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Vol. 303, Issue 3, 1095-1101, December 2002
/) Mouse Intestine
Gut Barrier Group, University of Manchester and Salford Hospitals Trust, Hope Hospital, Salford, (R.H.S., J.T.-H., N.B.H., G.W.), and Pharmaceutical Sciences, Pfizer Global Research & Development, Kent, United Kingdom (M.H.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Information on the extent to which xenobiotics interact with
P-glycoprotein (PGP) during transit through the intestine is crucial in
determining the influence of PGP on oral drug absorption. We have
recently described a novel use of isolated ileum from PGP-deficient
mdr1a(
/) mice to resolve PGP- and non-PGP-dependent drug efflux and
provide a definitive measure of intrinsic drug permeability without
recourse to inhibitors (Stephens et al., 2002
). The present study uses
this approach to investigate the impact of PGP on intestinal
permeability of paclitaxel and digoxin in different regions of the
mouse intestine (jejunum, ileum, and proximal and distal colon).
Absorption of paclitaxel and digoxin in tissues from wild-type mice was
low and showed little regional variation. In contrast, absorption of
both drugs was markedly higher in mdr1a(/) intestine, although the
increase was highly region-dependent, with the ileum and distal colon
showing the greatest effect and much smaller changes in the jejunum and
proximal colon. These effects were accompanied by the abolition of
paclitaxel and digoxin secretion in mdr1a(/) mice, suggesting that
regional variations in intestinal permeability are masked by
differential PGP expression, confirmed by immunoblotting studies.
Propranolol permeability, which is not influenced by PGP, showed
similar regional variation in both wild-type and mdr1a(/) tissues,
suggesting that differences are at the level of transcellular
permeability. These data suggest that the ileum and the distal colon
are regions of relatively high transcellular permeability for
xenobiotics that are compensated by enhanced expression of PGP.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The
xenobiotic transporter P-glycoprotein (PGP), the protein product of the
MDR1 gene, was initially associated with the development of multidrug
resistance in tumor cells (Hunter and Hirst, 1997). It is now
understood that this ABC protein forms an integral part of the
intestinal barrier together with several other polyspecific efflux
transporters, including members of the multidrug resistance-related protein (MRP) family and breast cancer resistance protein (BCRP) (Hunter and Hirst, 1997; Makhey et al., 1998; Gotoh et al., 2000; Jonker et al., 2000; Litman et al., 2001). The ability of PGP to
interact with a broad range of natural and synthetic xenobiotics, drugs, and naturally occurring toxins or food constituents (Hunter and
Hirst, 1997; Walle and Walle, 1999; Litman et al., 2001) strongly suggests that its ability to limit oral drug absorption is part of a
broader protective role as a modulator of intestinal permeability. Indeed, growing awareness of the wider physiological context in which
PGP resides has linked this and other efflux transport proteins to the
regulation of electrolyte transport, apoptosis, and inflammation (Johnstone et al., 2000).
Clearer definition of these physiological and pharmacological roles,
especially those directly related to xenobiotic transport, requires a
better understanding of the extent to which compounds interact with PGP
as they move sequentially along the cephalocaudal axis of the gut. This
will depend on how transporter activity varies along the gut and
whether non-PGP transporters contribute to efflux. MRP and BCRP can
interact with PGP substrates and appear to be expressed heterogeneously
in the intestine (Jonker et al., 2000; Mottino et al., 2000; Litman et
al., 2001; Maliepaard et al., 2001). Several other variables including
membrane composition and passive epithelial permeability may also be
important in determining which xenobiotics are transported effectively
(Brasitus and Schachter, 1984; Fagerholm et al., 1997; Ungell et al.,
1998).
