Cytochalasin B binding to rabbit proximal tubular basolateral membranes

Kidney International, Vol. 35 (/989), pp. 1290—1294

Cytochalasin B binding to rabbit proximal tubular basolateral

membranes PETER T. CHEUNG and MARC R. HAMMERMAN Renal and Endocrinology Divisions, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri, USA

Cytochalasin B binding to rabbit proximal tubular basolateral membranes. Cytochalasin B binds to the Na*independent D-glucose transporter in non-renal tissues. We have shown previously that the Naindependent D-glucose transporter of the rabbit renal proximal tubular cell is localized exclusively in the basolateral membrane. To determine whether cytochalasin B binds to this renal transporter we measured binding of [3Hlcytochalasin B to proximal tubular basolateral membranes isolated from rabbit kidneys. A steady state of binding is reached

by IS minutes at 20°C over a concentration range of 0.01 to 50 M. Non-linear regression analysis of cytochalasin B binding from 0.01 to 20

g plotted according to Scatchard reveals two classes of binding sites

with Kd 5.88 x I0 si, Bmax 16.1 pniol/mg protein; and Kd 5.62 X M,

l0-

2816 pmol/mg protein. [3H}cytochalasin B (0.1 .tM) binding to

basolateral membranes is a reversible process; it is displacable by excess unlabeled cytochalasin B with a time course similar to binding of [1H]cytochalasin B. Binding of [3Hlcytochalasin B is inhibited by 500 mM D-glucose (21%), 2-deoxy-D-glucose (57%) and 3-O-methyl-D-

glucose (64%), but not by L-glucose. [3H]cytochalasin B binding is reduced 71% by 0.1 msi phloretin, but only 26% by 0.1 mt phlorizin. Such substrate specificity and inhibitor sensitivity are similar to those previously demonstrated in non-renal tissues by others as well as in rabbit renal proximal tubular basolateral membranes by us. Our data suggest that cytochalasin B binds to the Na-independent D-glucose transporter or a component of the transporter in the renal proximal tubular basolateral membrane.

transporter but not the luminal Nat-glucose co-transporter. Phlorizin at 10 M inhibits the luminal, but not the antiluminal transporter, while phloretin and cytochalasin B, a compound isolated from the mold Helminthosporin dematioideum, at the same concentration are inhibitory to the antiluminal, but not the luminal D-glucose transport. The lack of Na-dependency, the substrate specificity, and sensitivity to cytochalasin B of the antiluminal D-glucose transporter are similar to characteristics described for Na-independent transporters in non-polar cells such as erythrocytes [5—7], adipocytes [8, 9], and in the basolateral membrane of the polar intestinal enterocyte [10, 11]. Cytochalasin B binds to multiple sites in membranes prepared from these cells. Binding to sites of the highest affinity correlates with inhibition of D-glucose

transport. These sites are thought to correspond to the Naindependent D-glucose transporter itself, or to a component of the transporter [5—9]. Binding of cytochalasin B to high affinity receptors in membranes prepared from erythrocytes and adi-

