Inhibitory Effect Of EGTA Research Articles

Effects of calcium on the activities of cytosolic antioxidative enzymes and IAA oxidase during in vitro adventitious rooting of mung bean seedlings

The effects of Ca2+ on antioxidative enzymes and indole-3-acetic acid (IAA) oxidase during adventitious rooting were investigated in mung bean (Vigna radiata). CaCl2 significantly promoted the formation and growth of adventitious roots. EGTA (a Ca2+ chelator) or ruthenium red (a Ca2+-channel blocker) significantly inhibited root formation and growth, but these inhibitory effects could be partially reversed by CaCl2. Furthermore, inclusion of 5 mM CaCl2 significantly increased superoxide dismutase (SOD) activity by 10% at 3 h and catalase (CAT) activity by an average of 29.6% at each time point. CaCl2 decreased peroxidase (POD) activity by 9.4% and 21% at 12 and 24 h, respectively, and ascorbate peroxidase (APX) activity by an average of 13.9% at each time point. These CaCl2-induced changes in enzymatic activities were similar to changes caused by indole-3-butyric acid (IBA). Treatment with EGTA or ruthenium red decreased SOD activity by an average of 18.4% and 15.2%, respectively; POD activity by 27.4% and 57.6%, respectively; APX activity by 10.3% and 15.6%, respectively; and CAT activity by 19.3% and 5.2%, respectively, when compared with CaCl2. In addition, CaCl2 increased IAA oxidase activity by an average of 5.5% beginning at 6 h, whereas EGTA significantly decreased IAA oxidase activity by 29.2%, 22.9%, and 13.5% at 6, 9, and 12 h, respectively. The inhibitory effects of EGTA could be partially suppressed by addition of CaCl2. These results imply that the stimulative effect of Ca2+ on adventitious rooting is partially related to Ca2+-induced changes in the activities of antioxidative enzymes and IAA oxidase.

Histone 2A stimulates glucose-6-phosphatase activity by permeabilization of liver microsomes.

Histone 2A increases glucose-6-phosphatase activity in liver microsomes. The effect has been attributed either to the conformational change of the enzyme, or to the permeabilization of microsomal membrane that allows the free access of substrate to the intraluminal glucose-6-phosphatase catalytic site. The aim of the present study was the critical reinvestigation of the mechanism of action of histone 2A. It has been found that the dose-effect curve of histone 2A is different from that of detergents and resembles that of the pore-forming alamethicin. Inhibitory effects of EGTA on glucose-6-phosphatase activity previously reported in histone 2A-treated microsomes have been also found in alamethicin-permeabilized vesicles. The effect of EGTA cannot therefore simply be an antagonization of the effect of histone 2A. Histone 2A stimulates the activity of another latent microsomal enzyme, UDP-glucuronosyltransferase, which has an intraluminal catalytic site. Finally, histone 2A renders microsomal vesicles permeable to non-permeant compounds. Taken together, the results demonstrate that histone 2A stimulates glucose-6-phosphatase activity by permeabilizing the microsomal membrane.

Métaux de transition et production de monoxyde d’azote par les cellules endothéliales humaines

The bioavailability of endothelial nitric oxide (NO) is regulated by transition metals but their mechanisms of action on NO synthesis and degradation are not clearly understood. Using differential pulse amperometry and NO microelectrodes, local NO concentration was measured at the surface of cultured human umbilical vein endothelial cells (HUVECs) stimulated by histamine or thrombin in the presence of transition metal chelators. The agonist-activated NO release required both extracellular Ca 2+ and transition metals. In the presence of 1 mM external Ca 2+, a low concentration of EGTA (5 μM) inhibited by 40 % the NO release from stimulated HUVECs. In the presence of extracellular L-arginine, the inhibitory effect of EGTA was even more marked and, in its absence, it was suppressed by adding exogenous superoxide dismutase. The decrease in NO release induced by the copper chelators, cuprizone and DETC, suggests that extracellular traces of Cu 2+ could regulate NO availability.

