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Regulation of the plasma membrane potential in Pneumocystis carinii

Nicole VanderHeyden , Gerald L. McLaughlin , Roberto Docampo
DOI: http://dx.doi.org/10.1111/j.1574-6968.2000.tb08979.x 327-330 First published online: 1 February 2000

Abstract

Many protists use a H+ gradient across the plasma membrane, the proton motive force, to drive nutrient uptake. This force is generated in part by the plasma membrane potential (ΔΨ). We investigated the regulation of the ΔΨ in Pneumocystis carinii using the potentiometric fluorescent dye bisoxonol. The steady state ΔΨ in a buffer containing Na+ and K+ (standard buffer) was found to be −78±8 mV. In the absence of Na+ and K+ (NMG buffer) or Cl (gluconate buffer), ΔΨ was not significantly changed suggesting that cation and anion conductances do not play a significant role in the regulation of ΔΨ in P. carinii. The ΔΨ was also not affected by inhibitors of the Na+/K+-ATPase, ouabain (1 mM), and the K+/H+-ATPase, omeprazole (1 mM). In contrast, inhibitors of the plasma membrane H+-ATPase, dicyclohexylcarbodiimide (100 μM), N-ethylmaleimide (100 μM) and diethylstilbestrol (25 μM), significantly depolarized the ΔΨ to −43±7, −56±5 and −40±12 mV, respectively. The data support that the plasma membrane H+-ATPase plays a significant role in the regulation of ΔΨ in P. carinii.

Keywords
  • Bisoxonol
  • H+-ATPase
  • Membrane potential
  • Pneumocystis carinii

1 Introduction

The fungus Pneumocystis carinii is the etiologic agent of P. carinii pneumonia (PcP). P. carinii is an opportunistic pathogen of the lung that has been recognized as a significant cause of morbidity and mortality in AIDS and other immunosuppressed patients. PcP remains a problem because of limited therapeutic choices and adverse reactions to the two standard treatment regimens, trimethropin-sulfamethoxazole and pentamidine isethionate [1,2]. Therefore, it is important to identify enzymes and metabolic processes in P. carinii that might be potential targets for drug development.

The plasma membrane potential (ΔΨ) of eukaryotic cells is generated in part by the concentration gradients of H+, Na+, K+ and Cl across the plasma membrane and by the operation of electrogenic pumps [3]. In most mammalian cells, the ΔΨ is generated by the electrogenic Na+/K+-ATPase, which pumps three Na+ out for every two K+ it brings in, creating the high cytosolic K+ and inside negative ΔΨ [4]. The activity of this ATPase as well as the permeability of the membrane to various ions then determines the actual resting ΔΨ, that for various mammalian cells ranges between −10 and −90 mV. In plants, yeast and many lower eukaryotes which lack a Na+/K+-ATPase, a plasma membrane P-type H+-ATPase generates the ΔΨ through electrogenic proton efflux [5]. In eukaryotic cells, ΔΨ is believed to play a role in signal transduction between the cell surface and the interior via voltage- or ligand-gated ion channels [6]. Furthermore, H+-ATPases and the Na+/K+-ATPase generate electrochemical gradients, the protonmotive and sodiomotive forces, respectively, that drive secondary transporters involved in moving ions and metabolites across the cell membrane. In P. carinii, an H+-ATPase has been shown to regulate intracellular pH (pHi) [7] and a gene for a putative P-type H+-ATPase has been identified [8].

In this work, we studied the regulation of the ΔΨ in short-term culture P. carinii using the potentiometric fluorescent dye bisoxonol. ΔΨ was found to be highly negative and diminished by inhibitors of H+-ATPases, supporting the role of these pumps in the regulation of ΔΨ in P. carinii.

