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1.
Selective Changes in Cell Bodies and Growth Cones of Nerve Growth Factor-Differentiated PC12 Cells Induced by Chemical Hypoxia 总被引:2,自引:1,他引:1
Abstract: Cytosolic free Ca2+ concentration ([Ca2+]i) was measured in differentiated PC12 cells to test whether chemical hypoxia selectively alters intracellular Ca2+ in growth cones and cell bodies. Hypoxia increased [Ca2+]i and exaggerated its response to K+ depolarization in both parts of the cells. [Ca2+]i in the cell bodies was greater than that in the growth cones under resting conditions and in response to K+ or hypoxia. Ca2+-channel blockers selectively altered these responses. The L-channel blocker nifedipine reduced [Ca2+]i following K+ depolarization by 67% in the cell bodies but only 25% in the growth cones. In contrast, the N-channel blocker ω-conotoxin GVIA (ω-CgTX) diminished K+-induced changes in [Ca2+]i only in the growth cones. During hypoxia, nifedipine was more effective in the cell bodies than in the growth cones. During hypoxia, ω-CgTX diminished K+-induced changes by 50–75% in both parts of the cell, but only immediately after depolarization. The combination of nifedipine and ω-CgTX diminished the [Ca2+]i response to K+ with or without hypoxia by >90% in the cell body and 70% in the growth cones. Thus, the increased Ca2+ entry with K+ during hypoxia is primarily through L channels in the cell bodies, whereas in growth cones influx through L and N channels is about equal. The results show that chemical hypoxia selectively alters Ca2+ regulation in the growth cone and cell body of the same cell. 相似文献
2.
The interaction between ATP- and high K+-evoked increase in intracellular free calcium concentration ([Ca2+]i) was investigated to gain an insight into the mechanism of interaction of ATP with voltage-sensitive calcium channels. [Ca2+]i was measured in the neuronal model, neuroblastoma × glioma hybrid cells (NG 108–15), using the fluorescence indicator fura-2. In the presence of 1.8 mM extracellular Ca2+, ATP induced a rapid, concentration-dependent increase in [Ca2+]i. High K+ (50 mM) evoked a [Ca2+]i rise from 109 ± 11 nM to 387 ± 81 nM (n = 16). The application of either of these two [Ca2+]i-increase provoking agents in sequence with the other caused impairment of the latter effect. The mutual desensitization of the responses to ATP and high K+ strongly suggests that both agents rely at least in part on the same source of Ca2+ for elevation of [Ca2+]i in NG 108–15 cells. 相似文献
3.
Robert S. Siegel Shaowu Xue Yoshiyuki Murata Yingzhen Yang Noriyuki Nishimura Angela Wang Julian I. Schroeder 《The Plant journal : for cell and molecular biology》2009,59(2):207-220
Stomatal closure in response to abscisic acid depends on mechanisms that are mediated by intracellular [Ca2+] ([Ca2+]i), and also on mechanisms that are independent of [Ca2+]i in guard cells. In this study, we addressed three important questions with respect to these two predicted pathways in Arabidopsis thaliana. (i) How large is the relative abscisic acid (ABA)‐induced stomatal closure response in the [Ca2+]i‐elevation‐independent pathway? (ii) How do ABA‐insensitive mutants affect the [Ca2+]i‐elevation‐independent pathway? (iii) Does ABA enhance (prime) the Ca2+ sensitivity of anion and inward‐rectifying K+ channel regulation? We monitored stomatal responses to ABA while experimentally inhibiting [Ca2+]i elevations and clamping [Ca2+]i to resting levels. The absence of [Ca2+]i elevations was confirmed by ratiometric [Ca2+]i imaging experiments. ABA‐induced stomatal closure in the absence of [Ca2+]i elevations above the physiological resting [Ca2+]i showed only approximately 30% of the normal stomatal closure response, and was greatly slowed compared to the response in the presence of [Ca2+]i elevations. The ABA‐insensitive mutants ost1‐2, abi2‐1 and gca2 showed partial stomatal closure responses that correlate with [Ca2+]i‐dependent ABA signaling. Interestingly, patch‐clamp experiments showed that exposure of guard cells to ABA greatly enhances the ability of cytosolic Ca2+ to activate S‐type anion channels and down‐regulate inward‐rectifying K+ channels, providing strong evidence for a Ca2+ sensitivity priming hypothesis. The present study demonstrates and quantifies an attenuated and slowed ABA response when [Ca2+]i elevations are directly inhibited in guard cells. A minimal model is discussed, in which ABA enhances (primes) the [Ca2+]i sensitivity of stomatal closure mechanisms. 相似文献
4.
