Furosemide Inhibition of Chloride Transport in Human Red Blood Cells PETER C. BRAZY and ROBERT B. GUNN F r o m the D e p a r t m e n t o f Physiology a n d P h a r m a c o l o g y , Duke University Medical C e n t e r , D u r h a m , N o r t h Carolina 27710, a n d t h e D e p a r t m e n t o f Pharmacological a n d Physiological Sciences, University o f Chicago, Pritzker School o f Medicine, Chicago, Illinois 60637

ABSTRACT The chloride self-exchange,flux across the human red cell membrane is rapidly and reversibly inhibited by 10-4 M furosemide, a potent chloruretic agent. Furosemide reduces the chloride flux at all chloride concentrations and increases the cellular chloride concentration at which the flux is half-maximum. Kinetic analysis of the flux measurements made at several furosemide and chloride concentrations yields a pattern of mixed inhibition with a dissociation constant for the inhibitor-transport mechanism complex of 5 × 10-s M. From this pattern of inhibition and other observations, including that the percent inhibition is independent of pH (range 5.6-8.9), we conclude that the anionic form of furosemide interacts primarily with the chloride transport mechanism at a site separate from both the transport site and the halide-reactive modifier site. INTRODUCTION

Furosemide (Fig. 1) is a rapidly acting, potent diuretic that is c o m m o n l y used in clinical medicine. Its administration causes a m a r k e d increase in the excretion o f sodium, potassium, calcium, m a g n e s i u m , chloride, p h o s p h a t e , and water by the' kidney in rats, dogs, a n d man (15). T h e major site of furosemide's diuretic effect is the ascending limb o f Henle's loop (8). Because H o o k a n d Williamson (21) had f o u n d that f u r o s e m i d e decreased the ATPase activity in microsomal p r e p a r a tions f r o m rat kidney, and Sachs (26) had f o u n d that f u r o s e m i d e decreased the ouabain-sensitive a n d insensitive sodium permeability in h u m a n red blood cells, the mechanism o f action o f this d r u g was t h o u g h t to be an inhibition o f cation transport. More recently, attention has been focused on the inhibitory effects of furosemide on the t r a n s p o r t o f inorganic anions. Furosemide was one o f n u m e r o u s inhibitors that decreased p h o s p h a t e tracer efflux across intact h u m a n red blood cells (14). T h e [32P]phosphate efflux in solutions with 1 mM total p h o s p h a t e , p H 7.35, at 37°C was inhibited 50% by 5 × 10-4 M f u r o s e m i d e . This inhibition was reversible and noncompetitive. Recently B u r g and Green (3) f o u n d that chloride was actively t r a n s p o r t e d out o f the l u m e n of perfused tubules isolated f r o m the rabbit kidney. F u r o s e m i d e (10 -5 M) in the lumen o f the thick ascending limb o f Henle's loop decreased the electrical potential difference f r o m +5 mV (lumen positive) to near zero a n d decreased the net flux o f NaCI f r o m the l u m e n (4). This observation is consistent with active electrogenic t r a n s p o r t o f chloride THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 68, 1976 " pages 583-599

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which is inhibited by furosemide and passive diffusion o f Na f r o m l u m e n to peritubular fluid. Furosemide can cause a 50% reduction in the short circuit c u r r e n t in frog c o r n e a (6), in which active chloride t r a n s p o r t accounts for 90% o f the short circuit c u r r e n t (31). I n man and dogs systemic f u r o s e m i d e decreased the volume and the bicarbonate concentration of the fluid collected f r o m cannulated pancreatic ducts (25, 16). An anion exchange mechanism has been rep o r t e d in m a m m a l i a n pancreatic ducts (24); the observed decrease in bicarbonate concentration o f pancreatic fluid caused by f u r o s e m i d e may be a result o f an inhibited anion e x c h a n g e mechanism. In this p a p e r we r e p o r t the results o f o u r studies on the effect of f u r o s e m i d e on the chloride t r a n s p o r t system in the h u m a n red blood cell. T h e chloride selfexchange t r a n s p o r t in erythrocytes shows saturation with increasing chloride concentration, competitive inhibition by o t h e r inorganic anions, noncompetitive inhibition by certain organic c o m p o u n d s , a characteristic p H d e p e n d e n c e (19), H

H

H

Cl

FIGURE 1. Furosemide (Lasix). 4-Chloro-N-furfuryl-5-sulfamoyl anthranilic acid. Molecular weight 330.8 daltons. and a high a p p a r e n t activation e n e r g y (13). This anion e x c h a n g e mechanism appears to d e p e n d u p o n the integrity of an intrinsic erythrocyte m e m b r a n e protein (5, 20) and can be described by a titratable carrier model (17). We studied the interaction o f f u r o s e m i d e with the red cell anion exchange mechanism in an effort to increase o u r u n d e r s t a n d i n g of both the d r u g ' s m o d e of action and the chloride t r a n s p o r t mechanism in h u m a n red blood cells. MATERIALS

AND

METHODS

The methods are fully described in Gunn et al. (19); a brief summary is given here. Media with the following millimolar concentrations were prepared from reagent grade chemicals: chloride medium-140 NaCl, 5 KC1, 1.5 CaCIz, 1.0 MgCiz, 5 d-glucose, and 27 glycylglycine; acetate medium-140 Na-acetate, 5 K-acetate, 1.5 Ca-(acetate)2, 1.0 Mg(acetate)2, 5 d-glucose, and 27 glycylglycine; nystatin medium-25-600 KC1, 10 NaCI, and 27 sucrose. Furosemide, a white powder received as a gift from Hoechst Parmaceuticals, Inc., (Cincinnati, Ohio) was made into a 50 mM stock solution in 0.1 N KOH. Aliquots of the stock solution were added to the appropriate medium before final adjustment of the pH at 0°C. Nystatin (E. R. Squibb and Sons, Inc., Princeton, N.J.) was dissolved in dimethyl sulfoxide to make a stock solution. Red blood cell suspensions were prepared from freshly drawn heparinized whole

