Concentrations Of EGTA Research Articles

Hypoxia-induced inhibition of calcium channels in guinea-pig taenia caeci smooth muscle cells.

1. The effects of hypoxia on whole-cell current in single smooth muscle cells and on a high K(+)-induced contraction of strips of the guinea-pig taenia caeci were studied. 2. In physiological salt solution (PSS) and K(+)-based pipette solution, hypoxia (PO2 = 20 mmHg) reversibly inhibited both the inward Ca2+ current (ICa) and outward Ca(2+)-activated K+ current (IK(Ca)) components of the whole-cell current. 3. In PSS and Cs(+)-based pipette solution, hypoxia reversibly suppressed ICa by 30 +/- 5% at 0 mV. 4. When Ba2+ was used as a charge carrier, the IBa was suppressed by hypoxia in a potential-dependent manner, with the maximum of 40 +/- 7% at +10 mV. Alterations of concentrations of EGTA, GDB beta S or ATP in the pipette solution did not change the inhibitory effects of hypoxia on ICa and IBa. 5. In PSS with 2 mM CaCl2 replaced by CoCl2, hypoxia did not affect the Ca2+ influx-independent potassium current. 6. In cells voltage clamped at -20 mV hypoxia reversibly inhibited the spontaneous transient outward currents. 7. The response of high K(+)-contracted taenia caeci to hypoxia was composed of an initial rapid relaxation followed by a small transient contraction and slow relaxation. The transient contraction was blocked by atropine (1-10 microM), while relaxations were unaffected by atropine and guanethidine (10 microM). 8. The results show that hypoxia reversibly inhibits ICa and secondarily suppresses IK(Ca) due to decreased Ca2+ influx through Ca2+ channels. 9. It is suggested that inhibition of ICa was responsible for the rapid relaxation, whereas transient contraction may have been due to release of acetylcholine from nerve terminals upon hypoxia.

CaEGTA uncompetitively inhibits calcium activation of whorl morphogenesis inAcetabularia

The spacing between adjacent hairs in vegetative whorls ofAcetabularia acetabulum (formerlyA. mediterranea) was earlier reported as being quantitatively responsive to calcium ion concentration in the culture medium. We here report a quantitative response to the concentration of the calcium-chelator EGTA, in the opposite sense to the effect of calcium. (Increasing [Ca2+] diminishes the spacing; increasing [EGTA] increases it.) The earlier work was interpreted in terms of control of the spacing by a putative reaction-diffusion mechanism in the cell membrane, in which a receptor R was activated by calcium-binding to initiate the process. We extend this interpretation by treating CaEGTA as an uncompetitive inhibitor of the effect of calcium on R. This leads to thermodynamic constants for CaEGTA binding to the CaR complex: ΔH 298 0 =−250 ± 60 kJ/mol; ΔS 298 0 =−820 ± 200 J/mol · K. Consistency of the concentration and temperature dependences reported here with the postulated dynamic mechanism increases the probability that this mechanism is correct.

Ca2+ and Calmodulin Dynamics during Photopolarization in Fucus serratus Zygotes.

The role of Ca2+ in zygote polarization in fucoid algae (Fucus, Ascophyllum, and Pelvetia species) zygote polarization is controversial. Using a local source of Fucus serratus, we established that zygotes form a polar axis relative to unilateral light (photopolarization) between 8 and 14 h after fertilization (AF), and become committed to this polarity at approximately 15 to 18 h AF. We investigated the role of Ca2+, calmodulin, and actin during photopolarization by simultaneously exposing F. serratus zygotes to polarizing light and various inhibitors. Neither removal of Ca2+ from the culture medium or high concentrations of EGTA and LaCl3 had any effect on photopolarization. Bepridil, 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester, nifedipine, and verapamil, all of which block intracellular Ca2 release, reduced photopolarization from 75 to 30%. The calmodulin antagonists N-(6-aminohexyl)-5-chloro-L-naphthalenesulfonamide and trifluoperazine inhibited photopolarization in all zygotes, whereas N-(6-aminohexyl)-L-naphthalenesulfonamide had no effect. Cytochalasin B, cytochalasin D, and latrunculin B, all of which inhibit actin polymerization, had no effect on photopolarization, but arrested polar axis fixation. The role of calmodulin during polarization was investigated further. Calmodulin mRNA from the closely related brown alga Macrocystis pyrifera was cloned and the protein was expressed in bacteria. Photopolarization was enhanced following microinjections of this recombinant calmodulin into developing zygotes. Confocal imaging of fluorescein isothiocyanate-labeled recombinant calmodulin in photopolarized zygotes showed a homogenous signal distribution at 13 h AF, which localized to the presumptive rhizoid site at 15 h AF.

