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1.
Intracellular Ca2+ ([Ca2+]i) and membrane properties were measured in fura-2 dialysed dorsal vagal neurons (DVN) spontaneously active at a frequency of 0.5–5 Hz. [Ca2+]i increased by about 30 nm upon rising spike frequency by more than 200% due to 20–50 pA current pulses or 10 μm serotonin. It fell by 30 nm upon block of spiking by current-injection, tetrodotoxin or Ni2+ and also during hyperpolarization due to γ-aminobutyric acid or opening of adenosine triphosphate (ATP) -sensitive K+ (KATP) channels with diazoxide. KATP channel-mediated hyperpolarizations during anoxia or cyanide produced an initial [Ca2+]i decrease which reversed into a secondary Ca2+ rise by less than 100 nm . Similar moderate rises of [Ca2+]i were observed during block of aerobic metabolism under voltage-clamp as well as in intact cells, loaded with fura-2 AM. The magnitude of the metabolism-related [Ca2+]i transients did not correlate with the amplitude of the KATP channel-mediated outward current. [Ca2+]i did not change during diazoxide-induced or spontaneous activation of KATP outward current observed in 10% of cells after establishing whole-cell recording. Increasing [Ca2+]i with cyclopiazonic acid did not activate KATP channels. [Ca2+]i was not affected upon block of outward current with sulphonylureas, but these KATP channel blockers were effective to reverse inhibition of spike discharge and, thus, the initial [Ca2+]i fall upon spontaneous or diazoxide-, anoxia- and cyanide-induced KATP channel activation. A sulphonylurea-sensitive hyperpolarization and [Ca2+]i fall was also revealed in the early phase of iodoacetate-induced metabolic arrest, whereas after about 20 min, occurrence of a progressive depolarization led to an irreversible rise of [Ca2+]i to more than 1 μm . The results indicate that KATP channel activity in DVN is not affected by physiological changes of intracellular Ca2+ and the lack of a major perturbance of Ca2+ homeostasis contributes to their high tolerance to anoxia. 相似文献
2.
3.
P. B. Simpson R. A. J. Challiss S. R. Nahorski 《The European journal of neuroscience》1995,7(5):831-840
In this study the rate of Mn2+ quench of fura-2 fluorescence evoked by glutamatergic and cholinergic agonists, depolarization and Ca2+ store modulators was measured in cultured cerebellar granule cells, in order to study their effects on Ca2+ entry in isolation from effects on Ca2+ store release. The rate of fluorescence quench by 0.1 mM Mn2+ was markedly increased by 25 mM K+- evoked depolarization or by 200 μM N-methyl-D-aspartate (NMDA), with a significantly greater increase occurring during the rapid-onset peak phase compared to the plateau phase of the K+- or NMDA-evoked [Ca2+]i response. The stimulatory effect of NMDA on Mn2+ quench was abolished by dizocilpine (10 μM), but nitrendipine (2 μM), while decreasing the rate of basal quench, did not affect NMDA-stimulated Mn2+ entry. This suggests that nitrendipine may not act on NMDA channels in granule cells, at least under these conditions, and that voltage-operated Ca2+ channels are involved in control quench whereas the NMDA-evoked quench is dependent on entry through the receptor channel. The t1/2 of quench was unaffected by α-amino-hydroxyisoxazole propionic acid (200 μM) and carbamyl choline (1 mM). Neither thapsigargin (10 μM) nor dantrolene (30 μM) significantly affected the rate of quench under control or NMDA- or K+-stimulated conditions, which confirms that the previously reported inhibitory effects on [Ca2+]i elevations evoked by these agents are due to actions on Ca2+ stores. However, thapsigargin elevated [Ca2+Ii in the presence of normal [Ca2+]i, but not in nominally Ca2+-free medium, indicating that it evokes Ca2+ entry in cerebellar granule cells, probably subsequent to store depletion, which appears to be either too small to be detected by Mn2+ quench or to occur via Mn2+-impermeant channels. 相似文献
4.
