transepithelial secretion, cellular accumulation and cytotoxicity of vinblastine in defined mdck...

Biochimica et Biophysica Acta, 1179 (1993) 1-10 1 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4889/93/$06.00

BBAMCR 13444

Transepithelial secretion, cellular accumulation and cytotoxicity of vinblastine in defined MDCK cell strains

Janice Hunter, Barry H. Hirst and Nicholas L. Simmons Gastrointestinal Drug Delivery Research Centre and Department of Physiological Sciences, Medical School,

University of Newcastle upon Tyne, Newcastle upon Tyne (UK)

(Received 29 October 1992) (Revised manuscript received 10 March 1993)

Key words: Vinblastine; Transepithelial transport; P-glycoprotein; Multidrug resistance; Cytotoxicity; (Kidney cell-line); (MDCK cell)

Transepithelial vinblastine secretion in two defined MDCK strains displays saturation kinetics; (Strain 1) K m = 2.8 + 0.6 #M (six experiments), Vma x 35.9 ± 1.93 pmol/cm 2 per h (six experiments), Strain 2 K m 0.78_ 0.36 izM (three experiments), Vma x 12.1 -1- 4.5 pmol/cm 2 per h (three experiments). Concentrations of vinblastine > 1/zM are associated with an increased passive vinblastine permeability (PA-B). This correlates with an increased transepithelial conductance/decreased permselectivity, suggesting that this may in part result from increased paracellular conductance. Verapamil inhibits vinblastine secretion, half-maximal inhibition of basal-to-apical flux (JB-A) is observed at 3.4 + 0.3 and 1.7 ± 0.05 t~M verapamil for Strain-1 and Strain-2 epithelial layers, respectively. Cellular accumulation of vinblastine across the apical membrane is small with respect to that across the basolateral surfaces. This polarity is unaffected by verapamil. The apical membranes, therefore, possess a low intrinsic permeability to vinblastine. Inhibition of cell growth by vinblastine is enhanced by verapamil. Both the effect of vinblastine, and its enhancement by verapamil, upon cell growth are reduced as initial cell seeding density increases.

Introduction

P-glycoprotein is a 170-180-kDa membrane glycoprotein associated with the phenomenon of pleiotropic (multidrug) resistance (MDR) [1,2]. Immunohistochem- ical techniques have demonstrated the presence of P-glycoprotein in the apical regions of several natural epithelia [3-6], especially in the gastrointestinal tract and kidney. Epithelial cell-lines capable of reforming intact epithelial layers when grown upon permeable matrices (e.g., MDCK, LLC-PK1), including those derived from human gastrointestinal tumours (T84, HCT- 8), have been shown to express P-glycoprotein and to mediate epithelial secretion (from basal-to-apical cell surfaces) of P-glycoprotein substrates such as vinblastine [7-10]. Retroviral transfection of the dog kidney epithelial cell-line (MDCK) with MDR1 cDNA [11] results in polarised expression of P-glycoprotein to the apical plasma membrane domain and an enhanced

Correspondence to: J. Hunter, Gastrointestinal Drug Delivery Re- search Centre, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK.

transepithelial secretion of vinblastine above intrinsic levels.

A reduced intraceUular drug accumulation resulting from increased active (ATP-dependent) drug efflux is accepted as an important factor in the mechanism of pleiotropic drug resistance [12-15]. For human colonic adenocarcinoma ceil-lines, cellular vinblastine accumulation depends not only on P-glycoprotein function but also upon the site of drug presentation (to the apical or basolateral bathing compartments) [7]. This suggests that other properties of the apical and basolateral membrane domains, together with the presence of the diffusion restriction afforded by the apical junctional complexes (zonae occludentes or tight junction) are factors in determining the exact sensitivity of cells of epithelial origin to cytotoxic regimes.

Epithelia, broadly, may be divided into two classes on the basis of their transepithelial conductance, namely 'leaky' (10-50 mS/cm 2) and 'tight' (< 1 mS/ cm2), depending upon the conductance of the paracellular pathway [16]. The purpose of the present study has been to investigate whether epithelial biophysical parameters may explain, in part, the relative insensitivity of cells derived from epithelia to cytotoxic regimes.

We have chosen two well-defined strains of the dog kidney cellqine MDCK, which display marked differences in transepithelial (paracellular) conductance [17], and characterised the kinetics of transepithelial vinblastine secretion, cellular vinblastine accumulation, together with the modulation by verapamil, and corre- lated these observations with a standard assessment of cytotoxicity. We find that both cell strains maintain marked vectorial vinblastine secretion, consistent with P-glycoprotein expression. Cellular vinblastine uptake across the apical cell border, even in the absence of P-glycoprotein function, is restricted compared to that across the basolateral domains. Finally, we provide evidence that the apparent cytotoxic insensitivity of MDCK cells grown upon plastic reflects, in part, the relative impermeability of the apical plasma membranes to vinblastine together with the diffusion restriction to the basolateral surfaces afforded by the paracellular route even with epithelial layers of high conductance (MDCK Strain 2).