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blood from one of the researchers (P. C. B.). T h e red cells were separated from the plasma and buffy coat, washed in 170 mM NaC1, titrated to the desired p H with CO2 and bicarbonate at 0°C, and then washed several times in cold m e d i u m until the chloride was in steady state between cells and solution. T h e n nnC1 (ICN Pharmaceuticals, Inc., Cleveland, Ohio, 10/zi of 462/zCi/ml) was a d d e d to the suspension and allowed to equilibrate. This suspension was packed in thin nylon tubes and centrifuged to separate cells from medium. 0.25 g of packed cells was a d d e d to 25 ml of cold medium at time zero in a thermostated chamber. T h e extracellular fluid was sampled by the rapid filtration technique of Dalmark and Wieth (13). An "infinity" sample was taken from this suspension after the tracer had reached a steady-state distribution. A semilog g r a p h of the fraction of tracer remaining in the cells as a function of time was a straight line whose slope is the rate constant. T h e product of this rate constant and the intracellular chloride concentration in milliequivalents per kilogram cell solids is the self-exchange flux in milliequivalents per (kilogram cell solids- minute). T h e intraceilular chloride concentration for each flux was calculated from the steady-state ratio of intracellular to extracellular n6Cl activity (re1) times the measured extraceUular chloride concentration and the kilograms cell water per kilogram cell solids (determined by drying cells to constant weight). Duplicate flux measurements were p e r f o r m e d for each set of conditions. T h e average range between duplicate measurements was 5% of the flux magnitude. T h e nystatin method of equilibrium dialysis of red cells was used to alter, the intracellular chloride concentration in one series of experiments, (7, 10). RESULTS

Furosemide Inhibition of Chloride Self-Exchange Flux T h e s e l f - e x c h a n g e o f c h l o r i d e i s o t o p e s a c r o s s r e d b l o o d cells was m e a s u r e d in t h e p r e s e n c e o f g r a d e d c o n c e n t r a t i o n s o f f u r o s e m i d e at p H 7.80, 0°C. T h e r e d cells w e r e first s u s p e n d e d in c h l o r i d e m e d i u m with a g i v e n c o n c e n t r a t i o n o f f u r o s e m i d e , l o a d e d with r a d i o a c t i v e 36C1, a n d i n j e c t e d i n t o a n o n r a d i o a c t i v e c h l o r i d e m e d i u m with t h e s a m e f u r o s e m i d e c o n c e n t r a t i o n . T h e r e s u l t s a r e s h o w n in Fig. 2. T h e s e l f - e x c h a n g e f l u x was i n h i b i t e d 50% by 2 × 10 -4 M f u r o s e m i d e . I n 5 × 10 -n M f u r o s e m i d e t h e r e s i d u a l i n s e n s i t i v e f l u x was o n l y 12 m e q / ( k g cell s o l i d s , m i n ) o r 1.6% o f t h e c o n t r o l f l u x v a l u e . B o t h t h e r a p i d i t y with w h i c h f u r o s e m i d e i n h i b i t e d c h l o r i d e s e l f - e x c h a n g e a n d t h e r e v e r s i b i l i t y o f its e f f e c t w e r e s h o w n by t h e e x p e r i m e n t s p r e s e n t e d in F i g . 3. F i r s t t h e e f f l u x o f c h l o r i d e f r o m cells p r e w a s h e d with s o l u t i o n s c o n t a i n i n g 5 × 10 -4 M f u r o s e m i d e was c o m p a r e d with t h e e f f l u x o f cells w i t h o u t p r i o r e x p o s u r e to f u r o s e m i d e . B o t h t y p e s o f cells w e r e i n j e c t e d i n t o c h l o r i d e m e d i a c o n t a i n i n g 5 × 10 -4 M f u r o s e m i d e . T h e e f f l u x r a t e s w e r e i d e n t i c a l a n d t h e e f f l u x o f t h e cells w h i c h h a d n o p r i o r e x p o s u r e to f u r o s e m i d e f o l l o w e d t h e s a m e f i r s t - o r d e r k i n e t i c s . T h i s i n d i c a t e s t h a t t h e s a m e level o f i n h i b i t i o n was a t t a i n e d b e f o r e t h e first s a m p l e (at 6 s) b y t h e r e a c t i o n o f t h e cells w i t h t h e f u r o s e m i d e in s o l u t i o n . T h e r e v e r s i b i l i t y o f i n h i b i t i o n was d e m o n s t r a t e d b y p r e w a s h i n g cells in 10 -n M f u r o s e m i d e t h e n w a s h i n g a n a l i q u o t o f cells six t i m e s in 4 vol o f d r u g - f r e e m e d i u m b e f o r e m e a s u r i n g t h e e f f l u x . T h e r a t e c o n s t a n t f o r t h e c o n t r o l f l u x was 0.053 s -1 a n d f o r t h e f l u x in 10 -3 M f u r o s e m i d e 0.0051 s -1 while t h e e f f l u x r a t e c o e f f i c i e n t o f t h e p r e t r e a t e d cells w a s h e d in f u r o s e m i d e - f r e e m e d i u m was 0.050 s -1. T h i s v a l u e was n o t s i g n i f i c a n t l y less t h a n t h e c o n t r o l .

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80c -/

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60¢ -6 --

75 ~_ uJ

=

o~ 40C

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2,5

.4/ Control

I -40 -30 Log Furosemide C0ncentrotion

-2,0

FIGURE 2. D o s e - r e s p o n s e c u r v e for f u r o s e m i d e inhibition of the chloride selfe x c h a n g e flux. T h e inhibitory effect o f f u r o s e m i d e , plotted as the l o g a r i t h m o f the d r u g ' s c o n c e n t r a t i o n , on the chloride self-exchange flux was m e a s u r e d at p H 7.80, 0°C, in chloride m e d i u m . T h e c u r v e was d r a w n by eye. T h e u n i n h i b i t e d c h l o r i d e flux has a value o f 740 m e q / ( k g cell solids, min). 50% inhibition o f the c h l o r i d e flux o c c u r r e d at 2 x 10 -4 M f u r o s e m i d e . 1.0 0.8

I

0.4

0.3 1" 0

J I0

t 20

I J 30 40 Time S

J 50

I 60

FIGURE 3. Effects o f f u r o s e m i d e on the efflux of cellular 36C1. T h e rate constant for the e f f l u x o f chloride tracer was d e t e r m i n e d f r o m the slope o f the a p p e a r a n c e o f tracer in the extracellular c o m p a r t m e n t (1-at~a=, w h e r e at is the specific activity at time t; and a= is the specific activity at isotopic steady state) as a function o f time in seconds. T h e plot shows first-order kinetics for the tracer efflux of control cells (O), cells treated with f u r o s e m i d e (10 -3 M) t h e n washed in d r u g - f r e e m e d i u m (Q), cells p r e i n c u b a t e d a n d m e a s u r e d in f u r o s e m i d e (5 x 10 -4 M) (O), and d r u g - f r e e cells that were first e x p o s e d to f u r o s e m i d e (5 × 10 -4 M) at the b e g i n n i n g o f the e f f l u x m e a s u r e m e n t s ( I ) . T h e e f f l u x m e a s u r e m e n t s were m a d e in chloride m e d i u m , p H 8.00, 0°C. T h e slope of the g r a p h was d e t e r m i n e d by linear regression analysis.

