共查询到20条相似文献,搜索用时 31 毫秒
1.
The repetitive spiking of free cytosolic [Ca2+] ([Ca2+]i) during hormonal activation of hepatocytes depends on the activation and subsequent inactivation of InsP3-evoked Ca2+ release. The kinetics of both processes were studied with flash photolytic release of InsP3 and time resolved measurements of [Ca2+]i in single cells. InsP3 evoked Ca2+ flux into the cytosol was measured as d[Ca2+]i/dt, and the kinetics of Ca2+ release compared between hepatocytes and cerebellar Purkinje neurons. In hepatocytes release occurs at InsP3 concentrations greater than 0.1–0.2 μM. A comparison with photolytic release of metabolically stable 5-thio-InsP3 suggests that metabolism of InsP3 is important in determining the minimal concentration needed to produce Ca2+ release. A distinct latency or delay of several hundred milliseconds after release of low InsP3 concentrations decreased to a minimum of 20–30 ms at high concentrations and is reduced to zero by prior increase of [Ca2+]i, suggesting a cooperative action of Ca2+ in InsP3 receptor activation. InsP3-evoked flux and peak [Ca2+]i increased with InsP3 concentration up to 5–10 μM, with large variation from cell to cell at each InsP3 concentration. The duration of InsP3-evoked flux, measured as 10–90% risetime, showed a good reciprocal correlation with d[Ca2+]i/dt and much less cell to cell variation than the dependence of flux on InsP3 concentration, suggesting that the rate of termination of the Ca2+ flux depends on the free Ca2+ flux itself. Comparing this data between hepatocytes and Purkinje neurons shows a similar reciprocal correlation for both, in hepatocytes in the range of low Ca2+ flux, up to 50 μM · s−1 and in Purkinje neurons at high flux up to 1,400 μM · s−1. Experiments in which [Ca2+]i was controlled at resting or elevated levels support a mechanism in which InsP3-evoked Ca2+ flux is inhibited by Ca2+ inactivation of closed receptor/channels due to Ca2+ accumulation local to the release sites. Hepatocytes have a much smaller, more prolonged InsP3-evoked Ca2+ flux than Purkinje neurons. Evidence suggests that these differences in kinetics can be explained by the much lower InsP3 receptor density in hepatocytes than Purkinje neurons, rather than differences in receptor isoform, and, more generally, that high InsP3 receptor density promotes fast rising, rapidly inactivating InsP3-evoked [Ca2+]i transients. 相似文献
2.
We have demonstrated that soft substrate induced apoptosis in polarized cells, but not in transformed cells by disturbance of Ca2+ homeostasis. This study aims to further investigate the regulatory mechanisms underlying the disruption of Ca2+-signaling integrity in soft substrate–induced epithelial apoptosis. Soft substrate up-regulated the store-operated Ca2+ (SOC) entry across the plasma membrane of normal cervical epithelial cells, which resulted in increased cytosolic Ca2+ levels. Concomitantly, soft substrate induced the aggregation and translocation of stromal interacting molecule 1 (STIM1) toward the cell periphery to colocalize with Orai1, an essential pore subunit of SOC channel, detected by fluorescence resonance energy transfer approach and confocal image analyses. The disturbed Ca2+ homeostasis resulted in the activation of μ-calpain, which cleaved α-spectrin, induced actin disorganization, and caused apoptosis. In contrast, soft substrate did not disturb Ca2+ homeostasis or induce apoptosis in cervical cancer cells. Chelating extracellular Ca2+ by EGTA and down-regulated SOC entry by small interfering RNA targeting STIM1 or inhibitors targeting Ca2+-binding site of calpain significantly inhibited soft substrate–induced activation of μ-calpain and epithelial cell apoptosis. Thus, soft substrate up-regulates the interaction of STIM1 with SOC channels, which results in the activation of μ-calpain and subsequently induces normal epithelial cell apoptosis. 相似文献
3.