Materials and Methods

Cell culture. Strain 1 (60-75 serial passages) and Strain-2 MDCK (120 serial passages) cell-lines were used [17,18] and were maintained in serial culture in Minimum Essential Medium Eagle (Earle's salts) sup- plemented with 10% (v/v) foetal calf serum, kanamycin (1 /zg/ml), 1% non-essential amino acids and 2 mM glutamine. Confluent monolayers were subcultured ev- ery 7 days, by treatment with 0.05% trypsin and 0.02% EDTA in C a 2+- and MgZ+-free phosphate-buffered saline (PBS). For experimental purposes MDCK cells were grown as epithelial layers by high-density seeding (10 6 cells/cm 2) onto permeable culture inserts (Nunc 25-ram culture inserts, 4 c m 2 growth area). All cultures were incubated at 37°C in a humidified atmosphere of 5% CO2/95% air.

Culture inserts were incubated in 6-well plates for 2-3 days. The formation of functional epithelial layers was monitored visually and by the development of a significant transepithelial resistance (Rx), as measured using a WPI Evometer fitted with 'chopstick' elec- trodes to allow transepithelial current passage and potential sensing (for Strain 1) [17]. Since transepithelial resistance is low in Strain-2 cell-layers and the paracellular pathway is cation-selective [18], conflu- ence was assessed by the magnitude of the choline : Na + bi-ionic electrical potential difference (p.d.) [19] gener- ated by replacement of the basolateral bathing solution medium for one in which NaC1 was iso-osmotically replaced by choline chloride. Cell monolayers were used when the transepithelial resistance typically exceeded 1 k J2 • cm 2 Strain-1 cells, or 30 mV (basolateral solution electropositive) for (the choline:Na + bi-ionic

electrical p.d.) Strain-2 cells. Note that for Strain-1 epithelial layers values of resistance for the culture inserts, approx. 300 g2 • c m 2 is subtracted from all data.

Measurement of bidirectional transepithelial [3H] vinblastine fluxes. Measurements of transepithelial solute flux were made essentially as described previously [7,17]. Functional epithelial layers in culture inserts were washed with 2 × 3 ml serum-free medium and placed into fresh 6-well plates containing 3 ml serum- free medium (basolateral solution), a further 3 ml serum-free medium was then pipetted into the upper chamber (apical solution) of the filter cup. Trans- epithelial resistance was measured following 30 min incubation of the cells at 37°C, as described above.

The medium on either the apical, or basolateral side of the monolayers was then removed and replaced with 3 ml serum-free medium containing 10 nM [3H]vinblastine sulphate as tracer. Variable amounts of unlabelled vinblastine sulphate were added in the apical (A) and basal (B) solutions, thus the final vinblastine concentration was equal on both sides of the culture insert. Verapamil (10 mg/ml stock in DMSO) was added, unless stated otherwise, so the concentration was also equal on both sides of the culture inserts (and DMSO concentration did not exceed 1% (v/v)). Vin- blastine fluxes were also studied in ATP-depleted cells, these fluxes were carried out in the presence of 50 mM 2-deoxy-D-glucose and 15 mM Na azide, in glucose-free Krebs buffer. Control fluxes for this experiment were carried out in normal Krebs buffer with glucose (no significant difference was observed between control fluxes carried out in either the serum-free medium or Krebs buffer solution). The cell layers were then incubated at 37°C. In order to measure the bidirectional fluxes of vinblastine sulphate (JA-B, flux from apical- to-basal solutions, and JB-A, flux from basal-to-apical solutions), 100/zl samples of medium from each side of the monolayer were taken at regular intervals, 3H-activities in these samples were determined by liquid scintillation counting (Beckman LS5000CE scintillation counter) using LKB Optiphase scintillation cocktail. Appropriate corrections were made for quenching effects. Each incubation was performed at least in tripli- cate. On completion of the flux experiments epithelial integrity was determined either by measurement of transepithelial resistance, or by determination of the choline:Na + bi-ionic p.d as described above. In addition, cellular accumulation of vinblastine was determined. The filter cups were washed by immersion in 1 litre PBS, the filters removed from their holders and placed into scintillation vials and 3H activities determined.

[3H]Mannitol and [14C] inul in a n d [ 1 4 C ] P E G 4000 are inert molecules normally excluded from cells (ex- cluding fluid-phase endocytosis and transcytosis), and were used as a measure of extracellular permeability

pathways. Bidirectional extracellular marker fluxes were determined exactly as for vinblastine fluxes. Transepithelial solute permeabilities (PA-B and PB-A) were calculated as PA_B=JA_B/CA and PB_A = JB_A/CB, where CA and CB denote the respective solute concentrations in the apical or basolateral bathing compartments, respectively.

