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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 2, 2005 as doi:10.1096/fj.04-3371fje. |
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* Department of Physiology I, University of Tübingen, Tübingen, Germany;
Etablissement Francais du Sang-Alsace, INSERM U.311, Strasbourg, France;
Department of Medicine, University of Sydney, Nepean Hospital, Penrith, New South Wales, Australia; and
Department of Anatomy and Histology, University of Sydney, Sydney, New South Wales, Australia
1Correspondence: Department of Physiology I, Gmelinstrasse 5, Tübingen 72076, Germany. E-mail: stephan.huber{at}uni-tuebingen.de
SPECIFIC AIMS
The intraerythrocytic development of P. falciparum induces the so-called new permeability pathways (NPP) that appear in the membrane of the parasitized red blood cells (RBCs). Functionally, the NPP are organic osmolyte and anion channels that supply the parasite with nutrients and dispose of parasite-derived waste products. Until now, the signaling cascades that trigger the increase in the plasma membrane permeability of the host RBC have been ill defined. P. falciparum infection has been demonstrated to confer a high oxidative stress on the host RBC and experimental oxidation of noninfected RBCs induces membrane permeabilities similar to the infection-induced NPP. Physiologically, the NPP contribute to the regulatory volume decrease (RVD) of the volume-stressed host erythrocyte. RVD in nucleated cells has been demonstrated to employ ATP release and autocrine purinergic signaling. Therefore, the present work tested for autocrine purinoceptor signaling in human RBCs during induction of the NPP by P. falciparum infection or experimental membrane oxidation.
PRINCIPAL FINDINGS
1. ATP stimulates and purinoceptor antagonists inhibit the induction of the organic osmolyte permeability in infected RBCs
The NPP are permeable for neutral organic osmolytes such as sorbitol. Therefore, infected RBCs expressing the NPP hemolyzed colloidosmotically within 15 min when suspended in isosmotic sorbitol solution, while noninfected RBCs stayed intact. The parasite-induced hemolysis was blocked in the presence of NPPB (100 µM), an inhibitor of the NPP. In contrast, the nonspecific purinoceptor antagonist suramin (100 µM), the P2Y1-specific antagonist MRS2179, or the purinergic agonist ATP had no effect. This suggests that neither suramin nor MRS2179 nor ATP directly interfere with the sorbitol uptake by the NPP. In contrast, addition of ATP (100 µM for 6 h prior to the experiment) and suramin (50 µM free concentration for 6–18 h) to the synchronized in vitro culture significantly increased and decreased sorbitol hemolysis, respectively. In addition to suramin, preincubation with MRS2179 (6–18 h at 10–100 µM) decreased isosmotic sorbitol hemolysis of the infected RBCs.
2. ATP and P2Y purinoceptor agonists stimulate the induction of the organic osmolyte permeability in oxidized noninfected RBCs
Similar to infection, oxidation of noninfected human RBCs with t-BHP (1 mM for 15 min) induced NPPB-sensitive hemolysis in isosmotic sorbitol solution indicating activation of the organic osmolyte permeability (Fig. 1
A). This oxidation-induced hemolysis required a 2.5 h postincubation period to develop. ATP (100 µM) enhanced the oxidation-induced hemolysis by 2- to 3-fold. Similar to the oxidation-induced hemolysis, the ATP-stimulated oxidation-induced hemolysis was inhibited by NPPB. Most important, ATP did not induce hemolysis in nonoxidized (or noninfected) RBCs suggesting that both oxidation (or infection) and purinoceptor activation were required for the induction of the sorbitol permeability.
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ATP stimulated the oxidation-induced sorbitol permeability with an apparent EC50 of 
30 µM further pointing to a purinoceptor-triggered signal transduction pathway (Fig. 1B
). The nonhydrolyzable form of ATP, ATP
S, as well as other P2 receptor agonists such as 2-MeSATP, UTP, ADP, and ADPßS increased the hemolysis in a dose-dependent manner with an apparent EC50 of 100 µM or greater. In contrast, BzATP, an effective agonist of the P2X7, P2X1, and P2Y11 receptors had no significant effect on the oxidation-induced sorbitol hemolysis (Fig. 1B
). Addition of the ecto-ATPase inhibitor ARL67156 (100 µM, Fig. 1C
) induced a left shift by an order of magnitude in the EC50 for the ATP-induced increase of the sorbitol hemolysis of oxidized RBCs, suggesting enzymatic degradation of ATP in the medium.
3. Purinoceptor antagonists inhibit the oxidation-induced and ATP-stimulated organic osmolyte permeability of noninfected RBCs
Suramin and two further nonspecific P2 receptor antagonists (PPADS and reactive blue-2) inhibited the oxidation-induced hemolysis, as well as the oxidation-induced ATP-stimulated hemolysis in a dose-dependent manner with IC50s of less than 1 to 10 µM. Moreover, the P2Y1-specific antagonist MRS2179 inhibited the oxidation-induced hemolysis and the oxidation-induced ATP-stimulated hemolysis with IC50s of 1 µM and 0.1 µM, respectively, suggesting involvement of P2Y1 purinoceptor subtypes. However, ATP still had a stimulatory effect on oxidation-induced hemolysis at high MRS2179 and reactive blue 2 concentrations, strongly suggesting involvement of other purinoceptor subtypes that are insensitive to MRS2179 and reactive blue 2.
