bilayer are shown in Table 1 . The baseline value of ER (but not Isc) was significantly (P < 0.001) different between ciliary processes and epithelial bilayer preparations.


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  		TABLE 1. Baseline Electrophysiological Parameters

 
Effect of 8-Bromo-cAMP on Isc, PD, and ER
When the effect of a single concentration (10 µM, 100 µM, or 1 mM) of 8-bromo-cAMP on Isc and ER was studied over time, a difference in the Isc response esd observed between the NPE and PE side (Fig. 1) . On the NPE side, each concentration of 8-bromo-cAMP induced a significant Isc increase that was characterized by a transient (~2-minute) peak followed by a sustained (≥20-minute) plateau Isc increase. On the PE side, 8-bromo-cAMP induced no Isc increase at 10 µM, a slight sustained plateau- (but no peak-) Isc increase at 100 µM, and a delayed peak followed by a plateau Isc increase at 1 mM. In addition, although there was a time delay for approximately 4 minutes, the peak-Isc increase induced by applying 1 mM 8-bromo-cAMP on the PE side (10.94 ± 1.22 µA/cm2) apparently did not significantly differ (P = 0.25) from the one observed when this drug at the same concentration was applied on the NPE side (13.53 ± 2.18 µA/cm2; Fig. 1C ). Furthermore, applied simultaneously on both (NPE + PE) sides, each concentration (10 µM, 100 µM, and 1 mM) of 8-bromo-cAMP induced a very similar but not larger Isc increase than the one observed when same concentration was applied solely on the NPE side. No significant ER change was detected at any time after 8-bromo-cAMP application (Fig. 1) .


Figure 1
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  		FIGURE 1. Effect of a single concentration (A: 10 µM, B: 100 µM, or C: 1 mM) of 8-bromo-cAMP on Isc (left) and ER (right) over time in isolated ciliary processes when the drug was applied either on the PE side, or on the NPE side, or simultaneously on both (NPE + PE) sides of the preparation. When the drug was applied on the NPE side, at each concentration tested, 8-bromo-cAMP induced a peak- followed by a plateau-Isc increase that did not differ significantly from the Isc-increase observed when the drug was applied on both sides of the preparation. In contrast when 8-bromo-cAMP was applied on the PE side, at 10 µM, no Isc increase was noted, at 100 µM a small sustained plateau-Isc (but no peak-Isc) increase was observed, and at 1 mM a peak- followed by a plateau-Isc increase occurred. The latter peak-Isc increase was not significantly different, although delayed by approximately 4 minutes, from the one observed when the drug at the same concentration was applied to the NPE or simultaneously on both sides of the preparation. No significant changes in ER over time were ever observed. *P < 0.05, **P < 0.01, ***P < 0.001, before versus after drug application.

 
At the concentrations tested, when 8-bromo-cAMP was applied in an increasing cumulative manner either on the NPE or the PE side of the preparation, an increase in Isc and a change in PD (that became more negative toward the NPE side) was observed (Figs. 2 3) . The cumulative 8-bromo-cAMP-induced peak-Isc increase became significant (in comparison with that in vehicle control experiments) at a concentration that was 10,000 times lower when the drug was applied on the NPE (0.1 µM) than on the PE (1 mM) side. On the NPE side, the cumulative 8-bromo-cAMP-induced peak-Isc increase reached its maximum at 10 µM. In contrast, on the PE side at the same concentration (10 µM), 8-bromo-cAMP did not induce a significant Isc increase. In addition, the changes in Isc evoked by 8-bromo-cAMP were proportionally closely related to the changes in PD, but not associated with any statistically significant (P = 0.42–0.89) change in ER. Finally, after the application of the highest concentration of 8-bromo-cAMP, the changes in Isc and PD remained stable for at least 20 minutes (Fig. 2) .


