Impairment of the Plasmodium falciparum Erythrocytic Cycle Induced by Angiotensin Peptides

Plasmodium falciparum causes the most serious complications of malaria and is a public health problem worldwide with over 2 million deaths each year. The erythrocyte invasion mechanisms by Plasmodium sp. have been well described, however the physiological aspects involving host components in this process are still poorly understood. Here, we provide evidence for the role of renin-angiotensin system (RAS) components in reducing erythrocyte invasion by P. falciparum. Angiotensin II (Ang II) reduced erythrocyte invasion in an enriched schizont culture of P. falciparum in a dose-dependent manner. Using mass spectroscopy, we showed that Ang II was metabolized by erythrocytes to Ang IV and Ang-(1–7). Parasite infection decreased Ang-(1–7) and completely abolished Ang IV formation. Similar to Ang II, Ang-(1–7) decreased the level of infection in an A779 (specific antagonist of Ang-(1–7) receptor, MAS)-sensitive manner. 10−7 M PD123319, an AT2 receptor antagonist, partially reversed the effects of Ang-(1–7) and Ang II. However, 10−6 M losartan, an antagonist of the AT1 receptor, had no effect. Gs protein is a crucial player in the Plasmodium falciparum blood cycle and angiotensin peptides can modulate protein kinase A (PKA) activity; 10−8 M Ang II or 10−8 M Ang-(1–7) inhibited this activity in erythrocytes by 60% and this effect was reversed by 10−7 M A779. 10−6 M dibutyryl-cAMP increased the level of infection and 10−7 M PKA inhibitor decreased the level of infection by 30%. These results indicate that the effect of Ang-(1–7) on P. falciparum blood stage involves a MAS-mediated PKA inhibition. Our results indicate a crucial role for Ang II conversion into Ang-(1–7) in controlling the erythrocytic cycle of the malaria parasite, adding new functions to peptides initially described to be involved in the regulation of vascular tonus

Impairment of the Plasmodium falciparum Erythrocytic Cycle Induced by Angiotensin Peptides

Plasmodium falciparum causes the most serious complications of malaria and is a public health problem worldwide with over 2 million deaths each year. The erythrocyte invasion mechanisms by Plasmodium sp. have been well described, however the physiological aspects involving host components in this process are still poorly understood. Here, we provide evidence for the role of renin-angiotensin system (RAS) components in reducing erythrocyte invasion by P. falciparum. Angiotensin II (Ang II) reduced erythrocyte invasion in an enriched schizont culture of P. falciparum in a dose-dependent manner. Using mass spectroscopy, we showed that Ang II was metabolized by erythrocytes to Ang IV and Ang-(1–7). Parasite infection decreased Ang-(1–7) and completely abolished Ang IV formation. Similar to Ang II, Ang-(1–7) decreased the level of infection in an A779 (specific antagonist of Ang-(1–7) receptor, MAS)-sensitive manner. 10−7 M PD123319, an AT2 receptor antagonist, partially reversed the effects of Ang-(1–7) and Ang II. However, 10−6 M losartan, an antagonist of the AT1 receptor, had no effect. Gs protein is a crucial player in the Plasmodium falciparum blood cycle and angiotensin peptides can modulate protein kinase A (PKA) activity; 10−8 M Ang II or 10−8 M Ang-(1–7) inhibited this activity in erythrocytes by 60% and this effect was reversed by 10−7 M A779. 10−6 M dibutyryl-cAMP increased the level of infection and 10−7 M PKA inhibitor decreased the level of infection by 30%. These results indicate that the effect of Ang-(1–7) on P. falciparum blood stage involves a MAS-mediated PKA inhibition. Our results indicate a crucial role for Ang II conversion into Ang-(1–7) in controlling the erythrocytic cycle of the malaria parasite, adding new functions to peptides initially described to be involved in the regulation of vascular tonus

Regulation of extracellular ATP in human erythrocytes infected with Plasmodium falciparum.

In human erythrocytes (h-RBCs) various stimuli induce increases in [cAMP] that trigger ATP release. The resulting pattern of extracellular ATP accumulation (ATPe kinetics) depends on both ATP release and ATPe degradation by ectoATPase activity. In this study we evaluated ATPe kinetics from primary cultures of h-RBCs infected with P. falciparum at various stages of infection (ring, trophozoite and schizont stages). A "3V" mixture containing isoproterenol (β-adrenergic agonist), forskolin (adenylate kinase activator) and papaverine (phosphodiesterase inhibitor) was used to induce cAMP-dependent ATP release. ATPe kinetics of r-RBCs (ring-infected RBCs), t-RBCs (trophozoite-infected RBCs) and s-RBCs (schizont-infected RBCs) showed [ATPe] to peak acutely to a maximum value followed by a slower time dependent decrease. In all intraerythrocytic stages, values of ΔATP1 (difference between [ATPe] measured 1 min post-stimulus and basal [ATPe]) increased nonlinearly with parasitemia (from 2 to 12.5%). Under 3V exposure, t-RBCs at parasitemia 94% (t94-RBCs) showed 3.8-fold higher ΔATP1 values than in h-RBCs, indicative of upregulated ATP release. Pre-exposure to either 100 µM carbenoxolone, 100 nM mefloquine or 100 µM NPPB reduced ΔATP1 to 83-87% for h-RBCs and 63-74% for t94-RBCs. EctoATPase activity, assayed at both low nM concentrations (300-900 nM) and 500 µM exogenous ATPe concentrations increased approx. 400-fold in t94-RBCs, as compared to h-RBCs, while intracellular ATP concentrations of t94-RBCs were 65% that of h-RBCs. In t94-RBCs, production of nitric oxide (NO) was approx. 7-fold higher than in h-RBCs, and was partially inhibited by L-NAME pre-treatment. In media with L-NAME, ΔATP1 values were 2.7-times higher in h-RBCs and 4.2-times higher in t94-RBCs, than without L-NAME. Results suggest that P. falciparum infection of h-RBCs strongly activates ATP release via Pannexin 1 in these cells. Several processes partially counteracted ATPe accumulation: an upregulated ATPe degradation, an enhanced NO production, and a decreased intracellular ATP concentration

Effect of Pannexin 1 inhibitors on [ATPe] kinetics of a highly enriched population of trophozoite infected erythrocytes.

