Localization of ion transporters and channels in the bovine ciliary epithelium

Abstract

Glaucoma is one of the leading causes of blindness in the world. Most of the current pharmaceutical treatments of glaucoma aim to lower the intraocular pressure (IOP) by decreasing the formation of aqueous humor (AH). However, the mechanism of AH formation and its regulation are still unknown. Therefore, it is crucial to elucidate the mechanism of AH secretion so that effective methods can be devised to regulate IOP in glaucoma patients. It is believed that the aqueous humour formation is driven by active ion transport across the ciliary epithelium and water is dragged into the posterior chamber in the process of secretion. The ciliary epithelium is a bilayer epithelium, which consists of an inner non-pigmented epithelium (NPE), facing the aqueous humor and an outer pigmented epithelium (PE), facing the stroma. A net chloride (Cl-) transport across the bovine ciliary body epithelium (CBE) has recently been reported. Transepithelial (vectorial) transport of Cl- ions across the CBE may involve 3 steps: (1) influx of ions from the stroma into PE at the basolateral membrane of PE; (2) diffusion of Cl- through gap junctions between the PE and NPE; (3) Cl- efflux from the NPE into the posterior chamber. At least two groups of ion transporters have been suggested to take up Cl- in step 1: (a) sodium-proton, Na+/H+ (NHE) and bicarbonate-chloride, HCO3-/Cl- (AE) exchangers or (b) via the sodium-potassium-chloride co-transporter, Na-K-2Cl (NKCC). They were suggested to couple with the sodium potassium ATPase, Na/K-ATPase in step 1, to take up Cl- and Na+ from stroma to the CBE. Cl- is then pass through the gap junction in step 2 and then exit through the Cl- channel in step 3. The present study aimed at localizing these ion transporters in bovine CBE using immunohistochemistry and western blotting. Their localization at this bilayer epithelium is crucial in modeling vectorial ion transport that drives the aqueous humor formation. Bovine CBE was snap-frozen in liquid nitrogen or embedded in paraffin wax. The tissue sections were reacted with eight antibodies: a5 (for detecting all a-subunit of Na pump) and a6F (for a1 subunit of Na-pump), T4 (for NKCC), NHE3 (for NHE-3 isoform of NHE), NHE1 (for NHE1 isoform of NHE), AE2 (for AE), ClC2 and ClC3 (for ClC2 and ClC3 isoforms of Chloride channel respectively) in immunohistochemistry. Western blotting was carried out to estimate the molecular size of the ion transporters by the above antibodies to confirm the result in immunohistochemistry. The results have shown that: 1. all a subunit (a5) staining was found at the basolateral membranes of both epithelia with an intricate pattern where there was more staining in NPE than in PE, and only the basolateral membrane of the PE was stained with a1 subunit (a6F) of the Na pump. 2. T4/NKCC was found at the basolateral membrane of PE predominantly. 3. There was no NHE3 staining in the bilayer, although it was present in its positive control (rabbit kidney). 4. AE2 and NHE1 staining were found in the bilayer (PE and NPE) diffusely. 5. ClC2 staining was found in the NPE diffusely, and ClC3 was seen in the bilayer diffusely, with more staining at the basolateral membrane of PE. The staining pattern of Na pump suggested its pumping activity in the NPE is more active than in the PE. The basolateral location of NKCC at the PE is consistent with its role in up-taking sodium chloride from the stroma. NHE1 and AE2 were found diffusely in the bilayer and were not specific to any membrane. Their roles in vectorial Cl- uptake from the stroma cannot be corroborated from our study. ClC2 was localized diffusely in the NPE only and it may play a specific but unknown role in the Cl exit pathway. The role of Cl exit pathway for ClC3 as suggested by previous investigators could not be substantiated with our result.