Use of protein kinase C and phospholipase A2 inhibitors in bovine endometrial cells treated with estradiol and calcium ionophore / Uso de inibidores de proteína quinase C e fosfolipase A2 em células endometriais bovinas tratadas com estradiol e ionóforo de cálcio

The release of endometrial prostaglandin-F2α (PGF2α) in bovine females can be induced in vivo by estradiol (E2). However, its role in this mechanism has not been clarified. We hypothesized that E2 stimulates the activity and abundance of protein kinase C (PKC) and phospholipase A2 (PLA2). Our objective in this study was to analyze the effects of PKC and PLA2 inhibitors on PGF2α synthesis induced by E2 and calcium ionophore (CI) in bovine endometrial cells (BEND cells; Experiment 1). Additionally, we evaluated the abundance of PKC and PLA2 in endometrial explants of cows treated or not with E2 17 days after estrus (D17, D0 = estrus; Experiment 2). In Experiment 1, BEND cells were submitted to a PKC inhibitor (10 µM of C25H24N4O2; bisindolylmaleimide I, or BIS I), a PLA2 inhibitor (20 µM of arachydoniltrifluoromethane or AACOCF3), or none. The BEND cells were subsequently treated with E2 and CI, and PGF2α concentrations were measured in the culture medium through radioimmunoassay. For DIF-12 (PGF2α concentration 12 h after treatment subtracted from PGF2α concentration at hour 0), no PKC inhibitor effect was observed (P= 0.2709). However, DIF-12 was lower (P < 0.05) for groups treated with the PLA2 inhibitor and PLA2 inhibitor + CI + E2 groups than the control and CI + E2 groups. Thus, AACOCF3 was an efficient PLA2 inhibitor in the BEND cells culture system, and E2 did not stimulate the synthesis of PKC and PLA2. In Experiment 2, cyclic Nellore heifers received none (n = 5) or 3 mg (n = 6) of 17ß-E2 on D17 and were slaughtered 2 h after administration. The abundance of PKC and PLA2 in the endometrial tissue was evaluated using Western blotting analysis. No E2 effect was observed on PKC (P = 0.08) and PLA2 (P = 0.56). We concluded that E2 did not stimulate the activity and abundance of PKC and PLA2.(AU)

A liberação endometrial de prostaglandina-F2α (PGF2α) em fêmeas bovinas pode ser induzida in vivo pelo estradiol (E2). Entretanto o seu mecanismo de ação ainda não foi bem esclarecido. Nossa hipótese é que o E2 estimula a atividade e a abundância da proteína quinase C (PKC) e da fosfolipase A2 (PLA2). Nosso objetivo com este estudo foi analizar os efeitos de inibidores de PKC e PLA2 na síntese de PGF2α induzida por E2 e ionóforo de cálcio (CI) em células endometriais bovinas (células BEND; Experimento 1). Adicionalmente, nós avaliamos a abundância de PKC e PLA2 em explantes endometriais de vacas tratadas com ou sem E2 17 dias após o estro (D17, D0 = estro; Experimento 2). No Experimento 1, células BEND foram submetidas ao inibidor de PKC (10 µM de C25H24N4O2; bisindolylmaleimide I, ou BIS I), e ao inibidor de PLA2 (20 µM de arachydoniltrifluoromethane ou AACOCF3) ou a nenhum inibidor. As células BEND foram subsequentemente tratadas com E2 e CI e concentrações de PGF2α foram mensuradas no meio de cultura por radioimunoenssaio. Para DIF-12 (concentração de PGF2α 12 horas depois do tratamento, subtraída da concentração de PGF2α na hora 0), não foi observado efeito do inibidor de PKC (P = 0.2709). Entretanto DIF-12 foi menor (P < 0.05) nos grupos tratados com inibidor de PLA2 e inibidor de PLA2 + CI + E2 quando comparados com o grupo controle e o grupo CI + E2. O AACOCF3 foi um eficiente inibidor de PLA2 em sistema de cultura de células BEND e o E2 não estimulou a síntese de PKC e PLA2. No Experimento 2, novilhas Nelore cíclicas receberam 3 mg de 17ß-E2 (n = 6) ou nenhum tratamento (n = 5) no D17 e foram abatidas duas horas depois da administração dos tratamentos. A quantidade de PKC and PLA2 no tecido endometrial foi avaliada pela técnica de Western Blotting. Não foi observado efeito do E2 sobre a PKC (P= 0.08) e nem sobre a PLA2 (P= 0.56). Conclui-se que o E2 não estimulou a atividade e abundância de PKC e PLA2.(AU)

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.