We have examined how NGF-dependent rat sympathetic neurons maintain Ca2+ homeostasis when challenged with high K+ or 8-(4-chlorophenylthio)cyclic AMP (CPTcAMP), two survival factors. In the presence of NGF, high K+ (55 mM) caused a stable, 65% reduction in the density of cell soma voltage-sensitive Ca2+ channels within 2 days. Although resting [Ca2+]i was elevated by 1.6-fold, this was 50% less than the rise in [Ca2+]i measured before down-regulation occurred, suggesting that down-regulation may help prevent the toxic effects of persistently elevated [Ca2+]i. Inhibition of protein synthesis by cycloheximide blocked recovery from down-regulation. Moreover, treatment with cycloheximide or actinomycin-D caused a 2-fold rise in the peak Ca2+ current, suggesting that voltage-sensitive Ca2+ channel activity may be tonically attenuated during normal growth. In the absence of NGF, neurons survived for several days in high K+ medium with no significant rise in resting [Ca2+]i, although neurites did not grow. Neither Ca2+ channel density nor resting [Ca2+]i were altered in neurons surviving with CPTcAMP. Moreover, CPTcAMP lowered the dependence on extracellular Ca2+. However, the dihydropyridine antagonist nitrendipine blocked both high K+- and CPTcAMP-dependent survival although it had no effect in the presence of NGF. Thus, in the absence of NGF, sympathetic neurons do not require elevation of [Ca2+]i above resting levels to survive with either high K+ or CPTcAMP, but dihydropyridine-sensitive Ca2+ channel activity may be essential for their survival promoting actions. 相似文献
5.
We measured neurotransmitter release and motor nerve terminal currents in mouse phrenic nerve-diaphragm and triangularis sterni preparations, to evaluate the role of Ca2+-channel subtypes in regulating transmitter release. Saturated concentrations of either ωagatoxin IVA [ω-Aga-IVA (0.3 μM), a blocker of P-type Ca2+channels] or ω-conotoxin MVIIC [ω-CTx-MVIIC (2 μM), a P-and Q-type Ca2+-channel blocker], inhibited nerve-evoked muscle contractions and the amplitude of endplate potentials respectively. In contrast, combined treatment with nifedipine (50 μM, a blocker of L-type Ca2+ channels) plus ω-conotoxin GVIA [ω-CTx-GVIA (2 μM), a blocker of N-type Ca2+ channels] did not elicit inhibitory effects on nerve-evoked muscle contractions, endplate potentials or nerve terminal waveforms. Because of the non-linear relationship between endplate potentials and Ca2+ signals, a small decrease in presynaptic Ca2+ entry can significantly reduce the amplitude of the endplate potential. Thus, we applied 3, 4-diaminopyridine (3, 4-DAP, a k+-channel blocker) or high Ca2+(10 mM) to accelerate and amplify the endplate potentials and Ca2+ currents. The endplate potentials amplified by 3, 4-DAP or by high Ca2+ correspondingly proved to be quite resistant to both ω-Aga-IVA and ω-CTx-MVIIC; ωAga-IVA exerted only a partial inhibitory effect on endplate potentials, and the ω-Aga-IVA-resistant component was further inhibited by ω-CTx-MVIIC. The component that was resistant to the two toxins could be completely blocked by the non-selective Ca2+ channel blocker Cd2+ (300 μM). A combination of the two toxins had no significant effects on either spontaneous transmitter release or postsynaptic resting membrane potentials of the diaphragm preparation and the Na+ and K+ waveforms of the triangularis sterni preparations. This finding suggests a preferential inhibitory effect at a presynaptic site. Measuring the Ca2+ currents in the triangularis sterni also revealed partial inhibition by ω-CTx-MVIIC with further incomplete inhibition by ω-Aga-IVA. Cd2+ (300 μM) abolished the toxin-resistant component of the Ca2+ current. In contrast, a combination of nifedipine (50 μM) with ω-CTx-GVIA (2 μM) was without inhibitory effect. We conclude that multiple types of Ca2+channels, i.e. ω-Aga-IVA-sensitive, ω-CTx-MVIIC-sensitive and toxin-resistant Ca2+ channels, coexist in mouse motor nerve terminals. 相似文献
6.
