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 microEq x h(-1) x 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 approximately 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 (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.