stereoselective transport and uptake of propranolol across human intestinal caco-2 cell monolayers

Stereoselective Transport and Uptake of Propranolol AcrossHuman Intestinal Caco-2 Cell Monolayers

YI WANG,1 JIANG CAO,2* XIAODAN WANG,1 AND SU ZENG1*1Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences,

Zhejiang University, Hangzhou, Zhejiang 310031, People’s Republic of China2Sir Run Run Shaw Institute of Clinical Medicine, Zhejiang University, Hangzhou, Zhejiang 310016, People’s Republic of China

ABSTRACT The transport and uptake of individual propranolol (PPL) enantiomerswere studied in human intestinal Caco-2 cell monolayers, and a reversed-phase HPLC-UV assay was used for quantitative analysis. S-PPL and R-PPL across Caco-2 cell mono-layers was determined in the concentrations range of 10–500 lM in both apical (AP) tobasolateral (BL) and BL to AP directions. S-PPL exhibited greater permeability thanR-PPL in the AP to BL direction, whereas in the BL to AP direction S-enantiomer trans-ported less than R-enantiomer. Uptake of R-PPL was significantly higher than that ofS-PPL either from AP side or from BL side. The statistically significant differences inuptake were observed at the concentrations range from 10 to 50 lM. Furthermore, theapparent Michaelis constant (Km) and maximal velocity (Vmax) also showed significantdifference between the two enantiomers. Moreover, the AP to BL transport of PPL enan-tiomer was markedly decreased by lowering the pH of the apical side but it did notaffect the stereoselectivity of PPL across Caco-2 cell monolayers. The transport anduptake of PPL in the BL to AP direction was not influenced by several protein inhibitors.The results suggest that PPL enantiomers showed stereoselective transport and uptakeacross the Caco-2 cell monolayers. A special transport mechanism capable of directingthe PPL enantiomers might be present in the Caco-2 monolayers. Chirality 22:361–368,2010. VVC 2009 Wiley-Liss, Inc.

KEY WORDS: b-adrenoceptor blocker; enantiomers; accumulation; HPLC; apparentpermeability coefficients; Km; Vmax; inhibitor

INTRODUCTION

Propranolol [1-isopropylamino-3-(1-naphthoxy)-2-propa-nol] is a nonselective b-adrenoceptor blocker for the treat-ment of hypertension and cardiovascular disorders.1 It ishydrophobic in nature and the logP value is 1.5. Proprano-lol hydrochloride (molecular weight 295.8), a commer-cially available form of propranolol (PPL), contains a chiralcarbon in its structure (Fig. 1). It is used clinically as aracemic mixture despite its stereoselective pharmacologi-cal activity and disposition.2,3 For instance, the (S)-(2)-pro-pranolol is 100 times more potent than the (R)-(1)-pro-pranolol.4 In humans, the Cmax and AUC (area under thecurve) values of the (S)-isomer were greater than those ofthe (R)-isomer.5 Moreover, pharmacokinetic studiesindicated that there is up to 20-fold variation between indi-viduals in plasma PPL concentrations after oral administra-tion.6–8 Therefore, it is of clinical value to clarify anyenantiomeric differences in the absorption and dispositionof PPL. It has been demonstrated that there are stereose-lective differences for PPL in binding to a-1 acid glycopro-tein9 and metabolism.10,11 However, the human intestinalabsorption of the PPL enantiomers was rare stated.

Caco-2 cell, derived from a human colonic adeno-carcinoma, is a widely used model to study intestinal

absorption or secretion of various drugs.12,13 They differ-entiate spontaneously in culture into polarized cell mono-layers with many enterocyte-like properties of transportingepithelia.14 For example, they express membranetransporters (P-glycoprotein, multidrug resistant-associ-ated protein, lung cancer-associated resistance protein),15

phase I enzyme (cytochrome P450 (CYP) 1A1/1A2,CYP2B6, CYP2E1, CYP3A5),16 and phase II enzyme(UDP-glucuronosyltransferase (UGT) 1A1, UGT1A6,UGT2B7).17 The transport of PPL racemates across theCaco-2 was observed18 but the stereoselective transport ofPPL was overlooked.

