Summary
Loop diuretic-sensitive (Na+,K+,Cl−)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of grandients of all three ions was essential for optimal transport activity (Na+,K+) gradien-driven36Cl fluxes weree half-maximally inhibited by 0.14 μm bumetanide and 44 μm furosimide. While86Rb uptake rates showed hyperbolic dependencies on Na+ and K+ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl− concentration, Hill coefficient 1.8, indicating a 1 Na+: 1 K+:2 Cl− stoichiometry of ion transport.
The interaction of putative (Na+, K+, Cl−)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [3H]-bumetanide. The requirement for the simulataneous presence of Na+,K+, and Cl−, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na+, K+, Cl−)-cotransporter. [3H]-bumetanide recognizes a minimum of two classes of diuretic receptors sites. high-affinity (K D1=0.13 μm;B max1 =6.4 pmol/mg of protein) and low-affinity (K D2=34 μm;B max2=153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl− (K 1=60mm), whereas low-affinity sites seem to be unaffected by Cl−. We have shown that only high-affinity [3H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.
Similar content being viewed by others
References
Brown C.D.A., Murer, H. 1985. Characterization of a Na∶K∶2Cl cotransport system in the apical membrane of a renal epithelial cell line (LLC/PK1).J. Membrane Biol. 87:131–139
Burnham, C., Karlish, S.J.D., Jørgensen, P.L. 1985. Identification and reconstitution of a Na+/K+/Cl− cotransproter and K+ channel from luminal membranes of renal red outer medulla.Biochim. Biophys. Acta 821:461–469
Cabantchik, Z.I., Knauf, P.A., Rothstein, A. 1978. The anion transport system of the red blood cell. The role of membrane protein evaluated by the use of “probes”.Biochim. Biophys. Acta 515:239–302
Chou, T. 1976. Derivation and properties of Michaelis/Menten type and Hill type equations for reference ligands.J. Theor. Biol. 59:253–276
DeMeyts, P., Roth, J. 1975. Cooperativity in ligand binding: A new graphic analysis.Biochem. Biophys. Res. Commun. 66:11118–11125
Ellory, J.C., Dunham, P.B., Logue, P.J., Stewart, G.W. 1982. Anion-dependent cation transport in erythrocytes.Phil. Trans. R. Soc. London B 299:483–495
Epstein, F.H., Silva, P. 1985. Na-K-Cl cotransport in chloride-transporting epithelia.Ann. NY Acad. Sci. 456:187–197
Forbush, B. III., Palfrey, H.C. 1983. [3H] bumetanide binding to membranes isolated from dog kidney outer medulla.J. Biol. Chem. 258:11787–11792
Frizzell, R.A., Schultz, S.G. 1979. Models of electrolyte absorption and secretion by gastrointestinal epithelia.Int. Rev. Physiol. 19:205–225
Geck, P., Heinz, E. 1986. The Na-K-2Cl cotransport system.J. Membrane Biol. 91:97–105
Geck, P., Pfeiffer, B. 1985. Na++K++2Cl− cotransport in animal cell-Its role in volume regulation.Ann. Ny Acad. Sci. 456:166–182
Giesen-Crouse, E.M., McRoberts, J.A. 1987. Coordinate expression of piretanide receptors and Na−, K+, CKl− cotransport activity in Madin-Darby canine kidney cell mutants.J. Biol. Chem. 262:17393–17397
Greger, R. 1981. Cation selectivity of the isolated perfused cortical thick ascending limb of Henle's loop of rabbit kidney.Pfluegers Arch. 390:38–43
Gunn, R.B. 1985. Bumetanide inhibition of anion exchange in human red blood cells.Biophys. J. 47:326a
Gustin, M.C., Goodman, D.B.P. 1981. Isolation of the brush-border membrane from the rabbit descending colon epithelium.J. Biol. Chem. 256:10651–10656
Haas, M., Forbush, B., III. 1986. [3H] bumetanide binding to duck red cells. Correlation with inhibition of (Na+K+2Cl) co-transport.J. Biol. Chem. 261:8434–8441
Haas, M., Forbush, B., III. 1987. Na,K,Cl-cotransport system: Characterization by bumetanide binding and photolabeling.Kidney Int. 32:S134-S140
Haas, M., Forbush, B., III. 1987. Photolabeling of a 150-kDa (Na+K+Cl) cotransport protein from dog kidney with a bumetanide analogue.Am. J. Physiol. 253:C243-C250
Haas, M., McManus, T.J. 1983. Bumetanide inhibits (Na+K+2Cl) cotransport at a chloride site.Am. J. Physiol. 245:C235-C240
Hannafin, J., Kinne-Saffran, E., Friedman, D., Kinne, R. 1983. Presence of a sodium-potassium chloride cotransport system in the rectal gland ofSqualus acanthias.J. Membrane Biol. 75:73–83
Heintze, K., Stewart, C.P., Frizzell, R.A. 1983. Sodium-dependent chloride secretion across rabbit descending colon.Am. J. Physiol. 244:G357-G365
