a Departments of Clinical Chemistry and b Oncology, Umeå University, S-901 87 Umeå, Sweden
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Abstract |
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Introduction |
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Amphotericin B creates cell membrane pores13 inducing K+ efflux and apoptosis in eukaryotic cells.4,5 We have shown previously5 that amphotericin B-induced apoptosis and cytotoxicity were considerably reduced by the Na+, K+, 2Cl- cotransport blocker bumetanide.
Efflux of K+ plays an important role during the apoptotic process. The transition of an apoptotic cell from a state of a high towards a low K+ content permits both the loss in cell volume and the activation of enzymes that mediate induction of apoptosis.6 K+ uptake mechanisms, such as Na+, K+ ATPase or Na+, K+, 2Cl- cotransport, are activated to compensate the loss of intracellular K+,7 and thereby counteract apoptosis.8 Modulation of potassium pumps could thus be expected to decrease or increase drug-induced apoptosis and cytotoxicity.
To clarify further the role of K+ flux in amphotericin B-induced apoptosis and cytotoxicity, we used the potassium analogue rubidium (86Rb+) to measure direct effects on K+ efflux and influx. The Na+, K+ ATPase blocker ouabain was used as a probe for determination of Na+, K+ ATPase and Na+, K+, 2Cl- cotransport activity on the studied human mesothelioma cell line (P31) known to express these two K+ uptake mechanisms.9 The question of whether addition of bumetanide influenced amphotericin B-induced cytotoxicity to Candida albicans fungal cells was examined with a plate diffusion method.
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Materials and methods |
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A human pulmonary mesothelioma cell line (P31) propagated under standard tissue culture conditions was used. The cells were grown as monolayer culture in Eagle's minimal essential medium (EMEM) in Earl's saline supplemented with 10% fetal calf serum, 200 µmol/L l-glutamine, penicillin and streptomycin. They were incubated at 37°C in a humidified atmosphere containing 5% CO2.
86Rb+ efflux
The P31 cells were grown for 24 h in the wells of 24-well plates as monolayer culture in EMEM in Earl's saline supplemented with 10% fetal calf serum, 200 µmol/L l-glutamine and 1 µmol/L 86RbCl (500 Ci/mol). They were incubated at 37°C in a humidified atmosphere containing 5% CO2. The medium was then discarded by aspiration and the plates washed once with EMEM without fetal calf serum. EMEM was then added to the wells, with or without test substances (final concentrations: 3 mg/L amphotericin B and/or 100 µmol/L bumetanide), and the incubation continued for 15 or 60 min. The supernatant of each well was then aspirated and added to labelled scintillation vials. The remaining adherent cells were trypsinized, the well content transferred to scintillation vials, and radioactivity determined by liquid scintillation counting. All wells were handled in parallel in each experiment and with the same number of cells/well.
86Rb+ influx and methodology for interpretation of results with channel-blocking agents
The pulmonary carcinoma cells were grown in 24-well cluster plates for 0 or 60 min in EMEM (containing 5.3 mmol/L K+), without (control) or with amphotericin B (3 mg/L) alone or combined with bumetanide (100 µmol/L), and then incubated for 15 min in the same medium supplemented with 1 µmol/L 86RbCl (500 Ci/mol), with or without ouabain (100 µmol/L), alone or in combination. The concentrations of the channel-blocking agents were chosen because of the total inhibition of Na+, K+, 2Cl- cotransport and Na+, K+ ATPase activity.9 The cells were rinsed, trypsinized and transferred to scintillation vials, and radioactivity was determined by liquid scintillation counting. Isotope influx in drug-treated cells was expressed as a percentage of the influx of controls (basal influx) handled in parallel and without test substances. Counting ensured that the number of cells treated with drugs at 60 min were not reduced in number compared with untreated control cells. From a previous study9 we learned that three types of potassium flux pathway exist in this pulmonary carcinoma cell line: Na+, K+, 2Cl- cotransport (blocked by bumetanide), Na+, K+ ATPase activity (blocked by ouabain) and high conductance K+ channels (blocked by tetraethylammonium, TEA). As the TEA effect on 86Rb+ influx of P31 cells was small (and in the present experiments almost negligible), with the remnant 86Rb+ influx c. 3% of control when combining test substances with ouabain and bumetanide simultaneously, we interpreted the reduction of 86Rb+ influx (in relation to untreated control) when adding ouabain (ouabain-sensitive 86Rb+ influx) as being due to Na+, K+ ATPase activity, and the 86Rb+ influx remaining (ouabain-insensitive 86Rb+ influx) minus the remnant 86Rb+ influx in the presence of both ouabain and bumetanide as being due solely to Na+, K+, 2Cl- cotransport activity.
Plate diffusion method
To measure fungal susceptibility to amphotericin B alone and in combination with bumetanide we used a plate diffusion method. Two colonies of C. albicans were suspended in 5 mL of Milli-Q and transferred to a buffered YNB plate. Amphotericin B (100 µL, final concentration 3 mg/L; n = 6), alone or in combination with rising concentrations of bumetanide (1, 10, 100 µmol/L; n = 2), standard solution and internal standard solution were added to 10 mm holes punched in the plate. The plates were incubated for 30 min at room temperature, then further incubated at 37°C overnight. The size of the inhibition zones produced by the different solutions was measured and compared with amphotericin B alone.
