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1.
Properties of Single Calcium-activated Potassium Channels of Large Conductance in Rat Hippocampal Neurons in Culture 总被引:3,自引:0,他引:3
Patch-clamp recordings were made on rat hippocampal neurons maintained in culture. In cell-attached and excised inside-out and outside-out patches a large single-channel current was observed. This channel had a conductance of 220 and 100 pS in 140 mM [K+]i/140 mM [K+]o and 140 mM [K+]i/3 mM [K+]o respectively. From the reversal potential the channel was highly selective for K+, the PK+/Pna+ ratio being 50/1. Channel activity was voltage-dependent, the open probability at 100 mM [Ca2+]i increasing by e-fold for a 22 mV depolarization. It was also dependent on [Ca2+]i at both resting and depolarized membrane potentials. Channel open states were best described by the sum of two exponentials with time constants that increased as the membrane potential became more positive. Channel activity was sensitive to both external (500 μM) and internal (5 mM) tetraethylammonium chloride. These data are consistent with the properties of maxi-K+ channels described in other preparations, and further suggest a role for maxi-channel activity in regulating neuronal excitability at the resting membrane potential. Channel activity was not altered by 8-chlorophenyl thio cAMP, concanavalin A, pH reduction or neuraminidase. In two of five patches lemakalim (BRL 38227) increased channel activity. Internal ruthenium red (10 μM) blocked the channel by shortening the duration of both open states. This change in channel gating was distinct from the ‘mode switching’ seen in two patches, where a channel switched spontaneously from normal activity typified by two open states to a mode where only short openings were represented. 相似文献
2.
Membrane vesicles from rat cerebellum were reconstituted into lipid bilayers. The activity of two different potassium channels was recorded: (1) a small conducting voltage dependent potassium channel insensitive to [Ca2+]i, (2) a calcium and voltage dependent potassium channel (KCa). KCa channels had a conductance of (302±15) pS (n=5) and were activated by [Ca2+]i and membrane depolarizations. They were blocked by tetraethylamonium (TEA) and charybdotoxin (CTX) but insensitive to noxiustoxin (NTX). Finally, we showed the blocking effect of Androctonus australis Hector (AaH) scorpion venom on KCa channels from rat cerebellum. 相似文献
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.
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
5.
We performed patch-clamp experiments to identify distinct K+ channels underlying the high K+ conductance and K+ uptake mechanism of the neuropile glial cell membrane on the single-channel level. In the soma membrane four different types of K+ channels were characterized, which were found to be distributed in clusters. Since no other types of K+ channels were observed, these appear to be the complete repertoire of K+ channels expressed in the soma region of this cell type. The outward rectifying 42 pS K+ channel could markedly contribute to the high K+ conductance and the maintenance of the membrane potential, since it shows the highest open probability of all channels. The channel gating occurred in bursts and patch excision decreased the open probability. The outward rectifying 74 pS K+ channel was rarely active in the cell-attached configuration; however, patch excision enhanced its open probability considerably. This type of channel may be involved in neuron-glial crosstalk, since it is activated by both depolarizations and increases in the intracellular Ca2+ concentration, which are known to be induced by neurotransmitter release following the activation of neurons. The 40 pS and 83 pS K+ channels showed inward rectifying properties, suggesting their involvement in the regulation of the extracellular K+ content. The 40 pS K+ channel could only be observed in the inside-out configuration. The 83 pS channel was activated following patch excision. At membrane potentials more negative than −60 mV, flickering events indicated voltage-dependent gating. 相似文献
6.
