Application of a physiologically-based pharmacokinetic model for the prediction of bumetanide plasma and brain concentrations in the neonate.

Bumetanide is a loop diuretic that is proposed to possess a beneficial effect on disorders of the central nervous system, including neonatal seizures. Therefore, prediction of unbound bumetanide concentrations in the brain is relevant from a pharmacological prospective. A physiologically-based pharmacokinetic (PBPK) model was developed for the prediction of bumetanide disposition in plasma and brain in adult and paediatric populations. A compound file was built for bumetanide integrating physicochemical data and in vitro data. Bumetanide concentration profiles were simulated in both plasma and brain using the Simcyp PBPK model. Simulations of plasma bumetanide concentrations were compared against plasma levels published in the literature. The model performance was verified with data from adult studies before predictions in the paediatric population were undertaken. The adult and paediatric intravenous models predicted pharmacokinetic factors, namely area under the concentration-time curve, maximum concentration in plasma and time to maximum plasma concentration, within two-fold of observed values. However, predictions of plasma concentrations within the neonatal intravenous model did not produce a good fit with the observed values. The PBPK approach used in this study produced reasonable predictions of plasma concentrations of bumetanide, except in the critically ill neonatal population. This PBPK model requires more information regarding metabolic intrinsic clearance and transport parameters prior to further validation of drug disposition predictions in the neonatal population. Given the lack of information surrounding certain parameters in this special population, the model is not appropriately robust to support the recommendation of a suitable dose of bumetanide for use as an adjunct antiepileptic in neonates.

Pilot evaluation of the population pharmacokinetics of bumetanide in term newborn infants with seizures.

Recent experimental data suggest bumetanide as a possible therapeutic option in newborn infants with seizures after birth asphyxia. Because pharmacokinetic (PK) data are lacking in this population, who very often benefit from therapeutic cooling, which can modify the PK behavior of a drug, a PK study was conducted in term infants with seizures caused by hypoxic-ischemic encephalopathy. Fourteen infants were included, 13 of them being cooled. Forty-nine blood samples were available for the determination of the plasma concentration of bumetanide. Concentration-time data were analyzed by the use of a population approach performed with Monolix Software. Bumetanide was found to follow a 2-compartment model. The mean values were 0.063 L/h for clearance, 0.28 and 0.44 L for the central and peripheral distribution volumes, respectively, and 0.59 L/h for the distribution clearance. Birth body weight explained the interindividual variability of bumetanide clearance via an allometric model. No relationship was found between bumetanide exposure and its efficacy (reduction in seizure burden) or its toxicity (hearing loss). This study describes the first PK model of bumetanide in hypothermia-treated infants with seizures.

The organic anion transport inhibitor probenecid increases brain concentrations of the NKCC1 inhibitor bumetanide.

Bumetanide is increasingly being used for experimental treatment of brain disorders, including neonatal seizures, epilepsy, and autism, because the neuronal Na-K-Cl cotransporter NKCC1, which is inhibited by bumetanide, is implicated in the pathophysiology of such disorders. However, use of bumetanide for treatment of brain disorders is associated with problems, including poor brain penetration and systemic adverse effects such as diuresis, hypokalemic alkalosis, and hearing loss. The poor brain penetration is thought to be related to its high ionization rate and plasma protein binding, which restrict brain entry by passive diffusion, but more recently brain efflux transporters have been involved, too. Multidrug resistance protein 4 (MRP4), organic anion transporter 3 (OAT3) and organic anion transporting polypeptide 2 (OATP2) were suggested to mediate bumetanide brain efflux, but direct proof is lacking. Because MRP4, OAT3, and OATP2 can be inhibited by probenecid, we studied whether this drug alters brain levels of bumetanide in mice. Probenecid (50 mg/kg) significantly increased brain levels of bumetanide up to 3-fold; however, it also increased its plasma levels, so that the brain:plasma ratio (~0.015-0.02) was not altered. Probenecid markedly increased the plasma half-life of bumetanide, indicating reduced elimination of bumetanide most likely by inhibition of OAT-mediated transport of bumetanide in the kidney. However, the diuretic activity of bumetanide was not reduced by probenecid. In conclusion, our study demonstrates that the clinically available drug probenecid can be used to increase brain levels of bumetanide and decrease its elimination, which could have therapeutic potential in the treatment of brain disorders.

