Interaction between Phloretin and the Red B l o o d Cell M e m b r a n e MICHAEL

L. J E N N I N G S

a n d A. K. S O L O M O N

From the Biophysical Laboratory, Harvard Medical School, Boston, Massachusetts 0211'5

A B S T R A C T Phloretin binding to red blood cell components has been characterized at p H 6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturable processes approximately equal in magnitude, which strongly suggests that most o f the red cell binding may be ascribed to hemoglobin. This conclusion is s u p p o r t e d by the fact that hemoglobin-free red cell ghosts can bind only about 10% as much phloretin as an equivalent n u m b e r of red cells. T h e permeability of the red cell m e m b r a n e to phloretin has been d e t e r m i n e d by a direct m e a s u r e m e n t at the time-course o f the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7 s, corresponding to a permeability coefficient of 2 x 10-4 cm/s. T h e concentration d e p e n d e n c e of the binding to ghosts reveals at least two saturable components. Phloretin binds with high affinity (Kol~ = 1.5/.tM) to about 2.5 x 10~ sites per cell; it also binds with lower affinity (Koiss = 54/,tM) to a second (5.5 x 107 per cell) set of sites. In sonicated total lipid extracts o f red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total n u m b e r of sites are not significantly different from those o f the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. T h e r e f o r e , the high affinity phloretin binding sites are related to m e m b r a n e proteins, and the low affinity sites result from phloretin binding to lipid. T h e identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane. P h l o r e t i n a n d p h l o r i z i n a r e i n h i b i t o r s o f m e m b r a n e t r a n s p o r t p r o c e s s e s in a v a r i e t y o f s y s t e m s . P h l o r i z i n is t h e m o r e p o t e n t i n h i b i t o r o f t h e active t r a n s p o r t o f h e x o s e s w h i c h o c c u r s in t h e k i d n e y (1) a n d s m a l l i n t e s t i n e (2). P h l o r e t i n is m o r e p o t e n t in t h e i n h i b i t i o n o f e n e r g y - i n d e p e n d e n t t r a n s p o r t p r o c e s s e s : s u g a r t r a n s p o r t in t h e e r y t h r o c y t e (3), fat cells (4), a n d r a b b i t h e a r t m u s c l e (5), a n i o n e x c h a n g e in t h e e r y t h r o c y t e (6), a n d u r e a t r a n s p o r t in r e d cells (7) a n d t o a d b l a d d e r (8). S p e c i f i c p h l o r i z i n r e c e p t o r s , o b s e r v e d in t h e m e m b r a n e s o f fat cells (9) a n d in k i d n e y b r u s h b o r d e r m e m b r a n e s (10, 11), a r e b e l i e v e d to b e a s s o c i a t e d with p h l o r i z i n i n h i b i t i o n o f s u g a r t r a n s p o r t a c r o s s t h e s e m e m b r a n e s . L e F e v r e a n d M a r s h a l l (12) h a v e s h o w n t h a t p h l o r e t i n is b o u n d s t r o n g l y to r e d cells a n d t h a t t h e e x t e n t o f b i n d i n g , in p h l o r e t i n a n a l o g s , p a r a l l e l s p o t e n c y as g l u c o s e t r a n s p o r t i n h i b i t o r s . H o w e v e r , L e F e v r e a n d M a r s h a l l w e r e n o t a b l e to d e t e c t s p e c i f i c , s a t u r a b l e b i n d i n g sites f o r p h l o r e t i n o n t h e r e d cell m e m b r a n e . W e h a v e s t u d i e d t h e d e t a i l s o f p h l o r e t i n b i n d i n g to t h e r e d cell m e m b r a n e in a n a t t e m p t T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y " V O L U M E

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to i d e n t i f y the i n t e r a c t i o n s r e s p o n s i b l e f o r t h e v a r i o u s effects o f p h l o r e t i n o n r e d cell t r a n s p o r t processes. MATERIALS

AND

METHODS

Phioretin b i n d i n g to red cells was measured by the method described by LeFevre and Marshall (12). Fresh blood from healthy donors was drawn by venipuncture into heparin and used within 2 days. Red cells were washed four times in isotonic phosphate buffer before use. Phloretin was obtained from K and K Laboratories (Ptainview, N. Y.) and its purity was checked by thin-layer chromatography in 3:1 chloroform:propanol (13). Phloretin was dissolved first in a small volume of 0.1 N NaOH, added to buffer, and titrated to pH 6. No ethanol was used to dissolve the phloretin. All binding experiments were performed at 22 + 2°C. Hemoglobin-free red cell ghosts were prepared by the method of Dodge et al. (14). Cells were lysed in 20 vol of 20 mosM phosphate buffer, pH 8, 4°C, washed three times. and resealed by restoring normal osmolality with NaCI and incubating at 37°C for 1 h. 6070% of the ghosts were resealed by this procedure, as determined by the accessibility of the internal marker enzyme glyceraldehyde-3-phosphate dehydrogenase, as used by Steck (15). T h e ghost concentration in each experiment was assayed by the acetylcholinesterase content (acetylcho[inesterase activity is not lost d u r i n g ghost preparation) by the method of Ellman et al. (16). Since the acetylcholinesterase activity per cell varied somewhat a m o n g donors, a known quantity of red cells was assayed for this enzyme just before lysis, and the enzyme activity per cell from a given d o n o r was used to determine the ghost concentration in suspensions from the same donor. Phioretin binding to ghosts was measured optically by absorbance loss from the supernatant after a 10-min centrifugation at 35,000 g. At concentrations above about 10 v.M phioretin, the optical density was measured directly at 286 nm, the absorption maximum for phloretin at pH 6. At lower concentrations, the supernatant phloretin concentration was measured by extraction into ether, drying, and redissolving in a smaller volume of pH 10 carbonate buffer. T h e phloretin concentration was then measured at 323 nm, which is the absorbance peak at pH 10. T h e high pH was used in the final step of the assay because phloretin is quite soluble at this pH and also because the extinction coefficient at the high pH peak is higher than that at the low pH absorbance peak. T h i s e t h e r extraction method recovered at least 97.5% of the phloretin present, as j u d g e d from extraction of phloretin from solutions of known concentration. Such controls were performed for each experiment, and the losses due to the extraction method did not depend on phloretin concentration. Phloretin binding to total lipid extracts of red blood cells was measured by the column chromatography method of H u m m e l and Dreyer (17) using a 30 x 1-cm column containing Sephadex G50 (Pharmacia Fine Chemicals, Inc., Piscataway, N. J.). Lipids were extracted from ghosts by a modification of the method of Burger et al. (18). Five volumes (95 ml) of methanol were added with stirring to 19 ml of packed ghosts in a 250-ml centrifuge bottle. T h e suspension was allowed to stand 5 min at room temperature. T h e n 95 ml of chloroform were added, and the protein precipitate was spun out after 10 min. The supernatant was decanted into two centrifuge bottles, and chloroform and water were added to a final ratio, chloroform:methanol:water = 10:5:1. T h e resultant twophase system was mixed by vortexing and centrifuged for 10 min at 1,000 g. T h e top (aqueous) phase was removed and discarded, and the organic phase, which contained the lipid, was saved. Thin-layer chromatography in chloroform:methanol:water (65:25:4) revealed that all of the major lipid components of the red cell m e m b r a n e were present. The lipid extract was dried in a flash evaporator, dispersed in a small volume of distilled

