Quantitative Targeted Absolute Proteomics of Transporters and

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Quantitative Targeted Absolute Proteomics of Transporters and Pharmacoproteomics-based Reconstruction of P-Glycoprotein Function in Mouse Small Intestine TAKANORI AKAZAWA, Yasuo Uchida, Masanori Tachikawa, Sumio Ohtsuki, and Tetsuya Terasaki Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00196 • Publication Date (Web): 08 Jun 2016 Downloaded from http://pubs.acs.org on June 14, 2016

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Molecular Pharmaceutics

1

Quantitative

Targeted

Absolute

Proteomics

of

Transporters

and

2

Pharmacoproteomics-based Reconstruction of P-Glycoprotein Function in Mouse

3

Small Intestine

4 5

Takanori Akazawa†, Yasuo Uchida†, Masanori Tachikawa†, Sumio Ohtsuki‡, and

6

Tetsuya Terasaki*,†

7 8

†

9

Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai

Division of Membrane Transport and Drug Targeting, Graduate School of

10

980-8578, Miyagi, Japan

11

‡

12

University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Kumamoto, Japan

Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto

13 14

KEYWORDS: small intestine, drug absorption, transporter, P-glycoprotein/multidrug

15

resistance protein 1a, protein quantification, quantitative targeted absolute proteomics,

16

pharmacoproteomics

17

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ABSTRACT: The purpose of this study was to investigate whether a pharmacokinetic

2

model integrating in vitro mdr1a efflux activity (which we previously reported) with in

3

vitro/in vivo differences in protein expression level can reconstruct intestinal mdr1a

4

function. In situ intestinal permeability-surface area product ratio between wild-type

5

and mdr1a/1b (-/-) mice is one of the parameters used to describe intestinal mdr1a

6

function. The reconstructed ratios of 6 mdr1a substrates (dexamethasone, digoxin,

7

loperamide, quinidine, verapamil, vinblastine) and 1 non-substrate (diazepam) were

8

consistent with the observed values reported by Adachi et al. within 2.1-fold difference.

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Thus, intestinal mdr1a function can be reconstructed by our pharmacoproteomic

10

modeling approach. Furthermore, we evaluated regional differences in protein

11

expression levels of mouse intestinal transporters. Sixteen (mdr1a, mrp4, bcrp, abcg5,

12

abcg8, glut1, 4f2hc, sglt1, lat2, pept1, mct1, slc22a18, ostβ, villin1, Na+/K+-ATPase,

13

γ-gtp) out of 46 target molecules were detected by employing our established

14

quantitative targeted absolute proteomics technique. The protein expression amounts of

15

mdr1a and bcrp increased progressively from duodenum to ileum. Sglt1, lat2, and 4f2hc

16

were highly expressed in jejunum and ileum. Mct1 and ostβ were highly expressed in

17

ileum. The quantitative expression profiles established here should be helpful to

18

understand and predict intestinal transporter functions.


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INTRODUCTION

2

In drug development, it is important to predict the rate of drug absorption in the small

3

intestine quantitatively. Various transporters are expressed in small intestinal epithelial

4

cells, and are key players in determining the rate of oral drug absorption.1 Therefore, a

5

quantitative understanding of the transport activity of individual transporters in small

6

intestine is required for predicting the rate of drug absorption.

7

Previous studies of the activities of individual transporters in small intestine

8

have generally employed gene-knockout rodents2-13 or measured changes of the plasma

9

concentration of probe substrates coadministered with inhibitors in humans.14-18 To

10

predict in vivo transporter functions from in vitro findings, transporter-transfected cells

11

have often been used. Adachi et al. have evaluated the PS product, which is defined as

12

the product of apical-to-basolateral permeability coefficient and surface area, by means

13

of transcellular transport study and in situ intestinal perfusion study, respectively.2 They

14

reported that in vitro MDR1 activity (in vitro PS product ratio between

15

MDR1-transfected cells and parental cells) was correlated with intestinal MDR1 activity

16

(in situ PS product ratio between mdr1a/1b (-/-) and wild-type mice).2 However, the

17

absolute values of the in vitro PS product ratio were not the same as those of in situ PS

18

product ratio.2 One of the causes of differences of MDR1 activity between in vitro and

19

in vivo is considered to be differences of MDR1 protein expression levels in the two

20

situations. Therefore, it is important to take account of this difference of protein

21

expression levels of transporters for quantitative prediction.

22

To address this problem, we have proposed a pharmacoproteomics (PPx)-based

23

reconstruction method.19-21 In vivo transporter function is the outcome of both intrinsic

24

activity (transport activity per transporter protein) and protein expression level. The 3 ACS Paragon Plus Environment


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former can be obtained by means of in vitro transcellular transport studies and the latter

2

by means of measurements of in vivo protein expression level in cells expressing the

3

transporter. The concept of our PPx-based reconstruction method is therefore to

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reconstruct the in vivo function of a targeted transporter by integration of the separately

5

determined intrinsic activity and in vivo protein expression level. It has been

6

demonstrated that the efflux activities of several MDR1/mdr1a substrates at the

7

blood-brain barrier (BBB) could be accurately reconstructed in mouse and monkey by

8

employing this method.19-21 Hence, we anticipated that the intrinsic transport activities

9

of individual intestinal transporters could be identified by applying this method to the

10

transporters expressed in small intestine.

11

An important issue for such reconstruction in the small intestine is what kind of

12

samples should be used to obtain the values of the absolute protein expression levels. In

13

previous work, various methods, such as scraping the mucosa from the underlying

14

lamina propria mucosae and muscular layer with a glass microscope side22 or blocking

15

Ca2+-dependent epithelial cell adhesion with EDTA,23 have been employed to isolate

16

small intestinal epithelial cells, in which transporters are expressed. In isolation of

17

intestinal epithelial cells, the EDTA procedure affords epithelial cells with relatively

18

little contamination from lamina propria mucosae or muscular layer, as compared with

19

scraping the mucosa from the underlying lamina propria mucosae and muscular layer.

20

Further, absolute protein expression analyses of transporters in isolated intestinal

21

epithelial cells has been performed with samples of crude membrane fractions.24,25 In

22

the present study, we isolated intestinal epithelial cells with EDTA and quantified the

23

absolute protein expression levels in the plasma membrane fractions, because the EDTA


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1

procedure afforded epithelial cells with higher purity and the plasma membrane is the

2

site of functional activity of transporters.

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To verify whether the PPx-based reconstruction strategy is applicable to

4

intestinal transporters, it is important to choose a model transporter for which the

5

observed activity can be determined in vivo without interference from the activities of

6

the other transporters. MDR1/mdr1a is a major contributor to drug transport in the small

7

intestine.2-4,9,13-16,26 Adachi et al. reported that efflux transport of several mdr1a

8

substrates was not affected by other transport systems in mouse jejunum.2 Therefore, if

9

we use mdr1a and its substrates as employed in Adachi’s study, we can evaluate whether

10

the PPx-based method is able to reconstruct the transport functions of intestinal

11

transporters.

12

The primary aim of this study was to examine whether transport activities of

13

mdr1a in mouse small intestine can be reconstructed from in vitro transport activities by

14

integrating the protein expression levels in the plasma membrane fractions of mouse

15

jejunal epithelial cells isolated with the EDTA method23 and in vitro mdr1a-transfected

16

cells. Indeed, we found the mdr1a transport activities could be successfully

17

reconstructed, indicating that the absolute protein expression levels of transporters in

18

plasma membrane fraction of epithelial cells isolated with the EDTA method are

19

suitable to reconstruct and understand the transport activities in small intestine. Several

20

transporters exhibit regional differences in intestinal expression.3,7,10,27-32 Thus, it is also

21

important to clarify the absolute expression levels of multiple transporters in each

22

intestinal region for a better understanding of absorption throughout the entire small

23

intestine. Therefore, a secondary aim of this study was to determine the absolute protein

24

expression levels of 46 molecules, including 43 transporters, in mouse duodenum, 5 ACS Paragon Plus Environment


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jejunum, and ileum by using plasma membrane fractions of epithelial cells isolated from

2

those sites by the EDTA method.

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EXPERIMENTAL SECTION

2

Chemicals. All peptides (Tables S1 and S2) were chosen by employing the in

3

silico selection criteria described previously,33 and synthesized by Thermo Electron

4

Corporation (Sedanstrabe, Germany) with > 95% peptide purity. The concentrations of

5

peptide solutions were determined by quantitative amino acid analysis (Lachrom Elite,

6

Hitachi, Tokyo, Japan). Other chemicals were commercial products of analytical grade.

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Animals. Ten male ddY mice (9–10 weeks of age) were purchased from

9

Charles River (Yokohama, Japan). Mice were maintained on a 12-h light/dark cycle in a

10

temperature-controlled environment with free access to food and water; they were

11

denied access to food for 16 h before experiments. All experiments were approved by

12

the Institutional Animal Care and Use Committee in Tohoku University, and were

13

performed in accordance with the guidelines of Tohoku University.

14 15

Preparation of Plasma Membrane Fractions of Mouse Small Intestinal

16

Epithelial Cells and L-mdr1a Cells. We prepared pooled plasma membrane fractions

17

from ten mice for each intestinal segment (duodenum, jejunum, and ileum). Intestinal

18

epithelial cells were isolated by agitation in phosphate-buffered saline (PBS) with 2 mM

19

EDTA and 0.5 mM dithiothreitol as reported with minor modifications (Figure S1).23

20

Under anesthesia with pentobarbital (25.9 mg/kg), the mouse was decapitated and the

21

small intestine was removed. All of the following procedures were performed at 4 °C.