BRAZV AND GUNN FurosemideInhibition of Chloride Transport

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The Effect of Cellular Chloride Concentration on Furosemide Inhibition T h e chloride self-exchange flux o f red cells has b e e n s h o w n to d e p e n d u p o n the cellular chloride c o n c e n t r a t i o n (19, 9). T w o m e t h o d s were used to alter the intracellular chloride concentration o f erythrocytes. I n the first, the red cells were washed r e p e a t e d l y in Na-acetate m e d i u m to which a m m o n i u m chloride had been a d d e d to achieve the desired chloride concentration. T h e high p e r m e ability o f a m m o n i a a n d c a r b o n dioxide c o m b i n e d with the r a p i d chlorideb i c a r b o n a t e e x c h a n g e p e r m i t t e d the a m m o n i u m chloride rapidly to distribute itself across the r e d cell m e m b r a n e . O n c e the chloride h a d r e a c h e d steady state between the cells a n d the m e d i u m , the cells were t r e a t e d with f u r o s e m i d e a n d the tracer efflux into the s a m e Na-acetate-NH4Cl m e d i u m was m e a s u r e d . Using a second m e t h o d we looked for an effect o f a m m o n i u m a n d acetate ions on either f u r o s e m i d e or the chloride t r a n s p o r t m e c h a n i s m . I n this m e t h o d (7, 10) nystatin was used to reversibly increase red cell permeability while the ionic c o n t e n t was c h a n g e d (20-600 m M KCI, 10 m M NaC1). O n c e the cells were in steady state with the new m e d i u m , the nystatin was washed off. T h e s e cells were then t r e a t e d with f u r o s e m i d e a n d the chloride content a n d tracer efflux measured. Fig. 4a shows the effect o f cellular chloride c o n c e n t r a t i o n on the self-exchange flux in the p r e s e n c e o f 5 × 10 -4 M f u r o s e m i d e at p H 7.80, 0°C, in an Naacetate-NH4Cl m e d i u m . T h e solid c u r v e was d r a w n f r o m a Michaelis-Menten equation. In the absence o f f u r o s e m i d e the c o n c e n t r a t i o n d e p e n d e n c e o f the chloride self-exchange u n d e r these conditions (Fig. 5 in r e f e r e n c e 19) h a d a s h a p e of a Michaelis-Menten equation at cellular chloride concentrations below 75 m M . At high cellular chloride concentrations the flux decreased. This decrease was not seen in the p r e s e n c e o f f u r o s e m i d e , as s h o w n in Fig. 4a. Fig. 4b shows the c o n c e n t r a t i o n d e p e n d e n c e o f the chloride flux in cells p r e p a r e d by the nystatin m e t h o d o f altering cellular chloride. T h e c u r v e of the flux values in the absence o f f u r o s e m i d e was c o m p a r a b l e to that f o u n d by D a l m a r k (9) with a m a x i m u m n e a r 150 m M chloride a n d a decline in the flux value with f u r t h e r increases in the cellular chloride concentration. T h e c u r v e in the p r e s e n c e o f f u r o s e m i d e has the s a m e s h a p e as the curve f o u n d in Na-acetate-NH4Cl med i u m . A c o m p a r i s o n o f Fig. 4 a a n d b shows that the m a g n i t u d e o f the chloride self-exchange flux is lower in the Na-acetate-NH4C1 m e d i u m d u e to the n o n c o m petitive inhibition o f the chloride flux by acetate (19). H o w e v e r , the calculated m a x i m u m chloride flux (see below) f r o m the data in Fig. 4a is the s a m e as that o b s e r v e d in Fig. 4b. F u r t h e r m o r e , the presence o f f u r o s e m i d e (5 × 10 -4 M) w h e n studied by either m e t h o d p r e v e n t e d the decrease in the self-exchange flux at high cellular chloride concentrations which was o b s e r v e d in the absence o f f u r o s e m i d e with both m e t h o d s (19, 9). Fig. 5 is a double reciprocal plot showing the data f r o m Fig. 4 a (5 x 10 -4 M f u r o s e m i d e ) t o g e t h e r with similar data f r o m e x p e r i m e n t s in 10 -4 M f u r o s e m i d e and in the absence o f f u r o s e m i d e (only data f r o m cells with 0-75 m M cellular chloride were c o n s i d e r e d , to minimize the contribution o f the self-inhibition a p p a r e n t at h i g h e r chloride concentrations). T h e kinetic p a r a m e t e r s , Vm for the m a x i m u m flux a n d Kl1~ for the value o f cellular chloride concentration when

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the flux is h a l f - m a x i m u m , were e s t i m a t e d by the statistical m e t h o d o f W i l k i n s o n (30). F o r t h e c o n t r o l d a t a Vm = 800 - 40 (SE) m e q / ( k g cell solids, m i n ) a n d Kxt2 = 65 - 5 raM1; for the e x p e r i m e n t s in 10 -4 M f u r o s e m i d e V,, = 490 -+ 70, Kv2 = 106 - 30; a n d f o r t h o s e in 5 x 10 -4 M f u r o s e m i d e Vm = 213 - 6, K~/2 = 132 _-4-8. By a n a l o g y to e n z y m e k i n e t i c systems this f i g u r e i n d i c a t e s t h a t f u r o s e m i d e c a u s e d a m i x e d - t y p e ( c o m p e t i t i v e a n d n o n c o m p e t i t i v e ) i n h i b i t i o n (28). I n a D i x o n plot (Fig. 6), the lines t h r o u g h the d a t a p o i n t s f o r each o f t h r e e

