“非抗菌药物剂量和频次智能化监控系统”的研发

目的研究3,5,4'-三甲基白藜芦醇(trans-resveratrol derivative3,5,4'-trimethoxystilbene,TMS)对豚鼠心室肌细胞钠电流(INa)和钾电流(IK1)的直接作用,探讨其心肌保护作用。方法用全细胞膜片钳技术记录TMS对单个心室肌细胞INa和IK1的作用。结果TMS(10μmol·L-1)可快速抑制豚鼠心室肌细胞INa,用药后3min左右即开始起效,10min时抑制率为(36.8±5.6)%(P<0.005),洗脱后可完全恢复;1,3μmol·L-1TMS未影响INa大小。TMS不改变INa的最大激活电压,也不影响IK1的大小。10μmol·L-1使半数最大失活电压(V1/2)由(-87.0±3.3)mV变化到(-96.7±3.5)mV(P<0.001),使失活曲线斜率(S)由(4.9±0.3)mV变化到(5.4±0.3)mV(P<0.01);使半数最大激活电压(V1/2)(-38.9±1.4)mV变化到(-47.3±1.3)mV(P<0.001),未改变激活S。结论TMS可直接作用于豚鼠心室肌细胞,快速抑制INa,且此作用快速、可逆。     

3,5,4＇-三甲基白藜芦醇对豚鼠心室肌细胞钠电流和钾电流的影响

目的研究3,5,4＇-三甲基白藜芦醇（trans-resveratrol derivative3,5,4＇-trimethoxystilbene,TMS）对豚鼠心室肌细胞钠电流（INa）和钾电流（IK1）的直接作用,探讨其心肌保护作用。方法用全细胞膜片钳技术记录TMS对单个心室肌细胞INa和IK1的作用。结果TMS（10μmol·L^-1）可快速抑制豚鼠心室肌细胞INa,用药后3min左右即开始起效,10min时抑制率为（36.8±5.6）%（P〈0.005）,洗脱后可完全恢复;1,3μmol·L^-1TMS未影响INa大小。TMS不改变INa的最大激活电压,也不影响IK1的大小。10μmol·L-1使半数最大失活电压（V1/2）由（-87.0±3.3）mV变化到（-96.7±3.5）mV（P〈0.001）,使失活曲线斜率（S）由（4.9±0.3）mV变化到（5.4±0.3）mV（P〈0.01）;使半数最大激活电压（V1/2）（-38.9±1.4）mV变化到（-47.3±1.3）mV（P〈0.001）,未改变激活S。结论TMS可直接作用于豚鼠心室肌细胞,快速抑制INa,且此作用快速、可逆。     

雌二醇抑制豚鼠心室肌细胞内向整流和延迟整流钾通道电流

目的:研究雌二醇(Estradiol,Est)对心室肌细胞动作电位(AP)、内向整流钾通道电流(I_(K1))及延迟整流钾通道电流(I_K)的影响。方法:全细胞膜片箝技术。结果:EST 10μmol·L~(-1)使豚鼠心室肌细胞AP时程明显缩短,APD_(50)由给药前(474±71)ms缩短至(330±75)ms(P<0.05),Est 100μmol·L~(-1)使APD_(50)缩短至(229±67)ms(P<0.01),使APD_(90)由(587±60)ms缩短至(418±79)ms(P<0.05)。Est浓度依赖性地抑制I_K尾电流(I_K·tail),10μmol·L~(-1)浓度下,I_K·tail减少53％(P<0.05),100μmol·L~(-1)浓度下,I_K·tail减少80％(P<0.05)。10μmol·L~(-1)以上浓度Est明显抑制I_(K1),在-100mV刺激电压下,内向电流最大抑制为49％(P<0.01);在-40mV刺激电压下,外向电流最大抑制为72％(P<0.01)。同时,Est使I_(K1)翻转电位向负电位方向移位(由-70mV变为-76mV)。结论:Est对豚鼠心室肌细胞I_(K1)和I_K通道具有明显的抑制作用。     

四氢巴马汀对豚鼠心室肌细胞跨膜内向Ca~(2 )电流的抑制作用(英文)

