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1.
Sarcoplasmic reticulum contains the internal Ca2+ store in smooth muscle cells and its lumen appears to be a continuum that lacks diffusion barriers. Accordingly, the free luminal Ca2+ level is the same all throughout the SR; however, whether the Ca2+ buffer capacity is the same in all the SR is unknown. We have estimated indirectly the luminal Ca2+ buffer capacity of the SR by comparing the reduction in SR Ca2+ levels with the corresponding increase in [Ca2+]i during activation of either IP3Rs with carbachol or RyRs with caffeine, in smooth muscle cells from guinea pig urinary bladder. We have determined that carbachol-sensitive SR has a 2.4 times larger Ca2+ buffer capacity than caffeine-sensitive SR. Rapid inhibition of SERCA pumps with thapsigargin revealed that this pump activity accounts for 80% and 60% of the Ca2+ buffer capacities of carbachol- and caffeine-sensitive SR, respectively. Moreover, the Ca2+ buffer capacity of carbachol-sensitive SR was similar to caffeine-sensitive SR when SERCA pumps were inhibited. Similar rates of Ca2+ replenishments suggest similar levels of SERCA pump activities for either carbachol- or caffeine-sensitive SR. Paired pulses of caffeine, in conditions of low Ca2+ influx, indicate the relevance of luminal SR Ca2+ buffer capacity in the [Ca2+]i response. To further study the importance of luminal SR Ca2+ buffer capacity in the release process we used low levels of heparin to partially inhibit IP3Rs. This condition revealed carbachol-induced transient increase of luminal SR Ca2+ levels provided that SERCA pumps were active. It thus appears that SERCA pump activity keeps the luminal SR Ca2+-binding proteins in the high-capacity, low-affinity conformation, particularly for IP3R-mediated Ca2+ release.  相似文献   

2.
In this study, we investigated the role of elevated sarcoplasmic reticulum (SR) Ca2+ leak through ryanodine receptors (RyR2s) in heart failure (HF)-related abnormalities of intracellular Ca2+ handling, using a canine model of chronic HF. The cytosolic Ca2+ transients were reduced in amplitude and slowed in duration in HF myocytes compared with control, changes paralleled by a dramatic reduction in the total SR Ca2+ content. Direct measurements of [Ca2+]SR in both intact and permeabilized cardiac myocytes demonstrated that SR luminal [Ca2+] is markedly lowered in HF, suggesting that alterations in Ca2+ transport rather than fractional SR volume reduction accounts for the diminished Ca2+ release capacity of SR in HF. SR Ca2+ ATPase (SERCA2)-mediated SR Ca2+ uptake rate was not significantly altered, and Na+/Ca2+ exchange activity was accelerated in HF myocytes. At the same time, SR Ca2+ leak, measured directly as a loss of [Ca2+]SR after inhibition of SERCA2 by thapsigargin, was markedly enhanced in HF myocytes. Moreover, the reduced [Ca2+]SR in HF myocytes could be nearly completely restored by the RyR2 channel blocker ruthenium red. The effects of HF on cytosolic and SR luminal Ca2+ signals could be reasonably well mimicked by the RyR2 channel agonist caffeine. Taken together, these results suggest that RyR2-mediated SR Ca2+ leak is a major factor in the abnormal intracellular Ca2+ handling that critically contributes to the reduced SR Ca2+ content of failing cardiomyocytes.  相似文献   

3.
The endoplasmic reticulum is the main intracellular Ca2+ store for Ca2+ release during cell signaling. There are different strategies to avoid ER Ca2+ depletion. Release channels utilize first Ca2+-bound to proteins and this minimizes the reduction of the free luminal [Ca2+]. However, if release channels stay open after exhaustion of Ca2+-bound to proteins, then the reduction of the free luminal ER [Ca2+] (via STIM proteins) activates Ca2+ entry at the plasma membrane to restore the ER Ca2+ load, which will work provided that SERCA pump is active. Nevertheless, there are several noxious conditions that result in decreased activity of the SERCA pump such as oxidative stress, inflammatory cytokines, and saturated fatty acids, among others. These conditions result in a deficient restoration of the ER [Ca2+] and lead to the ER stress response that should facilitate recovery of the ER. However, if the stressful condition persists then ER stress ends up triggering cell death and the ensuing degenerative process leads to diverse pathologies; particularly insulin resistance, diabetes and several of the complications associated with diabetes. This scenario suggests that limiting ER stress should decrease the incidence of diabetes and the mobility and mortality associated with this illness.  相似文献   

