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Sample records for calcium sensitization induced

  1. Polyamines Transduce the Nongenomic, Androgen-Induced Calcium Sensitization in Intestinal Smooth Muscle

    PubMed Central

    González-Montelongo, María C.; Marín, Raquel; Pérez, José A.; Gómez, Tomás

    2013-01-01

    Androgens regulate body development and differentiation through a variety of genotropic mechanisms, mostly in reproductive organs. In recent years a different scenario for sex hormone actions has emerged: the intestinal muscle. Thus, although estrogens relax intestinal muscle, androgens are powerful inducers of mechanical potentiation. This effect of androgens was intriguing because it is observed at physiological concentrations, is mediated by nongenomic mechanisms, and involves a phenomenon of calcium sensitization of contractile machinery by stimulating phosphorylation of 20 kDa myosin light chain by Rho-associated kinase. Here we have deciphered the molecular mechanisms underlying calcium sensitization and mechanical potentiation by androgens in male intestinal muscle as well as its tight relationship to polyamine metabolism. Thus, androgens stimulate polyamine synthesis, and the inhibition of polyamine synthesis abolishes androgen-induced calcium sensitization and 20 kDa myosin light chain phosphorylation. We demonstrate that the first molecular step in the induction of calcium sensitization is a nonconventional activation of the adaptor protein RhoA, triggered by a transglutaminase-catalyzed polyamination of RhoA, which is then targeted to the membrane to activate Rho-associated kinase. Altogether, these results demonstrate that the physiological levels of androgens, through the modulation of polyamine metabolism and posttanslational modification of RhoA, activate a new signal transduction pathway in the intestinal smooth muscle to induce calcium sensitization. Furthermore, apart from being one of the few physiologically relevant nongenomic effects of androgens, these results might underlie the well-known gender differences in intestinal transits, thus expanding the nature's inventory of sex hormones effects. PMID:24002652

  2. Calcium influences sensitivity to growth inhibition induced by a cell surface sialoglycopeptide

    NASA Technical Reports Server (NTRS)

    Betz, N. A.; Fattaey, H. K.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    While studies concerning mitogenic factors have been an important area of research for many years, much less is understood about the mechanisms of action of cell surface growth inhibitors. We have purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) which can reversibly inhibit the proliferation of diverse cell types. The studies discussed in this article show that three mouse keratinocyte cell lines exhibit sixty-fold greater sensitivity than other fibroblasts and epithelial-like cells to CeReS-18-induced growth inhibition. Growth inhibition induced by CeReS-18 treatment is a reversible process, and the three mouse keratinocyte cell lines exhibited either single or multiple cell cycle arrest points, although a predominantly G0/G1 cell cycle arrest point was exhibited in Swiss 3T3 fibroblasts. The sensitivity of the mouse keratinocyte cell lines to CeReS-18-induced growth inhibition was not affected by the degree of tumorigenic progression in the cell lines and was not due to differences in CeReS-18 binding affinity or number of cell surface receptors per cell. However, the sensitivity of both murine fibroblasts and keratinocytes could be altered by changing the extracellular calcium concentration, such that increased extracellular calcium concentrations resulted in decreased sensitivity to CeReS-18-induced proliferation inhibition. Thus the increased sensitivity of the murine keratinocyte cell lines to CeReS-18 could be ascribed to the low calcium concentration used in their propagation. Studies are currently under way investigating the role of calcium in CeReS-18-induced growth arrest. The CeReS-18 may serve as a very useful tool to study negative growth control and the signal transduction events associated with cell cycling.

  3. Calcium influences sensitivity to growth inhibition induced by a cell surface sialoglycopeptide

    NASA Technical Reports Server (NTRS)

    Betz, N. A.; Fattaey, H. K.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    While studies concerning mitogenic factors have been an important area of research for many years, much less is understood about the mechanisms of action of cell surface growth inhibitors. We have purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) which can reversibly inhibit the proliferation of diverse cell types. The studies discussed in this article show that three mouse keratinocyte cell lines exhibit sixty-fold greater sensitivity than other fibroblasts and epithelial-like cells to CeReS-18-induced growth inhibition. Growth inhibition induced by CeReS-18 treatment is a reversible process, and the three mouse keratinocyte cell lines exhibited either single or multiple cell cycle arrest points, although a predominantly G0/G1 cell cycle arrest point was exhibited in Swiss 3T3 fibroblasts. The sensitivity of the mouse keratinocyte cell lines to CeReS-18-induced growth inhibition was not affected by the degree of tumorigenic progression in the cell lines and was not due to differences in CeReS-18 binding affinity or number of cell surface receptors per cell. However, the sensitivity of both murine fibroblasts and keratinocytes could be altered by changing the extracellular calcium concentration, such that increased extracellular calcium concentrations resulted in decreased sensitivity to CeReS-18-induced proliferation inhibition. Thus the increased sensitivity of the murine keratinocyte cell lines to CeReS-18 could be ascribed to the low calcium concentration used in their propagation. Studies are currently under way investigating the role of calcium in CeReS-18-induced growth arrest. The CeReS-18 may serve as a very useful tool to study negative growth control and the signal transduction events associated with cell cycling.

  4. Increased pressure-induced tone in rat parenchymal arterioles vs. middle cerebral arteries: role of ion channels and calcium sensitivity.

    PubMed

    Cipolla, Marilyn J; Sweet, Julie; Chan, Siu-Lung; Tavares, Matthew J; Gokina, Natalia; Brayden, Joseph E

    2014-07-01

    Brain parenchymal arterioles (PAs) are high-resistance vessels that branch off pial arteries and perfuse the brain parenchyma. PAs are the target of cerebral small vessel disease and have been shown to have greater pressure-induced tone at lower pressures than pial arteries. We investigated mechanisms by which brain PAs have increased myogenic tone compared with middle cerebral arteries (MCAs), focusing on differences in vascular smooth muscle (VSM) calcium and ion channel function. The amount of myogenic tone and VSM calcium was measured using Fura 2 in isolated and pressurized PAs and MCAs. Increases in intraluminal pressure caused larger increases in tone and cytosolic calcium in PAs compared with MCAs. At 50 mmHg, myogenic tone was 37 ± 5% for PAs vs. 6.5 ± 4% for MCAs (P < 0.01), and VSM calcium was 200 ± 20 nmol/l in PAs vs. 104 ± 15 nmol/l in MCAs (P < 0.01). In vessels permeabilized with Staphylococcus aureus α-toxin, PAs were not more sensitive to calcium, suggesting calcium sensitization was not at the level of the contractile apparatus. PAs were 30-fold more sensitive to the voltage-dependent calcium channel (VDCC) inhibitor nifedipine than MCAs (EC50 for PAs was 3.5 ± 0.4 vs. 82.1 ± 2.1 nmol/l for MCAs;P < 0.01); however, electrophysiological properties of the VDCC were not different in VSM. PAs had little to no response to the calcium-activated potassium channel inhibitor iberiotoxin, whereas MCAs constricted ∼15%. Thus increased myogenic tone in PAs appears related to differences in ion channel activity that promotes VSM membrane depolarization but not to a direct sensitization of the contractile apparatus to calcium.

  5. ATP-sensitive Potassium Channels and L-type Calcium Channels are Involved in Morphine-induced Hyperalgesia after Nociceptive Sensitization in Mice

    PubMed Central

    Ahmadi, Shamseddin; Azarian, Shaho; Ebrahimi, Sayede Shohre; Rezayof, Ameneh

    2014-01-01

    Introduction We investigated the role of ATP-sensitive potassium channels and L-type calcium channels in morphine-induced hyperalgesia after nociceptive sensitization. Methods We used a hotplate apparatus to assess pain behavior in male NMRI mice. Nociceptive sensitization was induced by three days injection of morphine and five days of drug free. On day 9 of the schedule, pain behavior test was performed for evaluating the effects of morphine by itself and along with nimodipine, a blocker of L-type calcium channels and diazoxide, an opener of ATP-sensitive potassium channels. All drugs were injected through an intraperitoneal route. Results The results showed that morphine (7.5, 10 and 15 mg/kg) induced analgesia in normal mice, which was prevented by naloxone (1 mg/kg). After nociceptive sensitization, analgesic effect of morphine (10 and 15 mg/kg) was significantly decreased in sensitized mice. The results showed that nimodipine (2.5, 5, 10 and 20 mg/kg) had no significant effect on pain behavior test in either normal or sensitized mice. However, nimodipine (20 mg/ kg) along with morphine (10 and 15 mg/kg) caused more decrease in morphine analgesia in sensitized mice. Furthermore, diazoxide by itself (0.25, 1, 5 and 20 mg/kg) had also no significant effect on pain behavior in both normal and sensitized mice, but at dose of 20 mg/kg along with morphine (10 and 15 mg/kg) decreased analgesic effect of morphine in sensitized mice. Discussion It can be concluded that potassium and calcium channels have some roles in decrease of analgesic effect of morphine after nociceptive sensitization induced by pretreatment of morphine. PMID:25337379

  6. Involvement of Rho kinase and protein kinase C in carbachol-induced calcium sensitization in beta-escin skinned rat and guinea-pig bladders.

    PubMed

    Durlu-Kandilci, N Tugba; Brading, Alison F

    2006-06-01

    1. The signal transduction pathways involved in carbachol (CCh)-induced calcium sensitization in beta-escin permeabilized rat and guinea-pig bladder smooth muscles were investigated and the results were compared with guinea-pig taenia caecum. 2. Calcium contractions elicited cumulatively (pCa 7.5-5) in the presence of calmodulin were significantly increased in all three tissues when CCh (50 microM) was added to the medium. 3. Under constant [Ca2+]i conditions (pCa 6), calmodulin (1 microM) and then GTP (100 microM) initiated significant contractions. CCh (50 microM) added to the bath caused a further contraction in all three tissues - calcium sensitization. This sensitization was significantly inhibited by atropine (50 microM). 4. The incubation of the tissues with the IP3-receptor blocker 2-APB (30 microM) reduced the subsequent development of calcium sensitization by CCh in rat bladder but did not affect it in guinea-pig bladder and taenia ceacum. 5. The Rho kinase (ROK) inhibitor Y-27632 (5 microM) added in the presence of CCh reversed the calcium sensitization in rat bladder, whereas a transient contraction followed by a relaxation to a level not significantly different from the CCh contraction was seen in both guinea-pig bladder and taenia caecum. Y-27632 (1 microM) continuously present significantly inhibited the CCh-induced Ca2+ sensitization in rat bladder but not in guinea-pig bladder or taenia caecum. 6. In the presence of cyclopiazonic acid (CPA) (1 microM) and calmodulin (1 microM), Y-27632 (5 microM) did not change the calcium response curve (3 x 10(-7)-10(-5) M) in rat bladder but increased the contractile responses significantly in both guinea-pig bladder and taenia caecum. 7. The protein kinase C (PKC) inhibitor GF 109203X (5 microM) added in the presence of CCh inhibited the calcium sensitization induced by this muscarinic agonist in all three tissues in different ratios. 8. In conclusion, muscarinic receptor activation induces calcium

  7. Involvement of Rho kinase and protein kinase C in carbachol-induced calcium sensitization in β-escin skinned rat and guinea-pig bladders

    PubMed Central

    Durlu-Kandilci, N Tugba; Brading, Alison F

    2006-01-01

    The signal transduction pathways involved in carbachol (CCh)-induced calcium sensitization in β-escin permeabilized rat and guinea-pig bladder smooth muscles were investigated and the results were compared with guinea-pig taenia caecum. Calcium contractions elicited cumulatively (pCa 7.5–5) in the presence of calmodulin were significantly increased in all three tissues when CCh (50 μM) was added to the medium. Under constant [Ca2+]i conditions (pCa 6), calmodulin (1 μM) and then GTP (100 μM) initiated significant contractions. CCh (50 μM) added to the bath caused a further contraction in all three tissues – calcium sensitization. This sensitization was significantly inhibited by atropine (50 μM). The incubation of the tissues with the IP3-receptor blocker 2-APB (30 μM) reduced the subsequent development of calcium sensitization by CCh in rat bladder but did not affect it in guinea-pig bladder and taenia ceacum. The Rho kinase (ROK) inhibitor Y-27632 (5 μM) added in the presence of CCh reversed the calcium sensitization in rat bladder, whereas a transient contraction followed by a relaxation to a level not significantly different from the CCh contraction was seen in both guinea-pig bladder and taenia caecum. Y-27632 (1 μM) continuously present significantly inhibited the CCh-induced Ca2+ sensitization in rat bladder but not in guinea-pig bladder or taenia caecum. In the presence of cyclopiazonic acid (CPA) (1 μM) and calmodulin (1 μM), Y-27632 (5 μM) did not change the calcium response curve (3 × 10−7–10−5 M) in rat bladder but increased the contractile responses significantly in both guinea-pig bladder and taenia caecum. The protein kinase C (PKC) inhibitor GF 109203X (5 μM) added in the presence of CCh inhibited the calcium sensitization induced by this muscarinic agonist in all three tissues in different ratios. In conclusion, muscarinic receptor activation induces calcium sensitization in rat and guinea

  8. Androgens Induce Nongenomic Stimulation of Colonic Contractile Activity through Induction of Calcium Sensitization and Phosphorylation of LC20 and CPI-17

    PubMed Central

    González-Montelongo, María C.; Marín, Raquel; Gómez, Tomás; Marrero-Alonso, Jorge; Díaz, Mario

    2010-01-01

    We show that androgens, testosterone and 5α-dihydrotestosterone (DHT), acutely (∼40 min) provoke the mechanical potentiation of spontaneous and agonist-induced contractile activity in mouse colonic longitudinal smooth muscle. The results using flutamide, finasteride, cycloheximide, and actinomycin D indicate that androgen-induced potentiation is dependent on androgen receptors, requires reduction of testosterone to DHT, and occurs independently of transcriptional and translational events. Using permeabilized colonic smooth muscle preparations, we could demonstrate that mechanical potentiation is entirely due to calcium sensitization of contractile machinery. In addition, DHT (10 nm) increased phosphorylation of both 20-kDa myosin light chain (LC20) [regulatory myosin light chain, (MLC)] and CPI-17 (an endogenous inhibitor of MLC phosphatase). Paralleling these findings, inhibition of Rho-associated Rho kinase (ROK) and/or protein kinase C (PKC) with, respectively, Y27632 and chelerythrine, prevented LC20 phosphorylation and abolished calcium sensitization. In addition, inhibition of ROK prevents CPI-17 phosphorylation, indicating that ROK is located upstream PKC-mediated CPI-17 modulation in the signalling cascade. Additionally, androgens induce a rapid activation of RhoA and its translocation to the plasma membrane to activate ROK. The results demonstrate that androgens induce sensitization of colonic smooth muscle to calcium through activation of ROK, which in turn, activates PKC to induce CPI-17 phosphorylation. Activation of this pathway induces a potent steady stimulation of LC20 by inhibiting MLC phosphatase and displacing the equilibrium of the regulatory subunit towards its phosphorylated state. This is the first demonstration that colonic smooth muscle is a physiological target for androgen hormones, and that androgens modulate force generation of smooth muscle contractile machinery through nongenomic calcium sensitization pathways. PMID:20207835

  9. Mepivacaine-induced contraction involves increased calcium sensitization mediated via Rho kinase and protein kinase C in endothelium-denuded rat aorta.

    PubMed

    Ok, Seong-Ho; Kwon, Seong-Chun; Yeol Han, Jeong; Yu, Jongsun; Shin, Il-Woo; Lee, Heon-Keun; Chung, Young-Kyun; Choi, Mun-Jeoung; Sohn, Ju-Tae

    2014-01-15

    Mepivacaine is an aminoamide local anesthetic that produces vasoconstriction in vivo and in vitro. The goals of this in vitro study were to determine whether mepivacaine-induced contraction involves calcium sensitization in isolated endothelium-denuded aortas, and to investigate the specific protein kinases involved. The effects of mepivacaine and potassium chloride on intracellular calcium concentrations ([Ca(2+)]i) and tension in the presence or absence of Y-27632 or GF 109203X were measured simultaneously using the acetoxymethyl ester of fura-2-loaded aortic strips. Cumulative mepivacaine concentration-response curves were generated in the presence or absence of the following inhibitors: Rho kinase inhibitor Y-27632, protein kinase C (PKC) inhibitor GF 109203X, extracellular signal-regulated kinase (ERK) inhibitor PD 98059, c-Jun NH2-terminal kinase (JNK) inhibitor SP600125, and p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580. Phosphorylation of PKC and MAPK, and membrane translocation of Rho kinase were detected in vascular smooth muscle cells by Western blotting. The slope of the mepivacaine-induced [Ca(2+)]i-tension curve was higher than that of the KCl-induced [Ca(2+)]i-tension curve. Pretreatment with Y-27632 or GF 109203X shifted the mepivacaine-induced [Ca(2+)]i-tension curve to the lower right. Pretreatment with Y-27632, GF 109203X, PD 98059, or SP600125 attenuated mepivacaine-induced contraction in a concentration-dependent manner. Y-27632 and GF 109203X attenuated mepivacaine-induced Rho kinase membrane translocation and PKC phosphorylation, respectively. PD 98059 and SP600125 attenuated mepivacaine-induced ERK and JNK phosphorylation, respectively. Taken together, these results indicate that mepivacaine-induced contraction involves increased calcium sensitization mediated by Rho kinase and PKC. Such contraction mainly involves activation of ERK- and JNK-mediated pathways. © 2013 Published by Elsevier B.V.

  10. Novel effect of 2-aminoethoxydiphenylborate through inhibition of calcium sensitization induced by Rho kinase activation in human detrusor smooth muscle.

    PubMed

    Shahab, Nouval; Kajioka, Shunichi; Takahashi, Ryosuke; Hayashi, Maya; Nakayama, Shinsuke; Sakamoto, Kazuyuki; Takeda, Masahiro; Masuda, Noriyuki; Naito, Seiji

    2013-05-15

    Since the introduction of 2-aminoethoxydiphenylborate (2-APB) as a membrane permeable modulator of inositol (1,4,5)-trisphosphate receptors, subsequent studies have revealed additional actions of this chemical on multiple Ca(2+)-permeable ionic channels in the plasma membrane. However, no reports have yet examined 2-APB as a modulator targeting contractile machinery in smooth muscle, independent of Ca(2+) mobilization, namely Ca(2+) sensitization. Here, we assessed whether or not 2-APB affects intracellular signaling pathways of Ca(2+) sensitization for contraction using α-toxin permeabilized human detrusor smooth muscle. Although contractions were induced by application of Ca(2+)-containing bath solutions, 2-APB had little effect on contractions induced by 1 µM Ca(2+) alone but significantly reversed the carbachol-induced augmentation of Ca(2+)-induced contraction in the presence of guanosine triphosphate (carbachol-induced Ca(2+) sensitization). The rho kinase inhibitor Y-27632 and protein kinase C inhibitor GF-109203X also reversed the carbachol-mediated Ca(2+) sensitization. Additional application of 2-APB caused a small but significant further attenuation of the contraction in the presence of GF-109203X but not in the presence of Y-27632. Like carbachol, the rho kinase activator; sphingosylphosphorylcholine, protein kinase C activator; phorbol 12,13 dibutyrate, and myosin light chain phosphatase inhibitor; calyculin-A all induced Ca(2+) sensitization. However, the inhibitory activity of 2-APB was limited with sphingosylphosphorylcholine-induced Ca(2+) sensitization. This study revealed a novel inhibitory effect of 2-APB on smooth muscle contractility through inhibition of the rho kinase pathway.

  11. Stretch-activated calcium channels relay fast calcium waves propagated by calcium-induced calcium influx.

    PubMed

    Jaffe, Lionel F

    2007-03-01

    For nearly 30 years, fast calcium waves have been attributed to a regenerative process propagated by CICR (calcium-induced calcium release) from the endoplasmic reticulum. Here, I propose a model containing a new subclass of fast calcium waves which is propagated by CICI (calcium-induced calcium influx) through the plasma membrane. They are called fast CICI waves. These move at the order of 100 to 1000 microm/s (at 20 degrees C), rather than the order of 3 to 30 microm/s found for CICR. Moreover, in this proposed subclass, the calcium influx which drives calcium waves is relayed by stretch-activated calcium channels. This model is based upon reports from approx. 60 various systems. In seven of these reports, calcium waves were imaged, and, in five of these, evidence was presented that these waves were regenerated by CICI. Much of this model involves waves that move along functioning flagella and cilia. In these systems, waves of local calcium influx are thought to cause waves of local contraction by inducing the sliding of dynein or of kinesin past tubulin microtubules. Other cells which are reported to exhibit waves, which move at speeds in the fast CICI range, include ones from a dozen protozoa, three polychaete worms, three molluscs, a bryozoan, two sea urchins, one arthropod, four insects, Amphioxus, frogs, two fish and a vascular plant (Equisetum), together with numerous healthy, as well as cancerous, mammalian cells, including ones from human. In two of these systems, very gentle local mechanical stimulation is reported to initiate waves. In these non-flagellar systems, the calcium influxes are thought to speed the sliding of actinomyosin filaments past each other. Finally, I propose that this mechanochemical model could be tested by seeing if gentle mechanical stimulation induces waves in more of these systems and, more importantly, by imaging the predicted calcium waves in more of them.

  12. Characterization of dihydropyridine-sensitive calcium channels

    SciTech Connect

    Horne, W.A.

    1989-01-01

    The structural and regulatory properties of the dihydropyridine-sensitive calcium channel were studied by isolating protein components of the channel complex from both cardiac and skeletal muscle. Hydrodynamic characterization of the (+)-({sup 3}H)PN200-110-labeled cardiac calcium channel revealed that the protein components of the complex had a total molecular mass of 370,000 daltons, a Stokes radius of 86 {angstrom}, and a frictional ratio of 1.3. A technique is described for the rapid incorporation of the CHAPS solubilized skeletal muscle calcium channel complex into phospholipid vesicles. {sup 45}Ca{sup 2+} uptake into phospholipid vesicles containing calcium channels was inhibited by phenylalkalamine calcium antagonists. Wheat germ lectin followed by DEAE chromatography of the CHAPS solubilized complex resulted in the dissociation of regulatory components of the complex from channel components. The DEAE preparation gave rise to {sup 45}Ca{sup 2+} uptake that was not inhibited by verapamil but was inhibited by GTPgS activated G{sub 0}. The inhibition of {sup 45}Ca{sup 2+} uptake by verapamil was restored by co-reconstitution of wash fractions from wheat germ lectin chromatography. Phosphorylation of polypeptides in this fraction by polypeptide-dependent protein kinase prevented the restoration of verapamil sensitivity. The partial purification of an endogenous skeletal muscle ADP-ribosyltransferase is also described. ADP-ribosylation of the {alpha}{sub 2} subunit of the calcium channel complex is enhanced by polylysine and inhibited by GTP{gamma}S, suggesting that regulation of this enzyme is under the control of GTP binding proteins. These results suggest a complex model, involving a number of different protein components, for calcium channel regulation in skeletal muscle.

  13. Peroxyacetyl nitrate-induced oxidative and calcium signaling events leading to cell death in ozone-sensitive tobacco cell-line

    PubMed Central

    Yukihiro, Masaru; Hiramatsu, Takuya; Bouteau, Francois; Kadono, Takashi; Kawano, Tomonori

    2012-01-01

    It has long been concerned that some secondary air pollutants such as smog components, ozone (O3) and peroxyacetyl nitrate (PAN), are highly phytotoxic even at low concentrations. Compared with the biology of O3, we largely lack the information on the toxicity model for PAN at the cellular signaling levels. Here, we studied the cell-damaging impact of PAN using suspension culture of smog-sensitive tobacco variety (Bel-W3). The cells were exposed to freshly synthesized PAN and the induced cell death was assessed under microscope after staining with Evans blue. Involvement of reactive oxygen species (ROS) in PAN toxicity was suggested by PAN-dependently increased intracellular H2O2 and also by the cell-protective effects of ROS scavengers and related inhibitors. Calcium chelator also lowered the level of PAN-induced cell death, indicating that Ca2+ is also involved. Using a transgenic cell line expressing aequorin, an increase in cytosolic Ca2+ concentration responsive to the pulse of PAN, but sensitive to Ca2+ channel blockers, was recorded, indicating that Ca2+ channels are activated by PAN or PAN-derived signals. Above data show some similarity between the signaling mechanisms responsive to O3 and PAN. PMID:22301977

  14. The opening of maitotoxin-sensitive calcium channels induces the acrosome reaction in human spermatozoa: differences from the zona pellucida

    PubMed Central

    Chávez, Julio C; de Blas, Gerardo A; de la Vega-Beltrán, José L; Nishigaki, Takuya; Chirinos, Mayel; González-González, María Elena; Larrea, Fernando; Solís, Alejandra; Darszon, Alberto; Treviño, Claudia L

    2011-01-01

    The acrosome reaction (AR), an absolute requirement for spermatozoa and egg fusion, requires the influx of Ca2+ into the spermatozoa through voltage-dependent Ca2+ channels and store-operated channels. Maitotoxin (MTx), a Ca2+-mobilizing agent, has been shown to be a potent inducer of the mouse sperm AR, with a pharmacology similar to that of the zona pellucida (ZP), possibly suggesting a common pathway for both inducers. Using recombinant human ZP3 (rhZP3), mouse ZP and two MTx channel blockers (U73122 and U73343), we investigated and compared the MTx- and ZP-induced ARs in human and mouse spermatozoa. Herein, we report that MTx induced AR and elevated intracellular Ca2+ ([Ca2+]i) in human spermatozoa, both of which were blocked by U73122 and U73343. These two compounds also inhibited the MTx-induced AR in mouse spermatozoa. In disagreement with our previous proposal, the AR triggered by rhZP3 or mouse ZP was not blocked by U73343, indicating that in human and mouse spermatozoa, the AR induction by the physiological ligands or by MTx occurred through distinct pathways. U73122, but not U73343 (inactive analogue), can block phospholipase C (PLC). Another PLC inhibitor, edelfosine, also blocked the rhZP3- and ZP-induced ARs. These findings confirmed the participation of a PLC-dependent signalling pathway in human and mouse zona protein-induced AR. Notably, edelfosine also inhibited the MTx-induced mouse sperm AR but not that of the human, suggesting that toxin-induced AR is PLC-dependent in mice and PLC-independent in humans. PMID:20835262

  15. Osmotic induction of calcium accumulation in human embryonic kidney cells detected with a high sensitivity FRET calcium sensor.

    PubMed

    Hou, Bi-Huei; Takanaga, Hitomi; Griesbeck, Oliver; Frommer, Wolf B

    2009-08-01

    Calcium serves as a second messenger in glucose-triggered insulin secretion of pancreatic cells. Less is known about sugar signaling in non-excitable cells. Here, the high sensitivity FRET calcium sensor TN-XXL was used to characterize glucose-induced calcium responses in non-excitable human embryonic kidney HEK293T cells. HEK293T cells responded to perfusion with glucose with a sustained and concentration-dependent increase in cytosolic calcium levels. Sucrose and mannitol triggered comparable calcium responses, suggesting that the increase of the calcium concentration was caused by osmotic effects. HEK293T cells are characterized by low endogenous glucose uptake capacity as shown with a high sensitivity glucose sensor. Consistently, when glucose influx was artificially increased by co-expression of GLUT glucose transporters, the glucose-induced calcium increase was significantly reduced. Neither calcium depletion, nor gadolinium or thapsigargin were able to inhibit the calcium accumulation. Taken together, membrane impermeable osmolytes such as sucrose and mannitol lead to an increase in calcium levels, while the effect of glucose depends on the cell's glucose uptake capacity and will thus vary between cell types in the body that differ in their glucose uptake capacity.

  16. Osmotic induction of calcium accumulation in human embryonic kidney cells detected with a high sensitivity FRET calcium sensor

    PubMed Central

    Hou, Bi-Huei; Takanaga, Hitomi; Griesbeck, Oliver; Frommer, Wolf B.

    2013-01-01

    Calcium serves as a second messenger in glucose-triggered insulin secretion of pancreatic cells. Less is known about sugar signaling in non-excitable cells. Here, the high sensitivity FRET calcium sensor TN-XXL was used to characterize glucose-induced calcium responses in non-excitable human embryonic kidney HEK293T cells. HEK293T cells responded to perfusion with glucose with a sustained and concentration-dependent increase in cytosolic calcium levels. Sucrose and mannitol triggered comparable calcium responses, suggesting that the increase of the calcium concentration was caused by osmotic effects. HEK293T cells are characterized by low endogenous glucose uptake capacity as shown with a high sensitivity glucose sensor. Consistently, when glucose influx was artificially increased by co-expression of GLUT glucose transporters, the glucose-induced calcium increase was significantly reduced. Neither calcium depletion, nor gadolinium or thapsigargin were able to inhibit the calcium accumulation. Taken together, membrane impermeable osmolytes such as sucrose and mannitol lead to an increase in calcium levels, while the effect of glucose depends on the cell's glucose uptake capacity and will thus vary between cell types in the body that differ in their glucose uptake capacity. PMID:19628278

  17. Role of Apamin-Sensitive Calcium-Activated Small-Conductance Potassium Currents on the Mechanisms of Ventricular Fibrillation in Pacing-Induced Failing Rabbit Hearts.

    PubMed

    Yin, Dechun; Hsieh, Yu-Cheng; Tsai, Wei-Chung; Wu, Adonis Zhi-Yang; Jiang, Zhaolei; Chan, Yi-Hsin; Xu, Dongzhu; Yang, Na; Shen, Changyu; Chen, Zhenhui; Lin, Shien-Fong; Chen, Peng-Sheng; Everett, Thomas H

    2017-02-01

    Ventricular fibrillation (VF) during heart failure is characterized by stable reentrant spiral waves (rotors). Apamin-sensitive small-conductance calcium-activated potassium currents (IKAS) are heterogeneously upregulated in failing hearts. We hypothesized that IKAS influences the location and stability of rotors during VF. Optical mapping was performed on 9 rabbit hearts with pacing-induced heart failure. The epicardial right ventricular and left ventricular surfaces were simultaneously mapped in a Langendorff preparation. At baseline and after apamin (100 nmol/L) infusion, the action potential duration (APD80) was determined, and VF was induced. Areas with a >50% increase in the maximum action potential duration (ΔAPD) after apamin infusion were considered to have a high IKAS distribution. At baseline, the distribution density of phase singularities during VF in high IKAS distribution areas was higher than in other areas (0.0035±0.0011 versus 0.0014±0.0010 phase singularities/pixel; P=0.004). In addition, high dominant frequencies also colocalized to high IKAS distribution areas (26.0 versus 17.9 Hz; P=0.003). These correlations were eliminated during VF after apamin infusion, as the number of phase singularities (17.2 versus 11.0; P=0.009) and dominant frequencies (22.1 versus 16.2 Hz; P=0.022) were all significantly decreased. In addition, reentrant spiral waves became unstable after apamin infusion, and the duration of VF decreased. The IKAS current influences the mechanism of VF in failing hearts as phase singularities, high dominant frequencies, and reentrant spiral waves all correlated to areas of high IKAS. Apamin eliminated this relationship and reduced VF vulnerability. © 2017 American Heart Association, Inc.

  18. Voltage sensitive calcium channels (VSCC) in cultured neuronal hybrid cells

    SciTech Connect

    Richard, C.L.; U'Prichard, D.C.; Noronha-Blob, L.

    1986-03-01

    Calcium entry via VSCC has been identified in selected, neuronal clonal cell lines using /sup 45/Ca uptake and the fluorescent calcium indicator, quin 2. VSCC in NG108-15 hybrid cells, differentiated with dibutyryl cyclic AMP (1 mM, 4 days) have been further characterized. Depolarization (50 mM K/sup +/, dp) resulted in a rapid (15 sec) influx of Ca/sup 2 +/. Intracellular calcium concentrations were elevated approx. 3 fold from 223 +- 68 nM to 666 +- 74 nM. Dp-sensitive calcium entry was voltage dependent, independent of Na/sup +/, stimulated (40%) by the agonist Bay K 8644 (1..mu..M) and blocked by divalent cations (..mu..M range) and organic calcium channel antagonists (nM range) Bay K 8644, in the absence of KCl, failed to stimulate Ca/sup 2 +/ influx. Tetrodotoxin (TTX) and tetraethylammonium had no effect on VSCC activity. Blockage of VSCC by nimodipine was reversed by increasing Ca/sup 2 +/ ions. IC/sub 50/ values were right shifted from 6.5 nM (1mM/sup 0/Ca/sup 2 +/) to 840 nM (10 mM Ca/sup 2 +/). Ca/sup 2 +/ entry was also stimulated by veratridine (VE), in a Na/sup +//sub 0/-sensitive manner. VE-induced Ca/sup 2 +/ entry was voltage-independent, TTX-sensitive, and was only 25% of dp-sensitive Ca/sup 2 +/ entry. These results together indicate that VSCC in neuronal cells offer a useful system for studying ion channel regulation.

  19. Influence of calcium-induced aggregation on the sensitivity of aminobis(methylenephosphonate)-containing potential MRI contrast agents.

    PubMed

    Henig, Jörg; Mamedov, Ilgar; Fouskova, Petra; Tóth, Éva; Logothetis, Nikos K; Angelovski, Goran; Mayer, Hermann A

    2011-07-18

    A novel class of 1,4,7,10-tetraazacyclododecane-1,4,7-tris(methylenecarboxylic) acid (DO3A)-based lanthanide complexes with relaxometric response to Ca(2+) was synthesized, and their physicochemical properties were investigated. Four macrocyclic ligands containing an alkyl-aminobis(methylenephosphonate) side chain for Ca(2+)-chelation have been studied (alkyl is propyl, butyl, pentyl, and hexyl for L(1), L(2), L(3), and L(4), respectively). Upon addition of Ca(2+), the r(1) relaxivity of their Gd(3+) complexes decreased up to 61% of the initial value for the best compounds GdL(3) and GdL(4). The relaxivity of the complexes was concentration dependent (it decreases with increasing concentration). Diffusion NMR studies on the Y(3+) analogues evidenced the formation of agglomerates at higher concentrations; the aggregation becomes even more important in the presence of Ca(2+). (31)P NMR experiments on EuL(1) and EuL(4) indicated the coordination of a phosphonate to the Ln(3+) for the ligand with a propyl chain, while phosphonate coordination was not observed for the analogue bearing a hexyl linker. Potentiometric titrations yielded protonation constants of the Gd(3+) complexes. log K(H1) values for all complexes lie between 6.12 and 7.11 whereas log K(H2) values are between 4.61 and 5.87. Luminescence emission spectra recorded on the Eu(3+) complexes confirmed the coordination of a phosphonate group to the Ln(3+) center in EuL(1). Luminescence lifetime measurements showed that Ca-induced agglomeration reduces the hydration number which is the main cause for the change in r(1). Variable temperature (17)O NMR experiments evidenced high water exchange rates on GdL(1), GdL(2), and GdL(3) comparable to that of the aqua ion.

  20. Oxytocin Inhibits the Membrane Depolarization-Induced Increase in Intracellular Calcium in Capsaicin Sensitive Sensory Neurons: A Peripheral Mechanism of Analgesic Action

    PubMed Central

    Hobo, Shotaro; Hayashida, Ken-ichiro; Eisenach, James C.

    2011-01-01

    Background Lumbar intrathecal injection of oxytocin produces antinociception in rats and analgesia in humans. Classically, oxytocin receptors couple to stimulatory G proteins, increase inositol-3-phosphate production, and result in neuronal excitation. Most work to date has focused on a spinal site of oxytocin to excite γ-aminobutyric acid interneurons to produce analgesia. Here we ask whether oxytocin might also affect primary sensory afferents by modulating high voltage-gated calcium channels, such as it does in the brain. Methods Dorsal root ganglion cells from adult rats were acutely dissociated and cultured, and changes in intracellular calcium determined by fluorescent microscopy using an indicator dye. The effects of oxytocin alone and in the presence of transient depolarization from increased extracellular KCl concentration were determined, then the pharmacology of these effects were studied. Cells from injured dorsal root ganglion cells after spinal nerve ligation were also studied. Results Oxytocin produced a concentration-dependent inhibition of the increase in intracellular calcium from membrane depolarization, an effect blocked more efficiently by oxytocin- than vasopressin-receptor selective antagonists. Oxytocin-induced inhibition was present in cells responding to capsaicin, and when internal stores of calcium were depleted with thapsigargin. Oxytocin produced similar inhibition in cells from animals with spinal nerve ligation. Conclusions These data suggest that oxytocin produces antinociception after intrathecal delivery in part by reducing excitatory neurotransmitter release from the central terminals of nociceptors. PMID:22104073

  1. Basal and Activated Calcium Sensitization Mediated by RhoA/Rho Kinase Pathway in Rats with Genetic and Salt Hypertension

    PubMed Central

    Bencze, Michal; Vaněčková, Ivana; Kuneš, Jaroslav; Zicha, Josef

    2017-01-01

    Calcium sensitization mediated by RhoA/Rho kinase pathway can be evaluated either in the absence (basal calcium sensitization) or in the presence of endogenous vasoconstrictor systems (activated calcium sensitization). Our aim was to compare basal and activated calcium sensitization in three forms of experimental hypertension with increased sympathetic tone and enhanced calcium entry—spontaneously hypertensive rats (SHR), heterozygous Ren-2 transgenic rats (TGR), and salt hypertensive Dahl rats. Activated calcium sensitization was determined as blood pressure reduction induced by acute administration of Rho kinase inhibitor fasudil in conscious rats with intact sympathetic nervous system (SNS) and renin-angiotensin system (RAS). Basal calcium sensitization was studied as fasudil-dependent difference in blood pressure response to calcium channel opener BAY K8644 in rats subjected to RAS and SNS blockade. Calcium sensitization was also estimated from reduced development of isolated artery contraction by Rho kinase inhibitor Y-27632. Activated calcium sensitization was enhanced in all three hypertensive models (due to the hyperactivity of vasoconstrictor systems). In contrast, basal calcium sensitization was reduced in SHR and TGR relative to their controls, whereas it was augmented in salt-sensitive Dahl rats relative to their salt-resistant controls. Similar differences in calcium sensitization were seen in femoral arteries of SHR and Dahl rats. PMID:28197417

  2. Basal and Activated Calcium Sensitization Mediated by RhoA/Rho Kinase Pathway in Rats with Genetic and Salt Hypertension.

    PubMed

    Behuliak, Michal; Bencze, Michal; Vaněčková, Ivana; Kuneš, Jaroslav; Zicha, Josef

    2017-01-01

    Calcium sensitization mediated by RhoA/Rho kinase pathway can be evaluated either in the absence (basal calcium sensitization) or in the presence of endogenous vasoconstrictor systems (activated calcium sensitization). Our aim was to compare basal and activated calcium sensitization in three forms of experimental hypertension with increased sympathetic tone and enhanced calcium entry-spontaneously hypertensive rats (SHR), heterozygous Ren-2 transgenic rats (TGR), and salt hypertensive Dahl rats. Activated calcium sensitization was determined as blood pressure reduction induced by acute administration of Rho kinase inhibitor fasudil in conscious rats with intact sympathetic nervous system (SNS) and renin-angiotensin system (RAS). Basal calcium sensitization was studied as fasudil-dependent difference in blood pressure response to calcium channel opener BAY K8644 in rats subjected to RAS and SNS blockade. Calcium sensitization was also estimated from reduced development of isolated artery contraction by Rho kinase inhibitor Y-27632. Activated calcium sensitization was enhanced in all three hypertensive models (due to the hyperactivity of vasoconstrictor systems). In contrast, basal calcium sensitization was reduced in SHR and TGR relative to their controls, whereas it was augmented in salt-sensitive Dahl rats relative to their salt-resistant controls. Similar differences in calcium sensitization were seen in femoral arteries of SHR and Dahl rats.

  3. Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels.

    PubMed

    Jiang, Shaojuan Amy; Campusano, Jorge M; Su, Hailing; O'Dowd, Diane K

    2005-07-01

    Spontaneous calcium oscillations in mushroom bodies of late stage pupal and adult Drosophila brains have been implicated in memory consolidation during olfactory associative learning. This study explores the cellular mechanisms regulating calcium dynamics in Kenyon cells, principal neurons in mushroom bodies. Fura-2 imaging shows that Kenyon cells cultured from late stage Drosophila pupae generate spontaneous calcium transients in a cell autonomous fashion, at a frequency similar to calcium oscillations in vivo (10-20/h). The expression of calcium transients is up regulated during pupal development. Although the ability to generate transients is a property intrinsic to Kenyon cells, transients can be modulated by bath application of nicotine and GABA. Calcium transients are blocked, and baseline calcium levels reduced, by removal of external calcium, addition of cobalt, or addition of Plectreurys toxin (PLTX), an insect-specific calcium channel antagonist. Transients do not require calcium release from intracellular stores. Whole cell recordings reveal that the majority of voltage-gated calcium channels in Kenyon cells are PLTX-sensitive. Together these data show that influx of calcium through PLTX-sensitive voltage-gated calcium channels mediates spontaneous calcium transients and regulates basal calcium levels in cultured Kenyon cells. The data also suggest that these calcium transients represent cellular events underlying calcium oscillations in the intact mushroom bodies. However, spontaneous calcium transients are not unique to Kenyon cells as they are present in approximately 60% of all cultured central brain neurons. This suggests the calcium transients play a more general role in maturation or function of adult brain neurons.

  4. Calcium deficiency cannot induce obesity in rats.

    PubMed

    Paradis, S; Cabanac, M

    2005-06-30

    If intake of a required nutrient--here calcium--affects body weight, the effect must be mediated by a change in the body weight set-point. Thus, the controversial 'anti-obesity' influence of high calcium intake should decrease the body weight set-point. Diets differing in calcium content were assigned to three groups of rats. The effects of the diets on body weight, BMI, fat content, plasma calcium, body weight set-point, food intake, and preference for various calcium solutions were measured after 6 weeks of calcium deprivation or supplementation, and again after a further 6 weeks of recovery on a regular diet. After 6 weeks, the low-calcium diet had induced calcium deficiency but had failed to raise the body weight set-point. Nor had it produced obesity or fat accumulation. After 6 weeks of recovery, body weight and fat content were no higher in calcium-deprived rats than in the control or supplemented rats. In this experiment, low-calcium intake failed to cause obesity and did not raise the body weight set-point. The results indicate that calcium intake probably does not affect body weight.

  5. Induced calcium carbonate precipitation using Bacillus species.

    PubMed

    Seifan, Mostafa; Samani, Ali Khajeh; Berenjian, Aydin

    2016-12-01

    Microbially induced calcium carbonate precipitation is an emerging process for the production of self-healing concrete. This study was aimed to investigate the effects and optimum conditions on calcium carbonate biosynthesis. Bacillus licheniformis, Bacillus sphaericus, yeast extract, urea, calcium chloride and aeration were found to be the most significant factors affecting the biomineralization of calcium carbonate. It was noticed that the morphology of microbial calcium carbonate was mainly affected by the genera of bacteria (cell surface properties), the viscosity of the media and the type of electron acceptors (Ca(2+)). The maximum calcium carbonate concentration of 33.78 g/L was achieved at the optimum conditions This value is the highest concentration reported in the literature.

  6. A model of propagating calcium-induced calcium release mediated by calcium diffusion

    PubMed Central

    1989-01-01

    The effect of sudden local fluctuations of the free sarcoplasmic [Ca++]i in cardiac cells on calcium release and calcium uptake by the sarcoplasmic reticulum (SR) was calculated with the aid of a simplified model of SR calcium handling. The model was used to evaluate whether propagation of calcium transients and the range of propagation velocities observed experimentally (0.05-15 mm s(-1)) could be predicted. Calcium fluctuations propagate by virtue of focal calcium release from the SR, diffusion through the cytosol (which is modulated by binding to troponin and calmodulin and sequestration by the SR), and subsequently induce calcium release from adjacent release sites of the SR. The minimal and maximal velocities derived from the simulation were 0.09 and 15 mm s(-1) respectively. The method of solution involved writing the diffusion equation as a difference equation in the spatial coordinates. Thus, coupled ordinary differential equations in time with banded coefficients were generated. The coupled equations were solved using Gear's sixth order predictor-corrector algorithm for stiff equations with reflective boundaries. The most important determinants of the velocity of propagation of the calcium waves were the diastolic [Ca++]i, the rate of rise of the release, and the amount of calcium released from the SR. The results are consistent with the assumptions that calcium loading causes an increase in intracellular calcium and calcium in the SR, and an increase in the amount and rate of calcium released. These two effects combine to increase the propagation velocity at higher levels of calcium loading. PMID:2738577

  7. Role of calcium in growth inhibition induced by a novel cell surface sialoglycopeptide

    NASA Technical Reports Server (NTRS)

    Betz, N. A.; Westhoff, B. A.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    Our laboratory has purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) from intact bovine cerebral cortex cells. Evidence presented here demonstrates that sensitivity to CeReS-18-induced growth inhibition in BALB-c 3T3 cells is influenced by calcium, such that a decrease in the calcium concentration in the growth medium results in an increase in sensitivity to CeReS-18. Calcium did not alter CeReS-18 binding to its cell surface receptor and CeReS-18 does not bind calcium directly. Addition of calcium, but not magnesium, to CeReS-18-inhibited 3T3 cells results in reentry into the cell cycle. A greater than 3-hour exposure to increased calcium is required for escape from CeReS-18-induced growth inhibition. The calcium ionophore ionomycin could partially mimic the effect of increasing extracellular calcium, but thapsigargin was ineffective in inducing escape from growth inhibition. Increasing extracellular calcium 10-fold resulted in an approximately 7-fold increase in total cell-associated 45Ca+2, while free intracellular calcium only increased approximately 30%. However, addition of CeReS-18 did not affect total cell-associated calcium or the increase in total cell-associated calcium observed with an increase in extracellular calcium. Serum addition induced mobilization of intracellular calcium and influx across the plasma membrane in 3T3 cells, and pretreatment of 3T3 cells with CeReS-18 appeared to inhibit these calcium mobilization events. These results suggest that a calcium-sensitive step exists in the recovery from CeReS-18-induced growth inhibition. CeReS-18 may inhibit cell proliferation through a novel mechanism involving altering the intracellular calcium mobilization/regulation necessary for cell cycle progression.

  8. Teaching Calcium-Induced Calcium Release in Cardiomyocytes Using a Classic Paper by Fabiato

    ERIC Educational Resources Information Center

    Liang, Willmann

    2008-01-01

    This teaching paper utilizes the materials presented by Dr. Fabiato in his review article entitled "Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum." In the review, supporting evidence of calcium-induced calcium release (CICR) is presented. Data concerning potential objections to the CICR theory are discussed as well. In…

  9. Teaching Calcium-Induced Calcium Release in Cardiomyocytes Using a Classic Paper by Fabiato

    ERIC Educational Resources Information Center

    Liang, Willmann

    2008-01-01

    This teaching paper utilizes the materials presented by Dr. Fabiato in his review article entitled "Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum." In the review, supporting evidence of calcium-induced calcium release (CICR) is presented. Data concerning potential objections to the CICR theory are discussed as well. In…

  10. Calcium precipitate induced aerobic granulation.

    PubMed

    Wan, Chunli; Lee, Duu-Jong; Yang, Xue; Wang, Yayi; Wang, Xingzu; Liu, Xiang

    2015-01-01

    Aerobic granulation is a novel biotechnology for wastewater treatment. This study refined existing aerobic granulation mechanisms as a sequencing process including formation of calcium precipitate under alkaline pH to form inorganic cores, followed by bacterial attachment and growth on these cores to form the exopolysaccharide matrix. Mature granules comprised an inner core and a matrix layer and a rim layer with enriched microbial strains. The inorganic core was a mix of different crystals of calcium and phosphates. Functional strains including Sphingomonas sp., Paracoccus sp. Sinorhizobium americanum strain and Flavobacterium sp. attached onto the cores. These functional strains promote c-di-GMP production and the expression by Psl and Alg genes for exopolysaccharide production to enhance formation of mature granules.

  11. Calcium-induced calcium release in crayfish skeletal muscle.

    PubMed Central

    Györke, S; Palade, P

    1992-01-01

    1. Cut crayfish skeletal muscle fibres were mounted in a triple Vaseline-gap voltage clamp with the Ca(2+)-sensing dye Rhod-2 allowed to diffuse in via the cut ends. Ca2+ currents across the surface/T-tubule membranes (ICa) were recorded simultaneously with changes in myoplasmic Ca2+ concentration (Ca2+ transients). 2. Excitation-contraction coupling in crayfish skeletal muscle fibres is abolished when calcium in the extracellular solution is replaced by Mg2+. 3. The amplitude of the Ca2+ transients elicited by voltage clamp pulses closely followed the amplitude of the peak calcium currents recorded simultaneously across the surface/T-tubule membranes. This included decreases in both parameters as the pulse potential approached ECa (reversal potential for Ca2+), as well as secondary Ca2+ transients accompanying large tail calcium currents occurring upon repolarization from very large depolarizations. 4. A large contribution of sarcoplasmic reticulum (SR) Ca2+ release to the Ca2+ transients was revealed by a large decrease in the transient caused by the calcium-induced calcium release (CICR) blockers procaine and tetracaine. 5. Short pulses which interrupted the calcium current while SR Ca2+ release was in progress at high rates caused the Ca2+ transient to stop rising nearly immediately after the end of the pulse in most fibres. In about 15% of the fibres the Ca2+ transients continued to rise, albeit at a slower rate, for 10-20 ms after the end of the pulse, as if released Ca2+ was able to elicit some further Ca2+ release from the SR for a while. 6. Even with fibres displaying little sign of continued release after termination of short pulses under control conditions, procaine accelerated the decay of Ca2+ transients elicited by short pulses, indicating that continued release was taking place even as the transient was declining. 7. These results suggest that CICR in crayfish fibres is more closely controlled by a small entry of Ca2+ via surface/T-tubule membrane Ca

  12. Calcium Oxalate Induces Renal Injury through Calcium-Sensing Receptor

    PubMed Central

    Li, Xiaoran; Ma, Junhai; Shi, Wei; Su, Yu; Fu, Xu; Yang, Yanlin; Lu, Jianzhong

    2016-01-01

    Objective. To investigate whether calcium-sensing receptor (CaSR) plays a role in calcium-oxalate-induced renal injury. Materials and Methods. HK-2 cells and rats were treated with calcium oxalate (CaOx) crystals with or without pretreatment with the CaSR-specific agonist gadolinium chloride (GdCl3) or the CaSR-specific antagonist NPS2390. Changes in oxidative stress (OS) in HK-2 cells and rat kidneys were assessed. In addition, CaSR, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal protein kinase (JNK), and p38 expression was determined. Further, crystal adhesion assay was performed in vitro, and the serum urea and creatinine levels and crystal deposition in the kidneys were also examined. Results. CaOx increased CaSR, ERK, JNK, and p38 protein expression and OS in vitro and in vivo. These deleterious changes were further enhanced upon pretreatment with the CaSR agonist GdCl3 but were attenuated by the specific CaSR inhibitor NPS2390 compared with CaOx treatment alone. Pretreatment with GdCl3 further increased in vitro and in vivo crystal adhesion and renal hypofunction. In contrast, pretreatment with NPS2390 decreased in vitro and in vivo crystal adhesion and renal hypofunction. Conclusions. CaOx-induced renal injury is related to CaSR-mediated OS and increased mitogen-activated protein kinase (MAPK) signaling, which subsequently leads to CaOx crystal adhesion. PMID:27965733

  13. Calcium Sensitization Mechanisms in Gastrointestinal Smooth Muscles.

    PubMed

    Perrino, Brian A

    2016-04-30

    An increase in intracellular Ca(2+) is the primary trigger of contraction of gastrointestinal (GI) smooth muscles. However, increasing the Ca(2+) sensitivity of the myofilaments by elevating myosin light chain phosphorylation also plays an essential role. Inhibiting myosin light chain phosphatase activity with protein kinase C-potentiated phosphatase inhibitor protein-17 kDa (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation is considered to be the primary mechanism underlying myofilament Ca(2+) sensitization. The relative importance of Ca(2+) sensitization mechanisms to the diverse patterns of GI motility is likely related to the varied functional roles of GI smooth muscles. Increases in CPI-17 and MYPT1 phosphorylation in response to agonist stimulation regulate myosin light chain phosphatase activity in phasic, tonic, and sphincteric GI smooth muscles. Recent evidence suggests that MYPT1 phosphorylation may also contribute to force generation by reorganization of the actin cytoskeleton. The mechanisms responsible for maintaining constitutive CPI-17 and MYPT1 phosphorylation in GI smooth muscles are still largely unknown. The characteristics of the cell-types comprising the neuroeffector junction lead to fundamental differences between the effects of exogenous agonists and endogenous neurotransmitters on Ca(2+) sensitization mechanisms. The contribution of various cell-types within the tunica muscularis to the motor responses of GI organs to neurotransmission must be considered when determining the mechanisms by which Ca(2+) sensitization pathways are activated. The signaling pathways regulating Ca(2+) sensitization may provide novel therapeutic strategies for controlling GI motility. This article will provide an overview of the current understanding of the biochemical basis for the regulation of Ca(2+) sensitization, while also discussing the functional importance to different smooth muscles of the GI tract.

  14. Calcium Sensitization Mechanisms in Gastrointestinal Smooth Muscles

    PubMed Central

    Perrino, Brian A

    2016-01-01

    An increase in intracellular Ca2+ is the primary trigger of contraction of gastrointestinal (GI) smooth muscles. However, increasing the Ca2+ sensitivity of the myofilaments by elevating myosin light chain phosphorylation also plays an essential role. Inhibiting myosin light chain phosphatase activity with protein kinase C-potentiated phosphatase inhibitor protein-17 kDa (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation is considered to be the primary mechanism underlying myofilament Ca2+ sensitization. The relative importance of Ca2+ sensitization mechanisms to the diverse patterns of GI motility is likely related to the varied functional roles of GI smooth muscles. Increases in CPI-17 and MYPT1 phosphorylation in response to agonist stimulation regulate myosin light chain phosphatase activity in phasic, tonic, and sphincteric GI smooth muscles. Recent evidence suggests that MYPT1 phosphorylation may also contribute to force generation by reorganization of the actin cytoskeleton. The mechanisms responsible for maintaining constitutive CPI-17 and MYPT1 phosphorylation in GI smooth muscles are still largely unknown. The characteristics of the cell-types comprising the neuroeffector junction lead to fundamental differences between the effects of exogenous agonists and endogenous neurotransmitters on Ca2+ sensitization mechanisms. The contribution of various cell-types within the tunica muscularis to the motor responses of GI organs to neurotransmission must be considered when determining the mechanisms by which Ca2+ sensitization pathways are activated. The signaling pathways regulating Ca2+ sensitization may provide novel therapeutic strategies for controlling GI motility. This article will provide an overview of the current understanding of the biochemical basis for the regulation of Ca2+ sensitization, while also discussing the functional importance to different smooth muscles of the GI tract. PMID:26701920

  15. Calcium-dependent inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells

    PubMed Central

    1988-01-01

    The inactivation of calcium channels in mammalian pituitary tumor cells (GH3) was studied with patch electrodes under voltage clamp in cell- free membrane patches and in dialyzed cells. The calcium current elicited by depolarization from a holding potential of -40 mV passed predominantly through one class of channels previously shown to be modulated by dihydropyridines and cAMP-dependent phosphorylation (Armstrong and Eckert, 1987). When exogenous calcium buffers were omitted from the pipette solution, the macroscopic calcium current through those channels inactivated with a half time of approximately 10 ms to a steady state level 40-75% smaller than the peak. Inactivation was also measured as the reduction in peak current during a test pulse that closely followed a prepulse. Inactivation was largely reduced or eliminated by (a) buffering free calcium in the pipette solution to less than 10(-8) M; (b) replacing extracellular calcium with barium; (c) increasing the prepulse voltage from +10 to +60 mV; or (d) increasing the intracellular concentration of cAMP, either 'directly' with dibutyryl-cAMP or indirectly by activating adenylate cyclase with forskolin or vasoactive intestinal peptide. Thus, inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells only occurs when membrane depolarization leads to calcium ion entry and intracellular accumulation. PMID:2849631

  16. Frequency-dependent acceleration of relaxation involves decreased myofilament calcium sensitivity.

    PubMed

    Varian, Kenneth D; Janssen, Paul M L

    2007-05-01

    The force-frequency relationship is an intrinsic modulator of cardiac contractility and relaxation. Force of contraction increases with frequency, while simultaneously a frequency-dependent acceleration of relaxation occurs. While frequency dependency of calcium handling and sarcoplasmic reticulum calcium load have been well described, it remains unknown whether frequency-dependent changes in myofilament calcium sensitivity occur. We hypothesized that an increase in heart rate that results in acceleration of relaxation is accompanied by a proportional decrease in myofilament calcium sensitivity. To test our hypothesis, ultrathin right ventricular trabeculae were isolated from New Zealand White rabbit hearts and iontophorically loaded with the calcium indicator bis-fura 2. Twitch and intracellular calcium handling parameters were measured and showed a robust increase in twitch force, acceleration of relaxation, and rise in both diastolic and systolic intracellular calcium concentration with increased frequency. Steady-state force-intracellular calcium concentration relationships were measured at frequencies 1, 2, 3, and 4 Hz at 37 degrees C using potassium-induced contractures. EC(50) significantly and gradually increased with frequency, from 475 +/- 64 nM at 1 Hz to 1,004 +/- 142 nM at 4 Hz (P < 0.05) and correlated with the corresponding changes in half relaxation time. No significant changes in maximal active force development or in the myofilament cooperativity coefficient were found. Myofilament protein phosphorylation was assessed using Pro-Q Diamond staining on protein gels of trabeculae frozen at either 1 or 4 Hz, revealing troponin I and myosin light chain-2 phosphorylation associated with the myofilament desensitization. We conclude that myofilament calcium sensitivity is substantially and significantly decreased at higher frequencies, playing a prominent role in frequency-dependent acceleration of relaxation.

  17. Calcium sensitive ring-like oligomers formed by synaptotagmin

    PubMed Central

    Wang, Jing; Bello, Oscar; Auclair, Sarah M.; Wang, Jing; Coleman, Jeff; Pincet, Frederic; Krishnakumar, Shyam S.; Sindelar, Charles V.; Rothman, James E.

    2014-01-01

    The synaptic vesicle protein synaptotagmin-1 (SYT) is required to couple calcium influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear. Here we report an unexpected circular arrangement (ring) of SYT’s cytosolic domain (C2AB) formed on lipid monolayers in the absence of free calcium ions as revealed by electron microscopy. Rings vary in diameter from 18–43 nm, corresponding to 11–26 molecules of SYT. Continuous stacking of the SYT rings occasionally converts both lipid monolayers and bilayers into protein-coated tubes. Helical reconstruction of the SYT tubes shows that one of the C2 domains (most likely C2B, based on its biochemical properties) interacts with the membrane and is involved in ring formation, and the other C2 domain points radially outward. SYT rings are disrupted rapidly by physiological concentrations of free calcium but not by magnesium. Assuming that calcium-free SYT rings are physiologically relevant, these results suggest a simple and novel mechanism by which SYT regulates neurotransmitter release: The ring acts as a spacer to prevent the completion of the soluble N-ethylmaleimide–sensitive factor activating protein receptor (SNARE) complex assembly, thereby clamping fusion in the absence of calcium. When the ring disassembles in the presence of calcium, fusion proceeds unimpeded. PMID:25201968

  18. Calcium-induced calcium release and gap junctions mediate large-scale calcium waves in olfactory ensheathing cells in situ.

    PubMed

    Stavermann, Maren; Meuth, Patrick; Doengi, Michael; Thyssen, Anne; Deitmer, Joachim W; Lohr, Christian

    2015-08-01

    Olfactory ensheathing cells (OECs) are a specialised type of glial cells, supporting axon growth and guidance during development and regeneration of the olfactory nerve and the nerve layer of the olfactory bulb. We measured calcium signalling in OECs in olfactory bulb in-toto preparations using confocal and epifluorescence microscopy and the calcium indicator Fluo-4. We identified two subpopulations of olfactory bulb OECs: OECs in the outer sublamina of the nerve layer responded to purinergic neurotransmitters such as adenosine triphosphate with calcium transients, while OECs in the inner sublamina of the nerve layer did not respond to neurotransmitters. However, the latter generated spontaneous calcium waves that covered hundreds of cells. These calcium waves persisted in the presence of tetrodotoxin and in calcium-free saline, but were abolished after calcium store depletion with cyclopiazonic acid or inositol trisphosphate receptor blockage with 2-APB. Calcium waves could be triggered by laser photolysis of caged inositol trisphosphate. Blocking purinoceptors with PPADS had no effect on calcium wave propagation, whereas blocking gap junctions with carbenoxolone or meclofenamic acid entirely suppressed calcium waves. Increasing calcium buffer capacity in OECs with NP-EGTA ("caged" Ca(2+)) prevented calcium wave generation, and laser photolysis of NP-EGTA in a small group of OECs resulted in a calcium increase in the irradiated cells followed by a calcium wave. We conclude that calcium waves in OECs can be initiated by calcium-induced calcium release via InsP3 receptors and propagate through gap junctions, while purinergic signalling is not involved. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Intracellular calcium affects prestin's voltage operating point indirectly via turgor-induced membrane tension

    NASA Astrophysics Data System (ADS)

    Song, Lei; Santos-Sacchi, Joseph

    2015-12-01

    Recent identification of a calmodulin binding site within prestin's C-terminus indicates that calcium can significantly alter prestin's operating voltage range as gauged by the Boltzmann parameter Vh (Keller et al., J. Neuroscience, 2014). We reasoned that those experiments may have identified the molecular substrate for the protein's tension sensitivity. In an effort to understand how this may happen, we evaluated the effects of turgor pressure on such shifts produced by calcium. We find that the shifts are induced by calcium's ability to reduce turgor pressure during whole cell voltage clamp recording. Clamping turgor pressure to 1kPa, the cell's normal intracellular pressure, completely counters the calcium effect. Furthermore, following unrestrained shifts, collapsing the cells abolishes induced shifts. We conclude that calcium does not work by direct action on prestin's conformational state. The possibility remains that calcium interaction with prestin alters water movements within the cell, possibly via its anion transport function.

  20. Detection of calcium activity in human monocytes by the fura-2 fluorescence method: in vitro differentiation sensitizes cells to dihydropyridine calcium channel modulators

    NASA Astrophysics Data System (ADS)

    Oraevsky, Alexander A.; Cabello, Olga A.; Shan, Qin; Tittel, Frank K.; Henry, Philip D.

    1994-07-01

    Dihydropyridine (DHP) calcium channel blockers have been shown to suppress atherogenesis in various species and controlled angiographic trials suggest that these drugs may retard the progression of occlusive coronary disease in humans. Because mononuclear leukocytes play a key role in the formation of early and advanced atheromatous lesions, we determined effects of DHP calcium channel modulators on calcium uptake by cells of the monocytic lineage. Human peripheral blood monocytes were evaluated before and after undergoing in vitro differentiation induced by two days of culture with fetal calf serum and FMLP. Changes in intracellular calcium activity were estimated with fura-2, a fluorescent calcium indicator. Freshly isolated (unactivated) monocytes were insensitive to DHP drugs both in the presence and absence of high potassium membrane depolarization. In contrast, nisoldipine, a DHP calcium channel blocker, and BAY K 8644, a DHP calcium channel activator, decreased and increased calcium uptake by KC1-depolarized differentiated monocytes. Results suggest that differentiation of monocytes to macrophages may involve a change in the expression and/or regulation of DHP- sensitive calcium channels.

  1. Influence of calcium sources on microbially induced calcium carbonate precipitation by Bacillus sp. CR2.

    PubMed

    Achal, Varenyam; Pan, Xiangliang

    2014-05-01

    Stimulation of microbially induced calcium carbonate precipitation (MICCP) is likely to be influenced by calcium sources. In order to study such influences, we performed MICCP using Bacillus sp. CR2 in nutrient broth containing urea, supplemented with different calcium sources (calcium chloride, calcium oxide, calcium acetate and calcium nitrate). The experiment lasted 7 days, during which bacterial growth, urease activity, calcite production and pH were measured. Our results showed that calcium chloride is the better calcium source for MICCP process, since it provides higher urease activity and more calcite production. The influences of calcium sources on MICCP were further studied using Fourier transform-infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and X-ray diffraction (XRD) analyses. These analyses confirmed that the precipitate formed was CaCO3 and composed of predominantly calcite crystals with a little amount of aragonite and vaterite crystals. The maximum yield of calcite precipitation was achievable with calcium chloride followed by calcium nitrate as a calcium source. The results of present study may be applicable to media preparation during efficient MICCP process.

  2. Neuropharmacological characterization of voltage-sensitive calcium channels: possible existence of neomycin-sensitive, omega-conotoxin GVIA- and dihydropyridines-resistant calcium channels in the rat brain.

    PubMed

    Yamada, K; Teraoka, T; Morita, S; Hasegawa, T; Nabeshima, T

    1993-12-01

    We attempted to characterize the functional roles of subtypes of voltage-sensitive calcium channels in the brain. The maximal number of [125I]omega-conotoxin GVIA (omega-CTX) binding sites in rat brain associated with N-type calcium channels (N-channels) was approximately 10 times more than that of [3H]-PN200-110 associated with L-type calcium channels (L-channels). [125I]omega-CTX binding was inhibited by aminoglycoside antibiotics, neomycin and dynorphin A(1-13), but not by various classes of L-channel antagonists. A 6-hydroxydopamine-induced lesion of the striatum resulted in a marked reduction of both [125I]-omega-CTX and [3H]PN200-110 binding. Kainic acid-induced lesion of the striatum reduced [3H]PN200-110 binding by 57%, but did not reduce [125I]omega-CTX binding. Omega-CTX produced a small (18%) but significant reduction of potassium-stimulated Ca2+ influx into rat brain synaptosomes, although it produced a concentration-dependent inhibition in chick brain synaptosomes. Neomycin inhibited Ca2+ influx in both preparations in a concentration-dependent manner. Both omega-CTX and neomycin inhibited potassium-stimulated [3H]dopamine (DA) release from rat striatal slices. The L-channel antagonists had no effect on either Ca2+ influx or [3H]DA release. These results suggest that DA release in the striatum is regulated by Ca2+ influx through N-channels located in presynaptic nerve terminals, and that the most of the Ca2+ influx in rat brain appears to be governed by neomycin-sensitive, omega-CTX- and DHP-resistant calcium channels.

  3. Differing calcium sensitivities of human cerebral and digital arteries, human metatarsal veins, and rat aorta.

    PubMed Central

    Iwanov, V; Moulds, R F

    1991-01-01

    1. The effects of the voltage dependent calcium channel blocking agent nifedipine, and of a calcium free bathing medium, on the responses of human blood vessels obtained postmortem to various agonists have been compared with those of the rat aorta. The human vessels studied were digital arteries, basilar arteries and metatarsal veins. 2. Responses to potassium chloride (5-80 mM), noradrenaline (10(-9)-10(-4) M), 5-hydroxytryptamine (10(-8)-10(-4) M) and U46619 (10(-11)-10(-6) M), in the presence and absence of nifedipine (1, 10, and 100 nM) or in a calcium-free bathing medium, were assessed using an area-under-curve analysis. 3. In general, the order of sensitivity of the vessels to inhibition of agonist induced contractures by nifedipine was basilar arteries greater than metatarsal veins = digital arteries = rat aorta. 4. For all the vessels, the order of sensitivity for antagonism of responses to the agonists by nifedipine was potassium chloride greater than 5-hydroxytryptamine = noradrenaline greater than U46619. 5. A calcium free bath inhibited responses of digital arteries to potassium chloride more than noradrenaline, 5-hydroxytryptamine or U46619, and responses of rat aorta to a greater extent than responses of the digital arteries. 6. In the rat aorta, a calcium-free bath inhibited responses to all agonists (except KCl) to a greater degree than did nifedipine. 7. We conclude that inhibition of extracellular calcium entry through voltage dependent calcium channels affects contractile responses of different blood vessels to different extents, and, within the same blood vessel, responses to different contractile agonists to different extents. PMID:2015170

  4. Reversible loss of gravitropic sensitivity in maize roots after tip application of calcium chelators

    NASA Technical Reports Server (NTRS)

    Lee, J. S.; Mulkey, T. J.; Evans, M. L.

    1983-01-01

    The application of calcium chelating agents (EDTA or EGTA) to the tips of maize roots caused a loss of gravitropic sensitivity. When the chelator was replaced with calcium chloride, gravitropic sensitivity was restored. Asymmetric application of calcium chloride near the tip of a vertical root caused curvature toward the calcium source. When the calcium was applied to the upper surface of the tip of a root oriented horizontally, the root curved upward even though control roots exhibited strong downward curvature. Application of calcium chloride to the tips of decapped roots, which are known to be gravitropically insensitive, did not restore gravitropic sensitivity. However, asymmetric application of calcium chloride near the tips of decapped roots caused curvature toward the calcium source. Calcium may play a key role in linking gravity detection to gravitropic curvature in roots.

  5. Reversible loss of gravitropic sensitivity in maize roots after tip application of calcium chelators

    NASA Technical Reports Server (NTRS)

    Lee, J. S.; Mulkey, T. J.; Evans, M. L.

    1983-01-01

    The application of calcium chelating agents (EDTA or EGTA) to the tips of maize roots caused a loss of gravitropic sensitivity. When the chelator was replaced with calcium chloride, gravitropic sensitivity was restored. Asymmetric application of calcium chloride near the tip of a vertical root caused curvature toward the calcium source. When the calcium was applied to the upper surface of the tip of a root oriented horizontally, the root curved upward even though control roots exhibited strong downward curvature. Application of calcium chloride to the tips of decapped roots, which are known to be gravitropically insensitive, did not restore gravitropic sensitivity. However, asymmetric application of calcium chloride near the tips of decapped roots caused curvature toward the calcium source. Calcium may play a key role in linking gravity detection to gravitropic curvature in roots.

  6. Glutamate Induces Calcium Waves in Cultured Astrocytes: Long-Range Glial Signaling

    NASA Astrophysics Data System (ADS)

    Cornell-Bell, Ann H.; Finkbeiner, Steven M.; Cooper, Mark S.; Smith, Stephen J.

    1990-01-01

    The finding that astrocytes possess glutamate-sensitive ion channels hinted at a previously unrecognized signaling role for these cells. Now it is reported that cultured hippocampal astrocytes can respond to glutamate with a prompt and oscillatory elevation of cytoplasmic free calcium, visible through use of the fluorescent calcium indicator fluo-3. Two types of glutamate receptor-one preferring quisqualate and releasing calcium from intracellular stores and the other preferring kainate and promoting surface-membrane calcium influx-appear to be involved. Moreover, glutamate-induced increases in cytoplasmic free calcium frequently propagate as waves within the cytoplasm of individual astrocytes and between adjacent astrocytes in confluent cultures. These propagating waves of calcium suggest that networks of astrocytes may constitute a long-range signaling system within the brain.

  7. Selective calcium sensitivity in immature glioma cancer stem cells.

    PubMed

    Wee, Shimei; Niklasson, Maria; Marinescu, Voichita Dana; Segerman, Anna; Schmidt, Linnéa; Hermansson, Annika; Dirks, Peter; Forsberg-Nilsson, Karin; Westermark, Bengt; Uhrbom, Lene; Linnarsson, Sten; Nelander, Sven; Andäng, Michael

    2014-01-01

    Tumor-initiating cells are a subpopulation in aggressive cancers that exhibit traits shared with stem cells, including the ability to self-renew and differentiate, commonly referred to as stemness. In addition, such cells are resistant to chemo- and radiation therapy posing a therapeutic challenge. To uncover stemness-associated functions in glioma-initiating cells (GICs), transcriptome profiles were compared to neural stem cells (NSCs) and gene ontology analysis identified an enrichment of Ca2+ signaling genes in NSCs and the more stem-like (NSC-proximal) GICs. Functional analysis in a set of different GIC lines regarding sensitivity to disturbed homeostasis using A23187 and Thapsigargin, revealed that NSC-proximal GICs were more sensitive, corroborating the transcriptome data. Furthermore, Ca2+ drug sensitivity was reduced in GICs after differentiation, with most potent effect in the NSC-proximal GIC, supporting a stemness-associated Ca2+ sensitivity. NSCs and the NSC-proximal GIC line expressed a larger number of ion channels permeable to potassium, sodium and Ca2+. Conversely, a higher number of and higher expression levels of Ca2+ binding genes that may buffer Ca2+, were expressed in NSC-distal GICs. In particular, expression of the AMPA glutamate receptor subunit GRIA1, was found to associate with Ca2+ sensitive NSC-proximal GICs, and decreased as GICs differentiated along with reduced Ca2+ drug sensitivity. The correlation between high expression of Ca2+ channels (such as GRIA1) and sensitivity to Ca2+ drugs was confirmed in an additional nine novel GIC lines. Calcium drug sensitivity also correlated with expression of the NSC markers nestin (NES) and FABP7 (BLBP, brain lipid-binding protein) in this extended analysis. In summary, NSC-associated NES+/FABP7+/GRIA1+ GICs were selectively sensitive to disturbances in Ca2+ homeostasis, providing a potential target mechanism for eradication of an immature population of malignant cells.

  8. Calcium accentuates injury induced by ethanol in human gastric cells.

    PubMed

    Kokoska, E R; Smith, G S; Deshpande, Y; Wolff, A B; Rieckenberg, C; Miller, T A

    1999-01-01

    The mechanism(s) whereby ethanol induces cellular injury remains poorly understood. Furthermore, the role of calcium in gastric mucosal injury under in vitro conditions is poorly defined. The major objectives of this study were to (1) define the temporal relationship between intracellular calcium accumulation induced by ethanol and cellular injury, (2) characterize the mechanism(s) whereby ethanol increases cellular calcium content, and (3) determine whether calcium removal would attenuate ethanol-induced cellular injury. Human gastric cells (AGS) were used for all experiments. Sustained intracellular calcium accumulation induced by ethanol, but not transient changes, preceded and directly correlated with cellular injury. Cells exposed to damaging concentrations of ethanol demonstrated an initial calcium surge that appeared to be a consequence of inositol 1,4,5-triphosphate (IP3) generation and subsequent internal store release followed by a sustained plateau resulting from extracellular calcium influx through store-operated calcium channels. Finally, both morphologic (cellular injury) and functional (clearance of bovine serum albumin) changes induced by ethanol were significantly attenuated when extracellular Ca(+&plus) influx was prevented, and further decreased when intracellular Ca(++) stores were depleted. These data indicate that calcium plays a significant role in cellular injury induced by ethanol.

  9. Modulation of iron metabolism by iron chelation regulates intracellular calcium and increases sensitivity to doxorubicin

    PubMed Central

    Yalcintepe, Leman; Halis, Emre

    2016-01-01

    Increased intracellular iron levels can both promote cell proliferation and death, as such; iron has a “two-sided effect” in the delicate balance of human health. Though the role of iron in the development of cancer remains unclear, investigations of iron chelators as anti-tumor agents have revealed promising results. Here, we investigated the influence of iron and desferrioxamine (DFO), the iron chelating agent on intracellular calcium in a human leukemia cell line, K562. Iron uptake is associated with increased reactive oxygen species (ROS) generation. Therefore, we showed that iron also caused dose-dependent ROS generation in K562 cells. The measurement of intracellular calcium was determined using Furo-2 with a fluorescence spectrophotometer. The iron delivery process to the cytoplasmic iron pool was examined by monitoring the fluorescence of cells loaded with calcein-acetoxymethyl. Our data showed that iron increased intracellular calcium, and this response was 8 times higher when cells were incubated with DFO. K562 cells with DFO caused a 3.5 times increase of intracellular calcium in the presence of doxorubicin (DOX). In conclusion, DFO induces intracellular calcium and increases their sensitivity to DOX, a chemotherapeutic agent. PMID:26773173

  10. Speract induces calcium oscillations in the sperm tail.

    PubMed

    Wood, Chris D; Darszon, Alberto; Whitaker, Michael

    2003-04-14

    Sea urchin sperm motility is modulated by sperm-activating peptides. One such peptide, speract, induces changes in intracellular free calcium concentration ([Ca2+]i). High resolution imaging of single sperm reveals that speract-induced changes in [Ca2+]i have a complex spatiotemporal structure. [Ca2+]i increases arise in the tail as periodic oscillations; [Ca2+]i increases in the sperm head lag those in the tail and appear to result from the summation of the tail signal transduction events. The period depends on speract concentration. Infrequent spontaneous [Ca2+]i transients were also seen in the tail of unstimulated sperm, again with the head lagging the tail. Speract-induced fluctuations were sensitive to membrane potential and calcium channel blockers, and were potentiated by niflumic acid, an anion channel blocker. 3-isobutyl-1-methylxanthine, which potentiates the cGMP/cAMP-signaling pathways, abolished the [Ca2+]i fluctuations in the tail, leading to a very delayed and sustained [Ca2+]i increase in the head. These data point to a model in which a messenger generated periodically in the tail diffuses to the head. Sperm are highly polarized cells. Our results indicate that a clear understanding of the link between [Ca2+]i and sperm motility will only be gained by analysis of [Ca2+]i signals at the level of the single sperm.

  11. Decreased expression of ryanodine receptors alters calcium-induced calcium release mechanism in mdx duodenal myocytes.

    PubMed

    Morel, Jean-Luc; Rakotoarisoa, Lala; Jeyakumar, Loice H; Fleischer, Sidney; Mironneau, Chantal; Mironneau, Jean

    2004-05-14

    It is generally believed that alterations of calcium homeostasis play a key role in skeletal muscle atrophy and degeneration observed in Duchenne's muscular dystrophy and mdx mice. Mechanical activity is also impaired in gastrointestinal muscles, but the cellular and molecular mechanisms of this pathological state have not yet been investigated. We showed, in mdx duodenal myocytes, that both caffeine- and depolarization-induced calcium responses were inhibited, whereas acetylcholine- and thapsigargin-induced calcium responses were not significantly affected compared with control mice. Calcium-induced calcium release efficiency was impaired in mdx duodenal myocytes depending only on inhibition of ryanodine receptor expression. Duodenal myocytes expressed both type 2 and type 3 ryanodine receptors and were unable to produce calcium sparks. In control and mdx duodenal myocytes, both caffeine- and depolarization-induced calcium responses were dose-dependently and specifically inhibited with the anti-type 2 ryanodine receptor antibody. A strong inhibition of type 2 ryanodine receptor in mdx duodenal myocytes was observed on the mRNA as well as on the protein level. Taken together, our results suggest that inhibition of type 2 ryanodine receptor expression in mdx duodenal myocytes may account for the decreased calcium release from the sarcoplasmic reticulum and reduced mechanical activity.