Abstract: The role of voltage-sensitive Ca2+ channels in mediating Ca2+ influx during ischemia was investigated in NG108-15 cells, a neuronal cell line that does not express glutamate-sensitive receptor-mediated Ca2+ channels. Concurrent 31P/19F and 23Na double-quantum filtered (DQF) NMR spectra were used to monitor cellular energy status, intracellular [Ca2+] ([Ca2+]i), and intracellular Na+ content in cells loaded with the calcium indicator 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5FBAPTA) during ischemia and reperfusion. Cells loaded with 5FBAPTA were indistinguishable from unloaded cells except for small immediate decreases in levels of phosphocreatine (PCr) and ATP. Ischemia induced a steady decrease in intracellular pH and PCr and ATP levels, and a steady increase in intracellular Na+ content; however, a substantial increase in [Ca2+]i (about threefold) was seen only following marked impairment of cellular energy status, when PCr was undetectable and ATP content was reduced to 55% of control levels. A depolarization-induced increase in [Ca2+]i could be completely blocked by 1 µM nifedipine, whereas up to 20 µM nifedipine had no effect on the increase in [Ca2+]i seen during ischemia. These data demonstrate that voltage-gated Ca2+ channels do not mediate significant Ca2+ flux during ischemia in this cell line and suggest an important role for Ca2+i stores, the Na+/Ca2+ antiporter, or other processes linked to cellular energy status in the increase in cytosolic Ca2+ level during ischemia. 相似文献
5.
Rosa Luisi Elisabetta Panza Vincenzo Barrese Fabio Arturo Iannotti† Davide Viggiano† Agnese Secondo Lorella Maria Teresa Canzoniero Maria Martire‡ Lucio Annunziato Maurizio Taglialatela† 《Journal of neurochemistry》2009,109(1):168-181
In this study, the functional consequences of the pharmacological modulation of the M‐current (IKM) on cytoplasmic Ca2+ intracellular Ca2+concentration ([Ca2+]i) changes and excitatory neurotransmitter release triggered by various stimuli from isolated rat cortical synaptosomes have been investigated. Kv7.2 immunoreactivity was identified in pre‐synaptic elements in cortical slices and isolated glutamatergic cortical synaptosomes. In cerebrocortical synaptosomes exposed to 20 mM [K+]e, the IKM activator retigabine (RT, 10 μM) inhibited [3H]d ‐aspartate ([3H]d ‐Asp) release and caused membrane hyperpolarization; both these effects were prevented by the IKM blocker XE‐991 (20 μM). The IKM activators RT (0.1–30 μM), flupirtine (10 μM) and BMS‐204352 (10 μM) inhibited 20 mM [K+]e‐induced synaptosomal [Ca2+]i increases; XE‐991 (20 μM) abolished RT‐induced inhibition of depolarization‐triggered [Ca2+]i transients. The P/Q‐type voltage‐sensitive Ca2+channel (VSCC) blocker ω‐agatoxin IVA prevented RT‐induced inhibition of depolarization‐induced [Ca2+]i increase and [3H]d ‐Asp release, whereas the N‐type blocker ω‐conotoxin GVIA failed to do so. Finally, 10 μM RT did not modify the increase of [Ca2+]i and the resulting enhancement of [3H]d ‐Asp release induced by [Ca2+]i mobilization from intracellular stores, or by store‐operated Ca2+channel activation. Collectively, the present data reveal that the pharmacological activation of IKM regulates depolarization‐induced [3H]d ‐Asp release from cerebrocortical synaptosomes by selectively controlling the changes of [Ca2+]i occurring through P/Q‐type VSCCs. 相似文献
6.