Contract grant sponsor: National Natural Science Foundation of China;Contract grant number: 30225047.Contract grant sponsor: Technology Foundation of China; Contract grantnumber: 2005C13026.*Correspondence to: Jiang Cao, Sir Run Run Shaw Institute of ClinicalMedicine, Zhejiang University, Hangzhou, Zhejiang 310016, People’sRepublic of China. E-mail: [email protected] or Su Zeng, Department ofPharmaceutical Analysis and Drug Metabolism, College of PharmaceuticalSciences, Zhejiang University, Hangzhou, Zhejiang 310058, People’sRepublic of China. E-mail: [email protected] for publication 12 November 2007; Accepted 13 May 2009DOI: 10.1002/chir.20753Published online 2 July 2009 in Wiley InterScience(www.interscience.wiley.com).

CHIRALITY 22:361–368 (2010)

VVC 2009 Wiley-Liss, Inc.

The aims of the present studies were to investigate thetransport and uptake of individual PPL enantiomers acrossthe Caco-2 cell monolayers. Furthermore, extensiveresearch was performed by treating the cells with differ-ence transporter inhibitors to propose the possible mecha-nism of stereoselective transport and uptake.

MATERIALS AND METHODSMaterials

(S)-(2)-propranolol hydrochloride (S-PPL), (R)-(1)-pro-pranolol hydrochloride (R-PPL), (R)-(1)-propafenonehydrochloride, cyclosporine A, cimetidine, and ethylenediamine tetraacetic acid (EDTA) were purchased fromSigma Chem Co. (St. Louis, MO), verapamil and indo-methacin were purchased from China National Institutefor the Quality Control of Pharmaceutical and BiologicalProducts. Ouabain and Lucifer yellow CH were purchasedfrom JK Chemical. Caco-2 cells at passage 17 wereobtained from Chinese Academy of Medical Science. Dul-becco’s modified eagle’s medium (DMEM, high glucose),fetal bovine serum (FBS), nonessential amino acids werepurchased from Gibco BRL (Grand Island, NY). Penicillin,streptomycin, and trypsin were purchased from InvitrogenLife Technologies. Bio-Rad DC Protein Assay kit was pur-chased from Bio-Rad Labotatories. The 12-well Transwell1

plate with clear polyester membrane insert (0.4 lm porediameter, 12 mm diameter) was purchased from CorningCostar Corp. (Acton, MA). Millicell-ERS voltohmmeterwas purchased from Millipore Corp. (Billerica, MA). All

other chemicals and solvents were of analytical orchromatographic grade and obtained from commercialsources.

Cell Culture

Caco-2 cells were cultured in DMEM containing 4.5 g/lglucose and supplemented with 10% FBS, 100 U/ml ofpenicillin, 100 lg/ml of streptomycin, and 1% nonessentialamino acid. Cultures were incubated at 378C in a humidi-fied atmosphere with 5% CO2 in air, and the cells were pas-saged at a split ratio of 1:3 with 0.25% trypsin/0.02% EDTAsolution after reaching 80% confluence. Caco-2 cells wereseeded, at passage 25 to 40, on transwell1 clear polyestermembrane insert with a seeding density of 1 3 105 cells/cm2. Medium was changed every 2 days for the first14 days and every day thereafter. Confluence was reachedwithin 3–4 days after seeding, and the cells were used ondays 21 to 25 postseeding to obtain differentiated mono-layers and a higher expression of transport proteins.19

Transepithelial electrical resistance-values (TEER values)were measured with a Millicell-ERS apparatus at roomtemperature. The integrity of Caco-2 monolayers was con-firmed when the TEER exceeded 350 X/cm2, and the Papp,A-B of Lucifer yellow CH was less than 0.5 3 1026 cm/s.20

It was processed before and after each experiment.