Neuronal calcium sensor-1 (NCS-1) interacts with many membranes and cytosolic proteins, both in a Ca2+-dependent and in a Ca2+-independent manner, and its physiological role is governed by its N-terminal myristoylation. To understand the role of myristoylation
in altering Ca2+ response and other basic biophysical properties, we have characterized the Ca2+ filling pathways in both myristoylated (myr) and non-myristoylated (non-myr) forms of NCS-1. We have observed that Ca2+ binds simultaneously to all three active EF-hands in non-myr NCS-1, whereas in the case of myr NCS-1, the process is sequential,
where the second EF-hand is filled first, followed by the third and fourth EF-hands. In the case of myr NCS-1, the observed
sequential Ca2+ binding process becomes more prominent in the presence of Mg2+. Besides, the analysis of 15N-relaxation data reveals that non-myr NCS-1 is more dynamic than myr NCS-1. The overall molecular tumbling correlation time
increases by approximately 20% upon myristoylation. Comparing the apo forms of non-myr NCS-1 and myr NCS-1, we found the possibility
of existence of some substates, which are structurally closer to the holo form of the protein. There are more such substates
in the case of non-myr NCS-1 than in the case of the myr NCS-1, suggesting that the former accesses larger volumes of conformational
substates compared with the latter. Further, the study reveals that the possibility of Ca2+ binding simultaneously to different parts of the protein is more favourable in non-myr NCS-1 than in myr NCS-1. 相似文献
4.
Summary Neuropeptide tachykinins, present within sensory nerves, have been implicated as neurotransmitters involved in nonadrenergic and noncholinergic airway muscle contraction. The signal transduction pathways of tachykinins on muscle contraction and Ca2+ mobilization were investigated in swine trachea. Tachykinins, substance P (SP) and neurokinin A (NKA), concentration (1 nM to 1 μM)-dependently induced contractile responses with removal of epithelium, whereas neurokinin B (NKB) did not alter the muscle tension. The SP- and NKA-evoked muscle contractions were inhibited by NK1-R antagonist L732138, but not by either NK2-R antagonist MDL29913 or NK3-R antagonist SB218795. Consistently, SP-elicited increase in [Ca2+]i was abolished by NK1-R antagonist, neither by NK2-R nor NK3-R antagonists. The SP-induced muscular responses were significantly inhibited by L-type Ca2+ channel blocker verapamil and withdrawal of external Ca2+. Caffeine (10 mM) or ryanodine (50 μM) also partly suppressed the SP-induced muscle responses. Inhibition of inositol 1,4,5-trisphosphate (InsP3) receptor with 2-APB (75 μM) potently attenuated SP-evoked Ca2+ mobilization and muscle contraction, which was further inhibited by 2-APB under Ca2+-free external solution, but not completely. Unexpectedly, simultaneous blockade of InsP3 receptor and ryanodine receptor (RyR) by 2-APB and ryanodine enhanced SP-evoked muscle contraction and Ca2+ mobilization. This potentiation was virtually abolished by removal of external Ca2+, suggesting native Ca2+ channels may contribute to this phenomenon. These results demonstrate that tachykinins produce a potent muscle contraction associated with Ca2+ mobilization via tachykinin NK1- R-dependent activation of multiple signal transduction pathways involving Ca2+ influx and release of Ca2+ from InsP3- and ryanodine-sensitive Ca2+ stores. Blockade of both InsP3 receptor and RyR enhances the Ca2+ influx through native Ca2+ channels in plasma membrane, which is crucial to Ca2+ signaling in response to NK1 receptor activation. 相似文献
5.
Horia Vais J. Kevin Foskett Ghanim Ullah John E. Pearson Don-On Daniel Mak 《The Journal of general physiology》2012,140(6):697-716
The ubiquitous inositol 1,4,5-trisphosphate (InsP3) receptor
(InsP3R) Ca2+ release channel plays a central
role in the generation and modulation of intracellular Ca2+
signals, and is intricately regulated by multiple mechanisms including
cytoplasmic ligand (InsP3, free Ca2+, free
ATP4−) binding, posttranslational modifications, and
interactions with cytoplasmic and endoplasmic reticulum (ER) luminal proteins.
However, regulation of InsP3R channel activity by free
Ca2+ in the ER lumen
([Ca2+]ER) remains poorly understood because of
limitations of Ca2+ flux measurements and imaging techniques.
