Participation of the inositol 1,4,5-trisphosphate-gated calcium channel in the zona pellucida- and progesterone-induced acrosome reaction and calcium influx in human spermatozoa / 亚洲男科学杂志(英文版)

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.

Protein kinase A inhibition induces EPAC-dependent acrosomal exocytosis in human sperm / 亚洲男科学杂志(英文版)

To interact with the egg, the spermatozoon must undergo several biochemical and motility modifications in the female reproductive tract, collectively called capacitation. Only capacitated sperm can undergo acrosomal exocytosis, near or on the egg, a process that allows the sperm to penetrate and fertilize the egg. In the present study, we investigated the involvement of cyclic adenosine monophosphate (cAMP)-dependent processes on acrosomal exocytosis. Inhibition of protein kinase A (PKA) at the end of capacitation induced acrosomal exocytosis. This process is cAMP-dependent; however, the addition of relatively high concentration of the membrane-permeable 8-bromo-cAMP (8Br-cAMP, 0.1 mmol l-1) analog induced significant inhibition of the acrosomal exocytosis. The induction of acrosomal exocytosis by PKA inhibition was significantly inhibited by an exchange protein directly activated by cAMP (EPAC) ESI09 inhibitor. The EPAC selective substrate activated AE at relatively low concentrations (0.02-0.1 μmol l-1), whereas higher concentrations (>5 μmol l-1) were inhibitory to the AE induced by PKA inhibition. Inhibition of PKA revealed about 50% increase in intracellular cAMP levels, conditions under which EPAC can be activated to induce the AE. Induction of AE by activating the actin severing-protein, gelsolin, which causes F-actin dispersion, was inhibited by the EPAC inhibitor. The AE induced by PKA inhibition was mediated by phospholipase C activity but not by the Ca2+-channel, CatSper. Thus, inhibition of PKA at the end of the capacitation process induced EPAC/phospholipase C-dependent acrosomal exocytosis. EPAC mediates F-actin depolymerization and/or activation of effectors downstream to F-actin breakdown that lead to acrosomal exocytosis.

Transglutaminase 2 inhibits apoptosis induced by calciumoverload through down-regulation of Bax

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.

Calcium-influx increases SOD1 aggregates via nitric oxide in cultured motor neurons

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.

Inhibition of tryptase release from human colon mast cells by histamine receptor antagonists.

The main objective of this study was to investigate the ability of histamine receptor antagonists to modulate tryptase release from human colon mast cells induced by histamine. Enzymatically dispersed cells from human colon were challenged with histamine in the absence or presence of the histamine receptor antagonists, and the tryptase release was determined. It was found that histamine induced tryptase release from colon mast cells was inhibited by up to approximately 61.5% and 24% by the H1 histamine receptor antagonist terfenadine and the H2 histamine receptor antagonist cimetidine, respectively, when histamine and its antagonists were added to cells at the same time. The H3 histamine receptor antagonist clobenpropit had no effect on histamine induced tryptase release from colon mast cells at all concentrations tested. Preincubation of terfenadine, cimetidine or clobenpropit with cells for 20 minutes before challenging with histamine did not enhance the ability of these antihistamines to inhibit histamine induced tryptase release. Apart from terfenadine at 100 microg/ml, the antagonists themselves did not stimulate tryptase release from colon mast cells following both 15 minutes and 35 minutes incubation periods. It was concluded that H1 and H2 histamine receptor antagonists were able to inhibit histamine induced tryptase release from colon mast cells. This not only added some new data to our hypothesis of self-amplification mechanisms of mast cell degranulation, but also suggested that combining these two types of antihistamine drugs could be useful for the treatment of inflammatory bowel disease (IBD).

Involvement of throm box aneA2 and tyrosine kinase in the synergistic interaction of platelet activating factor and calcium ionophore A23187 in human platelet aggregation

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.

