Chloride Secretion by Porcine Ciliary Epithelium: New Insight into Species Similarities and Differences in Aqueous Humor Formation

Abstract

purpose. To investigate the electrophysiology and mechanisms of chloride (Cl⁻) transport across the ciliary body-epithelium (CBE) of the porcine eye. The pig is widely believed to be a good model for studying human physiology. Current results strengthen our understanding of the physiology of aqueous humor formation (AHF).

methods. Freshly isolated porcine CBE were maintained in modified Ussing-Zerahn-type chambers. The effects of the bathing anion substitution (Cl⁻ and HCO₃ ⁻) and transport inhibitors including bumetanide, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt (DIDS), heptanol, and two chloride channel inhibitors, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), and niflumic acid, on the electrical properties and transepithelial Cl⁻ transport were investigated.

results. Viable porcine CBE preparations were maintained in vitro. A spontaneous transepithelial potential difference (PD) of approximately 1 mV was found across the CBE (aqueous side negative). The magnitudes of the PD and short-circuit current (I _sc) were found to be dependent on both the bathing Cl⁻ and HCO₃ ⁻ concentrations. In short-circuited conditions, a significant net Cl⁻ transport (1.01 μEq · h⁻¹ · cm⁻²; n = 109; P < 0.001) in the stromal-to-aqueous direction (J _netCl) was detected. The magnitude of the Cl⁻ current carried by the J _netCl was approximately 2.2 times the measured I _sc, suggesting there was cation (e.g., Na⁺) transport along with Cl⁻ and/or anion transport (e.g., HCO₃ ⁻) in the opposite direction. Bilateral bumetanide (0.1 mM) reduced the J _netCl by ∼56% while stromal DIDS (0.1 mM) produced no inhibition. Instead, aqueous DIDS (0.1 mM) triggered a sustained stimulation of both I _sc and J _netCl. Even if bilateral DIDS was used at a higher concentration (1 mM), together with bilateral dimethylamiloride (DMA, 0.1 mM), no inhibition of the I _sc was observed. Bilateral heptanol (3.5 mM) drastically reduced the I _sc and J _netCl. NPPB (0.1 mM), a common chloride channel inhibitor, did not inhibit the J _netCl, whereas NFA (1.0 mM) virtually abolished it.

conclusions. In the porcine eye, active secretion of Cl⁻ into aqueous was identified that may act as a driving force for AHF. The bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter (NKCC) clearly contributes to the Cl⁻ uptake into the pigmented epithelium (PE), whereas the DIDS-sensitive Cl⁻/HCO₃ ⁻ anion exchanger (AE) may exert a minor role. The intercellular gap junctions couple the porcine ciliary bilayers and thus the transepithelial Cl⁻ transport, as in other species. The Cl⁻ channel/efflux pathway located in the nonpigmented epithelium (NPE) is niflumic acid-sensitive but NPPB-insensitive. We also hypothesize that the AE located on the NPE may regulate the activity of a putative Cl⁻ channel on the basolateral membrane facing aqueous via modulation of the intracellular pH (pH_i). This work reinforces the general consensus that active secretion of Cl⁻ is the major driving force of AHF in mammalian eye and further substantiates the existence of species differences in the mechanism that accomplishes transepithelial Cl⁻ transport.

Elevated intraocular pressure (IOP), which causes damage to the optic nerve fibers in patients with glaucoma, is caused by altered aqueous humor (AH) dynamics. Theoretically, excessive aqueous inflow and obstructive outflow are both possible causes of the altered AH dynamics. The latter is generally believed to be the major cause of the elevated IOP. Currently, the primary antiglaucoma treatment relies on topical pharmacological agents that mostly lower IOP by reducing AHF, although the mechanism of AHF is still not fully understood. Nonetheless, a general consensus is that active Cl⁻ transport across the ciliary epithelium (CE) in the stromal-to-aqueous direction is the major driving force of AHF in several species. ^{1 2 3}  

To achieve transepithelial transport across the CE, a functional syncytium model has been proposed. ⁴ Transepithelial Cl⁻ transport across the CE follows the same route. ⁵ Numerous molecular transporters, which may participate in the vectorial Cl⁻ transport mechanism, have been identified in the CE. ^{6 7 8 9 10 11} The existing models of the ionic mechanism of AHF were deduced mainly from the observations in the two most extensively studied species: ox and rabbit. Contrasting findings have been obtained from these two species. There is a major controversy regarding the machinery responsible for the uptake of Cl⁻ into the pigmented epithelium (PE) cells. Both the Na⁺/K⁺/2Cl⁻ cotransporter (NKCC) and paired Cl⁻/HCO₃ ⁻ (AE) and Na⁺/H⁺ (NHE) double exchangers have been suggested as the major uptake mechanisms. In addition, the identity of the Cl⁻ channel/efflux pathway in the nonpigmented epithelium (NPE) remains unknown. 

The domestic pig has been considered a good experimental model of humans because there are considerable anatomic and physiological similarities. ^{12 13} These factors have in part contributed to the widespread interest in using pig organs for xenotransplantation. ¹² The availability and the comparable size of the porcine eye to human eyes have also made it an established model for studying various aspects of the aqueous outflow system. ^{14 15 16}  

In the present work, we studied the effects of various transport inhibitors on the electrical properties and Cl⁻ transport across the isolated porcine ciliary body/epithelium (CBE) in an attempt to elucidate the transmembrane events that govern the vectorial transport of Cl⁻ in the CE of this species. 

