CFTR upregulates the expression of the basolateral Na(+)-K(+)-2Cl(-) cotransporter in cultured pancreatic duct cells.
The purpose of the current experiments was 1) to assess basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) expression and 2) to ascertain the role of cystic fibrosis transmembrane conductance regulator (CFTR) in the regulation of this transporter in a prototypical pancreatic duct epithelial cell line. Previously validated human pancreatic duct cell lines (CFPAC-1), which exhibit physiological features prototypical of cystic fibrosis, and normal pancreatic duct epithelia (stable recombinant CFTR-bearing CFPAC-1 cells, termed CFPAC-WT) were grown to confluence before molecular and functional studies. High-stringency Northern blot hybridization, utilizing specific cDNA probes, confirmed that NKCC1 was expressed in both cell lines and its mRNA levels were twofold higher in CFPAC-WT cells than in CFPAC-1 cells (P < 0.01, n = 3). Na(+)-K(+)-2Cl(-) cotransporter activity, assayed as the bumetanide-sensitive, Na(+)- and Cl(-)-dependent NH(+)(4) entry into the cell (with NH(+)(4) acting as a substitute for K(+)), increased by approximately 115% in CFPAC-WT cells compared with CFPAC-1 cells (P < 0.01, n = 6). Reducing the intracellular Cl(-) by incubating the cells in a Cl(-)-free medium increased Na(+)-K(+)-2Cl(-) cotransporter activity by twofold (P < 0.01, n = 4) only in CFPAC-WT cells. We concluded that NKCC1 is expressed in pancreatic duct cells and mediates the entry of Cl(-). NKCC1 activity is enhanced in the presence of an inward Cl(-) gradient. The results further indicate that the presence of functional CFTR enhances the expression of NKCC1. We speculate that CFTR regulates this process in a Cl(-)-dependent manner.
Shumaker
H
H
Department of Medicine, University of Cincinnati, and Veterans Affairs Medical Center at Cincinnati, Cincinnati, Ohio 45267, USA.
Soleimani
M
M
D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics*; D002478:Cells, Cultured; D002712:Chlorides / Q000493:pharmacokinetics; D005576:Colforsin / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D010183:Pancreatic Ducts / Q000737:chemistry / Q000166:cytology*; D000644:Quaternary Ammonium Compounds / Q000493:pharmacokinetics; D012333:RNA, Messenger / Q000032:analysis; D012964:Sodium / Q000493:pharmacokinetics; D017693:Sodium Bicarbonate / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters
C505032:CFTR protein, human; D002352:Carrier Proteins; D002712:Chlorides; D000644:Quaternary Ammonium Compounds; D012333:RNA, Messenger; D028021:Sodium-Potassium-Chloride Symporters; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D017693:Sodium Bicarbonate; D012964:Sodium