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Vol. 181, No. 2, 1991
December 16, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 671-676
TIUNS-VlNBLASTINESECRETIONMEDIATEDBY
P-GLYCOPROTEIN Is INHIBITED BY FORSKOLIN DERIVATIVES
Janice Hunter, Barry H. Hint and Niiolas L. Simmons
Gastrointestinal Drug Delivery Research Centre, and Department of Physiological Sciences, University of Newcastle upon Tyne,
Medical School, Newcastle upon Tyne NE2 4HH, UK
Received October 28, 1991
Summary: [3HjVinblastine transport across MDCK (renal epithelial) cell layers has been character&d. The basal-to-apical FHlvinblastine flux (J& (at 10 nM) exceeded apical-to- basal flux by 19.6 fold. Net vinblastine secretion (JBA - J,,-B) was inhibited by verapamil(O.1 mM) primarily by a reduction in JB-*, consistent with net vinblastine secretion resulting from an inhibition of P-glycoprotein. 1,9-Dideoxy-forskolin and forskolin (0.1 mM) both resulted in significant inhibition of JBA and net vinblastine secretion of 64.3 f 3.1% and 29.1 f 4.8% respectively. 7~-deactyl-7/3-(~-N-methylpiperaxino)-butyryl-forskolin was ineffective. Half-maximal inhibition of vinblastine secretion by 1,9dideoxy-forskolin was observed at 65 PM. 1,9dideoxy-forskolin is unable to stimulate adenylate cyclase, suggesting that this forskolin derivative is a potentially important lead antagonist of P-glycoprotein for circumvention of pleiotropic drug resistance. (D 1991 Academic Press, Inc.
P-glycoprotein is a 170-180 kDa membrane glycoprotein associated with the phenomenon of
pleiotropic (multidrug) resistance (MDR) (reviewed in reference 1). Immunohistochemical
techniques have demonstrated the presence of P-glycoprotein in the apical regions of several
natural epithelia (2-5), 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-PK,), 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 (6-8). Retroviral
transfection of the Madin-Darby canine kidney (MDCK) epithelial cell line with mdrl cDNA
results in polarised expression of P-glycoprotein to the apical plasma membrane domain (9)
and an enhanced transepithelial secretion of vinblastine above intrinsic levels (6). We have
used the vinblastine secretion by native MDCK epithelia (6,7) as a direct functional measure
of P-glycoprotein to investigate its modulation by intrinsic stimuli and pharmacological
0006-291X/91 $1.50
671 Copyright 0 1991 by Academic Press, Inc.
All rights of reproduction in any fom reserved.
Vol. 181, No. 2, 1991 BIOCHEMtCAL AND BtOPHYSlCAL RESEARCH COMMUNlCATtONS
agents. Here we report that forskolin derivatives are effective inhibitors of P-glycoprotein
function. l,PDideoxy-forskolin has no effect upon stimulation of adenylate cyclase,
suggesting that such an otherwise inert biological molecule may be an important lead
compound in the development of specific inhibitors of P-glycoprotein function that will be
useful therapeutically.
Cell Culture MATERI[AIs AND METHODS
Strain 1 (60-75 serial passages) MDCK cells (10) were maintained in serial culture in Eagle’s Minimum Essential Medium supplemented with 2% v/v foetal calf serum, 8% v/v donor horse serum, kanamycin antibiotic (1 pg/ml), non-essential amino acids (1% v/v) and 2 mM glutamine. Confluent monolayers were subcultured every 7 days, by treatment with 0.05% trypsin and 0.2% EDTA in Ca”- and Mg*+-free phosphate-buffered saline (PBS). All cultures were incubated at 37°C in a humidified atmosphere of 5% C@/95% air.
For experimental purposes MDCK cells were grown as epithelial layers by high- density seeding (106 cellslcm2) onto permeable filter matrices (Anocel125mm culture inserts, 4 cm* growth area). Culture inserts were cultured in &-well plates for 2-3 days. The formation of functional epithelial layers was monitored visually and by the development of a significant transepithelial resistance (IQ, as measured using a WPI Evometer fitted with “chopstick” electrodes to allow transepithelial current passage and potential sensing (11). Cell monolayers were used when the transepithelial resistance exceeded 1 kQ *cm*. Values of resistance for filter inserts alone, - 300 fl l cm2, are subtracted from all data.
Measurement of bidirectional transepithelial13H/vinblasrim sulphate flures
Measurements of transepithelial solute flux were made essentially as described by Hunter et al. (7). Functional epithelial layers in filter cups were washed with 2 x 3 ml serum-free medium and placed into fresh 6-well plates containing 3 ml serum-free medium (basal solution), a further 3 ml serum-free medium was then pipetted into the upper chamber (apical solution) of the filter cup. Transepithelial resistance was measured following 10 min incubation of the cells at 37”C, as described above.
