Search

Dysferlin-null A/J myofibers generate abnormal Ca2+ transients that are slightly reduced in amplitude compared to controls. These are further reduced in amplitude by hypoosmotic shock and often appear as Ca2+ waves (Lukyanenko et al., J. Physiol., 2017). Ca2+ waves are typically associated with Ca2+-induced Ca2+ release, or CICR, which can be myopathic. We tested the ability of a permeable Ca2+ chelator, BAPTA-AM, to inhibit CICR in injured dysferlin-null fibers and found that 10–50 nM BAPTA-AM suppressed all Ca2+ waves. The same concentrations of BAPTA-AM increased the amplitude of the Ca2+ transient in A/J fibers to wild type levels and protected transients against the loss of amplitude after hypoosmotic shock, as also seen in wild type fibers. Incubation with 10 nM BAPTA-AM led to intracellular BAPTA concentrations of ~60 nM, as estimated with its fluorescent analog, Fluo-4AM. This should be sufficient to restore intracellular Ca2+ to levels seen in wild type muscle. Fluo- 4AM was ~10-fold less effective than BAPTA-AM, however, consistent with its lower affinity for Ca2+. EGTA, which has an affinity for Ca2+ similar to BAPTA, but with much slower kinetics of binding, was even less potent when introduced as the -AM derivative. By contrast, a dysferlin variant with GCaMP6fu in place of its C2A domain accumulated at triad junctions, like wild type dysferlin, and suppressed all abnormal Ca2+ signaling. GCaMP6fu introduced as a Venus chimera did not accumulate at junctions and failed to suppress abnormal Ca2+ signaling. Our results suggest that leak of Ca2+ into the triad junctional cleft underlies dysregulation of Ca2+ signaling in dysferlin-null myofibers, and that dysferlin’s C2A domain suppresses abnormal Ca2+ signaling and protects muscle against injury by binding Ca2+ in the cleft. [ABSTRACT FROM AUTHOR]

The physiological significance of calcium ions such as the role in cellular signalling, cell growth, etc. have driven the development of methods to detect and monitor the level of Ca2+ ions, both in vivo and in vitro. Although various approaches for the detection of calcium ions have been reported, methods based on small molecular fluorescent probes have unique advantages including small probe size, easy monitoring of detection processes and applicability in biological systems. In this review article, we will discuss the progress in the development of Ca2+‐binding fluorescent probes by taking into account the types of chelating groups that have been employed for Ca2+ binding. [ABSTRACT FROM AUTHOR]

Background: Our previous finding showed that brain ischemic preconditioning mediates neuroprotection through endoplasmic reticulum (ER) stress-induced autophagy. This study was aimed at exploring the role of ER chaperone GRP78 in IPC induced autophagy activation in neural cells. Results: Ischemic preconditioning (IPC) and oxygen glucose deprivation (OGD) models were established in rat pheochromocytoma (PC12) cells and primary cultured murine cortical neurons. IPC exerted neuroprotection against subsequent OGD injury in both PC12 cells and primary cortical neurons. IPC increased GRP78 expression and activated autophagy, as evidenced by upregulated LC3 and Beclin1, increased autophagic flux and formation of autophagosomes. BAPTA(dibromo-1,2-bis(aminophenoxy)ethane N,N,N9,N9 - tetra acetic acid, 0.125-2 μM) and small interfering RNA targeted GRP78 abrogated IPC induced neuroprotection and decreased the expression of GRP78, LC3II/LC3I and Beclin1. In contrast, lentiviral vector mediated GRP78 overexpression (LV-GRP78) strengthened resistance of PC12 cells to OGD injury and increased LC3 and Beclin1 expression. Moreover, knockdown of GRP78 in stable GRP78 overexpressing PC12 cells abolished the upregulation of LC3II/LC3I. GRP78 might activate autophagy through AMPK - mTOR pathway. Conclusion: These results suggest that IPC- induced GRP78 upregulation is involved in autophagy activation, and hence exerts protection against ischemic injury in neural cells. [ABSTRACT FROM AUTHOR]

