Effects Of BAPTA-AM Research Articles

Effect of the intracellular calcium concentration chelator BAPTA acetoxy-methylester on action potential duration in canine ventricular myocytes.

Intracellular calcium concentration ([Ca2+]i) is often buffered by using the cell-permeant acetoxy-methylester form of the Ca2+ chelator BAPTA (BAPTA-AM) under experimental conditions. This study was designed to investigate the time-dependent actions of extracellularly applied BAPTA-AM on action potential duration (APD) in cardiac cells. Action potentials were recorded from enzymatically isolated canine ventricular myocytes with conventional sharp microelectrodes. The effect of BAPTA-AM on the rapid delayed rectifier K+ current (IKr) was studied using conventional voltage clamp and action potential voltage clamp techniques. APD was lengthened by 5 μM BAPTA-AM - but not by BAPTA - and shortened by the Ca2+ ionophore A23187 in a time-dependent manner. The APD-lengthening effect of BAPTA-AM was strongly suppressed in the presence of nisoldipine, and enhanced in the presence of BAY K8644, suggesting that a shift in the [Ca2+]i-dependent inactivation of L-type Ca2+ current may be an important underlying mechanism. However, in the presence of the IKr-blocker dofetilide or E-4031 APD was shortened rather than lengthened by BAPTA-AM. Similarly, the APD-lengthening effect of 100 nM dofetilide was halved by the pretreatment with BAPTA-AM. In line with these results, IKr was significantly reduced by extracellularly applied BAPTA-AM under both conventional voltage clamp and action potential voltage clamp conditions. This inhibition of IKr was partially reversible and was not related to the Ca2+ chelator effect BAPTA-AM. The possible mechanisms involved in the APD-modifying effects of BAPTA-AM are discussed. It is concluded that BAPTA-AM has to be applied carefully to control [Ca2+]i in whole cell systems because of its direct inhibitory action on IKr.

The role of hydroxyl radicals and calcium ions in the priming of a respiratory burst in neutrophils and the increase in luminol-dependent blood chemiluminescence on exposure to combined magnetic fields with a very weak low-frequency alternating component

The intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA AM) used at low concentrations (1.0 and 2.5 μM) was shown to block the priming effect of weak combined static (42 μT) and low-frequency collinear alternating (1.0, 4.4, and 16.5 Hz; 0.86 μT) magnetic fields. This blockage was inferred from a greater increase in chemiluminescence observed for a mouse neutrophil suspension exposed to combined magnetic fields in response to the bacterial peptide N-formyl–Met–Leu–Phe added in the presence of luminol. Similar results were obtained for the effect of BAPTA AM on luminol-dependent chemiluminescence of whole blood. The priming effect of weak combined magnetic fields on the respiratory burst in neutrophils did not depend on the presence of extracellular Ca2+ and was not affected by the hydroxyl radical scavenger dimethyl sulfoxide used at 0.025–1.0 mM.

Effect of BAPTA-AM on Thawed Stallion Spermatozoa Extended in INRA 96 or Tyrode's Medium

We studied the effect of 1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) on the outcome of cryopreservation of stallion spermatozoa and whether reextension of thawed sperm in a more physiological and Ca2+-containing medium might improve the characteristics of thawed stallion spermatozoa. Individual ejaculates from six stallions were collected and split into three subsamples. The first two samples were supplemented with the membrane-permeable Ca2+ chelator BAPTA-AM at final concentrations of 5 and 10 μM, respectively, while the third subsample served as control. After 4 weeks of storage, samples were thawed in a water bath at 37°C and evaluated using flow cytometry and computer-assisted sperm analysis (CASA). In a second experiment, in order to determine whether restoring Ca2+ could improve sperm quality after cryopreservation, thawed semen was washed by centrifugation and resuspended in Tyrode's complete medium. BAPTA-AM supplementation did not modify the outcome of cryopreservation; however, changing the spermatozoa from INRA 96 to Tyrode's complete medium resulted in significant improvements in the percentages of live sperm and total motility post thaw.

