Calcium ionophore A23187 induces expression of glucose-regulated genes and their heterologous fusion genes

1985 ◽  
Vol 5 (6) ◽  
pp. 1212-1219
Author(s):  
E Resendez ◽  
J W Attenello ◽  
A Grafsky ◽  
C S Chang ◽  
A S Lee
Keyword(s):  
Gene Transfection ◽  
Calcium Ionophore ◽  
Fold Increase ◽  
Calcium Ionophore A23187 ◽  
Induced Response ◽  
Specific Gene ◽  
Regulatory Sequence ◽  
Cdna Clones ◽  
Ionophore A23187 ◽  
Flanking Sequence

Using two cDNA clones which encode hamster genes specifically induced by glucose starvation, we demonstrated that an 8- and 30-fold increase, respectively, in the transcription rates of these genes was coordinately effected by calcium ionophore A23187 treatment, resulting in a similar increase in the steady-state levels of their mRNAs. This response was observed within several hours of ionophore treatment in several mammalian cell types and appeared to be specifically mediated by A23187 but not by other ionophores in general. To define the regulatory sequence which mediates this Ca2+-induced response, we showed by gene transfection techniques that the 5' flanking sequence of a rat glucose-regulated gene contained the region for induction by A23187. The system reported here offers attractive features for the study of specific gene regulation by Ca2+.

scholarly journals Calcium ionophore A23187 induces expression of glucose-regulated genes and their heterologous fusion genes.

1985 ◽  
Vol 5 (6) ◽  
pp. 1212-1219 ◽  
Author(s):  
E Resendez ◽  
J W Attenello ◽  
A Grafsky ◽  
C S Chang ◽  
A S Lee
Keyword(s):  
Gene Transfection ◽  
Calcium Ionophore ◽  
Fold Increase ◽  
Calcium Ionophore A23187 ◽  
Induced Response ◽  
Specific Gene ◽  
Regulatory Sequence ◽  
Cdna Clones ◽  
Ionophore A23187 ◽  
Flanking Sequence

Using two cDNA clones which encode hamster genes specifically induced by glucose starvation, we demonstrated that an 8- and 30-fold increase, respectively, in the transcription rates of these genes was coordinately effected by calcium ionophore A23187 treatment, resulting in a similar increase in the steady-state levels of their mRNAs. This response was observed within several hours of ionophore treatment in several mammalian cell types and appeared to be specifically mediated by A23187 but not by other ionophores in general. To define the regulatory sequence which mediates this Ca2+-induced response, we showed by gene transfection techniques that the 5' flanking sequence of a rat glucose-regulated gene contained the region for induction by A23187. The system reported here offers attractive features for the study of specific gene regulation by Ca2+.

Amplification of LTB4 generation in AM-PMN cocultures: transcellular 5-lipoxygenase metabolism

1991 ◽  
Vol 261 (2) ◽  
pp. L195-L203 ◽  
Author(s):  
F. Grimminger ◽  
U. Sibelius ◽  
W. Seeger
Keyword(s):  
Hydrolysis Product ◽  
Calcium Ionophore ◽  
Fold Increase ◽  
Metabolite Profile ◽  
Product Formation ◽  
Calcium Ionophore A23187 ◽  
Oxidation Products ◽  
Ionophore A23187 ◽  
Lipoxygenase Product ◽  
Isolated Cell