pocytes has been a useful tool for characterization of Na-

independent D-glucose transport activity. Silverman has reported preliminary data that show binding of cytochalasin B to basolateral membranes prepared from dog kidney [121. Binding to multiple sites was suggested, however, The renal proximal tubular cell is a polar epithelial cell, the details such as kinetic constants relating to binding were not luminal and antiluminal membranes of which serve different presented. Accordingly, the present studies were conducted to transport functions. Reabsorption of D-glucose from glomerular further characterize the binding of cytochalasin B to isolated ultrafiltrate to peritubular capillary blood is effected through the proximal tubular basolateral membranes. Our data are similar activities of two different types of transporters. D-glucose to findings in non-renal cell membranes and are consistent with crosses the luminal brush border membrane through the action the identity of the high-affinity cytochalasin B binding site in of a Natdependent co-transporter and enters the blood across proximal tubular basolateral membranes as the Na-indepenthe antiluminal basolateral membrane via a Na-independent dent D-glucose transporter, or a component of the transporter. transporter. Each of these processes has been characterized in Methods membrane vesicles isolated from proximal tubular cells. Data reported from a number of laboratories [1—3], including our own Basolateral membranes from kidneys of female New Zealand [4], have shown that the luminal and antiluminal D-glucose White rabbits (2.5 kg) were used in these experiments. The transporters differ not only in their Na-dependency but also in kidneys were removed surgically following pentobarbital-intheir substrate specificity and inhibitor sensitivity. Thus, the duced anaesthesia and basolateral membranes prepared exactly two hexose analogues, 2-deoxy-D-glucose and 3-0-methyl- as previously described using a Percoll density gradient ultraD-glucose are substrates only for the antiluminal D-glucose centrifugation technique. A complete enzymatic characterization of this preparation has been published [4]. Enrichment for activity of the basolateral membrane marker enzyme, Na*Kf ATPase, in basolateral membrane suspensions over activity in Received for publication May 25. 1988 and in revised form August 12, 1988 cortex homogenates is approximately 17-fold. Parathyroid horAccepted for publication December 28. 1988 mone-stimulated adenylate cyclase activity of the basolateral membrane is enhanced 6.8-fold over that of crude homogenate © 1989 by the International Society of Nephrology 1290

1291

C/,eung and Ha,nmer,nan: Cvtochalasin B binding to hasolateral membranes

which is consistent with the proximal tubular origin of the preparation [4]. During the last two centrifugations of the isolation procedure, basolateral membranes were suspended in a Krebs-Ringers phosphate buffer containing 125 mM NaCI, 5 mM KCI, 1.5 mri CaCl2, 1.2 mivi MgSO4, and 10 mt Na2HPO4

at pH 7.5. Membranes were stored at —70°C and thawed immediately prior to use in experiments. Binding of [4-3H] cytochalasin B (18.5 Ci/mmol, New England Nuclear, Boston, Massachusetts, USA) to isolated basolateral membranes was measured using a rapid centrifugation technique. Basolateral membranes were suspended in 100 1d of Krebs-Ringers phosphate buffer containing 0.1% bovine serum albumin (to reduce "background" radioactivity), [3HJcytochalasin B (10,000 to 60,000 cpm) and varying concentrations of unlabeled cytochalasin B. Within a given series of experiments,

0.20

C C

0.16 0.12

cc

cc -C U

0 0.08 >-

I 0

0.04

0

10

20

30

40

50

60

Time, minutes

either 0.05 or 0.10 mg of protein was used. Binding of

1. Binding of [3HJcvtochalasin B to renal basolateral membranes [3H]cytochalasin B, as expressed in pmol/mg protein, is unaf- Fig. as a function of time of incubation. Data are expressed as the ratio of

fected by the amount of membrane protein present in the

incubation medium over this range. Cytochalasin B was dissolved in ethanol such that the final concentrations of ethanol in the suspensions were 0.4 to 1.0%. Within a given series of experiments, the concentration of ethanol was always the same. Binding assays were performed in triplicate in 1.5 ml polypropylene Eppendorf tubes. Following incubation at 10°C for varying periods of time, tubes were centrifuged for one minute in a Beckman microfuge (Beckman Instruments, Fullerton, California, USA). Supernatants containing unbound radioactivity were discarded and the remaining membrane pellets were solubilized in 100 M' of 1.5% sodium-dodecyl sulfate. Radioactivity in solubilized pellets was counted using liquid scintillation

spectrometry. The recovery of basolateral membrane protein from the pellets was approximately 65% for both 0.05 mg and 0.10mg samples. Data are expressed as binding per mg protein (measured for each experiment) and reflect protein present in pellets. Identical experiments were performed in the absence of basolateral membranes to determine background radioactivities. Aqueous space within the membrane pellets was determined by measuring [fructose-l-3H(N)-sucrose (11.4 Ci/mmol, New England Nuclear) "trapped" within pellets for each experiment. Measurements of [3H]cytochalasin B bound to membranes were corrected for both background radioactivity (4.4% of total) and "trapped" radioactivity (1.2% of total). Protein measurements were made by the method of Lowry et al [13] using bovine serum albumin as a standard. Results Binding of 0.01, 0.1 and 50 MM [3H]cytochalasin B to baso-