JENNINGSANDSOLOMON Interaction between Phloretin and Red Cell Membrane

383

water, and sonicated for 25 min at 50 W in a Branson sonicator (Branson Sonic Power Co., Danbury, Conn.) u n d e r N2, with the temperature controlled at 4°C. T h e HummelDreyer b i n d i n g experiments were performed as follows: between 1 a n d 200/xM phloretin in phosphate buffer at pH 6 was dripped through the column at a constant flow rate. T h e absorbance at 286 n m of the material emerging from the column was recorded continuously. At the beginning of an experiment, flow was interrupted, a known quantity of lipid, measured as organic phosphate by the method of Gomori (19), was added to the top of the column, and flow was resumed. As the lipid emerged (in the void volume) from the column, the absorbance increased rapidly to a value too large to measure. After the lipid had all passed out of the column, the absorbance returned to its initial value for a minute or two, then decreased to a m i n i m u m value, and subsequently r e t u r n e d to the original value. T h e area of this "depletion trough" was found to be directly proportional to the quantity of lipid added to the column. These control experiments show that, though phloretin is retarded by the column (that is, elutes at a volume larger than the total column volume), the depletion trough area is a quantitative measure of the a m o u n t of phloretin b o u n d by the lipid, after a small correction for the volume occupied by the added lipid suspension. T h e b o u n d phloretin was calculated from the trough area as follows: Bound phloretin (nanomoles) = acbf/s, where a = trough area (square centimeters), c = phioretin concentration (nanomoles/milliliter)/absorbance unit, b = n u m b e r of absorbance units per c e n t i m e t e r , f = flow rate (milliliters per minute), s = chart speed (centimeters per minute). Phloretin b i n d i n g to h u m a n hemoglobin (crystalline from Sigma Chemical Co., St. Louis, Mo.) was measured by equilibrium dialysis. Dialysis tubing (I/4 inch, A. H. Thomas, Philadelphia, Pa.) was washed three times by soaking for 1 h in distilled water at 80°C, and then filled with 2 ml of hemoglobin solution, with a known initial phioretin concentration in isosmolar phosphate, at pH 6. T h e contents of the dialysis bag were then dialyzed overnight against 23 ml of the same phloretin solution in phosphate buffer at pH 6.0, 22 -+ 2°C. Equilibrium was completely established by the next morning. Phloretin binding by the hemoglobin was measured as the disappearance of phloretin from the medium external to the bag. Corrections were necessary because of phloretin b i n d i n g to the rubber stopper in the test tube containing the dialysis tubing and to the dialysis tubing itself. The validity of the dialysis method was checked by performing a binding experiment by the dialysis method, using whole red cells instead of hemoglobin. At 35/zM free phloretin, pH 6, the cell:medium distribution ratio determined by dialysis is 120 +- 15 (two experiments), which agrees well with the value of 124, which we obtained by the direct method of LeFevre and Marshall (12). T h e rate of phloretin uptake by red cells was measured by three methods. Two of the methods employed rapid sampling of the extracellular medium after red cells had been suspended in a phloretin solution. Red cells, washed in pH 6 isotonic phosphate, were suspended in 100/zM phloretin, in pH 6 phosphate buffer. In the first method, the cells were rapidly sedimented in a B r i n k m a n n centrifuge 3200 (Brinkmann Instruments, Inc., Westbury, N. Y.) at various time intervals after the resuspension. The supernatants were then assayed (by absorbance at 286 nm) for phloretin. A satisfactory separation of cells from medium could be obtained in 3 s, but the exact time points had an error of about 2 s. The second method of sampling the supernatant was the Millipore filtration technique described by Dalmark and Wieth (20). Filtration assemblies were constructed exactly as specified by Dalmark and Wieth (20) with the omission of the ring of ice in the filter holders. Because phloretin adsorbs to the filters, only a very rough time-course for the uptake could be determined by this method. T h e third method for measuring the rate of phloretin uptake relied on the fact that the