Verónica Alvarez Maubecin Viviana N. Sanchez Marcelo D. Rosato Siri Bruce D. Cherksey Mutzuyuki Sugimori Rodolfo Llinás Osvaldo D. Uchitel 《Journal of neurochemistry》1995,64(6):2544-2551
Abstract: The voltage-dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L-type channels (1,4-dihydropyridines), N-type channels (ω-conotoxin GVIA), and P-type channels [funnel web toxin (FTX) and ω-agatoxin IVA]. K+-induced Ca2+ uptake by chicken synaptosomes was blocked by ω-conotoxin GVIA (IC50 = 250 nM). This toxin at 5 µM did not block Ca2+ entry into rat frontal cortex synaptosomes. FTX and ω-agatoxin IVA blocked Ca2+ uptake by rat synaptosomes (IC50 = 0.17 µl/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and ω-agatoxin IVA affected Ca2+ uptake. FTX (3 µl/ml) exerted a maximal inhibition of 40% with an IC50 similar to the one obtained in rat preparations, whereas with ω-agatoxin IVA saturation was not reached even at 5 µM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 µl/ml) and different concentrations of ω-conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and ω-conotoxin GVIA was never greater than the one observed with ω-conotoxin GVIA. We also found that 60% of the Ca2+ uptake by rat and chicken synaptosomes was inhibited by ω-conotoxin MVIID (1 µM), a toxin that has a high index of discrimination against N-type channels. Conversely, nitrendipine (10 µM) had no significant effect on Ca2+ uptake in either the rat or the chicken. In conclusion, Ca2+ uptake by rat synaptosomes is potently inhibited by different P-type Ca2+ channel blockers, thus indicating that P-type channels are predominant in this preparation. In contrast, Ca2+ uptake by chicken synaptosomes is sensitive to ω-conotoxin GVIA, FTX, ω-agatoxin IVA, and ω-conotoxin MVIID. This suggests that a channel subtype with a mixed pharmacology is present in chicken synaptosomes. 相似文献
7.
Marc Gleichmann Leon P. Collis† Peter J. S. Smith† Mark P. Mattson 《Journal of neurochemistry》2009,109(2):644-655
In order to determine the sequence of cellular processes in glutamate toxicity, we simultaneously recorded O2 consumption, cytosolic Ca2+ concentration ([Ca2+]i), and mitochondrial membrane potential (mΔψ) in single cortical neurons. Oxygen consumption was measured using an amperometric self‐referencing platinum electrode adjacent to neurons in which [Ca2+]i and mΔψ were monitored with Fluo‐4 and TMRE+, respectively, using a spinning disk laser confocal microscope. Excitotoxic doses of glutamate caused an elevation of [Ca2+]i followed seconds afterwards by an increase in O2 consumption which reached a maximum level within 1–5 min. A modest increase in mΔψ occurred during this time period, and then, shortly before maximal O2 consumption was reached, the mΔψ, as indicated by TMRE+ fluorescence, dissipated. Maximal O2 consumption lasted up to 5 min and then declined together with mΔψ and ATP levels, while [Ca2+]i further increased. mΔψ and [Ca2+]i returned to baseline levels when neurons were treated with an NMDA receptor antagonist shortly after the [Ca2+]i increased. Our unprecedented spatial and time resolution revealed that this sequence of events is identical in all neurons, albeit with considerable variability in magnitude and kinetics of changes in O2 consumption, [Ca2+]i, and mΔψ. The data obtained using this new method are consistent with a model where Ca2+ influx causes ATP depletion, despite maximal mitochondrial respiration, minutes after glutamate receptor activation. 相似文献
8.