Application of a rapid and sensitive liquid chromatography-tandem mass spectrometry method for determination of bumetanide in human plasma for a bioequivalence study.

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been proposed for the determination of bumetanide in human plasma using tamsulosin as internal standard (IS). The analyte and IS were extracted from 200 µL of human plasma via solid phase extraction and the chromatographic separation was achieved on Peerless Basic C18 (100 mm × 4.6 mm, 3 µm) column under isocratic conditions. Detection of bumetanide and IS was done by tandem mass spectrometry, operating in positive ionization and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for bumetanide and IS were m/z 365.2→240.2 and 409.2→228.2 respectively. The method was fully validated as per the US FDA guidelines. The limit of detection and lower limit of quantitation of the method were 0.03 and 0.30 ng/mL respectively with a linear dynamic range of 0.30-200.0 ng/mL for bumetanide. The intra-batch and inter-batch precision (% CV) was ≤6.9% while the mean extraction recovery was >90% across quality control levels. The method is selective in presence of four diuretic drugs and some commonly used medications by healthy volunteers. It was successfully applied to a bioequivalence study of 2mg bumetanide tablet formulation in 10 healthy Indian male subjects under fasting condition. The reproducibility in the measurement of study data was demonstrated by reanalysis of 42 incurred samples.

Sensitive isotope dilution liquid chromatography/tandem mass spectrometry method for quantitative analysis of bumetanide in serum and brain tissue.

We have developed and validated a simple and sensitive stable isotope dilution liquid chromatography/tandem mass spectrometric (LC-MS/MS) method for the quantification of bumetanide in human serum. Samples were prepared with a simple acetonitrile based protein precipitation. The supernatant was then analyzed directly using LC-MS/MS. Chromatographic separation was achieved on a C18 reversed phase column using a methanol and water gradient. The detection was performed in selected reaction monitoring (SRM) mode via a positive electrospray ionization (ESI) interface. The method had a lower limit of quantification (LLOQ) of 1 ng/mL, linearity up to 1250 ng/mL, intra- and inter-day precision less than 10%, and accuracy within ±10%. This method was also demonstrated to be suitable for the analysis of bumetanide in rat serum and brain tissue. Bumetanide concentrations in rat serum and brain were determined for samples collected at several intervals following intraperitoneal (i.p.) injection of bumetanide, and were used to calculate bumetanide permeability through the blood-brain barrier.

Intranasal administration of different liquid formulations of bumetanide to rabbits.

The bioavailability of bumetanide in rabbits after intranasal administration of eight formulations intended for use in acute situations has been studied. The vehicles tested were combinations of phosphate buffer, pH 7.4, glycofurol 75. polyethylene glycol 200 and coconut oil. A mixture of 51% glycofurol in polyethylene glycol 200 was administered containing doses of 1 and 8 mg bumetanide respectively. For all other formulations the lower dose level only was studied. The tmax obtained ranged from 3 to 10 min. The vehicles resulting in the highest rate of absorption were 60% glycofurol in coconut oil and pure glycofurol. The observed bioavailability for the different formulations ranged from 16 to 37% for the time period 0-120 min. The bioavailability was also calculated omitting the initial peak seen after i.v. injection, which may be undesirable. Using this method bioavailabilities of 33-82, for the time interval 5-120 min was found. The study also demonstrated that the total amount of bumetanide absorbed increased proportionally to the dose administered. The rate of absorption of bumetanide from all formulations tested may be relevant for the treatment of acute oedematous states. The tmax obtained after intranasal administration was shorter than reported for other non-parenteral routes of administration.

The pharmacokinetics of bumetanide in the newborn infant.