The way in which the activity of PGP and other efflux transporters
varies along the intestine remains poorly defined. Several studies have
noted significant regional differences in PGP expression at the mRNA
level with expression increasing from the small intestine to the colon
(Fojo et al., 1987; Fricker et al., 1996; Li et al., 1999). However,
other studies have reported a different distribution (Cordon-Cardo et
al., 1990; Chianale et al., 1995), which may in part be due to species
differences in transporter expression. It is also noteworthy that
little work has been done to examine regional variation of any
transporter within the large intestine, a physiologically important
site for xenobiotic efflux (Penny and Campbell, 1994).
We have recently described a new in vitro approach using isolated ileum
from wild-type mice and mdr1a(/) mice, which express no functional
PGP in the intestine (Stephens et al., 2002). This model provides
functional analysis of PGP without recourse to inhibitors and allows a
compound's true passive permeability across the gut epithelium to be
defined. In addition, the contribution of non-PGP transporters can be
more readily identified. Using an extension of this approach, the
present study has sought to produce a functional profile of PGP
activity and expression along the cephalocaudal axis of the mouse small
and large intestine. We have also sought to investigate aspects of the
broader role that PGP might play in controlling the passive
permeability of the gut epithelium to xenobiotics.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Materials.
[G-3H]Digoxin and
L-[4-3H]propranolol were purchased
from PerkinElmer Life Sciences (Hounslow, Middlesex, UK), and
[G-3H]paclitaxel was obtained from Moravek
Biochemicals, Inc. (Brea, CA).
D-[1-14C]Mannitol was obtained from
Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK).
MK571
(3-([{3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl}-{(3-dimethyl)-amino3-oxopropyl)-thio}-methyl]thio}-methyl]thio)propionic acid) was purchased from Affiniti Research Products Ltd.
(Exeter, UK). All other compounds were obtained from Sigma-Aldrich
Chemical Co. Ltd. (Poole, UK). Wild-type FVB [mdr1a(+/+)] mice were
obtained from local barrier maintained stock. Transgenic mdr1a(/)
mice were obtained from Taconic M&B A/S (Bomholtgärd, Denmark).
Animals and Tissues.
Nonfasting male mice (10-20 weeks,
20-36 g) were killed by cervical dislocation. Intestinal tissue from
the region of interest was then immediately removed and the lumen
flushed with ice-cold, bicarbonate-buffered Ringer solution containing
146 mM
1 Na+, 4.2 mM1 K+, 1.2 mM1 Ca2+, 1.2 mM1 Mg2+, 125.8 mM1 Cl, 26.6 mM1
HCO3, 1.2 mM1
HPO42, 0.2 mM1
H2PO4,
and 10 mM D-glucose, which had been equilibrated to pH 7.4 by bubbling with 5% CO2/95%
O2.
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). Mounting was completed
within 20 min of removal from the animal. All procedures involving
animals conformed to current UK Home Office legislation.
Permeability Studies.
Drug transport across intestinal
tissues was measured by methods similar to those described previously
(Collett et al., 1999; Stephens et al., 2002). Intestinal mucosa was
bathed on the mucosal (apical) and serosal (basolateral) surfaces with
5 ml of bicarbonate-buffered Ringer, pH 7.4 (as above). Spontaneous
tissue open-circuit potential difference, short-circuit current
(ISC), and transepithelial electrical resistance
(RT) were monitored periodically throughout the
experiment; otherwise, tissues were maintained under open-circuit
conditions. A 30-min equilibration period was allowed prior to
beginning permeability measurements to allow stabilization of
electrical parameters. Unidirectional apical (A) to basolateral (B) and
B-A permeability of digoxin, paclitaxel, or propranolol was measured
following addition of radiolabeled (0.2 µCi · ml1, 7.4 kBq · ml1), and unlabeled drug (20-100 µM) to A or
B chambers. Drugs were added as stock solutions in dimethyl sulfoxide
giving a final solvent concentration of 0.02 to 0.3%. In the case of
digoxin, unlabeled drug was added to an aliquot of transport buffer
from the "donor" chamber, which was vortexed for 1 min and returned to the chamber to ensure thorough mixing. For all three compounds, 1-ml
samples were removed from the "receiver" chamber at
t = 0 and after each of six 40-min periods and replaced
with fresh transport buffer. Samples (100 µl) were also taken from
the donor chamber at the beginning of the first period and at
the end of the experiment to monitor any changes in donor drug
concentrations during the experiment and to safeguard mass balance.