Methods

Tissues Preparation

Fresh porcine eyes were obtained from a local abattoir and transported on ice (4–8°C) to the laboratory (postmortem time ∼70 minutes). The procedures for tissue preparation, mounting, and electrical measurements were adopted from our previous works with bovine CBE and have been described in detail. ^{17 18 19} Briefly, the cornea was removed. A sector of sclera was separated from the choroid by starting an incision at the anterior angle and continuing it tangentially to the globe. Two isolated CBE sectors (superior and inferior) were obtained from an eyeball and bathed in cool Ringer’s solution before mounting in a chamber. 

The CBE was carefully mounted on the chamber so that only the ciliary body band region was exposed to the aperture area. The transepithelial electrical parameters were monitored by dual voltage current clamp unit (DVC-1000; World Precision Instruments, Sarasota, FL). The signal outputs were fed into a dual-channel flatbed chart recorder (BD-12E; Kipp & Zonen Inc., Saskatoon, Saskatchewan, Canada). The spontaneous transepithelial electrical potential (PD) across the CBE was recorded with a pair of Ag/AgCl electrodes (EKV; World Precision Instruments) filled with 154 mM NaCl polyacrylamide gel. A potential-sensing device ^{17 18} that allows offsetting of the potential drift of the pair of voltage-sensing electrodes whenever necessary completed the connection from the chamber to the electrodes. Tissue resistance (R _t) of the CBE was determined by applying an external current (5 μA) via another pair of electrodes connected to the back of the chamber. The potential changes thus generated were used to calculate the R _t. By definition, the short-circuit current (I _sc) can be obtained eventually as the amount of externally applied current that clamps the PD to 0. In our experiment, I _sc is calculated as PD/R _t. When the electrical parameters were stable for 15 minutes, experimental maneuvers such as bathing anion substitutions or application of transport inhibitors were implemented, and the changes in the electrical parameters were continuously monitored. 

The Modified Ussing-Zerahn-Type Chamber

Two types of custom-made Ussing-Zerahn-type chamber, conceptually originating from the original version, ²⁰ were used. The blank resistance in all chambers (in the absence of CBE) was predetermined and compensated for appropriately. In both chamber types, the areas of the CBE exposed to the bathing solution were identical (0.10 cm²). All stages for CBE mounting were fitted with an O-ring and filled with a thin sheet of silicone grease to minimize the pressure for achieving a leak-proof condition. Experiments were conducted at 25°C to 27°C that were maintained by a precalibrated DC heating pad in the continuous perfusion chamber (CP) and water reservoir in the open recirculating chamber (ORC). 

The structure of the CP has been described in previous studies of bovine CBE. ²¹ CP was used for the preliminary study of the spontaneous electrical parameters and the effects of bathing anion substitutions. The perfusion rate of bathing solution was 10 mL · h⁻¹. 

The ORC was used for studying the effects of various transport inhibitors on the electrical parameters and the isotopic Cl⁻ fluxes across the CBE in short-circuited conditions. ²⁰ Twenty milliliters of bathing solution was filled in each bathing side and recirculated by the gentle bubbling of air. 

Study of Isotopic Cl⁻ Fluxes in the ORC

Only paired preparations from the same eye, attaining comparable electrical parameters, were used in the study. The measurements of unidirectional isotopic fluxes in both the basal- and drug-treated conditions were performed in the same preparation sequentially. This protocol allows a direct comparison of the unidirectional ion fluxes in the drug-treated condition to its basal fluxes, thus minimizing the error due to variations among different preparations. The net fluxes (J _net) were the differences between the two unidirectional fluxes: stromal-to-aqueous fluxes (J _sa) and aqueous-to-stromal fluxes (J _as) obtaining from the paired preparations. The unidirectional fluxes were stable for more than 3 hours, if no experimental maneuver was implemented (data not shown). 

Isotopic flux measurements were conducted only if the background counts in all baths were within normal limits. For the J _sa measurement, radiolabeled ³⁶[Cl] HCl (0.67 μCi · mL⁻¹) and ³[H] l-glucose (0.6 μCi · mL⁻¹) were pipetted into the stromal-side bath. For the J _as measurement, equal amounts of radioisotopes were added into the aqueous-side bath. The bathing side with radioisotopes added was designated the “hot” side, and the one without, the “cold” side. After equilibration of the radiolabels, the CBE was short circuited for 20 minutes before the first data sampling was taken. 

The procedures of samples taking for the cold and hot bath were different. For the cold side, samples were taken in every 20 minutes with the bubbling briefly interrupted. Two milliliters of the bathing solution in the cold side was pipetted into a scintillation vial (986542; Wheaton Science Products, Millville, NJ). Simultaneously, identical volume in the hot side was temporarily withdrawn into a 2.5-mL plastic syringe, to maintain an equal hydrostatic pressure on the tissue. Two milliliters of fresh Ringer’s refilling solution was then instilled into the cold side and the hot solution in the plastic syringe was also reintroduced into the recirculating bath. During the drug-treated conditions, the refilling solution contained the same concentration of the drugs as in the bath. Bubbling was resumed until the next sampling. For the hot-side samples, a small amount (20 μL each) was taken only at the beginning and the end of both the basal and drug-treated conditions and no refill was required. The 20 μL sample was injected into 1980 μL of fresh Ringer’s solution preloaded in a scintillation vial to become 2 mL. The radioactivity of the two samples collected from the hot bath would indicate if there was leakage during the experiment. 

