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

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.

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.

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