This study characterizes the pharmacokinetics of bumetanide after an intravenous dose of 0.05 or 0.10 mg/kg to 14 neonates (weight range 820-4,000 g; gestational age 26-40 weeks) during the first week of life. Blood samples and urine were collected for up to 12 h after dosing. Estimated serum clearance was 0.2-1.0 ml/min.kg (range), volume of distribution was 0.22 l/kg (range 0.11-0.32 l/kg), and the harmonic mean half-life was 6-7 h (range of 4-19 h). Nonrenal clearance accounted for 58-97% of the serum clearance with the presence of certain oxidative metabolites of bumetanide in the urine. These findings suggest higher dosing requirements and prolonged intervals as compared to adults. Utilizing these pharmacokinetic data, pharmacodynamic and ototoxicity studies should be conducted to establish a safe and effective neonatal dose.

Arterial and venous blood sampling in pharmacokinetic studies: bumetanide in rabbits.

The pharmacokinetics of bumetanide were evaluated simultaneously using both arterial and venous plasma data in 4 rabbits after a rapid 5 sec intravenous (iv) bolus dosing. Initial arterial to venous concentration ratios at 5 sec after injection were the highest with the values of 1410, 246, 4.25, and 351 for rabbits 1-4, respectively. Both curves decayed paralleling each other at the terminal phase with the higher venous levels than the arterial levels by 21.6, 48.2, 17.0, and 47.9% for rabbits 1-4, respectively. An exponential term with a negative coefficient was used to account for the short and steep rising phase of venous plasma levels after injection. Detailed analysis showed apparent volume of distribution at steady state (VSS) and mean residence time (MRT) values calculated from venous plasma data were higher than those form arterial plasma data. A plot of 1/Q (urine flow rate) versus 1/CLR (renal clearance) of bumetanide yielded a straight line in 4 rabbits, indicating that the CLR of bumetanide is urine flow dependent in rabbits.

The effect of transport-blocking drugs on secretion of fluid and electrolytes by the mandibular gland of red kangaroos, Macropus rufus.

Mechanisms of primary fluid formation by macropodine mandibular glands were investigated in anaesthetized red kangaroos using ion-transport and carbonic anhydrase inhibitors. Bumetanide at carotid plasma concentrations of 0.005-0.1 mmol/l progressively reduced a stable, acetylcholine-evoked flow rate of 1.02 +/- 0.024 ml/min to 0.16 +/- 0.016 ml/min (mean +/- SEM). Concurrently, saliva [Na], [Cl] and osmolality decreased, [K] and [HCO3] increased and HCO3 excretion was unaffected. High-rate cholinergic stimulation was unable to increase salivary flow above 12 +/- 1.5% of that for equivalent pre-bumetanide stimulation. Furosemide (1.0 mmol/l) and ethacrynate (0.5 mmol/l) caused depression of salivary flow and qualitatively similar effects on ion concentrations to those of bumetanide. Amiloride (up to 0.5 mmol/l) caused no reduction in salivary flow rates or [Na] but decreased [K] and [Cl] and increased [HCO3]. When compared with bumetanide alone, amiloride combined with bumetanide further augmented [K] and [HCO3] and lowered [Cl], but had no additional effects on Na or flow. At the higher level, 4-acetamido-4'- isothiocyanatostilbene-2,2'disulphonic acid (SITS) (0.05 and 0.5 mmol/l) stimulated fluid output, increased [HCO3] and [protein], and depressed [Na], [K] and [Cl]. Relative to bumetanide alone, SITS given with bumetanide had no additional effects on salivary flow or electrolytes. Methazolamide (0.5 mmol/l) in combination with bumetanide curtailed the decrease in [Cl] and the increases in [K] and [HCO3] associated with bumetanide. The residual methazolamide-resistant HCO3 excretion was sufficient to support 2-6% of primary fluid secretion. It was concluded that secretion of primary fluid by the kangaroo mandibular gland is initiated mainly (> 90%) by Cl transport resulting from Na-K-2Cl symport activity. A small proportion of the fluid secretion (up to 6%) appears to be supported by HCO3 secretion. No evidence was found for fluid secretion being dependent on Cl transport involving Na/H and Cl/HCO3 antiports or on HCO3 synthesis involving carbonic anhydrase.