Figure 2
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  		FIGURE 2. Original recordings of Isc- and PD-peak responses over time induced by 8-bromo-cAMP in isolated ciliary process preparations. The drug was applied (vertical arrows) in a cumulative and increasing concentration manner either (A) on the nonpigmented epithelium (NPE; 10 nM–30 µM) or (B) on the pigmented epithelium (PE; 10 nM–1 mM) side of the preparation. During Isc measurements, before and after drug application, ER was determined from the current deflections induced by offsetting the short-circuited condition by 500 µV (spikes).

 

Figure 3
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  		FIGURE 3. Concentration–response curves of peak Isc (A), PD (B), and ER (C) induced by applying different concentrations of 8-bromo-cAMP in a cumulative and increasing manner either on the nonpigmented epithelium (NPE; 10 nM–30 µM) or on the pigmented epithelial (PE; 10 nM–1 mM) side of ciliary process preparations. In comparison to vehicle control experiments, the 8-bromo-cAMP-induced peak Isc and PD increases became significant at a much lower concentration on the NPE side (≥100 nM) than on the PE side (1 mM). The maximum peak Isc and PD increases were observed when 10 µM 8-bromo-cAMP was applied on the NPE side. Application of 8-bromo-cAMP did not induce any significant change in ER. *P < 0.05, **P < 0.01, ***P < 0.001, vs. vehicle control. {dagger}P < 0.05, {dagger}{dagger}P < 0.01, {dagger}{dagger}{dagger}P < 0.001, NPE side versus PE side drug application.

 
Effect on Isc of cAMP Analogues with Different Degrees of Lipophilicity
Experiments were further conducted to investigate whether the permeability of the drug and/or the stroma underlying the ciliary epithelium could be responsible for the difference in the Isc change observed when 8-bromo-cAMP (10 nM–30 µM) was applied on either the NPE or the PE side of the preparations. To investigate, experiments were conducted in ciliary processes and in the ciliary epithelial bilayer with cAMP analogues (10 nM–30 µM) that had, in comparison to cAMP, different degrees (0.7–400) of lipophilicity (Fig. 4) . At the concentration tested and despite their different degrees of lipophilicity, these drugs induced a significant peak-Isc increase again only when they were applied on the NPE side of either the ciliary processes or the ciliary epithelial bilayer preparations. This peak-Isc increase tended to be more pronounced with 8-bromo-cAMP than with 8-OH-cAMP and finally with 8-CPT-6-Phe-cAMP in ciliary processes (10 µM: 17.72 ± 2.69, 13.63 ± 1.22, and 10.60 ± 1.22 µA/cm2, respectively; Fig 4A ). A similar observation was made in the ciliary epithelial bilayer with 8-bromo-cAMP, 8-OH-cAMP, and 8-CPT-6-Phe-cAMP applied on the NPE side (10 µM: 15.21 ± 1.56, 14.52 ± 1.42, and 8.22 ± 0.97 µA/cm2, respectively; Fig 4B ). These peak-Isc increases evoked by each drug applied on the NPE side did not differ significantly (P = 0.27–0.91) between ciliary processes and ciliary epithelial bilayer preparations. In contrast, on the PE side of either the ciliary processes or the ciliary epithelial bilayer preparations, again none of these cAMP analogues at the concentrations tested (10 nM–30 µM) induced any significant Isc increase in comparison to the vehicle control experiments, although a slight but persistently insignificant peak-Isc increase was evoked by the highest membrane-permeable agent, 8-CPT-6-Phe-cAMP (10 µM: 1.16 ± 0.20 and 1.31 ± 0.17 µA/cm2, respectively).


Figure 4
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  		FIGURE 4. In isolated ciliary processes (A) and in ciliary epithelial bilayer (B) preparations, the effect on Isc-peak response of cumulative increasing concentrations (10 nM–30 µM) of cAMP analogues that had different degrees of lipophilicity (cell membrane permeability) in comparison to cAMP (8-CPT-6-Phe-cAMP: 400; 8-bromo-cAMP: ~ 2, or 8-OH-cAMP: ~ 0.7). In these experiments, a significant peak-Isc increase was observed only when these drugs were applied on the NPE side but not on the PE side of the preparations. *P < 0.05, **P < 0.01, ***P < 0.001, vs. vehicle control.