  12. Sensitive red protein calcium indicators for imaging neural activity

    PubMed Central

    Dana, Hod; Mohar, Boaz; Sun, Yi; Narayan, Sujatha; Gordus, Andrew; Hasseman, Jeremy P; Tsegaye, Getahun; Holt, Graham T; Hu, Amy; Walpita, Deepika; Patel, Ronak; Macklin, John J; Bargmann, Cornelia I; Ahrens, Misha B; Schreiter, Eric R; Jayaraman, Vivek; Looger, Loren L; Svoboda, Karel; Kim, Douglas S

    2016-01-01

    Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging. DOI: http://dx.doi.org/10.7554/eLife.12727.001 PMID:27011354

  13. Electroporation loading of calcium-sensitive dyes into the CNS.

    PubMed

    Bonnot, Agnès; Mentis, George Z; Skoch, Jesse; O'Donovan, Michael J

    2005-03-01

    Calcium imaging of neural network function has been limited by the extent of tissue labeled or the time taken for labeling. We now describe the use of electroporation-an established technique for transfecting cells with genes-to load neurons with calcium-sensitive dyes in the isolated spinal cord of the neonatal mouse in vitro. The dyes were injected subdurally, intravascularly, or into the central canal. This technique results in rapid and extensive labeling of neurons and their processes at all depths of the spinal cord, over a rostrocaudal extent determined by the position and size of the electrodes. Our results suggest that vascular distribution of the dye is involved in all three types of injections. Electroporation disrupts local reflex and network function only transiently (approximately 1 h), after which time they recover. We describe applications of the method to image activity of neuronal populations and individual neurons during antidromic, reflex, and locomotor-like behaviors. We show that these different motor behaviors are characterized by distinct patterns of activation among the labeled populations of cells.

  14. Levosimendan improves calcium sensitivity of diaphragm muscle fibres from a rat model of heart failure.

    PubMed

    van Hees, H W H; Andrade Acuña, Gl; Linkels, M; Dekhuijzen, P N R; Heunks, L M A

    2011-02-01

    Diaphragm muscle weakness occurs in patients with heart failure (HF) and is associated with exercise intolerance and increased mortality. Reduced sensitivity of diaphragm fibres to calcium contributes to diaphragm weakness in HF. Here we have investigated the ability of the calcium sensitizer levosimendan to restore the reduced calcium sensitivity of diaphragm fibres from rats with HF. Coronary artery ligation in rats was used as an animal model for HF. Sham-operated rats served as controls. Fifteen weeks after induction of HF or sham operations animals were killed and muscle fibres were isolated from the diaphragm. Diaphragm fibres were skinned and activated with solutions containing incremental calcium concentrations and 10 µM levosimendan or vehicle (0.02% DMSO). Developed force was measured at each calcium concentration, and force-calcium concentration relationships were plotted. Calcium sensitivity of force generation was reduced in diaphragm muscle fibres from HF rats, compared with fibres from control rats (P < 0.01). Maximal force generation was ∼25% lower in HF diaphragm fibres than in control fibres (P < 0.05). Levosimendan significantly increased calcium sensitivity of force generation in diaphragm fibres from HF and control rats, without affecting maximal force generation. Levosimendan enhanced the force generating capacity of diaphragm fibres from HF rats by increasing the sensitivity of force generation to calcium concentration. These results provide strong support for testing the effect of calcium sensitizers on diaphragm muscle weakness in patients with HF. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  15. Direct Current-Induced Calcium Trafficking in Different Neuronal Preparations

    PubMed Central

    Ahmed, Zaghloul

    2016-01-01

    The influence of direct current (DC) stimulation on radioactive calcium trafficking in sciatic nerve in vivo and in vitro, spinal cord, and synaptosomes was investigated. The exposure to DC enhanced calcium redistribution in all of these preparations. The effect was dependent on the strength of the stimulation and extended beyond the phase of exposure to DC. The DC-induced increase in calcium sequestration by synaptosomes was significantly reduced by cobalt and rupture of synaptosomes by osmotic shock. Although both anodal and cathodal currents were effective, the experiments with two electrodes of different areas revealed that cathodal stimulation exerted stronger effect. The exposure to DC induced not only relocation but also redistribution of calcium within segments of the sciatic nerve. Enzymatic removal of sialic acid by preincubation of synaptosomes with neuroaminidase, or carrying out the experiments in sodium-free environment, amplified DC-induced calcium accumulation. PMID:28074161

  16. High-pressure studies on the calcium-ion-sensitive fluorophore Fluo-4

    NASA Astrophysics Data System (ADS)

    Frey, Eric W.; Urayama, Paul

    2007-10-01

    Fluorescence-based methods for intracellular calcium ion sensing are well established at ambient pressure. Because calcium ions play a ubiquitous role in cellular signaling, extending techniques of intracellular calcium-sensing to high pressures would play an important role in understanding the large variety of piezophysiologic effects. Here, we characterize the intracellular calcium-ion-sensitive fluorophore Fluo-4 under hydrostatic pressures up to 500 atm (50 MPa). Using an EGTA/MOPS solution as a calcium-buffer reference, we investigate the pressure dependence of the reaction pK and determine the thermodynamic volume change associated with the Fluo-4 calcium-binding reaction.

  17. Noise induced calcium oscillations in a cell exposed to electromagnetic fields.

    PubMed

    Zhang, Yuhong; Zhao, Yongli; Chen, Yafei; Yuan, Changqing; Zhan, Yong

    2015-01-01

    The effects of noise on the calcium oscillations in a cell exposed to electromagnetic fields are described by a dynamic model. Noise is a very important factor to be considered in the dynamic research on the calcium oscillations in a cell exposed to electromagnetic fields. Some meaningful results have been obtained here based on the discussion. The results show that the pattern of intracellular calcium oscillations exposure to electromagnetic fields can be influenced by noise. Furthermore, the intracellular calcium oscillations exposure to electromagnetic fields can also be induced by noise. And the work has also studied the relationships between the voltage sensitive calcium channel's open probability and electromagnetic field. The result can provide new insights into constructive roles and potential applications of selecting appropriate electromagnetic field frequency during the research of biological effect of electromagnetic field.

  18. Calcium-induced calcium release supports recruitment of synaptic vesicles in auditory hair cells

    PubMed Central

    Schnee, Michael E.; Ricci, Anthony J.

    2015-01-01

    Hair cells from auditory and vestibular systems transmit continuous sound and balance information to the central nervous system through the release of synaptic vesicles at ribbon synapses. The high activity experienced by hair cells requires a unique mechanism to sustain recruitment and replenishment of synaptic vesicles for continuous release. Using pre- and postsynaptic electrophysiological recordings, we explored the potential contribution of calcium-induced calcium release (CICR) in modulating the recruitment of vesicles to auditory hair cell ribbon synapses. Pharmacological manipulation of CICR with agents targeting endoplasmic reticulum calcium stores reduced both spontaneous postsynaptic multiunit activity and the frequency of excitatory postsynaptic currents (EPSCs). Pharmacological treatments had no effect on hair cell resting potential or activation curves for calcium and potassium channels. However, these drugs exerted a reduction in vesicle release measured by dual-sine capacitance methods. In addition, calcium substitution by barium reduced release efficacy by delaying release onset and diminishing vesicle recruitment. Together these results demonstrate a role for calcium stores in hair cell ribbon synaptic transmission and suggest a novel contribution of CICR in hair cell vesicle recruitment. We hypothesize that calcium entry via calcium channels is tightly regulated to control timing of vesicle fusion at the synapse, whereas CICR is used to maintain a tonic calcium signal to modulate vesicle trafficking. PMID:26510758

  19. Sensitivity and specificity of 24-hour urine chemistry levels for detecting elevated calcium oxalate and calcium phosphate supersaturation

    PubMed Central

    Rossi, M. Adrian; Singer, Eric A; Golijanin, Dragan J; Monk, Rebeca D; Erturk, Erdal; Bushinsky, David A

    2008-01-01

    Objectives The gold standard for determining likelihood of calcium oxalate (CaOx) and calcium phosphate (CaPhos) stone formation in urine is supersaturation of CaOx and CaPhos. Our objective was to investigate whether traditional measurement of total calcium, oxalate and phosphate in a 24-hour urine collection is sufficiently sensitive and specific for detecting elevated supersaturation to preclude the more expensive supersaturation test. Methods We performed a retrospective review of 150 consecutive patients with nephrolithiasis who underwent measurement of CaOx supersaturation (CaOxSS) and CaPhos supersaturation (CaPhosSS), as well as total calcium, oxalate and phosphate in a 24-hour urine collection. We used various cut-off values to determine sensitivity and specificity of 24-hour urine measurements for detecting elevated CaOxSS and CaPhosSS. Results In men and women, the sensitivity of 24-hour calcium for detecting elevated CaOxSS was 71% and 79%, respectively; for oxalate, sensitivity was 59% and 36%, respectively. In men and women, the sensitivity of 24-hour calcium for detecting elevated CaPhosSS was 74% and 88%, respectively; for phosphate, sensitivity was 57% and 8%, respectively. In men and women, the specificity of 24-hour calcium for detecting elevated CaOxSS was 55% and 48%, respectively; it was 60% for detecting elevated CaPhosSS in both men and women. Conclusion Traditional 24-hour urine analysis is sensitive, but not specific, for detecting elevated CaOxSS and CaPhosSS. Most patients with abnormal 24-hour urine analysis have normal supersaturation, and treatment decisions based on traditional urine analysis would lead to overtreatment in these patients. PMID:18542745

  20. PYK2: A Calcium-sensitive Protein Tyrosine Kinase Activated in Response to Fertilization of the Zebrafish Oocyte

    PubMed Central

    Sharma, Dipika; Kinsey, William H.

    2012-01-01

    Fertilization begins with binding and fusion of a sperm with the oocyte, a process that triggers a high amplitude calcium transient which propagates through the oocyte and stimulates a series of preprogrammed signal transduction events critical for zygote development. Identification of the pathways downstream of this calcium transient remains an important step in understanding the basis of zygote quality. The present study demonstrates that the calcium-calmodulin sensitive protein tyrosine kinase PYK2 is a target of the fertilization-induced calcium transient in the zebrafish oocyte and that it plays an important role in actin-mediated events critical for sperm incorporation. At fertilization, PYK2 was activated initially at the site of sperm-oocyte interaction and was closely associated with actin filaments forming the fertilization cone. Later PYK2 activation was evident throughout the entire oocyte cortex, however activation was most intense over the animal hemisphere. Fertilization-induced PYK2 activation could be blocked by suppressing calcium transients in the ooplasm via injection of BAPTA as a calcium chelator. PYK2 activation could be artificially induced in unfertilized oocytes by injection of IP3 at concentrations sufficient to induce calcium release. Functionally, suppression of PYK2 activity by chemical inhibition or by injection of a dominant-negative construct encoding the N-terminal ERM domain of PKY2 inhibited formation of an organized fertilization cone and reduced the frequency of successful sperm incorporation. Together, the above findings support a model in which PYK2 responds to the fertilization-induced calcium transient by promoting reorganization of the cortical actin cytoskeleton to form the fertilization cone. PMID:23084926

  1. Sphingomyelinase depresses force and calcium sensitivity of the contractile apparatus in mouse diaphragm muscle fibers

    PubMed Central

    Ferreira, Leonardo F.; Moylan, Jennifer S.; Stasko, Shawn; Smith, Jeffrey D.; Campbell, Kenneth S.

    2012-01-01

    Diseases that result in muscle weakness, e.g., heart failure, are characterized by elevated sphingomyelinase (SMase) activity. In intact muscle, SMase increases oxidants that contribute to diminished muscle force. However, the source of oxidants, specific processes of muscle contraction that are dysfunctional, and biochemical changes underlying the weakness elicited by SMase remain unknown. We tested three hypotheses: 1) SMase-induced depression of muscle force is mediated by mitochondrial reactive oxygen species (ROS), 2) SMase depresses force and calcium sensitivity of the contractile apparatus, and 3) SMase promotes oxidation and phosphorylation of myofibrillar proteins. Our experiments included intact muscle bundles, permeabilized single fibers, and isolated myofibrillar proteins. The mitochondrial-targeted antioxidant d-Arg-2′,6′-dimethyl-Tyr-Lys-Phe-NH2, decreased cytosolic oxidants and protected intact muscle bundles from weakness stimulated by SMase. SMase depressed maximal calcium-activated force by 20% in permeabilized single fibers (in kN/m2: control 117 ± 6; SMase 93 ± 8; P < 0.05). Calcium sensitivity of permeabilized single fibers decreased from 5.98 ± 0.03 (control) to 5.91 ± 0.02 (SMase; P < 0.05). Myofibrillar protein nitrotyrosines, carbonyls, and phosphorylation were unaltered by SMase. Our study shows that the fall in specific force of intact muscle elicited by SMase is mediated by mitochondrial ROS and can be attributed largely to dysfunction of the contractile apparatus. PMID:22362402

  2. Calcium-Induced calcium release during action potential firing in developing inner hair cells.

    PubMed

    Iosub, Radu; Avitabile, Daniele; Grant, Lisa; Tsaneva-Atanasova, Krasimira; Kennedy, Helen J

    2015-03-10

    In the mature auditory system, inner hair cells (IHCs) convert sound-induced vibrations into electrical signals that are relayed to the central nervous system via auditory afferents. Before the cochlea can respond to normal sound levels, developing IHCs fire calcium-based action potentials that disappear close to the onset of hearing. Action potential firing triggers transmitter release from the immature IHC that in turn generates experience-independent firing in auditory neurons. These early signaling events are thought to be essential for the organization and development of the auditory system and hair cells. A critical component of the action potential is the rise in intracellular calcium that activates both small conductance potassium channels essential during membrane repolarization, and triggers transmitter release from the cell. Whether this calcium signal is generated by calcium influx or requires calcium-induced calcium release (CICR) is not yet known. IHCs can generate CICR, but to date its physiological role has remained unclear. Here, we used high and low concentrations of ryanodine to block or enhance CICR to determine whether calcium release from intracellular stores affected action potential waveform, interspike interval, or changes in membrane capacitance during development of mouse IHCs. Blocking CICR resulted in mixed action potential waveforms with both brief and prolonged oscillations in membrane potential and intracellular calcium. This mixed behavior is captured well by our mathematical model of IHC electrical activity. We perform two-parameter bifurcation analysis of the model that predicts the dependence of IHCs firing patterns on the level of activation of two parameters, the SK2 channels activation and CICR rate. Our data show that CICR forms an important component of the calcium signal that shapes action potentials and regulates firing patterns, but is not involved directly in triggering exocytosis. These data provide important insights

  3. Calcium-Induced Calcium Release during Action Potential Firing in Developing Inner Hair Cells

    PubMed Central

    Iosub, Radu; Avitabile, Daniele; Grant, Lisa; Tsaneva-Atanasova, Krasimira; Kennedy, Helen J.

    2015-01-01

    In the mature auditory system, inner hair cells (IHCs) convert sound-induced vibrations into electrical signals that are relayed to the central nervous system via auditory afferents. Before the cochlea can respond to normal sound levels, developing IHCs fire calcium-based action potentials that disappear close to the onset of hearing. Action potential firing triggers transmitter release from the immature IHC that in turn generates experience-independent firing in auditory neurons. These early signaling events are thought to be essential for the organization and development of the auditory system and hair cells. A critical component of the action potential is the rise in intracellular calcium that activates both small conductance potassium channels essential during membrane repolarization, and triggers transmitter release from the cell. Whether this calcium signal is generated by calcium influx or requires calcium-induced calcium release (CICR) is not yet known. IHCs can generate CICR, but to date its physiological role has remained unclear. Here, we used high and low concentrations of ryanodine to block or enhance CICR to determine whether calcium release from intracellular stores affected action potential waveform, interspike interval, or changes in membrane capacitance during development of mouse IHCs. Blocking CICR resulted in mixed action potential waveforms with both brief and prolonged oscillations in membrane potential and intracellular calcium. This mixed behavior is captured well by our mathematical model of IHC electrical activity. We perform two-parameter bifurcation analysis of the model that predicts the dependence of IHCs firing patterns on the level of activation of two parameters, the SK2 channels activation and CICR rate. Our data show that CICR forms an important component of the calcium signal that shapes action potentials and regulates firing patterns, but is not involved directly in triggering exocytosis. These data provide important insights

  4. Multiple receptors mobilize calcium through a pertussis toxin (PT) sensitive GTP-binding protein in human neutrophils (PMN's)

    SciTech Connect

    Lad, P.M.; Olson, C.V.; Grewal, I.S.; Frolich, M.; Scott, S.J.

    1986-03-05

    Treatment of PMN's with PT causes an abolition of chemotaxis, enzyme release, superoxide generation and aggregation caused by f-met-leu-phe (FMLP),C5a and platelet activating factor (PAF). Lectin (Con-A) induced capping and receptor induced shape change are abolished, but phagocytosis is unaltered. In whole cells, calcium mobilization induced by FMLP, PAF and Con-A inhibited by PT although the FMLP-mediated effect is more susceptible to PT's effects. Treatment of PMN's with phorbol 12-myristate 13 acetate (PMA) causes an abolition of calcium mobilization by all agents in a range which also inhibits cap formation. Investigation of calcium uptake reveals PT sensitive and insensitive components. Reciprocal interactions between Ns and Ni proteins are also observed since pretreatment with FMLP and PAF causes a stimulation of Ns-mediated cyclic AMP enhancement while pretreatment with Ns linked receptors (PGE/sub 1/ and beta receptor agonists) inhibits calcium mobilization. Comparative peptide mapping studies indicate substantial similarity between Ni proteins in PMN's, platelets and human erythrocytes. The authors results suggest that the Ni linked calcium mobilization sensitive to PMA is important to the regulation of the human neutrophil.

  5. Mechanically Induced Intercellular Calcium Communication in Confined Endothelial Structures

    PubMed Central

    Junkin, Michael; Lu, Yi; Long, Juexuan; Deymier, Pierre A.; Hoying, James B.; Wong, Pak Kin

    2012-01-01

    Calcium signaling in the diverse vascular structures is regulated by a wide range of mechanical and biochemical factors to maintain essential physiological functions of the vasculature. To properly transmit information, the intercellular calcium communication mechanism must be robust against various conditions in the cellular microenvironment. Using plasma lithography geometric confinement, we investigate mechanically induced calcium wave propagation in networks of human umbilical vein endothelial cells organized. Endothelial cell networks with confined architectures were stimulated at the single cell level, including using capacitive force probes. Calcium wave propagation in the network was observed using fluorescence calcium imaging. We show that mechanically induced calcium signaling in the endothelial networks is dynamically regulated against a wide range of probing forces and repeated stimulations. The calcium wave is able to propagate consistently in various dimensions from monolayers to individual cell chains, and in different topologies from linear patterns to cell junctions. Our results reveal that calcium signaling provides a robust mechanism for cell-cell communication in networks of endothelial cells despite the diversity of the microenvironmental inputs and complexity of vascular structures. PMID:23267827

  6. Cadmium Induces Transcription Independently of Intracellular Calcium Mobilization

    PubMed Central

    Tvermoes, Brooke E.; Bird, Gary S.; Freedman, Jonathan H.

    2011-01-01

    Background Exposure to cadmium is associated with human pathologies and altered gene expression. The molecular mechanisms by which cadmium affects transcription remain unclear. It has been proposed that cadmium activates transcription by altering intracellular calcium concentration ([Ca2+]i) and disrupting calcium-mediated intracellular signaling processes. This hypothesis is based on several studies that may be technically problematic; including the use of BAPTA chelators, BAPTA-based fluorescent sensors, and cytotoxic concentrations of metal. Methodology/Principal Finding In the present report, the effects of cadmium on [Ca2+]i under non-cytotoxic and cytotoxic conditions was monitored using the protein-based calcium sensor yellow cameleon (YC3.60), which was stably expressed in HEK293 cells. In HEK293 constitutively expressing YC3.60, this calcium sensor was found to be insensitive to cadmium. Exposing HEK293::YC3.60 cells to non-cytotoxic cadmium concentrations was sufficient to induce transcription of cadmium-responsive genes but did not affect [Ca2+]i mobilization or increase steady-state mRNA levels of calcium-responsive genes. In contrast, exposure to cytotoxic concentrations of cadmium significantly reduced intracellular calcium stores and altered calcium-responsive gene expression. Conclusions/Significance These data indicate that at low levels, cadmium induces transcription independently of intracellular calcium mobilization. The results also support a model whereby cytotoxic levels of cadmium activate calcium-responsive transcription as a general response to metal-induced intracellular damage and not via a specific mechanism. Thus, the modulation of intracellular calcium may not be a primary mechanism by which cadmium regulates transcription. PMID:21694771

  7. Cytokinin stimulates dihydropyridine-sensitive calcium uptake in moss protoplasts.

    PubMed Central

    Schumaker, K S; Gizinski, M J

    1993-01-01

    Ca2+ influx through dihydropyridine (DHP)-sensitive Ca2+ channels is thought to be an early event in cytokinin-induced bud formation in moss protonema because DHP antagonists inhibit bud formation in the presence of cytokinin and DHP agonists stimulate bud formation in the absence of cytokinin [Conrad, P. A. & Helper, P. K. (1988) Plant Physiol. 86, 684-687]. In the present study, we established the presence of a DHP-sensitive Ca2+ transport system by measuring 45Ca2+ influx into moss protoplasts. Ca2+ influx was stimulated by external KCl (up to 5 mM), indicating that transport is voltage-dependent. K(+)-induced Ca2+ influx was DHP-sensitive with > 50% inhibition at 500 nM nifedipine. Ca2+ influx was stimulated by increasing concentrations of the DHP Ca2+ channel agonist Bay K8644 with half-maximal effects at 25 nM; this stimulation was seen only in the absence of K+, suggesting that the agonist works preferentially on polarized membranes. Ca2+ influx was also inhibited by phenylalkylamines (verapamil) and benzothiazepines (diltiazem). The phytohormone 6-benzylaminopurine consistently stimulated Ca2+ influx with a Km value of 1 nM, whereas adenine, indoleacetic acid, and gibberellic acid had no effect on Ca2+ transport. The cytokinins kinetin and trans-zeatin caused a greater stimulation of Ca2+ influx and induced more bud formation than did 6-benzylaminopurine. These results indicate that Ca2+ is taken up into moss protoplasts through voltage-dependent DHP-sensitive Ca2+ channels on the plasma membrane and that one of the cytokinin effects in the induction of bud formation is regulation of this plasma membrane Ca2+ channel. PMID:7504288

  8. 4-aminopyridine-induced contracture in frog ventricle is due to calcium released from intracellular stores.

    PubMed

    Bhaskar, A; Subbanna, P K; Arasan, S; Rajapathy, J; Rao, J P; Subramani, S

    2008-01-01

    The aim of the study is to demonstrate the presence of intracellular calcium store in frog ventricle based on contractures induced by 4-aminopyridine in calcium-free media. Frog-ventricular strips were subjected to field stimulation at 0.2 Hz and the force of contraction was recorded after stabilization. The preparation was then kept quiescent for some time in solutions with different sodium concentrations, containing 0 or 1 mmol/L calcium. Caffeine, 4-aminopyridine (4-AP), or tetraethylammonium chloride was then added. Frog skeletal muscle preparations were used as positive controls for the caffeine experiments. Frog ventricular preparations did not develop contractures (sustained contractions) in the presence of caffeine (25 mmol/L), while frog skeletal muscle preparations developed caffeine-induced contractures. However, 4-AP (16 mmol/L) was able to induce contractures in quiescent frog ventricular preparations, even when they were superfused with calcium-free solution. 4-AP contractures in frog ventricle were seen in the presence of nifedipine also. Amplitude of 4-AP evoked contractures in frog ventricle were much larger in low sodium (30 mmol/L) and sodium-free (sodium substituted by lithium) solutions than in normal sodium solution, suggesting that the route of extrusion of the cytosolic calcium (released from intracellular stores by 4-AP) is the sodium calcium exchanger, which gets reversed in low sodium solutions. Tetraethylammonium chloride (TEA) was not able to induce contractures in frog ventricle suggesting that the contracture evoked by 4-AP is not due to its potassium channel blocking effect. In quiescent frog skeletal muscle preparations, caffeine as well as 4-AP induced contractures in calcium-free solutions. We therefore conclude that there is a caffeine-insensitive, 4-AP sensitive intracellular calcium store in the frog ventricle.

  9. Bitter tasting compounds dilate airways by inhibiting airway smooth muscle calcium oscillations and calcium sensitivity

    PubMed Central

    Tan, Xiahui; Sanderson, Michael J

    2014-01-01

    Background and Purpose While selective, bitter tasting, TAS2R agonists can relax agonist-contracted airway smooth muscle (ASM), their mechanism of action is unclear. However, ASM contraction is regulated by Ca2+ signalling and Ca2+ sensitivity. We have therefore investigated how the TAS2R10 agonists chloroquine, quinine and denotonium regulate contractile agonist-induced Ca2+ signalling and sensitivity. Experimental Approach Airways in mouse lung slices were contracted with either methacholine (MCh) or 5HT and bronchodilation assessed using phase-contrast microscopy. Ca2+ signalling was measured with 2-photon fluorescence microscopy of ASM cells loaded with Oregon Green, a Ca2+-sensitive indicator (with or without caged-IP3). Effects on Ca2+ sensitivity were assessed on lung slices treated with caffeine and ryanodine to permeabilize ASM cells to Ca2+. Key Results The TAS2R10 agonists dilated airways constricted by either MCh or 5HT, accompanied by inhibition of agonist-induced Ca2+ oscillations. However, in non-contracted airways, TAS2R10 agonists, at concentrations that maximally dilated constricted airways, did not evoke Ca2+ signals in ASM cells. Ca2+ increases mediated by the photolysis of caged-IP3 were also attenuated by chloroquine, quinine and denotonium. In Ca2+-permeabilized ASM cells, the TAS2R10 agonists dilated MCh- and 5HT-constricted airways. Conclusions and Implications TAS2R10 agonists reversed bronchoconstriction by inhibiting agonist-induced Ca2+ oscillations while simultaneously reducing the Ca2+ sensitivity of ASM cells. Reduction of Ca2+ oscillations may be due to inhibition of Ca2+ release through IP3 receptors. Further characterization of bronchodilatory TAS2R agonists may lead to the development of novel therapies for the treatment of bronchoconstrictive conditions. PMID:24117140

  10. Intracellular Calcium Mobilization in Response to Ion Channel Regulators via a Calcium-Induced Calcium Release Mechanism

    PubMed Central

    Petrou, Terry; Olsen, Hervør L.; Thrasivoulou, Christopher; Masters, John R.; Ashmore, Jonathan F.

    2017-01-01

    Free intracellular calcium ([Ca2+]i), in addition to being an important second messenger, is a key regulator of many cellular processes including cell membrane potential, proliferation, and apoptosis. In many cases, the mobilization of [Ca2+]i is controlled by intracellular store activation and calcium influx. We have investigated the effect of several ion channel modulators, which have been used to treat a range of human diseases, on [Ca2+]i release, by ratiometric calcium imaging. We show that six such modulators [amiodarone (Ami), dofetilide, furosemide (Fur), minoxidil (Min), loxapine (Lox), and Nicorandil] initiate release of [Ca2+]i in prostate and breast cancer cell lines, PC3 and MCF7, respectively. Whole-cell currents in PC3 cells were inhibited by the compounds tested in patch-clamp experiments in a concentration-dependent manner. In all cases [Ca2+]i was increased by modulator concentrations comparable to those used clinically. The increase in [Ca2+]i in response to Ami, Fur, Lox, and Min was reduced significantly (P < 0.01) when the external calcium was reduced to nM concentration by chelation with EGTA. The data suggest that many ion channel regulators mobilize [Ca2+]i. We suggest a mechanism whereby calcium-induced calcium release is implicated; such a mechanism may be important for understanding the action of these compounds. PMID:27980039

  11. Neutron-Induced Cross Sections Measurements of Calcium

    SciTech Connect

    Guber, Klaus H; Kopecky, S.; Schillebeeckx, P.; Kauwenberghs, K.; Siegler, P.

    2013-01-01

    To support the US Department of Energy Nuclear Criticality Safety Program neutron induced cross section experiments were performed at the Geel Electron Linear Accelerator of the Institute for Reference Material and Measurements of the Joint Research Centers, European Union. Neutron capture and transmission measurements were carried out using a metallic calcium sample. The obtained data will be used for a new calcium evaluation, which will be submitted with its covariances to the ENDBF/B nuclear data base.

  12. Neutron-induced Cross Section Measurements of Calcium

    NASA Astrophysics Data System (ADS)

    Guber, K.; Kopecky, S.; Schillebeeckx, P.; Kauwenberghs, K.; Siegler, P.

    2014-05-01

    To support the US Department of Energy Nuclear Criticality Safety Program, neutron-induced cross section experiments were performed at the Geel Electron Linear Accelerator of the Institute for Reference Material and Measurements of the Joint Research Centers, European Union. Neutron capture and transmission measurements were carried out using a metallic calcium sample. The measured data will be used for a new calcium evaluation, which will be submitted with covariances to the ENDF/B nuclear data library.

  13. Mechanisms by which smooth muscle sensitivity may be altered by calcium.

    PubMed

    Kaiman, M; Shibata, S

    1978-01-01

    6-Hydroxydopamine (6-OHDA) increased the sensitivity of rat, rabbit and guinea pig portal veins to norepinephrine (NE), methoxamine (ME), barium (Ba++) and calcium (Ca++) but not to potassium (K+). Reserpine potentiated the responses of NE, ME and Ca++ but not Ba++ or K+ in rabbit and guinea pig veins and did not alter the sensitivity of rat veins to all agonists tested. Cocaine only potentiated the NE responses of the veins and decreased the sensitivity of rabbit and guinea pig veins to K+. 6-OHDA increased 48Ca-influx in all veins whereas reserpine increased 45Ca-influx only in rabbit and guinea pig veins. Cocaine failed to increase 45Ca-influx in all veins tested. NE, ME and K+ increased 45Ca-influx in the veins from the different animals. The agonist-induced 45Ca-influx was greater in most of the supersensitive veins than in the control veins. In veins that failed to develop supersensitivity, agonist-induced 45Ca-influx did not differ from that of the control veins. It is concluded that the development of super- and subsensitivity exhibits species variation and that the alteration in Ca++ influx would be consistent with the changes in sensitivity of the venous smooth muscle.

  14. The involvement of calcium carriers and of the vacuole in the glucose-induced calcium signaling and activation of the plasma membrane H(+)-ATPase in Saccharomyces cerevisiae cells.

    PubMed

    Bouillet, L E M; Cardoso, A S; Perovano, E; Pereira, R R; Ribeiro, E M C; Trópia, M J M; Fietto, L G; Tisi, R; Martegani, E; Castro, I M; Brandão, R L

    2012-01-01

    Previous work from our laboratories demonstrated that the sugar-induced activation of plasma membrane H(+)-ATPase in Saccharomyces cerevisiae is dependent on calcium metabolism with the contribution of calcium influx from external medium. Our results demonstrate that a glucose-induced calcium (GIC) transporter, a new and still unidentified calcium carrier, sensitive to nifedipine and gadolinium and activated by glucose addition, seems to be partially involved in the glucose-induced activation of the plasma membrane H(+)-ATPase. On the other hand, the importance of calcium carriers that can release calcium from internal stores was analyzed in glucose-induced calcium signaling and activation of plasma membrane H(+)-ATPase, in experimental conditions presenting very low external calcium concentrations. Therefore the aim was also to investigate how the vacuole, through the participation of both Ca(2+)-ATPase Pmc1 and the TRP homologue calcium channel Yvc1 (respectively, encoded by the genes PMC1 and YVC1) contributes to control the intracellular calcium availability and the plasma membrane H(+)-ATPase activation in response to glucose. In strains presenting a single deletion in YVC1 gene or a double deletion in YVC1 and PMC1 genes, both glucose-induced calcium signaling and activation of the H(+)-ATPase are nearly abolished. These results suggest that Yvc1 calcium channel is an important component of this signal transduction pathway activated in response to glucose addition. We also found that by a still undefined mechanism Yvc1 activation seems to correlate with the changes in the intracellular level of IP(3). Taken together, these data demonstrate that glucose addition to yeast cells exposed to low external calcium concentrations affects calcium uptake and the activity of the vacuolar calcium channel Yvc1, contributing to the occurrence of calcium signaling connected to plasma membrane H(+)-ATPase activation. Copyright © 2011 Elsevier Ltd. All rights reserved.

  15. Wind-induced plant motion immediately increases cytosolic calcium.

    PubMed Central

    Knight, M R; Smith, S M; Trewavas, A J

    1992-01-01

    Wind is one of the most unusual and more dramatic of the environmental signals to modify plant development. Wind-stimulated crops are also known to experience considerable reductions in growth and subsequent yield. There is at present no experimental data to suggest how wind signals are perceived and transduced by plant cells. We have genetically transformed Nicotiana plumbaginifolia to express aequorin and thus produced luminous plants that directly report cytosolic calcium by emitting blue light. With these plants we have found wind stimulation to cause immediate increases in cytosolic calcium and our evidence, based on the use of specific inhibitors, suggests that this calcium is mobilized from organelle sources. Our data further suggest that wind-induced movement of tissues, by mechanically stimulating and stressing constituent plant cells, is responsible for the immediate elevation of cytosolic calcium; increases occur only when the plant tissue is actually in motion. Repeated wind stimulation renders the cells refractory to further calcium signaling but responsiveness is rapidly recovered when stimulation is subsequently diminished. Our data suggest that mechanoperception in plant cells may possibly be transduced through intracellular calcium. Since mechanoperception and transduction are considered crucial to plant morphogenesis, our observations suggest that calcium could be central in the control and generation of plant form. Images PMID:11536497

  16. Calcium fingerprints induced by calmodulin interactors in eukaryotic cells.

    PubMed

    Dagher, Rania; Brière, Christian; Fève, Marie; Zeniou, Maria; Pigault, Claire; Mazars, Christian; Chneiweiss, Hervé; Ranjeva, Raoul; Kilhoffer, Marie-Claude; Haiech, Jacques

    2009-06-01

    Calcium (Ca2+) is a ubiquitous second messenger which promotes cell responses through transient changes in intracellular concentrations. The prominent role of Ca2+ in cell physiology is mediated by a whole set of proteins constituting a Ca2+-signalling toolkit involved in Ca2+-signal generation, deciphering and arrest. The different Ca2+-signalosomes deliver Ca2+-signals with spatial and temporal dynamics to control the function of specific cell types. Among the intracellular proteins involved in Ca2+-signal deciphering, calmodulin (CaM) plays a pivotal role in controlling Ca2+-homeostasis and downstream Ca2+-based signalling events. Due to its ubiquitous expression in eukaryotic cells and the variety of proteins it interacts with, CaM is central in Ca2+-signalling networks. For these reasons, it is expected that disrupting or modifying CaM interactions with its target proteins will affect Ca2+-homeostasis and cellular responses. The resulting calcium response will vary depending on which interactions between CaM and target proteins are altered by the molecules and on the specific Ca2+-toolkit expressed in a given cell, even in the resting state. In the present paper, the effect of six classical CaM interactors (W5, W7, W12, W13, bifonazole and calmidazolium) was studied on Ca2+-signalling in tumor initiating cells isolated from human glioblastoma (TG1) and tobacco cells (BY-2) using the fluorescent Ca2+-sensitive Indo-1 dye and aequorin, respectively. Various Ca2+-fingerprints were obtained depending both on the CaM interactor used and the cell type investigated. These data demonstrate that interaction between the antagonists and CaM results in a differential inhibition of CaM-dependent proteins involved in Ca2+-signal regulation. In addition, the distinct Ca2+-fingerprints in tobacco and human tumor initiating glioblastoma cells induced by a given CaM interactor highlight the specificity of the Ca2+-signalosome in eukaryotic cells.

  17. Effectiveness of nano-calcium phosphate paste on sensitivity during and after bleaching: a randomized clinical trial.

    PubMed

    Loguercio, Alessandro Dourado; Tay, Lidia Yileng; Herrera, Daniel Rodrigo; Bauer, Jose; Reis, Alessandra

    2015-01-01

    The study aimed to evaluate the effectiveness of in-office bleaching and associated tooth sensitivity on application of nano-calcium phosphate paste as desensitizing agent. Bleaching was performed with 35% hydrogen peroxide gel in 40 patients who were randomly divided into placebo and nano-calcium phosphate paste groups. Bleaching efficacy (BE) was evaluated using a value-oriented Vita shade guide. Tooth sensitivity was recorded using a numeric rating scale (0-4) during bleaching and up to 48 h after each session. The primary outcome of absolute risk of tooth sensitivity was compared using the Fisher's exact test (α = 0.05). The intensity of tooth sensitivity and the efficacy of in-office bleaching were also statistically evaluated. No significant differences in absolute risk and intensity of tooth sensitivity were detected between the groups (p = 1.0 and p = 0.53, respectively). BE was also found to be similar between the groups (p = 0.67). Although the use of a nano-calcium phosphate paste associated with fluoride and potassium nitrate did not influence the whitening outcome, but it also did not reduce bleaching-induced tooth sensitivity.

  18. TRICHLOROETHYLENE IHIBITS VOLTAGE-SENSITIVE CALCIUM CURRENTS IN DIFFERENTIATED PC 12 CELLS.

    EPA Science Inventory

    ABSTRACT BODY: It has been demonstrated recently that volatile organic compounds (VOCs)such as toluene, perchloroethylene and trichloroethylene inhibit function of voltage-sensitive calcium channels (VSSC). Such actions are hypothesized to contribute to the acute neurotoxicity of...

  19. Hypotonic stress-induced calcium signaling in Saccharomyces cerevisiae involves TRP-like transporters on the endoplasmic reticulum membrane.

    PubMed

    Rigamonti, M; Groppi, S; Belotti, F; Ambrosini, R; Filippi, G; Martegani, E; Tisi, R

    2015-02-01

    Saccharomyces cerevisiae cells respond to hypotonic stress (HTS) by a cytosolic calcium rise, either generated by an influx of calcium from extracellular medium, when calcium is available, or by a release from intracellular stores in scarcity of extracellular calcium. Calcium release from intracellular compartments is peculiarly inhibited by external calcium in a calcineurin-independent and Cch1-, but not Mid1-, driven manner. HTS-induced calcium release is also negatively regulated by the ER protein Cls2 and involves a poorly characterized protein, FLC2/YAL053W gene product, previously proposed to be required for FAD transport in the ER, albeit, due to its molecular features, it was also previously classified as an ion transporter. A computational analysis revealed that this gene and its three homologs in S. cerevisiae, together with previously identified Schizosaccharomyces pombe pkd2 and Neurospora crassa calcium-related spray protein, belong to a fungal branch of TRP-like ion transporters related to human mucolipin and polycystin 2 calcium transporters. Moreover, disruption of FLC2 gene confers severe sensitivity to Calcofluor white and hyper-activation of the cell wall integrity MAPK cascade, suggesting a role in cell wall maintenance as previously suggested for the fission yeast homolog. Perturbation in cytosolic resting calcium concentration and hyper-activation of calcineurin in exponentially growing cells suggest a role for this transporter in calcium homeostasis in yeast. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Selective use of a reserved mechanism for inducing calcium oscillations.

    PubMed

    Ma, Chun Yan; Chen, Chun Ying; Cui, Zong Jie

    2004-12-01

    Concentration-dependent transformation of hormone- and neurotransmitter-induced calcium oscillation is a common phenomenon in diverse types of cells especially of the secretory type. The rodent submandibular acinar cells are an exception to this rule, which show elevated plateau increase in intracellular calcium under all stimulatory concentrations of both norepinephrine and acetylcholine. However, under depolarized state this cell type could also show a variation of periodic calcium changes. This reserved mechanism of calcium oscillation is jump-started by depolarization only with muscarinic cholinergic stimulation, but not with adrenergic stimulation. This latter effect is attributable to alpha receptor activation, not due to simultaneous activation of alpha and beta receptors, with beta receptor activation only serving to enhance the magnitude. These data suggest that this reserved mechanism for inducing calcium oscillation can be selectively used only by specific receptor-signaling pathways, and may therefore partly explain the long-known differences between secretion induced by sympathetic and parasympathetic stimulation in the submandibular gland.

  1. Calcium regulation in aortic smooth muscle cells during the initial phase of tunicamycin-induced endo/sarcoplasmic reticulum stress.

    PubMed

    Ziomek, Gabriela; Cheraghi Zanjani, Parisa; Arman, Darian; van Breemen, Cornelis; Esfandiarei, Mitra

    2014-07-15

    Endo/sarcoplasmic reticulum stress and the unfolded protein response have been implicated as underlying mechanisms of cell death in many pathological conditions. We have confirmed that long-term exposure to 10µM tunicamycin induced the endo/sarcoplasmic reticulum stress in cultured vascular smooth muscle cells. Since tunicamycin is reported to induce the stress response by inhibiting protein glycosylation, we attempted to investigate a causal link between accumulation of unfolded proteins and dysregulation of cellular calcium transport. However, we found that tunicamycin caused an immediate release of calcium from the endo/sarcoplasmic reticulum, which was sensitive to thapsigargin, and an influx of calcium through the plasma membrane, resulting in a significant increase in cytoplasmic calcium and depletion of endo/sarcoplasmic reticulum calcium. Furthermore, we observed that tunicamycin also induced contraction in intact vascular smooth muscle. By applying established procedures and antagonists, we established that tunicamycin did not directly activate physiological calcium channels, such as store-operated channels, voltage gated calcium channels, ryanodine receptors or inositol trisphosphate receptors. Instead, we found that its effects on cellular calcium fluxes closely resembled those of the known calcium ionophore, ionomycin. We have concluded that tunicamycin directly permeabilizes the plasma membrane and endo/sarcoplasmic reticulum to calcium, and is, therefore, inappropriate for studying the relationship between accumulation of unfolded proteins and endo/sarcoplasmic reticulum calcium dysregulation during the endo/sarcoplasmic reticulum stress response. In contrast, we also report that two other well-known endo/sarcoplasmic reticulum stress inducers, brefeldin A and dithiothreitol, did not exhibit similar increases in calcium permeability. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Myofilament Calcium Sensitivity: Role in Regulation of In vivo Cardiac Contraction and Relaxation

    PubMed Central

    Chung, Jae-Hoon; Biesiadecki, Brandon J.; Ziolo, Mark T.; Davis, Jonathan P.; Janssen, Paul M. L.

    2016-01-01

    Myofilament calcium sensitivity is an often-used indicator of cardiac muscle function, often assessed in disease states such as hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). While assessment of calcium sensitivity provides important insights into the mechanical force-generating capability of a muscle at steady-state, the dynamic behavior of the muscle cannot be sufficiently assessed with a force-pCa curve alone. The equilibrium dissociation constant (Kd) of the force-pCa curve depends on the ratio of the apparent calcium association rate constant (kon) and apparent calcium dissociation rate constant (koff) of calcium on TnC and as a stand-alone parameter cannot provide an accurate description of the dynamic contraction and relaxation behavior without the additional quantification of kon or koff, or actually measuring dynamic twitch kinetic parameters in an intact muscle. In this review, we examine the effect of length, frequency, and beta-adrenergic stimulation on myofilament calcium sensitivity and dynamic contraction in the myocardium, the effect of membrane permeabilization/mechanical- or chemical skinning on calcium sensitivity, and the dynamic consequences of various myofilament protein mutations with potential implications in contractile and relaxation behavior. PMID:28018228

  3. Sulfhydryl oxidation overrides Mg(2+) inhibition of calcium-induced calcium release in skeletal muscle triads.

    PubMed Central

    Donoso, P; Aracena, P; Hidalgo, C

    2000-01-01

    We studied the effect of oxidation of sulfhydryl (SH) residues on the inhibition by Mg(2+) of calcium-induced calcium release (CICR) in triad-enriched sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. Vesicles were either passively or actively loaded with calcium before eliciting CICR by dilution at pCa 4.6-4.4 in the presence of 1.2 mM free [ATP] and variable free [Mg(2+)]. Native triads exhibited a significant inhibition of CICR by Mg(2+), with a K(0.5) approximately 50 microM. Partial oxidation of vesicles with thimerosal produced a significant increase of release rate constants and initial release rates at all [Mg(2+)] tested (up to 1 mM), and shifted the K(0.5) value for Mg(2+) inhibition to 101 or 137 microM in triads actively or passively loaded with calcium, respectively. Further oxidation of vesicles with thimerosal completely suppressed the inhibitory effect of [Mg(2+)] on CICR, yielding initial rates of CICR of 2 micromol/(mg x s) in the presence of 1 mM free [Mg(2+)]. These effects of oxidation on CICR were fully reversed by SH reducing agents. We propose that oxidation of calcium release channels, by decreasing markedly the affinity of the channel inhibitory site for Mg(2+), makes CICR possible in skeletal muscle. PMID:10866954

  4. Calcium

    MedlinePlus

    ... You'll also find calcium in broccoli and dark green, leafy vegetables (especially collard and turnip greens, ... can enjoy good sources of calcium such as dark green, leafy vegetables, broccoli, chickpeas, and calcium-fortified ...

  5. Influence of extracellular calcium on allyl alcohol-induced hepatotoxicity.

    PubMed

    Strubelt, O; Younes, M; Pentz, R

    1986-07-01

    The role of calcium in allyl alcohol-induced hepatotoxicity was investigated in the isolated haemoglobin-free perfused rat liver. At a Ca++ concentration of 2.5 mmol/l in the perfusate, allyl alcohol (initial concentration 1.17 mmol/l) produced an enhanced release of GPT and SDH from the liver, an increase in the lactate/pyruvate ratio of the perfusate, a decrease in hepatic oxygen consumption and an increase of both hepatic calcium and malondialdehyde content. In the absence of Ca++ in the perfusate, no hepatic calcium accumulation occurred with allyl alcohol, but all other signs of hepatic damage were as severe as with 2.5 mmol/l Ca++. On the other hand, high extracellular Ca++ (5 mmol/l) alone led to a threefold increase of liver calcium but produced only marginal hepatotoxicity and only slightly enhanced the hepatotoxic effects of allyl alcohol. The concentrations of allyl alcohol in the perfusate were not altered at different Ca++ concentrations. In conclusion, the primary allyl alcohol-induced hepatotoxic injury does not appear to depend upon an influx of extracellular calcium.

  6. Intracellular calcium elevation induced by extracellular application of cyclic-ADP-ribose or oxytocin is temperature-sensitive in rodent NG108-15 neuronal cells with or without exogenous expression of human oxytocin receptors.

    PubMed

    Amina, S; Hashii, M; Ma, W-J; Yokoyama, S; Lopatina, O; Liu, H-X; Islam, M S; Higashida, H

    2010-05-01

    ADP-ribosyl cyclase and/or CD38 are activated after oxytocin receptor stimulation in the hypothalamus and pituitary in adult mice, leading to facilitation of oxytocin secretion. Although cyclic adenosine 5'-diphosphoribose (cADPR) primarily acts as an intracellular second messenger, it has been suggested that extracellular cADPR stimulates intracellular ryanodine receptors after internalisation via the nucleotide-transporting capacity of CD38 in fibroblasts and astrocytes. However, little is known about whether extracellular cADPR activates neurones. To address this question, we used a model neuronal cell line, NG108-15 mouse neuroblastoma x rat glioma hybrid cells possessing CD38 but not oxytocin receptors, and measured cytosolic free calcium concentrations ([Ca(2+)](i)). Extracellular application of cADPR to NG108-15 cells elevated [Ca(2+)](i) at 35 degrees C. The elevation was significantly enhanced when measured at 40 degrees C. The cADPR and heat-induced [Ca(2+)](i) increase were blocked under extracellular Ca(2+)-free conditions and by 2-aminoethoxydiphenyl borate, an antagonist of melastatin-related transient receptor potential channel 2 (TRPM2) cation channels. Reverse transcriptation-polymerase chain reaction analyses indicated that TRPM2 channels were expressed in NG108-15 cells. Application of oxytocin elevated [Ca(2+)](i) in NG108-15 cells transformed to transiently express cloned human oxytocin receptors. The oxytocin-induced [Ca(2+)](i) response was also enhanced by heat. These results indicate that the extracellular application of cADPR, together with heat, activates cation influx downstream of oxytocin receptor signalling in NG108-15 neuronal cells, and suggest the possible involvement of TRPM2 channels in oxytocin release in the mammalian brain.

  7. Alternative splicing, muscle calcium sensitivity, and the modulation of dragonfly flight performance

    PubMed Central

    Marden, James H.; Fitzhugh, Gail H.; Wolf, Melisande R.; Arnold, Kristina D.; Rowan, Barry

    1999-01-01

    Calcium sensitivity of myosin cross-bridge activation in striated muscles commonly varies during ontogeny and in response to alterations in muscle usage, but the consequences for whole-organism physiology are not well known. Here we show that the relative abundances of alternatively spliced transcripts of the calcium regulatory protein troponin T (TnT) vary widely in flight muscle of Libellula pulchella dragonflies, and that the mixture of TnT splice variants explains significant portions of the variation in muscle calcium sensitivity, wing-beat frequency, and an index of aerodynamic power output during free flight. Two size-distinguishable morphs differ in their maturational pattern of TnT splicing, yet they show the same relationship between TnT transcript mixture and calcium sensitivity and between calcium sensitivity and aerodynamic power output. This consistency of effect in different developmental and physiological contexts strengthens the hypothesis that TnT isoform variation modulates muscle calcium sensitivity and whole-organism locomotor performance. Modulating muscle power output appears to provide the ecologically important ability to operate at different points along a tradeoff between performance and energetic cost. PMID:10611380

  8. Hydrogen sulfide induces calcium waves in astrocytes.

    PubMed

    Nagai, Yasuo; Tsugane, Mamiko; Oka, Jun-Ichiro; Kimura, Hideo

    2004-03-01

    Hydrogen sulfide (H2S) modifies hippocampal long-term potentiation (LTP) and functions as a neuromodulator. Here, we show that H2S increases intracellular Ca2+ and induces Ca2+ waves in primary cultures of astrocytes as well as hippocampal slices. H2S increases the influx of Ca2+ and to a lesser extent causes the release from intracellular Ca2+ stores. Ca2+ waves induced by neuronal excitation as well as responses to exogenously applied H2S are potently blocked by La3+ and Gd3+, inhibitors of Ca2+ channels. These observations suggest that H2S induces Ca2+ waves that propagate to neighboring astrocytes.

  9. High calcium diet reduces blood pressure in Dahl salt-sensitive rats by neural mechanisms.

    PubMed

    Peuler, J D; Morgan, D A; Mark, A L

    1987-06-01

    We tested the hypothesis that high dietary calcium attenuates hypertension in Dahl salt-sensitive rats by neural as opposed to vascular mechanisms. Four-week-old Dahl salt-sensitive rats were fed a high salt diet (3.3% sodium) with either high (4.0%; n = 21) or normal (0.4%; n = 21) calcium content until they were 10 to 11 weeks old. Total plasma calcium concentration was increased and plasma phosphorus concentration was decreased by the high calcium diet. At 10 weeks, food intake and intestinal absorption of sodium were not altered by the high calcium diet. There were three major observations. First, mean arterial pressure was lower in awake rats fed a high versus normal calcium diet (137 +/- 7, n = 11, vs 165 +/- 6 mm Hg, n = 10, respectively; p less than 0.05). This pressure difference was dependent on intact autonomic transmission, since ganglionic blockade eliminated the significant difference between pressures in rats fed high (78 +/- 5 mm Hg) and normal (85 +/- 6 mm Hg) calcium diets. Second, high calcium intake augmented baroreceptor reflex inhibition of renal sympathetic nerve activity and heart rate during ramp increase in arterial pressure produced by infusion of phenylephrine. Reflex suppression of renal sympathetic nerve activity was twofold greater in rats fed the high (vs normal) calcium diet (-2.79 +/- 0.25 vs -1.34 +/- 0.14% delta/delta mm Hg, respectively; n = 9 rats per group; p less than 0.05). Third, high calcium intake did not attenuate vascular responsiveness, since pressor responses to norepinephrine and angiotensin II did not differ between rats fed high and normal calcium diets after ganglionic blockade.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Spermine selectively inhibits high-conductance, but not low-conductance calcium-induced permeability transition pore.

    PubMed

    Elustondo, Pia A; Negoda, Alexander; Kane, Constance L; Kane, Daniel A; Pavlov, Evgeny V

    2015-02-01

    The permeability transition pore (PTP) is a large channel of the mitochondrial inner membrane, the opening of which is the central event in many types of stress-induced cell death. PTP opening is induced by elevated concentrations of mitochondrial calcium. It has been demonstrated that spermine and other polyamines can delay calcium-induced swelling of isolated mitochondria, suggesting their role as inhibitors of the mitochondrial PTP. Here we further investigated the mechanism by which spermine inhibits the calcium-induced, cyclosporine A (CSA) -sensitive PTP by using three indicators: 1) calcium release from the mitochondria detected with calcium green, 2) mitochondrial membrane depolarization using TMRM, and 3) mitochondrial swelling by measuring light absorbance. We found that despite calcium release and membrane depolarization, indicative of PTP activation, mitochondria underwent only partial swelling in the presence of spermine. This was in striking contrast to the high-amplitude swelling detected in control mitochondria and in mitochondria treated with the PTP inhibitor CSA. We conclude that spermine selectively prevents opening of the high-conductance state, while allowing activation of the lower conductance state of the PTP. We propose that the existence of lower conductance, stress-induced PTP might play an important physiological role, as it is expected to allow the release of toxic levels of calcium, while keeping important molecules (e.g., NAD) within the mitochondrial matrix.

  11. Failure of Elevating Calcium Induces Oxidative Stress Tolerance and Imparts Cisplatin Resistance in Ovarian Cancer Cells

    PubMed Central

    Ma, Liwei; Wang, Hongjun; Wang, Chunyan; Su, Jing; Xie, Qi; Xu, Lu; Yu, Yang; Liu, Shibing; Li, Songyan; Xu, Ye; Li, Zhixin

    2016-01-01

    Cisplatin is a commonly used chemotherapeutic drug, used for the treatment of malignant ovarian cancer, but acquired resistance limits its application. There is therefore an overwhelming need to understand the mechanism of cisplatin resistance in ovarian cancer, that is, ovarian cancer cells are insensitive to cisplatin treatment. Here, we show that failure of elevating calcium and oxidative stress tolerance play key roles in cisplatin resistance in ovarian cancer cell lines. Cisplatin induces an increase in oxidative stress and alters intracellular Ca2+ concentration, including cytosolic and mitochondrial Ca2+ in cisplatin-sensitive SKOV3 cells, but not in cisplatin-resistant SKOV3/DDP cells. Cisplatin induces mitochondrial damage and triggers the mitochondrial apoptotic pathway in cisplatin-sensitive SKOV3 cells, but rarely in cisplatin-resistant SKOV3/DDP cells. Inhibition of calcium signaling attenuates cisplatin-induced oxidative stress and intracellular Ca2+ overload in cisplatin-sensitive SKOV3 cells. Moreover, in vivo xenograft models of nude mouse, cisplatin significantly reduced the growth rates of tumors originating from SKOV3 cells, but not that of SKOV3/DDP cells. Collectively, our data indicate that failure of calcium up-regulation mediates cisplatin resistance by alleviating oxidative stress in ovarian cancer cells. Our results highlight potential therapeutic strategies to improve cisplatin resistance. PMID:27330840

  12. AHR-16303B, a novel antagonist of 5-HT2 receptors and voltage-sensitive calcium channels

    SciTech Connect

    Barrett, R.J.; Appell, K.C.; Kilpatrick, B.F.; Proakis, A.G.; Nolan, J.C.; Walsh, D.A. )

    1991-01-01

    In vivo and in vitro methods were used to characterize AHR-16303B, a novel compound with antagonistic action at 5-HT2 receptors and voltage-sensitive calcium channels. The 5-HT2 receptor-antagonistic properties of AHR-16303B were demonstrated by inhibition of (a) (3H)ketanserin binding to rat cerebral cortical membranes (IC50 = 165 nM); (b) 5-hydroxytryptamine (5-HT)-induced foot edema in rats (minimum effective dose, (MED) = 0.32 mg/kg orally, p.o.); (c) 5-HT-induced vasopressor responses in spontaneously hypertensive rats (SHR) (ID50 = 0.18 mg/kg intravenously (i.v.), 1.8 mg/kg p.o.), (d) 5-HT-induced antidiuresis in rats (MED = 1 mg/kg p.o.), and (e) platelet aggregation induced by 5-HT + ADP (IC50 = 1.5 mM). The calcium antagonist properties of AHR-16303B were demonstrated by inhibition of (a) (3H)nimodipine binding to voltage-sensitive calcium channels on rabbit skeletal muscle membranes (IC50 = 15 nM), (b) KCl-stimulated calcium flux into cultured PC12 cells (IC50 = 81 nM), and (c) CaCl2-induced contractions of rabbit thoracic aortic strips (pA2 = 8.84). AHR-16303B had little or no effect on binding of radioligands to dopamine2 (DA2) alpha 1, alpha 2, H1, 5-HT1 alpha, beta 2, muscarinic M1, or sigma opioid receptors; had no effect on 5-HT3 receptor-mediated vagal bradycardia; and had only minor negative inotropic, chronotropic, and dromotropic effects on isolated guinea pig atria. In conscious SHR, 30 mg/kg p.o. AHR-16303B completely prevented the vasopressor responses to i.v. 5-HT, and decreased blood pressure (BP) by 24% 3 h after dosing.

  13. Increased calcium/calmodulin-dependent protein kinase II activity by morphine-sensitization in rat hippocampus.

    PubMed

    Kadivar, Mehdi; Farahmandfar, Maryam; Ranjbar, Faezeh Esmaeli; Zarrindast, Mohammad-Reza

    2014-07-01

    Repeated exposure to drugs of abuse, such as morphine, elicits a progressive enhancement of drug-induced behavioral responses, a phenomenon termed behavioral sensitization. These changes in behavior may reflect long-lasting changes in some of the important molecules involved in memory processing such as calcium/calmodulin-dependent protein kinase II (CaMKII). In the present study, we investigated the effect of morphine sensitization on mRNA expression of α and β isoforms and activity of CaMKII in the hippocampus of male rats. Animals were treated for 3 days with saline or morphine (20mg/kg) and following a washout period of 5 days, a challenge dose of morphine (5mg/kg) were administered. The results indicate that morphine administration in pre-treated animals produces behavioral sensitization, as determined by significant increase in locomotion and oral stereotypy behavior. In addition, repeated morphine treatment increased mRNA expression of both α and β isoforms of CaMKII in the hippocampus. The present study also showed that induction of morphine sensitization significantly increased both Ca2+/calmodulin-independent and Ca2+/calmodulin-dependent activities of CaMK II in the rat hippocampus. However, acute administration of morphine (5mg/kg) did not alter either α and β CaMKII mRNA expression or CaMKII activity in the hippocampus. The stimulation effects of morphine sensitization on mRNA expression and activity of CaMKII were completely abolished by administration of naloxone, 30min prior to s.c. injections of morphine (20mg/kg/day×3 days). Our data demonstrated that induction of morphine sensitization could effectively modulate the activity and the mRNA expression of CaMKII in the hippocampus and this effect of morphine was exerted by the activation of opioid receptors.

  14. Statins lower calcium-induced oxidative stress in isolated mitochondria.

    PubMed

    Parihar, A; Parihar, M S; Zenebe, W J; Ghafourifar, P

    2012-04-01

    Statins are widely used cholesterol-lowering agents that exert cholesterol-independent effects including antioxidative. The present study delineates the effects of statins, atorvastatin, and simvastatin on oxidative stress and functions of mitochondria that are the primary cellular sources of oxidative stress. In isolated rat liver mitochondria, both the statins prevented calcium-induced cytochrome c release, lipid peroxidation, and opening of the mitochondrial membrane permeability transition (MPT). Both the statins decreased the activity of mitochondrial nitric oxide synthase (mtNOS), lowered the intramitochondrial ionized calcium, and increased the mitochondrial transmembrane potential. Our findings suggest that statins lower intramitochondrial ionized calcium that decreases mtNOS activity, lowers oxidative stress, prevents MPT opening, and prevents the release of cytochrome c from the mitochondria. These results provide a novel framework for understanding the antioxidative properties of statins and their effects on mitochondrial functions.

  15. Glutathione-Induced Calcium Shifts in Chick Retinal Glial Cells

    PubMed Central

    Freitas, Hercules R.; Ferraz, Gabriel; Ferreira, Gustavo C.; Ribeiro-Resende, Victor T.; Chiarini, Luciana B.; do Nascimento, José Luiz M.; Matos Oliveira, Karen Renata H.; Pereira, Tiago de Lima; Ferreira, Leonardo G. B.; Kubrusly, Regina C.; Faria, Robson X.

    2016-01-01

    Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1–10mM) showed that 5–10mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50mM KCl (labeled as βIII tubulin positive cells). BBG 100nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70μM and MK-801 20μM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5mM failed to induce calcium mobilization in glia cells, indicating that the antioxidant and/or structural features of GSH are essential to promote elevations in cytoplasmic calcium levels. Indeed, a short GSH pulse (60s) protects Müller glia from oxidative damage after 30 min of incubation with 0.1% H2O2. Finally, GSH induced GABA release from chick embryonic retina, mixed neuron-glia or from Müller cell cultures, which were inhibited by BBG or in the absence of sodium. GSH also induced propidium iodide uptake in Müller cells in culture in a P2X7 receptor dependent manner. Our data suggest that GSH, in addition to antioxidant effects, could act signaling calcium shifts at the millimolar range particularly in Müller glia, and could regulate the release of GABA, with additional protective effects on retinal neuron-glial circuit. PMID:27078878

  16. Glutathione-Induced Calcium Shifts in Chick Retinal Glial Cells.

    PubMed

    Freitas, Hercules R; Ferraz, Gabriel; Ferreira, Gustavo C; Ribeiro-Resende, Victor T; Chiarini, Luciana B; do Nascimento, José Luiz M; Matos Oliveira, Karen Renata H; Pereira, Tiago de Lima; Ferreira, Leonardo G B; Kubrusly, Regina C; Faria, Robson X; Herculano, Anderson Manoel; Reis, Ricardo A de Melo

    2016-01-01

    Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1-10 mM) showed that 5-10 mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50 mM KCl (labeled as βIII tubulin positive cells). BBG 100 nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70 μM and MK-801 20 μM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5 mM failed to induce calcium mobilization in glia cells, indicating that the antioxidant and/or structural features of GSH are essential to promote elevations in cytoplasmic calcium levels. Indeed, a short GSH pulse (60s) protects Müller glia from oxidative damage after 30 min of incubation with 0.1% H2O2. Finally, GSH induced GABA release from chick embryonic retina, mixed neuron-glia or from Müller cell cultures, which were inhibited by BBG or in the absence of sodium. GSH also induced propidium iodide uptake in Müller cells in culture in a P2X7 receptor dependent manner. Our data suggest that GSH, in addition to antioxidant effects, could act signaling calcium shifts at the millimolar range particularly in Müller glia, and could regulate the release of GABA, with additional protective effects on retinal neuron-glial circuit.

  17. Calcium release induced by 2-pyridinecarboxaldehyde thiosemicarbazone and its copper complex contributes to tumor cell death.

    PubMed

    Fu, Yun; Liu, Youxun; Wang, Jiangang; Li, Cuiping; Zhou, Sufeng; Yang, Yun; Zhou, Pingxin; Lu, Chengbiao; Li, Changzheng

    2017-03-01

    Thiosemicarbazones display significant antitumor activity and their copper complexes also exhibit enhanced biological activities in most situations, but the underlying mechanism is poorly understood. Therefore, investigation of the mechanism involved in the change upon chelation is required to extend our understanding of the effects of thiosemicarbazones. In the present study, the inhibitory effect of 2-pyridinecarboxaldehyde thiosemicarbazone (PCT) and its copper complex (PCT-Cu) on cell proliferation was investigated. The copper chelate exhibited a 3- to 10-fold increase in antitumor activity (with an IC50 <5 µM). The results showed that both PCT and PCT-Cu induced reactive oxygen species (ROS) generation in vitro and in vivo, caused cellular DNA fragmentation, depolarization of the mitochondrial membrane and cell cycle arrest. Western blotting showed that both PCT and PCT-Cu induced apoptosis. Upregulation of GRP78 in HepG2 cells following treatment with the agents indicated that endoplasmic reticulum (ER) stress occurred. Furthermore calcium release was revealed in this study, suggesting that PCT and PCT-Cu disturbed calcium homeostasis. It was noted that PCT-Cu sensitized thapsigargin‑stimulated calcium release from the ER, which was correlated with the ROS level they induced, implying that the antitumor activity of PCT and PCT-Cu partly stemmed from calcium mobilization, a situation that was reported in few studies. Our findings may significantly contribute to the understanding of the anti‑proliferative effect of the derivatives of thiosemicarbazones along with their antitumor mechanism.

  18. Increased intracellular free calcium and sensitivity to angiotensin II in platelets of preeclamptic women.

    PubMed

    Haller, H; Oeney, T; Hauck, U; Distler, A; Philipp, T

    1989-04-01

    Preeclampsia is characterized by a generalized vasoconstriction and increased vascular sensitivity to angiotensin II. Intracellular free calcium, implicated in vascular smooth muscle contraction, has been found to be elevated in platelets of other hypertensive disorders. We therefore measured intracellular free calcium concentrations by using the fluorescent probe quin-2 in platelets of six patients with preeclampsia and compared them to measurements in ten normotensive pregnant women and ten age-matched nonpregnant women. Intracellular free calcium was also determined in the preeclamptic women after delivery. We found that intracellular free calcium was slightly elevated in normal pregnancy (102 +/- 13 nmol/L v 87 +/- 17 nmol/L) but was markedly increased in preeclampsia (138 +/- 13 nmol/L, P less than .05). This increase disappeared six weeks after delivery (84 + 10 nmol/L, P less than .01). To investigate whether the increased intracellular free calcium was related to angiotensin II, the platelets were exposed to thrombin and angiotensin II in vitro. Exposure to thrombin and angiotensin II caused a dose-dependent increase in intracellular free calcium. The intracellular response to thrombin was not significantly different in the three groups. However, stimulation with angiotensin II revealed an increased response in intracellular free calcium in preeclampsia (P less than .05) that disappeared after delivery. Our findings show a sustained increase in platelet intracellular free calcium in preeclampsia and suggest a functional alteration of the angiotensin II receptor in this disease.

  19. Low-calcium diet prevents fructose-induced hyperinsulinemia and ameliorates the response to glucose load in rats.

    PubMed

    Voznesenskaya, Anna; Tordoff, Michael G

    2015-01-01

    Consuming a fructose-rich diet leads to hyperinsulinemia, impaired glucose tolerance, and insulin resistance. In humans, the consumption of high levels of refined sugars often coincides with a diet containing suboptimal levels of calcium. Calcium and carbohydrate metabolism interact, so there is potential for fructose to have different health outcomes depending on whether the diet is calcium-rich or calcium-poor. We evaluated the metabolic effects of feeding fructose to rats that were maintained on either a calcium-replete diet or a low-calcium diet. Growing male Sprague Dawley rats were fed diets based on the AIN-93G formulation, with the main source of carbohydrate derived either from a mixture of cornstarch and sucrose or from fructose. Half the rats given each carbohydrate source were fed calcium at recommended levels (125 mmol/kg Ca(2+)); the others were fed a diet low in calcium (25 mmol/kg Ca(2+)). At various times, glucose and insulin tolerance tests were conducted to assess glucose metabolism. Rats fed low-calcium diet had lower fasting insulin levels irrespective of the carbohydrate source they ate. They had a normal glycemic response to a glucose load and did not develop hyperinsulinemia under conditions of fructose feeding. The drop in blood glucose levels in response to insulin injection was larger in rats fed low-calcium diet than in those fed calcium-replete diet. Low-calcium diet prevented fructose-induced hyperinsulinemia and improved glucose handling under conditions of fructose feeding. Potential mechanisms underlying these effects of the low-calcium diet remain to be determined, but possibilities include impairment of insulin release from the pancreas and improved peripheral insulin sensitivity.

  20. The coupling of acetylcholine-induced BK channel and calcium channel in guinea pig saccular type II vestibular hair cells.

    PubMed

    Kong, Wei-Jia; Guo, Chang-Kai; Zhang, Xiao-Wen; Chen, Xiong; Zhang, Song; Li, Guan-Qiao; Li, Zhi-Wang; Van Cauwenberge, Paul

    2007-01-19

    Molecular biological studies and electrophysiological data have demonstrated that acetylcholine (ACh) is the principal cochlear and vestibular efferent neurotransmitter among mammalians. However, the functional roles of ACh in type II vestibular hair cells (VHCs II) among mammalians are still unclear, with the exception of the well-known alpha9-containing nicotinic ACh receptor (alpha9-containing nAChR)-activated small conductance, calcium-dependent potassium current (SK) in cochlear hair cells and frog saccular hair cells. The activation of SK current was necessary for the calcium influx through the alpha9-containing nAChR. Recently, we have demonstrated that ACh-induced big conductance, calcium-dependent potassium current (BK) was present in VHCs II of the vestibular end-organ of guinea pig. In this study, the nature of calcium influx for the activation of ACh-induced BK current in saccular VHCs II of guinea pig was investigated. Following extracellular perfusion of ACh, saccular VHCs II displayed a sustained outward current, which was sensitive to iberiotoxin (IBTX). High concentration of apamin failed to inhibit the current amplitude of ACh-induced outward current. Intracellular application of Cs(+) completely abolished the current evoked by ACh. ACh-induced current was potently inhibited by nifedipine, nimodipine, Cd(2+) and Ni(2+), respectively. The inhibition potency of these four calcium channel antagonists was nimodipine>nifedipine>cadmium>nickel. The L-type Ca(2+) channels agonist, (-)-Bay-K 8644 mimicked the effect of ACh and activated an IBTX-sensitive current. In addition, partial VHCs II displayed a biphasic waveform. In conclusion, the present data showed that in the guinea pig saccular VHCs II, ACh-induced BK channel was coupled with the calcium channel, but not the receptor. The perfusion of ACh will drive the opening of calcium channels; the influx of calcium ions will then activate the BK current.

  1. Cisplatin-induced peptic ulcers, vagotomy, adrenal and calcium modulation.

    PubMed

    Aggarwal, S K; San Antonio, J D; Sokhansanj, A; Miller, C

    1994-04-01

    Cytochemical and autoradiographic studies in Wistar rats [Crl:(WI)BR] show that cisplatin treatment (9 mg/kg) inhibits the release of acetylcholine from the axonal endings of the stomach smooth muscle resulting in bloating of the stomach and ulceration. Cisplatin also induces corticosteroid release from the adrenal gland stimulating peptic ulceration. Vagotomy helps ameliorate the effect but not eliminate it. Calcium supplementation restores normal neuromuscular function to gastric smooth muscle, thereby eliminating the gastro-intestinal toxicity due to cisplatin.

  2. Calcium-induced conidiation in Penicillium cyclopium: calcium triggers cytosolic alkalinization at the hyphal tip.

    PubMed Central

    Roncal, T; Ugalde, U O; Irastorza, A

    1993-01-01

    Addition of Ca2+ (1 to 10 mM) to submerged cultures of Penicillium cyclopium induces conidiation. Ca2+ induced an increase in cytosolic pH from approximately 7.00 to > 7.60 in less than 10 min, as determined with the fluorescent pH probe fluorescein. Measurement of the H(+)-ATPase activity in total membrane fractions did not show any stable activation in vivo as a result of Ca2+ treatment. By fluorescence ratio imaging microscopy, it was observed that vegetative hyphae exhibit a tip-to-base pH gradient, with the tip being more acidic. Ca2+ caused this gradient to dissipate within 10 min. The effect of several agents that are supposed to cause internal acidification, by different means, on conidiation was tested. Concentrations of these agents that did not significantly affect growth but inhibited Ca(2+)-induced conidiation also prevented the intracellular alkalinization observed after exposure to the cation. Calcium channel blockers (lanthanum, cobalt, verapamil, and nifedipine) were not able to inhibit Ca(2+)-induced conidiation, although their effect on calcium uptake was not evaluated. However, the combined results point towards externally bound Ca2+ as the primary agent of conidiation induction, causing changes in plasma membrane function which disrupt the pH gradient observed during apical growth. Images PMID:8380805

  3. Calcium

    MedlinePlus

    ... in luck if you like sardines and canned salmon with bones. Almond milk. previous continue Working Calcium ... drinks, and cereals. Other Considerations for Building Bones Vitamin D is essential for calcium absorption, so it's ...

  4. Myofilament Calcium Sensitivity: Consequences of the Effective Concentration of Troponin I

    PubMed Central

    Siddiqui, Jalal K.; Tikunova, Svetlana B.; Walton, Shane D.; Liu, Bin; Meyer, Meredith; de Tombe, Pieter P.; Neilson, Nathan; Kekenes-Huskey, Peter M.; Salhi, Hussam E.; Janssen, Paul M. L.; Biesiadecki, Brandon J.; Davis, Jonathan P.

    2016-01-01

    Control of calcium binding to and dissociation from cardiac troponin C (TnC) is essential to healthy cardiac muscle contraction/relaxation. There are numerous aberrant post-translational modifications and mutations within a plethora of contractile, and even non-contractile, proteins that appear to imbalance this delicate relationship. The direction and extent of the resulting change in calcium sensitivity is thought to drive the heart toward one type of disease or another. There are a number of molecular mechanisms that may be responsible for the altered calcium binding properties of TnC, potentially the most significant being the ability of the regulatory domain of TnC to bind the switch peptide region of TnI. Considering TnI is essentially tethered to TnC and cannot diffuse away in the absence of calcium, we suggest that the apparent calcium binding properties of TnC are highly dependent upon an “effective concentration” of TnI available to bind TnC. Based on our previous work, TnI peptide binding studies and the calcium binding properties of chimeric TnC-TnI fusion constructs, and building upon the concept of effective concentration, we have developed a mathematical model that can simulate the steady-state and kinetic calcium binding properties of a wide assortment of disease-related and post-translational protein modifications in the isolated troponin complex and reconstituted thin filament. We predict that several TnI and TnT modifications do not alter any of the intrinsic calcium or TnI binding constants of TnC, but rather alter the ability of TnC to “find” TnI in the presence of calcium. These studies demonstrate the apparent consequences of the effective TnI concentration in modulating the calcium binding properties of TnC. PMID:28066265

  5. Inhibition of Paclitaxel-induced Decreases in Calcium Signaling*

    PubMed Central

    Benbow, Jennifer H.; Mann, Taylor; Keeler, Camille; Fan, Chengpeng; Hodsdon, Michael E.; Lolis, Elias; DeGray, Brenda; Ehrlich, Barbara E.

    2012-01-01

    Peripheral neuropathy is one of the most severe and irreversible side effects caused by treatment from several chemotherapeutic drugs, including paclitaxel (Taxol®) and vincristine. Strategies are needed that inhibit this unwanted side effect without altering the chemotherapeutic action of these drugs. We previously identified two proteins in the cellular pathway that lead to Taxol-induced peripheral neuropathy, neuronal calcium sensor-1 (NCS-1) and calpain. Prolonged treatment with Taxol induces activation of calpain, degradation of NCS-1, and loss of intracellular calcium signaling. This paper has focused on understanding the molecular basis for prevention of peripheral neuropathy by testing the effects of addition of two candidate compounds to the existing chemotherapeutic drug regime: lithium and ibudilast. We found that the co-administration of either lithium or ibudilast to neuroblastoma cells that were treated with Taxol or vincristine inhibited activation of calpain and the reductions in NCS-1 levels and calcium signaling associated with these chemotherapeutic drugs. The ability of Taxol to alter microtubule formation was unchanged by the addition of either candidate compound. These results allow us to suggest that it is possible to prevent the unnecessary and irreversible damage caused by chemotherapeutic drugs while still maintaining therapeutic efficacy. Specifically, the addition of either lithium or ibudilast to existing chemotherapy treatment protocols has the potential to prevent chemotherapy-induced peripheral neuropathy. PMID:22988235

  6. Laser-Induced Damage of Calcium Fluoride

    SciTech Connect

    Espana, A.; Joly, A.G.; Hess, W.P.; Dickinson, J.T.

    2004-01-01

    As advances continue to be made in laser technology there is an increasing demand for materials that have high thresholds for laser-induced damage. Laser damage occurs when light is absorbed, creating defects in the crystal lattice. These defects can lead to the emission of atoms, ions and molecules from the sample. One specific field where laser damage is of serious concern is semiconductor lithography, which is beginning to use light at a wavelength of 157 nm. CaF2 is a candidate material for use in this new generation of lithography. In order to prevent unnecessary damage of optical components, it is necessary to understand the mechanisms for laser damage and the factors that serve to enhance it. In this research, we study various aspects of laser interactions with CaF2, including impurity absorbance and various forms of damage caused by incident laser light. Ultraviolet (UV) laser light at 266 nm with both femtosecond (fs) and nanosecond (ns) pulse widths is used to induce ion and neutral particle emission from cleaved samples of CaF2. The resulting mass spectra show significant differences suggesting that different mechanisms for desorption occur following excitation using the different pulse durations. Following irradiation by ns pulses at 266 nm, multiple single-photon absorption from defect states is likely responsible for ion emission whereas the fs case is driven by a multi-photon absorption process. This idea is further supported by the measurements made of the transmission and reflection of fs laser pulses at 266 nm, the results of which reveal a non-linear absorption process in effect at high incident intensities. In addition, the kinetic energy profiles of desorbed Ca and K contaminant atoms are different indicating that a different mechanism is responsible for their emission as well. Overall, these results show that purity plays a key role in the desorption of atoms from CaF2 when using ns pulses. On the other hand, once the irradiance reaches high

  7. Broiler chicks fed low-calcium diets. 2. Increased sensitivity to copper toxicity.

    PubMed

    Leach, R M; Rosenblum, C I; Amman, M J; Burdette, J

    1990-11-01

    Young broiler chicks were more sensitive to copper toxicity when they were fed diets deficient or marginal in calcium content. Growth rate was depressed and liver copper concentration was increased under these conditions. Chicks fed a casein-gelatin diet were more sensitive to copper toxicity than those fed a corn-soybean meal diet. Addition of phytic acid to the casein-gelatin basal diet enhanced copper toxicity as evidenced by effects on growth rate and liver copper content. Measurements of intestinal and biliary copper content suggested that the influence of calcium on copper toxicity was mediated via intestinal absorption rather than through influences on copper excretion.

  8. Calcium

    MedlinePlus

    ... such as canned sardines and salmon Calcium-enriched foods such as breakfast cereals, fruit juices, soy and rice drinks, and tofu. Check the product labels. The exact amount of calcium you need depends on your age and other factors. Growing children and teenagers need more calcium than ...