Inhibition of N-type Calcium Channels: The Only Mechanism by which Presynaptic α2 -Autoreceptors Control Sympathetic Transmitter Release 总被引:3,自引:0,他引:3
α2-Adrenoceptors are known to inhibit voltage-dependent Ca2+ channels located at neuronal cell bodies; the present study investigated whether this or alternative mechanisms, possibly downstream of Ca2+ entry, underlie the presynaptic α2-adrenergic modulation of transmitter release from chick sympathetic neurons. Using chick sympathetic neurons, overflow of previously incorporated [3H]noradrenaline was elicited in the presence of extracellular Ca2+ by electrical pulses, 25 mM K+ or 10μM nicotine, or by adding Ca2+ to otherwise Ca2+-free medium when cells had been made permeable by the calcium ionophore A23187 or by α-latrotoxin. Pretreatment of neurons with the N-type Ca2+ channel blocker ω-conotoxin GVIA and application of the α2-adrenergic agonist UK 14304 reduced the overflow elicited by electrical pulses, K+ or nicotine, but not the overflow caused by Ca2+ after permeabilization with α-latrotoxin or A23187. In contrast, the L-type Ca2+ channel blocker nitrendipine reduced the overflow due to K+ and nicotine, but not the overflow following electrical stimulation or α-latrotoxin- and A23187-permeabilization. The inhibition of electrically evoked overflow by UK 14304 persisted in the presence of nitrendipine and the L-type Ca2+ channel agonist BayK 8644, which per se enhanced overflow. In ω-conotoxin GVIA-treated cultures, electrically evoked overflow was also enhanced by BayK 8644 and almost reached the value obtained in untreated neurons. However, UK 14304 lost its effect under these conditions. Whole-cell recordings of voltage-activated Ca2+ currents corroborated these results: UK 14304 inhibited Ca2+ currents by 33%, nitrendipine caused a 7% reduction, and BayK 8644 increased the currents by 30%. Moreover, the dihydropyridines failed to abolish the inhibition by UK 14304, but pretreatment with ω-conotoxin GVIA, which reduced mean amplitude from 0.95 to 0.23 nA, entirely prevented α2-adrenergic effects. Our results indicate that the α2-autoreceptor-mediated modulation of noradrenaline release from chick sympathetic neurons relies exclusively on the inhibition of ω-conotoxin GVIA-sensitive N-type Ca2+ channels. Mechanisms downstream of these channels and voltage-sensitive Ca2+ channels other than N-type appear not to be important. 相似文献
7.
Virginia Soares Lemos Bernard Bucher Steyner F. Côrtes Kenneth Takeda 《The European journal of neuroscience》1997,9(6):1144-1152
The effects of neuropeptide Y on the intracellular level of Ca2+ ([Ca2+]i) were studied in cultured rat adrenal chromaffin cells loaded with fura-2. A proportion (16%) of cells exhibited spontaneous rhythmic [Ca2+]i oscillations. In silent cells, oscillations could be induced by forskolin and 1,9–dideoxyforskolin. This action of forskolin was not modified by H-89, an inhibitor of protein kinase A. Spontaneous [Ca2+i fluctuations and [Ca2+]i fluctuations induced by forskolin- and 1,9-dideoxyforskolin were inhibited by neuropeptide Y. Increases in [Ca2+]i induced by 10 and 20 mM KCI but not by 50 mM KCI were diminished by neuropeptide Y. However, neuropeptide Y had no effect on [Ca2+]i increases evoked by (-)BAY K8644 and the inhibitory effect of neuropeptide Y on responses induced by 20 mM KCI was not modified by o-conotoxin GVIA, consistent with neither L- nor N-type voltage-sensitive Ca2+ channels being affected by neuropeptide Y. Rises in [Ca2+]i provoked by 10 mM tetraethylammonium were not decreased by neuropeptide Y, suggesting that K+ channel blockade reduces the effect of neuropeptide Y. However, [Ca2+]i transients induced by 1 mM tetraethylammonium and charybdotoxin were still inhibited by neuropeptide Y, as were those to 20 mM KCI in the presence of apamin. The actions of neuropeptide Y on [Ca2+]i transients provoked by 20 and 50 mM KCI, 1 mM tetraethylammonium, (-)BAY K8644 and charybdotoxin were mimicked by 8–bromo-cGMP. In contrast, 8–bromo-CAMP did not modify responses to 20 mM KCI or 1 mM tetraethylammonium. The inhibitory effects of neuropeptide Y and 8–bromo-cGMP on increases in [Ca2+]i induced by 1 mM tetraethylammonium were abolished by the Rp-8–pCPT-cGMPS, an inhibitor of protein kinase G, but not by H-89. A rapid, transient increase in cGMP level was found in rat adrenal medullary tissues stimulated with 1 μM neuropeptide Y. Rises in [Ca2+]i produced by DMPP, a nicotinic agonist, but not by muscarine, were decreased by neuropeptide Y. Our data suggest that neuropeptide Y activates a K+ conductance via a protein kinase G-dependent pathway, thereby opposing the depolarizing action of K+ channel blocking agents and the associated rise in [Ca2+]i. 相似文献
8.