 
In addition, no significant change in ER was ever detected before or after the experiments in either the ciliary process preparations (8-bromo-cAMP: 66.14 ± 3.36 vs. 65.12 ± 3.58 {Omega} · cm2; 8-OH-cAMP: 61.72 ± 3.85 vs. 62.52 ± 3.76 {Omega} · cm2; 8-CPT-6-Phe-cAMP: 61.95 ± 3.59 vs. 61.46 ± 3.25 {Omega} · cm2) or in the ciliary epithelial bilayer preparations (8-bromo-cAMP: 39.91 ± 2.73 vs. 40.96 ± 2.16 {Omega} · cm2; 8-OH-cAMP: 41.60 ± 2.77 vs. 40.40 ± 2.83 {Omega} · cm2; 8-CPT-6-Phe-cAMP: 43.28 ± 3.41 vs. 43.54 ± 3.71 {Omega} · cm2; Fig. 4 ).

Effect of IBMX on the Peak-Isc Increase Induced by Applying 8-Bromo-cAMP on the PE Side
The changes in baseline Isc and/or ER induced by a single concentration (1, 5, or 10 µM) of IBMX (a nonspecific phosphodiesterase inhibitor) applied on both (NPE and PE) sides of ciliary process preparations are shown in Table 2 . When it was applied simultaneously to both sides of ciliary process preparations, apparently in a concentration-dependent manner, IBMX tended to increase significantly the peak-Isc concentration-response curve induced by applying 8-bromo-cAMP on the PE side and to shift this curve to the left (Fig. 5) .


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  		TABLE 2. Effects of IBMX on Isc and ER

 

Figure 5
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  		FIGURE 5. Effect of the nonspecific phosphodiesterase inhibitor IBMX on the 8-bromo-cAMP-induced Isc-peak increase in isolated porcine ciliary processes. In the presence of IBMX on both NPE and PE sides, the peak-Isc concentration–response curve evoked by applying 8-bromo-cAMP on the PE side was significantly increased and tended to shift to the left. *P < 0.05, **P < 0.01, ***P < 0.001, vs. vehicle control. {dagger}P < 0.05, {dagger}{dagger}P < 0.01, {dagger}{dagger}{dagger}P < 0.001, vs. 8-bromo-cAMP applied on the PE side. {ddagger}P < 0.05, {ddagger}{ddagger}P < 0.01, {ddagger}{ddagger}{ddagger}P < 0.001, vs. 1 µM IBMX applied on both sides + 8-bromo-cAMP applied on the PE side. §P < 0.05, §§P < 0.01, §§§P < 0.001, vs. 5 µM IBMX applied on both sides + 8-bromo-cAMP applied on the PE side.

 
Baseline Transepithelial Cl– and Na+ Fluxes
As shown in Table 3 , in baseline short-circuited conditions, the PE-to-NPE Cl– influx (J'Cl PE-NPE) was larger than the NPE-to-PE Cl– outflux (J'Cl NPE-PE), thus leading to a significant (P < 0.05 vs. zero) net transepithelial Cl– flux (J'Cl net: 0.04 ± 0.02 µEq/min per square centimeter) from the PE (stromal) to the NPE (aqueous humor) side. In contrast, Na+ was transported in an equivalent manner in two opposite directions (influx J'Na PE-NPE and outflux J'Na PE-NPE), leading to no detectable net transepithelial flux (J'Na net).