  9. Evidence for a distinct light-induced calcium-dependent potassium current in Hermissenda crassicornis.

    PubMed

    Blackwell, K T

    2000-01-01

    A model of phototransduction is developed as a first step toward a model for investigating the critical interaction of light and turbulence stimuli within the type B photoreceptor of Hermissenda crassicronis. The model includes equations describing phototransduction, release of calcium from intracellular stores, and other calcium regulatory mechanisms, as well as equations describing ligand-gating of a rhabdomeric sodium current. The model is used to determine the sources of calcium in the soma, whether calcium or IP3 is a plausible ligand of the light-induced sodium current, and whether the light-induced potassium current is equivalent to the calcium-dependent potassium current activated by light-induced calcium release. Simulations show that the early light-induced calcium elevation is due to influx through voltage-dependent channels, whereas the later calcium elevation is due to release from intracellular stores. Simulations suggest that the ligand of the fast, light-induced sodium current is IP3 but that there is a smaller, prolonged component of the light-induced sodium current that is activated by calcium. In the model, the calcium-dependent potassium current, located in the soma, is activated only slightly by light-induced calcium elevation, leading to the prediction that a calcium-dependent potassium current, active at resting potential, is located in the rhabdomere and is responsible for the light-induced potassium current.

  10. Prolonged calcium influx after termination of light-induced calcium release in invertebrate photoreceptors

    PubMed Central

    Nasi, Enrico

    2009-01-01

    In microvillar photoreceptors, light stimulates the phospholipase C cascade and triggers an elevation of cytosolic Ca2+ that is essential for the regulation of both visual excitation and sensory adaptation. In some organisms, influx through light-activated ion channels contributes to the Ca2+ increase. In contrast, in other species, such as Lima, Ca2+ is initially only released from an intracellular pool, as the light-sensitive conductance is negligibly permeable to calcium ions. As a consequence, coping with sustained stimulation poses a challenge, requiring an alternative pathway for further calcium mobilization. We observed that after bright or prolonged illumination, the receptor potential of Lima photoreceptors is followed by the gradual development of an after-depolarization that decays in 1–4 minutes. Under voltage clamp, a graded, slow inward current (Islow) can be reproducibly elicited by flashes that saturate the photocurrent, and can reach a peak amplitude in excess of 200 pA. Islow obtains after replacing extracellular Na+ with Li+, guanidinium, or N-methyl-d-glucamine, indicating that it does not reflect the activation of an electrogenic Na/Ca exchange mechanism. An increase in membrane conductance accompanies the slow current. Islow is impervious to anion replacements and can be measured with extracellular Ca2+ as the sole permeant species; Ba can substitute for Ca2+ but Mg2+ cannot. A persistent Ca2+ elevation parallels Islow, when no further internal release takes place. Thus, this slow current could contribute to sustained Ca2+ mobilization and the concomitant regulation of the phototransduction machinery. Although reminiscent of the classical store depletion–operated calcium influx described in other cells, Islow appears to diverge in some significant aspects, such as its large size and insensitivity to SKF96365 and lanthanum; therefore, it may reflect an alternative mechanism for prolonged increase of cytosolic calcium in photoreceptors. PMID

  11. Sensitization effect of thimerosal is mediated in vitro via reactive oxygen species and calcium signaling.

    PubMed

    Migdal, Camille; Foggia, Lucie; Tailhardat, Magalie; Courtellemont, Pascal; Haftek, Marek; Serres, Mireille

    2010-01-01

    Thimerosal, a mercury derivative composed of ethyl mercury chloride (EtHgCl) and thiosalicylic acid (TSA), is widely used as a preservative in vaccines and cosmetic products and causes cutaneous reactions. Since dendritic cells (DCs) play an essential role in the immune response, the sensitization potency of chemicals was studied in vitro using U937, a human promyelomonocytic cell line that is used as a surrogate of monocytic differentiation and activation. Currently, this cell line is under ECVAM (European Center for the Validation of Alternative Methods) validation as an alternative method for discriminating chemicals. Thimerosal and mercury derivatives induced in U937 an overexpression of CD86 and interleukin (IL)-8 secretion similarly to 1-chloro-2,4-dinitrobenzene (DNCB), a sensitizer used as a positive control for DC activation. Non-sensitizers, dichloronitrobenzene (DCNB), TSA and sodium dodecyl sulfate (SDS), an irritant, had no effect. U937 activation was prevented by cell pretreatment with N-acetyl-L-cysteine (NAC) but not with thiol-independent antioxidants except vitamin E which affected CD86 expression by preventing lipid peroxidation of cell membranes. Thimerosal, EtHgCl and DNCB induced glutathione (GSH) depletion and reactive oxygen species (ROS) within 15 min; another peak was detected after 2h for mercury compounds only. MitoSOX, a specific mitochondrial fluorescent probe, confirmed that ROS were essentially produced by mitochondria in correlation with its membrane depolarization. Changes in mitochondrial membrane permeability induced by mercury were reversed by NAC but not by thiol-independent antioxidants. Thimerosal and EtHgCl also induced a calcium (Ca2+) influx with a peak at 3h, suggesting that Ca2+ influx is a secondary event following ROS induction as Ca2+ influx was suppressed after pretreatment with NAC but not with thiol-independent antioxidants. Ca2+ influx was also suppressed when culture medium was deprived of Ca2+ confirming the

  12. Synaptotagmin restores kinetic properties of a syntaxin-associated N-type voltage sensitive calcium channel.

    PubMed

    Wiser, O; Tobi, D; Trus, M; Atlas, D

    1997-03-10

    The voltage sensitive N-type calcium channel interacts functionally and biochemically with synaptotagmin (p65). N-type channel interaction with p65 is demonstrated in the Xenopus oocyte expression system, where p65 alters the steady state voltage inactivation of the N-channel, and fully restores the syntaxin-modified current amplitude and inactivation kinetics in a calcium dependent manner. In agreement with the functional results, GST-p65 fusion protein binds to a cytosolic region, amino acids 710-1090 of the N-type channel (N-loop(710-1090)). The results of the combined approach provide a functional and biochemical basis for proposing that p65 interaction with the N-type channel brings p65 into a close association with a syntaxin-coupled channel. In turn, calcium entry through the liberated channel initiates fusion of the primed vesicles with the cell membrane at a short distance from the site of calcium entry.

  13. [Therapy of glucocorticoid-induced osteoporosis with alfacalcidol/calcium and vitamin D/calcium].

    PubMed

    Ringe, J D; Cöster, A; Meng, T; Schacht, E; Umbach, R

    2000-06-01

    Calcium/vitamin D supplementation is generally used as a first step treatment of glucocorticoid-induced osteoporosis (GIOP). The aim of this trial was to compare the efficacy of the D-hormone alfacalcidol with plain vitamin D in patients with established GIOP with or without vertebral fractures. Patients on long-term glucocorticoid-therapy were treated either with 1 microgram alfacalcidol plus 5000 mg calcium (group A: n = 43) or with 1000 IU vitamin D plus 500 mg calcium (group B: n = 42). The two groups were not different in respect to initial characteristics such as age, sex distribution, concomittant diseases, bone mineral density (mean T-score values at lumbar spine and femoral neck: -3.29 and -3.25 resp.), and in the number of prevalent vertebral and non-vertebral fractures. During the 3 years of treatment we found a significant increase in lumbar spine density in group A (+2.0%, p < 0.0001), while no significant changes could be documented in group B at both measuring sites. After 3 years 12 new vertebral fractures had occurred in 10 patients of group A and 21 in 17 patients in group B (ns). Correspondingly we registered a significant decrease of back pain only in group A (p < 0.0001). We conclude that alfacalcidol treatment in superior to plain vitamin D in GIOP.

  14. Hysteresis and the length dependence of calcium sensitivity in chemically skinned rat cardiac muscle.

    PubMed Central

    Harrison, S M; Lamont, C; Miller, D J

    1988-01-01

    1. The relationship between pCa (-log10[Ca2+]) and steady-state isometric tension has been investigated in saponin- or Triton-treated (chemically 'skinned') cardiac muscle of rat. 2. Hysteresis exists in the relationship such that the muscle is less sensitive to Ca2+ during increasing activation (as [Ca2+] is stepped upward) than during reducing activation (as [Ca2+] is stepped downward). 3. The extent of the hysteresis is insensitive to interventions that increase overall calcium sensitivity by chemical means, such as caffeine, carnosine or increased pH. 4. The extent of the hysteresis is sensitive to sarcomere length. The phenomenon is virtually absent above sarcomere lengths of about 2.2-2.3 microns but becomes progressively greater at shorter sarcomere lengths. 5. The effect of sarcomere length on calcium sensitivity is restricted to the upward-going (increasing activation) part of the pCa-tension loop below 2.2 microns. The downward-going (decreasing activation) part of the hysteretic relationship is virtually unaffected by sarcomere length up to 2.2 microns. 6. Significant alterations in sarcomere length do not occur during tension development in the experiments described here: the phenomenon is not attributable to experimental artifacts of this kind. 7. Hysteresis develops sufficiently rapidly to be consistent with a physiological relevance during the normal heart beat. 8. The effects of sarcomere length show that the phenomenon is not due to force per se since, for example, greater peak force produces less hysteresis as sarcomere length is increased towards 2.2 microns. 9. Tonicity increase (by high-molecular-weight dextran), which shrinks the myofilament lattice, increases calcium sensitivity but reduces the effect of sarcomere length on calcium sensitivity. 10. The results suggest that lattice shrinkage is the mechanism which accounts for hysteresis in, and the sarcomere length dependence of, calcium sensitivity in cardiac muscle. Images Fig. 1 Fig. 11

  15. Intracellular calcium oscillations in articular chondrocytes induced by basic calcium phosphate crystals lead to cartilage degradation.

    PubMed

    Nguyen, C; Lieberherr, M; Bordat, C; Velard, F; Côme, D; Lioté, F; Ea, H-K

    2012-11-01

    Basic calcium phosphate (BCP) crystals, including octacalcium phosphate (OCP), carbonated-apatite (CA) and hydroxyapatite (HA) crystals are associated with destructive forms of osteoarthritis. Mechanisms of BCP-induced cartilage breakdown remain incompletely understood. We assessed the ability of BCP to induce changes in intracellular calcium (iCa(2+)) content and oscillations and the role of iCa(2+) in BCP-induced cartilage degradation. Bovine articular chondrocytes (BACs) and bovine cartilage explants (BCEs) were stimulated with BCP or monosodium urate (MSU) crystals. iCa(2+) levels were determined by spectrofluorimetry and oscillations by confocal microscopy. mRNA expression of matrix metalloproteinase 3 (MMP-3), a disintegrin and metalloprotease with thrombospondin-like motifs 4 (ADAMTS-4) and ADAMTS-5 was assessed by quantitative real-time PCR. Glycosaminoglycan (GAG) release was measured in the supernatants of BCE cultures. All three BCP crystals significantly increased iCa(2+) content. OCP also induced iCa(2+) oscillations. Rate of BACs displaying iCa(2+) oscillations increased over time, with a peak after 20 min of stimulation. OCP-induced iCa(2+) oscillations involved both extracellular Ca(2+) (eCa(2+)) influx and iCa(2+) stores. Indeed, OCP-induced iCa(2+) oscillations decreased rapidly in Ca(2+)-free medium. Both voltage- and non-voltage-dependent Ca(2+) channels were involved in eCa(2+) influx. BCP crystal-induced variation in iCa(2+) content was associated with BCP crystal-induced cartilage matrix degradation. However, iCa²(+) was not associated with OCP crystal-induced mRNA expression of MMP-3, ADAMTS-4 or ADAMTS-5. BCP crystals can induce variation in iCa(2+) content and oscillations in articular chondrocytes. Furthermore, BCP crystal-induced changes in iCa(2+) content play a pivotal role in BCP catabolic effects on articular cartilage. Copyright © 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  16. PKC-mediated modulation of L-type calcium channels may contribute to fat-induced insulin resistance.

    PubMed

    McCarty, Mark F

    2006-01-01

    Increased intracellular free calcium [Ca2+]i has been noted in adipocytes, platelets, and leukocytes of subjects with insulin resistance syndrome or allied disorders. In rodent studies, measures which increase [Ca2+]i in adipocytes and skeletal muscle are associated with impaired insulin signaling, attributable at least in part to diminished ability of insulin to activate phosphoserine phosphatase-1 (PP-1). In fat-fed insulin resistant rats, pre-treatment with a drug that selectively chelates intracellular calcium eliminates about half of the decrement in insulin-stimulated glucose uptake induced by fat feeding; since this chelator does not influence the insulin sensitivity of chow-fed rats, it is reasonable to suspect that fat feeding boosts [Ca2+]i in skeletal muscle, and that this effect is partially responsible for the associated reduction in insulin sensitivity. Clinical insulin resistance is associated with increased levels of triglycerides and other fatty acid metabolites in muscle fibers; this can give rise to diacylglycerol-mediated activation of PKC, which in turn compromises insulin signaling by triggering kinase cascades that phosphorylate IRS-1 on key serine residues. Yet there is also evidence that, in skeletal muscle, PKC activity up-regulates the function of L-type calcium channels, increasing their maximal conductance while left-shifting their voltage dependence. Thus, the PKC activation associated with fat overexposure might be expected to boost basal [Ca2+]i in skeletal muscle, potentially impeding insulin-mediated activation of PP-1. This hypothesis is consistent with several clinical studies demonstrating that long-acting inhibitors of L-type calcium channels can improve insulin sensitivity in overweight hypertensives; it should be readily testable in rodent models of fat-induced insulin resistance. Since parathyroid hormone can act on adipocytes and muscle to boost [Ca2+]i, mild secondary hyperparathyroidism associated with low calcium intakes

  17. Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes*

    PubMed Central

    Chen, Xi; Khambu, Bilon; Zhang, Hao; Gao, Wentao; Li, Min; Chen, Xiaoyun; Yoshimori, Tamotsu; Yin, Xiao-Ming

    2014-01-01

    Calcium phosphate precipitates (CPPs) form complexes with DNA, which enter cells via endocytosis. Under this condition CPPs induce autophagy via the canonic autophagy machinery. Here we showed that CPP-induced autophagy was also dependent on endocytosis as the process was significantly inhibited by methyl-β-cyclodextrin and dynasore, which suppress clathrin-dependent endocytosis. Consistently, CPP treatment triggered the formation of filipin-positive intracellular vesicles whose membranes are rich in cholesterol. Unexpectedly, these vesicles were also positive for galectin 3, suggesting that they were damaged and the membrane glycans became accessible to galectins to bind. Endosome damage was caused by endocytosis of CPPs and was reversed by calcium chelators or by endocytosis inhibitors. Notably, CPP-induced LC3-positive autophagosomes were colocalized with galectin 3, ubiquitin, and p62/SQSTM1. Inhibition of galectin 3 reduced p62 puncta and autophagosome formation. Knockdown of p62 additionally inhibited the colocalization of autophagosomes with galectins. Furthermore, most of the galectin 3-positive vesicles were colocalized with Rab7 or LAMP1. Agents that affect endosome/lysosome maturation and function, such as bafilomycin A1, also significantly affected CPP-induced tubulovesicular autophagosome formation. These findings thus indicate that endocytosed CPPs caused endosome damage and recruitment of galectins, particularly at the later endosome stage, which led to the interaction of the autophagosomal membranes with the damaged endosome in the presence of p62. PMID:24619419

  18. Calcium Influx Induced by Stimulation of ATP Receptors on Neurons Cultured from Rat Dorsal Root Ganglia.

    PubMed

    Bouvier, M. M.; Evans, M. L.; Benham, C. D.

    1991-01-01

    A combination of microspectrofluorimetry and single cell voltage-clamp was used to examine the response to ATP of cultured neurons from rat dorsal root ganglia. ATP activated an inward current and a rise in internal calcium concentration that was dependent on the external calcium concentration and on the magnitude of the ATP-induced current response. The response was not affected by prerelease of internal calcium stores with caffeine. The rise in internal calcium was increased at hyperpolarized membrane potentials as the calcium driving force was increased. These results demonstrate that the ATP-gated channels in these cells can admit a significant amount of calcium in a physiological calcium gradient. This alternative calcium entry pathway could provide an internal calcium signal that is spatially distinct to that generated by voltage-gated calcium entry.

  19. Calcium homeostasis and sensitization in pulmonary arterial smooth muscle.

    PubMed

    Jernigan, Nikki L; Resta, Thomas C

    2014-04-01

    The pulmonary circulation is a low-pressure, low-resistance vascular bed with little to no resting tone under normal conditions. An increase in the [Ca(2+) ]i in PASMCs is an important determinant of contraction, migration, and proliferation. Both Ca(2+) influx through plasma membrane Ca(2+) channels and Ca(2+) release from the SR contribute to a rise in [Ca(2+) ]i . Additionally important in the pulmonary circulation are several kinase-mediated signaling pathways that act to increase the sensitivity of the contractile apparatus to [Ca(2+) ]i . Similarly, cytoskeletal processes resulting in dynamic remodeling of the actin cytoskeleton can further contribute to contractility in the pulmonary circulation. In addition to endocrine, paracrine, and autocrine factors, alveolar hypoxia is an important stimulus for pulmonary vasoconstriction. However, prolonged hypoxia is a critical pathological stimulus associated with the development of pulmonary hypertension and cor pulmonale. In this review, we will discuss recent advances in our understanding of how Ca(2+) homeostasis and sensitization regulate PASMC contractility under both physiological and pathophysiological conditions.

  20. Fast activation of dihydropyridine-sensitive calcium channels of skeletal muscle. Multiple pathways of channel gating

    PubMed Central

    1996-01-01

    Dihydropyridine (DHP) receptors of the transverse tubule membrane play two roles in excitation-contraction coupling in skeletal muscle: (a) they function as the voltage sensor which undergoes fast transition to control release of calcium from sarcoplasmic reticulum, and (b) they provide the conducting unit of a slowly activating L-type calcium channel. To understand this dual function of the DHP receptor, we studied the effect of depolarizing conditioning pulse on the activation kinetics of the skeletal muscle DHP-sensitive calcium channels reconstituted into lipid bilayer membranes. Activation of the incorporated calcium channel was imposed by depolarizing test pulses from a holding potential of -80 mV. The gating kinetics of the channel was studied with ensemble averages of repeated episodes. Based on a first latency analysis, two distinct classes of channel openings occurred after depolarization: most had delayed latencies, distributed with a mode of 70 ms (slow gating); a small number of openings had short first latencies, < 12 ms (fast gating). A depolarizing conditioning pulse to +20 mV placed 200 ms before the test pulse (-10 mV), led to a significant increase in the activation rate of the ensemble averaged-current; the time constant of activation went from tau m = 110 ms (reference) to tau m = 45 ms after conditioning. This enhanced activation by the conditioning pulse was due to the increase in frequency of fast open events, which was a steep function of the intermediate voltage and the interval between the conditioning pulse and the test pulse. Additional analysis demonstrated that fast gating is the property of the same individual channels that normally gate slowly and that the channels adopt this property after a sojourn in the open state. The rapid secondary activation seen after depolarizing prepulses is not compatible with a linear activation model for the calcium channel, but is highly consistent with a cyclical model. A six- state cyclical model is

  1. Paclitaxel Induces Apoptosis in Breast Cancer Cells through Different Calcium—Regulating Mechanisms Depending on External Calcium Conditions

    PubMed Central

    Pan, Zhi; Avila, Andrew; Gollahon, Lauren

    2014-01-01

    Previously, we reported that endoplasmic reticulum calcium stores were a direct target for paclitaxel initiation of apoptosis. Furthermore, the actions of paclitaxel attenuated Bcl-2 resistance to apoptosis through endoplasmic reticulum-mediated calcium release. To better understand the calcium-regulated mechanisms of paclitaxel-induced apoptosis in breast cancer cells, we investigated the role of extracellular calcium, specifically; whether influx of extracellular calcium contributed to and/or was necessary for paclitaxel-induced apoptosis. Our results demonstrated that paclitaxel induced extracellular calcium influx. This mobilization of extracellular calcium contributed to subsequent cytosolic calcium elevation differently, depending on dosage. Under normal extracellular calcium conditions, high dose paclitaxel induced apoptosis-promoting calcium influx, which did not occur in calcium-free conditions. In the absence of extracellular calcium an “Enhanced Calcium Efflux” mechanism in which high dose paclitaxel stimulated calcium efflux immediately, leading to dramatic cytosolic calcium decrease, was observed. In the absence of extracellular calcium, high dose paclitaxel’s stimulatory effects on capacitative calcium entry and apoptosis could not be completely restored. Thus, normal extracellular calcium concentrations are critical for high dose paclitaxel-induced apoptosis. In contrast, low dose paclitaxel mirrored controls, indicating that it occurs independent of extracellular calcium. Thus, extracellular calcium conditions only affect efficacy of high dose paclitaxel-induced apoptosis. PMID:24549172

  2. Sandia National Laboratories Small-Scale Sensitivity Testing (SSST) Report: Calcium Nitrate Mixtures with Various Fuels.

    SciTech Connect

    Phillips, Jason Joe

    2014-07-01

    Based upon the presented sensitivity data for the examined calcium nitrate mixtures using sugar and sawdust, contact handling/mixing of these materials does not present hazards greater than those occurring during handling of dry PETN powder. The aluminized calcium nitrate mixtures present a known ESD fire hazard due to the fine aluminum powder fuel. These mixtures may yet present an ESD explosion hazard, though this has not been investigated at this time. The detonability of these mixtures will be investigated during Phase III testing.

  3. N-methyl-D-aspartate receptors and large conductance calcium-sensitive potassium channels inhibit the release of opioid peptides that induce mu-opioid receptor internalization in the rat spinal cord.

    PubMed

    Song, B; Marvizón, J C G

    2005-01-01

    Endogenous opioids in the spinal cord play an important role in nociception, but the mechanisms that control their release are poorly understood. To simultaneously detect all opioids able to activate the mu-opioid receptor, we measured mu-opioid receptor internalization in rat spinal cord slices stimulated electrically or chemically to evoke opioid release. Electrical stimulation of the dorsal horn in the presence of peptidase inhibitors produced mu-opioid receptor internalization in half of the mu-opioid receptor neurons. This internalization was rapidly abolished by N-methyl-D-aspartate (IC50=2 microM), and N-methyl-D-aspartate antagonists prevented this effect. mu-Opioid receptor internalization evoked by high K+ or veratridine was also inhibited by N-methyl-D-aspartate receptor activation. N-methyl-D-aspartate did not affect mu-opioid receptor internalization induced by exogenous endomorphins, confirming that the effect of N-methyl-D-aspartate was on opioid release. We hypothesized that this inhibition was mediated by large conductance Ca2+-sensitive K+ channels BK(Ca2+). Indeed, inhibition by N-methyl-D-aspartate was prevented by tetraethylammonium and by the selective BK(Ca2+) blockers paxilline, penitrem A and verruculogen. Paxilline did not increase mu-opioid receptor internalization in the absence of N-methyl-D-aspartate, indicating that it does not produce an increase in opioid release unrelated to the inhibition by N-methyl-d-aspartate. The BK(Ca2+) involved appears to be a subtype with slow association kinetics for iberiotoxin, which was effective only with long incubations. The BK(Ca2+) opener NS-1619 also inhibited the evoked mu-opioid receptor internalization, and iberiotoxin prevented this effect. We concluded that Ca2+ influx through N-methyl-D-aspartate receptors causes the opening of BK(Ca2+) and hyperpolarization in opioid-containing dorsal horn neurons, resulting in the inhibition of opioid release. Since mu-opioid receptors in the dorsal horn

  4. N-METHYL-d-ASPARTATE RECEPTORS AND LARGE CONDUCTANCE CALCIUM-SENSITIVE POTASSIUM CHANNELS INHIBIT THE RELEASE OF OPIOID PEPTIDES THAT INDUCE μ-OPIOID RECEPTOR INTERNALIZATION IN THE RAT SPINAL CORD

    PubMed Central

    SONG, B.; MARVIZÓN, J. C. G.

    2006-01-01

    Endogenous opioids in the spinal cord play an important role in nociception, but the mechanisms that control their release are poorly understood. To simultaneously detect all opioids able to activate the μ-opioid receptor, we measured μ-opioid receptor internalization in rat spinal cord slices stimulated electrically or chemically to evoke opioid release. Electrical stimulation of the dorsal horn in the presence of peptidase inhibitors produced μ-opioid receptor internalization in half of the μ-opioid receptor neurons. This internalization was rapidly abolished by N-methyl-d-aspartate (IC50=2 μM), and N-methyl-d-aspartate antagonists prevented this effect. μ-Opioid receptor internalization evoked by high K+ or veratridine was also inhibited by N-methyl-d-aspartate receptor activation. N-methyl-d-aspartate did not affect μ-opioid receptor internalization induced by exogenous endomorphins, confirming that the effect of N-methyl-d-aspartate was on opioid release. We hypothesized that this inhibition was mediated by large conductance Ca2+-sensitive K+ channels BK(Ca2+). Indeed, inhibition by N-methyl-d-aspartate was prevented by tetraethylammonium and by the selective BK(Ca2+) blockers paxilline, penitrem A and verruculogen. Paxilline did not increase μ-opioid receptor internalization in the absence of N-methyl-d-aspartate, indicating that it does not produce an increase in opioid release unrelated to the inhibition by N-methyl-d-aspartate. The BK(Ca2+) involved appears to be a subtype with slow association kinetics for iberiotoxin, which was effective only with long incubations. The BK(Ca2+) opener NS-1619 also inhibited the evoked μ-opioid receptor internalization, and iberiotoxin prevented this effect. We concluded that Ca2+ influx through N-methyl-d-aspartate receptors causes the opening of BK(Ca2+) and hyperpolarization in opioid-containing dorsal horn neurons, resulting in the inhibition of opioid release. Since μ-opioid receptors in the dorsal horn

  5. Treatment of glucocorticoid-induced osteoporosis with alfacalcidol/calcium versus vitamin D/calcium.

    PubMed

    Ringe, J D; Cöster, A; Meng, T; Schacht, E; Umbach, R

    1999-10-01

    Vitamin D/calcium substitution is generally regarded as an effective first step treatment for glucocorticoid-induced osteoporosis (GIOP). The aim of our study was to evaluate the efficacy of the active vitamin D metabolite alfacalcidol (1alpha) compared with the native vitamin D(3) in patients with established GIOP with or without vertebral fractures. Patients on long-term corticoid therapy were given either 1 microg alfacalcidol plus 500 mg calcium per day (group A, n = 43) or 1000 IU vitamin D(3) plus 500 mg calcium (group B, n = 42). The two groups were alike in age range, sex ratio, percentages of underlying diseases, average initial bone density values (lumbar spine: mean T-score -3.28 and -3.25, respectively), and rates of vertebral and nonvertebral fractures. During the 3-year study we found a small but significant increase of lumbar spine density in group 1alpha (+2.0%, P < 0.0001) and no significant changes at the femoral neck. In the D(3) group, there were no significant changes at both sites. At the end of the study, 12 new vertebral fractures had occurred in 10 patients of the group 1alpha and 21 in 17 patients of the D(3) group. In accordance with the observed fracture rates, the alfacalcidol group showed a significant decrease in back pain (P < 0.0001) whereas no change was seen in the vitamin D group. We conclude that with the doses used in this trial, alfacalcidol is superior to vitamin D in the treatment of established GIOP.

  6. The ethanol withdrawal syndrome: A role for dihydropyridine-sensitive calcium channels in neuronal hyperexcitability states

    SciTech Connect

    Whittington, M.A.

    1990-01-01

    This project investigated the effects of dihydropyridine calcium channel blockers on behavioral and electrophysiological aspects of ethanol withdrawal. The effects of the dihydropyridine (+)-PN 200-110, on changes in neuronal function during ethanol withdrawal, were compared with effects on changes caused by the GABAergic convulsant drug bicuculline. Behavioral correlates of ethanol withdrawal were measured in two strains of mice using a rating of handling-induced convulsions. Concurrent chronic treatment with ethanol and the dihydropyridine calcium channel blockers ([plus minus])-nitrendipine, ([plus minus])-nimodipine or ([plus minus])-PN 200-110 prevented withdrawal-induced increased in convulsive behavior. This effect was dose dependent. The duration of chronic treatment with calcium channel blocker affected the degree of protection against increases in convulsive behavior seen during ethanol withdrawal. Concurrent chronic treatment with ethanol, and the mixed calcium channel activator/blocker ([plus minus])-BAY K 8644, prevented ethanol withdrawal-induced increases in convulsive behavior. Single acute injections of nitrendipine immediately on cessation of chronic treatment with ethanol, or 2h later, reduced withdrawal-induced increases in convulsive behavior in a dose-dependent manner throughout the 12h test period. Slices isolated from mice after chronic ethanol treatment showed a complex, time-dependent pattern of changes in the above measurements, culminating in epileptiform discharges seen from 4h to 7h into withdrawal.

  7. The dihydropyridine-sensitive calcium channel subtype in cone photoreceptors

    PubMed Central

    1996-01-01

    High-voltage activated Ca channels in tiger salamander cone photoreceptors were studied with nystatin-permeabilized patch recordings in 3 mM Ca2+ and 10 mM Ba2+. The majority of Ca channel current was dihydropyridine sensitive, suggesting a preponderance of L- type Ca channels. However, voltage-dependent, incomplete block (maximum 60%) by nifedipine (0.1-100 microM) was evident in recordings of cones in tissue slice. In isolated cones, where the block was more potent, nifedipine (0.1-10 microM) or nisoldipine (0.5-5 microM) still failed to eliminate completely the Ca channel current. Nisoldipine was equally effective in blocking Ca channel current elicited in the presence of 10 mM Ba2+ (76% block) or 3 mM Ca2+ (88% block). 15% of the Ba2+ current was reversibly blocked by omega-conotoxin GVIA (1 microM). After enhancement with 1 microM Bay K 8644, omega-conotoxin GVIA blocked a greater proportion (22%) of Ba2+ current than in control. After achieving partial block of the Ba2+ current with nifedipine, concomitant application of omega-conotoxin GVIA produced no further block. The P-type Ca channel blocker, omega-agatoxin IVA (200 nM), had variable and insignificant effects. The current persisting in the presence of these blockers could be eliminated with Cd2+ (100 microM). These results indicate that photoreceptors express an L-type Ca channel having a distinguishing pharmacological profile similar to the alpha 1D Ca channel subtype. The presence of additional Ca channel subtypes, resistant to the widely used L-, N-, and P-type Ca channel blockers, cannot, however, be ruled out. PMID:8740375

  8. Calcium feedback and sensitivity regulation in primate rods

    PubMed Central

    1991-01-01

    Membrane current was recorded from a single primate rod with a suction pipette while the cell was bath perfused with solutions maintained at a temperature of approximately 38 degrees C. A transient inward current was observed at the onset of bright illumination after briefly exposing the outer segment in darkness to Ringer's (Locke) solution containing 3- isobutyl-1-methylxanthine (IBMX), an inhibitor of cGMP phosphodiesterase. After briefly removing external Na+ from around the outer segment in darkness, a similar current was observed upon Na+ restoration in bright light. By analogy to amphibian rods, this inward current was interpreted to represent the activity of an electrogenic Na(+)-dependent Ca2+ efflux, which under physiological conditions in the light is expected to reduce the free Ca2+ in the outer segment and provide negative feedback (the "Ca2+ feedback") to the phototransduction process. The exchange current had a saturated amplitude of up to approximately 5 pA and a decline time course that appeared to have more than one exponential component. In the absence of the Ca2+ feedback, made possible by removing the Ca2+ influx and efflux at the outer segment using a 0 Na(+)-0 Ca2+ external solution, the response of a rod to a dim flash was two to three times larger and had a longer time to peak than in physiological solution. These changes can be approximately accounted for by a simple model describing the Ca2+ feedback in primate rods. The dark hydrolytic rate for cGMP was estimated to be 1.2 s-1. The incremental hydrolytic rate, beta*(t), activated by one photoisomerization was approximately 0.09 s-1 at its peak, with a time-integrated activity, integral of beta*(t)dt, of approximately 0.033, both numbers being derived assuming spatial homogeneity in the outer segment. Finally, we have found that primate rods adapt to light in much the same way as amphibian and other mammalian rods, such as showing a Weber-Fechner relation between flash sensitivity and

  9. Calcium oxalate calculi-induced clusterin expression in kidney.

    PubMed

    Li, Jin-Yi; Liu, Junjiang; Jiang, Junyi; Pumill, Chris; Elaiho, Cordelia; Zhang, Yunxia; Li, Shoubin; Zhou, Tie

    2015-10-01

    The aim of the study was to investigate clusterin expression in the kidney and evaluate the urine clusterin level in the kidney stone formers. (1) In vitro, we treated the Madin-Darby canine kidney (MDCK) cell line with different concentrations of calcium oxalate (CaOx), and then the clusterin protein expression in the cells was evaluated by Western blotting. (2) Kidney stone patients who received percutaneous nephrolithotomy were enrolled in our study. Urine samples were collected before surgery, the kidney punctured to obtain kidney tissue guided by ultrasound intraoperatively. Clusterin expression in the human kidney tissue was evaluated by immunochemistry. The urine clusterin level was determined by enzyme-linked immunosorbent assay. Non-kidney disease subjects were chosen as controls. In vitro, the clusterin expression was up-regulated in the MDCK cells induced by CaOx. The study included 49 patients and 41 non-kidney disease subjects. All calculi were composed of calcium oxalate monohydrate or calcium oxalate dihydrate and a few also contained protein or uric acid. Mean ± SD urine clusterin level was 17.47 ± 18.61 μg/ml in patients, and 3.31 ± 5.42 μg/ml in non-kidney disease subjects, respectively (p < 0.001). Immunohistochemistry revealed the clusterin was located in the cytoplasm of the renal distal and collecting tubular epithelial cells. Also the tissue clusterin expression increased significantly in the kidney stone formers compared to the control groups (p = 0.001). CaOx could induce clusterin expression in renal tubular cells, and increase clusterin levels in the kidney and urine from the kidney stone formers.

  10. The Impact of Ovariectomy on Calcium Homeostasis and Myofilament Calcium Sensitivity in the Aging Mouse Heart

    PubMed Central

    Fares, Elias; Pyle, W. Glen; Ray, Gibanananda; Rose, Robert A.; Denovan-Wright, Eileen M.; Chen, Robert P.; Howlett, Susan E.

    2013-01-01

    This study determined whether deficiency of ovarian estrogen starting very early in life promoted age-associated Ca2+ dysregulation and contractile dysfunction in isolated ventricular myocytes. Myocytes were isolated from anesthetized C57BL/6 female mice. Animals received an ovariectomy or sham-operation at one month and were aged to ~24 months. Excitation-contraction coupling parameters were compared in fura-2 loaded myocytes (37°C). While Ca2+ transients were larger and faster in field-stimulated myocytes from ovariectomized mice, ovariectomy had no effect on peak fractional shortening. Similarly, ovariectomy had no effect on fractional shortening measured in vivo by echocardiography (values were 60.5 ± 2.9 vs. 60.3 ± 2.5% in sham and ovariectomized, respectively; n=5 mice/group). Ovariectomy did decrease myofilament Ca2+ sensitivity, as evidenced by a 26% increase in the Ca2+ required to activate actomyosin MgATPase in ovariectomized hearts. Larger Ca2+ transients were attributable to a 48% increase in peak Ca2+ current, along with an increase in the amplitude, width and frequency of Ca2+ sparks measured in fluo-4 loaded myocytes. These changes in Ca2+ handling were not due to increased expression of Ca2+ channels (Cav1.2), sarcoplasmic reticulum Ca2+ ATPase (SERCA2) or Na+-Ca2+ exchanger in ovariectomized hearts. However, ovariectomy increased sarcoplasmic reticulum Ca2+ stores by ~90% and promoted spontaneous Ca2+ release from the sarcoplasmic reticulum when compared to sham controls. These observations demonstrate that long-term ovariectomy promotes intracellular Ca2+ dysregulation, reduces myofilament Ca2+ sensitivity and increases spontaneous Ca2+ release in the aging female heart. PMID:24058623

  11. PYRETHROID INDUCED ALTERATIONS IN TRANSCRIPTION OF CALCIUM RESPONSIVE AND IMMEDIATE EARLY GENES IN VIVO.

    EPA Science Inventory

    Multiple molecular targets for pyrethroid insecticides have been evaluated in in vitro preparations, including but not limited to voltage-sensitive sodium channels (VSSCs), voltage-sensitive calcium channels (VSCCs), GABAergic receptors, ATPases and mitochondrial respiratory chai...

  12. Protection against methoxyacetic-acid-induced spermatocyte apoptosis with calcium channel blockers in cultured rat seminiferous tubules: possible mechanisms.

    PubMed

    Li, L H; Wine, R N; Miller, D S; Reece, J M; Smith, M; Chapin, R E

    1997-05-01

    A calcium-mediated mechanism underlying spermatocyte apoptosis induced by 2-methoxyethanol (2-ME) has been previously proposed. This hypothesis was tested in vitro in the present study using cultured juvenile (25 days old) and adult rat seminiferous tubules (JRST and ARST, respectively) with methoxyacetic acid (MAA, the active metabolite of 2-ME). In JRST, spermatocyte degeneration was morphologically obvious 19 hr after a 5-hr exposure to 5 mM MAA. The lesion was unaffected by the presence or absence of extratubular Ca2+. However, MAA-induced cell death was significantly prevented by cotreatment with the dihydropyridines (DHP) nifedipine (50 microM) and nicardipine (20 microM), as well as verapamil (50 microM) and TMB-8 (50 microM), all of which are able to inhibit calcium movement through plasma membranes. However, neither ryanodine, dantrolene, nor cyclosporin A and ruthenium red, which inhibit Ca2+ mobilization from intracellular stores (endoplasmic reticulum and mitochondria), affected the MAA-induced cell death. Inhibition of calcium mobilization through IP3-sensitive pathways by blocking the product of IP3 with manoalide, neomycin, and U73122 did not block the MAA-induced lesion. The protective effects of 50 microM nifedipine and 50 microM TMB-8 were also observed in ARSTs treated with 10 mM MAA for 5 hr. However, when rat testicular sections were immunohistochemically stained with monoclonal antibodies specific for the alpha 1 (the DHP receptor) or the alpha 2 subunits of DHP-sensitive calcium channels, no positive staining was found. Finally, in an attempt to see whether the intracellular free calcium concentrations ([Ca2+]i) in germ cells were increased after the MAA treatment, intact seminiferous tubules were loaded with indo-1 and were measured using laser-scanning confocal microscopy. No detectable increase in the signal in MA A-sensitive spermatocytes was observed, while a 34-54% increase in the signal could be detected in the same cell types when

  13. Fast calcium and voltage-sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge.

    PubMed

    Michel, K; Michaelis, M; Mazzuoli, G; Mueller, K; Vanden Berghe, P; Schemann, M

    2011-12-15

    Slow changes in [Ca(2+)](i) reflect increased neuronal activity. Our study demonstrates that single-trial fast [Ca(2+)](i) imaging (≥200 Hz sampling rate) revealed peaks each of which are associated with single spike discharge recorded by consecutive voltage-sensitive dye (VSD) imaging in enteric neurones and nerve fibres. Fast [Ca(2+)](i) imaging also revealed subthreshold fast excitatory postsynaptic potentials. Nicotine-evoked [Ca(2+)](i) peaks were reduced by -conotoxin and blocked by ruthenium red or tetrodotoxin. Fast [Ca(2+)](i) imaging can be used to directly record single action potentials in enteric neurones. [Ca(2+)](i) peaks required opening of voltage-gated sodium and calcium channels as well as Ca(2+) release from intracellular stores.

  14. Binding of ( sup 125 I)iodipine to parathyroid cell membranes: Evidence of a dihydropyridine-sensitive calcium channel

    SciTech Connect

    Jones, J.I.; Fitzpatrick, L.A. )

    1990-04-01

    The parathyroid cell is unusual, in that an increase in extracellular calcium concentrations inhibits PTH release. Calcium channels are glycoproteins that span cell membranes and allow entry of extracellular calcium into cells. We have demonstrated that the calcium channel agonist (+)202-791, which opens calcium channels, inhibits PTH release and that the antagonist (-)202-791, which closes calcium channels, stimulates PTH release. To identify the calcium channels responsible for these effects, we used a radioligand that specifically binds to calcium channels. Bovine parathyroid cell membranes were prepared and incubated under reduced lighting with (125I) iodipine (SA, 2000 Ci/mmol), which recognizes 1,4-dihydropyridine-sensitive calcium channels. Bound ligand was separated from free ligand by rapid filtration through Whatman GF/B filters. Nonspecific binding was measured by the inclusion of nifedipine at 10 microM. Specific binding represented approximately 40% of the total binding. The optimal temperature for (125I) iodipine binding was 4 C, and binding reached equilibrium by 30 min. The equilibrium dissociation constant (Kd) was approximately 550 pM, and the maximum number of binding sites was 780 fmol/mg protein. Both the calcium channel agonist (+)202-791 and antagonist (-)202-791 competitively inhibited (125I) iodipine binding, with 50% inhibition concentrations of 20 and 300 nM, respectively. These data indicate the presence of dihydropyridine-sensitive calcium channels on parathyroid cell membranes.

  15. The role of L-type calcium channels in the development and expression of behavioral sensitization to ethanol.

    PubMed

    Broadbent, Julie

    2013-10-11

    Behavioral sensitization is thought to play a significant role in drug addiction. L-type calcium channels have been implicated in sensitization to stimulant and opiate drugs but it is unclear if these channels also contribute to sensitization to ethanol. The effects of three L-type calcium channel blockers, nifedipine (1-7.5 mg/kg), diltiazem (12.5-50 mg/kg), and verapamil (12.5 and 25 mg/kg), on sensitization to ethanol (2 g/kg) were examined in DBA/2J mice. All three blockers reduced but did not prevent expression of sensitization. Only nifedipine blocked acquisition of sensitization. Nifedipine and verapamil decreased blood ethanol levels. The current findings suggest L-type calcium channels do not play a substantial role in sensitization to ethanol and that the neural mechanisms underlying sensitization to ethanol are distinct from those mediating sensitization to stimulants and opiates.

  16. The Role of L-Type Calcium Channels in the Development and Expression of Behavioral Sensitization to Ethanol

    PubMed Central

    Broadbent, Julie

    2013-01-01

    Behavioral sensitization is thought to play a significant role in drug addiction. L-type calcium channels have been implicated in sensitization to stimulant and opiate drugs but it is unclear if these channels also contribute to sensitization to ethanol. The effects of three L-type calcium channel blockers, nifedipine (1 – 7.5 mg/kg), diltiazem (12.5 – 50 mg/kg), and verapamil (12.5 and 25 mg/kg), on sensitization to ethanol (2 g/kg) were examined in DBA/2J mice. All three blockers reduced but did not prevent expression of sensitization. Only nifedipine blocked acquisition of sensitization. Nifedipine and verapamil decreased blood ethanol levels. The current findings suggest L-type calcium channels do not play a substantial role in sensitization to ethanol and that the neural mechanisms underlying sensitization to ethanol are distinct from those mediating sensitization to stimulants and opiates. PMID:23994059

  17. Immature thymocytes become sensitive to calcium-mediated apoptosis with the onset of CD8, CD4, and the T cell receptor expression: a role for bcl-2?

    PubMed

    Andjelić, S; Jain, N; Nikolić-Zugić, J

    1993-11-01

    During intrathymic negative selection by clonal deletion, crosslinking of the T cell receptor (TCR) induces cell death by delivering an apoptotic signal(s) to the nucleus along a calcium-dependent pathway. We investigated the reactivity of early precursor-containing thymocytes to Ca(2+)-induced signals, and discovered a breakpoint in their sensitivity to calcium-mediated cell death (CMCD). CD25+CD8-4- TCR- (triple negative [TN]) thymocytes stimulated with a calcium ionophore maintain their viability and precursor activity. By contrast, their immediate progeny, CD25-CD8lo4loTCR alpha beta lo (triple low [TL]) cells react to calcium elevation by abrogation of precursor activity and apoptotic cell death. This developmental difference is specific for CMCD, since both CD25+TN and CD25-TL cells are susceptible to steroid-induced apoptosis. The presence of bcl-2 mRNA correlates directly to the resistance to CMCD-CD25+ TN cells express it and CD25-TL cells do not. These experiments show that thymocytes become sensitive to Ca(2+)-induced apoptosis as soon as they begin to express molecules that mediate thymic selection, and suggest that a concomitant downregulation of bcl-2 may mediate this phenomenon.

  18. Immature thymocytes become sensitive to calcium-mediated apoptosis with the onset of CD8, CD4, and the T cell receptor expression: a role for bcl-2?

    PubMed Central

    1993-01-01

    During intrathymic negative selection by clonal deletion, crosslinking of the T cell receptor (TCR) induces cell death by delivering an apoptotic signal(s) to the nucleus along a calcium-dependent pathway. We investigated the reactivity of early precursor-containing thymocytes to Ca(2+)-induced signals, and discovered a breakpoint in their sensitivity to calcium-mediated cell death (CMCD). CD25+CD8-4- TCR- (triple negative [TN]) thymocytes stimulated with a calcium ionophore maintain their viability and precursor activity. By contrast, their immediate progeny, CD25-CD8lo4loTCR alpha beta lo (triple low [TL]) cells react to calcium elevation by abrogation of precursor activity and apoptotic cell death. This developmental difference is specific for CMCD, since both CD25+TN and CD25-TL cells are susceptible to steroid- induced apoptosis. The presence of bcl-2 mRNA correlates directly to the resistance to CMCD-CD25+ TN cells express it and CD25-TL cells do not. These experiments show that thymocytes become sensitive to Ca(2+)- induced apoptosis as soon as they begin to express molecules that mediate thymic selection, and suggest that a concomitant downregulation of bcl-2 may mediate this phenomenon. PMID:8228820

  19. Pseudopterosin A inhibits phagocytosis and alters intracellular calcium turnover in a pertussis toxin sensitive site in Tetrahymena thermophila.

    PubMed

    Moya, Claudia E; Jacobs, Robert S

    2006-08-01

    The free living ciliate Tetrahymena thermophila was chosen as a cellular model in order to investigate the mode of action of the anti-inflammatory marine natural product Pseudopterosin A (PsA). In this paper we present evidence that PsA inhibits phagosome formation (KD=10.5 microM) and triggers a discrete intracellular calcium release (depletion) from a site in T. thermophila cells (KD=6.4 microM). Pre-treatment with the Gi/o protein inhibitor, pertussis toxin (PTX), inhibits PsA activity of both responses providing pharmacological evidence that the site of action for PsA is at a PTX sensitive G protein or a G protein coupled receptor (GPCR). Addition of extracellular calcium induced a concentration dependent increase in the incidence of phagosome formation (KD=30.3 microM) and was blocked by PsA pre-treatment. This particular effect of PsA on extracellular calcium was not blocked by PTX pre-treatment.

  20. Yeast Gdt1 is a Golgi-localized calcium transporter required for stress-induced calcium signaling and protein glycosylation

    PubMed Central

    Colinet, Anne-Sophie; Sengottaiyan, Palanivelu; Deschamps, Antoine; Colsoul, Marie-Lise; Thines, Louise; Demaegd, Didier; Duchêne, Marie-Clémence; Foulquier, François; Hols, Pascal; Morsomme, Pierre

    2016-01-01

    Calcium signaling depends on a tightly regulated set of pumps, exchangers, and channels that are responsible for controlling calcium fluxes between the different subcellular compartments of the eukaryotic cell. We have recently reported that two members of the highly-conserved UPF0016 family, human TMEM165 and budding yeast Gdt1p, are functionally related and might form a new group of Golgi-localized cation/Ca2+ exchangers. Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. Using an assay based on the heterologous expression of GDT1 in the bacterium Lactococcus lactis, we demonstrated the calcium transport activity of Gdt1p. We observed a Ca2+ uptake activity in cells expressing GDT1, which was dependent on the external pH, indicating that Gdt1p may act as a Ca2+/H+ antiporter. In yeast, we found that Gdt1p controls cellular calcium stores and plays a major role in the calcium response induced by osmotic shock when the Golgi calcium pump, Pmr1p, is absent. Importantly, we also discovered that, in the presence of a high concentration of external calcium, Gdt1p is required for glycosylation of carboxypeptidase Y and the glucanosyltransferase Gas1p. Finally we showed that glycosylation process is restored by providing more Mn2+ to the cells. PMID:27075443

  1. Calcium occupancy of N-terminal sites within calmodulin induces inhibition of the ryanodine receptor calcium release channel.

    PubMed

    Boschek, Curt B; Jones, Terry E; Squier, Thomas C; Bigelow, Diana J

    2007-09-18

    -domain of CaM mirrors the calcium dependence of RyR1 inhibition observed at activating calcium levels, where [Ca]1/2 = 4.3 +/- 0.4 microM, suggesting a direct regulation of RyR1 function upon the calcium-dependent activation of CaM. These results indicate that occupancy of the N-terminal domain calcium binding sites in CaM bound to the identified CaM-binding sequence K3614-N3643 induces conformational rearrangements within the complex between CaM and RyR1 responsible for the CaM-dependent modulation of the RyR1 calcium release channel.

  2. Calcium phosphate bioceramics induce mineralization modulated by proteins.

    PubMed

    Wang, Kefeng; Leng, Yang; Lu, Xiong; Ren, Fuzeng

    2013-08-01

    Proteins play an important role in the process of biomineralization, which is considered the critical process of new bone formation. The calcium phosphate (Ca-P) mineralization happened on hydroxyapatite (HA), β-tricalcium phosphate (β-TCP) and biphasic calcium phosphate (BCP) when proteins presented were investigated systematically. The results reveal that the presence of protein in the revised simulated body fluid (RSBF) did not alter the shape and crystal structure of the precipitated micro-crystals in the Ca-P layer formed on the three types of bioceramics. However, the morphology of the Ca-P precipitates was regulated but the structure of Ca-P crystal was unchanged in vivo. The presence of proteins always inhibits Ca-P mineralization in RSBF and the degree of inhibitory effect is concentration dependent. Furthermore, Protein presence can increase the possibility of HA precipitation in vitro and in vivo. The results obtained in this study can be helpful for better understanding the mechanism of biomineralization induced by the Ca-P bioceramics.

  3. Stimulation of high affinity gamma-aminobutyric acidB receptors potentiates the depolarization-induced increase of intraneuronal ionized calcium content in cerebellar granule neurons.

    PubMed

    De Erausquin, G; Brooker, G; Costa, E; Wojcik, W J

    1992-09-01

    In the treatment of spasticity, the therapeutic cerebrospinal fluid levels of (+/-)-baclofen, a gamma-aminobutyric acid (GABA)B receptor agonist, are below 1 microM. However, the mechanism of the therapeutic action of (+/-)-baclofen remains unknown, because, for the most part, the action of (+/-)-baclofen on GABAB receptors requires micromolar concentrations. Using fura-2 fluorescence microscopy, intracellular ionized calcium was measured in cerebellar granule neurons. Stimulation of a high affinity GABAB receptor potentiated by 2-3-fold the rise in intracellular calcium observed after depolarization of the cell with a Krebs Ringer's buffered solution containing 40 mM K+. Both GABA (100 nM) and (+/-)-baclofen (10-100 nM) stimulated this high affinity receptor. The potentiation of the depolarization-induced rise in intracellular calcium by (+/-)-baclofen (100 nM) was completely blocked by the GABAB receptor antagonist CGP 35348 (200 microM). Also, the intracellular calcium response induced by the activation of high affinity GABAB receptors was prevented by dantrolene (10 microM). The cerebellar granule neurons contained calcium-induced calcium release (CICR) stores. Caffeine (3 mM) and ryanodine (100 microM) potentiated the depolarization-induced rise in intracellular calcium, and this response to both drugs was blocked by dantrolene (10 microM). Because dantrolene does not prevent the rise in intracellular calcium after cell depolarization (this calcium originated from the influx of extracellular calcium), (+/-)-baclofen acting via the high affinity GABAB receptor indirectly activates the CICR stores, allowing the influx of extracellular calcium to trigger the release of calcium from these dantrolene-sensitive CICR stores. Thus, this high affinity GABAB receptor might become activated during persistent depolarization caused by pathological states and could be a mechanism to be studied for the therapeutic action of (+/-)-baclofen in spasticity.

  4. G protein-induced trafficking of voltage-dependent calcium channels.

    PubMed

    Tombler, Eugene; Cabanilla, Nory Jun; Carman, Paul; Permaul, Natasha; Hall, John J; Richman, Ryan W; Lee, Jessica; Rodriguez, Jennifer; Felsenfeld, Dan P; Hennigan, Robert F; Diversé-Pierluissi, María A

    2006-01-20

    Calcium channels are well known targets for inhibition by G protein-coupled receptors, and multiple forms of inhibition have been described. Here we report a novel mechanism for G protein-mediated modulation of neuronal voltage-dependent calcium channels that involves the destabilization and subsequent removal of calcium channels from the plasma membrane. Imaging experiments in living sensory neurons show that, within seconds of receptor activation, calcium channels are cleared from the membrane and sequestered in clathrin-coated vesicles. Disruption of the L1-CAM-ankyrin B complex with the calcium channel mimics transmitter-induced trafficking of the channels, reduces calcium influx, and decreases exocytosis. Our results suggest that G protein-induced removal of plasma membrane calcium channels is a consequence of disrupting channel-cytoskeleton interactions and might represent a novel mechanism of presynaptic inhibition.

  5. Role of calcium in gonadotropin releasing hormone-induced luteinizing hormone secretion from the bovine pituitary

    SciTech Connect

    Kile, J.P.

    1986-01-01

    The hypothesis was tested that GnRH acts to release LH by increasing calcium uptake by gonadotroph which in turn stimulates calcium-calmodulin activity and results in LH release from bovine pituitary cells as it does in the rat. Pituitary glands of calves (4-10 months of age) were enzymatically dispersed (0.2% collagenase) and grown for 5 days to confluency in multiwell plates (3 x 10/sup 5//well). Cells treated with GnRH Ca/sup + +/ ionophore A23187, and ouabain all produced significant releases of LH release in a pronounced all or none fashion, while thorough washing of the cells with 0.5 mM EGTA in Ca/sup + +/-free media prevented the action of GnRH. GnRH caused a rapid efflux of /sup 45/Ca/sup + +/. Both GnRH-stimulated /sup 45/Ca efflux and LH release could be partially blocked by verapamil GnRH-induced LH release could also be blocked by nifedipine and tetrodotoxin, although these agents did not affect /sup 45/Ca efflux. The calmodulin antagonists calmidazolium and W7 were found to block GnRH induced LH release, as well as LH release induced by theophylline, KC PGE/sub 2/ and estradiol. These data indicated that: (1) calcium is required for GnRH action, but extracellular Ca/sup + +/ does not regulate LH release; (2) GnRH elevates intracellular Ca/sup + +/ by opening both voltage sensitive and receptor mediated Ca/sup + +/ channels; (3) activation of calmodulin is one mechanism involved in GnRH-induced LH release.

  6. Microdamage induced calcium efflux from bone matrix activates intracellular calcium signaling in osteoblasts via L-type and T-type voltage-gated calcium channels.

    PubMed

    Jung, Hyungjin; Best, Makenzie; Akkus, Ozan

    2015-07-01

    Mechanisms by which bone microdamage triggers repair response are not completely understood. It has been shown that calcium efflux ([Ca(2+)]E) occurs from regions of bone undergoing microdamage. Such efflux has also been shown to trigger intracellular calcium signaling ([Ca(2+)]I) in MC3T3-E1 cells local to damaged regions. Voltage-gated calcium channels (VGCCs) are implicated in the entry of [Ca(2+)]E to the cytoplasm. We investigated the involvement of VGCC in the extracellular calcium induced intracellular calcium response (ECIICR). MC3T3-E1 cells were subjected to one dimensional calcium efflux from their basal aspect which results in an increase in [Ca(2+)]I. This increase was concomitant with membrane depolarization and it was significantly reduced in the presence of Bepridil, a non-selective VGCC inhibitor. To identify specific type(s) of VGCC in ECIICR, the cells were treated with selective inhibitors for different types of VGCC. Significant changes in the peak intensity and the number of [Ca(2+)]I oscillations were observed when L-type and T-type specific VGCC inhibitors (Verapamil and NNC55-0396, respectively) were used. So as to confirm the involvement of L- and T-type VGCC in the context of microdamage, cells were seeded on devitalized notched bone specimen, which were loaded to induce microdamage in the presence and absence of Verapamil and NNC55-0396. The results showed significant decrease in [Ca(2+)]I activity of cells in the microdamaged regions of bone when L- and T-type blockers were applied. This study demonstrated that extracellular calcium increase in association with damage depolarizes the cell membrane and the calcium ions enter the cell cytoplasm by L- and T-type VGCCs.

  7. Biofilm-induced calcium carbonate precipitation: application in the subsurface

    NASA Astrophysics Data System (ADS)

    Phillips, A. J.; Eldring, J.; Lauchnor, E.; Hiebert, R.; Gerlach, R.; Mitchell, A. C.; Esposito, R.; Cunningham, A. B.; Spangler, L.

    2012-12-01

    We have investigated mitigation strategies for sealing high permeability regions, like fractures, in the subsurface. This technology has the potential to, for example, improve the long-term security of geologically-stored carbon dioxide (CO2) by sealing fractures in cap rocks or to mitigate leakage pathways to prevent contamination of overlying aquifers from hydraulic fracturing fluids. Sealing technologies using low-viscosity fluids are advantageous since they potentially reduce the necessary injection pressures and increase the radius of influence around injection wells. In this technology, aqueous solutions and suspensions are used to promote microbially-induced mineral precipitation which can be applied in subsurface environments. To this end, a strategy was developed to twice seal a hydraulically fractured, 74 cm (2.4') diameter Boyles Sandstone core, collected in North-Central Alabama, with biofilm-induced calcium carbonate (CaCO3) precipitates under ambient pressures. Sporosarcina pasteurii biofilms were established and calcium and urea containing reagents were injected to promote saturation conditions favorable for CaCO3 precipitation followed by growth reagents to resuscitate the biofilm's ureolytic activity. Then, in order to evaluate this process at relevant deep subsurface pressures, a novel high pressure test vessel was developed to house the 74 cm diameter core under pressures as high as 96 bar (1,400 psi). After determining that no impact to the fracture permeability occurred due to increasing overburden pressure, the fractured core was sealed under subsurface relevant pressures relating to 457 meters (1,500 feet) below ground surface (44 bar (650 psi) overburden pressure). After fracture sealing under both ambient and subsurface relevant pressure conditions, the sandstone core withstood three times higher well bore pressure than during the initial fracturing event, which occurred prior to biofilm-induced CaCO3 mineralization. These studies suggest

  8. Calcium.

    PubMed

    Williams, Robert J P

    2002-01-01

    This chapter describes the chemical and biological value of the calcium ion. In calcium chemistry, our main interest is in equilibria within static, nonflowing systems. Hence, we examined the way calcium formed precipitates and complex ions in solution. We observed thereafter its uses by humankind in a vast number of materials such as minerals, e.g., marble, concrete, mortars, which parallel the biological use in shells and bones. In complex formation, we noted that many combinations were of anion interaction with calcium for example in the uses of detergents and medicines. The rates of exchange of calcium from bound states were noted but they had little application. Calcium ions do not act as catalysts of organic reactions. In biological systems, interest is in the above chemistry, but extends to the fact that Ca2+ ions can carry information by flowing in one solution or from one solution to another through membranes. Hence, we became interested in the details of rates of calcium exchange. The fast exchange of this divalent ion from most organic binding sites has allowed it to develop as the dominant second messenger. Now the flow can be examined in vitro as calcium binds particular isolated proteins, which it activates as seen in physical mechanical changes or chemical changes and this piece-by-piece study of cells is common. Here, however, we have chosen to stress the whole circuit of Ca2+ action indicating that the cell is organized both at a basal and an activated state kinetic level by the steady state flow of the ion (see Fig. 11). Different time constants of exchange utilizing very similar binding constants lead to: 1) fast responses as in the muscle of an animal; or 2) slower change as in differentiation of an egg or seed. Many other changes of state may relate to Ca2+ steady-state levels of flow in the circuitry and here we point to two: 1) dormancy in reptiles and animals; and 2) sporulation in both bacteria and lower plants. In the other chapters of

  9. Mepivacaine-induced intracellular calcium increase appears to be mediated primarily by calcium influx in rat aorta without endothelium.

    PubMed

    Ok, Seong-Ho; Kwon, Seong-Chun; Kang, Sebin; Choi, Mun-Jeoung; Sohn, Ju-Tae

    2014-12-01

    Mepivacaine induces contraction or decreased blood flow both in vivo and in vitro. Vasoconstriction is associated with an increase in the intracellular calcium concentration ([Ca(2+)]i). However, the mechanism responsible for the mepivacaine-evoked [Ca(2+)]i increase remains to be determined. Therefore, the objective of this in vitro study was to examine the mechanism responsible for the mepivacaine-evoked [Ca(2+)]i increment in isolated rat aorta. Isometric tension was measured in isolated rat aorta without endothelium. In addition, fura-2 loaded aortic muscle strips were illuminated alternately (48 Hz) at two excitation wavelengths (340 and 380 nm). The ratio of F340 to F380 (F340/F380) was regarded as an amount of [Ca(2+)]i. We investigated the effects of nifedipine, 2-aminoethoxydiphenylborate (2-APB), gadolinium chloride hexahydrate (Gd(3+)), low calcium level and Krebs solution without calcium on the mepivacaine-evoked contraction in isolated rat aorta and on the mepivacaine-evoked [Ca(2+)]i increment in fura-2 loaded aortic strips. We assessed the effect of verapamil on the mepivacaine-evoked [Ca(2+)]i increment. Mepivacaine produced vasoconstriction and increased [Ca(2+)]i. Nifedipine, 2-APB and low calcium attenuated vasoconstriction and the [Ca(2+)]i increase evoked by mepivacaine. Verapamil attenuated the mepivacaine-induced [Ca(2+)]i increment. Calcium-free solution almost abolished mepivacaine-induced contraction and strongly attenuated the mepivacaineinduced [Ca(2+)]i increase. Gd(3+) had no effect on either vasoconstriction or the [Ca(2+)]i increment evoked by mepivacaine. The mepivacaine-evoked [Ca(2+)]i increment, which contributes to mepivacaine-evoked contraction, appears to be mediated mainly by calcium influx and partially by calcium released from the sarcoplasmic reticulum.

  10. Mepivacaine-induced intracellular calcium increase appears to be mediated primarily by calcium influx in rat aorta without endothelium

    PubMed Central

    Ok, Seong-Ho; Kwon, Seong-Chun; Kang, Sebin; Choi, Mun-Jeoung

    2014-01-01

    Background Mepivacaine induces contraction or decreased blood flow both in vivo and in vitro. Vasoconstriction is associated with an increase in the intracellular calcium concentration ([Ca2+]i). However, the mechanism responsible for the mepivacaine-evoked [Ca2+]i increase remains to be determined. Therefore, the objective of this in vitro study was to examine the mechanism responsible for the mepivacaine-evoked [Ca2+]i increment in isolated rat aorta. Methods Isometric tension was measured in isolated rat aorta without endothelium. In addition, fura-2 loaded aortic muscle strips were illuminated alternately (48 Hz) at two excitation wavelengths (340 and 380 nm). The ratio of F340 to F380 (F340/F380) was regarded as an amount of [Ca2+]i. We investigated the effects of nifedipine, 2-aminoethoxydiphenylborate (2-APB), gadolinium chloride hexahydrate (Gd3+), low calcium level and Krebs solution without calcium on the mepivacaine-evoked contraction in isolated rat aorta and on the mepivacaine-evoked [Ca2+]i increment in fura-2 loaded aortic strips. We assessed the effect of verapamil on the mepivacaine-evoked [Ca2+]i increment. Results Mepivacaine produced vasoconstriction and increased [Ca2+]i. Nifedipine, 2-APB and low calcium attenuated vasoconstriction and the [Ca2+]i increase evoked by mepivacaine. Verapamil attenuated the mepivacaine-induced [Ca2+]i increment. Calcium-free solution almost abolished mepivacaine-induced contraction and strongly attenuated the mepivacaineinduced [Ca2+]i increase. Gd3+ had no effect on either vasoconstriction or the [Ca2+]i increment evoked by mepivacaine. Conclusions The mepivacaine-evoked [Ca2+]i increment, which contributes to mepivacaine-evoked contraction, appears to be mediated mainly by calcium influx and partially by calcium released from the sarcoplasmic reticulum. PMID:25558341

  11. Asbestos-induced disruption of calcium homeostasis induces endoplasmic reticulum stress in macrophages.

    PubMed

    Ryan, Alan J; Larson-Casey, Jennifer L; He, Chao; Murthy, Shuhba; Carter, A Brent

    2014-11-28

    Although the mechanisms for fibrosis development remain largely unknown, recent evidence indicates that endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) may act as an important fibrotic stimulus in diseased lungs. ER stress is observed in lungs of patients with idiopathic pulmonary fibrosis. In this study we evaluated if ER stress and the UPR was present in macrophages exposed to chrysotile asbestos and if ER stress in macrophages was associated with asbestos-induced pulmonary fibrosis. Macrophages exposed to chrysotile had elevated transcript levels of several ER stress genes. Macrophages loaded with the Ca(2+)-sensitive dye Fura2-AM showed that cytosolic Ca(2+) increased significantly within minutes after chrysotile exposure and remained elevated for a prolonged time. Chrysotile-induced increases in cytosolic Ca(2+) were partially inhibited by either anisomycin, an inhibitor of passive Ca(2+) leak from the ER, or 1,2-bis(2-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), an intracellular Ca(2+) chelator known to deplete ER Ca(2+) stores. Anisomycin inhibited X-box-binding protein 1 (XBP1) mRNA splicing and reduced immunoglobulin-binding protein (BiP) levels, whereas BAPTA-AM increased XBP1 splicing and BiP expression, suggesting that ER calcium depletion may be one factor contributing to ER stress in cells exposed to chrysotile. To evaluate ER stress in vivo, asbestos-exposed mice showed fibrosis development, and alveolar macrophages from fibrotic mice showed increased expression of BiP. Bronchoalveolar macrophages from asbestosis patients showed increased expression of several ER stress genes compared with normal subjects. These findings suggest that alveolar macrophages undergo ER stress, which is associated with fibrosis development.

  12. Cell swelling-induced ATP release is tightly dependent on intracellular calcium elevations

    PubMed Central

    Boudreault, Francis; Grygorczyk, Ryszard

    2004-01-01

    Mechanical stresses release ATP from a variety of cells by a poorly defined mechanism(s). Using custom-designed flow-through chambers, we investigated the kinetics of cell swelling-induced ATP secretion, cell volume and intracellular calcium changes in epithelial A549 and 16HBE14o− cells, and NIH/3T3 fibroblasts. Fifty per cent hypotonic shock triggered transient ATP release from cell confluent monolayers, which consistently peaked at around 1 min 45 s for A549 and NIH/3T3, and at 3 min for 16HBE14o− cells, then declined to baseline within the next 15 min. Whereas the release time course had a similar pattern for the three cell types, the peak rates differed significantly (294 ± 67, 70 ± 22 and 17 ± 2.8 pmol min−1 (106 cells)−1, for A549, 16HBE14o− and NIH/3T3, respectively). The concomitant volume changes of substrate-attached cells were analysed by a 3-dimensional cell shape reconstruction method based on images acquired from two perpendicular directions. The three cell types swelled at a similar rate, reaching maximal expansion in 1 min 45 s, but differed in the duration of the volume plateau and regulatory volume decrease (RVD). These experiments revealed that ATP release does not correlate with either cell volume expansion and the expected activation of stretch-sensitive channels, or with the activation of volume-sensitive, 5-nitro-2-(3-phenylpropylamino) benzoic acid-inhibitable anion channels during RVD. By contrast, ATP release was tightly synchronized, in all three cell types, with cytosolic calcium elevations. Furthermore, loading A549 cells with the calcium chelator BAPTA significantly diminished ATP release (71% inhibition of the peak rate), while the calcium ionophore ionomycin triggered ATP release in the absence of cell swelling. Lowering the temperature to 10°C almost completely abolished A549 cell swelling-induced ATP release (95% inhibition of the peak rate). These results strongly suggest that calcium-dependent exocytosis plays a

  13. Annexin A4 induces platinum resistance in a chloride-and calcium-dependent manner

    PubMed Central

    Morimoto, Akiko; Serada, Satoshi; Enomoto, Takayuki; Kim, Ayako; Matsuzaki, Shinya; Takahashi, Tsuyoshi; Ueda, Yutaka; Yoshino, Kiyoshi; Fujita, Masami; Fujimoto, Minoru; Kimura, Tadashi; Naka, Tetsuji

    2014-01-01

    Platinum resistance has long been a major issue in the treatment of various cancers. We previously reported that enhanced annexin A4 (ANXA4) expression, a Ca2+-regulated phospholipid-binding protein, induces chemoresistance to platinum-based drugs. In this study, we investigated the role of annexin repeats, a conserved structure of all the annexin family, responsible for platinum-resistance as well as the effect of knockdown of ANXA4. ANXA4 knockdown increased sensitivity to platinum-based drugs both in vitro and in vivo. To identify the domain responsible for chemoresistance, ANXA4 deletion mutants were constructed by deleting annexin repeats one by one from the C terminus. Platinum resistance was induced both in vitro and in vivo in cells expressing either full-length ANXA4 or the deletion mutants, containing at least one intact annexin repeat. However, cells expressing the mutant without any calcium-binding sites in the annexin repeated sequence, which is essential for ANXA4 translocation from the cytosol to plasma membrane, failed to acquire platinum resistance. After cisplatin treatment, the intracellular chloride ion concentration, whose channel is partly regulated by ANXA4, significantly increased in the platinum-resistant cells. These findings indicate that the calcium-binding site in the annexin repeat induces chemoresistance to the platinum-based drug by elevating the intracellular chloride concentration. PMID:25277200

  14. Host-specific Nod-factors associated with Medicago truncatula nodule infection differentially induce calcium influx and calcium spiking in root hairs

    PubMed Central

    Morieri, Giulia; Martinez, Eduardo A; Jarynowski, Andrzej; Driguez, Hugues; Morris, Richard; Oldroyd, Giles E D; Downie, J Allan

    2013-01-01

    Rhizobial nodulation (Nod) factors activate both nodule morphogenesis and infection thread development during legume nodulation. Nod factors induce two different calcium responses: intra-nuclear calcium oscillations and a calcium influx at the root hair tip. Calcium oscillations activate nodule development; we wanted to test if the calcium influx is associated with infection. Sinorhizobium meliloti nodL and nodF mutations additively reduce infection of Medicago truncatula. Nod-factors made by the nodL mutant lack an acetyl group; mutation of nodF causes the nitrogen (N)-linked C16:2 acyl chain to be replaced by C18:1. We tested whether these Nod-factors differentially induced calcium influx and calcium spiking. The absence of the NodL-determined acetyl group greatly reduced the induction of calcium influx without affecting calcium spiking. The calcium influx was even further reduced if the N-linked C16:2 acyl group was replaced by C18:1. These additive effects on calcium influx correlate with the additive effects of mutations in nodF and nodL on legume infection. Infection thread development is inhibited by ethylene, which also inhibited Nod-factor-induced calcium influx. We conclude that Nod-factor perception differentially activates the two developmental pathways required for nodulation and that activation of the pathway involving the calcium influx is important for efficient infection. PMID:24015832

  15. Development of a sensitive setup for laser spectroscopy studies of very exotic calcium isotopes

    NASA Astrophysics Data System (ADS)

    Garcia Ruiz, R. F.; Gorges, C.; Bissell, M.; Blaum, K.; Gins, W.; Heylen, H.; Koenig, K.; Kaufmann, S.; Kowalska, M.; Krämer, J.; Lievens, P.; Malbrunot-Ettenauer, S.; Neugart, R.; Neyens, G.; Nörtershäuser, W.; Yordanov, D. T.; Yang, X. F.

    2017-04-01

    An experimental setup for sensitive high-resolution measurements of hyperfine structure spectra of exotic calcium isotopes has been developed and commissioned at the COLLAPS beam line at ISOLDE, CERN. The technique is based on the radioactive detection of decaying isotopes after optical pumping and state selective neutralization (ROC) (Vermeeren et al 1992 Phys. Rev. Lett. 68 1679). The improvements and developments necessary to extend the applicability of the experimental technique to calcium isotopes produced at rates as low as few ions s-1 are discussed. Numerical calculations of laser-ion interaction and ion-beam simulations were explored to obtain the optimum performance of the experimental setup. Among the implemented features are a multi-step optical pumping region for sensitive measurements of isotopes with hyperfine splitting, a high-voltage platform for adequate control of low-energy ion beams and simultaneous β-detection of neutralized and remaining ions. The commissioning of the experimental setup, and the first online results on neutron-rich calcium isotopes are presented.

  16. Inflammation of peripheral tissues and injury to peripheral nerves induce differing effects in the expression of the calcium-sensitive N-arachydonoylethanolamine-synthesizing enzyme and related molecules in rat primary sensory neurons.

    PubMed

    Sousa-Valente, João; Varga, Angelika; Torres-Perez, Jose Vicente; Jenes, Agnes; Wahba, John; Mackie, Ken; Cravatt, Benjamin; Ueda, Natsuo; Tsuboi, Kazuhito; Santha, Peter; Jancso, Gabor; Tailor, Hiren; Avelino, António; Nagy, Istvan

    2017-06-01

    Elevation of intracellular Ca(2+) concentration induces the synthesis of N-arachydonoylethanolamine (anandamide) in a subpopulation of primary sensory neurons. N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) is the only known enzyme that synthesizes anandamide in a Ca(2+) -dependent manner. NAPE-PLD mRNA as well as anandamide's main targets, the excitatory transient receptor potential vanilloid type 1 ion channel (TRPV1), the inhibitory cannabinoid type 1 (CB1) receptor, and the main anandamide-hydrolyzing enzyme fatty acid amide hydrolase (FAAH), are all expressed by subpopulations of nociceptive primary sensory neurons. Thus, NAPE-PLD, TRPV1, the CB1 receptor, and FAAH could form an autocrine signaling system that could shape the activity of a major subpopulation of nociceptive primary sensory neurons, contributing to the development of pain. Although the expression patterns of TRPV1, the CB1 receptor, and FAAH have been comprehensively elucidated, little is known about NAPE-PLD expression in primary sensory neurons under physiological and pathological conditions. This study shows that NAPE-PLD is expressed by about one-third of primary sensory neurons, the overwhelming majority of which also express nociceptive markers as well as the CB1 receptor, TRPV1, and FAAH. Inflammation of peripheral tissues and injury to peripheral nerves induce differing but concerted changes in the expression pattern of NAPE-PLD, the CB1 receptor, TRPV1, and FAAH. Together these data indicate the existence of the anatomical basis for an autocrine signaling system in a major proportion of nociceptive primary sensory neurons and that alterations in that autocrine signaling by peripheral pathologies could contribute to the development of both inflammatory and neuropathic pain.

  17. Real-time imaging of neurons retrogradely and anterogradely labelled with calcium-sensitive dyes.

    PubMed

    O'Donovan, M J; Ho, S; Sholomenko, G; Yee, W

    1993-02-01

    Membrane-impermeant calcium indicator dyes were used to retrogradely label dorsal root ganglia, spinal motoneurons and interneurons in the spinal cord of the chick embryo. The dyes were also used to label anterogradely primary afferent axons in the spinal cord and synaptic endings in the ciliary ganglion. Labelled neurons were imaged using digital videomicroscopy. Motoneurons and dorsal root ganglion cells exhibited a frequency-dependent change in fluorescence during antidromic stimulation. Single antidromic stimuli resulted in fluorescence transients that could be resolved in individual cells in real time. In addition, fluorescence changes could be recorded in motoneurons during episodes of bursting generated by rhythmic synaptic inputs from premotor networks. Stimulus-induced fluorescence signals were also detected in axons and synaptic endings labelled anterogradely. Optical signals were largely abolished in the absence of extracellular calcium. The results show that calcium changes can now be measured in identified populations of neurons and presynaptic terminals. The strong dependence of these signals on impulse activity suggests that the technique will be useful for monitoring the activity of identified neuronal populations. The calcium-dependent fluorescence signal probably results from cytosolic dye derived from diffusion which may limit the technique to situations in which the dye can be applied close (< 1 cm) to cell bodies.

  18. Noise-induced sensitization of human brain

    NASA Astrophysics Data System (ADS)

    Yamamoto, Yoshiharu; Hidaka, Ichiro; Nozaki, Daichi; Iso-o, Noriko; Soma, Rika; Kwak, Shin

    2002-11-01

    In the past decade, it has been recognized that noise can enhance the response of nonlinear systems to weak signals, via a mechanism known as stochastic resonance (SR). Particularly, the concept of SR has generated considerable interest in sensory biology, because it has been shown in several experimental studies that noise can assist neural systems in detecting weak signals which could not be detected in its absence. Recently, we have shown a similar type of noise-induced sensitization of human brain; externally added noise to the brain stem baroreflex centers sensitized their responses in maintaining adequate blood perfusion to the brain itself. Furthermore, the addition of noise has also shown to be useful in compensating for dysfunctions of the baroreflex centers in certain neurological diseases. It is concluded that the statistical physics concept of SR could be useful in sensitizing human brain in health and disease.

  19. Reductions in Calcium Uptake Induced in Rat Brain Synaptosomes by Ionizing Radiation

    DTIC Science & Technology

    1991-01-01

    was also reduced by radiation exposure. Nimodipine binding to dihydropyridine (DHP) L-type calcium uptake after irradiation in wh31e-brain, cortical...TERIz. and L.. GANDIA. Dihydropyridine Bay K 8644 aetivates chro- 2-32-286 (1950). mall’in cell calcium channels.Nature 309. 69-71 (1984). 33. E. L...TRIGGLE. Bay K 8644. a I .4-dihv- of dihydropyridine -sensitive calcium channels in rat brain synapto- dropyridinc Ca> channel activator: Dissociation

  20. Fluorescence Based Characterization of Calcium Sensitizer Action on the Troponin Complex.

    PubMed

    Schlecht, William; Li, King-Lun; Hu, Dehong; Dong, Wenji

    2016-02-01

    Calcium sensitizers enhance the transduction of the Ca(2+) signal into force within the heart and have found use in treating heart failure. However the mechanisms of action for most Ca(2+) sensitizers remain unclear. To address this issue an efficient fluorescence based approach to Ca(2+) sensitizer screening was developed which monitors cardiac troponin C's (cTnC's) hydrophobic cleft. This approach was tested on four common Ca(2+) -sensitizers, EMD 57033, levosimendan, bepridil and pimobendan with the aim of elucidating the mechanisms of action for each as well as proving the efficacy of the new screening method. Ca(2+) -titration experiments were employed to determine the effect on Ca(2+) sensitivity and cooperativity of cTnC opening, while stopped flow experiments were used to investigate the impact on cTnC relaxation kinetics. Bepridil was shown to increase the sensitivity of cTnC for Ca(2+) under all reconstitution conditions, sensitization by the other drugs was context dependent. Levosimendan and pimobendan reduced the rate of cTnC closing consistent with a stabilization of cTnC's open conformation while bepridil increased the rate of relaxation. Experiments were also run on samples containing cTnT(T204E), a known Ca(2+) -desensitizing phosphorylation mimic. Levosimendan, bepridil, and pimobendan were found to elevate the Ca(2+) -sensitivity of cTnT(T204E) containing samples in this context.