Regulation of plastid gene expression in rice involves calcium and protein phosphatases/kinases for signal transduction

Spatial control of expression of plastid-encoded photosynthesis-related genes, psbA, psaA and rbcL, has been studied in rice ( Oryza sativa L.) seedlings. Whereas roots accumulate negligible amount of mRNAs of these genes, shoots show high transcript levels. A gradient in expression of these genes is seen even in lower, middle and upper segments of leaf sheath, with lower segment showing the least amount of mRNA—being nearer to the meristematic zone. The steady-state transcript levels in lower segment are significantly reduced on treatment with EGTA. The inhibitory effect of EGTA could be reversed by addition of calcium during treatment. Nifedipine and verapamil—the calcium channel blockers—also decrease the levels of transcripts. Okadaic acid, the specific inhibitor of protein phosphatases 1 and 2A (PPI and PP2A), and a general inhibitor of protein phosphatases, NaF, inhibit the steady-state transcript levels of psaA, psbA and rbcL significantly. Staurosporine, the protein kinase inhibitor, increases the steady-state transcript levels of these genes. These results suggest the importance of calcium and protein phosphorylation in expression of genes during chloroplast biogenesis. However, the inter-relationship of calcium effects and protein phosphorylation remains to be worked out.

Calcium Chelator and Channel Blockers Suppress the IAA-Induced Membrane Hyperpolarization without Inhibiting the Following Growth Promotion in Hypocotyl Sections of Vigna unguiculata under Xylem Perfusion

The effects of the Ca chelator EGTA and the Ca channel blockers La', Gd, nifedipine, and verapamil on the IAA-induced early responses of hypocotyl sections of Vigna unguiculata were studied to assess the involvement of Ca in the IAA-induced hyperpolarization followed by growth promotion using the xylem perfusion method. The IAA-induced hyperpolarization was distinctly inhibited by pretreatment with EGTA, La, and/or Gd (5 mM each). EGTA inhibited by about 80-90% while La and Gd inhibited by about 60-75%. The sustained hyperpolarization after IAA application was also inhibited by these reagents. Nifedipine and verapamil (50 //M each) had little effect on the IAA-induced hyperpolarization and growth promotion. The inhibitory effects of EGTA, La and Gd on the membrane potentials were not due to changes in a passive component, but mainly to decreases in an electrogenic component. The effect of EGTA was reversible, while those of La and Gd were not. Present results suggest that the influx of Ca through the plasma membrane might be required not only for the initiation of hyperpolarization by IAA but also for the following sustained hyperpolarization and may therefore play an essential role in the IAA-induced activation of the electrogenic proton pumps at the plasma membrane in Vigna hypocotyls. Inhibition of the hyperpolarization by EGTA, La, and Gd, however, did not involve inhibition of the promotion of growth by IAA. It seems that activation of the electrogenic proton pumps at the plasma membrane is not always the indispensable primary step of the IAA-induced promotion of elongation growth. Some alternative pathway of proton extrusion might take part.

Inhibitory Effect of EGTA on Serotonin Transport into Rabbit Blood Platelets: Possible Involvement of the Glycoprotein IIb/IIIa Complex

We reported previously that serotonin (5-HT) transport was attenuated by treatment of platelets with EGTA, and that this inhibitory effect of EGTA was restored by CaCl2. In the present study, the inhibitory effect of EGTA was found to be uncompetitive, and no inhibitory effect was observed when EGTA was added at 20°C. Genistein and thyrphostin A47, both protein tyrosine kinase inhibitors, inhibited Ca2+-induced restoration of 5-HT transport. In contrast, the protein tyrosine phosphatase inhibitor phenylarsine oxide significantly augmented Ca2+-induced restoration of 5-HT transport. These results might support the hypothesis that the glycoprotein (GP) IIb/IIIa complex, a platelet membrane integrin protein, might regulate 5-HT transport into blood platelets. It is conceivable that Ca2+ chelation by EGTA might cause temperature-dependent dissociation of the GP IIb/IIIa complex, which results in the reduction of 5-HT transport. Rearrangement of the GP IIb/IIIa complex by replenishment of the Ca2+ binding sites might restore the EGTA-induced reduction of 5-HT transport.

The lysine-dependent stimulation of lysine catabolism in tobacco seed requires calcium and protein phosphorylation.