Application of calcium ionophore A23187 in ICSI for globozoospermia: A report of 2 cases and review of the literature / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the pathogenesis of globozoospermia, fertilization ability of round-headed sperm, and the application value of assisted oocyte activation in intracytoplasmic sperm injection (ICSI) for the wives of glohozoospermia men.</p><p><b>METHODS</b>We collected oocytes from the wives of 2 globozoospermia patients and randomly divided them into two groups after ICSI to receive calcium ionophore A23187-activation and conventional treatment, respectively. We reviewed the relevant literature published at home and abroad, and discussed the etiology of globozoospermia, fertilization ability of round-headed sperm, and treatment options for this disease.</p><p><b>RESULTS</b>Quality embryos were obtained in the A23187-activation group while no fertilized oocytes, oocyte cleavage, quality embryos, or blastular formation were found in the conventional treatment group. Both women achieved pregnancy and gave birth to healthy neonates after transfer of the quality embryos from the A23187-activation group.</p><p><b>CONCLUSION</b>Calcium ionophore A23187 can be applied to ICSI for the wives of globozoospermia men and bring about desirable clinical outcomes. Meanwhile, attention should be paid to its safety.</p>

Effect of calcium on medium alkalinization induced by salicylic acid in Salvia miltiorrhiza suspension cultures / 生物工程学报

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.

Effect of oocyte activation with calcium ionophore on ICSI outcomes in teratospermia: a randomized clinical trial

Chemical activation is the most frequently used method for artificial oocyte activation [AOA], results in high fertilization rate. This prospective, randomized, unblinded, clinical study aimed to evaluate the efficiency of oocyte activation with calcium ionophore on fertilization and pregnancy rate after intracytoplasmic sperm injection [ICSI] in infertile men suffer from teratoospermia. Thirty eight women with teratoospermic partner underwent ICSI with antagonist protocol. A total of 313 metaphase 2 [M2] oocytes were randomly divided into two groups: In the oocytes of the control group [n=145], routine ICSI was applied. Oocytes in the AOA group [n=168] immediately after ICSI, were entered in culture medium supplemented with 5 Micro calcium ionophore [A23187] for 5 minutes and then washed at least five times with MOPS solution. In both groups, the fertilization was evaluated 16-18 hours after ICSI. The number of fertilized oocytes and embryos obtained were significantly different between two groups [p=0.04]. There was no significant difference between the two studied groups regarding the fertilization and cleavage rate [95.33% vs. 84.4%, p=0.11; and 89.56% vs. 87.74%, p=0.76, respectively]. Implantation rate was higher in AOA group than in control group, but the difference was not significant [17.64% vs. 7.4%, p=0.14]. No significant differences were observed in chemical and clinical pregnancy rate between groups [47.1% vs. 16.7%, p=0.07; and 41.2% vs. 16.7%; p=0.14, respectively]. We didn't find significant difference in the implantation, fertilization, cleavage and pregnancy rates between the two groups but could significantly increase the number of fertilized oocytes and embryos obtained. Finally oocyte activation with calcium ionophore may improve ICSI outcomes in infertile men suffer from teratoospermia. Further study with more cases can provide greater value

Effect of calcium ionophore A23187 plus IFN-γ on dendritic cells derived from peripheral blood mononuclear cells / 中南大学学报(医学版)

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.

