BAPTA‐AM dramatically improves maturation and development of bovine oocytes from grade‐3 cumulus‐oocyte complexes

Intracellular free calcium ([Ca²⁺]_i) is essential for oocyte maturation and early embryonic development. Here, we investigated the role of [Ca²⁺]_i in oocytes from cumulus‐oocyte complexes (COCs) with respect to maturation and early embryonic development, using the calcium‐buffering agent BAPTA‐AM (1,2‐bis[2‐aminophenoxy]ethane‐N,N,N′,N′‐tetraacetic acid tetrakis [acetoxymethyl ester]). COCs were graded based on compactness of the cumulus mass and appearance of the cytoplasm, with Grade 1 indicating higher quality and developmental potential than Grade 3. Results showed that: (i) [Ca²⁺]_i in metaphase‐II (MII) oocytes from Grade‐3 COCs was significantly higher than those from Grade‐1 COCs, and was significantly reduced by BAPTA‐AM; (ii) nuclear maturation of oocytes from Grade‐3 COCs treated with BAPTA‐AM was enhanced compared to untreated COCs; (iii) protein abundance of Cyclin B and oocyte‐specific Histone 1 (H1FOO) was improved in MII oocytes from Grade‐3 COCs treated with BAPTA‐AM; (iv) Ca²⁺ transients were triggered in each group upon fertilization, and the amplitude of [Ca²⁺]_i oscillations increased in the Grade‐3 group upon treatment with BAPTA‐AM, with the magnitude approaching that of the Grade‐1 group; and (v) cleavage rates and blastocyst‐formation rates were improved in the Grade‐3 group treated with BAPTA‐AM compared to untreated controls following in vitro fertilization and parthenogenetic activation. Therefore, BAPTA‐AM dramatically improved oocyte maturation, oocyte quality, and embryonic development of oocytes from Grade‐3 COCs.     

Taurine prevents intracellular calcium overload during calcium paradox of cultured cardiomyocytes

Summary The effect of taurine on the cellular distribution of [Ca²⁺]_i, during the calcium paradox was examined by digital imaging of a single fura-2-loaded cell. Cardiomyocytes superfused with control medium containing 2mM Ca²⁺ exhibited typical transients associated with spontaneous beating. When the cells were exposed to Ca²⁺-free buffer, immediate cessation of both spontaneous contractions and calcium transients was observed as [Ca²⁺]; rapidly fell to a level of 3–6 × 10^–8M. Subsequent restoration of medium calcium increased [Ca²⁺]_i to level 4–7 times normal. Large increases in [Ca²⁺]_i were observed in most cells and were associated with the development of contracture and bleb formation.Taurine pretreatment (20mM) caused no significant effect on [Ca²⁺]_i during Ca²⁺ depletion. However, it inhibited excessive accumulation of [Ca²⁺]_i during the Ca²⁺ repletion. Moreover, taurine treated cells recovered their Ca²⁺-transients and beating pattern earlier than non-treated cells. Finally morphological abnormalities commonly associated with calcium overload were attenuated by taurine treatment.     

Agomelatine modulates calcium signaling through protein kinase C and phospholipase C-mediated mechanisms in rat sensory neurons

Agomelatine, a novel antidepressant exerting its effects through melatonergic and serotonergic systems, implicated to be effective against pain including neuropathic pain but without any knowledge of mechanism of action. To explore the possible role of agomelatine on nociceptive transmission at the peripheral level, the effects of agomelatine on intracellular calcium ([Ca²⁺]_i) signaling in peripheral neurons were investigated in cultured rat dorsal root ganglion (DRG) neurons. Using the fura-2-based calcium imaging technique, the effects of agomelatine on [Ca²⁺]_i and roles of the second messenger-mediated pathways were assessed. Agomelatine caused [Ca²⁺]_i signaling in a dose-dependent manner when tested at 10 and 100 μM concentration. Luzindole, a selective melatonin receptor antagonist, almost completely blocked the agomelatine-induced calcium signals. The agomelatine-induced calcium transients were also nearly abolished following pretreatment with the 100 ng/ml pertussis toxin, a Gi/o protein inhibitor. The stimulatory effects of agomelatine on [Ca²⁺]_i transients were significantly reduced by applications of phospholipase C (PLC) and protein kinase C (PKC) blockers, 10 μM U73122, and 10 μM chelerythrine chloride, respectively. The obtained results of agomelatine-induced [Ca²⁺]_i signals indicates that peripheral mechanisms are involved in analgesic effects of agomelatine. These mechanisms seems to involve G-protein-coupled receptor activation and PLC and PKC mediated mechanisms.     