Chemicals
Amphotericin B (CAS 1397-89-3) lot no. 70316 was from Bristol-Myers Squibb, New York, NY, USA. Ouabain (CAS 11018-89-6), bumetanide (CAS 28395-03-1) and tetraethylammonium chloride (CAS 56-34-8) were from Sigma Chemical Company, St Louis, MO, USA. 86RbCl (CAS 7791-11-9) was purchased from Amersham International, Amersham, UK. EMEM and l-glutamine were from Gibco Ltd, Paisley, UK. Fetal calf serum was purchased from Biochrom KG, Berlin, Germany. Cell culture 24-well cluster plates and polystyrene dishes were from Costar, Cambridge, MA, USA. All other chemicals were of analytical grade.
Statistics
Statistical significance of the differences between surviving clones was tested with one-way ANOVA. The level of significance for rejecting the null hypothesis of zero treatment effect was P = 0.05.
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Results |
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The 86Rb+ efflux in P31 pulmonary mesothelioma cells was run for 15 or 60 min. Therefore, we assumed that the efflux during the first 15 min reflects the efflux rate of 86Rb+ and is not affected by re-uptake of extracellular accumulation of the isotope. 86Rb+ efflux during 60 min, however, could possibly be counteracted by 86Rb+ influx due to extracellular accumulation.
Amphotericin B (3 mg/L) or bumetanide (100 µmol/L) significantly (P < 0.05) stimulated the immediate (15 min) 86Rb+ efflux from 72.2 ± 2.4 (control) to 78.8 ± 1.9 and 86.1 ± 5.3 pmol/105 cells/15 min, respectively (Figure). However, when added together, amphotericin B/bumetanide initially reduced cellular 86Rb+ efflux markedly and significantly (P < 0.001) to 57.8 ± 2.4 pmol/105 cells/15 min, which is even below the level of untreated control cells. When the 86Rb+ efflux was run for 60 min, bumetanide reduced (P < 0.05) efflux [from 164.8 ± 4.6 (control) to 151.5 ± 3.0 pmol/105 cells/60 min]. The augmented efflux induced by amphotericin B was even more pronounced (P < 0.001) after 60 min efflux, but now the amphotericin B/bumetanide mixture augmented (from 197.8 ± 5.6 to 238.3 ± 3.9 pmol/105 cells/60 min; P < 0.001) the efflux seen with amphotericin B alone (Figure
).
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The 86Rb+ influx in P31 lung mesothelioma cells was linear for 15 min. Therefore, it was assumed that the influx during this time period reflects the influx rate of 86Rb+ and is not affected by intracellular accumulation of the isotope.9 Addition of TEA (a blocker of K+ channels with high conductance) to the ouabain and bumetanide combination only marginally affected the 86Rb+ influx (results not shown). The absolute influx of 86Rb+ of control cells was 74.4 ± 0.8 pmol/105 cells/15 min. Ouabain (100 µmol/L) inhibited the influx to 23.0 ± 3.6 pmol/105 cells/15 min, thus yielding an Na+, K+ ATPase activity of 51.4 (74.423.0) pmol/105 cells/15 min and an Na+, K+, 2Cl- cotransport activity (ouabain-insensitive 86Rb+ influx) of 20.4 (23.02.6) pmol/105 cells/15 min, when remnant influx (2.6 pmol/105 cells/15 min) of the combination of ouabain and bumetanide was deducted. This remnant 86Rb+ influx was not affected (influx 2.63.5 pmol/105 cells/15 min) by the addition of amphotericin B. This indicates that Na+, K+, 2Cl- cotransport and Na+, K+ ATPase activity are responsible for almost all 86Rb+ influx in this cell line and that K+ channels with high conductance (TEA-blockable) were not activated during experiments. Bumetanide (100 µmol/L) inhibited 86Rb+ influx to 51.8 ± 3.6 pmol/105 cells/15 min, indicating a (bumetanide-sensitive) Na+, K+, 2Cl- cotransport activity of 22.6 (74.451.8) and an Na+, K+ ATPase activity of 49.2 (51.82.6) pmol/105 cells/15 min (Table 1). The similar results obtained on cotransport and ATPase activity, whether bumetanide or ouabain was used as the probe for the activity determination, shows that the probes are specific inhibitors of cotransport and ATPase activity in the control situation without amphotericin B.
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86Rb+ influx after 60 min incubation with amphotericin B
Addition of TEA (a blocker of K+ channels with high conductance) to the ouabain and bumetanide combination only marginally reduced the 86Rb+ influx (from 4.0 ± 0.07 to 3.5 ± 0.20 pmol/105 cells/15 min; n = 6) after 60 min incubation.
