丁咯地尔抑制NE和GIu引起的单个脑细胞游离钙增高

目的：研究丁咯地尔对去甲肾上腺素（ＮＥ）和谷氨酸（Ｇｌｕ）引起大鼠单个脑细胞内游离钙增高的影响。方法：应用ＡＲ－ＣＭ－ＭＩＣ阳离子测定系统测量细胞内游离钙（［Ｃａ２＋］１）．结果：细胞外钙为 １．３ ｍｍｏｌ·Ｌ－１,丁咯地尔 ０．ｌ,ｌ．０,１０．０μｍｏｌ·Ｌ－１对细胞静息［Ｃａ２＋］．无明显影响,对ＮＥ诱导的［Ｃａ２＋］增高明显抑制,对Ｇｌｕ 诱导的［Ｃａ２＋］ 增高具有一定的抑制作用。结论：丁咯地尔能抑制ＮＥ和Ｇｌｕ 引起的单个脑细胞游离钙增高。     

高糖对缺氧神经元的影响及钙相关机制研究

研究高糖对神经元缺氧的影响,并探讨其钙相关机制.利用SD大鼠大脑皮质神经元体外缺氧模型,通过对细胞活力的检测,观察浓度分别为22.7(对照组),30,40,50,60(高糖组)mmol/L的葡萄糖对神经元缺氧的影响;以Fura-2/Am为荧光指示剂,测定细胞内游离钙离子浓度([Ca2+]i.结果发现当培养基中葡萄糖浓度达到60 mmol/L时,高糖可引起缺氧神经元损伤;与对照组相比,有钙介质与无钙介质中静息[Ca2+]i均升高,P＜0.01;但对照组与高糖组在有钙介质中对氯化钾(KCl)、谷氨酸(Glu)刺激引起的[Ca2+]i升高无明显差异,P＞0.05;在无钙介质中,对氯化钙(CaCl2)引起的[Ca2+]i增高率无明显差别,P＞0.05.本研究表明:高糖对神经元的损伤作用可能与其促进细胞内钙离子释放,诱发细胞内钙超载有关.     

阿魏酸钠对培养的皮质神经细胞内游离Ca2+的影响

目的 :研究阿魏酸钠对谷氨酸诱导培养的皮质神经细胞损伤的作用。方法 :采用新生大鼠皮质神经细胞原代培养建立谷氨酸神经细胞损伤模型 ,用Ca2 +指示剂Fura 2 /AM检测神经细胞内游离钙浓度 ( [Ca2 +] i)的变化 ,并观察反映神经细胞受损程度的培养液中乳酸脱氢酶 (LDH)的释放量的变化。结果 :阿魏酸钠 40～ 10 0 μmol·L-1能剂量依赖性抑制谷氨酸钠所引起的 [Ca2 +] i 升高及LDH释放。结论 :阿魏酸钠通过抑制谷氨酸钠所引起的 [Ca2 +] i 升高可能是其抗氧化性神经损伤作用的重要机制     

大鼠小直径痛觉三叉神经节神经元同时存在三磷酸肌醇敏感性钙库和阿诺碱敏感性钙库？P2X和P2Y嘌呤受体介导细胞内钙信号转导通路的变化

背景： 面部创伤或面部手术等可能损伤三叉神经系统而导致三叉神经疼痛,由于其疼痛剧烈难忍、易复发,长期以来一直为口腔临床治疗的一大难题。现有大量研究发现嘌呤类受体与三叉神经痛相关,目前对其作用机制知之甚少。 目的：探讨在小直径三叉神经节神经元中嘌呤类受体介导钙信号途径。 方法：用Fura-2荧光染料通过显微镜荧光测定技术实时检测急性分离成年大鼠小直径三叉神经节神经元的细胞内钙离子浓度。 结果：用正常外液或去除细胞外Ca2+灌流细胞,分别给予thapsigargin（1 μmol/L）,内质网钙泵ATP酶抑制剂,和咖啡因（20 mmol/L）,ryanodine受体激动剂,均能够引起细胞内游离钙离子浓度（[Ca2+]i）不同程度地升高。ATP（100 μmol/L）也能够产生类似的效应。在去除细胞外Ca2+条件下,ATP引起的[Ca2+]i升高可被thapsigargin可逆性地抑制,而不能被咖啡因抑制;然而在正常外液环境中,ATP引起的细胞内[Ca2+]i 升高不能完全地被thapsigargin抑制。 结论：在痛觉三叉神经节神经元中,嘌呤类受体介导的[Ca2+]i升高有两条途径,一种途径是通过代谢型P2Y受体作用于三磷酸肌醇敏感性钙库;另一种途径是通过离子型受体P2X受体引起外钙内流。     

