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• W2025605230 abstract "At present, approaches to studying mitochondrial functions in malarial parasites are quite limited because of the technical difficulties in isolating functional mitochondria in sufficient quantity and purity. We have developed a flow cytometric assay as an alternate means to study mitochondrial functions in intact erythrocytes infected with Plasmodium yoelii, a rodent malaria parasite. By using a very low concentration (2 nM) of a lipophilic cationic fluorescent probe, 3,3′dihexyloxacarbocyanine iodide, we were able to measure mitochondrial membrane potential(ΔΨm) in live intact parasitized erythrocytes through flow cytometry. The accumulation of the probe into parasite mitochondria was dependent on the presence of a membrane potential since inclusion of carbonyl cyanide m-chlorophenylhydrazone, a protonophore, dissipated the membrane potential and abolished the probe accumulation. We tested the effect of standard mitochondrial inhibitors such as myxothiazole, antimycin, cyanide and rotenone. All of them except rotenone collapsed the ΔΨm and inhibited respiration. The assay was validated by comparing the EC50 of these compounds for inhibiting ΔΨm and respiration. This assay was used to investigate the effect of various antimalarial drugs such as chloroquine, tetracycline and a broad spectrum antiparasitic drug atovaquone. We observed that only atovaquone collapsed ΔΨm and inhibited parasite respiration within minutes after drug treatment. Furthermore, atovaquone had no effect on mammalian ΔΨm. This suggests that atovaquone, shown to inhibit mitochondrial electron transport, also depolarizes malarial mitochondria with consequent cellular damage and death. At present, approaches to studying mitochondrial functions in malarial parasites are quite limited because of the technical difficulties in isolating functional mitochondria in sufficient quantity and purity. We have developed a flow cytometric assay as an alternate means to study mitochondrial functions in intact erythrocytes infected with Plasmodium yoelii, a rodent malaria parasite. By using a very low concentration (2 nM) of a lipophilic cationic fluorescent probe, 3,3′dihexyloxacarbocyanine iodide, we were able to measure mitochondrial membrane potential(ΔΨm) in live intact parasitized erythrocytes through flow cytometry. The accumulation of the probe into parasite mitochondria was dependent on the presence of a membrane potential since inclusion of carbonyl cyanide m-chlorophenylhydrazone, a protonophore, dissipated the membrane potential and abolished the probe accumulation. We tested the effect of standard mitochondrial inhibitors such as myxothiazole, antimycin, cyanide and rotenone. All of them except rotenone collapsed the ΔΨm and inhibited respiration. The assay was validated by comparing the EC50 of these compounds for inhibiting ΔΨm and respiration. This assay was used to investigate the effect of various antimalarial drugs such as chloroquine, tetracycline and a broad spectrum antiparasitic drug atovaquone. We observed that only atovaquone collapsed ΔΨm and inhibited parasite respiration within minutes after drug treatment. Furthermore, atovaquone had no effect on mammalian ΔΨm. This suggests that atovaquone, shown to inhibit mitochondrial electron transport, also depolarizes malarial mitochondria with consequent cellular damage and death. Plasmodium spp. are obligate intracellular parasites, spending a major portion of their life cycle within erythrocytes and converting these relatively inactive cells into metabolically thriving active hosts. At present, our knowledge of mechanisms by which the parasite accomplishes these changes is limited, as is our understanding of metabolic processes associated with parasitism. It is generally believed that glycolysis is the main source of ATP in erythrocytic stages of malarial parasites with little or no contribution by mitochondria to the cellular ATP pool (1Scheibel L.W. Wernsdorfer W.H. McGregor I.A. Malaria: Principles and Practice of Malariology. Churchill and