Phosphorylation at conserved carboxyl-terminal hydrophobic motif regulates the catalytic and regulatory domains of protein kinase C.

Mature protein kinase C is phosphorylated at a conserved carboxyl-terminal motif that contains a Ser (or Thr) bracketed by two hydrophobic residues; in protein kinase C betaII, this residue is Ser-660 (Keranen, L. M., Dutil, E. M., and Newton, A. C. (1995) Curr. Biol. 5, 1394-1403). This contribution examines how negative charge at this position regulates the function of protein kinase C. Specifically, Ser-660 in protein kinase C betaII was mutated to Ala or Glu and the enzyme's stability, membrane interaction, Ca2+ regulation, and kinetic parameters were compared with those of wild-type protein phosphorylated at residue 660. Negative charge at this position had no significant effect on the enzyme's diacylglycerol-stimulated membrane interaction nor the conformational change accompanying membrane binding. In contrast, phosphate caused a 10-fold increase in the enzyme's affinity for Ca2+ and a comparable increase in its affinity for phosphatidylserine, two interactions that are mediated by the C2 domain. Negative charge also increased the protein's thermal stability and decreased its Km for ATP and peptide substrate. These data indicate that phosphorylation at the extreme carboxyl terminus of protein kinase C structures the active site so that it binds ATP and substrate with higher affinity and structures determinants in the regulatory region enabling higher affinity binding of Ca2+. The motif surrounding Ser-660 in protein kinase C betaII is found in a number of other kinases, suggesting interactions promoted by phosphorylation of the carboxyl terminus may provide a general mechanism for stabilizing kinase structure.

The Regulatory Ser262 of Microtubule-associated Protein Tau Is Phosphorylated by Phosphorylase Kinase

Abnormally phosphorylated tau is the major component of paired helical filaments found in the brains of patients suffering from Alzheimer's disease. Therefore, the identification of kinases that phosphorylate tau is of considerable interest. A DEAE-Sepharose column resolved porcine brain extract into five tau kinase activity peaks. Among these peaks, two were completely inhibited by EGTA, indicating that these two activity peaks contained Ca2+-dependent tau kinases. One of the above two Ca2+-dependent tau kinase activity peaks also contained phosphorylase kinase activity. The tau kinase and phosphorylase kinase activities associated with this peak could not be separated from each other by Superose 12 gel filtration, hydroxylapatite, and calmodulin-agarose affinity chromatographies. Phosphorylase kinase, purified from rabbit skeletal muscle, phosphorylated tau to a stoichiometry of 2.1 mol of phosphate/mol of tau and converted tau to a species with a retarded mobility on SDS-polyacrylamide gel electrophoresis. The apparent Km and kcat values for tau phosphorylation by muscle phosphorylase kinase were 6.9 microM and 47.4 min-1, respectively. As a substrate of muscle phosphorylase kinase, phosphorylase was eight times better than tau. Sequence analyses of tryptic and thermolytic phosphopeptides derived from tau phosphorylated by muscle phosphorylase kinase revealed five phosphorylation sites, Ser237, Ser262, Ser285, Ser305, and Ser352. Among these sites, Ser262 was previously shown to be phosphorylated in human tau from fetal, adult, and Alzheimer's diseased brains (Seubert, P., Mawal-Dewan, M., Barbour, R., Jakes, R., Goedert, M., Johnson, G. V. W., Litersky, J. M., Schenk, D., Lieberburg, I., Trojanowski, J. Q., and Lee, V. M. Y. (1995) J. Biol. Chem. 270, 18917-18922); and its phosphorylation abolished tau's binding to microtubules (Drewes, G., Trinczek, B., Illenberger, S., Biernat, J., Schmitt-Ulms, G., Meyer, H. E., Mandelkow, E.-M., and Mandelkow, E. (1995) J. Biol. Chem. 270, 7679-7688). Slot-blot analysis using a monoclonal antibody against muscle phosphorylase kinase and an activity assay using phosphorylase revealed that phosphorylase kinase was present in microtubules extensively purified by repeated cycles of polymerization and depolymerization. Taken together, these results suggest that in neurons, phosphorylase kinase may be one of the kinases that participate in the phosphorylation of tau.

