Regulation of Heme Polymerizing Activity and the Antimalarial Action of Chloroquine

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1997, p Vol. 41, No /97/$ Copyright 1997, American Society for Microbiology Regulation of Heme Polymerizing Activity and the Antimalarial Action of Chloroquine COY D. FITCH* AND ALBERT C. CHOU Department of Internal Medicine, Saint Louis University School of Medicine, St. Louis, Missouri Received 17 April 1997/Returned for modification 16 June 1997/Accepted 14 August 1997 Mice infected with Plasmodium berghei served as donors of erythrocytes with a high level of parasitemia for the study of ferriprotoporphyrin IX (FP) polymerization. Six hours after treatment of these mice with 3 mol of chloroquine per 25 g of body weight, there were significant losses of heme polymerase I (HPA I). For chloroquine-susceptible (CS) P. berghei, the rate of FP polymerization decreased from (mean standard deviation; n 12) to (n 8) nmol of FP polymerized per h per ml of packed erythrocytes (normalized to represent 1,000 parasites per 1,000 erythrocytes). For chloroquine-resistant (CR) P. berghei, the rate decreased from (n 16) to (n 6) nmol per h per ml. The chloroquine-induced loss of HPA I was accompanied by the accumulation of unpolymerized FP in CS P. berghei but not in CR P. berghei, which is consistent with the hypothesis that FP mediates the antimalarial action of chloroquine. Quinine treatment partially reversed the effects of chloroquine in CS P. berghei but not in CR P. berghei. Cycloheximide treatment antagonized the effects of chloroquine in both lines of parasites. To explain these findings, we propose that chloroquine, quinine, and cycloheximide perturb a regulatory process for HPA I. Furthermore, we propose that when chloroquine engages its target in the regulatory process, it initiates a chain of events which culminates in increased production, accessibility, or reactivity of a regulator (inactivator) of HPA I. Malaria parasites polymerize heme and store it in hemozoin (13). This activity allows them to digest hemoglobin without accumulating excess amounts of nonhemozoin (unpolymerized) ferriprotoporphyrin IX (FP), an oxidized form of heme. Chloroquine treatment of susceptible Plasmodium berghei reduces FP polymerization (5, 6) and causes unpolymerized FP to accumulate in vivo (6). Since unpolymerized FP can kill malaria parasites (10, 16), interference with FP polymerization in vivo probably is a fundamental pharmacological action of chloroquine (5). In erythrocytes infected with P. berghei, there are at least two heme polymerizing activities, HPA I and HPA II (12). Heme polymerizing activities have been called heme polymerases in the past, although they have not been proven to be proteins (1, 7, 12). HPA I is heat labile, resistant to proteolysis, and active in a low-acetate incubation medium (0.08 M) at ph 5. It is considered to be the physiologically important heme polymerizing activity. HPA II is heat stimulable, susceptible to proteolysis, and inactive in 0.08 M acetate at ph 5. Both activities catalyze the formation of a polymer (hemozoin FP) (12) that is insoluble in water at ph 8 and below (12, 13). Hemozoin FP is a naturally occurring form of -hematin (2, 13, 18), which is composed of FP monomers linked through their iron atoms and the carboxyl groups of their propionate side chains (2, 18). Chloroquine reduces FP polymerization in vivo by inducing the loss of HPA I (5, 6). Since chloroquine-induced loss of HPA I probably is central to the antimalarial action of chloroquine, we ask two questions in the present paper. Does chloroquine have the same effect on HPA I in chloroquine-resistant (CR) P. berghei as it does in chloroquine-susceptible (CS) P. berghei? And is the effect of chloroquine on HPA I antagonized by the same agents that prevent the manifestations of chloroquine toxicity in CS P. * Corresponding author. Mailing address: Department of Internal Medicine, Saint Louis University School of Medicine, 1402 South Grand Blvd. St. Louis, MO Phone: (314) Fax: (314) fitchcd@wpogate.slu.edu. berghei? Quinine and diverse metabolic inhibitors, including cycloheximide, prevent, for example, chloroquine-induced autophagic vacuole formation (19). Therefore, we included quinine and cycloheximide in the present studies. In answering the first question, we obtained new evidence that chloroquine resistance in P. berghei is due to the lack of accumulation of unpolymerized FP. In answering the second question, we obtained evidence of a novel target for chloroquine in the regulation of HPA I. MATERIALS AND METHODS Male Swiss mice (CF-1) weighing approximately 25 g were purchased from