Abstract: The effect of replacement of extracellular Na+ with N-methyl-d -glucamine (NMG) on P2 receptor signaling pathways was investigated in dibutyryl cyclic AMP-differentiated NG108-15 cells. Benzoylbenzoic ATP (BzATP) dose-dependently increased the cytosolic Ca2+ concentration ([Ca2+]i) with an EC50 value of 230 µM. Replacement of Na+ with NMG as well as removal of Mg2+ from the bathing buffer potentiated ethidium bromide uptake, [Ca2+]i increase, and 45Ca2+ uptake in response to ATP or BzATP. In contrast, in the presence of 5 mM Mg2+ to limit the amount of ATP4?, replacement of Na+ with NMG had no effect on the ATP-induced [Ca2+]i increase but caused a markedly larger [Ca2+]i increase when the calculated concentration of ATP4? was >10 µM. The calculated EC50 value for ATP4? stimulation of the [Ca2+]i increase was 23 µM in NG108-15 cells. In vascular smooth muscle cells, intracellular Ca2+ release was the major pathway for the ATP-induced [Ca2+]i increase; both removal of Mg2+ and replacement of Na+ with NMG did not affect the action of ATP. These data suggest that ATP4?-promoted pores are antagonized by Na+ and Mg2+ in dibutyryl cyclic AMP-differentiated NG108-15 cells. 相似文献
9.
Hong Shan Zhang Thomas V. McDonald Herbert B. Tanowitz Murray Wittner Louis M. Weiss John P. Bilezikian Stephen A. Morris 《The Journal of eukaryotic microbiology》1998,45(1):80-86
ABSTRACT Trypomastigotes of Trypanosoma cruzi maintain an intracellular Ca2+ concentration([Ca2+]i) of 64 ± 30 nM. Equilibration of trypomastigotes in an extracellular buffer containing 0.5 mM [Ca2+]o (preloaded cells) increased [Ca2+]i < 20 nM whereas total cell Ca2+ increased by 1.5 to 2.0 pmole/cell. This amount of Ca2+ would be expected to increase [Ca2+]i to > 10 μM suggesting active sequestration of Ca2+. We tested the hypothesis that maintenance of [Ca2+]i involved both the sequestration into intracellular storage sites and extrusion into the extracellular space. Pharmacological probes known to influence [Ca2+]i through well characterized pathways in higher eukaryotic cells were employed. [Ca2+], responses in the presence or absence of [Ca2+]o were measured to asses the relative contribution of sequestration or extrusion processes in [Ca2+]i homeostasis. In the presence of 0.5 mM [Ca2+]o, the ability of several agents to increase [Ca2+]i was magnified in the order ionomycin ? nigericin > thapsigargin > monensin > valinomycin. In contrast, preloading markedly enhanced the increase in [Ca2+], observed only in response to monensin. Manoalide, an inhibitor of phospholipase A2, enhanced the accumulation of [Ca2+]i due to all agents tested, particularly ionomycin and thapsigargin. Our results suggest that sequestration of [Ca2+]i involved storage sites sensitive to monensin and ionomycin whereas extrusion of Ca2+ may involve phospholipase A2 activity. A Na+/Ca2+ exchange mechanism did not appear to contribute to Ca2+ homeostasis. 相似文献
10.