Ca2+‐stimulated adenylyl cyclases regulate the L‐type Ca2+ current in guinea‐pig atrial myocytes

Ca(2+)-stimulated adenylyl cyclases (ACs) have recently been shown to play important roles in pacemaking in the sino-atrial node. Here we present evidence that Ca(2+)-stimulated ACs are functionally active in guinea-pig atrial myocytes. Basal activity of an AC in isolated atrial myocytes was demonstrated by the observations that MDL 12,330A (10 μm), an AC inhibitor, reduced L-type Ca(2+) current (I(CaL)) amplitude, while inhibition of phosphodiesterases with IBMX (100 μm) increased I(CaL) amplitude. Buffering of cytosolic Ca(2+) by exposure of myocytes to BAPTA-AM (5 μm) reduced I(CaL) amplitude, as did inhibition of Ca(2+) release from the sarcoplasmic reticulum with ryanodine (2 μm) and thapsigargin (1 μm). [Ca(2+)]i-activated calmodulin kinase II (CaMKII) inhibition with KN-93 (1 μm) reduced I(CaL), but subsequent application of BAPTA-AM further reduced I(CaL). This effect of BAPTA-AM, in the presence of CaMKII inhibition, demonstrates that there is an additional Ca(2+)-modulated pathway (not dependent on CaMKII) that regulates I(CaL) in atrial myocytes. The effects of BAPTA could be reversed by forskolin (10 μm), a direct stimulator of all AC isoforms, which would restore cAMP levels. In the presence of BAPTA-AM, the actions of IBMX were reduced. In addition, inclusion of cAMP in the patch electrode in the whole-cell configuration prevented the effects of BAPTA. These effects are all consistent with a role for Ca(2+)-stimulated AC in the regulation of atrial myocyte I(CaL).

Intracellular calcium chelation and pharmacological SERCA inhibition of Ca2+ pump in the insular cortex differentially affect taste aversive memory formation and retrieval

Variation in intracellular calcium concentration regulates the induction of long-term synaptic plasticity and is associated with a variety of memory/retrieval and learning paradigms. Accordingly, impaired calcium mobilization from internal deposits affects synaptic plasticity and cognition in the aged brain. During taste memory formation several proteins are modulated directly or indirectly by calcium, and recent evidence suggests the importance of calcium buffering and the role of intracellular calcium deposits during cognitive processes. Thus, the main goal of this research was to study the consequence of hampering changes in cytoplasmic calcium and inhibiting SERCA activity by BAPTA-AM and thapsigargin treatments, respectively, in the insular cortex during different stages of taste memory formation. Using conditioned taste aversion (CTA), we found differential effects of BAPTA-AM and thapsigargin infusions before and after gustatory stimulation, as well as during taste aversive memory consolidation; BAPTA-AM, but not thapsigargin, attenuates acquisition and/or consolidation of CTA, but neither compound affects taste aversive memory retrieval. These results point to the importance of intracellular calcium dynamics in the insular cortex during different stages of taste aversive memory formation.

Pretreatment of BAPTA-AM Suppresses the Genesis of Repetitive Endocardial Focal Discharges and Pacing-Induced Ventricular Arrhythmia During Global Ischemia

In isolated rabbit hearts, repetitive endocardial focal discharges (REFDs) were consistently observed during ventricular fibrillation (VF) with prolonged (>5 minutes) global ischemia (GI). We hypothesized that BAPTA-AM, a calcium chelator, can suppress these REFDs. Using a two-camera optical mapping system, we simultaneously mapped endocardial (left ventricle, LV) and epicardial (both ventricles) activations during ventricular arrhythmia with GI. In 5 hearts (protocol I), we infused Tyrode's solution (no BAPTA-AM) for ≥30 minutes before the onset of no-flow GI. In 7 additional hearts (protocol II), BAPTA-AM (20 μmol/L) was infused for ≥30 minutes before the initiation of GI. In protocol I, sustained VF (>30 seconds) was successfully induced in all 5 hearts with prolonged GI. REFDs were present in >85 % of recording time. In protocol II, however, ventricular arrhythmia was not inducible and REFDs were not observed after 5-minute GI in 5 hearts. Effects of BAPTA-AM on intracellular calcium (Ca(i) ) at the LV endocardium were also evaluated in 5 hearts (protocol III) using dual Ca(i) /membrane potential mapping. GI, both without and with BAPTA-AM pretreatment, caused a decrease of Ca(i) amplitude during S(1) pacing. However, this effect was more pronounced in the hearts with BAPTA-AM pretreatment (P < 0.001). GI, without BAPTA-AM pretreatment, caused broadening of Ca(i) transient. In contrast, GI, with BAPTA-AM pretreatment, caused narrowing of Ca(i) transient. BAPTA-AM pretreatment attenuates Ca(i) transient, suppressing the genesis of REFDs and pacing-induced ventricular arrhythmia during GI. These findings support the notion that Ca(i) dynamics is important in the maintenance of REFDs.

