共查询到20条相似文献,搜索用时 31 毫秒
1.
《Biochemical and biophysical research communications》2014,445(3):629-632
Elevation of glucose induces transient inhibition of insulin release by lowering cytoplasmic Ca2+ ([Ca2+]i) below baseline in pancreatic β-cells. The period of [Ca2+]i decrease (phase 0) coincides with increased glucagon release and is therefore the starting point for antisynchronous pulses of insulin and glucagon. We now examine if activation of adrenergic α2A and muscarinic M3 receptors affects the initial [Ca2+]i response to increase of glucose from 3 to 20 mM in β-cells situated in mouse islets. In the absence of receptor stimulation the elevation of glucose lowered [Ca2+]i during 90–120 s followed by rise due to opening of voltage-dependent Ca2+ channels. The period of [Ca2+]i decrease was prolonged by activation of the α2A adrenergic receptors (1 μM epinephrine or 100 nM clonidine) and shortened by stimulation of the muscarinic M3 receptors (0.1 μM acetylcholine). The latter effect was mimicked by the Na/K pump inhibitor ouabain (10–100 μM). The results indicate that prolonged initial decrease (phase 0) is followed by slow [Ca2+]i rise and shorter decrease followed by fast rise. It is concluded that the period of initial decrease of [Ca2+]i regulates the subsequent β-cell response to glucose. 相似文献
2.
Patrizia Rubini Javorina Milosevic Johannes Engelhardt Mahmoud Al-Khrasani Heike Franke Attilla Heinrich Beata Sperlagh Sigrid C. Schwarz Johannes Schwarz Wolfgang Nörenberg Peter Illes 《Cell calcium》2009,45(5):485-498
Nucleotides play an important role in brain development and may exert their action via ligand-gated cationic channels or G protein-coupled receptors. Patch-clamp measurements indicated that in contrast to AMPA, ATP did not induce membrane currents in human midbrain derived neuronal progenitor cells (hmNPCs). Various nucleotide agonists concentration-dependently increased [Ca2+]i as measured by the Fura-2 method, with the rank order of potency ATP > ADP > UTP > UDP. A Ca2+-free external medium moderately decreased, whereas a depletion of the intracellular Ca2+ storage sites by cyclopiazonic acid markedly depressed the [Ca2+]i transients induced by either ATP or UTP. Further, the P2Y1 receptor antagonistic PPADS and MRS 2179, as well as the nucleotide catalyzing enzyme apyrase, allmost abolished the effects of these two nucleotides. However, the P2Y1,2,12 antagonistic suramin only slightly blocked the action of ATP, but strongly inhibited that of UTP. In agreement with this finding, UTP evoked the release of ATP from hmNPCs in a suramin-, but not PPADS-sensitive manner. Immunocytochemistry indicated the co-localization of P2Y1,2,4-immunoreactivities (IR) with nestin-IR at these cells. In conclusion, UTP may induce the release of ATP from hmNPCs via P2Y2 receptor-activation and thereby causes [Ca2+]i transients by stimulating a P2Y1-like receptor. 相似文献
3.
Microbubble facilitated ultrasound (US) application can enhance intracellular delivery of drugs and genes in endothelial cells cultured in static condition by transiently disrupting the cell membrane, or sonoporation. However, endothelial cells in vivo that are constantly exposed to blood flow may exhibit different sonoporation characteristics. This study investigates the effects of shear stress cultivation on sonoporation of endothelial cells in terms of membrane disruption and changes in the intracellular calcium concentration ([Ca2+]i). Sonoporation experiments were conducted using murine brain microvascular endothelial (bEnd.3) cells and human umbilical vein endothelial cells (HUVECs) cultured under static or shear stress (5 dyne/cm2 for 5 days) condition in a microchannel environment. The cells were exposed to a short US tone burst (1.25 MHz, 8 μs duration, 0.24 MPa) in the presence of DefinityTM microbubbles to facilitate sonoporation. Membrane disruption was assessed by propidium iodide (PI) and changes in [Ca2+]i measured by fura-2AM. Results from this study show that shear stress cultivation significantly reduced the impact of ultrasound-driven microbubbles activities on endothelial cells. Cells cultured under shear stress condition exhibited much lower percentage with membrane disruption and changes in [Ca2+]i compared to statically cultured cells. The maximum increases of PI uptake and [Ca2+]i were also significantly lower in the shear stress cultured cells. In addition, the extent of [Ca2+]i waves in shear cultured HUVECs was reduced compared to the statically cultured cells. 相似文献
4.