Livingstone, New York1988: 171-217Google Scholar, 2Fry M. Webb E. Pudney M. Comp. Biochem. Physiol. B. 1990; 96: 775-782Crossref PubMed Scopus (46) Google Scholar). A lack of tricarboxylic acid cycle enzymes (3Sherman I.W. Microbiol. Rev. 1979; 43: 453-495Crossref PubMed Google Scholar, 4Oelshlegel Jr., F.J. Brewer G.J. Surgenor D.M. The Red Cell. Academic Press, San Diego1975: 1264Google Scholar, 5Homewood C.A. Neame K.D. Kreier J.P. Malaria. Academic Press, New York1980: 345-405Google Scholar, 6Trager W. Living Together: The Biology of Animal Parasitism. Plenum Press, New York1986: 147-169Crossref Google Scholar) and an acristate mitochondrial morphology has led to the suggestion that mitochondria in malaria parasite act mainly to serve as an electron disposal sink for dihydroorotate dehydrogenase, a critical enzyme in pyrimidine biosynthesis (7Gutteridge W.E. Dave D. Richards W.H.G. Biochim. Biophys. Acta. 1979; 582: 390-401Crossref PubMed Scopus (132) Google Scholar, 8Gero A.M. Brown G.V. O'Sullivan W.J. J. Parasitol. 1984; 70: 536-541Crossref PubMed Scopus (50) Google Scholar, 9Prapunwattana P. O'Sullivan W.J. Yuthavong Y. Mol. Biochem. Parasitol. 1988; 27: 119-124Crossref PubMed Scopus (54) Google Scholar). It is well established through studies in other systems that, in addition to oxidative phosphorylation, mitochondria are also central to many other physiological activities such as the metabolism of molecules such as amino acids, lipids, and heme, as well as intracellular Ca2+ homeostasis (10Alberts B. Bray D. Lewis J. Raff M. Roberts K. Watson J.D. Molecular Biology of the Cell. Garland, New York1994: 655-683Google Scholar). These functions are achieved by the action of gene products encoded by both mitochondrial and nuclear genomes. Because most of the mitochondrial proteins are encoded by the nuclear genome and imported into mitochondria, an active import mechanism is necessary for mitochondrial functions. Both metabolites and protein transport require maintenance of membrane potential across the inner mitochondrial membrane (11Mitchell P. Science. 1979; 206: 1148-1159Crossref PubMed Scopus (556) Google Scholar, 12Schwarz E. Neupert W. Biochim. Biophys. Acta. 1994; 1187: 270-274Crossref PubMed Scopus (38) Google Scholar). The mitochondrial electron transport chain serves to generate this membrane potential (11Mitchell P. Science. 1979; 206: 1148-1159Crossref PubMed Scopus (556) Google Scholar). Hence, maintenance of ΔΨm 1The abbreviations used are: ΔΨmmitochondrial membrane potentialFACSfluorescence-activated cell scannerDiOC6(3)3,3′-dihexyloxacarbocyaninePCDprogrammed cell deathCCCPcarbonyl cyanide m-chlorophenylhydrazone. is critical not only for ATP synthesis but also for the maintenance of additional metabolic activities of mitochondria. While it is not established which of the non-ATP synthesis functions are present in malarial parasites, it is safe to assume that these are likely to be critical for parasite physiology. mitochondrial membrane potential fluorescence-activated cell scanner 3,3′-dihexyloxacarbocyanine programmed cell death carbonyl cyanide m-chlorophenylhydrazone. Approaches for investigating mitochondrial functions in malarial parasite are quite limited at present. Mitochondrial DNAs of various Plasmodium spp. have been sequenced and found to encode at least three components of the electron transport chain, viz. subunits 1 and 3 of cytochrome c oxidase, and apocytochrome b (13Vaidya A.B. Akella R. Suplick K. Mol. Biochem. Parasitol. 1989; 35: 97-108Crossref PubMed Scopus (145) Google Scholar, 14Aldritt S.M. Joseph J.T. Wirth D.F. Mol. Cell. Biol. 1989; 9: 3614-3620Crossref PubMed Scopus (63) Google Scholar, 15Feagin J.E. Mol. Biochem. Parasitol. 1992; 52: 145-148Crossref PubMed Scopus (128) Google Scholar, 16Vaidya A.B. Lashgari M.S. Pologe L.G. Morrisey J. Mol. Biochem. Parasitol. 1993; 58: 33-42Crossref PubMed Scopus (99) Google Scholar). In addition, mitochondrial preparations have been shown to contain ubiquinone cytochrome c oxidoreductase (bc1 complex) (17Fry M. Beesley J.E. Parasitology. 