Biochemical and Biophysical Research Communications
Transepithelial vinblastine secretion mediated by P-glycoprotein is inhibited by forskolin derivatives
Abstract
[3H]Vinblastine transport across MDCK (renal epithelial) cell layers has been characterised. The basal-to-apical [3H]vinblastine flux (JA-B) (at 10 nM) exceeded apical-to-basal flux by 19.6 fold. Net vinblastine secretion (JB-A-JA-B)was inhibited by verapamil (0.1 mM) primarily by a reduction in JB-A, 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 JB-A and net vinblastine secretion of 64.3 ± 3.1% and 29.1 ± 4.8% respectively. 7β-deactyl-7β-(γ-N-methylpiperazino)-butyryl-forskolin was ineffective. Half-maximal inhibition of vinblastine secretion by 1,9-dideoxy-forskolin was observed at 65 μM. 1,9-dideoxy-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.
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Cited by (16)
Intestinal secretion of drugs. The role of P-glycoprotein and related drug efflux systems in limiting oral drug absorption
1997, Advanced Drug Delivery ReviewsOral bioavailability requires absorption of drugs across the intestinal epithelium. This may be mediated by either the paracellular and/or transcellular routes. Passive transcellular absorption requires the appropriate physicochemical properties to allow permeation across the apical and basolateral membrane domains. Compounds demonstrating these properties are more likely to be recognised as substrates for intracellular metabolism, such as by cytochrome P450 isozymes, and/or secretory drug efflux systems, including P-glycoprotein, such that oral bioavailability will be limited. P-glycoprotein, which leads to multidrug resistance in tumour cells, is an ATP-dependent secretory drug efflux pump, encoded by the MDR1 gene in humans. It acts to clear the membrane lipid bilayer of lipophilic drugs, in the manner of a flippase. In the intestine, as well as at specific other epithelial and endothelial sites, P-glycoprotein expression is localised to the apical membrane, consistent with secretory detoxifying and absorption limitation functions. Other secretory efflux systems, such as multidrug-resistance associated protein (a glutathione S-conjugate transporter), fluorochrome efflux systems and the methotrexate efflux system, together with drug ionic charge and the intestinal pH microclimate, may mediate intestinal secretion of a wide variety of drugs. Direct evidence for P-glycoprotein limiting drug absorption comes from studies in vitro with human Caco-2 cells and includes non-linear dependence of absorption on substrate (vinblastine) concentration, increased absorption upon saturation of secretion and increased absorption upon inhibition of P-glycoprotein function, with modulators such as verapamil. P-glycoprotein-like mechanisms are implicated in the intestinal secretion of a variety of drugs, in addition to classical P-glycoprotein substrates, including cyclosporin, certain peptides, digoxin, fluoroquinolones, ranitidine and β-adrenoceptor antagonists. These drug interactions with P-glycoprotein may explain the pharmacokinetics of absorption in vivo. P-glycoprotein function may be integrated with drug metabolism, with several drugs being common substrates for P-glycoprotein and cytochrome P-450 3A. Recognising the interactions of drugs with intestinal secretory and metabolic systems that limit absorption will lead to novel strategies of overcoming problems of poor oral bioavailability.
Renal secretion of xenobiotics mediated by P-glycoprotein: Importance to renal function in health and exploitation for targeted drug delivery to epithelial cysts in polycystic kidney disease
1997, Advanced Drug Delivery ReviewsThe proximal renal tubule is the major site of P-glycoprotein expression within the kidney; there is good evidence that P-glycoprotein expression is not restricted to the proximal tubule occurring in thick ascending limb and more distal segments. P-glycoprotein is expressed at the apical brush-border membrane where it participates in ATP-dependent extrusion of substrates to urine.
The Madin-Darby canine kidney (MDCK) dog-renal epithelial cell-line is a model for renal tubular P-glycoprotein mediated secretion. The apical membrane is rendered effectively impermeable to archetype substrates such as vinblastine by a combination of low intrinsic permeability and the operation of ATP-dependent export from cytosol to the apical (lumen) solution. Since the MDCK cell-line possesses features characteristic of the distal/collecting duct, this suggests a possible role for tubular expression at distal sites in limiting back-diffusion and trapping of moderately lipophilic P-glycoprotein substrates within the renal medulla after secretion in the proximal tubule and subsequent concentration resulting from tubular fluid reabsorption.
The relative role of P-glycoprotein in mediating renal clearance of archetype P-glycoprotein substrates is discussed. Due to the absence of inhibitory agents of absolute specificity a definite assessment is not yet possible. The pharmacological importance of P-glycoprotein mediated transport in renal cells is discussed in relation to renal transplantation and to treatment of renal carcinomas. The utility of transgenic “knockout” animal models for assessing P-glycoprotein function in renal clearance studies is highlighted.
Renal polycystic disease is a major cause of renal insufficiency. Cysts arise from tubules, but are closed structures with the epithelium oriented so that the brush-border is adjacent to the closed lumen. Bodipy-verapamil has been tested as a fluorescent substrate for P-glycoprotein mediated transepithelial secretion in MDCK-C5A epithelia (selected for their ability to form cysts in vitro) reconstituted first as a monolayer on permeable substrates. Net transepithelial secretion (Jnet) of [3H]-vinblastine from basal to apical surfaces is inhibited by taxotere, verapamil and bodipy-verapamil. Bodipy-verapamil is itself subject to a saturable transepithelial net secretion by MDCK-C5A epithelia. In MDCK-C5A cysts grown in hydrated collagen gel laser scanning confocal microscopy shows that bodipy-verapamil is accumulated within the cyst lumen above medium concentrations. Renal cystic epithelial P-glycoprotein expression is exploitable to target cytotoxic P-glycoprotein substrates to renal cysts. The possible advantages of targeting cytotoxic substrates to renal cysts is discussed.
