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 mu Eq center dot h(-1)center dot cm(-2); n=109; P < 0.001) in the stromal-to-aqueous direction (J(net) Cl) was detected. The magnitude of the Cl- current carried by the J(net)Cl 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(net)Cl by similar to 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(net)Cl. 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(net)Cl. NPPB ( 0.1 mM), a common chloride channel inhibitor, did not inhibit the J(net)Cl, 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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