SPECIAL COMMUNICATION
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 |
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 |
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 |
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).
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 |
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.
 |
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