ABSTRACT
Marine mussels Mytilus californianus and Mytilus edulis, hardy
invertebrates resistant to pollution, were found to possess a multi¬
xenobiotic resistance mechanism analogous to multi-drug resistance
(MDR) found in resistant mammalian tumor cell lines. This MDR-like
efflux pump may provide defense against natural toxins in the
aquatic environment. An assay using the fluorescent dye rhodamine
B was adapted to measure MDR activity in Mytilus sp. gill tissue in
vivo. Four pieces of evidence support the existence of a multi¬
xenobiotic resistance in Mytilus sp.: (1) accumulation of rhodamine B
is sensitive to known inhibitors of MDR, such as verapamil; (2)
verapamil inhibits an efflux pump; (3) Western blot analysis shows
the presence of a protein immunologically related and similar in
molecular weight to mammalian MDR P-glycoprotein; and (4) the
Mytilus sp. efflux pump is sensitive to nanomolar concentrations of
rhodamine B.
MDR in Mytilus; Bard and Cornwall
199.
INTRODUCTION
Mussels (Mytilus sp.) are hardy organisms which are able to
successfully colonize heavily polluted waters. This character
suggests that these bivalves may possess a mechanism to resist the
toxicity of the diverse array of compounds found in aquatic pollution.
We have found in the marine mussels, Mytilus californianus and
Mytilus edulis, a mechanism of toxin resistance similar to the multi¬
xenobiotic resistance found in the mussel Mytilus galloprovincialis by
Kurelec and Pivcevic (1991), and analogous to the phenomenon of
multi-drug resistance (MDR) found in mammalian tumor cell lines.
MDR was first discovered in cancer patients undergoing
chemotherapy who were found to become simultaneously insensitive
to a wide range of structurally unrelated, cytotoxic drugs (Beck,
1987. Hofli and Nissen-Meyer, 1990). The clinical resistance to these
drugs, marked by a decrease in drug accumulation within the tumor
cells, was found to correspond to an increased expression of a 170 kD
membrane P-glycoprotein (P-gp) (Pearce, 1990). The P-gp was later
found to bind and export a wide array of compounds from the cell in
an energy-dependent, saturable process (Pearce, 1990).
Previous research has shown that certain drugs reverse the
MDR activity in tumor cells (Hofsli and Nissen-Meyer, 1990). This
observation has led to the discovery that certain compounds will
competitively inhibit the efflux of other compounds from cells with
P-glycoprotein. Hence the latter compounds will accumulate in the
cell and, in effect, the cell is no longer resistant to them. Such MDR
inhibitors include verapamil (Yusa and Tsuruo, 1989), forskolin
MDR in Mytilus; Bard and Cornwall, 1992; 2
(Morris et al., 1991), quinidine (Cornwell et al., 1987), emetine
(Samuelson et al., 1990), vinblastine (Tamai and Safa, 1991), and
trifluoperazine (Cano-Gauci and Riordan, 1987), among many others.
This competitive inhibition phenomenon has been used to detect
MDR activity.
Neyfakh (1988) found that multi-drug resistant tumor cells
under normal conditions resisted staining by a number of fluorescent
dyes. However, the cells accumulated high concentrations of the
dyes in the presence of known inhibitors of MDR, such as verapamil,
trifluoperazine, and weak detergents. This information was used to
develop an assay to observe and characterize MDR-like activity: a
marked increase in accumulation of rhodamine in the presence of
MDR inhibitors, as measured by fluorescence microscopy, may signify
the presence of an MDR-like mechanism.
We modified this assay for our study of Mytilus sp., using
epifluorescence microscopy to measure rhodamine B accumulation in
single cells of intact Mytilus sp. gill tissue in vivo. The purpose of
this study is to determine if the marine mussels Mytilus
californianus and Mytilus edulis exhibit an efflux mechanism similar
to the multi-drug resistance phenomenon described in mammalian
tumor cells, and to determine if this mechanism could be effective
against the low concentrations of toxins found in the aquatic
environment.
Four pieces of evidence suggest that the Mytilus possesses an
MDR-like mechanism to efflux xenobiotics and avoid bioaccumulation
of these toxins. First, accumulation of rhodamine B in the gill tissue
is sensitive to known inhibitors of the MDR protein, such as
MDR in Mytilus; Bard and Cornwall, 1992; 3
verapamil. Second, verapamil inhibits an efflux pump. Third,
western blot analysis shows the presence of a protein
immunologically related and similar in molecular weight to the
mammalian MDR transport protein. Finally, the Mytilus sp. efflux
pump is sensitive to nanomolar concentrations of rhodamine B.
MATERIALS AND METHODS
MDR ACTIVITY DETECTION ASSAYS
MDR activity was measured in Mytilus sp. gill tissue. Gills were
removed from freshly dissected Mytilus californianus and Mytilus