22

The intestine was divided into the duodenum (first 5 cm from pylorus), jejunum

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(proximal two-fifths of the rest of the small intestine), and ileum (distal three-fifths of

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the rest of the small intestine). The duodenum, jejunum, and ileum were cut into 1 cm 7 ACS Paragon Plus Environment


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pieces and incised, respectively. The pieces of the small intestine were agitated in PBS

2

with 2 mM EDTA and 0.5 mM dithiothreitol for 1 hour at 1,000 rpm. The suspension

3

was passed through a 210 nylon mesh to remove the intestinal pieces, and the filtrate

4

was centrifuged at 100,000 × g for 40 min. In the step of isolation of epithelial cells

5

with EDTA, cells were partially broken, and the obtained pellet contained intestinal

6

epithelial cells and cellular structures, including cell membranes. To collect the cells and

7

structures together, we performed centrifugation at high speed (100,000 × g), although

8

cells are typically collected by centrifugation at low speed (e.g. 100-500 × g). We

9

considered that there would be no effect on the purity of the plasma membrane fraction

10

even if the pellet contained the cellular structures, because the obtained pellet was

11

subsequently homogenized by rotated strokes with a Potter-Elvehjem homogenizer and

12

nitrogen cavitation according to our previous report.34 The isolated mouse intestinal

13

epithelial cells were homogenized by 15 up-and-down rotated strokes (1,000 rpm) of a

14

Potter-Elvehjem homogenizer in hypotonic buffer [10 mM Tris–HCl (pH 7.4), 10 mM

15

NaCl, 1.5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, and 1 % (v/v) protease

16

inhibitor cocktails (Sigma Chemical Co., St. Louis, MO, USA)]. The homogenates were

17

kept in ice for 30 min, then homogenized again with 20 up-and-down rotated strokes

18

(1,000 rpm) on ice, and finally subjected to nitrogen cavitation at 450 psi for 15 min at

19

4°C. The resulting homogenates were centrifuged at 10,000 g for 10 min at 4°C, and the

20

supernatants were centrifuged again at 10,000 g for 10 min at 4°C to remove as much

21

debris as possible. The resultant supernatants were centrifuged at 100,000 g for 40 min

22

at 4°C, and then the pellets were suspended in suspension buffer [10 mM Tris-HCl (pH

23

7.4), 250 mM sucrose], layered on top of a 38 % (w/v) sucrose solution, and centrifuged

24

at 100,000 g for 40 min at 4°C. The turbid layer at the interface was recovered, 8 ACS Paragon Plus Environment

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suspended in suspension buffer, and centrifuged at 100,000 g for 40 min at 4°C. The

2

resultant pellets were suspended in the suspension buffer to obtain the plasma

3

membrane fractions. Protein concentrations were measured by the Lowry method using

4

the DC protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA).

5

Mouse mdr1a-transfected LLC-PK1 cells (L-mdr1a) were kindly provided by

6

Dr. Alfred H. Schinkel (The Netherlands Cancer Institute, Amsterdam, The Netherlands)

7

and used under license. L-mdr1a cells were cultured according to the reported method.19

8

On the 4th day after seeding on Transwell filters, L-mdr1a cells were harvested. The

9

plasma membrane fractions of L-mdr1a cells were prepared as described previously;35

10

we did not prepare the plasma membrane fraction of parental LLC-PK1 cells. The

11

harvested L-mdr1a cells were collected by centrifugation at 230 g for 5 min at 4 °C. The

12

cells were suspended in suspension buffer with 1 % (v/v) protease inhibitor cocktails

13

and then subjected to nitrogen cavitation at 450 psi for 15 min at 4 °C. The subsequent

14

procedure for preparation of plasma membrane fraction of L-mdr1a cells was the same

15

as that described for mouse intestinal epithelial cells.

16 17

Protein

Quantification

by

Multiplexed Selected/Multiple

Reaction

18

Monitoring (SRM/MRM) by LC-MS/MS. We quantified the protein expression levels

19

of target molecules with one pooled sample from ten mice for each intestinal segment

20

(duodenum, jejunum, and ileum). The protein expression levels were determined by

21

multiplexed selected/multiple reaction monitoring (SRM/MRM) analysis as described

22

previously.33,36 Samples were digested with a combination of lysyl endopeptidase,

23

trypsin, and a trypsin enhancer (ProteaseMax) (Promega, Madison, Wisconsin, USA)

24

according to our previously reported method.36 9 ACS Paragon Plus Environment


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The digested samples were mixed with stable isotope-labeled peptide mixture

2

as internal standard peptides, then acidified with formic acid, and centrifuged at 17,360

3

× g for 5 min at 4 oC. The supernatants were subjected to LC-MS/MS. The mass

4

spectrometer made use of a triple quadrupole mass spectrometer or Triple TOF5600.

5

Triple quadrupole mass spectrometry was performed with an Agilent 1100

6

HPLC system (Agilent Technologies, Santa Clara, CA, USA) coupled to a triple

7

quadrupole mass spectrometer (API5000 or QTRAP5500; AB SCIEX, Framingham,

8

MA, USA) equipped with Turbo V ion source (AB SCIEX). HPLC was performed with

9

C18 columns (XBridge BEH130 C18, 1.0 mm ID x 100 mm, 3.5 µm particles) (Waters,

10

Milford, USA). Probe peptides of the quantified molecules and their SRM/MRM

11

transitions are listed in Table S1. The measurement and analysis were carried out

12

according to our previous report.37

13

When the signal peak of the target peptide was occluded by background noise

14

in analysis using a triple quadrupole mass spectrometer (API5000 or Qtrap5500), the

15

peptide was measured by employing Triple TOF5600, which is a high-resolution MS

16

analyzer. Triple TOF5600 system analysis was performed with a NanoLC-Ultra 1D plus

17

system (Eksigent Technologies, Dublin, CA, USA) coupled with a cHiPLC-nanoflex

18

system (Eksigent Technologies) and a Triple-TOF5600 (AB SCIEX) equipped with a

19

NanoSpray III ion source (AB SCIEX). The measurement and data analysis were

20

conducted according to our previous reports with minor modifications in the LC

21

conditions as follows.34 Mobile phases A and B consisted of 0.1% formic acid in 98%

22

water and 2% acetonitrile, and 0.1% formic acid in 80% acetonitrile and 20% water,

23

respectively. A 10 µL aliquot of each sample was injected and loaded on the trap column

24

(Nano cHiPLC Trap column 200 µm x 0.5 mm ChromXP C18-CL 3 µm 120Å) 10 ACS Paragon Plus Environment

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(Eksigent Technologies) with mobile phase A (5 µL/min) for 10 min after injection for

2

sample loading (at 0 to 10 min). The trap column was then placed in line with the Nano

3

cHiPLC analytical column (Nano cHiPLC column 75 µm x 15 cm ChromXP C18-CL 3

4

µm 120Å) (Eksigent Technologies). The peptides were separated and eluted from the

5

column at a flow rate of 200 nL/min with a linear gradient as follows: 2% B for 1 min

6

(at 10 to 11 min), 10% B for 5 min (at 11 to 16 min), 10% B to 50% B for 114 min (at

7

16 to 130 min), 50% B to 90% B for 3 min (at 130 to 133 min), 90% B for 6 min (at 133

8

to 139 min), 90% B to 2% B for 1 min (at 139 to 140 min), and 2% B for 20 min (at 140

9

to 160 min). The targeted peptides were simultaneously measured with multiple reaction

10

monitoring (MRM)-high resolution (MRM-HR) mode, known as parallel reaction

11

monitoring in the TripleTOF5600, by using the m/z values of Q1 in Table S2. Twenty

12

peptides (10 unlabeled and the corresponding 10 stable-isotope-labeled ones) were

13

simultaneously measured at maximum in a run. In data analysis by MultiQuant software

14

(AB SCIEX), four SRM/MRM transitions of each targeted peptide were extracted from

15

the raw data (Table S2).

16

For measurement and analysis, each peptide for a target protein was monitored

17

with four kinds of SRM/MRM transitions. When positive peaks were observed in three

18

or four sets of transitions, the amount (fmol) of the target peptide was calculated from

19

the peak area ratio (target peptide/corresponding internal standard peptide) and

20

calibration curve for each transition. Then, the protein expression level (fmol/µg plasma

21

membrane protein) of the targeted protein at each transition was calculated by dividing

22

the amount (fmol) of the target peptide by that of plasma membrane fraction examined

23

(µg plasma membrane protein); this procedure afforded three or four values of protein

24

expression level per target molecule in one sample. The protein expression levels in 11 ACS Paragon Plus Environment


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three or four sets of transitions for one pooled sample from ten mice were used to

2

calculate the average (mean) and variability (S.E.M.), which are shown as mean ±

3

S.E.M. in Table 2.

