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1.
Arachidonic acid causes an increase in free cytoplasmic calcium concentration ([Ca2+]i) in differentiated skeletal multinucleated myotubes C2C12 and does not induce calcium response in C2C12 myoblasts. The same reaction of myotubes to arachidonic acid is observed in Ca2+-free medium. This indicates that arachidonic acid induces release of calcium ions from intracellular stores. The blocker of ryanodine receptor channels of sarcoplasmic reticulum dantrolene (20 μM) inhibits this effect by 68.7 ± 6.3% (p < 0.001). The inhibitor of two-pore calcium channels of endolysosomal vesicles trans-NED19 (10 μM) decreases the response to arachidonic acid by 35.8 ± 5.4% (p < 0.05). The phospholipase C inhibitor U73122 (10 μM) has no effect. These data indicate the involvement of ryanodine receptor calcium channels of sarcoplasmic reticulum in [Ca2+]i elevation in skeletal myotubes caused by arachidonic acid and possible participation of two-pore calcium channels from endolysosomal vesicles in this process. 相似文献
2.
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
3.
Marnie L. Olson Susan Chalmers John G. McCarron 《The Journal of biological chemistry》2010,285(3):2040-2050
Smooth muscle activities are regulated by inositol 1,4,5-trisphosphate (InsP3)-mediated increases in cytosolic Ca2+ concentration ([Ca2+]c). Local Ca2+ release from an InsP3 receptor (InsP3R) cluster present on the sarcoplasmic reticulum is termed a Ca2+ puff. Ca2+ released via InsP3R may diffuse to adjacent clusters to trigger further release and generate a cell-wide (global) Ca2+ rise. In smooth muscle, mitochondrial Ca2+ uptake maintains global InsP3-mediated Ca2+ release by preventing a negative feedback effect of high [Ca2+] on InsP3R. Mitochondria may regulate InsP3-mediated Ca2+ signals by operating between or within InsP3R clusters. In the former mitochondria could regulate only global Ca2+ signals, whereas in the latter both local and global signals would be affected. Here whether mitochondria maintain InsP3-mediated Ca2+ release by operating within (local) or between (global) InsP3R clusters has been addressed. Ca2+ puffs evoked by localized photolysis of InsP3 in single voltage-clamped colonic smooth muscle cells had amplitudes of 0.5–4.0 F/F0, durations of ∼112 ms at half-maximum amplitude, and were abolished by the InsP3R inhibitor 2-aminoethoxydiphenyl borate. The protonophore carbonyl cyanide 3-chloropheylhydrazone and complex I inhibitor rotenone each depolarized ΔΨM to prevent mitochondrial Ca2+ uptake and attenuated Ca2+ puffs by ∼66 or ∼60%, respectively. The mitochondrial uniporter inhibitor, RU360, attenuated Ca2+ puffs by ∼62%. The “fast” Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acted like mitochondria to prolong InsP3-mediated Ca2+ release suggesting that mitochondrial influence is via their Ca2+ uptake facility. These results indicate Ca2+ uptake occurs quickly enough to influence InsP3R communication at the intra-cluster level and that mitochondria regulate both local and global InsP3-mediated Ca2+ signals. 相似文献
4.
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
5.
《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
6.
Maria Teresa Alonso Maria José Barrero Pedro Michelena Estela Carnicero Inmaculada Cuchillo Antonio G. García Javier García-Sancho Mayte Montero Javier Alvarez 《The Journal of cell biology》1999,144(2):241-254
The presence and physiological role of Ca2+-induced Ca2+ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca2+] ([Ca2+]c). Using targeted aequorin, we have directly monitored [Ca2+] changes inside the ER ([Ca2+]ER) in bovine adrenal chromaffin cells. Ca2+ entry induced by cell depolarization triggered a transient Ca2+ release from the ER that was highly dependent on [Ca2+]ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca2+ release was quantal in nature due to modulation by [Ca2+]ER. Whereas caffeine released essentially all the Ca2+ from the ER, inositol 1,4,5-trisphosphate (InsP3)- producing agonists released only 60–80%. Both InsP3 and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca2+ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca2+]c measurements showed that the wave of [Ca2+]c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca2+ pool that can release Ca2+ both via InsP3 receptors or CICR. 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.
