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Inhibition of P-glycoprotein-mediated transport by a hydrophobic contaminant in commercial gluconate salts

Carlos G. Vanoye, Guillermo A. Altenberg, and Luis Reuss

Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0641


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES

The substitution of gluconate for Cl- is commonly used to characterize Cl- transport or Cl--dependent transport mechanisms. We evaluated the effects of substituting gluconate for Cl- on the transport of the P-glycoprotein substrate rhodamine 123 (R123). The replacement of Ringer solution containing Cl- (Cl--Ringer) with gluconate-Ringer inhibited R123 efflux, whereas the replacement of Cl- by other anions (sulfate or cyclamate) had no effect. The inhibition of R123 efflux by gluconate-Ringer was absent after chloroform extraction of the sodium gluconate salt. The readdition of the sodium gluconate-chloroform extract to the extracted gluconate-Ringer or to cyclamate-Ringer inhibited R123 efflux, whereas its addition to Cl--Ringer had no effect. These observations indicate that the inhibition of P-glycoprotein-mediated R123 transport by gluconate is due to one or more chloroform-soluble contaminants and that the inhibition is absent in the presence of Cl-. The results are consistent with the fact that P-glycoprotein substrates are hydrophobic. Care should be taken when replacing ions to evaluate membrane transport mechanisms because highly pure commercial preparations may still contain potent contaminants that affect transport.

multidrug resistance; rhodamine 123; ion substitution; transport; chloride


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES

ONE OF THE BASIC experimental procedures utilized to characterize membrane transport mechanisms is the replacement of small inorganic ions with larger organic ions that are not transported via the specific pathway under study. It is generally assumed that the effects produced by the ion substitution are the result of the removal of the inorganic ion. However, there is the possibility that the replacement ion has additional effects on transport mechanisms, e.g., it is known that the replacement of HCO-3 by HEPES inhibits some Cl- channels (9). It is also possible that contaminants in the experimental solution affect the function of transport proteins. An example is the presence of the ATPase inhibitor vanadate in some commercial preparations of ATP (4).

The replacement of Cl- by gluconate is commonly used to characterize Cl- transport or Cl--dependent transport mechanisms. It is thought that gluconate is a large and inert anion that has a very limited permeability through Cl- channels and that cannot substitute for Cl- in Cl--dependent transporters such as the Na+-K+-Cl- cotransporter and the Cl-/HCO-3 exchanger. In this paper, we present evidence that a hydrophobic contaminant in commercial gluconate preparations blocks the transport of drugs by the multidrug resistance protein P-glycoprotein.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES

The transport of rhodamine 123 (R123) in the V79 cell line was measured (6). V79 cells are Chinese hamster lung fibroblasts that express pgp1 (7), a drug-transporting hamster P-glycoprotein. The efflux of R123 in V79 cells is P-glycoprotein dependent and has been characterized in detail (2). The efflux of R123 was measured as previously described (2). Briefly, 80%-confluent monolayers of V79 cells grown on round coverslips were loaded with 10 µM R123 in Ringer solution containing Cl- (Cl--Ringer) for 1 h at 37°C. After being loaded, the cells were placed in a chamber on the stage of an inverted microscope (Nikon Diaphot) coupled to a fluorometer (2). The fluorescence of R123 (FR123) was measured using an excitation light of 495 nm, and emitted light was detected at 525 nm (2). The cells were superfused with experimental solutions devoid of R123 at 37°C, and the rate of decrease in intracellular FR123 was measured. Under the conditions of these experiments, the fall in FR123 depends on functional P-glycoprotein expression (nonfluorescent P-glycoprotein substrates reduce R123 efflux to negligible values; Fig. 1; see also Ref. 2). The composition of the experimental solutions is shown in Table 1. Free Ca2+ concentration ([Ca2+]) was measured with a Ca2+-sensitive electrode (World Precision Instruments, Sarasota, FL).

                              
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Table 1.   Composition of bath solutions

A chloroform extract of sodium gluconate was made as follows. Equal volumes of a 1 M sodium gluconate aqueous solution and chloroform (Sigma) were placed in a glass separation funnel and thoroughly mixed by shaking. The chloroform phase was then removed, and another volume of chloroform was added to the water phase. The procedure was repeated 10 times. The chloroform phases were pooled and evaporated to dryness in a chemical hood. After the completion of this procedure, the extracted sodium gluconate was used to make experimental solutions. In some experiments, the dried extract was resuspended in extracted gluconate-Ringer, cyclamate-Ringer, or Cl--Ringer to evaluate its inhibitory effect on R123 efflux.


