劉小妮， 曹冬青， 張娜娜， 陶 然， 丁瀅泂， 金惠銘， 盧 寧△

(1復(fù)旦大學(xué)基礎(chǔ)醫(yī)學(xué)院生理與病理生理系，上海 200032； 2復(fù)旦大學(xué)附屬華山醫(yī)院神經(jīng)外科，上海 200040)

活性氧簇介導(dǎo)血管緊張素 II對(duì)延髓神經(jīng)元胞內(nèi)游離Ca2+水平的調(diào)節(jié)作用*

劉小妮1， 曹冬青2， 張娜娜1， 陶 然1， 丁瀅泂1， 金惠銘1， 盧 寧1△

(1復(fù)旦大學(xué)基礎(chǔ)醫(yī)學(xué)院生理與病理生理系，上海 200032；2復(fù)旦大學(xué)附屬華山醫(yī)院神經(jīng)外科，上海 200040)

目的： 探討活性氧簇(reactive oxygen species, ROS)介導(dǎo)血管緊張素II(angiotensin II, Ang II)對(duì)延髓神經(jīng)元胞內(nèi)游離Ca2+的調(diào)節(jié)作用及其機(jī)制。方法：原代培養(yǎng)延髓神經(jīng)元；免疫熒光雙標(biāo)法鑒定培養(yǎng)神經(jīng)元的特征；給予Ang II處理后，用二氫乙啶熒光探針?lè)y(cè)定神經(jīng)元ROS水平；同時(shí)或單獨(dú)給予Ang II和NADPH氧化酶抑制劑apocynin或自由基清除劑TEMPOL后，F(xiàn)ura-2/AM鈣瞬變法記錄神經(jīng)元胞內(nèi)游離Ca2+的水平；CCK-8法檢測(cè)神經(jīng)元活性。結(jié)果：原代培養(yǎng)的延髓神經(jīng)元多數(shù)為谷氨酸陽(yáng)性的神經(jīng)元；Ang II(5 μmol/L)可在10 min內(nèi)顯著升高神經(jīng)元ROS水平(P<0.01)；給予Ang II處理后延髓神經(jīng)元胞內(nèi)Ca2+水平顯著升高(P<0.01)；給予apocynin/TEMPOL預(yù)處理后，Ang II引起的延髓神經(jīng)元胞內(nèi)Ca2+的升高則被抑制(P<0.05)。實(shí)驗(yàn)濃度的Ang II對(duì)神經(jīng)元無(wú)毒性作用。結(jié)論：ROS介導(dǎo)Ang II誘導(dǎo)的延髓神經(jīng)元胞內(nèi)Ca2+的升高作用，可能是Ang II在中樞誘導(dǎo)氧化應(yīng)激作用的潛在細(xì)胞內(nèi)信號(hào)機(jī)制。

延髓神經(jīng)元； 活性氧簇； 細(xì)胞內(nèi)Ca2+； 血管緊張素II

已知血管緊張素II(angiotensin II, Ang II)可通過(guò)中樞調(diào)控對(duì)體液及心血管調(diào)節(jié)發(fā)揮重要調(diào)節(jié)作用。Ang II 可通過(guò)作用于心血管中樞所在部位的AT1受體，而產(chǎn)生升高血壓、促進(jìn)飲水、促進(jìn)抗利尿激素的釋放和引起交感神經(jīng)興奮等[1-2]。中樞Ang II信號(hào)機(jī)制的紊亂與高血壓、心力衰竭等多種心血管疾病的病理過(guò)程相關(guān)[3]。但Ang II在心血管調(diào)控中樞介導(dǎo)的細(xì)胞內(nèi)信號(hào)機(jī)制仍不明確。