Modulation by Calcium of Deiters' Neuron Gabaa Receptors in the Rabbit

The function of classical GABAA receptors of the rabbit Deiters' neurons has been studied at the single membrane level by a biochemical micromethod involving the study of labelled chloride permeation. In particular, labelled chloride permeation across microdissected fresh single membranes was studied in a microchamber system. The stimulation of 36Cl− out in permeation by “extracellular” GABA was determined under different conditions in the respect of Ca++. When the conditions were such that “intracellular” Ca++ was 0.02 μ there appeared to be an optimal effect by GABA on chloride passage. Conditions presumably resulting in an increase of [Ca++]i beyond the level reported above led to a decreased GABA effect, especially at the highest GABA concentrations used (± lO−4M). However, complete removal of Ca++ by a high (12mM) intracellular EGTA concentration erased completely the GABA effect.

The effect of extracellular Ca2+ on responses to purinoceptor agonists in cultured swine tracheal smooth muscle cells.

The effects of extracellular Ca2+ ions on purinergic responses were examined in swine tracheal smooth muscle cells (TSMCs) in primary culture. ATP (1 microM to 1 mM) and alpha, beta-methylene adenosine 5' triphosphate (alpha, beta-Me ATP) (100 microM and 1 mM) concentration-dependently increased [Ca2+]i in the presence and the absence of extracellular Ca2+. Responses to ATP (10 microM to 1 mM) in the presence of extracellular Ca2+ were significantly larger than those in its absence (n=8), whereas those to alpha, beta-Me ATP were not significantly different between the presence (n=7) and the absence (n=8) of extracellular Ca2+. Responses to ATP (1 mM) at extracellular Ca2+ concentration ([Ca2+]o) of 10 and 5 mM were significantly larger than that at extracellular EGTA concentration ([EGTA]o) of 1 mM (p< 0.01, n=5), whereas the responses to alpha, beta-Me ATP (1 mM) at 10 and 5 mM [Ca2+]o were significantly smaller than that at [EGTA]o of 1 mM (p<0.05, n=5). Increasing [Ca2+]o to 5 mM after the application of either 1 mM ATP (n=4) or 1 mM alpha, beta-Me ATP (n=4) in the absence of extracellular Ca2+ (1 mM [EGTA]o) further increased [Ca2+]i, though the increases in [Ca2+]i by agonists in 1 mM [EGTA]o had been already maximal. Incubating cells for 300 s in 5 mM [Ca2+]o before the application of ATP (1 mM) significantly increased the response to the drug than that obtained by incubating cells for 6 sec in 5 mM [Ca2+]o before the drug application (p< 0.01, n=4). However, alpha, beta-Me ATP (1 mM) induced similar responses by incubating cells for 30 or 300 s in 5 mM [Ca2+]o to that by incubating them for 6 s. These results suggest that the effect of alpha, beta-Me ATP in swine TSMCs in primary culture is mainly through Ca2+ release and that its effect on Ca2+ entry is smaller than other nucleotides.

Ca 2+-independent contraction induced by hyperosmolar K +-rich solutions in rat uterus

