Departments of 1Ophthalmology and 2Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York
Submitted 15 April 2004 ; accepted in final form 3 February 2005
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ABSTRACT |
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Ussing chamber; short-circuit current; electrolyte transport; chloride secretagogue; potassium conductance; 1-ethyl-2-benzimidazolinone; 1,10-phenanthroline
In an early concept, two types of secretion by the secretory epithelia of the ocular surface and the various orbital glands were proposed: basic secretion and reflex secretion. Basic secretion was regarded as a baseline rate of production, and reflex secretion was considered an increased rate caused by neuronal stimulation of the main lacrimal gland (12, 13). Overall, the conjunctival epithelium has adequate water permeability (2) and the transporters necessary to contribute significant fluid to the tear film (50 µl·h1 on the basis of its total surface area) (18, 30). This level of fluid flow is sufficiently large that it may represent most of the baseline tear secretion not attributed to the lacrimal gland as suggested by Li et al. (18).
The major transporters of the rabbit conjunctival epithelium are identical to those in other Cl-secreting epithelia; that is, it has a basolateral bumetanide-sensitive Cl uptake process (mediated by the Na+-K+-2Cl cotransporter, NKCC1) positioned in series with apical Cl channels, including CFTR (34, 35). In addition, Na+/H+ and Cl/HCO3 exchangers exist in parallel in the basolateral membrane and also can mediate Cl uptake (33).
Oppositely directed, electrogenic Na+ reabsorption is amiloride insensitive (29), indicating the absence of epithelial Na+ channels at the apical surface, and occurs via Na+-dependent cotransporters such as those carrying glucose (9) and amino acids (16) in series with the basolaterally located Na+-K+ pump. Furthermore, nonselective cation channels (NSCC) were identified in whole cell patch clamping of freshly isolated conjunctival epithelial cells (39) and the possibility that such channels reside at the apical surface has been suggested (32). As commonly found among Cl-secreting epithelia (4, 22), apical Cl and basolateral K+ conductances are simultaneously increased by cAMP-elevating agents (32).
Spontaneous fluid transport across the conjunctival epithelium engenders a net flow in the basolateral-to-apical direction (18, 30), a property consistent with the more dominant Cl secretory activity of the tissue. These studies demonstrated that the measured fluid secretion was dependent on transepithelial electrolyte transport, given its abolition by ouabain, sensitivity to K+ channel blockade, and Cl dependency. In addition, fluid transport was 1) markedly inhibited in experiments that increased the Na+ absorptive activity by raising the glucose concentration (to 25 mM) of the apical bath, an inhibition that did not occur with a similar concentration of mannitol (30); and 2) increased (50100%) by Cl secretagogues that included purinergic agonists acting via P2Y2 receptors (17, 18, 30). Furthermore, on the basis of the stimulatory effects of purinergics on the short-circuited epithelium, these studies also found that the increase in the transepithelial transport current correlated with the increase in fluid transport. This latter observation implied that P2Y2 receptor activation leads to a selective stimulation of only the Cl secretory activity of the epithelium (17, 30).
However, as observed with other Cl-secreting epithelia (19), the reported stimulation of Cl secretion via purinergic receptors appears to be transitory. This led us to pose the question whether so-called Cl channel openers might prove useful in the conjunctival epithelium. To address this prospect, in the present study, we have examined the effects of 1-ethyl-2-benzimidazolinone (EBIO), an agent previously shown to be an effective Cl secretagogue because of its activation of membrane channels (27, 31). The data reported herein, garnered from experiments with isolated rabbit conjunctivae in Ussing-type chambers under various conditions, demonstrate the effectiveness of the compound in activating conductances in the apical and basolateral aspects for Cl and K+, respectively. These results should encourage further examination of the effects of this agent on fluid transport, which might be of potential value in developing dry-eye therapies.
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METHODS |
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The hemichambers included the necessary arrangements for electrical determinations and vigorous stirring. The transconjunctival potential difference (PD) was short-circuited, with the current needed to maintain 0 mV across the tissue (Isc) being continuously recorded (26). Transmural electrical resistance (Rt) was determined by measuring the amount of current necessary to offset the short-circuited condition by 3 mV for a few seconds.