In the resting state, the Ca2+ concentration in agonist-sensitive intracellular stores reflects the balance between active uptake of Ca2+, which is mediated by Ca2+-ATPase (SERCA), and passive leakage of Ca2+. The mechanisms underlying such a leakage in cells of the submaxillary salivary gland were not studied. In our experiments,
we examined possible pathways of passive leakage of Ca2+ from the endoplasmic reticulum (ER) of acinar cells obtained from the rat submaxillary salivary gland; direct measurements
of the concentration of Ca2+ in the ER ([Ca2+]ER) using a low-affinity calcium-sensitive dye, mag-fura 2/AM, were performed. The cellular membrane was permeabilized with
the help of β-escin (40 μg/ml); the Ca2+ concentration in the cytoplasm ([Ca2+]
i
) was clamped at its level typical of the resting state (∼100 nM) using an EGTA/Ca2+ buffer. Incubation of permeabilized acinar cells in a calcium-free intracellular milieu, as well as application of thapsigargin,
resulted in complete inhibition of the uptake of Ca2+ with the involvement of SERCA. This effect was observed 1 min after the beginning of superfusion of the cells with the corresponding
solutions and was accompanied by the leakage of Ca2+ from the ER; this is confirmed by a gradual drop in the [Ca2+]ER. Such a leakage of Ca2+ remained unchanged in the presence of thapsigargin, heparin, and ruthenium red; therefore, it is not mediated by SERCA, inositol
1,4,5-trisphosphate-sensitive receptors (InsP3R), or ryanodine receptors (RyRs). At the same time, an antibiotic, puromycin (0.1 to 1.0 mM), which disconnects polypeptides
from the ER-ribosome translocon complex, caused intensification of passive leakage of Ca2+ from the ER. This effect did not depend on the functioning of SERCA, InsP3R, or RyR. Therefore, passive leakage of Ca2+ from the ER in acinar cells of the submaxillary salivary gland is realized through pores of the translocon complex of the
ER membrane.
Neirofiziologiya/Neurophysiology, Vol. 37, No. 4, pp. 339–346, July–August, 2005. 相似文献
9.
SantoDomingo J Fonteriz RI Lobatón CD Montero M Moreno A Alvarez J 《Cellular and molecular neurobiology》2010,30(8):1267-1274
We have investigated the dynamics of the free [Ca2+] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca2+-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca2+] ([Ca2+]SG] was around 20–40 μM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type
Ca2+ pumps with thapsigargin largely blocked Ca2+ uptake by the granules in Ca2+-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consistently, the SERCA-type
Ca2+ pump inhibitor benzohydroquinone induced a rapid release of Ca2+ from the granules both in intact and permeabilized cells, suggesting that the continuous activity of SERCA-type Ca2+ pumps is essential to maintain the steady-state [Ca2+]SG. Both inositol 1,4,5-trisphosphate (InsP3) and caffeine produced a rapid Ca2+ release from the granules, suggesting the presence of InsP3 and ryanodine receptors in the granules. The response to high-K+ depolarization was different in both cell types, a decrease in [Ca2+]SG in PC12 cells and an increase in [Ca2+]SG in INS1 cells. The difference may rely on the heterogeneous response of different vesicle populations in each cell type.
Finally, increasing the glucose concentration triggered a decrease in [Ca2+]SG in INS1 cells. In conclusion, our data show that the secretory granules of PC12 and INS1 cells take up Ca2+ through SERCA-type Ca2+ pumps and can release it through InsP3 and ryanodine receptors, supporting the hypothesis that secretory granule Ca2+ may be released during cell stimulation and contribute to secretion. 相似文献
10.