細(xì)胞內(nèi)鈣離子濃度(intracellular Ca2+concentration, [Ca2+]i)在神經(jīng)細(xì)胞內(nèi)和神經(jīng)細(xì)胞之間的生理與病理過(guò)程中都起著非常關(guān)鍵的作用，神經(jīng)元胞內(nèi)[Ca2+]i的增加可產(chǎn)生促進(jìn)神經(jīng)遞質(zhì)的釋放、加強(qiáng)神經(jīng)元之間的聯(lián)系和調(diào)節(jié)突觸的可塑性、促進(jìn)基因轉(zhuǎn)錄等效應(yīng)[9-10]。神經(jīng)元胞內(nèi)Ca2+水平的變化可直接影響神經(jīng)元的興奮性[11]。由Ang II介導(dǎo)產(chǎn)生ROS的潛在信號(hào)機(jī)制是Ca2+的相關(guān)機(jī)制[12-13]。研究表明，Ang II可引起Ca2+電流的增加和K+電流的降低，從而通過(guò)調(diào)節(jié)動(dòng)作電位的產(chǎn)生而增強(qiáng)神經(jīng)元放電頻率[14]。ROS介導(dǎo)的神經(jīng)元胞內(nèi)[Ca2+]i的增加可能與細(xì)胞毒性及神經(jīng)系統(tǒng)退行性疾病相關(guān)。但是，由Ang II介導(dǎo)的ROS及Ca2+的相關(guān)信號(hào)機(jī)制在心血管的中樞調(diào)控過(guò)程中仍有待進(jìn)一步闡明。

因此，本研究主要通過(guò)給予Ang II觀察其對(duì)延髓神經(jīng)元的ROS及[Ca2+]i的調(diào)節(jié)，并進(jìn)一步對(duì)可能的作用機(jī)制進(jìn)行探討，為闡明Ang II介導(dǎo)的ROS在心血管的中樞調(diào)控過(guò)程中發(fā)揮作用的細(xì)胞內(nèi)信號(hào)機(jī)制提供實(shí)驗(yàn)依據(jù)。

材 料 和 方 法

1 主要試劑與儀器

Ang II、apocynin、TEMPOL和二氫乙啶(dihydroethidium, DHE)購(gòu)自Sigma；Fura-2/AM和 CCK-8試劑盒購(gòu)自Dojindo；小鼠來(lái)源MAP-2抗體購(gòu)自Abcam；兔來(lái)源谷氨酸抗體購(gòu)自Sigma-Aldrich；神經(jīng)元培養(yǎng)液、B27、胎牛血清(fetal bovine serum, FBS)和2.5%胰酶購(gòu)自Gibco；DAPI、FITC標(biāo)記山羊抗兔IgG (H+L)和Cy3標(biāo)記山羊抗小鼠IgG (H+L)購(gòu)自碧云天。CO2恒溫細(xì)胞培養(yǎng)箱(上海力申科學(xué)儀器有限公司)；無(wú)菌超凈臺(tái)(上海博迅實(shí)業(yè)有限公司醫(yī)療設(shè)備廠)；激光共聚焦系統(tǒng)(ZEISS)；酶標(biāo)儀(TECAN)；鈣瞬變系統(tǒng)(PTI)。

2 方法

2.1 原代延髓神經(jīng)元培養(yǎng)[5]取孕14～17 d的SD大鼠(購(gòu)自上海斯萊克實(shí)驗(yàn)動(dòng)物中心)，取胎鼠斷頭；在解剖顯微鏡下分離延髓，剪碎(約1 mm3)；0.125%胰蛋白酶溶液于37 ℃細(xì)胞培養(yǎng)箱中消化10 min左右；10% 的FBS終止消化；取上清至新的離心管；1 000 r/min離心8 min；棄上清，加入Neurobasal medium 與B27的混合培養(yǎng)基(比例為49∶1)，重懸細(xì)胞。將細(xì)胞接種到35 mm的培養(yǎng)皿或96孔板(前1 d用多聚賴氨酸處理)中，使細(xì)胞在37 ℃、 5% CO2條件下貼壁生長(zhǎng)；每2 d半量換液1次，培養(yǎng)7 d左右，待神經(jīng)元發(fā)育成熟，進(jìn)行實(shí)驗(yàn)。

