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1.
In pancreatic β-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca2+ influx, which triggers insulin exocytosis. The mitochondrial Ca2+ uniporter (MCU) mediates Ca2+ uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilized clonal β-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca2+ rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCU-silenced cells. In contrast, nutrient-induced hyperpolarization of the electrical gradient was not altered. In permeabilized cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca2+. Suppression of the putative Ca2+/H+ antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca2+-induced matrix acidification. These results demonstrate that MCU-mediated Ca2+ uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells.  相似文献   

2.
3.
How Ca2+ oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca2+ oscillations report signal strength via frequency, whereas Ca2+ spike amplitude and wave velocity remain constant. IP3 uncaging also triggers oscillatory Ca2+ release, but, in contrast to hormones, Ca2+ spike amplitude, width, and wave velocity were dependent on [IP3] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP3 uncaging are driven by the biphasic regulation of the IP3 receptor by Ca2+, and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca2+ did not perturb Ca2+ oscillations elicited by IP3 uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca2+ influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca2+ oscillations but had no effect on Ca2+ increases induced by uncaging IP3. Importantly, PKC activation and inhibition differentially affected Ca2+ spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP3 metabolism to attenuate IP3 levels and suppress the generation of Ca2+ oscillations. Inhibition of PKC relieves negative feedback regulation of IP3 accumulation and, thereby, shifts Ca2+ oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca2+ wave velocity, whereas responses to IP3 uncaging are enhanced. The ability to assess Ca2+ responses in the absence of PLC activity indicates that IP3 receptor modulation by PKC regulates Ca2+ release and wave velocity.  相似文献   

4.
Physiological signaling by reactive oxygen species (ROS) and their pathophysiological role in cell death are well recognized. This review focuses on two ROS targets that are key to local Ca2+ signaling at the ER/mitochondrial interface – notably, inositol trisphosphate receptors (IP3Rs) and the mitochondrial calcium uniporter (MCU). Both transport systems are central to molecular mechanisms in cell survival and death. Methods for the measurement of the redox state of these proteins and for the detection of ROS nanodomains are described. Recent results on the redox regulation of these proteins are reviewed.  相似文献   

5.
1. Gastrulating chick embryo cells (stages 3–5 by HH) possess Ca2+-mobilizing receptors for ACh and ATP; insulin and noradrenaline have a weaker effect on [Ca2+], mobilization.2. The ed50 value for ACh is 4 (±0.5)· 10−6M and for ATP 20 (±5)· 10−6M.3. Addition of ACh and ATP to dissociated chick embryo cells causes rapid accumulation of IP3.4. The stimulatory effects of ACh and ATP on [Ca2+], mobilization and IP3 rapid formation are both additive.  相似文献   

6.
The effect of inositol 1,4,5-trisphosphate (IP3) on Ca2+ release in the transformed murine mast cells, mastocytoma P-815 cells permeabilized with digitonin was studied. Ca2+ was sequestered by intracellular organelles in the presence of ATP until the medium free Ca2+ concentration was lowered to a new steady-state level. The subsequent addition of IP3 caused a rapid Ca2+ release, which was followed by a slow re-uptake of Ca2+. Fifty percent of the sequestered Ca2+ was released by 10 μM IP3. Maximal Ca2+ release occurred at 10 μM and half maximal activity was at 1.3 μM. These results indicate that IP3 may function as a messenger of intracellular Ca2+ mobilization in mastocytoma cells.  相似文献   

7.
Previous studies have suggested that the cellular Ca2+ and iron homeostasis, which can be regulated by mitochondrial calcium uniporter (MCU), is associated with oxidative stress, apoptosis and many neurological diseases. However, little is known about the role of MCU‐mediated Ca2+ and iron accumulation in traumatic brain injury (TBI). Under physiological conditions, MCU can be inhibited by ruthenium red (RR) and activated by spermine (Sper). In the present study, we used RR and Sper to reveal the role of MCU in mouse and neuron TBI models. Our results suggested that the Ca2+ and iron concentrations were obviously increased after TBI. In addition, TBI models showed a significant generation of reactive oxygen species (ROS), decrease in adenosine triphosphate (ATP), deformation of mitochondria, up‐regulation of deoxyribonucleic acid (DNA) damage and increase in apoptosis. Blockage of MCU by RR prevented Ca2+ and iron accumulation, abated the level of oxidative stress, improved the energy supply, stabilized mitochondria, reduced DNA damage and decreased apoptosis both in vivo and in vitro. Interestingly, Sper did not increase cellular Ca2+ and iron concentrations, but suppressed the Ca2+ and iron accumulation to benefit the mice in vivo. However, Sper had no significant impact on TBI in vitro. Taken together, our data demonstrated for the first time that blockage of MCU‐mediated Ca2+ and iron accumulation was essential for TBI. These findings indicated that MCU could be a novel therapeutic target for treating TBI.  相似文献   

