不同浓度褪黑激素对新生大鼠海马神经元电压门控性瞬时外向钾电流的影响

目的 探讨不同浓度褪黑激素对新生大鼠海马神经元电压门控性瞬时外向钾电流(I_A)的影响.方法 原代培养新生Wistar大鼠(出生时间<12 h)海马锥体神经元7～12 d.配制褪黑激素溶液,终浓度依次为1 nmol/L、10 nmol/L、100 nmol/L、1 μmol/L、10 μmol/L、100 μmol/L和1 mmol/L,选择胞体清晰、光晕良好、轴突明显的锥体神经元采用膜片钳全细胞记录模式记录I_A的基本电生理特点,记录不同浓度褪黑激素作用下I_A的动力学.结果 与褪黑激素作用前比较,1、10 nmol/L褪黑激素作用后海马锥体神经元I_A幅度升高(P<0.05),其余浓度褪黑激素作用前后海马锥体神经元I_A幅度比较差异无统计学意义(P>0.05);10 nmol/L褪黑激素作用后海马椎体神经元电压门控性瞬时外向钾通道激活曲线的半数激活膜电位及曲线斜率因子与褪黑激素作用前比较差异无统计学意义(P>0.05).结论 生理浓度褪黑激素可增加体外新生大鼠海马椎体神经元I_A.     

吗啡对大鼠海马锥体神经元L-型Ca2+通道电流的影响

目的观察吗啡对大鼠海马锥体神经元L-型Ca^2＋通道电流的影响。方法成年SD大鼠,体重230～270 g,麻醉后快速断头取脑,急性分离海马锥体神经元,12个海马（椎体神经元）随机分为2组（n=6）,吗啡组应用细胞贴附式膜片钳技术记录依次加入不同浓度吗啡[0（未加吗啡）、10^-8、10^-7、10^-6、10^-5mol/L]后海马锥体神经元L-型Ca^2＋通道电流及电导;纳洛酮组加入10^-5mol/L纳洛酮后30min,再依次加入上述不同浓度吗啡,记录此通道电流及电导。结果与未加吗啡比较,吗啡组10^-6、10^-5mol/L吗啡可抑制海马神经元L-型Ca^2＋通道电流,10^-5 mol/L的抑制作用更明显（P＜0．01）;预先加入纳洛酮可阻断吗啡对该通道电流的抑制作用（P＜0．01）;但L-型Ca^2＋通道电导无变化。结论通过作用于μ片受体,10^-6、10^-5 mol/L吗啡可抑制大鼠海马锥体神经元L-型Ca^2＋通道电流,10^-5mol/L的抑制作用更强。     

异丙酚对大鼠海马CA1缺血神经元持续钠电流的影响

目的 探讨异丙酚对大鼠海马CA1缺血神经元持续钠电流的影响。方法 酶消化法急性分离SD大鼠海马CA1锥体细胞,通过低氧和无糖法制备神经元缺血模型,全细胞膜片钳技术记录异丙酚对缺血神经元持续钠电流的影响。结果 神经元缺血5 min后持续钠电流显著增强。异丙酚10μmol/L和100μmol/L均能明显抑制缺血引起的持续钠电流增强(与0μmmol/L组比,P<0.01),此作用为异丙酚100μmol/L较10μmol/L儿更强(P<0.05)。结论 异丙酚能够抑制体外脑缺血时海马神经元持续钠电流,这可能是其产生脑保护作用的机制之一。     

异丙酚对海马锥体神经元钠通道电流的抑制作用

目的 研究异丙酚对海马锥体神经元钠通道电流的影响,探讨脑钠通道在异丙酚麻醉中的作用。方法 酶消化法急性分离SD大鼠(10～14 d)海马锥体神经元,全细胞膜片钳技术记录异丙酚和脂肪乳剂对其钠通道电流的影响。结果 在钳制电位-100 mV时,4组浓度(10、30、50、100μmol/L)的异丙酚分别抑制峰钠电流为:14.4％±8.7％、42.9％±8.8％、67.2％±18.1％和85.1％±14.9％,抑制程度与浓度呈正相关(r=0.993,P<0.01),IC50为32.5 μmol/L。与本研究中较高浓度异丙酚组(50和100μmol/L)对应浓度的脂肪乳剂(ILP50组和ILP100组)对峰钠电流无明显影响。结论异丙酚对脑钠通道电流有明显的抑制作用,且呈剂量依赖性,提示脑钠通道的抑制在异丙酚的麻醉中可能起一定作用。     