Bcl-2 Protects Against Apoptosis in Neuronal Cell Line Caused by Thapsigargin-Induced Depletion of Intracellular Calcium Stores

Abstract: The toxicity of thapsigargin, a selective inhibitor of endoplasmic reticular Ca²⁺-ATPase, was investigated in GT1-7 cells, a murine hypothalamic cell line. Treatment of these cells with 50 or 100 nM thapsigargin greatly reduced cell viability at 24 and 48 h. These doses of thapsigargin induced a rapid rise in free cytosolic Ca²⁺ ([Ca²⁺]_i), followed by a sustained increase. Addition of EGTA to chelate extracellular Ca²⁺ diminished somewhat the size of the initial increase of [Ca²⁺]_i caused by thapsigargin, and abolished the sustained increase. The sustained increase could also be abolished by addition of La³⁺ and by SKF 96365, a drug selective for receptor-mediated calcium entry, but not by verapamil or flunarizine. Pretreatment with 50 µM BAPTA/AM, a cytosolic Ca²⁺ chelator, inhibited the peak [Ca²⁺]_i caused by thapsigargin but did not inhibit the sustained elevation of [Ca²⁺]_i. Neither EGTA nor BAPTA/AM inhibited the cell death induced by thapsigargin. The cell death was characterized by DNA fragmentation (“laddering”), nuclear condensation and fragmentation, and was inhibited by protein synthesis inhibitor cycloheximide, all characteristic of apoptotic cell death. Overexpression of the proto-oncogene bcl-2 in GT1-7 cells inhibited significantly DNA fragmentation, nuclear condensation and fragmentation, and cell death induced by thapsigargin. However, Bcl-2 did not alter either basal [Ca²⁺]_i or the elevation of [Ca²⁺]_i induced by thapsigargin. Our results suggest that abnormal Ca²⁺ release from endoplasmic reticulum caused by thapsigargin induces GT1-7 death by apoptosis and that this effect does not depend on Ca²⁺ influx from the extracellular space. Bcl-2 inhibited apoptosis induced by thapsigargin, but the mechanism is unlikely to be inhibition of endoplasmic reticular Ca²⁺ release in GT1-7 neuronal cells.     

Orexin A-induced extracellular calcium influx in prefrontal cortex neurons involves L-type calcium channels

Orexins, novel excitatory neuropeptides from the lateral hypothalamus, have been strongly implicated in the regulation of sleep and wakefulness. In this study, we explored the effects and mechanisms of orexin A on intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of freshly dissociated neurons from layers V and VI in prefrontal cortex (PFC). Changes in [Ca²⁺]_i were measured with fluo-4/AM using confocal laser scanning microscopy. The results revealed that application of orexin A (0.1 ≈1 μM) induced increase of [Ca²⁺]_i in a dose-dependent manner. This elevation of [Ca²⁺]_i was completely blocked by pretreatment with selective orexin receptor 1 antagonist SB 334867. While depletion of intracellular Ca²⁺ stores by the endoplasmic reticulum inhibitor thapsigargin (2 μM), [Ca²⁺]_i in PFC neurons showed no increase in response to orexin A. Under extracellular Ca²⁺-free condition, orexin A failed to induce any changes of Ca²⁺ fluorescence intensity in these acutely dissociated cells. Our data further demonstrated that the orexin A-induced increase of [Ca²⁺]_i was completely abolished by the inhibition of intracellular protein kinase C or phospholipase C activities using specific inhibitors, BIS II (1 μM) and D609 (10 μM), respectively. Selective blockade of L-type Ca²⁺ channels by nifedipine (5 μM) significantly suppressed the elevation of [Ca²⁺]_i induced by orexin A. Therefore, these findings suggest that exposure to orexin A could induce increase of [Ca²⁺]_i in neurons from deep layers of PFC, which depends on extracellular Ca²⁺ influx via L-type Ca²⁺ channels through activation of intracellular PLC-PKC signaling pathway by binding orexin receptor 1.     

