Hypothermia increases aquaporin 4 (AQP4) plasma membrane abundance in human primary cortical astrocytes via a calcium/transient receptor potential vanilloid 4 (TRPV4)- and calmodulin-mediated mechanism

Mootaz M. Salman, Philip Kitchen, M. Nicola Woodroofe, James E. Brown, Roslyn M Bill, Alex C. Conner, Matthew T. Conner

Research output: Contribution to journalArticle

Abstract

Human aquaporin 4 (AQP4) is the primary water channel protein in brain astrocytes. Hypothermia is known to cause astrocyte swelling in culture, but the precise role of AQP4 in this process is unknown. Primary human cortical astrocytes were cultured under hypothermic (32 °C) or normothermic (37 °C) conditions. AQP4 transcript, total protein and surface-localized protein were quantified using RT-qPCR, sandwich ELISA with whole cell lysates or cell surface biotinylation, followed by ELISA analysis of the surface-localized protein, respectively. Four-hour mild hypothermic treatment increased the surface localization of AQP4 in human astrocytes to 155 ± 4% of normothermic controls, despite no change in total protein expression levels. The hypothermia-mediated increase in AQP4 surface abundance on human astrocytes was blocked using either calmodulin antagonist (trifluoperazine, TFP); TRPV4 antagonist, HC-067047 or calcium chelation using EGTA-AM. The TRPV4 agonist (GSK1016790A) mimicked the effect of hypothermia compared with untreated normothermic astrocytes. Hypothermia led to an increase in surface localization of AQP4 in human astrocytes through a mechanism likely dependent on the TRPV4 calcium channel and calmodulin activation. Understanding the effects of hypothermia on astrocytic AQP4 cell surface expression may help develop new treatments for brain swelling based on an in-depth mechanistic understanding of AQP4 translocation.
Original languageEnglish
Pages (from-to)2542-2547
Number of pages6
JournalEuropean Journal of Neuroscience
Volume46
Issue number9
Early online date13 Oct 2017
DOIs
Publication statusPublished - 13 Oct 2017

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Aquaporin 4
Calcium-Sensing Receptors
Calmodulin
Hypothermia
Astrocytes
Cell Membrane
TRPV Cation Channels
Membrane Proteins
Enzyme-Linked Immunosorbent Assay
Biotinylation
Trifluoperazine
Aquaporins
Brain Edema
Calcium Channels
Proteins
Calcium

Bibliographical note

© The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Funding: Sheffield Hallam University, University of Wolverhampton, School of Life and Health Sciences Aston University and the HCED/Iraq grant number GD-13-3 (M Salman.

Keywords

  • TRPV4
  • aquaporin 4
  • astrocyte
  • calcium
  • calmodulin
  • mild therapeutic hypothermia

Cite this

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title = "Hypothermia increases aquaporin 4 (AQP4) plasma membrane abundance in human primary cortical astrocytes via a calcium/transient receptor potential vanilloid 4 (TRPV4)- and calmodulin-mediated mechanism",
abstract = "Human aquaporin 4 (AQP4) is the primary water channel protein in brain astrocytes. Hypothermia is known to cause astrocyte swelling in culture, but the precise role of AQP4 in this process is unknown. Primary human cortical astrocytes were cultured under hypothermic (32 °C) or normothermic (37 °C) conditions. AQP4 transcript, total protein and surface-localized protein were quantified using RT-qPCR, sandwich ELISA with whole cell lysates or cell surface biotinylation, followed by ELISA analysis of the surface-localized protein, respectively. Four-hour mild hypothermic treatment increased the surface localization of AQP4 in human astrocytes to 155 ± 4{\%} of normothermic controls, despite no change in total protein expression levels. The hypothermia-mediated increase in AQP4 surface abundance on human astrocytes was blocked using either calmodulin antagonist (trifluoperazine, TFP); TRPV4 antagonist, HC-067047 or calcium chelation using EGTA-AM. The TRPV4 agonist (GSK1016790A) mimicked the effect of hypothermia compared with untreated normothermic astrocytes. Hypothermia led to an increase in surface localization of AQP4 in human astrocytes through a mechanism likely dependent on the TRPV4 calcium channel and calmodulin activation. Understanding the effects of hypothermia on astrocytic AQP4 cell surface expression may help develop new treatments for brain swelling based on an in-depth mechanistic understanding of AQP4 translocation.",
keywords = "TRPV4 , aquaporin 4 , astrocyte , calcium , calmodulin , mild therapeutic hypothermia",
author = "Salman, {Mootaz M.} and Philip Kitchen and Woodroofe, {M. Nicola} and Brown, {James E.} and Bill, {Roslyn M} and Conner, {Alex C.} and Conner, {Matthew T.}",
note = "{\circledC} The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. Funding: Sheffield Hallam University, University of Wolverhampton, School of Life and Health Sciences Aston University and the HCED/Iraq grant number GD-13-3 (M Salman.",
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T1 - Hypothermia increases aquaporin 4 (AQP4) plasma membrane abundance in human primary cortical astrocytes via a calcium/transient receptor potential vanilloid 4 (TRPV4)- and calmodulin-mediated mechanism

