Gene expression in rat lacrimal gland duct cells collected using laser capture microdissection: evidence for K+ secretion by duct cells.
OBJECTIVE
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, IK(Ca)1, 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.
Ubels
John L
JL
Department of Biology, Calvin College, Grand Rapids, Michigan 49546, USA. jubels@calvin.edu
Hoffman
Holly M
HM
Srikanth
Sujata
S
Resau
James H
JH
Webb
Craig P
CP
D000818:Animals; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression / Q000502:physiology*; D020869:Gene Expression Profiling; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000235:genetics / Q000378:metabolism; D007765:Lacrimal Apparatus / Q000166:cytology / Q000378:metabolism*; D007834:Lasers; D008297:Male; D018613:Microscopy, Confocal; D020411:Oligonucleotide Array Sequence Analysis; D011188:Potassium / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D043587:Receptor, Muscarinic M3 / Q000235:genetics / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism; D064506:Solute Carrier Family 12, Member 2; D027981:Symporters / Q000235:genetics / Q000378:metabolism; D000096922:K Cl- Cotransporters
D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; D011188:Potassium; D043587:Receptor, Muscarinic M3; D028021:Sodium-Potassium-Chloride Symporters; D000254:Sodium-Potassium-Exchanging ATPase; D064506:Solute Carrier Family 12, Member 2; D027981:Symporters; D000096922:K Cl- Cotransporters; C491575:Kcnn4 protein, rat; C578879:Slc12a2 protein, rat