scholarly journals Calcium ionophore A23187 as a regulator of gene expression in mammalian cells.

1986 ◽  
Vol 103 (6) ◽  
pp. 2145-2152 ◽  
Author(s):  
E Resendez ◽  
J Ting ◽  
K S Kim ◽  
S K Wooden ◽  
A S Lee
Keyword(s):  
Mammalian Cells ◽  
De Novo ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Similar Response ◽  
Calmodulin Antagonist ◽  
Induction Process ◽  
Continuous Presence ◽  
Lower Magnitude

The calcium ionophore A23187 can reversibly induce the expression of two glucose-regulated genes, p3C5 and p4A3. This induction requires a continuous presence of the ionophore for over 2 h. Although extracellular Ca2+ is important for the optimal effect of A23187, it is not necessary for the induction, since a similar response with a lower magnitude can be triggered in cells cultured in low Ca2+ medium buffered with EGTA. Both the basal and induced levels of p3C5 and p4A3 transcripts can be modulated by the calmodulin antagonist W-7, indicating the involvement of Ca2+/calmodulin-associated pathways. In addition, the sensitivity of the A23187 induction to cycloheximide suggests that the induction process is dependent on de novo protein synthesis.

scholarly journals Endothelial Cell “Memory” of Inflammatory Stimulation: Human Venular Endothelial Cells Store Interleukin 8 in Weibel-Palade Bodies

1998 ◽  
Vol 188 (9) ◽  
pp. 1757-1762 ◽  
Author(s):  
Barbara Wolff ◽  
Alan R. Burns ◽  
James Middleton ◽  
Antal Rot
Keyword(s):  
Endothelial Cells ◽  
Secretory Pathway ◽  
De Novo ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Inflammatory Stimuli ◽  
Regulated Secretory Pathway ◽  
Von Willebrand ◽  
Willebrand Factor

The expression and secretion of interleukin (IL)-8, the prototype member of the C-X-C subfamily of chemokines, can be induced by diverse inflammatory stimuli in many cells, including endothelial cells (EC). Upon de novo synthesis, IL-8 localizes intracellularly in the Golgi apparatus, from where it is secreted. In addition to this constitutive secretory pathway, we describe a depot storage and separate regulated secretory pathway of IL-8 in EC. The prolonged stimulation of primary human EC with inflammatory mediators resulted in the accumulation of IL-8 in Weibel-Palade bodies, where it colocalized with von Willebrand factor. IL-8 was retained in these storage organelles for several days after the removal of the stimulus and could be released by EC secretagogues such as phorbol myristate acetate, the calcium ionophore A23187, and histamine. These findings suggest that storage of IL-8 in Weibel-Palade bodies may serve as the EC “memory” of a preceding inflammatory insult, which then enables the cells to secrete IL-8 immediately without de novo protein synthesis.

The role of calcium and calmodulin in mediating release of thyrotrophin-releasing hormone by cultured hypothalamic cells

1987 ◽  
Vol 115 (2) ◽  
pp. 255-262 ◽  
Author(s):  
M. D. Lewis ◽  
S. M. Foord ◽  
M. F. Scanlon
Keyword(s):  
Cell Cultures ◽  
Calcium Influx ◽  
Calcium Ionophore ◽  
Reversed Phase ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Calmodulin Antagonist ◽  
Thyrotrophin Releasing Hormone ◽  
Fetal Rat ◽  
Basal Release

ABSTRACT We have developed a fetal rat hypothalamic cell culture system for the study of factors controlling the acute release of TRH. Release of TRH by the cells has been characterized by reversed-phase high pressure liquid chromatography and about 86% of the total immunoreactivity in the medium co-eluted with synthetic TRH. Release of TRH by the cells in response to 56 mmol K+/l increased between days 5 and 9 of culture but reached a plateau thereafter. Cell contents of TRH did not change significantly between days 5 and 14 of culture. Release of TRH from the cells was stimulated by K+ (56 mmol/l), veratridine (100 μmol/l) and ouabain (100 μmol/l) to 550, 480 and 335% of basal release respectively over a 1-h period. Release of TRH was dependent upon calcium in that it was absent when calcium-free medium was used and could be blocked by verapamil (20 μmol/l); however it could not be blocked by nifedipine (50 μmol/l). The calcium ionophore A23187 (1 μmol/l) stimulated TRH release to 340% of basal release. Tetrodotoxin (1 μmol/l) completely abolished the release in response to veratridine but had no effect on the release stimulated by K+ (56 mmol/l). The calmodulin antagonists trifluoperazine and triflupromazine (50 μmol/l) inhibited veratridine-stimulated TRH release. This was at a site after calcium influx as they also inhibited A23187-stimulated TRH release. The highly specific calmodulin antagonist W7 (10 μmol/l) also inhibited both veratridine and A23187-stimulated TRH release whereas, at the same concentration, its inactive analogue W5 did not significantly inhibit TRH release in response to either stimulus. These results confirm that fetal rat hypothalamic cell cultures release authentic TRH which can be stimulated by a number of depolarizing agents. Calcium is essential for depolarization-induced release which is also dependent on calmodulin. Fetal rat hypothalamic cell cultures are a valid model for the study of factors controlling the release of TRH. J. Endocr. (1987) 115, 255–262