2.2 神經(jīng)元的鑒定[5]細(xì)胞經(jīng)培養(yǎng)7 d左右，用0.01 mol/L PBS洗3次，4%多聚甲醛室溫固定細(xì)胞20 min；棄固定液，0.01 mol/L PBS洗3次；5%～7%胎牛血清封閉30 min；棄封閉液，加入Ⅰ抗：小鼠抗MAP-2抗體(1∶200,1%FBS配制)，37℃孵育1 h后轉(zhuǎn)入4 ℃過(guò)夜；棄Ⅰ抗，0.01 mol/L PBS洗3次；加入Ⅱ抗：Cy3結(jié)合的山羊抗小鼠IgG (H+L)(1∶100，Ⅱ抗稀釋液配制)，避光，37 ℃孵育1 h； 0.01 mol/L PBS洗3次；加入DAPI染核5 min； 0.01 mol/L PBS洗3次；采用激光共聚焦系統(tǒng)拍照鑒定。

2.3 延髓谷氨酸陽(yáng)性神經(jīng)元的鑒定 方法同2.2。第一Ⅰ抗為小鼠抗MAP-2抗體(1∶200)和第二Ⅰ抗為兔抗谷氨酸抗體(1∶100)。Ⅱ抗為FITC標(biāo)記山羊抗兔IgG (H+L); Cy3標(biāo)記山羊抗小鼠IgG (H+L)。

2.5 神經(jīng)元胞內(nèi)游離Ca2+的測(cè)定 以鈣離子敏感的熒光探針Fura-2/AM來(lái)檢測(cè)細(xì)胞內(nèi)[Ca2+]i。Fura-2能以1∶1的比例特異性地與Ca2+結(jié)合，并且結(jié)合Ca2+后，其最大激發(fā)波長(zhǎng)由原來(lái)Fura-2的380 nm變?yōu)槠渑cCa2+結(jié)合后的復(fù)合物的340 nm，通過(guò)鈣瞬變系統(tǒng)檢測(cè)延髓神經(jīng)元F340/380的比值(R)，采用R/R0來(lái)評(píng)估給藥前后細(xì)胞內(nèi)Ca2+的變化，其中R0代表藥物處理前的熒光信號(hào)，R代表藥物處理后的熒光信號(hào)。實(shí)驗(yàn)時(shí)，將已孵育好Fura-2/AM的神經(jīng)元培養(yǎng)皿固定在載物臺(tái)上，用HBSS溶液灌流細(xì)胞，待鈣瞬變系統(tǒng)基線穩(wěn)定后，用不同藥物灌流細(xì)胞。以上操作由FelixGX-4.2.2軟件執(zhí)行。

2.6 CCK-8實(shí)驗(yàn)檢測(cè)神經(jīng)元的活性 神經(jīng)元種于96孔板；培養(yǎng)的延髓神經(jīng)元給予不同藥物處理后，棄含藥物的培養(yǎng)基，D-Hank’s溶液洗3遍；每孔加入110 μL CCK-8與培養(yǎng)基的混合液(二者體積比為1∶10)，置于37 ℃細(xì)胞培養(yǎng)箱中孵育2 h；用酶標(biāo)儀測(cè)定450 nm處的吸光度。

3 統(tǒng)計(jì)學(xué)處理

采用SPSS 19.0統(tǒng)計(jì)軟件進(jìn)行分析。實(shí)驗(yàn)結(jié)果用均數(shù)±標(biāo)準(zhǔn)誤(mean±SEM)表示。兩組實(shí)驗(yàn)數(shù)據(jù)采用獨(dú)立樣本t檢驗(yàn)，多組數(shù)據(jù)采用單因素方差分析(one-way ANOVA)。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 原代延髓神經(jīng)元的培養(yǎng)及鑒定