Transport Studies

Caco-2 cell monolayers were washed three times withwarm (378C) transport buffer21 (PBS containing 25 mMHEPES, pH 7.4) and equilibrated in transport buffer at378C for 30 min before the experiment. Transport studieswere conducted at 378C in a humidified incubator withshaking (50 rpm) and completed before the receiver con-centration exceeds 10% of the donor concentration pertime interval to maintain sink conditions. All volumesamounted to 0.5 ml at the apical (AP) side of the mono-layer and 1.5 ml at the basolateral side. The transport wasinitiated by replacing the donor buffer with the transportbuffer containing individual concentration of S-PPL orR-PPL (10 lM, 50 lM, 100 lM, 500 lM). At 20, 40, 60,and 80 min, 250 ll of the incubation medium wereremoved from the receiver compartment followed by add-ing the same volume of warm buffer as replenishment.22

The effect of apical pH on the transport of PPL enan-tiomers was conducted at 378C. The pH of the basolateralside was 7.4 (25 mM HEPES in PBS), and the pH of theapical side was either 6.5 (25 mM MES in PBS) or 7.4.The cells were preincubated with PBS at pH 6.5 or 7.4 onthe apical side for 30 min. The PBS in the basolateral sideremained at pH 7.4. The transport study was initiated byreplacing the donor solution with transport buffer contain-ing 75 lM PPL enantiomer. The incubation medium werethen collected every 20 min up to 60 min followed by add-ing warm transport buffer with the same volume and pHas replenishment. All the transport experiments wereconducted in triplicate.

For the transporter inhibition studies, individual stocksof the inhibitors verapamil, cyclosporine A, indomethacin,cimetidine, and ouabain were prepared in dimethyl sulfox-ide (DMSO) and added to the transport buffer at the given

Fig. 1. Chemical structure of (S)- and (R)-propranolol hydrochloride.

362 WANG ET AL.

Chirality DOI 10.1002/chir

concentrations (100 lM of verapamil,23 10 lM of cyclospo-rine A,24 10 lM of indomethacin,13 50 lM of cimetidine,25

and 1 mM of ouabain26). The final concentration of the sol-vent DMSO was set to 0.1% in all experiments. The cellmonolayers were pretreated with each inhibitor for 30 minat 378C. The transport experiments were then initiated byreplacing basolateral (BL) solution with 1.5 ml of the trans-port buffer containing 75 lM of PPL enantiomer and theinhibitor. At 20 min and 40 min, 250 ll of incubation me-dium were removed from the AP side followed by addingthe same volume buffer containing the inhibitor as replen-ishment. The samples were kept at 2208C until they wereanalyzed.

Uptake Studies

At the end of the transport studies, the cells werescraped off and washed three times with ice-cold PBS (pH7.4) to stop further uptake. After addition of 0.25 ml PBS,cells were lysed by sonic oscillation at 48C. The concentra-tion of the compound in the lysis solution was determinedby HPLC and normalized with cellular protein content.Protein content was determined using a Bio-Rad DCProtein Assay kit, with bovine serum albumin as thestandard.20,27

HPLC Analysis of Propranolol

The (S) or (R)-enantiomers of propranolol in the sam-ples was quantified by reversed-phase HPLC. Forty-micro-liter of 0.02 mg/ml (R)-(1)-propafenone were added to250 ll of samples obtained from transport or uptake stud-ies in Caco-2 cell monolayers as an internal standard. Afteralkalifying with 40 ll of concentrated aqueous ammonia,the mixture was extracted with 1 ml of dichloromethane,vortexed for 2 min, and then centrifuged for 3 min at13,000 rpm. The upper organic layer (800 ll) was trans-ferred into another test tube and evaporated to drynesswith N2. The residue was reconstituted in 60 ll mobilephase before analysis. Twenty microliters of the samplewas injected into the HPLC system.