Christian Lohr Eric Tucker Lynne A. Oland Leslie P. Tolbert 《Developmental neurobiology》2002,52(2):85-98
Changes in the intracellular Ca2+ concentration ([Ca2+]i) induced by depolarization have been measured in glial cells acutely isolated from antennal lobes of the moth Manduca sexta at different postembryonic developmental stages. Depolarization of the glial cell membrane was elicited by increasing the external K+ concentration from 4 to 25 mM. At midstage 5 and earlier stages, less than 20% of the cells responded to 25 mM K+ (1 min) with a transient increase in [Ca2+]i of approximately 40 nM. One day later, at late stage 5, 68% of the cells responded to 25 mM K+, the amplitude of the [Ca2+]i transients averaging 592 nM. At later stages, all cells responded to 25 mM K+ with [Ca2+]i transients with amplitudes not significantly different from those at late stage 5. In stage 6 glial cells isolated from deafferented antennal lobes, i.e., from antennal lobes chronically deprived of olfactory receptor axons, only 30% of the cells responded with [Ca2+]i transients. The amplitudes of these [Ca2+]i transients averaged 93 nM and were significantly smaller than those in normal stage 6 glial cells. [Ca2+]i transients were greatly reduced in Ca2+‐free, EGTA‐buffered saline, and in the presence of the Ca2+ channel blockers cadmium and verapamil. The results suggest that depolarization of the cell membrane induces Ca2+ influx through voltage‐activated Ca2+ channels into antennal lobe glial cells. The development of the depolarization‐induced Ca2+ transients is rapid between midstage 5 and stage 6, and depends on the presence of afferent axons from the olfactory receptor cells in the antenna. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 85–98, 2002 相似文献
5.
《Cell calcium》2015,58(5-6):366-375
In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca2+]i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca2+]i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48 h to a variety of stressors: cytokines (low-grade inflammation), 28 mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca2+]i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca2+]i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3–11 mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca2+]i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11 mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3 mM glucose) observed for FFAs and also for 28G. We also clamped [Ca2+]i using 30 mM KCl + 250 μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3–11 mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca2+]i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca2+]i but not conventional insulin secretion and ‘metabolic’ stressors (FFAs, 28G, rotenone) impacted insulin secretion. 相似文献
6.
7.
Giuseppe Cassano Giuseppe Gasparre Mauro Niso Marialessandra Contino Vito Scalera Nicola Antonio Colabufo 《Cell calcium》2009,45(4):340-345
We demonstrate that F281, a synthetic agonist of the sigma-2 receptor (s2R), induces a non transient increase in intracellular [Ca2+] ([Ca2+]i) and cell death in SK-N-SH cells. Sigma receptors are classified into two subtypes, with different molecular weight and tissue distribution. While the sigma-1 receptor has been cloned, the s2r is less characterized and its physiological ligand and role need further investigation. In tumour cell lines, synthetic agonists of the s2R trigger apoptosis and modulate [Ca2+]i. In particular, CB-64D induces a Ca2+ response while PB28 supresses Ca2+ signalling. We have recently synthesized F281, by replacing the 5-methoxytetraline moiety of PB28 with a carbazole nucleus. Although this bioisosteric substitution should not affect the ligand affinity at the receptor, F281 (after 24 h incubation) was more cytotoxic than PB28 (EC50 values 65.4 nM and 8.13 μM, respectively) in SK-N-SH cells. We used the fluorescent probes fura-2, rhod-2 and JC-1. F281 mobilizes Ca2+ from mitochondria and from the endoplasmic reticulum, by opening its inositol 1,4,5-trisphosphate receptor; Ca2+-entry through the channels activated by store depletion was also observed. After the increase in [Ca2+]i and within 10 min, we observed a sudden drop in metabolic activity and intracellular [ATP] leading to cell death. 相似文献
8.