The accumulation of free lysine in tobacco seed triggers the stimulation of lysine-ketoglutarate reductase, an enzyme that acts in lysine catabolism. The mechanism of lysine-ketoglutarate reductase stimulation was studied in two different systems: (1) developing seeds of wild-type plants in which the low basal lysine-ketoglutarate reductase activity can be stimulated by the exogenous addition of lysine; and (2) developing seeds of transgenic tobacco plants expressing a bacterial dihydrodipicolinate synthase in which lysine-ketoglutarate reductase activity is stimulated by endogenous lysine overproduction. In both systems, the stimulation of lysine-ketoglutarate reductase activity was significantly reduced when treated with the Ca2+ chelator EGTA. Moreover, the inhibitory effect of EGTA was overcome by the addition of Ca2+ but not Mg2+, suggesting that the lysine-dependent activation of lysine-ketoglutarate reductase requires Ca2+. This was further confirmed by a significant stimulation of lysine-ketoglutarate reductase activity following the treatment of wild-type seeds with ionomycin (an ionophore that increases Ca2+ flow into the cytoplasm). In addition, treatment of wild-type seeds with the protein phosphatase inhibitor okadaic acid triggered a significant induction in lysine-ketoglutarate reductase activity, whereas treatment of the transgenic seeds with the protein kinase inhibitor K-252a caused a significant reduction in its activity. Thus, we conclude that the stimulation of lysine-ketoglutarate reductase activity by lysine in tobacco seed operates through an intracellular signaling cascade mediated by Ca2+ and protein phosphorylation.

Comparison of ?1A- and ?1B-adrenoceptor coupling to inositol phosphate formation in rat kidney

We have compared the coupling mechanisms of rat renal alpha 1A- and alpha 1B-like adrenoceptors to inositol phosphate formation. The experiments were performed in parallel in native renal tissue preparations and in those where alpha 1B-adrenoceptors had been inactivated by treatment with 10 mumol/l chloroethylclonidine for 30 min at 37 degrees C; renal slices were used in most experiments but isolated renal cells were also used in some cases. The Ca2+ chelating agent, EGTA (5 mmol/l), reduced noradrenaline-stimulated inositol phosphate formation in native but enhanced it in chloroethylclonidine-treated renal slices. The inhibitory effect of EGTA was not mimicked by 100 nmol/l nifedipine. Inactivation of 87% of cellular Gi by 16-20 h treatment with 500 ng/ml pertussis toxin did not significantly affect noradrenaline-stimulated inositol phosphate formation in isolated renal cells but abolished the inhibitory effect of chloroethylclonidine. The adenylate cyclase activator, forskolin (20 mumol/l), inhibited noradrenaline-stimulated inositol phosphate formation in native and chloroethylclonidine-treated slices, and the inhibitory effects of chloroethylclonidine treatment and forskolin were additive. We conclude that in rat kidney inositol phosphate formation via alpha 1B-like adrenoceptors may involve the influx of extracellular Ca2+ and a pertussis toxin-sensitive G-protein but is insensitive to inhibition by forskolin. In contrast alpha 1A-like adrenoceptor-mediated inositol phosphate formation does not require the presence of extracellular Ca2+ or of Gi and is sensitive to inhibition by forskolin. In comparison to published data from other model systems we further conclude that the signaling mechanisms of alpha 1-adrenoceptor subtypes may depend on their cellular environment.

Signal transduction mechanisms of C1q-mediated superoxide production. Evidence for the involvement of temporally distinct staurosporine-insensitive and sensitive pathways.

C1q, a plasma glycoprotein and the recognition component of the classical complement pathway, interacts with specific cells of the immune system resulting in the enhancement of cell function. For example, interaction of C1q with its cell-surface receptor on neutrophils induces the activation of the respiratory burst, a finding previously documented using a chemiluminescent assay to detect oxygen radical formation. In an alternative approach we have now used a modified cytochrome c reduction assay to characterize C1q-mediated production of superoxide anion (O2-) in more detail. C1q coated to microtiter wells induced O2- release, which occurred microtiter wells induced O2- release, which occurred after a lag period of 10 to 20 min, and was then sustained over approximately 1 h. O2- production could be triggered by the purified pepsin-resistant, collagen-like fragment of C1q, but not by mannose-binding protein and pulmonary surfactant protein A, proteins that also contain collagen-like domains. Concentrations of C1q which promoted a vigorous O2- generation did not induce release of neutrophil primary granules and caused little or no secondary granule release. Investigation of the biochemical events mediating C1q stimulated O2- production by neutrophils revealed that the response invoked two biochemical pathways with distinct sensitivities to previously described inhibitors. A role for Ca2+ in initiation of the response was suggested by the inhibitory effect of EGTA, the calmodulin antagonist W7, and the intracellular Ca2+ chelator BAPTA. The protein kinase inhibitor staurosporine did not inhibit the induction of the response, but did block that component of the response occurring after approximately 30 min. Neither phase of C1q-mediated O2- production was inhibited by pertussis toxin, a strong inhibitor of the G-protein-coupled FMLP-mediated response. In summary, C1q-triggered O2- production is relatively unique both in terms of the kinetics of the response and the biochemical pathways evoked. These data support the hypothesis that more than one biochemical pathway induced by ligand-receptor interaction can activate the neutrophil NADPH oxidase.