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PappA-B).
If the ratio of
PappB-A/PappA-B is greater
than 1, this indicates that net secretion (B-A asymmetry or efflux) is
taking place. Where values for net Papp are
shown, positive values (i.e., B-A > A-B) indicate secretion,
whereas negative values represent net absorption in the A-B direction (i.e., B-A < A-B).
The results as expressed are the mean of the Papp
values over the indicated number of flux periods, usually three. In the case of all three drugs (digoxin, paclitaxel, and propranolol), the
flux was shown to remain linear over the time course of the experiment.
In some experiments using mdr1a(+/+) tissues, the PGP inhibitor
quinidine (200 µM) was added to both apical and basolateral chambers
after the third 40-min flux period. The data from three to six tissue
sections in which Papp has been measured in each
of the A-B or B-A directions are then pooled.
Integrity of the paracellular route in mouse tissues was assessed using
mannitol. Following tissue equilibration, 100 µM mannitol, containing
0.2 µCi · ml1
[14C]mannitol, was added to the apical chamber,
and samples were taken from the basolateral chamber at 60-min
intervals. Papp was calculated as described above.
Viability of Mouse Tissues.
Changes in transepithelial
resistance, RT, were used as the primary
indicator of tissue viability, and RT values were
monitored throughout the course of the experiment. Distal colon
exhibited the highest RT [mdr1a(+/+), 124.2 ± 5.; mdr1a(/, 130.5 ± 7.6 
· cm2], whereas the other three segments were
lower and similar to each other [mdr1a(+/+), DJ: 64.9 ± 2.7, I:
72.7 ± 1.5, PC: 86.84 ± 2.9 · cm2; mdr1a(/), DJ: 86.3 ± 4.3, I:
75.0 ± 1.6, PC: 81.3 ± 3.0 · cm2]. RT values did not
vary significantly during the experiment, remaining within 15% of the
starting value, and tissues were excluded where
RT fell by more than this. As a further test of
tissue viability, the cAMP agonist, forskolin (1 µM), was added
basolaterally at the conclusion of each experiment. This elicited a
sharp and sustained rise in Isc (>25% increase)
in viable tissues, indicating stimulation of electrogenic
Cl secretion (Warhurst et al., 1996). Any
tissue segments that failed to respond to forskolin at the end of the
experiment were discarded.
Immunoblot Analysis of P-Glycoprotein Expression.
Epithelial
cells were isolated from the same four regions of mouse intestine
described above using the procedures below, which gave maximum cell
yield. Segments of small and large intestine were flushed twice with
ice-cold Ca2+- and
Mg2+-free bicarbonate-buffered Ringer solution
[121 mM1 NaCl, 25 mM1
NaHCO3, 1.6 mM1
KHCO3, 1.2 mM1
K2HPO4, 0.2 mM1
KH2PO4, 10 mM1
D-glucose, 0.05 dithiothreitol, pH 7.4].
Segments were everted over a glass rod and incubated for 30 min (small
bowel) or 60 min (large bowel) in Ca2+- and
Mg2+-free Ringer as above, continuously gassed
with 5% CO2/95% O2, at
4°C. Tissue was then transferred to Ca2+- and
Mg2+-free bicarbonate-buffered Ringer solution
(as above) containing 5 mM EDTA (jejunum, ileum) or 10 mM EDTA (colon)
and incubated for 15 min at 4°C. Following this, segments were
attached to a Vibromixer (Chemap AG, Volketswil, Switzerland)
and vibrated at 60 Hz for 2 to 5 min in 30-s bursts, replacing the EDTA
solution after every two or three bursts. Light microscopy of the
bathing solution fractions revealed that this procedure isolated
primarily villus cells, mainly as intact units. Once fractions began to contain significant numbers of crypts and little or no villus material
was evident, the bathing solution was warmed to room temperature and
the surface of the tissue was subjected to gentle mechanical disruption
by rubbing it with a fine glass rod. This resulted in copious
production of free crypts. Once more, sequential fractions of bathing
solution were examined by light microscopy to determine the point at
which little or no additional material could be removed. At this point,
all the collected fractions were pooled, washed three times by
centrifugation at 2000g/4°C in complete bicarbonate-buffered Ringer solution without dithiothreitol (see Animals and Tissues) and stored at 70°C until required.