edulis. The gills are comprised of four monolayer sheets of cells.
Small (« 25 mm2), single cell layer tissue samples were cut from the
dorsal and ventral rims of the medial layer of the gill. The gills of M.
edulis had to be incubated in sea water for 10-15 minutes to flush
out the large amounts of mucus.
To assess MDR substrate competition, seven 5mL sea water
solutions were prepared in 30mL beakers with 1 uM rhodamine B
and one each of the following MDR-inhibiting drugs at the following
concentrations: verapamil (22 uM), emetine (40 ug/mL), vinblastine
(50ug/mL), forskolin (5 uM), trifluoperazine (22 uM), quinidine (22
UM), and sodium azide (10 mM). The control solution contained 1
MM rhodamine B only. Two tissue samples were incubated in each
solution at 18-20 °C for one hour. The tissues were then removed
from the incubation and swirled gently in 10-20 mL sea water for a
MDR in Mytilus; Bard and Cornwall, 1992; 4
few seconds to remove excess rhodamine from interstitial spaces.
The tissues were then placed, free-floating, in sea water in 5 mL
petri dishes for examination. The rhodamine epifluorescence in
individual cells in each tissue sample was observed with a 40X
water-immersion lens on a Zeiss epifluorescence microscope. The
intensity was measured by a light meter and recorded in volts by a
digital multimeter. Measurements for each sample were completed
in less than 10 minutes. In each tissue, 25-40 cells were measured,
and means and standard deviations were computed.
MDR ACTIVITY CHARACTERIZATION ASSAYS
To assess rhodamine uptake over time, tissue samples were
incubated in 5 mL of luM rhodamine/sea water solution with and
without 22 uM verapamil for 0, 15, 30, 60, 90, and 120 minutes.
The tissues were then rinsed and examined as described above. To
assess rhodamine efflux over time, tissue samples were incubated for
one hour in 1 uM rhodamine and were then placed in 5 mL sea
water with and without 22 uM verapamil for 0, 15, 30, 60, 90, and
120 minutes. This protocol was repeated for tissue samples
incubated for one hour in 2 uM rhodamine and 22 uM verapamil.
Fluorescence measurements were made as described above. All sea
water and solutions were kept at 18-20 °C, except during
examination when the microscope light warmed the sea water dish.
MDR in Mytilus; Bard and Cornwall, 1992; 5
WESTERN BLOTS
Mytilus californianus gill tissue samples were tested by
western blot analysis for the presence of a protein related to the
mammalian MDR P-glycoprotein. Chopped samples of M.
californianus gill tissue were homogenized in hypotonic lysis buffer
(10 mM KCl, 1.5 mM MgCl2, 10mM Tris HCl, and 2 mM Phenylmethyl
Sulfonyl Fluoride) and then sonicated in 5% SDS. Proteins from gill
tissue samples and from human tumor K 562 R7 cells that
overexpress the MDR P-glycoprotein were resolved on a 7.5% SDS
polyacrylamide BioRad Mini-Protean II gel and transferred to
nitrocellulose as described by Otter et al. (1987). The P-glycoprotein
specific monoclonal antibody C219 from Centocor, the epitope
sequence of which was determined by Georges et al. (1990), was
used to locate the P-glycoprotein in the MDR-positive R7 cell and gill
samples. The blots were developed by the alkaline phosphatase
assay described by Mierendorf et al. (1987).
SOURCES OF REAGENTS
Rhodamine B, Quinidine HCl, and Vinblastine were from Sigma
Chemical Company (St. Louis, MO); Verapamil HCl from Knoll
Pharmaceutical Company (Whippany, NJ); Emetine HCl from K&K
Laboratories, Inc. (Plainview, NY); 1,9-dideoxy Forskolin from
Calbiochem (San Diego, CA); Trifluoperazine from Smith Kline and
French Labs (Philadelphia, PA); and Sodium Azide from Fisher
Scientific Company (Fair Lawn, NJ).
MDR in Mytilus; Bard and Cornwall, 1992; 6
RESULTS
RHODAMINE UPTAKE WITH AND WITHOUT VERAPAMIL
We incubated M. californianus gill tissues in 1 uM rhodamine
with and without 22 uM verapamil for 15, 30, 60, 90, and 120
minutes to characterize rhodamine accumulation over time. As seen
in Figure 1, a much greater accumulation of rhodamine occurred in
the tissues which were incubated in the presence of verapamil. With
verapamil, the fluorescence appeared to level off after one hour in
the solution at approximately a 500% higher level than the tissues in
the absence of verapamil. Without verapamil, the fluorescence
remained low, peaking at around 30 minutes, decreasing slightly, and
levelling off after one hour.
INHIBITION OF RHODAMINE ACCUMULATION BY OTHER DRUGS
Since rhodamine accumulation levelled off after one hour of
incubation, we tested the other competitive inhibitors by measuring
rhodamine fluorescence at the 60 minute time point only. As seen in
Figures 2 and 3, gill tissue samples from both Mytilus californianus
and Mytilus edulis showed increased accumulation of rhodamine in