The Chromatographic conditions were listed below: Agi-lent 1100 System equipped with a G1314A VWD UV detec-tor (Set at 220 nm), a Platinum EPS C18 100A (250 3 4.6mm i.d., 5 lm, purchased from Alltech Associates) columnwith a guard column (10 mm 3 4.6 mm i.d., 5 lm, packedwith YWG-C18H37, purchased from Jiangsu Hanbon Sci-ence and Technology Co.), and the chromatographic datawere collected and processed on Agilent Chemstationversion (No. G2170AA). The mobile phase consisted ofmethanol-acetonitrile-phosphate buffer (pH 4.0; 10 mM)(10:45:45, v/v/v) at a flow rate of 0.9 ml/min. The phos-phate buffer used for the mobile phase was prepared from10 mM potassium dihydrogen phosphate and adjusted topH 4.0 with concentrated phosphoric acid. The concentra-tions of PPL enantiomers were calculated from thecalibration curves.

Data Analysis

The permeability of PPL enantiomers in each directionwas measured as apparent permeability coefficients (Papp)(cm s21) obtained according to the following equation:

Papp ¼ ðV=AC0ÞðdC=dtÞ ¼ cm s�1 ð1Þ

where V is the volume of the solution in the receiving com-partment (ml), A is the surface area of the Caco-2 mono-layers (cm2), C0 is the initial concentration in the donorcompartment (lM), and dC/dt is the change in drug con-centration in the receiver solution over time (lM/s).28,29

The kinetics parameters for PPL enantiomers uptake inCaco-2 cells were estimated as eq. 2.

V0 ¼ Vmax 3 S=ðKm þ SÞ þ Kd 3 S ð2Þ

where V0 is the uptake rate of the PPL enantiomers(lmol/mg protein/h), S is the initial concentrations ofPPL enantiomers in the transport buffer (lM), Vmax is themaximum uptake rate by the saturable process (lmol/mgprotein/h), Km is the Michaelis constant (lM), and Kd isthe coefficient of simple diffusion (l/mg protein/h). Theuptake measurements were fitted to the above equation bynonlinear least-squares regression analysis.30

Statistical Analysis

All values were presented as a mean 6 standard devia-tion (S.D.) throughout the article. Student’s t-test forunpaired data was used to compare the apical-to-basolat-eral and the basolateral-to-apical transepithelial transportand accumulation, the apical vs. the basolateral efflux, andthe efflux at different pH conditions.31 Satistical signifi-cance of differences between R- and S-enantiomer was cal-culated using paired t-test.32 P < 0.05 was considered tobe statistically significant.

RESULTS AND DISCUSSIONValidation of HPLC Methods

Representative chromatograms for S-PPL or R-PPL withinternal standard (R-propafenone) in transport buffer areshown in Figure 2. The retention time for propranol enan-tiomer and R-propafenone were about 11.8 min and 18.2min. The interfering peaks coeluting with the compoundsof interest was not observed. The presence of compoundssuch as verapamil, cyclosporine A, indomethacin, cimeti-dine, and ouabain also did not cause any interference inthe assay.

Calibration curves for S-PPL and R-PPL were linear overthe concentration range of 0.2–20 lM. Peak area ratios (y)of S-PPL or R-PPL versus the internal standard were meas-ured and plotted against the concentration (x) of each iso-mer. The regression equations of the calibration curveswere y 5 3.6538x 2 0.1843 (r 5 0.9998) for (S)-(2)-pro-pranolol, y 5 3.6220x 2 0.1882 (r 5 0.9996) for (R)-(1)-propranolol.

The precision and accuracy of the method wereassessed by intra- and interassay validations (n 5 5) atconcentrations of 1, 5, and 10 lM. The results showed thatthe intra- and interday coefficients of variation were lessthan 10% (R.S.D.). The recovery of the assay was between98.8% and 101.8%.