ATP-induced calcium signalling in acinar cells of the submandibular salivary gland

To study changes in the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and the total amount of calcium in cells, we used, respectively, the fluorescent dye fura 2/AM and the metallochrome dye arsenazo III. The total amount of calcium in acinar cells after their incubation in calcium-free ATP-containing extracellular solution decreased. The action of ATP induced a dose-dependent increase in the [Ca²⁺]_i; the EC₅₀ was, on average, 130 ± ± 36 μM. Calcium transients induced by ATP demonstrated no desensitization. Against the background of a blocker of ionotropic P2X receptors, pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid, we observed a decrease in the ATP-induced calcium transients by 72%. In addition, these transients were reduced by 65% in the calcium-free milieu, while after thapsigargin-induced exhaustion of the endoplasmic reticulum store they disappeared. This is indicative of the involvement of metabotropic P2Y receptors in the formation of the above calcium transients. Therefore, P2X and P2Y receptors participate in ATP-induced calcium signalling in acinar cells of the submandibular salivary gland; activation of these channels results in a rise in the [Ca²⁺]_i. The P2X receptors to a higher extent contribute to the formation of calcium signals; the P2Y-determined increase in the [Ca²⁺]_i is smaller (equal to about 35%). Therefore, the functionally active ligand-operated ionotropic P2Y receptors and metabotropic G protein-related P2Y receptors do exist in acinar cells of the submandibular salivary gland and play an important role in the control of functioning of this gland. Neirofiziologiya/Neurophysiology, Vol. 37, Nos. 5/6, pp. 395–402, September–December, 2005.     

A novel fluorescence chamber for the determination of volume changes in human CaSki cell cultures attached on filters

The objective of the study was to test the hypothesis that, in the cultured human cervical epithelium, CaSki, the effect of calcium mobilizing agents on transepithelial electrical conductance (G_TE), is the result of cell volume decrease. CaSki cells attached on filters were loaded with fura-2, and measurements of fluorescence at the isosbestic wavelength 360 nm (excitation/emission [F_360/510]) were made in a newly designed fluorescence chamber; this design allowed us also to determine changes in cytosolic calcium ([Ca²⁺]_i). The experimental conditions were similar to those used to measure changes in paracellular permeability in the Ussing chamber, and they enabled us to compare the time-course of changes in [Ca²⁺]_i, in F_360/510, and in G_TE. Hypertonicity increased, and hypotonicity decreased F_360/510 and G_TE, without having an effect on [Ca²⁺]_i, and the changes in F_360/510 and in G_TE correlated linearly. Metabolism, bleaching, and extrusion of intracellular fura-2 were minimal, indicating that the changes in F_360/510 reflect changes in dye concentration. Hypertonicity decreased, and hypotonicity increased the size of dispersed CaSki cells, suggesting that osmolarity-induced changes in F_360/510 reflect changes in size of the attached cells. Ionomycin increased [Ca²⁺]_i, F_360/510, and G_TE, but the increases in [Ca²⁺]_i preceded those in F_360/510 and G_TE. The calcium chelator BAPTA blocked the ionomycin-induced increase in [Ca²⁺]_i, F_360/510, and in G_TE. Preincubation with 4-acetamido-4′isothiocyanatostilbene-2,2′disulfonic acid (SITS) augmented the ionomycin-induced increase in [Ca²⁺]_i, but blocked the increases in F_360/510 and in G_TE. Pretreatment of cells with hypertonic solution abrogated the increases in F_360/510 and in G_TE in response to ionomycin, but had little effect on the ionomycin-induced increase in [Ca²⁺]_i. On the basis of these results we suggest that the ionomycin-induced increase in G_TE is mediated by [Ca²⁺]_i-dependent chloride secretion and osmotic water loss.     

Bergmann glial cells in situ express endothelinB receptors linked to cytoplasmic calcium signals