the presence of known MDR competitive inhibitors: verapamil,
vinblastine, trifluoperazine, emetine, quinidine, and forskolin. M.
californianus tissue samples exposed to the drugs displayed
increased fluorescence greater than 500% of the control with a
maximum of 1700% in verapamil (see Figure 2). The M. edulis tissue
MDR in Mytilus; Bard and Cornwall, 1992; 7
samples incubated in these drugs demonstrated fluorescence greater
than 350% of the control with a maximum 440% with forskolin (see
Figure 3).
In similar rhodamine assays, tissues were incubated in the
presence of sodium azide (data not shown), an electron transport
chain blocker (Neyfakh, 1988), to test for the ATP dependence of the
efflux mechanism. No significant increase in rhodamine
accumulation was found in either species.
RHODAMINE EFFLUX
We next attempted to determine whether verapamil increased
rhodamine accumulation by inhibiting an efflux pump. To examine
efflux, we loaded tissues with fluorescent dye and then measured
efflux in the presence of verapamil. M. californianus tissues were
incubated in 1 uM rhodamine for one hour and then incubated in
sea water with and without 22 uM verapamil for 30, 60, 90, and 120
minutes. As shown in Figure 4, the tissues incubated in plain sea
water showed a decrease in fluorescence to slightly above natural
fluorescence levels after about 30 minutes. However, tissues
incubated in sea water containing 22 uM verapamil showed a much
slower decrease in fluorescence, remaining over seven times higher
than natural fluorescence at two hours. (See Figure 6)
We also incubated tissues in 2 uM rhodamine and 22 uM
verapamil for one hour, and then incubated them in sea water with
and without 22 uM verapamil for 15, 30, 60, 90, and 120 minutes.
Under these conditions, unlike the 1 uM rhodamine incubation, there
MDR in Mytilus; Bard and Cornwall, 1992; 8
was no significant difference between rates of fluorescence decrease
in the presence or absence of verapamil over two hours. (See Figure
5; see Discussion section for further explanation.)
WESTERN BLOT
We then tested for the presence of a protein related to the P-
glycoprotein responsible for mammalian MDR. Western blot analysis
of M. californianus gill tissue with the monoclonal antibody C219
revealed a single band, parallel to a 150 kD band from human tumor
R7 cells which over express the MDR P-glycoprotein. (See Figure 6)
RHODAMINE CONCENTRATION DEPENDENCE
To determine the range of rhodamine concentrations over
which activity of the efflux pump is detectable, we incubated M.
californianus gill tissues in varying concentrations of rhodamine with
and without 22 uM verapamil for one hour. As shown in Figure 8,
there was no significant increase in rhodamine accumulation in the
presence of verapamil at 2.5 uM rhodamine. Likewise, we found no
difference in rhodamine accumulation with and without verapamil at
0.1 nM rhodamine, as shown in Figure 7. However, between 1 nM
and 2uM rhodamine, there was at least a 100%, and up to a 500%,
increase in rhodamine accumulation in the presence of verapamil,
suggesting the presence of MDR-like activity. (See Figure 7)
MDR in Mytilus; Bard and Cornwall, 1992; 9
MDR-LIKE ACTIVITY IN OTHER ORGANISMS
A preliminary survey of MDR-like activity in other marine
organisms was conducted. Eggs of the sand crab Emerita analoga and
the solitary tunicate Ascidia ceratodes exhibited a 200% increase in
rhodamine accumulation in the presence of verapamil. No increase
in rhodamine accumulation on addition of verapamil was detected in
eggs of the following species, Aplysia californica (sea hare), Patiria
miniata (bat star), and Pisaster ochraceus (purple sea star).
Eggs of Parastichopus parvimenesis (California sea cucumber)
were incubated in rhodamine solutions containing verapamil,
emetine, trifluoperazine, quinidine, sodium azide and forskolin.
Rhodamine accumulation in the presence of these drugs was 20% and
40% below that of the control.
We found no evidence for the MDR P-glycoprotein on the
Western blot for any of the aforementioned species (all tested except
Aplysia eggs).
DISCUSSION
From the results of the rhodamine assays and Western Blot
analysis, we conclude that the marine mussels, Mytilus californianus
and Mytilus edulis, possess an MDR-like mechanism which may
provide defense against toxins in the aquatic environment.
MDR in Mytilus; Bard and Cornwall, 1992; 10
COMPETITIVE INHIBITION ASSAYS
The first piece of evidence is the enhanced accumulation of
rhodamine B in the presence of MDR inhibitors such as verapamil.
Rhodamine accumulation in sea water is low, but in the presence of
verapamil, intracellular rhodamine content increases to over 800% of
control. Since the concentration of verapamil in the incubation is

