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1.
In muscle, ATP is required for the powerstroke of the myosin head, the detachment of actin and myosin filaments, and the reuptake of Ca2+ into the sarcoplasmic reticulum. During contraction-relaxation, large amounts of ATP are consumed at the sites of action of the myosin-ATPase and sarcoplasmic reticulum Ca2+-ATPase. The present study addresses the consequences of a reduction in mitochondrial ATP production capacity on sarcoplasmic Ca2+ handling. To this end, myotubes were cultured from patient quadriceps with a biochemically defined decrease in the maximal rate of mitochondrial ATP production and were loaded with indo 1 for imaging of sarcoplasmic Ca2+ changes in real time by confocal microscopy. Myotubes were field-stimulated with 10-ms pulses of 16 V to evoke transient rises in sarcoplasmic Ca2+ concentration ([Ca2+]S). Three single pulses, two pulse trains (1 Hz), and one single pulse were applied in succession to mimic changing workloads. Control myotubes displayed [Ca2+]S transients with an amplitude that was independent of the strength of the stimulus. Intriguingly, the rate of sarcoplasmic Ca2+ removal (CRR) was significantly upregulated during the second and subsequent transients. In myotubes with a reduced mitochondrial ATP production capacity, the amplitude of the [Ca2+]S transients was markedly increased at higher stimulus intensities. Moreover, upregulation of the CRR was significantly decreased compared with control. Taken together, these results are in good agreement with a tight coupling between mitochondrial ATP production and sarcoplasmic Ca2+ handling. Moreover, they support the existence of a relatively long-lasting mitochondrial memory for sarcoplasmic [Ca2+] rises. This memory, which manifested itself as an increase in CRR upon recurrent stimulation, was impaired in patient myotubes with a reduced mitochondrial ATP production capacity. sarcoplasmic Ca2+ removal; video-rate imaging; indo 1; electrical stimulation; mitochondrial memory  相似文献   

2.
Decoding of fast cytosolic Ca2+ concentration ([Ca2+]i) transients by mitochondria was studied in permeabilized cat ventricular myocytes. Mitochondrial [Ca2+] ([Ca2+]m) was measured with fluo-3 trapped inside mitochondria after removal of cytosolic indicator by plasma membrane permeabilization with digitonin. Elevation of extramitochondrial [Ca2+] ([Ca2+]em) to >0.5 µM resulted in a [Ca2+]em-dependent increase in the rate of mitochondrial Ca2+ accumulation ([Ca2+]em resulting in half-maximal rate of Ca2+ accumulation = 4.4 µM) via Ca2+ uniporter. Ca2+ uptake was sensitive to the Ca2+ uniporter blocker ruthenium red and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and depended on inorganic phosphate concentration. The rates of [Ca2+]m increase and recovery were dependent on the extramitochondrial [Na+] ([Na+]em) due to Ca2+ extrusion via mitochondrial Na+/Ca2+ exchanger. The maximal rate of Ca2+ extrusion was observed with [Na+]em in the range of 20–40 mM. Rapid switching (0.25–1 Hz) of [Ca2+]em between 0 and 100 µM simulated rapid beat-to-beat changes in [Ca2+]i (with [Ca2+]i transient duration of 100–500 ms). No [Ca2+]m oscillations were observed, either under conditions of maximal rate of Ca2+ uptake (100 µM [Ca2+]em, 0 [Na+]em) or with maximal rate of Ca2+ removal (0 [Ca2+]em, 40 mM [Na+]em). The slow frequency-dependent increase of [Ca2+]m argues against a rapid transmission of Ca2+ signals between cytosol and mitochondria on a beat-to-beat basis in the heart. [Ca2+]m changes elicited by continuous or pulsatile exposure to elevated [Ca2+]em showed no difference in mitochondrial Ca2+ uptake. Thus in cardiac myocytes fast [Ca2+]i transients are integrated by mitochondrial Ca2+ transport systems, resulting in a frequency-dependent net mitochondrial Ca2+ accumulation. mitochondrial Ca2+; excitation-contraction coupling; cardiomyocytes  相似文献   