  1. Temperature dependence of calcium-induced fusion of sonicated phosphatidylserine vesicles.

    PubMed Central

    Sun, S T; Day, E P; Ho, J T

    1978-01-01

    We have measured the temperature dependence calcium-induced fusion of sonicated phosphatidylserine vesicles. The vesicles were incubated in the presence of calcium at a specified temperature until the resulting aggregation or fusion process had gone to completion. EDTA was then added and the resulting final size of the vesicle population was measured by using dynamic light scattering. This final size was plotted against incubation temperature to show the temperature dependence of calcium-induced fusion. This curve has a peak near 11 degrees C which may be associated with the phase transition of the sonicated phosphatidylserine vesicles in the presence of calcium prior to the aggregation or fusion process. PMID:279918

  2. Temperature sensitivity of calcium binding for parvalbumins from Antarctic and temperate zone teleost fishes.

    PubMed

    Erickson, Jeffrey R; Sidell, Bruce D; Moerland, Timothy S

    2005-02-01

    Parvalbumin (PV) is a soluble calcium-binding protein that is especially abundant in fast-twitch muscles of fish and other lower vertebrates. Despite its prevalence in ectothermic taxa, few data address the effects of temperature on PV binding function. In this study, calcium dissociation constants (KD) were measured as a function of temperature (0-25 degrees C) for PV from two Antarctic (Gobionotothen gibberifrons and Chaenocephalus aceratus) and two temperate zone fish species (Cyprinus carpio and Micropterus salmoides). Measurements by fluorometric competitive binding assay show that KD values for PVs from the Antarctic species were significantly higher at all assay temperatures and were less sensitive to temperature relative to carp and bass. However, estimates of KD are fundamentally similar for PVs from the Antarctic and temperate zone species when examined at their native physiological temperature. Variation in pH and ionic strength within a physiologically relevant range had only modest effects on KD. Thermodynamics of calcium binding to PV from G. gibberifrons and C. carpio was measured by isothermal microcalorimetry. When measured at 15 degrees C, the Gibbs free energy change (deltaG) was significantly greater for calcium binding to PV from G. gibberifrons than from carp (-43.4+/-1.5 kJ mol(-1) and -46.6+/-3.0 kJ mol(-1), respectively), and the relative contribution of entropy to deltaG for calcium binding to PV from the Antarctic species was about twice that of carp (deltaS=16.0+/-0.8 J degrees C(-1) mol(-1) for G. gibberifrons; deltaS=7.5+/-0.8 J degrees C(-1) mol(-1) for C. carpio).

  3. Calcium sensitizers isolated from the edible pine mushroom, Tricholoma matsutake (S. Ito & Imai) Sing.

    PubMed

    Hou, Yunlong; Sun, Shichao; Wu, Lijun; Wang, Xiaodan; Li, Ting; Zhang, Minyun; Wang, Jianwei; Wang, Libo

    2013-01-01

    Three lactam compounds were isolated from the fruiting body of Tricholoma matsutake (S. Ito & Imai) Sing., an edible mushroom, and their structures were identified as cyclo-S-proline-R-leucine (1), hexahydro-2H-azepin-2-one (2), and butyl 5-oxo-2-pyrrolidine carboxylate (3) by chemical, physicochemical, and spectral evidence. In in vitro screening tests, compounds 1 and 2 acted as calcium sensitizers in ventricular cells from rat. Further studies on compounds 1 and 2 in ex vivo isolated right atria showed positive inotropic effects without disturbing the spontaneous beating rate. The inotropic effect of compounds 1 and 2 could be greatly abolished by pretreating the myocardium in Ca(2+)-free solution. These findings indicate that compounds 1 and 2 can significantly increase the calcium ion concentration ([Ca2+]i) in myocytes, which is greatly dependent on the influx of extracellular Ca2+.

  4. Ethanol suppresses carbamylcholine-induced intracellular calcium oscillation in mouse pancreatic acinar cells.

    PubMed

    Yoon, Mi Na; Kim, Min Jae; Koong, Hwa Soo; Kim, Dong Kwan; Kim, Se Hoon; Park, Hyung Seo

    2017-09-01

    Oscillation of intracellular calcium levels is closely linked to initiating secretion of digestive enzymes from pancreatic acinar cells. Excessive alcohol consumption is known to relate to a variety of disorders in the digestive system, including the exocrine pancreas. In this study, we have investigated the role and mechanism of ethanol on carbamylcholine (CCh)-induced intracellular calcium oscillation in murine pancreatic acinar cells. Ethanol at concentrations of 30 and 100 mM reversibly suppressed CCh-induced Ca(2+) oscillation in a dose-dependent manner. Pretreatment of ethanol has no effect on the store-operated calcium entry induced by 10 μM of CCh. Ethanol significantly reduced the initial calcium peak induced by low concentrations of CCh and therefore, the CCh-induced dose-response curve of the initial calcium peak was shifted to the right by ethanol pretreatment. Furthermore, ethanol significantly dose-dependently reduced inositol 1,4,5-trisphosphate-induced calcium release from the internal stores in permeabilized acinar cells. These results provide evidence that excessive alcohol intake could impair cytosolic calcium oscillation through inhibiting calcium release from intracellular stores in mouse pancreatic acinar cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Fluoride induced endoplasmic reticulum stress and calcium overload in ameloblasts.

    PubMed

    Zhang, Ying; Zhang, KaiQiang; Ma, Lin; Gu, HeFeng; Li, Jian; Lei, Shuang

    2016-09-01

    The aim of the study was to evaluate the involvement of endoplasmic reticulum stress and intracellular calcium overload on the development of dental fluorosis. We cultured and exposed rat ameloblast HAT-7 cells to various concentrations of fluoride and measured apoptosis with flow cytometry and intracellular Ca2+ changes using confocal microscopy, investigated the protein levels of GRP78, calreticulin, XBP1 and CHOP by western blotting, and their transcriptional levels with RT-PCR. We also created an in vivo model of dental fluorosis by exposing animals to various concentrations of fluoride. Subsequently, thin dental tissue slices were analyzed with H&E staining, immunohistochemical staining, and transmission electron microscopy, TUNEL assay was also performed on dental tissue slices for assessment of apoptosis. High fluoride concentration was associated with decreased ameloblast proliferation, elevated ameloblast apoptosis, and increased intracellular Ca2+ in vitro. The translation and transcription of the proteins associated with endoplasmic reticulum stress were significantly elevated with high concentrations of fluoride. Based on immunohistochemical staining, these proteins were also highly expressed in animals exposed to high fluoride concentrations. Histologically, we found significant fluorosis-like changes in tissues from animals exposed to high fluoride concentrations. Transmission electron microscopy cytology indicated significant apoptotic changes in tissues exposed to high concentrations of fluoride. These results indicate that exposure to high levels of fluoride led to endoplasmic reticulum stress which induced apoptosis in cultured ameloblasts and in vivo rat model, suggesting an important role of calcium overload and endoplasmic reticulum stress triggered by high concentrations of fluoride in the development of dental fluorosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Dynamic and static calcium gradients inside large snail (Helix aspersa) neurones detected with calcium-sensitive microelectrodes.

    PubMed

    Thomas, Roger C; Postma, Marten

    2007-04-01

    We have used quartz Ca2+-sensitive microelectrodes (CASMs) in large voltage-clamped snail neurones to investigate the inward spread of Ca2+ after a brief depolarisation. Both steady state and [Ca2+]i transients changed with depth of penetration. When the CASM tip was within 20 microm of the far side of the cell the [Ca2+]i transient time to peak was 4.4+/-0.5s, rising to 14.7+/-0.7s at a distance of 80 microm. We estimate that the Ca2+ transients travelled centripetally at an average speed of 6 microm2 s(-1) and decreased in size by half over a distance of about 45 microm. Cyclopiazonic acid had little effect on the size and time to peak of Ca2+ transients but slowed their recovery significantly. This suggests that the endoplasmic reticulum curtails rather than reinforces the transients. Injecting the calcium buffer BAPTA made the Ca2+ transients more uniform in size and increased their times to peak and rates of recovery near the membrane. We have developed a computational model for the transients, which includes diffusion, uptake and Ca2+ extrusion. Good fits were obtained with a rather large apparent diffusion coefficient of about 90+/-20 microm2 s(-1). This may assist fast recovery by extrusion.

  7. CXCL12 induces hepatic stellate cell contraction through a calcium-independent pathway.

    PubMed

    Saiman, Yedidya; Agarwal, Ritu; Hickman, DaShawn A; Fausther, Michel; El-Shamy, Ahmed; Dranoff, Jonathan A; Friedman, Scott L; Bansal, Meena B

    2013-09-01

    Liver fibrosis, with subsequent development of cirrhosis and ultimately portal hypertension, results in the death of patients with end-stage liver disease if liver transplantation is not performed. Hepatic stellate cells (HSCs), central mediators of liver fibrosis, resemble tissue pericytes and regulate intrahepatic blood flow by modulating pericapillary resistance. Therefore, HSCs can contribute to portal hypertension in patients with chronic liver disease (CLD). We have previously demonstrated that activated HSCs express functional chemokine receptor, CXCR4, and that receptor engagement by its ligand, CXCL12, which is increased in patients with CLD, leads to further stellate cell activation in a CXCR4-specific manner. We therefore hypothesized that CXCL12 promotes HSC contraction in a CXCR4-dependent manner. Stimulation of HSCs on collagen gel lattices with CXCL12 led to gel contraction and myosin light chain (MLC) phosphorylation, which was blocked by addition of AMD3100, a CXCR4 small molecule inhibitor. These effects were further mediated by the Rho kinase pathway since both Rho kinase knockdown or Y-27632, a Rho kinase inhibitor, blocked CXCL12 induced phosphorylation of MLC and gel contraction. BAPTA-AM, a calcium chelator, had no effect, indicating that this pathway is calcium sensitive but not calcium dependent. In conclusion, CXCL12 promotes stellate cell contractility in a predominantly calcium-independent fashion. Our data demonstrates a novel role of CXCL12 in stellate cell contraction and the availability of small molecule inhibitors of the CXCL12/CXCR4 axis justifies further investigation into its potential as therapeutic target for portal hypertension.

  8. Peptidoglycan Induces the Production of Interleukin-8 via Calcium Signaling in Human Gingival Epithelium

    PubMed Central

    Son, Aran; Hong, Jeong Hee

    2015-01-01

    The etiology of periodontal disease is multifactorial. Exogenous stimuli such as bacterial pathogens can interact with toll-like receptors to activate intracellular calcium signaling in gingival epithelium and other tissues. The triggering of calcium signaling induces the secretion of pro-inflammatory cytokines such as interleukin-8 as part of the inflammatory response; however, the exact mechanism of calcium signaling induced by bacterial toxins when gingival epithelial cells are exposed to pathogens is unclear. Here, we investigate calcium signaling induced by bacteria and expression of inflammatory cytokines in human gingival epithelial cells. We found that peptidoglycan, a constituent of gram-positive bacteria and an agonist of toll-like receptor 2, increases intracellular calcium in a concentration-dependent manner. Peptidoglycan-induced calcium signaling was abolished by treatment with blockers of phospholipase C (U73122), inositol 1,4,5-trisphosphate receptors, indicating the release of calcium from intracellular calcium stores. Peptidoglycan-mediated interleukin-8 expression was blocked by U73122 and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester). Moreover, interleukin-8 expression was induced by thapsigargin, a selective inhibitor of the sarco/endoplasmic reticulum calcium ATPase, when thapsigargin was treated alone or co-treated with peptidoglycan. These results suggest that the gram-positive bacterial toxin peptidoglycan induces calcium signaling via the phospholipase C/inositol 1,4,5-trisphosphate pathway, and that increased interleukin-8 expression is mediated by intracellular calcium levels in human gingival epithelial cells. PMID:25605997

  9. Differential calcium sensitivity in NaV 1.5 mixed syndrome mutants.

    PubMed

    Abdelsayed, Mena; Baruteau, Alban-Elouen; Gibbs, Karen; Sanatani, Shubhayan; Krahn, Andrew D; Probst, Vincent; Ruben, Peter C

    2017-09-15

    SCN5a mutations may express gain-of-function (Long QT Syndrome-3), loss-of-function (Brugada Syndrome 1) or both (mixed syndromes), depending on the mutation and environmental triggers. One such trigger may be an increase in cytosolic calcium, accompanying exercise. Many mixed syndromes mutants, including ∆KPQ, E1784K, 1795insD and Q1909R, are found in calcium-sensitive regions. Elevated cytosolic calcium attenuates gain-of-function properties in ∆KPQ, 1795insD and Q1909R, but not in E1784K. By contrast, elevated cytosolic calcium further exacerbates gain-of-function in E1784K by destabilizing slow inactivation. Action potential modelling, using a modified O'Hara Rudy model, suggests that elevated heart rate rescues action potential duration in ∆KPQ, 1795insD and Q1909R, but not in E1784K. Action potential simulations suggest that E1784K carriers have an increased intracellular sodium-to-calcium ratio under bradycardia and tachycardia conditions. Elevated cytosolic calcium, which is common during high heart rates, ameliorates or exacerbates the mixed syndrome phenotype depending on the genetic signature. Inherited arrhythmias may arise from mutations in the gene for SCN5a, which encodes the cardiac voltage-gated sodium channel, NaV 1.5. Mutants in NaV 1.5 result in Brugada Syndrome (BrS1), Long-QT Syndrome (LQT3) or mixed syndromes (an overlap of BrS1/LQT3). Exercise is a potential arrhythmogenic trigger in mixed syndromes. We aimed to determine the effects of elevated cytosolic calcium, which is common during exercise, in mixed syndrome NaV 1.5 mutants. We used whole-cell patch clamp to assess the biophysical properties of NaV 1.5 wild-type (WT), ∆KPQ, E1784K, 1795insD and Q1909R mutants in human embryonic kidney 293 cells transiently transfected with the NaV 1.5 α subunit (WT or mutants), β1 subunit and enhanced green fluorescent protein. Voltage-dependence and kinetics were measured at cytosolic calcium levels of approximately 0, 500 and 2500 nm. In

  10. Nanoelectropulse intracellular perturbation and electropermeabilization technology: phospholipid translocation, calcium bursts, chromatin rearrangement, cardiomyocyte activation, and tumor cell sensitivity.

    PubMed

    Vernier, P Thomas; Sun, Yinghua; Wang, Jingjing; Thu, Mya Mya; Garon, Edward; Valderrabano, Miguel; Marcu, Laura; Koeffler, H Phillip; Gundersen, Martin A

    2005-01-01

    Nanosecond, megavolt-per-meter pulsed electric fields scramble the asymmetric arrangement of phospholipids in the plasma membrane, release intracellular calcium, trigger cardiomyocyte activity, and induce apoptosis in mammalian cancer cells, without the permeabilizing effects associated with longer, lower-field pulses. Dose dependencies with respect to pulse width, amplitude, and repetition rate, and total pulse count are observed for all of these phenomena. Sensitivities vary among cell types; cells of lymphoid origin growing in suspension are more susceptible to nanoelectropulse exposure than solid tumor lines. Simple electrical models of the cell are useful for first-order explanations, but more sophisticated treatments will be required for analysis and prediction at both biomolecular and tissue levels.

  11. CCK-4-induced calcium mobilization in T cells is enhanced in panic disorder.

    PubMed

    Akiyoshi, J; Moriyama, T; Isogawa, K; Miyamoto, M; Sasaki, I; Kuga, K; Yamamoto, H; Yamada, K; Fujii, I

    1996-04-01

    We investigated the effects of brain cholecystokinin (CCK) receptors on the intracellular calcium concentration and protein kinase C in human T cells. CCK-4 produced a transient increase in calcium in the absence of extracellular calcium. CCK-B agonists stimulated calcium mobilization in a dose-dependent manner in T cells. CCK-B antagonists suppressed CCK-4-induced calcium mobilization more potently than CCK-A antagonist. The recovery of desensitization of the CCK-4-induced response was delayed by phosphoserine/phosphothreonine phosphatase inhibitor, calyculin A. The responsiveness to CCK-4 was also reduced by phorbol 12,13-dibutyrate (PDBu), and this effect of PDBu was abolished completely by preincubation with staurosporine. CCK-4-induced calcium mobilization was too small to attribute the desensitization to the protein kinase C transduction pathway. T cells from patients with untreated panic disorder exhibited significantly higher cholecystokinin-4-induced calcium mobilization than those from healthy controls or patients with treated panic disorder. These results suggest that cholecystokinin-B receptor function in T cells of patients with panic disorder is enhanced. Cholecystokinin-4-induced calcium mobilization in T cells may be state dependent and useful as a biological marker of panic disorder.

  12. Carpal tunnel syndrome induced by two types of calcium deposition.

    PubMed

    Ikawa, H; Hashizume, H; Inoue, H

    1997-12-01

    Two rare cases of carpal tunnel syndrome caused by calcification in the carpal tunnel are reported. One case involved a tumorous calcification consisting of basic calcium phosphate, and the other involved a diffuse calcification consisting of a mixture of calcium pyrophosphate dihydrate and basic calcium phosphate. These cases suggest that the shape of carpal tunnel calcifications is influenced by the nature of calcifying substance itself, i.e., whether it is heterogenous or homogenous.

  13. Ultra-sensitive Flow Injection Analysis (FIA) determination of calcium in ice cores at ppt level.

    PubMed

    Traversi, R; Becagli, S; Castellano, E; Maggi, V; Morganti, A; Severi, M; Udisti, R

    2007-07-02

    A Flow Injection Analysis (FIA) spectrofluorimetric method for calcium determination in ice cores was optimised in order to achieve better analytical performances which would make it suitable for reliable calcium measurements at ppt level. The method here optimised is based on the formation of a fluorescent compound between Ca and Quin-2 in buffered environment. A careful evaluation of operative parameters (reagent concentration, buffer composition and concentration, pH), influence of interfering species possibly present in real samples and potential favourable effect of surfactant addition was carried out. The obtained detection limit is around 15 ppt, which is one order of magnitude lower than the most sensitive Flow Analysis method for Ca determination currently available in literature and reproducibility is better than 4% for Ca concentrations of 0.2 ppb. The method was validated through measurements performed in parallel with Ion Chromatography on 200 samples from an alpine ice core (Lys Glacier) revealing an excellent fit between the two chemical series. Calcium stratigraphy in Lys ice core was discussed in terms of seasonal pattern and occurrence of Saharan dust events.

  14. Antagonism of T-type calcium channels inhibits high-fat diet-induced weight gain in mice.

    PubMed

    Uebele, Victor N; Gotter, Anthony L; Nuss, Cindy E; Kraus, Richard L; Doran, Scott M; Garson, Susan L; Reiss, Duane R; Li, Yuxing; Barrow, James C; Reger, Thomas S; Yang, Zhi-Qiang; Ballard, Jeanine E; Tang, Cuyue; Metzger, Joseph M; Wang, Sheng-Ping; Koblan, Kenneth S; Renger, John J

    2009-06-01

    The epidemics of obesity and metabolic disorders have well-recognized health and economic burdens. Pharmacologic treatments for these diseases remain unsatisfactory with respect to both efficacy and side-effect profiles. Here, we have identified a potential central role for T-type calcium channels in regulating body weight maintenance and sleep. Previously, it was shown that mice lacking CaV3.1 T-type calcium channels have altered sleep/wake activity. We found that these mice were also resistant to high-fat diet-induced weight gain, without changes in food intake or sensitivity to high-fat diet-induced disruptions of diurnal rhythm. Administration of a potent and selective antagonist of T-type calcium channels, TTA-A2, to normal-weight animals prior to the inactive phase acutely increased sleep, decreased body core temperature, and prevented high-fat diet-induced weight gain. Administration of TTA-A2 to obese rodents reduced body weight and fat mass while concurrently increasing lean muscle mass. These effects likely result from better alignment of diurnal feeding patterns with daily changes in circadian physiology and potentially an increased metabolic rate during the active phase. Together, these studies reveal what we believe to be a previously unknown role for T-type calcium channels in the regulation of sleep and weight maintenance and suggest the potential for a novel therapeutic approach to treating obesity.

  15. Effects of dietary calcium on Helicobacter pylori-induced gastritis in Mongolian gerbils.

    PubMed

    Iimuro, Masaki; Nakamura, Shiro; Arakawa, Tetsuo; Wakabayashi, Keiji; Mutoh, Michihiro

    2013-09-01

    Helicobacter pylori (Hp) infection causes gastritis and is considered a gastric cancer risk factor. We have previously reported that codfish meal markedly enhanced Hp-induced gastritis in Mongolian gerbils. In the present study, we sought the responsible components in codfish meal. Codfish were divided into three parts (meat, viscera and 'other parts', including bone), and administered to Hp-infected gerbils. Subsequently, cod bone, sardine bone and prawn shell were tested, along with major calcium components, hydroxyapatite and calcium carbonate, in bone and shell, respectively. 'Other parts' and cod bone enhanced Hp-induced gastritis, as was observed for whole codfish. Similarly, sardine bone and prawn shell, as well as 0.22-0.88% hydroxyapatite and calcium carbonate, enhanced gastritis. In contrast, administration of a higher dose of the calcium compounds exerted protective effects. Intake of calcium compounds may contribute to enhancement of Hp-induced gastritis.

  16. Intracellular Calcium Spikes in Rat Suprachiasmatic Nucleus Neurons Induced by BAPTA-Based Calcium Dyes

    PubMed Central

    Hong, Jin Hee; Min, Cheol Hong; Jeong, Byeongha; Kojiya, Tomoyoshi; Morioka, Eri; Nagai, Takeharu; Ikeda, Masayuki; Lee, Kyoung J.

    2010-01-01

    Background Circadian rhythms in spontaneous action potential (AP) firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN). Also reported is the existence of “Ca2+ spikes” (i.e., [Ca2+]c transients having a bandwidth of 10∼100 seconds) in SCN neurons, but it is unclear if these SCN Ca2+ spikes are related to the slow circadian rhythms. Methodology/Principal Findings We addressed this issue based on a Ca2+ indicator dye (fluo-4) and a protein Ca2+ sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca2+ spikes in 18% of rat SCN cells in acute brain slices, but the Ca2+ spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse) SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca2+ spike was barely observed (<3%). When fluo-4 AM or BAPTA-AM was loaded in addition to the cameleon-expressing SCN cultures, however, the number of cells exhibiting Ca2+ spikes was increased to 13∼14%. Conclusions/Significance Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca2+ spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca2+ spiking activity is caused by the Ca2+ chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca2+]c in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca2+ spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca2+ spikes in the function of SCN. PMID:20224788

  17. Intracellular calcium spikes in rat suprachiasmatic nucleus neurons induced by BAPTA-based calcium dyes.

    PubMed

    Hong, Jin Hee; Min, Cheol Hong; Jeong, Byeongha; Kojiya, Tomoyoshi; Morioka, Eri; Nagai, Takeharu; Ikeda, Masayuki; Lee, Kyoung J

    2010-03-10

    Circadian rhythms in spontaneous action potential (AP) firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN). Also reported is the existence of "Ca(2+) spikes" (i.e., [Ca(2+)](c) transients having a bandwidth of 10 approximately 100 seconds) in SCN neurons, but it is unclear if these SCN Ca(2+) spikes are related to the slow circadian rhythms. We addressed this issue based on a Ca(2+) indicator dye (fluo-4) and a protein Ca(2+) sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca(2+) spikes in 18% of rat SCN cells in acute brain slices, but the Ca(2+) spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse) SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca(2+) spike was barely observed (<3%). When fluo-4 AM or BAPTA-AM was loaded in addition to the cameleon-expressing SCN cultures, however, the number of cells exhibiting Ca(2+) spikes was increased to 13 approximately 14%. Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca(2+) spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca(2+) spiking activity is caused by the Ca(2+) chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca(2+)](c) in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca(2+) spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca(2+) spikes in the function of SCN.

  18. Differential oxygen sensitivity of calcium channels in rabbit smooth muscle cells of conduit and resistance pulmonary arteries.

    PubMed Central

    Franco-Obregón, A; López-Barneo, J

    1996-01-01

    1. Calcium currents were recorded from smooth muscle cells dispersed from conduit and resistance rabbit pulmonary arteries. We tested the hypothesis that Ca2+ channel activity was regulated by environmental O2 tension. 2. Conduit (proximal) and resistance (distal) myocytes differ in their Ca2+ channel density and responses to low PO2. Ca2+ current density in distal myocytes (20.7 +/- 7.4 pA pF-1, n = 10) is almost twice the value in proximal myocytes (12.6 +/- 5.5 pA pF-1, n = 39). In proximal myocytes, the predominant response to reductions in PO2 is inhibition of the calcium current (n = 12) at membrane potentials below 0 mV, whereas potentiation of current amplitude is observed in distal myocytes (n = 24). 3. Hypoxia also produces opposite shifts in the conductance-voltage relationships along the voltage axis. The average displacements induced by low PO2 are +5.05 +/- 2.98 mV (n = 5) in proximal myocytes and -6.06 +/- 2.45 (n = 10) in distal myocytes. 4. These findings demonstrate longitudinal differences in Ca2+ channel density and O2 sensitivity in myocytes along the pulmonary arterial tree. These results may help to understand the differential reactivity to hypoxia of the pulmonary vasculature: vasodilatation in conduit arteries and vasoconstriction in resistance vessels. Images Figure 4 PMID:8866874

  19. Reductions in calcium uptake induced in rat brain synaptosomes by ionizing radiation

    SciTech Connect

    Kandasamy, S.B.; Howerton, T.C.; Hunt, W.A. )

    1991-02-01

    Gamma irradiation (60Co) reduced KCl-stimulated voltage-dependent 45Ca2+ uptake in whole-brain, cortical, and striatal synaptosomes. The time course (3, 10, 30, and 60 s) of calcium uptake by irradiated (3 Gy) and nonirradiated synaptosomes, as well as the effect of KCl (15-65 mM), was measured in whole-brain synaptosomes. The fastest and highest rate of depolarization-dependent calcium uptake occurred at 3 s with 65 mM KCl. Irradiation reduced calcium uptake at all incubation times and KCl concentrations. Bay K 8644 enhancement of KCl-stimulated calcium influx was also reduced by radiation exposure. Nimodipine binding to dihydropyridine (DHP) L-type calcium channel receptors was not altered following radiation exposure. These results demonstrate an inhibitory effect of ionizing radiation on the voltage-sensitive calcium channels in rat brain synaptosomes that are not mediated by DHP receptors.

  20. Pressure induced reactions amongst calcium aluminate hydrate phases

    SciTech Connect

    Moon, Ju-hyuk; Oh, Jae Eun; Balonis, Magdalena; Glasser, Fredrik P.; Clark, Simon M.; Monteiro, Paulo J.M.

    2011-06-15

    The compressibilities of two AFm phases (straetlingite and calcium hemicarboaluminate hydrate) and hydrogarnet were obtained up to 5 GPa by using synchrotron high-pressure X-ray powder diffraction with a diamond anvil cell. The AFm phases show abrupt volume contraction regardless of the molecular size of the pressure-transmitting media. This volume discontinuity could be associated to a structural transition or to the movement of the weakly bound interlayer water molecules in the AFm structure. The experimental results seem to indicate that the pressure-induced dehydration is the dominant mechanism especially with hygroscopic pressure medium. The Birch-Murnaghan equation of state was used to compute the bulk modulus of the minerals. Due to the discontinuity in the pressure-volume diagram, a two stage bulk modulus of each AFm phase was calculated. The abnormal volume compressibility for the AFm phases caused a significant change to their bulk modulus. The reliability of this experiment is verified by comparing the bulk modulus of hydrogarnet with previous studies.

  1. Correlation between oxidative stress and alteration of intracellular calcium handling in isoproterenol-induced myocardial infarction.

    PubMed

    Díaz-Muñoz, Mauricio; Alvarez-Pérez, Marco Antonio; Yáñez, Lucía; Vidrio, Susana; Martínez, Lidia; Rosas, Gisele; Yáñez, Mario; Ramírez, Sotero; de Sánchez, Victoria Chagoya

    2006-09-01

    Myocardial Ca(2+) overload and oxidative stress are well documented effects associated to isoproterenol (ISO)-induced myocardial necrosis, but information correlating these two issues is scarce. Using an ISO-induced myocardial infarction model, 3 stages of myocardial damage were defined: pre-infarction (0-12 h), infarction (12-24 h) and post-infarction (24-96 h). Alterations in Ca(2+) homeostasis and oxidative stress were studied in mitochondria, sarcoplasmic reticulum and plasmalemma by measuring the Ca(2+) content, the activity of Ca(2+) handling proteins, and by quantifying TBARs, nitric oxide (NO) and oxidative protein damage (changes in carbonyl and thiol groups). Free radicals generated system, antioxidant enzymes and oxidative stress (GSH/GSSG ratio) were also monitored at different times of ISO-induced cardiotoxicity. The Ca(2+) overload induced by ISO was counterbalanced by a diminution in the ryanodine receptor activity and the Na(+)-Ca(+2) exchanger as well as by the increase in both calcium ATPases activities (vanadate- and thapsigargine-sensitive) and mitochondrial Ca(2+) uptake during pre-infarction and infarction stages. Pro-oxidative reactions and antioxidant defences during the 3 stages of cardiotoxicity were observed, with maximal oxidative stress during the infarction. Significant correlations were found among pro-oxidative reactions with plasmalemma and sarcoplasmic reticulum Ca(2+) ATPases, and ryanodine receptor activities at the onset and development of ISO-induced infarction. These findings could be helpful in the design of antioxidant therapies in this pathology.

  2. Calcium-sensitive and insensitive transient outward current in rabbit ventricular myocytes.

    PubMed Central

    Hiraoka, M; Kawano, S

    1989-01-01

    1. A suction pipette whole-cell voltage-clamp technique was used to record membrane currents and potentials of isolated ventricular myocytes from rabbit hearts. 2. Transient outward current (Ito) was activated by voltage steps positive to -20 mV, increasing in amplitude with further depolarization to reach a maximum around +70 mV. The current attained its peak within 10 ms and then it inactivated for 100-200 ms. 3. A large portion of Ito still remained after the calcium current (ICa) was blocked when depolarizing pulses were applied at a frequency of 0.1 Hz or less. Therefore, this current component is referred to as calcium-insensitive Ito or It. 4. It showed voltage- and time-dependent inactivation similar to that observed in Purkinje fibres and other cardiac preparations. 5. The reversal potential of It depended on external K+ concentration, [K+]o, with a slope of 32 mV per 10-fold change in the presence of a normal [Na+]o (143 mM), while the slope was 48 mV per 10-fold change in low [Na+]o (1.0 mM). 6. It was completely inhibited by 2-4 mM-4-aminopyridine. Ito in the presence of ICa was also partially blocked by 4-aminopyridine and the remainder was abolished by 5 mM-caffeine. 7. The calcium-insensitive and caffeine-sensitive Ito differed in their decay rates as well as in their recovery time courses. The former was predominantly available at a slow pulsing rate, while the latter increased its amplitude with high-frequency depolarization. 8. The caffeine-sensitive Ito was inhibited by a blockade of ICa, by replacing Ca2+ with Sr2+, by external application of ryanodine and by internal application of EGTA. This indicates that the current is calcium-sensitive and is dependent on increased myoplasmic Ca2+ through Ca2+ influx via the sarcolemma and Ca2+ release from the sarcoplasmic reticulum. The current is therefore designated as IK, Ca. 9. The physiological functions of IK, Ca and It are indicated by their contribution to ventricular repolarization at fast and

  3. Calcium-dependent glutamate release during neuronal development and synaptogenesis: different involvement of omega-agatoxin IVA- and omega-conotoxin GVIA-sensitive channels.

    PubMed Central

    Verderio, C; Coco, S; Fumagalli, G; Matteoli, M

    1995-01-01

    Hippocampal neurons maintained in primary culture recycle synaptic vesicles and express functional glutamate receptors since early stages of neuronal development. By analyzing glutamate-induced cytosolic calcium changes to sense presynaptically released neurotransmitter, we demonstrate that the ability of neurons to release glutamate in the extracellular space is temporally coincident with the property of synaptic vesicles to undergo exocytotic-endocytotic recycling. Neuronal differentiation and maturation of synaptic contacts coincide with a change in the subtype of calcium channels primarily involved in controlling neurosecretion. Whereas omega-agatoxin IVA-sensitive channels play a role in controlling neurotransmitter secretion at all stages of neuronal differentiation, omega-conotoxin GVIA-sensitive channels are primarily involved in mediating glutamate release at early developmental stages only. PMID:7604011

  4. [Involvement of protein kinase C in enhancement of vascular calcium sensitivity by blocking mesenteric lymph return in hemorrhagic shock rats].

    PubMed

    Niu, Chun-Yu; Zhao, Zi-Gang; Wei, Yan-Ling; Zhang, Yu-Ping; Zhang, Jing

    2012-04-25

    The aim of the present study was to investigate whether protein kinase C (PKC) was involved in the effect of mesenteric lymph duct ligation or mesenteric lymph drainage on vascular calcium sensitivity in hemorrhagic shock rats. Male Wistar rats were randomly divided into Sham, Shock (hemorrhagic shock), Shock+Ligation (mesenteric lymph duct ligation plus shock) and Shock+Drainage (mesenteric lymph drainage plus shock) groups. After being in shock (hypotension 40 mmHg) for 3 h, the tissue of superior mesenteric artery (SMA) was taken out for detecting the PKC expression and phospho-PKC (p-PKC) activity, and the vascular rings of SMA were prepared and used to measure the response to gradient calcium concentration for assaying the calcium sensitivity, the parameters of which including tension, maximum tension (E(max)) and negative logarithm of EC(50), called the pD(2). Other vascular rings from Shock+Ligation and Shock+Drainage groups were incubated with PKC regulator PMA or Staurosporine before the measurement of calcium sensitivity. The results showed that, PKC expression, p-PKC activity and calcium sensitivity of SMA in Shock group was significantly lower than that of Sham group, whereas the above-mentioned indexes were significantly elevated in Shock+Ligation and Shock+Drainage groups compared with those in Shock group. PKC agonist PMA enhanced the contractile activity of vascular rings to gradient calcium ions, and increased E(max) of SMA in Shock+Ligation and Shock+Drainage groups. On the contrary, PKC inhibitor Staurosporine significantly decreased the response to gradient calcium ions and E(max) of SMA in Shock+Ligation and Shock+Drainage groups. These results suggest that PKC plays a role in the improvement of vascular calcium sensitivity by blockade of mesenteric lymph return in hemorrhagic shock rats.

  5. Apamin-sensitive, small-conductance, calcium-activated potassium channels mediate cholinergic inhibition of chick auditory hair cells.

    PubMed

    Yuhas, W A; Fuchs, P A

    1999-11-01

    Acetylcholine released from efferent neurons in the cochlea causes inhibition of mechanosensory hair cells due to the activation of calcium-dependent potassium channels. Hair cells are known to have large-conductance, "BK"-type potassium channels associated with the afferent synapse, but these channels have different properties than those activated by acetylcholine. Whole-cell (tight-seal) and cell-attached patch-clamp recordings were made from short (outer) hair cells isolated from the chicken basilar papilla (cochlea equivalent). The peptides apamin and charybdotoxin were used to distinguish the calcium-activated potassium channels involved in the acetylcholine response from the BK-type channels associated with the afferent synapse. Differential toxin blockade of these potassium currents provides definitive evidence that ACh activates apamin-sensitive, "SK"-type potassium channels, but does not activate carybdotoxin-sensitive BK channels. This conclusion is supported by tentative identification of small-conductance, calcium-sensitive but voltage-insensitive potassium channels in cell-attached patches. The distinction between these channel types is important for understanding the segregation of opposing afferent and efferent synaptic activity in the hair cell, both of which depend on calcium influx. These different calcium-activated potassium channels serve as sensitive indicators for functionally significant calcium influx in the hair cell.

  6. Calcium and gibberellin-induced elongation of lettuce hypocotyl sections.

    PubMed

    Moll, C; Jones, R L

    1981-08-01

    The relationship between calcium ions and gibberellic acid (GA3)-induced growth in the excised hypocotyl of lettuce (Lactuca sativa L.) was investigated. The short-term kinetics of growth responses were measured using a linear displacement transducer. Test solutions were added either as drops to the filter paper on which the hypocotyl stood ("non-flow-past") or by switching solution flowing past the base of hypocotyl ("flow-past"), resulting in differences in growth behavior. Drops of CaCl2 added at a high concentration (10 mM) inhibited growth within a few minutes. This inhibition was reversed by ethylenediaminetetraacetic acid (EDTA). Drops of EDTA or ethyleneglycol-bis(2-aminoethylether)-tetraacetic acid caused a rapid increase in growth rate. Growth induced by EDTA was not further promoted by GA3. A continuous H2O flow resulted in growth rates comparable to those in response to GA3. Addition of CaCl2 to the flow-past medium inhibited growth and this inhibition was reversed by a decrease in CaCl2 concentration. The growth rate was found to be a function of CaCl2 concentration. When a constant CaCl2 concentration was maintained by the flow-past medium, a shift in pH from 5.5 to 4.25 had no obvious effect on hypocotyl elongation. Gibberellic acid was found to reverse the inhibitory effect of CaCl2, causing an increase in growth rate similar to that found previously when GA3 was added to hypocotyls grown in H2O under non-flow-past conditions. We propose that gibberellin controls extension growth in lettuce hypocotyl sections by regulating the uptake of Ca(2+) by the hypocotyl cells.

  7. Fluid Flow Induced Calcium Response in Bone Cell Network

    PubMed Central

    Huo, Bo; Lu, Xin L.; Hung, Clark T.; Costa, Kevin D.; Xu, Qiaobing; Whitesides, George M.; Guo, X. Edward

    2010-01-01

    In our previous work, bone cell networks with controlled spacing and functional intercellular gap junctions had been successfully established by using microcontact printing and self assembled monolayers technologies [Guo, X. E., E. Takai, X. Jiang, Q. Xu, G. M. Whitesides, J. T. Yardley, C. T. Hung, E. M. Chow, T. Hantschel, and K. D. Costa. Mol. Cell. Biomech. 3:95–107, 2006]. The present study investigated the calcium response and the underlying signaling pathways in patterned bone cell networks exposed to a steady fluid flow. The glass slides with cell networks were separated into eight groups for treatment with specific pharmacological agents that inhibit pathways significant in bone cell calcium signaling. The calcium transients of the network were recorded and quantitatively evaluated with a set of network parameters. The results showed that 18α-GA (gap junction blocker), suramin (ATP inhibitor), and thapsigargin (depleting intracellular calcium stores) significantly reduced the occurrence of multiple calcium peaks, which were visually obvious in the untreated group. The number of responsive peaks also decreased slightly yet significantly when either the COX-2/PGE2 or the NOS/nitric oxide pathway was disrupted. Different from all other groups, cells treated with 18α-GA maintained a high concentration of intracellular calcium following the first peak. In the absence of calcium in the culture medium, the intracellular calcium concentration decreased slowly with fluid flow without any calcium transients observed. These findings have identified important factors in the flow mediated calcium signaling of bone cells within a patterned network. PMID:20852730

  8. Respiratory metabolism and calorie restriction relieve persistent endoplasmic reticulum stress induced by calcium shortage in yeast

    PubMed Central

    Busti, Stefano; Mapelli, Valeria; Tripodi, Farida; Sanvito, Rossella; Magni, Fulvio; Coccetti, Paola; Rocchetti, Marcella; Nielsen, Jens; Alberghina, Lilia; Vanoni, Marco

    2016-01-01

    Calcium homeostasis is crucial to eukaryotic cell survival. By acting as an enzyme cofactor and a second messenger in several signal transduction pathways, the calcium ion controls many essential biological processes. Inside the endoplasmic reticulum (ER) calcium concentration is carefully regulated to safeguard the correct folding and processing of secretory proteins. By using the model organism Saccharomyces cerevisiae we show that calcium shortage leads to a slowdown of cell growth and metabolism. Accumulation of unfolded proteins within the calcium-depleted lumen of the endoplasmic reticulum (ER stress) triggers the unfolded protein response (UPR) and generates a state of oxidative stress that decreases cell viability. These effects are severe during growth on rapidly fermentable carbon sources and can be mitigated by decreasing the protein synthesis rate or by inducing cellular respiration. Calcium homeostasis, protein biosynthesis and the unfolded protein response are tightly intertwined and the consequences of facing calcium starvation are determined by whether cellular energy production is balanced with demands for anabolic functions. Our findings confirm that the connections linking disturbance of ER calcium equilibrium to ER stress and UPR signaling are evolutionary conserved and highlight the crucial role of metabolism in modulating the effects induced by calcium shortage. PMID:27305947

  9. Multiple Modes of Calcium-Induced Calcium Release in Sympathetic Neurons II

    PubMed Central

    Hongpaisan, Jarin; Pivovarova, Natalia B.; Colegrove, Stephen L.; Leapman, Richard D.; Friel, David D.; Andrews, S. Brian

    2001-01-01

    CICR from an intracellular store, here directly characterized as the ER, usually refers to net Ca2+ release that amplifies evoked elevations in cytosolic free calcium ([Ca2+]i). However, the companion paper (Albrecht, M.A., S.L. Colegrove, J. Hongpaisan, N.B. Pivovarova, S.B. Andrews, and D.D. Friel. 2001. J. Gen. Physiol. 118:83–100) shows that in sympathetic neurons, small [Ca2+]i elevations evoked by weak depolarization stimulate ER Ca accumulation, but at a rate attenuated by activation of a ryanodine-sensitive CICR pathway. Here, we have measured depolarization-evoked changes in total ER Ca concentration ([Ca]ER) as a function of [Ca2+]i, and found that progressively larger [Ca2+]i elevations cause a graded transition from ER Ca accumulation to net release, consistent with the expression of multiple modes of CICR. [Ca]ER is relatively high at rest (12.8 ± 0.9 mmol/kg dry weight, mean ± SEM) and is reduced by thapsigargin or ryanodine (5.5 ± 0.7 and 4.7 ± 1.1 mmol/kg, respectively). [Ca]ER rises during weak depolarization (to 17.0 ± 1.6 mmol/kg over 120s, [Ca2+]i less than ∼350 nM), changes little in response to stronger depolarization (12.1 ± 1.1 mmol/kg, [Ca2+]i ∼700 nM), and declines (to 6.5 ± 1.0 mmol/kg) with larger [Ca2+]i elevations (>1 μM) evoked by the same depolarization when mitochondrial Ca2+ uptake is inhibited (FCCP). Thus, net ER Ca2+ transport exhibits a biphasic dependence on [Ca2+]i. With mitochondrial Ca2+ uptake enabled, [Ca]ER rises after repolarization (to 16.6 ± 1.8 mmol/kg at 15 min) as [Ca2+]i falls within the permissive range for ER Ca accumulation over a period lengthened by mitochondrial Ca2+ release. Finally, although spatially averaged [Ca]ER is unchanged during strong depolarization, net ER Ca2+ release still occurs, but only in the outermost ∼5-μm cytoplasmic shell where [Ca2+]i should reach its highest levels. Since mitochondrial Ca accumulation occurs preferentially in peripheral cytoplasm, as demonstrated

  10. Sources of activator calcium for potassium- and serotonin-induced constriction of isolated bovine cerebral arteries

    SciTech Connect

    Not Available

    1986-03-01

    Previous in vitro studies with the calcium channel blockers (CCB) indirectly suggest that K/sup +/ and serotonin (5HT) constrict bovine middle cerebral arteries (BMCA) by promoting the influx of extracellular calcium (Ca) through CCB-sensitive channels. In this study, the authors directly determined the sources of activator Ca for K/sup +/- and 5HT-induced constriction of BMCA, using radiolabelled /sup 4/)2%Ca and /sup 3/H-sorbitol. EGTA-resistant Ca uptake, an estimate of Ca influx into vascular smooth muscle, was determined by exposure to Ca-deficient 2 mM EGTA solutions at 1/sup 0/C. The total Ca content of BMCA was 4.4 nmole/mg (wet wt.) after equilibration at 37/sup 0/C. The total exchangeable Ca content was 1.64 nmole/mg after 1 hr of /sup 45/Ca loading; the Ca content of the extracellular water was 0.30 nmole/mg, as estimated from the /sup 3/H-sorbitol space (0.25 ul/mg). The EGTA-resistant Ca uptake at 1 hr was 134 pmole/mg. K/sup +/ and 5HT significantly increased EGTA-resistant Ca uptake during 5 min of /sup 45/Ca loading; for K/sup +/, Ca uptake increased from 71 to 202 pmole/mg, and for 5HT, from 65 to 102 pmole/mg. Verapamil (10/sup -5/ M) or nifedipine (3.3 x 10/sup -7/ M) significantly blocked the increase in EGTA-resistant Ca uptake induced by K/sup +/ or 5HT. These results provide direct evidence that K/sup +/ or 5HT may constrict BMCA by promoting the influx of extracellular Ca through CCB-sensitive channels.

  11. Spontaneous and CRH-Induced Excitability and Calcium Signaling in Mice Corticotrophs Involves Sodium, Calcium, and Cation-Conducting Channels.

    PubMed

    Zemkova, Hana; Tomić, Melanija; Kucka, Marek; Aguilera, Greti; Stojilkovic, Stanko S

    2016-04-01

    Transgenic mice expressing the tdimer2(12) form of Discosoma red fluorescent protein under control of the proopiomelanocortin gene's regulatory elements are a useful model for studying corticotrophs. Using these mice, we studied the ion channels and mechanisms controlling corticotroph excitability. Corticotrophs were either quiescent or electrically active, with a 22-mV difference in the resting membrane potential (RMP) between the 2 groups. In quiescent cells, CRH depolarized the membrane, leading to initial single spiking and sustained bursting; in active cells, CRH further facilitated or inhibited electrical activity and calcium spiking, depending on the initial activity pattern and CRH concentration. The stimulatory but not inhibitory action of CRH on electrical activity was mimicked by cAMP independently of the presence or absence of arachidonic acid. Removal of bath sodium silenced spiking and hyperpolarized the majority of cells; in contrast, the removal of bath calcium did not affect RMP but reduced CRH-induced depolarization, which abolished bursting electrical activity and decreased the spiking frequency but not the amplitude of single spikes. Corticotrophs with inhibited voltage-gated sodium channels fired calcium-dependent action potentials, whereas cells with inhibited L-type calcium channels fired sodium-dependent spikes; blockade of both channels abolished spiking without affecting the RMP. These results indicate that the background voltage-insensitive sodium conductance influences RMP, the CRH-depolarization current is driven by a cationic conductance, and the interplay between voltage-gated sodium and calcium channels plays a critical role in determining the status and pattern of electrical activity and calcium signaling.

  12. CALCIUM PLAYS A CENTRAL ROLE IN THE SENSITIZATION OF TRPV3 CHANNEL TO REPETITIVE STIMULATIONS

    PubMed Central

    Xiao, Rui; Tang, Jisen; Wang, Chunbo; Colton, Craig K.; Tian, Jinbin; Zhu, Michael X.

    2008-01-01

    Transient Receptor Potential (TRP) channels are involved in sensing chemical and physical changes inside and outside of cells. TRPV3 is highly expressed in skin keratinocytes, where it forms a non-selective cation channel activated by hot temperatures in the innocuous and noxious range. The channel has also been implicated in flavor sensation in oral and nasal cavities as well as being a molecular target of some allergens and skin sensitizers. TRPV3 is unique in that its activity is sensitized upon repetitive stimulations. Here, we investigated the role of calcium ions in the sensitization of TRPV3 to repetitive stimulations. We show that the sensitization is accompanied with a decrease of Ca2+-dependent channel inhibition mediated by calmodulin acting at an N-terminal site (aa 108-130) and by an acidic residue (Asp641) at the pore loop of TRPV3. These sites also contribute to the voltage dependence of TRPV3. During sensitization, the channel displayed a gradual shift of the voltage dependence to more negative potentials as well as uncoupling from voltage sensing. The initial response to ligand stimulation was increased and sensitization to repetitive stimulations was decreased by increasing the intracellular Ca2+ buffering strength, inhibiting calmodulin, or disrupting the calmodulin-binding site. Mutation of Asp641 to Asn abolished the high affinity extracellular Ca2+-mediated inhibition and greatly facilitated the activation of TRPV3. We conclude that Ca2+ inhibits TRPV3 from both the extracellular and intracellular sides. The inhibition is sequentially reduced, appearing as sensitization to repetitive stimulations. PMID:18178557

  13. Deficiency of calcium and magnesium induces apoptosis via scavenger receptor BI

    PubMed Central

    Feng, Hong; Guo, Ling; Gao, Haiqing; Li, Xiang-An

    2011-01-01

    Aims Cell undergoes apoptosis in stressed status such as intracellular calcium overload or extracellular calcium/magnesium deficiency. The mechanisms of how deficiency of the divalent metal ions induces apoptosis remain to be defined. Scavenger receptor BI (SRBI) is a high density lipoprotein (HDL) receptor. Recent studies demonstrated that SR-BI is a stress response molecule which induces apoptosis upon serum deprivation. In this study, we assessed our hypothesis that the deficiency of calcium/magnesium induces apoptosis via SR-BI apoptotic pathway. Main methods We employed CHO cell lines expressing vector and SR-BI to test the effect of SR-BI on apoptosis induced by deficiency of calcium, magnesium and zinc in culture medium. The regain of different metal ions in deficient medium was also performed, respectively. Cell death was detected by morphological changes and quantified by LDH cytotoxicity assay. Apoptosis was also assessed by DNA ladder assay and DNA condensation assay. The SR-BIC323G mutant cells which lack the apoptotic activity of SR-BI were employed to verify the SR-BI-dependent effect on calcium/magnesium induced apoptosis. Key findings The deficiency of calcium/magnesium induced cell apoptosis CHO-SR-BI cells, but not in CHO-vector cells. Moreover, no apoptotic cell death was observed in SR-BIC323G mutant cells, indicating that the deficiency of divalent metal ions induces apoptosis in a SR-BI-dependent manner. Furthermore, the restoration of calcium or magnesium, but not zinc, protected CHO-SR-BI cells from apoptotic cell death, in a dose-dependent fashion. Significance These findings extend our understanding about how calcium and magnesium deficiency induces apoptosis. PMID:21291896

  14. Mediation of growth factor induced DNA synthesis and calcium mobilization by Gq and Gi2

    PubMed Central

    1993-01-01

    A newly identified subclass of the heterotrimeric GTP binding regulatory protein family, Gq, has been found to be expressed in a diverse range of cell types. We investigated the potential role of this protein in growth factor signal transduction pathways and its potential relationship to the function of other G alpha subclasses. Recent biochemical studies have suggested that Gq regulates the beta 1 isozyme of phospholipase C (PLC beta 1), an effector for some growth factors. By microinjection of inhibitory antibodies specific to distinct G alpha subunits into living cells, we have determined that G alpha q transduces bradykinin- and thrombin-stimulated intracellular calcium transients which are likely to be mediated by PLC beta 1. Moreover, we found that G alpha q function is required for the mitogenic action of both of these growth factors. These results indicate that both thrombin and bradykinin utilize Gq to couple to increases in intracellular calcium, and that Gq is a necessary component of the mitogenic action of these factors. While microinjection of antibodies against G alpha i2 did not abolish calcium transients stimulated by either of these factors, such microinjection prevented DNA synthesis in response to thrombin but not to bradykinin. These data suggest that thrombin- induced mitogenesis requires both Gq and Gi2, whereas bradykinin needs only the former. Thus, different growth factors operating upon the same cell type use overlapping yet distinct sets of G alpha subtypes in mitogenic signal transduction pathways. The direct identification of the coupling of both a pertussis toxin sensitive and insensitive G protein subtype in the mitogenic pathways utilized by thrombin offers an in vivo biochemical clarification of previous results obtained by pharmacologic studies. PMID:8458876

  15. Ryanodine receptor sensitivity governs the stability and synchrony of local calcium release during cardiac excitation-contraction coupling.

    PubMed

    Wescott, Andrew P; Jafri, M Saleet; Lederer, W J; Williams, George S B

    2016-03-01

    Calcium-induced calcium release is the principal mechanism that triggers the cell-wide [Ca(2+)]i transient that activates muscle contraction during cardiac excitation-contraction coupling (ECC). Here, we characterize this process in mouse cardiac myocytes with a novel mathematical action potential (AP) model that incorporates realistic stochastic gating of voltage-dependent L-type calcium (Ca(2+)) channels (LCCs) and sarcoplasmic reticulum (SR) Ca(2+) release channels (the ryanodine receptors, RyR2s). Depolarization of the sarcolemma during an AP stochastically activates the LCCs elevating subspace [Ca(2+)] within each of the cell's 20,000 independent calcium release units (CRUs) to trigger local RyR2 opening and initiate Ca(2+) sparks, the fundamental unit of triggered Ca(2+) release. Synchronization of Ca(2+) sparks during systole depends on the nearly uniform cellular activation of LCCs and the likelihood of local LCC openings triggering local Ca(2+) sparks (ECC fidelity). The detailed design and true SR Ca(2+) pump/leak balance displayed by our model permits investigation of ECC fidelity and Ca(2+) spark fidelity, the balance between visible (Ca(2+) spark) and invisible (Ca(2+) quark/sub-spark) SR Ca(2+) release events. Excess SR Ca(2+) leak is examined as a disease mechanism in the context of "catecholaminergic polymorphic ventricular tachycardia (CPVT)", a Ca(2+)-dependent arrhythmia. We find that that RyR2s (and therefore Ca(2+) sparks) are relatively insensitive to LCC openings across a wide range of membrane potentials; and that key differences exist between Ca(2+) sparks evoked during quiescence, diastole, and systole. The enhanced RyR2 [Ca(2+)]i sensitivity during CPVT leads to increased Ca(2+) spark fidelity resulting in asynchronous systolic Ca(2+) spark activity. It also produces increased diastolic SR Ca(2+) leak with some prolonged Ca(2+) sparks that at times become "metastable" and fail to efficiently terminate. There is a huge margin of safety for

  16. The role of bound calcium in supersensitivity induced by cocaine.

    PubMed Central

    Greenberg, R; Innes, I R

    1976-01-01

    1 Noradrenaline caused a small contraction of the cat isolated spleen strip bathed in a calcium-free solution; this contraction was greatly potentiated by cocaine. This potentiation was also present in isolated spleen strips where noradrenaline stores were depleted by reserpine. The maximum response of the spleen strip to noradrenaline in the absence of extracellular calcium was also increased by cocaine. 2 The disodium salt of ethylenediaminetetraacetic acid (Na-EDTA) but not Ca-EDTA antagonized the potentiation of the response to noradrenaline by cocaine in a calcium-free solution, and greatly reduced the magnitude of subsequent responses to noradrenaline and cocaine. 3 Strontium caused equivalent contractions of normal and reserpine-treated spleen strips bathed in a calcium-free solution. These responses were potentiated by cocaine. 4 Histamine caused a small contraction of the isolated spleen strip bathed in a Ca-free solution. Cocaine failed to potentiate these very small histamine contractions, but did potentiate the contraction of these same strips in response to noradrenaline. 5 It is concluded that the potentiation of the response of the isolated spleen strip to noradrenaline by cocaine in the absence of extracellular calcium is due to a mechanism other than decreased neuronal uptake of noradrenaline. It is suggested that cocaine makes a bound store of calcium more available to promote contraction of the spleen strip by noradrenaline. PMID:823996

  17. Calcium-induced precipitate formation in brain mitochondria: composition, calcium capacity, and retention

    PubMed Central

    Kristian, Tibor; Pivovarova, Natalia B.; Fiskum, Gary; Andrews, S. Brian

    2008-01-01

    Both isolated brain mitochondria and mitochondria in intact neurons are capable of accumulating large amounts of calcium, which leads to formation in the matrix of calcium- and phosphorus-rich precipitates, the chemical composition of which is largely unknown. Here, we have used inhibitors of the mitochondrial permeability transition (MPT) to determine how the amount and rate of mitochondrial calcium uptake relate to mitochondrial morphology, precipitate composition, and precipitate retention. Using isolated rat brain (RBM) or liver mitochondria (RLM) Ca2+-loaded by continuous cation infusion, precipitate composition was measured in situ in parallel with Ca2+ uptake and mitochondrial swelling. In RBM, the endogenous MPT inhibitors adenosine 5′-diphosphate (ADP) and adenosine 5′-triphosphate (ATP) increased mitochondrial Ca2+ loading capacity and facilitated formation of precipitates. In the presence of ADP, the Ca/P ratio approached 1.5, while ATP or reduced infusion rates decreased this ratio towards 1.0, indicating that precipitate chemical form varies with the conditions of loading. In both RBM and RLM, the presence of cyclosporine A in addition to ADP increased the Ca2+ capacity and precipitate Ca/P ratio. Following MPT and/or depolarization, the release of accumulated Ca2+ is rapid but incomplete; significant residual calcium in the form of precipitates is retained in damaged mitochondria for prolonged periods. PMID:17663756

  18. Calcium-induced precipitate formation in brain mitochondria: composition, calcium capacity, and retention.

    PubMed

    Kristian, Tibor; Pivovarova, Natalia B; Fiskum, Gary; Andrews, S Brian

    2007-08-01

    Both isolated brain mitochondria and mitochondria in intact neurons are capable of accumulating large amounts of calcium, which leads to formation in the matrix of calcium- and phosphorus-rich precipitates, the chemical composition of which is largely unknown. Here, we have used inhibitors of the mitochondrial permeability transition (MPT) to determine how the amount and rate of mitochondrial calcium uptake relate to mitochondrial morphology, precipitate composition, and precipitate retention. Using isolated rat brain (RBM) or liver mitochondria (RLM) Ca(2+)-loaded by continuous cation infusion, precipitate composition was measured in situ in parallel with Ca(2+) uptake and mitochondrial swelling. In RBM, the endogenous MPT inhibitors adenosine 5'-diphosphate (ADP) and adenosine 5'-triphosphate (ATP) increased mitochondrial Ca(2+) loading capacity and facilitated formation of precipitates. In the presence of ADP, the Ca/P ratio approached 1.5, while ATP or reduced infusion rates decreased this ratio towards 1.0, indicating that precipitate chemical form varies with the conditions of loading. In both RBM and RLM, the presence of cyclosporine A in addition to ADP increased the Ca(2+) capacity and precipitate Ca/P ratio. Following MPT and/or depolarization, the release of accumulated Ca(2+) is rapid but incomplete; significant residual calcium in the form of precipitates is retained in damaged mitochondria for prolonged periods.

  19. Calcium regulates cyclic compression-induced early changes in chondrocytes during in vitro cartilage tissue formation.

    PubMed

    Raizman, Igal; De Croos, J N Amritha; Pilliar, Robert; Kandel, Rita A

    2010-10-01

    A single application of cyclic compression (1kPa, 1Hz, 30min) to bioengineered cartilage results in improved tissue formation through sequential catabolic and anabolic changes mediated via cell shape changes that are regulated by α5β1 integrin and membrane-type metalloprotease (MT1-MMP). To determine if calcium was involved in this process, the role of calcium in regulating cell shape changes, MT1-MMP expression and integrin activity in response to mechanical stimulation was examined. Stimulation-induced changes in cell shape and MT1-MMP expression were abolished by chelation of extracellular calcium, and this effect was reversed by re-introduction of calcium. Spreading was inhibited by blocking stretch-activated channels (with gadolinium), while retraction was prevented by blocking the L-Type voltage-gated channel (with nifedipine); both compounds inhibited MT1-MMP upregulation. Calcium A23187 ionophore restored cellular response further supporting a role for these channels. Calcium regulated the integrin-mediated signalling pathway, which was facilitated through Src kinase. Both calcium- and integrin-mediated pathways converged on ERK-MAPK in response to stimulation. While both integrins and calcium signalling mediate chondrocyte mechanotransduction, calcium appears to play the major regulatory role. Understanding the underlying molecular mechanisms involved in chondrocyte mechanotransduction may lead to the development of improved bioengineered cartilage.

  20. Fluid shear stress induces calcium transients in osteoblasts through depolarization of osteoblastic membrane.

    PubMed

    Sun, Junqing; Liu, Xifang; Tong, Jie; Sun, Lijun; Xu, Hao; Shi, Liang; Zhang, Jianbao

    2014-12-18

    Intracellular calcium transient ([Ca(2+)]i transient) induced by fluid shear stress (FSS) plays an important role in osteoblastic mechanotransduction. Changes of membrane potential usually affect [Ca(2+)]i level. Here, we sought to determine whether there was a relationship between membrane potential and FSS-induced [Ca(2+)]i transient in osteoblasts. Fluorescent dyes DiBAC4(3) and fura-2AM were respectively used to detect membrane potential and [Ca(2+)]i. Our results showed that FSS firstly induced depolarization of membrane potential and then a transient rising of [Ca(2+)]i in osteoblasts. There was a same threshold for FSS to induce depolarization of membrane potential and [Ca(2+)]i transients. Replacing extracellular Na(+) with tetraethylammonium or blocking stretch-activated channels (SACs) with gadolinium both effectively inhibited FSS-induced membrane depolarization and [Ca(2+)]i transients. However, voltage-activated K(+) channel inhibitor, 4-Aminopyridine, did not affect these responses. Removing extracellular Ca(2+) or blocking of L-type voltage-sensitive Ca(2+) channels (L-VSCCs) with nifedipine inhibited FSS-induced [Ca(2+)]i transients in osteoblasts too. Quantifying membrane potential with patch clamp showed that the resting potential of osteoblasts was -43.3mV and the depolarization induced by FSS was about 44mV. Voltage clamp indicated that this depolarization was enough to activated L-VSCCs in osteoblasts. These results suggested a time line of Ca(2+) mobilization wherein FSS activated SACs to promote Na(+) entry to depolarize membrane that, in turn, activated L-VSCCs and Ca(2+) influx though L-VSCCs switched on [Ca(2+)]i response in osteoblasts.

  1. Effect of high calcium diet on cadmium-induced hypertension in rat.

    PubMed

    Chen, K S

    1992-04-01

    Effect of high calcium diet on the formation of hypertension induced by chronic cadmium chloride (CdCl2) treatment was investigated in Wistar rats. Intraperitoneal injection of CdCl2 solution (0.5 and 1.0 mg/kg/day) into female rats for two weeks resulted in an elevation of mean blood pressure. Bilateral adrenalectomy failed to prevent the CdCl2-induced hypertension. However, elevation of blood pressure from CdCl2 was not be found in rats on high calcium diet (4% Wt/Wt). Administration of high calcium diet influenced neither the blood pressure nor body weight of normal rats. My results suggest that high calcium diet may prevent the development of CdCl2-induced hypertension in rats.

  2. A pH-Sensitive, Biobased Calcium Carbonate Aragonite Nanocrystal as a Novel Anticancer Delivery System

    PubMed Central

    Ismail, Maznah; Tengku Ibrahim, Tengku Azmi; Zakaria, Zuki Abu Bakar

    2013-01-01

    The synthesised biobased calcium carbonate nanocrystals had demonstrated to be an effective carrier for delivery of anticancer drug doxorubicin (DOX). The use of these nanocrystals displayed high levels of selectivity and specificity in achieving effective cancer cell death without nonspecific toxicity. These results confirmed that DOX was intercalated into calcium carbonate nanocrystals at high loading and encapsulation efficiency (4.8 and 96%, resp.). The CaCO3/DOX nanocrystals are relatively stable at neutral pH (7.4), resulting in slow release, but the nanocrystals progressively dissociated in acidic pH (4.8) regimes, triggering faster release of DOX. The CaCO3/DOX nanocrystals exhibited high uptake by MDA MB231 breast cancer cells and a promising potential delivery of DOX to target cells. In vitro chemosensitivity using MTT, modified neutral red/trypan blue assay, and LDH on MDA MB231 breast cancer cells revealed that CaCO3/DOX nanocrystals are more sensitive and gave a greater reduction in cell growth than free DOX. Our findings suggest that CaCO3 nanocrystals hold tremendous promise in the areas of controlled drug delivery and targeted cancer therapy. PMID:24324966

  3. Cross-linking of Phospholipid Membranes is a Conserved Property of Calcium-sensitive Synaptotagmins

    PubMed Central

    Connell, Emma; Giniatullina, Asiya; Lai-Kee-Him, Joséphine; Tavare, Richard; Ferrari, Enrico; Roseman, Alan; Cojoc, Dan; Brisson, Alain R.; Davletov, Bazbek

    2008-01-01

    Synaptotagmins are vesicular proteins implicated in many membrane trafficking events. They are highly conserved in evolution and the mammalian family contains 16 isoforms. We now show that the tandem C2 domains of several calcium-sensitive synaptotagmin isoforms tested, including Drosophila synaptotagmin, rapidly cross-link phospholipid membranes. In contrast to the tandem structure, individual C2 domains failed to trigger membrane cross-linking in several novel assays. Large-scale liposomal aggregation driven by tandem C2 domains in response to calcium was confirmed by the following techniques: turbidity assay, dynamic light-scattering and both confocal and negative stain electron microscopy. Firm cross-linking of membranes was evident from laser trap experiments. High-resolution cryo-electron microscopy revealed that membrane cross-linking by tandem C2 domains results in a constant distance of ∼9 nm between the apposed membranes. Our findings show the conserved nature of this important property of synaptotagmin, demonstrate the significance of the tandem C2 domain structure and provide a plausible explanation for the accelerating effect of synaptotagmins on membrane fusion. PMID:18508081

  4. Cross-linking of phospholipid membranes is a conserved property of calcium-sensitive synaptotagmins.

    PubMed

    Connell, Emma; Giniatullina, Asiya; Lai-Kee-Him, Joséphine; Tavare, Richard; Ferrari, Enrico; Roseman, Alan; Cojoc, Dan; Brisson, Alain R; Davletov, Bazbek

    2008-06-27

    Synaptotagmins are vesicular proteins implicated in many membrane trafficking events. They are highly conserved in evolution and the mammalian family contains 16 isoforms. We now show that the tandem C2 domains of several calcium-sensitive synaptotagmin isoforms tested, including Drosophila synaptotagmin, rapidly cross-link phospholipid membranes. In contrast to the tandem structure, individual C2 domains failed to trigger membrane cross-linking in several novel assays. Large-scale liposomal aggregation driven by tandem C2 domains in response to calcium was confirmed by the following techniques: turbidity assay, dynamic light-scattering and both confocal and negative stain electron microscopy. Firm cross-linking of membranes was evident from laser trap experiments. High-resolution cryo-electron microscopy revealed that membrane cross-linking by tandem C2 domains results in a constant distance of approximately 9 nm between the apposed membranes. Our findings show the conserved nature of this important property of synaptotagmin, demonstrate the significance of the tandem C2 domain structure and provide a plausible explanation for the accelerating effect of synaptotagmins on membrane fusion.

  5. Complex voltage-dependent behavior of single unliganded calcium-sensitive potassium channels.

    PubMed Central

    Talukder, G; Aldrich, R W

    2000-01-01

    study and characterization of unliganded openings is of central significance for the elucidation of gating mechanisms for allosteric ligand-gated ion channels. Unliganded openings have been reported for many channel types, but their low open probability can make it difficult to study their kinetics in detail. Because the large conductance calcium-activated potassium channel mSlo is sensitive to both intracellular calcium and to membrane potential, we have been able to obtain stable unliganded single-channel recordings of mSlo with relatively high opening probability. We have found that the single-channel gating behavior of mSlo is complex, with multiple open and closed states, even when no ligand is present. Our results rule out a Monod-Wyman-Changeux allosteric mechanism with a central voltage-dependent concerted step, and they support the existence of quaternary states with less than the full number of voltage sensors activated, as has been suggested by previous work involving measurements of gating currents. PMID:10653789

  6. Caffeine-Induced Suppression of GABAergic Inhibition and Calcium-Independent Metaplasticity

    PubMed Central

    Isokawa, Masako

    2016-01-01

    GABAergic inhibition plays a critical role in the regulation of neuron excitability; thus, it is subject to modulations by many factors. Recent evidence suggests the elevation of intracellular calcium ([Ca2+]i) and calcium-dependent signaling molecules underlie the modulations. Caffeine induces a release of calcium from intracellular stores. We tested whether caffeine modulated GABAergic transmission by increasing [Ca2+]i. A brief local puff-application of caffeine to hippocampal CA1 pyramidal cells transiently suppressed GABAergic inhibitory postsynaptic currents (IPSCs) by 73.2 ± 6.98%. Time course of suppression and the subsequent recovery of IPSCs resembled DSI (depolarization-induced suppression of inhibition), mediated by endogenous cannabinoids that require a [Ca2+]i rise. However, unlike DSI, caffeine-induced suppression of IPSCs (CSI) persisted in the absence of a [Ca2+]i rise. Intracellular applications of BAPTA and ryanodine (which blocks caffeine-induced calcium release from intracellular stores) failed to prevent the generation of CSI. Surprisingly, ruthenium red, an inhibitor of multiple calcium permeable/release channels including those of stores, induced metaplasticity by amplifying the magnitude of CSI independently of calcium. This metaplasticity was accompanied with the generation of a large inward current. Although ionic basis of this inward current is undetermined, the present result demonstrates that caffeine has a robust Ca2+-independent inhibitory action on GABAergic inhibition and causes metaplasticity by opening plasma membrane channels. PMID:26998364

  7. Caffeine-Induced Suppression of GABAergic Inhibition and Calcium-Independent Metaplasticity.

    PubMed

    Isokawa, Masako

    2016-01-01

    GABAergic inhibition plays a critical role in the regulation of neuron excitability; thus, it is subject to modulations by many factors. Recent evidence suggests the elevation of intracellular calcium ([Ca(2+)]i) and calcium-dependent signaling molecules underlie the modulations. Caffeine induces a release of calcium from intracellular stores. We tested whether caffeine modulated GABAergic transmission by increasing [Ca(2+)]i. A brief local puff-application of caffeine to hippocampal CA1 pyramidal cells transiently suppressed GABAergic inhibitory postsynaptic currents (IPSCs) by 73.2 ± 6.98%. Time course of suppression and the subsequent recovery of IPSCs resembled DSI (depolarization-induced suppression of inhibition), mediated by endogenous cannabinoids that require a [Ca(2+)]i rise. However, unlike DSI, caffeine-induced suppression of IPSCs (CSI) persisted in the absence of a [Ca(2+)]i rise. Intracellular applications of BAPTA and ryanodine (which blocks caffeine-induced calcium release from intracellular stores) failed to prevent the generation of CSI. Surprisingly, ruthenium red, an inhibitor of multiple calcium permeable/release channels including those of stores, induced metaplasticity by amplifying the magnitude of CSI independently of calcium. This metaplasticity was accompanied with the generation of a large inward current. Although ionic basis of this inward current is undetermined, the present result demonstrates that caffeine has a robust Ca(2+)-independent inhibitory action on GABAergic inhibition and causes metaplasticity by opening plasma membrane channels.

  8. Calcium-induced generation of reactive oxygen species in brain mitochondria is mediated by permeability transition.

    PubMed

    Hansson, Magnus J; Månsson, Roland; Morota, Saori; Uchino, Hiroyuki; Kallur, Thérese; Sumi, Tetsuo; Ishii, Nagao; Shimazu, Motohide; Keep, Marcus F; Jegorov, Alexandr; Elmér, Eskil

    2008-08-01

    Mitochondrial uptake of calcium in excitotoxicity is associated with subsequent increase in reactive oxygen species (ROS) generation and delayed cellular calcium deregulation in ischemic and neurodegenerative insults. The mechanisms linking mitochondrial calcium uptake and ROS production remain unknown but activation of the mitochondrial permeability transition (mPT) may be one such mechanism. In the present study, calcium increased ROS generation in isolated rodent brain and human liver mitochondria undergoing mPT despite an associated loss of membrane potential, NADH and respiration. Unspecific permeabilization of the inner mitochondrial membrane by alamethicin likewise increased ROS independently of calcium, and the ROS increase was further potentiated if NAD(H) was added to the system. Importantly, calcium per se did not induce a ROS increase unless mPT was triggered. Twenty-one cyclosporin A analogs were evaluated for inhibition of calcium-induced ROS and their efficacy clearly paralleled their potency of inhibiting mPT-mediated mitochondrial swelling. We conclude that while intact respiring mitochondria possess powerful antioxidant capability, mPT induces a dysregulated oxidative state with loss of GSH- and NADPH-dependent ROS detoxification. We propose that mPT is a significant cause of pathological ROS generation in excitotoxic cell death.

  9. Loss of β2-laminin alters calcium sensitivity and voltage-gated calcium channel maturation of neurotransmission at the neuromuscular junction.

    PubMed

    Chand, Kirat K; Lee, Kah Meng; Schenning, Mitja P; Lavidis, Nickolas A; Noakes, Peter G

    2015-01-01

    Neuromuscular junctions from β2-laminin-deficient mice exhibit lower levels of calcium sensitivity. Loss of β2-laminin leads to a failure in switching from N- to P/Q-type voltage-gated calcium channel (VGCC)-mediated transmitter release that normally occurs with neuromuscular junction maturation. The motor nerve terminals from β2-laminin-deficient mice fail to up-regulate the expression of P/Q-type VGCCs clusters and down-regulate N-type VGCCs clusters, as they mature. There is decreased co-localisation of presynaptic specialisations in β2-laminin-deficient neuromuscular junctions as a consequence of lesser P/Q-type VGCC expression. These findings support the idea that β2-laminin is critical in the organisation and maintenance of active zones at the neuromuscular junction via its interaction with P/Q-type VGCCs, which aid in stabilisation of the synapse. β2-laminin is a key mediator in the differentiation and formation of the skeletal neuromuscular junction. Loss of β2-laminin results in significant structural and functional aberrations such as decreased number of active zones and reduced spontaneous release of transmitter. In vitro β2-laminin has been shown to bind directly to the pore forming subunit of P/Q-type voltage-gated calcium channels (VGCCs). Neurotransmission is initially mediated by N-type VGCCs, but by postnatal day 18 switches to P/Q-type VGCC dominance. The present study investigated the changes in neurotransmission during the switch from N- to P/Q-type VGCC-mediated transmitter release at β2-laminin-deficient junctions. Analysis of the relationship between quantal content and extracellular calcium concentrations demonstrated a decrease in the calcium sensitivity, but no change in calcium dependence at β2-laminin-deficient junctions. Electrophysiological studies on VGCC sub-types involved in transmitter release indicate N-type VGCCs remain the primary mediator of transmitter release at matured β2-laminin-deficient junctions

  10. Calcium sensitivity and myofilament lattice structure in titin N2B KO mice.

    PubMed

    Lee, Eun-Jeong; Nedrud, Joshua; Schemmel, Peter; Gotthardt, Michael; Irving, Thomas C; Granzier, Henk L

    2013-07-01

    The cellular basis of the Frank-Starling "Law of the Heart" is the length-dependence of activation, but the mechanisms by which the sarcomere detects length changes and converts this information to altered calcium sensitivity has remained elusive. Here the effect of titin-based passive tension on the length-dependence of activation (LDA) was studied by measuring the tension-pCa relation in skinned mouse LV muscle at two sarcomere lengths (SLs). N2B KO myocardium, where the N2B spring element in titin is deleted and passive tension is elevated, was compared to WT myocardium. Myofilament lattice structure was studied with low-angle X-ray diffraction; the myofilament lattice spacing (d1,0) was measured as well as the ratio of the intensities of the 1,1 and 1,0 diffraction peaks (I1,1/I1,0) as an estimate of the degree of association of myosin heads with the thin filaments. Experiments were carried out in skinned muscle in which the lattice spacing was reduced with Dextran-T500. Experiments with and without lattice compression were also carried out following PKA phosphorylation of the skinned muscle. Under all conditions that were tested, LDA was significantly larger in N2B KO myocardium compared to WT myocardium, with the largest differences following PKA phosphorylation. A positive correlation between passive tension and LDA was found that persisted when the myofilament lattice was compressed with Dextran and that was enhanced following PKA phosphorylation. Low-angle X-ray diffraction revealed a shift in mass from thin filaments to thick filaments as sarcomere length was increased. Furthermore, a positive correlation was obtained between myofilament lattice spacing and passive tension and the change in I1,1/I1,0 and passive tension and these provide possible explanations for how titin-based passive tension might regulate calcium sensitivity.

