- Email: [email protected]
Effect of phloretin on transport processes in guinea-pig kidney cortex slices
Gen. Pharmu(. Vol. 12. pp. 273 to 277. 1981 Printed in Great Britain All riEhts rc~cr~ed
03(~-3fi23 X l 070273-05502 (X) 0 i C o p ) r i g h t 19gl Pcr~.amon Prc~, Lid
EFFECT OF PHLORETIN ON TRANSPORT PROCESSES IN GUINEA-PIG KIDNEY CORTEX SLICES J. W. L. ROBINSON Departement de Chirurgie Exl~rimentale. Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland (Receired 4 December 1980)
Abstract--l. Phloretin inhibits the transport processes in the basolateral membrane of the renal proximal cell at a lower concentration than that which affects brush-border transport mechanisms. 2. It is proposed that phloretin interacts with membrane lipid components to alter membrane permeability.
INTRODUCTION The dihydro-chalcone, phloretin, is a well-known inhibitor of the sugar transport system in erythrocytes (Lefevre, 1961). More recently, the drug has been shown to inhibit a N a ' - i n d e p e n d e n t sugar transport system in the basolateral membrane of the isolated epithelial cells of chicken intestine (Kimmich & Randies, 1975). These authors followed up their studies with a survey of the effects of different flavonoids in the same system and found that a wide variety of such compounds were inhibitory (Kimmich & Randles, 1978); they concluded that flavonoids and chalcones directly affect the carrier responsible for this transport process, while they apparently had no effect on the transport mechanisms present in the brush-border membranes of the same cells. Studies with intact tissue have provided somewhat different results, since phloretin has been shown to inhibit the influx of both sugars and amino-acids into mammalian small intestinal epithelial cells (Alvarado, 1967, 1970; Robinson, 1979). The flavonoid, naringenin (4',5,7-trihydroxy-ttavanone), inhibits both the influx and the accumulation of the same substrates in the intestine (Robinson et al., 1979), though the fact that the kinetic characteristics of its inhibition of phenylalanine influx apparently did not correspond to those exhibited by phloretin (Robinson. 1979) implied that the mechanism of action of the two compounds might be different. In the kidney, naringenin inhibited the transport processes in both peritubular and brush-border membranes, but the former were much more sensitive to the drug than the latter (Robinson et al., 1979). Since flavonoids are known to interfere with membrane components in bacterial cells (Ring et al., 1977), it was postulated that naringenin interacted more readily with fatty acid residues in the peritubular membrane of the proximal cell than with those of the brush-border membrane. In an attempt to ascertain whether the inhibitory pattern exhibited by phloretin in the kidney corresponded to that of naringenin--which would provide evidence in favour of an identical mode of action--the effects of the chaicone on the accumulation of different substrates in kidney cortex slices were compared; the results show that, as in the case of naringenin, the processes in the peritubular membrane are much more sensitive to the (.,.P 12 4. -E
273
drug than those situated in the brush border. These observations are interpreted in terms of an interaction of phloretin with lipidic components of membranes, as has also been suggested by Cousin & Motais (1978) from an examination of the effects of chalcones on chloride permeability in red cells.
METHODS
The experiments were performed with guinea-pig kidneys which were cut, after removal of the capsule, into slices of thickness 0.4 mm, using a spring-loaded guillotine (Ganguillet et ul., 1973}. The medullary region of the slices was then dissected away by hand. The tissue accumulation of radioactive solutes was assessed by incubating the cortical slices for one hour at 3 7 C in Krebs bicarbonate buffer containing a 0.1 mM solution of 14C-labelled substrate. For the measurement of the uptake of p-amino-hippurate, 10 mM sodium acetate was also added to the incubation medium. In view of the low solubility of phloretin, a stock solution (100 mM) was prepared in a mixture of water and polyethylene-glycol 300 (1:1 by volumek and then diluted into the incubation medium. The final concentration of polyethylene-glycol in all incubation flasks was maintained constant at 2.50 (v/v}. Preliminary experiments showed that the solvent, at this concentration, had no effect on the uptake of the substrates used in the present study. After the incubation, the slices were rinsed, weighed and dissolved individually in 0.1 ml 307,,; KOH before being counted in a liquid scintillation spectrometer, using a toluene/ethanol scintillation fluid (Robinson, 1972}. Oxygen consumption by tissue slices in the absence and presence of phloretin was determined in a Gilson differential respirometer, using a Krebs phosphate buffer containing 11.1 mM glucose, as described earlier {Robinson, 1976). Again the concentration of solvent was maintained constant in all flasks. To assess the sensitivity of a given transport system to phloretin, the results of a log dose/response curve were subjected to probit analysis. First, the raw data for uptake, expressed in pmol/g fresh tissue, were corrected for tissue water (80?Jg) and entry into the extracellular space (40'~o}in the usual manner (Rosenberg et al., 1961) to provide values in pmol/ml intracellular water. Since the inhibition of transport systems by phloretin has often been shown to be an incomplete process, the inhibition tending towards a limiting value beyond which the drug has no further effect (Alvarado, 1967, 1973), it seemed likely that the chalcone would not completely inhibit the renal transport systems" under study, an assumption confirmed by visual inspection
274
J . W . L . ROBINSON
of the log dose/response curves expressing the inhibition of the entry into the intracellular water at different concentrations of the drug. Accordingly, it was necessary to estimate the most probable asymptote of these curves. This was done by an iterative procedure consisting of systematically testing different values of the asymptote until the best linear regression was obtained when the observations were subjected to a probit transformation (Robinson, 1972). The concentration causing a 50"0 inhibition of the transport process was then read directly from the best probit line. To determine the lowest concentration that caused a significant inhibition, the data set was subjected to a randomblock analysis of variance to extract the least significant difference between means (Lison, 1968). This value was subtracted from the control uptake, and the phloretin concentration that reduced the uptake to this level was read from the probit line.
100, 90. 80. 70. 00. SO. 40. 30. 20. lo.