4

On the other hand, when two or more among the four sets of transitions gave

5

no signal peak in the chromatogram of the target protein samples, the value of the limit

6

of quantification (LQ) was calculated as follows. In the analysis using a triple

7

quadrupole mass spectrometer (API5000 or QTRAP5500), the LQ was defined as the

8

protein concentration (fmol/µg plasma membrane protein) that yields a peak area count

9

of 5000 in the chromatogram, according to our previous report.37 In the analysis using

10

Triple TOF 5600, the LQ was defined as the value giving a peak area count of 1000 in

11

the chromatogram, according to our previous report.34

12 13

Theory for Reconstruction of in situ PSa to b Ratio. PS product is defined as the

14

product of the permeability coefficient in the apical-to-basolateral direction and the

15

surface area of the cell membrane. The in situ intestinal PS product ratio in the

16

apical-to-basolateral direction between wild-type and mdr1a/1b (-/-) mice determined

17

by in situ intestinal perfusion study (in situ PSa to b ratio) is one of the parameters used to

18

reflect intestinal mdr1a function. In this section, we described the theory for

19

reconstruction of

20

direction between mdr1a-transfected LLC-PK1 cell monolayers (L-mdr1a cells) and

21

parental cell monolayers (LLC-PK1 cells) based on in vitro transcellular transport study

22

(in

23

expression level.

vitro

PSa

to b

in situ

PSa to b ratio from PS product ratio in the apical-to-basolateral

ratio), combined with in vivo/in vitro difference in mdr1a protein


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In subsection 1, we define

1

in situ

PSa to b ratio and

in vitro PSa to b

ratio, which were

2

previously reported by Adachi et al.2 In order to reconstruct in situ PSa to b ratio from in vitro

3

PSa to b ratio, in subsection 2, we described the theory for integration of in situ PSa to b ratio

4

and

5

of the assumptions in Supporting Information. In subsection 3, we show that in situ PSa to b

6

ratio can be reconstructed from in vitro mdr1a efflux activity obtained by in vitro

7

transcellular transport study and mdr1a protein expression levels in mouse small

8

intestine and L-mdr1a cell monolayers.

in vitro

PSa to b ratio, together with some assumptions. We discuss the appropriateness

9

Subsection 1; Definition of

10

in situ PSa to b

Ratio and

in vitro

PSa to b Ratio. In the

11

present study, we define

12

product of the permeability coefficient in the apical-to-basolateral direction and the

13

surface area per 1 µg plasma membrane protein of mouse small intestinal epithelial cells.

14

We also define in vitro PSa to b (µL/min/µg plasma membrane protein) as the product of the

15

permeability coefficient in the apical-to-basolateral direction and the surface area per 1

16

µg plasma membrane protein of L-mdr1a cells and LLC-PK1 cells. According to the

17

pharmacokinetic model illustrated in Scheme 1,

18

following equation (eq. 1), reported previously by Adachi et al.:2

in situ

PSa to b (µL/min/µg plasma membrane protein) as the

in situ

PSa

to b

ratio is given by the

19

in situ

PSa to b ratio =

in situ PSa to b in mdr1a/1b (-/-) in situ

PSa to b in WT

= 1+

PSmdr1a in situ PSa, eff + in situ PSb, eff in situ

(1)

20 21

where

22

WT

in situ

PSa to b in mdr1a/1b (-/-) (µL/min/µg plasma membrane protein) and

in situ

PSa to b in

(µL/min/µg plasma membrane protein) represent the PS product in the



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Page 14 of 59

1

apical-to-basolateral direction in mdr1a/1b (-/-) mice and that in wild-type mice,

2

respectively;

3

(µL/min/µg plasma membrane protein) represent the PS products for the

4

mdr1a-mediated efflux in wild-type mice and the apical efflux in mouse small intestine

5

excluding mdr1a-mediated efflux, respectively;

6

membrane protein) represents the PS product for basolateral efflux in mouse small

7

intestine.

PSmdr1a (µL/min/µg plasma membrane protein) and

In the same manner,

8 9

in situ

in vitro

in situ

in situ

PSa,eff

PSb,eff (µL/min/µg plasma

PSa to b ratio is given by the following equation (eq.

2), reported previously by Adachi et al.:2

10

in vitro

PSa to b ratio =

PSa to b in LLC-PK1 = 1+ in vitro PSa to b in L-mdr1a

in vitro

PSmdr1a in vitro PSa, eff + in vitro PSb, eff in vitro

(2)

11 12

where

in vitro PSa to b in LLC-PK1

(µL/min/µg plasma membrane protein) and

13

L-mdr1a

14

apical-to-basolateral direction in LLC-PK1 cells and in L-mdr1a cells, respectively;

15

vitro

16

membrane protein) represent the PS products for the mdr1a-mediated efflux in L-mdr1a

17

cells and the apical efflux of in vitro cell monolayers excluding mdr1a-mediated efflux,

18

respectively;

19

products for the basolateral efflux of in vitro cell monolayers.

in vitro

PSa to b in

(µL/min/µg plasma membrane protein) represent the PS product in the

PSmdr1a (µL/min/µg plasma membrane protein) and

in vitro

in vitro

in

PSa,eff (µL/min/µg plasma

PSb,eff (µL/min/µg plasma membrane protein) represents the PS

20

Unlike the previous pharmacokinetic model,2 the present study takes account

21

of not only transport across the apical and basal membranes, but also that across the

22

lateral membrane (Scheme 1). Tight junction proteins exist in the border between the


Page 15 of 59

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1

apical and lateral membranes and restrict paracellular diffusion.38,39 By contrast, other

2

lateral intercellular junctions (e.g. gap junctions or adherence junctions) are looser than

3

tight junctions, and lateral intercellular spaces are observed.38,39 Farquhar and Palade

4

have shown that intraperitoneally administered hemoglobin penetrated into the lateral

5

intercellular spaces, but did not penetrate beyond the border between the apical and

6

lateral membranes in rat nephron lumen.39 This suggests that the apical membrane

7

should be separated from the lateral one, but the lateral one should not be separated

8

from the basal one in the pharmacokinetic model. Hence, we dealt with the lateral and

9

basal membranes as “basolateral” membrane and constructed an apical-to-“basolateral”

10

direction model (Scheme 1), not an apical-to-“basal” direction model as described by

11

Adachi et al.2

12

Subsection 2; Integration of

13

in situ

PSa to b Ratio and

in vitro

PSa to b Ratio. In

14

this subsection, we integrate eqs. 1 and 2 to reconstruct the

15

vitro PSa to b

16

If test compounds are selective substrates for mdr1a, membrane permeability clearance,

17

except for mdr1a-mediated efflux, can be ascribed to passive diffusion, and eq. 3 is

18

obtained (the process of deriving eq. 3 is described in Supporting Information).

in situ

PSa to b ratio from the

in

ratio obtained by in vitro transcellular transport study across cell monolayers.

19 20

in situ

PSa, eff + in situ PSb, eff = in vitro PSa, eff + in vitro PSb, eff

21 22

Then, eqs. 1 and 2 can be combined to obtain eq. 4 using eq. 3.

23


(3)


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in situ PSa to b

ratio = 1 + in vitro PS a to b ratio - 1 ×

PSmdr1a in vitro PSmdr1a in situ

Page 16 of 59

(4)

1 2

Hoffmeyer et al. and Shirasaka et al. have suggested that MDR1 efflux activity

3

is proportional to its protein expression level in human small intestine and in vitro cell

4

monolayers.26,40 Hence, if there is no difference of intrinsic transport activity of mdr1a

5

(transport rate per mdr1a protein) between in situ and in vitro, the ratio of mdr1a efflux

6

activities in situ and in vitro is identical to the ratio of mdr1a protein expression levels

7

in situ and in vitro, as shown in eq. 5 (the process of deriving eq. 5 is described in

8

Supporting Information):

9

PSmdr1a in situ Amdr1a = in vitro PSmdr1a in vitro Amdr1a in situ

(5)

10 11

where

in situ Amdr1a

(fmol/µg plasma membrane protein) and in vitro Amdr1a (fmol/µg plasma

12

membrane protein) represent the mdr1a expression levels per 1 µg protein of the whole

13

plasma membrane of mouse jejunum and L-mdr1a cells, respectively. In the present

14

study, we quantified mdr1a protein expression levels in the duodenum, jejunum, and

15

ileum in mouse. For reconstruction of

16

because Adachi et al. performed their in situ intestinal perfusion study in jejunum.2

in situ

PSa to b ratio, we focused on the jejunum,

17 18

According to eqs. 4 and 5, in situ PSa to b ratio is converted to eq. 6.

19

in situ PSa to b

ratio = 1 + in vitro PSa to b ratio - 1 ×

in situ Amdr1a in vitro Amdr1a


(6)

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1

Subsection 3; Reconstruction of

2

in situ

PSa

to b

Ratio from In Vitro

3

Experiments. To calculate

4

apical-to-basolateral direction (in

5

monolayers,19 eq. 6 is converted to eq. 7 (the process of deriving eq. 7 is described in

6

Supporting Information)

in vitro

PSa to b ratios from the reported values of Papp in the vitro

Papp,

a to b)

in LLC-PK1 and L-mdr1a cell

7

in situ PSa to b

in vitro Papp, a to b in LLC-PK1

ratio = 1 +

in vitro Papp, a to b in L-mdr1a

- 1 ×

in situ Amdr1a

(7)

in vitro Amdr1a

8 9

where

in vitro

Papp, a to b

in LLC-PK1

and

in vitro

Papp, a to b

in L-mdr1a

represent the permeability

10

coefficient in the apical-to-basolateral direction across LLC-PK1 and L-mdr1a cell

11

monolayers, respectively, determined by in vitro transcellular transport study.

12

Here, in vitro Papp, a to b

in LLC-PK1

and

in vitro

Papp, a to b

in L-mdr1a

are determined by in

13

vitro transcellular transport study across L-mdr1a and parental cell monolayers,

14

respectively. The

15

protein expression levels in mouse small intestine and L-mdr1a cells, respectively.