1991; 102: 17-26Crossref PubMed Scopus (157) Google Scholar), and cytochrome c oxidase activities (18Scheibel L.W. Miller J. Mil. Med. 1969; 134: 1074-1080Crossref PubMed Scopus (24) Google Scholar, 19Scheibel L.W. Pflaum W.K. J. Parasitol. 1970; 56: 1054Crossref PubMed Scopus (16) Google Scholar, 20Krungkrai J. Krungkrai S.R. Bhumiratana A. Exp. Parasitol. 1993; 77: 136-146Crossref PubMed Scopus (32) Google Scholar). However, detailed studies of mitochondrial functions and their response to antimalarial drugs have been hampered by the technical difficulties of obtaining workable quantities of functional mitochondria. To circumvent these problems, we have explored the possibility of studying mitochondrial functioning in intact parasitized erythrocytes with a fluorescent activated cell scanner (FACS). We have used the ability of the lipophilic cationic fluorescent probe DiOC6(3) to partition into energized mitochondria as a measure of ΔΨm. By using this assay, we were able to demonstrate that a new antiparasitic drug, atovaquone, rapidly collapses ΔΨm in erythrocytes infected with a rodent malaria parasite Plasmodium yoelii An animal colony of BALB/cByJ mice was maintained in our American Association of Laboratory Animal Care accredited animal facility. P. yoelii 17XL was maintained in vivo in either male or female BALB/cByJ mice. Blood was collected in Hanks' balanced salt solution (HBSS) containing 10 units of heparin/ml of HBSS at approximately 60% parasitemia. Red blood cells were washed three times with HBSS at low speed centrifugation (800 × g for 10 min each time). Washed erythrocytes were diluted 1:1 in phosphate-buffered saline, pH 7.4, and passed over a microcrystalline cellulose column to remove leukocytes and platelets (21Eling W. Bull W. H. O. 1977; 55: 105-114PubMed Google Scholar). Red blood cells depleted of leukocytes and platelets were washed and resuspended in phosphate-buffered saline, and infected red blood cells were enriched by centrifugation over a discontinuous Percoll density gradient as described elsewhere (22McNally J. O'Donovan S.M. Dalton J.P. Parasitology. 1992; 105: 355-362Crossref PubMed Scopus (21) Google Scholar). Infected erythrocytes within 65% Percoll and 65-75% Percoll interphase were collected and contained predominantly trophozoites and schizonts as determined by Giemsa-stained thin blood smears. These preparations were free of leukocytes and platelets as judged by Giemsa staining. These fractions were pooled, washed twice with RPMI 1640 medium containing 1% fetal calf serum and used in all of the experiments described here. All of the mitochondrial respiratory chain inhibitors, e.g. antimycin, myxothiazole, rotenone, and cyanide; uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP); and antimalarials tetracycline and chloroquine, were purchased from Sigma. The antimalarial atovaquone was a gift from Glaxo Wellcome, Research Triangle Park, NC. Flow cytometric assays were carried out using a FACScan (Becton Dickinson Cellular Imaging). The flow cytometer was adjusted for forward scattering profile, side scattering profile and fluorescence detection of infected red blood cells at channel 1. Parasitized cells at a concentration of 5 × 106/ml in RPMI 1640 medium containing 1% fetal calf serum were incubated with a 2 nM final concentration of DiOC6(3) for 20 min at 37°C. At the end of the incubation period the suspension was aliquoted into eight tubes of 250 μl each. Different concentrations of the compound to be tested were added, and the mixture was incubated for an additional 20 min. At the end of the incubation period, each sample was subjected to flow cytometric analysis. For each sample, 10,000 events were counted at the same flow cytometer setting. The results were calculated from the mean fluorescence of 104 cells in a histogram. In each experiment, measurements of fluorescence in infected erythrocytes in the presence of dye (F+D) and in absence of dye (F−D) were carried out to establish baselines. Mitochondrial uptake of O2 was measured in a closed system using a Clark's oxygen electrode and K-IC oxygraph (Gilson Medical Electronics Inc., Middleton, WI) in a reaction volume of 1.5 ml at 37°C following the method of Chance and Williams (23Chance B. Williams G.R. Nature. 