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1.
《Cell calcium》2015,57(6):504-512
Imaging with Ca2+-sensitive fluorescent dye has provided a wealth of insight into the dynamics of cellular Ca2+ signaling. The spatiotemporal evolution of intracellular free Ca2+ observed in imaging experiments is shaped by binding and unbinding to cytoplasmic Ca2+ buffers, as well as the fluorescent indicator used for imaging. These factors must be taken into account in the interpretation of Ca2+ imaging data, and can be exploited to investigate endogenous Ca2+ buffer properties. Here we extended the use of Ca2+ fluorometry in the characterization of Ca2+ binding molecules within cells, building on a method of titration of intracellular Ca2+ binding sites in situ with measured amounts of Ca2+ entering through voltage-gated Ca2+ channels. We developed a systematic procedure for fitting fluorescence data acquired during a series of voltage steps to models with multiple Ca2+ binding sites. The method was tested on simulated data, and then applied to 2-photon fluorescence imaging data from rat posterior pituitary nerve terminals patch clamp-loaded with the Ca2+ indicator fluo-8. Focusing on data sets well described by a single endogenous Ca2+ buffer and dye, this method yielded estimates of the endogenous buffer concentration and Kd, the dye Kd, and the fraction of Ca2+ inaccessible cellular volume. The in situ Kd of fluo-8 thus obtained was indistinguishable from that measured in vitro. This method of calibrating Ca2+-sensitive fluorescent dyes in situ has significant advantages over previous methods. Our analysis of Ca2+ titration fluorometric data makes more effective use of the experimental data, and provides a rigorous treatment of multivariate errors and multiple Ca2+ binding species. This method offers a versatile approach to the study of endogenous Ca2+ binding molecules in their physiological milieu. 相似文献
2.
Intracellular Ca2+ release is a versatile second messenger system. It is modeled here by reaction-diffusion equations for the free Ca2+ and Ca2+ buffers, with spatially discrete clusters of stochastic IP3 receptor channels (IP3Rs) controlling the release of Ca2+ from the endoplasmic reticulum. IP3Rs are activated by a small rise of the cytosolic Ca2+ concentration and inhibited by large concentrations. Buffering of cytosolic Ca2+ shapes global Ca2+ transients. Here we use a model to investigate the effect of buffers with slow and fast reaction rates on single release spikes. We find that, depending on their diffusion coefficient, fast buffers can either decouple clusters or delay inhibition. Slow buffers have little effect on Ca2+ release, but affect the time course of the signals from the fluorescent Ca2+ indicator mainly by competing for Ca2+. At low [IP3], fast buffers suppress fluorescence signals, slow buffers increase the contrast between bulk signals and signals at open clusters, and large concentrations of buffers, either fast or slow, decouple clusters. 相似文献
3.
Ca2+ binding proteins (CaBPs), calbindin-D28k (calbindin) and calretinin, are thought to contribute to the regulation of intracellular Ca2+ in many neuronal populations and perhaps more importantly, signal functional modulation in neuronal activity. In the present experiments, light microscopic immunohistochemistry revealed that the immunoreactivity of calbindin and calretinin was contained in varicose axons in the posterior pituitary. The dual labeling study with confocal microscopy demonstrated that calbindin immunoreactivity was present in the terminals of both oxytocin (OXT) and arginine-vasopressin (AVP) neurons. However, calretinin immunoreactivity was exclusively seen in the OXT terminals. Moreover, the dual labeling study showed that most calretinin-positive terminals contained calbindin immunoreactivity, demonstrating the colocalization of calbindin and calretinin in the same OXT nerve terminals. By electron microscopy, calbindin and calretinin immunoreactivities were seen in the neurosecretory axons and nerve terminals. These immunoreactive nerve terminals were seen to contain more clear microvesicles than dense-core neurosecretory granules. This immunoelectron microscopic observation suggests that both calbindin and calretinin localize preferentially in the active zone of the nerve terminals, which usually face the perivascular space around fenestrated capillaries. In spite of similar localization of calbindin and calretinin within the posterior pituitary, Western blot analysis showed some differences between the two CaBPs. Calbindin was present mostly in the soluble fraction with little in the insoluble fraction, but a substantial portion of calretinin was present in both the insoluble and soluble fractions. Moreover, dehydration induced by drinking 2% NaCl solution and deprivation of drinking water increased calretinin levels in the posterior pituitary as compared with control, but the calbindin level was not changed. The present findings demonstrate that calbindin and calretinin colocalize in the active zones of OXT nerve terminals, but only calretinin is upregulated with dehydration, suggesting different physiological role of calbindin and calretinin in the nerve terminals. 相似文献
4.