RESULTS Phloretin inhibits the accumulation of p-amino-hippurate, 2-deoxy-glucose, fl-methyl-glucoside and glycine by guinea-pig renal cortex slices (Table 1). it is clear that the sensitivity of the first two transport systems to the chalcone is greater than that of the other two. In order to quantify this difference, the log dose response curves were constructed, after correction of the uptake for entry into the extracellular space; these are presented in Fig. 1. The probit analysis, shown in the lower panel of the figure, yielded the concentrations capable of inhibiting each transport system semi-maximally: these are listed in Table 2. In an attempt to obtain the confidence limits of the estimates of the concentration responsible for a semimaximal inhibition, the probit analysis was applied to the results of each individual animal separately, and then the values were averaged, as r e c o m m e n d e d by Atkins & N i m m o (1980). In this way, the phloretin concentration that causes a 5000 inhibition of p-aminohippurate transport was found to be 0.126 + 0.0259mM, the corresponding values for the inhibition of 2-deoxy-glucose and fl-methyl-glucoside being 0.172 + 0.0153 m M and 0.252 + 0 . 0 2 0 0 m M respectively. By applying a t-test to these means, it is evident that there is a significant difference (P < 0.01)
Probit .7
.6
-4
-3
-2
log [phk:xetin] Fig. I. Above: Log dose/response curves of the inhibition by phloretin of the accumulation of p-amino-hippurate (open circles), 2-deoxy-glucose (squares), /Lmethyl-glucoside (triangles) and glycine (closed circles) in guinea-pig kidney cortex slices. The ordinate represents the percentage of the control uptake, after correction of the results given in Table I for the entry in the extracellular space. Below: Probit analysis of the results presented. The lines represent the regressions after iteration to find the best asymptote.
Table I. Influence of graded concentrations of phloretin on the uptake of different organic substrates by guinea-pig renal cortex slices Substrate uptake (,umols, g wet tissue) Phloretin concentration (mM) Glycine
/Lmethyl-glucoside 2-deoxy-glucose p-amino-hippurate(PAH)
0 0.04 0.1 0.25 0.5 1 2.5 5
0.363 0.364 0.332 0.290 0.237 0.186 0.109 0.085
0.465 0.451 0.365 0.293 0.198 0.102 0.067 0.067
0.414 0.364 0.266 0.196 0.128 0.088 0.055 0.056
0.370 0.280 0.229 0.170 0.126 0.092 0.064 0.060
Do o s Do. 01 Do.o0 ~
0.032 0.042 0.055
0.050 0.066 0.086
0.043 0.058 0.075
0.044 0.059 0.077
Tissues were incubated for 1 hr at 37=C in Krebs bicarbonate buffer with 0.1 mM substrate and the desired concentration of phloretin (10 mM sodium acetate was added in the case of PAH). The results are the means of 8 experiments in each case, presented with the least significant difference at different probability levels (Lison, 1968).
Phloretin and kidney transport
275
Table 2. Sensitivities of different transport systems to phloretin as evaluated from the probit analyses shown in Fig. I Glycine
/3-methyl-glucoside
2-deoxy-glucose
p-amino-hippurate (PAH)
Concentration causing 500/o inhibition
0.710
0.266
0.168
0.126
Concentration causing a significant inhibition at 5°~,probability level
0.107
0.075
0.030
0.0168
Concentrations expressed in mM.
between the sensitivity of/3-methyl-glucoside uptake on the one hand and the sensitivities of 2-deoxy-glucose and p-amino-hippurate transport on the other; there is no significant difference between the sensitivities of the transport of the two latter substrates to phloretin. A similar analysis could not be applied to the results for glycine, since the inhibition elicited was generally very slight, and the number of points available for the evaluation was too small in the case of some animals. Table 2 also lists the lowest concentrations of phloretin that cause a significant inhibition of the uptake of each of the four substrates. Again, the sensitivity of the mechanisms for the transport of p-amino-hippurate and of 2-deoxy-glucose is seen to be greater than that of the systems that transport /J-methyl-glucoside and glycine. Phloretin is a powerful metabolic inhibitor (Randies & Kimmich, 1978). This observation is confirmed by the results listed in Table 3, where it is clear that a concentration of 0.1 mM phloretin causes a severe inhibition of oxygen consumption by guinea-pig renal cortex slices. DISCUSSION Flavonoids, especially certain hydroxylated flavanones, inhibit a number of membrane transport processes (Lefevre, 1959; Diedrich & Stringham, 1970; Ring et al., 1977; Kimmich & Randles, 1978; Robinson et al., 1979). Since the most potent effects in the intestine were observed in the case of sugar movement across the basolateral cell membrane, it was believed that such compounds might interact directly with an equilibrating carrier mechanism at that locus (KimTable 3. Effect of phloretin on oxygen consumption by guinea-pig renal cortex slices Phloretin Concentration ImMI 0 0.1 0.4 I
Do 0~ Do oi Do ooi
Oxygen consumption (,ul/min.mg dry tissue) 156 4- I 1.4 119 + 9.9 73 4- 5.8 48 4- 5.8 14.8 20.2 27.6
Tissues were incubated in phosphate buffer containing 11.1 mM glucose in the presence of the requisite concentration of the inhibitor. Results are means ___SEM of 7 experiments, presented together with the values of the least significant difference at various probability levels.