Abstract 5328: Intracellular Calcium Mediated Late-Phase 3 Early Afterdepolarization Induces Ventricular Fibrillation in Isolated Rabbit Hearts with Activated K ATP Channel

Introduction: The mechanism and importance of late-phase 3 early after depolarization (EAD) in ventricular arrhythmogenesis and the corresponding intracellular calcium dynamics are unknown. Methods: We studied 22 Langendorff-perfused isolated normal rabbit hearts with and without K ATP channels opener, pinacidil (80 μM). In group I (n=10), we performed simultaneous dual optical mapping of transmembrane potential (V m ) and intracellular calcium (Ca i ) transient on left ventricular endocardium. In group II (n=6), glass microelectrode single cell recording of transmembrane potential (TMP) was performed. In group III (n=6), we examined the effects of BAPTA-AM, a calcium chelator, on late-phase 3 EAD and VF induction. Results: Ventricular endocardium was paced at PCL between 60 ms and 350 ms for 29 beats. Pinacidil shortened both APD 90 (192±11 ms at baseline to 98±9 ms) and duration of Ca i at 50% of amplitude (DCaT 50 , 152±10 ms at baseline to 84±9 ms) at PCL of 250 ms. At pacing rate &lt; 200 ms, the first post-pacing spontaneous beat showed persistent APD shortening and late-phase 3 EAD accompanied by large Ca i elevation. There was a positive correlation between the EAD amplitudes and the amplitude of Ca i elevation (r=0.899). At a critical Ca i elevation magnitude, the late-phase 3 EAD then induced triggered activity and VF in 10 episodes. The single cell TMP recording confirmed the optical mapping data of late-phase 3 EAD-induced VF. Infusion of BAPTA-AM significantly reduced the maximal Ca i amplitude in the mapped region (26.4±3.5% of the control; p&lt;0.001). Due to the lowered Ca i level, neither EAD in the first post-pacing beat nor VF induction was observed after pacing train at any PCLs. Conclusions: K ATP channel activation leads to shortened APD with correspondingly large Ca i elevation, resulting in late-phase 3 EAD in first spontaneous beat at the cessation of rapid pacing. Reducing intracellular calcium handling by BAPTA-AM prevented EAD and VF induction/reinitiation. Because acute myocardial ischemia activates the K ATP channel, the calcium mediated late-phase 3 EAD induced triggered activity may be a novel mechanism for VF and sudden death during acute myocardial ischemia. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Increased Cell Proliferation is Attenuated by Ca2+ Chelation in a Mouse Model of ARPKD

Increased renal tubular epithelial cell proliferation has been demonstrated to precede cyst development in various models of polycystic kidney disease. Our lab has previously shown, in cilia-deficient cortical collecting duct cells from the Oak Ridge Polycystic Kidney mouse (orpk cilia(−)), that there is increased apical Ca2+ permeability which causes elevated sub-apical Ca2+ levels compared with cilia-competent counterparts (orpk cilia(+)). We hypothesize that elevated Ca2+ levels in orpk cilia(−) cells may lead to increased cell proliferation. In the present studies, cell proliferation rates were measured in orpk cilia(+) and cilia(−) cells by cell counting using a hemacytometer. Cells from both groups were seeded at equal density, and allowed to attach for 24 hours. Treatments to lower [Ca2+]i using BAPTA-AM were initiated 24 hours post-seeding, and repeated at 24 hour intervals. We found that untreated orpk cilia(−) cells grew at a 36% faster rate than cilia(+) cells (2.1*10^5 vs. 1.5*10^5 cells/(plate*day)). 10 μM BAPTA-AM attenuated the growth rate by nearly 57% in cilia(−) cells (2.1*10^5 vs. 0.9*10^5 cells/(plate*day)). The effect of BAPTA-AM on the proliferation rate of orpk cilia(+) cells was less pronounced, as Ca2+ chelation inhibited growth by approximately 22% (1.5*10^5 vs. 1.2*10^5 cells/(plate*day)). Furthermore, proliferation rates of BAPTA-AM treated orpk cilia(−) and cilia(+) cells were comparable (0.9*10^5 vs. 1.2*10^5 cells/(plate*day)). Thus, renal cystogenesis may result from increased proliferation due to elevated sub-apical Ca2+ levels in the orpk cilia(−) model.