[3Hlcytochalasin B cpm bound/total cpm added to incubations and represent the mean SE of 3 experiments. Symbols are: (D 0.01 /SM; (A) 0.1 ,sM; (0) 50 tM.

pmol/mg protein; and Kd 5.62 x IO M, Bmax 2816 pml/mg protein (N 3). No concentration-independent non-specific binding component was identified which confirms the adequacy of correction for background and "trapped" radioactivities. For all subsequent binding experiments, the concentration of 0.1 MM [3H]cytochalasin B was used as this approximates the K of the high affinity site. At this concentration, the occupancy of the high affinity binding site is estimated to be 59% while that

of the second site is 0.1%.

To determine whether binding of [5H]cytochalasin B to basolateral membranes is a reversible process, 10 MM unlabeled

cytochalasin B was added to membranes that had been equilibrated with 0.1 MM [1H]cytochalasin B and binding of radioactivity to membranes was measured over time following addition. As a control, an equal volume of ethanol-containing buffer

was added. As shown in Figure 3, "displacement" of [3H]cytochalasin B was reversible with a time course similar to

that of binding. Maximal displacement was attained by IS minutes following addition of unlabeled cytochalasin B. The effect of varying concentrations of known glucose trans-

port inhibitors, phlorizin, phloretin, cytochalasin B, and its analogue cytochalasin E, on steady state binding of 0.1 MM 13H]cytochalasin B to basolateral membranes is illustrated in Figure 4. Data are expressed as percent [3H]cytochalasin B

lateral membranes was determined as a function of time of incubation at 20°C. At each concentration, a steady state of binding was reached by 15 minutes of incubation (Fig. I). Accordingly, binding of [3H]cytochalasin B measured at IS

bound in the presence of inhibitor divided by that in the absence of inhibitor (B/Bo). Binding of [3H]cytochalasin B is inhibited more completely by phloretin than by phlorizin. Thus binding is 16% by io— M phloretin (N 4, P < 0.01 by reduced 71 Dunnett's multiple comparison procedure [15]), but only 26

minutes was used to reflect steady state binding in subsequent experiments. Steady state binding to basolateral membranes of varying concentrations of cytochalasin B (0.01 to 20 MM) were plotted according to Scatchard (Fig. 2). Non-linear regression analysis of data was conducted using an interactive computer program in order to isolate all binding sites as described by Munson and Rodbard [14]. The result of such analysis is most compatible with a two-binding site model: K 5.88 x lO_8 M. 16.1

[3H]cytochalasin B binding is inhibited to a greater extent by cytochalasin E and by unlabeled cytochalasin B itself than by either phlorizin or phloretin. In order to ascertain whether binding of [3H]cytochalasin B to basolateral membranes is affected by D-glucose or hexose analogues, we measured binding of 0.1 MM [3H]cytochalasin B in the presence of 500 mrvi L-glucose, D-glucose, 2-deoxyD-glucose or 3-O-methyl-D-glucose. In preliminary experi-

10% by the same concentration of phlorizin (P =

NS).

1292

Cheang and Hammerinan: Cytochalasin B binding to ha.colateral membranes

0.3

0 0.2

0.3

5: 0

C

0

.