Peter S. Freestone Kenny K. H. Chung Ezia Guatteo Nicola B. Mercuri Louise F.B. Nicholson Janusz Lipski 《The European journal of neuroscience》2009,30(10):1849-1859
Rotenone is a toxin used to generate animal models of Parkinson’s disease; however, the mechanisms of toxicity in substantia nigra pars compacta (SNc) neurons have not been well characterized. We have investigated rotenone (0.05–1 μm ) effects on SNc neurons in acute rat midbrain slices, using whole‐cell patch‐clamp recording combined with microfluorometry. Rotenone evoked a tolbutamide‐sensitive outward current (94 ± 15 pA) associated with increases in intracellular [Ca2+] ([Ca2+]i) (73.8 ± 7.7 nm ) and intracellular [Na+] (3.1 ± 0.6 mm ) (all with 1 μm ). The outward current was not affected by a high ATP level (10 mm ) in the patch pipette but was decreased by Trolox. The [Ca2+]i rise was abolished by removing extracellular Ca2+, and attenuated by Trolox and a transient receptor potential M2 (TRPM2) channel blocker, N‐(p‐amylcinnamoyl) anthranilic acid. Other effects included mitochondrial depolarization (rhodamine‐123) and increased mitochondrial reactive oxygen species (ROS) production (MitoSox), which was also abolished by Trolox. A low concentration of rotenone (5 nm ) that, by itself, did not evoke a [Ca2+]i rise resulted in a large (46.6 ± 25.3 nm ) Ca2+ response when baseline [Ca2+]i was increased by a ‘priming’ protocol that activated voltage‐gated Ca2+ channels. There was also a positive correlation between ‘naturally’ occurring variations in baseline [Ca2+]i and the rotenone‐induced [Ca2+]i rise. This correlation was not seen in non‐dopaminergic neurons of the substantia nigra pars reticulata (SNr). Our results show that mitochondrial ROS production is a key element in the effect of rotenone on ATP‐gated K+ channels and TRPM2‐like channels in SNc neurons, and demonstrate, in these neurons (but not in the SNr), a large potentiation of rotenone‐induced [Ca2+]i rise by a small increase in baseline [Ca2+]i. 相似文献
9.
Noriko Tamura Kunihiko Yokotani Yasunobu Okuma Miyuki Okada Hisayuki Ueno Yoshitsugu Osumi 《Brain research》1995,676(2):363
We examined the properties of voltage-gated calcium channels mediating endogenous dopamine (DA) and acetylcholine (ACh) release in the isolated rat retina. Application of 30 mM KCl elicited the release of DA and ACh, and these releases were abolished in Ca2+-free medium. The high K+-evoked DA release was largely blocked by both of ω-agatoxin IVA and ω-conotoxin MVIIC, P- and Q-type calcium channel antagonists, and partly blocked by isradipine, an L-type calcium channel antagonist, and ω-conotoxin GVIA, an N-type calcium channel antagonist, ω-Agatoxin IVA at a small dose, sufficient to block P-type channels alone, was however without effect. On the other hand, the high K+-evoked ACh release was partly blocked by ω-agatoxin IVA and ω-conotoxin MVIIC, but was resistant to isradipine and ω-conotoxin GVIA. Flunarizine, a non-selective T-type calcium channel antagonist, did not inhibit the release of DA and ACh. Cd2+ markedly blocked the release of both DA and ACh, Co2+ and Ni2+ slightly blocked the release of DA, and the release of ACh was not blocked by these two divalent cations. These results suggest that the high K+-evoked release of retinal DA is largely mediated by ω-agatoxin IVA and ω-conotoxin MVIIC sensitive calcium channels (probably Q-type channels), while the release of retinal ACh is largely mediated by as yet uncharacterized Cd2+ sensitive calcium channels. The properties of voltage-gated calcium channels involved in the release of ACh in the rat retina differ from those of DA. 相似文献
10.