mich & Randles, 1978). When it was later demonstrafed that active transport mechanisms in the brushborder membrane were also sensitive to high concentrations of naringenin (Robinson et al., 1979), this hypothesis became untenable, and it was suggested that the flavonoids might interfere with membrane lipid components, thereby changing the permeability characteristics of the membranes. This idea was based on results of Ring et al. (1977) who showed that flavonoids markedly stabilised the lipid components of bacterial cell walls, presumably by interacting with fatty acid residues in the membrane matrix. The question now arises as to whether phloretin and naringenin exert similar actions on membranes. Kimmich & Randles (1975, 1978) maintained that the effects of phloretin and naringenin on the basolateral membrane of isolated chicken enterocytes were very similar and proposed an identical mode of action for the two compounds. In our earlier work, we were more cautious in our interpretations for several reasons (Robinson et al., 1979): First, naringenin is a compact, non-planar molecule, whereas phloretin is freely pliable, despite the superficial resemblance between the two structures; if their mode of action consisted of an interaction with membrane components, phloretin would have to take up a configuration close to that of naringenin in order to exert its effect. Secondly, the kinetic characteristics of the inhibition of phenylalanine influx in the intestine were different for the two inhibitors, naringenin appearing as a fully non-competitive and phloretin as a partially competitive inhibitor (Robinson, 1979); however, this divergence may be more apparent than real, since the interpretation depends on the results obtained at high concentrations of naringenin. Since saturating concentrations of naringenin were not used, the possibility must remain open that raising the concentration even more might have revealed a limit to the inhibitory capacity of this substance, an event which would have permitted naringenin to have been classified as partially non-competitive inhibitor; in this case a mode of action analogous to that of phloretin could have been envisaged. In addition, no additivity between the effect of phloretin and naringenin in this system could be demonstrated (Robinson et al., 1979), thus providing a strong indication that the modes of action of the compounds might be similar. In the present work, we have examined the effect of phloretin on the accumulation of four substrates by renal cortex slices. Two of them (p-amino-hippurate and 2-deoxy-glucose) are believed to be transported across the peritubular membrane of the renal cell (Kleinzeller & McAvoy, 1973), and two (glycine and
276
J.W.L. ROaINSON
fl-methyl-glucoside) principally across the brushborder membrane. The other typical substrate for transport across the peritubular membrane, N l-methylnicotinamide, which we have used for previous studies of this type (Septilveda & Robinson, 1976; Robinson et al., 1979), could not be tested in the present work, since its uptake was very strongly inhibited, for some unknown reason, by the solvent used to solubilise the phloretin, polyethylene-glycol 300. The results in this study show that the transport systems in the peritubular membrane are significantly more sensitive to phloretin than those situated in the luminal membrane. Thus the actions of the chalcone are analogous to those of the flavanone, naringenin, which also has a much more powerful effect on the transport of p-amino-hippurate than on that of glycine. These observations permit the suggestion that, by analogy with naringenin, phloretin exerts its inhibitory effects by interacting with lipid components in membrane matrices, rendering them less permeable to organic solutes. The greater sensitivity of the transport systems located in the basolateral membranes is probably related to the composition of the membranes themselves which may be more amenable to modification by phloretin. It is noteworthy that there are differences in the sensitivities of the different systems located in the same membrane (Table 2), glycine transport, for instance, being distinctly less susceptible to inhibition by phloretin than that of fl-methyl-glucoside. This divergence may simply be due to the topography of the membranes and the relationships between the individual binding sites and the phloretin-sensitive membrane components. There are several observations in the literature which uphold the interpretation in terms of an interaction between phloretin and membrane lipids. First. Alvarado (1967. 1970, 1973) has proposed the existence of a phenol-binding site in brush-border membranes in order to explain the phenomenal inhibitory potency of phloridzin, the glucoside of phloretin, on intestinal and renal transport systems, and to account for the action of high concentrations of phloretin on transport processes in the same membrane. Our only modification to this point of view is that the action of phloretin would be of a more general nature than simple binding to a specific membrane site. Secondly, studies with membrane vesicles from small intestine have shown that the transport processes in the basolateral membrane are more sensitive to phloretin than those situated in the brush-border membrane (Hopfer et al., 1976; Wright et al., 1980); such observations agree with those reported in the present work. Our interpretation contrasts with that of Kimmich & Randies (1975, 1978) who proposed that phloretin specifically inhibits a Na+-independent monosaccharide transport system located in the basolateral membranes of chicken enterocytes. They based their conclusion on the fact that the same concentration of phloretin (100/am) had no effect on Na*-independent valine transport under identical conditions. However, since they could not be certain that the valine was in fact entering the cell across the basolateral membrane under the conditions of their experiment (Na*-independent transport of amino-acids across the brushborder membrane not being negligible) and since
100/~M phloretin would not be expected to inhibit brush-border transport mechanisms, their results could equally well be interpreted in terms of a nonspecific effect on membrane permeability, especially since--as discussed above--different transport mechanisms in the same membrane may be affected to greater or lesser extents by the interference of phloretin with membrane permeability properties. Finally, one other observation of Kimmich & Randles (1978) is particularly compatible with the permeability theory: They observed that in the presence of phloretin or of different flavonoids, the accumulation of monosaccharides that are transported across the brush-border membrane was enhanced. There was also a slight stimulation of the accumulation of amino-acids by some flavonoids, but this point was ignored by the authors, and in any case, the flavonoids used in this test were not those likely to produce the most significant effects. Now, if phloretin and flavanones reduce the permeability of the basolateral membrane by interacting with lipid components, then the solutes accumulated by transport across the opposing membrane will have greater difficulty in leaving the cell across the affected membrane, and will thus be accumulated to a greater extent. An exactly analogous effect was demonstrated in kidney cortex slices by Septilveda & Robinson (1976) who demonstrated that the flavonoid, (+)-catechin, inhibited the uptake of p-amino-hippurate and of Nt-methyl-nicotinamide, substrates that are transported across the peritubular membrane, but enhanced the accumulation of glycine and /~-methyl-glucoside which are reabsorbed across the luminal membrane. In this case, the flavonoid was assumed to impermeabilise the peritubular membrane without affecting that of the brush border. One last point should be emphasised. Phloretin is a strong metabolic inhibitor, as shown in Table 3, in confirmation of the results of other investigators (Randles & Kimmich, 1978). Since all the transport systems studied are at least partly sodium- and energydependent, phloretin might be expected to inhibit these transport systems as a result of its effect on cellular metabolism. The great difference in sensitivity between p-amino-hippurate transport on the one hand and that of glycine uptake on the other suggests that the metabolic effects of the chalcone cannot account for most of the present results. However, it must be admitted that the inhibition of glycine accumulation could be simply a result of metabolic effects, and there is no compelling reason, on the basis of the present results alone, to postulate a direct interference with the amino-acid transport system in the kidney. Nevertheless, by analogy with the intestine, where an inhibition of amino-acid influx has been demonstrated (Alvarado, 1970; Robinson, 1979), it seems likely that this glycine transport is indeed directly affected.
