l-Menthol-induced [Ca2+]i increase and impulses in cultured sensory neurons

We investigated the effects of l-menthol on cultured dorsal root ganglion (DRG) cells, instead of free nerve endings of sensory fibers. Using Fura-2 microfluorimetry, we identified a few DRG neurons that showed an increase in intracellular free Ca2+ concentration ([Ca2+]i) in response to l-menthol. They made up only 10% of the neurons activated by a high K+ solution. l-Menthol induced the [Ca2+]i increase in a dose-dependent manner, with an EC50 of 37.9 microM and a Hill coefficient of 0.97. A related compound, cyclohexanol, had no effect. When extracellular Ca2+ was removed, l-menthol did not induce the [Ca2+]i increase. Whole-cell current-clamp recordings revealed that l-menthol induced depolarization (13.2 mV, receptor potential) leading to impulses. We conclude that l-menthol induced the impulses through activation of menthol receptors in a small subset of the cultured sensory neurons.     

Mechanism of hemolysate-induced [Ca2+]i elevation in cultured fibroblasts.

Erythrocyte lysate (hemolysate) released from blood clot after subarachnoid hemorrhage is the causative agent for chronic cerebral vasospasm, a prolonged contraction of cerebral arteries. Fibroblasts, the outer layer cells of vessel wall that in contact with blood clot directly, may contribute to cerebral vasospasm. However, the effect of hemolysate on intracellular Ca2+ ([Ca2+]i) mobilization in fibroblasts has not been studied. We investigated hemolysate-induced [Ca2+]i mobilization in cultured neonatal human dermal and canine middle cerebral arterial fibroblasts by using fura-2 microfluorimetry. Hemolysate increased [Ca2+]i by releasing internal Ca2+ stores and promoting Ca2+ entry. Tyrosine kinase inhibitors partially but significantly reduced the effect of hemolysate. The major components of hemolysate, oxyhemoglobin (OxyHb) and adenosine triphosphate (ATP) failed to mimic the effect of hemolysate. In cultured canine middle cerebral arterial fibroblasts, hemolysate produced similar Ca2+ mobilization to that of dermal cells. OxyHb and ATP failed again to reproduce the effect of hemolysate. We conclude that hemolysate increases [Ca2+]i in fibroblasts and this effect of hemolysate is not mediated by OxyHb or ATP but by some unknown factors.     

Effects of superoxide generating systems on muscle tone, cholinergic and NANC responses in cat airway

To study the possible role of reactive oxygen species in airway hyperreactivity, we examined the effects of the superoxide anion radical (O(2)(-)) generating systems, pyrogallol and xanthine with xanthine oxidase, on muscle tone, excitatory and inhibitory neurotransmission in the cat airway. Smooth muscle contraction or non-adrenergic non-cholinergic (NANC) relaxation evoked by electrical field stimulation (EFS) were measured before or after O(2)(-) generating systems with or without diethydithiocarbamic acid (DEDTCA), an inhibitor of endogenous superoxide dismutase (SOD). Resting membrane potential or excitatory junction potential (EJP) were also measured in vitro. Both pyrogallol and xanthine/xanthine oxidase produced biphasic changes in basal and elevated (by 5-HT) muscle tone. After SOD pretreatment, both systems consistently produced a prolonged contraction, thereby indicating that O(2)(-) was converted to H(2)O(2) by the action of SOD and as a result the actions of O(2)(-) were lost but those of H(2)O(2) introduced. The O(2)(-) showed no significant effect on smooth muscle contraction or EJP evoked by EFS, however after DEDTCA pretreatment, it evoked initial enhancement followed by suppression of the contraction and EJP. DEDTCA pretreatment ameliorated the inhibitory action of pyrogallol and xanthine/xanthine oxidase on the NANC relaxation, probably because O(2)(-) could combine with endogenous NO to form peroxynitrite. These results indicate that the O(2)(-) generating systems have multiple actions, presumably due to the presence and simultaneous action of at least two different reactive oxygen species (O(2)(-) and H(2)O(2)). While H(2)O(2) seems to be responsible for elevation of muscle tone and augmentation of smooth muscle contraction by EFS, O(2)(-) inhibits muscle tone, cholinergic and NANC neurotransmission.     