采用激光共聚焦結(jié)合顯微鏡觀察的方法對(duì)培養(yǎng)的細(xì)胞進(jìn)行鑒定，圖1為培養(yǎng)5～7 d的延髓細(xì)胞，免疫熒光染色可見(jiàn)紅色的特異性MAP-2標(biāo)記細(xì)胞，且可見(jiàn)原代培養(yǎng)的延髓細(xì)胞90%以上為神經(jīng)元。

Figure 1.Identification of the primarily cultured medullary neurons. A: the white-light graph of the primarily cultured medullary neurons; B: fluorescence micrograph of the neurons stained with anti-MAP-2 antibody, fluorescence micrograph of neurons stained with DAPI, and merged graph of the former 2 photos.

圖1 原代延髓神經(jīng)元的培養(yǎng)及鑒定

2 延髓谷氨酸陽(yáng)性神經(jīng)元的鑒定

由于中樞調(diào)節(jié)心血管活動(dòng)的神經(jīng)元大多為谷氨酸陽(yáng)性的神經(jīng)元，因此我們用谷氨酸去標(biāo)記培養(yǎng)的延髓神經(jīng)元。圖2可見(jiàn)紅色為MAP-2標(biāo)記的神經(jīng)元，綠色為谷氨酸陽(yáng)性的細(xì)胞，藍(lán)色為DAPI標(biāo)記的細(xì)胞核。結(jié)果顯示原代培養(yǎng)的延髓神經(jīng)元80%以上為谷氨酸陽(yáng)性的神經(jīng)元。

Figure 2.Identification of glutamatergic neurons. The confocal images were the fluorescence micrographs of the neurons stained with anti-MAP-2 antibody (a neuronal maker), anti-glutamate (Glu) antibody and DAPI, and the merged graph of the former 3 photos.

圖2 延髓谷氨酸陽(yáng)性神經(jīng)元的鑒定

3 Ang II升高延髓神經(jīng)元ROS的水平

前期研究顯示，Ang II(5 μmol/L)可顯著升高小鼠神經(jīng)母細(xì)胞瘤細(xì)胞ROS水平[12]。在本研究中，給予Ang II(5 μmol/L)，37 ℃分別孵育5、10 和30 min后，采用DHE熒光探針?lè)z測(cè)延髓神經(jīng)元ROS水平。結(jié)果顯示Ang II可在10 min內(nèi)顯著升高神經(jīng)元的ROS水平，見(jiàn)圖3。

4 Ang II 引起神經(jīng)元胞內(nèi)游離Ca2+水平的升高

為研究Ang II對(duì)延髓神經(jīng)元胞內(nèi)[Ca2+]i的影響，我們首先研究單獨(dú)給予Ang II對(duì)神經(jīng)元包內(nèi)游離Ca2+水平的影響。結(jié)果顯示：Ang II可在10 min左右顯著升高神經(jīng)元胞內(nèi)[Ca2+]i，并在其升高達(dá)到穩(wěn)定并持續(xù)約2 min后，再給予HBSS沖洗后，神經(jīng)元胞內(nèi)[Ca2+]i開(kāi)始下降,見(jiàn)圖4。

Figure 3.The effects of Ang II on the ROS levels in the primarily cultured medullary neurons treated with or without Ang II (5 μmol/L) for 5, 10 and 30 min. ROS level was determined using the oxidant-sensitive fluorogenic probe dihydroethidium (DHE). Mean±SEM.n=12.*P<0.05,**P<0.01vs0 min group.

