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Volume regulation in in situ human airway epithelial (calu‐3) cells: Focal Adhesion Kinase (FAK) and NKCC1
Author(s) -
MacLellan Amber L,
Marshall William S
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.728.1
Subject(s) - bumetanide , cotransporter , chemistry , focal adhesion , microbiology and biotechnology , ion transporter , cystic fibrosis transmembrane conductance regulator , osmotic concentration , tonicity , biophysics , cell , biochemistry , biology , sodium , membrane , organic chemistry , gene
Ion transport by FAK‐activated Na + ‐K + ‐2Cl − co‐transporter (NKCC1) and cystic fibrosis transmembrane conductance regulators (CFTR) is essential in cell regulatory volume responses in teleost epithelia (Marshall et al. 2005,2009). FAK‐related protein tyrosine kinase 2 (PYK2) activates CFTR in Calu‐3 cells (Liang et al. 2011), however, FAK regulation of NKCC or CFTR has not been investigated in a mammalian system. We aim to examine the possible role of FAK in regulating NKCC or CFTR ion‐transport during cell volume regulation in human airway (Calu‐3) cells. To model in vivo airway conditions, Calu‐3 cells were cultured into confluent monolayers on glass coverslips in minimal essential medium (MEM) at 37°C with 5% CO 2 /95% air. Cell volume regulation was examined using, 4–7 day‐old monolayers stained with BODIPY‐ceramide vital dye (1:1000 in isotonic MEM solution for 1hr) and mounted in a perfusion chamber. Monolayers were perfused with combinations of isotonic MEM 285 mOsm/kg (control), and NaCl‐equilibrated 360 mOsm/kg hypertonic MEM (hypersaline lung lavages use 3–7% NaCl aq ). Cell height (volume) changes were recorded over 1hr using XZt confocal micrographs. Hypertonic shock caused Calu‐3 cells shrinkage over 1–7 mins followed by regulatory volume increase (RVI) (contrasting previous reports) over 45‐mins (n=18 cells; 6 monolayers). To determine if NKCC1 or FAK‐facilitated ion transport facilitated RVI, monolayers were treated with NKCC1 inhibitor bumetanide and a FAK‐inhibitor TAE226. Inhibition of NKCC1 and FAK altered volume regulation patterns suggesting a critical mediation of airway cell volume regulation by NKCC1 and FAK. Funded by NSERC and NSHRF.