Acknowledoements--This work was supported by grants from Zyma S. A., Nyon. The technical assistance of Pierrette Ganguillet, Sylvianne Henriot and Dominique Mettraux is gratefully acknowledged.
Phloretin and kidney transport SUMMARY The effect of different concentrations of phloretin on the accumulation of p.amino-hippurate, 2-deoxyglucose, fl-methyl-glucoside a n d glycine by guinea-pig kidney cortex slices was determined. The t r a n s p o r t systems of the peritubular m e m b r a n e (p-amino-hippurate and 2-deoxy-glucose) were significantly more sensitive to the drug t h a n those located in the brushborder m e m b r a n e (fl-methyl-glucoside and glycine}. A l t h o u g h phloretin is a powerful metabolic inhibitor, the large differences in sensitivity of different transport systems that have been observed suggest that the chalcone has direct m e m b r a n e effects. The results are interpreted in terms of a hypothesis whereby phioretin interacts directly with m e m b r a n e lipids, those of basolateral m e m b r a n e s being more sensitive than those of b r u s h - b o r d e r membranes.
REFERENCES ALW,RAOO F. (1967) Hypothesis for the interaction of phlorizin and phloretin with membrane carriers for sugars. Biochim biophys. Acta 135, 483-495. ALVAR^OO F. 11970l Effect of phloretin and phlorizin on sugar and amino acid transport systems in small intestine. FEBS Syrup. 20, 131-139. ALVARAOO F. (1973) Comparative aspects of the effect of phenols on sugar transport. In Comparative Physiology (Edited by BOLLSL., SCHMIDT-NIELSEN K. & MADDRELL S. H. P.). pp. 451~63. North Holland. Amsterdam. ATKtNS G. L. & NtMMO i. A. (1980) Current trends in the estimation of Michaelis-Menten parameters. Analyt. Biochem. 104, 1-9. COUStN J. L. & MOTAtS R. 11978l Effect of phloretin on chloride permeability: a structure-activity study. Biochim. biophys. Acta ,¢~07, 531--538. DtEDgtcrt D. F. & STRINGHAM C. H. (1970) Active site comparison of mutarotase with the glucose carrier in human erythrocytes. Arch. Biochera. Biophys. 138, 499- 505. GANGUILLE'r F., MIRKOVITCH V, ROBINSON J. W. L. & GOMSA SZ. (1973) V&ification de l'ei~cacit6 d'une guillotine multilames pour la pr6paration standardis6e de tranches tissulaires. Pfliiyer.s Arch. 341, 171-178. HOPFER e., SIGRIST-NELSON K., AMMANN E. R, MURER H, (1976) Differences in neutral amino acid and glucose transport between brush border and basolateral plasma
277
membrane of intestinal epithelial cells, d. cell. Physiol. 89, 805-810. KIMMICn G. A. & RANDLES J. (1975) A Na+-independent, phloretin-sensitive monosaccbaride transport system in isolated intestinal epithelial cells. J. mere. Biol. 23, 57-76. KIUMICH G. A. & RANOLESJ. (1978) Phloretin-like action of bioflavonoids on sugar accumulation capability of isolated intestinal cells. Mere. Biochem. I, 221-237. KLEtNZELLER A. & McAvov E. M. (1973) Sugar transport across the peritubular face of renal cells of the flounder. J. gen. Physiol. 62, 169-184. LEFEVRE P. G. (1959) Molecular structural factors in competitive inhibition of sugar transport. Science 130, 104-105. LEFEVRE P. G. (1961) Sugar transport in the red blood cells: structure-activity relationships in substrates and antagonists. Pharmac. Rev. 13, 39-70. LISON U (1968) Statistique Appliqu(e fi la Bioloyie Experimentale, p. 169. Gauthier-Villars, Paris. RANDLESJ. & KIMMICHG. A. (1978) Effect of phloretin and theophylline on 3-O-methyl-glucose transport by intestinal epithelial cells. Am. J. Physiol. 234, C64--C72. RSNG K., EHLE H., FOIT B. & SCHWARZ M. (1977) The cytoplasmic membrane as a target for (+)-catechin and some other flavonoids. Proc. 3th Huny. Bioflavonoid Syrup., MftraJ-ured, pp. 427-437. Akad6miai Kiad6, Budapest. ROmNSON J. W. L. (1972) The inhibition of glycine and fl-methyl-glucoside transport in dog kidney cortex slices by ouabain and ethacrynic acid: contribution to the understanding of sodium-pumping mechanisms. Comp. gen. Pharmacol. 3, 145-159. ROmNSOr~ J. W. L. 119761 Mouvements ioniques et consommation d'oxyg6ne dans les tranches de cortex renal de chien. Effets de I'ouaba'me, de I'acide &acrynique et de la buformine. J. Physiol., Paris 72, 857-870. ROmNSON J. W. U (19791 Inhibition of phenylalanine influx in guinea-pig small intestine by naringenin. J. Physiol. (Lond.) 289, 44P-45P. ROBINSON J. W. L., L'HERMINIER M. & CLAUDET H. G. A. (1979} The effect of naringenin on the intestinal and renal transport of organic solutes. Naunyn-Schmiedeberg's Archs Pharmac. 307, 78-89. ROSENBERG L. E., BLAIR A. & SEGAL S. (1961) Transport of amino-acids by slices of rat-kidney-cortex. Biochim. hiophys. Acta 54, 479-488. SEPULVEDA F. V. & ROBINSON J. W. L. (1976) Effect of ( + bcatechin on renal and intestinal transport. Experientia 32, 87-88. WRIGHT E. M.. VAN Os C. H. & MmCHEFF A. K. (1980) Sugar uptake by intestinal basolateral membrane vesicles. Biochim. hiophys. Acto 597, 112- 124.
