马尾松花粉硫酸酯化多糖对大鼠主动脉平滑肌细胞[Ca2＋]i调控及增殖的影响

目的研究马尾松花粉多糖PPM60．A及其硫酸酯化物SPPM60-A对大鼠动脉平滑肌细胞[Ca2＋]i调控及增殖的影响。方法常规水提醇沉法制备马尾松花粉多糖,SephacrylS-400HR色谱分离得PPM60-A,氯磺酸一吡啶法得硫酸酯化物SPPM60-A,酯化度为1．28。酶解法分离制备大鼠动脉平滑肌细胞,测定酯化前后多糖对其胞内[Ca2＋]i和细胞增殖的影响。结果PPM60．A和SPPM60-A均可以降低[Ca2＋]i,抑制高K＋和去甲肾上腺素（NE）诱导的钙离子升高,降低高K＋引起的钙离子水平上升,对NE诱导的血管主动脉平滑肌细胞增殖具有显著的抑制作用。PPM60．A作用效果好于SPPM60-A。结论PPM60．A及SPPM60．A均能抑制细胞外Ca2＋内流,抑制血管平滑肌细胞增殖。     

神经肽DGAVP和Org2766对神经细胞内游离Ca2+的影响

运用Ca²⁺指示剂Fura-2作为细胞内钙离子的荧光探针,利用AR—CM—MIC阳离子测定系统,检测了分离的神经细胞内游离钙及其变化,并观测了DGAVP和Org2766对蛋白质合成抑制剂茴香霉素(ANI)引起细胞内钙离子浓度([Ca²⁺]_i)变化的影响。结果表明茴香霉素可使[Ca²⁺]_i显著升高,且有量效关系;DGAVP本身并不引起[Ca²⁺]_i发生显著变化,但适当剂量的DGAVP可显著对抗一定剂量范围内ANI升高[Ca²⁺]_i的作用,提示DGAVP对抗ANI的蛋白质合成抑制效应可能是通过拮抗ANI升高[Ca²⁺]_i这一途径实现的,另一神经肽Org2766则可能不是通过这一机制发生作用。从细胞内Ca²⁺的角度看,这两种肽的作用机理显然是不同的。     

眼睛蛇毒心脏毒素对大鼠心肌细胞收缩及细胞内钙离子的作用

目的研究眼睛蛇毒心脏毒素(Cardiotoxin,CTX)对心肌细胞的形态、收缩幅度和细胞内钙离子([Ca²⁺]_i)的作用。方法应用荧光计量法(以Fura-2/AM为荧光染料)及光学成像系统来测定单个心肌细胞[Ca²⁺]_i和收缩幅度。结果0.001～1μmol/L的CTX使心肌细胞由杆状变成圆形,药物的作用从第1分钟时开始,到第20分钟时趋于稳定。在电刺激存在的情况下,1μmol/L的CTX最初导致电诱导的[Ca²⁺]_i和收缩幅度瞬间增加,接下来[Ca²⁺]_i时程延长,最终细胞对电刺激不敏感、突然收缩、[Ca²⁺]_i持续增高。在缺乏电刺激的情况下,1μmol/L的CTX可诱导Ca²⁺震荡波、持续性[Ca²⁺]_i增高,这种作用与40mmol/L的KC l和10mmol/L咖啡因所引起的[Ca²⁺]_i瞬间增加不同。结论CTX作用初期使[Ca²⁺]_i增高,使细胞[Ca²⁺]_i超载,同时伴随细胞形状的改变。     