AU - Salman, Mootaz M.

AU - Kitchen, Philip

AU - Woodroofe, M. Nicola

AU - Brown, James E.

AU - Bill, Roslyn M

AU - Conner, Alex C.

AU - Conner, Matthew T.

N1 - © The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. Funding: Sheffield Hallam University, University of Wolverhampton, School of Life and Health Sciences Aston University and the HCED/Iraq grant number GD-13-3 (M Salman.

PY - 2017/10/13

Y1 - 2017/10/13

N2 - Human aquaporin 4 (AQP4) is the primary water channel protein in brain astrocytes. Hypothermia is known to cause astrocyte swelling in culture, but the precise role of AQP4 in this process is unknown. Primary human cortical astrocytes were cultured under hypothermic (32 °C) or normothermic (37 °C) conditions. AQP4 transcript, total protein and surface-localized protein were quantified using RT-qPCR, sandwich ELISA with whole cell lysates or cell surface biotinylation, followed by ELISA analysis of the surface-localized protein, respectively. Four-hour mild hypothermic treatment increased the surface localization of AQP4 in human astrocytes to 155 ± 4% of normothermic controls, despite no change in total protein expression levels. The hypothermia-mediated increase in AQP4 surface abundance on human astrocytes was blocked using either calmodulin antagonist (trifluoperazine, TFP); TRPV4 antagonist, HC-067047 or calcium chelation using EGTA-AM. The TRPV4 agonist (GSK1016790A) mimicked the effect of hypothermia compared with untreated normothermic astrocytes. Hypothermia led to an increase in surface localization of AQP4 in human astrocytes through a mechanism likely dependent on the TRPV4 calcium channel and calmodulin activation. Understanding the effects of hypothermia on astrocytic AQP4 cell surface expression may help develop new treatments for brain swelling based on an in-depth mechanistic understanding of AQP4 translocation.

AB - Human aquaporin 4 (AQP4) is the primary water channel protein in brain astrocytes. Hypothermia is known to cause astrocyte swelling in culture, but the precise role of AQP4 in this process is unknown. Primary human cortical astrocytes were cultured under hypothermic (32 °C) or normothermic (37 °C) conditions. AQP4 transcript, total protein and surface-localized protein were quantified using RT-qPCR, sandwich ELISA with whole cell lysates or cell surface biotinylation, followed by ELISA analysis of the surface-localized protein, respectively. Four-hour mild hypothermic treatment increased the surface localization of AQP4 in human astrocytes to 155 ± 4% of normothermic controls, despite no change in total protein expression levels. The hypothermia-mediated increase in AQP4 surface abundance on human astrocytes was blocked using either calmodulin antagonist (trifluoperazine, TFP); TRPV4 antagonist, HC-067047 or calcium chelation using EGTA-AM. The TRPV4 agonist (GSK1016790A) mimicked the effect of hypothermia compared with untreated normothermic astrocytes. Hypothermia led to an increase in surface localization of AQP4 in human astrocytes through a mechanism likely dependent on the TRPV4 calcium channel and calmodulin activation. Understanding the effects of hypothermia on astrocytic AQP4 cell surface expression may help develop new treatments for brain swelling based on an in-depth mechanistic understanding of AQP4 translocation.

KW - TRPV4

KW - aquaporin 4

KW - astrocyte

KW - calcium

KW - calmodulin

KW - mild therapeutic hypothermia

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DO - 10.1111/ejn.13723

M3 - Article

C2 - 28925524

VL - 46

SP - 2542

EP - 2547

JO - European Journal of Neuroscience

JF - European Journal of Neuroscience

SN - 0953-816X

IS - 9

ER -