1955; 175: 1120-1121Crossref PubMed Scopus (268) Google Scholar). The instrument was calibrated following the manufacturer's instructions. Briefly, 1.5 × 108 parasitized cells in 1.5 ml of RPMI 1640 medium containing 1% fetal calf serum were added into the chamber, and the rate of O2 consumption by infected erythrocytes was followed for 5 min. A desired concentration of the compound to be tested was added into the chamber using a Hamilton syringe. The rate of O2 consumption by infected erythrocytes was followed for the next 5 min. The rate of O2 consumption was expressed as nAO/108 infected erythrocytes/min. The difference in the rate of O2 consumption by infected erythrocytes in the presence versus the absence of the compound was calculated as the measure of respiration inhibition. All the compound concentrations were tested individually in a separate set of experiments. DiOC6(3) is a cationic, lipophilic fluorescent compound. When incubated with infected erythrocytes, it diffuses into cells and is concentrated several orders of magnitude into negative-inside mitochondria. A collapsed ΔΨm will result in diffusion of the probe out of the mitochondria resulting in dissipation of the signal. As shown in Fig. 1A, incubation of parasitized erythrocytes with 2 nM DiOC6(3) led to the accumulation of the probe into parasites, which was reflected by the increase in fluorescence intensity. The fact that the accumulation of the probe within the mitochondria was dependent upon a membrane potential was shown by the dissipation of the signal intensity after incubation of infected erythrocytes with the protonophore CCCP (Fig. 1B). As shown in Fig. 2A), cell-associated DiOC6(3) fluorescence intensity increased as a function of the probe concentration up to 50 nM. Incubation with CCCP dissipated cell-associated DiOC6(3) fluorescence. Maximal dissipation of about 70% was obtained at the probe concentration of 2 nM. At higher probe concentrations the cell-associated fluorescence became relatively resistant to CCCP-mediated dissipation. The inhibition of the fluorescence above 50 nM appears to be the result of the inhibition of electron transport by the dye (see below). The decrease in the extent of quenching by CCCP above 2 nM appears to result from the self-quenching of the dye on the interface of the mitochondrial membrane, which thus saturates the signal. Hence, we decided to use 2 nM DiOC6(3) as the final probe concentration for the ΔΨm assay for malarial parasite. We also measured the kinetics of probe accumulation at 2 nM concentration in infected erythrocytes as shown in Fig. 2B A rapid accumulation of the probe in infected erythrocytes was observed, reaching maximum levels within 30 min. The slight reduction of fluorescent intensity after 30 min suggested that some self quenching occurred even at 2 nM dye concentration. Continued incubation of infected erythrocytes with the probe for longer than 60 min did not result in any significant change of fluorescence profile of the probe, indicating that partitioning of the probe into mitochondria had reached its equilibrium.Fig. 2Optimization of ΔΨm assay using DiOC6(3). a, to determine the optimum probe concentration to be used in ΔΨm assay, infected erythrocytes were incubated with different probe concentrations for the same period of time and subjected to FACS analysis. Results are presented both as the ratio of fluorescence intensity in the presence of the probe (F+D) and the background fluorescence (F−D) of the probe at different probe concentrations (•) and as the percent inhibition of fluorescence intensity with 40 μM CCCP at different probe concentrations (○). B, the kinetics of probe accumulation. Infected erythrocytes were incubated with 2 nM DiOC6(3) for 2 h. For the first 30 min an aliquot was removed every 5 min and subjected to FACS analysis. Subsequently samples were removed at 15-30 min intervals and analyzed by FACS. Results are presented as the ratio of F+D/F−D at the indicated time points.