ACTIVATION OF AN NA+/K+/2CL(-) COTRANSPORT SYSTEM BY PHOSPHORYLATION IN CRYPT CELLS ISOLATED FROM GUINEA-PIG DISTAL COLON

Background and 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 Kf influx was observed. Pretreatment with the protein-phosphatase inhibitor calyculin A (50% effective concentration, 23 nmol/L) or ionomycin showed a bumetanide-sensitive Kf 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+.