Acidic Food Vacuole Research Articles

Potentiation of an antimalarial oxidant drug.

In a previous report we described the synergistic antimalarial interaction between two structurally similar compounds, rufigallol and exifone. To explain this phenomenon, we proposed that exifone is transformed inside the parasitized erythrocyte into a xanthone with potent antimalarial properties. We speculated that the transformation process was induced by the prooxidant activity of rufigallol. On the basis of this model we hypothesized that exifone would act synergistically with other oxidant drugs. In the present study we have found a similar synergistic interaction between exifone and ascorbic acid (vitamin C) against both chloroquine-susceptible and multidrug-resistant strains of Plasmodium falciparum. The prooxidant activity of ascorbic acid against Plasmodium-infected erythrocytes is believed to result from an intraerythrocytic Fenton reaction occurring in the acidic food vacuole of the parasite. The hydroxyl radicals produced during this process are believed to attack exifone, which undergoes cyclodehydration to become 2,3,4,5,6-pentahydroxyxanthone (X5). Evidence presented to support this "xanthone hypothesis" includes the demonstration that the exifone ==> X5 transformation occurs readily in vitro under mildly acidic conditions in the presence of iron, ascorbic acid, and oxygen.

Role of protozoan grazing in relieving iron limitation of phytoplankton

RECENT evidence indicates that iron is a limiting factor in primary production in some areas of the oceans1,2. In sea water, iron is largely present in the form of particulate and colloidal phases which are apparently unavailable for uptake by phytoplankton3–5. Several mechanisms have been proposed whereby non-reactive iron may be converted into more labile forms (for example, thermal dissolution6, photochemical reactions7,8 and ligand complexation9). Here we report that digestion of colloidal iron in the acidic food vacuoles of protozoan grazers may be a mechanism for the generation of 'bioavailable' iron from refractory iron phases. We have demonstrated several grazer-mediated effects on colloidal ferrihydrite, including a decrease in colloid size, an increase in colloid lability as determined by competitive ligand-exchange techniques, and an increase in the bioavailability of colloids to iron-limited diatoms. These results indicate that protozoan grazers may significantly enhance the supply of iron to marine phytoplankton from terrestrial sources.

Inhibition by anti-malarial drugs of haemoglobin denaturation and iron release in acidified red blood cell lysates--a possible mechanism of their anti-malarial effect?

Intraerythrocytic malaria parasites ingest the cytosol of their host cell and digest it inside their acid food vacuoles. Acidified (pH 4-5.5, 37 degrees C) human red blood cell lysates were used to simulate this process, measuring the denaturation of haemoglobin (Hb) and the release of iron, in the absence or presence of exogenous protease. Spontaneous Hb denaturation and appearance of non-heme iron were observed upon lysate acidification, their rates decreasing with increasing pH, and increasing in the presence of protease. Both processes were inhibited by the quinoline-containing anti-malarial drugs (QCDs) chloroquine, quinine, mefloquine and amodiaquine at concentrations well below those expected in the acidic food vacuole of the parasite. Spectrophotometric analysis indicated that chloroquine complexes with heme in acid-denatured haemoglobin. Other weak bases as well as verapamil and diltiazem, known to reverse the resistance of malarial parasites to chloroquine, were without effect indicating that the action of QCDs is specific. Based on our previous results and the present report, we suggest that iron release in acidified lysates is mediated through the formation of ferryl (Fe(IV)) radicals. QCDs possibly complex with this radical, as they do with heme, and prevent its contact with an adjacent heme molecule which is required for ring opening and iron release. These results may suggest that one of the anti-malarial effects of QCDs is to deprive the parasite of an adequate iron supply. Addition of iron to cultures of Plasmodium falciparum was expected to circumvent the deprivation of iron and reduce the anti-malarial effect of QCDs. However, adding iron as penetrating fructose or nitrilotriacetate complexes did not alter the parasite's susceptibility to chloroquine. Ascorbate markedly increased the release of iron in acidified lysates, and this effect was not reduced by chloroquine. Ascorbate was found to decrease parasite susceptibility to chloroquine, suggesting that iron deprivation may be an important factor in the anti-malarial action of QCDs.