363STEREOSELECTIVITY OF PROPRANOLOL IN Caco-2


The limit of detection, defined as the lowest sample con-centration which can be detected (signal-to-noise ratio 53) was 0.005 lM for each enantiomer. The limit of quantifi-cation, defined as the lowest sample concentration whichcan be quantitatively determined with suitable precisionand accuracy (signal-to-noise ration >10) was 0.2 lM(R.S.D. <10%) for each enantiomer.

Transcellular Transport and Cellular Accumulation ofPropranolol Enantiomers

The transport of S-PPL or R-PPL across Caco-2 cellmonolayers was measured at increasing concentrations(10–500 lM) in both AP to BL and BL to AP directions.According to the Ref. 18, the estimated concentrations ofthe propranolol reached in the gut lumen and the plasmawere 617 lM and 2.2 lM, respectively, at the dose of 160mg (b.i.d., twice daily). Therefore, the concentrations ofPPL used in our studies were of physiologic relevance.The stereoselective binding of the propranolol enantiom-ers to plasma proteins is reported,33 but whether these

enantiomers bind to Caco-2 monolayers selectively isunknown. To avoid the interactions between the enantiom-ers, individual enantiomers of PPL were applied to thisstudy.

The stereoselective transcellular transport of PPL enan-tiomers is showed in Figure 3. The BL to AP transportsfor each PPL enantiomer were greater than that of the APto BL transport (Fig. 3A). Importantly, the two enantiom-ers performed differently in the same transport direction.The transport rate of S-PPL was lower than that of R-PPLin BL to AP direction. Whereas in the opposite direction,the transport rate of S-PPL was greater than that of R-PPL.The transport rates of PPL enantiomers in both directionswere concentration dependent.

The apparent permeability coefficient (Papp) is anotherpermeability parameter, which represents the compositeeffects of traversing all permeation pathways across thetransport systems. The Papp values of each enantiomer arepresented in Figure 3B. Similar to the observations intransport rate of PPL enantiomers, there were statistically

Fig. 2. Representative chromatograms of the blank transport buffer (A) and the transport buffer spiked with propranolol enantiomer and internalstandard R-propafenone (B). Peak 1: S-propranolol (S-PPL) or R-propranolol (R-PPL) (1 lM); Peak 2: R-propafenone.

364 WANG ET AL.


significant differences in Papp values between S-PPL and R-PPL for both transport directions. In AP to BL direction,the permeability of S-PPL was greater than that of R-PPL,and the statistically significant differences were observedat the concentration range of 10–100 lM. In contrast, Pappvalues of S-PPL were smaller than that of R-PPL in secre-tory direction, and the statistically significant differenceswere observed at the lowest and highest concentrations.Furthermore, the permeability of R-PPL in BL to AP direc-tion was greater than that in AP to BL direction. The statis-tically significant differences were seen over the entireconcentration range. S-PPL had the similar secretory per-formance, and the statistically differences were observedwhen the concentrations were 50 and 500 lM. Moreover,the results demonstrated that the Papp across Caco-2monolayers increased with propanolol concentration, indi-cating that saturate process may be involved. In general,apparent permeability (Papp) decreases when the drug con-centration increases if the active transport is involved. Butthere are some papers suggested that many permeabilityequations fail to take into account cellular retention and fil-ter adsorption during the transport experiments, especially

for the high permeability drugs and basic drugs.34,35 Col-lectively, these observations strongly suggested thatstereoselective transport of PPL might happen acrossCaco-2 monolayers.

Caco-2 cells were derived from human colorectal adeno-carcinoma. When cultured as a monolayer, they differenti-ate to form tight junctions between cells and express trans-porter proteins, efflux proteins, and phase II conjugationenzymes to model a variety of transcellular pathways. Inmany respects, the Caco-2 cell monolayer mimics thehuman intestinal epithelium.12 Therefore, a specific carriermight mediate the stereoselective transport. Indeed, a fewreports have showed that intestinal absorption of some chi-ral drugs might be stereoselective.36–40 In instance, a se-ries of aryloxy phosphoramidate prodrugs of d4T werereported to having stereoselective polarized permeabilityin Caco-2 and madin-darby canine kidney cell mono-layers.41 The metabolism of propranolol is mainly affectedby phase I enzymes (CYP1A2 and CYP2D6) and phase IIenzymes (UDPG1A9 and UDPG1A10) based on in vitroand in vivo studies.42–44 Although Caco-2 monolayersexpressed low level of these enzymes, the possibility thatstereoselective metabolism of PPL might make some con-tribution can’t be excluded.