Sox-4 is a positive regulator of Hep3B and HepG2 cells' apoptosis induced by prostaglandin (PG)A2 and 12-PGJ2

We reported earlier that expression of Sox-4 was found to be elevated during prostaglandin (PG) A2 and delta(12)-PGJ(12) induced apoptosis in human hepatocarcinoma Hep3B cells. In this study, the role of Sox-4 was examined using human Hep3B and HepG2 cell lines. Sox-4 induction by several apoptotic inducer such as A23187 (Ca(2+) ionophore) and etoposide (topoisomerase II inhibitor) and Sox-4 transfection into the cells were able to induce apoptosis as observed by the cellular DNA fragmentation. Antisense oligonucleotide of Sox-4 inhibited the induction of Sox-4 expression and blocked the formation of DNA fragmentation by PGA(2) and delta(12)-PGJ(12) in Hep3B and HepG2 cells. Sox-4-induced apoptosis was accompanied with caspase-1 activation indicating that caspase cascade was involved in this apoptotic pathway. These results indicate that Sox-4 is involved in Hep3B and HepG2 cells apoptosis as an important apoptotic mediator.

Comparative functional characterization of mouse bone marrow-derived mast cells and peritoneal mast cells in response to non-immunological stimuli.

The cultured mouse mast cells that are dependent on spleen-derived factor for their proliferation and maintenance and have been shown to be similar to mucosal mast cells in terms of their T-cell dependence and histochemical staining characteristics. Mast cell heterogeneity has been confirmed by functional characterization of mouse bone marrow-derived mast cells (MBMMC) and mouse peritoneal mast cells (MPMCs). MPMCs released around 30% of histamine when stimulated with compound 48/80 whereas MBMMC were almost unresponsive to the same stimulus. Calcium Ionophore A23187 on the other hand, released histamine in dose-dependent manner from MBMMC. The study was undertaken to investigate the effect of antiallergic drug, disodium cromoglycate (DSCG), a synthetic cromone and quercetin, a plant-derived flavonoid on Ca ionophore A23187 induced histamine release from MBMMC. MBMMCs were almost unresponsive to DSCG whereas Ca Ionophore induced histamine release was blocked by Quercetin. The results indicate that response of mast cells at one anatomic site to a given stimulus does not necessarily predict the response of mast cells at a different anatomic location to the same stimulus. It shows functional heterogeneity within a single species. So, it cannot be assumed that antiallergic compounds stabilizing mast cells in one tissue site or organ will be equally efficacious against mast cells in other sites.

Comparison of basophil histamine releasability between atopic and nonatopic thmatics

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.

Cell-mediated immunosuppression in mice by street rabies virus not restored by calcium ionophore or PMA.

It is known that rabies virus can suppress the host immune system. In this study we demonstrate a depression of cell-mediated immunity in mice, peripherally infected with Thai street rabies virus. The cell-mediated cytolysis of spleen cells from mice increased transiently on day 5 after infection and declined rapidly thereafter until death. The proliferation of spleen cells stimulated with a T-cell mitogen such as phytohemagglutinin or concanavalin A, was significantly suppressed during the course of infection. There was also a marked suppression of IL-2 secretion in parallel with a decrease of the T-cell proliferative response to mitogen. The suppression of T-cell proliferation was not restored by treatment with a calcium ionophore (A 23187) or phorbol 12-myristate-13 acetate (PMA).

Effect of Ca(2+)-channel blockers, Ca(2+)-ionophore and increased pyrene excimer formation on the microsomal glucose-6-phosphatase.

