醒脑益智组分中药对β-淀粉样蛋白（1-40）诱导SH-SY5Y细胞损伤的保护作用

目的：探讨醒脑益智组分中药对β-淀粉样蛋白（Aβ_1-40）诱导SH-SY5Y细胞损伤的影响。方法：以SH-SY5Y细胞体外培养为研究对象,Aβ_1-40损伤细胞建立模型,石杉碱甲（6 μg·mL^-1）和不同浓度（70,35,17.5 μg·mL^-1）药物干预,四甲基偶氮唑盐（MTT）法检测细胞存活率;细胞膜损伤测定乳酸脱氢酶（LDH）漏出率;酶联免疫吸附法检测脑源性神经营养因子（BDNF）和肿瘤坏死因子（TNF-α）含量;Hochest 33258染色观察药物对损伤细胞的作用;AnnexinⅤ-FITC/PI双染流式细胞术检测细胞凋亡情况。结果：醒脑益智组分中药能明显改善Aβ_1-40（15 μmol·L^-1）诱导的神经细胞存活率,降低LDH漏出率,改善细胞内BDNF含量,降低TNF-α水平,并抑制细胞的凋亡。结论：醒脑益智组分中药可以改善Aβ_1-40诱导的SH-SY5Y细胞损伤,并抑制其凋亡,其机制可能与改善BDNF水平,调节免疫及相关凋亡基因的表达有关。     

苦参碱对阿霉素所致H9c2心肌细胞损伤的保护作用及与线粒体Na+-K+-ATP酶、Ca2+-ATP酶关系的研究

目的：研究苦参碱对阿霉素所致H9c2心肌细胞损伤的保护作用及其机制。方法：将H9c2心肌细胞培养,取2~3代细胞进行试验,共分为4组。对照组：接受生理盐水作为干预因素;阿霉素组：接受0.5 mg·L^-1阿霉素作为损伤模型;苦参碱+阿霉素组：接受0.5 mg·L^-1阿霉素和不同浓度苦参碱（50,150,200 mg·L^-1）作为干预因素;苦参碱组：接受不同浓度苦参碱（50,150,200 mg·L^-1）作为干预因素。以上各组相应干预24 h后,应用流式细胞仪检测H9c2心肌细胞凋亡水平,应用分光光度法检测线粒体Na⁺-K⁺-ATP酶、Ca²⁺-ATP酶活性,利用JC-1法检测线粒体膜电位。结果：与阿霉素组相比,苦参碱+阿霉素组H9c2心肌细胞凋亡显著减少、线粒体Na⁺-K⁺-ATP酶、Ca²⁺-ATP酶活性显著升高（P<0.05）、线粒体膜电位显著改善。结论：苦参碱对阿霉素所致H9c2心肌细胞有保护作用,减轻心肌细胞凋亡,改善线粒体膜电位、提高线粒体Na⁺-K⁺-ATP酶、Ca²⁺-ATP酶活性。     

PKC/HSP70通路介导白藜芦醇对心肌细胞缺氧/复氧损伤的保护作用

目的：探讨白藜芦醇（resveratrol,RSVL）对心肌细胞缺氧/复氧损伤（anoxia/reoxygenation injury,ARI）的保护作用及机制。方法：分离、纯化SD乳鼠原代心肌细胞,建立缺氧/复氧损伤模型,采用随机数字量表法分为6组：正常对照（CON）组、ARI组、ARI+不同浓度RSVL（25,50,75 μmol·L^-1）预处理组、ARI+RSVL（75 μmol·L^-1）+PKC抑制剂白屈菜红碱（1 μmol·L^-1）组。倒置相差显微镜观察心肌细胞生长形态,自发搏动频率和节律;Hoechst 33258染色计算细胞凋亡率;比色法测定细胞培养液中LDH、CK漏出量及细胞内Ca²⁺-ATPase活性;荧光分光光度计测定细胞内游离钙离子浓度;Western blot检测心肌细胞PKC、HSP70及Ca²⁺-ATPase蛋白水平。结果：不同浓度RSVL与ARI组相比,心肌细胞折光性好,搏动有力而规则,频率在90~115次/分（P<0.01）;呈剂量依赖性减少细胞凋亡（P<0.01）,降低培养液中LDH和CK漏出量（P<0.05或P<0.01）,提高心肌细胞Ca²⁺-ATPase活性及降低细胞内[Ca²⁺]_i量（P<0.05或P<0.01）;心肌细胞PKC、HSP70及Ca²⁺-ATPase蛋白表达增加（P<0.05或P<0.01）。而RSVL的保护作用可被PKC抑制剂白屈菜红碱所取消（P<0.05或P<0.01）。结论： RSVL可能通过PKC/HSP70通路产生对心肌细胞缺氧/复氧损伤的保护作用。     

