BINDING OF LOOP DIURETICS TO THEIR RENAL RECEPTORS: USE AS A SCREENING MODEL FOR POTENTIAL DIURETIC ACTIVITY
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Summary— Loop diuretics of the benzoic acid and aryloxyacetic acid families inhibit Na+K+Cl − cotransport. The ranking order of potencies measured in the thick ascending limb of Henle's loop and the ranking order of affinities for [ 3 H]piretanide receptors on renal plasma membranes are the same. Potencies and affinities correlate well (correlation coefficient r = 0.959 for the medulla and r = 0.951 for the cortex). Therefore, measurement of [ 1 H]piretanide binding is proposed to facilitate screening for loop diuretic action.Keywords:
Loop of Henle
Affinities
Bumetanide
The authors present the results of a simultaneous assay of: intracellular Na+ and K+ concentrations, Na+ and K+ outward bumetanide-sensitive effluxes (Na+, K+ cotransport), Na+ efflux stimulated by extracellular Li+ (Na+, Li+ countertransport), and ouabain- and bumetanide-resistant Na+ and K+ effluxes (passive membrane permeability) performed in red blood cells from 15 uremic patients an regular hemodialysis and from 12 normal subjects, with an established flux assay. Na+ and K+ effluxes by the Na+, K+ cotransport system were significantly (p less than 0.01) lower in uremic patients then in normals (219 +/- 37 vs 82 +/- 17 mumol/l RBC/h and 251 +/- 29 vs 139 +/- 14 mumol/l RBC/h respectively). In normal subjects the bumetanide sensitive Na+ and K+ effluxes were strongly (r = 0.89; p less than 0.01) intercorrelated; and the intracellular Na+ concentration was related to the outward Na+ cotransport flux (r = 0.53; p approximately 0.05). Among uremic patients these correlations were not found. Na+ and K+ intracellular concentrations, passive Na+ and K+ permeability, and Na+, Li+ countertransport activity were not different among uremic patients and normal controls. In conclusion, in uremic dialyzed patients, red blood cell Na+, K+ cotransport activity is quite uniformly suppressed. The possible pathogenesis of this disfunction is still speculative and deserves further studies.
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The effect of bumetanide (6 and 12 mg) was compared with that of furosemide (250 and 500 mg) in 13 patients with chronic renal insufficiency in a double-blind study using each patient as his own control. In the dose ratio of 1:40 previously obtained in normal subjects furosemide showed a significantly higher diuretic response than bumetanide. Comparing the relative natriuretic effects in chronic renal insufficiency a ratio of 1:10 to 1:12 (B:F) seemed to be adequate. More than 12 mg bumetanide cannot be recommended because of the severe muscle pains which occurred in all patients with that dosage.
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The relationship between cell volume and Na-K-2Cl cotransport was studied in cultured bovine aortic endothelial cells. Hypertonic cell shrinkage increased bumetanide-sensitive, Na- or Cl-dependent K influx without altering bumetanide-insensitive influx. Greater stimulation of cotransport was observed in cells shrunken isosmotically either by preincubation in K-free and Na-free medium or by preincubation in hypotonic medium. Cell swelling, produced by preincubation in isotonic high-K medium, inhibited bumetanide-sensitive K influx. Simultaneous measurements of [3H]bumetanide binding and K influx revealed an increased number of binding sites without an increased influx per binding site in shrunken cells. Bumetanide did not alter the volume or ion content of cells in isotonic or hypertonic medium, indicating that no net influx of ions occurs through cotransport under these conditions. In isosmotically shrunken cells, there was greater stimulation of bumetanide-sensitive influx than of bumetanide-sensitive efflux, resulting in net bumetanide-sensitive influx. Rapid recovery of cell K, Na, and water occurred over 10-20 min and was inhibited by bumetanide or by the removal of external Na or Cl. These data demonstrate that Na-K-2Cl cotransport in aortic endothelial cells is regulated by cell volume, possibly through changes in the number of functional cotransporters, and mediates a brisk regulatory volume increase in isosmotically shrunken cells. Although thermodynamically favored, no net influx occurs through Na-K-2Cl cotransport in cells of normal volume or in hypertonically shrunken cells. This suggests additional regulation of cotransport, perhaps through trans-inhibition by intracellular Cl. Regulation of cell volume by Na-K-2Cl cotransport may be important in maintaining endothelial integrity.