The generation of arachidonic acid (AA) metabolites by human polymorphonuclear leukocytes (PMN) and by rabbit alveolar macrophages (AM) was investigated and compared with that produced under conditions of coculture. Incubation of PMN with the calcium ionophore A23187 resulted in rapid generation of leukotriene (LT) B4 and its omega-oxidation products, paralleled by substantial secretion of 5-hydroxyeicosatetraenoic acid (HETE) and intact LTA4. Rapid LTA4 decay to nonenzymatic hydrolysis products in the extracellular space ensued. Exogenous AA, offered simultaneously with the ionophore, markedly increased 5-lipoxygenase product formation. Incubation of AM with A23187 evoked protracted generation of LTB4 in the absence of omega-oxidation, with concomitant liberation of 5-HETE, 15-HETE, free AA, and minor amounts of AA cyclooxygenase products. Exogenously offered LTA4 was avidly taken up and converted into LTB4 by these cells. Costimulation of AM and PMN with the ionophore resulted in an approximately 2.5-fold increase in the generation of LTB4 and its metabolites (compared with the summed amounts of the isolated cell experiments), whereas 5-HETE and nonenzymatic LTA4, hydrolysis product formation were markedly reduced. This change in metabolite profile was dependent on the AM-to-PMN ratio. Acetylsalicylic acid increased 5-lipoxygenase product formation in the coculture studies but not in the isolated cell experiments. AA prelabeling of either PMN or AM resulted in radioactivity detection in all AA lipoxygenase products except for 15-HETE.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Phosphorylase a activity as an indicator of neutrophil activation by chemotactic peptide.

1985 ◽  
Vol 101 (4) ◽  
pp. 1191-1197 ◽  
Author(s):  
J L Slonczewski ◽  
M W Wilde ◽  
S H Zigmond
Keyword(s):  
Glycogen Phosphorylase ◽  
Calcium Ionophore ◽  
Fold Increase ◽  
Dose Dependence ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Cytoplasmic Calcium ◽  
Chemotactic Peptide ◽  
Transient Activation ◽  
Fold Activation

The activity of glycogen phosphorylase, an enzyme that is activated by both cAMP and calcium, was used as an indicator of the state of the cytoplasm after chemotactic stimulation of polymorphonuclear leukocytes (neutrophils). The activity of the enzyme showed a clear dependence on cytoplasmic calcium. Addition of the calcium ionophore A23187 caused a 4-5-fold increase in activity of phosphorylase a. In the absence of external Ca2+, A23187 caused only brief transient activation of phosphorylase; probably reflecting release of sequestered intracellular Ca2+. Addition of the chemotactic peptide N-formylnorleucylleucylphenylalanine (FNLLP) caused a transient 2-3-fold activation of the enzyme. The dose-dependence of activation by FNLLP showed a peak at 10(-8) M, near the Kd of the receptor for FNLLP. The phosphorylase activity peaks by 90 s and then declines, returning to basal levels by 20 min after stimulation with 10(-8) M peptide and by 60 min with 10(-7) M peptide. This finding suggests that the cells do not need to maintain elevated cytoplasmic calcium levels to exhibit stimulated locomotion. Thus, if calcium continues to modulate the motility, there either must be highly localized changes that are not detected in measures of the total cytoplasm, or the sensitivity to calcium must be variable such that basal levels are sufficient to maintain locomotion. Cells loaded with the fluorescence calcium probe quin2 (0.6 mM) in the presence or absence of external Ca2+ had elevated phosphorylase levels before addition of FNLLP. Thus, the presence of quin2 may alter the cytoplasmic Ca2+ level, and it clearly alters some aspects of the neutrophil physiology. Phosphorylase a appears to be a sensitive, nonperturbing indicator of the cytoplasmic calcium levels.

scholarly journals The activation of protein degradation in muscle by Ca2+ or muscle injury does not involve a lysosomal mechanism

1986 ◽  
Vol 237 (3) ◽  
pp. 859-864 ◽  
Author(s):  
K Furuno ◽  
A L Goldberg
Keyword(s):  
Muscle Injury ◽  
Calcium Ionophore ◽  
Fold Increase ◽  
Calcium Ionophore A23187 ◽  
Muscle Fibres ◽  
Complete Medium ◽  
Ionophore A23187 ◽  
Protein Breakdown ◽  
Thiol Proteinases ◽  
Thiol Proteinase