圖3 Ang II升高延髓神經(jīng)元ROS水平

5 ROS 介導(dǎo)Ang II對(duì)延髓神經(jīng)元[Ca2+]i的升高作用

延髓神經(jīng)元給予NADPH氧化酶抑制劑apocynin或自由基清除劑TEMPOL預(yù)處理后，觀察Ang II 對(duì)神經(jīng)元[Ca2+]i的效應(yīng)，結(jié)果顯示apocynin和TEMPOL均可抑制Ang II對(duì)延髓神經(jīng)元[Ca2+]i的升高作用,見(jiàn)圖5。

6 Ang II對(duì)延髓神經(jīng)元無(wú)毒性作用

考慮到Ang II可能對(duì)神經(jīng)元產(chǎn)生毒性作用，我們用CCK-8法檢測(cè)了實(shí)驗(yàn)濃度的Ang II孵育神經(jīng)元30 min后的細(xì)胞活力，結(jié)果顯示實(shí)驗(yàn)中所用濃度的Ang II (5 μmol/L)不影響神經(jīng)元的細(xì)胞活力，見(jiàn)圖6。

討 論

研究表明，NADPH氧化酶來(lái)源的ROS在Ang II依賴的心血管疾病的中樞調(diào)控過(guò)程中起著非常重要的調(diào)節(jié)作用[8, 15]。在高血壓的發(fā)病過(guò)程中，中樞及外周Ang II 可通過(guò)穹窿下區(qū)(suofornial organ，SFO)-室旁核(paraventricularis nucleus，PVN)-延髓頭端腹外側(cè)(the rostral ventrolateral medulla, RVLM)的通路，最后通過(guò)脊髓中間外側(cè)柱(intermediolateral column of spinal cord)的傳出將興奮傳遞至外周，引起交感神經(jīng)興奮，而導(dǎo)致高血壓的發(fā)生[16]。在此過(guò)程中，RVLM區(qū)作為心血管交感活動(dòng)的基本中樞，接受中樞心血管效應(yīng)相關(guān)核團(tuán)SFO、PVN等的纖維投射，在維持心血管中樞緊張性活動(dòng)中起關(guān)鍵作用[16]。本課題組前期研究表明Ang II 可顯著升高延髓神經(jīng)元的ROS水平，且主要是通過(guò)激活NADPH氧化酶產(chǎn)生[5-6]；RVLM區(qū)微量注射N(xiāo)aHS可顯著降低SHR大鼠的血壓的心率，其機(jī)制可能是通過(guò)抑制NADPH氧化酶p47phox亞單位的磷酸化從而降低酶的活性，使ROS生成減少而所致[17]。因此研究中樞ROS的細(xì)胞內(nèi)信號(hào)機(jī)制對(duì)闡明Ang II誘導(dǎo)的心血管疾病中樞調(diào)控機(jī)制具有重要的應(yīng)用價(jià)值。

Figure 4.The effect of Ang II on [Ca2+]iin the primarily cultured medullary neurons. A: typical elevation of [Ca2+]iinduced by Ang II at 5 μmol/L; B: typical elevation of [Ca2+]iinduced by Ang II at 5 μmol/L and washout of Ang II led [Ca2+]ito decline; C: the quantitative analysis of the peak increase in [Ca2+]iin the neurons stimulated with Ang II (5 μmol/L). Mean±SEM.n=8.**P<0.01vscontrol.

圖4 Ang II引起延髓神經(jīng)元胞內(nèi)游離Ca2+水平的升高

在前期研究的基礎(chǔ)上，本工作主要采用離體實(shí)驗(yàn)在細(xì)胞水平觀察ROS是否介導(dǎo)Ang II對(duì)細(xì)胞內(nèi)行了鑒定，結(jié)果顯示培養(yǎng)的細(xì)胞中90%以上為神經(jīng)元。由于中樞調(diào)節(jié)心血管活動(dòng)的神經(jīng)元大多為谷氨酸陽(yáng)性的神經(jīng)元，本研究重點(diǎn)觀察了培養(yǎng)的延髓神經(jīng)元中谷氨酸的表達(dá)變化。結(jié)果顯示原代培養(yǎng)的延髓神經(jīng)元80%以上為谷氨酸陽(yáng)性神經(jīng)元，為進(jìn)一步研究奠定了細(xì)胞學(xué)基礎(chǔ)。