  11. Differential Effect of Renal Cortical and Medullary Interstitial Fluid Calcium on Blood Pressure Regulation in Salt-Sensitive Hypertension

    PubMed Central

    Eley, Shaleka; Anderson, Lauren; Waters, Brittany; Royall, Brittany; Nichols, Sheena; Wells, Candace

    2015-01-01

    BACKGROUND Hypercalciuria is a frequent characteristic of hypertension. In this report we extend our earlier studies investigating the role of renal interstitial fluid calcium (ISFCa)2+ as a link between urinary calcium excretion and blood pressure in the Dahl salt-sensitive (DS) hypertensive model. METHODS Dahl salt-sensitive and salt-resistant (DR) rats were placed on control (0.45%) and high (8%) salt diets to determine if changes in renal cortical and medullary ISFCa 2+correlated with changes in urinary calcium excretion and blood pressure. RESULTS We observed that renal ISFCa 2+ was predicted by urinary calcium excretion (P < 0.05) in DS rats but not DR rats. Renal cortical ISFCa 2+ was negatively associated with blood pressure (P < 0.03) while renal medullary ISFCa 2+ was positively associated with blood pressure in DS rats (P < 0.04). In contrast, neither urinary calcium excretion nor renal ISFCa 2+ was associated with blood pressure in the DR rats under the conditions of this study. CONCLUSION We interpret these findings to suggest that decreased renal cortical ISFCa 2+ plays a role in the increase in blood pressure following a high salt diet in salt hypertension perhaps by mediating renal vasoconstriction; the role of medullary calcium remains to be fully understood. Further studies are needed to determine the mechanism of the altered renal ISFCa 2+ and its role in blood pressure regulation. PMID:25552516

  12. A highly sensitive fluorescent indicator dye for calcium imaging of neural activity in vitro and in vivo

    PubMed Central

    Tada, Mayumi; Takeuchi, Atsuya; Hashizume, Miki; Kitamura, Kazuo; Kano, Masanobu

    2014-01-01

    Calcium imaging of individual neurons is widely used for monitoring their activity in vitro and in vivo. Synthetic fluorescent calcium indicator dyes are commonly used, but the resulting calcium signals sometimes suffer from a low signal-to-noise ratio (SNR). Therefore, it is difficult to detect signals caused by single action potentials (APs) particularly from neurons in vivo. Here we showed that a recently developed calcium indicator dye, Cal-520, is sufficiently sensitive to reliably detect single APs both in vitro and in vivo. In neocortical neurons, calcium signals were linearly correlated with the number of APs, and the SNR was > 6 for in vitro slice preparations and > 1.6 for in vivo anesthetised mice. In cerebellar Purkinje cells, dendritic calcium transients evoked by climbing fiber inputs were clearly observed in anesthetised mice with a high SNR and fast decay time. These characteristics of Cal-520 are a great advantage over those of Oregon Green BAPTA-1, the most commonly used calcium indicator dye, for monitoring the activity of individual neurons both in vitro and in vivo. PMID:24405482

  13. A highly sensitive fluorescent indicator dye for calcium imaging of neural activity in vitro and in vivo.

    PubMed

    Tada, Mayumi; Takeuchi, Atsuya; Hashizume, Miki; Kitamura, Kazuo; Kano, Masanobu

    2014-06-01

    Calcium imaging of individual neurons is widely used for monitoring their activity in vitro and in vivo. Synthetic fluorescent calcium indicator dyes are commonly used, but the resulting calcium signals sometimes suffer from a low signal-to-noise ratio (SNR). Therefore, it is difficult to detect signals caused by single action potentials (APs) particularly from neurons in vivo. Here we showed that a recently developed calcium indicator dye, Cal-520, is sufficiently sensitive to reliably detect single APs both in vitro and in vivo. In neocortical neurons, calcium signals were linearly correlated with the number of APs, and the SNR was > 6 for in vitro slice preparations and > 1.6 for in vivo anesthetised mice. In cerebellar Purkinje cells, dendritic calcium transients evoked by climbing fiber inputs were clearly observed in anesthetised mice with a high SNR and fast decay time. These characteristics of Cal-520 are a great advantage over those of Oregon Green BAPTA-1, the most commonly used calcium indicator dye, for monitoring the activity of individual neurons both in vitro and in vivo. © 2014 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  14. The Calcium-Sensing Receptor Mediates Bone Turnover Induced by Dietary Calcium and Parathyroid Hormone in Neonates

    PubMed Central

    Shu, Lei; Ji, Ji; Zhu, Qi; Cao, Guofan; Karaplis, Andrew; Pollak, Martin R; Brown, Edward; Goltzman, David; Miao, Dengshun

    2011-01-01

    We have investigated, in neonates, whether the calcium-sensing receptor (CaR) mediates the effects of dietary calcium on bone turnover and/or modulates parathyroid hormone (PTH)–induced bone turnover. Wild-type (WT) pups and pups with targeted deletion of the Pth (Pth–/–) gene or of both Pth and CaR (Pth–/–CaR–/–) genes were nursed by dams on a normal or high-calcium diet. Pups nursed by dams on a normal diet received daily injections of vehicle or of PTH(1–34) (80 µg/kg) for 2 weeks starting from 1 week of age. In pups receiving vehicle and fed by dams on a normal diet, trabecular bone volume, osteoblast number, type 1 collagen–positive area, and mineral apposition rate, as well as the expression of bone-formation-related genes, all were reduced significantly in Pth–/– pups compared with WT pups and were decreased even more dramatically in Pth–/–CaR–/– pups. These parameters were increased in WT and Pth–/– pups but not in Pth–/–CaR–/– pups fed by dams on a high-calcium diet compared with pups fed by dams on a normal diet. These parameters also were increased in WT, Pth–/–, and Pth–/–CaR–/– pups following exogenous PTH treatment; however, the percentage increase was less in Pth–/–CaR–/– pups than in WT and Pth–/– pups. In vehicle-treated pups fed by dams on either the normal or high-calcium diet and in PTH-treated pups fed by dams on a normal diet, the number and surfaces of osteoclasts and the ratio of RANKL/OPG were reduced significantly in Pth–/– pups and less significantly in Pth–/–CaR–/– pups compared with WT pups. These parameters were further reduced significantly in WT and Pth–/– pups from dams fed a high-calcium diet but did not decrease significantly in similarly treated Pth–/–CaR–/– pups, and they increased significantly in PTH-treated pups compared with vehicle-treated, genotype-matched pups fed by dams on the normal diet. These results indicate that in neonates

  15. External copper inhibits the activity of the large-conductance calcium- and voltage-sensitive potassium channel from skeletal muscle.

    PubMed

    Morera, F J; Wolff, D; Vergara, C

    2003-03-01

    We have characterized the effect of external copper on the gating properties of the large-conductance calcium- and voltage-sensitive potassium channel from skeletal muscle, incorporated into artificial bilayers. The effect of Cu2+ was evaluated as changes in the gating kinetic properties of the channel after the addition of this ion. We found that, from concentrations of 20 microM and up, copper induced a concentration- and time-dependent decrease in channel open probability. The inhibition of channel activity by Cu2+ could not be reversed by washing or by addition of the copper chelator, bathocuproinedisulfonic acid. However, channel activity was appreciably restored by the sulfhydryl reducing agent dithiothreitol. The effect of copper was specific since other transition metal divalent cations such as Ni2+, Zn2+ or Cd2+ did not affect BK(Ca) channel activity in the same concentration range. These results suggest that external Cu2+-induced inhibition of channel activity was due to direct or indirect oxidation of key amino-acid sulfhydryl groups that might have a role in channel gating.

  16. Omega-conotoxin GVIA binding to a high-affinity receptor in brain: characterization, calcium sensitivity, and solubilization

    SciTech Connect

    Wagner, J.A.; Snowman, A.M.; Biswas, A.; Olivera, B.M.; Snyder, S.H.

    1988-09-01

    We describe unique, high-affinity binding sites for omega(/sup 125/I)conotoxin GVIA in membranes from rat brain and rabbit sympathetic ganglia which appear to be primarily associated with N-type voltage-dependent calcium channels. The dissociation constant (KD) for the toxin in rat brain membranes is 60 pM. Physiologic extracellular concentrations of calcium inhibit toxin binding noncompetitively (IC50 = 0.2 mM). The regional distribution of the binding sites in rat brain differs markedly from that of dihydropyridine calcium antagonist receptors associated with L-type calcium channels. In detergent-solubilized brain membranes, toxin binding retains the same affinity, specificity, and ionic sensitivity as in particulate preparations.

  17. A gene encoding multidrug resistance (MDR)-like protein is induced by aluminum and inhibitors of calcium flux in wheat.

    PubMed

    Sasaki, Takayuki; Ezaki, Bunichi; Matsumoto, Hideaki

    2002-02-01

    A cDNA clone exclusively induced by aluminum (Al) was isolated from root apices of wheat (Triticum aestivum L.) by the differential display method. The predicted amino acid sequence exhibited homology to the multidrug resistance (MDR) proteins that is known as a member of the ATP-binding cassette (ABC) protein superfamily. Thus this gene was named TaMDR1 (Triticum aestivum MDR). TaMDR1 was induced as a function of Al concentration in the range from 5 to 50 microM, which is in the range of Al content in natural acid soil environment. The concentration required for the induction was lower in the Al-sensitive cultivar than in the Al-tolerant cultivar, indicating that the accumulation of TaMDR1 mRNA was associated with the events caused by Al toxicity rather than Al tolerance. TaMDR1 was significantly induced by the exposure to lanthanum, gadolinium and ruthenium red, which are known as inhibitors of calcium channels. Furthermore, decreasing of calcium ion in growth medium caused stimulation of the gene expression. These results suggested that the induction of TaMDR1 is caused by the breaking of calcium homeostasis which occurred at early stage of Al toxicity.

  18. Calcium responses induced by acetylcholine in submucosal arterioles of the guinea-pig small intestine

    PubMed Central

    Fukuta, Hiroyasu; Hashitani, Hikaru; Yamamoto, Yoshimichi; Suzuki, Hikaru

    1999-01-01

    Calcium responses induced by brief stimulation with acetylcholine (ACh) were assessed from the fluorescence changes in fura-2 loaded submucosal arterioles of the guinea-pig small intestine. Initially, 1–1.5 h after loading with fura-2 (fresh tissues), ACh increased [Ca2+]i in a concentration-dependent manner. This response diminished with time, and finally disappeared in 2–3 h (old tissues). Ba2+ elevated [Ca2+]i to a similar extent in both fresh and old tissues. ACh further increased the Ba2+-elevated [Ca2+]i in fresh tissues, but reduced it in old tissues. Responses were not affected by either indomethacin or nitroarginine. In fresh mesenteric arteries, mechanical removal of endothelial cells abolished the ACh-induced increase in [Ca2+]i, with no alteration of [Ca2+]i at rest and during elevation with Ba2+. In the presence of indomethacin and nitroarginine, high-K+ solution elevated [Ca2+]i in both fresh and old tissues. Subsequent addition of ACh further increased [Ca2+]i in fresh tissues without changing it in old tissues. Proadifen, an inhibitor of the enzyme cytochrome P450 mono-oxygenase, inhibited the ACh-induced changes in [Ca2+]i in both fresh and Ba2+-stimulated old tissues. It also inhibited the ACh-induced hyperpolarization. In fresh tissues, the ACh-induced Ca2+ response was not changed by apamin, charybdotoxin (CTX), 4-aminopyridine (4-AP) or glibenclamide. In old tissues in which [Ca2+]i had previously been elevated with Ba2+, the ACh-induced Ca2+ response was inhibited by CTX but not by apamin, 4-AP or glibenclamide. It is concluded that in submucosal arterioles, ACh elevates endothelial [Ca2+]i and reduces muscular [Ca2+]i, probably through the hyperpolarization of endothelial or smooth muscle membrane by activating CTX-sensitive K+ channels. PMID:10050015

  19. Bradykinin and histamine-induced cytosolic calcium increase in capillary endothelial cells of bovine adrenal medulla.

    PubMed

    Vinet, Raúl; Cortés, Magdalena P; Alvarez, Rocío; Delpiano, Marco A

    2014-09-01

    We have assessed the effect of bradykinin and histamine on the cytosolic free calcium concentration ([Ca(2+)]i ) of bovine adrenal medulla capillary endothelial cells (BAMCECs). To measure [Ca(2+)]i changes in BAMCECs the intracellular fluorescent probe, fluo-3 AM, was used. Bradykinin (3 µM) produced a transient monophasic increase in [Ca(2+)]i , which was depressed by B1650 (0.1 µM), a B2-bradykinin receptor antagonist (D-Arg-[Hyp(3), Thi(5,8) , D-Phe(7)]-Bradykinin). Similarly, increase in [Ca(2+)]i induced by histamine was also depressed by tripolidine (0.1 µM), an H1-histamine receptor antagonist. [Ca(2+)]i increase induced by both agonists was unaffected in the absence of extracellular Ca(2+) or presence of antagonists of voltage operated Ca(2+) channels (VOCCs). Thapsigargin (1 µM) did not abolish the increase of [Ca(2+)]i produced by bradykinin, but abolished that of histamine. In contrast, caffeine (100 µM), abolished the [Ca(2+)]i response induced by bradykinin (3 µM), but did not affect the [Ca(2+)]i increase induced by histamine (100 µM). The results indicate the presence of B2 bradykinin- and H1 histamine-receptors in BAMCECs. Liberation of Ca(2+) induced by both agonists occurs through 2 different intracellular mechanisms. While bradykinin activates a sarco(endo) plasmic reticulum (SER) containing a SER Ca(2+) -ATPase (SERCA) thapsigargin-insensitive, histamine activates a SER containing a SERCA thapsigargin-sensitive. We suggest that the increase in [Ca(2+)]i induced by bradykinin and histamine could be of physiological relevance, modulating adrenal gland microcirculation.

  20. Loss of β2-laminin alters calcium sensitivity and voltage-gated calcium channel maturation of neurotransmission at the neuromuscular junction

    PubMed Central

    Chand, Kirat K; Lee, Kah Meng; Schenning, Mitja P; Lavidis, Nickolas A; Noakes, Peter G

    2015-01-01

    β2-laminin is a key mediator in the differentiation and formation of the skeletal neuromuscular junction. Loss of β2-laminin results in significant structural and functional aberrations such as decreased number of active zones and reduced spontaneous release of transmitter. In vitro β2-laminin has been shown to bind directly to the pore forming subunit of P/Q-type voltage-gated calcium channels (VGCCs). Neurotransmission is initially mediated by N-type VGCCs, but by postnatal day 18 switches to P/Q-type VGCC dominance. The present study investigated the changes in neurotransmission during the switch from N- to P/Q-type VGCC-mediated transmitter release at β2-laminin-deficient junctions. Analysis of the relationship between quantal content and extracellular calcium concentrations demonstrated a decrease in the calcium sensitivity, but no change in calcium dependence at β2-laminin-deficient junctions. Electrophysiological studies on VGCC sub-types involved in transmitter release indicate N-type VGCCs remain the primary mediator of transmitter release at matured β2-laminin-deficient junctions. Immunohistochemical analyses displayed irregularly shaped and immature β2-laminin-deficient neuromuscular junctions when compared to matured wild-type junctions. β2-laminin-deficient junctions also maintained the presence of N-type VGCC clustering within the presynaptic membrane, which supported the functional findings of the present study. We conclude that β2-laminin is a key regulator in development of the NMJ, with its loss resulting in reduced transmitter release due to decreased calcium sensitivity stemming from a failure to switch from N- to P/Q-type VGCC-mediated synaptic transmission. PMID:25556799

  1. Effects of caulophine on caffeine-induced cellular injury and calcium homeostasis in rat cardiomyocytes.

    PubMed

    Si, Kai-Wei; Liu, Jun-Tian; He, Lang-Chong; Li, Xi-Kuan; Gou, Wei; Liu, Chuan-Hao; Li, Xiao-Qi

    2010-12-01

    Caulophine is a novel fluorenone alkaloid isolated from the radix of Caulophyllum robustum Maxim. Caulophine showed high affinity for the rat myocardial cell membrane as assessed by cell membrane chromatography, suggesting that the compound may exert bioactivity in the heart. It is known that calcium plays an important role in the pathogenesis of ischaemic heart disease, and caffeine can cause calcium overload in cardiomyocytes by inducing calcium release from the sarcoplasmic reticulum. Therefore, the present study evaluated the effects of caulophine on caffeine-induced injury and calcium homeostasis in cardiomyocytes. Cardiomyocytes were pre-treated with caulophine before exposure to caffeine or potassium chloride (KCl). Cell viability was assayed using the MTT method, and lactate dehydrogenase (LDH) and malondialdehyde (MDA) were measured spectrophotometrically. Caulophine-pre-treated cardiomyocytes were incubated with Fluo-3/AM, and then caffeine or KCl was used to induce Ca(2+) overload. The total intracellular Ca(2+) concentration was measured by flow cytometry. Fluorescence densities of single cardiomyocytes were detected using a confocal microscope. Caulophine increased the viability of caffeine-injured cardiomyocytes and decreased LDH activity and MDA level in cardiomyocytes. Furthermore, caulophine significantly decreased the total intracellular free Ca(2+) concentration and intracellular calcium release in cardiomyocytes in response to caffeine. However, the same concentrations of caulophine did not affect KCl-induced calcium influx. Our results suggest that caulophine protects cardiomyocytes from caffeine-induced injury as a result of calcium antagonism. This finding provides a basis for further study and development of caulophine as a new calcium antagonist for treating ischaemic cardiovascular diseases.

  2. [Changes induced by hypertonic solutions in the transportation of calcium by the cardiac reticular sarcoplasma].

    PubMed

    Sierra, M; Holguín, J A

    1979-01-01

    In the sarcoplasmic reticulum of the myocardium, celular organell which function is to regulate the cytoplasmic concentration of calcium in contraction and relaxation, we have studied the effect of hypertonic solutions of sucrose between 1 and 6.96 times the normal tonicity in order to observe the behavior of the internal linked or free calcium of this structure, as well as to prove the hypothesis that hypertonic solutions encourage the calcium exit of the sarcoplasmatic reticulum with the resulting signs of contractures. The following results were obtained: 1. The ATP hydrolisis and calcium transport rate are 14% and 90% respectively of the maximum speeds of 10(-5) M in calcium, while for concentrations of 10(-7) M or ess of the said cation, the transport rates and the ATPase do not reach 5% of the maximum values. 2. Between 1 and 2.54 times of the normal tonicity the calcium uptake remains between 400 and 500 nmoles of calcium/mg protein/min, the transported amount of calcium varies between 14 and 16 nmoles/mg protein and the rate of the ATP hydrolysis increases a 37% to 0.4 M in sucrose. 3. Between 0.4 and 1.2 M in sucrose of 2.54 to 6.96 times the isotonicity, the calcium transport rate velocity as well as the ATP hydrolisis are strongly inhibited. The vesicles volume minimizes and the amount of linked calcium remains within the control values, proving that the capacity of linking this cathion is independent from sarcoplasmic reticulum volume. These results show that the sarcoplasmic reticulum is involved in the contractures induced by hypertonic solutions in intact cells, since the osmolarity increase produces changes of volume which results in a decrease of the calcium transportation velocity or in an increase of the exit of said cathion.

  3. Intracellular calcium changes induced by the endozepine triakontatetraneuropeptide in human polymorphonuclear leukocytes: role of protein kinase C and effect of calcium channel blockers.

    PubMed

    Marino, Franca; Cosentino, Marco; Ferrari, Marco; Cattaneo, Simona; Frigo, Giuseppina; Fietta, Anna M; Lecchini, Sergio; Frigo, Gian Mario

    2004-06-30

    BACKGROUND: The endozepine triakontatetraneuropeptide (TTN) induces intracellular calcium ([Ca++]i) changes followed by activation in human polymorphonuclear leukocytes (PMNs). The present study was undertaken to investigate the role of protein kinase (PK) C in the modulation of the response to TTN by human PMNs, and to examine the pharmacology of TTN-induced Ca++ entry through the plasma membrane of these cells. RESULTS: The PKC activator 12-O-tetradecanoylphorbol-13-acetate (PMA) concentration-dependently inhibited TTN-induced [Ca++]i rise, and this effect was reverted by the PKC inhibitors rottlerin (partially) and Ro 32-0432 (completely). PMA also inhibited TTN-induced IL-8 mRNA expression. In the absence of PMA, however, rottlerin (but not Ro 32-0432) per se partially inhibited TTN-induced [Ca++]i rise. The response of [Ca++]i to TTN was also sensitive to mibefradil and flunarizine (T-type Ca++-channel blockers), but not to nifedipine, verapamil (L-type) or omega-conotoxin GVIA (N-type). In agreement with this observation, PCR analysis showed the expression in human PMNs of the mRNA for all the alpha1 subunits of T-type Ca++ channels (namely, alpha1G, alpha1H, and alpha1I). CONCLUSIONS: In human PMNs TTN activates PKC-modulated pathways leading to Ca++ entry possibly through T-type Ca++ channels.

  4. Intracellular calcium changes induced by the endozepine triakontatetraneuropeptide in human polymorphonuclear leukocytes: role of protein kinase C and effect of calcium channel blockers

    PubMed Central

    Marino, Franca; Cosentino, Marco; Ferrari, Marco; Cattaneo, Simona; Frigo, Giuseppina; Fietta, Anna M; Lecchini, Sergio; Frigo, Gian Mario

    2004-01-01

    Background The endozepine triakontatetraneuropeptide (TTN) induces intracellular calcium ([Ca++]i) changes followed by activation in human polymorphonuclear leukocytes (PMNs). The present study was undertaken to investigate the role of protein kinase (PK) C in the modulation of the response to TTN by human PMNs, and to examine the pharmacology of TTN-induced Ca++ entry through the plasma membrane of these cells. Results The PKC activator 12-O-tetradecanoylphorbol-13-acetate (PMA) concentration-dependently inhibited TTN-induced [Ca++]i rise, and this effect was reverted by the PKC inhibitors rottlerin (partially) and Ro 32-0432 (completely). PMA also inhibited TTN-induced IL-8 mRNA expression. In the absence of PMA, however, rottlerin (but not Ro 32-0432) per se partially inhibited TTN-induced [Ca++]i rise. The response of [Ca++]i to TTN was also sensitive to mibefradil and flunarizine (T-type Ca++-channel blockers), but not to nifedipine, verapamil (L-type) or ω-conotoxin GVIA (N-type). In agreement with this observation, PCR analysis showed the expression in human PMNs of the mRNA for all the α1 subunits of T-type Ca++ channels (namely, α1G, α1H, and α1I). Conclusions In human PMNs TTN activates PKC-modulated pathways leading to Ca++ entry possibly through T-type Ca++ channels. PMID:15228623

  5. Cholate and deoxycholate counteract the calcium-induced lowering of fat digestion in rats.

    PubMed

    Yuangklang, C; Wensing, Th; Lankhorst, Ae; Lemmens, A G; Fielmich-Bouman, X M; Jittakhot, S; Beynen, A C

    2005-10-01

    The objective of the present experiment was to investigate whether deoxycholate and cholate would differ in their effectiveness of counteracting the inhibitory effect of calcium on fat digestibility in rats. Rats were fed one of four experimental diets, a diet low in calcium, high in calcium or high in calcium with either 0.5% sodium cholate or 0.5% sodium deoxycholate. Both deoxycholate and cholate supplementation of the high-calcium diet reduced feed intake and body-weight gain. Low-calcium intake increased fat digestibility. Supplemental bile acids partially counteracted the calcium-induced inhibition of fat digestion, cholate being more effective than deoxycholate. The outcome is explained by the suggestion that cholate is bound to the calcium phosphate sediment in the small intestinal lumen with less affinity than deoxycholate. As a result, more cholate than deoxycholate would be available to support the process of fat digestion. Rats fed cholate had higher liver and serum cholesterol concentrations than did the rats fed deoxycholate.

  6. Potassium-induced contraction in the lamb proximal urethra: Involvement of norepinephrine and different calcium entry pathways

    SciTech Connect

    Garcia-Pascual, A.; Costa, G.; Isla, M.; Jimenez, E.; Garcia-Sacristan, A. )

    1991-01-01

    The purpose of this work was to investigate the mechanisms involved in the peculiar biphasic response of the lamb urethral smooth muscle to high K+ solutions. The relative amplitude of the phasic and tonic components of the contraction and its reproducibility were dependent on the concentration of K+ used. Only concentrations higher than 80 mM (i.e., 120 mM) showed a tonic component greater in amplitude than the phasic one and manifested a tachyphylactic effect. Phentolamine (10(-6) M), prazosin (10(-6) M) and chemical denervation with 6-hydroxydopamine significantly inhibited the tonic component of the K+ (120 mM)-induced contraction, modifying its morphology. Reproducible contractions to K+ (120 mM) could be obtained in the presence of prazosin (10(-6) M) or cocaine (10(-6) M). The preparations were also shown to accumulate (3H)noradrenaline and release it upon depolarization with K+ (60 and 120 mM). Calcium removal inhibited the K+ (120 mM)-induced contraction. After addition of calcium (0.5-5 mM) the contractile activity was restored. Nifedipine (10(-6) M) and verapamil (10(-6) M) but not sodium nitroprusside (10(-6) M) significantly blocked the contractile response for calcium as well as the phasic component of the K+ contraction in calcium-containing medium. In preparations treated with prazosin (10(-6) M) the tonic component of the K+ (120 mM) contraction was more sensitive to nifedipine and removal of extracellular calcium than the phasic one.

  7. Vasorelaxation induced by dodoneine is mediated by calcium channels blockade and carbonic anhydrase inhibition on vascular smooth muscle cells.

    PubMed

    Carre, Grégoire; Ouedraogo, Maurice; Magaud, Christophe; Carreyre, Hélène; Becq, Frédéric; Bois, Patrick; Supuran, Claudiu T; Thibaudeau, Sébastien; Vandebrouck, Clarisse; Bescond, Jocelyn

    2015-07-01

    Dodoneine (Ddn) is one of the active compounds identified from Agelanthus dodoneifolius (DC.) Polhill and Wiens, a medicinal plant used in traditional medicine for the treatment of hypertension. This dihydropyranone exerts hypotensive and vasorelaxant effects on rats, and two molecular targets have been characterized: the carbonic anhydrase and the L-type calcium channel in cardiomyocytes with biochemical and electrophysiological techniques, respectively. To further evaluate the involvement of these two molecular targets in vasorelaxation, the effect of Ddn on rat vascular smooth muscle was investigated. The effects of Ddn on L-type calcium current and on resting membrane potential were characterized in A7r5 cell line using the whole-cell patch-clamp configuration. The molecular identities of carbonic anhydrase isozymes in smooth muscle cells were examined with RT-PCR. Vascular response was measured on rat aortic rings in an organ bath apparatus and the effect of Ddn on intracellular pH was determined by flow cytometry using the pH-sensitive fluorescent probe BCECF-AM [2,7-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester]. 100µM Ddn reduced calcium current density of about 30%. In addition, carbonic anhydrase II, III, XIII and XIV were shown to be expressed in rat aorta and inhibited in smooth muscle cells by Ddn. This inhibition resulted in a rise in pHi of about 0.31, leading to KCa channel activation, thereby inducing membrane hyperpolarization and vasorelaxation. The results of vascular reactivity experiments obtained with pharmacological tools acting on the L-type calcium current and carbonic anhydrase suggest that Ddn produces its vasorelaxant effect via the inhibition of these two molecular targets. This study demonstrates that Ddn induced vasorelaxation by targeting two proteins involved in the modulation of excitation-contraction coupling: L-type calcium channels and carbonic anhydrase. Copyright © 2015 Elsevier Ireland Ltd. All

  8. Polymer surface interacts with calcium in aqueous media to induce stem cell assembly.

    PubMed

    Hung, Kun-Che; Hsu, Shan-Hui

    2015-10-28

    Bioinspired surface with functional group rearrangement abilities are highly desirable for designing functional materials. Calcium ion (Ca(2+) ) is a pivotal life element and the ion transport is tightly regulated through calcium channels. It is demonstrated here that Ca(2+) can be transported by polymer surface to induce cell assembly. A series of polyurethane materials is synthesized with different abilities to rearrange the surface functional groups in response to aqueous environment. It is observed that surface recruitment of carboxyl and amino groups from the bulk material can interact with Ca(2+) and facilitate its translocation from aqueous media into cells. The surface rearrangement of functional group triggers the calcium trafficking and turns on signals involving cell merging and assembly. This observation provides an insight on adjusting material-calcium interaction to design nature-inspired smart interfaces to induce cell organization and tissue regeneration. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Stereocontrolled synthesis of rosuvastatin calcium via iodine chloride-induced intramolecular cyclization.

    PubMed

    Xiong, Fangjun; Wang, Haifeng; Yan, Lingjie; Han, Sheng; Tao, Yuan; Wu, Yan; Chen, Fener

    2016-01-28

    A novel, stereoselective approach towards rosuvastatin calcium from the known (S)-homoallylic alcohol has been developed. The synthesis is highlighted by a regio- and stereocontrolled ICl-induced intramolecular cyclization of chiral homoallylic carbonate to deliver the C6-formyl statin side chain with a syn-1,3-diol moiety. An improved synthesis of the rosuvastatin pyrimidine core moiety is also included. Moreover, this methodology is useful in the asymmetric synthesis of structural variants of statins such as pitavastatin calcium and atorvastatin calcium and their related analogs.

  10. [Calcium polystyrene sulfonate induced colonic necrosis in patient with chronic kidney disease].

    PubMed

    Lee, Sung Hoa; Kim, Sung Jung; Kim, Go Eun; Lee, Woo Jin; Hong, Won Ki; Baik, Gwang Ho; Choi, Young Hee; Kim, Dong Joon

    2010-04-01

    A 63-year-old woman was admitted due to right upper quadrant abdominal pain. She was going through hemodialysis due to end stage renal disease and taking calcium polystyrene sulfonate orally and rectally due to hyperkalemia. Colonoscopy showed a circular ulcerative mass on the proximal ascending colon. Biopsy specimen from the mass showed inflammation and necrotic debris. It also revealed basophilic angulated crystals which were adherent to the ulcer bed and normal mucosa. These crystals were morphologically consistent with calcium polystyrene sulfonate. She was diagnosed with calcium polystyrene phosphate induced colonic necrosis and improved with conservative treatment.

  11. Calcium channel antagonists increase morphine-induced analgesia and antagonize morphine tolerance.

    PubMed

    Contreras, E; Tamayo, L; Amigo, M

    1988-04-13

    The influence of calcium channel blockers on morphine-induced analgesia and on tolerance to the chronic administration of the opiate was investigated in mice. The effects of a test dose of morphine were significantly increased by the administration of diltiazem, flunarizine, nicardipine and verapamil. In contrast, nifedipine induced an antagonistic effect. The calcium channel antagonists did not change the reaction time to thermal stimulation in mice (hot plate test). The administration of nifedipine, flunarizine and verapamil reduced the intensity of the tolerance induced by a single dose of morphine administered in a slow release preparation. Diltiazem induced a non-significant decrease of the process. The present results are in accordance with the known interaction of acute and chronic morphine administration with the intracellular calcium concentration in neurones of the central nervous system.

  12. Effect of subcutaneous administration of calcium channel blockers on nerve injury-induced hyperalgesia.

    PubMed

    White, D M; Cousins, M J

    1998-08-10

    Recent studies suggest that calcium contributes to peripheral neural mechanisms of hyperalgesia associated with nerve damage. In this animal behavioural study, we examined further the contribution of calcium in neuropathic pain by testing whether subcutaneous administration of either a calcium chelating agent or voltage-dependent calcium channel blockers attenuate nerve injury-induced hyperalgesia to mechanical stimulation. Studies were carried out in animals with partially ligated sciatic nerves, an established animal model of neuropathic pain. The nociceptive flexion reflex was quantified using an Ugo Basile Analgesymeter. Partial nerve injury induced a significant decrease in mechanical threshold compared to the sham operated controls. Daily subcutaneous injections of the calcium chelating agent, Quin 2 (20 microgram/2.5 microliter), significantly attenuated the nerve injury-induced hyperalgesia. Similarly, SNX-111, a N-type channel blocker, also significantly attenuated the nerve injury-induced hyperalgesia. SNX-230, a P and/or Q-type channel blocker, and nifedipine, a L-type channel blocker, had no effect on the hyperalgesia to mechanical stimulation. In control experiments, SNX-111 had no effect on mechanical thresholds when administered subcutaneously in either the hindpaw of normal animals or the back of the neck in nerve injury animals. This study shows that neuropathic pain involves a local calcium-dependent mechanism in the receptive field of intact neurons of an injured nerve, since it can be alleviated by subcutaneous injections of either a calcium chelating agent or SNX-111, a N-type calcium channel blocker. These agents may be effective, peripherally acting therapeutic agents for neuropathic pain.

  13. Accumulation of cytoplasmic calcium, but not apamin-sensitive afterhyperpolarization current, during high frequency firing in rat subthalamic nucleus cells

    PubMed Central

    Teagarden, Mark; Atherton, Jeremy F; Bevan, Mark D; Wilson, Charles J

    2008-01-01

    The autonomous firing pattern of neurons in the rat subthalamic nucleus (STN) is shaped by action potential afterhyperpolarization currents. One of these is an apamin-sensitive calcium-dependent potassium current (SK). The duration of SK current is usually considered to be limited by the clearance of calcium from the vicinity of the channel. When the cell is driven to fire faster, calcium is expected to accumulate, and this is expected to result in accumulation of calcium-dependent AHP current. We measured the time course of calcium transients in the soma and proximal dendrites of STN neurons during spontaneous firing and their accumulation during driven firing. We compared these to the time course and accumulation of AHP currents using whole-cell and perforated patch recordings. During spontaneous firing, a rise in free cytoplasmic calcium was seen after each action potential, and decayed with a time constant of about 200 ms in the soma, and 80 ms in the dendrites. At rates higher than 10 Hz, calcium transients accumulated as predicted. In addition, there was a slow calcium transient not predicted by summation of action potentials that became more pronounced at high firing frequency. Spike AHP currents were measured in voltage clamp as tail currents after 2 ms voltage pulses that triggered action currents. Apamin-sensitive AHP (SK) current was measured by subtraction of tail currents obtained before and after treatment with apamin. SK current peaked between 10 and 15 ms after an action potential, had a decay time constant of about 30 ms, and showed no accumulation. At frequencies between 5 and 200 spikes s−1, the maximal SK current remained the same as that evoked by a single action potential. AHP current did not have time to decay between action potentials, so at frequencies above 50 spikes s−1 the apamin-sensitive current was effectively constant. These results are inconsistent with the view that the decay of SK current is governed by calcium dynamics. They

  14. Accumulation of cytoplasmic calcium, but not apamin-sensitive afterhyperpolarization current, during high frequency firing in rat subthalamic nucleus cells.

    PubMed

    Teagarden, Mark; Atherton, Jeremy F; Bevan, Mark D; Wilson, Charles J

    2008-02-01

    The autonomous firing pattern of neurons in the rat subthalamic nucleus (STN) is shaped by action potential afterhyperpolarization currents. One of these is an apamin-sensitive calcium-dependent potassium current (SK). The duration of SK current is usually considered to be limited by the clearance of calcium from the vicinity of the channel. When the cell is driven to fire faster, calcium is expected to accumulate, and this is expected to result in accumulation of calcium-dependent AHP current. We measured the time course of calcium transients in the soma and proximal dendrites of STN neurons during spontaneous firing and their accumulation during driven firing. We compared these to the time course and accumulation of AHP currents using whole-cell and perforated patch recordings. During spontaneous firing, a rise in free cytoplasmic calcium was seen after each action potential, and decayed with a time constant of about 200 ms in the soma, and 80 ms in the dendrites. At rates higher than 10 Hz, calcium transients accumulated as predicted. In addition, there was a slow calcium transient not predicted by summation of action potentials that became more pronounced at high firing frequency. Spike AHP currents were measured in voltage clamp as tail currents after 2 ms voltage pulses that triggered action currents. Apamin-sensitive AHP (SK) current was measured by subtraction of tail currents obtained before and after treatment with apamin. SK current peaked between 10 and 15 ms after an action potential, had a decay time constant of about 30 ms, and showed no accumulation. At frequencies between 5 and 200 spikes s(-1), the maximal SK current remained the same as that evoked by a single action potential. AHP current did not have time to decay between action potentials, so at frequencies above 50 spikes s(-1) the apamin-sensitive current was effectively constant. These results are inconsistent with the view that the decay of SK current is governed by calcium dynamics. They

  15. Mobilization of dantrolene-sensitive intracellular calcium pools is involved in the cytotoxicity induced by quisqualate and N-methyl-D-aspartate but not by 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate and kainate in cultured cerebral cortical neurons.

    PubMed Central

    Frandsen, A; Schousboe, A

    1992-01-01

    By using primary cultures of cerebral cortical neurons, it has been demonstrated that the antihyperthermia drug dantrolene protects against cytotoxicity induced by the excitatory amino acids quisqualate (QA) and N-methyl-D-aspartate (NMDA), whereas no effect was observed on cell damage mediated by kainate (KA) or 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate (AMPA). In parallel it was shown that KA and AMPA increased the concentration of intracellular free calcium ([Ca2+]i) mainly by influx, whereas the increase in [Ca2+]i stimulated by NMDA and QA predominantly was caused by release of Ca2+ from intracellular stores, which for NMDA seemed to be mediated at least partly by Ca2+ influx. In accordance with the effects on cytotoxicity, dantrolene blocked the increase in [Ca2+]i elicited by QA and NMDA leaving the increase induced by KA and AMPA unaffected. The finding that 2-amino-3-[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propionate, which regarding toxicity is a selective KA antagonist, only reduced the KA-stimulated increase in [Ca2+]i by 30% may suggest that the elevation of [Ca2+]i is not the only element in KA-induced cytotoxicity. On the other hand, the present study underlines the importance of Ca2+ for cytotoxicity induced by some excitatory amino acids (glutamate, NMDA, and QA) and supports the current proposal that multiple mechanisms are operating, even concerning calcium homeostasis. Because excitatory amino acid-induced cytotoxicity is thought to be involved in neuropathological conditions such as ischemia, it is possible that dantrolene might be of therapeutic interest. PMID:1372982

  16. Calcium transients and the effect of a photolytically released calcium chelator during electrically induced contractions in rabbit rectococcygeus smooth muscle.

    PubMed Central

    Arner, A; Malmqvist, U; Rigler, R

    1998-01-01

    Intracellular Ca2+ was determined with the fura-2 technique during electrically induced contractions in the rabbit rectococcygeus smooth muscle at 22 degreesC. The muscles were electrically activated to give short, reproducible contractions. Intracellular [Ca2+] increased during activation; the increase in [Ca2+] preceded force development by approximately 2 s. After cessation of stimulation Ca2+ fell, preceding the fall in force by approximately 4 s. The fluorescence properties of fura-2 were determined with time-resolved spectroscopy using synchrotron light at the MAX-storage ring, Lund, Sweden. The fluorescence decay of free fura-2 was best described by two exponential decays (time constants approximately 0.5 and 1.5 ns) at low Ca2+ (pCa 9). At high Ca2+ (pCa 4.5), fluorescence decay became slower and could be fitted by one exponential decay (1.9 ns). Time-resolved anisotropy of free fura-2 was characteristic of free rotational motion (correlation time 0.3 ns). Motion of fura-2 could be markedly inhibited by high concentrations of creatine kinase. Time-resolved spectroscopy measurements of muscle fibers loaded with fura-2 showed that the fluorescence lifetime of the probe was longer, suggesting an influence of the chemical environment. Anisotropy measurements revealed, however, that the probe was mobile in the cells. The Ca2+-dependence of contraction and relaxation was studied using a photolabile calcium chelator, diazo-2, which could be loaded into the muscle cells in a similar manner as fura-2. Photolysis of diazo-2 leads to an increase in its Ca2+-affinity and a fall in free Ca2+. When muscles that had been loaded with diazo-2 were illuminated with UV light flashes during the rising phase of contraction, the rate of contraction became slower, suggesting a close relation between intracellular Ca2+ and the cross-bridge interaction. In contrast, photolysis during relaxation did not influence the rate of force decay, suggesting that relaxation of these

  17. [HOMOCYSTEINE-INDUCED MEMBRANE CURRENTS, CALCIUM RESPONSES AND CHANGES OF MITOCHONDRIAL POTENTIAL IN RAT CORTICAL NEURONS].

    PubMed

    Abushik, P A; Karelina, T V; Sibarov, D A; Stepanenko, J D; Giniatullin, R; Antonov, S M

    2015-01-01

    Homocysteine, a sulfur-containing amino acid, exhibits neurotoxic effects and is involved in the pathogenesis of several major neurodegenerative disorders. In contrast to well studied excitoxicity of glutamate, the mechanism of homocysteine neurotoxicity is not clearly understood. By using whole-cell patch-clamp, calcium imaging (fluo-3) and measurements of mitochondrial membrane potential (rhodamine 123) we studied transmembrane currents, calcium signals and changes in mitochondrial membrane potential induced by homocysteine versus responses induced by NMDA and glutamate in cultured rat cortical neurons. L-homocysteine (50 µM) induced inward currents that could be completely blocked by the selective antagonist of NMDA receptors - AP-5. In contrast to NMDA-induced currents, homocysteine-induced currents had a smaller steady-state amplitude. Comparison of calcium responses to homocysteine, NMDA or glutamate demonstrated that in all cortical neurons homocysteine elicited short, oscillatory-type calcium responses, whereas NMDA or glutamate induced sustained increase of intracellular calcium. Analysis of mitochondrial changes demonstrated that in contrast to NMDA homocysteine did not cause a drop of mitochondrial membrane potential at the early stages of action. However, after its long-term action, as in the case of NMDA and glutamate, the changes in mitochondrial membrane potential were comparable with the full drop of respiratory chain induced by protonophore FCCP. Our data suggest that in cultured rat cortical neuron homocysteine at the first stages of action induces neurotoxic effects through activation of NMDA-type ionotropic glutamate receptors with strong calcium influx through the channels of these receptors. The long-term action of homocysteine may lead to mitochondrial disfuction and appears as a drop of mitochondrial membrane potential.

  18. Involvement of dihydropyridine-sensitive calcium channels in high asynchrony of transmitter release in neuromuscular synapses of newborn rats.

    PubMed

    Khuzakhmetova, V F; Nurullin, L F; Bukharaeva, E A; Nikolsky, E E

    2016-09-01

    Experiments on neuromuscular synapses of rats at different stages of ontogenesis have been performed. It has been found that one of the reasons of higher asynchrony of the release of single quanta of acetylcholine in the synapses of newborn animals is the activity of the presynaptic dihydropyridine-sensitive calcium channels of the L-type.

  19. Surface acidity and solid-state compatibility of excipients with an acid-sensitive API: case study of atorvastatin calcium.

    PubMed

    Govindarajan, Ramprakash; Landis, Margaret; Hancock, Bruno; Gatlin, Larry A; Suryanarayanan, Raj; Shalaev, Evgenyi Y

    2015-04-01

    The objectives of this study were to measure the apparent surface acidity of common excipients and to correlate the acidity with the chemical stability of an acid-sensitive active pharmaceutical ingredient (API) in binary API-excipient powder mixtures. The acidity of 26 solid excipients was determined by two methods, (i) by measuring the pH of their suspensions or solutions and (ii) the pH equivalent (pHeq) measured via ionization of probe molecules deposited on the surface of the excipients. The chemical stability of an API, atorvastatin calcium (AC), in mixtures with the excipients was evaluated by monitoring the appearance of an acid-induced degradant, atorvastatin lactone, under accelerated storage conditions. The extent of lactone formation in AC-excipient mixtures was presented as a function of either solution/suspension pH or pHeq. No lactone formation was observed in mixtures with excipients having pHeq > 6, while the lactone levels were pronounced (> 0.6% after 6 weeks at 50°C/20% RH) with excipients exhibiting pHeq < 3. The three pHeq regions (> 6, 3-6, and < 3) were consistent with the reported solution pH-stability profile of AC. In contrast to the pHeq scale, lactone formation did not show any clear trend when plotted as a function of the suspension/solution pH. Two mechanisms to explain the discrepancy between the suspension/solution pH and the chemical stability data were discussed. Acidic excipients, which are expected to be incompatible with an acid-sensitive API, were identified based on pHeq measurements. The incompatibility prediction was confirmed in the chemical stability tests using AC as an example of an acid-sensitive API.

  20. Disrupted calcium homeostasis is involved in elevated zinc ion-induced photoreceptor cell death.

    PubMed

    Guo, Dadong; Du, Yuxiang; Wu, Qiuxin; Jiang, Wenjun; Bi, Hongsheng

    2014-10-15

    Zinc (Zn), the second abundant trace element in living organisms, plays an important role in regulating cell metabolism, signaling, proliferation, gene expression and apoptosis. Meanwhile, the overload of Zn will disrupt the intracellular calcium homeostasis via impairing mitochondrial function. However, the specific molecular mechanism underlying zinc-induced calcium regulation remains poorly understood. In the present study, using zinc chloride (ZnCl2) as a stressor, we investigated the effect of exogenous Zn(2+) in regulating murine photoreceptor cell viability, reactive oxygen species (ROS), cell cycle distribution and calcium homeostasis as well as plasma membrane calcium ATPase (PMCA) isoforms (PMCA1 and PMCA2, i.e., ATP2B1, ATP2B2) expression. We found that the exogenous Zn(2+) in the exposure range (31.25-125.0 μmol/L) results in the overgeneration of ROS, cell cycle arrest at G2/M phases, elevation of cytosolic [Ca(2+)], inactivation of Ca(2+)-ATPase and reduction of both PMCA1 and PMCA2 in 661 W cells, and thus induces cell death. In conclusion, ZnCl2 exposure can elevate the cytosolic [Ca(2+)], disrupt the intracellular calcium homeostasis, further initiate Ca(2+)-dependent signaling pathway in 661 W cells, and finally cause cell death. Our results will facilitate the understanding of cell death induced by the zinc ion-mediated calcium homeostasis disruption. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. [Effect of calcium on medium alkalinization induced by salicylic acid in Salvia miltiorrhiza suspension cultures].

    PubMed

    Liu, Liancheng; Wang, Cong; Dong, Juan'e; Su, Hui; Zhuo, Zequn; Xue, Yaxin

    2013-07-01

    We studied medium alkalinization in Salvia miltiorrhiza suspension cultures treated with salicylic acid and the effect of Ca2+ in this process through application of calcium channel antagonists (Verapamil, LaCl3, LiCl, 2-APB) and ionophore A23187. The results show that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture. Verapamil and LaCl3 or LiCl and 2-APB, two different groups of calcium channel antagonist, significantly inhibited the medium alkalinization induced by salicylic acid. However, the suppression effect of verapamil or LaCl3 on medium alkalinization induced by salicylic acid was higher than that of LiCl or 2-APB. When two types of calcium channel inhibitor (LaCl3 and 2-APB) were used together, the medium alkalinization induced by salicylic acid was completely suppressed and even reduced the pH in medium. On the other hand, A23187 could promote the medium alkalinization. Based on the results above, we speculated that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture, depending on the calcium from both extracell and intracell. Moreover, calcium from extracell plays a more dominant role in this process. Reveal of relationship in this research between Ca2+ and medium alkalinization can provide theory evidence for mechanism of the plant secondary metabolism.

  2. Determination of the calcium antagonist benidipine hydrochloride in plasma by sensitive radioimmunoassay.

    PubMed

    Akinaga, S; Kobayashi, H; Kobayashi, S; Inoue, A; Nakamizo, N; Oka, T

    1988-11-01

    A sensitive radioimmunoassay of the 1,4-dihydropyridine calcium channel blocker (+/-)-(R*)-2,6-dimethyl-4-(m-nitrophenyl)-1,4-dihydropyridine-3,5-dicarb oxylic acid (R*)-1-benzyl-3-piperidinyl ester, methyl ester hydrochloride (benidipine hydrochloride, KW-3049) has been developed. Antiserum against KW-3049 was produced in rabbits by immunization with an immunogen prepared by conjugating a derivative of KW-3049 to bovine serum albumin. This antiserum was found to specifically bind to [3H]-KW 3049, while the recognition to [3H]-nitrendipine, another well-known dihydropyridine calcium channel blocker, was less pronounced. With the antiserum, [3H]-KW-3049 and dextran coated charcoal, this radioimmunoassay could detect 39 approximately equal to 500 pg/tube of KW-3049 in a buffer system, and 156 to 5000 pg/ml of KW-3049 in plasma by using 0.5 ml of the plasma which was pretreated with MeOH for deproteinization and extracted with diethyl ether under alkaline condition. To assess the specificity of the radioimmunoassay, the inhibition of [3H]-KW-3049 binding to the antiserum by the presumable metabolites was examined. Though three of these presumable metabolites could slightly inhibit the binding of [3H]-KW-3049, they were not detected in rat and dog plasma at 0.5 h after oral administration of KW-3049. Plasma levels of KW-3049 in rats receiving a single oral dose (1 mg/kg) determined by the radioimmunoassay show good agreement with those obtained by gas chromotography.

  3. Interaction of GCAP1 with retinal guanylyl cyclase and calcium: sensitivity to fatty acylation

    PubMed Central

    Peshenko, Igor V.; Olshevskaya, Elena V.; Dizhoor, Alexander M.

    2012-01-01

    Guanylyl cyclase activating proteins (GCAPs) are calcium/magnesium binding proteins within neuronal calcium sensor proteins group (NCS) of the EF-hand proteins superfamily. GCAPs activate retinal guanylyl cyclase (RetGC) in vertebrate photoreceptors in response to light-dependent fall of the intracellular free Ca2+ concentrations. GCAPs consist of four EF-hand domains and contain N-terminal fatty acylated glycine, which in GCAP1 is required for the normal activation of RetGC. We analyzed the effects of a substitution prohibiting N-myristoylation (Gly2 → Ala) on the ability of the recombinant GCAP1 to co-localize with its target enzyme when heterologously expressed in HEK293 cells. We also compared Ca2+ binding and RetGC-activating properties of the purified non-acylated G2A mutant and C14:0 acylated GCAP1 in vitro. The G2A GCAP1 expressed with a C-terminal GFP tag was able to co-localize with the cyclase, albeit less efficiently than the wild type, but much less effectively stimulated cyclase activity in vitro. Ca2+ binding isotherm of the G2A GCAP1 was slightly shifted toward higher free Ca2+ concentrations and so was Ca2+ sensitivity of RetGC reconstituted with the G2A mutant. At the same time, myristoylation had little effect on the high-affinity Ca2+-binding in the EF-hand proximal to the myristoyl residue in three-dimensional GCAP1 structure. These data indicate that the N-terminal fatty acyl group may alter the activity of EF-hands in the distal portion of the GCAP1 molecule via presently unknown intramolecular mechanism. PMID:22371697

  4. Synthetic Aβ oligomers (Aβ(1-42) globulomer) modulate presynaptic calcium currents: prevention of Aβ-induced synaptic deficits by calcium channel blockers.

    PubMed

    Hermann, David; Mezler, Mario; Müller, Michaela K; Wicke, Karsten; Gross, Gerhard; Draguhn, Andreas; Bruehl, Claus; Nimmrich, Volker

    2013-02-28

    Alzheimer's disease is accompanied by increased brain levels of soluble amyloid-β (Aβ) oligomers. It has been suggested that oligomers directly impair synaptic function, thereby causing cognitive deficits in Alzheimer's disease patients. Recently, it has been shown that synthetic Aβ oligomers directly modulate P/Q-type calcium channels, possibly leading to excitotoxic cascades and subsequent synaptic decline. Using whole-cell recordings we studied the modulation of recombinant presynaptic calcium channels in HEK293 cells after application of a stable Aβ oligomer preparation (Aβ1-42 globulomer). Aβ globulomer shifted the half-activation voltage of P/Q-type and N-type calcium channels to more hyperpolarized values (by 11.5 and 7.5 mV). Application of non-aggregated Aβ peptides had no effect. We then analyzed the potential of calcium channel blockers to prevent Aβ globulomer-induced synaptic decline in hippocampal slice cultures. Specific block of P/Q-type or N-type calcium channels with peptide toxins completely reversed Aβ globulomer-induced deficits in glutamatergic neurotransmission. Two state-dependent low molecular weight P/Q-type and N-type calcium channel blockers also protected neurons from Aβ-induced alterations. On the contrary, inhibition of L-type calcium channels failed to reverse the deficit. Our data show that Aβ globulomer directly modulates recombinant P/Q-type and N-type calcium channels in HEK293 cells. Block of presynaptic calcium channels with both state-dependent and state-independent modulators can reverse Aβ-induced functional deficits in synaptic transmission. These findings indicate that presynaptic calcium channel blockers may be a therapeutic strategy for the treatment of Alzheimer's disease.

  5. Engineered Troponin C Constructs Correct Disease-related Cardiac Myofilament Calcium Sensitivity*

    PubMed Central

    Liu, Bin; Lee, Ryan S.; Biesiadecki, Brandon J.; Tikunova, Svetlana B.; Davis, Jonathan P.

    2012-01-01

    Aberrant myofilament Ca2+ sensitivity is commonly observed with multiple cardiac diseases, especially familial cardiomyopathies. Although the etiology of the cardiomyopathies remains unclear, improving cardiac muscle Ca2+ sensitivity through either pharmacological or genetic approaches shows promise of alleviating the disease-related symptoms. Due to its central role as the Ca2+ sensor for cardiac muscle contraction, troponin C (TnC) stands out as an obvious and versatile target to reset disease-associated myofilament Ca2+ sensitivity back to normal. To test the hypothesis that aberrant myofilament Ca2+ sensitivity and its related function can be corrected through rationally engineered TnC constructs, three thin filament protein modifications representing different proteins (troponin I or troponin T), modifications (missense mutation, deletion, or truncation), and disease subtypes (familial or acquired) were studied. A fluorescent TnC was utilized to measure Ca2+ binding to TnC in the physiologically relevant biochemical model system of reconstituted thin filaments. Consistent with the pathophysiology, the restrictive cardiomyopathy mutation, troponin I R192H, and ischemia-induced truncation of troponin I (residues 1–192) increased the Ca2+ sensitivity of TnC on the thin filament, whereas the dilated cardiomyopathy mutation, troponin T ΔK210, decreased the Ca2+ sensitivity of TnC on the thin filament. Rationally engineered TnC constructs corrected the abnormal Ca2+ sensitivities of the thin filament, reconstituted actomyosin ATPase activity, and force generation in skinned trabeculae. Thus, the present study provides a novel and versatile therapeutic strategy to restore diseased cardiac muscle Ca2+ sensitivity. PMID:22511780

  6. Levetiracetam Inhibits Both Ryanodine and IP3 Receptor Activated Calcium Induced Calcium Release in Hippocampal Neurons in Culture

    PubMed Central

    Nagarkatti, Nisha; Deshpande, Laxmikant S.; DeLorenzo, Robert J.

    2010-01-01

    Epilepsy affects approximately 1% of the population worldwide, and there is a pressing need to develop new anti-epileptic drugs (AEDs) and understand their mechanisms of action. Levetiracetam (LEV) is a novel AED and despite its increasingly widespread clinical use, its mechanism of action is as yet undetermined. Intracellular calcium ([Ca2+]i) regulation by both inositol 1,4,5-triphosphate receptors (IP3R) and ryanodine receptors (RyR) has been implicated in epileptogenesis and the maintenance of epilepsy. To this end, we investigated the effect of LEV on RyR and IP3R activated calcium-induced calcium release (CICR) in hippocampal neuronal cultures. RyR-mediated CICR was stimulated using the well-characterized RyR activator, caffeine. Caffeine (10mM) caused a significant increase in [Ca2+]i in hippocampal neurons. Treatment with LEV (33μM) prior to stimulation of RyR-mediated CICR by caffeine led to a 61% decrease in the caffeine induced peak height of [Ca2+]i when compared to the control. Bradykinin stimulates IP3R-activated CICR—to test the effect of LEV on IP3R-mediated CICR, bradykinin (1μM) was used to stimulate cells pre-treated with LEV (100μM). The data showed that LEV caused a 74% decrease in IP3R-mediated CICR compared to the control. In previous studies we have shown that altered Ca2+ homeostatic mechanisms play a role in seizure activity and the development of spontaneous recurrent epileptiform discharges (SREDs). Elevations in [Ca2+]i mediated by CICR systems have been associated with neurotoxicity, changes in neuronal plasticity, and the development of AE. Thus, the ability of LEV to modulate the two major CICR systems demonstrates an important molecular effect of this agent on a major second messenger system in neurons. PMID:18406528

  7. PKCα mediates acetylcholine-induced activation of TRPV4-dependent calcium influx in endothelial cells

    PubMed Central

    Adapala, Ravi K.; Talasila, Phani K.; Bratz, Ian N.; Zhang, David X.; Suzuki, Makoto; Meszaros, J. Gary

    2011-01-01

    Transient receptor potential vanilloid channel 4 (TRPV4) is a polymodally activated nonselective cationic channel implicated in the regulation of vasodilation and hypertension. We and others have recently shown that cyclic stretch and shear stress activate TRPV4-mediated calcium influx in endothelial cells (EC). In addition to the mechanical forces, acetylcholine (ACh) was shown to activate TRPV4-mediated calcium influx in endothelial cells, which is important for nitric oxide-dependent vasodilation. However, the molecular mechanism through which ACh activates TRPV4 is not known. Here, we show that ACh-induced calcium influx and endothelial nitric oxide synthase (eNOS) phosphorylation but not calcium release from intracellular stores is inhibited by a specific TRPV4 antagonist, AB-159908. Importantly, activation of store-operated calcium influx was not altered in the TRPV4 null EC, suggesting that TRPV4-dependent calcium influx is mediated through a receptor-operated pathway. Furthermore, we found that ACh treatment activated protein kinase C (PKC) α, and inhibition of PKCα activity by the specific inhibitor Go-6976, or expression of a kinase-dead mutant of PKCα but not PKCε or downregulation of PKCα expression by chronic 12-O-tetradecanoylphorbol-13-acetate treatment, completely abolished ACh-induced calcium influx. Finally, we found that ACh-induced vasodilation was inhibited by the PKCα inhibitor Go-6976 in small mesenteric arteries from wild-type mice, but not in TRPV4 null mice. Taken together, these findings demonstrate, for the first time, that a specific isoform of PKC, PKCα, mediates agonist-induced receptor-mediated TRPV4 activation in endothelial cells. PMID:21705673

  8. Alpha Hemolysin Induces an Increase of Erythrocytes Calcium: A FLIM 2-Photon Phasor Analysis Approach

    PubMed Central

    Sanchez, Susana; Bakás, Laura; Gratton, Enrico; Herlax, Vanesa

    2011-01-01

    α-hemolysin (HlyA) from Escherichia coli is considered as the prototype of a family of toxins called RTX (repeat in toxin), a group of proteins that share genetic and structural features. HlyA is an important virulence factor in E. coli extraintestinal infections, such as meningitis, septicemia and urinary infections. High concentrations of the toxin cause the lysis of several cells such as erythrocytes, granulocytes, monocytes, endothelial and renal epithelial cells of different species. At low concentrations it induces the production of cytokines and apoptosis. Since many of the subcytolytic effects in other cells have been reported to be triggered by the increase of intracellular calcium, we followed the calcium concentration inside the erythrocytes while incubating with sublytic concentrations of HlyA. Calcium concentration was monitored using the calcium indicator Green 1, 2-photon excitation, and fluorescence lifetime imaging microscopy (FLIM). Data were analyzed using the phasor representation. In this report, we present evidence that, at sublytic concentrations, HlyA induces an increase of calcium concentration in rabbit erythrocytes in the first 10 s. Results are discussed in relation to the difficulties of measuring calcium concentrations in erythrocytes where hemoglobin is present, the contribution of the background and the heterogeneity of the response observed in individual cells. PMID:21698153

  9. Dietary Fructose Inhibits Intestinal Calcium Absorption and Induces Vitamin D Insufficiency in CKD

    PubMed Central

    Douard, Veronique; Asgerally, Abbas; Sabbagh, Yves; Sugiura, Shozo; Shapses, Sue A.; Casirola, Donatella

    2010-01-01

    Renal disease leads to perturbations in calcium and phosphate homeostasis and vitamin D metabolism. Dietary fructose aggravates chronic kidney disease (CKD), but whether it also worsens CKD-induced derangements in calcium and phosphate homeostasis is unknown. Here, we fed rats diets containing 60% glucose or fructose for 1 mo beginning 6 wk after 5/6 nephrectomy or sham operation. Nephrectomized rats had markedly greater kidney weight, blood urea nitrogen, and serum levels of creatinine, phosphate, and calcium-phosphate product; dietary fructose significantly exacerbated all of these outcomes. Expression and activity of intestinal phosphate transporter, which did not change after nephrectomy or dietary fructose, did not correlate with hyperphosphatemia in 5/6-nephrectomized rats. Intestinal transport of calcium, however, decreased with dietary fructose, probably because of fructose-mediated downregulation of calbindin 9k. Serum calcium levels, however, were unaffected by nephrectomy and diet. Finally, only 5/6-nephrectomized rats that received dietary fructose demonstrated marked reductions in 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 levels, despite upregulation of 1α-hydroxylase. In summary, excess dietary fructose inhibits intestinal calcium absorption, induces marked vitamin D insufficiency in CKD, and exacerbates other classical symptoms of the disease. Future studies should evaluate the relevance of monitoring fructose consumption in patients with CKD. PMID:19959720

  10. Serotonin-induced intercellular calcium waves in salivary glands of the blowfly Calliphora erythrocephala.

    PubMed Central

    Zimmermann, B; Walz, B

    1997-01-01

    1. Blowfly salivary glands have been used extensively as a model system for the analysis of inositol phosphate-dependent signal transduction. To detect and characterize changes in intracellular free calcium ([Ca2+]i) that might be expected to be triggered by stimulation with serotonin (5-HT), we have carried out digital calcium-imaging experiments on intact glands using the Ca2+-sensitive dye fura-2. 2. 5-HT (1-10 nM) induced repetitive transient increases in [Ca2+]i, i.e. Ca2+ spikes whose frequency was a function of agonist concentration (EC50 = 2.8 nM). 3. Pre-incubation in EGTA decreased the frequency but did not inhibit spiking. Thapsigargin abolished periodic spike activity indicating that the [Ca2+]i rise results from Ca2+ release. Neither caffeine (10 mM) nor ryanodine (10 and 50 microM) induced increases in [Ca2+]i. 4. Oscillatory activity in individual cells was synchronized by regenerative intercellular Ca2+ waves that propagated over distances greater than 400 microm. Colliding waves annihilated each other. 5. Desynchronization of the oscillation pattern by 100 microM 1-octanol suggests the involvement of gap junctions and an intracellular messenger in wave propagation. 6. Local stimulation of glands elicited [Ca2+]i elevations in the stimulated area, but not in adjacent cells, indicating that local increases in [Ca2+]i are not sufficient to trigger Ca2+ waves. However, local stimulation was capable of evoking propagating Ca2+ waves when combined with low-dose 5-HT stimulation of the whole gland. 7. The data are consistent with the hypothesis that: (1) Ca2+ acts as the intercellular messenger and modulates its own release via positive and negative feedback on the inosital 1,4,5-trisphosphate (InsP3) receptor, and (2) sensitization of the InsP3 receptor to Ca2+ by InsP3 is required for the propagation of intercellular Ca2+ waves, as proposed for intracellular Ca2+ waves in Xenopus oocytes. Images Figure 5 Figure 6 Figure 7 Figure 8 PMID:9097929

  11. Mathematical Modeling of Calcium Waves Induced by Mechanical Stimulation in Keratinocytes

    PubMed Central

    Kobayashi, Yasuaki; Sanno, Yumi; Sakai, Akihiko; Sawabu, Yusuke; Tsutsumi, Moe; Goto, Makiko; Kitahata, Hiroyuki; Nakata, Satoshi; Kumamoto, Junichi; Denda, Mitsuhiro; Nagayama, Masaharu

    2014-01-01

    Recent studies have shown that the behavior of calcium in the epidermis is closely related to the conditions of the skin, especially the differentiation of the epidermal keratinocytes and the permeability barrier function, and therefore a correct understanding of the calcium dynamics is important in explaining epidermal homeostasis. Here we report on experimental observations of in vitro calcium waves in keratinocytes induced by mechanical stimulation, and present a mathematical model that can describe the experimentally observed wave behavior that includes finite-range wave propagation and a ring-shaped pattern. A mechanism of the ring formation hypothesized by our model may be related to similar calcium propagation patterns observed during the wound healing process in the epidermis. We discuss a possible extension of our model that may serve as a tool for investigating the mechanisms of various skin diseases. PMID:24663805

  12. Individual aggregates of amyloid beta induce temporary calcium influx through the cell membrane of neuronal cells

    PubMed Central

    Drews, Anna; Flint, Jennie; Shivji, Nadia; Jönsson, Peter; Wirthensohn, David; De Genst, Erwin; Vincke, Cécile; Muyldermans, Serge; Dobson, Chris; Klenerman, David

    2016-01-01

    Local delivery of amyloid beta oligomers from the tip of a nanopipette, controlled over the cell surface, has been used to deliver physiological picomolar oligomer concentrations to primary astrocytes or neurons. Calcium influx was observed when as few as 2000 oligomers were delivered to the cell surface. When the dosing of oligomers was stopped the intracellular calcium returned to basal levels or below. Calcium influx was prevented by the presence in the pipette of the extracellular chaperone clusterin, which is known to selectively bind oligomers, and by the presence a specific nanobody to amyloid beta. These data are consistent with individual oligomers larger than trimers inducing calcium entry as they cross the cell membrane, a result supported by imaging experiments in bilayers, and suggest that the initial molecular event that leads to neuronal damage does not involve any cellular receptors, in contrast to work performed at much higher oligomer concentrations. PMID:27553885

  13. Combination therapy of Chinese herbal medicine Fructus Ligustri Lucidi with high calcium diet on calcium imbalance induced by ovariectomy in mice.

    PubMed

    Zhang, Yan; Mukwaya, Emmanuel; Pan, Hai; Li, Xiao-Min; Yang, Jiu-Lin; Ge, Jun; Wang, Hai-Ying

    2015-07-01

    Our previous biological study demonstrated that Fructus Ligustri Lucidi (FLL), the fruit of Ligustrum lucidum Ait. (Oleaceae), could be used to maintain calcium balance and prevent age-related osteoporosis since it effectively decreased calcium loss and increased calcium retention in rats. This study investigates the combination effect of the Chinese herbal medicine FLL and a high calcium diet on calcium imbalance induced by ovariectomy in mice. The ovariectomized (OVX) mice were orally treated with vehicle, FLL extract (700 mg/kg), milk powder (5 g/mice) fortified with calcium (1.0% Ca) and the combination of FLL with milk powder. After 6 weeks of treatment, urine, serum, and tibia were preserved for biochemical analysis and kidneys were taken for gene expression analysis. The combination treatment of FLL and a high calcium diet significantly increased bone calcium content (6.80 ± 0.34 mg) by 22% (p < 0.05) and decreased urine calcium excretion (0.099 ± 0.009 mg/mg) by 62% (p < 0.01) as compared with those of the OVX group (bone Ca, 5.57 ± 0.31 mg; urine Ca/Cr, 0.261 ± 0.017 mg/mg). The mRNA expression of renal calcium-binding protein-9k (CaBP-9k) and calcium-sensing receptor (CaSR) in combination treatment group was significantly up-regulated and down-regulated, respectively, as compared with those of the OVX group. The beneficial effects of this combination therapy on calcium balance of OVX mice were, at least partially, attributed to its regulation on mRNA expression of CaBP-9k and CaSR in kidney.

  14. 3,4-Methylenedioxymethamphetamine (Ecstasy) increases the sensitivity of the contractile apparatus to calcium ions in both malignant hyperthermia-susceptible and normal skeletal muscle fibres.

    PubMed

    Gerbershagen, Mark U; Missler, Goetz; Schütte, Jan K; Starosse, Alexander; Graf, Bernhard M; Wappler, Frank; Zink, Wolfgang

    2012-01-01

    The present study was designed to investigate whether 3,4-methylenedioxymethamphetamine (MDMA, 'Ecstasy') increases the sensitivity of the contractile apparatus to calcium in muscle fibres from malignant hyperthermia-susceptible and malignant hyperthermia-negative pigs, whether it causes calcium ion release from the sarcoplasmic reticulum and whether it inhibits calcium reuptake into the sarcoplasmic reticulum. Experimental study, using a model of porcine saponin-skinned fibres. Administration of MDMA in concentrations of 1, 2 and 4 mmol l(-1)l did not result in relevant force transients in skinned muscle fibres of malignant hyperthermia-susceptible or malignant hyperthermia-negative pigs. Furthermore, MDMA in these concentrations did not alter calcium ion loading of the sarcoplasmic reticulum in either group. With regard to changes in the calcium ion sensitivity of the contractile proteins, however, MDMA dose-dependently increased (pCa50) values (negative decadic logarithm of [Ca2+] at which isometric force is half-maximal) in both groups. In the present study, we were able to demonstrate that MDMA dose-dependently increases the sensitivity of the contractile apparatus to calcium in both malignant hyperthermia-susceptible and malignant hyperthermia-negative fibres. Consequently, the malignant hyperthermia status should not affect the calcium sensitivity of the contractile apparatus. However, the increased calcium sensitivity is an important finding that must be appreciated, particularly in relation to the agonistic effect of MDMA at the nicotinic acetylcholine receptor, which increases intracellular calcium ion concentrations.

  15. Clinicopathology of gout in growing layers induced by high calcium and high protein diets.

    PubMed

    Guo, X; Huang, K; Tang, J

    2005-10-01

    1. An experiment was conducted to test the independent and combined effects of high dietary calcium and protein concentrations on the induction of visceral gout in growing birds of a layer strain. 2. One hundred and sixty healthy birds were randomly divided into 4 groups at 35 d of age. The different groups were given 4 diets containing normal or high concentrations of dietary calcium or crude protein in a 2 x 2 factorial experiment for 30 d. The diets contained normal calcium (Ca) and crude protein (CP) (NCNP, 8.5 g Ca/kg and 175g CP/kg), high calcium and normal protein (HC, 36.3 g Ca/kg and 175 g CP/kg), normal calcium and high protein (HP, 8.8 g Ca/kg and 245 g CP/kg) or high calcium and high protein (HCHP, 36.8 g Ca/kg and 242 g CP/kg), respectively. 3. Typical visceral gout was induced by the HCHP diet. The HCHP and HC diet caused severe kidney damage. The HP diet did not cause kidney damage, but significantly increased plasma uric acid and inorganic phosphorus concentrations. 4. The HC diet significantly increased plasma uric acid, calcium and sodium, but significantly decreased plasma inorganic phosphorus, potassium and magnesium concentrations. The HCHP diet significantly increased plasma uric acid, calcium and sodium. 5. Urine volumes were significantly higher on the HCHP and HC diets than on the control. The growers raised on HC and HCHP diets had significantly higher total quantity of 24 h urinary excretion of uric acid, calcium, magnesium, inorganic phosphorus and potassium and a significantly lower 24 h urinary excretion of sodium. The growers fed on the HP diet had a higher 24 h urinary excretion of uric acid and inorganic phosphorus than the control. 6. It is concluded that growing layer birds should not be fed on layer rations.

  16. Levosimendan: a calcium-sensitizing agent for the treatment of patients with decompensated heart failure.

    PubMed

    Lehtonen, Lasse

    2004-09-01

    Levosimendan is a new inodilator. Its mechanism of action includes calcium sensitization of contractile proteins and the opening of adenosine triphosphate-dependent K channels. The combination of positive inotropy with anti-ischemic effects of K-channel opening offers potential benefits in comparison with currently available intravenous inotropes, which are contraindicated in patients with ongoing myocardial ischemia. Levosimendan has been extensively studied in various animal models of heart failure, in which the drug has increased contractility without adverse effects on diastolic function. These results have been repeated in patients with heart failure, in whom levosimendan dose-dependently increases cardiac output and reduces pulmonary capillary wedge pressure. The active metabolite of levosimendan (OR-1896) significantly prolongs the duration of the hemodynamic effects of the therapeutic 24-hour levosimendan infusion. Levosimendan has been studied in two major trials with decompensated patients (LIDO and RUSSLAN), in which it showed outcome benefits in comparison with dobutamine and placebo, respectively. A third comparative study (CASINO) recently suggested mortality benefits with levosimendan over placebo and dobutamine. Currently, two large prospective trials (SURVIVE and REVIVE) in patients who are hospitalized because of worsening heart failure are underway. These trials will conclusively prove whether levosimendan should be added to the standard treatment in patients who are hospitalized because of cardiac decompensation.

  17. Perovskite-type calcium titanate nanoparticles as novel matrix for designing sensitive electrochemical biosensing.

    PubMed

    Wang, Lei; Li, Juan; Feng, Mengjie; Min, Lingfeng; Yang, Juan; Yu, Suhua; Zhang, Yongcai; Hu, Xiaoya; Yang, Zhanjun

    2017-10-15

    In this work, novel perovskite-type calcium titanate nanoparticles (CaTiO3NPs) were for the first time exploited for the immobilization of proteins and the development of electrochemical biosensor. The CaTiO3NPs were synthesized with a simple and cost-effective route at low temperature, and characterized by scanning electron microscopy, X-ray photoelectron spectroscopic spectrum, electrochemical impedance spectrum, UV-visible spectroscopy, Fourier transform infrared spectrum, and cyclic voltammetry, respectively. The results indicated that CaTiO3NPs exhibited large surface area, and greatly promoted the direct electron transfer between enzyme molecules and electrode surface. The immobilized enzymes on this matrix retained its native bioactivity and exhibited a surface controlled, quasi-reversible two-proton and two-electron transfer reaction with an electron transfer rate of 3.35s(-1). Using glucose oxidase as model, the prepared glucose biosensor showed a high sensitivity of 14.10±0.5mAM(-1) cm(-2), a wide linear range of 7.0×10(-6) to 1.49×10(-3)M, and a low detection limit of 2.3×10(-6)M at signal-to-noise of 3. Moreover, the biosensor also possessed good reproducibility, excellent selectivity and acceptable storage life. This research provided a new-type and promising perovskite nanomaterials for the development of efficient biosensors. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. TRPV3 is a calcium-permeable temperature-sensitive cation channel.

    PubMed

    Xu, Haoxing; Ramsey, I Scott; Kotecha, Suhas A; Moran, Magdalene M; Chong, Jayhong A; Lawson, Deborah; Ge, Pei; Lilly, Jeremiah; Silos-Santiago, Inmaculada; Xie, Yu; DiStefano, Peter S; Curtis, Rory; Clapham, David E

    2002-07-11

    Transient receptor potential (TRP) proteins are cation-selective channels that function in processes as diverse as sensation and vasoregulation. Mammalian TRP channels that are gated by heat and capsaicin (>43 degrees C; TRPV1 (ref. 1)), noxious heat (>52 degrees C; TRPV2 (ref. 2)), and cooling (< 22 degrees C; TRPM8 (refs 3, 4)) have been cloned; however, little is known about the molecular determinants of temperature sensing in the range between approximately 22 degrees C and 40 degrees C. Here we have identified a member of the vanilloid channel family, human TRPV3 (hTRPV3) that is expressed in skin, tongue, dorsal root ganglion, trigeminal ganglion, spinal cord and brain. Increasing temperature from 22 degrees C to 40 degrees C in mammalian cells transfected with hTRPV3 elevated intracellular calcium by activating a nonselective cationic conductance. As in published recordings from sensory neurons, the current was steeply dependent on temperature, sensitized with repeated heating, and displayed a marked hysteresis on heating and cooling. On the basis of these properties, we propose that hTRPV3 is thermosensitive in the physiological range of temperatures between TRPM8 and TRPV1.

  19. New Conotoxin SO-3 Targeting N-type Voltage-Sensitive Calcium Channels

    PubMed Central

    Wen, Lei; Yang, Sheng; Zhou, Wenxia; Zhang, Yongxiang; Huang, Peitang

    2006-01-01

    Selective blockers of the N-type voltage-sensitive calcium (CaV) channels are useful in the management of severe chronic pain. Here, the structure and function characteristics of a novel N-type CaV channel blocker, SO-3, are reviewed. SO-3 is a 25- amino acid conopeptide originally derived from the venom of Conus striatus, and contains the same 4-loop, 6-cysteine framework (C-C-CC-C-C) as O-superfamily conotoxins. The synthetic SO-3 has high analgesic activity similar to ω-conotoxin MVIIA (MVIIA), a selective N-type CaV channel blocker approved in the USA and Europe for the alleviation of persistent pain states. In electrophysiological studies, SO-3 shows more selectivity towards the N-type CaV channels than MVIIA. The dissimilarity between SO-3 and MVIIA in the primary and tertiary structures is further discussed in an attempt to illustrate the difference in selectivity of SO-3 and MVIIA towards N-type CaV channels.

  20. Improved sensitivity in flow cytometric intracellular ionized calcium measurement using fluo-3/Fura Red fluorescence ratios.

    PubMed

    Novak, E J; Rabinovitch, P S

    1994-10-01

    Measurement of changes in intracellular ionized calcium concentrations ([Ca2+]i) has proved to be of wide use in the study of cellular responses to activating stimuli. The fluorescent dye Indo-1 has successfully been used in flow cytometry for this purpose, and when used as a ratiometric indicator it provides optimum sensitivity and accuracy. Unfortunately, this dye requires ultraviolet (UV) excitation which is often not available. We show here that similar results can be obtained using a ratio of green to red fluorescence from the simultaneous loading of the dyes Fura Red and fluo-3. Both Fura Red and fluo-3 are excited using the commonly available blue 488 nm laser line. With appropriate concentrations of the two dyes, the magnitude of response with the fluo-3/Fura Red ratio is greater than that achieved with indo-1, while the intercellular variation in measurement is similar to that seen with indo-1. Analyses can be simultaneously combined with immunofluorescent detection of PE-labeled antibodies to enable [Ca2+]i measurement within cell subsets.

  1. Dehydration-induced amorphous phases of calcium carbonate.

    PubMed

    Saharay, Moumita; Yazaydin, A Ozgur; Kirkpatrick, R James

    2013-03-28

    Amorphous calcium carbonate (ACC) is a critical transient phase in the inorganic precipitation of CaCO3 and in biomineralization. The calcium carbonate crystallization pathway is thought to involve dehydration of more hydrated ACC to less hydrated ACC followed by the formation of anhydrous ACC. We present here computational studies of the transition of a hydrated ACC with a H2O/CaCO3 ratio of 1.0 to anhydrous ACC. During dehydration, ACC undergoes reorganization to a more ordered structure with a significant increase in density. The computed density of anhydrous ACC is similar to that of calcite, the stable crystalline phase. Compared to the crystalline CaCO3 phases, calcite, vaterite, and aragonite, the computed local structure of anhydrous ACC is most-similar to those of calcite and vaterite, but the overall structure is not well described by either. The strong hydrogen bond interaction between the carbonate ions and water molecules plays a crucial role in stabilizing the less hydrated ACC compositions compared to the more hydrated ones, leading to a progressively increasing hydration energy with decreasing water content.

  2. Shear stress-induced NO production is dependent on ATP autocrine signaling and capacitative calcium entry

    PubMed Central

    Andrews, Allison M.; Jaron, Dov; Buerk, Donald G.; Barbee, Kenneth A.

    2014-01-01

    Flow-induced production of nitric oxide (NO) by endothelial cells plays a fundamental role in vascular homeostasis. However, the mechanisms by which shear stress activates NO production remain unclear due in part to limitations in measuring NO, especially under flow conditions. Shear stress elicits the release of ATP, but the relative contribution of autocrine stimulation by ATP to flow-induced NO production has not been established. Furthermore, the importance of calcium in shear stress-induced NO production remains controversial, and in particular the role of capacitive calcium entry (CCE) has yet to be determined. We have utilized our unique NO measurement device to investigate the role of ATP autocrine signaling and CCE in shear stress-induced NO production. We found that endogenously released ATP and downstream activation of purinergic receptors and CCE plays a significant role in shear stress-induced NO production. ATP-induced eNOS phophorylation under static conditions is also dependent on CCE. Inhibition of protein kinase C significantly inhibited eNOS phosphorylation and the calcium response. To our knowledge, we are the first to report on the role of CCE in the mechanism of acute shear stress-induced NO response. In addition, our work highlights the importance of ATP autocrine signaling in shear stress-induced NO production. PMID:25386222

  3. Effects of extracellular calcium and sodium on depolarization-induced automaticity in guinea pig papillary muscle.

    PubMed

    Katzung, B G

    1975-07-01

    Regenerative discharge of action potentials is induced in mammalian papillary muscles by passage of small depolarizing currents. In this paper, the effects of various extracellular calcium and sodium concentrations and of tetrodotoxin on this phenomenon were studied in guinea pig papillary muscles in a sucrose gap chamber. Phase 4 diastolic depolarization was found to be associated with an increase in membrane resistance. The slope of phase 4 depolarization was decreased by reductions in extracellular calcium or sodium concentration. The range of maximum diastolic potentials and the thresholds from which regenerative potentials arose were reduced, especially at the positive limit of potentials, by a reduction in either ion. It was concluded that both calcium and sodium influence diastolic depolarization and participate in the regenerative action potentials of depolarization-induced ventricular automaticity.