目的:研究四氢巴马汀(THP)对豚鼠心室肌细胞膜Ca~(2 )通道的作用。方法:分离豚鼠心室肌细胞,利用膜片箝技术观察、记录心室肌细胞膜跨膜内向钙电流(I_(Ca))。结果:THP灌流5min后明显抑制豚鼠心室肌细胞内向I_(Ca),其作用呈现剂量-效应关系,并具有频率依赖性。0.1,1,10μmol·L~(-1)的THP使I_(ca)从1.15±0.22,0.91±0.18,1.60±0.42nA(control,n=5)分别降低到0.90±0.21(P<0.01),0.56±0.21(P<0.01),0.83±0.21(P<0.05)nA,抑制率分别为22％,38％和48％,THP的作用易于洗脱。峰值电压为0mV,此时THP作用最明显,THP对较高频率刺激产生的电流抑制作用较强,2Hz时的抑制率高于0.1Hz时的抑制率(P<0.01)。结论:THP具有Ca~(2 )通道阻滞作用。     

KB-R7943对豚鼠心室肌细胞Na~ -Ca~(2 )交换电流的作用

目的　观察KB R7943对豚鼠心室肌细胞Na Ca2 交换电流 (INa Ca)的内向电流成分和外向电流成分的影响。方法　采用缺血再灌时胞内Na 超载的细胞模型 ,在同时记录内向、外向电流的双向离子条件下 ,用膜片钳全细胞技术 ,记录INa Ca的电流 电压关系曲线。结果　 10 -6和 10 -5mol·L-1KB R7943 ,在 5 0mV时 ,对INa Ca的抑制率分别是 2 9 4%和 6 1 7% ;在 - 80mV时抑制率分别是 2 2 1%和 5 6 9%。结论　KB R7943对豚鼠心室肌细胞INa Ca有抑制作用 ,但对外向成分和内向成分的抑制不具选择性。     

盐酸戊乙奎醚对离体豚鼠心室肌细胞IKM3的影响

目的 建立膜片钳实验中适于记录延迟整流钾电流(IKM3)的单个豚鼠心室肌细胞快速分离方法,研究盐酸戊乙奎醚对IKM3的作用.方法 单纯Ⅱ型胶原酶二步灌流法分离豚鼠心室肌细胞;膜片钳全细胞记录模式记录盐酸戊乙奎醚对IKM3的影响.结果 分离的心肌细胞质量高,形态结构完整,成功记录了IKM3;0.2 μmol·L-1盐酸戊乙奎醚在不同钳制电压下对IKM3有不同程度的抑制,试验电压为+50 mV时,可使心室肌细胞上IKM3的电流密度由588.63±189.32 PA降至72.85±41.91 PA,抑制率为86.95%.结论 二步灌流法分离得到的豚鼠心室肌细胞可用于IKM3的研究;盐酸戊乙奎醚能作用于心室肌细胞上的M3受体,减小IKM3的电流密度.     

牛磺酸镁配合物对豚鼠心室肌细胞钠离子和钙离子通道的影响

目的研究牛磺酸镁配合物(TMC)对正常豚鼠心室肌细胞钠电流(INa)和L-钙电流(ICa,L)的影响,旨在探讨其抗心律失常作用的可能机制。方法酶解法分离豚鼠单个心室肌细胞,全细胞膜片钳技术记录单个心室肌细胞的INa和ICa,L。结果TMC50μmol·L-1不影响INa,而100～200μmol·L-1浓度依赖性地抑制INa;TMC50～200μmol·L-1浓度依赖性地增加ICa,L,使ICa,L的稳态失活曲线右移,对ICa,L的稳态激活曲线无影响。结论TMC对心室肌细胞INa的阻滞可能是其抗心律失常作用的机制之一;对ICa,L的促进可能有利于其发挥正性肌力作用。     