ABSTRACT Osteoclasts are multinucleated cells responsible for the resorption of bone and other mineralized tissues during development, physiological remodeling, and pathological bone loss. Osteoclasts have the ability to resorb substrate while concurrently migrating. However, the subcellular processes underlying migration are not well understood. It has been proposed that, in other cell types, cytosolic free Ca2+ concentration ([Ca2+]i) regulates cell protrusion as well as retraction. Integration of these distinct events would require precise spatiotemporal patterning of subcellular Ca2+. The large size of osteoclasts offers a unique opportunity to monitor patterns of Ca2+ during cell migration. We used ratiometric imaging to map [Ca2+]i within rat and mouse osteoclasts. Migration was characterized by lamellipodial outgrowth at the leading edge, along with intermittent retraction of the uropod. Migrating osteoclasts displayed elevation of [Ca2+]i in the uropod, that began prior to retraction. Dissipation of this [Ca2+]i gradient by loading osteoclasts with the Ca2+ chelator BAPTA abolished uropod retraction, on both glass and mineralized substrates. In contrast, elevation of [Ca2+]i using ionomycin initiated prompt uropod retraction. To investigate downstream effectors, we treated cells with calpain inhibitor-1, which impaired uropod retraction. In contrast, lamellipodial outgrowth at the leading edge of osteoclasts was unaffected by any of these interventions, indicating that the signals regulating outgrowth are distinct from those triggering retraction. The large size of mature, multinucleated osteoclasts allowed us to discern a novel spatiotemporal pattern of Ca2+ involved in cell migration. Whereas localized elevation of Ca2+ is necessary for uropod retraction, lamellipod outgrowth is independent of Ca2+-a heretofore unrecognized degree of specificity underlying the regulation of osteoclast migration. © 2014 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

NMDA receptor (NMDAR) activation controls long-term potentiation (LIP) as well as long-term depression (LTD) of synaptic transmission, cellular models of learning and memory. A long-standing view proposes that a high level of Ca2+ entry through NMDAR5 triggers LTP; lower Ca2+ entry triggers LTD. Here we show that ligand binding to NMDAR5 is sufficient to induce LID; neither ion flow through NMDARs nor Ca2+ rise is required. However, basal levels of Ca2+ are permissively required. Lowering, but not maintaining, basal Ca2+ levels with Ca2+ chelators blocks LTD and drives strong synaptic potentiation, indicating that basal Ca2+ levels control NMDAR-dependent LTD and basal synaptic transmission. Our findings indicate that metabotropic actions of NMDARs can weaken active synapses without raising postsynaptic calcium, thereby revising and expanding the mechanisms controlling syn- aptic plasticity. [ABSTRACT FROM AUTHOR]

The American alligator can hibernate during winter, which may lead to osmotic imbalance because of reduced kidney function and lack of food consumption during this period. Accordingly, we hypothesized that their red blood cells would have a well-developed regulatory volume decrease (RVD) to cope with the homeostatic challenges associated with torpor. Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca²+ concentration were visualized using fluorescence microscopy and fluo-4-AM. Osmotic fragility increased and the ability to regulate volume was inhibited when extracellular Na+ was replaced with K+, or when cells were exposed to the K+ channel inhibitor quinine, indicating a requirement of K+ efflux for RVD. Addition of the ionophore gramicidin to the extracellular medium decreased osmotic fragility and also potentiated volume recovery, even in the presence of quinine. In addition, hypotonic shock (0.5 x Ringer) caused an increase in cytosolic Ca²+, which resulted from Ca²+ influx because it was not observed when extracellular Ca²+ was chelated with EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid). Furthermore, cells loaded with BAPTA-AM (1,2-bis(2-aminophenoxymethyl)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester) or exposed to a low Ca²+-EGTA hypotonic Ringer had a greater osmotic fragility and also failed to recover from cell swelling, indicating that extracellular Ca²+ was needed for RVD. Gramicidin reversed the inhibitory effect of low extracellular Ca²+. Finally, and surprisingly, the Ca²+ ionophore A23187 increased osmotic fragility and inhibited volume recovery. Taken together, our results show that cell swelling activated a K+ permeable pathway via a Ca²+-dependent mechanism, and this process mediated K+ loss during RVD. [ABSTRACT FROM AUTHOR]