Pharmacokinetics of bumetanide in critically ill infants.

OBJECTIVE: Define the pharmacokinetics of bumetanide after single intravenous doses in volume-overloaded critically ill infants. METHODS: A prospective, open-label study was carried out in a group of 58 infants aged 0 to 6 months who required diuretic therapy. Each patient received a single dose of intravenous bumetanide. Doses selected in sequential order ranged from 0.005 to 0.10 mg/kg. Hematologic and serum chemistry studies were performed before and at 6 and 24 hours after bumetanide administration. Determinations of urine volume and chemistries were performed before (collected from -2 to -4 hours to time 0) and at 1, 2, 3, 4, 6, and 12 hours after bumetanide dosing. Serum samples collected at time 0 and at 5, 15, 30, 60, 120, 180, 240, 360, and 480 minutes and urine collected at time 0 and at 0 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 6, and 6 to 12 hours were analyzed for bumetanide concentration. Data were evaluated by standard noncompartmental pharmacokinetic techniques. RESULTS: Peak serum bumetanide concentrations occurred at 5 minutes after bumetanide administration. Area under the curve and peak serum bumetanide concentrations showed linear increases over the twentyfold dose range; whereas beta volume of distribution, volume of distribution at steady state, clearance, renal clearance, half-life, and mean residence time values were independent of dose. Peak urinary excretion rates of bumetanide increased linearly with increasing doses. The mean percent of bumetanide recovered in the urine from 0 to 12 hours was 40% +/- 15% of the administered dose. CONCLUSIONS: Distribution and elimination kinetics of bumetanide were similar in all patients. Elimination kinetics were first order over the dose range of 0.005 to 0.10 mg/kg. Pharmacokinetic parameter estimates (beta volume of distribution, volume of distribution at steady state, clearance, renal clearance, half-life, and mean residence time) were independent of the dose of bumetanide administered. Single doses of bumetanide up to 0.10 mg/kg appear to be well tolerated in acutely ill volume-overloaded infants aged 0 to 6 months.

Dose-ranging evaluation of bumetanide pharmacodynamics in critically ill infants.

OBJECTIVES: Determine the diuretic effects of single intravenous doses of bumetanide in volume-overloaded critically ill infants. METHODS: A prospective, open-label study was carried out in 56 infants aged 0 to 6 months who required diuretic therapy. Each patient received a single intravenous dose of bumetanide. Doses selected in sequential order ranged from 0.005 to 0.10 mg/kg. Determinations of urine volume, electrolytes, creatinine levels, and osmolality were performed before (collected from -2 to -4 hours to time 0) and at 1, 2, 3, 4, 6, and 12 hours after bumetanide dosing. Serum samples collected at time 0 and at 5, 15, 30, 60, 120, 180, 240, 360, and 480 minutes and urine aliquots collected at time 0, 0 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 6, and 6 to 12 hours were analyzed for bumetanide concentration. Individual changes in urine flow rate and electrolyte excretion were plotted against corresponding bumetanide excretion rates, taken as the effective dose of the drug. RESULTS: Peak bumetanide excretion rates increased linearly with increasing doses of drug. Time course patterns for urine flow rate and electrolyte excretion were similar for all dosage groups. Urine flow rate and electrolyte excretion increased linearly up to a bumetanide excretion rate of approximately 7 micrograms/kg/hr and either plateaued (urine flow rate) or declined at a bumetanide excretion rate of > 10 micrograms/kg/hr. Diuretic efficiency of bumetanide was maximal at doses of 0.005 to 0.010 mg/kg but decreased at higher doses. CONCLUSIONS: Maximal diuretic responses occurred at a bumetanide excretion rate of about 7 micrograms/kg/hr, corresponding to doses of 0.035 to 0.040 mg/kg. Higher doses produced a proportionately higher bumetanide excretion rate but no increased diuretic effect. Lower doses of bumetanide had the greatest diuretic efficiency, suggesting that continuous infusion of low doses of bumetanide or intermittent low-dose boluses may produce optimal diuretic responses in critically ill infants.