3.
The role of mitochondria inCa2+ homeostasis is controversial.We employed the Ca2+-sensitive dyerhod 2 with novel, high temporal and spatial resolution imaging toevaluate changes in the matrix freeCa2+ concentration of individualmitochondria([Ca2+]m)in agonist-stimulated, primary cultured aortic myocytes. Stimulation with 10 µM serotonin (5-HT) evoked modest cytosolicCa2+ transients[cytosolic freeCa2+ concentration([Ca2+]cyt)<500 nM; measured with fura 2] and triggered contractions inshort-term cultured myocytes. However, 5-HT triggered a large mitochondrial rhod 2 signal (indicating pronounced elevation of [Ca2+]m)in only 4% of cells. This revealed heterogeneity in the responses ofindividual mitochondria, all of which stained with MitoTracker GreenFM. In contrast, stimulation with 100 µM ATP evoked large cytosolicCa2+ transients (>1,000 nM) andinduced pronounced, reversible elevation of[Ca2+]m(measured as rhod 2 fluorescence) in 60% of cells. This mitochondrial Ca2+ uptake usually lagged behindthe cytosolic Ca2+ transient peakby 3-5 s, and[Ca2+]mdeclined more slowly than did bulk[Ca2+]cyt.The uptake delay may prevent mitochondria from interfering with rapidsignaling events while enhancing the mitochondrial response to large,long-duration elevations of[Ca2+]cyt.The responses of arterial myocytes to modest physiological stimulationdo not, however, depend on such marked changes in [Ca2+]m.  相似文献   

4.
Physiological and pathologicalCa2+ loads are thought to be takenup by mitochondria via a process dependent on aerobic metabolism. Wesought to determine whether human diploid fibroblasts from a patientwith an inherited defect in pyruvate dehydrogenase (PDH) exhibit adecreased ability to sequester cytosolicCa2+ into mitochondria.Mobilization of Ca2+ stores withbradykinin (BK) increased the cytosolicCa2+ concentration([Ca2+]c)to comparable levels in control and PDH-deficient fibroblasts. Innormal fibroblasts transfected with plasmid DNA encodingmitochondrion-targeted apoaequorin, BK elicited an increase inCa2+-dependent aequorinluminescence corresponding to an increase in the mitochondrialCa2+ concentration([Ca2+]mt)of 2.0 ± 0.2 µM. The mitochondrial uncoupling agent carbonyl cyanidep-(trifluoromethoxy)phenylhydrazoneblocked the BK-induced [Ca2+]mtincrease, although it did not affect the[Ca2+]ctransient. Basal[Ca2+]cand[Ca2+]mtin control and PDH-deficient cells were similar. However, confocalimaging of the potential-sensitive dye JC-1 indicated that thepercentage of highly polarized mitochondria was reduced from 30 ± 1% in normal cells to 19 ± 2% in the PDH-deficient fibroblasts. BK-elicited[Ca2+]mttransients in PDH-deficient cells were reduced to 4% of control, indicating that PDH-deficient mitochondria have a decreased ability totake up cytosolic Ca2+. Thus cellswith compromised aerobic metabolism have a reduced capacity tosequester Ca2+.

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5.
Cytoplasmic Ca2+concentration ([Ca2+]i) variation is akey event in myoblast differentiation, but the mechanism by which itoccurs is still debated. Here we show that increases of extracellular Ca2+ concentration ([Ca2+]o)produced membrane hyperpolarization and a concentration-dependent increase of [Ca2+]i due to Ca2+influx across the plasma membrane. Responses were not related toinositol phosphate turnover and Ca2+-sensing receptor.[Ca2+]o-induced[Ca2+]i increase was inhibited byCa2+ channel inhibitors and appeared to be modulated byseveral kinase activities. [Ca2+]i increasewas potentiated by depletion of intracellular Ca2+ storesand depressed by inactivation of the Na+/Ca2+exchanger. The response to arginine vasopressin (AVP), which inducesinositol 1,4,5-trisphosphate-dependent[Ca2+]i increase in L6-C5 cells, was notmodified by high [Ca2+]o. On the contrary,AVP potentiated the [Ca2+]i increase in thepresence of elevated [Ca2+]o. Other clones ofthe L6 line as well as the rhabdomyosarcoma RD cell line and thesatellite cell-derived C2-C12 line expressed similar responses to high[Ca2+]o, and the amplitude of the responseswas correlated with the myogenic potential of the cells.