    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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P-glycoprotein-mediated R123 efflux is blocked by exposure to gluconate-Ringer. Figure 1 illustrates the effect of replacing Cl--Ringer with gluconate-, cyclamate-, or sulfate-Ringer on P-glycoprotein-mediated R123 efflux. Exposure to gluconate-Ringer inhibits the R123 efflux reversibly, whereas replacing Cl--Ringer with cyclamate- or sulfate-Ringer had no effect. The reduction in R123 efflux during exposure to gluconate-Ringer is unlikely to be due to the inhibition of Cl--dependent R123 efflux because the replacement of Cl- by other anions, both inorganic (sulfate) and organic (cyclamate), had no effect, in agreement with previous observations (1). The experiments shown above were carried out with gluconate-Ringer made with gluconate salts from Sigma. Experiments performed with gluconate salts from Fluka yielded similar results (data not shown). In separate experiments, we tested the inhibitory effect of replacing sodium and potassium cyclamate salts with the corresponding gluconate salts on R123 efflux. Both sodium and potassium gluconate inhibited R123 efflux. However, when the blocking effects of sodium and potassium gluconate salts from Sigma and Fluka were compared, the inhibition of R123 efflux by the Sigma salts was found to be somewhat larger (30-40%; not shown). Figure 1 also shows that vinblastine (10 µM), a well-known P-glycoprotein substrate, reduced R123 efflux reversibly to near zero. This observation, together with others previously published (2), indicates that essentially all the R123 efflux is via P-glycoprotein.


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Fig. 1.   Effects of exchanging Ringer solution containing Cl- (Cl--Ringer) for sulfate- (SO4-R; A), cyclamate- (Cyc-R; B), or gluconate-Ringer (Glu-R; C) or Cl--Ringer containing 10 µM vinblastine (R-VLB; D) on rhodamine 123 (R123) efflux. Shown are time courses of decay of intracellular R123 fluorescence (FR123) in V79 cells loaded for 1 h with 10 µM R123 (see METHODS for additional details). Each trace is representative of at least 3 experiments. a.u., Arbitrary units.

The effect of gluconate is not due to Ca2+ chelation. A possible explanation for the inhibition of R123 transport by gluconate-Ringer is that P-glycoprotein-mediated R123 efflux is reduced by lowering extracellular free [Ca2+]. It is known that many organic anions at millimolar concentrations can reduce free [Ca2+] by chelation. In fact, free [Ca2+] was reduced from 2 mM in Cl--Ringer to 0.14 mM in gluconate-Ringer and to 0.89 mM in cyclamate-Ringer (Fig. 2A). However, the decrease in R123 efflux by exposure to gluconate-Ringer is not due to Ca2+ chelation because lowering free [Ca2+] to 0.1 mM in Cl--Ringer had no effect on R123 efflux (Fig. 2B).


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Fig. 2.   Effects of lowering free Ca2+ concentration ([Ca2+]) on R123 efflux. A: decrease in free [Ca2+] in cyclamate- (Cyc-R) and gluconate-Ringer (Glu-R) compared with Cl--Ringer (Cl--R). B: lowering Ringer [Ca2+] from 2 to 0.1 mM has no appreciable effect on R123 efflux. Experiments were performed as described in legend for Fig. 1. Fluorescence trace is representative of 3 experiments.

The block by gluconate is produced by a contaminant that can be extracted with chloroform. Figure 3 depicts the effects of sodium gluconate from Sigma without or with a prior extraction with chloroform. In the experiment shown, the control solution was Cl--Ringer and the effects of sodium gluconate were tested with gluconate-Ringer extracted with chloroform. Extracted gluconate-Ringer had no effect on R123 efflux, but the addition of the chloroform extract resuspended in the inactive extracted gluconate-Ringer blocked R123 efflux completely. The inhibition of R123 efflux was reduced with nonextracted gluconate-Ringer (extract concentration was likely >10-fold that in gluconate-Ringer because the extract from 1 liter of 1 M sodium gluconate was resuspended in 300 ml of extracted gluconate-Ringer). Finally, R123 efflux was faster in cyclamate-Ringer. Because gluconic acid, gluconate salts, and metal-gluconate complexes are insoluble in organic solvents (5), we can rule them out as the inhibitors of P-glycoprotein-mediated R123 efflux.


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Fig. 3.   Effect of chloroform extract of sodium gluconate on R123 efflux. Bath substitutions were performed at the times indicated by the bars. Cl--R, Cl--Ringer; Eglu-R, gluconate-Ringer extracted with chloroform; Eglu-R + CE, gluconate-Ringer extracted with chloroform + chloroform extract; Glu-R, gluconate-Ringer. Trace is representative of 3 experiments.

Similar inhibition of R123 efflux was obtained when the chloroform extract was added to cyclamate-Ringer, but no effect was observed by the addition of the gluconate extract to Cl--Ringer. We found that 10 mM Cl- is sufficient to abolish the effect of the gluconate extract, whereas 1 mM Cl- reduced the inhibition by about 50% (not shown). These observations indicate that the decrease in P-glycoprotein-dependent R123 efflux is inhibited by Cl-, even though Cl- is not required for R123 transport by P-glycoprotein (2). Boiling the gluconate extract had no effect on the block of R123 efflux, suggesting that the blocker is not a hydrophobic peptide. In addition, amino acids could not be detected in the chloroform extract of sodium gluconate.