毒毛旋花子苷元对豚鼠心室肌细胞内钙浓度的影响

观察毒毛旋花子苷元(strophanthidin, Str)对分离豚鼠心室肌细胞内游离钙浓度(［Ca²⁺］_i)的影响。酶解分离豚鼠心室肌细胞, 用Fluo 3-AM负载, 激光共聚焦显微镜法测定单个豚鼠心室肌细胞［Ca²⁺］_i的荧光密度。Str可浓度依赖性地升高［Ca²⁺］_i, Str (10 μmol·L^-1)在［Ca²⁺］_i升高达峰值时, 可使细胞挛缩, 而Str (1和10 nmol·L^-1)对细胞形态无影响。TTX、 尼索地平或升高细胞外钙可影响Str (1和100 nmol·L^-1)对［Ca²⁺］_i的升高作用,而对Str (10 μmol·L^-1)无明显影响。在外液中加入ryanodine或去除细胞外钙, 则3个检测浓度的Str升高［Ca²⁺］_i作用均被明显抑制。在无K⁺、 无Na⁺液中, 10 μmol·L^-1 Str升高［Ca²⁺］_i的作用减弱, 而Str (1和100 nmol·L^-1)升高［Ca²⁺］_i的作用无明显影响。加入TTX、 尼索地平或增加细胞外的钙离子浓度, 则3个检测浓度Str的作用均受到影响。提示低浓度Str对［Ca²⁺］_i的升高作用与抑制Na⁺、K⁺-ATP酶活性无关, 而与促进L-型钙通道和TTX敏感性钠通道的“slip-mode”钙电导有关; 高浓度Str升高［Ca²⁺］_i的作用则是抑制Na⁺、K⁺-ATP酶的结果。此外, Str对［Ca²⁺］_i的升高作用还与直接作用于ryanodine受体促进内钙释放有关。     

阿米洛利对大鼠压力超负荷性心肌肥厚的抑制作用

目的　观察钠氢交换体(NHE)抑制剂阿米洛利(Ami)对压力超负荷左室肥厚(LVH)大鼠心功能、心肌细胞内游离钙浓度([Ca²⁺]_i)及心肌细胞膜Na⁺ 、K⁺-ATP酶活性的影响。方法　①同步记录离体工作心脏LVSP、LVEDP、±dp/dt_max及T值;②测定Fura-2/A负载后的单个心室肌细胞的[Ca²⁺]_i;③光电比     

前胡丙素对培养大鼠心肌细胞内游离Ca2+的影响

用Fura-2/AM技术直接观察前胡丙素(Pra-C)对培养大鼠心室肌细胞内游离钙的影响。结果显示Pra-C浓度为0.1～1.0μmol·L^-1可明显抑制CaCl₂,高K⁺和Bay K 8644引起[Ca²⁺]i增加,并且有剂量—效应关系,对ouabain引起的[Ca²⁺]i增加无明显作用。结果提示Pra-C降低心肌细胞[Ca²⁺]i的作用与抑制电压依赖性钙通道有关。     

小檗碱对培养大鼠神经细胞内游离Ca2+的影响

以Fura-2/AM为细胞内钙离子的荧光指示剂,用AR-CM-MIC阳离子测定系统,直接测定了体外培养的新生大鼠神经细胞内游离钙([Ca²⁺]i)值,并观察了小檗碱(Ber)的影响。结果表明,Ber对神经细胞静息[Ca²⁺]i无明显影响,Ber1～100μmol·L^-1能剂量依赖地抑制去甲肾上腺素和H₂O₂引起的[Ca²⁺]i升高,其IC₅₀分别为39.9和17.9μmol·L^-1。高剂量Ber(10～100μmol·L^-1)能抑制高K+引起的[Ca²⁺]i升高。姐果提示,Ber对去甲肾上腺素,高K⁺及H₂O₂引起的[Ca²⁺]i升高的抑制作用可能是其抗脑缺血作用机制之一。     

小檗碱对培养大鼠心肌细胞胞内游离Ca2+的作用

利用Fura-2技术和AR-CM-MIC阳离子测定系统,直接观察了小檗碱对培养大鼠心肌细胞胞内[Ca²⁺]i的影响。结果显示:小檗碱可明显升高心肌细胞静息[Ca²⁺]i且具饱合性,维拉帕米和CoCl₂对其有一定的抑制作用;小檗碱与高K⁺,高Ca²⁺,去甲肾上腺素,哇巴因合用比单用上述激动剂更能明显增高[Ca²⁺]i;维拉帕米对其有抑制作用;在胞外无外Ca²⁺和无外Ca²⁺,外K⁺,外Na⁺时,小檗碱30～200μmol·L^-1仍能升高静息[Ca²⁺]i,维拉帕米只对前者有一定抑制作用。结果提示:小檗碱可能通过促胞外Ca²⁺内流和胞内Ca²⁺释放等途径有限度地增高心肌细胞内游离Ca²⁺浓度,显示强心作用。     