4.
The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca2+ release during systole. However, it remains obscure how the dyadic organization affects SR Ca2+ handling during diastole. By measuring intraluminal SR Ca2+ ([Ca2+]SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca2+ is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca2+ that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca2+-ATPase (SERCA). Detubulation decreased [Ca2+]SR decline during rest, thus making the leaked SR Ca2+ more accessible for SERCA. These results suggest that Ca2+ extrusion systems are localized in T-tubules. Inhibition of Na+-Ca2+ exchanger (NCX) slowed [Ca2+]SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca2+]SR decline. Despite a significant SR Ca2+ leak, Ca2+ sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca2+ spark activity independent of SR Ca2+ load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca2+ that leaks from the SR. This can be explained if the majority of SR Ca2+ leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca2+] by NCX play a critical role in suppressing Ca2+ sparks during rest.  相似文献   

5.
It has been shown that 2-APB is a nonspecific modulator of ion channel activity, while most of the channels are inhibited by this compound, there are few examples of channels that are activated by 2-APB. Additionally, it has been shown that, 2-APB leads to a reduction in the luminal endoplasmic reticulum Ca2+ level ([Ca2+]ER) and we have carried out simultaneous recordings of both [Ca2+]i and the [Ca2+]ER in HeLa cell suspensions to assess the mechanism involved in this effect. This approach allowed us to determine that 2-APB induces a reduction in the [Ca2+]ER by activating an ER-resident Ca2+ permeable channel more than by inhibiting the activity of SERCA pumps. Interestingly, this effect of 2-APB of reducing the [Ca2+]ER is auto-limited because depends on a replete ER Ca2+ store; a condition that thapsigargin does not require to decrease the [Ca2+]ER. Additionally, our data indicate that the ER Ca2+ permeable channel activated by 2-APB does not seem to participate in the ER Ca2+ leak revealed by inhibiting SERCA pump with thapsigargin. This work suggests that, prolonged incubations with even low concentrations of 2-APB (5 μM) would lead to the reduction in the [Ca2+]ER that might explain the inhibitory effect of this compound on those signals that require Ca2+ release from the ER store.  相似文献   

6.
The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca2+ release during systole. However, it remains obscure how the dyadic organization affects SR Ca2+ handling during diastole. By measuring intraluminal SR Ca2+ ([Ca2+]SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca2+ is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca2+ that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca2+-ATPase (SERCA). Detubulation decreased [Ca2+]SR decline during rest, thus making the leaked SR Ca2+ more accessible for SERCA. These results suggest that Ca2+ extrusion systems are localized in T-tubules. Inhibition of Na+-Ca2+ exchanger (NCX) slowed [Ca2+]SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca2+]SR decline. Despite a significant SR Ca2+ leak, Ca2+ sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca2+ spark activity independent of SR Ca2+ load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca2+ that leaks from the SR. This can be explained if the majority of SR Ca2+ leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca2+] by NCX play a critical role in suppressing Ca2+ sparks during rest.  相似文献   

7.
Experiments were performed to characterize the properties of the intrinsic Ca2+ buffers in the sarcoplasmic reticulum (SR) of cut fibers from frog twitch muscle. The concentrations of total and free calcium ions within the SR ([CaT]SR and [Ca2+]SR) were measured, respectively, with the EGTA/phenol red method and tetramethylmurexide (a low affinity Ca2+ indicator). Results indicate SR Ca2+ buffering was consistent with a single cooperative-binding component or a combination of a cooperative-binding component and a linear binding component accounting for 20% or less of the bound Ca2+. Under the assumption of a single cooperative-binding component, the most likely resting values of [Ca2+]SR and [CaT]SR are 0.67 and 17.1 mM, respectively, and the dissociation constant, Hill coefficient, and concentration of the Ca-binding sites are 0.78 mM, 3.0, and 44 mM, respectively. This information can be used to calculate a variable proportional to the Ca2+ permeability of the SR, namely d[CaT]SR/dt ÷ [Ca2+]SR (denoted release permeability), in experiments in which only [CaT]SR or [Ca2+]SR is measured. In response to a voltage-clamp step to −20 mV at 15°C, the release permeability reaches an early peak followed by a rapid decline to a quasi-steady level that lasts ∼50 ms, followed by a slower decline during which the release permeability decreases by at least threefold. During the quasi-steady level of release, the release amplitude is 3.3-fold greater than expected from voltage activation alone, a result consistent with the recruitment by Ca-induced Ca2+ release of 2.3 SR Ca2+ release channels neighboring each channel activated by its associated voltage sensor. Release permeability at −60 mV increases as [CaT]SR decreases from its resting physiological level to ∼0.1 of this level. This result argues against a release termination mechanism proposed in mammalian muscle fibers in which a luminal sensor of [Ca2+]SR inhibits release when [CaT]SR declines to a low level.  相似文献   