《Brain research》1997,769(2):1194
We have found earlier that the depolarization-induced release of acetylcholine from the brain could be inhibited by tacrine (tetrahydroaminoacridine) but the mechanism of this action of tacrine was not clarified (S. Tu?ek, V. Dole?al, J. Neurochem. 56 (1991) 1216). We have now investigated whether tacrine has an effect on the changes in the intracellular concentration of calcium ions ([Ca2+]i) induced by depolarization. Experiments were performed on the cholinergic SN56 neuronal cell line with Fura-2 fluorescence technique of calcium imaging. The depolarization by 71 mmol/l K+ evoked minimum increases of [Ca2+]i up to day 5 in culture. Then the response gradually increased and reached a plateau after 7 days in culture. A similar time course was observed for acetylcholinesterase activity. The effect of K+ ions was concentration-dependent and the concentration of 71 mmol/l K+ evoked maximum [Ca2+]i responses. The increases of [Ca2+]i did not occur in the absence of extracellular calcium. They were mediated by high voltage-activated calcium channels of the L-type and the N-type. Nifedipine (2 μmol/l; L-type calcium channel blocker) and ω-conotoxin GVIA (100 nmol/l; N-type calcium channel blocker) diminished the response to 71 mmol/l K+ by 53% and 39%, respectively, and their effects were additive (decrease to 8% of controls). Non-selective inorganic blocker of voltage-activated calcium channels LaCl3 (0.1 mmol/l) decreased the response by 83%. Tacrine attenuated the [Ca2+]i response in a concentration-dependent manner. At a concentration of 10 μmol/l it inhibited the [Ca2+]i response by 55% and its inhibitory effect was additive with that of ω-conotoxin GVIA but not with that of nifedipine. An equimolar concentration of paraoxon, an irreversible inhibitor of cholinesterases, had no influence on [Ca2+]i response. Tacrine exhibited the same inhibitory effect when paraoxon was present. In conclusion, our data indicate that high-voltage-activated calcium channels of the L-type and the N-type are both present in the SN56 cells but that they are fully expressed only after 6–7 days in culture. Tacrine attenuates the influx of calcium by inhibiting the L-type calcium channels. This inhibitory effect is not a consequence of the anticholinesterase activity of tacrine. The finding that low micromolar concentrations of tacrine may interfere with calcium-dependent events is likely to be of importance for the evaluation of the therapeutic potential of the drug. 相似文献
11.
In leech Retzius neurones the inhibition of the Na+–K+ pump by ouabain causes an increase in the cytosolic free calcium concentration ([Ca2+]i). To elucidate the mechanism of this increase we investigated the changes in [Ca2+]i (measured by Fura-2) and in membrane potential that were induced by inhibiting the Na+–K+ pump in bathing solutions of different ionic composition. The results show that Na+–K+ pump inhibition induced a [Ca2+]i increase only if the cells depolarized sufficiently in the presence of extracellular Ca2+. Specifically, the relationship between [Ca2+]i and the membrane potential upon Na+–K+ pump inhibition closely matched the corresponding relationship upon activation of the voltage-dependent Ca2+ channels by raising the extracellular K+ concentration. It is concluded that the [Ca2+]i increase caused by inhibiting the Na+–K+ pump in leech Retzius neurones is exclusively due to Ca2+ influx through voltage-dependent Ca2+ channels. 相似文献
12.
We determined the relationships between the intracellular free Ca2+ concentration ([Ca2+]i) and the membrane potential (Em) of six different neurones in the leech central nervous system: Retzius, 50 (Leydig), AP, AE, P, and N neurones. The [Ca2+]i was monitored by using iontophoretically injected fura-2. The membrane depolarization evoked by raising the extracellular K+ concentration ([K+]o) up to 89 mM caused a persistent increase in [Ca2+]i, which was abolished in Ca2+-free solution indicating that it was due to Ca2+ influx. The threshold membrane potential that must be reached in the different types of neurones to induce a [Ca2+]i increase ranged between −40 and −25 mV. The different threshold potentials as well as differences in the relationships between [Ca2+]i and Em were partly due to the cell-specific generation of action potentials. In Na+-free solution, the action potentials were suppressed and the [Ca2+]i/Em relationships were similar. The K+-induced [Ca2+]i increase was inhibited by the polyvalent cations Co2+, Ni2+, Mn2+, Cd2+, and La3+, as well as by the cyclic alcohol menthol. Neither the polyvalent cations nor menthol had a significant effect on the K+-induced membrane depolarization. Our results suggest that different leech neurones possess voltage-dependent Ca2+ channels with similar properties. 相似文献
13.
Opioid agonists induced an increase in the intracellular free calcium concentration ([Ca2+]i) or an inhibition of K+ (25 mM)-stimulated increase in [Ca2+]i in different subsets of mouse dorsal root ganglion (DRG) neurons. The total neuronal population was grouped into three classes according to somatic diameter and defined as small (<16 μm), intermediate (16–25 μm), or large (>25 μm) neurons. Substance P-like immunoreactivity was detected mainly in the small and intermediate neurons. The δ, κ, and μ opioid receptor agonists [D-Ser2, Leu5]enkephalin-Thr (DSLET), U69593, and [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAMGO) each induced a transient increase in [Ca2+]i in a small fraction (<30%) of neurons. The increases in [Ca2+]i were blocked by the opioid antagonist naloxone. The dihydropyridine-sensitive calcium channel blocker nifedipine also blocked the increase in [Ca2+]i induced by 1 μM DSLET. The rank order of potency (percentage of cells responding to each opioid agonist) was DSLET > U69593 > DAMGO. The opioid-induced increase in [Ca2+]i was observed mainly in large neurons, with a low incidence in small and intermediate neurons. Opioid agonists also caused inhibition of K+-stimulated increases in [Ca2+]i, which were blocked by naloxone (1 μM). Inhibition of the K+-stimulated increase by 1 μM DSLET or U69593 was greater in small and intermediate neurons than in large neurons. © 1996 Wiley-Liss, Inc. 相似文献
14.