A K+ single channel and whole-cell clamp study on the effects of levcromakalim in guinea pig portal vein cells

Single channel cell-attached patch and whole-cell clamp experiments on the mode of action of the K⁺ channel opener (KCO), levcromakalim, were performed in guinea pig isolated portal vein cells. At +20 mV (135/23 mM K⁺ in bath/pipette), 10 μM levcromakalim activated K⁺ channels with a chord conductance of 23.2 pS (K_KCO), which were sensitive to the blocker of ATP-dependent K⁺ channels (K_ATP), glibenclamide. Voltage steps from –80 mV to +20 mV activated 4-aminopyridine-sensitive K⁺ channels of 6.5 pS with properties of delayed rectifier K⁺ channels (K_v). In patches which upon a previous voltage step had revealed the existence of K_v, levcromakalim reduced the open-probability of K_v, but it did not concomitantly activate K_KCO. During the course of the experiments, but unrelated to the presence of levcromakalim, large conductance K⁺ channels (BK_Ca) appeared which could be inhibited by iberiotoxin, a selective blocker of BK_Ca, and by the membrane-permeant calcium buffer, BAPTA/AM, but not by glibenclamide. Whole-cell current-voltage (i-V) relations were established in response to voltage ramps from +50 mV to –100 mV; on subtraction of control i-V curves from i-V curves obtained in the presence of 10 μM levcromakalim, the KCO-induced K⁺ current remained which was proportional to voltage. This is not compatible with the upward-bent curvature predicted by the GHK current equation for purely resistive channels at high [K⁺]_i versus low [K⁺]_o. In conclusion, in the guinea pig portal vein cells, no evidence could be established for the hypotheses that KCOs may act via conversion of K_v to K_ATP (Beech and Bolton 1989; Edwards et al. 1993) or by activation of BK_Ca (Balwierczak et al. 1995). In these cells, mild inward rectification of the levcromakalim-induced current was observed which underlines their relationship to K_ATP in other tissues. Received: 1 August 1997 / Accepted: 7 June 1998     

间尼索地平对5-羟色胺诱导的大鼠肺动脉平滑肌细胞增殖中Ca2+/CaM/CaN通路的影响

探讨Ca²⁺/CaM/CaN信号通路在5-羟色胺 (5-HT) 诱导的大鼠肺动脉平滑肌细胞 (PASMCs) 增殖中的作用以及间尼索地平 (m-Nis) 对此的影响。采用细胞培养、噻唑蓝 (MTT) 比色检测、激光共聚焦显微镜及反转录聚合酶链反应 (RT-PCR) 等方法, 研究5-HT (1 μmol·L⁻¹) 对大鼠PASMCs细胞增殖的影响以及m-Nis对此的抑制作用, 并通过检测m-Nis对5-HT诱导的大鼠PASMCs增殖中Ca²⁺/CaM/CaN通路的影响深入探讨其作用机制。结果显示, 5-HT (1 μmol·L⁻¹) 可明显诱导大鼠PASMCs增殖 (P < 0.01), m-Nis对此有明显的抑制作用, 并呈现一定的浓度依赖性 (P < 0.05或P < 0.01); 另外, m-Nis还明显抑制了5-HT引起的[Ca²⁺]_i的升高 (P < 0.01)、CaM和CaN mRNA的表达以及CaN活性的升高 (P < 0.05或P < 0.01)。结果表明, Ca²⁺/CaM/CaN信号通路在5-HT诱导的大鼠PASMCs增殖中起重要作用, m-Nis抗5-HT诱导的增殖作用可能与抑制[Ca²⁺]_i增高从而阻断了Ca²⁺/CaM/CaN信号通路有关。     