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6.
The effects ofendurance run training onNa+-dependentCa2+ regulation in rat leftventricular myocytes were examined. Myocytes were isolated fromsedentary and trained rats and loaded with fura 2. Contractile dynamicsand fluorescence ratio transients were recorded during electricalpacing at 0.5 Hz, 2 mM extracellular Ca2+ concentration, and 29°C.Resting and peak cytosolic Ca2+concentration([Ca2+]c)did not change with exercise training. However, resting and peak[Ca2+]cincreased significantly in both groups during 5 min of continuous pacing, although diastolic[Ca2+]cin the trained group was less susceptible to this elevation ofintracellular Ca2+. Run trainingalso significantly reduced the rate of[Ca2+]cdecay during relaxation. Myocytes were then exposed to 10 mM caffeinein the absence of external Na+ orCa2+ to trigger sarcoplasmicreticular Ca2+ release and tosuppress cellular Ca2+ efflux.This maneuver elicited an elevated steady-state[Ca2+]c.External Na+ was then added, andthe rate of[Ca2+]cclearance was determined. Run training significantly reduced the rateof Na+-dependent clearance of[Ca2+]cduring the caffeine-induced contractures. These data demonstrate thatthe removal of cytosolic Ca2+ wasdepressed with exercise training under these experimental conditionsand may be specifically reflective of a training-induced decrease inthe rate of cytosolic Ca2+ removalviaNa+/Ca2+exchange and/or in the amount ofCa2+ moved across the sarcolemmaduring a contraction.  相似文献   

7.
In a variety of disorders, overaccumulation of lipid in nonadipose tissues, including the heart, skeletal muscle, kidney, and liver, is associated with deterioration of normal organ function, and is accompanied by excessive plasma and cellular levels of free fatty acids (FA). Increased concentrations of FA may lead to defects in mitochondrial function found in diverse diseases. One of the most important regulators of mitochondrial function is mitochondrial Ca2+ ([Ca2+]m), which fluctuates in coordination with intracellular Ca2+ ([Ca2+]i). Polyunsaturated FA (PUFA) have been shown to cause [Ca2+]i mobilization albeit by unknown mechanisms. We have found that PUFA but not monounsaturated or saturated FA cause [Ca2+]i mobilization in NT2 human teratocarcinoma cells. Unlike the [Ca2+]i response to the muscarinic G protein-coupled receptor agonist carbachol, PUFA-mediated [Ca2+]i mobilization in NT2 cells is independent of phospholipase C and inositol-1,4,5-trisphospate (IP3) receptor activation, as well as IP3-sensitive internal Ca2+ stores. Furthermore, PUFA-mediated [Ca2+]i mobilization is inhibited by the mitochondria uncoupler carboxyl cyanide m-chlorophenylhydrozone. Direct measurements of [Ca2+]m with X-rhod-1 and 45Ca2+ indicate that PUFA induce Ca2+ efflux from mitochondria. Further studies show that ruthenium red, an inhibitor of the mitochondrial Ca2+ uniporter, blocks PUFA-induced Ca2+ efflux from mitochondria, whereas inhibitors of the mitochondrial permeability transition pore cyclosporin A and bongkrekic acid have no effect. Thus PUFA-gated Ca2+ release from mitochondria, possibly via the Ca2+ uniporter, appears to be the underlying mechanism for PUFA-induced [Ca2+]i mobilization in NT2 cells. arachidonic acid; mitochondrial Ca2+ uniporter; G protein-coupled receptor; IP3 receptor  相似文献   

