MODULATORY EFFECT OF CYCLOSPORIN A ON TERT-BUTYL HYDROPEROXIDE–INDUCED OXIDATIVE DAMAGE IN HEPATOCYTES

In the present work, we followed an in vitro protective action of cyclosporin A (CsA) against tert-butyl hydroperoxide (t-BHP)–induced oxidative damage in hepatocytes. Various parameters (cell viability, cytosolic calcium level, rhodamine 123 accumulation as indicator of mitochondrial membrane potential and alanine-aminotransferase leakage from cells) were measured as an index of cytotoxicity. Tert-butyl hydroperoxide (1 mM) significantly increased cytosolic Ca²⁺ and affected mitochondrial membrane potential. Pretreatment with cyclosporin A (0.5 μM) reduced t-BHP-induced cytosolic Ca²⁺ increase and ALT (alanine-aminotransferase) leakage, but had no protective effect on t-BHP-induced changes of mitochondrial membrane potential. Our data thus suggest that the mechanism of cytoprotection of CsA on the cytosolic Ca²⁺ changes and ALT leakage induced by t-BHP, does not directly correlate with protection of t-BHP-induced changes of mitochondrial membrane potential.     

心肌细胞钙稳态失调在心力衰竭中作用的研究进展

心肌细胞的钙稳态依赖于质膜、肌/内质网(SR)、线粒体等对钙(Ca2+)转运的调节.胞质钙超载、SR钙渗漏等在内的钙稳态失调,使心肌细胞钙瞬变减低,收缩力下降是心力衰竭的主要特征之一.     

Aging does not alter cytosolic calcium levels of cortical synaptosomes in Fischer 344 rats

Several lines of experimental evidence support an association between altered Ca²⁺ regulation and aging. It has been supposed that free cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) may decrease or increase in aged animals. In this study, both resting and KCl-stimulated [Ca²⁺]_i were measured in purified cortical synaptosomes from young (3 mo.), middle-aged (12 mo.), and old (24 mo.) Fischer 344 rats. Two additional groups of rats were included, one middle-aged and one old which were trained on a treadmill for 6 months prior to experimentation. The [Ca²⁺]_i was determined using the fluorescent Ca²⁺ chelator fura-2. Net KCl-dependent changes (ΔK) in [Ca²⁺]_i were determined by the difference between stimulatory (100 μM Ca²⁺/60 mM KCl) and resting (100 μM Ca²⁺/5 mM KCl buffer) conditions among the 3 age groups. Significant increases in [Ca²⁺]_i were observed in each age group upon depolarization with 60 mM KCl. However, there were no significant age-dependent differences in either resting [Ca²⁺]_i or KCl-stimulated [Ca²⁺]_i.     

Vip Modulates Intracellular Calcium Oscillations in Human Lymphoblasts

Vasoactive intestinal polypeptide (VIP) has been shown to stimulate adenylate cyclase in a human lymphoblast cell line (MOLT 4). In the present study, we monitored fluorescence in cell suspensions and in single fura-2 loaded MOLT 4 lymphoblasts to determine if VIP modulates intracellular calcium concentrations ([Ca²⁺]_i), and if this modulation is mediated by adenylate cyclase. The distribution of [Ca²⁺]_i in resting and stimulated cells was non-homogeneous, with gradients of high [Ca²⁺]_i present in the subplasmalemmal space. In a subset of cells (10-30% of all cells studied), [Ca²⁺]_i showed La³⁺-sensitive, temporal changes in the form of [Ca²⁺]_i oscillations with a baseline [Ca²⁺]_i value of 115±10 nM, an oscillation amplitude of 150±18 nM and a mean period of 9.2±2s. The remaining non-oscillating cells showed a constant [Ca²⁺]_i level of 75±5 nM (n=65 cells from 4 experiments). In the subset of cells with spontaneous [Ca²⁺]_i oscillations, VIP dose-dependendy (10^-12 to 10^-8M) increased the amplitude of oscillations but did not stimulate their frequency. The stimulatory effect of VIP was correlated with baseline [Ca²⁺]_i in these cells, was attenuated in the presence of La³⁺ (25 μM), but was unaffected by cell depolarization (126 mM KC1). Dibutyryl cyclic AMP (10^-4 to 10^-3 M) and forskolin (10^-4M) had no effect on [Ca²⁺]_i oscillations, or on [Ca²⁺]_i in cells without oscillations. In cell suspensions, baseline [Ca²⁺]_i was found to be 55. 1±11.2 nM (meanS.E.M., n=11); VIP, cyclic AMP analogues or forskolin had no significant effect on [Ca²⁺]_i. These findings suggest that: a) VIP modulates the amplitude of [Ca²⁺]_i oscillations generated by a cytosolic [Ca²⁺] oscillator in a subset of cells at a concentration of 10^-12M, a thousand-fold below the K_D for the VIP receptor; b) baseline [Ca²⁺] values may be related to both the ability of cells to generate spontaneous [Ca²⁺] oscillations and of oscillating cells to respond to VIP; c) due to the small number of responding cells, VIP-induced [Ca²⁺]_i changes are not detectable when studied in cell suspensions.     

