观察肺动脉平滑肌单细胞舒缩活动的实验模型

为观察肺动脉平滑肌细胞(SMC)的反应性建立了单细胞收缩模型。猪肺动脉SMC在经传代培养后接种于载玻片上,放入实验灌流浴槽,并以胰蛋白酶轻度消化制成单个SMC。高钾(40mmol/L,n=17)、苯肾上腺素(PE,10~(-5)mol/L,n=16)、钙离子携带剂A23187(10~(-7)mol/L,n=15)及PGF_(2α)(10~(-6)mol/L,n=16)可引起单个SMC的收缩,用药后细胞表面积分别缩小29％、15％、23％及15％,其中PE的作用在酚妥拉明(10~(-5)mol/L)存在时被抑制(n=10,P<0.05)。结果提示,培养的肺动脉SMC可作为观察单个细胞舒缩活动的实验模型。     

增强Na+-Ca2+交换对大鼠心脏的正性变力作用及对哇巴因效应的加强

本文采用大鼠乳头肌张力测定及离体心脏灌流技术,研究大鼠心肌Na -Ca2 交换对乳头肌及离体灌流心肌变力性的影响。采用大鼠特异性Na -Ca2 交换激动剂E-4031能剂量依赖性地增加大鼠乳头肌的发展张力(P<0.05,n=6)及离体心脏的心泵功能(P<0.05,n=4);特异性Na -Ca2 交换抑制剂KB-R7943具有相反的效应,并可完全消除E-4031引起的正性变力作用。哇巴因(ouabain,0.5μmol/L)与E-4031(3μmol/L)联合使用,可使乳头肌发展张力由单独使用哇巴因时的0.25±0.03 g升高至0.29±0.04g(P<0.05,n=6);联合用药对大鼠离体心脏心泵功能的影响也强于哇巴因单独作用的效果。本研究结果证实,E-4031通过增强心肌Na -Ca2 交换,对大鼠乳头肌和离体心脏产生正性变力作用;与哇巴因合用时,它们的正性变力作用有相加作用。     

木犀草素改善低温下离体大鼠心脏保存效果的研究

目的:研究木犀草素是否能改善心脏停搏保存液(UW液)对离体大鼠心脏的低温保存效果。方法:将40只成年SD大鼠随机分成4组(n=10):对照组(UW组)、7.5μmol/L木犀草素小剂量组,15μmol/L木犀草素中剂量组及30μmol/L木犀草素大剂量组。利用Langendorff离体心脏灌流法,观察心脏在4℃含或不含木犀草素的UW液中保存12 h复灌60 min后心脏功能及超微结构变化,比较心脏冠脉流量(CF)、心肌含水量及冠脉流出液中磷酸肌酸激酶(CK)的释放量。结果:与对照组比较,添加木犀草素后,复灌期心脏的收缩功能(LVPSP,+dp/dtmax)与心脏舒张功能(-dp/dtmax)、冠脉流量在多个复灌时间点均优于对照组,心率在复灌60 min时也显著优于对照组;复灌过程中磷酸肌酸激酶的漏出量及低温保存后心脏超微结构的损伤也均明显低于对照组;随灌注时间延长木犀草素组心脏结构和功能的改善有剂量依赖性趋势;木犀草素对心肌含水量没有影响。结论:木犀草素能显著改善UW液对离体大鼠心脏的低温保存效果,对心脏有明显的保护作用,以30μmol/L的木犀草素大剂量组作用最显著。     

