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Characterization of Rat Meibomian Gland Ion and Fluid Transport
PURPOSE: We establish novel primary rat meibomian gland (MG) cell culture systems and explore the ion transport activities of the rat MG. METHODS: Freshly excised rat MG tissues were characterized as follows: (1) mRNA expression of selected epithelial ion channels/transporters were measured by RT-PC...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Association for Research in Vision and Ophthalmology
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4855829/ https://www.ncbi.nlm.nih.gov/pubmed/27127933 http://dx.doi.org/10.1167/iovs.15-17945 |
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author | 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 |
author_facet | 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 |
author_sort | Yu, Dongfang |
collection | PubMed |
description | PURPOSE: We establish novel primary rat meibomian gland (MG) cell culture systems and explore the ion transport activities of the rat MG. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. |
format | Online Article Text |
id | pubmed-4855829 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | The Association for Research in Vision and Ophthalmology |
record_format | MEDLINE/PubMed |
spelling | pubmed-48558292016-10-01 Characterization of Rat Meibomian Gland Ion and Fluid Transport 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 Invest Ophthalmol Vis Sci Physiology and Pharmacology PURPOSE: We establish novel primary rat meibomian gland (MG) cell culture systems and explore the ion transport activities of the rat MG. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. The Association for Research in Vision and Ophthalmology 2016-04-29 2016-04 /pmc/articles/PMC4855829/ /pubmed/27127933 http://dx.doi.org/10.1167/iovs.15-17945 Text en http://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. |
spellingShingle | Physiology and Pharmacology 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 Characterization of Rat Meibomian Gland Ion and Fluid Transport |
title | Characterization of Rat Meibomian Gland Ion and Fluid Transport |
title_full | Characterization of Rat Meibomian Gland Ion and Fluid Transport |
title_fullStr | Characterization of Rat Meibomian Gland Ion and Fluid Transport |
title_full_unstemmed | Characterization of Rat Meibomian Gland Ion and Fluid Transport |
title_short | Characterization of Rat Meibomian Gland Ion and Fluid Transport |
title_sort | characterization of rat meibomian gland ion and fluid transport |
topic | Physiology and Pharmacology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4855829/ https://www.ncbi.nlm.nih.gov/pubmed/27127933 http://dx.doi.org/10.1167/iovs.15-17945 |
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