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*

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

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

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

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

OBJECTIVETo investigate the main proteinases responsible for CD16b shedding under different stimulators.

METHODSHEK293 cell line stably expressing CD16b was constructed by lentivirus system. The cell line was then overexpressed with a disintegrin and metalloproteinase 10 (ADAM10) or ADAM17, suppressed with short hairpin RNA of ADAM10 or ADAM17, and reconstituted with ADAM10 or ADAM17, respectively. After each treatment, the cell line was stimulated with ionomycin or phorbol 12-myristate- 13-acetate (PMA) for 12 hours. The soluble CD16b released from cell membrane was detected by immunoprecipition and immunoblot. Quantitation was then implemented to compare the amount of soluble CD16b in cell supernatant after stimulation.

RESULTSHEK293 cell line stably expressing CD16b was successfully established. When CD16b expressing cell line was overexpressed with ADAM10, shedding of CD16b was increased after stimulation with ionomycin but not PMA; when the cell line overexpressed with ADAM17, shedding of CD16b was increased after stimulation with PMA but not ionomycin. Similarly, when ADAM10 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with ionomycin; when ADAM17 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with PMA. The shedding of CD16b was increased again when CD16b expressing cell line was reconstituted with ADAM10 and stimulated by ionomycin or reconstituted with ADAM17 and stimulated by PMA.

CONCLUSIONSBoth ADAM10 and ADAM17 could shed CD16b, but they possess differed preferences. ADAM10 is the main sheddase under stimulation of ionomycin, while ADAM17 is the main sheddase under stimulation of PMA.

ADAM Proteins ; genetics ; metabolism ; physiology ; ADAM10 Protein ; ADAM17 Protein ; Amyloid Precursor Protein Secretases ; genetics ; metabolism ; physiology ; Calcium Ionophores ; pharmacology ; Carcinogens ; pharmacology ; Cells, Cultured ; Drug Evaluation, Preclinical ; GPI-Linked Proteins ; metabolism ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Ionomycin ; pharmacology ; Membrane Proteins ; genetics ; metabolism ; physiology ; Protein Processing, Post-Translational ; drug effects ; Protein Transport ; drug effects ; Proteolysis ; drug effects ; Receptors, IgG ; metabolism ; Tetradecanoylphorbol Acetate ; pharmacology ; Transfection

An abrupt increase of intracellular Ca2+ is observed in cells under hypoxic or oxidatively stressed conditions. The dysregulated increase of cytosolic Ca2+ triggers apoptotic cell death through mitochondrial swelling and activation of Ca2+-dependent enzymes. Transglutaminase 2 (TG2) is a Ca2+-dependent enzyme that catalyzes transamidation reaction producing cross-linked and polyaminated proteins. TG2 activity is known to be involved in the apoptotic process. However, the pro-apoptotic role of TG2 is still controversial. In this study, we investigate the role of TG2 in apoptosis induced by Ca2+-overload. Overexpression of TG2 inhibited the A23187-induced apoptosis through suppression of caspase-3 and -9 activities, cytochrome c release into cytosol, and mitochondria membrane depolarization. Conversely, down-regulation of TG2 caused the increases of cell death, caspase-3 activity and cytochrome c in cytosol in response to Ca2+-overload. Western blot analysis of Bcl-2 family proteins showed that TG2 reduced the expression level of Bax protein. Moreover, overexpression of Bax abrogated the anti-apoptotic effect of TG2, indicating that TG2-mediated suppression of Bax is responsible for inhibiting cell death under Ca2+-overloaded conditions. Our findings revealed a novel anti-apoptotic pathway involving TG2, and suggested the induction of TG2 as a novel strategy for promoting cell survival in diseases such as ischemia and neurodegeneration.
*Apoptosis ; Apoptosis Regulatory Proteins/metabolism ; Calcimycin/pharmacology ; Calcium/*metabolism ; Caspases/metabolism ; Cell Death ; Cell Survival ; Cytochromes c/metabolism ; Down-Regulation ; GTP-Binding Proteins/*metabolism ; HEK293 Cells ; Hela Cells ; Humans ; Ionophores/pharmacology ; Mitochondria/metabolism ; Transglutaminases/*metabolism ; bcl-2-Associated X Protein/genetics/*metabolism

Familial amyotrophic lateral sclerosis (fALS) is caused by mutations in Cu/Zn-superoxide dismutase (SOD1), and SOD1 aggregation and calcium toxicity are involved in neuronal death. However, the effect of altered calcium homeostasis on the SOD1 aggregation is unknown. To investigate whether calcium triggers mutant SOD1 aggregation in vitro, human mutant SOD1 (G93A) was transfected into motor neuronal cell line (VSC 4.1 cells). These cells were then treated with calcium ionophore A23187 or agents that induce intracellular calcium release like cyclic ADP ribose, ryanodine or thapsigargin. A23187 was found to increase mutant SOD1 aggregation and neuronal nitric oxide synthase (nNOS) expression. Moreover, the NOS inhibitor (L-NAME) and a NO-dependent cyclic GMP cascade inhibitor (ODQ) reduced SOD1 aggregation, whereas an exogenous NO donor (GSNO) increased mutant SOD1 aggregation, which was also prevented by NOS or cGMP cascade inhibitor. Our data demonstrate that calcium-influx increases SOD1 aggregation by upregulating NO in cultured motor neuronal cells.
Amyotrophic Lateral Sclerosis/genetics/metabolism ; Animals ; Calcimycin/pharmacology ; Calcium/*metabolism ; Calpain/metabolism ; Caspase 3/metabolism ; Cell Line ; Humans ; Ionophores/pharmacology ; Motor Neurons/*metabolism ; Multiprotein Complexes ; Mutation ; Nitric Oxide/*metabolism ; Rats ; Recombinant Proteins/chemistry/genetics/metabolism ; Superoxide Dismutase/chemistry/genetics/*metabolism ; Transfection