-actin antibody (clone AC15, 1: 5000 dilution in TBS-T; Sigma-Aldrich). The secondary antibody was goat
anti-mouse IgG horseradish peroxidase-conjugated antibody (Bio-Rad
Laboratories Ltd., Hemel Hempstead, UK) used at a dilution of 1:5000.
Blots were developed using the ECL system (Amersham Biosciences UK, Ltd.).
Statistical Methods. Values are expressed as mean ± S.E.M. (n). Statistical analyses were carried out using Arcus Quickstat (Research Solutions, Cambridge, UK) Papp values and the effects of inhibitors on substrate fluxes were tested for significance using one-way ANOVA with an appropriate post hoc test (Bonferroni) with a significance level of 5%.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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PGP Ablation Unmasks Differences in Regional Permeability of
Paclitaxel and Digoxin along Mouse Intestine.
Initial studies used
two compounds known to interact with PGP, paclitaxel and digoxin, to
investigate the effects of PGP on drug permeability in different
regions of the mouse intestine. In mdr1a(+/+) mice, paclitaxel
exhibited a low absorptive permeability (A-B) along the intestine with
relatively small regional variations, although ileal permeability was
significantly higher (2.26 ± 0.2 × 106 cm · s1,
n = 5) than any other region (p < 0.05) with proximal colon having the lowest permeability (1.08 ± 0.2 × 106 cm · s1, n = 5) (Fig.
2A). Tissues from mdr1a(/) mice
showed significantly higher paclitaxel absorptive permeability
throughout the intestine, although the degree of increase varied
markedly from region to region (Fig. 2A). Large increases in paclitaxel
absorption were observed in the ileum and distal colon with
permeability 
5-fold higher than in the equivalent tissues from
mdr1a(+/+) animals. Increases in permeability in distal jejunum and
proximal colon were more modest, being only 2-fold higher. Comparison
of paclitaxel's efflux in mdr1a(+/+) and mdr1a(/) tissues show an
opposite profile (Fig. 2B). mdr1a(+/+) tissues showed high levels of
paclitaxel secretion, which varied markedly along the intestine, being
greatest in ileum and distal colon and lowest in distal jejunum and
proximal colon, corresponding with regions of high A-B permeability in mdr1a(/) intestine. In contrast, paclitaxel secretion was
completely abolished in mdr1a(/) tissues from all intestinal
regions (Fig. 2B). These data confirm PGP as the sole mediator of
paclitaxel secretion in small and large intestine tissues. Removal of
PGP reveals marked, region-dependent differences in intestinal
permeability of this compound.
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/) mice are due specifically to the
removal of PGP, we reasoned that it should be possible to reproduce the
region-dependent increases in paclitaxel absorption in mdr1a(+/+)
tissues using the PGP inhibitor quinidine. Figure 3 shows the effect of 200 µM quinidine
on the absorptive permeability of paclitaxel in mdr1a(+/+) tissues.
Although there are quantitative differences between the
inhibitor-induced increases in permeability and those seen in
mdr1a(/) mice, the region-specific increases in paclitaxel
permeability observed in mdr1a(/) tissues are clearly reproduced by
treatment with quinidine.
