MYOINOSITOL TRISPHOSPHATE-MEDIATED CALCIUM-RELEASE FROM INTERNAL STORES OF ENTAMOEBA-HISTOLYTICA

Calcium mobilisation from internal stores of the parasitic protozoan E ntamoeba histolytica was studied by fluorescence measurements of the c alcium indicator quin 2 and Ca-45(2+) incorporation studies in saponin -permeabilised amoebae. Prior energy-dependent calcium sequestration w as 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 a ddition of a lower concentration of Ins(1,4,5)P-3. cAMP failed to infl uence inositol trisphosphate induced calcium release, indicating the a bsence of control mechanisms through cAMP-dependent phosphorylation. G TP neither induced calcium release nor could potentiate inositol trisp hosphate mediated calcium mobilisation. A saturating concentration of Ins(1,4,5)P-3 could release 50% of radiolabelled calcium sequestered b y energy-dependent mechanisms in E. histolytica. The energy-dependent calcium sequestration was inhibited by vanadate and the calcium antago nist Diltiazem but not by dicyclohexylcarbodiimide (DCCD), suggesting the involvement of an endoplasmic reticulum-like structure in calcium storage. Binding studies showed specific association of [H-3]Ins(1,4,5 )P-3 to crude membrane fractions of E. histolytica, which was signific antly inhibited by heparin in a dose-dependent manner. IC50 (concentra tion which produced half-maximal inhibition) values for displacement o f 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 mu M for bath isomers. Our res ults suggested that Ins(1,4,5)P-3-mediated calcium release from intern al stores of E. histolytica most probably occurred in an inositol tris phosphate receptor-dependent manner.