Properties of voltage-gated Ca2+ currents measured from mouse pancreatic β-cells in situ

We used the single-microelectrode voltage-clamp technique to record ionic currents from pancreatic beta-cells within intact mouse islets of Langerhans at 37 degrees C, the typical preparation for studies of glucose-induced "bursting" electrical activity. Cells were impaled with intracellular microelectrodes, and voltage pulses were applied in the presence of tetraethylammonium. Under these conditions, a voltage-dependent Ca2+ current (I(Cav)), containing L-type and non-L-type components, was observed. The current measured in situ was larger than that measured in single cells with whole-cell patch clamping, particularly at membrane potentials corresponding to the action potentials of beta-cell electrical activity. The temperature dependence of I(Cav) was not sufficient to account for the difference in size of the currents recorded with the two methods. During prolonged pulses, the voltage-dependent Ca2+ current measured in situ displayed both rapid and slow components of inactivation. The rapid component was Ca2+-dependent and was inhibited by the membrane-permeable Ca2+ chelator, BAPTA-AM. The effect of BAPTA-AM on beta-cell electrical activity then demonstrated that Ca2+-dependent inactivation of I(Cav) contributes to action potential repolarization and to control of burst frequency. Our results demonstrate the utility of voltage clamping beta-cells in situ for determining the roles of ion channels in electrical activity and insulin secretion.

The role of intracellular Ca2+ in the regulation of proteinase‐activated receptor‐2 mediated nuclear factor kappa B signalling in keratinocytes

1 In this study, we examined the role of Ca2+ in linking proteinase-activated receptor-2 (PAR2) to the nuclear factor kappa B (NFkappaB) pathway in a skin epithelial cell line NCTC2544 stably expressing PAR2 (clone G). 2 In clone G, PAR2-mediated NFkappaB luciferase reporter activity and NFkappaB DNA-binding activity was reduced by preincubation with BAPTA-AM but not BAPTA. Trypsin stimulation of inhibitory kappa B kinases, IKKalpha and IKKbeta, was also inhibited following pretreatment with BAPTA-AM. 3 BAPTA/AM also prevented PAR2-mediated IKKalpha activation in cultured primary human keratinocytes. 4 The effect of BAPTA-AM was also selective for the IKK/NFkappaB signalling axis; PAR2 coupling to ERK, or p38 MAP kinase was unaffected. 5 Pharmacological inhibition of the Ca2+-dependent regulatory protein calcineurin did not inhibit trypsin-stimulated IKK activity or NFkappaB-DNA binding; however, inhibition of Ca2+-dependent protein kinase C isoforms or InsP3 formation using GF109203X or the phospholipase C inhibitor U73122, respectively, reduced both IKK activity and NFkappaB-DNA binding. 6 Mutation of PAR2 within the C-terminal to produce a mutant receptor, which does not couple to Ca2+ signalling, but is able to activate ERK, abrogated NFkappaB-DNA binding and IKK activity stimulated by trypsin. 7 These results suggest a predominant role for the InsP3/Ca2+ axis in the regulation of IKK signalling and NFkappaB transcriptional activation.

Effects of BAPTA-AM, Forskolin, DSF and Z.VAD.fmk on PDT-induced apoptosis and m-THPC phototoxicity on B16 cells.

As many types of cells exposed to photodynamic therapy (PDT) appear to undergo apoptosis, various apoptosis inhibitors have already been used in studies of PDT-induced apoptosis. Although these inhibitors decrease apoptosis, their real effect on the phototoxic efficacy of photosensitisers is unclear. The good phototoxicity of m-THPC was confirmed on murine melanoma B16-A45 cells. Toxicity and phototoxicity studies were then carried out using four apoptosis inhibitors: BAPTA-AM, Forskolin, DSF, and Z.VAD.fmk. Although all inhibitors tested blocked PDT-induced apoptosis, none produced a significant modification of the phototoxic effect of m-THPC on B16 cells. It has been suggested that apoptosis and necrosis share common initiation pathways and that the final outcome is determined by the presence of an active caspase. This implies that apoptosis inhibition reorients cells to necrosis, i.e. those cells sufficiently damaged by PDT appear to be killed, regardless of the mechanism involved.