<p>A. The time course of [ATPe] (pmol/10⁶ cells) was assessed for trophozoite-infected erythrocytes at 94% parasitemia (denoted as t94-RBCs) in the absence and presence of 100 µM carbenoxolone (CBX), 100 nM mefloquine (MFQ), or 100 µM of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), inhibitors of Pannexin 1. Exposure to 3V is indicated by the arrow. In some experiments, prior to 3V exposure cells were pre-incubated 10 min with either CBX, MFQ or NPPB. For a comparison, similar experiments with noninfected RBCs (h-RBCs) are shown. t94-RBCs (N = 14, n = 19), t94-RBCs +CBX (N = 6, n = 7), t94-RBCs+MFQ (N = 4, n = 4), t94-RBCs+NPPB (N = 4, n = 4), h-RBCs (N = 15; n = 19), h-RBCs+CBX (N = 6, n = 9), h-RBCs+MFQ (N = 4, n = 4), h-RBCs+NPPB (N = 3, n = 3). N = independent preparations, n = replicates. B. 3V-dependent increase of [ATPe] calculated from A. Values are expressed as ΔATP₁, i.e., the difference between [ATPe] at 1 min post-stimulus and basal [ATPe]. Results are means ± SEM. (*p<0.05, ***p<0.001).</p

3V-dependent ATP kinetics of <i>P. falciparum</i> infected RBCs.

<p>A, C. Time course of ATPe concentration (pmol/10⁶ cells) for r-RBCs (ring-infected erythrocytes), t-RBCs (trophozoite-infected erythrocytes) and s-RBCs (schizont-infected erythrocytes) at low parasitemia (<5%, A) and high parasitemia (5–12.5%, C). In the time indicated by the arrow, cells were exposed to “3V”, a cAMP activating cocktail containing 10 mM isoproterenol, 30 mM forskolin and 100 mM papaverine. For a comparison, similar experiments with h-RBCs are shown. B, D. 3V-dependent increases of [ATPe] calculated from A and C. Values are expressed as ΔATP₁ for low parasitemia (B) and high parasitemia (D). Results are means ± SEM. (*p<0.05, ***p<0.001). (N, n), with N = independent preparations, n = replicates. E. Initial rate of [ATPe] decay (pmol/10⁶ cells/min) taken from data of C.</p

3V-dependent increase of [ATPe] of trophozoite-infected RBCs.

<p>Values of ΔATP₁ as a function of parasitemia (5–12.5%) for trophozoite-infected RBCs (N = 4, n = 4–5). Prior to experiments, cells were pre-incubated 3 hours in the absence (black circles) or presence (green squares) of 2 mM L-NAME. Hyperbolic functions were fitted to experimental data. Results are means ± SEM with N = 3 and n = 5–10. N = independent preparations, n = replicates. The red symbols illustrate an estimate of ΔATP₁ under a hypothetical situation where ectoATPase activity is blocked. It was calculated by: 1- estimating the concentration of ATPe hydrolyzed during the first minute post-stimulus (using results of Fig. 5); 2- adding that value to the experimentally obtained ΔATP₁.</p

Apparent maximal ectoATPase activities of h-RBCs and t94-RBCs.

<p>A, B. Rates of Pi accumulation ([³²P]Pi) released from exogenous 500 µM [γ-³²P]ATP, using suspensions of noninfected RBCs (h-RBCs, N = 4, n = 4) and trophozoite-infected RBCs at 94% parasitemia (t94-RBCs; N = 3, n = 4). The dotted lines represent the fitting of exponential functions to experimental data, with values of the corresponding rate constant (k) given in brackets. Values of best fit were used to calculate apparent maximal ectoATPase activities as described in Materials and Methods. C. Apparent maximal ectoATPase activities at 500 µM ATP were determined from exponential fits of A and B. For a comparison, ectoATPase activities of h- and t94-RBCs at 900 nM ATP, taken from Fig. 5, are shown. Significant differences are indicated (*, p<0,05, **, p<0,01). Results are means ± SEM.</p

Kinetics of ATPe from cAMP-stimulated human erythrocytes (h-RBCs).

<p>The time course of ATPe concentration ([ATPe]) from h-RBCs was quantified by real-time luminometry, as described in Materials and Methods. In the time indicated by the arrow, cells were exposed to “3V”, a cAMP activating cocktail containing 10 mM isoproterenol, 30 mM forskolin and 100 mM papaverine. Levels of ATPe were expressed both as pmol ATP/(10⁶ cells) (left axis) or as ATPe concentration (nM) with 10⁶ cells in 60 µl assay volume (right axis). Data represent mean values ± SEM from N = 14 independent preparations.</p

Effect of L-NAME on 3V-dependent increase of [ATPe] in h-RBCs and t94-RBCs.

<p>Values are expressed as ΔATP₁, i.e., the difference between [ATPe] at 1 min post-stimulus and basal [ATPe]. Before the experiments, cells were pre-incubated 3 hs in the absence and presence of 2 mM L-NAME. A. ΔATP₁ values for noninfected and trophozoite-infected RBCs (h- and t94-RBCs) in the absence and presence of L-NAME. Results are means ± SEM with N = 4 and n = 4. (*p<0.05, ***p<0.001).</p