scholarly journals Induction and Signaling of an Apoptotic Response Typified by DNA Laddering in the Defense Response of Oats to Infection and Elicitors

2001 ◽  
Vol 14 (4) ◽  
pp. 477-486 ◽  
Author(s):  
Yasuomi Tada ◽  
Shingo Hata ◽  
Yoko Takata ◽  
Hitoshi Nakayashiki ◽  
Yukio Tosa ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dna Cleavage ◽  
De Novo ◽  
Calcium Ionophore ◽  
Cysteine Proteases ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Dna Laddering ◽  
Chitin And Chitosan ◽  
Induced Apoptosis

Cells in the primary leaves of oats displayed internucleosomal DNA cleavage in response to incompatible crown rust infection. DNA laddering also was evident in leaves treated with calcium ionophore A23187, nonspecific elicitors such as chitin and chitosan oligomers, and victorin, which functions as a specific elicitor in Pc-2/Vb containing oat leaves. The nuclei in a victorin-treated susceptible oat line were positive for the TUNEL assay. These elicitors clearly induced a 28-kDa nuclease (p28) in addition to three constitutive nucleases of 33, 24, and 22 kDa. Activation of p28 preceded the appearance of DNA laddering and possibly was mediated by de novo synthesis and/or cysteine protease activity. Pharmacological studies showed that the induction of DNA laddering was associated with oxidative stress, Ca2+ influx, and serine and cysteine proteases. Protein kinase and calmodulin activities did not seem to be involved in the induction of DNA laddering by victorin, whereas kinase-mediated signals were involved in DNA laddering induced by A23187. Protein kinase, calmodulin, G-protein activities, and Ca2+ influx, however, are involved in phytoalexin production. Our results imply that p28 is a possible nuclease candidate responsible for the induction of DNA laddering. The results also demonstrated that the mediators involved in the induction of apoptosis depended on the type of stimuli, whereas p28 and serine and cysteine proteases commonly are associated with each elicitor-induced apoptosis.

Transcriptional activation of the glucose-regulated protein genes and their heterologous fusion genes by beta-mercaptoethanol

1987 ◽  
Vol 7 (8) ◽  
pp. 2974-2976
Author(s):  
Y K Kim ◽  
A S Lee
Keyword(s):  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Protein Glycosylation ◽  
Temperature Sensitive ◽  
Ionophore A23187 ◽  
Grp78 Promoter ◽  
Steady State Levels ◽  
Tata Sequence

The sulfhydryl-reducing agent beta-mercaptoethanol preferentially stimulates the synthesis of glucose-regulated proteins (GRPs) in mammalian cells. The rapid and large increase in GRPs is due to transcriptional activation of GRP94 and GRP78 genes, resulting in a rapid increase in the steady-state levels of GRP transcripts. From analysis of 5'-deletion mutants, the region of beta-mercaptoethanol responsiveness in the GRP78 promoter was mapped within 450 nucleotides upstream of the TATA sequence. This same general region was demonstrated to be important for induction of the GRP78 gene by the calcium ionophore A23187, glucose starvation, and a temperature-sensitive mutation in a K12 cell line defective in protein glycosylation.

scholarly journals Transcriptional activation of the glucose-regulated protein genes and their heterologous fusion genes by beta-mercaptoethanol.