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/)
tissues (Fig. 4A). Digoxin permeability
decreased from small intestine to colon in mdr1a(+/+) animals [distal
jejunum, 2.84 ± 0.3; ileum, 3.06 ± 0.6; proximal colon,
1.73 ± 0.7, and distal colon, 1.64 ± 0.4 × 106 cm · s1
(n = 3-4 in each group)]. Digoxin absorptive
permeability was significantly higher in mdr1a(/) tissues with a
regional profile similar to that seen with paclitaxel, with the
greatest increase occurring in ileum and distal colon. It was
noticeable, however, that increases in digoxin absorptive permeability
(ranging from 1.5-3 fold) in mdr1a(/) tissues were lower than
those seen with paclitaxel (2- to 5-fold). The abolition of digoxin
secretion in mdr1a(/) tissues (Fig. 4B) confirms the importance of
this transporter in modulating digoxin absorption along the gut. Once again, the distribution of secretory activity corresponded with regions
of high A-B permeability and mirrored that seen with paclitaxel.
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Molecular Expression of PGP in Mouse Intestine.
To complement
the functional studies described above, the molecular expression of PGP
was measured in epithelial cell preparations from the four regions by
Western blotting. Figure 5 shows regional expression of PGP for epithelial preparations from mdr1a(+/+) mice
standardized against -actin. In the small intestine, the ileum
exhibits the highest PGP expression while in the large intestine maximum expression occurs in the distal colon. Therefore, the distribution of PGP expression is consistent with the functional data
on paclitaxel and digoxin permeability.
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Propranolol Exhibits Regional Differences in Absorptive
Permeability in Both mdr1a(+/+) and mdr1a(/) Tissues.
The
above data indicate that PGP is the only efflux transporter involved in
modulating paclitaxel and digoxin permeability along the mouse
intestine. As a result, the absorptive permeability of these compounds
measured in mdr1a(/) tissues should be indicative of their
true passive permeability across the gut epithelium. One
explanation for the regional differences in digoxin and paclitaxel permeability in mdr1a(/) tissues is that this represents inherent differences in transcellular permeability along the gut epithelium, which are unmasked when PGP is removed. If this is the case,
high-permeability compounds, such as propranolol, that cross the
epithelium by a passive transcellular process and are unaffected by PGP
(Stephens et al., 2002) would be expected to exhibit a similar pattern
of regionality in both mdr1a(+/+) and mdr1a(/) tissues. Figure 6A shows that the absorptive permeability
of propranolol exhibits regional differences in absorptive permeability
in both mouse strains that are qualitatively similar to those seen with
paclitaxel and digoxin in mdr1a(/) tissues. Interestingly,
absorptive propranolol permeability was significantly higher in
mdr1a(+/+) than in mdr1a(/) intestine in all regions except the
distal colon (p < 0.001, Fig. 6A).
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/) tissues. Mannitol permeability was
significantly higher in jejunum than in ileum in both mouse strains
(p < 0.05), but with no further reduction in
permeability in colonic segments. The fact that mannitol and propranolol have clearly different permeability profiles supports the
contention that the regional variation in propranolol permeability, and
by inference that of digoxin and paclitaxel, is specific to the
transcellular route.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Information on the extent of interaction of drug molecules
with PGP and other efflux transporters as they move sequentially along
the cephalocaudal axis of the intestine is crucial to understanding the
likely impact of these transporters on drug absorption. This is
particularly true of controlled release drugs with target sites in
the lower ileum or colon. We investigated the role of PGP in regulating
passive transcellular permeability along the mouse intestine in vitro.