高糖对缺氧神经元的影响及钙相关机制研究

研究高糖对神经元缺氧的影响 ,并探讨其钙相关机制。利用 SD大鼠大脑皮质神经元体外缺氧模型 ,通过对细胞活力的检测 ,观察浓度分别为 2 2 .7(对照组 ) ,30 ,40 ,50 ,60 (高糖组 ) mmol/ L的葡萄糖对神经元缺氧的影响 ;以 Fura- 2 / Am为荧光指示剂 ,测定细胞内游离钙离子浓度 ([Ca2 ]i。结果发现当培养基中葡萄糖浓度达到 60 mmol/ L时 ,高糖可引起缺氧神经元损伤 ;与对照组相比 ,有钙介质与无钙介质中静息[Ca2 ]i 均升高 ,P<0 .0 1 ;但对照组与高糖组在有钙介质中对氯化钾 (KCl)、谷氨酸 (Glu)刺激引起的[Ca2 ]i 升高无明显差异 ,P>0 .0 5;在无钙介质中 ,对氯化钙 (Ca Cl2 )引起的 [Ca2 ]i 增高率无明显差别 ,P>0 .0 5。本研究表明 :高糖对神经元的损伤作用可能与其促进细胞内钙离子释放 ,诱发细胞内钙超载有关     

国产与进口丁咯地尔对新生大鼠单个脑细胞游离钙影响的比较

脑缺血后发生一系列病理生理变化 ,其中导致脑细胞死亡的中心环节是神经细胞内钙超载。故降低神经细胞内钙浓度是阻止神经细胞损伤、死亡的重要途径。丁咯地尔是一种血管活性药物 ,在临床上用于治疗缺血性脑血管疾病。本实验应用 AR- CM- MIC阳离子测定系统观察国产与进口丁咯地尔对新生大鼠脑细胞内游离钙 ([Ca2 ]i)增高的影响。1　材料与方法药品与仪器 :国产盐酸丁咯地尔 5 0 m g/支 (商品名“赛莱乐”,南京医药股份有限公司 )。进口盐酸丁咯地尔 5 0 mg/支(商品名“活脑灵”,法国 L ABORATOIRESL L AFON 公司 )。取新生 S…     

15-HETE影响大鼠脑动脉平滑肌细胞内钙离子浓度的机制

目的 观察15-羟化二十烷四烯酸(15-HETE)对脑动脉平滑肌细胞内钙离子浓度([Ca2+]i)的影响,进一步探讨15-HETE引起[Ca2+]i变化的钙来源,从而明确15-HETE引起脑动脉平滑肌收缩的机制. 方法 酶法分离大鼠脑动脉平滑肌细胞,分为15-HETE组与对照组,15-HETE组添加15-HETE处理;对照组正常培养,不做其他处理.激光共聚焦技术测定15-HETE对[Ca2+]i的影响;进一步通过阻断外钙内流和耗竭内钙,探明15-HETE引起钙动员的来源;应用血管环技术从功能上判定细胞外钙对15-HETE引起的颈内动脉环收缩有无影响. 结果 15-HETE组与对照组相比,[Ca2+]i明显增加,差异有统计学意义(P＜0.05);预先加入硝苯地平、镧离子及改用无钙液阻断外钙内流后,15-HETE组[Ca2+]i仍明显高于对照组,差异有统计学意义(P＜0.05);而预先加入咖啡因耗竭细胞内钙后,15-HETE组[Ca2+]i较对照组差异无统计学意义(P＞0.05);采用无钙液去除细胞外钙后,15-HETE引起的血管环张力增加与有钙液中比较差异无统计学意义(P＞0.05). 结论 15-HETE可通过促使内钙释放而使[Ca2+]i增加,进而引起大鼠脑动脉平滑肌收缩.     