Here, we used nuclear patch-clamp experiments in excised luminal-side-out
configuration with perfusion solution exchange to study the effects of
[Ca2+]ER on homotetrameric rat type 3
InsP3R channel activity. In optimal
[Ca2+]i and subsaturating [InsP3],
jumps of [Ca2+]ER from 70 nM to 300 µM
reduced channel activity significantly. This inhibition was abrogated by
saturating InsP3 but restored when
[Ca2+]ER was raised to 1.1 mM. In suboptimal
[Ca2+]i, jumps of
[Ca2+]ER (70 nM to 300 µM) enhanced
channel activity. Thus, [Ca2+]ER effects on channel
activity exhibited a biphasic dependence on
[Ca2+]i. In addition, the effect of high
[Ca2+]ER was attenuated when a voltage was
applied to oppose Ca2+ flux through the channel. These
observations can be accounted for by Ca2+ flux driven through
the open InsP3R channel by [Ca2+]ER,
raising local [Ca2+]i around the channel to
regulate its activity through its cytoplasmic regulatory
Ca2+-binding sites. Importantly,
[Ca2+]ER regulation of InsP3R
channel activity depended on cytoplasmic Ca2+-buffering
conditions: it was more pronounced when [Ca2+]i was
weakly buffered but completely abolished in strong
Ca2+-buffering conditions. With strong cytoplasmic buffering
and Ca2+ flux sufficiently reduced by applied voltage, both
activation and inhibition of InsP3R channel gating by physiological
levels of [Ca2+]ER were completely abolished.
Collectively, these results rule out Ca2+ regulation of
channel activity by direct binding to the luminal aspect of the channel. 相似文献
6.
T. J. Shuttleworth 《Cell calcium》1995,17(6)
It has been suggested that the release of Ca2+ from intracellular stores by inositol 1,4,5-trisphosphate (InsP3) is modulated by the luminal Ca2+ content of the stores and that such an effect could underlie the apparent ‘quantal’ nature of InsP3-induced release. Although initial studies failed to find evidence in support of such a modulation, several subsequent reports have indicated luminal Ca2+ effects that become apparent only after a greater than 70–75% depletion of Ca2+ stores. In these studies, Ca2+ release was expressed as a percentage of an A23187-releasable pool which comprised both InsP3-sensi-tive and InsP3-insensitive components. In model calculations we have found that the presence of even a minor InsP3-insensitive component in the total Ca2+ pool significantly distorts interpretation of the data. We show that the published results can be accurately duplicated without any requirement for a shift in the true InsP3 sensitivity of Ca2+ release if either: (a) the InsP3-insensitive component does not remain a constant proportion of the total pool during depletion (i.e. depletion disproportionally affects the InsP3-sensitive component); or (b) during generation of InsP3-response curves, additional Cal 2+ is released from the InsP3-insensitive component as the InsP3-sensitive component is progressively emptied. Examination indicates that either, or both, of these conditions apply in the published reports and we conclude that the demonstrated effects of luminal Ca2+ may be artifacts. 相似文献
7.
M. Tshipamba H. De Smedt L. Missiaen B. Himpens L. Van Den Bosch R. Borghgraef 《Journal of cellular physiology》1993,155(1):96-103
We have studied arginine vasopressin (AVP)-, thapsigargin- and inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release in renal epithelial LLC-PK1 cells. AVP-induced changes in the intracellular free calcium concentration ([Ca2+]i) were studied in indo-1 loaded single cells by confocal laser cytometry. AVP-mediated Ca2+ mobilization was also observed in the absence of extracellular Ca2+, but was completely abolished after depletion of the intracellular Ca2+ stores by 2 μM thapsigargin. Using 45Ca2+ fluxes in saponin-permeabilized cell monolayers, we have analysed how InsP3 affected the Ca2+ content of nonmitochondrial Ca2+ pools in different loading and release conditions. Less than 10% of the Ca2+ was taken up in a thapsigargin-insensitive pool when loading was performed in a medium containing 0.1 μM Ca2+. The thapsigargin-insensitive compartment amounted to 35% in the presence of 110 μM Ca2+, but Ca2+ sequestered in this pool could not be released by InsP3. The thapsigargin-sensitive Ca2+ pool, in contrast, was nearly completely InsP3 sensitive. A submaximal [InsP3], however, released only a fraction of the sequestered Ca2+. This fraction was dependent on the cytosolic as well as on the luminal [Ca2+]. The cytosolic free [Ca2+] affected the InsP3-induced Ca2+ release in a biphasic way. Maximal sensitivity toward InsP3 was found at a free cytosolic [Ca2+] between 0.1 and 0.5 μM, whereas higher cytosolic [Ca2+] decreased the InsP3 sensitivity. Other divalent cations or La3+ did not provoke similar inhibitory effects on InsP3-induced Ca2+ release. The luminal free [Ca2+] was manipulated by varying the time of incubation of Ca2+ -loaded cells in an EGTA-containing medium. Reduction of the Ca2+ content to one-third of its initial value resulted in a fivefold decrease in the InsP3 sensitivity of the Ca2+ release. © 1993 Wiley-Liss, Inc. 相似文献
8.