Infection efficiency is the key issue for gene delivery using adenovirus vector and usually unsatisfactory. In this study, recombinant adenoviruses encoding recombinant human EPO were prepared using the Adeasy system, and injected into the mammary gland of goats via the teat canal. BAPTA was used to treat the mammary gland to facilitate adenoviruses infection compared with EGTA. Milk serum was collected from the infected mammary gland and characterized by ELISAs and Western blotting. Expression level of rhEPO from the group treated by BAPTA was higher than that treated by EGTA. [ABSTRACT FROM AUTHOR]

An increase in cytosolic Ca2+ concentration in coronary artery smooth muscle causes a contraction but in endothelium it causes relaxation. Na+-Ca2+-exchanger (NCX) may play a role in Ca2+ dynamics in both the cell types. Here, the NCX-mediated 45Ca2+ uptake was compared in Na+-loaded pig coronary artery smooth muscle and endothelial cells. In both the cell types, this uptake was inhibited by KB-R7943, SEA 0400 and by monensin, but not by cariporide. Prior loading of the cells with the Ca2+ chelator BAPTA increased the NCX-mediated 45Ca2+ uptake in smooth muscle but not in endothelial cells. In the presence or absence of BAPTA loading, the Na+-mediated 45Ca2+ uptake was greater in endothelial than in smooth muscle cells. In smooth muscle cells without BAPTA loading, thapsigargin diminished the NCX-mediated 45Ca2+ entry. This effect was not observed in endothelial cells or in either cell type after BAPTA loading. The results in the smooth muscle cells are consistent with a limited diffusional space model in which the NCX-mediated 45Ca2+ uptake was enhanced by chelation of cytosolic Ca2+ or by its sequestration by the sarco/endoplasmic reticulum Ca2+ pump (SERCA). They suggest a functional linkage between NCX and SERCA in the smooth muscle but not in the endothelial cells. The concept of a linkage between NCX and SERCA in smooth muscle was also confirmed by similar distribution of NCX and SERCA2 proteins when detergent-treated microsomes were fractionated by flotation on sucrose density gradients. Thus, the coronary artery smooth muscle and endothelial cells differ not only in the relative activities of NCX but also in its functional linkage to SERCA. [ABSTRACT FROM AUTHOR]

A series of new 1,2-bis-(2-aminophenoxyethane) N,N,N',N'-tetraacetic acid (BAPTA) chelators were designed and synthesized to form novel and improved fluorescent indicators. Substituted 2-nitrophenol, acting as the material, reacted in five steps to give BAPTA-derived chelators. The Ca2+ affinity of BAPTA chelators could increase significantly by addition of electron-donating groups and decrease by electron-withdrawing groups. Compounds 3a-e were treated with iron powder instead of hydrogenation, obtaining a satisfying yield. The structures of all compounds were characterized by infrared, 1H NMR, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra. [ABSTRACT FROM AUTHOR]

Glial cells can be activated by neurotransmitters via metabotropic, G protein-coupled receptors. We have studied the effects of `global' G protein activation by GTP--S on the membrane potential, membrane conductance, intracellular Ca2+ and Na+ of the giant glial cell in isolated ganglia of the leech Hirudo medicinalis. Uncaging GTP--S (injected into a giant glial cell as caged compound) by moderate UV illumination hyperpolarized the membrane due to an increase in K+ conductance. Uncaging GTP--S also evoked rises in cytosolic Ca2+ and Na+, both of which were suppressed after depleting the intracellular Ca2+ stores with cyclopiazonic acid (20 μmol l-1). Uncaging inositol-trisphosphate evoked a transient rise in cytosolic Ca2+ and Na+ but no change in membrane potential. Injection of the fast Ca2+ chelator BAPTA or depletion of intracellular Ca2+ stores did not suppress the membrane hyperpolarization induced by uncaging GTP--S. Our results suggest that global activation of G proteins in the leech giant glial cell results in a rise of Ca2+-independent membrane K+ conductance, a rise of cytosolic Ca2+, due to release from intracellular stores, and a rise of cytosolic Na+, presumably due to increased Na+/Ca2+ exchange. [ABSTRACT FROM AUTHOR]