Valeria Magnelli Pietro Baldelli & Emilio Carbone 《The European journal of neuroscience》1998,10(5):1810-1825
Ca2+ channels diversity of cultured rat embryo motoneurons was investigated with whole-cell current recordings. In 5–20 mm Ba2+, the whole-cell currents were separated in low- (LVA) and high-voltage-activated (HVA) current. The LVA current was evident since the first day in culture, while the HVA component was small and increased with time. Recordings after 4 days revealed ≈ 20% L-, ≈ 45% N- and ≈ 35% P- and R-type currents. P-type currents were revealed only in 40% of motoneurons, in which 20–200 nm ω-Aga-IVA caused 20% irreversible block of total current. The remaining 60% of cells were insensitive even to higher doses of the toxin (500 nm in 5 mm Ba2+), suggesting weak expression and heterogeneous distribution of P-type channels compensated by high densities of HVA Ca2+ channels resistant to all the antagonists (R-type). A significant residual current could also be resolved after prolonged applications of 5 μm ω-CTx-MVIIC, which allowed separation of N- and P-type currents by the distinct onset of toxin block. The antagonists-resistant current reveals biophysical characteristics typical of HVA channels, but distinct from the α1E channel. The current activates around ?20 mV in 20 mm Ba2+; inactivates slowly and independently of Ca2+; is blocked by low [Cd2+] and high [Ni2+]; and is larger with Ba2+ than Ca2+. The uncovered R-type calcium current can account for part of the presynaptic Ca2+ current controlling neurotransmitter release at the mammalian neuromuscular junction whose activity is resistant to DHP- and ω-CTx-GVIA, and displays anomalous sensitivity to ω-Aga-IVA and ω-CTx-MVIIC ( 1 ) J. Physiol. (Lond.), 482, 283–290; 2 ) Eur. J. Neurosci., 9, 817–823]. 相似文献
15.
Jian Wang David B. Reichling reas Kyrozis Amy B. MacDermott 《The European journal of neuroscience》1994,6(8):1275-1280
More than 90% of dorsal horn neurons from embryonic day 15–16 rats responded to the inhibitory amino acids GABA and glycine by a transient elevation of intracellular Ca2+ concentration ([Ca2+]i) when maintained in culture for <1 week. This [Ca2+]i response has previously been shown to be due to depolarization and subsequent Ca2+ entry through voltage-gated Ca2+ channels following activation of bicuculline-sensitive GABAA receptors and strychnine-sensitive glycine receptors. Both the number of cells responding to GABA and glycine and the amplitude of the [Ca2+]i response diminished over time in culture. By 30 days in culture, none of the cells responded to GABA, muscimol or glycine by elevation of [Ca2+]i. The loss of the [Ca2+]i response was not due to a change in the abundance or the properties of voltage-gated Ca2+ channels, since over the same period of time dorsal horn neurons showed a large increase in the amplitude of the [Ca2+]i transient in response to 30 mM K+. Nor was the loss of the [Ca2+]i response due to a loss of GABA and glycine receptors. Instead, the decrease in the [Ca2+]i response over time paralleled a similar change in the electrophysiological responses. More than 90% of the neurons tested were depolarized in response to inhibitory amino acids during the first week in culture. After 30 days, all neurons tested responded to GABA and glycine with a hyperpolarization. These observations add support to the suggestion that GABA and glycine may excite dorsal horn neurons earlyin development and play a role in postmitotic differentiation. 相似文献
16.
Cytoplasmic calcium concentration was measured in neurosecretory nerve terminals (neurosecretosomes) isolated from rat neurohypophyses by fura-2 fluorescence measurements and digital video microscopy. Hormone release and cytoplasmic calcium concentration were measured during depolarizations induced by elevated extracellular potassium concentration. During prolonged depolarizations with 55 mM [K+]0, the cytoplasmic calcium concentration remained elevated as long as depolarization persisted, while secretion inactivated after the initial sharp rise. The amplitude and duration of the increase in [Ca2+]i was dependent on the degree of depolarization such that upon low levels of depolarizations (12.5 mM or 25 mM [K+]0), the calcium responses were smaller and relatively transient, and with higher levels of depolarization (55 mM [K+]0) the responses were sustained and were higher in amplitude. Responses to low levels of depolarization were less sensitive to the dihydropyridine calcium channel blocker, nimodipine, while the increase in [Ca2+]i induced by 55 mM [K+]0 became transient, and was significantly smaller. These observations suggest that these peptidergic nerve terminals possess at least two different types of voltage-gated calcium channels. Removal of extracellular sodium resulted in a significant increase in [Ca2+]i and secretion in the absence of depolarizing stimulus, suggesting that sodium-calcium exchange mechanism is operative in these nerve terminals. Although the [Ca2+]i increase was of similar magnitude to the depolarization-induced changes, the resultant secretion was 10-fold lower, but the rate of inactivation of secretion, however, was comparable. 相似文献
17.