The medium on either the apical or basal side of the monolayers was then removed and replaced with 3 ml serum-free medium containing 10 nM [3avinblastine sulphate as tracer, and that on the contralateral side replaced with 3 ml serum-free medium containing 10 nM vinblastine sulphate, in the presence or absence of varying concentrations of verapamil or forskolin derivatives, followed by incubation at 37°C. In order to measure the bidirectional fluxes of vinblastine sulphate (JxeB, flux from apical to basal solutions, and JBA, flux from basal to apical solutions), 100~1 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. Each incubation was performed at least in triplicate. On completion of the flux experiments epithelial integrity was determined by measurement of transepithelial resistance.
Measurement of adenylate cyclase activities in MDCK cell homogenates
Adenylate cyclase was measured in trip&ate on homogenates of MDCK cells (12) in a buffer containing 80 mM Tris-maleate, 4 mM MgSO,, 0.2 mM EGTA, 1 mM isobutylmethyl- xanthine, 1 mM ATP, 0.1 mM GTP, 10 mM creatine phosphate and 10 pg cm-3 creatine kinase. Incubations were terminated by HCl and after neutralisation, samples were assayed for their CAMP content using a competitive protein binding assay (13).
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Vol. 181, No. 2, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Data are expressed as mean f SEM of n replicates (within experiment) or N experiments (between experiment). Tests of significance of differences between mean values were made using a two-tailed Student’s I test, where appropriate. Inhibition constants for verapamil inhibition of vinblastine flux were determined by non-linear regression with the method of least-squares fitting for a logistic sigmoid (P.Fit, Biosoft, Cambridge UK)
Materials
[3H]vinblastine sulphate was obtained from Amersham International (Little Chalfont, Bucks, UK). All tissue culture media and reagents (Gibco BRL) and tissue culture plastics and Anocell tissue culture inserts (Nunc) were supplied by Life Technologies Ltd. (Paisley, Scotland). Forskolin, 1,9-dideoxy-forskolin (DiD-forskolin) and 7/3-deactyl-7/3-(7-N- methylpipcrazino)-butyryl-forskolin dihydrochloride (DMPB-forskolin) were obtained from Calbiochem (Novabiochem, Nottingham, UK). All other chemicals were obtained from Sigma Chemical Co. (Poole, Dorset) or BDH Chemicals Ltd. (Poole, Dorset).
RESULTS
Strain 1 MDCK epithelia have a high transepithelial electrical resistance (3354 + 214
@I= 18) Q *cm’> typical of right epithelia. With tracer concentrations of vinblastine (10 nM)
a marked asymmetry in the bidirectional transepithelial fluxes of vinblastine is observed (Fig.
1); the basal-to-apical flux JB,, (0.253 + 0.014 pmol crnm2 he1 (N=15)) being considerably
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Fr~uan 1. Transepithelial vinblastine sulphate fluxes. [31-Ijvinblastine flux was measured in the basolateral-*apical (B-A), apical-to-basolatera1 (A-B), and in the basolateml-to-apical direction in the presence 0.2 mM verapamil (Verqamil) or 10 CM DiD-forskolin @ii Forslcolin), added to both apical and basal bathing solutions. The lines illustrate the least squares best fit to the data, the slopes of which give the vinblastine flux rate. Data arc mean values f SE (n=3) from 5, 2 and 1 experiment! for control, vesapamil and DiD-forskolin respectively. Data for A-B fluxes plus veraPamiI and DiD-forskolin superimpose on control data and are omitted for clarity (see text).
Fmr.utn 2. Concentration-dependence of forskolin (B). DiD-forskolin (0) and veqxunil @,) inhibition of J&,, vinblastine flux in MDCK epithelial cell layers. Vinblastine flux rates are expressed as a percentage of the control flux in the absence of any modulator in the same experiment. Data arc the mean f SE of three observations per data point.
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Vol. 181, No. 2, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
larger than flux in the reverse direction (JAea; 0.013 f 0.003 pmol crne2 h-’ (N= 15)). A net
secretion (0.24 pmol cmq h-l) of vinblastine thus occurs from basal-to-apical surfaces. Under
control conditions and in the presence of vinblastine the spontaneous transepithelial electrical
potential difference is low (l-3 mV, basal solution electropositive). The observed
asymmetries for vinblastine flux (JbA/JAm8 of 19.5 fold), therefore, cannot be accounted by
passive electrica? forces and must represent active transport by the epithelium. Net
vinblastine flux (Jad = JBA - J,& demonstrates saturation kinetics; K,,, = 2.8 f 0.6 (N=6)
PM, V, 35.9 f 1.93 (N=6) pmol cmS2 h-l.