much higher than that of rhodamine (11-22,000 -fold), verapamil
competes successfully for binding sites on the P-glycoprotein.
Therefore, verapamil is effluxed and rhodamine B is allowed to
accumulate within the cells.

The same result is found with the inhibitors vinblastine,
trifluoperazine, emetine, quinidine, forskolin as shown for Mytilus
californianus in Figure 2, and Mytilus edulis in Figure 3.
ATP-DEPENDENCE
We found that the metabolic inhibitor, NaAz did not inhibit
MDR activity in Mytilus, although mammalian MDR has been found to
be energy dependent (Horio et al, 1988). NaAz prevents reduction of

cytochrome oxidase, thereby blocking the electron transport chain
and the production of ATP via aerobic metabolism. Bivalves such as
M. edulis are able to function under anaerobic conditions for several
days producing over 60% of the ATP produced under normal

conditions (de Zwaan and Wijsman, 1975). This ability would allow
the MDR efflux pump to continue functioning and would account for
MDR in Mytilus; Bard and Cornwall, 1992; 11
the low intracellular rhodamine concentration in the presence of
NaAz.
VERAPAMIL INHIBITS AN EFFLUX PUMP
We found that verapamil enhances rhodamine accumulation by
inhibiting an efflux pump. When tissues were incubated in a low
concentration of rhodamine and then placed in sea water, verapamil
significantly slowed the rate of rhodamine efflux. Suppose, as in
Figure 9, that in sea water a concentration gradient and an efflux
pump together decrease intracellular rhodamine concentration.
the efflux pump is inhibited one would expect a slower rate of
decrease of intracellular rhodamine, as seen in Figure 4.
However, at high initial intracellular rhodamine concentrations,
we see no decrease in rhodamine efflux rate in the presence of
verapamil. The higher intracellular rhodamine concentration
increases the concentration gradient when the tissues are placed in
the sea water. Thus the concentration gradient provides a much
larger fraction of the total efflux force than it does at low
concentrations. Since the pump then represents only a very small
fraction of the total efflux force, we can see no difference in efflux
rate when the pump is inhibited. (See Figure 10)
WESTERN BLOT ANALYSIS
We employed western blot analysis to detect a protein in the
M. californianus gill related to the P-glycoprotein found in resistant
MDR in Mytilus; Bard and Cornwall, 1992; 12
mammalian tumor cells. The MDR-specific monoclonal antibody C219
(Georges et al., 1990) indicated a single band of similar molecular
weight (about 150 KD) as a band resolved from a sample of tumor
cells that over-express the MDR phenotype. (See Figure 6) From
these results we conclude that the MDR-like activity in M.
californianus involves a P-glycoprotein related mechanism.
RHODAMINE CONCENTRATION DEPENDENCE
Finally, we found that this MDR-like efflux pump is sensitive to
extremely low concentrations of rhodamine. At 1 nM extracellular
rhodamine, we were able to detect a 100% increase in rhodamine
accumulation in the presence of verapamil. This result suggests that
the MDR-like efflux pump is sensitive to very low concentrations of

substrate. Thus, even though most toxins in the aquatic environment
are found at nanomolar concentrations, the Mytilus efflux pump
would be able to effectively prevent their accumulation.
However, at extremely low concentrations (1 pM), virtually no
rhodamine was accumulated either with or without the presence of

