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Although the functionality of the lens water channels aquaporin 1 (AQP1; epithelium) and AQP0 (fiber cells) is well established, less is known about the role of AQP5 in the lens. Since in other tissues AQP5 functions as a regulated water channel with a water permeability (P H2O ) some 20 times higher than AQP0, AQP5 could function to modulate P H2O in lens fiber cells. To test this possibility, a fluorescence dye dilution assay was used to calculate the relative P H2O of epithelial cells and fiber membrane vesicles isolated from either the mouse or rat lens, in the absence and presence of HgCl 2 , an inhibitor of AQP1 and AQP5. Immunolabeling of lens sections and fiber membrane vesicles from mouse and rat lenses revealed differences in the subcellular distributions of AQP5 in the outer cortex between species, with AQP5 being predominantly membranous in the mouse but predominantly cytoplasmic in the rat. In contrast, AQP0 labeling was always membranous in both species. This species-specific heterogeneity in AQP5 membrane localization was mirrored in measurements of P H2O , with only fiber membrane vesicles isolated from the mouse lens, exhibiting a significant Hg 2+ -sensitive contribution to P H2O . When rat lenses were first organ cultured, immunolabeling revealed an insertion of AQP5 into cortical fiber cells, and a significant increase in Hg 2+ -sensitive P H2O was detected in membrane vesicles. Our results show that AQP5 forms functional water channels in the rodent lens, and they suggest that dynamic membrane insertion of AQP5 may regulate water fluxes in the lens by modulating P H2O in the outer cortex.

Extracellular ATP has been shown to mobilize intracellular Ca(2+) in cultured ovine lens epithelial cells and in human lens epithelium, suggesting a role for purines in the modulation of lens transparency. In this study, we characterized the expression profiles of P2Y receptor isoforms throughout the rat lens at both the molecular and the functional levels. RT-PCR indicated that P2Y(1), P2Y(2), P2Y(4) and P2Y(6) are expressed in the lens, while P2Y(12), P2Y(13) and P2Y(14) are not. Immunohistochemistry, using isoform specific antibodies, indicated that the epithelium does not express P2Y(1) and P2Y(2), but that the underlying fiber cells, which differentiate from the epithelial cells, exhibit strong membranous labeling. Although co-expressed in fiber cells, differences in P2Y(1) and P2Y(2) expression were apparent. P2Y(1) expression extended deeper into the lens than P2Y(2), and its expression co-localized with Cx50 gap junction plaques, while P2Y(2) did not. Labeling for P2Y(4) and P2Y(6) receptors were observed in both epithelial cells and fiber cells, but the labeling was predominantly cytoplasmic in nature. While purine agonist (ATP, ADP, UTP and UDP) application to the lens induced mobilization of intracellular Ca(2+) in cortical fiber cells, little to no effect was observed in the anterior and equatorial epithelium. Thus the inability of UTP and UDP to mobilize intracellular Ca(2+) in the epithelium and the predominately cytoplasmic location of P2Y(4) and P2Y(6) suggests that these receptors may represent an inactive pool of receptors that may be activated under non-physiological conditions. In contrast, our results indicated that P2Y(1) and P2Y(2) are functionally active in fiber cells and their differential subcellular expression patterns suggest they may regulate distinct processes in the lens under steady state conditions.

Impedance measurements in whole lenses showed that lens fiber cells possess different permeability properties to the epithelial cells from which they differentiate. To confirm these observations at the cellular level, we analyzed the membrane properties of fiber cells isolated in the presence of the nonselective cation channel inhibitor Gd3+. Isolated fiber cells were viable in physiological [Ca2+] and exhibited a range of lengths that reflected their stage of differentiation. Analysis of a large population of fiber cells revealed a subgroup of cells whose conductivity matched values measured in the whole lens (1). In this group of cells, membrane resistance, conductivity, and reversal potential all varied with cell length, suggesting that the process of differentiation is associated with a change in the membrane properties of fiber cells. Using pharmacology and ion substitution experiments, we showed that newly differentiated fiber cells (<150 microm) contained variable combinations of Ba2+-and tetraethylammonium-sensitive K+ currents. Longer fiber cells (150-650 microm) were dominated by a lyotropic anion conductance, which also appears to plays a role in the intact lens. Longer cells also exhibited a low-level, nonselective conductance that was eliminated by the replacement of extracellular Na+ with N-methyl-d-glucamine, indicating that the lens contains both Gd3+-sensitive and -insensitive nonselective cation conductances. Fiber cell differentiation is therefore associated with a shift in membrane permeability from a dominant K+ conductance(s) toward larger contributions from anion and nonselective cation conductances as fiber cells elongate.