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because incubation with lipophilic compounds can also affect parasite physiology, we determined the effect of DiOC6(3) on mitochondrial functioning by measuring the rate of respiration in the presence and absence of DiOC6(3). As shown in Fig. 3, there was no significant effect of the probe at 2 nM upon the rate of O2 consumption. However, at the higher concentrations inhibition of respiration became apparent, reaching up to 50% at 150 nM DiOC6(3) concentration. Hence, for the rest of the study, DiOC6(3) was used at the final concentration of 2 nM. Having established a way to assay ΔΨm in intact parasitized erythrocytes, we tested the effect of various known mitochondrial inhibitors on ΔΨm. Inhibitors used were a protonophore (CCCP), a site I inhibitor (rotenone), site II inhibitors (antimycin and myxothiazole), and a site III inhibitor (cyanide). Fig. 4 shows representative histograms showing effects of these inhibitors (at the highest concentration tested) on mitochondrial accumulation of DiOC6(3) in infected erythrocytes. Fig. 5A shows the concentration-dependent dissipation of the fluorescence for each of these inhibitors. Rotenone did not have any significant effect on ΔΨm, which is consistent with the earlier findings regarding the lack of NADH-ubiquinone oxidoreductase (site I) (17Fry M. Beesley J.E. Parasitology. 1991; 102: 17-26Crossref PubMed Scopus (157) Google Scholar) in malarial parasites. Myxothiazole and antimycin collapsed ΔΨm with an EC50 of 2 × 10−7M and 6 × 10−7M, respectively, consistent with the presence of the bc1 complex (site II) in malarial parasites. A much higher concentration of cyanide (6 mM) was required to completely collapse the ΔΨm and dissipate the DiOC6(3) accumulation with an EC50 of 3 × 10−4M.Fig. 5The concentration-dependent effect of mitochondrial inhibitors on ΔΨm and respiration. A, concentration-dependent effect of mitochondrial inhibitors on ΔΨm. Fluorescence intensity was quantitated by FACS analysis in the presence of various concentrations of CCCP (○), antimycin (•), myxothiazole (▪), rotenone (▴), and cyanide (□). B, concentration-dependent effect of mitochondrial inhibitors on parasite respiration. Respiration rate by P. yoelii-infected erythrocytes was measured in the presence of various concentrations of antimycin (•), myxothiazole (▪), cyanide (□), and CCCP (⋄) and is plotted as percent inhibition.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also tested the effect of these inhibitors on rate of respiration by infected erythrocytes as shown in Fig. 5B. Both antimycin and myxothiazole inhibited respiration with EC50 of 2.5 × 10−7M and 9 × 10−8M, respectively. At higher concentrations, i.e. 1 × 10−6M and above, both these drugs almost completely inhibited respiration. Rotenone had no apparent effect upon the rate of O2 consumption in the range of concentrations tested in this study. Surprisingly, CCCP did not have any appreciable effect on the respiration rate of infected red blood cells. This observation is counter to the classical effect of CCCP where a collapsed membrane potential leads to a release from respiration control over electron transport, resulting in an increased rate of O2 consumption. Having demonstrated a good correlation between the DiOC6(3) assay of ΔΨm and respiration by intact infected erythrocytes, we decided to assess the effect of various antimalarial drugs on mitochondrial physiology. Three different drugs were tested: atovaquone, a new broad spectrum antiparasitic drug shown to inhibit the bc1 complex of the malarial parasite (24Fry M. Pudney M. Biochem. Pharmacol. 1992; 43: 1545-1553Crossref PubMed Scopus (405) Google Scholar); tetracycline, an antibiotic thought to inhibit organellar protein synthesis (9Prapunwattana P. O'Sullivan W.J. Yuthavong Y. Mol. Biochem. Parasitol. 