Mobilization of intracellular Ca2+ and suppression of inward currents in a neuronal hybrid cell line triggered by bradykinin

Bradykinin triggered intracellular Ca mobilizations and ionic conductance changes were studied in the neuroblastoma x glioma hybrid cell line NG108-15 using Ca-sensitive fluorescent indicator fura-2 under patch pipette whole cell voltage clamp condition. The time course of outward current induced by bradykinin was closely related to the time-course of [Ca2+]i change. Following application of bradykinin, [Ca2+]i increased transiently and then decreased below the basal level before bradykinin application. The inward currents activated by step-depolarization were suppressed after bradykinin application, but the time-course of the suppression did not go in parallel with the [Ca2+]i changes: the suppression started before the [Ca2+]i change emerged and outlasted the phase of [Ca2+]i increase. Both transient type and long-lasting type Ca current were suppressed by bradykinin. [Ca2+]i increase induced by high potassium depolarization was suppressed by bradykinin. Pertussis toxin did not affect the Ca transient nor the suppression of Ca channel induced by bradykinin. Our results suggest that the modifications of ionic channels by bradykinin could be through the other mechanisms than the well established activation of the G-protein leading to the IP3 mechanisms and that the bradykinin receptor might couple with the pertussis toxin-insensitive G protein which regulates the calcium channels.     

Modulation of intracellular Ca++ in cultured astrocytes by influx through voltage-activated Ca++ channels.

Fura-2 and indo-1 fluorescence measurements were used to examine intracellular Ca++ concentration ([Ca++]i) and its modulation by voltage-activated influx in murine cortical astrocytes in primary cell culture. Extracellular K+ was increased from 5 to 50 mM to depolarize cells to determine if Ca++ influx through voltage activated Ca++ channels could alter [Ca++]i. In confluent 4 to 6 weeks in vitro astrocyte cultures 50 mM K+ increased [Ca++]i 3-4-fold (from 150 nM up to 550 nM); this increase was blocked by nifedipine and enhanced by BayK 8644 indicating that influx was through L-type channels. However, in 1 to 2 weeks in vitro astrocyte cultures, high K+ reduced [Ca++]i. L-type channels were apparently present in these cells because high K+ in combination with BayK 8644 increased [Ca++]i. Following pretreatment of 1 to 2 weeks in vitro astrocytes with dibutyryl cAMP (dbcAMP) high K+ increased [Ca++]i in the absence of BayK 8644 indicating enhanced activity of Ca++ channels in agreement with previous voltage-clamp studies. Ca++ influx through voltage-activated channels in cultured cortical astrocytes can substantially increase [Ca++]i and these channels can be dynamically modulated by dihydropyridines. Immature astrocytes may express 'silent' or inactive Ca++ channels or have a much lower number of channels.     

Bradykinin induces hyperpolarizations in rat glioma cells and in neuroblastoma × glioma hybrid cells

The effect of the nonapeptide bradykinin on the membrane potential of permanent cell lines from neural origin was studied. A hyperpolarizing response of 10–30 s duration was produced when bradykinin was iontophoretically applied onto polyploid rat glioma cells (clone C6-4-2). Starting from the resting membra potential the peak value of the hyperpolarizing response was reached within 0.5–1.5 s. Then the potential returned more slowly to the origin value. The hyperpolarization was associated with an approximately 50% decrease in membrane resistance. Neither Na⁺ nor Cl⁻ seemed to be important for the hyperpolarizing response, since bradykinin elicited similar hyperpolarizations in cells exposed to media in which Na⁺ or Cl⁻ were replaced by choline or isethionate, respectively. Ca²⁺ fluxes are unlikely to be involved, since the addition of D600 did not affect the hyperpolarizations induced by bradykinin. However, a 10-fold increase in the concentration of K⁺ in the medium reduced the amplitude of the hyperpolarization by 40 mV. Thus, the hyperpolarization induced by bradykinin is associated with a decrease in membrane resistance which is likely to be caused by an increased K⁺-conductance. The glioma cells showed a desensitization upon repeated application of bradykinin. However, the sensitivity of the cells to bradykinin was restored after 3–8 min of incubation in the absence of bradykinin. Since an antagonist of bradykinin is not known, the specificity of the action of bradykinin is difficult to assess. Nevertheless, the hyperpolarizing response to bradykinin appears to be specific insofar as other peptides, i.e. lutoliberin, thyroliberin, neurotensin, substance P and apamin, exerted no effect on the membrane potential of the glioma cells. Bradikynin-elicited hyperpolarizations with characteristics similar to those described above could also be demonstrated in neuroblastoma × glioma hybrid cells, but not in multinucleated fibroblast cells.     

