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The purpose of the study was to develop a per-6-thiolated α-cyclodextrin (α-CD) by substituting all primary hydroxyl groups of α-CD with thiol groups and to assess its solubility-improving and permeation-enhancing properties for a BCS Class IV drug in vitro as well as in vivo. The primary hydroxyl groups of α-CD were replaced by iodine, followed by substitution with -SH groups. The structure of per-6-thiolated α-CD was approved by FT-IR and 1 H NMR spectroscopy. The per-6-thiolated was characterized for thiol content, -SH stability, cytotoxicity, and solubility-improving properties by using the model BCS Class IV drug furosemide (FUR). The mucoadhesive properties of the thiolated oligomer were investigated via viscoelastic measurements with porcine mucus, whereas permeation-enhancing features were evaluated on the Caco-2 cell monolayer and rat gut mucosa. Furthermore, oral bioavailability studies were performed in rats. The per-6-thiolated α-CD oligomer displayed 4244 ± 402 μmol/g thiol groups. These -SH groups were stable at pH ≤ 4, exhibiting a p K a value of 8.1, but subject to oxidation at higher pH. Per-6-thiolated α-CD was not cytotoxic to Caco-2 cells in 0.5% (m/v) concentration within 24 h. It improved the solubility of FUR in the same manner as unmodified α-CD. The addition of per-6-thiolated α-CD (0.5% m/v) increased the mucus viscosity up to 5.8-fold at 37 °C within 4 h. Because of the incorporation in per-6-thiolated α-CD, the apparent permeability coefficient ( P app ) of FUR was 6.87-fold improved on the Caco-2 cell monolayer and 6.55-fold on the intestinal mucosa. Moreover, in vivo studies showed a 4.9-fold improved oral bioavailability of FUR due to the incorporation in per-6-thiolated α-CD. These results indicate that per-6-thiolated α-CD would be a promising auxiliary agent for the mucosal delivery of, in particular, BCS Class IV drugs.

The in vitro glucuronidation of a range of structurally diverse chemicals has been studied in hepatic and renal microsomes from human donors and the beagle dog. These studies were undertaken to improve on the limited knowledge of glucuronidation by the dog and to assess its suitability as a model species for pharmacokinetic studies. In general, the compounds studied were glucuronidated severalfold more rapidly (based on intrinsic clearance estimates) by DLM than by HLM. Intrinsic clearance values for human UGT1A1 and UGT2B7 substrates were an order of magnitude higher in DLM than in HLM (e.g., gemfibrozil: 31 microl/min/mg versus 3.0 microl/min/mg; ketoprofen: 2.4 microl/min/mg versus 0.2 microl/min/mg). There were also drug-specific differences. HLM readily glucuronidated propofol (2.4 microl/min/mg) whereas DLM appeared unable to glucuronidate this drug directly. Regioselective differences in morphine glucuronidation were also apparent. Human kidney microsomes catalyzed the glucuronidation of many xenobiotics, although glucuronidation of the endobiotic bilirubin was not detectable in this tissue. In direct contrast, dog kidney microsomes glucuronidated bilirubin only (no glucuronidation of all other xenobiotics was detected). These preliminary studies indicated significant differences in the glucuronidation of xenobiotics by microsomes from the livers and kidneys of human and dog and should be confirmed using a larger panel of tissues from individual dogs. Early knowledge of the relative rates of in vitro glucuronidation, the UGTs responsible for drug glucuronidation, and their tissue distribution in different species could assist the design and analysis of preclinical pharmacokinetic and safety evaluation studies.

