AJP - Cell Physiology, Vol 266, Issue 1 C284-C292, Copyright © 1994 by American Physiological Society

ARTICLES

Characterization of the endogenous Na(+)-K(+)-2Cl- cotransporter in Xenopus oocytes

W. Suvitayavat, H. C. Palfrey, M. Haas, P. B. Dunham, F. Kalmar and M. C. Rao
Department of Physiology and Biophysics, University of Illinois at Chicago 60612.

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)

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