To investigate the safety, tolerability and pharmacokinetics of DP-b99 in healthy volunteers. DP-b99 is a newly developed lipophilic, cell permeable derivative of BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid), which selectively modulates the distribution of metal ions in hydrophobic milieu, and is in clinical development as a neuroprotectant for cerebral ischaemic stroke. To our knowledge no BAPTA derivative has ever been administered to man. Here we report the first human administration of DP-b99 in a phase I, two-part, double-blind, randomized placebo controlled study, with single IV doses of 0.003–1.0 mg kg−1 day−1 DP-b99 (part 1) or multiple ascending doses of 0.03–1.0 mg kg−1 day−1 DP-b99 over 4 days (part 2). A double-blind, dose escalating tolerability study of DP-b99 in normal (young – aged between 18 and 40 years and elderly – aged between 65 and 85 years) healthy adult male volunteers was conducted. Part 1 of the study investigated single administration of ascending intravenous doses, and part 2 examined the effects of ascending doses given repeatedly over 4 days . Twenty-four young volunteers in part 1 received single dose administrations and 26 young volunteers in part 2 received repeated ascending dose administrations of either intravenous DP-b99 or placebo. Subsequently, 10 elderly volunteers received repeated intravenous DP-b99 (1 mg kg−1) or placebo in part 2 over 4 days. Adverse events were identified by either subject self reporting or based upon laboratory parameters (blood chemistry, complete blood cell count, prothrombin time (PT), activated partial thromboplastin (PTT), physical examination, vital signs (blood pressure, pulse rate, respiratory rate, body temperature) and urinalysis. A comprehensive set of cardiovascular parameters was assessed as well (blood pressure, 12 lead-ECG recordings and continuous bedside cardiac monitoring for 6 h on day 1). The administration of DP-b99 up to the highest dose of 1.0 mg kg−1 was well tolerated and had an acceptable safety profile up to the highest dose of 1.0 mg kg−1 tested in both study parts. No serious or severe adverse events were encountered. Eight mild to moderate adverse events were observed in six of the seven young subjects treated with four repeated doses of 1.0 mg kg−1, with reversible phlebitis being the most frequently reported adverse event. The drug was tolerated better at the injection site by the elderly group compared with the younger subjects. No adverse effects were observed in cardiovascular parameters sensitive to trans-membranous calcium concentrations. The pharmacokinetic parameters were derived by noncompartmental analysis. On day 1 following administration of 1 mg kg−1 the mean half-life of DP-b99 in young volunteers was 3.47 ± 0.90 h and in the elderly was 2.11 ± 0.09 h. On day 4 following the same administration of DP-b99 the mean half-life was 4.36 ± 1.49 and 2.10 ± 1.14 h in the young and elderly, respectively. There was higher systemic exposure in the elderly, for example Cmax, had a mean 1.6-fold higher exposure on day 1 (95% CI Lower 0.90, Upper 2.74) and 2.5-fold on day 4 (95%CI 1.70, 3.68). This increase is in line with the presumed central role of hepatic blood flow in the elimination of DP-b99. No accumulation was observed after repeated dosing with 1 mg kg−1 (mean accumulation calculated by AUC(0, 24 h) (day 4) : AUC(0, 24 h) (day 1) and was observed to be between 0.9 and 1.3 (young, elderly). This study suggests that DP-b99 is well tolerated in healthy young and elderly volunteers within the dose range evaluated. Studies to investigate further the efficacy of the compound are in progress. [ABSTRACT FROM AUTHOR]

In living organisms, Ca2+ signalling is central to cell physiology. The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA) has been widely used as a probe to test the role of calcium in a large variety of cell functions. Here we show that in most cell types BAPTA has a potent actin and microtubule depolymerizing activity and that this activity is completely independent of Ca2+ chelation. Thus, the depolymerizing effect of BAPTA is shared by a derivative (D-BAPTA) showing a dramatically reduced calcium chelating activity. Because the extraordinary depolymerizing activity of BAPTA could be due to a general depletion of cell fuel molecules such as ATP, we tested the effects of BAPTA on cellular ATP levels and on mitochondrial function. We find that BAPTA depletes ATP pools and affects mitochondrial respiration in vitro as well as mitochondrial shape and distribution in cells. However, these effects are unrelated to the Ca2+ chelating properties of BAPTA and do not account for the depolymerizing effect of BAPTA on the cell cytoskeleton. We propose that D-BAPTA should be systematically introduced in calcium signalling experiments, as controls for the known and unknown calcium independent effects of BAPTA. Additionally, the concomitant depolymerizing effect of BAPTA on both tubulin and actin assemblies is intriguing and may lead to the identification of a new control mechanism for cytoskeleton assembly. [ABSTRACT FROM AUTHOR]