Aims: To study reaction of photoactivated frusemide (F) and F glucuronide (Fgnd metabolite) with human serum albumin in order to find a clue to clarify a mechanism of phototoxic blisters from high frusemide dosage., Methods: F was exposed to light in the presence of human serum albumin (HSA). HSA treated with this method (TR-HSA) was characterized by fluorescence spectroscopic experiment, alkali treatment and reversible binding experiment., Results: Less 4-hydroxyl-N-furfuryl-5-sulphamoylanthranilic acid (4HFSA, a photodegradation product of F) was formed in the presence of HSA than in the absence of HSA. A new fluorescence spectrum excited at 320 nm was observed for TR-HSA. Alkali treatment of TR-HSA released 4HFSA. Quenching of the fluorescence due to the lone tryptophan near the warfarin-binding site of HSA was observed in TR-HSA. The reversible binding of F or naproxen to the warfarin-binding site of TR-HSA was less than to that of native HSA. These results indicate the photoactivated F was covalently bound to the warfarin-binding site of HSA. The covalent binding of Fgnd, which is also reversibly bound to the warfarin-binding site of HSA, was also induced by exposure to sunlight. Fgnd was more photoactive than F, indicating that F could be activated by glucuronidation to become a more photoactive compound., Conclusions: The reactivity of photoactivated F and Fgnd to HSA and/or to other endogenous compounds may cause the phototoxic blisters that result at high F dosage.

Furosemide 1-O-acyl glucuronide (Fgnd) was reversibly bound to a single class of binding sites on human serum albumin (HSA), and the binding of Fgnd decreased with increasing F concentrations, suggesting that Fgnd binds to the same warfarin binding sites on HSA as F binds. The rate of Fgnd degradation (hydrolysis and acyl migration) decreased in the presence of HSA. Although the formation of acyl migration isomers of Fgnd was slower in the presence of HSA than in its absence, hydrolysis of Fgnd to F was faster in the presence of HSA. Rapid minor irreversible binding of Fgnd to HSA within 30 min was followed by slow major irreversible binding. Slow irreversible binding of Fgnd to HSA was decreased by F, though not significantly. This suggests that major irreversible binding may proceed via reversible binding. It has been reported that acyl migration is a prerequisite for irreversible binding. Therefore, these results indicate that HSA decreases irreversible binding of Fgnd to protein by suppressing acyl migration. Furthermore, these results suggest that HSA may prevent irreversible binding of Fgnd to other proteins in the body by decreasing the concentration of reactive Fgnd in the unbound form. HSA eliminates reactive Fgnd by hydrolysis to F. Therefore, it is concluded that HSA works as a scavenger to decrease reactive compounds by reversible binding or eliminates reactive compounds by irreversible binding.

This review discusses the possibility of whether furosemide acyl glucuronide, a metabolite of furosemide, contributes to the clinical effect of diuresis. First an analytical method (e.g. HPLC) must be available to measure both parent drug and furosemide acyl glucuronide. Then, with correctly treated plasma and urine samples (light protected, pH 5) from volunteers and furosemide-treated patients, the kinetic curves of both furosemide as well as its acyl glucuronide can be measured. The acyl glucuronide is formed in part by the kidney tubules and it is possible that the compound is pharmacologically active through inhibition of the Na+/2Cl-/K+ co-transport system; up to now the mechanism of action has been solely attributed to furosemide. The total body clearance of furosemide occurs by hepatic and renal glucuronidation (50%) and by renal excretion (50%). Enterohepatic cycling of furosemide acyl glucuronide, followed by hydrolysis, results in a second and slow elimination phase with a half-life of 20-30 h. This slow elimination phase coincides with a pharmacodynamic rebound phase of urine retention. After each dosage of furosemide, there is first a short stimulation of urine flow (4 h), which is followed by a 3-day recovery period of the body. The following clinical implications arise from study of the elimination kinetics of furosemide. Repetitive dosing must result in accumulation of the recovery period. Accumulation of furosemide and its acyl glucuronide in patients with end-stage renal failure results from infinite hepatic cycling. Impaired kidney function may result in impaired glucuronidation and diuresis. While kidney impairment normally requires a dose reduction for those compounds which are mainly eliminated by renal excretion, for diuretics, a dose increment is required in order to maintain a required level of diuresis. The full clinical impact of the accumulation of furosemide and its acyl glucuronide in patients with end-stage renal failure has to be determined.