Intervention of sulfur mustard toxicity by downregulation of cell proliferation and metabolic rates.

Metabolically active and proliferating basal cells in the skin are most sensitive to the potent skin blistering chemical warfare compound HD (bis-(2-chloroethyl) sulfide). We previously described a Ca2+-dependent mechanism of HD (0.3-1 mM) toxicity that was inhibited by the cell-permeant Ca2+ chelator BAPTA AM (1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester). We describe some cellular effects of BAPTA AM that suggest a mechanism for its protective action. Monolayer log-phase normal human epidermal keratinocytes were incubated (37 degrees C) first in keratinocyte growth medium (KGM) containing BAPTA AM (10-40 microM) for 30 min and then in KGM alone overnight prior to evaluation. The BAPTA AM inhibited cell growth in a concentration-dependent manner with some cellular degeneration above 30 microM (light microscopy). At 20-30 microM, BAPTA AM also inhibited cellular metabolic processes, as evidenced by a lower incorporation of [3H]-thymidine (DNA synthesis, 54 +/- 5%), [3H]-uridine (RNA synthesis, 29 +/- 6%) and [14C]-valine (protein synthesis, 12 +/- 2%) as well as a lower protein content per culture (30 +/- 3%) compared with corresponding untreated controls. However, 20-30 microM BAPTA AM did not cause any demonstrable cytopathology based on morphological (electron microscopy) as well as biochemical (lactate dehydrogenase release, an indicator of cell viability loss) criteria, indicating a lack of acute toxicity. These results suggest that a mechanism of protection by BAPTA AM against HD may be via decreasing some metabolic, and therefore proliferative, rates.

Effects of BAPTA-AM and Forskolin on Apoptosis and Cytochrome c Release in Photosensitized Chinese Hamster V79 Cells

The mechanism of caspase-3-dependent apoptosis induced by photodynamic therapy (PDT) of cultured Chinese hamster V79 cells with pheophorbide a (PPa) was investigated. The PPa-PDT induced rapid apoptosis within 30 min after irradiation of cells. This apoptosis was inhibited by the 1O2 quencher N3-and caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde, suggesting that 1O2 activated caspase-3 and then caused apoptosis. The intracellular calcium [Ca2+]1 chelator (acetoxymethyl)-l,2-bis(o-aminophenoxy)ethane N,N,N',N,'-tetraacetic acid (BAPTA-AM) and the cyclic adenosine monophosphate (cAMP)-increasing agent forskolin also inhibited not only the PPa-PDT-induced activation of caspase-3 but also apoptosis in V79 cells. Furthermore, PPa-PDT-induced cytochrome c release from mitochondria was found to be inhibited by the treatment with BAPTA-AM but not forskolin. These results indicated that [Ca2+]1 and cAMP independently serve as regulators for PPa-PDT-induced apoptosis in the upstream of caspase-3.

Effects of BAPTA-AM and forskolin on apoptosis and cytochrome C release in photosensitized chinese hamster V79 cells : O. Inanami, A. Yoshito, K. Takahashi, W. Hiraoka and M. Kuwabara. Lab. Radiat. Biol., Dept. Environ. Vet. Med., Graduate School of Vet. Med., Hokkaido Univ., Sapporo, 060–0818. Dept. Physics, School of Science and Technol., Meiji Univ., Kawasaki, 214–8571 Japan

The mechanism of caspase-3-dependent apoptosis induced by photodynamic therapy (PDT) of cultured Chinese hamster V79 cells with pheophorbide a (PPa) was investigated. The PPa-PDT induced rapid apoptosis within 30 min after irradiation of cells. This apoptosis was inhibited by the 1O2 quencher N3-and caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde, suggesting that 1O2 activated caspase-3 and then caused apoptosis. The intracellular calcium [Ca2+]1 chelator (acetoxymethyl)-l,2-bis(o-aminophenoxy)ethane N,N,N',N,'-tetraacetic acid (BAPTA-AM) and the cyclic adenosine monophosphate (cAMP)-increasing agent forskolin also inhibited not only the PPa-PDT-induced activation of caspase-3 but also apoptosis in V79 cells. Furthermore, PPa-PDT-induced cytochrome c release from mitochondria was found to be inhibited by the treatment with BAPTA-AM but not forskolin. These results indicated that [Ca2+]1 and cAMP independently serve as regulators for PPa-PDT-induced apoptosis in the upstream of caspase-3.