Regulation of intracellular pH in Calu-3 human airway cells.
The Calu-3 human cell line exhibits features of submucosal gland serous cells and secretes HCO(3)(-). The aim of this study was to identify the HCO(3)(-) transporters present in these cells by studying their role in the regulation of intracellular pH (pH(i)). Calu-3 cells were grown on coverslips, loaded with the pH-sensitive fluorescent dye BCECF, and their fluorescence intensity monitored as an indication of pH(i). Cells were acidified with NH(4)Cl (25 mM, 1 min) and pH(i) recovery recorded. In the absence of HCO(3)(-), initial recovery was 0.208 +/- 0.016 pH units min(-1) (n = 37). This was almost abolished by removal of extracellular Na(+) and by amiloride (1 mM), consistent with the activity of a Na(+)-H(+) exchanger (NHE). In the presence of HCO(3)(-) and CO(2), recovery (0.156 +/- 0.018 pH units min(-1)) was abolished (reduced by 91.8 +/- 6.7 %, n = 7) by removal of Na(+) but only attenuated (by 63.3 +/- 5.8 %, n = 9) by amiloride. 4,4-Dinitrostilbene-2,2-disulfonic acid (DNDS) inhibited recovery by 45.8 +/- 5.0 % (n = 7). The amiloride-insensitive recovery was insensitive to changes in membrane potential, as confirmed by direct microelectrode measurements, brought about by changing extracellular [K(+)] in the presence of either valinomycin or the K(+) channel opener 1-EBIO. In addition, forskolin (10 microM), which activates the cystic fibrosis transmembrane conductance regulator Cl(-) conductance in these cells and depolarises the cell membrane, had no effect on recovery. Removal of extracellular Cl(-) trebled pH(i) recovery rates, suggesting that an electroneutral, DNDS-sensitive, Cl(-)-HCO(3)(-) exchanger together with a NHE may be involved in pH(i) regulation and HCO(3)(-) secretion in these cells. RT-PCR detected the expression of the electrogenic Na(+)-HCO(3)(-) cotransporter NBC1 and the Cl(-)-HCO(3)(-) exchanger (AE2) but not the electroneutral Na(+)-HCO(3)(-) cotransporter NBCn1.
Inglis
S K
SK
Lung Membrane Transport Group, Tayside Institute of Child Health, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK. s.k.inglis@dundee.ac.uk
Finlay
L
L
Ramminger
S J
SJ
Richard
K
K
Ward
M R
MR
Wilson
S M
SM
Olver
R E
RE
D000584:Amiloride / Q000494:pharmacology; D001639:Bicarbonates / Q000494:pharmacology; D002021:Buffers; D002245:Carbon Dioxide / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D005576:Colforsin / Q000494:pharmacology; D006531:HEPES / Q000494:pharmacology; D006706:Homeostasis; D006801:Humans; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D012137:Respiratory System / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D012704:Serous Membrane / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D012964:Sodium / Q000502:physiology; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000502:physiology; D012996:Solutions; D013267:Stilbenes / Q000494:pharmacology
D001639:Bicarbonates; D002021:Buffers; D017923:Sodium-Hydrogen Exchangers; D012996:Solutions; D013267:Stilbenes; D002245:Carbon Dioxide; D005576:Colforsin; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; D000584:Amiloride; D006859:Hydrogen; D012964:Sodium; D006531:HEPES