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Sample records for a1d l-type ca

  1. L-type Ca2+ channels in the heart: structure and regulation.

    PubMed

    Treinys, Rimantas; Jurevicius, Jonas

    2008-01-01

    This review analyzes the structure and regulation mechanisms of voltage-dependent L-type Ca(2+) channel in the heart. L-type Ca(2+) channels in the heart are composed of four different polypeptide subunits, and the pore-forming subunit alpha(1) is the most important part of the channel. In cardiac myocytes, Ca(2+) enter cell cytoplasm from extracellular space mainly through L-type Ca(2+) channels; these channels are very important system in heart Ca(2+) uptake regulation. L-type Ca(2+) channels are responsible for the activation of sarcoplasmic reticulum channels (RyR2) and force of muscle contraction generation in heart; hence, activity of the heart depends on L-type Ca(2+) channels. Phosphorylation of channel-forming subunits by different kinases is one of the most important ways to change the activity of L-type Ca(2+) channel. Additionally, the activity of L-type Ca(2+) channels depends on Ca(2+) concentration in cytoplasm. Ca(2+) current in cardiac cells can facilitate, and this process is regulated by phosphorylation of L-type Ca(2+) channels and intracellular Ca(2+) concentration. Disturbances in cellular Ca(2+) transport and regulation of L-type Ca(2+) channels are directly related to heart diseases, life quality, and life span.

  2. Pharmacological preconditioning by diazoxide downregulates cardiac L-type Ca2+ channels

    PubMed Central

    González, G; Zaldívar, D; Carrillo, ED; Hernández, A; García, MC; Sánchez, JA

    2010-01-01

    BACKGROUND AND PURPOSE Pharmacological preconditioning (PPC) with mitochondrial ATP-sensitive K+ (mitoKATP) channel openers such as diazoxide, leads to cardioprotection against ischaemia. However, effects on Ca2+ homeostasis during PPC, particularly changes in Ca2+ channel activity, are poorly understood. We investigated the effects of PPC on cardiac L-type Ca2+ channels. EXPERIMENTAL APPROACH PPC was induced in isolated hearts and enzymatically dissociated cardiomyocytes from adult rats by preincubation with diazoxide. We measured reactive oxygen species (ROS) production and Ca2+ signals associated with action potentials using fluorescent probes, and L-type currents using a whole-cell patch-clamp technique. Levels of the α1c subunit of L-type channels in the cellular membrane were measured by Western blot. KEY RESULTS PPC was accompanied by a 50% reduction in α1c subunit levels, and by a reversible fall in L-type current amplitude and Ca2+ transients. These effects were prevented by the ROS scavenger N-acetyl-L-cysteine (NAC), or by the mitoKATP channel blocker 5-hydroxydecanoate (5-HD). PPC signficantly reduced infarct size, an effect blocked by NAC and 5-HD. Nifedipine also conferred protection against infarction when applied during the reperfusion period. Downregulation of the α1c subunit and Ca2+ channel function were prevented in part by the protease inhibitor leupeptin. CONCLUSIONS AND IMPLICATIONS PPC downregulated the α1c subunit, possibly through ROS. Downregulation involved increased degradation of the Ca2+ channel, which in turn reduced Ca2+ influx, which may attenuate Ca2+ overload during reperfusion. PMID:20636393

  3. Coupling of SK channels, L-type Ca2+ channels, and ryanodine receptors in cardiomyocytes.

    PubMed

    Zhang, Xiao-Dong; Coulibaly, Zana A; Chen, Wei Chun; Ledford, Hannah A; Lee, Jeong Han; Sirish, Padmini; Dai, Gu; Jian, Zhong; Chuang, Frank; Brust-Mascher, Ingrid; Yamoah, Ebenezer N; Chen-Izu, Ye; Izu, Leighton T; Chiamvimonvat, Nipavan

    2018-03-16

    Small-conductance Ca 2+ -activated K + (SK) channels regulate the excitability of cardiomyocytes by integrating intracellular Ca 2+ and membrane potentials on a beat-to-beat basis. The inextricable interplay between activation of SK channels and Ca 2+ dynamics suggests the pathology of one begets another. Yet, the exact mechanistic underpinning for the activation of cardiac SK channels remains unaddressed. Here, we investigated the intracellular Ca 2+ microdomains necessary for SK channel activation. SK currents coupled with Ca 2+ influx via L-type Ca 2+ channels (LTCCs) continued to be elicited after application of caffeine, ryanodine or thapsigargin to deplete SR Ca 2+ store, suggesting that LTCCs provide the immediate Ca 2+ microdomain for the activation of SK channels in cardiomyocytes. Super-resolution imaging of SK2, Ca v 1.2 Ca 2+ channel, and ryanodine receptor 2 (RyR2) was performed to quantify the nearest neighbor distances (NND) and localized the three molecules within hundreds of nanometers. The distribution of NND between SK2 and RyR2 as well as SK2 and Ca v 1.2 was bimodal, suggesting a spatial relationship between the channels. The activation mechanism revealed by our study paved the way for the understanding of the roles of SK channels on the feedback mechanism to regulate the activities of LTCCs and RyR2 to influence local and global Ca 2+ signaling.

  4. Oestrogen directly inhibits the cardiovascular L-type Ca{sup 2+} channel Ca{sub v}1.2

    SciTech Connect

    Ullrich, Nina D.; Koschak, Alexandra; MacLeod, Kenneth T.

    2007-09-21

    Oestrogen can modify the contractile function of vascular smooth muscle and cardiomyocytes. The negative inotropic actions of oestrogen on the heart and coronary vasculature appear to be mediated by L-type Ca{sup 2+} channel (Ca{sub v}1.2) inhibition, but the underlying mechanisms remain elusive. We tested the hypothesis that oestrogen directly inhibits the cardiovascular L-type Ca{sup 2+} current, I {sub CaL}. The effect of oestrogen on I {sub CaL} was measured in Ca{sub v}1.2-transfected HEK-293 cells using the whole-cell patch-clamp technique. The current revealed typical activation and inactivation profiles of nifedipine- and cadmium-sensitive I {sub CaL}. Oestrogen (50 {mu}M) rapidly reduced Imore » {sub CaL} by 50% and shifted voltage-dependent activation and availability to more negative potentials. Furthermore, oestrogen blocked the Ca{sup 2+} channel in a rate-dependent way, exhibiting higher efficiency of block at higher stimulation frequencies. Our data suggest that oestrogen inhibits I {sub CaL} through direct interaction of the steroid with the channel protein.« less

  5. Molecular basis of proton block of L-type Ca2+ channels.

    PubMed

    Chen, X H; Bezprozvanny, I; Tsien, R W

    1996-11-01

    Hydrogen ions are important regulators of ion flux through voltage-gated Ca2+ channels but their site of action has been controversial. To identify molecular determinants of proton block of L-type Ca2+ channels, we combined site-directed mutagenesis and unitary current recordings from wild-type (WT) and mutant L-type Ca2+ channels expressed in Xenopus oocytes. WT channels in 150 mM K+ displayed two conductance states, deprotonated (140 pS) and protonated (45 pS), as found previously in native L-type Ca2+ channels. Proton block was altered in a unique fashion by mutation of each of the four P-region glutamates (EI-EIV) that form the locus of high affinity Ca2+ interaction. Glu(E)-->Gln(Q) substitution in either repeats I or III abolished the high-conductance state, as if the titration site had become permanently protonated. While the EIQ mutant displayed only an approximately 40 pS conductance, the EIIIQ mutant showed the approximately 40 pS conductance plus additional pH-sensitive transitions to an even lower conductance level. The EIVQ mutant exhibited the same deprotonated and protonated conductance states as WT, but with an accelerated rate of deprotonation. The EIIQ mutant was unusual in exhibiting three conductance states (approximately 145, 102, 50 pS, respectively). Occupancy of the low conductance state increased with external acidification, albeit much higher proton concentration was required than for WT. In contrast, the equilibrium between medium and high conductance levels was apparently pH-insensitive. We concluded that the protonation site in L-type Ca2+ channels lies within the pore and is formed by a combination of conserved P-region glutamates in repeats I, II, and III, acting in concert. EIV lies to the cytoplasmic side of the site but exerts an additional stabilizing influence on protonation, most likely via electrostatic interaction. These findings are likely to hold for all voltage-gated Ca2+ channels and provide a simple molecular explanation

  6. Role of L-type Ca2+ channel isoforms in the extinction of conditioned fear.

    PubMed

    Busquet, Perrine; Hetzenauer, Alfred; Sinnegger-Brauns, Martina J; Striessnig, Jörg; Singewald, Nicolas

    2008-05-01

    Dihydropyridine (DHP) L-type Ca(2+) channel (LTCC) antagonists, such as nifedipine, have been reported to impair the extinction of conditioned fear without interfering with its acquisition. Identification of the LTCC isoforms mediating this DHP effect is an essential basis to reveal their role as potential drug targets for the treatment of specific anxiety disorders. Ca(V)1.2 and Ca(V)1.3 are the predominant LTCCs in the mammalian brain. However, since no isoform-selective DHP blockers are available, their individual contribution to fear memory extinction is unknown. We used a novel mouse model expressing DHP-insensitive Ca(V)1.2 LTCCs (Ca(V)1.2DHP(-/-) mice) to address this question. In line with previous studies, wild-type (WT) mice treated with systemic nifedipine displayed markedly impaired fear extinction. This DHP effect was completely abolished in Ca(V)1.2DHP(-/-) mice, indicating that it is mediated by Ca(V)1.2, but not by Ca(V)1.3 LTCCs. Supporting this conclusion, Ca(V)1.3-deficient mice (Ca(V)1.3(-/-)) showed extinction identical to the respective WT mice. The inhibition of fear extinction was not observed after intracerebroventricular (i.c.v.) application of different doses of nifedipine, suggesting that this effect is secondary to inhibition of peripheral Ca(V)1.2 channels. The LTCC activator BayK, which lacks neurotoxic effects in Ca(V)1.2DHP(-/-) mice, did not influence the extinction time course. In summary, we demonstrate that LTCC signaling through the Ca(V)1.2 isoform of LTCCs interferes with fear memory extinction, presumably via a peripherally mediated mechanism. Activation of other LTCC isoforms (predominantly Ca(V)1.3) is not sufficient to accelerate extinction of conditioned fear in mice.

  7. Role of L-type Ca2+ channel isoforms in the extinction of conditioned fear

    PubMed Central

    Busquet, Perrine; Hetzenauer, Alfred; Sinnegger-Brauns, Martina J.; Striessnig, Jörg; Singewald, Nicolas

    2008-01-01

    Dihydropyridine (DHP) L-type Ca2+ channel (LTCC) antagonists, such as nifedipine, have been reported to impair the extinction of conditioned fear without interfering with its acquisition. Identification of the LTCC isoforms mediating this DHP effect is an essential basis to reveal their role as potential drug targets for the treatment of specific anxiety disorders. CaV1.2 and CaV1.3 are the predominant LTCCs in the mammalian brain. However, since no isoform-selective DHP blockers are available, their individual contribution to fear memory extinction is unknown. We used a novel mouse model expressing DHP-insensitive CaV1.2 LTCCs (CaV1.2DHP−/− mice) to address this question. In line with previous studies, wild-type (WT) mice treated with systemic nifedipine displayed markedly impaired fear extinction. This DHP effect was completely abolished in CaV1.2DHP−/− mice, indicating that it is mediated by CaV1.2, but not by CaV1.3 LTCCs. Supporting this conclusion, CaV1.3-deficient mice (CaV1.3−/−) showed extinction identical to the respective WT mice. The inhibition of fear extinction was not observed after intracerebroventricular (i.c.v.) application of different doses of nifedipine, suggesting that this effect is secondary to inhibition of peripheral CaV1.2 channels. The LTCC activator BayK, which lacks neurotoxic effects in CaV1.2DHP−/− mice, did not influence the extinction time course. In summary, we demonstrate that LTCC signaling through the CaV1.2 isoform of LTCCs interferes with fear memory extinction, presumably via a peripherally mediated mechanism. Activation of other LTCC isoforms (predominantly CaV1.3) is not sufficient to accelerate extinction of conditioned fear in mice. PMID:18441296

  8. Qiliqiangxin Affects L Type Ca2+ Current in the Normal and Hypertrophied Rat Heart

    PubMed Central

    Wei, Yidong; Liu, Xiaoyu; Hou, Lei; Che, Wenliang; The, Erlinda; Jhummon, Muktanand Vikash

    2012-01-01

    Qiliqiangxin capsule is newly developed Chinese patent drug and proved to be effective and safe for the treatment of patients with chronic heart failure. We compared the effects of different dose Qiliqiangxin on L type Ca2+ current (I Ca-L) between normal and hypertrophied myocytes. A total of 40 healthy Sprague—Dawley rats were used in the study. The rats were randomly divided into two groups (control group and hypertrophy group). Cardiac hypertrophy was induced by pressure overload produced by partial ligation of the abdominal aorta. The control group was the sham-operated group. After 1 month, cardiac ventricular myocytes were isolated from the hearts of rats. Ventricular myocytes were exposed to 10 and 50 μmol/L Qiliqiangxin, and whole cell patch-clamp technique was used to study the effects of Qiliqiangxin on I Ca-L. The current densities of I Ca-L were similar in control group (−12.70 ± 0.53 pA/pF, n = 12) and in hypertrophy group (−12.39 ± 0.62 pA/pF, n = 10). They were not statistically significant. 10 and 50 μmol/L Qiliqiangxin can decrease I Ca-L peak current 48.6%±16.8% and 59.0%±4.4% in control group. However, the peak current was only reduced 16.73%±8.03% by 50 μmol/L Qiliqiangxin in hypertrophied myocytes. The inhibited action of Qiliqiangxin on I Ca-L of hypertrophy group was lower than in control group. Qiliqiangxin affected L-type Ca2+ channel and blocked I Ca-L, as well as affected cardiac function finally. Qiliqiangxin has diphasic action that is either class IV antiarrhythmic agent or the agent of effect cardiac function. PMID:22536279

  9. Conservation of cardiac L-type Ca2+ channels and their regulation in Drosophila: A novel genetically-pliable channelopathic model.

    PubMed

    Limpitikul, Worawan B; Viswanathan, Meera C; O'Rourke, Brian; Yue, David T; Cammarato, Anthony

    2018-04-21

    Dysregulation of L-type Ca 2+ channels (LTCCs) underlies numerous cardiac pathologies. Understanding their modulation with high fidelity relies on investigating LTCCs in their native environment with intact interacting proteins. Such studies benefit from genetic manipulation of endogenous channels in cardiomyocytes, which often proves cumbersome in mammalian models. Drosophila melanogaster, however, offers a potentially efficient alternative as it possesses a relatively simple heart, is genetically pliable, and expresses well-conserved genes. Fluorescence in situ hybridization confirmed an abundance of Ca-α1D and Ca-α1T mRNA in fly myocardium, which encode subunits that specify hetero-oligomeric channels homologous to mammalian LTCCs and T-type Ca 2+ channels, respectively. Cardiac-specific knockdown of Ca-α1D via interfering RNA abolished cardiac contraction, suggesting Ca-α1D (i.e. A1D) represents the primary functioning Ca 2+ channel in Drosophila hearts. Moreover, we successfully isolated viable single cardiomyocytes and recorded Ca 2+ currents via patch clamping, a feat never before accomplished with the fly model. The profile of Ca 2+ currents recorded in individual cells when Ca 2+ channels were hypomorphic, absent, or under selective LTCC blockage by nifedipine, additionally confirmed the predominance of A1D current across all activation voltages. T-type current, activated at more negative voltages, was also detected. Lastly, A1D channels displayed Ca 2+ -dependent inactivation, a critical negative feedback mechanism of LTCCs, and the current through them was augmented by forskolin, an activator of the protein kinase A pathway. In sum, the Drosophila heart possesses a conserved compendium of Ca 2+ channels, suggesting that the fly may serve as a robust and effective platform for studying cardiac channelopathies. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Endogenous testosterone increases L-type Ca2+ channel expression in porcine coronary smooth muscle.

    PubMed

    Bowles, D K; Maddali, K K; Ganjam, V K; Rubin, L J; Tharp, D L; Turk, J R; Heaps, C L

    2004-11-01

    Evidence indicates that gender and sex hormonal status influence cardiovascular physiology and pathophysiology. We recently demonstrated increased L-type voltage-gated Ca2+ current (ICa,L) in coronary arterial smooth muscle (CASM) of male compared with female swine. The promoter region of the L-type voltage-gated Ca2+ channel (VGCC) (Cav1.2) gene contains a hormone response element that is activated by testosterone. Thus the purpose of the present study was to determine whether endogenous testosterone regulates CASM ICa,L through regulation of VGCC expression and activity. Sexually mature male and female Yucatan swine (7-8 mo; 35-45 kg) were obtained from the breeder. Males were left intact (IM, n=8), castrated (CM, n=8), or castrated with testosterone replacement (CMT, n=8; 10 mg/day Androgel). Females remained gonad intact (n=8). In right coronary arteries, both Cav1.2 mRNA and protein were greater in IM compared with intact females. Cav1.2 mRNA and protein were reduced in CM compared with IM and restored in CMT. In isolated CASM, both peak and steady-state ICa were reduced in CM compared with IM and restored in CMT. In males, a linear relationship was found between serum testosterone levels and ICa. In vitro, both testosterone and the nonaromatizable androgen, dihydrotestosterone, increased Cav1.2 expression. Furthermore, this effect was blocked by the androgen receptor antagonist cyproterone. We conclude that endogenous testosterone is a primary regulator of Cav1.2 expression and activity in coronary arteries of males.

  11. Decreased cardiac L-type Ca2+ channel activity induces hypertrophy and heart failure in mice

    PubMed Central

    Goonasekera, Sanjeewa A.; Hammer, Karin; Auger-Messier, Mannix; Bodi, Ilona; Chen, Xiongwen; Zhang, Hongyu; Reiken, Steven; Elrod, John W.; Correll, Robert N.; York, Allen J.; Sargent, Michelle A.; Hofmann, Franz; Moosmang, Sven; Marks, Andrew R.; Houser, Steven R.; Bers, Donald M.; Molkentin, Jeffery D.

    2011-01-01

    Antagonists of L-type Ca2+ channels (LTCCs) have been used to treat human cardiovascular diseases for decades. However, these inhibitors can have untoward effects in patients with heart failure, and their overall therapeutic profile remains nebulous given differential effects in the vasculature when compared with those in cardiomyocytes. To investigate this issue, we examined mice heterozygous for the gene encoding the pore-forming subunit of LTCC (calcium channel, voltage-dependent, L type, α1C subunit [Cacna1c mice; referred to herein as α1C–/+ mice]) and mice in which this gene was loxP targeted to achieve graded heart-specific gene deletion (termed herein α1C-loxP mice). Adult cardiomyocytes from the hearts of α1C–/+ mice at 10 weeks of age showed a decrease in LTCC current and a modest decrease in cardiac function, which we initially hypothesized would be cardioprotective. However, α1C–/+ mice subjected to pressure overload stimulation, isoproterenol infusion, and swimming showed greater cardiac hypertrophy, greater reductions in ventricular performance, and greater ventricular dilation than α1C+/+ controls. The same detrimental effects were observed in α1C-loxP animals with a cardiomyocyte-specific deletion of one allele. More severe reductions in α1C protein levels with combinatorial deleted alleles produced spontaneous cardiac hypertrophy before 3 months of age, with early adulthood lethality. Mechanistically, our data suggest that a reduction in LTCC current leads to neuroendocrine stress, with sensitized and leaky sarcoplasmic reticulum Ca2+ release as a compensatory mechanism to preserve contractility. This state results in calcineurin/nuclear factor of activated T cells signaling that promotes hypertrophy and disease. PMID:22133878

  12. Role of voltage-gated L-type Ca2+ channel isoforms for brain function.

    PubMed

    Striessnig, J; Koschak, A; Sinnegger-Brauns, M J; Hetzenauer, A; Nguyen, N K; Busquet, P; Pelster, G; Singewald, N

    2006-11-01

    Voltage-gated LTCCs (L-type Ca2+ channels) are established drug targets for the treatment of cardiovascular diseases. LTCCs are also expressed outside the cardiovascular system. In the brain, LTCCs control synaptic plasticity in neurons, and DHP (dihydropyridine) LTCC blockers such as nifedipine modulate brain function (such as fear memory extinction and depression-like behaviour). Voltage-sensitive Ca2+ channels Cav1 .2 and Cav1.3 are the predominant brain LTCCs. As DHPs and other classes of organic LTCC blockers inhibit both isoforms, their pharmacological distinction is impossible and their individual contributions to defined brain functions remain largely unknown. Here, we summarize our recent experiments with two genetically modified mouse strains, which we generated to explore the individual biophysical features of Cav1.2 and Cav1.3 LTCCs and to determine their relative contributions to various physiological peripheral and neuronal functions. The results described here also allow predictions about the pharmacotherapeutic potential of isoform-selective LTCC modulators.

  13. The inhibitory effect of BIM (I) on L-type Ca²⁺ channels in rat ventricular cells.

    PubMed

    Son, Youn Kyoung; Hong, Da Hye; Choi, Tae-Hoon; Choi, Seong Woo; Shin, Dong Hoon; Kim, Sung Joon; Jung, In Duk; Park, Yeong-Min; Jung, Won-Kyo; Kim, Dae-Joong; Choi, Il-Whan; Park, Won Sun

    2012-06-22

    We investigated the effect of a specific protein kinase C (PKC) inhibitor, bisindolylmaleimide I [BIM (I)], on L-type Ca(2+) channels in rat ventricular myocytes. BIM (I) alone inhibited the L-type Ca(2+) current in a concentration-dependent manner, with a K(d) value of 3.31 ± 0.25 μM, and a Hill coefficient of 2.34 ± 0.23. Inhibition was immediate after applying BIM (I) in the bath solution and then it partially washed out. The steady-state activation curve was not altered by applying 3μ M BIM (I), but the steady-state inactivation curve shifted to a more negative potential with a change in the slope factor. Other PKC inhibitors, PKC-IP and chelerythrine, showed no significant effects either on the L-type Ca(2+) current or on the inhibitory effect of BIM (I) on the L-type Ca(2+) current. The results suggest that the inhibitory effect of BIM (I) on the L-type Ca(2+) current is independent of the PKC pathway. Thus, our results should be considered in studies using BIM (I) to inhibit PKC activity and ion channel modulation. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Direct interaction of CaVβ with actin up-regulates L-type calcium currents in HL-1 cardiomyocytes.

    PubMed

    Stölting, Gabriel; de Oliveira, Regina Campos; Guzman, Raul E; Miranda-Laferte, Erick; Conrad, Rachel; Jordan, Nadine; Schmidt, Silke; Hendriks, Johnny; Gensch, Thomas; Hidalgo, Patricia

    2015-02-20

    Expression of the β-subunit (CaVβ) is required for normal function of cardiac L-type calcium channels, and its up-regulation is associated with heart failure. CaVβ binds to the α1 pore-forming subunit of L-type channels and augments calcium current density by facilitating channel opening and increasing the number of channels in the plasma membrane, by a poorly understood mechanism. Actin, a key component of the intracellular trafficking machinery, interacts with Src homology 3 domains in different proteins. Although CaVβ encompasses a highly conserved Src homology 3 domain, association with actin has not yet been explored. Here, using co-sedimentation assays and FRET experiments, we uncover a direct interaction between CaVβ and actin filaments. Consistently, single-molecule localization analysis reveals streaklike structures composed by CaVβ2 that distribute over several micrometers along actin filaments in HL-1 cardiomyocytes. Overexpression of CaVβ2-N3 in HL-1 cells induces an increase in L-type current without altering voltage-dependent activation, thus reflecting an increased number of channels in the plasma membrane. CaVβ mediated L-type up-regulation, and CaVβ-actin association is prevented by disruption of the actin cytoskeleton with cytochalasin D. Our study reveals for the first time an interacting partner of CaVβ that is directly involved in vesicular trafficking. We propose a model in which CaVβ promotes anterograde trafficking of the L-type channels by anchoring them to actin filaments in their itinerary to the plasma membrane. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Direct Interaction of CaVβ with Actin Up-regulates L-type Calcium Currents in HL-1 Cardiomyocytes*

    PubMed Central

    Stölting, Gabriel; de Oliveira, Regina Campos; Guzman, Raul E.; Miranda-Laferte, Erick; Conrad, Rachel; Jordan, Nadine; Schmidt, Silke; Hendriks, Johnny; Gensch, Thomas; Hidalgo, Patricia

    2015-01-01

    Expression of the β-subunit (CaVβ) is required for normal function of cardiac L-type calcium channels, and its up-regulation is associated with heart failure. CaVβ binds to the α1 pore-forming subunit of L-type channels and augments calcium current density by facilitating channel opening and increasing the number of channels in the plasma membrane, by a poorly understood mechanism. Actin, a key component of the intracellular trafficking machinery, interacts with Src homology 3 domains in different proteins. Although CaVβ encompasses a highly conserved Src homology 3 domain, association with actin has not yet been explored. Here, using co-sedimentation assays and FRET experiments, we uncover a direct interaction between CaVβ and actin filaments. Consistently, single-molecule localization analysis reveals streaklike structures composed by CaVβ2 that distribute over several micrometers along actin filaments in HL-1 cardiomyocytes. Overexpression of CaVβ2-N3 in HL-1 cells induces an increase in L-type current without altering voltage-dependent activation, thus reflecting an increased number of channels in the plasma membrane. CaVβ mediated L-type up-regulation, and CaVβ-actin association is prevented by disruption of the actin cytoskeleton with cytochalasin D. Our study reveals for the first time an interacting partner of CaVβ that is directly involved in vesicular trafficking. We propose a model in which CaVβ promotes anterograde trafficking of the L-type channels by anchoring them to actin filaments in their itinerary to the plasma membrane. PMID:25533460

  16. Estimates of the location of L-type Ca2+ channels in motoneurons of different sizes: a computational study.

    PubMed

    Grande, Giovanbattista; Bui, Tuan V; Rose, P Ken

    2007-06-01

    In the presence of monoamines, L-type Ca(2+) channels on the dendrites of motoneurons contribute to persistent inward currents (PICs) that can amplify synaptic inputs two- to sixfold. However, the exact location of the L-type Ca(2+) channels is controversial, and the importance of the location as a means of regulating the input-output properties of motoneurons is unknown. In this study, we used a computational strategy developed previously to estimate the dendritic location of the L-type Ca(2+) channels and test the hypothesis that the location of L-type Ca(2+) channels varies as a function of motoneuron size. Compartmental models were constructed based on dendritic trees of five motoneurons that ranged in size from small to large. These models were constrained by known differences in PIC activation reported for low- and high-conductance motoneurons and the relationship between somatic PIC threshold and the presence or absence of tonic excitatory or inhibitory synaptic activity. Our simulations suggest that L-type Ca(2+) channels are concentrated in hotspots whose distance from the soma increases with the size of the dendritic tree. Moving the hotspots away from these sites (e.g., using the hotspot locations from large motoneurons on intermediate-sized motoneurons) fails to replicate the shifts in PIC threshold that occur experimentally during tonic excitatory or inhibitory synaptic activity. In models equipped with a size-dependent distribution of L-type Ca(2+) channels, the amplification of synaptic current by PICs depends on motoneuron size and the location of the synaptic input on the dendritic tree.

  17. Physiological role of L-type Ca2+ channels in marginal cells in the stria vascularis of guinea pigs.

    PubMed

    Inui, Takaki; Mori, Yoshiaki; Watanabe, Masahito; Takamaki, Atsuko; Yamaji, Junko; Sohma, Yoshiro; Yoshida, Ryotaro; Takenaka, Hiroshi; Kubota, Takahiro

    2007-10-01

    Using immunohistochemical and electrophysiological methods, we investigated the role of L-type Ca(2+) channels in the regulation of the endocochlear potential (EP) of the endolymphatic surface cells (ESC) of the guinea pig stria vascularis. The following findings were made: (1) Administration of 30 microg/ml nifedipine via a vertebral artery significantly suppressed the transient asphyxia-induced decrease in the EP (TAID) and the transient asphyxia-induced increase in the Ca(2+), referred to as TAIICa, concentration in the endolymph ([Ca](e)). (2) The endolymphatic administration of 1 microg/ml nifedipine significantly inhibited the TAID as well as the TAIICa. The endolymphatic administration of nifedipine (0.001-10 microg/ml) inhibited the TAID in a dose-dependent manner. (3) The endolymphatic administration of (+)-Bay K8644, an L-type Ca(2+) channel closer, significantly inhibited the TAID, whereas (-)-Bay K8644, an L-type Ca(2+) channel opener, caused a large decrease in the EP from approximately +75 mV to approximately +20 mV at 10 min after the endolymphatic administration. (4) By means of immunohistochemistry, a positive staining reaction with L-type Ca(2+) channels was detected in the marginal cells of the stria vascularis. (5) Under the high [Ca](e) condition, we examined the mechanism of the TAIICa and hypothesized that the TAIICa might have been caused by the decrease in the EP through a shunt pathway in the ESC. (6) The administration of nifedipine to the endolymph significantly inhibited the Ba(2+)-induced decrease in the EP. These findings support the view that L-type Ca(2+) channels in the marginal cells regulate the EP, but not directly the TAIICa.

  18. Temperature-dependence of L-type Ca(2+) current in ventricular cardiomyocytes of the Alaska blackfish (Dallia pectoralis).

    PubMed

    Kubly, Kerry L; Stecyk, Jonathan A W

    2015-12-01

    To lend insight into the overwintering strategy of the Alaska blackfish (Dallia pectoralis), we acclimated fish to 15 or 5 °C and then utilized whole-cell patch clamp to characterize the effects of thermal acclimation and acute temperature change on the density and kinetics of ventricular L-type Ca(2+) current (I Ca). Peak I Ca density at 5 °C (-1.1 ± 0.1 pA pF(-1)) was 1/8th that at 15 °C (-8.8 ± 0.6 pA pF(-1)). However, alterations of the Ca(2+)- and voltage-dependent inactivation properties of L-type Ca(2+) channels partially compensated against the decrease. The time constant tau (τ) for the kinetics of inactivation of I Ca was ~4.5 times greater at 5 °C than at 15 °C, and the voltage for half-maximal inactivation was shifted from -23.3 ± 1.0 mV at 15 °C to -19.8 ± 1.2 mV at 5 °C. These modifications increase the open probability of the channel and culminate in an approximate doubling of the L-type Ca(2+) window current, which contributes to approximately 15% of the maximal Ca(2+) conductance at 5 °C. Consequently, the charge density of I Ca (Q Ca) and the total Ca(2+) transferred through the L-type Ca(2+) channels (Δ[Ca(2+)]) were not as severely reduced at 5 °C as compared to peak I Ca density. In combination, the results suggest that while the Alaska blackfish substantially down-regulates I Ca with acclimation to low temperature, there is sufficient compensation in the kinetics of the L-type Ca(2+) channel to support the level of cardiac performance required for the fish to remain active throughout the winter.

  19. Rock Tea extract (Jasonia glutinosa) relaxes rat aortic smooth muscle by inhibition of L-type Ca(2+) channels.

    PubMed

    Valero, Marta Sofía; Oliván-Viguera, Aida; Garrido, Irene; Langa, Elisa; Berzosa, César; López, Víctor; Gómez-Rincón, Carlota; Murillo, María Divina; Köhler, Ralf

    2015-12-01

    In traditional herbal medicine, Rock Tea (Jasonia glutinosa) is known for its prophylactic and therapeutic value in various disorders including arterial hypertension. However, the mechanism by which Rock Tea exerts blood pressure-lowering actions has not been elucidated yet. Our aim was to demonstrate vasorelaxing effects of Rock Tea extract and to reveal its possible action mechanism. Isometric myography was conducted on high-K+-precontracted rings from rat thoracic aorta and tested extracts at concentrations of 0.5-5 mg/ml. Whole-cell patch-clamp experiments were performed in rat aortic vascular smooth muscle cells (line A7r5) to determine blocking effects on L-type Ca(2+) channels. Rock Tea extract relaxed the aorta contracted by high [K+] concentration dependently with an EC50 of ≈2.4 mg/ml and produced ≈75 % relaxation at the highest concentration tested. The L-type Ca(2+) channel blocker, verapamil (10(-6) M), had similar effects. Rock Tea extract had no effect in nominally Ca(2+)-free high-K(+) buffer but significantly inhibited contractions to re-addition of Ca(2+). Rock Tea extract inhibited the contractions induced by the L-type Ca(2+) channel activator Bay K 8644 (10(-5) M) and by phenylephrine (10(-6) M). Rock Tea extract and Y-27632 (10(-6) M), Rho-kinase inhibitor, had similar effects and the respective effects were not additive. Patch-clamp experiments demonstrated that Rock Tea extract (2.5 mg/ml) virtually abolished L-type Ca(2+) currents in A7r5. We conclude that Rock Tea extract produced vasorelaxation of rat aorta and that this relaxant effect is mediated by inhibition of L-type Ca(2+) channels. Rock Tea extracts may be of phytomedicinal value for prevention and adjuvant treatment of hypertension and other cardiovascular diseases.

  20. OxLDL enhances L-type Ca2+ currents via lysophosphatidylcholine-induced mitochondrial reactive oxygen species (ROS) production.

    PubMed

    Fearon, Ian M

    2006-03-01

    To examine the mechanisms underlying oxidised LDL- (oxLDL)-induced alterations in Ca(2+) currents, an effect which underlies altered vascular contractility and cardiac myocyte function. Ca(2+) currents (I(Ca)) were recorded by whole-cell patch-clamp in HEK293 cells expressing L-type Ca(2+) channel alpha(1C) subunits or isolated rat ventricular myocytes. oxLDL (but not native LDL) significantly enhanced recombinant I(Ca), an effect mimicked by 1 microM lysophosphatidylcholine (LPC). LPC failed to enhance I(Ca) either in mitochondrial electron transport chain-depleted rho(0) cells, or in the presence of rotenone (1 microM), or MPP(+) (10 microM). The LPC response was similarly ablated by ascorbate (200 microM) or TROLOX (500 microM) and by the mitochondria-targeted antioxidant, MitoQ (250 nM). In myocytes, enhancement of I(Ca) due to LPC was similarly abrogated with rotenone and MitoQ. These data suggest that LPC enhanced recombinant Ca(2+) currents due to increased mitochondrial ROS production. In support with this, LPC enhanced fluorescence in HEK293 cells and cardiac myocytes loaded with a ROS-sensitive mitochondrial dye, reduced mitotracker red. LPC up-regulates L-type Ca(2+) currents due to altered mitochondrial ROS production, an effect which mediates the response of the native I(Ca) in cardiac myocytes to oxLDL.

  1. Role of L-Type Ca[superscript 2+] Channel Isoforms in the Extinction of Conditioned Fear

    ERIC Educational Resources Information Center

    Busquet, Perrine; Hetzenauer, Alfred; Sinnegger-Brauns, Martina J.; Striessnig, Jorg; Singewald, Nicolas

    2008-01-01

    Dihydropyridine (DHP) L-type Ca[superscript 2+] channel (LTCC) antagonists, such as nifedipine, have been reported to impair the extinction of conditioned fear without interfering with its acquisition. Identification of the LTCC isoforms mediating this DHP effect is an essential basis to reveal their role as potential drug targets for the…

  2. Temperature-dependence of L-type Ca2+ current in ventricular cardiomyocytes of the Alaska blackfish (Dallia pectoralis)

    PubMed Central

    Kubly, Kerry L.; Stecyk, Jonathan A.W.

    2016-01-01

    Summary To lend insight into the overwintering strategy of the Alaska blackfish (Dallia pectoralis), we acclimated fish to 15°C or 5°C and then utilized whole-cell patch-clamp to characterize the effects of thermal acclimation and acute temperature change on the density and kinetics of ventricular L-type Ca2+ current (ICa). Peak ICa density at 5°C (−1.1± 0.1 pA pF−1) was 1/8th that at 15°C (−8.8 ± 0.6 pA pF−1). However, alterations of the Ca2+- and voltage-dependent inactivation properties of L-type Ca2+ channels partially compensated against the decrease. The time constant tau (τ) for the kinetics of inactivation of ICa was ~4.5-times greater at 5°C than at 15°C, and the voltage for half-maximal inactivation was shifted from −23.3 ± 1.0 mV at 15°C to - 19.8 ± 1.2 mV at 5°C. These modifications increase the open probability of the channel and culminated in an approximate doubling of the L-type Ca2+ window current, which contributed to approximately 15% of the maximal Ca2+ conductance at 5°C. Consequently, the charge density of ICa (QCa) and the total Ca2+ transferred through the L-type Ca channels (Δ[Ca2+]) were not as severely reduced at 5°C as compared to peak ICa density. In combination, the results suggest that while the Alaska blackfish substantially down-regulates ICa with acclimation to low temperature, there is sufficient compensation in the kinetics of the L-type Ca2+ channel to support the level of cardiac performance required for the fish to remain active throughout the winter. PMID:26439127

  3. CaV1.3 L-type Ca2+ channels modulate depression-like behaviour in mice independent of deaf phenotype.

    PubMed

    Busquet, Perrine; Nguyen, Ngoc Khoi; Schmid, Eduard; Tanimoto, Naoyuki; Seeliger, Mathias W; Ben-Yosef, Tamar; Mizuno, Fengxia; Akopian, Abram; Striessnig, Jörg; Singewald, Nicolas

    2010-05-01

    Mounting evidence suggests that voltage-gated L-type Ca2+ channels can modulate affective behaviour. We therefore explored the role of CaV1.3 L-type Ca2+ channels in depression- and anxiety-like behaviours using CaV1.3-deficient mice (CaV1.3-/-). We showed that CaV1.3-/- mice displayed less immobility in the forced swim test as well as in the tail suspension test, indicating an antidepressant-like phenotype. Locomotor activity in the home cage or a novel open-field test was not influenced. In the elevated plus maze (EPM), CaV1.3-/- mice entered the open arms more frequently and spent more time there indicating an anxiolytic-like phenotype which was, however, not supported in the stress-induced hyperthermia test. By performing parallel experiments in Claudin 14 knockout mice (Cldn14-/-), which like CaV1.3-/- mice are congenitally deaf, an influence of deafness on the antidepressant-like phenotype could be ruled out. On the other hand, a similar EPM behaviour indicative of an anxiolytic phenotype was also found in the Cldn14-/- animals. Using electroretinography and visual behavioural tasks we demonstrated that at least in mice, CaV1.3 channels do not significantly contribute to visual function. However, marked morphological changes were revealed in synaptic ribbons in the outer plexiform layer of CaV1.3-/- retinas by immunohistochemistry suggesting a possible role of this channel type in structural plasticity at the ribbon synapse. Taken together, our findings indicate that CaV1.3 L-type Ca2+ channels modulate depression-like behaviour but are not essential for visual function. The findings raise the possibility that selective modulation of CaV1.3 channels could be a promising new therapeutic concept for the treatment of mood disorders.

  4. Reduced density and altered regulation of rat atrial L-type Ca2+ current in heart failure.

    PubMed

    Bond, Richard C; Bryant, Simon M; Watson, Judy J; Hancox, Jules C; Orchard, Clive H; James, Andrew F

    2017-03-01

    Constitutive regulation by PKA has recently been shown to contribute to L-type Ca 2+ current ( I CaL ) at the ventricular t-tubule in heart failure. Conversely, reduction in constitutive regulation by PKA has been proposed to underlie the downregulation of atrial I CaL in heart failure. The hypothesis that downregulation of atrial I CaL in heart failure involves reduced channel phosphorylation was examined. Anesthetized adult male Wistar rats underwent surgical coronary artery ligation (CAL, N =10) or equivalent sham-operation (Sham, N =12). Left atrial myocytes were isolated ~18 wk postsurgery and whole cell currents recorded (holding potential=-80 mV). I CaL activated by depolarizing pulses to voltages from -40 to +50 mV were normalized to cell capacitance and current density-voltage relations plotted. CAL cell capacitances were ~1.67-fold greater than Sham ( P ≤ 0.0001). Maximal I CaL conductance ( G max ) was downregulated more than 2-fold in CAL vs. Sham myocytes ( P < 0.0001). Norepinephrine (1 μmol/l) increased G max >50% more effectively in CAL than in Sham so that differences in I CaL density were abolished. Differences between CAL and Sham G max were not abolished by calyculin A (100 nmol/l), suggesting that increased protein dephosphorylation did not account for I CaL downregulation. Treatment with either H-89 (10 μmol/l) or AIP (5 μmol/l) had no effect on basal currents in Sham or CAL myocytes, indicating that, in contrast to ventricular myocytes, neither PKA nor CaMKII regulated basal I CaL Expression of the L-type α 1C -subunit, protein phosphatases 1 and 2A, and inhibitor-1 proteins was unchanged. In conclusion, reduction in PKA-dependent regulation did not contribute to downregulation of atrial I CaL in heart failure. NEW & NOTEWORTHY Whole cell recording of L-type Ca 2+ currents in atrial myocytes from rat hearts subjected to coronary artery ligation compared with those from sham-operated controls reveals marked reduction in current density

  5. Polycystin-1 Is a Cardiomyocyte Mechanosensor That Governs L-Type Ca2+ Channel Protein Stability.

    PubMed

    Pedrozo, Zully; Criollo, Alfredo; Battiprolu, Pavan K; Morales, Cyndi R; Contreras-Ferrat, Ariel; Fernández, Carolina; Jiang, Nan; Luo, Xiang; Caplan, Michael J; Somlo, Stefan; Rothermel, Beverly A; Gillette, Thomas G; Lavandero, Sergio; Hill, Joseph A

    2015-06-16

    L-type calcium channel activity is critical to afterload-induced hypertrophic growth of the heart. However, the mechanisms governing mechanical stress-induced activation of L-type calcium channel activity are obscure. Polycystin-1 (PC-1) is a G protein-coupled receptor-like protein that functions as a mechanosensor in a variety of cell types and is present in cardiomyocytes. We subjected neonatal rat ventricular myocytes to mechanical stretch by exposing them to hypo-osmotic medium or cyclic mechanical stretch, triggering cell growth in a manner dependent on L-type calcium channel activity. RNAi-dependent knockdown of PC-1 blocked this hypertrophy. Overexpression of a C-terminal fragment of PC-1 was sufficient to trigger neonatal rat ventricular myocyte hypertrophy. Exposing neonatal rat ventricular myocytes to hypo-osmotic medium resulted in an increase in α1C protein levels, a response that was prevented by PC-1 knockdown. MG132, a proteasomal inhibitor, rescued PC-1 knockdown-dependent declines in α1C protein. To test this in vivo, we engineered mice harboring conditional silencing of PC-1 selectively in cardiomyocytes (PC-1 knockout) and subjected them to mechanical stress in vivo (transverse aortic constriction). At baseline, PC-1 knockout mice manifested decreased cardiac function relative to littermate controls, and α1C L-type calcium channel protein levels were significantly lower in PC-1 knockout hearts. Whereas control mice manifested robust transverse aortic constriction-induced increases in cardiac mass, PC-1 knockout mice showed no significant growth. Likewise, transverse aortic constriction-elicited increases in hypertrophic markers and interstitial fibrosis were blunted in the knockout animals PC-1 is a cardiomyocyte mechanosensor that is required for cardiac hypertrophy through a mechanism that involves stabilization of α1C protein. © 2015 American Heart Association, Inc.

  6. Methylene blue counteracts H2S toxicity-induced cardiac depression by restoring L-type Ca channel activity

    PubMed Central

    Zhang, Xue-Qian; Sonobe, Takashi; Song, Jianliang; Rannals, Matthew D.; Wang, JuFang; Tubbs, Nicole; Cheung, Joseph Y.; Haouzi, Philippe

    2016-01-01

    We have previously reported that methylene blue (MB) can counteract hydrogen sulfide (H2S) intoxication-induced circulatory failure. Because of the multifarious effects of high concentrations of H2S on cardiac function, as well as the numerous properties of MB, the nature of this interaction, if any, remains uncertain. The aim of this study was to clarify 1) the effects of MB on H2S-induced cardiac toxicity and 2) whether L-type Ca2+ channels, one of the targets of H2S, could transduce some of the counteracting effects of MB. In sedated rats, H2S infused at a rate that would be lethal within 5 min (24 μM·kg−1·min−1), produced a rapid fall in left ventricle ejection fraction, determined by echocardiography, leading to a pulseless electrical activity. Blood concentrations of gaseous H2S reached 7.09 ± 3.53 μM when cardiac contractility started to decrease. Two to three injections of MB (4 mg/kg) transiently restored cardiac contractility, blood pressure, and V̇o2, allowing the animals to stay alive until the end of H2S infusion. MB also delayed PEA by several minutes following H2S-induced coma and shock in unsedated rats. Applying a solution containing lethal levels of H2S (100 μM) on isolated mouse cardiomyocytes significantly reduced cell contractility, intracellular calcium concentration ([Ca2+]i) transient amplitudes, and L-type Ca2+ currents (ICa) within 3 min of exposure. MB (20 mg/l) restored the cardiomyocyte function, ([Ca2+]i) transient, and ICa. The present results offer a new approach for counteracting H2S toxicity and potentially other conditions associated with acute inhibition of L-type Ca2+ channels. PMID:26962024

  7. Modeling regulation of cardiac KATP and L-type Ca2+ currents by ATP, ADP, and Mg2+.

    PubMed

    Michailova, Anushka; Saucerman, Jeffrey; Belik, Mary Ellen; McCulloch, Andrew D

    2005-03-01

    Changes in cytosolic free Mg(2+) and adenosine nucleotide phosphates affect cardiac excitability and contractility. To investigate how modulation by Mg(2+), ATP, and ADP of K(ATP) and L-type Ca(2+) channels influences excitation-contraction coupling, we incorporated equations for intracellular ATP and MgADP regulation of the K(ATP) current and MgATP regulation of the L-type Ca(2+) current in an ionic-metabolic model of the canine ventricular myocyte. The new model: 1), quantitatively reproduces a dose-response relationship for the effects of changes in ATP on K(ATP) current, 2), simulates effects of ADP in modulating ATP sensitivity of K(ATP) channel, 3), predicts activation of Ca(2+) current during rapid increase in MgATP, and 4), demonstrates that decreased ATP/ADP ratio with normal total Mg(2+) or increased free Mg(2+) with normal ATP and ADP activate K(ATP) current, shorten action potential, and alter ionic currents and intracellular Ca(2+) signals. The model predictions are in agreement with experimental data measured under normal and a variety of pathological conditions.

  8. Modeling regulation of cardiac KATP and L-type Ca2+ currents by ATP, ADP, and Mg2+

    NASA Technical Reports Server (NTRS)

    Michailova, Anushka; Saucerman, Jeffrey; Belik, Mary Ellen; McCulloch, Andrew D.

    2005-01-01

    Changes in cytosolic free Mg(2+) and adenosine nucleotide phosphates affect cardiac excitability and contractility. To investigate how modulation by Mg(2+), ATP, and ADP of K(ATP) and L-type Ca(2+) channels influences excitation-contraction coupling, we incorporated equations for intracellular ATP and MgADP regulation of the K(ATP) current and MgATP regulation of the L-type Ca(2+) current in an ionic-metabolic model of the canine ventricular myocyte. The new model: 1), quantitatively reproduces a dose-response relationship for the effects of changes in ATP on K(ATP) current, 2), simulates effects of ADP in modulating ATP sensitivity of K(ATP) channel, 3), predicts activation of Ca(2+) current during rapid increase in MgATP, and 4), demonstrates that decreased ATP/ADP ratio with normal total Mg(2+) or increased free Mg(2+) with normal ATP and ADP activate K(ATP) current, shorten action potential, and alter ionic currents and intracellular Ca(2+) signals. The model predictions are in agreement with experimental data measured under normal and a variety of pathological conditions.

  9. Estrogen attenuates glutamate-induced cell death by inhibiting Ca2+ influx through L-type voltage-gated Ca2+ channels

    PubMed Central

    Sribnick, Eric A.; Del Re, Angelo M.; Ray, Swapan K.; Woodward, John J.; Banik, Naren L.

    2009-01-01

    Estrogen-mediated neuroprotection is observed in neurodegenerative disease and neurotrauama models; however, determining a mechanism for these effects has been difficult. We propose that estrogen may limit cell death in the nervous system tissue by inhibiting increases in intracellular free Ca2+. Here, we present data using VSC 4.1 cell line, a ventral spinal motoneuron and neuroblastoma hybrid cell line. Treatment with 1 mM glutamate for 24 h induced apoptosis. When cells were pre-treated with 100 nM 17β-estradiol (estrogen) for 1 h and then co-treated with glutamate, apoptotic death was significantly attenuated. Estrogen also prevented glutamate-mediated changes in resting membrane potential and membrane capacitance. Treatment with either 17α-estradiol or cell impermeable estrogen did not mimic the findings seen with estrogen. Glutamate treatment significantly increased both intracellular free Ca2+ and the activities of downstream proteases such as calpain and caspase-3. Estrogen attenuated both the increases in intracellular free Ca2+ and protease activities. In order to determine the pathway responsible for estrogen-mediated inhibition of these increases in intracellular free Ca2+, cells were treated with several Ca2+ entry inhibitors, but only the L-type Ca2+ channel blocker nifedipine demonstrated cytoprotective effects comparable to estrogen. To expand these findings, cells were treated with the L-type Ca2+ channel agonist FPL 64176, which increased both cell death and intracellular free Ca2+, and estrogen inhibited both effects. From these observations, we conclude that estrogen limits glutamate-induced cell death in VSC 4.1 cells through effects on L-type Ca2+ channels, inhibiting Ca2+ influx as well as activation of the pro-apoptotic proteases calpain and caspase-3. PMID:19389388

  10. A dynamic alpha-beta inter-subunit agonist signaling complex is a novel feedback mechanism for regulating L-type Ca2+ channel opening.

    PubMed

    Zhang, Rong; Dzhura, Igor; Grueter, Chad E; Thiel, William; Colbran, Roger J; Anderson, Mark E

    2005-09-01

    L-type Ca2+ channels are macromolecular protein complexes in neurons and myocytes that open in response to cell membrane depolarization to supply Ca2+ for regulating gene transcription and vesicle secretion and triggering cell contraction. L-type Ca2+ channels include a pore-forming alpha and an auxiliary beta subunit, and alpha subunit openings are regulated by cellular Ca2+ through a mechanism involving the Ca2+-sensing protein calmodulin (CaM) and CaM binding motifs in the alpha subunit cytoplasmic C terminus. Here we show that these CaM binding motifs are "auto-agonists" that increase alpha subunit openings by binding the beta subunit. The CaM binding domains are necessary and sufficient for the alpha subunit C terminus to bind the beta subunit in vitro, and excess CaM blocks this interaction. Addition of CaM binding domains to native cardiac L-type Ca2+ channels in excised cell membrane patches increases openings, and this agonist effect is prevented by excess CaM. Recombinant LTCC openings are also increased by exogenous CaM binding domains by a mechanism requiring the beta subunit, and excess CaM blocks this effect. Thus, the bifunctional ability of the alpha subunit CaM binding motifs to competitively associate with the beta subunit or CaM provides a novel paradigm for feedback control of cellular Ca2+ entry.

  11. Tetrodotoxin Blockade on Canine Cardiac L-Type Ca2+ Channels Depends on pH and Redox Potential

    PubMed Central

    Hegyi, Bence; Komáromi, István; Kistamás, Kornél; Ruzsnavszky, Ferenc; Váczi, Krisztina; Horváth, Balázs; Magyar, János; Bányász, Tamás; Nánási, Péter P.; Szentandrássy, Norbert

    2013-01-01

    Tetrodotoxin (TTX) is believed to be one of the most selective inhibitors of voltage-gated fast Na+ channels in excitable tissues. Recently, however, TTX has been shown to block L-type Ca2+ current (ICa) in canine cardiac cells. In the present study, the TTX-sensitivity of ICa was studied in isolated canine ventricular myocytes as a function of (1) channel phosphorylation, (2) extracellular pH and (3) the redox potential of the bathing medium using the whole cell voltage clamp technique. Fifty-five micromoles of TTX (IC50 value obtained under physiological conditions) caused 60% ± 2% inhibition of ICa in acidic (pH = 6.4), while only a 26% ± 2% block in alkaline (pH = 8.4) milieu. Similarly, the same concentration of TTX induced 62% ± 6% suppression of ICa in a reductant milieu (containing glutathione + ascorbic acid + dithiothreitol, 1 mM each), in contrast to the 31% ± 3% blockade obtained in the presence of a strong oxidant (100 μM H2O2). Phosphorylation of the channel protein (induced by 3 μM forskolin) failed to modify the inhibiting potency of TTX; an IC50 value of 50 ± 4 μM was found in forskolin. The results are in a good accordance with the predictions of our model, indicating that TTX binds, in fact, to the selectivity filter of cardiac L-type Ca channels. PMID:23771047

  12. Polycystin-1 is a Cardiomyocyte Mechanosensor That Governs L-type Ca2+ Channel Protein Stability

    PubMed Central

    Pedrozo, Zully; Criollo, Alfredo; Battiprolu, Pavan K.; Morales, Cyndi R.; Contreras, Ariel; Fernández, Carolina; Jiang, Nan; Luo, Xiang; Caplan, Michael J.; Somlo, Stefan; Rothermel, Beverly A.; Gillette, Thomas G.; Lavandero, Sergio; Hill, Joseph A.

    2015-01-01

    Background L-type calcium channel (LTCC) activity is critical to afterload-induced hypertrophic growth of the heart. However, mechanisms governing mechanical stress-induced activation of LTCC activity are obscure. Polycystin-1 (PC-1) is a G-protein-coupled receptor-like protein that functions as a mechanosensor in a variety of cell types and is present in cardiomyocytes. Methods and Results We subjected neonatal rat ventricular myocytes (NRVMs) to mechanical stretch by exposing them to hypo-osmotic (HS) medium or cyclic mechanical stretch, triggering cell growth in a manner dependent on LTCC activity. RNAi-dependent knockdown of PC-1 blocked this hypertrophy. Over-expression of a C-terminal fragment of PC-1 was sufficient to trigger NRVM hypertrophy. Exposing NRVMs to HS medium resulted in an increase in α1C protein levels, a response that was prevented by PC-1 knockdown. MG132, a proteasomal inhibitor, rescued PC-1 knockdown-dependent declines in α1C protein. To test this in vivo, we engineered mice harboring conditional silencing of PC-1 selectively in cardiomyocytes (PC-1 KO) and subjected them to mechanical stress in vivo (transverse aortic constriction, TAC). At baseline, PC-1 KO mice manifested decreased cardiac function relative to littermate controls, and α1C LTCC protein levels were significantly lower in PC-1 KO hearts. Whereas control mice manifested robust TAC-induced increases in cardiac mass, PC-1 KO mice showed no significant growth. Likewise, TAC-elicited increases in hypertrophic markers and interstitial fibrosis were blunted in the knockout animals Conclusions PC-1 is a cardiomyocyte mechanosensor and is required for cardiac hypertrophy through a mechanism that involves stabilization of α1C protein. PMID:25888683

  13. Inhibitory effects of purified antibody against α-1 repeat (117-137) on Na(+)-Ca(2+) exchange and L-type Ca(2+) currents in rat cardiomyocytes.

    PubMed

    Feng, Qi-Long; Wu, Dong-Mei; Cui, Xiang-Li; Zhao, Hua-Chen; Lin, Yuan-Yuan; Zhao, Lu-Ying; Wu, Bo-Wei

    2010-10-25

    Considering that α-1 repeat region may be involved in the ion binding and translocation of Na(+)-Ca(2+) exchanger (NCX), it is possible that the antibodies against NCX α-1 repeat may have a crucial action on NCX activity. The aim of the present study is to investigate the effect of antibody against α-1 repeat (117-137), designated as α-1(117-137), on NCX activity. The antibody against the synthesized α-1(117-137) was prepared and affinity-purified. Whole-cell patch clamp technique was used to study the change of Na(+)-Ca(2+) exchange current (I(Na/Ca)) in adult rat cardiomyocytes. To evaluate the functional specificity of this antibody, its effects on L-type Ca(2+) current (I(Ca,L)), voltage-gated Na(+) current (I(Na)) and delayed rectifier K(+) current (I(K)) were also observed. The amino acid sequences of α-1(117-137) in NCX and residues 1 076-1 096 within L-type Ca(2+) channel were compared using EMBOSS Pairwise Alignment Algorithms. The results showed that outward and inward I(Na/Ca) were decreased by the antibody against α-1(117-137) dose-dependently in the concentration range from 10 to 160 nmol/L, with IC(50) values of 18.9 nmol/L and 22.4 nmol/L, respectively. Meanwhile, the antibody also decreased I(Ca,L) in a concentration-dependent manner with IC(50) of 22.7 nmol/L. No obvious effects of the antibody on I(Na) and I(K) were observed. Moreover, comparison of the amino acid sequences showed there was 23.8% sequence similarity between NCX α-1(117-137) and residues 1 076-1 096 within L-type Ca(2+) channel. These results suggest that antibody against α-1(117-137) is a blocking antibody to NCX and can also decrease I(Ca,L) in a concentration-dependent manner, while it does not have obvious effects on I(Na) and I(K).

  14. Contractile dysfunctions in ATP-dependent K+ channel-deficient mouse muscle during fatigue involve excessive depolarization and Ca2+ influx through L-type Ca2+ channels.

    PubMed

    Cifelli, Carlo; Boudreault, Louise; Gong, Bing; Bercier, Jean-Philippe; Renaud, Jean-Marc

    2008-10-01

    Muscles deficient in ATP-dependent potassium (KATP) channels develop contractile dysfunctions during fatigue that may explain their apparently faster rate of fatigue compared with wild-type muscles. The objectives of this study were to determine: (1) whether the contractile dysfunctions, namely unstimulated force and depressed force recovery, result from excessive membrane depolarization and Ca2+ influx through L-type Ca2+ channels; and (2) whether reducing the magnitude of these two contractile dysfunctions reduces the rate of fatigue in KATP channel-deficient muscles. To reduce Ca2+ influx, we lowered the extracellular Ca2+ concentration ([Ca2+]o) from 2.4 to 0.6 mM or added 1 microM verapamil, an L-type Ca2+ channel blocker. Flexor digitorum brevis (FDB) muscles deficient in KATP channels were obtained by exposing wild-type muscles to 10 microM glibenclamide or by using FDB from Kir6.2-/- mice. Fatigue was elicited with one contraction per second for 3 min at 37 degrees C. In wild-type FDB, lowered [Ca2+]o or verapamil did not affect the decrease in peak tetanic force and unstimulated force during fatigue and force recovery following fatigue. In KATP channel-deficient FDB, lowered [Ca2+]o or verapamil slowed down the decrease in peak tetanic force recovery, reduced unstimulated force and improved force recovery. In Kir6.2-/- FDB, the rate of fatigue became slower than in wild-type FDB in the presence of verapamil. The cell membrane depolarized from -83 to -57 mV in normal wild-type FDB. The depolarizations in some glibenclamide-exposed fibres were similar to those of normal FDB, while in other fibres the cell membrane depolarized to -31 mV in 80 s, which was also the time when these fibres supercontracted. It is concluded that: (1) KATP channels are crucial in preventing excessive membrane depolarization and Ca2+ influx through L-type Ca2+ channels; and (2) they contribute to the decrease in force during fatigue.

  15. Voltage inactivation of Ca2+ entry and secretion associated with N- and P/Q-type but not L-type Ca2+ channels of bovine chromaffin cells

    PubMed Central

    Villarroya, Mercedes; Olivares, Román; Ruíz, Ana; Cano-Abad, María F; de Pascual, Ricardo; Lomax, Richard B; López, Manuela G; Mayorgas, Inés; Gandía, Luis; García, Antonio G

    1999-01-01

    In this study we pose the question of why the bovine adrenal medullary chromaffin cell needs various subtypes (L, N, P, Q) of the neuronal high-voltage activated Ca2+ channels to control a given physiological function, i.e. the exocytotic release of catecholamines. One plausible hypothesis is that Ca2+ channel subtypes undergo different patterns of inactivation during cell depolarization. The net Ca2+ uptake (measured using 45Ca2+) into hyperpolarized cells (bathed in a nominally Ca2+-free solution containing 1·2 mM K+) after application of a Ca2+ pulse (5 s exposure to 100 mM K+ and 2 mM Ca2+), amounted to 0·65 ± 0·02 fmol cell−1; in depolarized cells (bathed in nominally Ca2+-free solution containing 100 mM K+) the net Ca2+ uptake was 0·16 ± 0·01 fmol cell−1. This was paralleled by a dramatic reduction of the increase in the cytosolic Ca2+ concentration, [Ca2+]i, caused by Ca2+ pulses applied to fura-2-loaded single cells, from 1181 ± 104 nM in hyperpolarized cells to 115 ± 9 nM in depolarized cells. A similar decrease was observed when studying catecholamine release. Secretion was decreased when K+ concentration was increased from 1·2 to 100 mM; the Ca2+ pulse caused, when comparing the extreme conditions, the secretion of 807 ± 35 nA of catecholamines in hyperpolarized cells and 220 ± 19 nA in depolarized cells. The inactivation by depolarization of Ca2+ entry and secretion occluded the blocking effects of combined ω-conotoxin GVIA (1 μM) and ω-agatoxin IVA (2 μM), thus suggesting that depolarization caused a selective inactivation of the N- and P/Q-type Ca2+ channels. This was strengthened by two additional findings: (i) nifedipine (3 μM), an L-type Ca2+ channel blocker, suppressed the fraction of Ca2+ entry (24 %) and secretion (27 %) left unblocked by depolarization; (ii) FPL64176 (3 μM), an L-type Ca2+ channel ‘activator’, dramatically enhanced the entry of Ca2+ and the secretory response in depolarized cells. In voltage

  16. Interactions between N and C termini of α1C subunit regulate inactivation of CaV1.2 L-type Ca2+ channel

    PubMed Central

    Benmocha Guggenheimer, Adva; Almagor, Lior; Tsemakhovich, Vladimir; Tripathy, Debi Ranjan; Hirsch, Joel A; Dascal, Nathan

    2016-01-01

    The modulation and regulation of voltage-gated Ca2+ channels is affected by the pore-forming segments, the cytosolic parts of the channel, and interacting intracellular proteins. In this study we demonstrate a direct physical interaction between the N terminus (NT) and C terminus (CT) of the main subunit of the L-type Ca2+ channel CaV1.2, α1C, and explore the importance of this interaction for the regulation of the channel. We used biochemistry to measure the strength of the interaction and to map the location of the interaction sites, and electrophysiology to investigate the functional impact of the interaction. We show that the full-length NT (amino acids 1-154) and the proximal (close to the plasma membrane) part of the CT, pCT (amino acids 1508-1669) interact with sub-micromolar to low-micromolar affinity. Calmodulin (CaM) is not essential for the binding. The results further suggest that the NT-CT interaction regulates the channel's inactivation, and that Ca2+, presumably through binding to calmodulin (CaM), reduces the strength of NT-CT interaction. We propose a molecular mechanism in which NT and CT of the channel serve as levers whose movements regulate inactivation by promoting changes in the transmembrane core of the channel via S1 (NT) or S6 (pCT) segments of domains I and IV, accordingly, and not as a kind of pore blocker. We hypothesize that Ca2+-CaM-induced changes in NT-CT interaction may, in part, underlie the acceleration of CaV1.2 inactivation induced by Ca2+ entry into the cell. PMID:26577286

  17. The L-Type Voltage-Gated Calcium Channel Ca [subscript V] 1.2 Mediates Fear Extinction and Modulates Synaptic Tone in the Lateral Amygdala

    ERIC Educational Resources Information Center

    Temme, Stephanie J.; Murphy, Geoffrey G.

    2017-01-01

    L-type voltage-gated calcium channels (LVGCCs) have been implicated in both the formation and the reduction of fear through Pavlovian fear conditioning and extinction. Despite the implication of LVGCCs in fear learning and extinction, studies of the individual LVGCC subtypes, Ca[subscript V]1.2 and Ca[subscript V] 1.3, using transgenic mice have…

  18. L-type Ca2+ channel blockade with antihypertensive medication disrupts VTA synaptic plasticity and drug-associated contextual memory

    PubMed Central

    Degoulet, Mickael; Stelly, Claire E.; Ahn, Kee-Chan; Morikawa, Hitoshi

    2015-01-01

    Drug addiction is driven, in part, by powerful and enduring memories of sensory cues associated with drug intake. As such, relapse to drug use during abstinence is frequently triggered by an encounter with drug-associated cues, including the drug itself. L-type Ca2+ channels (LTCCs) are known to regulate different forms of synaptic plasticity, the major neural substrate for learning and memory, in various brain areas. Long-term potentiation (LTP) of NMDA receptor (NMDAR)-mediated glutamatergic transmission in the ventral tegmental area (VTA) may contribute to the increased motivational valence of drug-associated cues triggering relapse. In this study, using rat brain slices, we found that isradipine, a general LTCC antagonist used as antihypertensive medication, not only blocks the induction of NMDAR LTP but also promotes the reversal of previously induced LTP in the VTA. In behaving rats, isradipine injected into the VTA suppressed the acquisition of cocaine-paired contextual cue memory assessed using a conditioned place preference (CPP) paradigm. Furthermore, administration of isradipine or a CaV1.3 subtype-selective LTCC antagonist (systemic or intra-VTA) before a single extinction or reinstatement session, while having no immediate effect at the time of administration, abolished previously acquired cocaine and alcohol (ethanol) CPP on subsequent days. Notably, CPP thus extinguished cannot be reinstated by drug re-exposure, even after 2 weeks of withdrawal. These results suggest that LTCC blockade during exposure to drug-associated cues may cause unlearning of the increased valence of those cues, presumably via reversal of glutamatergic synaptic plasticity in the VTA. PMID:26100537

  19. Ser1928 phosphorylation by PKA stimulates the L-type Ca2+ channel CaV1.2 and vasoconstriction during acute hyperglycemia and diabetes.

    PubMed

    Nystoriak, Matthew A; Nieves-Cintrón, Madeline; Patriarchi, Tommaso; Buonarati, Olivia R; Prada, Maria Paz; Morotti, Stefano; Grandi, Eleonora; Fernandes, Julia Dos Santos; Forbush, Katherine; Hofmann, Franz; Sasse, Kent C; Scott, John D; Ward, Sean M; Hell, Johannes W; Navedo, Manuel F

    2017-01-24

    Hypercontractility of arterial myocytes and enhanced vascular tone during diabetes are, in part, attributed to the effects of increased glucose (hyperglycemia) on L-type Ca V 1.2 channels. In murine arterial myocytes, kinase-dependent mechanisms mediate the increase in Ca V 1.2 activity in response to increased extracellular glucose. We identified a subpopulation of the Ca V 1.2 channel pore-forming subunit (α1 C ) within nanometer proximity of protein kinase A (PKA) at the sarcolemma of murine and human arterial myocytes. This arrangement depended upon scaffolding of PKA by an A-kinase anchoring protein 150 (AKAP150) in mice. Glucose-mediated increases in Ca V 1.2 channel activity were associated with PKA activity, leading to α1 C phosphorylation at Ser 1928 Compared to arteries from low-fat diet (LFD)-fed mice and nondiabetic patients, arteries from high-fat diet (HFD)-fed mice and from diabetic patients had increased Ser 1928 phosphorylation and Ca V 1.2 activity. Arterial myocytes and arteries from mice lacking AKAP150 or expressing mutant AKAP150 unable to bind PKA did not exhibit increased Ser 1928 phosphorylation and Ca V 1.2 current density in response to increased glucose or to HFD. Consistent with a functional role for Ser 1928 phosphorylation, arterial myocytes and arteries from knockin mice expressing a Ca V 1.2 with Ser 1928 mutated to alanine (S1928A) lacked glucose-mediated increases in Ca V 1.2 activity and vasoconstriction. Furthermore, the HFD-induced increases in Ca V 1.2 current density and myogenic tone were prevented in S1928A knockin mice. These findings reveal an essential role for α1 C phosphorylation at Ser 1928 in stimulating Ca V 1.2 channel activity and vasoconstriction by AKAP-targeted PKA upon exposure to increased glucose and in diabetes. Copyright © 2017, American Association for the Advancement of Science.

  20. Ser1928 phosphorylation by PKA stimulates the L-type Ca2+ channel CaV1.2 and vasoconstriction during acute hyperglycemia and diabetes

    PubMed Central

    Nystoriak, Matthew A.; Nieves-Cintrón, Madeline; Patriarchi, Tommaso; Buonarati, Olivia R.; Prada, Maria Paz; Morotti, Stefano; Grandi, Eleonora; Fernandes, Julia Dos Santos; Forbush, Katherine; Hofmann, Franz; Sasse, Kent C.; Scott, John D.; Ward, Sean M.; Hell, Johannes W.; Navedo, Manuel F.

    2017-01-01

    Hypercontractility of arterial myocytes and enhanced vascular tone during diabetes are, in part, attributed to the effects of increased glucose (hyperglycemia) on L-type CaV1.2 channels. In murine arterial myocytes, kinase-dependent mechanisms mediate the increase in CaV1.2 activity in response to increased extracellular glucose. We identified a subpopulation of the CaV1.2 channel pore-forming subunit (α1C) within nanometer proximity of protein kinase A (PKA) at the sarcolemma of murine and human arterial myocytes. This arrangement depended upon scaffolding of PKA by an A-kinase anchoring protein 150 (AKAP150) in mice. Glucose-mediated increases in CaV1.2 channel activity were associated with PKA activity, leading to α1C phosphorylation at Ser1928. Compared to arteries from low-fat diet (LFD)–fed mice and nondiabetic patients, arteries from high-fat diet (HFD)–fed mice and from diabetic patients had increased Ser1928 phosphorylation and CaV1.2 activity. Arterial myocytes and arteries from mice lacking AKAP150 or expressing mutant AKAP150 unable to bind PKA did not exhibit increased Ser1928 phosphorylation and CaV1.2 current density in response to increased glucose or to HFD. Consistent with a functional role for Ser1928 phosphorylation, arterial myocytes and arteries from knockin mice expressing a CaV1.2 with Ser1928 mutated to alanine (S1928A) lacked glucose-mediated increases in CaV1.2 activity and vasoconstriction. Furthermore, the HFD-induced increases in CaV1.2 current density and myogenic tone were prevented in S1928A knockin mice. These findings reveal an essential role for α1C phosphorylation at Ser1928 in stimulating CaV1.2 channel activity and vasoconstriction by AKAP-targeted PKA upon exposure to increased glucose and in diabetes. PMID:28119464

  1. Insulin-like growth factor-1 enhances rat skeletal muscle charge movement and L-type Ca2+ channel gene expression

    PubMed Central

    Wang, Zhong-Min; Laura Messi, María; Renganathan, Muthukrishnan; Delbono, Osvaldo

    1999-01-01

    We investigated whether insulin-like growth factor-1 (IGF-1), an endogenous potent activator of skeletal muscle proliferation and differentiation, enhances L-type Ca2+ channel gene expression resulting in increased functional voltage sensors in single skeletal muscle cells. Charge movement and inward Ca2+ current were recorded in primary cultured rat myoballs using the whole-cell configuration of the patch-clamp technique. Ca2+ current and maximum charge movement (Qmax) were potentiated in cells treated with IGF-1 without significant changes in their voltage dependence. Peak Ca2+ current in control and IGF-1-treated cells was -7·8 ± 0·44 and -10·5 ± 0·37 pA pF−1, respectively (P < 0·01), whilst Qmax was 12·9 ± 0·4 and 22·0 ± 0·3 nC μF−1, respectively (P < 0·01). The number of L-type Ca2+ channels was found to increase in the same preparation. The maximum binding capacity (Bmax) of the high-affinity radioligand [3H]PN200-110 in control and IGF-1-treated cells was 1·21 ± 0·25 and 3·15 ± 0·5 pmol (mg protein)−1, respectively (P < 0·01). No significant change in the dissociation constant for [3H]PN200-110 was found. Antisense RNA amplification showed a significant increase in the level of mRNA encoding the L-type Ca2+ channel α1-subunit in IGF-1-treated cells. This study demonstrates that IGF-1 regulates charge movement and the level of L-type Ca2+ channel α1-subunits through activation of gene expression in skeletal muscle cells. PMID:10087334

  2. Intracellular Cs+ activates the PKA pathway, revealing a fast, reversible, Ca2+-dependent inactivation of L-type Ca2+ current.

    PubMed

    Brette, Fabien; Lacampagne, Alain; Sallé, Laurent; Findlay, Ian; Le Guennec, Jean-Yves

    2003-08-01

    Inactivation of the L-type Ca2+ current (ICaL) was studied in isolated guinea pig ventricular myocytes with different ionic solutions. Under basal conditions, ICaL of 82% of cells infused with Cs+-based intracellular solutions showed enhanced amplitude with multiphasic decay and diastolic depolarization-induced facilitation. The characteristics of ICaL in this population of cells were not due to contamination by other currents or an artifact. These phenomena were reduced by ryanodine, caffeine, cyclopiazonic acid, the protein kinase A inhibitor H-89, and the cAMP-dependent protein kinase inhibitor. Forskolin and isoproterenol increased ICaL by only approximately 60% in these cells. Cells infused with either N-methyl-d-glucamine or K+-based intracellular solutions did not show multiphasic decay or facilitation under basal conditions. Isoproterenol increased ICaL by approximately 200% in these cells. In conclusion, we show that multiphasic inactivation of ICaL is due to Ca2+-dependent inactivation that is reversible on a time scale of tens of milliseconds. Cs+ seems to activate the cAMP-dependent protein kinase pathway when used as a substitute for K+ in the pipette solution.

  3. Effects of induced Na+/Ca2+ exchanger overexpression on the spatial distribution of L-type Ca2+ channels and junctophilin-2 in pressure-overloaded hearts.

    PubMed

    Ujihara, Yoshihiro; Mohri, Satoshi; Katanosaka, Yuki

    2016-11-25

    The Na + /Ca 2+ exchanger 1 (NCX1) is an essential Ca 2+ efflux system in cardiomyocytes. Although NCX1 is distributed throughout the sarcolemma, a subpopulation of NCX1 is localized to transverse (T)-tubules. There is growing evidence that T-tubule disorganization is a causal event that shifts the transition from hypertrophy to heart failure (HF). However, the detailed molecular mechanisms have not been clarified. Previously, we showed that induced NCX1 expression in pressure-overloaded hearts attenuates defective excitation-contraction coupling and HF progression. Here, we examined the effects of induced NCX1 overexpression on the spatial distribution of L-type Ca 2+ channels (LTCCs) and junctophilin-2 (JP2), a structural protein that connects the T-tubule and sarcoplasmic reticulum membrane, in pressure-overloaded hearts. Quantitative analysis showed that the regularity of NCX1 localization was significantly decreased at 8 weeks after transverse aortic constriction (TAC)-surgery; however, T-tubule organization and the regularities of LTCC and JP2 immunofluorescent signals were maintained at this time point. These observations demonstrated that release of NCX1 from the T-tubule area occurred before the onset of T-tubule disorganization and LTCC and JP2 mislocalization. Moreover, induced NCX1 overexpression at 8 weeks post-TAC not only recovered NCX1 regularity but also prevented the decrease in LTCC and JP2 regularities at 16 weeks post-TAC. These results suggested that NCX1 may play an important role in the proper spatial distribution of LTCC and JP2 in T-tubules in the context of pressure-overloading. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.

    PubMed

    Bock, Gabriella; Gebhart, Mathias; Scharinger, Anja; Jangsangthong, Wanchana; Busquet, Perrine; Poggiani, Chiara; Sartori, Simone; Mangoni, Matteo E; Sinnegger-Brauns, Martina J; Herzig, Stefan; Striessnig, Jörg; Koschak, Alexandra

    2011-12-09

    An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Ca(v)1.3 L-type Ca(2+) channels (Ca(v)1.3(L)) is a major determinant of their voltage- and Ca(2+)-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Ca(v)1.3(42A) channels that activate at a more negative voltage range and exhibit more pronounced Ca(2+)-dependent inactivation. Here we describe the discovery of a novel short splice variant (Ca(v)1.3(43S)) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Ca(v)1.3(42A), still contains PCRD. When expressed together with α2δ1 and β3 subunits in tsA-201 cells, Ca(v)1.3(43S) also activated at more negative voltages like Ca(v)1.3(42A) but Ca(2+)-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Ca(v)1.3(L). The presence of the proximal C terminus in Ca(v)1.3(43S) channels preserved their modulation by distal C terminus-containing Ca(v)1.3- and Ca(v)1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca(2+) influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Ca(v)1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca(2+) channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca(2+) accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca(2+)-induced neurodegenerative processes.

  5. Apo calmodulin binding to the L-type voltage-gated calcium channel Ca{sub v}1.2 IQ peptide

    SciTech Connect

    Lian Luyun; Myatt, Daniel; Kitmitto, Ashraf

    2007-02-16

    The influx of calcium through the L-type voltage-gated calcium channels (LTCCs) is the trigger for the process of calcium-induced calcium release (CICR) from the sarcoplasmic recticulum, an essential step for cardiac contraction. There are two feedback mechanisms that regulate LTCC activity: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF), both of which are mediated by calmodulin (CaM) binding. The IQ domain (aa 1645-1668) housed within the cytoplasmic domain of the LTCC Ca{sub v}1.2 subunit has been shown to bind both calcium-loaded (Ca{sup 2+}CaM ) and calcium-free CaM (apoCaM). Here, we provide new data for the structural basis for the interaction ofmore » apoCaM with the IQ peptide using NMR, revealing that the apoCaM C-lobe residues are most significantly perturbed upon complex formation. In addition, we have employed transmission electron microscopy of purified LTCC complexes which shows that both apoCaM and Ca{sup 2+}CaM can bind to the intact channel.« less

  6. Thiamine Deficiency Increases Ca2+ Current and CaV1.2 L-type Ca2+ Channel Levels in Cerebellum Granular Neurons.

    PubMed

    Moreira-Lobo, Daniel C; Cruz, Jader S; Silva, Flavia R; Ribeiro, Fabíola M; Kushmerick, Christopher; Oliveira, Fernando A

    2017-04-01

    Thiamine (vitamin B1) is co-factor for three pivotal enzymes for glycolytic metabolism: pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase. Thiamine deficiency leads to neurodegeneration of several brain regions, especially the cerebellum. In addition, several neurodegenerative diseases are associated with impairments of glycolytic metabolism, including Alzheimer's disease. Therefore, understanding the link between dysfunction of the glycolytic pathway and neuronal death will be an important step to comprehend the mechanism and progression of neuronal degeneration as well as the development of new treatment for neurodegenerative states. Here, using an in vitro model to study the effects of thiamine deficiency on cerebellum granule neurons, we show an increase in Ca 2+ current density and Ca V 1.2 expression. These results indicate a link between alterations in glycolytic metabolism and changes to Ca 2+ dynamics, two factors that have been implicated in neurodegeneration.

  7. Effects of itopride hydrochloride on the delayed rectifier K+ and L-type CA2+ currents in guinea-pig ventricular myocytes.

    PubMed

    Morisawa, T; Hasegawa, J; Hama, R; Kitano, M; Kishimoto, Y; Kawasaki, H

    1999-01-01

    The effects of itopride hydrochloride, a new drug used to regulate motility in the gastrointestinal tract, on the delayed rectifier K+ current (I(K)) and the L-type Ca2+ current (I(Ca)) were evaluated in guinea-pig ventricular myocytes at concentrations of 1, 10 and 100 microM to determine whether the drug has a proarrhythmic effect through blockade of I(K). Itopride did not affect I(K) at concentrations of 100 microM or less, and no significant effects of 1, 10 or 100 microM itopride were observed on the inward rectifier K+ current (I(K1)) responsible for the resting potential and final repolarization phase of the action potential. We next investigated the effects of itopride on L-type Ca2+ current (I(Ca)). Significant inhibition of I(Ca) was observed at itopride concentrations greater than 10 microM. These results suggested that itopride hydrochloride has an inhibitory effect on I(Ca) at concentrations much higher than those in clinical use.

  8. L-type Ca(2+) currents overlapping threshold Na(+) currents: could they be responsible for the "slip-mode" phenomenon in cardiac myocytes?

    PubMed

    Piacentino, Valentino; Gaughan, John P; Houser, Steven R

    2002-03-08

    Phosphorylation of Na channels has been suggested to increase their Ca permeability. Termed "slip-mode conductance" (SMC), this hypothesis predicts that Ca influx via protein kinase A (PKA)-modified Na channels can induce sarcoplasmic reticulum (SR) Ca release. We tested this hypothesis by determining if SR Ca release is graded with I(Na) in the presence of activated PKA (with Isoproterenol, ISO). V(m), I(m), and [Ca](i) were measured in feline (n=26) and failing human (n=19) ventricular myocytes. Voltage steps from -70 through -40 mV were used to grade I(Na). Na channel antagonists (tetrodotoxin), L-type Ca channel (I(Ca,L)) antagonists (nifedipine, cadmium, verapamil), and agonists (Bay K 8644, FPL 64176) were used to separate SMC from I(Ca,L). In the absence of ISO, I(Na) was associated with SR Ca release in human but not feline myocytes. After ISO, graded I(Na) was associated with small amounts of SR Ca release in feline myocytes and the magnitude of release increased in human myocytes. I(Na)-related SR Ca release was insensitive to tetrodotoxin (n=10) but was blocked by nifedipine (n=10) and cadmium (n=3). SR Ca release was induced over the same voltage range in the absence of ISO with Bay K 8644 and FPL 64176 (n=9). Positive voltage steps (to 0 mV) to fully activate Na channels (SMC) in the presence of ISO and Verapamil only caused SR Ca release when block of I(Ca,L) was incomplete. We conclude that PKA-mediated increases in I(Ca,L) and SR Ca loading can reproduce many of the experimental features of SMC.

