OBJECTIVETo discuss the important role of actin polymerization in calcium ionophore A23187-induced human acrosome reaction and its mechanism.

METHODSEach spermatozoon specimen was divided into five groups, treated with A23187 3 micromol/L in Group A, Phalloidin 40 micromol/L and A23187 3 micromol/L in Group B, SLO 0.5 U/ml and A23187 3 micromol/L in Group C, SLO 0.5 U/ ml, Phalloidin 40 micromol/L and A23187 3 micromol/L in Group D, and nothing added in Grpup E. Then the percentage of the human acrosome reaction was assessed with Rhodamine-PSA (10 microg/ml).

RESULTSThe difference of the human spermatozoon acrosome reaction was significant (P < 0.01) among the 5 groups with or without SLO, Phalloidin and calcium ionophore A23187 but not between Groups A and B (P > 0.01).

CONCLUSIONPhalloidin does not work on the acrosome reaction of intact human spermatozoa, but in an SLO-permeabilized human spermatozoal model, it can obviously decrease the percentage of human spermatozoon acrosome reaction, which indicates that the polymerization of actin plays an important role in the course of human spermatozoon acrosome reaction, and mostly acts on the acrosome inside.

Acrosome Reaction ; drug effects ; Actins ; physiology ; Bacterial Proteins ; pharmacology ; Calcimycin ; pharmacology ; Cells, Cultured ; Humans ; Ionophores ; pharmacology ; Male ; Phalloidine ; pharmacology ; Spermatozoa ; drug effects ; physiology ; Streptolysins ; pharmacology

We studied medium alkalinization in Salvia miltiorrhiza suspension cultures treated with salicylic acid and the effect of Ca2+ in this process through application of calcium channel antagonists (Verapamil, LaCl3, LiCl, 2-APB) and ionophore A23187. The results show that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture. Verapamil and LaCl3 or LiCl and 2-APB, two different groups of calcium channel antagonist, significantly inhibited the medium alkalinization induced by salicylic acid. However, the suppression effect of verapamil or LaCl3 on medium alkalinization induced by salicylic acid was higher than that of LiCl or 2-APB. When two types of calcium channel inhibitor (LaCl3 and 2-APB) were used together, the medium alkalinization induced by salicylic acid was completely suppressed and even reduced the pH in medium. On the other hand, A23187 could promote the medium alkalinization. Based on the results above, we speculated that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture, depending on the calcium from both extracell and intracell. Moreover, calcium from extracell plays a more dominant role in this process. Reveal of relationship in this research between Ca2+ and medium alkalinization can provide theory evidence for mechanism of the plant secondary metabolism.
Calcimycin ; pharmacology ; Calcium ; chemistry ; Calcium Channel Blockers ; pharmacology ; Calcium Ionophores ; pharmacology ; Cell Culture Techniques ; Culture Media ; chemistry ; Salicylic Acid ; pharmacology ; Salvia miltiorrhiza ; metabolism ; Verapamil ; pharmacology

AIMTo examine the ability of calcium ionophore (CI) to induce tryptase and histamine release from human mast cells and its mechanisms.

METHODSEnzymatically dispersed cells from human colons were challenged with CI, and the cell supernatants after challenge were collected. Tryptase release was determined with a sandwich ELISA procedure and histamine release was measured using a glass fibre-based fluorometric assay.

RESULTSCI was able to induce a concentration dependent release of histamine and tryptase from human colon mast cells following 15 min incubation. The maximum of induced histamine and tryptase release were approximately 5.3 and 2.8 fold more than the levels of spontaneous release, respectively. CI at the concentrations higher than 1.0 micromol/L was able to induce significantly more histamine than tryptase release from mast cells. The time course revealed that the action of CI on mast cells started from 10 s, peaked at 6 min and lasted at least 15 min following incubation. Pertussis toxin and metabolic inhibitors were able to inhibit mast cell response to CI.

CONCLUSIONHuman colon mast cells were able to release tryptase and histamine in response to CI. The process seemed to be associated with the activation of a G-protein coupled receptor on the membrane of mast cells and requires cell energy supply.