The endothelin (ET) isoforms ET-1, ET-2 and ET-3 applied at 100 nM triggered a transient increase in [Ca²⁺]_i in Bergmann glial cells in cerebellar slices acutely isolated from 20–25 day-old mice. The intracellular calcium concentration ([Ca²⁺]_i) was monitored using Fura-2-based ([Ca²⁺]_i) microfluorimetry. The ET-triggered ([Ca²⁺]_i) transients were mimicked by ET, receptor agonist BO-3020 and were inhibited by ETB receptor antagonist BQ-788. ET elevated [Ca²⁺]_i in Ca2+-free extracellular solution and the ET-triggered [Ca²⁺]_i elevation was blocked by 500 nM thapsigargin indicating that the [Ca²⁺]_i was released from InsP3 sensitive intracellular pools. The ET-triggered [Ca²⁺]_i increase in Ca²⁺-free solution was shorter in duration. Restoration of normal extracellular [Ca²⁺] briefly after the ET application induced a second [Ca²⁺]_i increase indicating the presence of a secondary Ca²⁺ influx which prolongs the Ca²⁺ signal. Pre-application of 100 μM ATP or 10 μM noradrenaline blocked the ET response suggesting the involvement of a common Ca²⁺ depot. The expression of ETB receptor mRNAs in Bergmann glial cells was revealed by single-cell RT-PCR. The mRNA was also found in Purkinje neurones, but no Ca²⁺ signalling was triggered by ET. We conclude that Bergmann glial cells are endowed with functional ET_B receptors which induce the generation of intracellular [Ca²⁺]_i signals by activation of Ca²⁺ release from InsP₃-sensitive intracellular stores followed by a secondary Ca²⁺ influx.     

Ketamine inhibition of cytoplasmic calcium signalling in rat pheochromocytoma (PC-12) cells

This study examines the mechanism of action of ketamine, a dissociative anesthetic, with a specific focus on its ability to inhibit changes in the concentration of intracellular free calcium, [Ca²⁺]_i, in PC-12 cells. The resting [Ca²⁺]_i as measured with the fluorescent probe Fura-2 AM in control cells is 184.8±8.6 nM (mean±SEM, n = 15). Changes in [Ca²⁺]_i via influx through voltage-gated calcium channels after membrane depolarization with potassium chloride were monitored in the absence and presence of various concentrations of ketamine. Potassium-depolarization caused a dose-dependent rapid increase in [Ca²⁺]_i, averaging 62±5%, 33±2% and 18±3% (n = 10 each) above control levels for 70 mM, 50 mM and 35 mM KCl, respectively. Ketamine, in the dosage range studied (5 – 500 μM), inhibited the increase in [Ca²⁺]_i stimulated by potassium-depolarization in a dose-dependent manner. The computer-fitted dose-response curve of the pooled data yielded a half maximal suppression concentration, ED₅₀, of 33 μM. In conclusion, this study demonstrates that ketamine inhibits Ca²⁺ influx through voltage-gated Ca²⁺ channels in PC-12 cells at clinically relevant doses, and may play a role in ketamine's action as a general anesthetic agent.     

Effects of levobupivacaine and bupivacaine on intracellular calcium signaling in cultured rat dorsal root ganglion neurons

Bupivacaine and levobupivacaine have been shown to be effective in the treatment of pain as local anesthetics, although the mechanisms mediating their antinociceptive actions are still not well understood. The aim of this study was to investigate the effects of bupivacaine and levobupivacaine on intracellular calcium ([Ca²⁺]_i) signaling in cultured rat dorsal root ganglion (DRG) neurons. DRG neuronal cultures loaded with 5?μM Fura-2/AM and [Ca²⁺]_i transients for stimulation with 30?mM KCl (Hi K⁺) were assessed by using fluorescent ratiometry. DRGs were excited at 340 and 380?nm, emission was recorded at 510?nm, and responses were determined from the change in the 340/380 ratio (basal-peak) for individual DRG neurons. Data were analyzed by using Student’s t-test. Levobupivacaine and bupivacaine attenuated the KCl-evoked [Ca²⁺]_i transients in a reversible manner. [Ca²⁺]_i increase evoked by Hi K⁺ was significantly reduced to 99.9?±?5.1% (n?=?18) and 62.5?±?4.2% (n?=?15, P?<?0.05) after the application of 5 and 50?µM levobupivacaine, respectively. Bupivacaine also inhibited Hi K⁺-induced [Ca²⁺]_i responses, reduced to 98.7?±?4.8% (n?=?10) and 69.5?±?4.5% (n?=?9, P?<?0.05) inhibition of fluorescence ratio values of Hi K⁺-induced responses at 5 and 50?μM, respectively. Our results indicate that bupivacaine and levobupivacaine, with no significant differences between both agents, attenuated KCl-evoked calcium transients in a reversible manner. The inhibition of calcium signals in DRG neurons by levobupivacaine and bupivacaine might contribute to the antinociceptive effects of these local anesthetics.     