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  		TABLE 3. Baseline Transepithelial 36Cl– and 22Na+ Fluxes

 
Effect of 10 µM 8-bromo-cAMP on Transepithelial Cl– and Na+ Fluxes over Time
NPE Side.
Shortly after the application of a single concentration (10 µM) of 8-bromo-cAMP to the NPE side, in comparison to the paralleled vehicle control experiments, a significant transient (~2 minutes) increase in Cl– influx (J'Cl PE-NPE) but not in Cl– outflux (J'Cl NPE-PE) led to a significant transient increase in the net transepithelial Cl– transport (J'Cl net) from the PE to the NPE side. Afterward, no significant change of unidirectional transepithelial Cl– fluxes was observed (Fig. 6A) . No significant change in the unidirectional transepithelial Na+ transport was ever observed after drug exposure (Fig. 6B) .


Figure 6
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  		FIGURE 6. Changes in transepithelial fluxes (J') of Cl– and Na+ induced by 10 µM 8-bromo-cAMP applied on the NPE or the PE side of isolated porcine ciliary process preparations. The ionic flux from the PE to the NPE side (influx, J'PE-NPE), from the NPE to the PE side (outflux, J'NPE-PE), and the net ionic flux (J'net = J'PE-NPE – J'NPE-PE) were calculated. (A) When 10 µM 8-bromo-cAMP was applied on the NPE side, it transiently induced a significant increase in net Cl– flux from the PE to the NPE side. However, when 10 µM 8-bromo-cAMP was applied to the PE side, it transiently evoked an equivalent increase in both Cl– influx and outflux, resulting in no significant increase in net transepithelial Cl– flux. (B) Either on the NPE side or on the PE side, no significant changes of unidirectional Na+ flux were induced by 10 µM 8-bromo-cAMP. *P < 0.05, **P < 0.01, ***P < 0.001, vs. vehicle control.

 
PE Side.
Shortly after the application of 10 µM 8-bromo-cAMP on the PE side, in comparison to the paralleled vehicle control experiments, significant and transient (approximately 2 minutes) increases of both Cl– influx (J'Cl PE-NPE) and Cl– outflux (J'Cl NPE-PE) were observed (Fig. 6A) . As the amounts of Cl– influx and outflux increase were almost equivalent, the result was no detectable significant change in the transepithelial Cl– net transport (J'Cl net). As was the case when 8-bromo-cAMP was applied on the NPE side, no significant effect on the unidirectional transepithelial Na+ transport was observed when the drug was applied on the PE side (Fig. 6B) .


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Transepithelial Cl– Flux, Na+ Flux, and Isc in Baseline Short-Circuited Conditions
The present study appears to be the first report in the literature in which Cl– transport has been assessed in isolated ciliary processes of the pig. In line with other studies conducted in the ciliary epithelium of cats,15 rabbits,16 and bovines,9 we found that, in baseline short-circuited conditions, there was a net transepithelial transport of Cl– (0.04 ± 0.02 µEq/min per square centimeter) from the PE to the NPE side. Such a net PE-to-NPE transport of Cl– in the baseline condition is also in agreement with the hypothesis that the net transepithelial transport of Cl– is a rate-limiting mechanism in the process of aqueous humor production.11

The average (7.60 ± 1.28 µA/cm2, n = 10) of the baseline Isc observed in the radioactive experiments was equivalent to a net PE-to-NPE anion flux of approximately 0.005 µEq/min per square centimeter, which was much smaller than the observed net transport of Cl– (approximately 0.04 µEq/min per square centimeter). This observation suggests that most of the Cl– movements across the ciliary epithelium take place in an electroneutral manner, probably either with a countertransport of other anion(s) (i.e., HCO3–) and/or with a cotransport of cation(s). However, as has been reported in the rabbit17 or bovine,18 in the present study conducted in porcine ciliary processes, no net transepithelial transport of Na+ has ever been detected to account for the discrepancy between the Isc (the algebraic sum of all the ion flux) and net Cl– flux observed in baseline short-circuited conditions. This observation suggests that ion(s) other than Cl– and Na+ (e.g., HCO3– and/or K+) is involved in the Isc baseline build-up. For example, in the rabbit ciliary body epithelium with a cell-entrapable fluorescent PH probe, BCECF-AM, it has been shown that an electroneutral Cl–/base exchange that does not depend mainly on Na+ can exist.19 In another study performed in human NPE cells with whole-cell patch clamping, it has also been demonstrated that a K+ conductance can exist in baseline conditions.20