03(~-3fi23 X l 070273-05502 (X) 0 i C o p ) r i g h t 19gl Pcr~.amon Prc~, Lid
EFFECT OF PHLORETIN ON TRANSPORT PROCESSES IN GUINEA-PIG KIDNEY CORTEX SLICES J. W. L. ROBINSON Departement de Chirurgie Exl~rimentale. Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland (Receired 4 December 1980)
Abstract--l. Phloretin inhibits the transport processes in the basolateral membrane of the renal proximal cell at a lower concentration than that which affects brush-border transport mechanisms. 2. It is proposed that phloretin interacts with membrane lipid components to alter membrane permeability.
INTRODUCTION The dihydro-chalcone, phloretin, is a well-known inhibitor of the sugar transport system in erythrocytes (Lefevre, 1961). More recently, the drug has been shown to inhibit a N a ' - i n d e p e n d e n t sugar transport system in the basolateral membrane of the isolated epithelial cells of chicken intestine (Kimmich & Randies, 1975). These authors followed up their studies with a survey of the effects of different flavonoids in the same system and found that a wide variety of such compounds were inhibitory (Kimmich & Randles, 1978); they concluded that flavonoids and chalcones directly affect the carrier responsible for this transport process, while they apparently had no effect on the transport mechanisms present in the brush-border membranes of the same cells. Studies with intact tissue have provided somewhat different results, since phloretin has been shown to inhibit the influx of both sugars and amino-acids into mammalian small intestinal epithelial cells (Alvarado, 1967, 1970; Robinson, 1979). The flavonoid, naringenin (4',5,7-trihydroxy-ttavanone), inhibits both the influx and the accumulation of the same substrates in the intestine (Robinson et al., 1979), though the fact that the kinetic characteristics of its inhibition of phenylalanine influx apparently did not correspond to those exhibited by phloretin (Robinson. 1979) implied that the mechanism of action of the two compounds might be different. In the kidney, naringenin inhibited the transport processes in both peritubular and brush-border membranes, but the former were much more sensitive to the drug than the latter (Robinson et al., 1979). Since flavonoids are known to interfere with membrane components in bacterial cells (Ring et al., 1977), it was postulated that naringenin interacted more readily with fatty acid residues in the peritubular membrane of the proximal cell than with those of the brush-border membrane. In an attempt to ascertain whether the inhibitory pattern exhibited by phloretin in the kidney corresponded to that of naringenin--which would provide evidence in favour of an identical mode of action--the effects of the chaicone on the accumulation of different substrates in kidney cortex slices were compared; the results show that, as in the case of naringenin, the processes in the peritubular membrane are much more sensitive to the (.,.P 12 4. -E
273
drug than those situated in the brush border. These observations are interpreted in terms of an interaction of phloretin with lipidic components of membranes, as has also been suggested by Cousin & Motais (1978) from an examination of the effects of chalcones on chloride permeability in red cells.
METHODS
The experiments were performed with guinea-pig kidneys which were cut, after removal of the capsule, into slices of thickness 0.4 mm, using a spring-loaded guillotine (Ganguillet et ul., 1973}. The medullary region of the slices was then dissected away by hand. The tissue accumulation of radioactive solutes was assessed by incubating the cortical slices for one hour at 3 7 C in Krebs bicarbonate buffer containing a 0.1 mM solution of 14C-labelled substrate. For the measurement of the uptake of p-amino-hippurate, 10 mM sodium acetate was also added to the incubation medium. In view of the low solubility of phloretin, a stock solution (100 mM) was prepared in a mixture of water and polyethylene-glycol 300 (1:1 by volumek and then diluted into the incubation medium. The final concentration of polyethylene-glycol in all incubation flasks was maintained constant at 2.50 (v/v}. Preliminary experiments showed that the solvent, at this concentration, had no effect on the uptake of the substrates used in the present study. After the incubation, the slices were rinsed, weighed and dissolved individually in 0.1 ml 307,,; KOH before being counted in a liquid scintillation spectrometer, using a toluene/ethanol scintillation fluid (Robinson, 1972}. Oxygen consumption by tissue slices in the absence and presence of phloretin was determined in a Gilson differential respirometer, using a Krebs phosphate buffer containing 11.1 mM glucose, as described earlier {Robinson, 1976). Again the concentration of solvent was maintained constant in all flasks. To assess the sensitivity of a given transport system to phloretin, the results of a log dose/response curve were subjected to probit analysis. First, the raw data for uptake, expressed in pmol/g fresh tissue, were corrected for tissue water (80?Jg) and entry into the extracellular space (40'~o}in the usual manner (Rosenberg et al., 1961) to provide values in pmol/ml intracellular water. Since the inhibition of transport systems by phloretin has often been shown to be an incomplete process, the inhibition tending towards a limiting value beyond which the drug has no further effect (Alvarado, 1967, 1973), it seemed likely that the chalcone would not completely inhibit the renal transport systems" under study, an assumption confirmed by visual inspection
274
J . W . L . ROBINSON
of the log dose/response curves expressing the inhibition of the entry into the intracellular water at different concentrations of the drug. Accordingly, it was necessary to estimate the most probable asymptote of these curves. This was done by an iterative procedure consisting of systematically testing different values of the asymptote until the best linear regression was obtained when the observations were subjected to a probit transformation (Robinson, 1972). The concentration causing a 50"0 inhibition of the transport process was then read directly from the best probit line. To determine the lowest concentration that caused a significant inhibition, the data set was subjected to a randomblock analysis of variance to extract the least significant difference between means (Lison, 1968). This value was subtracted from the control uptake, and the phloretin concentration that reduced the uptake to this level was read from the probit line.
100, 90. 80. 70. 00. SO. 40. 30. 20. lo.