Abstract: We studied the effect of α-latrotoxin (αLTX) on [14C]acetylcholine ([14C]ACh) release, intracellular Ca2+ concentration ([Ca2+]i), plasma membrane potential, and high-affinity choline uptake of synaptosomes isolated from guinea pig cortex. αLTX (10?10-10?8M) caused an elevation of the [Ca2+]i as detected by Fura 2 fluorescence and evoked [14C]ACh efflux. Two components in the action of the toxin were distinguished: one that required the presence of Na+ in the external medium and another that did not. Displacement of Na+ by sucrose or N-methylglucamine in the medium considerably decreased the elevation of [Ca2+]i and [14C]ACh release by αLTX. The Na+-dependent component of the αLTX action was obvious in the inhibition of the high-affinity choline uptake of synaptosomes. Some of the toxin action on both [Ca2+]i and [14C]ACh release remained in the absence of Na+. Both the Na+-dependent and the Na+-independent components of the αLTX-evoked [14C]ACh release partly required the presence of either Mg2+ or Ca2+. The nonneurotransmitter [14C]choline was released along with [14C]ACh, but this release did not depend on the presence of either Na+ or Ca2+, indicating nonspecific leakage through the plasma membrane. We conclude that there are two factors in the release of ACh from synaptosomes caused by the toxin: (1) cation-dependent ACh release, which is related to (a) Na+-dependent divalent cation entry and (b) Na+-independent divalent cation entry, and (2) nonspecific Na+- and divalent cation-independent leakage. 相似文献
11.
12.
Jörg Dreessen Claudia Lutum Beat W. Schäfer Claus W. Heizmann Thomas Knöpfel 《Cell calcium》1996,19(6):527-533
We investigated whether the expression of human α-parvalbumin affects depolarization-induced elevations of the cytosolic free calcium concentration ([Ca2+]i) in human neuroblastoma SKNBE2 cells. A full length human parvalbumin cDNA was cloned by PCR from human cerebellum and transiently transfected into SKNBE2 cells. Immunofluorescence staining using an antibody raised against parvalbumin revealed a transfection efficacy of about 14%. In parvalbumin-expressing SKNBE2 cells, parvalbumin concentration determined by quantitative Western blotting amounted to 0.42 mM.Transfected SKNBE2 cells were depolarized for 2 min by 50 mM K+. During this period, [Ca2+]i was monitored by video microfluorimetry using the Ca2+ indicator Fura-2. In a fraction of cells, depolarization induced a transient elevation in [Ca2+]i The size of this elevation was compared with the immunofluorimetrically determined expression of parvalbumin on a cell-to-cell basis. Cells with a significant parvalbumin immunofluorescence responded to depolarization with smaller elevations in [Ca2+]i than non-parvalbumin-expressing cells. Resting [Ca 2+], did not differ between parvalbumin-expressing and control cells. These observations indicate that large depolarization-induced transient elevations of [Ca2+]i in neuroblastoma cells can be suppressed by parvalbumin. 相似文献
13.
Kazuhiro Takuma Toshio Matsuda Hitoshi Hashimoto Junichi Kitanaka Shoichi Asano Yoko Kishida Akemichi Baba 《Journal of neurochemistry》1996,67(5):1840-1845
Abstract: We have previously demonstrated that activation of the Na+-Ca2+ exchanger in the reverse mode causes Ca2+ influx in astrocytes. In addition, we showed that the exchange activity was stimulated by nitric oxide (NO)/cyclic GMP and inhibited by ascorbic acid. The present study demonstrates that the Na+-Ca2+ exchanger is involved in agonist-induced Ca2+ signaling in cultured rat astrocytes. The astrocytic intracellular Ca2+ concentration ([Ca2+]i) was increased by l -glutamate, noradrenaline (NA), and ATP, and the increases were all attenuated by the NO generator sodium nitroprusside (SNP). SNP also reduced the ionomycin-induced increase in [Ca2+]i. The Na-induced Ca2+ signal was also attenuated by S-nitroso-l -cysteine and 8-bromo cyclic GMP, whereas it was enhanced by 3,4-dichlorobenzamil, an inhibitor of the Na+-Ca2+ exchanger. Treatment of astrocytes with antisense, but not sense, deoxynucleotides to the sequence encoding the Na+-Ca2+ exchanger enhanced the ionomycin-induced increase in [Ca2+]i and blocked the effects of SNP and 8-bromo cyclic GMP in reducing the NA-induced Ca2+ signal. Furthermore, the ionomycin-induced Ca2+ signal was enhanced by removal of extracellular Na+ and pretreatment with ascorbic acid. These findings indicate that the Na+-Ca2+ exchanger is a target for NO modulation of elevated [Ca2+]i and that the exchanger plays a role in Ca2+ efflux when [Ca2+]i is raised above basal levels in astrocytes. 相似文献
14.
