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

Abstract

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 mumol/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.(ABSTRACT TRUNCATED AT 250 WORDS)