Bacterial endotoxin induces [Ca2+]i transients and changes the organization of actin in microglia

We have employed amoeboid microglia purified from primary cultures of neonatal rat brain to examine the effect of bacterial lipopolysaccharide (LPS), a potent activator of immune cells, on intracellular calcium concentration ([Ca²⁺]i) in brain macrophages. In single brain macrophages loaded with indo 1, pulse administration of LPS elicited a rapid and transient increase in [Ca²⁺]i From a total of 70 cells examined, all responded to LPS with a similar [Ca²⁺]i transient, indicating a good homogeneity of the cell population with regard to the LPS response. It was concluded that the rise of cytosolic [Ca²⁺]i originated from intracellular stores because the response to LPS occured similarly in the presence or in the absence of extracellular Ca²⁺. A second administration of LPS to the same cells resulted in a second but reduced [Ca²⁺]i transient. In contrast to the first response to LPS, this second response was totally dependent on the presence of Ca²⁺ in the extracellular medium. The first response to LPS was strongly inhibited by ruthenium red and could be suppressed in a reversible manner by pre incubating the cells with caffeine in the absence of Ca²⁺ in the extracellular medium. These results indicate that caffeinesensitive intracellular Ca²⁺ stores may be the major source of Ca²⁺ in the response of brain macrophages to LPS. The possible release of Ca²⁺ from phosphatidylinositol(3,4,5)-trisphosphate (IP₃)-sensitive stores in brain macrophages was also evaluated by stimulating cells with the IP₃-mobilizing agonist histamine. Brain macrophages were heterogeneous with regard to the histamine response since histamine induced a [Ca²⁺]i rise in only 30% of cells examined. The increase in [Ca²⁺]i triggered by LPS may in turn activate several intracellular events involved in the transition from one microglial functional state to another. We examined the effect of LPS on the state of organization of actin, which is an essential component of chemoattractant signal transduction. Immunofluorescence staining with anti-actin antibody which recognizes actin in both filamentous and nonfilamentous configurations, indicated that LPS produced drastic changes in the actin organization. LPS-treated cells appeared more intensely fluorescent than untreated cells due to the concentration of diffuse fluorescence near the center of the cell. In addition, prominent fluorescent dots were present in the subplasmalemmal region in cells stimulated with LPS. This specific LPS-induced reorganization of actin was also observed in cells preincubated in the external medium without Ca²⁺. Thus, it is likely that the LPS-induced mobilization of Ca²⁺ from intracellular stores may be responsible for the changes observed in the actin organization. © 1994 Wiley-Liss, Inc.     

Dantrolene sodium is able to reduce the resting ionic [Ca2+]i in muscle from humans with malignant hyperthermia

Malignant hyperthermia (MH) is a hereditary myopathy, triggered when susceptible patients are exposed to a depolarizing muscle relaxant and/or potent volatile anesthetics. We have studied the effects of dantrolene on the free [Ca2+]i of intercostal muscle biopsies obtained from two MH-susceptible patients before and after administration of dantrolene orally (2.5 mg/kg for 3 days) and intravenously (1.0 mg/kg 2 hours before the biopsy). The free [Ca2+]i was measured by Ca2+-selective microelectrodes. The mean resting free [Ca2+]i in the MH-susceptible muscle before dantrolene treatment was 0.42 +/- 0.01 microM (mean +/- SEM, n = 12). The administration of dantrolene reduced this value to 0.27 +/- 0.01 microM (n = 14). There was no detectable difference in the resting membrane potential after dantrolene. These results represent the first direct demonstration that dantrolene is able to reduce the resting free [Ca2+]i in skeletal muscle of MH-susceptible patients.     