RESULTS Phloretin inhibits the accumulation of p-amino-hippurate, 2-deoxy-glucose, fl-methyl-glucoside and glycine by guinea-pig renal cortex slices (Table 1). it is clear that the sensitivity of the first two transport systems to the chalcone is greater than that of the other two. In order to quantify this difference, the log dose response curves were constructed, after correction of the uptake for entry into the extracellular space; these are presented in Fig. 1. The probit analysis, shown in the lower panel of the figure, yielded the concentrations capable of inhibiting each transport system semi-maximally: these are listed in Table 2. In an attempt to obtain the confidence limits of the estimates of the concentration responsible for a semimaximal inhibition, the probit analysis was applied to the results of each individual animal separately, and then the values were averaged, as r e c o m m e n d e d by Atkins & N i m m o (1980). In this way, the phloretin concentration that causes a 5000 inhibition of p-aminohippurate transport was found to be 0.126 + 0.0259mM, the corresponding values for the inhibition of 2-deoxy-glucose and fl-methyl-glucoside being 0.172 + 0.0153 m M and 0.252 + 0 . 0 2 0 0 m M respectively. By applying a t-test to these means, it is evident that there is a significant difference (P < 0.01)
Probit .7
.6
-4
-3
-2
log [phk:xetin] Fig. I. Above: Log dose/response curves of the inhibition by phloretin of the accumulation of p-amino-hippurate (open circles), 2-deoxy-glucose (squares), /Lmethyl-glucoside (triangles) and glycine (closed circles) in guinea-pig kidney cortex slices. The ordinate represents the percentage of the control uptake, after correction of the results given in Table I for the entry in the extracellular space. Below: Probit analysis of the results presented. The lines represent the regressions after iteration to find the best asymptote.
Table I. Influence of graded concentrations of phloretin on the uptake of different organic substrates by guinea-pig renal cortex slices Substrate uptake (,umols, g wet tissue) Phloretin concentration (mM) Glycine
/Lmethyl-glucoside 2-deoxy-glucose p-amino-hippurate(PAH)
0 0.04 0.1 0.25 0.5 1 2.5 5
0.363 0.364 0.332 0.290 0.237 0.186 0.109 0.085
0.465 0.451 0.365 0.293 0.198 0.102 0.067 0.067
0.414 0.364 0.266 0.196 0.128 0.088 0.055 0.056
0.370 0.280 0.229 0.170 0.126 0.092 0.064 0.060
Do o s Do. 01 Do.o0 ~
0.032 0.042 0.055
0.050 0.066 0.086
0.043 0.058 0.075
0.044 0.059 0.077
Tissues were incubated for 1 hr at 37=C in Krebs bicarbonate buffer with 0.1 mM substrate and the desired concentration of phloretin (10 mM sodium acetate was added in the case of PAH). The results are the means of 8 experiments in each case, presented with the least significant difference at different probability levels (Lison, 1968).
Phloretin and kidney transport
275
Table 2. Sensitivities of different transport systems to phloretin as evaluated from the probit analyses shown in Fig. I Glycine
/3-methyl-glucoside
2-deoxy-glucose
p-amino-hippurate (PAH)
Concentration causing 500/o inhibition
0.710
0.266
0.168
0.126
Concentration causing a significant inhibition at 5°~,probability level
0.107
0.075
0.030
0.0168
Concentrations expressed in mM.
between the sensitivity of/3-methyl-glucoside uptake on the one hand and the sensitivities of 2-deoxy-glucose and p-amino-hippurate transport on the other; there is no significant difference between the sensitivities of the transport of the two latter substrates to phloretin. A similar analysis could not be applied to the results for glycine, since the inhibition elicited was generally very slight, and the number of points available for the evaluation was too small in the case of some animals. Table 2 also lists the lowest concentrations of phloretin that cause a significant inhibition of the uptake of each of the four substrates. Again, the sensitivity of the mechanisms for the transport of p-amino-hippurate and of 2-deoxy-glucose is seen to be greater than that of the systems that transport /J-methyl-glucoside and glycine. Phloretin is a powerful metabolic inhibitor (Randies & Kimmich, 1978). This observation is confirmed by the results listed in Table 3, where it is clear that a concentration of 0.1 mM phloretin causes a severe inhibition of oxygen consumption by guinea-pig renal cortex slices. DISCUSSION Flavonoids, especially certain hydroxylated flavanones, inhibit a number of membrane transport processes (Lefevre, 1959; Diedrich & Stringham, 1970; Ring et al., 1977; Kimmich & Randles, 1978; Robinson et al., 1979). Since the most potent effects in the intestine were observed in the case of sugar movement across the basolateral cell membrane, it was believed that such compounds might interact directly with an equilibrating carrier mechanism at that locus (KimTable 3. Effect of phloretin on oxygen consumption by guinea-pig renal cortex slices Phloretin Concentration ImMI 0 0.1 0.4 I
Do 0~ Do oi Do ooi
Oxygen consumption (,ul/min.mg dry tissue) 156 4- I 1.4 119 + 9.9 73 4- 5.8 48 4- 5.8 14.8 20.2 27.6
Tissues were incubated in phosphate buffer containing 11.1 mM glucose in the presence of the requisite concentration of the inhibitor. Results are means ___SEM of 7 experiments, presented together with the values of the least significant difference at various probability levels.