The peptide hormone calcitonin is a potent inhibitor of osteoclastic resorption, but it is unstable and poorly absorbed following oral administration. Conjugates of salmon calcitonin covalently linked to low-molecular-weight amphiphilic polymers show improved stability and absorption. The purpose of this study was to investigate the biological activity of these conjugates in vitro using rat osteoclasts and HEK-293 cells transfected with the C1a isoform of the calcitonin receptor. Salmon calcitonin or its conjugates (10 pM-10 nM) caused rapid arrest of osteoclast membrane ruffling and subsequent retraction. The same amphiphilic polymer attached to an unrelated protein had no effect on osteoclast morphology or motility. Since calcitonin-induced retraction of osteoclasts is thought to be mediated by Ca2+ signaling, we investigated the effects of calcitonin and its conjugates on cytosolic free Ca2+ concentration ([Ca24]i). In HEK-293 cells transfected with the calcitonin receptor, these agents induced transient elevations of [Ca2+]i. However, the rise of [Ca2+]i in HEK-293 cells occurred at concentrations 100-1000-fold higher than those required to elicit osteoclast retraction. To investigate the role of Ca2+ in osteoclast retraction, we preloaded cells with BAPTA to buffer changes in [Ca2+]i. BAPTA decreased the initial rate of calcitonin-induced osteoclast retraction, but it did not affect the degree of retraction 2-3 hours following calcitonin, indicating that retraction is mediated primarily by Ca(2+)-independent processes. We conclude that calcitonin conjugates cause osteoclast retraction and [Ca2+]i signaling in a manner similar to that elicited by calcitonin. Thus, orally bioavailable calcitonin conjugates show potential for use as antiresorptive agents. [ABSTRACT FROM AUTHOR]

Different types of voltage-dependent calcium channels (VDCCs) have been recognized based on their molecular structure as well as their pharmacological and biophysical properties. One of these, the P/Q type, is the main channel involved in nerve evoked neurotransmitter release at neuromuscular junctions (NMJs) and many central nervous system synapses. However, under particular experimental or biological conditions, other channels can be involved. L-type VDCC presence at the NMJ has been demonstrated by the contribution to the perineural calcium currents (I[sub Ca] ) at adult mice Bapta-loaded NMJs. This is probably a result of a reduction in Ca[sup 2+] inactivation. The L-type current was not coupled to neurotransmitter release, but became coupled, as demonstrated by the release of acetylcholine, after the inhibition of serine/threonine protein phosphatases with okadaic acid (OA). Thus, under these conditions, L-type channels were unmasked at Bapta- but not at Egta-loaded NMJs. This suggests that the speed, not the capacity, of the calcium chelator was decisive in preventing Ca[sup 2+] -inactivation and facilitating the contribution to neurotransmitter release. At neonatal rat NMJs, N-type VDCCs were involved early during development whereas P/Q-type VDCCs play a main role at all stages of development. Furthermore, P/Q-type VDCCs were more efficiently coupled to neurotransmitter release than N-type VDCCs. This difference could be accounted for by a differential location of these channels at the release site. Neuromuscular transmission in P/Q-type calcium channel knock out ataxic mice jointly depends on both N-type and R-type channels and shows several altered properties including low quantal content. Thus, calcium channels may be recruited to mediate neurotransmitter release with a functional hierarchy where the P/Q channel seems to be the channel most suited to mediate exocytosis at NMJs. [ABSTRACT FROM AUTHOR]