Sasco, Inc. (Omaha, Neb.). They were given Purina Laboratory Chow and water ad libitum, and their care was provided in accordance with Saint Louis University guidelines. Some of the mice were infected by intraperitoneal passage of approximately a million erythrocytes parasitized either with the CS NYU-2 strain of P. berghei or with a CR line derived from the NYU-2 strain (14). The CR line has been passaged every 2 weeks and kept under chloroquine pressure for 24 years. Chloroquine pressure is applied by intraperitoneal injection of individual infected mice with 3 mol of chloroquine daily on Monday through Friday of each week. It should be noted, however, that mice infected with malaria parasites for the purposes of the present experiments received no chloroquine treatment unless it was part of the experimental protocol. The methods used to maintain the parasites and the characteristics of the infections have been described previously (14). The CS line infects erythrocytes of all ages and is heavily pigmented. The CR line infects only immature, polychromatophilic erythrocytes and is unpigmented. Multiparasitism is common in both, and both infections are asynchronous. Parasitemia was determined by counting the number of individual parasites in 1,000 erythrocytes in Geimsa-stained blood films. When parasitemias reached values between 800 and 2,000 parasites per 1,000 erythrocytes at approximately 6 days for CS P. berghei and 14 days for CR P. berghei, individual mice were injected intraperitoneally with cycloheximide or various antimalarial drugs singly or in certain combinations. Doses of quinine and cycloheximide similar to those which have previously been shown to prevent chloroquine-induced vacuole formation in P. berghei were chosen for the present work. When used in combinations, the drugs were administered separately. The drugs were dissolved either in 0.1 ml of 0.9% NaCl in water or in 0.1 ml of a 1:2 mixture of absolute ethanol and 0.9% NaCl. Control mice received injections with one or the other of these solvents until it was determined that there was no effect on FP polymerization. To obtain parasitized erythrocytes, the mice were anesthetized with ether, an incision was made to sever the axillary artery and form a pouch in which to pool blood, and the blood was collected and diluted with an equal volume of a standard, isotonic medium (6) containing 5 mm glucose, 50 mm phosphate (ph 2461

2 2462 FITCH AND CHOU ANTIMICROB. AGENTS CHEMOTHER. 7.4), and approximately 1 mg of heparin per ml. Erythrocytes then were separated from plasma by centrifugation, washed three times with ice-cold standard medium to remove plasma and buffy coat, suspended in standard medium to achieve a hematocrit of 25%, and lysed by freezing and thawing (5). The unpolymerized FP and preformed hemozoin FP (hemozoin FP content prior to incubation) contents in these lysates were measured as previously described (6). To assay heme polymerizing activity, 0.2 ml of a lysate was included in a 2-ml incubation mixture which also contained 0.08 M acetate (ph 5) and 300 M FP. The low acetate concentration was used to keep HPA II inactive and thus allow only HPA I activity to be measured. Incubations were conducted at 37 C for 4 h in capped tubes which were attached to a slowly rotating wheel. The incubations were terminated by centrifugation and removal of the colorless supernatant fluid, and the pellets were used for measurement of hemozoin FP content. The increase in the amount of hemozoin FP was taken as a measure of heme polymerizing activity. Under these conditions, the increase in hemozoin FP is linear for 6 h (5). To evaluate heat lability (12), some of the lysates were heated in a boiling water bath for 2 min at ph 7.4 prior to the measurement of heme polymerizing activity. To measure hemozoin FP content after incubation, the incubation mixtures were centrifuged, the supernatant fluids were discarded, and the pellets were extracted with aqueous sodium dodecyl sulfate (SDS) to remove unpolymerized FP (12). To ensure that the unpolymerized FP had been extracted quantitatively, the pellets remaining after extraction were resuspended in 2.5% SDS and their visible absorption spectra were compared to the spectrum of authentic hemozoin FP (13). Sufficient NaOH was added to the hemozoin FP suspensions to achieve a concentration of 0.02 N, and FP content was measured spectrophotometrically (5). Under alkaline conditions, hemozoin FP depolymerizes, and the unpolymerized FP is solubilized in the SDS solution (13). For measurement of chloroquine accumulation in parasitized erythrocytes, individual mice were injected intraperitoneally with 3 mol of