title: Myo-inositol trisphosphate-mediated calcium release from internal stores of Entamoeba histolytica.
abstract: Calcium mobilisation from internal stores of the parasitic protozoan Entamoeba histolytica was studied by fluorescence measurements of the calcium indicator quin 2 and 45Ca2+ 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)P3). Both Ins(1,4,5)P3 and inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) could release calcium equally well from permeabilised E. histolytica with similar EC50 (concentration which produced half maximal release) values for calcium release. Ins(1,4,5)P3-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)P3. 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)P3 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)P3 to crude membrane fractions of E. histolytica, which was significantly inhibited by heparin in a dose-dependent manner. IC50 (concentration which produced half-maximal inhibition) values for displacement of radiolabelled Ins(1,4,5)P3 binding by unlabelled Ins(1,4,5)P3 and Ins(2,4,5)P3 were estimated to be 0.99 microM for both isomers. Our results suggested that Ins(1,4,5)P3-mediated calcium release from internal stores of E. histolytica most probably occurred in an inositol trisphosphate receptor-dependent manner.
authors.affiliation: Department of Biophysics, Molecular Biology and Genetics, Calcutta University, India.
authors.forename: S
authors.initials: S
authors.lastname: Raha
authors.forename: B
authors.initials: B
authors.lastname: Dalal
authors.forename: S
authors.initials: S
authors.lastname: Biswas
authors.forename: B B
authors.initials: BB
authors.lastname: Biswas
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D004110:Diltiazem / Q000494:pharmacology; D004748:Entamoeba histolytica / Q000187:drug effects* / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism / Q000494:pharmacology*; D007295:Inositol Phosphates / Q000494:pharmacology; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D015290:Second Messenger Systems; D014638:Vanadates / Q000494:pharmacology
chemical_list: D007295:Inositol Phosphates; D014638:Vanadates; D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D006493:Heparin; C062616:inositol 2,4,5-trisphosphate; D000242:Cyclic AMP; D004110:Diltiazem; D002118:Calcium