Calcium Ionophores ; pharmacology ; Cells, Cultured ; Colon ; cytology ; Histamine ; metabolism ; Humans ; Mast Cells ; drug effects ; metabolism ; secretion ; Tryptases ; metabolism

The acrosome reaction is a prerequisite for fertilization, and its signaling pathway has been investigated for decades. Regardless of the type of inducers present, the acrosome reaction is ultimately mediated by the elevation of cytosolic calcium. Inositol 1,4,5-trisphosphate-gated calcium channels are important components of the acrosome reaction signaling pathway and have been confirmed by several researchers. In this study, we used a novel permeabilization tool BioPORTER^® and first demonstrated its effectiveness in spermatozoa. The inositol 1,4,5-trisphosphate type-1 receptor antibody was introduced into spermatozoa by BioPORTER^® and significantly reduced the calcium influx and acrosome reaction induced by progesterone, solubilized zona pellucida, and the calcium ionophore A23187. This finding indicates that the inositol 1,4,5-trisphosphate type-1 receptor antibody is a valid inositol 1,4,5-trisphosphate receptor inhibitor and provides evidence of inositol 1,4,5-trisphosphate-gated calcium channel involvement in the acrosome reaction in human spermatozoa. Moreover, we demonstrated that the transfer of 1,4,5-trisphosphate into spermatozoa induced acrosome reactions, which provides more reliable evidence for this process. In addition, by treating the spermatozoa with inositol 1,4,5-trisphosphate/BioPORTER^® in the presence or absence of calcium in the culture medium, we showed that the opening of inositol 1,4,5-trisphosphate-gated calcium channels led to extracellular calcium influx. This particular extracellular calcium influx may be the major process of the final step of the acrosome reaction signaling pathway.
Acrosome Reaction/physiology* ; Calcimycin/pharmacology* ; Calcium/pharmacology* ; Calcium Ionophores/pharmacology* ; Drug Delivery Systems ; Humans ; Inositol 1,4,5-Trisphosphate Receptors/metabolism* ; Male ; Progesterone/pharmacology* ; Spermatozoa/metabolism* ; Zona Pellucida/metabolism*

To compare the mediator releasability between atopic and nonatopic asthmatics, we measured basophil histamine releasability (BaHR) using a calcium-ionophore A23187 and anti-IgE in 137 subjects who were treated at Seoul National University Hospital. Subjects were categorized into atopic (group AA, n=77) or nonatopic asthmatics (group NA, n=32), or normal controls (group NC, n=28). Serum total IgE levels were determined and correlation with BaHR was assessed. Anti-IgE-induced maximal BaHR in groups AA, NA, and NC was 41.0+/-3.2, 23.1+/-4.5, and 16.8+/-3.8, respectively (mean+/-SE, %). Anti-IgE-induced BaHR in group AA was significantly higher than that in groups NA and NC (p<0.05). Calcium ionophore A23187-induced maximal BaHR was 43.1+/-2.8, 40.8+/-4.4, and 50.5+/-5.2, respectively (mean+/-SE, %), and there was no significant difference among the groups. Serum total IgE level correlated significantly with anti-IgE-induced maximal BaHR (r=0.281, p<0.01) but not with that induced by calcium ionophore A23187. In conclusion, IgE receptor-related BaHR is higher in atopic asthmatics than in nonatopic asthmatics, and this increased BaHR in atopics is significantly associated with increased serum total IgE level.
Asthma/immunology* ; Basophils/immunology* ; Basophils/drug effects ; Calcimycin/pharmacology ; Child ; Comparative Study ; Histamine Release/immunology* ; Histamine Release/drug effects ; Human ; IgE/immunology* ; IgE/blood ; Ionophores/pharmacology