In the short-circuited conditions in the present study, although no net transepithelial transport of Na+ was detected, this cation was apparently transported in both the PE-to-NPE and NPE-to-PE directions in an equivalent manner. Similar observations have already been reported in rabbits.10 17 In addition, it cannot be excluded that, in a more physiological open-circuited condition, the small electrical transepithelial gradient across the epithelium (in part generated by the transport of Cl–) may ultimately lead to a net transfer of Na+.

Different Isc Responses after Application of cAMP on the NPE or PE Side
In the literature, there is another study in which the effect of 8-bromo-cAMP on the Isc change has been reported, but in rabbits.21 This report, in which only a single concentration (1 mM) of 8-bromo-cAMP was studied, 8-bromo-cAMP induced an Isc increase that did not differ significantly when the drug was applied to the NPE or the PE side of isolated rabbit ciliary bodies (1.2 ± 0.33 µA/cm2, n = 8 vs. 1.86 ± 0.47 µA/cm2; n = 4; P = 0.38). To a certain extent, a similar observation was also made in the present study performed in porcine ciliary processes with 1 mM 8-bromo-cAMP. However, when the time course of the Isc response was further investigated, the Isc increase induced by 1 mM 8-bromo-cAMP occurred approximately 4 minutes later, when the drug was applied on the PE side than when it was applied to the NPE side. In addition, the present study also further demonstrated a significant difference in the Isc response when lower concentrations (10 or 100 µM) of 8-bromo-cAMP were applied, either on the NPE or on the PE side. Indeed, on the NPE side, as was the case at 1 mM, 8-bromo-cAMP at a concentration of 10 or 100 µM induced a significant biphasic (a transient peak- followed by a sustained plateau-) Isc increase. In contrast, on the PE side, 8-bromo-cAMP induced virtually no Isc increase at 10 µM and only a slight plateau- (but no peak-) Isc increase at 100 µM. Furthermore, simultaneous application on both the NPE and PE sides with each concentration of 8-bromo-cAMP always induced very similar but no larger Isc increase than the one observed when the drug at the same concentration was solely applied on the NPE side. Taken together, the results in the present study indicate that 8-bromo-cAMP triggers an ionic transport mechanism that is likely to be located on the NPE side of the ciliary processes.

Increasing Effect of IBMX on Isc Responses Induced by cAMP on the PE Side
In the concentration–response curve, the present study again indicated that the NPE side was more sensitive to 8-bromo-cAMP for an Isc increase than the PE side. It also highlights that, at the concentrations tested, this 8-bromo-cAMP-induced peak-Isc response difference between NPE and PE side reached its maximum at 10 µM (NPE: 15.15 ± 1.17 µA/cm2 vs. PE: 0.29 ± 0.21 µA/cm2, n = 5, P < 0.0001). Such a difference in Isc responses could be explained by the fact that, when 8-bromo-cAMP was applied on the PE side, it did not reach its target at the same concentration as when it was applied on the NPE side, possibly due to a lack of membrane permeability and/or the intense metabolism of this drug.

To address the first hypothesis, we tested the effect of cAMP analogues (10 nM–30 µM), which differed in their degrees of lipophilicity by a factor of approximately 600, when applied to the PE side of both ciliary processes and ciliary epithelial bilayer preparations. In comparison to vehicle control experiments, none of these drugs could induce any significant Isc increase. In these experiments, only the drug that had the highest membrane-permeability (8-CPT-6-Phe-cAMP) slightly, although not significantly, increased the Isc.