芬太尼对大鼠海马锥体神经元GABAA受体的作用

目的探讨芬太尼对大鼠海马锥体神经元GABAA受体的作用。方法采用酶-机械法急性分离出生3-10 d SD大鼠的海马锥体神经元,用全细胞膜片钳技术记录锥体神经元GABAA受体介导的Cl-电流(GABA电流,IGABA),不同浓度的GABA和1 μmol/L GABAA受体特异性拮抗剂荷包牡丹碱诱发并确定IGABA;用不同浓度的芬太尼和1．0 μmol/L μ受体特异性拮抗剂CTAP评价芬太尼对30 μmol/L GABA诱发IGABA的影响。结果 GABA诱发内向电流(IGABA),其EC50为23．73μmol/L,荷包牡丹碱阻断IGABA;芬太尼剂量依赖性地抑制IGABA,其EC50为0．011 μmol/L,并缩短IGABA的脱敏感时间常数τdes;CTAP使芬太尼抑制GABA诱发IGABA的EC50升至0．414μmol/L;芬太尼不改变IGABA的翻转电位 ECl-(-3．0mV)。结论芬太尼可抑制大鼠海马锥体神经元GABAA受体的功能。     

普鲁卡因对鼠大脑皮层锥体神经元延迟整流型钾离子通道电流的影响

目的　观察不同浓度普鲁卡因对新生鼠大脑皮层锥体神经元延迟整流型K+ 通道电流的影响 ,以探讨其中枢作用机理。方法　采取神经元急性分离法分离SD大鼠皮层锥体细胞 ,运用膜片钳内面向外式记录技术 ,记录不同浓度普鲁卡因对延迟整流型K+ 通道电流参数。根据普鲁卡因浓度不同分为 8个组 ,其浓度分别为 0、0 2、0 4、0 8、1 6、2 4、3 2和 4 0mmol/L ,每组以 7个不同钳制电压观测 1 1个样本。结果　当普鲁卡因浓度增至 0 4mmol/L时 ,其Ⅰ Ⅴ关系线斜率最小 ,通道的电导减至最低 [(36± 1 4) pS] ;此后随药物浓度的增加 ,Ⅰ Ⅴ关系线斜率增大 ,电导增加 ,并在2 4mmol/L增至最高 [(60± 2 0 )pS]。结论　普鲁卡因对鼠大脑皮层锥体神经元延迟整流型K+ 离子通道电流、电导的影响随其浓度不同表现为抑制与兴奋双相作用     

雄激素对前列腺癌细胞内游离钙离子浓度的影响

目的 探讨5α-双氢睾酮(DHT)对前列腺癌LNCaP细胞内游离钙离子浓度([Ca2+]i)的影响及其机制.方法 应用Fura-2/乙酸甲酯(Fura-2/AM)Ca2+荧光探针法结合MiraCal荧光成像系统实时检测不同浓度的DHT刺激以及Ca2+通道阻滞剂干预后LNCaP细胞[Ca2+]i的变化.结果 DHT能快速诱导[Ca2+]i升高,在20 s～3 min升至峰值.DHT浓度为1、10、100和1000nmol/L时能诱导[Ca2+]i分别从基础值(28±5)、(29±5)、(28±4)和(28±9)nmol/L上升至峰值31±3(P＞0.05)、65±9(P＜0.01)、193±33(P＜0.001)和(208±42)nmol/L(P＜0.001).DHT浓度为100和1000 mol/L时,[Ca2+]i峰值间差异无统计学意义(P＞0.05).细胞外液无Ca2+时,1000 nmol/L DHT未能诱导[Ca2+]i升高.细胞膜L-型电压门控Ca2+通道阻滞剂维拉帕米(50μmol/L)、地尔硫卓(100 μmol/L)或硝苯地平(5 mmol/L)37℃孵育细胞5 min后,能完全抑制1000nmol/L DHT诱导的[Ca2+]i升高.磷脂酶C抑制剂新霉素(1 mmol/L)37 ℃孵育细胞5 min或兰尼定受体阻滞剂普鲁卡因(50 mmol/L)37℃孵育细胞3 min后,对1000 nmol/L DHT诱导的[Ca2+]i升高没有影响.结论 DHT可快速、剂量依赖性诱导LNCaP细胞[Ca2+]i升高;DHT诱导LN-CaP细胞[Ca2+]i的升高是细胞外Ca2+经细胞膜L-型电压门控Ca2+通道流入细胞内实现的,细胞内贮钙库未释放Ca2+.     