Figure 5.The effect of Ang II on [Ca2+]iin the primarily cultured medullary neurons pretreated with apocynin (an inhibitor of NADPH oxidase) or TEMPOL (a cell membrane-permeable superoxide dismutase mimetic). A, B: typical suppression of [Ca2+]ilevel induced by Ang II (5 μmol/L) with or without apocynin (100 μmol/L) or TEMPOL (100 μmol/L); C: the quantitative analysis showed the effects of Ang II were atte-nuated by apocynin or tempol. Mean±SEM.n=6.**P<0.01vscontrol group;#P<0.05vsAng II group.

圖5 ROS 介導(dǎo)Ang II對(duì)延髓神經(jīng)元[Ca2+]i的升高作用

Ca2+水平的調(diào)節(jié)作用。并采用免疫熒光雙標(biāo)結(jié)合激光共聚焦顯微鏡的方法對(duì)原代培養(yǎng)的延髓神經(jīng)元進(jìn)已證明，Ca2+作為重要的第二信使，參與神經(jīng)遞質(zhì)的釋放、突觸可塑性的調(diào)節(jié)、神經(jīng)元的興奮性及基因轉(zhuǎn)錄的調(diào)節(jié)[9-10]；神經(jīng)元[Ca2+]i的變化可直接影響神經(jīng)的興奮性[11]；而[Ca2+]i在神經(jīng)元之間的信號(hào)傳遞中起著非常重要的作用，并受細(xì)胞膜上的Ca2+通道和細(xì)胞內(nèi)鈣庫(kù)的調(diào)控[18]，提示研究神經(jīng)元Ca2+的相關(guān)機(jī)制對(duì)其功能具有重要意義。為明確Ang II對(duì)延髓神經(jīng)元[Ca2+]i的影響，我們采用Fura-2/AM法記錄神經(jīng)元胞內(nèi)[Ca2+]i的變化。結(jié)果顯示Ang II可在10 min左右顯著升高神經(jīng)元[Ca2+]i，并在其升高達(dá)到穩(wěn)定后，再給予HBSS沖洗后，神經(jīng)元[Ca2+]i開(kāi)始下降，提示Ang II可顯著升高延髓神經(jīng)元[Ca2+]i。由于ROS參與了中樞Ang II所致的交感興奮與心血管效應(yīng)，且主要與NADPH氧化酶途徑的激活有關(guān)[3-4, 7-8]。因此，為明確Ang II對(duì)神經(jīng)元ROS的影響，我們首先給予Ang II處理后檢測(cè)延髓神經(jīng)元ROS水平。結(jié)果顯示Ang II可在10 min內(nèi)顯著升高神經(jīng)元ROS水平，提示Ang II可顯著升高延髓神經(jīng)元ROS水平。研究表明，apocynin可通過(guò)特異性地抑制NADPH氧化酶(ROS生成的關(guān)鍵酶)亞單位gp91ds-tat而選擇性地抑制NADPH氧化酶活性，從而使ROS生成減少[8]；而TEMPOL則作為一種自由基清除劑可降低細(xì)胞ROS水平[19]。在本研究中，我們給予apocynin (NADPH氧化酶抑制劑)或TEMPOL(自由基清除劑)預(yù)處理后，再觀察Ang II 對(duì)神經(jīng)元[Ca2+]i的效應(yīng)，結(jié)果顯示apocynin和TEMPOL均可抑制Ang II對(duì)延髓神經(jīng)元[Ca2+]i的升高作用，提示ROS 介導(dǎo)Ang II對(duì)延髓神經(jīng)元[Ca2+]i的升高作用。此外，考慮到Ang II可能對(duì)神經(jīng)元產(chǎn)生毒性作用，本工作采用CCK-8法檢測(cè)了實(shí)驗(yàn)濃度的Ang II孵育神經(jīng)元30 min后的細(xì)胞活力，結(jié)果顯示實(shí)驗(yàn)中所用濃度的Ang II不影響神經(jīng)元的細(xì)胞活力，提示實(shí)驗(yàn)中所用濃度的Ang II對(duì)神經(jīng)元無(wú)毒性作用。