800 E

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FIGURE 4. Concentration dependence of chloride self-exchange flux in the presence of furosemide (5 × 10-4 M). Left, T h e intracellular chloride was altered by equilibrating red cells in Na-acetate-NH4Cl medium, pH 7.80, 0°C. T h e flux measurements were performed when cells were in steady state with respect to chloride (rot was 0.78-0.93). T h e curve was drawn from a Michaelis-Menten equation: Mcl = Vm" Cleen/(Klt2 + C l c e l l ) , whose constants were determined by statistical analysis (30) of a double reciprocal plot (Vm = 213 meq/(kg cell solids" min) and Kin = 130 mM cellular chloride). Right, The chloride self-exchange flux vs. cellular chloride concentration plot with the intracellular chloride altered by the nystatin method of Cass and Dalmark (7). In this medium chloride is the only anion and the effluxes were performed at pH 7.20, 0°C, rcl = 1.0-0.90. T h e self-exchange flux for the control curve as well as the furosemide (5 × 10-4 M) curve are shown over a cellular chloride range of 25-550 mM. c h l o r i d e c o n c e n t r a t i o n s i n t e r s e c t at a p o i n t a b o v e t h e abscissa a n d to the left o f the o r d i n a t e , a g a i n c o m p a t i b l e with a m i x e d type o f i n h i b i t i o n (28). T h e p o i n t o f i n t e r s e c t i o n o f t h e s e lines c o r r e s p o n d s to the KI (dissociation c o n s t a n t o f t h e t These values from a statistical evaluation of data at only low CI concentrations (where selfinhibition is not prominent) are different from the observed maximum flux of 460 and concentration of chloride at which the flux is half of 460, namely 35 mM, as reported earlier (19). We believe that the values in the text more accurately reflect the underlying phenomena of the transport system than the simple observables reported earlier.

BRAZV AND GUNN Furosemide Inhibition of Chloride Transport

qa .01

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.04 ,08 I / Cell Chloride, (meq/kg cell w0ter)-I

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FIGURE 5. Lineweaver-Burk plot. T h e reciprocal chloride self-exchange flux is g r a p h e d as a function of the reciprocal cellular chloride concentration. All fluxes were p e r f o r m e d at 0°C, pH 7.80, and rm = 0.75-0.92 in Na-acetate-NH~C1 m e d i u m . T h e data from the saturation curves of the uninhibited flux and the inhibited flux at two different furosemide concentrations are plotted here. Statistical analysis (30) was used to d e t e r m i n e the intercepts (values are given in the text). T h e pattern of this inhibition is that of a mixed type (competitive-noncompetitive).

0/ ~ -~

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I t t I J 20 40 60 80 I00 Furosemide Cc*'~entroti~ ( M X IOs }

FIGURE 6. Dixon plot. T h e reciprocal of the chloride self-exchange flux is g r a p h e d as a function of the furosemide concentration at three cellular chloride concentrations. In this graph the Na-acetate-NH4Cl m e d i u m method was used to alter intracellular chloride at pH 7.80, 0°C. T h e lines were drawn by eye. T h e pattern of the graph is compatible with mixed-type inhibition and the lines intersect at a point equal to the negative of the dissociation constant (K1) which is 5 × 10-5 M for furosemide.

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i n h i b i t o r - e n z y m e complex) which was 5 × 10 -5 M in this case. T h e deviation f r o m linearity seen at the lowest chloride concentration (26 mM) a n d the highest f u r o s e m i d e c o n c e n t r a t i o n reflects the presence o f the furosemide-insensitive chloride flux which b e c o m e s a g r e a t e r portion of the m e a s u r e d flux u n d e r these conditions. Because o f this furosemide-insensitive flux, the reciprocal o f the flux has a m a x i m u m value which the lines on the Dixon plot m u s t asymptotically a p p r o a c h at high d r u g concentrations.

The pH and Temperature Dependence of the Chloride Self-Exchange Flux in the Presence of Furosemide T w o characteristic features o f the chloride t r a n s p o r t system in red blood cells are the decreasing self-exchange flux at p H values below 7.8 at 0°C (19, 9) a n d the high a p p a r e n t activation e n e r g y o f 30 kcal/mol (12, 13, 9). T h e s e p a r a m e t e r s were restudied in the presence o f 5 × 10 -4 M f u r o s e m i d e ( a p p r o x i m a t e l y 75% inhibition o f the control flux) to d e t e r m i n e if changes in p H or t e m p e r a t u r e would alter the effectiveness o f f u r o s e m i d e inhibition. T h e p H d e p e n d e n c e of the chloride self-exchange flux was d e t e r m i n e d in red cells equilibrated at 0°C in chloride m e d i u m at the desired p H value. Fig. 7 shows the relationship o f the self-exchange flux to the p H o f the m e d i u m for uninhibited cells a n d cells inhibited by 5 × 10 -4 M f u r o s e m i d e . T h e curves for both conditions are concave with a m a x i m u m a r o u n d p H 7.8. T h e p e r c e n t inhibition o f the control flux at the d i f f e r e n t p H values was nearly constant (75-80%) over this p H r a n g e (5.6-8.9). T h u s , the reaction of the t r a n s p o r t m e c h a n i s m with h y d r o g e n ions which causes inhibition did not significantly alter the inhibitory effect of f u r o s e m i d e . This observation is consistent with either a model in which H 3 0 + and f u r o s e m i d e c o m p e t e for a c o m m o n locus or a m o d e l in which they have s e p a r a t e loci a n d i n d e p e n d e n t inhibitory actions. Theoretically, in both of these models the fractional inhibition caused by one inhibitor is d e p e n d e n t u p o n the concentration o f the second inhibitor, because the reactions are reversible a n d obey the law o f mass action (reference 18, p. 322). H o w e v e r , u n d e r the e x p e r i m e n t a l conditions of Fig. 7 with a fixed f u r o s e m i d e concentration, very little c h a n g e in the p e r c e n t inhibition would occur if either m o d e l were o p e r a tive. 2 T h e r e f o r e the relationship between the sites o f H 3 0 + inhibition a n d 2 First consider a single site for n o n c o m p e t i t i v e inhibition by both H 3 0 + (H) a n d f u r o s e m i d e (F).