In general, we observed that conjunctival preparations from heavier rabbits (at least 3 kg) were less delicate and easier to handle and place in the chambers, given the larger areas of tissue that could readily be procured from such animals. The frailty of the preparation, however, appeared to contribute to a spontaneous, gradual decline in Rt that was commonly observed after a prolonged period in the chamber (1030% reduction in Rt in control preparations during 34 h of observation). This decline is best explained as a loss in paracellular resistance, because it occurred in the presence of a steady Isc (authors unpublished data). Under the short-circuited conditions, increases in paracellular ion movement did not result in a net flow across this pathway, given the absence of a potential difference across the epithelium and identical electrolyte concentrations on each side of the preparation. Thus, although the Isc measured net transcellular flow in experiments with symmetrical solutions, conjunctival Rt changes elicited by the addition of various agents frequently underestimated changes in membrane resistance; that is, because of the proportionally larger transcellular than paracellular resistance, large changes in transcellular resistance were observed as smaller changes when measuring Rt. Nevertheless, unless indicated otherwise, the Rt changes described in this report, although proportionally small in some sets of experiments, were statistically significant as paired data (P < 0.05; Student's t-test) and reflect the Rt values elicited by experimental agencies at the point of their introduction. The illustrations of the electrical changes that are shown were acquired by scanning the chart recordings with a page scanner so that the background chart grids could be removed using commercially available software.
The medium used during the dissection and bathing of the tissue in the chambers in most experiments was a modified Tyrode solution composed of (in mM) 1.8 Ca2+ gluconate, 1.2 MgCl2, 4 KCl, 103 NaCl, 30 NaHCO3, 1 NaH2PO4, 5.7 glucose, 0.3 glutathione, and 10 sucrose. The pH of this solution when bubbled with 5% CO2-95% air was 7.5. It measured 280 mosmol/kgH2O.
In some experiments (see Fig. 3), gluconate was used as a Cl substitute along with MgSO4 as a replacement for MgCl2. In others in which transepithelial Cl gradients were established (see Figs. 5 and 6), the Cl-rich side of the preparation contained 103 mM KCl in lieu of NaCl, with all other components unaltered, while SO42 salts were used to replace Cl in the opposite-side bath, with the osmolality being maintained with sucrose.
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To measure unidirectional Cl fluxes, 5 µCi of 36Cl was added to one chamber compartment (final concentration 0.42 µCi·ml1), and periodic samples were obtained from the opposite compartment. The specific activity of the labeled solution remained constant throughout the experiment, and the activity of the opposite solution was always
0.001 of the labeled side. Two-milliliter samples were taken every 15 min from the unlabeled side, the volume of which was kept constant by immediate addition of fresh medium. Twenty-five-microliter samples were taken from the labeled side and diluted up to 2 ml with bathing medium to determine the specific activity of the labeled solution. The samples were counted with a Wallac scintillation counter.
Chemicals.
H36Cl was obtained from PerkinElmer Life Sciences (Boston, MA). EBIO was purchased from Tocris Cookson (Ballwin, MO), stored at 5°C as a 1 M stock in dimethyl sulfoxide (DMSO), and consumed within 2 wk. Calbiochem (La Jolla, CA) was the source of calmidazolium, which was prepared as a 10 mM stock in DMSO, as well as the supplier of the isoquinolinesulfonamide H-89, which was stored at 5°C in aqueous solution (10 mM). All other chemicals were purchased from Sigma (St. Louis, MO). Agents solubilized in DMSO, stored at 5°C, and used within a few days included forskolin, glibenclamide, and A23187
[GenBank]
, each in 1,000-fold solutions. Also stored at refrigerator temperatures were aqueous solutions of amphotericin B (10 mM) and tetraethyl ammonium (TEA; 1 M). 1,10-Phenanthroline (0.2 M) was solubilized in methanol, kept at 5°C, and applied within a few days. Nystatin and UTP were freshly prepared immediately before dilution into the hemichambers, the latter as an aqueous 10 mM solution and the former as a 40 mg·ml1 DMSO suspension followed by 30 s of sonication.
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RESULTS |
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Apical additions of EBIO elicited prompt Isc increases and reductions in Rt, changes that occurred in a concentration-dependent manner (Fig. 1). Although 1 mM concentration produced the largest stable change, for the purpose of economy, most experiments in this study restricted applications of EBIO to 0.5 mM. Additional observations indicated that adding this agent to the stromal side bathing solution resulted in slower and more variable responses than those obtained apically (data not shown). Presumably, EBIO does not readily traverse the stroma; it solely affected channels in the apical domain and/or reached channels in lateral membranes when applied from the apical direction, owing to its lipophilicity.