8.
HL-1 cells are the adult cardiac cell lines available that continuously divide while maintaining an atrial phenotype. Here we examined the expression and localization of inositol 1,4,5-trisphosphate receptor (IP3R) subtypes, and investigated how pattern of IP3-induced subcellular local Ca2+ signaling is encoded by multiple IP3R subtypes in HL-1 cells. The type 1 IP3R (IP3R1) was expressed in the perinucleus with a diffuse pattern and the type 2 IP3R (IP3R2) was expressed in the cytosol with a punctate distribution. Extracellular ATP (1 mM) elicited transient intracellular Ca2+ releases accompanied by a Ca2+ oscillation, which was eliminated by the blocker of IP3Rs, 2-APB, and attenuated by ryanodine. Direct introduction of IP3 into the permeabilized cells induced Ca2+ transients with Ca2+ oscillations at ⩾ 20 μM of IP3, which was removed by the inhibition of IP3Rs using 2-APB and heparin. IP3-induced local Ca2+ transients contained two distinct time courses: a rapid oscillation and a monophasic Ca2+ transient. The magnitude of Ca2+ oscillation was significantly larger in the cytosol than in the nucleus, while the monophasic Ca2+ transient was more pronounced in the nucleus. These results provide evidence for the molecular and functional expression of IP3R1 and IP3R2 in HL-1 cells, and suggest that such distinct local Ca2+ signaling may be correlated with the punctate distribution of IP3R2s in the cytosol and the diffuse localization of IP3R1 in the peri-nucleus.  相似文献   

9.
《Biophysical journal》2020,118(5):1196-1204
Intercellular bridges are plasma continuities formed at the end of the cytokinesis process that facilitate intercellular mass transport between the two daughter cells. However, it remains largely unknown how the intercellular bridge mediates Ca2+ communication between postmitotic cells. In this work, we utilize BV-2 microglial cells planted on dumbbell-shaped micropatterned assemblies to resolve spatiotemporal characteristics of Ca2+ signal transfer over the intercellular bridges. With the use of such micropatterns, considerably longer and more regular intercellular bridges can be obtained than in conventional cell cultures. The initial Ca2+ signal is evoked by mechanical stimulation of one of the daughter cells. A considerable time delay is observed between the arrivals of passive Ca2+ diffusion and endogenous Ca2+ response in the intercellular-bridge-connected cell, indicating two different pathways of the Ca2+ communication. Extracellular Ca2+ and the paracrine pathway have practically no effect on the endogenous Ca2+ response, demonstrated by application of Ca2+-free medium, exogenous ATP, and P2Y13 receptor antagonist. In contrast, the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin and inositol trisphosphate (IP3) receptor blocker 2-aminoethyl diphenylborate significantly inhibit the endogenous Ca2+ increase, which signifies involvement of IP3-sensitive calcium store release. Notably, passive Ca2+ diffusion into the connected cell can clearly be detected when IP3-sensitive calcium store release is abolished by 2-aminoethyl diphenylborate. Those observations prove that both passive Ca2+ diffusion and IP3-mediated endogenous Ca2+ response contribute to the Ca2+ increase in intercellular-bridge-connected cells. Moreover, a simulation model agreed well with the experimental observations.  相似文献   

10.
This work shows that ATP activates JNK1, but not JNK2, in rat osteoblasts and ROS-A 17/2.8 osteoblast-like cells. In ROS-A 17/2.8 cells ATP induced JNK1 phosphorylation in a dose- and time-dependent manner. JNK1 phosphorylation also increased after osteoblast stimulation with ATPγS and UTP, but not with ADPβS. RT-PCR studies supported the expression of P2Y2 receptor subtype. ATP-induced JNK1 activation was reduced by PI-PLC, IP3 receptor, PKC and Src inhibitors and by gadolinium, nifedipine and verapamil or a Ca2+-free medium. ERK 1/2 or p38 MAPK inhibitors diminished JNK1 activation by ATP, suggesting a cross-talk between these pathways. ATP stimulated osteoblast-like cell proliferation consistent with the participation of P2Y2 receptors. These results show that P2Y2 receptor stimulation by ATP induces JNK1 phosphorylation in ROS-A 17/2.8 cells in a way dependent on PI-PLC/IP3/intracellular Ca2+ release and Ca2+ influx through stress activated and L-type voltage-dependent calcium channels and involves PKC and Src kinases.  相似文献   