All samples collected were mixed with 15 mL of scintillation cocktail (NBCS104; GE Healthcare, Amersham, UK) and their respective radioactivity was counted by a liquid scintillation counter (model 1414 Winspectral DSA; Wallac, Helsinki, Finland). The quenching effects of some transport inhibitors used were predetermined and corrected. The ³[H] l-glucose fluxes concurrently monitored indicated whether there was substantial paracellular leakage across the preparations. 

For calculation of the unidirectional fluxes, the differences in radioactivity between two consecutive samples from the cold bath were first translated into the increase of radioactivity (ΔC) in the whole cold bath during the period (T) between samplings. The radioactivity in the two samples taken in the same condition from the hot bath were averaged and converted to average total radioactivity (H). The unidirectional ion fluxes were calculated according to the following equation:  

\[J{=}\frac{{\Delta}C{\times}I{\times}V}{H{\times}T{\times}A}\]

where J is unidirectional flux of the studied ion; ΔC is increase in radioactivity in cold bath during period T (in counts per minute); H is the average total radioactivity in the hot bath (in counts per minute); I is concentration of the studied ion in the bathing solution (in millimolar); V is volume of cold sample taken (in milliliters); A is the cross-sectional area of the chamber aperture (in square centimeters); and T is the period between two consecutive samples (in hours). 

Four samples taken from the cold side during the basal condition were therefore translated into Cl⁻ flux data in three consecutive 20-minute periods, while eight samples taken in the drug-treated condition were translated into fluxes data in seven consecutive 20-minute periods. The mean unidirectional Cl⁻ fluxes across a CBE during the basal and drug-treated conditions was averaged from the data of at least three stable consecutive fluxes. The unit of unidirectional Cl⁻ flux is microequivalents per hour per square centimeter (μEq · h⁻¹ · cm⁻²). 

Bathing Solution and Preparation of Transport Inhibitors

All chemical reagents were obtained from Sigma-Aldrich, Inc. (St. Louis. MO), unless otherwise stated. The bathing solution was standard normal ringer (NRR) containing (mM): NaCl 113.0, KCl 4.56, NaHCO₃ 21.0, MgSO₄ 0.6, d-glucose 7.5, reduced glutathione 1.0, Na₂HPO₄ 1.0, HEPES 10.0, and CaCl₂ 1.4. The pH was adjusted to 7.4 with NaOH/HCl. In the anion substitution experiments, the respective ion was substituted with an equimolar amount of gluconate. The standard and low Cl⁻ bathing solutions were bubbled with 95% O₂-5% CO₂ and the HCO₃ ⁻-free solution was bubbled with air for 15 minutes before use. Bumetanide (BMT), 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt (DIDS), heptanol, niflumic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) were dissolved in dimethyl sulfoxide (DMSO; maximum concentration, 0.1%). The solvent alone has no effect on the electrical parameters and Cl⁻ fluxes. [³⁶Cl] HCl and [³H] l-glucose was purchased from NEN Life Science Products, Inc. (Boston, MA) and Sigma-Aldrich, Inc., respectively. 

Statistical Analysis

All data are presented as the mean ± SEM. Statistical analyses were performed with commercial software (InStat ver. 3.00; GraphPad Software, San Diego CA). Student’s t-test was used to compare the mean values of two groups. 

Results

Steady State Basal Transepithelial Electrical Parameters

Our preliminary data showed that preparations with PD smaller than −0.2 mV or R _t smaller than 50 Ω · cm² were less responsive to various pharmacological agents and the electrical parameters dissipated eventually. Those preparations were therefore excluded from our analysis. The steady state basal transepithelial electrical parameters of porcine CBE are summarized in Table 1 . The PD was approximately 1 mV with the aqueous side consistently negative with respect to the stromal side. The electrical parameters remained stable for at least 3 hours in the CP and 5 hours in the ORC. To functionally validate the viability of our isolated porcine CBE, the effects of bilateral ouabain (1 mM) were tested. Ouabain produced typical biphasic I _sc changes, as observed previously in different species. ^{18 22 23} The I _sc was first stimulated to 136% ± 4% of the basal value (Student’s paired t-test, P < 0.01, n = 32) in approximately 30 minutes. Afterward, the I _sc was reduced toward 0 gradually. The half-life of the I _sc reduction was approximately 100 minutes. 

Effects of Anion Substitutions on the Transepithelial Electrical Parameters

The effects of bilateral bathing Cl⁻ and HCO₃ ⁻ substitutions on the transepithelial electrical parameters are summarized in Tables 2 and 3 , respectively. Our results showed that the I _sc across the porcine CBE was dependent on both the bathing Cl⁻ and HCO₃ ⁻ concentrations. 

It is interesting to note that low bathing Cl⁻ induced a transient stimulation of the I _sc, which was followed by a steady state inhibition (Fig. 1) . 

Effects of Transport Inhibitors on the Transepithelial Electrical Parameters

Bumetanide, heptanol, and niflumic acid all significantly inhibited the I _sc, whereas stromal DIDS, aqueous, and bilateral NPPB had no effect. Unexpectedly, aqueous DIDS increased the I _sc drastically (86%). All these results are summarized in Table 4 . Typical I _sc time-courses of the action of various transport inhibitors are shown as Figure 2 . 