糖皮质激素对单个大鼠肾上腺髓质嗜铬细胞（Ca^2＋）i的快速作用

为探讨糖皮质激素（glucocorticoids,GC）快速地抑制肾上腺髓质嗜铬细胞（adrenalmedullarychromaffincells，AMCcells）受刺激时分泌儿茶酚胺的机制，用Fura-2作Ca(2 )指示剂．用Spex阳离子测定系统检测单个AMC细胞，观察了对AMC细胞的［Ca(2 )］．的影响。发现皮质酮（B．corticosterone）对AMC细胞静息［Ca(2 )］i无影响，但B可以抑制AMC细胞从无钙介质换到含钙介质时［Ca(2 )］i的升高，可以抑制ACh、Nico（nicotine）、Muse（muscarine）及高钾引起的[Ca(2 )]i升高，RU38486可以部分阻断B对ACh刺激引起的[Ca(2 )]i升高的抑制作用。B还可抑制在无钙介质中Musc引起的［Ca(2 )]i升高。Dex（dexamethasone）与B有相似的作用，而E2(17β-estradiol）、P（progesterone）、Ald（aldosterone）和Andro（androstenedion）无作用。     

氯氮平及其代谢产物对大鼠胰岛细胞胞内钙的影响

目的从胞内钙离子水平探讨氯氮平及其代谢产物对离体大鼠胰岛细胞内分泌功能的影响。方法分别用低浓度(3.3mmol/L)和高浓度(16.7mmol/L)的葡萄糖Hank′s液经灌流装置孵育诱导,以终浓度为1μmol/L的氯氮平、去甲基氯氮平和N-氧化氯氮平分别作用于分离培养的大鼠胰岛细胞,并设两种浓度葡萄糖的空白对照组,以Fluo-4/AM为荧光探针,应用激光扫描共聚焦显微镜,动态监测不同药物作用于胰岛细胞后胞内钙荧光强度的变化。结果(1)低糖条件下,氯氮平组[(22±4)%]和去甲基氯氮平组[(49±6)%]胞内[Ca2+]i均低于空白对照组[(93±6)%;P<0.01];与未加处理因素前(0min)相比,氯氮平组和去甲基氯氮平组的胞内[Ca2+]i随时间的延长而降低(P<0.05~0.01),且氯氮平的抑制作用强于去甲基氯氮平(P<0.01)。(2)高糖条件下,氯氮平组[(62±10)%]和去甲基氯氮平组[(18±8)%]胞内[Ca2+]i亦均低于空白对照组[(94±5)%;P<0.01];与未加处理因素前(0min)相比,氯氮平组和去甲基氯氮平组的胞内[Ca2+]i随时间的延长而降低(P<0.05~0.01),其中去甲基氯氮平的抑制作用强于氯氮平(P<0.01)。N-氧化氯氮平组对胞内[Ca2+]i的影响不大(P>0.05)。结论氯氮平及去甲基氯氮平均降低胰岛细胞胞内[Ca2+]i,从而抑制胰岛素分泌。     