Ionic composition of cisternal CSF in acute respiratory acidosis: lack of effect of large dose bumetanide.

Sodium/chloride cotransport carrier is known to be involved in transepithelial fluid absorption and secretion in various tissues. Recent studies indicate that Na,K,2Cl cotransport carrier also exists in the choroid plexus cells and inhibition of the carrier alters ionic composition of the choroidal tissue. In this study, we report the effects of large dose intravenous bumetanide, a potent inhibitor of Na,K,2Cl carrier, on cisternal CSF ionic composition in acute respiratory acidosis in pentobarbital-anesthetized mechanically ventilated dogs. Renal pedicles were ligated to prevent bumetanide-induced diuresis. The experimental group (Group II, n = 7) received 50 mg/kg of bumetanide intravenously and Group I (the control group, n = 7) received the vehicle. Analysis of serum and choroidal plexus tissue revealed bumetanide concentration of approximately 10(-5) mol/L in Group II. During 5 h of acute respiratory acidosis in both groups, the mean PaCO2 increased approximately 25 mm Hg, with comparable changes in CSF PCO2. In both groups, CSF [HCO3-] and [H+] increased approximately 3 mEq/L and 20 nEq/L, respectively. Furthermore, changes in CSF [Na+], [K+], [Ca2+], [Mg2+], [Cl-], and [Na(+)-Cl-] were also similar and were not significantly different from each other. These data show that bumetanide, at the dose that inhibits NaCl cotransport carrier, does not significantly affect ionic composition of cisternal CSF.

Effects of water deprivation on the pharmacokinetics and pharmacodynamics of bumetanide in rats.

The effects of temporary water deprivation for 48 h on the pharmacokinetics and pharmacodynamics of bumetanide were examined after intravenous (i.v.) administration of bumetanide, 8 mg kg-1 to control and water deprived rats (n = 7). The values of AUC, t1/2 and MRT increased 79.0, 417, and 633 per cent, respectively, and CL and CLNR decreased 44.0 and 41.2 per cent, respectively, in water deprived rats. They were all significantly different. The decreased CLNR in water deprived rats could be due to decreased nonrenal metabolism of bumetanide; it could be supported that the amounts of glucuronide conjugate of bumetanide (52.5 vs 12.9 micrograms), desbutylbumetanide (170 vs 113 micrograms) and its glucuronide conjugation (191 vs 125 micrograms), and sum of the three metabolites (414 vs 229 micrograms), which are expressed in terms of bumetanide excreted in 24 h urine, decreased significantly in water deprived rats. The 8-h urine outputs per 100 g body weight (4.32 vs 1.34 ml) also reduced significantly in water deprived rats, and it might be due to significantly reduced amounts of bumetanide excreted in 8 h urine (90.9 vs 25.7 micrograms) and/or reduced kidney function in water deprived rats. The kidney function based on CLIot (9.87 vs 2.14 ml min-1 kg-1) reduced significantly in water deprived rats. The 8-h urinary excretions of sodium (0.430 vs 0.0818 mmol), potassium (0.567 vs 0.270 mmol), and chloride (0.549 vs 0.0624 mmol) per 100 g body weight also reduced significantly in water deprived rats.

Pharmacokinetics and pharmacodynamics of bumetanide after intravenous and oral administration to spontaneously hypertensive rats and DOCA-salt induced hypertensive rats.

The pharmacokinetics and pharmacodynamics of bumetanide were investigated after intravenous (i.v.) administration, 10 mg kg-1, and oral administration, 20 mg kg-1, to spontaneously hypertensive rats (SHRs) and deoxycorticosterone acetate-salt induced hypertensive rats (DOCA-salt rats). After i.v. administration, the pharmacokinetic and pharmacodynamic parameters of bumetanide did not vary significantly between SHRs and the control Wistar rats. Similar results were also shown between DOCA-salt rats and the control Sprague-Dawley (SD) rats. After oral administration, the AUC0-12 h decreased significantly (186 versus 335 micrograms min ml-1) in SHRs and this resulted in decreased F(15.4 versus 23.6 and 2.78 versus 5.76% using two equations) in SHRs when compared with the control Wistar rats, although none of the other pharmacokinetic parameters varied significantly between SHRs and Wistar rats. This effect seemed to be due to the decreased enterohepatic recirculation of bumetanide in SHRs: the amounts of both bumetanide and its glucuronide product, which are capable of enterohepatic recirculation, excreted in 8 h bile juice decreased significantly in SHRs (11.3 versus 37.4 micrograms as expressed in terms of bumetanide) when compared with Wistar rats. The pharmacodynamic parameters did not vary significantly between SHRs and Wistar rats after oral administration of bumetanide. The pharmacokinetic and pharmacodynamic parameters of bumetanide did not vary significantly between DOCA-salt rats and SD rats after oral administration of the drug. The liver weights compared to body weight increased significantly in SHRs when compared with Wistar rats and the corresponding values for the kidney increased significantly in DOCA-salt rats when compared with SD rats.

Inhibition of Na-K-2Cl cotransport and bumetanide binding by ethacrynic acid, its analogues, and adducts.

The inhibitory effect of ethacrynic acid (EA) and a variety of its derivatives on Na-K-2Cl cotransport in avian erythrocytes was investigated. The most potent compound tested was the adduct of EA with L-cysteine, with an IC50 of 7.2 x 10(-7) M. EA itself, dihydro-EA, EA-D-cysteine, and adducts of EA with other sulfhydryl (-SH) compounds were much less potent. The mechanism of action of EA and EA-L-cysteine differed in several respects: 1) EA-L-cysteine acted more rapidly than EA (half times of < 1 and 4 min, respectively, at 37 degrees C); 2) the action of EA-L-cysteine was reversible by washing, whereas that of EA was not; and 3) the degree of inhibition by EA-L-cysteine varied with medium [K], whereas that of EA did not. The inhibitory effects of both EA-L-cysteine and EA were affected by medium [Na] and [Cl]. We conclude that EA-L-cysteine does not "deliver" EA to transport-related -SH residues or act as an alkylating agent but has some stereospecific effect on cotransport that is a property of the entire molecule. EA does appear to inhibit cotransport by alkylating -SH residues, as closely related compounds lacking the ability to covalently react with such groups were reversible, and other -SH reagents (e.g., N-ethylmaleimide) also inhibited cotransport. EA, EA-L-cysteine, and EA-D-cysteine all inhibited [3H]bumetanide binding to membranes from activated avian erythrocytes at concentrations similar to those that inhibited cotransport. It is possible that the EA and bumetanide types of diuretics interact with closely apposed sites on the Na-K-2Cl cotransporter.

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition.

A high-performance liquid chromatographic method for the measurement of bumetanide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C18 column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C18 Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol-water-glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5-2000 ng/ml.

The regulation of Na/K/2Cl cotransport and bumetanide binding in avian erythrocytes by protein phosphorylation and dephosphorylation. Effects of kinase inhibitors and okadaic acid.

