Characterization of intracellular calcium oscillations induced by extracellular nucleotides in HEp-2 cells

The effect of extracellular nucleotides on the cytosolic calcium concentration of fluo-3-loaded HEp-2 cells was examined using confocal microscopy. Extracellular ATP and UTP at micromolar concentration induced cytosolic calcium oscillations in 42-66% of the cells. Oscillations were usually sinusoid and their frequency depended only slightly on agonist concentration. Oscillations developed in calcium-free medium but were diminished by depletion of intracellular calcium stores with thapsigargin, indicating periodic calcium release from internal stores. Inhibition of phospholipase C with U73122 prevented the development of oscillations, while ryanodine did not abolish the response to extracellular nucleotides. Activation of protein kinase C with 4beta-phorbol 12-myristate 13-acetate also prevented the development of oscillations. These results indicate that extracellular nucleotides induce periodic calcium release from inositol 1,4,5-trisphosphate-sensitive pools in HEp-2 cells and that the inhibitory effect of protein kinase C on the phosphatidylinositol signaling pathway can contribute to the development of intracellular calcium oscillations.     

A confocal study of mechanism of 5-hydroxytryptamino induced intracellular calcium dynamics in cultured rat stomach fundus smooth muscle cells with a new Ca~(2+) indicator STDIn-AM

A new fluorescent Ca2+ indicator STDIn-AM for detecting [Ca2+]i transients in cultured smooth muscle cells is presented. By making a comparison, the difference between STDIn and fluo-3 is discussed in detail. Using the new Ca2+ indicator, the mechanism of 5-hydroxytryptamino (5-HT) induced intracellular calcium dynamics in stomach fundus smooth muscle cells (SFSMC) of rats is investigated. It is shown that in contrast with fluo-3, STDIn is uniformly distributed in the cytosolic compartment but excluded from the nucleus, when it is transfected into cells. This feature makes it a real cytosol Ca2+ indicator and can reflect changes of cytosol [Ca2+] more accurately than that of fluo-3. In addition, STDIn responds to the [Ca2+]i transients more sensitive and faster than fluo-3. The results also show that, the L-type Ca2+channel inhibitor Mn9202 and the PLC inhibitor Compound 48/80 can significantly inhibit the [Ca2+]i elevation induced by 5-HT, while the PKC inhibitor D-Sphingosine can enhance the effect of     

5-Hydroxytryptamino-induced calcium sparks in cultured rat stomach fundus smooth muscle cells

With the imaging fluorescence probe of Ca2+ (fluo-3) and a laser scanning confocal micro-scope, the spontaneous localized calcium release event was first discovered in resting rat cardiac myocytes by Cheng[1] in 1993. A mathematical simulation is developed with computer in order to reveal the effect, which is immediately suggested that these events are likely to reflect the local-ized release of Ca2+ from a small cluster of ryanodine-sensitive Ca2+ release channels in sar-coplasmic reticulum …     

Effects of low power laser irradiation on intracellular calcium and histamine release in RBL-2H3 mast cells

Although laser irradiation has been reported to promote skin wound healing, the mechanism is still unclear. As mast cells are found to accumulate at the site of skin wounds we hypothesized that mast cells might be involved in the biological effects of laser irradiation. In this work the mast cells, RBL-2H3, were used in vitro to investigate the effects of laser irradiation on cellular responses. After laser irradiation, the amount of intracellular calcium ([Ca2+]i) was increased, followed by histamine release, as measured by confocal fluorescence microscopy with Fluo-3/AM staining and a fluorescence spectrometer with o-phthalaldehyde staining, respectively. The histamine release was mediated by the increment of [Ca2+]i from the influx of the extracellular buffer solution through the cation channel protein, transient receptor potential vanilloid 4 (TRPV4). The TRPV4 inhibitor, Ruthenium Red (RR) can effectively block such histamine release, indicating that TRPV4 was the key factor responding to laser irradiation. These induced responses of mast cells may provide an explanation for the biological effects of laser irradiation on promoting wound healing, as histamine is known to have multi-functions on accelerating wound healing.     