This study investigates the alterations in the spatiotemporal distribution pattern of the free intracellular Ca2+ concentration ([Ca2+]i) during axotomy and throughout the recovery process of cultured Aplysia neurons, and correlates these alterations with changes in the neurons input resistance and trans-membrane potential. For the experiments, the axons were transected while imaging the changes in [Ca2+]i with fura-2, and monitoring the neurons’resting potential and input resistance (Ri) with an intracellular microelectrode inserted into the cell body. The alterations in the spatiotemporal distribution pattern of [Ca2+]i were essentially the same in the proximal and the distal segments, and occurred in two distinct steps: concomitantly with the rupturing of the axolemma, as evidenced by membrane depolarization and a decrease in the input resistance, [Ca2+]i increased from resting levels of 0.05 – 0.1 μM to 1 – 1.5 μM along the entire axon. This is followed by a slower process in which a [Ca2+]i front propagates at a rate of 11 – 16 μm/s from the point of transection towards the intact ends, elevating [Ca2+]i to 3 – 18 μM. Following the resealing of the cut end 0.5 – 2 min post-axotomy, [Ca2+]i recovers in a typical pattern of a retreating front, travelling from the intact ends towards the cut regions. The [Ca2+]i recovers to the control level 7 – 10 min post-axotomy. In Ca2+-free artificial sea water (2.5 mM EGTA) axotomy does not lead to increased [Ca2+]i and a membrane seal is not formed over the cut end. Upon reperfusion with normal artificial sea water, [Ca2+]i is elevated at the tip of the cut axon and a membrane seal is formed. This experiment, together with the observations that injections of Ca2+, Mg2+ and Na+ into intact axons do not induce the release of Ca2+ from intracellular stores, indicates that Ca2+ influx through voltage gated Ca2+ channels and through the cut end are the primary sources of [Ca2+]i following axotomy. However, examination of the spatiotemporal distribution pattern of [Ca2+]i following axotomy and during the recovery process indicates that diffusion is not the dominating process in shaping the [Ca2+]i gradients. Other Ca2+ regulatory mechanisms seem to be very effective in limiting these gradients, thus enabling the neuron to survive the injury. 相似文献
18.
Toshio Matsuda Kazuhiro Takuma Emi Nishiguchi Shoichi Asano Hitoshi Hashimoto Junichi Azuma Akemichi Baba 《The European journal of neuroscience》1996,8(5):951-958
In some cells, Ca2+ depletion induces an increase in intracellular Ca2+ ([Ca2+]i) after reperfusion with Ca2+-containing solution, but the mechanism for the reperfusion injury is not fully elucidated. Using an antisense strategy we studied the role of the Na+-Ca2+ exchanger in reperfusion injury in cultured rat astrocytes. When astrocytes were perfused in Ca2+-free medium for 15–60 min, a persistent increase in [Ca2+]i was observed immediately after reperfusion with Ca2+-containing medium, and the number of surviving cells decreased 3–5 days latter. The increase in [Ca2+]i was enhanced by low extracellular Na+ ([Na+]o) during reperfusion and blocked by the inhibitors of the Na+-Ca2+ exchanger amiloride and 3,4-dichlorobenzamil, but not by the Ca2+ channel antagonists nifedipine, Cd2+ and Ni2+. Treatment of astrocytes with antisense, but not sense, oligodeoxynucleotide to the Na+-Ca2+ exchanger decreased Na+–Ca2+ exchanger protein level and exchange activity. The antisense oligomer attenuated reperfusion-induced increase in [Ca2+]i and cell toxicity. The Na+-Ca2+ exchange inhibitors 3,4-dichlorobenzamil and ascorbic acid protected astrocytes from reperfusion injury partially, while the stimulators sodium nitroprusside and 8-bromo-cyclic GMP and low [Na+]o exacerbated the injury. Pretreatment of astrocytes with ouabain and monensin caused similar delayed glial cell death. These findings suggest that Ca2+ entry via the Na+–Ca2+ exchanger plays an important role in reperfusion-induced delayed glial cell death. 相似文献
19.