Spinorphin inhibits membrane depolarization- and capsaicin-induced intracellular calcium signals in rat primary nociceptive dorsal root ganglion neurons in culture

Abstract

Objective: Spinorphin is a potential endogenous antinociceptive agent although the mechanism(s) of its analgesic effect remain unknown. We conducted this study to investigate, by considering intracellular calcium concentrations as a key signal for nociceptive transmission, the effects of spinorphin on cytoplasmic Ca²⁺ ([Ca²⁺]_i) transients, evoked by high-K⁺ (30?mM) depolariasation or capsaicin, and to determine whether there were any differences in the effects of spinorphin among subpopulation of cultured rat dorsal root ganglion (DRG) neurons. Methods: DRG neurons were cultured on glass coverslips following enzymatic digestion and mechanical agitation, and loaded with the calcium sensitive dye fura-2 AM (1?µM). Intracellular calcium responses in individual DRG neurons were quantified using standard fura-2 based ratiometric calcium imaging technique. All data were analyzed by using unpaired t test, p?<?0.05 defining statistical significance. Results: Here we found that spinorphin inhibited cytoplasmic Ca²⁺ ([Ca²⁺]_i) transients, evoked by depolarization and capsaicin selectively in medium and small cultured rat DRG neurons. Spinorphin (10–300?µM) inhibited the Ca²⁺ signals in concentration dependant manner in small- and medium diameter DRG neurons. Capsaicin produced [Ca²⁺]_i responses only in small- and medium-sized DRG neurons, and pre-treatment with spinorphin significantly attenuated these [Ca²⁺]_i responses. Conclusion: Results from this study indicates that spinorphin significantly inhibits [Ca²⁺]_i signaling, which are key for the modulation of cell membrane excitability and neurotransmitter release, preferably in nociceptive subtypes of this primary sensory neurons suggesting that peripheral site is involved in the pain modulating effect of this endogenous agent.     

Role of calcium and ROS in cell death induced by polyunsaturated fatty acids in murine thymocytes

We investigated the mechanisms whereby omega‐3 and ‐6 polyunsaturated fatty acids (PUFAs) cause cell death of mouse thymocytes using flow cytometry, focusing on the respective roles of intracellular calcium concentration, [Ca²⁺]_i and reactive oxygen species (ROS). We applied the C‐22, 20, and 18 carbon omega‐3 (DHA, EPA, ALA) and omega‐6 (DTA, ARA, and LNA) fatty acids to isolated thymocytes and monitored cell death using the DNA‐binding dye, propidium iodide. When applied at 20 µM concentration, omega‐3 fatty acids killed thymocytes over a period of 1 h with a potency of DHA > EPA > ALA. The omega‐6 PUFAs were more potent. The C18 omega‐6 fatty acid, LNA, was the most potent, followed by DHA and ARA. Cell death was always accompanied by an increase in the levels of [Ca²⁺]_i and ROS. Both increases were in proportion to the potency of the PUFAs in inducing cell death. Removing extracellular calcium did not prevent the elevation in [Ca²⁺]_i nor cell death. However, the intracellular calcium chelator, BAPTA, almost totally reduced both the elevation in [Ca²⁺]_i and cell death, while vitamin E reduced the elevation in ROS and cell death. BAPTA also prevented the elevation in ROS, but vitamin E did not prevent the elevation in [Ca²⁺]_i. Thapsigargin, which depletes endoplasmic reticulum calcium, blocked the elevation in [Ca²⁺]_i, but CCCP, a mitochondrial calcium uptake inhibitor, did not. These results suggest that the six PUFAs we studied kill thymocytes by causing release of calcium from endoplasmic reticulum, which causes release of ROS from mitochondria which leads to cell death. J. Cell. Physiol. 225: 829–836, 2010. © 2010 Wiley‐Liss, Inc.     