不同温度对谷氨酸诱发原代培养皮质神经元损伤的保护机制

目的探讨不同温度对谷氨酸诱发皮质神经元损伤的保护机制。方法体外培养新生24 h内Wistar大鼠的皮质神经元,毒性剂量的谷氨酸(200μmol·L-1)诱致皮质神经元损伤,随机分为37℃组(常温)和33℃组(亚低温),两组再根据谷氨酸持续作用时间点(10 min,3 h,6 h,24 h)分为4个亚组,标记为T1,T2,T3,T4。两组细胞均在损伤后24 h,通过激光扫描共聚焦显微镜测量细胞内Ca2+浓度([Ca2+]i)、线粒体内Ca2+浓度([Ca2+]m)及线粒体跨膜电位(ΔΨm)。结果 37℃组和33℃组的[Ca2+]i及[Ca2+]m均随损伤时间的延长而显著性增加,同时伴有ΔΨm显著下降(P0.05);33℃组较37℃组[Ca2+]i及[Ca2+]m的增加明显减少,同时显著减低ΔΨm下降幅度(P0.05)。结论 33℃可抑制谷氨酸诱发皮质神经元损伤时的[Ca2+]m内流,减少[Ca2+]m,维持ΔΨm。     

Fluorescence measurement of changes in intracellular calcium induced by excitatory amino acids in cultured cortical astrocytes

Population response of [Ca2+]i in cultured cortical astrocytes to excitatory amino acids was measured at room temperature using the calcium-sensitive dye fura-2. Quisqualic acid (QA), glutamate (Glu), and kainic acid (KA) caused a peak increase in [Ca2+]i in the order QA greater than Glu greater than KA. No response to N-methyl-D-aspartic acid (NMDA) was observed whether or not Mg2+ was present externally. Both QA and Glu (100 microM) frequently elicited a decaying oscillatory [Ca2+]i response during sustained agonist application; the period of oscillations initially was 23.5 sec and increased as the response was damped. Comparatively, the [Ca2+]i response to KA was nonoscillatory. Both responses to Glu and KA were reduced slightly by antagonist gamma-D-glutamylaminomethyl-sulfonic acid (1 mM), but virtually were abolished by kynurenic acid (3 mM). Replacement of external Na+ by choline had no significant effect on the Glu response. Removal of external Ca2+ reduced the peak response to QA, Glu, and KA to 40, 34, and 18%, respectively; and markedly reduced the degree of QA- and Glu-induced [Ca2+]i oscillations. Pretreatment with phorbol esters, a potent activator of protein kinase C, blocked the [Ca2+]i response to Glu but not KA. It is concluded that cortical astrocytes express Glu receptors of the non-NMDA type in culture and that receptor activation leads to Ca2+ influx and release of internal Ca2+. Mobilization of Ca2+ apparently occurs via the known Glu-mediated hydrolysis of inositol lipids, which may come under negative-feed-back control by protein kinase C activation. Oscillatory [Ca2+]i signaling offers the possibility of a dynamic population response in an electrically coupled glial network.     

CCKB receptor stimulation mediates [Ca2+]i increase but no PKC activation in Jurkat T-cells.

We have investigated the effect of cholecystokinin-octapeptide (CCK-8) on [Ca2+]i and protein kinase C (PKC) activity in Jurkat T-cells. CCK-8 produced a transient [Ca2+]i increase in the presence of extracellular Ca2+. While CCKB receptor antagonist L-365,260 abolished the elevation of [Ca2+]i, CCKA receptor antagonist L-364,718 was without effect. Moreover, the dihydropyridine calcium channel blocker nitrendipine was shown to block the observed calcium response. Results suggest that the calcium effect is caused by an interaction of CCK-8 with CCKB binding sites and an influx of external Ca2+ via dihydropyridine sensitive calcium channels might serve as a source for the increased [Ca2+]i. Because CCK-8 induced no PKC activation CCKB receptor mediated rise of intracellular calcium seems not to include activation of phospholipase C.     

The bradykinin receptor--a putative receptor-operated channel in PC12 cells: studies of neurotransmitter release and inositol phosphate accumulation.