16

Therefore, in situ PSa to b ratio can be reconstructed from in vitro experiments.

in situ

Amdr1a and

in vitro Amdr1a

are obtained by QTAP analysis of mdr1a

17 18

Prediction of Fa with

in situ

PSa to b Ratio. According to the tube model,41

in situ

19

PSa to b and the fraction absorbed (Fa), which is defined as the fraction absorbed into

20

intestinal epithelial cells from the lumen, can be integrated with the parameter of in situ

21

intestinal perfusion by applying the following equation (eq. 8);42

22



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in situ

PSa to b = -

Q × ln1 - Fa L

Page 18 of 59

(8)

1 2

where Q is the perfusion rate and L is the length of perfused segments. Hence, the PS product ratio between in the presence of MDR1 (wild-type) and

3 4

in the absence of MDR1 (knockout) can be represented by eq. 9;

5

in situ

PSa to b ratio =

ln1 - Fawithout MDR1 ln1 - Fawith MDR1

(9)

6 7

where Fawithout MDR1 and Fawith MDR1 represent the absorbed fraction in the absence of

8

MDR1 (knockout) and that in the presence of MDR1 (wild-type), respectively.

9

Furthermore, eq. 9 can be converted to eqs. 10 and 11.

10

Fawith MDR1 = 1 - 1 - Fawithout MDR11/in situ PSa to b ratio

(10)

Fawithout MDR1 Fawithout MDR1 = Fawith MDR1 1 - 1 - Fawithout MDR11/in situ PSa to b ratio

(11)

11

12 13

Theory for Reconstruction of Effective Absorption Ratio. In the present

14

study, we defined “effective absorption ratio” according to the following equations (eqs.

15

12 and 13).

16

effective absorption ratio =

in situ PSa to b in WT in situ

PSa to b in mdr1a/1b (-/-)

=

1 (12) in situ PSa to b ratio

17


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1

in situ

PSa to b in WT = effective absorption ratio × in situ PSa to b in mdr1a/1b(-/-) (0 < effective absorption ratio ≤ 1)

2

(13)

3 4

Eq. 13 indicates that effective absorption ratio is a parameter reflecting the

5

degree of reduction of intestinal absorption by mdr1a-mediated efflux, and the value of

6

effective absorption ratio is smaller as the compound is a better substrate for mdr1a.

7

Statistical Analysis. All data represent the mean ± S.E.M. The values of S.E.M.

8 9

of

in vitro

PSa to b ratio, reconstructed

in situ

PSa to b ratio, and effective absorption ratio were

10

calculated according to the law of propagation of error as described previously.19 In

11

protein expression analysis, the values of the mean and S.E.M. of the protein expression

12

levels were calculated from the values in three or four sets of transitions in one pooled

13

sample from ten mice. The statistical significance of differences in protein expression

14

levels in the three segments of mouse small intestine was determined by one-way

15

ANOVA followed by Tukey’s post hoc test. A value of p < 0.05 was considered

16

statistically significant.



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1

Page 20 of 59

RESULTS

2

Quantification of Protein Expression Levels of Mdr1a in Plasma

3

Membrane Fraction of Mouse Jejunal Epithelial Cells and L-mdr1a Cell

4

Monolayer. The protein expression levels of mdr1a in the plasma membrane fraction of

5

epithelial cells isolated from mouse jejunum (Figure. S1) and L-mdr1a cell monolayers

6

were quantified by LC-MS/MS. Here, we quantified mdr1a protein expression levels in

7

the duodenum, jejunum, and ileum in mouse. We subsequently focused on the jejunum,

8

because Adachi et al. performed their in situ intestinal perfusion study with mouse

9

jejunum segments.2 The quantitative values were 7.57 ± 0.52 fmol/µg plasma membrane

10

protein in mouse jejunum and 45.7 ± 1.4 fmol/µg plasma membrane protein in L-mdr1a

11

cell monolayers (mean ± S.E.M.).

12

Reconstruction of

13

in situ

PSa

to b

Ratio from

in vitro

PSa

to b

Ratio and In

14

Vitro/In Vivo Difference in Protein Expression Levels of Mdr1a. The

15

ratios for 7 compounds, including 6 substrates of mdr1a (dexamethasone, digoxin,

16

loperamide, quinidine, verapamil, and vinblastine) and 1 non-substrate (diazepam), in

17

mouse jejunum were reconstructed by using eq. 7 (Table 1). The mean values of the

18

absolute protein expression levels of mdr1a in mouse jejunum (7.57 fmol/µg plasma

19

membrane protein) and L-mdr1a cell monolayers (45.7 fmol/µg plasma membrane

20

protein) were adopted for

21

permeability coefficient in the apical-to-basolateral direction across LLC-PK1 cell

22

monolayers (in vitro Papp, a to b in LLC-PK1) and L-mdr1a cell monolayers (in vitro Papp, a to b in

23

L-mdr1a)

24

reconstructed in situ PSa to b ratios of the 7 compounds agreed within a 2.1-fold difference

in situ

Amdr1a and

in vitro

in situ

PSa to b

Amdr1a, respectively. The values of the

for the 7 compounds were taken from our previous report19 (see Table 1). The


Page 21 of 59

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1

with the observed values in mouse jejunum, as reported by Adachi et al.2 (Figure 1,

2

correlation coefficient was 0.853).

3 4

Reconstruction of Effective Absorption Ratio. The observed and

5

reconstructed effective absorption ratios for 7 compounds, including 6 substrates of

6

mdr1a (dexamethasone, digoxin, loperamide, quinidine, verapamil, and vinblastine) and

7

1 non-substrate (diazepam), in mouse jejunum were calculated by using eq. 12 (Figure 2

8

and Table S3). The observed

9

reconstructed

in situ

in situ

PSa to b ratios were taken from Adachi et al.,2 and the

PSa to b ratios were taken from Table 1. The reconstructed effective

10

absorption ratios of the 7 compounds agreed within a 2.1-fold difference with the

11

observed values in mouse jejunum (Figure 2 and Table S3, correlation coefficient was

12

0.824).

13 14

Quantitative Analysis of Membrane Protein Expression in Mouse Small

15

Intestine. Intestinal epithelial cells were isolated from mouse duodenum, jejunum, and

16

ileum and the plasma membrane fractions were prepared. The protein expression levels

17

of 46 proteins in the plasma membrane fraction were examined by LC-MS/MS, and 16

18

of them, including 5 ABC transporters and 8 SLC transporters, were detected in all three

19

intestinal segments (Table 2). The other 30 molecules were not detected in any intestinal

20

segment; their limits of quantification (LQ) were calculated according to our previous

21

reports34,37 as described in experimental section (Table 3).

22

Among ABC transporters, mdr1a and bcrp were expressed at the highest levels

23

in all intestinal segments, followed by abcg5, abcg8, and mrp4. Among SLC

24

transporters, glut1, sglt1, lat2 and its associated protein (4f2hc), pept1, mct1, slc22a18, 21 ACS Paragon Plus Environment


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1

and ostβ were detected. The absolute protein expression levels of both mdr1a and bcrp

2

increased from the proximal to distal intestine and the levels of mdr1a and bcrp in the

3

ileum were 1.8-, and 1.5-fold higher than those in the duodenum, respectively. Sglt1,

4

lat2, and 4f2hc were more highly expressed in jejunum and ileum than in duodenum.

5

Mct1 and ostβ were more strongly expressed in ileum than in duodenum or jejunum.

6

The expression of mrp2, which reportedly plays a role in mouse small

7

intestine,8,9 was under the limit of quantification. Similarly, mrp3 and asbt, which have

8

been immunohistochemically detected in mouse small intestine,43,30 were not detected in

9

the present study.

10 11

In addition to the ABC and SLC transporters, expression of 3 marker proteins, villin1, Na+/K+-ATPase, and gamma-glutamyl transpeptidase (γ-gtp), was detected.

12


Page 22 of 59

Page 23 of 59

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1

DISCUSSION

2

This is the first study to demonstrate that the contribution of mdr1a to lumen-to-blood

3

absorption in mouse small intestine can be reconstructed from in vitro mdr1a transport

4

activity and mdr1a protein expression levels in mouse small intestine and

5

mdr1a-transfected cell monolayers. This result indicates that the previously reported

6

theory underlying PPx-based reconstruction of the function of MDR1/mdr1a at the

7

BBB19-21 is also applicable to mdr1a in mouse small intestine.

8

In the previous reported reconstruction of mdr1a function at the BBB,19 the

9

reconstructed values were calculated using the mdr1a expression levels in whole cell

10

lysate of both brain capillaries and L-mdr1a cells. On the other hand, in the present

11

study, we employed the mdr1a expression in plasma membrane of both small intestine

12

and L-mdr1a cells for reconstruction of the mdr1a functions in small intestine. In the

13

present theory for reconstruction, we defined

14

product in the apical-to-basolateral direction per 1 µg protein of the plasma membrane

15

in mouse small intestinal epithelial cells and L-mdr1a cells, respectively. Accordingly,

16

we assumed that the surface area per 1 µg protein of the plasma membrane in mouse

17

small intestine is equal to that in LLC-PK1 cells, as described in eq. S4 (Supporting

18

Information). If we employ the mdr1a expression levels in whole cell lysate in mouse

19

small intestine and L-mdr1a cells for reconstruction in small intestine, eq. S4 would

20

represent the assumption that there is no difference of surface area of plasma membrane

21

per 1 µg protein of the whole cell lysate between mouse small intestine and LLC-PK1

22

cells. However, microvilli greatly increase the apical surface area per unit cell in small

23

intestine,44 whereas the microvilli in LLC-PK1 cells are sparse.45 Hence, in the present

24

reconstruction, we considered that using the protein expression levels in plasma

in situ

PSa to b and


in vitro

PSa to b as the PS


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1

membrane would reduce the difference of surface area per unit cell between small

2

intestine and LLC-PK1 cells compared with the use of expression levels in whole cell

3

lysate. Therefore, we quantified the mdr1a expression levels in plasma membrane of

4

mouse small intestine and L-mdr1a cells, and established suitable theory for

5

reconstruction of

6

small intestinal epithelial cells.

in situ

PSa to b ratio per 1 µg protein of the plasma membrane in mouse

7

Our achievement opens up the possibility of investigating the intrinsic

8

functions of individual transporters in intestinal absorption. Like mdr1a, other ABC

9

transporters, such as bcrp and mrp2, play important roles in intestinal drug

10

absorption.5-9,17,18 The efflux activities of not only mdr1a, but also bcrp and mrp2 can be

11

obtained by in vitro transcellular transport study across monolayers of cells expressing

12

these transporters.46-49 Therefore, the efflux activities of bcrp and mrp2 for their

13

substrates in small intestine could be also clarified by employing the same PPx-based

14

reconstruction strategy as used for mdr1a.