Cl⁻ Transport across the Isolated Porcine CBE

The steady state basal Cl⁻ fluxes across the porcine CBE are summarized in Table 5 . The stromal-to-aqueous Cl⁻ fluxes (J _saCl) were significantly larger than the aqueous-to-stromal Cl⁻ fluxes (J _as Cl). Therefore, a net Cl⁻ transport (J _netCl) of 1.01 μEq · h⁻¹ · cm⁻² was found in the stromal-to-aqueous direction. This net Cl⁻ transport corresponds to a current of approximately 27.1 μA · cm⁻², which is approximately 2.2 times the average measured I _sc (Table 1) . 

The effects of various transport inhibitors on the Cl⁻ transport across the porcine CBE are summarized in Table 6 . Similar to the I _sc responses, bumetanide significantly reduced the J _netCl, regardless of its side of addition. Heptanol and niflumic acid almost abolished the J _netCl. Moreover, stromal DIDS and aqueous NPPB did not inhibit the J _netCl, whereas aqueous DIDS stimulated it by 59%. 

Discussion

Aqueous Negative PD and Its Dependence on the Bathing Cl⁻ and HCO₃ ⁻

Our in vitro porcine CBE possesses a spontaneous aqueous-negative PD of approximately 1 mV. The negative PD is consistent with most recent findings. Interesting findings have been reported in shark ²³ and pig. ²⁴ Aqueous-negative PD was found in half of the shark CE and two thirds of the porcine CBE. The rest of the preparations in both species possessed aqueous-positive PD of comparable magnitude. It was postulated that the relative activities of Na⁺ and Cl⁻ transport across the cell membrane of the PE and NPE cells may have determined the polarity and magnitude of the electrical parameters. ²³ In our porcine CBE, we noticed that the aqueous-negative PD depolarized toward 0 when the bathing temperature was high (>30°C) or when the perfusion in the CP chamber was interrupted. Those preparations with deteriorating PD were believed to be physiologically compromised, as they were less responsive to various pharmacological reagents. We therefore performed our later experiments in a lower temperature (25–27°C). Presumably lower bathing temperature can reduce the accumulation of metabolic wastes and preserve a better in vitro physiology. 

The aqueous-negative PD found across porcine CBE can be explained by anionic transport in the stromal-to-aqueous direction and/or cationic transport in the opposite direction. Considering the direction of AHF, the anionic transport is more plausible. Our observations that both bathing Cl⁻ reduction and HCO₃ ⁻ depletion substantially inhibited the I _sc are consistent with the existence of the anionic transport. 

In porcine CBE, the steady state I _sc was inhibited by 35% and 63% when the bathing Cl⁻ concentration was reduced from 120 mM to 60 mM and 30 mM, respectively. These findings were consonant with those in the toad ²⁵ and ox. ¹⁷ Conflicting results have been obtained from rabbit preparations. Kishida et al. ²⁶ first demonstrated the Cl⁻-dependent PD but later work by Krupin et al. ²² failed to reproduce the ionic dependency. It is intriguing that we also observed a transient stimulation of the I _sc by reducing the bathing Cl⁻ concentration. An apparent direct explanation for the transient phenomenon is that the Cl⁻ in the NPE has immediately flushed into aqueous along its electrochemical gradient, as the bathing Cl⁻ concentration in the aqueous side was reduced suddenly. However, the fact that the transient stimulation of the I _sc required almost 30 minutes to reach its peak renders this direct explanation unlikely. In 30 minutes, the amount of Cl⁻ exiting the NPE probably exceeds the ions originally accumulated in the cell. Another possible explanation for the transient I _sc stimulation is that the reduction of bathing Cl⁻ may have reduced the exchange of extracellular Cl⁻ with intracellular HCO₃ ⁻ by a putative anion exchanger (AE) located on the basolateral membrane of the NPE. As the intracellular HCO₃ ⁻ increases, intracellular pH (pH_i) increases and it may open up a Cl⁻ conductive pathway facing the aqueous, and thus the Cl⁻ efflux is enhanced and a transient stimulation of the I _sc occurs. Observations supporting such speculation will be discussed further later. 

With the bathing HCO₃ ⁻ depleted, the I _sc of our porcine CBE was nearly abolished. Removing HCO₃ ⁻ reversed the polarity of the PD in rabbit preparations, ^{22 26} whereas the same maneuver only inhibited the I _sc by approximately 30% in the ox. ¹⁷ The extent of the PD dependence of our porcine CBE on the bathing HCO₃ ⁻ apparently falls between the rabbit and ox. This result implies that HCO₃ ⁻ may play a more important role in the AHF in the pig than in the ox, although recent findings of higher Cl⁻ (not HCO₃ ⁻) concentration in the aqueous humor than in the plasma in both species may indicate predominant transport of Cl⁻. ²⁷  

Mechanisms of Transepithelial Cl⁻ Transport

Our preliminary findings of net stromal-to-aqueous Cl⁻ transport (J _netCl) across the isolated porcine CBE had been reported elsewhere previously (Kong MCW et al. IOVS 2003;44:ARVO E-Abstract 3248). The magnitude of the J _netCl found across the porcine CBE is of the same order of magnitude as in the cat, ²⁸ toad, ²⁹ rabbit, ³⁰ and ox. ¹⁷ Present results corroborate the importance of active Cl⁻ transport as a driving force for the AHF in porcine eye, as in other species. 