Mitochondrial Ca2+ buffering in hypoglossal motoneurons from mouse

A variety of studies demonstrated a crucial role of mitochondria for clearance of Ca²⁺ loads in motoneurons. However, previous reports rarely addressed the potential influence of cell dialysis during patch-clamp recordings or temperature on mitochondrial processes. We therefore developed a protocol allowing investigation of Ca²⁺ dynamics in “undisturbed” AM-ester loaded hypoglossal motoneurons in a slice preparation. By comparing our findings to previous results, we argue against a significant disturbance of mitochondrial buffering by cell dialysis. By varying bath temperatures between 19 and 32 °C, we show that temperature alters the rate of mitochondrial uptake but not the relative contribution to maintenance of Ca²⁺ homeostasis. The results further indicate that mitochondria in hypoglossal motoneurons participate in intracellular Ca²⁺ regulation at concentrations much lower than has been generally observed for other neurons or neuroendocrine cells. Taken together, our findings further support the important role of mitochondria as regulators of Ca²⁺ homeostasis in motoneurons.     

Brain oxidation is an initial process in sleep induction

CNS activity is generally coupled to the vigilance state, being primarily active during wakefulness and primarily inactive during deep sleep. During periods of high neuronal activity, a significant volume of oxygen is used to maintain neuronal membrane potentials, which subsequently produces cytotoxic reactive oxygen species (ROS). Glutathione, a major endogenous antioxidant, is an important factor protecting against ROS-mediated neuronal degeneration. Glutathione has also been proposed to be a sleep-promoting substance, yet the relationship between sleep and cerebral oxidation remains unclear. Here we report that i.c.v. infusion of the organic peroxide t-butyl-hydroperoxide at a concentration below that triggering neurodegeneration (0.1 μmol/100 μl/10 h) promotes sleep in rats. Also, microinjection (2 nmol, 2 μl) or microdialysis (100 μM, 20 min) oft-butyl-hydroperoxide into the preoptic/anterior hypothalamus (POAH) induces the release of the sleep-inducing neuromodulators, nitric oxide and adenosine, without causing neurodegeneration. Nitric oxide and adenosine release was inhibited by co-dialysis of the N-methyl-d-aspartate receptor antagonist, d(−)-2-amino-5-phosphonopentanoic acid (D-AP5; 1 mM), suggesting that glutamate-induced neuronal excitation mediates the peroxide-induced release of nitric oxide and adenosine. Indeed, Ca²⁺ release from mitochondria and delayed-onset Ca²⁺ influx via N-methyl-d-aspartate receptors was visualized during peroxide exposure using Ca²⁺ indicator proteins (YC-2.1 and mitochondrial-targeted Pericam) expressed in organotypic cultures of the POAH. In the in vitro models, t-butyl-hydroperoxide (50 μM) causes dendritic swelling followed by the intracellular Ca²⁺ mobilization, and D-AP5 (100 μM) or glutathione (500 μM) inhibited t-butyl-hydroperoxide-induced intracellular Ca²⁺ mobilization and protected POAH neurons from oxidative stress.

These data suggest that low-level subcortical oxidation under the control of an antioxidant system may trigger sleep via the Ca²⁺-dependent release of sleep-inducing neuromodulators in the POAH, and thus we propose that a moderate increase of ROS during wakefulness in the neuronal circuits regulating sleep may be an initial trigger in sleep induction.     