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acid (uncharged) form, but not the dissociated (anionic) form of phioretin is taken up by the cells. Uptake of phloretin from an unbuffered phloretin solution therefore results in a pH rise. The rate of pH rise is a measure of the rate of phloretin uptake. Red cells were washed four times in 154 mM NaCI, titrated with HCi to an external pH of 6.90, and resuspended in 200/~M phloretin, pH 6.90, 26 - I°C. The extraceilular pH was measured continuously with a pH meter and strip chart recorder. The response time of the system to a pulse of NaOH was 1.5-2.2 s. This finite response time introduced negligible error in the measured time-course of phloretin entry into the red cells. We also attempted to measure the time-course of phloretin binding to lipid model membranes by this method. The lecithin used was prepared from egg yolks by the procedure of Singleton et al. (21), lyophilized, dispersed in 154 mM NaC1, and sonicated for 40 min at 4°C under N2 in a Branson sonicator. RESULTS AND D I S C U S S I O N Phloretin Binding to Red Cells Phloretin, a weak acid (pK = 7.3), is p r e s e n t in two f o r m s in neutral p H : u n c h a r g e d a n d negatively c h a r g e d . It is the u n c h a r g e d f o r m which inhibits red cell glucose t r a n s p o r t (12) a n d chloride e x c h a n g e (22). T h e p H d e p e n d e n c e o f phloretin b i n d i n g to red cells indicates that it is the u n c h a r g e d f o r m which binds to the cells (12). For these reasons, phloretin b i n d i n g e x p e r i m e n t s were perf o r m e d at p H 6, w h e r e over 90% o f the phloretin is u n c h a r g e d . T h e results are plotted in Fig. 1, showing b o u n d phloretin in molecules per cell as a function o f free concentration. T h e s e results are in quantitative a g r e e m e n t with those obtained by LeFevre a n d Marshall; the average c e l l : m e d i u m distribution ratio for phloretin in o u r e x p e r i m e n t s is 124 +- 4, c o m p a r e d with 110 r e p o r t e d by LeFevre a n d Marshall at p H 6. T h e n u m b e r o f phloretin molecules b o u n d per red cell is a linear function o f free concentration, with no saturable b i n d i n g process detectable within the accuracy o f o u r m e a s u r e m e n t s . Phloretin Binding to Hemoglobin and to Red Cell Ghosts T h e binding o f phloretin to h e m o g l o b i n - f r e e red cell ghosts a n d to purified h u m a n h e m o g l o b i n was d e t e r m i n e d in o r d e r to assess the relative contributions o f the m e m b r a n e a n d the m a j o r intracellular constituent to the total phloretin binding capacity o f the intact red cell. T h e conditions o f the b i n d m g experiments were identical with those in Fig. 1: p H 6 a n d 22 - 2°C. T h e results are plotted in Fig. 2, in the same coordinates as Fig. 1, using 328-mg H b / m l packed cells (1.1 × 10 l° cells) (23) to normalize the data for h e m o g l o b i n . T h e data in Fig. 2 show that phloretin binding to h e m o g l o b i n is an a p p a r e n t l y nonsaturable process o f very similar m a g n i t u d e to phloretin binding by intact cells. T h e e r r o r bars are a reflection o f the uncertainty o f the phloretin b i n d i n g by the dialysis a p p a r a t u s used in the e x p e r i m e n t . T h e similarity between phloretin binding by red cells a n d h e m o g l o b i n suggests that the majority o f the phloretin b i n d i n g to red cells results f r o m phloretin b i n d i n g to h e m o g l o b i n . I f most o f the binding is intracellular, t h e n isolated m e m b r a n e s should bind only a small fraction o f the phloretin b o u n d by intact cells. T h i s is the case, as shown for ghosts in the lower portion o f Fig. 2. T h e s e data show that a b o u t 90% o f the

385

JENNINGS AND SOLOMON Interaction between Phloretin and Red Cell Membrane

80C pH6,22°C '..,.. ~ 60C _m 40C ,~ 20C

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FIGURE 1, Phloretin binding to intact human red cells. Bound phloretin (10 8 molecules per cell) is plotted as a function o f free phloretin concentration (micromolar). The data represent the results of 12 separate experiments. T h e slope of the line is 8.7 + 0.3 (10 6 molecules/cell, micromolar).

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FIGUR~ 2. Phloretin binding to human hemoglobin (x) and to hemoglobin-free red cell ghosts (O). Bound phloretin (10 6 molecules per equivalent cell) is plotted as a function o f free phloretin concentration (micromolar). The straight line represents the phloretin binding to intact red cells from Fig. 1. T h e data points for hemoglobin are results o f two sets of dialysis experiments. Ghost data points are from 11 separate experiments on three different ghost preparations.

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• 1976

phloretin b i n d i n g capacity o f red cells is lost w h e n h e m o g l o b i n a n d the o t h e r intracellular constituents are r e m o v e d , a n d that this lost binding can be acc o u n t e d for by phloretin b i n d i n g to h e m o g l o b i n . T h i s implies that p h l o r e t i n is able to p e n e t r a t e the red cell m e m b r a n e . T h e fact that the total phloretin b i n d i n g to ghosts is a small fraction o f the binding to red cells means that studies o f the b i n d i n g o f phloretin to ghosts may reveal specific, saturable b i n d i n g sites which were undetectable in r e d cells because o f the large h e m o g l o b i n contribution. T h e Scatchard plot o f phloretin binding to red cell ghosts shown in Fig. 3 has been m a d e f r o m the data for ghosts in Fig. 2. It reveals at least two classes of saturable b i n d i n g sites for p h l o r e t i n on the m e m b r a n e . T h e steeply sloping left-hand region o f the plot indicates the presence o f high affinity binding sites. T h e m o d e r a t e l y sloping m i d r e g i o n o f the plot indicates saturable binding sites o f lower affinity. At the u p p e r m o s t b o u n d levels, the b o u n d / f r e e ratio tends toward a constant, suggesting that t h e r e is also a n o n s a t u r a b l e binding process. As shown in the horizontal line, the contribution o f this a p p a r e n t l y n o n s a t u r a b l e process is small except at the highest phloretin concentrations. T h e b i n d i n g p a r a m e t e r s for the two saturable proc-

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FIGURE 3. Scatchard plot of phloretin bincling to hemoglobin-free red cell ghosts. The abscissa is bound phloretin (106 molecules per equivalent cell), and the ordinate is bound phloretin (106 molecules per equivalent cell) divided by a free phloretin concentration (micromolar). The separation of the Scatchard plot into high affinity, low affinity, and nonsaturable binding processes is discussed in the text, and the curve through the data represents the sum of the three separate binding processes shown. Data were obtained using both direct absorbance assay (×) and ether extraction assay (+).

