Abstract
Calcium mobilisation from internal stores of the parasitic protozoan Entamoeba histolytica was studied by fluorescence measurements of the calcium indicator quin 2 and 45Ca 2+ incorporation studies in saponin-permeabilised amoebae. Prior energy-dependent calcium sequestration was found to be necessary for subsequent release of calcium by inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3). Both Ins(1,4,5)P 3 and inositol 2,4,5-trisphosphate (Ins(2,4,5)P 3) could release calcium equally well from permeabilised E. histolytica with similar EC 50 (concentration which produced half maximal release) values for calcium release. Ins(1,4,5)P 3-mediated calcium release occurred from a vesicular store, was sensitive to prior treatment by heparin and was attenuated by prior addition of a lower concentration of Ins(1,4,5)P 3. cAMP failed to influence inositol trisphosphate induced calcium release, indicating the absence of control mechanisms through cAMP-dependent phosphorylation. GTP neither induced calcium release nor could potentiate inositol trisphosphate mediated calcium mobilisation. A saturating concentration of Ins(1,4,5)P 3 could release 50% of radiolabelled calcium sequestered by energy-dependent mechanisms in E. histolytica. The energy-dependent calcium sequestration was inhibited by vanadate and the calcium antagonist Diltiazem but not by dicyclohexylcarbodiimide (DCCD), suggesting the involvement of an endoplasmic reticulum-like structure in calcium storage. Binding studies showed specific association of [ 3H]Ins(1,4,5)P 3 to crude membrane fractions of E. histolytica, which was significantly inhibited by heparin in a dose-dependent manner. IC 50 (concentration which produced half-maximal inhibition) values for displacement of radiolabelled Ins(1,4,5)P 3 binding by unlabelled Ins(1,4,5)P 3 and Ins(2,4,5)P 3 were estimated to be 0.99 μM for both isomers. Our results suggested that Ins(1,4,5)P 4-mediated calcium release from internal stores of E. histolytica most probably occurred in an inositol trisphosphate receptor-dependent manner.
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