Characterization of Rat Meibomian Gland Ion and Fluid Transport

• Published in: Investigative ophthalmology & visual science 2016-04, Vol.57 (4), p.2328-2343
• Main Authors: Yu, Dongfang, Davis, Richard M, Aita, Megumi, Burns, Kimberlie A, Clapp, Phillip W, Gilmore, Rodney C, Chua, Michael, O'Neal, Wanda K, Schlegel, Richard, Randell, Scott H, C Boucher, Richard
• Format: Article
• Language: English
• Subjects: 3T3 Cells - physiology; Amides - pharmacology; Animals; Cells, Cultured; Coculture Techniques; Fluorescent Antibody Technique; In Situ Hybridization; Ion Channels - physiology; Ion Transport - physiology; Male; Meibomian Glands - cytology; Meibomian Glands - metabolism; Meibomian Glands - physiology; Mice; Microscopy, Electron, Transmission; Physiology and Pharmacology; Pyridines - pharmacology; Rats; Rats, Sprague-Dawley; rho-Associated Kinases - antagonists & inhibitors; Sodium-Potassium-Chloride Symporters - physiology; Sodium-Potassium-Exchanging ATPase - physiology
• ISSN: 1552-5783; 0146-0404; 1552-5783
• DOI: 10.1167/iovs.15-17945

We establish novel primary rat meibomian gland (MG) cell culture systems and explore the ion transport activities of the rat MG. Freshly excised rat MG tissues were characterized as follows: (1) mRNA expression of selected epithelial ion channels/transporters were measured by RT-PCR, (2) localization of epithelial sodium channel (ENaC) mRNAs was performed by in situ hybridization, and (3) protein expression and localization of βENaC, the Na+/K+/Cl- cotransporter (NKCC), and the Na+/K+ ATPase were evaluated by immunofluorescence. Primary isolated rat MG cells were cocultured with 3T3 feeder cells and a Rho-associated kinase (ROCK) inhibitor (Y-27632) for expansion. Passaged rat MG cells were cultured as planar sheets under air-liquid interface (ALI) conditions for gene expression and electrophysiologic studies. Passaged rat MG cells also were cultured in matrigel matrices to form spheroids, which were examined ultrastructurally by transmission electron microscopy (TEM) and functionally using swelling assays. Expression of multiple ion channel/transporter genes was detected in rat MG tissues. β-ENaC mRNA and protein were localized more to MG peripheral acinar cells than central acinar cells or ductular epithelial cells. Electrophysiologic studies of rat MG cell planar cultures demonstrated functional sodium, chloride, and potassium channels, and cotransporters activities. Transmission electron microscopic analyses of rat MG spheroids revealed highly differentiated MG cells with abundant lysosomal lamellar bodies. Rat MG spheroids culture-based measurements demonstrated active volume regulation by ion channels. This study demonstrates the presence and function of ion channels and volume transport by rat MG. Two novel primary MG cell culture models that may be useful for MG research were established.