The Na/K/2Cl cotransport system in the avian erythrocyte can be activated by agents that raise intracellular cAMP suggesting the involvement of cAMP-dependent protein kinase (cAMP-PK) in its regulation. Another group of stimuli including fluoride and hypertonicity stimulate cotransport via cAMP-independent means. To further investigate the role of phosphorylation in these processes, we examined the effects of protein kinase inhibitors of 8 (p-Cl-phenylthio)-cAMP (cpt-cAMP), fluoride and hypertonic activation of cotransport in duck red cells, and [3H]bumetanide binding to isolated membranes. Preincubation of cells with the kinase inhibitors K-252a (Ki approximately 1.6 microM) and H-9 (Ki approximately 100 microM) blocked cpt-cAMP activation of bumetanide-sensitive 86Rb influx and bumetanide binding. These inhibitors also led to a rapid deactivation of cotransport and decrease in bumetanide binding when added to cells maximally stimulated by cpt-cAMP. K-252a and H-9 inhibited cotransport activation by cAMP-independent stimuli, but 10-fold higher concentrations were required, implying the involvement of a cAMP-independent phosphorylation process in the mechanism of action of these agents. Removal of stimuli that elevate cAMP leads to a rapid reversal of cotransport indicating the presence of active protein phosphatases in these cells. The protein phosphatase inhibitor okadaic acid (OA, EC50: 630 nM) stimulated both Na/K/2Cl cotransport and bumetanide binding to membranes. As with fluoride and hypertonic stimulation, the OA effect was inhibited only at relatively high concentrations of K-252a. Phosphorylation of the membrane skeletal protein goblin (Mr 230,000) at specific cAMP-dependent sites was used as an in situ marker for the state of activation of cAMP-PK. Goblin phosphorylation at these sites was increased by norepinephrine and cpt-cAMP and rapidly reversed by K-252a and H-9, confirming that both inhibitors do block cAMP-PK activity. While OA markedly increased overall phosphorylation of many erythrocyte membrane proteins, including goblin, it did not affect goblin phosphorylation at specific cAMP-dependent sites. These results implicate a cAMP-independent protein kinase in the mediation of the OA effect on cotransport and bumetanide binding. The bumetanide-binding component of the avian erythrocyte cotransporter, an Mr approximately 150,000 protein that can be photolabeled with the bumetanide analog [3H]4-benzoyl-5-sulfamoyl-3-(3-thenyloxy)-benzoic acid was found to be a phosphoprotein. These results strongly support the hypothesis that phosphorylation and dephosphorylation, possibly of the Na/K/2Cl cotransporter itself, regulates the activity of

[3H]bumetanide binding to avian erythrocyte membranes. Correlation with activation and deactivation of Na/K/2Cl cotransport.

The relationship between Na/K/2Cl cotransport activation in duck erythrocytes and binding of the diuretic [3H]bumetanide to isolated membranes from stimulated cells has been assessed. Cotransport was activated by either cAMP-dependent (norepinephrine) or -independent (fluoride, hypertonicity) pathways. Membranes isolated from unstimulated cells possessed no specific bumetanide binding. In the presence of norepinephrine, cotransport and saturable binding rose in parallel, reaching a maximum after 5-7 min. In membranes from maximally stimulated cells the K1/2 and Bmax for bumetanide binding were 100 nM and 1.7 pmol/mg protein, respectively. The diuretic binding properties of these membranes were characteristic of interactions of ligands with the Na/K/2Cl cotransporter: specific binding required the presence of all three cotransported ions (Na, K, and Cl), and the rank order of potency for diuretic competition with bumetanide for binding sites was benzmetanide greater than bumetanide greater than furosemide. The appearance of specific bumetanide binding was also seen in membranes from erythrocytes activated by non-cAMP-dependent stimuli, with an excellent temporal correlation between cotransport activation and diuretic binding. On removal of all stimuli both cotransport and bumetanide binding declined in parallel. Duck erythrocytes treated with norepinephrine in a solution containing 15 mM K+ swell to a new stable cell volume after 60 min, during which time cotransport becomes inoperative. Bumetanide binding to both whole cells and isolated membranes paralleled the decline in cotransport activity. It is concluded that bumetanide binding to isolated membranes faithfully reflects the state of activation of the Na/K/2Cl cotransporter in intact cells under a variety of conditions.

Simultaneous analysis of furosemide and bumetanide in horse plasma using high performance liquid chromatography.

A high performance liquid chromatographic method is described for the simultaneous determination of furosemide and bumetanide in horse plasma. The C8 (3 microns) reversed phase column (4.8 x 150 mm) provided clear separation of furosemide and bumetanide with other components present in the horse plasma. The detection limit for both the drugs was 10 ng/mL. Both drugs were stable in plasma (at natural or acidic pH) for up to 24 h. The method is sufficiently sensitive to detect furosemide levels in plasma obtained from horses receiving a therapeutic dose of furosemide.