Conclusions. From the present results, we can conclude the following. First, care should be taken when replacing ions to evaluate membrane transport mechanisms because commercial preparations that are >99% pure may still contain potent contaminants that can affect transport mechanisms. It is important to use several different anions to replace Cl- when evaluating the effects of anions on membrane transport processes. Second, gluconate salts contain one or more hydrophobic contaminants that inhibit P-glycoprotein-mediated transport. Although we do not know the chemical nature of the P-glycoprotein inhibitor present in the commercial gluconate salts, we suggest that the contaminants are hydrophobic organic compounds produced by fermentation of glucose during the production of gluconic acid (3). This speculation is based on the observation that the P-glycoprotein substrates are hydrophobic organic substances (10). Recently, it has been found that unidentified grapefruit components that are soluble in organic solvents inhibit P-glycoprotein-mediated drug transport (8). If our reasoning is correct, it is likely that many commercial chemical preparations that are produced by fermentation (e.g., lactic acid, itaconic acid, citric acid) contain organic substances produced by microorganisms. In many cases, it will be advisable to consider the possibility that some of the effects of these substances are not due to themselves but to contaminants in the commercial preparations. In these cases, it may be useful to extract the commercial preparation with organic solvents before use.


    ACKNOWLEDGEMENTS

We thank Drs. S. Lewis and M. Brodwick for comments on a preliminary version of the manuscript and F. Bavarian and K. Spilker for technical help. V79 cells were generously provided by Dr. J. A. Belli.


    FOOTNOTES

This work was supported in part by grants from Searle Research and Development and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-08865.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: L. Reuss, Dept. of Physiology and Biophysics, Univ. of Texas Medical Branch, 301 Univ. Blvd., Galveston, TX 77555-0641 (E-mail: lreuss{at}utmb.edu).

Received 2 February 1999; accepted in final form 19 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES

1.   Altenberg, G. A., C. G. Vanoye, E. S. Han, J. W. Deitmer, and L. Reuss. Relationships between rhodamine 123 transport, cell volume and ion channel function of P-glycoprotein. J. Biol. Chem. 269: 7145-7149, 1994[Abstract/Free Full Text].

2.   Altenberg, G. A., C. G. Vanoye, J. K. Horton, and L. Reuss. Unidirectional fluxes of rhodamine 123 in multidrug-resistant cells: evidence against direct drug extrusion from the plasma membrane. Proc. Natl. Acad. Sci. USA 91: 4654-4657, 1994[Abstract].

3.   Budavary, S., M. J. O'Neil, A. Smith, and P. E. Heckelman (Editors). The Merck Index. An Encyclopedia of Chemicals, Drugs, and Biologicals (11th ed.). Rahway, NJ: Merck, 1989Budavary, S., M. J. O'Neil, A. Smith, and P. E. Heckelman (Editors). The Merck Index. An Encyclopedia of Chemicals, Drugs, and Biologicals (11th ed.). Rahway, NJ: Merck, 1989.

4.   Cantley, L. C., Jr., L. Josephson, R. Warner, M. Yanagisawa, C. Lechene, and G. Guidotti. Vanadate is a potent (Na,K)-ATPase inhibitor found in ATP derived from muscle. J. Biol. Chem. 252: 7421-7423, 1977[Abstract].

5.   Prescott, F. J., J. K. Shaw, J. P. Bilello, and G. O. Cragwall. Gluconic acid and its derivatives. Industrial Engineering Chem. 45: 338-342, 1953.

6.   Sognier, M. A., Y. Zhang, R. L. Eberle, and J. A. Belli. Characterization of adriamycin-resistant and radiation-sensitive Chinese hamster cell lines. Biochem. Pharmacol. 44: 1859-1868, 1992[Medline].

7.   Sognier, M. A., Y. Zhang, R. L. Eberle, K. M. Sweet, G. A. Altenberg, and J. A. Belli. Sequestration of doxorubicin in vesicles in a multidrug-resistant cell line (LZ-100). Biochem. Pharmacol. 48: 391-401, 1994[Medline].

8.   Takanaga, H., A. Ohnishi, H. Matsuo, and Y. Sawada. Inhibition of vinblastine efflux mediated by P-glycoprotein by grapefruit juice components in caco-2 cells. Biol. Pharm. Bull. 21: 1062-1066, 1998[Medline].

9.   Yamamoto, D., and N. Suzuki. Blockage of chloride channels by HEPES buffer. Proc. R. Soc. Lond. B Biol. Sci. 230: 93-100, 1987[Medline].

10.   Zamora, J. M., H. L. Pearce, and W. T. Beck. Physical-chemical properties shared by compounds that modulate multidrug resistance in human leukemic cells. Mol. Pharmacol. 33: 454-462, 1988[Abstract].


Am J Physiol Cell Physiol 276(6):C1439-C1442
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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