吗啡对人神经母细胞瘤SK-N-SH细胞L-Ca2+ 通道的作用

目的 观察吗啡对人神经母细胞瘤SK-N-SH细胞L-Ca2+通道的影响.方法 培养人神经母细胞瘤SK-N-SH细胞,应用细胞贴附式膜片钳技术记录在不同浓度吗啡(0、10-8 mol/L、10-7 mol/L、10-6 mol/L、10-5 mol/L)作用下SK-N-SH细胞L-Ca2+通道电流和电导变化.预先加入10-5 mol/L纳洛酮后,再依次加入上述不同浓度吗啡,记录此通道电流和电导.结果 10-7 mol/L、10-6 mol/L、10-5 mol/L吗啡可抑制SK-N-SH细胞L-型Ca2+通道电流,吗啡浓度为10-6mol/L、10-5 mol/L时抑制作用更明显(P<0.01),具有浓度依赖性;各浓度吗啡对L-Ca2+通道电导无影响;预先加入纳络酮,可阻断吗啡对该通道的抑制作用.结论 吗啡可抑制SK-N-SH细胞上L-Ca2+通道电流,对该通道电导无作用.纳络酮可翻转吗啡的作用,其抑制作用可能通过μ阿片受体产生.     

皮质酮复合异丙酚对大鼠海马 CA1区长时程抑制的影响

目的探讨皮质酮复合异丙酚对大鼠海马CA1区锥体神经元产生的长时程抑制(LTD)的影响。方法制备Wistar大鼠400μm厚度的海马脑片,随机分为5组:对照组、脂肪乳剂组、异丙酚组、皮质酮组、皮质酮 异丙酚组。对照组不加任何药物,脂肪乳剂组、异丙酚组、皮质酮组、皮质酮 异丙酚组分别以100μmol/L脂肪乳剂、100μmol／L异丙酚、10μmol／L皮质酮、10μmol／L皮质酮复合100μmol／L异丙酚预孵脑片60min,然后给予低频刺激(LFS),记录LTD的表达情况。结果各组海马CAI区锥体神经元给予LFS后,都产生LTD,与对照组相比,脂肪乳剂组给予LPS后10-40minEPSC值没有明显变化,异丙酚组、皮质酮组和皮质酮 异丙酚组给予LPS后10-40min的EPSC值明显降低(P<0.05),且皮质酮 异丙酚组给予LPS后10-40min兴奋性突触后电流与异丙酚组和皮质酮组相比,差异有统计学意义(P<0.05)。结论100μmol／L异丙酚或10μmol/L皮质酮都使大鼠海马CA1区锥体神经元LTD表达易化,二药复合应用时则进一步增强LTD的表达。     

大鼠结肠cajal细胞库容性Ca^2＋内流检测的临床价值

目的:通过检测大鼠结肠cajat细胞中库容性Ca2+内流(capacitative Ca2+ entry,CCE),探讨导致大鼠结肠cajal细胞内Ca2+稳态失衡的信号途径,为治疗相关疾病提供新的思路.方法:荧光探针Fu-ra-2/AM标记细胞内游离Ca2+后,用荧光分光光度计检测乙二醇-双(2-氨基乙基醚)四乙酸(EGTA)耗竭胞内钙库后对分离的大鼠结肠cajal细胞Ca2+浓度([Ca2+])的影响.结果:在无Ca2+缓冲液中加入EGTA(10 mmol/L),细胞内[Ca2+]由静息时的(69.37±3.02)nmol/L升高至(272.52±5.21)nmol/L;继之,向细咆外液中引入1.5 mmol/L和3.0 mmol/L的氯化钙溶液,导致细胞内Ca2+进一步升高为(466.43±4.63)nmol/L和(977.43±3.27)nmol/L;且此升高效应对维拉帕米(5 μmol/L)不敏感,但可被2APB(20～100 μmol/L)抑制.结论:酶解法并结合密度离心法分离的大鼠结肠cajal细胞上存在胞内钙库耗竭激活的Ca2+内流.这对于研究钙通道活性改变、预防和控制因胃肠动力紊乱所致的消化系统疾病具有重要意义.     