《Cell calcium》2011,49(6):324-332
Multiple mechanisms that maintain Ca2+ homeostasis and provide for Ca2+ signalling operate in the somatas and neurohypophysial nerve terminals of supraoptic nucleus (SON) neurones. Here, we examined the Ca2+ clearance mechanisms of SON neurones from adult rats by monitoring the effects of the selective inhibition of different Ca2+ homeostatic molecules on cytosolic Ca2+ ([Ca2+]i) transients in isolated SON neurones. In addition, we measured somatodendritic vasopressin (AVP) release from intact SON tissue in an attempt to correlate it with [Ca2+]i dynamics. When bathing the cells in a Na+-free extracellular solution, thapsigargin, cyclopiazonic acid (CPA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and the inhibitor of plasma membrane Ca2+-ATPase (PMCA), La3+, all significantly slowed down the recovery of depolarisation (50 mM KCl)-induced [Ca2+]i transients. The release of AVP was stimulated by 50 mM KCl, and the decline in the peptide release was slowed by Ca2+ transport inhibitors. In contrast to previous reports, our results show that in the fully mature adult rats: (i) all four Ca2+ homeostatic pathways, the Na+/Ca2+ exchanger, the endoplasmic reticulum Ca2+ pump, the plasmalemmal Ca2+ pump and mitochondria, are complementary in actively clearing Ca2+ from SON neurones; (ii) somatodendritic AVP release closely correlates with intracellular [Ca2+]i dynamics; (iii) there is (are) Ca2+ clearance mechanism(s) distinct from the four outlined above; and (iv) Ca2+ homeostatic systems in the somatas of SON neurones differ from those expressed in their terminals. 相似文献
9.
A novel staining and quantification method to investigate changes in intracellular calcium levels [Ca2+]i and morphology in filamentous fungus is presented. Using a simple protocol, two fluorescent dyes, Fluo-4-AM and Cell trace calcein red-orange-AM were loaded into the filamentous fungus Penicillium chrysogenum. The present study investigates the applicability of using Ca2+-sensitive dye to quantify and image [Ca2+]i in P. chrysogenum cultures chosen for its potential as an experimental system to study Ca2+ signalling in elicited cultures. The dye loading was optimised and investigated at different pH loading conditions. It was observed that the fluorophore was taken up throughout the hyphae, retaining cell membrane integrity and no dye compartmentalisation within organelles was observed. From the fluorescent plate-reader studies a significant rise (p < 0.001) in the relative fluorescence levels corresponding to [Ca2+]i levels in the hyphae was observed when challenged with an elicitor (mannan oligosaccharide, 150 mg L?1) which was dependent upon extracellular calcium. Concurrently a novel application of dye-loaded hyphae for morphological analysis was also examined using the imaging software Filament Tracer (Bitplane). Essential quantitative mycelial information including the length and diameter of the segments and number of branch points was obtained using this application based on the three-dimensional data. 相似文献
10.
AimsThis study examined whether the essential oil component carvacrol altered cytosolic free Ca2+ level ([Ca2+]i) and viability in human glioblastoma cells.Main methodsThe Ca2+-sensitive fluorescent dye fura-2 was applied to measure [Ca2+]i. Cell viability was measured by detecting reagent WST-1. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry.Key findingsCarvacrol at concentrations of 400–1000 μM induced a [Ca2+]i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca2+. Carvacrol-induced Ca2+ signal was not altered by nifedipine, econazole, SK&;F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca2+ was removed, incubation with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished carvacrol-induced [Ca2+]i rise. Incubation with carvacrol also abolished thapsigargin or BHQ-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 abolished carvacrol-induced [Ca2+]i rise. At concentrations of 200–800 μM, carvacrol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N–-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that carvacrol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. At concentrations of 200, 400 and 600 μM, carvacrol induced production of ROS.SignificanceIn human glioblastoma cells, carvacrol induced a [Ca2+]i rise by inducing phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via protein kinase C-sensitive, non store-operated Ca2+ channels. Carvacrol induced cell death that might involve ROS-mediated apoptosis. 相似文献
11.
