Piretanide-dextran and piretanide-polyethylene glycol interact with high affinity with the Na+ 2 Cl− K+ cotransporter in the thick ascending limb of the loop of Henle
- 25 Downloads
Piretanide blocks the Na+ 2Cl− K+ cotransporter protein in the thick ascending limb (TAL) of the loop of Henle reversibly. When tested from the luminal side in isolated perfused cTAL segments it leads to a half maximal inhibition (IC50) of the equivalent short circuit current (Isc) at a concentration of 10−6 mol/l. From the basolateral side it has no effect on Isc up to 10−4 mol/l. The present study was designed to search for high affinity blockers of the Na+ 2Cl− K+ cotransporter with large molecular weight in an attempt to use these macromolecules for antibody-labelling or affinity separation of this transport-protein. Amino-ethyl-dextran or amino-ethyl-polyethylene glycol (M.W. 5kd) were coupled to isothiocyanato-piretanide (ISO-PIR) at room temperature in DMSO. The resulting compounds dextran-sulfonylurea-piretanide (PIR-DEX) and polyethylene glycol-sulfonylurea-piretanide (PIR-PEG) (M.W. 5.38kd) were purified and tested in isolated perfused cTAL segments. IC50 values for ISO-PIR, PIR-DEX and PIR-PEG were estimated from dose response curves after their addition to the lumen or bath perfusate, respectively. ISO-PIR, PIR-DEX and PIR-PEG acted from the lumen side at 3·10−6, 6·10−6 and 2·10−6 mol/l. The inhibitory effect was easily reversible. From the basolateral side no effect for any compound was seen at up to 10−4 mol/l. In clearance experiments PIR-DEX was given to female Wistar rats as an i.v. bolus (25 μmol/kg) and the diuretic urine was collected. After dialysis (exclusion limit 2.5kd) the dialysed urine and the dialysate were tested in isolated perfused cTAL segments. The dialysates had no effect on Isc, but the dialysed urine inhibited Isc by 35% from the luminal side. The present data show: High molecular derivatives of piretanide with dextran or polyethylene glycol moieties block the Na+ 2Cl− K+ cotransporter in cTAL segments at roughly the same low concentration as piretanide itself. Our data exclude a metabolism of these piretanide compounds in the kidney. Since these macromolecular probes can probably not enter the cell their inhibitory effect indicates that the binding site for piretanide diuretics on the Na+ 2Cl− K+ cotransporter is exposed on the surface of the luminal cell membrane.
Key wordsIsolated perfused tubule cTAL Na+ 2Cl− K+ cotransporter piretanide macromolecular probe
Unable to display preview. Download preview PDF.
- 1).Amsler, K. and Kinne, R.: Photoinactivation of sodium-potassium-chloride cotransport in LLC-PK1/Cl 4 cells by bumetanide. AM. J. Physiol. 250:C799-C806 (1986)Google Scholar
- 2).Feit, P.W., Hofmann, E.K., Schiodt, Kristensen, P. Jessen, F. and Dunham, P.B.: Purification of proteins of the Na/Cl cotransporter from membranes of Ehrlich ascites cells using a bumetanide-sepharose affinity column. J. Mem. Biol. 103:135–147 (1988)Google Scholar
- 3).Greger, R.: Cation selectivity of the isolated perfused cortical thick ascending limb of Henle's loop of rabbit kidney. Pflügers Arch. 390:30–37 (1981)Google Scholar
- 4).Greger, R. and Schlatter, E.: Properties of the basolateral membrane of the cortical thick ascending limb of Henle's loop of rabbit kidney. A model for secondary active chloride transport. Pflügers Arch. 396:325–334 (1983)Google Scholar
- 5).Haas, M. and Forbush III, B.: Photolabeling of a 150-kDa (Na+K+Cl) cotransport protein from dog kidney with a bumetanide analog. Am. J. Physiol. 253:C243-C250 (1987)Google Scholar
- 6).Reeves, W.A. and Guthrie, J.D.: Aminization of cotton. Text. Res. J. 23:522–527 (1953)Google Scholar
- 7).Schlatter, E., Greger, R. and Weidtke, C.: Effect of “High Ceiling” diuretics on active salt transport in the cortical thick ascending limb of Henle's loop of rabbit kidney. Pflüger Arch. 396:210–217 (1983)Google Scholar
- 8).Di Stefano, A., Wangemann, P., Friedrich, T., Burckhardt, G., Ökonomopoulos, R.R., Englert, H.C., Lang, H.J. and Greger, R.: Photoaffinity labelling of the Na−2Cl−K carrier. Pflügers Arch. 406:R59 (1986)Google Scholar
- 9).Wangemann, Ph., Braitsch, R. and Greger, R.: The diuretic effect of muzolimine. Pflügers Arch. 410:674–676 (1987)Google Scholar
- 10).Zalipsky, S., Gilon, C. and Zlkha, A.: Attachment of drugs to polyethylene glycols. Eur. Polym. J. 19:1177–1183 (1983)Google Scholar