Bradykinin (BK) induced [3H]norepinephrine [( 3H]NE) release and phosphatidylinositol turnover were investigated in PC12 cells. Induction of [3H]NE release by BK is mediated by activation of BK-B2-receptors, as determined using type specific BK receptor antagonists. BK induces [3H]NE release with a half maximal effective concentration of 30 +/- 0.5 nM, and reaches maximal net fractional release of 9.0 +/- 1% with 200 nM BK. The BK-induced release is Ca2+ dependent, reaching maximal release at 1.0 mM Ca2+, is pertussis toxin insensitive (1 microgram/ml), slightly increased by a dibutyryl cAMP (1 mM) and not affected by inhibitors of the cyclooxygenase or lipoxygenase pathways. Voltage-sensitive Ca2+ channel blockers, verapamil (10 microM), nifedipine (10 microM), and omega-conotoxin (CgTx 10 nM), do not block the BK-induced release. However, a considerable inhibitory effect was obtained by divalent cations Co2+ (ED50 = 0.2 mM) and Ni2+ (ED50(2)+ = 1 mM). These results indicate the involvement of a Ca2+ channel in the BK-mediated release which is different from the L- or N-type voltage sensitive calcium channels. Whereas [Ca2+]ex is essential for the BK-induction of catecholamine release, the rise in level of InsP's induced by BK in the presence or in the absence of [Ca2+]ex is similar up to concentration of 1 microM. This indicates that the rise in InsP's induced by BK is not sufficient to cause neurotransmitter release. Moreover, subsequent addition of Ca2+ to BK-stimulated cells in Ca(2+)-free medium yields no release. Hence, no activity triggered by BK alone could be further stimulated by Ca2+ for induction of release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium signalling and removal mechanisms in myenteric neurones.

To characterize further the Ca2+ signalling mechanisms of myenteric neurones, we studied the effect of thapsigargin, a blocker of the Ca2+-store ATPase, and the mechanisms involved in restoring the intracellular Ca2+ concentration ([Ca2+]i) after activation. Thapsigargin (5 x 10(-6) mol L(-1)) induced an oscillatory [Ca2+]i response in 86.6% of the neurones (n=276), which was blocked by the removal of extracellular Ca2+ and by omega-conotoxin MVIIA (5 x 10(-7) mol L(-1)). The IP3-blocker, 2-aminoethyl-diphenyl-borate (75 x 10(-6) mol L(-1)), blocked or reduced the responses in 74.5% of the neurones. The oscillatory responses induced by the depletion of Ca2+ stores suggest that myenteric neurones might recruite N-type Ca2+ channels as a refill mechanism. Thapsigargin pretreatment increased the amplitude, the upstroke and duration of the K+-induced [Ca2+]i responses. Mitochondrial blockers (rotenone and antimycin/oligomycin) also prolonged the responses, but without affecting the amplitude. Furthermore, it was found that for high [Ca2+]i, the thapsigargin-sensitive Ca2+ uptake was crucial, while mitochondrial blockade affected the Ca2+ uptake over a wide range of concentrations. The Ca2+-sequestering components might also have been compensating for each other, as most drugs only delayed and not inhibited Ca2+ removal.     

Bradykinin-induced intracellular Ca2+ elevation in neuroblastoma X glioma hybrid NG108-15 cells; relationship to the action of inositol phospholipids metabolites

The effect of bradykinin on the intracellular Ca2+ concentration ([Ca2+]i) in NG108-15 cells was studied using a Ca2+ indicator quin 2. Bradykinin induced two phases of change in [Ca2+]i. Bradykinin induced a spike phase of [Ca2+]i increase which was detectable within 15 s and decayed to near-basal concentration in 3 min and then a prolonged plateau phase of [Ca2+]i increase which continued for 15 min. The bradykinin-induced spike phase was not diminished by decreasing extracellular Ca2+ concentration ([Ca2+]o) to 1 microM. On the contrary, the plateau phase was dependent on [Ca2+]o and inhibited by Ca2+ blockers, verapamil (50 microM), nifedipine (1 microM). The iontophoretic injection of inositol-trisphosphate (IP3) into the single cell induced the increase of [Ca2+]i, which was independent of [Ca2+]o. These results indicate that the bradykinin-induced spike phase is mediated by the release of intracellular Ca2+ stores induced by IP3, while the plateau phase is mediated by influx of extracellular Ca2+ probably through voltage-sensitive Ca2+ channels.     