The dependence of microsomal glucose-6-phosphatase (G-6-Pase) activity on Ca2+ as well as the membrane lipid microviscosity was studied by the effect of Ca(2+)-channel blockers (namely verapamil and nifedipine), Ca(2+)-ionophore, A23187 and pyrene excimer formation. Channel blockers depressed the G-6-Pase and Ca(2+)-ATPase while the ionophore increased these activities. Dimethyl sulfoxide, a known membrane surface active agent showed no change. Ca(2+)-uptake into the membrane has expectedly been lowered by the channel blockers while the ionophores facilitated the ion flux. Excimer formation of the fluorescent probe, pyrene as an indicator of increased membrane fluidity, and microviscosity calculated from there on, showed that Ca(2+)- and lipid microenvironment in the membrane significantly influenced the activity of G-6-Pase. Membrane lipid composition such as phospholipid/cholesterol molar ratio which also indicates an increased membrane fluidity is markedly increased with the ionophore but decreased with the channel blockers, while protein/phospholipid ratio remained unchanged. Microsomal G-6-Pase is a multicomponent multifunctional protein. It is argued that Ca2+ may play the role of an obligatory cofactor not only for the hydrolysis of G-6-P (catalytic part of the enzyme) but also involved in the regulation of substrate and product transport in or out of the endoplasmic reticulum lumen.

Heparan sulfate proteoglycan and control of cell proliferation: enhanced synthesis induced by phorbol ester (PMA) during G1-phase

The effect of phorbol 12-myristate-13-acetate (PMA), a tumor-promoting phorbol ester, on the synthesis of proteoglycans of endothelial cells in culture was investigated. This phorbol activates protein kinase C (PKC) when added to cells in culture. PKC, in turn, modulates the activity of growth factors. Using [35S]-sulfate or [3H]-glucosamine to label the proteglycans we have observed a 4-24-fold increase of the heparan sulfate (HS) synthesis in a dose-dependent manner (0-100 ng/ml). Chondroitin sulfate (CS) synthesis was not affected by PMA. The effect of PMA could be completely abolished by a calcium ionophore (A23187). By the use of synchronized cells and PMA pulses at different periods of the cell cycle, as well as [3H]-thymidine incorporation, we were able to show that the enhancement of heparan sulfate synthesis is most prominent during G1. Our data suggest that the release of HS to the medium could be one of the responses of the cell to a mitogenic stimulus

Role of calcium in secretion of chorionic gonadotropin by first trimester human placenta.

The role of calcium in regulation of secretion of human chorionic gonadotropin (hCG) by first trimester human placental minces in vitro has been investigated. Depletion of calcium in the medium by addition of EGTA resulted in a drastic decrease in the levels of immunoreactive hCG in the medium with consequent of accumulation of hCG in the tissue. Addition of A 23187 which is a calcium ionophore resulted in a dose dose dependent increase in the hCG in the medium and this stimulatory response could not be observed in the absence of calcium. Use of lanthanum (a calcium antagonist) in place of calcium in the medium used resulted in a significant decrease in the levels of hCG in the medium. Addition of veratridine (a sodium channel activator) stimulated hCG secretion in a dose dependent manner. These results suggest that calcium is essential for normal secretion of hCG by human placenta.

Membrane skeletal protein structure and interactions in human erythrocytes after their treatment with diamide and calcium.

To analyse the role of native structures of membrane proteins in their structural modifications induced by the elevated intracellular free Ca2+ levels, we have studied the Ca(2+)-mediated effects on membrane skeletal proteins in human erythrocytes that were loaded with Ca2+ using the ionophore A23187 after their pretreatment with the sulphydryl oxidizing agent, diamide. The diamide treatment not only induced polymerization of the major membrane skeletal protein, spectrin, in the erythrocytes, but it also promoted intersubunit crosslinking within the tetramers and dimers of this protein. Loading of these diamide-treated cells with Ca2+ failed to induce significant structural modifications of spectrin as well as polypeptide 4.1, another major membrane skeletal protein, as compared to the erythrocytes that were loaded with Ca2+ without the diamide pretreatment. These results have been interpreted to suggest that the Ca(2+)-induced membrane skeletal protein changes in erythrocytes depend on both the shape and relative orientation of these proteins within the membrane skeleton.