chloroquine containing 1 Ci of ring-labeled 3-[ 14 C]chloroquine (New England Nuclear Corp., Boston, Mass.). Six hours later, the mice were killed by exsanguination under ether anesthesia. For each data point shown in Fig. 4, blood from two or three mice was pooled and centrifuged to collect erythrocytes, which were divided into two fractions, one for measurement of unpolymerized FP content (6) and the other for radiochemical measurement of chloroquine content (8). The radiochemical method involves dissolving intact erythrocytes in 1 N NaOH and extracting the [ 14 C]chloroquine into heptane for counting. An LS 7500 liquid scintillation spectrometer (Beckman Scientific Instruments Division, Irvine, Calif.) and an Opti-Fluor scintillation cocktail (Packard Instrument Co., Meriden, Conn.) were used to count the samples. The counting error did not exceed 2%. Within4hofinjection of [ 14 C]chloroquine, the concentration of chloroquine was 100 times greater in parasitized erythrocytes than in plasma (15). When comparisons between CS and CR P. berghei were of interest, the data were expressed as a function of packed erythrocytes, with parasitemia normalized to represent 1,000 parasites per 1,000 erythrocytes. Otherwise, the data were expressed as a function of preformed hemozoin FP because its measurement was easier and more reproducible than the counting of parasites (6). Mefloquine was provided by the Walter Reed Army Institute of Research (Washington, D.C.), and amodiaquine and amopyroquine were provided by Parke-Davis, a Division of Warner-Lambert Co. (Morris Plains, N.J.). FP in the form of hematin and the remaining drugs were purchased from Sigma Chemical Co. (St. Louis, Mo.). Reagent grade chemicals were purchased from Sigma Chemical Co. or Fisher Scientific Co. (Fair Lawn, N.J.). FIG. 1. Chloroquine-induced loss of HPA I in CS P. berghei. Mice were individually injected intraperitoneally with one of three doses of chloroquine, and blood from groups of three to five mice was pooled at the time intervals shown in the figure for measurement of heme polymerizing activity as described in the text. The doses of chloroquine were 3 ( ), 0.3 (F), and 0.12 mol ( ). Heme polymerizing activity is expressed as nanomoles of FP polymerized per hour per milliliter of packed erythrocytes, normalized to represent a parasitemia of 1,000 parasites per 1,000 erythrocytes. The average of duplicate measurements of a separate pool of blood for each point is shown. We next studied the antagonism between chloroquine, quinine, and cycloheximide (Fig. 2). In these experiments, chloroquine was administered to mice infected with CS P. berghei, and the level of HPA I was allowed to decrease for 2 h. Then RESULTS The time course of the effect of chloroquine treatment on FP polymerization in lysates is presented for CS P. berghei in Fig. 1 through 3. Within 30 min of administration of 3 mol of chloroquine, there was a 30% loss of heme polymerizing activity, and the loss continued approximately in a linear fashion for another hour. Thereafter, the rate of loss decreased rapidly, and a plateau was reached by the sixth hour (Fig. 2 and 3). The activity remaining after 6 h was not significantly reduced by heating for 2 min in a boiling water bath (42 22 versus nmol of FP polymerized per h per mol of preformed hemozoin FP [means standard deviations {SD}; n 3] for unheated versus heated samples, respectively), indicating that all of the HPA I in the parasites had been lost as a consequence of chloroquine treatment (12). A limited dose response study also is shown in Fig. 1. For the first hour after treatment, the effect of 0.3 mol of chloroquine was nearly as great as the effect of 3 mol. By 4 h, the loss was less than that induced by 3 mol (67 versus 80%). Treatment with 0.12 mol of chloroquine induced a 31% loss of HPA I at 4h. FIG. 2. Reversal by quinine of the chloroquine-induced loss of heme polymerizing activity in CS P. berghei. Mice were individually injected intraperitoneally with 3 mol of chloroquine, 6 mol of quinine, or 16 mol of cycloheximide singly or in certain combinations, and blood from groups of three to five mice was pooled at the time intervals shown in the figure for measurement of heme polymerizing activity. F, chloroquine alone at 0 h; E, chloroquine alone at 0 h then cycloheximide at 2 h;, chloroquine alone at 0 h then quinine at 2 h;, chloroquine alone at 0 h then quinine and cycloheximide at 2 h. Heme polymerizing activity is expressed as nanomoles of FP polymerized per hour per micromole of preformed hemozoin FP. The mean SD and the number of experiments are shown at each point for which there were three or more experiments.

