Na+, K+, Cl- cotransport and its regulation in Ehrlich ascites tumor cells. Ca2+/calmodulin and protein kinase C dependent pathways.
Net Cl- uptake as well as unidirectional 36Cl influx during regulatory volume increase (RVI) require external K+. Half-maximal rate of bumetanide-sensitive 36Cl uptake is attained at about 3.3 mM external K+. The bumetanide-sensitive K+ influx found during RVI is strongly dependent on both Na+ and Cl-. The bumetanide-sensitive unidirectional Na+ influx during RVI is dependent on K+ as well as on Cl-. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na+, K+ and Cl-. In the presence of ouabain and Ba+ the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na+, 0.8 K+, 2.0 Cl- or approximately 1:Na, 1:K, 2:Cl. Under these circumstances the K+ and Cl- flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 +/- 0.08 and 1.82 +/- 0.15 which should be compared to the gradient for the Na+, K+, 2Cl- cotransport system at 1.75 +/- 0.24. Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K+ influx is activated. During hypotonic conditions a small bumetanide-sensitive K+ influx is observed, indicating that the cotransport system is already activated. The cotransport is activated 10-15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca2+ and activation of protein kinase C. The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K+ influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na+, K+, Cl- cotransport involves both Ca2+/calmodulin and protein kinase C.
Jensen
B S
BS
Institute of Biological Chemistry A, August Krogh Institute, University of Copenhagen, Denmark.
Jessen
F
F
Hoffmann
E K
EK
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002034:Bumetanide / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000502:physiology*; D002286:Carcinoma, Ehrlich Tumor / Q000737:chemistry / Q000473:pathology* / Q000503:physiopathology; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D006982:Hypertonic Solutions; D051379:Mice; D011493:Protein Kinase C / Q000502:physiology*; D028021:Sodium-Potassium-Chloride Symporters; D013395:Sucrose / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000502:physiology
D002147:Calmodulin; D002352:Carrier Proteins; D006982:Hypertonic Solutions; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D013395:Sucrose; D011493:Protein Kinase C; D010738:Type C Phospholipases; D001920:Bradykinin; D002118:Calcium