Jurij Dolen?ek Andra? Sto?er Ma?a Skelin Klemen Evan W. Miller Marjan Slak Rupnik 《PloS one》2013,8(12)
Oscillatory electrical activity is regarded as a hallmark of the pancreatic beta cell glucose-dependent excitability pattern. Electrophysiologically recorded membrane potential oscillations in beta cells are associated with in-phase oscillatory cytosolic calcium activity ([Ca2+]i) measured with fluorescent probes. Recent high spatial and temporal resolution confocal imaging revealed that glucose stimulation of beta cells in intact islets within acute tissue slices produces a [Ca2+]i change with initial transient phase followed by a plateau phase with highly synchronized [Ca2+]i oscillations. Here, we aimed to correlate the plateau [Ca2+]i oscillations with the oscillations of membrane potential using patch-clamp and for the first time high resolution voltage-sensitive dye based confocal imaging. Our results demonstrated that the glucose-evoked membrane potential oscillations spread over the islet in a wave-like manner, their durations and wave velocities being comparable to the ones for [Ca2+]i oscillations and waves. High temporal resolution simultaneous records of membrane potential and [Ca2+]i confirmed tight but nevertheless limited coupling of the two processes, with membrane depolarization preceding the [Ca2+]i increase. The potassium channel blocker tetraethylammonium increased the velocity at which oscillations advanced over the islet by several-fold while, at the same time, emphasized differences in kinetics of the membrane potential and the [Ca2+]i. The combination of both imaging techniques provides a powerful tool that will help us attain deeper knowledge of the beta cell network. 相似文献
12.
AimsThis study was designed to investigate the effects of sodium ferulate (SF) on rat isolated thoracic aortas and the possible mechanisms.Main methodsIsometric tension was recorded in response to drugs in organ bath. Cytosolic free Ca2+ concentration ([Ca2+]i) was measured using Fluo-3 in cultured rat aortic smooth muscle cells (RASMC).Key findingsSF (0.1–30 mM) relaxed the isolated aortic rings precontracted with phenylephrine (PE) and high-K+ in a concentration-dependent manner with respective pD2 of 2.7 ± 0.02 and 2.6 ± 0.06. Mechanical removal of endothelium did not significantly modify the SF-induced relaxation. In Ca2+-free solution, SF noticeably inhibited extracellular Ca2+-induced contraction in high-K+ and PE pre-challenged rings, and suppressed the transient contraction induced by PE and caffeine. The vasorelaxant effect of SF was unaffected by various K+ channel blockers such as tetraethylammonium, glibenclamide, 4-aminopyridine, and barium chloride. In addition, SF concentration-dependently reduced the contraction induced by phorbol-12-myristate-13-acetate, an activator of protein kinase C (PKC), in the absence of extracellular Ca2+, with the pD2 of 2.9 ± 0.03. In RASMC, SF had no effect on PE- or KCl-induced [Ca2+]i increase either in the presence or in the absence of external Ca2+.SignificanceThese results indicate that SF acts directly as a non-selective relaxant to vascular smooth muscle. The direct inhibition of the common pathway after [Ca2+]i increase may account for the SF-induced relaxation in Ca2+-dependent contraction, while the blockage of the PKC-mediated contractile mechanism is likely responsible for the SF-induced relaxation in Ca2+-independent contraction. 相似文献
13.