右美托咪啶后处理对大鼠离体心脏缺血再灌注时线粒体损伤的影响

目的 探讨右美托咪啶后处理对大鼠离体心脏缺血再灌注时线粒体损伤的影响.方法 健康雌性Wistar大鼠,体重220～250 g,成功制备Langendorff离体灌注模型的40个心脏随机分为5组(n=8):缺血再灌注组(A组)、右美托咪啶10 nmol/L组(B组)、右美托咪啶100 nmol/L组(C组)、线粒体通透性转换孔开放剂苍术苷组(D组)及右美托咪啶联合苍术苷组(E组).离体心脏经K-H液平衡灌注20 min后,采用全心停灌40 min再灌注60 min的方法制备离体心脏缺血再灌注模型.于再灌注即刻B组、C组、D组和E组分别灌注含10 nmol/L右美托咪啶、100 nmol/L右美托咪啶、20μmol/L苍术苷、100 nmol/L右美托咪啶和20 μmol/L苍术苷的K-H液10 min.再灌注结束即刻取心尖组织,分离线粒体,测定SOD、Na+ -K+ -ATP酶、Ca2+-ATP酶活性和MDA和Ca2+含量.结果 与A组比较,B组和C组线粒体SOD、Na+ -K+ -ATP酶和Ca2+ -ATP酶活性升高,MDA和Ca2+含量降低(P＜0.05),D组和E组上述指标比较差异无统计学意义(P＞0.05);与C组比较,D组和E组线粒体SOD、Na+-K+-ATP酶和Ca2+ -ATP酶活性降低,MDA和Ca2+含量升高(P＜0.05),B组上述指标比较差异无统计学意义(P＞0.05).结论 右美托咪啶后处理可减轻大鼠离体心脏缺血再灌注时的线粒体损伤,其机制可能与抑制线粒体通透性转换孔开放有关.     

婴幼儿体外循环对红细胞内钙离子浓度的影响

目的 通过比较先天性心脏病患儿体外循环(cardiepulmonary bypass,CPB)过程中红细胞内外钙离子浓度的变化,探讨CPB对红细胞形态和功能的影响.方法 分别于CPB前、中、后抽取26例先天性心脏病患儿的中心静脉血检测血浆游离钙离子浓度,同时采用Fluo-3-AM测定红细胞内的钙离子浓度.结果 CPB中血浆钙离子浓度由CPB前的(1.24±0.06)mmol/L降低至(0.99±0.05)mmol/L,CPB后钙离子浓度又恢复至(1.28±0.08)mmol/L;CPB过程中红细胞内钙离子浓度则维持了相对稳定[CPB前(138±72)nmol/L;CPB中(145±70)nmol/L;CPB后(115±83)nmol/L].结论 CPB期间红细胞内钙离子浓度无显著变化.     

Ryanodine receptor and capacitative Ca2+ entry in fresh preglomerular vascular smooth muscle cells

BACKGROUND: A multiplicity of hormonal, neural, and paracrine factors regulates preglomerular arterial tone by stimulating calcium entry or mobilization. We have previously provided evidence for capacitative (store-operated) Ca2+ entry in fresh renal vascular smooth muscle cells (VSMCs). Ryanodine-sensitive receptors (RyRs) have recently been identified in a variety of nonrenal vascular beds. METHODS: We isolated fresh rat preglomerular VSMCs with a magnetized microsphere/sieving technique; cytosolic Ca2+ ([Ca2+]i) was measured with fura-2 ratiometric fluorescence. RESULTS: Ryanodine (3 micromol/L) increased [Ca2+]i from 79 to 138 nmol/L (P = 0.01). Nifedipine (Nif), given before or after ryanodine, was without effect. The addition of calcium (1 mmol/L) to VSMCs in calcium-free buffer did not alter resting [Ca2+]i. In Ca-free buffer containing Nif, [Ca2+]i rose from 61 to 88 nmol/L after the addition of the Ca2+-ATPase inhibitor cyclopiazonic acid and to 159 nmol/L after the addition of Ca2+ (1 mmol/L). Mn2+ quenched the Ca/fura signal, confirming divalent cation entry. In Ca-free buffer with Nif, [Ca2+]i increased from 80 to 94 nmol/L with the addition of ryanodine and further to 166 nmol/L after the addition of Ca2+ (1 mmol/L). Mn2+ quenching was again shown. Thus, emptying of the sarcoplasmic reticulum (SR) with ryanodine stimulated capacitative Ca2+ entry. CONCLUSION: Preglomerular VSMCs have functional RyR, and a capacitative (store-operated) entry mechanism is activated by the depletion of SR Ca2+ with ryanodine, as is the case with inhibitors of SR Ca2+-ATPase.     

