Cyclic AMP and calcium interplay as second messengers in melatonin-dependent regulation of Plasmodium falciparum cell cycle

Abstract

The host hormone melatonin increases cytoplasmic Ca(2+) concentration and synchronizes Plasmodium cell cycle (Hotta, C.T., M.L. Gazarini, F.H. Beraldo, F.P. Varotti, C. Lopes, R.P. Markus, T. Pozzan, and C.R. Garcia. 2000. Nat. Cell Biol. 2:466-468). Here we show that in Plasmodium falciparum melatonin induces an increase in cyclic AMP (cAMP) levels and cAMP-dependent protein kinase (PKA) activity (40 and 50%, respectively). When red blood cells infected with P. falciparum are treated with cAMP analogue adenosine 3',5'-cyclic monophosphate N6-benzoyl/PKA activator (6-Bz-cAMP) there is an alteration of the parasite cell cycle. This effect appears to depend on activation of PKA (abolished by the PKA inhibitors adenosine 3',5'-cyclic monophosphorothioate/8 Bromo Rp isomer, PKI [cell permeable peptide], and H89). An unexpected cross talk was found to exist between the cAMP and the Ca(2+)-dependent signaling pathways. The increases in cAMP by melatonin are inhibited by blocker of phospholipase C U73122, and addition of 6-Bz-cAMP increases cytosolic Ca(2+) concentration, through PKA activation. These findings suggest that in Plasmodium a highly complex interplay exists between the Ca(2+) and cAMP signaling pathways, but also that the control of the parasite cell cycle by melatonin requires the activation of both second messenger controlled pathways.

Figure 1.

Measurement of cAMP levels in P. falciparum parasites. Experiments were performed by using isolated parasites at trophozoite stage treated with 100 nM melatonin in the presence or absence of 100 μM IBMX. Cells were incubated with: 10 μM U73122, 10 μM ionomycin, 25 μM BAPTA, 10 μM calmidazolium, 1 μM staurosporin, and 10 μM U73343. After treatment, samples were analyzed with cAMP enzyme immunoassay kit (Amersham Biosciences). There was a significant variation with melatonin addition (one-way analysis of variance vs. Newman-Keuls test, P < 0.05). Results represent duplicated data from three different experiments.

Figure 2.

Effect of cAMP signaling drugs on P. falciparum cell cycle development in vitro. Incubation for 24 h at 37°C in 24-well plates with reagents: 100 nM MLT, 20 μM PKI, 20 μM 8-BrcAMP-RP isomer, and 20 μM 6-Bz-cAMP. Values are the percentage of parasitemia variation to control: (A) R, ring; T, trophozoite; and S, Schizont parasites stages; and (B) total parasitemia. The parasitemia (percentage of red cells infected) was determined by count of infected cells (1,000) from three different experiments. In these experiments the initial parasitemia was around 5% and the maximal parasitemia was around 15%. Data were compared by one-way analysis of variance and a Newman-Keuls test. *, Statistical significance with respect to control values P < 0.01.

Figure 3.

PKA activity levels in P. falciparum parasites. Isolated parasites at trophozoite stage treated with 100 nM MLT, 100 μM IBMX, or MLT and IBMX. Data was calculated from duplicated data from three different experiments assays, as a percentage of total PKA activity obtained with an additional 1 μM cAMP.

Figure 4.

Ca^{2 +} fluorescence measurement in P. falciparum parasites analyzed by confocal microscopy. Parasites at trophozoite stage were loaded with Fluo-4 AM. (see Materials and methods) and its fluorescence ratio (F1/F0) intensity was plotted as a function of time. (A–C) Addition of 100 nM melatonin in buffer with 2 mM Ca²⁺ promotes an increase of 1.4 ± 0.4 (n = 18) and similar results was observed with melatonin 100 nM in the presence of PKI in a Ca²⁺ medium or in a Ca²⁺-free medium, respectively. (D) 20 μM 6-Bz-cAMP resulted in 1.5 ± 0.1 (n = 6). (E) 20 μM 6-Bz-cAMP was added in Ca²⁺-free medium (EGTA containing) and causes a transient fluorescence increase of 1.36 ± 0.2 (n = 4). (F) 20 μM 6-Bz-cAMP did not induce an increase of Ca²⁺ when added in the presence of PKA inhibitor PKI. (G) 20 μM 6-Bz-cAMP induced an increase of Ca²⁺ of 1.41 ± 0.3 (n = 3) in the presence of PLC inhibitor U73122 (10 μM).

Figure 5.

Ca^{2 +} fluorescence measurement in P. falciparum parasites analyzed by confocal microscopy. Parasites at trophozoite stage were loaded with Fluo-4 AM (see Materials and methods) and its fluorescence ratio (F1/F0) intensity was plotted as a function of time. (A) Addition of 100 nM melatonin in buffer with 2 mM Ca²⁺: the mean increase of fluorescence ratio was 1.32 ± 0.2 (n = 11) no effect was observed with a further addition of 20 μM 6-Bz-cAMP. (B) Addition of 20 μM 6-Bz-cAMP caused an average an increase in fluorescence ratio of 1.40 ± 0.3 (n = 5) and no effect was observed with a further addition of melatonin. (C) Same conditions as above. When indicated 10 μM THG was added. (D and E) Where indicated THG and 6-Bz-cAMP were added. In D the medium contained 2 mM CaCl₂ whereas in E no Ca²⁺ was added and 100 μM EGTA was included instead.

Figure 6.

Schematic model of signaling events in Plasmodium with data provided from literature and our results. AC, adenylyl cyclase; PLC, phospholipase C; PDE, phosphodiesterase; PKA, protein kinase A; ER, endoplasmic reticulum; R, hypothetical melatonin receptor.