Arachidonic acid (AA), an endogenous lipid signal molecule released from membrane upon cell activation, modulates intracellular Ca2 + ([Ca2 +]i) signaling positively and negatively. However, the mechanisms underlying the biphasic effects of AA are rather obscure. Using probes for measurements of [Ca2 +]i and fluidity of plasma membrane (PM)/endoplasmic reticulum (ER), immunostaining, immunoblotting and shRNA interference approaches, we found that AA at low concentration, 3 μM, reduced the PM fluidity by activating PKCα and PKCβII translocation to PM and also the ER fluidity directly. In accordance, 3 μM AA did not impact the basal [Ca2 +]i but significantly suppressed the thapsigargin-induced Ca2 + release and Ca2 + influx. Inhibition of PKC with Gö6983 or knockdown of PKCα or PKCβ using shRNA significantly attenuated the inhibitory effects of 3 μM AA on PM fluidity and agonist-induced Ca2 + signal. However, AA at high concentration, 30 μM, caused robust release and entry of Ca2 + accompanied by a facilitated PM fluidity but decreased ER fluidity and dramatic PKCβI and PKCβII redistribution in the ER. Compared with ursodeoxycholate acid, a membrane stabilizing agent that only inhibited the 30 μM AA-induced Ca2 + influx by 45%, Gd3 + at concentration of 10 μM could completely abolish both release and entry of Ca2 + induced by AA, suggesting that the potentiated PM fluidity is not the only reason for AA eliciting Ca2 + signal. Therefore, the study herein demonstrates that a lowered PM fluidity by PKC activation and a direct ER stabilization contribute significantly for AA downregulation of [Ca2 +]i response, while Gd3 +-sensitive ‘pores’ in PM/ER play an important role in AA-induced Ca2 + signal in HEK293 cells. 相似文献
14.
Although pulsed electromagnetic field (PEMF) exposure has been reported to promote neuronal differentiation, the mechanism is still unclear. Here, we aimed to examine the effects of PEMF exposure on brain-derived neurotrophic factor (Bdnf) mRNA expression and the correlation between the intracellular free calcium concentration ([Ca2+]i) and Bdnf mRNA expression in cultured dorsal root ganglion neurons (DRGNs). Exposure to 50 Hz and 1 mT PEMF for 2 h increased the level of [Ca2+]i and Bdnf mRNA expression, which was found to be mediated by increased [Ca2+]i from Ca2+ influx through L-type voltage-gated calcium channels (VGCCs). However, calcium mobilization was not involved in the increased [Ca2+]i and BDNF expression, indicating that calcium influx was one of the key factors responding to PEMF exposure. Moreover, PD098059, an extracellular signal-regulated kinase (Erk) inhibitor, strongly inhibited PEMF-dependant Erk1/2 activation and BDNF expression, indicating that Erk activation is required for PEMF-induced upregulation of BDNF expression. These findings indicated that PEMF exposure increased BDNF expression in DRGNs by activating Ca2+- and Erk-dependent signaling pathways. 相似文献
15.
Soo-Jin Park Kyoung-Pil Lee Saeromi Kang Hae-Young Chung Yoe-Sik Bae Fumikazu Okajima Dong-Soon Im 《Cellular signalling》2013,25(11):2147-2154
Lysophosphatidylethanolamine (LPE) is a lyso-type metabolite of phosphatidylethanolamine (a plasma membrane component), and its intracellular Ca2 + ([Ca2 +]i) increasing actions may be mediated through G-protein-coupled receptor (GPCR). However, GPCRs for lysophosphatidic acid (LPA), a structurally similar representative lipid mediator, have not been implicated in LPE-mediated activities in SK-OV3 or OVCAR-3 ovarian cancer cells or in receptor over-expression systems. In the present study, LPE-induced [Ca2 +]i increase was observed in MDA-MB-231 cells but not in other breast cancer cell lines. In addition, LPE- and LPA-induced responses showed homologous and heterologous desensitization. Furthermore, VPC32183 and Ki16425 (antagonists of LPA1 and LPA3) inhibited LPE-induced [Ca2 +]i increases, and knockdown of LPA1 by transfection with LPA1 siRNA completely inhibited LPE-induced [Ca2 +]i increases. Furthermore, the involvement of CD97 (an adhesion GPCR) in the action of LPA1 in MDA-MB-231 cells was demonstrated by siRNA transfection. Pertussis toxin (a specific inhibitor of Gi/o proteins), edelfosine (an inhibitor of phospholipase C), or 2-APB (an inhibitor of IP3 receptor) completely inhibited LPE-induced [Ca2 +]i increases, whereas HA130, an inhibitor of autotaxin/lysophospholipase D, did not. Therefore, LPE is supposed to act on LPA1-CD97/Gi/o proteins/phospholipase C/IP3/Ca2 + rise in MDA-MB-231 breast cancer cells. 相似文献
16.