mich & Randles, 1978). When it was later demonstrafed that active transport mechanisms in the brushborder membrane were also sensitive to high concentrations of naringenin (Robinson et al., 1979), this hypothesis became untenable, and it was suggested that the flavonoids might interfere with membrane lipid components, thereby changing the permeability characteristics of the membranes. This idea was based on results of Ring et al. (1977) who showed that flavonoids markedly stabilised the lipid components of bacterial cell walls, presumably by interacting with fatty acid residues in the membrane matrix. The question now arises as to whether phloretin and naringenin exert similar actions on membranes. Kimmich & Randles (1975, 1978) maintained that the effects of phloretin and naringenin on the basolateral membrane of isolated chicken enterocytes were very similar and proposed an identical mode of action for the two compounds. In our earlier work, we were more cautious in our interpretations for several reasons (Robinson et al., 1979): First, naringenin is a compact, non-planar molecule, whereas phloretin is freely pliable, despite the superficial resemblance between the two structures; if their mode of action consisted of an interaction with membrane components, phloretin would have to take up a configuration close to that of naringenin in order to exert its effect. Secondly, the kinetic characteristics of the inhibition of phenylalanine influx in the intestine were different for the two inhibitors, naringenin appearing as a fully non-competitive and phloretin as a partially competitive inhibitor (Robinson, 1979); however, this divergence may be more apparent than real, since the interpretation depends on the results obtained at high concentrations of naringenin. Since saturating concentrations of naringenin were not used, the possibility must remain open that raising the concentration even more might have revealed a limit to the inhibitory capacity of this substance, an event which would have permitted naringenin to have been classified as partially non-competitive inhibitor; in this case a mode of action analogous to that of phloretin could have been envisaged. In addition, no additivity between the effect of phloretin and naringenin in this system could be demonstrated (Robinson et al., 1979), thus providing a strong indication that the modes of action of the compounds might be similar. In the present work, we have examined the effect of phloretin on the accumulation of four substrates by renal cortex slices. Two of them (p-amino-hippurate and 2-deoxy-glucose) are believed to be transported across the peritubular membrane of the renal cell (Kleinzeller & McAvoy, 1973), and two (glycine and
276
J.W.L. ROaINSON
fl-methyl-glucoside) principally across the brushborder membrane. The other typical substrate for transport across the peritubular membrane, N l-methylnicotinamide, which we have used for previous studies of this type (Septilveda & Robinson, 1976; Robinson et al., 1979), could not be tested in the present work, since its uptake was very strongly inhibited, for some unknown reason, by the solvent used to solubilise the phloretin, polyethylene-glycol 300. The results in this study show that the transport systems in the peritubular membrane are significantly more sensitive to phloretin than those situated in the luminal membrane. Thus the actions of the chalcone are analogous to those of the flavanone, naringenin, which also has a much more powerful effect on the transport of p-amino-hippurate than on that of glycine. These observations permit the suggestion that, by analogy with naringenin, phloretin exerts its inhibitory effects by interacting with lipid components in membrane matrices, rendering them less permeable to organic solutes. The greater sensitivity of the transport systems located in the basolateral membranes is probably related to the composition of the membranes themselves which may be more amenable to modification by phloretin. It is noteworthy that there are differences in the sensitivities of the different systems located in the same membrane (Table 2), glycine transport, for instance, being distinctly less susceptible to inhibition by phloretin than that of fl-methyl-glucoside. This divergence may simply be due to the topography of the membranes and the relationships between the individual binding sites and the phloretin-sensitive membrane components. There are several observations in the literature which uphold the interpretation in terms of an interaction between phloretin and membrane lipids. First. Alvarado (1967. 1970, 1973) has proposed the existence of a phenol-binding site in brush-border membranes in order to explain the phenomenal inhibitory potency of phloridzin, the glucoside of phloretin, on intestinal and renal transport systems, and to account for the action of high concentrations of phloretin on transport processes in the same membrane. Our only modification to this point of view is that the action of phloretin would be of a more general nature than simple binding to a specific membrane site. Secondly, studies with membrane vesicles from small intestine have shown that the transport processes in the basolateral membrane are more sensitive to phloretin than those situated in the brush-border membrane (Hopfer et al., 1976; Wright et al., 1980); such observations agree with those reported in the present work. Our interpretation contrasts with that of Kimmich & Randies (1975, 1978) who proposed that phloretin specifically inhibits a Na+-independent monosaccharide transport system located in the basolateral membranes of chicken enterocytes. They based their conclusion on the fact that the same concentration of phloretin (100/am) had no effect on Na*-independent valine transport under identical conditions. However, since they could not be certain that the valine was in fact entering the cell across the basolateral membrane under the conditions of their experiment (Na*-independent transport of amino-acids across the brushborder membrane not being negligible) and since
100/~M phloretin would not be expected to inhibit brush-border transport mechanisms, their results could equally well be interpreted in terms of a nonspecific effect on membrane permeability, especially since--as discussed above--different transport mechanisms in the same membrane may be affected to greater or lesser extents by the interference of phloretin with membrane permeability properties. Finally, one other observation of Kimmich & Randles (1978) is particularly compatible with the permeability theory: They observed that in the presence of phloretin or of different flavonoids, the accumulation of monosaccharides that are transported across the brush-border membrane was enhanced. There was also a slight stimulation of the accumulation of amino-acids by some flavonoids, but this point was ignored by the authors, and in any case, the flavonoids used in this test were not those likely to produce the most significant effects. Now, if phloretin and flavanones reduce the permeability of the basolateral membrane by interacting with lipid components, then the solutes accumulated by transport across the opposing membrane will have greater difficulty in leaving the cell across the affected membrane, and will thus be accumulated to a greater extent. An exactly analogous effect was demonstrated in kidney cortex slices by Septilveda & Robinson (1976) who demonstrated that the flavonoid, (+)-catechin, inhibited the uptake of p-amino-hippurate and of Nt-methyl-nicotinamide, substrates that are transported across the peritubular membrane, but enhanced the accumulation of glycine and /~-methyl-glucoside which are reabsorbed across the luminal membrane. In this case, the flavonoid was assumed to impermeabilise the peritubular membrane without affecting that of the brush border. One last point should be emphasised. Phloretin is a strong metabolic inhibitor, as shown in Table 3, in confirmation of the results of other investigators (Randles & Kimmich, 1978). Since all the transport systems studied are at least partly sodium- and energydependent, phloretin might be expected to inhibit these transport systems as a result of its effect on cellular metabolism. The great difference in sensitivity between p-amino-hippurate transport on the one hand and that of glycine uptake on the other suggests that the metabolic effects of the chalcone cannot account for most of the present results. However, it must be admitted that the inhibition of glycine accumulation could be simply a result of metabolic effects, and there is no compelling reason, on the basis of the present results alone, to postulate a direct interference with the amino-acid transport system in the kidney. Nevertheless, by analogy with the intestine, where an inhibition of amino-acid influx has been demonstrated (Alvarado, 1970; Robinson, 1979), it seems likely that this glycine transport is indeed directly affected.