The approach used has been to compare the regional permeability of
two compounds we know to be "pure" PGP substrates (Litman et al.,
2001; Stephens et al., 2002) in intestinal tissues from mdr1a(+/+) (WT)
and mdr1a(/) mice. This approach offers several advantages over
existing in vitro systems: the influence of PGP on drug permeability
can be characterized without recourse to inhibitors, the role of
non-PGP transporters can be identified, and mdr1a(/) tissues can
provide a definitive measure of a compound's passive A-B permeability
in the absence of PGP-mediated secretion (Stephens et al., 2002). The
increase in absorptive permeability when moving from WT to mdr1a(/)
tissues may be used as an indicator of the level of expression and
functional distribution of PGP in WT tissues. In WT intestine, PGP
effectively restricts paclitaxel and digoxin permeability resulting in
low levels of absorption that vary little from jejunum to distal colon. However, similar studies in tissues from mdr1a(/) animals unmask a
marked increase in the absorptive permeability of both drugs which is
highly region-dependent with the rank order ileum > distal colon > jejunum > proximal colon. Such findings are
consistent with regional differences in passive transcellular
permeability of the intestinal epithelium that are presumably masked by
the parallel expression of PGP in WT tissues.
The regional distribution of PGP activity observed here conflicts with
the generally accepted view that expression of PGP increases
progressively from duodenum to colon, at least at the level of mRNA
(Fojo et al., 1987; Fricker et al., 1996). However, other studies have
observed an apparent expression peak in the ileum (Trezise et al.,
1992; Chianale et al., 1995) but did not suggest a second PGP
expression peak in the distal colon like that reported here. There have
been relatively few studies of regional PGP transporter activity,
arguably a more reliable measure. In vitro measurements of drug efflux
along the rat intestine have suggested peak PGP activity in ileum with
lower levels in upper small intestine and colon (Chianale et al., 1995;
Yumoto et al., 1999; Kunta et al., 2000; Stephens et al., 2001). The
present study, utilizing a more unequivocal measure of PGP-mediated
drug transport using mdr1a(/) mouse intestine, offers clear
evidence that PGP-mediated activity increases markedly from jejunum to ileum. As a result, when mdr1a(/) mouse intestine is studied, which
expresses no PGP, the ileum is almost 3 times more permeable to
paclitaxel than the jejunum, whereas in WT intestine, paclitaxel permeability is similar in both regions. Subdividing the colon into
proximal and distal segments also highlighted a significant, and
previously unsuspected, heterogeneity in PGP activity in this region.
The transition from ileum to proximal colon is accompanied by a fall in
PGP activity to a level similar to that observed in jejunum. However,
there was a clear increase in PGP activity in distal colon, borne out
by molecular evidence. The effect was particularly noticeable with
paclitaxel, with a 3-fold increase in absorptive permeability compared
with proximal colon in mdr1a(/) tissues. This finding may have
implications for delivery systems that aim to target drug release to
the distal intestine (Yang et al., 2002), although this would clearly
depend on similar regional differences in PGP expression and drug
permeability occurring in other species, particularly human. It is
interesting in this respect that coadministration of the PGP inhibitor
GF120918
(N-{4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]phenyl}-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide) caused a 3-fold increase in the apical to basolateral absorption of somatostatin receptor peptidomimetics in rat distal colon
in vitro but had no effect in proximal colon (Emery et al., 2002).
The higher levels of absorption of paclitaxel and digoxin observed in
mdr1a(/) intestine are expected given that these compounds are
subject to PGP-mediated efflux. The complete abolition of efflux of
both compounds in mdr1a(/) intestine shows that their permeability
is unaffected by other intestinal transporters and, as a result,
permeability measured in mdr1a(/) conditions should be a measure of
their "true" passive permeability in intestine. The regional
variation in permeability under these conditions suggests that there
are inherent differences in transcellular permeability in the gut,
which are masked by the action of efflux transporters. If this is the
case, a compound that is absorbed by a passive transcellular route but
with intestinal permeability that is not significantly influenced by
efflux transporters should exhibit a similar regional profile in both
mdr1a(/) and WT tissues. Propranolol meets these criteria(Ungell et
al., 1998; Letrent et al., 1999) and does indeed show regional
differences in permeability in WT tissues qualitatively similar to
those seen for paclitaxel and digoxin in mdr1a(/) tissues. The high
permeability of propranolol compared with the other compounds may
explain why the regional differences are more modest, since it would be
expected to be nearer the maximal absorptive capacity of the tissue.