The observed J _netCl (1.01 μEq · h⁻¹ · cm⁻², n = 109) corresponds to a Cl⁻ current of approximately 2.2 times the measured I _sc. The discrepancy is a common observation among different species ^{17 28 29 30} and implies cation (e.g., Na⁺) transport accompanying Cl⁻ and/or anion (e.g., HCO₃ ⁻) transport proceeding in the opposite direction. The existence of other active ion transport across the porcine CBE is yet to be determined. 

The uptake pathways for loading of NaCl into the PE cells have been a longstanding research interest. The importance of the Na⁺/K⁺/2Cl⁻ cotransporter (NKCC) ^{17 31 32 33} and the paired Cl⁻/HCO₃ ⁻ (AE) and Na⁺/H⁺ (NHE) double exchangers ^{4 19 34 35} has been suggested. 

Na⁺/K⁺/2Cl⁻ Cotransporter

Na⁺/K⁺/2Cl⁻ cotransporter (NKCC) transports Na⁺, K⁺ and Cl⁻ ions in a coupled and electroneutral manner across membranes. ³⁶ The 5-sulfamoylbenzoic acid loop diuretics bumetanide is a relatively specific inhibitor of the transport of NKCC. ³⁷ In the CE, evidences demonstrating the role of NKCC for solute uptakes into PE cells are substantial. ^{17 33 38} Our findings support NKCC as a Cl⁻ uptake pathway into the porcine PE, since bumetanide inhibited both the I _sc and J _netCl. In that, bilateral bumetanide reduced both parameters by approximately 56%. The inhibitory effect of bumetanide on the J _netCl across the porcine CBE was comparable to that of the rabbit ³¹ (52%) but smaller than that of the ox ¹⁷ (86%). 

Presumably, stromal bumetanide would have produced a larger inhibitory effect if NKCC were more abundant on the basolateral membrane of the PE cells, as has been demonstrated in previous immunologic studies in rabbit ³¹ and ox. ³² Our observations that aqueous bumetanide inhibited the I _sc and J _netCl to a larger extent than its stromal addition were at odds with those immunologic findings. A possible explanation is that the ciliary stromal tissues in our CBE may have hindered the diffusion of the reagent from the stromal side. Moreover, we could not exclude the existence of an unknown bumetanide-sensitive anion transport system which is located on the NPE cells and contributes to Cl⁻ efflux into AH. 

The function of NKCC on the NPE cells is less well defined. In cultured human NPE cells, NKCC participated in the ion absorption from AH leading to regulatory volume increase (RVI) when the external K⁺ was elevated to 20 mM, whereas paired Cl⁻/HCO₃ ⁻ and Na⁺/H⁺ double exchanger, Na⁺/Cl⁻ symport, and Na⁺ channel subserved the RVI in normal physiologic conditions. ³⁹ In an interesting model developed by Macknight et al., ⁴⁰ the net outward movement of ions (Na⁺, K⁺ and Cl⁻) from the CE to both the stromal and aqueous sides was assigned to the NKCC located on the basolateral membrane of both the PE and NPE. The NKCC on the NPE cells were also proposed as the major conduit for solute efflux into the aqueous. Our data argue against such outwardly directed NKCC on the bilayers. With such arrangements, the effects of unilateral bumetanide on both the I _sc and Cl⁻ transport should not have been coherent, as in our observations. For instance, stromal bumetanide should have reduced the J _as Cl flux if NKCC were responsible for extrusion of solutes back into the stroma, which has never been observed. Furthermore, our data do not support the proposition that NKCC is the major conduit for solute efflux from the NPE cells into the aqueous, because the stromal-to-aqueous Cl⁻ transport can only be slightly inhibited by bilateral bumetanide while drastically reduced by a Cl⁻ channel inhibitor, niflumic acid. 

Paired Cl⁻/HCO₃ ⁻ and Na⁺/H⁺ Double Exchangers

Cl⁻/HCO₃ ⁻ exchanger (AE) and Na⁺/H⁺ exchanger (NHE) are primarily physically independent entities that exist virtually in all cells. They are crucial for a number of physiological functions including the regulation of intracellular pH (pH_i), cell volume and transepithelial transport. ^{41 42} In the CE, AE and NHE have been functionally demonstrated in both PE ^{6 7 43 44} and NPE cells. ^{45 46 47} Wiederholt et al. ⁴ proposed that AE and NHE could facilitate NaCl uptake into the PE cells by functionally coupling to carbonic anhydrase (CA). Data from electron probe x-ray microanalysis (EPMA) confirmed the importance of the AE and NHE for the NaCl uptake. ^{35 40} In our study, stromal DIDS (0.1 mM) neither inhibited the I _sc nor J _netCl. Instead, aqueous DIDS stimulated the I _sc (86%) and J _netCl (59%) across the porcine CBE. Our results apparently dismiss the role of AE for Cl⁻ uptake into the porcine PE. The effects of DIDS observed in previous electrophysiological studies of the CE were variable. ^{17 31 48} In cultured bovine PE cells, although 0.1 mM DIDS, which inhibits the AE, was able to hinder the intracellular pH shifts elicited by reducing bathing Cl⁻, a higher concentration of DIDS (0.5 mM) together with bumetanide (10 μM) was necessary to inhibit the RVI. ³⁴ Considering the variable effects from previous studies and the existence of the ciliary stroma in our CBE, it is possible that the 0.1 mM DIDS used was insufficient in inhibiting the AE or simply unable to reach the AE located on the PE due to the existence of a physical barrier. Moreover, the porcine Cl⁻ and K⁺ channels may be exceptionally sensitive to intracellular pH shifts so that reduced Cl⁻ uptake by inhibiting the AE might have been masked by the concurrent enhanced Cl⁻ channel activity secondary to intracellular alkalosis. As a result, we tested the combined effects of bilateral dimethylamiloride (DMA, 0.1 mM), a NHE inhibitor, and higher concentration of DIDS (1 mM). Even under such a condition, no significant inhibition of the I _sc was noted (Table 4) . However, the stimulation of the I _sc was indeed diminished compared with that induced by aqueous DIDS (0.1 mM). Therefore, a minor role of AE for the Cl⁻ uptake into the porcine PE cells could not be excluded. Further investigation is needed to clarify the situation. 