细胞外信号调节蛋白激酶介导醛固酮诱导的肾小球系膜细胞增殖

Objective To determine the role of extracellular signal-regulated kinases (ERK1/2) in aldosterone-induced rat mesangial cells (RMCs) proliferation. Methods RMCs were obtained from intact glomeruli of 4- to 6-week-old Sprague-Dawley rats and characterized according to published methods. RMCs between passages 5 and passages 10 were used. Protein levels of mineralocorticoid receptor(MR) in RMCs were analyzed by Western blotting. The cells were divided into the following groups: control group, PD98059(10 ?滋mol/L) group, eplerenone (1 ?滋mol/L) group, aldosterone (100 nmol/L) group, aldosterone (100 nmol/L) ＋PD98059 (10 ?滋mol/L) group, aldosterone(100 nmol/L)＋eplerenone (1 ?滋mol/L) group. ERK1/2 activity was measured by Western blotting. Cell proliferation of RMCs was evaluated by [3H]-thymidine uptake measurements. Results MR protein expression in RMCs was confirmed by Western blotting. Aldosterone activated ERK1/2, and the maximal ERK1/2 activation induced by aldosterone was at a concentration of 100 nmol/L. Aldosterone (100 nmol/L)-induced activation of ERK1/2 peaked at 10 minutes (P<0.05). Pretreatment with a selective MR antagonist eplerenone (1 ?滋mol/L) significantly attenuated aldosterone-induced ERK1/2 phosphorylation. Aldosterone (100 nmol/L) treatment for 30 hours increased [3H]-thymidine incorporation of RMCs (135%±8% of controls, P<0.05). Cellular proliferation induced by aldosterone could be prevented by pretreatment with eplerenone or an ERK (MEK) inhibitor PD988059. Conclusion Aldosterone induces RMCs proliferation through MR and ERK1/2 activation, which may contribute to the pathogenesis of glomerular mesangial injury.     

T-型钙通道在利多卡因致神经母细胞瘤SH-SY5Y细胞损伤中的作用

目的 探讨T-型钙通道在利多卡因致神经母细胞瘤SH-SY5Y细胞损伤中的作用.方法 SH-SY5Y细胞离体培养后,采用随机数字表法,将SH-SY5Y细胞随机分为4组,每组66孔,正常对照组(C组):SH-SY5Y细胞继续培养24 h;米贝地尔+SH-SY5Y细胞组(M组):培养液中加入T-型钙通道阻断剂米贝地尔,终浓度5 μmol/L,孵育24 h;利多卡因+SH-SY5Y细胞组(L组):培养液中加入利多卡因,终浓度10 mmol/L,孵育24 h;米贝地尔+利多卡因+SH-SYSY细胞组(ML组):培养液中加入米贝地尔和利多卡因,终浓度分别为5 μmol/L、10 mmol/L,孵育24 h.于开始培养或孵育时(T0)、培养或孵育1、6、12和24 h时测定SH-SY5Y细胞活力和凋亡率,孵育24 h时观察SH-SY5Y细胞形态学.结果 与C组比较,L组和ML组SH-SY5Y细胞活力降低,凋亡率升高(P＜0.05),M组差异无统计学意义(P＞0.05);与M组比较,L组和ML组SH-SY5Y细胞活力降低,凋亡率升高(P＜0.05);与L组比较,ML组SH-SYSY细胞活力升高,凋亡率降低(P＜0.05).L组SH-SY5Y细胞形态学发生改变,ML组SH-SY5Y细胞形态学改变较L组减轻.结论 T-型钙通道参与了利多卡因导致的神经细胞损伤.