马尾松花粉硫酸酯化多糖调控脾细胞[Ca~(2+)]_i的研究

目的探讨马尾松花粉多糖硫酸酯化物(SPPM60-A)对小鼠脾细胞内游离钙离子浓度([Ca2+]i)调控的机制。方法热水浸提醇沉淀法提取马尾松花粉粗多糖,SephacrylS-400HR分离纯化,氯磺酸-吡啶法进行硫酸酯化,荧光分光光度计测定脾淋巴细胞[Ca2+]i。结果 LPS与SPPM60-A都能促进脾淋巴细胞[Ca2+]i升高,与对照相比升高率分别为11.7%和15.4%(P<0.01)。TAK-242、LY294002、U73122、低分子肝素与维拉帕米均可以抑制[Ca2+]i的升高。结论推测硫酸酯化马尾松花粉多糖可通过TLR4-PI3K-PLC-IP3R信号通路促进脾淋巴细胞内[Ca2+]i升高。     

The Vasodilator Effect and Its Mechanism of Sulfur Dioxide-Derivatives on Isolated Aortic Rings of Rats

The vasodilator effect of exogenous sulfur dioxide (SO₂) derivatives (mixture of sodium bisulfite and sodium sulfite, 3:1 M/M in neutral solution) on rat vascular system was studied in order to explore the mechanism of blood pressure lowered by SO₂ and its derivatives. Isolated rat aortic rings were perfused in bath tubes containing various chemicals and their tensions were recorded. The results showed: (1) The SO₂ derivatives could relax isolated aorta precontracted by norepinephrine (NE) or potassium chloride (KCl) in a dose-dependent manner. (2) This vasodilator effect was attenuated after preincubation with indomethacin, but was not affected by N-L-nitro-arginine, methylene blue, and propranolol, and was independent of the aorta endothelium. (3) The vasoconstriction responses induced by NE, KCl, or Ca²⁺ were antagonized by SO₂ derivatives in a noncompetitive manner. (4) The vasoconstrictions of two components (initial fast vasoconstriction induced by intracellular Ca²⁺ release and sustained vasoconstriction evoked by extracellular Ca²⁺ influx) were also inhibited by SO₂ derivatives. These results led to the conclusions: The SO₂ derivatives could cause vasorelaxation by a direct role of the chemicals on aortic smooth muscle cells. It was not dependent on vascular endothelium and was independent of nitric oxide (NO). It is suggested that SO₂ and its derivatives might be also vasoactive substances that modulate changes of blood pressure, like other gasotransmitters. The vasorelaxation might be related to the inhibition effects of SO₂ derivatives on Ca²⁺ entry through both potential-dependent calcium channels and receptor-operating calcium channels, and also to the inhibition of intracellular Ca²⁺ release. The vasorelaxation was at partly related to the increase of prostacyclin (PGI₂) induced by SO₂ derivatives.     

Cyanidin-3-glucoside Inhibits ATP-induced Intracellular Free Ca2+ Concentration,ROS Formation and Mitochondrial Depolarization in PC12 Cells