Mobilization of intracellular calcium by substance p in a human astrocytoma cell line (U-373 MG)

Variations in intracellular free calcium concentration (Δ[Ca²⁺]_i) were measured in intact and isolated human astrocytoma cells (U373 MG) loaded with fura-2 acetoxymethylester. Microperfusion of 50 nM substance P (SP), applied for 1 s, increased [Ca²⁺]_i by 351 nM from a stable basal level of [Ca²⁺]_i of 26 nM. The peak Δ[Ca²⁺]_i induced by SP was dose dependent with a threshold of 10_-3 nM, an ED₅₀ of 1.3 nM and a maximal effect for concentrations of SP greater than 100 nM. The NKI receptor agonist, [Sar⁹Met(O₂)¹¹]SP, mimicked the effect of SP, while the NK₂ and NK₃ selective receptor agonists, [N1¹⁰]NKA(4-10) and senktide, respectively, had no effect. The Δ[Ca²⁺]_i induced by SP was unaffected by 100 μM cadmium or by removal of extracellular calcium ions. Caffeine up to 30 mM had no effect on [Ca²⁺]_i. In contrast, thapsigargin increased resting [Ca²⁺]_i by 92 nM and reduced the Δ[Ca²⁺]_i induced by SP. A pertussis treatment (500 ng/ml-24 h) did not modify the Δ[Ca²⁺]_i induced by SP. We conclude that SP, acting on a NK1 receptor, mobilizes cytosolic calcium from an intracellular calcium pool which can be partially depleted by thapsigargin. © 1994 Wiley-Liss, Inc.     

Modulation of Ca2+ currents in rat thalamocortical relay neurons by activity and phosphorylation

Rhythmic low and high frequency activity in thalamocortical networks depend critically on activation of low- and high-voltage-activated (LVA, HVA) Ca2+ currents. In order to test whether Ca2+ currents are modified during repetitive activation, acutely isolated thalamocortical relay neurons of rats, at postnatal days 12 (P12) to P20, were investigated using patch-clamp, Ca2+ imaging and Western blot techniques. High-voltage-activated, but not LVA Ca2+ currents were reduced significantly during 2 Hz stimulation. Ca2+ imaging experiments demonstrated a close correlation between the increase in intracellular Ca2+ levels and the decrease in HVA Ca2+ current amplitudes. Further examination of HVA Ca2+ currents revealed a 'U-shaped' inactivation curve and a time-dependent inactivation process that could be described by a two-exponential function. The 'U-shape' was significantly reduced, current amplitude was increased significantly and time-dependent inactivation revealed a one-exponential decline with Ba2+ as the charge carrier, following activation of the cAMP/PKA pathway, and following application of phosphatase inhibitors (ascomycin, calyculin A). Western blot analysis and the effect of ascomycin indicated an involvement of calcineurin in the inactivation process. Isolation of HVA Ca2+ current components by subtype-specific blockers revealed that changes in time-dependent inactivation, inactivation curve and current amplitude were carried mainly by L-type and N-type Ca2+ currents. Furthermore, Ca2+-dependent inactivation was operative during stimulation protocols mimicking tonic action potential firing. These data indicate a modulation of L- and N-type Ca2+ channels by phosphorylation, resulting jointly in an increased intracellular Ca2+ influx during activity of the ascending brainstem system, the latter occurring during states of wakefulness.     