Abstract：

Objective To investigate the role of T-type calcium channel in lidocaine-induced neuronal cytotoxicity . Methods SH-SYSY cell line was a gift from cell biology laboratory of our medical university. The cells were cultured in DMEM liquid culture medium at 37℃ in incubator filled with 5% CO2 , and randomly divided into 4 groups ( n = 66 each) : control group (group C)and M, L and ML groups were exposed to 5 μmol/L mibefradil (a T-type calcium channel blocker), 10 mmol/L lidocaine and 5 μmoL/L mibefradil + 10 mmol/L lidocaine for 24 h. Cell morphology was examined by electronic microscopy at 24 h of drug exposure. Cell viability (by MTT) and neuronal apoptosis (by flow cytometry) were detected immediately before and at 1, 6, 12 and 24 h of exposure to mibefradil or/and lidocaine.Results In C and M groups, the cells demonstrated dendritic protrusions, enlarged nerve processes and dense lattice. After being exposed to lidocaine for 24 h, the dendritic protrusions disappeared,the cells decreased in size, shrinked and became round; the cell viability was significantly decreased while the neuronal apoptosis increased. The lidocaine-induced changes were significantly attenuated by co-incubation with mibefradil. ConclusionT-type calcium channel is involved in lidocaine-induced neuronal cytotoxicity.     

异丙酚对大鼠顶叶皮层神经元延迟整流性钾通道电流的影响

目的 探讨异丙酚对大鼠顶叶皮层神经元延迟整流性钾通道电流(IK)的影响.方法 酶消化法急性分离Wistar大鼠顶叶皮层神经元,随机分为4组,分别为不同浓度异丙酚组(P1-4组):培养皿加入异丙酚,终浓度分别为10、30、100和300 μmol/L.于异丙酚给药前,给药后1 min时采用全细胞膜片钳技术,记录顶叶皮层神经元IK,计算IK抑制率,绘制100 μmol/L异丙酚作用下大鼠皮层神经元延迟整流性钾通道电流-电压曲线、延迟整流性钾通道激活曲线和失活曲线.结果 与给药前比较,各组给药后顶叶皮层神经元IK降低(P<0.01);异丙酚对顶叶皮层神经元IK的抑制率呈浓度依赖性(P<0.01);100 μmol/L异丙酚给药后大鼠顶叶皮层神经元延迟整流性钾通道电流-电压曲线下移,但波形、阈电位没有改变;与给药前比较,100 μmol/L异丙酚给药后延迟整流性钾通道激活和失活曲线的半数激活膜电位、曲线斜率因子差异无统计学意义(P>0.05),激活曲线向右移动大约12 mV,失活曲线向左移动大约6 mV.结论 异丙酚可抑制大鼠顶叶皮层神经元IK,且呈浓度依赖性;100 μmol/L异丙酚可减慢延迟整流性钾通道激活,加速其失活.     

瞬时感受器电位香草酸受体1阻断剂减轻利多卡因所致的背根神经节细胞毒性

目的验证瞬时感受器电位香草酸受体1(TRPV1)的阻断剂Capsazepine(CPZ)对利多卡因所致的大鼠背根神经节(DRG)神经元毒性的保护作用。方法取出生后3d的乳大鼠DRG神经元进行体外培养,培养过程中较文献报道减低了胰酶浓度至0.125%以取得较高的细胞活力;然后采用CCK-8法检测0 mmol/L(C1组)、2.5 mmol/L(L1组)、5 mmol/L(L2组)、10 mmol/L(L3组)、20mmol/L(L4组)和40mmol/L(L5组)的利多卡因10min时对DRG神经元活力的影响,计算其半数致死浓度(LC50);再用CCK-8法检测0μmol/L(LC0组)、1μmol/L(LC1组)、10μmol/L(LC2组)及100μmol/L(LC3组)CPZ对10min时LC_(50)的利多卡因所致DRG神经元毒性的影响。结果原代DRG神经元经0.125%胰酶消化后培养纯度达91%;L1、L2、L3、L4、L5组DRG神经元活力明显低于C1组,L3、L4、L5组DRG神经元活力明显低于L1组,L4、L5组DRG神经元活力明显低于L2组,L5组DRG神经元活力明显低于L3和L4组(P0.05),利多卡因作用于DRG神经元10min的LC50为30mmol/L;10μmol/L和100μmol/L CPZ明显减轻LC50利多卡因所致的DRG神经元毒性(P0.05),LC0、LC1、LC2、LC3组DRG神经元活力明显低于C2组(P0.05);LC2、LC3组DRG神经元活力明显高于LC0和LC1组(P0.05),10μmol/L CPZ的效应已达最大,将利多卡因所致的细胞活力下降幅度由50%下调至35%。结论经改进的DRG培养方法可获得高质量原代神经元;利多卡因作用于乳大鼠DRG神经元10min时的LC50为30mmol/L;TRPV1受体阻断剂CPZ能减轻利多卡因对体外培养DRG神经元的毒性。     