《Cell calcium》1996,20(3):303-314
In Fura-2 loaded-single guinea pig adrenal chromaffin cells, muscarine, nicotine and KCl all caused an early peak rise in intracellular Ca concentration ([Ca2+]i) followed by a sustained rise. In Ca2+-free solution, muscarine, but neither nicotine nor KCl, caused a transient increase in [Ca2+]i, which was partially reduced by preceding application of caffeine or by treatment with ryanodine plus caffeine. In voltage-clamped cells at a holding potential of −60 mV, the muscarine-induced [Ca2+]i, rise, especially its sustained phase, decreased in magnitude. intracellular application of inositol 1,4,5-trisphosphate caused a transient increase in [Ca2+]i and inhibited the following [Ca2+]i response to muscarine without affecting responses to nicotine and a depolarizing pulse. Muscarine evoked membrane depolarization following brief hyperpolarization in most cells tested. There was a significant positive correlation between the amplitude of the depolarization and the magnitude of the sustained rise in [Ca2+]i. Muscarine-induced sustained [Ca2+]i rise was much greater in the current-clamp mode than that in the voltage-clamp mode. The sustained phase of [Ca2+]i rise and Mn2+ influx in response to muscarine were suppressed by a voltage-dependent Ca2+ channel blocker, methoxyverapamil. These results suggest that stimulation of muscarinic receptors causes not only extracellular Ca2+ entry, but also Ca2+ mobilization from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Voltage-dependent Ca2+ channels may function as one of the Ca2+ entry pathways activated by muscarinic receptor in guinea pig adrenal chromaffin cells. 相似文献
17.
Using a Ca2+-sensitive fluorescent indicator, fura-2/AM, we recorded calcium transients in secretory cells of isolated acini of the rat submandibular salivary gland; these transients were induced by hyperpotassium-induced depolarization (after an increase in [K+]
e
up to 50 mM) of the plasma membrane of the above cells. Calcium transients were significantly suppressed by 50 M nifedipine. Addition of 10 M carbonyl cyanide m-chlorophenylhydrazone to the normal extracellular solution was accompanied by a rise in [Ca2+]
i
, whereas when hyperpotassium solution is used the effect was less expressed. Blockers of CA2+-ATPase in the cellular membrane and in the endoplasmic reticulum, eosin Y (5 M) and cyclopiazonic acid (CPA, 5 M), respectively, evoked a significant increase in [Ca2+]
i
and a decrease in the K+-depolarization-induced calcium transient. Extracellular application of caffeine (2, 10, or 30 mM) was accompanied by a concentration-dependent rise in [Ca2+]
i
. Therefore, potassium depolarization of the plasma membrane of acinar cells of the rat submandibular salivary gland activates both the voltage-dependent Ca2+ influx and Ca2+-induced Ca2+ release from the endoplasmic reticulum; the initial level of [Ca2+]
i
was restored at the joint involvement of Ca2+-ATPases in the plasma membrane and the membranes of the endoplasmic reticulum and mitochondria. 相似文献
18.
Liron Boyman Aristide C. Chikando George S.B. Williams Ramzi J. Khairallah Sarah Kettlewell Christopher W. Ward Godfrey L. Smith Joseph P.Y. Kao W. Jonathan Lederer 《Biophysical journal》2014
Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca2+]i) in heart. These buffers can remove up to one-third of the Ca2+ that enters the cytosol during the [Ca2+]i transients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca2+ movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca2+ signals (i.e., Ca2+ sparks and [Ca2+]i transients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (ΔΨm), the role of the mitochondria in buffering cytosolic Ca2+ signals was investigated. We show here that rapid loss of ΔΨm leads to no significant changes in cytosolic Ca2+ signals. Second, we make direct measurements of mitochondrial [Ca2+] ([Ca2+]m) using a mitochondrially targeted Ca2+ probe (MityCam) and these data suggest that [Ca2+]m is near the [Ca2+]i level (∼100 nM) under quiescent conditions. These two findings indicate that although the mitochondrial matrix is fully buffer-capable under quiescent conditions, it does not function as a significant dynamic buffer during physiological Ca2+ signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca2+ cycling suggests that mitochondrial Ca2+ uptake would need to be at least ∼100-fold greater than the current estimates of Ca2+ influx for mitochondria to influence measurably cytosolic [Ca2+] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca2+ uptake does not significantly alter cytosolic Ca2+ signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca2+]i under physiological conditions in heart. 相似文献
19.