Vanadate-stimulated release of hepatic lipase activity from liver.

Vanadate stimulated the release of rat hepatic lipase activity from liver slices into an incubation medium in a time- and dose-dependent manner. Insulin, however, failed to have this stimulatory action, and the release by heparin was recognized, but was not additive to that by vanadate. Amiloride, an inhibitor of tyrosine kinase in some receptors and of the Na+/H+ exchange system suppressed the vanadate-stimulated release. Biochanin A, a different type of tyrosine kinase inhibitor than amiloride, also suppressed the effect of vanadate. The stimulation by vanadate was clearly preserved in Na(+)-, K(+)-, or Ca(2+)-free medium, suggesting that neither the Na+/H+ exchange system, Na+, K(+)-adenosine triphosphatase, nor Ca(2+)-influx into cells is involved in the action of this substance. These results suggest that vanadate-stimulated release of the enzyme activity is associated with the activation of the tyrosine kinase activity.     

Vascular dilatory action of the Chinese crude drug. II. Effects of scoparone on calcium mobilization

Further experiments were conducted to examine the effect of scoparone (6,7-dimethoxycoumarin), a coumarin derivative found in the Chinese crude drug "Capillaris Flos," on calcium mobilization. Scoparone does not affect Ca2+ influx through the voltage-dependent channel due to membrane depolarization. Its inhibitory action may be dependent not only on the inhibition of Ca2+ release from sarcoplasmic reticulum but also on the inhibition of extracellular Ca2+ influx through the receptor-operated channel.     

Na+-Ca2+ exchanger modulates Ca2+ content in intracellular Ca2+ stores in rat osteoblasts

Na+-Ca2+ exchanger (NCX) transports Ca2+ coupled with Na+ across the plasma membrane in a bi-directional mode. Ca2+ flux via NCX mediates osteogenic processes, such as formation of extracellular matrix proteins and bone nodules. However, it is not clearly understood how the NCX regulates cellular Ca2+ movements in osteogenic processes. In this study, the role of NCX in modulating Ca2+ content of intracellular stores ([Ca2+]ER) was investigated by measuring intracellular Ca2+ activity in isolated rat osteoblasts. Removal of extracellular Na+ elicited a transient increase of intracellular Ca2+ concentration ([Ca2+]i). Pretreatment of antisense oligodeoxynucleotide (AS) against NCX depressed this transient Ca2+ rise and raised the basal level of [Ca2+]i. In AS-pretreated cells, the expression and activity of alkaline phosphatase (ALP), an osteogenic marker, were decreased. However, the cell viability was not affected by AS-pretreatment. Suppression of NCX activity by the AS-pretreatment decreased ATP-activated Ca2+ release from intracellular stores and significantly enhanced Ca2+ influx via store operated calcium influx (SOCI), compared to those of S-pretreated or control cells. These results strongly suggest that NCX has a regulatory role in cellular Ca2+ pathways in osteoblasts by modulating intracellular Ca2+ content.     

Role of Ca(2+) in diallyl disulfide-induced apoptotic cell death of HCT-15 cells

Diallyl disulfide (DADS) induced apoptosis through the caspase-3 dependent pathway in leukemia cells was earlier reported from this laboratory. In this study, we investigated the involvement of Ca(2+) in DADS-induced apoptotic cell death of HCT-15, human colon cancer cell line. DADS induced the elevation of cytosolic Ca(2+) by biphasic pattern; rapid Ca(2+) peak at 3 min and following slow and sustained elevation till 3 h after the addition of DADS. Production of H(2)O(2) was also observed with its peak value at 4 h. Apoptotic pathways including the sequence of caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation by DADS were completely blocked by various inhibitors such as specific caspase-3 inhibitor, free radical scavenger, and intracellular Ca(2+) chelator. N-acetylcystein and catalase treatment prevented the accumulation of H2O2 and later caspase-3 dependent apoptotic pathway. However, these radical scavengers did not block the elevation of intracellular Ca(2+). Treatment of cells with 1, 2-bis (2-aminophenoxyethane)-N, N, N-tetraacetic acid tetrakis -acetoxymethyl ester (BAPTA-AM), cellular Ca(2+) chelator, resulted in a complete blockage of the caspase-3 dependent apoptotic pathway of HCT-15 cells. It abolished the elevation of intracellular Ca(2+), and furthermore, completely inhibited the production of H(2)O(2). These results indicate that cytosolic Ca(2+) elevation is an earlier signaling event in apoptosis of HCT-15 cells. Collectively, our data demonstrate that DADS can induce apoptosis in HCT-15 cells through the sequential mechanism of Ca(2+) homeostasis disruption, accumulation of H(2)O(2), and resulting caspase-3 activation.     