胍丁胺对大鼠心室肌细胞L—钙通道电流的影响

目的:观察胍丁胺(Agm)对大鼠心室肌细胞L-型钙通道电流(I_(Ca-L))的影响.方法:以酶解法制备单个心室肌细胞.应用全细胞膜片箝技术记录大鼠单个心室肌细胞钙通道电流.结果:(1)Agm(0.5,1,2mmol/L)可浓度依赖性地降低电压依赖性激活I_(Ca-L)(pA)峰值,其值从1451±236 (对照组)到937±105(n=8,P<0.05),585±74(n=8,P<0.01),和301±156(n=8,P<0.01).(2)Agm 1 mmol/L使用依赖性地阻滞I_(Ca-L)·1 Hz时抑制率为53％±12％(P<0.05),3Hz时为69％±11％(P<0.01).(3)Agm使I-V曲线上移,但对I_(Ca-L)的电压依赖特征、最大激活电压以及I_(Ca-L)稳态激活无明显影响.在Agm 1 mmol/L作用下,半数激活电压(V_(0.5)和斜率参数(k)与对照组相比均无显著性差异.V_(0.5)分别为(-20.2±2.5)mV和(-20.5±2.7)mV,k分别为(3.2±0.4)mV和(3.0±0.5)mV.(4)Agm 1 mmol/L可明显使钙电流稳态失活曲线左移,加速钙通道电压依赖性稳态失活.V_(0.5)分别为(-32±6)mV和(-40±5)mV,k分别为(7.6±O.9)mV和(12.5±1.1)mV(P<0.05).(5)Agm 1mmol/L还使I_(Ca)从失活状态下恢复明显减慢.结论:Agm抑制I_(Ca-L),并主要作用于L-型钙通道的失活状态,表现为钙通道失活加速和从失活状态下恢复减慢.     

Dofetilide对成年豚鼠心室肌细胞钠钙交换的影响

目的　研究dofetilide对豚鼠心室肌细胞Na+ /Ca2 + 交换电流的影响。方法　酶法分离成年豚鼠心室肌细胞 ,全细胞膜片钳方式、斜坡电压刺激程序 (从钳制电位 - 4 0mV去极化至 +6 0mV ,然后以 90mV/s的速率超极化至 - 12 0mV)记录Na+ /Ca2 + 交换电流 (INa/Ca)及dofetilide 0 0 3～ 1 0μmol·L-1对该电流的影响。结果　dofetilide 0 0 3～ 1 0μmol·L-1浓度依赖性地增强Na+ /Ca2 + 交换外向及内向电流 ,对外向电流的EC50 (5 0 %最大效应浓度 )为 0 178μmol·L-1(95 %可信限为 0 0 4 0～ 0 787μmol·L-1) ,对内向电流的EC50 为 0 178μmol·L-1(95 %可信限为 0 0 38～ 0 84 2μmol·L-1)。结论　dofetilide对豚鼠心室肌细胞Na+ /Ca2 +交换外向及内向电流均有浓度依赖性的明显增强作用。     

四肽FMRFa对大鼠心室肌Na^＋／Ca^2＋交换的抑制

目的　研究四肽FMRFa对大鼠单个心室肌细胞Na /Ca2 交换的作用。方法　用膜片钳全细胞记录法测定成年大鼠心室肌细胞Na /Ca2 交换电流 (INa /Ca2 )和其他离子通道电流。结果　FMRFa对大鼠心室肌细胞INa /Ca2 呈浓度依赖性抑制 ,10 0 μmol·L-1浓度时抑制内向和外向INa /Ca2 密度分别达 6 0 1%和 5 6 5 % ,对内向电流及外向电流的IC50 分别为 2 0 μmol·L-1和 34μmol·L-1。FMRFa 5 μmol·L-1抑制INa /Ca2 内向和外向电流密度分别为 38 7%和 34 9% ,但FMRFa 5 μmol·L-1及 2 0 μmol·L-1对L型钙电流、钠电流、瞬时外向电流和内向整流钾电流均无显著抑制作用。结论　FMRFa对大鼠心室肌细胞是一个特异性Na /Ca2 交换抑制剂。     

Adeno-associated virus-mediated vascular endothelial growth factor gene transfer into cardiac myocytes

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that stimulates endothelial cell proliferation, increases endothelial permeability, and promotes collateral vessel formation. We transferred human VEGF gene into rat cardiac myocytes using adeno-associated virus (AAV) vectors and investigated whether VEGF secreted from the transduced cardiac myocytes promoted proliferation of endothelial cells. We produced VEGF-expressing AAV vectors (AAV-VEGF) by the adenovirus-free method. Immunoblotting revealed VEGF protein expression in AAV-VEGF-transduced rat cardiac myocytes. More than 60% of cardiac myocytes were stained positively on immunohistochemical staining using anti-VEGF antibody. Concentration of VEGF in the culture medium of AAV-VEGF-transduced myocytes was increased in a vector dose-dependent manner, and VEGF secretion from the transduced myocytes persisted for > or = 14 days. Thymidine incorporation into human vascular endothelial cells was significantly increased by incubation with the conditioned medium from AAV-VEGF-transduced myocytes. This increased thymidine uptake was significantly inhibited by anti-VEGF antibody. We demonstrated here that AAV-mediated VEGF gene transfer into cardiac myocytes induces the secretion of functional VEGF.     