Supplementation of IVF medium with melatonin: effect on sperm functionality and in vitro produced bovine embryos

Gamete co‐incubation generates high free radical levels surrounding growing zygotes which may impair subsequent embryo viability. Melatonin eliminates a wide variety of free radicals; hence, we tried to improve in vitro embryo production by adding melatonin to in vitro fertilisation (IVF) media in high (Exp. 1) and low concentrations (Exp. 2), and we evaluated its effect on bull sperm function during IVF co‐incubation time (Exp. 3). In Experiment 1, we supplemented IVF media culture with 0.01, 0.1 and 1 mmol of melatonin, along with a no melatonin control group. In Experiment 2, melatonin levels were reduced to 10, 100 and 1000 nmol, with a no melatonin control group. In Experiment 3, spermatozoa were incubated in IVF media with melatonin (as Exp. 2) and functional parameters were analysed at 0, 4 and 18 h. In Experiment 1, only 1 mmol melatonin showed lesser blastocyst rates than control (C: 23.2 ± 6.7% versus 1 mmol: 2.0 ± 1.7%). In Experiment 2, no statistical differences were found in cleavage percentage, blastocyst percentage and total cell count for any melatonin treatment. In Experiment 3, sperm samples with 1000 nmol melatonin had a significantly higher wobbler (WOB) coefficient, a lower percentage of intact acrosomes, a lower percentage of viable spermatozoa with ROS, greater DNA fragmentation and higher DNA oxidation than controls. Total fluorescence intensity for ROS at 10 nmol melatonin was significantly greater than controls (P < 0.05). IVF media with 1 mmol melatonin is deleterious for embryo development, and in lower concentrations, it modulated sperm functionality, but had no effects on embryo production.     

Remifentanil induces l-type ca2+ channel inhibition in human mesenteric arterial smooth muscle cells

PURPOSE: Remifentanil is known to cause vasodilation at standard anesthetic concentrations. The intracellular mechanisms underlying its vasodilator action may involve the activation of ion channels. The purpose of this study was to examine whether remifentanil inhibits L-type calcium channels (Ca.(L)) and provides dose-dependent effects on L-type calcium channel Ba(2+) currents (I(Ba.L)) in human mesenteric arterial smooth muscle cells. METHODS: Using the whole-cell patch-clamp method, an in depth analysis of the mechanism of the I(Ba.L) induced by remifentanil was performed in cells which were enzymatically isolated from human mesenteric arterial smooth muscle. Ten millimolars Ba(2+) was used to replace 1.5 mM Ca(2+) to increase the amplitude of the inward current through Ca(2+)channels. L-type calcium channel Ba(2+) was elicited during 50 msec depolarizing test pulses (150 msec duration) to +80 mV (10 mV increments) from a holding potential of -60 mV. The effects of remifentanil on Ca.(L) were observed at the following concentrations: 1.21, 4.84, and 19.4 nmol.L(-1) and were compared with control. RESULTS: Remifentanil produced a concentration-dependent block of I(Ba,L) with IC(50) values of 38.90 +/- 3.96 x 10(-3) micromol.L(-1). The L-type calcium channel blocker, nifedipine, antagonized these remifentanil-induced currents. Remifentanil, at all concentrations, shifted the maximum of the current-voltage relationship in the hyperpolarizing direction of I(Ba.L). CONCLUSION: Remifentanil significantly inhibits Ca.(L) channels in a concentration-dependent manner in human mesenteric arteriolar smooth muscle cells.