Intracellular calcium plays a critical role in the alcohol‐mediated death of cerebellar granule neurons

Alcohol is a potent neuroteratogen that can trigger neuronal death in the developing brain. However, the mechanism underlying this alcohol‐induced neuronal death is not fully understood. Utilizing primary cultures of cerebellar granule neurons (CGN), we tested the hypothesis that the alcohol‐induced increase in intracellular calcium [Ca²⁺]_i causes the death of CGN. Alcohol induced a dose‐dependent (200–800 mg/dL) neuronal death within 24 h. Ratiometric Ca²⁺ imaging with Fura‐2 revealed that alcohol causes a rapid (1–2 min), dose‐dependent increase in [Ca²⁺]_i, which persisted for the duration of the experiment (5 or 7 min). The alcohol‐induced increase in [Ca²⁺]_i was observed in Ca²⁺‐free media, suggesting intracellular Ca²⁺ release. Pre‐treatment of CGN cultures with an inhibitor (2‐APB) of the inositol‐triphosphate receptor (IP₃R), which regulates Ca²⁺ release from the endoplasmic reticulum (ER), blocked both the alcohol‐induced rise in [Ca²⁺]_i and the neuronal death caused by alcohol. Similarly, pre‐treatment with BAPTA/AM, a Ca²⁺‐chelator, also inhibited the alcohol‐induced surge in [Ca²⁺]_i and prevented neuronal death. In conclusion, alcohol disrupts [Ca²⁺]_i homeostasis in CGN by releasing Ca²⁺ from intracellular stores, resulting in a sustained increase in [Ca²⁺]_i. This sustained increase in [Ca²⁺]_i may be a key determinant in the mechanism underlying alcohol‐induced neuronal death.     

Slow oscillations of free intracellular calcium ion concentration in human fibroblasts responding to mechanical stretch

Calcium transients in single, human gingival fibroblasts were studied after mechanical stretching of flexible culture substrates. A model system was developed to reproducibly stretch and rapidly (< 1 sec) refocus cells in the same focal plane so that changes in the concentration of free intracellular calcium ions ([Ca²⁺]_i) were monitored without delay. Attached cells were grown on flexible bottom Petriperm dishes, loaded with fura-2/AM, and stretched by 1% or 2.8% of substrate area. The stretch caused no significant cell detachment or membrane lesions. A 1% stretch induced no calcium response, but a 2.8% stretch stimulated an initial calcium transient and the subsequent generation of [Ca²⁺]_i oscillations of up to 2,000 sec. At 1% stretch, there was no calcium response. Cell shape and plating time were important determinants in the calcium response to mechanical stimulation: the responder cells were small and round without long processes. Major calcium transients were inhibited completely by 5 mM EGTA or by 10 μM gadolinium ions, by 50 μM nifedipine, or 250 μM verapamil, suggesting an influx of calcium through stretch-activated (SA) channels and L-type calcium channels. Depolarization by high KCl (144 mM) in the extracellular medium enhanced the amplitude of calcium transients by 54%. Calcium oscillations were not inhibited by preincubation with thapsigargin, caffeine, cholera toxin, staurosporine or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), indicating that IP₃ sensitive pools, IP₃ insensitive pools, G₅α subunits, and protein kinase C, respectively, were not involved in the generation of calcium oscillations. Pretreatment with genistein, a specific tyrosine kinase inhibitor or cytochalasin D, an inhibitor of actin polymerization, or pertussis toxin, an inhibitor of G_iα and G_oα subunits, completely abolished calcium transients and oscillations. These results indicate that Ca²⁺ flux due to mechanical stretching is likely mediated through SA ion channe s and is dependent on tyrosine kinases, pertussis toxin-sensitive subunits of G-proteins, and actin filaments. © 1994 Wiley-Liss, Inc.     

Flow cytometry reveals different lag times in rapid cytoplasmic calcium elevations in human neutrophils in response to N-formyl peptide

Flow cytometric analyses were performed to study intracellular single-cell calcium transients ([Ca²⁺]_i) in suspended human neutrophils during the initial phase of N-formyl peptide stimulation. Thereby, two neutrophil populations became apparent. Early maximally Ca²⁺-responding (high fluorescence) neutrophils and not-yet Ca²⁺-responding (low fluorescence) neutrophils, but no neutrophils with intermediate levels of [Ca²⁺]_i, were detected. Within 7 s the number of low fluorescence neutrophils decreased and the number of high fluorescence neutrophils increased maximally. This suggests that [Ca²⁺]_i transients occurred abruptly in individual neutrophils within a time interval below 1 s. At lower N-formyl peptide concentrations the lag times of individual neutrophils and the interval time of maximal activation of the [Ca²⁺]_i-responding neutrophil population increased, however the percentage of [Ca²⁺]_i-responding cells decreased. Surprisingly, no influence of the N-formyl peptide concentration on the [Ca²⁺]_i-induced fluorescence signal of the individual cell was observed: it was always in an almost maximal range or not responding. In parallel, binding studies performed with fluorescein-labeled N-formyl peptide revealed that the heterogeneity of [Ca²⁺]_i-responding cells cannot be explained by different receptor occupancy. In summary, this study demonstrates that [Ca²⁺]_i transients induced by N-formyl peptides in suspended individual human neutrophils occur very rapidly in an almost “all-or-none manner” and that the mean increasing fluorescence signal of a calcium indicator within a whole neutrophil population results from varying lag times of the individual cells, rather than from the mean simultaneous progress of many cells. © 1993 Wiley-Liss, Inc.     