Effect of halothane on the release of [Ca2+]i in dorsal root ganglion neurons

We investigated the effect of the volatile anaesthetic halothane on [Ca2+]i of dorsal root ganglion neurons. Halothane was able to increase [Ca2+]i in those neurons in a dose-dependent manner and independent of extracellular calcium. However, halothane action was inhibited by BAPTA-AM, suggesting the involvement of intracellular calcium stores. Dantrolene, an inhibitor of ryanodine-sensitive calcium stores had no effect while 2-APB, an inhibitor of IP3-sensitive calcium store reduced by 78% the halothane-evoked increase on [Ca2+]i. These data suggests that halothane increased [Ca2+]i of ganglion neurons through calcium release from IP3-sensitive calcium store. One possible consequence of the halothane action is to alter presynaptic activity and signaling pathways that influence neurotransmission.     

GABA Triggers a [Ca2+]i Increase in Murine Precursor Cells of the Oligodendrocyte Lineage

The development of oligodendrocytes from their precursor cells can be studied in vitro by using a culture system in which cells can be identified at different developmental stages. We used this culture system to compare the effect of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) on intracellular Ca2+ concentration ([Ca2+]i) using fura-2 fluorescence systems. Application of GABA evoked transient [Ca2+]i increases in precursor cells. In contrast, [Ca2+]i levels were not affected in oligodendrocytes, which were identified by their positive labelling with the antibody O1. The precursor cells, identified by a lack of O1 staining, responded to GABA in the concentration range between 10-6 and 10-4 M. Since muscimol mimicked and bicuculline as well as picrotoxin blocked the GABA response, we conclude that the response is mediated by activation of GABAA receptors. The involvement of Ca2+ channels is inferred from the observation that the [Ca2+]i changes could be blocked by nifedipine or by omitting Ca2+ from the bath solution. Both GABAA receptors and Ca2+ channels have been previously identified on these precursor cells with the aid of the patch-clamp technique. We thus propose the following mechanism to explain our observations: the Cl- efflux via the GABA receptor depolarizes precursor cells, and this depolarization leads to activation of Ca2+ channels, resulting in an influx of Ca2+ and the observed rise in cytosolic [Ca2+]. Although its physiological importance is speculative, this event could serve as a signal from GABAergic neurons to glial precursor cells.     

钙离子在颞叶癫癎大鼠海马神经元内的动态变化

目的探讨海马神经元内钙离子浓度的动态变化在颞叶癫癎中的可能作用. 方法应用新一代钙荧光指示剂Fluo-3/AM,采用激光共聚焦扫描显微镜技术对颞叶癫癎大鼠海马神经元内的钙离子浓度变化进行动态观察. 结果各时间点癫癎组大鼠海马神经细胞的平均荧光像素数(3 h634 942±27 735;12 h697 066±14 863;7 d732 844±23 107;60 d800 030±16 450)均明显高于对照组(3 h396 499±31 951;12 h389 498±33 257;7 d389 809±29 486;60 d392 758±35 197),差异有统计学意义(P＜0.01);癫癎大鼠海马神经细胞内钙离子浓度的变化可分3个阶段急性期细胞内的钙离子浓度先急剧升高,然后缓慢升高;静止期开始时钙离子浓度较急性期后期先稍有下降,然后再次缓慢上升;到慢性复发期,钙离子浓度达到最高峰,并维持在高水平. 结论在颞叶癫癎的发生发展过程中海马细胞内存在钙稳态失调,该变化导致了神经元内游离钙离子浓度的急性和持久性升高,后者可能诱发了颞叶癫癎的自发性复发性发作.     