丹参诱导大鼠骨髓间充质干细胞分化为神经元样细胞中的钙离子检测

目的 检测大鼠骨髓间充质干细胞经丹参注射液诱导分化的神经元样细胞内钙离子浓度,以期为骨髓间充质干细胞应用于神经系统疾病的治疗提供理论依据.方法 从成年大鼠骨髓中获取骨髓间充质干细胞,体外扩增培养,经碱性成纤维生长因子预诱导后施加10mL/L丹参注射液于骨髓间充质干细胞培养液中.运用免疫荧光检测神经元特异性核蛋白(NeuN)在诱导后细胞与经新生大鼠海马获取的体外培养海马神经元中的表达.激光共聚焦技术检测诱导后的细胞内钙离子浓度.并与原代培养海马神经元内的钙离子浓度进行比较.结果 大鼠骨髓间充质干细胞经碱性成纤维生长因子和丹参注射液处理后,可表达NeuN,并具有神经元样的表型.诱导分化的神经元样细胞内钙离子浓度为984.75±79.51,原代培养海马神经元内钙离子浓度为769.42±60.93,两者比较差异无统计学意义(P>0.05).结论 丹参注射液诱导骨髓间充质干细胞分化的神经元样细胞具有神经元的某些特征.     

Proliferation and synapse formation of neuroblastoma × glioma hybrid cells: Effect of glia maturation factor

Glia maturation factor (GMF), extracted from bovine brain, stimulated DNA synthetis and proliferation of glioma cells and hybrid cells derived from glioma and neuroblastoma cells (NG108-15), but had no effect on neuroblastoma cells. The synapse formation of NG108-15 cells with rat striated myotubes was lower in the presence of GMF than the control and also lower after treatment with prostaglandin E₁ (PGE₁) plus theophylline, indicating that GMF did not induce functional differentiation of NG108-15 cells. The results show that expression of mitogenic action for GMF in the hybrid cells is a property derived from the glioma parent, and that NG108-15 is therefore an excellent model for studying glial-neuronal interactions.     

Relation of exocytosis and Ca-activated K current during Ca release from intracellular stores in individual rat chromaffin cells

Measurement of the change in cell membrane capacitance (C_m) along with the change in I_K(Ca) was used to investigate the effects of bradykinin and caffeine on the secretory process in rat adrenal chromaffin cells. In a Ca²⁺-free external solution, bradykinin (100 nM) caused a transient increase in C_m with a concurrent change in I_K(Ca). Extracellular application of neomycin as an inhibitor of phospholipase C activity reversibly inhibited the bradykinin-activated event, implying an IP₃-mediated increase of submembrane-free Ca²⁺. The increases in C_m and I_K(Ca) caused by bradykinin were transient even with the sustained application of bradykinin. Caffeine also caused exocytosis in the Ca²⁺-free solution, and this was irreversibly blocked by ryanodine (1 μM) in a use-dependent manner. Caffeine-sensitive intracellular Ca²⁺ stores were also depleted in several seconds and recovered by an influx of external Ca²⁺. The sequential application of bradykinin and caffeine showed that these are likely to activate Ca²⁺ release from the same or distinct but rapidly equilibrating intracellular Ca²⁺ stores. The single cell assay of exocytosis and the increase in I_K(Ca) revealed cell-to-cell variability in bradykinin- and caffeine-induced exocytotic response. Our results suggest that Ca²⁺ release from intracellular stores potentially increases submembrane Ca²⁺ concentration and modulates simultaneously two submembrane Ca²⁺-dependent processes, exocytosis and I_K(Ca), in rat adrenal chromaffin cells.     