《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
5.
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
6.
Jason J. Lefkowitz Kevin E. Fogarty Lawrence M. Lifshitz Karl D. Bellve Richard A. Tuft Ronghua ZhuGe John V. Walsh Jr. Valerie De Crescenzo 《The Journal of general physiology》2009,134(4):267-280
A central concept in the physiology of neurosecretion is that a rise in cytosolic [Ca2+] in the vicinity of plasmalemmal Ca2+ channels due to Ca2+ influx elicits exocytosis. Here, we examine the effect on spontaneous exocytosis of a rise in focal cytosolic [Ca2+] in the vicinity of ryanodine receptors (RYRs) due to release from internal stores in the form of Ca2+ syntillas. Ca2+ syntillas are focal cytosolic transients mediated by RYRs, which we first found in hypothalamic magnocellular neuronal terminals. (scintilla, Latin for spark; found in nerve terminals, normally synaptic structures.) We have also observed Ca2+ syntillas in mouse adrenal chromaffin cells. Here, we examine the effect of Ca2+ syntillas on exocytosis in chromaffin cells. In such a study on elicited exocytosis, there are two sources of Ca2+: one due to influx from the cell exterior through voltage-gated Ca2+ channels, and that due to release from intracellular stores. To eliminate complications arising from Ca2+ influx, we have examined spontaneous exocytosis where influx is not activated. We report here that decreasing syntillas leads to an increase in spontaneous exocytosis measured amperometrically. Two independent lines of experimentation each lead to this conclusion. In one case, release from stores was blocked by ryanodine; in another, stores were partially emptied using thapsigargin plus caffeine, after which syntillas were decreased. We conclude that Ca2+ syntillas act to inhibit spontaneous exocytosis, and we propose a simple model to account quantitatively for this action of syntillas. 相似文献
7.
Summary Recently Plieth et al. [Protoplasma (1997) 198: 107–124; 199: 223] gave a quantitative picture of the Ca2+ and H+ buffers in green algae which we would like to comment. In that paper a mechanistic model was derived which describes the relationship between cytosolic Ca2+ and H+ assuming that Ca2+ and H+ interact with the same binding site of a Ca2+-H+-exchange buffer. But the increase of the cytosolic free Ca2+ concentration observed upon acidification can alternatively be described by a co-operative (n=2) protonation of a Ca2+/H+-binding buffer pointing to an allosteric mechanism of Ca2+ liberation. Furthermore we present evidences that the cytosolic buffer capacities for H+ (90 mM/pH) and Ca2+ (20 mM/pCa) given for Eremosphaera viridis were overestimated by a factor of three and three orders of magnitude, respectively.Abbreviations [Ca2+]c
free cytosolic
- Ca2+
concentration 相似文献
8.
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》2014,107(6):1289-1301
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
9.
