The potential role of cyclic 3', 5'-adenosine monophosphate in regulating aqueous humour secretion

Abstract

Glaucoma is one of the leading causes of irreversible blindness. It is often associated with elevated intraocular pressure (IOP), resulting from an imbalance in aqueous humour production and drainage. Clinically,lowering IOP has been the only effective treatment adopted to attenuate the progression of glaucomatous vision impairment. However,the detailed mechanisms regulating aqueous humour formation have not been fully elucidated. The aim of this study was to investigate the role of cyclic 3', 5'-adenosine monophosphate (cAMP) in the regulation of aqueous humour production using an integrated approach. This study was conducted in three main parts.In the first part,Ussing-type (UT) chamber system was used to monitor transepithelial electrical parameters across freshly isolated porcine ciliary epithelium,and fluid flow (FF) chamber system was employed to measure transepithelial fluid movement across the tissue preparations. Results showed that administration of 1,10,and 100 {471}M 8-Br-cAMP (cAMP) to the non-pigmented epithelium (NPE) side triggered a sustained stimulation of short-circuit current (Isc) by approximately 70 90%. When applied to the pigmented epithelium (PE) side, it was only effective at 100 {471}M.Forskolin was also shown to elicit a sustained stimulation of Isc. Similarly,it acted more effectively when applied to the NPE side than PE side. Concomitant with Isc stimulation,aqueous administration of cAMP or forskolin significantly increased the spontaneous fluid flow across porcine ciliary body.Aqueous addition of niflumic acid (NFA), a Cl-channel blocker, inhibited cAMP-induced Isc stimulation. Substituting bathing Cl-concentration or applying heptanol, a gap junction blocker, to the bath significantly inhibited the baseline Isc, as well as the subsequent cAMP-induced Isc stimulation.Pretreating the ciliary epithelium preparations with protein kinase A (PKA) blockers (H89 and KT5720) inhibited the stimulatory effect of cAMP on Isc. Taken together,these results suggest that cAMP stimulates aqueous humour secretion,and its effects may be mediated through Cl-channels in the NPE cells and gap junctions linking between PE and NPE cells. In the second part, the effect of cAMP on NPE Cl-channel activity was investigated using whole-cell patch clamping technique. In addition, intracellular Cl-concentration was monitored by N-(Ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) fluorescence imaging technique.Whole-cell Cl-current of porcine NPE cells was significantly increased by both cAMP and forskolin. The stimulation of Cl-whole-cell current was effectively blocked by H89. In addition,intracellular Cl-concentration of isolated NPE cells and PE-NPE cell couplets were significantly reduced in the presence of cAMP.In contrast, cAMP had no significant effect on the whole-cell current in porcine PE cells. These results imply that cAMP stimulates Cl-efflux from NPE cell via the PKA pathway, leading to a reduction of intracellular Cl-concentration. In the third part,the effect of cAMP on porcine PE-NPE gap junction permeability was studied by dye transfer technique.Results showed that cAMP and forskolin enhanced dye diffusion from PE to NPE cells across gap junctions. The effect of forskolin was unaffected by the pretreatment with H89,suggesting that cAMP increases gap junction permeability independent of the PKA pathway. The effects of cAMP on aqueous humour inflow have been studied by many researchers,but conflicting results were reported.Species variations are likely to contribute to the discrepancy.Pig appears to be a good model to mimic human physiology.Our results show that,in porcine eye,cAMP stimulates aqueous humour secretion by stimulating Cl-channels activity in NPE cells and enhancing PE-NPE gap junctional permeability. These findings may aid development of novel anti-glaucoma drugs.