  9. Magnolol inhibits colonic motility through down-regulation of voltage-sensitive L-type Ca2+ channels of colonic smooth muscle cells in rats.

    PubMed

    Zhang, Man; Zang, Kai-Hong; Luo, Jia-Lie; Leung, Fung-Ping; Huang, Yu; Lin, Cheng-Yuan; Yang, Zhi-Jun; Lu, Ai-Ping; Tang, Xu-Dong; Xu, Hong-Xi; Sung, Joseph Jao-yiu; Bian, Zhao-Xiang

    2013-11-15

    This study aimed to investigate the effect of magnolol (5,5'-diallyl-2,2'-biphenyldiol) on contraction in distal colonic segments of rats and the underlying mechanisms. Colonic segments were mounted in organ baths for isometric force measurement. Whole-cell voltage-sensitive L-type Ca(2+) currents were recorded on isolated single colonic smooth muscle cells using patch-clamp technique. The spontaneous contractions and acetylcholine (ACh)- and Bay K 8644-induced contractions were inhibited by magnolol (3-100 μM). In the presence of Bay K8644 (100 nM), magnolol (10-100 μM) inhibited the contraction induced by 10 μM ACh. By contrast, tetrodotoxin (100 nM) and Nώ-nitro-L-arginine methyl ester (L-NAME 100 μM) did not change the inhibitory effect of magnolol (10 μM). In addition, magnolol (3-100 μM) inhibited the L-type Ca(2+) currents. The present results suggest that magnolol inhibits colonic smooth muscle contraction through downregulating L-type Ca(2+) channel activity. Copyright © 2013 Elsevier GmbH. All rights reserved.

  10. Carbon monoxide stimulates astrocytic mitochondrial biogenesis via L-type Ca{sup 2+} channel-mediated PGC-1α/ERRα activation

    SciTech Connect

    Choi, Yoon Kyung; Park, Joon Ha; Baek, Yi-Yong

    Carbon monoxide (CO), derived by the enzymatic reaction of heme oxygenase (HO), is a cellular regulator of energy metabolism and cytoprotection; however, its underlying mechanism has not been clearly elucidated. Astrocytes pre-exposed to the CO-releasing compound CORM-2 increased mitochondrial biogenesis, mitochondrial electron transport components (cytochrome c, Cyt c; cytochrome c oxidase subunit 2, COX2), and ATP synthesis. The increased mitochondrial function was correlated with activation of AMP-activated protein kinase α and upregulation of HO-1, peroxisome proliferators-activated receptor γ-coactivator-1α (PGC-1α), and estrogen-related receptor α (ERRα). These events elicited by CORM-2 were suppressed by Ca{sup 2+} chelators, a HO inhibitor, and anmore » L-type Ca{sup 2+} channel blocker, but not other Ca{sup 2+} channel inhibitors. Among the HO byproducts, combined CORM-2 and bilirubin treatment effectively increased PGC-1α, Cyt c and COX2 expression, mitochondrial biogenesis, and ATP synthesis, and these increases were blocked by Ca{sup 2+} chelators. Moreover, cerebral ischemia significantly increased HO-1, PGC-1α, and ERRα levels, subsequently increasing Cyt c and COX2 expression, in wild-type mice, compared with HO-1{sup +/−} mice. These results suggest that HO-1-derived CO enhances mitochondrial biogenesis in astrocytes by activating L-type Ca{sup 2+} channel-mediated PGC-1α/ERRα axis, leading to maintenance of astrocyte function and neuroprotection/recovery against damage of brain function. - Highlights: • CORM-pretreated astrocytes induces mitochondrial biogenesis by activating L-type Ca{sup 2+} channel-mediated PGC-1α stabilization. • Cerebral ischemia increased electron transport chain proteins (e.g. Cyt c and COX2), in WT mice, compared with HO-1{sup +/−} mice. • CO/HO-1 pathway increases astrocytic mitochondrial functions via a PGC-1α/ERRα axis.« less

  11. Modulation of intracellular Ca2+ via L-type calcium channels in heart cells by the autoantibody directed against the second extracellular loop of the alpha1-adrenoceptors.

    PubMed

    Bkaily, Ghassan; El-Bizri, Nesrine; Bui, Michel; Sukarieh, Rami; Jacques, Danielle; Fu, Michael L X

    2003-03-01

    The effects of methoxamine, a selective alpha1-adrenergic receptor agonist, and the autoantibody directed against the second extracellular loop of alpha1-adrenoceptors were studied on intracellular free Ca2+ levels using confocal microscopy and ionic currents using the whole-cell patch clamp technique in single cells of 10-day-old embryonic chick and 20-week-old fetal human hearts. We observed that like methoxamine, the autoantibody directed against the second extracellular loop of alpha1-adrenoreceptors significantly increased the L-type calcium current (I(Ca(L))) but had no effect on the T-type calcium current (I(Ca(T))), the delayed outward potassium current, or the fast sodium current. This effect of the autoantibody was prevented by a prestimulation of the receptors with methoxamine and vice versa. Moreover, treating the cells with prazosin, a selective alpha1-adrenergic receptor antagonist blocked the methoxamine and the autoantibody-induced increase in I(Ca(L)), respectively. In absence of prazosin, both methoxamine and the autoantibody showed a substantial enhancement in the frequency of cell contraction and that of the concomitant cytosolic and nuclear free Ca2+ variations. The subsequent addition of nifedipine, a specific L-type Ca2+ channel blocker, reversed not only the methoxamine or the autoantibody-induced effect but also completely abolished cell contraction. These results demonstrated that functional alpha1-adrenoceptors exist in both 10-day-old embryonic chick and 20-week-old human fetal hearts and that the autoantibody directed against the second extracellular loop of this type of receptors plays an important role in stimulating their activity via activation of L-type calcium channels. This loop seems to have a functional significance by being the target of alpha1-receptor agonists like methoxamine.

  12. Ion channel mechanisms of rat tail artery contraction-relaxation by menthol involving, respectively, TRPM8 activation and L-type Ca2+ channel inhibition

    PubMed Central

    Melanaphy, Donal; Kustov, Maxim V.; Watson, Conall A.; Borysova, Lyudmyla; Burdyga, Theodor V.; Zholos, Alexander V.

    2016-01-01

    Transient receptor potential melastatin 8 (TRPM8) is the principal cold and menthol receptor channel. Characterized primarily for its cold-sensing role in sensory neurons, it is expressed and functional in several nonneuronal tissues, including vasculature. We previously demonstrated that menthol causes variable mechanical responses (vasoconstriction, vasodilatation, or biphasic reactions) in isolated arteries, depending on vascular tone. Here we aimed to dissect the specific ion channel mechanisms and corresponding Ca2+ signaling pathways underlying such complex responses to menthol and other TRPM8 ligands in rat tail artery myocytes using patch-clamp electrophysiology, confocal Ca2+ imaging, and ratiometric Ca2+ recording. Menthol (300 μM, a concentration typically used to induce TRPM8 currents) strongly inhibited L-type Ca2+ channel current (L-ICa) in isolated myocytes, especially its sustained component, most relevant for depolarization-induced vasoconstriction. In contraction studies, with nifedipine present (10 μM) to abolish L-ICa contribution to phenylephrine (PE)-induced vasoconstrictions of vascular rings, a marked increase in tone was observed with menthol, similar to resting (i.e., without α-adrenoceptor stimulation by PE) conditions, when L-type channels were mostly deactivated. Menthol-induced increases in PE-induced vasoconstrictions could be inhibited both by the TRPM8 antagonist AMTB (thus confirming the specific role of TRPM8) and by cyclopiazonic acid treatment to deplete Ca2+ stores, pointing to a major contribution of Ca2+ release from the sarcoplasmic reticulum in these contractile responses. Immunocytochemical analysis has indeed revealed colocalization of TRPM8 and InsP3 receptors. Moreover, menthol Ca2+ responses, which were somewhat reduced under Ca2+-free conditions, were strongly reduced by cyclopiazonic acid treatment to deplete Ca2+ store, whereas caffeine-induced Ca2+ responses were blunted in the presence of menthol. Finally, two

  13. Ethanol-mediated relaxation of guinea pig urinary bladder smooth muscle: involvement of BK and L-type Ca2+ channels

    PubMed Central

    Malysz, John; Afeli, Serge A. Y.; Provence, Aaron

    2013-01-01

    Mechanisms underlying ethanol (EtOH)-induced detrusor smooth muscle (DSM) relaxation and increased urinary bladder capacity remain unknown. We investigated whether the large conductance Ca2+-activated K+ (BK) channels or L-type voltage-dependent Ca2+ channels (VDCCs), major regulators of DSM excitability and contractility, are targets for EtOH by patch-clamp electrophysiology (conventional and perforated whole cell and excised patch single channel) and isometric tension recordings using guinea pig DSM cells and isolated tissue strips, respectively. EtOH at 0.3% vol/vol (∼50 mM) enhanced whole cell BK currents at +30 mV and above, determined by the selective BK channel blocker paxilline. In excised patches recorded at +40 mV and ∼300 nM intracellular Ca2+ concentration ([Ca2+]), EtOH (0.1–0.3%) affected single BK channels (mean conductance ∼210 pS and blocked by paxilline) by increasing the open channel probability, number of open channel events, and open dwell-time constants. The amplitude of single BK channel currents and unitary conductance were not altered by EtOH. Conversely, at ∼10 μM but not ∼2 μM intracellular [Ca2+], EtOH (0.3%) decreased the single BK channel activity. EtOH (0.3%) affected transient BK currents (TBKCs) by either increasing frequency or decreasing amplitude, depending on the basal level of TBKC frequency. In isolated DSM strips, EtOH (0.1–1%) reduced the amplitude and muscle force of spontaneous phasic contractions. The EtOH-induced DSM relaxation, except at 1%, was attenuated by paxilline. EtOH (1%) inhibited L-type VDCC currents in DSM cells. In summary, we reveal the involvement of BK channels and L-type VDCCs in mediating EtOH-induced urinary bladder relaxation accommodating alcohol-induced diuresis. PMID:24153429

  14. Regulation of L-type CaV1.3 channel activity and insulin secretion by the cGMP-PKG signaling pathway

    PubMed Central

    Sandoval, Alejandro; Duran, Paz; Gandini, María A.; Andrade, Arturo; Almanza, Angélica; Kaja, Simon; Felix, Ricardo

    2018-01-01

    cGMP is a second messenger widely used in the nervous system and other tissues. One of the major effectors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG), which catalyzes the phosphorylation of a variety of proteins including ion channels. Previously, it has been shown that the cGMP-PKG signaling pathway inhibits Ca2+ currents in rat vestibular hair cells and chromaffin cells. This current allegedly flow through voltage-gated CaV1.3L-type Ca2+ channels, and is important for controlling vestibular hair cell sensory function and catecholamine secretion, respectively. Here, we show that native L-type channels in the insulin-secreting RIN-m5F cell line, and recombinant CaV1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG signaling cascade. Our results indicate that the CaVα1 ion-conducting subunit of the CaV1.3 channels is highly expressed in RIN-m5F cells and that the application of 8-Br-cGMP, a membrane-permeable analogue of cGMP, significantly inhibits Ca2+ macroscopic currents and impair insulin release stimulated with high K+. In addition, KT-5823, a specific inhibitor of PKG, prevents the current inhibition generated by 8-Br-cGMP in the heterologous expression system. Interestingly, mutating the putative phosphorylation sites to residues resistant to phosphorylation showed that the relevant PKG sites for CaV1.3 L-type channel regulation centers on two amino acid residues, Ser793 and Ser860, located in the intracellular loop connecting the II and III repeats of the CaVα1 pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate CaV1.3 channels and contribute to regulate insulin secretion. PMID:28807144

  15. Regulation of L-type CaV1.3 channel activity and insulin secretion by the cGMP-PKG signaling pathway.

    PubMed

    Sandoval, Alejandro; Duran, Paz; Gandini, María A; Andrade, Arturo; Almanza, Angélica; Kaja, Simon; Felix, Ricardo

    2017-09-01

    cGMP is a second messenger widely used in the nervous system and other tissues. One of the major effectors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG), which catalyzes the phosphorylation of a variety of proteins including ion channels. Previously, it has been shown that the cGMP-PKG signaling pathway inhibits Ca 2+ currents in rat vestibular hair cells and chromaffin cells. This current allegedly flow through voltage-gated Ca V 1.3L-type Ca 2+ channels, and is important for controlling vestibular hair cell sensory function and catecholamine secretion, respectively. Here, we show that native L-type channels in the insulin-secreting RIN-m5F cell line, and recombinant Ca V 1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG signaling cascade. Our results indicate that the Ca V α 1 ion-conducting subunit of the Ca V 1.3 channels is highly expressed in RIN-m5F cells and that the application of 8-Br-cGMP, a membrane-permeable analogue of cGMP, significantly inhibits Ca 2+ macroscopic currents and impair insulin release stimulated with high K + . In addition, KT-5823, a specific inhibitor of PKG, prevents the current inhibition generated by 8-Br-cGMP in the heterologous expression system. Interestingly, mutating the putative phosphorylation sites to residues resistant to phosphorylation showed that the relevant PKG sites for Ca V 1.3 L-type channel regulation centers on two amino acid residues, Ser793 and Ser860, located in the intracellular loop connecting the II and III repeats of the Ca V α 1 pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate Ca V 1.3 channels and contribute to regulate insulin secretion. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Functional Properties of a Newly Identified C-terminal Splice Variant of Cav1.3 L-type Ca2+ Channels*

    PubMed Central

    Bock, Gabriella; Gebhart, Mathias; Scharinger, Anja; Jangsangthong, Wanchana; Busquet, Perrine; Poggiani, Chiara; Sartori, Simone; Mangoni, Matteo E.; Sinnegger-Brauns, Martina J.; Herzig, Stefan; Striessnig, Jörg; Koschak, Alexandra

    2011-01-01

    An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Cav1.3 L-type Ca2+ channels (Cav1.3L) is a major determinant of their voltage- and Ca2+-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Cav1.342A channels that activate at a more negative voltage range and exhibit more pronounced Ca2+-dependent inactivation. Here we describe the discovery of a novel short splice variant (Cav1.343S) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Cav1.342A, still contains PCRD. When expressed together with α2δ1 and β3 subunits in tsA-201 cells, Cav1.343S also activated at more negative voltages like Cav1.342A but Ca2+-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Cav1.3L. The presence of the proximal C terminus in Cav1.343S channels preserved their modulation by distal C terminus-containing Cav1.3- and Cav1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca2+ influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Cav1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca2+ channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca2+ accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca2+-induced neurodegenerative processes. PMID:21998310

  17. Chronic treatment with otilonium bromide induces changes in L-type Ca²⁺ channel, tachykinins, and nitric oxide synthase expression in rat colon muscle coat.

    PubMed

    Traini, C; Cipriani, G; Evangelista, S; Santicioli, P; Faussone-Pellegrini, M-S; Vannucchi, M-G

    2013-11-01

    Otilonium bromide (OB) is a quaternary ammonium derivative used for the treatment of intestinal hypermotility and is endowed with neurokinin2 receptor (NK2r) antagonist and Ca²⁺ channel blocker properties. Therefore, the possibility that OB might play a role in the neurokinin receptor/Substance-P/nitric oxide (NKr/SP/NO) circuit was investigated after chronic exposition to the drug. Rats were treated with OB 2-20 mg kg⁻¹ for 10 and 30 days. In the proximal colon, the expression and distribution of muscle NOsynthase 1 (NOS1), NK1r, NK2r, SP and Cav 1.2 subunit (for L-type Ca²⁺ channel) and the spontaneous activity and stimulated responses to NK1r and NK2r agonists were investigated. Immunohistochemistry showed a redistribution of NK1r and L-type Ca²⁺ channel in muscle cells with no change of NK2r at 30 days, a significant increase in muscle NOS1 expression at 10 days and a significant decrease in the SP content early in the ganglia and later in the intramuscular nerve fibers. Functional studies showed no change in spontaneous activity but a significant increase in maximal contraction induced by NK1r agonist. Chronic exposition to OB significantly affects the NKr/SP/NO circuit. The progressive decrease in SP-expression might be the consequence of the persistent presence of OB, the increase of NOS1 expression in muscle cells at 10 days in an attempt to guarantee an adequate NO production, and, at 30 days, the redistribution of the L-type Ca²⁺ channel and NK1r as a sign to compensate the drug channel block by re-cycling both of them. The physiological data suggest NK1r hypersensitivity. © 2013 John Wiley & Sons Ltd.

  18. Mechanism of auxiliary β-subunit-mediated membrane targeting of L-type (CaV1.2) channels

    PubMed Central

    Fang, Kun; Colecraft, Henry M

    2011-01-01

    Abstract Ca2+ influx via CaV1/CaV2 channels drives processes ranging from neurotransmission to muscle contraction. Association of a pore-forming α1 and cytosolic β is necessary for trafficking CaV1/CaV2 channels to the cell surface through poorly understood mechanisms. A prevalent idea suggests β binds the α1 intracellular I–II loop, masking an endoplasmic reticulum (ER) retention signal as the dominant mechanism for CaV1/CaV2 channel membrane trafficking. There are hints that other α1 subunit cytoplasmic domains may play a significant role, but the nature of their potential contribution is unclear. We assessed the roles of all intracellular domains of CaV1.2-α1C by generating chimeras featuring substitutions of all possible permutations of intracellular loops/termini of α1C into the β-independent CaV3.1-α1G channel. Surprisingly, functional analyses demonstrated α1C I–II loop strongly increases channel surface density while other cytoplasmic domains had a competing opposing effect. Alanine-scanning mutagenesis identified an acidic-residue putative ER export motif responsible for the I–II loop-mediated increase in channel surface density. β-dependent increase in current arose as an emergent property requiring four α1C intracellular domains, with the I–II loop and C-terminus being essential. The results suggest β binding to the α1C I–II loop causes a C-terminus-dependent rearrangement of intracellular domains, shifting a balance of power between export signals on the I–II loop and retention signals elsewhere. PMID:21746784

  19. Participation of IP3R, RyR and L-type Ca2+ channel in the nuclear maturation of Rhinella arenarum oocytes.

    PubMed

    Toranzo, G Sánchez; Bühler, M C Gramajo; Bühler, M I

    2014-05-01

    During meiosis resumption, oocytes undergo a series of nuclear and cytosolic changes that prepare them for fertilization and that are referred to as oocyte maturation. These events are characterized by germinal vesicle breakdown (GVBD), chromatin condensation and spindle formation and, among cytosolic changes, organelle redistribution and maturation of Ca2+-release mechanisms. The progression of the meiotic cell cycle is regulated by M phase/maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Changes in the levels of intracellular free Ca2+ ion have also been implicated strongly in the triggering of the initiation of the M phase. Ca2+ signals can be generated by Ca2+ release from intracellular Ca2+ stores (endoplasmic reticulum; ER) or by Ca2+ influx from the extracellular space. In this sense, the L-type Ca2+ channel plays an important role in the incorporation of Ca2+ from the extracellular space. Two types of intracellular Ca2+ receptor/channels are known to mediate the intracellular Ca2+ release from the ER lumen. The most abundant, the inositol 1,4,5-trisphosphate receptor (IP3R), and the other Ca2+ channel, the ryanodine receptor (RyR), have also been reported to mediate Ca2+ release in several oocytes. In amphibians, MPF and MAPK play a central role during oocyte maturation, controlling several events. However, no definitive relationships have been identified between Ca2+ and MPF or MAPK. We investigated the participation of Ca2+ in the spontaneous and progesterone-induced nuclear maturation in Rhinella arenarum oocytes and the effect of different pharmacological agents known to produce modifications in the Ca2+ channels. We demonstrated that loading competent and incompetent oocytes with the intracellular calcium chelator BAPTA/AM produced suppression of spontaneous and progesterone-induced GVBD. In our results, the capacity of progesterone to trigger meiosis reinitiation in Rhinella in the presence of L-type Ca2+ channel blockers

  20. The carboxyl-terminal region of ahnak provides a link between cardiac L-type Ca2+ channels and the actin-based cytoskeleton.

    PubMed

    Hohaus, Annette; Person, Veronika; Behlke, Joachim; Schaper, Jutta; Morano, Ingo; Haase, Hannelore

    2002-08-01

    Ahnak is a ubiquitously expressed giant protein of 5643 amino acids implicated in cell differentiation and signal transduction. In a recent study, we demonstrated the association of ahnak with the regulatory beta2 subunit of the cardiac L-type Ca2+ channel. Here we identify the most carboxyl-terminal ahnak region (aa 5262-5643) to interact with recombinant beta2a as well as with beta2 and beta1a isoforms of native muscle Ca2+ channels using a panel of GST fusion proteins. Equilibrium sedimentation analysis revealed Kd values of 55 +/- 11 nM and 328 +/- 24 nM for carboxyl-terminal (aa 195-606) and amino-terminal (aa 1-200) truncates of the beta2a subunit, respectively. The same carboxyl-terminal ahnak region (aa 5262-5643) bound to G-actin and cosedimented with F-actin. Confocal microscopy of human left ventricular tissue localized the carboxyl-terminal ahnak portion to the sarcolemma including the T-tubular system and the intercalated disks of cardiomyocytes. These results suggest that ahnak provides a structural basis for the subsarcolemmal cytoarchitecture and confers the regulatory role of the actin-based cytoskeleton to the L-type Ca2+ channel.

  1. The effect of hypercholesterolemia on carbachol-induced contractions of the detrusor smooth muscle in rats: increased role of L-type Ca2+ channels.

    PubMed

    Balkanci, Zeynep Dicle; Pehlivanoğlu, Bilge; Bayrak, Sibel; Karabulut, Ismail; Karaismailoğlu, Serkan; Erdem, Ayşen

    2012-11-01

    To investigate a possible relation between hypercholesterolemia and detrusor smooth muscle function, we studied the contractile response to potassium challenge, carbachol (CCh), and the components of CCh-induced contractile mechanism in high-cholesterol diet-fed rats. Adult male Sprague-Dawley rats were fed with standard (control group, N = 17) or 4 % cholesterol diet (hypercholesterolemia group (HC), N = 16) for 4 weeks. Spontaneous contractions of detrusor muscle strips and their responses to potassium chloride (KCl) or cumulative dose-contraction curves to CCh were recorded. The effects of muscarinic receptor antagonists (methoctramin and/or 4-diphenylacetoxy-N-methylpiperidine), L-type Ca(+2) channel blocker (nifedipine), and/or rho-kinase inhibitor Y-27632 were investigated. Blood cholesterol level was increased in the HC group with no sign of atherosclerosis. The KCl-induced detrusor smooth muscle contractions were higher in HC, whereas spontaneous and CCh-induced responses were similar in both groups. Preincubation with receptor antagonist for M(3) but not for M(2) attenuated contraction significantly, shifting the dose-response curve to the right. This response was similar in both groups. Among two effector mechanisms of M(3)-mediated detrusor smooth muscle contraction, rho-kinase pathway was not affected by hypercholesterolemia, whereas blockade of L-type Ca(+2) channels potently reduced contractions. The results of this study point out a relation between hypercholesterolemia and contractile mechanism of detrusor smooth muscle likely to change urinary bladder function, via altering L-type Ca(+2) channels. Taken together with escalating incidence of hypercholesterolemia and lower urinary tract symptoms, it is a field which deserves to be investigated further.

  2. The L-type voltage-gated calcium channel CaV1.2 mediates fear extinction and modulates synaptic tone in the lateral amygdala.

    PubMed

    Temme, Stephanie J; Murphy, Geoffrey G

    2017-11-01

    L-type voltage-gated calcium channels (LVGCCs) have been implicated in both the formation and the reduction of fear through Pavlovian fear conditioning and extinction. Despite the implication of LVGCCs in fear learning and extinction, studies of the individual LVGCC subtypes, Ca V 1.2 and Ca V 1.3, using transgenic mice have failed to find a role of either subtype in fear extinction. This discontinuity between the pharmacological studies of LVGCCs and the studies investigating individual subtype contributions could be due to the limited neuronal deletion pattern of the Ca V 1.2 conditional knockout mice previously studied to excitatory neurons in the forebrain. To investigate the effects of deletion of Ca V 1.2 in all neuronal populations, we generated Ca V 1.2 conditional knockout mice using the synapsin1 promoter to drive Cre recombinase expression. Pan-neuronal deletion of Ca V 1.2 did not alter basal anxiety or fear learning. However, pan-neuronal deletion of Ca V 1.2 resulted in a significant deficit in extinction of contextual fear, implicating LVGCCs, specifically Ca V 1.2, in extinction learning. Further exploration on the effects of deletion of Ca V 1.2 on inhibitory and excitatory input onto the principle neurons of the lateral amygdala revealed a significant shift in inhibitory/excitatory balance. Together these data illustrate an important role of Ca V 1.2 in fear extinction and the synaptic regulation of activity within the amygdala. © 2017 Temme and Murphy; Published by Cold Spring Harbor Laboratory Press.

  3. L-type Ca2+ channels in mood, cognition and addiction: integrating human and rodent studies with a focus on behavioural endophenotypes.

    PubMed

    Kabir, Z D; Lee, A S; Rajadhyaksha, A M

    2016-10-15

    Brain Ca v 1.2 and Ca v 1.3 L-type Ca 2+ channels play key physiological roles in various neuronal processes that contribute to brain function. Genetic studies have recently identified CACNA1C as a candidate risk gene for bipolar disorder (BD), schizophrenia (SCZ), major depressive disorder (MDD) and autism spectrum disorder (ASD), and CACNA1D for BD and ASD, suggesting a contribution of Ca v 1.2 and Ca v 1.3 Ca 2+ signalling to the pathophysiology of neuropsychiatric disorders. Once considered sole clinical entities, it is now clear that BD, SCZ, MDD and ASD share common phenotypic features, most likely due to overlapping neurocircuitry and common molecular mechanisms. A major future challenge lies in translating the human genetic findings to pathological mechanisms that are translatable back to the patient. One approach for tackling such a daunting scientific endeavour for complex behaviour-based neuropsychiatric disorders is to examine intermediate biological phenotypes in the context of endophenotypes within distinct behavioural domains. This will better allow us to integrate findings from genes to behaviour across species, and improve the chances of translating preclinical findings to clinical practice. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

  4. CaLecRK-S.5, a pepper L-type lectin receptor kinase gene, confers broad-spectrum resistance by activating priming

    PubMed Central

    Woo, Joo Yong; Jeong, Kwang Ju; Kim, Young Jin; Paek, Kyung-Hee

    2016-01-01

    In Arabidopsis, several L-type lectin receptor kinases (LecRKs) have been identified as putative immune receptors. However, to date, there have been few analyses of LecRKs in crop plants. Virus-induced gene silencing of CaLecRK-S.5 verified the role of CaLecRK-S.5 in broad-spectrum resistance. Compared with control plants, CaLecRK-S.5-silenced plants showed reduced hypersensitive response, reactive oxygen species burst, secondary metabolite production, mitogen-activated protein kinase activation, and defense-related gene expression in response to Tobacco mosaic virus pathotype P0 (TMV-P0) infection. Suppression of CaLecRK-S.5 expression significantly enhanced the susceptibility to Pepper mild mottle virus pathotype P1,2,3, Xanthomonas campestris pv. vesicatoria, Phytophthora capsici, as well as TMV-P0. Additionally, β-aminobutyric acid treatment and a systemic acquired resistance assay revealed that CaLecRK-S.5 is involved in priming of plant immunity. Pre-treatment with β-aminobutyric acid before viral infection restored the reduced disease resistance phenotypes shown in CaLecRK-S.5-silenced plants. Systemic acquired resistance was also abolished in CaLecRK-S.5-silenced plants. Finally, RNA sequencing analysis indicated that CaLecRK-S.5 positively regulates plant immunity at the transcriptional level. Altogether, these results suggest that CaLecRK-S.5-mediated broad-spectrum resistance is associated with the regulation of priming. PMID:27647723

  5. Aberrant Splicing Promotes Proteasomal Degradation of L-type CaV1.2 Calcium Channels by Competitive Binding for CaVβ Subunits in Cardiac Hypertrophy.

    PubMed

    Hu, Zhenyu; Wang, Jiong-Wei; Yu, Dejie; Soon, Jia Lin; de Kleijn, Dominique P V; Foo, Roger; Liao, Ping; Colecraft, Henry M; Soong, Tuck Wah

    2016-10-12

    Decreased expression and activity of Ca V 1.2 calcium channels has been reported in pressure overload-induced cardiac hypertrophy and heart failure. However, the underlying mechanisms remain unknown. Here we identified in rodents a splice variant of Ca V 1.2 channel, named Ca V 1.2 e21+22 , that contained the pair of mutually exclusive exons 21 and 22. This variant was highly expressed in neonatal hearts. The abundance of this variant was gradually increased by 12.5-folds within 14 days of transverse aortic banding that induced cardiac hypertrophy in adult mouse hearts and was also elevated in left ventricles from patients with dilated cardiomyopathy. Although this variant did not conduct Ca 2+ ions, it reduced the cell-surface expression of wild-type Ca V 1.2 channels and consequently decreased the whole-cell Ca 2+ influx via the Ca V 1.2 channels. In addition, the Ca V 1.2 e21+22 variant interacted with Ca V β subunits significantly more than wild-type Ca V 1.2 channels, and competition of Ca V β subunits by Ca V 1.2 e21+22 consequently enhanced ubiquitination and subsequent proteasomal degradation of the wild-type Ca V 1.2 channels. Our findings show that the resurgence of a specific neonatal splice variant of Ca V 1.2 channels in adult heart under stress may contribute to heart failure.

  6. Vasoconstriction triggered by hydrogen sulfide: Evidence for Na+,K+,2Cl-cotransport and L-type Ca2+ channel-mediated pathway.

    PubMed

    Orlov, Sergei N; Gusakova, Svetlana V; Smaglii, Liudmila V; Koltsova, Svetlana V; Sidorenko, Svetalana V

    2017-12-01

    This study examined the dose-dependent actions of hydrogen sulfide donor sodium hydrosulphide (NaHS) on isometric contractions and ion transport in rat aorta smooth muscle cells (SMC). Isometric contraction was measured in ring aortas segments from male Wistar rats. Activity of Na + /K + -pump and Na + ,K + ,2Cl - cotransport was measured in cultured endothelial and smooth muscle cells from the rat aorta as ouabain-sensitive and ouabain-resistant, bumetanide-sensitive components of the 86 Rb influx, respectively. NaHS exhibited the bimodal action on contractions triggered by modest depolarization ([K + ] o =30 mM). At 10 -4 M, NaHS augmented contractions of intact and endothelium-denuded strips by ~ 15% and 25%, respectively, whereas at concentration of 10 -3  M it decreased contractile responses by more than two-fold. Contractions evoked by 10 -4  M NaHS were completely abolished by bumetanide, a potent inhibitor of Na + ,K + ,2Cl - cotransport, whereas the inhibition seen at 10 -3  M NaHS was suppressed in the presence of K + channel blocker TEA. In cultured SMC, 5×10 -5  M NaHS increased Na + ,K + ,2Cl - - cotransport without any effect on the activity of this carrier in endothelial cells. In depolarized SMC, 45 Ca influx was enhanced in the presence of 10 -4  M NaHS and suppressed under elevation of [NaHS] up to 10 -3  M. 45 Ca influx triggered by 10 -4  M NaHS was abolished by bumetanide and L-type Ca 2+ channel blocker nicardipine. Our results strongly suggest that contractions of rat aortic rings triggered by low doses of NaHS are mediated by activation of Na + ,K + ,2Cl - cotransport and Ca 2+ influx via L-type channels.

  7. Effects of caffeine on cytoplasmic free Ca2+ concentration in pancreatic beta-cells are mediated by interaction with ATP-sensitive K+ channels and L-type voltage-gated Ca2+ channels but not the ryanodine receptor.

    PubMed Central

    Islam, M S; Larsson, O; Nilsson, T; Berggren, P O

    1995-01-01

    In the pancreatic beta-cell, an increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i) by caffeine is believed to indicate mobilization of Ca2+ from intracellular stores, through activation of a ryanodine receptor-like channel. It is not known whether other mechanisms, as well, underlie caffeine-induced changes in [Ca2+]i. We studied the effects of caffeine on [Ca2+]i by using dual-wavelength excitation microfluorimetry in fura-2-loaded beta-cells. In the presence of a non-stimulatory concentration of glucose, caffeine (10-50 mM) consistently increased [Ca2+]i. The effect was completely blocked by omission of extracellular Ca2+ and by blockers of the L-type voltage-gated Ca2+ channel, such as D-600 or nifedipine. Depletion of agonist-sensitive intracellular Ca2+ pools by thapsigargin did not inhibit the stimulatory effect of caffeine on [Ca2+]i. Moreover, this effect of caffeine was not due to an increase in cyclic AMP, since forskolin and 3-isobutyl-1-methylxanthine (IBMX) failed to raise [Ca2+]i in unstimulated beta-cells. In beta-cells, glucose and sulphonylureas increase [Ca2+]i by causing closure of ATP-sensitive K+ channels (KATP channels). Caffeine also caused inhibition of KATP channel activity, as measured in excised inside-out patches. Accordingly, caffeine (> 10 mM) induced insulin release from beta-cells in the presence of a non-stimulatory concentration of glucose (3 mM). Hence, membrane depolarization and opening of voltage-gated L-type Ca2+ channels were the underlying mechanisms whereby the xanthine drug increased [Ca2+]i and induced insulin release. Paradoxically, in glucose-stimulated beta-cells, caffeine (> 10 mM) lowered [Ca2+]i. This effect was due to the fact that caffeine reduced depolarization-induced whole-cell Ca2+ current through the L-type voltage-gated Ca2+ channel in a dose-dependent manner. Lower concentrations of caffeine (2.5-5.0 mM), when added after glucose-stimulated increase in [Ca2+]i, induced fast oscillations in [Ca2

  8. Somatostatin receptor subtypes SSTR2 and SSTR5 couple negatively to an L-type Ca2+ current in the pituitary cell line AtT-20.

    PubMed

    Tallent, M; Liapakis, G; O'Carroll, A M; Lolait, S J; Dichter, M; Reisine, T

    1996-04-01

    The somatostatin receptor subtypes SSTR2 and SSTR5 mediate distinct endocrine and exocrine functions of somatostatin and may also be involved in mediating the neuromodulatory actions of somatostatin in the brain. To investigate whether these receptors couple to voltage-sensitive Ca2+ channels, SSTR2 and SSTR5 selective agonists were tested for their effects on AtT-20 cells using whole cell patch clamp techniques. The SSTR2 selective agonist MK 678 inhibited Ca2+ currents in AtT-20 cells. The effects of MK 678 were reversible and blocked by pertussis toxin pretreatment, suggesting that SSTR2 couples to the L-type Ca2+ channels via G proteins. Other SSTR2-selective agonists, including BIM 23027 and NC8-12, were able to inhibit the Ca2+ currents in these cells. The SSTR5 selective agonist BIM 23052 also inhibited the Ca2+ currents in these cells and this effect was reversible and blocked by pertussis toxin treatment. The ability of SSTR5 to mediate inhibition of the Ca2+ current was greatly attenuated by pretreatment with the SSTR5-selective agonist BIM 23052, whereas SSTR2-mediated inhibition of the Ca2+ current was not altered by pretreatment with the SSTR2-selective agonist MK 678. Thus, the SSTR2 and SSTR5 couplings to the Ca2+ current are differentially regulated. The peptide L362,855, which we previously have shown to have high affinity for the cloned SSTR5, had minimal effects on Ca2+ currents in AtT-20 cells at concentrations up to 100 nM and did not alter the ability of MK 678 to inhibit Ca2+ currents. However, it completely antagonized the effects of the SSTR5-selective agonist BIM 23052 on the Ca2+ currents. L362,855 is an antagonist/partial agonist at SSTR5 since it can reduce Ca2+ currents in these cells at concentrations above 100 nM. L362,855 is also an antagonist/partial agonist at the cloned rat SSTR5 expressed in CHO cells since it is able to block the inhibition of cAMP accumulation induced by somatostatin at concentrations below 100 nM but at

  9. Significance of KATP channels, L-type Ca2+ channels and CYP450-4A enzymes in oxygen sensing in mouse cremaster muscle arterioles In vivo

    PubMed Central

    2013-01-01

    Background ATP-sensitive K+ channels (KATP channels), NO, prostaglandins, 20-HETE and L-type Ca2+ channels have all been suggested to be involved in oxygen sensing in skeletal muscle arterioles, but the role of the individual mechanisms remain controversial. We aimed to establish the importance of these mechanisms for oxygen sensing in arterioles in an in vivo model of metabolically active skeletal muscle. For this purpose we utilized the exteriorized cremaster muscle of anesthetized mice, in which the cremaster muscle was exposed to controlled perturbation of tissue PO2. Results Change from “high” oxygen tension (PO2 = 153.4 ± 3.4 mmHg) to “low” oxygen tension (PO2 = 13.8 ± 1.3 mmHg) dilated cremaster muscle arterioles from 11.0 ± 0.4 μm to 32.9 ± 0.9 μm (n = 28, P < 0.05). Glibenclamide (KATP channel blocker) caused maximal vasoconstriction, and abolished the dilation to low oxygen, whereas the KATP channel opener cromakalim caused maximal dilation and prevented the constriction to high oxygen. When adding cromakalim on top of glibenclamide or vice versa, the reactivity to oxygen was gradually restored. Inhibition of L-type Ca2+ channels using 3 μM nifedipine did not fully block basal tone in the arterioles, but rendered them unresponsive to changes in PO2. Inhibition of the CYP450-4A enzyme using DDMS blocked vasoconstriction to an increase in PO2, but had no effect on dilation to low PO2. Conclusions We conclude that: 1) L-type Ca2+ channels are central to oxygen sensing, 2) KATP channels are permissive for the arteriolar response to oxygen, but are not directly involved in the oxygen sensing mechanism and 3) CYP450-4A mediated 20-HETE production is involved in vasoconstriction to high PO2. PMID:23663730

  10. Interaction of gonadal steroids and the glucocorticoid corticosterone in the regulation of the L-type Ca(2+) current in rat left ventricular cardiomyocytes.

    PubMed

    Wagner, M; Moritz, A; Volk, T

    2011-08-01

    Gonadal steroids as well as glucocorticoids have been shown to regulate the cardiac L-type Ca(2+) current (I(CaL) ). Herein, we compare the effects of the gonadal steroids testosterone and 17β-estradiol with the glucocorticoid corticosterone on I(CaL) , and investigate the interaction between the gonadal steroids and corticosterone. Myocytes were isolated from the left ventricular free wall of female and male Wistar rats and investigated using the ruptured-patch whole-cell patch-clamp technique. In myocytes isolated from female rats, 24 h incubation with 100 nm testosterone led to a 33% increase in I(CaL) compared with control (-8.8 ± 0.5 pA pF(-1) , n = 25 vs. -6.6 ± 0.4 pA pF(-1) , n = 26, P < 0.01, V(Pip) = 0 mV). Incubation with 1 μm corticosterone resulted in a 79% increase in I(CaL) (-11.8 ± 0.7 pA pF(-1) , n = 29, P < 0.001). However, the combination of testosterone and corticosterone did not have any additional effect compared with corticosterone alone (-11.7 ± 0.6 pA pF(-1) , n = 25, ns). In cardiomyocytes from male rats, I(CaL) was not affected by testosterone, whereas the effect of corticosterone was preserved (P < 0.05). 24 h incubation with 17β-estradiol increased I(CaL) by 32% from -7.6 ± 0.5 pA pF(-1) (n = 15) to 10.0 ± 0.9 pA pF(-1) (n = 15, P < 0.05). 17β-estradiol did not exert an additional effect upon co-incubation with corticosterone and did not have an effect on I(CaL) in cardiomyocytes from female rats. Higher concentrations of the gonadal steroids did not result in increased effects. When compared with corticosterone, the in vitro effects of the gonadal steroids are small. However, under conditions in which I(CaL) is not fully activated by glucocorticoids, gonadal steroids may significantly contribute to I(CaL) regulation. © 2011 The Authors. Acta Physiologica © 2011 Scandinavian Physiological Society.

  11. Modulation of voltage- and Ca2+-dependent gating of CaV1.3 L-type calcium channels by alternative splicing of a C-terminal regulatory domain.

    PubMed

    Singh, Anamika; Gebhart, Mathias; Fritsch, Reinhard; Sinnegger-Brauns, Martina J; Poggiani, Chiara; Hoda, Jean-Charles; Engel, Jutta; Romanin, Christoph; Striessnig, Jörg; Koschak, Alexandra

    2008-07-25

    Low voltage activation of Ca(V)1.3 L-type Ca(2+) channels controls excitability in sensory cells and central neurons as well as sinoatrial node pacemaking. Ca(V)1.3-mediated pacemaking determines neuronal vulnerability of dopaminergic striatal neurons affected in Parkinson disease. We have previously found that in Ca(V)1.4 L-type Ca(2+) channels, activation, voltage, and calcium-dependent inactivation are controlled by an intrinsic distal C-terminal modulator. Because alternative splicing in the Ca(V)1.3 alpha1 subunit C terminus gives rise to a long (Ca(V)1.3(42)) and a short form (Ca(V)1.3(42A)), we investigated if a C-terminal modulatory mechanism also controls Ca(V)1.3 gating. The biophysical properties of both splice variants were compared after heterologous expression together with beta3 and alpha2delta1 subunits in HEK-293 cells. Activation of calcium current through Ca(V)1.3(42A) channels was more pronounced at negative voltages, and inactivation was faster because of enhanced calcium-dependent inactivation. By investigating several Ca(V)1.3 channel truncations, we restricted the modulator activity to the last 116 amino acids of the C terminus. The resulting Ca(V)1.3(DeltaC116) channels showed gating properties similar to Ca(V)1.3(42A) that were reverted by co-expression of the corresponding C-terminal peptide C(116). Fluorescence resonance energy transfer experiments confirmed an intramolecular protein interaction in the C terminus of Ca(V)1.3 channels that also modulates calmodulin binding. These experiments revealed a novel mechanism of channel modulation enabling cells to tightly control Ca(V)1.3 channel activity by alternative splicing. The absence of the C-terminal modulator in short splice forms facilitates Ca(V)1.3 channel activation at lower voltages expected to favor Ca(V)1.3 activity at threshold voltages as required for modulation of neuronal firing behavior and sinoatrial node pacemaking.

  12. Bioactive Natural Product and Superacid Chemistry for Lead Compound Identification: A Case Study of Selective hCA III and L-Type Ca2+ Current Inhibitors for Hypotensive Agent Discovery.

    PubMed

    Carreyre, Hélène; Carré, Grégoire; Ouedraogo, Maurice; Vandebrouck, Clarisse; Bescond, Jocelyn; Supuran, Claudiu T; Thibaudeau, Sébastien

    2017-05-31

    Dodoneine (Ddn) is one of the active compounds identified from Agelanthus dodoneifolius , which is a medicinal plant used in African pharmacopeia and traditional medicine for the treatment of hypertension. In the context of a scientific program aiming at discovering new hypotensive agents through the original combination of natural product discovery and superacid chemistry diversification, and after evidencing dodoneine's vasorelaxant effect on rat aorta, superacid modifications allowed us to generate original analogues which showed selective human carbonic anhydrase III (hCA III) and L-type Ca 2+ current inhibition. These derivatives can now be considered as new lead compounds for vasorelaxant therapeutics targeting these two proteins.

  13. Caveolae-localized L-type Ca2+ channels do not contribute to function or hypertrophic signalling in the mouse heart.

    PubMed

    Correll, Robert N; Makarewich, Catherine A; Zhang, Hongyu; Zhang, Chen; Sargent, Michelle A; York, Allen J; Berretta, Remus M; Chen, Xiongwen; Houser, Steven R; Molkentin, Jeffery D

    2017-06-01

    L-type Ca2+ channels (LTCCs) in adult cardiomyocytes are localized to t-tubules where they initiate excitation-contraction coupling. Our recent work has shown that a subpopulation of LTCCs found at the surface sarcolemma in caveolae of adult feline cardiomyocytes can also generate a Ca2+ microdomain that activates nuclear factor of activated T-cells signaling and cardiac hypertrophy, although the relevance of this paradigm to hypertrophy regulation in vivo has not been examined. Here we generated heart-specific transgenic mice with a putative caveolae-targeted LTCC activator protein that was ineffective in initiating or enhancing cardiac hypertrophy in vivo. We also generated transgenic mice with cardiac-specific overexpression of a putative caveolae-targeted inhibitor of LTCCs, and while this protein inhibited caveolae-localized LTCCs without effects on global Ca2+ handling, it similarly had no effect on cardiac hypertrophy in vivo. Cardiac hypertrophy was elicited by pressure overload for 2 or 12 weeks or with neurohumoral agonist infusion. Caveolae-specific LTCC activator or inhibitor transgenic mice showed no greater change in nuclear factor of activated T-cells activity after 2 weeks of pressure overload stimulation compared with control mice. Our results indicate that LTCCs in the caveolae microdomain do not affect cardiac function and are not necessary for the regulation of hypertrophic signaling in the adult mouse heart. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.

  14. Prolonged post-inhibitory rebound firing in the cerebellar nuclei mediated by group I mGluR potentiation of L-type Ca currents

    PubMed Central

    Zheng, Nan; Raman, Indira M.

    2011-01-01

    Neurons in the cerebellar nuclei fire at accelerated rates for prolonged periods after trains of synaptic inhibition that interrupt spontaneous firing. Both in vitro and in vivo, however, this prolonged rebound firing is favored by strong stimulation of afferents, suggesting that neurotransmitters other than GABA may contribute to the increased firing rates. Here, we tested whether metabotropic glutamate receptors modulate excitability of nuclear cells in cerebellar slices from mouse. In current clamp, the prolonged rebound firing rate after high-frequency synaptic stimulation was reduced by a variety of group I mGluR antagonists, including CPCCOEt (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester), JNJ16259685 ((3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone)+MPEP, or 3-MATIDA (α-amino-5-carboxy-3-methyl-2-thiopheneacetic acid) +MPEP, as long as both mGluR1 and mGluR5 were blocked. This mGluR-dependent acceleration of firing was reduced but still evident when IPSPs were prevented by GABAA receptor antagonists. In voltage clamp, voltage ramps revealed a non-inactivating, low-voltage-activated, nimodipine-sensitive current that was enhanced by the selective group I mGluR agonist s-DHPG ((S)-3,5-dihydroxyphenylglycine). This putative L-type current also increased when mGluRs were activated by trains of evoked synaptic currents instead of direct application of agonist. In current clamp, blocking L-type Ca channels with the specific blocker nifedipine greatly reduced prolonged post-stimulus firing and occluded the effect of adding group I mGluR antagonists. Thus, potentiation of a low-voltage-activated L-type current by synaptically released glutamate accounted nearly fully for the mGluR-dependent acceleration of firing. Together, these data suggest that prolonged rebound firing in the cerebellar nuclei in vivo is most likely to occur when GABAA and mGluRs are simultaneously activated by concurrent excitation and

  15. Deoxycholic acid inhibits smooth muscle contraction via protein kinase C-dependent modulation of L-type Ca2+ channels in rat proximal colon.

    PubMed

    Hu, Liu-Dan; Yu, Bao-Ping; Yang, Bin

    2012-10-01

    The aim of this study was to investigate the effects of deoxycholic acid (DCA) on the contractions of rat proximal colonic smooth muscle (PCSM) in vitro. The contractile response of rat PCSM strips was tested using a polyphysio-graph. The whole cell patch-clamp technique was also used in rat colonic smooth muscle cells (SMCs) isolated by an enzymatic procedure to record the L-type calcium current (I(Ca-L)) prior to and following the application of various concentrations of DCA. The application of DCA (10(-6)-10(-4) M) decreased the amplitude of spontaneous contractions of the PCSM strips in a dose-dependent manner. The administration of DCA (10(-5) M) caused the relaxation of isolated smooth muscle strips pre-contracted by acetylcholine (Ach) or KCl (by 12.2±1.5 and 16.3±6.9%, respectively). The concentration-response curve of CaCl2 was shifted to the right. Pre-treatment of the strips with the protein kinase C (PKC) inhibitor chelerythrine (1 µM) significantly attenuated the effects of DCA on the strips pre-contracted by Ach. DCA reduced the peak I(Ca-L) by 6.02±0.87% at 10(-6) M, 15.02±1.73% at 10(-5) M and 47.14±3.79% at 10(-4) M. DCA shifted the current-voltage (I-V) curve of ICa-L upward, but the contour of the I-V curve was unchanged, and the peak current-induced voltage remained at 0 mV. Pre-treatment with chelerythrine (1 µM) blocked the actions of DCA on the I(Ca-L). Taken together, the actions of DCA on I(Ca-L) in rat colonic SMCs contributed to a negative inotropic effect. These actions appear to be mediated through protein kinase C. Furthermore, this study suggests another possible mechanism for the DCA-related modulation of gastrointestinal motility.

  16. Electrophysiological properties of myocytes isolated from the mouse atrioventricular node: L-type ICa, IKr, If, and Na-Ca exchange

    PubMed Central

    Choisy, Stéphanie C; Cheng, Hongwei; Orchard, Clive H; James, Andrew F; Hancox, Jules C

    2015-01-01

    The atrioventricular node (AVN) is a key component of the cardiac pacemaker-conduction system. This study investigated the electrophysiology of cells isolated from the AVN region of adult mouse hearts, and compared murine ionic current magnitude with that of cells from the more extensively studied rabbit AVN. Whole-cell patch-clamp recordings of ionic currents, and perforated-patch recordings of action potentials (APs), were made at 35–37°C. Hyperpolarizing voltage commands from −40 mV elicited a Ba2+-sensitive inward rectifier current that was small at diastolic potentials. Some cells (Type 1; 33.4 ± 2.2 pF; n = 19) lacked the pacemaker current, If, whilst others (Type 2; 34.2 ± 1.5 pF; n = 21) exhibited a clear If, which was larger than in rabbit AVN cells. On depolarization from −40 mV L-type Ca2+ current, ICa,L, was elicited with a half maximal activation voltage (V0.5) of −7.6 ± 1.2 mV (n = 24). ICa,L density was smaller than in rabbit AVN cells. Rapid delayed rectifier (IKr) tail currents sensitive to E-4031 (5 μmol/L) were observed on repolarization to −40 mV, with an activation V0.5 of −10.7 ± 4.7 mV (n = 8). The IKr magnitude was similar in mouse and rabbit AVN. Under Na-Ca exchange selective conditions, mouse AVN cells exhibited 5 mmol/L Ni-sensitive exchange current that was inwardly directed negative to the holding potential (−40 mV). Spontaneous APs (5.2 ± 0.5 sec−1; n = 6) exhibited an upstroke velocity of 37.7 ± 16.2 V/s and ceased following inhibition of sarcoplasmic reticulum Ca2+ release by 1 μmol/L ryanodine, implicating intracellular Ca2+ cycling in murine AVN cell electrogenesis. PMID:26607172

  17. Electrophysiological properties of myocytes isolated from the mouse atrioventricular node: L-type ICa, IKr, If, and Na-Ca exchange.

    PubMed

    Choisy, Stéphanie C; Cheng, Hongwei; Orchard, Clive H; James, Andrew F; Hancox, Jules C

    2015-11-01

    The atrioventricular node (AVN) is a key component of the cardiac pacemaker-conduction system. This study investigated the electrophysiology of cells isolated from the AVN region of adult mouse hearts, and compared murine ionic current magnitude with that of cells from the more extensively studied rabbit AVN. Whole-cell patch-clamp recordings of ionic currents, and perforated-patch recordings of action potentials (APs), were made at 35-37°C. Hyperpolarizing voltage commands from -40 mV elicited a Ba(2+)-sensitive inward rectifier current that was small at diastolic potentials. Some cells (Type 1; 33.4 ± 2.2 pF; n = 19) lacked the pacemaker current, If, whilst others (Type 2; 34.2 ± 1.5 pF; n = 21) exhibited a clear If, which was larger than in rabbit AVN cells. On depolarization from -40 mV L-type Ca(2+) current, IC a,L, was elicited with a half maximal activation voltage (V0.5) of -7.6 ± 1.2 mV (n = 24). IC a,L density was smaller than in rabbit AVN cells. Rapid delayed rectifier (IK r) tail currents sensitive to E-4031 (5 μmol/L) were observed on repolarization to -40 mV, with an activation V0.5 of -10.7 ± 4.7 mV (n = 8). The IK r magnitude was similar in mouse and rabbit AVN. Under Na-Ca exchange selective conditions, mouse AVN cells exhibited 5 mmol/L Ni-sensitive exchange current that was inwardly directed negative to the holding potential (-40 mV). Spontaneous APs (5.2 ± 0.5 sec(-1); n = 6) exhibited an upstroke velocity of 37.7 ± 16.2 V/s and ceased following inhibition of sarcoplasmic reticulum Ca(2+) release by 1 μmol/L ryanodine, implicating intracellular Ca(2+) cycling in murine AVN cell electrogenesis. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  18. L-type Ca²⁺ channel blockade with antihypertensive medication disrupts VTA synaptic plasticity and drug-associated contextual memory.

    PubMed

    Degoulet, M; Stelly, C E; Ahn, K-C; Morikawa, H

    2016-03-01

    Drug addiction is driven, in part, by powerful and enduring memories of sensory cues associated with drug intake. As such, relapse to drug use during abstinence is frequently triggered by an encounter with drug-associated cues, including the drug itself. L-type Ca(2+) channels (LTCCs) are known to regulate different forms of synaptic plasticity, the major neural substrate for learning and memory, in various brain areas. Long-term potentiation (LTP) of NMDA receptor (NMDAR)-mediated glutamatergic transmission in the ventral tegmental area (VTA) may contribute to the increased motivational valence of drug-associated cues triggering relapse. In this study, using rat brain slices, we found that isradipine, a general LTCC antagonist used as antihypertensive medication, not only blocks the induction of NMDAR LTP but also promotes the reversal of previously induced LTP in the VTA. In behaving rats, isradipine injected into the VTA suppressed the acquisition of cocaine-paired contextual cue memory assessed using a conditioned place preference (CPP) paradigm. Furthermore, administration of isradipine or a CaV1.3 subtype-selective LTCC antagonist (systemic or intra-VTA) before a single extinction or reinstatement session, while having no immediate effect at the time of administration, abolished previously acquired cocaine and alcohol (ethanol) CPP on subsequent days. Notably, CPP thus extinguished cannot be reinstated by drug re-exposure, even after 2 weeks of withdrawal. These results suggest that LTCC blockade during exposure to drug-associated cues may cause unlearning of the increased valence of those cues, presumably via reversal of glutamatergic synaptic plasticity in the VTA.

  19. M3 cholinoreceptors alter electrical activity of rat left atrium via suppression of L-type Ca2+ current without affecting K+ conductance.

    PubMed

    Filatova, Tatiana S; Naumenko, Nikolay; Galenko-Yaroshevsky, Pavel A; Abramochkin, Denis V

    2017-05-01

    Electrophysiological effects produced by selective activation of M3 cholinoreceptors were studied in isolated left atrium preparations from rat using the standard sharp glass microelectrode technique. The stimulation of M3 receptors was obtained by application of muscarinic agonist pilocarpine (10 -5  M) in the presence of selective M2 antagonist methoctramine (10 -7  M). Stimulation of M3 receptors induced marked reduction of action potential duration by 14.4 ± 2.4% and 16.1 ± 2.5% of control duration measured at 50 and 90% of repolarization, respectively. This effect was completely abolished by selective M3 blocker 4-DAMP (10 -8  M). In isolated myocytes obtained from the rat left atrium, similar pharmacological stimulation of M3 receptors led to suppression of peak L-type calcium current by 13.9 ± 2.6% of control amplitude (measured at +10 mV), but failed to affect K + currents I to , I Kur , and I Kir . In the absence of M2 blocker methoctramine, pilocarpine (10 -5  M) produced stronger attenuation of I CaL and induced an increase in I Kir . This additive inward rectifier current could be abolished by highly selective blocker of K ir 3.1/3.4 channels tertiapin-Q (10 -6  M) and therefore was identified as I KACh . Thus, in the rat atrial myocardium activation of M3 receptors leads to shortening of action potentials via suppression of I CaL , but does not enhance the major potassium currents involved in repolarization. Joint stimulation of M2 and M3 receptors produces stronger action potential shortening due to M2-mediated activation of I KACh.

  20. C-terminal modulatory domain controls coupling of voltage-sensing to pore opening in Cav1.3 L-type Ca(2+) channels.

    PubMed

    Lieb, Andreas; Ortner, Nadine; Striessnig, Jörg

    2014-04-01

    Activity of voltage-gated Cav1.3 L-type Ca(2+) channels is required for proper hearing as well as sinoatrial node and brain function. This critically depends on their negative activation voltage range, which is further fine-tuned by alternative splicing. Shorter variants miss a C-terminal regulatory domain (CTM), which allows them to activate at even more negative potentials than C-terminally long-splice variants. It is at present unclear whether this is due to an increased voltage sensitivity of the Cav1.3 voltage-sensing domain, or an enhanced coupling of voltage-sensor conformational changes to the subsequent opening of the activation gate. We studied the voltage-dependence of voltage-sensor charge movement (QON-V) and of current activation (ICa-V) of the long (Cav1.3L) and a short Cav1.3 splice variant (Cav1.342A) expressed in tsA-201 cells using whole cell patch-clamp. Charge movement (QON) of Cav1.3L displayed a much steeper voltage-dependence and a more negative half-maximal activation voltage than Cav1.2 and Cav3.1. However, a significantly higher fraction of the total charge had to move for activation of Cav1.3 half-maximal conductance (Cav1.3: 68%; Cav1.2: 52%; Cav3.1: 22%). This indicated a weaker coupling of Cav1.3 voltage-sensor charge movement to pore opening. However, the coupling efficiency was strengthened in the absence of the CTM in Cav1.342A, thereby shifting ICa-V by 7.2 mV to potentials that were more negative without changing QON-V. We independently show that the presence of intracellular organic cations (such as n-methyl-D-glucamine) induces a pronounced negative shift of QON-V and a more negative activation of ICa-V of all three channels. These findings illustrate that the voltage sensors of Cav1.3 channels respond more sensitively to depolarization than those of Cav1.2 or Cav3.1. Weak coupling of voltage sensing to pore opening is enhanced in the absence of the CTM, allowing short Cav1.342A splice variants to activate at lower voltages

  1. Rotenone-stimulated superoxide release from mitochondrial complex I acutely augments L-type Ca2+ current in A7r5 aortic smooth muscle cells

    PubMed Central

    Dhagia, Vidhi; Lakhkar, Anand; Patel, Dhara; Wolin, Michael S.; Gupte, Sachin A.

    2016-01-01

    Voltage-gated L-type Ca2+ current (ICa,L) induces contraction of arterial smooth muscle cells (ASMCs), and ICa,L is increased by H2O2 in ASMCs. Superoxide released from the mitochondrial respiratory chain (MRC) is dismutated to H2O2. We studied whether superoxide per se acutely modulates ICa,L in ASMCs using cultured A7r5 cells derived from rat aorta. Rotenone is a toxin that inhibits complex I of the MRC and increases mitochondrial superoxide release. The superoxide content of mitochondria was estimated using mitochondrial-specific MitoSOX and HPLC methods, and was shown to be increased by a brief exposure to 10 μM rotenone. ICa,L was recorded with 5 mM BAPTA in the pipette solution. Rotenone administration (10 nM to 10 μM) resulted in a greater ICa,L increase in a dose-dependent manner to a maximum of 22.1% at 10 μM for 1 min, which gradually decreased to 9% after 5 min. The rotenone-induced ICa,L increase was associated with a shift in the current-voltage relationship (I-V) to a hyperpolarizing direction. DTT administration resulted in a 17.9% increase in ICa,L without a negative shift in I–V, and rotenone produced an additional increase with a shift. H2O2 (0.3 mM) inhibited ICa,L by 13%, and additional rotenone induced an increase with a negative shift. Sustained treatment with Tempol (4-hydroxy tempo) led to a significant ICa,L increase but it inhibited the rotenone-induced increase. Staurosporine, a broad-spectrum protein kinase inhibitor, partially inhibited ICa,L and completely suppressed the rotenone-induced increase. Superoxide released from mitochondria affected protein kinases and resulted in stronger ICa,L preceding its dismutation to H2O2. The removal of nitric oxide is a likely mechanism for the increase in ICa,L. PMID:26873970

  2. Crystal structure of dimeric cardiac L-type calcium channel regulatory domains bridged by Ca[superscript 2+]·calmodulins

    SciTech Connect

    Fallon, Jennifer L.; Baker, Mariah R.; Xiong, Liangwen

    2009-11-10

    Voltage-dependent calcium channels (Ca(V)) open in response to changes in membrane potential, but their activity is modulated by Ca(2+) binding to calmodulin (CaM). Structural studies of this family of channels have focused on CaM bound to the IQ motif; however, the minimal differences between structures cannot adequately describe CaM's role in the regulation of these channels. We report a unique crystal structure of a 77-residue fragment of the Ca(V)1.2 alpha(1) subunit carboxyl terminus, which includes a tandem of the pre-IQ and IQ domains, in complex with Ca(2+).CaM in 2 distinct binding modes. The structure of the Ca(V)1.2 fragment is anmore » unusual dimer of 2 coiled-coiled pre-IQ regions bridged by 2 Ca(2+).CaMs interacting with the pre-IQ regions and a canonical Ca(V)1-IQ-Ca(2+).CaM complex. Native Ca(V)1.2 channels are shown to be a mixture of monomers/dimers and a point mutation in the pre-IQ region predicted to abolish the coiled-coil structure significantly reduces Ca(2+)-dependent inactivation of heterologously expressed Ca(V)1.2 channels.« less

  3. Compensatory T-type Ca2+ channel activity alters D2-autoreceptor responses of Substantia nigra dopamine neurons from Cav1.3 L-type Ca2+ channel KO mice.

    PubMed

    Poetschke, Christina; Dragicevic, Elena; Duda, Johanna; Benkert, Julia; Dougalis, Antonios; DeZio, Roberta; Snutch, Terrance P; Striessnig, Joerg; Liss, Birgit

    2015-09-18

    The preferential degeneration of Substantia nigra dopamine midbrain neurons (SN DA) causes the motor-symptoms of Parkinson's disease (PD). Voltage-gated L-type calcium channels (LTCCs), especially the Cav1.3-subtype, generate an activity-related oscillatory Ca(2+) burden in SN DA neurons, contributing to their degeneration and PD. While LTCC-blockers are already in clinical trials as PD-therapy, age-dependent functional roles of Cav1.3 LTCCs in SN DA neurons remain unclear. Thus, we analysed juvenile and adult Cav1.3-deficient mice with electrophysiological and molecular techniques. To unmask compensatory effects, we compared Cav1.3 KO mice with pharmacological LTCC-inhibition. LTCC-function was not necessary for SN DA pacemaker-activity at either age, but rather contributed to their pacemaker-precision. Moreover, juvenile Cav1.3 KO but not WT mice displayed adult wildtype-like, sensitised inhibitory dopamine-D2-autoreceptor (D2-AR) responses that depended upon both, interaction of the neuronal calcium sensor NCS-1 with D2-ARs, and on voltage-gated T-type calcium channel (TTCC) activity. This functional KO-phenotype was accompanied by cell-specific up-regulation of NCS-1 and Cav3.1-TTCC mRNA. Furthermore, in wildtype we identified an age-dependent switch of TTCC-function from contributing to SN DA pacemaker-precision in juveniles to pacemaker-frequency in adults. This novel interplay of Cav1.3 L-type and Cav3.1 T-type channels, and their modulation of SN DA activity-pattern and D2-AR-sensitisation, provide new insights into flexible age- and calcium-dependent activity-control of SN DA neurons and its pharmacological modulation.

  4. Ca2+–calmodulin-dependent protein kinase II represses cardiac transcription of the L-type calcium channel α1C-subunit gene (Cacna1c) by DREAM translocation

    PubMed Central

    Ronkainen, Jarkko J; Hänninen, Sandra L; Korhonen, Topi; Koivumäki, Jussi T; Skoumal, Reka; Rautio, Sini; Ronkainen, Veli-Pekka; Tavi, Pasi

    2011-01-01

    Abstract Recent studies have demonstrated that changes in the activity of calcium–calmodulin-dependent protein kinase II (CaMKII) induce a unique cardiomyocyte phenotype through the regulation of specific genes involved in excitation–contraction (E–C)-coupling. To explain the transcriptional effects of CaMKII we identified a novel CaMKII-dependent pathway for controlling the expression of the pore-forming α-subunit (Cav1.2) of the L-type calcium channel (LTCC) in cardiac myocytes. We show that overexpression of either cytosolic (δC) or nuclear (δB) CaMKII isoforms selectively downregulate the expression of the Cav1.2. Pharmacological inhibition of CaMKII activity induced measurable changes in LTCC current density and subsequent changes in cardiomyocyte calcium signalling in less than 24 h. The effect of CaMKII on the α1C-subunit gene (Cacna1c) promoter was abolished by deletion of the downstream regulatory element (DRE), which binds transcriptional repressor DREAM/calsenilin/KChIP3. Imaging DREAM–GFP (green fluorescent protein)-expressing cardiomyocytes showed that CaMKII potentiates the calcium-induced nuclear translocation of DREAM. Thereby CaMKII increases DREAM binding to the DRE consensus sequence of the endogenous Cacna1c gene. By mathematical modelling we demonstrate that the LTCC downregulation through the Ca2+–CaMKII–DREAM cascade constitutes a physiological feedback mechanism enabling cardiomyocytes to adjust the calcium intrusion through LTCCs to the amount of intracellular calcium detected by CaMKII. PMID:21486818

  5. Alpha-latrotoxin induces exocytosis by inhibition of voltage-dependent K+ channels and by stimulation of L-type Ca2+ channels via latrophilin in beta-cells.

    PubMed

    Lajus, Sophie; Vacher, Pierre; Huber, Denise; Dubois, Mathilde; Benassy, Marie-Noëlle; Ushkaryov, Yuri; Lang, Jochen

    2006-03-03

    The spider venom alpha-latrotoxin (alpha-LTX) induces massive exocytosis after binding to surface receptors, and its mechanism is not fully understood. We have investigated its action using toxin-sensitive MIN6 beta-cells, which express endogenously the alpha-LTX receptor latrophilin (LPH), and toxin-insensitive HIT-T15 beta-cells, which lack endogenous LPH. alpha-LTX evoked insulin exocytosis in HIT-T15 cells only upon expression of full-length LPH but not of LPH truncated after the first transmembrane domain (LPH-TD1). In HIT-T15 cells expressing full-length LPH and in native MIN6 cells, alpha-LTX first induced membrane depolarization by inhibition of repolarizing K(+) channels followed by the appearance of Ca(2+) transients. In a second phase, the toxin induced a large inward current and a prominent increase in intracellular calcium ([Ca(2+)](i)) reflecting pore formation. Upon expression of LPH-TD1 in HIT-T15 cells just this second phase was observed. Moreover, the mutated toxin LTX(N4C), which is devoid of pore formation, only evoked oscillations of membrane potential by reversible inhibition of iberiotoxin-sensitive K(+) channels via phospholipase C, activated L-type Ca(2+) channels independently from its effect on membrane potential, and induced an inositol 1,4,5-trisphosphate receptor-dependent release of intracellular calcium in MIN6 cells. The combined effects evoked transient increases in [Ca(2+)](i) in these cells, which were sensitive to inhibitors of phospholipase C, protein kinase C, or L-type Ca(2+) channels. The latter agents also reduced toxin-induced insulin exocytosis. In conclusion, alpha-LTX induces signaling distinct from pore formation via full-length LPH and phospholipase C to regulate physiologically important K(+) and Ca(2+) channels as novel targets of its secretory activity.

  6. Pediatric Dilated Cardiomyopathy-Associated LRRC10 (Leucine-Rich Repeat-Containing 10) Variant Reveals LRRC10 as an Auxiliary Subunit of Cardiac L-Type Ca2+ Channels.

    PubMed

    Woon, Marites T; Long, Pamela A; Reilly, Louise; Evans, Jared M; Keefe, Alexis M; Lea, Martin R; Beglinger, Carl J; Balijepalli, Ravi C; Lee, Youngsook; Olson, Timothy M; Kamp, Timothy J

    2018-02-03

    Genetic causes of dilated cardiomyopathy (DCM) are incompletely understood. LRRC10 (leucine-rich repeat-containing 10) is a cardiac-specific protein of unknown function. Heterozygous mutations in LRRC10 have been suggested to cause DCM, and deletion of Lrrc10 in mice results in DCM. Whole-exome sequencing was carried out on a patient who presented at 6 weeks of age with DCM and her unaffected parents, filtering for rare, deleterious, recessive, and de novo variants. Whole-exome sequencing followed by trio-based filtering identified a homozygous recessive variant in LRRC10 , I195T. Coexpression of I195T LRRC10 with the L-type Ca 2+ channel (Ca v 1.2, β 2CN2 , and α 2 δ subunits) in HEK293 cells resulted in a significant ≈0.5-fold decrease in I Ca,L at 0 mV, in contrast to the ≈1.4-fold increase in I Ca,L by coexpression of LRRC10 (n=9-12, P <0.05). Coexpression of LRRC10 or I195T LRRC10 did not alter the surface membrane expression of Ca v 1.2. LRRC10 coexpression with Ca v 1.2 in the absence of auxiliary β 2CN2 and α 2 δ subunits revealed coassociation of Ca v 1.2 and LRRC10 and a hyperpolarizing shift in the voltage dependence of activation (n=6-9, P <0.05). Ventricular myocytes from Lrrc10 -/- mice had significantly smaller I Ca,L , and coimmunoprecipitation experiments confirmed association between LRRC10 and the Ca v 1.2 subunit in mouse hearts. Examination of a patient with DCM revealed homozygosity for a previously unreported LRRC10 variant: I195T. Wild-type and I195T LRRC10 function as cardiac-specific subunits of L-type Ca 2+ channels and exert dramatically different effects on channel gating, providing a potential link to DCM. © 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

  7. Central Nervous System-Toxic Lidocaine Concentrations Unmask L-Type Ca²⁺ Current-Mediated Action Potentials in Rat Thalamocortical Neurons: An In Vitro Mechanism of Action Study.

    PubMed

    Putrenko, Igor; Ghavanini, Amer A; Meyer Schöniger, Katrin S; Schwarz, Stephan K W

    2016-05-01

    High systemic lidocaine concentrations exert well-known toxic effects on the central nervous system (CNS), including seizures, coma, and death. The underlying mechanisms are still largely obscure, and the actions of lidocaine on supraspinal neurons have received comparatively little study. We recently found that lidocaine at clinically neurotoxic concentrations increases excitability mediated by Na-independent, high-threshold (HT) action potential spikes in rat thalamocortical neurons. Our goal in this study was to characterize these spikes and test the hypothesis that they are generated by HT Ca currents, previously implicated in neurotoxicity. We also sought to identify and isolate the specific underlying subtype of Ca current. We investigated the actions of lidocaine in the CNS-toxic concentration range (100 μM-1 mM) on ventrobasal thalamocortical neurons in rat brain slices in vitro, using whole-cell patch-clamp recordings aided by differential interference contrast infrared videomicroscopy. Drugs were bath applied; action potentials were generated using current clamp protocols, and underlying currents were identified and isolated with ion channel blockers and electrolyte substitution. Lidocaine (100 μM-1 mM) abolished Na-dependent tonic firing in all neurons tested (n = 46). However, in 39 of 46 (85%) neurons, lidocaine unmasked evoked HT action potentials with lower amplitudes and rates of de-/repolarization compared with control. These HT action potentials remained during the application of tetrodotoxin (600 nM), were blocked by Cd (50 μM), and disappeared after superfusion with an extracellular solution deprived of Ca. These features implied that the unmasked potentials were generated by high-voltage-activated Ca channels and not by Na channels. Application of the L-type Ca channel blocker, nifedipine (5 μM), completely blocked the HT potentials, whereas the N-type Ca channel blocker, ω-conotoxin GVIA (1 μM), had little effect. At clinically CNS

  8. Estradiol up-regulates L-type Ca2+ channels via membrane-bound estrogen receptor/phosphoinositide-3-kinase/Akt/cAMP response element-binding protein signaling pathway.

    PubMed

    Yang, Xiaoyan; Mao, Xiaofang; Xu, Gao; Xing, Shasha; Chattopadhyay, Ansuman; Jin, Si; Salama, Guy

    2018-05-01

    In long QT syndrome type 2, women are more prone than men to the lethal arrhythmia torsades de pointes. We previously reported that 17β-estradiol (E2) up-regulates L-type Ca 2+ channels and current (I Ca,L ) (∼30%) in rabbit ventricular myocytes by a classic genomic mechanism mediated by estrogen receptor-α (ERα). In long QT syndrome type 2 (I Kr blockade or bradycardia), the higher Ca 2+ influx via I Ca,L causes Ca 2+ overload, spontaneous sarcoplasmic reticulum Ca 2+ release, and reactivation of I Ca,L that triggers early afterdepolarizations and torsades de pointes. The purpose of this study was to investigate the molecular mechanisms whereby E2 up-regulates I Ca,L , which are poorly understood. H9C2 and rat myocytes were incubated with E2 ± ER antagonist, or inhibitors of downstream transcription factors, for 24 hours, followed by western blots of Cav1.2α1C and voltage-clamp measurements of I Ca,L . Incubation of H9C2 cells with E2 (10-100 nM) increased I Ca,L density and Cav1.2α1C expression, which were suppressed by the ER antagonist ICI182,780 (1 μM). Enhanced I Ca,L and Cav1.2α1C expression by E2 was suppressed by inhibitors of phosphoinositide-3-kinase (Pi3K) (30 μM LY294002; P <.05) and Akt (5 μM MK2206) but not of mitogen-activated protein kinase (5 μM U0126) or protein kinase A (1 μM KT5720). E2 incubation increased p-CREB via the Pi3K/Akt pathway, reached a peak in 20 minutes (3-fold), and leveled off to 1.5-fold 24 hours later. Furthermore, a CREB decoy oligonucleotide inhibited E2-induced Cav1.2α1C expression, whereas membrane-impermeable E2 (E2-bovine serum albumin) was equally effective at Cav1.2α1C up-regulation as E2. Estradiol up-regulates Cav1.2α1C and I Ca,L via plasma membrane ER and by activating Pi3K, Akt, and CREB signaling. The promoter regions of the CACNA1C gene (human-rabbit-rat) contain adjacent/overlapping binding sites for p-CREB and ERα, which suggests a synergistic regulation by these pathways. Copyright © 2018

  9. PDGF-induced migration of synthetic vascular smooth muscle cells through c-Src-activated L-type Ca2+ channels with full-length CaV1.2 C-terminus.

    PubMed

    Guo, Xiaoguang; Kashihara, Toshihide; Nakada, Tsutomu; Aoyama, Toshifumi; Yamada, Mitsuhiko

    2018-06-01

    In atherosclerosis, vascular smooth muscle cells (VSMC) migrate from the media toward the intima of the arteries in response to cytokines, such as platelet-derived growth factor (PDGF). However, molecular mechanism underlying the PDGF-induced migration of VSMCs remains unclear. The migration of rat aorta-derived synthetic VSMCs, A7r5, in response to PDGF was potently inhibited by a Ca V 1.2 channel inhibitor, nifedipine, and a Src family tyrosine kinase (SFK)/Abl inhibitor, bosutinib, in a less-than-additive manner. PDGF significantly increased Ca V 1.2 channel currents without altering Ca V 1.2 protein expression levels in A7r5 cells. This reaction was inhibited by C-terminal Src kinase, a selective inhibitor of SFKs. In contractile VSMCs, the C-terminus of Ca V 1.2 is proteolytically cleaved into proximal and distal C-termini (PCT and DCT, respectively). Clipped DCT is noncovalently reassociated with PCT to autoinhibit the channel activity. Conversely, in synthetic A7r5 cells, full-length Ca V 1.2 (Ca V 1.2FL) is expressed much more abundantly than truncated Ca V 1.2. In a heterologous expression system, c-Src activated Ca V 1.2 channels composed of Ca V 1.2FL but not truncated Ca V 1.2 (Ca V 1.2Δ1763) or Ca V 1.2Δ1763 plus clipped DCT. Further, c-Src enhanced the coupling efficiency between the voltage-sensing domain and activation gate of Ca V 1.2FL channels by phosphorylating Tyr1709 and Tyr1758 in PCT. Compared with Ca V 1.2Δ1763, c-Src could more efficiently bind to and phosphorylate Ca V 1.2FL irrespective of the presence or absence of clipped DCT. Therefore, in atherosclerotic lesions, phenotypic switching of VSMCs may facilitate pro-migratory effects of PDGF on VSMCs by suppressing posttranslational Ca V 1.2 modifications.

  10. Psychostimulants, antidepressants and neurokinin-1 receptor antagonists ('motor disinhibitors') have overlapping, but distinct, effects on monoamine transmission: the involvement of L-type Ca2+ channels and implications for the treatment of ADHD.

    PubMed

    Stanford, S Clare

    2014-12-01

    Both psychostimulants and antidepressants target monoamine transporters and, as a consequence, augment monoamine transmission. These two groups of drugs also increase motor activity in preclinical behavioural screens for antidepressants. Substance P-preferring receptor (NK1R) antagonists similarly increase both motor activity in these tests and monoamine transmission in the brain. In this article, the neurochemical and behavioural responses to these three groups of drugs are compared. It becomes evident that NK1R antagonists represent a distinct class of compounds ('motor disinhibitors') that differ substantially from both psychostimulants and antidepressants, especially during states of heightened arousal or stress. Also, all three groups of drugs influence the activation of voltage-gated Ca(v)1.2 and Ca(v)1.3 L-type channels (LTCCs) in the brain, albeit in different ways. This article discusses evidence that points to disruption of these functional interactions between NK1R and LTCCs as a contributing factor in the cognitive and behavioural abnormalities that are prominent features of Attention Deficit Hyperactivity Disorder (ADHD). Arising from this is the interesting possibility that the hyperactivity and impulsivity (as in ADHD) and psychomotor retardation (as in depression) reflect opposite poles of a behavioural continuum. A better understanding of this pharmacological network could help explain why psychostimulants augment motor behaviour during stress (e.g., in preclinical screens for antidepressants) and yet reduce locomotor activity and impulsivity in ADHD. This article is part of the Special Issue entitled 'CNS Stimulants'. Copyright © 2014 The Author. Published by Elsevier Ltd.. All rights reserved.

  11. Down-regulation of L-type calcium channel and sarcoplasmic reticular Ca(2+)-ATPase mRNA in human atrial fibrillation without significant change in the mRNA of ryanodine receptor, calsequestrin and phospholamban: an insight into the mechanism of atrial electrical remodeling.

    PubMed

    Lai, L P; Su, M J; Lin, J L; Lin, F Y; Tsai, C H; Chen, Y S; Huang, S K; Tseng, Y Z; Lien, W P

    1999-04-01

    We investigated the gene expression of calcium-handling genes including L-type calcium channel, sarcoplasmic reticular calcium adenosine triphosphatase (Ca(2+)-ATPase), ryanodine receptor, calsequestrin and phospholamban in human atrial fibrillation. Recent studies have demonstrated that atrial electrical remodeling in atrial fibrillation is associated with intracellular calcium overload. However, the changes of calcium-handling proteins remain unclear. A total of 34 patients undergoing open heart surgery were included. Atrial tissue was obtained from the right atrial free wall, right atrial appendage, left atrial free wall and left atrial appendage, respectively. The messenger ribonucleic acid (mRNA) amount of the genes was measured by reverse transcription-polymerase chain reaction and normalized to the mRNA levels of glyceraldehyde 3-phosphate dehydrogenase. The mRNA of L-type calcium channel and of Ca(2+)-ATPase was significantly decreased in patients with persistent atrial fibrillation for more than 3 months (0.36+/-0.26 vs. 0.90+/-0.88 for L-type calcium channel; 0.69+/-0.42 vs. 1.21+/-0.68 for Ca(2+)-ATPase; both p < 0.05, all data in arbitrary unit). We further demonstrated that there was no spatial dispersion of the gene expression among the four atrial tissue sampling sites. Age, gender and underlying cardiac disease had no significant effects on the gene expression. In contrast, the mRNA levels of ryanodine receptor, calsequestrin and phospholamban showed no significant change in atrial fibrillation. L-type calcium channel and the sarcoplasmic reticular Ca(2+)-ATPase gene were down-regulated in atrial fibrillation. These changes may be a consequence of, as well as a contributory factor for, atrial fibrillation.