8.
Cytosolic calcium concentration ([Ca2+]c) is fundamental for regulation of many cellular processes such metabolism, proliferation, muscle contraction, cell signaling and insulin secretion. In resting conditions, the sarco/endoplasmic reticulum (ER/SR) Ca2+ ATPase's (SERCA) transport Ca2+ from the cytosol to the ER or SR lumen, maintaining the resting [Ca2+]c about 25–100 nM. A reduced activity and expression of SERCA2 protein have been described in heart failure and diabetic cardiomyopathy, resulting in an altered Ca2+ handling and cardiac contractility. In the diabetic pancreas, there has been reported reduction in SERCA2b and SERCA3 expression in β-cells, resulting in diminished insulin secretion. Evidence obtained from different diabetes models has suggested a role for advanced glycation end products formation, oxidative stress and increased O-GlcNAcylation in the lowered SERCA2 expression observed in diabetic cardiomyopathy. However, the role of SERCA2 down-regulation in the pathophysiology of diabetes mellitus and diabetic cardiomyopathy is not yet well described. In this review, we make a comprehensive analysis of the current knowledge of the role of the SERCA pumps in the pathophysiology of insulin-dependent diabetes mellitus type 1 (TIDM) and type 2 (T2DM) in the heart and β-cells in the pancreas.  相似文献   

9.
TRPV1 represents a non-selective cation channel activated by capsaicin, acidosis and high temperature. In the central nervous system where TRPV1 is highly expressed, its physiological role in nociception is clearly identified. In skeletal muscle, TRPV1 appears implicated in energy metabolism and exercise endurance. However, how as a Ca2+ channel, it contributes to intracellular calcium concentration ([Ca2+]i) maintenance and muscle contraction remains unknown. Here, as in rats, we report that TRPV1 is functionally expressed in mouse skeletal muscle. In contrast to earlier reports, our analysis show TRPV1 presence only at the sarcoplasmic reticulum (SR) membrane (preferably at the longitudinal part) in the proximity of SERCA1 pumps. Using intracellular Ca2+ imaging, we directly accessed to the channel functionality in intact FDB mouse fibers. Capsaicin and resiniferatoxin, both agonists as well as high temperature (45°C) elicited an increase in [Ca2+]i. TRPV1-inhibition by capsazepine resulted in a strong inhibition of TRPV1-mediated functional responses and abolished channel activation. Blocking the SR release (with ryanodine or dantrolene) led to a reduced capsaicin-induced Ca2+ elevation suggesting that TRPV1 may participate to a secondary SR Ca2+ liberation of greater amplitude. In conclusion, our experiments point out that TRPV1 is a functional SR Ca2+ leak channel and may crosstalk with RyR1 in adult mouse muscle fibers.  相似文献   

10.
Studies with electron microscopy have shown that sarcoplasmic reticulum (SR) andmitochondria locate close to each other in cardiac muscle cells. We investigated the hypothesis thatthis proximity results in a transient exposure of mitochondrial Ca2+ uniporter (CaUP) to highconcentrations of Ca2+ following Ca2+ release from the SR and thus an influx of Ca2+into mitochondria. Single ventricular myocytes of rat were skinned by exposing them to aphysiological solution containing saponin (0.2 mg/ml). Cytosolic Ca2+ concentration ([Ca2+]c)and mitochondrial Ca2+ concentration ([Ca2+]m) were measured with fura-2 and rhod2,respectively. Application of caffeine (10 mM) induced a concomitant increase in[Ca2+]c and [Ca2+]m.Ruthenium red, at concentrations that block CaUP but not SR release, diminished thecaffeine-induced increase in [Ca2+]m but not[Ca2+]c. In the presence of 1 mM BAPTA, a Ca2+ chelator,the caffeine-induced increase in [Ca2+]m was reduced substantially less than [Ca2+]c. Moreover,inhibition of SR Ca2+ pump with two different concentrations of thapsigargin caused anincrease in [Ca2+]m, which was related to the rate of [Ca2+]c increase. Finally, electronmicroscopy showed that sites of junctions between SR and T tubules from which Ca2+ is released,or Ca2+ release units, CRUs, are preferentially located in close proximity to mitochondria.The distance between individual SR Ca2+ release channels (feet or ryanodine receptors) isvery short, ranging between approximately 37 and 270 nm. These results are consistent withthe idea that there is a preferential coupling of Ca2+ transport from SR to mitochondria incardiac muscle cells, because of their structural proximity.  相似文献   