1. To determine the effects of diuresis and changes in electrolyte balance on kallikrein gene expression, renal kallikrein mRNA levels were correlated with urine volumes, urinary electrolyte levels, haematocrit and plasma electrolyte levels in rats treated with substances with a range of diuretic activities. 2. Furosemide and related compounds, benzyl furosemide and isofurosemide, as well as amiloride hydrochloride, chlorothiazide or the vehicle (saline) were administered twice daily for 24 or 72 h to rats housed in metabolic cages. 3. Diuresis occurred after each treatment with furosemide, after the initial treatment with benzyl furosemide and did not occur after isofurosemide, amiloride hydrochloride or chlorothiazide. 4. Kallikrein gene expression in kidney was increased after 72 h treatment with furosemide, after 24 or 72 h treatment with benzyl furosemide or amiloride hydrochloride and was unchanged after 24 or 72 h treatment with isofurosemide or chlorothiazide, compared with vehicle-treated controls. 5. Plasma urea levels were elevated after 72 h treatment with furosemide, benzyl furosemide and chlorothiazide and plasma chloride was decreased after 24 and 72 h benzyl furosemide. Haematocrits were unchanged. There were no changes in urinary electrolyte levels 72 h after treatment with any of the diuretics. 6. Neither diuresis nor measurable changes in plasma or urinary electrolytes correlate with changes in renal kallikrein gene expression after diuretic treatment of rats.

Stability of furosemide glucuronide, the major metabolite of furosemide, was studied in order to accurately assess the glucuronidation of furosemide. Furosemide glucuronide was purified by high-performance liquid chromatography, and the mass spectrum of furosemide glucuronide showed the molecular ion peaks [M-H]- at 505 and 507 (m/z). Furosemide glucuronide was photodegraded to the compound, which was shown more hydrophilic than furosemide glucuronide by high-performance liquid chromatography assay. The photodegradation product of furosemide glucuronide was hydrolyzed to one of the photodegradation products of furosemide by beta-glucuronidase, indicating that the photodegradation product of furosemide glucuronide possessed a glucuronic acid moiety. Furthermore, the mass spectrum of the photodegradation product of furosemide glucuronide exhibited molecular ion peaks [M-H]- at 487 and [M-2H+2Na]- at 509, indicating the chlorine displacement of furosemide glucuronide by a hydroxyl group. Furosemide glucuronide was unstable in an aqueous solution (pH=7.4), and presumed acyl migration isomers of furosemide glucuronide (furosemide glucuronide-isomers) were detected by high-performance liquid chromatography equipped with photodiode array UV detector. The UV spectra of seven furosemide glucuronide-isomers were closely similar to that of furosemide glucuronide but not furosemide. Exposing a mixture of furosemide glucuronide and furosemide glucuronide-isomers to light resulted in the production of new compounds. UV spectra of photodegradation products of furosemide glucuronide-isomers were closely similar to those of photodegradation product of furosemide glucuronide. These results suggested that furosemide glucuronide-isomers were also photodegraded, resulting in the displacement of chlorine by a hydroxyl group as in furosemide glucuronide.

The Na+-K+-2Cl- cotransporter blocker furosemide inhibits gamma-aminobutyric acid (GABA)-gated chloride currents and reverses GABA-mediated inhibition of [35S]-t-butylbicyclophosphorothionate ([35S]TBPS) binding of the cerebellar alpha6 subunit-containing GABA(A) receptors much more potently than the cerebrocortical non-alpha6 subunit-containing receptors. Of the 44 compounds studied, all precursors or derivatives of diuretics, one compound [hydrazinosulfonyl-furosemide (PF 1885)] reversed 5-microM GABA-induced inhibition of [35S]TBPS binding to cerebellar and cerebrocortical receptors. Three other compounds, all of which are structurally closely related to furosemide, were selective antagonists for the cerebellar receptors comparable to the lead compound. Still, the diuretic and GABAergic structure-activity relationships differ, since we found potent diuretic structures lacking GABA antagonistic activity. Further development of the GABAergic potency of furosemide derivatives can now focus on the modification of the carboxyl group, replaceable by tetrazole but not by sulfonic or phosphinic acids and the furanyl moiety which could be substituted by thienyl and benzyl groups.