活性维生素D3对肾小球上皮间质转化的影响研究

目的:观察活性维生素D₃（1,25-（OH）₂D₃）对糖尿病大鼠肾小球足细胞标志蛋白Nephrin,间质转化标志蛋白Snail及间质标志蛋白Desmin表达的影响,探讨1,25-（OH）₂D₃在上皮间质转化中的作用。方法:24只链脲菌素诱导的糖尿病大鼠随机分为糖尿病组（DM）和1,25-（OH）₂D₃治疗组（DD）,后者给与1,25-（OH）₂D₃按3 ng·100 g^-1·d^-1皮下注射。另以10只大鼠作为对照组（NC）。于6周检测血糖（BG）、24 h尿蛋白（24 h UP）、尿液足细胞（UPC）。处死大鼠,PT-PCR及Westernbolt分别测定肾小球Nephrin、Snail、Desmin mRNA和蛋白质的表达。结果:DM组BG、24 h UP较NC组显著升高,而UPC与NC组相比无显著差异。DM组肾小球Nephrin mRNA和蛋白质的表达水平较NC组显著降低,而Snail、Desmin mRNA和蛋白表达显著增加。DD组BG、UPC与DM组相比无显著差异,而24 h UP较DM组显著降低,肾小球Nephrin mRNA和蛋白质的表达较DM组升高,而Snail、Desmin mRNA和蛋白表达降低。结论:1,25（OH）₂D₃上调Nephrin的表达,并减少Snail、Desmin的表达,可抑制上皮间质转化,减轻肾损伤。     

1,25（OH）2D3对人肾小球系膜细胞增殖及PCNA表达的影响

目的探讨1,25-二羟基维生素D3[1,25（OH）₂D₃]对人肾小球系膜细胞中增殖细胞核抗原（PCNA）表达及细胞增殖的影响。方法体外培养人肾小球系膜细胞,取传代培养至3⁸代细胞随机分为4组:正常对照组（加含5%胎牛血清DMEM培养基）,增殖对照组（EGF组,加10μg/L的EGF）,一般干预组[VD组,加10^-8mol/L 1,25（OH）₂D₃],增殖干预组[EGF+VD组,加10μg/L EGF及10^-8mol/L1,25（OH）₂D₃],均作用48 h。流式细胞术检测各组细胞周期;Western blot检测各组PCNA表达的情况。结果 （1）细胞周期。与正常对照组相比,EGF组G₁期细胞明显减少,S、G2/M期细胞增多,增殖指数（PI）较高;VD组G₁期细胞明显增多,S、G₂/M期细胞减少,PI较低;与EGF组相比,VD组和EGF+VD组G₁期细胞增多,S、G₂/M期细胞减少,PI较低,差异均有统计学意义。（2）系膜细胞中PCNA蛋白的表达。与正常对照组相比,EGF组PCNA的表达较高,VD组PCNA的表达较低;与EGF组相比,VD组和EGF+VD组PCNA的表达较低。结论 1,25（OH）₂D₃通过阻滞细胞周期、抑制PCNA的表达,从而抑制人肾小球系膜细胞的增殖。     