E4031对慢性心衰大鼠离体心脏功能和心肌细胞内静息Ca^2＋水平的影响

目的：研究具有钠钙交换（NCX）激动作用的药物E 4031对慢性心衰大鼠离体心脏功能和心肌细胞内静息Ca2＋水平的影响。方法：通过腹主动脉缩窄建立大鼠慢性心力衰竭模型;利用Langendorff装置进行离体心脏灌流,检测大鼠心功能及E 4031对血流动力学指标的影响;急性分离心衰大鼠心肌细胞,与钙荧光指示剂fluo3/AM共同孵育后,用激光共聚焦显微镜系统观察E 4031对心肌细胞内荧光强度的影响。结果：缩窄大鼠腹主动脉12周后,langendorff离体灌流检测显示大鼠心功能明显降低;在灌流液中加入10μmol/L E 4031可以使心衰大鼠心脏左室发展压（LVDP）和左室收缩/舒张最大速率（±dp/dtmax）提高;与正常组和伪手术组相比,心衰大鼠心肌细胞内静息钙荧光强度明显升高,和10μmol/L E 4031共孵育后,心衰大鼠心肌细胞静息钙荧光强度呈现短期先升后降过程,然后在较低的水平保持稳定。结论：E 4031可以增强慢性心衰大鼠离体心功能,可能与其增强心肌细胞膜NCX活动,稳定细胞内Ca2＋水平有关。     

不同剂量维拉帕米和普奈洛尔钾停搏液对幼大鼠心功能影响

目的观察含不同剂量维拉帕米和普奈洛尔的钾停搏液对未成年缺血心脏保护效应并与高钾停搏液比较,探讨适宜剂量.方法幼大鼠离体心脏Langendorff法灌流,分6组(n=8)正常组(CON)连续灌流170 min;缺血-复灌组(I-R)灌流(稳定)20min,无糖不充氧台氏液灌3 min停灌27 min连续3阵(缺血90 min),恢复正常灌流(复灌)60min;高钾停搏液(ST)和低(L)、中(M)、高(H)剂量"钾维普"保护组缺血期每阵3 min灌注用不含(ST)和含维拉帕米、普奈洛尔(×10-7mol·L-1)分别为2.0、0.34(L),6.8、1.1(M),20、3.4(H)的ST.Thomas Ⅱ号停搏液.实验过程实时动态检测心肌张力、心率、收缩力、最大收缩和舒张速度、冠脉流量、复搏时间评价,心功能.结果CON组灌流150 min心脏张力稳定,心功能降低;与CON组相比,I-R组缺血40 min后心脏挛缩,复灌后张力高,心搏功能丧失;ST组缺血60 min心肌张力升高,复灌后心功能减弱.与ST组相比,L、M、H"钾维普"呈剂量依赖性降低缺血心肌张力,复灌后心搏强;H组复搏延迟.与CON组相比,L组缺血60 min心脏张力升高,复灌后心搏弱;H组缺血40 min心脏张力低,但复灌后心搏弱;M组缺血90 min心脏张力稳定,复灌后心功能好,心搏幅度超过稳定值.结论含维拉帕米6.8×10-7mol·L-1、普奈洛尔1.1×10-7mol·L-1的钾停搏液保护常温缺血90 min幼大鼠心脏效果最佳.     

血管钠肽对大鼠肠系膜动脉平滑肌细胞Ca2+激活K+通道的作用

目的研究血管钠肽(VNP)对大鼠肠系膜动脉血管平滑肌细胞(VSMCs)Ca2+激活K+通道(Kca)的作用及其机制.方法采用全细胞膜片钳技术观察VNP对Kca的影响,以及HS-142-1、8-Br-cGMP和美蓝(MB)在这一过程中的作用.结果①VNP(10-6 mol/L)显著增强Kca(P＜0.05,n=5).②8-Br-CGMP(10-3mol/L)模拟VNP增强Kca的作用(P＜0.05,n=6).③HS-142-1(2×10-5mol/L)或MB(10-5mol/L)完全阻断VNP增加Kca电流密度的作用.结论VNP通过作用于VSMCs的钠尿肽GC耦联受体,升高细胞内的cGMP水平,激活Kca.     

