In situ measurement of airway surface liquid [K+] using a ratioable K+-sensitive fluorescent dye.
The airway surface liquid (ASL) is the thin fluid layer lining airway surface epithelial cells, whose volume and composition are tightly regulated and may be abnormal in cystic fibrosis (CF). We synthesized a two-color fluorescent dextran to measure ASL [K(+)], TAC-Lime-dextran-TMR, consisting of a green-fluorescing triazacryptand K(+) ionophore-Bodipy conjugate, coupled to dextran, together with a red fluorescing tetramethylrhodamine reference chromophore. TAC-Lime-dextran-TMR fluorescence was K(+)-selective, increasing >4-fold with increasing [K(+)] from 0 to 40 mm. In well differentiated human airway epithelial cells, ASL [K(+)] was 20.8 +/- 0.3 mm and decreased by inhibition of the Na(+)/K(+) pump (ouabain), ENaC (amiloride), CF transmembrane conductance regulator (CFTR(inh)-172), or K(+) channels (TEA or XE991). ASL [K(+)] was increased by forskolin but not affected by Na(+)/K(+)/2Cl(-) cotransporter inhibition (bumetanide). Functional and expression studies indicated the involvement of [K(+)] channels KCNQ1, KCNQ3, and KCNQ5 as determinants of ASL [K(+)]. [K(+)] in CF cultures was similar to that in non-CF cultures, suggesting that abnormal ASL [K(+)] is not a factor in CF lung disease. In intact airways, ASL [K(+)] was also well above extracellular [K(+)]: 22 +/- 1 mm in pig trachea ex vivo and 16 +/- 1 mm in mouse trachea in vivo. Our results provide the first noninvasive measurements of [K(+)] in the ASL and indicate the involvement of apical and basolateral membrane ion transporters in maintaining a high ASL [K(+)].
Namkung
Wan
W
Departments of Medicine and Physiology, University of California, San Francisco, California 94143, USA.
Song
Yuanlin
Y
Mills
Aaron D
AD
Padmawar
Prashant
P
Finkbeiner
Walter E
WE
Verkman
A S
AS
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001980:Bronchi / Q000502:physiology*; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000503:physiopathology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology; D017931:DNA Primers; D004594:Electrophysiology / Q000379:methods; D004847:Epithelial Cells / Q000502:physiology*; D005456:Fluorescent Dyes; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D011188:Potassium / Q000502:physiology*; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014132:Trachea / Q000166:cytology / Q000502:physiology*
C505032:CFTR protein, human; D017931:DNA Primers; D005456:Fluorescent Dyes; D015221:Potassium Channels; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D012313:RNA; D000584:Amiloride; D000255:Adenosine Triphosphate; D011188:Potassium