Assessment of uinblastine cytotoxicity. Cytotoxicity was measured by assaying mitochondrial reductive function using the MTT assay [20]. MDCK cells were seeded onto 96-well plates at various seeding densities (cells were counted using a Coulter Counter ZM, Coul- ter Electronics, Luton, UK) and the monolayers were allowed to grow over the following 24 h. The cells were washed with 100/xl PBS before addition of vinblastine (0-500 /~M) with or without verapamil (100 tzM). Stock solutions of verapamil were freshly prepared in DMSO at a concentration of 10 mg/ml, followed by dilution in serum-free medium. Control wells were included in each plate (each solution contained the same concentration of DMSO). Cells were then incubated overnight under cell culture conditions. On completion of the incubation, the drugs were removed, the cells washed with 100 ~1 PBS, 100 ~I fresh cell culture medium added, and re-incubated for 48 h. Following this incubation 50 ~I of MTT reagent (1 mg/ml 3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro- mide in PBS) was added to each well. The plates were then incubated for a further 3 h at 37°C. The ceils were then washed and the formazan product was extracted by addition of 75 ~1 DMSO to each well before ab- sorbance at 570 nm was measured with a Dynatech MR5000 ELISA plate reader.

Statistical methods. Data are expressed as mean + S.E. of n individual monolayers or N experiments (for kinetic constants and ECs0 values). Tests of signifi- cances of differences between mean values were made using two-tailed unpaired or paired Student's t-test or Mann-Whitney U tests, where appropriate. Signifi- cance was set at P < 0.05. Kinetic constants for Michaelis-Menton kinetics were calculated by non-linear regression with the method of least squares fitting (Enzfitter, Elsevier Publishing UK). Inhibition constants for verapamil inhibition of vinblastine flux and ECso values for cytotoxicity assays were determined by non-linear regression with the method of least-squares fitting for a logistic sigmoid (Pfit, Biosoft, Cambridge, UK; GraphPAD Inplot, San Diego, CA, USA).

Materials. [3H]Vinblastine sulphate, [3H]mannitol, [14C]inulin and [14C]PEG 4000 were purchased from Amersham International (Little Chalfont, Amersham, UK). Unlabelled vinblastine sulphate was obtained from Lederle Laboratories (Gosport, UK). The scintillation cocktail Optiphase safe was obtained from Phamacia LKB Biotechnology (Milton Keynes, UK). All tissue culture media and reagents (Gibco BRL) and

tissue culture plastics including tissue culture inserts (Nunc) were supplied by Life Technologies (Paisley, UK). All other chemicals were obtained from Sigma (Poole, UK) or Merck (Poole, UK).

Results

Functional expression of P-glycoprotein in separate MDCK strains

Strain-1 MDCK epithelia have a high resistance (2132 + 445 (S.E.)/2. cm 2, n = 9) typical of 'tight' epithelia [17]; in contrast, Strain-2 MDCK epithelia have a low transepithelial resistance ( < 100 O" cm 2, n --- 15) due to the presence of a cation-selective paracellular shunt pathway (choline : Na ÷ bi-ionic p.d = - 29.3 _+ 1.7 mV, n = 15) [17]. With tracer concentrations of vinblastine (10-20 nM) a marked asymmetry in the bidirectional transepithelial fluxes of vinblastine was observed, JB-A being considerably larger than JA-~ for both cell strains (Fig. 1). A significant net secretion of vinblastine thus occurs from basal-to-apical surfaces. Verapamil (0.1 mM) present in both bathing solutions effectively abolishes the net secretion of vinblastine by reducing J s - a towards JA-B (Fig. 1). The effect of verapamil was more evident when administered to the apical surface. In Strain-1 cells apical verapamil (0.1 raM) reduced JB-A from 0.35 + 0.02 pmol/cm 2 per h to 0.024 + 0.003 pmol/cm 2 per h (n = 3), whereas basolateral verapamil resulted in a smaller reduction in JB-A to 0.24 + 0.01 pmol/cm 2 per h (n = 3). Vera- pamil (at 0.1 mM) had no effects on JA-B" In epithelial layers treated concurrently with 50 mM 2-deoxy-o-glucose and 15 mM Na azide to reduce intracellular ATP, vinblastine secretion was abolished. JB-A was significantly reduced from 0.15 + 0.01 pmol/cm 2 per h to 0.03 + 0.01 pmol/cm 2 per h (n = 3). There was no significant effect of 2-deoxy-D-glucose/azide on JA-B (control, 0.016 _+ 0.006 pmol/cm e per h; 2-deoxy-o-glucose/azide, 0.006 + 0.003 pmol/cm 2 per h, n = 3).

In both control conditions and in the presence of vinblastine the spontaneous transepithelial electrical p.d. is low (1-3 or 0.1-0.3 mV (range) basolateral solution electro-positive for Strain 1 and 2, respectively). The observed assymmetries for vinblastine flux (JB_A/JA_B were 34.4 + 7.5, n = 9, and 20.5 ___ 2.9, n = 14 for Strain 1 and Strain 2, respectively) cannot therefore be accounted for by passive electrical forces and must represent active transport by the epithelium. The bidirectional permeabilities PA-B for Strain 1 and 2 at 10-20 nM vinblastine were (1.0 + 0.21). 10 -3 (n = 9)

and (2.2 + 0.5)" 10 -3 cm/h (n = 14), respectively, indi- cating that despite at least a 20-fold difference in conductance between the MDCK epithelia, the formation of an intact epithelium with tight junctions effectively limits trans-monolayer vinblastine diffusion. A comparison was made between the unidirectional per-