Ca2+/calpain信号调控纤连蛋白诱导的MCF-10A乳腺上皮细胞-间质转化的作用

目的：观察Ca²⁺/calpain信号通路在纤连蛋白（FN）诱导MCF-10A乳腺上皮细胞-间质转化（EMT）中的作用。方法：以正常培养MCF-10A乳腺上皮细胞为实验对象。采用比色法检测细胞内Ca²⁺浓度;采用荧光分光光度法检测钙蛋白酶-2（calpain-2）活性;Western blot法检测calpain-2、波形蛋白（vimentin）、E-钙黏着蛋白（E-cadherin）的表达;采用划痕修复实验检测细胞迁移能力;Transwell小室实验检测细胞侵袭能力。结果：FN诱导MCF-10A细胞中Ca²⁺浓度、calpain-2活性表达明显升高,上调vimentin、calpain-2蛋白表达,下调E-cadherin蛋白水平;FN诱导MCF-10A细胞迁移和侵袭能力显著增强。钙离子螯合剂（BAPTA-AM）和氨氯地平（amlodipine）均能显著抑制FN诱导的细胞Ca²⁺浓度、calpain-2活性及表达升高;抑制vimentin蛋白表达上调及E-cadherin蛋白表达下调;抑制FN诱导MCF-10A细胞迁移和侵袭能力增强。结论：BAPTA-AM和Amlodipine均能够抑制FN诱导的上皮间质转化。FN能够诱导MCF-10A乳腺上皮细胞发生EMT,可能与Ca²⁺/calpain信号通路激活有关。     

牡荆素对CVB3诱导的病毒性心肌炎大鼠心肌损伤的保护作用及机制研究

目的：探究牡荆素（Vitexin）对大鼠心肌损伤的保护作用及机制。方法：选择Balb/c新生乳鼠腹腔注射柯萨奇病毒B3（Coxsackie virus B₃，CVB3）建立大鼠病毒性心肌炎模型，分别设置正常未感染组（对照组）、感染CVB3组（模型组）、CVB3+Vitexin（10 mg·kg^-1）组[简称Vitexin（10 mg·kg^-1）组]、CVB3+Vitexin（20 mg·kg^-1）组[简称Vitexin（20 mg·kg^-1）组]和CVB3+Vitexin（50 mg·kg^-1）组[简称Vitexin（50 mg·kg^-1）组]，连续腹腔注射给药1周。HE染色观察心肌细胞损伤水平，TUNEL染色检测肿瘤细胞凋亡。免疫组织化学法和酶联免疫吸附实验（enzyme-linked immunosorbent assay，ELISA）检测心肌炎标记分子和心肌损伤标记物。利用蛋白质免疫印迹（Western Blot）技术检测肿瘤凋亡及其下游靶基因等相关分子表达。结果：与对照组比较，模型组细胞受损严重，炎症因子、心肌损伤标志物及细胞凋亡因子显著升高（P<0.05），TGF-β1及TNF-α蛋白活性显著升高（P<0.05）。相较于模型组，Vitexin处理组心肌细胞形态正常，心肌纤维排列整齐；细胞凋亡显著减少；Vitexin处理组心肌炎症因子、心肌损伤标志物及细胞凋亡因子显著低于模型组（P<0.05）。TGF-β1及TNF-α蛋白活性显著降低（P<0.05）；并且Vitexin高剂量优于低剂量组。结论：Vitexin可以减少细胞凋亡，降低炎症因子水平来保护心肌炎症细胞受到CVB3病毒诱导的损伤；Vitexin可能是通过抑制TGF-β1及TNF-α蛋白活性来发挥影响。     