Development of intracellular calcium measurement by time-resolved photon-counting fluorescence

Calcium green I, a ratiometric probe based on fluorescence lifetime measurements, was used to monitor intracellular calcium activity ([Ca2+]i) in RINm5F cells using a time-resolved fluorescence confocal microscope. The probe affinity constant has been recalibrated in single cells using ionomycin as a calcium ionophore and ethylenebis(oxyethylenenitrilo)tetraacetic acid as a calcium buffer; Kd was found to equal 150 nmol/L. The kinetics of ionomycin equilibration showed that the calcium release from calcium stores occurs before equilibration with extracellular calcium. The response to the muscarinic agonist carbachol, measured on 17 cells receiving three consecutive applications was characterized both by a [Ca2+]i peak lasting 50 s without any trailing plateau and by desensitization with a 30% decrease in the response. The dose-dependent response was obtained for a carbachol concentration from 5 mumol/L to 0.5 mmol/L. The ability of our set-up to obtain a value every 10 ms enabled us to record asynchronous spikes of [Ca2+]i in the RINm5F cells. The spikes, lasting less than 1 s, are significantly bigger than the noise, and they are not observed in the colonic HT29 cells.     

Bioactive constituents of Chinese natural medicines. VII. Inhibitors of degranulation in RBL-2H3 cells and absolute stereostructures of three new diarylheptanoid glycosides from the bark of Myrica rubra

Three new diarylheptanoid glycosides, named (+)-S-myricanol 5-0-beta-D-glucopyranoside, myricanene A 5-O-alpha-L-arabinofuranosyl(1-->6)-beta-D-glucopyranoside, and myricanene B 5-0-alpha-L-arabinofuranosyl(1-->6)-beta-D-glucopyranoside, were isolated from the bark of Chinese Myrica rubra, together with twenty known compounds. The absolute stereostructures of the new diarylheptanoid glycosides were elucidated on the basis of chemical and physicochemical evidence, including the application of the modified Mosher's method. The inhibitory effects of isolated constituents on the release of beta-hexosaminidase from RBL-2H3 cells were examined, and several diarylheptanoids, myricanol, (+)-S-myricanol, myricanone, and myricanenes A and B, and a flavonol, myricetin, were found to show the inhibitory activity.     

Medicinal flowers. XIV. New acylated oleanane-type triterpene oligoglycosides with antiallergic activity from flower buds of chinese tea plant (Camellia sinensis)

The methanolic extract from the flower buds of Chinese tea plant (Camellia sinensis (L.) O. KUNTZE) was found to inhibit release of beta-hexosaminidase from RBL-2H3 cells. From the methanolic extract, six new acylated oleanane-type triterpene oligoglycosides, floratheasaponins D-I, were isolated together with 21 known compounds including floratheasaponins A-C. The chemical structures of floratheasaponins D-I were elucidated on the basis of chemical and physicochemical evidence. The principal constituents, floratheasaponins A-F, were found to show the inhibitory activity on the release of beta-hexosaminidase from RBL-2H3 cells.     