8.
In cultured porcine aortic smooth muscle cells,sphingosylphosphorylcholine (SPC), ATP, or bradykinin (BK) induced arapid dose-dependent increase in the cytosolicCa2+ concentration([Ca2+]i)and also stimulated inositol 1,4,5-trisphosphate(IP3) generation. Pretreatmentof cells with pertussis toxin blocked the SPC-induced IP3 generation and[Ca2+]iincrease but had no effect on the action of ATP or BK. In addition, SPCstimulated the mitogen-activated protein kinase (MAPK) and increasedDNA synthesis, whereas neither ATP nor BK produced such effects. Boththe SPC-induced MAPK activation and DNA synthesis were pertussis toxinsensitive. SPC-induced MAPK activation was blocked by treatment ofcells with the phospholipase C inhibitor, U-73122, or the intracellularCa2+-ATPase inhibitor,thapsigargin, but not by removal of extracellular Ca2+. Lysophosphatidic acidinduced cellular responses similar to SPC in a pertussistoxin-sensitive manner in terms of[Ca2+]iincrease, IP3 generation, MAPKactivation, and DNA synthesis. Platelet-derived growth factor (PDGF)also induced a[Ca2+]iincrease, MAPK activation, and DNA synthesis in the same cells; however, the PDGF-induced MAPK activation was not sensitive to pertussis toxin and changes in[Ca2+]i.SPC-induced MAPK activation was inhibited by pretreatment of cells withstaurosporine, W-7, or calmidazolium. Our results suggest that, inporcine aortic smooth muscle cells, MAPK is not activated by theincrease in[Ca2+]iunless a pertussis toxin-sensitive G protein is simultaneously stimulated, indicating the role ofCa2+ in pertussis toxin-sensitiveG protein-mediated MAPK activation.

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9.
Mitochondrial reactive oxygen species and Ca2+ signaling   总被引:1,自引:0,他引:1  
Mitochondria are an important source of reactive oxygen species (ROS) formed as a side product of oxidative phosphorylation. The main sites of oxidant production are complex I and complex III, where electrons flowing from reduced substrates are occasionally transferred to oxygen to form superoxide anion and derived products. These highly reactive compounds have a well-known role in pathological states and in some cellular responses. However, although their link with Ca2+ is well studied in cell death, it has been hardly investigated in normal cytosolic calcium concentration ([Ca2+]i) signals. Several Ca2+ transport systems are modulated by oxidation. Oxidation increases the activity of inositol 1,4,5-trisphosphate and ryanodine receptors, the main channels releasing Ca2+ from intracellular stores in response to cellular stimulation. On the other hand, mitochondria are known to control [Ca2+]i signals by Ca2+ uptake and release during cytosolic calcium mobilization, specially in mitochondria situated close to Ca2+ release channels. Mitochondrial inhibitors modify calcium signals in numerous cell types, including oscillations evoked by physiological stimulus. Although these inhibitors reduce mitochondrial Ca2+ uptake, they also impair ROS production in several systems. In keeping with this effect, recent reports show that antioxidants or oxidant scavengers also inhibit physiological calcium signals. Furthermore, there is evidence that mitochondria generate ROS in response to cell stimulation, an effect suppressed by mitochondrial inhibitors that simultaneously block [Ca2+]i signals. Together, the data reviewed here indicate that Ca2+-mobilizing stimulus generates mitochondrial ROS, which, in turn, facilitate [Ca2+]i signals, a new aspect in the biology of mitochondria. Finally, the potential implications for biological modeling are discussed. mitochondria; calcium  相似文献   

10.
In vascular endothelial cells, depletion of intracellularCa2+ stores elicited capacitativeCa2+ entry (CCE) that resulted inbiphasic changes of intracellular Ca2+ concentration([Ca2+]i)with a rapid initial peak of[Ca2+]ifollowed by a gradual decrease to a sustained plateau level. Weinvestigated the rates of Ca2+entry, removal, and sequestration during activation of CCE and theirrespective contributions to the biphasic changes of[Ca2+]i.Ca2+ buffering by mitochondria,removal byNa+/Ca2+exchange, and a fixed electrical driving force forCa2+ (voltage-clamp experiments)had little effect on the CCE signal. The rates of entry ofMn2+ andBa2+, used as unidirectionalsubstitutes for Ca2+ entry throughthe CCE pathway, were constant and did not follow the concomitantchanges of[Ca2+]i.Pharmacological inhibition of the plasma membraneCa2+ pump, however, abolished thesecondary decay phase of the CCE transient. The disparity between thebiphasic changes of[Ca2+]iand the constant rate of Ca2+entry during CCE was the result of a delayed,Ca2+-dependent activation of thepump. These results suggest an important modulatory role of the plasmamembrane Ca2+ pump in the netcellular gain of Ca2+ during CCE.