meabilities of vinblastine with mannitol (MW = 180), inulin (MW = 5000) and P E G (MW = 4000) in Strain-1 epithelia. PA-a was (2.41 + 0.06)" 10 -4 c m / h (n = 6) for mannitol , (9.2 _+ 1.8). 10 -5 c m / h (n = 6) for inulin and (5.1 + 0.5). 10 -5 c m / h (n = 6) for P E G 4000, suggesting that a significant por t ion of non-active vinblastine permeabil i ty comprises a transcellular lipid route. In contrast to the bidirectional vinblastine fluxes, there were no assymmetries in the bidirectional mannitol , inulin or P E G 4000 fluxes (mannitol JB_A/JA_B = 1.2 + 0.08 (n = 6), inulin JB_A//JA_B = 1.03 + 0.10 (n = 5) and P E G 4000 JB_A/JA_B = 1.15 + 0.04 (n = 6)).

Net uinblastine secretion displays saturation kinetics Fig. 2 shows vinblastine bidirectional fluxes mea-

sured over an extended range of vinblastine concentrations. For both cell strains, net vinblastine flux (J , et =

3.5

? 3.0

"6 2.5 E - 2.0

= 1.5 =- ~. 1.o

.~ o.s

(A) Strain 1.

JB-PA

~ jII.~A +ver = / = _ _ = J ..,

" U mnu , ~ _ _ ~ A B u i u n l u i

0 60 120 180 240 300 360

Time (mind

(B) Strain 2.

3.5

3.0

2.5

"-~ 2.0

= 1.5

~ 1.0

0.5 ~ ~ J B - * A +ver

J i n i 1 u n

0 60 120 180 2 4 0 3 0 0 360

Time (mini Fig. 1. Transepithelial vinblastine fluxes measured in (A) high-resistance Strain-1 (B) low resistance Strain-2 MDCK cells. [3H]vinblastine flux was measured in the basolateral-to-apical (B-A), apical-to- basolateral (A-B) and in the basolateral-to-apical direction in the presence 0.1 mM verapamil, present on both sides of the cell layer, (B-A+ver ) . The lines illustrate the least squares best fit to the data, the slopes of which give the vinblastine flux rate. Data are mean

values_+ S.E. from three separate epithelial layers.

(AI 75

" e0 Y E o ._:, 45 0

E 3 0 -

15-

O- 0,0

B - * A

A ~ B

Net flux

i

5.0 10.0 15.0

[ v i n b l a s t i n e l jaM

(a) 25

~. 20 B ~ A

R 15 O

E

10 A-*B

5 Net flux

0 [ i i t i

o,o 1.o 2.0 a.o 4.0 5.o

[vinbilstine] MM

Fig. 2. Transepithelial vinblastine fluxes over an extended range of vinblastine concentration. (A) High-resistance Strain 1, (B) Low-resistance Strain 2. Details as for Fig. 1, net flux Jnet = J B - A - - JA-B" Data are the mean + S.E. of at least three determinations from six separate experiments. Kinetic constants were K m 2.8:1:0.6 (S.E.) /~M, 0.78_+0.36 /xM, I/ma x 35.9+1.93 pmol/cm 2 per h, 12.1_+4.5 pmol/cm 2 per h for Strain-] and Strain-2 epithelia, respectively.

JB-A- - JA-B) demons t ra ted saturat ion kinetics. Kinetic constants derived f rom such direct measurements were: Strain 1, K m = 2.8 + 0.6 ( N = 6) /zM (range 0.32-3.9 /~M), Vm~ x 35.9 + 1.93 ( N = 6) p m o l / c m 2 per h (range 30.6-40.4 p m o l / c m 2 per hi; Strain 2, K m = 0.78 -I- 0.36 ( N = 3 ) / x M range 0 .16-1 .4 /zM) , lima ~ 12.1 _+ 4.5 ( N = 3) p m o l / c m 2 per h (range 5.8-20.9 p m o l / c m 2 per hi. These K m values are similar to those repor ted for vinblastine stimulation of the ATPase activity of P- glycoprotein in isolated membranes of both t ransfected Sf9 insect cells and KB-V1 cells [21,22]. In Strain-2 cell-monolayers JA-B shows a marked increase at 5 /xM vinblastine (see below).

Concentration-dependent increase in apical-to-basal vinblastine permeability

The unidirectional flux JA-B was not linearly de- penden t upon vinblastine concentra t ion in all experiments as would be expected f rom a simple diffusional

0.6

Concentration dependence of verapamil-inhibition of vinblastine secretion

Inhibition of vinblastine secretory flux JB-A by verapamil is concentration-dependent (Fig. 4), half-maxi-

? o

X

I

E 0

? Ct_

0.5 I

~: 0.4

0.3 E D.

< 0.2

0.1

0 1 10 100 1000

[Verapamil] /JM

Fig. 4. Concentration-dependence of verapamil inhibition of JB-A in MDCK epithelial cell layers, when verapamil is present on both sides of the cell layers. Data are the mean + S.E. of three observations per data point. Vinblastine was present at 20 nM, (o) Strain 1, high-resistance epithelial layers, (o) Strain 2, low-resistance layers. JA-B was 0.02 + 0.01 and 0.02_+ 0.005 pmol/cm 2 per h for Strain-1 and Strain-2

layers, respectively.

mal inhibition being observed at 4.1 and 1.6/zM verapamil for Strain-1 and -2 MDCK cells, respectively (when verapamil is added to both sides of the cell layers). Increasing concentration of verapamil progressively reduced JB-A flux to minimal values similar to

( ~ ) 6 -

( ~ ) 8.0

5,0

5 -

4 " x

7 s.

