Evidence for functional ryanodine receptors in Schistosoma mansoni and their putative role in the control of calcium homeostasis

ryanodine; Schistosoma mansoni; calcium; praziquantel

RESEARCH NOTE

Evidence for Functional Ryanodine Receptors in Schistosoma mansoni and their Putative Role in the Control of Calcium Homeostasis

CLM Silva, DL Mendonça-Silva, 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: ryanodine - Schistosoma mansoni - calcium - praziquantel

^{RESEARCH NOTE}

^{RESEARCH NOTE}

The main musculature present in adult male Schistosoma mansoni is mainly of the smooth muscle type (MH Silk & IM Spence 1969 S Afr J Med Sci 34: 11-20). As we have previously demonstrated, the contraction of S. mansoni presents a tonic component, dependent on extraworm calcium, and a phasic one, dependent on intracellular/intraworm calcium (S da Silva & F Noël 1995 Parasitol Res 81: 543-548). Since intracellular calcium channels sensitive to the alkaloid ryanodine and therefore known as ryanodine receptors (RyR) (R Coronado et al. 1994 Am J Physiol 266: C1485-C1504) have been demonstrated in a variety of mammalian as well as non-mammalian (chicken, frog) and non-vertebrate cells (Caenorhabditis elegans, lobster and drosophila) (G Meissner 1994 Annu Rev Physiol 56: 485-508), the main objective of this work was to investigate the presence of RyR in S. mansoni, as well as their subcellular distribution profile, pharmacological modulation and their putative involvement in S. mansoni contractility.

About 2,000 worms were homogenized in a Dounce homogenizer at 4^oC in 0.25 M sucrose solution (5 mM Tris-HCl pH 7.4) using three sequences of ten passes of the pestle. The homogenate was centrifuged to obtain four pellets (P₁, P₂, P₃, P₄) sedimenting respectively at 300 g_av (5 min); 1000 g_av (10 min); 8000 g_av (10 min) and 100,000 g_av (1 hr) (VMN Cunha et al. 1988 FEBS Lett 241: 65-68).

In the binding assays, 100-150 mg of proteins of subcellular fractions were incubated for 2 hr at 37^oCin a medium (0.5 ml) containing 1.5 M KCl, 10 mM Na₂ATP, 0.8 mM CaCl₂ (107 mM free Ca²⁺), HEPES 10 mM, NaOH pH 7.4 and 0.3 nM [³H]ryanodine (NEN, 84 Ci/mmol) in the presence or absence of 0.2-10 nM unlabelled ryanodine. Incubations were terminated by dilution of the samples with 5 ml of ice-cold buffer (150 mM KCl, 10 mM Tris, pH 7.4) followed by rapid filtration on glass fibre filters under vacuum (Whatman GF/C) in order to separate bound and free radioligand. The non-specific binding was determined in the presence 10 mM unlabelled ryanodine. Data from saturation experiments were treated by a computerised non-linear regression analysis (EBDA-LIGAND; Elsevier-Biosoft, Cambridge, UK).

For the in vitro studies, the worms were carefully recovered from mice portal veins, washed and placed in a glass dish containing 3 ml Tyrode's solution maintained at 37^oC. After 10 min for equilibration, 100 mM ryanodine or vehicle were added (time zero) and the grading of worm shortening was performed based on the worm length (da Silva & Noël loc. cit.).

The [³H]ryanodine specific binding sites were mainly recovered in the heterogeneous (P₁) and microsomal (P₄) fractions of S. mansoni (Table I). Analysis of the data from four saturation experiments revealed an equilibrium dissociation constant (K_d) in the low nanomolar range (6.9 ± 0.6 nM), a maximal number of receptors (B_max) of 78 ± 18 fmol/mg protein. We tested the influence of Ca²⁺ and Mg²⁺ on the binding of [³H]ryanodine to the P₁ fraction. As the free concentration of Ca²⁺ was increased from 0.3 to 107 mM there was a corresponding increase in the [³H]ryanodine specific binding (Table II). On the other hand 5 mM free Mg²⁺, in the absence of ATP, inhibited about 50% the [³H]ryanodine specific binding (Table II). Praziquantel, even at a concentration 10-fold higher than its therapeutic plasma concentration, had no effect on [³H]ryanodine binding (Table II). Finally ryanodine was able to contract the whole S. mansoni in a time-dependent manner (Fig.).

[³H]ryanodine specific binding sites were mainly recovered in the heterogeneous (P₁) and microsomal (P₄) fractions of S. mansoni. These binding sites have a similar pattern of subcellular distribution as the (Ca²⁺-Mg²⁺)ATPase sensitive to thapsigargin, that corresponds to a SERCA ATPase (VMN Cunha et al. 1996, Comp Biochem Physiol 114B: 199-205). The saturation experiment at equilibrium revealed the labelling of only one homogeneous population of receptors in the range of concentrations used, and a dissociation constant (K_d) in the nanomolar range, as observed in higher animals (Coronado loc. cit.) or even in the nematode C. elegans (YK Kim et al. 1992 Biophys J 63: 1379-1384). [³H]ryanodine binding correlates with the functional state of the ionic channel (Coronado loc. cit.) so that conditions increasing the open channel probability usually favour the [³H]ryanodine binding whereas conditions that close the channel decrease this binding, as for Ca²⁺ and Mg²⁺ respectively. With respect to the ryanodine receptors present in S. mansoni they possess some characteristics qualitatively very similar to those present in higher animals regarding the affinity for ryanodine and the modulatory effect of ions (Ca²⁺ and Mg²⁺). Recently, CA Redman et al. (1996 Parasitol Today 12: 14-20) suggested that the mechanism of action of praziquantel could be related to mobilisation of intracellular Ca²⁺ stores sensitive to IP₃ or Ca²⁺. Here we observed that praziquantel was not able to alter the [³H]ryanodine binding to the subcellular P₁ fraction. Other possibilities have just been discharged such as inhibition of (Ca²⁺- Mg²⁺) ATPase and (Na⁺/ K⁺)ATPase (VMN Cunha & F Noël 1997 Life Sci 60: PL289-294), thus the exact mechanism of action of praziquantel-induced contraction still remains to be elucidated. Finally, a high concentration of ryanodine (100 mM) was able to induce contractions of the whole adult worms, indicating that these receptors may have a role in the control of calcium homeostasis within the worm musculature.

to Dr Lygia dos R Corrêa, Instituto Oswaldo Cruz, who kindly provided the infected mice; to Eliana Freitas and José Ferreira Oliveira for their skilful technical assistance.

Figure | Table I | Table II

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

⁺Corresponding author. Fax [phone]. E-mail: [e-mail]

Received 4 May 1998

Accepted 31 August 1998

Table I

Table II

Figure

Time-course of ryanodine-induced contractions of whole Schistosoma mansoni. Grading of shortening was set according to the worm length (see Methods). Each point represents the median value for 8-14 experiments. (

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)100 mM ryanodine.

^*

p < 0.05 versus control using Mann-Whitney's nonparametric

U-test.

Publication Dates

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

History

• Accepted
  31 Aug 1998
• Received
  04 May 1998