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

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

OBJECTIVETo investigate the effect and mechanism of Huanglian Jiedu Tang active fraction (HIJDTAF) on calcium overloading in neurons.

METHODCerebral ischemia was imitated by hypoxia/hypoglycemia damage on fetal rat neurons. Double wavelength fluorospectrophotometry was used to assay the content of calcium in neurons in order to evaluate the effect of HLJDTAF on calcium overloading. Neurons were treated with glutamic acid, potassium chloride (KCl), A23187, caffeine(CAF) and methacholine (Mch) to analysis the related mechanism of HLJDTAF on calcium overloading in neurons.

RESULTHLJDTAF 0.3, 0.15 g x k(-1) could remarkably inhibit the calcium overloading in neurons caused by hypoxia/hypoglycemia, glutamic acid, KCl and A23187. HLJDTAF 0.3 g x kg(-1) could inhibit the increasing of calcium caused by CAF and Mch in the presence of and in the absence of extra-calcium.

CONCLUSIONHLJDTAF could remarkably inhibit the calcium overloading in neurons after cerebral ischemia injury, it probably plays the function via several pathways.

Animals ; Caffeine ; pharmacology ; Calcimycin ; pharmacology ; Calcium ; metabolism ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Glutamic Acid ; pharmacology ; Male ; Methacholine Chloride ; pharmacology ; Neurons ; cytology ; drug effects ; metabolism ; Potassium Chloride ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Spectrometry, Fluorescence

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

OBJECTIVETo explore a simple, rapid and efficient way to generate dendritic cells from leukemic cells.

METHODSK562 cells were cultured with calcium ionosphere A23187 alone, A23187 plus GM-CSF, or a DC differentiation cocktail consisting of GM-CSF, IL-4 and TNF-alpha, respectively. The expression of surface markers of induced DCs was analyzed by flow cytometry. The K562-DCs stimulating the proliferation of allo-genetic naive T cells and inducing cytotoxicity of T cells were determined by MTT assay.

RESULTSMicroscopic examination revealed that under all the three culture conditions, K562 cells became displaying DC morphology. At 72 hours in the two culture systems containing A23187, there were higher proportions of cells with dendritic morphology [(69.5 +/- 17.2)% and (73.1 +/- 13.9)%, respectively] than that in the cocktail system [(28.5 +/- 12.3)%] (P < 0.05). And the same did when cultured for 7 days [(69.5 +/- 17.2)%, (73.1 +/- 13.9)% respectively vs (51.2 +/- 10.7)%, P < 0.05]. In the 7-day cultures, the percentage of CD1a expressing cells was lower [(8.2 +/- 2.3)% and (10.3 +/- 5.1)% vs (17.2 +/- 1.6)%, respectively] while the CD83 expressing cells was higher [(85.6 +/- 8.8)% and (82.4 +/- 9.1)% vs (77.4 +/- 12.9)%, respectively] compared with that in the cocktail system (P < 0.05). No significant difference was found in the allogeneic T cell proliferation response and induced T cell cytotoxicity between A23187 containing and cocktail groups (P > 0.05).

CONCLUSIONSA23187 treatment is a simple, rapid and efficient in vitro strategy for inducing dendritic cell from leukemic cells.

Calcimycin ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Coculture Techniques ; Dendritic Cells ; cytology ; immunology ; metabolism ; Humans ; K562 Cells ; cytology ; drug effects

OBJECTIVE: To investigate the relationship between sperm acrosome reaction (AR) and the clinical pregnancy rate of intrauterine insemination (IUI). METHODS: We detected the sperm spontaneous AR rate and Ca2+ ionophore A23187-induced AR rate in 128 patients who accepted IUI treatment, collected their clinical data and analysed the relationship between sperm AR rate and clinical pregnancy rate of IUI. RESULTS: There was no statistical difference between the spontaneous AR rates in the pregnant group and the non-pregnant group (7.7% vs. 7.0%, P>0.05), but there was statistical difference between the induced AR rates(51.9 % vs. 43.5%, P<0.05). There was statistical difference in the clinical pregnancy rate among the 3 IUI groups divided by induced AR rate (≤20.0%, 20.1%-49.9%, and ≥50.0%; 4.8% vs. 12.5% vs. 18.6%, P<0.05). CONCLUSION The spontaneous AR rate has nothing to do with the clinical pregnancy rate of IUI, but the induced AR rate is associated with the clinical pregnancy rate of IUI.
Acrosome Reaction ; drug effects ; physiology ; Adult ; Calcimycin ; pharmacology ; Female ; Humans ; Infertility ; therapy ; Insemination, Artificial, Homologous ; methods ; Male ; Pregnancy ; Pregnancy Rate ; Spermatozoa ; physiology

BACKGROUNDGlucose regulated protein 78 (GRP78), an endoplasmic reticulum (ER) chaperone, plays a critical role in chemotherapy resistance in a variety of cancers. In this study, we investigated the up-regulation of GRP78 induced by A23187 and its association with the chemotherapeutical sensibility to cisplatin in human lung cancer cell line SPCA1.

METHODSSPCA1 cells were pretreated with A23187 at different concentrations. The expression of GRP78 at the mRNA level was analyzed by RT-PCR; the expression of GRP78 at the protein level was determined by Western blotting and immunofluorescence assay. Cell survival was determined by MTT assay. Cell apoptosis was analyzed by flow cytometry.

RESULTSThe expression of GRP78 at both the mRNA and protein levels was obviously induced by A23187 in SPCA1 cells, with an elevation of GRP78 by 2.1-fold at the mRNA level and by 3.8-fold at the protein level compared to the control. There was a dose-dependent response. Survival curve analysis demonstrated that A23187 induction caused a significant reduction of survival for the cells subjected to cisplatin treatment (P < 0.05). After treatment by cisplatin, the percentage of apoptotic cells in the A23187 pretreated group increased about three fold compared with the control group ((27.53 ± 4.32)% vs. (9.25 ± 3.64)%, P < 0.05).

CONCLUSIONSA23187 treatment was fairly effective for the induction of GRP78 in SPCA1 cells at both the mRNA and protein levels. To a certain extent, GRP78 up-regulation by A23187 was associated with the enhancement of drug sensitivity to cisplatin in human lung cancer cell line SPCA1.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; Calcimycin ; pharmacology ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Flow Cytometry ; Heat-Shock Proteins ; genetics ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction