瑞芬太尼对人肠系膜小动脉平滑肌细胞钙激活钾电流的影响

目的观察瑞芬太尼对人肠系膜小动脉平滑肌细胞钙激活钾电流(IKCa)的影响,探讨其扩张血管的作用机制。方法用二步酶消化法急性分离人肠系膜小动脉平滑肌细胞,以细胞内离钙、细胞外低钙及细胞外液中加入5mol/L 4-AP分离IKCa,采用全细胞膜片钳技术,观察不同浓度瑞芬太尼(1.2、4.8、19.4 nmol·L~(-1))在不同指令电压下IKCa的变化,并计算激活电压。结果三种浓度瑞芬太尼均可使IKCa增加(P＜0.05或0.01),细胞外液洗脱后IKCa可恢复至给药前水平,但不改变激活电压(P＞0.05)。结论瑞芬太尼激活人肠系膜动脉平滑肌细胞钙激活钾通道,产生扩张血管作用。     

瑞芬太尼对人肠系膜小动脉平滑肌细胞L-型钙通道电流的影响

目的观察瑞芬太尼对人肠系膜小动脉平滑肌细胞(MASMCs)L-型钙通道电流的影响,探讨其扩张血管的机制。方法酶解法分离人肠系膜小动脉单个平滑肌细胞,采用全细胞膜片钳技术,以钡电流作为载流子,观察19.4nmol／L瑞芬太尼在不同钳制电压下以及不同浓度瑞芬太尼在最大激活电压下对MASMCs L-型钡电流(TBa-L)的影响。结果19．4 nmol／L瑞芬太尼可抑制TBa-L,使电流密度-电压曲线上移,最大激活电压及翻转电压均向膜的负电位方向移动(P<0．01)。瑞芬太尼浓度依赖性地抑制TBa-L,其半数最大抑制效应的浓度为(39±4)nmol／L。结论瑞芬太尼浓度依赖性地抑制人肠系膜小动脉平滑肌细胞L-型钙通道,从而产生扩张血管的作用。     

异丙酚对人动脉平滑肌细胞膜大电导钙激活钾通道的影响

异丙酚引起低血压的电生理机制有两种可能，一种可能是抑制交感神经肌接头活动，降低血浆儿茶酚胺的浓度，从而降低血管外周阻力；另一种可能是对内皮的影响和对血管平滑肌细胞的直接作用，Wanersted等在器官水平上证实了异丙酚扩张血管可能与大电导钙激活钾通道（BKCa）有关。本研究拟观察不同浓度异丙酚对人肠系膜动脉血管平滑肌细胞膜BKCa的影响，从分子水平探讨其扩张血管的机制。     

羟丁酸钠对大电导钙激活钾通道的作用

目的:应用膜片钳技术研究羟丁酸钠对新生鼠海马锥体神经元大电导钙激活钾通道的作用。方法:实验选用培养3～7天的锥体神经细胞。在对称性高钾溶液中(140mM),应用细胞贴附式和内面向外式膜片记录单通道电流。结果:(1)该通道电导为190pS左右,随着细胞内钙离子浓度的增加,通道开放概率明显增加,当膜内侧加入钾通道阻断剂TEA后通道活动被阻断;(2)在细胞贴附式膜片下,随着羟丁酸钠浓度的增加,通道开放概率和平均开放时间逐渐增加(P<0.05)。结论:说明羟丁酸钠对该通道具有明显的激活作用。这种激活作用可能与羟丁酸钠发挥全麻作用的分子机制有关。     

大电导钙激活钾通道与心血管保护

背景 大电导钙激活钾通道(large conductance Ca2+-activated K+ channels,BKCa)可能是各类心脏疾病的重要治疗靶点. 目的 主要阐述如何通过调节心肌及血管平滑肌的BKCa通道而发挥保护心肌作用及其相关机制. 内容 在心肌细胞的线粒体上,BKCa通道能够有效地调节线粒体的活性氧、Ca2+和呼吸作用.在血管平滑肌细胞,BKCa通道能调节血管紧张度,促进血管扩张. 趋向 BKCa通道在心肌缺血/再灌注(ischemia/reperfusion,I/R)损伤中有着重要的作用,包括改善心肌功能和减少梗死面积.     

氯胺酮对海马神经细胞大电导钙激活钾通道的作用

目的:研究氯胺酮对海马神经细胞大电导钙激活钾(BK)通道的作用。方法:将培养的神经元置于对称性高钾溶液中,应用膜片钳技术,记录其单通道电流。用循环灌流的方法,观察了不同浓度氯胺酮对BK通道的作用。结果:(1)应用内面向外式膜片,该通道电导为170pS左右,随着胞内钙离子浓度的增加,通道开放概率明显增加(P<0.05),当膜内侧加入钾通道阻断剂(TEA)后,通道活动被阻断;(2)应用细胞贴附式膜片,当浴液内氯胺酮浓度为0.01～0.20mmol/L时,通道开放概率及电流幅值均降低(P<0.05);当其浓度为0.50～1.00mmol/L时,通道开放概率增加(P<0.05),而电流幅值与加药前相比无明显变化(P<0.05)。结论:氯胺酮对BK通道具有双向作用,其抑制作用可能是发挥麻醉效用时产生不良反应的原因之一,而其激活作用可能与全麻作用的分子机制有关。     

不同浓度右美托咪定对大鼠肠系膜动脉平滑肌细胞BKCa通道的影响

目的 评价不同浓度右美托咪定对大鼠肠系膜动脉平滑细胞大电导钙激活钾通道(BKCa)的影响.方法 SD大鼠,雌雄不拘,体重180～220 g,经两步法酶消化分离肠系膜动脉平滑肌细胞.选取10个肠系膜动脉平滑肌细胞,采用单通道内面向外式膜片钳技术进行记录,钳制电压为40 mV,游离钙离子浓度为10-7 mol/L,采用浓度累积法加入右美托咪定,分别于0(基础值)、10-9、10-8、10-7、10-6、10-5 mol/L右美托咪定时记录BKCa开放概率(NPo)、电流幅度(Am)、平均开放时间(To)和平均关闭时间(Tc).结果 与基础值比较,10-7、10-6、10-5 mol/L右美托咪定时NPo升高,且呈浓度依赖性,10-9、10-8、10-7、10-6、10-5 mol/L右美托咪定时Tc缩短(P＜0.05或0.01);与10-9mol/L右美托咪定比较,10-8mol/L右美托咪定时Tc缩短(P＜0.05),不同浓度右美托咪定时Am和To 差异无统计学意义(P＞0.05).结论 10-7、10-6、10-5 mol/L右美托咪定可呈浓度依赖性地激活大鼠肠系膜动脉平滑肌细胞BKCa通道,是其降压作用的机制之一.     

瑞芬太尼对正常和高血压大鼠基底动脉平滑肌细胞的不同作用

目的评价瑞芬太尼对正常和高血压大鼠基底动脉平滑肌细胞上大电导钙激活钾通道(BKCa)和电压门控钾通道(Kv)激活电流的影响。方法自发性高血压大鼠(spontaneously hypertensive rats,SHR)和同源正常血压(wistar-kyoto,WKY)大鼠,采用酶消化法急性分离基底动脉平滑肌细胞,每种大鼠选择6个基底动脉平滑肌细胞,采用全细胞膜片钳技术记录外向电流幅度。加入瑞芬太尼3×10-7mol/L,分别记录所设置的方波刺激(step刺激)方案中所有刺激电压下给药前(基础水平)和给药后电流幅度,并计算净电流=给药后电流幅度-基础值;采用浓度累积法给药,分别记录+60 m V刺激电压下给药前(基础值)和给予10-10、10-9、10-8、10-7、10-6、10-5mol/L瑞芬太尼后电流幅度,计算电流增加率和瑞芬太尼增加基底动脉平滑肌细胞电流幅度的半数有效浓度(EC50);另取每种大鼠6个基底动脉平滑肌细胞,加入瑞芬太尼3×10-7mol/L后分别给予BKCa阻滞剂四乙胺(tetraethylammonium,TEA)和Kv阻滞剂4-氨基吡啶(4-aminopyridine,4-AP),再分别加入其相应的瑞芬太尼混合液,记录每一次给药后的电流幅度。结果两种大鼠基底动脉平滑肌细胞给瑞芬太尼后在0、+20、+40和+60 m V刺激电压下产生的净电流依次明显增大(P0.05);10-10、10-9、10-8、10-7mol/L瑞芬太尼作用下,两种大鼠基底动脉平滑肌细胞外向电流增加率依次明显升高(P0.05);与WKY大鼠比较,瑞芬太尼增加SHR基底动脉平滑肌细胞电流幅度的(EC50)明显升高(P0.05);与基础值比较,两种大鼠基底动脉平滑肌细胞瑞芬太尼给药后电流幅度明显升高,TEA给药后或4-AP给药后电流幅度明显降低(P0.05);与TEA给药后或4-AP给药后比较,TEA+瑞芬太尼给药后或4-AP+瑞芬太尼给药后两种大鼠基底动脉平滑肌细胞电流幅度明显升高(P0.05)。结论瑞芬太尼呈电压依赖性和浓度依赖性激活两种大鼠基底动脉平滑肌细胞BKCa和Kv电流,瑞芬太尼对SHR基底动脉平滑肌细胞上BKCa和Kv激活电流的作用较WKY大鼠弱。     

依托咪酯对鼠皮质神经元大电导钙激活性钾通道的作用

依托咪酯对鼠皮质神经元大电导钙激活性钾通道的作用李明星王泉云曾晓荣作者单位：６１００４１成都市，华西医科大学附属一院〔李明星（现在上海市第一肺科医院麻醉科）、王泉云、曾晓荣〕采用膜片钳技术的吸附式构型和内面向外式构型，实验了依托咪酯对体外培养ＳＤ新生...     

BKCa和PKG在氯胺酮舒张哮喘大鼠离体气管平滑肌中的作用

目的 评价大电导钙激活钾通道(BKCa)和蛋白激酶G(PKG)在氯胺酮舒张哮喘大鼠离体气管平滑肌中的作用.方法 健康SD大鼠,体重250 ～ 300 g,采用卵蛋白致敏法建立哮喘模型,取哮喘大鼠15只,每只大鼠制备2～3条离体气管环.取哮喘大鼠离体气管环36条,采用随机数字表法,将其分为3组(n=12):氯胺酮处理组(AK组)、BKCa阻断剂IBTX+氯胺酮处理组(AKI组)和PKG抑制剂KT-58232+氯胺酮处理组(AKK组);AK组采用0.1 mmol/L乙酰胆碱预收缩大鼠气管环达稳态后,用0.4 g/L氯胺酮孵育15 min; AKI组用乙酰胆碱和氯胺酮孵育前,用3 μmol/L IBTX孵育30 min;AKK组用入乙酰胆碱和氯胺酮孵育前,用2μmol/L KT-5823孵育30 min;采用气管环相连的力-位移换能器测定气管环舒张幅度.结果 与AK组比较,AKI组和AKK组大鼠离体气管平滑肌舒张幅度降低(P＜0.05).结论 氯胺酮可通过激活BKCa和PKG信号通路舒张哮喘大鼠离体气管平滑肌.     

内毒素/脂多糖及烧伤血清对豚鼠肠道平滑肌细胞离子通道的影响

目的观察内毒素/脂多糖(LPS)及烧伤血清对豚鼠结肠带平滑肌细胞钙激活钾通道(KCa)的影响,探讨烧伤后发生胃肠动力障碍的分子电生理机制。方法用急性酶分离法获取单个健康豚鼠结肠带平滑肌细胞,在对称性高钾溶液中采用细胞膜片钳单通道记录技术,分别记录细胞贴附式膜片(电极位于细胞膜外面)和内面向外式膜片(电极位于细胞膜内面)上的电流。引出的电流信号经转换器转换,输入计算机,用离子通道计算机分析系统进行数据处理,并测定以下指标：(1)电流幅值(CA)；(2)开放概率(PO)；(3)开放时间(OT)；(4)关闭时间(CT),以检测其特性并鉴定是否为KCa。确定为KCa后,向浴液中分别加入20、40、60、80、100 mg/L的LPS,观察LPS对两种膜片方式上KCa的影响。在浴液中分别加入正常血清和烧伤血清,观察血清对KCa的影响。结果在对称性高钾溶液中,豚鼠结肠带平滑肌细胞内面向外式膜片上的KCa电导值为(271±7)ps,是高电导的离子通道。随着膜去极化及细胞内Ca~(2+)增加,通道PO增加,该通道可被细胞外低浓度的钾通道阻断剂四乙胺(TEA,1 mmol/L)所阻断,而膜内面较高浓度的TEA(40 mmol/L)对该通道无阻断作用,证实为KCa。当Ca~(2+)为0 mol/L时,向浴液中分别加入20、40、60、80、100 mg/L的LPS,两种膜片方式KCa活性随LPS浓度的增加而增加。40 mg/L以上的LPS对KCa具有明显激活作用,通道PO显著增加(P＜0．05或0．01),用不含LPS的浴液灌流后亦不能回转。两种膜片方式下烧伤血清对KCa有激活作用,而正常血清无此作用。结论LPS和烧伤血清可通过激活肠道平滑肌细胞KCa抑制肠道蠕动。     

Effects of Schisandra chinensis extract on the contractility of corpus cavernosal smooth muscle (CSM) and Ca2+ homeostasis in CSM cells

What's known on the subject? and What does the study add? Schisandra chinensis extract (SCE) has been known to have relaxative effects on penile smooth muscle. A recent study showed that SCE could enhance slidenafil citrate‐induced relaxation of penile corpus cavernosum. The current study investigated the mechanism of action of SCE and its constituents on corporal smooth muscle cells. And this study shows that SCE induced relaxation of CSM primarily through an endothelium independent pathway and the relaxation effects of SCE on corporal smooth muscle are, in part, due to the activation of K⁺ channels and inhibition of TRPC6 channels, resulting in decreased [Ca²⁺].

OBJECTIVE

• ? To evaluate the relaxant effects of Schisandra chinensis extract (SCE) on corporal tissue in the penis and to investigate the mechanism of action of SCE and its constituents on corporal smooth muscle (CSM) cells.

MATERIALS AND METHODS

• ? The fruit of SC was collected and extracted with ethanol. Six SC lignans (schisandrol A, schisandrol B, schisandrin A, schisandrin B, gomisin N, and schisandrin C) were isolated and purified, and the chemical structures were confirmed by ¹H‐nuclear magnetic resonance (NMR) and ¹³C‐NMR data.
• ? Isolated rabbit CSM strips were mounted in an organ‐bath system, and the effects of SCE were evaluated.
• ? To estimate the intracellular Ca²⁺ level ([Ca²⁺]_i), we used a Fura‐2 fluorescent technique, and a conventional whole‐cell patch‐clamp technique was used to measure the calcium‐sensitive K⁺ channels (K_Ca), inward rectifier K⁺ channels (K_IR), and canonical transient receptor potential cation channel 6 (TRPC6) currents.

RESULTS

• ? SCE induced concentration‐dependent relaxation in contracted CSM tissue, and the removal of the endothelium did not significantly affect their relaxation potencies.
• ? In CSM cells, extracellular application of SCE significantly increased whole‐cell K_Ca currents (117.4%) and K_IR currents (110.0%). These effects were completely abolished by charybdotoxin or BaCl₂.
• ? In contrast, carbachol‐induced TRPC6 channel activity was significantly inhibited (87.3%) by SCE in green fluorescent protein‐TRPC6 pcDNA transfected HEK 293 cells. [Ca²⁺]_i measurements showed that SCE effectively reduced basal [Ca²⁺]_i in both cell lines (CSM cells and A7r5 cells) and the [Arg8]‐vasopressin (AVP)‐induced [Ca²⁺]_i increase in A7r5 cells.
• ? Among the six SC lignans, schisandrin A and schisandrin B most effectively attenuated the AVP‐induced [Ca²⁺]_i increase.

CONCLUSIONS

• ? SCE induced relaxation of CSM that occurred primarily via an endothelium‐independent pathway.
• ? The relaxation effects of SCE on CSM were, in part, due to the activation of K⁺ channels and inhibition of TRPC6 channels, resulting in decreased [Ca²⁺]_i.

     

Potassium channels and human corporeal smooth muscle cell tone: further evidence of the physiological relevance of the Maxi-K channel subtype to the regulation of human corporeal smooth muscle tone in vitro

PURPOSE: Recent evidence indicates that the large conductance, voltage dependent, Ca2+ sensitive K channel or Maxi-K has an important role in the modulation of human corporeal smooth muscle tone and, thus, in erectile capacity. We further clarified the contribution of the Maxi-K channel subtype to the generation of contractile responses in isolated human corporeal tissue strips. MATERIALS AND METHODS: We performed pharmacological studies of phenylephrine contracted isolated corporeal tissue strips in the presence and absence of the 2 Maxi-K channel blockers tetraethylammonium chloride (TEA) and charybdotoxin, and the Maxi-K opener NS1619. K channel treatment effects were evaluated using 2 parameters, including 1) the steady state parameter of the empirically determined peak magnitude of the steady state contractile response and 2) the kinetic parameter of time required to achieve half of the peak steady state contractile response or half-time. Electrophysiological studies in freshly isolated and cultured myocytes were performed in parallel to corroborate findings further at the tissue level. RESULTS: Pre-incubating isolated human corporeal tissue strips with 1 mM. TEA and 1 microM. charybdotoxin was associated with an approximate 20% increase in the peak steady state contractile response and a corresponding approximate 20% decrease in the half-time of the phenylephrine induced contractile response. Conversely, pre-incubation with 10 microM. NS1619 produced a significant, approximately 20% decrease in the peak steady state contractile response and an approximate 38% increase in the half-time of the phenylephrine induced contractile response. Adding 30 to 180 microM. NS1619 to phenylephrine pre-contracted smooth muscle strips resulted in a 30% to 50% reduction in steady state contractile tension. No detectable effect of NS1619 was observed in 120 mM. KCl or 100 mM. TEA pre-contracted corporeal tissue strips. Whole cell recordings of freshly isolated and cultured corporeal myocytes confirmed that 30 microM. NS1619 induced a charybdotoxin sensitive hyperpolarizing current mediated by the Maxi-K channel. CONCLUSIONS: These in vitro studies confirm and extend previous observations indicating the importance of the Maxi-K channel for regulating human corporeal smooth muscle tone, and by extension, erectile capacity and function.     

异丙酚对小鼠脑动脉血管平滑肌细胞自发性瞬时外向钾电流的影响

目的 探讨异丙酚对小鼠脑动脉血管平滑肌细胞自发性瞬时外向钾电流的影响.方法 昆明小鼠,体重18～22 g,雌雄不拘,分离脑底部Willis动脉环,经两步法酶消化分离血管平滑肌细胞,选取5个血管平滑肌细胞,采用穿孔全细胞膜片钳技术,于钳制电位为- 30 mV时记录加入56μmol/L异丙酚前后自发性瞬时外向钾电流,分析其幅度、频率、曲线下面积和时间半宽.结果 与给药前比较,给药后自发性瞬时外向钾电流的幅度、频率和曲线下面积升高(P＜0.05或0.01),时间半宽差异无统计学意义(P＞0.05).结论 异丙酚可激活小鼠脑动脉血管平滑肌细胞自发性瞬时外向钾电流,诱发血管平滑肌舒张.