  12. High-Frequency Stimulation-Induced Synaptic Potentiation in Dorsal and Ventral CA1 Hippocampal Synapses: The Involvement of NMDA Receptors, mGluR5, and (L-Type) Voltage-Gated Calcium Channels

    ERIC Educational Resources Information Center

    Papatheodoropoulos, Costas; Kouvaros, Stylianos

    2016-01-01

    The ability of the ventral hippocampus (VH) for long-lasting long-term potentiation (LTP) and the mechanisms underlying its lower ability for shortlasting LTP compared with the dorsal hippocampus (DH) are unknown. Using recordings of field excitatory postsynaptic potentials (EPSPs) from the CA1 field of adult rat hippocampal slices, we found that…

  13. Blocking the L-type Ca2+ channel (Cav 1.2) is the key mechanism for the vascular relaxing effect of Pterodon spp. and its isolated diterpene methyl-6α-acetoxy-7β-hydroxyvouacapan-17β-oate.

    PubMed

    de Fátima Reis, Carolina; de Andrade, Daniela Medeiros Lobo; Junior Neves, Bruno; de Almeida Ribeiro Oliveira, Leandra; Pinho, José Felippe; da Silva, Leidiane Pinha; Dos Santos Cruz, Jader; Bara, Maria Teresa Freitas; Andrade, Carolina Horta; Rocha, Matheus Lavorenti

    2015-10-01

    Pterodon spp. Vogel (Fabaceae), popularly known as "sucupira", has ethnopharmacological application which is described as having antispasmodic and relaxant effects. Hence, it was hypothesized that sucupira oil-resin (SOR) could induce smooth muscle relaxation. So, this study investigated the mechanisms involved in the vasorelaxant effect of SOR and its isolated diterpene (methyl-6α-acetoxy-7β-hydroxyvouacapan-17β-oate). Vascular reactivity experiments were performed using rat aortic rings (n=5-8) with (E+) or without endothelium (E-) in an isolated bath organ. The SOR (0-56 μg/mL) relaxed phenylephrine (E+: 86.7±7.1%; E-: 92.3±4.7%) and KCl contracted rings (E-: 97.1±2.8%). In the same way, diterpene (0-48 μg/mL) also relaxed phenylephrine (E+: 94.5±3.6%; E-: 92.2±3.4%) and KCl contracted rings (E-: 99.7±0.2%). The pre-incubation of arterial rings with cyclopiazonic acid (reticular Ca2+-ATPase inhibitor), tetraethylammonium (K+ channels blocker) or MDL-12,330A (adenylyl cyclesinhibitor) did not modify either SOR- or diterpeneinduced vasorelaxation. However, ODQ (guanylyl cyclase inhibitor) impaired only diterpene-induced vasorelaxation. SOR and diterpene significantly reduced CaCl2-induced contraction stimulated by Bay K8644 (1 μM), phenylephrine (0.1 μM) or KCl solution (40 mM). Computational molecular docking studies demonstrated that the vasodilator effect of diterpene relies on blocking the Cav 1.2 channel, and patch clamp results showed that diterpene substantially decreased the ionic current through Cav 1.2 in freshly dissociated vascular smooth muscle cells. These findings suggest that SOR and its isolated diterpene induce endothelium-independent vascular relaxation by blocking the L-type Ca2+ channel (Cav 1.2). Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. A Critical Neurodevelopmental Role for L-Type Voltage-Gated Calcium Channels in Neurite Extension and Radial Migration.

    PubMed

    Kamijo, Satoshi; Ishii, Yuichiro; Horigane, Shin-Ichiro; Suzuki, Kanzo; Ohkura, Masamichi; Nakai, Junichi; Fujii, Hajime; Takemoto-Kimura, Sayaka; Bito, Haruhiko

    2018-06-13

    Despite many association studies linking gene polymorphisms and mutations of L-type voltage-gated Ca 2+ channels (VGCCs) in neurodevelopmental disorders such as autism and schizophrenia, the roles of specific L-type VGCC during brain development remain unclear. Calcium signaling has been shown to be essential for neurodevelopmental processes such as sculpting of neurites, functional wiring, and fine tuning of growing networks. To investigate this relationship, we performed submembraneous calcium imaging using a membrane-tethered genetically encoded calcium indicator (GECI) Lck-G-CaMP7. We successfully recorded s pontaneous regenerative calcium transients (SRCaTs) in developing mouse excitatory cortical neurons prepared from both sexes before synapse formation. SRCaTs originated locally in immature neurites independently of somatic calcium rises and were significantly more elevated in the axons than in dendrites. SRCaTs were not blocked by tetrodoxin, a Na + channel blocker, but were strongly inhibited by hyperpolarization, suggesting a voltage-dependent source. Pharmacological and genetic manipulations revealed the critical importance of the Ca v 1.2 (CACNA1C) pore-forming subunit of L-type VGCCs, which were indeed expressed in immature mouse brains. Consistently, knocking out Ca v 1.2 resulted in significant alterations of neurite outgrowth. Furthermore, expression of a gain-of-function Ca v 1.2 mutant found in Timothy syndrome, an autosomal dominant multisystem disorder exhibiting syndromic autism, resulted in impaired radial migration of layer 2/3 excitatory neurons, whereas postnatal abrogation of Ca v 1.2 enhancement could rescue cortical malformation. Together, these lines of evidence suggest a critical role for spontaneous opening of L-type VGCCs in neural development and corticogenesis and indicate that L-type VGCCs might constitute a perinatal therapeutic target for neuropsychiatric calciochannelopathies. SIGNIFICANCE STATEMENT Despite many association

  15. Quercetin induces insulin secretion by direct activation of L-type calcium channels in pancreatic beta cells

    PubMed Central

    Bardy, G; Virsolvy, A; Quignard, J F; Ravier, M A; Bertrand, G; Dalle, S; Cros, G; Magous, R; Richard, S; Oiry, C

    2013-01-01

    Background and Purpose Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca2+]i) in beta cells, in the absence of any co-stimulating factor. Experimental Approach Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca2+]i were measured using the ratiometric fluorescent Ca2+ indicator Fura-2. Ca2+ channel currents were recorded with the whole-cell patch-clamp technique. Key Results Quercetin concentration-dependently increased insulin secretion and elevated [Ca2+]i. These effects were not modified by the SERCA inhibitor thapsigargin (1 μmol·L−1), but were nearly abolished by the L-type Ca2+ channel antagonist nifedipine (1 μmol·L−1). Similar to the L-type Ca2+ channel agonist Bay K 8644, quercetin enhanced the L-type Ca2+ current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca2+]i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 μmol·L−1), with the two drugs having cumulative effects on [Ca2+]i. Conclusions and Implications Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca2+ influx through an interaction with L-type Ca2+ channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin's mechanism of action on insulin secretion. PMID:23530660

  16. L-type calcium channel blockade attenuates morphine withdrawal: in vivo interaction between L-type calcium channels and corticosterone.

    PubMed

    Esmaeili-Mahani, Saeed; Fathi, Yadollah; Motamedi, Fereshteh; Hosseinpanah, Farhad; Ahmadiani, Abolhassan

    2008-02-01

    Both opioids and calcium channel blockers could affect hypothalamic-pituitary-adrenal (HPA) axis function. Nifedipine, as a calcium channel blocker, can attenuate the development of morphine dependence; however, the role of the HPA axis in this effect has not been elucidated. We examined the effect of nifedipine on the induction of morphine dependency in intact and adrenalectomized (ADX) male rats, as assessed by the naloxone precipitation test. We also evaluated the effect of this drug on HPA activity induced by naloxone. Our results showed that despite the demonstration of dependence in both groups of rats, nifedipine is more effective in preventing of withdrawal signs in ADX rats than in sham-operated rats. In groups that received morphine and nifedipine concomitantly, naloxone-induced corticosterone secretion was attenuated. Thus, we have shown the involvement of the HPA axis in the effect of nifedipine on the development of morphine dependency and additionally demonstrated an in vivo interaction between the L-type Ca2+ channels and corticosterone.

  17. Inactivation of Gating Currents of L-Type Calcium Channels

    PubMed Central

    Shirokov, Roman; Ferreira, Gonzalo; Yi, Jianxun; Ríos, Eduardo

    1998-01-01

    In studies of gating currents of rabbit cardiac Ca channels expressed as α1C/β2a or α1C/β2a/α2δ subunit combinations in tsA201 cells, we found that long-lasting depolarization shifted the distribution of mobile charge to very negative potentials. The phenomenon has been termed charge interconversion in native skeletal muscle (Brum, G., and E. Ríos. 1987. J. Physiol. (Camb.). 387:489–517) and cardiac Ca channels (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1992. J. Gen. Physiol. 99:863–895). Charge 1 (voltage of half-maximal transfer, V1/2 ≃ 0 mV) gates noninactivated channels, while charge 2 (V1/2 ≃ −90 mV) is generated in inactivated channels. In α1C/β2a cells, the available charge 1 decreased upon inactivating depolarization with a time constant τ ≃ 8, while the available charge 2 decreased upon recovery from inactivation (at −200 mV) with τ ≃ 0.3 s. These processes therefore are much slower than charge movement, which takes <50 ms. This separation between the time scale of measurable charge movement and that of changes in their availability, which was even wider in the presence of α2δ, implies that charges 1 and 2 originate from separate channel modes. Because clear modal separation characterizes slow (C-type) inactivation of Na and K channels, this observation establishes the nature of voltage-dependent inactivation of L-type Ca channels as slow or C-type. The presence of the α2δ subunit did not change the V1/2 of charge 2, but sped up the reduction of charge 1 upon inactivation at 40 mV (to τ ≃ 2 s), while slowing the reduction of charge 2 upon recovery (τ ≃ 2 s). The observations were well simulated with a model that describes activation as continuous electrodiffusion (Levitt, D. 1989. Biophys. J. 55:489–498) and inactivation as discrete modal change. The effects of α2δ are reproduced assuming that the subunit lowers the free energy of the inactivated mode. PMID:9607938

  18. Aging-associated changes in L-type calcium channels in the left atria of dogs.

    PubMed

    Gan, Tian-Yi; Qiao, Weiwei; Xu, Guo-Jun; Zhou, Xian-Hui; Tang, Bao-Peng; Song, Jian-Guo; Li, Yao-Dong; Zhang, Jian; Li, Fa-Peng; Mao, Ting; Jiang, Tao

    2013-10-01

    Action potential (AP) contours vary considerably between the fibers of normal adult and aged left atria. The underlying ionic and molecular mechanisms that mediate these differences remain unknown. The aim of the present study was to investigate whether the L-type calcium current (I Ca.L ) and the L-type Ca 2+ channel of the left atria may be altered with age to contribute to atrial fibrillation (AF). Two groups of mongrel dogs (normal adults, 2-2.5 years old and older dogs, >8 years old) were used in this study. The inducibility of AF was quantitated using the cumulative window of vulnerability (WOV). A whole-cell patch-clamp was used to record APs and I Ca.L in left atrial (LA) cells obtained from the two groups of dogs. Protein and mRNA expression levels of the a1C (Cav1.2) subunit of the L-type calcium channel were assessed using western blotting and quantitative PCR (qPCR), respectively. Although the resting potential, AP amplitude and did not differ with age, the plateau potential was more negative and the APD 90 was longer in the aged cells compared with that in normal adult cells. Aged LA cells exhibited lower peak I Ca.L current densities than normal adult LA cells (P<0.05). In addition, the Cav1.2 mRNA and protein expression levels in LA cells were decreased in the aged group compared with those in the normal adult group. The lower AP plateau potential and the decreased I Ca.L of LA cells in aged dogs may contribute to the slow and discontinuous conduction of the left atria. Furthermore, the reduction of the expression levels of Cav1.2 with age may be the molecular mechanism that mediates the decline in I Ca.L with increasing age.

  19. Mini-dystrophin restores L-type calcium currents in skeletal muscle of transgenic mdx mice

    PubMed Central

    Friedrich, O; Both, M; Gillis, J M; Chamberlain, J S; Fink, RHA

    2004-01-01

    L-type calcium currents (iCa) were recorded using the two-microelectrode voltage-clamp technique in single short toe muscle fibres of three different mouse strains: (i) C57/SV129 wild-type mice (wt); (ii) mdx mice (an animal model for Duchenne muscular dystrophy; and (iii) transgenically engineered mini-dystrophin (MinD)-expressing mdx mice. The activation and inactivation properties of iCa were examined in 2- to 18-month-old animals. Ca2+ current densities at 0 mV in mdx fibres increased with age, but were always significantly smaller compared to age-matched wild-type fibres. Time-to-peak (TTP) of iCa was prolonged in mdx fibres compared to wt fibres. MinD fibres always showed similar TTP and current amplitudes compared to age-matched wt fibres. In all three genotypes, the voltage-dependent inactivation and deactivation of iCa were similar. Intracellular resting calcium concentration ([Ca2+]i) and the distribution of dihydropyridine binding sites were also not different in young animals of all three genotypes, whereas iCa was markedly reduced in mdx fibres. We conclude, that dystrophin influences L-type Ca2+ channels via a direct or indirect linkage which may be disrupted in mdx mice and may be crucial for proper excitation–contraction coupling initiating Ca2+ release from the sarcoplasmic reticulum. This linkage seems to be fully restored in the presence of mini-dystrophin. PMID:14594987

  20. Calcium influx through L-type channels attenuates skeletal muscle contraction via inhibition of adenylyl cyclases.

    PubMed

    Menezes-Rodrigues, Francisco Sandro; Pires-Oliveira, Marcelo; Duarte, Thiago; Paredes-Gamero, Edgar Julian; Chiavegatti, Tiago; Godinho, Rosely Oliveira

    2013-11-15

    Skeletal muscle contraction is triggered by acetylcholine induced release of Ca(2+) from sarcoplasmic reticulum. Although this signaling pathway is independent of extracellular Ca(2+), L-type voltage-gated calcium channel (Cav) blockers have inotropic effects on frog skeletal muscles which occur by an unknown mechanism. Taking into account that skeletal muscle fiber expresses Ca(+2)-sensitive adenylyl cyclase (AC) isoforms and that cAMP is able to increase skeletal muscle contraction force, we investigated the role of Ca(2+) influx on mouse skeletal muscle contraction and the putative crosstalk between extracellular Ca(2+) and intracellular cAMP signaling pathways. The effects of Cav blockers (verapamil and nifedipine) and extracellular Ca(2+) chelator EGTA were evaluated on isometric contractility of mouse diaphragm muscle under direct electrical stimulus (supramaximal voltage, 2 ms, 0.1 Hz). Production of cAMP was evaluated by radiometric assay while Ca(2+) transients were assessed by confocal microscopy using L6 cells loaded with fluo-4/AM. Ca(2+) channel blockers verapamil and nifedipine had positive inotropic effect, which was mimicked by removal of extracellular Ca(+2) with EGTA or Ca(2+)-free Tyrode. While phosphodiesterase inhibitor IBMX potentiates verapamil positive inotropic effect, it was abolished by AC inhibitors SQ22536 and NYK80. Finally, the inotropic effect of verapamil was associated with increased intracellular cAMP content and mobilization of intracellular Ca(2+), indicating that positive inotropic effects of Ca(2+) blockers depend on cAMP formation. Together, our results show that extracellular Ca(2+) modulates skeletal muscle contraction, through inhibition of Ca(2+)-sensitive AC. The cross-talk between extracellular calcium and cAMP-dependent signaling pathways appears to regulate the extent of skeletal muscle contraction responses. © 2013 Published by Elsevier B.V.

  1. Influence of infrasound exposure on the whole L-type calcium currents in rat ventricular myocytes.

    PubMed

    Pei, Zhaohui; Zhuang, Zhiqiang; Xiao, Pingxi; Chen, Jingzao; Sang, Hanfei; Ren, Jun; Wu, Zhenbiao; Yan, Guangmei

    2009-06-01

    This study was designed to examine the effect of infrasound exposure (5 Hz at 130 dB) on whole-cell L-type Ca2+ currents (WLCC) in rat ventricular myocytes and the underlying mechanism(s) involved. Thirty-two adult Sprague-Dawley rats were randomly assigned to infrasound exposure and control groups. [Ca2+](i), WLCC, mRNA expression of the a(1c) subunit of L-type Ca2+ channels (LCC), and SERCA2 protein were examined on day 1, 7, and 14 after initiation of infrasound exposure. Fluo-3/AM fluorescence and the laser scanning confocal microscope techniques were used to measure [Ca2+](i) in freshly isolated ventricular myocytes. The Ca2+ fluorescence intensity (FI), denoting [Ca2+](i) in cardiomyocytes, was significantly elevated in a time-dependent manner in the exposure groups. There was a significant increase in WLCC in the 1-day group and a further significant increase in the 7- and 14-day groups. LCC mRNA expression measured by RT-PCR revealed a significant rise in the 1-day group and a significant additional rise in the 7- and 14-day groups compared with control group. SERCA2 expression was significantly upregulated in the 1-day group followed by an overt decrease in the 7- and 14-day groups. Prolonged exposure of infrasound altered WLCC in rat cardiomyocytes by shifting the steady-state inactivation curves to the right (more depolarized direction) without altering the slope and biophysical properties of I (Ca,L). Taken together, our data suggest that changes in [Ca2+](I) levels as well as expression of LCC and SERCA2 may contribute to the infrasound exposure-elicited cardiac response.

  2. The Contribution of L-Type Cav1.3 Channels to Retinal Light Responses.

    PubMed

    Shi, Liheng; Chang, Janet Ya-An; Yu, Fei; Ko, Michael L; Ko, Gladys Y-P

    2017-01-01

    L-type voltage-gated calcium channels (LTCCs) regulate tonic neurotransmitter release from sensory neurons including retinal photoreceptors. There are three types of LTCCs (Ca v 1.2, Ca v 1.3, and Ca v 1.4) expressed in the retina. While Ca v 1.2 is expressed in all retinal cells including the Müller glia and neurons, Ca v 1.3 and Ca v 1.4 are expressed in the retinal neurons with Ca v 1.4 exclusively expressed in the photoreceptor synaptic terminals. Mutations in the gene encoding Ca v 1.4 cause incomplete X-linked congenital stationary night blindness in humans. Even though Ca v 1.3 is present in the photoreceptor inner segments and the synaptic terminals in various vertebrate species, its role in vision is unclear, since genetic alterations in Ca v 1.3 are not associated with severe vision impairment in humans or in Ca v 1.3-null (Ca v 1.3 -/- ) mice. However, a failure to regulate Ca v 1.3 was found in a mouse model of Usher syndrome, the most common cause of combined deafness and blindness in humans, indicating that Ca v 1.3 may contribute to retinal function. In this report, we combined physiological and morphological data to demonstrate the role of Ca v 1.3 in retinal physiology and function that has been undervalued thus far. Through ex vivo and in vivo electroretinogram (ERG) recordings and immunohistochemical staining, we found that Ca v 1.3 plays a role in retinal light responses and synaptic plasticity. Pharmacological inhibition of Ca v 1.3 decreased ex vivo ERG a- and b-wave amplitudes. In Ca v 1.3 -/- mice, their dark-adapted ERG a-, b-wave, and oscillatory potential amplitudes were significantly dampened, and implicit times were delayed compared to the wild type (WT). Furthermore, the density of ribbon synapses was reduced in the outer plexiform layer of Ca v 1.3 -/- mice retinas. Hence, Ca v 1.3 plays a more prominent role in retinal physiology and function than previously reported.

  3. Calcium/calmodulin-dependent serine protein kinase CASK modulates the L-type calcium current.

    PubMed

    Nafzger, Sabine; Rougier, Jean-Sebastien

    2017-01-01

    The L-type voltage-gated calcium channel Ca v 1.2 mediates the calcium influx into cells upon membrane depolarization. The list of cardiopathies associated to Ca v 1.2 dysfunctions highlights the importance of this channel in cardiac physiology. Calcium/calmodulin-dependent serine protein kinase (CASK), expressed in cardiac cells, has been identified as a regulator of Ca v 2.2 channels in neurons, but no experiments have been performed to investigate its role in Ca v 1.2 regulation. Full length or the distal C-terminal truncated of the pore-forming Ca v 1.2 channel (Ca v 1.2α1c), both present in cardiac cells, were expressed in TsA-201 cells. In addition, a shRNA silencer, or scramble as negative control, of CASK was co-transfected in order to silence CASK endogenously expressed. Three days post-transfection, the barium current was increased only for the truncated form without alteration of the steady state activation and inactivation biophysical properties. The calcium current, however, was increased after CASK silencing with both types of Ca v 1.2α1c subunits suggesting that, in absence of calcium, the distal C-terminal counteracts the CASK effect. Biochemistry experiments did not reveals neither an alteration of Ca v 1.2 channel protein expression after CASK silencing nor an interaction between Ca v 1.2α1c subunits and CASK. Nevertheless, after CASK silencing, single calcium channel recordings have shown an increase of the voltage-gated calcium channel Ca v 1.2 open probability explaining the increase of the whole-cell current. This study suggests CASK as a novel regulator of Ca v 1.2 via a modulation of the voltage-gated calcium channel Ca v 1.2 open probability. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Permeation and gating properties of the L-type calcium channel in mouse pancreatic beta cells

    PubMed Central

    1993-01-01

    Ba2+ currents through L-type Ca2+ channels were recorded from cell- attached patches on mouse pancreatic beta cells. In 10 mM Ba2+, single- channel currents were recorded at -70 mV, the beta cell resting membrane potential. This suggests that Ca2+ influx at negative membrane potentials may contribute to the resting intracellular Ca2+ concentration and thus to basal insulin release. Increasing external Ba2+ increased the single-channel current amplitude and shifted the current-voltage relation to more positive potentials. This voltage shift could be modeled by assuming that divalent cations both screen and bind to surface charges located at the channel mouth. The single- channel conductance was related to the bulk Ba2+ concentration by a Langmuir isotherm with a dissociation constant (Kd(gamma)) of 5.5 mM and a maximum single-channel conductance (gamma max) of 22 pS. A closer fit to the data was obtained when the barium concentration at the membrane surface was used (Kd(gamma) = 200 mM and gamma max = 47 pS), which suggests that saturation of the concentration-conductance curve may be due to saturation of the surface Ba2+ concentration. Increasing external Ba2+ also shifted the voltage dependence of ensemble currents to positive potentials, consistent with Ba2+ screening and binding to membrane surface charge associated with gating. Ensemble currents recorded with 10 mM Ca2+ activated at more positive potentials than in 10 mM Ba2+, suggesting that external Ca2+ binds more tightly to membrane surface charge associated with gating. The perforated-patch technique was used to record whole-cell currents flowing through L-type Ca2+ channels. Inward currents in 10 mM Ba2+ had a similar voltage dependence to those recorded at a physiological Ca2+ concentration (2.6 mM). BAY-K 8644 (1 microM) increased the amplitude of the ensemble and whole-cell currents but did not alter their voltage dependence. Our results suggest that the high divalent cation solutions usually used to

  5. Novel 1, 4-dihydropyridines for L-type calcium channel as antagonists for cadmium toxicity

    PubMed Central

    Saddala, Madhu Sudhana; Kandimalla, Ramesh; Adi, Pradeepkiran Jangampalli; Bhashyam, Sainath Sri; Asupatri, Usha Rani

    2017-01-01

    The present study, we design and synthesize the novel dihydropyridine derivatives, i.e., 3 (a-e) and 5 (a-e) and evaluated, anticonvulsant activity. Initially due to the lacuna of LCC, we modeled the protein through modeller 9.15v and evaluated through servers. Docking studies were performed with the synthesized compounds and resulted two best compounds, i.e., 5a, 5e showed the best binding energies. The activity of intracellular Ca2+ measurements was performed on two cell lines: A7r5 (rat aortic smooth muscle cells) and SH-SY5Y (human neuroblastoma cells). The 5a and 5e compounds was showing the more specific activity on L-type calcium channels, i.e. A7r5 (IC50 = 0.18 ± 0.02 and 0.25 ± 0.63 μg/ml, respectively) (containing only L-type channels) than SH-SY5Y (i.e. both L-type and T-type channels) (IC50 = 8 ± 0.23 and 10 ± 0.18 μg/ml, respectively) with intracellular calcium mobility similar to amlodipine. Finally, both in silico and in vitro results exploring two derivatives 5a and 5e succeeded to treat cadmium toxicity. PMID:28345598

  6. A novel dihydropyridine with 3-aryl meta-hydroxyl substitution blocks L-type calcium channels in rat cardiomyocytes.

    PubMed

    Galvis-Pareja, David; Zapata-Torres, Gerald; Hidalgo, Jorge; Ayala, Pedro; Pedrozo, Zully; Ibarra, Cristián; Diaz-Araya, Guillermo; Hall, Andrew R; Vicencio, Jose Miguel; Nuñez-Vergara, Luis; Lavandero, Sergio

    2014-08-15

    Dihydropyridines are widely used for the treatment of several cardiac diseases due to their blocking activity on L-type Ca(2+) channels and their renowned antioxidant properties. We synthesized six novel dihydropyridine molecules and performed docking studies on the binding site of the L-type Ca(2+) channel. We used biochemical techniques on isolated adult rat cardiomyocytes to assess the efficacy of these molecules on their Ca(2+) channel-blocking activity and antioxidant properties. The Ca(2+) channel-blocking activity was evaluated by confocal microscopy on fluo-3AM loaded cardiomyocytes, as well as using patch clamp experiments. Antioxidant properties were evaluated by flow cytometry using the ROS sensitive dye 1,2,3 DHR. Our docking studies show that a novel compound with 3-OH substitution inserts into the active binding site of the L-type Ca(2+) channel previously described for nitrendipine. In biochemical assays, the novel meta-OH group in the aryl in C4 showed a high blocking effect on L-type Ca(2+) channel as opposed to para-substituted compounds. In the tests we performed, none of the molecules showed antioxidant properties. Only substitutions in C2, C3 and C5 of the aryl ring render dihydropyridine compounds with the capacity of blocking LTCC. Based on our docking studies, we postulate that the antioxidant activity requires a larger group than the meta-OH substitution in C2, C3 or C5 of the dihydropyridine ring. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. L-type calcium channels refine the neural population code of sound level.

    PubMed

    Grimsley, Calum Alex; Green, David Brian; Sivaramakrishnan, Shobhana

    2016-12-01

    The coding of sound level by ensembles of neurons improves the accuracy with which listeners identify how loud a sound is. In the auditory system, the rate at which neurons fire in response to changes in sound level is shaped by local networks. Voltage-gated conductances alter local output by regulating neuronal firing, but their role in modulating responses to sound level is unclear. We tested the effects of L-type calcium channels (Ca L : Ca V 1.1-1.4) on sound-level coding in the central nucleus of the inferior colliculus (ICC) in the auditory midbrain. We characterized the contribution of Ca L to the total calcium current in brain slices and then examined its effects on rate-level functions (RLFs) in vivo using single-unit recordings in awake mice. Ca L is a high-threshold current and comprises ∼50% of the total calcium current in ICC neurons. In vivo, Ca L activates at sound levels that evoke high firing rates. In RLFs that increase monotonically with sound level, Ca L boosts spike rates at high sound levels and increases the maximum firing rate achieved. In different populations of RLFs that change nonmonotonically with sound level, Ca L either suppresses or enhances firing at sound levels that evoke maximum firing. Ca L multiplies the gain of monotonic RLFs with dynamic range and divides the gain of nonmonotonic RLFs with the width of the RLF. These results suggest that a single broad class of calcium channels activates enhancing and suppressing local circuits to regulate the sensitivity of neuronal populations to sound level. Copyright © 2016 the American Physiological Society.

  8. L-type calcium channels refine the neural population code of sound level

    PubMed Central

    Grimsley, Calum Alex; Green, David Brian

    2016-01-01

    The coding of sound level by ensembles of neurons improves the accuracy with which listeners identify how loud a sound is. In the auditory system, the rate at which neurons fire in response to changes in sound level is shaped by local networks. Voltage-gated conductances alter local output by regulating neuronal firing, but their role in modulating responses to sound level is unclear. We tested the effects of L-type calcium channels (CaL: CaV1.1–1.4) on sound-level coding in the central nucleus of the inferior colliculus (ICC) in the auditory midbrain. We characterized the contribution of CaL to the total calcium current in brain slices and then examined its effects on rate-level functions (RLFs) in vivo using single-unit recordings in awake mice. CaL is a high-threshold current and comprises ∼50% of the total calcium current in ICC neurons. In vivo, CaL activates at sound levels that evoke high firing rates. In RLFs that increase monotonically with sound level, CaL boosts spike rates at high sound levels and increases the maximum firing rate achieved. In different populations of RLFs that change nonmonotonically with sound level, CaL either suppresses or enhances firing at sound levels that evoke maximum firing. CaL multiplies the gain of monotonic RLFs with dynamic range and divides the gain of nonmonotonic RLFs with the width of the RLF. These results suggest that a single broad class of calcium channels activates enhancing and suppressing local circuits to regulate the sensitivity of neuronal populations to sound level. PMID:27605536

  9. L-Type Calcium Channels Modulation by Estradiol.

    PubMed

    Vega-Vela, Nelson E; Osorio, Daniel; Avila-Rodriguez, Marco; Gonzalez, Janneth; García-Segura, Luis Miguel; Echeverria, Valentina; Barreto, George E

    2017-09-01

    Voltage-gated calcium channels are key regulators of brain function, and their dysfunction has been associated with multiple conditions and neurodegenerative diseases because they couple membrane depolarization to the influx of calcium-and other processes such as gene expression-in excitable cells. L-type calcium channels, one of the three major classes and probably the best characterized of the voltage-gated calcium channels, act as an essential calcium binding proteins with a significant biological relevance. It is well known that estradiol can activate rapidly brain signaling pathways and modulatory/regulatory proteins through non-genomic (or non-transcriptional) mechanisms, which lead to an increase of intracellular calcium that activate multiple kinases and signaling cascades, in the same way as L-type calcium channels responses. In this context, estrogens-L-type calcium channels signaling raises intracellular calcium levels and activates the same signaling cascades in the brain probably through estrogen receptor-independent modulatory mechanisms. In this review, we discuss the available literature on this area, which seems to suggest that estradiol exerts dual effects/modulation on these channels in a concentration-dependent manner (as a potentiator of these channels in pM concentrations and as an inhibitor in nM concentrations). Indeed, estradiol may orchestrate multiple neurotrophic responses, which open a new avenue for the development of novel estrogen-based therapies to alleviate different neuropathologies. We also highlight that it is essential to determine through computational and/or experimental approaches the interaction between estradiol and L-type calcium channels to assist these developments, which is an interesting area of research that deserves a closer look in future biomedical research.

  10. A novel dihydropyridine with 3-aryl meta-hydroxyl substitution blocks L-type calcium channels in rat cardiomyocytes

    SciTech Connect

    Galvis-Pareja, David; Centro Estudios Moleculares de la Célula; Zapata-Torres, Gerald

    2014-08-15

    Rationale: Dihydropyridines are widely used for the treatment of several cardiac diseases due to their blocking activity on L-type Ca{sup 2+} channels and their renowned antioxidant properties. Methods: We synthesized six novel dihydropyridine molecules and performed docking studies on the binding site of the L-type Ca{sup 2+} channel. We used biochemical techniques on isolated adult rat cardiomyocytes to assess the efficacy of these molecules on their Ca{sup 2+} channel-blocking activity and antioxidant properties. The Ca{sup 2+} channel-blocking activity was evaluated by confocal microscopy on fluo-3AM loaded cardiomyocytes, as well as using patch clamp experiments. Antioxidant properties were evaluated by flowmore » cytometry using the ROS sensitive dye 1,2,3 DHR. Results: Our docking studies show that a novel compound with 3-OH substitution inserts into the active binding site of the L-type Ca{sup 2+} channel previously described for nitrendipine. In biochemical assays, the novel meta-OH group in the aryl in C4 showed a high blocking effect on L-type Ca{sup 2+} channel as opposed to para-substituted compounds. In the tests we performed, none of the molecules showed antioxidant properties. Conclusions: Only substitutions in C2, C3 and C5 of the aryl ring render dihydropyridine compounds with the capacity of blocking LTCC. Based on our docking studies, we postulate that the antioxidant activity requires a larger group than the meta-OH substitution in C2, C3 or C5 of the dihydropyridine ring. - Highlights: • Dihydropyridine (DHP) molecules are widely used in cardiovascular disease. • DHPs block Ca{sup 2+} entry through LTCC—some DHPs have antioxidant activity as well. • We synthesized 6 new DHPs and tested their Ca{sup 2+} blocking and antioxidant activities. • 3-Aryl meta-hydroxyl substitution strongly increases their Ca{sup 2+} blocking activity. • 3-Aryl meta-hydroxyl substitution did not affect the antioxidant properties.« less

  11. Differential Roles for L-Type Calcium Channel Subtypes in Alcohol Dependence

    PubMed Central

    Uhrig, Stefanie; Vandael, David; Marcantoni, Andrea; Dedic, Nina; Bilbao, Ainhoa; Vogt, Miriam A; Hirth, Natalie; Broccoli, Laura; Bernardi, Rick E; Schönig, Kai; Gass, Peter; Bartsch, Dusan; Spanagel, Rainer; Deussing, Jan M; Sommer, Wolfgang H; Carbone, Emilio; Hansson, Anita C

    2017-01-01

    It has previously been shown that the inhibition of L-type calcium channels (LTCCs) decreases alcohol consumption, although the contribution of the central LTCC subtypes Cav1.2 and Cav1.3 remains unknown. Here, we determined changes in Cav1.2 (Cacna1c) and Cav1.3 (Cacna1d) mRNA and protein expression in alcohol-dependent rats during protracted abstinence and naive controls using in situ hybridization and western blot analysis. Functional validation was obtained by electrophysiological recordings of calcium currents in dissociated hippocampal pyramidal neurons. We then measured alcohol self-administration and cue-induced reinstatement of alcohol seeking in dependent and nondependent rats after intracerebroventricular (i.c.v.) injection of the LTCC antagonist verapamil, as well as in mice with an inducible knockout (KO) of Cav1.2 in Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα)-expressing neurons. Our results show that Cacna1c mRNA concentration was increased in the amygdala and hippocampus of alcohol-dependent rats after 21 days of abstinence, with no changes in Cacna1d mRNA. This was associated with increased Cav1.2 protein concentration and L-type calcium current amplitudes. Further analysis of Cacna1c mRNA in the CA1, basolateral amygdala (BLA), and central amygdala (CeA) revealed a dynamic regulation over time during the development of alcohol dependence. The inhibition of central LTCCs via i.c.v. administration of verapamil prevented cue-induced reinstatement of alcohol seeking in alcohol-dependent rats. Further studies in conditional Cav1.2-KO mice showed a lack of dependence-induced increase of alcohol-seeking behavior. Together, our data indicate that central Cav1.2 channels, rather than Cav1.3, mediate alcohol-seeking behavior. This finding may be of interest for the development of new antirelapse medications. PMID:27905406

  12. The Involvement of Ser1898 of the Human L-Type Calcium Channel in Evoked Secretion

    PubMed Central

    Bachnoff, Niv; Cohen-Kutner, Moshe; Atlas, Daphne

    2011-01-01

    A PKA consensus phosphorylation site S1928 at the α 11.2 subunit of the rabbit cardiac L-type channel, CaV1.2, is involved in the regulation of CaV1.2 kinetics and affects catecholamine secretion. This mutation does not alter basal CaV1.2 current properties or regulation of CaV1.2 current by PKA and the beta-adrenergic receptor, but abolishes CaV1.2 phosphorylation by PKA. Here, we test the contribution of the corresponding PKA phosphorylation site of the human α 11.2 subunit S1898, to the regulation of catecholamine secretion in bovine chromaffin cells. Chromaffin cells were infected with a Semliki-Forest viral vector containing either the human wt or a mutated S1898A α 11.2 subunit. Both subunits harbor a T1036Y mutation conferring nifedipine insensitivity. Secretion evoked by depolarization in the presence of nifedipine was monitored by amperometry. Depolarization-triggered secretion in cells infected with either the wt α 11.2 or α 11.2/S1898A mutated subunit was elevated to a similar extent by forskolin. Forskolin, known to directly activate adenylyl-cyclase, increased the rate of secretion in a manner that is largely independent of the presence of S1898. Our results are consistent with the involvement of additional PKA regulatory site(s) at the C-tail of α 11.2, the pore forming subunit of CaV1.2. PMID:22216029

  13. Glucose-6-phosphate dehydrogenase and NADPH redox regulates cardiac myocyte L-type calcium channel activity and myocardial contractile function.

    PubMed

    Rawat, Dhwajbahadur K; Hecker, Peter; Watanabe, Makino; Chettimada, Sukrutha; Levy, Richard J; Okada, Takao; Edwards, John G; Gupte, Sachin A

    2012-01-01

    We recently demonstrated that a 17-ketosteroid, epiandrosterone, attenuates L-type Ca(2+) currents (I(Ca-L)) in cardiac myocytes and inhibits myocardial contractility. Because 17-ketosteroids are known to inhibit glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, and to reduce intracellular NADPH levels, we hypothesized that inhibition of G6PD could be a novel signaling mechanism which inhibit I(Ca-L) and, therefore, cardiac contractile function. We tested this idea by examining myocardial function in isolated hearts and Ca(2+) channel activity in isolated cardiac myocytes. Myocardial function was tested in Langendorff perfused hearts and I(Ca-L) were recorded in the whole-cell patch configuration by applying double pulses from a holding potential of -80 mV and then normalized to the peak amplitudes of control currents. 6-Aminonicotinamide, a competitive inhibitor of G6PD, increased pCO(2) and decreased pH. Additionally, 6-aminonicotinamide inhibited G6PD activity, reduced NADPH levels, attenuated peak I(Ca-L) amplitudes, and decreased left ventricular developed pressure and ±dp/dt. Finally, dialyzing NADPH into cells from the patch pipette solution attenuated the suppression of I(Ca-L) by 6-aminonicotinamide. Likewise, in G6PD-deficient mice, G6PD insufficiency in the heart decreased GSH-to-GSSG ratio, superoxide, cholesterol and acetyl CoA. In these mice, M-mode echocardiographic findings showed increased diastolic volume and end-diastolic diameter without changes in the fraction shortening. Taken together, these findings suggest that inhibiting G6PD activity and reducing NADPH levels alters metabolism and leads to inhibition of L-type Ca(2+) channel activity. Notably, this pathway may be involved in modulating myocardial contractility under physiological and pathophysiological conditions during which the pentose phosphate pathway-derived NADPH redox is modulated (e.g., ischemia-reperfusion and heart failure).

  14. Rescuing cardiac automaticity in L-type Cav1.3 channelopathies and beyond.

    PubMed

    Mesirca, Pietro; Bidaud, Isabelle; Mangoni, Matteo E

    2016-10-15

    Pacemaker activity of the sino-atrial node generates the heart rate. Disease of the sinus node and impairment of atrioventricular conduction induce an excessively low ventricular rate (bradycardia), which cannot meet the needs of the organism. Bradycardia accounts for about half of the total workload of clinical cardiologists. The 'sick sinus' syndrome (SSS) is characterized by sinus bradycardia and periods of intermittent atrial fibrillation. Several genetic or acquired risk factors or pathologies can lead to SSS. Implantation of an electronic pacemaker constitutes the only available therapy for SSS. The incidence of SSS is forecast to double over the next 50 years, with ageing of the general population thus urging the development of complementary or alternative therapeutic strategies. In recent years an increasing number of mutations affecting ion channels involved in sino-atrial automaticity have been reported to underlie inheritable SSS. L-type Ca v 1.3 channels play a major role in the generation and regulation of sino-atrial pacemaker activity and atrioventricular conduction. Mutation in the CACNA1D gene encoding Ca v 1.3 channels induces loss-of-function in channel activity and underlies the sino-atrial node dysfunction and deafness syndrome (SANDD). Mice lacking Ca v 1.3 channels (Ca v 1.3 -/- ) fairly recapitulate SSS and constitute a precious model to test new therapeutic approaches to handle this disease. Work in our laboratory shows that targeting G protein-gated K + (I KACh ) channels effectively rescues SSS of Ca v 1.3 -/- mice. This new concept of 'compensatory' ion channel targeting shines new light on the principles underlying the pacemaker mechanism and may open the way to new therapies for SSS. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

  15. Methamphetamine acutely inhibits voltage-gated calcium channels but chronically up-regulates L-type channels.

    PubMed

    Andres, Marilou A; Cooke, Ian M; Bellinger, Frederick P; Berry, Marla J; Zaporteza, Maribel M; Rueli, Rachel H; Barayuga, Stephanie M; Chang, Linda

    2015-07-01

    In neurons, calcium (Ca(2+) ) channels regulate a wide variety of functions ranging from synaptic transmission to gene expression. They also induce neuroplastic changes that alter gene expression following psychostimulant administration. Ca(2+) channel blockers have been considered as potential therapeutic agents for the treatment of methamphetamine (METH) dependence because of their ability to reduce drug craving among METH users. Here, we studied the effects of METH exposure on voltage-gated Ca(2+) channels using SH-SY5Y cells as a model of dopaminergic neurons. We found that METH has different short- and long-term effects. A short-term effect involves immediate (< 5 min) direct inhibition of Ca(2+) ion movements through Ca(2+) channels. Longer exposure to METH (20 min or 48 h) selectively up-regulates the expression of only the CACNA1C gene, thus increasing the number of L-type Ca(2+) channels. This up-regulation of CACNA1C is associated with the expression of the cAMP-responsive element-binding protein (CREB), a known regulator of CACNA1C gene expression, and the MYC gene, which encodes a transcription factor that putatively binds to a site proximal to the CACNA1C gene transcription initiation site. The short-term inhibition of Ca(2+) ion movement and later, the up-regulation of Ca(2+) channel gene expression together suggest the operation of cAMP-responsive element-binding protein- and C-MYC-mediated mechanisms to compensate for Ca(2+) channel inhibition by METH. Increased Ca(2+) current density and subsequent increased intracellular Ca(2+) may contribute to the neurodegeneration accompanying chronic METH abuse. Methamphetamine (METH) exposure has both short- and long-term effects. Acutely, methamphetamine directly inhibits voltage-gated calcium channels. Chronically, neurons compensate by up-regulating the L-type Ca(2+) channel gene, CACNA1C. This compensatory mechanism is mediated by transcription factors C-MYC and CREB, in which CREB is linked to the

  16. Attenuated response of L-type calcium current to nitric oxide in atrial fibrillation.

    PubMed

    Rozmaritsa, Nadiia; Christ, Torsten; Van Wagoner, David R; Haase, Hannelore; Stasch, Johannes-Peter; Matschke, Klaus; Ravens, Ursula

    2014-03-01

    Nitric oxide (NO) synthesized by cardiomyocytes plays an important role in the regulation of cardiac function. Here, we studied the impact of NO signalling on calcium influx in human right atrial myocytes and its relation to atrial fibrillation (AF). Right atrial appendages (RAAs) were obtained from patients in sinus rhythm (SR) and AF. The biotin-switch technique was used to evaluate endogenous S-nitrosylation of the α1C subunit of L-type calcium channels. Comparing SR to AF, S-nitrosylation of Ca(2+) channels was similar. Direct effects of the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) on L-type calcium current (ICa,L) were studied in cardiomyocytes with standard voltage-clamp techniques. In SR, ICa,L increased with SNAP (100 µM) by 48%, n/N = 117/56, P < 0.001. The SNAP effect on ICa,L involved activation of soluble guanylate cyclase and protein kinase A. Specific inhibition of phosphodiesterase (PDE)3 with cilostamide (1 µM) enhanced ICa,L to a similar extent as SNAP. However, when cAMP was elevated by PDE3 inhibition or β-adrenoceptor stimulation, SNAP reduced ICa,L, pointing to cGMP-cAMP cross-regulation. In AF, the stimulatory effect of SNAP on ICa,L was attenuated, while its inhibitory effect on isoprenaline- or cilostamide-stimulated current was preserved. cGMP elevation with SNAP was comparable between the SR and AF group. Moreover, the expression of PDE3 and soluble guanylate cyclase was not reduced in AF. NO exerts dual effects on ICa,L in SR with an increase of basal and inhibition of cAMP-stimulated current, and in AF NO inhibits only stimulated ICa,L. We conclude that in AF, cGMP regulation of PDE2 is preserved, but regulation of PDE3 is lost.

  17. The Calmodulin-Binding, Short Linear Motif, NSCaTE Is Conserved in L-Type Channel Ancestors of Vertebrate Cav1.2 and Cav1.3 Channels

    PubMed Central

    Taiakina, Valentina; Boone, Adrienne N.; Fux, Julia; Senatore, Adriano; Weber-Adrian, Danielle

    2013-01-01

    NSCaTE is a short linear motif of (xWxxx(I or L)xxxx), composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM) which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca2+ concentrations, promoting calcium-dependent inactivation of L-type calcium channels. NSCaTE is absent in some arthropod species, and is also lacking in vertebrate L-type isoforms, Cav1.1 and Cav1.4 channels. The pervasiveness of a methionine just downstream from NSCaTE suggests that L-type channels could generate alternative N-termini lacking NSCaTE through the choice of translational start sites. Long N-terminus with an NSCaTE motif in L-type calcium channel homolog LCav1 from pond snail Lymnaea stagnalis has a faster calcium-dependent inactivation than a shortened N-termini lacking NSCaTE. NSCaTE effects are present in low concentrations of internal buffer (0.5 mM EGTA), but disappears in high buffer conditions (10 mM EGTA). Snail and mammalian NSCaTE have an alpha-helical propensity upon binding Ca2+-CaM and can saturate both CaM N-terminal and C-terminal domains in the absence of a competing IQ motif. NSCaTE evolved in ancestors of the first animals with internal organs for promoting a more rapid, calcium-sensitive inactivation of L-type channels. PMID:23626724

  18. Perturbed atrial calcium handling in an ovine model of heart failure: Potential roles for reductions in the L-type calcium current

    PubMed Central

    Clarke, Jessica D.; Caldwell, Jessica L.; Horn, Margaux A.; Bode, Elizabeth F.; Richards, Mark A.; Hall, Mark C.S.; Graham, Helen K.; Briston, Sarah J.; Greensmith, David J.; Eisner, David A.; Dibb, Katharine M.; Trafford, Andrew W.

    2015-01-01

    Heart failure (HF) is commonly associated with reduced cardiac output and an increased risk of atrial arrhythmias particularly during β-adrenergic stimulation. The aim of the present study was to determine how HF alters systolic Ca2 + and the response to β-adrenergic (β-AR) stimulation in atrial myocytes. HF was induced in sheep by ventricular tachypacing and changes in intracellular Ca2 + concentration studied in single left atrial myocytes under voltage and current clamp conditions. The following were all reduced in HF atrial myocytes; Ca2 + transient amplitude (by 46% in current clamped and 28% in voltage clamped cells), SR dependent rate of Ca2 + removal (kSR, by 32%), L-type Ca2 + current density (by 36%) and action potential duration (APD90 by 22%). However, in HF SR Ca2 + content was increased (by 19%) when measured under voltage-clamp stimulation. Inhibiting the L-type Ca2 + current (ICa-L) in control cells reproduced both the decrease in Ca2 + transient amplitude and increase of SR Ca2 + content observed in voltage-clamped HF cells. During β-AR stimulation Ca2 + transient amplitude was the same in control and HF cells. However, ICa-L remained less in HF than control cells whilst SR Ca2 + content was highest in HF cells during β-AR stimulation. The decrease in ICa-L that occurs in HF atrial myocytes appears to underpin the decreased Ca2 + transient amplitude and increased SR Ca2 + content observed in voltage-clamped cells. PMID:25463272

  19. Bradykinin induced a positive chronotropic effect via stimulation of T- and L-type calcium currents in heart cells.

    PubMed

    El-Bizri, Nesrine; Bkaily, Ghassan; Wang, Shimin; Jacques, Danielle; Regoli, Domenico; D'Orléans-Juste, Pedro; Sukarieh, Rami

    2003-03-01

    Using Fluo-3 calcium dye confocal microscopy and spontaneously contracting embryonic chick heart cells, bradykinin (10(-10) M) was found to induce positive chronotropic effects by increasing the frequency of the transient increase of cytosolic and nuclear free Ca2+. Pretreatment of the cells with either B1 or B2 receptor antagonists (R126 and R817, respectively) completely prevented bradykinin (BK) induced positive chronotropic effects on spontaneously contracting single heart cells. Using the whole-cell voltage clamp technique and ionic substitution to separate the different ionic current species, our results showed that BK (10(-6) M) had no effect on fast Na+ inward current and delayed outward potassium current. However, both L- and T-type Ca2+ currents were found to be increased by BK in a dose-dependent manner (10(-10)-10(-7) M). The effects of BK on T- and L-type Ca2+ currents were partially blocked by the B1 receptor antagonist [Leu8]des-Arg9-BK (R592) (10(-7) M) and completely reversed by the B2 receptor antagonist D-Arg[Hyp3,D-Phe7,Leu8]BK (R-588) (10(-7) M) or pretreatment with pertussis toxin (PTX). These results demonstrate that BK induced a positive chronotropic effect via stimulation of T- and L-type Ca2+ currents in heart cells mainly via stimulation of B2 receptor coupled to PTX-sensitive G-proteins. The increase of both types of Ca2+ current by BK in heart cells may explain the positive inotropic and chronotropic effects of this hormone.

  20. A 1D radiative transfer benchmark with polarization via doubling and adding

    NASA Astrophysics Data System (ADS)

    Ganapol, B. D.

    2017-11-01

    Highly precise numerical solutions to the radiative transfer equation with polarization present a special challenge. Here, we establish a precise numerical solution to the radiative transfer equation with combined Rayleigh and isotropic scattering in a 1D-slab medium with simple polarization. The 2-Stokes vector solution for the fully discretized radiative transfer equation in space and direction derives from the method of doubling and adding enhanced through convergence acceleration. Updates to benchmark solutions found in the literature to seven places for reflectance and transmittance as well as for angular flux follow. Finally, we conclude with the numerical solution in a partially randomly absorbing heterogeneous medium.

  1. Enhanced effect of VEGF165 on L-type calcium currents in guinea-pig cardiac ventricular myocytes.

    PubMed

    Xing, Wenlu; Gao, Chuanyu; Qi, Datun; Zhang, You; Hao, Peiyuan; Dai, Guoyou; Yan, Ganxin

    2017-01-01

    The mechanisms of vascular endothelial growth factor 165 (VEGF165) on electrical properties of cardiomyocytes have not been fully elucidated. The aim of this study is to test the hypothesis that VEGF165, an angiogenesis-initiating factor, affects L-type calcium currents (I Ca,L ) and cell membrane potential in cardiac myocytes by acting on VEGF type-2 receptors (VEGFR2). I Ca,L and action potentials (AP) were recorded by the whole-cell patch clamp method in isolated guinea-pig ventricular myocytes treated with different concentrations of VEGF165 proteins. Using a VEGFR2 inhibitor, we also tested the receptor of VEGF165 in cardiomyocytes. We found that VEGF165 increased I Ca,L in a concentration-dependent manner. SU5416, a VEGFR2 inhibitor, almost completely eliminated VEGF165-induced I Ca,L increase. VEGF165 had no significant influence on action potential 90 (APD90) and other properties of AP. We conclude that in guinea-pig ventricular myocytes, I Ca,L can be increased by VEGF165 in a concentration-dependent manner through binding to VEGFR2 without causing any significant alteration to action potential duration. Results of this study may further expound the safety of VEGF165 when used in the intervention of heart diseases. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  2. Vitamin E isomer δ-tocopherol enhances the efficiency of neural stem cell differentiation via L-type calcium channel.

    PubMed

    Deng, Sihao; Hou, Guoqiang; Xue, Zhiqin; Zhang, Longmei; Zhou, Yuye; Liu, Chao; Liu, Yanqing; Li, Zhiyuan

    2015-01-12

    The effects of the vitamin E isomer δ-tocopherol on neural stem cell (NSC) differentiation have not been investigated until now. Here we investigated the effects of δ-tocopherol on NSC neural differentiation, maturation and its possible mechanisms. Neonatal rat NSCs were grown in suspended neurosphere cultures, and were identified by their expression of nestin protein and their capacity for self-renewal. Treatment with a low concentration of δ-tocopherol induced a significant increase in the percentage of β-III-tubulin-positive cells. δ-Tocopherol also stimulated morphological maturation of neurons in culture. We further observed that δ-tocopherol stimulation increased the expression of voltage-dependent Ca(2+) channels. Moreover, a L-type specific Ca(2+) channel blocker verapamil reduced the percentage of differentiated neurons after δ-tocopherol treatment, and blocked the effects of δ-tocopherol on NSC differentiation into neurons. Together, our study demonstrates that δ-tocopherol may act through elevation of L-type calcium channel activity to increase neuronal differentiation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. 75 FR 3160 - Corporate Reorganizations; Distributions Under Sections 368(a)(1)(D) and 354(b)(1)(B); Correction

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-20

    ... DEPARTMENT OF THE TREASURY Internal Revenue Service 26 CFR Part 1 [TD 9475] RIN 1545-BF83 Corporate Reorganizations; Distributions Under Sections 368(a)(1)(D) and 354(b)(1)(B); Correction AGENCY... reorganizations described in section 368(a)(1)(D) where no stock and/or securities of the acquiring corporation is...

  4. 75 FR 3159 - Corporate Reorganizations; Distributions Under Sections 368(a)(1)(D) and 354(b)(1)(B); Correction

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-20

    ... DEPARTMENT OF THE TREASURY Internal Revenue Service 26 CFR Part 1 [TD 9475] RIN 1545-BF83 Corporate Reorganizations; Distributions Under Sections 368(a)(1)(D) and 354(b)(1)(B); Correction AGENCY... transactions as reorganizations described in section 368(a)(1)(D) where no stock and/or securities of the...

  5. 76 FR 49300 - Corporate Reorganizations; Distributions Under Sections 368(a)(1)(D) and 354(b)(1)(B); Correction

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-10

    ... Corporate Reorganizations; Distributions Under Sections 368(a)(1)(D) and 354(b)(1)(B); Correction AGENCY... in section 368(a)(1)(D) where no stock and/or securities of the acquiring corporation is issued and... guidance regarding the determination of the basis of stock or securities in a reorganization described in...

  6. Quantum simulation of 2D topological physics in a 1D array of optical cavities

    PubMed Central

    Luo, Xi-Wang; Zhou, Xingxiang; Li, Chuan-Feng; Xu, Jin-Shi; Guo, Guang-Can; Zhou, Zheng-Wei

    2015-01-01

    Orbital angular momentum of light is a fundamental optical degree of freedom characterized by unlimited number of available angular momentum states. Although this unique property has proved invaluable in diverse recent studies ranging from optical communication to quantum information, it has not been considered useful or even relevant for simulating nontrivial physics problems such as topological phenomena. Contrary to this misconception, we demonstrate the incredible value of orbital angular momentum of light for quantum simulation by showing theoretically how it allows to study a variety of important 2D topological physics in a 1D array of optical cavities. This application for orbital angular momentum of light not only reduces required physical resources but also increases feasible scale of simulation, and thus makes it possible to investigate important topics such as edge-state transport and topological phase transition in a small simulator ready for immediate experimental exploration. PMID:26145177

  7. Quantum simulation of 2D topological physics in a 1D array of optical cavities.

    PubMed

    Luo, Xi-Wang; Zhou, Xingxiang; Li, Chuan-Feng; Xu, Jin-Shi; Guo, Guang-Can; Zhou, Zheng-Wei

    2015-07-06

    Orbital angular momentum of light is a fundamental optical degree of freedom characterized by unlimited number of available angular momentum states. Although this unique property has proved invaluable in diverse recent studies ranging from optical communication to quantum information, it has not been considered useful or even relevant for simulating nontrivial physics problems such as topological phenomena. Contrary to this misconception, we demonstrate the incredible value of orbital angular momentum of light for quantum simulation by showing theoretically how it allows to study a variety of important 2D topological physics in a 1D array of optical cavities. This application for orbital angular momentum of light not only reduces required physical resources but also increases feasible scale of simulation, and thus makes it possible to investigate important topics such as edge-state transport and topological phase transition in a small simulator ready for immediate experimental exploration.

  8. Orexin-A potentiates L-type calcium/barium currents in rat retinal ganglion cells.

    PubMed

    Liu, F; Weng, S-J; Yang, X-L; Zhong, Y-M

    2015-10-01

    Two neuropeptides, orexin-A and orexin-B (also called hypocretin-1 and -2), have been implicated in sleep/wake regulation, feeding behaviors via the activation of two subtypes of G-protein-coupled receptors: orexin 1 and orexin 2 receptors (OX1R and OX2R). While the expression of orexins and orexin receptors is immunohistochemically revealed in retinal neurons, the function of these peptides in the retina is largely unknown. Using whole-cell patch-clamp recordings in rat retinal slices, we demonstrated that orexin-A increased L-type-like barium currents (IBa,L) in ganglion cells (GCs), and the effect was blocked by the selective OX1R antagonist SB334867, but not by the OX2R antagonist TCS OX2 29. The orexin-A effect was abolished by intracellular dialysis of GDP-β-S/GPAnt-2A, a Gq protein inhibitor, suggesting the mediation of Gq. Additionally, during internal dialysis of the phosphatidylinositol (PI)-phospholipase C (PLC) inhibitor U73122, orexin-A did not change the IBa,L of GCs, whereas the orexin-A effect persisted in the presence of the phosphatidylcholine (PC)-PLC inhibitor D609. The orexin-A-induced potentiation was not seen with internal infusion of Ca(2+)-free solution or when inositol 1,4,5-trisphosphate (IP3)-sensitive Ca(2+) release from intracellular stores was blocked by heparin/xestospongins-C. Moreover, the orexin-A effect was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate, but was eliminated when PKC was inhibited by bisindolylmaleimide IV (Bis-IV)/Gö6976. Neither adenosine 3',5'-cyclic monophosphate (cAMP)-protein kinase A (PKA) nor guanosine 3',5'-cyclic monophosphate (cGMP)-protein kinase G (PKG) signaling pathway was likely involved, as orexin-A persisted to potentiate the IBa,L of GCs no matter these two pathways were activated or inhibited. These results suggest that, by activating OX1R, orexin-A potentiates the IBa,L of rat GCs through a distinct Gq/PI-PLC/IP3/Ca(2+)/PKC signaling pathway. Copyright

  9. Cav1.2, but not Cav1.3, L-type calcium channel subtype mediates nicotine-induced conditioned place preference in miceo.

    PubMed

    Liu, Yudan; Harding, Meghan; Dore, Jules; Chen, Xihua

    2017-04-03

    Nicotine use is one of the most common forms of drug addiction. Although L-type calcium channels (LTCCs) are involved in nicotine addiction, the contribution of the two primary LTCC subtypes (Ca v 1.2 and 1.3) is unknown. This study aims to determine the contribution of these two LTCC subtypes to nicotine-induced conditioned place preference (CPP) responses by using transgenic mouse models that do not express Ca v 1.3 (Ca v 1.3 -/- ) or contain a mutation in the dihydropyridine (DHP) site of the Ca v 1.2 (Ca v 1.2DHP -/- ). We found a hyperbolic dose dependent nicotine (0.1-1mg/kg; 0.5mg/kg optimum) effect on place preference in wild type (WT) mice, that could be prevented by the DHP LTCC blocker nifedipine pretreatment. Similarly, Ca v 1.3 -/- mice showed nicotine-induced place preference which was antagonized by nifedipine. In contrast, nifedipine pretreatment of Ca v 1.2DHP -/- mice had no effect on nicotine-induced CPP responses, suggesting an involvement of Ca v 1.2 subtype in the nicotine-induced CPP response. Nifedipine alone failed to produce either conditioned place aversion or CPP in WT mice. These results collectively indicate Ca v 1.2, but not Ca v 1.3 LTCC subtype regulates, at least in part, the reinforcing effects of nicotine use. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Dissipative NEGF methodology to treat short range Coulomb interaction: Current through a 1D nanostructure.

    PubMed

    Martinez, Antonio; Barker, John R; Di Prieto, Riccardo

    2018-06-13

    A methodology describing Coulomb blockade in the Non-equilibrium Green Function formalism is presented. We carried out ballistic and dissipative simulations through a 1D quantum dot using an Einstein phonon model. Inelastic phonons with different energies have been considered. The methodology incorporates the short-range Coulomb interaction between two electrons through the use of a two-particle Green's function. Unlike previous work, the quantum dot has spatial resolution i.e. it is not just parameterized by the energy level and coupling constants of the dot. Our method intends to describe the effect of electron localization while maintaining an open boundary or extended wave function. The formalism conserves the current through the nanostructure. A simple 1D model is used to explain the increase of mobility in semi-crystalline polymers as a function of the electron concentration. The mechanism suggested is based on the lifting of energy levels into the transmission window as a result of the local electron-electron repulsion inside a crystalline domain. The results are aligned with recent experimental findings. Finally, as a proof of concept, we present a simulation of a low temperature resonant structure showing the stability diagram in the Coulomb blockade regime. . © 2018 IOP Publishing Ltd.

  11. Sensitivity of transpiration to subsurface properties: Exploration with a 1-D model

    NASA Astrophysics Data System (ADS)

    Vrettas, Michail D.; Fung, Inez Y.

    2017-06-01

    The amount of moisture transpired by vegetation is critically tied to the moisture supply accessible to the root zone. In a Mediterranean climate, integrated evapotranspiration (ET) is typically greater in the dry summer when there is an uninterrupted period of high insolation. We present a 1-D model to explore the subsurface factors that may sustain ET through the dry season. The model includes a stochastic parameterization of hydraulic conductivity, root water uptake efficiency, and hydraulic redistribution by plant roots. Model experiments vary the precipitation, the magnitude and seasonality of ET demand, as well as rooting profiles and rooting depths of the vegetation. The results show that the amount of subsurface moisture remaining at the end of the wet winter is determined by the competition among abundant precipitation input, fast infiltration, and winter ET demand. The weathered bedrock retains ˜30% of the winter rain and provides a substantial moisture reservoir that may sustain ET of deep-rooted (>8 m) trees through the dry season. A small negative feedback exists in the root zone, where the depletion of moisture by ET decreases hydraulic conductivity and enhances the retention of moisture. Hence, hydraulic redistribution by plant roots is impactful in a dry season, or with a less conductive subsurface. Suggestions for implementing the model in the CESM are discussed.

  12. Sensitivity of transpiration to subsurface properties: Exploration with a 1-D model

    DOE PAGES

    Vrettas, Michail D.; Fung, Inez Y.

    2017-05-04

    The amount of moisture transpired by vegetation is critically tied to the moisture supply accessible to the root zone. In a Mediterranean climate, integrated evapotranspiration (ET) is typically greater in the dry summer when there is an uninterrupted period of high insolation. We present a 1-D model to explore the subsurface factors that may sustain ET through the dry season. The model includes a stochastic parameterization of hydraulic conductivity, root water uptake efficiency, and hydraulic redistribution by plant roots. Model experiments vary the precipitation, the magnitude and seasonality of ET demand, as well as rooting profiles and rooting depths ofmore » the vegetation. The results show that the amount of subsurface moisture remaining at the end of the wet winter is determined by the competition among abundant precipitation input, fast infiltration, and winter ET demand. The weathered bedrock retains math formula of the winter rain and provides a substantial moisture reservoir that may sustain ET of deep-rooted (>8 m) trees through the dry season. A small negative feedback exists in the root zone, where the depletion of moisture by ET decreases hydraulic conductivity and enhances the retention of moisture. Hence, hydraulic redistribution by plant roots is impactful in a dry season, or with a less conductive subsurface. Suggestions for implementing the model in the CESM are discussed.« less

  13. Diffusive Public Goods and Coexistence of Cooperators and Cheaters on a 1D Lattice

    PubMed Central

    Scheuring, István

    2014-01-01

    Many populations of cells cooperate through the production of extracellular materials. These materials (enzymes, siderophores) spread by diffusion and can be applied by both the cooperator and cheater (non-producer) cells. In this paper the problem of coexistence of cooperator and cheater cells is studied on a 1D lattice where cooperator cells produce a diffusive material which is beneficial to the individuals according to the local concentration of this public good. The reproduction success of a cell increases linearly with the benefit in the first model version and increases non-linearly (saturates) in the second version. Two types of update rules are considered; either the cooperative cell stops producing material before death (death-production-birth, DpB) or it produces the common material before it is selected to die (production-death-birth, pDB). The empty space is occupied by its neighbors according to their replication rates. By using analytical and numerical methods I have shown that coexistence of the cooperator and cheater cells is possible although atypical in the linear version of this 1D model if either DpB or pDB update rule is assumed. While coexistence is impossible in the non-linear model with pDB update rule, it is one of the typical behaviors in case of the non-linear model with DpB update rule. PMID:25025985

  14. Photoexcitations in a 1D manganite model: From quasiclassical light absorption to quasiparticle relaxations

    NASA Astrophysics Data System (ADS)

    Köhler, T.; Schumann, O.; Biebl, F.; Kramer, S.; Kehrein, S.; Manmana, S.; Rajpurohit, S.; Sotoudeh, M.; Blöchl, P.

    We investigate 1D correlated systems following a photoexcitation by combining ab-initio methods, time-dependent matrix product state (MPS) approaches, analytical insights from linearized quantum Boltzmann equations (LBE), and molecular dynamics (MD) simulations to describe the dynamics on different time scales ranging from femto- up to nanoseconds. This is done for manganite systems in the material class Pr1-xCaxMnO3. We derive 1D ab-initio model Hamiltonians for which we compute the ground states at different values of the doping using MD simulations. At half doping, we obtain a magnetic microstructure of alternating dimers from which we derive a 1D Hubbard-type model. The dynamics is analyzed concerning the formation and lifetime of such quasiparticles via a LBE. We find that the magnetic microstructure strongly enhances the lifetime of the excitations. In this way, our work constitutes a first step to building a unifying theoretical framework for the description of photoexcitations in strongly correlated materials over a wide range of time scales, capable of making predictions for ongoing experiments investigating pump-probe situations and unconventional photovoltaics. Financial support from the Deutsche Forschungsgemeinschaft (DFG) through SFB/CRC1073 (Projects B03 and C03) is gratefully acknowledged.

  15. Trapping and patterning of large particles and cells in a 1D ultrasonic standing wave.

    PubMed

    Habibi, Ruhollah; Devendran, Citsabehsan; Neild, Adrian

    2017-09-26

    The use of ultrasound for trapping and patterning particles or cells in microfluidic systems is usually confined to particles which are considerably smaller than the acoustic wavelength. In this regime, the primary forces result in particle clustering at certain locations in the sound field, whilst secondary forces, those arising due to particle-particle interaction forces, assist this clustering process. Using a wavelength closer to the size of the particles allows one particle to be held at each primary force minimum. However, to achieve this, the influence of secondary forces needs to be carefully studied, as inter-particle attraction is highly undesirable. Here, we study the effect of particle size and material properties on both the primary and secondary acoustic forces as the particle diameter is increased towards the wavelength of the 1-dimensional axisymmetric ultrasonic field. We show that the resonance frequencies of the solid sphere have an important role in the resulting secondary forces which leads to a narrow band of frequencies that allow the patterning of large particles in a 1-D array. Knowledge regarding the naturally existent secondary forces would allow for system designs enabling single cell studies to be conducted in a biologically safe manner.

  16. A Co16 cluster and a 1-D Mn chain complex supported by benzohydroxamic acid.

    PubMed

    Cao, Yanyuan; Chen, Yanmei; Li, Lei; Gao, Dandan; Liu, Wei; Hu, Hailiang; Li, Wu; Li, Yahong

    2013-08-14

    The syntheses, crystal structures and magnetic properties are described for a {Co16} cluster [Co(II)16O(OH)2(bha)12(PhCO2)4(Phen)2(MeOH)4]·2MeOH (1) and a 1-D Mn(II) chain complex [Mn(Hbha)2]n·(2MeOH)n (2) (H2bha = benzohydroxamic acid; Phen = 1,10-phenanthroline). The 1 : 1 : 0.5 reaction of Co(O2CMe)2·4H2O, H2bha and 1,10-phenanthroline in MeOH at 100 °C under autogenous pressure gave cluster 1. Complex 2 was obtained from the 1 : 1 reaction mixture of Mn(O2CMe)2·2H2O and H2bha in MeOH under solvothermal conditions. The {Co16} cluster can be thought as a face-centered cube with two wings. The H2bha ligands show hydroximic form in 1 and exhibit hydroxamic mode in 2. The hydroximate ligands in 1 display three distinct binding modes, one of which is novel. Variable-temperature solid-state dc magnetic susceptibility studies have been performed in the 2.0-300 K range for complexes 1 and 2. Antiferromagnetic M(II)···M(II) exchange interactions were found for both 1 and 2. This work also demonstrates that solvothermal method is a potential synthetic approach for the design and growth of high nuclearity clusters or chain complexes of the H2bha ligand.

  17. Testing the early Mars H2-CO2 greenhouse hypothesis with a 1-D photochemical model

    NASA Astrophysics Data System (ADS)

    Batalha, Natasha; Domagal-Goldman, Shawn D.; Ramirez, Ramses; Kasting, James F.

    2015-09-01

    A recent study by Ramirez et al. (Ramirez, R.M. et al. [2014]. Nat. Geosci. 7(1), 59-63. http://www.nature.com/doifinder/10.1038/ngeo2000 (accessed 16.09.14)) demonstrated that an atmosphere with 1.3-4 bar of CO2 and H2O, in addition to 5-20% H2, could have raised the mean annual and global surface temperature of early Mars above the freezing point of water. Such warm temperatures appear necessary to generate the rainfall (or snowfall) amounts required to carve the ancient martian valleys. Here, we use our best estimates for early martian outgassing rates, along with a 1-D photochemical model, to assess the conversion efficiency of CO, CH4, and H2S to CO2, SO2, and H2. Our outgassing estimates assume that Mars was actively recycling volatiles between its crust and interior, as Earth does today. H2 production from serpentinization and deposition of banded iron-formations is also considered. Under these assumptions, maintaining an H2 concentration of ˜1-2% by volume is achievable, but reaching 5% H2 requires additional H2 sources or a slowing of the hydrogen escape rate below the diffusion limit. If the early martian atmosphere was indeed H2-rich, we might be able to see evidence of this in the rock record. The hypothesis proposed here is consistent with new data from the Curiosity Rover, which show evidence for a long-lived lake in Gale Crater near Mt. Sharp. It is also consistent with measured oxygen fugacities of martian meteorites, which show evidence for progressive mantle oxidation over time.

  18. A 1-D evolutionary model for icy satellites, applied to Enceladus

    NASA Astrophysics Data System (ADS)

    Prialnik, Dina; Malamud, Uri

    2015-11-01

    A 1-D long-term evolution code for icy satellites is presented, which couples multiple processes: water migration, geochemical reactions, water and silicate phase transitions, crystallization, compaction by self-gravity, and ablation. The code takes into account various energy sources: tidal heating, radiogenic heating, geochemical energy released by serpentinization or absorbed by mineral dehydration, gravitational energy, and insolation. It includes heat transport by conduction, convection, and advection.The code is applied to Enceladus, by guessing the initial conditions that would render a structure compatible with present-day observations, and adopting a homogeneous initial structure. Assuming that the satellite has been losing water continually along its evolution, it follows that it was formed as a more massive, more ice-rich and more porous object, and gradually transformed into its present day state, due to sustained tidal heating. Several initial compositions and evolution scenarios are considered, and the evolution is simulated for the age of the Solar System. The results corresponding to the present configuration are confronted with the available observational constraints. The present configuration is shown to be differentiated into a pure icy mantle, several tens of km thick, overlying a rocky core, composed of dehydrated rock in the central part and hydrated rock in the outer part. Such a differentiated structure is obtained not only for Enceladus, but for other medium size ice-rich bodies as well.Predictions for Enceladus are a higher rock/ice mass ratio than previously assumed, and a thinner ice mantle, compatible with recent estimates based on gravity field measurements. Although, obviously, the 1-D model cannot be used to explain local phenomena, it sheds light on the internal structure invoked in explanations of localized features and activities.

  19. Modelling turbulent vertical mixing sensitivity using a 1-D version of NEMO

    NASA Astrophysics Data System (ADS)

    Reffray, G.; Bourdalle-Badie, R.; Calone, C.

    2014-08-01

    Through two numerical experiments, a 1-D vertical model called NEMO1D was used to investigate physical and numerical turbulent-mixing behaviour. The results show that all the turbulent closures tested (k + l from Blanke and Delecluse, 1993 and two equation models: Generic Lengh Scale closures from Umlauf and Burchard, 2003) are able to correctly reproduce the classical test of Kato and Phillips (1969) under favourable numerical conditions while some solutions may diverge depending on the degradation of the spatial and time discretization. The performances of turbulence models were then compared with data measured over a one-year period (mid-2010 to mid-2011) at the PAPA station, located in the North Pacific Ocean. The modelled temperature and salinity were in good agreement with the observations, with a maximum temperature error between -2 and 2 °C during the stratified period (June to October). However the results also depend on the numerical conditions. The vertical RMSE varied, for different turbulent closures, from 0.1 to 0.3 °C during the stratified period and from 0.03 to 0.15 °C during the homogeneous period. This 1-D configuration at the PAPA station (called PAPA1D) is now available in NEMO as a reference configuration including the input files and atmospheric forcing set described in this paper. Thus, all the results described can be recovered by downloading and launching PAPA1D. The configuration is described on the NEMO site (http://www.nemo-ocean.eu/Using-NEMO/Configurations/C1D_PAPA). This package is a good starting point for further investigation of vertical processes.

  20. Modelling turbulent vertical mixing sensitivity using a 1-D version of NEMO

    NASA Astrophysics Data System (ADS)

    Reffray, G.; Bourdalle-Badie, R.; Calone, C.

    2015-01-01

    Through two numerical experiments, a 1-D vertical model called NEMO1D was used to investigate physical and numerical turbulent-mixing behaviour. The results show that all the turbulent closures tested (k+l from Blanke and Delecluse, 1993, and two equation models: generic length scale closures from Umlauf and Burchard, 2003) are able to correctly reproduce the classical test of Kato and Phillips (1969) under favourable numerical conditions while some solutions may diverge depending on the degradation of the spatial and time discretization. The performances of turbulence models were then compared with data measured over a 1-year period (mid-2010 to mid-2011) at the PAPA station, located in the North Pacific Ocean. The modelled temperature and salinity were in good agreement with the observations, with a maximum temperature error between -2 and 2 °C during the stratified period (June to October). However, the results also depend on the numerical conditions. The vertical RMSE varied, for different turbulent closures, from 0.1 to 0.3 °C during the stratified period and from 0.03 to 0.15 °C during the homogeneous period. This 1-D configuration at the PAPA station (called PAPA1D) is now available in NEMO as a reference configuration including the input files and atmospheric forcing set described in this paper. Thus, all the results described can be recovered by downloading and launching PAPA1D. The configuration is described on the NEMO site (http://www.nemo-ocean.eu/Using-NEMO/Configurations/C1D_PAPA). This package is a good starting point for further investigation of vertical processes.

  1. Improving the Performance of a 1-D Ultrasound Transducer Array by Subdicing.

    PubMed

    Janjic, Jovana; Shabanimotlagh, Maysam; van Soest, Gijs; van der Steen, Antonius F W; de Jong, Nico; Verweij, Martin D

    2016-08-01

    In medical ultrasound transducer design, the geometry of the individual elements is crucial since it affects the vibration mode of each element and its radiation impedance. For a fixed frequency, optimal vibration (i.e., uniform surface motion) can be achieved by designing elements with very small width-to-thickness ratios. However, for optimal radiation impedance (i.e., highest radiated power), the width should be as large as possible. This leads to a contradiction that can be solved by subdicing wide elements. To systematically examine the effect of subdicing on the performance of a 1-D ultrasound transducer array, we applied finite-element simulations. We investigated the influence of subdicing on the radiation impedance, on the time and frequency response, and on the directivity of linear arrays with variable element widths. We also studied the effect of varying the depth of the subdicing cut. The results show that, for elements having a width greater than 0.6 times the wavelength, subdicing improves the performance compared with that of nonsubdiced elements: the emitted pressure may be increased up to a factor of three, the ringing time may be reduced by up to 50%, the bandwidth increased by up to 77%, and the sidelobes reduced by up to 13 dB. Moreover, this simulation study shows that all these improvements can already be achieved by subdicing the elements to a depth of 70% of the total element thickness. Thus, subdicing can improve important transducer parameters and, therefore, help in achieving images with improved signal-to-noise ratio and improved resolution.

  2. Testing the accuracy of a 1-D volcanic plume model in estimating mass eruption rate

    USGS Publications Warehouse

    Mastin, Larry G.

    2014-01-01

    During volcanic eruptions, empirical relationships are used to estimate mass eruption rate from plume height. Although simple, such relationships can be inaccurate and can underestimate rates in windy conditions. One-dimensional plume models can incorporate atmospheric conditions and give potentially more accurate estimates. Here I present a 1-D model for plumes in crosswind and simulate 25 historical eruptions where plume height Hobs was well observed and mass eruption rate Mobs could be calculated from mapped deposit mass and observed duration. The simulations considered wind, temperature, and phase changes of water. Atmospheric conditions were obtained from the National Center for Atmospheric Research Reanalysis 2.5° model. Simulations calculate the minimum, maximum, and average values (Mmin, Mmax, and Mavg) that fit the plume height. Eruption rates were also estimated from the empirical formula Mempir = 140Hobs4.14 (Mempir is in kilogram per second, Hobs is in kilometer). For these eruptions, the standard error of the residual in log space is about 0.53 for Mavg and 0.50 for Mempir. Thus, for this data set, the model is slightly less accurate at predicting Mobs than the empirical curve. The inability of this model to improve eruption rate estimates may lie in the limited accuracy of even well-observed plume heights, inaccurate model formulation, or the fact that most eruptions examined were not highly influenced by wind. For the low, wind-blown plume of 14–18 April 2010 at Eyjafjallajökull, where an accurate plume height time series is available, modeled rates do agree better with Mobs than Mempir.

  3. [Aging-related ionic remodeling of L-type voltage dependent calcium channel in left atria of canine].

    PubMed

    Zhou, Xian-hui; Zhang, Jian; Gan, Tian-yi; Xu, Guo-jun; Tang, Bao-peng

    2012-04-01

    To investigate aging-related ionic remodeling of L-type voltage dependent calcium channel (LVDCC) in left atria of canine. Seven adult (2.0 - 2.5 years) and 10 aged (> 8 years) dogs were used. The current of LVDCC was recorded by patch clamp technique in the whole cell mode. The action potential duration (APD(90)), amplitude of action potential plateau (APA), I(Ca-L) peak current density of LVDCC were recorded. The mRNA and protein expressions of α1c subunit (Ca(V1.2)), sarcoplasmic reticulum Ca(2+)-ATPase (SECRA(2)), Calpain-I, ryanodine receptor (RYR(2)) were detected by quantitative RT-PCR and Western blot, respectively. I(Ca-L) peak current density [(-8.11 ± 0.54) pA/pF vs. (-14.04 ± 0.82) pA/pF, P < 0.05] was significantly reduced and action potential duration to 90% repolarization (APD(90)) significantly prolonged [(340.5 ± 10.1) ms vs. (320.0 ± 7.9) ms, P < 0.05] in aged group than in adult group. The mRNA gene expression level of Ca(V1.2) was significantly lower (0.90 ± 0.35 vs. 2.38 ± 0.40, P < 0.05) while mRNA expression of RYR(2) was significantly higher (4.39 ± 4.68 vs. 1.49 ± 1.69, P < 0.05) in the aged dogs than in the adult dogs. mRNA expression of SECRA(2) and Calpain-I was similar between the two groups. Similarly, the protein expression level of Ca(V1.2) was significantly lower (0.13 ± 0.10 vs. 0.29 ± 0.12, P < 0.05) while the protein expression level of RYR(2) was significantly higher (0.18 ± 0.21 vs. 0.08 ± 0.36, P < 0.05) in the aged dogs than in the adult dogs. Again, protein expression of SECRA(2), PLN(1) and Calpain-I was similar between the two groups. These data suggest that aging could induce mRNA and protein expression changes of Ca(V1.2) and RYR(2) of LVDCC which might serve as the molecular basis of I(Ca-L) remodeling in aged dogs and might be linked to the increased likelihood of developing atrial fibrillation (AF) in aged dogs.

  4. On the interpretation of the optical spectra of L-type dwarfs

    NASA Astrophysics Data System (ADS)

    Pavlenko, Ya.; Zapatero Osorio, M. R.; Rebolo, R.

