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(Investigative Ophthalmology and Visual Science. 2006;47:1876-1885.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0363
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Gene Expression in Rat Lacrimal Gland Duct Cells Collected Using Laser Capture Microdissection: Evidence for K+ Secretion by Duct Cells

John L. Ubels,1,2 Holly M. Hoffman,1 Sujata Srikanth,2 James H. Resau,2 and Craig P. Webb2

1From the Department of Biology, Calvin College, and the 2Van Andel Research Institute, Grand Rapids, Michigan.

PURPOSE. To compare gene expression profiles of lacrimal gland duct and acinar cells after laser capture microdissection (LCM) and identify molecular networks related to K+ secretion, testing the hypothesis that duct cells are responsible for high K+ levels in tears.

METHODS. Frozen sections of lacrimal glands from five rats were subjected to LCM to isolate pure samples of duct and acinar cells. RNA was extracted, amplified, reverse transcribed, and hybridized to rat cDNA microarrays. Paired arrays from ducts and acini of the five animals were scanned and analyzed with in-house software. Gene expression was confirmed with fluorescent antibodies and confocal microscopy.

RESULTS. A list of 10,294 genes expressed in ducts and acini was searched using gene ontologies related to ion transport. From a list of 55 genes that were expressed in ducts, a panel of genes hypothesized to be involved in basolateral-to-apical transport of K+ and Cl– was chosen for validation by immunofluorescence and confocal microscopy. This analysis confirmed translation of the genes of interest and showed that NKCC1, Na+,K+-ATPase and the M3 cholinergic receptor are expressed on the basolateral membrane of duct cells, whereas KCC1, IKCa1, CFTR, and ClC3 are apically localized.

CONCLUSIONS. Laser capture microdissection in conjunction with gene expression analysis provides an excellent approach for studying lacrimal gland duct cells about which relatively little is known at the molecular level. As demonstrated in a proposed model, the polarized expression of transporters and channels on lacrimal gland duct membranes is consistent with the hypothesis that duct cells secrete the relatively high K+ in lacrimal fluid.




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E. Toth-Molnar, V. Venglovecz, B. Ozsvari, Z. Rakonczay Jr, A. Varro, J. G. Papp, A. Toth, J. Lonovics, T. Takacs, I. Ignath, et al.
New Experimental Method to Study Acid/Base Transporters and Their Regulation in Lacrimal Gland Ductal Epithelia
Invest. Ophthalmol. Vis. Sci., August 1, 2007; 48(8): 3746 - 3755.
[Abstract] [Full Text] [PDF]


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