To determine the concentration dependent manner ofPPL enantiomers uptake in Caco-2 cells, cellular accumula-tion of each enantiomer was examined following the trans-port experiment. As illustrated in Figure 4, uptake rates ofeach PPL enantiomer were concentration dependence andaffected by the donor sides. Moreover, the uptake of each

Fig. 3. Effects of the concentration on the transport rate (A) and Papp(B) of Propranolol enantiomers from apical (AP) to basolateral (BL) andBL to AP side. S-PPL or R-PPL was loaded on either AP or BL side andincubated at 378C. Samples from receiving side were collected and S-PPLor R-PPL was determined by HPLC. Data are the mean 6 S.D. from fourdeterminations. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4. Cellular uptake characteristics of S-PPL and R-PPL across theapical (AP) or basolateral (BL) membrane of Caco-2 cells. S-PPL or R-PPLwas loaded on either apical (AP) or basolateral (BL) side and incubated at378C for 80 min. The vertical bars represent 6 S.D. of three individualexperiments conducted in triplicate. *p < 0.05.

TABLE 1. Kinetic parameters for S-propranolol (S-PPL)and R-propranolol (R-PPL) uptake by Caco-2 cells

Apical side Basolateral side

S-PPL R-PPL S-PPL R-PPL

Vmax (lmol/mg protein/h) 0.51 0.35 1.03 0.30Km (lM) 287 50 1037 147Kd (l/mg protein/h) 0.0003 0.0002 0.0001 0.0001

Kinetic constants were estimated as described in the Materials and Meth-ods section.



enantiomer across AP membrane of Caco-2 cells was sig-nificantly greater than that across BL membrane at theconcentrations range from 10 to 100 lM. The accumula-tion difference gradually diminished with the increase ofdrug concentration. Comparing the enantiomers uptakefrom the same donor side, the uptake rates of R-PPL weresignificantly higher than those of S-PPL. The statisticallysignificant differences were observed in the concentrationrange of 10–50 lM. The observations are consistent withthe presence of a special transport mechanism capable ofdirecting the PPL enantiomers across the Caco-2 mono-layers. Some carrier might enhance R-PPL uptake orinhibit S-PPL accumulated in Caco-2 cells, and thetransport would be saturated.

Furthermore, the kinetic parameters obtained fromeach enantiomer uptake were calculated in the concentra-tion range of 10–500 lM (Table 1). The apparent Michae-lis constant (Km), maximal velocity (Vmax), and the coeffi-

cient of simple diffusion (Kd) were estimated from theMichaelis-Menten equation using nonlinear least-squaresanalysis. The Vmax and Km for S-PPL uptake were signifi-cantly greater than those of R-PPL, and the stereoselectiveuptake was not influenced by the donor side. Theseresults indicated that some carrier might prefer S-PPL toR-PPL as a substrate and pump them out of the cells eitherin the AP side or BL sides. Whereas the Kd value of S-PPLwas not significantly different from the one of R-PPL at thesame donor side. Interestingly, the Vmax and Km of S-PPLuptake from the BL side were significantly greater thanthose from AP side. The results suggested that the carrierbefore transport S-enantiomer might be major distributedin the BL side. It was important to note that the Vmax/Km

valueof R-PPL, which reflects a high-uptake rate, wasgreater than that of S-PPL from both sides. The resultsalso indicated that a carrier prefer S- to R-enantiomer fortransport prevent S-PPL from accumulating in Caco-2 cells.