11.
Wataru Masuda Matthew J. Betzenhauser David I. Yule 《The Journal of biological chemistry》2010,285(48):37927-37938
Ca2+ release through inositol 1,4,5-trisphosphate receptors (InsP3R) can be modulated by numerous factors, including input from other signal transduction cascades. These events shape the spatio-temporal characteristics of the Ca2+ signal and provide fidelity essential for the appropriate activation of effectors. In this study, we investigate the regulation of Ca2+ release via InsP3R following activation of cyclic nucleotide-dependent kinases in the presence and absence of expression of a binding partner InsP3R-associated cGMP kinase substrate (IRAG). cGMP-dependent kinase (PKG) phosphorylation of only the S2+ InsP3R-1 subtype resulted in enhanced Ca2+ release in the absence of IRAG expression. In contrast, IRAG bound to each InsP3R subtype, and phosphorylation of IRAG by PKG attenuated Ca2+ release through all InsP3R subtypes. Surprisingly, simply the expression of IRAG attenuated phosphorylation and inhibited the enhanced Ca2+ release through InsP3R-1 following cAMP-dependent protein kinase (PKA) activation. In contrast, IRAG expression did not influence the PKA-enhanced activity of the InsP3R-2. Phosphorylation of IRAG resulted in reduced Ca2+ release through all InsP3R subtypes during concurrent activation of PKA and PKG, indicating that IRAG modulation is dominant under these conditions. These studies yield mechanistic insight into how cells with various complements of proteins integrate and prioritize signals from ubiquitous signaling pathways. 相似文献
12.
13.
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
14.
15.
The influence of Earth magnetic field shielded down to 0.3 μT and static magnetic field (60–160 μT) on the proliferation and
differentiation of satellite muscle cells in primary culture has been investigated. A stimulatory effect of static magnetic
fields on the rate of the formation of massive multinucleate myotubes and an increase in the intracellular calcium concentration
([Ca2+]
i
) have been detected for magnetic fields of the microtesla range. On the other hand, it was shown that the reduction of earth
magnetic fields to 0.3 μT leads to inhibition of proliferation and differentiation of skeletal muscle cells in primary culture.
Since the formation of contractile myotubes during in vitro experiments is similar to the regeneration of skeletal muscle
fibers under muscle damage in vivo, it may be concluded that weak magnetic fields have a strong effect on intracellular processes
by influencing all phases of muscle fiber formation. It is necessary to take this fact into consideration when forecasting
probable complications of skeletal muscle regeneration during long-term exposure of man to low-intensity magnetic fields and
also for the potential use of low static magnetic fields as a tool to recover the affected myogenesis. 相似文献
16.
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
17.
Vronique Machelon Franoise Nom Brigitte Grosse Michle Lieberherr 《Journal of cellular biochemistry》1996,61(4):619-628
We investigated the early effects (5–60 s) of progesterone (1 pM–0.1 μM) on cytosolic free calcium concentration ([Ca2+]i) and inositol 1,4,5-trisphosphate (InsP3) formation in nonluteinized and in vitro luteinized porcine granulosa cells (pGCs). Progesterone increased [Ca2+]i and InsP3 formation within 5 s in both cell types. Progesterone induced calcium mobilization from the endoplasmic reticulum via the activation of a phospholipase C linked to a pertussis-insensitive G-protein. This process was controlled by protein kinases C and A. In contrast, only nonluteinized pGCs showed a Ca2+ influx via dihydropyridine-insensitive calcium channel. In both cell types, the nuclear progesterone receptor antagonist RU-38486 did not inhibit the progesterone-induced increase in [Ca2+]i; progesterone immobilized on bovine serum albumin, which did not enter the cell, increased [Ca2+]i within 5 s and was a full agonist, but less potent than the free progesterone; pertussis toxin did not inhibit progesterone effect on InsP3. In conclusion, progesterone may interact with membrane unconventional receptors that belong to the class of membrane receptors coupled to a phospholipase C via a pertussis toxin-insensitive G-protein. The source of the Ca2+ for the progesterone-induced increase in [Ca2+]i also depends on the stage of cell luteinization. © 1996 Wiley-Liss, Inc. 相似文献
18.