Emília P. Duarte Graça Baltazar Arsélio P. Carvalho 《The European journal of neuroscience》1994,6(7):1128-1135
We compared the effectiveness of Ca2+ entering by Na+/Ca2+ exchange with that of Ca2+ entering by channels produced by membrane depolarization with K+ in inducing catecholamine release from bovine adrenal chromaffin cells. The Ca2+ influx through the Na+/Ca2+ exchanger was promoted by reversing the normal inward gradient of Na+ by preincubating the cells with ouabain to increase the intracellular Na+ and then removing Na+ from the external medium. In this way we were able to increase the cytosolic free Ca2+ concentration ([Ca2+]c) by Na+/Ca2+ exchange to 325 ± 14 nM, which was similar to the rise in [Ca2+]c observed upon depolarization with 35 mM K+ of cells not treated with ouabain. After incubating the cells with ouabain, K+ depolarization raised the [Ca2+]c to 398 ± 31 nM, and the recovery of [Ca2+]c to resting levels was significantly slower. Reversal of the Na+ gradient caused an −6-fold increase in the release of noradrenaline or adrenaline, whereas K+ depolarization induced a 12-fold increase in noradrenaline release but only a 9-fold increase in adrenaline release. The ratio of noradrenaline to adrenaline release was 1.24 ± 0.23 upon reversal of the Na+/Ca2+ exchange, whereas it was 1.83 ± 0.19 for K+ depolarization. Reversal of the Na+/Ca2+ exchange appeared to be as efficient as membrane depolarization in inducing adrenaline release, in that the relation of [Ca2+]c to adrenaline release was the same in both cases. In contrast, we found that for the same average [Ca2+]c, the Ca2+ influx through voltage-gated channels was much more efficient than the Ca2+ entering through the Na+/Ca2+ exchanger in inducing noradrenaline release from chromaffin ceils. This greater effectiveness of membrane depolarization in stimulating noradrenaline release suggests that there is a pool of noradrenaline vesicles which is more accessible to Ca2+ entering through voltage-gated Ca2+ channels than to Ca2+ entering through the Na+/Ca2+ exchanger, whereas the adrenaline vesicles do not distinguish between the source of Ca2+. 相似文献
20.
Carlos B. Duarte Ildete L. Ferreira Paulo F. Santos Catarina R. Oliveira Arslio P. Carvalho 《Brain research》1993,611(1)
The effect of glutamate of [Ca2+]i and on [3H]γ-aminobutyric acid (GABA) release was studied on cultured chick embryonic retina cells. It was observed that glutamate (100 μM) increases the [Ca2+]i by Ca2+ influx through Ca2+ channels sensitive to nitrendipine, but not to ω-conotoxin GVIA (ω-Cg Tx) (50%), and by other channels insensitive to either Ca2+ channel blocker. Mobilization of Ca2+ by glutamate required the presence of external Na+, suggesting that Na+ mobilization through the ionotropic glutamate receptors is necessary for the Ca2+ channels to open. The increase in [Ca2+]i was not related to the release of [3H]GABA induced by glutamate, suggesting that the pathway for the entry of Ca2+ triggered by glutamate does not lead to exocytosis. In fact, the glutamate-induced release of [3H]GABA was significantly depressed by Cao2+, but it was dependent on Nao+, just as was observed for the [3H]GABA release induced by veratridine (50 μM). The veratridine-induced release could be fully inhibited by TTX, but this toxin had no effect on the glutamate-induced [3H]GABA release. Both veratridine- and glutamate-induced [3H]GABA release were inhibited by 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid (NNC-711), a blocker of the GABA carrier. Blockade of the NMDA and non-NMDA glutamate receptors with MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, almost completely blocked the release of [3H]GABA evoked by glutamate. Continuous depolarization with 50 mM K+ induced maximal release of [3H]GABA of about 1.5%, which is much smaller than the release evoked by glutamate under the same conditions (6.0–6.5%). Glycine (3 μM) stimulated [3H]GABA release induced by 50 mM K+, and this effect was blocked by MK-801, suggesting that the effect of K+ on [3H]GABA release was partially mediated through the NMDA receptor which probably was stimulated by glutamate released by K+ depolarization. We conclude that glutamate induces Ca2+-independent release of [3H]GABA through reversal of the GABA carrier due to Na+ entry through the NMDA and non-NMDA, TTX-insensitive, channels. Furthermore the GABA carrier seems to be inhibited by Ca2+ entering by the pathways open by glutamate. This Ca2+ does not lead to exocytosis, probably because the Ca2+ channels used are located at sites far from the active zones. 相似文献