11.
The expression of protein kinase C (PKC) isoforms and the modulation of Ca2+ mobilization by PKC were investigated in the human submandibular duct cell line A253. Three new PKC (nPKC) isoforms (, , and ) and one atypical PKC (aPKC) isoform () are expressed in this cell line. No classical PKC (cPKC) isoforms were present. The effects of the PKC activator phorbol 12-myristate-13-acetate (PMA) and of the PKC inhibitors calphostin C (CC) and bisindolymaleimide I (BSM) on inositol 1,4,5-trisphosphate (IP3) and Ca2+ responses to ATP and to thapsigargin (TG) were investigated. Pre-exposure to PMA inhibited IP3 formation, Ca2+ release and Ca2+ influx in response to ATP. Pre-exposure to CC or BSM slightly enhanced IP3 formation but inhibited the Ca2+ release and the Ca2+ influx induced by ATP. In contrast, pre-exposure to PMA did not modify the Ca2+ release induced by TG, but reduced the influx of Ca2+ seen in the presence of this Ca2+-ATPase inhibitor. These results suggest that PKC modulates elements of the IP3/Ca2+ signal transduction pathway in A253 cells by (1) inhibiting phosphatidylinositol turnover and altering the sensitivity of the Ca2+ channels to IP3, (2) altering the activity, the sensitivity to inhibitors, or the distribution of the TG-sensitive Ca2+ ATPase, and (3) modulating Ca2+ entry pathways.  相似文献   

12.
《Cell calcium》2016,59(6):638-648
Localized subcellular changes in Ca2+ serve as important cellular signaling elements, regulating processes as diverse as neuronal excitability and gene expression. Studies of cellular Ca2+ signaling have been greatly facilitated by the availability of fluorescent Ca2+ indicators. The respective merits of different indicators to monitor bulk changes in cellular Ca2+ levels have been widely evaluated, but a comprehensive comparison for their use in detecting and analyzing local, subcellular Ca2+ signals is lacking. Here, we evaluated several fluorescent Ca2+ indicators in the context of local Ca2+ signals (puffs) evoked by inositol 1,4,5-trisphosphate (IP3) in cultured human neuroblastoma SH-SY5Y cells, using high-speed video-microscopy. Altogether, nine synthetic Ca2+ dyes (Fluo-4, Fluo-8, Fluo-8 high affinity, Fluo-8 low affinity, Oregon Green BAPTA-1, Cal-520, Rhod-4, Asante Calcium Red, and X-Rhod-1) and three genetically-encoded Ca2+-indicators (GCaMP6-slow, -medium and -fast variants) were tested; criteria include the magnitude, kinetics, signal-to-noise ratio and detection efficiency of local Ca2+ puffs. Among these, we conclude that Cal-520 is the optimal indicator for detecting and faithfully tracking local events; that Rhod-4 is the red-emitting indicator of choice; and that none of the GCaMP6 variants are well suited for imaging subcellular Ca2+ signals.  相似文献   

13.
Background information. The IP3R (inositol 1,4,5‐trisphosphate receptor) is a tetrameric channel that accounts for a large part of the intracellular Ca2+ release in virtually all cell types. We have previously demonstrated that caspase‐3‐mediated cleavage of IP3R1 during cell death generates a C‐terminal fragment of 95 kDa comprising the complete channel domain. Expression of this truncated IP3R increases the cellular sensitivity to apoptotic stimuli, and it was postulated to be a constitutively active channel. Results. In the present study, we demonstrate that expression of the caspase‐3‐cleaved C‐terminus of IP3R1 increased the rate of thapsigargin‐mediated Ca2+ leak and decreased the rate of Ca2+ uptake into the ER (endoplasmic reticulum), although it was not sufficient by itself to deplete intracellular Ca2+ stores. We detected the truncated IP3R1 in different cell types after a challenge with apoptotic stimuli, as well as in aged mouse oocytes. Injection of mRNA corresponding to the truncated IP3R1 blocked sperm factor‐induced Ca2+ oscillations and induced an apoptotic phenotype. Conclusions. In the present study, we show that caspase‐3‐mediated truncation of IP3R1 enhanced the Ca2+ leak from the ER. We suggest a model in which, in normal conditions, the increased Ca2+ leak is largely compensated by enhanced Ca2+‐uptake activity, whereas in situations where the cellular metabolism is compromised, as occurring in aging oocytes, the Ca2+ leak acts as a feed‐forward mechanism to divert the cell into apoptosis.  相似文献   