The effect of Ca2+ applied in high concentrations (50 and 300 µM) was addressed on the generation of reactive oxygen species in isolated mitochondria from guinea-pig brain. The experiments were performed in the presence of ADP, a very effective inhibitor of mitochondrial permeability transition. Moderate increase in H2O2 release from mitochondria was induced by Ca2+ applied in 50 µM, but not in 300 µM concentration as measured with Amplex red fluorescent assay starting with a delay of 100-150 sec after exposure to Ca2+. Parallel measurements of membrane potential (ΔΨm) by safranine fluorescence showed a transient depolarization by Ca2+ followed by the recovery of ΔΨm to a value, which was more negative than that observed before addition of Ca2+ indicating a relative hyperpolarization. NAD(P)H fluorescence was also increased by Ca2+ given in 50 µM concentration. In mitochondria having high ΔΨm in the presence of oligomycin or ATP, the basal rate of release of H2O2 was significantly higher than that observed in a medium containing ADP and Ca2+ no longer increased but rather decreased the rate of H2O2 release. With 300 µM Ca2+ only a loss but no tendency of a recovery of ΔΨm was detected and H2O2 release was unchanged. It is suggested that in the presence of nucleotides the effect of Ca2+ on mitochondrial ROS release is related to changes in ΔΨm; in depolarized mitochondria, in the presence of ADP, moderate increase in H2O2 release is induced by calcium, but only in ≤ 100 µM concentration, when after a transient Ca2+-induced depolarization mitochondria became more polarized. In highly polarized mitochondria, in the presence of ATP or oligomycin, where no hyperpolarization follows the Ca2+-induced depolarization, Ca2+ fails to stimulate mitochondrial ROS generation. These effects of calcium (≤ 300 µM) are unrelated to mitochondrial permeability transition. 相似文献
20.
Laura Vay Esther Hernández-SanMiguel Carmen D. Lobatón Alfredo Moreno Mayte Montero Javier Alvarez 《Cell calcium》2009,45(3):243-250
Mitochondrial Ca2+ activates many processes, from mitochondrial metabolism to opening of the permeability transition pore (PTP) and apoptosis. However, there is considerable controversy regarding the free mitochondrial [Ca2+] ([Ca2+]M) levels that can be attained during cell activation or even in mitochondrial preparations. Studies using fluorescent dyes (rhod-2 or similar), have reported that phosphate precipitation precludes [Ca2+]M from increasing above 2–3 μM. Instead, using low-Ca2+-affinity aequorin probes, we have measured [Ca2+]M values more than two orders of magnitude higher. We confirm here these values by making a direct in situ calibration of mitochondrial aequorin, and we show that a prolonged increase in [Ca2+]M to levels of 0.5–1 mM was actually observed at any phosphate concentration (0–10 mM) during continuous perfusion of 3.5–100 μM Ca2+-buffers. In spite of this high and maintained (>10 min) [Ca2+]M, mitochondria retained functionality and the [Ca2+]M drop induced by a protonophore was fully reversible. In addition, this high [Ca2+]M did not induce PTP opening unless additional activators (phenyl arsine oxide, PAO) were present. PAO induced a rapid, concentration-dependent and irreversible drop in [Ca2+]M. In conclusion [Ca2+]M levels of 0.5–1 mM can be reached and maintained for prolonged periods (>10 min) in phosphate-containing medium, and massive opening of PTP requires additional pore activators. 相似文献