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M. M. Civan, C. W. McLaughlin, S. Zellhuber-Mcmillan, A. D. C. Macknight; Electron Probe X-Ray Microanalysis of NaCl Reabsorption at Aqueous Humor Surface of Anterior Rabbit Ciliary Epithelium. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2829.
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To determine the site and mode of ouabain-triggered Na+/K+ exchange and NaCl uptake by rabbit ciliary epithelium.
Excised iris-ciliary bodies were divided into quarters, incubated in Ussing chambers and then quick-frozen in liquid nitrogen-cooled propane. Frozen tissues are freeze-dried, cryosectioned and later irradiated with electron beams to generate the X-ray spectra. The characteristic spectral signals allow determination of Na, K, Cl and P contents. Electrolyte contents are normalized to P content, which primarily reflects covalently-linked phosphorus.
Incubation in 0.1 mM ouabain on both surfaces produced a much larger increase in Na and Cl and loss of K in the anterior than posterior region. The changes in adjoining nonpigmented (NPE) and pigmented (PE) epithelial cells were similar. Na/P, K/P and Cl/P contents were 0.88±0.02, 0.50±0.01 and 0.305±0.004 in NPE (N=197), and 0.81±0.02, 0.49±0.01 and 0.247±0.003 in PE (N=197). The changes in Na/P and K/P were reduced by 39-66% when the tissues were preincubated in amiloride (N=444) or dimethylamiloride (N=410), which selectively blocks Na+/H+ exchange, and reduced by 16-24% when preincubated in benzamil (N=410), which selectively blocks epithelial Na+ channels. After preincubation in 3 mM heptanol, which interrupts gap-junctional communication, ouabain produced far greater changes in Na/P, K/P and Cl/P in the NPE than in the PE cells. With heptanol, Na/P, K/P and Cl/P ratios were 0.95±0.04, 0.38±0.02 and 0.42±0.01 in NPE (N=157), and 0.51±0.03, 0.71±0.02 and 0.196±0.006 in PE (N=157).
The net turnover of Na+, K+ and Cl- contents produced by ouabain is far greater in the anterior than posterior region of the ciliary epithelium. A major mode of Na+ entry into the tissue is through Na+/H+ antiports at the basolateral (aqueous humor) surface of the NPE cells. The presence of transporters that underlie reabsorption, as well as secretion, permits tight regulation of net transport, and may potentially lead to net reabsorption at that membrane.
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