Suppression of sodium pump activity and an increase in the intracellular Ca concentration by dexamethasone in acidotic mouse brain

The effects of dexamethasone on adenosine 5'-triphosphatase (ATPase) activity and the intracellular Ca(2+) concentration ([Ca(2+)](i)) were investigated in acidotic mouse brain. Dexamethasone (3 mg/kg, i.p.) or vehicle was administered 3 h before decapitation ischemia, and the brain concentration of adenosine 5'-triphosphate (ATP) was determined 0.5-2 min after ischemia. The effects of dexamethasone (0.3-3 mg/kg, i.p.) on Na(+),K(+)-activated ATPase (Na(+),K(+)-ATPase) and Ca(2+)-ATPase activities were evaluated at pH 7.4 and 6.8. Changes in [Ca(2+)](i) in an acidic medium were determined in hippocampal slices by microfluorometry using rhod-2 acetoxymethyl ester as a Ca(2+) marker, and the effects of dexamethasone (240 microg/l) was evaluated. Decapitation ischemia for 0.5 and 1 min reduced the brain ATP contents to 32% and 16% of the basal level, respectively. Dexamethasone slightly suppressed the extent of the decrease in the ATP level. Although dexamethasone did not affect Na(+),K(+)-ATPase activity at pH 7.4, the activity was suppressed by dexamethasone (3 mg/kg) to 68% at pH 6.8. The activity of Ca(2+)-ATPase was not affected by dexamethasone at either pH 7.4 or pH 6.8. When the pH of the medium of the brain slices was changed from 7.4 to 6.8, almost no increase in [Ca(2+)](i) was observed in the control group. The dexamethasone treatment increased [Ca(2+)](i) in the CA1 field and dentate gyrus immediately after induction of the acidic medium, the effect being significant after 150 s. Because anaerobic glucose metabolism in the early stage of ischemia enhances intracellular lactic acidosis, the findings may suggest a mechanism for the aggravation of ischemic neuronal damage by glucocorticoids.     

Dopamine D1 receptor‐mediated upregulation of BKCa currents modifies Müller cell gliosis in a rat chronic ocular hypertension model

Müller cell gliosis is a common response in many retinal pathological conditions. We previously demonstrated that downregulation of Kir channels contributes to Müller cell gliosis in a rat chronic ocular hypertension (COH) model. Here, the possible involvement of outward K⁺ currents in Müller cell gliosis was investigated. Outward K⁺ current densities in Müller cells isolated from COH rats, as compared with those in normal rats, showed a significant increase, which was mainly contributed by large‐conductance Ca²⁺‐activated K⁺ (BK_Ca) channels. The involvement of BK_Ca channels in Müller cell gliosis is suggested by the fact that glial fibrillary acidic protein (GFAP) levels were augmented in COH retinas when these channels were suppressed by intravitreal injections of iberiotoxin. In COH retinas an increase in dopamine (DA) D1 receptor (D1R) expression in Müller cells was revealed by both immunohistochemistry and Western blotting. Moreover, protein levels of tyrosine hydroxylase were also increased, and consistent to this, retinal DA contents were elevated. SKF81297, a selective D1R agonist, enhanced BK_Ca currents of normal Müller cells through intracellular cAMP‐PKA signaling pathway. Furthermore, GFAP levels were increased by the D1R antagonist SCH23390 injected intravitreally through eliminating the BK_Ca current upregulation in COH retinas, but partially reduced by SKF81297. All these results strongly suggest that the DA‐D1R system may be activated to a stronger extent in COH rat retinas, thus increasing BK_Ca currents of Müller cells. The upregulation of BK_Ca channels may antagonize the Kir channel inhibition‐induced depolarization of Müller cells, thereby attenuating the gliosis of these cells.     

Intracellular acidification induced by membrane depolarization in rat hippocampal slices: roles of intracellular Ca and glycolysis

To elucidate the mechanism of pH_i changes induced by membrane depolarization, the variations in pH_i and [Ca²⁺]_i induced by a number of depolarizing agents, including high K⁺, veratridine, N-methyl-