Sravan Pandalaneni Vijaykumar Karuppiah Muhammad Saleem Lee P. Haynes Robert D. Burgoyne Olga Mayans Jeremy P. Derrick Lu-Yun Lian 《The Journal of biological chemistry》2015,290(30):18744-18756
Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca2+-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca2+/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca2+/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178–Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca2+/NCS-1·D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178–Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1. 相似文献
9.
《Channels (Austin, Tex.)》2013,7(1):67-71
Cardiac hypertrophy is associated with profound remodelling of Ca2+ signalling pathways. During the early, compensated stages of hypertrophy, Ca2+ fluxes may be enhanced to facilitate greater contraction, whereas as the hypertrophic heart decompensates, Ca2+ homeostatic mechanisms are dysregulated leading to decreased contractility, arrhythmia and death. Although ryanodine receptor Ca2+ release channels (RyR) on the sarcoplasmic reticulum (SR) intracellular Ca2+ store are primarily responsible for the Ca2+ flux that induces myocyte contraction, a role for Ca2+ release via the inositol 1,4,5-trisphosphate receptor (InsP3R) in cardiac physiology has also emerged. Specifically, InsP3-induced Ca2+ signals generated following myocyte stimulation with an InsP3-generating agonist (e.g. endothelin, ET-1), lead to modulation of Ca2+ signals associated with excitation-contraction coupling (ECC) and the induction of spontaneous Ca2+ release events that cause cellular arrhythmia. Using myocytes from spontaneously hypertensive rats (SHR), we recently reported that expression of the type 2 InsP3R (InsP3R2) is significantly increased during hypertrophy. Notably, this increased expression was restricted to the junctional SR in close proximity to RyRs. There, enhanced Ca2+ release via InsP3Rs serves to sensitise neighbouring RyRs causing an augmentation of Ca2+ fluxes during ECC as well as an increase in non-triggered Ca2+ release events. Although the sensitization of RyRs may be a beneficial consequence of elevated InsP3R expression during hypertrophy, the spontaneous Ca2+ release events are potentially of pathological significance giving rise to cardiac arrhythmia. InsP3R2 expression was also increased in hypertrophic hearts from patients with ischemic dilated cardiomyopathy and aortically-banded mice demonstrating that increased InsP3R expression may be a general phenomenon that underlies Ca2+ changes during hypertrophy. 相似文献
10.