Acknowledoements--This work was supported by grants from Zyma S. A., Nyon. The technical assistance of Pierrette Ganguillet, Sylvianne Henriot and Dominique Mettraux is gratefully acknowledged.
Phloretin and kidney transport SUMMARY The effect of different concentrations of phloretin on the accumulation of p.amino-hippurate, 2-deoxyglucose, fl-methyl-glucoside a n d glycine by guinea-pig kidney cortex slices was determined. The t r a n s p o r t systems of the peritubular m e m b r a n e (p-amino-hippurate and 2-deoxy-glucose) were significantly more sensitive to the drug t h a n those located in the brushborder m e m b r a n e (fl-methyl-glucoside and glycine}. A l t h o u g h phloretin is a powerful metabolic inhibitor, the large differences in sensitivity of different transport systems that have been observed suggest that the chalcone has direct m e m b r a n e effects. The results are interpreted in terms of a hypothesis whereby phioretin interacts directly with m e m b r a n e lipids, those of basolateral m e m b r a n e s being more sensitive than those of b r u s h - b o r d e r membranes.
REFERENCES ALW,RAOO F. (1967) Hypothesis for the interaction of phlorizin and phloretin with membrane carriers for sugars. Biochim biophys. Acta 135, 483-495. ALVAR^OO F. 11970l Effect of phloretin and phlorizin on sugar and amino acid transport systems in small intestine. FEBS Syrup. 20, 131-139. ALVARAOO F. (1973) Comparative aspects of the effect of phenols on sugar transport. In Comparative Physiology (Edited by BOLLSL., SCHMIDT-NIELSEN K. & MADDRELL S. H. P.). pp. 451~63. North Holland. Amsterdam. ATKtNS G. L. & NtMMO i. A. (1980) Current trends in the estimation of Michaelis-Menten parameters. Analyt. Biochem. 104, 1-9. COUStN J. L. & MOTAtS R. 11978l Effect of phloretin on chloride permeability: a structure-activity study. Biochim. biophys. Acta ,¢~07, 531--538. DtEDgtcrt D. F. & STRINGHAM C. H. (1970) Active site comparison of mutarotase with the glucose carrier in human erythrocytes. Arch. Biochera. Biophys. 138, 499- 505. GANGUILLE'r F., MIRKOVITCH V, ROBINSON J. W. L. & GOMSA SZ. (1973) V&ification de l'ei~cacit6 d'une guillotine multilames pour la pr6paration standardis6e de tranches tissulaires. Pfliiyer.s Arch. 341, 171-178. HOPFER e., SIGRIST-NELSON K., AMMANN E. R, MURER H, (1976) Differences in neutral amino acid and glucose transport between brush border and basolateral plasma
277
membrane of intestinal epithelial cells, d. cell. Physiol. 89, 805-810. KIMMICn G. A. & RANDLES J. (1975) A Na+-independent, phloretin-sensitive monosaccbaride transport system in isolated intestinal epithelial cells. J. mere. Biol. 23, 57-76. KIUMICH G. A. & RANOLESJ. (1978) Phloretin-like action of bioflavonoids on sugar accumulation capability of isolated intestinal cells. Mere. Biochem. I, 221-237. KLEtNZELLER A. & McAvov E. M. (1973) Sugar transport across the peritubular face of renal cells of the flounder. J. gen. Physiol. 62, 169-184. LEFEVRE P. G. (1959) Molecular structural factors in competitive inhibition of sugar transport. Science 130, 104-105. LEFEVRE P. G. (1961) Sugar transport in the red blood cells: structure-activity relationships in substrates and antagonists. Pharmac. Rev. 13, 39-70. LISON U (1968) Statistique Appliqu(e fi la Bioloyie Experimentale, p. 169. Gauthier-Villars, Paris. RANDLESJ. & KIMMICHG. A. (1978) Effect of phloretin and theophylline on 3-O-methyl-glucose transport by intestinal epithelial cells. Am. J. Physiol. 234, C64--C72. RSNG K., EHLE H., FOIT B. & SCHWARZ M. (1977) The cytoplasmic membrane as a target for (+)-catechin and some other flavonoids. Proc. 3th Huny. Bioflavonoid Syrup., MftraJ-ured, pp. 427-437. Akad6miai Kiad6, Budapest. ROmNSON J. W. L. (1972) The inhibition of glycine and fl-methyl-glucoside transport in dog kidney cortex slices by ouabain and ethacrynic acid: contribution to the understanding of sodium-pumping mechanisms. Comp. gen. Pharmacol. 3, 145-159. ROmNSOr~ J. W. L. 119761 Mouvements ioniques et consommation d'oxyg6ne dans les tranches de cortex renal de chien. Effets de I'ouaba'me, de I'acide &acrynique et de la buformine. J. Physiol., Paris 72, 857-870. ROmNSON J. W. U (19791 Inhibition of phenylalanine influx in guinea-pig small intestine by naringenin. J. Physiol. (Lond.) 289, 44P-45P. ROBINSON J. W. L., L'HERMINIER M. & CLAUDET H. G. A. (1979} The effect of naringenin on the intestinal and renal transport of organic solutes. Naunyn-Schmiedeberg's Archs Pharmac. 307, 78-89. ROSENBERG L. E., BLAIR A. & SEGAL S. (1961) Transport of amino-acids by slices of rat-kidney-cortex. Biochim. hiophys. Acta 54, 479-488. SEPULVEDA F. V. & ROBINSON J. W. L. (1976) Effect of ( + bcatechin on renal and intestinal transport. Experientia 32, 87-88. WRIGHT E. M.. VAN Os C. H. & MmCHEFF A. K. (1980) Sugar uptake by intestinal basolateral membrane vesicles. Biochim. hiophys. Acto 597, 112- 124.