The existence of voltage-sensitive Ca2+ channels in type I vestibular hair cells of mammals has not been conclusively proven. Furthermore, Ca2+ channels present in type II vestibular hair cells of mammals have not been pharmacologically identified. Fura-2 fluorescence was used to estimate, in both cell types, intracellular Ca2+ concentration ([Ca2+]i) variations induced by K+ depolarization and modified by specific Ca2+ channel agonists and antagonists. At rest, [Ca2+]i was 90 ± 20 nm in both cell types. Microperifusion of high-K+ solution (50 mm ) for 1 s increased [Ca2+]i to 290 ± 50 nm in type I (n = 20) and to 440 ± 50 nm in type II cells (n = 10). In Ca2+-free medium, K+ did not alter [Ca2+]i. The specific L-type Ca2+ channel agonist, Bay K, and antagonist, nitrendipine, modified in a dose-dependent manner the K+-induced [Ca2+]i increase in both cell types with maximum effect at 2 μm and 400 nm , respectively. Ni2+, a T-type Ca2+ channel blocker, reduced K+-evoked Ca2+ responses in a dose-dependent manner. For elevated Ni2+ concentrations, the response was differently affected by Ni2+ alone, or combined to nitrendipine (500 nm ). In optimal conditions, nitrendipine and Ni2+ strongly depressed by 95% the [Ca2+]i increases. By contrast, neither ω-agatoxin IVA (1 μm ), a specific P- and Q-type blocker, nor ω-conotoxin GVIA (1 μm ), a specific N-type blocker, affected K+-evoked Ca2+i responses. These results provide the first direct evidence that L- and probably T-type channels control the K+-induced Ca2+ influx in both types of sensory cells. 相似文献
7.
A potassium-selective channel, characterized by a single channel conductance of 160 pS was demonstrated to be present in rat freshly dispersed ventromedial hypothalamic nucleus neurons. The single channel activity was shown to be dependent, using inside-out membrane patches, upon the presence of intracellular calcium ions, with maximal sensitivity between 10?6 and 10?6 M[Ca2+], and to be modulated by membrane voltage, depolarization causing an increase in open-state probability in the presence of an activating concentration of calcium. Therefore these properties place this channel into the category of a large conductance (maxi-K+) calcium-activated potassium (Ca2+-K+) channel. This channel is active in cell-attached recordings from glucoreceptive cells when depolarized by glucose or tolbutamide with openings often associated with action current repolarization. These openings were shown to be abolished in the presence of extracellular Cd2+ and La3+ ions, which block calcium channels, suggesting that extracellular calcium entry upon cell depolarization is responsible for their activation. On a few occasions, a larger conductance (250 pS) Ca2+-K+ channel was observed in inside-out membrane patches isolated from ventromedial hypothalamic nucleus neurons. In contrast to the 160 pS channel, the presence of intracellularly-applied ATP caused a concentration-dependent, reversible inhibition of its open-state probability. 相似文献
8.
It has been reported previously that the neuronal excitability persistently suppresses and the amplitude of fast afterhyperpolarization (fAHP) increases in CA1 pyramidal cells of rat hippocampus following transient forebrain ischemia. To understand the conductance mechanisms underlying these post-ischemic electrophysiological alterations, we compared differences in activities of large conductance Ca2+-activated potassium (BKCa) channels in CA1 pyramidal cells acutely dissociated from hippocampus before and after ischemia by using inside-out configuration of patch clamp techniques. (1) The unitary conductance of BKCa channels in post-ischemic neurons (295 pS) was higher than that in control neurons (245 pS) in symmetrical 140/140 mM K+ in inside-out patch; (2) the membrane depolarization for an e-fold increase in open probability (Po) showed no significant differences between two groups while the membrane potential required to produce one-half of the maximum Po was more negative after ischemia, indicating no obvious changes in channel voltage dependence; (3) the [Ca2+]i required to half activate BKCa channels was only 1 μM in post-ischemic whereas 2 μM in control neurons, indicating an increase in [Ca2+]i sensitivity after ischemia; and (4) BKCa channels had a longer open time and a shorter closed time after ischemia without significant differences in open frequency as compared to control. The present results indicate that enhanced activity of BKCa channels in CA1 pyramidal neurons after ischemia may partially contribute to the post-ischemic decrease in neuronal excitability and increase in fAHP. 相似文献
9.