Flavonoids have an ability to suppress various ion channels. We determined whether one of flavonoids, cyanidin-3-glucoside, affects adenosine 5''-triphosphate (ATP)-induced calcium signaling using digital imaging methods for intracellular free Ca²⁺ concentration ([Ca²⁺]_i), reactive oxygen species (ROS) and mitochondrial membrane potential in PC12 cells. Treatment with ATP (100µM) for 90 sec induced [Ca²⁺]_i increases in PC12 cells. Pretreatment with cyanidin-3-glucoside (1µ g/ml to 100µg/ml) for 30 min inhibited the ATP-induced [Ca²⁺]_i increases in a concentration-dependent manner (IC₅₀=15.3µg/ml). Pretreatment with cyanidin-3-glucoside (15µg/ml) for 30 min significantly inhibited the ATP-induced [Ca²⁺]_i responses following removal of extracellular Ca²⁺ or depletion of intracellular [Ca²⁺]_i stores. Cyanidin-3-glucoside also significantly inhibited the relatively specific P2X2 receptor agonist 2-MeSATP-induced [Ca²⁺]_i responses. Cyanidin-3-glucoside significantly inhibited the thapsigargin or ATP-induced store-operated calcium entry. Cyanidin-3-glucoside significantly inhibited the ATP-induced [Ca²⁺]_i responses in the presence of nimodipine and ω-conotoxin. Cyanidin-3-glucoside also significantly inhibited KCl (50 mM)-induced [Ca²⁺]_i increases. Cyanidin-3-glucoside significantly inhibited ATP-induced mitochondrial depolarization. The intracellular Ca²⁺ chelator BAPTA-AM or the mitochondrial Ca²⁺ uniporter inhibitor RU360 blocked the ATP-induced mitochondrial depolarization in the presence of cyanidin-3-glucoside. Cyanidin-3-glucoside blocked ATP-induced formation of ROS. BAPTA-AM further decreased the formation of ROS in the presence of cyanidin-3-glucoside. All these results suggest that cyanidin-3-glucoside inhibits ATP-induced calcium signaling in PC12 cells by inhibiting multiple pathways which are the influx of extracellular Ca²⁺ through the nimodipine and ω-conotoxin-sensitive and -insensitive pathways and the release of Ca²⁺ from intracellular stores. In addition, cyanidin-3-glucoside inhibits ATP-induced formation of ROS by inhibiting Ca²⁺-induced mitochondrial depolarization.     

M3受体在脑心综合征心律失常中的作用

观察M₃受体(M₃R)在脑心综合征(CCS)模型大鼠心室肌中的作用及其表达，以探讨其与CCS心律失常的关系。线栓法建立CCS模型，监测心电图，激光共聚焦扫描显微镜记录激动M₃R后心肌细胞内钙的变化，Western blotting检测心室肌中M₃R表达水平的改变。同对照组相比，模型组心电图QRS、QT间期显著延长(P<0.01)；Western blotting结果显示模型组心室肌中M₃R的表达水平较对照组显著降低(P<0.05)；胆碱激动M₃R可以抑制KCl除极诱导的模型组心肌细胞内钙的升高幅度(P<0.01)，其特异阻断剂4-DAMP可以阻断这一作用。M₃R表达降低可能是CCS心律失常的重要原因之一。激动M₃R可以降低心肌细胞内钙，M₃R可能是治疗CCS心律失常新的靶点。     