Effects of hypoxia and putative transmitters on [Ca2+]i of rat glomus cells

Dissociated rat glomus cells were loaded with Fura-2 AM to study the effects of hypoxia, and carotid body transmitters on intracellular calcium, [Ca2+]i. The mean control [Ca2+]i was 55 nM in isolated cells and 67 nM in clusters. The following procedures changed [Ca2+]i:0[Ca2+]o+EGTA reduced [Ca2+]i by about 50%, suggesting that the remaining calcium originated from intracellular organelles. [Ca2+]i increased when [Ca2+]o was doubled.Hypoxia by sodium dithionite (Na2S2O4) induced large [Ca2+]i increases in clustered and isolated cells. Smaller rises occurred with 100% N2 hypoxia. The augmented [Ca2+]i, induced by Na2S2O4, was reduced (not eliminated) in 0[Ca2+]o+EGTA, suggesting that some calcium was intracellularly released. Nifedipine depressed (did not block) the Na2S2O4-induced calcium increase, implying some inflow via other (N, T or P/Q) voltage-dependent or voltage-independent calcium channels.Cholinergic agents (ACh, nicotine, muscarine, bethanechol and pilocarpine) increased [Ca2+]i. The ACh effect was produced exclusively by calcium inflow since it was eliminated in 0[Ca2+]o+EGTA. Cholinergic effects were depressed (not obliterated) by D-tubocurarine (D-TC), hexamethonium (C6) and atropine.ACh, nicotine and pilocarpine potentiated the excitatory effect of Na2S2O4 on [Ca2+]i. Bethanechol depressed this excitation whereas muscarine had inconsistent effects.Atropine and C6 depressed [Ca2+]i increases elicited by Na2S2O4 but the effects of D-TC were variable.Dopamine (DA) had variable effects. It increased [Ca2+]i in 75% of cases, and reduced the Na2S2O4 -induced calcium increase.Thus, calcium increases during Na2S2O4 occur by direct effects on the glomus cells and feedback action through released ACh and DA.     

The ouabain-induced [Ca2+]i increase in leech Retzius neurones is mediated by voltage-dependent Ca2+ channels

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.     

利用激光共聚焦图像系统检测实验性变态反应性脑脊髓炎动物淋巴细胞内游离钙浓度

目的　进一步探讨多发性硬化 (MS)和实验性变态反应性脑脊髓炎 (EAE)的发病机制。方法　取EAE动物的脾细胞 ,制备淋巴细胞悬液 ,以荧光探针Fluo 3 AM负载后 ,通过激光扫描共聚焦显微镜 (InsightplusIQ ,Meridian ,USA)检测细胞荧光强度 ,其荧光强度与细胞内钙离子浓度成正比。结果　EAE组淋巴细胞内钙离子浓度明显高于正常对照组。结论　细胞内钙在EAE动物淋巴细胞的过度活化方面发挥重要作用     

Variation in the pattern of [Ca2+]i change induced by acetylcholine in cultured hippocampal neurons

Acetylcholine (ACh) caused various patterns of change in the intracellular Ca2+ concentration ([Ca2+]i) in cultured rat hippocampal neurons. We studied the underlying mechanisms of the [Ca2+]i changes with simultaneous recording of [Ca2+]i and membrane potential/current. In most cases, [Ca2+]i rise was accompanied by a membrane depolarization. The [Ca2+]i change was significantly reduced when the membrane was voltage clamped, which implies that most of the [Ca2+]i rise results from the Ca2+ influx through the voltage-gated Ca2+ channel activated by the membrane depolarization. The membrane depolarizations were classified into two types, one associated with membrane conductance decrease and the other associated with membrane conductance increase. The former results from potassium conductance ((gK+) decrease, and the latter may result from the activation of a Na(+)-permeable channel. However, [Ca2+]i elevation was also observed in some neurons showing membrane hyperpolarization in response to ACh. This seems to show that ACh liberates Ca2+ from the intracellular Ca2+ store, resulting in the activation of a calcium-dependent K+ channel (KCa). The variations of ACh response in the hippocampal neurons seem to result from a variety of muscarinic acetylcholine receptors and various species of ion channels governed by those receptors.     