The aqueous DIDS stimulatory effects on the I _sc and J _netCl were unexpected and have not been previously reported. Our unidirectional flux data clearly showed that the increase of the J _netCl (0.61 μEq · h⁻¹ · cm⁻²) was predominantly due to a stimulated stromal-to-aqueous Cl⁻ transport (0.51 μEq · h⁻¹ · cm⁻²). Among other possibilities, we hypothesize that the stimulation of the J _netCl could be explained by the presence of a putative, pH-sensitive Cl⁻ channel on the basolateral membrane of the NPE. This putative Cl⁻ channel is opened at alkaline intracellular pH (pH_i) but closed at acidic pH_i. Aqueous DIDS may have inhibited the putative AE on the basolateral membrane of the NPE ⁴⁶ and increased the intracellular HCO₃ ⁻ [HCO₃ ⁻]_i and thus the pH_i. This alkalosis then caused the opening of the putative Cl⁻ channel and both the I _sc and J _netCl were increased. It should also be noted that intracellular alkalosis could also activate the K⁺ efflux from the CE, which causes cellular hyperpolarization and in turn stimulates Cl⁻ efflux from the NPE secondarily. ⁴⁹ Our observation that the DIDS stimulated J _netCl corresponds to an equivalent current (16.4 μA · cm⁻²) larger than the measured I _sc changes (13.2 μA · cm⁻²) are consistent with this possibility. 

Our results in anion substitution experiments may also be explained similarly although they do not rigorously prove this speculation. On the one hand, in our study, lowering of the bathing Cl⁻ concentration may have altered the activity of the AE and led to an increase in the [HCO₃ ⁻]_i and pH_i (alkalosis), which eventually opened the putative Cl⁻ and K⁺ channel. Thus, transient increase of the I _sc was noticed. On the other hand, depletion of the bathing HCO₃ ⁻ may have favored HCO₃ ⁻ efflux via the AE and the pH_i was reduced (acidosis) so that the two channels were closed. pH_i reduction due to HCO₃ ⁻ depletion was detected in cultured rabbit NPE cells. ⁴⁷ Cell acidification caused by aqueous HCO₃ ⁻ depletion has also been proposed to close the Cl⁻ channel and reduce the I _sc and J _netCl in bovine CBE. ¹⁹ Although direct evidence suggesting regulation of the Cl⁻ channels on the NPE cells by pH_i is yet to be available, similar regulatory mechanism had been shown in parotid ⁵⁰ and lacrimal acinar cells. ⁵¹ In those cells, their Ca²⁺-dependent Cl⁻ channel was shown to be modulated by pH_i. 

Gap Junctions

Once Cl⁻ is taken up into the PE cells, the ions can freely diffuse between the PE and NPE cells via the gap junctions, which has been well characterized in structural, ⁵² immunologic ^{53 54} and functional studies. ^{33 55 56} In porcine CBE, heptanol, a gap junction inhibitor, almost abolished the I _sc and inhibited the J _netCl by 82%. These effects are comparable to previous findings of strong heptanol inhibition on the I _sc (85%–90%) across rabbit ICB ⁵⁷ and both I _sc (80%) and J _netCl (80%) in bovine CBE. ¹⁷ Our results confirm the coupling function of gap junction in the CE and also highlight the transcellular nature of the J _netCl found across the isolated porcine CBE. 

Efflux of Cl⁻ from the NPE

The final step of the transepithelial Cl⁻ secretion in the CE is widely believed to go through the Cl⁻ channels on the basolateral membrane of the NPE cells. ⁵ This Cl⁻ efflux has been proposed as the rate-limiting step of AHF. ^{9 58} NPPB is a common Cl⁻ channel blocker that inhibits Cl⁻ channel activity in a number of CE studies. ^{21 49 59 60} In bovine tissues, it reduced the AHF rate (25%) in in vitro perfused eye ⁶¹ and drastically reduced the I _sc and J _netCl across the CBE by more than 90%. ¹⁷ However, we did not observe any inhibitory effect of NPPB on the I _sc and J _netCl across the porcine CBE. Instead, we found niflumic acid, another Cl⁻ channel inhibitor, virtually abolished the I _sc and J _netCl. These results show that the Cl⁻ efflux mechanism on the porcine NPE is niflumic acid-sensitive but NPPB-insensitive and strongly suggest that the Cl⁻ efflux pathways in the two species are of different types. At present, the molecular identity of the Cl⁻ channel on the NPE is still unknown although several candidates have been proposed from several regulatory volume decrease (RVD) studies. ^{9 59 62} It warrants further research to disclose the identity of this Cl⁻ channel. 