15

The theory used for PPx-based reconstruction would also be applicable to

16

transporters expressed in colon. Some drugs are absorbed in colon as well as small

17

intestine,50 suggesting that the effect of transporters expressed in colon should also be

18

taken into consideration. The protein expression level and activity of mdr1a in colon are

19

almost the same as those of jejunum and ileum in mouse small intestine.3 Therefore, it

20

would be important to evaluate the effect of mdr1a on colonic absorption, if compounds

21

absorbed in colon are substrates of mdr1a. The contribution of mdr1a to colonic

22

absorption could be evaluated by employing a PPx-based reconstruction model with

23

mdr1a protein expression levels in colon.

24

The theory for PPx-based reconstruction would be applicable to human small 24 ACS Paragon Plus Environment

Page 24 of 59

Page 25 of 59

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1

intestinal absorption as well. Human MDR1-mediated transport activities in small

2

intestine (in

3

activity across human MDR1-transfected and parental cell monolayers together with

4

protein expression levels of MDR1 in plasma membrane fractions of human small

5

intestine and human MDR1-transfected cell monolayers. Furthermore, PPx-based

6

reconstruction allows us to predict Fa for MDR1 substrates. The in situ PSa to b ratio can be

7

predicted from in vitro data by using the PPx-based reconstruction method. Additionally,

8

for selective substrates for MDR1, the values of Fawithout MDR1 follow the kinetics of

9

passive diffusion and can be predicted from in vitro analysis, such as PAMPA

10

models.51,52 Therefore, the Fa values of selective substrates for MDR1 in human can be

11

predicted from in vitro data, using eq. 10. Furthermore, by using eq. 11, it is possible to

12

predict quantitatively the contribution of MDR1-mediated efflux to oral absorption and

13

the increase in the Fa values with maximum inhibition of MDR1 caused by drug-drug

14

interaction (DDI). This is expected to be useful for predicting the absorbed amounts or

15

the risk of toxicity of drug candidates in drug discovery.

situ

PSa

to b

ratio) could be reconstructed from in vitro MDR1 transport

16

In the present theory for reconstruction, we assumed that the permeability

17

coefficients of passive diffusion in apical and basolateral efflux in mouse small

18

intestinal epithelial cells and in LLC-PK1 cells are all equal (eq. S1) as described in

19

Supporting Information. The proportions of phospholipids constructing the plasma

20

membrane have been reported to be different in the apical and basolateral membranes in

21

small intestinal epithelial cells, and plasma membrane in LLC-PK1 cells.53,54 In rat and

22

monkey small intestine, Nishimura et al demonstrated that the permeability coefficients

23

for apical and basolateral efflux of midazolam, which penetrates the cell membrane by

24

passive diffusion, were approximately equal.55 In addition, the proportions of 25 ACS Paragon Plus Environment


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Page 26 of 59

1

phospholipids constructing the plasma membrane have been reported to be different

2

between LLC-PK1 and MDCKII (Madin–Darby canine kidney type II) cell

3

monolayers.54,56 However, Kodaira et al. showed that there is little difference in the

4

passive permeability coefficients of various compounds between LLC-PK1 cell and

5

MDCKII cell monolayers.46 These results indicate that the permeability coefficients of

6

passive diffusion are almost the same, regardless of differences in the proportions of

7

phospholipids constructing cell membranes, and thus underpin our assumption of

8

equivalence of all permeability coefficients in the four kinds of cell membranes in

9

mouse small intestinal epithelial cells and LLC-PK1 cells.

10

There was a large difference in surface area between the apical and basolateral

11

membrane in small intestinal epithelial cells, because abundant microvilli exist at the

12

apical membrane. In rat small intestine, it has been reported that the mucosal surface

13

area taking account of the microvilli was almost 20-fold greater than that without

14

consideration of the microvilli by electron-microscopic morphometry.44 On the other

15

hand, in cultured kidney epithelial cell monolayers, such as LLC-PK1 and MDCK cell

16

monolayers, few microvilli were observed at the apical membranes,45 and the surface

17

area of the apical membrane was reported to be approximately equal to that of the basal

18

membrane in MDCK cells by electron-microscopic morphometry.57 These results

19

suggested that the ratios of the surface areas of the apical and the basolateral membranes

20

per unit cell are greatly different between the small intestine and cultured epithelial cell

21

monolayers. For this reason, in the present study, we took account of the proportion of

22

each cell membrane (% of in situ AM, % of in situ BM, % of in vitro AM, and % of in vitro BM),

23

as described in subsections S1 and S2 in the theory for reconstruction of

24

ratio (Supporting Information). However, the proportion of each cell membrane was not 26 ACS Paragon Plus Environment

in situ

PSa to b

Page 27 of 59

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1

included in the definitive equation for reconstruction (eq. 7). This suggested that the

2

reconstruction of mdr1a efflux activity was not affected by the differences in the

3

proportions of the surface areas in the apical and the basolateral membranes between

4

mouse small intestinal epithelial cells and LLC-PK1 cells.

5

The apical influx (PSinf) might also be different between mouse small intestine

6

and LLC-PK1 cells. Functional analyses of mdr1a was carried out at different pH values

7

in the in situ intestinal perfusion study (pH 6.4)2 and in vitro transcellular transport

8

study (pH 7.4).19 The difference of the pH value could affect the proportions of the ionic

9

and molecular forms of compounds. Various anionic or cationic compounds exhibit

10

different permeability coefficients under different pH conditions in transcellular

11

transport across Caco-2 cell monolayers.58 Thus, the PSinf values might also be different

12

between in situ intestinal perfusion and in vitro transcellular transport. In addition to the

13

difference in pH value, the effects of the unstirred water layer have been reported to be

14

different between small intestine and in vitro cell monolayers.59-61 The influx

15

permeability of compounds with high membrane permeability could be affected by the

16

unstirred water layer. It has been reported that the difference of unstirred water layer

17

thickness caused a ~5 fold difference of the permeability coefficient of compounds with

18

high passive membrane permeability between human small intestine and Caco-2 cell

19

monolayers.62 Thus, PSinf could be different between mouse small intestine and in vitro

20

cell monolayers due to differences in pH or in the unstirred water layer. However, these

21

effects could be offset in the process of deriving the equations for the

22

and the

23

mdr1a (-/-) mice and wild-type mice or L-mdr1a cells and parental LLC-PK1 cells,

24

respectively.

in vitro PSa to b

in situ

PSa to b ratio

ratio (eqs. 1 and 2), unless the PSinf values are different between



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Page 28 of 59

1

The purity of isolated intestinal epithelial cells and the prepared plasma

2

membrane is critical for successful reconstruction of intestinal transporter activity. The

3

isolated cells could contain not only epithelial cells, but also lamina propria mucosae or

4

muscular layer to some extent. In addition, it is possible that the purity of plasma

5

membrane is different between mouse intestinal epithelial cells and L-mdr1a cells.

6

Membrane properties, such as the proportions of phospholipids constructing the plasma

7

membrane, have been reported to be different between mouse intestinal epithelial cells

8

and LLC-PK1 cells.53,54 Therefore, the ratios of plasma membrane proteins in the

9

obtained fractions could be different between small intestine and L-mdr1a cells, and

10

there could be differences in the levels of contamination by other fractions, such as

11

microsomal fraction. If the purity of isolated intestinal epithelial cells and the prepared

12

plasma membrane is low, the reconstructed

13

observed values due to underestimation of protein expression levels of transporters in

14

mouse intestinal epithelial cells. However, the reconstructed values of

15

were in good agreement with observed ones, indicating that there is little difference in

16

the purity of isolated intestinal epithelial cells and prepared plasma membrane between

17

mouse small intestine and L-mdr1a cells. Among the 7 compounds we examined,

18

quinidine, which is the best substrate for mdr1a, showed the most underestimated value

19

of reconstructed

20

2.1-fold smaller than the observed value.2 The effect of the second term of the right side

21

of eq. 6 becomes greater for compounds that are better substrates of mdr1a, because

22

good substrates of mdr1a have high values of in vitro PSa to b ratio. Therefore, even a small

23

difference of purity between mouse small intestine and L-mdr1a cells could contribute

24

to underestimation of the reconstructed values for the best substrates of mdr1a, such as

in situ

in situ

PSa to b ratios would be lower than the

in situ

PSa to b ratio

PSa to b ratio (Figure 1); the reconstructed value of quinidine was


Page 29 of 59

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1

quinidine. To obtain intestinal epithelial cells with extremely high purity, a method such

2

as FACS (fluorescence-activated cell sorter) sorting with epithelial cell marker protein

3

could be used in future studies.