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Chronic renal failure
Chronic renal insufficiency
Loop of Henle
Loop diuretic
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DIDS
Stoichiometry
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Two analogues of the loop diuretics furosemide and bumetanide have been identified as differential inhibitors of KCl and NaKCl cotransport systems, assayed by measuring K + influx in ‘young’ human red cells. H25 inhibited both NaKCl and KCl cotransport, with I 50% values of 0.03 and 30 μM respectively; H74 had no effect on NaKCl cotransport, even at 0.3 mM, but inhibited KCl cotransport with an I 50% of 75 μM. These compounds are therefore useful for resolving the two transport systems.
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In confluent primary cultures of dog tracheal epithelium, we tested whether Cl entry across the basolateral membrane is by cotransport with K. Two approaches were taken. First, we measured the inhibition of short-circuit current (Isc) by the K channel inhibitor, Ba2+. Consistent with Na-K-2Cl cotransport, maximal doses of Ba2+ inhibited five-sixths of Isc in tissues previously stimulated to secrete Cl; only two-thirds of Isc should be sensitive to Ba2+ if NaCl cotransport is the entry mechanism. Second, we measured basolateral 86Rb uptake and demonstrated inhibition by bumetanide, an inhibitor of Na-K-2Cl cotransport in other tissues. The degree of inhibition by bumetanide was consistent with the levels of Cl secretion measured as Isc. Uptake of 86Rb was also reduced by removal of Na or Cl, and under these conditions Rb uptake was not further inhibited by bumetanide. These results suggest that the process responsible for Cl entry across the basolateral membrane of tracheal epithelium during Cl secretion is Na-K-2Cl rather than Na-Cl cotransport.
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Simultaneous measurements were made of net Cl influxes and [3H]bumetanide binding to Ehrlich ascites tumor cells in which the chloride-cation cotransport pathway had been activated by hypertonic challenge. There was a good linear correlation between inhibition of Cl influx during regulatory volume increase and numbers of bumetanide molecules bound per cell, consistent with high specificity of bumetanide binding to cotransport sites. The extrapolation to the number of bumetanide binding sites per cell at maximal inhibition of Cl transport thus gives the number of cotransport sites per cell as 2.0 X 10(6). From this, and the fluxes measured (not necessarily maximum fluxes), the turnover number is calculated at 50 Cl ions per site per second. Unstimulated cells in isotonic medium, with negligible bumetanide-inhibitable fluxes, have the same number of bumetanide binding sites as the activated cells undergoing volume regulation.
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Rb+ uptake into LLC-PK1/Cl 4 cells can be subdivided into three components: 1) ouabain-sensitive uptake, 2) bumetanide-sensitive uptake, and 3) ouabain- and bumetanide-insensitive uptake. Exposure of cells to near-UV light in the presence of low concentrations of bumetanide produces a specific, irreversible inhibition of the bumetanide-sensitive uptake component, while not affecting the other two uptake components. Irreversible inhibition of bumetanide-sensitive transport is observed when measuring either cellular uptake or efflux and also when measuring 86Rb+ uptake into membrane vesicles. The irreversible inhibition is both concentration and time dependent and is blocked under conditions where the interaction of bumetanide with the Na+-K+-Cl- cotransporter is disturbed. We conclude that bumetanide, at low concentrations, can specifically and irreversibly inhibit the Na+-K+-Cl- cotransporter of LLC-PK1/Cl 4 cells. We suggest that this irreversible inhibition is the result of the photoactivation of an ether linkage in the bumetanide molecule, leading to a covalent binding of bumetanide to the Na+-K+-Cl- cotransporter.
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