In vitro Bovine Ciliary Body/Epithelium in a Small Continuously Perfused Ussing Type Chamber.

Abstract

ABSTRACT. Our goal is to assess the viability of an in vitro preparation of bovine ciliary body/epithelium (CBE) in a small volume Ussing-type chamber. A new small volume Ussing-type chamber with continuous perfusion was developed for bovine CBE. The trans-CBE electrical parameters were monitored and the electrical responses of the CBE to ouabain (1 and 0.01 mM) were recorded. The trans-CBE fluxes of [14C]-L-ascorbate and [3H]-L-glucose were also studied. The bovine CBEpreparation was stable inside the chamber in terms of its potential difference (PD), short circuit current (SCC) and trans-CBE resistance.They were -0.51 ±0.05 mV (aqueous side negative), -5.43±0.04 //Acm~2 and 94±2 Q.cm2 (mean±s.e.m., n=35), respectively.The preparation hyperpolarised when 0.01 mMouabain was administered to the aqueous side, depolarised when ouabain was applied to the stromal side.[3H]-L-glucose diffusion was about 74 nEq h~1cm~2 in either direction (n=12). Taking the area magnification factor of the CBEinto consideration, the diffusional L-glucose flux across the bovine CBEwas comparable to other tight epithelia. A significant net ascorbate flux (0.26 ± 0.05 nEq h~1cm~2, n=4, p<0.01) was found in the stroma to aqueous direction. Wehave developed a viable in vitro bovine CBE preparation which was (1) electrically stable, (2) responsive to ouabain, (3) tight to L-glucose diffusion, and (4) capable of actively secreting ascorbate. A net trans-CBE chloride transport (0.81 ±0.30 /*Eq h~1cm~2, n=12, p=0.01) from stromal to aqueous side was found in the present in vitro model under short-circuited conditions. The aqueous humour is important for the normal functioning of the eye. It not only supplies nutrients to the avascular crystalline lens and cornea but also maintains the intraocular pressure of the eye which is crucial for the optical system to function. An abnormally high intraocular pressure is frequently associated with the sight threatening eye disease, glaucoma (6). It is widely believed that a component of the aqueous humour is actively secreted by the ciliary epithelium of the ciliary body (4). The bilayer ciliary epithelium is composed of non-pigmented (NPE) and pigmented (PE) ciliary epithelial cells which form a functional syncytium which produces the aqueous humour. However, the ionic mechanism behind the production of aqueous humour is not fully understood. It is generally accepted that fluid secretion is driven by active transport of ions. The active ion flow creates an osmotic gradient by which bulk flow of water follows. Therefore in order to understand how aqueous humour is produced, it is important to identify any active ion transport across the ciliary epithelium. Electrophysiological techniques with Ussing-Zerahn type chambersaim to understand mechanisms of ion or substrate transport across cells and epithelia. Isolated mammalian iris ciliary body/epithelium preparations have been mountedin Ussing-type chambers previously (3, 8). Based on the positive polarity of the bovine ciliary epithelium on the aqueous side...

Inhibitions of Chloride Transport and Gap Junction Reduce Fluid Flow across the Whole Porcine Ciliary Epithelium

“…Although our in vitro FF of the ciliary body preparation represents only a small fraction of the in vivo AH formation rate, our present and previous experiments have shown that the CE preparation is viable. 4,19 The in vitro preparation demonstrated typical characteristics of a tight epithelium, with high transepithelial resistance (taking the area folding ratio into The FF rate measured for 90 minutes before drug addition was taken as the baseline. After the stromal addition of DMA, 60 minutes was allowed for stabilization and FF rate was measured thereafter for 60 minutes as the drug-treatment recording.…”

Effects of Ion Transport and Channel-Blocking Drugs on Aqueous Humor Formation in Isolated Bovine Eye

“…It inhibits the electrical parameters of isolated ciliary body of shark, 26 rabbit, 27 and ox. 22 It also abolishes chloride secretion by bovine CE. 22 In the present study, stromal furosemide inhibited 45.5% of in vitro AHF, which therefore suggests that the AHF in the whole-eye perfusion is at least in part driven by anionic transport, and chloride secretion is likely to be a major candidate.…”

“…22 It also abolishes chloride secretion by bovine CE. 22 In the present study, stromal furosemide inhibited 45.5% of in vitro AHF, which therefore suggests that the AHF in the whole-eye perfusion is at least in part driven by anionic transport, and chloride secretion is likely to be a major candidate. In addition to blocking the Na-K-2Cl cotransporter with a potency equal to that of bumetanide, 28 additional inhibitory effects of furosemide on the K-Cl cotransporter, 29 -31 and on the Cl Ϫ channel 32 have been reported.…”

“…27 Bumetanide also decreased the short-circuited current (SCC) in bovine ciliary body epithelium in an Ussing chamber. 22 However, McLaughlin et al 25 found that bumetanide did not inhibit the intracellular Cl Ϫ accumulation in isolated rabbit PE cells. They concluded that NKCC is not important in salt uptake by the PE cells.…”