    2000-03-01

    We present synthetic optical spectra in the red and far-red (640-930 nm) of a sample of field L dwarfs suitably selected to cover this new spectral class, and the brown dwarf GL 229B. We have used the recent ``dusty'' atmospheres by Tsuji (\\cite{tsuji00}) and by Allard (\\cite{allard99}), and a synthesis code (Pavlenko et al. \\cite{pav95}) working under LTE conditions which considers the chemical equilibrium of more than 100 molecular species and the detailed opacities for the most relevant bands. Our computations show that the alkali elements Li, Na, K, Rb, and Cs govern the optical spectra of the objects in our sample, with Na and K contributing significantly to block the optical emergent radiation. Molecular absorption bands of oxides (TiO and VO) and hydrides (CrH, FeH and CaH) also dominate at these wavelengths in the early L-types showing a strength that progressively decreases for later types. We find that the densities of these molecules in the atmospheres of our objects are considerably smaller by larger factors than those predicted by chemical equilibrium considerations. This is consistent with Ti and V atoms being depleted into grains of dust. In order to reproduce the overall shape of the optical spectra of our observations an additional opacity is required to be implemented in the computations. We have modelled it with a simple law of the form adeg \\ (nu / nu deg)N, with N = 4, and found that this provides a sufficiently good fit to the data. This additional opacity could be due to molecular/dust absorption or to dust scattering. We remark that the equivalent widths and intensities of the alkali lines are highly affected by this opacity. In particular, the lithium resonance line at 670.8 nm, which is widely used as a substellarity discriminator, is more affected by the additional opacity than by the natural depletion of neutral lithium atoms into molecular species. Our theoretical spectra displays a rather strong resonance feature even at very cool

  5. Viscous shear heating instabilities in a 1-D viscoelastic shear zone

    NASA Astrophysics Data System (ADS)

    Homburg, J. M.; Coon, E. T.; Spiegelman, M.; Kelemen, P. B.; Hirth, G.

    2010-12-01

    Viscous shear instabilities may provide a possible mechanism for some intermediate depth earthquakes where high confining pressure makes it difficult to achieve frictional failure. While many studies have explored the feedback between temperature-dependent strain rate and strain-rate dependent shear heating (e.g. Braeck and Podladchikov, 2007), most have used thermal anomalies to initiate a shear instability or have imposed a low viscosity region in their model domain (John et al., 2009). By contrast, Kelemen and Hirth (2007) relied on an initial grain size contrast between a predetermined fine-grained shear zone and coarse grained host rock to initiate an instability. This choice is supported by observations of numerous fine grained ductile shear zones in shallow mantle massifs as well as the possibility that annealed fine grained fault gouge, formed at oceanic transforms, subduction related thrusts and ‘outer rise’ faults, could be carried below the brittle/ductile transition by subduction. Improving upon the work of Kelemen and Hirth (2007), we have developed a 1-D numerical model that describes the behavior of a Maxwell viscoelastic body with the rheology of dry olivine being driven at a constant velocity at its boundary. We include diffusion and dislocation creep, dislocation accommodated grain boundary sliding, and low-temperature plasticity (Peierls mechanism). Initial results suggest that including low-temperature plasticity inhibits the ability of the system to undergo an instability, similar to the results of Kameyama et al. (1999). This is due to increased deformation in the background allowing more shear heating to take place, and thus softening the system prior to reaching the peak stress. However if the applied strain rate is high enough (e.g. greater than 0.5 x 10-11 s-1 for a domain size of 2 km, an 8 m wide shear zone, a background grain size of 1 mm, a shear zone grain size of 150 μm, and an initial temperature of 650°C) dramatic

  6. [Alterations of cardiac hemodynamics, sodium current and L-type calcium current in rats with L-thyroxine-induced cardiomyopathy].

    PubMed

    Wang, Jing; Zhang, Wei-Dong; Lin, Mu-Sen; Zhai, Qing-Bo; Yu, Feng

    2010-08-25

    The aim of the present study is to investigate the alterations of cardiac hemodynamics, sodium current (I(Na)) and L-type calcium current (I(Ca-L)) in the cardiomyopathic model of rats. The model of cardiomyopathy was established by intraperitoneal injection of L-thyroxine (0.5 mg/kg) for 10 d. The hemodynamics was measured with biological experimental system, and then I(Na) and I(Ca-L) were recorded by using whole cell patch clamp technique. The results showed that left ventricular systolic pressure (LVSP), left ventricular developed pressure (LVDP), +/-dp/dt(max) in cardiomyopathic group were significantly lower than those in the control group, while left ventricular end-diastolic pressure (LVEDP) in cardiomyopathic group was higher than that in the control group. Intraperitoneal injection of L-thyroxine significantly increased the current density of I(Na) [(-26.2+/-3.2) pA/pF vs (-21.1+/-6.3) pA/pF, P<0.01], shifted steady-state activation and inactivation curves negatively, and markedly prolonged the time constant of recovery from inactivation. On the other hand, the injection of L-thyroxine significantly increased the current density of I(Ca-L) [(-7.9+/-0.8) pA/pF vs (-5.4+/-0.6) pA/pF, P<0.01)], shifted steady-state activation and inactivation curves negatively, and obviously shortened the time constant of recovery from inactivation. In conclusion, the cardiac performance of cardiomyopathic rats is similar to that of rats with heart failure, in which the current density of I(Na) and especially the I(Ca-L) are enhanced, suggesting that calcium channel blockade and a decrease in Na(+) permeability of membrane may play an important role in the treatment of cardiomyopathy.

  7. Distribution of L-type calcium channels in rat thalamic neurones.

    PubMed

    Budde, T; Munsch, T; Pape, H C

    1998-02-01

    One major pathway for calcium entry into neurones is through voltage-activated calcium channels. The distribution of calcium channels over the membrane surface is important for their contribution to neuronal function. Electrophysiological recordings from thalamic cells in situ and after acute isolation demonstrated the presence of high-voltage activated calcium currents. The use of specific L-type calcium channel agonists and antagonists of the dihydropyridine type revealed an about 40% contribution of L-type channels to the total high-voltage-activated calcium current. In order to localize L-type calcium channels in thalamic neurones, fluorescent dihydropyridines were used. They were combined with the fluorescent dye RH414, which allowed the use of a ratio technique and thereby the determination of channel density. The distribution of L-type channels was analysed in the three main thalamic cell types: thalamocortical relay cells, local interneurones and reticular thalamic neurones. While channel density was highest in the soma and decreased significantly in the dendritic region, channels appeared to be clustered differentially in the three types of cells. In thalamocortical cells, L-type channels were clustered in high density around the base of dendrites, while they were more evenly distributed on the soma of interneurones. Reticular thalamic neurones exhibited high density of L-type channels in more central somatic regions. The differential localization of L-type calcium channels found in this study implies their predominate involvement in the regulation of somatic and proximal dendritic calcium-dependent processes, which may be of importance for specific thalamic functions, such as those mediating the transition from rhythmic burst activity during sleep to single spike activity during wakefulness or regulating the relay of visual information.

  8. The saponin monomer of dwarf lilyturf tuber, DT-13, reduces L-type calcium currents during hypoxia in adult rat ventricular myocytes.

    PubMed

    Tao, Jin; Wang, Hongyi; Zhou, Hong; Li, Shengnan

    2005-10-28

    The saponin monomer 13 of dwarf lilyturf tuber (DT-13), one of the saponin monomers of dwarf lilyturf tuber, has been found to have potent cardioprotective effects. In order to investigate the effects of DT-13 on L-type calcium currents (I(Ca,L)), exploring the mechanisms of DT-13's cardioprotective effects in the condition of pathophysiology, we directly measured the I(Ca,L) during hypoxia in the adult rat cardiac myocytes exposed to DT-13 using standard whole-cell patch-clamp recording technique. Our previous results showed that DT-13 exerted decreasing effects on the I(Ca,L) of the single adult rat cardiac myocytes. In the condition of hypoxia, the current density was inhibited by about 29% after exposure of the cells to DT-13 (0.1 micromol L(-1)) for 10 min, from 6.96+/-1.05 pA/pF to 4.38+/-0.35 pA/pF (n=5, P<0.05). This I(Ca,L)-inhibiting action of DT-13 was concentration-dependent and showed no frequency-dependence. DT-13 up-shifted the current-voltage (I-V) curve. Steady-state activation of I(Ca,L) was not affected markedly, and the half activation potential (V(0.5)) in the presence of DT-13 (0.1 micromol L(-1)) was also not significantly different. DT-13 at 0.1 micromol L(-1) markedly accelerated the voltage-dependent steady-state inactivation of calcium current and shifted the steady-state inactivation curve of I(Ca,L) to the left. In combination with previous reports, these results suggest that there might be a close relationship between the cardioprotective effects of DT-13 and L-type calcium channels in the condition of hypoxia.

  9. GLP-2 potentiates L-type CA2+ channel activity associated with stimulated glucose uptake in hippocampal neurons

    USDA-ARS?s Scientific Manuscript database

    Glucagon-like peptide-2 (GLP-2) is a neuropeptide secreted from endocrine cells in the gut and neurons in the brain. GLP-2 stimulates intestinal crypt cell proliferation and mucosal blood flow while decreasing gastric emptying and gut motility. However, a GLP-2-mediated signaling network has not bee...

  10. Ion permeation and glutamate residues linked by Poisson-Nernst-Planck theory in L-type calcium channels.

    PubMed Central

    Nonner, W; Eisenberg, B

    1998-01-01

    L-type Ca channels contain a cluster of four charged glutamate residues (EEEE locus), which seem essential for high Ca specificity. To understand how this highly charged structure might produce the currents and selectivity observed in this channel, a theory is needed that relates charge to current. We use an extended Poisson-Nernst-Planck (PNP2) theory to compute (mean) Coulombic interactions and thus to examine the role of the mean field electrostatic interactions in producing current and selectivity. The pore was modeled as a central cylinder with tapered atria; the cylinder (i.e., "pore proper") contained a uniform volume density of fixed charge equivalent to that of one to four carboxyl groups. The pore proper was assigned ion-specific, but spatially uniform, diffusion coefficients and excess chemical potentials. Thus electrostatic selection by valency was computed self-consistently, and selection by other features was also allowed. The five external parameters needed for a system of four ionic species (Na, Ca, Cl, and H) were determined analytically from published measurements of thre limiting conductances and two critical ion concentrations, while treating the pore as a macroscopic ion-exchange system in equilibrium with a uniform bath solution. The extended PNP equations were solved with these parameters, and the predictions were compared to currents measured in a variety of solutions over a range of transmembrane voltages. The extended PNP theory accurately predicted current-voltage relations, anomalous mole fraction effects in the observed current, saturation effects of varied Ca and Na concentrations, and block by protons. Pore geometry, dielectric permittivity, and the number of carboxyl groups had only weak effects. The successful prediction of Ca fluxes in this paper demonstrates that ad hoc electrostatic parameters, multiple discrete binding sites, and logistic assumptions of single-file movement are all unnecessary for the prediction of permeation in

  11. Antidepressants Rescue Stress-Induced Disruption of Synaptic Plasticity via Serotonin Transporter-Independent Inhibition of L-Type Calcium Channels.

    PubMed

    Normann, Claus; Frase, Sibylle; Haug, Verena; von Wolff, Gregor; Clark, Kristin; Münzer, Patrick; Dorner, Alexandra; Scholliers, Jonas; Horn, Max; Vo Van, Tanja; Seifert, Gabriel; Serchov, Tsvetan; Biber, Knut; Nissen, Christoph; Klugbauer, Norbert; Bischofberger, Josef

    2017-10-19

    Long-term synaptic plasticity is a basic ability of the brain to dynamically adapt to external stimuli and regulate synaptic strength and ultimately network function. It is dysregulated by behavioral stress in animal models of depression and in humans with major depressive disorder. Antidepressants have been shown to restore disrupted synaptic plasticity in both animal models and humans; however, the underlying mechanism is unclear. We examined modulation of synaptic plasticity by selective serotonin reuptake inhibitors (SSRIs) in hippocampal brain slices from wild-type rats and serotonin transporter (SERT) knockout mice. Recombinant voltage-gated calcium (Ca 2+ ) channels in heterologous expression systems were used to determine the modulation of Ca 2+ channels by SSRIs. We tested the behavioral effects of SSRIs in the chronic behavioral despair model of depression both in the presence and in the absence of SERT. SSRIs selectively inhibited hippocampal long-term depression. The inhibition of long-term depression by SSRIs was mediated by a direct block of voltage-activated L-type Ca 2+ channels and was independent of SERT. Furthermore, SSRIs protected both wild-type and SERT knockout mice from behavioral despair induced by chronic stress. Finally, long-term depression was facilitated in animals subjected to the behavioral despair model, which was prevented by SSRI treatment. These results showed that antidepressants protected synaptic plasticity and neuronal circuitry from the effects of stress via a modulation of Ca 2+ channels and synaptic plasticity independent of SERT. Thus, L-type Ca 2+ channels might constitute an important signaling hub for stress response and for pathophysiology and treatment of depression. Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  12. L-type Voltage-Gated Calcium Channels in Conditioned Fear: A Genetic and Pharmacological Analysis

    ERIC Educational Resources Information Center

    McKinney, Brandon C.; Sze, Wilson; White, Jessica A.; Murphy, Geoffrey G.

    2008-01-01

    Using pharmacological approaches, others have suggested that L-type voltage-gated calcium channels (L-VGCCs) mediate both consolidation and extinction of conditioned fear. In the absence of L-VGCC isoform-specific antagonists, we have begun to investigate the subtype-specific role of LVGCCs in consolidation and extinction of conditioned fear…

  13. Oral Transmission of L-Type Bovine Spongiform Encephalopathy Agent among Cattle.

    PubMed

    Okada, Hiroyuki; Iwamaru, Yoshifumi; Imamura, Morikazu; Miyazawa, Kohtaro; Matsuura, Yuichi; Masujin, Kentaro; Murayama, Yuichi; Yokoyama, Takashi

    2017-02-01

    To determine oral transmissibility of the L-type bovine spongiform encephalopathy (BSE) prion, we orally inoculated 16 calves with brain homogenates of the agent. Only 1 animal, given a high dose, showed signs and died at 88 months. These results suggest low risk for oral transmission of the L-BSE agent among cattle.

  14. Trans-Golgi network localized small GTPase RabA1d is involved in cell plate formation and oscillatory root hair growth.

    PubMed

    Berson, Tobias; von Wangenheim, Daniel; Takáč, Tomáš; Šamajová, Olga; Rosero, Amparo; Ovečka, Miroslav; Komis, George; Stelzer, Ernst H K; Šamaj, Jozef

    2014-09-27

    Small Rab GTPases are important regulators of vesicular trafficking in plants. AtRabA1d, a member of the RabA1 subfamily of small GTPases, was previously found in the vesicle-rich apical dome of growing root hairs suggesting a role during tip growth; however, its specific intracellular localization and role in plants has not been well described. The transient expression of 35S::GFP:RabA1d construct in Allium porrum and Nicotiana benthamiana revealed vesicular structures, which were further corroborated in stable transformed Arabidopsis thaliana plants. GFP-RabA1d colocalized with the trans-Golgi network marker mCherry-VTI12 and with early FM4-64-labeled endosomal compartments. Late endosomes and endoplasmic reticulum labeled with FYVE-DsRed and ER-DsRed, respectively, were devoid of GFP-RabA1d. The accumulation of GFP-RabA1d in the core of brefeldin A (BFA)-induced-compartments and the quantitative upregulation of RabA1d protein levels after BFA treatment confirmed the association of RabA1d with early endosomes/TGN and its role in vesicle trafficking. Light-sheet microscopy revealed involvement of RabA1d in root development. In root cells, GFP-RabA1d followed cell plate expansion consistently with cytokinesis-related vesicular trafficking and membrane recycling. GFP-RabA1d accumulated in disc-like structures of nascent cell plates, which progressively evolved to marginal ring-like structures of the growing cell plates. During root hair growth and development, GFP-RabA1d was enriched at root hair bulges and at the apical dome of vigorously elongating root hairs. Importantly, GFP-RabA1d signal intensity exhibited an oscillatory behavior in-phase with tip growth. Progressively, this tip localization dissapeared in mature root hairs suggesting a link between tip localization of RabA1d and root hair elongation. Our results support a RabA1d role in events that require vigorous membrane trafficking. RabA1d is located in early endosomes/TGN and is involved in vesicle

  15. Nicergoline inhibits T-type Ca2+ channels in rat isolated hippocampal CA1 pyramidal neurones.

    PubMed Central

    Takahashi, K.; Akaike, N.

    1990-01-01

    1. The effects of nicergoline on the T- and L-type Ca2+ currents in pyramidal cells freshly isolated from rat hippocampal CA1 region were investigated by use of a 'concentration-clamp' technique. The technique combines a suction-pipette technique, which allows intracellular perfusion under a single-electrode voltage-clamp, and rapid exchange of extracellular solution within 2 ms. 2. T-type Ca2+ currents were evoked by step depolarizations from a holding potential of -100 mV to potentials more positive than -70 to -60 mV, and reached a peak at about -30 mV in the current-voltage relationship. Activation and inactivation of T-type Ca2+ currents were highly potential-dependent. 3. Nicergoline and other Ca2+ antagonists dose-dependently blocked the T-type Ca2+ channel with an order of potency nicardipine greater than nicergoline greater than diltiazem. 4. The L-type Ca2+ channel was also blocked in the order nicardipine greater than nicergoline greater than diltiazem, although the T-type Ca2+ channel was more sensitive to nicergoline. 5. The inhibitory effects of nicergoline and nicardipine on the T-type Ca2+ current were voltage-, time-, and use-dependent, and the inhibition increased with a decrease in the external Ca2+ concentration. Diltiazem showed only a use-dependent block. PMID:2169937

  16. Nicergoline inhibits T-type Ca2+ channels in rat isolated hippocampal CA1 pyramidal neurones.

    PubMed

    Takahashi, K; Akaike, N

    1990-08-01

    1. The effects of nicergoline on the T- and L-type Ca2+ currents in pyramidal cells freshly isolated from rat hippocampal CA1 region were investigated by use of a 'concentration-clamp' technique. The technique combines a suction-pipette technique, which allows intracellular perfusion under a single-electrode voltage-clamp, and rapid exchange of extracellular solution within 2 ms. 2. T-type Ca2+ currents were evoked by step depolarizations from a holding potential of -100 mV to potentials more positive than -70 to -60 mV, and reached a peak at about -30 mV in the current-voltage relationship. Activation and inactivation of T-type Ca2+ currents were highly potential-dependent. 3. Nicergoline and other Ca2+ antagonists dose-dependently blocked the T-type Ca2+ channel with an order of potency nicardipine greater than nicergoline greater than diltiazem. 4. The L-type Ca2+ channel was also blocked in the order nicardipine greater than nicergoline greater than diltiazem, although the T-type Ca2+ channel was more sensitive to nicergoline. 5. The inhibitory effects of nicergoline and nicardipine on the T-type Ca2+ current were voltage-, time-, and use-dependent, and the inhibition increased with a decrease in the external Ca2+ concentration. Diltiazem showed only a use-dependent block.

  17. Characterisation of marrubenol, a diterpene extracted from Marrubium vulgare, as an L-type calcium channel blocker

    PubMed Central

    El Bardai, Sanae; Wibo, Maurice; Hamaide, Marie-Christine; Lyoussi, Badiaa; Quetin-Leclercq, Joëlle; Morel, Nicole

    2003-01-01

    The objective of the present study was to investigate the mechanism of the relaxant activity of marrubenol, a diterpenoid extracted from Marrubium vulgare. In rat aorta, marrubenol was a more potent inhibitor of the contraction evoked by 100 mM KCl (IC50: 11.8±0.3 μM, maximum relaxation: 93±0.6%) than of the contraction evoked by noradrenaline (maximum relaxation: 30±1.5%). In fura-2-loaded aorta, marrubenol simultaneously inhibited the Ca2+ signal and the contraction evoked by 100 mM KCl, and decreased the quenching rate of fura-2 fluorescence by Mn2+. Patch-clamp data obtained in aortic smooth muscle cells (A7r5) indicated that marrubenol inhibited Ba2+ inward current in a voltage-dependent manner (KD: 8±2 and 40±6 μM at holding potentials of −50 and −100 mV, respectively). These results showed that marrubenol inhibits smooth muscle contraction by blocking L-type calcium channels. PMID:14597602

  18. Microdomain-Specific Modulation of L-Type Calcium Channels Leads to Triggered Ventricular Arrhythmia in Heart Failure

    PubMed Central

    Sanchez-Alonso, Jose L.; Bhargava, Anamika; O’Hara, Thomas; Glukhov, Alexey V.; Schobesberger, Sophie; Bhogal, Navneet; Sikkel, Markus B.; Mansfield, Catherine; Korchev, Yuri E.; Lyon, Alexander R.; Punjabi, Prakash P.; Nikolaev, Viacheslav O.; Trayanova, Natalia A.

    2016-01-01

    Rationale: Disruption in subcellular targeting of Ca2+ signaling complexes secondary to changes in cardiac myocyte structure may contribute to the pathophysiology of a variety of cardiac diseases, including heart failure (HF) and certain arrhythmias. Objective: To explore microdomain-targeted remodeling of ventricular L-type Ca2+ channels (LTCCs) in HF. Methods and Results: Super-resolution scanning patch-clamp, confocal and fluorescence microscopy were used to explore the distribution of single LTCCs in different membrane microdomains of nonfailing and failing human and rat ventricular myocytes. Disruption of membrane structure in both species led to the redistribution of functional LTCCs from their canonical location in transversal tubules (T-tubules) to the non-native crest of the sarcolemma, where their open probability was dramatically increased (0.034±0.011 versus 0.154±0.027, P<0.001). High open probability was linked to enhance calcium–calmodulin kinase II–mediated phosphorylation in non-native microdomains and resulted in an elevated ICa,L window current, which contributed to the development of early afterdepolarizations. A novel model of LTCC function in HF was developed; after its validation with experimental data, the model was used to ascertain how HF-induced T-tubule loss led to altered LTCC function and early afterdepolarizations. The HF myocyte model was then implemented in a 3-dimensional left ventricle model, demonstrating that such early afterdepolarizations can propagate and initiate reentrant arrhythmias. Conclusions: Microdomain-targeted remodeling of LTCC properties is an important event in pathways that may contribute to ventricular arrhythmogenesis in the settings of HF-associated remodeling. This extends beyond the classical concept of electric remodeling in HF and adds a new dimension to cardiovascular disease. PMID:27572487

  19. Multiple mobility edges in a 1D Aubry chain with Hubbard interaction in presence of electric field: Controlled electron transport

    NASA Astrophysics Data System (ADS)

    Saha, Srilekha; Maiti, Santanu K.; Karmakar, S. N.

    2016-09-01

    Electronic behavior of a 1D Aubry chain with Hubbard interaction is critically analyzed in presence of electric field. Multiple energy bands are generated as a result of Hubbard correlation and Aubry potential, and, within these bands localized states are developed under the application of electric field. Within a tight-binding framework we compute electronic transmission probability and average density of states using Green's function approach where the interaction parameter is treated under Hartree-Fock mean field scheme. From our analysis we find that selective transmission can be obtained by tuning injecting electron energy, and thus, the present model can be utilized as a controlled switching device.

  20. Effect of Ca2+ Efflux Pathway Distribution and Exogenous Ca2+ Buffers on Intracellular Ca2+ Dynamics in the Rat Ventricular Myocyte: A Simulation Study

    PubMed Central

    Šimurda, Jiří; Orchard, Clive H.

    2014-01-01

    We have used a previously published computer model of the rat cardiac ventricular myocyte to investigate the effect of changing the distribution of Ca2+ efflux pathways (SERCA, Na+/Ca2+ exchange, and sarcolemmal Ca2+ ATPase) between the dyad and bulk cytoplasm and the effect of adding exogenous Ca2+ buffers (BAPTA or EGTA), which are used experimentally to differentially buffer Ca2+ in the dyad and bulk cytoplasm, on cellular Ca2+ cycling. Increasing the dyadic fraction of a particular Ca2+ efflux pathway increases the amount of Ca2+ removed by that pathway, with corresponding changes in Ca2+ efflux from the bulk cytoplasm. The magnitude of these effects varies with the proportion of the total Ca2+ removed from the cytoplasm by that pathway. Differences in the response to EGTA and BAPTA, including changes in Ca2+-dependent inactivation of the L-type Ca2+ current, resulted from the buffers acting as slow and fast “shuttles,” respectively, removing Ca2+ from the dyadic space. The data suggest that complex changes in dyadic Ca2+ and cellular Ca2+ cycling occur as a result of changes in the location of Ca2+ removal pathways or the presence of exogenous Ca2+ buffers, although changing the distribution of Ca2+ efflux pathways has relatively small effects on the systolic Ca2+ transient. PMID:24971358

  1. Gestational hypothyroidism-induced changes in L-type calcium channels of rat aorta smooth muscle and their impact on the responses to vasoconstrictors.

    PubMed

    Sedaghat, Katayoun; Zahediasl, Saleh; Ghasemi, Asghar

    2015-02-01

    Thyroid hormones play an essential role in fetal growth and maternal hypo-thyroidism which leads to cardiovascular deficiency in their offspring. Considering this, we intended to investigate the impact of gestational hypothyroidism on offspring vascular contractibility and possible underlying mechanisms. Hypothyroidism was induced in female rats by administration of 6-n-propyl-2-thiouracil in drinking water (0.02%) till delivery. The offspring aorta smooth muscle (without endothelium) contractile response to KCl (10-100 mM), KCl in the presence of nifedipine (10(-4)-10(-1) µM), phenylephrine (10(-9)-10(-6) M) and finally, phenylephrine and caffeine 100 mM in Ca(2+)-free Krebs were measured. KCl and phenylephrine-induced contractions were considerably lower in gestational hypothyroid (GH) than euthyroid offspring. GH responded to nifedipine with less sensitivity than control. The GH and control groups produced almost equal contraction in respond to phenylephrine and caffeine in Ca(2+)-free Krebs. This study suggests that in hypothyroid offspring L-type Ca(2+) channels are less functional, while intracellular Ca(2+) handling systems are less modified by low levels of maternal thyroid hormones.

  2. Role of different types of Ca2+ channels and a reticulum-like Ca2+ pump in neurotransmitter release.

    PubMed

    Fossier, P; Baux, G; Tauc, L

    1993-01-01

    The factors controlling the Ca2+ concentration directly responsible for triggering acetylcholine (ACh) release were investigated at an identified neuro-neuronal synapse of the Aplysia buccal ganglion. The types of presynaptic voltage-gated Ca2+ channels associated with transmitter release were determined by using selective blockers such as nifedipine, omega-conotoxin and a partially purified extract from the venom of a funnel web spider (FTx). L-type, N-type and P-type Ca2+ channels are present in the presynaptic neuron. The influx of Ca2+ through both N- and P-types induces the release of ACh whereas Ca2+ flowing through L-type channels modulates the duration of the presynaptic action potential by controlling the Ca(2+)-dependent K+ current. tBuBHQ, a blocker of the reticulum Ca2+ pump, induces a potentiation of evoked release without modifying the presynaptic Ca2+ influx. This seems to indicate that a part of the Ca2+ entering the presynaptic terminal through N- and P-type Ca2+ channels is sequestered in a presynaptic reticulum-like Ca2+ buffer preventing these ions from contributing to ACh release. To exert its control, this Ca2+ buffer must be located close to both the presynaptic Ca2+ channels and the transmitter release mechanism.

  3. Arterial Smooth Muscle Mitochondria Amplify Hydrogen Peroxide Microdomains Functionally Coupled to L-Type Calcium Channels

    PubMed Central

    Chaplin, Nathan L.; Nieves-Cintrón, Madeline; Fresquez, Adriana M.; Navedo, Manuel F.; Amberg, Gregory C.

    2015-01-01

    Rationale Mitochondria are key integrators of convergent intracellular signaling pathways. Two important second messengers modulated by mitochondria are calcium and reactive oxygen species. To date, coherent mechanisms describing mitochondrial integration of calcium and oxidative signaling in arterial smooth muscle are incomplete. Objective To address and add clarity to this issue we tested the hypothesis that mitochondria regulate subplasmalemmal calcium and hydrogen peroxide microdomain signaling in cerebral arterial smooth muscle. Methods and Results Using an image-based approach we investigated the impact of mitochondrial regulation of L-type calcium channels on subcellular calcium and ROS signaling microdomains in isolated arterial smooth muscle cells. Our single cell observations were then related experimentally to intact arterial segments and to living animals. We found that subplasmalemmal mitochondrial amplification of hydrogen peroxide microdomain signaling stimulates L-type calcium channels and that this mechanism strongly impacts the functional capacity of the vasoconstrictor angiotensin II. Importantly, we also found that disrupting this mitochondrial amplification mechanism in vivo normalized arterial function and attenuated the hypertensive response to systemic endothelial dysfunction. Conclusions From these observations we conclude that mitochondrial amplification of subplasmalemmal calcium and hydrogen peroxide microdomain signaling is a fundamental mechanism regulating arterial smooth muscle function. As the principle components involved are fairly ubiquitous and positioning of mitochondria near the plasma membrane is not restricted to arterial smooth muscle, this mechanism could occur in many cell types and contribute to pathological elevations of intracellular calcium and increased oxidative stress associated with many diseases. PMID:26390880

  4. A 1D-2D Shallow Water Equations solver for discontinuous porosity field based on a Generalized Riemann Problem

    NASA Astrophysics Data System (ADS)

    Ferrari, Alessia; Vacondio, Renato; Dazzi, Susanna; Mignosa, Paolo

    2017-09-01

    A novel augmented Riemann Solver capable of handling porosity discontinuities in 1D and 2D Shallow Water Equation (SWE) models is presented. With the aim of accurately approximating the porosity source term, a Generalized Riemann Problem is derived by adding an additional fictitious equation to the SWEs system and imposing mass and momentum conservation across the porosity discontinuity. The modified Shallow Water Equations are theoretically investigated, and the implementation of an augmented Roe Solver in a 1D Godunov-type finite volume scheme is presented. Robust treatment of transonic flows is ensured by introducing an entropy fix based on the wave pattern of the Generalized Riemann Problem. An Exact Riemann Solver is also derived in order to validate the numerical model. As an extension of the 1D scheme, an analogous 2D numerical model is also derived and validated through test cases with radial symmetry. The capability of the 1D and 2D numerical models to capture different wave patterns is assessed against several Riemann Problems with different wave patterns.

  5. [Characteristics of electrophysiology and effects of ouabain on transient outward potassium current and L-type calcium current of left atrium posterior wall in rabbits].

    PubMed

    Wang, Teng; Huang, Cong-xin; Jiang, Hong; Tang, Qi-zhu; Yang, Bo; Li, Geng-shan

    2009-12-01

    To investigate the properties of electrophysiology and effects of ouabain upon transient outward potassium current (I(to)) and L-type calcium current (I(Ca-L)) of left atrium posterior wall (LAPW) and left atrium appendage tissue (LAA)in rabbit so as to provide the scientific explanations that LAPW and ouabain can enhance atrial fibrillation (AF) vulnerability through increasing electrophysiological heterogeneity and electrical remodeling of different regions of left atrium in rabbits. Atrial myocytes from LAPWs and LAAs of rabbits on an in vitro heart perfusion system were obtained by enzymatic dissociation. The whole-cell patch-clamp technique was used to assess the effects of ouabain upon I(to) and I(Ca-L). The current-voltage (I-V) curves of I(to) and I(Ca-L) in LAPW and LAA myocytes were fitted before and after ouabain administration. (1) With holding potential +50 mV and commanding potential +50 mV, the current densities of LAPW I(to) decreased slightly less than that of LAA I(to) in control groups (P > 0.05). After ouabain administration, the current densities of LAPW I(to) were significantly larger than that of LAA I(to) [(10.97 +/- 0.58) pA/pF vs (9.39 +/- 0.83) pA/pF, P < 0.05]. The I-V curve of LAPW I(to) was slightly lowered to I-V curve of LAA I(to) in control groups. But with perfusion of ouabain, the I-V curve of LAPW I(to) opposed to I-V curve of LAA I(to) significantly changed from the bottom to the top with the same upward direction. (2) With the voltage clamp protocol of I(Ca-L), the current densities of LAPW I(Ca-L) markedly decreased compared with that of LAA I(Ca-L) in control groups (P < 0.05). With the addition of ouabain, the peak of amplitude of LAPW I(Ca-L) at +20 mV obviously increased to that of LAA I(Ca-L) [(-11.13 +/- 0.99) pA/pF vs (-8.86 +/- 0.51) pA/pF, P < 0.01]. In the control groups, the I-V curve of LAPW I(Ca-L) was shifted to the bottom of all I-V curves of I(Ca-L). Through the effects of ouabain, the I-V curve of LAPW I(Ca

  6. Use of a 1-dB decrease in C/N\\0x2080AS the GPS interference protection criterion : global positioning systems directorate.

    DOT National Transportation Integrated Search

    2017-03-30

    A 1 dB decrease in the carrier-to-noise (C/N) ratio is equivalent to a -6 dB interference-to-noise (I/N) ratio and a 1 dB increase in the noise floor ((I+N)/N). Regulations alternate between referencing the 1 dB decrease in C/N, -6 dB I/N ratio, and ...

  7. Development of a Radiolabeled Amlodipine Analog for L-type Calcium Channel Imaging.

    PubMed

    Firouzyar, Tahereh; Jalilian, Amir Reza; Aboudzadeh, Mohammad Reza; Sadeghpour, Hossein; Pooladi, Mehrban; Shafiee-Ardestani, Mahdi; Khalaj, Ali

    2017-01-01

    The non-invasive imaging and quantification of L-type calcium channels (also known as dihydropyridine channels) in living tissues is of great interest in diagnosis of congestive heart failure, myocardial hypertrophy, irritable bowel syndrome etc. Technetium-99m labeled amlodipine conjugate ([99mTc]-DTPA-AMLO) was prepared starting freshly eluted (<1 h) 99mTechnetium pertechnetate (86.5 MBq) and conjugated DTPAAMLO at pH 5 in 30 min at room temperature in high radiochemical purity (>99%, RTLC; specific activity: 55-60 GBq/mmol). The calcium channel blockade activity (CCBA) and apoptosis/necrosis assay of DTPA-amlodipine conjugate evaluations were performed for the conjugate. Log P, stability, bio-distribution and imaging studies were performed for the tracer followed by biodistribution studies as well as imaging. The conjugate demonstrated low toxicity on MCF-7 cells and CCBA (at µm level) compared to the amlodipine. The tracer was stable up to 4 h in final production and presence of human serum and log P (-0.49) was consistent with a water soluble complex. The tracer was excreted through kidneys and liver as expected for dihydropyridines; excluding excretory organs, calcium channel rich smooth muscle cells; including colon, intestine and lungs which demonstrated significant uptake. SPECT images supported the bio-distribution data up to 4 h. significant uptake of [99mTc]-DTPA-AMLO was obtained in calcium channel rich organs. The complex can be a candidate for further SPECT imaging for L-type calcium channels. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  8. Vascular smooth muscle-specific knockdown of the noncardiac form of the L-type calcium channel by microRNA-based short hairpin RNA as a potential antihypertensive therapy.

    PubMed

    Rhee, Sung W; Stimers, Joseph R; Wang, Wenze; Pang, Li

    2009-05-01

    In different rodent models of hypertension, vascular voltage-gated L-type calcium channel (Ca(L)) current and vascular tone is increased because of increased expression of the noncardiac form of the Ca(L) (Ca(v)1.2). The objective of this study was to develop a small interfering RNA (siRNA) expression system against the noncardiac form of Ca(v)1.2 to reduce its expression in vascular smooth muscle cells (VSMCs). siRNAs expressing plasmids and appropriate controls were constructed and first screened in human embryonic kidney (HEK) 293 cells cotransfected with a rat Ca(v)1.2 expression vector. The most effective gene silencing was achieved with a modified mir-30a-based short hairpin RNA (shRNAmir) driven by the cytomegalovirus promoter. In A7r5 cells, a vascular smooth muscle cell line, two copies of shRNAmir driven by a chimeric VSMC-specific enhancer/promoter reduced endogenous Ca(v)1.2 expression by 61% and decreased the Ca(L) current carried by barium by 47%. Moreover, the chimeric vascular smooth muscle-specific enhancer/promoter displayed almost no activity in non-VSMCs (PC-12 and HEK 293). Because the proposed siRNA was designed to only target the noncardiac form of Ca(v)1.2, it did not affect the Ca(L) expression and function in cultured cardiomyocytes, even when driven by a stronger cytomegalovirus promoter. In conclusion, vascular Ca(v)1.2 expression and function were effectively reduced by VSMC-specific delivery of the noncardiac form of Ca(v)1.2 siRNA without similarly affecting cardiac Ca(L) expression and function. When coupled with a viral vector, this molecular intervention in vivo may provide a novel long-term vascular-specific gene therapy for hypertension.

  9. l-Type Amino Acid Transporter-1 Overexpression and Melphalan Sensitivity in Barrett's Adenocarcinoma1

    PubMed Central

    Lin, Jules; Raoof, Duna A; Thomas, Dafydd G; Greenson, Joel K; Giordano, Thomas J; Robinson, Gregory S; Bourner, Maureen J; Bauer, Christopher T; Orringer, Mark B; Beer, David G

    2004-01-01

    Abstract The L-type amino acid transporter-1 (LAT-1) has been associated with tumor growth. Using cDNA microarrays, overexpression of LAT-1 was found in 87.5% (7/8) of esophageal adenocarcinomas relative to 12 Barrett's samples (33% metaplasia and 66% dysplasia) and was confirmed in 100% (28/28) of Barrett's adenocarcinomas by quantitative reverse transcription polymerase chain reaction. Immunohistochemistry revealed LAT-1 staining in 37.5% (24/64) of esophageal adenocarcinomas on tissue microarray. LAT-1 also transports the amino acid-related chemotherapeutic agent, melphalan. Two esophageal adenocarcinoma and one esophageal squamous cell line, expressing LAT-1 on Western blot analysis, were sensitive to therapeutic doses of melphalan (P < .001). Simultaneous treatment with the competitive inhibitor, BCH [2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid], decreased sensitivity to melphalan (P < .05). In addition, confluent esophageal squamous cultures were less sensitive to melphalan (P < .001) and had a decrease in LAT-1 protein expression. Tumors from two esophageal adenocarcinoma cell lines grown in nude mice retained LAT-1 mRNA expression. These results demonstrate that LAT-1 is highly expressed in a subset of esophageal adenocarcinomas and that Barrett's adenocarcinoma cell lines expressing LAT-1 are sensitive to melphalan. LAT-1 expression is also retained in cell lines grown in nude mice providing a model to evaluate melphalan as a chemotherapeutic agent against esophageal adenocarcinomas expressing LAT-1. PMID:15068672

  10. The CaV2.3 R-type voltage-gated Ca2+ channel in mouse sleep architecture.

    PubMed

    Siwek, Magdalena Elisabeth; Müller, Ralf; Henseler, Christina; Broich, Karl; Papazoglou, Anna; Weiergräber, Marco

    2014-05-01

    Voltage-gated Ca(2+) channels (VGCCs) are key elements in mediating thalamocortical rhythmicity. Low-voltage activated (LVA) CaV 3 T-type Ca(2+) channels have been related to thalamic rebound burst firing and to generation of non-rapid eye movement (NREM) sleep. High-voltage activated (HVA) CaV 1 L-type Ca(2+) channels, on the opposite, favor the tonic mode of action associated with higher levels of vigilance. However, the role of the HVA Non-L-type CaV2.3 Ca(2+) channels, which are predominantly expressed in the reticular thalamic nucleus (RTN), still remains unclear. Recently, CaV2.3(-/-) mice were reported to exhibit altered spike-wave discharge (SWD)/absence seizure susceptibility supported by the observation that CaV2.3 mediated Ca(2+) influx into RTN neurons can trigger small-conductance Ca(2+)-activated K(+)-channel type 2 (SK2) currents capable of maintaining thalamic burst activity. Based on these studies we investigated the role of CaV2.3 R-type Ca(2+) channels in rodent sleep. The role of CaV2.3 Ca(2+) channels was analyzed in CaV2.3(-/-) mice and controls in both spontaneous and artificial urethane-induced sleep, using implantable video-EEG radiotelemetry. Data were analyzed for alterations in sleep architecture using sleep staging software and time-frequency analysis. CaV2.3 deficient mice exhibited reduced wake duration and increased slow-wave sleep (SWS). Whereas mean sleep stage durations remained unchanged, the total number of SWS epochs was increased in CaV2.3(-/-) mice. Additional changes were observed for sleep stage transitions and EEG amplitudes. Furthermore, urethane-induced SWS mimicked spontaneous sleep results obtained from CaV2.3 deficient mice. Quantitative Real-time PCR did not reveal changes in thalamic CaV3 T-type Ca(2+) channel expression. The detailed mechanisms of SWS increase in CaV2.3(-/-) mice remain to be determined. Low-voltage activated CaV2.3 R-type Ca(2+) channels in the thalamocortical loop and extra

  11. Forskolin Regulates L-Type Calcium Channel through Interaction between Actinin 4 and β3 Subunit in Osteoblasts.

    PubMed

    Zhang, Xuemei; Li, Fangping; Guo, Lin; Hei, Hongya; Tian, Lulu; Peng, Wen; Cai, Hui

    2015-01-01

    Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α1c subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of β3 subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells.

  12. Forskolin Regulates L-Type Calcium Channel through Interaction between Actinin 4 and β3 Subunit in Osteoblasts

    PubMed Central

    Guo, Lin; Hei, Hongya; Tian, Lulu; Peng, Wen; Cai, Hui

    2015-01-01

    Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α1c subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of β3 subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells. PMID:25902045

  13. Clinical, pathological, and molecular features of classical and L-type atypical-BSE in goats

    PubMed Central

    D’Angelo, Antonio; Mazza, Maria; Meloni, Daniela; Baioni, Elisa; Maurella, Cristiana; Colussi, Silvia; Martinelli, Nicola; Lo Faro, Monica; Favole, Alessandra; Grifoni, Silvia; Gallo, Marina; Lombardi, Guerino; Iulini, Barbara; Casalone, Cristina; Corona, Cristiano

    2018-01-01

    Monitoring of small ruminants for transmissible spongiform encephalopathies (TSEs) has recently become more relevant after two natural scrapie suspected cases of goats were found to be positive for classical BSE (C-BSE). C-BSE probably established itself in this species unrecognized, undermining disease control measures. This opens the possibility that TSEs in goats may remain an animal source for human prion diseases. Currently, there are no data regarding the natural presence of the atypical BSE in caprines. Here we report that C-BSE and L-type atypical BSE (L-BSE) isolates from bovine species are intracerebrally transmissible to goats, with a 100% attack rate and a significantly shorter incubation period and survival time after C-BSE than after L-BSE experimental infection, suggesting a lower species barrier for classical agentin goat. All animals showed nearly the same clinical features of disease characterized by skin lesions, including broken hair and alopecia, and abnormal mental status. Histology and immunohistochemistry showed several differences between C-BSE and L-BSE infection, allowing discrimination between the two different strains. The lymphoreticular involvement we observed in the C-BSE positive goats argues in favour of a peripheral distribution of PrPSc similar to classical scrapie. Western blot and other currently approved screening tests detected both strains in the goats and were able to classify negative control animals. These data demonstrate that active surveillance of small ruminants, as applied to fallen stock and/or healthy slaughter populations in European countries, is able to correctly identify and classify classical and L-BSE and ultimately protect public health. PMID:29795663

  14. L-type calcium channel blockers and substance P induce angiogenesis of cortical vessels associated with beta-amyloid plaques in an Alzheimer mouse model.

    PubMed

    Daschil, Nina; Kniewallner, Kathrin M; Obermair, Gerald J; Hutter-Paier, Birgit; Windisch, Manfred; Marksteiner, Josef; Humpel, Christian

    2015-03-01

    It is well established that L-type calcium channels (LTCCs) are expressed in astroglia. However, their functional role is still speculative, especially under pathologic conditions. We recently showed that the α1 subunit-like immunoreactivity of the CaV1.2 channel is strongly expressed in reactive astrocytes around beta-amyloid plaques in 11-month-old Alzheimer transgenic (tg) mice with the amyloid precursor protein London and Swedish mutations. The aim of the present study was to examine the cellular expression of all LTCC subunits around beta-amyloid plaques by in situ hybridization using (35)S-labeled oligonucleotides. Our data show that messenger RNAs (mRNAs) of the LTCC CaV1.2 α1 subunit as well as all auxiliary β and α2δ subunits, except α2δ-4, were expressed in the hippocampus of age-matched wild-type mice. It was unexpected to see, that cells directly located in the plaque core in the cortex expressed mRNAs for CaV1.2 α1, β2, β4, and α2δ-1, whereas no expression was detected in the halo. Furthermore, cells in the plaque core also expressed preprotachykinin-A mRNA, the precursor for substance P. By means of confocal microscopy, we demonstrated that collagen-IV-stained brain vessels in the cortex were associated with the plaque core and were immunoreactive for substance P. In cortical organotypic brain slices of adult Alzheimer mice, we could demonstrate that LTCC blockers increased angiogenesis, which was further potentiated by substance P. In conclusion, our data show that brain vessels associated with beta-amyloid plaques express substance P and an LTCC and may play a role in angiogenesis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Characterization of L-type calcium channel activity in atrioventricular nodal myocytes from rats with streptozotocin-induced Diabetes mellitus

    PubMed Central

    Yuill, Kathryn H; Al Kury, Lina T; Howarth, Frank Christopher

    2015-01-01

    Cardiovascular complications are common in patients with Diabetes mellitus (DM). In addition to changes in cardiac muscle inotropy, electrical abnormalities are also commonly observed in these patients. We have previously shown that spontaneous cellular electrical activity is altered in atrioventricular nodal (AVN) myocytes, isolated from the streptozotocin (STZ) rat model of type-1 DM. In this study, utilizing the same model, we have characterized the changes in L-type calcium channel activity in single AVN myocytes. Ionic currents were recorded from AVN myocytes isolated from the hearts of control rats and from those with STZ-induced diabetes. Patch-clamp recordings were used to assess the changes in cellular electrical activity in individual myocytes. Type-1 DM significantly altered the cellular characteristics of L-type calcium current. A reduction in peak ICaL density was observed, with no corresponding changes in the activation parameters of the current. L-type calcium channel current also exhibited faster time-dependent inactivation in AVN myocytes from diabetic rats. A negative shift in the voltage dependence of inactivation was also evident, and a slowing of restitution parameters. These findings demonstrate that experimentally induced type-1 DM significantly alters AVN L-type calcium channel cellular electrophysiology. These changes in ion channel activity may contribute to the abnormalities in cardiac electrical function that are associated with high mortality levels in patients with DM. PMID:26603460

  16. Conditional Deletion of the L-Type Calcium Channel Cav1.2 in NG2-Positive Cells Impairs Remyelination in Mice.

    PubMed

    Santiago González, Diara A; Cheli, Veronica T; Zamora, Norma N; Lama, Tenzing N; Spreuer, Vilma; Murphy, Geoffrey G; Paez, Pablo M

    2017-10-18

    Exploring the molecular mechanisms that drive the maturation of oligodendrocyte progenitor cells (OPCs) during the remyelination process is essential to developing new therapeutic tools to intervene in demyelinating diseases such as multiple sclerosis. To determine whether L-type voltage-gated calcium channels (L-VGCCs) are required for OPC development during remyelination, we generated an inducible conditional knock-out mouse in which the L-VGCC isoform Cav1.2 was deleted in NG2-positive OPCs (Cav1.2 KO ). Using the cuprizone (CPZ) model of demyelination and mice of either sex, we establish that Cav1.2 deletion in OPCs leads to less efficient remyelination of the adult brain. Specifically, Cav1.2 KO OPCs mature slower and produce less myelin than control oligodendrocytes during the recovery period after CPZ intoxication. This reduced remyelination was accompanied by an important decline in the number of myelinating oligodendrocytes and in the rate of OPC proliferation. Furthermore, during the remyelination phase of the CPZ model, the corpus callosum of Cav1.2 KO animals presented a significant decrease in the percentage of myelinated axons and a substantial increase in the mean g-ratio of myelinated axons compared with controls. In addition, in a mouse line in which the Cav1.2 KO OPCs were identified by a Cre reporter, we establish that Cav1.2 KO OPCs display a reduced maturational rate through the entire remyelination process. These results suggest that Ca 2+ influx mediated by L-VGCCs in oligodendroglial cells is necessary for normal remyelination and is an essential Ca 2+ channel for OPC maturation during the remyelination of the adult brain. SIGNIFICANCE STATEMENT Ion channels implicated in oligodendrocyte differentiation and maturation may induce positive signals for myelin recovery. Voltage-gated Ca 2+ channels (VGCCs) are important for normal myelination by acting at several critical steps during oligodendrocyte progenitor cell (OPC) development. To

  17. Technical Note: Direct measurement of continuous TMR data with a 1D tank and automated couch movements.

    PubMed

    Knutson, Nels C; Schmidt, Matthew C; Belley, Matthew D; Nguyen, Ngoc B; Li, H Harold; Sajo, Erno; Price, Michael J

    2017-07-01

    Real-time dynamic control of the linear accelerator, couch, and imaging parameters during radiation delivery was investigated as a novel technique for acquiring tissue maximum ratio (TMR) data. TrueBeam Developer Mode (Varian Medical Systems, Palo Alto, CA, USA) was used to control the linear accelerator using the Extensible Markup Language (XML). A single XML file was used to dynamically manipulate the machine, couch, and imaging parameters during radiation delivery. A TG-51 compliant 1D water tank was placed on the treatment couch, and used to position a detector at isocenter at a depth of 24.5 cm. A depth scan was performed towards the water surface. Via XML control, the treatment couch vertical position was simultaneously lowered at the same rate, maintaining the detector position at isocenter, allowing for the collection of TMR data. To ensure the detector remained at isocenter during the delivery, the in-room camera was used to monitor the detector. Continuous kV fluoroscopic images during 10 test runs further confirmed this result. TMR data at multiple Source to Detector Distances (SDD) and scan speeds were acquired to investigate their impact on the TMR data. Percentage depth dose (PDD) scans (for conversion to TMR) along with traditional discrete TMR data were acquired as a standard for comparison. More than 99.8% of the measured points had a gamma value (1%/1 mm) < 1 when compared with discrete or PDD converted TMR data. Fluoroscopic images showed that the concurrent couch and tank movements resulted in SDD errors < 1 mm. TMRs acquired at SDDs of 99, 100, and 101 cm showed differences less than 0.004. TrueBeam Developer Mode was used to collect continuous TMR data with the same accuracy as traditionally collected discrete data, but yielded higher sampled resolution and reduced acquisition time. This novel method does not require the modification of any equipment and does not use a 3D tank or reservoir. © 2017 American Association of Physicists in

  18. The Contribution of L-Type Cav1.3 Channels to Retinal Light Responses

    PubMed Central

    Shi, Liheng; Chang, Janet Ya-An; Yu, Fei; Ko, Michael L.; Ko, Gladys Y.-P.

    2017-01-01

    L-type voltage-gated calcium channels (LTCCs) regulate tonic neurotransmitter release from sensory neurons including retinal photoreceptors. There are three types of LTCCs (Cav1.2, Cav1.3, and Cav1.4) expressed in the retina. While Cav1.2 is expressed in all retinal cells including the Müller glia and neurons, Cav1.3 and Cav1.4 are expressed in the retinal neurons with Cav1.4 exclusively expressed in the photoreceptor synaptic terminals. Mutations in the gene encoding Cav1.4 cause incomplete X-linked congenital stationary night blindness in humans. Even though Cav1.3 is present in the photoreceptor inner segments and the synaptic terminals in various vertebrate species, its role in vision is unclear, since genetic alterations in Cav1.3 are not associated with severe vision impairment in humans or in Cav1.3-null (Cav1.3−/−) mice. However, a failure to regulate Cav1.3 was found in a mouse model of Usher syndrome, the most common cause of combined deafness and blindness in humans, indicating that Cav1.3 may contribute to retinal function. In this report, we combined physiological and morphological data to demonstrate the role of Cav1.3 in retinal physiology and function that has been undervalued thus far. Through ex vivo and in vivo electroretinogram (ERG) recordings and immunohistochemical staining, we found that Cav1.3 plays a role in retinal light responses and synaptic plasticity. Pharmacological inhibition of Cav1.3 decreased ex vivo ERG a- and b-wave amplitudes. In Cav1.3−/− mice, their dark-adapted ERG a-, b-wave, and oscillatory potential amplitudes were significantly dampened, and implicit times were delayed compared to the wild type (WT). Furthermore, the density of ribbon synapses was reduced in the outer plexiform layer of Cav1.3−/− mice retinas. Hence, Cav1.3 plays a more prominent role in retinal physiology and function than previously reported. PMID:29259539

  19. Retinoschisin, a New Binding Partner for L-type Voltage-gated Calcium Channels in the Retina*

    PubMed Central

    Shi, Liheng; Jian, Kuihuan; Ko, Michael L.; Trump, Dorothy; Ko, Gladys Y.-P.

    2009-01-01

    The L-type voltage-gated calcium channels (L-VGCCs) are activated under high depolarization voltages. They are vital for diverse biological events, including cell excitability, differentiation, and synaptic transmission. In retinal photoreceptors, L-VGCCs are responsible for neurotransmitter release and are under circadian influences. However, the mechanism of L-VGCC regulation in photoreceptors is not fully understood. Here, we show that retinoschisin, a highly conserved extracellular protein, interacts with the L-VGCCα1D subunit and regulates its activities in a circadian manner. Mutations in the gene encoding retinoschisin (RS1) cause retinal disorganization that leads to early onset of macular degeneration. Since ion channel activities can be modulated through interactions with extracellular proteins, disruption of these interactions can alter physiology and be the root cause of disease states. Co-immunoprecipitation and mammalian two-hybrid assays showed that retinoschisin and the N-terminal fragment of the L-VGCCα1 subunit physically interacted with one another. The expression and secretion of retinoschisin are under circadian regulation with a peak at night and nadir during the day. Inhibition of L-type VGCCs decreased membrane-bound retinoschisin at night. Overexpression of a missense RS1 mutant gene, R141G, into chicken cone photoreceptors caused a decrease of L-type VGCC currents at night. Our findings demonstrate a novel bidirectional relationship between an ion channel and an extracellular protein; L-type VGCCs regulate the circadian rhythm of retinoschisin secretion, whereas secreted retinoschisin feeds back to regulate L-type VGCCs. Therefore, physical interactions between L-VGCCα1 subunits and retinoschisin play an important role in the membrane retention of L-VGCCα1 subunits and photoreceptor-bipolar synaptic transmission. PMID:19074145

  20. [Human calcium channelopathies. Voltage-gated Ca(2+) channels in etiology, pathogenesis, and pharmacotherapy of neurologic disorders].

    PubMed

    Weiergräber, M; Hescheler, J; Schneider, T

    2008-04-01

    Voltage-gated calcium channels are key components in a variety of physiological processes. Within the last decade an increasing number of voltage-gated Ca(2+) channelopathies in both humans and animal models has been described, most of which are related to the neurologic and muscular system. In humans, mutations were found in L-type Ca(v)1.2 and Ca(v)1.4 Ca(2+) channels as well as the non-L-type Ca(v)2.1 and T-type Ca(v)3.2 channels, resulting in altered electrophysiologic properties. Based on their widespread distribution within the CNS, voltage-gated calcium channels are of particular importance in the etiology and pathogenesis of various forms of epilepsy and neuropsychiatric disorders. In this review we characterise the different human Ca(2+) channelopathies known so far, further illuminating basic pathophysiologic mechanisms and clinical aspects.

  1. L-type voltage-dependent calcium channel is involved in the snake venom group IA secretory phospholipase A2-induced neuronal apoptosis.

    PubMed

    Yagami, Tatsurou; Yamamoto, Yasuhiro; Kohma, Hiromi; Nakamura, Tsutomu; Takasu, Nobuo; Okamura, Noboru

    2013-03-01

    Snake venom group IA secretory phospholipase A2 (sPLA2-IA) is known as a neurotoxin. Snake venom sPLA2s are neurotoxic in vivo and in vitro, causing synergistic neurotoxicity to cortical cultures when applied with toxic concentrations of glutamate. However, it has not yet been cleared sufficiently how sPLA2-IA exerts neurotoxicity. Here, we found sPLA2-IA induced neuronal cell death in a concentration-dependent manner. This death was a delayed response requiring a latent time for 6h. sPLA2-IA-induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. NMDA receptor blockers suppressed the neurotoxicity of sPLA2-IA, but an AMPA receptor blocker did not. Interestingly, L-type voltage-dependent Ca(2+) channel (L-VDCC) blocker significantly protected neurons from the sPLA2-IA-induced apoptosis. On the other hand, neither N-VDCC blockers nor P/Q-VDCC blocker did. In conclusion, we demonstrated that sPLA2-IA induced neuronal cell death via apoptosis. Furthermore, the present study suggests that not only NMDA receptor but also L-VDCC contributed to the neurotoxicity of snake venom sPLA2-IA. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. (2R,3S,2”R,3”R)-manniflavanone, a new gastrointestinal smooth muscle L-type calcium channel inhibitor, which underlies the spasmolytic properties of Garcinia buchananii stem bark extract

    PubMed Central

    Balemba, Onesmo B.; Stark, Timo D.; Lösch, Sofie; Patterson, Savannah; McMillan, John S.; Mawe, Gary M.; Hofmann, Thomas

    2014-01-01

    Garcinia buchananii Baker stem bark extract (GBB) is a traditional medication of diarrhea and dysentery in sub-Saharan Africa. It is believed that GBB causes gastrointestinal smooth muscle relaxation. The aim of this study was to determine whether GBB has spasmolytic actions and identify compounds underlying these actions. Calcium (Ca2+) imaging was used to analyze the effect of GBB on Ca2+ flashes and Ca2+ waves in guinea pig gallbladder and distal colon smooth muscle. Intracellular microelectrode recording was used to determine the effect of GBB, six fractions of GBB, M1–5 and M7, and (2R,3S,2”R,3”R)-manniflavanone, a compound isolated from M3 on action potentials in gallbladder smooth muscle. The technique was also used to analyze the effect of GBB, M3, and (2R,3S,2”R,3”R)-manniflavanone on action potentials in the circular muscle of mouse and guinea pig distal colons, and the effect of GBB and (2R,3S,2”R,3”R)-manniflavanone on slow waves in porcine ileum. GBB inhibited Ca2+ flashes and Ca2+ waves. GBB, M3 and (2R,3S,2”R,3”R)-manniflavanone inhibited action potentials. L-type Ca2+ channel activator Bay K 8644 increased the discharge of action potentials in mouse colon but did not trigger or increase action potentials in the presence of GBB and (2R,3S,2”R,3”R)-manniflavanone. GBB and (2R,3S,2”R,3”R)-manniflavanone inhibited action potentials in the presence of Bay K 8644. GBB and (2R,3S,2”R,3”R)-manniflavanone reduced the amplitude but did not alter the frequency of slow waves in the porcine ileum. In conclusion, GBB and (2R,3S,2”R,3”R)-manniflavanone relax smooth muscle by inhibiting L-type Ca2+ channels, thus have potential for use as therapies of gastrointestinal smooth muscle spasms, and arrhythmias. PMID:26081368

  3. Inhibition of l-type amino acid transporter 1 activity as a new therapeutic target for cholangiocarcinoma treatment.

    PubMed

    Yothaisong, Supak; Dokduang, Hasaya; Anzai, Naohiko; Hayashi, Keitaro; Namwat, Nisana; Yongvanit, Puangrat; Sangkhamanon, Sakkarn; Jutabha, Promsuk; Endou, Hitoshi; Loilome, Watcharin

    2017-03-01

    Unlike normal cells, cancer cells undergo unlimited growth and multiplication, causing them to require massive amounts of amino acid to support their continuous metabolism. Among the amino acid transporters expressed on the plasma membrane, l-type amino acid transporter-1, a Na + -independent neutral amino acid transporter, is highly expressed in many types of human cancer including cholangiocarcinoma. Our previous study reported that l-type amino acid transporter-1 and its co-functional protein CD98 were highly expressed and implicated in cholangiocarcinoma progression and carcinogenesis. Therefore, this study determined the effect of JPH203, a selective inhibitor of l-type amino acid transporter-1 activity, on cholangiocarcinoma cell inhibition both in vitro and in vivo. JPH203 dramatically suppressed [ 14 C]l-leucine uptake as well as cell growth in cholangiocarcinoma cell lines along with altering the expression of l-type amino acid transporter-1 and CD98 in response to amino acid depletion. We also demonstrated that JPH203 induced both G2/M and G0/G1 cell cycle arrest, as well as reduced the S phase accompanied by altered expression of the proteins in cell cycle progression: cyclin D1, CDK4, and CDK6. There was also cell cycle arrest of the related proteins, P21 and P27, in KKU-055 and KKU-213 cholangiocarcinoma cells. Apoptosis induction, detected by an increase in trypan blue-stained cells along with a cleaved caspase-3/caspase-3 ratio, occurred in JPH203-treated cholangiocarcinoma cells at the highest concentration tested (100 µM). As expected, daily intravenous administration of JPH203 (12.5 and 25 mg/kg) significantly inhibited tumor growth in KKU-213 cholangiocarcinoma cell xenografts in the nude mice model in a dose-dependent manner with no statistically significant change in the animal's body weight and with no differences in the histology and appearance of the internal organs compared with the control group. Our study demonstrates that

  4. Stimulation-induced Ca(2+) influx at nodes of Ranvier in mouse peripheral motor axons.

    PubMed

    Zhang, Zhongsheng; David, Gavriel

    2016-01-01

    In peripheral myelinated axons of mammalian spinal motor neurons, Ca(2+) influx was thought to occur only in pathological conditions such as ischaemia. Using Ca(2+) imaging in mouse large motor axons, we find that physiological stimulation with trains of action potentials transiently elevates axoplasmic [C(2+)] around nodes of Ranvier. These stimulation-induced [Ca(2+)] elevations require Ca(2+) influx, and are partially reduced by blocking T-type Ca(2+) channels (e.g. mibefradil) and by blocking the Na(+)/Ca(2+) exchanger (NCX), suggesting an important contribution of Ca(2+) influx via reverse-mode NCX activity. Acute disruption of paranodal myelin dramatically increases stimulation-induced [Ca(2+)] elevations around nodes by allowing activation of sub-myelin L-type (nimodipine-sensitive) Ca(2+) channels. The Ca(2+) that enters myelinated motor axons during normal activity is likely to contribute to several signalling pathways; the larger Ca(2+) influx that occurs following demyelination may contribute to the axonal degeneration that occurs in peripheral demyelinating diseases. Activity-dependent Ca(2+) signalling is well established for somata and terminals of mammalian spinal motor neurons, but not for their axons. Imaging of an intra-axonally injected fluorescent [Ca(2+)] indicator revealed that during repetitive action potential stimulation, [Ca(2+)] elevations localized to nodal regions occurred in mouse motor axons from ventral roots, phrenic nerve and intramuscular branches. These [Ca(2+)] elevations (∼ 0.1 μm with stimulation at 50 Hz, 10 s) were blocked by removal of Ca(2+) from the extracellular solution. Effects of pharmacological blockers indicated contributions from both T-type Ca(2+) channels and reverse mode Na(+)/Ca(2+) exchange (NCX). Acute disruption of paranodal myelin (by stretch or lysophosphatidylcholine) increased the stimulation-induced [Ca(2+)] elevations, which now included a prominent contribution from L-type Ca(2+) channels. These

  5. An integrative model of the cardiac ventricular myocyte incorporating local control of Ca2+ release.

    PubMed Central

    Greenstein, Joseph L; Winslow, Raimond L

    2002-01-01

    The local control theory of excitation-contraction (EC) coupling in cardiac muscle asserts that L-type Ca(2+) current tightly controls Ca(2+) release from the sarcoplasmic reticulum (SR) via local interaction of closely apposed L-type Ca(2+) channels (LCCs) and ryanodine receptors (RyRs). These local interactions give rise to smoothly graded Ca(2+)-induced Ca(2+) release (CICR), which exhibits high gain. In this study we present a biophysically detailed model of the normal canine ventricular myocyte that conforms to local control theory. The model formulation incorporates details of microscopic EC coupling properties in the form of Ca(2+) release units (CaRUs) in which individual sarcolemmal LCCs interact in a stochastic manner with nearby RyRs in localized regions where junctional SR membrane and transverse-tubular membrane are in close proximity. The CaRUs are embedded within and interact with the global systems of the myocyte describing ionic and membrane pump/exchanger currents, SR Ca(2+) uptake, and time-varying cytosolic ion concentrations to form a model of the cardiac action potential (AP). The model can reproduce both the detailed properties of EC coupling, such as variable gain and graded SR Ca(2+) release, and whole-cell phenomena, such as modulation of AP duration by SR Ca(2+) release. Simulations indicate that the local control paradigm predicts stable APs when the L-type Ca(2+) current is adjusted in accord with the balance between voltage- and Ca(2+)-dependent inactivation processes as measured experimentally, a scenario where common pool models become unstable. The local control myocyte model provides a means for studying the interrelationship between microscopic and macroscopic behaviors in a manner that would not be possible in experiments. PMID:12496068

  6. CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through upregulating L-type calcium channel activity.

    PubMed

    Sun, Meiqun; Liu, Hongli; Xu, Huanbai; Wang, Hongtao; Wang, Xiaojing

    2016-09-01

    A specialized culture medium termed ciliary neurotrophic factor-treated astrocyte-conditioned medium (CNTF-ACM) allows investigators to assess the peripheral effects of CNTF-induced activated astrocytes upon cultured neurons. CNTF-ACM has been shown to upregulate neuronal L-type calcium channel current activity, which has been previously linked to changes in mitochondrial respiration and oxidative stress. Therefore, the aim of this study was to evaluate CNTF-ACM's effects upon mitochondrial respiration and oxidative stress in rat cortical neurons. Cortical neurons, CNTF-ACM, and untreated control astrocyte-conditioned medium (UC-ACM) were prepared from neonatal Sprague-Dawley rat cortical tissue. Neurons were cultured in either CNTF-ACM or UC-ACM for a 48-h period. Changes in the following parameters before and after treatment with the L-type calcium channel blocker isradipine were assessed: (i) intracellular calcium levels, (ii) mitochondrial membrane potential (ΔΨm), (iii) oxygen consumption rate (OCR) and adenosine triphosphate (ATP) formation, (iv) intracellular nitric oxide (NO) levels, (v) mitochondrial reactive oxygen species (ROS) production, and (vi) susceptibility to the mitochondrial complex I toxin rotenone. CNTF-ACM neurons displayed the following significant changes relative to UC-ACM neurons: (i) increased intracellular calcium levels (p < 0.05), (ii) elevation in ΔΨm (p < 0.05), (iii) increased OCR and ATP formation (p < 0.05), (iv) increased intracellular NO levels (p < 0.05), (v) increased mitochondrial ROS production (p < 0.05), and (vi) increased susceptibility to rotenone (p < 0.05). Treatment with isradipine was able to partially rescue these negative effects of CNTF-ACM (p < 0.05). CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through elevating L-type calcium channel activity.

  7. Heterogeneous expression of Ca(2+) handling proteins in rabbit sinoatrial node.

    PubMed

    Musa, Hanny; Lei, Ming; Honjo, Hauro; Jones, Sandra A; Dobrzynski, Halina; Lancaster, Mathew K; Takagishi, Yoshiko; Henderson, Zaineb; Kodama, Itsuo; Boyett, Mark R

    2002-03-01

    We investigated the densities of the L-type Ca(2+) current, i(Ca,L), and various Ca(2+) handling proteins in rabbit sinoatrial (SA) node. The density of i(Ca,L), recorded with the whole-cell patch-clamp technique, varied widely in sinoatrial node cells. The density of i(Ca,L) was significantly (p<0.001) correlated with cell capacitance (measure of cell size) and the density was greater in larger cells (likely to be from the periphery of the SA node) than in smaller cells (likely to be from the center of the SA node). Immunocytochemical labeling of the L-type Ca(2+) channel, Na(+)-Ca(2+) exchanger, sarcoplasmic reticulum Ca(2+) release channel (RYR2), and sarcoplasmic reticulum Ca(2+) pump (SERCA2) also varied widely in SA node cells. In all cases there was significantly (p<0.05) denser labeling of cells from the periphery of the SA node than of cells from the center. In contrast, immunocytochemical labeling of the Na(+)-K(+) pump was similar in peripheral and central cells. We conclude that Ca(2+) handling proteins are sparse and poorly organized in the center of the SA node (normally the leading pacemaker site), whereas they are more abundant in the periphery (at the border of the SA node with the surrounding atrial muscle).

  8. L-type calcium channels play a critical role in maintaining lens transparency by regulating phosphorylation of aquaporin-0 and myosin light chain and expression of connexins.

    PubMed

    Maddala, Rupalatha; Nagendran, Tharkika; de Ridder, Gustaaf G; Schey, Kevin L; Rao, Ponugoti Vasantha

    2013-01-01

    Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels (LTCCs) and explored their role in morphological integrity and transparency of the mouse lens, using cDNA microarray, RT-PCR, immunoblot, pharmacological inhibitors and immunofluorescence analyses. The results revealed that Ca (V) 1.2 and 1.3 channels are expressed and distributed in both the epithelium and cortical fiber cells in mouse lens. Inhibition of LTCCs with felodipine or nifedipine induces progressive cortical cataract formation with time, in association with decreased lens weight in ex-vivo mouse lenses. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling the phenotype reported for altered aquaporin-0 activity without detectable cytotoxic effects. Analysis of both soluble and membrane rich fractions from felodipine treated lenses by SDS-PAGE in conjunction with mass spectrometry and immunoblot analyses revealed decreases in β-B1-crystallin, Hsp-90, spectrin and filensin. Significantly, loss of transparency in the felodipine treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and levels of connexin-50, together with decreases in myosin light chain phosphorylation and the levels of 14-3-3ε, a phosphoprotein-binding regulatory protein. Felodipine treatment led to a significant increase in gene expression of connexin-50 and 46 in the mouse lens. Additionally, felodipine inhibition of LTCCs in primary cultures of mouse lens epithelial cells resulted in decreased intracellular calcium, and decreased actin stress fibers and myosin light chain phosphorylation, without detectable cytotoxic response. Taken together, these observations reveal a crucial

  9. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

    NASA Technical Reports Server (NTRS)

    Jorgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne; Civitelli, Roberto; Sorensen, Ole Helmer; Steinberg, Thomas H.

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

  10. Preparation and preclinical evaluation of 68Ga-DOTA-amlodipine for L-type calcium channel imaging.

    PubMed

    Firuzyar, Tahereh; Jalilian, Amir Reza; Aboudzadeh, Mohammad Reza; Sadeghpour, Hossein; Shafiee-Ardestani, Mahdi; Khalaj, Ali

    2016-01-01

    In order to develop a possible tracer for L-type calcium channel imaging, we here report the development of a Ga-68 amlodipine derivative for possible PET imaging. Amlodipine DOTA conjugate was synthesized, characterized and went through calcium channel blockade, toxicity, apoptosis/necrosis tests. [ 68 Ga] DOTA AMLO was prepared at optimized conditions followed by stability tests, partition coefficient determination and biodistribution studies using tissue counting and co incidence imaging up to 2 h. [ 68 Ga] DOTA AMLO was prepared at pH 4-5 in 7-10 min at 95°C in high radiochemical purity (>99%, radio thin layer chromatography; specific activity: 1.9-2.1 GBq/mmol) and was stable up to 4 h with a log P of -0.94. Calcium channel rich tissues including myocardium, and tissues with smooth muscle cells such as colon, intestine, and lungs demonstrated significant uptake. Co incidence images supported the biodistribution data up to 2 h. The complex can be a candidate for further positron emission tomography imaging for L type calcium channels.

  11. Preparation and preclinical evaluation of 68Ga-DOTA-amlodipine for L-type calcium channel imaging

    PubMed Central

    Firuzyar, Tahereh; Jalilian, Amir Reza; Aboudzadeh, Mohammad Reza; Sadeghpour, Hossein; Shafiee-Ardestani, Mahdi; Khalaj, Ali

    2016-01-01

    Aim: In order to develop a possible tracer for L-type calcium channel imaging, we here report the development of a Ga-68 amlodipine derivative for possible PET imaging. Materials and Methods: Amlodipine DOTA conjugate was synthesized, characterized and went through calcium channel blockade, toxicity, apoptosis/necrosis tests. [68Ga] DOTA AMLO was prepared at optimized conditions followed by stability tests, partition coefficient determination and biodistribution studies using tissue counting and co incidence imaging up to 2 h. Results: [68Ga] DOTA AMLO was prepared at pH 4–5 in 7–10 min at 95°C in high radiochemical purity (>99%, radio thin layer chromatography; specific activity: 1.9–2.1 GBq/mmol) and was stable up to 4 h with a log P of −0.94. Calcium channel rich tissues including myocardium, and tissues with smooth muscle cells such as colon, intestine, and lungs demonstrated significant uptake. Co incidence images supported the biodistribution data up to 2 h. Conclusions: The complex can be a candidate for further positron emission tomography imaging for L type calcium channels. PMID:27833311

  12. Population Density and Moment-based Approaches to Modeling Domain Calcium-mediated Inactivation of L-type Calcium Channels.

    PubMed

    Wang, Xiao; Hardcastle, Kiah; Weinberg, Seth H; Smith, Gregory D

    2016-03-01

    We present a population density and moment-based description of the stochastic dynamics of domain [Formula: see text]-mediated inactivation of L-type [Formula: see text] channels. Our approach accounts for the effect of heterogeneity of local [Formula: see text] signals on whole cell [Formula: see text] currents; however, in contrast with prior work, e.g., Sherman et al. (Biophys J 58(4):985-995, 1990), we do not assume that [Formula: see text] domain formation and collapse are fast compared to channel gating. We demonstrate the population density and moment-based modeling approaches using a 12-state Markov chain model of an L-type [Formula: see text] channel introduced by Greenstein and Winslow (Biophys J 83(6):2918-2945, 2002). Simulated whole cell voltage clamp responses yield an inactivation function for the whole cell [Formula: see text] current that agrees with the traditional approach when domain dynamics are fast. We analyze the voltage-dependence of [Formula: see text] inactivation that may occur via slow heterogeneous domain [[Formula: see text

  13. The CaV2.3 R-Type Voltage-Gated Ca2+ Channel in Mouse Sleep Architecture

    PubMed Central

    Siwek, Magdalena Elisabeth; Müller, Ralf; Henseler, Christina; Broich, Karl; Papazoglou, Anna; Weiergräber, Marco

    2014-01-01

    Study Objectives: Voltage-gated Ca2+ channels (VGCCs) are key elements in mediating thalamocortical rhythmicity. Low-voltage activated (LVA) CaV 3 T-type Ca2+ channels have been related to thalamic rebound burst firing and to generation of non-rapid eye movement (NREM) sleep. High-voltage activated (HVA) CaV 1 L-type Ca2+ channels, on the opposite, favor the tonic mode of action associated with higher levels of vigilance. However, the role of the HVA Non-L-type CaV2.3 Ca2+ channels, which are predominantly expressed in the reticular thalamic nucleus (RTN), still remains unclear. Recently, CaV2.3−/− mice were reported to exhibit altered spike-wave discharge (SWD)/absence seizure susceptibility supported by the observation that CaV2.3 mediated Ca2+ influx into RTN neurons can trigger small-conductance Ca2+-activated K+-channel type 2 (SK2) currents capable of maintaining thalamic burst activity. Based on these studies we investigated the role of CaV2.3 R-type Ca2+ channels in rodent sleep. Methods: The role of CaV2.3 Ca2+ channels was analyzed in CaV2.3−/− mice and controls in both spontaneous and artificial urethane-induced sleep, using implantable video-EEG radiotelemetry. Data were analyzed for alterations in sleep architecture using sleep staging software and time-frequency analysis. Results: CaV2.3 deficient mice exhibited reduced wake duration and increased slow-wave sleep (SWS). Whereas mean sleep stage durations remained unchanged, the total number of SWS epochs was increased in CaV2.3−/− mice. Additional changes were observed for sleep stage transitions and EEG amplitudes. Furthermore, urethane-induced SWS mimicked spontaneous sleep results obtained from CaV2.3 deficient mice. Quantitative Real-time PCR did not reveal changes in thalamic CaV3 T-type Ca2+ channel expression. The detailed mechanisms of SWS increase in CaV2.3−/− mice remain to be determined. Conclusions: Low-voltage activated CaV2.3 R-type Ca2+ channels in the thalamocortical

  14. Vascular Smooth Muscle-Specific Knockdown of the Noncardiac Form of the L-Type Calcium Channel by MicroRNA-Based Short Hairpin RNA as a Potential Antihypertensive Therapy

    PubMed Central

    Rhee, Sung W.; Stimers, Joseph R.; Wang, Wenze; Pang, Li

    2009-01-01

    In different rodent models of hypertension, vascular voltage-gated L-type calcium channel (CaL) current and vascular tone is increased because of increased expression of the noncardiac form of the CaL (Cav1.2). The objective of this study was to develop a small interfering RNA (siRNA) expression system against the noncardiac form of Cav1.2 to reduce its expression in vascular smooth muscle cells (VSMCs). siRNAs expressing plasmids and appropriate controls were constructed and first screened in human embryonic kidney (HEK) 293 cells cotransfected with a rat Cav1.2 expression vector. The most effective gene silencing was achieved with a modified mir-30a-based short hairpin RNA (shRNAmir) driven by the cytomegalovirus promoter. In A7r5 cells, a vascular smooth muscle cell line, two copies of shRNAmir driven by a chimeric VSMC-specific enhancer/promoter reduced endogenous Cav1.2 expression by 61% and decreased the CaL current carried by barium by 47%. Moreover, the chimeric vascular smooth muscle-specific enhancer/promoter displayed almost no activity in non-VSMCs (PC-12 and HEK 293). Because the proposed siRNA was designed to only target the noncardiac form of Cav1.2, it did not affect the CaL expression and function in cultured cardiomyocytes, even when driven by a stronger cytomegalovirus promoter. In conclusion, vascular Cav1.2 expression and function were effectively reduced by VSMC-specific delivery of the noncardiac form of Cav1.2 siRNA without similarly affecting cardiac CaL expression and function. When coupled with a viral vector, this molecular intervention in vivo may provide a novel long-term vascular-specific gene therapy for hypertension. PMID:19244098

  15. Message-passing-interface-based parallel FDTD investigation on the EM scattering from a 1-D rough sea surface using uniaxial perfectly matched layer absorbing boundary.

    PubMed

    Li, J; Guo, L-X; Zeng, H; Han, X-B

    2009-06-01

    A message-passing-interface (MPI)-based parallel finite-difference time-domain (FDTD) algorithm for the electromagnetic scattering from a 1-D randomly rough sea surface is presented. The uniaxial perfectly matched layer (UPML) medium is adopted for truncation of FDTD lattices, in which the finite-difference equations can be used for the total computation domain by properly choosing the uniaxial parameters. This makes the parallel FDTD algorithm easier to implement. The parallel performance with different processors is illustrated for one sea surface realization, and the computation time of the parallel FDTD algorithm is dramatically reduced compared to a single-process implementation. Finally, some numerical results are shown, including the backscattering characteristics of sea surface for different polarization and the bistatic scattering from a sea surface with large incident angle and large wind speed.

  16. Oxidized Low-density Lipoprotein (ox-LDL) Cholesterol Induces the Expression of miRNA-223 and L-type Calcium Channel Protein in Atrial Fibrillation

    PubMed Central

    He, Fengping; Xu, Xin; Yuan, Shuguo; Tan, Liangqiu; Gao, Lingjun; Ma, Shaochun; Zhang, Shebin; Ma, Zhanzhong; Jiang, Wei; Liu, Fenglian; Chen, Baofeng; Zhang, Beibei; Pang, Jungang; Huang, Xiuyan; Weng, Jiaqiang

    2016-01-01

    Atrial fibrillation (AF) is the most common sustained arrhythmia causing high morbidity and mortality. While changing of the cellular calcium homeostasis plays a critical role in AF, the L-type calcium channel α1c protein has suggested as an important regulator of reentrant spiral dynamics and is a major component of AF-related electrical remodeling. Our computational modeling predicted that miRNA-223 may regulate the CACNA1C gene which encodes the cardiac L-type calcium channel α1c subunit. We found that oxidized low-density lipoprotein (ox-LDL) cholesterol significantly up-regulates both the expression of miRNA-223 and L-type calcium channel protein. In contrast, knockdown of miRNA-223 reduced L-type calcium channel protein expression, while genetic knockdown of endogenous miRNA-223 dampened AF vulnerability. Transfection of miRNA-223 by adenovirus-mediated expression enhanced L-type calcium currents and promoted AF in mice while co-injection of a CACNA1C-specific miR-mimic counteracted the effect. Taken together, ox-LDL, as a known factor in AF-associated remodeling, positively regulates miRNA-223 transcription and L-type calcium channel protein expression. Our results implicate a new molecular mechanism for AF in which miRNA-223 can be used as an biomarker of AF rheumatic heart disease. PMID:27488468

  17. Oxidized Low-density Lipoprotein (ox-LDL) Cholesterol Induces the Expression of miRNA-223 and L-type Calcium Channel Protein in Atrial Fibrillation

    NASA Astrophysics Data System (ADS)

    He, Fengping; Xu, Xin; Yuan, Shuguo; Tan, Liangqiu; Gao, Lingjun; Ma, Shaochun; Zhang, Shebin; Ma, Zhanzhong; Jiang, Wei; Liu, Fenglian; Chen, Baofeng; Zhang, Beibei; Pang, Jungang; Huang, Xiuyan; Weng, Jiaqiang

    2016-08-01

    Atrial fibrillation (AF) is the most common sustained arrhythmia causing high morbidity and mortality. While changing of the cellular calcium homeostasis plays a critical role in AF, the L-type calcium channel α1c protein has suggested as an important regulator of reentrant spiral dynamics and is a major component of AF-related electrical remodeling. Our computational modeling predicted that miRNA-223 may regulate the CACNA1C gene which encodes the cardiac L-type calcium channel α1c subunit. We found that oxidized low-density lipoprotein (ox-LDL) cholesterol significantly up-regulates both the expression of miRNA-223 and L-type calcium channel protein. In contrast, knockdown of miRNA-223 reduced L-type calcium channel protein expression, while genetic knockdown of endogenous miRNA-223 dampened AF vulnerability. Transfection of miRNA-223 by adenovirus-mediated expression enhanced L-type calcium currents and promoted AF in mice while co-injection of a CACNA1C-specific miR-mimic counteracted the effect. Taken together, ox-LDL, as a known factor in AF-associated remodeling, positively regulates miRNA-223 transcription and L-type calcium channel protein expression. Our results implicate a new molecular mechanism for AF in which miRNA-223 can be used as an biomarker of AF rheumatic heart disease.