Acute stress is known to enhance the memory of events that are potentially threatening to the organisms. Glutamate, the most
abundant excitatory neurotransmitter in the mammalian central nervous system, plays a critical role in learning and memory
formation and calcium (Ca2+) plays an essential role in transmitter release from nerve terminals (synaptosomes). In the present study, we investigated
the effects of acute restraint stress on cytosolic free Ca2+ concentration ([Ca2+]i) and glutamate release in cerebrocortical synaptosomes from mice. Acute restraint stress caused a significant increase in
resting [Ca2+]i and significantly enhanced the ability of the depolarizing agents K+ and 4-aminopyridine (4-AP) to increase [Ca2+]i. It also brought about a significant increase in spontaneous (unstimulated) glutamate release and significantly enhanced
K+- and 4-AP-induced Ca2+-dependent glutamate release. The pretreatment of synaptosomes with a combination of ω-agatoxin IVA (a P-type Ca2+ channel blocker) and ω-conotoxin GVIA (an N-type Ca2+ channel blocker) completely suppressed the enhancements of [Ca2+]i and Ca2+-dependent glutamate release in acute restraint-stressed mice. These results indicate that acute restraint stress enhances
K+- or 4-AP-induced glutamate release by increasing [Ca2+]i via stimulation of Ca2+ entry through P- and N-type Ca2+ channels. 相似文献
15.
Abstract: The potent nicotinic agonist anatoxin-a elicits mecamylamine-sensitive [3H]dopamine release from striatal synaptosomes, and this action is both Na+ and Ca2+ dependent and is blocked by Cd2+. This suggests that stimulation of presynaptic nicotinic receptors results in Na+ influx and local depolarisation that activates voltage-sensitive Ca2+ channels, which in turn provide the Ca2+ for exocytosis. Here we have investigated the subtypes of Ca2+ channels implicated in this mechanism. [3H]Dopamine release evoked by anatoxin-a (1 µM) was partially blocked by 20 µM nifedipine, whereas KCl-evoked release was insensitive to the dihydropyridine. However, a 86Rb+ efflux assay of nicotinic receptor function suggested that nifedipine has a direct effect on the receptor, discrediting the involvement of L-type channels. The N-type Ca2+ channel blocker ω-conotoxin GVIA (1 µM) blocked anatoxin-a-evoked [3H]dopamine release by 60% but had no significant effect on 86Rb+ efflux; release evoked by both 15 and 25 mM KCl was inhibited by only 30%. The P-type channel blocker ω-agatoxin IVA (90 nM) also inhibited KCl-evoked release by ~30%, whereas anatoxin-a-evoked release was insensitive. The Q-type channel blocker ω-conotoxin MVIIC (1 µM) had no effect on either stimulus. These results suggest that presynaptic nicotinic receptors on striatal nerve terminals promote [3H]dopamine release by activation of N-type Ca2+ channels. In contrast, KCl-evoked [3H]dopamine release appears to involve both N-type and P-type channels. 相似文献
16.