We study Ca2+ release through single and clustered IP3 receptor channels on the ER membrane under presence of buffer proteins. Our computational scheme couples reaction-diffusion equations and a Markovian channel model and allows our investigating the effects of buffer proteins on local calcium concentrations and channel gating. We find transient and stationary elevations of calcium concentrations around active channels and show how they determine release amplitude. Transient calcium domains occur after closing of isolated channels and constitute an important part of the channel's feedback. They cause repeated openings (bursts) and mediate increased release due to Ca2+ buffering by immobile proteins. Stationary domains occur during prolonged activity of clustered channels, where the spatial proximity of IP3Rs produces a distinct [Ca2+] scale (0.5-10 μM), which is smaller than channel pore concentrations (>100 μM) but larger than transient levels. While immobile buffer affects transient levels only, mobile buffers in general reduce both transient and stationary domains, giving rise to Ca2+ evacuation and biphasic modulation of release amplitude. Our findings explain recent experiments in oocytes and provide a general framework for the understanding of calcium signals. 相似文献
10.
Calcium (Ca2+)-induced Ca2+ release (CICR) is widely accepted as the principal mechanism linking electrical excitation and mechanical contraction in cardiac cells. The CICR mechanism has been understood mainly based on binding of cytosolic Ca2+ with ryanodine receptors (RyRs) and inducing Ca2+ release from the sarcoplasmic reticulum (SR). However, recent experiments suggest that SR lumenal Ca2+ may also participate in regulating RyR gating through calsequestrin (CSQ), the SR lumenal Ca2+ buffer. We investigate how SR Ca2+ release via RyR is regulated by Ca2+ and calsequestrin (CSQ). First, a mathematical model of RyR kinetics is derived based on experimental evidence. We assume that the RyR has three binding sites, two cytosolic sites for Ca2+ activation and inactivation, and one SR lumenal site for CSQ binding. The open probability (Po) of the RyR is found by simulation under controlled cytosolic and SR lumenal Ca2+. Both peak and steady-state Po effectively increase as SR lumenal Ca2+ increases. Second, we incorporate the RyR model into a CICR model that has both a diadic space and the junctional SR (jSR). At low jSR Ca2+ loads, CSQs are more likely to bind with the RyR and act to inhibit jSR Ca2+ release, while at high SR loads CSQs are more likely to detach from the RyR, thereby increasing jSR Ca2+ release. Furthermore, this CICR model produces a nonlinear relationship between fractional jSR Ca2+ release and jSR load. These findings agree with experimental observations in lipid bilayers and cardiac myocytes. 相似文献
11.
12.
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
13.
M. Tshipamba H. De Smedt L. Missiaen B. Himpens L. Van Den Bosch R. Borghgraef 《Journal of cellular physiology》1993,155(1):96-103
We have studied arginine vasopressin (AVP)-, thapsigargin- and inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release in renal epithelial LLC-PK1 cells. AVP-induced changes in the intracellular free calcium concentration ([Ca2+]i) were studied in indo-1 loaded single cells by confocal laser cytometry. AVP-mediated Ca2+ mobilization was also observed in the absence of extracellular Ca2+, but was completely abolished after depletion of the intracellular Ca2+ stores by 2 μM thapsigargin. Using 45Ca2+ fluxes in saponin-permeabilized cell monolayers, we have analysed how InsP3 affected the Ca2+ content of nonmitochondrial Ca2+ pools in different loading and release conditions. Less than 10% of the Ca2+ was taken up in a thapsigargin-insensitive pool when loading was performed in a medium containing 0.1 μM Ca2+. The thapsigargin-insensitive compartment amounted to 35% in the presence of 110 μM Ca2+, but Ca2+ sequestered in this pool could not be released by InsP3. The thapsigargin-sensitive Ca2+ pool, in contrast, was nearly completely InsP3 sensitive. A submaximal [InsP3], however, released only a fraction of the sequestered Ca2+. This fraction was dependent on the cytosolic as well as on the luminal [Ca2+]. The cytosolic free [Ca2+] affected the InsP3-induced Ca2+ release in a biphasic way. Maximal sensitivity toward InsP3 was found at a free cytosolic [Ca2+] between 0.1 and 0.5 μM, whereas higher cytosolic [Ca2+] decreased the InsP3 sensitivity. Other divalent cations or La3+ did not provoke similar inhibitory effects on InsP3-induced Ca2+ release. The luminal free [Ca2+] was manipulated by varying the time of incubation of Ca2+ -loaded cells in an EGTA-containing medium. Reduction of the Ca2+ content to one-third of its initial value resulted in a fivefold decrease in the InsP3 sensitivity of the Ca2+ release. © 1993 Wiley-Liss, Inc. 相似文献
14.