安心酮对心血管系统作用的实验研究

目的研究安心酮（麻黄苯丙酮,Ｏｘｙｆｅｄｒｉｎｅ）对心脏血流动力学、冠脉流量和急性心肌缺血的药理效应方法观察药物对大鼠离体灌流心脏和在体狗心脏血流动力学、冠脉流量和心肌缺血的影响结果安心酮在一定的剂量范围内明显地增强心肌收缩力和对抗戊巴比妥所致的心力衰竭。但是,高剂量的安心酮可导致心功能抑制,使心肌收缩力和血压降低,β受体阻断剂可阻断其对心脏的激动作用,而安心酮又可拮抗异丙肾上腺素对心脏的兴奋作用。因此认为,本品可能是一种β受体部分激动剂。此外,安心酮可明显增加冠脉流量,减轻结扎冠状动脉引起的心肌梗死,以及拮抗高剂量的脑垂体后叶素所致的急性心肌缺血,这些作用可能是激动血管β２受体、选择性扩张冠脉和降低心肌耗氧量的结果结论安心酮明显地增强心肌收缩力、增加冠脉流量和改善心肌缺血。     

维拉帕米拮抗内皮素所引起的心肌损伤

本实验用10~(-8)mol/L 内皮素灌流离体大鼠心脏,导致平均灌流压,心室内压急剧升高,±LV dP/dt_(max)值显著降低,心肌蛋白漏出和脂质过氧化物形成,心肌钙含量显著增加等严重心脏功能和组织损伤。钙通道阻滞剂维拉帕米,无论与内皮素同时灌流或在内皮素灌流以后应用,都能有效地拮抗内皮素的心脏损伤作用。     

ATP敏感性钾通道、酪氨酸激酶和NF-κB参与高铁血红素诱导的大鼠心肌保护作用

通过诱导血红素氧化酶1(Hemeoxygenase1,HO1)可增强大鼠对抗心肌缺血复灌损伤。本文探讨线粒体ATP敏感性钾通道(MitochondrialATPsensitivepotassiumchannel,mitoKATP)、酪氨酸激酶(Proteintyrosinekinases,PTK)和核因子κB(NuclearfactorkappaB,NFκB)是否参与其中。SD大鼠腹腔注射HO1的诱导剂高铁血红素(hemin)50mg/kg,24h后取离体心脏给予30min缺血和120min复灌。结果发现,hemin可改善缺血-复灌(Ischemiareperfusion,IS)心脏的收缩功能,缩小心肌梗死面积;而HO1的抑制剂ZnPP可抑制hemin引起的HO1活性增加,并抵消hemin诱导的心肌保护作用。在腹腔注射hemin前给予mitoKATP通道阻断剂5HD(5mg/kg),与hemin IS组相比,心脏的收缩功能明显下降,心肌梗死面积增大,LDH和CK释放增加。而在hemin预处理后24h,30min缺血前给予5HD灌流(100μmol/L)同样可阻断hemin诱导的心肌保护作用。hemin诱导的心肌保护作用亦可被PTK抑制剂genistein(10μmol/L)或NFκB抑制剂PDTC(100μmol/L)所取消。结果提示:hemin可诱导心肌HO1增加,保护心肌缺血-复灌性损伤,其作用可能与PTK和NFκB的激活有关,而mitoKATP通道在hemin诱导的心肌保护作用中可能扮演了启动因子和终末效应器双重角色。     

红景天对儿茶酚胺损伤大鼠心肌以及对HIF-1α表达的影响

目的:观察红景天对儿茶酚胺损伤大鼠心肌以及对HIF-1α mRNA表达的影响.方法:SD大鼠45只,随机分为异丙肾组(n=15),红景天组(n=15)和对照组(n=15).第六天大鼠处死,测定血清CK-MB、NO,组织病理学检查.RT-2PCR法检测损伤心肌HIF-1α、VEGF、eNOSmRNA的表达.结果:红景天组心肌坏死病理积分较异丙肾组明显降低(P＜0.01),可减轻异丙肾上腺素所致大鼠CK-MB升高(P＜0.05)并且可使异丙肾上腺素心肌损伤大鼠血清NO升高93.8%(P＜0.01).与对照组相比,异丙肾组HIF-1α、VEGF、eNOSmRNA的表达明显增加(P＜0.001);红景天组与异丙肾组比较,HIF-1α、VEGF、eNOSmRNA表达增高(P＜0.001).结论:红景天对儿茶酚胺所致的心肌损害具有明显保护作用,机制可能与上调HIF-1α表达,促进下游VEGF、eNOS等基因的表达,增加血清NO的释放有关.     

