calcimycin and inositol-1-3-4-5-tetrakisphosphate

Calcium release from internal stores of Entamoeba histolytica, a parasitic protozoan, was observed by measuring fluorescence of Fura-2. Emptying of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3)-sensitive calcium pools in permeabilized E. histolytica did not significantly affect subsequent calcium release by inositol(1,3,4,5)tetrakis-phosphate (Ins(1,3,4,5)P4). Similarly, prior depletion of Ins(1,3,4,5)P4-sensitive stores did not have any influence on subsequent calcium release by Ins(1,4,5)P3. The EC50 for calcium release was 0.15 microM with Ins(1,4,5)P3 and 0.68 microM with Ins(1,3,4,5)P4. In conclusion, the Ins(1,3,4,5)P4-sensitive calcium store in E. histolytica is separate and independent from the Ins(1,4,5)P3-sensitive pool.

Topics: Animals; Calcimycin; Calcium; Cell Membrane Permeability; Entamoeba histolytica; Fura-2; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Saponins

An investigation was made of the production of inositol tris- and tetrakisphosphates concomitant with ionophore-stimulated breakdown of PtdIns(4,5)P2 in ram spermatozoa. As spermatozoa displayed very low rates of incorporation of [3H]inositol into their phosphoinositides, the studies were carried out using 32P-labelled cells. Using a specially developed procedure, inositol tris- and tetrakisphosphates were isolated, free of labelled ATP and P(i); they were then separated from each other (and from other minor labelled compounds) and analysed, using ionophoresis and HPLC. Levels of 32P-labelled material with the chromatographic characteristics of Ins(1,4,5)P3 were very low in untreated cells, but rose sharply with ionophore treatment, in parallel with rapid PtdInsP2 breakdown. No 32P-labelled material with the characteristics of Ins(1,3,4,5)P4 or Ins(1,3,4)P3 was found, and there was no evidence for phosphorylation of Ins(1,4,5)P3 in sperm homogenates. The implications of our findings are discussed with respect to the physiological modulation of Ca2+ influx that is required to initiate the acrosome reaction at fertilization.

Topics: Acrosome; Animals; Calcimycin; Calcium; Chromatography, High Pressure Liquid; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Male; Phospholipids; Phosphotransferases; Phosphotransferases (Alcohol Group Acceptor); Sheep; Spermatozoa

The effects of extracellular ATP on inositol phospholipid breakdown and synthesis of eicosanoids were studied in mouse peritoneal macrophages. Addition of ATP to intact cells labelled with [3H]inositol stimulated a rapid (within 10 s) formation of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. In parallel there was also a substantial accumulation of inositol 1,3,4-trisphosphate and the monophosphate and bisphosphate derivatives of inositol. Within 10 s after the addition of 30 microM ATP there was a twofold increase in inositol trisphosphate (InsP3), which declined over 2 min. The ED50 for ATP-stimulated generation of InsP3 was approximately 12 microM. ADP and GTP showed only weak effects on InsP3 formation, while AMP and adenosine were completely ineffective at 30 microM. Furthermore, the rank order of potency of ATP analogues was ATP greater than ATP[S] greater than AdoPP[NH]P = AdoPP[CH2]P greater than AdoP[CH2]PP thus, indicating the presence of a P2y-purinergic receptor. Cells labelled with [3H]arachidonic acid showed a 50% increase of label in 1,2-diacylglycerol after 15 s upon stimulation with ATP. In parallel to the stimulation of inositol phospholipid hydrolysis, ATP also caused a marked synthesis of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) in mouse peritoneal macrophages. The rank order of potency of ATP analogues was identical with that of InsP3 generation. The effect on eicosanoid synthesis could be mimicked by the calcium ionophore A23187 and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. These results suggest that ATP-induced activation of P2y-purinergic receptors in mouse peritoneal macrophages triggers inositol phospholipid breakdown and eicosanoid synthesis.

Topics: Adenosine Triphosphate; Animals; Calcimycin; Cells, Cultured; Dinoprostone; Inositol; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Macrophages; Mice; Mice, Inbred Strains; Peritoneal Cavity; SRS-A; Structure-Activity Relationship; Tetradecanoylphorbol Acetate