11.
Of the many ongoing controversies regarding the workings of the sarcoplasmic reticulum (SR) in cardiac myocytes, two unresolved and interconnected topics are 1), mechanisms of calcium (Ca2+) wave propagation, and 2), speed of Ca2+ diffusion within the SR. Ca2+ waves are initiated when a spontaneous local SR Ca2+ release event triggers additional release from neighboring clusters of SR release channels (ryanodine receptors (RyRs)). A lack of consensus regarding the effective Ca2+ diffusion constant in the SR (DCa,SR) severely complicates our understanding of whether dynamic local changes in SR [Ca2+] can influence wave propagation. To address this problem, we have implemented a computational model of cytosolic and SR [Ca2+] during Ca2+ waves. Simulations have investigated how dynamic local changes in SR [Ca2+] are influenced by 1), DCa,SR; 2), the distance between RyR clusters; 3), partial inhibition or stimulation of SR Ca2+ pumps; 4), SR Ca2+ pump dependence on cytosolic [Ca2+]; and 5), the rate of transfer between network and junctional SR. Of these factors, DCa,SR is the primary determinant of how release from one RyR cluster alters SR [Ca2+] in nearby regions. Specifically, our results show that local increases in SR [Ca2+] ahead of the wave can potentially facilitate Ca2+ wave propagation, but only if SR diffusion is relatively slow. These simulations help to delineate what changes in [Ca2+] are possible during SR Ca2+release, and they broaden our understanding of the regulatory role played by dynamic changes in [Ca2+]SR.  相似文献   

12.
Lamboley CR  Pape PC 《Cell calcium》2011,50(6):530-547
One aim of this article was to determine the resting concentration of free Ca2+ in the sarcoplasmic reticulum (SR) of frog cut skeletal muscle fibers ([Ca2+]SR,R) using the calcium absorbance indicator dye tetramethylmurexide (TMX). Another was to determine the ratio of [Ca2+]SR,R to TMX's apparent dissociation constant for Ca2+ (Kapp) in order to establish the capability of monitoring [Ca2+]SR(t) during SR Ca2+ release – a signal needed to determine the Ca2+ permeability of the SR. To reveal the properties of TMX in the SR, the surface membrane was rapidly permeabilized with saponin to rapidly dissipate myoplasmic TMX. Results indicated that the concentration of Ca-free TMX in the SR was 2.8-fold greater than that in the myoplasm apparently due to binding of TMX to sites in the SR. Taking into account that such binding might influence Kapp as well as a dependence of Kapp on TMX concentration, the results indicate an average [Ca2+]SR,R ranging from 0.43 to 1.70 mM. The ratio [Ca2+]SR,R/Kapp averaged 0.256, a relatively low value which should not depend on factors influencing Kapp. As a result, the time course of [Ca2+]SR(t) in response to electrical stimulation is well determined by, and approximately linearly related to, the active TMX absorbance signal.  相似文献   

13.
Skeletal muscle stores Ca2+ in the sarcoplasmic reticulum (SR) and releases it to initiate contraction, but the concentration of luminal Ca2+ in the SR ([Ca2+]SR) and the amount that is released by physiological or pharmacological stimulation has been difficult to measure. Here we present a novel, yet simple and direct, method that provides the first quantitative estimates of static content and dynamic changes in [Ca2+]SR in mammalian skeletal muscle, to our knowledge. The method uses fluo-5N loaded into the SR of single, mammalian skeletal muscle cells (murine flexor digitorum brevis myofibers) and confocal imaging to detect and calibrate the signals. Using this method, we have determined that [Ca2+]SR, free is 390 μM. 4-Chloro-m-cresol, an activator of the skeletal muscle ryanodine receptor, reduces [Ca2+]SR, free to ∼8 μM, when values are corrected for background fluorescence from cytoplasmic pools of dye. Prolonged electrical stimulation (10 s) at 50 Hz releases 88% of the SR Ca2+ content, whereas stimulation at 1 Hz (10 s) releases only 20%. Our results lay the foundation for molecular modeling of the dynamics of luminal SR Ca2+ and for future studies of the role of SR Ca2+ in healthy and diseased mammalian muscle.  相似文献   

14.

