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Phosphatidylinositol and inositolphosphatide metabolism in hypertrophied rat heart
Published in H. Saito, Y. Yamori, M. Minami, S.H. Parvez, New Advances in SHR Research –, 2020
Hideaki Kawaguchi, Akira Kitabatake
Measurement of intracellular Ca2+. Cytosolic Ca2+ concentrations [Ca2+]¡ were also measured using fura 2-AM (acetoxymethl ester)-loaded myocytes. For loading with fura-2, cell suspensions (1 × 105 /ml) were incubated with 5 mM of fura 2-AM for 30 min in PB containing 0.1 % bovine serum albumin. Cells were then washed with Hanks’ balanced salt solution with 1.26 mM calcium and incubated in 0.7 ml of Hanks’ balanced salt solution (also containing 1.26 mM Ca2+). The ratio of excitation (340 nM) and emission (380 nm) wavelengths was monitored by fluorospectrometer. The [Ca2+]¡ was calibrated by exposing cells to 4-10 mM ionomycin followed by the addition of 6.6 mM EGTA.
Stimulus-Secretion Coupling: Intracellular Proteins and Nucleotides
Published in Stephen W. Carmichael, Susan L. Stoddard, The Adrenal Medulla 1986 - 1988, 2017
Stephen W. Carmichael, Susan L. Stoddard
Tachikawa, Takahashi, Shimizu et al. (1987) examined the effect of the specific protein kinase C inhibitor polymyxin B on secretagogue-evoked secretion of catecholamines from cultured bovine adrenal chromaffin cells. Polymyxin B inhibited the phorbol ester-induced secretion of catecholamines and also inhibited secretion induced by the calcium ionophore ionomycin and acetylcholine. Polymyxin B blocked the increase in intracellular calcium concentration induced by acetylcholine or potassium. In contrast, it did not affect the ionomycin-induced increase in calcium. They strongly suggested that catecholamine secretion induced by phorbol esters or ionomycin is mediated via activation of protein kinase C. They further indicated that, in potassium- or acetylcholine-evoked secretion, polymyxin B inhibits secretion by blocking calcium influx into the cells.
Assisted oocyte activation Current understanding, practice, and future perspectives
Published in David K. Gardner, Ariel Weissman, Colin M. Howles, Zeev Shoham, Textbook of Assisted Reproductive Techniques, 2017
Ca2+ ionophores are usually lipid-soluble molecules that transport Ca2+ across cell membranes by increas- ing Ca2+ permeability. They can cause extracellular Ca2+ influx and also act on intracellular Ca2+ stores to release stored Ca2+ (82, 83). Such agents have been shown to acti- vate oocytes from all animals examined in studies dating back to the 1970s. Examples of well-used Ca2+ ionophores include ionomycin and A23187 (Figure 14.3). Ionomycin is the most specific Ca2+ ionophore and has value for some experimental studies in that it is non-fluorescent. However, A23187 is the most commonly used ionophore to artificially activate oocytes. These agents both result in a similar and single prolonged Ca2+ rise, and they do not elicit Ca2+ oscillations (40, 83). Figure 14.4 shows an exam- ple of a Ca2+ increase in a mouse oocyte exposed to the Ca2+ ionophore ionomycin (84).
Rituximab induces a flare-up of activated neutrophil extracellular traps under in vitro conditions
Published in Immunopharmacology and Immunotoxicology, 2022
Joerg Hoffmann, Samira Roesner, Andreas Neubauer
Complete blood count was obtained from all samples (Sysmex XS-1000i, Sysmex Corporation, Kobe, Japan). Afterwards 1 ml 6% hydroxyethyl starch (Fresenius Kabi, Bad Homburg, Germany) was added to the samples. Erythrocytes were allowed to sediment for 30 min at room temperature and leukocytes were harvested from the top layer. After a washing step white blood cells were seeded in 2 ml wells of a cell culture plate (Sarstedt, Nümbrecht, Germany) with prewarmed cell culture medium (RPMI 1640, Thermo Fisher Scientific, Waltham, MA) and 2% fetal calf serum (FCS; PAN-Biotech, Aidenbach, Germany). To stimulate NET formation, leukocytes were incubated 30 min with 4 µM ionomycin I3909 (Sigma-Aldrich, St. Louis, MO) at 37 °C and 5% CO2. Parallel to ionomycin incubation the leucocytes were exposed to different drugs as follows: 10 µM dexamethasone (Dexamethason-ratiopharm®, Ratiopharm, Ulm, Germany), 5 nM acetylsalicylic acid (Aspirin®, Bayer, Leverkusen, Germany), 20 µM etoposide (Eto-GRY®, Teva, Parsippany, NJ), 20 nM bortezomib (Velcade® Janssen Pharmaceutica NV, Beerse, Belgium), or 10 µg/ml rituximab (MabThera®, Hoffman-La Roche, Basel, Switzerland). For each drug and patient positive control was tested in replicates of two. Negative controls without drug exposure were performed with ionomycin stimulation and without ionomycin stimulation.