Furosemide 1-O-acyl glucuronide (Fgnd) was extracted from the urine following oral administration of furosemide. The crude Fgnd was applied to micronized Amberlite XAD-2 column (2.5 cm i.d. x 90 cm length, 75-500 microns particle size). The purified Fgnd was identified by mass spectrometry and beta-glucuronidase treatment. This method was also applicable to the purification of glucuronide of tolmetin (nonsteroidal anti-inflammatory drug, NSAID), suggesting that it was applicable to the other NSAIDs, most of which were known to be metabolized to acyl-glucuronides.

Two kinds of dosage forms (tablets and retarded capsules) of furosemide (F) were compared in vitro dissolution profile and in vivo absorption studies. The dissolution of F from retarded capsules was extremely restricted in the first fluid of the JP XII disintegration test (within 0.8%), while the dissolution of F from tablets and retarded capsules in the second fluid of JP XII disintegration test were both complete. Metabolite specific assay of F showed F, conjugation of F with glucuronic acid (FG) and acyl migration isomers of FG (FG-iso) in urine or plasma. The mean cumulative urinary excretion of F following administration of the tablets during 24 h was twice that of retarded capsules. The mean area under the plasma concentration-time curve (AUC) of F following administration of tablets was 1.5 times that of retarded capsules. The mean cumulative urine volume during 24 h, however, was not significantly different between the two dosage forms. Clockwise hysteresis relationships between the diuretic response and the urinary excretion rate of F was observed after administration of retarded capsules. A straight relation between logarithm of the diuresis and logarithm of the urinary excretion of F was observed after maximum excretion rate of F following administration of both dosage forms.

The stability of furosemide glucuronide (FG) was investigated in buffer solutions ranging from pH 1 through 10. This glucuronic acid conjugate was the major metabolite of furosemide (F) excreted in human urine. FG, obtained by extraction from human urine, was purified by ion-exchange chromatography. The concentration of FG, acyl migration isomers of FG (FG-iso), and F were determined simultaneously with an HPLC method that included fluorescence detection and gradient elution. FG was found to be unstable in highly acidic and in neutral to alkaline solutions. Hydrogen ion and hydroxy ion catalyzed the hydrolysis of FG below pH 2.8 and above pH 5.6, respectively. Above pH 3.7, FG instability led to the formation of eight FG-iso compounds. Though beta-glucuronidase cleaved FG, the FG-iso compounds were resistant to the enzyme. The half-life of FG in a buffer solution at pH 7.4 and 37 degrees C was 4.4 h. The maximum stability of FG (half-life about 62 d) occurred at approximately pH 3.2. Below pH 3.7, acyl migration products of FG were not detected. Instead, the hydrolysis of FG to F and glucuronic acid was followed by the formation of 4-chloro-5-sulfamoylanthranilic acid (CSA), a secondary product in acidic media.

Furosemide is metabolized in humans by acyl glucuronidation to the 1-O-glucuronide (Fgluc). Furosemide (F) and the conjugate can be measured directly by gradient high-performance liquid chromatographic analysis without enzymic deglucuronidation. The glucuronide conjugate was isolated by preparative HPLC from human urine samples. Furosemide and its acyl glucuronide were present in plasma. No isoglucuronides were present in acidic urine of a volunteer. Calibration curves were constructed by enzymic deconjugation of samples containing different concentrations of isolated F-acyl glucuronide. The limit of quantitation of F in plasma is 0.007 microgram/ml, Fgluc 0.010 microgram/ml. The limits of quantitation in urine are respectively: F 0.10 microgram/ml, Fgluc 0.15 microgram/ml. A pharmacokinetic profile of furosemide is shown, and some preliminary pharmacokinetic parameters of furosemide obtained from one human volunteer are given. Probenecid does not inhibit the formation of the acyl glucuronide of F, but inhibits the renal clearance of both compounds.