Microglial activation of p38 contributes to scorpion envenomation-induced hyperalgesia

Intraplantar (i.pl.) injection of BmK I, a receptor site 3-specific modulator of voltage-gated sodium channels (VGSCs) from the venom of scorpion Buthus martensi Karsch (BmK), was shown to induce long-lasting and spontaneous nociceptive responses as demonstrated through experiments utilizing primary thermal and mirror-imaged mechanical hypersensitivity with different time course of development in rats. In this study, microglia was activated on both sides of L4–L5 spinal cord by i.pl. injection of BmK I. Meanwhile, the activation of p38/MAPK in L4–L5 spinal cord was found to be co-expressed with OX-42, the cell marker of microglia. The unilateral thermal and bilateral mechanical pain hypersensitivity of rat induced by BmK I was suppressed in a dose-dependent manner following pretreatment with SB203580 (a specific inhibitor of p-p38). Interestingly, microglia activity was also reduced in the presence of SB203580, which suggests that BmK I-induced microglial activation is mediated by p38/MAPK pathway. Combined with previously published literature, the results of this study demonstrate that p38-dependent microglial activation plays a role in scorpion envenomation-induced pain-related behaviors.     

氨甲酰胆碱通过M2毒蕈碱受体增加豚鼠心肌细胞钠钙交换电流

本文旨在研究氨甲酰胆碱(carbachol, CCh)对豚鼠心肌的正性变力性机制。用Axon200A膜片钳放大器观察CCh 对电压钳制下的豚鼠心肌细胞L-型钙电流(ICa)和钠钙交换电流(INa/Ca)的效应。结果表明, CCh(100 μmol/L)分别使正向INa/Ca从对照组的(1.2 ± 0.1) pA/pF 增加到(2.0 ± 0.3) pA/pF,使反向 INa/Ca 从对照组的(1.3 ± 0.5) pA/pF 增加到(2.1 ± 0.8) pA/pF (P<0.01)。CCh对ICa无影响。CCh 对INa/Ca的激动作用可被阿托品和methoctramine所阻断。以上结果提示, CCh 对豚鼠心脏的正性变力作用是通过激动了钠钙交换,而且是 M2 毒蕈碱受体所介导的。     

The saponin monomer of dwarf lilyturf tuber, DT-13, reduces L-type calcium currents during hypoxia in adult rat ventricular myocytes

The saponin monomer 13 of dwarf lilyturf tuber (DT-13), one of the saponin monomers of dwarf lilyturf tuber, has been found to have potent cardioprotective effects. In order to investigate the effects of DT-13 on L-type calcium currents (I(Ca,L)), exploring the mechanisms of DT-13's cardioprotective effects in the condition of pathophysiology, we directly measured the I(Ca,L) during hypoxia in the adult rat cardiac myocytes exposed to DT-13 using standard whole-cell patch-clamp recording technique. Our previous results showed that DT-13 exerted decreasing effects on the I(Ca,L) of the single adult rat cardiac myocytes. In the condition of hypoxia, the current density was inhibited by about 29% after exposure of the cells to DT-13 (0.1 micromol L(-1)) for 10 min, from 6.96+/-1.05 pA/pF to 4.38+/-0.35 pA/pF (n=5, P<0.05). This I(Ca,L)-inhibiting action of DT-13 was concentration-dependent and showed no frequency-dependence. DT-13 up-shifted the current-voltage (I-V) curve. Steady-state activation of I(Ca,L) was not affected markedly, and the half activation potential (V(0.5)) in the presence of DT-13 (0.1 micromol L(-1)) was also not significantly different. DT-13 at 0.1 micromol L(-1) markedly accelerated the voltage-dependent steady-state inactivation of calcium current and shifted the steady-state inactivation curve of I(Ca,L) to the left. In combination with previous reports, these results suggest that there might be a close relationship between the cardioprotective effects of DT-13 and L-type calcium channels in the condition of hypoxia.     