Mastoparan induces Ca2+-dependent deflagellation of the unicellular green alga Chlamydomonas moewusii Gerloff, as well as the activation of phospholipase C and the production of inositol 1,4,5-trisphosphate (InsP3; T. Munnik et al., 1998, Planta 207: 133–145). Even in the absence of extracellular Ca2+, mastoparan still induces deflagellation (L.M. Quarmby and H.C. Hartzell, 1994, J Cell Biol 124: 807–815; J.A.J. van Himbergen
et al., 1999, J Exp Bot, in press) suggesting that InsP3 mediates Ca2+ release from intracellular stores. To test this hypothesis, cells were pre-loaded with 45Ca2+ and their plasma membranes permeabilized by digitonin. Subsequent treatment of the cells with mastoparan (3.5 μM) induced
release of intracellular 45Ca2+. Mastoparan also activated phospholipase C in permeabilized cells, as demonstrated by the breakdown of 32P-phosphatidylinositol 4,5-bisphosphate and the production of diacylglycerol. The mastoparan analogues mas7 and mas17 were
also effective and their efficacy was correlated with their biological activity. X-ray microanalysis showed that electron-dense
bodies (EDBs) are a major Ca2+ store in C. moewusii. Analysis of digitonin-permeabilized cells showed that EDBs lost calcium at digitonin concentrations that released radioactivity
from 45Ca2+-labelled cells, suggesting that 45Ca2+ monitored the content of EDBs. X-ray microanaysis of living cells treated with mastoparan also revealed that calcium was
released from EDBs.
Received: 30 December 1998 / Accepted: 25 June 1999 相似文献
19.
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
20.
Meenakshisundaram Ananthanarayanan Jesus M. Banales Mateus T. Guerra Carlo Spirli Patricia Munoz-Garrido Kisha Mitchell-Richards Denisse Tafur Elena Saez Michael H. Nathanson 《The Journal of biological chemistry》2015,290(1):184-196
The type III isoform of the inositol 1,4,5-trisphosphate receptor (InsP3R3) is apically localized and triggers Ca2+ waves and secretion in a number of polarized epithelia. However, nothing is known about epigenetic regulation of this InsP3R isoform. We investigated miRNA regulation of InsP3R3 in primary bile duct epithelia (cholangiocytes) and in the H69 cholangiocyte cell line, because the role of InsP3R3 in cholangiocyte Ca2+ signaling and secretion is well established and because loss of InsP3R3 from cholangiocytes is responsible for the impairment in bile secretion that occurs in a number of liver diseases. Analysis of the 3′-UTR of human InsP3R3 mRNA revealed two highly conserved binding sites for miR-506. Transfection of miR-506 mimics into cell lines expressing InsP3R3–3′UTR-luciferase led to decreased reporter activity, whereas co-transfection with miR-506 inhibitors led to enhanced activity. Reporter activity was abrogated in isolated mutant proximal or distal miR-506 constructs in miR-506-transfected HEK293 cells. InsP3R3 protein levels were decreased by miR-506 mimics and increased by inhibitors, and InsP3R3 expression was markedly decreased in H69 cells stably transfected with miR-506 relative to control cells. miR-506-H69 cells exhibited a fibrotic signature. In situ hybridization revealed elevated miR-506 expression in vivo in human-diseased cholangiocytes. Histamine-induced, InsP3-mediated Ca2+ signals were decreased by 50% in stable miR-506 cells compared with controls. Finally, InsP3R3-mediated fluid secretion was significantly decreased in isolated bile duct units transfected with miR-506, relative to control IBDU. Together, these data identify miR-506 as a regulator of InsP3R3 expression and InsP3R3-mediated Ca2+ signaling and secretion. 相似文献