Optical identification of calcium-dependent action potentials transiently expressed in the embryonic rat brainstem

Using multiple-site optical recording of transmembrane potential changes, we have found a new type of calcium-dependent action potential expressed transiently in the embryonic rat dorsal motor nucleus of the vagus nerve. Slice preparations with vagus nerve fibers attached were dissected from 12- to 16-day-old embryonic (E12–E16) rat brainstems, and they were stained with a voltage-sensitive merocyanine–rhodanine dye (NK2761). Electrical activities in response to vagal stimuli were optically recorded simultaneously from many sites using 1020- or 128-element photodiode array measuring systems. In brainstem preparations, two types of action potential-related optical signals were identified. One was detected from the dorsolateral region, and was related to sensory nerve activity (Type I). The other was detected from the dorsomedial region, and corresponded to the action potential in the dorsal motor nucleus of the vagus nerve (Type II). We found a difference in the ionic basis of the Type I vs Type II signals. The Type I signal was not altered in Ca²⁺-free bathing solution and was eliminated by tetrodotoxin, suggesting that the sensory nerve activity is mediated by Na⁺ currents. The Type II signal at early developmental stages (E12–E13, and some preparations in E14) was also independent of Ca²⁺. However, the Type II signal in later developmental stages (E15–E16, and some preparations in E14) did depend upon Ca²⁺: it was eliminated in Ca²⁺-free Ringer's solution, blocked by Cd²⁺, Ni²⁺ or Mn²⁺, and elicited in Sr²⁺-containing Ringer's solution, where CaCl₂ was replaced with SrCl₂. These results suggest that the cation which dominates the motoneuron action potential changes from Na⁺ to Ca²⁺ during development, and this change occurs around E14. With pharmacological analysis using Ca²⁺ channel blockers, we show that the Ca²⁺ channel mediating the motoneuron action potential is distinct from T-, L-, N-, P- or Q-type channels.

Because the vagal action potential in adult mammals is mainly mediated by Na⁺, we suggest that a Ca²⁺ action potential mediated by a new type of Ca²⁺ channel is expressed transiently in the rat dorsal motor nucleus of the vagus nerve at particular stages of development.     

Stimulation-induced changes in NADH fluorescence and mitochondrial membrane potential in lizard motor nerve terminals

To investigate mitochondrial responses to repetitive stimulation, we measured changes in NADH fluorescence and mitochondrial membrane potential (Ψ_m) produced by trains of action potentials (50 Hz for 10–50 s) delivered to motor nerve terminals innervating external intercostal muscles. Stimulation produced a rapid decrease in NADH fluorescence and partial depolarization of Ψ_m. These changes were blocked when Ca²⁺ was removed from the bath or when N-type Ca²⁺ channels were inhibited with ω-conotoxin GVIA, but were not blocked when bath Ca²⁺ was replaced by Sr²⁺, or when vesicular release was inhibited with botulinum toxin A. When stimulation stopped, NADH fluorescence and Ψ_m returned to baseline values much faster than mitochondrial [Ca²⁺]. In contrast to findings in other tissues, there was usually little or no poststimulation overshoot of NADH fluorescence. These findings suggest that the major change in motor terminal mitochondrial function brought about by repetitive stimulation is a rapid acceleration of electron transport chain (ETC) activity due to the Ψ_m depolarization produced by mitochondrial Ca²⁺ (or Sr²⁺) influx. After partial inhibition of complex I of the ETC with amytal, stimulation produced greater Ψ_m depolarization and a greater elevation of cytosolic [Ca²⁺]. These results suggest that the ability to accelerate ETC activity is important for normal mitochondrial sequestration of stimulation-induced Ca²⁺ loads.     