Binding characteristics of BmK I, an alpha-like scorpion neurotoxic polypeptide, on cockroach nerve cord synaptosomes.

In this study, the binding characteristics of BmK I, an alpha-like neurotoxic polypeptide purified from the venom of the Chinese scorpion Buthus martensi Karsch, were investigated on rat brain and cockroach nerve cord synaptosomes. The results showed that BmK I can bind to a single class of noninteracting binding sites on cockroach nerve cord synaptosomes with medium affinity (Kd = 16.5 +/ - 4.4 nM) and low binding capacity (Bmax = 1.05 +/- 0.23 pmol/mg protein), but lacks specific binding on rat brain synaptosomes. BmK AS, BmK AS-1 (two novel sodium channel-blocking ligands), BmK IT (an excitatory insect-selective toxin) and BmK IT2 (a depressant insect-selective toxin) from the same venom were found to be capable of depressing BmK I binding in cockroach nerve cord synaptosomes, which might be attributed to either allosteric modulation of voltage-gated Na+ channels by these toxic polypeptides or partial overlapping between the receptor binding sites of BmK I and these toxins. This thus supported the notion that alpha-like scorpion neurotoxic polypeptides bind to a distinct receptor site on sodium channels, which might be similar to the binding receptor site of alpha-type insect toxins, and also related to those of BmK AS type and insect-selective scorpion toxins on insect sodium channels.     

Electrophysiological characterization of BmK M1, an alpha-like toxin from Buthus martensi Karsch venom

The present study investigates the electrophysiological actions of BmK M1, an alpha-like toxin purified from the venom of the scorpion Buthus martensi Karsch, on voltage-gated Na+ channels. Using the voltage clamp technique, we assessed the BmK M1 activity on the cardiac Na+ channel (hH1) functionally expressed in Xenopus oocytes. The main actions of the toxin are a concentration-dependent slowing of the inactivation process and a hyperpolarizing shift of the steady-state inactivation. This work is the first electrophysiological characterization of BmK M1 on a cloned Na+ channel, demonstrating that this toxin belongs to the class of scorpion alpha-toxins. Our results also show that BmK M1 can be considered as a cardiotoxin.     

The anti-nociceptive effect of BmK AS, a scorpion active polypeptide, and the possible mechanism on specifically modulating voltage-gated Na+ currents in primary afferent neurons

In the present study, we investigated the anti-nociceptive effect and the underlying mechanism of BmK AS, an active peptide purified from scorpion Buthus martensi Karsch. The results showed that BmK AS can significantly relieve formalin-induced two-phase spontaneous flinching response and carrageenan-induced mechanical hyperalgesia. Using the whole-cell patch-clamp recording, exposure of acutely isolated sensory neurons to 500 nM BmK AS produced a one-fold decrease in the number of action potentials (APs) evoked by a ramp of depolarizing current. To investigate the mechanism of action of BmK AS, isolated membrane current and Ca2+ influx on rat primary sensory neurons were examined. BmK AS produced insignificant effect on voltage-dependent I(K) and KCl or caffeine-induced Ca2+ influx, but caused remarkable suppressive effect on tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) I(Na). Further experiments showed that BmK AS reduced the peak TTX-R and TTX-S Na+ conductance in a dose-dependent manner, prompted the voltage-dependent activation, and caused a negative shift of the steady-state inactivation of TTX-R and TTX-S I(Na). Thus, the present results indicate the anti-nociceptive response of BmK AS may be ascribed to its specific modulation of voltage-gated Na+ channels of sensory neurons.     

Cardiac effects of oxytocin: is there a role for this peptide in cardiovascular homeostasis?

