Presence Of BAPTA Research Articles

ATP from Subplasmalemmal Mitochondria Controls Ca2+-dependent Inactivation of CRAC Channels

A sustained Ca2+ entry is the primary signal for T lymphocyte activation after antigen recognition. This Ca2+ entry mainly occurs through store-operated Ca2+ channels responsible for a highly selective Ca2+ current known as I(CRAC). Ca2+ ions act as negative feedback regulators of I(CRAC), promoting its inactivation. Mitochondria, which act as intracellular Ca2+ buffers, have been proposed to control all stages of CRAC current and, hence, intracellular Ca2+ signaling in several types of non-excitable cells. Using the whole-cell configuration of the patch clamp technique, which allows control of the intracellular environment, we report here that respiring mitochondria located close to CRAC channels can regulate slow Ca2+-dependent inactivation of I(CRAC) by increasing the Ca2+-buffering capacity beneath the plasma membrane, mainly through the release of ATP.

Phosphorylation of serine 1928 in the distal C-terminal domain of cardiac Ca V 1.2 channels during β1-adrenergic regulation

During the fight-or-flight response, epinephrine and norepinephrine released by the sympathetic nervous system increase L-type calcium currents conducted by Ca(V)1.2a channels in the heart, which contributes to enhanced cardiac performance. Activation of beta-adrenergic receptors increases channel activity via phosphorylation by cAMP-dependent protein kinase (PKA) tethered to the distal C-terminal domain of the alpha(1) subunit via an A-kinase anchoring protein (AKAP15). Here we measure phosphorylation of S1928 in dissociated rat ventricular myocytes in response to beta-adrenergic receptor stimulation by using a phosphospecific antibody. Isoproterenol treatment increased phosphorylation of S1928 in the distal C-terminal domain, and a similar increase was observed with a direct activator of adenylyl cyclase, forskolin, confirming that the cAMP and PKA are responsible. Pretreatment with selective beta1- and beta2-adrenergic antagonists reduced the increase in phosphorylation by 79% and 42%, respectively, and pretreatment with both agents completely blocked it. In contrast, treatment with these agents in the presence of 1,2-bis(2-aminophenoxy)ethane-N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester to buffer intracellular calcium results in only beta1-stimulated phosphorylation of S1928. Whole-cell patch clamp studies with intracellular BAPTA demonstrated that 98% of the increase in calcium current was attributable to beta1-adrenergic receptors. Thus, beta-adrenergic stimulation results in phosphorylation of S1928 on the Ca(V)1.2 alpha1 subunit in intact ventricular myocytes via both beta1- and beta2-adrenergic receptor pathways, but the beta2-dependent increase in phosphorylation depends on elevated intracellular calcium and does not contribute to regulation of whole-cell calcium currents at basal calcium levels. Our results correlate phosphorylation of S1928 with beta1-adrenergic functional up-regulation of cardiac calcium channels in the presence of BAPTA in intact ventricular myocytes.

Regulation of Nicotinic Acetylcholine Receptor Desensitization by Ca2+

The relationship between the concentration of intracellular Ca2+ ([Ca2+](i)) and recovery from desensitization of nicotinic acetylcholine receptors (nAChRs) in rat medial habenula (MHb) neurons was investigated using the whole cell patch-clamp techniques in combination with microfluorescent [Ca2+](i) measurements. Recovery from desensitization was assessed with a paired-pulse agonist application protocol. Application of 100 microM nicotine (5 s) caused pronounced desensitization of nAChRs, after which recovery proceeded with two components. The relative weight of the two phases of recovery was sensitive to the nature of the intracellular Ca2+ chelator, with a greater fraction of channels recovering during the fast phase in the presence of BAPTA than EGTA. Recovery was affected by differential Ca2+ buffering only when Ca2+ was present in the extracellular solution, implying that Ca2+ influx through nAChRs was responsible for slowing the recovery. Simultaneous [Ca2+](i) measurements showed that recovery from desensitization was inversely correlated with the instantaneous [Ca2+](i), further supporting the suggestion that elevation of [Ca2+](i) limits the return of nAChRs to the resting state. In a separate set of experiments, activation of voltage-gated Ca2+ channels during the recovery phase produced a sufficiently large increase in [Ca2+](i) to reduce recovery from desensitization even in the absence of Ca2+ influx through nAChRs. Overall, it is suggested that Ca2+ entry through both nAChRs and voltage-gated Ca2+ channels exerts a negative feedback on nAChR activity through stabilization of desensitized states. The interaction of these two Ca2+ sources could form the basis of a coincidence detector under specific circumstances.