1988; 27: 119-124Crossref PubMed Scopus (54) Google Scholar, 25Gale E.F. Cundliff E. Reynolds P.E. Richmond M.H. Waring M.J. Molecular Basis of Antibiotic Action. Wiley Interscience, London1981: 448-453Google Scholar); and chloroquine, a drug likely to affect nonmitochondrial target(s) (26Yayon A. Cabatchik Z. Ginsburg H. EMBO J. 1984; 3: 2695-2700Crossref PubMed Scopus (269) Google Scholar, 27Krogstad D.J. Schlesinger P.H. Gluzman I.Y. J. Cell Biol. 1985; 101: 2302-2309Crossref PubMed Scopus (218) Google Scholar, 28Krogstad D.J. Schlesinger P.H. N. Engl. J. Med. 1987; 317: 542-549Crossref PubMed Scopus (90) Google Scholar, 29Slater A.F.G. Cerami A. Nature. 1992; 355: 167-169Crossref PubMed Scopus (538) Google Scholar). Representative fluorescence histograms of infected red blood cells in the presence and absence of antimalarial compounds are presented in Fig. 6. It is clear that only atovaquone caused the depolarization of plasmodial mitochondria and dissipated the dye accumulation as observed by a change in flow cytometric profile, while the other two antimalarials chloroquine (Fig. 6B) and tetracycline (Fig. 6C) had no effect on membrane potential. In order to ascertain that atovaquone specifically affects malarial mitochondria, we also tested the effect of atovaquone on mouse lymphocyte mitochondria. No effect of atovaquone was detected on membrane potential of lymphocyte mitochondria (Fig. 6D). It is known that atovaquone does not inhibit mammalian mitochondrial bc1 complex (24Fry M. Pudney M. Biochem. Pharmacol. 1992; 43: 1545-1553Crossref PubMed Scopus (405) Google Scholar). However, in lymphocytes other respiratory inhibitors also fail to collapse ΔΨm (see “Discussion”). Concentration dependence of the effect of the antimalarials tested on P. yoelii ΔΨm is presented in Fig. 7A Atovaquone had an EC50 of 2.6 × 10−7M, with the maximum inhibition observed at ∼2 × 10−6M. Neither chloroquine nor tetracycline had any effect on malarial ΔΨm even at millimolar concentrations. We also tested the effect of atovaquone on the rate of O2 consumption by parasitized red blood cells as shown in Fig. 7B Atovaquone inhibited P. yoelii respiration at an EC50 of 7 × 10−8M. At 2 × 10−5M and above, atovaquone inhibited 90% of the O2 consumption by infected red blood cells.Fig. 7The concentration-dependent effect of atovaquone and other antimalarial on ΔΨm and respiration. A, concentration dependence of the effect of atovaquone and other antimalarials on ΔΨm. Fluorescence intensity of P. yoelii-parasitized erythrocytes is shown in the presence of various concentrations of atovaquone (•), chloroquine (▪), and tetracycline (▴). The effect of atovaquone on mouse lymphocytes is also depicted (♦). B, concentration-dependent effect of atovaquone on respiration rate by P. yoelii-infected erythrocytes.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It has become apparent that mitochondria are quite heterogeneous with regard to the various functions they serve in different cell types; while ATP synthesis may be a common critical function for mitochondria in most eukaryotic cells, additional crucial functions may be determined by the differentiation status of the cell or the organism in which these organelles reside. Our knowledge of mitochondrial physiology, however, is based largely upon classical studies done with easily accessible systems derived from tissues such as the liver and the heart (30Petit P.X. O'Connor J.E. Grunwald D. Brown S.C. Eur. J. Biochem. 1990; 194: 389-397Crossref PubMed Scopus (213) Google Scholar, 31Reers M. Smith T.W. Chen L.B. Biochemistry. 1991; 30: 4480-4486Crossref PubMed Scopus (873) Google Scholar). To broaden our view it is necessary to study mitochondrial physiology in various cell types and organisms. In this report we describe a flow cytometric assay employing a cationic lipophilic fluorescent probe to monitor ΔΨm in a rodent malarial parasite. Fluorescence-based demonstration of mitochondrial membrane potential has been used for many years with fluorochromes such as rhodamine 123 (32Jhonson L.V. Walsh M.L. Chen L.B. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 990-994Crossref PubMed Scopus (1290) Google Scholar, 33Summerhayes I.C. Lampidis T.J. Bernal S.D. Nadakavukaren J.J. Nandakavukaren K.K. Sherperd E.L. Chen L.B. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 5292-5296Crossref PubMed Scopus (341) Google Scholar, 34Tanabe K. J. Protozool. 1983; 30: 707-710Crossref PubMed Scopus (38) Google Scholar, 35Divo A.A. Geary T.G. Jensen J.B. Ginsburg H. J. Protozool. 1985; 32: 442-446Crossref PubMed Scopus (89) Google Scholar). Most of these studies, however, involved fluorescence microscopy and employed micromolar concentration of the probe. Because the rhodamine dyes have adverse effects on mitochondrial respiration (36Modica-Napolitano J.S. Weiss M.J. Chen L.B. Aprille J.R. Biochem. Biophys. Res. Commun. 1984; 118: 717-723Crossref PubMed Scopus (124) Google Scholar, 37Mai M.S. Allison W.S Arch. Biochem. Biophys. 1983; 221: 467-476Crossref PubMed Scopus (50) Google Scholar) and do not appear to be strictly dependent on ΔΨm for intramitochondrial accumulation, 2H. Rottenberg and S. Wu, submitted for publication. their use in careful studies on mitochondrial physiology is problematic. A cationic lipophilic fluorochrome, DiOC6(3) originally used at high concentrations to monitor intracellular membranes (39Teraski M. Song J. Wong J.R. Weiss M.J. Chen L.B. Cell. 1984; 38: 101-108Abstract Full Text PDF PubMed Scopus (296) Google Scholar, 40Terasaki M. Chen L.B. Fujiwara K. J. Cell Biol. 1986; 103: 1557-1568Crossref PubMed Scopus (473) Google Scholar), appears to be a better alternative to assay ΔΨm when used at low concentrations. We empirically determined the optimal concentration of the probe for ΔΨm assay in intact malarial parasites as the concentration at which probe accumulation was essentially dependent on membrane potential and maximally dissipated by the protonophore CCCP. When used at the optimal concentration (2 nM), the probe did not dissipate significantly from the intact parasitized erythrocytes for up to 2 h. We also determined the effect of the probe on respiration by intact parasites, observing that at 10 nM and higher concentrations DiOC6(3) significantly affected respiration rate. We suggest that such parameters need to be established prior to utilization of this fluorochrome for studying mitochondrial physiology. The concentration of DiOC6(3) used for studying mitochondrial membrane potential in pre-apoptotic lymphocytes (40 nM) is 20-fold higher than what we have used in assaying ΔΨm in malarial parasites (41Zamzami N. Marchetti P. Castedo M. Zanin C. Vayssiere J.-L. Petit P.X. Kroemer G. J. Exp. Med. 1995; 181: 1661-1672Crossref PubMed Scopus (1093) Google Scholar). This concentration completely inhibited mitochondrial respiration in lymphocytes and the fluorescence level was highly quenched.2 The optimal concentration for the determination ofΔΨm in lymphocytes is 0.2 nM.2 Using this assay we determined the effects of various conventional mitochondrial inhibitors on ΔΨm of intact malarial parasites and compared them for their effects on respiration. Rotenone did not affect either ΔΨm or respiration, consistent with the previous studies that failed to observe NADH-ubiquinone oxidoreductase (site I) of the respiratory chain in malarial parasites (17Fry M. Beesley J.E. Parasitology. 1991; 102: 17-26Crossref PubMed Scopus (157) Google Scholar). Two inhibitors of ubiquinol-cytochrome c oxidoreductase (site II), myxothiazole and antimycin, as well as cyanide (a cytochrome c oxidase and site III inhibitor) collapsed ΔΨm and inhibited respiration. These components of the respiratory chain have been observed in malarial parasites. It is interesting to note that the inhibition of the respiratory chain seems to result in rapid dissipation of ΔΨm. This is in sharp contrast to what we observed in lymphocytes.2 For technical reasons, it was not possible to determine the effects of electron transport inhibitors on ΔΨm and respiration at the same inhibitor/cell ratios. It is possible that higher inhibitor/cell ratios are necessary to collapse ΔΨm than those required for respiration inhibition. Nevertheless, our results suggest that ΔΨm in the parasite cannot be supported by ATP synthase, which is in agreement with the notion that erythrocytic stages