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Effects of EBIO in the absence of Cl.
To determine the effects of EBIO on the Na+-absorptive properties of the epithelium, conjunctivae were bathed bilaterally under Cl-free conditions (gluconate substitution). In this situation, Isc is dependent on the presence of Na+ in the apical bath (29) and reflects the movement of Na+ across the apical face balanced by basolateral K+ efflux and the electrogenic Na+-K+ pump current (32). Under these conditions, the Isc stimulations evoked by EBIO addition were markedly smaller than those obtained in the presence of the halide (P < 0.05; unpaired data). This also was the case with preparations pretreated with the Ca2+ ionophore A23187
[GenBank]
(Fig. 3). The introduction of EBIO increased the A23187
[GenBank]
-pretreated Isc from 12.5 ± 3.8 to 16.7 ± 4.7 µA·cm2 (means ± SE; n = 4 conjunctivae), a 34% boost accompanied by a 9% decline in Rt from 0.79 ± 0.16 to 0.72 ± 0.15 k·cm2 (P < 0.05; paired data). As was true under more physiological conditions, the subsequent addition of forskolin increased the Isc (to 20.7 ± 4.7 µA·cm2, a 24% rise) and reduced Rt by
10% to 0.65 ± 0.13 k
·cm2 (P < 0.05; paired data) because of the activation of basolateral K+ channels gated by a PKA-dependent mechanism (32). The elevated current was returned to baseline by glibenclamide (27), which decreased the Isc to 5.1 ± 0.9 µA·cm2 and increased Rt to 0.71 ± 0.13 k
·cm2, changes of 75% and 8%, respectively. Under the present conditions, this inhibition apparently resulted from an effect on K+ channels in the lateral membranes, given the absence of a subsequent effect by the nonselective K+ channel blocker Ba2+. Presumably, glibenclamide either directly blocked K+ channels in the conjunctiva or inhibited PKA activity (27).
Because of the possibility that gluconate, the Cl substitute used in the above experiments, might have influenced the electrical response of the preparation due to its weak Ca2+ chelating properties, the Cl-free experiments were repeated with SO42 used as a replacement anion. In these experiments, the introduction of A23187
[GenBank]
did not evoke more substantial current changes than those observed with gluconate, suggesting that the cells had not been depleted of Ca2+ as a result of the combined presence of the ionophore and the chelator. More important, the subsequent addition of EBIO (0.5 mM) to conjunctivae bathed bilaterally with the SO42 solution and A23187
[GenBank]
pretreatment produced an Isc increase from 13.1 ± 2.9 to 18.0 ± 3.3 µA·cm2, a 37% rise, and an 11% Rt decline from 0.98 ± 2.1 to 0.87 ± 1.9 k·cm2 (n = 5 conjunctivae; P < 0.05). These changes were virtually identical to those obtained with gluconate as the Cl replacement (Fig. 3).
Evidence for EBIO modulation of basolateral K+ conductance. EBIO is recognized as an activator of Ca2+-dependent K+ channels (27), implying that the stimulatory effects of the compound under Cl-free conditions most likely resulted from activation of such elements in the conjunctival basolateral membrane. To examine this prospect, experiments were conducted in the presence of a transepithelial K+ gradient in the apical-to-basolateral direction. This entailed bathing the mucosal aspect of the tissue with a Cl-free, high-K+ solution with low [Na+], while the stromal side solution (also Cl free) contained physiological K+ levels with low Na+. Under these conditions, the Na+-K+ pump is quiescent (32, 40) and the Isc reflects solely the diffusion of K+ from the apical-to-basolateral baths across both transcellular and paracellular pathways. The apical introduction of the ionophore amphotericin B, which increases the membrane permeability to monovalent cations, eliminated a restriction to transcellular K+ diffusion as shown by the marked Isc increase that resulted (Fig. 4). In the presence of amphotericin B, the majority of the K+-dependent Isc is transcellular as judged by the fraction of the current remaining in the presence of the nonselective K+ channel blocker Ba2+, which was added at the end of these experiments (Fig. 4) to essentially reduce the Isc to a representation of K+ diffusion across the paracellular pathway.