Investigation of carvedilol-evoked Ca2+ movement and death in human oral cancer cells

The effect of carvedilol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in OC2 human oral cancer cells is unknown. This study examined if carvedilol altered basal [Ca²⁺]_i levels in suspended OC2 cells by using fura-2 as a Ca²⁺-sensitive fluorescent probe. Carvedilol at concentrations between 10 and 40 µM increased [Ca²⁺]_i in a concentration-dependent fashion. The Ca²⁺ signal was decreased by 50% by removing extracellular Ca²⁺. Carvedilol-induced Ca²⁺ entry was not affected by the store-operated Ca²⁺ channel blockers nifedipine, econazole, and SK&F96365, but was enhanced by activation or inhibition of protein kinase C. In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin did not change carvedilol-induced [Ca²⁺]_i rise; conversely, incubation with carvedilol did not reduce thapsigargin-induced Ca²⁺ release. Pretreatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) inhibited carvedilol-induced [Ca²⁺]_i release. Inhibition of phospholipase C with U73122 did not alter carvedilol-induced [Ca²⁺]_i rise. Carvedilol at 5–50 µM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca²⁺ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA/AM). Annexin V/propidium iodide staining assay suggests that apoptosis played a role in the death. Collectively, in OC2 cells, carvedilol induced [Ca²⁺]_i rise by causing phospholipase C-independent Ca²⁺ release from mitochondria and non-endoplasmic reticulum stores, and Ca²⁺ influx via protein kinase C-regulated channels. Carvedilol (up to 50 μM) induced cell death in a Ca²⁺-independent manner that involved apoptosis.     

Changes in cytosolic CA2+ are not involved in DDT-induced loss of gap junctional communication in WB-F344 cells

Recent studies have demonstrated that the insecticide DDT is a tumor promoting agent. Similar to many other tumor promoting agents, DDT has been shown to inhibit gap junctional intercellular communication (GJIC) between cells in culture, and it has been suggested that DDT-induced loss of communication between adjacent cells may depend on changes in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). In the present study, the role of[Ca²⁺]_i in DDT-induced loss of GJIC was investigated in WB-F344 rat liver cells using the scrape-loading/dye transfer assay (SLDT) and the Ca²⁺ fourescent indicator, furà-2. Our results show that DDT at non-cytotoxic concentrations caused a reversible loss of GJIC. Inhibition of GJIC was not associated with detectable increases in [Ca²⁺]_i, and was not prevented by loading cells with the intracellular Ca²⁺ chelator, BAPTA. In addition, the hydroquinone, tBuBHQ, which caused a 2+3 fold sustained increase in [Ca²⁺]_i, did not inhibit GJIC. Conversely, when untreated cells were loaded with increasing BAPTA concentrations, GJIC were lost. These results indicate that increases in [Ca²⁺]_i are not responsible for DDT-induced loss of communication and that, in general an increase in [Ca²⁺]_i, within physiological levels is not sufficient to abolish GJIC. However, Ca²⁺-dependent processes that are active at normal resting [Ca²⁺ _i appear to be required for the maintenance of GJIC.Abbreviations [Ca²⁺] cytosolic free Ca²⁺ concentration - GJIC gap junctional intercellular communication - SLDT scrape-loading/dye transfer assay - DDT 1,1,1-trichloro-2,2-di-(4-chlorophenyl)ethane - tBuBHQ 2,5-di(tert-butyl)-1,4-benzohydroquinone - LDH lactate dehydrogenase - ER endoplasmic reticulum - Fura-2 1-[2-(5carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxyl]-2-(2amino-5-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid - BAPTA bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraaceticacid - Fura-2/AM and BAPTA/AM are the cell permeant acetoxymethyl ester forms of fura-2 and BAPTA, respectively     

Development of depolarization‐induced calcium transients in insect glial cells is dependent on the presence of afferent axons