By use of different inhibitors, we distinguished three proteolytic processes in rat skeletal muscle. When soleus muscles maintained under tension were exposed to the calcium ionophore A23187 or were incubated under no tension in the presence of Ca2+, net protein breakdown increased by 50-80%. Although leupeptin and E-64 inhibit this acceleration of protein breakdown almost completely, other agents that prevent lysosomal function, such as methylamine or leucine methyl ester, did not inhibit this effect. A similar increase in net proteolysis occurred in muscle fibres injured by cutting, and this response was also inhibited by leupeptin, but not by methylamine. In contrast, all these inhibitors markedly decreased the 2-fold increase in protein breakdown induced by incubating muscles without insulin and leucine, isoleucine and valine. In addition, the low rate of proteolysis seen in muscles under passive tension in complete medium was not affected by any of these inhibitors. Thus the basal degradative process in muscle does not involve lysosomes or thiol proteinases, and muscle can enhance protein breakdown by two mechanisms: lack of insulin and nutrients enhances a lysosomal process in muscle, as in other cells, whereas Ca2+ and muscle injury activate a distinct pathway involving cytosolic thiol proteinase(s).

Polyphosphoinositide metabolism during the fertilization wave in sea urchin eggs

Development ◽  
1992 ◽  
Vol 115 (1) ◽  
pp. 187-195 ◽  
Author(s):  
B. Ciapa ◽  
B. Borg ◽  
M. Whitaker
Keyword(s):  
Sea Urchin ◽  
Calcium Release ◽  
Calcium Ionophore ◽  
Fold Increase ◽  
Calcium Ionophore A23187 ◽  
Calcium Signal ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Isotopic Equilibrium ◽  
Sea Urchin Eggs

A transient increase in intracellular free calcium is believed to be the signal responsible for the stimulation of the egg metabolism at fertilization and the resumption of the cell cycle. We have studied how the polyphosphoinositides (PPI) turn over at fertilization in sea urchin eggs, in order to determine the relationship between the metabolism of these lipids and the calcium signal. We compare the patterns of PPI turnover that occur during the first minute following fertilization in eggs in which PPI are labelled to steady state with [3H]inositol or [3H]arachidonate with that in which PPI are labelled for a shorter period with [3H]inositol. When eggs are labelled to apparent isotopic equilibrium with either [3H]inositol or [3H]arachidonate, no early increase in [3H]PtdInsP2 occurs while PtdIns decreases slightly. On the contrary, when not labelled to isotopic equilibrium, all [3H]PPI increase during the first 15 seconds following fertilization. We find that, within seconds, fertilization triggers a 600-fold increase in the turnover of PPI, producing an amount of InsP3 apparently sufficient to trigger calcium release. We suggest that phosphoinositidase C and PtdInsP kinase, responsible respectively for the hydrolysis and synthesis of PtdInsP2, are both stimulated to a comparable degree in the first 30 seconds following fertilization and that net changes in the amount of PtdInsP2 at fertilization are very sensitive to the relative levels of activation of the two enzymes. Activating the eggs with the calcium ionophore A23187 showed that both these enzymes are sensitive to calcium, suggesting that calcium-dependent InsP3 production might play a role in the initiation and/or the propagation of the fertilization calcium wave.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Oxygen-dependent 1,25-dihydroxycholecalciferol-induced calcium ion transport in rat intestine

1981 ◽  
Vol 194 (1) ◽  
pp. 178-186 ◽  
Author(s):  
N C Kendrick ◽  
B Kabakoff ◽  
H F DeLuca
Keyword(s):  
Vitamin D ◽  
Calcium Ionophore ◽  
Maximum Velocity ◽  
Fold Increase ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Mucosal Surfaces ◽  
Calcium Ion Transport ◽  
Disc Assay