Fig. 5. Effect pH on transport or accumulation of S-PPL or R-PPL. Caco-2 cell monolayers were incubated with S-PPL or R-PPL (75 lM), which wereadded to the apical side or basal side. The pH of the apical side was 6.5 or 7.4, and the pH of basal side was 7.4. Samples from receiving side were col-lected and S-PPL or R-PPL was determined by HPLC. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6. Effects of various potential inhibitors on permeabilities (A) and accumulations (B) of S-PPL and R-PPL. Each bar represents the means 6S.D. of at least three experiments.

366 WANG ET AL.


Effect of the Apical pH on Propranolol EnantiomersTranscellular Transport and Uptake

The human intestinal epithelial cells contain a mucinlayer, but it doesnot exit in the Caco-2 cell monolayersbecause apical solution contacts the cell surface directly.Therefore, the apical pH value can play a critical role inthe transcellular transport of drugs. To determine the pHdependent manner of PPL enantiomers transport anduptake in Caco-2 cells, the effects of apical pH on the trans-cellular transport and accumulations of PPL enantiomerswere examined. As illustrated in Figure 5, the AP to BLtransport of PPL enantiomer was markedly decreased bylowering the pH of the apical side (pH of the basolateralside was fixed to 7.4), and a statistically significantdecrease was also observed in the accumulation of R-PPLacross AP side. This pH-dependent transport of PPL mightbe partly explained by the passive diffusion of the nonion-ized form according to the pH-partition theory. Moreover,the apical pH change had no effects on the BL to AP trans-port and the stereoselective transcellular transport forboth enantiomers. It was important to note that the accu-mulation of S-PPL was not affected by changing pH from6.5 to 7.4 either in the AP uptake or in BL uptake. Thisresult strongly supported the hypothesis that some carrierwas responsible for the S-PPL transport in addition to thepassive diffusion in Caco-2 cells. Some metabolism inhibi-tors, such as a respiratory chain inhibitor, might beapplied in future study.

Effect of Inhibitor on Propranolol EnantiomersTranscellular Transport and Uptake

Since a stereoselective carrier was assumed to influencethe transport and accumulation of PPL enantiomers inCaco-2 cells, several different inhibitors were used toinvestigate whether the membrane transport protein(s) orthe Na1/K1 ATPase involved in the stereoselective trans-port and uptake of PPL (Fig. 6). Verapamil, a specific in-hibitor of P-glycoprotein, did not alter the stereoselectivepermeability of PPL, but weakly increased the cellularuptake without any chiral discrimination. Cyclosporine A(a P-glycoprotein inhibitor), indomethacin (a multidrug re-sistant associated protein inhibitor), cimetidine (an or-ganic cation transporter inhibitor), and ouabain (a Na1/K1 ATPase inhibitor) showed no inhibitory effect on thepermeability and accumulation of PPL enantiomers.Although propranolol was the substrate of P-glycoprotein,the transport in secretory direction of PPL was not influ-enced by introducing the P-glycoprotein (P-gp) inhibitors.The possibility was that passive transcellular diffusiondominated the absorptive transport behavior of PPL. Theobservations suggested that P-gp, multidrug resistantassociated protein, organic cation transporter, and Na1/K1 ATPase were not participate in the stereoselectivetranscellular transport of PPL enantiomers.

In summary, the present study demonstrated the trans-port and uptake of PPL across Caco-2 monolayers are ste-reoselective. PPL is a hydrophobic compound and used asa transcellular probe for studying intestinal drug absorp-tion in vitro. The results from present study suggest that a

carrier processing in a chirally discriminative way makesan important contribution to transport PPL enantiomers inCaco-2 cell monolayers. The research about plasma mem-brane proteome of Caco-2 cells was reported and morethan 1000 proteins were identified.45 It provides insightsinto the biology of Caco-2 cells and suggests potential tar-get proteins for the further studies about stereoselectivetransporters.

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