Activation of an Na+/K+/2Cl- cotransport system by phosphorylation in crypt cells isolated from guinea pig distal colon.
OBJECTIVE
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+.
Del Castillo
J R
JR
Agricultural and Food Research Council Babraham Institute, Babraham Hall, Cambridge, England.
Sepúlveda
F V
FV
D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D004988:Ethers, Cyclic / Q000494:pharmacology; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism; D017136:Ion Transport; D015759:Ionomycin / Q000494:pharmacology; D008297:Male; D008387:Marine Toxins; D019319:Okadaic Acid; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism*; D011494:Protein Kinases / Q000819:agonists; D012964:Sodium / Q000378:metabolism*
D002712:Chlorides; D004988:Ethers, Cyclic; D008387:Marine Toxins; D010080:Oxazoles; D002034:Bumetanide; D019319:Okadaic Acid; D015759:Ionomycin; C059041:calyculin A; D012964:Sodium; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D011188:Potassium; D002118:Calcium