Five sets of experiments were conducted using the K+-dependent Isc as a measure of basolateral K+ channel activity (Table 1), with representative traces of the first four conditions shown in Fig. 4. The introduction of EBIO (0.5 mM) as the initial test compound after amphotericin B permeabilization (Fig. 4A) enhanced the Isc by 4.6 ± 1.3 µA·cm2 (n = 12 conjunctivae; Table 1, protocol A), an 11% stimulation. In contrast, when the agent was added after A23187
[GenBank]
(Fig. 4B), the Isc rapidly increased by 29.6 ± 2.6 µA·cm2, a 90% enhancement (n = 16 conjunctivae; Table 1, protocol B), suggesting that a combination of high intracellular Ca2+ levels plus EBIO was required to produce a maximal change in basolateral K+ conductance. Consistent with this finding, the addition of A23187
[GenBank]
to tissues preexposed to EBIO (Fig. 4A and Table 1, protocol A) also evoked salient Isc stimulation. The Isc increases that were obtained using the combination of A23187
[GenBank]
and EBIO were statistically larger than those evoked using either agent alone (P < 0.01; unpaired data).
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To further examine the action of glibenclamide, two additional sets of experiments (Table 1, protocols D and E) were conducted to determine the relative effects of this channel blocker with H-89, a kinase inhibitor with a relatively high affinity for PKA (8), on the gross macroscopic K+ conductance of the tissue. Each agent induced inhibitions of the Isc sequential to that of the other (Table 1), suggesting that the effect of glibenclamide may not have arisen solely from an influence on PKA activity and/or that each agent produced only partial PKA inhibition. Nevertheless, the subsequent introduction of EBIO to conjunctivae simultaneously exposed to the two agents evoked relatively sustained increases in the K+-dependent current (Fig. 4D and Table 1), consistent with the prospect that the effect of this secretagogue on basolateral K+ conductances is via a Ca2+-dependent mechanism and not via one involving PKA.
All of the experimental protocols involving the transepithelial K+ gradient (Fig. 4) included the introduction of the channel blocker TEA before Ba2+, given the relatively high selectivity of the former for BK channels (37). When added after A23187
[GenBank]
plus EBIO (Fig. 4B; Table 1, protocol B), TEA produced a 9.2 ± 1.4 µA·cm2 current decline (n = 16 conjunctivae), an 15% inhibition. This degree of Isc reduction was not markedly affected in the protocols in which TEA was added to tissues preexposed to calmidazolium, glibenclamide, and H-89, indicating that these other drugs may not have affected conductances sensitive to this channel blocker and that the TEA-sensitive channels contributed only a small amount to the Isc generated by the K+ gradient (data not included for the sake of simplicity).
Evidence for EBIO modulation of apical Cl conductance.
To determine whether EBIO also affects channels in the conjunctival apical membrane, experiments analogous to those described above for K+ currents were conducted, except that in this case, the isolated tissues were bathed in the presence of an apical-to-basolateral Cl gradient (Fig. 5). For these experiments, the apical bath contained a high-KCl medium (107 mM), while the basolateral side was Cl-free (solution prepared with 53.5 mM K2SO4). Under these conditions, a negative current (11.3 ± 1.5 µA·cm2; n = 27 conjunctivae) representing transepithelial Cl movement from the apical to basolateral hemichambers was recorded. The cholesterol-binding antibiotic nystatin was then introduced to increase the permeability of the basolateral aspect. Preliminary experiments determined that the effects of this agent varied considerably among isolated preparations, presumably because of variations in the thickness of the underlying stroma that was retained from the dissection. As such, a relatively high level of 400 µg·ml1 was determined to produce the most consistent Isc change (an increase in the negative current of 8.5 ± 1.0 µA·cm2 to 19.8 ± 2.2 µA·cm2; n = 27 conjunctivae). Under these conditions, treatment with A23187
[GenBank]
(Fig. 5A) was ineffective (n = 12 conjunctivae), suggesting that intracellular Ca2+ levels were not rate limiting to transepithelial Cl movement. The addition of EBIO to the apical bath (Fig. 5A) induced an increase in the negative current of 4.6 ± 0.9 µA·cm2 (from 12.9 ± 2.2 to 17.5 ± 2.9 µA·cm2), a 35% change accompanied by a 17% Rt decline (from 0.53 ± 0.10 to 0.44 ± 0.07 k·cm2; n = 8 conjunctivae) (P < 0.05), effects consistent with activation of apical Cl channels. When EBIO was added directly after nystatin (i.e., omitting the addition of A23187
[GenBank]
), the increase in the negative current (
Isc = 3.7 ± 1.0 µA·cm2; n = 9 conjunctivae) (data not shown) was comparable to that observed in the presence of the Ca2+ ionophore A23187
[GenBank]
(P > 0.5; unpaired data).