k~

k,

kt

k~

k3

are t h e e q u a t i o n s for two n o n c o m p e t i t i v e inhibitors which can react with t h e u n l o a d e d (E) a n d chloride-loaded (E-CI) carrier. T h e chloride flux (v) equals k(E-CI). U s i n g conservation o f carriers (Y.E~ = T) o n e can calculate the chloride flux in the p r e s e n c e o f H only (VH) a n d in the presence o f both H a n d F (Vnv) a n d obtain their ratio. VnF _ 1 + kcH vH 1 + kzH + kzF

If H inhibits 80% alone ( p H 5.6) a n d F inhibits 75% alone as in Fig. 7, t h e n k2H = 0.25 a n d k~F = 0.34 a n d vnF/vn = 0.78. T h u s at p H 5.6 the flux in t h e p r e s e n c e o f f u r o s e m i d e is 78% o f that in the absence o f f u r o s e m i d e . If k2H = O as at p H 8, t h e n vnr/vn = 0.75. T h e fractional inhibition will be practically i n d e p e n d e n t o f p H r a n g i n g f r o m 0.75 to 0.78 while H + inhibition r a n g e s f r o m 0 to 80%. Second, c o n s i d e r two s e p a r a t e sites for n o n c o m p e t i t i v e inhibition by H a n d F. Vne/Vn = (1 +

591

BRAZV ANn GUNN Furoseraide Inhibition of Chloride Transport

f u r o s e m i d e inhibition c a n n o t be ascertained by this e x p e r i m e n t . T h e t e m p e r a t u r e d e p e n d e n c e of the chloride self-exchange flux was determ i n e d in chloride m e d i u m in which the p H was adjusted to 7.80 against s t a n d a r d b u f f e r s at the desired t e m p e r a t u r e . Again this e x p e r i m e n t was m a d e with red cells in the p r e s e n c e or absence o f 5 × 10 -4 M f u r o s e m i d e . Fig. 8 shows this data on an A r r h e n i u s d i a g r a m with the log o f the chloride flux plotted as a function o f the reciprocal t e m p e r a t u r e in d e g r e e s Kelvin. T h e slope o f this g r a p h is p r o p o r tional to the a p p a r e n t activation e n e r g y . T h e flux values at the h i g h e r t e m p e r a tures deviated f r o m the line t h r o u g h the values at lower t e m p e r a t u r e s . T h i s nonlinearity has recently been o b s e r v e d by B r a h m (2) who studied the chloride 800

Furosemicie=0 600

400 E

o

200

.

I 6.0

I t 7.0 80 Extnocellulot pH

90

FIGURE 7. E f f e c t o f e x t r a c e l l u l a r p H o n f u r o s e m i d e i n h i b i t i o n o f c h l o r i d e selfe x c h a n g e flux at 0°C. C h l o r i d e s e l f - e x c h a n g e fluxes w e r e p e r f o r m e d at several e x t r a c e l l u l a r p H values with a n d w i t h o u t f u r o s e m i d e (5 x 10 -4 M) in c h l o r i d e m e d i u m . T h e rcl v a r i e d linearly with t h e p H f r o m 1.3 at p H 5.6 to 0.3 at p H 8.9. T h e c u r v e was d r a w n by eye t h r o u g h d u p l i c a t e e f f l u x d e t e r m i n a t i o n s at e a c h p H value. T h e p e r c e n t o f i n h i b i t i o n by f u r o s e m i d e was 75% at t h e low a n d 80% at t h e h i g h p H values.

self-exchange between 0 ° a n d 38°C with a flow t u b e a p p a r a t u s a n d m a y reflect an increase o f K1/2 with t e m p e r a t u r e . Alternatively, we may have i n t r o d u c e d a systematic e r r o r at the highest t e m p e r a t u r e s d u e to the very rapid rate o f k2H)/(l + kaH + k~F + kak4H'F). If k4 is small [k4 = HFE/(HE.F) = (HFE-CI)/(HE-CI.F)], then the above calculation holds for a single site; and if not, a reasonable guess is that k, = kn or that the reaction of H or CI with their separate sites does not affect the binding affinity for furosemide (F) at its separate independent site. If so, then under the same experimental conditions as above, vHv/vn = 0.75 at pH 5.6 where k2H = 0.25, kaF = 0.34 and k2ka/-/.F = 0.085 and vn~/Vn = 0.75 at high pH where k~t--/ = k~I-IK,F = O. Thus under these experimental conditions both the single-site and the two-site models predict a pH-independent fractional inhibition by furosemide and cannot be distinguished by these data. Since at very low pH other transport mechanisms come into play, we cannot be sure that at even more extreme conditions these two model mechanisms can be separated by further experiments.

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sampling for these data points. I n o u r data the slope o f the g r a p h t h r o u g h the lower points yields an a p p a r e n t activation e n e r g y o f 29 kcal/mol for both the control and the furosemide-inhibited chloride flux. T h u s , there was no unusual t e m p e r a t u r e sensitivity of the inhibition o f the chloride flux by furosemide. DISCUSSION Anion t r a n s p o r t in h u m a n red blood cells was first shown to be f u r o s e m i d e

4.0~

~

pH=7.8

.~_ E

~ 30 g u..

e, Furosemide:5 x 10"4M~

2.0 34

I 3.5

I 3.6

\ 3.7

IO00/T °Kelvin

FmURE 8. Effect of temperature on furosemide inhibition of chloride self-exchange flux; Arrhenius plot. The chloride self-exchange flux was performed at different temperatures, at constant pH 7.80 in chloride medium, with an rCl of 0.'/1-0.77. The slope from the graph of the log chloride flux vs. the reciprocal temperature (degrees Kelvin) is proportional to the apparent activation energy. The line was drawn by eye through duplicate flux measurements at each temperature. The slope of the control and the furosemide-inhibited curves is the same and the apparent activation energy is 29 kcal/mol. sensitive by Deuticke and Gerlach (14). In their study the furosemide concentration required for 50% inhibition of [a2p]phosphate efflux was 5 × 10 -4 M. This value is only 2.5 times the concentration required to inhibit by 50% the chloride self-exchange flux r e p o r t e d in this study u n d e r quite different experimental conditions. Deuticke and Gerlach m e a s u r e d p h o s p h a t e self-exchange at 37°C at p H 7.35 in Locke's solution containing 1 mM p h o s p h a t e . T h e chloride and bicarbonate ions in Locke's solution are inhibitors (probably competitive) of divalent (29) and m o n o v a l e n t inorganic anion transport (19, 11). These inhibitors would decrease the inhibitory effect of f u r o s e m i d e if they reacted with the