14.
Ca2+ release from intracellular stores regulates muscle contraction and a vast array of cell functions, but its role in the central nervous system (CNS) has not been completely elucidated. A new method of blocking IP3 signaling by artificially expressing IP3 5-phosphatase has been used to clarify the functions of intracellular Ca2+ mobilization in CNS. Here I review two of such functions: the activity-dependent synaptic maintenance mechanism and the regulation of neuronal growth by spontaneous Ca2+ oscillations in astrocytes. These findings add new bases for better understanding CNS functions and suggest the presence of as yet unidentified neuronal and glial functions that are regulated by Ca2+ store-dependent Ca2+ signaling.  相似文献   

15.
《Cell calcium》2015,58(5-6):348-365
High environmental salt elicits an increase in cytosolic Ca2+ ([Ca2+]cyt) in plants, which is generated by extracellular Ca2+ influx and Ca2+ release from intracellular stores, such as vacuole and endoplasmic reticulum. This study aimed to determine the physiological mechanisms underlying Ca2+ release from vacuoles and its role in ionic homeostasis in Populus euphratica. In vivo Ca2+ imaging showed that NaCl treatment induced a rapid elevation in [Ca2+]cyt, which was accompanied by a subsequent release of vacuolar Ca2+. In cell cultures, NaCl-altered intracellular Ca2+ mobilization was abolished by antagonists of inositol (1, 4, 5) trisphosphate (IP3) and cyclic adenosine diphosphate ribose (cADPR) signaling pathways, but not by slow vacuolar (SV) channel blockers. Furthermore, the NaCl-induced vacuolar Ca2+ release was dependent on extracellular ATP, extracellular Ca2+ influx, H2O2, and NO. In vitro Ca2+ flux recordings confirmed that IP3, cADPR, and Ca2+ induced substantial Ca2+ efflux from intact vacuoles, but this vacuolar Ca2+ flux did not directly respond to ATP, H2O2, or NO. Moreover, the IP3/cADPR-mediated vacuolar Ca2+ release enhanced the expression of salt-responsive genes that regulated a wide range of cellular processes required for ion homeostasis, including cytosolic K+ maintenance, Na+ and Cl exclusion across the plasma membrane, and Na+/H+ and Cl/H+ exchanges across the vacuolar membrane.  相似文献   

16.
Purinergic signaling mediated by P2 receptors (P2Rs) plays important roles in embryonic and stem cell development. However, how it mediates Ca2+ signals in human embryonic stem cells (hESCs) and derived cardiovascular progenitor cells (CVPCs) remains unclear. Here, we aimed to determine the role of P2Rs in mediating Ca2+ mobilizations of these cells. hESCs were induced to differentiate into CVPCs by our recently established methods. Gene expression of P2Rs and inositol 1,4,5-trisphosphate receptors (IP3Rs) was analyzed by quantitative/RT-PCR. IP3R3 knockdown (KD) or IP3R2 knockout (KO) hESCs were established by shRNA- or TALEN-mediated gene manipulations, respectively. Confocal imaging revealed that Ca2+ responses in CVPCs to ATP and UTP were more sensitive and stronger than those in hESCs. Consistently, the gene expression levels of most P2YRs except P2Y1 were increased in CVPCs. Suramin or PPADS blocked ATP-induced Ca2+ transients in hESCs but only partially inhibited those in CVPCs. Moreover, the P2Y1 receptor-specific antagonist MRS2279 abolished most ATP-induced Ca2+ signals in hESCs but not in CVPCs. P2Y1 receptor-specific agonist MRS2365 induced Ca2+ transients only in hESCs but not in CVPCs. Furthermore, IP3R2KO but not IP3R3KD decreased the proportion of hESCs responding to MRS2365. In contrast, both IP3R2 and IP3R3 contributed to UTP-induced Ca2+ responses while ATP-induced Ca2+ responses were more dependent on IP3R2 in the CVPCs. In conclusion, a predominant role of P2Y1 receptors in hESCs and a transition of P2Y-IP3R coupling in derived CVPCs are responsible for the differential Ca2+ mobilization between these cells.  相似文献   

17.
The inositol 1,4,5-trisphosphate receptor/channel (IP3R) is a major regulator of intracellular Ca2+ signaling, and liberates Ca2+ ions from the endoplasmic reticulum in response to binding at cytosolic sites for both IP3 and Ca2+. Although the steady-state gating properties of the IP3R have been extensively studied and modeled under conditions of fixed [IP3] and [Ca2+], little is known about how Ca2+ flux through a channel may modulate the gating of that same channel by feedback onto activating and inhibitory Ca2+ binding sites. We thus simulated the dynamics of Ca2+ self-feedback on monomeric and tetrameric IP3R models. A major conclusion is that self-activation depends crucially on stationary cytosolic Ca2+ buffers that slow the collapse of the local [Ca2+] microdomain after closure. This promotes burst-like reopenings by the rebinding of Ca2+ to the activating site; whereas inhibitory actions are substantially independent of stationary buffers but are strongly dependent on the location of the inhibitory Ca2+ binding site on the IP3R in relation to the channel pore.  相似文献   

