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Published on: September 1, 2015
Activation of an Na+/K+/2Cl- cotransport system by phosphorylation in crypt cells isolated from guinea pig distal
J R Del Castillo1, F V Sepúlveda
1Agricultural and Food Research Council Babraham Institute, Babraham Hall, Cambridge, England.
Background & Aims:
K+ secretion is believed to require the presence of a basolateral Na+/K+/2Cl- cotransporter. The aim of this study was to identify this transport system in epithelial cells from guinea pig colon and to study its possible regulation by phosphorylation.
Methods:
Cells were selectively isolated from crypt or surface epithelium of proximal or distal colon. Radioisotopes were used to measure K+, Na+, or Cl- influx. Bumetanide was used to discriminate for influx mediated by Na+/K+/2Cl- cotransport.
Results:
Under basal conditions, no bumetanide-sensitive K+ influx was observed. Pretreatment with the protein-phosphatase inhibitor calyculin A (50% effective concentration, 23 nmol/L) or ionomycin showed a bumetanide-sensitive K+ influx specifically in distal colon crypt cells. Okadaic acid and protein kinases C or A activators did not have effect. Bumetanide-sensitive K+ uptake was abolished by the removal of external Na+ or Cl- and occurred by cotransport in a 1Na+/1K+/2Cl- stoichiometry.
Conclusions:
Evidence is presented for the presence of an Na+/K+/2Cl- cotransporter in crypt cells from distal colon epithelium. The activity of this transporter is proposed to be regulated by a phosphorylation/dephosphorylation cycle, controlled by a type I protein phosphatase. It is possible that this phosphatase(s) is modulated by intracellular Ca2+.
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