Background

Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. In this study, oxidative stress was produced by exposure to tert-butyl hydroperoxide (tBHP); cell viability was assessed using a dye reduction assay; receptor binding was characterized using [3H]N-methylscopolamine ([3H]MS); and cytosolic and luminal endoplasmic reticulum (ER) calcium concentrations ([Ca2+]i and [Ca2+]L, respectively) were measured by fluorescent imaging.

Results

Activation of M3 muscarinic receptors induced a biphasic increase in [Ca2+]i: an initial, inositol trisphosphate (IP3)-mediated release of Ca2+ from endoplasmic reticulum (ER) stores followed by a sustained phase of Ca2+ entry (i.e., store-operated calcium entry; SOCE). Under non-cytotoxic conditions, tBHP increased resting [Ca2+]i; a 90 minute exposure to tBHP (0.5-10 mM ) increased [Ca2+]i from 26 to up to 127 nM and decreased [Ca2+]L by 55%. The initial response to 10 μM carbamylcholine was depressed by tBHP in the absence, but not the presence, of extracellular calcium. SOCE, however, was depressed in both the presence and absence of extracellular calcium. Acute exposure to tBHP did not block calcium influx through open SOCE channels. Activation of SOCE following thapsigargin-induced depletion of ER calcium was depressed by tBHP exposure. In calcium-free media, tBHP depressed both SOCE and the extent of thapsigargin-induced release of Ca2+ from the ER. M3 receptor binding parameters (ligand affinity, guanine nucleotide sensitivity, allosteric modulation) were not affected by exposure to tBHP.

Conclusions

Oxidative stress induced by tBHP affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca2+]i, [Ca2+]L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling.  相似文献   

15.
Liberation of Ca2+ from the endoplasmic reticulum (ER) through inositol trisphosphate receptors (IP3R) is modulated by the ER Ca2+ content, and overexpression of SERCA2b to accelerate Ca2+ sequestration into the ER has been shown to potentiate the frequency and amplitude of IP3-evoked Ca2+ waves in Xenopus oocytes. Here, we examined the effects of SERCA overexpression on the elementary IP3-evoked puffs to elucidate whether ER [Ca2+] may modulate IP3R function via luminal regulatory sites in addition to simply determining the size of the available store and electrochemical driving force for Ca2+ release. SERCA2b and Ca2+ permeable nicotinic plasmalemmal channels were expressed in oocytes, and hyperpolarizing pulses were delivered to induce Ca2+ influx and thereby load ER stores. Puffs evoked by photoreleased IP3 were significantly potentiated in terms of numbers of responding sites, frequency and amplitude following transient Ca2+ influx in SERCA-overexpressing cells, whereas little change was evident with SERCA overexpression alone or following Ca2+ influx in control cells not overexpressing SERCA. Intriguingly, we observed the appearance of a new population of puffs that arose after long latencies and had prolonged durations supporting the notion of luminal regulation of IP3R gating kinetics.  相似文献   

16.
We have developed a quantitative model for the creation of cytoplasmic Ca2+ gradients near the inner surface of the plasma membrane (PM). In particular we simulated the refilling of the sarcoplasmic reticulum (SR) via PM–SR junctions during asynchronous [Ca2+]i oscillations in smooth muscle cells of the rabbit inferior vena cava. We have combined confocal microscopy data on the [Ca2+]i oscillations, force transduction data from cell contraction studies and electron microscopic images to build a basis for computational simulations that model the transport of calcium ions from Na+/Ca2+ exchangers (NCX) on the PM to sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps on the SR as a three-dimensional random walk through the PM–SR junctional cytoplasmic spaces. Electron microscopic ultrastructural images of the smooth muscle cells were elaborated with software algorithms to produce a very clear and dimensionally accurate picture of the PM–SR junctions. From this study, we conclude that it is plausible and possible for enough Ca2+ to pass through the PM–SR junctions to replete the SR during the regenerative Ca2+ release, which underlies agonist induced asynchronous Ca2+ oscillations in vascular smooth muscle.  相似文献   