  18. Overexpression of L-Type Amino Acid Transporter 1 (LAT1) and 2 (LAT2): Novel Markers of Neuroendocrine Tumors

    PubMed Central

    Barollo, Susi; Bertazza, Loris; Watutantrige-Fernando, Sara; Censi, Simona; Cavedon, Elisabetta; Galuppini, Francesca; Pennelli, Gianmaria; Fassina, Ambrogio; Citton, Marilisa; Rubin, Beatrice; Pezzani, Raffaele; Benna, Clara; Opocher, Giuseppe; Iacobone, Maurizio; Mian, Caterina

    2016-01-01

    Background 6-18F-fluoro-L-3,4-dihydroxyphenylalanine (18F-FDOPA) PET is a useful tool in the clinical management of pheochromocytoma (PHEO) and medullary thyroid carcinoma (MTC). 18F-FDOPA is a large neutral amino acid biochemically resembling endogenous L-DOPA and taken up by the L-type amino acid transporters (LAT1 and LAT2). This study was conducted to examine the expression of the LAT system in PHEO and MTC. Methods Real-time PCR and Western blot analyses were used to assess LAT1 and LAT2 gene and protein expression in 32 PHEO, 38 MTC, 16 normal adrenal medulla and 15 normal thyroid tissue samples. Immunohistochemistry method was applied to identify the proteins’ subcellular localization. Results LAT1 and LAT2 were overexpressed in both PHEO and MTC by comparison with normal tissues. LAT1 presented a stronger induction than LAT2, and their greater expression was more evident in PHEO (15.1- and 4.1-fold increases, respectively) than in MTC (9.9- and 4.1-fold increases, respectively). Furthermore we found a good correlation between LAT1/2 and GLUT1 expression levels. A positive correlation was also found between urinary noradrenaline and adrenaline levels and LAT1 gene expression in PHEO. The increased expression of LAT1 is also confirmed at the protein level, in both PHEO and MTC, with a strong cytoplasmic localization. Conclusions The present study is the first to provide experimental evidence of the overexpression in some NET cancers (such as PHEO or MTC) of L-type amino acid transporters, and the LAT1 isoform in particular, giving the molecular basis to explain the increase of the DOPA uptake seen in such tumor cells. PMID:27224648

  19. Group III metabotropic glutamate receptors and exocytosed protons inhibit L-type calcium currents in cones but not in rods.

    PubMed

    Hosoi, Nobutake; Arai, Itaru; Tachibana, Masao

    2005-04-20

    Light responses of photoreceptors (rods and cones) are transmitted to the second-order neurons (bipolar cells and horizontal cells) via glutamatergic synapses located in the outer plexiform layer of the retina. Although it has been well established that postsynaptic group III metabotropic glutamate receptors (mGluRs) of ON bipolar cells contribute to generating the ON signal, presynaptic roles of group III mGluRs remain to be elucidated at this synaptic connection. We addressed this issue by applying the slice patch-clamp technique to the newt retina. OFF bipolar cells and horizontal cells generate a steady inward current in the dark and a transient inward current at light offset, both of which are mediated via postsynaptic non-NMDA receptors. A group III mGluR-specific agonist, L-2-amino-4-phosphonobutyric acid (L-AP-4), inhibited both the steady and off-transient inward currents but did not affect the glutamate-induced current in these postsynaptic neurons. L-AP-4 inhibited the presynaptic L-type calcium current (ICa) in cones by shifting the voltage dependence of activation to more positive membrane potentials. The inhibition of ICa was most prominent around the physiological range of cone membrane potentials. In contrast, L-AP-4 did not affect L-type ICa in rods. Paired recordings from photoreceptors and the synaptically connected second-order neurons confirmed that L-AP-4 inhibited both ICa and glutamate release in cones but not in rods. Furthermore, we found that exocytosed protons also inhibited ICa in cones but not in rods. Selective modulation of ICa in cones may help broaden the dynamic range of synaptic transfer by controlling the amount of transmitter release from cones.

  20. Antiapolipoprotein A-1 IgG chronotropic effects require nongenomic action of aldosterone on L-type calcium channels.

    PubMed

    Rossier, Michel F; Pagano, Sabrina; Python, Magaly; Maturana, Andres D; James, Richard W; Mach, François; Roux-Lombard, Pascale; Vuilleumier, Nicolas

    2012-03-01

    Autoantibodies to apolipoprotein A-1 (antiapoA-1 IgG) have been shown to be associated with higher resting heart rate and morbidity in myocardial infarction patients and to behave as a chronotropic agent in the presence of aldosterone on isolated neonatal rat ventricular cardiomyocytes (NRVC). We aimed at identifying the pathways accounting for this aldosterone-dependent antiapoA-1 IgG-positive chronotropic effect on NRVC. The rate of regular spontaneous contractions was determined on NRVC in the presence of different steroid hormones and antagonists. AntiapoA-1 IgG chronotropic response was maximal within 20 min and observed only in aldosterone-pretreated cells but not in those exposed to other steroids. The positive antiapoA-1 IgG chronotropic effect was already significant after 5 min aldosterone preincubation, was dependent on 3-kinase and protein kinase A activities, was not inhibited by actinomycin D, and was fully abrogated by eplerenone (but not by spironolactone), demonstrating the dependence on a nongenomic action of aldosterone elicited through the mineralocorticoid receptor (MR). Under oxidative conditions (but not under normal redox state), corticosterone mimicked the permissive action of aldosterone on the antiapoA-1 IgG chronotropic response. Pharmacological and patch-clamp studies identified L-type calcium channels as crucial effectors of antiapoA-1 IgG chronotropic action, involving two converging pathways that increase the channel activity. The first one involves the rapid, nongenomic activation of the phosphatidylinositol 3-kinase enzyme by MR, and the second one requires a constitutive basal protein kinase A activity. In conclusion, our results indicate that, on NRVC, the aldosterone-dependent chronotropic effects of antiapoA-1 IgG involve the nongenomic activation of L-type calcium channels.

  1. Cytosolic Ca2+ Buffers

    PubMed Central

    Schwaller, Beat

    2010-01-01

    Ca2+ buffers,” a class of cytosolic Ca2+-binding proteins, act as modulators of short-lived intracellular Ca2+ signals; they affect both the temporal and spatial aspects of these transient increases in [Ca2+]i. Examples of Ca2+ buffers include parvalbumins (α and β isoforms), calbindin-D9k, calbindin-D28k, and calretinin. Besides their proven Ca2+ buffer function, some might additionally have Ca2+ sensor functions. Ca2+ buffers have to be viewed as one of the components implicated in the precise regulation of Ca2+ signaling and Ca2+ homeostasis. Each cell is equipped with proteins, including Ca2+ channels, transporters, and pumps that, together with the Ca2+ buffers, shape the intracellular Ca2+ signals. All of these molecules are not only functionally coupled, but their expression is likely to be regulated in a Ca2+-dependent manner to maintain normal Ca2+ signaling, even in the absence or malfunctioning of one of the components. PMID:20943758

  2. Spontaneous Ca2+ spiking in a vascular smooth muscle cell line is independent of the release of intracellular Ca2+ stores.

    PubMed

    Byron, K L; Taylor, C W

    1993-04-05

    Monolayers of fura-2-loaded A7r5 cells, a cell line derived from rat embryonic aorta, generated spontaneous Ca2+ spikes that were synchronized within the cell population. These Ca2+ spikes were abolished by removal of extracellular Ca2+ or addition of nimodipine (50 nM), and their frequency was increased by depolarization with high K+ or by treatment with BAYK 8644 (1 microM), indicating that Ca2+ entry through L-type Ca2+ channels is required for Ca2+ spiking. Several lines of evidence indicate that mobilization of intracellular Ca2+ stores is not necessary for this Ca2+ spiking. 1) Ryanodine (0.1-50 microM) neither stimulated Ca2+ mobilization nor affected Ca2+ spiking; 2) the complex effects of caffeine were mimicked by theophylline, 8-bromo-cyclic adenosine 3':5'-monophosphate (8-bromo-cAMP), and forskolin, suggesting that the caffeine effects may be mediated by cAMP and not by ryanodine receptors; 3) prolonged incubation with thapsigargin (50 nM), which depletes intracellular Ca2+ stores, did not affect the frequency of Ca2+ spiking; 4) Ba2+ or Sr2+ could substitute for Ca2+ in the spike-generating mechanism even when intracellular stores were depleted of Ca2+. Under conditions where the sarcoplasmic reticulum (SR) contained Ca2+, Ba2+ spikes did not cause Ca2+ mobilization. The mechanisms involved in generating spontaneous Ca2+ spiking in A7r5 cells are therefore likely to reside in the sarcolemma and to operate independently of SR Ca2+ uptake and release.

  3. A model of cardiac ryanodine receptor gating predicts experimental Ca2+-dynamics and Ca2+-triggered arrhythmia in the long QT syndrome

    NASA Astrophysics Data System (ADS)

    Wilson, Dan; Ermentrout, Bard; Němec, Jan; Salama, Guy

    2017-09-01

    Abnormal Ca2+ handling is well-established as the trigger of cardiac arrhythmia in catecholaminergic polymorphic ventricular tachycardia and digoxin toxicity, but its role remains controversial in Torsade de Pointes (TdP), the arrhythmia associated with the long QT syndrome (LQTS). Recent experimental results show that early afterdepolarizations (EADs) that initiate TdP are caused by spontaneous (non-voltage-triggered) Ca2+ release from Ca2+-overloaded sarcoplasmic reticulum (SR) rather than the activation of the L-type Ca2+-channel window current. In bradycardia and long QT type 2 (LQT2), a second, non-voltage triggered cytosolic Ca2+ elevation increases gradually in amplitude, occurs before overt voltage instability, and then precedes the rise of EADs. Here, we used a modified Shannon-Puglisi-Bers model of rabbit ventricular myocytes to reproduce experimental Ca2+ dynamics in bradycardia and LQT2. Abnormal systolic Ca2+-oscillations and EADs caused by SR Ca2+-release are reproduced in a modified 0-dimensional model, where 3 gates in series control the ryanodine receptor (RyR2) conductance. Two gates control RyR2 activation and inactivation and sense cytosolic Ca2+ while a third gate senses luminal junctional SR Ca2+. The model predicts EADs in bradycardia and low extracellular [K+] and cessation of SR Ca2+-release terminate salvos of EADs. Ca2+-waves, systolic cell-synchronous Ca2+-release, and multifocal diastolic Ca2+ release seen in subcellular Ca2+-mapping experiments are observed in the 2-dimensional version of the model. These results support the role of SR Ca2+-overload, abnormal SR Ca2+-release, and the subsequent activation of the electrogenic Na+/Ca2+-exchanger as the mechanism of TdP. The model offers new insights into the genesis of cardiac arrhythmia and new therapeutic strategies.

  4. Apo states of calmodulin and CaBP1 control CaV1 voltage-gated calcium channel function through direct competition for the IQ domain.

    PubMed

    Findeisen, Felix; Rumpf, Christine H; Minor, Daniel L

    2013-09-09

    In neurons, binding of calmodulin (CaM) or calcium-binding protein 1 (CaBP1) to the CaV1 (L-type) voltage-gated calcium channel IQ domain endows the channel with diametrically opposed properties. CaM causes calcium-dependent inactivation and limits calcium entry, whereas CaBP1 blocks calcium-dependent inactivation (CDI) and allows sustained calcium influx. Here, we combine isothermal titration calorimetry with cell-based functional measurements and mathematical modeling to show that these calcium sensors behave in a competitive manner that is explained quantitatively by their apo-state binding affinities for the IQ domain. This competition can be completely blocked by covalent tethering of CaM to the channel. Further, we show that Ca(2+)/CaM has a sub-picomolar affinity for the IQ domain that is achieved without drastic alteration of calcium-binding properties. The observation that the apo forms of CaM and CaBP1 compete with each other demonstrates a simple mechanism for direct modulation of CaV1 function and suggests a means by which excitable cells may dynamically tune CaV activity. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. Apo-states of calmodulin and CaBP1 control CaV1 voltage-gated calcium channel function through direct competition for the IQ domain

    PubMed Central

    Findeisen, Felix; Rumpf, Christine; Minor, Daniel L.

    2013-01-01

    In neurons, binding of calmodulin (CaM) or calcium-binding protein 1 (CaBP1) to the CaV1 (L-type) voltage-gated calcium channel IQ domain endows the channel with diametrically opposed properties. CaM causes calcium-dependent inactivation (CDI) and limits calcium entry, whereas CaBP1 blocks CDI and allows sustained calcium influx. Here, we combine isothermal titration calorimetry (ITC) with cell-based functional measurements and mathematical modeling to show that these calcium sensors behave in a competitive manner that is explained quantitatively by their apo-state binding affinities for the IQ domain. This competition can be completely blocked by covalent tethering of CaM to the channel. Further, we show that Ca2+/CaM has a sub-picomolar affinity for the IQ domain that is achieved without drastic alteration of calcium binding properties. The observation that the apo-forms of CaM and CaBP1 compete with each other demonstrates a simple mechanism for direct modulation of CaV1 function and suggests a means by which excitable cells may dynamically tune CaV activity. PMID:23811053

  6. Sensitivity of Rabbit Ventricular Action Potential and Ca2+ Dynamics to Small Variations in Membrane Currents and Ion Diffusion Coefficients

    PubMed Central

    Lo, Yuan Hung; Peachey, Tom; Abramson, David; McCulloch, Andrew

    2013-01-01

    Little is known about how small variations in ionic currents and Ca2+ and Na+ diffusion coefficients impact action potential and Ca2+ dynamics in rabbit ventricular myocytes. We applied sensitivity analysis to quantify the sensitivity of Shannon et al. model (Biophys. J., 2004) to 5%–10% changes in currents conductance, channels distribution, and ion diffusion in rabbit ventricular cells. We found that action potential duration and Ca2+ peaks are highly sensitive to 10% increase in L-type Ca2+ current; moderately influenced by 10% increase in Na+-Ca2+ exchanger, Na+-K+ pump, rapid delayed and slow transient outward K+ currents, and Cl− background current; insensitive to 10% increases in all other ionic currents and sarcoplasmic reticulum Ca2+ fluxes. Cell electrical activity is strongly affected by 5% shift of L-type Ca2+ channels and Na+-Ca2+ exchanger in between junctional and submembrane spaces while Ca2+-activated Cl−-channel redistribution has the modest effect. Small changes in submembrane and cytosolic diffusion coefficients for Ca2+, but not in Na+ transfer, may alter notably myocyte contraction. Our studies highlight the need for more precise measurements and further extending and testing of the Shannon et al. model. Our results demonstrate usefulness of sensitivity analysis to identify specific knowledge gaps and controversies related to ventricular cell electrophysiology and Ca2+ signaling. PMID:24222910

  7. RGK protein-mediated impairment of slow depolarization- dependent Ca2+ entry into developing myotubes

    PubMed Central

    Romberg, Christin F; Beqollari, Donald; Meza, Ulises; Bannister, Roger A

    2014-01-01

    Three physiological functions have been described for the skeletal muscle 1,4-dihydropyridine receptor (CaV1.1): (1) voltage-sensor for excitation-contraction (EC) coupling, (2) L-type Ca2+ channel, and (3) voltage-sensor for slow depolarization-dependent Ca2+ entry. Members of the RGK (Rad, Rem, Rem2, Gem/Kir) family of monomeric GTP-binding proteins are potent inhibitors of the former two functions of CaV1.1. However, it is not known whether the latter function that has been attributed to CaV1.1 is subject to modulation by RGK proteins. Thus, the purpose of this study was to determine whether Rad, Gem and/or Rem inhibit the slowly developing, persistent Ca2+ entry that is dependent on the voltage-sensing capability of CaV1.1. As a means to investigate this question, Venus fluorescent protein-fused RGK proteins (V-Rad, V-Rem and V-Gem) were overexpressed in “normal” mouse myotubes. We observed that such overexpression of V-Rad, V-Rem or V-Gem in myotubes caused marked changes in morphology of the cells. As shown previously for YFP-Rem, both L-type current and EC coupling were also impaired greatly in myotubes expressing either V-Rad or V-Gem. The reductions in L-type current and EC coupling were paralleled by reductions in depolarization-induced Ca2+ entry. Our observations provide the first evidence of modulation of this enigmatic Ca2+ entry pathway peculiar to skeletal muscle. PMID:24476902

  8. Myogenic tone is impaired at low arterial pressure in mice deficient in the low-voltage-activated CaV 3.1 T-type Ca(2+) channel.

    PubMed

    Björling, K; Morita, H; Olsen, M F; Prodan, A; Hansen, P B; Lory, P; Holstein-Rathlou, N-H; Jensen, L J

    2013-04-01

    Using mice deficient in the CaV 3.1 T-type Ca(2+) channel, the aim of the present study was to elucidate the molecular identity of non-L-type channels involved in vascular tone regulation in mesenteric arteries and arterioles. We used immunofluorescence microscopy to localize CaV 3.1 channels, patch clamp electrophysiology to test the effects of a putative T-type channel blocker NNC 55-0396 on whole-cell Ca(2+) currents, pressure myography and Ca(2+) imaging to test diameter and Ca(2+) responses of the applied vasoconstrictors, and Q-PCR to check mRNA expression levels of several Ca(2+) handling proteins in wild-type and CaV 3.1(-/-) mice. Our data indicated that CaV 3.1 channels are important for the maintenance of myogenic tone at low pressures (40-80 mm Hg), whereas they are not involved in high-voltage-activated Ca(2+) currents, Ca(2+) entry or vasoconstriction to high KCl in mesenteric arteries and arterioles. Furthermore, we show that NNC 55-0396 is not a specific T-type channel inhibitor, as it potently blocks L-type and non-L-type high-voltage-activated Ca(2+) currents in mouse mesenteric vascular smooth muscle cell. Our data using mice deficient in the CaV 3.1 T-type channel represent new evidence for the involvement of non-L-type channels in arteriolar tone regulation. We showed that CaV 3.1 channels are important for the myogenic tone at low arterial pressure, which is potentially relevant under resting conditions in vivo. Moreover, CaV 3.1 channels are not involved in Ca(2+) entry and vasoconstriction to large depolarization with, for example, high KCl. Finally, we caution against using NNC 55-0396 as a specific T-type channel blocker in native cells expressing high-voltage-activated Ca(2+) channels. Acta Physiologica © 2013 Scandinavian Physiological Society.

  9. Role of Ca2+ signaling in initiation of stretch-induced apoptosis in neonatal heart cells.

    PubMed

    Liao, Xu Dong; Tang, Ai Hui; Chen, Quan; Jin, Hai Jing; Wu, Cai Hong; Chen, Lan-Ying; Wang, Shi Qiang

    2003-10-17

    Abnormal mechanical load, as seen in hypertension, is found to induce heart cell apoptosis, yet the signaling link between cell stretch and apoptotic pathways is not known. Using an in vitro stretch model mimicking diastolic pressure stress, here we show that Ca(2+) signaling participates essentially in the early stage of stretch-induced apoptosis. In neonatal rat cardiomyocytes, the moderate 20% stretch resulted in tonic elevation of intracellular free Ca(2+) ([Ca(2+)](i)). Buffering [Ca(2+)](i) by EGTA-AM, suppressing ryanodine-sensitive Ca(2+) release, and blocking L-type Ca(2+) channels all prevented the stretch-induced apoptosis as assessed by phosphatidylserine exposure and nuclear fragmentation. Notably, Ca(2+) suppression also prevented known stretch-activated apoptotic events, including caspase-3/-9 activation, mitochondrial membrane potential corruption, and reactive oxygen species production, suggesting that Ca(2+) signaling is the upstream of these events. Since [Ca(2+)](i) did not change without activating mechanosensitive Ca(2+) entry, we conclude that stretch-induced Ca(2+) entry, via the Ca(2+)-induced Ca(2+) release mechanism, plays an important role in initiating apoptotic signaling during mechanical stress.

  10. Further characterization of the effect of ethanol on voltage-gated Ca(2+) channel function in developing CA3 hippocampal pyramidal neurons.

    PubMed

    Morton, Russell A; Valenzuela, C Fernando

    2016-02-15

    Developmental ethanol exposure damages the hippocampus, a brain region involved in learning and memory. Alterations in synaptic transmission and plasticity may play a role in this effect of ethanol. We previously reported that acute and repeated exposure to ethanol during the third trimester-equivalent inhibits long-term potentiation of GABAA receptor-dependent synaptic currents in CA3 pyramidal neurons through a mechanism that depends on retrograde release of brain-derived neurotrophic factor driven by activation of voltage-gated Ca(2+) channels (Zucca and Valenzuela, 2010). We found evidence indicating that voltage-gated Ca(2+) channels are inhibited in the presence of ethanol, an effect that may play a role in its mechanism of action. Here, we further investigated the acute effect of ethanol on the function of voltage-gated Ca(2+) channels in CA3 pyramidal neurons using Ca(2+) imaging techniques. These experiments revealed that acute ethanol exposure inhibits voltage-gated Ca(2+) channels both in somatic and proximal dendritic compartments. To investigate the long-term consequences of ethanol on voltage-gated Ca(2+) channels, we used patch-clamp electrophysiological techniques to assess the function of L-type voltage-gated Ca(2+) channels during and following ten days of vapor ethanol exposure. During ethanol withdrawal periods, the function of these channels was not significantly affected by vapor chamber exposure. Taken together with our previous findings, our results suggest that 3(rd) trimester-equivalent ethanol exposure transiently inhibits L-type voltage-gated Ca(2+) channel function in CA3 pyramidal neurons and that compensatory mechanisms restore their function during ethanol withdrawal. Transient inhibition of these channels by ethanol may be, in part, responsible for the hippocampal abnormalities associated with developmental exposure to this agent. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity*

    PubMed Central

    Tétreault, Marie-Philippe; Bourdin, Benoîte; Briot, Julie; Segura, Emilie; Lesage, Sylvie; Fiset, Céline; Parent, Lucie

    2016-01-01

    Alteration in the L-type current density is one aspect of the electrical remodeling observed in patients suffering from cardiac arrhythmias. Changes in channel function could result from variations in the protein biogenesis, stability, post-translational modification, and/or trafficking in any of the regulatory subunits forming cardiac L-type Ca2+ channel complexes. CaVα2δ1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type CaV1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant CaVα2δ1 proteins. This change was also observed upon simultaneous mutation of the 16 Asn sites. Nonetheless, the mutation of only 6/16 sites was sufficient to significantly 1) reduce the steady-state cell surface fluorescence of CaVα2δ1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the CaVα2δ1-mediated increase in the peak current density and voltage-dependent gating of CaV1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored CaVα2δ1 function. Single mutation N663Q and double mutations N348Q/N468Q, N348Q/N812Q, and N468Q/N812Q decreased protein stability/synthesis and nearly abolished steady-state cell surface density of CaVα2δ1 as well as the CaVα2δ1-induced up-regulation of L-type currents. These results demonstrate that Asn-663 and to a lesser extent Asn-348, Asn-468, and Asn-812 contribute to protein stability/synthesis of CaVα2δ1, and furthermore that N-glycosylation of CaVα2δ1 is essential to produce functional L-type Ca2+ channels. PMID:26742847

  12. Functional Characterization of CaVα2δ Mutations Associated with Sudden Cardiac Death*

    PubMed Central

    Bourdin, Benoîte; Shakeri, Behzad; Tétreault, Marie-Philippe; Sauvé, Rémy; Lesage, Sylvie; Parent, Lucie

    2015-01-01

    L-type Ca2+ channels play a critical role in cardiac rhythmicity. These ion channels are oligomeric complexes formed by the pore-forming CaVα1 with the auxiliary CaVβ and CaVα2δ subunits. CaVα2δ increases the peak current density and improves the voltage-dependent activation gating of CaV1.2 channels without increasing the surface expression of the CaVα1 subunit. The functional impact of genetic variants of CACNA2D1 (the gene encoding for CaVα2δ), associated with shorter repolarization QT intervals (the time interval between the Q and the T waves on the cardiac electrocardiogram), was investigated after recombinant expression of the full complement of L-type CaV1.2 subunits in human embryonic kidney 293 cells. By performing side-by-side high resolution flow cytometry assays and whole-cell patch clamp recordings, we revealed that the surface density of the CaVα2δ wild-type protein correlates with the peak current density. Furthermore, the cell surface density of CaVα2δ mutants S755T, Q917H, and S956T was not significantly different from the cell surface density of the CaVα2δ wild-type protein expressed under the same conditions. In contrast, the cell surface expression of CaVα2δ D550Y, CaVα2δ S709N, and the double mutant D550Y/Q917H was reduced, respectively, by ≈30–33% for the single mutants and by 60% for the latter. The cell surface density of D550Y/Q917H was more significantly impaired than protein stability, suggesting that surface trafficking of CaVα2δ was disrupted by the double mutation. Co-expression with D550Y/Q917H significantly decreased CaV1.2 currents as compared with results obtained with CaVα2δ wild type. It is concluded that D550Y/Q917H reduced inward Ca2+ currents through a defect in the cell surface trafficking of CaVα2δ. Altogether, our results provide novel insight in the molecular mechanism underlying the modulation of CaV1.2 currents by CaVα2δ. PMID:25527503

  13. Catecholamine-Independent Heart Rate Increases Require CaMKII

    PubMed Central

    Gao, Zhan; Singh, Madhu V; Hall, Duane D; Koval, Olha M.; Luczak, Elizabeth D.; Joiner, Mei-ling A.; Chen, Biyi; Wu, Yuejin; Chaudhary, Ashok K; Martins, James B; Hund, Thomas J; Mohler, Peter J; Song, Long-Sheng; Anderson, Mark E.

    2011-01-01

    Background Catecholamines increase heart rate by augmenting the cAMP responsive HCN4 ‘pacemaker current’ (If) and/or by promoting inward Na+/Ca2+ exchanger current (INCX), by a ‘Ca2+ clock’ mechanism in sinoatrial nodal cells (SANCs). The importance, identity and function of signals that connect If and Ca2+ clock mechanisms are uncertain and controversial, but the multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) is required for physiological heart rate responses to β-adrenergic receptor (β-AR) stimulation. The aim of this stuy is to measure the contribution of the Ca2+ clock and CaMKII to cardiac pacing independent of β-AR agonist stimulation. Methods and Results We used the L-type Ca2+ channel agonist BayK 8644 (BayK) to activate the SANC Ca2+ clock. BayK and isoproterenol were similarly effective in increasing rates in SANCs and Langendorff-perfused hearts from WT control mice. In contrast, SANCs and isolated hearts from mice with CaMKII inhibition by transgenic expression of an inhibitory peptide (AC3-I) were resistant to rate increases by BayK. BayK only activated CaMKII in control SANCs, but increased ICa equally in all SANCs, indicating that increasing ICa was insufficient and suggesting CaMKII activation was required for heart rate increases by BayK. BayK did not increase If or protein kinase A (PKA)-dependent phosphorylation of phospholamban (at Ser16), indicating that increased SANC Ca2+ by BayK did not augment cAMP/PKA signaling at these targets. Late diastolic intracellular Ca2+ release and INCX were significantly reduced in AC3-I SANCs and the response to BayK was eliminated by ryanodine in all groups. Conclusions The Ca2+ clock is capable of supporting physiological fight or flight responses, independent of β-AR stimulation or If increases. Complete Ca2+ clock and β-AR stimulation responses require CaMKII. PMID:21406683

  14. Increased Ca2+ signaling through CaV1.2 promotes bone formation and prevents estrogen deficiency-induced bone loss.

    PubMed

    Cao, Chike; Ren, Yinshi; Barnett, Adam S; Mirando, Anthony J; Rouse, Douglas; Mun, Se Hwan; Park-Min, Kyung-Hyun; McNulty, Amy L; Guilak, Farshid; Karner, Courtney M; Hilton, Matthew J; Pitt, Geoffrey S

    2017-11-16

    While the prevalence of osteoporosis is growing rapidly with population aging, therapeutic options remain limited. Here, we identify potentially novel roles for CaV1.2 L-type voltage-gated Ca2+ channels in osteogenesis and exploit a transgenic gain-of-function mutant CaV1.2 to stem bone loss in ovariectomized female mice. We show that endogenous CaV1.2 is expressed in developing bone within proliferating chondrocytes and osteoblasts. Using primary BM stromal cell (BMSC) cultures, we found that Ca2+ influx through CaV1.2 activates osteogenic transcriptional programs and promotes mineralization. We used Prx1-, Col2a1-, or Col1a1-Cre drivers to express an inactivation-deficient CaV1.2 mutant in chondrogenic and/or osteogenic precursors in vivo and found that the resulting increased Ca2+ influx markedly thickened bone not only by promoting osteogenesis, but also by inhibiting osteoclast activity through increased osteoprotegerin secretion from osteoblasts. Activating the CaV1.2 mutant in osteoblasts at the time of ovariectomy stemmed bone loss. Together, these data highlight roles for CaV1.2 in bone and demonstrate the potential dual anabolic and anticatabolic therapeutic actions of tissue-specific CaV1.2 activation in osteoblasts.

  15. Increased Ca2+ signaling through CaV1.2 promotes bone formation and prevents estrogen deficiency–induced bone loss

    PubMed Central

    Cao, Chike; Barnett, Adam S.; Mirando, Anthony J.; Rouse, Douglas; Mun, Se Hwan; Park-Min, Kyung-Hyun; McNulty, Amy L.; Karner, Courtney M.; Hilton, Matthew J.

    2017-01-01

    While the prevalence of osteoporosis is growing rapidly with population aging, therapeutic options remain limited. Here, we identify potentially novel roles for CaV1.2 L-type voltage–gated Ca2+ channels in osteogenesis and exploit a transgenic gain-of-function mutant CaV1.2 to stem bone loss in ovariectomized female mice. We show that endogenous CaV1.2 is expressed in developing bone within proliferating chondrocytes and osteoblasts. Using primary BM stromal cell (BMSC) cultures, we found that Ca2+ influx through CaV1.2 activates osteogenic transcriptional programs and promotes mineralization. We used Prx1-, Col2a1-, or Col1a1-Cre drivers to express an inactivation-deficient CaV1.2 mutant in chondrogenic and/or osteogenic precursors in vivo and found that the resulting increased Ca2+ influx markedly thickened bone not only by promoting osteogenesis, but also by inhibiting osteoclast activity through increased osteoprotegerin secretion from osteoblasts. Activating the CaV1.2 mutant in osteoblasts at the time of ovariectomy stemmed bone loss. Together, these data highlight roles for CaV1.2 in bone and demonstrate the potential dual anabolic and anticatabolic therapeutic actions of tissue-specific CaV1.2 activation in osteoblasts. PMID:29202453

  16. Quantitative insight into the design of compounds recognized by the L-type amino acid transporter 1 (LAT1).

    PubMed

    Ylikangas, Henna; Malmioja, Kalle; Peura, Lauri; Gynther, Mikko; Nwachukwu, Emmanuel O; Leppänen, Jukka; Laine, Krista; Rautio, Jarkko; Lahtela-Kakkonen, Maija; Huttunen, Kristiina M; Poso, Antti

    2014-12-01

    L-Type amino acid transporter 1 (LAT1) is a transmembrane protein expressed abundantly at the blood-brain barrier (BBB), where it ensures the transport of hydrophobic acids from the blood to the brain. Due to its unique substrate specificity and high expression at the BBB, LAT1 is an intriguing target for carrier-mediated transport of drugs into the brain. In this study, a comparative molecular field analysis (CoMFA) model with considerable statistical quality (Q(2) =0.53, R(2) =0.75, Q(2) SE=0.77, R(2) SE=0.57) and good external predictivity (CCC=0.91) was generated. The model was used to guide the synthesis of eight new prodrugs whose affinity for LAT1 was tested by using an in situ rat brain perfusion technique. This resulted in the creation of a novel LAT1 prodrug with L-tryptophan as the promoiety; it also provided a better understanding of the molecular features of LAT1-targeted high-affinity prodrugs, as well as their promoiety and parent drug. The results obtained will be beneficial in the rational design of novel LAT1-binding prodrugs and other compounds that bind to LAT1. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. The Role of Dopamine Receptors in the Neurobehavioral Syndrome Provoked by Activation of L-Type Calcium Channels in Rodents

    PubMed Central

    Kasim, Suhail; Blake, Bonita L.; Fan, Xueliang; Chartoff, Elena; Egami, Kiyoshi; Breese, George R.; Hess, Ellen J.; Jinnah, H.A.

    2010-01-01

    In rodents, activation of L-type calcium channels with ± BayK 8644 causes an unusual behavioral syndrome that includes dystonia and self-biting. Prior studies have linked both of these behaviors to dysfunction of dopaminergic transmission in the striatum. The current studies were designed to further elucidate the relationship between ± BayK 8644 and dopaminergic transmission in the expression of the behavioral syndrome. The drug does not appear to release presynaptic dopamine stores, since microdialysis of the striatum revealed dopamine release was unaltered by ± BayK 8644. In addition, the behaviors were preserved or even exaggerated in mice or rats with virtually complete dopamine depletion. On the other hand, pretreatment of mice with D3 or D1/5 dopamine receptor antagonists attenuated the behavioral effects of ± BayK 8644, while pretreatment with D2 or D4 antagonists had no effect. In D3 receptor knockout mice, ± BayK 8644 elicited both dystonia and self-biting, but these behaviors were less severe than in matched controls. In D1 receptor knockout mice, behavioral responses to ± BayK 8644 appeared exaggerated. These results argue that the behavioral effects of ± BayK 8644 are not mediated by a presynaptic influence. Instead, the behaviors appear to result from a postsynaptic activation of the drug, which does not require but can be modified by D3 or D1/5 receptors. PMID:17028428

  18. Spectral modification of seismic waves propagating through solids exhibiting a resonance frequency: a 1-D coupled wave propagation-oscillation model

    NASA Astrophysics Data System (ADS)

    Frehner, Marcel; Schmalholz, Stefan M.; Podladchikov, Yuri

    2009-02-01

    A 1-D model is presented that couples the microscale oscillations of non-wetting fluid blobs in a partially saturated poroelastic medium with the macroscale wave propagation through the elastic skeleton. The fluid oscillations are caused by surface tension forces that act as the restoring forces driving the oscillations. The oscillations are described mathematically with the equation for a linear oscillator and the wave propagation is described with the 1-D elastic wave equation. Coupling is done using Hamilton's variational principle for continuous systems. The resulting linear system of two partial differential equations is solved numerically with explicit finite differences. Numerical simulations are used to analyse the effect of solids exhibiting internal oscillations, and consequently a resonance frequency, on seismic waves propagating through such media. The phase velocity dispersion relation shows a higher phase velocity in the high-frequency limit and a lower phase velocity in the low-frequency limit. At the resonance frequency a singularity in the dispersion relation occurs. Seismic waves can initiate oscillations of the fluid by transferring energy from solid to fluid at the resonance frequency. Due to this transfer, the spectral amplitude of the solid particle velocity decreases at the resonance frequency. After initiation, the oscillatory movement of the fluid continuously transfers energy at the resonance frequency back to the solid. Therefore, the spectral amplitude of the solid particle velocity is increased at the resonance frequency. Once initiated, fluid oscillations decrease in amplitude with increasing time. Consequently, the spectral peak of the solid particle velocity at the resonance frequency decreases with time.

  19. Estimation of sum-to-one constrained parameters with non-Gaussian extensions of ensemble-based Kalman filters: application to a 1D ocean biogeochemical model

    NASA Astrophysics Data System (ADS)

    Simon, E.; Bertino, L.; Samuelsen, A.

    2011-12-01

    Combined state-parameter estimation in ocean biogeochemical models with ensemble-based Kalman filters is a challenging task due to the non-linearity of the models, the constraints of positiveness that apply to the variables and parameters, and the non-Gaussian distribution of the variables in which they result. Furthermore, these models are sensitive to numerous parameters that are poorly known. Previous works [1] demonstrated that the Gaussian anamorphosis extensions of ensemble-based Kalman filters were relevant tools to perform combined state-parameter estimation in such non-Gaussian framework. In this study, we focus on the estimation of the grazing preferences parameters of zooplankton species. These parameters are introduced to model the diet of zooplankton species among phytoplankton species and detritus. They are positive values and their sum is equal to one. Because the sum-to-one constraint cannot be handled by ensemble-based Kalman filters, a reformulation of the parameterization is proposed. We investigate two types of changes of variables for the estimation of sum-to-one constrained parameters. The first one is based on Gelman [2] and leads to the estimation of normal distributed parameters. The second one is based on the representation of the unit sphere in spherical coordinates and leads to the estimation of parameters with bounded distributions (triangular or uniform). These formulations are illustrated and discussed in the framework of twin experiments realized in the 1D coupled model GOTM-NORWECOM with Gaussian anamorphosis extensions of the deterministic ensemble Kalman filter (DEnKF). [1] Simon E., Bertino L. : Gaussian anamorphosis extension of the DEnKF for combined state and parameter estimation : application to a 1D ocean ecosystem model. Journal of Marine Systems, 2011. doi :10.1016/j.jmarsys.2011.07.007 [2] Gelman A. : Method of Moments Using Monte Carlo Simulation. Journal of Computational and Graphical Statistics, 4, 1, 36-54, 1995.

  20. A novel D-phenylalanine-derivative hypoglycemic agent A-4166 increases cytosolic free Ca2+ in rat pancreatic beta-cells by stimulating Ca2+ influx.

    PubMed

    Fujitani, S; Yada, T

    1994-03-01

    It has recently been shown that N-[(trans-4-isopropylcyclohexyl)-carbonyl]D-phenylalanine (A-4166), a new nonsulfonylurea oral hypoglycemic agent, reduces blood glucose levels in nondiabetic and diabetic animals in a quicker and shorter lasting manner than sulfonylureas, and that the hypoglycemic effect of A-4166 is due to the stimulation of insulin release. However, the mechanism by which A-4166 stimulates insulin release is still unknown. In the present study, we investigated the effect of A-4166 on the cytosolic free Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells from normal rats by dual wavelength fura-2 microfluorometry. In the presence of 2.8 mM glucose, A-4166 produced a rapid increase in [Ca2+]i in a concentration-dependent manner over the range of 3-30 microM. The increase in [Ca2+]i was transient, oscillatory, or sustained. A-4166 did not evoke any decrease in [Ca2+]i, whereas a high concentration of glucose (16.7 mM), a metabolized secretagogue, produced an initial decrease and a subsequent increase in [Ca2+]i. In the presence of 16.7 mM glucose, low concentrations (0.03-1 microM) of A-4166 produced an increase in [Ca2+]i in some of the beta-cells tested. The [Ca2+]i response to A-4166 was completely and reversibly inhibited under Ca(2+)-free conditions as well as by nitrendipine, a blocker of the L-type Ca2+ channel. Nitrendipine also inhibited insulin release from perfused rat pancreases stimulated by A-4166. Diazoxide, an opener of the ATP-sensitive K+ channel, blocked the [Ca2+]i response to A-4166. Sulfonylureas such as tolbutamide and glibenclamide increased [Ca2+]i in a manner similar to A-4166. These results indicate that at basal glucose concentrations, A-4166 increases [Ca2+]i in rat pancreatic beta-cells by stimulating Ca2+ influx through L-type Ca2+ channels, and that this effect is markedly augmented at elevated glucose concentrations. It appears that the increase in [Ca2+]i is related to the stimulation of insulin release by A-4166

  1. MO-FG-202-03: Efficient Data Collection of Continuous 2D and Discrete Relative Dosimetric Data for Annual LINAC QA Using TrueBeam Developer Mode and a 1D Scanning Tank

    SciTech Connect

    Knutson, N; Schmidt, M; University of Rhode Island, Kingston, RI

    2016-06-15

    Purpose: To develop a method to exploit real-time dynamic machine and couch parameter control during linear accelerator (LINAC) beam delivery to facilitate efficient performance of TG-142 suggested, Annual LINAC QA tests. Methods: Varian’s TrueBeam Developer Mode (Varian Medical Systems, Palo Alto, CA) facilitates control of Varian’s TrueBeam LINAC via instructions provided in Extensible Markup Language (XML) files. This allows machine and couch parameters to be varied dynamically, in real-time, during beam delivery. Custom XML files were created to allow for the collection of (1) continuous Tissue Maximum Ratios (TMRs), (2) beam profiles, and (3) continuous output factors using a 1D-scanningmore » tank. TMRs were acquired by orienting an ionization chamber (IC) at isocenter (depth=25cm) and synchronizing a depth scan towards the water surface while lowering the couch at 1mm/s. For beam profiles, the couch was driven laterally and longitudinally while logging IC electrometer readings. Output factors (OFs) where collected by continually varying field sizes (4×4 to 30×30-cm{sup 2}) at a constant speed of 6.66 mm/s. To validate measurements, comparisons were made to data collected using traditional methods (e.g. 1D or 3D tank). Results: All data collecting using the proposed methods agreed with traditionally collected data (TMRs within 1%, OFs within 0.5% and beam profile agreement within 1% / 1mm) while taking less time to collect (factor of approximately 1/10) and with a finer sample resolution. Conclusion: TrueBeam developer mode facilitates collection of continuous data with the same accuracy as traditionally collected data with a finer resolution in less time. Results demonstrate an order of magnitude increase in sampled resolution and an order of magnitude reduction in collection time compared to traditional acquisition methods (e.g. 3D scanning tank). We are currently extending this approach to perform other TG-142 tasks.« less

  2. Molecular characterization of thyroid hormone-inhibited atrial L-type calcium channel expression: implication for atrial fibrillation in hyperthyroidism.

    PubMed

    Chen, Wei-Jan; Yeh, Yung-Hsin; Lin, Kwang-Huei; Chang, Gwo-Jyh; Kuo, Chi-Tai

    2011-03-01

    Atrial fibrillation (AF) is a common complication in hyperthyroidism. Earlier studies demonstrate that thyroid hormone decreases L-type calcium channel (LCC) current expression with resultant shortening of action potential duration (APD), providing a substrate for AF. The aim of this study was to investigate the potential mechanism underlying the regulatory effect of thyroid hormone on LCC. In a hyperthyroid rat model, thyroid hormone (triiodothyronine [T3]) administration down-regulated atrial LCC expression. In vitro, treatment of murine atrial myocytes (HL-1) with T3 decreased the expression of LCC and its current, resulting in abbreviation of APD. Furthermore, T3 inhibited the activation of cyclic AMP response element (CRE)-binding protein (CREB), including phosphorylation at Ser133 and its nuclear translocation. Transient transfection studies in HL-1 cells indicated that T3 reduced LCC promoter activity. Deletion and mutation analysis of the LCC promoter region along with chromatin immunoprecipitation using anti-CREB antibody showed that CRE was essential for T3-mediated LCC gene expression. Transfection of dominant-negative CREB (mutated Ser133) and mutant thyroid hormone receptor (TR, mutated Cys51) abolished the T3-dependent effects, suggesting an association between both transcriptional factors. Co-immunoprecipitation documented an increased binding of TR with CREB after T3 treatment. The transcriptional cross-talk 3 between TR and CREB bound to CRE mediates T3-inhibited CREB activity and LCC expression. Thyroid hormone-induced TR binding of CREB inhibits CREB activity and LCC current expression, which may contribute to AF. These findings provide an important mechanistic insight into hyperthyroidism-induced AF.

  3. Melanopsin Phototransduction Contributes to Light-Evoked Choroidal Expansion and Rod L-Type Calcium Channel Function In Vivo.

    PubMed

    Berkowitz, Bruce A; Schmidt, Tiffany; Podolsky, Robert H; Roberts, Robin

    2016-10-01

    In humans, rodents, and pigeons, the dark → light transition signals nonretinal brain tissue to increase choroidal thickness, a major control element of choroidal blood flow, and thus of photoreceptor and retinal pigment epithelium function. However, it is unclear which photopigments in the retina relay the light signal to the brain. Here, we test the hypothesis that melanopsin (Opn4)-regulated phototransduction modulates light-evoked choroidal thickness expansion in mice. Two-month-old C57Bl/6 wild-type (B6), 4- to 5-month-old C57Bl/6/129S6 wild-type (B6 + S6), and 2-month-old melanopsin knockout (Opn4-/-) on a B6 + S6 background were studied. Retinal anatomy was evaluated in vivo by optical coherence tomography and MRI. Choroidal thickness in dark and light were measured by diffusion-weighted MRI. Rod cell L-type calcium channel (LTCC) function in dark and light (manganese-enhanced MRI [MEMRI]) was also measured. Opn4-/- mice did not show the light-evoked expansion of choroidal thickness observed in B6 and B6 + S6 controls. Additionally, Opn4-/- mice had lower than normal rod cell and inner retinal LTCC function in the dark but not in the light. These deficits were not due to structural abnormalities because retinal laminar architecture and thickness, and choroidal thickness in the Opn4-/- mice were similar to controls. First time evidence is provided that melanopsin phototransduction contributes to dark → light control of murine choroidal thickness. The data also highlight a contribution in vivo of melanopsin phototransduction to rod cell and inner retinal depolarization in the dark.

  4. Modeling ground thermal regime of an ancient buried ice body in Beacon Valley, Antarctica using a 1-D heat equation with latent heat effect

    NASA Astrophysics Data System (ADS)

    Liu, L.; Sletten, R. S.; Hallet, B.; Waddington, E. D.; Wood, S. E.

    2013-12-01

    An ancient massive ice body buried under several decimeters of debris in Beacon Valley, Antarctica is believed to be over one million years old, making it older than any known glacier or ice cap. It is fundamentally important as a reservoir of water, proxy for climatic information, and an expression of the periglacial landscape. It is also one of Earth's closest analog for widespread, near-surface ice found in Martian soils and ice-cored landforms. We are interested in understanding controls on how long this ice may persist since our physical model of sublimation suggests it should not be stable. In these models, the soil temperatures and the gradient are important because it determines the direction and magnitude of the vapor flux, and thus sublimation rates. To better understand the heat transfer processes and constrain the rates of processes governing ground ice stability, a model of the thermal behavior of the permafrost is applied to Beacon Valley, Antarctica. It calculates soil temperatures based on a 1-D thermal diffusion equation using a fully implicit finite volume method (FVM). This model is constrained by soil physical properties and boundary conditions of in-situ ground surface temperature measurements (with an average of -23.6oC, a maximum of 20.5oC and a minimum of -54.3oC) and ice-core temperature record at ~30 m. Model results are compared to in-situ temperature measurements at depths of 0.10 m, 0.20 m, 0.30 m, and 0.45 m to assess the model's ability to reproduce the temperature profile for given thermal properties of the debris cover and ice. The model's sensitivity to the thermal diffusivity of the permafrost and the overlaying debris is also examined. Furthermore, we incorporate the role of ice condensation/sublimation which is calculated using our vapor diffusion model in the 1-D thermal diffusion model to assess potential latent heat effects that in turn affect ground ice sublimation rates. In general, the model simulates the ground thermal

  5. BAG3 regulates contractility and Ca(2+) homeostasis in adult mouse ventricular myocytes.

    PubMed

    Feldman, Arthur M; Gordon, Jennifer; Wang, JuFang; Song, Jianliang; Zhang, Xue-Qian; Myers, Valerie D; Tilley, Douglas G; Gao, Erhe; Hoffman, Nicholas E; Tomar, Dhanendra; Madesh, Muniswamy; Rabinowitz, Joseph; Koch, Walter J; Su, Feifei; Khalili, Kamel; Cheung, Joseph Y

    2016-03-01

    Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na(+)-K(+)-ATPase and L-type Ca(2+) channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca(2+) channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca(2+)]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca(2+) current (ICa) and sarcoplasmic reticulum (SR) Ca(2+) content but not Na(+)/Ca(2+) exchange current (INaCa) or SR Ca(2+) uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyryl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD50 and APD90 were prolonged in shBAG3 myocytes. Protein levels of Ca(2+) entry molecules and other important excitation-contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca(2+) channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure. Copyright © 2016 Elsevier Ltd. All rights

  6. BAG3 regulates contractility and Ca2+ homeostasis in adult mouse ventricular myocytes

    PubMed Central

    Feldman, Arthur M.; Gordon, Jennifer; Wang, JuFang; Song, Jianliang; Zhang, Xue-Qian; Myers, Valerie D.; Tilley, Douglas G.; Gao, Erhe; Hoffman, Nicholas E.; Tomar, Dhanendra; Madesh, Muniswamy; Rabinowitz, Joseph; Koch, Walter J.; Su, Feifei; Khalili, Kamel; Cheung, Joseph Y.

    2016-01-01

    Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na+-K+-ATPase and L-type Ca2+ channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca2+ channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca2+]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca2+ current (ICa) and sarcoplasmic reticulum (SR) Ca2+ content but not Na+/Ca2+ exchange current (INaCa) or SR Ca2+ uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyrl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD50 and APD90 were prolonged in shBAG3 myocytes. Protein levels of Ca2+ entry molecules and other important excitation-contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca2+ channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure. PMID:26796036

  7. ß-Adrenoceptor Activation Enhances L-Type Calcium Channel Currents in Anterior Piriform Cortex Pyramidal Cells of Neonatal Mice: Implication for Odor Learning

    ERIC Educational Resources Information Center

    Ghosh, Abhinaba; Mukherjee, Bandhan; Chen, Xihua; Yuan, Qi

    2017-01-01

    Early odor preference learning occurs in one-week-old rodents when a novel odor is paired with a tactile stimulation mimicking maternal care. ß-Adrenoceptors and L-type calcium channels (LTCCs) in the anterior piriform cortex (aPC) are critically involved in this learning. However, whether ß-adrenoceptors interact directly with LTCCs in aPC…

  8. Epigallocatechin-3-gallate increases intracellular [Ca2+] in U87 cells mainly by influx of extracellular Ca2+ and partly by release of intracellular stores.

    PubMed

    Kim, Hee Jung; Yum, Keun Sang; Sung, Jong-Ho; Rhie, Duck-Joo; Kim, Myung-Jun; Min, Do Sik; Hahn, Sang June; Kim, Myung-Suk; Jo, Yang-Hyeok; Yoon, Shin Hee

    2004-02-01

    Green tea has been receiving considerable attention as a possible preventive agent against cancer and cardiovascular disease. Epigallocatechin-3-gallate (EGCG) is a major polyphenol component of green tea. Using digital calcium imaging and an assay for [3H]-inositol phosphates, we determined whether EGCG increases intracellular [Ca2+] ([Ca2+]i) in non-excitable human astrocytoma U87 cells. EGCG induced concentration-dependent increases in [Ca2+]i. The EGCG-induced [Ca2+]i increases were reduced to 20.9% of control by removal of extracellular Ca2+. The increases were also inhibited markedly by treatment with the non-specific Ca2+ channel inhibitors cobalt (3 mM) for 3 min and lanthanum (1 mM) for 5 min. The increases were not significantly inhibited by treatment for 10 min with the L-type Ca2+ channel blocker nifedipine (100 nM). Treatment with the inhibitor of endoplasmic reticulum Ca2+-ATPase thapsigargin (1 micro M) also significantly inhibited the EGCG-induced [Ca2+]i increases. Treatment for 15 min with the phospholipase C (PLC) inhibitor neomycin (300 micro M) attenuated the increases significantly, while the tyrosine kinase inhibitor genistein (30 micro M) had no effect. EGCG increased [3H]-inositol phosphates formation via PLC activation. Treatment for 10 min with mefenamic acid (100 micro M) and flufenamic acid (100 micro M), derivatives of diphenylamine-2-carboxylate, blocked the EGCG-induced [Ca2+]i increase in non-treated and thapsigargin-treated cells but indomethacin (100 micro M) did not affect the increases. Collectively, these data suggest that EGCG increases [Ca2+]i in non-excitable U87 cells mainly by eliciting influx of extracellular Ca2+ and partly by mobilizing intracellular Ca2+ stores by PLC activation. The EGCG-induced [Ca2+]i influx is mediated mainly through channels sensitive to diphenylamine-2-carboxylate derivatives.

  9. Lowering glucose level elevates [Ca2+]i in hypothalamic arcuate nucleus NPY neurons through P/Q-type Ca2+ channel activation and GSK3β inhibition

    PubMed Central

    Chen, Yu; Zhou, Jun; Xie, Na; Huang, Chao; Zhang, Jun-qi; Hu, Zhuang-li; Ni, Lan; Jin, You; Wang, Fang; Chen, Jian-guo; Long, Li-hong

    2012-01-01

    Aim: To identify the mechanisms underlying the elevation of intracellular Ca2+ level ([Ca2+]i) induced by lowering extracellular glucose in rat hypothalamic arcuate nucleus NPY neurons. Methods: Primary cultures of hypothalamic arcuate nucleus (ARC) neurons were prepared from Sprague-Dawley rats. NPY neurons were identified with immunocytochemical method. [Ca2+]i was measured using fura-2 AM. Ca2+ current was recorded using whole-cell patch clamp recording. AMPK and GSK3β levels were measured using Western blot assay. Results: Lowering glucose level in the medium (from 10 to 1 mmol/L) induced a transient elevation of [Ca2+]i in ARC neurons, but not in hippocampal and cortical neurons. The low-glucose induced elevation of [Ca2+]i in ARC neurons depended on extracellular Ca2+, and was blocked by P/Q-type Ca2+channel blocker ω-agatoxin TK (100 nmol/L), but not by L-type Ca2+ channel blocker nifedipine (10 μmol/L) or N-type Ca2+channel blocker ω-conotoxin GVIA (300 nmol/L). Lowering glucose level increased the peak amplitude of high voltage-activated Ca2+ current in ARC neurons. The low-glucose induced elevation of [Ca2+]i in ARC neurons was blocked by the AMPK inhibitor compound C (20 μmol/L), and enhanced by the GSK3β inhibitor LiCl (10 mmol/L). Moreover, lowering glucose level induced the phosphorylation of AMPK and GSK3β, which was inhibited by compound C (20 μmol/L). Conclusion: Lowering glucose level enhances the activity of P/Q type Ca2+channels and elevates [Ca2+]i level in hypothalamic arcuate nucleus neurons via inhibition of GSK3β. PMID:22504905

  10. Sarcoplasmic reticulum Ca(2+) atpase (SERCA) 1a structurally substitutes for SERCA2a in the cardiac sarcoplasmic reticulum and increases cardiac Ca(2+) handling capacity.

    PubMed

    Lalli, M J; Yong, J; Prasad, V; Hashimoto, K; Plank, D; Babu, G J; Kirkpatrick, D; Walsh, R A; Sussman, M; Yatani, A; Marbán, E; Periasamy, M

    2001-07-20

    Ectopic expression of the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca(2+) transients; however, the cellular mechanisms underlying altered Ca(2+) handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca(2+) load measurements revealed that TG myocytes have significantly enhanced SR Ca(2+) load. Confocal line-scan images of field-stimulated SR Ca(2+) release showed an increased rate of Ca(2+) removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by approximately 30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca(2+) current density was reduced by approximately 50%, whereas the time course of inactivation was unchanged in TG myocytes. These studies provide important evidence that SERCA1a can substitute both structurally and functionally for SERCA2a in the heart and that SERCA1a overexpression can be used to enhance SR Ca(2+) transport and cardiac contractility.

  11. The Ca2+ leak paradox and “rogue ryanodine receptors”: SR Ca2+ efflux theory and practice

    PubMed Central

    Sobie, Eric A.; Guatimosim, Silvia; Gómez-Viquez, Leticia; Song, Long-Sheng; Hartmann, Hali; Jafri, M. Saleet; Lederer, W.J.

    2006-01-01

    Ca2+ efflux from the sarcoplasmic reticulum (SR) is routed primarily through SR Ca2+ release channels (ryanodine receptors, RyRs). When clusters of RyRs are activated by trigger Ca2+ influx through L-type Ca2+ channels (dihydropyridine receptors, DHPR), Ca2+ sparks are observed. Close spatial coupling between DHPRs and RyR clusters and the relative insensitivity of RyRs to be triggered by Ca2+ together ensure the stability of this positive-feedback system of Ca2+ amplification. Despite evidence from single channel RyR gating experiments that phosphorylation of RyRs by protein kinase A (PKA) or calcium-calmodulin dependent protein kinase II (CAMK II) causes an increase in the sensitivity of the RyR to be triggered by [Ca2+]i there is little clear evidence to date showing an increase in Ca2+ spark rate. Indeed, there is some evidence that the SR Ca2+ content may be decreased in hyperadrenergic disease states. The question is whether or not these observations are compatible with each other and with the development of arrhythmogenic extrasystoles that can occur under these conditions. Furthermore, the appearance of an increase in the SR Ca2+ “leak” under these conditions is perplexing. These and related complexities are analyzed and discussed in this report. Using simple mathematical modeling discussed in the context of recent experimental findings, a possible resolution to this paradox is proposed. The resolution depends upon two features of SR function that have not been confirmed directly but are broadly consistent with several lines of indirect evidence: (1) the existence of unclustered or “rogue” RyRs that may respond differently to local [Ca2+]i in diastole and during the [Ca2+]i transient; and (2) a decrease in cooperative or coupled gating between clustered RyRs in response to physiologic phosphorylation or hyperphosphorylation of RyRs in disease states such as heart failure. Taken together, these two features may provide a framework that allows for an

  12. The Ca2+/Calmodulin/CaMKK2 Axis: Nature’s Metabolic CaMshaft

    PubMed Central

    Marcelo, Kathrina L.; Means, Anthony R.; York, Brian

    2016-01-01

    Calcium (Ca2+) is an essential ligand that binds its primary intracellular receptor Calmodulin (CaM) to trigger a variety of downstream processes and pathways. Central to the actions of Ca2+/CaM is the activation of a highly conserved Ca2+/CaM kinase (CaMK) cascade, which amplifies Ca2+ signals through a series of subsequent phosphorylation events. Proper regulation of Ca2+ flux is necessary for whole-body metabolism and disruption of Ca2+ homeostasis has been linked to a variety of metabolic diseases. Herein, we provide a synthesis of recent advances that highlight the roles of the Ca2+/CaM kinase axis in key metabolic tissues. An appreciation of this information is critical in order to understand the mechanisms by which Ca2+/CaM-dependent signaling contributes to metabolic homeostasis and disease. PMID:27449752

  13. Carbachol-induced long-term synaptic depression is enhanced during senescence at hippocampal CA3-CA1 synapses.

    PubMed

    Kumar, Ashok

    2010-08-01

    Dysregulation of the cholinergic transmitter system is a hallmark of Alzheimer's disease and contributes to an age-associated decline in memory performance. The current study examined the influence of carbachol, a cholinergic receptor agonist, on synaptic transmission over the course of aging. Extracellular excitatory postsynaptic field potentials were recorded from CA3-CA1 synapses in acute hippocampal slices obtained from young adult (5-8 mo) and aged (22-24 mo) male Fischer 344 rats. Bath application of carbachol elicited a transient depression of synaptic transmission, which was followed by a long-lasting depression (CCh-LTD) observed 90 min after carbachol cessation in both age groups. However, the magnitude of CCh-LTD was significantly larger in senescent animals and was attenuated by N-methyl-D-aspartate receptor blockade in aged animals. Blockade of L-type Ca(2+) channels inhibited CCh-LTD to a greater extent in aged animals compared to young adults. Finally, the expression of CCh-LTD was dependent on protein synthesis. The results indicate that altered Ca(2+) homeostasis or muscarinic activation of Ca(2+) signaling contribute to the enhanced CCh-LTD during senescence.

  14. Parallel activation of Ca(2+)-induced survival and death pathways in cardiomyocytes by sorbitol-induced hyperosmotic stress.

    PubMed

    Chiong, M; Parra, V; Eisner, V; Ibarra, C; Maldonado, C; Criollo, A; Bravo, R; Quiroga, C; Contreras, A; Vicencio, J M; Cea, P; Bucarey, J L; Molgó, J; Jaimovich, E; Hidalgo, C; Kroemer, G; Lavandero, S

    2010-08-01

    Hyperosmotic stress promotes rapid and pronounced apoptosis in cultured cardiomyocytes. Here, we investigated if Ca(2+) signals contribute to this response. Exposure of cardiomyocytes to sorbitol [600 mosmol (kg water)(-1)] elicited large and oscillatory intracellular Ca(2+) concentration increases. These Ca(2+) signals were inhibited by nifedipine, Cd(2+), U73122, xestospongin C and ryanodine, suggesting contributions from both Ca(2+) influx through voltage dependent L-type Ca(2+) channels plus Ca(2+) release from intracellular stores mediated by IP(3) receptors and ryanodine receptors. Hyperosmotic stress also increased mitochondrial Ca(2+) levels, promoted mitochondrial depolarization, reduced intracellular ATP content, and activated the transcriptional factor cyclic AMP responsive element binding protein (CREB), determined by increased CREB phosphorylation and electrophoretic mobility shift assays. Incubation with 1 mM EGTA to decrease extracellular [Ca(2+)] prevented cardiomyocyte apoptosis induced by hyperosmotic stress, while overexpression of an adenoviral dominant negative form of CREB abolished the cardioprotection provided by 1 mM EGTA. These results suggest that hyperosmotic stress induced by sorbitol, by increasing Ca(2+) influx and raising intracellular Ca(2+) concentration, activates Ca(2+) release from stores and causes cell death through mitochondrial function collapse. In addition, the present results suggest that the Ca(2+) increase induced by hyperosmotic stress promotes cell survival by recruiting CREB-mediated signaling. Thus, the fate of cardiomyocytes under hyperosmotic stress will depend on the balance between Ca(2+)-induced survival and death pathways.

  15. Ryanodine receptors regulate arterial diameter and wall [Ca2+] in cerebral arteries of rat via Ca2+-dependent K+ channels

    PubMed Central

    Knot, Harm J; Standen, Nicholas B; Nelson, Mark T

    1998-01-01

    The effects of inhibitors of ryanodine-sensitive calcium release (RyR) channels in the sarcoplasmic reticulum (SR) and Ca2+-dependent potassium (KCa) channels on the membrane potential, intracellular [Ca2+], and diameters of small pressurized (60 mmHg) cerebral arteries (100–200 μm) were studied using digital fluorescence video imaging of arterial diameter and wall [Ca2+], combined with microelectrode measurements of arterial membrane potential. Ryanodine (10 μm), an inhibitor of RyR channels, depolarized by 9 mV, increased intracellular [Ca2+] by 46 nm and constricted pressurized (to 60 mmHg) arteries with myogenic tone by 44 μm (∼22 %). Iberiotoxin (100 nm), a blocker of KCa channels, under the same conditions, depolarized the arteries by 10 mV, increased arterial wall calcium by 51 nm, and constricted by 37 μm (∼19 %). The effects of ryanodine and iberiotoxin were not additive and were blocked by inhibitors of voltage-dependent Ca2+ channels. Caffeine (10 mm), an activator of RyR channels, transiently increased arterial wall [Ca2+] by 136 ± 9 nm in control arteries and by 158 ± 12 nm in the presence of iberiotoxin. Caffeine was relatively ineffective in the presence of ryanodine, increasing [calcium] by 18 ± 5 nm. In the presence of blockers of voltage-dependent Ca2+ channels (nimodipine, diltiazem), ryanodine and inhibitors of the SR calcium ATPase (thapsigargin, cyclopiazonic acid) were without effect on arterial wall [Ca2+] and diameter. These results suggest that local Ca2+ release originating from RyR channels (Ca2+ sparks) in the SR of arterial smooth muscle regulates myogenic tone in cerebral arteries solely through activation of KCa channels, which regulate membrane potential through tonic hyperpolarization, thus limiting Ca2+ entry through L-type voltage-dependent Ca2+ channels. KCa channels therefore act as a negative feedback control element regulating arterial diameter through a reduction in global intracellular free [Ca2+]. PMID:9490841

  16. Carbachol induces burst firing of dopamine cells in the ventral tegmental area by promoting calcium entry through L-type channels in the rat

    PubMed Central

    Zhang, Lei; Liu, Yudan; Chen, Xihua

    2005-01-01

    Enhanced activity of the central dopamine system has been implicated in many psychiatric disorders including schizophrenia and addiction. Besides terminal mechanisms that boost dopamine levels at the synapse, the cell body of dopamine cells enhances terminal dopamine concentration through encoding action potentials in bursts. This paper presents evidence that burst firing of dopamine cells in the ventral tegmental area was under cholinergic control using nystatin-perforated patch clamp recording from slice preparations. The non-selective cholinergic agonist carbachol excited the majority of recorded neurones, an action that was not affected by blocking glutamate and GABA ionotropic receptors. Twenty per cent of dopamine cells responded to carbachol with robust bursting, an effect mediated by both muscarinic and nicotinic cholinoceptors postsynaptically. Burst firing induced as such was completely dependent on calcium entry as it could be blocked by cadmium and more specifically the L-type blocker nifedipine. In the presence of the sodium channel blocker tetrodotoxin, carbachol induced membrane potential oscillation that had similar kinetics and frequency as burst firing cycles and could also be blocked by cadmium and nifedipine. Direct activation of the L-type channel with Bay K8644 induced strong bursting which could be blocked by nifedipine but not by depleting internal calcium stores. These results indicate that carbachol increases calcium entry into the postsynaptic cell through L-type channels to generate calcium-dependent membrane potential oscillation and burst firing. This could establish the L-type channel as a target for modulating the function of the central dopamine system in disease conditions. PMID:16081481

  17. CAED Document Repository

    EPA Pesticide Factsheets

    Compliance Assurance and Enforcement Division Document Repository (CAEDDOCRESP) provides internal and external access of Inspection Records, Enforcement Actions, and National Environmental Protection Act (NEPA) documents to all CAED staff. The respository will also include supporting documents, images, etc.

  18. CA-125 blood test

    MedlinePlus

    ... above 35 U/mL is considered abnormal. Normal value ranges may vary slightly among different laboratories. Some ... 125 usually does not mean ovarian cancer is present. Most healthy women with an elevated CA-125 ...

  19. Voltage-dependent Ca2+ release from the SR of feline ventricular myocytes is explained by Ca2+-induced Ca2+ release.

    PubMed

    Piacentino, V; Dipla, K; Gaughan, J P; Houser, S R

    2000-03-15

    1. Direct voltage-gated (voltage-dependent Ca2+ release, VDCR) and Ca2+ influx-gated (Ca2+-induced Ca2+ release, CICR) sarcoplasmic reticulum (SR) Ca2+ release were studied in feline ventricular myocytes. The voltage-contraction relationship predicted by the VDCR hypothesis is sigmoidal with large contractions at potentials near the Ca2+ equilibrium potential (ECa). The relationship predicted by the CICR hypothesis is bell-shaped with no contraction at ECa. 2. The voltage dependence of contraction was measured in ventricular myocytes at physiological temperature (37 C), resting membrane potential and physiological [K+]. Experiments were performed with cyclic adenosine 3',5'-monophosphate (cAMP) in the pipette or in the presence of the beta-adrenergic agonist isoproterenol (isoprenaline; ISO). 3. The voltage-contraction relationship was bell-shaped in Na+-free solutions (to eliminate the Na+ current and Na+-Ca2+ exchange, NCX) but the relationship was broader than the L-type Ca2+ current (ICa,L)-voltage relationship. 4. Contractions induced with voltage steps from normal resting potentials to -40 mV are thought to represent VDCR rather than CICR. We found that cAMP and ISO shifted the voltage dependence of ICa,L activation to more negative potentials so that ICa,L was always present with steps to -40 mV. ICa,L at -40 mV inactivated when the holding potential was decreased (VŁ = -57.8 +/- 0.49 mV). 5. ISO increased inward current, SR Ca2+ load and contraction in physiological [Na+] and a broad bell-shaped voltage-contraction relationship was observed. Inhibition of reverse-mode NCX, decreasing ICa,L and decreasing SR Ca2+ loading all decreased contractions at strongly positive potentials near ECa. 6. The voltage-contraction relationship in 200 microM cadmium (Cd2+) was bell-shaped, supporting a role of ICa,L rather than VDCR. 7. All results could be accounted for by the CICR hypothesis, and many results exclude the VDCR hypothesis.

  20. Voltage-dependent Ca2+ release from the SR of feline ventricular myocytes is explained by Ca2+-induced Ca2+ release

    PubMed Central

    Piacentino, Valentino; Dipla, Konstantina; Gaughan, John P; Houser, Steven R

    2000-01-01

    Direct voltage-gated (voltage-dependent Ca2+ release, VDCR) and Ca2+ influx-gated (Ca2+-induced Ca2+ release, CICR) sarcoplasmic reticulum (SR) Ca2+ release were studied in feline ventricular myocytes. The voltage-contraction relationship predicted by the VDCR hypothesis is sigmoidal with large contractions at potentials near the Ca2+ equilibrium potential (ECa). The relationship predicted by the CICR hypothesis is bell-shaped with no contraction at ECa. The voltage dependence of contraction was measured in ventricular myocytes at physiological temperature (37 °C), resting membrane potential and physiological [K+]. Experiments were performed with cyclic adenosine 3′,5′-monophosphate (cAMP) in the pipette or in the presence of the β-adrenergic agonist isoproterenol (isoprenaline; ISO). The voltage-contraction relationship was bell-shaped in Na+-free solutions (to eliminate the Na+ current and Na+-Ca2+ exchange, NCX) but the relationship was broader than the L-type Ca2+ current (ICa,L)-voltage relationship. Contractions induced with voltage steps from normal resting potentials to -40 mV are thought to represent VDCR rather than CICR. We found that cAMP and ISO shifted the voltage dependence of ICa,L activation to more negative potentials so that ICa,L was always present with steps to -40 mV. ICa,L at -40 mV inactivated when the holding potential was decreased (V½ =−57·8 ± 0·49 mV). ISO increased inward current, SR Ca2+ load and contraction in physiological [Na+] and a broad bell-shaped voltage-contraction relationship was observed. Inhibition of reverse-mode NCX, decreasing ICa,L and decreasing SR Ca2+ loading all decreased contractions at strongly positive potentials near ECa. The voltage-contraction relationship in 200 μM cadmium (Cd2+) was bell-shaped, supporting a role of ICa,L rather than VDCR. All results could be accounted for by the CICR hypothesis, and many results exclude the VDCR hypothesis. PMID:10718736

  1. Effect of [6]-shogaol on cytosolic Ca2+ levels and proliferation in human oral cancer cells (OC2).

    PubMed

    Chen, Chung-Yi; Yang, Yu-Han; Kuo, Soong-Yu

    2010-08-27

    The effect of [6]-shogaol (1) on cytosolic free Ca(2+) concentrations ([Ca(2+)](i)) and viability has not been explored previously in oral epithelial cells. The present study has examined whether 1 alters [Ca(2+)](i) and viability in OC2 human oral cancer cells. Compound 1 at concentrations > or = 5 microM increased [Ca(2+)](i) in a concentration-dependent manner with a 50% effective concentration (EC(50)) value of 65 microM. The Ca(2+) signal was reduced substantially by removing extracellular Ca(2+). In a Ca(2+)-free medium, the 1-induced [Ca(2+)](i) elevation was mostly attenuated by depleting stored Ca(2+) with thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor). The [Ca(2+)](i) signal was inhibited by La(3+) but not by L-type Ca(2+) channel blockers. The elevation of [Ca(2+)](i) caused by 1 in a Ca(2+)-containing medium was not affected by modulation of protein kinase C activity, but was inhibited by 82% with the phospholipase A2 inhibitor aristolochic acid I (20 microM). U73122, a selective inhibitor of phospholipase C, abolished 1-induced [Ca(2+)](i) release. At concentrations of 5-100 microM, 1 killed cells in a concentration-dependent manner. These findings suggest that [6]-shogaol induces a significant rise in [Ca(2+)](i) in oral cancer OC2 cells by causing stored Ca(2+) release from the thapsigargin-sensitive endoplasmic reticulum pool in an inositol 1,4,5-trisphosphate-dependent manner and by inducing Ca(2+) influx via a phospholipase A2- and La(3+)-sensitive pathway.

  2. Ca(2+) influx and neurotransmitter release at ribbon synapses.

    PubMed

    Cho, Soyoun; von Gersdorff, Henrique

    2012-01-01

    Ca(2+) influx through voltage-gated Ca(2+) channels triggers the release of neurotransmitters at presynaptic terminals. Some sensory receptor cells in the peripheral auditory and visual systems have specialized synapses that express an electron-dense organelle called a synaptic ribbon. Like conventional synapses, ribbon synapses exhibit SNARE-mediated exocytosis, clathrin-mediated endocytosis, and short-term plasticity. However, unlike non-ribbon synapses, voltage-gated L-type Ca(2+) channel opening at ribbon synapses triggers a form of multiquantal release that can be highly synchronous. Furthermore, ribbon synapses appear to be specialized for fast and high throughput exocytosis controlled by graded membrane potential changes. Here we will discuss some of the basic aspects of synaptic transmission at different types of ribbon synapses, and we will emphasize recent evidence that auditory and retinal ribbon synapses have marked differences. This will lead us to suggest that ribbon synapses are specialized for particular operating ranges and frequencies of stimulation. We propose that different types of ribbon synapses transfer diverse rates of sensory information by expressing a particular repertoire of critical components, and by placing them at precise and strategic locations, so that a continuous supply of primed vesicles and Ca(2+) influx leads to fast, accurate, and ongoing exocytosis. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Excitation-contraction coupling in zebrafish ventricular myocardium is regulated by trans-sarcolemmal Ca2+ influx and sarcoplasmic reticulum Ca2+ release.

    PubMed

    Haustein, Moritz; Hannes, Tobias; Trieschmann, Jan; Verhaegh, Rabea; Köster, Annette; Hescheler, Jürgen; Brockmeier, Konrad; Adelmann, Roland; Khalil, Markus

    2015-01-01

    Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca(2+)-flux and sarcoplasmic reticulum Ca(2+)-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca(2+)-concentration, trans-sarcolemmal Ca(2+)-flux via L-type Ca(2+)-channels and Na(+)-Ca(2+)-exchanger, and Ca(2+)-release from the sarcoplasmic reticulum as well as beating frequency and β-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca(2+)-channels by verapamil (1 μM) decreased force of contraction to 22 ± 7% compared to baseline (n=4, p<0.05). Ni(2+) was the only substance to prolong relaxation (5 mM, time after peak to 50% relaxation: 73 ± 3 ms vs. 101 ± 8 ms, n=5, p<0.05). Surprisingly though, inhibition of the sarcoplasmic Ca(2+)-release decreased force development to 54 ± 3% in ventricular (n=13, p<0.05) and to 52 ± 8% in atrial myocardium (n=5, p<0.05) suggesting a substantial role of SR Ca(2+)-release in force generation. In line with this finding, we observed significant post pause potentiation after pauses of 5 s (169 ± 7% force compared to baseline, n=8, p<0.05) and 10 s (198 ± 9% force compared to baseline, n=5, p<0.05) and mildly positive lusitropy after β-adrenergic stimulation. In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca(2+)-cycling. In contrast to

  4. Excitation-Contraction Coupling in Zebrafish Ventricular Myocardium Is Regulated by Trans-Sarcolemmal Ca2+ Influx and Sarcoplasmic Reticulum Ca2+ Release

    PubMed Central

    Trieschmann, Jan; Verhaegh, Rabea; Köster, Annette; Hescheler, Jürgen; Brockmeier, Konrad; Adelmann, Roland; Khalil, Markus

    2015-01-01

    Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca2+-flux and sarcoplasmic reticulum Ca2+-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca2+-concentration, trans-sarcolemmal Ca2+-flux via L-type Ca2+-channels and Na+-Ca2+-exchanger, and Ca2+-release from the sarcoplasmic reticulum as well as beating frequency and β-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca2+-channels by verapamil (1 μM) decreased force of contraction to 22±7% compared to baseline (n=4, p<0.05). Ni2+ was the only substance to prolong relaxation (5 mM, time after peak to 50% relaxation: 73±3 ms vs. 101±8 ms, n=5, p<0.05). Surprisingly though, inhibition of the sarcoplasmic Ca2+-release decreased force development to 54±3% in ventricular (n=13, p<0.05) and to 52±8% in atrial myocardium (n=5, p<0.05) suggesting a substantial role of SR Ca2+-release in force generation. In line with this finding, we observed significant post pause potentiation after pauses of 5 s (169±7% force compared to baseline, n=8, p<0.05) and 10 s (198±9% force compared to baseline, n=5, p<0.05) and mildly positive lusitropy after β-adrenergic stimulation. In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca2+-cycling. In contrast to mammals, FFR is strongly negative in the

  5. Memory circuits: CA2.

    PubMed

    Piskorowski, Rebecca A; Chevaleyre, Vivien

    2018-04-26

    The hippocampus is a central region in the coding of spatial, temporal and episodic memory. Recent discoveries have revealed surprising and complex roles of the small area CA2 in hippocampal function. Lesion studies have revealed that this region is required for social memory formation. Area CA2 is targeted by extra-hippocampal paraventricular inputs that release vasopressin and can act to enhance social memory performance. In vivo recordings have revealed nonconventional activity by neurons in this region that act to both initiate hippocampal sharp-wave ripple events as well as encode spatial information during immobility. Silencing of CA2 pyramidal neurons has revealed that this area also acts to control hippocampal network excitability during encoding, and this balance of excitation and inhibition is disrupted in disease. This review summarizes recent findings and attempts to integrate these results into pre-existing models. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. Parathyroid hormone enhances fluid shear-induced [Ca2+]i signaling in osteoblastic cells through activation of mechanosensitive and voltage-sensitive Ca2+ channels

    NASA Technical Reports Server (NTRS)

    Ryder, K. D.; Duncan, R. L.

    2001-01-01

    Osteoblasts respond to both fluid shear and parathyroid hormone (PTH) with a rapid increase in intracellular calcium concentration ([Ca2+]i). Because both stimuli modulate the kinetics of the mechanosensitive cation channel (MSCC), we postulated PTH would enhance the [Ca2+]i response to fluid shear by increasing the sensitivity of MSCCs. After a 3-minute preflow at 1 dyne/cm2, MC3T3-E1 cells were subjected to various levels of shear and changes in [Ca2+]i were assessed using Fura-2. Pretreatment with 50 nM bovine PTH(1-34) [bPTH(1-34)] significantly enhanced the shear magnitude-dependent increase in [Ca2+]i. Gadolinium (Gd3+), an MSCC blocker, significantly inhibited the mean peak [Ca2+]i response to shear and shear + bPTH(1-34). Nifedipine (Nif), an L-type voltage-sensitive Ca2+ channel (VSCC) blocker, also significantly reduced the [Ca2+]i response to shear + bPTH(1-34), but not to shear alone, suggesting VSCC activation plays an interactive role in the action of these stimuli together. Activation of either the protein kinase C (PKC) or protein kinase A (PKA) pathways with specific agonists indicated that PKC activation did not alter the Ca2+ response to shear, whereas PKA activation significantly increased the [Ca2+]i response to lower magnitudes of shear. bPTH(1-34), which activates both pathways, induced the greatest [Ca2+]i response at each level of shear, suggesting an interaction of these pathways in this response. These data indicate that PTH significantly enhances the [Ca2+]i response to shear primarily via PKA modulation of the MSCC and VSCC.

  7. A toxin fraction (FTX) from the funnel-web spider poison inhibits dihydropyridine-insensitive Ca2+ channels coupled to catecholamine release in bovine adrenal chromaffin cells.