6 0

5

4

3

2

I

0

I m

•

f-y..

i l 4.0

I~l • .~ ~.o

m , ~ r im : 1.0

~ = == 'I,

0-

0 | , , , T i - 5 0 - 4 0 - $ 0 - 2 0 - 1 0 0

Choline : Nil + bl-lonic p.d. [mVl , , , i i ,

0.5 1.0 1 .S 2.0 2.5 3.0

permeability. Fig. 3 shows that in both strains of MDCK epithelia, the apparent permeability PA-B may increase as vinblastine concentration increases. For high-resistance Strain-1 epithelial layers incubation with 12 ~tM vinblastine (Fig. 2A) transepithelial conductance was significantly increased from 0.01 + 0.15 mS/cm 2 to 1.94 _+ 0.46 mS/cm 2 (n = 6). Similarly, for Strain-2 epithelia the choline:Na ÷ bi-ionic p.d. (an index of the cation selectivity of the paracellular pathway) of the MDCK layers significantly decreased upon exposure to high vinblastine concentrations, from 32.0 _+ 1.1 mV to 16.6 _+ 3.1 mV (n = 6) in the presence of 5 /zM vinblastine (Fig. 2B). Scatter graphs of epithelial conductance (Strain 1) with vinblastine permeability (PA-B), or choline:Na ÷ bi-ionic p.d. (Strain 2)with vinblastine permeability show highly significant correla- tions (insets Fig. 3A,B). These data are, therefore, consistent with part of vinblastine-dependent increase vinblastine permeability resulting from an increase in paracellular conductance.

8

7

x 5

1

i i 1 I I I I I I

0 1 2 3 0 1 2 3 4 S 8

[vinblastine] ~M [vinblastine] luM Fig. 3. Vinblastine-concentration-dependent increase in vinblastine permeability (PA-B = J A - a / C , where C = concentration of vinblastine in bathing solution). (A) High-resistance, Strain-1 epithelial layers, (B) Low-resistance Strain-2 epithelial layers. Mean data+S.E, from three replicates in individual representative experiments. Inset scatter plots show the correlation between apparent vinblastine permeabilities and epithelial conductance (Strain 1) (r = 0.70, dr= 43, P < 0.0001) and epithelial permselectivity (choline:Na ÷ bi-ionic p.d.) (r = 0.56, df = 49,

P < 0.0001).

>,

u

(A]

0.3

0.2

0.1

Ia)

Strain 1

\ \ \ \

\ \ \ ~ ,

\ \ \ \

Strain 2

Fig. 5. Cellular accumulation of vinblastine sulphate across the basal and apical cell borders of M DC K epithelial layers. (A) Strain 1; (B)

Strain 2. Data are the m e a n + S.E. (n = 3).

60 Basolateral loading

.,~

~ 4 0

.0,oa, , o . , . "

~ = ~ 2 0 -

N

~ ' i n i i

0.0 1.o a.0 3.0 4.0 s.0

[vinblastine] pM Fig. 6. Concentration dependence of cellular vinblastine accumulation across the apical (&) and basolateral borders (11) of Strain-] cells. Solid lines are linear regression lines for the data; apical loading, y = 5.24x -0 .49 , r = 0.98, P < 0.0001, df = 37. Basal loading y = 9.82x -0 .24 , r = 0.99, P < 0.0001, df = 34. Similar data were

obtained for Strain-2 cells.

those values for JA-B (see Fig. 1) between 50 and 100 /xM verapamil. These values are compatible with the K m for verapamil stimulation of P-glycoprotein ATP- ase activity in membranes from transfected insect cells [21] and the multidrug resistant cell line KB-V1 [22].

Cellular accumulation of vinblastine Fig. 5 illustrates the cellular accumulation of

[3H]vinblastine from either the apical or basolateral bathing solutions. It is clear that accumulation across the basolateral cell aspects exceeds that across the apical cell aspects in both MDCK-cell strains. These data contradict the predictions of cellular accumulation calculated according to Horio et al. [10]. The asymmetry observed in cellular uptake cannot be accounted for by binding of vinblastine to the Nunc culture inserts; Nunc filters bound only 0.018 or 0.027 (n = 2) pmol/cm 2 from apical or basolateral solutions containing 20 nM [3H]vinblastine. In contrast, Costar Transwell polycarbonate culture inserts bound 0.29 pmol/cm 2. For Nunc culture inserts, filter binding was subtracted from cell-associated vinblastine; note that the amount of [3H]vinblastine accumulated across the basolateral cell aspects exceeded filter binding by an order of magnitude. The uptake of the extracellular marker [14C]inulin was determined in Strain-1 epithelial layers and compared with that found for vinblastine. Inulin uptake across the apical cell border (expressed in equivalent solution volumes) amounted to 12 + 1.8 nl /cm 2 per 4 h (n = 6), whereas that across the basolateral border amounted to 940 + 56 nl /cm 2 per 4 h (n = 6). Equivalent volumes for vinblastine accumulation would be 1.9 and 10.4 g l / c m 2 per 4 h (Fig. 5).