Spatial and dynamic changes in intracellular Ca measured by confocal laser-scanning microscopy in bullfrog sympathetic ganglion cells

Confocal laser scanning microscopy (CLSM) was used to record spatial and dynamic changes in the intracellular Ca²⁺ ([Ca²⁺]_i) of bullfrog sympathetic ganglion cells in excised tissue or in culture. A CLSM utilizing Ar ion laser (488 nm) and recording fluo-3 fluorescence yielded the sliced image of ganglion cells, while conventional epifluorescence microscopy provided the cell image of a convex structure. A high K⁺ (50 mM) solution, caffeine (3–10 mM) and electrical stimulation (10–20 Hz, 0.5–10 s) caused a homogeneous increase in fluo-3 fluorescence with or without regional differences, possibly due to intracellular organelles and other constituents. Scanning a single horizontal line across the cytoplasm with He-Cd laser (325 nm) and recording indo-1 fluorescence demonstrated that the rate of rise in [Ca²⁺]_i following action potentials depends on the distance from the cell membrane and on the cytoplasmic constituents, showing an inward spread of ‘Ca²⁺-wave’ at variable speeds of 17–219 μm/s. These results suggest that heterogeneity of the cytoplasmic structures and constituents affects dynamic and spatial changes of [Ca²⁺]_i in response to stimuli in neurones. Such heterogenic changes in [Ca²⁺]_i would better be studied by CLMS.     

Modulation of Human T-Lymphocyte Plasma Membrane Ca +2 Permeability by Imidazole Antimycotics

The role of cytochrome P-450 in the regulation of plasma membrane Ca⁺² permeability of human peripheral T-lymphocytes by intracellular Ca⁺² was examined. We assessed the effect of imidazole inhibitors of cytochrome P-450 on the intracytoplasmic free Ca⁺² ([Ca⁺²]i) response generated using the microsomal ATPase inhibitor thapsigargin (THG) to deplete the intracellular Ca⁺² stores. Econazole, miconazole and clotrimazole dramatically inhibited the THG mediated increase in [Ca⁺²]i and indud an increase in [Ca⁺²]i themselves. This inhibitory effect was previously observed in other cell systems and was attributed to inhibition of cytochrome P-450 by these agents. However, we evaluated a variety of structurally dissimilar P-450 inhibitors and found that none affected [Ca⁺²]i, indicating that the mechanism of imidazole action does not involve P-450.     

Presence and functional significance of presynaptic ryanodine receptors

Ca²⁺-induced Ca²⁺ release (CICR) mediated by sarcoplasmic reticulum resident ryanodine receptors (RyRs) has been well described in cardiac, skeletal and smooth muscle. In brain, RyRs are localised primarily to endoplasmic reticulum (ER) and have been demonstrated in postsynaptic entities, astrocytes and oligodendrocytes where they regulate intracellular Ca²⁺ concentration ([Ca²⁺]_i), membrane potential and the activity of a variety of second messenger systems. Recently, the contribution of presynaptic RyRs and CICR to functions of central and peripheral presynaptic terminals, including neurotransmitter release, has received increased attention. However, there is no general agreement that RyRs are localised to presynaptic terminals, nor is it clear that RyRs regulate a large enough pool of intracellular Ca²⁺ to be physiologically significant. Here, we review direct and indirect evidence that on balance favours the notion that ER and RyRs are found in presynaptic terminals and are physiologically significant. In so doing, it became obvious that some of the controversy originates from issues related to (i) the ability to demonstrate conclusively the physical presence of ER and RyRs, (ii) whether the biophysical properties of RyRs are such that they can contribute physiologically to regulation of presynaptic [Ca²⁺]_i, (iii) how ER Ca²⁺ load and feedback gain of CICR contributes to the ability to detect functionally relevant RyRs, (iv) the distance that Ca²⁺ diffuses from plasma membranes to RyRs to trigger CICR and from RyRs to the Active Zone to enhance vesicle release, and (v) the experimental conditions used. The recognition that ER Ca²⁺ stores are able to modulate local Ca²⁺ levels and neurotransmitter release in presynaptic terminals will aid in the understanding of the cellular mechanisms controlling neuronal function.     