18.
The influence of infrared laser pulses on intracellular Ca2+ signaling was investigated in neural cell lines with fluorescent live cell imaging. The probe Fluo‐4 was used to measure Ca2+ in HT22 mouse hippocampal neurons and nonelectrically excitable U87 human glioblastoma cells exposed to 50 to 500 ms infrared pulses at 1470 nm. Fluorescence recordings of Fluo‐4 demonstrated that infrared stimulation induced an instantaneous intracellular Ca2+ transient with similar dose‐response characteristics in hippocampal neurons and glioblastoma cells (half‐maximal effective energy density EC50 of around 58 J.cm?2). For both type of cells, the source of the infrared‐induced Ca2+ transients was found to originate from intracellular stores and to be mediated by phospholipase C and IP3‐induced Ca2+ release from the endoplasmic reticulum. The activation of phosphoinositide signaling by IR light is a new mechanism of interaction relevant to infrared neural stimulation that will also be widely applicable to nonexcitable cell types. The prospect of infrared optostimulation of the PLC/IP3 cell signaling cascade has many potential applications including the development of optoceutical therapeutics.   相似文献   

19.
The 5' AMP-activated protein kinase (AMPK) is a nutrient-sensitive kinase that plays a key role in the control of cellular energy metabolism. We have explored here the relationship between AMPK and Ca2+ signaling by looking at the effect of an AMPK activator (A769662) and an AMPK inhibitor (dorsomorphin) on histamine-induced Ca2+-release from the endoplasmic reticulum (ER) in HeLa cells. Our data show that incubation with A769662 (EC50 = 29 μM) inhibited histamine-induced Ca2+-release from the ER in intact cells, as well as inositol-1,4,5-trisphosphate (IP3)-induced Ca2+ release in permeabilized cells. On the contrary, dorsomorphin (EC50 = 0.4 μM) activated both histamine and IP3-induced Ca2+-release and reversed the effect of A769662. These results suggest a direct effect of AMPK regulation on IP3 receptor (IP3R) function. A phosphoproteomic study did not reveal changes in IP3R phosphorylation, but showed significant changes in phosphorylation of proteins placed upstream in the IP3R interactome and in several proteins related with Ca2+ metabolism, which could be candidates to mediate the effects observed. In conclusion, our data suggest that AMPK negatively regulates IP3R. This effect constitutes a novel and very important link between Ca2+ signaling and the AMPK pathway.  相似文献   

20.
The large inner membrane electrochemical driving force and restricted volume of the matrix confer unique constraints on mitochondrial ion transport. Cation uptake along with anion and water movement induces swelling if not compensated by other processes. For mitochondrial Ca2+ uptake, these include activation of countertransporters (Na+/Ca2+ exchanger and Na+/H+ exchanger) coupled to the proton gradient, ultimately maintained by the proton pumps of the respiratory chain, and Ca2+ binding to matrix buffers. Inorganic phosphate (Pi) is known to affect both the Ca2+ uptake rate and the buffering reaction, but the role of anion transport in determining mitochondrial Ca2+ dynamics is poorly understood. Here we simultaneously monitor extra- and intra-mitochondrial Ca2+ and mitochondrial membrane potential (ΔΨm) to examine the effects of anion transport on mitochondrial Ca2+ flux and buffering in Pi-depleted guinea pig cardiac mitochondria. Mitochondrial Ca2+ uptake proceeded slowly in the absence of Pi but matrix free Ca2+ ([Ca2+]mito) still rose to ∼50 μm. Pi (0.001–1 mm) accelerated Ca2+ uptake but decreased [Ca2+]mito by almost 50% while restoring ΔΨm. Pi-dependent effects on Ca2+ were blocked by inhibiting the phosphate carrier. Mitochondrial Ca2+ uptake rate was also increased by vanadate (Vi), acetate, ATP, or a non-hydrolyzable ATP analog (AMP-PNP), with differential effects on matrix Ca2+ buffering and ΔΨm recovery. Interestingly, ATP or AMP-PNP prevented the effects of Pi on Ca2+ uptake. The results show that anion transport imposes an upper limit on mitochondrial Ca2+ uptake and modifies the [Ca2+]mito response in a complex manner.  相似文献   

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