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L-Type Calcium Channels Play a Critical Role in Maintaining Lens Transparency by Regulating Phosphorylation of Aquaporin-0 and Myosin Light Chain and Expression of Connexins

Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels...

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Autores principales: Maddala, Rupalatha, Nagendran, Tharkika, de Ridder, Gustaaf G., Schey, Kevin L., Rao, Ponugoti Vasantha
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3667166/
https://www.ncbi.nlm.nih.gov/pubmed/23734214
http://dx.doi.org/10.1371/journal.pone.0064676
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author Maddala, Rupalatha
Nagendran, Tharkika
de Ridder, Gustaaf G.
Schey, Kevin L.
Rao, Ponugoti Vasantha
author_facet Maddala, Rupalatha
Nagendran, Tharkika
de Ridder, Gustaaf G.
Schey, Kevin L.
Rao, Ponugoti Vasantha
author_sort Maddala, Rupalatha
collection PubMed
description Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels (LTCCs) and explored their role in morphological integrity and transparency of the mouse lens, using cDNA microarray, RT-PCR, immunoblot, pharmacological inhibitors and immunofluorescence analyses. The results revealed that Ca (V) 1.2 and 1.3 channels are expressed and distributed in both the epithelium and cortical fiber cells in mouse lens. Inhibition of LTCCs with felodipine or nifedipine induces progressive cortical cataract formation with time, in association with decreased lens weight in ex-vivo mouse lenses. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling the phenotype reported for altered aquaporin-0 activity without detectable cytotoxic effects. Analysis of both soluble and membrane rich fractions from felodipine treated lenses by SDS-PAGE in conjunction with mass spectrometry and immunoblot analyses revealed decreases in β-B1-crystallin, Hsp-90, spectrin and filensin. Significantly, loss of transparency in the felodipine treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and levels of connexin-50, together with decreases in myosin light chain phosphorylation and the levels of 14-3-3ε, a phosphoprotein-binding regulatory protein. Felodipine treatment led to a significant increase in gene expression of connexin-50 and 46 in the mouse lens. Additionally, felodipine inhibition of LTCCs in primary cultures of mouse lens epithelial cells resulted in decreased intracellular calcium, and decreased actin stress fibers and myosin light chain phosphorylation, without detectable cytotoxic response. Taken together, these observations reveal a crucial role for LTCCs in regulation of expression, activity and stability of aquaporin-0, connexins, cytoskeletal proteins, and the mechanical properties of lens, all of which have a vital role in maintaining lens function and cytoarchitecture.
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spelling pubmed-36671662013-06-03 L-Type Calcium Channels Play a Critical Role in Maintaining Lens Transparency by Regulating Phosphorylation of Aquaporin-0 and Myosin Light Chain and Expression of Connexins Maddala, Rupalatha Nagendran, Tharkika de Ridder, Gustaaf G. Schey, Kevin L. Rao, Ponugoti Vasantha PLoS One Research Article Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels (LTCCs) and explored their role in morphological integrity and transparency of the mouse lens, using cDNA microarray, RT-PCR, immunoblot, pharmacological inhibitors and immunofluorescence analyses. The results revealed that Ca (V) 1.2 and 1.3 channels are expressed and distributed in both the epithelium and cortical fiber cells in mouse lens. Inhibition of LTCCs with felodipine or nifedipine induces progressive cortical cataract formation with time, in association with decreased lens weight in ex-vivo mouse lenses. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling the phenotype reported for altered aquaporin-0 activity without detectable cytotoxic effects. Analysis of both soluble and membrane rich fractions from felodipine treated lenses by SDS-PAGE in conjunction with mass spectrometry and immunoblot analyses revealed decreases in β-B1-crystallin, Hsp-90, spectrin and filensin. Significantly, loss of transparency in the felodipine treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and levels of connexin-50, together with decreases in myosin light chain phosphorylation and the levels of 14-3-3ε, a phosphoprotein-binding regulatory protein. Felodipine treatment led to a significant increase in gene expression of connexin-50 and 46 in the mouse lens. Additionally, felodipine inhibition of LTCCs in primary cultures of mouse lens epithelial cells resulted in decreased intracellular calcium, and decreased actin stress fibers and myosin light chain phosphorylation, without detectable cytotoxic response. Taken together, these observations reveal a crucial role for LTCCs in regulation of expression, activity and stability of aquaporin-0, connexins, cytoskeletal proteins, and the mechanical properties of lens, all of which have a vital role in maintaining lens function and cytoarchitecture. Public Library of Science 2013-05-29 /pmc/articles/PMC3667166/ /pubmed/23734214 http://dx.doi.org/10.1371/journal.pone.0064676 Text en © 2013 Maddala et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Maddala, Rupalatha
Nagendran, Tharkika
de Ridder, Gustaaf G.
Schey, Kevin L.
Rao, Ponugoti Vasantha
L-Type Calcium Channels Play a Critical Role in Maintaining Lens Transparency by Regulating Phosphorylation of Aquaporin-0 and Myosin Light Chain and Expression of Connexins
title L-Type Calcium Channels Play a Critical Role in Maintaining Lens Transparency by Regulating Phosphorylation of Aquaporin-0 and Myosin Light Chain and Expression of Connexins
title_full L-Type Calcium Channels Play a Critical Role in Maintaining Lens Transparency by Regulating Phosphorylation of Aquaporin-0 and Myosin Light Chain and Expression of Connexins
title_fullStr L-Type Calcium Channels Play a Critical Role in Maintaining Lens Transparency by Regulating Phosphorylation of Aquaporin-0 and Myosin Light Chain and Expression of Connexins
title_full_unstemmed L-Type Calcium Channels Play a Critical Role in Maintaining Lens Transparency by Regulating Phosphorylation of Aquaporin-0 and Myosin Light Chain and Expression of Connexins
title_short L-Type Calcium Channels Play a Critical Role in Maintaining Lens Transparency by Regulating Phosphorylation of Aquaporin-0 and Myosin Light Chain and Expression of Connexins
title_sort l-type calcium channels play a critical role in maintaining lens transparency by regulating phosphorylation of aquaporin-0 and myosin light chain and expression of connexins
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3667166/
https://www.ncbi.nlm.nih.gov/pubmed/23734214
http://dx.doi.org/10.1371/journal.pone.0064676
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