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-aspartate (NMDA) and ouabain, were investigated in rat hippocampal slices by the fluorophotometrical technique using BCECF or fura-2. All of these depolarizing agents elicited a decrease in pH_i and an elevation of intracellular calcium ([Ca²⁺]_i) in the CA1 pyramidal cell layer. The increases in [Ca²⁺]_i caused by the depolarizing agents almost completely disappeared in the absence of Ca²⁺ (0 mM Ca²⁺ with 1 mM EGTA). In Ca²⁺ free media, pH_i acid shifts produced by high K⁺, veratridine or NMDA were attenuated by 10–25%, and those produced by ouabain decreased by 50%. Glucose-substitution with equimolar amounts of pyruvate suppressed by two-thirds the pH_i acid shifts induced by both high K⁺ and NMDA. Furthermore, lactate contents were significantly increased in hippocampal slices by exposure to high K⁺, veratridine or NMDA but not by ouabain. These results suggest that the intracellular acidification produced by these depolarizing agents, with the exception of ouabain, is mainly due to lactate accumulation which may occur as a result of accelerated glycolysis mediated by increased Na⁺–K⁺ ATPase activity. A Ca²⁺-dependent process may also contribute to the intracellular acidification induced by membrane depolarization. Since an increase in H⁺ concentration can attenuate neuronal activity, glycolytic acid production induced by membrane depolarization may contribute to the mechanism that prevents excessive neuronal excitation.     

Selective block of Ca2+-dependent K+ current in crayfish neuromuscular system and chromaffin cells by sea anemone Bunodosoma cangicum venom

The effects of the nematocyst venom of the sea anemone Bunodosoma cangicum on depolarization-activated currents were studied in opener crayfish muscle fibers and in cultured bovine chromaffin cells. The venom selectively and reversibly blocked the Ca²⁺ -dependent K⁺ current (I_K(Ca)) present in crayfish muscle in a dose-dependent manner without affecting voltage-gated Ca²⁺ or K⁺ currents. Furthermore, the venom also reduced I_K(Ca) in chromaffin cells, without modifying voltage-gated Na⁺, Ca²⁺, or K⁺ currents. Synaptic transmission in crayfish muscle was also affected by the venom. Repetitive excitatory and inhibitory postsynaptic currents (each associated with a presynaptic action potential) were evoked by each nerve stimulus, suggesting that presynaptic I_K(Ca) may control the electrical activity of excitatory and inhibitory presynaptic fibers. We conclude that B. cangicum venom includes a toxin that selectively and reversibly blocks Ca²⁺ -dependent K⁺ currents in crayfish muscle and in bovine chromaffin cells, and modifies excitatory and inhibitory synaptic transmission, probably abolishing a similar conductance at the presynaptic fibers. © 1995 Wiley-Liss, Inc.     

Transport pathways for lithium ions in neuroblastoma × glioma hybrid cells at ‘therapeutic’ concentrations of Li

The pathways of Li⁺ transport in neuroblastoma × glioma hybrid cells were studied at 2 mM external Li⁺. Five components of Li⁺ transport were identified. (1) A Na⁺-dependent Li⁺ countertransport system mediating Li⁺ transport in both directions across the plasma membrane. This transport pathway is insensitive to ouabain or external K⁺. It shows trans-stimulation (i.e. acceleration of Li⁺ extrusion by external Na⁺ and stimulation of Li⁺ uptake by internal Na⁺) and cis-inhibition (i.e. reduction of Li⁺ uptake by external Na⁺). (2) The Na⁺K⁺ pump mediates Li⁺ uptake but not Li⁺ release in cells with physiological Na⁺ and K⁺ content. Li⁺ uptake by the pump in choline media is inhibited by both external Na⁺ and K⁺. In Na⁺ media, external K⁺ exhibits a biphasic effect: in concentrations up to about 1 mM, K⁺ accelerates, and at higher concentrations, K⁺ inhibits, Li⁺ uptake by the pump. (3) Li⁺ can enter the voltage-dependent Na⁺ channel. Li⁺ uptake through this pathway is stimulated by veratridine and scorpion toxin, the stimulation being blocked by tetrodotoxin. Residual pathways comprise (4) a saturable component, which is comparable to basal Na⁺ uptake, and (5) a ouabain-resistant component promoting Li⁺ extrusion against an electrochemical gradient in choline media. The mechanisms for Li⁺ extrusion described here possibly explain how neuronal cells maintain the steady-state ratio of internal to external Li⁺ below 1 during chronic exposure to 1–2 mM external Li⁺.