Paraoxon suppresses Ca 2+ spike and afterhyperpolarization in snail neurons: Relevance to the hyperexcitability induction

The effects of organophosphate (OP) paraoxon, active metabolite of parathion, were studied on the Ca 2+ and Ba 2+ spikes and on the excitability of the neuronal soma membranes of land snail ( Caucasotachea atrolabiata). Paraoxon (0.3 μM) reversibly decreased the duration and amplitude of Ca 2+ and Ba 2+ spikes. It also reduced the duration and the amplitude of the afterhyperpolarization (AHP) that follows spikes, leading to a significant increase in the frequency of Ca 2+ spikes. Pretreatment with atropine and hexamethonium, selective blockers of muscarinic and nicotinic receptors, respectively, did not prevent the effects of paraoxon on Ca 2+ spikes. Intracellular injection of the calcium chelator BAPTA dramatically decreased the duration and amplitude of AHP and increased the duration and frequency of Ca 2+ spikes. In the presence of BAPTA, paraoxon decreased the duration of the Ca 2+ spikes without affecting their frequency. Apamin, a neurotoxin from bee venom, known to selectively block small conductance of calcium-activated potassium channels (SK), significantly decreased the duration and amplitude of the AHP, an effect that was associated with an increase in spike frequency. In the presence of apamin, bath application of paraoxon reduced the duration of Ca 2+ spike and AHP and increased the firing frequency of nerve cells. In summary, these data suggest that exposure to submicromolar concentration of paraoxon may directly affect membrane excitability. Suppression of Ca 2+ entry during the action potential would down regulate Ca 2+-activated K + channels leading to a reduction of the AHP and an increase in cell firing.

ENOS translocation but not eNOS phosphorylation is dependent on intracellular Ca2+ in human atrial myocardium

In endothelial cells, two ways of endothelial nitric oxide (NO) synthase (eNOS) activation are known: 1) translocation and 2) Akt-dependent phosphorylation of the enzyme at Ser(1177) (Ser(1177) eNOS). We have recently shown that agonist-induced Ser(1177) eNOS phosphorylation also occurs in human myocardium (10). In this study, we investigated the Ca(2+) dependency of these two mechanisms in human atrium. Therefore, atrial tissue was obtained from patients who underwent coronary artery bypass operations. In immunohistochemical experiments, the translocated form of eNOS and phosphorylated Ser(1177) eNOS were labeled using specific antibodies. eNOS translocation was measured in the absence and presence of the Ca(2+) chelator BAPTA before and after application of BRL 37344 (BRL), a beta(3)-adrenoceptor agonist that increases eNOS activity (34). In the absence of BAPTA, BRL time dependently increased the staining intensity of translocated eNOS, whereas in the presence of BAPTA, this effect was blunted. In contrast, BRL clearly increased the staining of phosphorylated Ser(1177) eNOS even in the presence of BAPTA. This observation was confirmed using Western blot analysis. Using the NO-sensitive dye diaminofluorescein, we have demonstrated that BRL induced a strong NO release. This effect was completely abolished in the presence of BAPTA but was unaffected by LY-292004, an inhibitor of phosphatidylinositol 3-kinase activity and eNOS phosphorylation. Although Ca(2+) dependent, neither the translocation of eNOS nor NO release was changed by the adenylate cyclase activator forskolin. In conclusion, 1) in human atrial myocardium, BRL-induced eNOS translocation but not Ser(1177) eNOS phosphorylation is dependent on intracellular Ca(2+). 2) In atrial myocardium, eNOS-translocation and not Ser(1177) eNOS phosphorylation is responsible for generating the main amount of NO. 3) Although Ca(2+) dependent, eNOS translocation and NO release could not be mimicked by adenylate cyclase activation as a mediator of beta-adrenergic stimulation.

