TY - JOUR
T1 - A model system using confocal fluorescence microscopy for examining real-time intracellular sodium ion regulation
AU - Lee, Jacqueline A.
AU - Collings, David A.
AU - Glover, Chris N.
N1 - Publisher Copyright:
© 2016 Elsevier Inc. All rights reserved.
PY - 2016/8/15
Y1 - 2016/8/15
N2 - The gills of euryhaline fish are the ultimate ionoregulatory tissue, achieving ion homeostasis despite rapid and significant changes in external salinity. Cellular handling of sodium is not only critical for salt and water balance but is also directly linked to other essential functions such as acid-base homeostasis and nitrogen excretion. However, although measurement of intracellular sodium ([Na+]i) is important for an understanding of gill transport function, it is challenging and subject to methodological artifacts. Using gill filaments from a model euryhaline fish, inanga (Galaxias maculatus), the suitability of the fluorescent dye CoroNa Green as a probe for measuring [Na+]i in intact ionocytes was confirmed via confocal microscopy. Cell viability was verified, optimal dye loading parameters were determined, and the dye-ion dissociation constant was measured. Application of the technique to freshwater- and 100% seawater-acclimated inanga showed salinity-dependent changes in branchial [Na+]i, whereas no significant differences in branchial [Na+]i were determined in 50% seawater-acclimated fish. This technique facilitates the examination of real-time changes in gill [Na+]i in response to environmental factors and may offer significant insight into key homeostatic functions associated with the fish gill and the principles of sodium ion transport in other tissues and organisms.
AB - The gills of euryhaline fish are the ultimate ionoregulatory tissue, achieving ion homeostasis despite rapid and significant changes in external salinity. Cellular handling of sodium is not only critical for salt and water balance but is also directly linked to other essential functions such as acid-base homeostasis and nitrogen excretion. However, although measurement of intracellular sodium ([Na+]i) is important for an understanding of gill transport function, it is challenging and subject to methodological artifacts. Using gill filaments from a model euryhaline fish, inanga (Galaxias maculatus), the suitability of the fluorescent dye CoroNa Green as a probe for measuring [Na+]i in intact ionocytes was confirmed via confocal microscopy. Cell viability was verified, optimal dye loading parameters were determined, and the dye-ion dissociation constant was measured. Application of the technique to freshwater- and 100% seawater-acclimated inanga showed salinity-dependent changes in branchial [Na+]i, whereas no significant differences in branchial [Na+]i were determined in 50% seawater-acclimated fish. This technique facilitates the examination of real-time changes in gill [Na+]i in response to environmental factors and may offer significant insight into key homeostatic functions associated with the fish gill and the principles of sodium ion transport in other tissues and organisms.
KW - Amphidromous
KW - Fish
KW - Gill
KW - Intracellular sodium concentration
KW - Laser scanning confocal microscopy
KW - Salinity acclimation
UR - http://www.scopus.com/inward/record.url?scp=84973889210&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2016.05.008
DO - 10.1016/j.ab.2016.05.008
M3 - Journal Article
C2 - 27235170
AN - SCOPUS:84973889210
SN - 0003-2697
VL - 507
SP - 40
EP - 46
JO - Analytical Biochemistry
JF - Analytical Biochemistry
ER -