The negative, Cl-dependent Isc was increased 21% by forskolin (from 25.9 ± 7.3 to 31.3 ± 7.2 µA·cm2; n = 5 conjunctivae) (P < 0.05; paired data) when the diterpenoid was added as the initial test compound after nystatin (not shown). This cAMP-elevating agent was always effective when introduced after EBIO (by increasing Isc an additional 8%; n = 4 conjunctivae) (P < 0.05) (Fig. 5A). However, in experiments in which the order of addition was reversed, EBIO did not increase the negative current beyond that produced by forskolin (n = 5 conjunctivae; data not shown) as found with intact epithelial preparations under physiological conditions (Fig. 2C).
When applied in conjunctivae bathed with the transepithelial Cl gradient, glibenclamide inhibited the negative current (Fig. 5, A and B), suggesting that under these conditions, the compound acted as a Cl channel blocker. Consistent with this finding, Ba2+ did not have a pronounced effect (n = 8 conjunctivae) when rapidly added in a sequential manner to one hemichamber and then the other.
In the presence of glibenclamide (Fig. 5B), the effects of EBIO and forskolin were markedly attenuated. When added as the initial compound after nystatin, glibenclamide decreased Isc from 12.6 ± 3.1 to 8.1 ± 3.7 µA·cm2, a 36% reduction, and increased Rt by 24%, from 0.66 ± 0.05 to 0.82 ± 0.04 k·cm2 (n = 4 conjunctivae; P < 0.05); an ensuing effect by EBIO was not discernible. The subsequent addition of forskolin (Fig. 5B) produced a transitory increase in negative current to 8.7 ± 1.2 µA·cm2 (n = 4 conjunctivae; P < 0.05), a 7% rise that was followed by a gradual current decline.
36Cl fluxes across conjunctivae in the direction of the Cl gradient.
To corroborate the electrical results with the transepithelial Cl gradient, 36Cl was added to the high-Cl apical side bath and unidirectional fluxes were measured in the apical-to-basolateral direction for 1 h under baseline conditions, followed by three sequential, 1-h periods in which nystatin, EBIO, and glibenclamide were introduced to the bathing solutions (Table 2). Although the changes in the measured fluxes in response to these drugs mimicked those of the Cl-dependent Isc (see, e.g., Fig. 5), it also was clear that the absolute values for unidirectional flux were markedly larger than the current. Thus an additional ionic flux was subtracting from the measured Isc. Because the bathing solutions had identical cationic concentrations on both sides of the epithelium, it seemed likely that the Cl substitute in the basal side bath (i.e., SO42) was subtracting from the current by diffusing paracellularly in the basolateral-to-apical direction, given its low membrane permeability, gradient in the opposite direction, and the relatively low Rt values (0.5 k
·cm2) that were obtained with tissues mounted under these conditions. Consistent with this possibility, in the presence of glibenclamide, which may have eliminated the transcellular component of the Isc, the change from the EBIO prestimulated values for both the calculated, theoretical Isc and the empirically measured current were nearly identical (14.7 and 15.1 µA·cm2, respectively; Table 2). Moreover, the ratio of the measured-to-calculated Isc declined in the presence of glibenclamide (Table 2), providing another indication that the measured fluxes in the presence of the blocker were largely paracellular and that the discrepancy between the unidirectional Cl fluxes and the Cl-dependent, diffusional Isc may have resulted from a backflux of SO42.
