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Immunolocalization of the Na-K-Cl Cotransporter in Bovine Ciliary Epithelium

¹ From the Beckman Vision Center, Department of Ophthalmology, University of California San Francisco; and ² Biomedical Sciences, University of California, Riverside.

PURPOSE. Recent evidence suggests that Na-K-Cl cotransport plays a major role in blood-to-aqueous anion transport across the ciliary body epithelium. The present study was undertaken to determine the location of the Na-K-Cl cotransporter in fixed sections of bovine eye.

METHODS. Sections of paraformaldehyde-fixed adult and calf bovine eyes were treated with a monoclonal antibody to mammalian Na-K-Cl cotransporter and a fluorescent secondary antibody and examined under a fluorescent microscope. Na-K-Cl cotransporter protein was detected on immunoblots of dissected tissue and purified nonpigmented ciliary epithelial (NPE) and pigmented ciliary epithelial (PE) cells.

RESULTS. Cotransporter immunofluorescence was most intense along the basolateral surfaces of the PE cells. Anterior pars plicata possessed the greatest PE immunofluorescence, and this diminished posteriorly toward the pars plana. Quantitation of immunofluorescence images indicated 7- to 10-fold more cotransporter protein in pars plicata PE than in pars plana PE. Diffuse cytoplasmic fluorescence was seen in the NPE cells, which was also brightest in anterior pars plicata. Immunoblots of separated PE and NPE cells from anterior pars plicata showed that PE contain four times more 170-kDa cotransporter protein than NPE. This confirmed fluorescence quantitation estimates. Cotransporter was barely detectable in cells isolated from pars plana in either cell layer. Immunoblots of the Na,K-ATPase catalytic (alpha) subunit in separated NPE and PE cells showed that NPE cells possessed approximately eight times more alpha subunit protein than PE. Immunofluorescence indicated a similar distribution of alpha subunits and indicated a basolateral membrane location for the subunit on both cell types. Na-K-Cl cotransporter fluorescence patterns showed more variability in adult animals than in calves, which may be related to aging and/or disease. Distinctive patterns of cotransporter fluorescence were also seen in the cornea, iris, and retina.

CONCLUSIONS. Localization of the Na-K-Cl cotransporter to the plasma membrane on the blood side of the ciliary epithelium tight junctions supports a role for the Na-K-Cl cotransporter in ciliary epithelium as a chloride entry mechanism involved in blood-to-aqueous chloride transport. The concentration of Na,K-ATPase catalytic subunits on NPE basolateral membranes could provide net Na⁺ efflux into the aqueous humor.

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