Conclusion

The porcine CBE possesses an aqueous-negative PD and actively secretes Cl⁻ into the AH. The Cl⁻ secretion may act as a driving force for the AHF in porcine eye. In this species, the bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter (NKCC) clearly contributes to the uptake of Cl⁻ into the PE cells, whereas the DIDS-sensitive Cl⁻/HCO₃ ⁻ AE may play a minor role. The intercellular gap junctions between the PE and NPE couple the transepithelial Cl⁻ transport. The Cl⁻ channel that contributes to the Cl⁻ efflux from the NPE is niflumic acid– sensitive but NPPB-insensitive. The exact identity of the Cl⁻ channel/efflux pathway on the NPE is still unknown. The observations that aqueous DIDS stimulated the I _sc and the J _netCl into AH were unexpected. We hypothesize that it may be due to a putative Cl⁻ channel on the basolateral membrane of the NPE which is regulated by pH_i. The validity of this proposition awaits further investigation. For clarity, Figure 3shows a model of the ionic mechanism of Cl⁻ secretion in porcine CE, according to our findings. 

This work reinforces the general consensus that active secretion of Cl⁻ is the major driving force of AHF in mammalian eye, and it further substantiates the existence of species differences in the machinery that accomplishes transepithelial Cl⁻ transport. 

 Supported by Hong Kong Polytechnic University Research Grants G-U028, G-T595, and B-Q04F. C-WK was supported by a PolyU Postgraduate Studentship G-V955 from The Hong Kong Polytechnic University.

 Submitted for publication February 20, 2006; revised July 16, 2006; accepted October 11, 2006.

 Disclosure: C.-W. Kong, None; K.-K. Li, None; C.-H. To, 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.

 

Table 1.

 

Steady State Basal Transepithelial Electrical Parameters of the Isolated Porcine CBE

Chamber Type	n	PD (mV)	I sc (μA · cm−2)	R t (Ω · cm2)
CP	226	−1.17 ± 0.03	−15.77 ± 0.44	75 ± 1
ORC	448	−1.03 ± 0.02	−12.36 ± 0.24	85 ± 1

  n, number of experiments. Data are the mean ± SEM.

Table 2.

 

Effects of the Bathing Cl⁻ Concentration on the Transepithelial Electrical Parameters across the Isolated Porcine CBE in the CP Chamber

Condition	n	PD (mV)			I sc (μA · cm−2)			R t (Ω · cm−2)
				Basal	Low Cl−	Basal	Low Cl−	Basal	Low Cl−
60 mM Cl−	15	−1.36 ± 0.09	−0.95 ± 0.09, **	−17.43 ± 1.23	−11.25 ± 1.14, **	79 ± 4	87 ± 8*
30 mM Cl−	6	−1.18 ± 0.25	−0.48 ± 0.23, **	−15.63 ± 2.91	−5.86 ± 2.66, **	76 ± 9	88 ± 9*

 The basal bathing solution contained 120 mM of Cl⁻. n, number of experiments. Values are the mean ± SEM.

*   P < 0.05,

**   P < 0.01, Student’s paired t-test, low Cl⁻ compared with basal.

Table 3.

 

Effects of the Bicarbonate-Free Condition on the Transepithelial Electrical Parameters across the Isolated Porcine CBE in the CP Chamber

n	PD (mV)			I sc (μA · cm−2)			R t (Ω · cm−2)
		Basal	BCF	Basal	BCF	Basal	BCF
6	−1.52 ± 0.07	0.00 ± 0.10**	−18.88 ± 1.85	−0.30 ± 1.41**	84 ± 6	77 ± 6**

  n, number of experiments. Data are the mean ± SEM. BCF, bicarbonate free.

**   P < 0.01, Student’s paired t-test, BCF compared with basal.

Figure 1.

 

A typical time-course of the I _sc changes across the isolated porcine CBE when the bathing Cl⁻ concentration was changed sequentially (arrows). The broken curve indicates the duration at which the solution is changed.

Table 4.

 

Effects of Transport Inhibitors on the I _sc across the Isolated Porcine CBE in the ORC Chamber

Condition	n	Basal I sc (μA · cm−2)	Drug-Treated I sc (μA · cm−2)	Change (%)
Bumetanide (ST)	22	−12.45 ± 1.04	−8.44 ± 1.06	−32, **
Bumetanide (AQ)	26	−11.33 ± 0.99	−5.48 ± 0.71	−52, **
Bumetanide (BS)	24	−11.15 ± 0.72	−4.85 ± 0.59	−56, **
DIDS (ST)	18	−14.32 ± 1.15	−14.16 ± 1.17	−1NS
DIDS (AQ)	22	−15.30 ± 0.85	−28.51 ± 2.35	+86, **
DMA (BS) + DIDS 1 mM (BS)	3	−18.20 ± 0.61	−21.16 ± 1.60	+16NS
Heptanol 3.5 mM (BS)	18	−15.00 ± 1.62	−1.50 ± 0.88	−90, **
NPPB (AQ)	30	−12.60 ± 0.70	−12.93 ± 0.81	+3NS
NPPB (BS)	4	−21.33 ± 1.45	−24.04 ± 1.11	+13*
Niflumic acid 1 mM (AQ)	18	−15.97 ± 1.65	−0.54 ± 0.93	−97, **

 The drug concentration is 0.1 mM, unless specified. n indicates number of experiments. Data are the mean ± SEM. ST, AQ, and BS stand for stromal, aqueous and bilateral addition of the reagent. NS, not significant.

*   P < 0.05

**   P < 0.01, Student’s paired t-test, drug-treated compared with basal.

Figure 2.