Mammalian vestibular type I hair cells (VIHCs) are innervated by an afferent synaptic calyx which contains vesicles and is immunoreactive for GABA. We describe here the effects of GABA on electrophysiological properties and on cytosolic free-calcium levels ([Ca2+]i) of VIHCs isolated from guinea-pig ampullae. Whole-cell tight-seal macroscopic currents recorded from VIHCs showed that 100 μM GABA induced a decrease in the outward currents elicited by depolarizing membrane potentials. These are known to comprise potassium calcium-dependent currents. This effect was mimicked by baclofen, a GABAB agonist, and was not affected by picrotoxin, a GABAA antagonist. GABA also induced an increase in the inward current elicited at hyperpolarized membrane potentials in 50% of the tested cells. Single channel recording in cell-attached patches revealed that externally applied GABA produced a decrease and an increase in the open probability of 170 pS and 45 pS and of 15 pS channels, respectively. In imaging experiments using the dye Fura-2 to measure [Ca2+]i, the only reversible modulation of [Ca2+]i observed in response to GABA application was a decrease. These results demonstrate a modulation of calcium and potassium conductances by GABA, via GABAB receptors, in guinea-pig VIHCs. 相似文献
10.
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
11.
J. M. Simard Y. Song K. Tewari S. Dunn K. Werrbach-Perez J. R. Perez-Polo H. M. Eisenberg 《Journal of neuroscience research》1993,34(2):170-178
Ionic channels in human cortical neurons have not been studied extensively. HCN-1 and HCN-1A cells, which recently were established as continuous cultures from human cortical tissue, have been shown by histochemical and immunochemical methods to exhibit a neuronal phenotype, but expression of functional ionic channels was not demonstrated. For the present study, HCN-1 and HCN-1A cells were cultured in Dulbecco's modified Eagle's medium with 15% fetal calf serum, in some cases supplemented with 10 ng/ml nerve growth factor, 10 μM forskolin, and 1 mM dibutyryl cyclic adenosine monophosphate to promote differentiation. Cells or membrane patches were voltage clamped using conventional patch clamp techniques. In HCN-1A cells, we identified a tetrodotoxin-sensitive Na+ current, two types of Ca2+ channel current, including L-type current and a second type that in some respects resembled N-type current, and four types of K+ current, including a delayed outward rectifier that showed voltage-dependent inactivation, two types of noninactivating Ca2+-activated K+ channels with slope conductances of 146 and 23 pS (K+ iK+ o 145 mM/5 mM), and less frequently, a noninactivating, intermediate conductance channel that was not sensitive to internal Ca2+. When HCN-1A cells were examined after 3 days of exposure to differentiating agents, pronounced morphological changes were evident but no differences in ionic currents were apparent. HCN-1 cells also exhibited K+ and Ca2+ channel currents, but Na+ currents were not detected in these cells. Our data provide additional evidence indicating a functional neuronal phenotype for HCN-1A cells, and represent the most comprehensive survey to date of the variety of ionic channels expressed by human cortical neurons. © 1993 Wiley-Liss, Inc. 相似文献
12.
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
13.
《Brain research》1994,639(2):333-336
The purpose of the present study was to describe, at the single-channel level, the activity of a calcium-sensitive potassium channel in rat visceral-sensory neurons which has been suggested to be involved in sensory neuron excitability. Single-channel recordings in the inside-out configuration identified a 220 pS conductance calcium-activated potassium channel (KCa). From a −20 mV holding potential, increasing [Ca2+]i from 0.01 μM to 1.0 μM increased the open probability of this channel 92% (from 0.12 to 0.23). However, from a +20 mV holding potential, increasing [Ca2+]i from 0.01 to 1.0 μM increased the open probability by 326% (from 0.15 to 0.64). In addition, this large conductance KCa channel was blocked by TEA (1.0 μM) and charybdotoxin (40 μM) when applied to the external surface. These results are the first to characterize a large conductance KCa channel in the sensory afferent neurons of the rat nodose ganglia and should further expand the understanding to the ionic currents involved in the regulation of sensory afferent neuronal activity. 相似文献
14.
