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Chi-Wing Kong, King-Kit Li, Chi-Ho To; Chloride Secretion by Porcine Ciliary Epithelium: New Insight into Species Similarities and Differences in Aqueous Humor Formation. Invest. Ophthalmol. Vis. Sci. 2006;47(12):5428-5436. doi: 10.1167/iovs.06-0180.
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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 (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.
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