(Ca2+-Mg2+)ATPase in Schistosoma mansoni: evidence for heterogeneity and resistance to praziquantel

ATPase; calcium; (Ca2+-Mg2+)ATPase; Schistosoma mansoni; thapsigargin; praziquantel

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

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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 g_av (5 min); 1000 x g_av (10 min); 8000 x g_av (10 min) and 100,000 x g_av (1hr). About 25 mg of protein from P₁ or P₄ fractions were incubated for 1 hr at 37^oC in 0.5 ml of a mixture containing (unless otherwise stated) 5 mM Na₂ATP, 0.3 mM EGTA, 10 mM NaN_3, 4 mM MgCl_2, 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 (I_max = 10³ ± 5.1%, n = 3). Under the same conditions, about 20% of the (Ca²⁺-Mg²⁺)ATPase activity present in P₁ was resistant to thapsigargin (I_max=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 (I_max = 84.8 ± 2.2%, n = 3) than P₁ (I_max = 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.

: 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: [phone]. E-mail: [e-mail]

Received 4 May 1998

Accepted 31 August 1998

Figure 1

Figure 2

) and P

₁ (

) from S

chistosoma mansoni. Fractions (16-30 mg protein) were incubated for 1 hr at 37

^oC. Each point is the mean of three assays performed in triplicate in different enzymatic preparations, with bars denoting SEM. The curves were drawn using the parameters obtained by non-linear regression analysis according to the Hill equation.

Publication Dates

• Publication in this collection
  14 July 2000
• Date of issue
  1998

History

• Accepted
  31 Aug 1998
• Received
  04 May 1998