3 VOL. 41, 1997 HEME POLYMERIZING ACTIVITY IN MALARIA PARASITES 2463 TABLE 1. Effect of chloroquine, quinine, and cycloheximide on accumulation of unpolymerized FP in CS P. berghei Treatment a FP b (no. of expts.) None (9) Chloroquine (7) Cycloheximide (3) Chloroquine and cycloheximide (3) Chloroquine and quinine (3) a Mice having a parasitemia of approximately 1,500 parasites per 1,000 erythrocytes were each injected intraperitoneally with 3 mol of chloroquine, 16 mol of cycloheximide, or 6 mol of quinine singly or in the combinations shown, and erythrocytes were obtained for measurement of unpolymerized FP 6 h later. The mice receiving the combination of chloroquine and quinine were given a second dose of 6 mol of quinine 2 h after the experiment began. b Nanomoles of unpolymerized FP per milliliter of packed erythrocytes normalized to represent a parasitemia of 1,000 parasites per 1,000 erythrocytes. Means SD are shown. FIG. 3. Effect of cycloheximide on the chloroquine-induced loss of HPA I in CS P. berghei. Mice were individually injected intraperitoneally with 3 mol of chloroquine or 16 mol of cycloheximide singly or in combination, and blood from groups of three to five mice was pooled at the time intervals shown in the figure for measurement of heme polymerizing activity. The times indicated on the abscissa represent the elapsed time after chloroquine treatment, except for cycloheximide alone, in which case the times after administration of cycloheximide are shown., no treatment; F, chloroquine alone;, cycloheximide alone;, cycloheximide 1 h before chloroquine;, cycloheximide and chloroquine simultaneously; Œ, cycloheximide 1 h after chloroquine. Heme polymerizing activity is expressed as nanomoles of FP polymerized per hour per micromole of preformed hemozoin FP. The mean SD and number of experiments are shown at each point for which there were three or more experiments. quinine and cycloheximide were administered singly or in combination. During the first hour after quinine administration, there was approximately a 50% recovery of the HPA I which had been lost in the first 2 h after chloroquine treatment. Cycloheximide treatment did not reverse the effect of chloroquine, and the combination of cycloheximide with quinine had essentially the same effect as quinine alone, i.e., cycloheximide did not prevent the reversal by quinine of the chloroquineinduced loss of HPA I. In related experiments, which are not shown, quinine in concentrations of 30 to 300 M added in vitro to lysates of erythrocytes parasitized with CS P. berghei did not reverse the effect of chloroquine treatment. On the contrary, quinine in vitro inhibited polymerization, as has been previously reported (6, 17). Although cycloheximide did not reverse the effect of chloroquine (Fig. 2), it nevertheless partially prevented the loss of HPA I (Fig. 3) when administered either prior to or simultaneously with chloroquine. In agreement with the data shown in Fig. 2, cycloheximide was relatively ineffective when administered 1 h after chloroquine (Fig. 3). Cycloheximide in concentrations up to 300 M had no effect on HPA I when added to lysates of parasitized erythrocytes in vitro (data not shown). Table 1 shows the effect of cycloheximide and quinine treatment on the accumulation of unpolymerized FP in erythrocytes infected with CS P. berghei. When administered in the absence of chloroquine, cycloheximide had no detectable effect on the accumulation of unpolymerized FP. When administered simultaneously with chloroquine, cycloheximide prevented the chloroquine-induced accumulation of unpolymerized FP. Similarly, quinine, which has no detectable effect in the absence of chloroquine (6), prevented the chloroquine-induced accumulation of unpolymerized FP (Table 1). Data analogous to those shown in Table 1 are unavailable for erythrocytes parasitized with CR P. berghei because unpolymerized FP was undetectable both before and after chloroquine treatment. Furthermore, in confirmation of the lack of pigmentation found by light microscopy (14), we found only (mean SD) nmol of preformed hemozoin FP per ml of packed erythrocytes parasitized with CR P. berghei (normalized to represent a parasitemia of 1,000 parasites per 1,000 erythrocytes) in 23 separate experiments. The comparable value for erythrocytes parasitized with CS P. berghei is 1,500 nmol per ml (13). The accessibility of unpolymerized FP which accumulated after chloroquine treatment was evaluated by correlating chloroquine and unpolymerized FP accumulation in vivo in erythrocytes of mice infected with CS P. berghei (Fig. 4). The wide range of unpolymerized FP contents shown in Fig. 4 was obtained by combining quinine or cycloheximide treatment with chloroquine treatment, as described for the experiments presented in Table 1 and Fig. 2 and 3, and by selecting mice with a wide range of parasitemias. Figure 4 demonstrates an excellent correlation between the chloroquine and unpolymerized FIG. 4. Correlation of chloroquine accumulation with unpolymerized FP content of erythrocytes parasitized with CS P. berghei. The methods used to measure chloroquine accumulation and unpolymerized FP content are given in the text. The amounts of unpolymerized FP and chloroquine are expressed as nanomoles per milliliter of packed erythrocytes.