Rhythmic Activity of Murine Neuromuscular Junctions upon Activation of Release of Stored Calcium in the Presence of a Calcium Buffer

Using a two-electrode voltage-clamp technique, we recorded end-plate currents (EPCs) in neuromuscular synaptic junctions of the murine diaphragm upon rhythmic stimulation of the n. phrenicus with frequencies of 7, 20, 50, 70, and 100 sec−1. Parameters of EPC series were analyzed against the background of the action of a mobilizer of intracellular calcium, ryanodine (0.5 μM), after the loading of terminals by 1.2 mM BAPTA (calcium buffer with rapid dynamics of binding of calcium), and upon the action of ryanodine in the presence of BAPTA. Under the action of ryanodine, the amplitude and quantum content of EPC within the plateau phase increased by 100 to 150% (P < 0.05). Loading with BAPTA evoked sharp decreases in the quantum content of unitary EPCs, the intensity of the initial facilitation, and the level of the EPC plateau in series within the entire range of stimulation frequencies used. Against the background of the action of BAPTA, the facilitatory effect of ryanodine increased; inhibitory effects of BAPTA with respect to the amplitude of unitary EPC and the level of the initial facilitation were completely compensated, whereas the level of EPC at the plateau stage increased to levels exceeding the control values by 50 to 70%. The ability of ryanodine to facilitate the transmitter (acetylcholine) release, which was enhanced in the presence of BAPTA, was completely neutralized by a blocker of L-type calcium channels, verapamil (5 μM). In the absence of BAPTA, verapamil did not influence the effects of ryanodine. We hypothesize that in the presence of BAPTA calcium channels of L type whose activity is resistive to the buffer action of BAPTA are disinhibited. The calcium current through L-type channels, perhaps, is capable of stimulating calcium release from the stores of nerve terminals and, as a consequence, of intensifying the facilitatory effect of ryanodine on the release of acetylcholine. After verapamil-induced blockade of this current, BAPTA demonstrates the ability to prevent the facilitatory effect of ryanodine on the transmitter release.

Ion Regulation of Homotypic Vacuole Fusion in Saccharomyces cerevisiae

Biological membrane fusion employs divalent cations as protein cofactors or as signaling ligands. For example, Mg2+ is a cofactor for the N-ethylmaleimide-sensitive factor (NSF) ATPase, and the Ca2+ signal from neuronal membrane depolarization is required for synaptotagmin activation. Divalent cations also regulate liposome fusion, but the role of such ion interactions with lipid bilayers in Rab- and soluble NSF attachment protein receptor (SNARE)-dependent biological membrane fusion is less clear. Yeast vacuole fusion requires Mg2+ for Sec18p ATPase activity, and vacuole docking triggers an efflux of luminal Ca2+. We now report distinct reaction conditions where divalent or monovalent ions interchangeably regulate Rab- and SNARE-dependent vacuole fusion. In reactions with 5 mm Mg2+, other free divalent ions are not needed. Reactions containing low Mg2+ concentrations are strongly inhibited by the rapid Ca2+ chelator BAPTA. However, addition of the soluble SNARE Vam7p relieves BAPTA inhibition as effectively as Ca2+ or Mg2+, suggesting that Ca2+ does not perform a unique signaling function. When the need for Mg2+, ATP, and Sec18p for fusion is bypassed through the addition of Vam7p, vacuole fusion does not require any appreciable free divalent cations and can even be stimulated by their chelators. The similarity of these findings to those with liposomes, and the higher ion specificity of the regulation of proteins, suggests a working model in which ion interactions with bilayer lipids permit Rab- and SNARE-dependent membrane fusion.

Docosahexaenoic acid and other fatty acids induce a decrease in pHi in Jurkat T-cells.