Cellular accumulation of vinblastine was linearly dependent upon vinblastine concentration across both

cell borders (Fig. 6) over the concentration range tested. This lack of saturation contrasts with the saturation of the secretory transepithelial flux within the concentration range tested (see above).

Effect of verapamil upon cellular vinblastme accumulation

Verapamil (when presented on both sides of the cell layers) produced a dose-dependent increase in cellular vinblastine accumulation across the basolateral cell surfaces for both MDCK strains (Fig. 7). The increase in cellular accumulation correlates with the dose-dependent inhibition of vinblastine JB-A flux (see above). At concentrations above 100 /zM verapamil, cellular vinblastine accumulation across the basolateral surface declined but was still greater than control accumulation. Verapamil at 100 /zM in the both bathing solutions increased apical vinblastine accumulation in

1.2

m • ~ 1.0

g f o.s

~ o.4 >

~ 0.2

Q

u 0

~ ' / / /

N

0 2 10 20 100 200 500

[verapamil] pM

Fig. 7. Effect of verapamil upon vinblastine accumulation. Vinblas- tine accumulation was measured from the basolateral cell a s p e c t s

(vinblastine concentration 19.4 nM) in the presence or absence of increasing concentrations of verapamil, in both bathing solutions. Data are for Strain-1 epithelial layers, similar data were obtained for

Strain-2 epithelial layers. Data are the mean +_ S.E. (n = 3).

Strain 1 from 0.048 _+ 0.005 pm o l / cm 2 (n = 30), in controls, to 0.103 _+ 0.015 pm o l / cm 2 (n -- 9). However, this increased accumulation never approached vinblastine levels achieved with basolateral loading (control values of 0.204 _+ 0.015 pm o l / cm 2 are increased with verapamil present in both bathing solutions to 0.478 +_ 0.055 pmo l / cm 2 (n = 9); Fig. 7). With 100 /zM verapamil present only in the apical solution, there was no significant effect on vinblastine accumulation across the apical membrane (0.045 + 0.009 pm o l / cm 2 (n = 3), compared with control values of 0.048 + 0.005 p m o l / c m 2 (n = 30)), despite a significant increase in vinblastine accumulation across the basolateral membrane (to 0.442 + 0.036 p m o l / c m 2 (n- -3) ) . Thus, despite maximal inhibition of vinblastine secretion, cellular [3H]vinblastine accumulation across the apical cell border remained restricted.

In the presence of 50 mM 2-deoxy-o-glucose and 15 mM Na azide to inhibit transepithelial vinblastine secretion by ATP depletion (see above), accumulation of vinblastine across the apical border also remained restricted (control, 0.032 _+ 0.007 pm o l / cm 2, 2-deoxyglu- cose/azide, 0.037 _+ 0.001 pm o l / cm 2 (n = 3)).

Inhibition o f cell growth by vinblastine: enhancement by verapamil

Inhibition of cell growth (cytotoxicity) by vinblastine was dependent upon cell density (Table I) decreasing as density increased. The effect of verapamil in poten- tiating vinblastine cytotoxicity was greatest at the low- est cell density (Table I) and decreased as density increased. Similar data were observed in both cell strains, though Strain-2 cells appeared more sensitive to vinblastine. Access of vinblastine to the basolateral surface via the solution will decrease as epithelial layers are formed on the plastic surface. In this case, vinblastine permeation and accumulation will become progressively dependent upon trans-apical membrane permeation.

D i s c u s s i o n

TABLE I

Inhibition of MDCK cell growth (cytotoxicity) by vinblastine is dependent upon cell density

MDCK cells were seeded at (A) 2.10 3, (B) 4"10 3 and (C) 1.10 4

cells/well and then treated as described in Materials and Methods. The sensitivity of cells to vinblastine alone (Vin) or vinblastine plus 100 ~M verapamil (Vin+Ver) was then determined. Data are the mean values of the concentration of vinblastine required for 50% inhibition (IC50) of cell growth determined with 8 vinblastine concentrations with 4 replicate wells per dose for N separate experiments (in parentheses). The ratio of IC50 values for vinblastine alone to vinblastine plus 100/xM verapamil is also indicated, aCell killing was 100% within the concentration tested.