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Interaction between Phloretin and Red Cell Membrane

esses were d e t e r m i n e d by a n o n l i n e a r regression analysis o f the data (after the n o n s a t u r a b l e c o m p o n e n t was estimated a n d subtracted f r o m the data): the high affinity sites n u m b e r 2.5 - 1.5 × 10 e p e r cell a n d have an a p p a r e n t dissociation constant for p h l o r e t i n o f 1.5 + 1 × 10 -6 M. T h e low affinity sites are m u c h m o r e n u m e r o u s : 5.5 - 0.5 × l0 T p e r cell, with an a p p a r e n t dissociation constant o f 5.4 -+ 0.5 × 10 -5 M. T h e large u n c e r t a i n t y in the total n u m b e r o f high affinity sites is a consequence o f the very m u c h l a r g e r n u m b e r o f low affinity sites, which m e a n s that a 10% e r r o r in the n u m b e r o f low affinity sites causes a m u c h h i g h e r uncertainty in the n u m b e r o f high affinity sites.

Phloretin Binding to Red Cell Lipids We m e a s u r e d the b i n d i n g o f phloretin to sonicated total lipid extracts o f r e d cell ghosts to d e t e r m i n e the contribution o f m e m b r a n e lipids to the total phloretin b i n d i n g by ghosts. As in the o t h e r b i n d i n g e x p e r i m e n t s , the phloretin b i n d i n g to lipid was d e t e r m i n e d at 22 - 2°C, p H 6, in isotonic p h o s p h a t e b u f f e r . A Scatchard plot o f the results is shown in Fig. 4, using 4.01 × 10 -]° # m o l lipid p h o s p h o r u s p e r ghost (24) to n o r m a l i z e the data to the same coordinates used in Fig. 3. T h e e x p e r i m e n t a l points fall on a single straight line, indicating that the binding is a simple, saturable process, with an a p p a r e n t dissociation constant o f 4.4 --- 0.3 x 10 .5 M a n d a total n u m b e r o f 5.2 - 0.2 × l0 T sites p e r equivalent cell. At m a x i m u m binding, t h e r e is o n e phloretin molecule for every 8.5 lipid molecules, including cholesterol. T h e b i n d i n g p a r a m e t e r s do not differ significantly f r o m those o f the low affinity b i n d i n g process f o u n d in ghosts. T h u s the low affinity sites can be identified with red cell lipids. T h e high affinity b i n d i n g is absent in r e d cell lipids, indicating that the high affinity b i n d i n g sites for phloretin are associated with m e m b r a n e proteins.

pH6, 22%

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FIGURE 4. Scatchard p l o t o f p h l o r e t i n b i n d i n g to sonicated r e d cel] lipids, in the same c o o r d i n a t e s as in Fig. 3. Each p o i n t is the result o f a single H u m m e l - D r e y e r

experiment. Two different lipid preparations were used. The solid line is the leastsquares fit for the experimental points. The dashed line is the Scatchard plot of the low affinity binding of phloretin to red cell ghosts, obtained from Fig. 3.

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67 • 1976

Rate of Phloretin Permeation O u r results show that only a small fraction o f the phloretin binding by red cells is associated with the m e m b r a n e . T h e rest o f the binding can be accounted for by phloretin binding to hemoglobin, indicating that phloretin is able to p e n e t r a t e the red cell m e m b r a n e . This result contradicts the conclusion o f Ben~s et al. (25) that the red cell m e m b r a n e is i m p e r m e a b l e to phloretin. T h e y based their conclusion in part on monosaccharide transport inhibition experiments which indicated that external phloretin has no effect on monosaccharide influx into ghosts. T a v e r n a and L a n g d o n (26), however, have shown that external phloretin strongly retards glucose entry into red cell ghosts. Ben~s et al. present f u r t h e r evidence derived from phloretin binding and trapping experiments which show phloretin binding to intact red cells to be similar in m a g n i t u d e to that in ghosts. This result is at variance with o u r results and also with earlier observations by LeFevre and Marshall (12), who r e p o r t e d that ghosts partially depleted o f hemoglobin bind much less phloretin than do intact cells. T h e belief that phloretin does not p e n e t r a t e the red cell m e m b r a n e was also based on the observations that phloretin uptake by red cells is rapid (12, 25) and that phloretin effects are exerted almost immediately after it is a d d e d to red cell suspensions (22, 27). O u r a r g u m e n t that phloretin penetrates the m e m b r a n e would be f u r t h e r s t r e n g t h e n e d if (a) a time-course o f phloretin uptake were measured, and (b) this time-course is shown to be m u c h slower than the rate o f phloretin binding to the m e m b r a n e surface. Initial e x p e r i m e n t s showed, as r e p o r t e d earlier by LeFevre and Marshall (12), that the phloretin uptake by red cells is too rapid to be studied by conventional centrifugation techniques. We f o u n d that the phloretin uptake is complete 2 min after the cells are suspended in the phloretin-containing solution. It was necessary, t h e r e f o r e , to employ techniques which are capable o f m o r e rapid sampling of the extracellular m e d i u m . T h e first m e t h o d we used was rapid centrifugation in a B r i n k m a n n centrifuge 3200, which is capable o f sedimenting red ceils in less than 3 s. By this m e t h o d we obtained a half time o f 5-10 s for the uptake o f phloretin by red cells (4% hematocrit), although there was a 2-s uncertainty in the exact times the cells were pelleted. T h e second m e t h o d for sampling the s u p e r n a t a n t was the Millipore filtration technique e m p l o y e d by Dalmark and Wieth (20) to measure anion efflux f r o m red cells. By this m e t h o d , the time points could be obtained with an e r r o r o f less than 1 s. However, phloretin adsorbs to the Millipore filters, creating an uncertainty in the phloretin concentration in the m e d i u m , a n d , t h e r e f o r e , in the half time for the uptake. T h e half time for phloretin penetration obtained by this m e t h o d was between 5 and 15 s, in a g r e e m e n t with the centrifugation m e t h o d . A more precise m e t h o d for following the phloretin uptake was based on the fact that phloretin is a weak acid (pK ~ 7.3). It is the undissociated form which is taken up by the cells. T h e r e f o r e , if red cells are s u s p e n d e d in an u n b u f f e r e d solution at a p H near the pK o f phloretin, the extracellular p H will rise. This p H rise results f r o m the fact that the acid (undissociated) f o r m o f phloretin is leaving the u n b u f f e r e d extracellular m e d i u m , which is the equivalent o f adding base to the m e d i u m . Specifically, suppose that initially 200 /~M phloretin is present,