本工作的實(shí)驗(yàn)結(jié)果提示ROS介導(dǎo)Ang II誘導(dǎo)的延髓神經(jīng)元胞內(nèi)Ca2+的升高作用，可能是Ang II在中樞誘導(dǎo)氧化應(yīng)激作用的潛在細(xì)胞內(nèi)信號(hào)機(jī)制。

Figure 6.The cell viability analyzed by CCK-8 assay showed no significant difference of the cell activity between Ang II group and control group. Mean±SEM.n=6.

圖6 Ang II對(duì)神經(jīng)元無(wú)毒性作用

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(責(zé)任編輯： 盧 萍， 羅 森)

ROS mediates regulation of intracellular Ca2+induced by angiotensin II in primarily cultured medullary neurons

LIU Xiao-ni1, CAO Dong-qing2, ZHANG Na-na1, TAO Ran1, DING Ying-jiong1, JIN Hui-ming1, LU Ning1

(1DepartmentofPhysiologyandPathophysiology,SchoolofBasicMedicalSciences,FudanUniversity,Shanghai200032,China;2DepartmentofNeurosurgery,HuashanHospital,FudanUniversity,Shanghai200040,China.E-mail:luning7@shmu.edu.cn)

AIM: To investigate the role of reactive oxygen species (ROS) in the regulation of intracellular Ca2 + induced by angiotensin II (Ang II) in the primarily cultured medullary neurons． METHODS: Primarily cultured medullary neurons were prepared from 14-day-old embryos of Sprague-Dawley rats in the study． The identification of medullary neurons was assessed by double-labeling immunofluorescence． To explore the role of ROS，mainly the superoxide ( O2 -· ) ， the O2 -· generation was measured using the fluorogenic probe dihydroethidium (DHE) ． To determine intracellular free calcium concentration ( [Ca2 +]i ) ，the neurons were loaded with the Ca2+-specific dye Fura-2 /AM． The cell viability(DHE).Todetermineintracellularfreecalciumconcentration([Ca2+]i),theneuronswereloadedwiththeCa2+-specificdyeFura-2/AM.ThecellviabilityafteraddingAngIIwasalsoexaminedusingCCK-8assay. RESULTS: Most of the cultured cells were medullary neurons, more than 80% of which were glutamate positive neurons. Ang II (5 μmol/L) increased the level of ROS within 10 min in the medullary neurons. Ang II at 5 μmol/L induced a significant [Ca2+]iincrease in the medullary neurons, and the effect of Ang II occurred rapidly and reached a peak within 20 min after administration. The level of [Ca2+]istarted to decline after washout. The Ca2+elevation induced by Ang II was significantly decreased by apocynin or TEMPOL. No significant difference in the cell viability between control group and 5 μmol/L Ang II treatment group was observed. CONCLUSION: ROS is involved in the regulation of [Ca2+]iinduced by Ang II in the primarily cultured medullary neurons, suggesting a potential intracellular signaling mechanism involved in the Ang II-mediated oxidant regulation of central neural control of blood pressure.

Medullary neurons; Reactive oxygen species; Intracellular Ca2+; Angiotensin II

1000- 4718(2016)12- 2133- 06

2016- 07- 12

2016- 09- 05

國(guó)家自然科學(xué)基金資助項(xiàng)目(No. 81170237)；國(guó)家基礎(chǔ)科學(xué)人才培養(yǎng)基金資助項(xiàng)目(No. J1210041)

R363.2

A

10.3969/j.issn.1000- 4718.2016.12.003

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△通訊作者 Tel: 021-54237452； E-mail: luning7@shmu.edu.cn