Ohkuma M Kawai F Miyachi E 《Journal of neural transmission (Vienna, Austria : 1996)》2002,109(11):1365-1371
Summary. Odorants are known to suppress voltage-gated channels not only in olfactory receptor cells but also in neurons of outside
of the olfactory system. Here we found that odorants suppress glutamate-gated channels in newt retinal neurons using the Ca2+ imaging technique. Bath application of 100 μM glutamate rose [Ca2+]i under application of the voltage-gated Ca2+ channel blocker. Thus, [Ca2+]i rises in the neurons were most likely attributable to Ca2+ influx via Ca2+-permeable glutamate-gated channels rather than voltage-gated Ca2+ channels. A similar increase of [Ca2+]i was observed by application of 100 μM NMDA and 50 μM kainate, suggesting that both NMDA and AMPA/kainate receptors were expressed
in newt retinal neurons. Application of odorants, 1 mM amyl acetate and acetophenone, reversibly reduced [Ca2+]i increased by glutamate, NMDA and kainate. This suggests that odorants can suppress not only voltage-gated channels but also
ligand-gated channels such as NMDA and AMPA/kainate receptors.
Received January 9, 2002; accepted February 25, 2002 Published online June 28, 2002
Acknowledgements We thank H. Kurajyo, A. Mamiya, and Y. Hikita for their technical assistance. This work was supported by Japan Society of
the Promotion of Science (No. 12780620 to F.K.), the SKYLARK Food Science Institute, the Fujisawa Foundation, Narishige Neuroscience
Research Foundation, the Research Foundation for Pharmaceutical Sciences, Daiko Foundation, and the Naito Foundation.
Authors' address: Dr. M. Ohkuma, Department of Physiology, School of Medicine, Fujita Health University, 1-98 Dengakugakubo,
Kutsukakechou, Toyoake, Aichi, 470-1192, Japan, e-mail: m-ohkuma@fujita-hu.ac.jp 相似文献
15.
The purpose of this work was to characterize the gap junctions between cultured glomus cells of the rat carotid body and to assess the effects of acidity and accompanying changes in [Ca2+]i on electric coupling. Dual voltage clamping of coupled glomus cells showed a mean macrojunctional conductance (Gj) of 1.16 nS±0.6 (S.E.), range 0.15–4.86 nS. At normal pHo (7.43), a steady transjunctional voltage (ΔVj=100.1±10.9 mV) showed multiple junction channel activity with a mean microconductance (gj) of 93.98±0.6 pS, range 0.3–324.5 pS. Single-channel conductances, calculated as variance/mean gj, gave a mean value of 16.7±0.2 pS, range 5.13–39.38 pS. Manual measurements of single-channel activity showed a mean gj of 22.03±0.2 pS, range 1.3–160 pS. Computer analysis of the noise spectral density distribution gave a channel mean open time of 12.7±1.5 ms, range 6.37–23.42 ms. The number of junction channels, estimated in each experiment from Gj/single-channel gj, showed a range of 7 to 258 channels (mean, 107.2). Optical measurements of [Ca2+]i gave a mean value of 80.2±4.27 nM at pHo of 7.43. Acidification of the medium with lactic acid (1 mM, pH 6.3) induced: 1) Variable changes in Gj (decreases and increases); 2) A significant decrease in mean gj (to 80.36±0.34 pS) and in single-channel conductance (gj=12.8±0.2 pS in computer analyses and 17.23±0.2 pS when measured by hand); 3) Variable changes in open times, resulting in a similar mean (12.8±1.5 ms) and 4) No change in the number of junction channels. When pHo was lowered to 6.3 [Ca2+]i did not change significantly (there were increases and decreases). However, when pHo was lowered to 4.4, [Ca2+]i increased significantly to 157.1±8.1 nM. It is concluded that saline acidification to pH 6.3 depresses the conductance of junction channels and this effect may be either a direct effect on channel proteins or synergistically enhanced by increases in [Ca2+]i. However, there are no studies correlating changes of [Ca2+]i and intercellular coupling in glomus cells. Stronger acidification (pHo 4.4), producing much larger changes in [Ca2+]i, may enhance this synergism. But, again, there are no studies correlating these effects. 相似文献
16.
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
17.