The present experiments were designed to investigate the mechanisms involved in the contractile responses evoked by KCl, added either isoosmotically or hyperosmotically, in the rat uterus. Exposure of uterine strips to a Ca 2+-free, 3 mM EGTA-containing solution abolished the responses induced by isoosmotic KCl solutions. Conversely, addition of hyperosmolar KCl induced concentration-dependent tonic responses in a Ca 2+-free, 3 mM EGTA-containing solution. The maximum increase in tension was reached with 210 mM K +. The response to hyperosmotic K + was unaffected by previous depletion of intracellular Ca 2+ stores with oxytocin (1 μM), by inhibition of refilling of the intracellular Ca 2+ stores using cyclopiazonic acid (10 μM) or by increasing the concentration of EGTA in the medium to 10 mM. Sucrose and mannitol (60–420 mM) induced concentration-dependent sustained contractions which were not reproducible and were significantly smaller in size than those evoked by the maximally effective concentration of hyperosmotic K + (210 mM). The contraction induced by hyperosmotic K + in Ca 2+-free solution was not altered by the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide hydrochloride (W-7, 100 μM), the Ca 2+ calmodulin protein kinase II inhibitor 1-[ N,O-bis(1,5-isoquinoline-sulphonyl)- N-methyl- l-tyrosyl]-4-phenylpiperazine (KN-62, 10 μM) or the tyrosine kinase inhibitor genistein (10 μM). The protein kinase C inhibitor calphostin C (1–3 μM) failed to modify the K +-effect curve, which was however partially inhibited in the presence of the non-selective protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine dihydrochloride (H-7, 3–100 μM). The protein kinase inhibitor staurosporine (30–300 nM) depressed the contraction induced by hyperosmolar K + in a concentration-dependent manner. The contraction induced by sucrose in Ca 2+-free solution was unaffected by W-7 (100 μM) and KN-62 (10 μM) but was partially reduced by calphostin C (1 μM), H-7 (30 μM), staurosporine (100 nM) and genistein (10 μM). These results suggest that different mechanisms are involved in the responses evoked by isoosmotic and hyperosmotic KCl in the rat uterus. A component of the contraction induced by hypertonic KCl seems mainly independent of both external and internal Ca 2+ and of hyperosmolar stress. This contraction is not mediated by protein kinase C, Ca 2+ calmodulin-dependent kinases or protein tyrosine kinases but involves activation of other, at the present unknown, staurosporine-sensitive protein kinase(s).

Regulation of the purified Ca2+ release channel/ryanodine receptor complex of skeletal muscle sarcoplasmic reticulum by luminal calcium

45Ca2+ flux and single channel measurements have related that the Ca2+ release channel/ryanodine receptor complex of striated muscle is regulated by micromolar cytoplasmic Ca2+. The effect of luminal Ca2+, however, remains controversial. In the experiments presented here, we reconstituted the isolated Ca2+ release channel of rabbit skeletal muscle sarcoplasmic reticulum into planar lipid bilayers in the presence of symmetrical K+ solutions. Using K+ as the charge carrier, we were able to examine the effect of changes in luminal calcium in the micro- to millimolar range. In the presence of activating cytoplasmic Ca2+, the release channel was activated and inactivated in a concentration-dependent manner by luminal Ca2+. Since increasing cytoplasmic EGTA concentrations shifted the dependence of channel open probability on luminal Ca2+ to higher Ca2+ concentrations, it is suggested that luminal Ca2+ exerts ist regulating effect by acting on Ca2+ binding sites accessible from the cytoplasmic side of the channel.

Selective activation of exocytosis by low concentrations of ACh in rat pancreatic acinar cells.

1. We have monitored changes in membrane capacitance (delta C) and conductance (delta G) induced by muscarinic acetylcholine stimulation in single rat pancreatic acinar cells. 2. Acetylcholine (ACh, 500 nM) induced simultaneous increases of delta C and delta G. In contrast, a low concentration (50 nM) of ACh exclusively induced delta C increases without delta G. These responses were abolished by the internal perfusion of heparin. This indicates that inositol 1,4,5-trisphosphate-mediated internal Ca2+ mobilization either simultaneously activates exocytosis and ion channels or exclusively initiates exocytosis. In comparison, a low concentration of A23187 selectively activated ion channels but a high concentration activated exocytosis and ion channels simultaneously. 3. These selective response patterns of delta C and delta G depend on the choice of agonist and the internal EGTA concentration. From this, we postulated two explanations for the selective action of muscarinic ACh stimulation on exocytosis. First, an area of high [Ca2+]i, spatially close to secretory granules, activates exocytosis. Second, an as yet unknown signalling factor sensitizes the Ca2+ affinity of the exocytotic apparatus.