Heytler, P.G. 1979. Uncouplers of oxidative phosphorylation.Methods Enzymol. LV:462–472
Hoffmann, E.K. 1986. Anion transport systems in the plasma membrane of vertebrate cells.Biochim. Biophys. Acta 864:1–31
Hoffmann, E.K., Schiodt, M., Dunham, P. 1986. The number of chloride-cation cotransport sites on Ehrlich ascites cells measured with [3H] bumetanide.Am. J. Physiol. 250:C688-C693
Jørgensen, P.L., Petersen, J., Rees, W.D. 1984. Identification of a Na−,K−,Cl−1 cotransport protein ofM r 34,000 from kidney by photolabeling with [3H] bumetanide.Biochim. Biophys. Acta 775:105–110
Kessler, M., Tannenbaum, V., Tannenbaum, C. 1978. A simple apparatus for performing short time uptake measurements in small volumes: Its application tod-glucose transport studies in brush border vesicles from rabbit jejunum.Biochim. Biophys. Acta 509:348–359
Kinne, R., Hannafin, J.A., König, B. 1985. Role of the NaCl−KCl cotransport system in active chloride absorption and secretion.Ann. NY Acad. Sci. 456:198–206
Kinne, R., Koenig, B., Hannafin, J., Kinne-Saffran, E., Scott, D.M., Zierold, K. 1985. The use of membrane vesicles to study the NaCl/KCl cotransporter involved in active chloride transport.Pfluegers Arch. 405:S101-S105
Kinsella, J.L., Aronson, P.S. 1981. Amiloride inhibition of the Na−/H− exchanger in renal microvillus membrane vesicles.Am. J. Physiol. 241:F374-F379
Klotz, I.M. 1982. Number of receptor sites from Scatchard graphs: Facts and fantasies.Science 217:1247–1249
Koenig, B., Ricapito, S., Kinne, R. 1983. Chloride transport in the thick ascending limb of Henle's loop: K dependence and stoichiometry of NaCl cotransport in plasma membrane vesicles.Pfluegers Arch. 399:173–179
Liedtke, C.M., Hopfer, U. 1982. Mechanisms of Cl− translocation across small intestinal brush-border membrane. II. Demonstration of Cl−−OH− exchange and Cl− conductance.Am. J. Physiol. 242:G272-G280
Loftfield, R.B., Eigner, E.A. 1969. Molecular order of participation of inhibitors (or activators) in biological systems.Science 164:305–308
Mercer, R.W., Hoffman, J.F. 1985. Bumetanide sensitive Na/K cotransport in ferret red blood cells.Biophys. J. 47:157a
Miyamoto, H., Ikehara, T., Yamaguchi, H., Hosokawa, K., Yonezu, T., Masuya, T. 1986. Kinetic mechanism of Na+,K+,Cl−-cotransport as studied by Rb+ influx into HeLa cells: Effects of extracellular monovalent ions.J. Membrane Biol. 92:135–150
Owen, N.E., Prastein, M.L. 1985. Na/K/Cl cotransport in cultured human fibroblasts.J. Biol. Chem. 260:1445–1451
Palfrey, H.C., Feit, P.W., Greengard, P. 1980. cAMP-stimulated cation cotransport in avian erythrocytes: Inhibition by “loop” diuretics.Am. J. Physiol. 238:C139-C148
Palfrey, H.C., Rao, M.C. 1983. Na/K/Cl cotransport and its regulation.J. Exp. Biol. 106:43–54
Palfrey, H.C., Silva, P., Epstein, F.H. 1984. Sensitivity of cAMP-stimulated salt secretion in shark rectal gland to “loop” diuretics.Am. J. Physiol. 246:C242-C246
Plass, H., Gridl, A., Turnheim, K. 1986. Absorption and secretion of potassium by rabbit descending colon.Pfluegers Arch. 406:509–519
Rugg, E.L., Simmons, N.L., Tivey, D.R. 1985. An investigation of [3H] bumetanide uptake in intact cultured renal epithelial cells (MDCK).J. Physiol. (London) 367:72P
Saier, M.H., Jr., Boyden, D.A. 1984. Mechanism-regulation and physiological significance of the loop diuretic sensitive NaCl/KKCl symport in animal cells.Mol. Cell Biochem. 59:11–32
Segel, I.H. 1975. Enzyme Kinetics. New York, Wiley
Turner, R.J. 1983. Quantitative studies of cotransport systems: Models and vesicles.J. Membrane Biol. 76:1–15
Turner, R.J., George, J.N., Baum, B.J. 1986. Evidence for a Na+/K+/Cl− cotransport system in basolateral membrane vesicles from the rabbit parotid.J. Membrane Biol. 94:143–152
Welsh, M.J., Smith, P.L., Fromm, M., Frizzell, R.A. 1982. Crypts are the site of intestinal third and electrolyte secretion.Science 218:1219–1221
Wiener, H., Turnheim, K., Os, C.H., van. 1989. Rabbit distal colon epithelium: I. Isolation and characterization of basolateral plasma membrane vesicles from surface and crypt cells.J. Membrane Biol. 110:147–162
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Wiener, H., van Os, C.H. Rabit distal colon epithelium: II. Characterization of (Na+, K+, Cl−)-cotransport and [3H]-bumetanide binding. J. Membrain Biol. 110, 163–174 (1989). https://doi.org/10.1007/BF01869471
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01869471