磷脂酰肌醇蛋白多糖3基因沉默联合三氟拉嗪对肝癌细胞增殖和凋亡的影响

目的 研究磷脂酰肌醇蛋白多糖3（GPC3）基因沉默联合三氟拉嗪（TFP）对肝癌细胞增殖和凋亡的影响。方法 将肝癌细胞HepG2分为对照组、TFP组、TFP+si-NC组和TFP+si-GPC3组。TFP组用20μmol·L^-1TFP处理,TFP+si-NC组转染si-NC且用20μmol·L^-1TFP处理,TFP+si-GPC3组转染si-GPC3且用20μmol·L^-1TFP处理,对照组不转染且不用TFP处理。用细胞计数试剂盒-8（CCK-8）法检测不同浓度TFP对HepG2细胞生长的抑制作用,计算药物的半抑制浓度(IC₅₀)并选择最适药物浓度。用流式细胞仪检测各组细胞凋亡情况;用蛋白质印迹（Western blot）法检测细胞凋亡相关蛋白裂解的胱天蛋白酶3（Cl-caspase-3）、B细胞淋巴瘤-2相关X蛋白（Bax）的表达;用实时荧光定量聚合酶链反应（qRT-PCR）和Western blot检测GPC3基因和蛋白的表达;用CCK-8法检测各组细胞活力。结果 对照组、TFP组、TFP+si-...     

半枝莲黄酮对复合Aβ所致大鼠皮层细胞凋亡抑制作用及线粒体凋亡通路的调节机制

目的:探讨半枝莲黄酮(SBF)对淀粉样蛋白25-35(Aβ_25-35)联合三氯化铝(AlCl₃)和人类重组转移因子-β1(RHTGF-β1)(复合Aβ)所致大鼠皮层细胞凋亡及线粒体凋亡通路中细胞色素-C及其下游凋亡因子Apaf-1、Caspase-9和Caspase-3的调节机制。方法:雄性SD大鼠,脑室注射复合Aβ建立记忆障碍模型,术后第45天进行记忆障碍模型筛选,模型成功大鼠随机分为模型对照组和SBF 35,70,140 mg·kg^-1剂量组。SBF药物组大鼠连续灌胃给药36 d,模型和假手术组连续灌胃生理盐水36 d。吉姆萨染色(Giemsa)法检测皮层细胞凋亡情况;RT-PCR法检测皮层细胞胞液中细胞色素-C(Cyt-C)、凋亡蛋白酶激活因子-1(Apaf-1)和半胱氨酸天冬氨酸蛋白酶-9(Caspase-9)mRNA水平;Western blotting检测皮层细胞胞液中半胱氨酸天冬氨酸蛋白酶(Caspase-3)蛋白水平。结果:大鼠脑室注射复合Aβ能够引起大鼠皮层细胞凋亡,伴随着胞液中Cyt-C、Apaf-1和Caspase-9 mRNA及Caspase-3蛋白表达的增加。而SBF可显著抑制复合Aβ所致大鼠皮层细胞凋亡、逆转胞液中Cyt-C、Apaf-1、Caspase-9和Caspase-3表达的增加(P<0.01)。结论:SBF通过减少线粒体对Cyt-C的释放及逆转Apaf-1、Caspase-9和Caspase-3的表达抑制复合Aβ所致大鼠皮层细胞凋亡。     

太白楤木总皂苷对H2O2诱导LO2细胞损伤的修复作用及机制研究

目的 观察太白楤木总皂苷(TSAT)对H₂O₂诱导的LO₂细胞损伤的修复作用，并探讨其作用机制。方法 将培养的LO₂细胞分为对照组、H₂O₂模型组和TSAT不同浓度处理组，通过MTT法进行TSAT浓度筛选，检测TSAT对LO₂细胞增殖的影响，倒置显微镜下观察LO₂细胞形态变化，检测TSAT对LO₂细胞丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)、丙二醛(MDA)和乳酸脱氢酶(LDH)水平的影响，Hoechst染色观察LO₂细胞核改变，流式细胞术检测凋亡率，Western blot法检测凋亡蛋白Bcl-2、Bax、caspase 9和caspase 3的表达。结果 当TSAT浓度小于20μg·mL^-1时对细胞具有保护作用，选用10、15、20μg·mL^-1 TSAT进行后续实验。...     