A variety of furosemide assays and their application to biological samples have been described over the past 2 decades. We describe development of a specific, sensitive assay appropriate for efficient measurement of large numbers of biological samples. With development of this methodology we have demonstrated that the putative 2-amino-4-chloro-5-sulfamoylanthranilic acid metabolite is an artifact of sample preparation. In addition, some preparative techniques overestimate furosemide because they hydrolyze the glucuronide conjugate back to the parent compound. Applications of this assay to studies of normal subjects and patients who have received kidney transplants demonstrate a relatively constant amount of glucuronide formation and evidence that this conjugation occurs in the kidney. We also show that patients with renal transplants who are resistant to the effects of furosemide appear to have decreased secretion of the drug into the urinary site of action, the mechanism of which is unknown.

The structure activity correlation of several groups of anthranilic acid derivatives was studied. 59 compounds, most of them possessing the anthranilic acid moiety, were synthesized and tested for diuretic and saluretic activities. Equations correlating the biological activities of these compounds with their physicochemical constants suggest positive dependence of the diuretic activity on log P (octanol:water partition coefficient). It is concluded that, within limits, the variation in biological activity is primarily governed by the lipophilicity of the molecule, and further increase in log P value will not enhance this activity.

A series of 4,N-disubstituted 5-sulfamoylorthanilic acids was synthesized by nucleophilic substitution reactions starting either from 2,4-dihalogeno-5-sulfamoylbenzenesulfonic acids or, in most cases, from phenyl 2,4-dihalogeno-5-sulfamoylbenzenesulfonates. The latter method is based on the relative stability of the phenoxysulfonyl group to nucleophiles, e.g., amines, phenols, and thiols, and the possibility of smooth hydrolytic or hydrogenolytic cleavage as a final step, with formation of the SO3H group. On evaluation of these compounds for salidiuretic activity in rats orally (po), and in dogs orally and intravenously (iv), a number of highly active substances was found; the best had a threshold dose of 0.02 mg/kg po in dogs. The results are given in tables, and the structure-activity relationships within the series are discussed. Besides the known effect of the phenoxy radical, an outstanding activating effect was shown by the butylsulfonyl and cycloalkylsulfonyl radicals and by the N-methylanilino radical in particular when they were located in the 4-position of the orthanilic acid molecule. The sulfanilic acid isomers corresponding to three of the most active compounds were synthesized and proved to be completely inactive in rats.

Urine from 5 renal transplant recipients treated with frusemide was analyzed for unchanged frusemide (F), glucuronidated frusemide (G) and 4-chloro-5-sulfamoylanthranilic acid (CSA) by HPLC. In 3 recipients, whose renal function recovered steadily and whose hepatic function was normal throughout, the ratio of frusemide to its metabolites, F/(F + G + CSA), increased steadily in conjunction with the recovery of renal function. In one patient, who received frusemide 200-400 mg/day i.v., the urinary CSA concentration was 64-102 micrograms X ml-1. In 2 patients who experienced shock and/or hepatic dysfunction after transplantation, the F/(F + G + CSA) ratio fluctuated.