Role of ADAM10 and ADAM17 in CD16b shedding mediated by different stimulators / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the main proteinases responsible for CD16b shedding under different stimulators.</p><p><b>METHODS</b>HEK293 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.</p><p><b>RESULTS</b>HEK293 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.</p><p><b>CONCLUSIONS</b>Both 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.</p>

Effects of selenium, calcium and calcium ionophore on human oocytes in vitro maturation in a chemically defined medium

In vitro maturation [IVM] of human oocytes is an emerging procedure quickly incorporated into the world of assisted reproductive technologies. As an effective method of in vitro maturation, several studies have reported the critical role of differentions on activating the complex process involved in both gamete maturation and fertilization. In this study, we supplemented a chemically defined medium with different combinations of selenium, calcium and calcium ionophore concentrations to obtain the best rate of human oocytes maturation, survival, and fertilization. As an experimental study, Three combinations of [selenium [5 microg/ml], calcium [5 microg/ml] and calcium ionophore [1 microg/ml]], [selenium [10 microg/ml], calcium [7 microg/ml] and calcium ionophore [2 microg/ml]] and [selenium [15 microg/ml], calcium [10 microg/ml] and calcium ionophore [5 microg/ml]] added to the chemically defined medium and the morphology of oocytes assessed after 22-24 hours in vitro maturation of the oocytes. The highest percentage of MII [meiosis II] oocytes [68%], developing beyond the morula [20.1%] and the blastocyst formation [11.1%] observed in oocytes treated with 15microg/ml selenium, 10microg/ml calcium and 5microg/ml calcium ionophore. Moreover, we showed the significant rate of survival in each three combinations after 36, 72 and 96 hours. Maturation and activation of oocytes may be triggered by changes in intracellular ion concentrations as second messengers in signal transduction pathways. Here, we received the highest percentage of in vitro maturation and fertilization among three combinations of selenium, calcium and calcium ionophore treatments. Using this combination of ions beside other factors might be useful for the enrichment of the human oocytes IVM medium

Calcium ionophore induced histamine and tryptase release from human mast cells / 中国应用生理学杂志

<p><b>AIM</b>To examine the ability of calcium ionophore (CI) to induce tryptase and histamine release from human mast cells and its mechanisms.</p><p><b>METHODS</b>Enzymatically 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.</p><p><b>RESULTS</b>CI 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.</p><p><b>CONCLUSION</b>Human 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.</p>

Effects of Halothane and Fentanyl Anesthesia on Coronary Endothelial Endothelin-1 Production During Myocardial Ischemia-Reperfusion in Dogs / 대한마취과학회지

Endothelin(ET), is the most potent endogenous vasoconstrictor. Myocardial ischemia and chemical stimuli including calcium ionophores are known to release ET-1. Recently, halothane has been shown to block calcium channel. Thus, halothane might attenuate coronary endothelial ET-1 production during myocardial ischemia-reperfusion. To test this hypothesis, we measured plasma ET-1 level continuously in open chest dogs subjected to 15 min of left anterior coronary arterial occlusion and 1 hour of reperfusion during fentanly(n=8) or halothane(n=7) anesthesia. The results were as follows. I) Baseline ET-1 levels of both femoral artery and great cardiac vein in the halothane group were lower than in the fentanly group(NS). 2) ET-1 level of femoral artery and great coronary vein in both halothane and fentanyl group remained unchanged 10 min into ischemia. 3) Coronary blood flow increased by 325, 250% in the halothane group and by 315, 258% in the fentanly group 2, 5 min into reperfusion, respectively. 4) ET-1 production increased from baseline of -2.9+/-1.7 pg/min to 66.0+/-21.5(p<0.05), 20.8+/-5.1 (p<0.01), 13.2+/-6.2(p<0.05) pg/min 5, 15, 30 min into reperfusion, respectively in the fentanyl group, but it remained unchanged from baseline of 0.8+/-3.1 pg/min in the halothane group. These findings suggest that ET-1 production or release is diminished by halothane during myocardial ischemia-reperfusion. Thus, halothane provides an advantage over fentanyl in patients with myocarial ischemic episodes.