4 2464 FITCH AND CHOU ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Effect of treatment with antimalarial drugs on HPA I HPA I activity d (no. of expts.) in: Drug a Dose b CS P. berghei c CR P. berghei None (12) (16) Chloroquine (8) (6) Quinacrine (3) (3) Amodiaquine (3) (3) Amopyroquine (3) (3) Quinine (5) (3) Mefloquine (4) (3) a The drugs were injected intraperitoneally 6 h before the mice were killed to obtain blood for the measurement of heme polymerizing activity. b The dose is given in micromoles of drug injected per mouse. c For CS P. berghei, pertinent data from a previous report (6) were recalculated to express heme polymerizing activity as a function of parasitemia instead of as a function of preformed hemozoin to facilitate comparisons with CR P. berghei. d Means SD are shown. Activity is expressed as nanomoles of FP polymerized per hour per milliliter of packed erythrocytes normalized to represent a parasitemia of 1,000 parasites per 1,000 erythrocytes. FP contents of these erythrocytes (r ), as would be expected if a significant fraction of the unpolymerized FP were available to bind chloroquine (3). The molar ratio of chloroquine to unpolymerized FP was 1:5. When chloroquine binds to FP at ph 7.4 in the absence of other ligands in vitro, the molar ratio of chloroquine to FP is 1:2 (3). Unpolymerized FP which binds chloroquine also is toxic to malaria parasites (10, 16). Table 2 compares the abilities of several different quinoline antimalarial drugs to reduce HPA I in CS and CR P. berghei.in both lines of parasites, chloroquine, quinacrine, amodiaquine, and amopyroquine caused losses of HPA I but quinine and mefloquine did not. Significantly, however, erythrocytes parasitized with CR P. berghei had a lower baseline heme polymerizing activity (P by t test), and the losses of activity induced by chloroquine and the other drugs were smaller than those observed in erythrocytes parasitized with CS P. berghei. To demonstrate the heat lability of the baseline heme polymerizing activity in CR P. berghei, seven different lysates were heated in a boiling water bath for 2 min (12). Heat treatment caused an 87 2% loss of activity, which is similar to the previously reported results from studies of lysates of erythrocytes infected with CS P. berghei (12). The effects of treatment of mice infected with CR P. berghei with chloroquine, quinine, and cycloheximide are shown in Fig. 5. After treatment with 3 mol of chloroquine, the initial rate of loss of HPA I from CR P. berghei was approximately onefifth of the initial rate of loss from CS P. berghei (42 versus 235 nmol of FP polymerized per h; cf. Fig. 5 with Fig. 1), but the rate of loss from CR P. berghei was relatively constant for 6 h. The total reduction in activity was 67% at 6 h after treatment with 3 mol of chloroquine. In experiments not shown, treatment with 0.3 mol of chloroquine resulted in a 30% reduction in HPA I after 6 h. Figure 5 also demonstrates that there is little if any antagonism between quinine and chloroquine in the control of HPA I in CR P. berghei and that cycloheximide treatment in the absence of chloroquine has little or no effect on HPA I. Nevertheless, cycloheximide treatment prevented the chloroquine-induced loss of HPA I from CR P. berghei (Fig. 5). DISCUSSION We found several significant differences between CS and CR P. berghei. First, our quantitative measurements confirmed earlier light microscopic studies (14) by demonstrating a virtual absence of hemozoin FP in CR P. berghei. In addition, we FIG. 5. Effects of chloroquine, quinine, and cycloheximide on HPA I in CR P. berghei. Mice were individually injected intraperitoneally with 3 mol of chloroquine, 6 mol of quinine, or 16 mol of cycloheximide singly or in certain combinations, and blood from three to five mice was pooled at the time intervals shown in the figure for measurement of heme polymerase activity., no treatment. F, chloroquine; E, chloroquine and quinine;, cycloheximide alone or chloroquine and cycloheximide. Heme polymerizing activity is expressed as nanomoles of FP polymerized per hour per milliliter of packed erythrocytes, normalized to represent a parasitemia of 1,000 parasites per 1,000 erythrocytes. Means SD and numbers of experiments are shown. found that the chloroquine-induced loss of HPA I in CR P. berghei occurred at a considerably slower rate, that quinine treatment did not prevent or reverse the chloroquine-induced loss of HPA I, and that unpolymerized FP did not accumulate despite the loss