Activation of the volume-sensitive chloride current in vascular endothelial cells requires a permissive intracellular Ca2+ concentration

Combined patch clamp and Ca2+-measurements (Fura-2) were used to study the dependence of volume-activated Cl--currents (ICl,vol) of endothelial cells from bovine pulmonary artery on the intracellular Ca2+-concentration [Ca2+]i. Loading the cells with high concentrations of EGTA or BAPTA via ruptured membrane patches or by preincubating them with 50 microM BAPTA-AM caused a substantial decrease of ICl,vol. This reduction was independent of the activation state of the current: the current amplitude was not only diminished if [Ca2+]i was lowered at the peak of the volume-activated current, but this low Ca2+-concentration also prevented activation of the current by a second hypotonic challenge.ICl,vol is already maximally activated at intracellular Ca2+-concentrations between 50 and 100 nmol/l, a further increase of [Ca2+]i does not affect the size of ICl,vol.These results indicate that a sustained full activation of ICl,vol in endothelial cells requires submicromolar concentrations of Ca2+, and that changes in [Ca2+]i do not modulate the current.

Analysis of interaction between cadmium and metallothionein isoforms by capillary zone electrophoresis.

The effects of cadmium on the peak area of metallothionein (MT) protein were studied by capillary zone electrophoresis with a polyacrylamide-coated tube at neutral pH. When cadmium was added to a commercial standard MT-1 isoform prepared from rabbit liver, the peak are of the MT-1 isoform decreased in a time- and dose-dependent manner. The MT-2 isoform also decreased with time, but the rate of decrease was lower than that of the MT-1 isoform. When ethylene glyco-bis-(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA) was added to a solution containing cadmium and MT-1, the peak area recovered with increasing concentration of EGTA. Zinc caused a slight decrease in the peak areas of MT-1 and MT-2 compared with cadmium, while addition of sodium did not decrease the areas. Furthermore, the peak areas of MT isoforms of the crude extract prepared from zinc-treated mice also decreased with increasing concentration of cadmium. These results indicate that cadmium may changed the charge of MT, which may account for the observed differences in electrophoretic behavior.

Modulation of Na +,Ca 2+ exchange current by EGTA calcium buffering in giant cardiac membrane patches

Effects of calcium buffering by EGTA were examined on sodium-calcium exchange currents ( I Naca) in inside-out giant cardiac membrane patches. Free calcium concentrations (Ca 2+) were monitored with a calcium electrode and a fluorescent calcium indicator (Calcium Green-5N). With 1.8 μM cytoplasmic Ca 2+, inward I Naca increased 2-fold at −120 mV when EGTA concentration was increased from 0.1 mM to 10 mM (37°C and 140 mM extracellular sodium). Stimulation by EGTA was decreased or abolished under conditions of attenuated exchanger turnover rate: temperature < 30°C, extracellular sodium < 70 mM, and membrane potential > + 60 mV. EGTA concentration had no effect on outward I Naca with 100 mM cytoplasmic Na + and 0.8 μM cytoplasmic Ca 2+, conditions under which the current inactivated by about 70%. EGTA (0.1–10 mM) and BAPTA (10 mM) inhibited the current by about 80% when the outward I Naca was stimulated by 2 mM cytoplasmic ATP or by phosphatidylserine. The apparent K i for EGTA was 0.2 mM. The electroneutral calcium ionophore, A23187, activated outward I Naca even in presence of 10 mM EGTA. Our results are consistent with EGTA acting as a simple calcium buffer with no direct effect on the exchanger. At low concentrations of EGTA, inhibition of the inward I Naca is expected due to submembrane calcium depletion by the exchanger; enhancement of the outward I Naca at low EGTA concentrations is expected because submembrane calcium accumulates and activates I Naca via regulatory calcium binding sites.