Ca2+ chelating agents are widely used in biological research for Ca2+ buffering. Here we report that BAPTA, EDTA and HEDTA produce fast, reversible, voltage-dependent inhibition of swelling-activated Cl− current (ICl,swell) in LNCaP prostate cancer epithelial cells that is unrelated to their Ca2+ binding. BAPTA was the most effective (maximal blockade 67%, IC50=70 μM, at +100 mV) followed by EDTA and HEDTA. ICl,swell blockade by EDTA was pH-dependent. BAPTA blocked ICl,swell also in other cell types. We conclude that Ca2+ chelating agents block ICl,swell by acting directly on the underlying channel, and that the negative charge of the free chelator form is critical for the blockade. [Copyright &y& Elsevier]

Rises in free [Ca2+]i in response to various tetanic stimuli (Ca2+ transient) in frog motor nerve terminals were measured by recording fluorescence changes of Ca2+ indicators and analyzed in relation to short-term synaptic plasticity. Ca2+ transients reached a plateau after 10–20 impulses at 100 Hz and decayed in a three-exponential manner, in which the fast component was predominant. The plateau and fast component of the Ca2+ transient were elevated infralinearly with an increase in tetanus frequency. Computer simulation showed that the Ca2+ transients estimated from fluorescence changes faithfully reflect the true changes in [Ca2+]i except for the initial 20 ms. A slow Ca2+ chelator, EGTA, loaded into the nerve terminal, decreased the magnitude of both the fast and slow components of facilitation of transmitter release and the time constant of the former. A fast Ca2+ chelator, BAPTA, decreased the magnitude of fast facilitation but slightly increased its time constant. These results suggest that Ca2+ transients in the frog motor nerve terminals are primarily caused by Ca2+ entry and are dissipated by three components, in which the rate of the fast component is equivalent to that of free Ca2+ diffusion. The residual Ca2+ in the nerve terminals after stimulation accounts for the fast component of facilitation. [ABSTRACT FROM AUTHOR]

We studied regulation of Cl–transport by cAMP and Ca2+ in renal epithelial A6 cells. Stimulation of A6 cells by 1 mM 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of phosphodiesterase), which increased cytosolic cAMP, elicited biphasic increases in short-circuit current ( Isc), i.e., a transient phase followed by a sustained one. Apical application of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, a Cl–channel blocker) markedly and dose-dependently inhibited the IBMX-induced Isc. Pretreatment with nifedipine (100 μM, a Ca2+ channel blocker) or 1,2-bis (o-aminophenoxy)-ethane- N, N, N′, N′-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA/AM, 10 μM, a Ca2+ chelator) partially but markedly inhibited the Isc. On the other hand, a cAMP-dependent protein kinase inhibitor, H89 (0.5 μM for 1 h), also reduced the IBMX-induced Isc to a level similar to that following nifedipine or BAPTA pretreatment. Nifedipine had no synergistic effects on the IBMX-induced Isc in cells treated with H89. Ionomycin (a Ca2+ ionophore) could mimic the transient increase dose dependently, and H89 did not block the ionomycin-induced Isc. Taken together, our observations suggest that: (1) part of the IBMX-stimulated Cl–release is regulated by an increased cytosolic Ca2+ through nifedipine-sensitive Ca2+ influx; (2) cAMP-dependent phosphorylation may be required for elevation of the cytosolic Ca2+ concentration but not for activation of Cl–channels, which are directly activated by cytosolic Ca2+; and (3) the IBMX-induced sustained Cl–release requires cAMP elevation in addition to an increase in the cytosolic Ca2+ concentration. [ABSTRACT FROM AUTHOR]

The early outward current in opener muscle fibres of crayfish ( Procambarus clarkii) was studied using the two-electrode voltage-clamp technique. This current was abolished in Ca-free and 5 mM Cd solutions, and was blocked by extra- or intracellular tetraethylammonium, indicating that it was a Ca-dependent K current [ I]. I was voltage dependent, apamin insensitive and sensitive to charybdotoxin (CTX), which, in addition to its tetraethylammonium sensitivity, suggests that the channels mediating I behave in a BK type manner. I activation was extremely fast, reaching a maximum within 5 ms, and the inactivation was incomplete, stabilizing at a persistent steady-state. I was insensitive to intracellular ethylenebis(oxonitrilo)tetraacetate (EGTA), but was abolished by injection of the faster Ca chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N∼'-tetraacetic acid (BAPTA), suggesting that voltage-dependent Ca channels and those mediating I should be clustered closely on the membrane. Under two-electrode current-clamp recording mode, low amplitude, graded responses were evoked under control conditions, whereas repetitive all-or-none spikes were elicited by application of CTX or after loading the cells with BAPTA. We conclude that I activates extremely quickly, is persistent and is responsible for the generation and control of the low amplitude, graded, active responses of opener muscle fibres. [ABSTRACT FROM AUTHOR]