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DISCUSSION |
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Yet, forskolin, which increases the baseline intracellular cAMP levels of the conjunctival epithelium 3-fold (authors unpublished data), serves as a more potent agent than EBIO in increasing the Isc. This finding was observed in intact epithelial preparations (Fig. 2), epithelial preparations bathed bilaterally in solutions lacking Cl (Fig. 3), and putatively apical-membrane preparations bathed in the presence of a transepithelial Cl gradient plus nystatin treatment of the basolateral aspect (Fig. 5). In contrast, EBIO was markedly effective when combined with the Ca2+ ionophore A23187
[GenBank]
in increasing the K+-dependent Isc of preparations bathed with a K+ gradient plus permeabilization of the apical membrane. Such current stimulations were salient for the nearly instantaneous response to the agents and for the magnitude of the current changes (Fig. 4, A and B), which superseded earlier descriptions of Isc increases obtained with cAMP-elevating maneuvers under identical conditions (32). These observations could indicate that the more limited Isc stimulations induced by EBIO with intact epithelia (Figs. 2 and 3) reflect rate-limiting conductances in the apical membrane.
Given the fact that gluconate was used as a Cl substitute for the experiments conducted in the absence of the halide with intact preparations (Fig. 3) and that gluconate is a weak Ca2+ chelator, it is possible that upon applying A23187 [GenBank] , the cells may have been depleted of Ca2+ rather than loaded with it. If so, such conditions might explain the reduced effect of EBIO in the Cl-free experiments. Alternatively, substantial Ca2+ chelation by gluconate may not have been expected a priori, because 1) Rt was not atypical under these conditions and this parameter did not decay dramatically, suggesting that bath Ca2+ levels were sufficient to maintain epithelial integrity; and 2) gluconate was present bilaterally in the K+ gradient experiments, which were performed in Cl-free conditions (Fig. 4). In the latter case, EBIO elicited significantly larger Isc stimulation when combined with A23187 [GenBank] , suggesting that the ionophore indeed increased cellular Ca2+ levels in a gluconate-rich bath. Consistent with this finding, the EBIO stimulatory effects observed under Cl-free conditions were empirically identical to those shown in Fig. 3, when SO42 was used as a Cl substitute, thereby inferring that transapical Na+ reabsorption limited the Isc increase observed in the absence of Cl (Fig. 3) relative to that obtained with the halide present (Fig. 2).
The direct examination of the effect of EBIO on apical membrane preparations (Figs. 5 and 6) resulted in relatively high paracellular 36Cl fluxes as well as underestimated Cl diffusional Isc in the direction of the Cl gradient (Tables 2 and 3). Nevertheless, these experiments indicated an increase in unidirectional 36Cl fluxes in the presence of EBIO and a reduction with glibenclamide, changes that are consistent with the putative involvement of CFTR (27), which is expressed by the conjunctival epithelium (35).
Although glibenclamide is recognized as a blocker of Cl conductances mediated by CFTR, this agent also inhibits a variety of other Cl and K+ channels and affects numerous intracellular enzymes, including PKA (27). Its inhibition of the conjunctival Isc under Cl-free conditions (Fig. 3), a situation in which only the Na+-absorptive component of the Isc is measured, could have resulted from K+ channel blockade as well as inhibition of PKA, given earlier evidence of the effectiveness of H-89 under these conditions (32).
Alternatively, one might suspect that glibenclamide could have inhibited a HCO3 current mediated by CFTR under Cl-free conditions (Fig. 3). However, a lack of evidence for such HCO3 transport by the conjunctival epithelium mitigates this possibility. It was observed earlier that in the bilateral absence of Cl, the Isc was reduced to zero upon unilaterally superfusing the apical side hemichamber with Na+-free solution, and it was completely restored upon reintroduction of the cation (29). In addition, parallel Na+/H+ and Cl/HCO3 exchangers on the basolateral side contribute to transepithelial Cl transport (indicating removal of HCO3 from the cell across the basolateral aspect) (33), and validation of a HCO3 pump such as a basolateral Na+-(n)HCO3 cotransporter was not successful with the rabbit conjunctiva (33). Moreover, introducing HCO3 plus CO2 bubbling to tissues bathed with HEPES buffer and air bubbling under Cl-free conditions did not result in an Isc increase (authors unpublished data). As such, the most likely explanation for the finding of an inhibitory effect by glibenclamide under Cl-free conditions (Fig. 3) is an effect on K+ channels, a possibility substantiated by demonstrating the negative influence of glibenclamide on the Isc generated with a K+ gradient (Fig. 4 and Table 1). However, further work is needed to identify the specific K+ channels affected by glibenclamide in the conjunctival epithelium as well as to determine the potency of the drug as a PKA inhibitor in this system.