Activation of Calcium-Sensitive Potassium Channels in L6 Skeletal Myocytes by Arginine Vasopressin Requires Extracellular Calcium

The effects of extracellular application of arginine vasopressin (AVP) upon membrane currents in L6 skeletal myocytes was investigated using the whole-cell configuration of the patch-clamp technique. At O mV AVP produced large amplitude, transient outward currents that reversed when the clamping potential was changed to −100 mV (negative to EK) The effects of alterations in the extracellular K+ concentration upon the current reversal potential suggested that the current elicited by AVP was carried mainly by K+ ions. Intracellular dialysis with 10 μM inositol 1,4,5-trisphosphate (InsP3) elicited similar currents but only in 6/14 cells. Inclusion of 5 mg ml−1 heparin in the intracellular solutions was ineffective at inhibiting the current responses to AVP. The AVP-induced current was totally abolished when the intracellular EGTA concentration was increased from 0.05 mM to 10 mM or Ca2+ was removed from the extracellular perfusing solution. These results suggest that AVP produces activation of a Ca2+-sensitive K+ conductance in L6 skeletal myocytes by a process dependent upon extracellular Ca2+ and not intracellular Ca2+ release.

Ionic currents and inhibitory effects of glibenclamide in seminal vesicle smooth muscle cells.

1. Whole-cell voltage-clamp recordings were made from smooth muscle cells isolated from guinea-pig seminal vesicle. 2. When the recording pipette solution contained 130 mM KCl and a low concentration of EGTA (0.2 mM), a dominant outward current was elicited by depolarization to positive of -30 mV from a holding potential of -50 mV. The current was non-inactivating, stimulated by intracellular Ca2+ and blocked by bath-applied 1 mM tetraethylammonium but not 1 mM 3,4 diaminopyridine. 3. If 10 mM EGTA was added to the KCl pipette solution and the holding potential was -50 mV, or more negative, the major current elicited by depolarization to positive of -30 mV was an A-type K(+)-current. This current inactivated rapidly (within 100 ms) and was blocked by bath-applied 1 mM 3,4-diaminopyridine but not 10 mM tetraethylammonium. 4. An inward voltage-gated Ca channel current was observed on depolarization to positive of -30 mV with 1.5 mM Ca2+ or 10 mM Ba2+ in the bath solution and when Ca+ replaced K+ in the pipette. The Ba(2+)-current was shown to be abolished by bath-applied 100 microM Cd2+ and inhibited by 90% by 1 microM nifedipine, and thus appeared to be carried by L-type Ca channels. 5. High concentrations of glibenclamide (10-500 microM) inhibited A-type K(+)-current, Ba(2+)-current and contraction of the whole tissue induced by noradrenaline or electrical field stimulation. 6. From these data we suggest that seminal vesicle smooth muscle cells express Ca2+ -dependent K channels, A-type K channels and L-type Ca channels which are inhibited by tetraethylammonium,3,4-diaminopyridine and nifedipine, respectively. In addition, an unexpected relaxant effect of high concentrations of glibenclamide may be explained by inhibition of the Ca channels.

The short transcript of Leishmania RNA virus is generated by RNA cleavage

Leishmania RNA virus 1 produces a short viral RNA transcript corresponding to the 5' end of positive-sense single-stranded RNAs both in virally infected cells and in in vitro polymerase assays. We hypothesized that this short transcript was generated via cleavage of full-length positive-sense single-stranded RNA. A putative cleavage site was mapped by primer extension analysis to nucleotide 320 of the viral genome. To address the hypothesis that the short transcript is generated via cleavage at this site, two substrate RNAs that possessed viral sequence encompassing the putative cleavage site were created. When incubated with sucrose-purified viral particles, these substrate RNAs were site-specifically cleaved. The cleavage site of the in vitro-processed RNAs also mapped to viral nucleotide 320. The short-transcript-generating activity could be specifically abolished by proteinase K treatment of sucrose-purified viral particles and high concentrations of EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], suggesting that the activity requires a proteinaceous factor and possibly intact viral particles. The cleavage activity is directly associated with short-transcript-generating activity, since only viral particle preparations which were capable of generating the short transcript in polymerase assays were also active in the cleavage assay. Furthermore, the short-transcript-generating activity is independent of the viral polymerase's transcriptase and replicase activities. We present a working model whereby cleavage of Leishmaniavirus RNA transcripts functions in the maintenance of a low-level persistent infection.

Mechanism of active DNA demethylation during embryonic development and cellular differentiation in vertebrates

Incubation of hemimethylated and labelled oligodeoxynucleotides with nuclear extracts from differentiating chicken embryos and mouse myoblasts resulted in the replacement of m 5C by C. One of the enzymes involved is mSCpG endonuclease. It cleaves only m 5CpG and not, m 5CpT, m 5CpA, m 5CpC or m 6ApT. The enzyme is not sequence specific and catalyses the reaction in the presence of high concentrations of EDTA or EGTA.