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u n b u f f e r e d , at, for e x a m p l e , pH0 = 6.9. At this p H , the ratio o f u n c h a r g e d phloretin (HA) to c h a r g e d phloretin (A-) is

HA/A-=

10 pK-pHo = 107.3-6.9

=

100.4

(1)

So, initially, t h e r e is 200/[1 + 10 -°'4] = 143 /zM u n c h a r g e d phloretin, a n d 57/xM c h a r g e d phloretin. I f red cells (whose equilibrium external p H is 6.90, unb u f f e r e d ) are s u s p e n d e d in this m e d i u m , they will take u p an a m o u n t , X, o f the u n c h a r g e d p h l o r e t i n , so that the final free c o n c e n t r a t i o n o f phloretin is 200 - X, a n d the final c o n c e n t r a t i o n o f u n c h a r g e d phloretin is 143 - X. T h e new p H is given by 10pK-pH = H A ~ A - = [143 - X]/57.

(2)

I f we define the c h a n g e in extracellular p H as Y = p H - 6.90, Eq. 2 becomes 10°'4-Y = [143 - X]/57.

(3)

I f Y is small, 10 - r can be e x p a n d e d to 10 -v = e -2"3v ~ 1 - 2.3Y,

(4)

a n d when Eq. 4 is substituted into Eq. 3 the result is Y = X/330.

(5)

So Y is a linear function o f X, which m e a n s that the time-course o f the u p t a k e X is the s a m e as the time-course o f the p H change Y. Note that this m e t h o d assumes that changes in the extracellular p H are i n d e p e n d e n t o f the s t r o n g b u f f e r i n g capacity o f the cell contents. This is not generally the'case in a r e d cell e x p e r i m e n t , because r a p i d chloride-bicarbonate e x c h a n g e p r o d u c e s p H equilibration between inside a n d outside media. H o w ever, phloretin itself is a p o w e r f u l inhibitor o f anion e x c h a n g e (6), a n d t h e r e f o r e should inhibit p H equilibration. In fact, we have f o u n d that in the p r e s e n c e o f 200 t~M p h l o r e t i n , the half time f o r p H equilibration is 10 min or l o n g e r (28), which is quite slow on the time scale o f the phloretin u p t a k e e x p e r i m e n t s . T h e r e f o r e , d u r i n g the phloretin influx, the external p H is i n d e p e n d e n t o f the intracellular p H . T h e phloretin influx e x p e r i m e n t s using the p H m e t h o d were carried out as follows. Red cells were washed f o u r times in 154 m M NaCI, a n d titrated with HCI to an equilibrium external p H o f 6.90. T h e s e cells were s u s p e n d e d in an u n b u f f e r e d , 200 g M phloretin solution in 154 m M NaCI, p H 6.90, 26 - I°C. T h e extracellular p H was m e a s u r e d by a glass electrode, p H m e t e r , a n d strip c h a r t r e c o r d e r . T h e e x t e r n a l p H , s u b s e q u e n t to the addition o f the cells, rises with a single e x p o n e n t i a l time-course (t = 10-20 s) to a value between 7.05 a n d 7.25, d e p e n d i n g on the n u m b e r o f cells a d d e d , a n d then falls with a m u c h slower time-course toward the equilibrium p H o f 6.90 (Fig. 5). T h e p H d r o p toward 6.9 results f r o m the fact that the b u f f e r i n g p o w e r o f the cell contents is s t r o n g e r than that o f the phloretin, so that the equilibrium p H is specified mainly by the cell contents. In o r d e r to c o n f i r m f u r t h e r that the time-course o f the p H rise r e p r e s e n t s

390

T H E JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 6 7

1976

Ceils

pH ~0

6.9 410

810

JJ

TIME (s)

20(3

3(~0

4~)0

FIGURE. 5. Time-course of the extracellular pH of an unbuffered 200 p.M phloretin solution to which red cells are added (final hematocrit 0.0092). The rapid pH rise represents the uptake of undissociated phloretin. The slower pH drop toward 6.9 results from pH equilibration (by CI-HCOa exchange) between the extracellular medium and the heavily buffered cell contents. The ceils had been previously titrated to an equilibrium extracellular pH of 6.9. p e n e t r a t i o n o f phloretin t h r o u g h the m e m b r a n e r a t h e r t h a n the time-course o f surface binding, we tried, using the same m e t h o d , to m e a s u r e the rate o f phloretin b i n d i n g to sonicated p h o s p h a t i d y l choline vesicles. Addition o f vesicles to the phloretin solution resulted in a p H rise the time-course o f which was not distinguishable f r o m a r a p i d pulse o f N a O H . T h a t is, the time-course o f the phloretin binding to the surface o f the lipid m e m b r a n e s is too r a p i d to be m e a s u r e d by this m e t h o d . T h e m u c h slower p H rise resulting f r o m red cell additions t h e r e f o r e does not r e p r e s e n t surface binding. T h e rate constant for the p H rise (and hence for the phloretin uptake) is shown in Fig. 6 as a function o f hematocrit. T h e rate is a linear function o f h e m a t o c r i t with a positive slope a n d positive intercept. I f the line is e x t r a p o l a t e d to a 4% h e m a t o c r i t (the h e m a t o c r i t used in the r a p i d centrifugation experiments), the rate constant is 0.125 -+ 0.013 s -~. This c o r r e s p o n d s to a half time o f 5.5 +- 0.5 s, in a g r e e m e n t with the centrifugation m e t h o d . T h e reason that the rate constant d e p e n d s on h e m a t o c r i t even at low cell concentrations is that the majority o f the intracellular phloretin is b o u n d to h e m o g l o b i n . F r o m the m e a s u r e d c e l h m e d i u m c o n c e n t r a t i o n ratio for unc h a r g e d p h l o r e t i n (120) a n d the m e a s u r e d b i n d i n g o f phloretin to the m e m b r a n e (10% o f the total cell binding), it is i n f e r r e d that the total intracellular phloretin concentration is a b o u t 108 times the free ( u n c h a r g e d ) phloretin concentration. This m e a n s that the intracellular pool o f phloretin is a b o u t 108 times larger (per unit volume) than the extracellular pool o f phloretin. Since cell water constitutes a b o u t 70% o f the cell v o l u m e , the internal c o m p a r t m e n t p e r unit solvent v o l u m e is 155 times as large as the external pool.