Bovine carbonic anhydrase B (CA) inhibitory effect of furosemide was established in two pH values at 26 degress. FFBu [N-furfuryl-4-chloro-5-(butoxymethylsulfamoyl)anthranilic acid] and FFMe [N-furfuryl-4-chloro-5-(methoxy-methylsulfamoyl)anthranilic acid], two of its alkoxymethyl derivatives, did not exert any CA inhibitory effect at those conditions but were found to inhibit the CA activity after their hydrolysis, which yielded the furosemide molecule. The CA inhibitory effect of furosemide was utilized for determining the kinetic rate constants for the hydrolysis of FFBu and FFMe at various pH and temperature levels. The hydrolysis rate constants of FFBu and FFMe were pH-independent in the pH range tested, and the temperature dependence for FFBu yielded an activation energy of 18 kcal/mol. It is pointed out that the hydrolysis rates of FFBu may be important for the explanation of its possible delayed diuretic effect.

After i.p. administration of 0,60 mg/100 g of furosemide, adult rats excreted 50 to 70% of this diuretic drug unchanged and 10% as desfurylmethylfurosemide. The renal excretion of furosemide is slower in 5- and 15-day-old rats. Age differences in absorption from the abdominal cavity or in the reabsorption rate in the kidney tubules can be excluded. The excretion of furosemide by the kidney is competitively inhibited by p-aminohippuric acid (PAH). The relative small inhibitory effect of PAH in young rats is caused by a smaller participation of tubular secretion in the renal excretion of furosemide in this age group. The half life time of furosemide is 83 min in 5-day-old rats and 47 min in 55-day-old rats. The distribution volume is 146% of body weight in both age groups. The concentration of furosemide in the kidney homogenate is higher in young rats than in adults. The slow renal excretion of furosemide in young rats is in accordance with the prolonged efficacy. The greater efficacy of furosemide in 5- and 15-day-old rats than in adults is caused particularly by differences in the conditions for diuretic effectiveness, especially by the retention of water and electrolytes in young rats.

In order to investigate whether the loop diuretic sensitive, sodium-chloride cotransport system described previously in shark rectal gland is in fact a sodium-potassium chloride cotransport system, plasma membrane vesicles were isolated from rectal glands of Squalus acanthias and sodium and rubidium uptake were measured by a rapid filtration technique. In addition, the binding of N-methylfurosemide to the membranes was investigated. Sodium uptake into the vesicles in the presence of a 170 mM KCl gradient was initially about five-fold higher than in the presence of a 170 mM KNO3 gradient. In the presence of chloride, sodium uptake was inhibited 56% by 0.4 mM bumetanide and 40% by 0.8 mM N-methylfurosemide. When potassium chloride was replaced by choline chloride or lithium chloride, sodium uptake decreased to the values observed in the presence of potassium nitrate. Replacement of potassium chloride by rubidium chloride, however, did not change sodium uptake. Initial rubidium uptake into the membrane vesicles was about 2.5-fold higher in the presence of a 170 mM NaCl gradient than in the presence of a 170 mM NaNO3 gradient. The effect of chloride was completely abolished by 0.4 mM bumetanide. Replacement of the sodium chloride gradient by a lithium chloride gradient decreased rubidium uptake by about 40%; replacement by a choline chloride gradient reduced the uptake even further. Rubidium uptake was also strongly inhibited by potassium. Sodium chloride dependence and bumetanide inhibition of rubidium flux were also found in tracer exchange experiments in the absence of salt gradients. The isolated plasma membranes bound 3[H]-N-methylfurosemide in a dose-dependent manner. In Scatchard plots, one saturable component could be detected with an apparent KD of 3.5 x 10(-6) M and a number of sites n of 104 pmol/mg protein. At 0.8 microM, N-methylfurosemide binding decreased 51% when sodium-free or low-potassium media were used. The same decrease was observed when the chloride concentration was increased from 200 to 600 mM or when 600 1 mM bumetanide or furosemide was added to the incubation medium. These studies indicate that the sodium-chloride cotransport system described previously in the rectal gland is in fact a sodium-potassium chloride cotransport system. It is postulated that this transport system plays an essential role in the secondary active chloride secretion of the rectal gland.