The mechanisms underlying the stimulatory effects of EBIO on epithelial transport are not well established. Benzimidazolone compounds (e.g., NS1619) were initially identified as openers of Ca2+-dependent K+ channels (KCa), exhibiting relatively large conductances (known as maxi-K or BK channels) (27). Another such analog, NS004, was the first described pharmacological opener of CFTR (27), but it did not stimulate transepithelial Cl transport in T84 cells, owing to its ineffectiveness on the KCa channels expressed by these cells (27, 29). Instead, it was serendipitously discovered that the EBIO analog served as a stimulator of Cl secretion in T84 cells because of its Cl channel-opening properties as well as its activation of the intermediate KCa, or IK, channels (27, 29).
It currently appears that EBIO increases the affinity for Ca2+ of both the small conductance KCa, known as SK channels, and the IK channels via a mechanism probably involving calmodulin (24, 25, 38). Although EBIO is ineffective in the absence of Ca2+, it reportedly can stimulate SK and IK channels with intracellular Ca2+ levels in the 3050 nM range (24, 25). In the presence of EBIO, current vs. Ca2+ activation curves were found to shift leftward and saturation occurred at lower Ca2+ concentration (25). On the basis of results obtained with macroscopic basolateral K+ conductance, we gained evidence consistent with a Ca2+-dependent mechanism in the conjunctival epithelium (Fig. 4).
Explanations of the mechanism by which EBIO activates CFTR are presently scant (31). It has been reported that EBIO increases intracellular cAMP levels in isolated colonic crypts, presumably by affecting adenylyl cyclase directly (3, 19), but the changes were relatively small compared with those induced by forskolin. Researchers in several studies have observed that EBIO does not increase intracellular Ca2+ levels (3, 19, 20, 28), while investigators in one study reported Ca2+ oscillations in the presence of EBIO (10).
Besides CFTR, the conjunctival epithelium expresses Ca2+-activated Cl channels such as CLCA2 (11), and these elements also may be involved in the EBIO-evoked stimulation of Cl conductance (Figs. 5 and 6). Moreover, in addition to these channels, very recent data derived using expression microarray assays (36) have indicated message for CLCA4 in the human conjunctiva, as well as mRNA for the human isoforms of the BK and SK4 K+ channels (Turner H and Wolosin M, personal communication). Assuming functional expression of these channels in the rabbit conjunctival membranes, such moieties could be responsive to EBIO. The inhibitory effects of TEA and calmidazolium on the K+-dependent Isc (Fig. 4) seem consistent with this prospect. To date, patch-clamp studies of rabbit conjunctiva have defined only nonselective cation channels and inwardly rectifying K+ conductances in the epithelium (39); there have been no additional studies conducted in other species.
Regardless of such limited information, it is tempting to speculate that EBIO or EBIO-like compounds may alleviate dry-eye symptoms in individuals with lacrimal gland dysfunction by stimulating fluid transport across the conjunctival epithelium. This suggestion is analogous to the hypotheses of others who have assessed the potential therapeutic value of Cl channel openers in the treatment of patients with cystic fibrosis (7, 27, 31). Currently, the most suitable approach to palliate dry-eye complications seems to involve the administration of purinergics (23), because of not only the stimulatory effects of such agonists on conjunctival Cl secretion and fluid transport (17, 18) but also the fact that purinergics serve as mucin secretagogues from conjunctival goblet cells (14), which are essentially unicellular mucus glands within the epithelium. As such, purinergics appear to have utility in conserving the composition of the tear film (23).
However, MacVinish et al. (19) noted that the stimulation of Cl transport in epithelia by P2Y2 receptor activation is often transitory because of the nature of the Ca2+ signal itself and the fact that purinergic agonists produce receptor desensitization, from which recovery is slow. Such considerations might explain the need to administer synthetic P2Y2 agonists four or five times daily in clinical trials as well as the time-dependent loss of efficacy that is observable in the data produced by such trials (23).
Currently, there is little information on the toxicity of EBIO. Clearly, an agent such as this one should be considered a potential adjunct to dry-eye therapies that are based on the administration of UTP or UTP derivatives.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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