JENNINGSA N D SOLOMON InteractionbetweenPhloretinandRedCellMembrane

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0.~2 k(s~ /

O.OE

/

/

/

004

0

'

'

062

HEMATOCRIT

FIGURE 6. Rate constant (s-I) for phloretin uptake by red cells, plotted as a function of suspension hematocrit. T h e solid line is a least-squares fit of the data.

I f there were no binding o f phloretin to the hemoglobin, the rate constant in s -1 for the uptake would be given by k = [PA][1]V,, + 1/Vcw],

(6)

where P is the permeability coefficient (centimeters per second), A is the memb r a n e area (square centimeters), Vcw is the volume o f cellular water (cubic centimeters), and Vm is the v o l u m e o f extracellular water (cubic centimeters). However, the b o u n d phloretin effectively increases Vow by a factor o f 155, so the actual rate constant for uptake is k = [PA][1/V,, + 1/155 Vcwl,

(7)

k = [PA/155 Vew][1 + 155 Vcw/Vm].

(8)

which can be written

Since P and A/Vow a r e constants, Eq. 8 shows that the rate o f phloretin u p t a k e should be a linear function o f the ratio Vcw/Vm. T h e slope divided by the intercept is 155. I f the data in Fig. 5 are plotted as K vs. Vcw/Vm, the slope:intercept ratio is 114 -+ 30, which is in reasonable a g r e e m e n t with the predicted value o f 155. T h e intercept is k = 0.030 s -~. T a k i n g A/Vcw = 2.33 x 104 cm -1 (22), P = 2.0 x 10 -4 cm/s. This permeability coefficient is comparable to the permeability coefficient o f the red cell m e m b r a n e for urea (3 x 10 -4 cm/s; 22) and is p e r h a p s surprisingly high for a solute whose molecular weight is 274. H o w e v e r , phloretin is very lipid soluble, as shown by its large binding by red cell lipids. Consistent with this high lipid solubility is the fact that phloretin has ether-water and octanol-water partition coefficients in excess o f 100 (28). It is this high lipid solubility which is responsible for the comparatively large permeability. Relationship Between Phloretin Binding and Transport Inhibition O u r results show two classes o f saturable binding sites for phloretin on the erythrocyte m e m b r a n e : high affinity protein sites and low affinity lipid sites.

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Hence, the various phloretin effects may be classified on the basis of the concentrations at which they occur. T h e low affinity lipid binding sites are half saturated with phloretin at 50/zM free phloretin at pH 6 (about 100/zM at pH 7.4), so that phloretin effects observed at concentrations of 100 ttM or above at pH 7.4 may be attributed to phloretin binding to the lipid region of the membrane. At high concentrations (100 ttM and above), ph!oretin has an accelerating effect on the transport o f several iipophilic nonelectrolytes (29), valinomycinmediated K exchange, and acetate self-exchange (6) across the red cell membrane. At these concentrations the low affinity binding dominates, indicating that these accelerating effects result from phloretin action on the lipid region of the membrane. T h e r e are two mechanisms by which phloretin may interact with the membrane lipids. It has been suggested by Cass et al. (30) that phloretin may change the electrostatic configuration of the lipid polar head groups. This mechanism was proposed to account for the fact that phloretin increases carriermediated cation conductance but decreases the conductance of lipophilic anions across planar lipid bilayer membranes. From the increase in membrane permeability to noncharged solutes a second mechanism may be inferred: an increase in the fluidity of the hydrocarbon chains of the lipid molecules. This increase is consistent with the observation of Cass et al. of an increased carrier-mediated cation conductance. A fluidity increase is also consistent with the fact that phloretin protects red cells against hypotonic lysis (28), as do anesthetics (31). In the case o f anesthetics this protection against lysis is believed to result from a hydrophobic expansion of the membrane interior, resulting in an increased membrane fluidity (32). Further evidence of the effect of phloretin in fluidizing the membrane is afforded by the results of Cass et al. (30) who have observed that phloretin at 100/zM doubles the permeability of lecithin-cholesterol bilayers to acetamide. Similar effects have been observed by Poznansky et al. (33) on the permeability of egg lecithin spherical bilayers to urea, formamide, acetamide, and valeramide. Phloretin effects which occur at concentrations lower than 10-20/xM at pH 7.4 are probably mediated by protein rather than lipid, since only 20% or less of the lipid binding sites are occupied at these concentrations. T he protein binding sites for phloretin number between 1 and 4 million per cell and exhibit an apparent dissociation constant of 0.5-2.5/~M at pH 6 (about 1-5/xM at pH 7.4). It is of interest to enumerate those transport processes which are associated with these high affinity binding sites. Phloretin inhibits red cell glucose transport competitively with an apparent K1 of 0.5-2.5/xM (34, 12). Under the conditions of our binding experiments (pH 6, 22 ± 2°C), we have found that phloretin inhibits glucose efflux from red cells half maximally at 0.6/~M (28). This is in reasonable agreement with the dissociation constant for the high affinity sites. Some subset of these sites is therefore responsible for the action of phloretin on glucose transport. Since phloretin is a competitive inhibitor of glucose transport, large concentrations of glucose should be able to displace phloretin from the glucose transport system. In two experiments, however, we have found that 100 mM glucose is unable to displace