2 Materials and methods

2.1 Short-term cultivation of P. carinii

Human embryonic lung fibroblasts (HEL-299, ATCC CCL137) were grown on Cytodex beads in spinner flasks containing minimum essential medium (MEM) and 10% fetal calf serum, as described before [9], and inoculated with P. carinii obtained from infected rat lungs at a concentration of 7×105 trophozoites ml−1. Cultures were incubated with stirring for up to 7 days at 37°C. P. carinii were obtained from the culture as described before [9]. The inocula were prepared by homogenizing P. carinii-infected rat lung in MEM, centrifuging the homogenates at 250×g to sediment large pieces of tissue, counting the numbers of trophozoites in 10-μl Giemsa-stained samples of supernatant, and adjusting the volume of the supernatant appropriately to achieve the desired concentration of trophozoites per ml. After harvesting, the cells were washed twice at 2000×g for 10 min in buffer A (116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 5.5 mM glucose, 50 mM HEPES) and resuspended in the same buffer to a final density of 1×109 cells ml−1 and maintained on ice.

2.2 Chemicals and solutions

MEM, fetal calf serum, dicyclohexylcarbodiimide (DCCD), diethylstilbestrol (DES), N-ethylmaleimide (NEM), gramicidin D, filipin and ouabain were purchased from Sigma Chemical. Omeprazole was a gift from Astra Hässle AB (Mölndal, Sweden) through the courtesy of Dr. K. Andersson. Bisoxonol was from Molecular Probes, Eugene, OR, USA. All other reagents were of analytical grade.

Standard buffer (135 mM NaCl, 5 mM KCl, 5 mM glucose, 1 mM CaCl2, 1 mM MgSO4 and 10 mM HEPES/Tris), gluconate buffer (135 mM Na gluconate, 5 mM K gluconate, 5 mM glucose, 1 mM Ca gluconate, 1 mM MgSO4 and 10 mM HEPES/Tris) and NMG buffer (140 mM N-methylglucamine, 5 mM glucose, 1 mM MgSO4, 1 mM CaCl2 and 10 mM HEPES/Tris) were adjusted to pH 7.4.

2.3 Plasma membrane potential measurements

The membrane potential was measured fluorometrically using the potentiometric dye bisoxonol whose fluorescence intensity increases with cell depolarization [10]. Bisoxonol is anionic and therefore has the advantage of not accumulating in the mitochondria. However, because it is lipophilic, it has the disadvantage of interacting with many hydrophobic compounds. Therefore, all inhibitors and carrier solutions were examined in a cell free system with bisoxonol first. For each experiment, 1×108 trophozoites were added to a cuvette containing 0.2 μM bisoxonol that had been allowed to equilibrate in 2.5 ml of the appropriate buffer for approximately 5 min. The fluorescent intensity was measured at 37°C in a Hitachi F-2000 spectrofluorometer with excitation set at 540 nm and emission at 580 nm. Background fluorescence was subtracted from the cell fluorescence for all measurements. Inhibitors were added to the cuvette after cell fluorescence had reached steady state. When large volumes of inhibitors were added (50 μl or more), an identical amount of buffer was subsequently added to determine the decrease in fluorescence (ΔF) due to dilution and this was subtracted from the ΔF resulting from changes in ΔΨ. The membrane permeabilizing agent filipin (25 μM) was added at the completion of each experiment to completely depolarize the parasites and act as an internal standard. The concentration of dye and the concentration of parasites were both found to be linearly related to total fluorescence in the range used for these experiments. Calibration was done by washing and resuspending the parasites in NMG buffer.

3 Results and discussion

The ΔΨ for any given cell is determined by the concentration gradient of various ions including protons across the plasma membrane. The energy for generating the gradients is provided by ATP through the operation of electrogenic pumps but ultimately, the ion gradients are the result of the activity of ion channels and transporters in the plasma membrane [3]. Therefore, to investigate the generation and regulation of ΔΨ, it is necessary to examine the effects of external ion concentrations as well as the role of electrogenic pumps.

The calibration of ΔΨ in trophozoites is shown in Fig. 1. Following signal stabilization, the Na+/K+ ionophore gramicidin D (0.8 μM) was added to the buffer. KCl was then incrementally added and the fluorescent signal recorded after each addition (Fig. 1A). The membrane potential values were calculated using the Nernst equation assuming an intracellular K+ concentration of 120 mM (Fig. 1B). The resting plasma membrane potential (ΔΨ) of P. carinii in a buffer containing Na+ and K+ (standard buffer) at 37°C was found to be −78±8 mV (Fig. 2). This value is less than that reported for many lower eukaryotes including plants, fungi and protists whose ΔΨ are highly negative (−120 to −150 mV) but similar to values observed in many non-excitable mammalian cells [10].

