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Title: Fluid transport and its regulation by cyclic 3',5'-adenosine monophosphate in the porcine ciliary body epithelium
Authors: Law, Cheung-sing
Degree: Ph.D.
Issue Date: 2010
Abstract: Glaucoma is the leading cause of irreversible blindness worldwide, frequently associated with elevated intraocular pressure (IOP). Currently, there is no cure for the disease, although its progression can be retarded by pharmacological reduction of IOP. Reducing IOP is the only medical treatment documented effective in delaying the onset and retarding the progression of visual loss. Anti-glaucoma drugs function to lower IOP by reducing the production of aqueous humour (AH). However, the transport mechanisms underlying AH formation are not yet fully understood, although many transporters are identified in the ciliary epithelium. Moreover, its regulation is of crucial importance for the development of novel pharmacological treatment of glaucoma. In the present study, a modified Ussing-type fluid chamber for the simultaneous measurement of both fluid flow (FF) and electrical parameters across the whole annulus of porcine ciliary body epithelium (CBE) preparation has been constructed. The porcine CBE preparation transported fluid in the blood-to-aqueous direction at an average rate of ~2.75 μL/h per preparation. The standing transepithelial potential difference (PD) declined gradually, but subsisted for at least 4 hours. The aqueous-negative PD indicated a net anion transport across the CBE from blood to aqueous. The in vitro FF was largely dependent on Cl¯ and HCO₃¯, to a lesser extent, indicating that the fluid movement was primarily driven by the transepithelial anion secretion across the preparation. Blocking Na⁺,K⁺-ATPase with ouabain remarkably reduced the FF (stromal ouabain nearly abolished the FF) and elicited a typical biphasic response across the CBE preparation, suggesting the fluid movement was active and derived from the Na⁺,K⁺-ATPase activity. The effects of several Cl¯ transport inhibitors on the FF rate and PD were examined. Blocking Cl¯/HCO₃¯ and Na⁺ /H⁺ antiporters with 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, 0.1 mM) and 5-(N,N-dimethyl)amiloride hydrochloride (DMA, 0.1 mM) applied on the stromal side respectively did not cause significant effects in both FF and PD across the CBE. However, the blockade of Na⁺-K⁺-2Cl¯ cotransporter with stromal bumetanide (0.1 mM) reduced the FF by 46%, and induced a slight depolarisation of PD, suggesting that Na⁺-K ⁺-2Cl¯ cotransporter was the predominant Cl¯ uptake pathway into the pigmented epithelium (PE), while the paired were less important. Heptanol, a blocker of gap junction, virtually abolished the PD and largely reduced the FF by nearly 80%, indicating that the FF was primarily driven by the transcellular Cl¯ transport. The Cl¯ channel blocker niflumic acid (1 mM), applied on aqueous-side bath, abolished the PD and markedly reduced the FF by 61%, suggesting that the Cl¯ release by the non-pigmented epithelium (NPE) was mediated through niflumic acid-sensitive Cl¯ channels. In addition, the potential regulation of AH formation by the second messenger cyclic 3′,5′-adenosine monophosphate (cAMP) were investigated. Aqueous application of forskolin (10 μM) and 8-Br-cAMP (100 μM) elicited a tremendous hyperpolarisation of PD by 100% and 171% respectively, and a concomitant increase in FF by 42% and 54% respectively, suggesting that cAMP was able to modulate the in vitro ion and fluid transport. Aqueous application of 3-isobutyl-1-methylxanthine (IBMX, 1 mM) induced a slight hyperpolarisation of PD by 22%, but did not cause significant change in FF. The pre-treatment of protein kinase A (PKA) inhibitor H-89 (50 μM) effectively blocked the cAMP stimulated PD and FF by 65% and 52% respectively, implying that PKA activation was essential, at least in part, for the cAMP-induced stimulation of ion and fluid transport. These results support a major role of net Cl¯ transport as the driving force for fluid formation across the ciliary epithelium. cAMP can stimulate the AH formation mainly via a PKA pathway.
Subjects: Hong Kong Polytechnic University -- Dissertations
Cyclic adenylic acid
Body fluid flow
Ciliary body
Pages: xviii, 155 p. : ill. ; 31 cm.
Appears in Collections:Thesis

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