1. Docosahexaenoic acid (DHA) induced rapid (t1/2=33 s) and dose-dependent decreases in pHi in BCECF-loaded human (Jurkat) T-cells. Addition of 5-(N,N-dimethyl)-amiloride, an inhibitor of Na+/H+ exchanger, prolonged DHA-induced acidification as a function of time, indicating that the exchanger is implicated in pHi recovery. 2. Other fatty acids like oleic acid, arachidonic acid, eicosapentaenoic acid, but not palmitic acid, also induced a fall in pHi in these cells. 3. To assess the role of calcium in the DHA-induced acidification, we conducted experiments in Ca2+-free (0% Ca2+) and Ca2+-containing (100% Ca2+) buffer. We observed that there was no difference in the degree of DHA-induced transient acidification in both the experimental conditions, though pHi recovery was faster in 0% Ca2+ medium than that in 100% Ca2+ medium. 4. In the presence of BAPTA, a calcium chelator, a rapid recovery of DHA-induced acidosis was observed. Furthermore, addition of CaCl2 into 0% Ca2+ medium curtailed DHA-evoked rapid pHi recovery. In 0% Ca2+ medium, containing BAPTA, DHA did not evoke increases in [Ca2+]i, though this fatty acid still induced a rapid acidification in these cells. These observations suggest that calcium is implicated in the long-lasting DHA-induced acidosis. 5. DHA-induced rapid acidification may be due to its deprotonation in the plasma membrane (flip-flop model), as suggested by the following observations: (1) DHA with a -COOH group induced intracellular acidification, but this fatty acid with a -COOCH3 group failed to do so, and (2) DHA, but not propionic acid, -induced acidification was completely reversed by addition of fatty acid-free bovine serum albumin in these cells. 6. These results suggest that DHA induces acidosis via deprotonation and Ca2+ mobilization in human T-cells.

Ca(2+)-independent caspase-3 but not Ca(2+)-dependent caspase-2 activation induced by oxidative stress leads to SH-SY5Y human neuroblastoma cell apoptosis.

Continuous and long-lasting exposure to tert-butylhydroperoxide (t-BOOH) increased the number of apoptotic SH-SY5Y human neuroblastoma cells both in the presence and in the absence of the intracellular Ca(2+) ion chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). In addition, t-BOOH exposure induced activation of CPP32, as demonstrated by poly-(ADP-ribose) polymerase (PARP) cleavage, and of ICH-1L caspases. Exposure to t-BOOH also induced a time-dependent release of cytochrome c. Interestingly, in the presence of BAPTA, CPP32 activation still occurred, whereas ICH-1L activation was blocked. Ac-DEVD-CHO, an inhibitor of CPP32 activity, prevented the appearance of apoptotic cells, whereas the inhibitor of ICH-1L activity Z-VDVAD-FMK did not. Collectively, these findings demonstrate that in SH-SY5Y neuroblastoma cells exposure to continuous and long-lasting oxidative stress induced activation of caspase-3 that was independent of intracellular Ca(2+) ion concentration ([Ca(2+)](i)) elevation but led to cell apoptosis. In contrast, caspase-2 activation was dependent on [Ca(2+)](i) increase but did not result in apoptosis.

Identification of a calcium/calmodulin-dependent protein kinase that phosphorylates the Neurospora circadian clock protein FREQUENCY.

Phosphorylation of circadian clock proteins represents a major regulatory step that controls circadian clocks. In Neurospora, the circadian clock protein FREQUENCY (FRQ) is progressively phosphorylated over time, and its level decreases when it is hyperphosphorylated. In this study, we showed that most of the kinase activity phosphorylating FRQ in vitro was calcium/calmodulin-dependent, and the endogenous FRQ in the Neurospora extracts was phosphorylated by a Ca/CaM-dependent kinase-like activity. From Neurospora cell extracts, an approximately 50-kDa Ca/CaM-dependent kinase (CAMK-1) that can specifically phosphorylate FRQ was purified. In vitro, this kinase accounts for near half of the FRQ kinase activity, and it can phosphorylate the FRQ region that contains the three known functionally important phosphorylation sites. To understand the function of camk-1 in vivo, it was disrupted in Neurospora by gene replacement. After germination from ascospores, the camk-1 null strains grew slowly, indicating that CAMK-1 plays an important role in growth and development of Neurospora. This phenotype was transient however, revealing redundancy in the system. Analysis of the camk-1 null strain revealed that the deletion of camk-1 affected phase, period, and light-induced phase shifting of the circadian conidiation rhythm. Taken together, our results suggest that multiple kinases may phosphorylate FRQ in vivo.

