Identification and membrane localization of electrogenic sodium bicarbonate cotransporters in Calu-3 cells.
Cystic fibrosis (CF) is a severely life-shortening genetic disease resulting from mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR). Impaired bicarbonate secretion is a key component of CF-related pancreatic disease, but the role of impaired bicarbonate secretion in CF lung disease is less well understood. The submucosal glands of the conducting airways produce and secrete a complex airway surface liquid that lines the airway epithelium and plays a significant role in mucociliary clearance. The serous cell is the predominant cell type of the submucosal gland and a predominant site of CFTR expression. Calu-3 cells are a model of airway submucosal gland serous cells that demonstrates vectorial bicarbonate secretion in response to elevations in cAMP. Based on previously published measurements of unidirectional ion flux, pharmacological inhibition of short-circuit current and ion substitution studies, one can hypothesize the existence of an electrogenic sodium bicarbonate cotransporter (NBC) in the basolateral membrane of Calu-3 cells that mediates bicarbonate entry from the interstitium. To test this hypothesis, we performed reverse-transcriptase PCR, western blotting, and surface biotinylation to identify and localize electrogenic NBCs in Calu-3 cells. Our data demonstrate that both pNBC1 and NBC4 mRNAs can be identified and that their protein products are expressed at the basolateral membrane of polarized Calu-3 cells. These data suggest that these transporters contribute to regulated bicarbonate secretion across Calu-3 cells and perhaps human airway submucosal glands.
Kreindler
James L
JL
Department of Pediatrics, Children's Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213, USA. james.kreindler@chp.edu
Peters
Kathryn W
KW
Frizzell
Raymond A
RA
Bridges
Robert J
RJ
D001483:Base Sequence; D001639:Bicarbonates / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D006801:Humans; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D020545:Respiratory Mucosa / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics* / Q000378:metabolism*
D001639:Bicarbonates; D017931:DNA Primers; D027982:Sodium-Bicarbonate Symporters; D012313:RNA; D012964:Sodium