BRAZ'Y AND GUNN Furoseraide Inhibition of Chloride Transport

593

anion t r a n s p o r t m e c h a n i s m at a c o m m o n site. O t h e r d i f f e r e n c e s , e.g., t e m p e r a ture a n d d e g r e e o f c a r r i e r saturation, m a y also play a~role in the slight d i f f e r e n c e in the c o n c e n t r a t i o n o f f u r o s e m i d e r e q u i r e d for 50% inhibition in these two systems. F u r o s e m i d e interacts with the r e d cell very rapidly a n d reversibly. In Fig. 3 o n e can see that the onset o f inhibition by this d r u g is less t h a n 6 s at 0°C. T h e rapidity o f this effect implies that f u r o s e m i d e acts at a superficial site on the cell. Since f u r o s e m i d e is a large organic acid ( p K a = 3.9) one would not expect it to rapidly cross the cell m e m b r a n e . For c o m p a r i s o n p a r a s u l f a m o y l benzoic acid which is similar to a p a r t o f the f u r o s e m i d e molecule (see Fig. 1) does not cross the ox red cell m e m b r a n e to any significant d e g r e e (1). T h u s we would not expect the c h a r g e d f o r m (see below) o f the larger f u r o s e m i d e molecule to cross into the red cell within 6 s. F u r o s e m i d e ' s effect on r e d cell chloride t r a n s p o r t was reversed by washing the cells six times with d r u g - f r e e m e d i u m . T h e relative ease of reversing the inhibition of this d r u g also suggests that f u r o s e m i d e ' s action is on the m e m b r a n e surface. So while f u r o s e m i d e has b e e n shown to inhibit glycolysis in cell-free p r e p a r a t i o n s (23), it a p p e a r s to inhibit anion t r a n s p o r t in red cells at the level o f the cell m e m b r a n e , a n d most likely the o u t e r surface o f this m e m b r a n e . F u r o s e m i d e has a negatively c h a r g e d carboxyl g r o u p at n e u t r a l p H . We meas u r e d the effect o f altering the p H o f the m e d i u m on the inhibitory action o f f u r o s e m i d e on the chloride self-exchange flux. O v e r a p H r a n g e o f 5.6-8.9 (Fig. 7) the p e r c e n t o f inhibition o f chloride flux caused by 5 x 10 -4 M f u r o s e m i d e was nearly constant (75-80%). Since the c o n c e n t r a t i o n o f u n c h a r g e d f u r o s e m i d e varied by a factor o f 10n while the negatively c h a r g e d f o r m was nearly constant, this result indicates that the anionic f o r m o f the d r u g is the active inhibitor. We cannot say w h e t h e r the n e u t r a l f o r m o f the d r u g is also an inhibitor. T h e kinetic analysis o f the e x p e r i m e n t a l data in this p a p e r reveals four points. In the absence o f acetate, that is in m e d i a containing only chloride a n d f u r o s e m ide anions, (a) the d o s e - r e s p o n s e curve shows an a p p a r e n t stoichiometry of m o r e than o n e f u r o s e m i d e molecule inhibiting each t r a n s p o r t unit, a n d (b) the curve o f the chloride flux g r a p h e d against chloride c o n c e n t r a t i o n (Fig. 4b) is c o m p l e x a n d a b o v e a certain c o n c e n t r a t i o n , chloride inhibits its own flux. I n the p r e s e n c e o f acetate anions, (c) the stoichiometry for f u r o s e m i d e inhibition o f t r a n s p o r t units is 1:1, a n d (d) f u r o s e m i d e behaves as a m i x e d - t y p e inhibitor o f the chloride self-exchange system (Figs. 5 and 6). T h e analyses s u p p o r t i n g points (a) a n d (c) are p r e s e n t e d below a n d in Fig. 9. D a l m a r k (11) has m a d e a detailed study o f the chloride saturation curve (Fig. 4b) with particular attention to the influences o f o t h e r halide anions on the selfinhibition by chloride. H e has p r o p o s e d that the chloride t r a n s p o r t m e c h a n i s m has two anion b i n d i n g sites, one for anion translocation a n d a n o t h e r for noncompetitive inhibition o f translocation. D a l m a r k o b s e r v e d chloride self-exc h a n g e between 10 a n d 400 m M chloride in the p r e s e n c e of o t h e r halides (10-300 mM) in cells p r e p a r e d by the nystatin m e t h o d , a n d f o u n d that halides d e c r e a s e d the calculated m a x i m u m flux, increased the K~/2, a n d d e c r e a s e d the chloride self-inhibition. H e c o n c l u d e d first that fluoride, b r o m i d e , a n d iodide act both as c o m p e t i t i v e ' a n d n o n c o m p e t i t i v e inhibitors of the chloride carrier, a n d are able