1987 ◽  
Vol 7 (8) ◽  
pp. 2974-2976 ◽  
Author(s):  
Y K Kim ◽  
A S Lee
Keyword(s):  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Protein Glycosylation ◽  
Temperature Sensitive ◽  
Ionophore A23187 ◽  
Grp78 Promoter ◽  
Steady State Levels ◽  
Tata Sequence

The sulfhydryl-reducing agent beta-mercaptoethanol preferentially stimulates the synthesis of glucose-regulated proteins (GRPs) in mammalian cells. The rapid and large increase in GRPs is due to transcriptional activation of GRP94 and GRP78 genes, resulting in a rapid increase in the steady-state levels of GRP transcripts. From analysis of 5'-deletion mutants, the region of beta-mercaptoethanol responsiveness in the GRP78 promoter was mapped within 450 nucleotides upstream of the TATA sequence. This same general region was demonstrated to be important for induction of the GRP78 gene by the calcium ionophore A23187, glucose starvation, and a temperature-sensitive mutation in a K12 cell line defective in protein glycosylation.

Calcium, calmodulin, and the production of prostacyclin by cultured vascular endothelial cells

1983 ◽  
Vol 3 (11) ◽  
pp. 1007-1015 ◽  
Author(s):  
J. M. Seid ◽  
S. Macneil ◽  
S. Tomlinson
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Vascular Endothelial ◽  
Calmodulin Antagonist ◽  
Aortic Endothelial Cells ◽  
Intracellular Calcium Mobilization ◽  
Porcine Aortic Endothelial Cells

The production of prostacyclin (PGI2) by cultured porcine aortic endothelial cells, in response to serum and the calcium ionophore A23187, was inhibited by TMB-8, an antagonist of intracellular calcium mobilization. The calcium-channel blocker methoxyverapamil (D600) inhibited serum-induced PGI2 production in but had little effect on A23187-induced PGI2 production. Calmodulin activity was detected in endothelial-cell Jysates and was inhibited by the calmodulin antagonist W7, which also inhibited PGI2 production in response to both agonists. Calcium and calmodulin appear to play an important role in mediating PGI2 production by the vascular endothelium.

A23187 stimulates translocation of 5-lipoxygenase from cytosol to membrane in human alveolar macrophages

1992 ◽  
Vol 262 (4) ◽  
pp. L454-L458
Author(s):  
R. J. Pueringer ◽  
C. C. Bahns ◽  
M. M. Monick ◽  
G. W. Hunninghake
Keyword(s):  
Alveolar Macrophages ◽  
Membrane Fraction ◽  
De Novo ◽  
Calcium Ionophore ◽  
Leukotriene B4 ◽  
Calcium Ionophore A23187 ◽  
Eicosatetraenoic Acid ◽  
Ionophore A23187 ◽  
Protein Synthesis Inhibitors ◽  
Human Alveolar Macrophages

Human alveolar macrophages stimulated with the calcium ionophore A23187 selectively release large amounts of leukotriene B4 (LTB4) and (+/-)-5-hydroxy-(6E, 8Z-11Z, 14Z)-eicosatetraenoic acid. To determine whether LTB4 release by human alveolar macrophages following A23187 stimulation required the de novo production of 5-lipoxygenase, alveolar macrophages were stimulated under conditions that would preclude a role for new enzyme production. We found that A23187-stimulated alveolar macrophages release LTB4 within 10 min following stimulation, that LTB4 release is not inhibited by protein synthesis inhibitors, and that release of LTB4 does not correlate with the de novo synthesis of the first committed enzyme, 5-lipoxygenase. In contrast, LTB4 release correlated with the translocation of 5-lipoxygenase from the cytosol to the membrane fraction of the cells following A23187 stimulation and was inhibited by MK-886. These findings show that A23187 stimulation of alveolar macrophages results in translocation of a preexistent 5-lipoxygenase from the cytosol to the membrane fraction of the cell and that this translocation of 5-lipoxygenase is associated with release of LTB4 from the cells.

scholarly journals 12-LOX catalyzes the oxidation of 2-arachidonoyl-lysolipids in platelets generating eicosanoid-lysolipids that are attenuated by iPLA2γ knockout

2020 ◽  
Vol 295 (16) ◽  
pp. 5307-5320 ◽  
Author(s):  
Xinping Liu ◽  
Harold F. Sims ◽  
Christopher M. Jenkins ◽  
Shaoping Guan ◽  
Beverly G. Dilthey ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Oxidation Products ◽  
Therapeutic Interventions ◽  
Enzymatic Oxidation ◽  
Ionophore A23187 ◽  
Genetic Ablation ◽  
Age Related ◽  
12 Lipoxygenase