L-β-N-oxalyl-α,β-diaminopropionic acid toxicity in motor neurons

The excitatory amino acid L-β-N-oxalyl-α,β-diaminopropionic acid (L-β-ODAP) in Lathyrus sativus L. is proposed as the causative agent of the neurodegenerative disease neurolathyrism. We investigated the effect of L-β-ODAP on [Ca2+]i handling, redox homeostasis, and cell death in rat spinal motor neurons. L-β-ODAP and L-glutamate triggered [Ca2+]i transients, which were inhibited by the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor blockers; 2,3-dioxo-6-nitro-1,2,3, 4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide and 1-naphthyl acetylspermine, the latter specifically blocking Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors. In addition, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide, and to a lesser extent 1-naphthyl acetylspermine, protected the neurons against cell death induced by L-β-ODAP or L-glutamate. Methionine and cysteine were also protective against neuronal cell death. We conclude that deregulation of [Ca2+]i homeostasis and oxidative stress contribute to motor neuron cell death in neurolathyrism.     

Orexin-a activates phospholipase C- and protein kinase C-mediated Ca2+ signaling in dopamine neurons of the ventral tegmental area.

The orexin-orexin receptor system has been implicated in the regulation of wakefulness/sleep states. Behavioral and psycho-stimulant effects of orexins have also been shown. Mesolimbic dopamine neurons in the ventral tegmental area (VTA) are implicated in the regulation of reward and wakefulness/sleep, In the present study, we examined the effect of orexin-A on cytosolic [Ca2+]i concentration ([Ca2+]) in the isolated rat VTA dopamine neurons. Orexin-A (10-12-10-8 M) concentration dependently increased [Ca2+]i in dopamine-containing neurons. The [Ca2+]i responses to orexin-A were inhibited under Ca2+-free conditions and by blockers of voltage-gated L- and N-type [Ca2+]i channels, nitrendipine and omega-conotoxin, respectively. The [Ca2+]i responses were also abolished by a phosphatidylcholine-specific phospholipase C inhibitor, D609, and a protein kinase C (PKC) inhibitor, calphostin C. A PKC activator, TPA, mimicked orexin-A in increasing [Ca2+]i. These results indicate that orexin-A increases [Ca2+]i in VTA dopamine neurons via phosphatidylcholine-specific PLC- and PKC-mediated activation of L- and N-type Ca2+ channels. This effect may serve as the mechanism by which orexin regulates wakefulness/sleep states and exerts its behavioral and psychostimulant effects.     

Inhibitory effect of CCK-8 and ceruletide on glutamate-induced rises in intracellular free calcium concentrations in rat neuron cultures

To study the mechanism by which cholecystokinin octapeptide (CCK-8) and its potent analogue, ceruletide, prevent glutamate-induced neuronal cell death in rat neuron cultures, we examined the effect of both peptides on glutamate-induced increases in the intracellular free calcium concentrations ([Ca2+]i), which are known to be a crucial trigger of the neurodegeneration induced by glutamate. CCK-8 itself did not alter [Ca2+]i in rat neuron cultures. Glutamate increased [Ca2+]i in neuron cultures rapidly and markedly. CCK-8 and ceruletide significantly suppressed the increases in [Ca2+]i induced by glutamate. The maximum inhibitory effects of CCK-8 and ceruletide at 10(-6) M reached 43 and 46% of the response to glutamate, respectively. Gastrin-I and CCK-4 also significantly attenuated the increases in [Ca2+]i induced by glutamate. The inhibitory effect of CCK-8 was completely blocked by the selective antagonist for CCK-B receptors, (+)L-365,260, but not by (-)L-364,718, which is a selective antagonist for CCK-A receptors. CCK-8 significantly suppressed [Ca2+]i response to kainate and high concentrations of extracellular K+, but not to N-methyl-D-aspartate. With cultured astrocytes, CCK-8 did not inhibit the increment of [Ca2+]i induced by glutamate. These findings clearly demonstrated that CCK-8 and ceruletide inhibit glutamate-induced increases in [Ca2+]i in neuron cultures through CCK-B receptors, suggesting that CCK-8 may participate in the central actions of glutamate.     