4

There is a possibility that normalization of the protein expression, by dividing

5

the protein expression level of the targeted transporter by that of the membrane marker,

6

could be done to correct for any difference of purity of plasma membrane between

7

mouse jejunum and L-mdr1a cells. We quantified γ-gtp as an apical membrane marker63,

8

and Na+/K+-ATPase as a basolateral membrane marker64 in mouse small intestine (Table

9

2). However, we did not quantify these membrane markers in L-mdr1a cells. LLC-PK1

10

cells are a porcine kidney epithelial cell line, and are not derived from intestine, and we

11

previously showed that the protein expression levels of γ-gtp and Na+/K+-ATPase differ

12

between tissues.33 Therefore, normalization of protein expression levels of transporters

13

with marker proteins appears not to be suitable for the reconstruction of intestinal

14

transporter functions.

15

High substrate selectivity of the targeted transporter in small intestine and

16

LLC-PK1 cells is important for successful reconstruction in our present theory, and

17

contributions of other transporters to transport activity of test compounds should be

18

excluded. In the present reconstruction theory, we assumed that the apical efflux, except

19

for mdr1a-mediated efflux, and the basolateral efflux are due only to passive diffusion.

20

If the transport activity of other transporters enhances the values of in situ PSa, eff and/or in

21

situ

22

according to eq. 1. Hence, reconstructed

23

with the observed ones, if test compounds are substrates of other transporters than

24

mdr1a in mouse small intestine or LLC-PK1 cells. We chose digoxin as a model

PSb, eff, the observed in situ PSa to b ratios would be smaller than the reconstructed ones, in situ

PSa to b ratios would not be in agreement



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1

substrate for mdr1a to verify the PPx-based reconstruction strategy, but digoxin is also a

2

substrate of ostα/β,65 which is a basolateral efflux transporter, and ostβ is expressed in

3

the jejunum (Table 2). According to eq. 1, the actual value of in situ PSa to b ratio would be

4

smaller when ostα/β transport is taken into consideration in basolateral efflux (in situ PSb,

5

eff),

6

assumed that the contribution of ostα/β transport to digoxin absorption in mouse

7

jejunum can be neglected for the following reason. Stephens et al. evaluated the

8

bidirectional (apical-to-basolateral and basolateral-to-apical) permeability of digoxin in

9

isolated jejunum of wild-type and mdr1a (-/-) mice by employing an Ussing chamber

10

technique.3 They found that the permeability of digoxin in the basolateral-to-apical

11

direction was higher than that in the apical-to-basolateral direction in jejunum of

12

wild-type mice, whereas there was no difference of the permeability of digoxin between

13

the basolateral-to-apical and apical-to-basolateral directions in jejunum of mdr1a (-/-)

14

mice. This result indicated that the absorption of digoxin was predominantly restricted

15

by mdr1a, and the contribution of other transporters including ostα/β would be small in

16

mouse jejunal absorption. In addition, in L-mdr1a and LLC-PK1 cells, the transport

17

activity of endogenous porcine kidney transporter can affect the result of reconstruction

18

of mdr1a, although we have not performed protein quantification of ABC and SLC

19

transporters, except for mouse mdr1a, in L-mdr1a cells. If the transport activity of

20

background transporters increases the value of PSa, eff and/or PSb, eff, the value of the

21

vitro PSa to b

22

b

23

which is the value of permeability coefficient in the basolateral-to-apical direction

24

divided by that in the apical-to-basolateral direction, was 0.5-2 for the compounds we

because the

in situ

PSb,

eff

value is increased by ostα/β transport. However, we

in

ratio would be smaller, according to eq. 2, so that the reconstructed in situ PSa to

ratio would be underestimated, according to eq. 6. However, the value of flux ratio,


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1

used (dexamethasone, digoxin, loperamide, quinidine, verapamil, vinblastine, and

2

diazepam) in parental LLC-PK1 cells, as described in our previous report.19 If the

3

transport of test compounds is not affected by other transporters, the value of the flux

4

ratio in parental LLC-PK1 cells would theoretically be 1, so the endogenous porcine

5

kidney transporter appears to have little impact on transport of test compounds in the

6

present study.

7

In the present study, we also defined “effective absorption ratio” as shown in

8

eqs. 12 and 13. Eq. 13 indicates that effective absorption ratio is a parameter reflecting

9

the degree of reduction of intestinal absorption by mdr1a-mediated efflux, and the value

10

of effective absorption ratio becomes smaller for compound that are better substrates for

11

mdr1a. We found that there was a good correspondence between observed and

12

reconstructed values of effective absorption ratio, suggesting that effective absorption

13

ratio can also be reconstructed from in vitro findings (Figure 2 and Table S3).

14

In the analysis of protein expression in small intestine, epithelial cells have

15

been obtained by various methods, such as scraping mucosa from the underlying

16

underlying lamina propria mucosae and muscular layer22 or blocking Ca2+-dependent

17

epithelial cell adhesion with EDTA.23 In the present study, in order to ensure that

18

information on transporter expression reflected transport function, we considered that it

19

would be important to obtain intestinal epithelial cells with high purity and to quantify

20

the absolute protein expression levels of transporters in the plasma membrane, which is

21

the site of action of transporters. Therefore, we isolated epithelial cells from mouse

22

small intestine with EDTA with little contamination of lamina propria mucosae or

23

muscular layer and quantified the absolute protein expression level of mdr1a in the

24

plasma membrane fraction. The fact that we were able to reconstruct the efflux activity 31 ACS Paragon Plus Environment


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1

of mdr1a accurately suggests that the absolute protein expression levels of transporters

2

obtained by the present methods well reflect the in vivo transport activities.

3

In addition to mdr1a, various other transporters are also expressed in small

4

intestine and it is important to understand the differences in their functions. The QTAP

5

methods can simultaneously quantify multiple transporters expressed in small

6

intestine.33 However, some transporters in small intestine exhibit regional differences in

7

expression.3,710,27-32 Hence, it is important to take these issues into account for

8

understanding the absorption in the entire small intestine. We elucidated the absolute

9

expression levels of 46 molecules, including 43 transporters, in mouse duodenum,

10

jejunum, and ileum, using plasma membrane fractions of epithelial cells isolated by the

11

EDTA method.

12

Among the target molecules, 13 transporters were detected (Table 2), while 30

13

transporters were not detected in mouse small intestine (Table 3). The latter 30

14

transporters include molecules such as mrp2, mrp3, and asbt, which have been reported

15

to play a role in intestinal absorption based on studies in knockout mice8,9 or have been

16

immunohistochemically detected in mouse small intestine.43,30 A possible reason why

17

these transporters were not detected in mouse small intestine is insufﬁcient digestion of

18

these molecules by lysyl endopeptidase and trypsin. The efficiency of enzyme digestion

19

is one of the key points for absolute quantification of target proteins. We previously

20

confirmed that no bands larger than 20 kDa were observed by SDS-PAGE after trypsin

21

digestion in brain capillary endothelial cells, liver plasma membrane, renal cortex and

22

medulla plasma membrane of mouse, suggesting that enzyme digestion proceeds

23

efficiently.33 Moreover, in that work, we performed enzyme digestion with only

24

trypsin,33 whereas in the present study we used lysyl endopeptidase and Protease Max in 32 ACS Paragon Plus Environment

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1

addition to trypsin. We reported elsewhere that combined digestion with lysyl

2

endopeptidase, trypsin, and Protease Max gave a dramatic improvement of the digestion

3

rate as compared with single digestion with trypsin.36 Thus, it is likely that enzyme

4

digestion proceeded efﬁciently in brain capillary endothelial cells, liver plasma

5

membrane, renal cortex and medulla plasma membrane of mouse; nevertheless, it would

6

be desirable to confirm the digestion efficiency in mouse small intestine samples in the

7

future.

8

Bcrp plays role in apical efflux of diverse drugs and sulfate conjugates,5-7 and

9

its absolute expression level was as high as that of mdr1a (Table 2). Pept1, which is

10

involved in the intestinal absorption of peptide-like drugs such as β-lactam

11

antibiotics,11,66 showed high-level expression, similar to mdr1a and bcrp (Table 2).

12

In contrast, expression of mrp2, which may be involved in apical efflux of

13

anionic drugs in small intestine8,9 was under the limit of quantification and its

14

expression level was at least 28- and 26-fold lower than those of mdr1a and bcrp,

15

respectively (Tables 2 and 3). In previous studies, mrp2 gene expression was observed

16

in mouse small intestine29 and its contribution to intestinal absorption was confirmed by

17

employing mrp2 (-/-) mice.8,9 In human intestine, MRP2 protein expression was

18

detected in fractions of crude membrane by employing the QTAP technique,24,25

19

suggesting that the intestinal efflux activity of MRP2 in human might be higher than

20

that in mouse. However, drugs for which mrp2 was reported to contribute to intestinal

21

absorption are also substrates of mdr1a or bcrp,8,9,49 making it difficult to elucidate

22

species differences of intrinsic efflux activity of MRP2/mrp2 in intestinal absorption.

23

Evaluation by means of the reconstruction method reported here, or functional analyses

24

with highly selective substrates or inhibitors of MRP2/mrp2 might be useful to address 33 ACS Paragon Plus Environment


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1

this question in the future.