Calcium-sensitivity of contraction, force-velocity relation and ATP hydrolysis rate at different pH (6.2-7.8) were investigated in skinned smooth muscle preparations from the guinea pig taenia coli. Varied free-calcium levels were buffered by 4 mM BAPTA (1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid) which has calcium binding properties little affected by pH. A small increase of calcium-sensitivity of contraction was seen at pH 6.2 compared to 6.9 and 7.8 (ED50 shift of about 0.15 pCa units). The isometric force and V in fibres activated either by calcium or by thiophosphorylation of the myosin light chains were each reduced by about 15% at pH 6.2 compared to 6.9 and 7.8. Following an isotonic quick release the shortening velocity decreases with time. This effect was more pronounced at pH 6.2 than at pH 6.9 or 7.8. The ATP hydrolysis rates in relaxed and thiophosphorylated fibres were essentially unaffected by alteration in pH between 6.2 and 7.8. Due to the lower force, energetic cost of force maintenance was thus somewhat increased at pH 6.2. These results suggest that pH alteration between 6.2 and 7.8 have effects on the properties of the contractile machinery of the smooth muscle in the skinned guinea pig taenia coli. The effects are however small and therefore probably of little functional importance over a pH range which should cover most cases of intracellular pH alteration under physiological or pathophysiological conditions. [ABSTRACT FROM AUTHOR]

In order to test the hypothesis that excitation in Drosophila photoreceptors is mediated by Ca released from internal stores, the Ca buffers EGTA, BAPTA and di-bromo-BAPTA (DBB) were introduced into dissociated photoreceptors via whole-cell recording pipettes. All buffers were preloaded with Ca to provide the same free Ca concentration (250 n M). EGTA (up to 18 m M free buffer) had only weak effects upon voltage-clamped flash responses in normal Ringer's solution (1.5 m M Ca), and no effect in Ca-free solution. The maximum BAPTA concentration tested (14.4 m M free BAPTA) reduced the initial rate of rise by ca. 5000-fold in normal Ringer's solution; by ca. 500-fold in Cafree solution; and only ca. 60-fold in the absence of Mg, which preferentially blocks one component of the light-sensitive current. Although BAPTA delayed the time-to-peak in normal Ringer's solution, responses in Ca free Ringer's solution were accelerated. These results support the role of Ca influx in regulating sensitivity and response kinetics; however, in view of the high concentrations required to attenuate responses in Ca free Ringer's solution, the role of Ca release in excitation remains unclear. DBB was ca. 2-3 fold more potent than BAPTA, and at concentrations > 5 m M had a qualitatively different action, greatly delaying the time-to-peak. This suggests DBB may have distinct pharmacological actions or access to compartments inaccessible to BAPTA. The only current activated by introducing 5-500 μ M Ca (buffered with nitrilo-triacetic acid) was electrogenic Na/Ca exchange. When this was blocked by removing Nao, a novel cationic conductance was activated. However, its properties did not resemble those the light-activated conductance, and thus do not support the hypothesis that Ca is sufficient for excitation. [ABSTRACT FROM AUTHOR]