Changes in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by depolarization have been measured in glial cells acutely isolated from antennal lobes of the moth Manduca sexta at different postembryonic developmental stages. Depolarization of the glial cell membrane was elicited by increasing the external K⁺ concentration from 4 to 25 mM. At midstage 5 and earlier stages, less than 20% of the cells responded to 25 mM K⁺ (1 min) with a transient increase in [Ca²⁺]_i of approximately 40 nM. One day later, at late stage 5, 68% of the cells responded to 25 mM K⁺, the amplitude of the [Ca²⁺]_i transients averaging 592 nM. At later stages, all cells responded to 25 mM K⁺ with [Ca²⁺]_i transients with amplitudes not significantly different from those at late stage 5. In stage 6 glial cells isolated from deafferented antennal lobes, i.e., from antennal lobes chronically deprived of olfactory receptor axons, only 30% of the cells responded with [Ca²⁺]_i transients. The amplitudes of these [Ca²⁺]_i transients averaged 93 nM and were significantly smaller than those in normal stage 6 glial cells. [Ca²⁺]_i transients were greatly reduced in Ca²⁺‐free, EGTA‐buffered saline, and in the presence of the Ca²⁺ channel blockers cadmium and verapamil. The results suggest that depolarization of the cell membrane induces Ca²⁺ influx through voltage‐activated Ca²⁺ channels into antennal lobe glial cells. The development of the depolarization‐induced Ca²⁺ transients is rapid between midstage 5 and stage 6, and depends on the presence of afferent axons from the olfactory receptor cells in the antenna. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 85–98, 2002     

Cardioprotective action of urocortin in postconditioning involves recovery of intracellular calcium handling

Ischemia/reperfusion (I/R) damage in the heart occurs mainly during the first minutes of reperfusion. Urocortin (Ucn) is a member of the corticotrophin-releasing factor that has been identified as a potent endogenous cardioprotector peptide when used in pre- and postconditioning protocols. However, the underlying mechanisms are not completely elucidated. Here, we focused on intracellular calcium ([Ca²⁺]_i) handling by Ucn when applied in early reperfusion. We used Langendorff-perfused rat hearts to determine hemodynamic parameters, and confocal microscopy to study global [Ca²⁺]_i transients evoked by electrical stimulation in isolated cardiomyocytes loaded with fluorescence Ca²⁺ dye fluo-3AM. We found that the acute application of Ucn at the onset of reperfusion, in isolated hearts submitted to ischemia, fully recovered the hearts contractility and relaxation. In isolated cardiac myocytes, following ischemia we observed that the diastolic [Ca²⁺]_i was increased, the systolic [Ca²⁺]_i transients amplitude were depressed and sarcoplasmic reticulum (SR) Ca²⁺ load was reduced. These effects were correlated to a decrease in the Na⁺/Ca²⁺ exchanger (NCX) activity. Importantly, Ucn applied at reperfusion produced a complete recovery in diastolic [Ca²⁺]_i and global [Ca²⁺]_i transient amplitude, which were due to NCX activity improvement. In conclusion, we demonstrated that [Ca²⁺]_i handling play an essential role in postconditioning action of Ucn.     

Tamoxifen-Induced [Ca2+]i Rises and Ca2+-Independent Cell Death in Human Oral Cancer Cells

The purpose of this study was to explore the effect of tamoxifen on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and cell viability in OC2 human oral cancer cells. [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Tamoxifen at concentrations above 2 μM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The tamoxifen-induced Ca²⁺ influx was sensitive to blockade of L-type Ca²⁺ channel blockers but insensitive to the estrogen receptor antagonist ICI 182,780 and protein kinase C modulators. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), tamoxifen-induced [Ca²⁺]_i rises were substantially inhibited; and conversely, tamoxifen pretreatment inhibited a part of thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change tamoxifen-induced [Ca²⁺]_i rises. At concentrations between 10 and 50 μM tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 23 μM tamoxifen was not reversed by prechelating cytosolic Ca²⁺ with BAPTA. Collectively, in OC2 cells, tamoxifen induced [Ca²⁺]_i rises, in a nongenomic manner, by causing Ca²⁺ release from the endoplasmic reticulum, and Ca²⁺ influx from L-type Ca²⁺ channels. Furthermore, tamoxifen-caused cytotoxicity was not via a preceding [Ca²⁺]_i rise.