17.
In cardiac muscle, intracellular Ca2+ and Mg2+ are potent regulators of calcium release from the sarcoplasmic reticulum (SR). It is well known that the free [Ca2+] in the SR ([Ca2+]L) stimulates the Ca2+ release channels (ryanodine receptor [RYR]2). However, little is known about the action of luminal Mg2+, which has not been regarded as an important regulator of Ca2+ release.  相似文献   

18.
Local Ca2+ transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca2+ release to activate mitochondrial Ca2+ uptake and to evoke a matrix [Ca2+] ([Ca2+]m) rise. [Ca2+]m exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca2+ release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca2+ sensitivity of both the Ca2+ release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca2+ accumulation in various apoptotic paradigms, methods are available for buffering of [Ca2+], for dissipation of the driving force of the mitochondrial Ca2+ uptake and for inhibition of the mitochondrial Ca2+ transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca2+ handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca2+ uptake on cytosolic [Ca2+] and [Ca2+]m in intact cultured cells.  相似文献   

19.
《Cell calcium》2016,59(6):577-588
Rises in cytosolic Ca2+ concentration ([Ca2+]cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca2+]cyt, but these only monitor the net effect of manipulations on the processes controlling [Ca2+]cyt (Ca2+ buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca2+ or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca2+ signalling, we here monitor Ca2+ flux around the platelet by measuring net Ca2+ fluxes to or from the extracellular space and the intracellular Ca2+ stores, which act as the major sources and sinks for Ca2+ influx into and efflux from the cytosol, as well as monitoring the cytosolic Na+ concentration ([Na+]cyt), which influences platelet Ca2+ fluxes via Na+/Ca2+ exchange. The intracellular store Ca2+ concentration ([Ca2+]st) was monitored using Fluo-5N, the extracellular Ca2+ concentration ([Ca2+]ext) was monitored using Fluo-4 whilst [Ca2+]cyt and [Na+]cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca2+]cyt in the absence of extracellular Ca2+. PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca2+ release and Ca2+ removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca2+]cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na+]cyt which would be expected to reduce Ca2+ removal via the Na+/Ca2+ exchanger (NCX). Thrombin-evoked rises in [Na+]cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non-selective cation permeability of the plasma membrane as assessed by Mn2+ quench of Fura-2 fluorescence. PKC inhibition was without effect on thrombin-evoked rises in [Ca2+]cyt following SERCA inhibition and either removal of extracellular Na+ or inhibition of Na+/K+-ATPase activity by removal of extracellular K+ or treatment with digoxin. These data suggest that PKC limits ADP-evoked rises in [Ca2+]cyt by acceleration of SERCA activity, whilst rises in [Ca2+]cyt evoked by the stronger platelet activator thrombin are limited by PKC through acceleration of both SERCA and Na+/K+-ATPase activity, with the latter limiting the effect of thrombin on rises in [Na+]cyt and so forward mode NCX activity. The use of selective PKC inhibitors indicated that conventional and not novel PKC isoforms are responsible for the inhibition of agonist-evoked Ca2+ signalling.  相似文献   

20.
We have studied in HeLa cells the molecular nature of the 2-APB induced ER Ca2+ leak using synthetic Ca2+ indicators that report changes in both the cytoplasmic ([Ca2+]i) and the luminal ER ([Ca2+]ER) Ca2+ concentrations. We have tested the hypothesis that Orai channels participate in the 2-APB-induced ER Ca2+ leak that was characterized in the companion paper. The expression of the dominant negative Orai1 E106A mutant, which has been reported to block the activity of all three types of Orai channels, inhibited the effect of 2-APB on the [Ca2+]ER but did not decrease the ER Ca2+ leak after thapsigargin (TG). Orai3 channel, but neither Orai1 nor Orai2, colocalizes with expressed IP3R and only Orai3 channel supported the 2-APB-induced ER Ca2+ leak, while Orai1 and Orai2 inhibited this type of ER Ca2+ leak. Decreasing the expression of Orai3 inhibited the 2-APB-induced ER Ca2+ leak but did not modify the ER Ca2+ leak revealed by inhibition of SERCA pumps with TG. However, reducing the expression of Orai3 channel resulted in larger [Ca2+]i response after TG but only when the ER store had been overloaded with Ca2+ by eliminating the acidic internal Ca2+ store with bafilomycin. These data suggest that Orai3 channel does not participate in the TG-revealed ER Ca2+ leak but forms an ER Ca2+ leak channel that is limiting the overloading with Ca2+ of the ER store.  相似文献   

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