The present study was carried out to examine the mechanisms of the synergistic interaction of PAF and A23187 mediated platelet aggregation. We found that platelet aggregation mediated by subthreshold concentrations of PAF (5 nM) and A23187 (1 micrometer) was inhibited by PAF receptor blocker (WEB 2086, IC50=0.65 micrometer) and calcium channel blockers, diltiazem (IC50=13 micrometer) and verapamil (IC50=18 micrometer). Pretreatment of platelets with PAF and A23187 induced rise in intracellular calcium and this effect was also blocked by verapamil. While examining the role of the down stream signaling pathways, we found that platelet aggregation induced by the co-addition of PAF and A23187 was also inhibited by low concentrations of phospholipase C (PLC) inhibitor (U73122; IC50 = 10 micrometer), a cyclooxygenase inhibitor (indomethacin; IC50=0.2 micrometer) and inhibitor of TLCK, herbimycin A with IC50 value of 5 micrometer. The effect was also inhibited by a specific TXA2 receptor antagonist, SQ 29548 with very low IC50 value of 0.05 micrometer. However, the inhibitors of MAP kinase, PD98059 and protein kinase C, chelerythrine had no effect on PAF and A23187-induced platelet aggregation. These data suggest that the synergism between PAF and A23187 in platelet aggregation involves activation of thromboxane and tyrosine kinase pathways.
Blood Platelets/*drug effects ; Calcimycin/*pharmacology ; Humans ; Indomethacin/pharmacology ; Ionophores/pharmacology ; Platelet Activating Factor/metabolism/*pharmacology ; Platelet Aggregation/*physiology ; Protein-Tyrosine Kinase/antagonists & inhibitors/*physiology ; Quinones/pharmacology ; Research Support, Non-U.S. Gov't ; Thromboxane A2/*physiology ; Verapamil/pharmacology

OBJECTIVE: To explore the effect of calcium ionophore (CI) A23187 plus IFN-γ on dendritic cells (DC) from healthy human peripheral blood mononuclear cells (PBMNC). METHODS: PBMNC from healthy donors were treated with GM-CSF plus IL-4, A23187, and A23187 plus IFN-γ, respectively. After culture for 72 h, the change of cellular morphology was observed under light microscope and electron microscope. Surface markers on DC were analyzed by flow cytometry. MTT colorimetry was used to detect the proliferation of allogeneic T cells. Plasma concentrations of IL-12 and IFN-γ were measured by ELISA. RESULTS: PBMNC treated with A23187 plus IFN-γ for 72 h presented DC with typical morphology effectively. The surface markers CD40, CD83, and CD86 were obviously increased in group A23187 plus IFN-γ (P<0.01), but decreased in CD1a (P<0.01). In addition, it evidently stimulated the proliferation of allogeneic T cells. The levels of IL-12 and IFN-γ were significantly increased compared with other groups (P<0.01). CONCLUSION A23187 plus IFN-γ can effectively enhance marked transformation of PBMNC into DC.
Calcimycin ; pharmacology ; Calcium Ionophores ; pharmacology ; Cell Proliferation ; Cells, Cultured ; Dendritic Cells ; drug effects ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Interferon-gamma ; metabolism ; pharmacology ; Interleukin-12 ; metabolism ; Interleukin-4 ; pharmacology ; Leukocytes, Mononuclear ; cytology ; T-Lymphocytes ; cytology

Unlike most secretory cells, high extra cellular calcium inhibits rather than stimulates hormonal secretion in several cells such as parathyroid cells, Juxtaglomerular cells and osteoclast. To gain further insight into the common but unique stimulus-secretion coupling mechanism in these cells, bovine parathyroid slices were incubated in various conditions of Krebs-Ringer (KR) solution containing essential amino acids. Parathyroid cells showed the inverse dependency of secretion on extra cellular calcium concentration as we expected. Ammonium acetate overcame the inhibitory effect of 2.5 mM of calcium and the maximum effect was as much as the five times of the basal value, while there was a little additive effect under 0 mM CaCl2. PTH secretion was biphasic according to the change of extra cellular osmolarity and the lowest response was observed at 300 mOsm/l. In Na-rich KR solution, high concentration of nigericin (> 10(-4)M) completely overcame the inhibitory effect of 2.5 mM CaCl2 and the maximum stimulatory effect was 8 times greater whereas it was only 2 times greater without CaCl2. In K-rich KR solution that abolished the K-gradient between the extra cellular solution and the cytoplasm, the rate of PTH secretion increased, and furthermore the addition of nigericin increased the rate of secretion significantly. The results above suggested that the osmotic swelling of the secretory vesicle in parathyroid cells might promote exocytosis as in Juxtaglomerular cells. We propose that the swelling of the vesicle is also prerequisite for secretion in several cells inhibited paradoxically by Ca++, whatever the signal transduction pathway for swelling of the secretory granules induced by the lowering of Ca++ in cytoplasm are.
Acetates/pharmacology ; Animal ; Body Fluids/*metabolism ; Cattle ; Cell Membrane Permeability ; Ionophores/pharmacology ; Mannitol/pharmacology ; Nigericin/pharmacology ; Osmosis ; Parathyroid Glands/drug effects/*secretion ; Parathyroid Hormones/*secretion ; Support, Non-U.S. Gov't

