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O.A. Candia, L.J. Alvarez, A.C. Zamudio; 1–EBIO serves as a Cl secretagogue in rabbit corneal and conjunctival epithelia. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1088.
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Purpose:To characterize the effects of 1–ethyl–2–benzimidazolinone (1–EBIO), which was initially identified as a Cl secretagogue in airway epithelia, on the transepithelial electrical parameters of the rabbit cornea and conjunctiva. Methods: Corneal and conjunctival epithelia were isolated in separate experiments in an Ussing–type chamber under short–circuit conditions. The effects of 1–EBIO on the short–circuit current (Isc) and transepithelial resistance (Rt) were measured under control, physiological conditions, as well as, in experiments with altered electrolyte concentrations. Results: Addition of 0.5 mM 1–EBIO to the apical–side baths of both the corneal and conjunctival epithelia stimulated the control Isc (ΔIsc= 2.9 ± 0.6 µA/cm2, a 70% rise, n= 4; and ΔIsc= 9.8 ± 1.4, a 53% increase, n= 4, respectively) and reduced Rt (–0.41 ± 0.20 kΩ cm2, a 25% reduction; and –0.15 ± 0.06, a 22% decline, respectively). The EC50 of the drug was about 10–4 M with the cornea and the conjunctiva. Under Cl–free conditions, the stimulatory effects of 1–EBIO were markedly attenuated (n= 4 for each tissue). In the presence of an apical–to–basolataral K gradient and permeabilization of the apical membrane with amphotericin B, the majority of the Isc reflects the transcellular movement of K via basolateral K channels. Under these conditions, 1–EBIO increased the transconjunctival Isc 2–3 fold (n= 6); the elevated K current was reverted upon the addition of 4 mM EGTA to the bathing solutions. With the corneal epithelium, which exhibits a basolateral K conductance that is inhibited by cAMP, 1–EBIO evoked paradoxical effects on tissues bathed with the K gradient. When added as the initial test compound, 1–EBIO mimicked the effects of forskolin and inhibited the K current. However, when applied after forskolin, which closes K channels negatively gated by cAMP, 1–EBIO elicited stimulations (ΔIsc= 5.1 ± 1.7 µA/cm2, a 180% change, n= 5) of the residual K–dependent Isc. In the presence of an apical–to–basolateral Cl gradient and nystatin permeabilization of the basolateral membrane, 1–EBIO increased the Cl–dependent Isc across both tissues, an effect that was prevented by the Cl–channel blocker, glibenclamide (0.3 mM). Conclusions: 1–EBIO can activate apical Cl channels and basolateral K channels (presumably those that are Ca–dependent) in both epithelia, thereby serving as a Cl secretagogue. This compound, or related derivatives, may have utility as topical agents to stimulate fluid transport across these tissues in individuals with lacrimal gland deficiencies.
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