羧乙基锗倍半氧化物对正常和异丙基肾上腺素损伤培养乳鼠心肌细胞的影响

羧乙基锗倍半氧化物(Ge—132)能促进培养乳鼠心肌细胞DNA和RNA的合成,提高超氧化物歧化酶(SOD)的活性,维持细胞膜结构的稳定。异丙基肾上腺素可使心肌细胞乳酸脱氢酶(LDH)释放增加,细胞搏动停止,超微结构显示肌膜和线粒体严重受损。Ge—132可显著减少异两基肾上腺素引起心肌细胞释放LDH,维持细胞的搏动功能和超微结构的完整。     

氢化可的松琥珀酸钠对人和豚鼠心肌细胞钠电流的影响氢化可的松琥珀酸钠对人和豚鼠心肌细胞钠电流的影响

目的研究氢化可的松琥珀酸钠对人心房肌细胞和豚鼠心室肌细胞钠电流的影响。方法应用酶解法分离单个心肌细胞,应用全细胞膜片钳技术记录氢化可的松琥珀酸钠对钠电流的作用。结果氢化可的松琥珀酸钠(1,3,10 μmol·L^-1)浓度依赖性地抑制人心房肌细胞和豚鼠心室肌细胞钠电流,IC₅₀分别为6.97和8.74 μmol·L^-1。氢化可的松琥珀酸钠不改变i_Na的最大激活电压。氢化可的松琥珀酸钠对钠电流的抑制作用起效快,见于用药后1～3 min。结论氢化可的松琥珀酸钠浓度依赖性地抑制钠电流,此作用出现快,提示可能与非基因组效应有关。     

Enhanced inhibition of L-type calcium currents by troglitazone in streptozotocin-induced diabetic rat cardiac ventricular myocytes

1. Troglitazone, an insulin-sensitizing agent shown to improve cardiac function in both experimental animals and patients with diabetes, inhibits voltage-dependent L-type Ca(2+) currents (I(Ca,L)) in cardiac myocytes, which may underlie its cardioprotective effects. However, inhibition by troglitazone of I(Ca,L) in diabetic cardiac myocytes has not been characterized. 2. Using whole-cell voltage-clamp techniques, I(Ca,L) was measured in ventricular myocytes isolated from 4-6 weeks streptozotocin (STZ)-induced diabetic rats and age-matched control rats. 3. Under control conditions with CsCl internal solution, diabetic myocytes did not differ from control myocytes in membrane capacitance, current density or voltage-dependent properties of I(Ca,L). 4. Troglitazone decreased amplitude of I(Ca,L) in both control and diabetic myocytes in a concentration-dependent manner. This inhibition was more potent in diabetic than in control myocytes; half-maximum inhibitory concentrations of troglitazone measured at a holding potential of -50 mV were 4.3 and 9.5 micromol l(-1), respectively. 5. Troglitazone at 5 micromol l(-1) did not significantly influence the voltage dependency of steady-state inactivation or the inactivation time course of I(Ca,L) in either control or diabetic myocytes. 6. Since troglitazone inhibits I(Ca,L) more effectively in STZ-induced diabetic ventricular myocytes, this agent may prevent cardiac dysfunction in diabetes.     

Taurine transporter in primary cultured neonatal rat heart cells: a comparison between cardiac myocytes and nonmyocytes

In the present study, we examined the characteristics of the taurine transporter and the intracellular taurine content in cultured neonatal heart cells. Primary cultures of cardiac myocytes and cardiac fibroblasts (nonmyocytes) were prepared from 1-day-old Wistar rats. The parameters examined were: (a) intracellular taurine content by the HPLC method, (b) the expression levels of taurine transporter mRNA and protein using northern and western blot analysis, and (c) transporter activity determined by the uptake of 3H-labeled taurine. The taurine content of myocytes was significantly higher (3-fold) than that of nonmyocytes. Taurine transporter mRNA was strongly expressed in both myocytes and nonmyocytes, whereas the magnitude [normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression] of the transporter mRNA expressed in myocytes was lower than that in nonmyocytes. The expression level of transporter protein in myocytes was also lower than that of nonmyocytes. Uptake of radiolabeled taurine into monolayer cultures of heart cells was stimulated markedly by the presence of Na+ in the medium, whereas this uptake was almost abolished in the absence of Na+. The Na+/taurine stoichiometry was 2:1 for both myocytes and nonmyocytes. Kinetic analysis showed that a single saturable system was involved in taurine uptake into both cell types. In myocytes, the apparent K(m) and V(max) values for the transporter were 20.7+/-0.5 microM and 1.07+/-0.01 nmol/10(6)cells/30 min, respectively. Similarly, those of nonmyocytes were 20.3+/-0.7 microM and 0.42+/-0.01 nmol/10(6)cells/30 min. These findings indicated that both myocytes and nonmyocytes expressed an identical taurine transporter with a Michaelis-Menten constant of 20-21 microM and that a higher taurine content in myocytes may be associated with a higher V(max).     

