J Li1, K Kuang, S Nielsen, J Fischbarg. 1. Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
Abstract
PURPOSE: Water channel proteins are important pathways for water movements across cell membranes, including those in the corneal endothelium that contribute to the fluid transport mechanism essential in maintaining corneal transparency. This study was conducted to identify and locate the water channel protein(s) in cultured bovine corneal endothelial cells (CBCECs). METHODS: Poly(A)+ RNA was isolated from CBCECs, and MMLV reverse transcriptase and random hexamer primers were used to generate a cDNA pool by reverse transcription-polymerase chain reaction (RT-PCR). Two specific degenerate primers were synthesized based on consensus sequences from the major intrinsic lens protein superfamily; a "touchdown" PCR protocol accommodated the degeneracy. Immunolocalization was performed by incubating sections of CBCECs with an antibody against human aquaporin 1 (AQP1). Cryosections (0.85 microm) of CBCECs were used for light microscopy, and 800-A ultrathin cryosections were used for electron microscopy (EM). RESULTS: A 372-bp fragment was isolated. Its encoded amino acid sequence was 100% identical with that of bovine AQP1 (AQP2_bovin). CBCECs reacted strongly with the anti-AQP1 antibody, and the labeling was selectively localized to the plasma membrane by light microscopy. Subcellular localization by EM revealed immunoreactivity with the inner leaflets of the plasma membrane. CONCLUSIONS: The identity of the aquaporin, its abundance, and its membrane location suggest that it is a major pathway for fluid flow across endothelial cell membranes. This is consistent with transcellular endothelial fluid transport.
PURPOSE:Water channel proteins are important pathways for water movements across cell membranes, including those in the corneal endothelium that contribute to the fluid transport mechanism essential in maintaining corneal transparency. This study was conducted to identify and locate the water channel protein(s) in cultured bovine corneal endothelial cells (CBCECs). METHODS:Poly(A)+ RNA was isolated from CBCECs, and MMLV reverse transcriptase and random hexamer primers were used to generate a cDNA pool by reverse transcription-polymerase chain reaction (RT-PCR). Two specific degenerate primers were synthesized based on consensus sequences from the major intrinsic lens protein superfamily; a "touchdown" PCR protocol accommodated the degeneracy. Immunolocalization was performed by incubating sections of CBCECs with an antibody against humanaquaporin 1 (AQP1). Cryosections (0.85 microm) of CBCECs were used for light microscopy, and 800-A ultrathin cryosections were used for electron microscopy (EM). RESULTS: A 372-bp fragment was isolated. Its encoded amino acid sequence was 100% identical with that of bovineAQP1 (AQP2_bovin). CBCECs reacted strongly with the anti-AQP1 antibody, and the labeling was selectively localized to the plasma membrane by light microscopy. Subcellular localization by EM revealed immunoreactivity with the inner leaflets of the plasma membrane. CONCLUSIONS: The identity of the aquaporin, its abundance, and its membrane location suggest that it is a major pathway for fluid flow across endothelial cell membranes. This is consistent with transcellular endothelial fluid transport.
Authors: Jorge Fischbarg; Julio A Hernandez; Andrey A Rubashkin; Pavel Iserovich; Veronica I Cacace; Carlos F Kusnier Journal: J Membr Biol Date: 2017-06-16 Impact factor: 1.843