2

For discussion of regional differences of transporter expression in duodenum,

3

jejunum, and ileum, it is important to consider the purity of the intestinal epithelial cells

4

and the prepared plasma membrane. We quantified γ-gtp as an apical membrane

5

marker,63 and Na+/K+-ATPase as a basolateral membrane marker64 in mouse small

6

intestine to confirm that the purity of intestinal epithelial cells and prepared plasma

7

membrane was consistent throughout the duodenum, jejunum, and ileum. In the present

8

study, expression of γ-gtp was highest in the jejunum and regional difference was

9

observed in mouse small intestine (Table 2). Ferraris et al. showed that the specific

10

activity of γ-gtp was highest in the upper jejunum in mouse small intestine.67 Hence, the

11

regional difference in γ-gtp protein expression appears to be well correlated with that in

12

its function. Furthermore, we showed that protein expression of Na+/K+-ATPase was

13

approximately equal in mouse duodenum, jejunum, and ileum (Table 2). The expression

14

levels of α-subunit of Na+/K+-ATPase were reported to be similar in three segments of

15

mouse small intestine.68 These results indicate that the distributions of membrane

16

markers in the apical and basolateral membranes in the present study were in agreement

17

with previous findings, supporting the consistency of purity level of intestinal epithelial

18

cells and prepared plasma membrane in duodenum, jejunum, and ileum, and thereby

19

supporting the validity of the present method for evaluating intestinal regional

20

differences in protein expression levels of membrane transporters.

21

As for protein expression levels in duodenum, jejunum, and ileum, expression

22

of mdr1a and bcrp increased from the duodenum to the ileum (1.8- and 1.5-fold greater

23

in ileum than in duodenum, respectively; Table 2). It has been reported that mRNA or

24

protein expression levels and efflux activities of mdr1a and bcrp increased from the 34 ACS Paragon Plus Environment

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1

proximal to the distal regions in mouse small intestine,3,7,27,28 in concordance with our

2

results. The protein expression level of P-gp in human small intestine has also been

3

reported to increase from the duodenum to the ileum.31,32 For example, Mouly and Paine

4

reported that the protein expression level of P-gp in the ileum was 1.5- to 3.0-fold

5

higher than that in the duodenum in human small intestine,32 implying that regional

6

differences of mdr1a/P-gp expression might be similar in mouse and human. In contrast,

7

unlike mouse small intestine, it has been reported that there was no significant regional

8

difference in BCRP protein expression level in human small intestine,31 implying that

9

there might be species differences in regional expression pattern of BCRP between

10

mouse and human. Ostβ was strongly expressed in the ileum, as compared with the

11

duodenum and the jejunum (Table 2), in accordance with a previous report.30 The

12

present study is the first to find that the protein expression level of ostβ is remarkably

13

higher than those of other transporters in mouse ileum. Ostβ is located at the basolateral

14

surface in mouse ileum; it forms a heteromeric complex with ostα and plays a role in

15

bile acid transport from the intracellular milieu to blood.30 Since we could not design a

16

suitable ostα specific peptide, we performed quantitative analysis of only ostβ.

17

Comprehensive screening to identify substrates of ostα/β has not yet been performed,

18

but it has been reported that ostα/β transports steroidal drugs, such as digoxin, and

19

ostα/β-mediated transport activity was inhibited by various organic anionic drugs

20

including indomethacin, sulfobromophthalein, probenecid, and spironolactone.65

21

Therefore, ostα/β can recognize various organic anionic drugs and might be involved in

22

their transport. Transportability by ostα/β could be an important factor in development

23

of drugs for rapid intestinal absorption.

24

In addition to drug transporters, various nutrient transporters (abcg5, abcg8, 35 ACS Paragon Plus Environment


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1

glut1, sglt1, lat2/4f2hc, pept1, mct1, and ostβ) were expressed at the protein level (Table

2

2). Among these transporters, sglt1, lat2/4f2hc, pept1, mct1, and ostβ showed

3

comparatively high expression levels (Table 2), implying that they play an important

4

role in nutrient absorption in mouse small intestine. Sglt1, lat2/4f2hc, and mct1 were

5

strongly expressed in the lower segment, as compared with the upper segment (Table 2).

6

Indeed, the protein expression levels of lat2 and 4f2hc have been reported to be higher

7

in jejunum and ileum than in duodenum in mouse small intestine.69 Similarly, Pept1

8

expression levels in jejunum and ileum tended to be higher than that in duodenum,

9

although this difference was not statistically significant (Table 2). Sglt1, lat2/4f2hc,

10

pept1, and mct1 recognize glucose, amino acids, di/tri-peptides, and short-chain fatty

11

acids as transport substrates, respectively. The pancreatic juice secreted into duodenum

12

digests food components to generate these substrates. Considering the digestion time,

13

higher expression of these transporters in the distal segment of small intestine could be

14

favorable for effective nutrient absorption, because the concentrations of these

15

substrates might be highest in the distal intestinal segment. In addition, ostα/β plays a

16

role in extraction of bile acids from the small intestine.30 Bile acids aid the absorption of

17

lipophilic substances by micellization. Since bile acids are secreted from the duodenum,

18

the localization of ostα/β in ileum could be favorable for maintaining high

19

concentrations of bile acids in the intestinal lumen to promote absorption of lipophilic

20

substances.

21

In conclusion, we have demonstrated that the contribution of mdr1a to

22

intestinal absorption can be reconstructed by integrating in vitro transport activity per

23

mdr1a molecule and protein expression level of mdr1a in mouse small intestine, using

24

the PPx-based reconstruction method. Furthermore, we determined the absolute protein 36 ACS Paragon Plus Environment

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1

expression levels of 13 transporters in mouse duodenum, jejunum, and ileum. It should

2

be possible to extend the PPx-based reconstruction strategy to establish the individual

3

contributions of other transporters to drug intestinal absorption, and also to predict

4

quantitatively the degree of DDI at intestinal transporters.



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1

ASSOCIATED CONTENT

2

Supporting Information

3

Additional supporting information may be found in the online version of this article at

4

the publisher's web-site:

5

Additional Theory for Reconstruction of in situ PSa to b Ratio.

6

Figure S1. Mouse small intestinal epithelial cells isolated by EDTA treatment.

7

Table S1. Peptide probes and SRM/MRM transitions for triple quadrupole mass

8

spectrometer.

9

Table S2. Peptide probes and SRM/MRM transitions for Triple TOF 5600.

10

Table S3. Comparison of Observed and Reconstruction Effective Absorption Ratios.

11 12

AUTHOR INFORMATION

13

Corresponding Author

14

*Division of Membrane Transport and Drug Targeting, Graduate School of

15

Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai,

16

980-8578, Miyagi, Japan. Voice, +81-22-795-6831; FAX, +81-22-795-6886.

17

E-mail, [email protected]

18 19

Note

20

Regarding conflicts of interest, Tetsuya Terasaki and Sumio Ohtsuki are full professors

21

at Tohoku University and Kumamoto University, respectively, and are also directors of

22

Proteomedix Frontiers Co., Ltd. This study was not supported by Proteomedix Frontiers

23

Co., Ltd., and their positions at Proteomedix Frontiers Co., Ltd. did not influence the

24

design of the study, the collection of the data, the analysis or interpretation of the data, 38 ACS Paragon Plus Environment

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1

the decision to submit the manuscript for publication, or the writing of the manuscript

2

and did not present any financial conflicts. The other authors declare no competing

3

interests.

4 5

ACKNOWLEDGEMENTS

6

We thank Dr. Alfred H. Schinkel for providing L-mdr1a and parental LLC-PK1 cells.

7

We thank Ms. Akiko Niitomi for her secretarial assistance. This study was supported in

8

part by two Grants-in-Aid from the Japanese Society for the Promotion of Science

9

(JSPS) for Scientific Research (S) [KAKENHI: 18109002] and for Scientific Research

10

(A) [KAKENHI: 24249011] and the Nakatomi Foundation.

11 12

ABBREVIATIONS USED

13

ABC, ATP-binding cassette; DDI, drug-drug interaction; LC-MS/MS, liquid

14

chromatography tandem mass spectrometry; LQ, limit of quantification; MDR1,

15

multidrug resistance protein 1; mdr1a, multidrug resistance protein 1a; Papp, apparent

16

permeability coefficient; PPx, pharmacoproteomics; PS, permeability-surface area;

17

QTAP, quantitative targeted absolute proteomics; SLC, solute carrier; SRM/MRM,

18

selected/multiple reaction monitoring; ULQ, under the limit of quantification.

19



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222-33. (54) Morita, S.; Ikeda, N.; Horikami, M.; Soda, K.; Ishihara, K.; Teraoka, R.; Terada, T.; Kitagawa, S. Effects of phosphatidylethanolamine N-methyltransferase on phospholipid composition, microvillus formation and bile salt resistance in LLC-PK1 cells. Febs J. 2011, 278, (24), 4768-81. (55) Nishimura, T.; Amano, N.; Kubo, Y.; Ono, M.; Kato, Y.; Fujita, H.; Kimura, Y.; Tsuji, A. Asymmetric intestinal first-pass metabolism causes minimal oral bioavailability of midazolam in cynomolgus monkey. Drug Metab. Dispos. 2007, 35, (8), 1275-84. (56) Delaunay, J. L.; Breton, M.; Trugnan, G.; Maurice, M. Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100. Biochim. Biophys. Acta. 2008, 1778, (1), 105-12. (57) von Bonsdorff, C. H.; Fuller, S. D.; Simons, K. Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters. Embo. J. 1985, 4, (11), 2781-92. (58) Yamashita, S.; Furubayashi, T.; Kataoka, M.; Sakane, T.; Sezaki, H.; Tokuda, H. Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells. Eur. J. Pharm. Sci. 2000, 10, (3), 195-204. (59) Anderson, B. W.; Levine, A. S.; Levitt, D. G.; Kneip, J. M.; Levitt, M. D. Physiological measurement of luminal stirring in perfused rat jejunum. Am. J. Physiol. 1988, 254, (6 Pt 1), G843-8. (60) Levitt, M. D.; Furne, J. K.; Strocchi, A.; Anderson, B. W.; Levitt, D. G. Physiological measurements of luminal stirring in the dog and human small bowel. J. Clin. Invest. 1990, 86, (5), 1540-7. 48 ACS Paragon Plus Environment

