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J.L. Ubels, H.M. Hoffman, S. Srikanth, J.H. Resau, C.P. Webb; Evidence From Laser Capture Microdissection and Cdna Microarrays for Potassium Secretion by Rat Lacrimal Gland Duct Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4424.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Relatively little is known about the function of the lacrimal duct (LD) epithelium. In this study duct and acinar cells were collected by laser capture microdissection (LCM) and gene expression was analyzed on cDNA microarrays with particular attention to expression of genes related to K+ secretion, testing the hypothesis that the duct cells are responsible for the high K+ levels in tears. Methods: Frozen sections of rat lacrimal glands were subjected to LCM using an Arcturus Pix–Cell system, capturing duct and acinar cells separately with 200–500 laser pulses. RNA was extracted, subjected to 2 rounds of amplification, reverse transcribed with Cy3 or Cy5 dCTP and hybridized to rat cDNA microarrays containing 20000 genes. The reference standard was 13–day rat embryo RNA. Arrays were scanned and analyzed with Gene–Pix and in–house software. Gene expression was confirmed by immunofluorescence and confocal microscopy using antibodies to ion channels, transporters and receptors to localize the proteins to specific cells and membranes. Results: About 150 genes were expressed at significantly higher levels in duct cells than in acini. Of particular interest was the expression in ducts of a K+–Cl– co–transporter (KCC1), a Na+–K+–2Cl– co–transporter (NKCC1) and Na+–K+ ATPase at significantly higher levels than in acini. Genes for an intermediate conductance Ca2+–activated K+ channel (KCa3.1), CFTR and other channels and transporters related to K+ and Cl– transport were also strongly expressed in duct cells. Confocal microscopy showed that NKCC1 is expressed only on the basolateral membrane of LD cells, while KCC1 is expressed only on the apical membrane. CFTR, ClC6 and KCa3.1 were expressed by duct and acinar cells but were more strongly expressed on apical membranes of duct cells, while the M3 cholinergic receptor was expressed on the basolateral membrane. AQP5 and pendrin (Cl–/HCO3–exchanger) were also identified in ducts on microarrays and by immunofluorescence. Conclusions: Laser capture microdissection is a powerful tool for the study of lacrimal gland duct cells. The strong, polarized expression of NKCC1, KCC1 and KCa3.1 by the duct cells is consistent with the hypothesis that duct cells secrete K+ and are the source of the relatively high K+ in lacrimal gland fluid.
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