Calcium (Ca2+) is an important intracellular messenger underlying cell physiology. Ca2+ channels are the main entry route for Ca2+ into excitable cells, and regulate processes such as neurotransmitter release and neuronal outgrowth. Neuronal Calcium Sensor-1
(NCS-1) is a member of the Calmodulin superfamily of EF-hand Ca2+ sensing proteins residing in the subfamily of NCS proteins. NCS-1 was originally discovered in Drosophila as an overexpression mutant (Frequenin), having an increased frequency of Ca2+-evoked neurotransmission. NCS-1 is N-terminally myristoylated, can bind intracellular membranes, and has a Ca2+ affinity of 0.3 μM. Over 10 years ago it was discovered that NCS-1 overexpression enhances Ca2+-evoked secretion in bovine adrenal chromaffin cells. The mechanism was unclear, but there was no apparent direct effect on
the exocytotic machinery. It was revealed, again in chromaffin cells, that NCS-1 regulates voltage-gated Ca2+ channels (Cavs) in G-Protein Coupled Receptor (GPCR) signaling pathways. This work in chromaffin cells highlighted NCS-1
as an important modulator of neurotransmission. NCS-1 has since been shown to regulate and/or directly interact with many
proteins including Cavs (P/Q, N, and L), TRPC1/5 channels, GPCRs, IP3R, and PI4 kinase type IIIβ. NCS-1 also affects neuronal
outgrowth having roles in learning and memory affecting both short- and long-term synaptic plasticity. It is not known if
NCS-1 affects neurotransmission and synaptic plasticity via its effect on PIP2 levels, and/or via a direct interaction with
Ca2+ channels or their signaling complexes. This review gives a historical account of NCS-1 function, examining contributions
from chromaffin cells, PC12 cells and other models, to describe how NCS-1’s regulation of Ca2+ channels allows it to exert its physiological effects. 相似文献
11.
Abou-Lovergne A Collado-Hilly M Monnet FP Koukoui O Prigent S Coquil JF Dupont G Combettes L 《Cell calcium》2011,50(1):62-72
In hepatocytes, as in other cell types, Ca2+ signaling is subject to complex regulations, which result largely from the intrinsic characteristics of the different inositol 1,4,5-trisphosphate receptor (InsP3R) isoforms and from their interactions with other proteins. Although sigma1 receptors (Sig-1Rs) are widely expressed in the liver, their involvement in hepatic Ca2+ signaling remains unknown. We here report that in this cell type Sig-1R interact with type 1 isoforms of the InsP3 receptors (InsP3R-1). These results obtained by immunoprecipitation experiments are confirmed by the observation that Sig-1R proteins and InsP3R-1 colocalize in hepatocytes. However, Sig-1R ligands have no effect on InsP3-induced Ca2+ release in hepatocytes. This can be explained by the rather low expression level expression of InsP3R-1. In contrast, we find that Sig-1R ligands can inhibit agonist-induced Ca2+ signaling via an inhibitory effect on InsP3 synthesis. We show that this inhibition is due to the stimulation of PKC activity by Sig-1R, resulting in the well-known down-regulation of the signaling pathway responsible for the transduction of the extracellular stimulus into InsP3 synthesis. The PKC sensitive to Sig-1R activity belongs to the family of conventional PKC, but the precise molecular mechanism of this regulation remains to be elucidated. 相似文献
12.
Aravind P Chandra K Reddy PP Jeromin A Chary KV Sharma Y 《Journal of molecular biology》2008,376(4):1100-1115
Neuronal calcium sensor-1 (NCS-1) is a major modulator of Ca2+ signaling with a known role in neurotransmitter release. NCS-1 has one cryptic (EF1) and three functional (EF2, EF3, and EF4) EF-hand motifs. However, it is not known which are the regulatory (Ca2+-specific) and structural (Ca2+- or Mg2+-binding) EF-hand motifs. To understand the specialized functions of NCS-1, identification of the ionic discrimination of the EF-hand sites is important. In this work, we determined the specificity of Ca2+ binding using NMR and EF-hand mutants. Ca2+ titration, as monitored by [15N,1H] heteronuclear single quantum coherence, suggests that Ca2+ binds to the EF2 and EF3 almost simultaneously, followed by EF4. Our NMR data suggest that Mg2+ binds to EF2 and EF3, thereby classifying them as structural sites, whereas EF4 is a Ca2+-specific or regulatory site. This was further corroborated using an EF2/EF3-disabled mutant, which binds only Ca2+ and not Mg2+. Ca2+ binding induces conformational rearrangements in the protein by reversing Mg2+-induced changes in Trp fluorescence and surface hydrophobicity. In a larger physiological perspective, exchanging or replacing Mg2+ with Ca2+ reduces the Ca2+-binding affinity of NCS-1 from 90 nM to 440 nM, which would be advantageous to the molecule by facilitating reversibility to the Ca2+-free state. Although the equilibrium unfolding transitions of apo-NCS-1 and Mg2+-bound NCS-1 are similar, the early unfolding transitions of Ca2+-bound NCS-1 are partially influenced in the presence of Mg2+. This study demonstrates the importance of Mg2+ as a modulator of calcium homeostasis and active-state behavior of NCS-1. 相似文献
13.