Modification of intracellular Ca2+ dynamics by laser inactivation of inositol 1,4,5-trisphosphate receptor using membrane-permeant probes

A membrane-permeant malachite green-conjugated IP3 analog (MGIP3/PM) was synthesized as a probe for small molecule-based CALI (smCALI), and its effect on the Ca2+ signaling in intact DT40 chicken B cells was examined. In DT40 B cells treated with the smCALI probe, laser irradiation inhibited IP3-induced Ca2+ oscillations in response to B cell receptor stimulation, demonstrating that IP3R was acutely inactivated. We then applied smCALI to clarify the mechanism of capacitative Ca2+ entry (CCE), in which involvement of IP3R has been suggested. Despite the inactivation of IP3R by smCALI, thapsigargin-induced CCE remained unaffected, providing evidence that functional IP3R is not required for CCE in DT40 cells. These results demonstrate the potency of the smCALI technique for the study of the roles of IP3R in complex intracellular Ca2+ dynamics.     

Inhibition of superoxide generation and of increase in intracellular Ca2+ concentration by zinc in rat neutrophils stimulated with zymosan

The effect of Zn2+ on the O2- generation and change in intracellular Ca2+ concentration ([Ca2+]i) of rat peritoneal neutrophils was studied. Zymosan (serum-treated zymosan (STZ))-induced O2- generation was inhibited by Zn2+ at concentrations as low as 10 microM. A large amount of the inhibition was observed in the absence of extracellular Ca2+ but the inhibition could not be restored by increasing the extracellular Ca2+ concentration, indicating that Zn2+ does not necessarily inhibit the O2- generation competitively with extracellular Ca2+. In the absence of extracellular Ca2+, Zn2+ inhibited STZ-induced transient increase in [Ca2+]i in the concentration range that evoked a marked inhibition in the O2- generation. On the other hand, Zn2+ did not inhibit significantly STZ-induced uptake of 45Ca2+ from extracellular medium by the cells. From these results, it is suggested that Zn2+ inhibits STZ-induced release of Ca2+ from intracellular storage sites, resulting in the suppression of the activation mechanism of neutrophils.     

Doxorubicin-induced reactive oxygen species generation and intracellular Ca2+ increase are reciprocally modulated in rat cardiomyocytes

Doxorubicin (DOX) is one of the most potent anticancer drugs and induces acute cardiac arrhythmias and chronic cumulative cardiomyopathy. Though DOX-induced cardiotoxicity is known to be caused mainly by ROS generation, a disturbance of Ca2+ homeostasis is also implicated one of the cardiotoxic mechanisms. In this study, a molecular basis of DOX-induced modulation of intracellular Ca2+ concentration ([Ca2+]i) was investigated. Treatment of adult rat cardiomyocytes with DOX increased [Ca2+]i irrespectively of extracellular Ca2+, indicating DOX-mediated Ca2+ release from intracellular Ca2+ stores. The DOX-induced Ca2+ increase was slowly processed and sustained. The Ca2+ increase was inhibited by pretreatment with a sarcoplasmic reticulum (SR) Ca2+ channel blocker, ryanodine or dantrolene, and an antioxidant, alpha-lipoic acid or alpha-tocopherol. DOX-induced ROS generation was observed immediately after DOX treatment and increased in a time-dependent manner. The ROS production was significantly reduced by the pretreatment of the SR Ca2+ channel blockers and the antioxidants. Moreover, DOX-mediated activation of caspase-3 was significantly inhibited by the Ca2+ channel blockers and a-lipoic acid but not a-tocopherol. In addition, cotreatment of ryanodine with alpha-lipoic acid resulted in further inhibition of the casapse-3 activity. These results demonstrate that DOX-mediated ROS opens ryanodine receptor, resulting in an increase in [Ca2+]i and that the increased [Ca2+]i induces ROS production. These observations also suggest that DOX/ROS-induced increase of [Ca2+]i plays a critical role in damage of cardiomyocytes.     