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detectable a m o u n t s o f phloretin f r o m the m e m b r a n e at free phloretin concentrations o f 0.42 a n d 0.6 /~M. C u r r e n t estimates o f the n u m b e r o f glucose transport sites per cell range f r o m 2 x 105 to 3.3 × 105 (35-37). T h e presence o f a total o f 300,000 glucose-inhibitable phloretin binding sites would have caused only a 10% reduction in phloretin binding at these concentrations. O u r experimental variation in such e x p e r i m e n t s is about I0%, so 300,000 glucose-inhibitable phloretin binding sites could be present but not detected. A n o t h e r phloretin effect which occurs at low concentrations is inhibition o f anion exchange across the red cell m e m b r a n e . At p H 7.7, 0°C, half-maximal inhibition o f chloride e x c h a n g e occurs at about 2 /~M (6). At p H 6.2, 23°C, phloretin inhibits chloride-bicarbonate exchange half maximally at 3-5 ~M (28). T h e fact that phloretin inhibits anion transport at such low concentrations indicates that some o f the high affinity phloretin sites are related to anion transport. Cabantchik and Rothstein (38) have shown that there are 300,000 anion t r a n s p o r t systems on the h u m a n red cell m e m b r a n e , and that anion transport is associated with a single m e m b r a n e polypeptide. T h e r e f o r e , 300,000 o f the high affinity sites for phloretin may be attributed to the anion t r a n s p o r t protein. T h e effect of phloretin on u r e a transport is less well d e f i n e d than that on glucose t r a n s p o r t and anion exchange. Most authors r e p o r t an inhibition (7, 29), t h o u g h Owen et al. (27) r e p o r t an acceleration at concentrations up to about 40 /~M and an inhibition at higher concentrations. Wieth et al. (22) and Kaplan et al. (39) have p r e s e n t e d evidence that there are two t r a n s p o r t pathways for urea across the h u m a n red cell m e m b r a n e . O n e is not saturable, is not inhibited by phloretin, and is t h o u g h t to be a result o f passive diffusion t h r o u g h the lipid portion o f the m e m b r a n e . T h e o t h e r pathway is saturable, at least for t h i o u r e a , exhibits mutual competition between urea and thiourea, and is inhibited by phloretin. Wieth et al. (22) have shown that the saturable pathway (in the case o f thiourea transport) is over 95% blocked by 250/~M phloretin at p H 7.2, suggesting that half-maximal inhibition occurs at concentrations well below 50/2M. In separate e x p e r i m e n t s in o u r own laboratory we have d e t e r m i n e d a K~ o f about 10 /.tM at p H 7 for phloretin inhibition o f thiourea efflux (28). This K l is m u c h lower than the a p p a r e n t dissociation constant for phloretin binding to red cell lipids, indicating that inhibition by phloretin o f thiourea t r a n s p o r t is not the result o f its action on the lipid, but r a t h e r results f r o m phloretin binding to a protein c o m p o n e n t o f the m e m b r a n e . T h e 2.5 - 1.5 × 106 high affinity sites for phloretin, t h e r e f o r e , r e p r e s e n t a h e t e r o g e n e o u s g r o u p o f transport systems, which include about 300,000 anion transport proteins, about 300,000 glucose transport proteins, and an u n k n o w n n u m b e r o f thiourea t r a n s p o r t pathways. In addition, the high affinity sites may include the transport pathways for the small hydrophilic nonelectrolytes glycerol, f o r m a m i d e , a n d acetamide, whose penetration across the h u m a n red cell m e m b r a n e is also inhibited by phloretin (7, 29). A complete dose-response relationship for phloretin inhibition o f the p e r m e a t i o n o f these solutes is necessary in o r d e r to establish w h e t h e r the high affinity phloretin sites are responsible. T h e detailed mechanism o f the action o f phloretin on these transport systems

394

THE JOURNAL OF GENERAL PHYSIOLOGy ' VOLUME 67 ' 1976

is as yet unclear. T h e phloretin binding is noncovalent, in contrast to the binding of amino or sulfhydryl reagents to m e m b r a n e proteins. T h e amphipathic character o f phloretin as well as the ability o f phenolic c o m p o u n d s to f o r m h y d r o g e n bonds are likely the i m p o r t a n t d e t e r m i n a n t s o f phloretin inhibition o f proteinmediated t r a n s p o r t processes. Because phloretin inhibits processes which are believed to be protein mediated (e.g., glucose t r a n s p o r t and anion exchange) and accelerates passive diffusion o f several solutes across the lipid region o f the m e m b r a n e , it is difficult to i n t e r p r e t phloretin effects on the transport o f solutes which may penetrate the m e m b r a n e by two pathways. In the case o f thiourea, phloretin inhibits the saturable fraction o f the transport but not the nonsaturable fraction. At 500 mM thiourea, i.e., when the nonsaturable transport dominates, 250 p.M phloretin has no effect on the transport rate, even t h o u g h it has completely inhibited the saturable fraction o f the flux (22). Phloretin, then, may a p p e a r to have very little effect on the transport o f a given substance because it inhibits one fraction o f the flux and accelerates the other. This may be the case for water, whose p e r m e a t i o n t h r o u g h the red cell m e m b r a n e is not affected by 250 or 500/xM phloretin (29, 7). T h e recent results o f Brown et al. (40) implicate a m e m b r a n e protein in the water p e r m e a t i o n process. T h e permeability coefficient o f water t h r o u g h lipid bilayers o f various compositions has been f o u n d to be o f the o r d e r o f 1.3 × 10 - a c m s -1 (41, 42) which is about 40% o f the diffusion permeability coefficient o f the h u m a n red cell to water. This indicates that t h e r e may be significant contributions to water transport across the m e m b r a n e f r o m both lipid and protein components. Since Poznansky et al. (33) have shown that 250/zM phloretin doubles the permeability o f egg lecithin bilayers to urea and short chain aliphatic amides, phloretin may also increase the i m p o r t a n c e o f the lipid pathway for water diffusion t h r o u g h the h u m a n red cell m e m b r a n e . T h e lack o f an effect on water transport by 250 /zM phloretin, t h e r e f o r e , could result f r o m an increase in the lipid fraction o f the flux and an inhibition o f the p r o t e i n - m e d i a t e d fraction. In o r d e r to establish whether this is the case, it will be necessary to c o m p a r e the effect o f various phloretin concentrations on water t r a n s p o r t t h r o u g h both lipid bilayers and the red cell m e m b r a n e . A f u r t h e r complication in i n t e r p r e t i n g phloretin effects on red cell m e m b r a n e transport results f r o m the presence o f the nonsaturable phloretin binding sites shown in Fig. 3. Since the nonsaturable binding is not present in isolated lipids, it probably results f r o m nonspecific a t t a c h m e n t o f phloretin to m e m b r a n e proteins, similar to the nonsaturable binding o f phloretin to hemoglobin. At a phloretin concentration o f 200 /~M, p H 6, this protein binding represents roughly 30% of the total binding to the m e m b r a n e . This indicates that, although the majority o f the phloretin binding is to the lipid, the nonspecific protein binding could contribute to phloretin effects which occur at concentrations o f 900 /zM and above. T h u s , interpretation o f the action o f phloretin on cell m e m b r a n e s requires a detailed study o f the dose-response curve in o r d e r to identify the m e m b r a n e c o m p o n e n t s responsible for the phloretin effects. W e w o u l d like to e x p r e s s o u r g r a t i t u d e to D r . E. A. D a w i d o w i c z f o r his a s s i s t a n c e in t h e p e r f o r m a n c e