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11.
The effects of inhibitors of CaMKII on intracellular Ca2+ signaling were examined in single calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorometry to measure cytoplasmic Ca2+ concentration ([Ca2+]i). The three CaMKII inhibitors, KN-93, KN-62, and autocamtide-2-related inhibitory peptide (AIP), all reduced the plateau phase of the [Ca2+]i transient evoked by stimulation with extracellular ATP. Exposure to KN-93 or AIP alone in the presence of 2 mM extracellular Ca2+ resulted in a dose-dependent increase of [Ca2+]i consisting of a rapid and transient Ca2+ spike followed by a small sustained plateau phase of elevated [Ca2+]i. Exposure to KN-93 in the absence of extracellular Ca2+ caused a transient rise of [Ca2+]i, suggesting that exposure to CaMKII inhibitors directly triggered release of Ca2+ from intracellular endoplasmic reticulum (ER) Ca2+ stores. Repetitive stimulation with KN-93 and ATP, respectively, revealed that both components released Ca2+ largely from the same store. Pretreatment of CPAE cells with the membrane-permeable inositol 1,4,5-trisphosphate (IP3) receptor blocker 2-aminoethoxydiphenyl borate caused a significant inhibition of the KN-93-induced Ca2+ response, suggesting that exposure to KN-93 affects Ca2+ release from an IP3-sensitive store. Depletion of Ca2+ stores by exposure to ATP or to the ER Ca2+ pump inhibitor thapsigargin triggered robust capacitative Ca2+ entry (CCE) signals in CPAE cells that could be blocked effectively with KN-93. The data suggest that in CPAE cells, CaMKII modulates Ca2+ handling at different levels. The use of CaMKII inhibitors revealed that in CPAE cells, the most profound effects of CaMKII are inhibition of release of Ca2+ from intracellular stores and activation of CCE. Ca2+/calmodulin-dependent kinase II; calcium regulation; capacitative calcium entry  相似文献   

12.
Despite their relevance for neuronal Ca2+-induced Ca2+ release (CICR), activation by Ca2+ of ryanodine receptor (RyR) channels of brain endoplasmic reticulum at the [ATP], [Mg2+], and redox conditions present in neurons has not been reported. Here, we studied the effects of varying cis-(cytoplasmic) free ATP concentration ([ATP]), [Mg2+], and RyR redox state on the Ca2+ dependence of endoplasmic reticulum RyR channels from rat brain cortex. At pCa 4.9 and 0.5 mM adenylylimidodiphosphate (AMP-PNP), increasing free [Mg2+] up to 1 mM inhibited vesicular [3H]ryanodine binding; incubation with thimerosal or dithiothreitol decreased or enhanced Mg2+ inhibition, respectively. Single RyR channels incorporated into lipid bilayers displayed three different Ca2+ dependencies, defined by low, moderate, or high maximal fractional open time (Po), that depend on RyR redox state, as we have previously reported. In all cases, cis-ATP addition (3 mM) decreased threshold [Ca2+] for activation, increased maximal Po, and shifted channel inhibition to higher [Ca2+]. Conversely, at pCa 4.5 and 3 mM ATP, increasing cis-[Mg2+] up to 1 mM inhibited low activity channels more than moderate activity channels but barely modified high activity channels. Addition of 0.5 mM free [ATP] plus 0.8 mM free [Mg2+] induced a right shift in Ca2+ dependence for all channels so that [Ca2+] <30 µM activated only high activity channels. These results strongly suggest that channel redox state determines RyR activation by Ca2+ at physiological [ATP] and [Mg2+]. If RyR behave similarly in living neurons, cellular redox state should affect RyR-mediated CICR. Ca2+-induced Ca2+ release; Ca2+ release channels; endoplasmic reticulum; thimerosal; 2,4-dithiothreitol; ryanodine receptor  相似文献   