G. Wu Z.-H. Lu K. Nakamura D.C. Spray R.W. Ledeen 《Journal of neuroscience research》1996,44(3):243-254
Survival of cerebellar granule cells (CGC) in culture was significantly improved in the presence of cholera toxin B subunit (Ctx B), a ligand which binds to GM1 with specificity and high affinity. This trophic effect was linked to elevation of intracellular calcium ([Ca2+]i), and was additive to that of high K+. Survival was optimized when Ctx B was present for several days during the early culture period. 45Ca2+ and cell survival studies indicated the mechanism to involve enhanced influx of Ca2+ through L-type voltage-sensitive channels, since the trophic effect was blocked by antagonists specific for that channel type. Inhibitors of N-methyl-D-aspartate receptor/channels were without effect. During the early stage of culture Ctx B, together with 25 mM K+, caused [Ca2+]i to rise to 0.2–0.7 μM in a higher proportion of cells than 25 mM K+ alone. A significant change in the nature of GM1 modulation of Ca2+ flux occurred after 7 days in culture, at which time Ctx B ceased to elevate and instead reduced [Ca2+]i below the level attained with 25 mM K+. GM1 thus appears to serve as intrinsic inhibitor of one or more L-type Ca2+ channels during the first 7 days in vitro, and then as intrinsic activator of (possibly other) L-type channels after that period. This is the first demonstration of a modulatory role for GM1 ganglioside affecting Ca2+ homeostasis in cultured neurons of the CNS. © 1996 Wiley-Liss, Inc. 相似文献
18.
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
19.
Measurement of intracellular Ca in the bullfrog sympathetic ganglion cells using fura-2 fluorescence
The intracellular free ([Ca2+]i) of the bullfrog sympathetic ganglion cell was measured with fura-2 fluorescence under various conditions, and compared with changes in membrane potential recorded with an intracellular electrode. The [Ca2+]i was 109 nM on average under the resting condition and increased by raising the extracellular K+, stimulating repetitively the pre- or post-ganglionic nerve, or by applying acetylcholine or muscarine. Since all these procedures depolarized the cell membrane, most of the rise in [Ca2+]i could be the result of opening of voltage-dependent Ca2+ channels. However, Ca2+ entries through nicotinic acetylcholine receptor channels and the channel activated by the muscarinic acetylcholine receptor were also indicated by considering the threshold for the opening of voltage-dependent Ca2+ channels (for both entries) or a limited number of the cells showing the latter response. 相似文献
20.
Tae-Ju Park So-Young Shin Byung-Chang Suh Eun-Kyung Suh Ihn-Soon Lee Yong-Sik Kim Kyong-Tai Kim 《Synapse (New York, N.Y.)》1998,29(3):248-256
We investigated the effects of amitriptyline, a tricyclic antidepressant, on [3H]norepinephrine ([3H]NE) secretion and ion flux in bovine adrenal chromaffin cells. Amitriptyline inhibited [3H]NE secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) and 70 mM K+. The half maximal inhibitory concentration (IC50) was 2 μM and 9 μM, respectively. Amitriptyline also inhibited the elevation of cytosolic calcium ([Ca2+]i) induced by DMPP and 70 mM K+ with IC50 values of 1.1 μM and 35 μM, respectively. The rises in cytosolic sodium ([Na+]i) and [Ca2+]i induced by the Na+ channel activator veratridine were also inhibited by amitriptyline with IC50 values of 7 μM and 30 μM, respectively. These results suggest that amitriptyline at micromolar concentrations inhibits both voltage-sensitive calcium (VSCCs) and sodium channels (VSSCs). Furthermore, submicromolar concentrations of amitriptyline significantly inhibited DMPP-induced [3H]NE secretion and [Ca2+]i rise, but not veratridine- or 70 mM K+-induced responses, suggesting that nicotinic acetylcholine receptors (nAChR) as well as VSCCs and VSSCs can be targeted by amitriptyline. DMPP-induced [Na+]i rise was much more sensitive to amitriptyline than the veratridine-induced rise, suggesting that the influx of Na+ and Ca2+ through the nAChR itself is blocked by amitriptyline. Receptor binding competition analysis showed that binding of [3H]nicotine to chromaffin cells was significantly affected by amitriptyline at submicromolar concentrations. The data suggest that amitriptyline inhibits catecholamine secretion by blocking nAChR, VSSC, and VSCC. Synapse 29:248–256, 1998. © 1998 Wiley-Liss, Inc. 相似文献