verapamil. Also, at rhodamine concentrations above 2uM, we were
unable to detect a significant increase in accumulation in the
presence of verapamil. (See Figure 8) At such high extracellular
concentrations, the inward diffusion may be so great that the fraction
of influxed rhodamine exported by the saturable efflux pump is
unresolvable by our assay, as depicted in Figure 10.
.
MDR in Mytilus; Bard and Cornwall, 1992; 13
„
CONCLUSION
From the results of this study, we conclude that the marine
mussels, Mytilus californianus and Mytilus edulis, possess a toxin
efflux mechanism analogous to the multi-drug resistance described
in mammalian tumor cells. Furthermore, since this efflux mechanism
is sensitive to nanomolar concentrations of substrate, it may provide
effective defense against natural toxins found in the marine
environment.
MDR in Mytilus; Bard and Co
ACKNOWLEDGEMENTS
We would like to express sincere gratitude to Dr. David Epel,
Barbara HollandiToomey, Dr. Rob Sweezy, Paul Sund, and Chris
Patton for the laboratories, knowledge, guidance, and enthusiasm;
and the faculty and staff of Hopkins Marine Station and the other
students of Biology 175H who provided us with an exhilarating
introduction to marine biology research. We also wish to express our
gratitude for a variety of loans, grants, scholarships, and work-study
jobs from the Stanford University Financial Aid Office.
MDR in Mytilus; Bard and Corny
REFERENCES
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Conference on the Health of Georgia Strait, 1991. B.C.: Quadra
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Beck, W.T. The cell biology of multiple drug resistance.1987.
Biochemical Pharmacology. Vol.36, No. 18, pp.2879-2887.
Cornwell, M. et al. Certain calcium channel blockers bind specifically
to multidrug-resistant human KB carcinoma membrane vesicles
and inhibit drug binding to P-glycoprotein. 1987. The Journal
of Biological Chemistry. Vol. 262, No. 5. pp. 2166-2170.
De Zwaan, A. and Wijsman, T.C.M. Review. Anaerobic metabolism in
bivalvia (Mollusca). Characteristics of anaerobic metabolism.
1976. Comp. Biochem. Physiol. 1976. Vol. 54B. pp.313-324.
Georges, E. et al. Detection of P-glycoprotein isoforms by gene specific
monoclonal antibodies. 1990. Proc. Natl. Acad. Sci. U.S.A., 87:
pp.152-156.
Hofsli, Eva and Jon Nissen-Meyer. Reversal of Multidrug Resistance
by Lipophilic Drugs. 1990. Cancer Research. 50, 3997-4002.
Horio, M., M.M. Gottesman, and I. Pastar, 1988. ATP-dependent
MDR in Mytilus; Bard and Cornwall, 1992; 16
transport of vinblastine in vesicles from human multi-drug
resistant cells. Proc. Natl. Acad. Sci. U.S.A. 85, 3580-3584.
Kessel, David et al. Characterization of multidrug resistance by
fluorescent dyes. 1991. Cancer Research. 51, pp 4665-4670.
Kurelec, B. and B. Pivcevic. Distinct Glutathione-dependent Enzyme
Activities and a Verapamil-sensitive Binding of Xenobiotics in a
Fresh-water Mussel Anodonta cygnea. (1989) Biochem.
Biophys. Res. Comm. Vol.164, No. 2, pp.934-940.
Kurelec, B. and B. Pivcevic. Evidence for a multi-xenobiotic resistance
mechanism in the mussel Mytilus galloprovincialis. (1991)
Aquatic Toxicology. 453, ppl-11.
Mierendorf et al. Gene isolation by screening lambda GTII libraries
with Ab's. 1987. Methods in Enz. 152: pp. 458-467.
Morris et al. Interaction of Forskolin with the P-Glycoprotein
Multidrug Transporter. 1991. Biochemistry. 30: pp. 8371-