13.
Pancreatitis is an inflammatory disease of pancreatic acinar cells whereby intracellular calcium concentration ([Ca2+]i) signaling and enzyme secretion are impaired. Increased oxidative stress has been suggested to mediate the associated cell injury. The present study tested the effects of the oxidant, hydrogen peroxide, on [Ca2+]i signaling in rat pancreatic acinar cells by simultaneously imaging fura-2, to measure [Ca2+]i, and dichlorofluorescein, to measure oxidative stress. Millimolar concentrations of hydrogen peroxide increased cellular oxidative stress and irreversibly increased [Ca2+]i, which was sensitive to antioxidants and removal of external Ca2+, and ultimately led to cell lysis. Responses were also abolished by pretreatment with (sarco)endoplasmic reticulum Ca2+-ATPase inhibitors, unless cells were prestimulated with cholecystokinin to promote mitochondrial Ca2+ uptake. This suggests that hydrogen peroxide promotes Ca2+ release from the endoplasmic reticulum and the mitochondria and that it promotes Ca2+ influx. Lower concentrations of hydrogen peroxide (10–100 µM) increased [Ca2+]i and altered cholecystokinin-evoked [Ca2+]i oscillations with marked heterogeneity, the severity of which was directly related to oxidative stress, suggesting differences in cellular antioxidant capacity. These changes in [Ca2+]i also upregulated the activity of the plasma membrane Ca2+-ATPase in a Ca2+-dependent manner, whereas higher concentrations (0.1–1 mM) inactivated the plasma membrane Ca2+-ATPase. This may be important in facilitating "Ca2+ overload," resulting in cell injury associated with pancreatitis. oxidant stress; pancreatitis; calcium pump  相似文献   

14.
We determined the effect of aromatic aminoacid stimulation of the human extracellular Ca2+-sensingreceptor (CaR) on intracellular Ca2+ concentration([Ca2+]i) in single HEK-293 cells. Additionof L-phenylalanine or L-tryptophan (at 5 mM)induced [Ca2+]i oscillations from a restingstate that was quiescent at 1.8 mM extracellular Ca2+concentration ([Ca2+]e). Each[Ca2+]i peak returned to baseline values, andthe average oscillation frequency was ~1 min1 at37°C. Oscillations were not induced or sustained if the[Ca2+]e was reduced to 0.5 mM, even in thecontinued presence of amino acid. Average oscillation frequency inresponse to an increase in [Ca2+]e (from 1.8 to 2.5-5 mM) was much higher (~4 min1) than thatinduced by aromatic amino acids. Oscillations in response to[Ca2+]e were sinusoidal whereas those inducedby amino acids were transient. Thus both amino acids andCa2+, acting through the same CaR, produce oscillatoryincreases in [Ca2+]i, but the resultantoscillation pattern and frequency allow the cell to discriminate whichagonist is bound to the receptor.

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15.
Hypotonicswelling increases the intracellular Ca2+ concentration([Ca2+]i) in vascular smooth muscle cells(VSMC). The source of this Ca2+ is not clear. To study thesource of increase in [Ca2+]i in response tohypotonic swelling, we measured [Ca2+]i infura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a40.7-nM increase in [Ca2+]i that was notinhibited by EGTA but was inhibited by 1 µM thapsigargin. Priordepletion of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores with vasopressin did not inhibit the increasein [Ca2+]i in response to hypotonic swelling.Exposure of 45Ca2+-loaded intracellular storesto hypotonic swelling in permeabilized VSMC produced an increase in45Ca2+ efflux, which was inhibited by 1 µMthapsigargin but not by 50 µg/ml heparin, 50 µM ruthenium red, or25 µM thio-NADP. Thus hypotonic swelling of VSMC causes a release ofCa2+ from the intracellular stores from a novel sitedistinct from the IP3-, ryanodine-, and nicotinic acidadenine dinucleotide phosphate-sensitive stores.