Effects of BAPTA on force and Ca2+ transient during isometric contraction of frog muscle fibers.

The effects of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) on force and intracellular Ca2+ transient were studied during isometric twitches and tetanuses in single frog muscle fibers. BAPTA was added to the bathing solution in its permeant AM form (50 and 100 microM). There was no clear correlation between the changes in force and the changes in Ca2+ transient. Thus during twitch stimulation BAPTA did not suppress the Ca2+ transient until the force had been reduced to <50% of its control value. At the same time, the peak myoplasmic free Ca2+ concentration reached during tetanic stimulation was markedly increased, whereas the force was slightly reduced by BAPTA. The effects of BAPTA were not duplicated by using another Ca2+ chelator, EGTA, indicating that BAPTA may act differently as a Ca2+ chelator. Stiffness measurements suggest that the decrease in mechanical performance in the presence of BAPTA is attributable to a reduced number of active cross bridges. The results could mean that BAPTA, under the conditions used, inhibits the binding of Ca2+ to troponin C resulting in a reduced state of activation of the contractile system.

Studies on Capacitative Calcium Entry in Vascular Smooth Muscle Cells by Measuring 45Ca2+ Influx

Capacitative calcium entry was studied in the A7r5 vascular smooth muscle cell line by measuring 45Ca2+ influx. Entry was induced by depletion of the Ca2+ pools by either the receptor agonist [Arg]8vasopressin (AVP) or the SR-Ca2+-ATPase inhibitor thapsigargin (TG). TG showed a higher efficacy for calcium influx than AVP. This is probably due to a larger Ca2+ release from the pools induced by TG compared to AVP and the irreversible inhibition of the SR-Ca2+-ATPase by TG causing influx to persist for a longer period of time. At maximally effective concentrations signals induced by AVP and TG were synergistic in the absence but not in the presence of the intracellular calcium chelator, 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Depolarisation with 55 mM KCl completely inhibited 45Ca2+ influx induced by TG but only slightly the one induced by AVP, both effects being less pronounced in the presence of BAPTA. [Ca2+]c signals induced by AVP and TG were both inhibited by depolarisation.In conclusion, although our results show differences between AVP- and TG-induced Ca2+ influx, they can be explained by their different mechanism of action and are in accordance with an activation of the same capacitative entry pathway by both agents.

Effects of EGTA on calcium signaling in airway epithelial cells

We have studied the effect of the extracellular calcium buffers ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) as well as varying extracellular calcium concentration on the intracellular free calcium response to histamine in a cystic fibrosis airway epithelial cell line (CF/T43). Histamine stimulates a rapid transient increase in cell calcium followed by a slower second peak lasting several minutes. Bathing cells in nominally calcium-free medium (no added calcium) did not abolish the second peak in the biphasic response to histamine. We examined the effect of including either 1 mM EGTA or BAPTA in the perfusate to investigate whether influx might have been supported by trace amounts of calcium in the nominally calcium-free medium. The second histamine-stimulated peak had a shorter duration but similar amplitude in the presence of BAPTA. In contrast, the second peak was completely abolished by EGTA. To examine if the different histamine responses were due to EGTA's lower dissociation constant or some pharmacological effect of unbound EGTA, extracellular calcium was removed by pretreating the saline with BAPTA covalently bound to polystyrene beads, and effluent from the imaging chamber was collected and analyzed for calcium contamination. Histamine stimulation still produced a biphasic calcium response when extracellular free calcium was approximately 7 nM, a concentration that should reverse the electrochemical gradient for calcium at the plasma membrane. These data suggest that the unbound form of EGTA can interfere with calcium signaling in cells through inhibition of its release from intracellular stores.