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to c o m p e t e with chloride at b o t h of the p r o p o s e d anion binding sites ( t r a n s p o r t site = Cl~; m o d i f i e r site = Cl2), a n d second, that the b i n d i n g o f a n o t h e r halide on the n o n c o m p e t i t i v e m o d i f i e r site (Cl2 site) p r e v e n t e d chloride f r o m binding t h e r e a n d p r e v e n t e d chloride f r o m inhibiting its own flux. In most o f the e x p e r i m e n t s in o u r study, chloride c o n c e n t r a t i o n was altered in cells by using an Na-acetate-NH4Cl m e d i u m . Acetate anions are n o n c o m p e t i t i v e inhibitors o f chloride self-exchange flux (19), a n d 141 m M acetate almost completely blocks chloride self-inhibition (see Figs. 5 a n d 6 in r e f e r e n c e 19). Acetate, t h e r e f o r e , behaves like the halides as a c o m p e t i t o r with chloride at the modifier (Cl2) site which is responsible for self-inhibition. I n the p r e s e n c e of acetate, the chloride self-exchange flux follows m o r e closely the equations o f MichaelisMenten kinetics, reflecting mainly the characteristics o f the t r a n s p o r t (Cll) site. T h u s the action o f acetate simplifies the analysis of f u r o s e m i d e inhibition (see Appendix). T h e stoichiometry o f the reaction between f u r o s e m i d e molecules a n d the chloride t r a n s p o r t m e c h a n i s m can be s u r m i s e d by g r a p h i n g a r e a r r a n g e m e n t o f the Michaelis-Menten kinetic equations in which the slope o f the plot indicates the n u m b e r of molecules o f inhibitor acting to inhibit each carrier unit (Fig. 9) (22). T h e slope in this g r a p h for f u r o s e m i d e inhibition in the absence of acetate (data f r o m Fig. 2) is 1.36, which indicates that the interaction o f m o r e t h a n one f u r o s e m i d e with each t r a n s p o r t unit is r e q u i r e d for inhibition. When one uses the data f r o m Fig. 6 (Dixon plot, CI = 146 raM) where f u r o s e m i d e inhibition is studied in the p r e s e n c e o f acetate, the slope o f the g r a p h on this plot is 1.03. T h u s , f u r o s e m i d e in the presence o f acetate requires only one molecule of inhibitor p e r t r a n s p o r t unit, but in the absence o f acetate, m o r e than one f u r o s e m i d e molecule binds to each t r a n s p o r t unit. T h e kinetic analyses in Figs. 5 a n d 6 indicate that f u r o s e m i d e in the presence o f 141 mM acetate behaves as a m i x e d - t y p e inhibitor o f the chloride selfe x c h a n g e t r a n s p o r t system. This result a p p e a r s to be similar to the halide inhibition of chloride t r a n s p o r t r e p o r t e d by D a l m a r k (11); however, the following c o m p a r i s o n o f results obtained in the absence and p r e s e n c e o f acetate in the e x p e r i m e n t a l m e d i u m shows this similarity to be illusory. T h e a p p a r e n t similarity between the action of f u r o s e m i d e a n d the halides is the observation that in the absence o f acetate they both block the self-inhibition by chloride (Fig. 4b in this p a p e r a n d Figs. 2 and 3 in r e f e r e n c e 11). In analogy with the halides a n d when o n e considers the two sites p r o p o s e d by D a l m a r k , f u r o s e m i d e could c o m p e t e with chloride at Cl~ a n d Cl2 to give, respectively, competitive a n d n o n c o m p e t i t i v e inhibition as seen with iodide, b r o m i d e , a n d fluoride. T h e stoichiometry o f m o r e than one f u r o s e m i d e molecule per transp o r t unit is consistent with this possibility. In the presence o f acetate, however, f u r o s e m i d e also behaves kinetically as a m i x e d - t y p e inhibitor a n d requires only one molecule to inhibit each chloride t r a n s p o r t unit. T h e r e f o r e , c o m p e t i t i o n between f u r o s e m i d e a n d chloride at two sites, Cl~ a n d Cl2, is u n t e n a b l e . C o m petition at only Cll would not p r o d u c e m i x e d - t y p e inhibition but p u r e competitive inhibition, so exclusive reaction at this site m a y be r e m o v e d f r o m consideration. We have a r g u e d that acetate c o m p e t i t i o n with chloride occurs at the Cl2 site a n d it does not seem possible that acetate c o m p e t i t i o n at Cl~ could block

BRazv A~D GtrN~r Furosemide Inhibition of Chloride Transport

595

f u r o s e m i d e c o m p e t i t i o n at Cll to l e a v e it o n l y a c t i n g at Cl2. W e t h e r e f o r e m u s t r e j e c t t h e n o t i o n t h a t f u r o s e m i d e r e a c t s with Cll a n d Cl2 as D a l m a r k p r o p o s e d f o r h a l i d e s a n d we m u s t p o s t u l a t e a t h i r d site, F~, w h i c h is n o t t h e c h l o r i d e t r a n s p o r t site a n d n o t t h e s e c o n d h a l i d e site b u t w h i c h is i n v o l v e d in i n h i b i t i o n by furosemide. The reaction scheme for mixed-type (competitive and noncompetitive) inhibi-

10.0 8.0

q.O

2/3 I

+1.0 0.8

0.4

0.2

' -4.0

* -3.0 1.0qFur0semide(1~1

- 0

FIGURE 9. Double logarithmic replot o f the dose-response curve. After the method o f J o h n s o n et al. (22) the data of the dose-response curve were replotted as the log o f the (control flux/inhibited flux)- t vs. the log o f the furosemide concentration. T h e slope o f this plot indicates the n u m b e r o f molecules o f inhibitor n e e d e d to inhibit one unit o f the transport mechanism. T h e solid line represents the data from Fig. 2 for furosemide in the absence of acetate and has a slope of 1.36. T h e b r o k e n line is from the data shown in Fig. 6, chloride concentration 146 raM, for furosemide in the presence o f 141 mM acetate and has a slope o f 1.03. In a separate e x p e r i m e n t using 5 x 10-~, 2 x 10-*, and 5 x 10-4 M furosemide, the slope in media without acetate was 1.23 while when 141 mM NH,-acetate was included the slope was 0.96. Acetate, therefore, reduces the stoichiometry of the inhibition o f chloride flux by furosemide to 1:1. t i o n b y a n i n h i b i t o r a c t i n g at a s i n g l e site (F1) is d i a g r a m m e d

E +S

.Ks.

E + I

. KI ~

ES + I

•

r

~agi.~.

ES IE + S

k )

as follows (27):

E +P

(aKs')

IES

IES

T h e i n h i b i t o r , I , b i n d s n o n c o m p e t i t i v e l y to t h e e n z y m e , E , a n d in d o i n g so a l t e r s t h e a f f i n i t y , Ks, o f t h e e n z y m e f o r t h e s u b s t r a t e b y a f a c t o r a a n d t h u s c o m p e t i -