Molecular characterization of L-type calcium channel splice variants expressed in human T lymphocytes

Calcium (Ca²⁺) influx is a fundamental intracellular signal that is required to initiate and sustain T lymphocyte activation. Dihydropyridine-sensitive, L-type Ca²⁺ channels appear to play a significant role in Ca²⁺ mobilization during T cell activation, but very little is known about the molecular structure of these channels in T lymphocytes. Here we identify two novel splice variants of the Ca_V1.4 (_1F) L-type Ca²⁺ channel that are expressed in human T lymphocytes, and also demonstrate expression of the Ca_V1.4 protein in the human Jurkat T cell leukemia line and human peripheral blood T lymphocytes (PBTs). The carboxy-termini of both Ca_V1.4 splice isoforms contain unique exon usages distinct from the Ca_V1.4 channel isolated from human retina that may render these channel variants insensitive to changes in membrane depolarization. Additional evidence of the importance of these new splice variants comes from the demonstration that the mRNA expression of the Ca_V1.4 splice isoforms is regulated by TCR-induced activation in Jurkat T cells, and to a lesser extent in human PBTs. Overall these results provide the first evidence that structurally unique L-type Ca²⁺ channels exist in T lymphocytes, which can contribute to a Ca²⁺ influx during T lymphocyte activation.     

Effects of free Ca on the [Ca + Mg-Dependent adenosinetriphosphatase (ATPase) of Alzheimer and normal fibroblasts

Differences between Alzheimer and control fibroblasts [Ca²⁺ + Mg²⁺]-dependent ATPase activity at free Ca²⁺ concentration considerably higher than physiologic concentrations were observed. At 50 μM free Ca²⁺, Alzheimer and control fibroblast homogenates exhibited maximum velocity values ranging from 8 to 25 nmoles phosphate released/min/mg protein. Higher free Ca²⁺ (350 μM) inhibited control fibroblast ATPase activity approximately 77%; whereas, Alzheimer fibroblasts retained greater than 75% starting activity. Although the pathophysiological significance of these findings is at present unclear, these data suggest the Ca²⁺ pump of Alzheimer fibroblasts behaves differently in the presence of high free Ca²⁺. Such behavior may be of potential diagnostic value.     

Effects of divalent cations on the time course of post-tetanic decay of miniature endplate potential frequency in frogs

Relatively high external Mg²⁺ specifically induces a shoulder in the post-tetanic decay of miniature endplate potential frequency at frog neuromuscular junctions. This effect is antagonized by Ca²⁺, but not by Sr²⁺ and Mn²⁺. This shoulder formation is not caused by Mn² or dinitrophenol in the medium.

It is suggested that Mg²⁺ enters nerve terminals and displaces Ca²⁺ from internal stores; Mg²⁺ subsequently interferes with Ca²⁺ removal until the Mg²⁺ itself has been removed. The dinitrophenol result suggests that ATP-dependent Ca²⁺ extrusion is not permanently slowed. An equally likely and simpler explanation is that Mg²⁺ enters nerve terminals and the Mg²⁺ itself causes an increase in miniature endplate potential frequency. External Ca²⁺ competes with Mg²⁺ for channel entry and prevents this effect. The time course of the decay of miniature endplate potential frequency reflects the processes involved in Mg²⁺ extrusion and/or uptake.     

Discrepancy between left ventricular mechanoenergetics and mitochondrial respiratory function in canine acute failing hearts

In canine excised cross-circulated hearts, we induced three different types of acute failure (time-dependently deteriorated, calcium (Ca²⁺) overloaded, and capsaicin-induced) to investigate the relation between left ventricular mechanoenergetics and myocardial subcellular (mitochondrial) energetics. First, we measured left ventricular pressure and volume and coronary arteriovenous oxygen content difference and blood flow. We analyzed these data by using the framework of the E_max (a contractility index)-PVA (pressure-volume area; total mechanical energy)-V_O2 (oxygen consumption per beat) relation. All acute failing hearts demonstrated similar changes in mechanoenergetics, that is, decreased E_max and decreasedV_O2 for the excitation-contraction coupling (presumably Ca²⁺ handling). We then examined the mitochondrial respiratory function by measuring their oxygen consumption for ATP synthesis polarographically. As indexes of the function, respiratory control index (RCI) and State III O₂ consumption were obtained with succinate as substrate. RCI and State III O₂ consumption behaved differently among the three different types of acute cardiac failure. We conclude that the left ventricular mechanoenergetics do not directly reflect changes in mitochondrial energetics in different types of acute failing.     