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of the Hummel-Dreyer experiments and to Dr. J. W. Peterson III, whose computer program was used to analyze the phloretin-ghost data. This work was supported in part by National Institutes of Health grants 5 R01 GM15692-08, 2 T01 GM00782-16 and 5 F01 GM48416-04. Receivedfor publication 29 July 1975. BIBLIOGRAPHY 1. CHAN, S. S., a n d W. D. LOTSPEICH. 1962. Comparative effects o f phlorizin and p h l o r e d n on glucose transport in the cat kidney. Am. J. Physiol. 203:975-979. 2. ALVARADO,F. 1967. Hypothesis for the interaction between phlorizin and phloretin with m e m b r a n e carriers for sugars. Biochim. Biophys. Acta. 135:483-495. 3. LEFEvRE, P. G. 1961. Sugar transport in the r e d blood cell: structure-activity relationships in substrates a n d antagonists. Pharmacol. Rev. 13:39-70. 4. CZECH, M. P., D. G. LYNN, and W. S. LYNN. 1973. Cytochalasin B-sensitive 2-Deoxyo-glucose t r a n s p o r t in adipose cell ghosts. J. Biol. Chem. 248:3636-3641. 5. BIHLER, I., H. M. CAVERT, and R. B. FISHER. 1965. A differential effect of inhibitors on sugar penetration into the isolated rabbit h e a r t . J . Physiol. (Lond.) 180:168-177. 6. WIETH, J. O., M. DALMARK,R. B. GUNN, a n d D. C. TOSTESON. 1972. T h e transfer o f monovalent inorganic anions t h r o u g h the r e d cell m e m b r a n e . In Erythrocytes, Thrombocytes, Leucocytes. E. Gerlach, K. Moser, E. Deutsch, and W. Wilmanns, editors. Georg T h i e m e Verlag, Stuttgart. 71-76. 7. MACEY, R. I., and R. E. L. FARMER. 1970. Inhibition of water and solute permeability in h u m a n red cells. Biochim. Biophys. Acta. 211:104-106. 8. LEVlNE, S., N. FRANKX, and R. M. HAYS. 1973. Effect of phloretin on water and solute movement in the toad bladder. J. Clin. Invest. 52:1435-1442. 9. CZECH, M. P., D. G. LYNN, and W. S. LYNN. 1973. Phlorizin-receptor interactions in fat cell plasma membranes. Biochim. Biophys. Acta. 323:639-642. 10. GLOSSMAN, H., and D. M. NEVILLE, JR. 1972. Phlorizin receptors in isolated kidney brush b o r d e r m e m b r a n e s . J . Biol. Chem. 247:7779-7789. 11. CHESNEY, R., B. SACKTOR, and A. KLE~NZELLER. 1974. T h e binding o f phloridzin to the isolated luminal m e m b r a n e o f the renal proximal tubule. Biochim. Biophys. Acta. 332:263-277. 12. LEFEVRE, P. G., and J. K. MARSHALL. 1959. T h e attachment o f phloretin a n d analogues to h u m a n erythrocytes in connection with inhibition o f sugar transport. J. Biol. Chem. 254:3022-3026. 13. DIEDRICH, D. F. 1968. Is phloretin the sugar transport inhibitor in intestine? Arch. Biochem. Biophys. 127:803-812. 14. DODGE, J. T., C. MITCHELL, and D . J . HANAHAN. 1963. T h e p r e p a r a t i o n and chemical characteristics of hemoglobin-free ghosts of h u m a n erythrocytes. Arch. Biochem. Biophys. 100:119-130. 15. STECK, T. L. 1974. Preparation of i m p e r m e a b l e inside-out and right-side-out vesicles from erythrocyte membranes. In Methods in Membrane Biology. Vol 2. E. D. Korn, editor. Plenum Press, New York. 245-281. 16. ELLMAN, G. L., K. D. COURTNEY, V. ANDRES,JR., and R. M. FEATHERSTONE. 1961. A new and rapid colorimetric determination o f acetylcholinesterase activity. Biochem. Pharmacol. 7:88-95. 17. HUMMEL, J. P., and W. J. DREYER. 1962. Measurement o f protein-binding p h e n o m ena by gel filtration. Biochim. Biophys. Acta. 63:530-532.

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