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Title: Characterization of chloride and bicarbonate transport across the isolated bovine ciliary body/epithelium (CBE)
Authors: Do, Chi-wai
Degree: Ph.D.
Issue Date: 2002
Abstract: The electrical properties and ion transport across the isolated bovine ciliary body / epithelium (CBE) were investigated by electrophysiological technique. A viable bovine CBE preparation was maintained in modified Ussing chambers. The functional viability of the preparation was demonstrated by the stable electrical parameters as well as the alteration of these parameters with metabolic inhibitors. The structural integrity of the preparation was shown by a low diffusional L-glucose flux. A spontaneous transepithelial potential difference (PD) (aqueous side negative) was found across the bovine CBE. The polarity and magnitude of PD and short-circuit current (Isc) were dependent primarily on the bathing Cl- concentration, and to a lesser extent on bathing Na+ and HCO3- concentration. The polarity of PD and Isc was reversed only when the bathing Cl- concentration was reduced to 30 mM or below. Removal of bathing HCO3- inhibited the electrical parameters but did not reverse their polarity. The presence of transepithelial HCO3- transport was investigated with a closed re-circulating type chamber which measured the unidirectional, radiolabeled fluxes of CO2 and HCO3-. No transepithelial HCO3- transport was detected under short-circuited condition. Acetazolamide, a carbonic anhydrase inhibitor, did not affect the unidirectional HCO3- transport in either direction although a substantial Isc inhibition was observed. This suggested that the inhibitory effects of acetazolamide and HCO3- depletion on electrical parameters might be due to their indirect effects on Cl- secretion. The transepithelial Cl- transport across the bovine CBE was studied in a continuous perfusing type chamber. A substantial net Cl- flux in blood-to-aqueous direction was detected under short-circuited condition. The net Cl- flux equates to approximately 3.5 times that of the measured Isc, suggesting a simultaneous cation secretion into the aqueous humor. The coupled Na+ and Cl- transport in bovine CBE was demonstrated by the abolishment of Cl- transport under reduced bathing Na+ concentration. To accomplish the transepithelial ion transport, three major transport steps are involved: (1) ion uptake from the ciliary stroma into the pigmented epithelium (PE), (2) ion transfer from the PE to non-pigmented epithelium (NPE), and (3) ion efflux from the NPE cells to the posterior chamber. In bovine CBE, there are two principle pathways responsible for the NaCl uptake into the PE cells: one is via Na+/K+/2Cl- cotransporter and the other one is via Na+/H+ and Cl-/HCO3- double exchangers. Bumetanide-sensitive Na+/K+/2Cl- cotransporter is the major pathway responsible for the Cl- uptake, whilst DIDS-sensitive Cl-/HCO3- exchanger provides an additional route for Cl- entry into the PE cells. The notion of Cl-/HCO3- exchange in mediating the transepithelial Cl- transport was further supported by the experiments that the Cl- transport was significantly inhibited either in HCO3- -free media or with acetazolamide in HCO3- -rich solution. The inhibition of Cl- transport in the latter two conditions was larger than that of DIDS, suggesting that additional mechanisms might be involved in reducing the Cl- transport across the bovine CBE. This may also explain the fact that acetazolamide reduces intraocular pressure in the absence of HCO3- transport across the bovine CBE. Intercellular gap junctions between the PE and NPE regulate the ion transfer across the ciliary epithelium. When the gap junctions were blocked by heptanol, the electrical parameters and Cl- transport were reduced by about 80%. This indicated that the uncoupling of gap junctions inhibited the Cl- secretion into the eye. The uptake of Cl- into the ciliary epithelium raises the intracellular Cl- concentration above the electrochemical equilibrium; thereby favoring its diffusion through Cl- channels down the electrochemical gradient. Cl- channel blocker (NPPB) inhibited the Cl- transport by 92% when added to the aqueous side, but had no effect on the blood side. These results suggested that the NPPB-sensitive Cl- channels provided a dominant pathway for Cl- efflux into the aqueous humor. Sodium azide, an activator for cGMP formation, inhibited the Cl- transport by half. In addition, a variety of agents which increased the intracellular cAMP formation, also inhibited the Cl- transport into the eye. These findings suggested that both cGMP and cAMP played significant roles in the modulation of transepithelial Cl- secretion. Timolol, a 帣-blocker commonly used in glaucoma treatment, did not inhibit, but stimulated the Cl- transport. Since a stimulation of Cl- transport reflected an increase in aqueous humor formation, our result suggested the hypotensive effect of timolol was not associated with the transepithelial Cl- secretion. The present study proposes a working model for ion transport across the bovine CBE.
Subjects: Hong Kong Polytechnic University -- Dissertations
Pages: xv, 168 leaves : ill. ; 30 cm
Appears in Collections:Thesis

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