1

Calibration of membrane potential (ΔΨ) in P. carinii trophozoites. An aliquot of 1×108 cells was equilibrated with 0.2 μM bisoxonol in NMG buffer. The traces in A are of fluorescence intensity (540 nm excitation and 580 nm emission) after background subtraction following addition of gramicidin D (0.8 μM) and KCl to give the final indicated concentrations. B depicts the relationship between fluorescence change (ΔF) and the calculated K+ equilibrium potential ΔΨ in mV.

2

Effect of ouabain on ΔΨ. Cells were suspended in standard buffer, pH 7.4, previously equilibrated with 0.2 μM bisoxonol. After signal stabilization was achieved, ouabain (1 mM) was added in B. A shows control cells. ΔΨ was measured as described in the legend to Fig. 1. Traces are representatives of three separate experiments.

Cation and anion conductance plays a role in the regulation of the ΔΨ in many mammalian cells. To examine the effect on ΔΨ of physiologic monovalent anions and cations, P. carinii trophozoites were centrifuged and resuspended in specific ion free buffers. In the absence of Na+ and K+ (NMG buffer) or Cl (gluconate buffer), ΔΨ was not significantly changed as compared to cells suspended in standard buffer (data not shown). The contribution of divalent cations was assessed by measuring the ΔΨ in standard buffer in the presence of the divalent cation chelator EGTA (1 mM). No significant differences on ΔΨ were noted (data not shown). These data support that cation and anion conductance do not appear to play a significant role in the maintenance of ΔΨ in P. carinii.

In most mammalian cells, a Na+/K+-ATPase generates the ΔΨ [4]. We then investigated the effect of ATPase inhibitors on the ΔΨ of P. carinii trophozoites. To examine the role of a Na+/K+-ATPase, we used the glycoside inhibitor ouabain. The addition of 1 mM ouabain after a steady state ΔΨ was reached had no effect suggesting that a Na+/K+-ATPase does not contribute significantly to the ΔΨ in P. carinii (Fig. 2). The addition of omeprazole (1 mM), a specific inhibitor of the gastric K+/H+-ATPase [12], had no significant effect on the steady state ΔΨ of trophozoites (data not shown) thus ruling out an important role of a similar enzyme in P. carinii.

Previously, we demonstrated that an electrogenic plasma membrane H+-ATPase regulated pHi in P. carinii trophozoites [7]. To examine the role of an H+-ATPase in the regulation of ΔΨ, we examined the effect of inhibitors of plasma membrane H+-ATPases [11]. When suspended in standard buffer, the ΔΨ was significantly depolarized by the general H+-ATPase inhibitors NEM (100 μM) and DCCD (100 μM) to −43±7 mV and −56±5 mV, respectively (Fig. 3A and B). The plant H+-ATPase inhibitor DES (25 μM) also significantly depolarized the ΔΨ to −40±12 mV (Fig. 3C).

3

Effect of H+-ATPase inhibitors on ΔΨ. Where indicated by the arrows, 100 μM NEM (A), 50 μM DCCD (B) and 25 μM DES (C) were added. Other experimental conditions are as in Fig. 2.

Taken together, these results suggest that the H+-ATPase of P. carinii plays a significant role in the regulation of pHi[7] and ΔΨ (this work). Interestingly, this H+ pump has been postulated as a potential target for the chemotherapy of several fungal diseases [13]. The likely plasma membrane location of the enzyme along with its role in the regulation of pH and ΔΨ make this enzyme an attractive target for drug development against P. carinii.

Acknowledgements

We thank Margaret Shaw and Pamela Durant for assistance with P. carinii growth. This work was supported by NIH Grant AI-41242 to R.D.

References

  1. [1]
  2. [2]
  3. [3]
  4. [4]
  5. [5]
  6. [6]
  7. [7]
  8. [8]
  9. [9]
  10. [10]
  11. [11]
  12. [12]
  13. [13]
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