MDCK strain Treatment IC50 (~M)

A B C

1 (high resistance)

2 (low resistance)

Vin 21.4+0.9 43.8 +1.2 72.5+1.0 (3) (3) (3)

Vin+Ver 1.2+0.4 2.7 +0.7 22.2+0.6 (3) (3) (3)

Ratio Vin/(Vin + Ver) 17.8 16.2 3.3

Vin < 10 nM a 2.2 :t: 1.4 50.0 (3) (2)

Vin+Ver < 10 nM a 0.24+0.09 24.1 (3) (2)

Ratio Vin/(Vin+Ver) - 9.4 2.1

renal cortex [25]. Thus, the present data prompt the suggestion that P-glycoprotein expression is not unique to proximal tubule cells and that it may be more widely expressed along the renal tubule.

Horio et al. [10] have developed a theoretical model which attempts to analyze transepithelial vinblastine flux data in the context of unidirectional fluxes across the apical and basolateral membranes. In the present study we have analysed secretory vinblastine fluxes

TABLE II

Comparison of transepithelial solute transport rates and membrane transport density in Strain-1 epithelia

The present study has confirmed the existence of a net transepithelial secretion of vinblastine sulphate by native MDCK epithelial layers. Moreover we have ex- amined secretory vinblastine fluxes in defined MDCK cell strains displaying different phenotypic properties [17,18]. MDCK epithelial cells display antigenic properties consistent with a distal tubular or collecting duct location [23]. Many of their physiological properties are consistent with this tubular origin [24]. Initial immuno- cytochemical evidence suggested that P-glycoprotein expression was confined to proximal renal tubules [4-6]. In situ hybridizations suggest that renal medullary tissue express P-glycoprotein m R N A levels in excess of

Transport system Site density/cell Vma x Turnover (mol/cm 2 per h) number

Na+/K+-ATPase 2.3-105 a 0.5.10 -6 c 100 Na+/K+/2C1 - 7.0-105b 0.5 ' 10 - 6 c 400 P-glycoprotein 1.2.10 3 d 12-35" 10-12 10

1.2.102 12-35-10-12 100

a Data from Ref. 27. b Data from Ref. 28. ¢ Data are maximal rate of CI- secretion dependent upon

Na+/K+-ATPase and Na+/K+/2CI - co-transport activity. This will underestimate transport rates as a significant portion of function of the Na + + K+-ATPase activity will result from 'housekeep- ing' non-transepithelial ion flux [40].

d Calculation made using a cell density of 0.5'10 6 cells/cm 2 and turnover numbers of 10 or 100 s -I.

over an extended vinblastine concentration range in order to quantify directly the affinity and capacity of vinblastine secretion. In both MDCK cell Strains, K m values of 0.8 and 2.8/zM are consistent with the data of Horio et al. [10,26] and with vinblastine stimulation of ATPase activity in membranes from MDR1 transfected insect cells [21] and the multidrug resistant cell line, KB-V1 [22]. In addition, we have determined the maximal secretory capacity to be 12 (Strain 2) to 35 (Strain 1) pmol/cm 2 per h. In Table II we have compared the rate of transepithelial C1- secretion in Strain-1 MDCK epithelia (together with the measured density of the Na+/K+-ATPase [27] and Na+ /K+/2 CI- co-transporter [28] which energise such transepithelial CI- secretion) with P-glycoprotein mediated vinblastine secretion. The turnover number of P-glycoprotein is unknown, however, the maximal levels of ATPase activity with substrate stimulation of membrane ATPase, in MDRl-transfected Sf9 cells [21] and multidrug resistant KB-V1 cells [22], suggests that this is similar to other vanadate-inhibited P-type ATPases. Assuming realistic turnover numbers of 10 or 100 s-1, the calculated site density implies that P-glycoprotein is present at a very low copy number compared to major ATP-dependent proteins such as the Na+/K +- ATPase. The existence of significant polarity in transepithelial vinblastine secretion must, therefore, not be confused with abundant pump expression. Clearly, the possibility that significant vinblastine secretory capacity and other functional effects (e.g., protection against cytotoxic regimes) are associated with only small levels of protein expression may place restrictions on conclu- sions drawn from biochemical and immunocyto- chemical identification of P-glycoprotein expression in normal and cancerous tissue.

In a study of vectorial transport of vinblastine by dog kidney cells transfected with a retroviral vector containing MDR1, increased vectorial transfer was seen upon transfection [9]. At 10 /~M vinblastine (i.e., at saturating concentrations for vinblastine secretion) Vma x was increased by only 4-fold, suggesting that the major increase in immunoprecipitated P-glycoprotein in transfected cells may represent, in part, non-functional protein.

Horio et al. show (Eqn. 11 in Ref. 10) that the relative accumulation of intracellular [3H]vinblastine is directly proportional to the relative passive permeabilities of the apical and basolateral membranes. This relative apical to basolateral membrane permeability was also calculated from the reduction or increase in JB-A or JA-B, respectively, of [3H]vinblastine flux with unlabelled vinblastine (Eqn. 28 in Ref. 10) resulting in the value for the relative permeability of basal/apical membranes, kb/k,, = 0.67. We have provided evidence that the increase in JA-B seen at high vinblastine concentrations may arise in part by a non-specific