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tively reduces the a p p e a r a n c e o f the end p r o d u c t , P, (in this case the t r a n s p o r t e d anion). T h u s f u r o s e m i d e can cause a mixed type of inhibition by binding to the chloride transport unit at a single modifier site, F1, discrete f r o m the t r a n s p o r t site. T h e molecular mechanism by which the affinity o f the t r a n s p o r t site, Cll, is c h a n g e d is not clear. T w o possibilities are that f u r o s e m i d e binding to the F1 site increases local negativity which reduces the chloride concentration at the transport site; a n d , alternatively, that the b i n d i n g to F1 decreases the chloride affinity t h r o u g h an allosteric c o n f o r m a t i o n a l c h a n g e at the Cll site. F u r o s e m i d e a p p a r e n t l y can also react with the Cl2 site. As pointed out earlier, the observation that acetate blocks chloride self-inhibition implies that acetate causes its noncompetitive inhibition at the Cl2 site. W h e n this conclusion is coupled with the observation that the stoichiometry o f f u r o s e m i d e inhibition is r e d u c e d f r o m 1.36:1 to 1.03:1 by acetate, one easily and, we believe, correctly is led to conclude that the acetate-inhibitory site and the second site o f f u r o s e m i d e b i n d i n g are the same, namely the C12 site. We t h e r e f o r e believe that f u r o s e m i d e reacts with two n o n t r a n s p o r t sites on the t r a n s p o r t mechanism and that acetate can displace f u r o s e m i d e at one o f these sites (see A p p e n d i x for this model). In conclusion, we believe that the negatively c h a r g e d f o r m o f f u r o s e m i d e inhibits the red cell chloride flux by reacting with the t r a n s p o r t m e c h a n i s m at two sites. O n e (C/2) is identical with the site at which chloride can cause selfinhibition. At the second site ( F 0 f u r o s e m i d e noncompetitively blocks chloride t r a n s p o r t a n d alters the affinity o f chloride for the t r a n s p o r t site (Cl0 to p r o d u c e the pattern o f mixed-type inhibition.

APPENDIX From Fig. 9 and for the reasons outlined in the Discussion, it appears that furosemide binds to two nontransport sites on the transport unit. At one of these sites furosemide is displaced by acetate. This is the Cl2 site which is complexed to the inhibitor when I stands to the right of E: El. The FI site is complexed when I stands to the left of E: IE. Although these sites may only be on the outside surface of the cell, the use of an asymmetric model and inclusion of the distribution of carriers between the inside and outside would introduce unwarranted complications at the present level of our knowledge. The analysis has, therefore, been restricted to a single set of reactions and the resulting parameters are, of course, only apparent dissociation constants. The kinetic model and equations for this situation are given below. k

Ks

E+S + I

(

)

ES + I

)

E+P Ks

I KI IE + S + I

aKs (

IflKt IEI + S

aKs ".

I aKl ) IES + I I ilK'

~. IEIS

= dissociation constant for carrier and substrate,

Kt = dissociation constant for carrier and inhibitor, a and /3 are coefficients both greater than 1.0.

BRAZY AND GUNN Furosemide Inhibition of Chloride Transport

597

Total carrier Et = E + ES + IE + IES + IEI + IEIS _ [ES]Ks

[s]

[ES][I]Ks

+ [ES] +

[S]K,

[ES][I] + [ES][I~]Ks

+~

[SleK,"

[ES][Iy

+ ,,.K,/3K,"

T h e a p p e a r a n c e o f p r o d u c t is as follows: k lEt][S] vo = k [ES] - (Ks + IS]) for the uninhibited reaction, and Ui =

Ks + [S] +

k [Et][S] ~ / [ / ] + [/][S]

~

Ks[lY

+~

[S][I]z

+ ,~/3K;

for the inhibited reaction. Let V,~o = k lEt]. T h e n the reciprocal plot will be 1 + v-]

1

[(Ks+[S])+[ I ] ( ~

+ Ks) ' [1]'

{[S]+ Ks)],

and the Dixon plot will not be linear. T h e presence o f acetate ions reduces the Dixon plot (Fig. 6) to a linear form and this implies that acetate ions cause a m a r k e d increase in the value o f 13. T h e double logarithmic plot (Fig. 9) has the following equation: V°-l+ v,

[S] +_ aKs [ [ I ] + [1]2 [S] + Ks \o~K, ~---~i2)

v~

[ S ] + _ a K s ( [I] + [S] + Ks \txK, otOK,/

T h e slope o f the double logarithmic plot will be 1.0 when/3 is large and [I] is small and 2.0 when [I] is large. Acetate again causes/3 to be a larger n u m b e r and makes the slope = 1.0. Without acetate over the concentration range studied the slope = 1.3. From the kinetic data and graphs (Figs. 5 and 6) values for the constants were d e t e r m i n e d . T h e values for Vmax a n d Ks (called Kin) are given in Results. Kt was d e t e r m i n e d to be 5 x 10-5 M from the Dixon plot; a comparable value o f 7.7 x 10-5 M was obtained from the Lineweaver-Burk plot. T h e coefficient a has a value of 2.44 from the data on the Lineweaver-Burk plot and 2.64 from the data on the Dixon plot. T h e coefficient/3 even in the absence of acetate must have a value of at least 100 for the 12 term in the above equations to be less than 10% o f the linear term in I. This will permit the graph in Fig. 9 o f the data obtained in the absence of acetate to a p p e a r linear. Furthermore, by using /3 = 100 the slope o f the calculated line will be 1.25. T h u s both the observed linearity (12 term small) and slope greater than unity (very close to the 1.36 and 1.30 values f o u n d by experiment) can be met having/3 = 100. I f one uses a larger value of /3 = 1,000 for the experiments in the presence o f acetate, one calculates a slope o f 1.03, the same as that f o u n d by experiment. In addition, this larger value of/3 is required for the Dixon plot (Fig. 6) to be linear up to furosemide concentrations o f 10-3 M. We have neglected the form E I (inhibitor b o u n d only to the nontransport C12 site) in the above model since/3 is estimated to be so much greater than tx. If/3 = 100, E I will be only 1% o f the free E form at 5 x 10-4 M furosemide, while the amount o f l E (inhibitor b o u n d only to the FI site) will be equal to that o f the free E form. If/3 = 1,000 the E I complex is only 0.1% o f the free E form and contributes even less. T h e inclusion o f the E1 complex would therefore complicate the equations without illuminating the mechanisms. Practically speaking, the C12 site is never complexed with furosemide unless the F1 site is already complexed with a n o t h e r furosemide.

598

THE J O U R N A L

OF

GENERAL

PHYSIOLOGY

" VOLUME

68

"

1976

This work was supported by grants HL-12157 and HL-18069 from the National Heart and Lung Institute. Peter C. Brazy was a recipient of a National Kidney Foundation Research Fellowship during this work. Robert B. Gunn is the recipient of a Research Career Development Award from the National Heart and Lung Institute (K4-HL-00208). This work was presented in part at the fall meeting of the American Physiological Society, 1975. The abstract was published in The Physiologist. 18:151.

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