    PubMed

    Duarte, C B; Rosario, L M; Sena, C M; Carvalho, A P

    1993-03-01

    In adrenal chromaffin cells, depolarization-evoked Ca2+ influx and catecholamine release are partially blocked by blockers of L-type voltage-sensitive Ca2+ channels. We have now evaluated the sensitivity of the dihydropyridine-resistant components of Ca2+ influx and catecholamine release to a toxin fraction (FTX) from the funnel-web spider poison, which is known to block P-type channels in mammalian neurons. FTX (1:4,000 dilution, with respect to the original fraction) inhibited K(+)-depolarization-induced Ca2+ influx by 50%, as monitored with fura-2, whereas nitrendipine (0.1-1 microM) and FTX (3:3), a synthetic FTX analogue (1 mM), blocked the [Ca2+]i transients by 35 and 30%, respectively. When tested together, FTX and nitrendipine reduced the [Ca2+]i transients by 70%. FTX or nitrendipine reduced adrenaline and noradrenaline release by approximately 80 and 70%, respectively, but both substances together abolished the K(+)-evoked catecholamine release, as measured by HPLC. The omega-conotoxin GVIA (0.5 microM) was without effect on K(+)-stimulated 45Ca2+ uptake. Our results indicate that FTX blocks dihydropyridine- and omega-conotoxin-insensitive Ca2+ channels that, together with L-type voltage-sensitive Ca2+ channels, are coupled to catecholamine release.

  8. ca_50mwind

    Science.gov Websites

    Publication_Date: January, 2003 Title: ca_50mwind Geospatial_Data_Presentation_Form: vector digital data and historical weather data under contract to Wind Powering America/NREL. This map has been validated with available surface data by NREL and wind energy meteorological consultants. Description: Abstract

  9. Ca2+ signaling and emesis: Recent progress and new perspectives.

    PubMed

    Zhong, Weixia; Picca, Andrew J; Lee, Albert S; Darmani, Nissar A

    2017-01-01

    Cisplatin-like chemotherapeutics cause vomiting via calcium (Ca 2+ )-dependent release of multiple neurotransmitters (dopamine, serotonin, substance P, etc.) from the gastrointestinal enterochromaffin cells and/or the brainstem. Intracellular Ca 2+ signaling is triggered by activation of diverse emetic receptors (including tachykininergic NK 1 , serotonergic 5-HT 3 , dopaminergic D 2 , cholinergic M 1 , or histaminergic H 1 ) , whose activation in vomit-competent species can evoke emesis. Other emetogens such as cisplatin, rotavirus NSP4 protein and bacterial toxins can also induce intracellular Ca 2+ elevation. Netupitant is a highly selective neurokinin NK 1 receptor (NK 1 R) antagonist and palonosetron is a selective second-generation serotonin 5-HT 3 receptor (5-HT 3 R) antagonist with a distinct pharmacological profile. An oral fixed combination of netupitant/palonosetron (NEPA; Akynzeo(®)) with >85% antiemetic efficacy is available for use in the prevention of acute and delayed chemotherapy-induced nausea and vomiting (CINV). Cannabinoid CB 1 receptor agonists possess broad-spectrum antiemetic activity since they prevent vomiting caused by a variety of emetic stimuli including the chemotherapeutic agent cisplatin, 5-HT 3 R agonists, and D 2 R agonists. Our findings demonstrate that application of the L-type Ca 2+ channel (LTCC) agonist FPL 64176 and the intracellular Ca 2+ mobilizing agent thapsigargin (a sarco/endoplasmic reticulum Ca 2+ -ATPase inhibitor) cause vomiting in the least shrew. On the other hand, blockade of LTCCs by corresponding antagonists (nifedipine or amlodipine) not only provide broad-spectrum antiemetic efficacy against diverse agents that specifically activate emetogenic receptors such as 5-HT 3 , NK 1 , D 2 , and M 1 receptors, but can also potentiate the antiemetic efficacy of palonosetron against the non-specific emetogen, cisplatin. In this review, we will provide an overview of Ca 2+ involvement in the emetic process; discuss the

  10. Effects of funnel web spider toxin on Ca2+ currents in neurohypophysial terminals.

    PubMed

    Wang, G; Lemos, J R

    1994-11-14

    Funnel web spider toxin (FTX) is reportedly a specific blocker of P-type Ca2+ channels. The effects of FTX on the Ca2+ currents of isolated neurohypophysial nerve terminals of the rat were investigated using the 'whole-cell' patch-clamp technique. Both the transient and long-lasting Ca2+ current components were maximally elicited by depolarization from a holding potential equal to the normal terminal resting potential (-90 mV). Externally applied FTX inhibited the high-voltage-threshold, transient component of the Ca2+ current in a concentration-dependent manner, with a half-maximal inhibition at a dilution of approximately 1:10000. FTX also shifted the peak current of the I-V relationship by +10 mV. The long-lasting Ca2+ current component, which is sensitive to L-type Ca2+ channel blockers, was insensitive to FTX. The transient current, which is sensitive to omega-conotoxin GVIA, was completely blocked by FTX. These results suggest that there could be a novel, inactivating Ca2+ channel in the rat neurohypophysial terminals which is affected by both N-type and P-type Ca2+ channel blockers.

  11. Phosphorylation sites in the Hook domain of CaVβ subunits differentially modulate CaV1.2 channel function.

    PubMed

    Brunet, Sylvain; Emrick, Michelle A; Sadilek, Martin; Scheuer, Todd; Catterall, William A

    2015-10-01

    Regulation of L-type calcium current is critical for the development, function, and regulation of many cell types. Ca(V)1.2 channels that conduct L-type calcium currents are regulated by many protein kinases, but the sites of action of these kinases remain unknown in most cases. We combined mass spectrometry (LC-MS/MS) and whole-cell patch clamp techniques in order to identify sites of phosphorylation of Ca(V)β subunits in vivo and test the impact of mutations of those sites on Ca(V)1.2 channel function in vitro. Using the Ca(V)1.1 channel purified from rabbit skeletal muscle as a substrate for phosphoproteomic analysis, we found that Ser(193) and Thr(205) in the HOOK domain of Ca(V)β1a subunits were both phosphorylated in vivo. Ser(193) is located in a potential consensus sequence for casein kinase II, but it was not phosphorylated in vitro by that kinase. In contrast, Thr(205) is located in a consensus sequence for cAMP-dependent phosphorylation, and it was robustly phosphorylated in vitro by PKA. These two sites are conserved in multiple Ca(V)β subunit isoforms, including the principal Ca(V)β subunit of cardiac Ca(V)1.2 channels, Ca(V)β2b. In order to assess potential modulatory effects of phosphorylation at these sites separately from the effects of phosphorylation of the α11.2 subunit, we inserted phosphomimetic or phosphoinhibitory mutations in Ca(V)β2b and analyzed their effects on Ca(V)1.2 channel function in transfected nonmuscle cells. The phosphomimetic mutation Ca(V)β2b(S152E) decreased peak channel currents and shifted the voltage dependence of both activation and inactivation to more positive membrane potentials. The phosphoinhibitory mutation Ca(V)β2b(S152A) had opposite effects. There were no differences in peak Ca(V)1.2 currents or voltage dependence between the phosphomimetic mutation Ca(V)β2b(T164D) and the phosphoinhibitory mutation Ca(V)β2b(T164A). However, calcium-dependent inactivation was significantly increased for the

  12. Model study of ATP and ADP buffering, transport of Ca(2+) and Mg(2+), and regulation of ion pumps in ventricular myocyte

    NASA Technical Reports Server (NTRS)

    Michailova, A.; McCulloch, A.

    2001-01-01

    We extended the model of the ventricular myocyte by Winslow et al. (Circ. Res 1999, 84:571-586) by incorporating equations for Ca(2+) and Mg(2+) buffering and transport by ATP and ADP and equations for MgATP regulation of ion transporters (Na(+)-K(+) pump, sarcolemmal and sarcoplasmic Ca(2+) pumps). The results indicate that, under normal conditions, Ca(2+) binding by low-affinity ATP and diffusion of CaATP may affect the amplitude and time course of intracellular Ca(2+) signals. The model also suggests that a fall in ATP/ADP ratio significantly reduces sarcoplasmic Ca(2+) content, increases diastolic Ca(2+), lowers systolic Ca(2+), increases Ca(2+) influx through L-type channels, and decreases the efficiency of the Na(+)/Ca(2+) exchanger in extruding Ca(2+) during periodic voltage-clamp stimulation. The analysis suggests that the most important reason for these changes during metabolic inhibition is the down-regulation of the sarcoplasmic Ca(2+)-ATPase pump by reduced diastolic MgATP levels. High Ca(2+) concentrations developed near the membrane might have a greater influence on Mg(2+), ATP, and ADP concentrations than that of the lower Ca(2+) concentrations in the bulk myoplasm. The model predictions are in general agreement with experimental observations measured under normal and pathological conditions.

  13. Ca2+ regulatory mechanisms of exercise protection against coronary artery disease in metabolic syndrome and diabetes.

    PubMed

    Sturek, Michael

    2011-08-01

    Chronic exercise attenuates coronary artery disease (CAD) in humans largely independent of reductions in risk factors; thus major protective mechanisms of exercise are directly within the coronary vasculature. Further, tight control of diabetes, e.g., blood glucose, can be detrimental. Accordingly, knowledge of mechanisms by which exercise attenuates diabetic CAD could catalyze development of molecular therapies. Exercise attenuates CAD (atherosclerosis) and restenosis in miniature swine models, which enable precise control of exercise parameters (intensity, duration, and frequency) and characterization of the metabolic syndrome (MetS) and diabetic milieu. Intracellular Ca(2+) is a pivotal second messenger for coronary smooth muscle (CSM) excitation-contraction and excitation-transcription coupling that modulates CSM proliferation, migration, and calcification. CSM of diabetic dyslipidemic Yucatan swine have impaired Ca(2+) extrusion via the plasmalemma Ca(2+) ATPase (PMCA), downregulation of L-type voltage-gated Ca(2+) channels (VGCC), increased Ca(2+) sequestration by the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA), increased nuclear Ca(2+) localization, and greater activation of K channels by Ca(2+) release from the SR. Endurance exercise training prevents Ca(2+) transport changes with virtually no effect on the diabetic milieu (glucose, lipids). In MetS Ossabaw swine transient receptor potential canonical (TRPC) channels are upregulated and exercise training reverses expression and TRPC-mediated Ca(2+) influx with almost no change in the MetS milieu. Overall, exercise effects on Ca(2+) signaling modulate CSM phenotype. Future studies should 1) selectively target key Ca(2+) transporters to determine definitively their causal role in atherosclerosis and 2) combine mechanistic studies with clinical outcomes, e.g., reduction of myocardial infarction.

  14. Ca currents activated by spontaneous firing and synaptic disinhibition in neurons of the cerebellar nuclei

    PubMed Central

    Zheng, Nan; Raman, Indira M.

    2009-01-01

    In neurons of the cerebellar nuclei, long-term potentiation of EPSCs is induced by high-frequency synaptic excitation by mossy fibers followed by synaptic inhibition by Purkinje cells. Induction requires activation of synaptic receptors as well as voltage-gated Ca channels. To examine how Purkinje-mediated inhibition of nuclear neurons affects Ca levels during plasticity-inducing stimuli, we have combined electrophysiology, Ca imaging, and pharmacology of cerebellar nuclear neurons in mouse cerebellar slices. We find that spontaneous firing generates tonic Ca signals in both somata and dendrites, which drop during 500-ms, 100-Hz trains of Purkinje IPSPs or hyperpolarizing steps. Although the presence of low-voltage-activated (T-type) Ca channels in nuclear neurons has fostered the inference that disinhibition activates these channels, synaptic inhibition with a physiological ECl (−75 mV) fails to hyperpolarize neurons sufficiently for T-type channels to recover substantially. Consequently, after IPSPs, Ca signals return to baseline, although firing is accelerated by ∼20 Hz for ∼300 ms. Only after hyperpolarizations beyond ECl does Ca rise gradually beyond baseline, as firing further exceeds spontaneous rates. Cd2+ (100 μM), which nearly eliminates L-type, N-type, P/Q-type, and R-type Ca currents while sparing about half the T-type current, prevents Ca changes during and after hyperpolarizations to ECl. Thus, high-frequency IPSPs in cerebellar nuclear neurons evoke little post-inhibitory current through T-type channels. Instead, inhibition regulates Ca levels simply by preventing action potentials, which usually permit Ca influx through high-voltage-activated channels. The decreases and restoration of Ca levels associated with Purkinje-mediated inhibition are likely to contribute to synaptic plasticity. PMID:19657035

  15. Protective effects of efonidipine, a T- and L-type calcium channel blocker, on renal function and arterial stiffness in type 2 diabetic patients with hypertension and nephropathy.

    PubMed

    Sasaki, Hidehisa; Saiki, Atsuhito; Endo, Kei; Ban, Noriko; Yamaguchi, Takashi; Kawana, Hidetoshi; Nagayama, Daizi; Ohhira, Masahiro; Oyama, Tomokazu; Miyashita, Yoh; Shirai, Kohji

    2009-10-01

    The three types of calcium channel blocker (CCB), L-, T- and N-type, possess heterogeneous actions on endothelial function and renal microvascular function. In the present study, we evaluated the effects of two CCBs, efonidipine and amlodipine, on renal function and arterial stiffness. Forty type 2 diabetic patients with hypertension and nephropathy receiving angiotensin receptor II blockers were enrolled and randomly divided into two groups: the efonidipine group was administered efonidipine hydrochloride ethanolate 40 mg/day and the amlodipine group was admin-istered amlodipine besilate 5 mg/day for 12 months. Arterial stiffness was evaluated by the cardio-ankle vascular index (CAVI). Changes in blood pressure during the study were almost the same in the two groups. Sig-nificant increases in serum creatinine and urinary albumin and a significant decrease in the esti-mated glomerular filtration rate were observed in the amlodipine group, but not in the efonidipine group. On the other hand, significant decreases in plasma aldosterone, urinary 8-hydroxy-2'-deoxy-guanosine and CAVI were observed after 12 months in the efonidipine group, but not in the amlo-dipine group. These results suggest that efonidipine, which is both a T-type and L-type calcium chan-nel blocker, has more favorable effects on renal function, oxidative stress and arterial stiffness than amlodipine, an L-type calcium channel blocker.

  16. Novel CPVT-Associated Calmodulin Mutation in CALM3 (CALM3-A103V) Activates Arrhythmogenic Ca Waves and Sparks

    PubMed Central

    Gomez-Hurtado, Nieves; Boczek, Nicole J.; Kryshtal, Dmytro O.; Johnson, Christopher N.; Sun, Jennifer; Nitu, Florentin R.; Cornea, Razvan L.; Chazin, Walter J.; Calvert, Melissa L.; Tester, David J.; Ackerman, Michael J.; Knollmann, Bjorn C.

    2016-01-01

    Background Calmodulin (CaM) mutations are associated with severe forms of long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). We recently reported that CaM mutations were found in 13% of genotype-negative LQTS patients, but the prevalence of CaM mutations in genotype-negative CPVT patients is unknown. Here, we identify and characterize CaM mutations in 12 patients with genotype-negative but clinically-diagnosed CPVT. Methods and Results Mutational analysis of CALM1, CALM2 and CALM3 coding regions, in vitro measurement of CaM-Ca2+ (Ca) binding affinity, RyR2-CaM binding, Ca handling, L-type Ca current (LTCC) and action potential duration (APD). We identified a novel CaM mutation – A103V – in CALM3 in 1 of 12 patients (8%), a female who experienced episodes of exertion-induced syncope since age 10, had normal QT interval, and displayed ventricular ectopy during stress testing consistent with CPVT. A103V modestly lowered CaM Ca-binding affinity (3-fold reduction vs WT-CaM), but did not alter CaM binding to RyR2. In permeabilized cardiomyocytes, A103V-CaM (100 nM) promoted spontaneous Ca wave and spark activity, a cellular phenotype of RyR2 activation. Even a 1:3 mixture of A103V-CaM:WT-CaM activated Ca waves, demonstrating functional dominance. Compared to LQTS D96V-CaM, A103V-CaM had significantly less effects on LTCC inactivation and APD, and caused delayed after depolarizations (DADs) and triggered beats in intact cardiomyocytes. Conclusions We discovered a novel CPVT mutation in the CALM3 gene that shares functional characteristics with established CPVT-associated mutations in CALM1. A small proportion of A103V-CaM is sufficient to evoke arrhythmogenic Ca disturbances via RyR2 dysregulation, which explains the autosomal dominant inheritance. PMID:27516456

  17. Spectrum and Prevalence of CALM1-, CALM2-, and CALM3-Encoded Calmodulin (CaM) Variants in Long QT Syndrome (LQTS) and Functional Characterization of a Novel LQTS-Associated CaM Missense Variant, E141G

    PubMed Central

    Calvert, Melissa L.; Tester, David J.; Kryshtal, Dmytro; Hwang, Hyun Seok; Johnson, Christopher N.; Chazin, Walter J.; Loporcaro, Christina G.; Shah, Maully; Papez, Andrew L.; Lau, Yung R.; Kanter, Ronald; Knollmann, Bjorn C.; Ackerman, Michael J.

    2016-01-01

    Background Calmodulin (CaM) is encoded by three genes, CALM1, CALM2, and CALM3, all of which harbor pathogenic variants linked to long QT syndrome (LQTS) with early and severe expressivity. These LQTS-causative variants reduce CaM affinity to Ca2+ and alter the properties of the cardiac L-type calcium channel (CaV1.2). CaM also modulates NaV1.5 and the ryanodine receptor, RyR2. All of these interactions may play a role in disease pathogenesis. Here, we determine the spectrum and prevalence of pathogenic CaM variants in a cohort of genetically elusive LQTS, and functionally characterize the novel variants. Methods and Results Thirty-nine genetically elusive LQTS cases underwent whole exome sequencing to identify CaM variants. Non-synonymous CaM variants were overrepresented significantly in this heretofore LQTS cohort (15.4%) compared to exome aggregation consortium (0.04%; p<0.0001). When the clinical sequelae of these 6 CaM-positive cases was compared to the 33 CaM-negative cases, CaM-positive cases had a more severe phenotype with an average age of onset of 8 months, an average QTc of 679 ms, and a high prevalence of cardiac arrest. Functional characterization of one novel variant, E141G-CaM, revealed an 11-fold reduction in Ca2+ binding affinity and a functionally-dominant loss of inactivation in CaV1.2, mild accentuation in NaV1.5 late current, but no effect on intracellular RyR2-mediated calcium release. Conclusions Overall, 15% of our genetically elusive LQTS cohort harbored non-synonymous variants in CaM. Genetic testing of CALM1-3 should be pursued for individuals with LQTS, especially those with early childhood cardiac arrest, extreme QT prolongation, and a negative family history. PMID:26969752

  18. Store-operated Ca²⁺ entry and depolarization explain the anomalous behaviour of myometrial SR: effects of SERCA inhibition on electrical activity, Ca²⁺ and force.

    PubMed

    Noble, Debbie; Borysova, Lyudmyla; Wray, Susan; Burdyga, Theodor

    2014-09-01

    In the myometrium SR Ca(2+) depletion promotes an increase in force but unlike several other smooth muscles, there is no Ca(2+) sparks-STOCs coupling mechanism to explain this. Given the importance of the control of contractility for successful parturition, we have examined, in pregnant rat myometrium, the effects of SR Ca(2+)-ATPase (SERCA) inhibition on the temporal relationship between action potentials, Ca(2+) transients and force. Simultaneous recording of electrical activity, calcium and force showed that SERCA inhibition, by cyclopiazonic acid (CPA 20 μM), caused time-dependent changes in excitability, most noticeably depolarization and elevations of baseline [Ca(2+)]i and force. At the onset of these changes there was a prolongation of the bursts of action potentials and a corresponding series of Ca(2+) spikes, which increased the amplitude and duration of contractions. As the rise of baseline Ca(2+) and depolarization continued a point was reached when electrical and Ca(2+) spikes and phasic contractions ceased, and a maintained, tonic force and Ca(2+) was produced. Lanthanum, a non-selective blocker of store-operated Ca(2+) entry, but not the L-type Ca(2+) channel blocker nifedipine (1-10 μM), could abolish the maintained force and calcium. Application of the agonist, carbachol, produced similar effects to CPA, i.e. depolarization, elevation of force and calcium. A brief, high concentration of carbachol, to cause SR Ca(2+) depletion without eliciting receptor-operated channel opening, also produced these results. The data obtained suggest that in pregnant rats SR Ca(2+) release is coupled to marked Ca(2+) entry, via store operated Ca(2+) channels, leading to depolarization and enhanced electrical and mechanical activity. Copyright © 2014. Published by Elsevier Ltd.

  19. Ca2+ current vs. Ca2+ channel cooperativity of exocytosis

    PubMed Central

    Matveev, Victor; Bertram, Richard; Sherman, Arthur

    2009-01-01

    Recently there has been significant interest and progress in the study of spatio-temporal dynamics of Ca2+ that triggers exocytosis at a fast chemical synapse, which requires understanding the contribution of individual calcium channels to the release of a single vesicle. Experimental protocols provide insight into this question by probing the sensitivity of exocytosis to Ca2+ influx. While varying extracellular or intracellular Ca2+ concentration assesses the intrinsic biochemical Ca2+ cooperativity of neurotransmitter release, varying the number of open Ca2+ channels using pharmacological channel block or the tail current titration probes the cooperativity between individual Ca2+ channels in triggering exocytosis. Despite the wide use of these Ca2+ sensitivity measurements, their interpretation often relies on heuristic arguments. Here we provide a detailed analysis of the Ca2+ sensitivity measures probed by these experimental protocols, present simple expressions for special cases, and demonstrate the distinction between the Ca2+ current cooperativity, defined by the relationship between exocytosis rate and the whole-terminal Ca2+ current magnitude, and the underlying Ca2+ channel cooperativity, defined as the average number of channels involved in the release of a single vesicle. We find simple algebraic expressions that show that the two are different but linearly related. Further, we use 3D computational modeling of buffered Ca2+ diffusion to analyze these distinct Ca2+ cooperativity measures, and demonstrate the role of endogenous Ca2+ buffers on such measures. We show that buffers can either increase or decrease the Ca2+ current cooperativity of exocytosis, depending on their concentration and the single-channel Ca2+ current. PMID:19793978

  20. A 1-D model of sinking particles

    NASA Astrophysics Data System (ADS)

    Jokulsdottir, T.; Archer, D.

    2006-12-01

    Acidification of the surface ocean due to increased atmospheric CO2 levels is altering its saturation state with respect to calcium carbonate (Orr et al., 2005) and the ability of calcifying phytoplankton to calcify (Riebesell et al., 2000). Sequestration of atmospheric carbon dioxide into the deep ocean is affected by this, because calcite is the key component in ballasting sinking particles (Klaas and Archer, 2001). The settling velocity of particles is not explicitly modeled but often represented as a constant in climate models. That is clearly inaccurate as the composition of particles changes with depth as bacteria and dissolution processes act on its different components, changing their ratio with depth. An idealized, mechanistic model of particles has been developed where settling velocity is calculated from first principles. The model is forced 100m below the surface with export ratios (organic carbon/calcium carbonate) corresponding to different CO2 levels according to Riebesell et al. The resulting flux is compared to the flux generated by the same model where the settling velocity is held constant. The model produces a relatively constant rain ratio regardless of the amount of calcite available to ballast the particle, which is what data suggests (Conte et al., 2001), whereas a constant velocity model does not. Comparing the flux of particulate organic carbon to the seafloor with increasing CO2 levels, the outcome of the constant velocity model is an increase whereas when the velocity is calculated a decrease results. If so, the change in export ratio with an increase in CO2 concentrations acts as a positive feedback: as increased atmospheric CO2 levels lead to the ocean pH being lowered, reduced calcification of marine organisms results and a decrease in particulate organic carbon flux to the deep ocean, which again raises CO2 concentrations. Conte, M.,, N. Ralph, E. Ross, Seasonal and interannual variability in deep ocean particle fluxes at the Oceanic Flux Program (OFP)/Bermuda Atlantic Time Series (BATS) site in the western Sargasso Sea near Bermuda, Deep-Sea Research II 48 1471-1505, 2001 Klaas, C., and D.E. Archer, Association of sinking organic matter with various types of mineral ballast in the deep sea: Implications for the rain ratio, Global Biogeochemical Cycles, 16, 2002. Orr, J. C. and et. al. Anthropogenic ocean acidification over calcifying organisms. Nature, 437(29):681 686, 2005. U. Riebesell, I. Zondervan, B. Rost, P.D. Tortell, R.E. Zeebe, and F.M.M.Morel. Reduced calcification of marine plankton in response to increased atmospheric CO2. Nature, 407:364 368, 2000.

  1. Regulation of voltage-gated Ca(2+) currents by Ca(2+)/calmodulin-dependent protein kinase II in resting sensory neurons.

    PubMed

    Kostic, Sandra; Pan, Bin; Guo, Yuan; Yu, Hongwei; Sapunar, Damir; Kwok, Wai-Meng; Hudmon, Andy; Wu, Hsiang-En; Hogan, Quinn H

    2014-09-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is recognized as a key element in encoding depolarization activity of excitable cells into facilitated voltage-gated Ca(2+) channel (VGCC) function. Less is known about the participation of CaMKII in regulating VGCCs in resting cells. We examined constitutive CaMKII control of Ca(2+) currents in peripheral sensory neurons acutely isolated from dorsal root ganglia (DRGs) of adult rats. The small molecule CaMKII inhibitor KN-93 (1.0μM) reduced depolarization-induced ICa by 16-30% in excess of the effects produced by the inactive homolog KN-92. The specificity of CaMKII inhibition on VGCC function was shown by the efficacy of the selective CaMKII blocking peptide autocamtide-2-related inhibitory peptide in a membrane-permeable myristoylated form, which also reduced VGCC current in resting neurons. Loss of VGCC currents is primarily due to reduced N-type current, as application of mAIP selectively reduced N-type current by approximately 30%, and prior N-type current inhibition eliminated the effect of mAIP on VGCCs, while prior block of L-type channels did not reduce the effect of mAIP on total ICa. T-type currents were not affected by mAIP in resting DRG neurons. Transduction of sensory neurons in vivo by DRG injection of an adeno-associated virus expressing AIP also resulted in a loss of N-type currents. Together, these findings reveal a novel molecular adaptation whereby sensory neurons retain CaMKII support of VGCCs despite remaining quiescent. Published by Elsevier Inc.

  2. Hypoxic augmentation of Ca2+ channel currents requires a functional electron transport chain.

    PubMed

    Brown, Stephen T; Scragg, Jason L; Boyle, John P; Hudasek, Kristin; Peers, Chris; Fearon, Ian M

    2005-06-10

    The incidence of Alzheimer disease is increased following ischemic episodes, and we previously demonstrated that following chronic hypoxia (CH), amyloid beta (Abeta) peptide-mediated increases in voltage-gated L-type Ca(2+) channel activity contribute to the Ca(2+) dyshomeostasis seen in Alzheimer disease. Because in certain cell types mitochondria are responsible for detecting altered O(2) levels we examined the role of mitochondrial oxidant production in the regulation of recombinant Ca(2+) channel alpha(1C) subunits during CH and exposure to Abeta-(1-40). In wild-type (rho(+)) HEK 293 cells expressing recombinant L-type alpha(1C) subunits, Ca(2+) currents were enhanced by prolonged (24 h) exposure to either CH (6% O(2)) or Abeta-(1-40) (50 nm). By contrast the response to CH was absent in rho(0) cells in which the mitochondrial electron transport chain (ETC) was depleted following long term treatment with ethidium bromide or in rho(+) cells cultured in the presence of 1 microm rotenone. CH was mimicked in rho(0) cells by the exogenous production of O2(-.). by xanthine/xanthine oxidase. Furthermore Abeta-(1-40) enhanced currents in rho(0) cells to a degree similar to that seen in cells with an intact ETC. The antioxidants ascorbate (200 microm) and Trolox (500 microm) ablated the effect of CH in rho(+) cells but were without effect on Abeta-(1-40)-mediated augmentation of Ca(2+) current in rho(0) cells. Thus oxidant production in the mitochondrial ETC is a critical factor, acting upstream of amyloid beta peptide production in the up-regulation of Ca(2+) channels in response to CH.

  3. Beyond Donor-Acceptor (D-A) Approach: Structure-Optoelectronic Properties-Organic Photovoltaic Performance Correlation in New D-A1 -D-A2 Low-Bandgap Conjugated Polymers.

    PubMed

    Chochos, Christos L; Drakopoulou, Sofia; Katsouras, Athanasios; Squeo, Benedetta M; Sprau, Christian; Colsmann, Alexander; Gregoriou, Vasilis G; Cando, Alex-Palma; Allard, Sybille; Scherf, Ullrich; Gasparini, Nicola; Kazerouni, Negar; Ameri, Tayebeh; Brabec, Christoph J; Avgeropoulos, Apostolos

    2017-04-01

    Low-bandgap near-infrared polymers are usually synthesized using the common donor-acceptor (D-A) approach. However, recently polymer chemists are introducing more complex chemical concepts for better fine tuning of their optoelectronic properties. Usually these studies are limited to one or two polymer examples in each case study so far, though. In this study, the dependence of optoelectronic and macroscopic (device performance) properties in a series of six new D-A 1 -D-A 2 low bandgap semiconducting polymers is reported for the first time. Correlation between the chemical structure of single-component polymer films and their optoelectronic properties has been achieved in terms of absorption maxima, optical bandgap, ionization potential, and electron affinity. Preliminary organic photovoltaic results based on blends of the D-A 1 -D-A 2 polymers as the electron donor mixed with the fullerene derivative [6,6]-phenyl-C 71 -butyric acid methyl ester demonstrate power conversion efficiencies close to 4% with short-circuit current densities (J sc ) of around 11 mA cm -2 , high fill factors up to 0.70, and high open-circuit voltages (V oc s) of 0.70 V. All the devices are fabricated in an inverted architecture with the photoactive layer processed in air with doctor blade technique, showing the compatibility with roll-to-roll large-scale manufacturing processes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. The effects of vasoactive peptide urocortin 2 on hemodynamics in spontaneous hypertensive rat and the role of L-type calcium channel and CRFR2.

    PubMed

    Liu, Chunna; Liu, Xinyu; Yang, Jing; Duan, Yan; Yao, Hongyue; Li, Fenghua; Zhang, Xia

    2015-04-01

    Urocortin (UCN) is a newly identified vascular-active peptide that has been shown to reverse cardiovascular remodeling and improve left ventricular (LV) function. The effects and mechanism of urocortin 2 (UCN2) in vivo on the electrical remodeling of left ventricle and the hemodynamics of hypertensive objectives have not been investigated. UCN2 (1 μg/kg/d, 3.5 μg/kg/d or 7 μg/kg/d) was intravenously injected for 2 weeks and its effects on hemodynamics in spontaneously hypertensive rats (SHRs) observed. The whole-cell patch clamp technique was used to explore the effects of UCN2 on the electrical remodeling of left ventricular cardiomyocytes. The flow cytometry method was used to determine the content of fluorescence calcium in myocardium. UCN2 improved the systolic and diastolic function of SHRs as demonstrated by decreased left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), increased +dp/dtmax and -dp/dtmax and decreased cAMP level. UCN2 inhibited the opening of L-type calcium channel and decreased the calcium channel current of cardiomyocytes. In addition, UCN2 also decreased the contents of fluorescence calcium in SHR myocardium. However, astressin2-B (AST-2B), the antagonist of corticotropin-releasing factor receptor 2 (CRFR2), could reverse the inhibitory effects of UCN2 on calcium channel. UCN2 can modulate electrical remodeling of the myocardium and hemodynamics in an experimental model of SHR via inhibition of L-type calcium channel and CRFR2 in cardiomyocytes. Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  5. FK506-binding protein 1b/12.6: a key to aging-related hippocampal Ca2+ dysregulation?

    PubMed Central

    Gant, JC; Blalock, EM; K-C, Chen; Kadish, I; Porter, NM; Norris, CM; Thibault, O; Landfield, PW

    2014-01-01

    It has been recognized for some time that the Ca2+-dependent slow afterhyperpolarization (sAHP) is larger in hippocampal neurons of aged compared with young animals. In addition, extensive studies since have shown that other Ca2+-mediated electrophysiological responses are increased in hippocampus with aging, including Ca2+ transients, L-type voltage-gated Ca2+ channel activity, Ca2+ spike duration and action potential accommodation. Elevated Ca2+-induced Ca2+ release from ryanodine receptors (RyRs) appears to drive amplification of the Ca2+ responses. Components of this Ca2+ dysregulation phenotype correlate with deficits in cognitive function and plasticity, indicating they may play critical roles in aging-related impairment of brain function. However, the molecular mechanisms underlying aging-related Ca2+ dysregulation are not well understood. FK506-binding proteins 1a and 1b (FKBP1a/1b, also known as FKBP12/12.6) are immunophilin proteins that bind the immunosuppressant drugs FK506 and rapamycin. In muscle cells, FKBP1a/1b also bind RyRs and inhibits Ca2+-induced Ca2+ release, but it is not clear whether FKBPs act similarly in brain cells. Recently, we found that selectively disrupting hippocampal FKBP1b function in young rats, either by microinjecting adeno-associated viral vectors containing siRNA, or by treatment with rapamycin, increases the sAHP and recapitulates much of the hippocampal Ca2+ dysregulation phenotype. Moreover, in microarray studies, we found FKBP1b gene expression was downregulated in hippocampus of aging rats and early-stage Alzheimer’s disease subjects. These results suggest the novel hypothesis that declining FKBP function is a key factor in aging-related Ca2+ dysregulation in the brain and point to potential new therapeutic targets for counteracting unhealthy brain aging. PMID:24291098

  6. Rapid Ca2+ flux through the transverse tubular membrane, activated by individual action potentials in mammalian skeletal muscle

    PubMed Central

    Launikonis, Bradley S; Stephenson, D George; Friedrich, Oliver

    2009-01-01

    Periods of low frequency stimulation are known to increase the net Ca2+ uptake in skeletal muscle but the mechanism responsible for this Ca2+ entry is not known. In this study a novel high-resolution fluorescence microscopy approach allowed the detection of an action potential-induced Ca2+ flux across the tubular (t-) system of rat extensor digitorum longus muscle fibres that appears to be responsible for the net uptake of Ca2+ in working muscle. Action potentials were triggered in the t-system of mechanically skinned fibres from rat by brief field stimulation and t-system [Ca2+] ([Ca2+]t-sys) and cytoplasmic [Ca2+] ([Ca2+]cyto) were simultaneously resolved on a confocal microscope. When initial [Ca2+]t-sys was ≥ 0.2 mm a Ca2+ flux from t-system to the cytoplasm was observed following a single action potential. The action potential-induced Ca2+ flux and associated t-system Ca2+ permeability decayed exponentially and displayed inactivation characteristics such that further Ca2+ entry across the t-system could not be observed after 2–3 action potentials at 10 Hz stimulation rate. When [Ca2+]t-sys was closer to 0.1 mm, a transient rise in [Ca2+]t-sys was observed almost concurrently with the increase in [Ca2+]cyto following the action potential. The change in direction of Ca2+ flux was consistent with changes in the direction of the driving force for Ca2+. This is the first demonstration of a rapid t-system Ca2+ flux associated with a single action potential in mammalian skeletal muscle. The properties of this channel are inconsistent with a flux through the L-type Ca2+ channel suggesting that an as yet unidentified t-system protein is conducting this current. This action potential-activated Ca2+ flux provides an explanation for the previously described Ca2+ entry and accumulation observed with prolonged, intermittent muscle activity. PMID:19332499

  7. Release from the cone ribbon synapse under bright light conditions can be controlled by the opening of only a few Ca(2+) channels.

    PubMed

    Bartoletti, Theodore M; Jackman, Skyler L; Babai, Norbert; Mercer, Aaron J; Kramer, Richard H; Thoreson, Wallace B

    2011-12-01

    Light hyperpolarizes cone photoreceptors, causing synaptic voltage-gated Ca(2+) channels to open infrequently. To understand neurotransmission under these conditions, we determined the number of L-type Ca(2+) channel openings necessary for vesicle fusion at the cone ribbon synapse. Ca(2+) currents (I(Ca)) were activated in voltage-clamped cones, and excitatory postsynaptic currents (EPSCs) were recorded from horizontal cells in the salamander retina slice preparation. Ca(2+) channel number and single-channel current amplitude were calculated by mean-variance analysis of I(Ca). Two different comparisons-one comparing average numbers of release events to average I(Ca) amplitude and the other involving deconvolution of both EPSCs and simultaneously recorded cone I(Ca)-suggested that fewer than three Ca(2+) channel openings accompanied fusion of each vesicle at the peak of release during the first few milliseconds of stimulation. Opening fewer Ca(2+) channels did not enhance fusion efficiency, suggesting that few unnecessary channel openings occurred during strong depolarization. We simulated release at the cone synapse, using empirically determined synaptic dimensions, vesicle pool size, Ca(2+) dependence of release, Ca(2+) channel number, and Ca(2+) channel properties. The model replicated observations when a barrier was added to slow Ca(2+) diffusion. Consistent with the presence of a diffusion barrier, dialyzing cones with diffusible Ca(2+) buffers did not affect release efficiency. The tight clustering of Ca(2+) channels, along with a high-Ca(2+) affinity release mechanism and diffusion barrier, promotes a linear coupling between Ca(2+) influx and vesicle fusion. This may improve detection of small light decrements when cones are hyperpolarized by bright light.

  8. Epileptogenesis causes an N-methyl-d-aspartate receptor/Ca2+-dependent decrease in Ca2+/calmodulin-dependent protein kinase II activity in a hippocampal neuronal culture model of spontaneous recurrent epileptiform discharges.

    PubMed

    Blair, Robert E; Sombati, Sompong; Churn, Severn B; Delorenzo, Robert J

    2008-06-24

    Alterations in the function of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) have been observed in both in vivo and in vitro models of epileptogenesis; however the molecular mechanism mediating the effects of epileptogenesis on CaM kinase II has not been elucidated. This study was initiated to evaluate the molecular pathways involved in causing the long-lasting decrease in CaM kinase II activity in the hippocampal neuronal culture model of low Mg2+-induced spontaneous recurrent epileptiform discharges (SREDs). We show here that the decrease in CaM kinase II activity associated with SREDs in hippocampal cultures involves a Ca2+/N-methyl-d-aspartate (NMDA) receptor-dependent mechanism. Low Mg2+-induced SREDs result in a significant decrease in Ca2+/calmodulin-dependent substrate phosphorylation of the synthetic peptide autocamtide-2. Reduction of extracellular Ca2+ levels (0.2 mM in treatment solution) or the addition of dl-2-amino-5-phosphonovaleric acid (APV) 25 microM blocked the low Mg2+-induced decrease in CaM kinase II-dependent substrate phosphorylation. Antagonists of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid receptor or L-type voltage sensitive Ca2+ channel had no effect on the low Mg2+-induced decrease in CaM kinase II-dependent substrate phosphorylation. The results of this study demonstrate that the decrease in CaM kinase II activity associated with this model of epileptogenesis involves a selective Ca2+/NMDA receptor-dependent mechanism and may contribute to the production and maintenance of SREDs in this model.

  9. Epileptogenesis causes an N-methyl-d-aspartate receptor/Ca2+-dependent decrease in Ca2+/calmodulin-dependent protein kinase II activity in a hippocampal neuronal culture model of spontaneous recurrent epileptiform discharges

    PubMed Central

    Blair, Robert E.; Sombati, Sompong; Churn, Severn B.; DeLorenzo, Robert J.

    2008-01-01

    Alterations in the function of Ca2+/calmodulin-dependent protein kinase II (CaM Kinase II) have been observed in both in vivo and in vitro models of epileptogenesis; however the molecular mechanism mediating the effects of epileptogenesis on CaM Kinase II have not been elucidated. This study was initiated to evaluate the molecular pathways involved in causing the long lasting decrease in CaM Kinase II activity in the hippocampal neuronal culture model of low Mg2+ induced spontaneous recurrent epileptiform discharges (SREDs). We show here that the decrease in CaM kinase II activity associated with SREDs in hippocampal cultures involves a Ca2+/N-methyl-d-aspartate (NMDA) receptor-dependent mechanism. Low Mg2+ induced SREDs results in a significant decrease in Ca2+/calmodulin-dependent substrate phosphorylation of the synthetic peptide autocamtide-2. Reduction of extracellular Ca2+ levels (0.2 mM in treatment solution) or the addition of DL-2-amino-5-phosphonovaleric acid (APV) 25 µM blocked the low Mg2+ induced decrease in CaM kinase II-dependent substrate phosphorylation. Antagonists of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid receptor or L-type voltage sensitive Ca2+ channel had no effect on the low Mg2+ induced decrease in CaM kinase II-dependent substrate phosphorylation. The results of this study demonstrate that the decrease in CaM kinase II activity associated with this model of epileptogenesis involves a selective Ca2+/NMDA receptor-dependent mechanism and may contribute to the production and maintenance of SREDs in this model. PMID:18495112

  10. Coachella Valley, CA

    NASA Image and Video Library

    2001-10-22

    These band composites, acquired on June 4, 2000, cover a 11 by 13.5 km sub-scene in the Coachella Valley, CA. The area is shown by the yellow box on the full scene in the LOWER RIGHT corner, northwest of the Salton Sea. This is a major agricultural region of California, growing fruit and produce throughout the year. Different combinations of ASTER bands help identify the different crop types. UPPER LEFT: bands 3, 2, 1 as red, green, and blue (RGB); UPPER RIGHT: bands 4, 2, 1 as RGB; LOWER LEFT: bands 4, 3, 2 as RGB. The image is centered at 33.6 degrees north latitude, 116.1 degrees west longitude. http://photojournal.jpl.nasa.gov/catalog/PIA11161

  11. Differential contribution of Ca2+ sources to day and night BK current activation in the circadian clock

    PubMed Central

    McNally, Beth A.

    2018-01-01

    Large conductance K+ (BK) channels are expressed widely in neurons, where their activation is regulated by membrane depolarization and intracellular Ca2+ (Ca2+i). To enable this regulation, BK channels functionally couple to both voltage-gated Ca2+ channels (VGCCs) and channels mediating Ca2+ release from intracellular stores. However, the relationship between BK channels and their specific Ca2+ source for particular patterns of excitability is not well understood. In neurons within the suprachiasmatic nucleus (SCN)—the brain’s circadian clock—BK current, VGCC current, and Ca2+i are diurnally regulated, but paradoxically, BK current is greatest at night when VGCC current and Ca2+i are reduced. Here, to determine whether diurnal regulation of Ca2+ is relevant for BK channel activation, we combine pharmacology with day and night patch-clamp recordings in acute slices of SCN. We find that activation of BK current depends primarily on three types of channels but that the relative contribution changes between day and night. BK current can be abrogated with nimodipine during the day but not at night, establishing that L-type Ca2+ channels (LTCCs) are the primary daytime Ca2+ source for BK activation. In contrast, dantrolene causes a significant decrease in BK current at night, suggesting that nighttime BK activation is driven by ryanodine receptor (RyR)–mediated Ca2+i release. The N- and P/Q-type Ca2+ channel blocker ω-conotoxin MVIIC causes a smaller reduction of BK current that does not differ between day and night. Finally, inhibition of LTCCs, but not RyRs, eliminates BK inactivation, but the BK β2 subunit was not required for activation of BK current by LTCCs. These data reveal a dynamic coupling strategy between BK channels and their Ca2+ sources in the SCN, contributing to diurnal regulation of SCN excitability. PMID:29237755

  12. Differential contribution of Ca2+ sources to day and night BK current activation in the circadian clock.

    PubMed

    Whitt, Joshua P; McNally, Beth A; Meredith, Andrea L

    2018-02-05

    Large conductance K + (BK) channels are expressed widely in neurons, where their activation is regulated by membrane depolarization and intracellular Ca 2+ (Ca 2+ i ). To enable this regulation, BK channels functionally couple to both voltage-gated Ca 2+ channels (VGCCs) and channels mediating Ca 2+ release from intracellular stores. However, the relationship between BK channels and their specific Ca 2+ source for particular patterns of excitability is not well understood. In neurons within the suprachiasmatic nucleus (SCN)-the brain's circadian clock-BK current, VGCC current, and Ca 2+ i are diurnally regulated, but paradoxically, BK current is greatest at night when VGCC current and Ca 2+ i are reduced. Here, to determine whether diurnal regulation of Ca 2+ is relevant for BK channel activation, we combine pharmacology with day and night patch-clamp recordings in acute slices of SCN. We find that activation of BK current depends primarily on three types of channels but that the relative contribution changes between day and night. BK current can be abrogated with nimodipine during the day but not at night, establishing that L-type Ca 2+ channels (LTCCs) are the primary daytime Ca 2+ source for BK activation. In contrast, dantrolene causes a significant decrease in BK current at night, suggesting that nighttime BK activation is driven by ryanodine receptor (RyR)-mediated Ca 2+ i release. The N- and P/Q-type Ca 2+ channel blocker ω-conotoxin MVIIC causes a smaller reduction of BK current that does not differ between day and night. Finally, inhibition of LTCCs, but not RyRs, eliminates BK inactivation, but the BK β2 subunit was not required for activation of BK current by LTCCs. These data reveal a dynamic coupling strategy between BK channels and their Ca 2+ sources in the SCN, contributing to diurnal regulation of SCN excitability. © 2018 Whitt et al.

  13. Effects of phloretin and phloridzin on Ca2+ handling, the action potential, and ion currents in rat ventricular myocytes.

    PubMed

    Olson, Marnie L; Kargacin, Margaret E; Ward, Christopher A; Kargacin, Gary J

    2007-06-01

    The effects of the phytoestrogens phloretin and phloridzin on Ca(2+) handling, cell shortening, the action potential, and Ca(2+) and K(+) currents in freshly isolated cardiac myocytes from rat ventricle were examined. Phloretin increased the amplitude and area and decreased the rate of decline of electrically evoked Ca(2+) transients in the myocytes. These effects were accompanied by an increase in the Ca(2+) load of the sarcoplasmic reticulum, as determined by the area of caffeine-evoked Ca(2+) transients. An increase in the extent of shortening of the myocytes in response to electrically evoked action potentials was also observed in the presence of phloretin. To further examine possible mechanisms contributing to the observed changes in Ca(2+) handling and contractility, the effects of phloretin on the cardiac action potential and plasma membrane Ca(2+) and K(+) currents were examined. Phloretin markedly increased the action potential duration in the myocytes, and it inhibited the Ca(2+)-independent transient outward K(+) current (I(to)). The inwardly rectifying K(+) current, the sustained outward delayed rectifier K(+) current, and L-type Ca(2+) currents were not significantly different in the presence and absence of phloretin, nor was there any evidence that the Na(+)/Ca(2+) exchanger was affected. The effects of phloretin on Ca(2+) handling in the myocytes are consistent with its effects on I(to). Phloridzin did not significantly alter the amplitude or area of electrically evoked Ca(2+) transients in the myocytes, nor did it have detectable effects on the sarcoplasmic reticulum Ca(2+) load, cell shortening, or the action potential.

  14. Altered Ca2+ signaling in skeletal muscle fibers of the R6/2 mouse, a model of Huntington’s disease

    PubMed Central

    Braubach, Peter; Orynbayev, Murat; Andronache, Zoita; Hering, Tanja; Landwehrmeyer, Georg Bernhard; Lindenberg, Katrin S.

    2014-01-01

    Huntington’s disease (HD) is caused by an expanded CAG trinucleotide repeat within the gene encoding the protein huntingtin. The resulting elongated glutamine (poly-Q) sequence of mutant huntingtin (mhtt) affects both central neurons and skeletal muscle. Recent reports suggest that ryanodine receptor–based Ca2+ signaling, which is crucial for skeletal muscle excitation–contraction coupling (ECC), is changed by mhtt in HD neurons. Consequently, we searched for alterations of ECC in muscle fibers of the R6/2 mouse, a mouse model of HD. We performed fluorometric recordings of action potentials (APs) and cellular Ca2+ transients on intact isolated toe muscle fibers (musculi interossei), and measured L-type Ca2+ inward currents on internally dialyzed fibers under voltage-clamp conditions. Both APs and AP-triggered Ca2+ transients showed slower kinetics in R6/2 fibers than in fibers from wild-type mice. Ca2+ removal from the myoplasm and Ca2+ release flux from the sarcoplasmic reticulum were characterized using a Ca2+ binding and transport model, which indicated a significant reduction in slow Ca2+ removal activity and Ca2+ release flux both after APs and under voltage-clamp conditions. In addition, the voltage-clamp experiments showed a highly significant decrease in L-type Ca2+ channel conductance. These results indicate profound changes of Ca2+ turnover in skeletal muscle of R6/2 mice and suggest that these changes may be associated with muscle pathology in HD. PMID:25348412

  15. Shear fluid-induced Ca2+ release and the role of mitochondria in rat cardiac myocytes.

    PubMed

    Belmonte, Steve; Morad, Martin

    2008-03-01

    Cardiac myocyte contraction occurs when Ca2+ influx through voltage-gated L-type Ca2+ channels causes Ca2+ release from ryanodine receptors of the sarcoplasmic reticulum (SR). Although mitochondria occupy about 35% of the cell volume in rat cardiac myocytes, and are thought to be located <300 nm from the junctional SR, their role in the beat-to-beat regulation of cardiac Ca2+ signaling remains unclear. We have recently shown that rapid ( approximately 20 ms) application of shear fluid forces ( approximately 25 dynes/cm2) to rat cardiac myocytes triggers slowly ( approximately 300 ms) developing Cai transients that were independent of activation of all transmembrane Ca2+ transporting pathways, but were suppressed by FCCP, CCCP, and Ru360, all of which are known to disrupt mitochondrial function. We have here used rapid 2-D confocal microscopy to monitor fluctuations in mitochondrial Ca2+ levels ([Ca2+]m) and mitochondrial membrane potential (Delta Psi m) in rat cardiac myocytes loaded either with rhod-2 AM or tetramethylrhodamine methyl ester (TMRM), respectively. Freshly isolated intact rat cardiac myocytes were plated on glass coverslips and incubated in 5 mM Ca2+ containing Tyrode's solution and 40 mM 2,3-butanedione monoxime (BDM) to inhibit cell contraction. Alternatively, myocytes were permeabilized with 10 microM digitonin and perfused with an "intracellular" solution containing 10 microM free [Ca2+], 5 mM EGTA, and 15 mM BDM. Direct [Ca2+]m measurements showed transient mitochondrial Ca2+ accumulation after exposure to 10 mM caffeine, as revealed by a 66% increase in the rhod-2 fluorescence intensity. Shear fluid forces, however, produced a 12% decrease in signal, suggesting that application of a mechanical force releases Ca2+ from the mitochondria. In addition, caffeine and CCCP or FCCP strongly reduced Delta Psi m, while application of a pressurized solution produced a transient Delta Psi m hyperpolarization in intact ventricular myocytes loaded with TMRM

  16. Effects of luminal flow and nucleotides on [Ca(2+)](i) in rabbit cortical collecting duct.

    PubMed

    Woda, Craig B; Leite, Maurilo; Rohatgi, Rajeev; Satlin, Lisa M

    2002-09-01

    Nucleotide binding to purinergic P2 receptors contributes to the regulation of a variety of physiological functions in renal epithelial cells. Whereas P2 receptors have been functionally identified at the basolateral membrane of the cortical collecting duct (CCD), a final regulatory site of urinary Na(+), K(+), and acid-base excretion, controversy exists as to whether apical purinoceptors exist in this segment. Nor has the distribution of receptor subtypes present on the unique cell populations that constitute Ca(2+) the CCD been established. To examine this, we measured nucleotide-induced changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura 2-loaded rabbit CCDs microperfused in vitro. Resting [Ca(2+)](i) did not differ between principal and intercalated cells, averaging approximately 120 nM. An acute increase in tubular fluid flow rate, associated with a 20% increase in tubular diameter, led to increases in [Ca(2+)](i) in both cell types. Luminal perfusion of 100 microM UTP or ATP-gamma-S, in the absence of change in flow rate, caused a rapid and transient approximately fourfold increase in [Ca(2+)](i) in both cell types (P < 0.05). Luminal suramin, a nonspecific P2 receptor antagonist, blocked the nucleotide- but not flow-induced [Ca(2+)](i) transients. Luminal perfusion with a P2X (alpha,beta-methylene-ATP), P2X(7) (benzoyl-benzoyl-ATP), P2Y(1) (2-methylthio-ATP), or P2Y(4)/P2Y(6) (UDP) receptor agonist had no effect on [Ca(2+)](i). The nucleotide-induced [Ca(2+)](i) transients were inhibited by the inositol-1,4,5-triphosphate receptor blocker 2-aminoethoxydiphenyl borate, thapsigargin, which depletes internal Ca(2+) stores, luminal perfusion with a Ca(2+)-free perfusate, or the L-type Ca(2+) channel blocker nifedipine. These results suggest that luminal nucleotides activate apical P2Y(2) receptors in the CCD via pathways that require both internal Ca(2+) mobilization and extracellular Ca(2+) entry. The flow-induced rise in [Ca(2+)](i) is

  17. Thermodynamic Linkage Between Calmodulin Domains Binding Calcium and Contiguous Sites in the C-Terminal Tail of CaV1.2

    PubMed Central

    Evans, T. Idil Apak; Hell, Johannes; Shea, Madeline A.

    2011-01-01

    Calmodulin (CaM) binding to the intracellular C-terminal tail (CTT) of the cardiac L-type Ca2+ channel (CaV1.2) regulates Ca2+ entry by recognizing sites that contribute to negative feedback mechanisms for channel closing. CaM associates with CaV1.2 under low resting [Ca2+], but is poised to change conformation and position when intracellular [Ca2+] rises. CaM binding Ca2+, and the domains of CaM binding the CTT are linked thermodynamic functions. To better understand regulation, we determined the energetics of CaM domains binding to peptides representing pre-IQ sites A1588, and C1614 and the IQ motif studied as overlapping peptides IQ1644 and IQ′1650 as well as their effect on calcium binding. (Ca2+)4-CaM bound to all four peptides very favorably (Kd ≤ 2 nM). Linkage analysis showed that IQ1644–1670 bound with a Kd ~1 pM. In the pre-IQ region, (Ca2+)2-N-domain bound preferentially to A1588, while (Ca2+)2-C-domain preferred C1614. When bound to C1614, calcium binding in the N-domain affected the tertiary conformation of the C-domain. Based on the thermodynamics, we propose a structural mechanism for calcium-dependent conformational change in which the linker between CTT sites A and C buckles to form an A-C hairpin that is bridged by calcium-saturated CaM. PMID:21757287

  18. cDNA cloning, molecular modeling and docking calculations of L-type lectins from Swartzia simplex var. grandiflora (Leguminosae, Papilionoideae), a member of the tribe Swartzieae.

    PubMed

    Maranhão, Paulo A C; Teixeira, Claudener S; Sousa, Bruno L; Barroso-Neto, Ito L; Monteiro-Júnior, José E; Fernandes, Andreia V; Ramos, Marcio V; Vasconcelos, Ilka M; Gonçalves, José F C; Rocha, Bruno A M; Freire, Valder N; Grangeiro, Thalles B

    2017-07-01

    The genus Swartzia is a member of the tribe Swartzieae, whose genera constitute the living descendants of one of the early branches of the papilionoid legumes. Legume lectins comprise one of the main families of structurally and evolutionarily related carbohydrate-binding proteins of plant origin. However, these proteins have been poorly investigated in Swartzia and to date, only the lectin from S. laevicarpa seeds (SLL) has been purified. Moreover, no sequence information is known from lectins of any member of the tribe Swartzieae. In the present study, partial cDNA sequences encoding L-type lectins were obtained from developing seeds of S. simplex var. grandiflora. The amino acid sequences of the S. simplex grandiflora lectins (SSGLs) were only averagely related to the known primary structures of legume lectins, with sequence identities not greater than 50-52%. The SSGL sequences were more related to amino acid sequences of papilionoid lectins from members of the tribes Sophoreae and Dalbergieae and from the Cladratis and Vataireoid clades, which constitute with other taxa, the first branching lineages of the subfamily Papilionoideae. The three-dimensional structures of 2 representative SSGLs (SSGL-A and SSGL-E) were predicted by homology modeling using templates that exhibit the characteristic β-sandwich fold of the L-type lectins. Molecular docking calculations predicted that SSGL-A is able to interact with D-galactose, N-acetyl-D-galactosamine and α-lactose, whereas SSGL-E is probably a non-functional lectin due to 2 mutations in the carbohydrate-binding site. Using molecular dynamics simulations followed by density functional theory calculations, the binding free energies of the interaction of SSGL-A with GalNAc and α-lactose were estimated as -31.7 and -47.5 kcal/mol, respectively. These findings gave insights about the carbohydrate-binding specificity of SLL, which binds to immobilized lactose but is not retained in a matrix containing D-GalNAc as

  19. Roles of Ca(v) channels and AHNAK1 in T cells: the beauty and the beast.

    PubMed

    Matza, Didi; Flavell, Richard A

    2009-09-01

    T lymphocytes require Ca2+ entry though the plasma membrane for their activation and function. Recently, several routes for Ca2+ entry through the T-cell plasma membrane after activation have been described. These include calcium release-activated channels (CRAC), transient receptor potential (TRP) channels, and inositol-1,4,5-trisphosphate receptors (IP3Rs). Herein we review the emergence of a fourth new route for Ca2+ entry, composed of Ca(v) channels (also known as L-type voltage-gated calcium channels) and the scaffold protein AHNAK1 (AHNAK/desmoyokin). Both helper (CD4+) and killer (CD8+) T cells express high levels of Ca(v)1 alpha1 subunits (alpha1S, alpha1C, alpha1D, and alpha1F) and AHNAK1 after their differentiation and require these molecules for Ca2+ entry during an immune response. In this article, we describe the observations and open questions that ultimately suggest the involvement of multiple consecutive routes for Ca2+ entry into lymphocytes, one of which may be mediated by Ca(v) channels and AHNAK1.

  20. Mitochondrial Ca2+ homeostasis during Ca2+ influx and Ca2+ release in gastric myocytes from Bufo marinus

    PubMed Central

    Drummond, Robert M; Mix, T Christian H; Tuft, Richard A; Walsh, John V; Fay, Fredric S

    2000-01-01

    The Ca2+-sensitive fluorescent indicator rhod-2 was used to monitor mitochondrial Ca2+ concentration ([Ca2+]m) in gastric smooth muscle cells from Bufo marinus. In some studies, fura-2 was used in combination with rhod-2, allowing simultaneous measurement of cytoplasmic Ca2+ concentration ([Ca2+]i) and [Ca2+]m, respectively. During a short train of depolarizations, which causes Ca2+ influx from the extracellular medium, there was an increase in both [Ca2+]i and [Ca2+]m. The half-time (t½) to peak for the increase in [Ca2+]m was considerably longer than the t½ to peak for the increase in [Ca2+]i. [Ca2+]m remained elevated for tens of seconds after [Ca2+]i had returned to its resting value. Stimulation with caffeine, which causes release of Ca2+ from the sarcoplasmic reticulum (SR), also produced increases in both [Ca2+]i and [Ca2+]m. The values of t½ to peak for the increase in [Ca2+] in both cytoplasm and mitochondria were similar; however, [Ca2+]i returned to baseline values much faster than [Ca2+]m. Using a wide-field digital imaging microscope, changes in [Ca2+]m were monitored within individual mitochondria in situ, during stimulation of Ca2+ influx or Ca2+ release from the SR. Mitochondrial Ca2+ uptake during depolarizing stimulation caused depolarization of the mitochondrial membrane potential. The mitochondrial membrane potential recovered considerably faster than the recovery of [Ca2+]m. This study shows that Ca2+ influx from the extracellular medium and Ca2+ release from the SR are capable of increasing [Ca2+]m in smooth muscle cells. The efflux of Ca2+ from the mitochondria is a slow process and appears to be dependent upon the amount of Ca2+ in the SR. PMID:10713963

  1. LecRK-V, an L-type lectin receptor kinase in Haynaldia villosa, plays positive role in resistance to wheat powdery mildew.

    PubMed

    Wang, Zongkuan; Cheng, Jiangyue; Fan, Anqi; Zhao, Jia; Yu, Zhongyu; Li, Yingbo; Zhang, Heng; Xiao, Jin; Muhammad, Faheem; Wang, Haiyan; Cao, Aizhong; Xing, Liping; Wang, Xiue

    2018-01-01

    Plant sense potential microbial pathogen using pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs). The Lectin receptor-like kinase genes (LecRKs) are involved in various cellular processes mediated by signal transduction pathways. In the present study, an L-type lectin receptor kinase gene LecRK-V was cloned from Haynaldia villosa, a diploid wheat relative which is highly resistant to powdery mildew. The expression of LecRK-V was rapidly up-regulated by Bgt inoculation and chitin treatment. Its transcript level was higher in the leaves than in roots, culms, spikes and callus. Single-cell transient overexpression of LecRK-V led to decreased haustorium index in wheat variety Yangmai158, which is powdery mildew susceptible. Stable transformation LecRK-V into Yangmai158 significantly enhanced the powdery mildew resistance at both seedling and adult stages. At seedling stage, the transgenic line was highly resistance to 18 of the tested 23 Bgt isolates, hypersensitive responses (HR) were observed for 22 Bgt isolates, and more ROS at the Bgt infection sites was accumulated. These indicated that LecRK-V confers broad-spectrum resistance to powdery mildew, and ROS and SA pathways contribute to the enhanced powdery mildew resistance in wheat. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  2. Cloud Atlas: Discovery of Rotational Spectral Modulations in a Low-mass, L-type Brown Dwarf Companion to a Star

    NASA Astrophysics Data System (ADS)

    Manjavacas, Elena; Apai, Dániel; Zhou, Yifan; Karalidi, Theodora; Lew, Ben W. P.; Schneider, Glenn; Cowan, Nicolas; Metchev, Stan; Miles-Páez, Paulo A.; Burgasser, Adam J.; Radigan, Jacqueline; Bedin, Luigi R.; Lowrance, Patrick J.; Marley, Mark S.

    2018-01-01

    Observations of rotational modulations of brown dwarfs and giant exoplanets allow the characterization of condensate cloud properties. As of now, rotational spectral modulations have only been seen in three L-type brown dwarfs. We report here the discovery of rotational spectral modulations in LP261-75B, an L6-type intermediate surface gravity companion to an M4.5 star. As a part of the Cloud Atlas Treasury program, we acquired time-resolved Wide Field Camera 3 grism spectroscopy (1.1–1.69 μm) of LP261-75B. We find gray spectral variations with the relative amplitude displaying only a weak wavelength dependence and no evidence for lower-amplitude modulations in the 1.4 μm water band than in the adjacent continuum. The likely rotational modulation period is 4.78 ± 0.95 hr, although the rotational phase is not well sampled. The minimum relative amplitude in the white light curve measured over the whole wavelength range is 2.41% ± 0.14%. We report an unusual light curve, which seems to have three peaks approximately evenly distributed in rotational phase. The spectral modulations suggests that the upper atmosphere cloud properties in LP261-75B are similar to two other mid-L dwarfs of typical infrared colors, but differ from that of the extremely red L-dwarf WISE0047.

  3. Circadian phase-dependent effect of nitric oxide on L-type voltage-gated calcium channels in avian cone photoreceptors

    PubMed Central

    Ko, Michael L.; Shi, Liheng; Huang, Cathy Chia-Yu; Grushin, Kirill; Park, So-Young; Ko, Gladys Y.-P.

    2014-01-01

    Nitric oxide (NO) plays an important role in phase-shifting of circadian neuronal activities in the suprachiasmatic nucleus and circadian behavior activity rhythms. In the retina, NO production is increased in a light-dependent manner. While endogenous circadian oscillators in retinal photoreceptors regulate their physiological states, it is not clear whether NO also participates in the circadian regulation of photoreceptors. In the present study, we demonstrate that NO is involved in the circadian phase-dependent regulation of L-type voltage-gated calcium channels (L-VGCCs). In chick cone photoreceptors, the L-VGCCα1 subunit expression and the maximal L-VGCC currents are higher at night, and both Ras-MAPK (mitogen-activated protein kinase)-Erk (extracellular-signal-regulated kinase) and Ras-phosphatidylinositol 3 kinase (PI3K)-protein kinase B (Akt) are part of the circadian output pathways regulating L-VGCCs. The NO-cGMP-protein kinase G (PKG) pathway decreases L-VGCCα1 subunit expression and L-VGCC currents at night, but not during the day, and exogenous NO donor or cGMP decreases the phosphorylation of Erk and Akt at night. The protein expression of neural NO synthase (nNOS) is also under circadian control, with both nNOS and NO production being higher during the day. Taken together, NO/cGMP/PKG signaling is involved as part of the circadian output pathway to regulate L-VGCCs in cone photoreceptors. PMID:23895452

  4. NMDA receptors in mouse anterior piriform cortex initialize early odor preference learning and L-type calcium channels engage for long-term memory.

    PubMed

    Mukherjee, Bandhan; Yuan, Qi

    2016-10-14

    The interactions of L-type calcium channels (LTCCs) and NMDA receptors (NMDARs) in memories are poorly understood. Here we investigated the specific roles of anterior piriform cortex (aPC) LTCCs and NMDARs in early odor preference memory in mice. Using calcium imaging in aPC slices, LTCC activation was shown to be dependent on NMDAR activation. Either D-APV (NMDAR antagonist) or nifedipine (LTCC antagonist) reduced somatic calcium transients in pyramidal cells evoked by lateral olfactory tract stimulation. However, nifedipine did not further reduce calcium in the presence of D-APV. In mice that underwent early odor preference training, blocking NMDARs in the aPC prevented short-term (3 hr) and long-term (24 hr) odor preference memory, and both memories were rescued when BayK-8644 (LTCC agonist) was co-infused. However, activating LTCCs in the absence of NMDARs resulted in loss of discrimination between the conditioned odor and a similar odor mixture at 3 hr. Elevated synaptic AMPAR expression at 3 hr was prevented by D-APV infusion but restored when LTCCs were directly activated, mirroring the behavioral outcomes. Blocking LTCCs prevented 24 hr memory and spared 3 hr memory. These results suggest that NMDARs mediate stimulus-specific encoding of odor memory while LTCCs mediate intracellular signaling leading to long-term memory.

  5. An Improved Targeted cAMP Sensor to Study the Regulation of Adenylyl Cyclase 8 by Ca2+ Entry through Voltage-Gated Channels

    PubMed Central

    Everett, Katy L.; Cooper, Dermot M. F.

    2013-01-01

    Here we describe an improved sensor with reduced pH sensitivity tethered to adenylyl cyclase (AC) 8. The sensor was used to study cAMP dynamics in the AC8 microdomain of MIN6 cells, a pancreatic β-cell line. In these cells, AC8 was activated by Ca2+ entry through L-type voltage-gated channels following depolarisation. This activation could be reconstituted in HEK293 cells co-expressing AC8 and either the α1C or α1D subunit of L-type voltage-gated Ca2+ channels. The development of this improved sensor opens the door to the study of cAMP microdomains in excitable cells that have previously been challenging due to the sensitivity of fluorescent proteins to pH changes. PMID:24086669

  6. Role of ion channels and subcellular Ca2+ signaling in arachidonic acid-induced dilation of pressurized retinal arterioles.

    PubMed

    Kur, Joanna; McGahon, Mary K; Fernández, Jose A; Scholfield, C Norman; McGeown, J Graham; Curtis, Tim M

    2014-05-02

    To investigate the mechanisms responsible for the dilatation of rat retinal arterioles in response to arachidonic acid (AA). Changes in the diameter of isolated, pressurized rat retinal arterioles were measured in the presence of AA alone and following pre-incubation with pharmacologic agents inhibiting Ca(2+) sparks and oscillations and K(+) channels. Subcellular Ca(2+) signals were recorded in arteriolar myocytes using Fluo-4-based confocal imaging. The effects of AA on membrane currents of retinal arteriolar myocytes were studied using whole-cell perforated patch clamp recording. Arachidonic acid dilated pressurized retinal arterioles under conditions of myogenic tone. Eicosatetraynoic acid (ETYA) exerted a similar effect, but unlike AA, its effects were rapidly reversible. Arachidonic acid-induced dilation was associated with an inhibition of subcellular Ca(2+) signals. Interventions known to block Ca(2+) sparks and oscillations in retinal arterioles caused dilatation and inhibited AA-induced vasodilator responses. Arachidonic acid accelerated the rate of inactivation of the A-type Kv current and the voltage dependence of inactivation was shifted to more negative membrane potentials. It also enhanced voltage-activated and spontaneous large-conductance calcium-activated K(+) (BK) currents, but only at positive membrane potentials. Pharmacologic inhibition of A-type Kv and BK currents failed to block AA-induced vasodilator responses. Arachidonic acid suppressed L-type Ca(2+) currents. These results suggest that AA induces retinal arteriolar vasodilation by inhibiting subcellular Ca(2+)-signaling activity in retinal arteriolar myocytes, most likely through a mechanism involving the inhibition of L-type Ca(2+)-channel activity. Arachidonic acid actions on K(+) currents are inconsistent with a model in which K(+) channels contribute to the vasodilator effects of AA.

  7. γCaMKII shuttles Ca2+/CaM to the nucleus to trigger CREB phosphorylation and gene expression

    PubMed Central

    Ma, Huan; Groth, Rachel D.; Cohen, Samuel M.; Emery, John F.; Li, Bo-Xing; Hoedt, Esthelle; Zhang, Guo-An; Neubert, Thomas A.; Tsien, Richard W.

    2014-01-01

    SUMMARY Activity-dependent CREB phosphorylation and gene expression are critical for long-term neuronal plasticity. Local signaling at CaV1 channels triggers these events but how information is relayed onward to the nucleus remains unclear. Here we report a novel mechanism that mediates long-distance communication within cells: a shuttle that transports Ca2+/calmodulin from the surface membrane to the nucleus. We show that the shuttle protein is γCaMKII, that its phosphorylation at Thr287 by βCaMKII protects the Ca2+/CaM signal, and that CaN triggers its nuclear translocation. Both βCaMKII and CaN act in close proximity to CaV1 channels, supporting their dominance, while γCaMKII operates as a carrier, not as a kinase. Upon arrival within the nucleus, Ca2+/CaM activates CaMKK and its substrate CaMKIV, the CREB kinase. This mechanism resolves longstanding puzzles about CaM/CaMK-dependent signaling to the nucleus. The significance of the mechanism is emphasized by dysregulation of CaV1, γCaMKII, βCaMKII and CaN in multiple neuropsychiatric disorders. PMID:25303525

  8. Aryl hydrocarbon receptor (AHR) regulation of L-Type Amino Acid Transporter 1 (LAT-1) expression in MCF-7 and MDA-MB-231 breast cancer cells.

    PubMed

    Tomblin, Justin K; Arthur, Subha; Primerano, Donald A; Chaudhry, Ateeq R; Fan, Jun; Denvir, James; Salisbury, Travis B

    2016-04-15

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that is regulated by environmental toxicants that function as AHR agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). L-Type Amino Acid Transporter 1 (LAT1) is a leucine transporter that is overexpressed in cancer. The regulation of LAT1 by AHR in MCF-7 and MDA-MB-231 breast cancer cells (BCCs) was investigated in this report. Ingenuity pathway analysis (IPA) revealed a significant association between TCDD-regulated genes (TRGs) and molecular transport. Overlapping the TCDD-RNA-Seq dataset obtained in this study with a published TCDD-ChIP-seq dataset identified LAT1 as a primary target of AHR-dependent TCDD induction. Short interfering RNA (siRNA)-directed knockdown of AHR confirmed that TCDD-stimulated increases in LAT1 mRNA and protein required AHR expression. TCDD-stimulated increases in LAT1 mRNA were also inhibited by the AHR antagonist CH-223191. Upregulation of LAT1 by TCDD coincided with increases in leucine uptake by MCF-7 cells in response to TCDD. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays revealed increases in AHR, AHR nuclear translocator (ARNT) and p300 binding and histone H3 acetylation at an AHR binding site in the LAT1 gene in response to TCDD. In MCF-7 and MDA-MB-231 cells, endogenous levels of LAT1 mRNA and protein were reduced in response to knockdown of AHR expression. Knockdown experiments demonstrated that proliferation of MCF-7 and MDA-MB-231 cells is dependent on both LAT1 and AHR. Collectively, these findings confirm the dependence of cancer cells on leucine uptake and establish a mechanism for extrinsic and intrinsic regulation of LAT1 by AHR. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. α-Actinin Promotes Surface Localization and Current Density of the Ca2+ Channel CaV1.2 by Binding to the IQ Region of the α1 Subunit.

    PubMed

    Tseng, Pang-Yen; Henderson, Peter B; Hergarden, Anne C; Patriarchi, Tommaso; Coleman, Andrea M; Lillya, Mark W; Montagut-Bordas, Carlota; Lee, Boram; Hell, Johannes W; Horne, Mary C

    2017-07-18

    The voltage-gated L-type Ca 2+ channel Ca V 1.2 is crucial for initiating heartbeat and control of a number of neuronal functions such as neuronal excitability and long-term potentiation. Mutations of Ca V 1.2 subunits result in serious health problems, including arrhythmia, autism spectrum disorders, immunodeficiency, and hypoglycemia. Thus, precise control of Ca V 1.2 surface expression and localization is essential. We previously reported that α-actinin associates and colocalizes with neuronal Ca V 1.2 channels and that shRNA-mediated depletion of α-actinin significantly reduces localization of endogenous Ca V 1.2 in dendritic spines in hippocampal neurons. Here we investigated the hypothesis that direct binding of α-actinin to Ca V 1.2 supports its surface expression. Using two-hybrid screens and pull-down assays, we identified three point mutations (K1647A, Y1649A, and I1654A) in the central, pore-forming α 1 1.2 subunit of Ca V 1.2 that individually impaired α-actinin binding. Surface biotinylation and flow cytometry assays revealed that Ca V 1.2 channels composed of the corresponding α-actinin-binding-deficient mutants result in a 35-40% reduction in surface expression compared to that of wild-type channels. Moreover, the mutant Ca V 1.2 channels expressed in HEK293 cells exhibit a 60-75% decrease in current density. The larger decrease in current density as compared to surface expression imparted by these α 1 1.2 subunit mutations hints at the possibility that α-actinin not only stabilizes surface localization of Ca V 1.2 but also augments its ion conducting activity.

  10. Modulation of subthalamic T-type Ca2+ channels remedies locomotor deficits in a rat model of Parkinson disease

    PubMed Central

    Tai, Chun-Hwei; Yang, Ya-Chin; Pan, Ming-Kai; Huang, Chen-Syuan; Kuo, Chung-Chin

    2011-01-01

    An increase in neuronal burst activities in the subthalamic nucleus (STN) is a well-documented electrophysiological feature of Parkinson disease (PD). However, the causal relationship between subthalamic bursts and PD symptoms and the ionic mechanisms underlying the bursts remain to be established. Here, we have shown that T-type Ca2+ channels are necessary for subthalamic burst firing and that pharmacological blockade of T-type Ca2+ channels reduces motor deficits in a rat model of PD. Ni2+, mibefradil, NNC 55-0396, and efonidipine, which inhibited T-type Ca2+ currents in acutely dissociated STN neurons, but not Cd2+ and nifedipine, which preferentially inhibited L-type or the other non–T-type Ca2+ currents, effectively diminished burst activity in STN slices. Topical administration of inhibitors of T-type Ca2+ channels decreased in vivo STN burst activity and dramatically reduced the locomotor deficits in a rat model of PD. Cd2+ and nifedipine showed no such electrophysiological and behavioral effects. While low-frequency deep brain stimulation (DBS) has been considered ineffective in PD, we found that lengthening the duration of the low-frequency depolarizing pulse effectively improved behavioral measures of locomotion in the rat model of PD, presumably by decreasing the availability of T-type Ca2+ channels. We therefore conclude that modulation of subthalamic T-type Ca2+ currents and consequent burst discharges may provide new strategies for the treatment of PD. PMID:21737877

  11. CaPTC Biennial Meetings

    Cancer.gov

    CaPTC hosts the 'Biennial Science of Global Prostate Cancer Disparities in Black Men' conference to address the growing global public health problem of prostate cancer among Black men in industrialized and developing countries.

  12. A new treatment for human malignant melanoma targeting L-type amino acid transporter 1 (LAT1): A pilot study in a canine model

    SciTech Connect

    Fukumoto, Shinya; Hanazono, Kiwamu; Fu, Dah-Renn

    2013-09-13

    Highlights: •LAT1 is highly expressed in tumors but at low levels in normal tissues. •We examine LAT1 expression and function in malignant melanoma (MM). •LAT1 expression in MM tissues and cell lines is higher than those in normal tissues. •LAT1 selective inhibitors inhibit amino acid uptake and cell growth in MM cells. •New chemotherapeutic protocols including LAT1 inhibitors are effective for treatment. -- Abstract: L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities such as cellular growth, proliferation and maintenance. This amino acid transportermore » recently has received attention because of its preferential and up-regulated expression in a variety of human tumors in contrast to its limited distribution and low-level expression in normal tissues. In this study, we explored the feasibility of using LAT1 inhibitor as a new therapeutic agent for human malignant melanomas (MM) using canine spontaneous MM as a model for human MM. A comparative study of LAT expression was performed in 48 normal tissues, 25 MM tissues and five cell lines established from MM. The study observed LAT1 mRNA levels from MM tissues and cell lines that were significantly (P < 0.01) higher than in normal tissues. Additionally, MM with distant metastasis showed a higher expression than those without distant metastasis. Functional analysis of LAT1 was performed on one of the five cell lines, CMeC-1. [{sup 3}H]L-Leucine uptake and cellular growth activities in CMeC-1 were inhibited in a dose-dependent manner by selective LAT1 inhibitors (2-amino-2-norbornane-carboxylic acid, BCH and melphalan, LPM). Inhibitory growth activities of various conventional anti-cancer drugs, including carboplatin, cyclophosphamide, dacarbazine, doxorubicin, mitoxantrone, nimustine, vinblastine and vincristine, were significantly (P < 0.05) enhanced by combination use with BCH or

  13. L-type Calcium Channel Blockers Enhance Trafficking and Function of Epilepsy-associated α1(D219N) Subunits of GABA(A) Receptors.

    PubMed

    Han, Dong-Yun; Guan, Bo-Jhih; Wang, Ya-Juan; Hatzoglou, Maria; Mu, Ting-Wei

    2015-09-18

    Gamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory ion channels in the mammalian central nervous system and play an essential role in regulating inhibition-excitation balance in neural circuits. The α1 subunit harboring the D219N mutation of GABAA receptors was reported to be retained in the endoplasmic reticulum (ER) and traffic inefficiently to the plasma membrane, leading to a loss of function of α1(D219N) subunits and thus idiopathic generalized epilepsy (IGE). We present the use of small molecule proteostasis regulators to enhance the forward trafficking of α1(D219N) subunits to restore their function. We showed that treatment with verapamil (4 μM, 24 h), an L-type calcium channel blocker, substantially increases the α1(D219N) subunit cell surface level in both HEK293 cells and neuronal SH-SY5Y cells and remarkably restores the GABA-induced maximal chloride current in HEK293 cells expressing α1(D219N)β2γ2 receptors to a level that is comparable to wild type receptors. Our drug mechanism study revealed that verapamil treatment promotes the ER to Golgi trafficking of the α1(D219N) subunits post-translationally. To achieve that, verapamil treatment enhances the interaction between the α1(D219N) subunit and β2 subunit and prevents the aggregation of the mutant protein by shifting the protein from the detergent-insoluble fractions to detergent-soluble fractions. By combining (35)S pulse-chase labeling and MG-132 inhibition experiments, we demonstrated that verapamil treatment does not inhibit the ER-associated degradation of the α1(D219N) subunit. In addition, its effect does not involve a dynamin-1 dependent endocytosis. To gain further mechanistic insight, we showed that verapamil increases the interaction between the mutant protein and calnexin and calreticulin, two major lectin chaperones in the ER. Moreover, calnexin binding promotes the forward trafficking of the mutant subunit. Taken together, our data indicate that

  14. Properties of spontaneous Ca2+ transients recorded from interstitial cells of Cajal-like cells of the rabbit urethra in situ

    PubMed Central

    Hashitani, Hikaru; Suzuki, Hikaru

    2007-01-01

    Interstitial cells of Cajal-like cells (ICC-LCs) in the urethra may act as electrical pacemakers of spontaneous contractions. However, their properties in situ and their interaction with neighbouring urethral smooth muscle cells (USMCs) remain to be elucidated. To further explore the physiological role of ICC-LCs, spontaneous changes in [Ca2+]i (Ca2+ transients) were visualized in fluo-4 loaded preparations of rabbit urethral smooth muscle. ICC-LCs were sparsely distributed, rather than forming an extensive network. Ca2+ transients in ICC-LCs had a lower frequency and a longer half-width than those of USMCs. ICC-LCs often exhibited Ca2+ transients synchronously with each other, but did not often show a close temporal relationship with Ca2+ transients in USMCs. Nicardipine (1 μm) suppressed Ca2+ transients in USMCs but not in ICC-LCs. Ca2+ transients in ICC-LCs were abolished by cyclopiazonic acid (10 μm), ryanodine (50 μm) and caffeine (10 mm) or by removing extracellular Ca2+, and inhibited by 2-aminoethoxydiphenyl borate (50 μm) and 3-morpholino-sydnonimine (SIN-1; 10 μm), but facilitated by increasing extracellular Ca2+ or phenylephrine (1–10 μm). These results indicated that Ca2+ transients in urethral ICC-LCs in situ rely on both Ca2+ release from intracellular Ca2+ stores and Ca2+ influx through non-L-type Ca2+ channel pathways. ICC-LCs may not act as a coordinated pacemaker electrical network as do ICC in the gastrointestinal (GI) tract. Rather they may randomly increase excitability of USMCs to maintain the tone of urethral smooth muscles. PMID:17615099

  15. Release from the cone ribbon synapse under bright light conditions can be controlled by the opening of only a few Ca2+ channels

    PubMed Central

    Bartoletti, Theodore M.; Jackman, Skyler L.; Babai, Norbert; Mercer, Aaron J.; Kramer, Richard H.