Although the underlying reason for observed differences in passive
membrane permeability in different regions is unclear, it could reflect previously described regional variations in membrane lipid composition and fluidity (Brasitus and Dudeja, 1985; Meddings 1989), which may
alter intestinal permeability to lipophilic compounds. Recent evidence
suggests that the level of P-glycoprotein may itself influence the
distribution of cholesterol within the plasma membrane (Garrigues et
al., 2002). Clearly, further studies on the lipid content of intestinal
regions in different species are needed.
There was no evidence that changes in paracellular permeability played
a role in these effects since mannitol displayed a regional
permeability profile completely different from that of propranolol,
digoxin, and paclitaxel. Mannitol permeability was significantly higher
in jejunal tissues, with little difference between ileum and the two
segments of the colon, and values from wild-type and mdr1a(/)
tissues were virtually identical. The absolute values reported in this
study are similar to those described by Ungell et al. (1998) in
isolated rat tissues, although the decrease in permeability from
jejunum to distal colon was greater than that seen in the present
study. Evidence suggests that the profile of mannitol permeability
along the intestine can vary considerably between species (van Meeteren
et al., 1998; Nejdfors et al., 2000).
Is there a physiological rationale for the observed differences in PGP
activity along the gut? PGP is considered to play an important role in
reducing the absorption of potentially toxic xenobiotics such as those
present in the gut lumen either as a result of microbial action in the
gut or by ingestion, e.g., in food (Blackmore et al., 2001). With
regard to the small intestine, the jejunum has a relatively short
transit time (e.g., 40 min in the rat; Kayne et al., 1993), favoring
absorption of lipophilic xenobiotics with high membrane permeability.
Such compounds may partition into the membrane at such a fast rate that
PGP-mediated efflux is balanced by passive influx and net absorption is
unaffected (Litman et al., 2001) In contrast, the transit time in the
ileum is considerably longer (140 min in the rat; Kayne et al.,
1993), and xenobiotics reaching this region will tend to be of lower permeability, making them more susceptible to PGP-mediated efflux. Given that many natural xenobiotics are likely to fall into this second
category, it could be argued that there is physiological merit in
having a higher PGP activity in the ileum. The reason for colonic
heterogeneity in PGP activity is not clear, although the ability of
distal colon to dehydrate the luminal contents against a high luminal
hydraulic resistance (Naftalin et al., 1999; Naftalin and Pedley, 1999)
may be a factor. This would be expected to concentrate xenobiotics
reaching the distal colon and may explain the observation of increased
PGP activity in this region. Although more information is needed on the
sites of absorption of natural xenobiotics, and the role of other
transporters such as MRP and BCRP, it is interesting to speculate that
the regional variations in PGP distribution are integral to the
protective function of PGP in the murine gut barrier.
In conclusion, by comparing the permeability of "pure" PGP
substrates in tissues from normal and mdr1a(/) mice, we have demonstrated significant regional variations in transporter activity within both small and large intestine with peaks of activity in distal
small intestine and distal colon. Ablation of PGP reveals inherent
differences in transcellular permeability along the gut, which are
masked under normal circumstances by the action of efflux transporters.
These data may provide a basis for a better understanding of the likely
impact of PGP on the absorption of the drugs as they move along the
intestine but will also be relevant in defining the physiological role
that PGP plays in protecting the gut epithelium from luminal xenobiotics.
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Footnotes |
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Accepted for publication August 27, 2002.
Received for publication July 9, 2002.
This work was supported by Pfizer Global Research and Development.