Ádám Fekete Laura Franklin Takeshi Ikemoto Balázs Rózsa Balázs Lendvai E. Sylvester Vizi Tibor Zelles 《Journal of neurochemistry》2009,111(3):745-756
Although the role of Na+ in several aspects of Ca2+ regulation has already been shown, the exact mechanism of intracellular Ca2+ concentration ([Ca2+]i) increase resulting from an enhancement in the persistent, non‐inactivating Na+ current (INa,P), a decisive factor in certain forms of epilepsy, has yet to be resolved. Persistent Na+ current, evoked by veratridine, induced bursts of action potentials and sustained membrane depolarization with monophasic intracellular Na+ concentration ([Na+]i) and biphasic [Ca2+]i increase in CA1 pyramidal cells in acute hippocampal slices. The Ca2+ response was tetrodotoxin‐ and extracellular Ca2+‐dependent and ionotropic glutamate receptor‐independent. The first phase of [Ca2+]i rise was the net result of Ca2+ influx through voltage‐gated Ca2+ channels and mitochondrial Ca2+ sequestration. The robust second phase in addition involved reverse operation of the Na+–Ca2+ exchanger and mitochondrial Ca2+ release. We excluded contribution of the endoplasmic reticulum. These results demonstrate a complex interaction between persistent, non‐inactivating Na+ current and [Ca2+]i regulation in CA1 pyramidal cells. The described cellular mechanisms are most likely part of the pathomechanism of certain forms of epilepsy that are associated with INa,P. Describing the magnitude, temporal pattern and sources of Ca2+ increase induced by INa,P may provide novel targets for antiepileptic drug therapy. 相似文献
17.
Joanne E. Millership Caroline Heard Ian M. Fearon Jason I. E. Bruce 《The Journal of membrane biology》2010,235(3):191-210
Calcium (Ca2+)-activated K+ (KCa) channels regulate membrane excitability and are activated by an increase in cytosolic Ca2+ concentration ([Ca2+]i), leading to membrane hyperpolarization. Most patch clamp experiments that measure KCa currents use steady-state [Ca2+] buffered within the patch pipette. However, when cells are stimulated physiologically, [Ca2+]i changes dynamically, for example during [Ca2+]i oscillations. Therefore, the aim of the present study was to examine the effect of dynamic changes in [Ca2+]i on small (SK3), intermediate (hIK1), and large conductance (BK) channels. HEK293 cells stably expressing each KCa subtype in isolation were used to simultaneously measure agonist-evoked [Ca2+]i signals, using indo-1 fluorescence, and current/voltage, using perforated patch clamp. Agonist-evoked [Ca2+]i oscillations induced a corresponding KCa current that faithfully followed the [Ca2+]i in 13–50% of cells, suggesting a good synchronization. However, [Ca2+]i and KCa current was much less synchronized in 50–76% of cells that exhibited Ca2+-independent current events (55% of SK3-, 50% of hIK1-, and 53% of BK-expressing cells) and current-independent [Ca2+]i events (18% SK3- and 33% of BK-expressing cells). Moreover, in BK-expressing cells, where [Ca2+]i and KCa current was least synchronized, 36% of total [Ca2+]i spikes occurred without activating a corresponding KCa current spike, suggesting that BKCa channels were either inhibited or had become desensitized. This desynchronization between dynamic [Ca2+]i and KCa current suggests that this relationship is more complex than could be predicted from steady-state [Ca2+]i and KCa current. These phenomena may be important for encoding stimulus–response coupling in various cell types. 相似文献
18.
G. Bkaily D. Econornos L. Potvin J. -L. Ardilouze C. Marriott J. Corcos D. Bonneau Chin Nok Fong 《Molecular and cellular biochemistry》1992,117(1):93-106
The effect of high K– concentration, insulin and the L-type Ca2– channel blocker PN 200-110 on cytosolic intracellular free calcium ([Ca2+]i) was studied in single ventricular myocytes of 10-day-old embryonic chick heart, 20-week-old human fetus and rabbit aorta (VSM) single cells using the Ca2+-sensitive fluorescent dye, Fura-2 microfluorometry and digital imaging technique. Depolarization of the cell membrane of both heart and VSM cells with continuous superfusion of 30 mM [K+]o induced a rapid transient increase of [Ca2+]i that was followed by a sustained component. The early transient increase of [Ca2+]i by high [+]o was blocked by the L-type calcium channel antagonist nifedipine. However, the sustained component was found to be insensitive to this drug. PN 200-110 another L-type Ca2+ blocker was found to decrease both the early transient and the sustained increase of [Ca2+]i induced by depolarization of the cell membrane with high [K+]o. Insulin at a concentration of 40 to 80 U/ml only produced a sustained increase of [Ca2+]i that was blocked by PN 200-110 or by lowering the extracellular Ca2+ concentration with EGTA. The sustained increase of [Ca2+], induced by high [K+]o or insulin was insensitive to metabolic inhibitors such as KCN and ouabain as well to the fast Na+ channel blocker, tetrodotoxin and to the increase of intracellular concentrations of cyclic nucleotides. Using the patch clamp technique, insulin did not affect the L-type Ca2+ current and the delayed outward K+ current. These results suggest that the early increase of (Ca2+]i during depolarization of the cell membrane of heart and VSM cells with high [K+]o is due to the opening and decay of an L-type Ca 2+ channel. However, the sustained increase of [Ca2+]i during a sustained depolarization is due to the activation of a resting (R) Ca 2+ channel that is insensitive to lowering [ATP]i and sensitive to insulin. 相似文献
19.