    2011-01-01

    Light hyperpolarizes cone photoreceptors, causing synaptic voltage-gated Ca2+ channels to open infrequently. To understand neurotransmission under these conditions, we determined the number of L-type Ca2+ channel openings necessary for vesicle fusion at the cone ribbon synapse. Ca2+ currents (ICa) were activated in voltage-clamped cones, and excitatory postsynaptic currents (EPSCs) were recorded from horizontal cells in the salamander retina slice preparation. Ca2+ channel number and single-channel current amplitude were calculated by mean-variance analysis of ICa. Two different comparisons—one comparing average numbers of release events to average ICa amplitude and the other involving deconvolution of both EPSCs and simultaneously recorded cone ICa—suggested that fewer than three Ca2+ channel openings accompanied fusion of each vesicle at the peak of release during the first few milliseconds of stimulation. Opening fewer Ca2+ channels did not enhance fusion efficiency, suggesting that few unnecessary channel openings occurred during strong depolarization. We simulated release at the cone synapse, using empirically determined synaptic dimensions, vesicle pool size, Ca2+ dependence of release, Ca2+ channel number, and Ca2+ channel properties. The model replicated observations when a barrier was added to slow Ca2+ diffusion. Consistent with the presence of a diffusion barrier, dialyzing cones with diffusible Ca2+ buffers did not affect release efficiency. The tight clustering of Ca2+ channels, along with a high-Ca2+ affinity release mechanism and diffusion barrier, promotes a linear coupling between Ca2+ influx and vesicle fusion. This may improve detection of small light decrements when cones are hyperpolarized by bright light. PMID:21880934

  16. Isosteviol prevents the prolongation of action potential in hypertrophied cardiomyoctyes by regulating transient outward potassium and L-type calcium channels.

    PubMed

    Fan, Zhuo; Lv, Nanying; Luo, Xiao; Tan, Wen

    2017-10-01

    Cardiac hypertrophy is a thickening of the heart muscle that is associated with cardiovascular diseases such as hypertension and myocardial infarction. It occurs initially as an adaptive process against increased workloads and often leads to sudden arrhythmic deaths. Studies suggest that the lethal arrhythmia is attributed to hypertrophy-induced destabilization of cardiac electrical activity, especially the prolongation of the action potential. The reduced activity of I to is demonstrated to be responsible for the ionic mechanism of prolonged action potential duration and arrhythmogeneity. Isosteviol (STV), a derivative of stevioside, plays a protective role in a variety of stress-induced cardiac diseases. Here we report effects of STV on rat ISO-induced hypertrophic cardiomyocytes. STV alleviated ISO-induced hypertrophy of cardiomyocytes by decreasing cell area of hypertrophied cardiomyocytes. STV application prevented the prolongation of action potential which was prominent in hypertrophied cells. The decrease and increase of current densities for I to and I CaL observed in hypertrophied myocytes were both prevented by STV application. In addition, the results of qRT-PCR suggested that the changes of electrophysiological activity of I to and I CaL are correlated to the alterations of the mRNA transcription level. Copyright © 2017. Published by Elsevier B.V.

  17. Divalent metal (Ca, Cd, Mn, Zn) uptake and interactions in the aquatic insect Hydropsyche sparna.

    PubMed

    Poteat, Monica D; Díaz-Jaramillo, Mauricio; Buchwalter, David B

    2012-05-01

    Despite their ecological importance and prevalent use as ecological indicators, the trace element physiology of aquatic insects remains poorly studied. Understanding divalent metal transport processes at the water-insect interface is important because these metals may be essential (e.g. Ca), essential and potentially toxic (e.g. Zn) or non-essential and toxic (e.g. Cd). We measured accumulation kinetics of Zn and Cd across dissolved concentrations ranging 4 orders of magnitude and examined interactions with Ca and Mn in the caddisfly Hydropsyche sparna. Here, we provide evidence for at least two transport systems for both Zn and Cd, the first of which operates at concentrations below 0.8 μmol l(-1) (and is fully saturable for Zn). We observed no signs of saturation of a second lower affinity transport system at concentrations up to 8.9 μmol l(-1) Cd and 15.3 μmol l(-1) Zn. In competition studies at 0.6 μmol l(-1) Zn and Cd, the presence of Cd slowed Zn accumulation by 35% while Cd was unaffected by Zn. At extreme concentrations (listed above), Cd accumulation was unaffected by the presence of Zn whereas Zn accumulation rates were reduced by 58%. Increasing Ca from 31.1 μmol l(-1) to 1.35 mmol l(-1) resulted in only modest decreases in Cd and Zn uptake. Mn decreased adsorption of Cd and Zn to the integument but not internalization. The L-type Ca(2+) channel blockers verapamil and nifedipine and the plasma membrane Ca(2+)-ATPase inhibitor carboxyeosin had no influence on Ca, Cd or Zn accumulation rates, while Ruthenium Red, a Ca(2+)-ATPase inhibitor, significantly decreased the accumulation of all three in a concentration-dependent manner.

  18. The impact of extracellular and intracellular Ca2+ on ethanol-induced smooth muscle contraction.

    PubMed

    Döndaş, Naciye Yaktubay; Kaplan, Mahir; Kaya, Derya; Singirik, Ergin

    2009-10-01

    To evaluate the impact of extracellular and intracellular Ca2+ on contractions induced by ethanol in smooth muscle. Longitudinal smooth muscle strips were prepared from the gastric fundi of mice. The contractions of smooth muscle strips were recorded with an isometric force displacement transducer. Ethanol (164 mmol/L) produced reproducible contractions in isolated gastric fundal strips of mice. Although lidocaine (50 and 100 micromol/L), a local anesthetic agent, and hexamethonium (100 and 500 micromol/L), a ganglionic blocking agent, failed to affect these contractions, verapamil (1-50 micromol/L) and nifedipine (1-50 micromol/L), selective blockers of L-type Ca2+ channels, significantly inhibited the contractile responses of ethanol. Using a Ca(2+)-free medium nearly eliminated these contractions in the same tissue. Ryanodine (1-50 micromol/L) and ruthenium red (10-100 micromol/L), selective blockers of intracellular Ca2+ channels/ryanodine receptors; cyclopiazonic acid (CPA; 1-10 mumol/L), a selective inhibitor of sarcoplasmic reticulum (SR) Ca(2+)-ATPase; and caffeine (0.5-5 mmol/L), a depleting agent of intracellular Ca2+ stores, significantly inhibited the contractile responses induced by ethanol. In addition, the combination of caffeine (5 mmol/L) plus CPA (10 micromol/L), and ryanodine (10 micromol/L) plus CPA (10 micromol/L), caused further inhibition of contractions in response to ethanol. This inhibition was significantly different from those associated with caffeine, ryanodine or CPA. Furthermore the combination of caffeine (5 mmol/L), ryanodine (10 micromol/L) and CPA(10 micromol/L) eliminated the contractions induced by ethanol in isolated gastric fundal strips of mice. Both extracellular and intracellular Ca2+ may have important roles in regulating contractions induced by ethanol in the mouse gastric fundus.

  19. Characteristics of single Ca(2+) channel kinetics in feline hypertrophied ventricular myocytes.

    PubMed

    Yang, Xiangjun; Hui, Jie; Jiang, Tingbo; Song, Jianping; Liu, Zhihua; Jiang, Wenping

    2002-04-01

    To explore the mechanism underlying the prolongation of action potential and delayed inactivation of the L-type Ca(2+) (I(Ca, L)) current in a feline model of left ventricular system hypertension and concomitant hypertrophy. Single Ca(2+) channel properties in myocytes isolated from normal and pressure overloaded cat left ventricles were studied, using patch-clamp techniques. Left ventricular pressure overload was induced by partial ligation of the ascending aorta for 4 - 6 weeks. The amplitude of single Ca(2+) channel current evoked by depolarizing pulses from -40 mV to 0 mV was 1.02 +/- 0.03 pA in normal cells and 1.05 +/- 0.03 pA in hypertrophied cells, and there was no difference in single channel current-voltage relationships between the groups since slope conductance was 26.2 +/- 1.0 pS in normal and hypertrophied cells, respectively. Peak amplitudes of the ensemble-averaged single Ca(2+) channel currents were not different between the two groups of cells. However, the amplitude of this averaged current at the end of the clamp pulse was significantly larger in hypertrophied cells than in normal cells. Open-time histograms revealed that open-time distribution was fitted by a single exponential function in channels of normal cells and by a two exponential function in channels of hypertrophied cells. The number of long-lasting openings was increased in channels of hypertrophied cells, and therefore the calculated mean open time of the channel was significantly longer compared to normal controls. Kinetic changes in the Ca(2+) channel may underlie both hypertrophy-associated delayed inactivation of the Ca(2+) current and, in part, the pressure overload-induced action potential lengthening in this cat model of ventricular left systolic hypertension and hypertrophy.

  20. Opposing Intermolecular Tuning of Ca2+ Affinity for Calmodulin by Neurogranin and CaMKII Peptides.

    PubMed

    Zhang, Pengzhi; Tripathi, Swarnendu; Trinh, Hoa; Cheung, Margaret S

    2017-03-28

    We investigated the impact of bound calmodulin (CaM)-target compound structure on the affinity of calcium (Ca 2+ ) by integrating coarse-grained models and all-atomistic simulations with nonequilibrium physics. We focused on binding between CaM and two specific targets, Ca 2+ /CaM-dependent protein kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaM-dependent Ca 2+ signaling pathways in neurons. It was shown experimentally that Ca 2+ /CaM (holoCaM) binds to the CaMKII peptide with overwhelmingly higher affinity than Ca 2+ -free CaM (apoCaM); the binding of CaMKII peptide to CaM in return increases the Ca 2+ affinity for CaM. However, this reciprocal relation was not observed in the Ng peptide (Ng 13-49 ), which binds to apoCaM or holoCaM with binding affinities of the same order of magnitude. Unlike the holoCaM-CaMKII peptide, whose structure can be determined by crystallography, the structural description of the apoCaM-Ng 13-49 is unknown due to low binding affinity, therefore we computationally generated an ensemble of apoCaM-Ng 13-49 structures by matching the changes in the chemical shifts of CaM upon Ng 13-49 binding from nuclear magnetic resonance experiments. Next, we computed the changes in Ca 2+ affinity for CaM with and without binding targets in atomistic models using Jarzynski's equality. We discovered the molecular underpinnings of lowered affinity of Ca 2+ for CaM in the presence of Ng 13-49 by showing that the N-terminal acidic region of Ng peptide pries open the β-sheet structure between the Ca 2+ binding loops particularly at C-domain of CaM, enabling Ca 2+ release. In contrast, CaMKII peptide increases Ca 2+ affinity for the C-domain of CaM by stabilizing the two Ca 2+ binding loops. We speculate that the distinctive structural difference in the bound complexes of apoCaM-Ng 13-49 and holoCaM-CaMKII delineates the importance of CaM's progressive mechanism of target binding on its Ca 2+ binding affinities. Copyright © 2017

  1. [Effects of nitric oxide on peritoneal lymphatic stomata and lymph drainage via NO-cGMP-Ca2+ pathway].

    PubMed

    Li, Yan-Yuan; Li, Ji-Cheng

    2005-02-25

    To study the cell signal transduction mechanism of nitric oxide (NO) on the peritoneal lymphatic stomata and lymph drainage in the rat, cGMP content were measured by a commercially available radioimmunoassay kit, and the [Ca(2+)](i) were observed by a confocal laser scanning microscope in the cultured peritoneal mesothelial cell. Animal experiment was practiced to study the effect of NO-cGMP-Ca(2+) pathway on the lymphatic stomata and lymph absorption. The results showed that: (1) Sper/NO increased cGMP of the rat peritoneal mesothelial cell (RPMC) in a dose-dependent manner (P<0.01) compared to the control group. This effect was blocked by 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) (P<0.05), a specific inhibitor of soluble guanylyl cyclase (sGC). The level of [Ca(2+)](i) in single RPMC decreased by adding Sper/NO (P<0.05). Pretreatment with ODQ for 10 min blocked the Sper/NO-induced decrease in [Ca(2+)](i). L-typed calcium channel blocker nifedipine induced an immediate and marked decrease in [Ca(2+)](i) (P<0.05).. After [Ca(2+)](i) reached a balance again, adding Sper/NO could not change [Ca(2+)](i) (P>0.05). (2) Sper/NO increased the area of the stomata (P<0.01) and the quantity of the tracer in a dose-dependent manner (P<0.05) compared to the control group. Pretreatment with ODQ significantly inhibited Sper/NO-induced change of lymphatic stomata and lymph drainage (P<0.01). Nifedipine increased the opening area of the lymphatic stomata (P< 0.01) and the concentration of absorbed trypan blue of the diaphragm (P<0.05). Sper/NO could not make a further change in the samples pretreated by nifedipine (P> 0.05). The results indicate that NO can decrease [Ca(2+)](i) in the RPMC through the NO-cGMP pathway. This procession is related with the L- type voltage-gated Ca(2+) channel. NO enlarges the opening area of the lymphatic stomata and enhances the lymph drainage of tracer by NO-cGMP-[Ca(2+)](i) pathway.

  2. Voltage-clamp analysis of the potentiation of the slow Ca2+-activated K+ current in hippocampal pyramidal neurons.

    PubMed

    Borde, M; Bonansco, C; Fernández de Sevilla, D; Le Ray, D; Buño, W

    2000-01-01

    Exploring the principles that govern activity-dependent changes in excitability is an essential step to understand the function of the nervous system, because they act as a general postsynaptic control mechanism that modulates the flow of synaptic signals. We show an activity-dependent potentiation of the slow Ca2+-activated K+ current (sl(AHP)) which induces sustained decreases in the excitability in CA1 pyramidal neurons. We analyzed the sl(AHP) using the slice technique and voltage-clamp recordings with sharp or patch-electrodes. Using sharp electrodes-repeated activation with depolarizing pulses evoked a prolonged (8-min) potentiation of the amplitude (171%) and duration (208%) of the sl(AHP). Using patch electrodes, early after entering the whole-cell configuration (<20 min), responses were as those reported above. However, although the sl(AHP) remained unchanged, its potentiation was markedly reduced in later recordings, suggesting that the underlying mechanisms were rapidly eliminated by intracellular dialysis. Inhibition of L-type Ca2+ current by nifedipine (20 microM) markedly reduced the sl(AHP) (79%) and its potentiation (55%). Ryanodine (20 microM) that blocks the release of intracellular Ca2+ also reduced sl(AHP) (29%) and its potentiation (25%). The potentiation of the sl(AHP) induced a marked and prolonged (>50%; approximately equals 8 min) decrease in excitability. The results suggest that sl(AHP) is potentiated as a result of an increased intracellular Ca2+ concentration ([Ca2+]i) following activation of voltage-gated L-type Ca2+ channels, aided by the subsequent release of Ca2+ from intracellular stores. Another possibility is that repeated activation increases the Ca2+-binding capacity of the channels mediating the sl(AHP). This potentiation of the sl(AHP) could be relevant in hippocampal physiology, because the changes in excitability it causes may regulate the induction threshold of the long-term potentiation of synaptic efficacy. Moreover, the

  3. Changes in Ca(2+) channel expression upon differentiation of SN56 cholinergic cells.

    PubMed

    Kushmerick, C; Romano-Silva, M A; Gomez, M V; Prado, M A

    2001-10-19

    The SN56 cell line, a fusion of septal neurons and neuroblastoma cells, has been used as a model for central cholinergic neurons. These cells show increased expression of cholinergic neurochemical features upon differentiation, but little is known about how differentiation affects their electrophysiological properties. We examined the changes in Ca(2+) channel expression that occur as these cells undergo morphological differentiation in response to serum withdrawal and exposure to dibutyryl-cAMP. Undifferentiated cells expressed a T-type current with biophysical and pharmacological properties similar, although not identical, to those reported for the current generated by the alpha(1H) (CaV3.2) Ca(2+) channel subunit. Differentiated cells expressed, in addition to this T-type current, high voltage activated currents which were inhibited 38% by the L-type channel antagonist nifedipine (5 microM), 37% by the N-type channel antagonist omega-conotoxin-GVIA (1 microM), and 15% by the P/Q-type channel antagonist omega-agatoxin-IVA (200 nM). Current resistant to these inhibitors accounted for 15% of the high voltage activated current in differentiated SN56 cells. Our data demonstrate that differentiation increases the expression of neuronal type voltage gated Ca(2+) channels in this cell line, and that the channels expressed are comparable to those reported for native basal forebrain cholinergic neurons. This cell line should thus provide a useful model system to study the relationship between calcium currents and cholinergic function and dysfunction.

  4. (-)-Terpinen-4-ol changes intracellular Ca2+ handling and induces pacing disturbance in rat hearts.

    PubMed

    Gondim, Antonio Nei Santana; Lara, Aline; Santos-Miranda, Artur; Roman-Campos, Danilo; Lauton-Santos, Sandra; Menezes-Filho, José Evaldo Rodrigues; de Vasconcelos, Carla Maria Lins; Conde-Garcia, Eduardo Antonio; Guatimosim, Silvia; Cruz, Jader S

    2017-07-15

    (-)-Terpinen-4-ol is a naturally occurring plant monoterpene and has been shown to have a plethora of biological activities. The objective of this study was to investigate the effects of (-)-terpinen-4-ol on the rat heart, a key player in the control and maintenance of arterial blood pressure. The effects of (-)-terpinen-4-ol on the rat heart were investigated using isolated left atrium isometric force measurements, in vivo electrocardiogram (ECG) recordings, patch clamp technique, and confocal microscopy. It was observed that (-)-terpinen-4-ol reduced contraction force in an isolated left atrium at millimolar concentrations. Conversely, it induced a positive inotropic effect and extrasystoles at micromolar concentrations, suggesting that (-)-terpinen-4-ol may have arrhythmogenic activity on cardiac tissue. In anaesthetized animals, (-)-terpinen-4-ol also elicited rhythm disturbance, such as supraventricular tachycardia and atrioventricular block. To investigate the cellular mechanism underlying the dual effect of (-)-terpinen-4-ol on heart muscle, experiments were performed on isolated ventricular cardiomyocytes to determine the effect of (-)-terpinen-4-ol on L-type Ca 2+ currents, Ca 2+ sparks, and Ca 2+ transients. The arrhythmogenic activity of (-)-terpinen-4-ol in vitro and in vivo may be explained by its effect on intracellular Ca 2+ handling. Taken together, our data suggest that (-)-terpinen-4-ol has cardiac arrhythmogenic activity. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Computational analysis of the regulation of Ca2+ dynamics in rat ventricular myocytes

    NASA Astrophysics Data System (ADS)

    Bugenhagen, Scott M.; Beard, Daniel A.

    2015-10-01

    Force-frequency relationships of isolated cardiac myocytes show complex behaviors that are thought to be specific to both the species and the conditions associated with the experimental preparation. Ca2+ signaling plays an important role in shaping the force-frequency relationship, and understanding the properties of the force-frequency relationship in vivo requires an understanding of Ca2+ dynamics under physiologically relevant conditions. Ca2+ signaling is itself a complicated process that is best understood on a quantitative level via biophysically based computational simulation. Although a large number of models are available in the literature, the models are often a conglomeration of components parameterized to data of incompatible species and/or experimental conditions. In addition, few models account for modulation of Ca2+ dynamics via β-adrenergic and calmodulin-dependent protein kinase II (CaMKII) signaling pathways even though they are hypothesized to play an important regulatory role in vivo. Both protein-kinase-A and CaMKII are known to phosphorylate a variety of targets known to be involved in Ca2+ signaling, but the effects of these pathways on the frequency- and inotrope-dependence of Ca2+ dynamics are not currently well understood. In order to better understand Ca2+ dynamics under physiological conditions relevant to rat, a previous computational model is adapted and re-parameterized to a self-consistent dataset obtained under physiological temperature and pacing frequency and updated to include β-adrenergic and CaMKII regulatory pathways. The necessity of specific effector mechanisms of these pathways in capturing inotrope- and frequency-dependence of the data is tested by attempting to fit the data while including and/or excluding those effector components. We find that: (1) β-adrenergic-mediated phosphorylation of the L-type calcium channel (LCC) (and not of phospholamban (PLB)) is sufficient to explain the inotrope-dependence; and (2) that

  6. The Influence of Ca2+ Buffers on Free [Ca2+] Fluctuations and the Effective Volume of Ca2+ Microdomains

    PubMed Central

    Weinberg, Seth H.; Smith, Gregory D.

    2014-01-01

    Intracellular calcium (Ca2+) plays a significant role in many cell signaling pathways, some of which are localized to spatially restricted microdomains. Ca2+ binding proteins (Ca2+ buffers) play an important role in regulating Ca2+ concentration ([Ca2+]). Buffers typically slow [Ca2+] temporal dynamics and increase the effective volume of Ca2+ domains. Because fluctuations in [Ca2+] decrease in proportion to the square-root of a domain’s physical volume, one might conjecture that buffers decrease [Ca2+] fluctuations and, consequently, mitigate the significance of small domain volume concerning Ca2+ signaling. We test this hypothesis through mathematical and computational analysis of idealized buffer-containing domains and their stochastic dynamics during free Ca2+ influx with passive exchange of both Ca2+ and buffer with bulk concentrations. We derive Langevin equations for the fluctuating dynamics of Ca2+ and buffer and use these stochastic differential equations to determine the magnitude of [Ca2+] fluctuations for different buffer parameters (e.g., dissociation constant and concentration). In marked contrast to expectations based on a naive application of the principle of effective volume as employed in deterministic models of Ca2+ signaling, we find that mobile and rapid buffers typically increase the magnitude of domain [Ca2+] fluctuations during periods of Ca2+ influx, whereas stationary (immobile) Ca2+ buffers do not. Also contrary to expectations, we find that in the absence of Ca2+ influx, buffers influence the temporal characteristics, but not the magnitude, of [Ca2+] fluctuations. We derive an analytical formula describing the influence of rapid Ca2+ buffers on [Ca2+] fluctuations and, importantly, identify the stochastic analog of (deterministic) effective domain volume. Our results demonstrate that Ca2+ buffers alter the dynamics of [Ca2+] fluctuations in a nonintuitive manner. The finding that Ca2+ buffers do not suppress intrinsic domain [Ca2

  7. The AKAP Cypher/Zasp contributes to β-adrenergic/PKA stimulation of cardiac CaV1.2 calcium channels.

    PubMed

    Yu, Haijie; Yuan, Can; Westenbroek, Ruth E; Catterall, William A

    2018-06-04

    Stimulation of the L-type Ca 2+ current conducted by Ca V 1.2 channels in cardiac myocytes by the β-adrenergic/protein kinase A (PKA) signaling pathway requires anchoring of PKA to the Ca V 1.2 channel by an A-kinase anchoring protein (AKAP). However, the AKAP(s) responsible for regulation in vivo remain unknown. Here, we test the role of the AKAP Cypher/Zasp in β-adrenergic regulation of Ca V 1.2 channels using physiological studies of cardiac ventricular myocytes from young-adult mice lacking the long form of Cypher/Zasp (LCyphKO mice). These myocytes have increased protein levels of Ca V 1.2, PKA, and calcineurin. In contrast, the cell surface density of Ca V 1.2 channels and the basal Ca 2+ current conducted by Ca V 1.2 channels are significantly reduced without substantial changes to kinetics or voltage dependence. β-adrenergic regulation of these L-type Ca 2+ currents is also significantly reduced in myocytes from LCyphKO mice, whether calculated as a stimulation ratio or as net-stimulated Ca 2+ current. At 100 nM isoproterenol, the net β-adrenergic-Ca 2+ current conducted by Ca V 1.2 channels was reduced to 39 ± 12% of wild type. However, concentration-response curves for β-adrenergic stimulation of myocytes from LCyphKO mice have concentrations that give a half-maximal response similar to those for wild-type mice. These results identify Cypher/Zasp as an important AKAP for β-adrenergic regulation of cardiac Ca V 1.2 channels. Other AKAPs may work cooperatively with Cypher/Zasp to give the full magnitude of β-adrenergic regulation of Ca V 1.2 channels observed in vivo. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

  8. Basal activity of voltage-gated Ca(2+) channels controls the IP3-mediated contraction by α(1)-adrenoceptor stimulation of mouse aorta segments.

    PubMed

    Leloup, Arthur J; Van Hove, Cor E; De Meyer, Guido R Y; Schrijvers, Dorien M; Fransen, Paul

    2015-08-05

    α1-Adrenoceptor stimulation of mouse aorta causes intracellular Ca(2+) release from sarcoplasmic reticulum Ca(2+) stores via stimulation of inositoltriphosphate (IP3) receptors. It is hypothesized that this Ca(2+) release from the contractile and IP3-sensitive Ca(2+) store is under the continuous dynamic control of time-independent basal Ca(2+) influx via L-type voltage-gated Ca(2+) channels (LCC) residing in their window voltage range. Mouse aortic segments were α1-adrenoceptor stimulated with phenylephrine in the absence of external Ca(2+) (0Ca) to measure phasic isometric contractions. They gradually decreased with time in 0Ca, were inhibited with 2-aminoethoxydiphenyl borate, and declined with previous membrane potential hyperpolarization (levcromakalim) or with previous inhibition of LCC (diltiazem). Former basal stimulation of LCC with depolarization (15 mM K(+)) or with BAY K8644 increased the subsequent phasic contractions by phenylephrine in 0Ca. Although exogenous NO (diethylamine NONOate) reduced the phasic contractions by phenylephrine, stimulation of endothelial cells with acetylcholine in 0Ca failed to attenuate these phasic contractions. Finally, inhibition of the basal release of NO with N(Ω)-nitro-L-arginine methyl ester also attenuated the phasic contractions by phenylephrine. Results indicated that α1-adrenoceptor stimulation with phenylephrine causes phasic contractions, which are controlled by basal LCC and endothelial NO synthase activity. Endothelial NO release by acetylcholine was absent in 0Ca. Given the growing interest in the active regulation of arterial compliance, the dependence of contractile SR Ca(2+) store-refilling in basal conditions on the activity of LCC and basal eNOS may contribute to a more thorough understanding of physiological mechanisms leading to arterial stiffness. Copyright © 2015. Published by Elsevier B.V.

  9. The site of net absorption of Ca from the intestinal tract of growing pigs and effect of phytic acid, Ca level and Ca source on Ca digestibility.

    PubMed

    González-Vega, J Caroline; Walk, Carrie L; Liu, Yanhong; Stein, Hans H

    2014-01-01

    An experiment was conducted to test the hypothesis that the standardised digestibility of Ca in calcium carbonate and Lithothamnium calcareum Ca is not different regardless of the level of dietary Ca, and that phytic acid affects the digestibility of Ca in these two ingredients to the same degree. The objectives were to determine where in the intestinal tract Ca absorption takes place and if there are measurable quantities of basal endogenous Ca fluxes in the stomach, small intestine or large intestine. Diets contained calcium carbonate or L. calcareum Ca as the sole source of Ca, 0% or 1% phytic acid and 0.4% or 0.8% Ca. A Ca-free diet was also formulated and used to measure endogenous fluxes and losses of Ca. Nine growing pigs (initial body weight 23.8 ± 1.3 kg) were cannulated in the duodenum and in the distal ileum, and faecal, ileal and duodenal samples were collected. Duodenal endogenous fluxes of Ca were greater (p < 0.05) than ileal endogenous fluxes and total tract endogenous losses of Ca, but ileal endogenous fluxes were less (p < 0.05) than total tract endogenous losses. Standardised digestibility of Ca was not affected by the level of phytic acid, but decreased (p < 0.05) as Ca level increased in L. calcareum Ca diets, but that was not the case if calcium carbonate was the source of Ca (interaction, p < 0.05). The standardised duodenal digestibility (SDD), standardised ileal digestibility (SID) and standardised total tract digestibility (STTD) of Ca were not different if calcium carbonate was the source of dietary Ca. However, the STTD of Ca in L. calcareum Ca was greater (p < 0.05) than the SID and SDD of Ca. The SDD, SID and STTD of Ca in calcium carbonate were greater (p < 0.05) than those of L. calcareum Ca. In conclusion, under the conditions of this experiment, standardised digestibility of Ca is not affected by the level of phytic acid, but may be affected by dietary Ca level depending on the Ca source. Calcium from calcium carbonate is mostly

  10. Regulation of Blood Pressure by Targeting CaV1.2-Galectin-1 Protein Interaction.

    PubMed

    Hu, Zhenyu; Li, Guang; Wang, Jiong-Wei; Chong, Suet Yen; Yu, Dejie; Wang, Xiaoyuan; Soon, Jia Lin; Liang, Mui Cheng; Wong, Yuk Peng; Huang, Na; Colecraft, Henry M; Liao, Ping; Soong, Tuck Wah

    2018-04-12

    Background -L-type Ca V 1.2 channels play crucial roles in regulation of blood pressure. Galectin-1 (Gal-1), has been reported to bind to the I-II loop of Ca V 1.2 channels to reduce their current density. However, the mechanistic understanding for the down-regulation of Ca V 1.2 channels by Gal-1, and whether Gal-1 plays a direct role in blood pressure regulation remain unclear. Methods - In vitro experiments involving co-IP, western blot, patch-clamp recordings, immunohistochemistry and pressure myography were used to evaluate the molecular mechanisms by which Gal-1 down-regulates Ca V 1.2 channel in transfected HEK 293 cells, smooth muscle cells, arteries from Lgasl1 -/- mice, rat and human patients. In vivo experiments involving delivery of Tat-e9c peptide and AAV5-Gal-1 into rats were performed to investigate the effect of targeting Ca V 1.2-Gal-1 interaction on blood pressure monitored by tail cuff or telemetry methods. Results -Our study reveals that Gal-1 is a key regulator for proteasomal degradation of Ca V 1.2 channels. Gal-1 competed allosterically with Ca V β subunit for binding to the I-II loop of Ca V 1.2 channel. This competitive disruption of Ca V β binding led to Ca V 1.2 degradation by exposing the channels to poly-ubiquitination. Notably, we demonstrated that the inverse relationship of reduced Gal-1 and increased Ca V 1.2 protein levels in arteries was associated with hypertension in hypertensive rats and patients, and Gal-1 deficiency induces higher blood pressure in mice due to up-regulated Ca V 1.2 protein level in arteries. To directly regulate blood pressure by targeting the Ca V 1.2-Gal-1 interaction, we administered Tat-e9c, a peptide that competed for binding of Gal-1, by a mini-osmotic pump and this specific disruption of Ca V 1.2-Gal-1 coupling increased smooth muscle Ca V 1.2 currents, induced larger arterial contraction and caused hypertension in rats. In contrasting experiments, over-expression of Gal-1 in smooth muscle by a

  11. Subpopulation of store-operated Ca2+ channels regulate Ca2+-induced Ca2+ release in non-excitable cells.

    PubMed

    Yao, Jian; Li, Qin; Chen, Jin; Muallem, Shmuel

    2004-05-14

    Ca2+-induced Ca2+ release (CICR) is a well characterized activity in skeletal and cardiac muscles mediated by the ryanodine receptors. The present study demonstrates CICR in the non-excitable parotid acinar cells, which resembles the mechanism described in cardiac myocytes. Partial depletion of internal Ca2+ stores leads to a minimal activation of Ca2+ influx. Ca2+ influx through this pathway results in an explosive mobilization of Ca2+ from the majority of the stores by CICR. Thus, stimulation of parotid acinar cells in Ca2+ -free medium with 0.5 microm carbachol releases approximately 5% of the Ca2+ mobilizable by 1 mm carbachol. Addition of external Ca2+ induced the same Ca2+ release observed in maximally stimulated cells. Similar results were obtained by a short treatment with 2.5-10 microm cyclopiazonic acid, an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase pump. The Ca2+ release induced by the addition of external Ca2+ was largely independent of IP(3)Rs because it was reduced by only approximately 30% by the inhibition of the inositol 1,4,5-trisphosphate receptors with caffeine or heparin. Measurements of Ca2+ -activated outward current and [Ca2+](i) suggested that most CICR triggered by Ca2+ influx occurred away from the plasma membrane. Measurement of the response to several concentrations of cyclopiazonic acid revealed that Ca2+ influx that regulates CICR is associated with a selective portion of the internal Ca2+ pool. The minimal activation of Ca2+ influx by partial store depletion was confirmed by the measurement of Mn2+ influx. Inhibition of Ca2+ influx with SKF96365 or 2-aminoethoxydiphenyl borate prevented activation of CICR observed on addition of external Ca2+. These findings provide evidence for activation of CICR by Ca2+ influx in non-excitable cells, demonstrate a previously unrecognized role for Ca2+ influx in triggering CICR, and indicate that CICR in non-excitable cells resembles CICR in cardiac myocytes with the exception that in

  12. Modulation of contraction by intracellular Na+ via Na(+)-Ca2+ exchange in single shark (Squalus acanthias) ventricular myocytes.

    PubMed Central

    Näbauer, M; Morad, M

    1992-01-01

    1. The effect of direct alteration of intracellular Na+ concentration on contractile properties of whole-cell clamped shark ventricular myocytes was studied using an array of 256 photodiodes to monitor the length of the isolated myocytes. 2. In myocytes dialysed with Na(+)-free solution, the voltage dependence of Ca2+ current (ICa) and contraction were similar and bell shaped. Contractions activated at all voltages were completely suppressed by nifedipine (5 microM), and failed to show significant tonic components, suggesting dependence of the contraction on Ca2+ influx through the L-type Ca2+ channel. 3. In myocytes dialysed with 60 mM Na+, a ICa-dependent and a ICa-independent component of contraction could be identified. The Ca2+ current-dependent component was prominent in voltages between -30 to +10 mV. The ICa-independent contractions were maintained for the duration of depolarization, increased with increasing depolarization between +10 to +100 mV, and were insensitive to nifedipine. 4. In such myocytes, repolarization produced slowly decaying inward tail currents closely related to the time course of relaxation and the degree of shortening prior to repolarization. 5. With 60 mM Na+ in the pipette solution, positive clamp potentials activated decaying outward currents which correlated to the size of contraction. These outward currents appeared to be generated by the Na(+)-Ca(2+)-exchanger since they depended on the presence of intracellular Na+, and were neither suppressed by nifedipine nor by K+ channel blockers. 6. The results suggest that in shark (Squalus acanthias) ventricular myocytes, which lack functionally relevant Ca2+ release pools, both Ca2+ channel and the Na(+)-Ca2+ exchanger deliver sufficient Ca2+ to activate contraction, though the effectiveness of the latter mechanism was highly dependent on the [Na+]i. PMID:1338467

  13. The BK(Ca) channels deficiency as a possible reason for radiation-induced vascular hypercontractility.

    PubMed

    Kyrychenko, Sergii; Tishkin, Sergey; Dosenko, Victor; Ivanova, Irina; Novokhatska, Tatiana; Soloviev, Anatoly

    2012-01-01

    It is likely that large-conductance Ca²⁺-activated K⁺ (BK(Ca)) channels channelopathy tightly involved in vascular malfunctions and arterial hypertension development. In the present study, we compared the results of siRNAs-induced α-BK(Ca) gene silencing and vascular abnormalities produced by whole-body ionized irradiation in rats. The experimental design comprised RT-PCR and patch clamp technique, thoracic aorta smooth muscle (SM) contractile recordings and arterial blood pressure (BP) measurements on the 30th day after whole body irradiation (6Gy) and following siRNAs KCNMA1 gene silencing in vivo. The expression profile of BK(Ca) mRNA transcripts in SM was significantly decreased in siRNAs-treated rats in a manner similar to irradiated SM. In contrast, the mRNA levels of K(v) and K(ATP) were significantly increased while L-type calcium channels mRNA transcripts demonstrated tendency to increment. The SMCs obtained from irradiated animals and after KCNMA1 gene silencing showed a significant decrease in total K⁺ current density amplitude. Paxilline (500 nM)-sensitive components of outward current were significantly decreased in both irradiated and gene silencing SMCs. KCNMA1 gene silencing increased SM sensitivity to norepinephrine while Ach-induced relaxation had decreased. The silencing of KCNMA1 had no significant effect on BP while radiation produced sustained arterial hypertension. Therefore, radiation alters the form and function of the BK(Ca) channel and this type of channelopathy may contribute to related vascular abnormalities. Nevertheless, it is unlikely that BK(Ca) can operate as a crucial factor for radiation-induced arterial hypertension. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts

    NASA Technical Reports Server (NTRS)

    Chen, N. X.; Ryder, K. D.; Pavalko, F. M.; Turner, C. H.; Burr, D. B.; Qiu, J.; Duncan, R. L.

    2000-01-01

    Osteoblasts subjected to fluid shear increase the expression of the early response gene, c-fos, and the inducible isoform of cyclooxygenase, COX-2, two proteins linked to the anabolic response of bone to mechanical stimulation, in vivo. These increases in gene expression are dependent on shear-induced actin stress fiber formation. Here, we demonstrate that MC3T3-E1 osteoblast-like cells respond to shear with a rapid increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) that we postulate is important to subsequent cellular responses to shear. To test this hypothesis, MC3T3-E1 cells were grown on glass slides coated with fibronectin and subjected to laminar fluid flow (12 dyn/cm(2)). Before application of shear, cells were treated with two Ca(2+) channel inhibitors or various blockers of intracellular Ca(2+) release for 0. 5-1 h. Although gadolinium, a mechanosensitive channel blocker, significantly reduced the [Ca(2+)](i) response, neither gadolinium nor nifedipine, an L-type channel Ca(2+) channel blocker, were able to block shear-induced stress fiber formation and increase in c-fos and COX-2 in MC3T3-E1 cells. However, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, an intracellular Ca(2+) chelator, or thapsigargin, which empties intracellular Ca(2+) stores, completely inhibited stress fiber formation and c-fos/COX-2 production in sheared osteoblasts. Neomycin or U-73122 inhibition of phospholipase C, which mediates D-myo-inositol 1,4,5-trisphosphate (IP(3))-induced intracellular Ca(2+) release, also completely suppressed actin reorganization and c-fos/COX-2 production. Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform of U-73122, did not inhibit these shear-induced responses. These results suggest that IP(3)-mediated intracellular Ca(2+) release is required for modulating flow-induced responses in MC3T3-E1 cells.

  15. Alcohol Withdrawal-Induced Seizure Susceptibility is Associated with an Upregulation of CaV1.3 Channels in the Rat Inferior Colliculus

    PubMed Central

    Akinfiresoye, Luli R.; Allard, Joanne S.; Lovinger, David M.

    2015-01-01

    Background: We previously reported increased current density through L-type voltage-gated Ca2+ (CaV1) channels in inferior colliculus (IC) neurons during alcohol withdrawal. However, the molecular correlate of this increased CaV1 current is currently unknown. Methods: Rats received three daily doses of ethanol every 8 hours for 4 consecutive days; control rats received vehicle. The IC was dissected at various time intervals following alcohol withdrawal, and the mRNA and protein levels of the CaV1.3 and CaV1.2 α1 subunits were measured. In separate experiments, rats were tested for their susceptibility to alcohol withdrawal–induced seizures (AWS) 3, 24, and 48 hours after alcohol withdrawal. Results: In the alcohol-treated group, AWS were observed 24 hours after withdrawal; no seizures were observed at 3 or 48 hours. No seizures were observed at any time in the control-treated rats. Compared to control-treated rats, the mRNA level of the CaV1.3 α1 subunit was increased 1.4-fold, 1.9-fold, and 1.3-fold at 3, 24, and 48 hours, respectively. In contrast, the mRNA level of the CaV1.2 α1 subunit increased 1.5-fold and 1.4-fold at 24 and 48 hours, respectively. At 24 hours, Western blot analyses revealed that the levels of the CaV1.3 and CaV1.2 α1 subunits increased by 52% and 32%, respectively, 24 hours after alcohol withdrawal. In contrast, the CaV1.2 and CaV1.3 α1 subunits were not altered at either 3 or 48 hours during alcohol withdrawal. Conclusions: Expression of the CaV1.3 α1 subunit increased in parallel with AWS development, suggesting that altered L-type CaV1.3 channel expression is an important feature of AWS pathogenesis. PMID:25556199

  16. Nitric Oxide Induces Ca2+-independent Activity of the Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)*

    PubMed Central

    Coultrap, Steven J.; Bayer, K. Ulrich

    2014-01-01

    Both signaling by nitric oxide (NO) and by the Ca2+/calmodulin (CaM)-dependent protein kinase II α isoform (CaMKIIα) are implicated in two opposing forms of synaptic plasticity underlying learning and memory, as well as in excitotoxic/ischemic neuronal cell death. For CaMKIIα, these functions specifically involve also Ca2+-independent autonomous activity, traditionally generated by Thr-286 autophosphorylation. Here, we demonstrate that NO-induced S-nitrosylation of CaMKIIα also directly generated autonomous activity, and that CaMKII inhibition protected from NO-induced neuronal cell death. NO induced S-nitrosylation at Cys-280/289, and mutation of either site abolished autonomy, indicating that simultaneous nitrosylation at both sites was required. Additionally, autonomy was generated only when Ca2+/CaM was present during NO exposure. Thus, generation of this form of CaMKIIα autonomy requires simultaneous signaling by NO and Ca2+. Nitrosylation also significantly reduced subsequent CaMKIIα autophosphorylation specifically at Thr-286, but not at Thr-305. A previously described reduction of CaMKII activity by S-nitrosylation at Cys-6 was also observed here, but only after prolonged (>5 min) exposure to NO donors. These results demonstrate a novel regulation of CaMKII by another second messenger system and indicate its involvement in excitotoxic neuronal cell death. PMID:24855644

  17. Activation of sodium channels by α-scorpion toxin, BmK NT1, produced neurotoxicity in cerebellar granule cells: an association with intracellular Ca2+ overloading.

    PubMed

    He, Yuwei; Zou, Xiaohan; Li, Xichun; Chen, Juan; Jin, Liang; Zhang, Fan; Yu, Boyang; Cao, Zhengyu

    2017-02-01

    Voltage-gated sodium channels (VGSCs) are responsible for the action potential generation in excitable cells including neurons and involved in many physiological and pathological processes. Scorpion toxins are invaluable tools to explore the structure and function of ion channels. BmK NT1, a scorpion toxin from Buthus martensii Karsch, stimulates sodium influx in cerebellar granule cells (CGCs). In this study, we characterized the mode of action of BmK NT1 on the VGSCs and explored the cellular response in CGC cultures. BmK NT1 delayed the fast inactivation of VGSCs, increased the Na + currents, and shifted the steady-state activation and inactivation to more hyperpolarized membrane potential, which was similar to the mode of action of α-scorpion toxins. BmK NT1 stimulated neuron death (EC 50  = 0.68 µM) and produced massive intracellular Ca 2+ overloading (EC 50  = 0.98 µM). TTX abrogated these responses, suggesting that both responses were subsequent to the activation of VGSCs. The Ca 2+ response of BmK NT1 was primary through extracellular Ca 2+ influx since reducing the extracellular Ca 2+ concentration suppressed the Ca 2+ response. Further pharmacological evaluation demonstrated that BmK NT1-induced Ca 2+ influx and neurotoxicity were partially blocked either by MK-801, an NMDA receptor blocker, or by KB-R7943, an inhibitor of Na + /Ca 2+ exchangers. Nifedipine, an L-type Ca 2+ channel inhibitor, slightly suppressed both Ca 2+ response and neurotoxicity. A combination of these three inhibitors abrogated both responses. Considered together, these data ambiguously demonstrated that activation of VGSCs by an α-scorpion toxin was sufficient to produce neurotoxicity which was associated with intracellular Ca 2+ overloading through both NMDA receptor- and Na + /Ca 2+ exchanger-mediated Ca 2+ influx.

  18. Thapsigargin-induced activation of Ca(2+)-CaMKII-ERK in brainstem contributes to substance P release and induction of emesis in the least shrew.

    PubMed

    Zhong, Weixia; Chebolu, Seetha; Darmani, Nissar A

    2016-04-01

    Cytoplasmic calcium (Ca(2+)) mobilization has been proposed to be an important factor in the induction of emesis. The selective sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin, is known to deplete intracellular Ca(2+) stores, which consequently evokes extracellular Ca(2+) entry through cell membrane-associated channels, accompanied by a prominent rise in cytosolic Ca(2+). A pro-drug form of thapsigargin is currently under clinical trial as a targeted cancer chemotherapeutic. We envisioned that the intracellular effects of thapsigargin could cause emesis and planned to investigate its mechanisms of emetic action. Indeed, thapsigargin did induce vomiting in the least shrew in a dose-dependent and bell-shaped manner, with maximal efficacy (100%) at 0.5 mg/kg (i.p.). Thapsigargin (0.5 mg/kg) also caused increases in c-Fos immunoreactivity in the brainstem emetic nuclei including the area postrema (AP), nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMNX), as well as enhancement of substance P (SP) immunoreactivity in DMNX. In addition, thapsigargin (0.5 mg/kg, i.p.) led to vomit-associated and time-dependent increases in phosphorylation of Ca(2+)/calmodulin kinase IIα (CaMKIIα) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) in the brainstem. We then explored the suppressive potential of diverse chemicals against thapsigargin-evoked emesis including antagonists of: i) neurokinin-1 receptors (netupitant), ii) the type 3 serotonin receptors (palonosetron), iii) store-operated Ca(2+) entry (YM-58483), iv) L-type Ca(2+) channels (nifedipine), and v) SER Ca(2+)-release channels inositol trisphosphate (IP3Rs) (2-APB)-, and ryanodine (RyRs) (dantrolene)-receptors. In addition, the antiemetic potential of inhibitors of CaMKII (KN93) and ERK1/2 (PD98059) were investigated. All tested antagonists/blockers attenuated emetic parameters to varying degrees except palonosetron, however a combination of non

  19. P-type Ca2+ channels mediate excitatory and inhibitory synaptic transmitter release in crayfish muscle.

    PubMed

    Araque, A; Clarac, F; Buño, W

    1994-05-10

    The toxin fraction (FTX) and peptide omega-Aga-IVA from the venom of the funnel-web spider Agelenopsis aperta, as well as a synthetic analogue of FTX, specifically block the P-type voltage-dependent Ca2+ channel (VDCC). The effects of these toxins on synaptic transmission were studied in the neuromuscular synapses of the crayfish opener muscle, which has a single excitatory and a single inhibitory motoneuron. FTX selectively and reversibly blocked excitatory and inhibitory postsynaptic currents and potentials in a dose-dependent manner. FTX had no effect on (i) resting and postsynaptic membrane conductance, (ii) postsynaptic L-type VDCC, and (iii) both glutamate- and gamma-aminobutyric acid-induced postsynaptic responses. Mean amplitude and frequency of miniature postsynaptic potentials were unchanged by FTX. The postsynaptic VDCC was inhibited by nifedipine, a selective dihydropyridine antagonist of L-type VDCC, whereas synaptic transmission was unaffected. Transmission was also undisturbed by omega-conotoxin, suggesting that N-type VDCCs are not involved. The peptide omega-Aga-IVA blocked excitatory and inhibitory transmission without affecting postsynaptic VDCC. Synaptic transmission was also blocked by synthetic FTX. We conclude that presynaptic P-type VDCCs are involved in both evoked excitatory and inhibitory transmitter release in crayfish neuromuscular synapses.

  20. P-type Ca2+ channels mediate excitatory and inhibitory synaptic transmitter release in crayfish muscle.

    PubMed Central

    Araque, A; Clarac, F; Buño, W

    1994-01-01

    The toxin fraction (FTX) and peptide omega-Aga-IVA from the venom of the funnel-web spider Agelenopsis aperta, as well as a synthetic analogue of FTX, specifically block the P-type voltage-dependent Ca2+ channel (VDCC). The effects of these toxins on synaptic transmission were studied in the neuromuscular synapses of the crayfish opener muscle, which has a single excitatory and a single inhibitory motoneuron. FTX selectively and reversibly blocked excitatory and inhibitory postsynaptic currents and potentials in a dose-dependent manner. FTX had no effect on (i) resting and postsynaptic membrane conductance, (ii) postsynaptic L-type VDCC, and (iii) both glutamate- and gamma-aminobutyric acid-induced postsynaptic responses. Mean amplitude and frequency of miniature postsynaptic potentials were unchanged by FTX. The postsynaptic VDCC was inhibited by nifedipine, a selective dihydropyridine antagonist of L-type VDCC, whereas synaptic transmission was unaffected. Transmission was also undisturbed by omega-conotoxin, suggesting that N-type VDCCs are not involved. The peptide omega-Aga-IVA blocked excitatory and inhibitory transmission without affecting postsynaptic VDCC. Synaptic transmission was also blocked by synthetic FTX. We conclude that presynaptic P-type VDCCs are involved in both evoked excitatory and inhibitory transmitter release in crayfish neuromuscular synapses. Images PMID:7910404

  1. Decoding Ca2+ signals in plants

    NASA Technical Reports Server (NTRS)

    Sathyanarayanan, P. V.; Poovaiah, B. W.

    2004-01-01

    Different input signals create their own characteristic Ca2+ fingerprints. These fingerprints are distinguished by frequency, amplitude, duration, and number of Ca2+ oscillations. Ca(2+)-binding proteins and protein kinases decode these complex Ca2+ fingerprints through conformational coupling and covalent modifications of proteins. This decoding of signals can lead to a physiological response with or without changes in gene expression. In plants, Ca(2+)-dependent protein kinases and Ca2+/calmodulin-dependent protein kinases are involved in decoding Ca2+ signals into phosphorylation signals. This review summarizes the elements of conformational coupling and molecular mechanisms of regulation of the two groups of protein kinases by Ca2+ and Ca2+/calmodulin in plants.

  2. A Devil in the Details: Matrix-Dependent 40Ca42Ca++/42Ca+ and Its Effects on Estimates of the Initial 41Ca/40Ca in the Solar System

    NASA Astrophysics Data System (ADS)

    McKeegan, K. D.; Liu, M.-C.

    2015-07-01

    Ian Hutcheon established that the molecular ion interference 40Ca42Ca++/42Ca+ on 41K+ is strongly dependent on the mineral analyzed. Correction for this "matrix effect" led to a downward revision of the initial 41Ca/40Ca of the solar system.

  3. CaFe interstellar clouds

    NASA Astrophysics Data System (ADS)

    Bondar, A.; Kozak, M.; Gnaciński, P.; Galazutdinov, G. A.; Beletsky, Y.; Krełowski, J.

    2007-07-01

    A new kind of interstellar cloud is proposed. These are rare (just a few examples among ~300 lines of sight) objects with the CaI 4227-Å, FeI 3720-Å and 3860-Å lines stronger than those of KI (near 7699 Å) and NaI (near 3302 Å). We propose the name `CaFe' for these clouds. Apparently they occupy different volumes from the well-known interstellar HI clouds where the KI and ultraviolet NaI lines are dominant features. In the CaFe clouds we have not found either detectable molecular features (CH, CN) or diffuse interstellar bands which, as commonly believed, are carried by some complex, organic molecules. We have found the CaFe clouds only along sightlines toward hot, luminous (and thus distant) objects with high rates of mass loss. In principle, the observed gas-phase interstellar abundances reflect the combined effects of the nucleosynthetic history of the material, the depletion of heavy elements into dust grains and the ionization state of these elements which may depend on irradiation by neighbouring stars. Based on data collected using the Maestro spectrograph at the Terskol 2-m telescope, Russia; and on data collected using the ESO Feros spectrograph; and on data obtained from the ESO Science Archive Facility acquired with the UVES spectrograph, Chile. E-mail: `arctur'@rambler.ru (AB); marizak@astri.uni.torun.pl (MK); pg@iftia.univ.gda.pl (PG); gala@boao.re.kr (GAG); ybialets@eso.org (YB); jacek@astri.uni.torun.pl (JK)

  4. Pharmacological characterization of the voltage-dependent Ca2+ channels present in synaptosomes from rat and chicken central nervous system.

    PubMed

    Alvarez Maubecin, V; Sanchez, V N; Rosato Siri, M D; Cherksey, B D; Sugimori, M; Llinás, R; Uchitel, O D

    1995-06-01

    The voltage-dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L-type channels (1,4-dihydropyridines), N-type channels (omega-conotoxin GVIA), and P-type channels [funnel web toxin (FTX) and omega-agatoxin IVA]. K(+)-induced Ca2+ uptake by chicken synaptosomes was blocked by omega-conotoxin GVIA (IC50 = 250 nM). This toxin at 5 microM did not block Ca2+ entry into rat frontal cortex synaptosomes. FTX and omega-agatoxin IVA blocked Ca2+ uptake by rat synaptosomes (IC50 = 0.17 microliter/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and omega-agatoxin IVA affected Ca2+ uptake, FTX (3 microliters/ml) exerted a maximal inhibition of 40% with an IC50 similar to the one obtained in rat preparations, whereas with omega-agatoxin IVA saturation was not reached even at 5 microM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 microliter/ml) and different concentrations of omega-conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and omega-conotoxin GVIA was never greater than the one observed with omega-conotoxin GVIA. We also found that 60% of the Ca2+ uptake by rat and chicken synaptosomes was inhibited by omega-conotoxin MVIID (1 microM), a toxin that has a high index of discrimination against N-type channels. Conversely, nitrendipine (10 microM) had no significant effect on Ca2+ uptake in either the rat or the chicken. In conclusion, Ca2+ uptake by rat synaptosomes is potently inhibited by different P-type Ca2+ channel blockers, thus indicating that P-type channels are predominant in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. CGP-37157 inhibits the sarcoplasmic reticulum Ca²+ ATPase and activates ryanodine receptor channels in striated muscle.

    PubMed

    Neumann, Jake T; Diaz-Sylvester, Paula L; Fleischer, Sidney; Copello, Julio A

    2011-01-01

    7-Chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one [CGP-37157 (CGP)], a benzothiazepine derivative of clonazepam, is commonly used as a blocker of the mitochondrial Na+/Ca²+ exchanger. However, evidence suggests that CGP could also affect other targets, such as L-type Ca²+ channels and plasmalemma Na+/Ca²+ exchanger. Here, we tested the possibility of a direct modulation of ryanodine receptor channels (RyRs) and/or sarco/endoplasmic reticulum Ca²+-stimulated ATPase (SERCA) by CGP. In the presence of ruthenium red (inhibitor of RyRs), CGP decreased SERCA-mediated Ca²+ uptake of cardiac and skeletal sarcoplasmic reticulum (SR) microsomes (IC₅₀ values of 6.6 and 9.9 μM, respectively). The CGP effects on SERCA activity correlated with a decreased V(max) of ATPase activity of SERCA-enriched skeletal SR fractions. CGP (≥ 5 μM) also increased RyR-mediated Ca²+ leak from skeletal SR microsomes. Planar bilayer studies confirmed that both cardiac and skeletal RyRs are directly activated by CGP (EC(50) values of 9.4 and 12.0 μM, respectively). In summary, we found that CGP inhibits SERCA and activates RyR channels. Hence, the action of CGP on cellular Ca²+ homeostasis reported in the literature of cardiac, skeletal muscle, and other nonmuscle systems requires further analysis to take into account the contribution of all CGP-sensitive Ca²+ transporters.

  6. CGP-37157 Inhibits the Sarcoplasmic Reticulum Ca2+ ATPase and Activates Ryanodine Receptor Channels in Striated Muscle

    PubMed Central

    Neumann, Jake T.; Diaz-Sylvester, Paula L.; Fleischer, Sidney

    2011-01-01

    7-Chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one [CGP-37157 (CGP)], a benzothiazepine derivative of clonazepam, is commonly used as a blocker of the mitochondrial Na+/Ca2+ exchanger. However, evidence suggests that CGP could also affect other targets, such as L-type Ca2+ channels and plasmalemma Na+/Ca2+ exchanger. Here, we tested the possibility of a direct modulation of ryanodine receptor channels (RyRs) and/or sarco/endoplasmic reticulum Ca2+-stimulated ATPase (SERCA) by CGP. In the presence of ruthenium red (inhibitor of RyRs), CGP decreased SERCA-mediated Ca2+ uptake of cardiac and skeletal sarcoplasmic reticulum (SR) microsomes (IC50 values of 6.6 and 9.9 μM, respectively). The CGP effects on SERCA activity correlated with a decreased Vmax of ATPase activity of SERCA-enriched skeletal SR fractions. CGP (≥5 μM) also increased RyR-mediated Ca2+ leak from skeletal SR microsomes. Planar bilayer studies confirmed that both cardiac and skeletal RyRs are directly activated by CGP (EC50 values of 9.4 and 12.0 μM, respectively). In summary, we found that CGP inhibits SERCA and activates RyR channels. Hence, the action of CGP on cellular Ca2+ homeostasis reported in the literature of cardiac, skeletal muscle, and other nonmuscle systems requires further analysis to take into account the contribution of all CGP-sensitive Ca2+ transporters. PMID:20923851

  7. Carcinogenesis of PIK3CA

    PubMed Central

    2013-01-01

    PIK3CA is the most frequently mutated oncogene in human cancers. PIK3CA is phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha. It controls cell growth, proliferation, motility, survival, differentiation and intracellular trafficking. In most of human cancer alteration occurred frequently in the alpha isoform of phosphatidylinositol 3 kinase. PIK3CA mutations were most frequent in endometrial, ovarian, colorectal, breast, cervical, squamous cell cancer of the head and neck, chondroma, thyroid carcinoma and in cancer family syndrome. Inhibition of PI3K signaling can diminish cell proliferation, and in some circumstances, promote cell death. Consequently, components of this pathway present attractive targets for cancer therapeutics. A number of PI3K pathway inhibitors have been developed and used. PI3K inhibitors (both pan-PI3K and isoform-specific PI3K inhibitors), dual PI3K-mTOR inhibitors that are catalytic site inhibitors of the p110 isoforms and mTOR (the kinase component of both mTORC1 and mTORC2), mTOR catalytic site inhibitors, and AKT inhibitors are the most advanced in the clinic. They are approved for the treatment of several carcinomas. PMID:23768168

  8. Manipulation of sarcoplasmic reticulum Ca2+ release in heart failure through mechanical intervention

    PubMed Central

    Ibrahim, Michael; Nader, Anas; Yacoub, Magdi H; Terracciano, Cesare

    2015-01-01

    Left ventricular assist devices (LVADs) were developed as a means of temporary circulatory support, but the mechanical unloading they offer also results in significant reverse remodelling. In selected patients, these improvements are sufficient to allow ultimate device explantation without requiring transplantation; this represents a fundamental shift in our understanding of heart failure. Like heart failure itself, LVADs influence multiple biological systems. The transverse tubules are a system of membrane invaginations in ventricular cardiomyocytes which allow rapid propagation of the action potential throughout the cell. Through their dense concentration of L-type Ca2+ channels in close proximity to intracellular ryanodine receptors, the t-tubules enable synchronous Ca2+ release throughout the cell. The t-tubules’ structure appears to be specifically regulated by mechanical load, such that either the overload of heart failure (or the spontaneously hypertensive rat model) or the profound unloading in a chronically unloaded heart result in impaired t-tubule structure, with ineffective Ca2+ release. While there are multiple molecular pathways which underpin t-tubule regulation, Telethonin (Tcap) appears to be important in regulating the effect of altered loading on the t-tubule system. PMID:25922157

  9. Arabidopsis transcriptional response to extracellular Ca2+ depletion involves a transient rise in cytosolic Ca2+.

    PubMed

    Wang, Jing; Tergel, Tergel; Chen, Jianhua; Yang, Ju; Kang, Yan; Qi, Zhi

    2015-02-01

    Ecological evidence indicates a worldwide trend of dramatically decreased soil Ca(2+) levels caused by increased acid deposition and massive timber harvesting. Little is known about the genetic and cellular mechanism of plants' responses to Ca(2+) depletion. In this study, transcriptional profiling analysis helped identify multiple extracellular Ca(2+) ([Ca(2+) ]ext ) depletion-responsive genes in Arabidopsis thaliana L., many of which are involved in response to other environmental stresses. Interestingly, a group of genes encoding putative cytosolic Ca(2+) ([Ca(2+) ]cyt ) sensors were significantly upregulated, implying that [Ca(2+) ]cyt has a role in sensing [Ca(2+) ]ext depletion. Consistent with this observation, [Ca(2+) ]ext depletion stimulated a transient rise in [Ca(2+) ]cyt that was negatively influenced by [K(+) ]ext , suggesting the involvement of a membrane potential-sensitive component. The [Ca(2+) ]cyt response to [Ca(2+) ]ext depletion was significantly desensitized after the initial treatment, which is typical of a receptor-mediated signaling event. The response was insensitive to an animal Ca(2+) sensor antagonist, but was suppressed by neomycin, an inhibitor of phospholipase C. Gd(3+) , an inhibitor of Ca(2+) channels, suppressed the [Ca(2+) ]ext -triggered rise in [Ca(2+) ]cyt and downstream changes in gene expression. Taken together, this study demonstrates that [Ca(2+) ]cyt plays an important role in the putative receptor-mediated cellular and transcriptional response to [Ca(2+) ]ext depletion of plant cells. © 2014 Institute of Botany, Chinese Academy of Sciences.

  10. Computer modeling of siRNA knockdown effects indicates an essential role of the Ca2+ channel alpha2delta-1 subunit in cardiac excitation-contraction coupling.

    PubMed

    Tuluc, Petronel; Kern, Georg; Obermair, Gerald J; Flucher, Bernhard E

    2007-06-26

    L-type Ca(2+) currents determine the shape of cardiac action potentials (AP) and the magnitude of the myoplasmic Ca(2+) signal, which regulates the contraction force. The auxiliary Ca(2+) channel subunits alpha(2)delta-1 and beta(2) are important regulators of membrane expression and current properties of the cardiac Ca(2+) channel (Ca(V)1.2). However, their role in cardiac excitation-contraction coupling is still elusive. Here we addressed this question by combining siRNA knockdown of the alpha(2)delta-1 subunit in a muscle expression system with simulation of APs and Ca(2+) transients by using a quantitative computer model of ventricular myocytes. Reconstitution of dysgenic muscle cells with Ca(V)1.2 (GFP-alpha(1C)) recapitulates key properties of cardiac excitation-contraction coupling. Concomitant depletion of the alpha(2)delta-1 subunit did not perturb membrane expression or targeting of the pore-forming GFP-alpha(1C) subunit into junctions between the outer membrane and the sarcoplasmic reticulum. However, alpha(2)delta-1 depletion shifted the voltage dependence of Ca(2+) current activation by 9 mV to more positive potentials, and it slowed down activation and inactivation kinetics approximately 2-fold. Computer modeling revealed that the altered voltage dependence and current kinetics exert opposing effects on the function of ventricular myocytes that in total cause a 60% prolongation of the AP and a 2-fold increase of the myoplasmic Ca(2+) concentration during each contraction. Thus, the Ca(2+) channel alpha(2)delta-1 subunit is not essential for normal Ca(2+) channel targeting in muscle but is a key determinant of normal excitation and contraction of cardiac muscle cells, and a reduction of alpha(2)delta-1 function is predicted to severely perturb normal heart function.

  11. Potentiation of inositol trisphosphate-induced Ca2+ mobilization in Xenopus oocytes by cytosolic Ca2+.

    PubMed Central

    Yao, Y; Parker, I

    1992-01-01

    1. The ability of cytosolic Ca2+ ions to modulate inositol 1,4,5-trisphosphate (Insp3)-induced Ca2+ liberation from intracellular stores was studied in Xenopus oocytes using light flash photolysis of caged InsP3. Changes in cytosolic free Ca2+ level were effected by inducing Ca2+ entry through ionophore and voltage-gated plasma membrane channels and by injection of Ca2+ through a micropipette. Their effects on Ca2+ liberation were monitored by video imaging of Fluo-3 fluorescence and by voltage clamp recording of Ca(2+)-activated membrane Cl- currents. 2. Treatment of oocytes with the Ca2+ ionophores A23187 and ionomycin caused a transient elevation of cytosolic Ca2+ level when cells were bathed in Ca(2+)-free solution, which probably arose because of release of Ca2+ from intracellular stores. 3. Membrane current and Fluo-3 Ca2+ signals evoked by photoreleased InsP3 in ionophore-treated oocytes were potentiated when the intracellular Ca2+ level was elevated by raising the Ca2+ level in the bathing solution. 4. Responses to photoreleased InsP3 were similarly potentiated following activation of Ca2+ entry through voltage-gated Ca2+ channels expressed in the plasma membrane. 5. Ca(2+)-activated membrane currents evoked by depolarization developed a delayed 'hump' component during sustained photorelease of InsP3, probably because Ca2+ ions entering through the membrane channels triggered liberation of Ca2+ from intracellular stores. 6. Ba2+ and Sr2+ ions were able to substitute for Ca2+ in potentiating InsP3-mediated Ca2+ liberation. 7. Gradual photorelease of InsP3 by weak photolysis light evoked Ca2+ liberation that began at particular foci and then propagated throughout, but not beyond that area of the oocyte exposed to the light. Local elevations of intracellular Ca2+ produced by microinjection of Ca2+ acted as new foci for the initiation of Ca2+ liberation by InsP3. 8. In resting oocytes, intracellular injections of Ca2+ resulted only in localized elevation of

  12. Inhibition of spontaneous activity of rabbit atrioventricular node cells by KB-R7943 and inhibitors of sarcoplasmic reticulum Ca2+ ATPase

    PubMed Central

    Cheng, Hongwei; Smith, Godfrey L.; Hancox, Jules C.; Orchard, Clive H.

    2011-01-01

    The atrioventricular node (AVN) can act as a subsidiary cardiac pacemaker if the sinoatrial node fails. In this study, we investigated the effects of the Na–Ca exchange (NCX) inhibitor KB-R7943, and inhibition of the sarcoplasmic reticulum calcium ATPase (SERCA), using thapsigargin or cyclopiazonic acid (CPA), on spontaneous action potentials (APs) and [Ca2+]i transients from cells isolated from the rabbit AVN. Spontaneous [Ca2+]i transients were monitored from undialysed AVN cells at 37 °C using Fluo-4. In separate experiments, spontaneous APs and ionic currents were recorded using the whole-cell patch clamp technique. Rapid application of 5 μM KB-R7943 slowed or stopped spontaneous APs and [Ca2+]i transients. However, in voltage clamp experiments in addition to blocking NCX current (INCX) KB-R7943 partially inhibited L-type calcium current (ICa,L). Rapid reduction of external [Na+] also abolished spontaneous activity. Inhibition of SERCA (using 2.5 μM thapsigargin or 30 μM CPA) also slowed or stopped spontaneous APs and [Ca2+]i transients. Our findings are consistent with the hypothesis that sarcoplasmic reticulum (SR) Ca2+ release influences spontaneous activity in AVN cells, and that this occurs via [Ca2+]i-activated INCX; however, the inhibitory action of KB-R7943 on ICa,L means that care is required in the interpretation of data obtained using this compound. PMID:21163524

  13. Hazardous Waste Minimization Assessment: Fort Ord, CA

    DTIC Science & Technology

    1991-06-01

    on microorganisms ( bacteria , fungi, etc.) to decompose and/or bioaccumulate the contaminants in wastes. As a HAZMIN technique, treatment, unlike source...washer X San Jose, CA manufacturer Kinsbursky Bros. Supply (714) 738-8516 North Lemon Street Recycler, Spent batteries x Anaheim. CA 92801 Lubrication Co...0863 2190 Main Street Processor X X San Diego, CA 92113 Pepper Oil Company. Inc. (619) 477-9336 2300 Tidelands Avenue Processor X X National City, CA

  14. Multiple C-terminal tail Ca(2+)/CaMs regulate Ca(V)1.2 function but do not mediate channel dimerization.

    PubMed

    Kim, Eun Young; Rumpf, Christine H; Van Petegem, Filip; Arant, Ryan J; Findeisen, Felix; Cooley, Elizabeth S; Isacoff, Ehud Y; Minor, Daniel L

    2010-12-01

    Interactions between voltage-gated calcium channels (Ca(V)s) and calmodulin (CaM) modulate Ca(V) function. In this study, we report the structure of a Ca(2+)/CaM Ca(V)1.2 C-terminal tail complex that contains two PreIQ helices bridged by two Ca(2+)/CaMs and two Ca(2+)/CaM-IQ domain complexes. Sedimentation equilibrium experiments establish that the complex has a 2:1 Ca(2+)/CaM:C-terminal tail stoichiometry and does not form higher order assemblies. Moreover, subunit-counting experiments demonstrate that in live cell membranes Ca(V)1.2s are monomers. Thus, contrary to previous proposals, the crystallographic dimer lacks physiological relevance. Isothermal titration calorimetry and biochemical experiments show that the two Ca(2+)/CaMs in the complex have different properties. Ca(2+)/CaM bound to the PreIQ C-region is labile, whereas Ca(2+)/CaM bound to the IQ domain is not. Furthermore, neither of lobes of apo-CaM interacts strongly with the PreIQ domain. Electrophysiological studies indicate that the PreIQ C-region has a role in calcium-dependent facilitation. Together, the data show that two Ca(2+)/CaMs can bind the Ca(V)1.2 tail simultaneously and indicate a functional role for Ca(2+)/CaM at the C-region site.

  15. Multiple C-terminal tail Ca2+/CaMs regulate CaV1.2 function but do not mediate channel dimerization

    PubMed Central

    Kim, Eun Young; Rumpf, Christine H; Van Petegem, Filip; Arant, Ryan J; Findeisen, Felix; Cooley, Elizabeth S; Isacoff, Ehud Y; Minor, Daniel L

    2010-01-01

    Interactions between voltage-gated calcium channels (CaVs) and calmodulin (CaM) modulate CaV function. In this study, we report the structure of a Ca2+/CaM CaV1.2 C-terminal tail complex that contains two PreIQ helices bridged by two Ca2+/CaMs and two Ca2+/CaM–IQ domain complexes. Sedimentation equilibrium experiments establish that the complex has a 2:1 Ca2+/CaM:C-terminal tail stoichiometry and does not form higher order assemblies. Moreover, subunit-counting experiments demonstrate that in live cell membranes CaV1.2s are monomers. Thus, contrary to previous proposals, the crystallographic dimer lacks physiological relevance. Isothermal titration calorimetry and biochemical experiments show that the two Ca2+/CaMs in the complex have different properties. Ca2+/CaM bound to the PreIQ C-region is labile, whereas Ca2+/CaM bound to the IQ domain is not. Furthermore, neither of lobes of apo-CaM interacts strongly with the PreIQ domain. Electrophysiological studies indicate that the PreIQ C-region has a role in calcium-dependent facilitation. Together, the data show that two Ca2+/CaMs can bind the CaV1.2 tail simultaneously and indicate a functional role for Ca2+/CaM at the C-region site. PMID:20953164

  16. Nimodipine, an L-type calcium channel blocker attenuates mitochondrial dysfunctions to protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism in mice.

    PubMed

    Singh, Alpana; Verma, Poonam; Balaji, Gillela; Samantaray, Supriti; Mohanakumar, Kochupurackal P

    2016-10-01

    Parkinson's disease (PD), the most common progressive neurodegenerative movement disorder, results from loss of dopaminergic neurons of substantia nigra pars compacta. These neurons exhibit Cav1.3 channel-dependent pacemaking activity. Epidemiological studies suggest reduced risk for PD in population under long-term antihypertensive therapy with L-type calcium channel antagonists. These prompted us to investigate nimodipine, an L-type calcium channel blocker for neuroprotective effect in cellular and animal models of PD. Nimodipine (0.1-10 μM) significantly attenuated 1-methyl-4-phenyl pyridinium ion-induced loss in mitochondrial morphology, mitochondrial membrane potential and increases in intracellular calcium levels in SH-SY5Y neuroblastoma cell line as measured respectively employing Mitotracker green staining, TMRM, and Fura-2 fluorescence, but only a feeble neuroprotective effect was observed in MTT assay. Nimodipine dose-dependently reduced 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonian syndromes (akinesia and catalepsy) and loss in swimming ability in Balb/c mice. It attenuated MPTP-induced loss of dopaminergic tyrosine hydroxylase positive neurons in substantia nigra, improved mitochondrial oxygen consumption and inhibited reactive oxygen species production in the striatal mitochondria measured using dichlorodihydrofluorescein fluorescence, but failed to block striatal dopamine depletion. These results point to an involvement of L-type calcium channels in MPTP-induced dopaminergic neuronal death in experimental parkinsonism and more importantly provide evidences for nimodipine to improve mitochondrial integrity and function. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Ca2+ Dependence of Synaptic Vesicle Endocytosis.

    PubMed

    Leitz, Jeremy; Kavalali, Ege T

    2016-10-01

    Ca(2+)-dependent synaptic vesicle recycling is essential for structural homeostasis of synapses and maintenance of neurotransmission. Although, the executive role of intrasynaptic Ca(2+) transients in synaptic vesicle exocytosis is well established, identifying the exact role of Ca(2+) in endocytosis has been difficult. In some studies, Ca(2+) has been suggested as an essential trigger required to initiate synaptic vesicle retrieval, whereas others manipulating synaptic Ca(2+) concentrations reported a modulatory role for Ca(2+) leading to inhibition or acceleration of endocytosis. Molecular studies of synaptic vesicle endocytosis, on the other hand, have consistently focused on the roles of Ca(2+)-calmodulin dependent phosphatase calcineurin and synaptic vesicle protein synaptotagmin as potential Ca(2+) sensors for endocytosis. Most studies probing the role of Ca(2+) in endocytosis have relied on measurements of synaptic vesicle retrieval after strong stimulation. Strong stimulation paradigms elicit fusion and retrieval of multiple synaptic vesicles and therefore can be affected by several factors besides the kinetics and duration of Ca(2+) signals that include the number of exocytosed vesicles and accumulation of released neurotransmitters thus altering fusion and retrieval processes indirectly via retrograde signaling. Studies monitoring single synaptic vesicle endocytosis may help resolve this conundrum as in these settings the impact of Ca(2+) on synaptic fusion probability can be uncoupled from its putative role on synaptic vesicle retrieval. Future experiments using these single vesicle approaches will help dissect the specific role(s) of Ca(2+) and its sensors in synaptic vesicle endocytosis. © The Author(s) 2015.

  18. Imaging Ca2+ with a Fluorescent Rhodol.

    PubMed

    Contractor, Alisha A; Miller, Evan W

    2018-01-16

    Ca 2+ mediates a host of biochemical and biophysical signaling processes in cells. The development of synthetic, Ca 2+ -sensitive fluorophores has played an instrumental role in our understanding of the temporal and spatial dynamics of Ca 2+ . Coupling Ca 2+ -selective ligands to fluorescent reporters has provided a wealth of excellent indicators that span the visible excitation and emission spectrum and possess Ca 2+ affinities suited to a variety of cellular contexts. One underdeveloped area is the use of hybrid rhodamine/fluorescein fluorophores, or rhodols, in the context of Ca 2+ sensing. Rhodols are bright and photostable and have good two-photon absorption cross sections (σ TPA ), making them excellent candidates for incorporation into Ca 2+ -sensing scaffolds. Here, we present the design, synthesis, and application of rhodol Ca 2+ sensor 1 (RCS-1), a chlorinated pyrrolidine-based rhodol. RCS-1 possesses a Ca 2+ binding constant of 240 nM and a 10-fold turn response to Ca 2+ . RCS-1 effectively absorbs infrared light and has a σ TPA of 76 GM at 840 nm, 3-fold greater than that of its fluorescein-based counterpart. The acetoxy-methyl ester of RCS-1 stains the cytosol of live cells, enabling observation of Ca 2+ fluctuations and cultured neurons using both one- and two-photon illumination. Together, these results demonstrate the utility of rhodol-based scaffolds for Ca 2+ sensing using two-photon illumination in neurons.

  19. Near-membrane [Ca2+] transients resolved using the Ca2+ indicator FFP18.

    PubMed

    Etter, E F; Minta, A; Poenie, M; Fay, F S

    1996-05-28

    (Ca2+)-sensitive processes at cell membranes involved in contraction, secretion, and neurotransmitter release are activated in situ or in vitro by Ca2+ concentrations ([Ca2+]) 10-100 times higher than [Ca2+] measured during stimulation in intact cells. This paradox might be explained if the local [Ca2+] at the cell membrane is very different from that in the rest of the cell. Soluble Ca2+ indicators, which indicate spatially averaged cytoplasmic [Ca2+], cannot resolve these localized, near-membrane [Ca2+] signals. FFP18, the newest Ca2+ indicator designed to selectively monitor near-membrane [Ca2+], has a lower Ca2+ affinity and is more water soluble than previously used membrane-associating Ca2+ indicators. Images of the intracellular distribution of FFP18 show that >65% is located on or near the plasma membrane. [Ca2+] transients recorded using FFP18 during membrane depolarization-induced Ca2+ influx show that near-membrane [Ca2+] rises faster and reaches micromolar levels at early times when the cytoplasmic [Ca2+], recorded using fura-2, has risen to only a few hundred nanomolar. High-speed series of digital images of [Ca2+] show that near-membrane [Ca2+], reported by FFP18, rises within 20 msec, peaks at 50-100 msec, and then declines. [Ca2+] reported by fura-2 rose slowly and continuously throughout the time images were acquired. The existence of these large, rapid increases in [Ca2+] directly beneath the surface membrane may explain how numerous (Ca2+)-sensitive membrane processes are activated at times when bulk cytoplasmic [Ca2+] changes are too small to activate them.

  20. Bradycardia alters Ca2+ dynamics enhancing dispersion of repolarization and arrhythmia risk

    PubMed Central

    Kim, Jong J.; Němec, Jan; Papp, Rita; Strongin, Robert; Abramson, Jonathan J.

    2013-01-01

    Bradycardia prolongs action potential (AP) durations (APD adaptation), enhances dispersion of repolarization (DOR), and promotes tachyarrhythmias. Yet, the mechanisms responsible for enhanced DOR and tachyarrhythmias remain largely unexplored. Ca2+ transients and APs were measured optically from Langendorff rabbit hearts at high (150 × 150 μm2) or low (1.5 × 1.5 cm2) magnification while pacing at a physiological (120 beats/min) or a slow heart rate (SHR = 50 beats/min). Western blots and pharmacological interventions were used to elucidate the regional effects of bradycardia. As a result, bradycardia (SHR 50 beats/min) increased APDs gradually (time constant τf→s = 48 ± 9.2 s) and caused a secondary Ca2+ release (SCR) from the sarcoplasmic reticulum during AP plateaus, occurring at the base on average of 184.4 ± 9.7 ms after the Ca2+ transient upstroke. In subcellular imaging, SCRs were temporally synchronous and spatially homogeneous within myocytes. In diastole, SHR elicited variable asynchronous sarcoplasmic reticulum Ca2+ release events leading to subcellular Ca2+ waves, detectable only at high magnification. SCR was regionally heterogeneous, correlated with APD prolongation (P < 0.01, n = 5), enhanced DOR (r = 0.9277 ± 0.03, n = 7), and was gradually reversed by pacing at 120 beats/min along with APD shortening (P < 0.05, n = 5). A stabilizer of leaky ryanodine receptors (RyR2), 3-(4-benzylcyclohexyl)-1-(7-methoxy-2,3-dihydrobenzo[f][1,4]thiazepin-4(5H)-yl)propan-1-one (K201; 1 μM), suppressed SCR and reduced APD at the base, thereby reducing DOR (P < 0.02, n = 5). Ventricular ectopy induced by bradycardia (n = 5/15) was suppressed by K201. Western blot analysis revealed spatial differences of voltage-gated L-type Ca2+ channel protein (Cav1.2α), Na+-Ca2+ exchange (NCX1), voltage-gated Na+ channel (Nav1.5), and rabbit ether-a-go-go-related (rERG) protein [but not RyR2 or sarcoplasmic reticulum Ca2+ ATPase 2a] that correlate with the SCR distribution

  1. Simulating LGM retreat of the Uummannaq Ice Stream and Rinks Isbrae, Western Greenland using a 1-D ice-stream model constrained by a suite of marine and terrestrial data

    NASA Astrophysics Data System (ADS)

    Jamieson, Stewart; Roberts, Dave; Rea, Brice; Lane, Timothy; Vieli, Andreas; Cofaigh, Colm Ó.