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(61) Korjamo, T.; Heikkinen, A. T.; Monkkonen, J. Analysis of unstirred water layer in in vitro permeability experiments. J. Pharm. Sci. 2009, 98, (12), 4469-79. (62) Lennernäs, H. Human intestinal permeability. J. Pharm. Sci. 1998, 87, (4), 403-10. (63) Curthoys, N. P.; Shapiro, R. gamma-Glutamyltranspeptidase in intestinal brush border membranes. FEBS Lett. 1975, 58, (1), 230-3. (64) Achler, C.; Filmer, D.; Merte, C.; Drenckhahn, D. Role of microtubules in polarized delivery of apical membrane proteins to the brush border of the intestinal epithelium. J. Cell Biol. 1989, 109, (1), 179-89. (65) Seward, D. J.; Koh, A. S.; Boyer, J. L.; Ballatori, N. Functional complementation between a novel mammalian polygenic transport complex and an evolutionarily ancient organic solute transporter, OSTalpha-OSTbeta. J. Biol. Chem. 2003, 278, (30), 27473-82. (66) Rubio-Aliaga, I.; Daniel, H. Peptide transporters and their roles in physiological processes and drug disposition. Xenobiotica. 2008, 38, (7-8), 1022-42. (67) Ferraris, R. P.; Villenas, S. A.; Diamond, J. Regulation of brush-border enzyme activities and enterocyte migration rates in mouse small intestine. Am. J. Physiol. 1992, 262, (6 Pt 1), G1047-59. (68) Lubarski, I.; Pihakaski-Maunsbach, K.; Karlish, S. J.; Maunsbach, A. B.; Garty, H. Interaction with the Na,K-ATPase and tissue distribution of FXYD5 (related to ion channel). J. Biol. Chem. 2005, 280, (45), 37717-24. (69) Dave, M. H.; Schulz, N.; Zecevic, M.; Wagner, C. A.; Verrey, F. Expression of heteromeric amino acid transporters along the murine intestine. J. Physiol. 2004, 558, (Pt 2), 597-610. 49 ACS Paragon Plus Environment


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1. Schematic diagram illustrating the PS product in the apical-to-basolateral direction in mouse small intestinal epithelial cells (in

situ

PSa

to b)

or in vitro cell

monolayers (in vitro PSa to b). This schematic diagram has been reported previously by Adachi et al.2

in situ

PSa,inf,

in situ

PSb,inf, and in situ PSb,eff represent the PS products for the

apical influx, basolateral influx, and basolateral efflux in mouse small intestinal epithelial cells, respectively.

in situ

PSmdr1a and in situ PSa, eff represent the PS products for

the mdr1a-mediated efflux in wild-type mice and the apical efflux, with the exception of mdr1a-mediated efflux, in mouse small intestinal epithelial cells, respectively. PSa,inf,

in vitro

PSb,inf, and

in vitro

PSb,eff represent the PS products for the apical influx,

basolateral influx, and basolateral efflux in vitro cell monolayers, respectively. PSmdr1a and

in vitro

in vitro

in vitro

PSa, eff represent the PS products for the mdr1a-mediated efflux in

L-mdr1a cells and the apical efflux, with the exception of mdr1a-mediated efflux, in vitro cell monolayers, respectively.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table 1. Reconstruction of

in situ PSa to b

Ratio from

Page 52 of 59

in vitro

PSa to b Ratio and Protein

Expression Levels of Mdr1a in the Plasma Membrane Fractions of Isolated Jejunal Epithelial Cells and Mdr1a-Transfected LLC-PK1 Cell Monolayers in vitro Papp, a to b

(×10-6 cm/s)*

Reconstructed

Compounds

in vitro

PSa to b ratio

LLC-PK1

L-mdr1a

Quinidine

57.2 ± 7.6

3.16 ± 0.10

18.1 ± 1.7

3.83 ± 0.23

Digoxin

11.8 ± 2.3

1.13 ± 0.11

10.4 ± 1.6

2.56 ± 0.20

Loperamide

49.7 ± 1.9

5.49 ± 0.51

9.05 ± 0.64

2.33 ± 0.09

Vinblastine

24.2 ± 7.7

2.83 ± 0.56

8.55 ± 2.27

2.25 ± 0.27

Verapamil

73.5 ± 6.6

11.9 ± 0.1

6.18 ± 0.39

1.86 ± 0.06

Dexamethasone

29.5 ± 0.2

6.91 ± 0.22

4.27 ± 0.10

1.54 ± 0.02

Diazepam

31.8 ± 2.1

57.3 ± 4.5

0.555 ± 0.040

0.926 ± 0.005

According to eq. 7, the values of reconstructed

in situ

in situ PSa to b

PSa to b ratio

ratio were calculated from

the Papp in apical-to-basolateral direction (in vitro Papp, a to b) in the parental LLC-PK1 cell monolayers and L-mdr1a cell monolayers with the protein expression levels of mdr1a in the plasma membrane fractions of isolated jejunal epithelial cells and L-mdr1a cell monolayers. The protein expression levels of mdr1a were 7.57 fmol/µg plasma membrane protein in mouse jejunum and 45.7 fmol/µg plasma membrane protein in L-mdr1a cell monolayers. Each value represents the mean ± S.E.M. The value of S.E.M. was calculated according to the law of propagation of errors as described by Uchida et al.19 *

Data were taken from Uchida et al.19


Page 53 of 59

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Comparison of observed and reconstructed reconstructed

in situ

PSa

to b

in situ

PSa

to b

ratios. The

ratios were taken from Table 1. The solid line passing

through the origin represents the line of identity. Each point represents the mean ± S.E.M. The value of S.E.M. was calculated according to the law of propagation of errors as described by Uchida et al.19 1, quinidine; 2, digoxin; 3, loperamide; 4, vinblastine; 5, verapamil; 6, dexamethasone; 7, diazepam. *

Data were taken from Adachi et al.2



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 54 of 59

Figure 2. Comparison of observed and reconstructed effective absorption ratios. According to eq. 12, the values of observed and reconstructed effective absorption ratio were calculated from the

in situ

PSa to b ratio. The observed

taken from Adachi et al.2 and the reconstructed

in situ

in situ

PSa to b ratios were

PSa to b ratios were taken from

Table 1. The solid line passing through the origin represents the line of identity. Each point represents the mean ± S.E.M. The value of S.E.M. was calculated according to the law of propagation of errors as described by Uchida et al.19 1, quinidine; 2, digoxin; 3, loperamide; 4, vinblastine; 5, verapamil; 6, dexamethasone; 7, diazepam.


Page 55 of 59

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2. Protein Expression Levels of Transporters and Marker Proteins in Plasma Membrane Fractions of Isolated Epithelial Cells in Mouse Small Intestine Absolute protein expression level (fmol/µg plasma membrane protein) Molecule Duodenum

Jejunum

Ileum

ABC transporter abcb1 / mdr1a

5.22

±

0.38† ##

7.57

±

0.52*

9.48

±

0.45**

abcc4 / mrp4

0.188

±

0.010#

0.168

±

0.000#

0.240

±

0.011* †

abcg2 / bcrp

4.79

±

0.18 ##

5.05

±

0.18##

6.97

±

0.22** ††

abcg5 / abcg5

2.62

±

0.34

3.62

±

0.30

3.11

±

0.25

abcg8 / abcg8

2.16

±

0.07††

3.16

±

0.17**

2.68

±

0.06**

slc2a1 / glut1

1.58

±

0.02†† ##

0.619

±

0.030** ##

1.20

±

0.01** ††

slc3a2 / 4f2hc

32.3

±

1.2†† ##

62.2

±

2.2**

59.3

±

0.9**

slc5a1 / sglt1

7.01

±

0.67†† #

10.6

±

0.1**

10.3

±

0.5*

slc7a8 / lat2

17.1

±

0.5† #

21.1

±

0.4*

19.9

±

0.4*

slc15a1 / pept1

4.95

±

0.77

6.92

±

0.69

6.71

±

1.54

slc16a1 / mct1

27.6

±

1.2#

25.1

±

1.1#

37.7

±

2.5* †

slc22a18

5.91

±

0.53

3.68

±

0.45

3.75

±

0.52

ostβ / slc51b

4.08

±

0.21##

5.31

±

0.54##

31.9

±

1.5** ††

villin1

12.8

±

0.3† ##

21.6

±

0.4*

23.2

±

2.1**

γ-gtp

4.39

±

0.16††

6.60

±

0.15** ##

4.62

±

0.13††

Na+/K+-ATPase

397

±

17

445

±

19

409

±

9

SLC transporter

Others



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 56 of 59

The plasma membrane fractions of isolated epithelial cells in mouse small intestine were digested with lysyl endopeptidase and trypsin, and then the protein expression levels were quantified by LC-MS/MS, using a triple quadrupole mass spectrometer or Triple TOF5600. Abcc4 / mrp4, slc2a1/ glut1, and γgtp were quantified by Triple TOF 5600 and the other molecules were quantified by a triple quadrupole mass spectrometer (API5000 or Qtrap5500). The protein expression levels in three or four sets of transitions in one pooled sample from ten mice were used to calculate the average (mean) and variability (S.E.M.), which are shown as mean ± S.E.M.

*

and

**

indicate

significant difference of the quantitative value compared with the duodenum (p

Quantitative Targeted Absolute Proteomics of Transporters and

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