GFAP-positive hippocampal astrocytes in situ respond to glutamatergic neuroligands with increases in [Ca2+]i

It is becoming increasingly clear that astrocytes play very dynamic and interactive roles that are important for the normal functioning of the central nervous system. In culture, astrocytes express many receptors coupled to increases in intracellular calcium ([Ca²⁺]_i). In vivo, it is likely that these receptors are important for the modulation of astrocytic functions such as the uptake of neurotransmitters and ions. Currently, however, very little is known about the expression or stimulation of such astrocytic receptors in vivo. To address this issue, confocal microscopy and calcium sensitive fluorescent dyes were used to examine the dynamic changes in astrocytic [Ca²⁺]_i, within acutely isolated hippocampal slices. Astrocytes were subsequently identified by immunocytochemistry for glial fibrillary acidic protein. In this paper, we present data indicating that hippocampal astrocytes in situ respond to glutamate, kainate, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), 1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), N-methyl-D-aspartate (NMDA), and depolarization with increases in [Ca²⁺]_i. The increases in [Ca²⁺]_i occurred in both the astrocytic cell bodies and the processes. Temporally the changes in [Ca²⁺]_i were very dynamic, and various patterns ranging from sustained elevations to oscillations of [Ca²⁺]_i were observed. Individual astrocytes responded to neuroligands selective for both ionotropic and metabotropic glutamate receptors with increases in [Ca²⁺]_i. These findings indicate that astrocytes in vivo contain glutamatergic receptors coupled to increases in [C²⁺]_i and are able to respond to neuronally released neurotransmitters. (c) 1995 Wiley-Liss, Inc.     

皮质酮对TNF-α致海马神经元细胞内钙变化的作用

应用荧光影像系统检测了原代培养海马神经元内钙离子的变化情况,并分析了皮质酮、肿瘤坏死因子(TNF-α)对谷氨酸引起的海马神经元内钙升高的调节作用及其皮质酮对TNF-α作用的调节.结果显示:(1)使用不同浓度的皮质酮处理海马神经元48 h后,观察到10-6 mol/L和10-7 mol/L的皮质酮可诱导海马神经元静息钙浓度升高,但10-8 mol/L和10-9 mol/L的皮质酮对海马神经元内钙无影响.(2)10-6 mol/L皮质酮处理海马神经元48 h后,对谷氨酸诱导的海马神经元内钙升高无调节作用.(3)使用100 ngTNF-α处理海马神经元48 h后,既可诱导海马神经元静息钙升高,也可抑制谷氨酸引起的海马神经元内钙升高.(4)皮质酮可逆转TNF-α对海马神经元静息钙浓度的调节作用,但对TNF-α抑制谷氨酸升钙效应无影响.     

Adenosine modulation of [Ca2+]i in cerebellar granular cells: multiple adenosine receptors involved

Elimination of adenosine by addition of adenosine deaminase (ADA) to the media leads to alterations in intracellular free calcium concentration ([Ca(2+)](i)) in cerebellar granular cells. Adenosine deaminase brings about increases or decreases in [Ca(2+)](i) depending on the previous activation state of the cell. These effects are dependent on the catalytic activity of adenosine deaminase, since its previous catalytic inactivation with Hg(2+) prevents the above-mentioned changes in intracellular calcium. Extracellular calcium is required for the increase in [Ca(2+)](i) promoted by ADA. This rise is insensitive to thapsigargin, but sensitive to micromolar concentrations of Ni(2+). Toxins specific for L, N and P/Q calcium channels do not overtly reduce this effect. N(6)-Cyclopentyl adenosine (CPA), an A(1) receptor agonist, produces a partial reversion of ADA effects, while CGS21680, A(2A)/A(2B) receptor agonist, slightly enhances them. Expression of A(1), A(2A), A(2B) and A(3) adenosine receptor mRNAs was detected in cerebellar granular cell cultures. These results suggest that adenosine modulate [Ca(2+)](i) in cerebellar granule cells through different adenosine receptor subtypes which, at least in part, seem to act through R-type calcium channels.     