    2014-05-01

    We aim to understand what controlled the retreat pattern of the Uummannaq Ice Stream (UIS) during the last deglaciation. Evidence for the pattern of retreat is found in both the marine and terrestrial realms, but because the evidence is temporally and spatially discontinuous, it is challenging to coherently reconstruct both grounding-line retreat and ice-surface thinning such that they are in agreement. Marine stratigraphic and geophysical evidence indicates that the ice stream was grounded close to the continental shelf edge at the Last Glacial Maximum, and retreated rapidly and nonlinearly after 14.8 ka. Cosmogenic nuclide exposure dating on Ubekendt Island at the convergence zone of multiple feeder ice streams show that the ice surface thinned progressively and that the island became ice-free by ca. 12.4 ka. The ice stream then collapsed over the next 1-1.6 kyrs and the ice stream separated into a series of distinct inland arms. In the northernmost Rinks system, there is a 'staircase' of evidence showing ice surface thinning over time, but it is unclear where the grounding line was located during this phase of thinning. Furthermore, it is currently unclear what controlled the nonlinear retreat pattern identified in the Uummannaq system. We develop a numerical model of ice-stream retreat using the marine geophysical data and measurements of sediment strength on the continental shelf to control the boundary conditions. The model has the capability to dynamically and robustly simulate grounding line-retreat behaviour over millennial timescales. We simulate the retreat of the UIS grounding line into the northernmost Rinks system in response to enhanced ocean warming, rising sea level and warming climate. We compare the simulated dynamic behaviour of the UIS against the geomorphological and cosmogenic exposure evidence for ice surface thinning onshore and against dated marine grounding line positions. Our model results enable us to match grounding-line positions in

  2. Synthesis, biological evaluation and molecular modelling of diversely functionalized heterocyclic derivatives as inhibitors of acetylcholinesterase/butyrylcholinesterase and modulators of Ca2+ channels and nicotinic receptors.

    PubMed

    Marco, José L; de los Ríos, Cristóbal; García, Antonio G; Villarroya, Mercedes; Carreiras, M Carmo; Martins, Carla; Eleutério, Ana; Morreale, Antonio; Orozco, M; Luque, F Javier

    2004-05-01

    The synthesis and the biological activity of compounds 5-40 as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), as well as modulators of voltage-dependent Ca(2+) channels and nicotinic receptors, are described. These molecules are tacrine analogues, which have been prepared from polyfunctionalized 6-amino-5-cyano-4H-pyrans, 6-amino-5-cyano-pyridines and 5-amino-2-aryl-3-cyano-1,3-oxazoles via Friedländer reaction with selected cycloalkanones. These compounds are moderate acetylcholinesterase and butyrylcholinesterase inhibitors, the BuChE/AChE selectivity of the most active molecules ranges from 10.0 (compound 29) to 76.9 (compound 16). Interestingly, the 'oxazolo-tacrine' derivatives are devoid of any activity. All compounds showed an important inhibitory effect on the nicotinic acetylcholine receptor. Most of them also blocked L-type Ca(2+) channels, and three of them, 64, 19 and 67, the non-L type of Ca(2+) channels. Molecular modelling studies suggest that these compounds might bind at the peripheral binding site of AChE, which opens the possibility to design inhibitors able to bind at both, the catalytic and peripheral binding sites of the enzyme.

  3. Evolutionary insights into T-type Ca2+ channel structure, function, and ion selectivity from the Trichoplax adhaerens homologue

    PubMed Central

    Smith, Carolyn L.; Abdallah, Salsabil; Le, Phuong; Harracksingh, Alicia N.; Artinian, Liana; Tamvacakis, Arianna N.; Rehder, Vincent; Reese, Thomas S.

    2017-01-01

    Four-domain voltage-gated Ca2+ (Cav) channels play fundamental roles in the nervous system, but little is known about when or how their unique properties and cellular roles evolved. Of the three types of metazoan Cav channels, Cav1 (L-type), Cav2 (P/Q-, N- and R-type) and Cav3 (T-type), Cav3 channels are optimized for regulating cellular excitability because of their fast kinetics and low activation voltages. These same properties permit Cav3 channels to drive low-threshold exocytosis in select neurons and neurosecretory cells. Here, we characterize the single T-type calcium channel from Trichoplax adhaerens (TCav3), an early diverging animal that lacks muscle, neurons, and synapses. Co-immunolocalization using antibodies against TCav3 and neurosecretory cell marker complexin labeled gland cells, which are hypothesized to play roles in paracrine signaling. Cloning and in vitro expression of TCav3 reveals that, despite roughly 600 million years of divergence from other T-type channels, it bears the defining structural and biophysical features of the Cav3 family. We also characterize the channel’s cation permeation properties and find that its pore is less selective for Ca2+ over Na+ compared with the human homologue Cav3.1, yet it exhibits a similar potent block of inward Na+ current by low external Ca2+ concentrations (i.e., the Ca2+ block effect). A comparison of the permeability features of TCav3 with other cloned channels suggests that Ca2+ block is a locus of evolutionary change in T-type channel cation permeation properties and that mammalian channels distinguish themselves from invertebrate ones by bearing both stronger Ca2+ block and higher Ca2+ selectivity. TCav3 is the most divergent metazoan T-type calcium channel and thus provides an evolutionary perspective on Cav3 channel structure–function properties, ion selectivity, and cellular physiology. PMID:28330839

  4. Electrogenic Na+/Ca2+ Exchange

    PubMed Central

    Danaceau, Jonathan P.; Lucero, Mary T.

    2000-01-01

    Olfactory receptor neurons (ORNs) from the squid, Lolliguncula brevis, respond to the odors l-glutamate or dopamine with increases in internal Ca2+ concentrations ([Ca2+]i). To directly asses the effects of increasing [Ca2+]i in perforated-patched squid ORNs, we applied 10 mM caffeine to release Ca2+ from internal stores. We observed an inward current response to caffeine. Monovalent cation replacement of Na+ from the external bath solution completely and selectively inhibited the caffeine-induced response, and ruled out the possibility of a Ca2+-dependent nonselective cation current. The strict dependence on internal Ca2+ and external Na+ indicated that the inward current was due to an electrogenic Na+/Ca2+ exchanger. Block of the caffeine-induced current by an inhibitor of Na+/Ca2+ exchange (50–100 μM 2′,4′-dichlorobenzamil) and reversibility of the exchanger current, further confirmed its presence. We tested whether Na+/Ca2+ exchange contributed to odor responses by applying the aquatic odor l-glutamate in the presence and absence of 2′,4′-dichlorobenzamil. We found that electrogenic Na+/Ca2+ exchange was responsible for ∼26% of the total current associated with glutamate-induced odor responses. Although Na+/Ca2+ exchangers are known to be present in ORNs from numerous species, this is the first work to demonstrate amplifying contributions of the exchanger current to odor transduction. PMID:10828249

  5. Rate-dependent Ca2+ signalling underlying the force-frequency response in rat ventricular myocytes: a coupled electromechanical modeling study

    PubMed Central

    2013-01-01

    Background Rate-dependent effects on the Ca2+ sub-system in a rat ventricular myocyte are investigated. Here, we employ a deterministic mathematical model describing various Ca2+ signalling pathways under voltage clamp (VC) conditions, to better understand the important role of calmodulin (CaM) in modulating the key control variables Ca2+/calmodulin-dependent protein kinase-II (CaMKII), calcineurin (CaN), and cyclic adenosine monophosphate (cAMP) as they affect various intracellular targets. In particular, we study the frequency dependence of the peak force generated by the myofilaments, the force-frequency response (FFR). Methods Our cell model incorporates frequency-dependent CaM-mediated spatially heterogenous interaction of CaMKII and CaN with their principal targets (dihydropyridine (DHPR) and ryanodine (RyR) receptors and the SERCA pump). It also accounts for the rate-dependent effects of phospholamban (PLB) on the SERCA pump; the rate-dependent role of cAMP in up-regulation of the L-type Ca2+ channel (ICa,L); and the enhancement in SERCA pump activity via phosphorylation of PLB. Results Our model reproduces positive peak FFR observed in rat ventricular myocytes during voltage-clamp studies both in the presence/absence of cAMP mediated β-adrenergic stimulation. This study provides quantitative insight into the rate-dependence of Ca2+-induced Ca2+-release (CICR) by investigating the frequency-dependence of the trigger current (ICa,L) and RyR-release. It also highlights the relative role of the sodium-calcium exchanger (NCX) and the SERCA pump at higher frequencies, as well as the rate-dependence of sarcoplasmic reticulum (SR) Ca2+ content. A rigorous Ca2+ balance imposed on our investigation of these Ca2+ signalling pathways clarifies their individual roles. Here, we present a coupled electromechanical study emphasizing the rate-dependence of isometric force developed and also investigate the temperature-dependence of FFR. Conclusions Our model provides

  6. Ca2+/Cation Antiporters (CaCA): Identification, Characterization and Expression Profiling in Bread Wheat (Triticum aestivum L.)

    PubMed Central

    Taneja, Mehak; Tyagi, Shivi; Sharma, Shailesh; Upadhyay, Santosh Kumar

    2016-01-01

    The Ca2+/cation antiporters (CaCA) superfamily proteins play vital function in Ca2+ ion homeostasis, which is an important event during development and defense response. Molecular characterization of these proteins has been performed in certain plants, but they are still not characterized in Triticum aestivum (bread wheat). Herein, we identified 34 TaCaCA superfamily proteins, which were classified into TaCAX, TaCCX, TaNCL, and TaMHX protein families based on their structural organization and evolutionary relation with earlier reported proteins. Since the T. aestivum comprises an allohexaploid genome, TaCaCA genes were derived from each A, B, and D subgenome and homeologous chromosome (HC), except chromosome-group 1. Majority of genes were derived from more than one HCs in each family that were considered as homeologous genes (HGs) due to their high similarity with each other. These HGs showed comparable gene and protein structures in terms of exon/intron organization and domain architecture. Majority of TaCaCA proteins comprised two Na_Ca_ex domains. However, TaNCLs consisted of an additional EF-hand domain with calcium binding motifs. Each TaCaCA protein family consisted of about 10 transmembrane and two α-repeat regions with specifically conserved signature motifs except TaNCL, which had single α-repeat. Variable expression of most of the TaCaCA genes during various developmental stages suggested their specified role in development. However, constitutively high expression of a few genes like TaCAX1-A and TaNCL1-B indicated their role throughout the plant growth and development. The modulated expression of certain genes during biotic (fungal infections) and abiotic stresses (heat, drought, salt) suggested their role in stress response. Majority of TaCCX and TaNCL family genes were found highly affected during various abiotic stresses. However, the role of individual gene needs to be established. The present study unfolded the opportunity for detail functional

  7. Ca2+/Cation Antiporters (CaCA): Identification, Characterization and Expression Profiling in Bread Wheat (Triticum aestivum L.).

    PubMed

    Taneja, Mehak; Tyagi, Shivi; Sharma, Shailesh; Upadhyay, Santosh Kumar

    2016-01-01

    The Ca 2+ /cation antiporters (CaCA) superfamily proteins play vital function in Ca 2+ ion homeostasis, which is an important event during development and defense response. Molecular characterization of these proteins has been performed in certain plants, but they are still not characterized in Triticum aestivum (bread wheat). Herein, we identified 34 TaCaCA superfamily proteins, which were classified into TaCAX, TaCCX, TaNCL, and TaMHX protein families based on their structural organization and evolutionary relation with earlier reported proteins. Since the T. aestivum comprises an allohexaploid genome, TaCaCA genes were derived from each A, B, and D subgenome and homeologous chromosome (HC), except chromosome-group 1. Majority of genes were derived from more than one HCs in each family that were considered as homeologous genes (HGs) due to their high similarity with each other. These HGs showed comparable gene and protein structures in terms of exon/intron organization and domain architecture. Majority of TaCaCA proteins comprised two Na_Ca_ex domains. However, TaNCLs consisted of an additional EF-hand domain with calcium binding motifs. Each TaCaCA protein family consisted of about 10 transmembrane and two α-repeat regions with specifically conserved signature motifs except TaNCL, which had single α-repeat. Variable expression of most of the TaCaCA genes during various developmental stages suggested their specified role in development. However, constitutively high expression of a few genes like TaCAX1-A and TaNCL1-B indicated their role throughout the plant growth and development. The modulated expression of certain genes during biotic (fungal infections) and abiotic stresses (heat, drought, salt) suggested their role in stress response. Majority of TaCCX and TaNCL family genes were found highly affected during various abiotic stresses. However, the role of individual gene needs to be established. The present study unfolded the opportunity for detail

  8. Activity and Ca2+ regulate the mobility of TRPV1 channels in the plasma membrane of sensory neurons

    PubMed Central

    Senning, Eric N; Gordon, Sharona E

    2015-01-01

    TRPV1 channels are gated by a variety of thermal, chemical, and mechanical stimuli. We used optical recording of Ca2+ influx through TRPV1 to measure activity and mobility of single TRPV1 molecules in isolated dorsal root ganglion neurons and cell lines. The opening of single TRPV1 channels produced sparklets, representing localized regions of elevated Ca2+. Unlike sparklets reported for L-type Ca2+ channels, TRPV4 channels, and AchR channels, TRPV1 channels diffused laterally in the plasma membrane as they gated. Mobility was highly variable from channel-to-channel and, to a smaller extent, from cell to cell. Most surprisingly, we found that mobility decreased upon channel activation by capsaicin, but only in the presence of extracellular Ca2+. We propose that decreased mobility of open TRPV1 could act as a diffusion trap to concentrate channels in cell regions with high activity. DOI: http://dx.doi.org/10.7554/eLife.03819.001 PMID:25569155

  9. Carbonic anhydrase inhibitors: in vitro inhibition of α isoforms (hCA I, hCA II, bCA III, hCA IV) by flavonoids.

    PubMed

    Ekinci, Derya; Karagoz, Lutfi; Ekinci, Deniz; Senturk, Murat; Supuran, Claudiu T

    2013-04-01

    A series of flavonoids, such as quercetin, catechin, apigenin, luteolin, morin, were investigated for their inhibitory effects against the metalloenzyme carbonic anhydrase (CA). The compounds were tested against four α-CA isozymes purified from human and bovine (hCA I, hCA II, bCA III, hCA IV) tissues. The four isozymes showed quite diverse inhibition profiles with these compounds. The flavonoids inhibited hCA I with K(I)-s in the range of 2.2-12.8 μM, hCA II with K(I)-s in the range of 0.74-6.2 μM, bCA III with K(I)-s in the range of 2.2-21.3 μM, and hCA IV with inhibition constants in the range of 4.4-15.7, with an esterase assay using 4-nitrophenyl acetate as substrate. Some simple phenols/sulfonamides were also investigated as standard inhibitors. The flavonoids incorporate phenol moieties which inhibit these CAs through a diverse, not yet determined inhibition mechanism, compared to classic inhibitors such as the sulfonamide/sulfamate ones.

  10. Bax regulates neuronal Ca2+ homeostasis.

    PubMed

    D'Orsi, Beatrice; Kilbride, Seán M; Chen, Gang; Perez Alvarez, Sergio; Bonner, Helena P; Pfeiffer, Shona; Plesnila, Nikolaus; Engel, Tobias; Henshall, David C; Düssmann, Heiko; Prehn, Jochen H M

    2015-01-28

    Excessive Ca(2+) entry during glutamate receptor overactivation ("excitotoxicity") induces acute or delayed neuronal death. We report here that deficiency in bax exerted broad neuroprotection against excitotoxic injury and oxygen/glucose deprivation in mouse neocortical neuron cultures and reduced infarct size, necrotic injury, and cerebral edema formation after middle cerebral artery occlusion in mice. Neuronal Ca(2+) and mitochondrial membrane potential (Δψm) analysis during excitotoxic injury revealed that bax-deficient neurons showed significantly reduced Ca(2+) transients during the NMDA excitation period and did not exhibit the deregulation of Δψm that was observed in their wild-type (WT) counterparts. Reintroduction of bax or a bax mutant incapable of proapoptotic oligomerization equally restored neuronal Ca(2+) dynamics during NMDA excitation, suggesting that Bax controlled Ca(2+) signaling independently of its role in apoptosis execution. Quantitative confocal imaging of intracellular ATP or mitochondrial Ca(2+) levels using FRET-based sensors indicated that the effects of bax deficiency on Ca(2+) handling were not due to enhanced cellular bioenergetics or increased Ca(2+) uptake into mitochondria. We also observed that mitochondria isolated from WT or bax-deficient cells similarly underwent Ca(2+)-induced permeability transition. However, when Ca(2+) uptake into the sarco/endoplasmic reticulum was blocked with the Ca(2+)-ATPase inhibitor thapsigargin, bax-deficient neurons showed strongly elevated cytosolic Ca(2+) levels during NMDA excitation, suggesting that the ability of Bax to support dynamic ER Ca(2+) handling is critical for cell death signaling during periods of neuronal overexcitation. Copyright © 2015 the authors 0270-6474/15/351706-17$15.00/0.

  11. Divergent biophysical properties, gating mechanisms, and possible functions of the two skeletal muscle Ca(V)1.1 calcium channel splice variants.

    PubMed

    Tuluc, Petronel; Flucher, Bernhard E

    2011-12-01

    Voltage-gated calcium channels are multi-subunit protein complexes that specifically allow calcium ions to enter the cell in response to membrane depolarization. But, for many years it seemed that the skeletal muscle calcium channel Ca(V)1.1 is the exception. The classical splice variant Ca(V)1.1a activates slowly, has a very small current amplitude and poor voltage sensitivity. In fact adult muscle fibers work perfectly well even in the absence of calcium influx. Recently a new splice variant of the skeletal muscle calcium channel Ca(V)1.1e has been characterized. The lack of the 19 amino acid exon 29 in this splice variant results in a rapidly activating calcium channel with high current amplitude and good voltage sensitivity. Ca(V)1.1e is the dominant channel in embryonic muscle, where the expression of this high calcium-conducting Ca(V)1.1 isoform readily explains developmental processes depending on L-type calcium currents. Moreover, the availability of these two structurally similar but functionally distinct channel variants facilitates the analysis of the molecular mechanisms underlying the unique current properties of the classical Ca(V)1.1a channel.

  12. Allometric constraints on Sr/Ca and Ba/Ca partitioning in terrestrial mammalian trophic chains.

    PubMed

    Balter, Vincent

    2004-03-01

    In biological systems, strontium (Sr) and barium (Ba) are two non-essential elements, in comparison to calcium (Ca) which is essential. The Sr/Ca and Ba/Ca ratios tend to decrease in biochemical pathways which include Ca as an essential element, and these processes are termed biopurification of Ca. The quantitative pathway of the biopurification of Ca in relation to Sr and Ba between two biological reservoirs ( Rn and R(n -1)) is measured with an observed ratio (OR) expressed by the (Sr/Ca) Rn /(Sr/Ca)( Rn-1) and (Ba/Ca) Rn /(Ba/Ca)( Rn-1) ratios. For a mammalian organism, during the whole biopurification of Ca starting with the diet to the ultimate reservoir of Ca which is the bone, the mean values for ORSr and ORBa are 0.25 and 0.2, respectively. In this study, published Sr/Ca and Ba/Ca ratios are used for three sets of soils, plants, and bones of herbivorous and carnivorous mammals, each comprising a trophic chain, to illustrate the biopurification of Ca at the level of trophic chains. Calculated ORSr and ORBa of herbivore bones in relation to plants and of bones of carnivores in relation to bones of herbivores give ORSr=0.30+/-0.08 and ORBa=0.16+/-0.08, thus suggesting that trophic chains reflect the Sr/Ca and Ba/Ca fluxes that are prevalent at the level of a mammalian organism. The slopes of the three regression equations of log(Sr/Ca) vs. log(Ba/Ca) are similar, indicating that the process of biopurification of Ca with respect to Sr and Ba is due to biological processes and is independent of the geological settings. Modifications of the logarithmic expression of the Sr/Ca and Ba/Ca relationship allow a new formula of the biopurification process to be deduced, leading to the general equation ORBa=ORSr(1.79+/-0.33), where the allometric coefficient is the mean of the slopes of the three regression equations. Some recent examples are used to illustrate this new analysis of predator-prey relations between mammals. This opens up new possibilities for the

  13. Assays of mitochondrial Ca2+ transport and Ca2+ efflux via the MPTP.

    PubMed

    Ben-Hail, Danya; Shoshan-Barmatz, Varda

    2014-02-01

    Studying Ca(2+) transport in mitochondria in connection with energy production, as well as cell death, is of great importance. Ca(2+) activates several key enzymes in the mitochondrial matrix to enhance ATP production. This provides an important mechanism for synchronizing energy production with the energy demands of Ca(2+)-activated processes, such as contraction, allowing important feedback effects to help shape cytosolic Ca(2+) signals. A rise in mitochondrial Ca(2+) can convey both apoptotic and necrotic death signals by inducing opening of the mitochondrial permeability transition pore (MPTP). Here, we present a protocol for measuring Ca(2+) transport and release in isolated mitochondria.

  14. Oxidized CaMKII Triggers Atrial Fibrillation

    PubMed Central

    Purohit, Anil; Rokita, Adam G.; Guan, Xiaoqun; Chen, Biyi; Koval, Olha M.; Voigt, Niels; Neef, Stefan; Sowa, Thomas; Gao, Zhan; Luczak, Elizabeth D.; Stefansdottir, Hrafnhildur; Behunin, Andrew C.; Li, Na; El-Accaoui, Ramzi N.; Yang, Baoli; Swaminathan, Paari Dominic; Weiss, Robert M.; Wehrens, Xander H.T.; Song, Long-Sheng; Dobrev, Dobromir; Maier, Lars S.; Anderson, Mark E.

    2013-01-01

    Background Atrial fibrillation is a growing public health problem without adequate therapies. Angiotensin II (Ang II) and reactive oxygen species (ROS) are validated risk factors for atrial fibrillation (AF) in patients, but the molecular pathway(s) connecting ROS and AF is unknown. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a ROS activated proarrhythmic signal, so we hypothesized that oxidized CaMKIIδ(ox-CaMKII) could contribute to AF. Methods and Results We found ox-CaMKII was increased in atria from AF patients compared to patients in sinus rhythm and from mice infused with Ang II compared with saline. Ang II treated mice had increased susceptibility to AF compared to saline treated WT mice, establishing Ang II as a risk factor for AF in mice. Knock in mice lacking critical oxidation sites in CaMKIIδ (MM-VV) and mice with myocardial-restricted transgenic over-expression of methionine sulfoxide reductase A (MsrA TG), an enzyme that reduces ox-CaMKII, were resistant to AF induction after Ang II infusion. Conclusions Our studies suggest that CaMKII is a molecular signal that couples increased ROS with AF and that therapeutic strategies to decrease ox-CaMKII may prevent or reduce AF. PMID:24030498

  15. The initial 41Ca/40Ca ratios in two type A Ca-Al-rich inclusions: Implications for the origin of short-lived 41Ca

    NASA Astrophysics Data System (ADS)

    Liu, Ming-Chang

    2017-03-01

    This paper reports new 41Ca-41K isotopic data for two Type A CAIs, NWA 3118 #1Nb (Compact Type A) and Vigarano 3138 F8 (Fluffy Type A), from reduced CV3 chondrites. The NWA CAI is found to have carried live 41Ca at the level of (4.6 ± 1.9) ×10-9 , consistent with the proposed Solar System initial 41Ca /40Ca = 4.2 ×10-9 by Liu et al. (2012a). On the other hand, the Vigarano CAI does not have resolvable radiogenic 41K excesses that can be attributed to the decay of 41Ca. Combined with the 26Al data that have been reported for these two CAIs, we infer that the 41Ca distribution was not homogeneous when 26Al was widespread at the canonical level of 26Al /27Al = 5.2 ×10-5 . Such a 41Ca heterogeneity can be understood under two astrophysical contexts: in situ charged particle irradiation by the protoSun in the solar nebula that had inherited some baseline 10Be abundance from the molecular cloud, and Solar System formation in a molecular cloud enriched in 26Al and 41Ca contaminated by massive star winds. That said, more high quality 41Ca data are still needed to better understand the origin of this radionuclide.

  16. Hypoxic pulmonary vasoconstriction in the absence of pretone: essential role for intracellular Ca2+ release

    PubMed Central

    Connolly, Michelle J; Prieto-Lloret, Jesus; Becker, Silke; Ward, Jeremy P T; Aaronson, Philip I

    2013-01-01

    Hypoxic pulmonary vasoconstriction (HPV) maintains blood oxygenation during acute hypoxia but contributes to pulmonary hypertension during chronic hypoxia. The mechanisms of HPV remain controversial, in part because HPV is usually studied in the presence of agonist-induced preconstriction (‘pretone’). This potentiates HPV but may obscure and distort its underlying mechanisms. We therefore carried out an extensive assessment of proposed mechanisms contributing to HPV in isolated intrapulmonary arteries (IPAs) in the absence of pretone by using a conventional small vessel myograph. Hypoxia elicited a biphasic constriction consisting of a small transient (phase 1) superimposed upon a sustained (phase 2) component. Neither phase was affected by the L-type Ca2+ channel antagonists diltiazem (10 and 30 μm) or nifedipine (3 μm). Application of the store-operated Ca2+ entry (SOCE) blockers BTP2 (10 μm) or SKF96365 (50 μm) attenuated phase 2 but not phase 1, whereas a lengthy (30 min) incubation in Ca2+-free physiological saline solution similarly reduced phase 2 but abolished phase 1. No further effect of inhibition of HPV was observed if the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (30 μm) was also applied during the 30 min incubation in Ca2+-free physiological saline solution. Pretreatment with 10 μm ryanodine and 15 mm caffeine abolished both phases, whereas treatment with 100 μm ryanodine attenuated both phases. The two-pore channel blocker NED-19 (1 μm) and the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist BZ194 (200 μm) had no effect on either phase of HPV. The lysosomal Ca2+-depleting agent concanamycin (1 μm) enhanced HPV if applied during hypoxia, but had no effect on HPV during a subsequent hypoxic challenge. The cyclic ADP ribose antagonist 8-bromo-cyclic ADP ribose (30 μm) had no effect on either phase of HPV. Neither the Ca2+-sensing receptor (CaSR) blocker NPS2390 (0.1 and 10 μm) nor FK506 (10

  17. Stimulation of ANP secretion by 2-Cl-IB-MECA through A(3) receptor and CaMKII.

    PubMed

    Yuan, Kuichang; Bai, Guang Yi; Park, Woo Hyun; Kim, Sung Zoo; Kim, Suhn Hee

    2008-12-01

    Adenosine is a potent mediator of myocardial protection against hypertrophy via A(1) or A(3) receptors that may be partly related to atrial natriuretic peptide (ANP) release. However, little is known about the possible involvement of the A(3) receptor on ANP release. We studied the effects of the A(3) receptor on atrial functions and its modification in hypertrophied atria. A selective A(3) receptor agonist, 2-chloro-N(6)-(3-iodobenzyl) adenosine-5'-N-methyluronamide (2-CI-IB-MECA), was perfused into isolated, beating rat atria with and without receptor modifiers. 2-CI-IB-MECA dose-dependently increased the ANP secretion, which was blocked by the A(3) receptor antagonist, but the increased atrial contractility and decreased cAMP levels induced by 30muM 2-CI-IB-MECA were not affected. The 100muM 2-(1-hexylnyl)-N-methyladenosine (HEMADO) and N(6)-(3-iodobenzyl) adenosine-5'-N-methyluronamide (IB-MECA), A(3) receptor agonist, also stimulated the ANP secretion without positive inotropy. The potency for the stimulation of ANP secretion was 2-CI-IB-MECA>IB-MECA=HEMADO. The inhibition of the ryanodine receptor or calcium/calmodulin-dependent kinase II (CaMKII) attenuated 2-CI-IB-MECA-induced ANP release, positive inotropy, and translocation of extracellular fluid. However, the inhibition of L-type Ca(2+) channels, sarcoplasmic reticulum Ca(2+)-reuptake, phospholipase C or inositol 1,4,5-triphosphate receptors did not affect these parameters. 2-CI-IB-MECA decreased cAMP level, which was blocked only with an inhibitor of CaMKII or adenylyl cyclase. These results suggest that 2-CI-IB-MECA increases the ANP secretion mainly via A(3) receptor activation and positive inotropy by intracellular Ca(2+) regulation via the ryanodine receptor and CaMKII.

  18. Biphasic decay of the Ca transient results from increased sarcoplasmic reticulum Ca leak

    PubMed Central

    Sankaranarayanan, Rajiv; Li, Yatong; Greensmith, David J.; Eisner, David A.

    2016-01-01

    Key points Ca leak from the sarcoplasmic reticulum through the ryanodine receptor (RyR) reduces the amplitude of the Ca transient and slows its rate of decay.In the presence of β‐adrenergic stimulation, RyR‐mediated Ca leak produces a biphasic decay of the Ca transient with a fast early phase and a slow late phase.Two forms of Ca leak have been studied, Ca‐sensitising (induced by caffeine) and non‐sensitising (induced by ryanodine) and both induce biphasic decay of the Ca transient.Only Ca‐sensitising leak can be reversed by traditional RyR inhibitors such as tetracaine.Ca leak can also induce Ca waves. At low levels of leak, waves occur. As leak is increased, first biphasic decay and then slowed monophasic decay is seen. The level of leak has major effects on the shape of the Ca transient. Abstract In heart failure, a reduction in Ca transient amplitude and contractile dysfunction can by caused by Ca leak through the sarcoplasmic reticulum (SR) Ca channel (ryanodine receptor, RyR) and/or decreased activity of the SR Ca ATPase (SERCA). We have characterised the effects of two forms of Ca leak (Ca‐sensitising and non‐sensitising) on calcium cycling and compared with those of SERCA inhibition. We measured [Ca2+]i with fluo‐3 in voltage‐clamped rat ventricular myocytes. Increasing SR leak with either caffeine (to sensitise the RyR to Ca activation) or ryanodine (non‐sensitising) had similar effects to SERCA inhibition: decreased systolic [Ca2+]i, increased diastolic [Ca2+]i and slowed decay. However, in the presence of isoproterenol, leak produced a biphasic decay of the Ca transient in the majority of cells while SERCA inhibition produced monophasic decay. Tetracaine reversed the effects of caffeine but not of ryanodine. When caffeine (1 mmol l−1) was added to a cell which displayed Ca waves, the wave frequency initially increased before waves disappeared and biphasic decay developed. Eventually (at higher caffeine concentrations), the

  19. Edward C. Little Water Recycling Plant, El Segundo, CA: CA0063401

    EPA Pesticide Factsheets

    Joint EPA and Los Angeles Regional Water Quality Control Board NPDES Permit and Waiver from Secondary Treatment for the West Basin Municipal Water District Edward C. Little Water Recycling Plant, El Segundo, CA: CA0063401

  20. Berberine alleviates the cerebrovascular contractility in streptozotocin-induced diabetic rats through modulation of intracellular Ca²⁺ handling in smooth muscle cells.

    PubMed

    Ma, Yu-Guang; Zhang, Yin-Bin; Bai, Yun-Gang; Dai, Zhi-Jun; Liang, Liang; Liu, Mei; Xie, Man-Jiang; Guan, Hai-Tao

    2016-04-12

    Vascular dysfunction is a distinctive phenotype in diabetes mellitus. Current treatments mostly focus on the tight glycemic control and few of these treatments have been designed to directly recover the vascular dysfunction in diabetes. As a classical natural medicine, berberine has been explored as a possible therapy for DM. In addition, it is reported that berberine has an extra-protective effect in diabetic vascular dysfunction. However, little is known whether the berberine treatment could ameliorate the smooth muscle contractility independent of a functional endothelium under hyperglycemia. Furthermore, it remains unknown whether berberine affects the arterial contractility by regulating the intracellular Ca(2+) handling in vascular smooth cells (VSMCs) under hyperglycemia. Sprague-Dawley rats were used to establish the diabetic model with a high-fat diet plus injections of streptozotocin (STZ). Berberine (50, 100, and 200 mg/kg/day) were intragastrically administered to control and diabetic rats for 8 weeks since the injection of STZ. The intracellular Ca(2+) handling of isolated cerebral VSMCs was investigated by recording the whole-cell L-type Ca(2+) channel (CaL) currents, assessing the protein expressions of CaL channel, and measuring the intracellular Ca(2+) in response to caffeine. Our results showed that chronic administration of 100 mg/kg/day berberine not only reduced glucose levels, but also inhibited the augmented contractile function of cerebral artery to KCl and 5-hydroxytryptamine (5-HT) in diabetic rats. Furthermore, chronic administration of 100 mg/kg/day berberine significantly inhibited the CaL channel current densities, reduced the α1C-subunit expressions of CaL channel, decreased the resting intracellular Ca(2+) ([Ca(2+)]i) level, and suppressed the Ca(2+) releases from RyRs in cerebral VSMCs isolated from diabetic rats. Correspondingly, acute application of 10 μM berberine could directly inhibit the hyperglycemia-induced CaL currents

  1. Pre-Steady-State Kinetics of Ba-Ca Exchange Reveals a Second Electrogenic Step Involved in Ca2+ Translocation by the Na-Ca Exchanger

    PubMed Central

    Haase, Andreas; Hartung, Klaus

    2009-01-01

    Kinetic properties of the Na-Ca exchanger (guinea pig NCX1) expressed in Xenopus oocytes were investigated with excised membrane patches in the inside-out configuration and photolytic Ca2+ concentration jumps with either 5 mM extracellular Sr2+ or Ba2+. After a Ca2+ concentration jump on the cytoplasmic side, the exchanger performed Sr-Ca or Ba-Ca exchange. In the Sr-Ca mode, currents are transient and decay in a monoexponential manner similar to that of currents in the Ca-Ca exchange mode described before. Currents recorded in the Ba-Ca mode are also transient, but the decay is biphasic. In the Sr-Ca mode the amount of charge translocated increases at negative potentials in agreement with experiments performed in the Ca-Ca mode. In the Ba-Ca mode the total amount of charge translocated after a Ca2+ concentration jump is ∼4 to 5 times that in Ca-Ca or Sr-Ca mode. In the Ba-Ca mode the voltage dependence of charge translocation depends on the Ca2+ concentration on the cytosolic side before the Ca2+ concentration jump. At low initial Ca2+ levels (∼0.5 μM), charge translocation is voltage independent. At a higher initial concentration (1 μM Ca2+), the amount of charge translocated increases at positive potentials. Biphasic relaxation of the current was also observed in the Ca-Ca mode if the external Ca2+ concentration was reduced to ≤0.5 mM. The results reported here and in previous publications can be described by using a 6-state model with two voltage-dependent conformational transitions. PMID:19486679

  2. Structures of Ca(V) Ca**2+/CaM-IQ Domain Complexes Reveal Binding Modes That Underlie Calcium-Dependent Inactivation And Facilitation

    SciTech Connect

    Kim, E.Y.; Rumpf, C.H.; Fujiwara, Y.

    2009-05-20

    Calcium influx drives two opposing voltage-activated calcium channel (Ca{sub V}) self-modulatory processes: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF). Specific Ca{sup 2+}/calmodulin (Ca{sup 2+}/CaM) lobes produce CDI and CDF through interactions with the Ca{sub V}{alpha}{sub 1} subunit IQ domain. Curiously, Ca{sup 2+}/CaM lobe modulation polarity appears inverted between Ca{sub V}1s and Ca{sub V}2s. Here, we present crystal structures of Ca{sub V}2.1, Ca{sub V}2.2, and Ca{sub V}2.3 Ca{sup 2+}/CaM-IQ domain complexes. All display binding orientations opposite to Ca{sub V}1.2 with a physical reversal of the CaM lobe positions relative to the IQ {alpha}-helix. Titration calorimetry reveals lobe competition for a high-affinitymore » site common to Ca{sub V}1 and Ca{sub V}2 IQ domains that is occupied by the CDI lobe in the structures. Electrophysiological experiments demonstrate that the N-terminal Ca{sub V}2 Ca{sup 2+}/C-lobe anchors affect CDF. Together, the data unveil the remarkable structural plasticity at the heart of Ca{sub V} feedback modulation and indicate that Ca{sub V}1 and Ca{sub V}2 IQ domains bear a dedicated CDF site that exchanges Ca{sup 2+}/CaM lobe occupants.« less

  3. 46 CFR 7.130 - Point Conception, CA to Point Sur, CA.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... LINES Pacific Coast § 7.130 Point Conception, CA to Point Sur, CA. (a) A line drawn from the... 46 Shipping 1 2014-10-01 2014-10-01 false Point Conception, CA to Point Sur, CA. 7.130 Section 7... Breakwater. (b) A line drawn from the outer end of Morro Bay Entrance East Breakwater to latitude 35°21.5′ N...

  4. 46 CFR 7.130 - Point Conception, CA to Point Sur, CA.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... LINES Pacific Coast § 7.130 Point Conception, CA to Point Sur, CA. (a) A line drawn from the... 46 Shipping 1 2011-10-01 2011-10-01 false Point Conception, CA to Point Sur, CA. 7.130 Section 7... Breakwater. (b) A line drawn from the outer end of Morro Bay Entrance East Breakwater to latitude 35°21.5′ N...

  5. 46 CFR 7.125 - Point Vincente, CA to Point Conception, CA.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... BOUNDARY LINES Pacific Coast § 7.125 Point Vincente, CA to Point Conception, CA. (a) A line drawn from Redondo Beach East Jetty Light “2” to Redondo Beach West Jetty Light “3”. (b) A line drawn from Marina Del... 46 Shipping 1 2010-10-01 2010-10-01 false Point Vincente, CA to Point Conception, CA. 7.125...

  6. 46 CFR 7.130 - Point Conception, CA to Point Sur, CA.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... LINES Pacific Coast § 7.130 Point Conception, CA to Point Sur, CA. (a) A line drawn from the... 46 Shipping 1 2013-10-01 2013-10-01 false Point Conception, CA to Point Sur, CA. 7.130 Section 7... Breakwater. (b) A line drawn from the outer end of Morro Bay Entrance East Breakwater to latitude 35°21.5′ N...

  7. 46 CFR 7.125 - Point Vincente, CA to Point Conception, CA.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... BOUNDARY LINES Pacific Coast § 7.125 Point Vincente, CA to Point Conception, CA. (a) A line drawn from Redondo Beach East Jetty Light “2” to Redondo Beach West Jetty Light “3”. (b) A line drawn from Marina Del... 46 Shipping 1 2012-10-01 2012-10-01 false Point Vincente, CA to Point Conception, CA. 7.125...

  8. 46 CFR 7.125 - Point Vincente, CA to Point Conception, CA.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... BOUNDARY LINES Pacific Coast § 7.125 Point Vincente, CA to Point Conception, CA. (a) A line drawn from Redondo Beach East Jetty Light “2” to Redondo Beach West Jetty Light “3”. (b) A line drawn from Marina Del... 46 Shipping 1 2013-10-01 2013-10-01 false Point Vincente, CA to Point Conception, CA. 7.125...

  9. 46 CFR 7.130 - Point Conception, CA to Point Sur, CA.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... LINES Pacific Coast § 7.130 Point Conception, CA to Point Sur, CA. (a) A line drawn from the... 46 Shipping 1 2012-10-01 2012-10-01 false Point Conception, CA to Point Sur, CA. 7.130 Section 7... Breakwater. (b) A line drawn from the outer end of Morro Bay Entrance East Breakwater to latitude 35°21.5′ N...

  10. 46 CFR 7.125 - Point Vincente, CA to Point Conception, CA.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... BOUNDARY LINES Pacific Coast § 7.125 Point Vincente, CA to Point Conception, CA. (a) A line drawn from Redondo Beach East Jetty Light “2” to Redondo Beach West Jetty Light “3”. (b) A line drawn from Marina Del... 46 Shipping 1 2014-10-01 2014-10-01 false Point Vincente, CA to Point Conception, CA. 7.125...

  11. 46 CFR 7.125 - Point Vincente, CA to Point Conception, CA.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... BOUNDARY LINES Pacific Coast § 7.125 Point Vincente, CA to Point Conception, CA. (a) A line drawn from Redondo Beach East Jetty Light “2” to Redondo Beach West Jetty Light “3”. (b) A line drawn from Marina Del... 46 Shipping 1 2011-10-01 2011-10-01 false Point Vincente, CA to Point Conception, CA. 7.125...

  12. 46 CFR 7.130 - Point Conception, CA to Point Sur, CA.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... LINES Pacific Coast § 7.130 Point Conception, CA to Point Sur, CA. (a) A line drawn from the... 46 Shipping 1 2010-10-01 2010-10-01 false Point Conception, CA to Point Sur, CA. 7.130 Section 7... Breakwater. (b) A line drawn from the outer end of Morro Bay Entrance East Breakwater to latitude 35°21.5′ N...

  13. Intracellular Ca2+ release and Ca2+ influx during regulatory volume decrease in IMCD cells.

    PubMed

    Tinel, H; Wehner, F; Sauer, H

    1994-07-01

    Volume changes and cytosolic Ca2+ concentration ([Ca2+]i) of inner medullary collecting duct (IMCD) cells under hypotonic stress were monitored by means of confocal laser scanning microscopy and fura 2 fluorescence, respectively. Reduction of extracellular osmolality from 600 to 300 mosmol/kgH2O by omission of sucrose led to an increase in cell volume within 1 min to 135 +/- 3% (n = 9), followed by a partial regulatory volume decrease (RVD) to 109 +/- 2% (n = 9) within the ensuring 5 min. In parallel, [Ca2+]i rose from 145 +/- 9 to 433 +/- 16 nmol/l (n = 9) and thereafter reached a lower steady state of 259 +/- 9 nmol/l. Under low-Ca2+ conditions (10 nmol/l) RVD was not impeded and reduction of osmolality evoked only a transient increase of [Ca2+]i by 182 +/- 22 nmol/l (n = 6). Preincubation with 100 mumol/l 8-(N,N-diethylamino)octyl-3,4,5-trimethoxy-benzoate hydrochloride (TMB-8) or 20 mmol/l caffeine, both effective inhibitors of Ca2+ release from intracellular stores, in low Ca2+ as well as in high Ca2+, inhibited the Ca2+ response and abolished RVD. The temporal relationship between Ca2+ release from intracellular stores and Ca2+ entry was analyzed by determining fura 2 quenching, using Mn2+ as a substitute for external Ca2+. Intracellular Ca2+ release preceded Mn2+ influx by 17 +/- 3 s (n = 10). Mn2+ influx persisted during the whole period of exposure to hypotonicity, indicating that there is no time-dependent Ca2+ channel inactivation. Preincubation with TMB-8 or caffeine reduced Mn2+ influx to the control level, indicating that activation of Ca2+ channels in the plasma membrane occurs via intracellular Ca2+ release.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. An Aqueous Ca-Ion Battery.

    PubMed

    Gheytani, Saman; Liang, Yanliang; Wu, Feilong; Jing, Yan; Dong, Hui; Rao, Karun K; Chi, Xiaowei; Fang, Fang; Yao, Yan

    2017-12-01

    Multivalent-ion batteries are emerging as low-cost, high energy density, and safe alternatives to Li-ion batteries but are challenged by slow cation diffusion in electrode materials due to the high polarization strength of Mg- and Al-ions. In contrast, Ca-ion has a low polarization strength similar to that of Li-ion, therefore a Ca-ion battery will share the advantages while avoiding the kinetics issues related to multivalent batteries. However, there is no battery known that utilizes the Ca-ion chemistry due to the limited success in Ca-ion storage materials. Here, a safe and low-cost aqueous Ca-ion battery based on a highly reversible polyimide anode and a high-potential open framework copper hexacyanoferrate cathode is demonstrated. The prototype cell shows a stable capacity and high efficiency at both high and low current rates, with an 88% capacity retention and an average 99% coloumbic efficiency after cycling at 10C for 1000 cycles. The Ca-ion storage mechanism for both electrodes as well as the origin of the fast kinetics have been investigated. Additional comparison with a Mg-ion cell with identical electrodes reveals clear kinetics advantages for the Ca-ion system, which is explained by the smaller ionic radii and more facile desolvation of hydrated Ca-ions.

  15. An Aqueous Ca-Ion Battery

    SciTech Connect

    Gheytani, Saman; Liang, Yanliang; Wu, Feilong

    Multivalent-ion batteries are emerging as low-cost, high energy density, and safe alternatives to Li-ion batteries but are challenged by slow cation diffusion in electrode materials due to the high polarization strength of Mg- and Al-ions. In contrast, Ca-ion has a low polarization strength similar to that of Li-ion, therefore a Ca-ion battery will share the advantages while avoiding the kinetics issues related to multivalent batteries. However, there is no battery known that utilizes the Ca-ion chemistry due to the limited success in Ca-ion storage materials. Here, a safe and low-cost aqueous Ca-ion battery based on a highly reversible polyimide anodemore » and a high-potential open framework copper hexacyanoferrate cathode is demonstrated. The prototype cell shows a stable capacity and high efficiency at both high and low current rates, with an 88% capacity retention and an average 99% coloumbic efficiency after cycling at 10C for 1000 cycles. The Ca-ion storage mechanism for both electrodes as well as the origin of the fast kinetics have been investigated. Finally, additional comparison with a Mg-ion cell with identical electrodes reveals clear kinetics advantages for the Ca-ion system, which is explained by the smaller ionic radii and more facile desolvation of hydrated Ca-ions.« less

  16. An Aqueous Ca-Ion Battery

    DOE PAGES

    Gheytani, Saman; Liang, Yanliang; Wu, Feilong; ...

    2017-10-26

    Multivalent-ion batteries are emerging as low-cost, high energy density, and safe alternatives to Li-ion batteries but are challenged by slow cation diffusion in electrode materials due to the high polarization strength of Mg- and Al-ions. In contrast, Ca-ion has a low polarization strength similar to that of Li-ion, therefore a Ca-ion battery will share the advantages while avoiding the kinetics issues related to multivalent batteries. However, there is no battery known that utilizes the Ca-ion chemistry due to the limited success in Ca-ion storage materials. Here, a safe and low-cost aqueous Ca-ion battery based on a highly reversible polyimide anodemore » and a high-potential open framework copper hexacyanoferrate cathode is demonstrated. The prototype cell shows a stable capacity and high efficiency at both high and low current rates, with an 88% capacity retention and an average 99% coloumbic efficiency after cycling at 10C for 1000 cycles. The Ca-ion storage mechanism for both electrodes as well as the origin of the fast kinetics have been investigated. Finally, additional comparison with a Mg-ion cell with identical electrodes reveals clear kinetics advantages for the Ca-ion system, which is explained by the smaller ionic radii and more facile desolvation of hydrated Ca-ions.« less

  17. AMP-activated protein kinase-mediated feedback phosphorylation controls the Ca2+/calmodulin (CaM) dependence of Ca2+/CaM-dependent protein kinase kinase β.

    PubMed

    Nakanishi, Akihiro; Hatano, Naoya; Fujiwara, Yuya; Sha'ri, Arian; Takabatake, Shota; Akano, Hiroki; Kanayama, Naoki; Magari, Masaki; Nozaki, Naohito; Tokumitsu, Hiroshi

    2017-12-01

    The Ca 2+ /calmodulin-dependent protein kinase kinase β (CaMKKβ)/5'-AMP-activated protein kinase (AMPK) phosphorylation cascade affects various Ca 2+ -dependent metabolic pathways and cancer growth. Unlike recombinant CaMKKβ that exhibits higher basal activity (autonomous activity), activation of the CaMKKβ/AMPK signaling pathway requires increased intracellular Ca 2+ concentrations. Moreover, the Ca 2+ /CaM dependence of CaMKKβ appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKKβ activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKKβ at multiple residues by CaMKKβ-activated AMPK in addition to autophosphorylation in vitro , leading to reduced autonomous, but not Ca 2+ /CaM-activated, CaMKKβ activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKKβ indicated that Thr 144 phosphorylation by activated AMPK converts CaMKKβ into a Ca 2+ /CaM-dependent enzyme as shown by completely Ca 2+ /CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKKβ mutant. CaMKKβ mutant analysis indicated that the C-terminal domain (residues 471-587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr 144 phosphorylation-dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr 144 antibody revealed phosphorylation of Thr 144 in CaMKKβ in transfected COS-7 cells that was further enhanced by exogenous expression of AMPKα. These results indicate that AMPK-mediated feedback phosphorylation of CaMKKβ regulates the CaMKKβ/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca 2+ -dependent AMPK activation by CaMKKβ. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Domain model for Ca2(+)-inactivation of Ca2+ channels at low channel density.

    PubMed Central

    Sherman, A; Keizer, J; Rinzel, J

    1990-01-01

    The "shell" model for Ca2(+)-inactivation of Ca2+ channels is based on the accumulation of Ca2+ in a macroscopic shell beneath the plasma membrane. The shell is filled by Ca2+ entering through open channels, with the elevated Ca2+ concentration inactivating both open and closed channels at a rate determined by how fast the shell is filled. In cells with low channel density, the high concentration Ca2+ "shell" degenerates into a collection of nonoverlapping "domains" localized near open channels. These domains form rapidly when channels open and disappear rapidly when channels close. We use this idea to develop a "domain" model for Ca2(+)-inactivation of Ca2+ channels. In this model the kinetics of formation of an inactivated state resulting from Ca2+ binding to open channels determines the inactivation rate, a mechanism identical with that which explains single-channel recordings on rabbit-mesenteric artery Ca2+ channels (Huang Y., J. M. Quayle, J. F. Worley, N. B. Standen, and M. T. Nelson. 1989. Biophys. J. 56:1023-1028). We show that the model correctly predicts five important features of the whole-cell Ca2(+)-inactivation for mouse pancreatic beta-cells (Plants, T. D. 1988. J. Physiol. 404:731-747) and that Ca2(+)-inactivation has only minor effects on the bursting electrical activity of these cells. PMID:2174274

  19. Theoretical study on the potential energy surfaces of CaNC and CaCN

    NASA Astrophysics Data System (ADS)

    Ishii, Keisaku; Taketsugu, Tetsuya; Hirano, Tsuneo

    2003-06-01

    The potential energy surfaces of CaNC ( overlineX2Σ+) and CaCN ( overlineX2Σ+) have been investigated by the highly correlated ab initio molecular orbital methods. The bending potential for CaNC is shallow, and shows quite anharmonic and anomalous character, which can explain why the centrifugal distortion constants up to the tenth order were required for the analysis of its rotational spectrum. The reaction path for the isomerization reaction of CaNC and CaCN was also determined: The activation barrier is 2111 cm -1 from the CaNC side, and 602 cm -1 from the CaCN side. Core-core and core-valence correlation contributions of Ca M-shell electrons make the Ca-N (for CaNC) and Ca-C (for CaCN) bond lengths shorter by 0.05 and 0.04 Å, respectively, which indicates the significance of these core-correlation effects.

  20. CaMKII in the Cardiovascular System: Sensing Redox States

    PubMed Central

    Erickson, Jeffrey R.; He, B. Julie; Grumbach, Isabella M.; Anderson, Mark E

    2013-01-01

    The multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) is now recognized to play a central role in pathological events in the cardiovascular system. CaMKII has diverse downstream targets that promote vascular disease, heart failure and arrhythmias, so improved understanding of CaMKII signaling has the potential to lead to new therapies for cardiovascular disease. CaMKII is a multimeric serine-threonine kinase that is initially activated by binding calcified calmodulin (Ca2+/CaM). Under conditions of sustained exposure to elevated Ca2+/CaM CaMKII transitions into a Ca2+/CaM-autonomous enzyme by two distinct but parallel processes. Autophosphorylation of threonine 287 in the CaMKII regulatory domain ‘traps’ CaMKII into an open configuration even after Ca2+/CaM unbinding. More recently, our group identified a pair of methionines (281/282) in the CaMKII regulatory domain that undergo a partially reversible oxidation which, like autophosphorylation, prevents CaMKII from inactivating after Ca2+/CaM unbinding. Here we review roles of CaMKII in cardiovascular disease with an eye to understanding how CaMKII may act as a transduction signal to connect pro-oxidant conditions into specific downstream pathological effects that are relevant to rare and common forms of cardiovascular disease. PMID:21742790

  1. The role of luminal Ca2+ in the generation of Ca2+ waves in rat ventricular myocytes

    PubMed Central

    Lukyanenko, Valeriy; Subramanian, Saisunder; Györke, Inna; Wiesner, Theodore F; Györke, Sandor

    1999-01-01

    We used confocal Ca2+ imaging and fluo-3 to investigate the transition of localized Ca2+ releases induced by focal caffeine stimulation into propagating Ca2+ waves in isolated rat ventricular myocytes. Self-sustaining Ca2+ waves could be initiated when the cellular Ca2+ load was increased by elevating the extracellular [Ca2+] ([Ca2+]o) and they could also be initiated at normal Ca2+ loads when the sensitivity of the release sites to cytosolic Ca2+ was enhanced by low doses of caffeine. When we prevented the accumulation of extra Ca2+ in the luminal compartment of the sarcoplasmic reticulum (SR) with thapsigargin, focal caffeine pulses failed to trigger self-sustaining Ca2+ waves on elevation of [Ca2+]o. Inhibition of SR Ca2+ uptake by thapsigargin in cells already preloaded with Ca2+ above normal levels did not prevent local Ca2+ elevations from triggering propagating waves. Moreover, wave velocity increased by 20 %. Tetracaine (0·75 mM) caused transient complete inhibition of both local and propagating Ca2+ signals, followed by full recovery of the responses due to increased SR Ca2+ accumulation. Computer simulations using a numerical model with spatially distinct Ca2+ release sites suggested that increased amounts of releasable Ca2+ might not be sufficient to generate self-sustaining Ca2+ waves under conditions of Ca2+ overload unless the threshold of release site Ca2+ activation was set at relatively low levels (< 1·5 μM). We conclude that the potentiation of SR Ca2+ release channels by luminal Ca2+ is an important factor in Ca2+ wave generation. Wave propagation does not require the translocation of Ca2+ from the spreading wave front into the SR. Instead, it relies on luminal Ca2+ sensitizing Ca2+ release channels to cytosolic Ca2+. PMID:10373699

  2. Thermoelectric Power Factor Limit of a 1D Nanowire.

    PubMed

    Chen, I-Ju; Burke, Adam; Svilans, Artis; Linke, Heiner; Thelander, Claes

    2018-04-27

    In the past decade, there has been significant interest in the potentially advantageous thermoelectric properties of one-dimensional (1D) nanowires, but it has been challenging to find high thermoelectric power factors based on 1D effects in practice. Here we point out that there is an upper limit to the thermoelectric power factor of nonballistic 1D nanowires, as a consequence of the recently established quantum bound of thermoelectric power output. We experimentally test this limit in quasiballistic InAs nanowires by extracting the maximum power factor of the first 1D subband through I-V characterization, finding that the measured maximum power factors conform to the theoretical limit. The established limit allows the prediction of the achievable power factor of a specific nanowire material system with 1D electronic transport based on the nanowire dimension and mean free path. The power factor of state-of-the-art semiconductor nanowires with small cross section and high crystal quality can be expected to be highly competitive (on the order of mW/m K^{2}) at low temperatures. However, they have no clear advantage over bulk materials at, or above, room temperature.

  3. Thermoelectric Power Factor Limit of a 1D Nanowire

    NASA Astrophysics Data System (ADS)

    Chen, I.-Ju; Burke, Adam; Svilans, Artis; Linke, Heiner; Thelander, Claes

    2018-04-01

    In the past decade, there has been significant interest in the potentially advantageous thermoelectric properties of one-dimensional (1D) nanowires, but it has been challenging to find high thermoelectric power factors based on 1D effects in practice. Here we point out that there is an upper limit to the thermoelectric power factor of nonballistic 1D nanowires, as a consequence of the recently established quantum bound of thermoelectric power output. We experimentally test this limit in quasiballistic InAs nanowires by extracting the maximum power factor of the first 1D subband through I -V characterization, finding that the measured maximum power factors conform to the theoretical limit. The established limit allows the prediction of the achievable power factor of a specific nanowire material system with 1D electronic transport based on the nanowire dimension and mean free path. The power factor of state-of-the-art semiconductor nanowires with small cross section and high crystal quality can be expected to be highly competitive (on the order of mW /m K2 ) at low temperatures. However, they have no clear advantage over bulk materials at, or above, room temperature.

  4. Ferromagnetic CaRuO3

    PubMed Central

    Tripathi, Shivendra; Rana, Rakesh; Kumar, Sanjay; Pandey, Parul; Singh, R. S.; Rana, D. S.

    2014-01-01

    The non-magnetic and non-Fermi-liquid CaRuO3 is the iso-structural analog of the ferromagnetic (FM) and Fermi-liquid SrRuO3. We show that an FM order in the orthorhombic CaRuO3 can be established by the means of tensile epitaxial strain. The structural and magnetic property correlations in the CaRuO3 films formed on SrTiO3 (100) substrate establish a scaling relation between the FM moment and the tensile strain. The strain dependent crossover from non-magnetic to FM CaRuO3 was observed to be associated with switching of non-Fermi liquid to Fermi-liquid behavior. The intrinsic nature of this strain-induced FM order manifests in the Hall resistivity too; the anomalous Hall component realizes in FM tensile-strained CaRuO3 films on SrTiO3 (100) whereas the non-magnetic compressive-strained films on LaAlO3 (100) exhibit only the ordinary Hall effect. These observations of an elusive FM order are consistent with the theoretical predictions of scaling of the tensile epitaxial strain and the magnetic order in tensile CaRuO3. We further establish that the tensile strain is more efficient than the chemical route to induce FM order in CaRuO3. PMID:24464302

  5. Calcium homeostasis in crustaceans: subcellular Ca dynamics.

    PubMed

    Wheatly, M G; Zanotto, F P; Hubbard, M G

    2002-05-01

    The molting cycle of crustaceans, associated with renewal and remineralization of the cuticle, has emerged as a model system to study regulation of genes that code for Ca(2+)-transporting proteins, common to all eukaryotic cells. This article reviews state-of-the-art knowledge about how crustacean transporting epithelia (gills, hepatopancreas and antennal gland) effect mass transcellular movement of Ca(2+) while preventing cytotoxicity. The current model proposed is based on in vitro research on the intermolt stage with extrapolation to other molting stages. Plasma membrane proteins involved in apical and basolateral Ca(2+) movement (NCX, PMCA) are contrasted between aquatic species of different osmotic origin and among transporting epithelia of an individual species. Their roles are assessed in the context of epithelial Ca(2+) flux derived from organismic approaches. Exchange with extracellular environments is integrated with Ca(2+) sequestration mechanisms across endomembranes of the ER/SR and mitochondria. Finally, the review postulates how new Ca(2+) imaging techniques will allow spatial and temporal resolution of Ca(2+) concentration in subcellular domains.

  6. Mibefradil (Ro 40-5967) inhibits several Ca2+ and K+ currents in human fusion-competent myoblasts

    PubMed Central

    Liu, Jian-Hui; Bijlenga, Philippe; Occhiodoro, Teresa; Fischer-Lougheed, Jacqueline; Bader, Charles R; Bernheim, Laurent

    1999-01-01

    The effect of mibefradil (Ro 40-5967), an inhibitor of T-type Ca2+ current (ICa(T)), on myoblast fusion and on several voltage-gated currents expressed by fusion-competent myoblasts was examined.At a concentration of 5 μM, mibefradil decreases myoblast fusion by 57%. At this concentration, the peak amplitudes of ICa(T) and L-type Ca2+ current (ICa(L)) measured in fusion-competent myoblasts are reduced by 95 and 80%, respectively. The IC50 of mibefradil for ICa(T) and ICa(L) are 0.7 and 2 μM, respectively.At low concentrations, mibefradil increased the amplitude of ICa(L) with respect to control.Mibefradil blocked three voltage-gated K+ currents expressed by human fusion-competent myoblasts: a delayed rectifier K+ current, an ether-à-go-go K+ current, and an inward rectifier K+ current, with a respective IC50 of 0.3, 0.7 and 5.6 μM.It is concluded that mibefradil can interfere with myoblast fusion, a mechanism fundamental to muscle growth and repair, and that the interpretation of the effect of mibefradil in a given system should take into account the action of this drug on ionic currents other than Ca2+ currents. PMID:10051142

  7. Spontaneous Ca2+ sparks and Ca2+ homeostasis in a minimal model of permeabilized ventricular myocytes

    PubMed Central

    Hartman, Jana M.; Sobie, Eric A.

    2010-01-01

    Many issues remain unresolved concerning how local, subcellular Ca2+ signals interact with bulk cellular concentrations to maintain homeostasis in health and disease. To aid in the interpretation of data obtained in quiescent ventricular myocytes, we present here a minimal whole cell model that accounts for both localized (subcellular) and global (cellular) aspects of Ca2+ signaling. Using a minimal formulation of the distribution of local [Ca2+] associated with a large number of Ca2+-release sites, the model simulates both random spontaneous Ca2+ sparks and the changes in myoplasmic and sarcoplasmic reticulum (SR) [Ca2+] that result from the balance between stochastic release and reuptake into the SR. Ca2+-release sites are composed of clusters of two-state ryanodine receptors (RyRs) that exhibit activation by local cytosolic [Ca2+] but no inactivation or regulation by luminal Ca2+. Decreasing RyR open probability in the model causes a decrease in aggregate release flux and an increase in SR [Ca2+], regardless of whether RyR inhibition is mediated by a decrease in RyR open dwell time or an increase in RyR closed dwell time. The same balance of stochastic release and reuptake can be achieved, however, by either high-frequency/short-duration or low-frequency/long-duration Ca2+ sparks. The results are well correlated with recent experimental observations using pharmacological RyR inhibitors and clarify those aspects of the release-reuptake balance that are inherent to the coupling between local and global Ca2+ signals and those aspects that depend on molecular-level details. The model of Ca2+ sparks and homeostasis presented here can be a useful tool for understanding changes in cardiac Ca2+ release resulting from drugs, mutations, or acquired diseases. PMID:20852058

  8. A beta1-adrenergic receptor CaM kinase II-dependent pathway mediates cardiac myocyte fetal gene induction.

    PubMed

    Sucharov, Carmen C; Mariner, Peter D; Nunley, Karin R; Long, Carlin; Leinwand, Leslie; Bristow, Michael R

    2006-09-01

    Beta-adrenergic signaling plays an important role in the natural history of dilated cardiomyopathies. Chronic activation of beta-adrenergic receptors (beta1-AR and beta2-AR) during periods of cardiac stress ultimately harms the failing heart by mechanisms that include alterations in gene expression. Here, we show that stimulation of beta-ARs with isoproterenol in neonate rat ventricular myocytes causes a "fetal" response in the relative activities of the human cardiac fetal and/or adult gene promoters that includes repression of the human and rat alpha-myosin heavy chain (alpha-MyHC) promoters with simultaneous activation of the human atrial natriuretic peptide (ANP) and rat beta-MyHC promoters. We also show that the promoter changes correlate with changes in endogenous gene expression as measured by mRNA expression. Furthermore, we show that these changes are specifically mediated by the beta1-AR, but not the beta2-AR, and are independent of alpha1-AR stimulation. We also demonstrate that the fetal gene response is independent of cAMP and protein kinase A, whereas inhibition of Ca2+/calmodulin-dependent protein kinase (CaMK) pathway blocks isoproterenol-mediated fetal gene program induction. Finally, we show that induction of the fetal program is dependent on activation of the L-type Ca2+ channel. We conclude that in neonatal rat cardiac myocytes, agonist-occupied beta1-AR mobilizes Ca2+ stores to activate fetal gene induction through cAMP independent pathways that involve CaMK.

  9. Ca Isotopes Fingerprinting the Earliest Crustal Evolution

    NASA Astrophysics Data System (ADS)

    Kreissig, K.; Elliott, T. R.

    2001-12-01

    The mechanisms of continent formation remain unclear and can be explained in two contrasting ways, using either a steady state crustal growth model involving massive crustal recycling or continuous crustal growth models. Recent developments in mass spectrometry manifest in the new Finnigan-Triton allow Ca isotopic measurements precise enough to use the K-Ca isotope system to address the problem of early Archaean crustal evolution. Due to a strong fractionation of 40K and 40Ca during continent formation and a non-linear growth of 40Ca, Archaean continental crust should show radiogenic initial Ca isotopic composition if large volumes of it have already been existed 3.6 Ga ago. Simple 15-step calculations predict a difference in 40Ca /44Ca of 9 epsilon units at 3.6 Ga between the two crustal growth models. To test this, as well as to study the earliest crust formation processes, plagioclase separates from Archaean provinces reflecting the initial Ca isotopic composition and a range of different whole rock samples have been analysed. Preliminary data for ~ 3.6 Ga old TTGs from Zimbabwe show 40Ca /44Ca indistinguishable from the mantle. This is in agreement with rather chondritic initial Sr and Nd data and might reflect a short residence time of the juvenile mafic oceanic crust before partial melting forming the first continental crust. In contrast, the first results for 3.65 Ga old samples from the Itsaq Gneiss Complex of southern West Greenland yield a more evolved radiogenic Ca signature. This can be interpreted in two different ways. Either as partial melting of juvenile mafic crust shortly after its formation but incorporating already existing crust as also suggested by the existence of older inherited zircons in these rocks and negative ɛ Hf values. Partial melting of mafic oceanic crust long after its formation so that 40K and 40Ca had time to evolve would be an alternative explanation. Importantly, there is no evidence so far for high growth and recycling rates

  10. Intercellular communication within the rat anterior pituitary gland. XV. Properties of spontaneous and LHRH-induced Ca2+ transients in the transitional zone of the rat anterior pituitary in situ.

    PubMed

    Hattori, Kazuki; Shirasawa, Nobuyuki; Suzuki, Hikaru; Otsuka, Takanobu; Wada, Ikuo; Yashiro, Takashi; Herbert, Damon C; Soji, Tsuyoshi; Hashitani, Hikaru

    2013-01-01

    In the transitional zone of the rat anterior pituitary, spontaneous and LHRH-induced Ca(2+) dynamics were visualized using fluo-4 fluorescence Ca(2+) imaging. A majority of cells exhibited spontaneous Ca(2+) transients, while small populations of cells remained quiescent. Approximately 70% of spontaneously active cells generated fast, oscillatory Ca(2+) transients that were inhibited by cyclopiazonic acid (10 μm) but not nicardipine (1 μm), suggesting that Ca(2+) handling by endoplasmic reticulum, but not Ca(2+) influx through voltage-dependent L-type Ca(2+) channels, plays a fundamental role in their generation. In the adult rat anterior pituitary, LHRH (100 μg/ml) caused a transient increase in the Ca(2+) level in a majority of preparations taken from the morning group rats killed between 0930 h and 1030 h. However, the second application of LHRH invariably failed to elevate Ca(2+) levels, suggesting that the long-lasting refractoriness to LHRH stimulation was developed upon the first challenge of LHRH. In contrast, LHRH had no effect in most preparations taken from the afternoon group rats euthanized between 1200 h and 1400 h. In the neonatal rat anterior pituitary, LHRH caused a suppression of spontaneous Ca(2+) transients. Strikingly, the second application of LHRH was capable of reproducing the suppression of Ca(2+) signals, indicating that the refractoriness to LHRH had not been established in neonatal rats. These results suggest that responsiveness to LHRH has a long-term refractoriness in adult rats, and that the physiological LHRH surge may be clocked in the morning. Moreover, LHRH-induced excitation and associated refractoriness appear to be incomplete in neonatal rats and may be acquired during development.

  11. Properties of the Ca2+ influx reveal the duality of events underlying the activation by vanadate and fluoride of the Gárdos effect in human red blood cells.

    PubMed

    Varecka, L; Peterajová, E; Písová, E

    1998-08-14

    The properties of the 45Ca2+ influx by human red blood cells (RBC) induced by NaVO3 or NaF were compared. The NaVO3-induced 45Ca2+ influx was slower and less extensive than that induced by NaF. Both processes were saturable with Ca2+. Substitution of Na+ by K+ inhibited the 45Ca2+ influx induced by NaVO3 but stimulated that by NaF. The NaVO3-induced Ca2+ influx was sensitive to nifedipine (IC50 = 50 mol/l), Cu2+ (IC50=9 mol/l), DTNB (5,5'-dithiobis-(dinitrobenzoic acid)) (IC50 = 12 mol/l) (maximal inhibition 16%, 18%, and 28%, respectively, if NaF was used as inducer). On the other hand, tetrodotoxin (TTX) and cyclosporin A inhibited only the NaF-induced 45Ca2+ influx (IC50 = 21 mol/l and 28 mol/l, respectively). Pig RBC, known not to display the NaVO3-induced Ca2+ influx, exhibited Ca2+ influx induced by NaF. The results show that NaVO3 activates the Ca2+ influx via a pathway homologous to the L-type Ca2+ channel while the NaF-induced Ca2+ influx is mediated via the TTX-sensitive Na+ channel in the presence of NaF with possible participation of calcineurin or cyclophilin. Thus, the Gardos effect induced by NaVO3 and NaF represents two phenomena activated by different mechanisms present in the cryptic state in the RBC membrane.

  12. Oncogenicity of L-type amino-acid transporter 1 (LAT1) revealed by targeted gene disruption in chicken DT40 cells: LAT1 is a promising molecular target for human cancer therapy

    SciTech Connect

    Ohkawa, Mayumi; Ohno, Yoshiya; Masuko, Kazue

    Highlights: {yields} We established LAT1 amino-acid transporter-disrupted DT40 cells. {yields} LAT1-disrupted cells showed slow growth and lost the oncogenicity. {yields} siRNA and mAb inhibited human tumor growth in vitro and in vivo. {yields} LAT1 is a promising target molecule for cancer therapy. -- Abstract: L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4 kb. We established five homozygous LAT1-disrupted (LAT1{supmore » -/-}) cell clones, derived from a heterozygous LAT1{sup +/-} clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1{sup -/-} DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1{sup -/-} cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1{sup -/-} DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1{sup -/-} DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1{sup +/-} DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy.« less

  13. β-Adrenergic stimulation increases the intra-sarcoplasmic reticulum Ca2+ threshold for Ca2+ wave generation

    PubMed Central

    Domeier, Timothy L; Maxwell, Joshua T; Blatter, Lothar A

    2012-01-01

    β-Adrenergic signalling induces positive inotropic effects on the heart that associate with pro-arrhythmic spontaneous Ca2+ waves. A threshold level of sarcoplasmic reticulum (SR) Ca2+ ([Ca2+]SR) is necessary to trigger Ca2+ waves, and whether the increased incidence of Ca2+ waves during β-adrenergic stimulation is due to an alteration in this threshold remains controversial. Using the low-affinity Ca2+ indicator fluo-5N entrapped within the SR of rabbit ventricular myocytes, we addressed this controversy by directly monitoring [Ca2+]SR and Ca2+ waves during β-adrenergic stimulation. Electrical pacing in elevated extracellular Ca2+ ([Ca2+]o= 7 mm) was used to increase [Ca2+]SR to the threshold where Ca2+ waves were consistently observed. The β-adrenergic agonist isoproterenol (ISO; 1 μm) increased [Ca2+]SR well above the control threshold and consistently triggered Ca2+ waves. However, when [Ca2+]SR was subsequently lowered in the presence of ISO (by lowering [Ca2+]o to 1 mm and partially inhibiting sarcoplasmic/endoplasmic reticulum calcium ATPase with cyclopiazonic acid or thapsigargin), Ca2+ waves ceased to occur at a [Ca2+]SR that was higher than the control threshold. Furthermore, for a set [Ca2+]SR level the refractoriness of wave occurrence (Ca2+ wave latency) was prolonged during β-adrenergic stimulation, and was highly dependent on the extent that [Ca]SR exceeded the wave threshold. These data show that acute β-adrenergic stimulation increases the [Ca2+]SR threshold for Ca2+ waves, and therefore the primary cause of Ca2+ waves is the robust increase in [Ca2+]SR above this higher threshold level. Elevation of the [Ca2+]SR wave threshold and prolongation of wave latency represent potentially protective mechanisms against pro-arrhythmogenic Ca2+ release during β-adrenergic stimulation. PMID:22988136

  14. Aging and CaMKII Alter Intracellular Ca2+ Transients and Heart Rhythm in Drosophila melanogaster

    PubMed Central

    Santalla, Manuela; Valverde, Carlos A.; Harnichar, Ezequiel; Lacunza, Ezequiel; Aguilar-Fuentes, Javier; Mattiazzi, Alicia; Ferrero, Paola

    2014-01-01

    Aging is associated to disrupted contractility and rhythmicity, among other cardiovascular alterations. Drosophila melanogaster shows a pattern of aging similar to human beings and recapitulates the arrhythmogenic conditions found in the human heart. Moreover, the kinase CaMKII has been characterized as an important regulator of heart function and an arrhythmogenic molecule that participate in Ca2+ handling. Using a genetically engineered expressed Ca2+ indicator, we report changes in cardiac Ca2+ handling at two different ages. Aging prolonged relaxation, reduced spontaneous heart rate (HR) and increased the occurrence of arrhythmias, ectopic beats and asystoles. Alignment between Drosophila melanogaster and human CaMKII showed a high degree of conservation and indicates that relevant phosphorylation sites in humans are also present in the fruit fly. Inhibition of CaMKII by KN-93 (CaMKII-specific inhibitor), reduced HR without significant changes in other parameters. By contrast, overexpression of CaMKII increased HR and reduced arrhythmias. Moreover, it increased fluorescence amplitude, maximal rate of rise of fluorescence and reduced time to peak fluorescence. These results suggest that CaMKII in Drosophila melanogaster acts directly on heart function and that increasing CaMKII expression levels could be beneficial to improve contractility. PMID:25003749

  15. Ca2+ signalling, voltage-gated Ca2+ channels and praziquantel in flatworm neuromusculature.

    PubMed

    Greenberg, R M

    2005-01-01

    Transient changes in calcium (Ca2+) levels regulate a wide variety of cellular processes, and cells employ both intracellular and extracellular sources of Ca2+ for signalling. Praziquantel, the drug of choice against schistosomiasis, disrupts Ca2+ homeostasis in adult worms. This review will focus on voltage-gated Ca2+ channels, which regulate levels of intracellular Ca2+ by coupling membrane depolarization to entry of extracellular Ca2+. Ca2+ channels are members of the ion channel superfamily and represent essential components of neurons, muscles and other excitable cells. Ca2+ channels are membrane protein complexes in which the pore-forming alpha1 subunit is modulated by auxiliary subunits such as beta and alpha2delta. Schistosomes express two Ca2+ channel beta subunit subtypes: a conventional subtype similar to beta subunits found in other vertebrates and invertebrates and a novel variant subtype with unusual structural and functional properties. The variant schistosome beta subunit confers praziquantel sensitivity to an otherwise praziquantel-insensitive mammalian Ca2+ channel, implicating it as a mediator of praziquantel action.

  16. Aging and CaMKII alter intracellular Ca2+ transients and heart rhythm in Drosophila melanogaster.

    PubMed

    Santalla, Manuela; Valverde, Carlos A; Harnichar, Ezequiel; Lacunza, Ezequiel; Aguilar-Fuentes, Javier; Mattiazzi, Alicia; Ferrero, Paola

    2014-01-01

    Aging is associated to disrupted contractility and rhythmicity, among other cardiovascular alterations. Drosophila melanogaster shows a pattern of aging similar to human beings and recapitulates the arrhythmogenic conditions found in the human heart. Moreover, the kinase CaMKII has been characterized as an important regulator of heart function and an arrhythmogenic molecule that participate in Ca2+ handling. Using a genetically engineered expressed Ca2+ indicator, we report changes in cardiac Ca2+ handling at two different ages. Aging prolonged relaxation, reduced spontaneous heart rate (HR) and increased the occurrence of arrhythmias, ectopic beats and asystoles. Alignment between Drosophila melanogaster and human CaMKII showed a high degree of conservation and indicates that relevant phosphorylation sites in humans are also present in the fruit fly. Inhibition of CaMKII by KN-93 (CaMKII-specific inhibitor), reduced HR without significant changes in other parameters. By contrast, overexpression of CaMKII increased HR and reduced arrhythmias. Moreover, it increased fluorescence amplitude, maximal rate of rise of fluorescence and reduced time to peak fluorescence. These results suggest that CaMKII in Drosophila melanogaster acts directly on heart function and that increasing CaMKII expression levels could be beneficial to improve contractility.

  17. Pericellular Ca2+ recycling potentiates thrombin-evoked Ca2+ signals in human platelets

    PubMed Central

    Sage, Stewart O; Pugh, Nicholas; Farndale, Richard W; Harper, Alan G S

    2013-01-01

    We have previously demonstrated that Na+/Ca2+ exchangers (NCXs) potentiate Ca2+ signaling evoked by thapsigargin in human platelets, via their ability to modulate the secretion of autocoids from dense granules. This link was confirmed in platelets stimulated with the physiological agonist, thrombin, and experiments were performed to examine how Ca2+ removal by the NCX modulates platelet dense granule secretion. In cells loaded with the near-membrane indicator FFP-18, thrombin stimulation was observed to elicit an NCX-dependent accumulation of Ca2+ in a pericellular region around the platelets. To test whether this pericellular Ca2+ accumulation might be responsible for the influence of NCXs over platelet function, platelets were exposed to fast Ca2+ chelators or had their glycocalyx removed. Both manipulations of the pericellular Ca2+ rise reduced thrombin-evoked Ca2+ signals and dense granule secretion. Blocking Ca2+-permeable ion channels had a similar effect, suggesting that Ca2+ exported into the pericellular region is able to recycle back into the platelet cytosol. Single cell imaging with extracellular Fluo-4 indicated that thrombin-evoked rises in extracellular [Ca2+] occurred within the boundary described by the cell surface, suggesting their presence within the open canalicular system (OCS). FFP-18 fluorescence was similarly distributed. These data suggest that upon thrombin stimulation, NCX activity creates a rise in [Ca2+] within the pericellular region of the platelet from where it recycles back into the platelet cytosol, acting to both accelerate dense granule secretion and maintain the initial rise in cytosolic [Ca2+]. PMID:24303163

  18. CaP CURE Initiatives and Projects

    PubMed Central

    Soule, Howard R

    2003-01-01

    CaP CURE was founded in 1993 to help find better treatments and a cure for prostate cancer. By reducing the time and complexity required to apply for funding, and by funding many first-time applicants, CaP CURE has attracted a large number of high-level investigators to the field of prostate cancer research. The organization’s Therapy Consortium meets regularly to address major issues that impede progress in clinical development of new treatments for prostate cancer. CaP CURE has also sponsored an initiative to standardize clinical trial design scenarios for the clinical state of rising prostate-specific antigen and intends to present them to the Food and Drug Administration in partnership with the National Dialogue on Cancer. Finally, CaP CURE’s efforts have resulted in a significant increase in federal funding of prostate cancer research programs. PMID:16986049

  19. Departure gate of acidic Ca2+ confirmed

    PubMed Central

    Jentsch, Thomas J; Hoegg-Beiler, Maja B; Vogt, Janis

    2015-01-01

    More potent, but less known than IP3 that liberates Ca2+ from the ER, NAADP releases Ca2+ from acidic stores. The notion that TPC channels mediate this Ca2+ release was questioned recently by studies suggesting that TPCs are rather PI(3,5)P2-activated Na+ channels. Ruas et al (2015) now partially reconcile these views by showing that TPCs significantly conduct both cations and confirm their activation by both NAADP and PI(3,5)P2. They attribute the failure of others to observe TPC-dependent NAADP-induced Ca2+ release in vivo to inadequate mouse models that retain partial TPC function. PMID:26022292

  20. Role of Ca++ in Shoot Gravitropism. [avena

    NASA Technical Reports Server (NTRS)

    Rayle, D. L.

    1985-01-01

    A cornerstone in the argument that Ca(2+) levels may regulate growth is the finding the EGTA promotes straight growth. The usual explanation for these results is that Ca(2+) chelation from cell walls results in wall loosening and thus accelerated straight growth. The ability of frozen-thawed Avena coleoptile tissue (subjected to 15g tension) to extend in response to EGTA and Quin II was examined. The EGTA when applied in weakly buffered (i.e., 0.1mM) neutral solutions initiates rapid extension. When the buffer strength is increased, similar concentrations of EGTA produce no growth response. This implies when EGTA liberated protons are released upon Ca(2+) chelation they can either initiate acid growth (low buffer conditions) or if consumed (high buffer conditions) have no effect. Thus Ca(2+) chelation in itself apparently does not result in straight growth.

  1. The caBIG Terminology Review Process

    PubMed Central

    Cimino, James J.; Hayamizu, Terry F.; Bodenreider, Olivier; Davis, Brian; Stafford, Grace A.; Ringwald, Martin

    2009-01-01

    The National Cancer Institute (NCI) is developing an integrated biomedical informatics infrastructure, the cancer Biomedical Informatics Grid (caBIG®), to support collaboration within the cancer research community. A key part of the caBIG architecture is the establishment of terminology standards for representing data. In order to evaluate the suitability of existing controlled terminologies, the caBIG Vocabulary and Data Elements Workspace (VCDE WS) working group has developed a set of criteria that serve to assess a terminology's structure, content, documentation, and editorial process. This paper describes the evolution of these criteria and the results of their use in evaluating four standard terminologies: the Gene Ontology (GO), the NCI Thesaurus (NCIt), the Common Terminology for Adverse Events (known as CTCAE), and the laboratory portion of the Logical Objects, Identifiers, Names and Codes (LOINC). The resulting caBIG criteria are presented as a ma