K. Narita T. Akita M. Osanai T. Shirasaki H. Kijima K. Kuba 《The Journal of general physiology》1998,112(5):593-609
The extent to which Ca2+-induced Ca2+ release (CICR) affects transmitter release is unknown. Continuous nerve stimulation (20–50 Hz) caused slow transient increases in miniature end-plate potential (MEPP) frequency (MEPP-hump) and intracellular free Ca2+ ([Ca2+]i) in presynaptic terminals (Ca2+-hump) in frog skeletal muscles over a period of minutes in a low Ca2+, high Mg2+ solution. Mn2+ quenched Indo-1 and Fura-2 fluorescence, thus indicating that stimulation was accompanied by opening of voltage-dependent Ca2+ channels. MEPP-hump depended on extracellular Ca2+ (0.05–0.2 mM) and stimulation frequency. Both the Ca2+- and MEPP-humps were blocked by 8-(N,N-diethylamino)octyl3,4,5-trimethoxybenzoate hydrochloride (TMB-8), ryanodine, and thapsigargin, but enhanced by CN−. Thus, Ca2+-hump is generated by the activation of CICR via ryanodine receptors by Ca2+ entry, producing MEPP-hump. A short interruption of tetanus (<1 min) during MEPP-hump quickly reduced MEPP frequency to a level attained under the effect of TMB-8 or thapsigargin, while resuming tetanus swiftly raised MEPP frequency to the previous or higher level. Thus, the steady/equilibrium condition balancing CICR and Ca2+ clearance occurs in nerve terminals with slow changes toward a greater activation of CICR (priming) during the rising phase of MEPP-hump and toward a smaller activation during the decay phase. A short pause applied after the end of MEPP- or Ca2+-hump affected little MEPP frequency or [Ca2+]i, but caused a quick increase (faster than MEPP- or Ca2+-hump) after the pause, whose magnitude increased with an increase in pause duration (<1 min), suggesting that Ca2+ entry-dependent inactivation, but not depriming process, explains the decay of the humps. The depriming process was seen by giving a much longer pause (>1 min). Thus, ryanodine receptors in frog motor nerve terminals are endowed with Ca2+ entry-dependent slow priming and fast inactivation mechanisms, as well as Ca2+ entry-dependent activation, and involved in asynchronous exocytosis. Physiological significance of CICR in presynaptic terminals was discussed. 相似文献
15.
Alloisio S Cervetto C Passalacqua M Barbieri R Maura G Nobile M Marcoli M 《FEBS letters》2008,582(28):3948-3953
The presynaptic P2X7 receptor (P2X7R) plays an important role in the modulation of transmitter release. We recently demonstrated that, in nerve terminals of the adult rat cerebral cortex, P2X7R activation induced Ca2+-dependent vesicular glutamate release and significant Ca2+-independent glutamate efflux through the P2X7R itself. In the present study, we investigated the effect of the new selective P2X7R competitive antagonist 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (A-438079) on cerebrocortical terminal intracellular calcium (intrasynaptosomal calcium concentration;[Ca2+]i signals and glutamate release, and evaluated whether P2X7R immunoreactivity was consistent with these functional tests. A-438079 inhibited functional responses. P2X7R immunoreactivity was found in about 45% of cerebrocortical terminals, including glutamatergic and non-glutamatergic terminals. This percentage was similar to that of synaptosomes showing P2X7R-mediated [Ca2+]i signals. These findings provide compelling evidence of functional presynaptic P2X7R in cortical nerve terminals. 相似文献
16.