The canonical pathway of eicosanoid production in most mammalian cells is initiated by phospholipase A2-mediated release of arachidonic acid, followed by its enzymatic oxidation resulting in a vast array of eicosanoid products. However, recent work has demonstrated that the major phospholipase in mitochondria, iPLA2γ (patatin-like phospholipase domain containing 8 (PNPLA8)), possesses sn-1 specificity, with polyunsaturated fatty acids at the sn-2 position generating polyunsaturated sn-2-acyl lysophospholipids. Through strategic chemical derivatization, chiral chromatographic separation, and multistage tandem MS, here we first demonstrate that human platelet-type 12-lipoxygenase (12-LOX) can directly catalyze the regioselective and stereospecific oxidation of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) and 2-arachidonoyl-lysophosphatidylethanolamine (2-AA-LPE). Next, we identified these two eicosanoid-lysophospholipids in murine myocardium and in isolated platelets. Moreover, we observed robust increases in 2-AA-LPC, 2-AA-LPE, and their downstream 12-LOX oxidation products, 12(S)-HETE-LPC and 12(S)-HETE-LPE, in calcium ionophore (A23187)-stimulated murine platelets. Mechanistically, genetic ablation of iPLA2γ markedly decreased the calcium-stimulated production of 2-AA-LPC, 2-AA-LPE, and 12-HETE-lysophospholipids in mouse platelets. Importantly, a potent and selective 12-LOX inhibitor, ML355, significantly inhibited the production of 12-HETE-LPC and 12-HETE-LPE in activated platelets. Furthermore, we found that aging is accompanied by significant changes in 12-HETE-LPC in murine serum that were also markedly attenuated by iPLA2γ genetic ablation. Collectively, these results identify previously unknown iPLA2γ-initiated signaling pathways mediated by direct 12-LOX oxidation of 2-AA-LPC and 2-AA-LPE. This oxidation generates previously unrecognized eicosanoid-lysophospholipids that may serve as biomarkers for age-related diseases and could potentially be used as targets in therapeutic interventions.

scholarly journals Activation of (arachidonyl) phosphatidylinositol turnover in rabbit neutrophils by the calcium ionophore A23187

1981 ◽  
Vol 194 (2) ◽  
pp. 497-505 ◽  
Author(s):  
R P Rubin ◽  
L E Sink ◽  
R J Freer
Keyword(s):  
Arachidonic Acid ◽  
Maximum Rate ◽  
Cytochalasin B ◽  
De Novo ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Enzyme Release ◽  
Secretory Product ◽  
Acid Incorporation

The addition of the Ca2+ ionophore A23187 to rabbit neutrophils stimulated [14C]arachidonic acid incorporation into phosphatidylinositol and lysosomal enzyme secretion. A significant increase in phosphatidylinositol labelling was observed after a 2 min exposure to 0.1 microM-ionophore A23187. Maximum increases in rate of labelling were obtained with 1 microM-ionophore A23187 within 1 min, declining to basal rates after 15 min. Similarly, maximum rate of enzyme release occurred during the first 2 min of exposure to ionophore and release was essentially complete by 15 min. Threshold and peak ionophore A23187 concentrations for stimulating both processes were identical. In contrast with the specificity of phosphatidylinositol labelling induced by 1 microM-ionophore A23187 in the absence of cytochalasin B, ionophore also significantly stimulated labelling of phosphatidylserine and phosphatidylethanolamine in the presence of cytochalasin B. With a threshold ionophore concentration (0.1 microM), the enhanced incorporation of arachidonate was relatively specific for phosphatidylinositol in cytochalasin-treated cells. Ionophore A23187 did not accelerate labelling of phosphatidylinositol by [14C]acetate or [14C]glycerol, indicating that ionophore A23187 does not stimulate phosphatidylinositol synthesis de novo, although it did promote [14C]palmitate and [32P]Pi incorporation into neutrophil phosphatidylinositol. However, the increase in phosphatidylinositol labelling with these latter precursors was generally slower in onset and much more modest in magnitude than that observed with arachidonic acid. These results support the hypothesis that a Ca2+-dependent phospholipase, which acts on the arachidonate moiety of phosphatidylinositol, is responsible for initiating at least certain of the membrane events coupled to the release of secretory product from the neutrophil.

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