effect of high concentrations of vinblastine upon epithelial paracellular permeability. Direct determination of the amount of [3H]vinblastine accumulated across the apical or basolateral borders even in the presence of verapamil show that basolateral accumulation exceeds apical accumulation. This demonstrates that the apical membranes of both MDCK epithelia have low relative permeabilities as compared to the basolateral membranes. Several studies have demonstrated that the apical and basolateral plasma membrane domains possess unique lipid and integral membrane protein compositions [29]. It is also known that Strain-1 and -2 MDCK cells have distinct glycosphingolipid compositions [29]. Morphometric data show that the basolateral surface area exceeds that of the apical membrane by 7-10-fold [27,30]. It seems highly probable that the apical membrane restricts cellular vinblastine accumulation by a combination of both low intrinsic permeability and the presence of a low-capacity active pump (P-glycoprotein). The vinblastine permeability observed at tracer vinblastine concentrations is (1-2). 10 -3

cm/h. In epithelial monolayers of Caco-2 cells, compounds with log D values of 1.5 and 1.9 (propranolol and corticosterone) give unidirectional permeabilities of 1.5 and 2.0-10 -1 cm/h, respectively [31]. Thus, vinblastine permeability is less than would be expected on the basis of its lipophilicity (log D = 2.9) [32]. As vinblastine concentration is increased, the apparent permeability increases. This may be due to saturation of the efflux mechanism or to increased permeability by a parallel route such as the paracellular route (see above). Since verapamil is without marked effect upon JA-B, it is likely that the increased unidirectional permeability seen with increasing vinblastine concentrations is due mainly to an action on the paracellular pathway.

The present data support the linkage between the intrinsic resistance of epithelial cells to cytotoxic regimes and the action of P-glycoprotein. Intrinsic sensitivity to vinblastine correlates with the capacity of P-glycoprotein in each cell strain; verapamil inhibits P-glycoprotein function, enhances vinblastine accumulation and enhances vinblastine cytotoxic sensitivity. The present data confirm that despite a large variation in epithelial conductance, the paracellular route (via tight-junctions) for transepithelial vinblastine permeation for vinblastine is restricted. The effective impermeability of the apical border combined with diffusion restriction afforded by the tight-junctions between the epithelial cells, has implications for cytotoxicity studies in which epithelial cells are grown upon impermeant matrices such as plastic. At confluent cell densities where access to the basolateral domain is restricted, vinblastine accumulation would be dependent mainly on trans-apical membrane permeation and thus would be curtailed compared to basolateral loading.

Cellular accumulation of vinblastine across both the apical and basolateral membranes is linearly dependent upon external vinblastine concentration. Since secretory vinblastine flux demonstrates saturation kinetics, this implies that the major portion of cellular vinblastine is bound and that the intracellular binding elements have a large capacity. Free vinblastine, thus, comprises only a minor fraction. Recent evidence as to the cytostatic mechanism of vinblastine action is consistent with binding to tubulin [33]. The dose-dependent increase in cell-associated vinblastine seen with verapamil must occur by an action upon tubulin [33], in addition to inhibition of P-glycoprotein-mediated secretory flux.

The exact cellular location of P-glycoprotein in MDCK epithelial cells has not been determined, but the presence of vectorial transepithelial transport of vinblastine is consistent with a polarised expression to the apical cell surface. This apical polarization is also supported by the studies with MDCK cells transfected with MDR1 [11]. Recently, it has been suggested that P-glycoprotein may function as a volume-activated CI- channel [34]. In Strain-1 MDCK epithelia in which CI- secretion is stimulated by adrenaline (acting through cAMP/Ca2+), cell swelling is associated with an inhibition of CI- secretion, consistent with a basolateral location of the volume-activated CI- conductance [35].

Broxterman et al. [36] localised a significant portion of cellular P-glycoprotein immunofluorescence in drug resistant cells to intracellular vesicular structures. Ac- tive drug accumulation into an endomembrane (acidic) vesicular compartment, with subsequent exocytotic re- lease at the cell surface has been implicated in P-glycoprotein-mediated drug efflux [37]. Vesicular accumulation of vinblastine would not preclude vectorial transfer across an MDCK monolayer, provided that such vesicular traffic was polarised with final delivery to the apical membrane. Such a mechanism might explain how magnified protein expression might produce only modest flux increments [11] if delivery of drug-filled vesicles to the apical cell surface were rate limiting. Polarisation in endocytotic trafficking mechanisms from the apical and basolateral surfaces has been demonstrated in MDCK [30] and Caco-2 colonic adenocarcinoma cells [38]. It has also been reported that a significant transcytosis (bulk directional transcellular transport by a population of endocytotic vesicles) of extraeellular marker compounds exists. Could such a mechanism account for the vectorial transfer of vinblastine reported here? Direct determination of transepithelial inulin fluxes showed that these are at least an order of magnitude less than those of vinblastine, and non-vectorial in nature, demonstrating that an active accumulation must exist even with a vesicular model for transepithelial vinblastine permeation.

In summary, it is now apparent that the polarity of

expression of P-glycoprotein within an epithelium al- ters the apparent response of such a tissue to cytotoxic agents. This contrasts to non-differentiated or fibro- blastic cells with uniform surface properties [39].

Acknowledgement

We wish to thank the North of England Cancer Research Campaign for generous financial support for this work.

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