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1From the Laboratory of Experimental Optometry, School of Optometry, The Hong Kong Polytechnic University, Hung Hom, Hong Kong; and the 2Department of Ophthalmology, Mount Sinai School of Medicine, New York, New York.
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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 (Isc) 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 (JnetCl) was detected. The magnitude of the Cl– current carried by the JnetCl was approximately 2.2 times the measured Isc, 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 JnetCl by 
56% while stromal DIDS (0.1 mM) produced no inhibition. Instead, aqueous DIDS (0.1 mM) triggered a sustained stimulation of both Isc and JnetCl. Even if bilateral DIDS was used at a higher concentration (1 mM), together with bilateral dimethylamiloride (DMA, 0.1 mM), no inhibition of the Isc was observed. Bilateral heptanol (3.5 mM) drastically reduced the Isc and JnetCl. NPPB (0.1 mM), a common chloride channel inhibitor, did not inhibit the JnetCl, 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 (pHi). 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.
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.
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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 device17 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 (Rt) 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 Rt. By definition, the short-circuit current (Isc) can be obtained eventually as the amount of externally applied current that clamps the PD to 0. In our experiment, Isc is calculated as PD/Rt. 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 (Jnet) were the differences between the two unidirectional fluxes: stromal-to-aqueous fluxes (Jsa) and aqueous-to-stromal fluxes (Jas) 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 Jsa 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 Jas 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 (
C) 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:
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C 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.
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· 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 Isc changes, as observed previously in different species.18 22 23 The Isc 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 Isc was reduced toward 0 gradually. The half-life of the Isc reduction was approximately 100 minutes.
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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 Isc are consistent with the existence of the anionic transport.
In porcine CBE, the steady state Isc 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 toad25 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 Isc 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 Isc 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 Isc 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 (pHi) 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 Isc occurs. Observations supporting such speculation will be discussed further later.
With the bathing HCO3– depleted, the Isc 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 Isc 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 (JnetCl) 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 JnetCl 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 JnetCl (1.01 µEq · h–1 · cm–2, n = 109) corresponds to a Cl– current of approximately 2.2 times the measured Isc. The discrepancy is a common observation among different species17 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 exchangers4 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 Isc and JnetCl. In that, bilateral bumetanide reduced both parameters by approximately 56%. The inhibitory effect of bumetanide on the JnetCl across the porcine CBE was comparable to that of the rabbit31 (52%) but smaller than that of the ox17 (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 rabbit31 and ox.32 Our observations that aqueous bumetanide inhibited the Isc and JnetCl 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 Isc and Cl– transport should not have been coherent, as in our observations. For instance, stromal bumetanide should have reduced the JasCl 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 (pHi), cell volume and transepithelial transport.41 42 In the CE, AE and NHE have been functionally demonstrated in both PE6 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 Isc nor JnetCl. Instead, aqueous DIDS stimulated the Isc (86%) and JnetCl (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 Isc was noted (Table 4) . However, the stimulation of the Isc 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 Isc and JnetCl were unexpected and have not been previously reported. Our unidirectional flux data clearly showed that the increase of the JnetCl (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 JnetCl 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 (pHi) but closed at acidic pHi. Aqueous DIDS may have inhibited the putative AE on the basolateral membrane of the NPE46 and increased the intracellular HCO3– [HCO3–]i and thus the pHi. This alkalosis then caused the opening of the putative Cl– channel and both the Isc and JnetCl 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 JnetCl corresponds to an equivalent current (16.4 µA · cm–2) larger than the measured Isc 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 pHi (alkalosis), which eventually opened the putative Cl– and K+ channel. Thus, transient increase of the Isc was noticed. On the other hand, depletion of the bathing HCO3– may have favored HCO3– efflux via the AE and the pHi was reduced (acidosis) so that the two channels were closed. pHi 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 Isc and JnetCl in bovine CBE.19 Although direct evidence suggesting regulation of the Cl– channels on the NPE cells by pHi is yet to be available, similar regulatory mechanism had been shown in parotid50 and lacrimal acinar cells.51 In those cells, their Ca2+-dependent Cl– channel was shown to be modulated by pHi.
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 immunologic53 54 and functional studies.33 55 56 In porcine CBE, heptanol, a gap junction inhibitor, almost abolished the Isc and inhibited the JnetCl by 82%. These effects are comparable to previous findings of strong heptanol inhibition on the Isc (85%–90%) across rabbit ICB57 and both Isc (80%) and JnetCl (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 JnetCl 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 eye61 and drastically reduced the Isc and JnetCl across the CBE by more than 90%.17 However, we did not observe any inhibitory effect of NPPB on the Isc and JnetCl across the porcine CBE. Instead, we found niflumic acid, another Cl– channel inhibitor, virtually abolished the Isc and JnetCl. 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.
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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.
Corresponding author: Chi-Ho To, School of Optometry, The Hong Kong Polytechnic University, Hung Hom, Hong Kong; sochto{at}polyu.edu.hk.
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