Mauricette Collado‐Hilly Jean‐François Coquil 《Biology of the cell / under the auspices of the European Cell Biology Organization》2009,101(8):469-480
Background information. Interconnections between the Ca2+ and cAMP signalling pathways can determine the specificity and diversity of the cellular effects mediated by these second messengers. Most cAMP effects are mediated by PKA (protein kinase A), which is anchored close to its membranous substrates by AKAPs (A kinase‐anchoring proteins). In many cell types, the activation of InsP3R (inositol 1,4,5‐trisphosphate receptor), an endoplasmic reticulum Ca2+ channel, is a key event of Ca2+ signalling. The phosphorylation of InsP3R1 by PKA stimulates Ca2+ mobilization. This control is thought to be tight, involving the association of PKA with InsP3R1. The InsP3R1 isoform predominates in central nervous tissue and its concentration is highest in the cerebellar microsomes. We investigated the complex formed by InsP3R1 and PKA in this fraction, vith a view to identifying its components and determining its distribution in the cerebellar cortex. Results. Immunoprecipitation experiments showed that InsP3R1 associated with PKA type IIβ and AKAP450, the longer variant of AKAP9, in sheep cerebellar microsomes. The co‐purification of AKAP450 with InsP3R1 on heparin‐agarose provided further evidence of the association of these proteins. Immunohistofluorescence experiments on slices of cerebellar cortex showed that AKAP450 was colocalized with InsP3R1 and RIIβ (regulatory subunit of PKA IIβ) in granule cells, but not in Purkinje cells. AKAP450 was localized in the Golgi apparatus of these two cell types whereas InsP3R1 was detected in this organelle only in granule cells. Conclusions. Taken together these results suggest that InsP3R1 forms a complex with AKAP450 and PKAIIβ, localized in the Golgi apparatus of cerebellar granule cells. In contrast, the association of InsP3R1 with PKA in Purkinje cells would require a different macromolecular complex. 相似文献
14.
Nidhi Kapoor Joshua T. Maxwell Gregory A. Mignery David Will Lothar A. Blatter Kathrin Banach 《PloS one》2014,9(1)
The functional role of inositol 1,4,5-trisphosphate (InsP3) signaling in cardiomyocytes is not entirely understood but it was linked to an increased propensity for triggered activity. The aim of this study was to determine how InsP3 receptors can translate Ca2+ release into a depolarization of the plasma membrane and consequently arrhythmic activity. We used embryonic stem cell-derived cardiomyocytes (ESdCs) as a model system since their spontaneous electrical activity depends on InsP3-mediated Ca2+ release. [InsP3]i was monitored with the FRET-based InsP3-biosensor FIRE-1 (Fluorescent InsP3 Responsive Element) and heterogeneity in sub-cellular [InsP3]i was achieved by targeted expression of FIRE-1 in the nucleus (FIRE-1nuc) or expression of InsP3 5-phosphatase (m43) localized to the plasma membrane. Spontaneous activity of ESdCs was monitored simultaneously as cytosolic Ca2+ transients (Fluo-4/AM) and action potentials (current clamp). During diastole, the diastolic depolarization was paralleled by an increase of [Ca2+]i and spontaneous activity was modulated by [InsP3]i. A 3.7% and 1.7% increase of FIRE-1 FRET ratio and 3.0 and 1.5 fold increase in beating frequency was recorded upon stimulation with endothelin-1 (ET-1, 100 nmol/L) or phenylephrine (PE, 10 µmol/L), respectively. Buffering of InsP3 by FIRE-1nuc had no effect on the basal frequency while attenuation of InsP3 signaling throughout the cell (FIRE-1), or at the plasma membrane (m43) resulted in a 53.7% and 54.0% decrease in beating frequency. In m43 expressing cells the response to ET-1 was completely suppressed. Ca2+ released from InsP3Rs is more effective than Ca2+ released from RyRs to enhance INCX. The results support the hypothesis that in ESdCs InsP3Rs form a functional signaling domain with NCX that translates Ca2+ release efficiently into a depolarization of the membrane potential. 相似文献
15.