Heptachlor epoxide induces a non-capacitative type of Ca2+ entry and immediate early gene expression in mouse hepatoma cells

The effects of the organochlorine (OC) liver tumor promoter heptachlor epoxide (HE) and a related non-tumor promoting OC, delta-hexachlorocyclohexane (δ-HCH), on the dynamics of intracellular calcium (Ca²⁺) were investigated in mouse 1c1c7 hepatoma cells. HE induced a non-capacitative, Ca²⁺ entry-like phenomenon, which was transient and concentration-dependent with 10 and 50 μM HE. The plasma membrane Ca²⁺ channel blocker SKF-96365 antagonized this HE-induced Ca²⁺ entry. δ-HCH failed to induce Ca²⁺ entry, rather it antagonized the HE-induced Ca²⁺ entry. Both HE and δ-HCH induced Ca²⁺ release from endoplasmic reticulum (ER) at treatment concentrations as low as 10 μM; at 50 μM, the former induced 5× as much Ca²⁺ release as the latter. The HE-induced Ca²⁺ release from the ER was antagonized using the IP₃ receptor/channel blocker xestospongin C, suggesting that HE induces ER Ca²⁺ release through the IP₃ receptor/channel pore. These results show that the effect of HE on cellular Ca²⁺ mimics that of mitogens such as epidermal and hepatocyte growth factors. They also provide insight into the similarities and differences between tumorigenic and non-tumorigenic OCs, in terms of the mechanisms and the extent of the [Ca²⁺]_i increased by these agents.     

Calcium-mobilizing receptors

In a wide variety of cells, activation of certain surface membrane receptors results in a rise in intracellular Ca²⁺ due to release of intracellular Ca²⁺ stores, and to an increased rate of Ca²⁺ entry. James Putney summarizes some current ideas about the mechanisms by which Ca²⁺-mobilizing receptors act. The intracellular Ca²⁺ release is believed to result from the action of inositol 1,4,5-trisphosphate, generated as a result of the activation by receptors of a polyphosphoinositide-specific phospholipase C. Evidence suggests that a guanine nucleotide-dependent regulatory protein may mediate this activation. The regulation of Ca²⁺ entry is less well understood, but may involve a mechanism whereby the emptying of the (1,4,5)IP₃-sensitive pool secondarily activates Ca²⁺ influx across the plasma membrane.     

Adenosine A2 receptor activation facilitates Ca uptake by rat brain synaptosomes

Adenosine has been shown to increase the release of neurotransmitters by stimulation of adenosine A₂ receptors. This effect probably depends on Ca²⁺ entry into presynaptic nerve terminals. In the present work the ability of the mixed adenosine A₁/A₂ agonist, 2-chloroadenosine, to stimulate Ca²⁺ uptake into rat brain synaptosomes was investigated. ⁴⁵Ca²⁺ uptake was induced by 20 μM veratridine. In the absence of other drugs, 2-chloroadenosine (1 μM) decreased ⁴⁵Ca²⁺ uptake into synaptosomes. Blocking the adenosine A₁ receptor with 100 nM of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), 2-chloroadenosine (1 μM) increased rather than decreased the uptake of ⁴⁵Ca²⁺ into synaptosomes. The excitatory effect of 2-chloroadenosine observed in the presence of DPCPX was reversed by 200 nM of ω-agatoxin-IVA, a specific P-type Ca²⁺ channel antagonist, but not by L-type (nifedipine, 100 nM to 1 μM; methoxyverapamil 1-10 μM) or N-type (ω-conotoxin GVIA, 500 nM) Ca²⁺ channel antagonists. The adenosine A_2A selective agonist, 2-p-(2-carboxyethyl)-phenethylamino-5′-N-ethyl-carboxamido-adenosine (CGS 21680), did not significantly modify Ca²⁺ uptake induced by veratridine. In contrast, the selective adenosine A₂ receptor agonist, N⁶-(2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl)-adenosine (DPMA), in concentrations ranging from 10 nM to 1 μM increased Ca²⁺ uptake induced by veratridine. The selective adenosine A₂ receptor antagonist 3,7-dimethyl-1-propargylxanthine (DPMX) at a concentration of 10 μM antagonized the stimulatory effect of DPMA (0.1 μM) on ⁴⁵Ca²⁺ uptake. In conclusion, activation of adenosine A₂ receptors increases Ca²⁺ uptake by synaptosomes depolarized by veratridine, which could explain the increase of neurotransmitter release observed when A₂ receptors are activated.     