of malarial parasite lack the machinery for oxidative phosphorylation. A puzzling observation was the lack of respiration stimulation following CCCP treatment. This is contrary to the general observation of increased oxygen consumption in mitochondria with collapsed membrane potential. This suggests that the electron transport in the malarial parasite is not controlled by the mitochondrial ΔH+. Atovaquone, a hydroxynaphthoquinone, is a drug initially developed as an antimalarial but now known to be also effective against several other eukaryotic microbial parasites such as toxoplasma and pneumocystis (42Hughes W.T. Gray V.L. Gutteridge W.T. Lalter V.S. Pudney M. Antimicrob. Agents Chemother. 1990; 34: 225-228Crossref PubMed Scopus (106) Google Scholar). Using cholate-lysed mitochondria from Plasmodium falciparum and P. yoelii, and externally provided heterologous (horse) cytochrome c at 100 micromolar concentration, Fry and Pudney (24Fry M. Pudney M. Biochem. Pharmacol. 1992; 43: 1545-1553Crossref PubMed Scopus (405) Google Scholar) observed that atovaquone inhibited cytochrome c reductase activity. Based upon these results, it was concluded that atovaquone acts at the cytochrome bc1 complex of the malarial respiratory chain. Unique structural features of the parasite cytochrome b a were speculated as being responsible for the therapeutic value of some of the hydroxynaphthoquinones as antimalarials (16Vaidya A.B. Lashgari M.S. Pologe L.G. Morrisey J. Mol. Biochem. Parasitol. 1993; 58: 33-42Crossref PubMed Scopus (99) Google Scholar). Because malarial mitochondria do not seem to contribute much to the ATP pool, it has long been suggested that the main purpose for these organelles was to dispose of electrons generated by dihydroorotate dehydrogenase (7Gutteridge W.E. Dave D. Richards W.H.G. Biochim. Biophys. Acta. 1979; 582: 390-401Crossref PubMed Scopus (132) Google Scholar, 8Gero A.M. Brown G.V. O'Sullivan W.J. J. Parasitol. 1984; 70: 536-541Crossref PubMed Scopus (50) Google Scholar, 9Prapunwattana P. O'Sullivan W.J. Yuthavong Y. Mol. Biochem. Parasitol. 1988; 27: 119-124Crossref PubMed Scopus (54) Google Scholar), an essential enzyme in pyrimidine biosynthesis. Since the parasites are unable to salvage pyrimidines, inhibition of dihydroorotate dehydrogenase has been suggested as the reason for antimalarial activity of compounds such as atovaquone (43Hammond D.J. Burchell J.R. Pudney M. Mol. Biochem. Parasitol. 1985; 14: 97-109Crossref PubMed Scopus (94) Google Scholar). 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Cell Sci. 1994; 107: 2691-2704Crossref PubMed Google Scholar), and Tetrahymena (53Christensen S.T. Wheatley D.N. Rasmussen M.I. Rasmussen L. Cell Death Differ. 1995; 2: 301-308PubMed Google Scholar) although underlying mechanisms are not known at present. We suggest that the inhibition of mitochondrial electron transport chain in malarial parasites also acts to initiate a cascade of events similar to PCD. It would be important to investigate the possibility that the PCD-like response is elicited by collapsed ΔΨm in malarial parasites. Nevertheless, maintenance of ΔΨm is likely to be critical for all stages of the malaria parasite's life cycle, and drugs that affect ΔΨm are likely to act on all such stages. In accordance with this proposal atovaquone has been shown also to act against liver (54Davies C.S. Pudney M. Matthew P.J. Sinden R.E. Acta Leiden. 1989; 58: 115-128PubMed Google Scholar, 55Davies C.S. Nicholas J.C. Sinden R.E. Parasitology. 1993; 106: 1-6Crossref PubMed Scopus (31) Google Scholar) and insect stages of malarial parasite (38Fowler R.E. Sinden R.E. Pudney M. J. Parasitol. 1995; 81: 452-458Crossref PubMed Scopus (32) Google Scholar). Further investigations on parasite mitochondrial physiology will be illuminating in our attempts to understand mechanisms of drug action and the development of newer strategies for chemotherapy of malaria. We thank Glaxo Wellcome for providing the antimalarial atovaquone and Joanne Morrisey for technical assistance." @default.
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