Adenosine Triphosphate and the Late Steps in Calcium-dependent Exocytosis at a Ribbon Synapse
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Ruth Heidelberger 《The Journal of general physiology》1998,111(2):225-241
The ATP dependence of the kinetics of Ca2+-dependent exocytosis after flash photolysis of caged Ca2+ was studied by capacitance measurements with submillisecond resolution in single synaptic terminals of retinal bipolar neurons. After control experiments verified that this combination of techniques is valid for the study of exocytosis in synaptic terminals, a comparison was made between the Ca2+ dependence of the rate of exocytosis in synaptic terminals internally dialyzed with MgATP, MgATP-γ-S, or no added Mg2+ or nucleotide. The Ca2+ threshold for release, the maximum rate of release, and the overall relationship between the rate of synaptic vesicle fusion and [Ca2+]i were found to be independent of MgATP. A decrease in the average rate at near-threshold [Ca2+]i was observed in terminals with MgATP-γ-S, but due to the small sample size is of unclear significance. The Ca2+ dependence of the delay between the elevation of [Ca2+]i and the beginning of the capacitance rise was also found to be independent of MgATP. In contrast, MgATP had a marked effect on the ability of terminals to respond to multiple stimuli. Terminals with MgATP typically exhibited a capacitance increase to a second stimulus that was >70% of the amplitude of the first response and to a third stimulus with a response amplitude that was >50% of the first, whereas terminals without MgATP responded to a second stimulus with a response <35% of the first and rarely responded to a third flash. These results suggest a major role for MgATP in preparing synaptic vesicles for fusion, but indicate that cytosolic MgATP may have little role in events downstream of calcium entry, provided that [Ca2+]i near release sites is elevated above ≈30 μM. 相似文献
17.
Carolina Ladera María Del Carmen Godino María José Cabañero Magdalena Torres Masahiko Watanabe Rafael Luján José Sánchez‐Prieto 《Journal of neurochemistry》2008,107(6):1506-1517
Neuronal G protein‐gated inwardly rectifying potassium (GIRK) channels mediate the slow inhibitory effects of many neurotransmitters post‐synaptically. However, no evidence exists that supports that GIRK channels play any role in the inhibition of glutamate release by GABAB receptors. In this study, we show for the first time that GABAB receptors operate through two mechanisms in nerve terminals from the cerebral cortex. As shown previously, GABAB receptors reduces glutamate release and the Ca2+ influx mediated by N‐type Ca2+ channels in a mode insensitive to the GIRK channel blocker tertiapin‐Q and consistent with direct inhibition of this voltage‐gated Ca2+ channel. However, by means of weak stimulation protocols, we reveal that GABAB receptors also reduce glutamate release mediated by P/Q‐type Ca2+ channels, and that these responses are reversed by the GIRK channel blocker tertiapin‐Q. Consistent with the functional interaction between GABAB receptors and GIRK channels at nerve terminals we demonstrate by immunogold electron immunohistochemistry that pre‐synaptic boutons of asymmetric synapses co‐express GABAB receptors and GIRK channels, thus suggesting that the functional interaction of these two proteins, found at the post‐synaptic level, also occurs at glutamatergic nerve terminals. 相似文献
18.
Michael D. Salmon 《Biochemical and biophysical research communications》2009,384(1):87-92
The aim of this study was to investigate the possible relationship between NADPH oxidase activity and changes in cytosolic Ca2+ in response to different agonists. Treatment of neutrophils with leukotriene B4 (LTB4) demonstrated characteristic changes to cytoslic Ca2+ yielding an EC50 of 4 nM. The pA2 values for the specific LTB4 receptor (BLT) antagonists, U-75302 and LY-255283 were 6.32 and 6.38, respectively. Similarly, neutrophils treated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and platelet activating factor (PAF) exhibited changes in cytoslic Ca2+ in a dose dependant manner with pD2 values of 9.0 and 9.9, respectively. The phorbol ester PMA prevented elevations in cytosolic Ca2+ in response to LTB4, FMLP and PAF with IC50 values of 5.88, 1.44 and 5.71 nM, respectively. In addition, potent NADPH oxidase inhibitors apocynin and diphenyleneiodonium (DPI) inhibited FMLP mediated cytosolic Ca2+ release. These results demonstrate that inhibition of the NADPH oxidase suppresses cytosolic Ca2+ release in FMLP activated human neutrophils. 相似文献
19.