西洋参茎叶总皂苷对缺血/再灌注大鼠心肌线粒体膜电位及细胞凋亡的影响

 目的：研究西洋参茎叶总皂苷 (PQS) 对大鼠缺血/再灌注 (I/R) 损伤后心肌细胞凋亡的影响,并从线粒体膜电位 (ΔΨm) 及线粒体凋亡通路探讨其可能的机制。方法：健康雄性SD大鼠90只,随机分为假手术(sham)组、模型(I/R)组、PQS (200 mg·kg-1·d-1, 灌胃6周)+I/R组、环孢霉素A (CsA;10 mg·kg-1,再灌前10 min腹腔注射)组、CsA+I/R组和PQS+CsA+I/R组,各组n=15。除sham组和CsA组大鼠开胸后冠状动脉左前降支（LAD）下穿线不结扎外,其余各组大鼠常规麻醉后,结扎LAD 30 min,再灌注120 min复制I/R模型。生化分析仪测血清乳酸脱氢酶（LDH）含量,氯化三苯基四氮唑（TTC）和伊文思蓝双染法测心梗面积,原位缺口末端标记法（TUNEL）检测各组大鼠心肌细胞凋亡情况,Western blotting检测细胞凋亡相关蛋白的表达。采用JC-1作为荧光探针,用激光共聚焦显微镜和荧光酶标仪测定ΔΨm水平。结果：与sham组比较,I/R组血清LDH活性、心梗面积和心肌细胞凋亡率均显著升高（P<0.05）;与I/R组相比,PQS+I/R组、CsA+I/R组和PQS+CsA+I/R组血清LDH活性、心梗面积和心肌细胞凋亡率均显著降低（P<0.05）。Western blotting结果显示,与sham组相比,I/R组心肌Bcl-2蛋白表达量降低,Bax、胞浆cytochrome C和cleaved caspase-3升高（均P<0.05）;与I/R组相比,PQS+I/R组、CsA+I/R组和PQS+CsA+I/R组心肌Bcl-2蛋白表达量升高,Bax、胞浆cytochrome C及心肌cleaved caspase-3蛋白表达量均降低（P<0.05）。激光共聚焦显微镜观察结果示,I/R组线粒体JC-1染色后红色荧光强度减弱,荧光酶标仪测相对荧光单位（RFU） 较sham组降低（P<0.05）;PQS+I/R组、CsA+I/R组和PQS+CsA+I/R组RFU均较I/R组升高（P<0.05）。结论：PQS显著降低大鼠I/R后心肌细胞损伤,减少细胞凋亡,其机制与维持再灌注期ΔΨm稳定、抑制线粒体凋亡通路的激活有关。     

周围神经损伤后脊髓前角运动神经元内游离Ca2+的浓度

背景：神经损伤后可引起细胞膜上Ca²⁺通道的开放,使细胞外Ca²⁺内流,造成细胞内的钙超载。 目的：观察臂丛神经根切断伤后相应脊髓前角运动神经元内游离Ca²⁺浓度的变化。 方法：健康成年雄性Wistar大鼠84只,分为3组：假手术组暴露臂丛神经不切断;对照组臂丛神经切断伤后不做其他处理;实验组臂丛神经切断伤后,给予腹腔注射Ca2+阻带剂维拉帕米4 mg/(kg•d)。于切断伤后12 h,24 h,48 h,72 h,1周,2周,4周每组随机以4只取材。 结果与结论：周围神经损伤开始,对照组及实验组大鼠损伤侧脊髓前角运动神经元细胞内Ca²⁺浓度开始升高,至伤后48 h达高峰,此后逐渐下降,伤后1周,实验组已基本回至正常水平,但仍较假手术组高。说明神经损伤后相应神经元细胞膜上L型Ca²⁺通道开放,Ca2+内流进入细胞内,导致神经细胞内游离Ca²⁺浓度增加,L型Ca²⁺通道可以被维拉帕米阻断,减少神经损伤后的Ca²⁺内流,减少神经细胞凋亡的数量。     

Metabolic Regulation of Ca2+ Release in Permeabilized Mammalian Skeletal Muscle Fibres