O2-dependent CA2+ uptake by rat duodenal discs has been characterized and used in a revised assay for 1,25-dihydroxycholecalciferol-induced intestinal Ca2+ transport. Although both muscle and mucosal surfaces are exposed in this free-floating-disc assay, the Ca2+ influx across the muscle surface is small, not O2- or vitamin D-dependent, and can be subtracted out. Depriving the animals of food for 9-14 h before assay increases the O2-dependent uptake by about 75%. Half-saturation values for O2-dependent Ca2+ uptake as determined with this assay are: 0.8mM-Ca2+ (fed) and 0.5mM-Ca2+ (food-deprived) for vitamin D-deficient rats, and 0.9mM-Ca2+ (fed) and 1.5mM-Ca2+ (food-deprived) for rats dosed with 1,25-dihydroxycholecalciferol. The maximum velocity of uptake varies from 6.7nmol of Ca2+ per cm2/min (fed) to 7.0nmol of Ca2+ per cm2/min (food-deprived) for vitamin D-deficient rats and 16.7nmol of Ca2+ per cm2/min (fed) to 29 nmol of Ca2+ per cm2/min (food-deprived) for 1,25-dihydroxycholecalciferol-treated rats. By using a 5 min preincubation and 15 min incubation with 1.0mM-Ca2+, duodenal tissue taken from vitamin D-treated rats shows about a 3-fold increase in O2-dependent Ca2+ uptake when compared with tissue taken from vitamin D-deficient animals. The calcium ionophore A23187, depending on concentration, either has no significant effect on or inhibits the O2-dependent uptake, rather than increasing it. Actinomycin D, at a dose of 2 micrograms/g, inhibits the O2-dependent uptake in intestinal discs from both vitamin D-deficient and vitamin D-treated rats by 58 and 80% respectively, when administered in vivo 3 1/2 h before assay.

Time course of endothelial dysfunction and myocardial injury during coronary arterial occlusion

1991 ◽  
Vol 261 (3) ◽  
pp. H874-H881 ◽  
Author(s):  
G. E. Viehman ◽  
X. L. Ma ◽  
D. J. Lefer ◽  
A. M. Lefer
Keyword(s):  
Time Course ◽  
Calcium Ionophore ◽  
Arterial Occlusion ◽  
Endothelial Damage ◽  
Calcium Ionophore A23187 ◽  
Induced Response ◽  
Ionophore A23187 ◽  
Myeloperoxidase Activity ◽  
Neutrophil Accumulation ◽  
Transmission Electron

The time course of the effects of permanent myocardial ischemia without reperfusion on the coronary vascular endothelium and myocardium were investigated in anesthetized cats. The left anterior descending (LAD) coronary artery was occluded for 1.5, 3.0, 4.5, or 6.0 h. Coronary rings from the ischemic LAD and the nonischemic left circumflex (LCX) arteries were tested for their responsiveness to the endothelium-dependent vasodilators acetylcholine (ACh, 0.1-100 nM) and the calcium ionophore A23187 (1-1,000 nM), and the endothelium-independent vasodilator sodium nitrite (NaNO2, 0.1-100 microM). Vasorelaxation was not significantly impaired in response to ACh after 1.5 h of ischemia and only moderately impaired after 3.0 h of ischemia (63 +/- 5% of control). However, after 4.5 h of ischemia the ACh-induced response was decreased to 33 +/- 4% of control and further declined to 31 +/- 4% of control after 6.0 h (P less than 0.001 from 1.5 h). There was no significant decrease in LCX ring vasorelaxant responses to vasodilators at all times, and the LAD rings only showed a moderately decreased response to NaNO2 after 6.0 h of ischemia (82 +/- 4% relaxation, P less than 0.05). Transmission electron microscopy revealed very little endothelial damage at 4.5 and 6.0 h, with only some subendothelial swelling noted. Damage to the myocardium did not become significant until after 4.5 h of ischemia, and cardiac myeloperoxidase activity, indicative of neutrophil accumulation, was not significant at any time.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Stress relaxation of fibroblasts activates a cyclic AMP signaling pathway.