Chloroquine: Mechanism of drug action and resistance in plasmodium falciparum

Quinoline-containing drugs such as chloroquine and quinine have had a long and successful history in antimalarial chemotherapy. Although these drugs are known to accumulate by a weak base mechanism in the acidic food vacuoles of intraerythrocytic trophozoites and thereby prevent hemoglobin degradation from occuring in that organelle, the mechanism by which their selective toxicity for lysosomes of malaria trophozoites is achieved has been subject to much discussion and argument. In this review the recent discovery that chloroquine and related quinolines inhibit the novel heme polymerase enzyme that is also present in the trophozoite food vacuole is introduced. The proposal that this inhibition of heme polymerase can explain the specific toxicity of these drugs for the intraerythrocytic malaria parasite is then developed by showing that it is consistent with much of the disparate information currently available. The clinical usefulness of chloroquine, and in some recent cases of quinine as well, has been much reduced by the evolution and spread of chloroquine resistant malaria parasites. The mechanism of resistance involves a reduced accumulation of the drug, although again the mechanism involved is controversial. Possible explanations include an energy-dependent efflux of preaccumulated drug via an unidentified transmembrane protein pump, or an increase in vacuolar pH such that the proton gradient responsible for drug concentration is reduced. New data are also presented which show that heme polymerase isolated from chloroquine resistant trophozoites retains full sensitivity to drug inhibition, consistent with the observation that resistance involves a reduced accumulation of the drug at the (still vulnerable) target site. The significance of this result is discussed in relation to developing new strategies to overcome the problem presented by chloroquine resistant malaria parasites.

Studies on the antimalarial mode of action of quinoline-containing drugs: Time-dependence and irreversibility of drug action, and interactions with compounds that alter the function of the parasite's food vacuole

The quinoline-containing antimalarial drugs chloroquine, quinine and mefloquine exert an irreversible inhibitory effect on erythrocytic stages of Plasmodium falciparum grown in culture. Inhibition is time-and and concentration-dependent and the full effect is observed after 2–6 hours of exposure to the drug. Washing of infected cells after drug exposure in the presence of NH 4Cl to accelerate drug efflux, intensifies the inhibitory effect of chloroquine, probably due to the pH-dependent release of highly concentrated drug from the acidic food vacuole of the parasite. When both antimalarials and NH 4Cl are present in the culture, drug effect is reduced, as expected from the demonstrable alkalinization of the food vacuole and the consequent reduction in drug accumulation. The protease inhibitor leupeptin inhibits digestion of ingested host cell cytosol, and thus inhibits parasite growth, though reversibly so (Rosenthal et al, J. Clin. Invest. 82 1560–1566 (1988)). Thus, although the antimalarials also inhibit the feeding process, this is not the cause of their irreversible action. Leupeptin is found to be antagonistic to antimalarials' action, suggesting that the drugs from complexes with products of host cell digestion that are responsible for irreversible inhibition of parasite growth.

Recent Developments in the Understanding of the Pharmacokinetics and Mechanism of Action of Chloroquine