Abstract: The human neuroblastoma cell line SH-SY5Y, maintained at confluence for 14 days, released [3H]-noradrenaline ([3H]NA) when stimulated with either the muscarinic receptor agonist methacholine or bradykinin. The major fraction of release was rapid, occurring in <10 s, whereas nicotine-evoked release was slower. When the extracellular [Ca2+] ([Ca2+]e) was buffered to ~50–100 nM, release evoked by nicotine was abolished, whereas that in response to methacholine or bradykinin was reduced by ~50% with EC50 values of ?5.46 ± 0.05 M and ?7.46 ± 0.06 M (log10), respectively. Methacholine and bradykinin also produced rapid elevations of both inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and intracellular free [Ca2+] ([Ca2+]i). These elevations were reduced at low [Ca2+]e and under these conditions the EC50 values for peak elevation of [Ca2+]i were ?6.00 ± 0.14 M for methacholine and ?7.95 ± 0.34 M for bradykinin (n = 3 for all EC50 determinations). At low [Ca2+]e, depletion of nonmitochondrial intracellular Ca2+ stores with the Ca2+-ATPase inhibitor thapsigargin produced a transient small elevation of [Ca2+]i and a minor release of [3H]NA. At low [Ca2+]e, thapsigargin abolished elevation of [Ca2+]i in response to methacholine and bradykinin and completely inhibited their stimulation of [3H]NA release. It is proposed, therefore, that Ca2+ release from Ins(1,4,5)P3-sensitive stores is a major trigger of methacholine- and bradykinin-evoked [3H]NA release in SH-SY5Y cells. 相似文献
20.
Imanishi T Matsushima K Kawaguchi A Wada T Yoshida S Ichida S 《Neurochemical research》2006,31(1):33-40
Characteristics of the increasing effect for the concentration of intracellular calcium ions ([Ca2+]i) by high-KCl application were investigated in the neuroblastoma×glioma hybrid NG108-15 cell line (NG108-15 cells). The present
study confirmed that the increasing effect of [Ca2+]i by high-KCl application in single NG108-15 cells, differentiated with dibutyryl cAMP (Bt2cAMP), was significantly enhanced, compared to undifferentiated cells. The following observations were made at first: (1)
The response to high-KCl application, in both undifferentiated and differentiated cells, was significantly inhibited by calciseptine
(CaS), an L-type Ca2+ channel blocker, but not by N-, P- and R-type Ca2+ channel blockers. The IC50 values for CaS in both undifferentiated and differentiated cell was almost identical. (2) The inhibitory effect of CaS was
irreversible. (3) The increasing effect for [Ca2+]i by high-KCl application was completely dependent on the presence of extracellular calcium ions. (4) The increased [Ca2+]i by high-KCl application under a plateau concentration was quickly decreased to basal levels when the high-KCl solution was
exchanged for a high-KCl solution containing EGTA (without CaCl2). Together, these results suggest that the enhancement of the response effect of [Ca2+]i by high-KCl application in differentiated single NG108-15 cells was mainly due to the quantitative increase of L-type voltage-sensitive
calcium channels (VSCCs), which were irreversibly inhibited by CaS. 相似文献