High affinity binding of ramiprilat on isolated human glomeruli

Evidence for angiotensin-converting enzyme (ACE) on isolated human glomeruli was furnished by specific binding of tritium-labeled ramiprilat, a potent inhibitor of ACE. 3H-ramprilat bound to isolated glomeruli, depending on time and temperature displaying a K D of 3.8 nmol/l and a B max of 853 fmol/mg protein. Specific binding represented more than 90% of total binding. Dissociation occurred rapidly after dilution of the sample with incubation buffer or after addition of an excess of unlabeled inhibitor. Binding of 3H-ramiprilat was also inhibited by increasing concentrations of enalaprilat, another ACE-inhibitor or by preincubation of the glomeruli with polyclonal antibodies against ACE. ACE is a zinc-containing enzyme. Addition of EGTA to the assay, which chelates zinc ions, completely inhibited binding. This inhibitory effect of EGTA was reversed by divalent Zn 2+ and Ca 2+ ions but not by magnesium. Binding of 3H-ramiprilat to isolated glomeruli was maximal when the pH of the assay medium was brought to pH 8. In conclusion, the binding of 3H-ramiprilat to isolated human glomeruli is specific and resembles the characteristics which have been found earlier for enzyme activity of ACE. Thus, binding of 3H-ramiprilat to isolated glomeruli can be assumed to be directed to ACE.

Inhibition of Reproduction of Phytophthora by the Calmodulin-interacting Compounds Trifluoperazine and Ophiobolin A

SUMMARY: Sporangium production in Phytophthora palmivora was inhibited by trifluoperazine (TFP) and ophiobolin A. TFP was inhibitory to a similar extent to cultures both in light and in darkness, although the stimulatory effect of calcium and the inhibitory effect of EGTA were much greater in the dark. Sporangium and oospore production in P. cactorum were inhibited by TFP. Thus development of both asexual and sexual reproductive structures involves a calcium/calmodulin interaction.

Role of ions in activation of human lymphocytes.

Phytohemagglutinin (PHA)-stimulated lymphocytes were cultured in media containing varying levels of K+, Mg2+, Ca2+. Cell activation was monitored by measuring nuclear diameter and by evaluating the area of nucleolus which reacted with silver nitrate. Decreasing extracellular K+ from normal levels (5.0 mM) to 14% (0.7 mM) and decreasing extracellular Mg2+ from normal levels (1.0 mM) to 14% (0.14 mM) did not affect nuclear diameter or silver nitrate reactivity of PHA-stimulated lymphocytes. Chelation of extracellular Ca2+ with EGTA during the first 24 h after PHA stimulation completely inhibited the increases in silver reactivity and nuclear diameter associated with stimulation. Chelation of extracellular Ca2+ 48 h after PHA stimulation did not inhibit lymphocyte stimulation. Inhibitory effects of EGTA were completely reversed if CaCl2 was added to the medium within 24 h of PHA stimulation. By 48 h the effects were irreversible.

The role of the mannose/N-acetylglucosamine receptor in the pinocytosis of horseradish peroxidase by mouse peritoneal macrophages.

Saccharomyces cerevisiae mannan inhibits the pinocytosis of horseradish peroxidase (HRP) by resident, thioglycollate-,proteose peptone-, and Corynebacterium parvum-elicited macrophages from 30 to 70% when 1 mg/ml HRP is used, and 65 to 87% when 250 micrograms/ml HRP is used. In contrast, HRP uptake by J774 cells, a macrophage cell line reported to have little mannose receptor activity, is inhibited only about 25% by mannan. HRP uptake by resident and thioglycollate-elicited (thio) macrophages is also inhibited 34 and 66% by addition of EGTA to the medium and 55 and 79% by trypsin treatment of the macrophages, respectively. The inhibitory effect of EGTA can be reversed by 1 mM excess Ca2+. High extracellular concentrations of Ca2+, in the range of 10-20 mM, however, inhibit pinocytosis in resident macrophages by about 50%. Sucrose uptake by resident macrophages is not appreciably affected by mannan. These results support the hypothesis that HRP uptake is mediated by the macrophage mannose/N-acetylglucosamine receptor. PMA stimulates fluid-phase pinocytosis of HRP by thio macrophages but does not affect receptor-mediated uptake of HRP, while the combination of adenosine, homocysteine, and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) selectively inhibits bulk-phase uptake by thio macrophages.