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16.
Transfected Chinese hamster ovary cells stably expressing thebovine cardiacNa+/Ca2+exchanger (CK1.4 cells) were used to determine the range of cytosolic Ca2+ concentrations([Ca2+]i)that activateNa+/Ca2+exchange activity. Ba2+ influx wasmeasured in fura 2-loaded, ionomycin-treated cells under conditions inwhich the intracellular Na+concentration was clamped with gramicidin at ~20 mM.[Ca2+]iwas varied by preincubating ionomycin-treated cells with either theacetoxymethyl ester of EGTA or medium containing 0-1 mM added CaCl2. The rate ofBa2+ influx increased in asaturable manner with[Ca2+]i,with the half-maximal activation value of 44 nM and a Hill coefficientof 1.6. When identical experiments were carried out with cellsexpressing a Ca2+-insensitivemutant of the exchanger, Ba2+influx did not vary with[Ca2+]i.The concentration for activation of exchange activity was similar tothat reported for whole cardiac myocytes but approximately an order ofmagnitude lower than that reported for excised, giant patches. Thereason for the difference in Ca2+regulation between whole cells and membrane patches is unknown.

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17.
A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) is a trigger for pulmonary vasoconstriction and a stimulus for PASMC proliferation and migration. Multiple mechanisms are involved in regulating [Ca2+]cyt in human PASMC. The resting [Ca2+]cyt and Ca2+ entry are both increased in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH), which is believed to be a critical mechanism for sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in these patients. Here we report that protein expression of NCX1, an NCX family member of Na+/Ca2+ exchanger proteins is upregulated in PASMC from IPAH patients compared with PASMC from normal subjects and patients with other cardiopulmonary diseases. The Na+/Ca2+ exchanger operates in a forward (Ca2+ exit) and reverse (Ca2+ entry) mode. By activating the reverse mode of Na+/Ca2+ exchange, removal of extracellular Na+ caused a rapid increase in [Ca2+]cyt, which was significantly enhanced in IPAH PASMC compared with normal PASMC. Furthermore, passive depletion of intracellular Ca2+ stores using cyclopiazonic acid (10 µM) not only caused a rise in [Ca2+]cyt due to Ca2+ influx through store-operated Ca2+ channels but also mediated a rise in [Ca2+]cyt via the reverse mode of Na+/Ca2+ exchange. The upregulated NCX1 in IPAH PASMC led to an enhanced Ca2+ entry via the reverse mode of Na+/Ca2+ exchange, but did not accelerate Ca2+ extrusion via the forward mode of Na+/Ca2+ exchange. These observations indicate that the upregulated NCX1 and enhanced Ca2+ entry via the reverse mode of Na+/Ca2+ exchange are an additional mechanism responsible for the elevated [Ca2+]cyt in PASMC from IPAH patients. transient receptor potential channel; reverse and forward mode; proliferation  相似文献   

18.
The subcellular spatial and temporal organization ofagonist-induced Ca2+ signals wasinvestigated in single cultured vascular endothelial cells.Extracellular application of ATP initiated a rapid increase ofintracellular Ca2+ concentration([Ca2+]i)in peripheral cytoplasmic processes from where activation propagated asa[Ca2+]iwave toward the central regions of the cell. The average propagation velocity of the[Ca2+]iwave in the peripheral processes was 20-60 µm/s, whereas in thecentral region the wave propagated at <10 µm/s. The time course ofthe recovery of[Ca2+]idepended on the cell geometry. In the peripheral processes (i.e.,regions with a high surface-to-volume ratio)[Ca2+]ideclined monotonically, whereas in the central region[Ca2+]idecreased in an oscillatory fashion. Propagating[Ca2+]iwaves were preceded by small, highly localized[Ca2+]itransients originating from 1- to 3-µm-wide regions. The average amplitude of these elementary events ofCa2+ release was 23 nM, and theunderlying flux of Ca2+ amountedto ~1-2 × 1018mol/s or ~0.3 pA, consistent with aCa2+ flux through a single orsmall number of endoplasmic reticulum Ca2+-release channels.