Cellular calcium regulatory machinery of vasorelaxation elicited by petasin

1. The present study examined the cytosolic Ca²⁺ regulatory machinery involved in the vasorelaxation produced by petasin, a sesquiterpene isolated from Petasites formosanus. 2. Aortic rings isolated from Sprague‐Dawley rats were exposed to petasin (0.01–100 μmol/L) to elucidate its vascular effects on isometric contraction elicited by vasoconstrictors, as well as the contribution of the endothelium and Ca²⁺ to the responses observed. In addition, L‐type voltage‐dependent Ca²⁺ channel (VDCC) activity and [Ca²⁺]_i were determined in cultured vascular smooth muscle cells (VSMCs) from Sprague‐Dawley rats in the presence of 1–100 μmol/L petasin using whole‐cell patch‐clamp recording and the fluorescent probe fura‐2/AM. The effects of petasin on vascular responses were compared between aortic rings from spontaneously hypertensive rats (SHR) and normotensive Wistar‐Kyoto (WKY) rats. 3. Petasin reduced isometric contraction elicited by KCl or the L‐type Ca²⁺ channel opener BayK 8644 (IC₅₀ 3.0 ± 0.4 and 4.1 ± 1.1 μmol/L, respectively) in aortic rings isolated from Sprague‐Dawley rats, independent of the endothelium. In addition, petasin triggered a rightward shift in the concentration–response curve to KCl while reducing the maximal response by 82%. In Ca²⁺‐depleted and high K⁺‐depolarized aortic rings, 1–100 μmol/L petasin pretreatment attenuated the Ca²⁺‐induced contraction in a concentration‐dependent manner. 4. In cultured VSMCs, whole‐cell patch‐clamp recording revealed that petasin inhibited VDCC activity. Measurement of [Ca²⁺]_i using fura‐2/AM fluorescence indicated that petasin suppressed the KCl‐induced increase in [Ca²⁺]_i. However, receptor binding assays failed to identify any significant interaction between petasin and the dihydropyridine binding sites of the L‐type VDCC. 5. In aortic rings from SHR and WKY rats, petasin inhibited Ca²⁺‐induced contractions in Ca²⁺‐depleted and high K⁺‐depolarized solution with a more pronounced effect in rings from SHR. 6. Together, the results suggest that direct Ca²⁺ antagonism of L‐type VDCC in vascular smooth muscle may account, at least in part, for petasin‐induced vasorelaxation. The more pronounced effect of the sesquiterpene in blood vessels from SHR suggests its possible therapeutic potential in the mangement of hypertension.     

Rhynchophylline-induced vasodilation in human mesenteric artery is mainly due to blockage of L-type calcium channels in vascular smooth muscle cells

Rhynchophylline (Rhy) is a pharmacologically active substance isolated from Uncaria rhynchophylla which has been used to treat cardiovascular diseases and has drawn considerable attention in recent years for its antihypertensive activities. We investigated the actions of Rhy on endothelium-denuded human mesenteric artery by tension measurement and its actions on high conductance Ca²⁺-activated K⁺ channels (BK_Ca) currents and calcium currents (I_Ca) in freshly isolated smooth muscle cells using perforated patch clamp technique. Intracellular Ca²⁺ level was measured in Fura-2-loaded cells. Rhy inhibited both the KCl and BayK-evoked mesenteric artery constrictions in a dose-dependent manner. K⁺ channel blockers (TEA, glibenclamide, IbTX, and 4-AP) did not affect the vasorelaxing effect of Rhy. Rhy inhibited L-type voltage-gated Ca²⁺ current (I_Ca,L) but had no significant effect on macroscopic BK_Ca current. Rhy preincubation markedly reduced the elevation of [Ca²⁺]_i level induced by KCl depolarization. Caffeine-stimulated [Ca²⁺]_i elevation was also decreased to some extent by pretreatment with Rhy for 20 min. Our results show that Rhy relaxes smooth muscles of human mesenteric resistance arteries, mainly due to inhibition of Ca²⁺ influx by blockage of L-type Ca²⁺ channels and thereby the decrease in [Ca²⁺]_i.     

Effects of Apigenin on Glutamate-induced [Ca2+]i Increases in Cultured Rat Hippocampal Neurons

Flavonoids have been shown to affect calcium signaling in neurons. However, there are no reports on the effect of apigenin on glutamate-induced calcium signaling in neurons. We investigated whether apigenin affects glutamate-induced increase of free intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cultured rat hippocampal neurons, using fura-2-based digital calcium imaging and microfluorimetry. The hippocampal neurons were used between 10 and 13 days in culture from embryonic day 18 rats. Pretreatment of the cells with apigenin (1 µM to 100 µM) for 5 min inhibited glutamate (100 µM, 1 min) induced [Ca²⁺]_i increase, concentration-dependently. Pretreatment with apigenin (30 µM) for 5 min significantly decreased the [Ca²⁺]_i responses induced by two ionotropic glutamate receptor agonists, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA, 10 µM, 1 min) and N-methyl-D-aspartate (NMDA, 100 µM, 1 min), and significantly inhibited the AMPA-induced peak currents. Treatment with apigenin also significantly inhibited the [Ca²⁺]_i response induced by 50 mM KCl solution, decreased the [Ca²⁺]_i responses induced by the metabotropic glutamate receptor agonist, (S)-3,5-dihydroxyphenylglycine (DHPG, 100 µM, 90 s), and inhibited the caffeine (10 mM, 2 min)-induced [Ca²⁺]_i responses. Furthermore, treatment with apigenin (30 µM) significantly inhibited the amplitude and frequency of 0.1 mM [Mg²⁺]_o-induced [Ca²⁺]_i spikes. These data together suggest that apigenin inhibits glutamate-induced calcium signaling in cultured rat hippocampal neurons.     