Action potentials evoke calcium transients in dendrites of neocortical pyramidal neurons with time constants of <100 ms at physiological temperature. This time period may not be sufficient for inflowing calcium ions to equilibrate with all present Ca2+-binding molecules. We therefore explored nonequilibrium dynamics of Ca2+ binding to numerous Ca2+ reaction partners within a dendritelike compartment using numerical simulations. After a brief Ca2+ influx, the reaction partner with the fastest Ca2+ binding kinetics initially binds more Ca2+ than predicted from chemical equilibrium, while companion reaction partners bind less. This difference is consolidated and may result in bypassing of slow reaction partners if a Ca2+ clearance mechanism is active. On the other hand, slower reaction partners effectively bind Ca2+ during repetitive calcium current pulses or during slower Ca2+ influx. Nonequilibrium Ca2+ distribution can further be enhanced through strategic placement of the reaction partners within the compartment. Using the Ca2+ buffer EGTA as a competitor of fluo-3, we demonstrate competitive Ca2+ binding within dendrites experimentally. Nonequilibrium calcium dynamics is proposed as a potential mechanism for differential and conditional activation of intradendritic targets. [ABSTRACT FROM AUTHOR]

Five BAPTA buffers with differential affinities for Ca have been examined for their effects on cell plate formation in stamen hair cells of Tradescantia. The five include 5,5′-dimethyl BAPTA (Kd=0.15 μM), BAPTA (Kd=0.22 μM), 5,5′-dibromo BAPTA (Kd=1.5 μM), 5-methyl,5′-nitro BAPTA (Kd=22 μM), and 5-nitro BAPTA (Kd=40 μM). At a concentration of 5 mM and 25 mM in the pipette, the buffers were iontophoretically microinjected into dividing stamen hair cells (2 nA for 1 min) prior to or at the onset of cell plate formation. At the lowest concentration (5 mM), only one buffer, 5,5′-dibromo BAPTA, inhibits cell plate formation, and is most effective if delivered at the moment of cell plate vesicle aggregation. The inhibitory effects appear as a slowing of cell plate expansion, the formation of distorted plates, or the complete dissolution of plates that might have initiated normally. When the pipette tip concentration is elevated to 25 mM, the effects of 5,5′-dibromo BAPTA become more profound. At these levels 5,5′-dimethyl BAPTA, BAPTA, and 5-nitro BAPTA also modulate cell plate formation, producing effects similar to that of 5,5′-dibromo BAPTA at the lower concentration. Independent studies using fura-2 as a fluorescent analogue of the BAPTA buffers, indicate that the apparent effective concentration for 5,5′-dibromo BAPTA is between 1.0-1.4 mM; its threshold concentration is not known but expected to be somewhat lower. For the other buffers the threshold concentration is between 1.5-2.2 mM. The concentration dependence supports the idea that the buffers facilitate diffusion of Ca away from regions of elevated concentration. The results thus provide evidence that local Ca gradients may be present in the vicinity of the cell plate and that they participate in the cytokinetic process. [ABSTRACT FROM AUTHOR]

Coupling distances between Ca2+ channels and release sensors regulate vesicular release probability (p v). Tight coupling is thought to provide a framework for high p v and loose coupling for high plasticity at low p v. At synapses investigated during development, coupling distances decrease, thereby increasing p v and transmission fidelity. We find that neocortical high-fidelity synapses deviate from these rules. Paired recordings from pyramidal neurons with "slow" and "fast" Ca2+ chelators combined with experimentally constrained simulations suggest that coupling tightens significantly during development. However, fluctuation analysis revealed that neither p v (∼0.63) nor the number of release sites (∼8) changes concomitantly. Moreover, the amplitude and time course of presynaptic Ca2+ transients are not different between age groups. These results are explained by high- p v release sites with Ca2+ microdomains in young synapses and nanodomains in mature synapses. Thus, at neocortical synapses, a developmental reorganization of the active zone leaves p v unaffected, emphasizing developmental and functional synaptic diversity. • Influx-release coupling tightens during development of L5 pyramidal neuron synapses • The vesicular release probability remains high during development • Ca2+ influx dynamics do not change during development • Ca v 2.1, Ca v 2.2 microdomains develop toward Ca v 2.1 nanodomains During development, coupling distances at synapses between Ca v channels and release sensors tighten, thereby increasing release probability. Bornschein et al. find that L5 pyramidal neurons deviate from this rule. Synapses develop from loose coupling with Ca v 2.1 and Ca v 2.2 microdomains to tight coupling with Ca v 2.1 nanodomains, but release probability remains unaffected. [ABSTRACT FROM AUTHOR]