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19.
In the present study, we used laser scanning confocal microscopy in combination with fluorescent indicator dyes to investigate the effects of nitric oxide (NO) produced endogenously by stimulation of the mitochondria-specific NO synthase (mtNOS) or applied exogenously through a NO donor, on mitochondrial Ca2+ uptake, membrane potential, and gating of mitochondrial permeability transition pore (PTP) in permeabilized cultured calf pulmonary artery endothelial (CPAE) cells. Higher concentrations (100–500 µM) of the NO donor spermine NONOate (Sper/NO) significantly reduced mitochondrial Ca2+ uptake and Ca2+ extrusion rates, whereas low concentrations of Sper/NO (<100 µM) had no effect on mitochondrial Ca2+ levels ([Ca2+]mt). Stimulation of mitochondrial NO production by incubating cells with 1 mM L-arginine also decreased mitochondrial Ca2+ uptake, whereas inhibition of mtNOS with 10 µM L-N5-(1-iminoethyl)ornithine resulted in a significant increase of [Ca2+]mt. Sper/NO application caused a dose-dependent sustained mitochondrial depolarization as revealed with the voltage-sensitive dye tetramethylrhodamine ethyl ester (TMRE). Blocking mtNOS hyperpolarized basal mitochondrial membrane potential and partially prevented Ca2+-induced decrease in TMRE fluorescence. Higher concentrations of Sper/NO (100–500 µM) induced PTP opening, whereas lower concentrations (<100 µM) had no effect. The data demonstrate that in calf pulmonary artery endothelial cells, stimulation of mitochondrial Ca2+ uptake can activate NO production in mitochondria that in turn can modulate mitochondrial Ca2+ uptake and efflux, demonstrating a negative feedback regulation. This mechanism may be particularly important to protect against mitochondrial Ca2+ overload under pathological conditions where cellular [NO] can reach very high levels. nitric oxide synthase; permeability transition pore; endothelium  相似文献   

20.
How the endoplasmic reticulum (ER) and mitochondria communicate with each other and how they regulate plasmalemmal Ca2+ entry were studied in cultured rat brown adipocytes. Cytoplasmic Ca2+ or Mg2+ and mitochondrial membrane potential were measured by fluorometry. The sustained component of rises in cytoplasmic Ca2+ concentration ([Ca2+]i) produced by thapsigargin was abolished by removing extracellular Ca2+, depressed by depleting extracellular Na+, and enhanced by raising extracellular pH. FCCP, dinitrophenol, and rotenone caused bi- or triphasic rises in [Ca2+]i, in which the first phase was accompanied by mitochondrial depolarization. The FCCP-induced first phase was partially inhibited by oligomycin but not by ruthenium red, cyclosporine A, U-73122, a Ca2+-free EGTA solution, and an Na+-free solution. The FCCP-induced second phase paralleling mitochondrial repolarization was partially blocked by removing extracellular Ca2+ and fully blocked by oligomycin but not by thapsigargin or an Na+-deficient solution, was accompanied by a rise in cytoplasmic Mg2+ concentration, and was summated with a high pH-induced rise in [Ca2+]i, whereas the extracellular Ca2+-independent component was blocked by U-73122 and cyclopiazonic acid. The FCCP-induced third phase was blocked by removing Ca2+ but not by thapsigargin, depressed by decreasing Na+, and enhanced by raising pH. Cyclopiazonic acid-evoked rises in [Ca2+]i in a Ca2+-free solution were depressed after FCCP actions. Thus mitochondrial uncoupling causes Ca2+ release, activating Ca2+ release from the ER and store-operated Ca2+ entry, and directly elicits a novel plasmalemmal Ca2+ entry, whereas Ca2+ release from the ER activates Ca2+ accumulation in, or release from, mitochondria, indicating bidirectional mitochondria-ER couplings in rat brown adipocytes. plasmalemmal calcium entry; calcium release; mitochondrial depolarization; FCCP  相似文献   

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