Adenine attenuates the Ca<Superscript>2+</Superscript> contraction-signaling pathway via adenine receptor-mediated signaling in rat vascular smooth muscle cells

Our previous study demonstrated that adenine (6-amino-6H-purine) relaxed contracted rat aorta rings in an endothelial-independent manner. Although adenine receptors (AdeRs) are expressed in diverse tissues, aortic AdeR expression has not been ascertained. Thus, the aims of this study were to clarify the expression of AdeR in rat vascular smooth muscle cells (VSMCs) and to investigate the adenine-induced vasorelaxation mechanism(s). VSMCs were isolated from 8-week-old male Wistar-Kyoto rats and used in this study. Phosphorylation of myosin light chain (p-MLC) was measured by western blot. AdeR mRNA was detected by RT-PCR. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured by using Fura-2/AM. Vasorelaxant adenine (10–100 μM) significantly reduced p-MLC by angiotensin II (Ang II, 10 μM) in VSMCs (P < 0.05). We confirmed the expression of aortic AdeR mRNA and the activation of PKA in VSMCs through stimulation of AdeR by adenine by ELISA. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) measurement demonstrated that adenine inhibits Ang II- and m-3M3FBS (PLC agonist)-induced [Ca²⁺]_i elevation. In AdeR-knockdown VSMCs, PKA activation and p-MLC reduction by adenine were completely abolished. These results firstly demonstrated that vasorelaxant adenine can suppress Ca²⁺ contraction signaling pathways via aortic AdeR/PKA activation in VSMCs.     

IBUDILAST REDUCES INTRACELLULAR CALCIUM ELEVATION INDUCED BY IN VITRO ISCHAEMIA IN GERBIL HIPPOCAMPAL SLICES

1. A microfluorometry was carried out to investigate the effect of 3-isobutyryl-2-isopropylpyrazolo[1,5-a]pyridine (ibudilast) on changes in levels of intracellular calcium concentration ([Ca²⁺]_i) induced by in vitro ischaemia in the CA1 field of gerbil hippocampal slices. 2. When slices, loaded with a calcium ion sensitive dye (rhod-2) were exposed to a glucose-free physiological medium equilibrated with a 95% N₂/5% CO₂ gas mixture (standard in vitro ischaemia), a large [Ca²⁺]_i elevation was detected approximately 5 min after the beginning of in vitro ischaemia. 3. When slices were perfused with the in vitro ischaemic medium containing 43 μmol/L ibudilast, a [Ca²⁺]_i elevation was still observed; however, the extent of the increase in [Ca²⁺]_i was significantly depressed in all subregions of the hippocampal slices. 4. The extent of this inhibitory effect of ibudilast on the in vitro ischaemia-induced [Ca²⁺]_i elevation was in a similar range as those of Ca²⁺ blockers, including (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptan-5,10-imine maleate (MK-801), flunarizine and dantrolene. 5. Similar [Ca²⁺]_i increases in the CA1 field were induced by a Ca²⁺-free in vitro ischaemia, a high concentration of KCl or by specific agonists for glutamate receptor subtypes (N-methyl-d -aspartate (NMDA), (s)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate); these increases were also depressed with 43 μmol/L ibudilast present in the perfusion medium. 6. These results indicate that ibudilast may act by depressing the Ca²⁺ accumulation during and shortly after ischaemia, a possible pharmacological action of ibudilast that leads to the amelioration of ischaemic injury in the central nervous system.