SciELO - Scientific Electronic Library Online

 
vol.93 suppl.1Immunization with PIII, a fraction of Schistosoma mansoni soluble adult worm antigenic preparation, affects nitric oxide production by murine spleen cellsIgE and IgG4 antibodies in subjects reinfected with Schistosoma mansoni in an endemic area of Northeast Brazil author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Memórias do Instituto Oswaldo Cruz

Print version ISSN 0074-0276

Mem. Inst. Oswaldo Cruz vol.93  s.1 Rio de Janeiro  1998

http://dx.doi.org/10.1590/S0074-02761998000700029 

RESEARCH NOTE

(Ca2+-Mg2+)ATPase in Schistosoma mansoni: Evidence for Heterogeneity and Resistance to Praziquantel

Vol. 93, Suppl. I: 181-182

VMN Cunha, F Noël+

Departamento de Farmacologia Básica e Clínica, ICB, Universidade Federal do Rio de Janeiro, Cidade Universitária, 21941-590 Rio de Janeiro, RJ, Brasil

Key words: ATPase - calcium - (Ca2+-Mg2+)ATPase - Schistosoma mansoni - thapsigargin - praziquantel


RESEARCH NOTE

ACKNOWLEDGEMENTS


RESEARCH NOTE

In mammals, Ca2+ pumps use the chemical energy derived from the hydrolysis of ATP to remove Ca2+ from the cytoplasm of a variety of cell types (L De Meis & G Inesi 1982, p. 141-169. In E Carafoli, Membrane Transport of Calcium, Academic Press, New York, AF Rega 1986, p. 13-20. In AF Rega, The Ca2+ Pump of Plasma Membrane, CRC Press, Boca Raton). The pumps of plasma membranes (PMCA family) and those of endoplasmic or sarcoplasmic reticulum (SERCA family) (NM Green 1992 Ann NY Acad Sci 671: 104-169) have the same physiological functions, despite differences in their structure, subcellular localization and modulatory mechanisms. Thapsigargin, a plant-derived sesquiterpene lactone, inhibits the activity of all of the SERCA isoenzymes (SERCA1, SERCA2 and SERCA3) with equal potency, but has no effect on the ATPase or transport activities of PMCA pumps. In previous reports we demonstrated a (Ca2+-Mg2+)ATPase activity coupled to the active transport of Ca2+ in subcellular fractions from Schistosoma mansoni (VMN Cunha et al. 1988 FEBS Lett 241: 65-68, 1992 Mol Biochem Parasitol 52: 167-174). The aim of the present work was, first, to investigate the subcellular localization of the (Ca2+-Mg2+)ATPase activities present in P1 and P4 fractions, using thapsigargin, cyclopiazonic acid (CPA) and tamoxifen as inhibitors of Ca2+ pump. Second, to investigate if an ATPase responsible for the control of Ca2+ homeostasis, could be involved in the molecular mechanism of praziquantel-induced contraction in S. mansoni.

Male cercariae of S. mansoni (BH strain) were obtained from snails (Biomphalaria glabrata) previously infected with a single miracidium (Cunha 1988 loc. cit.). The subcellular fractions were obtained by homogenizing and centrifuging about 2000 worms to obtain four pellets (P1 , P2, P3, P4) sedimenting respectively at 300 x gav (5 min); 1000 x gav (10 min); 8000 x gav (10 min) and 100,000 x gav (1hr). About 25 mg of protein from P1 or P4 fractions were incubated for 1 hr at 37oC in 0.5 ml of a mixture containing (unless otherwise stated) 5 mM Na2ATP, 0.3 mM EGTA, 10 mM NaN3, 4 mM MgCl2, 60 mM KCl, 5 mM A23187 (calcimycin, a calcium ionophore), 50 mM HEPES-Tris buffer (pH 7.4) and 0 or 370 mM CaCl2 (10 mM free Ca2+), for the determination of ATPase activity. The (Ca2+-Mg2+)ATPase was calculated by subtracting the basal ATPase activity measured in the absence of calcium from the total activity measured in the presence of calcium.

The effect of thapsigargin on (Ca2+-Mg2+)ATPase activity in the fractions P1 and P4 from S. mansoni is shown in Fig. 1. In fraction P4, the (Ca2+-Mg2+)ATPase activity was inhibited by the drug with a half-maximal effect (I50) at 250 nM. At a saturating concentration of thapsigargin (3 mM), the activity was completely blocked (Imax = 103 ± 5.1%, n = 3). Under the same conditions, about 20% of the (Ca2+-Mg2+)ATPase activity present in P1 was resistant to thapsigargin (Imax=78.3 ± 4.9%, n = 3; P<0.005, Student's t - test). There was no significant difference between fractions P1 and P4 in their sensitivity to the drug. However, a lower sensitivity of S. mansoni ATPase to thapsigargin in relation to the mammalian enzymes was observed. The same pattern of inhibition was observed using CPA, with the fraction P4 exhibiting greater inhibition at a saturating concentration (Imax = 84.8 ± 2.2%, n = 3) than P1 (Imax = 70.9 ± 2.8%, n = 3; P< 0.005, Student's t - test) whereas there was no significant difference in the I50 values. Thus, 22% to 29% of the (Ca2+-Mg2+)ATPase activity of fraction P1 is resistant to thapsigargin and CPA in these preparations. To explore the subcellular origin of the (Ca2+-Mg2+)ATPase activity that is resistant to thapsigargin and CPA, tamoxifen was employed as a non-selective inhibitor of (Ca2+-Mg2+)ATPase. This drug completely inhibits the (Ca2+-Mg2+)ATPase activity in both fractions whereas only the inhibitory effect on (Ca2+-Mg2+)ATPase activity from fraction P1 could be partially antagonized by the addition of 90 mg calmodulin to the incubation mixture (VMN Cunha et al. 1996 Comp Biochem Parasitol 114B: 199-205). Several lines of evidence suggest that praziquantel interacts specifically with a molecule involved in Ca2+ regulation within the worm to promote contraction. Fig. 2 shows that neither the (Ca2+-Mg2+)ATPase activities of P1 nor of P4 fractions were inhibited by increased concentrations of praziquantel. As a whole our data show that the majority of the (Ca2+-Mg2+)ATPases from S. mansoni should be a SERCA isoform and that therapeutic concentrations of praziquantel have no direct action on the (Ca2+-Mg2+)ATPases described in S. mansoni.

Acknowledgements: to Dr Lygia dos Reis Corrêa (Instituto Oswaldo Cruz, Rio de Janeiro, Brasil) who kindly provided the infected mice; to Eliana Freitas and José Ferreira Oliveira for their skilful technical assistance.


Figure 1 | Figure 2


This work was supported by CNPq, Faperj, Finep and Pronex 41.96.0888.00.

+Corresponding author. Fax: +55-21- 280.4694. E-mail: fnoel@pharma.ufrj.br 

Received 4 May 1998

Accepted 31 August 1998