  4. Calcium-induced patterns of calcium-oxalate crystals in isolated leaflets of Gleditsia triacanthos L. and Albizia julibrissin Durazz.

    PubMed

    Borchert, R

    1985-08-01

    For experimental induction of crystal cells (=crystal idioblasts) containing calcium-oxalate crystals, the lower epidermis was peeled from seedling leaflets of Gleditsia triacanthos L., exposing the crystal-free mesophyll and minor veins to the experimental solutions on which leaflets were floated for up to 10 d under continous light. On 0.3-2.0 mM Ca-acetate, increasing numbers of crystals, appearing 96 h after peeling, were induced. The pattern of crystal distribution changed with Ca(2+)-concentration ([Ca(2+)]): at low [Ca(2+)], crystals formed only in the non-green bundlesheath cells surrounding the veins, believed to have a relatively low Ca(2+)-extrusion capacity; at higher [Ca(2+)], crystals developed in up to 90% of the mesophyll cells, and at supraoptimal [Ca(2+)], large extracellular crystals formed on the tissue surface. By sequential treatments with solutions of different [Ca(2+)], the following three phases were identified in the induction of crystal cells: (1) during the initial 24-h period (adaptive aging), Ca(2+) is not required and crystal induction is not possible; (2) during the following 48 h (induction period), exposure to 1-2 mM Ca-acetate induces the differentiation of mesophyll cells into crystal cells; (3) crystal growth begins 72 h after the start of induction. In intact leaflets of Albizia julibrissin Durazz., calcium-oxalate crystals are found exclusively in the bundle-sheath cells of the veins, but crystals were induced in the mesophyll of peeled leaflets floating on 1 mM Ca-acetate. Exposure to inductive [Ca(2+)] will thus trigger the differentiation of mature leaf cells into crystal cells; the spatial distribution of crystals is determined by the external [Ca(2+)] and by the structural and functional properties of the cells in the tissue.

  5. Genetic analysis of a synaptic calcium channel in Drosophila: intragenic modifiers of a temperature-sensitive paralytic mutant of cacophony.

    PubMed Central

    Brooks, I M; Felling, R; Kawasaki, F; Ordway, R W

    2003-01-01

    Our previous genetic analysis of synaptic mechanisms in Drosophila identified a temperature-sensitive paralytic mutant of the voltage-gated calcium channel alpha1 subunit gene, cacophony (cac). Electrophysiological studies in this mutant, designated cac(TS2), indicated cac encodes a primary calcium channel alpha1 subunit functioning in neurotransmitter release. To further examine the functions and interactions of cac-encoded calcium channels, a genetic screen was performed to isolate new mutations that modify the cac(TS2) paralytic phenotype. The screen recovered 10 mutations that enhance or suppress cac(TS2), including second-site mutations in cac (intragenic modifiers) as well as mutations mapping to other genes (extragenic modifiers). Here we report molecular characterization of three intragenic modifiers and examine the consequences of these mutations for temperature-sensitive behavior, synaptic function, and processing of cac pre-mRNAs. These mutations may further define the structural basis of calcium channel alpha1 subunit function in neurotransmitter release. PMID:12750329

  6. ROLE OF INTRACELLULAR CALCIUM AND PHOSPHOLIPASE A2 IN ARACHIDONIC ACID-INDUCED TOXICITY IN LIVER CELLS OVEREXPRESSING CYP2E1*

    PubMed Central

    Caro, Andres A.; Cederbaum, Arthur I.

    2007-01-01

    Liver cells (HepG2 and primary hepatocytes) overexpressing CYP2E1 and exposed to arachidonic acid (AA) were previously shown to lose viability together with enhanced lipid peroxidation. These events were blocked in cells pre-incubated with antioxidants (α -tocopherol, glutathione ethyl ester), or in HepG2 cells not expressing CYP2E1. The goal of the current study was to evaluate the role of calcium and calcium-activated hydrolases in these CYP2E1-AA interactions. CYP2E1-expressing HepG2 cells treated with AA showed an early increase in cytosolic calcium and partial depletion of ionomycin-sensitive calcium stores. These changes in calcium were blocked by α -tocopherol. AA activated phospholipase A2 (PLA2) in CYP2E1-expressing liver cells, and this was inhibited by PLA2 inhibitors or α -tocopherol. PLA2 inhibitors prevented the cell death caused by AA, without affecting CYP2E1 activity or lipid peroxidation. AA toxicity and PLA2 activation were inhibited in calcium-depleted cells, but not by removal of extracellular calcium alone. Removal of extracellular calcium inhibited the early increase in cytosolic calcium caused by AA. CYP2E1 overexpressing HepG2 cells exposed to AA showed a decrease in mitochondrial membrane potential, which was prevented by the PLA2 inhibitors. These results suggest that AA-induced toxicity to CYPE1-expressing cells: (i) is associated with release of Ca2+ from intracellular stores that depends mainly on oxidative membrane damage; (ii) is associated with activation of PLA2 that depends on intracellular calcium and lipid peroxidation; iii) does not depend on increased influx of extracellular calcium, and iv) depends on the effect of converging events (lipid peroxidation, intracellular calcium, activation of PLA2) on mitochondria to induce bioenergetic failure and necrosis. These interactions may play a role in alcohol liver toxicity, which requires polyunsaturated fatty acids, and involves induction of CYP2E1. PMID:17118330

  7. Quantification of alginate by aggregation induced by calcium ions and fluorescent polycations.

    PubMed

    Zheng, Hewen; Korendovych, Ivan V; Luk, Yan-Yeung

    2016-01-01

    For quantification of polysaccharides, including heparins and alginates, the commonly used carbazole assay involves hydrolysis of the polysaccharide to form a mixture of UV-active dye conjugate products. Here, we describe two efficient detection and quantification methods that make use of the negative charges of the alginate polymer and do not involve degradation of the targeted polysaccharide. The first method utilizes calcium ions to induce formation of hydrogel-like aggregates with alginate polymer; the aggregates can be quantified readily by staining with a crystal violet dye. This method does not require purification of alginate from the culture medium and can measure the large amount of alginate that is produced by a mucoid Pseudomonas aeruginosa culture. The second method employs polycations tethering a fluorescent dye to form suspension aggregates with the alginate polyanion. Encasing the fluorescent dye in the aggregates provides an increased scattering intensity with a sensitivity comparable to that of the conventional carbazole assay. Both approaches provide efficient methods for monitoring alginate production by mucoid P. aeruginosa.

  8. Extracellular calcium triggers unique transcriptional programs and modulates staurosporine-induced cell death in Neurospora crassa

    PubMed Central

    Gonçalves, A. P.; Monteiro, João; Lucchi, Chiara; Kowbel, David J.; Cordeiro, J. M.; Correia-de-Sá, Paulo; Rigden, Daniel J.; Glass, N. L.; Videira, Arnaldo

    2014-01-01

    Alterations in the intracellular levels of calcium are a common response to cell death stimuli in animals and fungi and, particularly, in the Neurospora crassa response to staurosporine. We highlight the importance of the extracellular availability of Ca2+ for this response. Limitation of the ion in the culture medium further sensitizes cells to the drug and results in increased accumulation of reactive oxygen species (ROS). Conversely, an approximately 30-fold excess of external Ca2+ leads to increased drug tolerance and lower ROS generation. In line with this, distinct staurosporine-induced cytosolic Ca2+ signaling profiles were observed in the absence or presence of excessive external Ca2+. High-throughput RNA sequencing revealed that different concentrations of extracellular Ca2+ define distinct transcriptional programs. Our transcriptional profiling also pointed to two putative novel Ca2+-binding proteins, encoded by the NCU08524 and NCU06607 genes, and provides a reference dataset for future investigations on the role of Ca2+ in fungal biology. PMID:28357255

  9. Electrically Induced Calcium Handling in Cardiac Progenitor Cells

    PubMed Central

    Wagner, Mary B.

    2016-01-01

    For nearly a century, the heart was viewed as a terminally differentiated organ until the discovery of a resident population of cardiac stem cells known as cardiac progenitor cells (CPCs). It has been shown that the regenerative capacity of CPCs can be enhanced by ex vivo modification. Preconditioning CPCs could provide drastic improvements in cardiac structure and function; however, a systematic approach to determining a mechanistic basis for these modifications founded on the physiology of CPCs is lacking. We have identified a novel property of CPCs to respond to electrical stimulation by initiating intracellular Ca2+ oscillations. We used confocal microscopy and intracellular calcium imaging to determine the spatiotemporal properties of the Ca2+ signal and the key proteins involved in this process using pharmacological inhibition and confocal Ca2+ imaging. Our results provide valuable insights into mechanisms to enhance the therapeutic potential in stem cells and further our understanding of human CPC physiology. PMID:27818693

  10. Calcium induced ATP synthesis: Isotope effect, magnetic parameters and mechanism

    NASA Astrophysics Data System (ADS)

    Buchachenko, A. L.; Kuznetsov, D. A.; Breslavskaya, N. N.; Shchegoleva, L. N.; Arkhangelsky, S. E.

    2011-03-01

    ATP synthesis by creatine kinase with calcium ions is accompanied by 43Ca/ 40Ca isotope effect: the enzyme with 43Ca 2+ was found to be 2.0 ± 0.3 times more active than enzymes, in which Ca 2+ ions have nonmagnetic nuclei 40Ca. The effect demonstrates that primary reaction in ATP synthesis is electron transfer between reaction partners, Сa( HO)n2+ ( n ⩽ 3) and Ca 2+(ADP) 3-. It generates ion-radical pair, in which spin conversion results in the isotope effect. Magnetic parameters (g-factors and HFC constants a( 43Ca) and a( 31P)) confirm that namely terminal oxygen atom of the ADP ligand in the complex Ca 2+(ADP) 3- donates electron to the Ca( HO)n2+ ion.

  11. Conformational plasticity of the calcium-binding pocket in the Burkholderia glumae lipase: remodeling induced by mutation of calcium coordinating residues.

    PubMed

    Papaleo, Elena; Invernizzi, Gaetano

    2011-02-01

    Most bacterial lipases bind one or more Ca²+ atoms at different locations and are a suitable case of study for investigating structural effects related to calcium binding, depletion, or mutation of calcium-binding sites. Generally Ca²+ in microbial lipases can play a crucial role in the stabilization of the whole three-dimensional structure by mediating long-range effects. It has been recently demonstrated that calcium binding influences thermal stability of Burkholderia glumae lipase (BGL) through the restriction of conformational plasticity of specific regions. Moreover, calcium depletion results in a highly cooperative protein unfolding, eliciting protein aggregation. To further shed light on molecular mechanisms and structural features connected to calcium binding in microbial lipases, we present a molecular dynamics investigation, based on multiple-replica approach at different temperatures, of BGL mutants targeting the calcium-binding site. It turns out that additional acidic residues, which are conserved in other microbial lipases, help in overcoming effects induced by mutation of D241 Ca²+-coordinating residue, upon rearrangements induced in the calcium binding site. © 2010 Wiley Periodicals, Inc.

  12. Dihydropyridine type calcium channel blocker-induced turbid dialysate in patients undergoing peritoneal dialysis.

    PubMed

    Yoshimoto, K; Saima, S; Nakamura, Y; Nakayama, M; Kubo, H; Kawaguchi, Y; Nishitani, H; Nakamura, Y; Yasui, A; Yokoyama, K; Kuriyama, S; Shirai, D; Kugiyama, A; Hayano, K; Fukui, H; Horigome, I; Amagasaki, Y; Tsubakihara, Y; Kamekawa, T; Ando, R; Tomura, S; Okamoto, R; Miwa, S; Koyama, T; Echizen, H

    1998-08-01

    We previously reported that manidipine, a new dihydropyridine type calcium channel blocker, produced chylous peritoneal dialysate being visually indistinguishable from infective peritonitis in 5 patients undergoing continuous ambulatory peritoneal dialysis (CAPD) [Yoshimoto et al. 1993]. To study whether such an adverse drug reaction would also be elicited by other commonly prescribed calcium channel blockers in CAPD patients, we have conducted postal inquiry to 15 collaborating hospitals and an institutional survey in International Medical Center of Japan as to the possible occurrence of calcium channel blocker-associated non-infective, turbid peritoneal dialysate in CAPD patients. Our diagnostic criteria for drug-induced turbidity of dialysate as a) it developed within 48 h after the administration of a newly introduced calcium channel blocker to the therapeutic regimen, b) absence of clinical symptoms of peritoneal inflammation (i.e., pyrexia, abdominal pain, nausea or vomiting), c) the fluid containing normal leukocyte counts and being negative for bacterial and fungal culture of the fluid, and d) it disappeared shortly after the withdrawal of the assumed causative agent. Results showed that 19 out of 251 CAPD patients given one of the calcium channel blockers developed non-infective turbid peritoneal dialysis that fulfilled all the above criteria. Four calcium channel blockers were suspected to be associated with the events: benidipine [2 out of 2 (100%) patients given the drug], manidipine [15 out of 36 (42%) patients], nisoldipine [1 out of 11 (9%) patients] and nifedipine [1 out of 159 (0.6%)] in descending order of frequency. None of the patients who received nicardipine, nilvadipine, nitrendipine, barnidipine and diltiazem (25, 7, 2, 1 and 8 patients, respectively) exhibited turbid dialysate. In conclusion, we consider that certain dihydropyridine type calcium channel blockers would cause turbid peritoneal dialysate being similar to that observed in

  13. Calcium acetate induces calcium uptake and formation of calcium-oxalate crystals in isolated leaflets of Gleditsia triacanthos L.

    PubMed

    Borchert, R

    1986-09-01

    During treatment of isolated, peeled leaflets of Gleditsia triacanthos with 0.5-2 mM [(45)Ca]acetate, saturation of the cell-wall free space with Ca(2+) occurred within 10 min and was followed by a period of 6-10 h during which there was no significant Ca-uptake into the protoplast, but apoplastic Ca(2+) was periodically released into the medium. Later, Ca(2+) was absorbed for 3-4 d at rates of up to 2.2 μmol Ca(2+)·h(-1)·(g FW)(-1) to final concentrations of 350 μmol Ca(2+)· (g FW)(-1). The distribution of absorbed Ca(2+) between cell wall, vacuole and Ca-oxalate crystals was determined during Ca-uptake. Wheras intact, cut leaflets deposited absorbed Ca(2+) as Ca-oxalate in the crystal cells, peeled leaflets lacking crystal cells accumulated at least 40-50 μmol·(g FW)(-1) soluble Ca(2+) before the absorbed Ca(2+) was precipitated as Ca-oxalate. These observations indicate that the mechanisms for the continuous uptake of Ca(2+), the synthesis of oxalate and the precipitation of Ca(2+) as Ca-oxalate are operational in the crystal cells of intact leaflets, but not in the mesophyll cells of peeled leaflets where they must be induced by exposure to Ca(2+). The precipitation of absorbed Ca(2+) as Ca-oxalate by the crystal cells of isolated Gleditsia leaflets illustrates the role of these cells in the excretion of surplus Ca(2+) which enters normal, attached leaves with the transpiration stream.In addition to acetate, only Ca-lactate and Ca-carbonate lead to Ca-uptake, but at rates well below those observed with Ca-acetate. Other small organic anions (citrate, glycolate, glyoxalate, malate) and inorganic anions (chloride, nitrate, sulfate) did not permit Ca-uptake. Acetate-(14)C was rapidly absorbed during Ca-uptake, but less than 20% was incorporated into Ca-oxalate; the rest remained mostly in the soluble fraction or was metabolized to CO2. Acetate, as a permeable weak acid, may enable rapid Ca-uptake by stimulating proton extrusion at the plasmalemma and by

  14. Dust-induced degradation of pyranometer sensitivity

    SciTech Connect

    Feuermann, D.; Zemel, A. )

    1993-06-01

    The effect of dust accumulation on the sensitivity of pyranometers is studied in order to estimate the possible loss of accuracy associated with leaving the instruments unattended for extended periods of time. Under the and conditions of Sede Boqer, Israel, the dust effect reduces the sensitivity of a horizontal pyranometer by one permille per day. For periods extending up to several weeks, the degradation rate appears to be constant, and no saturation effects are observed. The propagation of the measurement errors associated with dust accumulation on pyranometers to the errors on the beam and global radiation values derived from a stationary multipyranometer system is discussed.

  15. Spermidine-Induced Improvement of Reconsolidation of Memory Involves Calcium-Dependent Protein Kinase in Rats

    ERIC Educational Resources Information Center

    Girardi, Bruna Amanda; Ribeiro, Daniela Aymone; Signor, Cristiane; Muller, Michele; Gais, Mayara Ana; Mello, Carlos Fernando; Rubin, Maribel Antonello

    2016-01-01

    In this study, we determined whether the calcium-dependent protein kinase (PKC) signaling pathway is involved in the improvement of fear memory reconsolidation induced by the intrahippocampal administration of spermidine in rats. Male Wistar rats were trained in a fear conditioning apparatus using a 0.4-mA footshock as an unconditioned stimulus.…

  16. Spermidine-Induced Improvement of Reconsolidation of Memory Involves Calcium-Dependent Protein Kinase in Rats

    ERIC Educational Resources Information Center

    Girardi, Bruna Amanda; Ribeiro, Daniela Aymone; Signor, Cristiane; Muller, Michele; Gais, Mayara Ana; Mello, Carlos Fernando; Rubin, Maribel Antonello

    2016-01-01

    In this study, we determined whether the calcium-dependent protein kinase (PKC) signaling pathway is involved in the improvement of fear memory reconsolidation induced by the intrahippocampal administration of spermidine in rats. Male Wistar rats were trained in a fear conditioning apparatus using a 0.4-mA footshock as an unconditioned stimulus.…

  17. Scrophularia orientalis extract induces calcium signaling and apoptosis in neuroblastoma cells

    PubMed Central

    LANGE, INGO; MOSCHNY, JULIA; TAMANYAN, KAMILLA; KHUTSISHVILI, MANANA; ATHA, DANIEL; BORRIS, ROBERT P.; KOOMOA, DANA-LYNN

    2016-01-01

    Effective neuroblastoma (NB) treatments are still limited despite treatment options available today. Therefore, this study attempted to identify novel plant extracts that have anticancer effects. Cytotoxicity and increased intracellular calcium levels were determined using the Sulforhodamine B (SRB) assay and Fluo4-AM (acetoxymethyl) staining and fluorescence microscopy in NB cells in order to screen a library of plant extracts. The current study examined the anticancer effects of a dichloromethane extract from Scrophularia orientalis L. (Scrophulariaceae), a plant that has been used in Traditional Chinese Medicine. This extract contained highly potent agents that significantly reduced cell survival and increased calcium levels in NB cells. Further analysis revealed that cell death induced by this extract was associated with intracellular calcium release, opening of the MPTP, caspase 3- and PARP-cleavage suggesting that this extract induced aberrant calcium signaling that resulted in apoptosis via the mitochondrial pathway. Therefore, agents from Scrophularia orientalis may have the potential to lead to new chemo therapeutic anticancer drugs. Furthermore, targeting intracellular calcium signaling may be a novel strategy to develop more effective treatments for NB. PMID:26848085

  18. Visual contrast sensitivity in drug-induced Parkinsonism.

    PubMed

    Bulens, C; Meerwaldt, J D; van der Wildt, G J; Keemink, C J

    1989-03-01

    The influence of stimulus orientation on contrast sensitivity function was studied in 10 patients with drug-induced Parkinsonism. Nine of the 10 patients had at least one eye with contrast sensitivity deficit for vertical and/or horizontal stimuli. Only generalised contrast sensitivity loss, observed in two eyes, was stimulus orientation independent. All spatial frequency-selective contrast deficits in 15 eyes were orientation dependent. The striking similarity between the pattern of contrast sensitivity loss in drug-induced Parkinsonism and that in idiopathic Parkinson's disease, suggests that generalised dopaminergic deficiency, from whatever cause, affects visual function in an analogous way.

  19. Visual contrast sensitivity in drug-induced Parkinsonism.

    PubMed Central

    Bulens, C; Meerwaldt, J D; van der Wildt, G J; Keemink, C J

    1989-01-01

    The influence of stimulus orientation on contrast sensitivity function was studied in 10 patients with drug-induced Parkinsonism. Nine of the 10 patients had at least one eye with contrast sensitivity deficit for vertical and/or horizontal stimuli. Only generalised contrast sensitivity loss, observed in two eyes, was stimulus orientation independent. All spatial frequency-selective contrast deficits in 15 eyes were orientation dependent. The striking similarity between the pattern of contrast sensitivity loss in drug-induced Parkinsonism and that in idiopathic Parkinson's disease, suggests that generalised dopaminergic deficiency, from whatever cause, affects visual function in an analogous way. PMID:2926418

  20. Multiple Modes of Calcium-Induced Calcium Release in Sympathetic Neurons I

    PubMed Central

    Albrecht, Meredith A.; Colegrove, Stephen L.; Hongpaisan, Jarin; Pivovarova, Natalia B.; Andrews, S. Brian; Friel, David D.

    2001-01-01

    Many cells express ryanodine receptors (RyRs) whose activation is thought to amplify depolarization-evoked elevations in cytoplasmic Ca2+ concentration ([Ca2+]i) through a process of Ca2+-induced Ca2+ release (CICR). In neurons, it is usually assumed that CICR triggers net Ca2+ release from an ER Ca2+ store. However, since net ER Ca2+ transport depends on the relative rates of Ca2+ uptake and release via distinct pathways, weak activation of a CICR pathway during periods of ER Ca accumulation would have a totally different effect: attenuation of Ca2+ accumulation. Stronger CICR activation at higher [Ca2+]i could further attenuate Ca2+ accumulation or trigger net Ca2+ release, depending on the quantitative properties of the underlying Ca2+ transporters. This and the companion study (Hongpaisan, J., N.B. Pivovarova, S.L. Colgrove, R.D. Leapman, and D.D. Friel, and S.B. Andrews. 2001. J. Gen. Physiol. 118:101–112) investigate which of these CICR “modes” operate during depolarization-induced Ca2+ entry in sympathetic neurons. The present study focuses on small [Ca2+]i elevations (less than ∼350 nM) evoked by weak depolarization. The following two approaches were used: (1) Ca2+ fluxes were estimated from simultaneous measurements of [Ca2+]i and ICa in fura-2–loaded cells (perforated patch conditions), and (2) total ER Ca concentrations ([Ca]ER) were measured using X-ray microanalysis. Flux analysis revealed triggered net Ca2+ release during depolarization in the presence but not the absence of caffeine, and [Ca2+]i responses were accelerated by SERCA inhibitors, implicating ER Ca2+ accumulation, which was confirmed by direct [Ca]ER measurements. Ryanodine abolished caffeine-induced CICR and enhanced depolarization-induced ER Ca2+ accumulation, indicating that activation of the CICR pathway normally attenuates ER Ca2+ accumulation, which is a novel mechanism for accelerating evoked [Ca2+]i responses. Theory shows how such a low gain mode of CICR can operate

  1. Human erythrocyte membranes exhibit a cooperative calmodulin-dependent Ca2+-ATPase of high calcium sensitivity

    NASA Astrophysics Data System (ADS)

    Downes, Peter; Michell, Robert H.

    1981-03-01

    It is thought that the ionized Ca2+ concentration in the cytosol of healthy erythrocytes is in the range 0.01-0.1 µM (ref. 1) and that this low concentration is maintained by an ATP-driven calmodulin-dependent Ca2+ pump in the plasma membrane2,3. The Ca2+-stimulated ATPase which is the enzymatic expression of this pump varies in its calcium sensitivity between different preparations of erythrocyte ghosts, with activation generally occurring in a concentration range between ~1 and 10 µM Ca2+ (refs 2-5). This is a higher range of Ca2+ concentrations than might be anticipated for activation of a pump that sustains intracellular concentrations of Ca2+ below 0.1 µM, and recent reports have suggested activation in some membrane preparations at Ca2+ concentrations in the range 0.1-1.0 µM (refs 6, 7). We report here a simple method for preparing human erythrocyte membranes in 2.5 mM HEPES/1 mM EGTA at pH 7.0 (see Fig. 1 legend) in which the activation of the Ca2+-ATPase by Ca2+ and intracellular concentrations of calmodulin is highly cooperative and is complete by ~1 µM Ca2+. Unlike other available erythrocyte membrane preparations, the pattern of activation by Ca2+ and calmodulin is not complicated by partial resealing of the ghosts during and after isolation. We suggest that this cooperative activation of the Ca2+ pump may explain how healthy erythrocytes maintain their normal cytosol Ca2+ concentration at a threshold value at or below ~0.1 µM. We also note that several other calmodulin-dependent enzymes display similar cooperative activation kinetics.

  2. Microscopic heat pulses induce contraction of cardiomyocytes without calcium transients

    SciTech Connect

    Oyama, Kotaro; Mizuno, Akari; Shintani, Seine A.; Itoh, Hideki; Serizawa, Takahiro; Fukuda, Norio; Suzuki, Madoka

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Infra-red laser beam generates microscopic heat pulses. Black-Right-Pointing-Pointer Heat pulses induce contraction of cardiomyocytes. Black-Right-Pointing-Pointer Ca{sup 2+} transients during the contraction were not detected. Black-Right-Pointing-Pointer Skinned cardiomyocytes in free Ca{sup 2+} solution also contracted. Black-Right-Pointing-Pointer Heat pulses regulated the contractions without Ca{sup 2+} dynamics. -- Abstract: It was recently demonstrated that laser irradiation can control the beating of cardiomyocytes and hearts, however, the precise mechanism remains to be clarified. Among the effects induced by laser irradiation on biological tissues, temperature change is one possible effect which can alter physiological functions. Therefore, we investigated the mechanism by which heat pulses, produced by infra-red laser light under an optical microscope, induce contractions of cardiomyocytes. Here we show that microscopic heat pulses induce contraction of rat adult cardiomyocytes. The temperature increase, {Delta}T, required for inducing contraction of cardiomyocytes was dependent upon the ambient temperature; that is, {Delta}T at physiological temperature was lower than that at room temperature. Ca{sup 2+} transients, which are usually coupled to contraction, were not detected. We confirmed that the contractions of skinned cardiomyocytes were induced by the heat pulses even in free Ca{sup 2+} solution. This heat pulse-induced Ca{sup 2+}-decoupled contraction technique has the potential to stimulate heart and skeletal muscles in a manner different from the conventional electrical stimulations.

  3. Herpesviral G protein-coupled receptors activate NFAT to induce tumor formation via inhibiting the SERCA calcium ATPase.

    PubMed

    Zhang, Junjie; He, Shanping; Wang, Yi; Brulois, Kevin; Lan, Ke; Jung, Jae U; Feng, Pinghui

    2015-03-01

    G protein-coupled receptors (GPCRs) constitute the largest family of proteins that transmit signal to regulate an array of fundamental biological processes. Viruses deploy diverse tactics to hijack and harness intracellular signaling events induced by GPCR. Herpesviruses encode multiple GPCR homologues that are implicated in viral pathogenesis. Cellular GPCRs are primarily regulated by their cognate ligands, while herpesviral GPCRs constitutively activate downstream signaling cascades, including the nuclear factor of activated T cells (NFAT) pathway. However, the roles of NFAT activation and mechanism thereof in viral GPCR tumorigenesis remain unknown. Here we report that GPCRs of human Kaposi's sarcoma-associated herpesvirus (kGPCR) and cytomegalovirus (US28) shortcut NFAT activation by inhibiting the sarcoplasmic reticulum calcium ATPase (SERCA), which is necessary for viral GPCR tumorigenesis. Biochemical approaches, entailing pharmacological inhibitors and protein purification, demonstrate that viral GPCRs target SERCA2 to increase cytosolic calcium concentration. As such, NFAT activation induced by vGPCRs was exceedingly sensitive to cyclosporine A that targets calcineurin, but resistant to inhibition upstream of ER calcium release. Gene expression profiling identified a signature of NFAT activation in endothelial cells expressing viral GPCRs. The expression of NFAT-dependent genes was up-regulated in tumors derived from tva-kGPCR mouse and human KS. Employing recombinant kGPCR-deficient KSHV, we showed that kGPCR was critical for NFAT-dependent gene expression in KSHV lytic replication. Finally, cyclosporine A treatment diminished NFAT-dependent gene expression and tumor formation induced by viral GPCRs. These findings reveal essential roles of NFAT activation in viral GPCR tumorigenesis and a mechanism of "constitutive" NFAT activation by viral GPCRs.

  4. Encapsulated calcium carbonate suspensions: A drug delivery vehicle sensitive to ultrasound disruption

    PubMed Central

    Lanting, Brent; Barfett, Joe

    2006-01-01

    A calcium carbonate suspension, encapsulated within particles of calcium alginate hydrogel, is proposed as a drug delivery device susceptible to ultrasound disruption. Spheres approximately 1mm in diameter were prepared by the coaxial airflow method from mixtures of 1% sodium alginate (m/v) and each of 50%, 75% and 100% calcium carbonate (m/v) in distilled water. This product was subjected to cycles of 85 Watt ultrasound in 1 second on/off bursts via a lab sonication system until fully disintegrated, a process requiring between 8 and 20 minutes depending upon initial calcium carbonate concentrations. The spheres subjected to vortex did not demonstrate any signs of mechanical degeneration after 30 minutes. Before use as a model implant, further work is required to develop a method of drying the particles to make them impermeable to drug diffusion before the time of their disruption with ultrasound. PMID:18523616

  5. Detection of light-induced changes of intracellular ionized calcium concentration in Limulus ventral photoreceptors using arsenazo III

    PubMed Central

    Brown, J. E.; Brown, P. K.; Pinto, L. H.

    1977-01-01

    1. The metallochromic indicator dye, arsenazo III, was injected intracellularly into Limulus ventral photoreceptor cells to concentrations greater than 1 mM. 2. The absorption spectrum (450-750 nm) of the dye in single dark-adapted cells was measured by a scanning microspectrophotometer. When a cell was light-adapted, the absorption of the dye changed; the difference spectrum had two maxima at about 610 and 660 nm, a broad minimum at about 540 nm and an isosbestic point at about 585 nm. 3. When intracellular calcium concentration was raised in dark-adapted cells previously injected with arsenazo III, the difference spectum had two maxima at about 610 and 660 nm, a broad minimum at about 530 nm and an isosbestic point at about 585 nm. The injection of Mg2+ into dark-adapted cells previously injected with the dye induced a difference spectrum that had a single maximum at about 620 nm. Also, decreasing the intracellular pH of cells previously injected with the dye induced a difference spectrum that had a minimum at about 620 nm. The evidence suggests that there is a rise of intracellular ionized calcium when a Limulus ventral photoreceptor is light-adapted. 4. The intracellular calcium concentration, [Ca2+]1, in light-adapted photoreceptors was estimated to reach at least 10-4 M by compaing the light-induced difference spectra measured in ventral photoreceptors with a standard curve determined in microcuvettes containing 2mM arsenazo III in 400 mM-KCl, 1 mM-MgCl2 and 25 mM MOPS at pH 7·0. 5. In cells injected to less than 3 mM arsenazo III, light induced a transient decrease in optical transmission at 660 nm (T660). This decrease in T660 indicates that illumination of a ventral photoreceptor normally causes a transient increase of [Ca2+]1. 6. Arsenazo III was found to be sensitive, selective and rapid enough to measure light-induced changes of intracellular ionized calcium in Limulus ventral photoreceptor cells. PMID:17732

  6. Testosterone induces an intracellular calcium increase by a nongenomic mechanism in cultured rat cardiac myocytes.

    PubMed

    Vicencio, Jose Miguel; Ibarra, Cristian; Estrada, Manuel; Chiong, Mario; Soto, Dagoberto; Parra, Valentina; Diaz-Araya, Guillermo; Jaimovich, Enrique; Lavandero, Sergio

    2006-03-01

    Androgens are associated with important effects on the heart, such as hypertrophy or apoptosis. These responses involve the intracellular androgen receptor. However, the mechanisms of how androgens activate several membrane signaling pathways are not fully elucidated. We have investigated the effect of testosterone on intracellular calcium in cultured rat cardiac myocytes. Using fluo3-AM and epifluorescence microscopy, we found that exposure to testosterone rapidly (1-7 min) led to an increase of intracellular Ca2+, an effect that persisted in the absence of external Ca2+. Immunocytochemical analysis showed that these effects occurred before translocation of the intracellular androgen receptor to the perinuclear zone. Pretreatment of the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethylester and thapsigargin blocked this response, suggesting the involvement of internal Ca2+ stores. U-73122, an inhibitor of phospholipase C, and xestospongin C, an inhibitor of inositol 1,4,5-trisphosphate receptor, abolished the Ca2+ signal. The rise in intracellular Ca2+ was not inhibited by cyproterone, an antagonist of intracellular androgen receptor. Moreover, the cell impermeant testosterone-BSA complex also produced the Ca2+ signal, indicating its origin in the plasma membrane. This effect was observed in cultured neonatal and adult rat cardiac myocytes. Pertussis toxin and the adenoviral transduction of beta- adrenergic receptor kinase carboxy terminal peptide, a peptide inhibitor of betagamma-subunits of G protein, abolished the testosterone-induced Ca2+ release. In summary, this is the first study of rapid, nongenomic intracellular Ca2+ signaling of testosterone in cardiac myocytes. Using various inhibitors and testosterone-BSA complex, the mechanism for the rapid, testosterone-induced increase in intracellular Ca2+ is through activation of a plasma membrane receptor associated with a Pertussis toxin-sensitive G protein-phospholipase C

  7. Comparison of Tooth Discoloration Induced by Calcium-Enriched Mixture and Mineral Trioxide Aggregate

    PubMed Central

    Rouhani, Armita; Akbari, Majid; Farhadi-faz, Aida

    2016-01-01

    Introduction: The aim of this in vitro study was to evaluate the tooth discoloration induced by calcium-enriched mixture (CEM) cement and mineral trioxide aggregate (MTA). Methods and Materials: Forty five endodontically treated human maxillary central incisors were selected and divided into three groups (n=15) after removing the coronal 3 mm of the obturating materials. In the MTA group, white MTA plug was placed in pulp chamber and coronal zone of the root canal. In CEM cement group, CEM plug was placed in the tooth in the same manner. In both groups, a wet cotton pellet was placed in the access cavity and the teeth were temporarily sealed. After 24 h the teeth were restored with resin composite. In the negative control group the teeth were also restored with resin composite. The color change in the cervical third of teeth was measured with a colorimeter and was repeated 3 times for each specimen. The teeth were kept in artificial saliva for 6 months. After this period, the color change was measured again. Data were collected by Commission International de I'Eclairage's L*a*b color values, and corresponding ΔE values were calculated. The results were analyzed using the one-way ANOVA and post-hoc Tukey’s test with the significance level defined as 0.05. Results: There was no significant differences between CEM group and control group in mean discoloration. The mean tooth discoloration in MTA group was significantly greater than CEM and control groups (P<0.05). Conclusion: According to the result of the present study CEM cement did not induce tooth discoloration after six months. Therefore it can be used in vital pulp therapy of esthetically sensitive teeth. PMID:27471526

  8. Studies on the in vivo sensitivity of spindle microtubules to calcium ions and evidence for a vesicular calcium-sequestering system

    PubMed Central

    1981-01-01

    I microinjected calcium ions into echinoderm eggs during mitosis to determine the calcium sensitivity of microtubules (Mts) in vivo. Spindle birefringence (BR), a measure of the number of aligned Mts in the spindle, is locally, rapidly, and reversibly abolished by small volumes of microinjected CaCl2 (1 mM). Rapid return of BR is followed by anaphase, and subsequent divisions are normal. Similar doses of MgCl2, BaCl2, KCl, NaCl, pH buffers, distilled water, or vegetable oil have no effect on spindle BR, whereas large doses of such agents sometimes cause slow, uniform loss in BR over the course of a minute or more. Of the ions tested, only Sr++ causes effects comparable to Ca++. Ca-EGTA buffers, containing greater than micromolar free Ca++, abolishes BR in a manner similar to millimolar concentrations of injected CaCl2. Caffeine, a potent uncoupler of the Ca++-pump/ATPase of sarcoplasmic reticulum, causes a local, transient depression in spindle BR in the injected region. Finally, injection of potassium oxalate results in the formation of small, highly BR crystals, presumably CA- oxalate, in Triton-sensitive compartments in the cytoplasm. Taken together, these findings demonstrate that spindle Mts are sensitive to levels of free Ca++ in the physiological range, provide evidence for the existence of a strong cytoplasmic Ca++-sequestering system, and support the notion that Mt assembly and disassembly in local regions of the spindle may be orchestrated by local changes in the cytoplasmic free Ca++ concentration during mitosis. An appendix offers the design of a new chamber for immobilizing echinoderm eggs for injection, a new method for determining the volume of the injected solution, and a description of the microinjection technique, which was designed, but never fully described, by Hiramoto (Y. Hiramoto, Exp. Cell. Res., 1962, 27:416-426.). PMID:7194345

  9. An investigation on the role of vacuolar-type proton pumps and luminal acidity in calcium sequestration by nonmitochondrial and inositol-1,4,5-trisphosphate-sensitive intracellular calcium stores in clonal insulin-secreting cells.

    PubMed

    Bode, H P; Eder, B; Trautmann, M

    1994-06-15

    To test whether in RINm5F rat insulinoma cells luminal acidity and the activity of a vacuolar-type proton pump are involved in calcium sequestration by intracellular calcium stores sensitive to inositol 1,4,5-trisphosphate (InsP3) we examined the effects of various proton-conducting ionophores and ammonium chloride, and of bafilomycin, a specific inhibitor of vacuolar proton pumps, on this parameter. Bafilomycin in concentrations up to 1 microM did not affect calcium sequestration by nonmitochondrial, InsP3-sensitive stores at all; 50 microM carbonylcyanide m-chlorophenylhydrazone, 50 microM monensin and 30 mM NH4Cl, which are diverse ways to dissipate transmembrane pH gradients, did not inhibit calcium sequestration. This argues against signficant involvement of internal acidity and vacuolar proton pumps in calcium sequestration by InsP3-sensitive stores in RINm5F cells. The proton-potassium-exchanging ionophore nigericin (20-100 microM), however, inhibited calcium sequestration by nonmitochondrial and InsP3-sensitive stores. This effect was dependent on the presence of potassium and could be reversed by inclusion of carbonylcyanide m-chlorophenylhydrazone or acetate in the incubation medium. Thus, the inhibitory effect of nigericin appears to be based on proton extrusion coupled to potassium influx across the membrane of calcium stores in RINm5F cells, creating an internal alkalinization of these stores. The effect of nigericin implies the continuous maintenance of an outside-to-inside potassium concentration gradient by nonmitochondrial calcium stores in RINm5F cells. This feature will be of potential interest in the identification of InsP3-sensitive calcium-storing organelles.

  10. ATP releasing connexin 30 hemichannels mediate flow-induced calcium signaling in the collecting duct.

    PubMed

    Svenningsen, Per; Burford, James L; Peti-Peterdi, János

    2013-01-01

    ATP in the renal tubular fluid is an important regulator of salt and water reabsorption via purinergic calcium signaling that involves the P2Y2 receptor, ENaC, and AQP2. Recently, we have shown that connexin (Cx) 30 hemichannels are localized to the non-junctional apical membrane of cells in the distal nephron-collecting duct (CD) and release ATP into the tubular fluid upon mechanical stimuli, leading to reduced salt and water reabsorption. Cx30(-/-) mice show salt-dependent elevations in BP and impaired pressure-natriuresis. Thus, we hypothesized that increased tubular flow rate leads to Cx30-dependent purinergic intracellular calcium ([Ca(2+)]i) signaling in the CD. Cortical CDs (CCDs) from wild type and Cx30(-/-) mice were freshly dissected and microperfused in vitro. Using confocal fluorescence imaging and the calcium-sensitive fluorophore pair Fluo-4 and Fura Red, we found that increasing tubular flow rate from 2 to 20 nl/min caused a significant 2.1-fold elevation in [Ca(2+)]i in wild type CCDs. This response was blunted in Cx30(-/-) CCDs ([Ca(2+)]i increased only 1.2-fold, p < 0.0001 vs. WT, n = 6 each). To further test our hypothesis we performed CD [Ca(2+)]i imaging in intact mouse kidneys in vivo using multiphoton microscopy and micropuncture delivery of the calcium-sensitive fluorophore Rhod-2. We found intrinsic, spontaneous [Ca(2+)]i oscillations in free-flowing CDs of wild type but not Cx30(-/-) mice. The [Ca(2+)]i oscillations were sensitive also to P2-receptor inhibition by suramin. Taken together, these data confirm that mechanosensitive Cx30 hemichannels mediate tubular ATP release and purinergic calcium signaling in the CD which mechanism plays an important role in the regulation of CD salt and water reabsorption.

  11. ATP Releasing Connexin 30 Hemichannels Mediate Flow-Induced Calcium Signaling in the Collecting Duct

    PubMed Central

    Svenningsen, Per; Burford, James L.; Peti-Peterdi, János

    2013-01-01

    ATP in the renal tubular fluid is an important regulator of salt and water reabsorption via purinergic calcium signaling that involves the P2Y2 receptor, ENaC, and AQP2. Recently, we have shown that connexin (Cx) 30 hemichannels are localized to the non-junctional apical membrane of cells in the distal nephron-collecting duct (CD) and release ATP into the tubular fluid upon mechanical stimuli, leading to reduced salt and water reabsorption. Cx30−/− mice show salt-dependent elevations in BP and impaired pressure-natriuresis. Thus, we hypothesized that increased tubular flow rate leads to Cx30-dependent purinergic intracellular calcium ([Ca2+]i) signaling in the CD. Cortical CDs (CCDs) from wild type and Cx30−/− mice were freshly dissected and microperfused in vitro. Using confocal fluorescence imaging and the calcium-sensitive fluorophore pair Fluo-4 and Fura Red, we found that increasing tubular flow rate from 2 to 20 nl/min caused a significant 2.1-fold elevation in [Ca2+]i in wild type CCDs. This response was blunted in Cx30−/− CCDs ([Ca2+]i increased only 1.2-fold, p < 0.0001 vs. WT, n = 6 each). To further test our hypothesis we performed CD [Ca2+]i imaging in intact mouse kidneys in vivo using multiphoton microscopy and micropuncture delivery of the calcium-sensitive fluorophore Rhod-2. We found intrinsic, spontaneous [Ca2+]i oscillations in free-flowing CDs of wild type but not Cx30−/− mice. The [Ca2+]i oscillations were sensitive also to P2-receptor inhibition by suramin. Taken together, these data confirm that mechanosensitive Cx30 hemichannels mediate tubular ATP release and purinergic calcium signaling in the CD which mechanism plays an important role in the regulation of CD salt and water reabsorption. PMID:24137132

  12. K7del is a common TPM2 gene mutation associated with nemaline myopathy and raised myofibre calcium sensitivity.

    PubMed

    Mokbel, Nancy; Ilkovski, Biljana; Kreissl, Michaela; Memo, Massimiliano; Jeffries, Cy M; Marttila, Minttu; Lehtokari, Vilma-Lotta; Lemola, Elina; Grönholm, Mikaela; Yang, Nan; Menard, Dominique; Marcorelles, Pascale; Echaniz-Laguna, Andoni; Reimann, Jens; Vainzof, Mariz; Monnier, Nicole; Ravenscroft, Gianina; McNamara, Elyshia; Nowak, Kristen J; Laing, Nigel G; Wallgren-Pettersson, Carina; Trewhella, Jill; Marston, Steve; Ottenheijm, Coen; North, Kathryn N; Clarke, Nigel F

    2013-02-01

    Mutations in the TPM2 gene, which encodes β-tropomyosin, are an established cause of several congenital skeletal myopathies and distal arthrogryposis. We have identified a TPM2 mutation, p.K7del, in five unrelated families with nemaline myopathy and a consistent distinctive clinical phenotype. Patients develop large joint contractures during childhood, followed by slowly progressive skeletal muscle weakness during adulthood. The TPM2 p.K7del mutation results in the loss of a highly conserved lysine residue near the N-terminus of β-tropomyosin, which is predicted to disrupt head-to-tail polymerization of tropomyosin. Recombinant K7del-β-tropomyosin incorporates poorly into sarcomeres in C2C12 myotubes and has a reduced affinity for actin. Two-dimensional gel electrophoresis of patient muscle and primary patient cultured myotubes showed that mutant protein is expressed but incorporates poorly into sarcomeres and likely accumulates in nemaline rods. In vitro studies using recombinant K7del-β-tropomyosin and force measurements from single dissected patient myofibres showed increased myofilament calcium sensitivity. Together these data indicate that p.K7del is a common recurrent TPM2 mutation associated with mild nemaline myopathy. The p.K7del mutation likely disrupts head-to-tail polymerization of tropomyosin, which impairs incorporation into sarcomeres and also affects the equilibrium of the troponin/tropomyosin-dependent calcium switch of muscle. Joint contractures may stem from chronic muscle hypercontraction due to increased myofibrillar calcium sensitivity while declining strength in adulthood likely arises from other mechanisms, such as myofibre decompensation and fatty infiltration. These results suggest that patients may benefit from therapies that reduce skeletal muscle calcium sensitivity, and we highlight late muscle decompensation as an important cause of morbidity.

  13. Chronic elevated calcium blocks AMPK-induced GLUT-4 expression in skeletal muscle.

    PubMed

    Park, S; Scheffler, T L; Gunawan, A M; Shi, H; Zeng, C; Hannon, K M; Grant, A L; Gerrard, D E

    2009-01-01

    Muscle contraction stimulates glucose transport independent of insulin. Glucose uptake into muscle cells is positively related to skeletal muscle-specific glucose transporter (GLUT-4) expression. Therefore, our objective was to determine the effects of the contraction-mediated signals, calcium and AMP-activated protein kinase (AMPK), on glucose uptake and GLUT-4 expression under acute and chronic conditions. To accomplish this, we used pharmacological agents, cell culture, and pigs possessing genetic mutations for increased cytosolic calcium and constitutively active AMPK. In C2C12 myotubes, caffeine, a sarcoplasmic reticulum calcium-releasing agent, had a biphasic effect on GLUT-4 expression and glucose uptake. Low-concentration (1.25 to 2 mM) or short-term (4 h) caffeine treatment together with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR), had an additive effect on GLUT-4 expression. However, high-concentration (2.5 to 5 mM) or long-term (4 to 30 h) caffeine treatment decreased AMPK-induced GLUT-4 expression without affecting cell viability. The negative effect of caffeine on AICAR-induced GLUT-4 expression was reduced by dantrolene, which desensitizes the ryanodine receptor. Consistent with cell culture data, increases in GLUT-4 mRNA and protein expression induced by AMPK were blunted in pigs possessing genetic mutations for both increased cytosolic calcium and constitutively active AMPK. Altogether, these data suggest that chronic exposure to elevated cytosolic calcium concentration blocks AMPK-induced GLUT-4 expression in skeletal muscle.

  14. Modelling biological and chemically induced precipitation of calcium phosphate in enhanced biological phosphorus removal systems.

    PubMed

    Barat, R; Montoya, T; Seco, A; Ferrer, J

    2011-06-01

    The biologically induced precipitation processes can be important in wastewater treatment, in particular treating raw wastewater with high calcium concentration combined with Enhanced Biological Phosphorus Removal. Currently, there is little information and experience in modelling jointly biological and chemical processes. This paper presents a calcium phosphate precipitation model and its inclusion in the Activated Sludge Model No 2d (ASM2d). The proposed precipitation model considers that aqueous phase reactions quickly achieve the chemical equilibrium and that aqueous-solid change is kinetically governed. The model was calibrated using data from four experiments in a Sequencing Batch Reactor (SBR) operated for EBPR and finally validated with two experiments. The precipitation model proposed was able to reproduce the dynamics of amorphous calcium phosphate (ACP) formation and later crystallization to hydroxyapatite (HAP) under different scenarios. The model successfully characterised the EBPR performance of the SBR, including the biological, physical and chemical processes.

  15. A comparison of the effects of nifedipine, verapamil, and low-molecular-weight heparin on SLIGRL-NH₂-induced calcium influx through proteinase-activated receptor 2 activation.

    PubMed

    Sun, Jun; Dou, Wei; Shen, Hong; Hu, Jinhong

    2014-01-01

    Proteinase-activated receptor 2 (PAR2) may be implicated in skin disorders. Intracellular calcium mobilization is a key step in PAR2-induced signaling. In this study, we investigated the effects of nifedipine, verapamil, and low-molecular-weight heparin (LMWH) on SLIGRL-NH₂-induced PAR2-mediated calcium mobilization within cells. The intracellular calcium concentration was measured with fluo-8, a fluorescence indicator for free Ca(2+). Our results showed that SLIGRL-NH₂ induced a dose-dependent calcium influx. This calcium influx was completely blocked in HaCaT cells and significantly blocked by LMWH in HEK293/PAR2 cells. However, both nifedipine and verapamil failed to inhibit the SLIGRL-NH₂-induced calcium influx in either cell line. These results indicate that the PAR2 activation-induced calcium mobilization was mediated by intracellular calcium stores but not by extracellular calcium present in the media. © 2014 S. Karger AG, Basel.

  16. Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

    PubMed Central

    Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

    2013-01-01

    Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0. PMID:23685703

  17. Direct imaging of ER calcium with targeted-esterase induced dye loading (TED).

    PubMed

    Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

    2013-05-07

    Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca(2+) indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca(2+) indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca(2+) indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca(2+) indicator and a hydrophilic fluorescent dye/Ca(2+) complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0.

  18. Vitamin D3 Prevents Calcium-Induced Progression of Early-Stage Prostate Tumors by Counteracting TRPC6 and Calcium Sensing Receptor Upregulation.

    PubMed

    Bernichtein, Sophie; Pigat, Natascha; Barry Delongchamps, Nicolas; Boutillon, Florence; Verkarre, Virginie; Camparo, Philippe; Reyes-Gomez, Edouard; Méjean, Arnaud; Oudard, Stéphane M; Lepicard, Eve M; Viltard, Mélanie; Souberbielle, Jean-Claude; Friedlander, Gérard; Capiod, Thierry; Goffin, Vincent

    2017-01-15

    Active surveillance has emerged as an alternative to immediate treatment for men with low-risk prostate cancer. Accordingly, identification of environmental factors that facilitate progression to more aggressive stages is critical for disease prevention. Although calcium-enriched diets have been speculated to increase prostate cancer risk, their impact on early-stage tumors remains unexplored. In this study, we addressed this issue with a large interventional animal study. Mouse models of fully penetrant and slowly evolving prostate tumorigenesis showed that a high calcium diet dramatically accelerated the progression of prostate intraepithelial neoplasia, by promoting cell proliferation, micro-invasion, tissue inflammation, and expression of acknowledged prostate cancer markers. Strikingly, dietary vitamin D prevented these calcium-triggered tumorigenic effects. Expression profiling and in vitro mechanistic studies showed that stimulation of PC-3 cells with extracellular Ca(2+) resulted in an increase in cell proliferation rate, store-operated calcium entry (SOCE) amplitude, cationic channel TRPC6, and calcium sensing receptor (CaSR) expression. Notably, administration of the active vitamin D metabolite calcitriol reversed all these effects. Silencing CaSR or TRPC6 expression in calcium-stimulated PC3 cells decreased cell proliferation and SOCE. Overall, our results demonstrate the protective effects of vitamin D supplementation in blocking the progression of early-stage prostate lesions induced by a calcium-rich diet. Cancer Res; 77(2); 355-65. ©2016 AACR. ©2016 American Association for Cancer Research.

  19. Parabrachial lesions in rats disrupt sodium appetite induced by furosemide but not by calcium deprivation.

    PubMed

    Grigson, P S; Colechio, E M; Power, M L; Schulkin, J; Norgren, R

    2015-03-01

    An appetite for CaCl2 and NaCl occurs in young rats after they are fed a diet lacking Ca or Na, respectively. Bilateral lesions of the parabrachial nuclei (PBN) disrupt normal taste aversion learning and essentially eliminate the expression of sodium appetite. Here we tested whether similar lesions of the PBN would disrupt the calcium-deprivation-induced appetite for CaCl2 or NaCl. Controls and rats with PBN lesions failed to exhibit a calcium-deprivation-induced appetite for CaCl2. Nevertheless, both groups did exhibit a significant calcium-deprivation-induced appetite for 0.5M NaCl. Thus, while damage to the second central gustatory relay in the PBN disrupts the appetite for 0.5M NaCl induced by furosemide, deoxycorticosterone acetate, and polyethylene glycol, the sodium appetite induced by dietary CaCl2 depletion remains intact. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Purinergically induced membrane fluidization in ciliary cells: characterization and control by calcium and membrane potential.

    PubMed

    Alfahel, E; Korngreen, A; Parola, A H; Priel, Z

    1996-02-01

    To examine the role of membrane dynamics in transmembrane signal transduction, we studied changes in membrane fluidity in mucociliary tissues from frog palate and esophagus epithelia stimulated by extracellular ATP. Micromolar concentrations of ATP induced strong changes in fluorescence polarization, possibly indicating membrane fluidization. This effect was dosage dependent, reaching a maximum at 10-microM ATP. It was dependent on the presence of extracellular Ca2+ (or Mg2+), though it was insensitive to inhibitors of voltage-gated calcium channels. It was inhibited by thapsigargin and by ionomycin (at low extracellular Ca2+ concentration), both of which deplete Ca2+ stores. It was inhibited by the calcium-activated potassium channel inhibitors quinidine, charybdotoxin, and apamine and was reduced considerably by replacement of extracellular Na+ with K+. Hyperpolarization, or depolarization, of the mucociliary membrane induced membrane fluidization. The degree of membrane fluidization depended on the degree of hyperpolarization or depolarization of the ciliary membrane potential and was considerably lower than the effect induced by extracellular ATP. These results indicate that appreciable membrane fluidization induced by extracellular ATP depends both on an increase in intracellular Ca2+, mainly from its internal stores, and on hyperpolarization of the membrane. Calcium-dependent potassium channels couple the two effects. In light of recent results on the enhancement of ciliary beat frequency, it would appear that extracellular ATP-induced changes both in ciliary beat frequency and in membrane fluidity are triggered by similar signal transduction pathways.

  1. H sup + -dependent calcium uptake into an IP sub 3 -sensitive calcium pool from rat parotid gland

    SciTech Connect

    Thevenod, F.; Schulz, I. )

    1988-10-01

    In permeabilized parotid cells and in isolated membrane vesicles from parotid endoplasmic reticulum (ER), Mg-ATP-dependent Ca{sup 2+} uptake was measured using a Ca{sup 2+}-specific macroelectrode and {sup 45}Ca{sup 2+}, respectively. Mg-ATP-dependent Ca{sup 2+} uptake was inhibited by vanadate by {approximately}45% in permeabilized cells and by {approximately}70% in membrane vesicles from ER during the initial 10 min. After this lag phase, Ca{sup 2+} uptake increased. Subsequent addition of inositol 1,4,5-trisphosphate (IP{sub 3}) caused a similar Ca{sup 2+} release compared with control. This indicates that in presence of vanadate an IP{sub 3}-sensitive Ca{sup 2+} pool was filled. However, when protonophores, such as nigericin or carbonyl cyanide-m-chlorophenylhydrazone, were added in addition to vanadate, this low steady-state free (Ca{sup 2+}) was not reached. {sup 45}Ca{sup 2+} uptake was reduced by {approximately}70% within 60 min, and IP{sub 3} did not cause {sup 45}Ca{sup 2+} release when given subsequently, indicating that filling of an IP{sub 3}-sensitive Ca{sup 2+} pool was prevented. Mg-ATP-driven H{sup +} uptake into ER vesicles was abolished by protonophores and by the H{sup +}-ATPase blockers N-ethylmaleimide and Dio 9 but was unaltered by vanadate. Preincubation of ER vesicles in a medium without Ca{sup 2+}, but with vanadate and with Mg-ATP to generate an H{sup +} gradient, allowed demonstration of {sup 45}Ca{sup 2+} uptake from a medium that did not contain ATP. The data indicate that both a Ca{sup 2+} and a H{sup +} pump are located in a compartment of ER that is also sensitive to IP{sub 3}. Ca{sup 2+} uptake is coupled to an H{sup +} gradient that is generated by the H{sup +} pump and most likely occurs via Mg-ATP-driven Ca{sup 2+}-H{sup +} countertransport but to some extent can also operate in absence of ATP at the expense of the H{sup +} gradient.

  2. IP3-dependent, post-tetanic calcium transients induced by electrostimulation of adult skeletal muscle fibers

    PubMed Central

    Casas, Mariana; Figueroa, Reinaldo; Jorquera, Gonzalo; Escobar, Matías; Molgó, Jordi

    2010-01-01

    Tetanic electrical stimulation induces two separate calcium signals in rat skeletal myotubes, a fast one, dependent on Cav 1.1 or dihydropyridine receptors (DHPRs) and ryanodine receptors and related to contraction, and a slow signal, dependent on DHPR and inositol trisphosphate receptors (IP3Rs) and related to transcriptional events. We searched for slow calcium signals in adult muscle fibers using isolated adult flexor digitorum brevis fibers from 5–7-wk-old mice, loaded with fluo-3. When stimulated with trains of 0.3-ms pulses at various frequencies, cells responded with a fast calcium signal associated with muscle contraction, followed by a slower signal similar to one previously described in cultured myotubes. Nifedipine inhibited the slow signal more effectively than the fast one, suggesting a role for DHPR in its onset. The IP3R inhibitors Xestospongin B or C (5 µM) also inhibited it. The amplitude of post-tetanic calcium transients depends on both tetanus frequency and duration, having a maximum at 10–20 Hz. At this stimulation frequency, an increase of the slow isoform of troponin I mRNA was detected, while the fast isoform of this gene was inhibited. All three IP3R isoforms were present in adult muscle. IP3R-1 was differentially expressed in different types of muscle fibers, being higher in a subset of fast-type fibers. Interestingly, isolated fibers from the slow soleus muscle did not reveal the slow calcium signal induced by electrical stimulus. These results support the idea that IP3R-dependent slow calcium signals may be characteristic of distinct types of muscle fibers and may participate in the activation of specific transcriptional programs of slow and fast phenotype. PMID:20837675

  3. Changes in calcium and iron levels in the brains of rats during kainate induced epilepsy

    NASA Astrophysics Data System (ADS)

    Ren, Min-Qin; Ong, Wei-Yi; Makjanic, Jagoda; Watt, Frank

    1999-10-01

    Epilepsy is a recurrent disorder of cerebral function characterised by sudden brief attacks of altered consciousness, motor activity or sensory phenomena, and affects approximately 1% of the population. Kainic acid injection induces neuronal degeneration in rats, is associated with glial hypertrophy and proliferation in the CA3-CA4 fields of hippocampal complex, and is a model for temporal lobe epilepsy. In this study we have applied Nuclear Microscopy to the investigation of the elemental changes within the hippocampus and the cortex areas of the rat brain following kainate injection. Analyses of unstained freeze dried tissue sections taken at 1 day and 1, 2, 3 and 4 weeks following injection were carried out using the Nuclear Microscopy facility at the Research Centre for Nuclear Microscopy, National University of Singapore. Quantitative analysis and elemental mapping indicates that there are significant changes in the calcium levels and distributions in the hippocampus as early as 1 day following injection. Preliminary results indicate a rapid increase in cellular calcium. High levels of calcium can activate calcium dependent proteins and phospholipases. Activation of phospholipase A 2 can be harmful to surrounding neurons through free radical damage. In addition to observed increases in calcium, there was evidence of increases in iron levels. This is consistent with measurements in other degenerative brain disorders, and may signal a late surge in free radical production.

  4. Plasma Calcium, Inorganic Phosphate and Magnesium During Hypocalcaemia Induced by a Standardized EDTA Infusion in Cows

    PubMed Central

    Mellau, LSB; Jørgensen, RJ; Enemark, JMD

    2001-01-01

    The intravenous Na2EDTA infusion technique allows effective specific chelation of circulating Ca2+ leading to a progressive hypocalcaemia. Methods previously used were not described in detail and results obtained by monitoring total and free ionic calcium were not comparable due to differences in sampling and analysis. This paper describes a standardized EDTA infusion technique that allowed comparison of the response of calcium, phosphorus and magnesium between 2 groups of experimental cows. The concentration of the Na2EDTA solution was 0.134 mol/l and the flow rate was standardized at 1.2 ml/kg per hour. Involuntary recumbency occurred when ionised calcium dropped to 0.39 – 0.52 mmol/l due to chelation. An initial fast drop of ionized calcium was observed during the first 20 min of infusion followed by a fluctuation leading to a further drop until recumbency. Pre-infusion [Ca2+] between tests does not correlate with the amount of EDTA required to induce involuntary recumbence. Total calcium concentration measured by atomic absorption remained almost constant during the first 100 min of infusion but declined gradually when the infusion was prolonged. The concentration of inorganic phosphate declined gradually in a fluctuating manner until recumbency. Magnesium concentration remained constant during infusion. Such electrolyte responses during infusion were comparable to those in spontaneous milk fever. The standardized infusion technique might be useful in future experimental studies. PMID:11503370

  5. Coagulation of β-conglycinin, glycinin and isoflavones induced by calcium chloride in soymilk

    NASA Astrophysics Data System (ADS)

    Hsiao, Yu-Hsuan; Yu, Chia-Jung; Li, Wen-Tai; Hsieh, Jung-Feng

    2015-08-01

    The coagulation of β-conglycinin (7S), glycinin (11S) and isoflavones induced by calcium chloride was investigated. Approximately 92.6% of the soymilk proteins were coagulated into the soymilk pellet fraction (SPF) after the addition of 5 mM calcium chloride. SDS-PAGE and two-dimensional electrophoresis analysis indicated that most of the 7S (α’, α and β), 11S acidic (A1a, A1b, A2, A3 and A4) and 11S basic (B1a) proteins in the SSF were coagulated into the SPF after treatment with 5 mM calcium chloride. Isoflavones, including daidzein and genistein, were also coagulated into the SPF after the addition of 5 mM calcium chloride. The amounts of daidzein and genistein in the SSF decreased to 39.4 ± 1.6 and 11.8 ± 7.0%, respectively. HPLC analysis suggested that daidzein and genistein were bound with 7S and 11S proteins and then were coprecipitated into the SPF by 5 mM calcium chloride.

  6. Coagulation of β-conglycinin, glycinin and isoflavones induced by calcium chloride in soymilk.

    PubMed

    Hsiao, Yu-Hsuan; Yu, Chia-Jung; Li, Wen-Tai; Hsieh, Jung-Feng

    2015-08-11

    The coagulation of β-conglycinin (7S), glycinin (11S) and isoflavones induced by calcium chloride was investigated. Approximately 92.6% of the soymilk proteins were coagulated into the soymilk pellet fraction (SPF) after the addition of 5 mM calcium chloride. SDS-PAGE and two-dimensional electrophoresis analysis indicated that most of the 7S (α', α and β), 11S acidic (A1a, A1b, A2, A3 and A4) and 11S basic (B1a) proteins in the SSF were coagulated into the SPF after treatment with 5 mM calcium chloride. Isoflavones, including daidzein and genistein, were also coagulated into the SPF after the addition of 5 mM calcium chloride. The amounts of daidzein and genistein in the SSF decreased to 39.4 ± 1.6 and 11.8 ± 7.0%, respectively. HPLC analysis suggested that daidzein and genistein were bound with 7S and 11S proteins and then were coprecipitated into the SPF by 5 mM calcium chloride.

  7. Hypotension induced by the concomitant use of a calcium-channel blocker and clarithromycin

    PubMed Central

    Takeuchi, Sayako; Tsujimoto, Toshihide

    2017-01-01

    In the elderly, calcium-channel blockers are the first-line treatment for hypertension, and macrolides are commonly prescribed antibiotics. Here we report a 78-year-old man taking nifedipine, diltiazem and carvedilol who presented with persistent hypotension and bradycardia after clarithromycin was prescribed. He was diagnosed with drug-induced hypotension and treated with fluid resuscitation and vasoactive agents. His symptoms gradually improved. He was transferred out of the intensive care unit 3 days after hospitalisation. Combining calcium-channel blockers and clarithromycin can cause vasodilatory hypotension. The concomitant use of calcium-channel blockers and macrolide antibiotics increases the levels of calcium-channel blockers in the blood as they are metabolised by cytochrome P450 3A4 (CYP3A4), which is inhibited by macrolide antibiotics. Moreover, the addition of another calcium-channel blocker and a β blocker can lower cardiac output due to bradycardia and worsen hypotension. Therefore, it is important to consider drug interactions when the cause of hypotension is unknown. PMID:28069789

  8. Coagulation of β-conglycinin, glycinin and isoflavones induced by calcium chloride in soymilk

    PubMed Central

    Hsiao, Yu-Hsuan; Yu, Chia-Jung; Li, Wen-Tai; Hsieh, Jung-Feng

    2015-01-01

    The coagulation of β-conglycinin (7S), glycinin (11S) and isoflavones induced by calcium chloride was investigated. Approximately 92.6% of the soymilk proteins were coagulated into the soymilk pellet fraction (SPF) after the addition of 5 mM calcium chloride. SDS-PAGE and two-dimensional electrophoresis analysis indicated that most of the 7S (α’, α and β), 11S acidic (A1a, A1b, A2, A3 and A4) and 11S basic (B1a) proteins in the SSF were coagulated into the SPF after treatment with 5 mM calcium chloride. Isoflavones, including daidzein and genistein, were also coagulated into the SPF after the addition of 5 mM calcium chloride. The amounts of daidzein and genistein in the SSF decreased to 39.4 ± 1.6 and 11.8 ± 7.0%, respectively. HPLC analysis suggested that daidzein and genistein were bound with 7S and 11S proteins and then were coprecipitated into the SPF by 5 mM calcium chloride. PMID:26260443

  9. Targeting Calcium Signaling Induces Epigenetic Reactivation of Tumor Suppressor Genes in Cancer.

    PubMed

    Raynal, Noël J-M; Lee, Justin T; Wang, Youjun; Beaudry, Annie; Madireddi, Priyanka; Garriga, Judith; Malouf, Gabriel G; Dumont, Sarah; Dettman, Elisha J; Gharibyan, Vazganush; Ahmed, Saira; Chung, Woonbok; Childers, Wayne E; Abou-Gharbia, Magid; Henry, Ryan A; Andrews, Andrew J; Jelinek, Jaroslav; Cui, Ying; Baylin, Stephen B; Gill, Donald L; Issa, Jean-Pierre J

    2016-03-15

    Targeting epigenetic pathways is a promising approach for cancer therapy. Here, we report on the unexpected finding that targeting calcium signaling can reverse epigenetic silencing of tumor suppressor genes (TSG). In a screen for drugs that reactivate silenced gene expression in colon cancer cells, we found three classical epigenetic targeted drugs (DNA methylation and histone deacetylase inhibitors) and 11 other drugs that induced methylated and silenced CpG island promoters driving a reporter gene (GFP) as well as endogenous TSGs in multiple cancer cell lines. These newly identified drugs, most prominently cardiac glycosides, did not change DNA methylation locally or histone modifications globally. Instead, all 11 drugs altered calcium signaling and triggered calcium-calmodulin kinase (CamK) activity, leading to MeCP2 nuclear exclusion. Blocking CamK activity abolished gene reactivation and cancer cell killing by these drugs, showing that triggering calcium fluxes is an essential component of their epigenetic mechanism of action. Our data identify calcium signaling as a new pathway that can be targeted to reactivate TSGs in cancer.

  10. Pulmonary edema induced by calcium-channel blockade for tocolysis.

    PubMed

    Bal, Laurence; Thierry, Stéphane; Brocas, Elsa; Adam, Marie; Van de Louw, Andry; Tenaillon, Alain

    2004-09-01

    Nicardipine is used in the treatment of premature labor. There are no previous reports in the anesthesia literature of serious side effects associated with this drug. We report a case of pulmonary edema induced by nicardipine therapy for tocolysis in a pregnant 27-yr-old patient admitted to our hospital for preterm labor with intact membranes at 27 wk of gestation.

  11. Potential Interactions of Calcium-Sensitive Reagents with Zinc Ion in Different Cultured Cells

    PubMed Central

    Fujikawa, Koichi; Fukumori, Ryo; Nakamura, Saki; Kutsukake, Takaya; Takarada, Takeshi; Yoneda, Yukio

    2015-01-01

    Background Several chemicals have been widely used to evaluate the involvement of free Ca2+ in mechanisms underlying a variety of biological responses for decades. Here, we report high reactivity to zinc of well-known Ca2+-sensitive reagents in diverse cultured cells. Methodology/Principal Findings In rat astrocytic C6 glioma cells loaded with the fluorescent Ca2+ dye Fluo-3, the addition of ZnCl2 gradually increased the fluorescence intensity in a manner sensitive to the Ca2+ chelator EGTA irrespective of added CaCl2. The addition of the Ca2+ ionophore A23187 drastically increased Fluo-3 fluorescence in the absence of ZnCl2, while the addition of the Zn2+ ionophore pyrithione rapidly and additionally increased the fluorescence in the presence of ZnCl2, but not in its absence. In cells loaded with the zinc dye FluoZin-3 along with Fluo-3, a similarly gradual increase was seen in the fluorescence of Fluo-3, but not of FluoZin-3, in the presence of both CaCl2 and ZnCl2. Further addition of pyrithione drastically increased the fluorescence intensity of both dyes, while the addition of the Zn2+ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) rapidly and drastically decreased FluoZin-3 fluorescence. In cells loaded with FluoZin-3 alone, the addition of ZnCl2 induced a gradual increase in the fluorescence in a fashion independent of added CaCl2 but sensitive to EGTA. Significant inhibition was found in the vitality to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide in a manner sensitive to TPEN, EDTA and BAPTA in C6 glioma cells exposed to ZnCl2, with pyrithione accelerating the inhibition. Similar inhibition occurred in an EGTA-sensitive fashion after brief exposure to ZnCl2 in pluripotent P19 cells, neuronal Neuro2A cells and microglial BV2 cells, which all expressed mRNA for particular zinc transporters. Conclusions/Significance Taken together, comprehensive analysis is absolutely required for the demonstration of a

  12. Potential interactions of calcium-sensitive reagents with zinc ion in different cultured cells.

    PubMed

    Fujikawa, Koichi; Fukumori, Ryo; Nakamura, Saki; Kutsukake, Takaya; Takarada, Takeshi; Yoneda, Yukio

    2015-01-01

    Several chemicals have been widely used to evaluate the involvement of free Ca(2+) in mechanisms underlying a variety of biological responses for decades. Here, we report high reactivity to zinc of well-known Ca(2+)-sensitive reagents in diverse cultured cells. In rat astrocytic C6 glioma cells loaded with the fluorescent Ca(2+) dye Fluo-3, the addition of ZnCl2 gradually increased the fluorescence intensity in a manner sensitive to the Ca(2+) chelator EGTA irrespective of added CaCl2. The addition of the Ca(2+) ionophore A23187 drastically increased Fluo-3 fluorescence in the absence of ZnCl2, while the addition of the Zn(2+) ionophore pyrithione rapidly and additionally increased the fluorescence in the presence of ZnCl2, but not in its absence. In cells loaded with the zinc dye FluoZin-3 along with Fluo-3, a similarly gradual increase was seen in the fluorescence of Fluo-3, but not of FluoZin-3, in the presence of both CaCl2 and ZnCl2. Further addition of pyrithione drastically increased the fluorescence intensity of both dyes, while the addition of the Zn(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) rapidly and drastically decreased FluoZin-3 fluorescence. In cells loaded with FluoZin-3 alone, the addition of ZnCl2 induced a gradual increase in the fluorescence in a fashion independent of added CaCl2 but sensitive to EGTA. Significant inhibition was found in the vitality to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide in a manner sensitive to TPEN, EDTA and BAPTA in C6 glioma cells exposed to ZnCl2, with pyrithione accelerating the inhibition. Similar inhibition occurred in an EGTA-sensitive fashion after brief exposure to ZnCl2 in pluripotent P19 cells, neuronal Neuro2A cells and microglial BV2 cells, which all expressed mRNA for particular zinc transporters. Taken together, comprehensive analysis is absolutely required for the demonstration of a variety of physiological and pathological responses

  13. Coronal Discoloration Induced by Calcium-Enriched Mixture, Mineral Trioxide Aggregate and Calcium Hydroxide: A Spectrophotometric Analysis

    PubMed Central

    Esmaeili, Behnaz; Alaghehmand, Homayoun; Kordafshari, Tavoos; Daryakenari, Ghazaleh; Ehsani, Maryam; Bijani, Ali

    2016-01-01

    Introduction: The aim of this study was to compare the discoloration potential of calcium-enriched mixture (CEM) cement, white mineral trioxide aggregate (WMTA) and calcium hydroxide (CH), after placement in pulp chamber. Methods and Materials: Access cavities were prepared in 40 intact maxillary central incisors. Then, a 2×2 mm box was prepared on the middle third of the inner surface on the buccal wall of the access cavity. The specimens were randomly assigned into four groups; the boxes in the control group were left empty, in groups 1 to 3, the boxes were filled with CH, WMTA and CEM cement, respectively. The access cavities and the apical openings were sealed using resin modified glass ionomer (RMGI). The color measurement was performed with a spectrophotometer at the following intervals: before (T0), immediately after placement of the filling material (T1), one week (T2), 1 month (T3), 3 months (T4) and 5 months (T5) after filling of the box and finally immediately after removing the material from the boxes (T6). Color change (ΔE) values were calculated using the sample Kolmogorov-Smirnov test to determine the normal distribution of data, followed by ANOVA, repeated measured ANOVA and post-hoc Tukey’s tests. Results: All materials led to clinically perceptible crown discoloration after 1 week. The highest ΔE value belonged to WMTA group. Discoloration induced by CEM cement was not significantly different from CH or the control group (P>0.05). Conclusion: CEM cement may be the material of choice in the esthetic region, specifically pertaining to its lower color changing potential compared to WMTA. PMID:26843873

  14. The calcium-dependent protein kinase CPK28 negatively regulates the BIK1-mediated PAMP-induced calcium burst

    PubMed Central

    Monaghan, Jacqueline; Matschi, Susanne; Romeis, Tina; Zipfel, Cyril

    2015-01-01

    Plants are protected from microbial infection by a robust immune system. Two of the earliest responses mediated by surface-localized immune receptors include an increase in cytosolic calcium (Ca2+) and a burst of apoplastic reactive oxygen species (ROS). The Arabidopsis plasma membrane-associated cytoplasmic kinase BIK1 is an immediate convergent substrate of multiple surface-localized immune receptors that is genetically required for the PAMP-induced Ca2+ burst and directly regulates ROS production catalyzed by the NADPH oxidase RBOHD. We recently demonstrated that Arabidopsis plants maintain an optimal level of BIK1 through a process of continuous degradation regulated by the Ca2+-dependent protein kinase CPK28. cpk28 mutants accumulate more BIK1 protein and display enhanced immune signaling, while plants over-expressing CPK28 accumulate less BIK1 protein and display impaired immune signaling. Here, we show that CPK28 additionally contributes to the PAMP-induced Ca2+ burst, supporting its role as a negative regulator of BIK1. PMID:26039480

  15. Calcium-sensitive MRI contrast agents based on superparamagnetic iron oxide nanoparticles and calmodulin.

    PubMed

    Atanasijevic, Tatjana; Shusteff, Maxim; Fam, Peter; Jasanoff, Alan

    2006-10-03

    We describe a family of calcium indicators for magnetic resonance imaging (MRI), formed by combining a powerful iron oxide nanoparticle-based contrast mechanism with the versatile calcium-sensing protein calmodulin and its targets. Calcium-dependent protein-protein interactions drive particle clustering and produce up to 5-fold changes in T2 relaxivity, an indication of the sensors' potency. A variant based on conjugates of wild-type calmodulin and the peptide M13 reports concentration changes near 1 microM Ca(2+), suitable for detection of elevated intracellular calcium levels. The midpoint and cooperativity of the response can be tuned by mutating the protein domains that actuate the sensor. Robust MRI signal changes are achieved even at nanomolar particle concentrations (<1 microM in calmodulin) that are unlikely to buffer calcium levels. When combined with technologies for cellular delivery of nanoparticulate agents, these sensors and their derivatives may be useful for functional molecular imaging of biological signaling networks in live, opaque specimens.

  16. Air bubble contact with endothelial cells in vitro induces calcium influx and IP3-dependent release of calcium stores

    PubMed Central

    Sobolewski, Peter; Kandel, Judith; Klinger, Alexandra L.

    2011-01-01

    Gas embolism is a serious complication of decompression events and clinical procedures, but the mechanism of resulting injury remains unclear. Previous work has demonstrated that contact between air microbubbles and endothelial cells causes a rapid intracellular calcium transient and can lead to cell death. Here we examined the mechanism responsible for the calcium rise. Single air microbubbles (50–150 μm), trapped at the tip of a micropipette, were micromanipulated into contact with individual human umbilical vein endothelial cells (HUVECs) loaded with Fluo-4 (a fluorescent calcium indicator). Changes in intracellular calcium were then recorded via epifluorescence microscopy. First, we confirmed that HUVECs rapidly respond to air bubble contact with a calcium transient. Next, we examined the involvement of extracellular calcium influx by conducting experiments in low calcium buffer, which markedly attenuated the response, or by pretreating cells with stretch-activated channel blockers (gadolinium chloride or ruthenium red), which abolished the response. Finally, we tested the role of intracellular calcium release by pretreating cells with an inositol 1,4,5-trisphosphate (IP3) receptor blocker (xestospongin C) or phospholipase C inhibitor (neomycin sulfate), which eliminated the response in 64% and 67% of cases, respectively. Collectively, our results lead us to conclude that air bubble contact with endothelial cells causes an influx of calcium through a stretch-activated channel, such as a transient receptor potential vanilloid family member, triggering the release of calcium from intracellular stores via the IP3 pathway. PMID:21633077

  17. Air bubble contact with endothelial cells in vitro induces calcium influx and IP3-dependent release of calcium stores.