Because of the binding of chloroquine to various tissue components and the lysosomotropism of chloroquine, its pharmacokinetics exhibit large apparent volumes of distribution, partial recoveries in urine, and persistence of low blood levels. These complexities have been reinvestigated with modern, highly sensitive methods for the determination of the drug and its major metabolite, desethylchloroquine, which reaches blood levels of about one-third those of the parent drug and constitutes about one-fifth of the 56% of drug accounted for by urinary recovery. Bioavailability is essentially complete, apparent volumes of distribution range up to 800 L/kg, and the pharmacokinetic data are generally accomodated by three compartment models. Half-lives for the terminal component are 1 to 2 months, but the terminal phases may be of minor importance in effectiveness. Dose dependence, i.e. nonlinearity in the relationship of dose and area under the plasma curve, apparently is not a factor. Some of the most recent studies, designed to provide a rationale for safer parenteral administration, have made possible computer-generated optimal infusion regimens. Revised schedules of loading and maintenance oral doses for antimalarial therapy have resulted from other pharmacokinetic studies. The in vitro antimalarial potencies of the two optical enantiomers of chloroquine are identical, but recent data suggest stereospecific differences in metabolic rates and renal secretion mechanisms. The marked uptake of chloroquine into the acidic food vacuoles of parasites resident in erythrocytes is assumed to underlie its antimalarial action, but mechanisms other than the previously assumed alkalization of the vacuole, possibly inhibition of phospholipid metabolism, now seem more likely to inhibit parasite function. Mild immunosuppression with inhibition of the elaboration of rheumatoid factor and acute phase reactants is a likely mechanism for the beneficial effects of chloroquine in rheumatoid arthritis. Blockade of interleukin-1 release could lead to these effects and is one of the few instances in which the low chloroquine levels attainable in human serum can be shown to affect immunological reactions.

Plasmodium falciparum: inhibitors of lysosomal cysteine proteinases inhibit a trophozoite proteinase and block parasite development

Trophozoites of Plasmodium falciparum obtain free amino acids for protein synthesis by degrading host erythrocyte hemoglobin in an acidic food vacuole. We previously reported that leupeptin and l- trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64), two inhibitors of the cysteine class of proteinases, blocked hemoglobin degradation in the trophozoite food vacuole, and we identified a 28-kDa trophozoite cysteine proteinase as a potential food vacuole hemoglobinase. We now report that the biochemical properties of the trophozoite cysteine proteinase closely resembled those of the lysosomal cysteine proteinases cathepsin B and cathepsin L. The trophozoite proteinase had a pH optimum of 5.5–6.0, near that of both lysosomal proteinases, and it was efficiently inhibited by highly specific diazomethylketone and fluoromethylketone inhibitors of cathepsin B and cathepsin L. The trophozoite proteinase preferred peptide substrates with arginine adjacent to hydrophobic amino acids, as does cathepsin L. Micromolar concentrations of the fluoromethylketone inhibitor Z-Phe-Ala-CH 2F blocked the degradation of hemoglobin in the trophozoite food vacuole and prevented parasite multiplication. In previous studies much higher concentrations of the inhibitor were not toxic for mice. Our results provide additional evidence that the 28-kDa trophozoite proteinase is a food vacuole hemoglobinase and suggest that specific inhibitors of the enzyme may have potential as antimalarial drugs.

Susceptibility of human malaria parasites to chloroquine is pH dependent.

Chloroquine (CQ) accumulates in the acidic food vacuole of intraerythrocytic malaria parasites (Plasmodium falciparum) by virtue of its weak base properties. In the present work, the extent of CQ accumulation was determined by the transvacuolar pH gradient: modification of the latter--either by changing the external pH or by adding the acidotropic agent NH4Cl--led to a corresponding change in CQ distribution between cells and medium. Changes in pH gradient provoked a change in the susceptibility of parasites to CQ: at external pH values of 8.0, 7.4, and 6.8, the IC50 values for CQ were 0.48 X 10(-7) M, 1.8 X 10(-7) M, and 3.3 X 10(-7) M, respectively. Marked resistance to CQ (IC50 = 9.8 X 10(-7) M) was conferred upon cells by exposing them simultaneously to CQ and 10 mM NH4Cl, at pH 7.4. The final concentration of CQ attained within the acidic compartment of the parasite was correlated with inhibition of parasite growth. At therapeutic drug levels, CQ accumulation caused minor changes in the food vacuole pH, whereas at higher CQ concentrations substantial alkalinization was observed. The antimalarial activity of CQ is suggested to be exerted by the interference of the high concentrations of the accumulated drug with vital functions of the food vacuole.