Extracellular calcium is not required for gliotoxin or dexamethasone-induced DNA fragmentation: A reappraisal of the use of EGTA

The immunomodulating agent gliotoxin and related toxins cause apoptotic cell death in a variety of cell types including macrophages and thymocytes [Waring et al. (1988) J. biol. Chem., 263, 18,493-18,499; Waring et al. (1990) Int. J. Immunopharmac., 12, 445–457]. The mechanism of induction of apoptosis by gliotoxin is not yet known, although it does not require protein synthesis [Waring (1990) J. biol. Chem., 14,476-14,480], unlike dexamethasone-induced apoptosis in thymocytes. Because of the reported requirement for extracellular calcium in apoptosis induced by dexamethasone, we studied the effects of extracellular calcium on gliotoxin-induced apoptosis in macrophages. Initial experiments using calcium-depleted media showed no inhibition of apoptotic DNA fragmentation by gliotoxin. By measuring residual 45Ca2+ remaining in cells pulsed with labelled calcium over the time period required for DNA fragmentation, we could demonstrate some uptake of extracellular calcium into treated cells as assessed by residual, slowly exchanging calcium. When cells were treated with the calcium chelator EGTA at 0.5-2 mM, calcium uptake was abolished but DNA fragmentation was unaffected. EGTA at higher concentrations, up to 8 mM, did inhibit DNA fragmentation without any additional inhibition of calcium uptake. Similar results were found for dexamethasone-treated thymocytes. Thymocytes treated with 8 mM EGTA, however, were not rescued from apoptosis but died by necrosis. These results indicate that extracellular calcium is not essential for apoptosis induced by these agents and that the use of high concentrations of EGTA to establish a requirement for extracellular calcium in apoptosis should be treated with caution.

Fixation induces differential tip morphology and immunolocalization of the cytoskeleton in pollen tubes

The effects of chemical fixation on tip morphology and immunolocalization of the cytoskeleton in Tradescantia virginiana pollen tubes were evaluated using lour different fixation protocols differing in fixative type/concentration, fixation time, buffer system and additives. Apical regions were much more sensitive to fixation manipulations than more basipetal areas. The presence of the calcium chelator EGTA at 5 mM led to tip rupture and/or swelling in over 80% of germinated grains. However, low or no EGTA levels during fixation resulted in poor immunolocalizations, although lips had more normal morphology. Double fixation in which pollen tubes were first treated for a short period with higher fixative and lower EGTA (0.5 mM) concentrations, followed by lower fixative and higher EGTA (5.0 mM) concentrations, resulted in both improved preservation of pollen lube tip morphology and microfilament/microtubule localizations.

Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback.

Rapid inactivation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole-cell patch clamp recording and [Ca2+]i measurement techniques. In the presence of 22 mM extracellular Ca2+, the Ca2+ current declined with a biexponential time course (time constants of 8-30 ms and 50-150 ms) during hyperpolarizing pulses to potentials more negative than -40 mV. Several lines of evidence suggest that the fast inactivation process is Ca2+ but not voltage dependent. First, the speed and extent of inactivation are enhanced by conditions that increase the rate of Ca2+ entry through open channels. Second, inactivation is substantially reduced when Ba2+ is present as the charge carrier. Third, inactivation is slowed by intracellular dialysis with BAPTA (12 mM), a rapid Ca2+ buffer, but not by raising the cytoplasmic concentration of EGTA, a slower chelator, from 1.2 to 12 mM. Recovery from fast inactivation is complete within 200 ms after repolarization to -12 mV. Rapid inactivation is unaffected by changes in the number of open CRAC channels or global [Ca2+]i. These results demonstrate that rapid inactivation of ICRAC results from the action of Ca2+ in close proximity to the intracellular mouths of individual channels, and that Ca2+ entry through one CRAC channel does not affect neighboring channels. A simple model for Ca2+ diffusion in the presence of a mobile buffer predicts multiple Ca2+ inactivation sites situated 3-4 nm from the intracellular mouth of the pore, consistent with a location on the CRAC channel itself.