Toxoplasma gondii is an obligate intracellular protozoan parasite, which invades a wide range of hosts including humans. The exact mechanisms involved in its invasion are not fully understood. This study focused on the roles of Ca2+ in host cell invasion and in T. gondii replication. We examined the invasion and replication of T. gondii pretreated with several calcium modulators, the conoid extrusion of tachyzoites. Calmodulin localization in T. gondii were observed using the immunogold method, and Ca2+ levels in tachyzoites by confocal microscopy. In light microscopic observation, tachyzoites co-treated with A23187 and EGTA showed that host cell invasion and intracellular replication were decreased. The invasion of tachyzoites was slightly inhibited by the Ca2+ channel blockers, bepridil and verapamil, and by the calmodulin antagonist, calmidazolium. We observed that calcium saline containing A23187 induced the extrusion of tachyzoite conoid. By immunoelectron microscopy, gold particles bound to anti-calmodulin or anti-actin mAb, were found to be localized on the anterior portion of tachyzoites. Remarkably reduced intracellular Ca2+ was observed in tachyzoites treated with BAPTA/AM by confocal microscopy. These results suggest that host cell invasion and the intracellular replication of T. gondii tachyzoites are inhibited by the calcium ionophore, A23187, and by the extracellular calcium chelator, EGTA.
Animals ; Calcium/*physiology ; Calcium Channel Blockers/pharmacology ; Calmodulin/antagonists & inhibitors ; Chelating Agents/pharmacology ; Hela Cells ; Host-Parasite Relations ; Humans ; Ionophores/pharmacology ; Research Support, Non-U.S. Gov't ; Toxoplasma/drug effects/pathogenicity/*physiology

The aim of this study was to investigate the ability of calcium ionophore (CI ) to induce the differentiation of CML cells into dendritic cells (DC), to analyze the P210 expression in DCs and to evaluate the stimulatory effect of CML-DC on production of cytotoxic activity against CML cells via activating the autologous T cells. The mononuclear cells were isolated from bone marrow of CML patients whose WBC counts were more than 30x10(9)/L when samples were collected, then the lymphocytes and monocytes were discarded by pouring out supernatant twice at different culture time point. Slightly adherent cells were cultured in RPMI 1640 containing 10% FCS, with or without CI (375 ng/ml) and GM-CSF (200 ng/ml) at 37 degrees C, 5% CO2, fully humidified atmosphere for 96 hours. The cell morphology was observed under the inverted microscope and electron microscope; the expression of CD antigens was analyzed with flow cytometry; the P210 expression was measured with Western blot. LDH assay was used to evaluate the effect of cultured CML cells (CML-DC) generating cytotoxic T lymphocyte (CTL) activity against CML cells. The results indicated that after treatment with calcium ionophore and GM-CSF for 96 hours, CML cells showed DC morphological characteristics under inverted microscope and electron microscope. The expression of CD83, CD86, CD40, CD80 and HLA-DR increased remarkably. P210 was expressed in the CML-DC, but the expression level was lower than that in CML cells without CI and GM-CSF treatment. LDH assay showed that the CTL activity against CML was found greater in autologous T cells activated by CML-DC than that by CML cells. It is concluded that the CML cells can be induced to quickly differentiate into DC when cultured with CI and GM-CSF. CML-DC expresses P210, but the expression level is lower than that in CML cells. CML-DC can stimulate autologous T cells to produce CTL against CML.
Antigens, CD ; metabolism ; Bone Marrow Cells ; cytology ; Calcium ; pharmacology ; Cell Differentiation ; Dendritic Cells ; cytology ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Ionophores ; pharmacology ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; pathology ; Male ; Monocytes ; cytology ; Tumor Cells, Cultured