Verapamil(O.1 mM) effectively abolishes the net secretion of vinblastine (Fig. 1) by
reducing J, to 0.045 f 0.001 (N=9) pmol crnm2 h-l (P<O.OOl vs control flux), that is
towards the levels of JAeB (0.023 f 0.009 pmol cm” h-r; P>O.O5 vs control data). The
inhibition of vinblastine secretory flux by verapamil is dose-dependent (Fig. 2), half-maximal
inhibition being observed at 3.2 f 0.3 PM (df=17).
Forskolin (10 PM in both apical and basal solutions) reduced vinblastine secretion,
by a reduction in JB,, to 0.216 + 0.017 (N=6) pmol cm” h-l, with no effect upon JAmB (0.015
f 0.003 pmol cm-’ h-‘) (see also Fig. 2). This inhibition showed variability from batch to
batch of MDCK layers; for 4 separate batches the mean inhibition of vinblastine secretion
with 10 FM forskolin was 31.2 f 8.4% (range 17-5556). DiD-forskolin at 10 FM gave a
pronounced inhibition of vinblastine secretion (Fig. 1); JBA was reduced to 0.145 f 0.02
(N=6) pmol cmm2 h-l (P< 0.01 vs controls) while JA.B was only slightly elevated to 0,025 f
0.016 (N=6) pmol cm3 h I. Again variation in the inhibition from batch to batch of MDCK
cell layers was observed (see Fig. 2). DMPB-forskolin, 10 PM, was without effect upon
vinblastine fluxes. Figure 2 illustrates the concentration-dependent effects of forskolin and
its derivative; DiD-forskolin was most potent, half-maximal inhibition of JBA being observed
at 65 * 32 PM (df=17). Forskolin at the maximal concentration used (0.3 mM) reduced
vinblastine flux by 35% in this experiment.
The inhibition of vinblastine flux by forskolin is unrelated to the ability of these
compounds to stimulate adenylate cyclase. At 10 PM forskolin increased adenylate cyclase
in MDCK cell homogenates from basal values of 464 f 41 to 5304 f 323 (n=3) pmol mg
protein-’ 15 min“, whereas DMPB-forskolin increased adenylate cyclase to 3317 f 167 pmol
mg protein” 15 min-‘, and DiD-forskolin did not increase adenylate cyclase above basal
values.
DISCUSSION
The present study has confumed the functional expression of P-glycoprotein in native
(untransfected) epithelial layers of MDCK cells. The observed net transepithelial secretion
of vinblastine sulphate by native MDCK epithelial layers, which is inhibited by verapamil,
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Vol. 181, No. 2, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
provides an in vitro test system by which endogenous and exogenous regulation of P-
glycoprotein function may be conveniently monitored. Forskolin and DiD-forskolin are
shown to inhibit P-glycoprotein mediated flux. The mechanism of this inhibition is unlikely
to correlate with stimulation of adenylate cyclase since DiD-forskolin is an ineffective activator of adenylate cyclase. Conversely, DMPB-forskolin stimulates adenylate cyclase
activity in MDCK cells, but is unable to modify vinblastine flux.
The inhibition of P-glycoprotein mediated vinblastine secretion by forskolin and DiD-
forskolin is likely to correlate with chemosensitisation of drug-resistant cells by these
compounds, since agents which reverse multi-drug resistance are associated with inhibition
of vinblastine transport in isolated membrane vesicles (14). Further studies will be aimed
at correlating the effects of forskolin and derivatives upon membrane transport, intracellular
drug accumulation and chemosensitisation. A chemosensitising action of forskolin and DiD-
forskolin for doxorubicin-resistance in murine sarcoma S 180 variants has been reported (15).
The present data also point to the apparent diversity of chemical agents that are
capable of modulating P-glycoprotein function (1); recently intrinsic multidrug resistance in
Chinese hamster ovary cells has been reversed by amiloride analogues (16). An advantage I
of DiD-forskolin may reside in a lack of a specific pharmacology unrelated to P-glycoprotein
inhibition; verapamil, calmodulin antagonists, steroids and amiloride are (clinical) entities
whose use in therapeutic concentrations (as chemosensitisers) is limited by their primary
actions (1) (e.g., as Ca-channel or Na-channel blockers).
ACKNOWLEDGMENT
We wish to thank the North of England Cancer Research Campaign for generous financial support for this work.
REFERENCES
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[9] Pastan, I., Gottesman, M.M., Kazumitsu, U., Lovelace, E., Rutherford, A.V., and Willingham, M.C. (1988) Proc. Narl. Acad. Sci. USA g&4486-4490
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