86Rb+ influx of control cells with or without ouabain and/or bumetanide was very similar to the results of the shorter 15 min incubation (Table 2). However, after incubation with amphotericin B for 60 min, 86Rb+ influx activity was increased to 88.8 ± 1.9 pmol/105 cells/15 min. Using ouabain as a probe showed that this was due to a huge (from 51.3 to 78.0 pmol/105 cells/15 min) activation of Na+, K+ ATPase, but a reduced (7.4 pmol/105 cells/15 min) Na+, K+, 2Cl- cotransport activity. The combination of amphotericin B with bumetanide incubated for 60 min showed similar effects on Na+, K+ ATPase and Na+, K+, 2Cl- cotransport activity results to amphotericin combined with ouabain (Table 2
).
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The mean value (n = 6) of the inhibition zones by amphotericin B (3 mg/L) was 23 mm. When bumetanide was added in rising concentrations (1, 10, 100 µmol/L), the inhibition zones were 23.7, 22.7 and 22.7 mm, respectively (mean value, n = 2), thus there was no significant difference compared with amphotericin B alone.
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Discussion |
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Despite its considerable toxic effects, amphotericin B is the drug of choice in the treatment of systemic fungal infections. During therapy there are difficulties in obtaining adequate fungistatic plasma levels of amphotericin B without causing undesired side effects such as nephrotoxicity,10 or electrolyte abnormalities. Clinically, amphotericin B levels of c. 4.3 µmol/L (4 mg/L) have been obtained.11 Amphotericin B produces pores in the membrane,13 inducing potassium efflux, and increases by a factor of six the opening probability of potassium channels.12 A non-toxic concentration of amphotericin B significantly increased the endothelial cell membrane permeability to K+.13 Leakage of K+ within a few minutes was also found in rat hepatocytes.14
Transmembrane fluxes of cations are involved in several important cell processes, including cell proliferation, volume regulation and apoptosis.8 Efflux of K+ is fundamental during the apoptotic process since potassium loss causes the volume reduction of the cell during apoptosis. Low intracellular potassium concentration seems to be important for the activation of enzymes mediating apoptosis, and similarly physiological levels of K+ inhibit enzyme activation.6,15 Potassium efflux provoked by using NMDA-receptor gated K+ efflux, K+ ionophores or K+ channel openers promotes neural apoptosis.16,17 Thus, modulation of transmembrane K+ flux and thereby the intracellular potassium content would influence the induction of apoptosis and cytotoxicity of various drugs, including amphotericin B.
The major ion transport system accomplishing K+ uptake in shrunken cells and thereby counteracting apoptosis is the Na+, K+, 2Cl- cotransporter. K+ accumulation is inhibited by blockers of cotransport, thereby leading to increased intracellular K+ loss.1820 Surprisingly we found that the Na+, K+, 2Cl- cotransport inhibitor bumetanide markedly reduced the cytotoxicity of amphotericin B from 64% to 86% surviving clones of P31 cells. The combination of amphotericin B with bumetanide almost annihilated the apoptotic index (P < 0.05) in the quantitative nucleosome ELISA assay.5 Unexpectedly, however, the combination of bumetanide and amphotericin B initially reduced 86Rb+ efflux even below the 86Rb+ efflux of control cells. Thus, the elimination of amphotericin B cytotoxicity and apoptosis by bumetanide may be due to inhibition of potassium efflux rather than to inhibition of Na+, K+, 2Cl- cotransport activity. Whether or not the annihilating interaction of bumetanide with amphotericin B cytotoxicity and apoptosis is specific for bumetanide and related to Na+, K+, 2Cl- cotransport activity has to be further elucidated by investigating the doseresponse relationship of bumetanide and analogues on cytotoxicity, apoptosis, cell size and 86Rb+ flux. The combinatory effect of bumetanide and amphotericin B could be interpreted as initial blocking of stimulated Na+, K+, 2Cl- cotransport activity with a simultaneous reduction of Na+, K+ ATPase activity. However, as bumetanide in some situations is also known to inhibit Na+, K+ ATPase activity, an alternative interpretation of stimulated Na+, K+, 2Cl- cotransport activity would be that the combination of bumetanide with amphotericin B in addition to blockage of Na+, K+, 2Cl- cotransport activity also partly inhibits Na+, K+ ATPase activity. The alternatives could perhaps be resolved only by recording cell volume measurements concomitantly with 86Rb+ flux determinations.
The preserved antifungal effect implies a clinical application of the Na+, K+, 2Cl- cotransport blocker bumetanide, or related drugs in antifungal therapy, to increase amphotericin B effectiveness without increasing its adverse effects. The induction of apoptosis seems to be of significant importance in amphotericin B-induced cytotoxicity. We have shown previously that the Na+, K+, 2Cl- cotransport inhibitor bumetanide eradicated amphotericin B-induced cytotoxicity and apoptosis.9 When Na+, K+, 2Cl- cotransport activity of amphotericin B-treated cells is blocked by bumetanide, K+ efflux is considerably reduced. Thus, bumetanide reduction of amphotericin B cytotoxicity and apoptosis requires preserved intracellular K+ levels, and may be due to an initial reduction of cellular K+ efflux as well as a derangement of cellular K+ influx.
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Acknowledgements |
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Notes |
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References |
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Received 11 April 2001; returned 5 July 2001; revised 21 August 2001; accepted 30 August 2001