In the present study, the link between cellular metabolism and Ca²⁺ signalling was investigated in permeabilized mammalian skeletal muscle. Spontaneous events of Ca²⁺ release from the sarcoplasmic reticulum were detected with fluo-3 and confocal scanning microscopy. Mitochondrial functions were monitored by measuring local changes in mitochondrial membrane potential (with the potential-sensitive dye tetramethylrhodamine ethyl ester) and in mitochondrial [Ca²⁺] (with the Ca²⁺ indicator mag-rhod-2). Digital fluorescence imaging microscopy was used to quantify changes in the mitochondrial autofluorescence of NAD(P)H. When fibres were immersed in a solution without mitochondrial substrates, Ca²⁺ release events were readily observed. The addition of l -glutamate or pyruvate reversibly decreased the frequency of Ca²⁺ release events and increased mitochondrial membrane potential and NAD(P)H production. Application of various mitochondrial inhibitors led to the loss of mitochondrial [Ca²⁺] and promoted spontaneous Ca²⁺ release from the sarcoplasmic reticulum. In many cases, the increase in the frequency of Ca²⁺ release events was not accompanied by a rise in global [Ca²⁺]_i. Our results suggest that mitochondria exert a negative control over Ca²⁺ signalling in skeletal muscle by buffering Ca²⁺ near Ca²⁺ release channels.     

Mechanism of histamine-induced calcium efflux from cultured bovine adrenal chromaffin cells: possible involvement of an Na/Ca exchange mechanism

The effect of stimulation of the histamine receptor on Ca²⁺ mobilization in cultured bovine adrenal chromaffin cells was examined. Histamine (10⁻⁵ M) increased the intracellular free Ca²⁺ ([Ca²⁺]_i) to a peak in the presence or absence of extracellular Ca²⁺, followed by decrease with time. Histamine (10⁻⁸–10⁻⁵ M) also stimulated ⁴⁵Ca²⁺ efflux from cultured bovine adrenal chromaffin cells in a concentration dependent manner. Its stimulatory effect on ⁴⁵Ca²⁺ efflux was inhibited by the specific histamine H₁ receptor antagonist mepyramine. The increase in histamine-stimulated ⁴⁵Ca²⁺ efflux was inhibited by deprivation of extracellular Na⁺ and by the Na⁺/Ca²⁺ exchange inhibitor amiloride. In addition, histamine stimulated ²²Na⁺ influx into the cells, and this action was inhibited by amiloride. These results suggest that stimulation of the histamine H₁ receptor regulates Na⁺/Ca²⁺ exchange in cultured bovine adrenal chromaffin cells.     

The role of Ca2+ in the generation of spontaneous astrocytic Ca2+ oscillations

Astrocytes in the rat thalamus display spontaneous [Ca²⁺]_i oscillations that are due to intracellular release, but are not dependent on neuronal activity. In this study we have investigated the mechanisms involved in these spontaneous [Ca²⁺]_i oscillations using slices loaded with Fluo-4 AM (5 μM) and confocal microscopy. Bafilomycin A1 incubation had no effect on the number of spontaneous [Ca²⁺]_i oscillations indicating that they were not dependent on vesicular neurotransmitter release. Oscillations were also unaffected by ryanodine. Phospholipase C (PLC) inhibition decreased the number of astrocytes responding to metabotropic glutamate receptor (mGluR) activation but did not reduce the number of spontaneously active astrocytes, indicating that [Ca²⁺]_i increases are not due to membrane-coupled PLC activation. Spontaneous [Ca²⁺]_i increases were abolished by an IP3 receptor antagonist, whilst the protein kinase C (PKC) inhibitor chelerythrine chloride prolonged their duration, indicating a role for PKC and inositol 1,4,5,-triphosphate receptor activation. BayK8644 increased the number of astrocytes exhibiting [Ca²⁺]_i oscillations, and prolonged the responses to mGluR activation, indicating a possible effect on store-operated Ca²⁺ entry. Increasing [Ca²⁺]_o increased the number of spontaneously active astrocytes and the number of transients exhibited by each astrocyte. Inhibition of the endoplasmic reticulum Ca²⁺ ATPase by cyclopiazonic acid also induced [Ca²⁺]_i transients in astrocytes indicating a role for cytoplasmic Ca²⁺ in the induction of spontaneous oscillations. Incubation with 20 μM Fluo-4 reduced the number of astrocytes exhibiting spontaneous increases.

This study indicates that Ca²⁺ has a role in triggering Ca²⁺ release from an inositol 1,4,5,-triphosphate sensitive store in astrocytes during the generation of spontaneous [Ca²⁺]_i oscillations.