of HPA I. The reason for the failure of quinine to prevent or reverse the chloroquine-induced loss of HPA I in CR P. berghei is a complete mystery, but the lack of FP accumulation is not. It could result either from lack of FP release from hemoglobin or from detoxification of unpolymerized FP by a mechanism other than polymerization. The first explanation is plausible since CR P. berghei parasitizes only immature erythrocytes and may not need to degrade hemoglobin to obtain amino acids. Either explanation for the lack of accumulation of unpolymerized FP would account for the failure of chloroquine treatment to kill CR P. berghei despite the fact that chloroquine has qualitatively the same effect on HPA I in CR as in CS P. berghei. Thus, we again conclude that chloroquine resistance in P. berghei is due to the failure to accumulate unpolymerized FP in response to chloroquine treatment (9). Chloroquine resistance is not due to the absence of the target for chloroquine in CR P. berghei. Unlike CR P. berghei,csp. berghei parasites unquestionably degrade hemoglobin and accumulate unpolymerized FP as a consequence of the chloroquine-induced loss of HPA I (Table 1). This unpolymerized FP is accessible to bind chloroquine in vivo (Fig. 4), as predicted by our hypothesis that unpolymerized FP or a complex of chloroquine with unpolymerized FP kills these parasites (5, 10, 16). It is important, therefore, to understand how HPA I in malaria parasites is regulated. Do the various agents under study interact directly with HPA I to control it, or do they affect a process which regulates HPA I? Previous studies have yielded no evidence that chloroquine interacts directly with HPA I (5, 6). Likewise, the present studies yielded no evidence that quinine or cycloheximide in-

5 VOL. 41, 1997 HEME POLYMERIZING ACTIVITY IN MALARIA PARASITES 2465 teracts directly with HPA I, as neither quinine nor cycloheximide treatment in vitro caused an increase in HPA I, although they prevented or reversed the chloroquine-induced loss of HPA I in vivo. Furthermore, neither quinine nor cycloheximide treatment caused an increase in HPA I in vivo except in chloroquine-treated animals. On the contrary, cycloheximide treatment in the absence of chloroquine caused a slow loss of HPA I in vivo and quinine inhibited HPA I in vitro, presumably by forming a complex with substrate FP (6). These observations indicate that chloroquine, quinine, and cycloheximide interact in vivo with a regulatory process proximal to HPA I rather than with HPA I itself. As mentioned in the introduction, quinine and cycloheximide not only prevent the chloroquine-induced loss of HPA I, they prevent chloroquine-induced autophagic vacuole formation (19). Actinomycin D, puromycin, and other metabolic inhibitors also prevent chloroquine-induced autophagic vacuole formation (19). Our present findings provide a basis for understanding this common response to such a diverse group of agents. The rationale is as follows. Chloroquine acts on the negative arm of the regulatory process to cause a loss of HPA I, and consequently unpolymerized FP accumulates and, through its membrane toxicity (4, 11), causes autophagic vacuole formation. In addition, diverse metabolic inhibitors disrupt the negative arm of the regulatory process and thereby antagonize the effect of chloroquine. According to this rationale, chloroquine-induced autophagic vacuole formation may be considered to be a histologic indicator of the chloroquineinduced loss of HPA I. To move a step closer to an understanding of the antimalarial action of chloroquine at the molecular level, we propose that when chloroquine engages its target in the regulatory process, it initiates a chain of events which culminates in increased production, accessibility, or reactivity of a regulator (inactivator) of HPA I. Both the regulatory process and the putative HPA regulator merit further study. ACKNOWLEDGMENT We thank Yi-Feng Chen for technical assistance. REFERENCES 1. Bendrat, K., B. J. Berger, and A. Cerami Haem polymerization in malaria. Nature 378: Bohle, D. S., R. E. Dinnebier, S. K. Madsen, and P. W. Stephens Characterization of the products of the heme detoxification pathway in malarial late trophozoites by X-ray diffraction. J. Biol. Chem. 272: Chou, A. C., R. Chevli, and C. D. Fitch Ferriprotoporphyrin IX fulfills the criteria for identification as the chloroquine receptor of malaria parasites. Biochemistry 19: Chou, A. C., and C. D. 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