A study of the calcium complex of a glucosylceramide, soya-cerebroside II

In order to study calcium ion complex of soya-cerebroside II (1), an ionophoretic glucosylceramide isolated from soybean, C8-cerebroside (3) and 3,3',6'-trideoxy-C8-cerebroside (4) are designed and synthesized. On the basis of extensive 1H-NMR studies in the presence of Ca2+ and a continuous variation method via (1)H-NMR, soya-cerebroside II is suggested to form a calcium complex with 1/Ca2+ ratio of 1 : 1. Soya-cerebroside II serves as a tridentate chelating ligand for Ca2+; the amide carbonyl, C2'-hydroxy, and C2'-hydroxy oxygens are responsible for the Ca2+ binding. Soya-cerebroside II is structurally analogous to a neural glucosylceramide. Thus, the accumulated neural glucosylceramide inside of endoplasmic reticulum (ER) membrane may serve as an endogenous Ca2+-binding and -transport molecule (ionophore) that result in mobilization of Ca2+ from intracellular calcium stores.     

Experimental investigation of the complete inner shell hydration energies of Ca2+: threshold collision-induced dissociation of Ca(2+)(H2O)x Complexes (x = 2-8)

The sequential bond energies of Ca(2+)(H(2)O)(x) complexes, where x = 1-8, are measured by threshold collision-induced dissociation (TCID) in a guided ion beam tandem mass spectrometer. From an electrospray ionization source that produces an initial distribution of Ca(2+)(H(2)O)(x) complexes where x = 6-8, complexes down to x = 2 are formed using an in-source fragmentation technique. Ca(2+)(H(2)O) cannot be formed in this source because charge separation into CaOH(+) and H(3)O(+) is a lower energy pathway than simple water loss from Ca(2+)(H(2)O)(2). The kinetic energy dependent cross sections for dissociation of Ca(2+)(H(2)O)(x) complexes, where x = 2-9, are examined over a wide energy range to monitor all dissociation products and are modeled to obtain 0 and 298 K binding energies. Analysis of both primary and secondary water molecule losses from each sized complex provides thermochemistry for the sequential hydration energies of Ca(2+) for x = 1-8 and the first experimental values for x = 1-4. Additionally, the thermodynamic onsets leading to the charge separation products from Ca(2+)(H(2)O)(2) and Ca(2+)(H(2)O)(3) are determined for the first time. Our experimental results for x = 1-6 agree well with previously calculated binding enthalpies as well as quantum chemical calculations performed here. Agreement for x = 1 is improved when the basis set on calcium includes core correlation.     

Phototropins and blue light-dependent calcium signaling in higher plants

Plants have several kinds of photoreceptors, which regulate growth and development. Recent investigations using Arabidopsis thaliana revealed that the newly found blue light receptor phototropins mediate phototropism, chloroplast relocation, stomatal opening, rapid inhibition of hypocotyl elongation and leaf expansion. Several physiological studies suggest that one of the intermediates in phototropin signaling is cytosolic Ca2+. Studies using phototropin mutants have demonstrated that phototropins induce an increase in cytosolic Ca2+ concentration. However, the function of Ca2+ in the phototropin-mediated signaling process remains largely unknown. This review presents findings about phototropin-mediated calcium mobilization and the involvement of calcium in blue light-dependent plant responses.     

Nanosecond electric pulse-induced calcium entry into chromaffin cells

Electrically excitable bovine adrenal chromaffin cells were exposed to nanosecond duration electric pulses at field intensities ranging from 2 MV/m to 8 MV/m and intracellular calcium levels ([Ca(2+)](i)) monitored in real time by fluorescence imaging of cells loaded with Calcium Green. A single 4 ns, 8 MV/m pulse produced a rapid, short-lived increase in [Ca(2+)](i), with the magnitude of the calcium response depending on the intensity of the electric field. Multiple pulses failed to produce a greater calcium response than a single pulse, and a short refractory period was required between pulses before another maximal increase in [Ca(2+)](i) could be triggered. The pulse-induced rise in [Ca(2+)](i) was not affected by depleting intracellular calcium stores with caffeine or thapsigargin but was completely prevented by the presence of EGTA, Co(2+), or the L-type calcium channel blocker nitrendipine in the extracellular medium. Thus, a single nanosecond pulse is sufficient to elicit a rise in [Ca(2+)](i) that involves entry of calcium via L-type calcium channels.