 

Typical I _sc time-courses of the action of various transport inhibitors used. Arrow: time at which the inhibitor was introduced into the bath. The concentration of inhibitors is stated in Table 4 . ST, AQ, and BS stand for stromal, aqueous,and bilateral addition of the reagent.

Table 5.

 

Steady State Basal Cl⁻ Fluxes across the Isolated Porcine CBE

Chamber Type	n	Stroma-to-Aqueous (J sa)					Aqueous-to-Stroma (J as)					Net Cl− Flux (J net) (μEq · h−1 · cm−2)
				Cl−	R t	LG	Cl−	R t	LG
ORC	109	5.15 ± 0.09	84 ± 1	54 ± 1	4.14 ± 0.07	85 ± 1	53 ± 1	1.01 ± 0.08***

  n indicates number of experiments. Data are the mean ± SEM. LG, l-glucose leak. The unit of the Cl⁻ fluxes, R _t and LG is μEq · h⁻¹ · cm⁻², Ω · cm², and nanomoles · h⁻¹ · cm⁻², respectively. The R _t and LG of the J _sa preparations are not significantly different from that of the J _as preparations.

***   P < 0.001, Student’s paired t-test, J _sa compared with J _as.

Table 6.

 

Effects of Transport Inhibitors on the Cl⁻ Transport across the Isolated Porcine CBE in the ORC Chamber

Condition	n		Stroma-to-Aqueous (J sa)					Aqueous-to-Stroma (J as)					J netCl
						Cl−	LG	R t	Cl−	LG	R t
Bumetanide (ST)	5	Basal	5.35 ± 0.77	54 ± 8	78 ± 9	4.03 ± 0.43	53 ± 7	84 ± 7	1.32 ± 0.43
		Drug	5.40 ± 0.80NS			4.56 ± 0.49*			0.84 ± 0.38*
Bumetanide (AQ)	8	Basal	4.91 ± 0.36	54 ± 6	88 ± 4	3.86 ± 0.27	59 ± 7	86 ± 4	1.06 ± 0.48
		Drug	4.88 ± 0.40NS			4.32 ± 0.37*			0.57 ± 0.50*
Bumetanide (BS)	7	Basal	5.56 ± 0.34	61 ± 7	80 ± 4	4.71 ± 0.34	61 ± 11	82 ± 7	0.85 ± 0.36
		Drug	5.41 ± 0.35*			5.05 ± 0.38*			0.37 ± 0.29, **
DIDS (ST)	5	Basal	5.15 ± 0.59	48 ± 6	87 ± 9	4.11 ± 0.37	43 ± 3	94 ± 9	1.03 ± 0.42
		Drug	5.46 ± 0.58NS			4.29 ± 0.29NS			1.14 ± 0.50NS
DIDS (AQ)	5	Basal	4.89 ± 0.52	55 ± 6	92 ± 8	3.85 ± 0.27	55 ± 3	94 ± 3	1.04 ± 0.47
		Drug	5.40 ± 0.53, **			3.75 ± 0.25NS			1.65 ± 0.41*
Heptanol 3.5 mM (BS)	6	Basal	5.15 ± 0.37	59 ± 6	78 ± 6	4.21 ± 0.19	62 ± 4	76 ± 3	0.94 ± 0.40
		Drug	4.19 ± 0.23, **			4.02 ± 0.12NS			0.17 ± 0.24, **
NPPB (AQ)	7	Basal	4.92 ± 0.48	43 ± 3	87 ± 7	3.73 ± 0.37	39 ± 4	86 ± 5	1.19 ± 0.44
		Drug	4.72 ± 0.34NS			3.57 ± 0.28NS			1.15 ± 0.28NS
Niflumic acid 1 mM (AQ)	4	Basal	5.43 ± 0.20	49 ± 6	83 ± 6	4.14 ± 0.55	43 ± 7	82 ± 7	1.29 ± 0.44
		Drug	3.17 ± 0.12, **			3.20 ± 0.05, **			−0.03 ± 0.15*

 The drug concentration is 0.1 mM, unless specified. n, number of experiments. Data are the mean ± SEM. ST, stromal; AQ, aqueous; and BS bilateral addition of the reagent. LG stands for l-glucose leak. The units of the Cl⁻ fluxes, R _t and LG are μEq · h⁻¹ · cm⁻², Ω · cm² and nanomoles · h⁻¹ · cm⁻², respectively. The R _t and LG of the J _sa preparations are not significantly different from that of the J _as preparations in all experiments. NS, not significant.

*   P < 0.05,

**   P < 0.01, Student’s paired t-test, drug-treated compared with basal.

Figure 3.

 

A schematic diagram summarizes the ionic mechanism of Cl⁻ secretion in porcine CE. Both bumetanide-sensitive NKCC (1) and AE and NHE double exchangers (2) are responsible for the Cl⁻ uptake from the stroma into the PE cells. Apparently, the NKCC plays a predominant role. Gap junctions (GJ) (3) couple between the bilayers as in other species. The Cl⁻ channel (5) on the basolateral membrane of the NPE facing AH is niflumic acid sensitive but NPPB insensitive. The Cl⁻ channel may be regulated by pHi changes modulated by the AE (4) and thus contributes to the DIDS induced I _sc and J _netCl stimulation in our findings. Further investigation is necessary to validate this speculation.

The authors thank Pang Cheng for valuable comments on the manuscript.