    PubMed

    Sobolewski, Peter; Kandel, Judith; Klinger, Alexandra L; Eckmann, David M

    2011-09-01

    Gas embolism is a serious complication of decompression events and clinical procedures, but the mechanism of resulting injury remains unclear. Previous work has demonstrated that contact between air microbubbles and endothelial cells causes a rapid intracellular calcium transient and can lead to cell death. Here we examined the mechanism responsible for the calcium rise. Single air microbubbles (50-150 μm), trapped at the tip of a micropipette, were micromanipulated into contact with individual human umbilical vein endothelial cells (HUVECs) loaded with Fluo-4 (a fluorescent calcium indicator). Changes in intracellular calcium were then recorded via epifluorescence microscopy. First, we confirmed that HUVECs rapidly respond to air bubble contact with a calcium transient. Next, we examined the involvement of extracellular calcium influx by conducting experiments in low calcium buffer, which markedly attenuated the response, or by pretreating cells with stretch-activated channel blockers (gadolinium chloride or ruthenium red), which abolished the response. Finally, we tested the role of intracellular calcium release by pretreating cells with an inositol 1,4,5-trisphosphate (IP3) receptor blocker (xestospongin C) or phospholipase C inhibitor (neomycin sulfate), which eliminated the response in 64% and 67% of cases, respectively. Collectively, our results lead us to conclude that air bubble contact with endothelial cells causes an influx of calcium through a stretch-activated channel, such as a transient receptor potential vanilloid family member, triggering the release of calcium from intracellular stores via the IP3 pathway.

  18. Synthetic peptides corresponding to human follicle-stimulating hormone (hFSH)-beta-(1-15) and hFSH-beta-(51-65) induce uptake of 45Ca++ by liposomes: evidence for calcium-conducting transmembrane channel formation

    SciTech Connect

    Grasso, P.; Santa-Coloma, T.A.; Reichert, L.E. Jr. )

    1991-06-01

    We have previously described FSH receptor-mediated influx of 45Ca++ in cultured Sertoli cells from immature rats and receptor-enriched proteoliposomes via activation of voltage-sensitive and voltage-independent calcium channels. We have further shown that this effect of FSH does not require cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding protein or activation of adenylate cyclase. In the present study, we have identified regions of human FSH-beta-subunit which appear to be involved in mediating calcium influx. We screened 11 overlapping peptide amides representing the entire primary structure of hFSH-beta-subunit for their effects on 45Ca++ flux in FSH receptor-enriched proteoliposomes. hFSH-beta-(1-15) and hFSH-beta-(51-65) induced uptake of 45Ca++ in a concentration-related manner. This effect of hFSH-beta-(1-15) and hFSH-beta-(51-65) was also observed in liposomes lacking incorporated FSH receptor. Reducing membrane fluidity by incubating liposomes (containing no receptor) with hFSH-beta-(1-15) or hFSH-beta-(51-65) at temperatures lower than the transition temperatures of their constituent phospholipids resulted in no significant (P greater than 0.05) difference in 45Ca++ uptake. The effectiveness of the calcium ionophore A23187, however, was abolished. Ruthenium red, a voltage-independent calcium channel antagonist, was able to completely block uptake of 45Ca++ induced by hFSH-beta-(1-15) and hFSH-beta-(51-65) whereas nifedipine, a calcium channel blocker specific for L-type voltage-sensitive calcium channels, was without effect. These results suggest that in addition to its effect on voltage-sensitive calcium channel activity, interaction of FSH with its receptor may induce formation of transmembrane aqueous channels which also facilitate influx of extracellular calcium.

  19. Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death.

    PubMed

    Parra, Valentina; Moraga, Francisco; Kuzmicic, Jovan; López-Crisosto, Camila; Troncoso, Rodrigo; Torrealba, Natalia; Criollo, Alfredo; Díaz-Elizondo, Jessica; Rothermel, Beverly A; Quest, Andrew F G; Lavandero, Sergio

    2013-08-01

    Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca(2+) overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca(2+) levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca(2+) influx, mitochondrial network fragmentation and loss of the mitochondrial Ca(2+) buffer capacity. These biochemical events increase cytosolic Ca(2+) levels and trigger cardiomyocyte death via the activation of calpains. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death

    PubMed Central

    Parra, Valentina; Moraga, Francisco; Kuzmicic, Jovan; López-Crisosto, Camila; Troncoso, Rodrigo; Torrealba, Natalia; Criollo, Alfredo; Díaz-Elizondo, Jessica; Rothermel, Beverly A.; Quest, Andrew F.G.; Lavandero, Sergio

    2014-01-01

    Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulatenumerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca2+ overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca2+ levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca2+ influx, mitochondrial network fragmentation and loss of the mitochondrial Ca2+ buffer capacity. These biochemical events increase cytosolic Ca2+ levels and trigger cardiomyocyte death via the activation of calpains. PMID:23602992

  1. Calcium in the Mechanism of Ammonia-Induced Astrocyte Swelling

    PubMed Central

    Jayakumar, A.R.; Rao, K.V. Rama; Tong, X.Y; Norenberg, M.D.

    2016-01-01

    Brain edema, due largely to astrocyte swelling, is an important clinical problem in patients with acute liver failure. While mechanisms underlying astrocyte swelling in this condition are not fully understood, ammonia and associated oxidative/nitrosative stress (ONS) appear to be involved. Mechanisms responsible for the increase in reactive oxygen/nitrogen species (RONS) and their role in ammonia-induced astrocyte swelling, however, are poorly understood. Recent studies have demonstrated a transient increase in intracellular Ca2+ in cultured astrocytes exposed to ammonia. As Ca2+ is a known inducer of RONS, we investigated potential mechanisms by which Ca2+ may be responsible for the production of RONS and cell swelling in cultured astrocytes after treatment with ammonia. Exposure of cultured astrocytes to ammonia (5 mM) increased the formation of free radicals, including nitric oxide, and such increase was significantly diminished by treatment with the Ca2+ chelator BAPTA-AM. We then examined the activity of Ca2+-dependent enzymes that are known to generate RONS and found that ammonia significantly increased the activities of NADPH oxidase (NOX), constitutive nitric oxide synthase (cNOS) and phospholipase A2 (PLA2) and such increases in activity were significantly diminished by BAPTA. Pretreatment of cultures with 7-nitroindazole, apocyanin and quinacrine, respective inhibitors of cNOS, NOX and PLA2, all significantly diminished RONS production. Additionally, treatment of cultures with BAPTA or with inhibitors of cNOS, NOX and PLA2 reduced ammonia-induced astrocyte swelling. These studies suggest that the ammonia-induced rise in intracellular Ca2+ activates free radical producing enzymes that ultimately contribute to the mechanism of astrocyte swelling. PMID:19393035

  2. [Effect of 2,3-butanedione monoxime on calcium paradox-induced heart injury in rats].

    PubMed

    Kong, Ling-Heng; Gu, Xiao-Ming; Su, Xing-Li; Sun, Na; Wei, Ming; Zhu, Juan-Xia; Chang, Pan; Zhou, Jing-Jun

    2016-05-01

    To investigate the Effect of 2,3-butanedione monoxime (BDM) on calcium paradox-induced heart injury and its underlying mechanisms. Thirty-two adult male SD rats were randomized into 4 groups, namely the control group, BDM treatment control group, calcium paradox group, and BDM treatment group. Isolated Sprague Dawley male rat hearts underwent Langendorff perfusion and the left ventricular pressure (LVP) and left ventricular end-diastolic pressure (LVEDP) were monitored. Left ventricular developed pressure (LVDP) was calculated to evaluate the myocardial performance. Lactate dehydrogenase (LDH) content in the coronary flow was determined. Triphenyltetrazolium chloride staining was used to measure the infarct size, and myocardial cell apoptosis was tested with TUNEL method. Western blotting was used to determine the expression of cleaved caspase-3 and cytochrome c. Compared with the control group, BDM at 20 mmol/L had no effect on cardiac performance, cell death, apoptotic index or the content of LDH, cleaved caspase-3 and cytochrome c at the end of perfusion under control conditions (P>0.05). Calcium paradox treatment significantly decreased the cardiac function and the level of LVDP and induced a larger infarct size (P<0.01), an increased myocardial apoptosis index (P<0.01), and up-regulated expressions of cleaved caspase-3 and cytochrome c (P<0.01). BDM (20 mmol/L) significantly attenuated these effects induced by calcium paradox, and markedly down-regulated the levels of LVEDP and LDH (P<0.01), lowered myocardial apoptosis index, decreased the content of cleaved caspase-3 and cytochrome c (P<0.01), increased LVDP, and reduced the infarct size (P<0.01). BDM suppresses cell apoptosis and contracture and improves heart function and cell survival in rat hearts exposed to calcium paradox, suggesting the value of BDM as an potential drug for myocardial ischemia reperfusion injur.

  3. The calcium-sensitive large-conductance potassium channel (BK/MAXI K) is present in the inner mitochondrial membrane of rat brain.

    PubMed

    Douglas, R M; Lai, J C K; Bian, S; Cummins, L; Moczydlowski, E; Haddad, G G

    2006-01-01

    Large-conductance voltage- and calcium-sensitive channels are known to be expressed in the plasmalemma of central neurons; however, recent data suggest that large-conductance voltage- and calcium-sensitive channels may also be present in mitochondrial membranes. To determine the subcellular localization and distribution of large-conductance voltage- and calcium-sensitive channels, rat brain fractions obtained by Ficoll-sucrose density gradient centrifugation were examined by Western blotting, immunocytochemistry and immuno-gold electron microscopy. Immunoblotting studies demonstrated the presence of a consistent signal for the alpha subunit of the large-conductance voltage- and calcium-sensitive channel in the mitochondrial fraction. Double-labeling immunofluorescence also demonstrated that large-conductance voltage- and calcium-sensitive channels are present in mitochondria and co-localize with mitochondrial-specific proteins such as the translocase of the inner membrane 23, adenine nucleotide translocator, cytochrome c oxidase or complex IV-subunit 1 and the inner mitochondrial membrane protein but do not co-localize with calnexin, an endoplasmic reticulum marker. Western blotting of discrete subcellular fractions demonstrated that cytochrome c oxidase or complex IV-subunit 1 was only expressed in the mitochondrial fraction whereas actin, acetylcholinesterase, cadherins, calnexin, 58 kDa Golgi protein, lactate dehydrogenase and microtubule-associated protein 1 were not, demonstrating the purity of the mitochondrial fraction. Electron microscopic examination of the mitochondrial pellet demonstrated gold particle labeling within mitochondria, indicative of the presence of large-conductance voltage- and calcium-sensitive channels in the inner mitochondrial membrane. These studies provide concrete morphological evidence for the existence of large-conductance voltage- and calcium-sensitive channels in mitochondria: our findings corroborate the recent

  4. Omega-conotoxin CVID inhibits a pharmacologically distinct voltage-sensitive calcium channel associated with transmitter release from preganglionic nerve terminals.

    PubMed

    Adams, David J; Smith, Amanda B; Schroeder, Christina I; Yasuda, Takahiro; Lewis, Richard J

    2003-02-07

    Neurotransmitter release from preganglionic parasympathetic neurons is resistant to inhibition by selective antagonists of L-, N-, P/Q-, R-, and T-type calcium channels. In this study, the effects of different omega-conotoxins from genus Conus were investigated on current flow-through cloned voltage-sensitive calcium channels expressed in Xenopus oocytes and nerve-evoked transmitter release from the intact preganglionic cholinergic nerves innervating the rat submandibular ganglia. Our results indicate that omega-conotoxin CVID from Conus catus inhibits a pharmacologically distinct voltage-sensitive calcium channel involved in neurotransmitter release, whereas omega-conotoxin MVIIA had no effect. omega-Conotoxin CVID and MVIIA inhibited depolarization-activated Ba(2+) currents recorded from oocytes expressing N-type but not L- or R-type calcium channels. High affinity inhibition of the CVID-sensitive calcium channel was enhanced when position 10 of the omega-conotoxin was occupied by the smaller residue lysine as found in CVID instead of an arginine as found in MVIIA. Given that relatively small differences in the sequence of the N-type calcium channel alpha(1B) subunit can influence omega-conotoxin access (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), it is likely that the calcium channel in preganglionic nerve terminals targeted by CVID is a N-type (Ca(v)2.2) calcium channel variant.

  5. Protective effect of a calcium channel blocker "diltiazem" on aluminum chloride-induced dementia in mice.

    PubMed

    Rani, Anu; Neha; Sodhi, Rupinder K; Kaur, Amanpreet

    2015-11-01

    Many studies report that heavy metals such as aluminum are involved in amyloid beta aggregation and neurotoxicity. Further, high concentration of aluminum in the brain deregulates calcium signaling which contributes to synaptic dysfunction and halts neuronal communication which ultimately leads to the development of Alzheimer's disease. Recently, diltiazem, a calcium channel blocker clinically used in angina, is reported to decrease amyloid beta production by inhibiting calcium influx, decreasing inflammation and oxidative stress. However, the probable role of this drug in aluminum chloride (AlCl3)-induced experimental dementia is yet to be explored. Therefore, the present study is designed to investigate the effect of AlCl3-induced dementia in mice. Morris water maze test and elevated plus maze were utilized to evaluate learning and memory. Various biochemical estimations including brain acetylcholinesterase activity (AChE), brain total protein, thiobarbituric acid-reactive species (TBARS) level, reduced glutathione (GSH) level, nitrate/nitrite, and superoxide dismutase (SOD) were measured. AlCl3 significantly impaired learning and memory and increased brain AChE, brain total protein, TBARS, and nitrate/nitrite and decreased brain GSH or SOD. On the other hand, treatment with diltiazem significantly reversed AlCl3-induced behavioral and biochemical deficits. The present study indicates the beneficial role of diltiazem in AlCl3-induced dementia.

  6. SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload

    PubMed Central

    Zhang, Bin; Ma, Jian-xing

    2008-01-01

    Background SERPINA3K, an extracellular serine proteinase inhibitor (serpin), has been shown to have decreased levels in the retinas of diabetic rats, which may contribute to diabetic retinopathy. The function of SERPINA3K in the retina has not been investigated. Methodology/Principal Findings The present study identified a novel function of SERPINA3K, i.e. it protects retinal cells against oxidative stress-induced cell death including retinal neuronal cells and Müller cells. Flow-cytometry showed that the protective effect of SERPINA3K on Müller cells is via reducing oxidation-induced necrosis. Measurements of intracellular calcium concentration showed that SERPINA3K prevented the intracellular calcium overload induced by H2O2. A similar protective effect was observed using a calcium chelator (BAPTA/AM). Further, SERPINA3K inhibited the phosphorylation of phospholipase C (PLC)-gamma1 induced by H2O2. Likewise, a specific PLC inhibitor showed similar protective effects on Müller cells exposed to H2O2. Furthermore, the protective effect of SERPINA3K was attenuated by a specific PLC activator (m-3M3FBS). Finally, in a binding assay, SERPINA3K displayed saturable and specific binding on Müller cells. Conclusion/Significance These results for the first time demonstrate that SERPINA3K is an endogenous serpin which protects cells from oxidative stress-induced cells death, and its protective effect is via blocking the calcium overload through the PLC pathway. The decreased retinal levels of SERPINA3K may represent a new pathogenic mechanism for the retinal Müller cell dysfunction and neuron loss in diabetes. PMID:19115003

  7. An Arabidopsis mutant impaired in intracellular calcium elevation is sensitive to biotic and abiotic stress

    PubMed Central

    2014-01-01

    Background Ca2+, a versatile intracellular second messenger in various signaling pathways, initiates many responses involved in growth, defense and tolerance to biotic and abiotic stress. Endogenous and exogenous signals induce cytoplasmic Ca2+ ([Ca2+]cyt) elevation, which are responsible for the appropriate downstream responses. Results Here we report on an ethyl-methane sulfonate-mediated Arabidopsis mutant that fails to induce [Ca2+]cyt elevation in response to exudate preparations from the pathogenic mibrobes Alternaria brassicae, Rhizoctonia solani, Phytophthora parasitica var. nicotianae and Agrobacterium tumefaciens. The cytoplasmic Ca2+elevation mutant1 (cycam1) is susceptible to infections by A. brassicae, its toxin preparation and sensitive to abiotic stress such as drought and salt. It accumulates high levels of reactive oxygen species and contains elevated salicylic acid, abscisic acid and bioactive jasmonic acid iso-leucine levels. Reactive oxygen species- and phytohormone-related genes are higher in A. brassicae-treated wild-type and mutant seedlings. Depending on the analysed response, the elevated levels of defense-related compounds are either caused by the cycam mutation and are promoted by the pathogen, or they are mainly due to the pathogen infection or application of pathogen-associated molecular patterns. Furthermore, cycam1 shows altered responses to abscisic acid treatments: the hormone inhibits germination and growth of the mutant. Conclusions We isolated an Arabidopsis mutant which fails to induce [Ca2+]cyt elevation in response to exudate preparations from various microbes. The higher susceptibility of the mutant to pathogen infections correlates with the higher accumulation of defense-related compounds, such as phytohormones, reactive oxygen-species, defense-related mRNA levels and secondary metabolites. Therefore, CYCAM1 couples [Ca2+]cyt elevation to biotic, abiotic and oxidative stress responses. PMID:24920452

  8. T-type calcium channel Cav3.2 deficient mice show elevated anxiety, impaired memory and reduced sensitivity to psychostimulants

    PubMed Central

    Gangarossa, Giuseppe; Laffray, Sophie; Bourinet, Emmanuel; Valjent, Emmanuel

    2014-01-01

    The fine-tuning of neuronal excitability relies on a tight control of Ca2+ homeostasis. The low voltage-activated (LVA) T-type calcium channels (Cav3.1, Cav3.2 and Cav3.3 isoforms) play a critical role in regulating these processes. Despite their wide expression throughout the central nervous system, the implication of T-type Cav3.2 isoform in brain functions is still poorly characterized. Here, we investigate the effect of genetic ablation of this isoform in affective disorders, including anxiety, cognitive functions as well as sensitivity to drugs of abuse. Using a wide range of behavioral assays we show that genetic ablation of the cacna1h gene results in an anxiety-like phenotype, whereas novelty-induced locomotor activity is unaffected. Deletion of the T-type channel Cav3.2 also triggers impairment of hippocampus-dependent recognition memories. Acute and sensitized hyperlocomotion induced by d-amphetamine and cocaine are dramatically reduced in T-type Cav3.2 deficient mice. In addition, the administration of the T-type blocker TTA-A2 prevented the expression of locomotor sensitization observed in wildtype mice. In conclusion, our data reveal that physiological activity of this specific Ca2+ channel is required for affective and cognitive behaviors. Moreover, our work highlights the interest of T-type channel blockers as therapeutic strategies to reverse drug-associated alterations. PMID:24672455

  9. Calcium-acting drugs modulate expression and development of chronic tolerance to nicotine-induced antinociception in mice.

    PubMed

    Damaj, M I

    2005-11-01

    Initial studies in our laboratory suggested that tolerance to nicotine is thought to involve neuronal adaptation not only at the level of the drug-receptor interaction but at postreceptor events such as calcium-dependent second messengers. The present study was undertaken to investigate the hypothesis that L-type calcium channels and calcium-dependent calmodulin protein kinase II are involved in the development and expression of nicotine tolerance. To that end, the effects of modulation of L-type calcium channels (through the use of inhibitors or activators) as well as calcium-dependent calmodulin protein kinase II inactivation were studied in a mouse model of tolerance where mice were infused with nicotine in minipumps (24 mg/kg/day) for 14 days. In addition, the activity of calcium-dependent calmodulin protein kinase II in the lumbar spinal cord region obtained from nicotine-tolerant mice was measured. Our data showed that chronic administration of L-type calcium channel antagonists nimodipine (1 and 5 mg/kg) and verapamil (10 mg/kg) prevented the development of tolerance to nicotine-induced antinociception. In contrast, chronic exposure of BAYK8644 [(+/-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester], a calcium channel activator, enhanced nicotine's tolerance. Moreover, a significant increase in both dependent and independent calcium-dependent calmodulin protein kinase II activity was seen in the spinal cord in nicotine-tolerant mice. Finally, spinal administration of 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-tyrosyl]-4-phenylpiperazine (KN-62), a calcium-dependent calmodulin protein kinase II antagonist, reduced the expression of tolerance to nicotine-induced antinociception in mice. In conclusion, our data indicate that calcium-dependent mechanisms such as L-type calcium channels and calcium-dependent calmodulin protein kinase II activation are involved in the expression and development of nicotine

  10. Sodium-dependent calcium extrusion and sensitivity regulation in retinal cones of the salamander.

    PubMed Central

    Nakatani, K; Yau, K W

    1989-01-01

    1. Membrane current was recorded from an isolated, dark-adapted salamander cone by sucking its inner segment into a tight-fitting glass pipette containing Ringer solution. The outer segment of the cell was exposed to a bath solution that could be changed rapidly. 2. After removing Na+ from the bath Ringer solution for a short period of time in darkness (the 'loading period'), a transient inward current was observed upon restoring it in bright light. A similar but longer-lasting current was observed when Na+ was restored in the light after a large Ca2+ influx was induced through the light-sensitive conductance in darkness. 3. The above transient current was not observed if Li+ or guanidinium was substituted for Na+ in the light, or if Ba2+ was substituted for Ca2+ during the dark loading period. However, a current was observed if Sr2+ was the substituting ion for Ca2+ during loading. These observations suggested that the current was associated with an electrogenic Na+-dependent Ca2+ efflux at the cone outer segment. 4. The saturated amplitude of the exchange current was 12-25 pA with a mean around 16 pA. This is very comparable to that measured in the outer segment of a salamander rod under similar conditions. 5. By comparing a known Ca2+ load in a cone outer segment to the subsequent charge transfer through the exchange, we estimated that the stoichiometry of the exchange was near 3Na+:1Ca2+. 6. With a small Ca2+ load, or in the presence of Cs+ around the inner segment, the final temporal decline of the Na+-Ca2+ exchange current was roughly exponential, with a mean time constant of about 100 ms. This decline is about four times faster than that measured in rods. We interpret the shorter time constant in cones to reflect a faster rate of decline of intracellular free Ca2+ in their outer segments resulting from the exchange activity. 7. In the absence of external Na+, and hence any Na+-dependent Ca2+ efflux, the absolute sensitivity of a cone to a dim flash was

  11. Dihydropyridine-sensitive and omega-conotoxin-sensitive calcium channels in a mammalian neuroblastoma-glioma cell line.

    PubMed Central

    Kasai, H; Neher, E

    1992-01-01

    of cellular differentiation induced by dibutyryl cyclic AMP, IDHP appeared earlier than I omega CgTX. 11. These results indicate that two classes of Ca2+ channels contribute to the HVA currents of this cell line. The DHP-sensitive channel is more apt to generate Ca2+ spikes and Ca2+ plateau potentials than the omega CgTX-sensitive channel. PMID:1375634

  12. Calcium channel blockade reduces mechanical strain-induced extracellular matrix gene response in lamina cribrosa cells.

    PubMed

    Quill, B; Irnaten, M; Docherty, N G; McElnea, E M; Wallace, D M; Clark, A F; O'Brien, C J

    2015-07-01

    This study examines the effect of the L-type calcium channel blocker verapamil on mechanical strain-induced extracellular matrix genes in optic nerve head lamina cribrosa (LC) cells. Changes in LC cell intracellular calcium [Ca(2+)]i following hypotonic cell membrane stretch were measured with the fluorescent probe fura-2/AM. Fluorescence intensity was measured, after labelling, by calcium (Ca2+) imaging confocal microscopy. Confluent human LC cell cultures were serum starved for 24 h prior to exposure to cyclical mechanical strain (1 Hz, 15%) for 24 h in the presence or absence of verapamil (10 mm). Transforming growth factor-β 1 (TGF-β1), collagen 6A3 (COL6A3) and chondroitin sulfate proteoglycan 2 (CSPG2) mRNA expression levels were assessed by quantitative RT-PCR. Hypotonic cell membrane stretch of LC cells from normal donors significantly increased [Ca2+]i (p<0.05). Exposure to cyclical mechanical strain (15% strain) produced a statistically significant increase in the three matrix genes that were examined (TGF-β1, COL6A3 and CSPG2). This response in both cyclical and mechanical stretch was significantly reduced by pretreating LC cells with the L-type calcium channel blocker verapamil (p<0.05). This study provides evidence of a novel mechanotransduction pathway linking mechanical strain, cation channel function and the induction of LC cell matrix gene transcription. This highlights the potential involvement of calcium influx in the activation of matrix remodelling responses in the optic nerve head and supports the rationale that calcium channel blockers may attenuate disease progression in glaucoma. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  13. Protective effects of resveratrol on calcium-induced oxidative stress in rat heart mitochondria.

    PubMed

    Gutiérrez-Pérez, Areli; Cortés-Rojo, Christian; Noriega-Cisneros, Ruth; Calderón-Cortés, Elizabeth; Manzo-Avalos, Salvador; Clemente-Guerrero, Mónica; Godínez-Hernández, Daniel; Boldogh, Istvan; Saavedra-Molina, Alfredo

    2011-04-01

    Trans-resveratrol is a nutraceutical with known antioxidant, anti-inflammatory, cardioprotective, and anti-apoptotic properties. The aim of this study was to evaluate the effects of resveratrol on heart mitochondria. Resveratrol significantly decreased Fe(2+) + ascorbate oxidant system-induced lipid peroxide levels, preserved physiological levels of glutathione, and increased nitric oxide (NO) levels in mitochondria. Under calcium-mediated stress, there was a 2.7-fold increase in the NO levels, and a mild decoupling in the mitochondrial respiratory chain. These results provide a mechanism for and support the beneficial effects of resveratrol under pathological conditions induced by oxidative stress and calcium overload. In addition, these findings underscore the usefulness of resveratrol in the prevention of cardiovascular diseases.

  14. The role of calcium and nitric oxide during liver enzyme release induced by increased physical forces as evidenced in partially hepatectomized rats.

    PubMed

    Díaz-Juárez, Julieta; Hernández-Muñoz, Rolando

    2011-03-01

    Although increased plasma enzyme activities could be diagnostic for tissue damage, the mechanisms controlling cellular enzyme release remain poorly understood. We found a selective and drastic elevation of serum enzyme activities accompanying rat liver regeneration after partial hepatectomy (PH), apparently controlled by a mechanism dependent on flow-bearing physical forces. In fact, this study assesses a putative role of calcium mobilization and nitric oxide (NO) production underlying rat liver enzyme release. The role of increased shear stress (by enhancing viscosity during perfusion) and the participation of cell calcium and NO were tested in isolated livers subjected to increasing flow rate. After PH, there was a drastic elevation of serum activities for liver enzyme markers, clearly predominating those of mitochondrial localization. Liver enzyme release largely depended on extracellular calcium entry, probably mediated by stretch-sensitive calcium channels, as well as by increasing NO production. However, these effects were differentially observed when comparing liver enzymes from cytoplasmic or mitochondrial compartments. Moreover, a possible role for cell-mediated mechanotransduction in liver enzyme release was suggested by increasing shear stress (high viscosity), which also selectively affected the release of the enzymes tested. Therefore, we show, for the first time, that flow-induced shear stress can control the amount of hepatic enzymes released into the bloodstream, which is largely regulated through modifications in cell calcium mobilization and production of liver NO, events markedly elevated in the proliferating rat liver. Copyright © 2011 American Association for the Study of Liver Diseases.

  15. [Calcium leak of sarcoplasmic reticulum induces degradation of troponin I in skeletal muscle fibers.].

    PubMed

    Li, Quan; Wang, Yun-Ying; Li, Hui; Jiao, Bo; Yu, Zhi-Bin

    2009-06-25

    The troponin I subunit (TnI) was used as a molecular marker to explore the relationship between the resting intracellular Ca(2+) concentration and myofibril degradation in muscle fibers. The isolated soleus muscle strips of rats were treated by caffeine and H2O2. Caffeine is an opener to increase the calcium release channel open probability of sarcoplasmic reticulum (SR) in contraction phase. H2O2 induces a calcium leak of SR calcium release channel in relaxation phase. The expression and degradation of TnI were detected by Western blot. The resting tension of tetanic contraction and expression of TnI were not changed, but the developed tension was lowered in isolated soleus muscle strips during 40 min of calcium-free Krebs perfusion. Low concentrations of caffeine (1 and 5 mmol/L) perfusion induced a transient increase in resting tension during fatigue period, but did not alter the extent of fatigue, recovery rate after fatigue and expression of TnI in muscle strips. High concentration of caffeine (10 mmol/L) perfusion induced a progressive increase in resting tension, a higher rate of fatigue and a decrease in recovery rate after fatigue in muscle strips. There was a detectable degradation of TnI in soleus after 10 mmol/L caffeine treatment. H2O2 perfusion facilitated a progressive increase in resting tension in a dose-dependent manner, but did not influence the fatigue rate of tetanic contraction. The recovery rate after fatigue showed a quick resumption before decline during H2O2 perfusion. Degradation of TnI occurred in 5 and 10 mmol/L H2O2-treated soleus muscles. Since resting tension is dependent on intracellular Ca(2+) concentration, the above-mentioned results suggest that SR Ca(2+) leakage in relaxation phase may induce a degradation of TnI in skeletal muscle fibers.

  16. Evidence for increased renal tubule and parathyroid gland sensitivity to serum calcium in human idiopathic hypercalciuria.

    PubMed

    Worcester, Elaine M; Bergsland, Kristin J; Gillen, Daniel L; Coe, Fredric L

    2013-09-15

    Patients with idiopathic hypercalciuria (IH) have decreased renal calcium reabsorption, most marked in the postprandial state, but the mechanisms are unknown. We compared 29 subjects with IH and 17 normal subjects (N) each fed meals providing identical amounts of calcium. Urine and blood samples were collected fasting and after meals. Levels of three candidate signalers, serum calcium (SCa), insulin (I), and plasma parathyroid hormone (PTH), did not differ between IH and N either fasting or fed, but all changed with feeding, and the change in SCa was greater in IH than in N. Regression analysis of fractional excretion of calcium (FECa) was significant for PTH and SCa in IH but not N. With the use of multivariable analysis, Sca entered the model while PTH and I did not. To avoid internal correlation we decomposed FECa into its independent terms: adjusted urine calcium (UCa) and UFCa molarity. Analyses using adjusted Uca and unadjusted Uca parallel those using FECa, showing a dominant effect of SCa with no effect of PTH or I. The effect of SCa may be mediated via vitamin D receptor-stimulated increased abundance of basolateral Ca receptor, which is supported by the fact PTH levels also seem more responsive to serum Ca in IH than in N. Although our data support an effect of SCa on FECa and UCa, which is more marked in IH than in N, it can account for only a modest fraction of the meal effect, perhaps 10-20%, suggesting additional mediators are also responsible for the exaggerated postprandial hypercalciuria seen in IH.

  17. Modification of caffeine-induced injury in Ca2+-free perfused rat hearts. Relationship to the calcium paradox.

    PubMed Central

    Vander Heide, R. S.; Altschuld, R. A.; Lamka, K. G.; Ganote, C. E.

    1986-01-01

    The pathogenesis of the calcium paradox has not been established. In calcium-free perfused hearts, caffeine, which releases calcium from the sarcoplasmic reticulum, causes severe myocardial injury, with creatine kinase (CK) release and contraction band necrosis similar in many respects to the calcium paradox. It has been postulated that contracture, initiated by a small rise in intracellular calcium, may cause sarcolemmal injury in both the calcium paradox and caffeine-induced myocardial injury. The present study was initiated to determine whether interventions which modulate caffeine-induced contracture will also correspondingly alter cellular injury. The effects of caffeine dose, procaine, extended calcium-free perfusion, elevated potassium, temperature, and increasing intracellular sodium on caffeine-induced contracture were examined in Langendorff-perfused adult rat hearts. Caffeine-induced contracture at 22 C increased over a dose range of 5-40 mM caffeine. Procaine, which inhibits caffeine-induced calcium release at doses between 5 and 20 mM, progressively reduced contracture caused by addition of 20 mM caffeine at 22 C. Hearts perfused with calcium-free solution containing 16 mM K+ showed a reduction in caffeine-induced contracture. Extended calcium-free perfusion (20 minutes) at temperatures from 18 to 37 C resulted in a progressive reduction of caffeine-induced contracture. Each of these interventions was also found to inhibit caffeine-induced injury at 37 C. Low temperature was found to have complex effects. Hypothermia enhanced caffeine contractures but also protected hearts from cell separations and CK release. Increasing intracellular sodium was found to enhance caffeine-induced contracture at 37 C. There was a direct correlation between measured intracellular sodium levels and the magnitude and duration of caffeine-induced contracture. These results demonstrate a direct correlation between the magnitude of contracture and myocardial injury in calcium

  18. Influence of zinc on calcium-dependent signal transduction pathways during aluminium-induced neurodegeneration.

    PubMed

    Singla, Neha; Dhawan, D K

    2014-10-01

    Metals perform important functions in the normal physiological system, and alterations in their levels may lead to a number of diseases. Aluminium (Al) has been implicated as a major risk factor, which is linked to several neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. On the other hand, zinc (Zn) is considered as a neuromodulator and an essential dietary element that regulates a number of biological activities in our body. The aim of the present study was to investigate the effects of Zn supplementation, if any, in ameliorating the changes induced by Al on calcium signalling pathway. Male Sprague Dawley rats weighing 140-160 g were divided into four different groups viz.: normal control, aluminium treated (100 mg/kg b.wt./day via oral gavage), zinc treated (227 mg/l in drinking water) and combined aluminium and zinc treated. All the treatments were carried out for a total duration of 8 weeks. Al treatment decreased the Ca(2+) ATPase activity whereas increased the levels of 3', 5'-cyclic adenosine monophosphate, intracellular calcium and total calcium content in both the cerebrum and cerebellum, which, however, were modulated upon Zn supplementation. Al treatment exhibited a significant elevation in the protein expressions of phospholipase C, inositol triphosphate and protein kinase A but decreased the expression of protein kinase C, which, however, was reversed upon Zn co-treatment. Al treatment also revealed alterations in neurohistoarchitecture in the form of calcium deposits, which were improved upon zinc co-administration. The present study, therefore, suggests that zinc regulates the intracellular calcium signalling pathway during aluminium-induced neurodegeneration.

  19. Participation of extracellular calcium in α-hederin-induced contractions of rat isolated stomach strips.

    PubMed

    Mendel, Marta; Chłopecka, Magdalena; Dziekan, Natalia; Karlik, Wojciech; Wiechetek, Maria

    2013-03-07

    The dry extract of Hedera helix leaves, due to its secretolytic and antispasmodic effects, is commonly used to produce pharmaceuticals applied in case of cough and other respiratory symptoms. The results of some in vitro studies as well as the clinical signs of poisoning caused by Hedera helix suggest however strong contractile effect on smooth muscle. In order to clarify the impact of α-hederin (the main active agent of ivy extract) on smooth muscle, the origin of activated calcium involved in α-hederin-induced contraction of gastric smooth muscle preparations was studied. The study was carried out on rat isolated stomach corpus and fundus strips, under isotonic conditions. The effect of α-hederin (100 μM) on smooth muscle preparations was measured before and after the treatment with verapamil during the incubation in modified Krebs-Henseleit solution (M K-HS). Besides, the effect of saponin was measured during the incubation of preparation in Ca2+-free modified Krebs-Henseleit solution or Ca2+-free EGTA-containing modified Krebs-Henseleit solution. The obtained results revealed that the application of verapamil significantly inhibited the reaction evoked by α-hederin. The incubation of stomach strips in calcium-free modified Krebs-Henseleit solution did not change the force of the observed contraction in comparison to the reaction of the preparations incubated in regular incubation solution (M K-HS). In contrary, the replacement of M K-HS by calcium-free chelator-containing solution inhibited totally the reaction to α-hederin. The results indicated that α-hederin-induced contraction results from the influx of calcium which is located in intercellular spaces or bound to the outside of the cell membrane. The Ca2+ influx occurs predominantly through voltage-dependent calcium channels of L-type. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  20. Renal Calcium Oxalate Deposits Induce a Pro-Atherosclerotic and Pro-Osteoporotic Response in Mice.

    PubMed

    Kusumi, Kirsten; Barr-Beare, Evan; Saxena, Vijay; Safedi, Fayez; Schwaderer, Andrew

    2017-09-01

    Urinary stone disease (USD) is increasing in adult and pediatric populations. Adult and pediatric studies have demonstrated decreased bone mineral density and increased fracture rates. USD has also been independently linked to increased rates of myocardial infarction and cerebral vascular accidents. Although USD is a multisystem disorder involving the kidneys, bone, and vasculature, the molecular mechanisms linking these three organs remain unknown. Calcium oxalate nephropathy was induced in C57BL/6J mice with intra-peritoneal (ip) injection of sodium glyoxolate. Half of each kidney underwent Pizzalato staining and half was snap frozen for RNA extraction. RT(2) Profiler Mouse Atherosclerosis, Osteoporosis, and Calcium Signaling PCR Arrays (Qiagen) were performed. Only results that passed quality checks in PCR array reproducibility and genomic DNA contamination were included. Genes had to show at least fourfold differential expression and P < 0.01 to be considered significant. Atherosclerosis array showed upregulation of 19 genes by fourfold, 10 of which were ≥10-fold. All 19 had P ≤ 0.002. The Osteoporosis array showed fourfold upregulation of 10 genes, five showed >10-fold increase. All 10 have P ≤ 0.003. The calcium signaling array showed significant fourfold upregulation of 10 genes, four of which were ≥10-fold. All 10 have P ≤ 0.03. We have demonstrated that calcium oxalate nephropathy can induce upregulation of atherosclerotic, metabolic bone, and calcium homeostasis genes in a murine model. This may be and initial step in identifying the molecular mechanisms linking stone, bone, and cardiovascular disease. J. Cell. Biochem. 118: 2744-2751, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  1. Study of the function of sarcoplasmic reticulum of vascular smooth muscle during activation due to depolarization-induced calcium influx

    SciTech Connect

    Hwang, K.S.

    1987-01-01

    The role of sarcoplasmic reticulum (SR) in vascular smooth muscle was evaluated with respect to regulation of myoplasmic Ca{sup 2+} during the Ca{sup 2+} entry induced by depolarization. Calcium agonist, Bay K8644, stimulated Ca{sup 2+} influx as well as tension in physiological salt solution, (PSS) in contrast to the priming effects due to the depolarization originally reported. Disparity, however, was found between the Ca{sup 2+} entered and tension developed. Correlation between the tension and {sup 45}Ca influx showed a typical threshold phenomenon; the basal Ca{sup 2+} influx can be raised to a certain level (25%) without tension induction, after which a minor increase in Ca{sup 2+} influx produced significant tension. This subthreshold Ca{sup 2+} influx was found accumulated in the caffeine-sensitive Ca stores, the SR. This confirmed the dependency of tension on the rate of Ca{sup 2+} entry demonstrated by a previous report.

  2. Mapping of dihydropyridine binding residues in a less sensitive invertebrate L-type calcium channel (LCa v 1).

    PubMed

    Senatore, Adriano; Boone, Adrienne; Lam, Stanley; Dawson, Taylor F; Zhorov, Boris; Spafford, J David

    2011-01-01

    Invertebrate L-type calcium channel, LCa(v) 1, isolated from the pond snail Lymnaea stagnalis is nearly indistinguishable from mammalian Ca(v) 1.2 (α1C) calcium channel in biophysical characteristics observed in vitro. These L-type channels are likely constrained within a narrow range of biophysical parameters to perform similar functions in the snail and mammalian cardiovascular systems. What distinguishes snail and mammalian L-type channels is a difference in dihydropyridine sensitivity: 100 nM isradipine exhibits a significant block of mammalian Ca(v) 1.2 currents without effect on snail LCa(v)1 currents. The native snail channel serves as a valuable surrogate for validating key residue differences identified from previous experimental and molecular modeling work. As predicted, three residue changes in LCa(v)1 (N_3o18, F_3i10, and I_4i12) replaced with DHP-sensing residues in respective positions of Ca(v) 1.2, (Q_3o18, Y_3i10, and M_4i12) raises the potency of isradipine block of LCa(v)1 channels to that of mammalian Ca(v) 1.2. Interestingly, the single N_3o18_Q mutation in LCa(v) 1 channels lowers DHP sensitivity even further and the triple mutation bearing enhanced isradipine sensitivity, still retains a reduced potency of agonist, (S)-Bay K8644.

  3. Application of confocal microscopy on glutamate-induced intracellular calcium transient in neurons

    NASA Astrophysics Data System (ADS)

    Zhu, Geng; Zhou, Wei; Zhang, Yuan; Liu, Xiuli; Wu, Yuxiang; Luo, Qingming

    2006-02-01

    Intracellular calcium, as an important second messenger, plays a significant role in cell signaling transduction and metabolism. Glutamate can induce the intracellular calcium transient through triggering diverse signaling pathways. To test the effect of glutamate to neurons, we loaded Fluo-3/Am in cultured rat hippocampal neurons, and then acquired two-dimensional fluorescent image by confocal microscopy and the analyzed fluorescent intensity. In cultured neurons, we observed two types of neurons that have different morphology: bipolar-type and pyramidal-type. Inducing [Ca 2+] i transient by glutamate, we found the amplitude and time constant of the response curves of bipolar neurons are larger than those of pyramidal neurons. Further, we induced [Ca 2+] ii transient under different concentrations of glutamate. Two different types of kinetic of the [Ca 2+] i transient have been found, corresponded to the two kinds of neuron. The amplitude of [Ca 2+] i transient increased when applying higher concentration of glutamate in pyramidal neurons; while it decreased in bipolar ones. Responses of neurons bathing in calcium-free extracellular solution to glutamate were different from those bathing in normal solution. [Ca 2+] i transient of pyramidal neurons caused by any concentration were totally blocked; while [Ca 2+] i transient in bipolar neurons caused by high concentration of glutamate (500μM) were partly inhibited. All of the phenomena suggest that different types of cultured hippocampal neurons may have different mechanism of the response to glutamate.

  4. Effects of Calcium Gluconate, a Water Soluble Calcium Salt on the Collagen-Induced DBA/1J Mice Rheumatoid Arthritis

    PubMed Central

    Sohn, Ki Cheul; Kang, Su Jin; Kim, Joo Wan; Kim, Ki Young; Ku, Sae Kwang; Lee, Young Joon

    2013-01-01

    This study examined the effects of calcium (Ca) gluconate on collagen-induced DBA mouse rheumatoid arthritis (CIA). A single daily dose of 200, 100 or 50 mg/kg Ca gluconate was administered orally to male DBA/1J mice for 40 days after initial collagen immunization. To ascertain the effects administering the collagen booster, CIA-related features (including body weight, poly-arthritis, knee and paw thickness, and paw weight increase) were measured from histopathological changes in the spleen, left popliteal lymph node, third digit and the knee joint regions. CIA-related bone and cartilage damage improved significantly in the Ca gluconate- administered CIA mice. Additionally, myeloperoxidase (MPO) levels in the paw were reduced in Ca gluconate-treated CIA mice compared to CIA control groups. The level of malondialdehyde (MDA), an indicator of oxidative stress, decreased in a dosedependent manner in the Ca gluconate group. Finally, the production of IL-6 and TNF-α, involved in rheumatoid arthritis pathogenesis, were suppressed by treatment with Ca gluconate. Taken together, these results suggest that Ca gluconate is a promising candidate anti-rheumatoid arthritis agent, exerting anti-inflammatory, anti-oxidative and immunomodulatory effects in CIA mice. PMID:24244814

  5. Dual pathways of calcium entry in spike and plateau phases of luteinizing hormone release from chicken pituitary cells: sequential activation of receptor-operated and voltage-sensitive calcium channels by gonadotropin-releasing hormone

    SciTech Connect

    Davidson, J.S.; Wakefield, I.K.; King, J.A.; Mulligan, G.P.; Millar, R.P.

    1988-04-01

    It has previously been shown that, in pituitary gonadotrope cells, the initial rise in cytosolic Ca2+ induced by GnRH is due to a Ca2+ mobilization from intracellular stores. This raises the possibility that the initial transient spike phase of LH release might be fully or partially independent of extracellular Ca2+. We have therefore characterized the extracellular Ca2+ requirements, and the sensitivity to Ca2+ channel blockers, of the spike and plateau phases of secretion separately. In the absence of extracellular Ca2+ the spike and plateau phases were inhibited by 65 +/- 4% and 106 +/- 3%, respectively. Both phases exhibited a similar dependence on concentration of extracellular Ca2+. However, voltage-sensitive Ca2+ channel blockers D600 and nifedipine had a negligible effect on the spike phase, while inhibiting the plateau phase by approximately 50%. In contrast, ruthenium red, Gd3+ ions, and Co2+ ions inhibited both spike and plateau phases to a similar extent as removal of extracellular Ca2+. A fraction (35 +/- 4%) of spike phase release was resistant to removal of extracellular Ca2+. This fraction was abolished after calcium depletion of the cells by preincubation with EGTA in the presence of calcium ionophore A23187, indicating that it depends on intracellular Ca2+ stores. Neither absence of extracellular Ca2+, nor the presence of ruthenium red or Gd3+ prevented mobilization of 45Ca2+ from intracellular stores by GnRH. We conclude that mobilization of intracellular stored Ca2+ is insufficient by itself to account for full spike phase LH release.

  6. Novel Resorbable and Osteoconductive Calcium Silicophosphate Scaffold Induced Bone Formation

    PubMed Central

    Ros-Tárraga, Patricia; Mazón, Patricia; Rodríguez, Miguel A.; Meseguer-Olmo, Luis; De Aza, Piedad N.

    2016-01-01

    This aim of this research was to develop a novel ceramic scaffold to evaluate the response of bone after ceramic implantation in New Zealand (NZ) rabbits. Ceramics were prepared by the polymer replication method and inserted into NZ rabbits. Macroporous scaffolds with interconnected round-shaped pores (0.5–1.5 mm = were prepared). The scaffold acted as a physical support where cells with osteoblastic capability were found to migrate, develop processes, and newly immature and mature bone tissue colonized on the surface (initially) and in the material’s interior. The new ceramic induced about 62.18% ± 2.28% of new bone and almost complete degradation after six healing months. An elemental analysis showed that the gradual diffusion of Ca and Si ions from scaffolds into newly formed bone formed part of the biomaterial’s resorption process. Histological and radiological studies demonstrated that this porous ceramic scaffold showed biocompatibility and excellent osteointegration and osteoinductive capacity, with no interposition of fibrous tissue between the implanted material and the hematopoietic bone marrow interphase, nor any immune response after six months of implantation. No histological changes were observed in the various organs studied (para-aortic lymph nodes, liver, kidney and lung) as a result of degradation products being released. PMID:28773906

  7. Novel Resorbable and Osteoconductive Calcium Silicophosphate Scaffold Induced Bone Formation.

    PubMed

    Ros-Tárraga, Patricia; Mazón, Patricia; Rodríguez, Miguel A; Meseguer-Olmo, Luis; De Aza, Piedad N

    2016-09-20

    This aim of this research was to develop a novel ceramic scaffold to evaluate the response of bone after ceramic implantation in New Zealand (NZ) rabbits. Ceramics were prepared by the polymer replication method and inserted into NZ rabbits. Macroporous scaffolds with interconnected round-shaped pores (0.5-1.5 mm = were prepared). The scaffold acted as a physical support where cells with osteoblastic capability were found to migrate, develop processes, and newly immature and mature bone tissue colonized on the surface (initially) and in the material's interior. The new ceramic induced about 62.18% ± 2.28% of new bone and almost complete degradation after six healing months. An elemental analysis showed that the gradual diffusion of Ca and Si ions from scaffolds into newly formed bone formed part of the biomaterial's resorption process. Histological and radiological studies demonstrated that this porous ceramic scaffold showed biocompatibility and excellent osteointegration and osteoinductive capacity, with no interposition of fibrous tissue between the implanted material and the hematopoietic bone marrow interphase, nor any immune response after six months of implantation. No histological changes were observed in the various organs studied (para-aortic lymph nodes, liver, kidney and lung) as a result of degradation products being released.

  8. The antidepressant fluoxetine induces necrosis by energy depletion and mitochondrial calcium overload

    PubMed Central

    Kischel, Philippe; Delcroix, Vanessa; Demaurex, Nicolas; Castelbou, Cyril; Vacher, Anne-Marie; Devin, Anne; Ducret, Thomas; Nunes, Paula; Vacher, Pierre

    2017-01-01

    Selective Serotonin Reuptake Inhibitor antidepressants, such as fluoxetine (Prozac), have been shown to induce cell death in cancer cells, paving the way for their potential use as cancer therapy. These compounds are able to increase cytosolic calcium concentration ([Ca2+]cyt), but the involved mechanisms and their physiological consequences are still not well understood. Here, we show that fluoxetine induces an increase in [Ca2+]cyt by emptying the endoplasmic reticulum (ER) through the translocon, an ER Ca2+ leakage structure. Our data also show that fluoxetine inhibits oxygen consumption and lowers mitochondrial ATP. This latter is essential for Ca2+ reuptake into the ER, and we postulated therefore that the fluoxetine-induced decrease in mitochondrial ATP production results in the emptying of the ER, leading to capacitative calcium entry. Furthermore, Ca2+ quickly accumulated in the mitochondria, leading to mitochondrial Ca2+ overload and cell death. We found that fluoxetine could induce an early necrosis in human peripheral blood lymphocytes and Jurkat cells, and could also induce late apoptosis, especially in the tumor cell line. These results shed light on fluoxetine-induced cell death and its potential use in cancer treatment. PMID:27911858

  9. The antidepressant fluoxetine induces necrosis by energy depletion and mitochondrial calcium overload.

    PubMed

    Charles, Emilie; Hammadi, Mehdi; Kischel, Philippe; Delcroix, Vanessa; Demaurex, Nicolas; Castelbou, Cyril; Vacher, Anne-Marie; Devin, Anne; Ducret, Thomas; Nunes, Paula; Vacher, Pierre

    2017-01-10

    Selective Serotonin Reuptake Inhibitor antidepressants, such as fluoxetine (Prozac), have been shown to induce cell death in cancer cells, paving the way for their potential use as cancer therapy. These compounds are able to increase cytosolic calcium concentration ([Ca2+]cyt), but the involved mechanisms and their physiological consequences are still not well understood. Here, we show that fluoxetine induces an increase in [Ca2+]cyt by emptying the endoplasmic reticulum (ER) through the translocon, an ER Ca2+ leakage structure. Our data also show that fluoxetine inhibits oxygen consumption and lowers mitochondrial ATP. This latter is essential for Ca2+ reuptake into the ER, and we postulated therefore that the fluoxetine-induced decrease in mitochondrial ATP production results in the emptying of the ER, leading to capacitative calcium entry. Furthermore, Ca2+ quickly accumulated in the mitochondria, leading to mitochondrial Ca2+ overload and cell death. We found that fluoxetine could induce an early necrosis in human peripheral blood lymphocytes and Jurkat cells, and could also induce late apoptosis, especially in the tumor cell line. These results shed light on fluoxetine-induced cell death and its potential use in cancer treatment.

  10. Increased intracellular calcium activates serum and glucocorticoid-inducible kinase 1 (SGK1) through a calmodulin-calcium calmodulin dependent kinase kinase pathway in Chinese hamster ovary cells.

    PubMed

    Imai, Seiji; Okayama, Naotsuka; Shimizu, Manabu; Itoh, Makoto

    2003-04-04

    SGK1 is one of the protein-serine/threonine kinases that is activated by insulin in a PI3K-dependent manner. Although SGK1 mediates a variety of biological activities, the mechanisms regulating its activity remain unclear. In this study, we examined the potential roles of calcium signaling in the activation of SGK1. Treatment of CHO-IR cells with a cell-permeable calcium chelator, BAPTA-AM, abolished the insulin-induced activation of SGK1. Increasing intracellular calcium concentration by treating cells with thapsigargin or ionomycin induced a 6-8 fold increase in SGK1 activation. This was not affected by a PI3K inhibitor, wortmannin, but was completely inhibited by the calmodulin inhibitors, W 7 and W 5. Co-transfection of CHO cells with FLAG-SGK1 and CaMKK revealed the direct association of CaMKK with SGK1. These results suggest a calcium-triggered signaling cascade in which an increase in intracellular calcium concentration directly stimulates SGK1 through CaMKK.

  11. Effect of calcium ionophore A23187 on the sensitivity of early sea urchin embryos to cytotoxic neuropharmacological drugs.

    PubMed

    Buznikov, G A; Mileusnić, R; Yurovskaya, M A; Rakić, L J

    1984-01-01

    The ability of cytotoxic neurochemicals (indole and amphetamine derivatives) to block first cleavage division in the embryos of the sea urchin Arbacia lixula abruptly increases when the embryos are incubated in calcium-free seawater and decreases when the external Ca concentration is raised up to 46.4 mM. Sensitivity of the embryos to these drugs decreases also in the presence of the Ca-ionophore A23187. It is suggested that Ca ions are involved in the realization of physiological effects of "prenervous" neurotransmitters whose presence in early sea urchin embryos was shown by us earlier.

  12. Calcium Occupancy of N-terminal Sites within Calmodulin Induces Inhibition of the Ryanodine Receptor Calcium Release Channel

    SciTech Connect

    Boschek, Curt B; Jones, Terry E; Squier, Thomas C; Bigelow, Diana J

    2007-08-01

    Calmodulin (CaM) regulates calcium release from intracellular stores in skeletal muscle through its association with the ryanodine receptor (RyR1) calcium release channel, where CaM association enhances channel opening at resting calcium levels and its closing at micromolar calcium levels associated with muscle contraction. A high-affinity CaM-binding sequence (RyRp) has been identified in RyR1, which corresponds to a 30-residue sequence (i.e., K3614 – N3643) located within the central portion of the primary sequence. However, it is currently unclear whether the identified CaM-binding sequence a) senses calcium over the physiological range of calcium-concentrations associated with RyR1 regulation or b) plays a structural role unrelated to the calcium-dependent modulation of RyR1 function. Therefore, we have measured the calcium-dependent activation of the individual domains of CaM in association with RyRp and their relationship to the CaM-dependent regulation of RyR1. These measurements utilize an engineered CaM, permitting the site-specific incorporation of N-(1-pyrene) maleimide at either T34C (PyN-CaM) or T110C (PyC-CaM) in the N- and C-domains, respectively. Consistent with prior measurements, we observe a high-affinity association between both apo- and calcium-activated CaM and RyRp. Upon association with RyRp, fluorescence changes in PyN-CaM or PyC-CaM permit the measurement of the calcium-activation of these individual domains. Fluorescence changes upon calcium-activation of PyC-CaM in association with RyRp are indicative of high-affinity calcium-dependent activation of the C-terminal domain of CaM bound to RyRp at resting calcium levels and the activation of the N-terminal domain at levels of calcium associated cellular activation. In comparison, occupancy of calcium-binding sites in the N-domain of CaM mirrors the calcium-dependence of RyR1 inhibition observed at activating calcium levels, where [Ca]1/2 = 4.3 0.4 μM, suggesting a direct regulation of Ry

  13. Parathyroid hormone suppression by intravenous 1,25-dihydroxyvitamin D. A role for increased sensitivity to calcium.

    PubMed Central

    Delmez, J A; Tindira, C; Grooms, P; Dusso, A; Windus, D W; Slatopolsky, E

    1989-01-01

    Numerous in vitro studies in experimental animals have demonstrated a direct suppressive effect of 1,25-dihydroxyvitamin D (1,25(OH)2D) on parathyroid hormone (PTH) synthesis. We therefore sought to determine whether such an effect could be demonstrated in uremic patients undergoing maneuvers designed to avoid changes in serum calcium concentrations. In addition, the response of the parathyroid gland in patients undergoing hypercalcemic suppression (protocol I) and hypocalcemic stimulation (protocol II) before and after 2 wk of intravenous 1,25(OH)2D was evaluated. In those enlisted in protocol I, PTH values fell from 375 +/- 66 to 294 +/- 50 pg (P less than 0.01) after 1,25(OH)2D administration. During hypercalcemic suppression, the "set point" (PTH max + PTH min/2) for PTH suppression by calcium fell from 5.24 +/- 0.14 to 5.06 +/- 0.15 mg/dl (P less than 0.05) with 1,25(OH)2D. A similar decline in PTH levels after giving intravenous 1,25(OH)2D was noted in protocol II patients. During hypocalcemic stimulation, the parathyroid response was attenuated by 1,25(OH)2D. We conclude that intravenous 1,25(OH)2D directly suppresses PTH secretion in uremic patients. This suppression, in part, appears to be due to increased sensitivity of the gland to ambient calcium levels. PMID:2703535

  14. Single Low-Dose Radiation Induced Regulation of Keratinocyte Differentiation in Calcium-Induced HaCaT Cells

    PubMed Central

    Hahn, Hyung Jin; Youn, Hae Jeong; Cha, Hwa Jun; Kim, Karam; An, Sungkwan

    2016-01-01

    Background We are continually exposed to low-dose radiation (LDR) in the range 0.1 Gy from natural sources, medical devices, nuclear energy plants, and other industrial sources of ionizing radiation. There are three models for the biological mechanism of LDR: the linear no-threshold model, the hormetic model, and the threshold model. Objective We used keratinocytes as a model system to investigate the molecular genetic effects of LDR on epidermal cell differentiation. Methods To identify keratinocyte differentiation, we performed western blots using a specific antibody for involucrin, which is a precursor protein of the keratinocyte cornified envelope and a marker for keratinocyte terminal differentiation. We also performed quantitative polymerase chain reaction. We examined whether LDR induces changes in involucrin messenger RNA (mRNA) and protein levels in calcium-induced keratinocyte differentiation. Results Exposure of HaCaT cells to LDR (0.1 Gy) induced p21 expression. p21 is a key regulator that induces growth arrest and represses stemness, which accelerates keratinocyte differentiation. We correlated involucrin expression with keratinocyte differentiation, and examined the effects of LDR on involucrin levels and keratinocyte development. LDR significantly increased involucrin mRNA and protein levels during calcium-induced keratinocyte differentiation. Conclusion These studies provide new evidence for the biological role of LDR, and identify the potential to utilize LDR to regulate or induce keratinocyte differentiation. PMID:27489424

  15. Single Low-Dose Radiation Induced Regulation of Keratinocyte Differentiation in Calcium-Induced HaCaT Cells.

    PubMed

    Hahn, Hyung Jin; Youn, Hae Jeong; Cha, Hwa Jun; Kim, Karam; An, Sungkwan; Ahn, Kyu Joong

    2016-08-01

    We are continually exposed to low-dose radiation (LDR) in the range 0.1 Gy from natural sources, medical devices, nuclear energy plants, and other industrial sources of ionizing radiation. There are three models for the biological mechanism of LDR: the linear no-threshold model, the hormetic model, and the threshold model. We used keratinocytes as a model system to investigate the molecular genetic effects of LDR on epidermal cell differentiation. To identify keratinocyte differentiation, we performed western blots using a specific antibody for involucrin, which is a precursor protein of the keratinocyte cornified envelope and a marker for keratinocyte terminal differentiation. We also performed quantitative polymerase chain reaction. We examined whether LDR induces changes in involucrin messenger RNA (mRNA) and protein levels in calcium-induced keratinocyte differentiation. Exposure of HaCaT cells to LDR (0.1 Gy) induced p21 expression. p21 is a key regulator that induces growth arrest and represses stemness, which accelerates keratinocyte differentiation. We correlated involucrin expression with keratinocyte differentiation, and examined the effects of LDR on involucrin levels and keratinocyte development. LDR significantly increased involucrin mRNA and protein levels during calcium-induced keratinocyte differentiation. These studies provide new evidence for the biological role of LDR, and identify the potential to utilize LDR to regulate or induce keratinocyte differentiation.

  16. Protective effect of melatonin against human leukocyte apoptosis induced by intracellular calcium overload: relation with its antioxidant actions.

    PubMed

    Espino, Javier; Bejarano, Ignacio; Paredes, Sergio D; Barriga, Carmen; Rodríguez, Ana B; Pariente, José A

    2011-09-01

    Apoptosis or programmed cell death plays a critical role in both inflammatory and immune responses. Recent evidence demonstrates that control of leukocyte apoptosis is one of the most striking immune system-related roles of melatonin. For this reason, this study evaluated the protective effects of melatonin on human leukocyte apoptosis induced by sustained cytosolic calcium increases. Such protective effects are likely mediated by melatonin's free-radical scavenging actions. Treatments with the specific inhibitor of cytosolic calcium re-uptake, thapsigargin (TG), and/or the calcium-mobilizing agonist, N-formyl-methionyl-leucyl-phenylalanine (FMLP), induced intracellular reactive oxygen species (ROS) production, caspase activation as well as DNA fragmentation in human leukocytes. Also, TG- and/or FMLP-induced apoptosis was dependent on both cytosolic calcium increases and calcium uptake into mitochondria, because when cells were preincubated with the cytosolic calcium chelator, dimethyl BAPTA, and the inhibitor of mitochondrial calcium uptake, Ru360, TG- and FMLP-induced apoptosis was largely inhibited. Importantly, melatonin treatment substantially prevented intracellular ROS production, reversed caspase activation, and forestalled DNA fragmentation induced by TG and FMLP. Similar results were obtained by preincubating the cells with another well-known antioxidant, i.e., N-acetyl-L-cysteine. To sum up, depletion of intracellular calcium stores induced by TG and/or FMLP triggers different apoptotic events in human leukocytes that are dependent on calcium signaling. The protective effects resulting from melatonin administration on leukocyte apoptosis likely depend on melatonin's antioxidant action because we proved that this protection is melatonin receptor independent. These findings help to understand how melatonin controls apoptosis in cells of immune/inflammatory relevance. © 2011 John Wiley & Sons A/S.

  17. Two Chromogranin A-Derived Peptides Induce Calcium Entry in Human Neutrophils by Calmodulin-Regulated Calcium Independent Phospholipase A2

    PubMed Central

    Zhang, Dan; Shooshtarizadeh, Peiman; Laventie, Benoît-Joseph; Colin, Didier André; Chich, Jean-François; Vidic, Jasmina; de Barry, Jean; Chasserot-Golaz, Sylvette; Delalande, François; Van Dorsselaer, Alain; Schneider, Francis; Helle, Karen; Aunis, Dominique; Prévost, Gilles; Metz-Boutigue, Marie-Hélène

    2009-01-01

    Background Antimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides. Methodology/Principal Findings PMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR) or Catestatin (CAT) induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate), a store operated channels (SOCs) blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ) and that the two sequences can be aligned with the two CaM-binding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity. Conclusions/Significance For the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated iPLA2 pathway. Our

  18. Pressure-induced semimetallic behavior of calcium from ab initio calculations

    NASA Astrophysics Data System (ADS)

    Magnitskaya, M. V.; Matsko, N. L.; Baturin, V. S.; Uspenskii, Yu A.

    2014-05-01

    A loss of metallic properties in fcc calcium under high pressure is studied ab initio using the density functional theory (DFT) and GW approximation. It is found that a more correct description of many-electron effects given by GW method does not provide significant changes in the behavior of electronic spectrum in comparison with DFT approach. We note that the obtained width of (pseudo)gap is highly sensitive to the k-point sampling used for density of states calculation. The analysis of fcc calcium's band structure at p ~ 20 GPa shows that the crossing of bands at the Fermi level is removed if the spin-orbit coupling is taken into account.

  19. Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells.

    PubMed

    Lachaud, Christophe; Da Silva, Daniel; Cotelle, Valérie; Thuleau, Patrice; Xiong, Tou Cheu; Jauneau, Alain; Brière, Christian; Graziana, Annick; Bellec, Yannick; Faure, Jean-Denis; Ranjeva, Raoul; Mazars, Christian

    2010-01-01

    Studies performed in animals have highlighted the major role of sphingolipids in regulating the balance between cell proliferation and cell death. Sphingolipids have also been shown to induce cell death in plants via calcium-based signalling pathways but the contribution of free cytosolic and/or nuclear calcium in the overall process has never been evaluated. Here, we show that increase in tobacco BY-2 cells of the endogenous content of Long Chain Bases (LCBs) caused by external application of d-erythro-sphinganine (DHS) is followed by immediate dose-dependent elevations of cellular free calcium concentration within the first minute in the cytosol and 10min later in the nucleus. Cells challenged with DHS enter a death process through apoptotic-like mechanisms. Lanthanum chloride, a general blocker of calcium entry, suppresses the cellular calcium variations and the PCD induced by DHS. Interestingly, dl-2-amino-5-phosphopentanoic acid (AP5) and [(+)-dizocilpine] (MK801), two inhibitors of animal and plant ionotropic glutamate receptors, suppress DHS-induced cell death symptoms by selectively inhibiting the variations of nuclear calcium concentration. The selective action of these compounds demonstrates the crucial role of nuclear calcium signature in controlling DHS-induced cell death in tobacco cells. 2009 Elsevier Ltd. All rights reserved.

  20. Salvia miltiorrhiza attenuates the changes in contraction and intracellular calcium induced by anoxia and reoxygenation in rat cardiomyocytes.

    PubMed

    Cao, Chun-Mei; Xia, Qiang; Zhang, Xiong; Xu, Wan-Hong; Jiang, Hui-Di; Chen, Jun-Zhu

    2003-04-18

    The aim of the present study is to investigate the effect of Salvia miltiorrhiza (SM) on contraction and the intracellular calcium of isolated ventricular myocytes during normoxia or anoxia and reoxygenation using a video tracking system and spectrofluorometry. Cardiac ventricular myocytes were isolated enzymatically by collagenase and exposed to 5 min of anoxia followed by 10 min of reoxygenation. SM (1-9 g/L) depressed both contraction and the [Ca(2+)](i) transient in a dose-dependent manner. SM did not affect the diastolic calcium level and the sarcolemmal Ca(2+) channel of myocytes but decreased the caffeine-induced calcium release. During anoxia, the +/-dL/dtmax, amplitudes of contraction (dL) of cell contraction and [Ca(2+)](i) transients were decreased, while the diastolic calcium level was increased. None of the parameters returned to the pre-anoxia level during reoxygenaton. However, SM (3 g/L) did attenuate the changes in cell contraction and intracellular calcium induced by anoxia and reoxygenation. It is concluded that SM has different effects on normoxic and anoxic cardiomyocytes. The SM-induced reduction of changes in contraction and intracellular calcium induced by anoxia/reoxygenation indicates that SM may be beneficial for cardiac tissue in recovery of mechanical function and intracellular calcium homeostasis.

  1. The relationship between calcium and the metabolism of plasma membrane phospholipids in hemolysis induced by brown spider venom phospholipase-D toxin.

    PubMed

    Chaves-Moreira, Daniele; Souza, Fernanda N; Fogaça, Rosalvo T H; Mangili, Oldemir C; Gremski, Waldemiro; Senff-Ribeiro, Andrea; Chaim, Olga M; Veiga, Silvio S

    2011-09-01

    Brown spider venom phospholipase-D belongs to a family of toxins characterized as potent bioactive agents. These toxins have been involved in numerous aspects of cell pathophysiology including inflammatory response, platelet aggregation, endothelial cell hyperactivation, renal disorders, and hemolysis. The molecular mechanism by which these toxins cause hemolysis is under investigation; literature data have suggested that enzyme catalysis is necessary for the biological activities triggered by the toxin. However, the way by which phospholipase-D activity is directly related with human hemolysis has not been determined. To evaluate how brown spider venom phospholipase-D activity causes hemolysis, we examined the impact of recombinant phospholipase-D on human red blood cells. Using six different purified recombinant phospholipase-D molecules obtained from a cDNA venom gland library, we demonstrated that there is a correlation of hemolytic effect and phospholipase-D activity. Studying recombinant phospholipase-D, a potent hemolytic and phospholipase-D recombinant toxin (LiRecDT1), we determined that the toxin degrades synthetic sphingomyelin (SM), lysophosphatidylcholine (LPC), and lyso-platelet-activating factor. Additionally, we determined that the toxin degrades phospholipids in a detergent extract of human erythrocytes, as well as phospholipids from ghosts of human red blood cells. The products of the degradation of synthetic SM and LPC following recombinant phospholipase-D treatments caused hemolysis of human erythrocytes. This hemolysis, dependent on products of metabolism of phospholipids, is also dependent on calcium ion concentration because the percentage of hemolysis increased with an increase in the dose of calcium in the medium. Recombinant phospholipase-D treatment of human erythrocytes stimulated an influx of calcium into the cells that was detected by a calcium-sensitive fluorescent probe (Fluo-4). This calcium influx was shown to be channel

  2. Mg-ATPase and Ca+ activated myosin AtPase activity in ventricular myofibrils from non-failing and diseased human hearts--effects of calcium sensitizing agents MCI-154, DPI 201-106, and caffeine.

    PubMed

    Okafor, Chukwuka; Liao, Ronglih; Perreault-Micale, Cynthia; Li, Xiaoping; Ito, Toshiro; Stepanek, Anna; Doye, Angelia; de Tombe, Pieter; Gwathmey, Judith K

    2003-03-01

    We investigated the effects of two purported calcium sensitizing agents, MCI-154 and DPI 201-106, and a known calcium sensitizer caffeine on Mg-ATPase (myofibrillar ATPase) and myosin ATPase activity of left ventricular myofibrils isolated from non-failing, idiopathic (IDCM) and ischemic cardiomyopathic (ISCM) human hearts (i.e. failing hearts). The myofibrillar ATPase activity of non-failing myofibrils was higher than that of diseased myofibrils. MCI-154 increased myofibrillar ATPase Ca2+ sensitivity in myofibrils from non-failing and failing human hearts. Effects of caffeine similarly increased Ca2+ sensitivity. Effects of DPI 201-106 were, however, different. Only at the 10(-6) M concentration was a significant increase in myofibrillar ATPase calcium sensitivity seen in myofibrils from non-failing human hearts. In contrast, in myofibrils from failing hearts, DPI 201-106 caused a concentration-dependent increase in myofibrillar ATPase Ca2+ sensitivity. Myosin ATPase activity in failing myocardium was also decreased. In the presence of MCI-154, myosin ATPase activity increased by 11, 19, and 24% for non-failing, IDCM, and ISCM hearts, respectively. DPI 201-106 caused an increase in the enzymatic activity of less than 5% for all preparations, and caffeine induced an increase of 4, 11, and 10% in non-failing, IDCM and ISCM hearts, respectively. The mechanism of restoring the myofibrillar Ca2+ sensitivity and myosin enzymatic activity in diseased human hearts is most likely due to enhancement of the Ca2+ activation of the contractile apparatus induced by these agents. We propose that myosin light chain-related regulation may play a complementary role to the troponin-related regulation of myocardial contractility.

  3. High dietary calcium intake does not counteract disuse-induced bone loss

    NASA Astrophysics Data System (ADS)

    Baecker, N.; Boese, A.; Smith, S. M.; Heer, M.

    Reduction of mechanical stress on bone inhibits osteoblast-mediated bone formation, increases osteoclast-mediated bone resorption, and leads to what has been called disuse osteoporosis. Prolonged therapeutic bed rest, immobilization and space flight are common causes of disuse osteoporosis. There are sufficient data supporting the use of calcium in combination with vitamin D in the prevention and treatment of postmenopausal osteoporosis. In our study we examined the potential of high dietary calcium intake as a nutrition therapy for disuse-induced bone loss during head-down bed rest in healthy young men. In 2 identical metabolic ward, head-down bed rest (HDBR) experiments (crossover design), we studied the effect of high dietary calcium intake (2000 mg/d) in comparison to the recommended calcium intake of 1000 mg/d on markers of bone turnover. Experiment A (EA) was a 6-day randomized, controlled HDBR study. Experiment B (EB) was a 14-day randomized, controlled HDBR study. In both experiments, the test subjects stayed under well-controlled environmental conditions in our metabolic ward. Subjects' diets in the relevant study phases (HDBR versus Ambulatory Control) of EA and EB were identical except for the calcium intake. The subjects obtained 2000 mg/d Calcium in EA and 2000 mg/d in EB. Blood was drawn at baseline, before entering the relevant intervention period, on day 5 in study EA, and on days 6, 11 and 14 in study EB. Serum calcium, bone formation markers - Procollagen-I-C-Propeptide (PICP) and bone alkaline phosphatase (bAP) were analyzed in serum. 24h-urine was collected throughout the studies for determination of the excretion of calcium (UCaV) and a bone resorption marker, C-terminal telopeptide of collagen type I (UCTX). In both studies, serum calcium levels were unchanged. PICP tended to decrease in EA (p=0.08). In EB PICP decreased significantly over time (p=0.003) in both the control and HDBR periods, and tended to further decrease in the HDBR period (p

  4. The metabolic impact of β-hydroxybutyrate on neurotransmission: Reduced glycolysis mediates changes in calcium responses and KATP channel receptor sensitivity.

    PubMed

    Lund, Trine M; Ploug, Kenneth B; Iversen, Anne; Jensen, Anders A; Jansen-Olesen, Inger

    2015-03-01

    Glucose is the main energy substrate for neurons, and ketone bodies are known to be alternative substrates. However, the capacity of ketone bodies to support different neuronal functions is still unknown. Thus, a change in energy substrate from glucose alone to a combination of glucose and β-hydroxybutyrate might change neuronal function as there is a known coupling between metabolism and neurotransmission. The purpose of this study was to shed light on the effects of the ketone body β-hydroxybutyrate on glycolysis and neurotransmission in cultured murine glutamatergic neurons. Previous studies have shown an effect of β-hydroxybutyrate on glucose metabolism, and the present study further specified this by showing attenuation of glycolysis when β-hydroxybutyrate was present in these neurons. In addition, the NMDA receptor-induced calcium responses in the neurons were diminished in the presence of β-hydroxybutyrate, whereas a direct effect of the ketone body on transmitter release was absent. However, the presence of β-hydroxybutyrate augmented transmitter release induced by the KATP channel blocker glibenclamide, thus giving an indirect indication of the involvement of KATP channels in the effects of ketone bodies on transmitter release. Energy metabolism and neurotransmission are linked and involve ATP-sensitive potassium (KATP ) channels. However, it is still unclear how and to what degree available energy substrate affects this link. We investigated the effect of changing energy substrate from only glucose to a combination of glucose and R-β-hydroxybutyrate in cultured neurons. Using the latter combination, glycolysis was diminished, NMDA receptor-induced calcium responses were lower, and the KATP channel blocker glibenclamide caused a higher transmitter release.

  5. Cocaine-induced locomotor sensitization in rats correlates with nucleus accumbens activity on manganese-enhanced MRI.

    PubMed

    Perrine, Shane A; Ghoddoussi, Farhad; Desai, Kirtan; Kohler, Robert J; Eapen, Ajay T; Lisieski, Michael J; Angoa-Perez, Mariana; Kuhn, Donald M; Bosse, Kelly E; Conti, Alana C; Bissig, David; Berkowitz, Bruce A

    2015-11-01

    A long-standing goal of substance abuse research has been to link drug-induced behavioral outcomes with the activity of specific brain regions to understand the neurobiology of addiction behaviors and to search for drug-able targets. Here, we tested the hypothesis that cocaine produces locomotor (behavioral) sensitization that correlates with increased calcium channel-mediated neuroactivity in brain regions linked with drug addiction, such as the nucleus accumbens (NAC), anterior striatum (AST) and hippocampus, as measured using manganese-enhanced MRI (MEMRI). Rats were treated with cocaine for 5 days, followed by a 2-day drug-free period. The following day, locomotor sensitization was quantified as a metric of cocaine-induced neuroplasticity in the presence of manganese. Immediately following behavioral testing, rats were examined for changes in calcium channel-mediated neuronal activity in the NAC, AST, hippocampus and temporalis muscle, which was associated with behavioral sensitization using MEMRI. Cocaine significantly increased locomotor activity and produced behavioral sensitization compared with saline treatment of control rats. A significant increase in MEMRI signal intensity was determined in the NAC, but not AST or hippocampus, of cocaine-treated rats compared with saline-treated control rats. Cocaine did not increase signal intensity in the temporalis muscle. Notably, in support of our hypothesis, behavior was significantly and positively correlated with MEMRI signal intensity in the NAC. As neuronal uptake of manganese is regulated by calcium channels, these results indicate that MEMRI is a powerful research tool to study neuronal activity in freely behaving animals and to guide new calcium channel-based therapies for the treatment of cocaine abuse and dependence.

  6. Construction of two ureolytic model organisms for the study of microbially induced calcium carbonate precipitation.

    PubMed

    Connolly, James; Kaufman, Megan; Rothman, Adam; Gupta, Rashmi; Redden, George; Schuster, Martin; Colwell, Frederick; Gerlach, Robin

    2013-09-01

    Two bacterial strains, Pseudomonas aeruginosa MJK1 and Escherichia coli MJK2, were constructed that both express green fluorescent protein (GFP) and carry out ureolysis. These two novel model organisms are useful for studying bacterial carbonate mineral precipitation processes and specifically ureolysis-driven microbially induced calcium carbonate precipitation (MICP). The strains were constructed by adding plasmid-borne urease genes (ureABC, ureD and ureFG) to the strains P. aeruginosa AH298 and E. coli AF504gfp, both of which already carried unstable GFP derivatives. The ureolytic activities of the two new strains were compared to the common, non-GFP expressing, model organism Sporosarcina pasteurii in planktonic culture under standard laboratory growth conditions. It was found that the engineered strains exhibited a lower ureolysis rate per cell but were able to grow faster and to a higher population density under the conditions of this study. Both engineered strains were successfully grown as biofilms in capillary flow cell reactors and ureolysis-induced calcium carbonate mineral precipitation was observed microscopically. The undisturbed spatiotemporal distribution of biomass and calcium carbonate minerals were successfully resolved in 3D using confocal laser scanning microscopy. Observations of this nature were not possible previously because no obligate urease producer that expresses GFP had been available. Future observations using these organisms will allow researchers to further improve engineered application of MICP as well as study natural mineralization processes in model systems. © 2013.

  7. In vitro evaluation of pH changes induced by calcium hydroxide liners.

    PubMed

    Gençay, Koray; Seymen, Figen; Selvi, Senem; Kiziltan, Başak

    2004-01-01

    Since the highly alkaline pH of calcium hydroxide is considered by many to be responsible for its biologic activity, the possible variations of pH induced by the different calcium hydroxide liners are accepted as a major concern. The aim of the present study was to determine the pH changes of five different calcium hydroxide liners and variations of pH levels at different time intervals. The materials tested were Dycal, Life, Calic, Dycal VLC, and Calcident 450. Samples were prepared according to manufacturer instructions and by using plastic molds; five standard samples from each material were prepared. The samples were then placed in separate vials, containing 10 mL deionized water (pH 7.0), and stored at room temperature (200C). The pH measurements were taken 1 hour, 24 hours, 3 days, and 7 days after mixing. The pH variations of each material at the given time intervals were recorded, and the means were calculated. Statistical analysis showed significantly high differences between the mean pH values induced by each material at all time intervals. The highest value for the first-hour measurement was for Dycal VLC, and the highest values for the other time intervals were for Calcident 450. The pH values of the materials exhibited statistically significant differences among all the time intervals. All materials changed the pH of deionized water toward alkaline.