Hyposmolarity evokes norepinephrine efflux from synaptosomes by a depolarization- and Ca2+ -dependent exocytotic mechanism

Osmolarity reduction (20%) elicited 3H-norepinephrine (NE) efflux from rat cortical synaptosomes. The hyposmotic NE release resulted from the following events: (i) a Na+-dependent and La3+-, Gd3+- and ruthenium red-sensitive depolarization; (ii) a cytosolic Ca2+ ([Ca2+]i) rise with contributions from external Ca2+ influx and internal Ca2+ release, probably through the mitochondrial Na+-Ca2+ exchanger; and (iii) activation of a [Ca2+]i-evoked, tetanus toxin (TeTX)-sensitive, PKC-modulated NE efflux mechanism. This sequence was established from results showing a drop in the hyposmotic [Ca2+]i rise by preventing depolarization with La3+, and by the inhibitory effects of Ca2+-free medium (EGTA; 50%), CGP37157 (the mitochondrial Na+-Ca2+ exchanger blocker; 48%), EGTA + CGP37157 or by EGTA-AM (> 95% in both cases). In close correspondence with these effects, NE efflux was 92% decreased by Na+ omission, 75% by La3+, 47% by EGTA, 50% by CGP37157, 90% by EGTA + CGP37157 and 88% by EGTA-AM. PKC influenced the intracellular Ca2+ release and, mainly through this action, modulated NE efflux. TeTX suppressed NE efflux. The K+-stimulated NE release, studied in parallel, was unaffected by Na+ omission, or by La3+, Gd3+ or ruthenium red. It was fully dependent on external Ca2+, insensitive to CGP37157 and abolished by TeTX. These results suggest that the hyposmotic events, although different from the K+-evoked depolarization and [Ca2+]i rise mechanisms, are able to trigger a depolarization-dependent, Ca2+-dependent and TeTX-sensitive mechanism for neurotransmitter release.     

Arg8]-vasopressin-induced increase in intracellular Ca2+ concentration in cultured rat hippocampal neurons.

Changes in intracellular Ca2+ concentration ([Ca2+]i) induced by [Arg8]-vasopressin (AVP) were studied in cultured rat hippocampal neurons by fura-2 fluorometry. AVP (10-1,000 nM) caused a dose-dependent increase in [Ca2+]i. The selective V1 vasopressin receptor agonist [Phe2, Ile3, Orn8]-vasopressin also induced a significant increase in [Ca2+]i, whereas the selective V2 vasopressin receptor agonist [deamino Cys1, D-Arg8]-vasopressin showed no effect. The AVP-induced increase in [Ca2+]i was inhibited by the selective V1 vasopressin receptor antagonist d(CH2)5[Tyr2(Me), Arg8]-vasopressin and nonpeptide V1 antagonist OPC-21268. On the other hand, no antagonistic effects were observed with the V2 vasopressin antagonist desglycinamide-[d(CH2)5, D-Ile2, Ile4, Arg8]-vasopressin and nonpeptide V2 antagonist OPC-31260. The increase in [Ca2+]i induced by AVP was abolished after removal of extracellular Ca2+. In addition, AVP-induced [Ca2+]i elevation was not affected by treatment with verapamil, which blocked the [Ca2+]i increase induced by an isotonic high K(+)-medium (50 mM). However, omega-conotoxin GVIA completely inhibited the effect of AVP. These results suggested that the AVP-induced [Ca2+]i increase in cultured rat hippocampal neurons is due to influx of Ca2+ through V1 VP receptors coupled with N-type calcium channels.