Homologous and heterologous desensitisation of somatostatin-induced increases in intracellular Ca and inositol 1,4,5-trisphosphate in CHO-K1 cells expressing human recombinant somatostatin sst5 receptors

The mechanisms responsible for somatostatin (SRIF)-induced increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and subsequent desensitisation were studied in CHO-K1 cells expressing human sst₅ receptors (CHOsst₅ cells). To study the nature of the desensitisation, interactions with uridine triphosphate (UTP) were examined. SRIF (pEC₅₀ 7.10) and UTP (pEC₅₀ 5.14) caused concentration-dependent increases in [Ca²⁺]_i but the SRIF maximum was about 40% of that to UTP. SRIF-, but not UTP-, induced increases in [Ca²⁺]_i were transient and abolished by pertussis toxin. SRIF and UTP caused sustained increases in Ins(1,4,5)P₃ but the SRIF maximum was about 30% of that to UTP. Removal of [Ca²⁺ ]_e attenuated the SRIF-induced peak rise in [Ca²⁺]_i but had no effect on the peak increases in Ins(1,4,5)P₃. UTP-induced increases in [Ca²⁺]_i and Ins(1,4,5)P₃ were attenuated in the absence of [Ca²⁺]_e. Following pre-exposure to SRIF (1 μM) or UTP (100 μM) for 5 min, subsequent SRIF responses were desensitised. Similar results were obtained in the absence of [Ca²⁺]_e. Pre-exposure to SRIF had no effect on subsequent responses to UTP but in the absence of [Ca²⁺]_e, responses to UTP were attenuated. The results suggest that SRIF but not UTP-induced increases in [Ca²⁺]_i in CHOsst₅ cells are mediated by pertussis toxin sensitive G proteins and are caused by an entry of extracellular Ca²⁺ and release from an Ins(1,4,5)P₃ sensitive Ca²⁺ store. Homologous or heterologous desensitisation of agonist-induced increases in [Ca²⁺]_i could be demonstrated in the presence or absence of extracellular Ca²⁺ respectively, and the latter appeared to involve depletion of a common intracellular Ca²⁺ store.     

Ca dependence of the response of three adenosine type receptors in rat hepatocytes

The effect of three different receptor-specific adenosine agonists on the rate of ureagenesis by isolated rat hepatocytes and the dependence on the external free Ca²⁺ concentration ([Ca²⁺]_e) were investigated. In the presence of high [Ca²⁺]_e all adenosine receptor agonists increased ureagenesis to similar levels. However, with low [Ca²⁺]_e the effects of each agonist varied as follows: (i) the adenosine A₁ receptor agonist, 2-chloro-N⁶-cyclopentyl-adenosine, increased ureagenesis depending partially on [Ca²⁺]_e, (ii) the adenosine receptor A₂ agonist, 2-p-(-2-carboxy-ethyl) phenethylamino-5′-N-ethylcarboxyamido adenosine hydrochloride, increased ureagenesis independently of [Ca²⁺]_e and (iii) in contrast, the adenosine receptor A₃ agonist N⁶-2-(-4-aminophenyl) ethyladenosine, increased ureagenesis only in the presence of high [Ca²⁺]_e. The adenosine receptor A₁ antagonist, 1-allyl-3,7-dimethyl-8-phenyl xanthine, inhibited the effect of the adenosine receptor A₁ agonist on ureagenesis, but not the effect of the adenosine A₂ or A₃ receptor agonists. The adenosine A₂ receptor antagonist, 3,7-dimethyl-1-propargylxanthine, inhibited only the effect of the adenosine A₂ receptor agonist. Thus, in addition to A₁ and A₂ type adenosine receptors, rat hepatocytes possess an A₃-like adenosine receptor which responds to the addition of an adenosine A₃ agonist by accelerating ureagenesis a [Ca²⁺]_e dependent manner. Moreover, it was observed that in the presence of extracellular Ca²⁺ each agonist increased [Ca²⁺]_i and this effect was inhibited by the appropriate specific antagonist.     