Deciphering the β-adrenergic response in human embryonic stem cell-derived-cardiac myocytes: closer to clinical use?

Human embryonic stem cells (hESCs) are a pluripotent cell type considered to have high potential for the treatment of cardiovascular disease by cell replacement therapies. Several groups have shown that hESCs can be differentiated in vitro into cardiac myocytes, which may be used to facilitate tissue regeneration by injection directly into damaged myocardium. However, several hurdles still need to be overcome before these cells can be used in clinical trials. In particular, because transplanted hESC-cardiac myocytes should integrate fully within the damaged heart, these cells must be functionally compatible with the host myocardium. To assess this aspect of hESC-cardiac myocytes, Brito-Martins et al. (2008) in this issue of the BJP, describe the responses of hESC-cardiac myocytes to beta-adrenoceptor stimulation, compared to those of myocytes from adult human hearts. Data obtained using specific beta-adrenoceptor agonists showed good compatibility of hESC-cardiac myocytes with adult human myocardium in terms of beta-adrenoceptor response.     

Carbon disulfide cytotoxicity on cultured cardiac myocytes of rats

Although epidemiological studies have suggested that carbon disulfide produces cardiovascular effects in occupationally exposed workers, little is known about its cellular mechanism. The purpose of the present study was to evaluate the functional and histological effects on cardiac myocytes cultured under a condition of carbon disulfide exposure. Cardiac myocytes were isolated from neonatal rat ventricles by trypsin, dispersed and cultured for 3 days in a full Dulbecco's Modified Eagle Medium containing 2% calf serum. Then the myocytes (10(6) myocytes/mL) were incubated with carbon disulfide at concentrations of 0, 20, 40, and 80 micromol/mL for 24 h. The beating arrest rate of myocytes for each group was examined, succinodehydrogenase (SDH) activity in the myocardial cells was assessed using a cytochemical method, and a morphological examination was performed. We found that the beating arrest rate of cardiac myocytes increased with increasing exposure levels. Vacuolization and pseudopodia could be seen in the cytoplasm of the exposed group. SDH activity decreased with increasing exposure levels. The results suggest that CS2 has a direct and dose-dependent cytotoxic effect. The biochemical mechanism may be a reduction of the availability of energy (adenosine triphosphate) to cardiac myocytes, resulting in a decrease of contractility by lack of energy.     

HYPERGLYCAEMIA ABOLISHES THE ANTIHYPERTROPHIC EFFICACY OF BRADYKININ IN RAT VENTRICULAR MYOCYTES

1. Bradykinin inhibits hypertrophy of rat ventricular myocytes, but only in the presence of endothelial cells. 2. The influence of hyperglycaemia on the ability of bradykinin to prevent hypertrophy was investigated in adult rat ventricular myocytes cocultured with bovine aortic endothelial cells (BAEC). 3. In myocytes cocultured with normal BAEC, angiotensin II (AngII; 1 mumol/L) significantly increased [3H]-phenylalanine incorporation (an in vitro marker of hypertrophy) by 32 +/- 2%. This was abolished by bradykinin (10 mumol/L). 4. Pretreatment of BAEC with high glucose (25 mmol/L for 24 h) prior to coculture with myocytes reduced the antihypertrophic effect of bradykinin, but did not modulate the hypertrophic effect of AngII. 5. Pretreatment of BAEC with hyperglycaemia abolishes the antihypertrophic efficacy of bradykinin in rat ventricular myocytes cocultured with BAEC. This has implications for the action of angiotensin-converting enzyme inhibitors.