Targeted delivery of a substrate for P-glycoprotein to renal cysts in vitro
1995, BBA - BiomembranesBodipy-verapamil has been tested as a fluorescent substrate for P-glycoprotein-mediated transepithelial secretion in MDCK-C5A epithelia. Net transepithelial secretion (Jnet) of [3H]vinblastine from basal-to-apical surfaces of monolayer epithelia is inhibited by taxotere, verapamil and Bodipy-verapamil primarily by a reduction in basal to apical vinblastine (Jb−a) transport. Bodipy-verapamil is itself subject to transepithelial net secretion by MDCK-C5A epithelia; at 5 μM a Jnet of −310 ± 32 nmol cm−2 h−1, (n = 3) was observed. When MDCK-C5A cells are grown to form enclosed cysts in hydrated collagen gel, Bodipy-verapamil is accumulated within the cyst lumen showing that epithelial P-glycoprotein function may be used to target substrates to renal cysts.
Influence of dexniguldipine-HCl on rhodamine-123 accumulation in a multidrug-resistant leukaemia cell line: Comparison with other chemosensitisers
1994, European Journal of CancerIn the clinical therapy of cancer, resistance to many cytostatic drugs is a major cause of treatment failure. Among other mechanisms, the expression and pumping activity of P-glycoprotein (PGP) in the membrane of resistant cancer cells is responsible for the reduced uptake of cytostatics. The blockade or inhibition of PGP activity by chemosensitisers seems to be a tenable way to restore sensitivity to antineoplastic drugs and therapeutic efficacy. In the present work the influence of the new chemosensitiser dexniguldipine on rhodamine-123 accumulation in multidrug-resistant leukaemia cells was investigated. Dexniguldipine increases cellular rhodamine-123 accumulation dose-dependently. pec50 values (−log concentration of drug showing a half maximal effect) in accumulation studies are dependent on pH of the test system and are in the range of 6.5 (pH 7.2) to 7.2 (pH 8.0) for dexniguldipine. In comparison with other chemosensitisers such as SDZ PSC 833, cyclosporin A, verapamil, dipyridamole, quinidine and amiodarone, dexniguldipine is the most potent drug in this test system. In addition to equilibrium measurements of rhodamine-123 accumulation, efflux of rhodamine-123 was analysed in the absence and presence of chemosensitisers. A clear dose-dependency was seen and, moreover, a dramatic decrease in efflux rates was achieved in the presence of chemosensitisers. The described system can be used to investigate PGP-mediated drug transport on a pharmacological and biochemical basis.
Transepithelial secretion, cellular accumulation and cytotoxicity of vinblastine in defined MDCK cell strains
1993, BBA - Molecular Cell ResearchTransepithelial vinblastine secretion in two defined MDCK strains displays saturation kinetics; (Strain 1) Km = 2.8 ± 0.6 μM (six experiments), Vmax 35.9 ± 1.93 pmol/cm2 per h (six experiments), Strain 2 Km 0.78 ± 0.36 μM (three experiments), Vmax 12.1 ± 4.5 pmol/cm2 per h (three experiments). Concentrations of vinblastine > 1 μM 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 μM 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 verapmil. 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.
Polarized efflux of 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein from cultured epithelial cell monolayers
1992, Biochemical PharmacologyWe have investigated the polarity of the efflux of the intracellular pH fluorochrome 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) from layers of epithelial Madin-Darby canine kidney (MDCK, Strains I and II) and human intestinal (Caco-2, HCT-8 and T84) cells grown on porous membranes. In Strain I MDCK cells, BCECF efflux was effectively reduced by indomethacin (50% inhibition with 100 μM) and 5-nitro-2-(3-phenylpropyl-amino)-benzoate (NPPB; 50% inhibition with 10 μM). Replacement of external Cl− with bromide, iodide or nitrate did not alter BCECF efflux, while substitution with methanesulphonate resulted in a small but significant reduction. All five cell lines form confluent epithelial layers when grown on porous membranes. Efflux of BCECF from Strain I MDCK epithelial layers into the apical solution was approximately three times greater than into the basal solution. Addition of indomethacin to the apical solution attenuated efflux into the apical but not the basal solution, while basal indomethacin was effective against basal efflux. NPPB has a similar specificity of action. Adrenaline, a stimulant of electrogenic Cl− secretion, did not alter the pattern of BCECF efflux. BCECF efflux was also polarized, with apical efflux greater than basal efflux, in MDCK Strain II and Caco-2 epithelial layers. In contrast, BCECF efflux into the basal and apical media was equivalent in layers formed from HCT-8 and T84 cells. However, indomethacin reduced efflux in all five epithelial lines, although the relative sensitivities of the apical and basal efflux rates to indomethacin varied, as did the sensitivity to the sidedness of application of indomethacin. In MDCK and HCT-8 epithelial layers, transepithelial vinblastine secretion mediated by P-glycoprotein was not inhibited by indomethacin. The data are consistent with the hypothesis that BCECF efflux is a manifestation of a novel ATP-dependent xenobiotic secretory efflux mechanism in renal and gastrointestinal epithelia. The factors regulating the polarity of BCECF efflux, both the indomethacin-sensitive and -insensitive components, have yet to be elucidated.