Mechanisms for resin acid effects on membrane currents and GABA(A) receptors in mammalian CNS

Resin acids from bleached wood pulp are toxic to fish. 12,14-Dichlorodehydroabietic acid (12,14-Cl₂DHA) raises cytoplasmic Ca²⁺ in synaptosomes and blocks neural GABA_A receptors; however, the underlying mechanism remains unclear in these earlier rodent studies. 12,14-Cl₂DHA (50 μM) almost completely blocked native GABA_A currents (rat cortical cultures) but had no significant effect on picrotoxin-sensitive recombinant human receptors in oocytes (1, β2 and γ2L: the most prevalent isoforms in mammalian brain). In oocytes, 12,14-Cl₂DHA failed to produce a calcium-activated chloride current, in contrast to the calcium ionophore ionomycin (10 μM). However, in cultured cortical pyramidal cells, both ionomycin and 12,14-Cl₂DHA produced chloride-selective currents of similar magnitude (presumably secondary to Ca²⁺ release). 12,14-Cl₂DHA was unable to stimulate phosphate labelling of [³H]-inositol in mouse synaptosomes, indicating that the study compound does not cause Ca²⁺ release via an IP₃ mechanism. Calcium pump ATPase inhibition also seems unlikely since thapsigargin did not elevate free calcium in synaptosomes. 12,14-Cl₂DHA clearly blocks GABA_A currents indirectly: we infer that its toxicity may be secondary to the elevations in cytoplasmic Ca²⁺ via an unidentified recognition site (or receptor) found in neuronal cells.     

Effects of isoquinoline derivatives, HA1077 and H-7, on cytosolic Ca level and contraction in vascular smooth muscle

The effects of isoquinoline derivatives, HA1077 (1-[5-isoquinolinesulfonyl]-homopiperazine) and H-7 (1-[5-isoquinoline-sulfonyl]-2-methylpiperazine), on cytosolic Ca²⁺ levels ([Ca²⁺]_i) and muscle tension were examined in vascular smooth muscle of rat aorta. High K⁺ (72.7 mM) and norepinephrine (1 μM) induced a sustained contraction with a sustained increase in [Ca²⁺]_i. HA1077 and H-7 (3–10 μM) inhibited the increse in muscle tension more strongly than the increase in [Ca²⁺]_i. Verapamil (10 μM) completely inhibited the increase in [Ca²⁺]_i and the contraction induced by K⁺ whereas it inhibited the increase in [Ca²⁺]_i more strongly than the contraction due to norepinephrine. The verapamil-insensitive portion of the norepinephrine-induced contraction was inhibited by HA1077 or H-7. In Ca²⁺-free solution, 0.1 μM norepinephrine induced a transient increase in [Ca²⁺]_i and muscle tension. The transient contraction was inhibited by 10 μM HA1077 or 10 μM H-7 without inhibiting the increase in [Ca²⁺]_i. 12-Deoxyphorbol 13-isobutyrate (DPB) (1 μM) caused a sustained contraction, and this contraction was inhibited by HA1077 and H-7 at similar concentrations needed to inhibit the contractions induced by high K⁺ or norepinephrine. In rabbit mesenteric artery permeabilized with Staphylococcus aureus -toxin, 100 μM HA1077 and 100 μM H-7 inhibited the contraction induced by 0.3 μM Ca²⁺. These results suggest that the inhibitory effects of isoquinoline derivatives, HA1077 and H-7, are due to a decrease in [Ca²⁺]_i and in the Ca²⁺ sensitivity of contractile elemenst in vascular smooth muscle.     