A biosynthetic acyl-type glucuronic acid conjugate of furosemide was isolated from in vitro incubation of pregnenolone-16 alpha-carbonitrile-induced rat liver microsomes containing UDP-glucuronyltransferase activity, furosemide, and UDP-glucuronic acid. Furosemide 1-O-acyl glucuronide (FG) was specifically hydrolyzed by beta-glucuronidase (BG) and was also labile to alkaline hydrolysis. FG concentration decreased at an apparent first order rate when incubated at 37 degrees C in buffer solution of pH values greater than 6.0 with only moderate hydrolysis of the conjugate at pH values less than 8.5. Formation of rearrangement forms of FG that were resistant to BG but labile to alkaline hydrolysis accounted for most of the disappearance of FG at this pH range. Radiochemical labeling of the conjugate with either 14C-furosemide or 14C-UDP-glucuronic acid was detected in the BG-resistant isomerization products of FG as they were separated by HPLC. The structure of FG and its isomerization products was further verified by negative ion thermospray liquid chromatography/mass spectrometry. The abundant (M - 1)-ion at mass 505, the aglycone fragment at m/z 329, and the characteristic sugar fragment ion of mass 175 were found in the spectra of FG and three additional isomers. An ion at m/z 221 was noted only in the case of the parent conjugate and thus may prove to be a characteristic ion for 1-O-acyl-linked glucuronides under negative ion thermospray. In vivo as well as in vitro rearrangement of FG to BG-resistant forms might affect the results of furosemide disposition studies which use BG hydrolysis to determine FG formation.

By the means of thin-layer chromatography (TLC) prior to fluorometry it was possible to determine furosemide (F) in plasma and urine with improved specificity. Antranilic acid (A) and 4-chlor-5-sulphamoyl-antranilic acid (CSA) could be determined simultaneously with F in plasma. In urine, only F and A could be determined quantitatively by this method. The sensitivity is 0.1 microgram/ml for F and 0.15-0.20 microgram/ml for A and CSA. By means of TLC prior to fluorometry the blank value for F was reduced in normal subjects and in different groups of patients, and its diurnal variation was eliminated. The average values of the plasma concentrations of F after the intravenous injection of 40 mg into 12 normal subjects were at all times found to be lower by the TLC method than by the fluorometric method without TLC. A higher plasma clearance and a lower volume of distribution were found for F when the values obtained by the TLC method were used for the calculation.

The effects of several sulfamoyl benzoic acid derivatives on Na-K-Cl cotransport were investigated in winter flounder intestine. The relative efficacy (IC50 values) and order of potency of these derivatives were benzmetanide, 5 X 10(-8) M greater than bumetanide 3 X 10(-7) M greater than piretanide 3 X 10(-6) M greater than furosemide 7 X 10(-6) M greater than amino piretanide 1 X 10(-5) 3-amino-4-penoxy-5-sulfamoyl benzoic acid. Binding of [3H] bumetanide was studied in microsomal membranes from winter flounder intestine and compared to that in bovine kidney outer medulla. Binding was also studied in brush-border membranes from winter flounder intestine. The estimated values for Kd and number of binding sites (n) were: bovine kidney, Kd = 1.6 X 10(-7), n = 10.5 pmol/mg protein; winter flounder intestine, Kd 1.2 X 10(-7), n = 7.3 pmol/mg protein, and brush-border membranes from winter flounder, Kd = 5.3 X 10(-7), n = 20.4 pmol/mg protein. The estimated Kd for bumetanide binding to winter flounder brush-border membranes derived from association and dissociation kinetics was 6.8 X 10(-7) M. The similarity in magnitudes of IC50 and Kd for bumetanide suggests that the brush-border cotransporter is ordinarily rate-limiting for transmural salt absorption and that bumetanide specifically binds to the cotransporter. Measurement of bumetanide binding at various concentrations of Na, K and Cl showed that optimal binding required all three ions to be present at about 5 mM concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)