DOI: 10.1124/jpet.102.041236
Address correspondence to: Dr. Geoff Warhurst, Gut Barrier Group, University of Manchester and Salford Hospitals Trust, Hope Hospital, Eccles Old Road, Salford M6 8HD, UK. E-mail: gwarhurs{at}fs1.ho.man.ac.uk
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Abbreviations |
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PGP, P-glycoprotein; mdr1, multidrug resistance protein 1; MRP, multidrug resistance-associated protein; BCRP, breast cancer resistance protein; ISC, short-circuit current; RT, transepithelial electrical resistance; A-B, apical-to-basolateral; B-A, basolateral-to-apical; Papp, apparent permeability; TBS-T, Tris-buffered saline/Tween 20; ANOVA, analysis of variance; WT, wild-type.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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/) mouse intestine in the study of drug transport mechanisms in vitro.
Br J Pharmacol
135:
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  C. MacLean, U. Moenning, A. Reichel, and G. Fricker Closing the Gaps: A Full Scan of the Intestinal Expression of P-Glycoprotein, Breast Cancer Resistance Protein, and Multidrug Resistance-Associated Protein 2 in Male and Female Rats Drug Metab. Dispos., July 1, 2008; 36(7): 1249 - 1254. [Abstract] [Full Text] [PDF]
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A. Collett, R. H. Stephens, M. D. Harwood, M. Humphrey, L. Dallman, J. Bennett, J. Davis, G. L. Carlson, and G. Warhurst Investigation of Regional Mechanisms Responsible for Poor Oral Absorption in Humans of a Modified Release Preparation of the {alpha}-Adrenoreceptor Antagonist, 4-Amino-6,7-dimethoxy-2-(5-methanesulfonamido-1,2,3,4 tetrahydroisoquinol-2-yl)-5-(2-pyridyl)quinazoline (UK-338,003): The Rational Use of ex Vivo Intestine to Predict in Vivo Absorption Drug Metab. Dispos., January 1, 2008; 36(1): 87 - 94. [Abstract] [Full Text] [PDF]
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 J. M. Schmitz, V. J. McCracken, R. A. Dimmitt, and R. G. Lorenz Expression of CXCL15 (Lungkine) in Murine Gastrointestinal, Urogenital, and Endocrine Organs J. Histochem. Cytochem., May 1, 2007; 55(5): 515 - 524. [Abstract] [Full Text] [PDF]
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M. Ballent, A. Lifschitz, G. Virkel, J. Sallovitz, and C. Lanusse MODULATION OF THE P-GLYCOPROTEIN-MEDIATED INTESTINAL SECRETION OF IVERMECTIN: IN VITRO AND IN VIVO ASSESSMENTS Drug Metab. Dispos., March 1, 2006; 34(3): 457 - 463. [Abstract] [Full Text] [PDF]
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K. W. Ward, G. J. Stelman, J. A. Morgan, K. S. Zeigler, L. M. Azzarano, J. R. Kehler, J. E. McSurdy-Freed, J. W. Proksch, and B. R. Smith DEVELOPMENT OF AN IN VIVO PRECLINICAL SCREEN MODEL TO ESTIMATE ABSORPTION AND FIRST-PASS HEPATIC EXTRACTION OF XENOBIOTICS. II. USE OF KETOCONAZOLE TO IDENTIFY P-GLYCOPROTEIN/CYP3A-LIMITED BIOAVAILABILITY IN THE MONKEY Drug Metab. Dispos., February 1, 2004; 32(2): 172 - 177. [Abstract] [Full Text] [PDF]
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K. Takara, N. Ohnishi, S. Horibe, and T. Yokoyama EXPRESSION PROFILES OF DRUG-METABOLIZING ENZYME CYP3A AND DRUG EFFLUX TRANSPORTER MULTIDRUG RESISTANCE 1 SUBFAMILY mRNAS IN RAT SMALL INTESTINE Drug Metab. Dispos., October 1, 2003; 31(10): 1235 - 1239. [Abstract] [Full Text] [PDF]
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 G. D. Leonard, T. Fojo, and S. E. Bates The Role of ABC Transporters in Clinical Practice Oncologist, October 1, 2003; 8(5): 411 - 424. [Abstract] [Full Text] [PDF]
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,
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p < 0.01 with respect to proximal colon, ANOVA.
Absorptive Papp was significantly higher in
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