Mechanisms of vasoinhibitory effect of cardioprotective agent, CP-060S, in rat aorta

Vasoinhibitory effects of (−)-(S)-2-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-3-[3-[N-methyl-N-[2-(3,4-methylenedioxyphenoxy)ethyl]amino]propyl]-1,3-thiazolidin-4-one hydrogen fumarate (CP-060S), a synthesized cardioprotective agent, were examined. In the rat aortic rings, the contractile responses to cumulative application of angiotensin II, [Arg⁸]-vasopressin (vasopressin), or prostaglandin F₂ were inhibited by CP-060S in a concentration-dependent manner. The Ca²⁺-induced contractions in the presence of vasopressin or prostaglandin F₂ were also inhibited by CP-060S in a concentration-dependent manner. The inhibitory effect of 10⁻⁵ M CP-060S on phenylephrine-induced contraction was as potent as that of 10⁻⁶ M nifedipine, and the combined addition of 10⁻⁶ M nifedipine and 10⁻⁵ M CP-060S showed the effect similar to that of 10⁻⁵ M CP-060S alone. In rat aorta loaded with a Ca²⁺ indicator, fura-PE3, 10⁻⁵ M CP-060S completely inhibited the high K⁺-induced increase in cytosolic Ca²⁺ level ([Ca²⁺]_i) and contraction. In contrast, 10⁻⁵ M CP-060S only partially inhibited the increase in [Ca²⁺]_i and contraction due to phenylephrine or prostaglandin F₂. In the presence of 10⁻⁶ M nifedipine, 10⁻⁵ M CP-060S did not inhibit the increase in [Ca²⁺]_i and contraction induced by prostaglandin F₂. In a Ca²⁺-free medium, the phasic increases in contraction and [Ca²⁺]_i induced by phenylephrine were not affected by 10⁻⁵ M CP-060S. These results suggest that the vasoinhibitory effect of CP-060S in rat aortic rings is due mainly to the inhibition of L-type voltage-dependent Ca²⁺-channels.     

Sarcoplasmic reticulum Ca2+ release channel ryanodine receptor (RyR2) plays a crucial role in aconitine-induced arrhythmias

The present study established a model of RyR₂ knockdown cardiomyocytes and elucidated the role of RyR₂ in aconitine-induced arrhythmia. Cardiomyocytes were obtained from hearts of neonatal Sprague–Dawlay rats. siRNAs were used to down-regulate RyR₂ expression. Reduction of RyR₂ expression was documented by RT-PCR, western blot, and immunofluorescence. Ca²⁺ signals were investigated by measuring the relative intracellular Ca²⁺ concentration, spontaneous Ca²⁺ oscillations, caffeine-induced Ca²⁺ release, and L-type Ca²⁺ currents. In normal cardiomyocytes, steady and periodic spontaneous Ca²⁺ oscillations were observed, and the baseline [Ca²⁺]_i remained at the low level. Exposure to 3 μM aconitine increased the frequency and decreased the amplitude of Ca²⁺ oscillations; the baseline [Ca²⁺]_i and the level of caffeine-induced Ca²⁺ release were increased but the L-type Ca²⁺ currents were inhibited after application of 3 μM aconitine for 5 min. In RyR₂ knockdown cardiomyocytes, the steady and periodic spontaneous Ca²⁺ oscillations almost disappeared, but were re-induced by aconitine without affecting the baseline [Ca²⁺]_i level; the level of caffeine-induced Ca²⁺ release was increased but L-type Ca²⁺ currents were inhibited. Alterations of RyR₂ are important consequences of aconitine-stimulation and activation of RyR₂ appear to have a direct relationship with aconitine-induced arrhythmias. The present study demonstrates a potential method for preventing aconitine-induced arrhythmias by inhibiting Ca²⁺ leakage through the sarcoplasmic reticulum RyR₂ channel.