Mycophenolate mofetil enhances the effects of tacrolimus on the inhibitory function of regulatory T cells in patients after liver transplantation via PD-1 and TIGIT receptors
Published in Immunopharmacology and Immunotoxicology, 2021
Qiang Zeng, Xiaoye Yuan, Jinglin Cao, Xin Zhao, Yang Wang, Baowang Liu, Wenpeng Liu, Zhijun Zhu, Jian Dou
A 96-well culture plate with eight wells was selected. For the 1st to the 4th wells, 3.0 × 103 cells of the Teffs suspension was added. For the 2nd to the 4th wells, 0.75 × 103 cells, 1.5 × 103 cells and 3.0 × 103 cells of the Tregs suspension was added, respectively. Thus, the mixture ratio of Tregs to Teffs in the 1st to the 4th wells were 0, 1:4, 1:2, 1:1, respectively. The 5th to the 8th wells were filled with both 3.0 × 103 cells of Teffs and Tregs. (The mixture Tregs to Teffs ratio was 1:1). Cells were treated with 50 ng/mL phorbol myristate acetate and 1.0 μg/mL ionomycin was also added into wells for 8 h. The levels of IFN-γ and TNF-α in the supernatant were measured by corresponding ELISA kits, according to the manufacturer’s instructions. Serum was added into the primary antibody-coated wells and incubated for 30 min. Cells were rinsed with buffer after serum was removed. Tetramethylbenzidine (TMB) substrate solution and stop solution were added to metabolize the colorless substrates into colored products. The absorbance was measured at a wavelength of 450 nm using a microplate reader (BioTek, Winooski, VT, USA).
The role of SCAMP5 in central nervous system diseases
Published in Neurological Research, 2022
Ye Chen, Jiali Fan, Dongqiong Xiao, Xihong Li
However, the role of SCAMP5 has only been recognized in recent years. Using human dendritic cell cDNA or SCAMP5 plasmid as a template, an expression vector was transfected into HeLa cells for analysis. Through methods such as subcellular separation and membrane vesicle immune separation, SCAMP5 was found to be triggered by ionomycin to rapidly translocate from the Golgi apparatus to the plasma membrane along the classical exocytosis pathway and promote cytokine exocytosis [15]. Cytokines can be classified into signal peptide-containing cytokines and signal peptide-free cytokines. The former includes RANTES/CCL5 and IL-6, and the latter includes IL-1β and fibroblast growth factor (FGF). Cytokines containing signal peptides are secreted through the classical pathway, that is, from the endoplasmic reticulum to the Golgi apparatus and then to the plasma membrane [24]. Cells without signal peptides rely on nonclassical pathways [25]. As a model of exocytosis, SCAMP5 can promote the exocytosis of signal peptides containing cytokines but does not have this effect on cytokines without signal peptides. Ionomycin is a calcium ionophore that can increase the level of intracellular calcium ions and then promote membrane target docking and fusion. Studies have shown that upon ionomycin stimulation, SCAMP5 can promote the release of cytokines from macrophages, and SCAMP5 together with CCL5can be quickly transported to the plasma membrane, confirming that SCAMP5 is involved in the classical secretory transport pathway [15]. Through its C-terminal tail, SCAMP5 can bind to Synaptotagmin 1 (SYT1). SYT1 is a calcium ion-bound vesicle membrane protein that acts as a calcium ion receptor during exocytosis [26]. The interaction between SCAMP5 and SYT1 may be a result of SCAMP5 participation in calcium-triggered exocytosis.