4. Human erythrocytes release ATP upon P. falciparum infection or oxidation
To test for oxidation- or infection-induced release of ATP, the time-dependent changes of the extracellular ATP concentration of control, oxidized and P. falciparum-parasitized human RBCs were analyzed by the luciferin-luciferase assay. Both oxidation by t-BHP (200 µM) and infection by Plasmodium induced an increase in extracellular ATP, indicating a release of ATP from the RBCs by either specific ATP release pathways or hemolysis. In contrast, nonoxidized or noninfected control RBCs did not release ATP under identical experimental conditions. ATP release was a prerequisite for the oxidation-induced sorbitol hemolysis because hemolysis did not occur upon degradation of extracellular ATP by apyrase.
5. Protein and functional expression of the P2Y1 purinoceptor subtype in human and mouse erythrocytes
Immunofluorescence staining with a polyclonal anti-P2Y1 antibody and confocal microscopy demonstrated P2Y1 protein expression in human RBC membranes, with levels of expression varying between subjects (Fig. 2
A, B). Immunoblot analyses performed with another polyclonal anti-P2Y1 antibody revealed two bands in human RBC membranes at 42 ± 1 and 59 ± 0.5 kDa (Fig. 2C
, left lane). Pre- and coincubation of the antibody with the P2Y1-specific peptide used to raise the antibody abolished the detection of the 42 kDa band and largely decreased the intensity of the 59 kDa band (right lane), suggesting P2Y1-specificity of the used antibody.
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To study the functional expression of P2Y1, RBCs from P2Y1–/– mice were compared in oxidation-induced sorbitol hemolysis experiments with those from sex- and age-matched wild-type controls. ATP stimulated the oxidation-induced hemolysis in noninfected RBCs from wild-type but not P2Y1–/– mice (Fig. 2D
). Sorbitol hemolysis(15 min) of P. berghei ANKA-infected RBCs (parasitemia between 40 and 48%) retrieved from P2Y1–/– mice was significantly delayed compared with those retrieved from P2Y1+/+ mice (Fig. 2E
). These experiments indicated functional expression of P2Y1 purinoceptors in RBCs and involvement of this receptor subtype in activation of the sorbitol permeability.
6. Delayed in vivo growth of P. berghei in P2Y1-deficient mice
P2Y1-deficient mice and sex- and age-matched wild-type mice with identical genetic background were infected with P. berghei ANKA (18 mice in each group) and the increase in parasitemia was monitored. In wild-type mice, parasitemia developed exponentially up to a parasitemia of 60–70%. In sharp contrast, parasitemia of P2Y1-deficient mice developed almost linearly, with the maximal slope of parasitemia increase being significantly lower than that in wild-type animals. The delayed parasite development in P2Y1–/– mice did not result in a better survival of these mice.
7. The purinoceptor antagonist suramin impairs intraerythrocytic P. falciparum growth in vitro and P. berghei growth in vivo
To study the functional significance of the purinoceptor signaling for P. falciparum development in human RBCs, ring stage-synchronized parasites were grown in vitro (48 h) in the presence of increasing concentrations of suramin. Parasitemia was monitored by flow cytometry using the DNA/RNA specific fluorescence dye Syto16. Suramin dose-dependently decreased the rise in parasitemia with an apparent IC50 of 10 µM. In short-term growth assays (16 h) with synchronized parasites, suramin inhibited the synthesis of DNA/RNA within the trophozoites/schizonts, which is a measure of intraerythrocytic parasite amplification. Similarly, suramin (150–500 mg/kg B.W. p.i.) decreased the parasite development in P. berghei ANKA-infected wild-type mice.
CONCLUSIONS AND SIGNIFICANCE
The present study demonstrates that activation of the organic osmolyte permeability in Plasmodium-infected human and mouse erythrocytes is dependent on ATP release from the host erythrocyte and autocrine purinergic signaling via P2Y1 and other purinoceptor subtypes. Reportedly, the intraerythrocytic parasite development depends on the organic osmolyte permeability in the host membrane. Accordingly, P2Y1 gene deficiency or pharmacological inhibition of the purinoceptor signaling resulted in an impaired Plasmodium development in vivo and in vitro, collectively suggesting the functional significance of purinoceptor signaling in intraerythrocytic parasite development. Oxidative stress mimicked those processes leading to the induction of the organic osmolytes permeability in the Plasmodium-infected erythrocyte, further suggesting that parasite-derived oxidative stress is involved in the induction of the organic osmolyte permeability.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3371fje;
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0.01. B) Relative oxidation-induced sorbitol hemolysis in the absence or in the presence of increasing concentrations of ATP (filled diamonds), ATP