E.È. Saftenku 《Journal of theoretical biology》2009,257(2):228-244
The spatiotemporal distribution of cytosolic free calcium concentration ([Ca2+]i) in cerebellar granule cells (GrCs) is thought to be critical in defining the occurrence and direction of long-term changes in synaptic strength at cerebellar mossy fiber-GrC synapses. Despite this, the mechanisms responsible for shaping Ca2+ transients in GrCs are not well understood. To investigate the interplay between Ca2+ entry, extrusion, buffering and dendritic morphology in shaping Ca2+ elevations in GrCs, we developed a model of Ca2+ regulation in these cells and examined the requirements for reproducing fluorescence responses to depolarization and synaptic stimulation previously described in the literature. Two conclusions can be drawn from our simulation results. First, a significant progressive decrease in the amplitudes of depolarization-evoked fluorescence transients from the dendritic endings (digits) toward the soma of GrCs, can be reproduced in the model only if the density of Ca2+ channels is considerably higher or the concentration of endogenous buffers is much lower in the digits than in the parent dendrites. In contrast, heterogeneities in the distribution of Ca2+ pumps or in cytosolic fractional volume cannot account for the formation of [Ca2+]i gradients in GrCs. Second, much lower amplitudes of fluorescence transients induced by depolarization and synaptic stimulation than expected from typical measurements of Ca2+ and NMDA receptor-mediated currents can be reconciled with a pronounced slowing of the decay of fluorescence responses in the digits of GrCs after introducing a high-affinity Ca2+ indicator if a high-capacity immobile Ca2+ buffer (presumably plasma membrane-associated) is suggested to be present in the soma and apical part of digits. Mitochondria also are likely to modulate synaptically evoked Ca2+ responses in GrCs. The alternative hypotheses are thoroughly discussed and research avenues for their testing in future experiments are proposed. 相似文献
20.
Of the many ongoing controversies regarding the workings of the sarcoplasmic reticulum (SR) in cardiac myocytes, two unresolved and interconnected topics are 1), mechanisms of calcium (Ca2+) wave propagation, and 2), speed of Ca2+ diffusion within the SR. Ca2+ waves are initiated when a spontaneous local SR Ca2+ release event triggers additional release from neighboring clusters of SR release channels (ryanodine receptors (RyRs)). A lack of consensus regarding the effective Ca2+ diffusion constant in the SR (DCa,SR) severely complicates our understanding of whether dynamic local changes in SR [Ca2+] can influence wave propagation. To address this problem, we have implemented a computational model of cytosolic and SR [Ca2+] during Ca2+ waves. Simulations have investigated how dynamic local changes in SR [Ca2+] are influenced by 1), DCa,SR; 2), the distance between RyR clusters; 3), partial inhibition or stimulation of SR Ca2+ pumps; 4), SR Ca2+ pump dependence on cytosolic [Ca2+]; and 5), the rate of transfer between network and junctional SR. Of these factors, DCa,SR is the primary determinant of how release from one RyR cluster alters SR [Ca2+] in nearby regions. Specifically, our results show that local increases in SR [Ca2+] ahead of the wave can potentially facilitate Ca2+ wave propagation, but only if SR diffusion is relatively slow. These simulations help to delineate what changes in [Ca2+] are possible during SR Ca2+release, and they broaden our understanding of the regulatory role played by dynamic changes in [Ca2+]SR. 相似文献