0.1

0.2

a) a)

HTII I

5: C

0

0

50

100

150

0.1

[

Fig. 2. Scatchard plot of HJcytochalasin B

0

binding to basolateral membranes. Data

EIJ 0

400

200

0

200

600

Bound, pmol/mg protein

represent concentration range from 0.01 to 20 /LM. Data from 3 experiments are pooled. Inset: 2-site model generated by non-linear regression of binding data. Kdl = 5.88 M; B,,,,x1 = 16.1 pmol/mg; Kd2 = 5.62 x M; B2 = 2816 pmol/mg.

x 10

0.08 0)

0.06 OC

-o a)

0.04

a) C.)

0

I C.,

0.02

0

10

20 Time, minutes

30

40

Fig. 3. Binding of [3HJcytochalasin B (0.! M) to basolateral membranes as a function of time of incubation. Data are expressed as the ratio of 13H]cytochalasin B cpm bound/total cpm added to incubations and represent the mean SE of 3 experiments. Symbols are: (U) control; (A) 10 M Cyto B.

ments we determined that binding was not affected by 500 mi L-glucose (12.9 0.7 vs. 12.9 1.1. pmol/mg protein, N = 4, P = NS). However, as shown in Figure 5, binding is significantly inhibited by 500 mrvi D-glucose (P < 0.01), 2-deoxyD-glucose (P < 0.01), and 3-O-methyl-D-glucose (P < 0.01) (Dunnett's multiple comparison procedure) in comparison with L-glucose. The magnitude of inhibition by D-glucose (21 3%) is less than that effected by 2-deoxy-D-glucose (57 2%) or by 3-O-methyl-D-glucose (64

1%).

Discussion

The exit of D-glucose across the basolateral membrane of the

renal proximal tubular cell occurs through the activity of a Na-independent transporter [12]. The Na'-independent na-

0

io

iO-

iO-

Concentration, M

Fig. 4. Binding qf /3HJcytocI,alasin B (0.1 p.s) to basolateral membranes. Data are expressed as mean SE of 4 experiments. Symbols

are: ( phlorizin; (A) phloretin; (LI) Cyto B: (A) Cyto E.

ture of this transport process is similar to that in the basolateral membrane of intestinal epithelial cells and plasma membranes

of a variety of non-renal cells such as erythrocytes and adipocytes [2, 5—12]. Cytochalasin B is a fungal metabolite that inhibits Na -independent D-glucose transport in erythrocytes and adipocytes. Transport inhibition is though to be related to binding of cytochalasin B to the D-glucose transporter itself or to a component of the transporter [5—121. Binding of cytochalasin B to canine renal proximal tubular basolateral membranes has been reported in a preliminary communication [12]. In the present studies, cytochalasin B binding to rabbit renal proximal tubular basolateral membranes is more extensively characterized with respect to kinetics, inhibitor sensitivity and D-glucose analogue specificity.

Cheung and Hammerman: Cvtochalusin B binding to basolateral membranes

1293

cyte, adipocyte and intestinal membranes [7—10]. In addition, high concentrations (500 mM) of D-glucose, as well as 2-deoxyD-glucose and 3-O-methyl-D-glucose, but not L-glucose, inhibit cytochalasin B binding. Thus hexoses that are substrates for the basolateral Nat-independent D-glucose transporter also inhibit cytochalasin B binding [4]. It should be noted that as was the case in studies that employed non-renal membranes [6, 11, 18, 201, concentrations of D-glucose far in excess of the Km for transport were required to inhibit cytochalasin B binding. Both 2-deoxy-D-glucose and 3-O-methyl-D-glucose are more inhibitory to cytochalasin B binding in basolateral membranes

0

than is D-glucose itself. This is in contrast to our previous findings that trans-stimulated uptake of D-['4C]glucose into basolateral membrane vesicles by D-glucose is greater than that by either 2-deoxy-D-glucose or 3-O-methyl-D-glucose [4]. Similar observations were noted in human erythrocyte membranes L-Glu

D-Glu

2-DG

3-0MG

Concentration = 500 mM

Fig. 5. Effect of hexose analogues on [3HJcyiochalasin B (0.1 zM) binding to renal basolateral membranes. Data are expressed as mean SE

of 4 experiments.