Hella Schttelndreier Barry V.L. Potter Georg W. Mayr Andreas H. Guse 《Cellular signalling》2001,13(12):770
Contact of Jurkat T-lymphocytes with the extracellular matrix (ECM) protein laminin resulted in long-lasting α6β1-integrin-mediated Ca2+ signalling. Both Ca2+ release from thapsigargin-sensitive Ca2+ stores and capacitative Ca2+ entry via Ca2+ channels sensitive to SKF 96365 constitute important parts of this process. Inhibition of α6β1-integrin-mediated Ca2+ signalling by (1) the src kinase inhibitor PP2, (2) the PLC inhibitor U73122, and (3) the cyclic adenosine diphosphoribose (cADPR) antagonist 7-deaza-8-Br-cADPR indicate the involvement of src tyrosine kinases and the Ca2+-releasing second messengers d-myo-inositol 1,4,5-trisphosphate (InsP3) and cADPR. 相似文献
16.
In the present work we have investigated the actions of the oxidizing sulfhydryl reagent thimerosal on different mechanisms which regulate intracellular free Ca2+ concentration ([Ca2+]i) in GH4C1 pituitary cells. In intact Fura-2 loaded cells, low concentrations of thimerosal potentiated the spike phase of the TRH-induced (thyrotropin-releasing hormone) rise in [Ca2+]i, whereas high thimerosal concentrations inhibited it. The effect of thimerosal on the plateau phase was always inhibitory.The effect of thimerosal on the IP3-induced calcium release (IICR) was studied in permeabilized cells using the Ca2+ indicator Fluo-3. A low concentration of thimerosal (10 μM) stimulated IICR: the Ca2+ release induced by 300 nM inositol-1,4,5-trisphosphate (IP3) was enhanced in cells treated with thimerosal for 1 or 6 min (67 ± 11 nM and 34 ± 5 nM, respectively) as compared to control cells (17 ± 2 nM). On the other hand, a high concentration of thimerosal (100 μ inhibited IICR: when IP3 (10 μM) was added after a 5 min preincubation with thimerosal, the IP3-induced rise in [Ca2+]i (46 ± 14 nM) was 57% smaller as compared with that seen in control cells (106 ± 10 nM).The effect of thimerosal on the voltage-operated Ca 2+ channels (VOCCs) was studied by depolarizing intact Fura-2 loaded cells by addition of 20 mM K+ to the cuvette. The depolarization-evoked increase in [Ca2+]i was inhibited in a dose-dependent manner by thimerosal. Direct evidence for an inhibitory effect of thimerosal on VOCCs was obtained by using the whole-cell configuration of the patch-clamp technique: thimerosal (100 μM) potently inhibited the Ba2+ currents through VOCCs.In addition, our results indicated that thimerosal inhibited the caffeine-induced increase in [Ca2+]i, and activated a capacitative Ca2+ entry pathway. The actions of thimerosal were apparently due to its oxidizing activity because the effects were mostly reversed by the thiol-reducing agent dithiothreitol (DTT).We conclude that, in GH4C1 pituitary cells, the mobilization of intracellular calcium and the different Ca2+ entry pathways are sensitive to redox modulation. 相似文献
17.
The inositol 1,4,5-trisphosphate (InsP3) receptor was purified from bovine cerebellum and reconstituted in liposomes composed of phosphatidylcholine (PC) and phosphatidylethanolamine
(PE) (1:1) successfully. No effect of Ca2+ concentration on [3H]-InsP3 binding to unreconstituted InsP3 receptor could be observed either at 4°C or at 25°C, whereas the effect of [Ca2+] on reconstituted InsP3 receptor depended on the temperature. The Ca2+ concentration outside the proteolipsome ([Ca2+]o) had no detectable effect on InsP3 binding to InsP3 receptor at 4°C. In contrast, with increase of [Ca2+]o from 0 to 100 nmol/L at 25°C, the InsP3 binding activity increased gradually. Then the InsP3 binding activity was decreased drastically at higher [Ca2+]o and inhibited entirely at 50 μmol/L [Ca2+]o. Conformational studies on intrinsic fluorescence of the reconstituted InsP3 receptor and its quenching by KI and HB indicated that the global conformation of reconstituted InsP3 receptor could not be affected by [Ca2+]o at 4°C. While at 25°C, the effects of 10 μmol/L [Ca2+]o on global, membrane and cytoplasmic conformation of the reconstituted InsP3 receptor were different significantly from that of 100 nmol/L [Ca2+]o. 相似文献
18.
