|Author:||Kong, Chi-wing Marco|
|Title:||Chloride (Cl-) transport and its regulation by nitric oxide (NO) in porcine ciliary body/epithelium (CBE)|
|Subject:||Hong Kong Polytechnic University -- Dissertations.|
|Department:||School of Optometry|
|Pages:||190 p. : ill. ; 30 cm.|
|Abstract:||Glaucoma is a potentially sight-threatening disease that is frequently associated with the aqueous humor (AH) dynamics of the eye. Elevated intraocular pressure (IOP) which causes damages to the optic nerve fibres in glaucoma patients, is resulted from an altered AH dynamics via an increase of aqueous humor formation (AHF) or a decrease of AH outflow or a combination of both. Clinically, reducing IOP appears to be the only way that can effectively slow down the progression of glaucomatous defect. At present, topical pharmacological agents are used as the mainstay of glaucoma treatment and these agents lower the IOP by reducing AHF. However, the mechanism of AHF is still poorly understood. In order to devise better glaucoma drugs that are potent and specific, a clear understanding of the AHF mechanism and regulation is of paramount importance. It is generally agreed that the fluid secretion in AHF is secondary to active transport of ions and solutes across the ciliary epithelium (CE) in the stromal-to-aqueous direction. Data in ox and rabbit have shown that active chloride (Cl-) transport across the CE may be the major driving force for AHF. However, the machineries that constitute and regulate the transepithelial Cl- transport are yet to be fully characterized. Therefore, we investigated the AHF mechanism and regulation by studying the ion transport and electrophysiology of porcine CBE. In the first part of the present work, the electrical properties and Cl- transport across the isolated porcine ciliary body / epithelium (CBE) were studied with the Ussing chamber technique. Viable porcine CBE preparations were maintained in vitro. A spontaneous transepithelial potential difference (TEP) of approximately 1 mV was found across the porcine CBE (aqueous side negative). The magnitudes of the TEP and short-circuit current (Isc) were dependent on both the bathing Cl- and bicarbonate (HCO3) concentrations. Under short-circuited condition, a significant net Cl- transport (1.01 uEqhr-1cm-2, n = 109, p <0.001) in the stromal-to-aqueous direction (JnetCl) was detected which may be a driving force for the AHF in pig eye. The magnitude of the Cl- current (Icl) carrying by the JnetCl was about 2.2 times of the measured Isc, suggesting either there was cation (e.g. Na+) transport along with Cl- or anion transport (e.g. HCO3) in the opposite direction or both. To characterize the machineries driving the Cl- transport across the porcine CBE, the effects of transport inhibitors on the transepithelial electrical parameters and Cl- transport were investigated. For the uptake of Cl- into the pigmented epithelium (PE), our results indicated that the bumetanide-sensitive Na+/K+/2Cl- cotransporter (NKCC) played a significant role (bilateral bumetanide reduced the JnetCl by 57%) while the DIDS-sensitive Cl-/HCO3 anion exchanger (AE) did not. The intercellular gap junctions between the nonpigmented epithelium (NPE) and PE were also important for the transepithelial Cl- transport. Blockage of the gap junction by its inhibitor, heptanol, abolished the Isc and dramatically reduced the JnetCl (-82%). The efflux of Cl- from the NPE into AH is believed to be via Cl- channels. The present results indicated that the Cl- channel on the NPE of the porcine CE was a niflumic acid (NFA)-sensitive but NPPB-insensitive type. In the second part of the present work, the modulatory roles of nitric oxide (NO) signalling on the transepithelial electrical parameters and Cl- transport across the porcine CBE were investigated. cGMP analog (8-pCPT-cGMP) triggered a sustained hyperpolarization of the Isc (102%). NO donors (SNAP and SNP) induced a transient hyperpolarization of the Isc that returned to baseline if SNAP was used but produced a sustained depolarization (-46%) if SNP was used. Furthermore, 8-pCPT-cGMP produced a sustained significant increase of the steady-state JnetCl (31%) while SNP reduced the steady-state JnetCl (-44%) and SNAP produced no significant change in the steady-state JnetCl. The reduction of the steady-state JnetCl by SNP (-63%) and SNAP (-25%) were further aggravated after the porcine CBE was pre-treated with ODQ, a soluble guanylate cyclase (sGC) inhibitor. These results indicated that NO might exert two opposite effects on the steady-state JnetCl. It might have enhanced the steady-state JnetCl via a cGMP-dependent pathway but inhibited it via a cGMP-independent pathway. In general, it was noted that the NO related steady-state Isc changes correlated well with the changes in the steady-state JnetCl. Therefore, the steady-state Isc changes were studied in details so as to elucidate the mechanism of the unknown cGMP-independent pathway. Being a heme-containing protein, cytochrome P450 enzyme (Cyt P450) is a likely target of NO in additional to sGC and is involved in lipid signalling via its functions in metabolizing arachidonic acid (AA). Inhibition of Cyt P450 by its inhibitor, ABT, alleviated the depolarization produced by the NO donors on the steady-state Isc. This result indicated that the activation of a Cyt P450-pathway by NO might be, at least in part, responsible for the reduction of NO on the steady-state JnetCl. NO may modulate the transepithelial Cl- secretion via two opposing pathways: the cGMP-dependent and cGMP-independent pathways. The cGMP-independent pathway was apparently the dominant pathway since the NO-cGMP-dependent hyperpolarization of the Isc was only transient and the steady-state JnetCl after treatment of NO donors was never increased, contrasting to a sustained increase of the Isc and steady-state JnetCl by cGMP analog. The downstream cascades and the final molecular targets of the cGMP-independent pathway including the Cyt P450-dependent pathway are yet to be determined.|
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