Increases in [Ca2+]i and changes in intracellular pH during chemical anoxia in mouse neocortical neurons in primary culture.

The effect of chemical anoxia (azide) in the presence of glucose on the free intracellular Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) in mouse neocortical neurons was investigated using Fura-2 and BCECF. Anoxia induced a reversible increase in [Ca2+]i which was significantly inhibited in nominally Ca2+-free medium. A change in pHo (8.2 or 6.6), or addition of NMDA and non-NMDA receptor antagonists (D-AP5 and CNQX) in combination, significantly reduced the increase in [Ca2+]i, pointing to a protective effect of extracellular alkalosis or acidosis, and involvement of excitatory amino acids. An initial anoxia-induced acidification was observed under all experimental conditions. In the control situation, this acidification was followed by a recovery/alkalinization of pHi in about 50% of the cells, a few cells showed no recovery, and some showed further acidification. EIPA, an inhibitor of Na+/H+ exchangers, prevented alkalinization, pointing towards anoxia-induced activation of a Na+/H+ exchanger. In a nominally Ca2+-free medium, the initial acidification was followed by a significant alkalinization. At pHo 8.2, the alkalinization was significantly increased, while at pHo 6.2, the initial acidification was followed by further acidification in about 50% of the cells, and by no further change in the remaining cells.     

Myoplasmic free [Ca2+] during a malignant hyperthermia episode in swine

Malignant hyperthermia (MH) is a genetic syndrome usually initiated by exposure to volatile anesthetic agents or depolarizing neuromuscular blocking agents. We have used Ca2+-selective microelectrodes to measure in vivo the intracellular ionized calcium ([Ca2+]i) in skeletal muscle fibers of MH-susceptible swines before and during hyperthermic episodes and also after dantrolene administration. The animals were anesthetized with thiopental and fentanyl and maintained with a mixture of nitrous oxide (66%) and oxygen (34%). The malignant hyperthermic episode was triggered by exposure to halothane. Determinations of [Ca2+]i during the episode show an increase from 0.44 +/- 0.01 microM +/- SEM, n = 20) to 8.44 +/- 0.68 microM (mean +/- SEM, n = 10). Administration of dantrolene (2 mg/kg) during the hyperthermic episode reduces [Ca2+]i to 0.17 + 0.01 microM (mean +/- SEM, n = 10) and reverses the clinical symptoms. These results show that the MH episode is associated with an increase in the myoplasmic free Ca2+ concentration and that the therapeutic effect of dantrolene is related to a decrease in [Ca2+]i.     

NMDA-induced increase in [Ca2+]i and 45Ca2+ uptake in acutely dissociated brain cells derived from adult rats.

A preparation of acutely dissociated brain cells derived from adult (3-month-old) rat has been developed under conditions preserving the metabolic integrity of the cells and the function of N-methyl-D-aspartate (NMDA) receptors. The effects of glutamate and NMDA on [Ca2+]i measured with fluo3 and 45Ca2+ uptake have been studied on preparations derived from hippocampus and cerebral cortex. Glutamate (100 microM) and N-methyl-DL-aspartate (200 microM) increased [Ca2+]i by 26-12 nM and 23-9 nM after 90 s in cerebral cortex and hippocampus, and stimulated 45Ca2+ uptake about 16-10% in the same regions. The increases in [Ca2+]i and 45Ca2+ uptake were inhibited by 40% in the presence of 1 mM MgCl2 and by 90-50% in the presence of MK-801. The results indicate (a) that a large fraction of the [Ca2+]i response to glutamate in freshly dissociated brain cells from the adult rat involves NMDA receptors, (b) when compared with results in newborn rats, there is a substantial blunting of the [Ca2+]i increase in adult age.