Pharmacology of aqueous humour formation

Al-Malki, Waleed Hassan (2006) Pharmacology of aqueous humour formation. PhD thesis, University of Glasgow.

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Abstract

This study is a continuation of ongoing research in our laboratories, aiming to investigate the mechanism by which BK acts in the eye, which started by investigating the effect of BK on the ciliary artery (McNeish et al., 2003). The present work extends this study to the ciliary body, by examining BK actions on aqueous humour formation in the bovine arterially perfused eye and on intracellular calcium movements in cultured ciliary epithelium. Aqueous humour was estimated using a fluorescein dilution technique. The objective of this research was to reveal the possible pathway(s) and mechanism(s) by which BK affects the aqueous humour formation. The effect of BK on aqueous humour formation was concentration- dependent over the range 10-9 to 10-7 M and was examined in the presence of various drugs which are known to interfere with BK s actions in other tissues including L-NAME, flurbiprofen and clotrimazole, which inhibit nitric oxide synthase, cycloxygenase and endothelium-dependent hyperpolarising factor(EDHF) respectively. The study revealed that of these inhibitors, only L-NAME did block the effect of BK, therefore it can be concluded that the pathway of BK in causing reduction in aqueous humour formation in bovine eye is through nitric oxide, and not through cycloxygenase or EDHF. The involvement of cGMP in the effect of BK on aqueous humour formation was also examined. The analogue of cGMP, 8-Br- cGMP, was tested and produced significant suppression of aqueous humour formation in a manner that was clearly concentration-dependent. It appears that we may be observing a truly physiological mechanism for cGMP. ODQ, a soluble guanylate cyclase inhibitor, was used to differentiate cGMP-mediated effects of NO from cGMP-independent effects. When used in conjunction with BK, ODQ did block the effect of BK. Therefore it can be concluded that the mechanistic pathway of BK causing reduction in aqueous humour formation is through cGMP. For further confirmation of the participation of cGMP in the BK pathway in aqueous humour formation, the effects of KT-5823 an inhibitor of proten kinase G were examined. KT-5823 alone did not have any effect on aqueous humour formation suggesting that there is no background influence of endogenous cGMP on the basal rate of aqueous humour formation. However, when KT-5823 was used with BK, it completely suppressed the BK effect on aqueous humour formation. This blockade of BK-induced suppression of aqueous humour formation strongly suggests that an increase in cGMP production by BK is the most likely mechanism of its effects on aqueous humour formation. The influence of endogenous cGMP and also its participation in the BK pathway was tested using UK-114,542 (a specific inhibitor of phosphodiesterase type V). UK- 114,542 alone had a small but significant effect on aqueous humour formation. This suggests that there is some baseline production of cGMP in the bovine ciliary body and that PDE inhibition enhances cGMP levels to a level at which aqueous humour formation is affected. When used with BK, UK-114,542 appeared to have enhanced the effect of BK, consistent with the result for UK-114542 alone and also strengthening further the argument that cGMP is the mediator of its effects on aqueous humour formation. Two other minor questions were answered during this part of the study. It was shown that the absence of ascorbate from the arterial perfusate had on effect on the reduction of on aqueous humour formation by 8-Bro-cGMP and BK. The results of the present study also suggested that when a high concentration of BK (10-7 M) was included in the perfusate there was no significant association between protein accumulation in aqueous humour and a reported rise in aqueous humour formation. Instead, the effect of this concentration of BK was to suppress aqueous humour formation by 41%, a slightly greater inhibition than was exerted by any of the lower concentrations of bradykinin tested. The method used Fura-2 as a calcium-sensitive fluorescent marker to monitor free intracellular calcium in individual cells. The objective of the next section was to investigate the effect of BK administration on calcium release in non-pigmented ciliary epithelial cells and ultimately reveal the possible pathway(s) and mechanism(s) by which BK affects the calcium release. Bradykinin (10-9 to 3x10-8) was found to have a concentration-dependent effect on calcium release in these cells. The effects on BK-induced calcium release in the presence of inhibitors such as L-NAME, flurbiprofen and of clotrimazole were examined. The study revealed that L-NAME in conjunction with BK (3 x10-8) it inhibited calcium release while with low concentration of BK (3x10-9) it increased calcium release. Therefore it can be concluded that the pathway of BK in causing calcium release in non-pigmented ciliary epithelium somhow involve nitric oxide. ODQ, a soluble guanylate cyclase inhibitor, was used to differentiate cGMP- mediated effects of NO from cGMP-independent effects and was found to have no effect on calcium release, when used on its own. When used in conjunction with a high concentration of BK (3x10-8 M) ODQ did block the effect of BK. On the other hand, as was observed with L-NAME, BK at low concentration (3x10-9 M) it increased calcium release. Therefore it can be concluded that the mechanistic pathway of BK causing calcium release in non-pigmented ciliary epithelial is influenced by cGMP. This would suggest that NO and cGMP play a part in the release of calcium induced by BK. The involvement of cGMP in the effect of BK on calcium release was also examined. cGMP ( 10-8 to 10-11 M) with BK was tested and produced significant suppression of calcium release in a manner that was clearly concentration-dependent. For further confirmation of the participation of cGMP in the bradykinin pathway in calcium release, the effects of KT-5823 were examined. KT-5823 alone did not have any effect on calcium release. However, when KT-5823 was used with BK and cGMP, it completely suppressed the ability of cGMP to inhibit the BK effect on calcium release. This suggests that an increase in cGMP production by BK acts as a negative feedback on its release of calcium. The influence of endogenous cGMP and also its participation in the bradykinin pathway was tested using UK-114,542 (a specific inhibitor of phosphodiesterase V). UK-114,542 alone had no significant effect on calcium release. This suggests that there is not sufficient baseline production of cGMP in the cultured ciliary epithelial cells such that inhibition could enhance cGMP to a level at which calcium release is affected. When used with BK, UK-114,542 appeared to have enhanced the effect of bradykinin, consistent with the result for KT-5823 and strengthening further the argument that cGMP is produced as a result of BK action and that cGMP then inhibits further release of calcium. A possible role of EDHF in bradykinin-induced calcium release was assessed using clotrimazole. The calcium release was unaffected by administration of clotrimazole on its own, nor when clotrimazole was combined with BK. It therefore appears that EDHF is not involved in the action of BK on calcium release in ciliary epitheial cells. The other option, which BK-induced calcium release in bovine eye was due to a product of cyclo-oxygenase was challenged using flurbiprofen. The calcium release was unaffected by administration of flurbiprofen on its own, noe when flurbiprofen was combined with BK. It therefore appears that cyclo-oxygenase is not involved in the action of BK on calcium release. The results suggest that in the bovine ciliary body, similarly low concentrations of BK are required to inhibit aqumous humour formation and to stimulate intracellular calcium release. It is argued that these two effects are connected and that cGMP is the mediator of the BK-induced suppression of aqumous humour formation. The results of the calcium experiment support the idea that BK raises endogenous cGMP levels via its ability to release intracellular calcium. This strengthens the status of calcium as a primary mediator of intracellular events. A further implication of the low concentration of BK required to elicit these effects is that endogenous BK may modulate aqueous production in vivo.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Wilson
Keywords: Pharmacology, Ophthalmology
Date of Award: 2006
Depositing User: Enlighten Team
Unique ID: glathesis:2006-71742
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 17 May 2019 09:31
Last Modified: 17 May 2019 09:31
URI: http://theses.gla.ac.uk/id/eprint/71742

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