1994 ◽  
Vol 126 (2) ◽  
pp. 457-464 ◽  
Author(s):  
Y He ◽  
F Grinnell
Keyword(s):  
Arachidonic Acid ◽  
Stress Relaxation ◽  
Cyclic Amp ◽  
Adenylyl Cyclase ◽  
Signaling Pathway ◽  
Calcium Ionophore ◽  
Fold Increase ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Signaling Mechanism

Mechanical force regulates gene expression and cell proliferation in a variety of cell types, but the mechanotransducers and signaling mechanisms involved are highly speculative. We studied the fibroblast signaling mechanism that is activated when cells are switched from mechanically stressed to mechanically relaxed conditions, i.e., stress relaxation. Within 10 min after initiation of stress relaxation, we observed a transient 10-20-fold increase in cytoplasmic cyclic AMP (cAMP) and a threefold increase in protein kinase A activity. The increase in cAMP depended on stimulation of adenylyl cyclase rather than inhibition of phosphodiesterase. Generation of cAMP was inhibited by indomethacin, and release of arachidonic acid was found to be an upstream step of the pathway. Activation of signaling also depended on influx of extracellular Ca2+ because addition of EGTA to the incubations at concentrations just sufficient to exceed Ca2+ in the medium inhibited the stress relaxation-dependent increase in free arachidonic acid and cAMP. This inhibition was overcome by adding CaCl2 to the medium. On the other hand, treating fibroblasts in mechanically stressed cultures with the calcium ionophore A23187-stimulated arachidonic acid and cAMP production even without stress relaxation. In summary, our results show that fibroblast stress relaxation results in activation of a Ca(2+)-dependent, adenylyl cyclase signaling pathway. Overall, the effect of stress relaxation on cAMP and PKA levels was equivalent to that observed after treatment of cells with forskolin.

Growth factors and the primary cilium in BALB/c 3T3 cells

1987 ◽  
Vol 45 ◽  
pp. 830-831
Author(s):  
R. W. Tucker ◽  
N. S. More ◽  
S. Jayaraman
Keyword(s):  
Growth Factors ◽  
Primary Cilium ◽  
Cultured Cells ◽  
Morphological Changes ◽  
Calcium Ionophore ◽  
Growth Stimulation ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
3T3 Cells ◽  
Microtubule Depolymerization

The mechanisms by which polypeptide growth factors Induce DNA synthesis in cultured cells is not understood, but morphological changes Induced by growth factors have been used as clues to Intracellular messengers responsible for growth stimulation. One such morphological change has been the transient disappearance of the primary cilium, a “9 + 0” cilium formed by the perinuclear centriole in interphase cells. Since calcium ionophore A23187 also produced both mitogenesis and ciliary changes, microtubule depolymerization might explain ciliary disappearance monitored by indirect immunofluorescence with anti-tubulin antibody. However, complete resorption and subsequent reformation of the primary cilium occurs at mitosis, and might also account for ciliary disappearance induced by growth factors. To settle this issue, we investigated the ultrastructure of the primary cilium using serial thin-section electron microscopy of quiescent BALB/c 3T3 cells before and after stimulation with serum.

Inhibition of Platelet Aggregation by Ethanol in Vitro Shows Specificity for Aggregating Agent Used and Is Influenced by Platelet Lipid Composition

1982 ◽  
Vol 48 (01) ◽  
pp. 049-053 ◽  
Author(s):  
C G Fenn ◽  
J M Littleton
Keyword(s):  
Platelet Aggregation ◽  
Lipid Composition ◽  
Saturated Fatty Acids ◽  
Calcium Ionophore ◽  
Cholesterol Content ◽  
Membrane Lipid ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Increased Sensitivity

SummaryEthanol at physiologically tolerable concentrations inhibited platelet aggregation in vitro in a relatively specific way, which may be influenced by platelet membrane lipid composition. Aggregation to collagen, calcium ionophore A23187 and thrombin (low doses) were often markedly inhibited by ethanol, adrenaline and ADP responses were little affected, and aggregation to exogenous arachidonic acid was actually potentiated by ethanol. Aggregation to collagen, thrombin and A23187 was inhibited more by ethanol in platelets enriched with saturated fatty acids than in those enriched with unsaturated fats. Platelets enriched with cholesterol showed increased sensitivity to ADP, arachidonate and adrenaline but this increase in cholesterol content did not appear to influence the inhibition by ethanol of platelet responses. The results suggest that ethanol may inhibit aggregation by an effect on membrane fluidity and/or calcium mobilization resulting in decreased activity of a membrane-bound phospholipase.

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