Table I. Kinetics of cytochalasin B binding of high-affinity site plasma membranes from various tissues Tissue

Adipocyte, rat Adipocyte, rat Adipocyte, rat RBC. human RBC, rat Intestine, rat Intestine, mouse

Kd

Bm,x

M

p,nol/mg

Reference

7.8—24.4

Cushman [171 Czech [91 Wardzala [18] Wang [19]

17 >< 108

61 x 10 11.1 x lO 6.6 x lO

4.5 x 108

8 x l0 —

28 6.3—24.7 178

12.5

8.3 13.6

Wang [191

Maenz [II) Uezato [tO]

in which 2-deoxy-D-glucose and 3-O-methyl-D-glucose were more inhibitory to cytochalasin B binding than D-glucose [20], while transport studies revealed that 3-O-methyl-D-glucose had a lower affinity for the transporter than did D-glucose [21]. In rat intestinal basolateral membranes, 2-deoxy-D-glucose as well as D-xylose and D-mannose were more inhibitory to cytochalasin B binding than D-glucose [10]. This is in distinction to stereospecific D-['4C]glucose transport where D-glucose was as inhibitory as 2-deoxy-D-glucose and was more inhibitory than D-mannose and D-xylose [221. The lack of effect of L-glucose on cytochalasin B binding reflects the absence of an effect on D-glucose transport reported by us and others [1, 4].

Our observations are consistent with the identity of the high-affinity cytochalasin B binding site in renal proximal tubular basolateral membranes as the Na4-independent D-glucose

transporter or a component of the transporter. These data suggest that the Na-independent D-glucose transporter of the renal proximal tubular cell has structural similarity to glucose transporters of non-renal cells. Such a conclusion is consistent with the findings of Mueckler et al who showed that human kidney cortex and medulla contain messenger RNA species homologous to Nat-independent glucose transporter messen-

In several studies, measurements of cytochalasin B binding to non-renal tissues were performed in the presence of cytochalasin E [11, 16—19]. The assumption is made that cytocha- ger RNA obtained from a human hepatoma cell line [23].

Measurement of cytochalasin B binding to the high affinity site other than the glucose transporter, However, interactions be- of proximal tubular basolateral membranes should provide a tween the two cytochalasin compounds with one another and useful marker of D-glucose transport activity at this site. with membrane receptor sites have never been clearly defined. lasin E eliminates non-specific binding of cytochalasin B to sites

Therefore, cytochalasin E was not used in this study as a

Acknowledgments

blocker. The use of non-linear regression analysis of Scatchard We acknowledge the excellent technical assistance of Ms. Virginia binding data was adopted instead to account for all possible Hansen and Ms. Sharon Rogers. P.T.C. and M.R.H. were supported by binding sites on basolateral membranes. grants DK20579 and DK27600 from the National Institute of Diabetes, As is the case in erythrocyte and adipocyte plasma mem- Digestive and Kidney Diseases, (NIDDK), National Institutes of branes, and enterocyte basolateral membranes, renal proximal Health. MRH was supported by a grant from the Juvenile Diabetes tubular basolateral membranes contain a "high affinity" binding Foundation and by an Established Investigatorship from the American site for cytochalasin B. As shown in Table 1, the Kd of 5.88 X Heart Association. 10—8 M for high affinity cytochalasin B binding in renal proximal

tubule basolateral membrane is in close agreement with that reported for binding in plasma membranes from adipocytes, erythrocytes, and intestinal enterocytes [II, 17—19]. Likewise our estimated high affinity receptor density of 16.1 pmol/mg

Reprint request to Marc R. Hammerman, M.D., Department of Internal Medicine, Box 8121, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, Missouri 631/0, USA. References

protein is similar to that found in these membranes [9—Il, 17— 19].

Binding of [3H]cytochalasin B to proximal tubular basolateral membranes is inhibited by phioretin, as is the case in erythro-

I.

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