To address the second hypothesis, we tested the effect of the nonspecific phosphodiesterase inhibitor IBMX on the Isc response induced by applying 8-bromo-cAMP (10 nM–1 mM) on the PE side in ciliary process preparations. In comparison to the one observed without IBMX, the cumulative Isc increase induced by applying 8-bromo-cAMP on the PE side became significant at a concentration that was 300 times lower in the presence of IBMX (10 µM). In fact, although 8-bromo-cAMP is often believed to be a stable compound, several studies have demonstrated that this drug could actually be significantly hydrolyzed by the phosphodiesterases.22 23 24 In addition, a high level of cAMP-selective phosphodiesterase expression has been reported in mammalian (e.g., human, rabbit, and baboon) ocular ciliary epithelium.25 The present findings indicate that, when it was applied on the PE side, 8-bromo-cAMP was probably subject to an intense metabolism within porcine ciliary processes before it reached its target which was likely mainly located on the NPE.

Relationship between Isc Baseline and the cAMP-Induced Isc Response
It should be mentioned that, although fluctuations in baseline Isc was observed from one set of experiments to the other, these fluctuations did not influence significantly the 8-bromo-cAMP-induced Isc responses. For example, in the experiments shown in Figures 3 and 4 , the cumulative 8-bromo-cAMP-induced Isc responses at 10 µM were very close (15.15 ± 1.17 µA/cm2 vs. 16.61 ± 2.79 µA/cm2, P = 0.70), whereas baseline Isc differed significantly (12.92 ± 2.21 µA/cm2 vs. 5.62 ± 0.58 µA/cm2, P < 0.001). The reason for these fluctuations in baseline Isc values is still unclear but could reflect some subtle differences in tissue preparations. These differences could also explain why, in the present study, the overall average (7.76 ± 0.60 µA/cm2; n = 86) of the baseline Isc was lower than the one reported earlier in the literature in a study in which the same procedure was used.14

Transepithelial Cl– Flux after Application of cAMP on the NPE or PE Side
The present study also appears to be the first report in which the effects of 8-bromo-cAMP on ciliary transepithelial Isc and 36Cl– and 22Na+ transport were assessed over time. After the application of 10 µM 8-bromo-cAMP on the NPE side, only during the time when the transient peak-Isc increase occurred, a significant but transient increase in the net 36Cl– (but not 22Na+) transport from the PE to the NPE side was observed. Afterward, during the time when the sustained plateau-Isc increase occurred, no significant changes in the unidirectional 36Cl– and 22Na+ fluxes were detected. These observations indicate that the ionic transport mechanism(s) activated by 8-bromo-cAMP underlying the peak-Isc increase is different from the one underlying the plateau Isc increase. In addition, the plateau Isc increase appears to involve other ions than Cl– and Na+.

It should also be noted that, as was the case in the baseline conditions, there also was a discrepancy between the 8-bromo-cAMP-induced increases in peak-Isc and the net PE-to-NPE Cl– flux. Indeed, when 10 µM 8-bromo-cAMP was applied on the NPE side, the increase in the Isc peak was approximately 22 µA/cm2 (equivalent to a net ion flux of 0.013 µEq/min per square centimeter) which was much smaller than the transient increase in the net transport of Cl– (approximately 0.943 µEq/min per square centimeter). Again, no significant increase in unidirectional Na+ flux was ever detected after drug application to account for this discrepancy. This observation indicates that other ions than Cl– and Na+ are likely to be involved in the 8-bromo-cAMP-induced peak-Isc increase. For example, this is the case in rabbit conjunctival epithelium where it has been reported that cAMP (Db-cAMP) coordinately modulates the K+ conductance together with an activation of a certain Cl– channel, thus leading to an increase in KCl secretion.26 In contrast, in the present study when 8-bromo-cAMP was applied on the PE side, no significant increase in net transepithelial transport of either Cl– or Na+ was detected, despite the fact that both unidirectional 36Cl– influx and outflux were equivalently increased transiently. This observation apparently indicates that, on the PE side, although as discussed earlier 8-bromo-cAMP could not easily reach its target on the NPE side, this drug may also modulate the unidirectional Cl– flux, even though it did not induce any change in the net ionic transport (Isc). Further investigations are needed to verify this assumption.