Extracellular free calcium and fluid secretion by the rabbit lacrimal gland in vivo.

A possible role of extracellular free Ca2+ in methacholine-induced fluid secretion in the in vivo rabbit lacrimal gland has been investigated. Lowering the extracellular Ca2+ concentration by either intra-arterial injection or infusion of EGTA at doses which caused no systemic effects produced a dose related and reversible inhibition of methacholine-induced secretion. The inhibitory effect of EGTA was diminished when EGTA chelated with CaCl2 in varying concentration ratios was administered. On the other hand, intra-arterial injection of CaCl2 potentiated fluid secretion stimulated by submaximal doses of methacholine. These results suggest that fluid secretion from the rabbit lacrimal gland is dependent on the extracellular free Ca2+ concentration.

The role of calcium in the regulation of cyclic nucleotide levels in brain slices of rat and guinea pig

1. In rat cerebral cortical slices, the addition of EGTA to acutely lower extracellular calcium concentrations greatly reduced the accumulation of cyclic AMP elicited by norepinephrine while having little effect on the response to adenosine or N6-phenylisopropyladenosine. The inhibitory effect of EGTA on the response to norepinephrine was reversible. A prolonged 30 min preincubation of rat cortical slices with EGTA or merely with calcium-free medium produced a marked increase in basal levels of cyclic AMP which was inhibited by theophylline or adenosine deaminase. The increase in basal levels of cyclic AMP thus appears due to increased release of adenosine. The released adenosine potentiated the response to exogenous biogenic amines and thus complicated studies on the direct effect of extracellular calcium in cyclic AMP-systems in brain sclices. Elimination of released adenosine with adenosine deaminase reduced responses to norepinephrine in control slices and blocked responses to norepinephrine in EGTA-treated slices. Adenosine deaminase also reduced responses to phenylisopropyladenosine. 2. In guinea pig cortical slices the addition of EGTA to acutely reduce extracellular calcium increased basal levels of cyclic AMP and the response to norepinephrine, adenosine and phenylisopropyl-adenosine. The response to histamine was unchanged. Elimination of released adenosine with adenosine deaminase prevented the EGTA-elicited increase in basal levels of cyclic AMP and completely blocked the response to norepinephrine or histamine. Adenosine deaminase reduced the response to the amines in control slices but not that due to phenylisopropyl-adenosine. Cyclic GMP levels were increased 2 to 3-fold in guinea pig cerebral cortical slices by norepinephrine, histamine, and adenosine. These responses were blocked by the addition of EGTA. 3. The effects of the potent phosphodiesterase inhibitor, isobutylmethylxanthine, on cyclic AMP levels in brain slices both in the presence and absence of extracellular calcium appear, like the effects of theophylline, to primarily reflect inhibition of adenosine-responses rather than inhibition of phosphodiesterase. The effects of another phosphodiesterase inhibitor, Ro 20-1724, on cyclic AMP levels appear to reflect both a Ro 20-1724-elicited increase in net efflux of adenosine and inhibition of phosphodiesterase. 4. The results indicate that a) the presence of extracellular calcium is critical for norepinephrine and histamine-elicited accumulations of cyclic AMP in brain slices, but not for responses to adenosine, and that b) released adenosine can, at least partially, substitute for extracellular calcium in maintaining responsiveness of amine-sensitive cyclic AMP-generating systems in brain slices.

Calcium requirement for stimulation of cyclic AMP accumulation in anterior pituitary gland by LH-RH

Removal of Ca 2+ from the incubation medium by addition of 2 mM ethylene glycol bis-(β-aminoethyl ether)-N,N′tetraacetic acid (EGTA) leads to at least 75% inhibition of the luteinizing hormone-releasing hormone (LH-RH)-induced accumulation of adenosine 3′5′monophosphate (cyclic AMP) in rat anterior pituitary gland in vitro. This inhibitory effect of EGTA is reversed by the addition of Ca 2+. A half-maximal effect of Ca 2+ on LH-RH-induced cyclic AMP accumulation is observed at 2–5 × 10 −5 M free Ca 2+. The LH-RH induced LH and FSH release is completely dependent upon the presence of Ca 2+ in the incubation medium, a half-maximal effect being measured at 1–2 × 10 −4 M free Ca 2+. The basal release of LH is increased upon Ca 2+ removal.