8379.
Neyfakh, Alexander. Use of fluorescent dyes as molecular probes for
the study of multidrug resistance. 1988. Experimental Cell
Research. 174, pp168-176.
Otter, T. et al. A two-step procedure for efficient transfer of both
MDR in Mytilus; Bard and Cornwall, 1992; 17
high mol. wght. (S400,000) and low MW («20,000) proteins.
1987. Anal. Biochem. 162: pp. 370-377.
Pearce, H.L. et al. Structural characteristics of compounds that
modulate P-glycoprotein-associated multidrug resistance. 1990.
Adv. Enz. Reg. 301. pp357-373.
Samuelson et al. Emetine-resistant mutants of Entamoeba histolytica
overexpress mRNAs for multidrug resistance. 1990. Molecular
and Biochemical Parasitology. 38: pp. 281-290.
Tamai, I. and Safa, A. Azidopine noncompetitively interacts with
vinblastine and cyclosporin A binding to P-glycoprotine in
multidrug resistant cells. 1991. The Journal of Biological
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Yusa, K. and Tsuruo, T. Reversal mechanism of multidrug resistance
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MDR in Mytilus; Bard and Cornwall, 1992; 1
FIGURE LEGENDS
Eigure 1 Rhodamine uptake kinetics. Verapamil enhances
rhodamine accumulation. Tissue samples were incubated in 1 UM
rhodamine with and without 22 uM verapamil for varying lengths of
time. Percent increases are indicated above each point.
Figure 2. Competitive inhibition by MDR inhibitors. MDR inhibitors
enhance rhodamine accumulation. Mytilus californianus gill tissues
were incubated for one hour in 1 uM rhodamine with and without
known MDR competitive inhibitors: verapamil, trifluoperazine,
emetine, forskolin, quinidine, and vinblastine. Fluorescence values
are represented as percentages of control fluorescence (without
inhibitors = 100%).
Eigure 3. Competitive inhibition by MDR inhibitors. MDR inhibitors
enhance rhodamine accumulation. Mytilus edulis gill tissues were
incubated for one hour in 1 uM rhodamine with and without known
MDR competitive inhibitors: verapamil, trifluoperazine, emetine,
forskolin, quinidine, and vinblastine. Fluorescence values are
represented as percentages of control fluorescence (without
inhibitors = 100%).
Eigure 4. Rhodamine efflux. Verapamil inhibits an efflux pump.
Mytilus californianus gill tissues were incubated in 1 uM rhodamine
for one hour and then placed in sea water with and without
MDR in Mytilus; Bard and Cornwall, 1992; 19
verapamil for varying lengths of time. Time = 0 represents

placement into the sea water rinses. Data reported as mean
fluorescences with standard deviations. Percent differences are
indicated above each point.

Eigure 5. Rhodamine efflux. High rhodamine concentrations mask
inhibition of efflux pump. Mytilus californianus gill tissues were
incubated in 2 uM rhodamine with 22 UM verapamil, and then
placed in sea water with and without 22 uM verapamil. Time = 0
represents placement into the sea water rinses. Data reported as
mean fluorescences with standard deviations.
Eigure 6. Western Blot. MDR-related protein found in Mytilus gill.
Single band in lane six represents P-glycoprotein from human tumor

R7 cells that over-express the MDR phenotype. Single band in lane
two represents protein found in sample found in Mytilus
californianus gill tissue. Proteins traveled toward bottom of page.

Figure 7. Rhodamine concentration dependence. MDR activity found
at 1 nM rhodamine. Mytilus californianus gill tissues were incubated
for one hour in low, varying concentrations of rhodamine with and
without 22 UM verapamil. Fluorescence represented as raw data.
Data reported as mean fluorescences with standard deviations.

Percent differences are indicated above each point.
Figure 8. Rhodamine concentration dependence. MDR activity is
unresolvable above 2.0 uM rhodamine. Mytilus californianus gill

MDR in Mytilus; Bard and Cornwall, 1992; 20
tissues were incubated for one hour in high, varying concentrations
of rhodamine with and without 22 uM verapamil. Fluorescence
represented as raw data. Data reported as mean fluorescences with
standard deviations. Percent differences are indicated above each
point.
Eigure 9. Efflux pump inhibition model. (a) Under normal conditions,
diffusion and an efflux pump work together to decrease intracellulai
rhodamine concentration. (b) With the efflux pump inhibited, as in
the presence of verapamil, diffusion is the only major force driving
the rhodamine out, so intracellular rhodamine concentration
decreases at a slower rate.
Eigure 10. Problems with high rhodamine concentrations. (a) When
rhodamine diffusion and MDR efflux are on the same order of
magnitude, the inhibition of the efflux pump is noticeable. (b) When
the original rhodamine concentration is increased and verapamil is
added, the initial (Time = 0) intracellular rhodamine concentration is
greatly increased. Thus the concentration gradient driving
rhodamine out of the cells in the rinse is on a higher order of
magnitude than the MDR efflux pump. Therefore, inhibition of the
efflux pump is not as noticeable.
MDR in Mytilus; Bard and Cornwall, 1992; 21
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