Role of diradylglycerol formation in H2O2 and lactoferrin release in adherent human polymorphonuclear leukocytes

Polymorphonuclear leukocytes (PMNs) adherent to fibrinogen exhibit a delay in the release of H2O2 in response to fMLP. Previously, we demonstrated that H2O2 release in adherent PMNs coincides with the exocytosis of lactoferrin-containing specific granules and activation of phospholipase D (PLD). We also found that chelation of intracellular calcium blocked both lactoferrin and H2O2 release in stimulated PMNs in spite of the fact that adhesion and spreading remained normal. Since diradylglycerol (DRG) formation has been implicated in PMN secretion and oxidant release, we determined the effect of intracellular calcium chelation on PLD activation and DRG formation to ascertain whether DRG formation was coupled to lactoferrin and H2O2 release. We observed that chelation of intracellular calcium with bis-(O-aminophenoxy)-ethanol-N,N;N'-tetraacetic acid (BAPTA) prevented PLD activation as monitored by inhibition of phosphatidylethanol formation. Formation of DRG derived from phosphatidic acid (PA) was also inhibited in the presence of BAPTA. Following the addition of the calcium ionophore ionomycin to the BAPTA-treated PMNs, lactoferrin and H2O2 release was coincident with the onset of DRG formation. Also the addition of sn-1,2-didecanoylglycerol to the BAPTA-treated PMNs stimulated them to release H2O2. Our studies support the hypothesis that DRG derived from PLD activation is required for degranulation of specific granules and associated H2O2 release from adherent PMNs.

Calcium mobilization is required for nuclear vesicle fusion in vitro: Implications for membrane traffic and IP3 receptor function

We studied the fusion of nuclear vesicles bound to chromatin in Xenopus egg extracts. Fusion was inhibited by 5 mM BAPTA, a Ca2+ buffer that suppresses cytosolic [Ca2+] gradients. The BAPTA-inhibited step in fusion was biochemically distinct from, and occurred later than, the GTPγS-sensitive step mediated by the monomeric GTPase, ADP-ribosylation factor. Exogenous inositol 1,4,5-trisphosphate (IP3), which triggers Ca2+ release from lumenal stores via IP3 receptors, stimulated fusion in the presence of BAPTA. This rescue was specific, because inositol 1,3,4-trisphosphate had no effect. Heparin, a potent antagonist of IP3 receptors, independently blocked fusion in an IP3-reversible manner. We suggest that phosphoinositide signaling may regulate nuclear vesicle fusion.

Properties of the early phases of crotoxin poisoning at frog neuromuscular junctions

L. Rodrigues-Simioni, B. J. Hawgood and I. C. H. Smith. Properties of the early phases of crotoxin poisoning at frog neuromuscular junctions. Toxicon 28, 1479–1489, 1990.—Addition of crotoxin to the frog neuromuscular junction in either 0.9 mM Ca 2+ plus tubocurarine or 0.5 mM Ca 2+ Ringer solution produced a triphasic change in the amplitude of nerve-evoked endplate potentials (e.p.p.s) and, with 0.5 mM Ca 2+, a biphasic change in miniature endplate potential (m.e.p.p.) frequency. The secondary phase of rising e.p.p. amplitude was associated with an increase in facilitation of e.p.p. amplitude with closely spaced twin impulses; the increase in spontaneous release lagged behind that of evoked release. When a calcium chelator, BAPTA, was loaded into presynaptic nerve terminals to buffer cytosolic free Ca 2+, both e.p.p. amplitude and twin-impulse facilitation were increased by crotoxin to a similar extent relative to that in the control without BAPTA. The duration of the increase in twin-impulse facilitation was reduced but the duration of the increase in e.p.p. amplitude was unaffected by BAPTA loading. The presence of BAPTA did not alter the characteristic changes in spontaneous release in response to crotoxin. These results suggest that the augmentation of evoked and spontaneous transmitter release by crotoxin is not primarily due to changes in cytosolic Ca 2+. The response time between stimulus and e.p.p. peak was lengthened in all phases due to prolongation of the interval between stimulus and e.p.p. onset and, in the secondary and tertiary phases, slowing of e.p.p. rise-time. The protein kinase C inhibitor, H-7, produced complex changes in e.p.p.s. under control conditions but did not alter the triphasic response characteristic of intoxication. These results suggest that crotoxin initiates a primary disturbance in the phasic release process leading to a series of time-gated changes which include transient facilitation then uncoupling of phasic release and generalized acceleration of spontaneous release.