Oxytocin is well known for its role in reproduction. However, evidence has emerged suggesting a role in cardiovascular and hydroelectrolytic homeostasis. Although its renal effects have been characterized, the cardiac ones have not been much studied. Therefore, we aimed to investigate the cardiac effects of oxytocin both in vivo and in vitro. In unanesthetized rats (n=6) intravenous oxytocin (1 mug) decreased dP/dt(max) by 15% (P<0.05) and heart rate by 20% (P<0.001), at the first minute after injection. dP/dt(max) was still lower in OT-treated rats than in controls (n=8) after 15 min (P<0.05), while heart rate returned to control values after 5 min. In isolated hearts, oxytocin was able to promote negative inotropic and chronotropic effects. Perfusion with 10(-5), 10(-6) and 10(-7)M oxytocin resulted in approximately 60% (P<0.01), 25% (P<0.01) and 10% (P<0.05) reduction of left ventricle developed pressure, without effect in lower concentrations (10(-10) to 10(-8) M). Also, dP/dt(max) was reduced by 45 and 20% (10(-5) e 10(-6) M; P<0.01), while diastolic pressure raised and heart rate fell only with 10(-5)M oxytocin (P<0.05). Intravenous oxytocin (1 mug; n=6) increased arterial pressure by 22% at the first minute (+23+/-3 mm Hg; P<0.001), returning to control value thereafter. Thus, oxytocin is able to promote directly negative inotropic and chronotropic effects, but its in vivo effect also involves a reflex mechanism, originated from its pressor effect.     

血小板活化因子对豚鼠心室肌细胞动作电位和钾通道的影响

应用全细胞膜片钳技术研究了血小板活化因子(platelet activatingfactor,PAF)对豚鼠心室肌细胞动作电位和钾电流的影响.结果发现,当电极内液ATP浓度为5 mmol/L(模拟正常条件)时,1 μmol/L PAF使APD90由对照的225.8±23.3 ms延长至352.8±29.8ms(n=5,P＜0.05);使IK尾电流在指令电压 30 mV由对照的173.5±16.7 pA降至152.1±11.5 pA(P＜0.05,n=4);使Ikl在指令电压为-120 mV时由对照组的-6.1±1.3 nA降至-5.6±1.1 nA(P＜0.05,n=5);但PAF在生理膜电位范围(-90mV～ 20mV)对IK1没有影响.当电极内液ATP浓度为0mmol/L时,IK·ATP开放(模拟缺血条件),1 μmol/LPAF却显著缩短APD90,由对照的153±24.6 ms缩短至88.2±19.4 ms(n=5,P＜0.01).而用1 μmol/L格列本脲(IK·ATP的特异阻断剂)预处理后,恢复了PAF可显著延长动作电位时程的作用.结果提示,PAF可能扩大缺血心肌和正常心肌细胞动作电位时程的不均一性,是缺血/再灌注性心律失常发生的重要原因.     

多不饱和脂肪酸对成年雪貂心肌钾通道的作用

本研究是在成年雪貂的心肌上研究多不饱和脂肪酸（PUFA）对电压门控钾通道的效应。我们观察到,n-3 PUFA能抑制短时性外向钾电流（Ito)和延迟整流钾电流（IK),而对内向整流钾电流（IK1）则没有明显影响。二十二碳六烯酸（DHA）对Ito和Ik能产生浓度依赖性的抑制作用,其IC50分别为7.5和20μmol/L,但不影响IK1。二十碳五烯酸（EPA）对这三种钾通道的作用与DHA相似。花生四烯酸（5或10μmol/L)先引起IK的抑制,然后引起IK,AA的激活;用环氧合酶抑制剂消炎痛可以阻断花生四烯酸激活IK,AA的作用。不具有抗心律失常作用的单不饱和脂肪酸和饱和脂肪酸都不明显影响这些钾通道的活性。上述实验结果证明,n-3 PUFA能抑制心肌细胞的Ito和IK,但和我们以前报道的PUFA对心肌钠电流和钙电流的作用相比,其对Ito和IK抑制作用的效能较低。n-3 PUFA的抗心律失常效应可能与它们抑制心肌钠、钙、钾通道的作用有关。