  8. Fracture Sealing with Microbially-Induced Calcium Carbonate Precipitation: A Field Study.

    PubMed

    Phillips, Adrienne J; Cunningham, Alfred B; Gerlach, Robin; Hiebert, Randy; Hwang, Chiachi; Lomans, Bartholomeus P; Westrich, Joseph; Mantilla, Cesar; Kirksey, Jim; Esposito, Richard; Spangler, Lee

    2016-04-05

    A primary environmental risk from unconventional oil and gas development or carbon sequestration is subsurface fluid leakage in the near wellbore environment. A potential solution to remediate leakage pathways is to promote microbially induced calcium carbonate precipitation (MICP) to plug fractures and reduce permeability in porous materials. The advantage of microbially induced calcium carbonate precipitation (MICP) over cement-based sealants is that the solutions used to promote MICP are aqueous. MICP solutions have low viscosities compared to cement, facilitating fluid transport into the formation. In this study, MICP was promoted in a fractured sandstone layer within the Fayette Sandstone Formation 340.8 m below ground surface using conventional oil field subsurface fluid delivery technologies (packer and bailer). After 24 urea/calcium solution and 6 microbial (Sporosarcina pasteurii) suspension injections, the injectivity was decreased (flow rate decreased from 1.9 to 0.47 L/min) and a reduction in the in-well pressure falloff (>30% before and 7% after treatment) was observed. In addition, during refracturing an increase in the fracture extension pressure was measured as compared to before MICP treatment. This study suggests MICP is a promising tool for sealing subsurface fractures in the near wellbore environment.

  9. Analyses of signal transduction cascades reveal an essential role of calcium ions for regulation of melatonin biosynthesis in the light-sensitive pineal organ of the rainbow trout (Oncorhynchus mykiss).

    PubMed

    Kroeber, S; Meissl, H; Maronde, E; Korf, H W

    2000-06-01

    Signal transduction processes regulating melatonin production in the light-sensitive trout pineal organ were investigated by immunocytochemical and immunochemical demonstration of phosphorylated cyclic AMP-responsive element-binding protein (pCREB) and measurements of cyclic AMP, melatonin, and calcium levels. Melatonin levels were tightly controlled by light and darkness. Elevation of cyclic AMP levels by 8-bromo-cyclic AMP, forskolin, and 3-isobutyl-1-methylxanthine increased the levels of pCREB and melatonin in light- or dark-adapted pineal organs in vitro. Without pharmacological treatment, the levels of pCREB and cyclic AMP remained constant for several hours before and after light onset. Inhibition of cyclic AMP-dependent proteasomal proteolysis by lactacystin, MG 132, and calpain inhibitor I did not prevent the rapid, light-induced suppression of melatonin biosynthesis. However, changes in the intracellular calcium concentration by drugs affecting voltage-gated calcium channels of the L type and intracellular calcium oscillations (cobalt chloride, nifedipine, Bay K 8644) had dramatic effects on the rapid, light-dependent changes in melatonin levels. These effects were not accompanied by changes in cyclic AMP levels. Thus, the rapid, light-dependent changes in melatonin levels in the trout pineal organ are regulated apparently by a novel calcium signaling pathway and do not involve changes in cyclic AMP levels, cyclic AMP-dependent proteasomal proteolysis, or phosphorylation of cyclic AMP-responsive element-binding protein.

  10. Chemoprevention with acetylsalicylic acid, vitamin D and calcium reduces risk of carcinogen-induced lung tumors.

    PubMed

    Pommergaard, Hans-Christian; Burcharth, Jakob; Rosenberg, J; Raskov, Hans

    2013-11-01

    Research has shown that chemoprevention may be effective against the development of lung cancer. The purpose of the present study was to evaluate the effect of oral chemoprevention in a mouse model of tobacco carcinogen-induced lung tumor. A total of 60 A/J mice were randomized to a normal diet, a diet with low calcium, or a chemoprevention diet with acetylsalicylic acid, 1-α 25(0H)2-vitamin D3 and calcium. In addition to the diet, mice received carcinogens by oral gavage for ten weeks. The chemoprevention diet significantly reduced the number of animals with tumors [1 vs. 13, (p<0.001)] and the median number (range) of tumors [0 (0-1) vs. 1 (0-4), (p<0.001)] compared to controls. No signs of toxicity in relation to the diets were observed. The chemoprevention diet had a protective effect against tumor development in the mouse lungs.

  11. Protective effects of selenium, calcium, and magnesium against arsenic-induced oxidative stress in male rats.

    PubMed

    Srivastava, Deepti; Subramanian, Ramlingam B; Madamwar, Datta; Flora, Swaran J S

    2010-06-01

    Inorganic arsenic is a potent carcinogen and environmental pollutant. More than one hundred million people are reported to be exposed to elevated concentrations of arsenic mainly via drinking water. Essential trace elements can affect toxicity of metals by interacting with metals at the primary site of action and can also modify the body's response to toxic metals by altering their metabolism and transport. This study investigates the effects of concomitant administration of selenium, magnesium, and calcium with arsenic on blood biochemistry and oxidative stress. Selenium was the most effective in reducing arsenic-induced inhibition of blood delta-aminolevulinic acid dehydratase (ALAD) activity and liver oxidative stress. Calcium and magnesium also showed favourable effects on haematological and other biochemical parameters. Because selenium was the most effective, it should be added to chelation therapy to achieve the best protective effects against arsenic poisoning in humans.

  12. High-calcium exposure to frog heart: a simple model representing hypercalcemia-induced ECG abnormalities

    PubMed Central

    KAZAMA, Itsuro

    2016-01-01

    By simply adding a high concentration of calcium solution to the surface of the bullfrog heart, we reproduced electrocardiogram (ECG) abnormalities representing those observed in hypercalcemia, such as Osborn waves and shortening of the QT interval. The rise in extracellular calcium concentration may have activated the outward potassium currents during phase 3 of the action potential, and thus decreased its duration. In addition to the known decrease in the duration of phase 2, such changes in phase 3 were also likely to contribute to the shortening of the QT interval. The dual recordings of the action potential in cardiomyocytes and the ECG waves enabled us to demonstrate the mechanisms of ECG abnormalities induced by hypercalcemia. PMID:27773880

  13. Calcium-regulated nuclear enzymes: potential mediators of phytochrome-induced changes in nuclear metabolism?

    NASA Technical Reports Server (NTRS)

    Roux, S. J.

    1992-01-01

    Calcium ions have been proposed to serve as important regulatory elements in stimulus-response coupling for phytochrome responses. An important test of this hypothesis will be to identify specific targets of calcium action that are required for some growth or development process induced by the photoactivated form of phytochrome (Pfr). Initial studies have revealed that there are at least two enzymes in pea nuclei that are stimulated by Pfr in a Ca(2+)-dependent fashion, a calmodulin-regulated nucleoside triphosphatase and a calmodulin-independent but Ca(2+)-dependent protein kinase. The nucleoside triphosphatase appears to be associated with the nuclear envelope, while the protein kinase co-purifies with a nuclear fraction highly enriched for chromatin. This short review summarizes the latest findings on these enzymes and relates them to what is known about Pfr-regulated nuclear metabolism.

  14. Calcium-regulated nuclear enzymes: potential mediators of phytochrome-induced changes in nuclear metabolism?

    NASA Technical Reports Server (NTRS)

    Roux, S. J.

    1992-01-01

    Calcium ions have been proposed to serve as important regulatory elements in stimulus-response coupling for phytochrome responses. An important test of this hypothesis will be to identify specific targets of calcium action that are required for some growth or development process induced by the photoactivated form of phytochrome (Pfr). Initial studies have revealed that there are at least two enzymes in pea nuclei that are stimulated by Pfr in a Ca(2+)-dependent fashion, a calmodulin-regulated nucleoside triphosphatase and a calmodulin-independent but Ca(2+)-dependent protein kinase. The nucleoside triphosphatase appears to be associated with the nuclear envelope, while the protein kinase co-purifies with a nuclear fraction highly enriched for chromatin. This short review summarizes the latest findings on these enzymes and relates them to what is known about Pfr-regulated nuclear metabolism.

  15. High-calcium exposure to frog heart: a simple model representing hypercalcemia-induced ECG abnormalities.

    PubMed

    Kazama, Itsuro

    2017-01-20

    By simply adding a high concentration of calcium solution to the surface of the bullfrog heart, we reproduced electrocardiogram (ECG) abnormalities representing those observed in hypercalcemia, such as Osborn waves and shortening of the QT interval. The rise in extracellular calcium concentration may have activated the outward potassium currents during phase 3 of the action potential, and thus decreased its duration. In addition to the known decrease in the duration of phase 2, such changes in phase 3 were also likely to contribute to the shortening of the QT interval. The dual recordings of the action potential in cardiomyocytes and the ECG waves enabled us to demonstrate the mechanisms of ECG abnormalities induced by hypercalcemia.

  16. Spontaneous calcium signals induced by gap junctions in a network model of astrocytes

    NASA Astrophysics Data System (ADS)

    Kazantsev, V. B.

    2009-01-01

    The dynamics of a network model of astrocytes coupled by gap junctions is investigated. Calcium dynamics of the single cell is described by the biophysical model comprising the set of three nonlinear differential equations. Intercellular dynamics is provided by the diffusion of inositol 1,4,5-trisphosphate (IP3) through gap junctions between neighboring astrocytes. It is found that the diffusion induces the appearance of spontaneous activity patterns in the network. Stability of the network steady state is analyzed. It is proved that the increase of the diffusion coefficient above a certain critical value yields the generation of low-amplitude subthreshold oscillatory signals in a certain frequency range. It is shown that such spontaneous oscillations can facilitate calcium pulse generation and provide a certain time scale in astrocyte signaling.

  17. Mimicry of the calcium-induced conformational state of troponin C by low temperature under pressure.

    PubMed Central

    Foguel, D; Suarez, M C; Barbosa, C; Rodrigues, J J; Sorenson, M M; Smillie, L B; Silva, J L

    1996-01-01

    Calcium binding to the N-domain of troponin C initiates a series of conformational changes that lead to muscle contraction. Calcium binding provides the free energy for a hydrophobic region in the core of N-domain to assume a more open configuration. Fluorescence measurements on a tryptophan mutant (F29W) show that a similar conformational change occurs in the absence of Ca2+ when the temperature is lowered under pressure. The conformation induced by subzero temperatures binds the hydrophobic probe bis-aminonaphthalene sulfonate, and the tryptophan has the same fluorescence lifetime (7 ns) as in the Ca2+-bound form. The decrease in volume (delta V = -25.4 ml/mol) corresponds to an increase in surface area. Thermodynamic measurements suggest an enthalpy-driven conformational change that leads to an intermediate with an exposed N-domain core and a high affinity for Ca2+. Images Fig. 1 PMID:8855232

  18. Calcium binding domains and calcium-induced conformational transition of SPARC/BM-40/osteonectin, an extracellular glycoprotein expressed in mineralized and nonmineralized tissues

    SciTech Connect

    Engel, J.; Taylor, W.; Paulsson, M.; Sage, H.; Hogan, B.

    1987-11-03

    PSARC, BM-40, and osteonectin are identical or very closely related extracellular proteins of apparent M/sub r/ 43,000 (M/sub r/ 33,000 predicted from sequence). They were originally isolated from parietal endoderm cells, basement membrane producing tumors, and bone, respectively, but are rather widely distributed in various tissues. In view of the calcium binding activity reported for osteonectin, the authors analyzed the SPARC sequence and found two putative calcium binding domains. One is an N-terminal acid region with clusters of glutamic acid residues. This region, although neither ..gamma..-carboxylated nor homologous, resembles the ..gamma..-carboxyglutamic acid (Gla) domain of vitamin K dependent proteins of the blood clotting system in charge density, size of negatively charged clusters, and linkage to the rest of the molecule by a cysteine-rich domain. The other region is an EF-hand calcium binding domain located near the C-terminus. A disulfide bond between the E and F helix is predicted from modeling the EF-hand structure with the known coordinates of intestinal calcium binding protein. The disulfide bridge apparently serves to stabilize the isolated calcium loop in the extracellular protein. As observed for cytoplasmic EF-hand-containing proteins and for Gla domain containing proteins, a major conformational transition is induced in BM-40 upon binding of several Ca/sup 2 +/ ions. This is accompanied by a 35% increase in ..cap alpha..-helicity. A pronounced sigmoidicity of the dependence of the circular dichroism signal at 220 nm on calcium concentration indicates that the process is cooperative. In view of its properties, abundance, and wide distribution, it is proposed that SPARC/BM-40/osteonectin has a rather general regulatory function in calcium-dependent processes of the extra-cellular matrix.

  19. Calcium binding domains and calcium-induced conformational transition of SPARC/BM-40/osteonectin, an extracellular glycoprotein expressed in mineralized and nonmineralized tissues.

    PubMed

    Engel, J; Taylor, W; Paulsson, M; Sage, H; Hogan, B

    1987-11-03

    SPARC, BM-40, and osteonectin are identical or very closely related extracellular proteins of apparent Mr 43,000 (Mr 33,000 predicted from sequence). They were originally isolated from parietal endoderm cells, basement membrane producing tumors, and bone, respectively, but are rather widely distributed in various tissues. In view of the calcium binding activity reported for osteonectin, we analyzed the SPARC sequence and found two putative calcium binding domains. One is an N-terminal acidic region with clusters of glutamic acid residues. This region, although neither gamma-carboxylated nor homologous, resembles the gamma-carboxyglutamic acid (Gla) domain of vitamin K dependent proteins of the blood clotting system in charge density, size of negatively charged clusters, and linkage to the rest of the molecule by a cysteine-rich domain. The other region is an EF-hand calcium binding domain located near the C-terminus. A disulfide bond between the E and F helix is predicted from modeling the EF-hand structure with the known coordinates of intestinal calcium binding protein. The disulfide bridge apparently serves to stabilize the isolated calcium loop in the extracellular protein. As observed for cytoplasmic EF-hand-containing proteins and for Gla domain containing proteins, a major conformational transition is induced in BM-40 upon binding of several Ca2+ ions. This is accompanied by a 35% increase in alpha-helicity. A pronounced sigmoidicity of the dependence of the circular dichroism signal at 220 nm on calcium concentration indicates that the process is cooperative. In view of its properties, abundance, and wide distribution, it is proposed that SPARC/BM-40/osteonectin has a rather general regulatory function in calcium-dependent processes of the extracellular matrix.

  20. Bacterially induced calcium carbonate precipitation and strontium coprecipitation in a porous media flow system.

    PubMed

    Lauchnor, Ellen G; Schultz, Logan N; Bugni, Steven; Mitchell, Andrew C; Cunningham, Alfred B; Gerlach, Robin

    2013-02-05

    Strontium-90 is a principal radionuclide contaminant in the subsurface at several Department of Energy sites in the Western U.S., causing a threat to groundwater quality in areas such as Hanford, WA. In this work, we used laboratory-scale porous media flow cells to examine a potential remediation strategy employing coprecipitation of strontium in carbonate minerals. CaCO(3) precipitation and strontium coprecipitation were induced via ureolysis by Sporosarcina pasteurii in two-dimensional porous media reactors. An injection strategy using pulsed injection of calcium mineralization medium was tested against a continuous injection strategy. The pulsed injection strategy involved periods of lowered calcite saturation index combined with short high fluid velocity flow periods of calcium mineralization medium followed by stagnation (no-flow) periods to promote homogeneous CaCO(3) precipitation. By alternating the addition of mineralization and growth media the pulsed strategy promoted CaCO(3) precipitation while sustaining the ureolytic culture over time. Both injection strategies achieved ureolysis with subsequent CaCO(3) precipitation and strontium coprecipitation. The pulsed injection strategy precipitated 71-85% of calcium and 59% of strontium, while the continuous injection was less efficient and precipitated 61% of calcium and 56% of strontium. Over the 60 day operation of the pulsed reactors, ureolysis was continually observed, suggesting that the balance between growth and precipitation phases allowed for continued cell viability. Our results support the pulsed injection strategy as a viable option for ureolysis-induced strontium coprecipitation because it may reduce the likelihood of injection well accumulation caused by localized mineral plugging while Sr coprecipitation efficiency is maintained in field-scale applications.

  1. Calcium signatures and signaling in cytosol and organelles of tobacco cells induced by plant defense elicitors.

    PubMed

    Manzoor, Hamid; Chiltz, Annick; Madani, Siham; Vatsa, Parul; Schoefs, Benoît; Pugin, Alain; Garcia-Brugger, Angela

    2012-06-01

    Calcium signatures induced by two elicitors of plant defense reactions, namely cryptogein and oligogalacturonides, were monitored at the subcellular level, using apoaequorin-transformed Nicotiana tabacum var Xanthi cells, in which the apoaequorin calcium sensor was targeted either to cytosol, mitochondria or chloroplasts. Our study showed that both elicitors induced specific Ca(2+) signatures in each compartment, with the most striking difference relying on duration. Common properties also emerged from the analysis of Ca(2+) signatures: both elicitors induced a biphasic cytosolic [Ca(2+)] elevation together with a single mitochondrial [Ca(2+)] elevation concomitant with the first cytosolic [Ca(2+)] peak. In addition, both elicitors induced a chloroplastic [Ca(2+)] elevation peaking later in comparison to cytosolic [Ca(2+)] elevation. In cryptogein-treated cells, pharmacological studies indicated that IP(3) should play an important role in Ca(2+) signaling contrarily to cADPR or nitric oxide, which have limited or no effect on [Ca(2+)] variations. Our data also showed that, depending on [Ca(2+)] fluxes at the plasma membrane, cryptogein triggered a mitochondrial respiration increase and affected excess energy dissipation mechanisms in chloroplasts. Altogether the results indicate that cryptogein profoundly impacted cell functions at many levels, including organelles.

  2. Pseudomonas, Pantoea and Cupriavidus isolates induce calcium carbonate precipitation for biorestoration of ornamental stone.

    PubMed

    Daskalakis, M I; Magoulas, A; Kotoulas, G; Catsikis, I; Bakolas, A; Karageorgis, A P; Mavridou, A; Doulia, D; Rigas, F

    2013-08-01

    Bacterially induced calcium carbonate precipitation from various isolates was investigated aiming at developing an environmentally friendly technique for ornamental stone protection and restoration. Micro-organisms isolated from stone samples and identified using 16S rDNA and biochemical tests promoted calcium carbonate precipitation in solid and novel liquid growth media. Biomineral morphology was studied on marble samples with scanning electron microscopy. Most isolates demonstrated specimen weight increase, covering partially or even completely the marble surfaces mainly with vaterite. The conditions under which vaterite precipitated and its stability throughout the experimental runs are presented. A growth medium that facilitated bacterial growth of different species and promoted biomineralization was formulated. Most isolates induced biomineralization of CaCO3 . Micro-organisms may actually be a milestone in the investigation of vaterite formation facilitating our understanding of geomicrobiological interactions. Pseudomonas, Pantoea and Cupriavidus strains could be candidates for bioconsolidation of ornamental stone protection. Characterization of biomineralization capacity of different bacterial species improves understanding of the bacterially induced mineralization processes and enriches the list of candidates for biorestoration applications. Knowledge of biomineral morphology assists in differentiating mineral from biologically induced precipitates. © 2013 The Society for Applied Microbiology.

  3. Hypergravity stimulation induces changes in intracellular calcium concentration in Arabidopsis seedlings

    NASA Astrophysics Data System (ADS)

    Toyota, M.; Furuichi, T.; Tatsumi, H.; Sokabe, M.

    Gravity affects growth and morphogenesis in higher plants. Recently, it has become clear that hypergravity induces morphological changes such as inhibition of elongation growth and promotion of lateral growth. Some indirect evidence suggests that changes in the cytoplasmic free calcium concentration ([Ca2+]c) play an important role in these hypergravity-induced modifications of growth. However, the hypothetical changes in [Ca2+]c under hypergravity have not been examined. Here, we report the measurement of the [Ca2+]c changes induced by hypergravity stimuli in Arabidopsis seedlings expressing the calcium reporter, aequorin. When the seedlings are subjected to 20g-hypergravity produced by centrifugation, [Ca2+]c transiently increased and decayed exponentially during the hypergravity stimulation. Larger [Ca2+]c-increase was observed when the magnitude of hypergravity was increased up to 300g. The [Ca2+]c-response showed a strong desensitization, and it could not be elicited even 45 min after the cessation of the first stimulation. The [Ca2+]c-increase was inhibited by externally applied La3+ or Gd3+, potential mechanosensitive Ca2+-permeable channel inhibitors, suggesting that the hypergravity-induced [Ca2+]c-increase is mediated by the activation of Ca2+-permeable channels in the plasma membrane.

  4. Cadmium induced Drp1-dependent mitochondrial fragmentation by disturbing calcium homeostasis in its hepatotoxicity.

    PubMed

    Xu, S; Pi, H; Chen, Y; Zhang, N; Guo, P; Lu, Y; He, M; Xie, J; Zhong, M; Zhang, Y; Yu, Z; Zhou, Z

    2013-03-14

    Mitochondria are critical targets in the hepatotoxicity of cadmium (Cd). Abnormal mitochondrial dynamics have been increasingly implicated in mitochondrial dysfunction in pathophysiological conditions. Therefore, our study aimed to investigate the effects and underlying mechanism of Cd on mitochondrial dynamics during hepatotoxicity. In the L02 liver cell lines, 12 μM cadmium chloride (CdCl₂) exposure induced excessive mitochondrial fragmentation as early as 3 h post-treatment with Cd, which preceded the mitochondrial dysfunction such as reactive oxygen species (ROS) overproduction, mitochondrial membrane potential (ΔΨm) loss and ATP reduction. Concurrent to mitochondrial fragmentation, CdCl₂ treatment increased the protein levels of dynamin-related protein (Drp1) and promoted the recruitment of Drp1 into mitochondria. Strikingly, mitochondrial fragmentation also occurred in the liver tissue of rats exposed to CdCl₂, accompanied by enhanced recruitment of Drp1 into mitochondria. Moreover, in L02 cells, Drp1 silencing could effectively reverse Cd-induced mitochondrial fragmentation and mitochondrial dysfunction. Furthermore, the increased expression and mitochondrial recruitment of Drp1 were tightly related to the disturbance of calcium homeostasis, which could be prevented by both chelating [Ca²⁺]i and inhibiting [Ca²⁺]m uptake. Overall, our study indicated that Cd induced Drp1-dependent mitochondrial fragmentation by disturbing calcium homeostasis to promote hepatotoxicity. Manipulation of Drp1 may be the potential avenue for developing novel strategies to protect against cadmium-induced hepatotoxicity.

  5. Iron Mediates N-Methyl-d-aspartate Receptor-dependent Stimulation of Calcium-induced Pathways and Hippocampal Synaptic Plasticity*

    PubMed Central

    Muñoz, Pablo; Humeres, Alexis; Elgueta, Claudio; Kirkwood, Alfredo; Hidalgo, Cecilia; Núñez, Marco T.

    2011-01-01

    Iron deficiency hinders hippocampus-dependent learning processes and impairs cognitive performance, but current knowledge on the molecular mechanisms underlying the unique role of iron in neuronal function is sparse. Here, we investigated the participation of iron on calcium signal generation and ERK1/2 stimulation induced by the glutamate agonist N-methyl-d-aspartate (NMDA), and the effects of iron addition/chelation on hippocampal basal synaptic transmission and long-term potentiation (LTP). Addition of NMDA to primary hippocampal cultures elicited persistent calcium signals that required functional NMDA receptors and were independent of calcium influx through L-type calcium channels or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors; NMDA also promoted ERK1/2 phosphorylation and nuclear translocation. Iron chelation with desferrioxamine or inhibition of ryanodine receptor (RyR)-mediated calcium release with ryanodine-reduced calcium signal duration and prevented NMDA-induced ERK1/2 activation. Iron addition to hippocampal neurons readily increased the intracellular labile iron pool and stimulated reactive oxygen species production; the antioxidant N-acetylcysteine or the hydroxyl radical trapper MCI-186 prevented these responses. Iron addition to primary hippocampal cultures kept in calcium-free medium elicited calcium signals and stimulated ERK1/2 phosphorylation; RyR inhibition abolished these effects. Iron chelation decreased basal synaptic transmission in hippocampal slices, inhibited iron-induced synaptic stimulation, and impaired sustained LTP in hippocampal CA1 neurons induced by strong stimulation. In contrast, iron addition facilitated sustained LTP induction after suboptimal tetanic stimulation. Together, these results suggest that hippocampal neurons require iron to generate RyR-mediated calcium signals after NMDA receptor stimulation, which in turn promotes ERK1/2 activation, an essential step of sustained LTP. PMID:21296883

  6. Mitochondrial calcium uniporter inhibition attenuates mouse bone marrow-derived mast cell degranulation induced by beta-1,3-glucan

    PubMed Central

    Cuong, Dang Van; Kim, Hyoung Kyu; Marquez, Jubert; Kim, Nari; Ko, Kyung Soo; Rhee, Byoung Doo

    2016-01-01

    Mast cells are primary mediators of allergic inflammation. Beta-1,3-glucan (BG) protects against infection and shock by activating immune cells. Activation of the BG receptor induces an increase in intracellular Ca2+, which may induce exocytosis. However, little is known about the precise mechanisms underlying BG activation of immune cells and the possible role of mitochondria in this process. The present study examined whether BG induced mast cell degranulation, and evaluated the role of calcium transients during mast cell activation. Our investigation focused on the role of the mitochondrial calcium uniporter (MCU) in BG-induced degranulation. Black mouse (C57) bone marrow-derived mast cells were stimulated with 0.5 µg/ml BG, 100 µg/ml peptidoglycan (PGN), or 10 µM A23187 (calcium ionophore), and dynamic changes in cytosolic and mitochondrial calcium and membrane potential were monitored. BG-induced mast cell degranulation occurred in a time-dependent manner, and was significantly reduced under calcium-free conditions. Ruthenium red, a mitochondrial Ca2+ uniporter blocker, significantly reduced mast cell degranulation induced by BG, PGN, and A23187. These results suggest that the mitochondrial Ca2+ uniporter has an important regulatory role in BG-induced mast cell degranulation. PMID:26937218

  7. Loss-of-function mutation of the calcium sensor CBL1 increases aluminum sensitivity in Arabidopsis.

    PubMed

    Ligaba-Osena, Ayalew; Fei, Zhangjun; Liu, Jiping; Xu, Yimin; Shaff, Jon; Lee, Sung-Chul; Luan, Sheng; Kudla, Jörg; Kochian, Leon; Piñeros, Miguel

    2017-04-01

    Despite the physiological importance of aluminum (Al) phytotoxicity for plants, it remained unknown if, and how, calcineurin B-like calcium sensors (CBLs) and CBL-interacting protein kinases (CIPKs) are involved in Al resistance. We performed a comparative physiological and whole transcriptome investigation of an Arabidopsis CBL1 mutant (cbl1) and the wild-type (WT). cbl1 plants exudated less Al-chelating malate, accumulated more Al, and displayed a severe root growth reduction in response to Al. Genes involved in metabolism, transport, cell wall modification, transcription and oxidative stress were differentially regulated between the two lines, under both control and Al stress treatments. Exposure to Al resulted in up-regulation of a large set of genes only in WT and not cbl1 shoots, while a different set of genes were down-regulated in cbl1 but not in WT roots. These differences allowed us, for the first time, to define a calcium-regulated/dependent transcriptomic network for Al stress responses. Our analyses reveal not only the fundamental role of CBL1 in the adjustment of central transcriptomic networks involved in maintaining adequate physiological homeostasis processes, but also that a high shoot-root dynamics is required for the proper deployment of Al resistance responses in the root.

  8. Medullary N-type and P/Q-type calcium channels contribute to neuropathy-induced allodynia.

    PubMed

    Urban, Mark O; Ren, Kunkun; Sablad, Marciano; Park, Kenneth T

    2005-04-25

    The present study was designed to determine the contribution of N-type, P/Q-type and L-type calcium channels in the rostral ventromedial medulla to tactile allodynia following peripheral nerve injury. L5/L6 spinal nerve ligation in rats produced tactile allodynia, which was dose-dependently inhibited by intrarostral ventromedial medulla microinjection of the N-type calcium channel antagonist omega-conotoxin MVIIA. Similarly, intrarostral ventromedial medulla microinjection of the P/Q-type calcium channel antagonist omega-agatoxin IVA inhibited spinal nerve ligation-induced tactile allodynia, whereas intrarostral ventromedial medulla microinjection of the L-type calcium channel antagonist nimodipine had no effect. These results demonstrate that N-type and P/Q-type calcium channels in the rostral ventromedial medulla contribute to tactile allodynia following peripheral neuropathy, likely via neurotransmitter-mediated activation of descending facilitatory systems from the rostral ventromedial medulla.

  9. Neuronal Expression of the Human Neuropeptide S Receptor NPSR1 Identifies NPS-Induced Calcium Signaling Pathways

    PubMed Central

    Erdmann, Frank; Kügler, Sebastian; Blaesse, Peter; Lange, Maren D.; Skryabin, Boris V.; Pape, Hans-Christian; Jüngling, Kay

    2015-01-01

    The neuropeptide S (NPS) system was discovered as a novel neurotransmitter system a decade ago and has since been identified as a key player in the modulation of fear and anxiety. Genetic variations of the human NPS receptor (NPSR1) have been associated with pathologies like panic disorders. However, details on the molecular fundamentals of NPSR1 activity in neurons remained elusive. We expressed NPSR1 in primary hippocampal cultures. Using single-cell calcium imaging we found that NPSR1 stimulation induced calcium mobilization from the endoplasmic reticulum via activation of IP3 and ryanodine receptors. Store-operated calcium channels were activated in a downstream process mediating entry of extracellular calcium. We provide the first detailed analysis of NPSR1 activity and the underlying intracellular pathways with respect to calcium mobilization in neurons. PMID:25714705

  10. Inhibition of Western-diet induced hyperproliferation and hyperplasia in mouse colon by two sources of calcium.

    PubMed

    Richter, F; Newmark, H L; Richter, A; Leung, D; Lipkin, M

    1995-11-01

    A Western-style diet containing high-fat and phosphate, and low calcium and vitamin D was fed to mice for 20 weeks. Starting at week 8, subgroups of animals received the Western-style diet supplemented by two different calcium sources: tricalcium phosphate and calcium citrate malate. Hyperproliferation (increased [3H]thymidine-labelled cells/colonic crypt) and hyperplasia (increased total epithelial cells/crypt) developed in the sigmoid colon after 8 weeks of feeding the Western-style diet confirming previous results, and these were reversed at later periods by the addition of the two calcium sources to the Western-style diet. Findings indicate that the modified colonic epithelial cell hyperproliferation and hyperplasia which have been associated with subsequent development of colonic neoplasia, are induced in mice fed a Western-style diet, and the addition of calcium to the diet inhibited their development in the colonic mucosa.

  11. Cinnamaldehyde and cinnamaldehyde-containing micelles induce relaxation of isolated porcine coronary arteries: role of nitric oxide and calcium

    PubMed Central

    Raffai, Gábor; Kim, Byungkuk; Park, Sanga; Khang, Gilson; Lee, Dongwon; Vanhoutte, Paul M

    2014-01-01

    Background and purpose Cinnamaldehyde, a major component of cinnamon, induces the generation of reactive oxygen species and exerts vasodilator and anticancer effects, but its short half-life limits its clinical use. The present experiments were designed to compare the acute relaxing properties of cinnamaldehyde with those of self-assembling polymer micelles either loaded with cinnamaldehyde or consisting of a polymeric prodrug [poly(cinnamaldehyde)] that incorporates the compound in its backbone. Methods Rings of porcine coronary arteries were contracted with the thromboxane A2 receptor agonist U46619 or 40 mM KCl, and changes in isometric tension were recorded. Results Cinnamaldehyde induced concentration-dependent but endothelium-independent, nitric oxide synthase (NOS)-independent, cyclooxygenase-independent, soluble guanylyl cyclase (sGC)-independent, calcium-activated potassium-independent, and TRPA1 channel-independent relaxations. Cinnamaldehyde also inhibited the contractions induced by 40 mM KCl Ca2+ reintroduction in 40 mM KCl Ca2+-free solution or by the Ca2+ channel opener Bay K8644. Cinnamaldehyde-loaded control micelles induced complete, partly endothelium-dependent relaxations sensitive to catalase and inhibitors of NOS or sGC, but not cyclooxygenase or TRPA1, channels. Cinnamaldehyde-loaded micelles also inhibited contractions induced by 40 mM KCl Ca2+ reintroduction or Bay K8644. Poly(cinnamaldehyde) micelles induced only partial, endothelium-dependent relaxations that were reduced by inhibitors of NOS or sGC and by catalase and the antioxidant tiron, but not by indomethacin or TRPA1 channel blockers. Conclusion The present findings demonstrate that cinnamaldehyde-loaded and poly(cinnamaldehyde) micelles possess vasodilator properties, but that the mechanism underlying the relaxation that they cause differs from that of cinnamaldehyde, and thus could be used both to relieve coronary vasospasm and for therapeutic drug delivery. PMID:24904214

  12. Roscovitine in combination with calcium ionophore induces oocyte activation through reduction of M-phase promoting factor activity in mice.

    PubMed

    Iba, Tomomi; Yano, Yuya; Umeno, Mayumi; Hinokio, Kenji; Kuwahara, Akira; Irahara, Minoru; Yamano, Shuji; Yasui, Toshiyuki

    2012-11-01

    The aim of the present study was to determine oocyte activation and change in M-phase promoting factor (MPF) activity induced by treatment with calcium ionophore and roscovitine in comparison with those induced by treatment with roscovitine alone and treatment with calcium ionophore and puromycin in mice. Freshly ovulated oocytes obtained from 6-8-week-old mice were divided into five groups (no activation treatment; 5 μM calcium ionophore A23187; 50 μM roscovitine; 5 μM calcium ionophore and 10 μg/ml puromycin; and 5 μM calcium ionophore and 50 μM roscovitine) and were incubated for 6 h. Oocyte activation, assessed by morphological changes, and changes in MPF activity in the five groups at 0, 2, 4 and 6 h of incubation were examined. Activated oocytes were defined as oocytes with at least one pronucleus. Oocytes treated with roscovitine alone were not activated during the 6-h incubation period. All of the oocytes in the calcium ionophore with puromycin group and in the calcium ionophore with roscovitine group were activated. The percentage activity of MPF in oocytes treated with roscovitine alone was decreased after 2 h and increased after 4 h of incubation. The percentage activity of MPF in oocytes treated with calcium ionophore and roscovitine was significantly decreased with suppression of MPF activity being maintained for 6 h, and this change was similar to that in oocytes treated with calcium ionophore and puromycin. Roscovitine with calcium ionophore is effective for induction of oocyte activation through suppression of MPF activity in mice.

  13. Inhibition of the mitochondrial calcium uniporter inhibits Aβ-induced apoptosis by reducing reactive oxygen species-mediated endoplasmic reticulum stress in cultured microglia.

    PubMed

    Xie, Nanchang; Wu, Chuanjie; Wang, Cui; Cheng, Xuan; Zhang, Lu; Zhang, Haifeng; Lian, Yajun

    2017-09-19

    Amyloid-beta (Aβ) has been shown to induce microglial apoptosis, which is itself sensitive to disturbed mitochondrial calcium (Ca(2+)) homeostasis. The mitochondrial calcium uniporter (MCU) plays an important regulatory role in mitochondrial Ca(2+) homeostasis, but its role in Aβ-induced microglia apoptosis is unknown. In this study, we found increased mitochondrial Ca(2+) concentration in Aβ-treated primary microglia and BV-2 cells; also, the MCU inhibitor Ru360 significantly attenuated Aβ-induced microglial apoptosis, whereas the MCU activator spermine augmented it. In addition, Ru360 significantly attenuated Aβ-induced mitochondrial reactive oxygen species (ROS) production, as well as endoplasmic reticulum (ER) stress characterized by glucose-regulated protein 78 (GRP78) and C/-EBP homologous protein (CHOP) expression. Spermine, however, exerted the opposite effects on mitochondrial ROS production and ER stress. We also found that mitochondria-targeted antioxidant (Mito-TEMPO) treatment decreased GRP78 and CHOP expression in Aβ-treated microglia. Moreover, blocking endogenous CHOP expression using a CHOP small interfering RNA (siRNA) attenuated Aβ-induced cell death. Altogether, our data suggested that 1) inhibition of MCU exerts a neuroprotective effect on Aβ-induced microglia apoptosis, and 2) that the underlying mechanism may be related to reducing mitochondrial ROS-mediated ER stress. Copyright © 2017. Published by Elsevier B.V.

  14. Calcium-induced Cytochrome c release from rat brain mitochondria is altered by digitonin.

    PubMed

    Brustovetsky, Nickolay; Jemmerson, Ronald; Dubinsky, Janet M

    2002-10-31

    To determine if calcium could release Cytochrome c (Cyt c) from brain mitochondria without activating the permeability transition (mPT), brain mitochondria were prepared in two different ways. Digitonin was used to lyse synaptosomes and release synaptosomal mitochondria or a Percoll gradient was used to separate non-synaptosomal mitochondria from the synaptosomes. In gradient-purified mitochondria, low levels of added digitonin produced swelling and Cyt c release. Digitonin augmented Ca(2+)-induced Cyt c release that was insensitive to the mPT inhibitors, cyclosporin A CsA and ADP. Similarly, in mitochondria prepared with digitonin, these inhibitors also failed to prevent Ca(2+)-induced Cyt c release. Thus the mPT-independent, Ca(2+)-induced Cyt c release pathway was attributable to alteration of the permeability properties of the outer mitochondrial membrane by digitonin. Copyright 2002 Elsevier Science Ireland Ltd.

  15. The mechanism of calcium oxalate crystal-induced haemolysis of human erythrocytes.

    PubMed Central

    Elferink, J. G.

    1987-01-01

    Calcium oxalate (CaOx) crystals cause membrane damage in human erythrocytes, evident from K+ leakage and haemoglobin release. Whereas the hydrogen acceptor polyvinylpyridine-N-oxide is without effect on CaOx crystal-induced haemolysis, polyanions and negative proteins are strongly inhibitory. This indicates that positive charges are of importance for induction of haemolysis. These positive charges are located on the CaOx crystals. Removal of the negatively charged sialic acid from the cell surface does not affect CaOx crystal-induced haemolysis. CaOx crystals are able to release glucose from negatively charged liposomes, but not from positively charged liposomes. The results are compatible with the view that positive charges on the crystals are of predominant importance in CaOx-induced haemolysis, and that their interactions with negative charges or polarizable structures in the lipid part of the membrane leads to membrane disruption. PMID:2443155

  16. Ultrastructural Alteration of Plant Plasma Membranes Induced by Auxin and Calcium Ions 1

    PubMed Central

    Morré, D. James; Bracker, Charles E.

    1976-01-01

    Ultrastructural changes in isolated and in situ plasma membranes of etiolated soybean hypocotyls (Glycine max L. cv. Wayne) were induced by indole-3-acetic acid (IAA), other auxins, and calcium chloride. Fixed and embedded preparations were stained by a phosphotungstate-chromate procedure to identify and accentuate plasma membrane. Measurements were on micrographs obtained with an electron optical system calibrated and corrected for reproducible and accurate size measurements. Plasma membranes treated for 20 minutes with 1 μm IAA were 10 to 15% thinner than controls. The response to IAA was rapid, reproducible, auxin-specific, temperature-dependent, and reversible. Comparable responses were obtained with isolated and in situ membranes. Membranes treated with 0.5 m calcium chloride for 20 minutes were 15 to 20% thicker than controls. Multiple cycles of alternating calcium and IAA treatments yielded membranes with dimensions that reflected the last treatment of the series. The findings show a direct response of plasma membranes to growth regulating agents and provide evidence for a cell-free response of isolated plasma membranes to a hormone. Images PMID:16659714

  17. Enhancement of crystal induced neutrophil responses by opsonisation of calcium pyrophosphate dihydrate crystals.

    PubMed Central

    Burt, H M; Jackson, J K

    1993-01-01

    OBJECTIVES--Little is known about the effect on crystal induced neutrophil responses of the opsonisation of calcium pyrophosphate dihydrate (CPPD) (triclinic) crystals with components of serum and plasma. The purpose of this study was to determine the effects of precoating CPPD crystals with plasma, serum, complement depleted serum, and IgG on a full range of crystal induced neutrophil responses (calcium mobilisation, chemiluminescence, superoxide anion production, non-cytolytic lysosomal enzyme release, and leukotriene synthesis). METHODS--Crystals were precoated with IgG, serum, plasma, or complement depleted serum (heated at 56 degrees C), incubated with neutrophils and the responses monitored with time. Measurement of the extent of neutrophil association with crystals was based on monitoring the decrease in fluorescence intensity of supernatants when crystals and diphenylhexatriene labelled neutrophils were allowed to settle under gravity. RESULTS--Precoating CPPD crystals with IgG, plasma, and serum significantly enhanced chemiluminescence, superoxide anion generation, increases in cytosolic free calcium levels, and non-cytolytic lysosomal enzyme release by neutrophils compared with uncoated CPPD crystals. The enhancement of neutrophil responses by crystals coated with complement depleted serum was less pronounced. The increased neutrophil responses induced by CPPD crystals coated with IgG might have been due to the observed increase in the association of IgG coated crystals with neutrophils. CONCLUSIONS--These data show that there is a marked potentiation of all neutrophil responses to IgG, plasma, and serum coated CPPD crystals. It is suggested that the adsorption of synovial fluid proteins, including IgG and C3b, to CPPD crystals in vivo, results in the opsonised crystals becoming a potent neutrophil stimulant and inflammatory agent. PMID:8215624

  18. Beneficial effects of calcium oral coadministration in gentamicin-induced nephrotoxicity in rats.

    PubMed

    Stojiljkovic, Nenad; Stoiljkovic, Milan; Mihailovic, Dragan; Randjelovic, Pavle; Ilic, Sonja; Gocmanac-Ignjatovic, Marija; Veljkovic, Milica

    2012-01-01

    Frequent therapeutical use of an aminoglycoside antibiotic gentamicin (GM) is limited by its nephrotoxic effects often characterized by both morphological and functional alterations of kidney leading to acute renal failure. The aim of this study was to examine the effect of dietary calcium supplementation on GM-induced nephrotoxicity in rats. Experiments were performed on 30 adult male Wistar rats divided into three groups of 10 animals each. G-group received GM intraperitoneally at a dose of 100 mg/kg; GCa-group received the same dose of GM concomitantly with 1 g/kg calcium carbonate given orally; and C-group, serving as control, received 1 mL/day of normal saline. All groups were treated during 8 consecutive days. Quantitative evaluation of GM-induced structural and functional changes of kidney was performed by histopathological, morphometrical, and biochemical analyses. Compared with control, G-group of rats were found to have diffusely and unequally thickened glomerular basement membrane with neutrophil cells infiltration. In addition, vacuolization of cytoplasm of proximal tubule cells with coagulation-type necrosis was observed. These GM-induced pathological lesions were significantly reduced in the rats of GCa-group. Morphometric analysis revealed statistically significant differences in the size of glomeruli (area, major and minor axes, perimeter), optical density, and roundness of glomeruli (p < 0.05) between G and GCa groups. Biochemical analysis showed significant elevation in blood urea and serum creatinine concentrations, whereas potassium concentration was lowered in G-group compared with the other groups (p < 0.01). It is concluded that oral supplementation of calcium during treatment with GM resulted in significant reduction of morphological and functional kidney alterations.

  19. Hydrogen peroxide-induced calcium influx in lung microvascular endothelial cells involves TRPV4

    PubMed Central

    Suresh, Karthik; Servinsky, Laura; Reyes, Jose; Baksh, Syeda; Undem, Clark; Caterina, Michael; Pearse, David B.

    2015-01-01

    In acute respiratory distress syndrome, both reactive oxygen species (ROS) and increased intracellular calcium ([Ca2+]i) are thought to play important roles in promoting endothelial paracellular permeability, but the mechanisms linking ROS and [Ca2+]i in microvascular endothelial cells are not known. In this study, we assessed the effect of hydrogen peroxide (H2O2) on [Ca2+]i in mouse and human lung microvascular endothelial cells (MLMVEC and HLMVEC, respectively). We found that in both MLMVECs and HLMVECs, exogenously applied H2O2 increased [Ca2+]i through Ca2+ influx and that pharmacologic inhibition of the calcium channel transient receptor potential vanilloid 4 (TRPV4) attenuated the H2O2-induced Ca2+ influx. Additionally, knockdown of TRPV4 in HLMVEC also attenuated calcium influx following H2O2 challenge. Administration of H2O2 or TRPV4 agonists decreased transmembrane electrical resistance (TER), suggesting increased barrier permeability. To explore the regulatory mechanisms underlying TRPV4 activation by ROS, we examined H2O2-induced Ca2+ influx in MLMVECs and HLMVECs with either genetic deletion, silencing, or pharmacologic inhibition of Fyn, a Src family kinase. In both MLMVECs derived from mice deficient for Fyn and HLMVECs treated with either siRNA targeted to Fyn or the Src family kinase inhibitor SU-6656 for 24 or 48 h, the H2O2-induced Ca2+ influx was attenuated. Treatment with SU-6656 decreased the levels of phosphorylated, but not total, TRPV4 protein and had no effect on TRPV4 response to the external agonist, GSK1016790A. In conclusion, our data suggest that application of exogenous H2O2 increases [Ca2+]i and decreases TER in microvascular endothelial cells via activation of TRPV4 through a mechanism that requires the Src kinase Fyn. PMID:26453519

  20. Hydrogen peroxide-induced calcium influx in lung microvascular endothelial cells involves TRPV4.

    PubMed

    Suresh, Karthik; Servinsky, Laura; Reyes, Jose; Baksh, Syeda; Undem, Clark; Caterina, Michael; Pearse, David B; Shimoda, Larissa A

    2015-12-15

    In acute respiratory distress syndrome, both reactive oxygen species (ROS) and increased intracellular calcium ([Ca(2+)]i) are thought to play important roles in promoting endothelial paracellular permeability, but the mechanisms linking ROS and [Ca(2+)]i in microvascular endothelial cells are not known. In this study, we assessed the effect of hydrogen peroxide (H2O2) on [Ca(2+)]i in mouse and human lung microvascular endothelial cells (MLMVEC and HLMVEC, respectively). We found that in both MLMVECs and HLMVECs, exogenously applied H2O2 increased [Ca(2+)]i through Ca(2+) influx and that pharmacologic inhibition of the calcium channel transient receptor potential vanilloid 4 (TRPV4) attenuated the H2O2-induced Ca(2+) influx. Additionally, knockdown of TRPV4 in HLMVEC also attenuated calcium influx following H2O2 challenge. Administration of H2O2 or TRPV4 agonists decreased transmembrane electrical resistance (TER), suggesting increased barrier permeability. To explore the regulatory mechanisms underlying TRPV4 activation by ROS, we examined H2O2-induced Ca(2+) influx in MLMVECs and HLMVECs with either genetic deletion, silencing, or pharmacologic inhibition of Fyn, a Src family kinase. In both MLMVECs derived from mice deficient for Fyn and HLMVECs treated with either siRNA targeted to Fyn or the Src family kinase inhibitor SU-6656 for 24 or 48 h, the H2O2-induced Ca(2+) influx was attenuated. Treatment with SU-6656 decreased the levels of phosphorylated, but not total, TRPV4 protein and had no effect on TRPV4 response to the external agonist, GSK1016790A. In conclusion, our data suggest that application of exogenous H2O2 increases [Ca(2+)]i and decreases TER in microvascular endothelial cells via activation of TRPV4 through a mechanism that requires the Src kinase Fyn. Copyright © 2015 the American Physiological Society.

  1. Fluorescence Based Characterization of Calcium Sensitizer Action on the Troponin Complex

    SciTech Connect

    Schlecht, William; Li, King-Lun; Hu, Dehong; Dong, Wen-Ji

    2016-02-01

    By examining the behavior of each Ca2+ -sensitizer on cTnC at different levels of reconstitution (cTnI-cTnC, full troponin, or full troponin in thin filament) the importance of these proteins on sensitizer efficacy was evaluated, lending insight into the mechanism of action behind each drug. A fluorescence based approach was used to monitor the opening and closing of cardiac troponin C's hydrophobic pocket in the presence and absence of four common Ca2+ -sensitizers: EMD 57033, levosimendan, bepridil and pimobendan. Ca2+ -titration experiments were employed to determine the effect on Ca2+- sensitivity and cooperativity of cTnC opening, while stopped flow experiments were used to investigate the impact on cTnC relaxation kinetics. This study shows EMD 57033 is unable to sensitize cTnC to Ca2+, and likely requires the presence of myosin to illicit a response. Levosimendan, bepridil, and pimobendan were all able to increase the sensitivity of cTnC for Ca2+ to varying degrees; levosimendan and pimobendan reduced the rate of cTnC closing, while bepridil increased this rate. Additionally the same experiments were run on thin filament samples containing cTnT (T204E), a known Ca2+- blunting phosphorylation mimic. Levosimendan, bepridil, and pimobendan were found to elevate the Ca2+-sensitivity of cTnT(T204E) containing thin filaments to within range of the wild type thin filaments.

  2. Effect of Topical Calcium Channel Blockers on Intraocular Pressure in Steroid-induced Glaucoma.

    PubMed

    Ganekal, Sunil; Dorairaj, Syril; Jhanji, Vishal; Kudlu, Krishnaprasad

    2014-01-01

    To evaluate the effect of 0.125% verapamil and 0.5% diltiazem eye drops on intraocular pressure (IOP) in steroid-induced glaucoma in rabbit eyes. A total of 18 rabbits with steroid-induced glaucoma were divided into three groups (A, B and C; n = 6 each). Right eyes in groups A, B and C received 0.5% diltiazem, 0.125% verapamil and 0.5% timolol eye drops twice daily for 12 days, respectively; whereas, left eyes received distilled water. IOP was measured with Tono-pen XL at baseline, day 4, day 8, and day 12 of treatment. Both 0.5% diltiazem and 0.125% verapamil eye drops significantly reduced IOP compared to control eyes (p < 0.05). Reduction of IOP by 0.5% diltiazem, 0.125% verapamil eye drops were comparable to 0.5% timolol. No surface toxicity or systemic side effects were noted during the study period. Calcium channel blockers, verapamil, and diltia-zem significantly reduced IOP in rabbiteyes. This group of drugs may have a potential role in treatment of glaucoma How to cite this article: Ganekal S, Dorairaj S, Jhanji V, Kudlu K. Effect of Topical Calcium Channel Blockers on Intraocular Pressure in Steroid-induced Glaucoma. J Current Glau Prac 2014;8(1):15-19.

  3. Cadmium Induces Apoptosis in Freshwater Crab Sinopotamon henanense through Activating Calcium Signal Transduction Pathway

    PubMed Central

    Wang, Jinxiang; Zhang, Pingping; Liu, Na; Wang, Qian; Luo, Jixian; Wang, Lan

    2015-01-01

    Calcium ion (Ca2+) is one of the key intracellular signals, which is implicated in the regulation of cell functions such as impregnation, cell proliferation, differentiation and death. Cadmium (Cd) is a toxic environmental pollutant that can disturb cell functions and even lead to cell death. Recently, we have found that Cd induced apoptosis in gill cells of the freshwater crab Sinopotamon henanense via caspase activation. In the present study, we further investigated the role of calcium signaling in the Cd-induced apoptosis in the animals. Our data showed that Cd triggered gill cell apoptosis which is evidenced by apoptotic DNA fragmentation, activations of caspases-3, -8 and -9 and the presence of apoptotic morphological features. Moreover, Cd elevated the intracellular concentration of Ca2+, the protein concentration of calmodulin (CaM) and the activity of Ca2+-ATPase in the gill cells of the crabs. Pretreatment of the animals with ethylene glycol-bis-(b-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), Ca2+ chelator, inhibited Cd-induced activation of caspases-3, -8 and -9 as well as blocked the Cd-triggered apoptotic DNA fragmentation. The apoptotic morphological features were no longer observed in gill cells pretreated with the Ca2+ signaling inhibitors before Cd treatment. Our results indicate that Cd evokes gill cell apoptosis through activating Ca2+-CaM signaling transduction pathway. PMID:26714174

  4. The effects of reactive oxygen species on calcium- and carbachol-induced contractile responses in beta-escin permeabilized rat bladder.

    PubMed

    Durlu-Kandilci, N Tugba; Sahin-Erdemli, Inci

    2008-12-01

    The effect of reactive oxygen species on contractions in beta-escin permeabilized rat detrusor was investigated. Cumulative calcium contractions were inhibited by hydrogen peroxide (H(2)O(2)) and hydroxyl (*OH) but not by superoxide (O(2) *). The sarcoplasmic reticulum calcium-ATPase inhibitor cyclopiazonic acid (CPA) and the mitochondrial blocker carbonyl cyanide p-trifluromethoxyphenylhydrazone (FCCP) decreased the calcium contractions, however in their presence, H(2)O(2) and *OH did not have further effect. Carbachol contractions were inhibited by either H(2)O(2)/*OH/O(2) * or CPA/FCCP. In the presence of CPA, carbachol contractions were not affected by H(2)O(2) and *OH but further decreased by O(2) *. On the other hand, only H(2)O(2) and *OH elicited additional inhibition in carbachol responses in the presence of FCCP. Inositol triphosphate contraction was inhibited by *OH whereas none of the radicals affect carbachol induced calcium sensitization. These results show that H(2)O(2) and *OH affects sarcoplasmic reticulum where O(2) * acts on mitochondria to change contractions in rat detrusor smooth muscle.

  5. Salvinorin A Inhibits Airway Hyperreactivity Induced by Ovalbumin Sensitization.

    PubMed

    Rossi, Antonietta; Caiazzo, Elisabetta; Bilancia, Rossella; Riemma, Maria A; Pagano, Ester; Cicala, Carla; Ialenti, Armando; Zjawiony, Jordan K; Izzo, Angelo A; Capasso, Raffaele; Roviezzo, Fiorentina

    2016-01-01

    Salvinorin A, a neoclerodane diterpene isolated from Salvia divinorum, exerts a number of pharmacological actions which are not solely limited to the central nervous system. Recently it has been demonstrated that Salvinorin A inhibits acute inflammatory response affecting leukotriene (LT) production. Since LTs are potent lipid mediators implicated in allergic diseases, we evaluated the effect of Salvinorin A on allergic inflammation and on airways following sensitization in the mouse. Mice were sensitized with s.c. injection of ovalbumin (OVA) on days 1 and 8. Sensitized mice received on days 9 and 12 on the shaved dorsal surface air administration to induce the development of the air-pouches. On day 15 animals were challenged by injection of OVA into the air-pouch. Salvinorin A, administered (10 mg/kg) before each allergen exposure, significantly reduced OVA-induced LT increase in the air pouch. This effect was coupled to a reduction in cell recruitment and Th2 cytokine production. In another set of experiments, mice were sensitized with OVA and both bronchial reactivity and pulmonary inflammation were assessed. Salvinorin A abrogated bronchial hyperreactivity and interleukin (IL)-13 production, without effect on pulmonary inflammation. Indeed cell infiltration and peribronchial edema were still present following diterpenoid treatment. Similarly, pulmonary IL-4 and plasmatic IgE levels were not modulated. Conversely, Salvinorin A significantly reduced LTC4 production in the lung of sensitized mice. Finally mast cell activity was evaluated by means of toluidine blue staining. Data obtained evidenced that Salvinorin A significantly inhibited mast cell degranulation in the lung. Our study demonstrates that Salvinorin A inhibits airway hyperreactivity induced by sensitization by inhibition of LT production and mast cell degranulation. In conclusion Salvinorin A could represent a promising candidate for drug development in allergic diseases such as asthma.

  6. Salvinorin A Inhibits Airway Hyperreactivity Induced by Ovalbumin Sensitization

    PubMed Central

    Rossi, Antonietta; Caiazzo, Elisabetta; Bilancia, Rossella; Riemma, Maria A.; Pagano, Ester; Cicala, Carla; Ialenti, Armando; Zjawiony, Jordan K.; Izzo, Angelo A.; Capasso, Raffaele; Roviezzo, Fiorentina

    2017-01-01

    Salvinorin A, a neoclerodane diterpene isolated from Salvia divinorum, exerts a number of pharmacological actions which are not solely limited to the central nervous system. Recently it has been demonstrated that Salvinorin A inhibits acute inflammatory response affecting leukotriene (LT) production. Since LTs are potent lipid mediators implicated in allergic diseases, we evaluated the effect of Salvinorin A on allergic inflammation and on airways following sensitization in the mouse. Mice were sensitized with s.c. injection of ovalbumin (OVA) on days 1 and 8. Sensitized mice received on days 9 and 12 on the shaved dorsal surface air administration to induce the development of the air-pouches. On day 15 animals were challenged by injection of OVA into the air-pouch. Salvinorin A, administered (10 mg/kg) before each allergen exposure, significantly reduced OVA-induced LT increase in the air pouch. This effect was coupled to a reduction in cell recruitment and Th2 cytokine production. In another set of experiments, mice were sensitized with OVA and both bronchial reactivity and pulmonary inflammation were assessed. Salvinorin A abrogated bronchial hyperreactivity and interleukin (IL)-13 production, without effect on pulmonary inflammation. Indeed cell infiltration and peribronchial edema were still present following diterpenoid treatment. Similarly, pulmonary IL-4 and plasmatic IgE levels were not modulated. Conversely, Salvinorin A significantly reduced LTC4 production in the lung of sensitized mice. Finally mast cell activity was evaluated by means of toluidine blue staining. Data obtained evidenced that Salvinorin A significantly inhibited mast cell degranulation in the lung. Our study demonstrates that Salvinorin A inhibits airway hyperreactivity induced by sensitization by inhibition of LT production and mast cell degranulation. In conclusion Salvinorin A could represent a promising candidate for drug development in allergic diseases such as asthma. PMID

  7. Calcium Flux between the Endoplasmic Reticulum and Mitochondrion Contributes to Poliovirus-Induced Apoptosis▿

    PubMed Central

    Brisac, Cynthia; Téoulé, François; Autret, Arnaud; Pelletier, Isabelle; Colbère-Garapin, Florence; Brenner, Catherine; Lemaire, Christophe; Blondel, Bruno

    2010-01-01

    We show that poliovirus (PV) infection induces an increase in cytosolic calcium (Ca2+) concentration in neuroblastoma IMR5 cells, at least partly through Ca2+ release from the endoplasmic reticulum lumen via the inositol 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR) channels. This leads to Ca2+ accumulation in mitochondria through the mitochondrial Ca2+ uniporter and the voltage-dependent anion channel (VDAC). This increase in mitochondrial Ca2+ concentration in PV-infected cells leads to mitochondrial dysfunction and apoptosis. PMID:20861253

  8. Involvement of calcium-sensing receptor in ischemia/reperfusion-induced apoptosis in rat cardiomyocytes

    SciTech Connect

    Zhang Weihua; Fu Songbin; Lu Fanghao . E-mail: lufanghao1973@yahoo.com.cn; Wu Bo; Gong Dongmei; Pan, Zhen-wei; Lv Yanjie; Zhao Yajun; Li Quanfeng; Wang Rui; Yang Baofeng; Xu Changqing . E-mail: xucq@163.com

    2006-09-08

    The calcium-sensing receptor (CaR) is a seven-transmembrane G-protein coupled receptor, which activates intracellular effectors, for example, it causes inositol phosphate (IP) accumulation to increase the release of intracellular calcium. Although intracellular calcium overload has been implicated in the cardiac ischemia/reperfusion (I/R)-induced apoptosis, the role of CaR in the induction of apoptosis has not been fully understood. This study tested the hypothesis that CaR is involved in I/R cardiomyocyte apoptosis by increasing [Ca{sup 2+}]{sub i}. The isolated rat hearts were subjected to 40-min ischemia followed by 2 h of reperfusion, meanwhile GdCl{sub 3} was added to reperfusion solution. The expression of CaR increased at the exposure to GdCl{sub 3} during I/R. By laser confocal microscopy, it was observed that the intracellular calcium was significantly increased and exhibited a collapsed {delta}{psi} {sub m}, as monitored by 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) during reperfusion with GdCl{sub 3}. Furthermore, the number of apoptotic cells was significantly increased as shown by TUNEL assay. Typical apoptotic cells were observed with transmission electron microscopy in I/R with GdCl{sub 3} but not in the control group. The expression of cytosolic cytochrome c and activated caspase-9 and caspase-3 was significantly increased whereas the expression of mitochondrial cytochrome c significantly decreased in I/R with GdCl{sub 3} in comparison to the control. In conclusion, these results suggest that CaR is involved in the induction of cardiomyocyte apoptosis during ischemia/reperfusion through activation of cytochrome c-caspase-3 signaling pathway.

  9. Involvement of calcium-sensing receptor in ischemia/reperfusion-induced apoptosis in rat cardiomyocytes.

    PubMed

    Zhang, Wei-hua; Fu, Song-bin; Lu, Fang-hao; Wu, Bo; Gong, Dong-mei; Pan, Zhen-wei; Lv, Yan-jie; Zhao, Ya-jun; Li, Quan-Feng; Wang, Rui; Yang, Bao-feng; Xu, Chang-qing

    2006-09-08

    The calcium-sensing receptor (CaR) is a seven-transmembrane G-protein coupled receptor, which activates intracellular effectors, for example, it causes inositol phosphate (IP) accumulation to increase the release of intracellular calcium. Although intracellular calcium overload has been implicated in the cardiac ischemia/reperfusion (I/R)-induced apoptosis, the role of CaR in the induction of apoptosis has not been fully understood. This study tested the hypothesis that CaR is involved in I/R cardiomyocyte apoptosis by increasing [Ca2+]i. The isolated rat hearts were subjected to 40-min ischemia followed by 2 h of reperfusion, meanwhile GdCl3 was added to reperfusion solution. The expression of CaR increased at the exposure to GdCl3 during I/R. By laser confocal microscopy, it was observed that the intracellular calcium was significantly increased and exhibited a Deltapsim, as monitored by 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) during reperfusion with GdCl3. Furthermore, the number of apoptotic cells was significantly increased as shown by TUNEL assay. Typical apoptotic cells were observed with transmission electron microscopy in I/R with GdCl3 but not in the control group. The expression of cytosolic cytochrome c and activated caspase-9 and caspase-3 was significantly increased whereas the expression of mitochondrial cytochrome c significantly decreased in I/R with GdCl3 in comparison to the control. In conclusion, these results suggest that CaR is involved in the induction of cardiomyocyte apoptosis during ischemia/reperfusion through activation of cytochrome c-caspase-3 signaling pathway.

  10. Capsaicin sensitizes TRAIL-induced apoptosis through Sp1-mediated DR5 up-regulation: Involvement of Ca{sup 2+} influx

    SciTech Connect

    Moon, Dong-Oh; Kang, Chang-Hee; Kang, Sang-Hyuck; Choi, Yung-Hyun; Hyun, Jin-Won; Chang, Weon-Young; Kang, Hee-Kyoung; Koh, Young-Sang; Maeng, Young-Hee; Kim, Young-Ree; Kim, Gi-Young

    2012-02-15

    Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various malignant cells, several cancers including human hepatocellular carcinoma (HCC) exhibit potent resistance to TRAIL-induced cell death. The aim of this study is to evaluate the anti-cancer potential of capsaicin in TRAIL-induced cancer cell death. As indicated by assays that measure phosphatidylserine exposure, mitochondrial activity and activation of caspases, capsaicin potentiated TRAIL-resistant cells to lead to cell death. In addition, we found that capsaicin induces the cell surface expression of TRAIL receptor DR5, but not DR4 through the activation Sp1 on its promoter region. Furthermore, we investigated that capsaicin-induced DR5 expression and apoptosis are inhibited by calcium chelator or inhibitors for calmodulin-dependent protein kinase. Taken together, our data suggest that capsaicin sensitizes TRAIL-mediated HCC cell apoptosis by DR5 up-regulation via calcium influx-dependent Sp1 activation. Highlights: ► Capsaicin sensitizes TRAIL-induced apoptosis through activation of caspases. ► Capsaicin induces expression of DR5 through Sp1 activation. ► Capsaicin activates calcium signaling pathway.

  11. Endoplasmic reticulum stress and calcium imbalance are involved in cadmium-induced lipid aberrancy in Saccharomyces cerevisiae.

    PubMed

    Rajakumar, Selvaraj; Bhanupriya, Nagaraj; Ravi, Chidambaram; Nachiappan, Vasanthi

    2016-09-01

    The endoplasmic reticulum is the key organelle which controls protein folding, lipid biogenesis, and calcium (Ca(2+)) homeostasis. Cd exposure in Saccharomyces cerevisiae activated the unfolded protein response and was confirmed by the increased Kar2p expression. Cd exposure in wild-type (WT) cells increased PC levels and the PC biosynthetic genes. Deletion of the two phospholipid methyltransferases CHO2 and OPI3 modulated PC, TAG levels and the lipid droplets with cadmium exposure. Interestingly, we noticed an increase in the calcium levels upon Cd exposure in the mutant cells. This study concluded that Cd interrupted calcium homeostasis-induced lipid dysregulation leading to ER stress.

  12. The use of size-defined DNA-functionalized calcium phosphate nanoparticles to minimise intracellular calcium disturbance during transfection.

    PubMed

    Neumann, Sebastian; Kovtun, Anna; Dietzel, Irmgard D; Epple, Matthias; Heumann, Rolf

    2009-12-01

    Calcium phosphate-based transfection methods are frequently used to transfer DNA into living cells. However, it has so far not been studied in detail to what extend the different transfection methods lead to a net calcium uptake. Upon subsequent resolution of the calcium phosphate, intracellular free ionic calcium-surges could result, inducing as side effect various physiological responses that may finally result in cell death. Here we investigated the overall calcium uptake by the human bladder carcinoma cell line T24 during the standard calcium phosphate transfection method and also during transfection with custom-made calcium phosphate/DNA nanoparticles by isotope labelling with (45)calcium. (45)Calcium uptake was strongly increased after 7h of standard calcium phosphate transfection but not if the transfection was performed with calcium phosphate nanoparticles. Time lapse imaging microscopy using the calcium-sensitive dye Fura-2 revealed large transient increases of the intracellular free calcium level during the standard calcium phosphate transfection but not if calcium phosphate nanoparticles were used. Consistently, the viability of cells transfected by calcium phosphate/DNA nanoparticles was not changed, in remarkable contrast to the standard method where considerable cell death occurred.

  13. Red meat and colon cancer: dietary haem-induced colonic cytotoxicity and epithelial hyperproliferation are inhibited by calcium.

    PubMed

    Sesink, A L; Termont, D S; Kleibeuker, J H; Van der Meer, R

    2001-10-01

    High intake of red meat is associated with increased colon cancer risk. We have shown earlier that this may be due to the high haem content of red meat, because dietary haem increased cytolytic activity of faecal water and colonic epithelial proliferation. Dietary calcium inhibits diet-induced epithelial hyperproliferation. Furthermore, it has been shown that supplemental calcium inhibited the recurrence of colorectal adenomas. Therefore, we studied whether dietary calcium phosphate can exert its protective effects by inhibiting the deleterious effects of haem. In vitro, calcium phosphate precipitated haem and inhibited the haem-induced cytotoxicity. Subsequently, rats were fed diets, differing in haem (0 or 1.3 micromol/g) and calcium phosphate content only (20 or 180 micromol/g). Faeces were collected for biochemical analyses. Cytolytic activity of faecal water was determined from the degree of lysis of erythrocytes by faecal water. Colonic epithelial proliferation was measured in vivo using [(3)H]thymidine incorporation. In rats fed low calcium diets, dietary haem increased cytolytic activity of faecal water (98 +/- 1 versus 1 +/- 1%, P < 0.001) and the concentration of cations in faeces (964 +/- 31 versus 254 +/- 20 micromol/g), when compared with controls. This indicates that dietary haem increased colonic mucosal exposure to luminal irritants. Colonic epithelial proliferation was increased compared with controls (70 +/- 4 versus 48 +/- 8 d.p.m./microg DNA, P < 0.001). This was accompanied by metabolism of the ingested haem and solubilization of haem compounds in the faecal water. A high calcium diet largely prevented this metabolism and solubilization. It also inhibited the haem-induced cytolytic activity of faecal water and increase in faecal cation concentration. In accordance, the haem-induced colonic epithelial hyperproliferation was prevented. We therefore suggest that dietary calcium phosphate acts as a chemopreventive agent in colon carcinogenesis by

  14. Capsaicin-induced central sensitization evokes segmental increases in trigger point sensitivity in humans.

    PubMed

    Srbely, John Z; Dickey, James P; Bent, Leah R; Lee, David; Lowerison, Mark

    2010-07-01

    This study investigated whether inducing central sensitization evokes segmental increases in trigger point pressure sensitivity. We evoked central sensitization at the C(5) segment and validated its presence via mechanical cutaneous sensitivity (brush allodynia) testing. Trigger point pressure sensitivity was quantified using the pain pressure threshold (PPT) value. A 50 cm(2) area of the C(5) dermatome at the right lateral elbow was pretreated with 45 degrees heat for 10 minutes. Test subjects (n = 20) then received topical capsaicin cream (0.075%; Medicis, Toronto, Canada) to the C(5) dermatome, whereas control subjects (n = 20) received a topical placebo cream (Biotherm Massage, Montreal, Canada). PPT readings were recorded from the infraspinatus (C(5,6)) and gluteus medius (L(4,5)S(1)) trigger points at zero (pre-intervention), 10, 20, and 30 minutes after intervention; all PPT readings were normalized to pre-intervention (baseline) values. The difference between the PPT readings at the 2 trigger point sites represents the direct influence of segmental mechanisms on the trigger point sensitivity at the infraspinatus site (PPT(seg)). Test subjects demonstrated statistically significant increases in Total Allodynia scores and significant decreases in PPT(seg) at 10, 20, and 30 minutes after application, when compared with control subjects. These results demonstrate that increases in central sensitization evoke increases in trigger point pressure sensitivity in segmentally related muscles. Myofascial pain is the most common form of musculoskeletal pain. Myofascial trigger points play an important role in the clinical manifestation of myofascial pain syndrome. Elucidating the role of central sensitization in the pathophysiology of trigger points is fundamental to developing optimal strategies in the management of myofascial pain syndrome.

  15. Acute exposure to progesterone attenuates cardiac contraction by modifying myofilament calcium sensitivity in the female mouse heart.

    PubMed

    Feridooni, Hirad A; MacDonald, Jennifer K; Ghimire, Anjali; Pyle, W Glen; Howlett, Susan E

    2017-01-01

    application of progesterone. In females, but not males, progesterone attenuates and slows cardiomyocyte contraction with no effect on calcium transients. Progesterone also reduces myofilament calcium sensitivity in female hearts. This may adversely affect heart function, especially when serum progesterone levels are high in pregnancy.Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/acute-progesterone-modifies-cardiac-contraction/. Copyright © 2017 the American Physiological Society.

  16. Effects of troponin T cardiomyopathy mutations on the calcium sensitivity of the regulated thin filament and the actomyosin cross-bridge kinetics of human β-cardiac myosin.

    PubMed

    Sommese, Ruth F; Nag, Suman; Sutton, Shirley; Miller, Susan M; Spudich, James A; Ruppel, Kathleen M

    2013-01-01

    Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) lead to significant cardiovascular morbidity and mortality worldwide. Mutations in the genes encoding the sarcomere, the force-generating unit in the cardiomyocyte, cause familial forms of both HCM and DCM. This study examines two HCM-causing (I79N, E163K) and two DCM-causing (R141W, R173W) mutations in the troponin T subunit of the troponin complex using human β-cardiac myosin. Unlike earlier reports using various myosin constructs, we found that none of these mutations affect the maximal sliding velocities or maximal Ca(2+)-activated ADP release rates involving the thin filament human β-cardiac myosin complex. Changes in Ca(2+) sensitivity using the human myosin isoform do, however, mimic changes seen previously with non-human myosin isoforms. Transient kinetic measurements show that these mutations alter the kinetics of Ca(2+) induced conformational changes in the regulatory thin filament proteins. These changes in calcium sensitivity are independent of active, cycling human β-cardiac myosin.

  17. Effects of Troponin T Cardiomyopathy Mutations on the Calcium Sensitivity of the Regulated Thin Filament and the Actomyosin Cross-Bridge Kinetics of Human β-Cardiac Myosin

    PubMed Central

    Sutton, Shirley; Miller, Susan M.; Spudich, James A.; Ruppel, Kathleen M.

    2013-01-01

    Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) lead to significant cardiovascular morbidity and mortality worldwide. Mutations in the genes encoding the sarcomere, the force-generating unit in the cardiomyocyte, cause familial forms of both HCM and DCM. This study examines two HCM-causing (I79N, E163K) and two DCM-causing (R141W, R173W) mutations in the troponin T subunit of the troponin complex using human β-cardiac myosin. Unlike earlier reports using various myosin constructs, we found that none of these mutations affect the maximal sliding velocities or maximal Ca2+-activated ADP release rates involving the thin filament human β-cardiac myosin complex. Changes in Ca2+ sensitivity using the human myosin isoform do, however, mimic changes seen previously with non-human myosin isoforms. Transient kinetic measurements show that these mutations alter the kinetics of Ca2+ induced conformational changes in the regulatory thin filament proteins. These changes in calcium sensitivity are independent of active, cycling human β-cardiac myosin. PMID:24367593

  18. A MUTANT PRION PROTEIN SENSITIZES NEURONS TO GLUTAMATE-INDUCED EXCITOTOXICITY

    PubMed Central

    Biasini, Emiliano; Unterberger, Ursula; Solomon, Isaac H.; Massignan, Tania; Senatore, Assunta; Bian, Hejiao; Voigtlaender, Till; Bowman, Frederick P.; Bonetto, Valentina; Chiesa, Roberto; Luebke, Jennifer; Toselli, Paul; Harris, David A.

    2013-01-01

    Growing evidence suggests that a physiological activity of the cellular prion protein (PrPC) plays a crucial role in several neurodegenerative disorders, including prion and Alzheimer’s diseases. However, how the functional activity of PrPC is subverted to deliver neurotoxic signals remains uncertain. Transgenic mice expressing PrP with a deletion of residues 105–125 in the central region (referred to as ΔCR PrP) provide important insights into this problem. Tg(ΔCR) mice exhibit neonatal lethality and massive degeneration of cerebellar granule neurons, a phenotype that is dose-dependently suppressed by the presence of wild-type PrP. When expressed in cultured cells, ΔCR PrP induces large, ionic currents that can be detected by patch-clamping techniques. Here, we have tested the hypothesis that abnormal ion channel activity underlies the neuronal death seen in Tg(ΔCR) mice. We find that ΔCR PrP induces abnormal ionic currents in neurons in culture and in cerebellar slices, and that this activity sensitizes the neurons to glutamate-induced, calcium-mediated death. In combination with ultrastructural and biochemical analyses, these results demonstrate a role for glutamate-induced excitotoxicity in PrP-mediated neurodegeneration. A similar mechanism may operate in other neurodegenerative disorders due to toxic, β-rich oligomers that bind to PrPC. PMID:23392670

  19. A mutant prion protein sensitizes neurons to glutamate-induced excitotoxicity.

    PubMed

    Biasini, Emiliano; Unterberger, Ursula; Solomon, Isaac H; Massignan, Tania; Senatore, Assunta; Bian, Hejiao; Voigtlaender, Till; Bowman, Frederick P; Bonetto, Valentina; Chiesa, Roberto; Luebke, Jennifer; Toselli, Paul; Harris, David A

    2013-02-06

    Growing evidence suggests that a physiological activity of the cellular prion protein (PrP(C)) plays a crucial role in several neurodegenerative disorders, including prion and Alzheimer's diseases. However, how the functional activity of PrP(C) is subverted to deliver neurotoxic signals remains uncertain. Transgenic (Tg) mice expressing PrP with a deletion of residues 105-125 in the central region (referred to as ΔCR PrP) provide important insights into this problem. Tg(ΔCR) mice exhibit neonatal lethality and massive degeneration of cerebellar granule neurons, a phenotype that is dose dependently suppressed by the presence of wild-type PrP. When expressed in cultured cells, ΔCR PrP induces large, ionic currents that can be detected by patch-clamping techniques. Here, we tested the hypothesis that abnormal ion channel activity underlies the neuronal death seen in Tg(ΔCR) mice. We find that ΔCR PrP induces abnormal ionic currents in neurons in culture and in cerebellar slices and that this activity sensitizes the neurons to glutamate-induced, calcium-mediated death. In combination with ultrastructural and biochemical analyses, these results demonstrate a role for glutamate-induced excitotoxicity in PrP-mediated neurodegeneration. A similar mechanism may operate in other neurodegenerative disorders attributable to toxic, β-rich oligomers that bind to PrP(C).

  20. Image-based Modeling of Biofilm-induced Calcium Carbonate Precipitation

    NASA Astrophysics Data System (ADS)

    Connolly, J. M.; Rothman, A.; Jackson, B.; Klapper, I.; Cunningham, A. B.; Gerlach, R.

    2013-12-01

    Pore scale biological processes in the subsurface environment are important to understand in relation to many engineering applications including environmental contaminant remediation, geologic carbon sequestration, and petroleum production. Specifically, biofilm induced calcium carbonate precipitation has been identified as an attractive option to reduce permeability in a lasting way in the subsurface. This technology may be able to replace typical cement-based grouting in some circumstances; however, pore-scale processes must be better understood for it to be applied in a controlled manor. The work presented will focus on efforts to observe biofilm growth and ureolysis-induced mineral precipitation in micro-fabricated flow cells combined with finite element modelling as a tool to predict local chemical gradients of interest (see figure). We have been able to observe this phenomenon over time using a novel model organism that is able to hydrolyse urea and express a fluorescent protein allowing for non-invasive observation over time with confocal microscopy. The results of this study show the likely existence of a wide range of local saturation indices even in a small (1 cm length scale) experimental system. Interestingly, the locations of high predicted index do not correspond to the locations of higher precipitation density, highlighting the need for further understanding. Figure 1 - A micro-fabricated flow cell containing biofilm-induced calcium carbonate precipitation. (A) Experimental results: Active biofilm is in green and dark circles are calcium carbonate crystals. Note the channeling behavior in the top of the image, leaving a large hydraulically inactive area in the biofilm mass. (B) Finite element model: The prediction of relative saturation of calcium carbonate (as calcite). Fluid enters the system at a low saturation state (blue) but areas of high supersaturation (red) are predicted within the hydraulically inactive area in the biofilm. If only effluent

  1. Cobra venom cardiotoxin induces perturbations of cytosolic calcium homeostasis and hypercontracture in adult rat ventricular myocytes.

    PubMed

    Wang, H X; Lau, S Y; Huang, S J; Kwan, C Y; Wong, T M

    1997-10-01

    The effects of Cobra venom cardiotoxin (CTX) on the cellular morphology, twitch amplitude and intracellular calcium ([Ca2+]i) of the ventricular myocytes were studied. [Ca2+]i and twitch amplitude were determined with a fluorometric ratio method using Fura-2/AM and Calcium