In the literature, is only one other report that has investigated the effect of 8-bromo-cAMP on both transepithelial Isc and 36Cl– transport together, but in bovine ciliary processes.9 Interestingly enough, in this report,9 in contrast to the present study conducted in porcine ciliary processes and the one mentioned earlier that was conducted in rabbit,21 8-bromo-cAMP did not induced an increase but a decrease9 in Isc. It has to be stressed that besides the fact that different species have been studied, to a certain extent, different experimental protocols have been followed. Indeed, in the experiments with radioactive tracers, in the report made in bovine ciliary processes 8-bromo-cAMP was applied simultaneously on both sides,9 whereas in the present study 8-bromo-cAMP was applied alternatively on only one side of the preparations. This discrepancy in experimental protocol may explain why 8-bromo-cAMP has been reported to decrease the PE-to-NPE 36Cl– net transport in bovine (although it was not expressly mentioned whether this decrease reached significance),9 but to increase the PE-to-NPE 36Cl– net transport significantly in porcine ciliary processes in the present study. Nevertheless, it should be noted that, as was the case in the present study, the report made in bovine ciliary processes9 also demonstrated that 8-bromo-cAMP tended to increase the unidirectional 36Cl– transports in both the PE-to-NPE and the NPE-to-PE directions.

Relationship between cAMP-Induced Isc Response and ER
In theory, the Isc increase observed after 8-bromo-cAMP application could be the result of a decrease in the tissue ER. This decrease in ER could result from an increase in passive paracellular ionic transport (leakage) due to a change in the configuration of the tight junctions (between NPE cells) induced by 8-bromo-cAMP. However, such a mechanism does not seem to occur in porcine ciliary processes after 8-bromo-cAMP application. Indeed, in the present study, 8-bromo-cAMP application did not lead to any detectable change in tissue ER. Furthermore, in the short-circuited conditions, when 8-bromo-cAMP was applied on the NPE side, it induced a transient increase in 36Cl– transport from the PE to the NPE side that was significantly higher than the one from the NPE to PE side. If 8-bromo-cAMP increases paracellular leakage, one would expect to see an equivalent increase in 36Cl– transport in both directions. In addition, when 8-bromo-cAMP was applied on the PE side, a transient and selective increase in 36Cl– transport (although equivalent in both the NPE-to-PE and PE-to-NPE directions) was observed, but no increase in 22Na+ transport. If one assumes that it is unlikely that 8-bromo-cAMP would only increase the paracellular leakage though tight junctions in a transient and selective manner for 36Cl–, one can deduce that it is also unlikely that the effect of 8-bromo-cAMP on 36Cl– transport mainly reflects an increase in paracellular leakage through tight junctions.

In conclusion, in isolated porcine ciliary processes, apparently mainly on the NPE side, cAMP triggers a biphasic (transient peak followed by sustained plateau) Isc increase. Only the peak-Isc increase involves an increase in net PE-to-NPE Cl– transport.


   Acknowledgements
 
The authors thank Daniel Storch, PhD, and Reto Allemann, MD, for their support and help.


   Footnotes
 
Supported by Swiss National Science Foundation 32-61495.00, Bern, Switzerland.

Submitted for publication February 21, 2005; revised July 15 and December 11, 2005, and January 19, 2006; accepted March 20, 2006.

Disclosure: Y. Ni, None; R. Wu, None; W. Xu, None; H. Maecke, None; J. Flammer, None; I.O. Haefliger, None

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

Corresponding author: Ying Ni, Laboratory of Ocular Pharmacology and Physiology, University Eye Clinic Basel, Mittlere Strasse 91, P.O. Box CH-4012 Basel, Switzerland; juneye.ying.ni{at}gmail.com.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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