Light-induced expression of the Gsa gene encoding the heme and chlorophyll biosynthetic enzyme glutamate 1-semialdehyde aminotransferase in Chlamydomonas reinhardtii was previously shown to involve Ca2+ and calmodulin (CaM) (C. lm et al. 1996, Plant Cell 8: 2245–2253). To further analyze the signal transduction pathway for
light-induced Gsa expression, the effects of several pharmacological agents were examined. Treatment of light-dark synchronized cells with
the heterotrimeric G-protein agonist Mas-7 caused partial induction of Gsa in the dark. The phospholipase C inhibitor U73122 inhibited light induction of Gsa. Exposure of cells to light caused a sustained 3-fold increase in cellular d-inositol 1,4,5-trisphosphate (InsP3) concentration. KN-93, a specific inhibitor of Ca2+/CaM-dependent protein kinase II, inhibited light induction of Gsa. In contrast, cyclosporin A, a specific inhibitor of the Ca2+/CaM-dependent phosphoprotein phosphatase calcineurin, did not affect light induction of Gsa. These results, together with the earlier results, suggest the involvement of a canonical signal transduction pathway for
light-regulated Gsa expression that involves a heterotrimeric G-protein activation, phospholipase C-catalyzed InsP3 formation, InsP3-dependent Ca2+ release, and activation of a downstream signaling pathway through a Ca2+/CaM-dependent protein kinase.
Received: 21 October 1999 / Accepted: 3 December 1999 相似文献
19.
Bridget S. Wilson Janet R. Pfeiffer Alexander J. Smith Janet M. Oliver Jon A. Oberdorf Richard J.H. Wojcikiewicz 《Molecular biology of the cell》1998,9(6):1465-1478
Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP3), which operates as an InsP3-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor (FcεR1) leads to activation of phospholipase C γ isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways, release of InsP3-sensitive intracellular Ca2+ stores, and a sustained phase of Ca2+ influx. These events are accompanied by a redistribution of type II InsP3 receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP3 receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP3 receptor clustering occurs within 5–10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 μM, and maximal clustering responses are dependent on the presence of extracellular calcium. InsP3 receptor aggregation may be a characteristic cellular response to Ca2+-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4–2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36M3R cells. Stimulation of these three cell types leads to a reduction in InsP3 receptor levels only in AR4–2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP3 receptors may contribute to the previously observed changes in affinity for InsP3 in the presence of elevated Ca2+ and/or may establish discrete regions within refilled stores with varying capacity to release Ca2+ when a subsequent stimulus results in production of InsP3. 相似文献
20.
The Mg2+-dependency of Ca2+-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg2+- and Ca2+-buffering ligands. ATP hydrolysis is strongly stimulated by Mg2+ with a Km of 13 μ M in the absence or presence of 1 μ M free Ca2+. At free Mg2+ concentrations of 1 μ M and lower, ATP hydrolysis is Mg2+ -independent, but is strongly stimulated by submicromolar Ca2+ concentrations Km = 0.25 μM, Vmax = 24 μmol Pi/h per mg protein). The Ca2+-stimulated ATP hydrolysis strongly decreases at higher Mg2+ concentrations. The Ca2+-stimulated Mg2+-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg2+ concentrations calmodulin and trifluoperazine affect the high affinity Ca2+-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca2+-pumping in renal basolaterals and on Ca2+-ATPase in erythrocyte ghosts suggest that the Ca2+-pumping enzyme requires Mg2+. In contrast, a role of the Ca2+-stimulated Mg2+-independent ATP hydrolysis in active Ca2+ transport across basolateral membranes is rather unlikely. 相似文献