Characterization of the endogenous Na(+)-K(+)-2Cl- cotransporter in Xenopus oocytes.
Over time, Xenopus laevis changed from producing stage V and VI oocytes with little native Na(+)-K(+)-2Cl- cotransport activity to those with substantial activity. In oocytes with high endogenous activity, K+ uptake, using the tracer 86Rb+ was approximately 20 pmol.min-1.oocyte-1 in the presence of blockers of Na(+)-K(+)-ATPase and conductive K+ transport. Bumetanide (10 microM) inhibited > 90% of this uptake, suggesting involvement of Na(+)-K(+)-2Cl- cotransport. This was confirmed by two observations that are found in this cotransporter in other tissues: 1) The related diuretics, thiobenzmetanide [50% inhibitory concentration (IC50), 2 x 10(-11) M] > bumetanide (IC50, 7 x 10(-8) M) > furosemide (IC50, 2.5 x 10(-6) M) inhibited the cotransporter in a dose-dependent manner. 2) There was little uptake of K+ in the absence of extracellular Na+ or Cl-. Halving medium osmolarity to 92 mosM decreased bumetanide-sensitive K+ uptake by approximately 75%, whereas a doubling of medium osmolarity increased it by approximately 50%. The cotransport activity was increased fourfold by the phosphatase inhibitor calyculin A (200 nM) but was unaffected by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, 8-bromoguanosine 3',5'-cyclic monophosphate, ATP, ionomycin, or okadaic acid. Both the photoaffinity bumetanide analogue, 4-[3H]benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid, and an antiserum raised against Ehrlich ascites cell cotransporter specifically labeled an approximately 140-kDa oocyte membrane protein. These results demonstrated that, in addition to the Na+ pump and K+ channels, K+ uptake in Xenopus oocytes occurs via a loop-diuretic-sensitive Na(+)-K(+)-2Cl- cotransporter.(ABSTRACT TRUNCATED AT 250 WORDS)
Suvitayavat
W
W
Department of Physiology and Biophysics, University of Illinois at Chicago 60612.
Palfrey
H C
HC
Haas
M
M
Dunham
P B
PB
Kalmar
F
F
Rao
M C
MC
D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D007425:Intracellular Membranes / Q000502:physiology; D008387:Marine Toxins; D009865:Oocytes / Q000378:metabolism*; D009994:Osmolar Concentration; D010080:Oxazoles / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters; D014982:Xenopus laevis / Q000378:metabolism*
D002352:Carrier Proteins; D008387:Marine Toxins; D010080:Oxazoles; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; C059041:calyculin A