Activation of an Na +/K +/2Cl − cotransport system by phosphorylation in crypt cells isolated from guinea pig distal colon

Abstract

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 Ca 2+.